Immunoelectrophoretic approach to the metabolic regulation of glutamine synthetase in Rhodopseudomonas capsulata E1F1: role of glutamine

Anti-glutamine synthetase serum was raised in rabbits by injecting purified glutamine synthetase (GS) of the phototrophic bacterium Rhodopseudomonas capsulata E1F1. The antibodies were purified to monospecificity by immunoaffinity chromatography in GS-sepharose gel. These anti-GS antibodies were used to measure the antigen levels in crude extracts from bacteria, grown phototrophically with dinitrogen, nitrate, nitrite, ammonia, glutamate, glutamine or alanine as nitrogen sources. The amount of GS detected by rocket immunoelectrophoresis was proportional to Mn²⁺-dependent transferase activity measured in the crude extracts. Addition of GS inhibitor l-methionine-d,l-sulfoximine (MSX) to the actively growing cells promoted increased antigen levels, that were not found in the presence of glutamine or chloramphenicol. The ammonia-induced decrease in GS relative levels was reverted by MSX. GS levels remained constant when phototrophically growing cells were kept in the dark.Abbreviations GS glutamine synthetase - MOPS 2-(N-morpholine) propane sulfonate - MSX l-methionine-d,l-sulfoximine     

Phosphorus nutrition of barley, buckwheat and rape seedlings. II. Influx and efflux of phosphorous by intact roots of different P status

Seedlings of barley (Hordeum vulgare L. cvs Salka and Zita), buckwheat (Fagopyrum esculentum Moench) and rape (Brassica napus L. ssp. napus cv. Line) were raised at 8 or 10 different extenral P concentrations in the range 0–2000 μM. Apart from P, the nutrient solutions were complete. Phosphate influx in roots of different P status was determined by use of a nutrient solution containing 0.1 mM³²P-labelled phosphate. A double labelling technique was used for simultaneous determination of influx (³³P) and efflux (³²P) of phosphorus by roots of barley and rape with three selected P levels. Flux determinations were also done in presence of a metabolic uncoupler (2,4-dinitrophenol) and a protein synthesis inhibitor (cycloheximide). Influx of phosphate was maximal at a certin optimal P level of the roots and decreased at both lower and higher P levels. Maximum phosphate influex [μmol (g root)-^?1 h^?1] were: rape 4,4, buckwheat 2.2, barley cv. Salka 1.6, barley cv. Zita 1.5. Both Hill plots and plots of the untransformed decreasing phosphate influx vs root P concentrations above the optimal were linear and had high correlation coefficients. The Hill coefficient varied between -3.1 and -4.2. The decrease of phosphate influx from the maximum to the lowest value at the highest P concentration of the root was 60–70%. Hence, phosphate influex appeared to be regulated through negative feedback by the internal level of phosphorous in the roots. The regulation mechanism seems bascially similar for the three species and may be of an allosteric type. P efflux from roots of low and optimal (with regard to P influx) P status was 15–20% of the simultaneous P influx. Contary to P influx, P efflux increased at high P status and almost eliminated (barley) or halved (rape) net P uptake. 2,4-Dinitrophenol reduced both P influx and P efflux by low P roots and gave linearly increasing P efflux with increasing root P status. This indicates that P efflux partly occurred by counter transport and ion exchange at the uptake sites, partly by passive P efflux along an electrochemical potential gradient. Phosphate influx was not affected by inhibition of barley root growth with cycloheximide, but P efflux increased considerably.     

Evidence for coupled transport of bicarbonate and sodium in cultured bovine corneal endothelial cells

Summary Usin gintracellular microelectrode technique, the response of the voltageV across the plasma membrane of cultured bovine corneal endothelial cells to changes in sodium and bicarbonate concentrations was investigated. (1) The electrical response to changes in [HCO ₃ ^– ] _o (depolarization upon lowering and hyperpolarization upon raising [HCO ₃ ^– ] _o ) was dependent on sodium. Lithium could fairly well be substituted for sodium, whereas potassium or choline were much less effective. (2) Removal of external sodium caused a depolarization, while a readdition led to a hyperpolarization, which increased with time of preincubation in the sodium-depleted medium. (3) The response to changes in [Na⁺] _o was dependent on bicarbonate. In a nominally bicarbonate-free medium, its amplitude was decreased or even reversed in sign. (4) Application of SITS or DIDS (10^–3 m) had a similar effect on the response to sodium as bicarbonate-depleted medium. (5) At [Na⁺] _o =151mm and [HCO ₃ ^– ] _o =46mm, the transients ofV depended, with 39.0±9.0 (sd) mV/decade, on bicarbonate and, with 15.3±5.8 (sd) mV/decade, on sodium. (6) After the preincubation of cells with lithium, replacement of Li by choline led to similar effects as the replacement of sodium by choline, though the response ofV was smaller with Li. This response could be reduced or reversed by the removal of bicarbonate or by the application of SITS. (7) Amiloride (10^–3 m) caused a reversible hyperpolarization of the steady-state potential by 8.5±2.6 mV (sd). It did not affect the immediate response to changes in [Na⁺] _o or [HCO ₃ ^– ] _o , but reduced the speed of regaining the steady-state potential after a change in [HCO ₃ ^– ] _o . (8) Ouabain (10^–4 m) caused a fast depolarization of –6.8±1.1 (sd) mV, which was followed by a continuing slower depolarization. The effect was almost identical at 10^–5 m. (9) It is suggested, that corneal endothelial cells possess a cotransport for sodium and bicarbonate, which transports net negative charage with these ions. It is inhibitable by stilbenes, but not directly affected by amiloride or ouabain. Lithium is a good substitute for sodium with respect to bicarbonate transport and is transported itself. In addition, the effect of amiloride provides indirect evidence for the existence of a Na⁺/H⁺-antiport. A model for the transepithelial transport of bicarbonate across the corneal endothelium is proposed.     

