Characterization of the subunit structure of gonadotropin receptor in luteinized rat ovary

Gonadotropin receptors with specificity, high affinity and low capacity for luteinizing hormone and human chorionic gonadotropin (hCG) have been identified in rat luteal cells. To investigate the nature of the receptor, we have employed disuccinimidyl suberate, a cross-linker noncleavable by reducing agents, and dithiobis(succinimidyl propionate), a cleavable cross-linker, to covalently cross-link the 125I-hCG . receptor complex. The molecular weight of 125I-hCG-linked receptor complex and the receptor subunit structure were determined by electrophoresis in either 10 or 4.5% acrylamide in the presence of 0.1% sodium dodecyl sulfate with or without reducing agents. Autoradiographic analysis of the 125I-hCG-linked receptor separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing condition revealed a single labeled band corresponding to Mr = 305,000 +/- 15,000. However, electrophoresis performed in the presence of 50 mM dithiothreitol and 2% beta-mercaptoethanol resulted in the appearance of four labeled bands corresponding to Mr = 105,000 +/- 4,000, 96,000 +/- 5,000, 74,000 +/- 4,000, and 62,000 +/- 4,000 concomitant with the loss of the labeled band in the Mr = 305,000 region. Further experiments demonstrated that these four labeled bands were derived from the same molecular species. In addition, the 125I-hCG-linked receptor in the absence of reducing agent was not dissociated into subunits even by treatment with strong denaturing agent (8 M urea). The appearance of the cross-linked 125I-hCG . receptor was effectively inhibited by the unlabeled beta-subunit of hCG, intact hCG, and luteinizing hormone and partially inhibited by the alpha-subunit of hCG but not by choleratoxin, gonadotropin-releasing hormone, insulin or bovine serum albumin. These data suggest that 1) the hCG/luteinizing hormone receptor is an oligomeric complex linked by disulfide bonds and 2) that under reducing conditions, the oligomeric receptor dissociates into four nonidentical subunits.     

Contribution of cardiogenic oscillations to gas exchange in constant-flow ventilation

The contribution of cardiogenic oscillations to gas exchange during constant-flow ventilation was examined in 11 dogs. With the use of two variations of cardiopulmonary bypass to maintain the systemic and pulmonary circulation, the influence of cardiogenic oscillations was removed by arresting the heart. Cardiac arrest by ventricular fibrillation was associated with a mean decrease in alveolar ventilation of 43% in five dogs on right and left heart bypass. However, successful defibrillation and return of the prearrest level of alveolar ventilation could not be achieved; thus we studied six dogs on left heart bypass. Alveolar ventilation decreased an average of 37% with cardiac arrest, and defibrillation resulted in a return of alveolar ventilation to 81% of the prearrest value. These results are consistent with previous predictions that cardiogenic oscillations are an important mechanism of gas transport during constant-flow ventilation.     

Transport and metabolism of thiamine in Ehrlich ascites-carcinoma cells

1. Aerobic or anaerobic incubation at 37 degrees of Ehrlich ascites-carcinoma cells in Krebs-Ringer bicarbonate medium containing glucose and labelled thiamine results in accumulation in the cell of labelled thiamine, so that the concentration of total labelled thiamine in the cells greatly exceeds (by a factor 7) that in the medium. This concentration ratio is approximately constant for small initial external concentrations of labelled thiamine but diminishes when the latter exceed 0.4mum. 2. All the labelled thiamine in the tumour cells is present as thiamine phosphates. 3. The uptake of labelled thiamine is markedly diminished by decrease of temperature. At 9 degrees concentration ratio (cells/medium) 0.5 is observed whereas at 37 degrees the concentration ratio is 8.6. 4. The extent of phosphorylation of labelled thiamine depends on the period of incubation. 5. The influx of labelled thiamine is diminished by the presence of its analogues, pyrithiamine and Amprol, and also by the presence of thiamine monophosphate and thiamine diphosphate, which are potent inhibitors of thiamine phosphorylation in Ehrlich ascites cells. 6. Labelled thiamine phosphates leak from the cell into the medium, so that eventually all the labelled thiamine, both in the cell and medium, is converted into thiamine phosphates. However, in the presence of 2,4-dinitrophenol (0.1mm) and iodoacetate (1mm) thiamine phosphorylation is diminished, the concentration ratio for labelled thiamine (cells/medium) falls to half its normal value and little or no labelled thiamine phosphates leaks into the medium. 7. In the presence of thiamine phosphates, free labelled thiamine accumulates in Ehrlich ascites cells against a concentration gradient, concentration ratios (cells/medium) greater than unity being evident. 8. The evidence supports the conclusion that thiamine is transferred into the Ehrlich ascites cell by a carrier-mediated energy-assisted process.     

