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Biomedical subjects

S Ramakrishnan

Publications and source records attributed to S Ramakrishnan.

At least 91 records · Page 5Linked to original sources
PubMed1997

Vascular endothelial growth factor expression in early stage ovarian carcinoma.

BACKGROUND: Tumor angiogenesis is essential for solid tumor growth. Yet, the importance of any particular factor in neoplastic proliferation is poorly defined. This study examines the clinical significance of increased expression of one of the angiogenic factors, vascular endothelial growth factor (VEGF), in early stage ovarian carcinoma. METHODS: Tumor specimens from 68 patients with International Federation of Gynecology and Obstetrics Stage I and II ovarian carcinoma were evaluated for VEGF expression. Antisense and corresponding sense (control) RNA probes were transcribed from the pCRII construct (Invitrogen, San Diego, CA), which contained human VEGF cDNA. The antisense probe was designed to include a highly conserved region of the VEGF coding sequence and thus detect all known variants. After in situ hybridization, sections were assessed for overexpression of VEGF. RESULTS: Twenty-nine of the tumor samples overexpressed VEGF, whereas 39 specimens did not. In patients whose tumors demonstrated elevated VEGF expression, 25% were without evidence of disease recurrence at last follow-up. In contrast, 75% of the patients whose tumors did not overexpress VEGF were without evidence of disease at last follow-up (P < 0.001). Median disease free survival for the VEGF positive group was 22 months, compared with > 108 months for the VEGF negative group (P < 0.001). When borderline tumors were excluded from the survival analysis, median disease free survival for the VEGF positive group was 18 months, compared with >120 months for the VEGF negative group (P < 0.001). Other possible prognostic variables had minimal impact on survival; these included age, stage, grade, cytology, and tumor size (P > 0.05). Assignment to a high risk group, as defined by the Gynecologic Oncology Group of the National Cancer Institute, was somewhat predictive of a shorter relapse free interval (P = 0.056). In a multivariate analysis, however, only elevated VEGF expression was associated with poorer survival. CONCLUSIONS: In this analysis, patients with early stage ovarian carcinoma with increased VEGF expression had a poorer prognosis. Further study of VEGF may ultimately lead to identification of patients with high risk lesions whose tumor biology portends a worse prognosis and who therefore may benefit from aggressive adjuvant therapy.

Adult↗
PubMed1997

A novel technique for culture of human dermal microvascular endothelial cells under either serum-free or serum-supplemented conditions: isolation by panning and stimulation with vascular endothelial growth factor.

Several physiological and pathophysiological events involving vascular endothelium occur at the microvascular level. Studies on human microvasculature require homogenous primary cultures of microvascular endothelial cells. However, procedures available for isolating and culturing human dermal microvascular cells (HDMEC) result in significant contamination with fibroblasts. To eliminate contamination with fibroblasts or other cells, we developed a procedure to isolate HDMEC from neonatal human foreskin by panning the cells using EN4, an anti-endothelial cell monoclonal antibody. Panned cells uniformly expressed von Willebrand factor and CD36, confirming their microvascular endothelial characteristics, whereas cells cultured without panning showed a significant degree of contamination with fibroblasts. In the presence of vascular endothelial growth factor (VEGF), HDMEC could be cultured under serum-free conditions. VEGF stimulated the growth of HDMEC in a dose-dependent manner in serum-free medium or in media supplemented with either human serum or newborn calf serum. Since differences exist between large vessel endothelial cells and microvascular endothelial cells, we compared the response to VEGF stimulation of HDMEC with human umbilical vein endothelial cells (HUVEC). The dose response of the two cell types to VEGF was different. This effect of VEGF on endothelial cells may be mediated by the VEGF receptor kdr, since mRNA for kdr was detected using RT-PCR in both HDMEC and HUVEC. The procedure described in this study will make possible the culture of highly enriched HDMEC without contamination with fibroblasts and facilitate studies with these cells under defined assay conditions in a serum-free environment.

Animals↗
PubMed1997

The in vitro effect on T cell function of soluble IL-2Ralpha from advanced ovarian cancer ascites.

