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S Pestka

Publications and source records attributed to S Pestka.

At least 19 recordsLinked to original sources
PubMed1992

Structural analysis of the human interferon gamma receptor: a small segment of the intracellular domain is specifically required for class I major histocompatibility complex antigen induction and antiviral activity.

Mutations of the human interferon gamma (IFN-gamma) receptor intracellular domain have permitted us to define a restricted region of that domain as necessary for both induction of class I major histocompatibility complex antigen by IFN-gamma and protection against encephalomyocarditis virus. This region consists of five amino acids (YDKPH), all of which are conserved in the human and murine receptors. Tyr-457 and His-461 are essential for activity. Approximately 80% of the amino acids of the intracellular domain of the receptor is not required for major histocompatibility complex class I antigen induction or for antiviral protection against encephalomyocarditis virus. The observation that there was no protection by IFN-gamma against vesiculostomatitis virus indicates that other factors, in addition to chromosome 21 accessory factor(s), are required to generate the full complement of transduction signals from the human IFN-gamma receptor.

Amino Acid Sequence↗
PubMed1992

Antisense RNA. History and perspective.

Antisense RNA was first an in vitro curiosity that was found to shut off protein synthesis in cell-free extracts. It was later shown to function in prokaryotic cells as a natural modulator of the synthesis of some proteins. Artificial antisense constructs can inhibit protein synthesis in prokaryotic and eukaryotic cells. To inhibit synthesis of proteins effectively, high ratios of antisense to sense RNAs are required. Thus, the challenge is to develop strategies to locate suitable targets and provide for amplification of the antisense RNA. This report provides a summary of our original work on antisense RNA.

History, 20th Century↗
PubMed1992

Chimeric interferon-gamma receptors demonstrate that an accessory factor required for activity interacts with the extracellular domain.

We determined the species specificity and function of structural domains of the interferon-gamma receptor (IFN-gamma R) by construction of human/murine chimeric IFN-gamma R cDNA clones and their expression in various cells. We demonstrate that we can reconstitute a biologically active IFN-gamma R in eukaryotic cells with chimeric receptors as long as the extracellular domain and an accessory factor are from the same species. These results indicate that the extracellular domain of the receptor interacts directly or indirectly with the species-specific accessory factor.

3T3 Cells↗
PubMed1992

Engineered disulfide bond greatly increases specific activity of recombinant murine interferon-beta.

Unlike other species of interferon-beta (IFN-beta) mouse (Mu) IFN-beta has no naturally occurring intramolecular disulfide bond. When expressed in Escherichia coli, MuIFN-beta appears to exhibit instability and low activity. To increase its activity, we engineered a pair of cysteines into recombinant MuIFN-beta to test whether this change would improve its antiviral activity. In the absence of detailed structural data, the optimal placement of cysteines was determined by sequence comparison with other species of IFN-beta. While MuIFN-beta has only a single cysteine (at position 17), other species of IFN-beta have three cysteines, at positions 17, 31, and 141 (numbering based on consensus sequence), a disulfide bond being formed between the latter two residues. Thus, we introduced cysteines into MuIFN-beta at positions 29 and 136, which correspond to positions 31 and 141 of the consensus sequence. When the variant form of MuIFN-beta was expressed in bacteria and purified, we found that the additional cysteines greatly increased the antiviral activity. Further improvement was obtained by replacement of the Cys-17 with a serine. In this manner a specific activity approximately 15-fold that of the wild-type recombinant molecule was achieved.

Animals↗
PubMed1992

Anti-HIV-1 activity of recombinant and hybrid species of interferon-alpha.

