Contraceptive potential of antibodies to the zona pellucida

The notion of a contraceptive vaccine based on gamete-specific surface antigens was first proposed over a decade ago, as the result of in-vitro and in-vivo studies, and in recent years has been the subject of intensive research. In particular, the zona pellucida has attracted much attention as a potential target for immunological intervention in the fertilization process. Such is the rapidly-expanding nature of research into the biochemical and biological characterization of this structure, that a review of the implications for the development of a contraceptive vaccine seems timely.     

The protein phosphatases involved in cellular regulation. Primary structure of inhibitor-2 from rabbit skeletal muscle

The complete primary structure of inhibitor-2, a specific inhibitor of protein phosphatase-1, has been determined. The protein consists of a single polypeptide chain of 203 residues, and has a relative molecular mass of 22835 Da. This molecular mass is significantly lower than earlier estimates based on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The threonyl residue phosphorylated by glycogen synthase kinase-3 is located at position 72. The molecule is very hydrophilic, lacks cysteine residues and the single tryptophanyl and phenylalanyl residues are at positions 46 and 139, respectively. The N-terminal alanyl residue is N-acetylated. Digestion with Staphylococcus aureus V8 proteinase, trypsin, or cleavage with cyanogen bromide, destroyed the biological activity of inhibitor-2, demonstrating that many large fragments (e.g. 1-49, 49-92, 67-101, 108-134, 142-182 and 163-197) are inactive. Digestion with clostripain generated a peptide comprising residues 25-114 which retained 2% of the inhibitory potency of the parent molecule. There is no sequence homology between inhibitor-2 and inhibitor-1.     

A simple and rapid method of quantitative analysis of phosphoamino acids by high-performance liquid chromatography

A high-performance liquid chromatographic system for the separation of nonradiolabeled phosphoamino acids and orthophosphate by ion-pair reverse-phase chromatography has been developed. By the use of low-ionic-strength phthalate buffers at pH 6.3, the phosphoamino acids can be visualized by virtue of this uv-active eluant. The technique is sensitive to 200 pmol of phosphoamino acid and has been shown to be directly applicable to the analysis of isolated phosphoproteins.     

Analysis of the direct effects of prostaglandins on human sperm function

Time-exposure photomicrography and interspecies in-vitro fertilization procedures have been used to examine the influence of prostaglandins on human sperm function. An analysis of variance indicated that the presence of PGs in the incubation media was associated with a significant increase in sperm velocity and the frequency of sperm head rotation, although there were no differences between individual PGs in the degree of stimulation observed. Changes in the penetrating ability of human spermatozoa were detected after exposure to PGs, particularly PGE-1 and PGE-2. PGE-2 induced a sustained increase in penetration rates at all doses of greater than 8.4 micrograms/ml, while exposure to PGE-1 gave a bell-shaped dose-response curve which exhibited a peak between 8.4 and 33.3 micrograms/ml and progressively fell to reach control levels at the maximum concentration tested of 270 micrograms/ml. A combination of PGE-2 and PGE-1 produced a dose-response curve similar to that for PGE-1 alone, while exposure to PGF-2 alpha was without effect. Seminal extracts containing predominantly 19-hydroxy PGE-1, or equal amounts of 19-hydroxy PGE-1 + 2 induced a slight, but significant, rise in penetration rates while a combination of PGs representing the major components of human seminal plasma was without significant effect. We conclude that certain prostaglandins may have a direct action on the functional competence of human spermatozoa.     

