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Biomedical subjects

M J Potash

Publications and source records attributed to M J Potash.

33 records · Page 2Linked to original sources
PubMed1992

A Tat antagonist inhibits HIV-1 induction in naturally infected and experimentally infected T cells.

Ro 5-3335, a novel antagonist of human immunodeficiency virus type 1 (HIV-1) Tat activity, inhibits acute and chronic HIV-1 infection in T lymphocytes. Here we describe the effects of Ro 5-3335 on the accumulation of viral DNA during primary infection, the induction of virus from a latently infected cell line, and the expression of virus upon activation of naturally infected T cells. Ro 5-3335 permitted initial DNA synthesis during primary infection, but inhibited the subsequent increase in viral DNA copy number. The induction of HIV-1, as determined by the synthesis of p24 core antigen, was inhibited by 99% by Ro 5-3335 in both the model cell line and naturally infected T cells.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Virus-cell membrane fusion does not predict efficient infection of alveolar macrophages by human immunodeficiency virus type 1 (HIV-1).

Alveolar macrophages (AM) are the principal target cells for HIV-1 in lung tissue. To investigate the mechanisms of HIV-1 infection and efficient replication in these cells we isolated AM from 14 HIV-1 negative donors and exposed them to two virus isolates, either N1T, which replicates well in T lymphocytic and monocytic cell lines, or ADA, a monocytotropic virus. Membrane fluorescence dequenching assays demonstrated that HIV-1/N1T fuses efficiently with AM plasma membranes at neutral pH and that this interaction requires cellular CD4. Despite efficient fusion, AM from eight of 14 donors were not susceptible to productive infection with N1T. In contrast, ADA replicated in all AM populations tested. Soluble CD4 blocked infection of AM by either N1T or ADA, indicating that, like membrane fusion, entry of infectious virus requires an interaction with cellular CD4. Analysis of HIV-1 DNA accumulation in infected cells by enzymatic amplification revealed that productive infection by ADA correlated with a high HIV-1 DNA copy number and abortive infection by N1T was characterized by little or no stable cDNA. These studies suggest that the differences between the two HIV-1 strains studied in their ability to replicate in AM reside in phases of the virus life cycle that follow virus-cell fusion.

CD4 Antigens↗
PubMed1992

Evaluation of multiple parameters of HIV-1 replication cycle in testing of AIDS drugs in vitro.

Evaluation of the activities of antiretroviral agents and an immunoregulatory compound has been made using two models of HIV-1 infection and three measurements of virus expression. Acute infection of Jurkat cells or chronic/inducible infection in U1.1 cells was monitored at multiple time points after drug treatment. The 50% effective concentrations (EC50) of the HIV-1 inhibitors suramin, 3'-azido-3'-deoxythymidine (AZT), and 2',3'-dideoxycytidine, as measured by HIV-1 RNA hybridization in Jurkat cells two days after infection, were comparable to EC50 values obtained in parallel measurements of extracellular p24 levels and percent HIV-1 IF-positive cells. However, these measurements diverged: at seven days after infection the EC50 of AZT was greater than 10 microM when intracellular HIV-1 RNA was assayed, 0.2 microM by IF, and 0.03 microM by p24 assay. Human thymic humoral factor displayed no direct inductive activity in chronic HIV-1 infection in U1.1 cells, while phorbol ester and lymphocyte supernatants induced all parameters. These observations warrant care when interpreting results of only a single assay and suggest that definitive assay of HIV-1 infection requires measurements of multiple parameters of virus expression.

Antiviral Agents↗
PubMed1992

Discovery and characterization of an HIV-1 Tat antagonist.

Ro 5-3335, 7-chloro-5-(2-pyrryl)-3H-1,4-benzo-diazepin-2-(H)-one, has been shown to inhibit gene expression controlled by the human immunodeficiency virus-1 (HIV-1) LTR promoter. The inhibition was specific for the viral transcriptional transactivator Tat. The compound did not inhibit the basal activity of the HIV-1 LTR or the activity of promoters not responsive to Tat. Consistent with its mode of action, Ro 5-3335 inhibited HIV-1 replication (IC50 = 0.1-1 microM) by reducing viral RNA synthesis in acutely, as well as chronically, infected cells in vitro. The compound was active against HIV-1 and HIV-2, and AZT-resistant clinical isolates.

