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Biomedical subjects

M A Norcross

Publications and source records attributed to M A Norcross.

At least 37 records · Page 2Linked to original sources
PubMed1991

Dextran sulfate blocks antibody binding to the principal neutralizing domain of human immunodeficiency virus type 1 without interfering with gp120-CD4 interactions.

The mechanism of the antiviral activity of sulfated polysaccharides on human immunodeficiency virus type 1 (HIV-1) was investigated by determining the effect of dextran sulfate on the binding of CD4 and several anti-gp120 monoclonal antibodies to both recombinant and cell surface gp120. Dextran sulfate did not interfere with the binding of sCD4 to rgp120 on enzyme-linked immunosorbent assay (ELISA) plates or in solution and did not block sCD4 binding to HIV-1-infected cells expressing gp120 on the cell surface. Dextran sulfate had minimal effects on rgp120 binding to CD4+ cells at concentrations which effectively prevent HIV replication. In contrast, it potently inhibited the binding of both rgp120 and cell surface gp120 to several monoclonal antibodies directed against the principal neutralizing domain of gp120 (V3). In an ELISA format, dextran sulfate enhanced the binding of monoclonal antibodies against amino-terminal regions of gp120 and had no effect on antibodies directed to other regions of gp120, including the carboxy terminus. The inhibitory effects of polyanionic polysaccharides on viral binding, viral replication, and formation of syncytia therefore appear mediated by interactions with positively charged amino acids concentrated in the V3 region. This high local positive charge density, unique to the V3 loop, leads us to propose that this property is critical to the function of the V3 region in mediating envelope binding and subsequent fusion between viral and cell membranes. The specific interaction of dextran sulfate with this domain suggests that structurally related molecules on the cell surface, such as heparan sulfate, may be additional targets for HIV binding and infection.

Animals↗
PubMed1990

New food safety initiatives in the Food Safety and Inspection Service, U.S. Department of Agriculture.

The production of meat and poultry products has become increasingly complex. Technological growth has contributed to the need for sophistication in determining the origin and risk of food-borne microbial infections as well as environmental contaminants. The increasing use of agricultural chemicals in animal production and to some extent in processed foods has led to the presence of chemical residues in meat and poultry. These changes have caused the Food Safety and Inspection Service (FSIS), a public health agency within the United States Department of Agriculture (USDA), to institute new food safety initiatives and procedures for inspection of meat and poultry products. The goal is to reduce risks to the public health from conditions observed during antemortem and postmortem inspection or detected during processing. FSIS is committed to scientific innovation and has implemented several rapid inplant tests that have given the Agency inexpensive, less disruptive methods to determine product adulteration contamination.

Animals↗
PubMed1990

Induction of nuclear factor-kappa B and the human immunodeficiency virus long terminal repeat by okadaic acid, a specific inhibitor of phosphatases 1 and 2A.

We have used a specific phosphatase inhibitor, okadaic acid, to examine the role of two phosphatases, PP1 and PP2A, in the induction of NF-kappa B and the long terminal repeat of the human immunodeficiency virus type 1 (HIV-LTR). Treatment of Jurkat cells with okadaic acid induced NF-kappa B in nuclear extracts. The rate of induction by okadaic acid was delayed compared to the induction of NF-kappa B by phorbol myristate acetate (PMA). The induction of NF-kappa B by okadaic acid was enhanced by cycloheximide or phytohemagglutinin (PHA). In contrast to PMA, okadaic acid appeared to induce NF-kappa B independently of protein kinase C (PKC). That the NF-kappa B induced by okadaic acid was functional was demonstrated by the marked increase in CAT activity that occurred in Jurkat, BJA-B, and U251 cells that were transfected with HIV-LTR-CAT and treated with okadaic acid. The increase in CAT activity triggered by okadaic acid was dependent on the presence of the NF-kappa B sites in the long terminal repeat of HIV as assessed by deletion and mutation analysis. Similarly to its effect on the induction of NF-kappa B, PHA added together with okadaic acid resulted in a further increase in CAT activity. Somewhat surprisingly, the addition of PMA inhibited the increase in CAT activity in response to okadaic acid, which suggests that the activation of PKC may also induce inhibitory factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗
PubMed1987

Phorbol ester enhances human immunodeficiency virus-promoted gene expression and acts on a repeated 10-base-pair functional enhancer element.

