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Biomedical subjects

G B Caughman

Publications and source records attributed to G B Caughman.

At least 37 records · Page 2Linked to original sources
PubMed1993

Characterization of the myristylated polypeptide encoded by the UL1 gene that is conserved in the genome of defective interfering particles of equine herpesvirus 1.

Equine herpesvirus 1 (EHV-1, Kentucky A strain) preparations enriched for defective interfering particles (DIPs) can readily establish persistent infection. The UL1 gene, which is conserved in the genome of DIPs that mediate persistent infection, maps between nucleotides 1418 and 2192 (258 amino acids) from the L (long) terminus. UL1 has no homology with any known gene encoded by herpes simplex virus type 1 but has limited homology to open reading frame 2 of varicella-zoster virus and the "circ" gene of bovine herpesvirus type 1. Previous work showed that the EHV-1 UL1 gene belongs to the early kinetic class and is transcribed as a 1.2-kb polyadenylated mRNA (R. N. Harty, R. R. Yalamanchili, and D. J. O'Callaghan, Virology 183:830-833, 1991). In this report, the UL1 protein was identified and characterized as a 33-kDa polypeptide in EHV-1-infected cells by using rabbit polyclonal antiserum raised against a TrpE-UL1 fusion protein (amino acids 7 to 258 of UL1) synthesized in Escherichia coli. Results from Western blot (immunoblot), immunoprecipitation, indirect immunofluorescence, and biochemical analyses indicated that the UL1 polypeptide (i) is more abundant in cells infected with DIP-enriched virus than in cells infected with standard EHV-1, (ii) is synthesized as early as 3 h postinfection (p.i.) in infection with standard virus or in infection with DIP-enriched virus preparations and increases in abundance up to 12 h p.i., (iii) appears to be associated with the rough endoplasmic reticulum-Golgi apparatus early in infection (3 to 4 h p.i.), while a diffuse cytoplasmic pattern of fluorescence is observed late in infection (7 to 8 h p.i.), (iv) is modified by myristic acid as it contains a consensus N-terminal myristylation site and is readily labeled with [3H]myristic acid, and (v) is associated with mature EHV-1 virions.

Amino Acid Sequence↗
PubMed1992

Characterization of the regulatory functions of the equine herpesvirus 1 immediate-early gene product.

Use of the translation-inhibiting drug cycloheximide has indicated that the equine herpesvirus 1 (EHV-1) immediate-early (IE) gene, the sole EHV-1 IE gene, encodes a major viral regulatory protein since IE mRNA translation is a prerequisite for all further viral gene expression (W.L. Gray, R. P. Baumann, A. T. Robertson, G. B. Caughman, D. J. O'Callaghan, and J. Staczek, Virology 158:79-87, 1987). An EHV-1 IE gene expression vector (pSVIE) in combination with chimeric EHV-1 promoter-chloramphenicol acetyltransferase (CAT) reporter constructs was used in transient transfection assays to characterize the regulatory functions of the IE gene product. These experiments demonstrated that (i) the EHV-1 IE gene product is a bifunctional protein capable of both positive and negative modulation of gene expression; (ii) the IE gene product possesses an autoregulatory function which represses the IE promoter; (iii) IE autoregulation is dependent on IE promoter sequences mapping within positions -288 to +73 relative to the transcription initiation site (+1) of the IE gene; (iv) the IE gene product can independently activate the EHV-1 tk promoter (an early promoter) by as much as 60-fold; (v) two EHV-1 beta-gamma (leaky late) promoters, those of IR5 (gene 5 in the inverted repeat) and the glycoprotein D gene, demonstrate a requirement for both the IE gene product as well as a gene product encoded within the EHV-1 XbaI G fragment for significant activation; and (vi) the IE gene product is capable of activating heterologous viral promoters.

Animals↗
PubMed1991

Correlation of cytotoxicity, filler loading and curing time of dental composites.