To identify candidate interferons (IFNs) for the treatment of human immunodeficiency virus type 1 (HIV-1) infection and to investigate sequence-function relationships, the antiviral activities of nine species of recombinant IFN-alpha [IFN-alpha A, IFN-alpha B, IFN-alpha C, IFN-alpha D, IFN-alpha J, [Ser116]IFN-alpha J1, IFN-alpha K, IFN-alpha J/C(Fnu4HI), and IFN-alpha A/D(BglII)] were evaluated against HIV-1. MT-2 cells were exposed to various concentrations of each IFN and were then infected with HIV. Protective effect was determined by cell viability using a tetrazolium dye assay. Activity against vesicular stomatitis virus (VSV) was assessed on MDBK and WISH cells. The 50% inhibitory concentration against HIV was 37 +/- 14 pg/ml for IFN-alpha A, and ranged from 15 +/- 3 pg/ml for IFN-alpha J/C(Fnu4HI) to > 90,000 pg/ml for IFN-alpha D. In general, relative activity against HIV was similar to relative activity against VSV on WISH cells. IFN-alpha D was notable for its decreased activity on human cells. The observations suggest that it may be possible to produce IFNs-alpha with more favorable therapeutic indices than currently available IFNs. Furthermore, the anti-HIV activity of IFNs-alpha is not determined solely by their linear amino acid sequence.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Human interferon omega (omega) binds to the alpha/beta receptor.

It was proposed that human interferon omega (omega) binds to the interferon alpha/beta receptor but not to the interferon gamma receptor. However, since no studies were performed to provide direct evidence for this hypothesis, we carried out cross-linking experiments and saturation binding assays between a 32P-labeled human interferon-alpha (Hu-IFN-alpha) and unlabeled Hu-IFN-alpha A, -beta, -gamma, and -omega. These assays demonstrated that Hu-IFN-alpha A, -beta, and -omega, but not Hu-IFN-gamma, were able to block binding of 32P-labeled Hu-IFN-alpha A to human cells. These results indicate that Hu-IFN-omega binds to the alpha/beta receptor.

Binding, Competitive↗
PubMed1991

Expression and reconstitution of a biologically active mouse interferon gamma receptor in hamster cells. Chromosomal location of an accessory factor.

In order to localize the chromosome encoding the accessory factor required for function of the murine interferon gamma (IFN-gamma) receptor, we transfected a cDNA expression vector encoding the receptor into several Chinese hamster x mouse somatic cell hybrids. As mouse Chromosome 10 carries the gene which encodes the interferon gamma-receptor (Ifgr), we used somatic cell hybrids that lack this chromosome. The presence of mouse Chromosome 16 was required to generate a response to murine IFN-gamma as assayed by stimulation of class I major histocompatibility complex antigen expression. These results demonstrate a species-specific accessory factor encoded on mouse Chromosome 16 (termed Ifgt) is necessary for mouse IFN-gamma to stimulate major histocompatibility complex expression through its receptor.

Animals↗
PubMed1990

Alpha-interferon structure and natural killer cell stimulatory activity.

Expression vectors for human alpha-interferon (Hu-IFN-alpha) J1, a site-specific mutant [Ser116]Hu-IFN-alpha J1, and Hu-IFN-alpha J/C or Hu-IFN-alpha C/J hybrids were constructed and expressed in Escherichia coli. These interferons and others were purified by immunoaffinity chromatography with a monoclonal antibody against human alpha-interferon. Their antiviral activity and ability to stimulate natural killer cell activity were determined in comparison to several other human interferons. These results provide some insight into structure-activity relationships for stimulating natural killer cells and confirm our previous conclusions that antiviral activity cannot be used to predict other activities for an individual IFN-alpha species. The observations suggest that the tertiary structure rather than any specific linear sequence of amino acids regulates the ability of the interferons to stimulate natural killer cell activity.

Amino Acid Sequence↗
PubMed1990

Expression and reconstitution of a biologically active human interferon-gamma receptor in hamster cells.

We report the expression of the human interferon-gamma (Hu-IFN-gamma) receptor from a cDNA clone in an animal cell where both stable expression of the cloned receptor as well as its biological activity is demonstrated for the first time. Biological activity of the receptor (i.e. expression of surface histocompatibility antigens in response to human interferon-gamma) requires the presence of human chromosome 21 demonstrating the requirement for at least one other species-specific factor in the modulation of receptor action. This system should now facilitate delineation of regions involved in the binding of human interferon-gamma and receptor signal transduction.

