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R R Golgher

Publications and source records attributed to R R Golgher.

At least 19 recordsLinked to original sources
PubMed2001

N-terminal amino acids of bovine alpha interferons are relevant for the neutralization of their antiviral activity.

The structure-function relationship of interferons (IFNs) has been studied by epitope mapping. Epitopes of bovine IFNs, however, are practically unknown, despite their importance in virus infections and in the maternal recognition of pregnancy. It has been shown that recombinant bovine (rBo)IFN-alphaC and rBoIFN-alpha1 differ only in 12 amino acids and that the F12 monoclonal antibody (mAb) binds to a linear sequence of residues 10 to 34. We show here that the antiviral activities of these two IFNs were neutralized by the F12 mAb to different extents using two tests. In residual activity tests the antiviral activity dropped by more than 99% with rBoIFN-alphaC and by 84% with rBoIFN-alpha1. In checkerboard antibody titrations, the F12 mAb titer was 12,000 with rBoIFN-alphaC and only 600 with rBoIFN-alpha1. Since these IFNs differ in their amino acid sequence at positions 11, 16 and 19 of the amino terminus, only these amino acids could account for the different neutralization titers, and they should participate in antibody binding. According to the three-dimensional structure described for human and murine IFNs, these amino acids are located in the alpha helix A; amino acids 16 and 19 of the bovine IFNs would be expected to be exposed and could bind to the antibody directly. The amino acid at position 11 forms a hydrogen bond in human IFNs-alpha and it is possible that, in bovine IFNs-alpha, the F12 mAb, binding near position 11, would disturb this hydrogen bond, resulting in the difference in the extent of neutralization observed.

Amino Acid Sequence↗
PubMed1999

Quantitative liquid-phase chemiluminescence ELISA: detection of subtle epitope differences in HuIFN-beta.

We have developed a new liquid-phase, chemiluminescence-enhanced, inhibition ELISA (LP-CEI-ELISA) to explore the binding sites recognized by two neutralizing monoclonal antibodies (mAb) against recombinant human IFN-(beta)ser (rHuIFN-(beta)ser). In this assay, the initial antigen-antibody reaction occurs in solution under more physiologic conditions than in a standard solid-phase ELISA. Subsequently, the reaction mixture is applied to a membrane that is exposed to a second, peroxidase-labeled mAb, chemiluminescent reagents are added, and the membrane is photographically recorded. Competitive inhibition of binding of a second, labeled mAb by the first mAb decreases the signal detected. Two well-characterized mAb A1 and A7, have been shown to recognize distinct epitopes on rHuIFN-(beta)ser and to neutralize its antiviral and antiproliferative activity (Proc. Natl. Acad. Sci. USA 88, 4040-4044, 1991). In conventional solid-phase ELISA, mAb A1 does not inhibit the binding of A7 to rHuIFN-(beta)ser, but we observed partial inhibition in the new liquid-phase assay. In contrast, A7 did not inhibit the binding of A1, consistent with the solid-phase ELISA results. This observation suggests that in the LP-CEI-ELISA, A1 and A7 may recognize epitopes differently than in solid-phase assays. Thus, the LP-CEI-ELISA, which is simple, sensitive, and quantifiable, appears also to be able to detect subtle, conformational differences of epitopes not evident in a standard solid-phase ELISA.

Binding, Competitive↗
PubMed1998

Morphological and molecular characterization of the poxvirus BeAn 58058.

BeAn 58058 virus (BAV) was isolated from an Oryzomis rodent in Brazil. BAV was shown to be antigenically related to another poxvirus also isolated in Brazil, the Cotia virus, but it remained ungrouped. Electron microscopy revealed that BAV has a typical poxvirus morphology. The Hind III DNA profile of BAV genome was similar with that of VV WR and Lister, but some differences in the profile were detected. We have also detected the presence of genes homologous to vaccinia virus (VV WR) genes in the genome of BAV. Genes related to vaccinia thymidine kinase (TK) gene and vaccinia growth factor (VGF) gene were found. The patterns of TK and VGF mRNA transcripts described for vaccinia virus infected cells were observed in BAV infected cells. Nucleotide sequence of BAV VGF homologous gene was similar to VV WR VGF sequences. This similarity was further seen when cross-hybridization of total genomes of BAV and VV was done. Polypeptide synthesis of BAV and vaccinia in infected cells also showed similar profiles. The genetic data was used to construct a phylogenetic tree where BAV and VV were placed at the same cluster. Based on our findings we propose that BAV is a vaccinia virus variant.

Amino Acid Sequence↗
PubMed1998

Culture of human amniotic cells: a system to study interferon production.

