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Biomedical subjects

S D Showalter

Publications and source records attributed to S D Showalter.

At least 37 records · Page 2Linked to original sources
PubMed1988

Expression and purification of protein segments encoded by the envelope and 3'-orf genes of human immunodeficiency virus type 1.

The pJL6 expression vector and its derivatives, pJLA16 and pANH-1, have been used for the synthesis and high-level expression in Escherichia coli of restriction enzyme fragments derived from the envelope and 3'-orf genes of the BH10 and BH8 clones, respectively, of the human immunodeficiency virus (HIV-1). These bacterially expressed proteins have been purified to apparent homogeneity by sequential detergent extraction, gel filtration, and reverse-phase high-performance liquid chromatography. The recombinant proteins have been used for the production of polyclonal and monoclonal antibodies, and the fusion proteins from the envelope gene are currently being evaluated for use as immunodiagnostic assay reagants.

Cloning, Molecular↗
PubMed1988

Comparative analysis of gp41 antigens by enzyme-linked immunosorbent assays for detecting antibodies to human immunodeficiency virus type 1.

We have compared the antigenic qualities of human immunodeficiency virus type 1 (HIV-1) gp41 glycoprotein with a synthetic oligopeptide (peptide R21S) and a bacterially synthesized protein (protein 566), which are homologous with the N-terminal region of gp41, in enzyme-linked immunosorbent assays (ELISA) for detecting antibodies to HIV-1 in sera of patients with the acquired immunodeficiency syndrome (AIDS) or the aids-related complex (ARC). Although the use of all three types of antigens readily allowed the detection of antibodies in human sera, ELISA employing purified gp41 glycoprotein and the protein 566 were more specific and sensitive than the peptide R21S ELISA.

AIDS-Related Complex↗
PubMed1988

Partial purification of native HIV transmembrane protein gp41: generation of polyclonal and monoclonal antibodies.

We partially purified the human immunodeficiency virus (HIV) glycoprotein gp41 from infected H9 cells by immunoaffinity chromatography using a column containing the M25 monoclonal antibody (diMarzo-Veronese et al., 1985). A pH 11.5 buffer worked best for eluting the glycoprotein from this column. The eluted gp41 was used in a sensitive slot blot immunoassay to detect antibodies to HIV in human sera and to prepare rabbit polyclonal antibodies and the 41-1S mouse monoclonal antibody. These antibodies reacted with gp41 in immunoprecipitation and in Western blot assays, but did not neutralize HIV in a syncytium-forming microassay. A pH 2.5 buffer was found to be the most effective solution for eluting gp41 from a 41-1S monoclonal antibody column.

Antibodies, Monoclonal↗
PubMed1988

Identification of the human papillomavirus E2 protein in genital tract tissues.

A 27-kilodalton protein representing approximately 60% of the E2 open reading frame of human papillomavirus type 6 (HPV-6) was synthesized in a bacterial expression system. Affinity-purified polyclonal antibody to this protein detected the probable E2 gene product as a 50-kilodalton protein in most condylomas by Western blot (immunoblot) analysis. The E2-positive condylomas were associated with HPV-6, HPV-11, HPV-16, or unidentified HPVs.

Condylomata Acuminata↗
PubMed1988

High expression of ets-1 gene in human thymocytes and immature T leukemic cells.

The cellular ets-1 gene homologous to the 5' region of the v-ets sequence of the E26 retrovirus codes for a 6.8-kb mRNA that is translated into a 51-kDa protein in human cells. A survey of mRNA from human tissues showed the thymus as the tissue with the highest level of ets-1 transcription, within other hematopoietic organs and tissues, including spleen, fetal liver, lymph nodes, bone marrow, and peripheral lymphocytes exhibiting low or undetectable levels of hybridization. A high level of ets-1 expression was found in murine thymocyte mRNA as well. Investigation of the ets-1 expression levels in human leukemic samples showed that primary malignant T cells (T-ALLs), corresponding to intrathymic stages of maturation, have a much higher level of ets-1 mRNA than malignant T lymphoid cells with a mature phenotype, such as adult T cell leukemias (ATLs). T-ALLs were also higher in ets-1 expression than the other lymphoid (pre-T-ALL, c-ALL, pre-B-ALL) malignant cells analyzed. Insignificant amounts of the specific ets-1 mRNA were detected in several acute myeloid leukemias representing various degrees of maturation. The elevated ets-1 mRNA in thymocytes suggests a biological role for the ets-1 product in these cells that could be explored to investigate ets-1 function. Finally, the exhibited expression of ets-1 in lymphoid cells and absence from malignant myeloid cells makes it a candidate marker for phenotyping human hematopoietic tumors.

