Mutants of Saccharomyces cerevisiae cell division cycle defective in cytokinesis. Biosynthesis of the cell wall and morphology

The four temperature-sensitive mutants of Saccharomyces cerevisiae in the cell division cycle defective in cytokinesis (cdc, 3, 10, 11 and 12), have been analyzed with respect to the biosynthesis of the cell wall polymers. After 3 hours of incubation at the non-permissive temperature (37°C) these strains stop growing. The synthesis of glucan, mannan and chitin (wall polymers) level off in a similar time, but glucan, mannan and chitin synthases remained active for at least 4 hours.If the mutants are analyzed by transmission and scanning electron microscopy different pictures emerge. Two of the mutants cdc 10 and cdc 12, after 3 hours of incubation at 37°C present apparently normal cytoplasms and cell wall surfaces with multiple elongated buds. The other two mutants, cdc 3 and cdc 11, present a completely disarranged cytoplasmic content and damage at the level of the plasma membrane is evident.These and other observations, suggest that between the execution points of cdc 3 (0.27) and cdc 10 (0.58), essential processes in the assembly of cell membrane occur.This work was supported in part by a grant from la Comisión de Investigación Científica y Técnica of the Spanish Ministerio de Educación y Ciencia (Project no. 4593-1980).     

Effects of Chlorine Concentration on the Structure of Poliovirus

Chlorine concentrations below 0.8 mg/liter inactivated poliovirus without causing separation of the viral components. These results indicate that the release of RNA from the capsids is the result, not the cause, of virus inactivation by chlorine.     

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.     

Fructose 1,6-bisphosphatase: Dissociation of AMP cooperativity and AMP inhibition by carbamylation

Selective treatment of pig kidney fructose 1,6-bisphosphatase with potassium cyanate leads to the formation of an active carbamylated enzyme that has lost the cooperative interactions among AMP sites, but retains sensitivity to inhibition of catalytic activity by the regulator AMP. Incorporation data on [¹⁴C]KNCO indicate that the loss of enzyme cooperativity at the AMP sites is related to selective carbamylation of four lysine residues per mole of tetrameric enzyme. Exhaustive carbamylation suggests that a second lysine residue per subunit is essential for AMP inhibition.     

Elevated levels of polyneuronal innervation persist for as long as two years in reinnervated frog neuromuscular junctions

When the nerve to an adult frog sartorius muscle is crushed, and axons are allowed to regenerate, the level of polyneuronal innervation at reinnervated neuromuscular junctions is higher than normal. With time, much of this polyneuronal innervation is reduced by the process of synapse elimination (Werle and Herrera, 1988). Using intracellular recording, we estimated the level of polyneuronal innervation in adult frog (Rana pipiens) sartorius muscles 2 years (range: 1.7-2.4 years) after crushing the sartorius nerve. We found that 27% (S.E. = 1.4%) of the junctions in muscles 2 years after reinnervation were polyneuronally innervated, whereas only 10% (S.E. = 1.2%) of the junctions in normal frog muscles were polyneuronally innervated. Thus, the synapse elimination that occurs following reinnervation does not restore the normal level of polyneuronal innervation. Histological comparisons of junctional structure between muscles 2 years after reinnervation and normal muscles revealed substantial differences. Reinnervated junctions had a greater length of synaptic gutter apposed by nerve terminal processes, more axonal inputs, more empty synaptic gutter, more instances of single synaptic gutters innervated by more than one axon, and longer lengths of nerve terminal processes that connect synaptic gutters within a junction. On the basis of this physiological and anatomical evidence, we conclude that nerve injury causes persistent changes in the pattern of muscle innervation.     

The influence of ionic composition on the distribution of nematode species in springs of the province of Granada (Spain)

The ionic composition of 38 mineral springs in the province of Granada (Spain), and the distribution of 45 species of nematodes belonging to orders Monhysterida, Araeolaimida, Chromadorida and Enoplida were examined. Water chemistry is used to make two diagrams representing anionic and cationic composition. Diagrams for anionic composition (given the greater variance seen in the springs considered) are used to illustrate the distribution of individual species. The results obtained from species distribution and the correlation between species made it possible to group species which could be associated with springs where each of the anions considered predominated. A greater number of species groups was found to inhabit springs in which chloride concentrations was less than 50% of the total concentration of anions.     

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.     

Effects of progesterone and estradiol on the proliferation of phytohemagglutinin-stimulated human lymphocytes

In this paper we report on a study to elucidate whether the response of human lymphocytes to mitogenic stimulation was modified by physiological changes which occur during the menstrual cycle. Experiments with untreated cultures showed intra-individual variation to mitogen stimulation in female lymphocyte cultures, but a significant correlation between the menstrual cycle and the proliferation kinetics of lymphocytes was not found. Consequently, we performed experiments in which two of the hormones that regulate the menstrual cycle in women, estradiol and progesterone, were added to cultured human lymphocytes obtained from both men and women. The results indicate that both hormones at physiological concentrations have the capacity to modify the proliferation of PHA-stimulated human lymphocytes. Therefore, both hormones could play a role in the induction of the intra-individual variation observed in the untreated female cultures. However, in vivo other factors could also modify the proliferation kinetics of human lymphocytes preventing the demonstration of the effects of a single factor, such as the hormonal changes occurring during the menstrual cycle.