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Biomedical subjects

A Srinivasan

Publications and source records attributed to A Srinivasan.

At least 181 records · Page 10Linked to original sources
PubMed1995

Mutagenesis of the putative alpha-helical domain of the Vpr protein of human immunodeficiency virus type 1: effect on stability and virion incorporation.

vpr is one of the auxiliary genes of human immunodeficiency virus type 1 (HIV-1) and is conserved in the related HIV-2/simian immunodeficiency virus lentiviruses. The unique feature of Vpr is that it is the only nonstructural protein incorporated into the virus particle. Secondary structural analysis predicted an amphipathic alpha-helical domain in the amino terminus of Vpr (residues 17-34) which contains five acidic and four leucine residues. To evaluate the role of specific residues of the helical domain for virion incorporation, mutagenesis of this domain was carried out. Substitution of proline for any of the individual acidic residues (Asp-17 and Glu-21, -24, -25, and -29) eliminated the virion incorporation of Vpr and also altered the stability of Vpr in cells. Conservative replacement of glutamic residues of the helical domain with aspartic residues resulted in Vpr characteristic of wild type both in stability and virion incorporation, as did substitution of glutamine for the acidic residues. In contrast, replacement of leucine residues of the helical domain (residues 20, 22, 23, and 26) by alanine eliminated virion incorporation function of Vpr. These data indicate that acidic and hydrophobic residues and the helical structure in this region are critical for the stability of Vpr and its efficient incorporation into virus-like particles.

Amino Acid Sequence↗
PubMed1995

Identification of residues in the N-terminal acidic domain of HIV-1 Vpr essential for virion incorporation.

Vpr is one of the auxiliary proteins encoded by the HIV-1 genome and is selectively incorporated into the virus particle. It has been shown that Vpr incorporation in the virus particle requires only the core protein Gag. In an effort to identify the domains of Vpr which are essential for incorporation into the HIV-1 virion, site-specific mutagenesis of vpr was carried out. Mutation of the highly conserved acidic residues in the N-terminal domain (amino acid positions 17-34) eliminated virion incorporation. These mutations disrupt a predicted amphipathic alpha-helical structure that is highly conserved among Vpr sequences. In contrast, alterations of the conserved cysteine (Cys76), basic domain (Arg87 and Lys95), and other residues (Gln65) did not impair the incorporation of Vpr into virus-like particles directed by HIV-1 Gag. The results presented here suggest that protein-protein interactions mediated through the putative helical domain of Vpr may participate in its incorporation into the virus particle.

Amino Acids↗
PubMed1995

Thiol oxidation by 1,2,3-oxadiazolinium ions, presumed carcinogens.

3-Alkyl-1,2,3-oxadiazolinium ions 1 have been proposed as reactive intermediates in the activation of (2-hydroxyethyl)nitrosamines. The reaction of 3-methyl-1,2,3-oxadiazolinium tosylate (1a), 2-ethyl-1-methoxy-2-phenyldiazenium tetrafluoroborate (3), and 3-phenyl-1,2,3-oxadiazolinium triflate (1b) with thiols was investigated to determine the behavior of these compounds toward typical "cellular nucleophiles". Each of these substances oxidizes benzenethiol to diphenyl disulfide. The reaction in aqueous buffer at pH 7.4 is rapid. Reaction of 1b with benzenethiol gives, in addition to the disulfide, benzene, biphenyl, azobenzene, diphenyl sulfide, aniline, and glycolaldehyde. Similar products are obtained from 3. Phenyldiazene is postulated as an intermediate in this process, and its generation from phenyldiazoformate in the presence of benzenethiol gives similar products. Diazenes are presumed to arise by proton abstraction from the CH adjacent to N. The kinetics of reaction of 1a with N-acetylcysteine to give the corresponding disulfide show first order dependence on each reactant and base catalysis. The data from these model chemical experiments suggest that 1,2,3-oxadiazolinium ions could react with abundant thiols in cells to lead to either their detoxification or radical processes emanating from diazenes. The occurrence of thiol-oxadiazolinium ion redox transformations could modulate the alkylation chemistry of these substances as well.

