Search PubMed⌕ Search

Biomedical subjects

J A Holowczak

Publications and source records attributed to J A Holowczak.

At least 19 recordsLinked to original sources
PubMed1989

The immune response to vaccinia virus infection in mice: analysis of the role of antibody.

Immune response to primary intraperitoneal infection with vaccinia virus (strain IHD-J) was studied in C3H/Hej mice. Antibodies reactive with virus structural proteins were detected 6 days and neutralizing antibodies 8 days after infection. Although serum antibodies from infected mice bound to vaccinia virus infected cells, these antibodies were ineffective in complement mediated lysis of infected cells and were only moderately active in experiments with antibody dependent cellular cytotoxicity (ADCC). Immunoblotting analysis showed that serum antibody reacted with a number of structural proteins of both intracellular and extracellular forms of vaccinia virus. Immunoprecipitation results showed antibody binding of nonstructural proteins and glycoproteins. Correlation of the kinetics of NK and CTL activities in infected mice with neutralizing antibodies indicated that the cellular functions clearly precede the appearance of serum neutralizing antibody. The resolution of primary infection in mice thus appears to be mediated by functions of cellular immunity while resistance to reinfection may be dependent on circulating neutralizing antibody.

Animals↗
PubMed1986

Infection of DBA/2 or C3H/HeJ mice by intraperitoneal injection of vaccinia virus elicits activated macrophages, cytolytic and cytostatic for S91-melanoma tumor cells.

Murine peritoneal macrophages harvested 3-4 days after IP injection of vaccinia virus lysed S91-melanoma tumor cells in vitro; enhanced tumoricidal activity was measured with effector macrophages prepared 5-6 days after vaccinia virus infection. Treatment of virus-elicited macrophages prepared from DBA/2 mice with anti-asialo-GM1 antiserum, anti-Thy 1.2 antiserum or anti-Iad antiserum in the presence of complement so that cells sensitized with antibodies were lysed, did not reduce the measured level of tumoricidal activity indicating that macrophages [Ia(-); asialo GM1(-)] and not natural killer cells [asialo GM1(+); Thy 1.2(+/-)] or T-cells [Thy 1.2(+)] were responsible for mediating the lysis of S91-melanoma tumor cells. When incubated with virus-elicited macrophages but not thioglycollate-elicited macrophages, the ability of S91-melanoma tumor cells. to synthesize DNA was completely blocked. The results of these experiments support the view that one aspect of antitumor immunity enhanced during immunotherapy with vaccinia virus is the activation of macrophages which have cytolytic as well as cytostatic effects on melanoma tumor cells.

Animals↗
PubMed1985

Vaccinia virus proteins on the plasma membrane of infected cells. III. Infection of peritoneal macrophages.

Primary macrophage cultures were prepared from the peritoneal exudate cell population harvested from mice challenged intraperitoneally with saline, thioglycollate, or vaccinia virus. Vaccinia virus was adsorbed and penetrated into primary macrophages and L-cells with similar kinetics. As evidenced by the expression of some "early" virus-specified proteins, partial uncoating and activation of the virion-associated DNA-dependent RNA polymerase occurred in the infected macrophages. Subsequently, the viral replication cycle in macrophages was aborted; with time after infection, viral DNA and virion proteins initially associated with infected cells could be detected in an acid-soluble form in the medium harvested from infected macrophage cultures. The results suggest that at the time that the final stages of virus uncoating should have occurred, intracellular subviral particles were, instead, degraded in the infected, primary macrophages. Viral DNA synthesis could not be measured in vaccinia virus-infected macrophages, no "late" virus functions were expressed, and progeny virions were not assembled. As measured by the binding of antiviral-antibody-125I-protein A complexes to the surface of vaccinia virus-infected cells, the expression of virus-specified antigens on the surfaces of infected macrophages was significantly reduced and never exceeded that measured at 2 hr after infection on the surfaces of infected L-cells. The expression of virus-specified polypeptides with mol mass of 48-50, 45-46, 36-37, and 25 kDa on the plasma membranes of vaccinia virus-infected, thioglycollate-elicited macrophages, rendered the infected macrophages susceptible to lysis by vaccinia virus-specific cytotoxic T-cells.