Cell Line↗
PubMed1988

Characterization and localization of the products of the human homologs of the v-ets oncogene.

The avian erythroblastosis virus, E26, an acute leukemia virus, contains a transforming gene composed of two cellular components, v-myb and v-ets. The v-ets related sequences of man and other mammals consist of two transcriptionally active genes, ets-1 and ets-2, located on separate chromosomes. By contrast, both of these genes are contiguous in birds, are located on the same chromosome, and are coordinately transcribed. The human ets-1 and ets-2 gene products were identified by means of antibodies directed against the ets-1 and ets-2 encoded products. A 51 kD protein has been identified as the ets-1 gene product, and a 56 kD protein as the ets-2 gene product. Cellular fractionation studies indicated that the ets-1 protein is located in the cytoplasm and the ets-2 protein is nuclear. By comparison, the chicken ets protein, which contains both the ets-1 and ets-2 domains, distributes equally between the cytoplasm and nucleus. The differential compartmentalization of the ets gene products and their non-coordinate expression suggest that these proteins have different biological functions.

Cell Compartmentation↗
PubMed1987

Herpes simplex virus-induced suppression of macrophage-mediated tumoricidal activity in murine macrophages.

Herpes simplex virus (HSV) infections can enhance the progression of neoplastic diseases. Since macrophages can be activated to become tumorilytic and may figure prominently in host defense against cancer, the ability of HSV to modify macrophage-mediated tumoricidal functions was evaluated. Murine peritoneal macrophages treated with HSV could not be activated to a tumoricidal state by mouse recombinant gamma-interferon (IFN-gamma). Addition of HSV 4 h after treatment with IFN-gamma, at a time when the macrophages are fully committed to developing the cytotoxic phenotype, blocked macrophage-mediated lysis of syngeneic melanoma target cells. This inhibition of activation and cytotoxicity was not due simply to uptake of virus particles, because treatment with heat-inactivated HSV at 4-h posttreatment with IFN-gamma had no effect. In addition, HSV did not undergo a productive infection within macrophages, suggesting that the observed inhibitory activity might be due to a virus-induced product. In this regard, treatment of macrophages with recombinant alpha-interferon suppressed the activation of these cells by IFN-gamma, suggesting that virus-induced alpha-interferon may be mediating all or part of the suppressive activity. These studies suggest that enhancement of tumor progression following HSV infection may be related to the virus-induced suppression of macrophage-mediated tumoricidal activity.

Animals↗
PubMed1987

Detection of heterologous fusion proteins in Escherichia coli with a monoclonal antibody.

Several laboratories have constructed expression vectors for the production of heterologous fusion proteins containing the N-terminal 13 amino acids of the bacteriophage lambda cII-coded protein in Escherichia coli. We have prepared a monoclonal antibody to a synthetic peptide having this CII amino acid sequence and have found that this antibody reacts with authentic CII protein in Western blot tests and with most CII peptide-containing fusion proteins in both radioimmunoprecipitation and Western blot assays. However, there are some CII-hybrid protein species with which the antibody does not react. Our findings indicate that this antibody is a valuable tool for detecting and purifying expressed proteins and in studying their structure and function.

Animals↗
PubMed1987

Expression of the bovine leukemia virus X region in virus-infected cells.

Bovine leukemia virus, like its closest relatives the human T-cell leukemia virus types I and II, contains a 1.8-kilobase X region between the env gene and the 3' long terminal repeat. In this communication, we report the detection and characterization of a subgenomic mRNA from which this X region is presumably translated. This mRNA was produced by a complex splicing mechanism which resulted in juxtaposition of the 5' end of the env gene and the two overlapping X-region open reading frames. Translation of this mRNA could yield at least two distinct proteins depending on which initiation codon is used. Detection of the protein encoded by the BLV X-region long open reading frame has been reported (N. Sagata, J. Tsuzuku-Kawamura, M. Nagayoshi-Aida, F. Shimizu, K.-I. Imagawa, and Y. Ikawa, Proc. Natl. Acad. Sci. USA 82:7879-7883, 1985). Using synthetic peptide antisera, we detected a protein encoded by the short open reading frame in virus-infected cells. The protein migrated in sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of 19,000. It is a nuclear phosphoprotein.