Previous studies have shown that dental resin composites tested in cell culture produce cytotoxic effects on human gingival tissues. In this study, the cytotoxic potential of resin composites on primary human gingival fibroblast cultures was evaluated, based on inhibition of cellular protein synthesis measured by [35S] methionine incorporation. Both resin content and percentage of monomer conversion were considered as potential causes of cytotoxicity. Three resin composites were selected to provide a range of filler content from 45 to 86 wt%. Duplicate sample discs (1 mm thick x 10 mm diameter) of each composite were polymerized for 15, 30 and 60 s, followed by heat (110 degrees C, 10 min), and the degree of monomer conversion for each sample group was measured using Fourier transform infrared spectrophotometry. Identically fabricated discs were placed into 35 mm culture dishes with gingival fibroblasts and incubated for 24 h at 37 degrees C. The cell monolayers then were labelled at 24 h with [35S] methionine, washed and solubilized; then incorporated radioactivity was quantitated by liquid scintillation spectrometry. For each composite, as the percentage of monomer conversion increased, cellular toxicity decreased. In comparing different composites having similar monomer conversions, it was found that the filler/resin ratio was not the only factor determining the composite's relative toxicity.

Biocompatible Materials↗
PubMed1991

Altered lipid metabolism in parvovirus-infected cells.

A broad spectrum of cell lipid alterations are known to occur as a consequence of various viral infections. These changes include inhibition of lipid synthesis, stimulation of lipid synthesis and changes in the proportions of various lipids. The current study examined the effects of two parvoviruses on lipids of rat kidney (NRK) cells. Cells were infected with H-1 or Kilham rat virus (KRV) and the effects on 14C-acetate incorporation determined. Results showed that H-1 virus rapidly inhibited lipid formation (in 1 h) while KRV produced a similar effect beginning around 8 h. Pretreatment of the cells with cycloheximide did not alter this response. Fatty acid analysis by gas chromatography did not reveal major alterations in this component of total cell lipids although some fatty acids became undetectable by 18 h post-infection. The data suggest that these parvoviruses, especially H-1 virus, are able to rapidly alter lipid formation following infection and that this effect may be mediated by a virion component.

Animals↗
PubMed1990

Glass ionomer and composite resin cements: effects on oral cells.

Because the cement interfaces of restorations can approximate the periodontium, it is critical to determine the biocompatibility of cements. In this study, the cytotoxic potential of resin luting agents on cultures of gingival fibroblasts and oral epithelial cells was evaluated for direct microscopic cytotoxicity, cell morbidity, impaired adherence, and inhibition of macromolecular synthesis. Visible effects ranged from severe toxicity with inadequately polymerized composite resin to no detectable morphologic cell damage by a glass ionomer cement, but inhibition of protein and RNA synthesis varied with the material and cell type. The glass ionomer cement demonstrated no morphologic damage, but exhibited inhibition of macromolecular synthesis in gingival fibroblasts. These results confirmed that in vitro metabolic assays are appropriate for examining the biologic effects of materials.

Cells, Cultured↗
PubMed1990

Temporal regulation of equine herpesvirus type 3 transcription.

The transcription of equine herpesvirus type 3 (EHV-3; equine coital exanthema virus) has been examined and found to be temporally regulated into three classes: immediate early (IE), early (E), and late (L). Hybridization of in vivo 32PO4-labeled transcripts revealed that IE transcript(s) are derived exclusively from the inverted repeat segments (IRs) of the viral genome, while E and L transcripts are not restricted to any specific region of the genome. Northern blot analysis of EHV-3 IE RNA revealed a single transcript of approximately 5.7 kb (3.8 MDa). We have previously shown that transcription of equine herpesvirus type 1 (EHV-1) DNA is temporally regulated and produces a single 6 kb IE RNA which is derived from the IRs segments. In this paper, we show that the EHV-1 and EHV-3 IE RNA species are homologous, reflecting the colinearity of the genomes of these two related viruses. While four IE polypeptides are synthesized in EHV-1 infected cells in the presence of actinomycin D following the removal of a cycloheximide block, only one major IE polypeptide (180 kDa) is detectable in EHV-3 infected cells under these conditions. However, immunoprecipitation of EHV-3 infected cell extracts with polyvalent rabbit antisera to IE1 of EHV-1 revealed at least two other viral specific IE polypeptides.

Animals↗
PubMed1989

Disinfection of visible-light-curing devices.

Assays were developed for evaluating disinfection of visible-light-curing devices which were deliberately contaminated with an indicator organism, Streptococcus mutans, and devices which were contaminated during routine clinical use. Results indicated that wiping the surface with a substituted phenolic agent followed by wrapping in gauze saturated with the same solution was most effective for disinfecting the device handle and tip surfaces. Longer contact with the disinfectant is recommended to ensure virucidal/sporicidal action.

Dental Equipment↗
PubMed1988

Characterization of equine herpesvirus type 1 immediate early proteins.