Adsorption↗
PubMed1985

Vaccinia virus proteins on the plasma membranes of infected cells. II. Expression of viral antigens and killing of infected cells by vaccinia virus-specific cytotoxic T cells.

Evidence is presented that virion-derived antigens as well as viral antigens expressed on cell surfaces after infection, may participate in the formation of "target-antigen complexes" (TACs) which render vaccinia virus-infected cells susceptible to recognition and killing by syngeneic, vaccinia virus-specific cytotoxic T cells (VV-CTLs). By employing L cells infected with trypsin-treated and untreated virions, evidence was obtained that proteins with molecular weights of 32K and 37K may be among the virion-derived antigens which participate in TAC formation. Following virus infection, a sequential expression of virus-specified antigens on the plasma membrane of infected cells could be detected. At 1 hr p.i., polypeptides with molecular weights of 48K-50K and 36K-37K were present on infected cell surfaces; by 2 hr p.i., polypeptides with molecular weights of 48K-50K, 42K-44K, 36K-37K, 29K-30K, and 16K-17K were detected on plasma membranes. As measured by in vitro, 51Cr-release assays, vaccinia virus-infected L cells were completely susceptible to lysis by VV-CTLs (greater than or equal to 50% measured specific lysis) when (a) "early" but not late viral functions were expressed as measured with virus-infected cells which had been treated with hydroxyurea (5 X 10(-3) M) to block DNA replication or (b) when active protein synthesis was allowed to proceed for 90 min postadsorption and the infected cells were then treated with cycloheximide (100 micrograms/ml) to block further protein synthesis. Under these experimental conditions, polypeptides with molecular weights of 58K, 48K-50K, 42K, 36K-37K, 34K, 32K-33K, 27K-29K, and 16K-17K were expressed on the plasma membranes of vaccinia virus-infected cells but not uninfected cells. Whether each of the virion-derived and (or) virus-encoded polypeptides can associate with Class I, major histocompatibility antigens on the surfaces of virus-infected cells to form a primary or cross-reacting TAC recognized by VV-CTLs remains to be investigated.

Animals↗
PubMed1985

Vaccinia virus antigens on the plasma membrane of infected cells. I. Viral antigens transferred from infecting virus particles and synthesized after infection.

SDS-polyacrylamide gel electrophoretic analysis of plasma membranes prepared from L cells infected with radioiodinated vaccinia virus particles showed that at 2.0 hr postinfection, 125I-labeled virion polypeptides with molecular weights of 58K-60K, 32K-34K, 17K, and 12K-14K were associated with infected cell plasma membranes. By 4 hr postinfection, only the 32- to 34-kDa polypeptide, derived from infecting virus particles, could be detected on infected cell surfaces. A variety of techniques were applied to analyzing purified plasma cell membranes to define the viral antigens expressed on cell surfaces after infection, including (a) surface radioiodination of infected cells; (b) immune or Western blotting; (c) specific immunoprecipitation of viral proteins present in nonionic detergent extracts of membranes purified from [35S]methionine-labeled, virus-infected cells. It was determined that vaccinia virus-specified polypeptides with molecular weights of 78K-82K, 65K, 50K, 42K-45K, 35K-37K, 32K-34K, 30K, 20K, and 17K-18K were expressed by 3 hr postadsorption, on the plasma membranes of infected cells and were accessible to binding by exogenous antiviral antibodies. Viral antigens with molecular weights similar to those expressed on cell surfaces were secreted or shed from infected cells and could be detected in the medium harvested from virus-infected mouse L-cell cultures.

Adsorption↗
PubMed1985

Biochemical and electron microscopic studies of the transcription of vaccinia DNA by RNA polymerase from Escherichia coli: localization and characterization of transcriptional complexes.

