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D Pennica

Publications and source records attributed to D Pennica.

At least 73 records · Page 4Linked to original sources
PubMed1991

Protection against endotoxic shock by a tumor necrosis factor receptor immunoadhesin.

Tumor necrosis factors (TNF) alpha and beta are structurally related cytokines that mediate a wide range of immunological, inflammatory, and cytotoxic effects. During bacterial infection of the bloodstream (sepsis), TNF-alpha induction by bacterial endotoxin is thought to be a major factor contributing to the cardiovascular collapse and critical organ failure that can develop. Despite antibiotic therapy, these consequences of sepsis continue to have a high mortality rate in humans. Here we describe a potent TNF antagonist, a TNF receptor (TNFR) immunoadhesin, constructed by gene fusion of the extracellular portion of human type 1 TNFR with the constant domains of human IgG heavy chain (TNFR-IgG). When expressed in transfected human cells, TNFR-IgG is secreted as a disulfide-bonded homodimer. Purified TNFR-IgG binds to both TNF-alpha and TNF-beta and exhibits 6- to 8-fold higher affinity for TNF-alpha than cell surface or soluble TNF receptors. In vitro, TNFR-IgG blocks completely the cytolytic effect of TNF-alpha or TNF-beta on actinomycin D-treated cells and is markedly more efficient than soluble TNFR (24-fold) or monoclonal anti-TNF-alpha antibodies (4-fold) in inhibiting TNF-alpha. In vitro, TNFR-IgG prevents endotoxin-induced lethality in mice when given 0.5 hr prior to endotoxin and provides significant protection when given up to 1 hr after endotoxin challenge. These results confirm the importance of TNF-alpha in the pathogenesis of septic shock and suggest a clinical potential for TNFR-IgG as a preventive and therapeutic treatment in sepsis.

Animals↗
PubMed1991

Measurement of transient cDNA expression in mammalian cells using flow cytometric cell analysis and sorting.

Generalized methods for quantitative and sensitive measurement of transient cDNA expression in mammalian cells using flow cytometry (FCM) are described. The techniques are applicable to a wide variety of cDNAs encoding intracellular or cell surface protein products through the use of immunofluorescence- or nonimmunofluorescence-based detection methods. The methods illustrated have been optimized for sensitive detection of transfectants and efficient recovery of the encoding plasmids from the sorted cells. Expression levels and heterogeneities were compared using four methods of DNA transfer in addition to description of a novel method to optimize single copy transfer probabilities by multiparameter analysis. The overall sensitivities are compared by reconstruction and molecular cloning experiments to other methods of selection, such as immunoselection by panning. Through the measurement of multiple heterologous products per cell, or the measurement of multiple epitopes or binding sites per heterologous protein, expression levels on a single cell basis can be measured and correlated with other endpoints for various purposes. The ability to detect and recover rare clones based on a number of single and multiparameter selection criteria should significantly extend the use of transient mammalian cDNA expression methods for applications involving novel FCM-based reporter cDNA assays and for cloning certain rare surface-bound or secreted proteins using FCM.

Animals↗
PubMed1991

The extracellular domain of the human interferon gamma receptor interacts with a species-specific signal transducer.

At least two species-specific gene products are required for signal transduction by interferon gamma (IFN-gamma). The first is the IFN-gamma receptor, which binds ligand with high affinity in a species-specific manner. The second is an undetermined species-specific signal transducer(s). To determine whether the human IFN-gamma receptor (hIFN-gamma R) interacts directly with this signal transducer(s) and, if so, with what functional domain(s), we constructed expression vectors for the hIFN-gamma R and three hybrid human-murine IFN-gamma receptors. The hybrid receptors contained the extracellular, human IFN-gamma (hIFN-gamma) binding domain of the hIFN-gamma R, either the human or murine transmembrane domain, and either the human or murine intracellular domain. The vectors encoding these receptors were stably transfected into two mouse cell lines, one of which (SCC-16-5) contains a single copy of human chromosome 21. The resulting cell lines were treated with hIFN-gamma, and murine major histocompatibility complex class I antigen expression was analyzed by immunofluorescence flow cytometry. All transfected cell lines lacking human chromosome 21 remained insensitive to hIFN-gamma. However, all four of the IFN-gamma receptors were able to signal when expressed in the cell line containing human chromosome 21. We conclude that the extracellular domain of the IFN-gamma receptor is involved not only in the species specificity of IFN-gamma binding but also in signalling through interaction with an as yet unidentified species-specific factor(s) encoded by a gene(s) on human chromosome 21.

