Inactivation of N-assimilating enzymes and proteolytic activities in wheat leaf extracts: Effect of pyridine nucleotides and of adenylates

Summary Nitrate reductase was protected from inactivation in wheat leaf extracts by NADH, while NADPH was less effective. NAD, NADP or adenylates did not affect nitrate reductase inactivation in vitro. Glutamine synthetase was more stable than nitrate reductase and was protected from inactivation by ATP. ADP, AMP or pyridine nucleotides had no or only a minor effect on the decrease of glutamine synthetase activity in extracts. The caseolytic activity extracted from senescing leaves was slightly decreased by NADH and NADPH but this effect was not sufficient to explain the stabilization of nitrate reductase by NADH. Oxidized pyridine nucleotides and adenylates had no major effect on the caseolytic activity under the conditions used.This work was supported by grant 3.067-0.81 from the Swiss National Science Foundation. Author for correspondence: U.F.     

Metabolism of amphibian spermatozoa in relation to their motility

Summary Bufo arenarum spermatozoa were able to sustain motility both under aerobic and anaerobic conditions. In aerobiosis, the oxygen consumption varies between 2.6 and 4.2 l O₂/10⁸ cells/h at 30°C. The synthesis of lactic acid by anaerobic spermatozoa demonstrated the existence of an active glycolytic pathway.This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas and the Latinoamerican Program of Human Reproduction (PLAMIRH), grant 34-95-2-75.     

Biotransformation of aloenin,a bitter glucoside constituent ofAloe arborescens,by rats

Summary Aloenin has been established to be 4-methoxy-6-(2--D-glucopyranosyloxy-4-hydroxy-6-methylphenyl)-2-pyrone; it shows an inhibitory activity for gastric juice secretion. Rats metabolized it to 4-methoxy-6-(2,4-dihydroxy-6-methylphenyl)-2-pyrone, 2,5-dimethyl-7-hydroxychromone and glucose, which were excreted in the feces and the urine. The distribution of the radioactivity originating from¹⁴C-labeled aloenin was studied. The tracer found in the kidney and the liver reached 60% of the amount administered 24 h after feeding and decreased rapidly in the next 24 h.     

Juvenile hormone catabolism inManduca sexta: Homologue selectivity of catabolism and identification of a diol-phosphate conjugate as a major end product

We studied time-dependent metabolism of (10R)-[³H] juvenile hormone (JH) III and (10R, 11S)-[³H]JH I injected intoManduca sexta larvae; the hormones are metabolized to polar metabolites, expecially the JH acid-diol, and an unknown. Products were analyzed using a reversed-phase liquid chromatography assay. (10R)-JH III is metabolized much more rapidly than (10R, 11S)-[³H]JH I, whether injected seperately or as a mixture of hormones. The unknown metabolites of JH I and JH III were identified as phosphate conjugates of JH I and JH III diol by tandem mass spectral analysis of isolated samples. The phosphate conjugate of JH I diol is the principle end product of JH I metabolism.     

Microbial potential in deep-sea sediments

Summary In spite of high pressures and low temperatures in abyssal sediments of the North Atlantic Ocean, bacterial activity is evident and highest in the top 10 cm. At these locations the input of degradable organic material to the deep-sea bottom is low. Oxygen, therefore, remains the dominant oxidant in surface sediments. Although alternative electron acceptors like nitrate, oxidized manganese and sulfate are present in large amounts, they are not utilized in this natural habitat. In sediment cores which were collected from the site for laboratory perturbation studies, it was possible to stimulate microbially mediated processes which are dormant in situ. When the oxygen supply was cut off, nitrate and manganese reduction occurred. Denitrification was the major process observed in the upper anoxic layers, while nitrate-ammonification and manganese reduction occurred in deeper sediment strata (4–8 cm). This is evidence for the presence of a variety of different bacteria and of an anaerobic heterotrophic potential. Most of the activity is located in the top 10 cm of these sediments. The shift to anaerobiosis initiates microbial activities through which metals are converted into their mobile species at the lowered redox potential. Evaluation of the suitability of the deep sea as a repository for waste materials will have to account for the large dormant potential of microbial activities and the consequences of their release by changing the environmental conditions at the sea floor.     

Microbial activity in the terrestrial subsurface

Summary Little is known about the layers under the earth's crust. Only in recent years have techniques for sampling the deeper subsurface been developed to permit investigation of the subsurface environment. Prevailing conditions in the subsurface habitat such as nutrient availability, soil composition, redox potential, permeability and a variety of other factors can influence the microflora that flourish in a given environment. Microbial diversity varies between geological formations, but in general sandy soils support growth better than soils rich in clay. Bacteria predominate in subsurface sediments, while eukaryotes constitute only 1–2% of the microorganisms. Recent investigations revealed that most uncontaminated subsurface soils support the growth of aerobic heteroorganotrophic bacteria, but obviously anaerobic microorganisms also exist in the deeper subsurface habitat. The microorganisms residing below the surface of the earth are capable of degrading both natural and xenobiotic contaminants and can thereby adapt to growth under polluted conditions.     