Base Sequence↗
PubMed1985

Protection of mice against fatal herpes simplex type 2 infection by liposomes containing muramyl tripeptide.

Intravenous administration of liposomes containing muramyl tripeptide phosphatidylethanolamine, a lipophilic derivative of muramyl dipeptide that activates macrophages to a cytolytic state in situ, significantly protected mice against lethal challenge with herpes simplex virus type 2. These findings suggest that the systemic activation of macrophages by liposomes containing an immunomodulator can lead to prophylaxis of severe infections caused by herpesviruses.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1985

Human monocyte-mediated cytotoxicity against herpes simplex virus-infected cells: activation of cytotoxic monocytes by free and liposome-encapsulated lymphokines.

Human peripheral blood monocytes were incubated with free or liposome-encapsulated human lymphokines containing macrophage-activating factor (MAF) and tested for their effect on herpes simplex virus (HSV)-infected target cells. Activated monocytes lysed allogeneic HSV type 2 (HSV-2)-infected whole human embryo cells and xenogeneic BALB/c mouse embryo cells (10E2) without any significant effect on uninfected cells, as measured by release of 51Cr from target cells after 18 h of cocultivation. Kinetic studies revealed that lysis of virus-infected cells occurred by 10 h following cocultivation with activated monocytes. The inability of free MAF or supernatants from MAF-activated monocytes to lyse HSV-2-infected cells suggested that direct monocyte-target cell contact is required for monocyte-mediated cytotoxicity of the virus-infected cells. Monocytes activated with MAF suppressed the production of HSV-2 and HSV-1 from virus-infected cells more than control monocytes did. In addition, monocytes treated with liposome-encapsulated MAF selectively destroyed HSV-2-infected cells but left uninfected cells unharmed. The capacity of liposome-encapsulated immunomodulators to activate human monocytes to selectively lyse HSV-2-infected cells has potential therapeutic benefit and should be evaluated in vivo.

Cells, Cultured↗
PubMed1985

Enzyme-linked immunosorbent assay for determination of antibodies against herpes simplex virus types 1 and 2 in human sera.

A rapid and reproducible enzyme-linked immunosorbent assay (ELISA) is described for determining antibodies in human sera against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The sera were absorbed for 30 min with heterologous virus-infected-cell extracts to remove cross-reacting antibodies and then were applied to ELISA plates containing the target antigens, immunoaffinity-purified HSV-1 glycoproteins gC and gD and HSV-2 glycoproteins gD and gF. The absorbance index, defined as the ratio of A414 generated by a serum sample absorbed with a heterologous virus-infected-cell extract versus the A414 of a serum sample absorbed with an uninfected-cell extract, was used to determine the presence or absence of antibodies to HSV-1 and HSV-2. Results of the ELISA for detecting antibodies against HSV-2, when compared with results obtained for the same sera by the microneutralization test, showed an index of overall agreement of 91%. Results of the ELISA for detecting antibodies against HSV-1, when compared with microneutralization test results for sera negative for HSV-2 antibodies but positive for HSV antibodies by ELISA, showed an index of agreement of 99%.

Antibodies, Viral↗
PubMed1985

Analysis of a transgenic mouse containing simian virus 40 and v-myc sequences.

Two plasmids, one containing the simian virus 40 (SV40) genome and the mouse metallothionein I gene and one containing the v-myc gene of avian myelocytomatosis virus MC29, were coinjected into mouse embryos. Of the 13 surviving mice, one, designated M13, contained both myc and SV40 sequences. This mouse developed a cranial bulge identified as a choroid plexus papilloma at 13 weeks and was subsequently sacrificed; tissue samples were taken for further analysis. Primary cell lines derived from these tissues contained both myc and SV40 DNA. No v-myc mRNA could be detected, although SV40 mRNA was present in all of the cell lines tested. T antigen also was expressed in all of the cell lines analyzed. These data suggest that SV40 expression was involved in the abnormalities of mouse M13 and was responsible for the transformed phenotype of the primary cell lines. Primary cell lines from this mouse were atypical in that the population rapidly became progressively more transformed with time in culture based on the following criteria: morphology, growth rate, and the ability to grow in soft agar and in serum-free medium. The data also suggest that factors present in the mouse regulated the ability of SV40 to oncogenically transform most cells and that in vitro culture of cells allowed them to escape those factors.

