A Role for the Disintegrin Domain of Cyritestin, a Sperm Surface Protein Belonging to the ADAM Family, in Mouse Sperm–Egg Plasma Membrane Adhesion and Fusion

Sperm–egg plasma membrane fusion is preceded by sperm adhesion to the egg plasma membrane. Cell–cell adhesion frequently involves multiple adhesion molecules on the adhering cells. One sperm surface protein with a role in sperm–egg plasma membrane adhesion is fertilin, a transmembrane heterodimer (α and β subunits). Fertilin α and β are the first identified members of a new family of membrane proteins that each has the following domains: pro-, metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane, and cytoplasmic domain. This protein family has been named ADAM because all members contain a disintegrin and metalloprotease domain. Previous studies indicate that the disintegrin domain of fertilin β functions in sperm–egg adhesion leading to fusion. Full length cDNA clones have been isolated for five ADAMs expressed in mouse testis: fertilin α, fertilin β, cyritestin, ADAM 4, and ADAM 5. The presence of the disintegrin domain, a known integrin ligand, suggests that like fertilin β, other testis ADAMs could be involved in sperm adhesion to the egg membrane. We tested peptide mimetics from the predicted binding sites in the disintegrin domains of the five testis-expressed ADAMs in a sperm–egg plasma membrane adhesion and fusion assay. The active site peptide from cyritestin strongly inhibited (80–90%) sperm adhesion and fusion and was a more potent inhibitor than the fertilin β active site peptide. Antibodies generated against the active site region of either cyritestin or fertilin β also strongly inhibited (80–90%) both sperm–egg adhesion and fusion. Characterization of these two ADAM family members showed that they are both processed during sperm maturation and present on mature sperm. Indirect immunofluorescence on live, acrosome-reacted sperm using antibodies against either cyritestin or fertilin β showed staining of the equatorial region, a region of the sperm membrane that participates in the early steps of membrane fusion. Collectively, these data indicate that a second ADAM family member, cyritestin, functions with fertilin β in sperm–egg plasma membrane adhesion leading to fusion.     

Interaction of Co,Mn, Mg and Al with d(GCGTACGC): a spectroscopic study

Spectroscopic study on the interactions of trace elements Co, Mn, Mg and Al with d(GCGTACGC) indicated the following: Al and Mg did not alter T_m values. Mn enhanced T_m at lower concentration and decreased it at higher concentrations. Interestingly Co at higher concentration elevated the T_m. These studies also showed lower concentrations of Mn displaced EtBr, whereas Al could displace it at higher ionic strength. Mg and Co displaced EtBr fluorescence at moderate concentrations. The binding constant values and CD spectra clearly indicated strong binding of these elements to DNA.     

Synthesis and evaluation of azaproline peptides as potential inhibitors of dipeptidyl peptidase IV and prolyl oligopeptidase

Summary A series of azaproline dipeptides with various N-substituents were synthesized as possible active-site-directed inhibitors of two proline-specific serine proteases, dipeptidyl peptidase IV and prolyl oligopeptidase. Compounds with semicarbazide, carbazate, acylhydrazine and sulphonylhydrazine structures were tested. Some compounds show moderate activity, i.e., in the millimolar range.     

Plasmodium falciparum protein associated with the invasion junction contains a conserved oxidoreductase domain

The merozoite cap protein-1 (MCP-1) of Plasmodium falciparum follows the distribution of the moving Junction during invasion of erythrocytes. We have cloned the gene encoding this protein from a cDNA library using a monoclonal antibody. The protein lacks a signal sequence and has no predicted trans-membrane domains; none of the antisera reacts with the surfaces of intact merozoites, indicating that the cap distribution is submembranous. MCP-1 is divided into three domains. The N-terminal domain includes a 52-amino-acid region that is highly conserved in a large family of bacterial and eukaryotic proteins. Based on the known functions of two proteins of this family and the pattern of amino acid conservation, it is predicted that this domain may possess oxido-reductase activity, since the active cysteine residue of this domain is invariant in all proteins of the family. The other two domains of MCP-1 are not found in any other members of this protein family and may reflect the specific function of MCP-1 in invasion. The middle domain is negatively charged and enriched in glutamate; the C-terminal domain is positively charged and enriched in lysine. By virtue of its positive charge, the C-terminal domain resembles domains in some cytoskeleton-associated proteins and may mediate the interaction of MCP-1 with cytoskeleton in Plasmodium.     

Chemical enthalpy-entropy compensation effect in the hydrolysis of p-nitrophenyl carboxylates catalyzed by alkaline mesentericopeptidase

The temperature dependence of the hydrolysis of p-nitrophenyl carboxylates with general formula H(CH₂)_nCOOC₆H₄NO₂ catalyzed by alkaline mesentericopeptidase has been studied (n varying from 1 to 7, temperature range 2–30°C, pH 8.80, 5 vol% dimethylsulfoxide). The activation parameters of the deacylation step depend on the length of the hydrophobic side chain of the substrate molecule ( , , and decrease by 2.0 kcal/mol, 4.9 kcal/mol, and 10 eu, respectively, as the length of the acyl carbon chain increases from n = 1 to n = 4). The following criteria were applied to establish a chemical enthalpy-entropy compensation effect: (a) Exner's plot of log vs : (b) Petersen's plot of log, k/T vs 1/T; (c) Exner's statistical treatment in coordinates log k vs 1/T; (d) according to Krug et al. (ΔH^≠ vs ΔG_Thm^≠). By use of all the above-mentioned criteria the existence of a chemical enthalpy-entropy compensation effect was proved with an isokinetic temperature β of about 470°K, which is significantly higher than the average experimental temperature.     

