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Biomedical subjects

W James

Publications and source records attributed to W James.

At least 55 records · Page 3Linked to original sources
PubMed1994

Definition of the range and distribution of human immunodeficiency virus macrophage tropism using PCR-based infectivity measurements.

The tropism of human immunodeficiency virus (HIV) for macrophages (m phi) is a well recognized phenomenon, but the range and distribution of m phi-tropic phenotypes have not been defined by quantitative means. This study uses a PCR-based infectivity assay to derive an index of m phi tropism for several common strains of HIV. The results show that m phi tropism varies over about six orders of magnitude and that the most m phi-tropic strains have a higher infectivity for m phi than for peripheral blood lymphocytes. Strains were distributed throughout this range, suggesting that m phi tropism is a continuously variable phenotypic property. Although the degree of tropism was strongly influenced by the mode of isolation and propagation of virus strains, there was no evidence for the existence of distinct m phi-tropic or non-m phi-tropic phenotypes. Finally, the tropism of two selected strains was found to be determined by an early step in replication, probably virus entry.

Base Sequence↗
PubMed1993

Inhibition of HIV-1 replication by ribozymes that show poor activity in vitro.

Self-cleaving RNAs (ribozymes) can be engineered to cleave target RNAs of choice in a sequence-specific manner (1). Consequently, they could be used to inhibit virus replication or to analyse host gene function in vivo. However, ribozymes that are catalytic in vitro are generally disappointing when analysed in cells unless expressed at high levels relative to their target RNAs (2, 3). Here we provide evidence that this can be overcome by optimizing ribozyme structure using cellular rather than cell-free assays. We show that ribozymes of relatively long flanking complementary regions (FCRs), while poor catalysts in vitro, can produce profound inhibition of HIV replication in cells. By examining a series of ribozymes in which the FCRs vary from 9 to 564 nucleotides, we establish that the optimum length for activity in the cell is > or = 33 nucleotides.

Base Sequence↗
PubMed1993

Interleukin 13 inhibits human immunodeficiency virus type 1 production in primary blood-derived human macrophages in vitro.

The mechanisms by which cellular immunity maintains the asymptomatic state after human immunodeficiency virus type 1 (HIV-1) infection are poorly understood. CD4+ T lymphocytes play a complex role in regulating anti-HIV effector pathways, including activation of macrophages, which are themselves implicated in clinical latency and pathogenesis of symptomatic acquired immune deficiency syndrome. We have found that a newly identified T helper type 2 lymphokine, interleukin 13 (IL-13), inhibits HIV-1ADA and Ba-L replication in primary tissue culture-derived macrophages but not in peripheral blood lymphocytes. Viral production in cells was measured by viral protein (p24) and reverse transcriptase levels, while entry was assessed by proviral DNA analysis at timed intervals after infection. Inhibition by IL-13 was dose and time dependent and not mediated through altered viral entry, reverse transcription, or viral release. IL-13 is therefore a candidate cytokine for the suppression of HIV infection within monocytes and macrophages in vivo.

Base Sequence↗
PubMed1993

A rat CD4 mutant containing the gp120-binding site mediates human immunodeficiency virus type 1 infection.

CD4 is the primary receptor for the human immunodeficiency virus type 1 (HIV-1). Early mutational studies implicated a number of residues of CD4, centered in the region 41-59, in binding to gp120. However, further mutational analyses, together with studies using inhibitory antibodies or CD4-derived peptides, have suggested that other regions of CD4 are also involved in binding or postbinding events during infection. To resolve these ambiguities, we used rat CD4 mutants in which particular regions were replaced with the corresponding sequence of human CD4. We have previously shown that some of these are able to bind HIV-1 gp120, and here we test their ability to act as functional receptors. We find that the presence of human CD4 residues 33-62 is enough to confer efficient receptor function to rat CD4, and we conclude that it is unlikely that regions of CD4 outside this sequence are involved in specific interactions with HIV-1 during either infection or syncytium formation.

Animals↗
PubMed1993

HIV-1 pseudotype virus containing a Cocal virus genome and an HIV envelope: construction, assay and use.