Animals↗
PubMed1989

Detection by flow cytometry of protoplast fusion and transient expression of transferred heterologous CD4 sequences in COS-7 cells.

Transfer and expression of a plasmid containing the gene encoding the human T-cell antigen CD4 by protoplast fusion was measured by flow cytometry (FCM). Protoplasts were prelabeled with fluorescein isothiocyanate (FITC) and fused to COS-7 cells. Nonspecific protoplast adsorption to the plasma membrane was differentiated from successful protoplast fusion by the addition of an antibody directed against fluorescein to quench extracellular protoplast fluorescence. Transfection efficiencies were defined as both percent CD4 expressing cells and CD4 expression levels on a single cell basis in the transient immunofluorescence assay. Cell sorting studies indicated that intracellular protoplast-associated fluorescence immediately after fusion exhibited a good correlation with transient CD4 transfection efficiencies as measured by indirect immunofluorescence. Reconstruction experiments comparing CD4 transfer efficiencies of protoplast fusion and calcium phosphate transfection showed that fusion resulted in a higher percentage of CD4 expressing transfectants, while calcium phosphate transfection yielded higher CD4 expression levels on a single cell basis. Thus, FCM appears to be useful as a new tool for sensitive detection of transient expression of heterologous reporter genes in COS-7 cells.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Identification of human uromodulin as the Tamm-Horsfall urinary glycoprotein.

The primary structure of human uromodulin, a 616-amino acid, 85-kilodalton glycoprotein with in vitro immunosuppressive properties, was determined through isolation and characterization of complementary DNA and genomic clones. The amino acid sequence encoded by one of the exons of the uromodulin gene has homology to the low-density-lipoprotein receptor and the epidermal growth factor precursor. Northern hybridization analyses demonstrate that uromodulin is synthesized by the kidney. Evidence is provided that uromodulin is identical to the previously characterized Tamm-Horsfall glycoprotein, the most abundant protein in normal human urine.

Amino Acid Sequence↗
PubMed1987

Human tumour necrosis factors: structure and receptor interactions.

Activation of lymphoid and myeloid cells causes the production of factors cytotoxic to various tumour cell types in vitro and in vivo. We have investigated the biochemistry, molecular biology and mechanism of action of two such factors. The factor derived from a myeloid cell line was named TNF-alpha (previously referred to as TNF) and that derived from lymphoid cells named TNF-beta (previously called lymphotoxin). Both proteins were purified from the conditioned media of the human cell lines and sequenced. Structural information revealed that TNF-alpha is 157 amino acid residues long and contains one disulphide bond. TNF-beta is a glycoprotein of 171 amino acids that contains no cysteine residues. Protein sequence information was used to isolate and characterize cDNAs for TNF-alpha and TNF-beta by recombinant DNA methods. The expression of the cDNAs in Escherichia coli made available large quantities of these proteins for biological studies. The two proteins are 31% identical and 52% homologous to each other. The genes for both cytokines are approximately three kilobases in size and are closely linked on human chromosome six. TNF-alpha and TNF-beta both bind to various cell types via a single class of high affinity receptors. On most cells the same receptor is recognized by both cytokines. The receptors for TNF-alpha can be up-regulated by both interferons and lectins. Up-regulation of receptors by interferons is accompanied by synergistic enhancement of the biological response whereas up-regulation by lectins results in an antagonistic response. Besides antiproliferative effects, both cytokines exhibit direct antiviral effects on infection by both DNA and RNA viruses.

Gene Expression Regulation↗
PubMed1986

Two interleukin 1 genes in the mouse: cloning and expression of the cDNA for murine interleukin 1 beta.

A human interleukin 1 beta (IL 1 beta) cDNA probe was utilized to identify a homologous murine cDNA clone. The murine cDNA encodes a 269-residue protein which is 67% homologous to human IL 1 beta. The murine sequence was engineered for expression in mammalian cells and directs the synthesis of biologically active IL 1. This protein, termed murine IL 1 beta, is only 22% homologous with the previously described murine IL 1 sequence. Both IL 1 alpha and IL 1 beta are encoded by single genes, but IL 1 beta mRNA is about fivefold more abundant in a stimulated macrophage cell line.

