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S M Chamow

Publications and source records attributed to S M Chamow.

34 records · Page 2Linked to original sources
PubMed1992

Reduction of alpha-naphthylisothiocyanate-induced hepatotoxicity by recombinant human hepatocyte growth factor.

Hepatocyte growth factor (HGF) is a potent stimulator of DNA synthesis in cultured hepatocytes. To determine whether HGF has any activity in vivo, we have tested HGF in rats in which intrahepatic cholestasis was induced by acute administration of alpha-naphthylisothiocyanate (ANIT). The hepatotoxic effects of a single injection of ANIT were manifested 48 h later as large increases in serum bilirubin, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase. These biochemical changes were accompanied by widespread periportal edema, hypertrophy of bile duct epithelium, and randomly scattered areas of liquifaction necrosis in the hepatic parenchyma. The increases in bilirubin, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase were markedly attenuated when HGF was administered 30 min before ANIT and again at 6, 12, 24, 30, and 36 h after ANIT. In addition, this HGF dosing regimen completely prevented the occurrence of parenchymal lesions, although it had no effect on periportal histopathology. The effect of ANIT was dose dependent; a maximal response was observed at 320 micrograms/kg per injection, with an intermediate response at 105 micrograms/kg. Delaying the administration of HGF until 12 h after ANIT was as effective as when administration was begun 30 min before ANIT. Taken together these results show that HGF can prevent some aspects of ANIT hepatotoxicity.

1-Naphthylisothiocyanate↗
PubMed1992

CD4 immunoadhesins in anti-HIV therapy: new developments.

CD4, the cell-surface receptor for the human immunodeficiency virus (HIV), is a member of the immunoglobulin (Ig) gene superfamily. It contains 4 extracellular sequences homologous to Ig variable domains, the first of which (V1) is sufficient for binding to HIV. To develop CD4 as an anti-HIV therapeutic, we engineered a CD4 immunoadhesin (CD4-IgG)--a fusion protein containing the V1 and V2 domains of CD4 with the hinge and Fc regions of human Ig heavy chain. A chimeric protein of this type has several advantages compared to the soluble receptor, including a greatly extended in vivo half-life and greater avidity for HIV; moreover, like an antibody, it performs effector functions via its Fc domains, such as complement activation and antibody-dependent cell-mediated cytotoxicity. In vivo experiments show that CD4-IgG protects against HIV-I IIIB infection of chimpanzees when administered prior to viral challenge. In addition, CD4-IgG is transferred efficiently across the placenta from mother to fetus in rhesus monkeys. To evaluate its safety in humans, we conducted a phase-I clinical trial in adult patients with AIDS and AIDS-related complex. We found that, in a total of 16 patients, administration of CD4-IgG was well tolerated at doses up to 1000 micrograms/kg of body weight, with no important clinical or immunological toxicities noted. Given its unique properties, particularly the ability of CD4-IgG to cross the placenta, we plan to focus future clinical efforts on preventing infection of newborns via maternal-fetal transfer of HIV.

Acquired Immunodeficiency Syndrome↗
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↗
PubMed1990

Enzymatic cleavage of a CD4 immunoadhesin generates crystallizable, biologically active Fd-like fragments.

CD4, the cell-surface receptor for the human immunodeficiency virus (HIV), is a member of the immunoglobulin (Ig) gene superfamily. It contains four extracellular sequences homologous to Ig VL domains. The first of these (V1) is sufficient for binding to HIV; however, the structural basis for this binding has yet to be elucidated. While several models for the structure of Ig-like domains in CD4 have been proposed on the basis of crystal structures of Ig VL domains, direct evidence that CD4 and VL domains fold similarly has not been obtained. To produce individual domains of CD4 for structural studies, we used molecular fusions of such domains with Ig heavy chain (CD4 immunoadhesins), which are very efficiently expressed and secreted in mammalian cells and can be easily isolated in single-step purification with protein A. Since these fusion molecules are antibody-like homodimeric proteins, we investigated the possibility that they might be cleaved enzymatically to produce Fd-like and Fc fragments. We found that cleavage with papain releases an Fd-like fragment containing the V1 and V2 CD4 domains; this fragment fully retains the ability to bind to the HIV-1 envelope glycoprotein gp120 and to block HIV infection in vitro. Moreover, folding of the CD4 domains in the Fd-like fragment and in the parent immunoadhesin is indistinguishable, as indicated by circular dichroism. Spectral analysis of the Fd-like fragment suggests that secondary structure content is identical with that predicted from the known structure of Ig VL domains; this directly supports the hypothesis that the V1 and V2 domains of CD4 fold similarly to Ig VL domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Biological properties of a CD4 immunoadhesin.

