Glutathione reductase fromSaccharomyces cerevisiae undergoes redox interconversionin situ andin vivo

Redox interconversion of glutathione reductase was studiedin situ withS. cerevisiae. The enzyme was more sensitive to redox inactivation in 24 hour-starved cells than in freshly-grown ones. While 5 μM NADPH or 100 μM NADH caused 50% inactivation in normal cells in 30 min, 0.75 μM NADPH or 50 μM NADH promoted a similar effect in starved cells. GSSG reactivated the enzyme previously inactivated by NADPH, ascertaining that the enzyme was subjected to redox interconversion. Low EDTA concentrations fully protected the enzyme from NADPH inactivation, thus confirming the participation of metals in such a process. Extensive inactivation was obtained in permeabilized cells incubated with glucose-6-phosphate or 6-phosphogluconate, in agreement with the very high specific activities of the corresponding dehydrogenases. Some inactivation was also observed with malate, L-lactate, gluconate or isocitrate in the presence of low NADP⁺ concentrations. The inactivation of yeast glutathione reductase has also been studiedin vivo. The activity decreased to 75% after 2 hours of growth with glucono-δ-lactone as carbon source, while NADPH rose to 144% and NADP⁺ fell to 86% of their initial values. Greater changes were observed in the presence of 1.5 μM rotenone: enzymatic activity descended to 23% of the control value, while the NADH/NAD⁺ and NADPH/NADP⁺ ratios rose to 171% and 262% of their initial values, respectively. Such results indicate that the lowered redox potential of the pyridine nucleotide pool existing when glucono-δ-lactone is oxidized promotesin vivo inactivation of glutathione reductase.     

In vivo inhibition of nitrogenase by hydroxylamine in Rhodospirillaceae Role of nitric oxide

Rhodobacter capsulatus strains E1F1 and B10 and Rhodobacter sphaeroides DSM 158 did not use hydroxylamine as nitrogen source for growth but metabolized it mainly through the glutamine synthetase reaction. Hydroxylamine had a high toxicity for cells growing either under phototrophic or dark-aerobic conditions. l-methionine-d,l-sulfoximine partially inhibited hydroxylamine uptake and increased the inhibition time of nitrogenase activity by this nitrogen compound. Nitric oxide was also a powerful inhibitor of nitrogenase in intact cells of R. capsulatus. Since low amounts of NO were produced from hydroxylamine, short-term inhibition of nitrogenase in the presence of this compound could be mediated in vivo by nitric oxide.Abbreviations GS glutamine synthetase - MSX l-methionine-d,l-sulfoximine - MTA mixed alkyltrimethylammonium bromide     

Behavioral analysis of the choice of oviposition site by single females ofDrosophila Melanogaster (Diptera: Drosophilidae)

The oviposition behavior of single females of Drosophila melanogasterwas studied in population cages over 24 h. Each female shows a different behavior, but they can be arbitrarily separated into those which concentrate their egg laying in only one tube and those which spread it over more than two tubes. A comparison is made between females extracted from the Valdivian population and flies from lines selected for high and low aggregation. When one-tube females were grouped and compared with more-than-one-tube females, the aggregation indices between these groups were significantly different.     

Human adenovirus type 9-induced rat mammary tumors.

Following subcutaneous inoculation of newborn Wistar-Furth rats with human adenovirus type 9 (Ad9), 16 of 16 female and 0 of 11 male rats developed mammary tumors. Tumor-positive animals usually developed tumors in multiple glands. Histopathological analyses indicated that three general categories of tumor could be identified. Mammary fibroadenomas were the most common tumor type encountered, but phyllodeslike tumors and solid sarcomas were also frequently found. In situ hybridization and immunohistochemical techniques established that benign fibroadenomas were derived from mammary fibroblasts (collagen type I- and vimentin-positive cells) and that malignant tumors were derived from myoepithelial cells (collagen type IV-, vimentin-, and muscle-specific actin-positive cells). The fact that mammary tumors were limited to female rats suggested that female hormones are essential for tumor growth and development. In this regard, ovariectomy of Ad9-infected female rats prevented tumor development, while subsequent diethylstilbestrol (DES) treatment elicited tumor formation. In addition, Ad9-infected and castrated male rats which received DES also developed mammary tumors. Established male mammary tumors regressed when DES treatment was stopped and reappeared after DES treatment was resumed. Together, these results indicate that estrogen is required for both initiation and maintenance of Ad9-induced mammary tumors. Southern blot analysis of high-molecular-weight tumor DNA showed that mammary tumor cells contained single or multiple integrated copies of the entire Ad9 genome. RNase protection experiments established that estrogen receptor as well as Ad9 E1a and E4 mRNAs were expressed in mammary tumors, but Ad9 E3 and, surprisingly, E1b mRNAs were not expressed at detectable levels.     

