Contraceptive responses of mice immunized with purified recombinant mouse zona pellucida subunit 3 (mZP3) proteins.

Mouse zona pellucida subunit 3 (mZP3) was tested for efficacy as an immunocontraceptive antigen by comparing the fertility of mice immunized with recombinant mZP3 proteins. Recombinant protein was expressed using either the vaccinia virus T7 mammalian (vmZP3 protein) or baculovirus insect cell (bmZP3 protein)-expression systems. Female BALB/c or wild mice were immunized by i.p. injection using Freund's complete adjuvant and boosted three times with affinity purified recombinant proteins in Freund's incomplete adjuvant. Most mice developed antibodies that crossreacted to the respective mZP3 antigens by ELISA or western blot. In BALB/c mice immunized with vmZP3, fertility and mean litter size were reduced transiently to 25% and 10%, respectively, of those of control mice. However, immunization with bmZP3 did not affect either the fertility or mean litter sizes in BALB/c or wild mice immunized with bmZP3. The results demonstrate that reduction in fertility can be achieved in female BALB/c mice immunized using Freund's adjuvants and recombinant mZP3 protein produced in a mammalian, but not an insect, cell-expression system. Arguments are presented for the likely role of glycosylation of the mZP3 antigen in inducing contraceptive immune responses.

Assessment of the immunocontraceptive effect of a zona pellucida 3 peptide antigen in wild mice.

Immunizing laboratory mice against a short peptide to mouse zona pellucida protein 3 (mZP3; amino acids 328-342) reduces fertility in some strains. This antigen was therefore tested to see if it is suitable for use in an immunocontraceptive vaccine to control wild mice. Mouse zona pellucida protein 3 peptide conjugated to a carrier protein (keyhole limpet hemocyanin) was considerably more immunogenic and effective in reducing fertility in wild mice when compared with inbred BALB/c mice. Fertility of the immunized wild mice was reduced by over 50% compared with controls, whereas BALB/c mice showed no reduction. Variation in the responses between individual animals to mZP3 peptide was observed and infertility correlated to the presence of cross-reacting antibodies to native zona pellucida in wild, but not BALB/c, mice.

Mouse-specific immunocontraceptive polyepitope vaccines.

Two mouse-specific polyepitope protein antigens comprising different combinations of sequences chosen from the mouse fertility antigens zona pellucida proteins 1 and 3 (ZP1 and ZP3), prolactin, proliferin, granulocyte-macrophage colony-stimulating factor (GM-CSF), sperm protein SP56 and T-helper cell-stimulating epitopes were produced in bacterial protein expression systems. The recombinant proteins were fused to maltose binding protein (MBP) and used to immunize female mice; their effects on fertility were assessed. Controls were immunized with either MBP only or phosphate buffered saline (PBS). One antigen construct (MBP-polyepitope B), containing mouse-specific epitopes for ZP1, ZP3, SP56 and proliferin, significantly reduced the fertility of female BALB/c mice. Fertility in this group was decreased by > 40% compared with the MBP control and the number of viable embryos was decreased by > 60%. This construct will now be used to produce the antigen in a recombinant murine cytomegalovirus for assessment as a potential mouse-specific anti-fertility vaccine for use in the control of mice in the field.

Cloning and characterization of the cDNA encoding the PH20 protein in the European red fox Vulpes vulpes.

The PH20 protein is thought to play a crucial role in mammalian fertilization. The fox PH20 homologue has been cloned from a testis cDNA library and the deduced protein sequence shows high levels of homology to PH20 proteins isolated from other species. Unlike other PH20 proteins the fox protein does not appear to be membrane associated through a GPI-linkage nor does it show the presence of a transmembrane domain at the C-terminus of the protein. It is in this region that the proteins appear to be least conserved. Immunolocalization studies on fox sperm show that the PH20 protein is located on the inner acrosomal membrane. Transcription of PH20 in the fox is seasonally regulated, with the mRNA expressed during those months when spermatogenesis is at its peak. The PH20 sequence described in this paper has been submitted to the Genbank database and has the accession number U41412.

Molecular characterization of spontaneous mutations at the scarlet locus of Drosophila melanogaster.

Six spontaneous mutations of the scarlet (st) locus of Drosophila melanogaster have been studied at the molecular level. Two of the mutants (st1 and stsp) arose in laboratory populations, while the other four (stcob, stct89, stdct and stdv) were isolated from natural populations. In five of these there is a DNA insertion within the st region and in four cases the insertion has been identified as being a transposable element; these include the retrotransposons 412 and B104/roo, and also jockey a member of the LINE family. In the other case (stdct), the insertion appears to consist of partially duplicated st sequences. In two of the mutants (st1 and stdv) the same transposable element (412) has inserted in the same orientation at exactly the same site within the st gene. The transposable element insertions are found in intron and exon regions of the st gene and also in the putative upstream regulatory region; insertions located in introns or exons result in the production of truncated st transcripts. The results show that the same types of transposable elements that cause spontaneous mutation in laboratory stocks of D. melanogaster also cause mutation in the wild.

The human and mammalian N-acetylmuramyl-L-alanine amidase: distribution, action on different bacterial peptidoglycans, and comparison with the human lysozyme activities.