A method is described for the production and assay of pseudotype viruses between human immunodeficiency virus type 1 (HIV-1) and Cocal virus (COV), containing an HIV-1 envelope and a COV genome (COV(HIV)). COV(HIV) pseudotype virus is a useful tool for the investigation of a variety of questions regarding HIV entry into susceptible cells, including steps in virus binding, fusion, and internalization, and the role of molecules which inhibit entry. COV, a rhabdovirus closely related to vesicular stomatitis virus (VSV), replicated and caused cytopathic effect in primary cultures of human peripheral blood lymphocytes (PBLs) and monocyte-derived macrophages (MDM), and in human cell lines of lymphocytoid or monocytoid origin, making it an ideal candidate for pseudotype production. 174XCEM cells, which were permissive for selected macrophage-tropic strains as well as most lymphocyte-tropic strains of HIV-1, were used to produce stocks of putative pseudotype virus. To neutralize parental COV in these stocks, a rabbit antiserum was produced which had a neutralization index of > 10(7) at a dilution of 1:100. Using these methods, pseudotype viruses were produced with a titer of about 10(4) PFU per ml; these same stocks contained HIV-1 at a titer of about 10(5) TCD50 per ml and COV at a titer of about 10(8) PFU per ml. CD4-expressing HeLa cells were used to assay pseudotype stocks made with lymphocyte-tropic strains of HIV-1. The authenticity of the pseudotype stocks was validated by several controls, including their failure to register on congenic CD4-negative HeLa cells and their inhibition by monoclonal anti-CD4 antibodies such as Leu 3a.

Animals↗
PubMed1993

A novel model of a metastatic human breast tumour xenograft line.

The GI-101 human breast tumour xenograft line is unique in that it spontaneously metastasizes to the lungs of athymic murine hosts from subcutaneous trochar implants. Both tumour and lung metastases are positive for normal human breast tissue markers. GI-101 also is positive for the p53 antigen but negative for the c-erbB-2 oncogene.

Animals↗
PubMed1993

Effects of acute ethanol administration on polyphosphoinositide turnover and levels of inositol 1,4,5-trisphosphate in mouse cerebrum and cerebellum.

Although ethanol is known for its central depressant action, its effect on the polyphosphoinositide (poly-PI) signal transduction activity in brain has not been examined in detail. In this study, C57Bl/6J mice were injected intracerebrally with [3H]inositol, and poly-PI turnover in brain was assessed by determining the levels of labeled inositol monophosphates (IP1) accumulated after intraperitoneal injection of LiCl (6 meq/kg body weight) 4 hr before killing. Using this experimental protocol, acute ethanol administration (by gavage) resulted in time- and dose-dependent decreases in the levels of labeled IP1 in both cerebrum and cerebellum as compared with controls. The ethanol-induced decrease in labeled IP1 correlated well with the decrease in levels of inositol 1,4,5-triphosphate (as measured by the radioreceptor assay) and the increase in blood ethanol concentration. Despite a 4-fold higher accumulation of labeled IP1 in the cerebrum compared with the cerebellum, there were no major differences in the steady-state levels of inositol 1,4,5-triphosphate (based on tissue weight) in either brain region. Intraperitoneal injection of atropine (50 mg/kg) (a muscarinic cholinergic receptor antagonist) to the lithium-treated mice resulted in a 34% decrease in labeled IP1 as compared with controls. This result suggests that a substantial proportion of the signals transduced were due to activation of the muscarinic cholinergic receptor. Administration of ethanol (5 g/kg) to the atropine-treated mice resulted in a further decrease in labeled IP1 and longer sleep time as compared with those given ethanol alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcoholism↗
PubMed1991

Inhibition of heterologous strains of HIV by antisense RNA.

Antisense RNA can inhibit the expression of messenger RNAs (mRNAs) to which they are complementary by a variety of mechanisms and might provide the basis for antiviral therapies of high selectivity. In a previous study of six retrovirally expressed antisense RNAs targeted to HIV-1IIIB, we found that two significantly reduced HIV-1IIIB replication. Here we test the degree to which this inhibitory effect tolerates the natural variation found in the nucleotide sequence of different strains of HIV-1. We show that the longer of the two inhibitory antisense RNAs (600 bases) inhibits replication of HIV strains RF, MN and SF2 to at least as great an extent as it does the homologous strain. In contrast, the shorter (71 bases) does not inhibit replication of the heterologous strains. An examination of the predicted positions of the mismatches in the duplexes formed between the IIIB antisense RNAs and the mRNAs of heterologous strains suggests that one requirement of an inhibitory antisense RNA is that it can form a perfect duplex with its target mRNA of at least some 51-64 base-pairs. Although the observations presented here are not definitive proof of this, they are reminiscent of the structural requirements deduced for the double-stranded RNA-mediated induction of interferon and the activation of interferon-induced 2', 5'-oligo(A) synthetase and protein kinase. We tested the ability of antisense RNA to inhibit HIV replication in Jurkat, CEM, U937 and HeLa-T4 cells. The level of inhibition of HIV-1IIIB replication varied according to the cell line in which it was expressed, but in all cases was significant.

