Influence of caffeine on movement characteristics, fertilizing capacity and ability to penetrate cervical mucus of human spermatozoa

When added to frozen-thawed human semen, the 3 doses of caffeine tested (2, 5 and 10 mM) induced a significant increase in the percentage of motile spermatozoa but did not influence the quality of movement. Considerable variability was noted between samples in their responsiveness to caffeine which, at the 5 and 10 mM doses, was significantly correlated with the degree of motility lost during cryostorage. Caffeine treatment of frozen-thawed human spermatozoa also increased the number of spermatozoa penetrating cervical mucus in unit time, by increasing the frequency rather than the success of collisions between spermatozoa and the cervical mucus interface. When caffeine-stimulated spermatozoa were washed free of seminal plasma containing this compound they were no longer at an advantage with respect to their motility or fertilizing ability. When 2 mM-caffeine was added to washed suspensions of capacitated spermatozoa it failed to stimulate motility but did significantly enhance the fertilizing ability of the spermatozoa, indicating a possible clinical role for this compound in in-vitro fertilization therapy.     

Reliable plantlet formation from embryos and seedling shoot tips of radiata pine

A method has been devised for the reliable production of plantlets from embryos and seedling shoot tips of Pinus radiata D.Don (radiata pine). Buds were induced on an agar or liquid Schenk and Hildebrandt (SH) medium containing 5.0 mg/l benzylaminopurine (BAP). Except for some abnormal buds, the buds grew into elongated shoots on an agar SH medium without cytokinin. The transfer of shoots from a SH medium to a Gresshoff and Doy (GD) medium was found to be an important pretreatment which increased the survival of the shoots when they were placed in a peat and pumice mix for root formation. Elongated shoots were induced to form roots under non-sterile conditions in a humid environment with occasional misting. An intervening 5-day treatment of shoots in an agar medium containing 2.0 mg/l indolebutyric acid (IBA) and 0.5 mg/l napthaleneacetic acid (NAA) significantly increased the percentage of shoots forming roots and the number of roots formed per shoot over control shoots placed directly in the peat:pumice mix. An enhanced level of CO₂ during root formation had no effect on the time of root formation or on the percentage of shoots forming roots. These results concerning the elongation, growth and rooting of adventitious shoots are now being applied to the development of very large numbers of plantlets starting from cotyledons from partially germinated seeds.     

Properties of halophil nicotinamide–adenine dinucleotide phosphate-specific isocitrate dehydrogenase. True Michaelis constants, reaction mechanisms and molecular weights

True values of Michaelis constants of the NADP(+)-specific isocitrate dehydrogenase from Halobacterium salinarium were not very different from those of the apparent constants reported by Aitken et al. (1970). The true constants were affected by salt in a similar manner to that of the apparent constants obtained with NADP(+) at fixed concentrations of 1.0-0.2mm and threo-d(s)-(+)-isocitrate at fixed concentrations of 2.0-0.125mm. The response of apparent V(max.) to salt concentration was highly dependent on fixed substrate concentration in solutions of sodium chloride but much less so in solutions of potassium chloride. At several levels the results emphasize the difficulty of generalizing about the salt relations of a halophil enzyme without adequate attention to substrate concentration. The enzyme has at least two different reaction mechanisms depending on salt concentration. In its ;physiological' form (i.e. in 1.0m-potassium chloride), and also in 1.0m-sodium chloride, the reaction mechanism is ordered with NADP(+) the first substrate added and NADPH the last product released. In 0.25m-sodium chloride, however, the mechanism is different and is probably non-sequential. In 4.0m-sodium chloride with low concentrations of either fixed substrate, there was evidence of a co-operative action of the variable substrate. The evidence suggests that salt participates in the reaction mechanism in two ways: one is the reversible addition to the enzyme in a manner analogous to that of a substrate; the other is dead-end complex-formation. The relative contributions of these two types of reaction determine whether salt activates or inhibits the enzyme. In addition, the inhibition caused by high concentrations of sodium chloride is more complex than the corresponding inhibition by potassium chloride. Gel-filtration experiments indicated that at very low salt concentrations the enzyme has an apparent molecular weight of about 70800. In ;physiological' concentrations of potassium chloride the enzyme appears to be a dimer (mol.wt. 122000-135000) and, in 1.0-4.0m-sodium chloride, it behaves as a trimer or tetramer (mol.wt. 224000-251000). A preliminary method of purifying the enzyme is described.     