Activated T cells not only secrete interleukin-2 (IL-2) and express cell surface interleukin 2 receptor alpha (IL-2R alpha), but also shed IL-2R alpha. This soluble receptor is a truncated form of the membrane-bound p55 receptor with a similar binding affinity. It has been proposed that soluble IL-2R alpha (sIL-2R alpha) could negatively modulate local immune response. High levels of sIL-2R alpha have been found in the serum and ascites of ovarian cancer patients. The purpose of this investigation is to determine the amount of in vitro T cell inhibition seen in ovarian cancer ascites that is attributable to high levels of sIL-2R alpha. Purified sIL-2R alpha at levels up to 100,000 pg/ml was placed in lymphocyte proliferation assays. Soluble IL-2R alpha was removed from the ascites of three patients with advanced ovarian cancer. Lymphocyte proliferation assays utilizing phytohemaglutin (PHA) stimulation were carried out with this ascites. Untreated ascites from each patient served as control. Addition of purified sIL-2R alpha to lymphocyte proliferation assays failed to demonstrate significant lymphocyte suppression. Addition of ascites to the lymphocyte assays resulted in up to an 80% decrease in lymphocyte proliferation. Neutralization of ascites sIL-2R alpha as well as removal of sIL-2R alpha via a protein G column failed to reverse any of the observed lymphocyte suppression. We conclude that although sIL2R alpha is elevated in ascites of patients with ovarian cancer, it does not account for the profound ascites-induced T cell suppression observed in vitro.

Adult↗
PubMed1997

Eales' disease: accumulation of reactive oxygen intermediates and lipid peroxides and decrease of antioxidants causing inflammation, neovascularization and retinal damage.

PURPOSE: To measure the blood levels of oxygen and lipid-free radicals as lipid peroxidation products and of vitamins E, C and A, in order to explain intraocular inflammation, retinal neovascularization and detachment in Eales' disease. METHODS: Levels of the lipid peroxidation products produced by oxygen and lipid free radicals (estimated as thiobarbituric acid reactive substances-TBARS), vitamin E with ferric chloride, vitamin C with 2,6-dichlorophenol-indophenol and vitamin A with antimony trichloride were all evaluated in three groups of subjects between 20 and 40 years of age. Twenty three were patients with Eales' disease in the active perivasculitis stage (Group I), 19 were patients with Eales' disease in the healed-perivasculitis stage (Group II), and 46 were healthy volunteers (Group III), used as normal controls. Patients in each group were subdivided into two age groups, 20-30 and 31-40 years, each group containing both sexes. RESULTS: The results showed a 4.6- and 5.6-fold increase in the levels of TBARS in erythrocytes of patients with Eales' disease in the active perivasculitis stage (Group I) and a 2-fold increase in patients with Eales' disease in the healed perivasculitis stage (Group II), when compared to levels in the controls (Group III). On the other hand, there was a decrease of 75 and 76.2% in the levels of vitamin E in serum, 34 and 40.9% of vitamin C in plasma and 72.8 and 67% of vitamin A in serum in patients with Eales' disease in the active perivasculitis stage (Group I), as compared to the controls (Group III). Also decreases of 56 and 43.5% of vitamin E in serum, 26.8 and 12.5% of vitamin C in plasma and 50.5 and 49.4% of vitamin A in serum were found in patients with Eales' disease in the healed perivasculitis stage (Group II) as compared to the healthy controls (Group III). The two values of variations between the patients and the normal control group given above for TBARS, vitamins E,C and A are for the two age groups, 20-30 and 31-40 respectively. The differences were statistically significant (P < 0.01 to 0.001). CONCLUSIONS: Lowered levels of antioxidant vitamins E and C and consequent accumulation of oxygen and lipid free radicals, or vice versa, could explain the inflammation, neovascularization and retinal pathology in patients with Eales' disease. Also, vitamin A deficiency could aggravate retinal illness.

Adult↗
PubMed1997

Sequence data analysis reveals a relationship between LSR2, the recombinant fusion protein mimicing M. Leprae and VIF of bovine immunodeficiency virus (BIV).

