Production of superoxide and activity of superoxide dismutase in rabbit epididymal spermatozoa

Mature rabbit spermatozoa from the cauda epididymidis suspended in potassium Tris phosphate buffer at 24 degrees C produced O2.-, as measured by reduction of acetylated ferricytochrome c, with an intrinsic rate of 0.20 nmol/min per 10(8) cells. This rate increased to 1.80 nmol/min per 10(8) cells in the presence of 10 mM cyanide. These spermatozoa contain 2.8 units per 10(8) cells of superoxide dismutase activity, 95% of which is sensitive, and 5% of which is insensitive, to cyanide inhibition. These activities correspond to the cytosolic Cu-Zn form and the mitochondrial Mn form of the dismutase, respectively. Only the cyanide-sensitive form is released from the sperm on hypo-osmotic treatment or sonication. Hypo-osmotically treated rabbit epididymal spermatozoa produced O2.- with an intrinsic rate of 0.24 nmol/min per 10(8) cells, which increased to 0.58 nmol/min per 10(8) cells in the presence of 10 mM cyanide. Both intact and hypo-osmotically treated cells react with O2.- in a second order reaction as inferred from the hyperbolic dependence on cell concentration of O2.- production rate in both the absence and presence of cyanide. The second order rate constant for this reaction with intact cells, kS, was calculated to be 22.9 X 10(-8) (cells/ml)-1 min-1 in its absence. For hypo-osmotically treated cells, the values of kS were 10.8 X 10(-8) (cells/ml)-1 min-1 and 8.2 X 10(-8) (cells/ml) -1 min-1, respectively. Since hypo-osmotically treated cells have lost much of their plasma membrane, the lower value of kS for the treated cells implies that this membrane is one site of reaction of O2.- with the cells. The increase in kS in the presence of cyanide, which inhibits superoxide dismutase and so increases O2.- production, suggests that the cells become more reactive with O2.- as its production rate increase, as would be expected for the occurrence of radical chain oxidation. This in turn suggests that superoxide dismutase plays a major role in protecting rabbit sperm against damage from lipid peroxidation.     

Pro-Leu-Ser/Thr-Pro is a consensus primary sequence for substrate protein phosphorylation. Characterization of the phosphorylation of c-myc and c-jun proteins by an epidermal growth factor receptor threonine 669 protein kinase.

A growth factor-stimulated (MAP2-related) protein kinase, ERT, that phosphorylates the epidermal growth factor receptor at Thr669 has been purified from KB human tumor cells by Northwood and co-workers (Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 15266-15276). The ERT protein kinase has a restricted substrate specificity, and the structural determinants employed for substrate recognition by this enzyme have not been defined. As an approach toward understanding the specificity of substrate phosphorylation, we have used an in vitro assay to identify additional substrates for the ERT protein kinase. In this report we describe two novel substrates: (a) the human c-myc protein at Ser62 and (b) the rat c-jun protein at Ser246. Alignment of the primary sequences surrounding the phosphorylation sites located within the epidermal growth factor receptor (Thr669), Myc (Ser62), and Jun (Ser246) demonstrated a marked similarity. The observed consensus sequence was Pro-Leu-Ser/Thr-Pro. We propose that this sequence forms part of a substrate structure that is recognized by the ERT protein kinase.     

Differential proliferative response of cultured fetal and regenerating hepatocytes to growth factors and hormones

Upon epidermal growth factor (EGF) stimulation, fetal (20 days of gestation) and regenerating (44-48 h after partial hepatectomy) rat hepatocytes, isolated and cultured under identical conditions, increased DNA synthesis and entered into S-phase and mitosis, measured as [3H]thymidine incorporation and DNA content per nucleus in a flow cytometer, respectively. Fetal hepatocytes consisted of a homogeneous population of diploid (2C) cells. Two different populations of cells were present in regenerating liver, diploid (2C) and tetraploid (4C) cells, that responded to EGF. Glucagon or norepinephrine did not affect EGF stimulation of DNA synthesis in fetal liver cells, but they potentiated EGF response in regenerating hepatocyte cultures. Glucocorticoid hormones (dexamethasone) inhibited DNA synthesis in fetal hepatocyte cultures, an effect potentiated by the presence of glucagon or norepinephrine. In contrast, in regenerating hepatocytes, dexamethasone increased EGF-induced proliferation. EGF-dependent DNA synthesis was inhibited by TGF-beta in both fetal and regenerating cultured hepatocytes. TGF-beta action was partially suppressed by norepinephrine in regenerating hepatocytes, but was without effect in fetal hepatocyte cultures, whereas a synergistic action between TGF-beta and dexamethasone inhibiting growth in fetal but not in regenerating hepatocytes was found. Taken together, these results may suggest that there are significant differences between fetal and regenerating hepatocyte growth in their response to various hormones.     

