Biomedical subjects

A Srinivasan

Publications and source records attributed to A Srinivasan.

At least 235 records · Page 13Linked to original sources

PubMed1989

Mutational analysis of sodium butyrate inducible elements in the human immunodeficiency virus type I long terminal repeat.

Sodium butyrate induces gene expression directed by the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in HeLa cells. Inducible regions of the HIV-1 LTR were elucidated by using 5' and 3' LTR deletion mutants and LTR site-directed mutants within the Sp1 binding sites and the trans-activation responsive (TAR) region. Two LTR regions inducible by sodium butyrate were located: one at -117 to -103 (distal site) and one at -65 to -17 (proximal site). In HeLa cells trans-fected with pZ6neo, a biologically active HIV-1 proviral clone, sodium butyrate stimulated virus production following a 3-day treatment. Inducibility of HIV-1 gene expression by sodium butyrate was unrestricted in many human cell types, including CD4+ lymphoid cells and non-CD+ brain cells and fibroblasts. Additionally, sodium butyrate transiently induced HIV-2 LTR-directed gene expression in HeLa cells. Using the HIV-1SF-2 tat gene cotransfected with pLTR-CAT site-directed TAR mutants in HeLa cells, the boundaries of tat-trans-activation were delineated more precisely. These results suggest that the induction of HIV-1 gene expression is mediated by the interaction of sodium butyrate with cellular transcription factors that bind to the HIV-LTR.

Base Sequence↗

PubMed1989

Mutants with changes within or near a hydrophobic region of simian virus 40 large tumor antigen are defective for binding cellular protein p53.

SV40 mutants bearing either amino acid substitution or in-frame deletion/insertion mutations in a region of the gene for large T antigen encoding a stretch of hydrophobic residues were analyzed for their behavior in permissive and nonpermissive cells. One of the mutants, with an Ile(573)-Phe substitution had a phenotype indistinguishable from that of wild-type SV40. The remaining three mutants were not viable and were defective for DNA replication. In addition, they displayed a cell-type specificity with respect to transformation; namely, they transformed the mouse C3H10T1/2 cell line, although with a reduced efficiency relative to wild-type, but were unable to transform the rat REF52 cell line. None of the T antigens from the defective mutants formed a complex with the cellular protein p53, indicating that the T-antigen-p53 complex is not required for the transformation of C3H10T1/2 cells.

Amino Acid Sequence↗

PubMed1989

Biological and molecular characterization of human immunodeficiency virus (HIV-1BR) from the brain of a patient with progressive dementia.

HIV-1BR was isolated from the autopsied brain tissue of a 57-year-old man who died of progressive dementing illness. This virus was shown to be HIV-1 by hybridization to HIV-specific DNA probes. The expression of viral proteins as tested by radioimmunoprecipitation assay revealed the presence of HIV-1-specific proteins. HIV-1BR replicated in cultures of CD4+ T-lymphoid cells and induced cytopathic effects in these cells. HIV-1BR also replicated in monocytoid cell lines. The genetic nature of this isolate was determined by molecular cloning and sequencing of the 3'-half of the genome. DNA sequence information established that HIV-1BR is a unique HIV-1 isolate. A stretch of approximately 30 bases in the nef gene of HIV-1BR was found duplicated when compared with the other sequenced HIV-1 genomes. The functional significance of this duplication remains to be determined.

Acquired Immunodeficiency Syndrome↗

PubMed1989

Generation of hybrid human immunodeficiency virus by homologous recombination.

Human immunodeficiency virus (HIV) type 1, isolated from diverse sources, exhibits genomic diversity. The mechanisms by which the genomic diversity takes place in individuals exposed to multiple virus isolates is yet to be elucidated. Genetic variation, in general, might result from mutagenic events such as point mutations, rearrangements (insertions and deletions), and recombination. In an attempt to evaluate the process of genetic diversity, we designed experiments to analyze recombination between HIV DNAs by using DNA transfection in cell cultures. Here we report the successful recombination between truncated HIV proviral DNAs with an overlap homology of 53 base pairs that leads to the formation of viable hybrid virus. Recombination was also seen between exogenous DNA introduced into cells and homologous HIV sequences resident in the cells. These results indicate that recombination among various HIV isolates may play a significant role in the generation of genetic diversity of HIV. Further, the method used here enables the construction of hybrid HIV genomes to identify the viral determinants responsible for tropism, replication, and cytopathic effects.

Base Sequence↗

PubMed1989

Molecular characterization of HIV-1 isolated from a serum collected in 1976: nucleotide sequence comparison to recent isolates and generation of hybrid HIV.

