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Biomedical subjects

S Balasubramanian

Publications and source records attributed to S Balasubramanian.

At least 19 recordsLinked to original sources
PubMed2003

Different activation treatments for successful development of bovine oocytes following intracytoplasmic sperm injection.

In this study, the developmental capacity and cytogenetic composition of different oocyte activation protocols was evaluated following intracytoplasmic sperm injection (ICSI) of in vitro matured bovine oocytes. Motile spermatozoa selected by Percoll density gradient were treated with 5 mM dithiothreitol (DTT) and analysed for ultrastructural changes of the head using transmission electron microscopy (TEM). The alterations in sperm morphology after DTT treatment for different times (15, 30 and 60 min) were 10%, 45-55% and 70-85%, respectively. Further, a partial decondensation of sperm heads was observed after DTT treatment for 30 min. Oocytes were injected with sperm treated with DTT for 30 min. In group 1, sperm injection was performed without any activation stimulus to the oocytes. In group 2, sham injection without sperm was performed without activating the oocytes. Oocytes injected with sperm exposed to 5 microM ionomycin for 5 min (group 3), 5 microM ionomycin + 1.9 mM dimethylaminopurine (DMAP) for 3 h (group 4) and 5 microM ionomycin + 3 h culture in M199 + 1.9 mM DMAP (group 5) were also evaluated for cleavage, development and chromosomal abnormality. Cleavage and development rates in groups 1, 2 and 3 were significantly (p < 0.05) lower than those in groups 4 and 5. The incidence of chromosomal abnormality in the embryos treated directly with DMAP after ionomycin (group 4) was higher than in group 5. We conclude that immediate DMAP treatment after ionomycin exposure of oocytes results in arrest of release of the second polar body, and thus leads to changes in chromosomal pattern. Therefore, the time interval between ionomycin and DMAP plays a crucial role in bovine ICSI.

Animals↗
PubMed2003

Morphine inhibits VEGF expression in myocardial ischemia.

BACKGROUND: Vascular endothelial growth factor (VEGF) may contribute to the resolution of myocardial ischemia by stimulating collateral circulation. Morphine analgesia after myocardial ischemia is thought to increase infarct size. We hypothesize that morphine inhibits myocardial VEGF expression by inhibiting hypoxia-induced factor 1alpha (HIF-1alpha) and the signal transduction mechanisms involving Erk-1,2 MAP kinase (p42/p44), and PI3 kinase activity (phospho-Akt). METHODS: (1) In vitro: primary cultures of rat cardiac myocytes; (2) in vivo: rat coronary ligation model; (3) mRNA measurement: real-time reverse transcriptase-polymerase chain reaction; (4) protein measurements: enzyme-linked immunosorbent assay, Western immunoblot, electromobility shift assay (EMSA), and immunohistochemistry. RESULTS: Using rat cardiac myocytes in vitro, we show that morphine: (1) decreases hypoxia-induced VEGF(121) and VEGF(165) mRNA expression and VEGF protein concentration through an opioid receptor mechanism; (2) decreases HIF-1alpha protein expression (immunoblot) and nuclear protein binding to the VEGF HIF-1alpha DNA response element (EMSA); and (3) inhibits phospho-Erk-1,2 MAP kinase (immunoblot) and phospho-Akt kinase activity (immunoblot). Using a rat coronary ligation model, we show that morphine treatment: (1) decreases myocardial VEGF protein expression (immunohistochemistry); (2) decreases HIF-1alpha protein expression (immunoblot); and (3) decreases phospho-Erk-1,2 and phospho-Akt expression. CONCLUSIONS: (1) Morphine inhibits hypoxia-induced VEGF transcription, in part, through an HIF-1alpha-mediated mechanism and (2) morphine inhibition of hypoxia-induced HIF-1alpha may be mediated by inhibition of ERK 1,2 MAP kinase activity and PI3 kinase activity.

Animals↗
PubMed2002

Microtubulin configuration and mitochondrial distribution after ultra-rapid cooling of bovine oocytes.

