Tracheal insufflation of tumor necrosis factor protects rats against oxygen toxicity

Tracheal insufflation of tumor necrosis factor (TNF; 5 micrograms or 1.2 x 10(5) U) markedly enhanced the survival of adult rats exposed to 100% O2: 12 of 17 rats (71%) survived for greater than 11 days, whereas 30 of 30 control (Hanks' balanced salt solution) insufflated rats (100%) died within 3 days of O2 exposure. Insufflation of gamma-interferon (5 micrograms) or intraperitoneal injection of up to 40 micrograms TNF did not afford any protection. At 55 h after O2 exposure, TNF-insufflated rats showed less pulmonary edema, as determined by the extravascular lung water content-to-bloodless lung dry weigh ratio and less alveolar capillary leak as determined by the protein content in the bronchoalveolar lavage fluid, than control insufflated rats similarly exposed. This protection against O2 toxicity by TNF insufflation was associated with increased lung superoxide dismutase, catalase, and glutathione peroxidase activities. The enhancement of lung antioxidant enzyme activities was noted at 55 h of O2 exposure, when control animals began to die of O2 toxicity. This temporal relationship suggests that TNF-induced increase in antioxidant enzyme activities contributes, at least in part, to the observed protection.     

Repeated-batch cultures of Baby Hamster Kidney cell aggregates in stirred vessels

Natural aggregates of Baby Hamster Kidney cells were grown in stirred vessels operated as repeated-batch cultures during more than 600 hours. Different protocols were applied to passaging different fractions of the initial culture: single cells, large size distributed aggregates and large aggregates. When single cells or aggregates with the same size distribution found in culture are used as inoculum, it is possible to maintain semi-continuous cultures during more than 600 hours while keeping cell growth and viability. These results suggest that aggregate culture in large scale might be feasible, since a small scale culture can easily be used as inoculum for larger vessels without noticeable modification of the aggregate chacteristics. However, when only the large aggregates are used as inoculum, it was shown that much lower cell concentrations are obtained, cell viability in aggregates dropping to less than 60%. Under this selection procedure, aggregates maintain a constant size, larger than under batch experiments, up to approximately 400 hours; after this time, aggregate size increases to almost twice the size expected from batch cultures.     

Follicle-stimulating hormone and human chorionic gonadotropin induced changes in granulosa cell glycosyl-phosphatidylinositol concentration