Dual effect of cyclic GMP on renal brush border Na-H antiporter.

The Na-H antiporter of renal-brush border membranes is inhibited by cyclic AMP and stimulated by protein kinase C. The proximal tubule contains guanylate cyclase and is capable of cyclic GMP production. The effect of cGMP on renal Na-H antiporter activity was analyzed in phosphorylated brush border membranes by 22Na uptake in the presence or absence of 1 mM amiloride. 8-Bromo cyclic GMP (1 microM) increased the amiloride-sensitive 22Na uptake in control from 1.26 +/- 0.13 to 1.54 +/- 0.12 nmol/mg/protein/10 sec, P less than 0.01, without altering the amiloride-insensitive component. In the absence of exogenous ATP, cGMP also stimulated the amiloride-sensitive 22Na uptake, which can be explained by the presence of endogenous ATP in concentrations of up to 50 microM in the membranes. In ATP-depleted membrane vesicles, however, cGMP inhibited the amiloride-sensitive 22Na uptake. These data indicate that cGMP acts on the Na-H antiporter by at least two different mechanisms, one of which is ATP dependent. It is likely that cGMP-dependent protein kinase mediates the stimulatory effects seen in the presence of ATP, and the inhibition seen in ATP-depleted membranes results from cGMP direct action on the Na-H antiporter.     

Physiologic role for enhanced renal thromboxane production in murine lupus nephritis.

To investigate the physiologic significance of enhanced renal thromboxane production in murine lupus nephritis, we measured renal hemodynamics and eicosanoid production in MRL-lpr/lpr mice from 8 to 20 weeks of age. Over this age range, MRL-lpr/lpr mice develop an autoimmune disease with nephritis similar to human systemic lupus erythematosus (SLE). In these studies, glomerular filtration rate (GFR) and PAH clearance (CPAH) decreased progressively with age in MRL-lpr/lpr mice, but not in controls. This impairment of renal hemodynamics was associated with increased renal thromboxane production, as well as increased excretion of both thromboxane B2 (TxB2) and 2,3-dinor TxB2 in urine. There was an inverse correlation between renal thromboxane production in MRL-lpr/lpr mice and both GFR and CPAH. Furthermore, there were positive correlations between thromboxane production by the kidney and both the severity of renal histopathology and serum anti-DNA antibody levels measured in individual animals. Enhanced urinary excretion of TxB2 and the development of renal dysfunction also coincided temporally with the appearance of increased levels of interleukin 1 beta (IL-1 beta) mRNA in renal cortex. Acute administration of the specific thromboxane receptor antagonist GR32191 to MRL-lpr/lpr mice restored GFR to normal in early stages of the autoimmune disease. However, in animals with more advanced nephritis, the effect of acute thromboxane receptor blockade on renal hemodynamics was less marked. We conclude that thromboxane A2 is an important mediator of reversible renal hemodynamic impairment in murine lupus, especially in the early phase of disease.     

Planar and vertical signals in the induction and patterning of the Xenopus nervous system.

The cellular mechanisms responsible for the formation of the Xenopus nervous system have been examined in total exogastrula embryos in which the axial mesoderm appears to remain segregated from prospective neural ectoderm and in recombinates of ectoderm and mesoderm. Posterior neural tissue displaying anteroposterior pattern develops in exogastrula ectoderm. This effect may be mediated by planar signals that occur in the absence of underlying mesoderm. The formation of a posterior neural tube may depend on the notoplate, a midline ectodermal cell group which extends along the anteroposterior axis. The induction of neural structures characteristic of the forebrain and of cell types normally found in the ventral region of the posterior neural tube requires additional vertical signals from underlying axial mesoderm. Thus, the formation of the embryonic Xenopus nervous system appears to involve the cooperation of distinct planar and vertical signals derived from midline cell groups.     

Nucleosomal organization of chromatin in sperm nuclei of the bivalve mollusc Aulacomya ater

The sperm nuclei of Aulacomya ater, family Mitylidae, contain three proteins (X, Aa5 and Aa6) which are specific to this cell type coexisting with a set of five somatic-type histones. Information about the chromatin structure resulting from this kind of association is scarce. Therefore, we have probed the structure of this sperm chromatin through digestion with micrococcal nuclease in combination with salt fractionation. The data obtained have allowed us to propose a nucleosomal arrangement for this chromatin. However, two types of nucleosomes would be present in agreement with their protein components.