Evidence for the activation of phospholipases during acrosome reaction of human sperm elicited by calcium ionophore A23187

Incubation of washed human sperm with [3H]- or [14C]arachidonic acid allowed a major incorporation of the label into phospholipids, provided that the final concentration of the fatty acid did not exceed 20 microM. A further challenge with calcium ionophore A23187 of spermatozoa suspended in a calcium-containing medium led to phospholipid hydrolysis, which could account for 10-12% of total cell radioactivity. Degradation products were identified as free, unconverted arachidonic acid, occurring with some diacylglycerol. Phospholipid hydrolysis was significant after 15 min of incubation and became maximal after 120 min. It was found to be calcium dependent, diacylglycerol and free arachidonate production occurring maximally at 2 mM and 5 mM CaCl2, respectively. Phosphatidylcholine and phosphatidylinositol were the most significantly degraded phospholipids after 60 min of incubation. Similar incubations conducted with 32P-labeled sperm confirmed the selective hydrolysis of phosphatidylcholine and revealed an increase production of phosphatidic acid probably due to a phosphorylation of diacylglycerol. Under the same conditions, one third of the cells remained motile and electron microscopy revealed that acrosome reaction was completed in 40% of the cells and displayed an intermediary state in 40-50% of the spermatozoa. Furthermore, a good parallelism was observed between the extent of the acrosome reaction and the extent of phospholipid hydrolysis promoted by increasing concentrations of A23187. It is concluded that calcium entry into the cells activates both a phospholipase A2 and a phospholipase C, leading to the production of substances, like lysophospholipid, diacylglycerol or phosphatidic acid, which may or may not be involved in acrosome reaction.     

Phospholipase C from human sperm specific for phosphoinositides

Human sperm lysates were incubated in the presence of 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphocholine, 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoethanolamine or 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoinositol. Only the latter substrate was hydrolyzed to a significant extent, with a concomitant formation of 1-[14C]stearoyl-2-acyl-sn-glycerol. Furthermore, incubation of phosphatidyl[3H]inositol under the same conditions was accompanied by the formation, in roughly equal amounts, of [3H]inositol 1-phosphate and [3H]inositol 1:2-cyclic monophosphate. Finally [32P]phosphatidylinositol 4-phosphate and [32P]phosphatidylinositol 4,5-bisphosphate were degraded into [32P]inositol 1,4-bisphosphate and [32P]inositol 1,4,5-trisphosphate, respectively. The phosphoinositide-specific phospholipase C was activated by calcium (optimal concentration 5-10 mM) and inhibited by EGTA, although endogenous calcium supported a half-maximal activity. The enzyme displayed an optimal pH of 6.0 and an apparent Km of 0.08 mM. Its specific activity was around 10 nmol/min per mg protein, which is approximately the same as that found in human blood platelets. Subcellular fractionation revealed that 55% of the enzyme was solubilized under conditions where 80% of acrosin appeared in the supernatants. The majority of the particulate phospholipase C activity (37% of total) was found in the 1000 X g pellet, which contained only 8% of total acrosin activity. Further fractionation of spermatozoa into heads and tails indicated no specific enrichment of phospholipase C activity in any of these two fractions. However, owing to a 4-fold higher protein content in the head compared to the tail fraction, it is concluded that about 80% of particulate phospholipase C activity is located in sperm head. The physiological significance of this enzyme is discussed in relation to a possible role in acrosome reaction and (or) in egg fertilization.     

The aluminium signal: New dimensions to mechanisms of aluminium tolerance

The physiological basis of plant reaction to and tolerance of aluminium (Al) is poorly understood. We review the results of investigations into Al toxicity and root physiology to develop a theoretical basis for explaining the reaction of the root to Al, including suggested roles for Ca²⁺, mucilaginous cap secretions and endogenous growth regulators in mediating a transmitted response between Al-damaged cap cells and the interacting cell populations of the cap and root. This information is used to identify possible mechanisms of Al tolerance, notably involving signal transduction, Al uptake pathways and root morphogenesis; and to briefly discuss how procedures selecting for Al tolerance may be improved by incorporating the concept of stimulus-response coupling. Similarities in the responses of roots to Al and other signals (e.g. gravity, light, mechanical impedance) are used to develop the hypothesis that roots respond to environmental signals by way of a common regulatory system. New research prospects for extending our perception of Al tolerance mechanisms are identified.     