T-cell activation pathways are involved in the regulation of human immunodeficiency virus (HIV) expression. Phorbol 12-myristate 13-acetate (PMA) is a potent inducer of T-cell immune functions and has recently been demonstrated to increase viral replication in cell lines infected with HIV. To define sequences required for viral induction by PMA. T-cell lines were transiently transfected with viral long terminal repeat (LTR) sequences directing chloramphenicol acetyltransferase (CAT) gene expression. PMA added to transfected cell cultures 24 h before harvest reproducibly increased both CAT mRNA and enzyme expression 2- to 2-fold. Sequences necessary for basal and PMA-induced levels of CAT expression were determined by deletion and enhancer reconstitution constructs with fragments and oligonucleotides from the original LTR-CAT expression plasmid. PMA-inducible and basal activity required tandem repeats of a core enhancer element (GGGACTTTCC) located in the LTR between -105 and -82 relative to the RNA start site. The enhancerlike sequence could be inserted at a site distant to the CAT gene open reading frame and functioned in a position- and orientation-independent manner. The data thus define a transcriptionally active regulatory-enhancer element critical to the control of HIV gene expression.

Chromosome Mapping↗
PubMed1986

Transfected human B lymphoblastoid cells express the mouse Ad beta-chain in association with DR alpha.

The human EBV-transformed cell line TOM was transfected with plasmid DNA containing a complete genomic copy of the murine class II Ad beta gene. Drug resistant transfectant clones showed cell surface expression of the Ad beta gene product, as detected by flow microfluorimetry when using a mouse monoclonal antibody (MKD6) specific for Ad beta. Immunoprecipitation and two-dimensional gel electrophoretic analysis of the Ad beta-containing molecules from the transfected cells revealed that the mouse beta-chain was expressed in noncovalent association with the human DR alpha-chain rather than with DQ alpha, the human A alpha homologue. The implications of this unexpected pairing for our understanding of the processes controlling the association of class II alpha and beta gene products and of the roles of Ia molecules in normal and pathologic immune function are discussed.

Animals↗
PubMed1985

Qualitative and quantitative studies of antigen-presenting cell function by using I-A-expressing L cells.

I-A-expressing transfected murine L cells were analyzed as model antigen-presenting cells. Four features of accessory cell function were explored: antigen processing, interaction with accessory molecules (LFA-1, L3T4), influence of Ia density, and ability to stimulate resting, unprimed T lymphocytes. I-A+ L cells could present complex protein antigens to a variety of T cell hybridomas and clones. Paraformaldehyde fixation before but not subsequent to antigen exposure rendered I-A+ L cells unable to present intact antigen. These results are consistent with earlier studies that made use of these methods to inhibit "processing" by conventional antigen-presenting cells. The ability of anti-L3T4 antibody to inhibit T cell activation was the same for either B lymphoma or L cell antigen-presenting cells. In striking contrast, anti-LFA-1 antibody, which totally blocked B lymphoma-induced responses, had no effect on L cell antigen presentation, measured as interleukin 2 (IL 2) release by T hybridomas, proliferation, IL 2 release, or IL 2 receptor upregulation by a T cell clone. I-A+ L cell transfectants were found to have a stable level of membrane I-A and I-A mRNA, even after exposure to interferon-gamma-containing T cell supernatants. In agreement with earlier reports, a proportional relationship between the (Ia) X (Ag) product and T cell response was found for medium or bright I-A+ cells. However, dull I-A+ cells had a disproportionately low stimulatory capacity, suggesting that there may be a threshold density of Ia per antigen-presenting cell necessary for effective T cell stimulation. Finally, I-A-bearing L cells were shown to trigger low, but reproducible primary allogeneic mixed lymphocyte responses with the use of purified responder T cells, indicating that they are capable of triggering even resting T cells. These studies confirm the importance of antigen processing and I-A density in antigen-presenting cell function, but raise questions about the postulated role of the LFA-1 accessory molecule in T cell-antigen-presenting cell interaction. They also illustrate the utility of the L cell transfection model for analysis and dissection of antigen-presenting cell function.

Animals↗
PubMed1985

Use of molecular techniques for analysis of Ia structure-function relationships.

