HNCCH-TOCSY,a triple resonance experiment for the correlation of backbone 13Cα and 15N resonances with aliphatic side-chain proton resonances and for measuring vicinal 13CO, 1Hβ coupling constants in proteins

Summary A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and -carbons in uniformly ¹³C¹⁵N-labeled proteins. In-phase ¹³C magnetization is transferred to the aliphatic side-chain protons via the side-chain carbons using a CC-TOCSY mixing sequence. Thus, the experiment alleviates the resonance assignment process by providing information about the amino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from ¹³C to ¹H leads to E.COSY-type cross peaks, from which the ³J_{H co} coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin.     

鲑鱼生长激素基因分泌型表达质粒的构建

生长激素(GH)是动物垂体前叶分泌的一种多肽类激素.应用分子重组及PCR等技术,构建了一种鲑鱼生长激素基因分泌型表达质粒pOsGH153,使编码鲑鱼生长激素成熟肽的序列克隆在大肠杆菌分泌型表达载体PIN-Ⅲ-ompA内,直接位于编码大肠杆菌外膜蛋白A信号肽序列的下游,在Lpp-Lac杂合启动子控制下,经IPTG诱导,分子量约23 000的鲑鱼生长激素在大肠杆菌中获得高效表达,该产物具有天然鲑鱼生长激素的免疫活性,直接分泌到细胞周质,而信号肽被自动剪除.     

Effect of nitrogen supply on frost resistance, nitrogen metabolism and carbohydrate content in white clover (Trifolium repens)

Effects of mineral nitrogen (2, 4, 6 and 8 m M NH₄NO₃) and nodulation with Rhizobium on frost hardiness in seedlings of white clover ( Trifolium repens ) have been studied. Seedlings of a population from Bodø (67°N lat.) were grown in Leonard jars under controlled conditions in a phytotron. For induction of frost hardening, plants were first exposed to 12 h photoperiod conditions for 2 weeks at 18°C, then for 2 weeks at 6°C and finally for 2 weeks at 0.5°C. Frost hardiness after treatments at 6 and 0.5°C was significantly enhanced by increasing nitrogen supply and was positively correlated with total nitrogen content of the stolons. Frost hardiness of nodulated plants correlated to the tissue nitrogen concentration. Content of soluble proteins in stolons decreased during hardening at 6°C but did not change during treatment at 0.5°C. There were minor changes in total amount of free amino acids during hardening. Both absolute and relative amounts of proline and arginine increased, and those of asparagine decreased during hardening. Absolute amounts of all free amino acids increased with increasing nitrogen supply, but the changes during hardening were similar in all treatments. There was a significant increase in the content of soluble carbohydrates during hardening. However, this increase was inversely related to nitrogen supply.     

Enhancement of symbiotic nitrogen fixation by vitamin-secreting fluorescent Pseudomonas

Fluorescent Pseudomonas sp. strain 267 promotes growth of nodulated clover plants under gnotobiotic conditions. In the growth conditions (60 M FeCl₃), the production of siderophores of the pseudobactin-pyoverdin group was repressed. Plant growth enhancement results from secretion of B vitamins by Pseudomonas sp. strain 267. This was proven by stimulation of clover growth by naturally auxotrophic strains of Rhizobium leguminosarum bv. trifolii and marker strains E. coli thi^- and R. meliloti pan^- in the presence of the supernatant of Pseudomonas sp. strain 267. The addition of vitamins to the plant medium increased symbiotic nitrogen fixation by the clover plants.     

Defining phosphorus efficiency in plants

The many different definitions for "nutrient efficiency" make the use of the term ambiguous. We evaluated nutrient efficiency using data from a study of response to phosphorus (P) supply in white clover (Trifolium repens L.) and lucerne (Medicago sativa L.). Application of various criteria identified in the literature as measures of nutrient efficiency did not clarify differences between purportedly P efficient and inefficient germplasm. Germplasm differed in maximum shoot and total dry mass and in solution P concentration ([P]_s) required to achieve 80% maximum yield, but not in P concentration of tissue ([P]_t), internal P utilization, or P uptake per unit of fine root dry mass. Differences in yield may have resulted from factors other than efficient use of P. To reduce the confounding effects that other factors have on nutrient efficiency, it is essential that equivalent yields of germplasm be demonstrated where nutrients are not limiting. Mechanisms that enable enhanced nutrient efficiency can be identified less ambiguously using this approach.Joint contribution of the Minn. Agric. Exp. Stn. and the USDA-ARS     

Nitrogen losses in puddled soils as affected by timing of water deficit and nitrogen fertilization

Erratic rainfall in rainfed lowlands and inadequate water supply in irrigated lowlands can results in alternate soil drying and flooding during a rice (Oryza sativa L.) cropping period. Effects of alternate soil drying and flooding on N loss by nitrification-denitrification have been inconsistent in previous field research. To determine the effects of water deficit and urea timing on soil NO₃ and NH₄, floodwater NO₃, and N loss from added ¹⁵N-labeled urea, a field experiment was conducted for 2 yr on an Andaqueptic Haplaquoll in the Philippines. Water regimes were continuously flooded, not irrigated from 15 to 35 d after transplanting (DT), or not irrigated from 41 to 63 DT. The nitrogen treatments in factorial combination with water regimes were no applied N and 80 kg urea-N ha^–1, either applied half basally and half at 37 DT or half at 11 DT and half at 65 DT. Water deficit at 15 to 35 DT and 41 to 63 DT, compared with continuous soil flooding, significantly reduced extractable NH₄ in the top 30-cm soil layer and resulted in significant but small (<1.0 kg N ha^–1) soil NO₃ accumulations. Soil NO₃, which accumulated during the water deficit, rapidly disappeared after reflooding. Water deficit at 15 to 35 DT, unlike that at 41 to 63 DT, increased the gaseous loss of added urea N as determined from unrecovered ¹⁵N in ¹⁵N balances. The results indicate that application of urea to young rice in saturated or flooded soil results in large, rapid losses of N (mean = 35% of applied N), presumably by NH₃ volatilization. Subsequent soil drying and flooding during the vegetative growth phase can result in additional N loss (mean = 14% of applied N), presumably by nitrification-denitrification. This additional N loss due to soil drying and flooding decreases with increasing crop age, apparently because of increased competition by rice with soil microorganisms for NH₄ and NO₃.     

Flight metabolism in the African fruit beetle,Pachnoda sinuata

Metabolite concentrations in flight muscles and in abdomen of beetles (Pachnoda sinuata) were measured after various periods of tethered flight and subsequent rest. Three distinct phases of energy metabolism are found in active flight muscles: (1) during the first minutes of flight proline is used as main substrate and concomitantly alanine accumulated as an end product; (2) the second phase is characterized by a large-scale degradation of glycogen; (3) after about 8 min of flight the metabolite levels stabilize, while flight performance appears unchanged. After the termination of flight the preflight proline concentration (70 mol·g^-1 fw) is re-established in less than 60 min, whereas restoration of resting levels of other metabolites requires longer. The pattern of maximal enzyme activities and the respiratory rates of mitochondria with different substrates confirm the significance of proline and carbohydrates as the main fuels of working flight muscles.Abbreviations CS citrate synthetase - Cytox cytochrome c oxidase - EDTA ethylenediaminetetra-acetate - fw fresh weight - GluDH glutamate dehydrogenase - GPT alanine aminotransferase - HOAD hydroxyacyl-coenzyme A dehydrogenase - HPLC high pressure liquid chromatography - ME malic enzyme - PCA perchloric acid - RQ repiratory quotient - TRA triethanolamine     

High altitude tissue adaptation in Andean coots: capillarity,fibre area,fibre type and enzymatic activities of skeletal muscle