Metabolic studies of Hg-203 onChlamydomonas reinhardi

Summary Sterile cultures ofChlamydomonas reinhardi, WT⁺, were treated with Hg-203 at 25°C to identify probably formed volatile mercury compounds. Experiments were performed with living and dead cells under aerobic or anaerobic conditions, respectively, and the mercury concentration was measured in the system algae/nutrient medium. We found a timerelated decrease of mercury concentration in the cell suspension and the cell-free nutrient medium due to a reduction of Hg⁺⁺ to Hg⁰, probably caused by extracellular enzymes; monomethyl or dimethyl mercury could not be detected.This work was supported by a grant of Österreichisches Bundesministerium für Gesundheit und Umweltschutz.     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Summary Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptornegative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [³H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10^–6 M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10^–5 M linoleic acid or 10^–5 M arachidonic acid but not by 10^–6 M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (K_d 0.54 nM±0.11 nM; n=6) than growth in medium supplemented with untreated serum (complete medium) (K_d=1.68 nM±0.48 nM; n=6) (p<0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10^–5 M linoleic acid or 10^–5 M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10^–5 M stearic acid or 10^–5 M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10^–5 M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [³H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [³H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

Influence de l'humidité sur les cocons du Ver à soie(Bombyx mori)

Summary Experiments were carried out to test the effects of relative humidity on prepupae and pupae of the Silkworm,Bombyx mori L., at 25⁰ C. It was found that:(1) duration of prepupal + pupal stages is the same at all relative humidities between 7 and 100%;(2) mortality seems unaffected in all these humidity conditions;(3) moths obtained in percentages between 55 and 100% R. H. have the same average wet weight; moths obtained under dryer conditions have a lower average weight (expressed in percentage of initial weights of prepupae);(4) pupae extracted from their cocoons develop as quickly and as well as pupae in their cocoons.     

湿地甲烷排放研究进展

甲烷是一种仅次于二氧化碳的重要温室气体,对全球变暖的贡献率为25%。近年来大气中甲烷浓度显著增加,湿地甲烷释放量约占全球甲烷通量的20%,是大气甲烷的主要自然来源之一。甲烷主要通过产甲烷菌在厌氧条件下产生,在氧化条件下,甲烷通过土壤中的甲烷氧化菌氧化,并通过气泡、扩散和植物传输三种途径释放到大气。湿地甲烷通量在时间和空间两个方面有较大的变化,这与甲烷产生、氧化和传输过程有关,同时受到不同环境因素的影响,如:土壤质地、氧化还原电位、有机物、土壤酸碱度、植被条件、气候因素、农业管理措施等。文章还对湿地甲烷排放的观测方法和排放模型进行了简单介绍,并对今后的研究提出了几点建议。     

Biodegradation of nitrilotriacetic acid (NTA) in the absence of oxygen

Summary A microbiol culture able to grow in the absence of oxygen with nitrilotriacetate (NTA) as the sole source of carbon and energy was enriched from river sediment. Growth was strictly dependent on the presence of nitrate and the results indicate that nitrate was exclusively used as the electron acceptor and was reduced to dinitrogen (N₂). The stoichiometry of anaerobic NTA degradation by the enrichment culture was determined.     

Synergistic effect of acute hypoxia on flow-induced release of ATP from cultured endothelial cells

Human umbilical vein endothelial cells (HUVECs) in primary cultures were perfused under normoxic or hypoxic conditions. These cells were stimulated twice for 3 min by increased flow (from 0.5 to 3.0 ml/min). Under hypoxic conditions the basal release of ATP was the same as under normoxic conditions, but during increased flow the release was greater (0.58±0.07>0.32±0.04 pmoles/ml/10⁶ cells (+78%), for the first period of stimulation; 0.39±0.05>0.22±0.03 pmoles/ml/10⁶ cells (+79%) for the second period). Further experiments with sequential increments in flow rate showed that under both normoxic and hypoxic conditions, a positive correlation existed between ATP release and the rate of flow but there was always more ATP released under hypoxic conditions regardless of the flow rate.HUVECs in secondary culture (second passage) were similarly stimulated. No differences were observed between normoxic and hypoxic conditions. In both cases, the quantity of ATP released during high flow (0.050±0.004 pmoles/ml/10⁶ cells) was significantly smaller than the quantity of ATP released during low flow (0.09±0.01 pmoles/ml/10⁶ cells).To conclude, since hypoxia alone did not affect ATP release, there appears to be a synergistic relationship between increased shear stress and hypoxia in the stimulation of ATP release from HUVECs. Moreover, the release of ATP under these conditions seems to be a property of highly differentiated endothelial cells.     

The coordination and function of the redox centres of the membrane-bound nitrate reductases

Under anaerobic conditions and in the presence of nitrate, the facultative anaerobe Escherichia coli synthesises an electron-transport chain comprising a primary dehydrogenase and the terminal membrane-bound nitrate reductase A (NarGHI). This review focuses on recent advances obtained on the structure and function of the three protein subunits of membrane-bound nitrate reductases. We discuss a global architecture for the Mo-bisMGD-containing subunit (NarG) and a coordination model for the four [Fe–S] centres of the electron-transfer subunit (NarH) and for the two b-type haems of the anchor subunit NarI.     