Animals↗
PubMed1990

Augmentation of tumor antigen expression by recombinant human interferons: enhanced targeting of monoclonal antibodies to carcinomas.

The use of Mabs for the detection and treatment of human carcinoma lesions can still be regarded in its infancy. As with other new approaches to cancer therapy, several conceptual as well as real problems exist when designing clinical protocols for Mab-directed immunotherapy. From the Mab standpoint, studies using the intact IgG have shown that, in a majority of patients injected with IgG, human anti-mouse IgG antibodies develop that hamper the effectiveness of subsequent antibody administration. It is believed that the human anti-mouse antibody response is directed against the Fc region of the IgG molecule. The elimination of this region through fractionation of the Mab to obtain the minimum binding site could result in a less immunogenic molecule. Another approach aimed at reducing the immunogenicity of the Mab would be to clone the genes encoding for individual Mabs, reduce them via restriction endonuclease techniques, and insert human immunoglobulin constant regions. The resulting chimeric antibodies are believed to reduce the development of human anti-mouse antibodies. Effective Mab therapy of human tumor lesions may also be achieved through the recruitment of a portion of the host's immunologic defense system. An example is the use of anti-idiotype Mabs that use as immunogen a Mab to a tumor antigen. The anti-idiotype antibodies are selected for binding to the antigen binding, or idiotype, region of the first Mab. The binding sites of the new anti-idiotype Mabs should reflect the 'internal image' of the original antigen. The anti-idiotype antibodies may be used to immunize patients (i.e., vaccines) in an attempt to mount an active immune response against the antigen-positive tumor cells. Recent studies have shown a synergism between interferon-alpha and an anti-idiotype Mab for the in-vivo antitumor activity in a murine B-cell lymphoma experimental model. Whether an interferon-mediated increase in the tumor antigen or the Fc receptor was part of the synergism was not investigated. Mabs alone have also been shown to elicit cytotoxic activity in vitro and tumoricidal activity in vivo. Antibodies of the IgG2a isotype can direct macrophage-mediated cytotoxicity. These studies revealed the importance of the number of antibody sites per cell as well as the number of cells that bind the IgG2a Mab, thus suggesting a 'threshold' requirement for the demonstration of effective tumor cell lysis in vitro and in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗
PubMed1990

Hybrid selection of mRNA with biotinylated DNA.

A novel, convenient, and highly efficient hybrid selection procedure is described. The method utilizes the polymerase chain reaction (PCR) in which one of two primers is biotinylated at the 5'-terminus. The concentration of the biotinylated primer is 100 times that of the other to synthesize biotinylated single-stranded DNA (asymmetric PCR). After hybridization of the biotinylated DNA with mRNA in solution, streptavidin agarose is used to trap the hybrid duplex of mRNA.DNA-biotin onto the solid matrix. The selected mRNA is then eluted from the streptavidin agarose. The quantitative physical recovery of selected mRNA is about 70% with about 33% retention of biological activity.

Animals↗
PubMed1990

Molecular genetic markers spanning mouse chromosome 10.

Somatic cell hybrids, recombinant inbred (RI) strains, and progeny of an intersubspecific backcross were typed by Southern blot analysis to prepare a linkage map of mouse chromosome 10. The seven genetic markers in this map, four of which had not previously been positioned, include genes involved in oncogenesis (Gli, Myb, Tra-1), proviral integration (Emv-25), and immune responses (Ifg, Ifgr, Pfp). The linkage map spans much of the chromosome and covers a region of the mouse genome with few molecular markers. The gene order established here demonstrates that the genes for murine interferon-gamma (Ifg) and its receptor (Ifgr) are at opposite ends of the chromosome and that Ifgr and the Myb oncogene are closely linked, a factor that may be related to their joint transcriptional enhancement in some plasmacytoid lymphosarcomas.

Animals↗