Regulation of Calmodulin- and Dopamine-Stimulated Adenylate Cyclase Activities by Light in Bovine Retina

Abstract: Neural retina from most species contains 3,4-dihydroxyphenylethylamine (dopamine) receptors coupled to stimulation of adenylate cyclase activity. It has been demonstrated that release of dopamine from its neurons and subsequent occupation of dopamine receptors is increased by light. In this study, we have shown that adenylate cyclase activity in bovine retina is highly responsive to the endogenous Ca²⁺-binding protein, cal-modulin, and that calmodulin can increase dopamine-sen-sitive adenylate cyclase activity in bovine retina. We further demonstrate that both dopamine- and calmodulin-stimulated adenylate cyclase activities can be regulated by alterations in light. Bovine retinas were dissected from the eye under a low-intensity red safety light, defined as dark conditions, and incubated for 20 min in an oxygenated Krebs Henseleit buffer under either dark or light conditions. The retinas were then homogenized and adenylate cyclase activity measured in a paniculate fraction washed to deplete it of endogenous Ca²⁺ and calmodulin. Activation of adenylate cyclase activity by calmodulin, dopamine, and the nonhydrolyzable GTP analog, gua-nosine-5′-(β,γ-imido)triphosphate (GppNHp), was significantly (60%) greater in paniculate fractions from retinas that had been incubated under dark conditions as compared to those incubated under light conditions. Basal, Mn²⁺-, and GTP-stimulated adenylate cyclase activities were not altered by changes in lighting conditions. Calmodulin could increase the maximum stimulation of adenylate cyclase by dopamine in retinas incubated under either dark or light conditions, but the degree of its effect was greater in retinas incubated under light conditions. Activation of adenylate cyclase by calmodulin, dopamine, and GppNHp in paniculate fractions from retinas incubated under light conditions was indistinguishable from the activation obtained when retinas were incubated in the dark in the presence of exogenous dopamine. These results suggest that an increased release of dopamine occurs in light. The decreased response of adenylate cyclase to exogenous dopamine can then be explained by a subsequent down-regulation of dopamine receptor activity. The down-regulation of dopamine receptor activity can also regulate activation of adenylate cyclase by GppNHp and calmodulin. The results suggest that dopamine, calmodulin, and GppNHp are modulators of a common component of adenylate cyclase activity, and this component is regulated by light.     

Hormonal Regulation of Adipose S-100 Protein Release

The release of S-100 protein from epididymal fat pads was enhanced by epinephrine in vitro, and about 50% of S-100 protein in the tissue was released into the medium after 2-h incubation at 37 degrees C with 10 microM epinephrine. Similar results were obtained with the incubation of isolated adipocytes. The S-100 protein release was also enhanced by isoproterenol, norepinephrine, ACTH, and dibutyryl cyclic AMP, which all increase the lipolysis by increasing cyclic AMP levels in the tissue. Propranolol, a beta-adrenergic blocker, could block the increase of S-100 protein release by catecholamines, indicating that the release was mediated by the beta-adrenergic effect of catecholamines. However propranolol had no suppressive effect on the enhancement of S-100 protein release by ACTH or dibutyryl cyclic AMP. Insulin had an inhibitory effect on the epinephrine-enhanced S-100 protein release. Epinephrine or ACTH could not stimulate the S-100 protein release in the absence of Ca2+, whereas the epinephrine-enhanced glycerol release was not affected under the same conditions. The increase in S-100 protein release was induced by only a pretreatment of the tissue with epinephrine. However, the lipolysis in the tissue was not enhanced by the pretreatment alone. These results indicate that the release of S-100 protein from adipocytes is regulated by the hormones that have been known to control the lipolysis with a manner slightly different from that of lipolysis.     

