Nucleotide and derived amino-acid sequences of the CRE-binding proteins from rat C6 glioma and HeLa cells.
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Amino Acid Sequence↗
Biomedical subjects
S M Wolinsky
Publications and source records attributed to S M Wolinsky.
Analysis of alternatively spliced human immunodeficiency virus type-1 mRNA species, one of which encodes a novel tat-env fusion protein.
A polymerase chain reaction-based analysis was used to define the structures of the mRNAs that encode human immunodeficiency virus type-1 (HIV-1) regulatory and structural proteins in infected H9 cells. Twenty alternatively spliced mRNAs encoding the vif, vpr, env, nef, tat, and rev proteins were characterized. An evaluation of the coding potentials of these transcripts recognized both leaky scanning and reinitiation at downstream initiation codons as mechanisms that may operate during translation of many of the polycistronic messages. Two new splice acceptor sites, one at nt 6018 defining a new mRNA coding for the env and vpu proteins and another at nt 8671 defining a novel tat-env fusion transcript, were characterized. The latter transcript expressed a novel protein p17tev that was immunoprecipitated by both polyclonal tat antibodies and monoclonals directed towards the C-terminal region of gp41. The p17tev protein was able to transactivate transcription from the HIV-1 LTR in transient transfection assays. The use of multiple alternative splice donor and acceptor sites and the generation of novel proteins may confer evolutionary advantages on the viral mutants encoding them and influence the course of clinical disease.
Animals↗
Rapid detection of cytomegalovirus DNA and RNA in blood of renal transplant patients by in vitro enzymatic amplification.
Cytomegalovirus infection causes significant morbidity and mortality in renal transplant patients. The only marker of CMV infection that appears to correlate with the development of symptomatic illness is viremia. However, CMV grows slowly in tissue culture, requiring 2-6 weeks of incubation for detection of characteristic cytopathic effect. The efficacy of antiviral therapy for CMV may be improved by earlier detection of viremia and institution of antiviral therapy. We performed amplification of CMV DNA and RNA from peripheral blood of renal transplant patients using the polymerase chain reaction (PCR) technique. We consistently detected CMV DNA by PCR earlier than CMV was detected by culture. Detection of CMV RNA in one patient confirmed the presence of actively replicating virus in peripheral blood. Amplification of peripheral blood from healthy CMV-seropositive and seronegative individuals, and from seropositive renal transplant patients without evidence of active CMV disease, was consistently negative. These preliminary data indicate that PCR may provide a means for earlier diagnosis of CMV viremia. Future prospective studies should determine if early detection of CMV DNA by PCR in peripheral blood does predict viremia and symptomatic illness, and if earlier institution of antiviral therapy based on PCR results improves outcome for the CMV-infected transplanted patient.
Adolescent↗
Rapid detection of herpes-simplex-virus DNA in cerebrospinal fluid of patients with herpes simplex encephalitis.
Herpes-simplex-virus (HSV) DNA in cerebrospinal fluid was amplified by use of the polymerase chain reaction and identified by hybridisation to a specific oligonucleotide probe. Specimens of cerebrospinal fluid (CSF) from 4 of 4 patients with herpes simplex encephalitis were positive for HSV DNA, whereas CSF specimens from 6 patients with other central-nervous-system infections were negative. This technique may expedite diagnosis of herpes simplex encephalitis.
Adult↗
Infectious cycle of human papillomavirus type 11 in human foreskin xenografts in nude mice.
