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Biomedical subjects

D Hoover

Publications and source records attributed to D Hoover.

At least 19 recordsLinked to original sources

Rat Sertoli and spermatogenic cells express a similar gene, and its product is antigenically related to an outer dense fiber-associated protein.

We have previously reported that a heterodimeric protein secreted by rat Sertoli cells is antigenically related to a protein associated with outer dense fibers of the sperm tail. Therefore, we have explored the possibility that Sertoli and spermatogenic cells express a similar gene encoding a homologous protein. A Sertoli cell heterodimeric protein cDNA probe recognizes specific mRNA in pachytene and round spermatids fractionated by centrifugal elutriation; however, this specific mRNA was less prominent than in cultured Sertoli cells. In agreement with these observations, in situ hybridization experiments show that Sertoli cells are predominantly engaged in active heterodimeric protein mRNA synthesis, while meiotic prophase spermatocytes and spermatids also show significant but less abundant specific mRNA. Immunoblotting experiments demonstrate that, while Sertoli cells synthesize a heterodimeric protein consisting of two disulfide-linked components with molecular masses of 45 and 35 kD, both primary spermatocytes and round spermatids synthesize single 30 kD monomers not associated by disulfide linkage but recognized by antisera to Sertoli cell heterodimeric protein. Immunoblotting and immunogold electron microscopic studies show that antisera to Sertoli cell heterodimeric protein recognize a protein associated with outer dense fibers. This immunoreactivity was abolished by a 5-min pronase treatment, without affecting the integrity of outer dense fibers. Results of this study and previous studies demonstrate that both Sertoli and spermatogenic cells express a similar gene and that an antigenically related product encoded by this gene becomes associated with outer dense fibers during their assembly at spermiogenesis.

Animals

Developmental expression of the S35-S45/SGP-2/TRPM-2 gene in rat testis and epididymis.

Testosterone-repressed prostate message-2 (TRPM-2) was originally isolated and cloned from the regressing ventral prostate of the rat. In this tissue, and in other hormone-dependent tissues such as the mammary gland, this gene is induced in the absence of the appropriate trophic hormone. Sequence analysis of the cDNA and genomic clones of TRPM-2 have demonstrated that the coding sequence of this gene is identical to S35-S45 (also known as SGP-2 and clusterin), which is constitutively expressed by the Sertoli cells of the adult testis. Using Northern, slot blot, S1-nuclease analysis, and in situ hybridization, we have investigated the regulation of TRPM-2 expression in the testis and epididymis during development. Slot blot analysis of RNA extracted from the testis and epididymis of 7-, 14-, 28-, 35-, and 91-day-old rats demonstrates that the gene is induced to detectable levels between days 7 and 14 and that the relative level of expression does not change significantly after day 14. In situ hybridization using frozen sections of testis from day 2-, 7-, 14-, 28-, 35-, and 91-day-old rats confirms that there is little expression of TPRM-2 in the seminiferous epithelium of 7-day-old rats, but this increases considerably after 14 days, primarily in Sertoli cells but also in association with meiotic developing spermatogenic cells. However, TRPM-2 mRNA is expressed in the rete testis at 2 days of age, reaches a peak at 35 days of age, and continues to be expressed in the adult. Slot blot analysis demonstrates that TRPM-2 is also induced in the epididymis between 7 and 14 days of age, although, as has been demonstrated by in situ hybridization, TRPM-2 mRNA is detectable in the epithelial cells in the head of the epididymis but is barely detectable in the midportion or tail regions. Northern analysis suggests that the size of the TRPM-2 transcript in the testis also changes during development. In the early stages of testicular development, the TRPM-2 transcript appears to be a broad band of approximately 1.5 kb, while the transcript in the adult appears to be approximately 1.8 kb in length. S1-nuclease protection assays suggest that this increase in size is not due to differential splicing of the first exon of TRPM-2/SGP-2 and most probably reflects a difference in the polyadenylation of the mRNA in the testis at different times during development.

Animals

The diagnostic utility of elevation in cerebrospinal fluid beta 2-microglobulin in HIV-1 dementia. Multicenter AIDS Cohort Study.