Molecular fusions of CD4, the receptor for human immunodeficiency virus (HIV), with immunoglobulin (termed CD4 immunoadhesins) possess both the gp120-binding and HIV-blocking properties of recombinant soluble CD4, and certain properties of IgG, notably long plasma half-life and Fc receptor binding. Here we show that a CD4 immunoadhesin can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) towards HIV-infected cells, although, unlike natural anti-gp120 antibodies, it does not allow ADCC towards uninfected CD4-expressing cells that have bound soluble gp120 to the CD4 on their surface. In addition, CD4 immunoadhesin, like natural IgG molecules, is efficiently transferred across the placenta of a primate. These observations have implications for the therapeutic application of CD4 immunoadhesins, particularly in the area of perinatal transmission of HIV infection.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Characterization of a soluble form of human CD4. Peptide analyses confirm the expected amino acid sequence, identify glycosylation sites and demonstrate the presence of three disulfide bonds.

CD4 is a glycoprotein that is expressed on the surface of a variety of cells of the immune system and is believed to participate in the interactions of these cells with antigen-presenting cells bearing the class II major histocompatibility (MHC) antigens. CD4 also acts as the receptor for the human immunodeficiency virus (HIV) by binding to the viral glycoprotein gp120. Recombinant soluble CD4 (rCD4) is a truncated form of human CD4 that is secreted from transfected Chinese hamster ovary cells. This 368-amino-acid glycoprotein contains two potential sites of N-linked glycosylation (Asn-271 and Asn-300) and six cysteine residues. Amino-terminal sequence analysis demonstrated that the sequence begins at the third residue of the polypeptide originally predicted from the cDNA analysis [Maddon, P.J. et al. (1985) Cell 42, 93-104]. The rest of the primary sequence was confirmed by analysis of peptides purified by reversed-phase HPLC after digestion of S-carboxymethylated rCD4 with trypsin. Anhydrotrypsin affinity chromatography of trypsin-digested rCD4 confirmed that the carboxy-terminus of the protein was Pro-368. Enzymatic digestion of non-reduced rCD4 generated disulfide-bonded fragments that demonstrated the presence of disulfide bonds between Cys-16 and Cys-84, Cys-130 and Cys-159, and between Cys-303 and Cys-345. The constituent monosaccharides of the carbohydrate structures of rCD4 were found to be fucose, mannose, galactose, N-acetylglucosamine and N-acetylneuraminic acid. Characterization of the tryptic map of rCD4 after treatment with peptide: N-glycosidase F demonstrated that both potential N-glycosylation sites are utilized. The tryptic map of rCD4 treated with endo-beta-N-acetylglucosamine H demonstrated that only complex-type oligosaccharides are attached to Asn-271, while Asn-300 has high-mannose or hybrid structures attached in addition to complex-type oligosaccharides. Glucosamine was observed only in glycopeptides that contain Asn-300 or Asn-271 while no galactosamine was observed. This suggests that rCD4 contains no O-linked oligosaccharides.

Amino Acid Sequence↗
PubMed1990

CD4 immunoadhesin, but not recombinant soluble CD4, blocks syncytium formation by human immunodeficiency virus type 2-infected lymphoid cells.

Recombinant soluble CD4 (rCD4) has been shown to be an effective inhibitor of human immunodeficiency virus type 1 (HIV-1) and HIV-2 infection of lymphoid cells in vitro. In this report, we characterized the effects of rCD4, the V1V2 fragment of CD4, and the immunoadhesin CD4-immunoglobulin G on syncytium formation between lymphoid cells infected by HIV-1 or HIV-2 and uninfected cells. All three molecules blocked HIV-1-mediated syncytium formation, but only CD4-immunoglobulin G blocked HIV-2-mediated syncytium formation. rCD4 and the V1V2 fragment of CD4 enhanced HIV-2-mediated syncytium formation. These results suggest that the process of cell fusion is significantly different between HIV-1- and HIV-2-infected cells.

CD4 Antigens↗
PubMed1990

Oocyte-specific expression of mouse Zp-2: developmental regulation of the zona pellucida genes.