High affinity inhibition of Ca(2+)-dependent K+ channels by cytochrome P-450 inhibitors.

The Ca(2+)-dependent K+ channel of human red cells was inhibited with high affinity by several imidazole antimycotics which are potent inhibitors of cytochrome P-450. IC50 values were (in microM): clotrimazole, 0.05; tioconazole, 0.3; miconazole, 1.5; econazole, 1.8. Inhibition of the channel was also found with other drugs with known cytochrome P-450 inhibitory effect. However, no inhibition was obtained with carbon monoxide (CO). This suggests that, given the high selectivity of the above inhibitors for the heme moiety, a different but closely related to cytochrome P-450 kind of hemoprotein may be involved in the regulation of the red cell Ca(2+)-dependent K+ channel. Clotrimazole also inhibited two other charybdotoxin-sensitive Ca(2+)-dependent K+ channels, those of rat thymocytes (IC50 = 0.1-0.2 microM) and of Ehrlich ascites tumor cells (IC50 = 0.5 microM). Imidazole antimycotics inhibit also receptor-operated Ca2+ channels (Montero, M., Alvarez, J. and García-Sancho, J. (1991) Biochem. J. 277, 73-79). This suggests that both Ca2+ and Ca(2+)-dependent K+ channels might have a similar regulatory mechanism involving a cytochrome.     

Prothymosin alpha mRNA levels are invariant throughout the cell cycle.

Prothymosin alpha (PT-alpha) mRNA levels were evaluated at different stages during the cell cycle. NIH 3T3 cells were synchronized: (a) by serum deprivation, (b) by mitotic shake off after nocodazole arrest, and (c) by double thymidine block. Cell synchronism was estimated by flow cytometry. In cells grown in serum-free medium, PT-alpha mRNA levels were almost undetectable. 14 h after serum restoration PT-alpha mRNA was induced as had been described by others (Eschendfeldt, W. H., and Berger, S. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 9403-9407). PT-alpha mRNA induction seems to require the synthesis of proteic factor(s) since PT-alpha mRNA response to serum restoration was abolished in the presence of cycloheximide. Interestingly, cycling cells that were synchronized at different stages of the cycle by means of mitotic shake off after nocodazole arrest or double thymidine block did not show variations in the levels of PT-alpha mRNA when progressed synchronously through the cycle. On the contrary, histone H4 mRNA was expressed only during the S phase. These data indicate that PT-alpha mRNA was present in roughly the same amount through all phases of the cell cycle, arguing against the concept that PT-alpha is a cell cycle-regulated gene.     

LEAFY controls floral meristem identity in Arabidopsis.

The first step in flower development is the generation of a floral meristem by the inflorescence meristem. We have analyzed how this process is affected by mutant alleles of the Arabidopsis gene LEAFY. We show that LEAFY interacts with another floral control gene, APETALA1, to promote the transition from inflorescence to floral meristem. We have cloned the LEAFY gene, and, consistent with the mutant phenotype, we find that LEAFY RNA is expressed strongly in young flower primordia. LEAFY expression procedes expression of the homeotic genes AGAMOUS and APETALA3, which specify organ identify within the flower. Furthermore, we demonstrate that LEAFY is the Arabidopsis homolog of the FLORICAULA gene, which controls floral meristem identity in the distantly related species Antirrhinum majus.     

The effects of 21 or 30% O2 plus umbilical cord occlusion on fetal breathing and behavior.