Animals↗
PubMed1991

Inhibition of HIV replication in acute and chronic infections in vitro by a Tat antagonist.

The human immunodeficiency virus-1 (HIV-1) trans-activator Tat is an attractive target for the development of antiviral drugs because inhibition of Tat would arrest the virus at an early stage. The drug Ro 5-3335 [7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepine-2(H)-one], inhibited gene expression by HIV-1 at the level of transcriptional trans-activation by Tat. The compound did not inhibit the basal activity of the promoter. Both Tat and its target sequence TAR were required for the observed inhibitory activity. Ro 5-3335 reduced the amount of cell-associated viral RNA and antigen in acutely, as well as in chronically infected cells in vitro (median inhibition concentration 0.1 to 1 micromolar). Effective inhibition of viral replication was also observed 24 hours after cells were transfected with infectious recombinant HIV-1 DNA. The compound was active against both HIV-1 and HIV-2 and against 3'-azido-3'-deoxythymidine (AZT)-resistant clinical isolates.

Antiviral Agents↗
PubMed1991

Contribution of multiple rounds of viral entry and reverse transcription to expression of human immunodeficiency virus type 1. A quantitative kinetic study.

Human immunodeficiency virus 1 (HIV-1) infection in vitro has been analyzed by the kinetics of expression of HIV-1 RNA and antigens during treatment with antiviral agents. Intracellular HIV-1 RNA rose from input values of 15 molecules per cell to 28 molecules per cell within 3 h after infection and reached a peak of 13,125 in 5 days. The first detectable increase in levels of HIV-1 capsid protein production was 1 day after infection. Virus infection was interrupted at different time points by the introduction of either 2',3'-dideoxycytidine, 3'-azido-3'-deoxythymidine, or suramin to block reverse transcription, or recombinant soluble CD4 to block binding and re-entry of progeny virus. Two results are noteworthy. First, the three inhibitors of reverse transcription blocked viral expression when added up to 48 h after infection. Second, the extent of infection, although postponed, is not greatly altered by culture of infected cells in recombinant soluble CD4. These data imply that reinfection with progeny virus, while necessary for rapid virus expression, is not required for the establishment of productive HIV-1 infection.

CD4 Antigens↗
PubMed1985

Consequences of frameshift mutations at the immunoglobulin heavy chain locus of the mouse.

From an IgM secreting hybridoma line we have isolated 16 spontaneous mutants that produce truncated IgM polypeptides. The size of the mu-mRNAs produced by these mutants is normal, but they express 3- to 100-fold less mu-mRNA and mutant mu-protein than the parental cell line. Nucleotide sequence analysis of cloned mu-genes and/or their mRNAs show frameshift mutations that generate in-phase chain termination codons. The extent of the reduction in mu-mRNA levels depends on the position of the nonsense codon within the gene.

Amino Acid Sequence↗
PubMed1984

Monoclonal antibodies specific for murine IgM I. Characterization of antigenic determinants on the four constant domains of the mu heavy chain.

Seventeen monoclonal rat antibodies with specificities for mouse mu heavy chain recognize seven distinguishable determinants that are located on the four constant region domains. All determinants are present on secreted, intracellular and membrane bound IgM, and all but two are expressed on isolated mu heavy chain. One antibody, specific for a site in the first constant region domain, recognizes a determinant that is present on IgM of AKR, C3H/HeJ, C57BL/6J, SJL, DBA/2, BALB/c, NZB and CBA/J mouse strains, but not on IgM of A.TH, A/J and A.CA strains of mice.

Animals↗
PubMed1984

Different ways to modify monoclonal antibodies.

In this paper we summarize experiments which were undertaken to create altered antibody molecules. Three different approaches were used. Established hybridoma lines were re-hybridized to mouse spleen cells to generate arrays of secondary hybridomas which express one particular heavy chain and one specificity together with a multitude of different light chains. In such hybrids the influence of light chains to the antibody combining site and the influence of affinity to antibody effector functions can be studied. Another way to obtain altered antibodies was the selection of cells producing less lytic IgM. With this technique we obtained (among many other variants) a series of mu-deletion products which were used to map the fine specificity of rat anti-mouse mu monoclonal antibodies. Both the anti-mu antibodies and the deletion variants were used to assign the Clq binding to the fourth C mu-domain demonstrating the power of mutant IgM in the structure-function analysis. In a third series of experiments we show the feasibility of generating new antibody combining sites by the methods of molecular genetics. The variable region gene of a heavy chain was placed in front of a kappa-constant region gene. The plasmid construct was transferred into mouse myeloma lines which stably express a variable heavy-constant light chain protein. Upon fusion with a light chain producing line, chimaeric light chain dimers with a functional antibody combining site were secreted. These experiments demonstrate that new series of man-made antibody molecules can be made in the future.

