Inhibition of human seminal fluid DNA polymerase by an IgG fraction of seminal plasma from vasectomized men.
(9/2200)
Immunoglobulin G (IgG) was isolated from ejaculates of intact and vasectomized men by precipitation with ammonium sulphate and DEAE-cellulose ionexchange chromatography. Velocity centrifugation revealed that all of the IgG from intact males was 7S protein while less than 40% of the seminal IgG of vasectomized men cosedimented with the 7S marker; the remaining, immunologically unidentifiable, protein was considerably smaller and heterogeneous in size. Only the 7S IgG from the post-vasectomy ejaculates inhibited the activity of a DNA polymerase from the seminal fluid of an intact male. These results suggest that formation of antibody reactive with the seminal fluid DNA polymerase is one manifestation of a vasectomy-associated autoimmune response in man. (+info)
Genetic divergence with emergence of novel phenotypic variants of equine arteritis virus during persistent infection of stallions.
(10/2200)
The persistently infected carrier stallion is the critical natural reservoir of equine arteritis virus (EAV), as venereal infection of mares frequently occurs after breeding to such stallions. Two Thoroughbred stallions that were infected during the 1984 outbreak of equine viral arteritis in central Kentucky subsequently became long-term EAV carriers. EAV genomes amplified from the semen of these two stallions were compared by sequence analysis of the six 3' open reading frames (ORFs 2 through 7), which encode the four known structural proteins and two uncharacterized glycoproteins. The major variants of the EAV population that sequentially arose within the reproductive tract of each carrier stallion varied by approximately 1% per year, and the heterogeneity of the viral quasispecies increased during the course of long-term persistent infection. The various ORFs of the dominant EAV variants evolved independently, and there was apparently strong selective pressure on the uncharacterized GP3 protein during persistent infection. Amino acid changes also occurred in the V1 variable region of the GL protein. This region has been previously identified as a crucial neutralization domain, and selective pressures exerted on the V1 region during persistent EAV infection led to the emergence of virus variants with distinct neutralization properties. Thus, evolution of the EAV quasispecies that occurs during persistent infection of the stallion clearly can influence viral phenotypic properties such as neutralization and perhaps virulence. (+info)
Detection of simian immunodeficiency virus Gag-specific CD8(+) T lymphocytes in semen of chronically infected rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I-peptide complex.
(11/2200)
Evaluation of human immunodeficiency virus type 1-specific mucosal cytotoxic T lymphocytes can be hampered by limited cell yields from mucosal sites. We sought to characterize virus-specific CD8(+) T lymphocytes with cytotoxic activity in the male genital tracts of SIVmac-infected rhesus monkeys by using a peptide epitope-specific functional T-cell assay and a tetrameric major histocompatibility complex class I-peptide complex. This tetrameric complex was constructed with the rhesus monkey HLA-A homolog molecule Mamu-A*01 and a dominant-epitope 9-amino-acid fragment of SIVmac Gag (p11C, C-M). The proportion of tetramer-positive CD8(+) T cells in semen of SIVmac-infected monkeys ranged from 5.9 to 22.0%. By the use of a standard 51Cr release assay, these cells were found to have peptide epitope-specific cytolytic activity after in vitro expansion. Four-color flow-cytometric analysis of these seminal tetramer-positive CD8(+) T cells demonstrated that they express memory-associated (CD62L- CD45RA-) and activation-associated (CD11a+ Fas+ HLA-DR+) molecules. The present experiments illustrate the power of tetramer technology for evaluating antigen-specific CD8(+) T lymphocytes in a mucosal tissue compartment. (+info)
Studies of genetic relationships between bovine, caprine, cervine, and rangiferine alphaherpesviruses and improved molecular methods for virus detection and identification.
(12/2200)
The glycoprotein B (gB) and D (gD) genes from five ruminant alphaherpesviruses, bovine herpesvirus 1 (BHV-1), bovine herpesvirus 5 (BHV-5), caprine herpesvirus 1 (CapHV-1), cervine herpesvirus 1, and rangiferine herpesvirus 1, were partially sequenced. The nucleotide sequence alignments revealed a highly conserved gB gene, with homologies ranging between 87.2 and 99.6%, and a more variable gD gene, with homologies ranging between 71.3 and 98.9%. The phylogenetic analysis of the gB and gD nucleotide and deduced amino acid sequences revealed that BHV-5 is the most closely related virus to the BHV-1 subtype 1 and BHV-1 subtype 2 cluster and that CapHV-1 is the most distantly related virus. The phylogenetic data showed a close relationship of all the studied viruses with suid herpesvirus 1. On the basis of sequence data for the gB gene, a nested PCR combined with restriction enzyme analysis (REA) of the PCR products was developed for the simultaneous detection and identification of the viruses that were studied. Nested primers from highly conserved sequence stretches were selected in order to amplify a region of 294 bp in all five viruses, and a subsequent REA of the PCR products allowed specific identification. A mimic molecule that served as an internal standard of the amplification efficiency was constructed. The practical diagnostic applicability of the assay was evaluated with clinical samples consisting of semen and organ specimens from experimentally infected animals. (+info)
Multicenter comparison of PCR assays for detection of human herpesvirus 8 DNA in semen.