Dehydroepiandrosterone sulfate-binding sites in plasma membrane from human uterine cervical fibroblasts

Dehydroepiandrosterone sulfate (DHA-S) plays a critical role in cervical dilation at labor. Incubation of cervical fibroblasts with [³H]DHA-S caused a rapid and saturable increase in cellular radioactivity: an apparent equilibrium was reached by 2 min. There was no detectable conversion of DHA-S into DHA or oestradiol. When the fibroblasts loaded with [³H]DHA-S were homogenized and fractionated, the specific radioactivity in the plasma membrane fraction was enriched approximately 8- to 9-fold compared with the whole homogenate; only low amounts of radioactivity were observed in the other subcellular fractions. The binding of DHA-S to plasma membrane preparations showed saturation kinetics with an apparent equilibrium dissociation constant (K _d) of 12 nM, and the binding capacity (B _max) was calculated to be 1.25 fmol/mg protein. Neither DHA nor oestrone sulfate affected [³H]DHA-S binding to the plasma membrane. The plasma membranes of skin fibroblasts did not show specific binding sites for DHA-S. These findings demonstrate the presence of specific binding sites for DHA-S in the plasma membrane of cervical stroma cells. The fetal adrenal steroid may exert its action on cervical ripening at least in part through membrane-associated binding sites, or receptors.     

Absorption and biotransformation of L(+)-tartaric acid in rats

Summary Oral or parenteral doses of monosodium¹⁴C-L(+)-tartrate (400 mg/kg) are rapidly excreted by rats and a proportion completely metabolized to CO₂. The oral dose was well-absorbed.     

Progesterone formation and metabolism by rabbit placenta in vitro

4-¹⁴C-Progesterone and 4-¹⁴C-pregnenolone are metabolized in vitro by rabbit placenta, at day 15 and 28 of gestation, exclusively to compounds reduced in ring A (5) and at carbon 3 and 20.     

A method for sequencing peptides: A co-operation of diphenyl phosphorazidate and 2-mercapto- or 2-hydroxypyridine for N-acyldiketopiperazine formation

Summary A new method for sequencing peptides is proposed. As a model experiment for this, Bz-Gly-Pro-OH and Bz-Gly-Sar-OH were conveniently converted to their diketopiperazine derivatives by a co-operation of diphenyl phosphorazidate and 2-mercapto- or 2-hydroxypyridine.     

Synthetic simulation of nonribosomal peptide biosynthesis. A dual role of alkylthiol esters in peptide synthesis

Summary Coupling of peptide alkylthiol esters with amino acid derivatives in the presence of pivalic acid or 2-hydroxypyridine proceeds without racemization. A dual role of alkylthiol esters as protective and reactive functions in peptide synthesis was well proved.     

Does ammonium uptake influence xylem sap composition inPhaseolus vulgaris L. andGlycine max (L.) Merill?

The question of whether ammonium uptake influences the occurrence of ureides in legumes has been addressed in this study by investigating threeP. vulgaris genotypes as well as one cultivar ofGlycine max. All plants were raised in sand culture during the dry season in northern Thailand and irrigated daily with nitrogen-free nutrient solution, or the same solution containing 12 mol m^–3 nitrogen in the form of (NH₄)₂SO₄ or KNO₃, each treatment consisting of different proportions of either compound. Regression analyses of xylem sap composition relative to ammonium vs. nitrate supply of plants harvested at V4, R1 and R6 indicated close positive correlations of xylem amino nitrogen content and negative correlations with xylem nitrate content and ammonium supply. Statistically significant correlations between relative xylem ureide content and ammonium availability could be established for theP. vulgaris cultivar Brilliant up to stage R1, but not for the other plants investigated. It was concluded that at least for some genotypes of common bean a relationship exists between ureide production and ammonium uptake by the root system. Since the extent to which ureide production is stimulated remains quite small, its relevance to the xylem solute technique for measurement of N₂ fixation may be limited. Nevertheless, due to the possibility of large genotypic differences in the impact of ammonium on ureide production, this factor must be considered in calculations if N₂ fixation is to be determined in soils containing significant amounts of ammonium, e.g. in paddy fields.     

Effect of xanthurenic acid on P-450-dependent biotransformation by molting glands in vitro

Incubation of molting glands from the crayfishProcambarus clarkii (Y-organ) and the silkwormBombyx mori (prothoracic gland) with 23,24-[²H₄]-2-deoxyecdysone resulted in the production of deutero-ecdysone; this biotransformation was inhibited in the presence of xanthurenic acid. When the experiments were performed under an¹⁸O₂ atmosphere, the¹⁸O atom was introduced into ecdysone, as confirmed by mass spectrometry. We therefore suggest that xanthurenic acid inhibits P-450-dependent hydroxylation of 2-deoxyecdysone. However, deutero-2-deoxyecdysone was not converted to 3-dehydroecdysone when using Y-organs in vitro, although it is a major product. We therefore conclude that the biosynthetic pathway of ecdysteroids inP. clarkii branches at an early step.