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Biomedical subjects

S Pestka

Publications and source records attributed to S Pestka.

At least 73 records · Page 4Linked to original sources
PubMed1994

Identification and sequence of an accessory factor required for activation of the human interferon gamma receptor.

Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon gamma (Hu-IFN-gamma), as measured by induction of class I human leukocyte antigen (HLA) and protection against encephalomyocarditis virus (EMCV) infection. Whereas human chromosome 6 encodes the Hu-IFN-gamma receptor, human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-gamma receptor. Probes from a genomic clone were used to identity cDNA clones expressing a species-specific accessory factor. These cDNA clones are able to substitute for human chromosome 21 to reconstitute the Hu-IFN-gamma receptor-mediated induction of class I HLA antigens. However, the factor encoded by the cDNA does not confer full antiviral protection against EMCV, confirming that an additional factor encoded on human chromosome 21 is required for reconstitution of antiviral activity against EMCV. We conclude that this accessory factor belongs to a family of such accessory factors responsible for different actions of IFN-gamma.

Amino Acid Sequence↗
PubMed1994

Sublocalization of the human interferon-gamma receptor accessory factor gene and characterization of accessory factor activity by yeast artificial chromosomal fragmentation.

A chromosomal fragmentation procedure was employed to produce a deletion set of yeast artificial chromosomes (YACs) from a parental YAC, GART D142H8, known to map to human chromosome 21q and to encode the human interferon-gamma receptor (Hu-IFN-gamma R) accessory factor gene as well as the phosphoribosylglycinamide formyltransferase (GART) gene. When expressed in Chinese hamster ovary cells, these deleted YACs retain accessory factor activity, as judged by major histocompatibility complex class I antigen inducibility, until the deletions from the acentric end exceed 390 kilobases (kb). Therefore, the accessory factor (AF-1) gene can be localized to a 150-kb region at the left (centric) end of the parental 540-kb GART YAC. Cells containing functional YACs are also able to induce the ISGF3 gamma and gamma-activated factor (GAF) transcription factors, but were not protected against encephalomyocarditis virus (EMCV) upon treatment with Hu-IFN-gamma. Therefore, the Hu-IFN-gamma R and the AF-1 are sufficient for some, but not all, of the actions of Hu-IFN-gamma. We postulate that an additional accessory factor (AF-2) required for antiviral activity against EMCV is encoded on chromosome 21q.

Acyltransferases↗
PubMed1994

Generation of random internal deletion derivatives of YACs by homologous targeting to Alu sequences.

To facilitate the manipulation of human genomic DNA in yeast artificial chromosome (YAC) clones, a plasmid to integrate the selective marker for antibiotic G418 resistance into YACs and to delete some of the human DNA fragments from YACs was constructed. The linearized integration/deletion plasmid, which contains Alu family sequences at both ends, can recombine with YACs containing human repetitive sequences via homologous recombination. The homologous recombination results in a random integration of the antibiotic G418-resistant gene into a human genomic Alu sequence, and in most cases, an internal deletion within the YAC. The YACs with internal deletions can be useful to identify the location of the genes if they produce functional knockouts. In those cases when the integration/deletion event disrupts the integrity of the gene so it no longer can produce a viable and functional mRNA in fused eukaryotic cells, the site of integration in the YAC thus serves as a marker for the inactivated gene. In this report we describe a model system to locate specific genes in YACs.

Animals↗
PubMed1994

Construction and activity of phosphorylatable human interferon-alpha B2 and interferon-alpha A/D.

The polymerase chain reaction (PCR) was used to introduce a phosphorylation site into human interferon-alpha B2 (Hu-IFN-alpha B2) and the chimeric human interferon-alpha A/D (Hu-IFN-alpha A/D). The phosphorylation sites were created by adding an amino acid consensus sequence for phosphorylation by the cAMP-dependent protein kinase to the carboxyl termini of the IFNs. The resultant modified IFNs (Hu-IFN-alpha B2-P and Hu-IFN-alpha A/D-P) were expressed in Escherichia coli and purified. The purified proteins exhibited antiviral activities similar to that of unmodified Hu-IFN-alpha B2 and Hu-IFN-alpha A/D. The Hu-IFN-alpha B2-P and Hu-IFN-alpha A/D-P can be phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and [gamma-32P]ATP with retention of biological activities. The introduction of phosphorylation sites into Hu-IFN-alpha B2 and Hu-IFN-alpha A/D provides new reagents for studies of receptor binding, pharmacokinetics, and other studies where labeled IFNs are useful.

Amino Acid Sequence↗
PubMed1994

Identification of functional type I and type II interferon receptors.