We measured serum and CSF beta 2-microglobulin (beta 2M) levels in HIV-1 seropositive individuals with and without dementia to determine the frequency and diagnostic utility of elevation of CSF beta 2M. We compared 34 samples from 27 patients with HIV-1 dementia with 110 samples from 54 HIV-1 seropositive participants in the Multicenter AIDS Cohort Study, none of whom had progressive dementia. Neurosyphilis and CNS opportunistic processes were excluded in all subjects. We stratified the nondemented subjects by duration of HIV seropositivity and peripheral blood CD4 count. Compared with the nondemented group, demented subjects had significantly higher CSF total protein, IgG%, and CSF albumin/serum albumin ratios. A highly significant association was found between elevated CSF beta 2M and reduced CD4 count (p less than 0.0001). No significant differences were noted between the demented and nondemented groups in CSF WBC count or in the frequency of CSF HIV-1 isolation. The mean CSF beta 2M was 1.9 mg/l in the nondemented subjects compared with 4.2 mg/l in those with dementia (p less than 0.0001). We derived a cutoff of 3.8 mg/l from the distribution of CSF beta 2M in the nondemented group. The determination of CSF beta 2M had a sensitivity of 44%, specificity of 90%, and a positive predictive value of 88% for diagnosis of HIV dementia when compared with nondemented subjects with CD4 counts less than 200. In those without dementia, there was a strong correlation between serum and CSF beta 2M (r = 0.50, p less than 0.0001), but in demented subjects CSF beta 2M was elevated independently of serum levels, suggesting that CSF beta 2M is produced within the brain in HIV dementia. In the absence of CNS opportunistic processes, elevated CSF beta 2M greater than 3.8 mg/l is a clinically useful marker for HIV dementia.

AIDS Dementia Complex

The significance of western blot assays indeterminate for antibody to HIV in a cohort of homosexual/bisexual men. The Multicenter AIDS Cohort Study.

The objective of this study was to determine the frequency and significance of nondiagnostic Western blot (WB) assays in homosexual/bisexual men at risk of infection with HIV-1. The presence of a positive enzyme-linked antibody assay (EIA) confirmed by a positive WB was used as evidence of infection and seroconversion. Indeterminate WB assays were defined as reactions to only one viral gene product of HIV-1. Three analyses were conducted to (a) determine the frequency of such reactions in men who, during a 4-year period, did not develop diagnostic serologic reactions; (b) determine, retrospectively, the preseroconversion frequency of indeterminate WB assays in 286 men who seroconverted; and (c) evaluate in vitro production of specific antibody by peripheral blood mononuclear cells (PBMCs) as a method of indicating whether or not an indeterminate WB assay represents HIV-1 infection. Reactions to products of gag, pol, or env were noted in 8.0, 4.0, and 6.7% of 1,595 first-visit tests of men who remained seronegative for 4 years. Indeterminate reactions occurred in 204 men with negative EIAs who subsequently seroconverted and in 82 men with positive EIAs preconversion. Supernatants harvested from PBMCs of 2 of 36 seroconverters obtained one or two visits preseroconversion and cultured with pokeweed mitogen were antibody-positive. All were positive at the visit, with diagnostic serology. None of the supernatants from cells of 19 men with EIA-negative WB-indeterminate serologic assays were antibody-positive. Our results suggest that persistently EIA-negative homosexual/bisexual men who have indeterminate WB assays are unlikely to be infected with HIV-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Bisexuality

Longitudinal study of homosexual couples discordant for HIV-1 antibodies in the Baltimore MACS Study.