A Thick-Walled Organism Isolated from the Cockroach Gut by Using a Spent Medium Technique

By using a conditioning technique whereby complex media are inoculated several times with bacteria from the hindgut of the cockroach Eublaberus posticus, a succession of bacterial types occurred. An obligately anaerobic, pleomorphic, thick-walled, gram-positive organism is described which was isolated by this culturing technique.     

DNA and RNA from Uninfected Vertebrate Cells Contain Nucleotide Sequences Related to the Putative Transforming Gene of Avian Myelocytomatosis Virus

The avian carcinoma virus MC29 (MC29V) contains a sequence of approximately 1,500 nucleotides which may represent a gene responsible for tumorigenesis by MC29V. We present evidence that MC29V has acquired this nucleotide sequence from the DNA of its host. The host sequence which has been incorporated by MC29V is transcribed into RNA in uninfected chicken cells and thus probably encodes a cellular gene. We have prepared radioactive DNA complementary to the putative MC29V transforming gene (cDNA(mc) (29)) and have found that sequences homologous to cDNA(mc) (29) are present in the genomes of several uninfected vertebrate species. The DNA of chicken, the natural host for MC29V, contains at least 90% of the sequences represented by cDNA(mc) (29). DNAs from other animals show significant but decreasing amounts of complementarity to cDNA(mc) (29) in accordance with their evolutionary divergence from chickens; the thermal stabilities of duplexes formed between cDNA(mc) (29) and avian DNAs also reflect phylogenetic divergence. Sequences complementary to cDNA(mc) (29) are transcribed into approximately 10 copies per cell of polyadenylated RNA in uninfected chicken fibroblasts. Thus, the vertebrate homolog of cDNA(mc) (29) may be a gene which has been conserved throughout vertebrate evolution and which served as a progenitor for the putative transforming gene of MC29V. Recent experiments suggest that the putative transforming gene of avian erythroblastosis virus, like that of MC29V, may have arisen by incorporation of a host gene (Stehelin et al., personal communication). These findings for avian erythroblastosis virus and MC29V closely parallel previous results, suggesting a host origin for src (D. H. Spector, B. Baker, H. E. Varmus, and J. M. Bishop, Cell 13:381-386, 1978; D. H. Spector, K. Smith, T. Padgett, P. McCombe, D. Roulland-Dussoix, C. Moscovici, H. E. Varmus, and J. M. Bishop, Cell 13:371-379, 1978; D. H. Spector, H. E. Varmus, and J. M. Bishop, Proc. Natl. Acad. Sci. U.S.A. 75:4102-4106, 1978; D. Stehelin, H. E. Varmus, J. M. Bishop, and P. K. Vogt, Nature [London] 260:170-173, 1976), the gene responsible for tumorigenesis by avian sarcoma virus. Avian sarcoma virus, avian erythroblastosis virus, and MC29V, however, induce distinctly different spectra of tumors within their host. The putative transforming genes of these viruses share no detectable homology, although sequences homologous to all three types of putative transforming genes occur and are highly conserved in the genomes of several vertebrate species. These data suggest that evolution of oncogenic retroviruses has frequently involved a mechanism whereby incorporation and perhaps modification of different host genes provides each virus with the ability to induce its characteristic tumors.     

Anti-H-2 hybridoma antibodies as immunoselective agents

Using immunoselection with an H-2K^k-specific monoclonal antibody following mutagenesis on an (H-2 ^k/H-2^d) F₁ cell line we have obtained variants that do not react with the selecting monoclonal antibody but continue to react with other monoclonal antibodies directed against the same gene product. The mutants fall into two classes based on their serological profile. This phenotype is suggestive of a structural mutation in the selected gene. If the genetic change involved is a point mutation (as opposed to a deletion), one should be able to obtain revertants. Using the fluorescence-activated cell sorter, we have been able to obtain from one of the monoclonal-antibody-nonseactive mutants cells that do bind the selecting antibody. In order to prove that the presumptive revertant is not a contaminant wild-type cell that inadvertantly got mixed into the resistant mutant, we first introduced an outside marker, resistance to the purine analogue 2-amino-6-mercaptopurine (6-thioguanine), into the monoclonal-antibody-resistant mutant. The revertants obtained using the cell sorter continue to express the nonselective phenotype of resistance to 6-thioguanine, showing that they are not wild-type cells. In addition, their serological characteristics are different from those of either the wild-type cells or the hybrid oma-resistant mutants from which they were derived. Based on the serological analyses, it would seem that we have isolated at least three variant forms of the H-2K^k-gene product.     

Identifying sites of simultaneous DNA replication in eukaryotes by N-methyl-N''-nitro-N-nitrosoguanidine multiple mutagenesis.

Summary N-methyl-N-nitro-N-nitrosoguanidine (NG) induces certain classes of multiple mutations in yeast at high frequency. By selecting for mutation at one locus (his4 or leu1) one frequently obtains double mutants where another mutation to temperature sensitivity has also been induced. This multiple mutagenesis exhibits a considerable specificity: for mutation at one particular locus there is a high chance that another mutation will be found in the same cell at one of a restricted number of other loci. For any given locus (e.g. his4) there is a spectrum of sites at which temperature-sensitivity mutations are coinduced. This spectrum differs for different loci, such that the spectrum of sites co-mutating with leul differs completely from that for sites co-mutating with his4. This NG-induced co-mutation is interpreted in terms of NG acting to enhance mutagenesis at sites of simultaneous DNA replication within the cell. The results so obtained indicate a very strict control over the order and timing of gene replication in Saccharomyces cerevisiae, and it is suggested that it is now possible to use NG double mutagenesis to try and locate origins of replication in yeast.