Although the interferons were the first cytokines purified and cloned, their receptors have remained an enigma as fully functional receptors have not been defined and characterized. We previously discovered that the IFN-gamma receptor (IFN-gamma R) was able to bind IFN-gamma, but required one or more accessory factors to exhibit functional activity. We have now identified a gene and sequence of one of these accessory factors. Both genomic and cDNA clones were used to reconstitute a functional receptor. The presence of the Hu-IFN-gamma R and the human accessory factor-1 (designated AF-1) are both necessary and sufficient to induce MHC class I antigens in response to Hu-IFN-gamma in hamster cells. AF-1 is a transmembrane protein with no significant amino acid homology to other sequences in nucleic acid data bases, but has overall structural homology to the class 2 cytokine receptor family. Whereas total human DNA was used to first isolate a Type I interferon receptor, the only genomic or cDNA clone reported exhibited activity essentially only in response to Hu-IFN-alpha B2 (Hu-IFN-alpha 8). Yet hamster or mouse cells containing human Chromosome 21 seemed to have the full complement of Hu-IFN-alpha activities and responded to all Hu-IFN-alpha species as well as Hu-IFN-beta. To investigate this dilemma, we extended our earlier studies with total human DNA to employ defined YAC clones. We have now isolated a genomic segment of human Chromosome 21 that exhibits the properties expected of the Type I interferon receptor: response to multiple species of Hu-IFN-alpha; and excellent binding of Hu-IFN-alpha A and Hu-IFN-alpha B2 species. Reconstitution of functional Type I and Type II interferon receptors now provides the first steps to link the receptors to the signal transduction mechanisms; and will help to integrate our understanding of the ligand receptor interactions to the molecular signals that activate specific transcription factors that in turn induce specific genes carrying the interferon regulatory elements.

Animals↗
PubMed1993

Identification of a yeast artificial chromosome clone encoding an accessory factor for the human interferon gamma receptor: evidence for multiple accessory factors.

Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon gamma (Hu-IFN-gamma), as measured by the induction of human HLA class I antigen. Human chromosome 6 encodes the receptor for Hu-IFN-gamma, and human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-gamma receptor. A small region of human chromosome 21 that is responsible for encoding such factors was localized with hamster-human somatic cell hybrids carrying an irradiation-reduced fragment of human chromosome 21. The cell line with the minimum chromosome 21-specific DNA is Chinese hamster ovary 3x1S. To localize the genes further, 10 different yeast artificial chromosome clones from six different loci in the vicinity of the 3x1S region were fused to a human-hamster hybrid cell line (designated 16-9) that contains human chromosome 6q (supplying the Hu-IFN-gamma receptor) and the human HLA-B7 gene. These transformed 16-9 cells were assayed for induction of class I HLA antigens upon treatment with Hu-IFN-gamma. Here we report that a 540-kb yeast artificial chromosome encodes the necessary species-specific factor(s) and can substitute for human chromosome 21 to reconstitute the Hu-IFN-gamma-receptor-mediated induction of class I HLA antigens. However, the factor encoded on the yeast artificial chromosome does not confer antiviral protection against encephalomyocarditis virus, demonstrating that an additional factor encoded on human chromosome 21 is required for the antiviral activity.

Animals↗
PubMed1993

A simple and efficient method for site-directed mutagenesis with double-stranded plasmid DNA.

A general, simple and efficient method for preparing site-specific mutations in double-stranded plasmid DNA without the need for special plasmids, bacterial strains or reagents is described. Only one synthetic oligonucleotide for each mutation is required, subcloning is unnecessary and a high efficiency of mutation (58-97%) was obtained. If two synthetic oligonucleotide primers are used, two separate mutations can be simultaneously created in a single reaction tube.

Base Sequence↗
PubMed1993

Human interferon-alpha A, -alpha 2, and -alpha 2(Arg) genes in genomic DNA.

The frequency in human genomic DNA of three human interferon (IFN) alpha genes which differ from each other by a single base substitution was examined in 11 normal individuals. The polymerase chain reaction (PCR) was used to amplify the coding sequence for the Hu-IFN-alpha 2, Hu-IFN-alpha A, and Hu-IFN-alpha 2(Arg) sequences from genomic DNA. The PCR products were then cloned and individual clones were sequenced. PCR products were also analyzed by restriction endonuclease analysis for the IFN-alpha 2 and IFN-alpha A genes by use of a HinfI site which is eliminated by the substitution of an A for a G in IFN-alpha A. The IFN-alpha A gene which was cloned from the myeloblastoid cell line KG-1 was not observed in any of the 201 clones sequenced from normal individuals or in the Namalwa cell line. It was detected in KG-1 cell genomic DNA where it represented 49% of the clones sequenced. Similarly the IFN-alpha 2(Arg) gene was not detected in normal individuals or in the KG-1 cell line but only in the lymphoblastoid cell line from which it was cloned. In Namalwa cells the IFN-alpha 2(Arg) sequence represented 35% of the clones sequenced while the IFN-alpha 2 sequence comprised 59%. Therefore, both the IFN-alpha A and IFN-alpha 2(Arg) sequences represent alleles of the IFN-alpha 2 gene.