Animals↗
PubMed1991

Physicians promoting bicycle helmets for children: a randomized trial.

Head injury is the leading cause of death and serious morbidity in bicycle accidents. There is good evidence to recommend helmets, yet few children wear them. We evaluated helmet promotion in a randomized trial targeting children presenting to primary care settings for routine ambulatory care. The intervention consisted of physician counseling and take-home pamphlets. The study involved 339 families, 167 in the intervention group and 172 in the control group. In a follow-up telephone call, 2 to 3 weeks later, only 7.2% of the intervention group had purchased helmets, compared with 7.0% of the control group (chi 2 = 0.0056, P = .94). During the latter half of the study, bicycle safety received considerable media attention in Ottawa, and the provincial medical society sponsored a $5 discount campaign. Therefore both groups were subject to community "co-intervention." Nonetheless, we were surprised that physician counseling made no additional impact. Our results and the success of certain community programs suggest that physicians interested in helmet promotion would do better to participate in the design and implementation of multidisciplinary campaigns.

Adolescent↗
PubMed1990

Inhibition of human immunodeficiency virus replication in cell culture by endogenously synthesized antisense RNA.

Antisense RNA, which has a sequence complementary to mRNA, may provide the basis for antiviral therapies of high selectivity. We have explored the inhibitory effect of six antisense RNAs upon the replication of human immunodeficiency virus (HIV) in cell culture. We chose regions of the HIV genome to test whether sequences required for splicing or for translation initiation were more susceptible to antisense RNA interference. Our results suggest that inhibitory antisense RNAs contain sequences complementary to the AUG initiation codon of the tat gene and have a comparatively low tendency to form intramolecular base pairs which would interfere with intermolecular duplex formation. Inhibition can be substantial (over 70%) but is transient. Transience does not result from mutation of the input virus. Inhibition was not a consequence of the induction of interferon by antisense RNA-mRNA duplex formation. Our results suggest that at least part of the inhibitory effect is at the posttranscriptional level.

Base Sequence↗
PubMed1990

Autoantibodies from patients with localized and generalized bullous pemphigoid immunoprecipitate the same 230-kd keratinocyte antigen.

Two patients demonstrating the typical clinical, histologic, and immunopathologic features of nonscarring localized bullous pemphigoid are described. These patients possess circulating IgG autoantibodies that bind the epidermal side of 1.0-mol/L sodium chloride-split human skin in indirect immunofluorescence microscopy. Immunnoprecipitation studies demonstrate that these patients have circulating autoantibodies that immunoprecipitate the same 230-kd bullous pemphigoid antigen that is precipitated by autoantibodies from patients with generalized bullous pemphigoid. These findings indicate that localized bullous pemphigoid is a true clinical variant of generalized pemphigoid rather than a separate nosologic entity.

Aged↗
PubMed1989

Purified, modified eel sodium channels are active in planar bilayers in the absence of activating neurotoxins.

A recent study showed that limited trypsin treatment of liposomes containing purified Electrophorus electricus sodium channels activates a sodium radiotracer flux. We now report that similarly treated sodium channels show voltage-gated, tetrodotoxin-sensitive and highly sodium-selective single-channel currents when incorporated into planar lipid membranes. The trypsinized channels opened repeatedly in bursts of several seconds duration, as would be expected for channels whose fast inactivation process had been removed. Furthermore, they have a higher conductance, different voltage-dependence of gating, and a remarkably higher selectivity (PNa/PK = 41) than sodium channels bound by batrachotoxin or other activating neurotoxins; these properties of the trypsinized channels are probably closer to those of channels in intact electrocytes.

Animals↗