This study investigated whether primary culture of human amniotic membrane cells (PCHAM) could be used as an in vitro model system for the study of interferon (IFN) production. PCHAM cells infected with Newcastle disease virus (NDV) produced the two antigenic types of IFN, previously shown in a amniotic membrane cells (HAM) system. PCHAM IFN was detected as early as 2 h after NDV infection and was composed by two antigenically distinct fractions, one neutralized with anti-HuIFN beta antibody and another that is not related to IFN beta, -alpha and -gamma. These fractions correspond respectively to 80 and 20 per cent of the IFN produced 4 h after virus induction, 55 and 45 per cent of the IFN produced from 4 to 12 h and 67 and 33 per cent of the IFN produced 12 h after virus induction. A cDNA library, established from PCHAM with or without NDV infection, was screened for IFN alpha and -beta using specific primers. The PCR product, amplified by IFN beta primers, was cloned, sequenced and expressed in Escherichia coli M15. The sequences of several cloned cDNAs were identical to HuIFN beta gene and the antiviral activity of the expressed protein was neutralized only by antiHuIFN-beta antibody. The other IFN fraction not neutralized by polyclonal antibodies anti-IFN beta, -alpha and -gamma is now being studied.

Amnion↗
PubMed1996

Distinct antigenic subtypes of human beta interferon can be distinguished by neutralization.

Different molecular configurations of human beta interferon were titrated with the standard reference antiserum of the National Institutes of Health (NIH) which had been prepared with natural beta fibroblast interferon in order to determine to what extent differences in these configurations would influence the neutralization of the antiviral action of interferon. Neutralization tests were carried out in Vero cells by diluting both interferon and antiserum. Encephalomyocarditis virus was employed as challenge virus. The neutralization titer was considered to have been reached when the effect of eight units of interferon was reduced to one. Two natural beta interferons prepared from fibroblasts and from amniotic membranes gave similar high titers. However, titers were reduced five-fold with recombinant interferons expressed in Escherichia coli, which do not contain carbohydrate, one with the natural sequence and a mutant with a single amino acid substitution (cysteine for serine). The NIH antiserum did not neutralize the effect of a protein fraction from amniotic membranes antigenically different from the human alpha, beta or gamma interferons but having the biological activity of interferon. We conclude that the carbohydrate moieties of human beta interferons are essential for their recognition by the NIH antiserum and that antibodies specific for human recombinant beta interferon, which does not contain carbohydrate, are needed.

Amniotic Fluid↗
PubMed1991

Partial characterization of human amniotic membrane interferon.

1. The molecular weight and the electrofocusing profile of human amniotic membrane interferon (IFN-AM) were determined. 2. When submitted to gel filtration, IFN-AM showed a single 26-28 kDa component; in polyacrylamide gel electrophoresis one component of 19,500. 3. In electrofocusing, IFN-AM displayed a heterogeneous profile with 5 to 7 peaks, but different from human alpha or beta IFNs. This heterogeneity was reduced by previous treatment of IFN-AM with neuraminidase. 4. IFN-AM is a sialoglycoprotein similar to human beta IFN in terms of antigenicity but different from it in electrofocusing profile.

Chromatography, Gel↗
PubMed1989

Mill Hill strain of parainfluenza 1 virus is a potent human interferon inducer.

The interferon (IFN)-inducing capacities of the widely used Cantell strain of Sendai and the Mill Hill strain of parainfluenza 1 were compared. The Mill Hill strain proved to be a more potent inducer of IFN than the Cantell strain when tested in human peripheral blood leukocytes and amniotic membrane cultures. The Mill Hill strain did not produce defective particles readily, nor did these particles appear necessary for IFN induction. The Mill Hill strain of parainfluenza 1 virus appears superior to the Cantell strain of Sendai virus for the production of natural human IFN.

Amnion↗
PubMed1989

Antigenic characterization of human interferon derived from amniotic membranes induced by virus.

The presence of protein(s) with interferon (IFN)-like activity in culture fluid from human amniotic membranes induced by viruses has been described by different groups. However, the antigenic structure of this protein is controversial. Here we report the presence of IFN activity in supernatants of human amniotic membranes induced by Sendai virus. The major component responsible for this antiviral activity seems to be the classical IFN-beta. However, we were able to demonstrate the presence of a protein fraction with antiviral activity that does not bind to an affinity column for IFN-beta. The antiviral activity of this unbound fraction cannot be neutralized by antibodies to IFN-alpha, -beta, gamma, or by a mixture of them. We called this unbound fraction IFN-AM. We also report the development of a monoclonal antibody that does not neutralize the antiviral activity of IFN-alpha or IFN-beta but reduces the antiviral activity of a partially purified preparation of Sendai virus-induced amniotic membrane supernatant. These observations suggest that the IFN-AM (the unbound fraction that lacks reactivity with antibodies against known IFNs) contains a unique antigenic determinant that is not present, or, if so, is not located at the functional domain of IFN-alpha, -beta, or -gamma.