Considerable attention has been focused on the cryopreservation of mammalian oocytes, as a consequence of poor development of cryopreserved bovine oocytes in vitro, in order to enhance the application of genetic engineering. Experiments were carried out to evaluate the viability and ultra-structural changes of bovine oocytes cryopreserved by ultra rapid cooling methods. Oocytes that had been allowed to mature for 22 hr were exposed to a mixture of cryoprotectants (3.2 M ethylene glycol, 2.36 M dimethyl sulfoxide (DMSO), 0.6 M sucrose), and were cryopreserved by very rapid cooling either within glass capillaries or as droplets on copper electron microscope grids. After being warmed, the oocytes were cultured in in vitro maturation (IVM) medium for an additional 2 hr. Viability was assessed by determining the development rate after fertilization with frozen-thawed semen from which motile sperm had been recovered using a Percoll density gradient, and by immunochemical evaluation of microtubule and mitochondrial morphology. Cleavage and development rates were significantly (P < 0.05) lower in oocytes cryopreserved by vitrification than in in vitro fertilization (IVF) control group, but did not differ in the open-pulled glass (OPG) or copper grid (CG) groups. In most oocytes cryopreserved by vitrification, the microtubules were partially or completely broken. Similarly mitochondria appeared to be abnormal compared to that of unfrozen oocytes. Oocytes cultured in IVM medium supplemented with both cytochalasin B (a protein synthesis inhibitor) and 2-mercaptoethanol (an antioxidant) showed less damage to microtubules, but not to mitochondria after cryopreservation. In conclusion, this study showed that bovine oocytes can be cryopreserved by vitrification within small droplets using CGs. While damage to microtubules and mitochondria may be involved in reduced viability, supplementation of IVM medium with cytochalasin B appears to enhance stabilization of microtubules during oocyte cryopreservation.

Animals↗
PubMed2001

Role of hypothalamic-pituitary axis in morphine-induced alteration in thymic cell distribution using mu-opioid receptor knockout mice.

Mu-opioid receptor knockout mice (MORKO), were used to address two questions: (1) if morphine induced decrease in thymic weight and cell distribution is mediated by the mu-opioid receptor and (2) the role of corticosteroids in morphine mediated alteration in thymic cell distribution. Our result show that morphine mediated increase in plasma corticosterone is mediated by the mu-opioid receptor since morphine at doses as high as 25 mg/kg-body weight does not increase plasma corticosterone levels in the MORKO. In addition, we have also shown that morphine treatment results in the differentiation of CD4+CD8+ (double positive cells) to single positive CD4+ cells while dexamethasone treatment results in the deletion of CD4+CD8+ (double positive) cells.

Adrenocorticotropic Hormone↗
PubMed2001

Non-Arrhenius kinetics for the loop closure of a DNA hairpin.

Intramolecular chain diffusion is an elementary process in the conformational fluctuations of the DNA hairpin-loop. We have studied the temperature and viscosity dependence of a model DNA hairpin-loop by FRET (fluorescence resonance energy transfer) fluctuation spectroscopy (FRETfs). Apparent thermodynamic parameters were obtained by analyzing the correlation amplitude through a two-state model and are consistent with steady-state fluorescence measurements. The kinetics of closing the loop show non-Arrhenius behavior, in agreement with theoretical prediction and other experimental measurements on peptide folding. The fluctuation rates show a fractional power dependence (beta = 0.83) on the solution viscosity. A much slower intrachain diffusion coefficient in comparison to that of polypeptides was derived based on the first passage time theory of SSS [Szabo, A., Schulten, K. & Schulten, Z. (1980) J. Chem. Phys. 72, 4350-4357], suggesting that intrachain interactions, especially stacking interaction in the loop, might increase the roughness of the free energy surface of the DNA hairpin-loop.

DNA↗
PubMed2001

Selection of zinc fingers that bind single-stranded telomeric DNA in the G-quadruplex conformation.

There is considerable interest in molecules that bind to telomeric DNA sequences and G-quadruplexes with specificity. Such molecules would be useful to test hypotheses for telomere length regulation, and may have therapeutic potential. The versatility and modular nature of the zinc finger motif makes it an ideal candidate for engineering G-quadruplex-binding proteins. Phage display technology has previously been widely used to screen libraries of zinc fingers for binding to novel duplex DNA sequences. In this study, a three-finger library has been screened for clones that bind to an oligonucleotide containing the human telomeric repeat sequence folded in the G-quadruplex conformation. The selected clones show a strong amino acid consensus, suggesting analogous modes of binding. Binding was found to be both sequence dependent and structure specific. This is the first example of an engineered protein that binds to G-quadruplex DNA, and represents a new type of binding interaction for a zinc finger protein.

Amino Acid Sequence↗
PubMed2001

Exploring a benzyloxyaniline linker utilizing ceric ammonium nitrate (CAN) as a cleavage reagent: solid-phase synthesis of N-unsubstituted beta-lactams and secondary amides.

[figure: see text] A novel benzyloxyaniline linker that uses ceric ammonium nitrate (CAN) as a cleavage reagent is described. Its application in the solid-phase synthesis of N-unsubstituted beta-lactams and secondary amides furnishes compounds in moderate to excellent yield (45-91%) and high purity (93-99%).