Thirty-six sexually active couples serologically discordant for human immunodeficiency virus, type 1 (HIV-1), within the Baltimore Multicenter AIDS Cohort Study (MACS) were assessed to determine whether evidence of HIV-1 infection could be detected in the HIV-1-antibody-negative partners and whether factors associated with lack of transmission of HIV from the seropositive to the seronegative partner could be ascertained. Six HIV-1 seropositive couples and 18 seronegative couples were followed concurrently for comparison. None of the seropositive subjects had an AIDS-defining illness at entry into the study, and all subjects were followed for 1 year. A separate evaluation of unprotected anal receptive and insertive intercourse between discordant couples indicated high-risk activities for a median of 40 months, as reported by the HIV seropositive partner. Despite this finding, none of the HIV-1 seronegative men in discordant couples had evidence of HIV-1 infection by viral culture, p24 antigen testing, or polymerase chain reaction for HIV-1 DNA. Discordant seronegatives and seropositives did not differ from concordant seronegatives and seropositives in numbers of circulating CD4, CD8, and natural killer lymphocytes or in prevalence of antibodies to herpes simplex virus, type 1, Epstein-Barr virus, or cytomegalovirus, except that discordant seronegative men were less likely than their seropositive partners to have antibodies to herpes simplex virus, type 2. The reason for the apparent lack of HIV-1 infection in seronegative discordant individuals remains unexplained and did not appear to be associated with type of sexual activity, T-lymphocyte subsets or natural killer cells, or early stage of HIV-1 disease.

Adult

Recombinant human pim-1 protein exhibits serine/threonine kinase activity.

The protein predicted by the sequence of the human pim-1 proto-oncogene shares extensive homology with known serine/threonine protein kinases, and yet the human Pim-1 enzyme has previously been reported to exhibit protein tyrosine kinase activity both in vitro and in vivo. Recently a new class of protein kinases has been identified which exhibits both protein-serine/threonine and protein-tyrosine kinase activities. We therefore investigated the possibility that the human Pim-1 kinase likewise possesses such bifunctional enzymatic phosphorylating activities. A full-length human pim-1 cDNA was subcloned into the bacterial vector pGEX-2T and the Pim-1 protein expressed as a fusion product with bacterial glutathione S-transferase (GST). The hybrid GST-Pim-1 fusion protein was affinity purified on a glutathione-Sepharose column prior to treatment with thrombin for cleavage of the Pim-1 protein from the transferase. Pim-1 was purified and the identity of recombinant protein confirmed by amino-terminal sequence analysis. Pim-1 was tested for kinase activity with a variety of proteins and peptides known to be substrates for either mammalian protein-serine/threonine or protein-tyrosine kinases and was found to phosphorylate serine/threonine residues exclusively in vitro. Both the Pim-1-GST fusion protein and the isolated Pim-1 protein exhibited only serine/threonine phosphorylating activity under all in vitro conditions tested. Pim-1 phosphorylated purified mammalian histone H1 with a Km of approximately 51 microM. Additionally, Pim-1 exhibited low levels of serine/threonine autophosphorylating activity. These observations place the human Pim-1 in a small select group of cytoplasmic transforming oncogenic kinases, including the protein kinase C, the Raf/Mil, and the Mos subfamilies, exhibiting serine/threonine phosphorylating activity.

Cloning, Molecular

The use of bladder mucosa and combined bladder mucosa/preputial skin grafts for urethral reconstruction.

Autologous bladder mucosa was used as a free graft for urethral reconstruction in 47 patients. However, in some cases the graft extended to the tip of the penis and became exposed to air. The resultant columnar metaplasia and hypertrophic changes in the mucosa produced stickiness and occlusion of the meatus. As a result a combined graft has superceded the simple mucosal graft for urethral substitution extending to the tip of the penis. The terminal few millimeters of such a graft are fashioned from preputial or penile skin, and they prevent exposure of the bladder mucosa to air. Our initial results are encouraging, although followup is short.

Adolescent

Autologous bladder mucosa graft for urethral substitution.

Bladder mucosa was used to construct a neourethra in 32 children. Twenty-five of them had had previous failed surgery for hypospadias and seven had severe congenital defects of the urethra. A satisfactory functional and cosmetic result was achieved in 30 patients.

Adolescent

Streptococcal enteropathy in infant rats.