In the course of computer simulation study looking for active sites for the interaction between MHC II and T7--a 12 residue long peptide of LSR2--a recombinant fusion protein mimicing the native bacillus M.Leprae--an interesting relationship between the antigenicity of LSR2 and VIF of BIF has come to light. Computer analysis study has revealed this stretch of residue from 36 to 48 of LSR2 is highly antigenic. The experimental observation seems to confirm the role of this 12 residue peptide in antibody response. In an effort to determine whether a significant sequence level relationship exists between this and any other known protein, the sequence homology of both protein and nucleic acid was studied. It is found that this 12 residue long peptide (T7) of LSR2 is homologous with Viral Infectivity Factor (VIF) of the Bovine Immunodeficiency Virus (BIV). Homology with translated nucleic acid sequence also indicate the same fact. The VIF gene which codes for this protein is known to be essential for ability of cell-free virus preparation to infect cells. These results lead to the question--whether this 12 residue long peptide which is common to both proteins play a role in their infectivity. Whether mutations in the peptide or elimination of this peptide from the protein and studying the effect of this on the diseases themselves may help in controlling them is another important question relevant to medical researchers.

Animals↗
PubMed1997

First report of congenital or infantile cataract in deranged proteoglycan metabolism with released xylose.

AIM: To investigate the chemical pathology in the blood and lens, in cases of congenital or infantile cataract in children excreting predominantly non-reducing carbohydrates in urine. METHODS: Urine samples from children with congenital or infantile cataract, and age and sex-matched controls, were analysed for (i) inherited errors of metabolism, (ii) paper chromatography of sugars, (iii) spectrophotometric assay of glycosaminoglycans (GAG), (iv) cetyl trimethyl ammonium bromide test, (v) electrophoresis using Alcian blue, (vi) ion exchange chromatography with IR 120 resin, and (vii) HPLC for xylose. Blood and lens material were also tested for GAG fragments and xylose. beta Glucuronidase was assayed in lymphocytes and urine. RESULTS: Of 220 children of both sexes below 12 years of age, with congenital or infantile cataract treated in Sankara Nethralaya, Madras, India, during a period of 2 years, 145 excreted fragments of GAG (heparan and chondroitin sulphates) in their urine. There was no such excretion among the control group of 50 children. The same was found accumulated in the blood and lenses of affected children. In addition, xylose was present in small amounts in the urine and blood and xylitol was present in the lens. There was a significant elevation in the activity of beta glucuronidase in lymphocytes and urine, when compared with normals. All the above findings suggest deranged proteoglycan metabolism. As the urine contained mostly GAG fragments and very little xylose, Benedict's reagent was not reduced. This ruled out galactosaemia. CONCLUSION: An increase of beta glucuronidase activity might have caused extensive fragmentation of GAG with resultant accumulation in the blood and lens and excretion in urine. Small amounts of xylose may have come from xylose links between GAG and core protein of proteoglycans. Owing to their polyanionic nature, GAG fragments in the lens might abstract sodium, and with it water, thereby increasing the hydration of the lens. Excessive hydration and the osmotic effect of xylitol from xylose might cause cataract. While corneal clouding has been reported in inborn acid mucopolysaccharidosis, congenital or infantile cataract with deranged metabolism of proteoglycans (acid mucopolysaccharide-xylose-protein complex) is reported in children for the first time.

Case-Control Studies↗
PubMed1997

Element density and the efficiency of binocular matching.

Constraints on binocular matching were investigated by comparing the thresholds for interocular correlation in random element displays for human and model observers, with element density manipulated as a parameter. The models consisted of ideal decision rules operating on the entire stimulus, only on the edges in the stimulus, or only on the sparse minority elements in the stimulus. The results indicate that the human visual system selectively attends to the stimulus edges or to the sparse elements under most circumstances. Efficiencies (human or model) were highest at very low element densities (approximately 20%) and decreased with increasing element density with a log-log slope of -0.5, indicating that dynamic random element stereograms at the traditional 50% element density are vastly undersampled.

Computer Simulation↗
PubMed1997

Free lysine, glycine, alanine, glutamic acid and aspartic acid reduce the glycation of human lens proteins by galactose.