EHV-1 immediate early (IE) gene expression in lytic infection results in the production of four high mol wt immediate early polypeptides (IEPs), designated IE1, IE2, IE2, and IE4; however, IE transcription is limited to the synthesis of a single 6-kb mRNA. Together, these findings raised questions as to whether the four IEPs were related products of the same gene. In the present study the IEPs were characterized with respect to their structural similarities, antigenic relatedness, and postsynthetic modifications. IE1 was the most abundant IEP, in that it accounted for approximately 80% of the IEP-incorporated radiolabel in infected rabbit kidney cells labeled under IE conditions with [35S]methionine or 14C-labeled amino acid mixtures. IE1 also was the major phosphorylated species. Limited proteolytic digestion of isolated radiolabeled IEP bands with Staph V8 protease yielded virtually identical fragment profiles in SDS-PAGE, as did digestions with chymotrypsin and N-chlorosuccinimide. Monospecific rabbit antisera raised against each of the four isolated IEPs reacted with all the IEP species in immunoblotting assays. Pulse-chase experiments indicated that all the IEPs were detectable immediately after a 15-min pulse and that several alterations in the IEP profile occurred during subsequent chase periods. Thus, the EHV-1 IEPs are closely related structurally and antigenically and appeared to be either produced simultaneously or processed to yield the individual forms immediately.

Antibodies, Viral↗
PubMed1988

Analysis of the in vitro translation products of the equine herpesvirus type 1 immediate early mRNA.

Equine herpesvirus type 1 (EHV-1) gene expression is coordinately regulated in an alpha, beta, gamma fashion. Viral alpha gene products include a 6.0-kb immediate early (IE) mRNA species (W. L. Gray et al., 1987, Virology 158, 79-87) and at least four closely related IE polypeptides (IEPs) (G.B. Caughman et al., 1985, Virology 145, 49-61). In this report, we describe results obtained from a series of in vitro translation experiments which were performed in an effort to characterize the IEPs and identify the mechanism by which individual IE protein species are generated. Our data indicate that a family of IEPs is generated in vitro from the 6.0-kb mRNA size class and that these IEPs correspond in overall size and antigenicity to those synthesized in infected cells. Using time-course/pulse-chase analyses, we show that production of three of the major IEPs [IE1' (193 kDa), IE3' (166 kDa), and IE4' (130 kDA)] occurs concomitantly, that none of these protein species can be chased completely into another, and that at least two additional minor species appear to be processed following synthesis. Finally, we show that the 6.0-kb mRNA species isolated during early or late stages of the infection cycle can be translated to yield all of the major IE proteins, indicating that production of the family of IEPs is not dependent upon accumulation of the IE mRNA which occurs during a cycloheximide blocked infection cycle. The implications of these findings are discussed as they relate to the origin and production of the IEPs both in vivo and in vitro.

Blotting, Northern↗
PubMed1987

Regulation of equine herpesvirus type 1 gene expression: characterization of immediate early, early, and late transcription.

The regulation of equine herpesvirus type 1 (EHV-1) transcription was examined in infected rabbit kidney cells using metabolic inhibitors. In order to map EHV-1 immediate early, early, and late transcripts, viral RNA was 32P-labeled in vivo and hybridized to EHV-1 DNA restriction fragments immobilized on nitrocellulose filters. Immediate early viral RNA was mapped to one region of the viral genome within the inverted repeat DNA sequences (map units 0.78-0.83 and 0.95-1.0). Northern blot hybridization analysis using a 32P-labeled cloned DNA probe from this region identified a single immediate early viral transcript (approximately 6 kb). Transcription of early and late genes was not restricted to any specific region on the viral genome as indicated by the ability of 32P-labeled early and late RNA to hybridize to EHV-1 restriction endonuclease fragments from both the long and short components of EHV-1 DNA. Additional experiments performed without the use of metabolic inhibitors confirmed that EHV-1 transcription is temporally regulated. The characterization of EHV-1 transcription during productive infection will serve as a reference for the analysis of viral transcripts in oncogenically transformed and persistently infected cells.

Animals↗
PubMed1986

Heat-inactivated frog virus 3 selectively inhibits equine herpesvirus type 1 translation in a temporal class-dependent manner.