卧床前后压力感受性反射机能变化的研究

许多数据表明长期失重以后立位耐力降低可能与压力感受性反射功能的改变有关。本文比较了两组被试者15天低动力卧床前后的立位耐力。以血压调节模型为基础分析了两种不同方式卧床前后单纯立位和下身负压加立位时压力感受性反射功能的改变,并用颈部加压及下身负压对中枢调节功能改变进行了观察。结果表明严格的头低位卧床后,立位耐力下降及压力感受性反射功能改变明显大于半日平卧半日倚坐者。而压力感受性反射功能的改变,特别是中枢神经系统调节功能的紊乱,是卧床后立位耐力降低的主要原因。从这种考虑为基础,作者提出了改变失重或模拟失重状态下的血液分布,调整对压力感受器的刺激,可能是预防心血管失调的有效方法。     

Murine “housekeeping” enzyme (genetic locus:Idh-1) is regulated in an allele-specific manner

The murine “housekeeping” enzyme, cytosolic NADP-isocitrate dehydrogenase (E.C. 1.1.1.42) (genetic locus:Idh-1), exhibited a complex pattern of allele-specific expression. Protein electrophoresis on cellulose-acetate gels and determination of relative enzymatic activity by means of densitometry revealed that in heart tissue (but not liver tissue) of certain hybrid crosses the AA-homodimer was underrepresented relative to total enzymatic activity, and the degree of underrepresentation changed during development. In mixtures of homozygous tissue extracts of heart tissue (but not liver tissue) the AA-homodimer was underrepresented relative to the BB-homodimer. Relative activity of allelic isozymes varied as a function of tissue (heart versus liver), age, and the parental source of the Idh-1^a allele, but did not vary as a function of sex. Allele-specific expression was also exhibited in kidney tissue of the same animals. In adult male kidney tissue extracts from heterozygotes, the AA-hornodimer was underrepresented relative to total enzymatic activity; in adult female kidney tissue extracts from heterozygotes, a more codominant phenotype was observed. Tissue extracts from immature hybrid animals exhibited a phenotype midway between the adult male and adult female phenotypes. Tissue extracts from castrated males exhibited a phenotype equivalent to that seen in females. Relative activity of allelic isozymes in kidney varied as a function of age and sex, but did not vary as a function of the parental source of the Idh-1^a allele. While cytosolic NADP-IDH is a “housekeeping” enzyme, expressed in multiple tissues of the mouse, differences in the relative intensities of allelic isozyme bands provide evidence for tissue- and stage-specific regulatory variation.     

Effect of calcium ion on cytoplasmic streaming during turgor regulation in a brackish water charophyte Lamprothamnium

Abstract The brackish water charophyte Lamprothamnium succinctum regulates its turgor pressure against changes in the external osmotic pressure. Upon hypotonic treatment, the rate of cytoplasmic streaming in the internodal cells fell to almost zero, and then recovered to the original value within 20 min. The decrease could be inhibited by lowering the external Ca²⁺ concentration in the hypotonic medium. Also, cytoplasmic streaming in tonoplast-free cells of L. succintum was sensitive to Ca²⁺ like freshwater charophyte. Thus, the concentration of free Ca²⁺ in the cytoplasm seems to increase transiently upon hypotonic treatment.     

The dopaminergic innervation of the goldfish pituitary

Summary The dopaminergic innervation of the goldfish pituitary gland was studied by immunocytochemistry at the electron-microscope level using highly specific antibodies against dopamine coupled to bovine serum albumin with glutaraldehyde. A satisfactory preservation of the tissue was achieved after immersion in 5% glutaraldehyde in phosphate buffer containing sodium metabisulfite to prevent oxidation of the endogenous dopamine. The immunocyto-chemical procedure was performed on Vibratome sections using the preembedding method. Immunoreactivity was restricted to part of the neurosecretory type-B fibers (diameter of the secretory vesicles lower than 100 nm) in which it was found to occupy the whole cytoplasm. Labeled fibers were observed within the neurohypophysis in the different parts of the gland and in the adenohypophyseal tissue where immunoreactive profiles were detected in close apposition to the different cell types. These data are in agreement with previous results obtained by means of radioautography and further support a role for dopamine in the neuroendocrine regulation of pituitary functions in teleosts.     

Isolation of auxotrophic mutants in support of ammonia assimilation via glutamine synthetase in Methanobacterium ivanovii

Various enzymes involved in the initial metabolic pathway for ammonia assimilation by Methanobacterium ivanovii were examined. M. ivanovii showed significant activity of glutamine synthetase (GS). Glutamate synthase (GOGAT) and alanine dehydrogenase (ADH) were present, wheras, glutamate dehydrogenase (GDH) was not detected. When M. ivanovii was grown with different levels of NH ₊ ⁴ (i.e. 2, 20 or 200 mM), GS, GOGAT and ADH activities varied in response to NH ₊ ⁴ concentration. ADH was not detected at 2 mM level, but its activity increased with increased levels of NH ₊ ⁴ in the medium. Both GS and GOGAT activities increased with decreasing concentrations of NH ₊ ⁴ and were maximum when ammonia was limiting, suggesting that at low NH ₊ ⁴ levels, GS and GOGAT are responsible for ammonia assimilation and at higher NH ₊ ⁴ levels, ADH might play a role. Metabolic mutants of M. ivanovii that were auxotrophic for glutamine were obtained and analyzed for GS activity. Results indicate two categories of mutants: i) GS-deficient auxotrophic mutants and ii) GS-impaired auxotrophic mutants.Abbreviations GS Glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase