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The goal of this study was to identify the genes coding for β-N-acetylhexosaminidases in the Mediterranean fruit fly (medfly) Ceratitis capitata, one of the most destructive agricultural pests, belonging to the Tephritidae family, order Diptera. Two dimeric β-N-acetylhexosaminidases, HEXA and HEXB, have been recently identified on Drosophila sperm. These enzymes are involved in egg binding through interactions with complementary carbohydrates on the surface of the egg shell. Three genes, Hexosaminidase 1 (Hexo1), Hexosaminidase 2 (Hexo2) and fused lobes (fdl), encode for HEXA and HEXB subunits. The availability of C. capitata EST libraries derived from embryos and adult heads allowed us to identify three sequences homologous to the D. melanogaster Hexo1, Hexo2 and fdl genes. Here, we report the expression profile analysis of CcHexo1, CcHexo2 and Ccfdld in several tissues, organs and stages. Ccfdl expression was highest in heads of both sexes and in whole adult females. In the testis and ovary the three genes showed distinct spatial and temporal expression patterns. All the mRNAs were detectable in early stages of spermatogenesis; CcHexo2 and Ccfdl were also expressed in early elongating spermatid cysts. All three genes are expressed in the ovarian nurse cells. CcHexo1 and Ccfdl are stage specific, since they have been observed in stages 12 and 13 during oocyte growth, when programmed cell death occurs in nurse cells. The expression pattern of the three genes in medfly gonads suggests that, as their Drosophila counterparts, they may encode for proteins involved in gametogenesis and fertilization., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Fucosylated glycoconjugates play a role in a wide variety of biological processes, including immune responses, signal transduction, ontogenic events and pathogenesis of several human diseases. Alpha-L-fucosidases, which are responsible for their processing, have been demonstrated to be involved in lysosomal storage disease, inflammation, cystic fibrosis, cancer development and in the interactions between gametes in vertebrates as well as invertebrates. The sequence and comparative genomic analysis of these glycosyl hydrolases and the study of their evolutionary relationships appear therefore to be of considerable interest. In this work we carried out extensive similarity searches and comparative analyses to identify sequences encoding alpha-L-fucosidases. We have identified novel alpha-L-fucosidase coding sequences in worms, insects, sea urchin, ascidians, fish, chicken, amphibians, mammals and various bacteria resulting in a total of 39 alpha-L-fucosidase sequences. Two alpha-L-fucosidases that are present in all vertebrates likely reflect a distinct biological role for paralogous genes. Comparative sequence analysis of all metazoan alpha-L-fucosidases reveals a broad conservation of features, including the aspartate residue that constitutes the catalytic nucleophile. However, a cysteine which is thought to be part of the active site is also conserved in metazoa but not in arthropods, where it is replaced by an alanine. Phylogenetic analysis suggests a gene duplication event very early in metazoan evolution with the subsequent differential loss of isoforms in various metazoan lineages.

Sperm surface beta-N-acetylhexosaminidases are among the molecules mediating early gamete interactions in invertebrates and vertebrates, including man. The plasma membrane of Drosophila spermatozoa contains two beta-N-acetylhexosaminidases, DmHEXA and DmHEXB, which are required for egg fertilization. Here, we demonstrate that three putative Drosophila melanogaster genes predicted to code for beta-N-acetylhexosaminidases, Hexo1, Hexo2, and fdl, are all expressed in the male germ line. fdl codes for a homolog of the alpha-subunit of the mammalian lysosomal beta-N-acetylhexosaminidase Hex A. Hexo1 and Hexo2 encode two homologs of the beta-subunit of all known beta-N-acetylhexosaminidases, which we have named beta(1) and beta(2), respectively. Immunoblot analysis of sperm proteins indicated that the gene products associate in different heterodimeric combinations forming DmHEXA, with an alphabeta(2) structure, and DmHEXB, with a beta(1)beta(2) structure. Immunofluorescence demonstrated that all the gene products localized to the sperm plasma membrane. Although none of the genes was testis-specific, fdl was highly and preferentially expressed in the testis, whereas Hexo1 and Hexo2 showed broader tissue expression. Enzyme assays carried out on testis and on a variety of somatic tissues corroborated the results of gene expression analysis. These findings for the first time show the in vivo expression in insects of genes encoding beta-N-acetylhexosaminidases, the only molecules so far identified as involved in sperm/egg recognition in this class, whereas in mammals, the organisms where these enzymes have been best studied, only two types of polypeptide chains forming dimeric functional beta-N-acetylhexosaminidases are present in Drosophila three different gene products are available that might generate numerous dimeric isoforms.

Considerable evidence suggests that human immunodeficiency virus (HIV)-infected macrophages and/or lymphocytes may mediate sexual transmission of HIV. We and others have previously demonstrated that when vitally stained donor mouse lymphocytes or macrophages are placed in the vaginas of mice, some of the stained cells can later be found in the iliac lymph nodes. The aim of this study was to assess the extent of mononuclear cell trafficking from the vagina and to test the possibility that carrageenan formulation, a sulfated polysaccharide formulation containing 3% PDR98-15 carrageenan (PC-515; FMC Biopolymer, Rockland, ME), a vaginal microbicide, would prevent vaginal transmigration of macrophages. When supravitally stained mouse macrophages and T cells were inoculated into the vagina of recipient mice, discrete numbers of donor cells migrated to the recipient iliac and inguinal lymph nodes and spleen. When recipient mice were preinoculated with the carrageenan formulation, the number of macrophages in lymph nodes and spleen was reduced by >90%. In contrast, a methylcellulose formulation, which is believed to be inactive, did not significantly reduce migration to the lymphoid organs. Our findings suggest that the carrageenan formulation blocks cell trafficking of macrophages from vagina and that blocking does not result from cytotoxicity. Blocking cell trafficking may help to prevent sexual transmission of HIV.