In the present investigation, a hCG sensitive glycosyl-phosphatidylinositol (GPI) was isolated from cultured rat granulosa cells obtained from the ovaries of diethylstilbestrol (DES) implanted immature rats. The inositol-phosphoglycan (IPG) moiety of the GPI-lipid contains galactose, glucosamine, and myoinositol as demonstrated by metabolic labelling of granulosa cells for different time periods (5–96 h) with [³H]galactose, [³H]glucosamine, or [³H]myoinositol and treatment of the purified [³H]GPI with phosphatidylinositol-specific phospholipase C. Labelling equilibrium of the GPI-lipid was achieved after 24 h ([³H]galactose and [³H]myoinositol) or 72 h ([³H]glucosamine) incubation, whereas incorporation of other labelled carbohydrates tested ([³H]galactosamine, [³H]mannose, and [³H]sorbitol) was negligible throughout the time period studied. The glucosamine C-1 appears to be linked through a glycosidic bond to the myoinositol molecule of the IPG moiety as revealed by the generation of phosphatidylinositol (PtdIns) after nitrous acid deamination of dual labelled ([³H]glucosamine/[¹⁴C]palmitate or [³H]glucosamine/[¹⁴C]myristate) glycosyl-phosphatidylinositol. To investigate the fatty acid composition of the diacylglycerol (DAG) backbone of the GPI, granulosa cells were also labelled (5–72 hr) with [¹⁴C]linoleate, [³H]myristate, [³H]-oleate, [³H]palmitate, or [³H]stearate and the radioactivity associated with the purified glycosyl-phosphatidylinositol determined. Incorporation of [³H]palmitate and [³H]myristate into the GPI-lipid peaked after 8 h and 24 h of labelling, respectively, and both fatty acids were partially released after PLA₂ treatment of the dual labelled ([³H]glucosamine/[¹⁴C]palmitate or [³H]glucosamine/[¹⁴C]myristate) GPI. In parallel experiments no significant incorporation of labelled stearate, oleate, or linoleic acid into the DAG backbone of the glycosylphosphatidylinositol could be detected. Granulosa cells were also labelled with [³H]glucosamine in the presence of FSH (30 ng/ml), cholera toxin (1 μg/ml), or the membrane permeable cAMP analog (but)₂ cAMP (1 mM). Time related increases in GPI-labelling were apparent after 48 h and reached a maximum level (3-, 5-, and 7-fold for FSH, CT, and (but)₂ cAMP, respectively) after 72 h in culture. In another set of experiments, granulosa cells were labelled for 72 h with [³H]glucosamine in the presence of (but)₂cAMP (1 mM), TPA (10^?7 M), or combination thereof. The effect of treatment with the membrane permeable cAMP analog on GPI labelling was prevented in the presence of TPA, whereas no differences in [³H]GPI content could be observed in untreated granulosa cells or cells cultured in the presence of the protein kinase C-activating phorbol ester alone. In cells differentiated with FSH (30 ng/ml for 3 days) to induce LH receptors, treatment with hCG (100 ng/ml) induced a rapid (60 sec) and transient (5 min) decrease in the GPI content, whereas no efect of the hormone on undifferentiated granulosa cells could be observed. The rapid effect elicited by hCG on GPI content and turnover may be an early transduction mechanism involved in the biological effects of LH/hCG in differentiated granulosa cells. © 1993 Wiley-Liss, Inc.     

Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325° to 333deg;

In the framework of the European project aimed at the sequencing of the Bacillus subtilis genome the DNA region located between gerB (314°) and sacXV (333°) was assigned to the Institut Pasteur. In this paper we describe the cloning and sequencing of a segment of 97 kb of contiguous DNA. Ninety-two open reading frames were predicted to encode putative proteins among which only forty-two were found to display significant similarities to known proteins present in databanks, e.g. amino acid permeases, proteins involved in cell wall or antibiotic biosynthesis, various regulatory proteins, proteins of several dehydrogenase families and enzymes II of the phosphotransferase system involved in sugar transport. Additional experiments led to the identification of the products of new B. subtilis genes, e.g. galactokinase and an operon involved in thiamine biosynthesis.     

Occurrence of subcutaneous zygomycosis (Entomophthoromycosis Basidiobolae) caused by Basidiobolus haptosporus with pulmonary involvement

A case of a two-year-old boy with multiple subcutaneous lesions caused by Basidiobolus haptosporus is presented.The child had also a non-toxic familial goiter and clinical and radiological features of a pulmonary illness. The pulmonary manifestations only disappeared with the treatment with potassium iodide. The authors think that the pulmonary lesions must have arisen by direct spread of the fungus from the subcutaneous lesions of the chest.     

Resistance and mitotic instability to chloroneb and 1,4-oxathiin in Aspergillus nidulans.

Mutants resistant to two fungicides, chloroneb (1,4-dichloro-2,5-dimethoxybenzene) and vitavax (2,3-dihydro-5-carboxanilido-6-methyl-1,4-oxathiin) were spontaneously obtained from a strain of Aspergillus nidulans with frequencies of 12.5 and 1.1 respectively, in 10(8) conidia. One chloroneb-resistant mutant (Chl 1) segregated as a single gene and was mapped in linkage group IV. It also caused a partial dependence of the strain on the fungicide and was semi-dominant. The mutant resistant to vitavax (Vit 1) also segregated as a single gene and was dominant. Both fungicides altered the instability of diploid and duplication strains. Chloroneb mainly increased haploidization, and vitavax reduced the mitotic recombination in diploids. Chloroneb increased the instability of duplication strains, and vitavax reduced such instability. The possible mode of action of such fungicides affecting stability is discussed.