An overview of a strategy for the molecular analysis of class II major histocompatibility complex (Ia) gene product structure-function relationships is presented, and results obtained to date by using this approach are summarized. The A beta, A alpha, E alpha, and E beta genes have been cloned and sequenced to yield information on gene organization and primary protein sequence. Comparison of sequences from allelic forms of these genes show the NH2-terminal domain to be the locus of most intraspecies polymorphism. Transfection of I-A alpha and A beta genes into B lymphoma cells or L cells has generated cells expressing the transfected gene products on their membrane. Such Ia+ transfectants present antigen to various T cells, which use the expressed I-A as a restriction element. Exon shuffling has shown the beta 1 domain of A beta to play a predominant role in such restricted antigen recognition. Preliminary data refining this analysis to sites within beta 1, as well as data on control of alpha: beta chain association, are reviewed, and future prospects for use of this approach in resolving questions of immunological interest are discussed.

Animals↗
PubMed1984

Membrane Ia expression and antigen-presenting accessory cell function of L cells transfected with class II major histocompatibility complex genes.

To study the relationship between the structure and function of Ia antigens, as well as the physiologic requirements for antigen presentation to major histocompatibility complex-restricted T cells, class II A alpha and A beta genes from the k and d haplotypes were transfected into Ltk- fibroblasts using the calcium phosphate coprecipitation technique. Individually transfected genes were actively transcribed in the L cells without covalent linkage to, or cotransformation with, viral enhancer sequences. However, cell surface expression of detectable I-A required the presence of transfected A alpha dA beta d or A alpha kA beta k pairs in a single cell. The level of I-A expression under these conditions was 1/5-1/10 that of Ia+ B lymphoma cells, or B lymphoma cells expressing transfected class II genes. These I-A-expressing transfectants were tested for accessory cell function and shown to present polypeptide and complex protein antigens to T cell clones and hybridomas in the context of the transfected gene products. One T cell clone, restricted to I-Ak plus GAT (L-glutamic acid60-L-alanine30-L-tyrosine10), had a profound cytotoxic effect on I-Ak- but not I-Ad-expressing transfectants in the presence of specific antigen. Assays of unprimed T cells showed that both Ia+ and Ia- L cells could serve as accessory cells for concanavalin A-induced proliferative responses. These data indicate that L cells can transcribe, translate, and express transfected class II genes and that such I-A-bearing L cells possess the necessary metabolic mechanisms for presenting these antigens to T lymphocytes in the context of their I-A molecules.

Animals↗
PubMed1984

Regulation of TCGF production in T cells. II. Early membrane events after anti-Thy-1 binding by the TCGF-producing T lymphoma EL-4 G-12.

The T cell lymphoma line EL-4 G-12 produces large quantities of TCGF (IL 2) shortly after exposure to anti-Thy-1 and cross-linking reagents. Early surface events that initiate nonconstitutive synthesis and secretion have been examined by fluorescent microscopy, EM analysis, flow cytometry of anti-Thy-1, and measurements of membrane polarization. The data show that anti-Thy-1 binding leads to immediate and rapid endocytosis and shedding of the antibody molecules, but with no change in membrane potential. Cross-linking of the anti-Thy-1 results in stabilization and accumulation of the antibody on the surface, and rapid hyperpolarization. The data suggest that cross-linking of ligand antibody interactions at the surface is a regulatory event involved in initiation of the signal for TCGF synthesis and secretion.

Animals↗
PubMed1982

Dimethoxyethyl phthalate: embryopathy, teratogenicity, fetal metabolism and the role of zinc in the rat.

A single intraperitoneal injection (0.6 ml/kg) of dimethoxyethyl phthalate (DMEP) was given to groups of Wistar strain rats on day 10, 11, 12, 13 or 14 of gestation. Control rats received 0.6 ml/kg of physiological saline intraperitoneally. In phthalate-treated rats, embryopathy was manifested by a high incidence (12-79%) of fetal deaths and fetal resorptions. Fetotoxic effects were expressed by a significant reduction in fetal weights. Hydrocephalus interna, a congenital malformation of the brain, was caused by DMEP. Congenital skeletal deformities (66-96%), with multiple skeletal (14-64%) and appendicular malformations (25-57%), were also induced by DMEP. Control rats exhibited no congenital malformations of the brain and no appendicular or multiple skeletal deformities. DMEP caused a significant decrease in the zinc content of the fetus. Fetoplacental metabolism 1 and 4 hr after intravenous administration of 14C-DMEP suggested rapid transfer of the parent compound to the fetus across the placenta and that DMEP is a teratogenic moiety. The possible role of zinc in phthalate-induced teratogenesis in rats is also discussed.

Animals↗