Superinfection of equine herpesvirus type 1 (EHV-1)-infected rabbit kidney cells with heat-inactivated frog virus 3 (delta FV3) differentially blocked EHV-1 protein synthesis. The extent of inhibition varied with the specific EHV-1 message, but in general late protein synthesis was inhibited more than early and immediate early translation. Since FV3 has been shown to block heterologous RNA and protein synthesis, it was necessary to determine whether the observed reduction in herpesvirus protein synthesis was primarily due to a block in translation or to an earlier inhibition of EHV-1 mRNA synthesis. To distinguish between these alternatives, replicate cultures of EHV-1 infected cells were either superinfected with delta FV3 or treated with 10 micrograms/ml actinomycin D at 6 hr after infection, and EHV-1 protein synthesis monitored 3 hr later. We found that addition of actinomycin D to EHV-1 infected cultures had only a slight effect on EHV-1 translation, whereas superinfection with delta FV3 markedly reduced EHV-1 protein synthesis. This result suggested that the observed decline in EHV-1 protein synthesis was not due to the inhibition of herpesvirus mRNA synthesis. In addition, we showed that RNA extracted from delta FV3-superinfected cells directed the synthesis of full-size EHV-1 proteins in vitro indicating that shut-off was not caused by the degradation of EHV-1 mRNAs. Taken together these results show that delta FV3 selectively inhibited EHV-1 protein synthesis and are consistent with earlier observations which suggest that translational shut-off occurs at initiation.

Animals↗
PubMed1986

Oncogenic transformation of primary hamster embryo cells by equine herpesvirus type 3.

Infection of nonpermissive primary hamster embryo cells with equine herpesvirus type 3 (EHV-3; multiplicity of infection = 10 pfu/cell) resulted in an abortive infection and the development of several hundred foci of rapidly growing cells. Five of these foci were chosen at random for the establishment of transformed cell lines, designated EVD-1 (equine venereal disease) through 5. These transformed cell lines exhibited altered biological properties typical of transformed cells, including immortality, growth to high saturation density, colony formation in soft agar, reduced serum requirements, aneuploid karyotype, and oncogenicity in syngeneic animals. Subsequently, five corresponding tumor cell lines (EVD-1T through 5T) with similar biological properties were established. All EHV-3 transformed and tumor cell lines have been shown to express EHV-3-specific proteins by indirect immunofluorescence assays employing rabbit antisera to EHV-3 infected equine cells. None of the transformed cell lines were found to release infectious virus by infectious center or cocultivation assay or to contain viral particles by electron microscopy.

Animals↗
PubMed1985

Equine herpesvirus type 1 infected cell polypeptides: evidence for immediate early/early/late regulation of viral gene expression.

EHV-1 polypeptide synthesis was examined in productively infected rabbit kidney and hamster embryo cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of extracts from [35S]methionine- and 3H-amino acid-labeled-infected and mock-infected cultures revealed the presence of 30 infected cell-specific polypeptides (ICPs) which ranged in apparent molecular weights from 16.5K to 213K. Twenty-two of these ICPs comigrated with virion structural proteins. Four ICPs (203K, 176K, 151K, 129K) were detected in extracts of infected cultures labeled in the presence or absence of actinomycin D (Act D) immediately after release from a 4-hr treatment with cycloheximide (CH). These polypeptides, which were designated as EHV-1 immediate early (alpha) ICPs, were not detected in unblocked (non-CH-treated) infected cells. The most abundant ICP was a 31.5K nonstructural protein which, in addition to a 74K protein, was detected in unblocked infected cells at 2-3 hr postinfection. These proteins appeared to be regulated as early (beta) ICPs, since neither protein was observed in Act D-treated cultures released from CH block. Twelve ICPs were classified as late (gamma) polypeptides on the basis of their reduced synthesis in cultures in which viral DNA replication was inhibited by phosphonoacetic acid. All but one (40K) of these late ICPs corresponded to virion structural proteins.

Animals↗
PubMed1984

Equine cytomegalovirus: structural proteins of virions and nucleocapsids.

Enveloped virions and nucleocapsids of equine cytomegalovirus (ECMV; equine herpesvirus type 2) have been purified from the supernatants and the nuclear extracts of infected rabbit kidney (RK) cells, respectively, and their structural protein compositions have been analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that ECMV nucleocapsids were composed of nine proteins (average molecular weights = 148K, 52K, 49.5K, 46K, 43.5K, 38.5K, 27K, 20K, and 18K), which together constituted 89% of the total nucleocapsid protein on the basis of incorporated 3H-labeled amino acids. The 148K protein comprised 47.3% of the total protein and thus appeared to be similar in molecular weight and proportional composition to the major capsid proteins of other herpesviruses. Purified virions were composed of 37 proteins whose average molecular weights ranged from 14K to greater than 200K. Three intense glycoprotein bands (83K, 78K, and 73.5K) as well as four less intensely labeled glycoproteins were detected in [3H]glucosamine-labeled virion preparations. At least 14 structural proteins were readily detected in extracts of infected cells which had been [35S]methionine labeled late in infection, and 11 of these were immunoprecipitated by rabbit antiserum against purified virions. The protein composition of ECMV differs substantially from those of equine herpesvirus type 1 and type 3 as well as from those of other herpesviruses.