Amides↗
PubMed2001

Solid phase chemical technologies for combinatorial chemistry.

This article describes the exploration of synthetic methodologies on solid phase for combinatorial chemistry. Examples will cover various cyclisation strategies that include; the intramolecular Heck cyclisation leading to the formation of oxindoles, the radical cyclisation mechanism in the synthesis of furans, and a stereoselective cyclisation to form oxopiperazines.

Benzofurans↗
PubMed2001

Morphine sulfate inhibits hypoxia-induced vascular endothelial growth factor expression in endothelial cells and cardiac myocytes.

Vascular endothelial growth factor (VEGF) is an angiogenic mitogen, specific for endothelial cells. Hypoxia-induced VEGF in endothelial cells and cardiomyocytes leads to autocrine and paracrine stimulation, respectively. During myocardial ischemia, VEGF is upregulated in the endothelium and myocardium, and may mediate angiogenesis. Morphine sulfate is commonly used in pain relief for patients with acute myocardial infarction. We investigated the effect of morphine sulfate on VEGF expression in cultured endothelial cells and cardiac myocytes subjected to hypoxia. Enzyme-linked immunosorbent assays showed that morphine sulfate significantly inhibited hypoxia-induced VEGF expression in mouse heart microvascular endothelial cells (SMHEC4), primary cultures of human umbilical vein endothelial cells (HUVECs) and in primary cultures of rat cardiac myocytes (P<0.05). Real time reverse transcriptase polymerase chain reaction showed that morphine treatment (100 ng/ml) of hypoxic HUVECs resulted in a significant reduction in mRNA levels of VEGF(121) and VEGF(165) isoforms. Transfection of HUVECs with a human VEGF promoter-luciferase construct showed that hypoxia-induced transcriptional activation of VEGF was markedly inhibited by morphine sulfate (P<0.05). Phosphatidyl inositol-3 kinase and protein kinase C-mediated activation of the VEGF promoter was also inhibited by morphine. The opioid antagonist naloxone significantly reversed the inhibitory effects of morphine in endothelial cells suggesting the involvement of opioid receptors. Our results show that the inhibitory effects of morphine on hypoxia-induced VEGF expression in endothelial cells and cardiac myocytes can lead to a decrease in the autocrine and paracrine stimulation and hence limit neovascularization of the ischemic myocardium.

Animals↗
PubMed2001

Morphine directs T cells toward T(H2) differentiation.

BACKGROUND: Failure of cell-mediated immunity is thought to increase the morbidity and mortality rates after trauma and major surgical procedures and to be the result, in part, of a redirection of CD4(+) T cells toward T(H2) differentiation. We tested the hypothesis that morphine treatment after injury promotes T(H2) differentiation of precursor T cells through the mu-opioid receptor. METHODS: Human peripheral blood mononuclear cells (PBMCs) or splenocytes from either wild type or mu-opioid receptor knock-out mice were treated in vitro with either vehicle or morphine and then stimulated with anti-CD3/anti-CD28. The supernatant was assayed for T(H1) (interleukin-2 [IL-2], interferon gamma [IFN gamma]) and T(H2) (IL-4, IL-5) cytokines (enzyme-linked immunosorbent assay). Morphine regulation of IL-4 transcription was investigated in PBMCs (IL-4 messenger RNA, nuclear factor of activated T-cells) and Jurkat T cells transfected with a murine IL-4 promoter-luciferase construct. Morphine-induced nuclear factor of activated T-cell (NFAT) binding was assayed with the electromobility shift assay in Jurkat T cells. RESULTS: Morphine treatment of PBMCs decreases IL-2 and IFN gamma and increases IL-4 and IL-5 as a function of morphine concentration. Morphine treatment in wild type splenocytes inhibited IFN gamma and stimulated IL-4 protein synthesis. Changes in cytokine synthesis were abolished in mu-opioid receptor knockout mice. Morphine treatment increases IL-4 messenger RNA accumulation in PBMCs and increases IL-4 promoter activity in Jurkat T cells. Morphine increases NFAT nuclear protein binding to an NFAT DNA response element. CONCLUSIONS: We conclude that morphine treatment promotes T(H2) differentiation through a mu-opioid receptor mechanism and that morphine treatment increases IL-4 transcription, in part, through an NFAT mechanism.

Analgesics, Opioid↗
PubMed2001

Morphine modulates lymph node-derived T lymphocyte function: role of caspase-3, -8, and nitric oxide.

