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Biomedical subjects

L Walker

Publications and source records attributed to L Walker.

At least 19 recordsLinked to original sources
PubMed1992

Localized conformational changes in the N-terminal domain of CD4 identified in competitive binding assay of monoclonal antibodies and HIV-1 envelope glycoprotein.

The CD4 antigen is established as a major cellular receptor for the human immunodeficiency virus (HIV). Previous studies have suggested that certain anti-CD4 monoclonal antibodies (MAbs) can inhibit or enhance the binding of the viral envelope glycoprotein gp120 to CD4 by allosteric effects. In the study reported here, 17 anti-CD4 MAbs were tested for their ability to influence the binding of each other to recombinant soluble CD4 in a solid-phase radioimmunoassay. Marked enhancement of binding between specific pairs of MAbs was seen, as well as inhibition or lack of interaction. Enhancement was seen less often when CD4+ cells were used as the target antigen. Information on patterns of enhancement and inhibition permitted grouping of MAbs on the basis of epitope specificity, and this grouping was in agreement with published findings based on X-ray crystallographic studies. These results demonstrate connectivity between epitopes in the first domain of recombinant CD4 and suggest a high degree of flexibility of surface structure. These findings may be of physiological significance both in the normal function of CD4 and in the interaction of CD4 with HIV. The data have implications for research or therapeutic strategies based on recombinant CD4 or CD4 mutants and highlight the problems of interpreting experimental findings based on abrogation of MAb binding.

Antibodies, Monoclonal↗
PubMed1992

Inhibitory activity of HIV envelope gp120 dominates over its antigenicity for human T cells.

HIV-1 envelope glycoprotein (gp120), as a CD4-binding reactant, has been shown to inhibit in its native form human T cell responses to several antigens. Here we show that gp120 in soluble form also inhibits activation of a specific human T cell line that responds to gp120-pulsed autologous antigen-presenting cells. In addition the inhibitory property of gp120 for antigen-driven T cell proliferation depends upon its ability to bind CD4 and is lost when CD4-binding capacity is abolished by denaturation, or blocked by complexing with soluble CD4 or with polyclonal antibodies. In contrast, antigenicity of denatured or complexed gp120 for specific human T cells is preserved. Similar effects are also observed with another CD4-binding reactant (i.e. anti-Leu 3a MoAb), which stimulates and/or inhibits human T cells specific for mouse immunoglobulins depending on native or denatured conformation.

Antibodies, Monoclonal↗
PubMed1991

Nucleotide sequence and expression of the human gene encoding apolipoprotein H (beta 2-glycoprotein I).

Human apolipoprotein H (ApoH), also called beta 2-glycoprotein I, is a 50-kDa serum glycoprotein whose function is not clearly defined. We have cloned and sequenced ApoH cDNAs both from human liver and from a human hepatoma cell line (HepG2). Both cDNA sequences predict a protein 345 amino acids (aa) in length. This sequence includes a 19-aa hydrophobic, N-terminal signal sequence which is not present in the mature protein [Lozier et al., Proc. Natl. Acad. Sci. USA 81 (1984) 3640-3644]. It differs from this previously reported aa sequence at two positions, both of which strengthen the conservation among the four short consensus repeats within the ApoH molecule. COS-1 cells transiently transfected with the ApoH cDNA in a eukaryotic expression vector produced a single species of ApoH mRNA and secreted in the ApoH protein. The level of ApoH mRNA expressed by HepG2 cells is downregulated by incubation with inflammatory mediators, implying that ApoH is a negative acute-phase protein.

Acute-Phase Reaction↗
PubMed1991

Phenotypic inhibition of the renin-angiotensin system, emergence of the Ren-2 gene, and adaptive radiation of mice.

The renin-angiotensin system in sub-genus Mus displays unique features including duplication of the renin gene in most strains, strong expression of the second gene in submandibular gland of males, and inhibited responses to injected renin. Our findings indicate that this inhibition results from a paucity of renin substrate and is consistent with first-order kinetics. We find substrate paucity to be a feature of both sexes and all sub-species and strains of Mus irrespective of gene duplication. Attempts to increase the level of substrate in blood by intravenous injection caused marked increases in blood pressure in Mus, suggesting that substrate paucity was a phenotypic prerequisite for successful emergence of enhanced renin expression in salivary gland. We propose that these phenomena are linked to salivary "lethal factor", possibly transferred by biting, in an evolutionary sequence that has provided a major selective advantage for Mus and influenced the ecology and evolution of rodents.

Angiotensinogen↗
PubMed1991

Rectal bioavailability of delta-9-tetrahydrocannabinol from various esters.