Carcinogens↗
PubMed1995

The effects of joint washout and steroid injection compared with either joint washout or steroid injection alone in rheumatoid knee effusion.

Patients with rheumatoid arthritis attending rheumatology out-patients who had a symptomatic knee effusion were randomly allocated to receive one of three treatments: group I, a steroid injection without washout; group II, a joint washout with normal saline; group III, a joint washout with normal saline and steroid injection. Sixty knees in all were studied. Laboratory parameters for disease activity (erythrocyte sedimentation rate, C-reactive protein) were monitored in all patients prior to the study and at 3 months. Clinical assessment of disease activity (pain, morning stiffness involving the knee, circumference of the knee, walking distance and range of movement) were recorded prior to the study, at 1 month and at 3 months. All three treatments, resulted in a reduction of pain and increased movement. However, patients who had a joint washout alone showed significantly less improvement as compared with the other two groups. Symptomatic improvement was marginally greater in patients following joint washout and injection than in those who had had joint injection alone. The results of the study indicate that the simple procedure of joint aspiration and steroid injection, which can be carried out in the out-patient clinic, provides satisfactory relief of symptoms in rheumatoid patients with knee effusions. Joint washout alone was less beneficial.

Adult↗
PubMed1995

Evidence that a cell cycle regulator, E2F1, down-regulates transcriptional activity of the human immunodeficiency virus type 1 promoter.

Proliferation of eukaryotic cells is orchestrated by a series of cellular proteins which participate in various stages of the cell cycle to guide the cell through mitosis. Some of these proteins, including E2F1, play a critical role in G1 and S phases by coordinately regulating expression of several important cell cycle-associated genes. On the basis of recent observations indicating a block in human immunodeficiency virus type 1 (HIV-1) replication in cells arrested in G1/S phase of the cell cycle, we sought to evaluate the regulatory action of E2F1 on transcription from the HIV-1 long terminal repeat (LTR). Results from transient transfection of cells with an E2F1 expression plasmid indicated that E2F1 has the ability to suppress basal transcriptional activity of the LTR and to diminish the extent of the Tat-induced activation of the viral promoter. Deletion analysis of the HIV-1 LTR in transfection studies revealed the presence of two major elements responsive to E2F1 repression located distally (-454 to -381) and proximally (-117 to -80) with respect to the +1 transcription start site. E2F1-mediated suppression of LTR activity was observed in a wide range of human cell lines. Expression of E2F1 by a transgene showed an inhibitory effect on the levels of reverse transcriptase activity obtained upon introduction of the proviral genome into cells. The data presented in this study suggest that cellular regulatory proteins involved in the progression of cells through the mitotic cycle could play crucial roles in determining the efficiency of HIV-1 replication during the various stages of infection. The possible roles of these factors in viral latency and activation are discussed.

Carrier Proteins↗
PubMed1995

V3-independent determinants of macrophage tropism in a primary human immunodeficiency virus type 1 isolate.

Human immunodeficiency virus type 1 isolates differ in their ability to productively infect macrophages, and several groups have mapped the genetic basis for macrophage tropism to regions of env that include the third hypervariable region (V3 loop). We recently described a primary isolate (89.6) which is highly macrophage tropic and yet differs from other macrophage-tropic strains studied in that it is cytopathic in T cells. Genetic mapping of macrophage tropism determinants in this virus was done by using chimeras generated with the prototypic non-macrophage-tropic strain HXB2. Replacement of a 2.7-kb env-containing region of HXB with corresponding sequences from 89.6 conferred the macrophage-tropic phenotype, but insertion of the 89.6 V3 loop along with V4/V5 sequences did not. Conversely, placement of HXB sequences that included V3 into 89.6 did not impair this strain's ability to replicate in macrophages. Sequence analysis of V3 shows that 89.6 differs markedly from previously described macrophage-tropic consensus sequences and that it is more similar to highly charged non-macrophage-tropic strains. This suggests either that macrophage tropism is defined by structural determinants resulting from complex interactions among multiple env regions rather than V3 sequence-specific requirements or that there are multiple mechanisms by which different strains may establish productive macrophage infection. In addition, because the HXB V3 loop supports productive macrophage infection in the background of 89.6, phenotypic characterization of V3 sequences should be considered specific to the viral context in which they are placed.