Multiple isoforms of a protein kinase C inhibitor (KCIP-1/14-3-3) from sheep brain. Amino acid sequence of phosphorylated forms.

A potent inhibitor of protein kinase C (PKC), inhibitor protein-1 (KCIP-1), isolated from sheep brain has been shown to consist of eight isoforms by reverse-phase HPLC. Direct protein sequence analysis has revealed these to be the same as those of 14-3-3 protein, described as an activator of tyrosine and tryptophan hydroxylases involved in neurotransmitter biosynthesis. The N-termini of KCIP-1 isoforms were shown to be acetylated, and secondary structure predictions revealed a high degree of alpha-helix with an amphipathic nature. KCIP-1 showed no inhibitory activity towards protein kinase M (the catalytic fragment of PKC) and had no effect on the activities of three other protein kinases, cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase II and casein kinase 2. Four forms of KCIP-1 were shown to be substrates for PKC in vitro, but none were phosphorylated by the other protein kinases mentioned above.     

Purification and characterization of thioredoxin from the N2-fixing cyanobacterium Anabaena cylindrica

Thioredoxin has been purified to homogeneity from the cyanobacterium Anabaena cylindrica. The protein consists of a single polypeptide chain with a relative molecular mass of about 11 680 which has two cysteine residues (residues 31 and 34) in the sequence-Cys-Gly-Pro-Cys- and an isoelectric point at pH 4.55. The N-terminal amino acid sequence of 39 residues shows distinct homologies with the sequences of Escherichia coli and Corynebacterium nephridii thioredoxins. Anti-(A. cylindrica thioredoxin) antiserum was used to quantify the thioredoxin which constituted about 0.22% of the soluble protein in cell-free extracts of N2-fixing, NO3- -grown or NH4+-grown A. cylindrica. Activation of fructose-1,6-bisphosphatase of A. cylindrica, activation of glutamine synthetase and NADP+-dependent malate dehydrogenase of the green alga Scenedesmus obliquus but not of A. cylindrica, and deactivation of glucose-6-P dehydrogenase of the cyanobacterium Anabaena variabilis were all achieved using the same thioredoxin species. No other thioredoxin species were detected in extracts of A. cylindrica when examined for the activation of these enzymes.     

Intracellular mechanisms in the activation of human platelets by low-density lipoproteins.

Low-density lipoproteins (LDL) have been shown to cause aggregation of human blood platelets at concentrations above 2 g of protein/l. The secretion of the contents of platelet dense granules was detected, but not that of the lysosomes. LDL gave rise to a mobilization of [3H]arachidonic acid from phospholipids and the appearance of products of the cyclo-oxygenase pathway after only 10 s. LDL-promoted aggregation was inhibited by both aspirin and indomethacin. There was an increase in 3H-labelled diacylglycerols and the phosphorylation of 47 kDa proteins. LDL therefore shares at least some of the mechanisms of stimulus/response coupling with those of other agonists.     

Application of Poisson distribution theory to the zona-free hamster oocyte penetration test to assess sperm function of men with asthenozoospermia

Assessments of the penetrating potential of human spermatozoa were carried out using the zona-free hamster oocyte penetration test on 4 groups of subjects exhibiting normal fertility, idiopathic asthenozoospermia(less than 40% motility), asthenozoospermia associated with varicocele and oligoaesthenozoospermia (less than 20 X 10(6) spermatozoa/ml and less than 40% motility). When the Poisson model was used to correct the results of the in-vitro penetration experiments for differences in motile sperm concentration, significant differences were apparent between the normal fertile controls and all 3 categories of asthenozoospermic patient. Furthermore, the penetrating ability of the motile spermatozoa from patients presenting with a varicocele or oligoaesthenozoospermia was significantly less than that for the group in which asthenozoospermia was the only detectable defect. These results emphasize the practical significance of the Poisson model in the analysis of male fertility and demonstrate that the asthenozoospermic condition is associated with a significant reduction in the fertilizing potential of the motile spermatozoa.