Amnion↗
PubMed1981

Affinity chromatography of primary human amnion interferon.

The chromatographic behavior of human amniotic interferon on various affinity chromatography ligands was studied. Most of this interferon bound strongly to bovine plasma albumin-agarose, cibacron blue F3GA-agarose, concanavalin A-agarose and L-tryptophyl-L-tyrosine-omega-carboxyl-pentyl-agarose. After binding most of the interferon activity was eluted only with 50 percent ethylene glycol, showing the high hydrophobicity of this interferon. Smaller quantities could be recovered after phosphate-buffered saline elution or with increased salt concentration. On BPA-omega-carboxy-pentyl-agarose and omega-amino-hexyl-agarose, the majority of the biological activity was found in the break-through fraction (eluted with phosphate buffered saline) while some interferon was displaced with high salt or ethylene glycol. Increasing the salt concentration and lowering the pH was necessary to elute interferon from zinc chelate-agarose. These patterns indicate that human amniotic interferon is similar to human fibroblast (beta) interferon but different from human leukocyte (alpha) interferon. However, the heterogeneity displayed by amniotic interferon on bovine plasma albumin-agarose requires further investigation.

Amnion↗
PubMed1980

Sensitivity of group C arboviruses (bunyaviridae) to human amnion interferon.

The sensitivity of several group C arboviruses (Bunyaviridae) to human amnion interferon was compared to that of vesicular stomatitis virus (VSV) and Sindbis virus. In CPE inhibition assays. Apeu virus was the most sensitive of the group C arboviruses tested; it was significantly more inhibited than VSV, but was in the same range as Sindbis virus. In plaque reduction assays, the increasing order of sensitivity was Apeu, Marituba, VSV and Sindbis viruses. Single-cycle yields of VSV and Apeu virus were reduced to the same extent with 1-10 interferon units; with 230 units, VSV growth was inhibited to a much greater extent (100-fold) than Apeu virus.

Amnion↗
PubMed1979

Natural occurrence of 2-5A in interferon-treated EMC virus-infected L cells.

Until now the interferon-mediated 2'-5' adenine oligonucleotide inhibitors (2-5A) of cell-free protein synthesis have not been detected in intact cells. Here we report their natural occurrence in interferon-treated, EMC virus-infected mouse L cells in amounts consistent with the idea that they play a part in the inhibition of virus growth.

Adenine Nucleotides↗
PubMed1979

Assay of human interferon in Vero cells by several methods.

Four methods for the assay of human interferon in Vero cells were compared based on the inhibition of viral cytopathic effect (CPE) in tubes, the inhibition of CPE in microplates, the reduction of plaques, and the inhibition of quantitative hemadsorption. For inhibition of CPE, Sindbis virus, vesicular stomatitis virus, poliovirus type 2, and vaccinia virus were used for challenge. In the plaque reduction method, Sindbis virus, vesicular stomatitis virus, and poliovirus were employed, and Newcastle disease virus was used in the quantitative hemadsorption assay. Sindbis virus was most susceptible to interferon in those tests measuring inhibition of CPE, but vesicular stomatitis virus was as sensitive in the plaque reduction method. Highest titers of interferon were recorded in microplates, especially with Sindbis virus as the challenge agent, followed by the quantitative inhibition assay. The CPE inhibition method was the simplest, and the quantitative hemadsorption assay was the most rapid to perform. Reproducibilities, as shown by the coefficient of variation, were 15, 39, and 59% for plaque reduction, CPE inhibition in tubes, and CPE inhibition in microplates, respectively.

Animals↗
PubMed1977

Intraspecific variation in Trypanosoma cruzi: effect of temperature on the intracellular differentiation in tissue culture.

Inhibition of T. cruzi amastigote-trypomastigote differentiation in tissue culture at 37 C is a strain-dependent event. When eight T. cruzi strains were submitted to two environmental temperatures (33 and 37 C), the following patterns of differentiation were obtained: in three strains, transformation was inhibited at 37 C but readily occurred at 33 C; in three other strains differentiation took place at both temperatures; finally, in the two remaining strains, a partial inhibition was detected at 37 C. The authors discuss the meaning of this intraspecific variation and the possible relationship with the occurrence of temperature-sensitive mutants among protozoa.

Animals↗