Amino Acid Sequence↗
PubMed1995

High resolution analysis of HIV-1 quasispecies in the brain.

OBJECTIVE: To characterize HIV-1 quasispecies at high resolution to determine the in vivo sequence heterogeneity of virus infecting the brain. METHODS: A 1 kilobase region of the envelope gene, which includes the five hypervariable regions, was amplified by polymerase chain reaction (PCR) using DNA obtained from brain tissue of an HIV-1-infected patient. PCR products were cloned and 50 clones sequenced. RESULTS: Thirty-nine unique nucleotide sequences producing 35 protein variants were found. A consensus sequence was identified along with three distinct subtypes, each present at a level of 12%. The sequence variation from the consensus was 0.1-2.1% at the nucleotide level with hypermutation and recombination responsible for the highest diversity. Sequence heterogeneity resulted in both the creation and the elimination of N-linked glycosylation sites. Only nine clones differed from the consensus sequence in the V3 loop. No inactivating mutations were found. CONCLUSIONS: HIV-1 proviruses found in the brain generally demonstrate a low level of genetic variability in env. However, genomes that vary considerably from the predominant species can be present at significant levels. This observation may be of importance for understanding viral pathogenesis in the central nervous system.

AIDS Dementia Complex↗
PubMed1994

Distinct effects in primary macrophages and lymphocytes of the human immunodeficiency virus type 1 accessory genes vpr, vpu, and nef: mutational analysis of a primary HIV-1 isolate.

Macrophages and lymphocytes are the two main targets for productive HIV-1 infection in vivo. To compare the effects of the "nonessential" HIV-1 accessory genes vpr, vpu, and nef on viral replication in these primary cell types, we generated a panel of mutant viruses derived from a molecularly cloned macrophage-tropic HIV-1 primary isolate. Mutant viruses had markedly different patterns of replication in macrophages, in contrast to lymphocytes in which differences were modest. Loss of vpr or vpu reduced viral antigen production in macrophages by up to 1000-fold, while replication in lymphocytes was only marginally affected. Loss of nef did not affect lymphocyte infection, but decreased replication in macrophages to a small extent. Mutation of multiple accessory genes restricted replication in both cell types, but to a much greater extent in macrophages, and frequently resulted in nonproductive infection. The degree to which replication depended on intact accessory genes varied in macrophages from different donors. The essential functions of these accessory genes in HIV-1 infection may be related to their combined effects in facilitating productive infection of macrophages.

Base Sequence↗
PubMed1994

Benzopurpurin and related compounds inhibit the binding of gp120 to galactosyl ceramide/sulfatide and infection of human immunodeficiency virus.

In an attempt to find compounds capable of inhibiting the binding and infection by human immunodeficiency virus type 1 (HIV-1) in neural cells, we studied the effect of benzopurpurin and related compounds on the binding of gp120 to galactosyl ceramide (GalCer) and sulfatide. In this report, we show that the binding of gp120 to GalCer and sulfatide is inhibited by benzopurpurin and related compounds. These compounds also inhibit the binding and entry of HIV-1 to neural cell line, SK-N-MC. Binding studies indicate that benzopurpurin and related compounds bind to gp120. Studies involving related compounds indicate that the minimal structure required for the inhibition is two naphthalene rings with amine or sulfonic acids attached to a central biphenyl molecule by an azo group. This approach will be useful for screening of potential anti-HIV compounds.

Antiviral Agents↗
PubMed1994

Establishment of a human retinal cell line by transfection of SV40 T antigen gene with potential to undergo neuronal differentiation.