Human immunodeficiency virus type 1 (Z321 designate, HIV-1Z321), the oldest known HIV, was isolated from a serum sample collected in Zaire in 1976 and was molecularly cloned. Restriction enzyme analysis of unintegrated viral DNA revealed the presence of conserved restriction enzyme cleavage sites in the long terminal repeat sequences. Nucleotide sequence analysis of the 3' end of the viral DNA revealed a pattern similar to other HIV-1 isolates described. However, some of the common restriction sites present in other isolates were absent in HIV-1Z321. The extent of differences between HIV-1Z321 and recent isolates from North America and Zaire was 17.86-18.36% on the nucleotide sequence level and 26.5-33.2% difference in the predicted amino acid sequence in the envelope gene. Differences were also noted in 3'-orf (nef: according to HIV gene nomenclature; see Ref. 42) gene and U3 region of the long terminal repeat sequences of HIV-1Z321 and other isolates. Nucleotide sequence of a HIV-1 isolate, 12 years apart from the present isolates, will provide an important time calibration point for the evolutionary divergence of HIV isolates. Hybrid HIV was also generated by transfecting HIV-1Z321 and HIV-1HTLV-III viral DNAs into cells.

Amino Acid Sequence↗

PubMed1989

The large tumor antigen of simian virus 40 encodes at least two distinct transforming functions.

The large tumor antigen (T antigen) of simian virus 40 is necessary and sufficient for the neoplastic transformation of a number of established cell lines. Mutational analysis has revealed that a biochemical activity residing within the amino-terminal 121 amino acids of T antigen is sufficient to induce the transformation of some cell lines, such as C3H10T1/2. The same domain of the molecule also encodes the transactivation function of T antigen and the ability to complex with the retinoblastoma susceptibility gene product. However, the transformation of other lines, such as REF52, requires an additional activity that is affected by mutations in other portions of the molecule.

Animals↗

PubMed1989

Properties of a simian virus 40 mutant T antigen substituted in the hydrophobic region: defective ATPase and oligomerization activities and altered phosphorylation accompany an inability to complex with cellular p53.

We have analyzed the biochemical properties of a nonviable simian virus 40 (SV40) mutant encoding a large T antigen (T) bearing an amino acid substitution (Pro-584-Leu) in its hydrophobic region. Mutant 5080 has an altered cell type specificity for transformation (transforming mouse C3H10T1/2 but not rat REF52 cells), is defective for viral DNA replication, and encodes a T that is unable to form a complex with the cellular p53 protein (K. Peden, A. Srinivasan, J. Farber, and J. Pipas, Virology 168:13-21, 1989). In this article, we show that 5080-transformed C3H10T1/2 cell lines express an altered T that is synthesized at a significantly higher rate but with a shorter half-life than normal T from wild-type SV40-transformed cells. 5080 T did not oligomerize beyond 5 to 10S in size compared with normal T, which oligomerized predominantly to 14 to 20S species. In addition, the 5080 T complex had significantly decreased ATPase activity and had a 10-fold-lower level of in vivo phosphorylation compared with that of normal T. Two-dimensional phosphopeptide analysis indicated several changes in the specific 32P labeling pattern, with altered phosphorylation occurring at both termini of the mutant protein compared with the wild-type T. Loss of p53 binding is therefore concomitant with changes in ATPase activity, oligomerization, stability, and in vivo phosphorylation of T and can be correlated with defective replication and restricted transformation functions. That so many biochemical changes are associated with a single substitution in the hydrophobic region of T is consistent with its importance in regulating higher-order structural and functional relationships in SV40 T.

Adenosine Triphosphatases↗

PubMed1989

Replication of HIV in human fetal retinal cultures and established pigment epithelial cell lines.

The ability of the human immunodeficiency virus type 1 (HIV-1) to replicate in cells derived from ocular tissue was studied. Primary retinal cultures (containing both glial and neuronal cells) were found to support the replication of HIV upon transfection with molecularly cloned proviral DNA. In addition, established retinal pigment epithelial (RPE) cell lines also produced HIV particles upon transfection. HIV released by these cell lines was able to infect and induce characteristic cytopathic effects in T4+ cells. An indicator plasmid containing the HIV long terminal repeat sequences (LTR) linked to the chloramphenicol acetyltransferase gene showed barely detectable activity in RPE cells and was transactivated by the addition of the HIV "tat" gene. Based on these observations, direct infection of ocular tissue derived cells such as RPE, fetal retinal cells, retinoblastoma cells (Y 79, WER1), choroidal endothelial cells (Chor 55) (mix culture) and corneal fibroblasts (K61) by HIV was attempted. HIV replication in these cells was not detected by reverse transcriptase, antigen and transactivation function assays.