The amino acids lysine, glycine, alanine, glutamate and aspartate formed adducts with galactose at physiological pH and temperature as shown by incorporation of U[14C] galactose. The percentage of galactose reacting with lysine, glycine, alanine, glutamate and aspartate was 4.5 to 7.8, 7.9 to 10.8, 3.2 to 4.6, 2.8 to 4.8 and 3 to 5.2, respectively. Studies with lysine showed that the extent of glycation of the free amino acid increased with time. Incubation of lens homogenate with galactose, effected glycation of proteins. Addition of lysine in concentrations of 5 and 10 mM to equimolar concentrations of galactose decreased the glycation of lens proteins by 64% to 71%; glycine, alanine, glutamate and aspartate decreased glycation by 23 to 68%, 32 to 61%, 35 to 56% and 26 to 61% respectively. Under similar conditions, glycine reacts to a greater extent than lysine, alanine, glutamic and aspartic acids. However, lysine was more effective than glycine, alanine, aspartic and glutamic acids in decreasing glycation of lens proteins by galactose. The decrease of glycation with added lysine increased with time. In general increase of amino acid concentration rather than that of sugar augmented the decrease of glycation of lens proteins.

Adult↗
PubMed1996

Structural and functional significance of aspartic acid 89 of the troponin C central helix in Ca2+ signaling.

The central helix of troponin C is highly conserved in length and amino acid sequence. In this region, D89 is conserved and specific to TnC. To investigate its significance, three mutations were made in avian fast troponin C: (1) D89 was replaced with A (D89A); (2) the central helix was replaced with a designed alpha-helix (alpha h89A) consisting of 87AEAALKAAMEA97; and (3) A89 of alpha h89A was replaced with D (alpha h89D). D89A and alpha h89A activated the regulated actomyosin ATPase poorly in the presence of Ca2+ (24 +/- 1.0% and 14 +/- 2.0%, respectively, of the wild type maximal activity) whereas alpha h89D had higher activity (113 +/- 3%). Both alpha h89A and D89A had apparently normal interactions with TnI and TnT whereas alpha h89D formed a complex with TnT even in the absence of Ca2+. The central helix was also replaced with a flexible random coil and rigid polyproline linkers in which D89 was Arg or Pro, respectively. Like alpha h89A and D89A, both mutants were defective in activation of the actomyosin ATPase in the presence of Ca2+. Changes in regulatory function of the mutants did not correlate with altered Ca2+ affinity, altered conformational changes upon binding divalent cations, or Ca(2+)-dependent binding to TnI or TnT. The results suggest that D89 is required for Ca(2+)-dependent signal transduction, an event that can be dissociated from Ca(2+)-dependent binding to TnC targets on the thin filament.

Animals↗
PubMed1996

Anti-graft-versus-host disease effect of DT390-anti-CD3sFv, a single-chain Fv fusion immunotoxin specifically targeting the CD3 epsilon moiety of the T-cell receptor.

In a recent study, we showed that an immunotoxin (IT) made with a conventional monoclonal antibody targeting the CD3 epsilon moiety of the T-cell receptor (TCR) had a potent, but partial, graft-versus-host disease (GVHD) effect (Vallera et al, Blood 86:4367, 1995). Therefore, in this current study, we determined whether a fusion immunotoxin made with anti-CD3 single-chain Fv (sFv), the smallest unit of antibody recognizing antigen, would have anti-GVHD activity. A fusion protein was synthesized from a construct made by splicing sFv cDNA from the hybridoma 145-2C11 to a truncated form of the diphtheria toxin (DT390) gene. DT390 encodes a molecule that retains full enzymatic activity, but excludes the native DT binding domain. The DT390-anti-CD3sFv hybrid gene was cloned into a vector under the control of an inducible promoter. The protein was expressed in Escherichia coli and then purified from inclusion bodies. The DT390 moiety of the protein had full enzymatic activity compared with native DT and DT390-anti-CD3sFv, with an IC50 of 1 to 2 nmol/L against phytohemagglutinin-stimulated and alloantigen-stimulated T cells. Specificity was shown (1) by blocking the IT with parental anti-CD3 antibody, but not with a control antibody; (2) by failure of DT390-anti-CD3sFv to inhibit lipopolysaccharide-stimulated murine B cells; (3) by failure of an Ig control fusion protein, DT390-Fc, to inhibit T-cell responses; and (4) with in vivo immunohistochemisty studies. GVHD was studied in a model in which C57BL/6 (H-2b)-purified lymph node T cells were administered to major histocompatibility complex (MHC) antigen disparate unirradiated C.B.-17 scid (H-2d) mice to assess GVHD effects in the absence of irradiation toxicity. Flow cytometry studies showed that donor T cells were expanded 57-fold and histopathologic analysis showed the hallmarks of a lethal model of GVHD. Control mice receiving phosphate-buffered saline showed 17% survival on day 80 after bone marrow transplantation, and mice receiving 2 micrograms DT390-Fc fusion toxin control administered in 2 daily doses for 6 days (days 0 through 5) had a 43% survival rate. In contrast, 86% of mice receiving the same dose of DT390-anti-CD3sFv were survivors on day 80, a significant improvement, although survivors still showed histopathologic signs of GVHD. These findings suggest that new anti-GVHD agents can be genetically engineered and warrant further investigation of fusion proteins for GVHD treatment.