The zona pellucida surrounds all mammalian oocytes and plays a vital role at fertilization and in early development. The genes that code for two of the mouse zona proteins (ZP2 and ZP3) represent a developmentally regulated set of genes whose expression serves as markers of mouse oocyte growth and differentiation. We previously characterized the single-copy Zp-3 gene and showed that its expression is oocyte specific and restricted to a narrow window of oocyte development. We now define the Zp-2 gene transcript and show that it is coordinately expressed with Zp-3 only during the 2-week growth phase of oogenesis that occurs prior to ovulation. Like Zp-3, the expression of Zp-2 is restricted to oocytes, and, although not detectable in resting oocytes, both ZP2 and ZP3 transcripts accumulate to become very abundant messengers in 50-microns-diameter oocytes. Ovulated eggs contain ZP2 and ZP3 transcripts which are 200 nucleotides shorter than those found in growing oocytes and have an abundance of less than 5% of the peak levels. In an attempt to understand the molecular details associated with the developmentally regulated, tissue-specific gene expression of the zona genes, the Zp-2 genetic locus has been characterized and its 5' flanking sequences have been compared with those of Zp-3. Both genes contain three short (8- to 12-base-pair) DNA sequences of 80 to 88% identity located within 250 base pairs of their transcription start sites.

Amino Acid Sequence↗
PubMed1989

Vaccination with a synthetic zona pellucida peptide produces long-term contraception in female mice.

The zona pellucida surrounding mouse oocytes is an extracellular matrix composed of three sulfated glycoproteins, ZP1, ZP2, and ZP3. It has been demonstrated that a monoclonal antibody to ZP3 injected into female mice inhibits fertilization by binding to the zona pellucida and blocking sperm penetration. A complementary DNA encoding ZP3 was randomly cleaved and 200- to 1000-base pair fragments were cloned into the expression vector lambda gt11. This epitope library was screened with the aforementioned contraceptive antibody, and the positive clones were used to map the seven-amino acid epitope recognized by the antibody. Female mice were immunized with a synthetic peptide containing this B cell epitope coupled to a carrier protein to provide helper T cell epitopes. The resultant circulating antibodies to ZP3 bound to the zona pellucida of immunized animals and produced long-lasting contraception. The lack of ovarian histopathology or cellular cytotoxicity among the immunized animals may be because of the absence of zona pellucida T cell epitopes in this vaccine.

Amino Acid Sequence↗
PubMed1989

Designing CD4 immunoadhesins for AIDS therapy.

A newly-constructed antibody-like molecule containing the gp120-binding domain of the receptor for human immunodeficiency virus blocks HIV-1 infection of T cells and monocytes. Its long plasma half-life, other antibody-like properties, and potential to block all HIV isolates, make it a good candidate for therapeutic use.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Characterization of in vitro inhibition of human immunodeficiency virus by purified recombinant CD4.

The first step in infection of human T cells with human immunodeficiency virus (HIV) is binding of viral envelope glycoprotein gp120 to its cellular receptor, CD4. The specificity of this interaction has led to the development of soluble recombinant CD4 (rCD4) as a potential antiviral and therapeutic agent. We have previously shown that crude preparations of rCD4 can indeed block infection of T cells by HIV type 1 (HIV-1). Here we present a more detailed analysis of this antiviral activity, using HIV-1 infection of the T lymphoblastoid cell line H9 as a model. Purified preparations of rCD4 blocked infection in this system at nanomolar concentrations; combined with the known affinity of the CD4-gp120 interaction, this finding suggests that the inhibition is simply due to competition for gp120 binding. As predicted, rCD4 had comparable activity against all strains of HIV-1 tested and significant activity against HIV-2. Higher concentrations of rCD4 blocked infection even after the virus had been adsorbed to the cells. These findings imply that the processes of viral adsorption and penetration require different numbers of gp120-CD4 interactions. Recombinant CD4 was able to prevent the spread of HIV infection in mixtures of uninfected and previously infected cells. Our studies support the notion that rCD4 is a potent antiviral agent, effective against a broad range of HIV-1 isolates, and demonstrate the value of purified rCD4 as an experimental tool for studying the mechanism of virus entry into cells.

Adsorption↗
PubMed1988

A micromethod for the estimation of oligosaccharides containing glycosidically linked sialic acid or hexoses, or both, in glycoproteins.