Adult↗

PubMed1989

Detection of human immunodeficiency virus (HIV) and human lymphotropic virus (HTLV) type I or II dual infections by polymerase chain reaction.

To type the specific human T-lymphotropic virus (HTLV) coinfection in acquired immunodeficiency syndrome (AIDS) patients, we have utilized the polymerase chain reaction technique. The primer pairs employed for HTLV-I and HTLV-II are derived from pol and env regions and gag region for human immunodeficiency virus (HIV). The data indicate that the selected primer pairs are specific for each virus and are able to detect HTLV-I and -II coinfection in lymphoid cells established from AIDS patients.

Acquired Immunodeficiency Syndrome↗

PubMed1989

Successful imaging of malignant melanoma with technetium-99m-labeled monoclonal antibodies.

F(ab')2 and Fab fragments of murine monoclonal antibody 9.2.27, that recognizes the 250 kD melanoma-associated antigen, were labeled with 99mTc using the bifunctional chelate method of Fritzberg et al. Twenty-seven (27) patients received, intravenously, 10 mg of either F(ab')2 (8), or the Fab (27), labeled with up to 30 mCi of 99mTc. These doses were preceded by an infusion of cold irrelevant antibody. The average serum T1/2 of the F(ab')2 and the Fab were 11 hr and 2 hr, respectively. Twenty-two percent (22%) of the total injected F(ab')2 dose was excreted in the urine in 20 hr, compared to 55% for the Fab group. Imaging was optimal 6-9 hr postinjection for the Fab patients. No nonspecific uptake in liver, spleen, bone marrow, or lung was observed for either antibody form. Overall, (43/53) 81% of known metastases were seen with visualization of tumors as small as 250 mg and tumor localization as high as 0.03% injected dose/g. Immunoperoxidase staining of freshly-frozen tumor nodules removed 24 hr postinjection confirmed antibody deposition in the tumor. Thirty-six previously unknown ("occult") metastatic sites were detected. To date, 12/36 of these sites have been confirmed. We conclude that 99mTc-labeled antibody to melanoma produces high resolution images with a high sensitivity of detecting metastatic melanoma. The detection of previously unknown sites of disease has proven helpful in directing additional diagnostic studies (i.e., CT) as well as planning of therapeutic options.

Antibodies, Monoclonal↗

PubMed1989

Serum & cerebrospinal fluid ferritin levels in children with acute leukaemia.

Serum and CSF ferritin were estimated in 35 consecutive patients of acute leukaemia at the time of admission and on induction of remission. Serum ferritin levels were significantly raised in 94 per cent patients of acute leukaemia. The mean (+/- SD) serum ferritin (314.36 +/- 158.4 micrograms/1) was significantly higher when compared with control values (P less than 0.001). Remission induction resulted in significant fall in serum ferritin values to a mean of 149 (+/- 98.7) micrograms/l (P less than 0.05). Serum ferritin is thus of value in assessing the state of remission and is a sensitive indicator of the leukaemic cell mass and the state of activity of the disease. CSF ferritin levels in acute leukaemia were comparable to normal control values. CSF ferritin did not reflect CNS involvement in acute leukaemia and therefore its value as a tumour marker of CNS infiltration is doubtful.

Acute Disease↗

PubMed1988

Homologous recombination between human immunodeficiency viral DNAs in cultured human cells: analysis of the factors influencing recombination.

Recombination between HIV DNAs was analyzed using DNA transfection in cell cultures and the optimal conditions for efficient recombination were determined. Recombinant plasmid DNA substrates were constructed from HIV proviral DNAs and the success of recombination was measured by the production of viable hybrid virus. The process of recombination between HIV DNAs was shown to be i) dependent on homology between the truncated HIV DNAs and ii) maximum with concentrations of the truncated DNAs 3ug and above. HIV isolates with heterogeneity in their primary sequence, thus offer an ideal system for the analysis of the requirement of homologous recombination. In addition, recombination methodology would be useful for generating hybrid HIVs for the analysis of specific viral gene functions.

Calcium Phosphates↗

PubMed1988

The phylogenetic history of immunodeficiency viruses.