Animals↗
PubMed1984

Structure and genetic complexity of the genomes of herpesvirus defective-interfering particles associated with oncogenic transformation and persistent infection.

The complexity and structural organization of defective-interfering (DI) particle DNA of equine herpesvirus type 1 (EHV-1) have been elucidated by using restriction enzyme and Southern blot hybridization analyses. DI particles were generated by serial high-multiplicity passage of EHV-1 in L-M cells, and total viral DNA was extracted from virus purified from supernatants of these serial passages. EHV-1 DI particle DNA was quantitatively separated from standard (STD) DNA by several cycles of CsCl isopycnic banding in a vertical rotor. Restriction endonuclease digestion profiles of pure DI DNA were completely different from the mapped patterns observed for EHV-1 STD DNA. Digestion of pure defective DNA with restriction enzymes (Bg/II, EcoRI, and XbaI), for which there are few or no cleavage sites within the S (short) region of the EHV-1 STD genome, yielded high-molecular-weight supermolar DNA bands, suggesting that a large subgenomic repeat unit was present in defective DNA. DNA blot hybridization analysis with the Bg/II supermolar fragment of defective DNA, intact DI particle genomic DNA, and EHV-1 STD DNA restriction enzyme fragments as 32P-labeled probes indicated that the EHV-1 DI particle genome originates predominately from the STD DNA S region (0.77 to 1.00 map units) and to a lesser extent from the left terminus of the unique long (UL) region (0.00 to 0.05 map units). None of the EHV-1 DNA sequences associated to date with EHV-1 oncogenesis (0.32 to 0.38 map units; O'Callaghan et al. in B. Roizman [ed.], Herpesviruses, in press; Robinson et al., Cell 32:204-219, 1983, and Proc. Natl. Acad. Sci., U.S.A., 78:6684-6688, 1981) were detected in the DI particle DNA. The importance of these data with regard to DNA replication of DI particles and the role of DI particles in one model system of EHV-1 oncogenic transformation are discussed.

Animals↗
PubMed1983

Potentiation of factor H by heparin: a rate-limiting mechanism for inhibition of the alternative complement pathway.

The mechanism by which heparin inhibits the alternative complement pathway (ACP) by a fluid-phase activator, CoVF, has been studied. Results presented here indicate that heparin's major (rate-limiting) effect on the fluid-phase activation of the ACP was to potentiate Factor H activity. Such an effect results in a very efficient inhibition of C3b and C3bBb function and restriction of subsequent complement activation and hemolytic activity. Evidence was obtained to indicate that soluble heparin H shifted anodally. Assuming that the rate-limiting inhibitory effect of heparin is to potentiate Factor H, then C3-converting complexes such as CoVF-Bb, which do not require C3b for activity, should not be effected by heparin. Indeed, the inhibitory effect of heparin on C3 conversion in EGTA-Mg2+ serum-CoVF mixtures was lost with a prolonged incubation time (i.e. 60-90 min at 37 C). This finding indicated that with time ACP-mediated cleavage of C3 was able to bypass the heparin-mediated inhibitory step. From these studies it is suggested that heparin restricts the C3-converting activity due to soluble C3bBb complexes but not the C3 conversion due to CoVF-Bb complexes. Heparin-mediated restriction of the ACP activation by CoVF was used to calculate the relative percentages of C3 conversion due to C3bBb or CoVF-Bb complexes. In carefully controlled experiments, heparin could not prevent the spontaneous conversion of C3 which occurs upon removing functional Factor H from the sera. Addition of isolated Factor H restored heparin's inhibitory effect on the ACP. Kinetic studies of heparin's inhibition of ACP-mediated lysis of rabbit erythrocytes indicated that heparin's inhibitor functions did not occur until after the addition of an ACP activator. Each of these findings is consistent with the postulate that the major (rate-limiting) effect of heparin on the ACP is to potentiate the function of Factor H on activated C3b.

Complement Activation↗