Energy-dependent transport of nickel by Clostridium pasteurianum.

The mechanism of nickel transport by Clostridium pasteurianum was investigated by using 63NiCl2 and a microfiltration transport assay. Nickel transport was energy dependent, requiring either glucose or sucrose; xylose and o-methyl glucose did not support growth, butyrogenesis, or transport. Transport was optimum at pH 7 and 37 degrees C, and early-stationary-phase cells had the highest propensity for nickel transport. The apparent Km and Vmax for nickel transport approximated 85 microM Ni and 1,400 pmol of Ni transported per min per mg (dry weight) of cells, respectively. On the basis of metal specificity, nickel appears to be transported primarily by a magnesium transporter, although an alternative nickel transporter may also be involved. ATPase inhibitors (N,N'-dicyclohexylcarbodiimide, tributyltin chloride, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and quercetin), protonophores (carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, and gramicidin D), metal ionophores (valinomycin, monensin, and nigericin), benzyl viologen, carbon monoxide, and oxygen inhibited nickel transport. Nickel transport was coupled indirectly to butyrogenesis and was dependent on the energy state of the cell.     

Impaired production of gamma-interferon by newborn cells in vitro is due to a functionally immature macrophage

The decreased production of gamma-(PHA-induced) interferon (IFN) by leukocytes of normal newborns could be due to functionally immature T cells, macrophages, or both. We studied gamma-IFN production by macrophages and T cells, alone and in combination, obtained from 50 cord blood samples and 14 adult blood samples in a series of experiments. Adherent macrophages were cultivated for 7 days before the addition of T cells. After 48 hr, PHA-stimulated macrophage-T cell supernatants were harvested and assayed for IFN by a microassay. Macrophage-T cell cultures of autologous and nonautologous cells in 14 adults showed enhanced IFN production (GMT 121 +/- 5 IU) as compared with Ficoll-Hypaque mononuclear cells (GMT 42 +/- 5 IU). No IFN was detected in supernatants from PHA-stimulated Ficoll-Hypaque cord cells alone or macrophage-T cord combined cultures. Combined cord macrophages and adult T cells produced minimal IFN (GMT 13 +/- 3 IU); however, cord T cells combined with adult macrophages showed enhanced IFN production (GMT 195 +/- 47 IU). This cord macrophage dysfunction was not due to an inhibitor and improved with the time of in vitro cultivation. These results indicate that the neonatal macrophage is primarily responsible for the impaired gamma-IFN response by the newborn cells.     

Twin seals in Scotland

Published accounts of twinning in seals are reviewed briefly and details are given of new records of twins in the Common and Grey seals in Scotland.     

Mutator Gene of Escherichia coli B

An azaserine-resistant derivative of Escherichia coli B/UV, AZA/R(1), was found to carry a mutator gene. This gene, designated mutS1, was mapped by means of conjugation and P1kc-mediated transduction. The mutS1 gene was cotransduced with argB at a frequency of 2.4%; the gene order in this region of the chromosome is thy argB mutS1. To determine whether a relationship commonly exists between azaserine resistance and the mutator property, 12 additional azaserine-resistant derivatives of B/UV were developed and tested for the mutator phenotype. None of the twelve was a mutator strain. The level of azaserine resistance was not increased over that of the recipient parent when mutS1 was transduced to an azaserine-susceptible strain. Reversion studies indicated that mutS1 induced adenosine-ribosylthymine to guanosine-cytidine and guanosine-cytidine to adenosine-ribosylthymine transitions. Because such mutational changes are suppressible with deoxynucleosides when induced by base analogues, an attempt was made to suppress the mutator activity of mutS1 by the addition of deoxyribonucleosides to the medium. No suppression was found. Recombinants were prepared containing mutS1 and the Treffers mutator gene of E. coli K-12. The effect of the mutator genes appears to be additive.