We used the prokaryotic Escherichia coli RNA polymerase to determine if vaccinia DNA might provide recognition sites for the bacterial binding and initiation. Electron microscopic studies of the interaction of E. coli RNA polymerase with vaccinia DNA and molecular hybridization analysis of the transcription products formed after 3 or 5 min of in vitro incubation showed that: there were 30-40 sites on the template where the polymerase could bind and initiate cRNA synthesis; the entire coding capacity of the genome was utilized for cRNA synthesis; transcription was asymmetric; cRNA molecules were similar in size to the transcripts synthesized by the vaccinia virus RNA polymerase in vitro and in vivo; cRNA contains sequences in common with 'pre-early', 'early', and 'late' in vivo RNA; 'self-annealing' of cRNA in the presence or absence of RNA synthesized in vitro by the virion associated RNA polymerase showed that less than 1% dsRNA product could be detected suggesting that initially the same strand(s) was copied by the viral and bacterial enzymes; no differences in the frequency with which sequences represented in the Hind III fragments of vaccinia DNA were transcripted with time of in vitro incubation could be detected. These findings strongly suggest that the bacterial enzyme might recognize truly viral promotors. With extended in vitro incubations of the E. coli RNA polymerase with vaccinia DNA the control of transcription was found to diminish. This was correlated with an increase in the size of the transcripts and the synthesis of significant amounts of self-complementary RNA, indicating that symmetrical transcription was occurring. The dsRNA species recovered after self-annealing the cRNA from a 30 min in vitro reaction mixture were found to contain sequences which hybridized to some portion of all the Hind III restriction fragments of vaccinia DNA. The methods described here might be useful for the localization and characterization of promotor sequences in the genome of vaccinia virus, as well as for studies on sequence conservation between members of the Poxvirus genus.

Animals↗
PubMed1983

Immune responses of DBA/2 mice bearing melanoma tumors: cell-mediated immune responses after challenge with vaccinia virus.

Cell-mediated immune responses in DBA/2 mice bearing melanoma tumors (TB-mice) were measured and compared to similar responses in mice without tumors (C-mice). Splenic lymphocytes from TB-mice had a reduced capacity to respond to both B and T-cell mitogens, but TB-mice responded to infection with vaccinia virus by developing a virus-specific cytotoxic T-cell response equal to that measured with splenic effectors prepared from virus-infected C-mice. NK-cell activity, as measured by the in vitro lysis of YAC-1 targets by splenic effectors, was significantly depressed in TB-mice but, after infection of the animals with vaccinia virus, was restored to levels equal to that measured with splenic effectors prepared from C-mice. Doses of vaccinia virus, strain WR which elicited vaccinia-virus-specific cytotoxic T cells or stimulated NK-cell activity, failed to elicit or stimulate cytotoxic effectors specific for S91-melanoma tumor cells.

Animals↗
PubMed1983

Electron microscopic studies of transcriptional complexes released from vaccinia cores during RNA-synthesis in vitro: methods for fractionation of transcriptional complexes.

Electron microscopic (EM) and biochemical methods were employed to study the transcriptional complexes present in detergent lysates of vaccinia virus cores actively synthesizing RNA in vitro. When processed and examined in the EM, 14 'transcriptional sites' could be observed on full-length DNA templates. Fractionation of lysates by equilibrium density centrifugation in CsSO4, chromatography on hydroxyapatite columns or by sedimentation in sucrose gradients, allowed isolation of DNA templates associated with transcripts but these manipulations often resulted in fragmentation of the DNA template or promoted the release of transcripts from the template. It is suggested that RNA transcripts remain associated with the template in regions of supercoiling. These regions, in turn, may be maintained by DNA-protein interactions which are compromised as the transcriptional complexes are fractionated and purified.

Centrifugation, Density Gradient↗
PubMed1983

Some enzymatic activities associated with purified parapoxvirions.