Analysis of the biological properties of antibodies raised against intact and deglycosylated porcine zonae pellucidae

Zonae pellucidae (ZP) were isolated from 1,500 porcine ovaries and heat solubilized to generate approximately 15 mg ZP glycoprotein. Analysis of this material by isoelectric focusing, one-dimensional electrophoresis, and gas chromatography indicated the presence of a major glycoprotein species that exhibited considerable microheterogeneity with respect to its charge (pI 7.5–3.5) and molecular mass (45–85 kDa) and that contained 39.6% carbohydrate, predominantly N-acetylglucosamine. Chemical deglycosylation of porcine ZP using trifluoromethanesulphonic acid (TFMS) resulted in the production of five discrete protein bands on one-dimensional sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) with molecular masses of 66, 52, 36, 32, and 16 kDa. Antisera raised in rabbits and marmosets to ZP and/or deglycosylated ZP (DGZP) were used in immunoblotting experiments to demonstrate the retention of immunogenicity by DGZP and the cross-reactivity of the antisera with their heterologous antigen. These studies indicated that antisera that were capable of inhibiting the fertility of primates in vivo and the penetration of the human ZP in vitro reacted preferentially with 3 of the 5 products of deglycosylation, with molecular masses of 66, 52, and 36 kDa. Anti-DGZP antibodies were also shown to interact with intact porcine and human ZP and, with the latter, to block the ability of human spermatozoa to both bind to and penetrate this structure.     

Experimental toxocariasis and hyperactivity in mice

An observational study using videorecordings and computer-assisted data analysis was undertaken in order to investigate the behaviour of mice infected with larvae of Toxocara canis. The findings indicated that the infection had a marked effect on five readily and reliably differentiable categories of murine behaviour. A marked increase in the number of shorter bouts of each of the five behaviours was also associated with the infection. These results support previous findings and further suggest that T. canis infection affects the way in which mice respond to their environment. In particular the infection appears to be associated with hyperactivity in mice. Possible causes of such behavioural abnormalities as well as implications of these findings for clinical studies concerned with relationships between T. canis infection and hyperactivity in children are discussed.     

The effect of soil water status on critical phosphorus concentrations inStylosanthes hamata cv. Verano

Summary The effect of soil water status on the critical phosphorus concentration (CPC) determined in apices and whole tops ofStylosanthes hamata cv. Verano was investigated in a glasshouse trial. The species was grown with six rates of P and three ranges of soil water potential and was harvested at 10 and 14 weeks after germination. The CPC of both whole tops and apices decliced between the two harvests. At the first harvest the CPC of both whole tops and apices increased as the soil water potential decreased but at the second harvest there was no effect of soil water potential on CPC. It is suggested that at the earlier harvest water stress was delaying physiological development, resulting in a CPC characteristic of chronologically younger tissue, but that by the second harvest the decline in CPC with age had ceased for all water treatments.     

A Protein Kinase-Dependent Block to Reinitiation of DNA Replication in G2 Phase in Mammalian Cells

Eukaryotic cells normally replicate their DNA only once between mitoses. Unlike G1 nuclei, intact G2 nuclei do not replicate during incubation inXenopusegg extract. However, artificial permeabilization of the nuclear membrane of G2 nuclei allows induction of new initiations byXenopusegg extract. This is consistent with the action of a replication licensing factor which is believed to enter the nucleus when the nuclear membrane breaks down at mitosis. Here, we show that G2 nuclei will initiate a new round of replication in the absence of nuclear membrane permeabilization, if they are preexposed to protein kinase inhibitorsin vivo.Competence to rereplicate is generated within 30 min of drug treatment, well before the scheduled onset of mitosis. This demonstrates that a protein kinase-dependent mechanism is continually active in G2 phase to actively prevent regeneration of replication capacity in mammalian cells. Kinase inhibition in G2 cells causes nuclear accumulation of replication protein A. Rereplication of kinase-inhibited G2 nuclei also depends on factors supplied byXenopusegg extract, which are distinct from those required for replication licensing.     

Cold requirement for maximal activity of the bacterial ice nucleation protein INAZ in transgenic plants

The bacterial ice nucleation gene inaZ confers production of ice nuclei when transferred into transgenic plants. Conditioning of the transformed plant tissue at temperatures near 0°C greatly increased the ice nucleation activity in plants, and maximum ice nucleation activity was achieved only after low-temperature conditioning for about 48 h. Although the transgenic plants contain similar amounts of inaZ mRNA at both normal and low temperatures, low temperatures are required for accumulation of INAZ protein. We propose that the stability of the INAZ protein and thus ice nucleation activity in the transgenic plants is enhanced by low-temperature conditioning.