The bioavailability of delta-9-tetrahydrocannabinol (delta 9-THC) from suppository formulations containing several polar esters was studied. The esters tested were the hemisuccinate, N-formyl alaninate, N-methyl carbamate, and methoxy acetate. These esters were administered to monkeys in both lipophilic and hydrophilic suppository bases, namely, Witepsol H15 and polyethylene glycol, respectively. Each suppository contained a dose equivalent to 10 mg delta 9-THC. Blood samples were analyzed for both delta 9-THC and its carboxylic acid metabolite (ll-nor-delta 9-THC-9-COOH) using gas chromatography/mass spectrometry. The data showed that, with the exception of the hemisuccinate, no delta 9-THC or its metabolite was detected in the blood samples using the Witepsol H15. Using polyethylene glycol, low levels of delta 9-THC and its metabolite were detected in blood for all esters tested. The levels, however, were lower than those observed with delta 9-THC hemisuccinate using Witepsol H15. Subsequent studies in the conscious dog using the hemisuccinate in Witepsol H15 showed 67% bioavailability of delta 9-THC with a linear response in the dose range equivalent to 5-20 mg of delta 9-THC. No significant bioavailability differences were found when delta 9-THC hemisuccinate ester was administered in various lipophilic bases (Hydrokote 25, Kaomel, Suppocire AIML, and Witepsol H15).

Administration, Rectal↗
PubMed1991

Ethanol influences class I and class II MHC antigen expression on human fetal islet-like cell clusters.

Several autoimmune diseases have been linked to an aberrant expression of major histocompatibility complex (MHC) products Ethanol enhances Class I and Class II products on a variety of cell types, and there is evidence for an autoimmune etiology in numerous pathologies associated with alcoholism. We examined whether ethanol alters the expression of Class I and Class II MHC products on human fetal islet-like cell clusters. Incubation of islet-like clusters for 48 hr in ethanol at a starting concentration of 1.5% increased the percentage of single cells expressing Class I. The percentage of cells expressing Class II did not change, but their relative mean fluorescence increased significantly. These findings suggest that alcohol ingestion could alter MHC expression on pancreatic islet cells in vivo perhaps affecting the development of diabetes in genetically predisposed individuals. These findings also support the hypothesis that the rising incidence of type 1 diabetes seen in areas of the world where the per capita consumption of alcohol is also increasing may be a consequence of the immunological effects of alcohol intake.

Ethanol↗
PubMed1991

Prohibitin, an evolutionarily conserved intracellular protein that blocks DNA synthesis in normal fibroblasts and HeLa cells.

Genes that act inside the cell to negatively regulate proliferation are of great interest because of their implications for such processes as development and cancer, but these genes have been difficult to clone. This report details the cloning and analysis of cDNA for prohibitin, a novel mammalian antiproliferative protein. Microinjection of synthetic prohibitin mRNA blocks entry into S phase in both normal fibroblasts and HeLa cells. Microinjection of an antisense oligonucleotide stimulates entry into S phase. By sequence comparison, the prohibitin gene appears to be the mammalian analog of Cc, a Drosophila gene that is vital for normal development.

Amino Acid Sequence↗
PubMed1991

Activation of mouse peritoneal macrophages by maintenance in serum-free medium.

Normal mouse peritoneal macrophages maintained in a serum-free medium for 48-72 h and then stimulated with phorbol myristate acetate, zymosan or bacteria, released large amounts of hydrogen peroxide. Opsonized zymosan and bacteria stimulated greater release than their unopsonized counterparts. Enhanced peroxide production was not a consequence of increased uptake of particles. Addition of serum to the serum-free medium abolished activation. The addition of interferon-gamma to the serum-free medium enhanced the effect of the serum-free treatment of macrophages from C3H/HeJ mice but abolished the effect of serum free treatment of macrophages from CFLP mice. The results are discussed in terms of negative regulation of receptor-oxidase linkage by serum.

Animals↗
PubMed1991

Effect of rapid alteration in glucose concentration and insulin on thymidine uptake in bovine retinal capillary endothelium.

The effect of insulin and rapid changes in glucose concentration on bovine retinal capillary endothelium (BRCE) was examined by use of tissue culture and 3H-thymidine incorporation. In the absence of insulin, no effect on thymidine uptake by BRCE was observed when glucose concentration was rapidly altered, either increased or decreased. The stimulating effect of insulin on retinal capillary endothelin was more pronounced in log-phase cells than in stationary-phase cells. The extent of stimulation by insulin decreased with increasing glucose concentrations. This effect was not observed when osmotic pressure was increased by mannitol. The results of our experiments make it unlikely that altered osmotic stress, rapidly changing or abnormal glucose levels would be primarily responsible for altered endothelial activity.

Animals↗
PubMed1990

Identification of an HSV-1/HSV-2 cross-reactive T cell determinant.