Purified virions of milker's nodule virus, a parapoxvirus, were shown to contain an RNA polymerase, a nucleotide phosphohydrolase, and a protein kinase associated with or encapsulated within the DNA-containing core of the virus. In vitro, the activated viral RNA polymerase transcribed only 7 to 8% of the genome, in the form of 8S to 14S polyadenylated RNA molecules which were complementary to sequences present in milker's nodule virus DNA but not vaccinia virus DNA or DNA prepared from the host cells in which the virus was propagated. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that in vitro, the activated viral protein kinase phosphorylated viral polypeptides of 95, 60, 33.5, 15, and 13.8 kilodaltons.

DNA-Directed RNA Polymerases↗
PubMed1982

Poxvirus DNA.

Explore the source record for details and available documents.

Base Sequence↗
PubMed1981

Characterization of supercoiled nucleoprotein complexes released from detergent-treated vaccinia virions.

Treatment of vaccinia virions with 1% sodium dodecyl sulfate in the absence of reducing agents resulted in the release of subviral particles termed "subnucleoids," which contained viral DNA in combination with four polypeptides with molecular weights of 90,000, 68,000, 58,000 and 10,000. Biochemical and electron microscopic studies showed that viral DNA in combination with these polypeptides was maintained in a superhelical configuration. When subnucleoids were "fixed" with glutaraldehyde and formaldehyde and then examined by electron microscopy, spherical particles were observed, in which the supercoiled DNA was folded into globular structures that were 20 to 60 nm in diameter and were interconnected by DNA-protein fibers resembling the nucleosome structures described for eucaryotic chromatin.

Centrifugation, Density Gradient↗
PubMed1980

Preparation of subviral particles from vaccinia virions irradiated with ultraviolet light.

Nucleoprotein complexes, containing proteins covalently bound to DNA, were prepared from vaccinia virions irradiated with ultraviolet light. In the electron microscope, these complexes were observed to have a spherical morphology with a densely staining central portion, apparently containing proteins cross-linked to DNA, from which loops or fibers of DNA or DNA complexed with protein emerged.

Chemical Phenomena↗
PubMed1980

Biochemical and electron microscopic studies of the replication and composition of milker's node virus.

Replication of milker's node virus (MNV) DNA begins 4 to 8 h postinfection, continues to 30 to 36 h postinfection in the cytoplasm of infected, primary bovine embryonic kidney cells, and is accompanied by an inhibition of host nuclear DNA synthesis. Between 20 and 24 h postinfection, newly replicated genomes are incorporated into particles which cosediment with purified MNV. These biochemical measurements could be correlated with the development of MN virions as revealed by electron microscopic analysis of thin sections prepared from infected cells. Analysis of the DNA in purified MNV showed that the virions contained a double-stranded DNA molecule with a molecular weight of 85 x 10(6) to 87 x 10(6) and a guanine-plus-cytosine content of about 63%. After denaturation and sedimentation analysis of MNV DNA in alkaline sucrose gradients, three major DNA species were resolved. These species appeared to represent intact, terminally cross-linked genomes (approximately 75 to 80S); genomes bearing one nick (or with one cross-link removed) (60 to 65S); and complementary, denatured DNA strands released from cross-linked genomes bearing two nicks (or with both cross-links removed) (52 to 55S). Forty [35S]methionine-labeled polypeptides, ranging from approximately 200,000 daltons to 10,000 to 15,000 daltons, were detected by radioautography after polyacrylamide gel electrophoresis of the proteins present in detergent-solubilized MNV preparations. Treatment of MN virions with Nonidet P-40, beta-mercaptoethanol, and sonication released 10 polypeptides, which were apparently located on the surface of virions. Further fractionation of these released polypeptides, followed by electron microscopy and polyacrylamide gel electrophoresis, indicated that a 42,000- to 45,000-dalton polypeptide is a major component of the threadlike tubule structure present on the surface of MN virions.

Animals↗