Animals↗
PubMed1996

Differential effects of opioids on the proliferation of a macrophage cell line, Bac 1.2F5.

Chronic treatment of mice with morphine selectively abrogates the terminal differentiation of committed bone marrow progenitor cells to form macrophage colony forming units. To understand the molecular mechanisms involved in morphine-mediated suppression of myeloid cell differentiation, we investigated the use of a macrophage cell line, Bac 1.2 F5. In vitro proliferation of this cell line is dependent on the exogenous supply of macrophage colony stimulating factor. Treatment of Bac 1.2F5 cells in vitro with morphine showed a dose-dependent inhibition of proliferation which was associated with morphological changes. Characterization of the binding site revealed that the binding site for morphine on these cells is different from the classical opioid receptors described in the brain. In addition to the putative novel class of morphine receptors, Bac 1.2F5 cells also expressed the delta opioid receptors as determined by RT-PCR analyses. These studies show that Bac 1.2F5 cells are suitable for the molecular characterization of opioid effects on the proliferation and differentiation of myeloid progenitor cells.

Animals↗
PubMed1996

Vascular endothelial growth factor-toxin conjugate specifically inhibits KDR/flk-1-positive endothelial cell proliferation in vitro and angiogenesis in vivo.

Inhibition of tumor neovascularization has profound effects on the growth of solid tumors. An endothelial cell-specific cytotoxic conjugate was prepared by chemically linking recombinant vascular endothelial growth factor (VEGF165) and a truncated diphtheria toxin molecule (DT385). The treatment of subconfluent cultures of human umbilical vein endothelial cells and human microvascular endothelial cells with the VEGF165-DT385 conjugate resulted in a selective, dose-dependent inhibition of growth. Parallel experiments with either the free toxin or a mixture of VEGF and the toxin polypeptide did not affect proliferation (DNA synthesis) of these cells. The selective cytotoxicity correlated with the appropriate receptor expression (KDR/flk-1 positive) on the target cells. VEGF-toxin conjugate inhibited the growth of a murine hemangioma-derived endothelial cell line (Py-4-1), which was positive for flk-1 expression. Under similar conditions, the conjugate did not affect the proliferation of a receptor-negative ovarian cancer cell line in vitro. In an in vivo model of angiogenesis, the VEGF165-DT385 conjugate blocked basic fibroblast growth factor-induced neovascularization of the chick chorioallantoic membrane. These studies demonstrate the successful targeting of a cytotoxic polypeptide to proliferating vascular endothelial cells (normal and tumorigenic) and the potential utility of such conjugates in blocking tumor neovascularization.

Animals↗
PubMed1996

Purification and characterization of a new enzyme dipeptidase from human lens.

A new enzyme dipeptidase has been purified to homogeneity from human lens tissue adopting isoelectric focusing, preparative electrophoresis and gel filtration HPLC. The purified enzyme hydrolyses a wide variety of dipeptides containing aliphatic as well as aromatic amino acids but does not act on tripeptides and proteins. The identity of this enzyme as a dipeptidase has been confirmed by the use of dipeptides with modified amino or carboxyl groups. The optimum temperature and pH for this enzyme are 25 +/- 2 degrees C and 5.5 respectively and pI is 6.5. The Km for different dipeptides varied from 0.04 mM to 4.2 mM. The molecular weight of the native enzyme as determined by gel permeation HPLC is 52 kDa. Preparative electrophoresis, followed by HPLC gave two active proteins, with molecular weights of 52 kDa and 13 kDa. That with the molecular weight of 52 kDa was found to be the tetramer of the other by SDS-PAGE, and peptide mapping of tryptic digests. Properties of this enzyme have been compared with those reported for other proteinases and peptidases of the lens and dipeptidases of Escherichia coli and mouse tumour cells and they render additional support to the finding that this is a new enzyme. The physiological function of this enzyme is also discussed.