Recently, a number of laboratories have been interested in developing cell lines of ocular tissues to understand the pathogenesis of ocular diseases. Toward this end, we report here the generation of cell lines of human retina by transfection of simian virus SV40 T antigen gene. Established retinal cells grow as a monolayer and exhibit limited serum dependence. Phase-contrast and electron microscopic studies revealed distinct morphological cell types. Immunofluorescence studies showed that the established retinal cells were positive for neuron-specific enolase, neurofilament protein, glycine receptor, synaptophysin, and secretogranin. Cells were negative for glial fibrillary acidic protein, glutamine synthetase, galactocerebroside, and carbonic anhydrase II. In addition to neuronal features, a small percentage of flat cells were, however, positive for cellular retinaldehyde binding protein, and cells with the phenotype of rod and cone photoreceptor coexpressed opsin and interphotoreceptor retinoid-binding protein. An important feature of this cell line is that addition of phorbol ester and cAMP induced dramatic changes, with 100% of the cells extending long, thin neuritic processes. Thus, the established retinal cells would be useful for studies dealing with differentiation and plasticity of the cells of the nervous system.

Antigens, Polyomavirus Transforming↗
PubMed1994

Rotavirus as an aetiological organism in acute watery diarrhoea in Delhi children: reappraisal of clinical and epidemiological characteristics.

Four-hundred children below 3 years of age with acute watery diarrhoea were studied over a 12-month period for detection of rotavirus (RV) antigen in the stool samples. Detection of RV antigen in the cases has been compared with 175 age-matched non-diarrhoea controls. Overall positivity for detection of RV antigen was 6 per cent which is much lower than earlier studies. Even during the peak season the RV antigen detection was positive only in 13 per cent cases. However, RV antigen detection was significantly higher in cases than in the controls (P < 0.05). No significant correlation was noticed between the detection of RV antigen in the stool and environmental temperature, rainfall, and relative humidity except for higher percentage of RV antigen detection between February and April. Clinical profile of cases with RV antigen-positive stools has been compared with RV antigen-negative controls. Preceding history of vomiting and respiratory symptoms were noticed in higher percentage of RV antigen-positive cases, but no reliable 'clinical predictor' could be identified to differentiate RV diarrhoea from non-RV diarrhoea. Presence or absence of fecal polymorphonuclear leucocytes do not seem to rule out or suggest RV diarrhoea.

Antigens, Viral↗
PubMed1994

Investigations into size heterogeneity of the alpha B-crystallin mRNA.

alpha B-crystallin is expressed in a variety of developmental, physiological and pathological conditions in a number of tissues. Based on northern blots of total RNA, the existence of at least two size classes of alpha B-crystallin mRNAs has been reported, one smaller predominant species, 0.8-0.9 kb, and the other 1.2 to 1.4 kb. This heterogeneity has been attributed to alternative upstream transcriptional initiation. We have investigated the origin of the size heterogeneity of alpha B-crystallin mRNA by using 5'-upstream-, coding- and 3'-untranslated-region probes in RNAse protection and northern blot assays. RNAse protection assays indicate that there is only one predominant initiation site as previously reported and that the second polyadenylation signal is not used in the rat gene. Importantly, northern blot data obtained with coding region-only probe shows that the size of alpha B mRNA detected in the heart and the lens is similar (0.78 kb) in poly A+ as well as in total RNA. On the other hand, in the brain and in the lung, the larger hybridizing species (1.05 kb and 1.22 kb respectively) seen in total RNA are not detected in poly A+RNA which shows a 0.95 kb species in both tissues. The 5' upstream probe (-1 to -499) produces weak hybridization patterns in the brain and the lung, similar to those obtained with coding region-only probe. The 5' probe did not show hybridization in the heart and the lens RNAs. These data suggest that upstream initiations represent a minor population of transcripts and that higher size transcripts (about 500 bp larger) actually represent non-polyadenylated RNAs that may not contribute to the generation of the actual gene product.

Animals↗
PubMed1993

Recombination between human immunodeficiency viruses (HIV) type 1 and 2 results in generation of defective hybrid viruses.

Dual infection by HIV-1 and HIV-2 is already documented. To test if this could lead to recombination and generation of altered viruses, recombination between HIV-1 and HIV-2 DNAs was studied. Release of recombinant viruses was detected following cotransfection of human rhabdomyosarcoma cells with truncated versions of these proviral DNAs. Linearization of plasmid DNAs was required for virus production. Analysis of viral particles by hybridization revealed the presence of viral RNA. However viral replication was not evident. A computer search of the overlap region between the substrate DNAs revealed 66% homology despite an overall genomic sequence homology of only 35%. These data suggest possible generation of replication-deficient hybrid viruses as a result of recombination between HIV-1 and -2.