Animals↗
PubMed1984

Human monocytes activated by immunomodulators in liposomes lyse herpesvirus-infected but not normal cells.

Highly purified peripheral blood monocytes from normal human donors were activated in vitro by incubation with liposomes containing immunomodulators such as recombinant human gamma interferon, human lymphokines, or muramyl dipeptide. The ability of liposomes containing immunomodulators to activate monocytes to a cytotoxic state capable of discriminating between virus-infected and uninfected cells was shown by activated monocytes recognizing and destroying herpes simplex virus type 2-infected cells while leaving uninfected cells unharmed .

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1984

Antibodies to a synthetic oligopeptide that react with herpes simplex virus type 1 and 2 glycoprotein C.

Nucleotide sequence and mRNA localization studies have allowed the prediction of the amino acid sequence of herpes simplex virus type 1 (HSV-1) glycoprotein C (gC). We immunized a rabbit with a conjugate of bovine serum albumin and a synthetic peptide having the same sequence as that deduced for amino acids 128 through 139 of HSV-1 gC. A very similar amino acid sequence has been predicted to exist in the related product, herpes simplex virus type 2 (HSV-2) gC, which was formerly designated gF. Preparations of crude antiserum and immunoaffinity-purified antibodies were obtained and shown to react in enzyme-linked immunosorbent assays with purified HSV-1 gC and HSV-2 gC. Although these antibodies did not detectably immunoprecipitate proteins from radiolabeled infected cell extracts, they reacted with HSV-1 gC and HSV-2 gC that were electrophoretically transferred to nitrocellulose membranes from polyacrylamide gels. These results confirm that HSV-1 gC and HSV-2 gC are immunologically related and also define a specific portion of HSV-1 gC that is conserved.

Animals↗
PubMed1983

Detection in antisera of antibodies that cross-react with herpes simplex virus type 1 glycoprotein gC.

Herpes simplex virus type 1 (HSV-1) glycoprotein gC was purified by affinity chromatography with an immunosorbent column containing monoclonal antibody to HSV-1 gC, and its reactivity with rabbit antisera was measured by enzyme-linked immunosorbent assay and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of radioimmunoprecipitates. Positive reactions were detected between HSV-1 gC and rabbit hyperimmune antisera to both HSV-1 and HSV-2. Electrophoretic analysis also revealed reactivity between the rabbit antisera and peptides of HSV-1 gC generated by partial digestion with trypsin. These findings indicate that HSV-1 gC has one or more cross-reactive or type-common determinants that are readily detected, and therefore, the presence of antibodies reacting with HSV-1 gC in sera may not necessarily be indicative of an earlier infection with HSV-1.

Animals↗
PubMed1983

Lysis of herpesvirus-infected cells by macrophages activated with free or liposome-encapsulated lymphokine produced by a murine T cell hybridoma.

Thioglycolate-induced mouse peritoneal macrophages were activated in vitro by the lymphokine designated macrophage-activating factor (MAF) produced by a murine T cell hybridoma to lyse herpes simplex virus type 2 (HSV-2)-infected murine target cells. Comparison of uninfected BALB/c 10E2 cells with HSV-2-infected 10E2 cells showed that macrophages activated with MAF selectively destroyed HSV-2-infected cells and left uninfected cells unharmed, as measured by an 18-h 51Cr-release assay. In contrast, macrophages treated with medium were as efficient as MAF-activated macrophages in suppressing the production of HSV-2 from virus-infected cells. These findings suggest that macrophages must attain an activated state to lyse HSV-2-infected cells. Finally, incubation of macrophages with liposomes containing MAF was shown to be a highly efficient method for activation of macrophages against HSV-2 infected cells. The ability to selectively destroy herpesvirus-infected cells in vitro by macrophages activated with liposome-encapsulated MAF suggests that the therapeutic efficacy of this treatment in vivo should be evaluated.

Animals↗