A spontaneous outbreak of enteric disease with high morbidity and mortality occurred in preweanling rats on a multigeneration toxicity study. The rats had a large number of gram-positive cocci on the surface of villi in the small intestine without associated inflammatory change. Ultrastructural study identified filamentous attachments between adjacent bacteria, and between bacteria and morphologically normal microvilli. The organism was isolated and identified as Streptococcus sp. The disease was reproduced in rat pups by inoculating dams and newborn rats with pure cultures of the Streptococcus. Streptococcal enteropathy is a previously unreported disease in rats.

Animals

Methicillin-resistant Staphylococcus epidermidis blepharitis.

Blepharoconjunctivitis caused by methicillin-resistant strains of Staphylococcus epidermidis occurred in two hospitalized patients, a 74-year-old man and a 19-year-old man. Both strains were resistant to multiple courses of topical antibiotic therapy. Successful treatment in both cases depended upon antibiotic sensitivity testing. These methicillin-resistant strains of S. epidermidis, like methicillin-resistant strains of Staphylococcus aureus, demonstrated almost complete cross-resistance to cephalosporins despite apparent sensitivity on Kirby-Bauer disk sensitivity testing. Topical vancomycin was curative in one case. In the other case, treatment with topical gentamicin, intravenous cefoperazone, and oral rifampin led to resolution of the symptoms.

Adult

Unusual complication of Swan-Ganz catheter requiring mediastinotomy.

We were consulted to assist in the removal of an anchored Swan-Ganz catheter. After failing in several attempts to remove the catheter, we elected to return the patient to the operating room in the event that the catheter had been sutured to the right atrium. No evidence of this was found. We believe the catheter was anchored to the pulmonary bed, but the definite cause of this problem is still obscure.

Cardiac Catheterization

Depressed macrophage functions at temperatures below 37 degrees C.

Immunobiological properties of guinea pig and human peritoneal macrophages were studied at temperatures ranging from 25 degrees C to 37 degrees C. Glass adherence, random migration, response to MIF and killing of Salmonella typhimurium and Saccharomyces cerevesiae by guinea pig macrophages were decreased with temperatures below 37 degrees C. Killing of Sacch. cerevesiae by human macrophages was also reduced at temperatures less than 37 degrees C. Acid phosphatase and beta-N-acetylglucosaminidase (NAG) activity assayed at 37 degrees C did not change when the cells were preincubated for 1 and 5 hours at various temperatures. Impaired macrophage function with subnormal temperatures may contribute to enhanced susceptibility to infection of patients with chronic diseases such as renal failure and cirrhosis.

Acetylglucosaminidase

Immunologic and ionophore-induced generation of leukotriene B4 from mouse bone marrow-derived mast cells.

Mouse bone marrow-derived mast cells differentiated in vitro and sensitized with monoclonal IgE respond to antigen-initiated activation-secretion with the generation of leukotriene B4 (LTB4), a highly potent chemotactic factor. The antigen-initiated net percent release of the secretory granule marker beta-hexosaminidase and the generation of immunoreactive LTB4 and of immunoreactive leukotriene C4 (LTC4) were not diminished by washing the cells before challenge, indicating that interaction of the antigen occurred with the IgE fixed on cell membranes and was not due to phagocytosis of immune complexes formed in the fluid phase. The parallel dose-response relationship for the antigen-induced release of the performed mediator and the generation of both leukotrienes along with the superimposable time courses of their extracellular appearance indicate the origin of these mediators from a common cell type with IgE receptors. Resolution by reverse phase-high performance liquid chromatography (RP-HPLC) of the leukotrienes released from unsensitized cells by ionophore A23187 and from sensitized cells by the specific antigen revealed that the generation of LTB4 was accompanied by the production of the two diastereoisomers of 6-trans-LTB4, which were not immunoreactive. The immunoreactive LTB4 eluted from RP-HPLC at the same retention time as synthetic LTB4 was present in similar nanogram quantities when measured by either radioimmunoassay or integrated absorbance at 269 nm and exhibited a chemotactic activity for human neutrophils on a weight basis comparable to that of synthetic LTB4. The overall recoveries after RP-HPLC of immunoreactive LTB4 released from ionophore A23187-activated, bone marrow-derived mast cells and of added tritiated synthetic LTB4 in separate experiments with ionophore-activated cells were comparable and greater than 78%. The maximum generation of LTB4 by ionophore A23187-activated cells of 7.9 +/- 2.5 ng/10(6) cells (mean +/- SD), occurring at 40 min, was greater than the maximum generation of 4.5 +/- 0.8 ng/10(6) cells, with immunologic stimulation occurring 5 min after antigen challenge of sensitized cells. The initial rate of LTB4 generation after perturbation of the IgE-Fc receptors, however, exceeded that following ionophore A23187 stimulation and may represent one important variable in establishing a significant chemotactic gradient.