The results from a number of studies have documented that the HSV glycoprotein gD is an important target for neutralizing antibodies. In contrast, little is known about the Th cell determinants present on HSV that are required for anti HSV gD antibody production. In our study we have immunized BALB/c mice with a recombinant source of HSV-1 gD lacking the carboxyl-terminal 93 amino acids. T cell hybridomas produced from the immunized animals recognized a single antigenic peptide (amino acids 246-261) in the context of I-Ad. The determinant expressed by gD peptide 246-261 was generated and presented by both HSV-1 and HSV-2 infected APC. Fine specificity analysis using truncated synthetic gD peptides revealed that the minimal amino acids recognized by the T hybrids were identical between HSV-1 and HSV-2. In addition, the minimal peptide-I-Ad binding analysis demonstrated that the minimal peptide sequence required for the binding to I-Ad and for T cell recognition contained two prolines. Thus, this important HSV antigenic determinant would not be expected to form an amphipathic alpha-helix and could therefore be missed by algorithms currently used to predict which amino acid sequences would be antigenic based on the propensity to form helices.

Amino Acid Sequence↗
PubMed1990

Characterization of the specificity of peptide binding to four DR haplotypes.

This study describes the establishment of a peptide-binding assay for purified, detergent-solubilized DR molecules. For each of the DR specificities and peptides studied, a unique pattern of interaction was observed. Excellent correlation was detected between the DR1-, 2-, 5-, and 52a-binding capacities and the known DR restrictions of a panel of synthetic peptides. This supports the immunologic relevance of the binding assay, and emphasizes the importance of determinant selection in defining the immune response of individuals. We have also examined the capacity of a panel of DR-restricted peptides to compete with one another for binding to DR1. The results obtained are compatible with a single peptide-binding site on DR molecules. The peptide-binding capacity of the four different DR types (DR1, DR2, DR5, and DR52a) has been further examined by testing a collection of 133 different peptides. This collection is unbiased with respect to previously known DR binding and restrictions, and includes peptides of eukaryotic, bacterial, and viral origins. It was found that: 1) approximately 15 to 35% of the peptides tested bound any given DR type; 2) DR-binding capacities appeared to correlate with each other, suggesting that different alleles of the DR isotype may recognize related structures on an Ag molecule; and 3) despite the statistical correlation between binding capacity of different DR types, approximately 50% of the peptides that were positive binders still were specific in that they could bind only one of the four DR molecules tested. Degenerate binding (i.e., binding to most or all the DR molecules tested) was detected in only a minority of the cases analyzed (approximately 25%).

Amino Acid Sequence↗
PubMed1990

The effect of glucose and insulin concentration on type IV collagen biosynthesis by bovine retinal capillary endothelial cells in vitro.

Qualitative and quantitative analyses of type IV collagen, newly synthesized by cultured retinal capillary endothelial cells were carried out. After 24-hour incubation in high glucose (25 mM) media with 14C proline there was less type IV collagen synthesis than after incubation in 5.6 mM glucose media. Insulin stimulated the biosynthesis of type IV collagen in both low and high glucose concentrations. The stimulating effect of insulin (on the biosynthesis of type IV collagen) was still observed after inhibition of cell growth with hydroxyurea. High ambient glucose and the insulin effect could play a role in the structural changes observed in diabetic retinal microvascular basement membranes.

Animals↗
PubMed1990

Differences in affinity of anti-CD4 monoclonal antibodies predict their effects on syncytium induction by human immunodeficiency virus.

A panel of 20 anti-CD4 monoclonal antibodies (mAb) was ranked in terms of affinity, using an inhibition radioimmunoassay. The ability of these antibodies to inhibit the induction of syncytia by human immunodeficiency virus (HIV) and to prevent binding of the HIV envelope glycoprotein 120 (gp120) to CD4 was also measured. Syncytium inhibition correlated strongly with affinity (P less than 0.001) but only weakly with inhibition of gp120 binding (P = 0.038). Some antibodies partially blocked binding of gp120 to CD4 but did not inhibit syncytia, and some antibodies inhibited syncytia but only weakly blocked binding of gp120. These results suggest that the syncytium inhibition assay is highly affinity-dependent, and that epitopes on CD4 concerned with virus binding are distinct from those involved in syncytium formation.

Antibodies, Monoclonal↗
PubMed1989

Initial hormonal and metabolic profile in critically ill patients with community-acquired lobar pneumonia.

A prospective study of 18 critically ill patients with community-acquired lobar pneumonia was undertaken at Hillbrow Hospital, Johannesburg, in order to document the initial plasma hormonal and substrate profile as part of the stress response to the infection. The results of these studies, carried out before therapy, were compared with the results in a group of healthy fasting adults. Highly significant (P less than or equal to 0.005) increases in the mean plasma levels of adrenaline, noradrenaline, human growth hormone, cortisol, glucose and free fatty acids were noted in the study group, with a lesser increase in the prolactin concentration (P less than or equal to 0.01). The levels of dopamine, glucagon, insulin and adrenocorticotrophin did not show any significant change. No significant differences were found in the hormonal profile when comparing survivors with non-survivors. The neuro-endocrine hormonal and metabolic responses in pneumonia appear to be similar to those seen in other stress situations and failure of the initial stress response does not appear to contribute to the mortality of critically ill patients with community-acquired lobar pneumonia.

Adult↗