Effects of negative upper airway pressure on pattern of breathing in sleeping infants

Negative upper airway (UAW) pressure inhibits diaphragm inspiratory activity in animals, but there is no direct evidence of this reflex in humans. Also, little is known regarding reflex latency or effects of varying time of stimulation during the breathing cycle. We studied effects of UAW negative pressure on inspiratory airflow and respiratory timing in seven tracheostomized infants during quiet sleep with a face mask and syringe used to produce UAW suction without changing lower airway pressure. Suction trials lasted 2-3 s. During UAW suction, mean and peak inspiratory airflow as well as tidal volume was markedly reduced (16-68%) regardless of whether stimulation occurred in inspiration or expiration. Reflex latency was 42 +/- 3 ms. When suction was applied during inspiration or late expiration, the inspiration and the following expiration were shortened. In contrast, suction applied during midexpiration prolonged expiration and tended to prolong inspiration. The changes in flow, tidal volume, and timing indicate a marked inhibitory effect of UAW suction on thoracic inspiratory muscles. Such a reflex mechanism may function in preventing pharyngeal collapse by inspiratory suction pressure.     

Nature of the oxygen species generated by xanthine oxidase involved in secretory histamine release from mast cells

The present studies were carried out to characterize the nature of reactive oxygen species generated by the xanthine-xanthine oxidase system involved in the release of histamine by noncytotoxic and cytotoxic mechanisms. To distinguish secretory release from lytic release, mast cells were loaded with 51Cr and the release of 51Cr into the incubation medium was used as a measure of cell lysis. The secretory release of histamine was not inhibited by superoxide dismutase or catalase alone. However, together these agents inhibited the release. This suggests that the combination of superoxide and hydrogen peroxide can evoke secretory release. The lytic release of histamine, as monitored by concomitant release of 51Cr from mast cells at higher concentration of xanthine oxidase or longer periods of incubation, seems to be related to hydrogen peroxide production since catalase inhibited the cell lysis. Since it has been reported that exogenously added hydrogen peroxide at concentrations below 10 mM did not induce cell lysis, the lytic release, although hydrogen peroxide dependent, may not be due to its direct effect on the cell surface. The cell lysis observed in the xanthine-xanthine oxidase system seems to be brought about by a complex mechanism involving the interactions of hydrogen peroxide and superoxide with cellular components. These studies indicate that the beneficial effects of superoxide dismutase seen in biological systems may partly be due to inhibition of the secretory processes stimulated by superoxide.     

Physical mapping of the von Recklinghausen neurofibromatosis region on chromosome 17

The von Recklinghausen neurofibromatosis (NF1) locus has been linked to chromosome 17, and recent linkage analyses place the gene on the proximal long arm. NF1 probably resides in 17q11.2, since two unrelated NF1 patients have been identified who possess constitutional reciprocal translocations involving 17q11.2 with chromosomes 1 and 22. We have used a somatic-cell hybrid from the t(17;22) individual, along with other hybrid cell lines, to order probes around the NF1 locus. An additional probe, 17L1, has been isolated from a NotI linking library made from flow-sorted chromosome 17 material and has been mapped to a region immediately proximal to the translocation breakpoint. While neither NF1 translocation breakpoint has yet been identified by pulse-field gel analysis, an overlap between two probes, EW206 and EW207, has been detected. Furthermore, we have identified the breakpoint in a non-NF1 translocation, SP-3, on the proximal side of the NF1 locus. This breakpoint has been helpful in creating a 1,000-kb pulsed-field map, which includes the closely linked NF1 probes HHH202 and TH17.19. The combined somatic-cell hybrid and pulsed-field gel analysis we report here favors the probe order D17Z1-HHH202-TH17.19-CRYB1-17L1-NF1- (EW206, EW207, EW203, L581, L946)-(ERBB2, ERBA1). The agreement in probe ordering between linkage analysis and physical mapping is excellent, and the availability of translocation breakpoints in NF1 should now greatly assist the cloning of this locus.     

Role ofCandida in indirect pathogenesis of antibiotic associated diarrhoea in infants

One hundred and thirty seven isolates ofCandida species were isolated from antiobiotic associated diarrhoea cases and were examined to study the role ofCandida in the pathogenesis of diarrhoea in infants. The quantitative estimation of yeast population by simple gram stain smear revealed more than 70% of the cases had 3+ score. The isolates further screened for detection of-lactamases. Among the isolatedCandida sp,-lactamases was secreted byC. albicans, C. tropicalis, C. krusei andC. parapsilosis. Further, 46% of theCandida isolates were found to be produced 741–1110 mU/ml of-lactamases, suggesting that these enzyme would inactivate penicillin group of drugs and cause failure in the therapy directed against other diarrhoegenic bacteria.Abbreviation AAD antibiotic associated diarrhoea     

Effect of lipid supplements on the production and glycosylation of recombinant interferon-γ expressed in CHO cells

The effects of lipids on the glycosylation of recombinant human interferon- expressed in a Chinese Hamster Ovary cell line were investigated in batch culture. Lipids form an essential part of the N-glycosylation pathway, and have been shown to improve cell viability. In control (serum-free) medium the proportion of fully-glycosylated interferon- deteriorated reproducibly with time in batch culture, but the lipoprotein supplement ExCyte was shown to minimise this trend. Partially substituting the bovine serum albumin content of the medium with a fatty-acid free preparation also improved interferon- glycosylation, possibly indicating that oxidised lipids carried on Cohn fraction V albumin may damage the glycosylation process.Abbreviations BSA bovine serum albumin - CHO chinese hamster ovary - DHFR dihydrofolate reductase - FCS foetal calf serum - IFN- human interferon-gamma - q _IFN specific interferon production rate - specific growth rate - 2N doubly-gycosylated - 1N singly-glycosylated - ON non-glycosylated