N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28) specifically hydrolyzes the bacterial cell wall peptidoglycans (or mureins) and the muropeptides. The enzyme splits these molecules into two parts: the peptide subunits and the glycan strands or moieties. The bacterial peptidoglycans and their derived muropeptides display a number of biological properties. Removal of the glycosidic part of these molecules abolishes their beneficial as well as their detrimental properties. We report the high level of enzymatic activity found in all mammalian (including human) sera tested. The enzyme also occurred in human saliva, milk, cerebrospinal fluid, and synovial liquid. Mucosal tissue from different parts of the mammalian digestive tract exhibited enzymatic activity, but the enzyme was not detectable in the lumen content. The range of substrate specificity of the human enzyme was evaluated by measuring its action on the peptidoglycans extracted from several bacterial strains and representing different chemotypes and structures. Time course of the muramylalanine amidase and of the lysozyme (both of human origin) activities on some of these peptidoglycans are also reported, with the enzymes acting separately or together. From these data, we would speculate that a probable physiological role of the muramylalanine amidase is the maintenance of adequate ratios between the biologically active muropeptides and their inactive derivatives in the organism, the amidase activity antagonizing the production of biologically active molecules by lysozyme.

Cloning and partial characterization of the cDNA encoding the fox sperm protein FSA-Acr.1 with similarities to the SP-10 antigen.

We have isolated and characterized a cDNA, cFSA-Acr.1, encoding a testis-specific fox sperm antigen. The antigen is located on the inner acrosomal compartment, and is expressed during spermatogenesis on the developing acrosome of round and elongating spermatids. Database searches with the deduced amino acid sequence of cFSA-Acr.1 revealed that the clone has high homology to both human and baboon sperm protein SP-10, and the mouse sperm protein, MSA-63. The region of highest homology is within the carboxyl terminus. In the middle of the open reading frame, the fox sequence shows unique sequences absent from both the human, baboon SP-10, and mouse MSA-63 sequences. In addition to cFSA-Acr.1, two other clones were also isolated from the same fox testis cDNA library, and sequence analysis shows that they may represent alternatively spliced mRNAs coding for other FSA-Acr proteins.

Cloning and characterization of a fox sperm protein FSA-1.

A monoclonal antibody was raised to a fox sperm protein (FSA-1) which was found to be localized to the inner acrosomal compartment of sperm fixed in methanol. Western blots of testicular germ cell membrane extracts probed with this antibody identified a major protein band with a molecular weight of 36,000. Immunofluorescent studies on fox testis sections showed that the antigen is expressed on round and elongating spermatids on a crescent-shaped structure, which probably represents the developing acrosome. An antibody specific for FSA-1 was used to screen a fox testis cDNA library for its cognate gene. An 875-bp cDNA clone was isolated and sequenced revealing an open reading frame. Searches of the GenBank and EMBL databases with the nucleic acid sequence revealed significant homology (86%) of FSA-1 with 406 bases of an unidentified RNA transcript from human fetal brain (EST02625). Northern blot analysis of fox testis RNA samples identified an RNA transcript of approximately 0.9 kb during the months when spermatogenesis is active. Zoo Northern blots (at high stringency) reveal an RNA transcript of a similar size present in testis RNA from dogs and mice. Zoo Southern analysis (high stringency) reveal genomic sequences present in dogs, mice, cattle and sheep. At present, the function of the FSA-1 gene product remains unknown, but it may play a role as a structural protein component of the acrosome.

Purification and characterization of N-acetylmuramoyl-L-alanine amidase from human serum.

Purification to homogeneity of the N-acetylmuramoyl-L-alanine amidase (mucopeptide amidohydrolase, EC 3.5.1.28) from human serum has been achieved with a high yield. By molecular sieving chromatography, a molecular weight of 120,000-130,000 has been found for the native enzyme. Polyacrylamide gel electrophoresis under native conditions gave a unique band of Mr = 125,000. The same technique performed under denaturing conditions revealed that the protein is a dimer composed of one subunit of Mr = 57,000 and another of Mr = 70,000. In isoelectrofocalization assays, the amidase behaved as an acidic protein. Ethylenediaminetetraacetate inhibited the enzyme activity; the Mg2+ requirement was confirmed. The simultaneous presence of sulfhydryl groups and disulfide bonds in the protein was evidenced by the inhibitions produced by different thiol-blocking reagents and by several thiol-bearing substances. Direct measurements established the presence of two accessible thiol groups and the occurrence of nine disulfide bonds per protein molecule. Studies of substrate hydrolyzing capacities showed a marked preference for the muramoyl tripeptide derived from the Escherichia coli or Bacillus cereus mureins, the disaccharide tetrapeptide and the bis disaccharide tetra-tetrapeptide from E. coli were also good substrates. Activities on small muropeptides of other composition are also reported. Whole (insoluble) peptidoglycans representing the main bacterial chemotypes were submitted to the enzyme action; although with weak specific activities, the human amidase was nevertheless able to release soluble peptides from some of them. A bacteriolytic capacity on some microorganisms cannot be excluded. Results are discussed and the human enzyme is compared to presently known microbial muramoyl amidases.

Regulation of compartmentation of amino acid pools in Saccharomyces cerevisiae and its effects on metabolic control.

Compartmentation of intracellular amino acid pools has been studied under various growth conditions in wild-type strains as well as in mutants. Aspartate, glutamate, leucine and isoleucine pools are present in high concentrations in the cytoplasm, while all the other amino acids are more vacuolar. The nature of the nitrogen source for growth, the effectiveness of nitrogen assimilation, the rate of protein synthesis and the presence of high internal basic amino acid pools are important factors in the repartition of amino acid pools between the cytoplasm and the vacuole.