The issue of how human immunodeficiency virus-1 (HIV-1) enters the body following sexual contact has been the subject of considerable controversy. Several possible routes for the initial infection have been suggested [1-6], including the possibility that the transmission is mediated by HIV-1-infected lymphocytes or macrophages in serum and female genital tract secretions, rather than by free virus. We recently reported that HIV-1-infected, activated primary monocytes can migrate between epithelial cells grown in confluent monolayer cultures in vitro [7]. We report here on experiments carried out in mice to test the hypothesis that mononuclear blood cells are capable of migrating through intact epithelia, and thus of carrying a virus into an animal. We placed double-stained, activated mononuclear blood cells into the vaginas of mice; four hours later, numerous double-stained cells were observed in the connective tissue beneath the vaginal epithelium and the iliac lymph nodes of the experimental mice. We speculate that such migration may be involved in the sexual transmission of HIV-1.

Time-lapse cinematography revealed that activated human immunodeficiency virus (HIV)-infected monocytes crawl along surfaces, putting forward a leading pseudopod. Scanning electron micrographs showed monocyte pseudopods associated with spherical structures the size of HIV virions, and transmission electron micrographs revealed HIV virions budding from pseudopods. Filamentous actin (F-actin) was localized by electron microscopy in the pseudopod by heavy meromyosin decoration. Colocalization of F-actin and p24 viral antigen by light microscopy immunofluorescence indicated that F-actin and virus were present on the same pseudopod. These observations indicate that monocytes produce virus from a leading pseudopod. We suggest that HIV secretion at the leading edges of donor monocytes/macrophages may be an efficient way for HIV to infect target cells.

After the addition of human T-cell lymphotropic virus type I (HTLV-I)-infected lymphocytes to enterocyte monolayers, the lymphocytes adhered via microvilli from both cell types and shed virus onto the enterocyte surface. Virus fused with the epithelial membrane and infected these cells as confirmed by electron microscopic immunocytochemistry, in situ hybridization, and amplification by polymerase chain reaction.

Cytochrome oxidase activity via cytochrome c, as demonstrated by the diaminobenzidine procedure, has been employed in this electron microscope cytochemical study to determine the respiratory potency, integrity and fate of the Arbacia sperm mitochondrion at fertilization and during early embryogenesis. The sperm mitochondrion remained intact and was intensely positive for cytochrome oxidase activity both during and after penetration into the egg. The mitochondrion remained highly reactive throughout zygote formation, up to the eight-cell stage. The sperm mitochondrion formed many projections and buds in the cytoplasm of immature oocytes, monospermic and polyspermic eggs, and in blastomeres. At all stages of early embryogenesis, close juxtaposition and structural contact were observed between the highly reactive sperm mitochondrion and the less reactive egg mitochondria. The results suggest that following fertilization the mitochondrion of the sea urchin spermatozoon retains some degree of metabolic autonomy within the ooplasm. The structural integrity of the paternal mitochondrion is maintained along with a functional respiratory enzyme system (cytochrome c-a3). The hypothesis that the fertilizing sperm mitochondrion may have some relevance to sea urchin development is discussed.

Events following the binding of Concanavalin A and cationized ferritin to the surface of normal human peripheral blood lymphocytes were followed using ultracytochemical methods. In this paper, we described a technique involving microdensitometry, which was employed in order to make a comparative cytochemical quantitation of the dense horseradish peroxidase-diaminobenzidine reaction products representing the states of Concanavalin A receptors under various conditions of distribution and redistribution. The technique presents a direct approach to the analysis of various probes that are currently being employed for studying the nature and distribution of various cell surface components. An attempt was also made to analyze the distribution of anionic sites, and their lateral redistribution is compared with the mobility of Concanavalin A receptors. The data presented suggests an independent mobility of receptors for Concanavalin A and cationized ferritin.

The rate constant (k) of the cytochrome oxidase reaction under optimal conditions for cytochemical staining (i.e., 15 min fixation, incubation for 180 min for heart, 120 min for pancreas) can be used as a measure of the enzyme concentration within mitochondria. The rate constant derived from microdensitometric measurements of the mass thickness of the 3,3'-diaminobenzidine (DAB) cytochrome oxidase reaction in cristae times correlated data derived from morphometry on the surface density of cristae (SVcristae/Vmit micron-1) and the volume density of mitochondria per cell (Vmit/Vcell) has been used to determine the respiratory index (RI) of these tissues according to the following equation: RI = k(SVcristae/Vcell). Using this formula, the RI of cardiac muscle tissue was computed to be 33 times the RI of pancreas under the conditions of our experiments. The greater cristae surface density and the large mitochondrial volume density in cardiac muscle and high k value accounted for the higher RI of cardiac muscle.

An infertile man presented a spermiogram in which 100% of the spermatozoa displayed separation of head from tail at the level of the proximal centriole. Most tails were normally structured and ended anteriorly with the proximal centriole covered by a continuous plasma membrane. In a small percentage of tails a rudimentary connecting piece was surrounded by a minute cytoplasmic mass and the middle piece was missing, whereas the chromatoid body and the spindle-shaped body were still present. Finally, a few tails had a large cytoplasmic mass surrounding either regular connecting and middle pieces or a rudimentary connecting piece continuous with the main piece. Tails of the first type had good forward motility, although the pattern of movement appeared altered. The other types were immotile or motile but without forward progression. In the loose heads the implantation fossa had failed to differentiate. The separation of heads from tails appeared to be the result of a specific morphogenetic defect and took place at different stages of spermatid differentiation, giving rise to the structurally different types of tails.