Amino Acid Sequence↗
PubMed1996

Expression and radiolabeling of recombinant proteins containing a phosphorylation motif.

Radiolabeled proteins are useful in basic and clinical research. Current methods available for radiolabeling proteins involve chemical derivatization, resulting in multiple additions of radionuclides at random sites. A method designed to specifically localize the radionuclide to a unique site will offer advantages of control and predictability in radiolabeling. We have studied the usefulness of a prokaryotic expression vector by incorporating the coding sequence of a consensus phosphorylation motif (Kemptide) for the cAMP-dependent protein kinase A immediately upstream to the multiple cloning site. This vector was used to express five different recombinant proteins with a phosphorylation site at the amino terminus. In addition, the phosphorylation motif was introduced into two other proteins and expressed in yeast. The genetically engineered proteins were purified to homogeneity by affinity chromatography and radiolabeled with [gamma32P]ATP in vitro. All seven proteins used in this study could be expressed with the phosphorylation sequence at their amino terminus and specifically labeled without loss of biological activity. This strategy allows the option of labeling proteins to high or low specific radioactivity and holds potential for in vitro binding and in vivo localization studies.

Adenosine Triphosphate↗
PubMed1996

Free alanine, aspartic acid, or glutamic acid reduce the glycation of human lens proteins.

The amino acids lysine and glycine are reported to react with glucose at physiological pH and temperature and undergo non-enzymic glycation. Three other amino acids present in relatively larger amounts in the lens i.e. alanine, aspartic acid and glutamic acid were also found to undergo non-enzymic glycation as found by incorporation of uniformly labelled (U-[14C]) glucose into the amino acids. The glucose incorporation was 1.6 to 2.5% for alanine, 35 to 50% for aspartic acid and 2.3 to 3.3% for glutamic acid. Each amino acid of varying concentrations lowered the extent of in vitro glycation of lens proteins significantly in glucose-treated homogenates of normal lens from humans. The decrease in glycation for alanine was between 32 and 69%, that for aspartate was between 18 and 74%, and for glutamate was between 52 to 74%. Decreased glycation was greater for higher concentrations of glucose. Scavenging of intracellular glucose and decreasing the extent of glycation of lens proteins could be the mechanism of action by which the amino acids alanine, aspartic acid and glutamic acid could exercise a beneficial effect on cataract and diabetic retinopathy.

Alanine↗
PubMed1996

Differential expression of opioid receptor genes in human lymphoid cell lines and peripheral blood lymphocytes.

The existence of receptors for opioid compounds on cells of the immune system has long been hypothesized, but has been very difficult to demonstrate unequivocally. We have used reverse-transcription polymerase chain reaction to obtain cDNA clones from the human MOLT-4 and CEM-3 T-leukemic cell lines which are nearly identical to portions of the delta and kappa opioid receptor cDNAs recently isolated from human brain and placenta, respectively. Northern analyses with riboprobes derived from the delta and kappa opioid receptor clones indicate these sequences are expressed at low levels in human peripheral blood lymphocytes and in several human lymphoid cell lines. Sequences corresponding to the mu opioid receptor cDNA were not detected in this study. The results suggest that delta and kappa opioid receptors may be responsible for mediating some direct effects of opioids in immune cells.

Base Sequence↗
PubMed1996

Expression cloning of a full-length cDNA encoding delta opioid receptor from mouse thymocytes.

A delta opioid receptor complementary DNA (cDNA) was cloned by expression of cDNA library from activated thymocytes in Cos 7 cells. The deduced amino acid sequence of this receptor was similar to that described in the brain. As analyzed by southern blot hybridization, the delta opioid receptor transcripts are constitutively expressed in unactivated thymocytes. In addition, neither kappa nor mu opioid receptor transcripts were detected in thymocytes, suggesting tissue-specific opioid receptor gene expression in the immune system. The studies represent the first report of a full-length opioid receptor in the immune system.

Amino Acid Sequence↗