(13/2200)
Reported prevalences of human herpesvirus 8 (HHV-8) (Kaposi's sarcoma-associated herpesvirus) in semen have ranged widely. This is possibly due to differences in assay sensitivity, geographic or population-based differences in the true presence of the virus in semen, and PCR contamination. This study assessed interlaboratory sensitivity and reproducibility in the analysis of blinded experimental panels, each consisting of 48 specimens and being composed of semen specimens from different healthy artificial-insemination donors (n = 30) and human immunodeficiency virus (HIV)-infected patients (n = 7) plus positive (n = 4) and negative (n = 7) controls. The experimental panels analyzed in each laboratory were identical except for being independently coded. Of 10 experiments done in five laboratories, 5 experiments from three laboratories had evidence of PCR contamination; all instances of contamination were in the context of nested PCR procedures. In the experiments with no false-positive results, HHV-8 DNA was detected in three (8%) of the 37 semen specimens (two from artificial-insemination donors and one from an HIV-positive patient) but in only 3 (1.6%) of the 184 PCRs in which these specimens were analyzed. This suggests that HHV-8 DNA is present in semen at concentrations that can be too low to allow its consistent detection. This study emphasizes the importance of performing blinded, multi-institution experiments to provide a coherent basis for comparing results and to motivate standardization of methods. (+info)
Bovine seminal plasma (BSP) contains a family of phospholipid-binding proteins (BSP-A1/-A2, BSP-A3 and BSP-30-kDa, collectively called BSP proteins) that potentiate sperm capacitation induced by high-density lipoproteins. We showed recently that BSP proteins stimulate cholesterol efflux from epididymal spermatozoa and play a role in capacitation. Here, we investigated whether or not BSP proteins could stimulate cholesterol and phospholipid efflux from fibroblasts. Cells were radiolabeled ([3H]cholesterol or [3H]choline) and the appearance of radioactivity in the medium was determined in the presence of BSP proteins. Alcohol precipitates of bovine seminal plasma (designated crude BSP, cBSP), purified BSP-A1/-A2, BSP-A3 and BSP-30-kDa proteins stimulated cellular cholesterol and choline phospholipid efflux from fibroblasts. Efflux mechanistic differences were observed between BSP proteins and other cholesterol acceptors. Preincubation of BSP-A1/-A2 proteins with choline prevented cholesterol efflux, an effect not observed with apolipoprotein A-I. Also, the rate of BSP-induced efflux was rapid during the first 20 min, but leveled off thereafter in contrast to a relatively slow, but constant, rate of cholesterol efflux mediated by apolipoprotein A-I, apolipoprotein A-I-containing reconstituted lipoproteins (LpA-I) and high-density lipoproteins. These results indicate that fibroblasts are a good cell model to study the mechanism of lipid efflux mediated by BSP proteins. (+info)
The cleavage site specificity of human prostate specific antigen for insulin-like growth factor binding protein-3.
(15/2200)
The cleavage site of human insulin-like growth factor binding protein-3 by urinary prostate specific antigen was examined. Human insulin-like growth factor binding protein-3 was incubated with urinary prostate specific antigen at 37 degrees C and its proteolyzed fragments were separated by a reversed phase HPLC followed by N-terminal amino acid sequence analysis, demonstrating that the cleavage mainly occurred at Tyr-159. The synthetic peptide including Tyr-159 was also cleaved at the same site, although its reaction rate was relatively low. These results indicate that human insulin-like growth factor binding protein-3 is specifically cleaved at Tyr-159 by prostate specific antigen. Human insulin-like growth factor binding protein-3 was previously reported to be cleaved at five sites including Arg-97, Arg-132, Tyr-159, Phe-173 and Arg-179 by another group, however, prostate specific antigen preparation is possibly contaminated by trypsin-like protease. In contrast, our purified urinary prostate specific antigen had only a chymotrypsin-like activity, demonstrating that prostate specific antigen has the high substrate specificity for human insulin-like growth factor binding protein-3. (+info)
Immature germ cell separation using a modified discontinuous Percoll gradient technique in human semen.
(16/2200)
The difficulty of identifying immature germ cells in unstained, fresh semen has led most laboratories to use the broad definition 'round cells' to indicate cells other than spermatozoa, thus grouping together both leukocytes and immature germ cells. This is also the case in research andrology, where very little attention has been given to immature germ cells in the semen apart from some rare exceptions, such as the attempts to study meiosis. Here we report on the use of a discontinuous Percoll gradient method modified to enable the best separation possible of immature germ cells from the other cells found in the ejaculate, in order to obtain a cellular suspension free of spermatozoa. Our technique (intra-assay variation in duplicates < 10%) demonstrated a high immature germ cell concentration in gradient fractions with 30% to 45% Percoll with a small contamination (1.5-6%) of leukocytes, confirmed by May-Grunwald-Giemsa staining, immunofluorescence and cytofluorimetry. The concentrations of immature germ cells ranged from zero in obstructive azoospermia to 2.0 x 10(6)/ml in oligozoospermia and genital tract infection. The purified immature germ cell suspensions obtained can be useful for diagnostic and research purposes. (+info)