Knowledge of the phylogenetic history of the human immunodeficiency viruses (HIV-1 and HIV-2) is important for our understanding of the epidemiology of AIDS, the disease caused by these viruses. Reconstruction of the evolutionary tree is hampered, however, by two problems. One is the high variation in nucleotide sequence between the known HIV isolates which can create formidable difficulties in identifying homologous genomic sites that may be used in a molecular phylogenetic reconstruction. Another impediment has been the lack of unequivocal time calibration points: there is only a sparse 'fossil record' for HIV and limited historical epidemiological data. We have largely overcome these difficulties by: (1) a thorough optimal-sequence alignment analysis; (2) the inclusion of sequences of an early (1976) HIV-1 isolate, a recent (1986) HIV-2 isolate and two simian immunodeficiency viruses (SIV) along with five other HIV-1 isolates; and (3) the reconstruction of a minimum-length evolutionary tree based on the envelope-gene variable positions. We conclude that HIV-1 may have evolved from its common ancestor with HIV-2 as recently as 40 years ago.

Animals↗

PubMed1988

Preliminary molecular characterization of a human immunodeficiency virus (HIV-I) associated with neuropathology.

A human immunodeficiency virus (HIV-I) was isolated from the brain of a patient with progressive dementia but no obvious immunosuppression. This isolate, designated as HIV-IBR, was molecularly cloned and sequenced, and its long terminal repeat (LTR) and envelope sequences were compared with those of other HIV isolates not uniquely associated with dementia. The HIV-IBR LTR showed marked homology with the LTR sequences of the other HIV-I isolates. The predicted amino acid sequence of the external glycoprotein (gp120) of HIV-IBR revealed a pattern of conserved and variable regions similar to that of other HIV isolates. The sequence of the transmembrane portion of envelope, gp 41, was highly homologous to the counterpart region of other isolates. Further analysis is required to determine whether specific sequence variation can account for neurological manifestations of HIV-IBR infection.

Acquired Immunodeficiency Syndrome↗

PubMed1988

Studies on human immunodeficiency virus-induced cytopathic effects: use of human rhabdomyosarcoma (RD) cells.

A full-length molecular clone of human immunodeficiency virus (HIV) proviral DNA was transferred to human rhabdomyosarcoma (RD) cells by gene transfer method. RD cells released infectious virus within 12 hours after transfection and the viral particles present in the culture medium could be quantitated by monitoring reverse transcriptase activity. Chronic low level viral producer cell lines of RD were also established. Southern hybridization analysis revealed the presence of HIV sequences in transfected RD cells. Electron microscopic studies of the transfected cell revealed intracellular budding of HIV and also showed structural abnormalities such as giant cell phenotype and vacuolation. These features qualify RD cells as a useful system for studying the regulation and cytopathic effects of HIV.

Cytopathogenic Effect, Viral↗

PubMed1988

Human immunodeficiency virus replication in human brain cells. Brief report.

Human immunodeficiency virus (HIV), the etiologic agent of AIDS, was found to infect and replicate in human brain cells. The extent of HIV replication was minimal in human brain-derived cells in comparison to T4 lymphoid cells. These results suggest that direct infection of glial/neuronal cells by HIV may contribute to the CNS dysfunction frequently observed in HIV infected individuals.

Brain↗

PubMed1988

Specific and stable labeling of antibodies with technetium-99m with a diamide dithiolate chelating agent.

Technetium-99m labeling of antibodies has been suboptimal because of low affinity adventitious binding, nonspecific labeling, and loss of immunoreactivity. The diamide dithiolate ligand system (N2S2) forms highly stable, well-defined tetradentate complexes with Tc(V). Antibodies and their fragments have been labeled by conjugation of preformed 99mTc-4,5-bis(thioacetamido)pentanoate active ester to protein amine groups to give a chemically known 99mTc-N2S2 complex covalently linked to antibody. Evaluations of the 99mTc-N2S2-bound antibodies and their fragments have shown high stability and retained immunoreactivity.

Animals↗

PubMed1988

Interaction between rifabutin and human immunodeficiency virus type 1: inhibition of replication, cytopathic effect, and reverse transcriptase in vitro.

Rifabutin (ansamycin; LM427), a semisynthetic rifamycin derivative, inhibits the replication of human immunodeficiency virus type 1, the virus etiologically linked to acquired immunodeficiency syndrome. The inhibition of virus production was observed in human immunodeficiency virus type 1-infected peripheral blood mononuclear cells both by reverse transcriptase assay and virus-antigen assay. In addition, rifabutin effectively reduced human immunodeficiency virus type 1-associated cytopathic effect to OKT4-positive cells. Virion-associated reverse transcriptase was also markedly inhibited by rifabutin. The inhibitory dose of rifabutin was nontoxic to lymphoid cells, which further suggests that it might serve as a useful agent in the therapeutic modalities of acquired immunodeficiency syndrome.

Cell Line↗