The major objective of this paper is to characterize the mechanism by which morphine modulates lymphocyte function and if these effects are mediated through the mu-opioid receptor. We evaluated the in vitro effects of morphine on lymphocytes that were freshly isolated from lymph nodes from wild type (WT) and mu-opioid receptor knock-out (MORKO) mice. Results show that morphine inhibits Con A-induced lymph node T-cell proliferation and IL-2 and IFN-gamma synthesis in a dose-dependent manner. This effect was abolished in lymph node cells isolated from MORKO mice. The inhibition of T-cell function with low-dose morphine was associated with an increase in caspase-3- and caspase-8-mediated apoptosis. The inhibition of T-cell function with high-dose morphine was associated with an increase in the inducible NO synthase mRNA expression. N(G)-nitro-L-arginine methyl ester (L-NAME) antagonized the apoptosis induced by high-dose morphine. Our results suggest that low-dose morphine, through the mu-opioid receptor, can induce lymph node lymphocyte apoptosis through the cleavage activity of caspase-3 and caspase-8. Morphine at high doses induces NO release. This effect of morphine is also mediated through the mu-opioid receptor present on the surface of macrophages.

Animals↗
PubMed2001

A two parameter Weibull distribution method for predicting cations in the ground water of Cauvery delta of Tamilnadu.

A two parameter Weibull distribution model was applied to evaluate the quality of ground water collected from 50 wells in four regions (CSB, VSB, RCA, NDA) of Thanjavur district. The data of the cations (Ca, Mg, K, Na) and EC were used to predict the ground water quality. The shape (a) and scale (b) along with mean and percentile were zoned. A hybrid approach of the Weibull distribution model was done to estimate the mean. This hybrid approach may be used to predict and forecast the ground water quality. From the study the cationic parameters in the wells were not uniform which may be attributed to contamination either by surface flow or sea water.

Environmental Monitoring↗
PubMed2000

Thapsigargin suppresses phorbol ester-dependent human involucrin promoter activity by suppressing CCAAT-enhancer-binding protein alpha (C/EBPalpha) DNA binding.

Human involucrin (hINV) is a keratinocyte differentiation marker expressed in the suprabasal epidermal layers. In cultured keratinocytes hINV mRNA levels are increased 10-fold by a 24-h treatment with 50 ng/ml PMA, an agent that promotes keratinocyte differentiation. Previous studies show that thapsigargin (TGN), an agent that depletes intracellular calcium stores, inhibits keratinocyte differentiation. In the present study we show that TGN inhibits the PMA-dependent, differentiation-associated, increase in hINV mRNA levels and hINV promoter activity. Inhibition is half-maximal at 10 nM and maximal at 100 nM TGN. Neither basal hINV promoter activity nor glyceraldehyde-3-phosphate dehydrogenase mRNA levels are inhibited. Mutation of a functionally important CAATT-enhancer-binding protein (C/EBP) site within the hINV promoter proximal regulatory region eliminates the regulation, suggesting that TGN may effect C/EBP-dependent promoter activation. Consistent with this hypothesis, TGN inhibits C/EBPalpha-dependent promoter activation via a mechanism that involves inhibition of C/EBPalpha binding to DNA without changing C/EBPalpha protein levels. These results suggest that TGN interferes with hINV expression by interfering with C/EBP transcription-factor function.

Base Sequence↗
PubMed2000

Proteomics of Mycoplasma genitalium: identification and characterization of unannotated and atypical proteins in a small model genome.

We present the results of a comprehensive analysis of the proteome of Mycoplasma genitalium (MG), the smallest autonomously replicating organism that has been completely sequenced. Our aim was to identify and characterize all soluble proteins in MG that are structurally and functionally uncharacterized. We were particularly interested in identifying proteins that differed significantly from typical globular proteins, for example, proteins which are unstructured in the absence of a 'partner' molecule or those that exhibit unusual thermodynamic properties. This work is complementary to other structural genomics projects whose primary aim is to determine the three-dimensional structures of proteins with unknown folds. We have identified all the full-length open reading frames (ORFs) in MG that have no homologs of known structure and are of unknown function. Twenty-five of the total 483 ORFs fall into this category and we have expressed, purified and characterized 11 of them. We have used circular dichroism (CD) to rapidly investigate their biophysical properties. Our studies reveal that these proteins have a wide range of structures varying from highly helical to partially structured to unfolded or random coil. They also display a variety of thermodynamic properties ranging from cooperative unfolding to no detectable unfolding upon thermal denaturation. Several of these proteins are highly conserved from mycoplasma to man. Further information about target selection and CD results is available at http://bioinfo.mbb.yale.edu/genome

Amino Acid Sequence↗