Amino Acid Sequence↗
PubMed1985

Human lymphotoxin and tumor necrosis factor genes: structure, homology and chromosomal localization.

Human Tumor Necrosis Factor and Lymphotoxin are cytotoxic proteins which have similar biological activities and share 30 percent amino acid homology. The single copy genes which encode these proteins share several structural features: each gene is approximately three kilobase pairs in length and is interrupted by three introns. In addition, these genes are closely linked and have been mapped to human chromosome 6. However, only the last exons of both genes, which code for more than 80 percent of each secreted protein, are significantly homologous (56 percent).

Amino Acid Sequence↗
PubMed1985

Cloning and expression in Escherichia coli of the cDNA for murine tumor necrosis factor.

A murine tumor necrosis factor (MuTNF) cDNA was isolated from a cDNA library prepared by using mRNA from the murine macrophage-like cell line PU5-1.8 induced with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate. The cDNA encodes a polypeptide consisting of a 79 amino acid pre sequence followed by a mature MuTNF sequence of 156 amino acids. The 235 amino acid murine pre-TNF polypeptide is 79% homologous to the human pre-TNF protein. There is one potential N-linked glycosylation site on MuTNF, in contrast to human TNF, which lacks any such site. The MuTNF cDNA, when engineered for expression in Escherichia coli, was found to direct the synthesis of biologically active MuTNF as determined by its cytotoxicity against several transformed cell lines.

Amino Acid Sequence↗
PubMed1984

The amino acid sequence of murine p53 determined from a c-DNA clone.

A c-DNA clone containing the complete sequence information for the murine p53 protein, from embryonal carcinoma cells, has been isolated. The nucleotide sequence of this clone reveals an open reading frame encoding a protein of 390 amino acids with a molecular weight of 43,364 Da. The NH2-terminal domain of this protein is acidic whereas the carboxyl terminus is rich in basic amino acid residues. These terminal domains are separated by a proline-rich, hydrophobic run of amino acids. Proline comprises approximately 10% of the total amino acid residues. Two tryptic peptides, derived from p53 protein radiolabeled with either methionine or proline, were purified and the position of these labeled residues in the peptide was determined. The positions of three methionine and five proline residues in these two peptides matched the amino acid sequence of the predicted open reading frame determined from the c-DNA clone.

Amino Acid Sequence↗
PubMed1984

Biological properties of human tissue-type plasminogen activator obtained by expression of recombinant DNA in mammalian cells.

Human tissue-type plasminogen activator (t-PA), obtained by expression in mammalian cells of recombinant DNA coding for the entire sequence of t-PA (rt-PA), was compared with natural activator from melanoma cell culture (mt-PA). In an in vitro system, composed of [125I]fibrinogen-labeled plasma clot suspended in circulating human plasma, rt-PA and mt-PA caused a very similar dose-related degree of fibrinolysis without causing extensive fibrinolytic activation and fibrinogen breakdown in the surrounding plasma. Urokinase only induced fibrinolysis at a 5- to 10-fold higher concentration and in association with extensive fibrinogenolysis. Intravenous injection of mixtures of labeled (0.4 microCi/kg) and unlabeled (2000 I.U./kg) mt-PA or rt-PA resulted in a rapid but similar disappearance of activity from plasma (T1/2 of 3 min) and specific accumulation of tracer in the liver. In rabbits with experimental jugular vein thrombosis, rt-PA and mt-PA caused a very similar dose-dependent thrombolysis without causing substantial systemic activation of the fibrinolytic system and fibrinogenolysis. Urokinase induced significant thrombolysis only at a 10-fold higher dose and this was associated with systemic fibrinolytic activation. Infusion of 96,000 I.U./kg (approximately equal to 1 mg/kg) of mt-PA or rt-PA over 4 hr induced approximately 70% lysis, whereas a 10-fold higher dose of urokinase yielded 35 to 40% lysis. Two subfractions of rt-PA differing in the extent of glycosylation had very similar thrombolytic properties. It is concluded that the potentially more readily available rt-PA could constitute a specific, fibrin-selective thrombolytic agent.

Animals↗