We have performed the first molecular analysis of a time course of infection by a papillomavirus. The Hershey isolate of the human papillomavirus type 11 was used to infect human foreskin tissues, which were then implanted under the renal capsules of nude mice. The xenografts were recovered every 2 weeks for 14 weeks, fixed in formalin, and embedded in paraffin. Four-micrometer serial sections were examined by light microscopy for morphological changes, by immunocytochemistry for virion antigen production, and by in situ hybridization with 3H-labeled RNA probes for viral DNA replication and expression of the major mRNA species. After a lag period, probes spanning the E4 and E5 open reading frames, which are present in all E region viral mRNAs, generated the first detectable signals at week 4. Signals of other E region probes were minimally detected at week 6. Between weeks 6 and 8, there was an abrupt change in the implant such that cellular proliferation, viral DNA replication, and E and L region mRNA transcription were robust and reached a plateau. By weeks 10 to 12, the experimental condylomata were morphologically and histologically indistinguishable from naturally occurring condylomata acuminata. These findings suggest that cellular hyperproliferation and the morphologic features of condylomata are direct results of viral genetic activities. Unlike other DNA viruses, the E region transcripts increased with cell age and cellular differentiation and persisted throughout the entire experiment. In particular, the mRNA encoding the E1iE4 and perhaps E5 proteins remained overwhelmingly abundant. In contrast, viral DNA replication, L region mRNA synthesis, and virion antigen production were restricted to the most differentiated, superficial cells.
Animals↗
Human immunodeficiency virus type 1 (HIV-1) infection a median of 18 months before a diagnostic western blot. Evidence from a cohort of homosexual men.
STUDY OBJECTIVE: To study the natural history of human immunodeficiency virus type 1 (HIV-1) infection, we used an in-vitro amplification technique to detect HIV-1 nucleic acid sequences in sequential aliquots of peripheral blood mononuclear cells from homosexual men enrolled in the Multicenter AIDS Cohort Study. DESIGN AND PATIENTS: Blinded, longitudinal study of 24 homosexual men who were positive for HIV-1 antibodies at a recent follow-up visit. MEASUREMENTS AND MAIN RESULTS: Coded clinical samples were evaluated using two enzyme-linked immunosorbent assays (whole virus and gp120-gp41 fragment), Western blot, a p24 antigen capture assay, virus cocultivation, and in-vitro amplification of conserved regions from the HIV-1 gag and env open-reading frames. In 20 of the 24 men an HIV-1 enzymatically amplified product was detected before HIV-1 antibody seroconversion: at 42 months before seroconversion in two cases; at 36 months in one case; at 30 months in one case; at 24 months in four cases; at 18 months in eight cases; at 12 months in one case; and at 6 months in three cases (median, 18 months). In the four other men, detection of an HIV-1 enzymatically amplified product was concurrent with confirmation of antibody seroconversion by Western blot. CONCLUSIONS: There is a long and variable interval between virus acquisition and a diagnostic serum antibody response, perhaps due to the prolonged, persistent infection characteristic of the lentiviruses (family Retroviridae).
AIDS Serodiagnosis↗
Human immunodeficiency virus type 1 infection in homosexual men who remain seronegative for prolonged periods.
Infection with the human immunodeficiency virus type 1 (HIV-1), as demonstrated by viral cultures, has been described in some patients before antibodies to HIV-1 can be detected, but the duration and frequency of such latent infections are uncertain. We selected prospectively a cohort of 133 seronegative homosexual men who continued to be involved in high-risk sexual activity, and we cultured 225 samples of their peripheral-blood lymphocytes, using mitogen stimulation to activate the integrated HIV-1 genome. HIV-1 was isolated in blood samples from 31 of the 133 men (23 percent), 27 of whom have remained seronegative for up to 36 months after the positive culture. The other four men seroconverted 11 to 17 months after the isolation of HIV-1. In three of them, we studied cryopreserved lymphocytes obtained earlier, using the polymerase chain reaction to amplify small amounts of viral DNA, and we demonstrated that HIV-1 provirus had been present 23, 35, and 35 months before seroconversion. We conclude that HIV-1 infection in homosexual men at high risk may occur at least 35 months before antibodies to HIV-1 can be detected. A prolonged period of latency in such infections may be more common than previously recognized; the degree of infectiousness during such periods is unknown.