We have shown previously that continuous fetal breathing can be induced by 100% O2 alone or combined with umbilical cord occlusion (Baier, Hasan, Cates, Hooper, Nowaczyk & Rigatto, 1990). To know whether it could also be induced by lower O2 concentrations plus cord occlusion, we studied 9 chronically instrumented fetal sheep (16 experiments) using our window model. After a baseline cycle [1 low voltage + 1 high voltage electrocortical activity (ECoG) epoch] the fetal lung was distended via an endotracheal tube to about 30 cm H2O. Inspired N2 (control) and 21 or 30% O2 were given for one cycle each. While on 21% or 30% O2 the umbilical cord was occluded (balloon cuff). In 10 out of 16 experiments breathing output (% maximum of integral of EMGdi x f) increased after cord occlusion from 80 +/- 48 (N2) to 2871 +/- 641 (SEM; P < 0.01); in 7 of them breathing became continuous. Arterial PO2 increased from 14 +/- 1 (N2) to 33.5 +/- 5 Torr (occlusion; P < 0.01). In the other 6 experiments breathing output decreased from 319 +/- 116 (N2) to 86 +/- 38 (occlusion; P < 0.01) and arterial PO2 changed from 18 +/- 1 (N2) to 22 +/- 5 Torr (occlusion; P = 0.4). Arterial PCO2 increased similarly after occlusion in both groups, those which did respond with increased breathing (to 46 +/- 2 Torr) and those which did not respond (to 48 +/- 3 Torr; P = 0.6). The percent low voltage ECoG and the behavioral score increased after occlusion in the responder group only.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of aerobic biodegradation of benzene and toluene in sandy aquifer material

Monod's equation adequately described aerobic biodegradation rates of benzene and toluene by the microbial population of a sandy aquifer when these compounds were initially present at concentrations lower than 100 mg/l each. Concentrations higher than 100 mg/l were inhibitory, and no benzene or toluene degradation was observed when these compounds were initially present at 250 mg/l each. The Monod coefficients were calculated as k = 8.3 g-benzene/g-cells/day and Ks = 12.2 mg/l for benzene, and k = 9.9 g-toluene/g-cells/day and Ks = 17.4 mg/l for toluene. Specific first-order coefficients would be 0.68 l/mg.day for benzene and 0.57 l.mg.day for toluene.     

Phospholipid turnover during phagocytosis in human polymorphonuclear leucocytes

We have previously observed that the phagocytosis of zymosan particles coated with complement by human polymorphonuclear leucocytes is accompanied by a time- and dose-dependent inhibition of phosphatidylcholine synthesis by transmethylation [García Gil, Alonso, Sánchez Crespo & Mato (1981) Biochem. Biophys. Res. Commun. 101, 740–748]. The present studies show that phosphatidylcholine synthesis by a cholinephosphotransferase reaction is enhanced, up to 3-fold, during phagocytosis by polymorphonuclear cells. This effect was tested by both measuring the incorporation of radioactivity into phosphatidylcholine in cells labelled with [Me-¹⁴C]choline, and by assaying the activity of CDP-choline:diacylglycerol cholinephosphotransferase. The time course of CDP-choline:diacylglycerol cholinephosphotransferase activation by zymosan mirrors the inhibition of phospholipid methyltransferase activity previously reported. The extent of incorporation of radioactivity into phosphatidylcholine induced by various doses of zymosan correlates with the physiological response of the cells to this stimulus. This effect was specific for phosphatidylcholine, and phosphatidyl-ethanolamine turnover was not affected by zymosan. The purpose of this enhanced phosphatidylcholine synthesis is not to provide phospholipid molecules rich in arachidonic acid. The present studies show that about 80% of the arachidonic acid generated in response to zymosan derives from phosphatidylinositol. A transient accumulation of arachidonoyldiacylglycerol has also been observed, which indicates that a phospholipase C is responsible, at least in part, for the generation of arachidonic acid. Finally, isobutylmethylxanthine and quinacrine, inhibitors of phosphatidylinositol turnover, inhibit both arachidonic acid generation and phagocytosis, indicating a function for this pathway during this process.     

A brief comparative study of four disposable bubble oxygenators

Four widely used bubble oxygenators-the Optiflo I, the Bentley Q 200 A, the Harvey 200, and the Shiley 100 A-were tested and compared in 182 patients undergoing cardiac valve surgery. Fifty-six cases were performed with normothermia and 126 cases incorporated mild hypothermia (28-30 degrees C). There was no significant difference in the average age of the patients (51 yrs) or the perfusion time (60 min). All components of the extracorporeal circuit were identical, and anesthetic regimens and surgical techniques were also similar. In this study, the Shiley 100 A oxygenator was found to be the most suitable for cases requiring mild hypothermia and was generally considered to be the oxygenator of choice.