Animals↗
PubMed1984

Biochemical genetics of the mouse IgM system.

Using a mouse hybridoma system, we have developed methods of isolating a variety of mutant cell lines in which immunoglobulin function or synthesis is defective. The analysis of mutants defective in kappa chain synthesis has defined a class of murine transposons. The deletion mutants produce immunoglobulin M (IgM) bearing mu heavy chain fragments and provide information on the requirements of IgM assembly and mu gene expression. We also describe a transfer system for the mu and kappa genes which will be useful in analyzing the structural basis of IgM function.

Animals↗
PubMed1982

Deletions in immunoglobulin mu chains.

Eight mutant hybridoma lines are described, which synthesize short immunoglobulin mu chains. Four internal deletions were mapped by Southern blot analysis. They are shown to remove DNA from either part or all of the first, and first and second, constant mu exons. The sizes of the deletions range between 0.6 and 5 kb, leaving an equal or unequal number of splice signals. Shorter mu RNA of one size was found irrespective of whether an exon was completely or only partially deleted. These results preclude exclusive 3' (constant region) to 5' (variable region) directional splicing of the mu RNA. No important signals seem to reside in the deleted DNA stretches affecting the transcription or the correct RNA splicing of the remaining exons. The internal mu protein deletions revealed unusual covalent light chain attachment demonstrating functional homology between the first (normally used) and fourth mu constant domain. The other mu protein deletions (10, 11, and 12 kd) involved neither gross DNA nor RNA lesions and are considered to be due to premature chain termination. Since secretion is found in most of the mutant IgM-producing lines, no single one of the four mu constant domains (including the C-terminal one which contains the so-called secretory piece) is necessary for secretion.

Animals↗
PubMed1981

Roles of protein and carbohydrate in glycoprotein processing and secretion. Studies using mutants expressing altered IgM mu chains.

The role of specific structural elements in glycoprotein metabolism and secretion was studied using the IgM mu chains from normal and mutant hybridoma cell lines. Five classes of altered mu chains were studied, all of which lacked various portions of the normal protein sequence, and three of which had one and the others two fewer carbohydrate units than wild type mu. One mutant secreted mu chains more rapidly than wild type cells. Two of the mutant cell lines secreted very little IgM, and both of these degraded intracellular mu chains at an abnormally high rate. Tunicamycin largely blocked IgM secretion in wild type and three mutant cell lines, but caused less inhibition in the two other mutant lines. When glycosylation in the two low secreting mutants was blocked by tunicamycin, degradation of mu chains was substantially reduced in one but unaffected in the other. All of the above properties were retained by the altered mu chains when the mutants were further hybridized with cells producing wild type mu. Overall, the various carbohydrate units and polypeptide sequences seem to play different roles in a single protein's metabolism and expression. The carbohydrate moieties may exert independent effects on protein degradation and on secretion.

Animals↗
PubMed1978

Differential sensitivity of memory cell subpopulations to anti-immunoglobulin and complement.

Evidence is presented for the unique sensitivity of memory cells bearing surface immunoglobulin G1 (IgG1) to functional elimination with anti-immunoglobulin (anti-Ig) sera and complement (C). Treatment of cells for adoptive transfer with C and anti-gamma1, anti-kappa, or anti-Ig significantly reduces the number of plaque-forming cells (PFC) of only the IgG1 isotype found in adoptive recipients. An increase in PFC of other isotypes accompanies the decrease in IgG1 PFC; there is no net change in the total PFC response. The depletion of IgG1 PFC requires treatment of transferred cells with both specific antisera and C; antisera directed against other isotypes show no significant effects. The maintenance of the magnitude of PFC response, compensation, is discussed.

Animals↗