Capillarity, fibre types, fibre area and enzyme activities of different skeletal muscles (pectoralis, extensor digitorum longus), tibialis anterior, plantaris and the myocardium were compared in Andean coot (Fulica americana peruviana) native to high altitude (Junín, Perú, 4200 m) and the same species nesting at sea level. Numbers of capillaries per square millimeter were higher in all high-altitude muscles when compared with sea-level muscles (P<0.0001). Moreover, values for capillaries per fibre and capillaries in contact with each fibre were higher in digitorum and tibialis high-altitude muscles. Muscle fibres were classified as Type I, Type IIA or Type IIB on the basis of their myofibrillar ATPase pH lability. Pectoralis muscle of high-altitude and sea-level coots presented only fibres of Type IIA. In contrast, all the leg muscles studied showed a mosaic pattern of the three fibre types. Fibre areas were determined using a Leitz Texture Analysis System. Significant differences in fibre area were observed (P<0.01) between high-altitude and sea-level muscles. Mean muscle fibre diameters were also lower in the high-altitude group than in the sea-level group. The enzyme activities studied were hexokinase, lactate dehydrogenase, citrate synthase and 3-hydroxyacyl-CoA-dehydrogenase. The oxidative capacity, as reflected by citrate synthetase and hydroxyacyl-CoA-dehydrogenase activities, was greater for myocardial and pectoralis than for leg muscles. However, analysis of maximal enzyme activities showed that there were no significant differences between the glycolytic and oxidative enzyme activities of high-altitude and sea-level coots. These results suggest that in Andean coots genetically adapted to high altitude, changes in muscle capillarity and fibre size, in addition to high haemoglobin O₂ affinity and low haemoglobin concentration, are sufficient to allow adequate energy production without increases in enzymatic activities.Abbreviations BSA bovine serum albumin - C:F ratio Capillaries per fibre - CAF Capillaries in contact with each fibre - CD capillary density (mm^-2) - CS citrate synthetase - EDL muscularis digitorum longus - fra fraction reduction area - HA high altitude - HAD hydroxyacyl-CoA-dehydrogenase - HK hexokinase - LDH lactate dehydrogenase - P ₅₀ PO₂ at which hemoglobin is half saturated with O₂ - P _aO₂ arterial partial pressure of oxygen - PAS periodic acid-schiff - PEC muscularis pectoralis - PLA muscularis planaris - P _tO₂ mean tissue oxygen pressure - P _vO₂ mixed venous partial pressure of oxygen - SD standard deviation - SL sea level - TA muscularis tibialis anterior - TAS texture analysis system     

Glucose metabolism by brown trout peripheral blood lymphocytes

Glucose metabolism in peripheral blood lymphocytes from the brown trout Salmo trutta has been studied. Glucose is taken up by means of a sodium-independent saturable process (K _m=10.8 mmol·l^-1), as well as by simple diffusion. Once within the cell, most of glucose is directed to lactate production through either the Embden-Meyerhof pathway or the hexose-monophosphate shunt. Rates of lactate formation are higher than rates of CO₂ formation. Glutamine does not exert an effect on either glucose uptake or glucose metabolism. The present study provides information regarding the nature of energy sources for different cell types in salmonids.Abbreviations 3-OMG 3-O-methyl glucose - EM Embden-Meyerhoff pathway - G6D glucose-6-phosphate dehydrogenase - HK hexokinase - HMS hexose monophosphate shunt - ICDH isocitrate dehydrogenase - K _m apparent Michaelis constant - LDH lactate dehydrogenase - MCB modified Cortland buffer - PBL peripheral blood lymphocytes - PFK fructose-6-phosphate kinase - PK pyruvate kinase - RBC red blood cells - V _max maximal rate of uptake     

Functional mitochondria in snake Bothrops alternatus erythrocytes and modulation of HbO2 affinity by mitochondrial ATP

Digitonin was applied to permeabilize the plasma membrane of Bothrops alternatus erythrocytes to study respiration, oxidative phosphorylation and Ca²⁺ transport by mitochondria in situ. These mitochondria oxidized added NAD-linked substrates, succinate and N,N,N, N-tetramethyl-p-phenylenediamine. Respiration was sensitive to rotenone and cyanide but not to antimycin A. This indicates that Bothrops mitochondria possess the respiratory complexes NADH-ubiquinone, succinate-ubiquinone, and ferrocytochrome c-oxygen oxidoreductases, although the lack of sensitivity to antimycin A raises doubt about the composition of the ubiquinol cytochrome c-reductase complex. An ability to build up and sustain a membrane potential was documented by their capacity to accumulate tetraphenylphosphonium and Ca²⁺ through an uncoupler-sensitive mechanism. Addition of ADP caused a transient decrease in the membrane potential, indicating that this is the predominant driving force for ATP synthesis as in most types of mitochondria. Uncoupling of phosphorylation from the oxidative process increased hemoglobin O₂ affinity, which suggests that ATP production by mitochondria may participate in modulation of O₂ transport by hemoglobin.Abbreviations membrane potential - BAE Bothrops alternatus erythrocytes - DNP 2,4-dinitrophenol - DPG 2,3-diphosphoglycerate - EGTA ethyleneglycol tetra-acetic acid - FCCP carbonylcyanide p-trifloromethoxyphenylhydrazone - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - TPP⁺ tetraphenylphosphonium - TRIS tris-(hydroxymethyl)aminomethane