Acquired Immunodeficiency Syndrome↗
Differentiation-linked human papillomavirus types 6 and 11 transcription in genital condylomata revealed by in situ hybridization with message-specific RNA probes.
Human papillomaviruses (HPVs) infect specific human epithelial tissues. Because viral propagation in cultured cells has not been achieved, studies of HPV genetic activities have been difficult and rely largely on analyses of patient specimens by conventional biochemical methods. HPV type 6 and type 11 infections often result in genital warts (condylomata acuminata). Structural mapping of RNAs from such warts reveals that they use alternative promoters, splice sites, and polyadenylation sites to produce complex families of overlapping mRNAs that span multiple open reading frames. Based on the mRNA structures, we have developed message-specific subgenomic clones of HPV-6 and HPV-11 in pGEM vectors. Tritium-labeled, single-stranded RNA probes were synthesized in vitro and applied to serial thin sections of patient specimens for in situ hybridization. Our results reveal the qualitative and quantitative transcription patterns of different viral messages in relationship to one another, to viral DNA replication, and to cellular differentiation. The viral "E region" is transcribed before the onset of vegetative DNA replication and continues to be expressed in increasing amounts in the maturing epithelium. Even in mature epithelia, E region messengers are far more abundant than "L region" mRNAs. The L region messages encoding capsid proteins are truly late in that they appear concomitant with or after the onset of vegetative viral DNA replication and are only present in the superficial strata of the epithelium, which contain the oldest and most differentiated keratinocytes. Abundant intron material derived from processing E region transcripts accumulates in the nuclei. Strictly nuclear signals from the L region transcripts in the midepithelium suggest that regulation of their expression is at the level of transcription elongation.
Condylomata Acuminata↗
Enzymatic amplification of the human immunodeficiency virus in peripheral blood mononuclear cells from pediatric patients.
The presence of the human immunodeficiency virus type 1 (HIV) provirus was assessed in peripheral blood mononuclear cells (PBMCs) from 27 offspring of HIV-infected mothers, 12 of these mothers, and 4 HIV-uninfected mother-infant control pairs. Enzymatic amplification of specific conserved regions of the gag and env genes was performed directly in PBMC lysates using the polymerase chain reaction (PCR) technique. The enzymatically amplified gene products were evaluated using the oligomer hybridization detection procedure. PBMCs from infected infants (as determined by Centers for Disease Control clinical criteria) and from HIV-infected mothers manifested a characteristic HIV oligomer hybridization product. Clinically uninfected seropositive infants with declining HIV antibody titers and infants who became seronegative lacked an enzymatically amplified HIV gene product. These preliminary data indicate that PCR is a valuable diagnostic technique to detect or exclude HIV infection in young infants and children.
Blotting, Western↗
Loss of human immunodeficiency virus type 1 (HIV-1) antibodies with evidence of viral infection in asymptomatic homosexual men. A report from the Multicenter AIDS Cohort Study.
Four asymptomatic homosexual men reverted from positive to negative serologic results for the human immunodeficiency virus, type 1 (HIV-1) over 2.5 years, as shown by enzyme-linked immunosorbent assay (ELISA) and Western blot. Antibody bands in the Western blot from three men were undetectable 6 to 12 months after being positive; gradual fading of the number and intensity of bands was seen in the other man. No HIV-1-p24 antigenemia was detected; cryopreserved peripheral blood mononuclear cells were negative for HIV-1 by standard culture assay. Polymerase chain reaction (gene amplification) assays were done on peripheral blood mononuclear cells and showed the HIV-1 provirus in all subjects 6 to 18 months after the last positive antibody test. Serum specimens from each participant were genetically identical. Polymerase chain reaction showed that peripheral blood mononuclear cells from one subject at different times matched by HLA DNA typing. Clinical and laboratory features of these four men were similar to those of other seronegative subjects. Rare, asymptomatic persons seropositive for HIV-1 may not remain seropositive, but may remain latently infected with HIV-1.
