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Biomedical subjects

R Pottathil

Publications and source records attributed to R Pottathil.

14 recordsLinked to original sources
PubMed1992

Improved blood sample processing for PCR.

To simplify our procedure for blood sample processing for PCR, we introduced a simpler, shorter, and more cost-effective method for the separation of peripheral blood lymphocytes (PBL) and DNA extraction for amplification with Taq polymerase. By this method, blood samples are processed in two simple 15-min steps: (1) separation of PBLs from whole blood by red blood cell lysis with the Roche Specimen Washing Solution, and (2) DNA extraction by heat-detergent treatment of separated PBLs. This new method is simpler than the standard Ficoll-Hypaque method for PBLs separation and Proteinase K digestion for DNA extraction. It is not inhibitory to DNA amplification and it allows effective processing of blood samples even after prolonged storage (as long as 8 days) at room temperature.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Constant and variable antigenic regions of the HIV.

Human immunodeficiency virus (HIV) is the etiological agent in acquired immunodeficiency syndrome (AIDS). The presence of antibodies against HIV is indicative of prior exposure to HIV. Currently available antibody screening tests use HIV-infected cells as a source of viral antigens. This approach has resulted in inherent flaws, including a high rate of false-positive results. In order to develop a highly specific and sensitive test, various proteins of HIV [envelope (env), gag, pol] were cloned and expressed. In addition, we synthesized peptides corresponding to various regions of envelope (gp120, gp41), tat, art, and the protease gene products were synthesized. Using these synthetic peptides and cloned proteins, several conserved and highly antigenic epitopes in viral envelope were identified. Fusion proteins were made from the highly conserved and antigenic epitopes of env and gag. One of these fusion proteins, env-gag, was used to develop an enzyme immunoassay that would detect the antibodies against both env and gag epitopes. A clinical study of 13,781 samples demonstrated the very high specificity (99.9%) and sensitivity (100%) of this assay.

Antigenic Variation↗
PubMed1988

Immunoregulatory activities of human immunodeficiency virus (HIV) proteins: effect of HIV recombinant and synthetic peptides on immunoglobulin synthesis and proliferative responses by normal lymphocytes.

Recombinant and synthetic peptides corresponding to envelope proteins of the human immunodeficiency virus (HIV) were examined for their effects on the activities of lymphocytes from normal donors in vitro. Although lymphocytes cultured with env-gag peptides produced significant amounts of IgG, addition of env-gag peptides to a pokeweed mitogen-induced B-cell activation system resulted in suppression of immunoglobulin synthesis by normal lymphocytes. Recombinant antigens, env-gag and env-80 dihydrofolate reductase (DHFR), produced a substantial proliferative response by peripheral blood mononuclear cells (PBMC) as determined by [3H]thymidine incorporation. PBMC precultured with HIV synthetic peptide env 578-608 also manifested significant proliferative responses as compared to control cultures. CD3+ lymphocytes precultured with recombinant HIV antigens, env-gag and env-80 DHFR, and synthetic HIV peptide, env 487-511, showed moderate but significant proliferative responses. Both recombinant antigens and synthetic peptides also produced a dose-dependent stimulatory effect on proliferation by CD3- lymphocytes. Stimulation of CD3+ and CD3- lymphocyte subpopulations induced by env-gag peptides was specifically inhibited by goat anti-env-gag polyclonal antibodies, demonstrating the specificity of the reaction. These studies demonstrate that recombinant and synthetic peptides of the HIV genome express immunoregulatory T- and B-cell epitopes. Identification of unique HIV epitopes with immunogenic and immunoregulatory activities is necessary for the development of an effective vaccine against HIV infection.

Adult↗
PubMed1987

Comparison of recombinant human immunodeficiency virus gag precursor and gag/env fusion proteins and a synthetic env peptide as diagnostic reagents.

Diagnostic reagents for detection of human immunodeficiency virus (HIV) exposure with improved reliability may be provided by viral encoded proteins produced by recombinant DNA techniques or by synthetic peptides corresponding to appropriate viral epitopes. We have expressed at high levels in E. coli a gag gene segment corresponding to approximately 97% of the p55 gag precursor protein, as well as a novel gag/env fusion protein that contains antigenic determinants in common with gag p24, env gp41, and env gp120. The gag and gag/env proteins were purified from insoluble inclusion bodies by sequential extraction with increasing concentrations of urea. These components were tested for reactivity with antisera to HIV proteins and peptides. We have also chemically synthesized a peptide corresponding to env residues 578-608, representing a portion of env gp41. The final preparation of gag and gag/env proteins in 8 M urea reacted with sheep anti-HTLV-III p24 gag antibodies and acquired immune deficiency syndrome (AIDS) patient sera. The gag/env fusion protein also reacted with rabbit anti-HIV env 500-511 peptide antibody. Both recombinant proteins and the env peptide were suitable as reagents for evaluation of serum samples by enzyme-linked immunosorbent assay (ELISA). Results of ELISA assays utilizing the recombinant viral proteins and synthetic peptide were in good agreement with results obtained using disrupted virus as antigen in ELISA assays and immunoblotting.

Acquired Immunodeficiency Syndrome↗
PubMed1986

Studies on an antineoplastic fraction from human urine. Characterization of the major protein in this fraction.