The peeling reaction, the process by which oligosaccharides are degraded in alkali, was used as the basis for an assay to provide structural information about glycosidically linked oligosaccharides in glycoproteins. Glycoproteins were treated with 0.05 M NaOH at 50 degrees to induce release, and subsequent degradation ("peeling"), of glycosidically linked, but not of N-glycosydically linked, oligosaccharides. Among the degradation products generated from O-linked chains were three 3-deoxy sugar acids whose formation was correlated with certain structural features of the oligosaccharides. N-Acetylneuraminic acid was released from terminal positions in the oligosaccharides, and iso- and meta-saccharinic acids were derived from the degradation of 4-O- and 3-O-substituted hexoses, respectively. All of these sugar acids were detected colorimetrically by periodate oxidation and reaction of the product with 2-thiobarbituric acid. The ability of the method to generate 3-deoxy sugar acids was tested in 8 alkali-treated glycoproteins. 3-Deoxy sugar acids were detected only in those glycoproteins whose glycosidically linked carbohydrates contained N-acetylneuraminic acid, or 3-O- or 4-O-substituted hexoses, or both. As little as 0.12 microgram of 3-deoxy sugar acid produced from 5 micrograms of human chorionic gonadotropin was sufficient for detection. This method is novel in its ability to distinguish sialylation of glycosidically linked carbohydrates. Furthermore, it combines the specificity of beta-elimination with the sensitivity of the 2-thiobarbituric acid assay in targeting degradation products of the peeling reaction as candidates for an assay method.

Glycoproteins↗
PubMed1986

Subunit structure of a cortical granule lectin involved in the block to polyspermy in Xenopus laevis eggs.

The cortical granule lectin of Xenopus laevis eggs is a large molecular mass glycoprotein involved in the post-fertilization block to polyspermy. We have investigated the subunit structure of this lectin and found that the native molecule contains 10-12 monomers, each of which has considerable charge and size heterogeneity due to glycosylated side chains. In addition, significant amino acid sequence homology is indicated by peptide mapping of subunits separated by isoelectric focusing.

Amino Acid Sequence↗
PubMed1986

Oocyte-specific gene expression: molecular characterization of a cDNA coding for ZP-3, the sperm receptor of the mouse zona pellucida.

The mouse zona pellucida genes are expressed uniquely during oogenesis and are developmentally regulated in the absence of cell division. Little is known about the mechanisms that control the expression of these germ-line-specific genes that play crucial roles in early mammalian development. We have constructed a lambda gt11 cDNA library from ovarian poly(A)+ mRNA and have isolated clones coding for ZP-3, the mouse sperm receptor. The identity of the clones was confirmed by comparing their DNA sequence with an amino acid sequence obtained from an isolated ZP-3 peptide. The ZP-3 gene is transcribed as a 1.7-kilobase poly(A) mRNA that is detected exclusively in ovarian tissue. This germ-line-specific expression is reflected in the observed hypomethylation of the ZP-3 locus in ovarian but not liver or brain DNAs. The ZP-3 gene is otherwise identically organized in somatic and germ-line DNA where it appears to be present as a low-copy-number or single-copy gene. Despite the fact that the mouse sperm receptor demonstrates species specificity, the ZP-3 cDNA cross-hybridized with DNA from a variety of mammalian species, including rat, rabbit, dog, pig, cow, and human.

Animals↗
PubMedDate not supplied

Modification of CD4 immunoadhesin with monomethoxypoly(ethylene glycol) aldehyde via reductive alkylation.

CD4 immunoadhesin (CD4-IgG) is a chimeric glycoprotein molecule comprised of the gp120-binding portion of human CD4 fused to the hinge and Fc portions of human IgG. As a candidate for human therapeutic use, CD4-IgG represents an important advance over soluble CD4, insofar as the systemic clearance in humans of CD4-IgG is significantly slower. In an effort to prolong its in vivo residence time even further, we have modified CD4-IgG chemically by attaching monomethoxypoly(ethylene glycol) (MePEG) moieties to lysine residues via reductive alkylation. We synthesized MePEG aldehyde and investigated reaction conditions for adding a range of MePEG moieties per protein molecule. At neutral pH in the presence of sodium cyanoborohydride, the reaction was sufficiently slow to allow for significant control over the extent of MePEGylation. Addition of 7.7 or 14.4 MePEG moieties to CD4-IgG resulted in an approximately 4- or 5-fold increase, respectively, in the persistence of the protein in rats, as compared with unmodified CD4-IgG. These results suggest that the therapeutic utility of a human receptor IgG chimera can be improved by MePEGylation technology, provided that the modified immunoadhesin retains its biological activity in vivo. Such modification can lead to a significant additional increase in the in vivo residence time of the protein.

Alkylation↗