Adult↗
Human papillomavirus types 6 and 11 mRNAs from genital condylomata acuminata.
We have identified and mapped a number of RNA species of human papillomavirus types 6 and 11 from condylomata acuminata by the electron microscopic R-loop technique. Each of the early (E)- and late (L)-region open reading frames (ORFs) deduced from the DNA sequences was represented in one or more transcripts. In addition, RNA species that could encode the modulator of DNA replication and the repressor of transcription, functions recently identified in the genetically similar bovine papillomavirus type 1, were also detected. Some ORFs were 5' proximal in one or more transcripts, whereas others were not 5' proximal in any species, suggesting that internal initiation of translation might be required to gain access to these latter ORFs. Virtually all transcripts had their 5' ends located in the E region and were polyadenylated at one of two sites, i.e., at the end of the E region or at the end of the L region. The great majority of the RNAs were derived from the E region of the genome, with one species approximately 50 to 100 times more abundant than the others. For most of the RNAs, the 5' end mapped near nucleotide 700; minor populations had 5' ends near nucleotide 100 or 1200. By correlating our mapping data with the genomic DNA sequences as well as available RNA structures and cDNA sequences of several papillomaviruses, we predict a number of mRNA splice donor and acceptor sites and suggest that the papillomaviruses have sophisticated usage of ORFs through alternative promoters, mRNA splice sites, and polyadenylation sites.
Anus Neoplasms↗
Epidemiology of genital herpes simplex virus infection.
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Acyclovir↗
Immune impairment and metastatic tumor growth: the need for an immunorestorative drug as an adjunct to surgery.
A spontaneous murine metastatic tumor system was used as a model to assess the effects of a major surgical procedure on tumor-specific immune reactivity and the growth of micrometastases. Any major surgical procedure resulted in impaired cell-mediated cytotoxicity postoperatively and an increase in the number of gross pulmonary metastases. The use of an immunorestorative drug, Thiabendazole, in the perioperative period resulted in an improved cytotoxic response and a significant decrease in pulmonary metastases. Perioperative immunotherapy can be an effective adjunct to surgery in preventing the growth of micrometastatic foci.
Animals↗
Comparative rates of sexually transmitted diseases among heterosexual men, homosexual men, and heterosexual women.
The prevalences of several sexually transmitted diseases (STD) among patients attending a public STD clinic were determined in a prospective study. Rates of gonorrhea, syphilis, genital herpes, trichomoniasis, nongonococcal urethritis, pediculosis pubis, scabies, and venereal warts were ascertained according to sex, sexual preference, and race. Numbers of cases of diseases not routinely reported to most public health agencies exceeded numbers of cases of diseases that are routinely reported. Rates of gonorrhea were higher among men than among women and higher among blacks than among whites. Syphilis was most common among homosexual men. Trichomoniasis was three times more frequent among blacks than among whites. Rates of nongonococcal urethritis among heterosexual men were almost twice those among homosexual men. Whites were more likely than blacks to have pediculosis pubis, scabies, and venereal warts. STD not routinely reported to most public health agencies are commonly seen in public clinics, and rates vary according to sex, sexual preference, and race.
Black or African American↗
Single-dose cefuroxime axetil in the treatment of uncomplicated gonorrhea: a controlled trial.
One hundred eighty-four patients were enrolled in a randomized study that evaluated the efficacy and toxicity of a single dose of orally administered cefuroxine axetil in the treatment of men and women with uncomplicated gonorrhea. Sixty-two patients received cefuroxine axetil alone, 62 received cefuroxine axetil and probenecid, and 60 received amoxicillin plus probenecid. Cure rates in the three groups were 98%, 98%, 96%, respectively. Only 2% of patients who received cefuroxine axetil alone complained of nausea, as compared with 11% of those who received a regimen that contained probenecid (P less than .05). The results show that cefuroxime axetil is effective and nontoxic in the treatment of uncomplicated gonococcal infections in adults.
Adult↗