Base Sequence↗
PubMed1993

Transdifferentiation of adult human pigment epithelium into retinal cells by transfection with an activated H-ras proto-oncogene.

The identification of homologs to viral oncogenes in normal cells coupled with development of techniques for DNA transfer into cells offers a powerful approach to dissect the processes associated with differentiation-specific oncogenes. We have derived cell lines by transfection of viral DNAs and proto-oncogenes into primary retinal pigment epithelial (RPE) cells. Establishment of cell lines was successfully achieved with the SV40 large T-antigen gene activated form of Harvey (H)-ras proto-oncogene, c-myc, and adenovirus E1A. The cell lines derived using the H-ras oncogene appeared to contain cells with a neuronal phenotype. This feature was not observed in cell lines established with the other oncogenes. Characteristically, H-ras-transfected cells all exhibited features associated with neurons around 10-14 passages. The transdifferentiated cells were biochemically characterized and found to express neuronal markers, such as neurofilament protein and neuron-specific enolases. The specific neuronal changes were restricted to only two primary cultures of RPE derived from carcinoma donors. Although transdifferentiation of pigmented cells of iris, or the retina, into the lens has been demonstrated, our studies presented in this report provide evidence that RPE cells from adults can transdifferentiate into neurons under the influence of a specific oncogene. To the best of our knowledge, this is the first report on transdifferentiation of adult human pigment epithelium into a neuronal cell type.

Cell Differentiation↗
PubMed1993

Changes in cellular proteins associated with the expression of human immunodeficiency virus type 1 trans-activator protein Tat.

Earlier studies have revealed a distinct class of regulatory proteins known as trans-activator proteins in diverse biological systems. These proteins have been shown to act on both homologous and heterologous promoter targets. Activation of heterologous targets is speculated to be an integral part of virus-induced pathogenesis. To verify this hypothesis, stable Tat-producing human rhabdomyosarcoma (RD) cell lines were generated. These cell lines produced significant levels of functional Tat, as measured by transfection with the reporter plasmid pLTR-CAT. Tat-producing cells, although morphologically similar to the control, exhibited a slower growth rate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the cellular proteins from control (tat-) and tat+ cells revealed increased quantities of 34- and 40-kD proteins along with the appearance of a new 74-kD protein in tat+ cells. Subsequent two-dimensional gel analysis revealed several additional differences. Tat+ cell lines produced two proteins of M(r) 19.5 and 44 kD anew, while proteins with M(r) 14.5, 42, and 52.5 kD were in greater abundance. Interestingly, a 26-kD protein that was originally present in the G418+/tat- (control) sample disappeared in the presence of Tat. These data support a possible modulator role for Tat in cellular gene expression.

Cell Line↗
PubMed1993

PEIRS: a pathologist-maintained expert system for the interpretation of chemical pathology reports.

Provision of a comprehensive interpretative service is an important challenge facing chemical pathologists. Attempts to automate report interpretation using expert systems have been limited in the past by the difficulties of rule base maintenance. We have applied a novel knowledge acquisition technique, ripple down rules, in the development of PEIRS (Pathology Expert Interpretative Reporting System), a user-maintained expert system for automating chemical pathology report interpretation. We created over 950 rules for thyroid function tests, arterial blood gases and other test sub-groups in 9 mths of operation. A staff pathologist performed all maintenance tasks as part of his routine duties without any need for computer programming skills. No clerical staff involvement was required. Duplication of rule addition for reports requiring multiple comments was the only limitation to coverage of other high volume test groups. PEIRS is the first expert system for the automated interpretation of a range of chemical pathology reports which operates in routine use without extra staffing requirements. PEIRS does not require "knowledge engineering" expertise. Thus, the knowledge base is flexible and can be easily maintained and updated by the pathologist. Expert systems based on ripple down rules should enable pathologists to provide a comprehensive automated interpretative service within the context of the total testing process.

Chemistry, Clinical↗