Animals

Radioimmunoassay for leukotriene B4.

A rabbit immunized with leukotriene B(4) [LTB(4); (5S,12R)-6, 14-cis-8, 10-trans-icosatetraenoic acid] coupled to bovine serum albumin via the 12-oxy function of the lipid produced antibodies having an average association constant (K(a)) for [14,15-(3)H]LTB(4) of 3.2 x 10(9) M(-1) at 37 degrees C and in a concentration of 0.37 mug/ml of the immune plasma. When 10 mul of anti-LTB(4) and 3.9 nCi of [14,15-(3)H]LTB(4) (28 Ci/mmol; 1 Ci = 3.7 x 10(10) becquerels) were incubated in a volume of 250 mul, 50% inhibition of radioligand binding was achieved with 0.31 ng of LTB(4) and with 1.95 ng of (5S,12S)-6-trans-8-cis-LTB(4). The sulfidopeptide leukotrienes, LTC(4) and LTD(4), displaced the radioligand from this antibody with less than 1/100th the activity of LTB(4), and the diastereoisomers of 6-trans-LTB(4), 5-L-hydroxy-6-trans-8,11,14-cis-icosatetraenoic acid (5-HETE), and three prostaglandins were minimally effective. The specificity of this radioimmunoassay was further shown by assessment of the immunoreactive products generated from calcium ionophore (A23187)-activated rat serosal mast cells and human neutrophils after reversed-phase HPLC. Resolution of the supernatants from each cell type yielded a single immunoreactive peak that coeluted with synthetic LTB(4) and quantitatively correlated with the physical measurement by integrated A(269) in that peak; UV-absorbing peaks eluting at other retention times were not immunoreactive. The immunoreactive LTB(4) generated averaged 4.6 ng per 10(6) rat mast cells and resolution of the supernatants by reversed-phase HPLC without a prior extraction step gave a recovery of 54%, validating the direct applicability of this sensitive and specific assay for LTB(4), a highly potent chemotactic factor, to unfractionated biologic fluids.

Animals

Immunity of cholera in man: relative role of antibacterial versus antitoxic immunity.

Purified cholera toxoid is antigenic when given enterally and orally. Purified toxoid fails to provide protection against experimental challenge. Clinical cholera confers formidable protection against homologous or heterologous rechallenge. Failure to culture vibrios from intestinal fluid or stool of re-challenge volunteers suggests that the predominant immune mechanism is antibacterial rather than antitoxic.

Antibodies, Bacterial

The problem of emesis during oral glucose-electrolytes therapy given from the onset of severe cholera.

In an attempt to obviate the need for intravenous fluids by preventing dehydration, 57 adult volunteers who experienced induced clinical cholera during a vaccine development programme were treated from the onset of diarrhoea with oral glucose-electrolytes therapy. 44 individuals with mild to moderately profuse diarrhoea (less than 8 L. total volume) were maintained in normal water and electrolyte balance with oral therapy alone. 13 individuals with severe diarrhoea (greater than 8 L. total volume) could not be maintained in balance with oral therapy alone, due chiefly to emesis during the first day of illness. Emesis occurred in the absence of significant dehydration or acidosis. Since emesis precludes effective early oral therapy in severe cases, domiciliary oral therapy is unlikely to eliminate cholera mortality. Rural diarrhoea treatment centres using oral therapy with limited amounts of intravenous fluids when needed, could reduce case fatality from cholera and related diarrhoeas virtually to zero with least expense.

Adolescent