Base Sequence↗
PubMed1993

Anti-rhinoviral activity of recombinant and hybrid species of interferon alpha.

To define further differences in antiviral activity as well as to identify candidate interferons for study in the prevention of rhinovirus colds, the antiviral activities of nine species of recombinant interferon alpha (IFN-alpha A, IFN-alpha B, IFN-alpha C, IFN-alpha D, IFN-alpha J, [Ser-116]IFN-alpha J1, IFN-alpha K, IFN-alpha J/C(Fnu4HI), and IFN-alpha A/D(BglII)) were evaluated against rhinovirus types 39 (RV 39) and 1A (RV 1A). WI-38 cells were exposed to various concentrations of each interferon and were then infected with RV 39, RV 1A, or VSV. Efficacy was determined by protection from cytopathic effect using a tetrazolium dye assay. The 50% inhibitory concentrations ranged from 4 +/- 3 pg/ml for IFN-alpha C to > 3000 pg/ml for IFN-alpha D against RV 39, and from 6 +/- 4 pg/ml for IFN-alpha J/C(Fnu4HI) to > 3000 pg/ml for IFN-alpha D against RV 1A. IFN-alpha J/C(Fnu4HI), [Ser-116]IFN-alpha J1, and IFN-alpha C were the most active of the interferons, and were all more active than IFN-alpha A, against RV 39, RV 1A, and VSV. These interferons warrant further study against rhinoviruses and other viruses.

Amino Acid Sequence↗
PubMed1993

Amplification of specific gene products from human serum.

The polymerase chain reaction (PCR) is an in vitro method for the primer-directed enzymatic amplification of specific DNA sequences. Ordinarily, sources with obvious DNA content such as cells, viruses, and plasmids serve as the origin of templates for the PCR. Here we report a simple and efficient method to obtain cellular DNA from serum suitable for use in PCR reactions or gene analysis. This procedure should facilitate the detection of disease and provide a basis for the examination of mutations in genes before the onset as well as during the progression of various diseases.

Base Sequence↗
PubMed1993

Yeast artificial chromosome fragmentation vectors that utilize URA3 selection.

Two fragmentation vectors, pSE1 and pSE2, were developed for targeting yeast artificial chromosomes (YACs) containing human genomic DNA. Ura- yeast cells containing YACs were selected with 5-fluoro-orotic acid. Fragmented YACs were subsequently generated by transformation to a Ura+ phenotype. Over 80% of the transformants contained YACs of reduced molecular size. These fragmented YACs will prove to be useful in mapping the region of human chromosomes covered by the parental YAC. Fragmentation utilizing URA3 transformation provides a method for producing YAC deletion sets from YACs contained in AB1380 and other ura3- yeast stains. Linkage of a neomycin resistance gene to the URA3 gene facilitates functional analysis of these YACs in eukaryotic cells.

Acyltransferases↗
PubMed1993

Low-dose oral recombinant interferon-alpha A in patients with HIV-1 infection: a blinded pilot study.

OBJECTIVE: To evaluate the efficacy of low-dose oral recombinant interferon-alpha (IFN-alpha A) on clinical parameters, body weight, CD4+ lymphocyte counts and natural killer cell cytolytic activity in HIV-infected patients. DESIGN: Blinded crossover trial with controls for the protein and diluent components of the drug preparation. SETTING: Medical school outpatient referral center. PATIENTS, PARTICIPANTS: Eight patients with HIV-1 infection and a CD4+ lymphocyte count between 150 and 600 x 10(6)/l. Concurrent use of zidovudine was permitted. INTERVENTIONS: Patients received (daily, by mouth) 10 ml of a study solution of 2.5% albumin for 6 weeks, 150 IU IFN-alpha A for 6 weeks, and normal saline for 6 weeks. MAIN OUTCOME MEASURES: After two baseline visits, clinical assessments, vital signs, body weight, and laboratory tests, including enumeration of number and percentage of CD4+ and CD8+ lymphocytes and natural killer cell cytolytic activity, were performed every 3 weeks. Complete physical examinations were conducted every 6 weeks. RESULTS: No significant clinical or laboratory changes were observed during treatment with IFN-alpha A. Peak CD4+ lymphocyte counts were achieved at baseline in one patient, during albumin treatment in two patients, during IFN-alpha A treatment in one patient, and during saline treatment in four patients. All patients remained HIV-seropositive. Treatments were well-tolerated. CONCLUSION: This blinded pilot study of orally administered IFN-alpha A (150 IU daily for 6 weeks) did not demonstrate clinical benefit in HIV-infected patients.

Administration, Oral↗
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↗