A fraction has been isolated from human urine which exhibits antiproliferative activity against human tumour cell lines without affecting the growth of several normal diploid cell lines or tumour cells of mouse or hamster origin. The major protein present in this fraction has been characterized and tentatively designated antineoplastic urinary protein (ANUP). An S020,W value of 3.69 S was obtained by sedimentation velocity analysis, and a subunit molecular mass of 16 300 Da was obtained by sedimentation equilibrium and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Centrifugation data also indicated that the protein self-associates. The amino acid analysis of ANUP was consistent with its low pI (4.2) as determined by chromatofocusing analysis. Furthermore, the amino acid composition exhibited some features similar to collagen, as shown by high levels of proline and glycine, the absence of cysteine, and the presence of low levels of hydroxyproline.

Amino Acids↗
PubMed1986

Enhanced replication of murine cytomegalovirus in murine leukemic lymphocytes.

Replication in vitro of murine cytomegalovirus was found to be enhanced in lymphoid cells from leukemic (AKR/J) mice as compared with similar cells from nonleukemic animals. Prolonged productive murine cytomegalovirus infection in lymphoid organs in vivo was demonstrable only in leukemic AKR/J mice. Latent nonproductive murine cytomegalovirus infection established in young nonleukemic AKR/J mice was invariably reactivated and expressed in salivary glands and lymphoid organs after these animals became leukemic.

Animals↗
PubMed1985

Role of cell membrane composition in receptor-mediated internalization of vesicular stomatitis virus in human HEp-2 cells.

The role of essential fatty acids in membrane functions related to receptor-mediated endocytosis of vesicular stomatitis virus (VSV) was investigated using a human laryngeal carcinoma cell line (HEp-2) grown in chemically defined serum-free medium (DM) to deplete their essential fatty acid contents. VSV replicated much less effectively in HEp-2 cells grown in DM as compared to serum containing complete medium (CM). Observed reduction in the rate of virus multiplication was, at least in part, due to reduced virus penetration which was monitored using VSV labeled with nitroxyl free radicals as electron spin probe. Surface proteins of VSV were labeled with maleimide spin-label, and succinimide spin-label. Ni2+ was used as a broadening agent to identify the spin-label signals from viruses inside the cell. HEp-2 cells and mouse leukemia cell line L1210 treated with 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) cadaverine, an agent previously shown to inhibit the uptake of VSV in vitro, was used as a positive control in some experiments. VSV penetrated less effectively in both DM-grown cells and in CM-grown cells in the presence of dansylcadaverine. Similar results were obtained by monitoring the uptake of 125I-labeled VSV. When HEp-2 cells grown for several generations in DM were incubated with 10% fetal calf serum for 16 h, the cells supported virus replication to a similar extent as the cells grown in CM. In contrast, addition of arachidonic acid restored VSV growth only partially. Continued growth of HEp-2 cells in DM resulted in a shift in fatty acyl chain composition of phospholipids. The results indicate a finite role for essential fatty acids in receptor-mediated internalization of virus particles.

Animals↗
PubMed1985

Essential fatty acids in diabetes and systemic lupus erythematosus (SLE) patients.

Sera obtained from normal subjects and juvenile-onset diabetes (JD) and systemic lupus erythromatus (SLE) patients were examined for free fatty acid composition and 6-keto-PGF1 alpha content. In addition, prostaglandins in urine samples from normal and diabetic individuals were separated by HPLC, and 6-keto-PGF1 alpha levels were monitored by RIA. Arachidonate (20:4) content in diabetic and SLE individuals were significantly lower than that of controls. Urine from diabetic individuals showed decreased levels of 6-keto-PG F1 alpha. The study also indicated that RIA measurements on crude biological samples may yield erroneous data due to immune cross reactivity with other compounds.

6-Ketoprostaglandin F1 alpha↗
PubMed1985

A 64,000 dalton matrix protein of human cytomegalovirus induces in vitro immune responses similar to those of whole viral antigen.

Human cytomegalovirus contains approximately 30 to 35 structural polypeptides. Although antibodies to several of these proteins are made during natural infection, their relationship to T cell recognition of this virus and subsequent control of infection is poorly understood. We have purified one of these proteins (HCMVgp64) that is found in abundance in infected cell lysates in order to delineate the relationship of single viral proteins to the immune response caused by the virus. HCMVgp64 induced T cell reactivity only in individuals with serologic evidence of past infection. In addition, HCMVgp64 elicited similar in vitro immune reactions as the whole virus including T cell proliferation, interleukin 2 production, and receptor expression as well as interferon production. These studies suggest that single proteins of HCMV such as HCMVgp64 are capable of inducing T cell responses and may be important in the development of immune reactivity to HCMV.

Antigens, Viral↗
PubMed1977

Antitumor effects of RNA isolated from murine tumors and embryos.

We describe the antitumor effects of a certain RNA(s) isolated from murine tumors and embryos. A single i.v. injected of 10 to 30 microgram of this RNA(s) induces necrosis, hemorrhages, and regression of solid tumors in the strain of origin; in pregnant mice it is embryotoxic, causing resorption of the embryos, but has no toxic effect upon the tumor-bearing or pregnant host. Its action is highly specific, and its presence in both tumors and embryos suggests that it may result from reexpression of embryonic genes in tumor cells.

Adenocarcinoma↗