PubMed Health⌕ Search

Biomedical subjects

R Bose

Publications and source records attributed to R Bose.

At least 37 records · Page 2Linked to original sources
PubMed1997

Spectroscopic determination of sarcoplasmic reticulum Ca2+ uptake and Ca2+ release.

In this report we describe the application of spectroscopic methods to the study of Ca2+ release by isolated native sarcoplasmic reticulum (SR) membranes from rabbit skeletal muscle. To date, dual-wavelength spectroscopy of arsenazo III and antipyrylazo III difference absorbance have been the most common spectroscopic methods for the assay of SR Ca2+ transport. The utility of these methods is the ability to manipulate intraluminal Ca2+ loading of SR vesicles. These methods have also been useful for studying the effect of both agonists and antagonists upon SR Ca2+ release and Ca2+ uptake. In this study, we have developed the application of Calcium Green-2, a long-wavelength excitable fluorescent indicator, for the study of SR Ca2+ uptake and release. With this method we demonstrate how ryanodine receptor Ca2+ channel opening and closing is regulated in a complex manner by the relative distribution of Ca2+ between extraluminal and intraluminal Ca2+ compartments. Intraluminal Ca2+ is shown to be a key regulator of Ca2+ channel opening. However, these methods also reveal that the intraluminal Ca2+ threshold for Ca2+-induced Ca2+ release varies as a function of extraluminal Ca2+ concentration. The ability to study how the relative distribution of a finite pool of Ca2+ across the SR membrane influences Ca2+ uptake and Ca2+ release may be useful for understanding how the ryanodine receptor is regulated, in vivo.

Animals↗
PubMed1996

Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis.

The induction of programmed cell death, or apoptosis, involves activation of a signalling system, many elements of which remain unknown. The sphingomyelin pathway, initiated by hydrolysis of the phospholipid sphingomyelin in the cell membrane to generate the second messenger ceramide, is thought to mediate apoptosis in response to tumour-necrosis factor (TNF)-alpha, to Fas ligand and to X-rays. It is not known whether it plays a role in the stimulation of other forms of stress-induced apoptosis. Given that environmental stresses also stimulate a stress-activated protein kinase (SAPK/JNK), the sphingomyelin and SAPK/JNK signalling systems may be coordinated in induction of apoptosis. Here we report that ceramide initiates apoptosis through the SAPK cascade and provide evidence for a signalling mechanism that integrates cytokine- and stress-activated apoptosis.

Animals↗
PubMed1996

Embryo associated immunosuppressor factor (EASF) and serum estradiol synergism in pre-embryonic development after in vitro fertilization.

The aim of this study was to test whether the level of serum immunosuppressor factor (EASF) and serum estradiol (E2) play a cooperative role in the pre-implantation early embryonic development in IVF-patients. Serum samples were collected from 80 patients undergoing IVF-ET and the level of EASF in patient's sera was assayed using a two-site sandwich enzyme linked immunosorbent assay (ELISA) system. Two-hundred fifty-one IVF-culture media were obtained from these patients and the EASF activity was measured using concanavalin A-induced lymphocyte proliferation assay. EASF activity in embryo growth media, quality of ova, number of cells in each pre-embryo at the time of embryo transfer, cycle day 3 serum E2 and peak E2 levels were correlated with pregnancy outcome. Thirty-three out of 80 patients had detectable levels of serum EASF. The number of pre-embryos with > 4 cell stage at the time of embryo transfer correlated with the total number of ova retrieved in patients positive for serum EASF (P = 0.01) and patients that had an ongoing pregnancy at 7-8 weeks (P < 0.01). Ratio of the number of pre-embryos with > 4 cell stage at the time of embryo transfer and total number of ova retrieved correlated with cycle day 3 serum E2 (P < 0.01) and peak E2 (P = 0.01) levels only in patients positive for EASF. The EASF activity of embryo growth media correlated with cycle day 3 serum E2 (P = 0.0001) and peak E2 (P = 0.007) levels only in patients that were positive for EASF and got pregnant after IVF-ET. This study suggests that the levels of serum EASF and E2 may act synergistically in the development of early embryo.

Antibodies, Monoclonal↗
PubMed1996

Effects of anion exchange on intracellular pH and tension in basilar artery of dogs.

We simultaneously measured intracellular pH (pHi) and isometric tension in canine basilar arteries. pHi was calculated from the ratio of fluorescence intensities at 540 nm of exciting wavelengths of 500 and 440 nm in the presence of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). pHi was 7.33 +/- 0.02 in Krebs-Henseleit solution (pH of 7.4 at 37 degrees C). Application of the anion exchange blocker 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) decreased the resting pHi by 0.25 pH units. Increasing extracellular pH (pHo) [by decreasing CO2 tension (PCO2)] from 7.4 to 7.8 increased pHi by 0.38 pH units and increased tension by 2.28 +/- 0.21 mN. Decreasing pHo from 7.8 to 7.4 (by increasing PCO2) restored the pHi and muscle tension to their baseline levels. SITS inhibited the increase in pHi and isometric tension in response to the increase in pHo in an endothelium-independent fashion. The Na+/H+ exchange blockers, amiloride or 5-(N-methyl-N-guanidinocarbonylmethyl)-amiloride, did not affect the pHo or tension changes. The results suggest that in the range of pH tested, anion exchange is more important than Na+/H+ exchange in the regulation of pHi and mechanical tone in the basilar artery.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1996

Mechanism of cardiac inotropy by phenamil, and epithelial sodium channel blocker.

Phenamil, an amiloride derivative, is a potent inhibitor of epithelial type sodium channels and a relaxant of smooth muscle. In canine cardiac ventricular trabeculae, which do not express epithelial type sodium channels, phenamil produces positive inotropy and prolongs twitch duration. Sarcoplasmic reticulum does not appear to be essential for phenamil-induced inotropy, because cyclopiazonic acid and ryanodine do not abolish this effect. Furthermore, in tissues made to contract biphasically with 90 to 98% substitution of calcium with strontium, phenamil enhanced the second phase of the contraction which is transsarcolemmal-calcium dependent. Phenamil did not alkalinize or acidify the cytosol (measured with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, BCECF) during the induction of positive inotropy, therefore the sodium-hydrogen exchange is not affected. Sodium-calcium exchange, as assessed by twin rapid cooling contractures, was not inhibited by phenamil. Direct inhibition of this exchanger is therefore not necessary for the inotropic action of phenamil. Phenamil did not inhibit the sodium pump in smooth muscle. Unlike ouabagenin, it significantly prolonged the action potential duration at 90% repolarization. We have demonstrated recently that prolongation of cardiac action potential duration with phenamil is due to inhibition of the inwardly rectifying potassium current without any direct effect on cardiac calcium channels. The resulting delay in repolarization of the terminal part of the action potential reduces the driving force for the forward mode of sodium-calcium exchange. This indirectly reduces the activity of the sodium-calcium exchanger and slows the extrusion of calcium from the cell at the end of the action potential leading to a gradual development of positive inotropy.

Amiloride↗
PubMed1995

Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals.

The sphingomyelin pathway, which is initiated by sphingomyelin hydrolysis to generate the second messenger ceramide, signals apoptosis for tumor necrosis factor alpha, Fas, and ionizing radiation. In the present studies, the anticancer drug daunorubicin also stimulated ceramide elevation and apoptosis in P388 and U937 cells. Cell-permeable analogs of ceramide, but not other lipid second messengers, mimicked daunorubicin in inducing apoptosis. Daunorubicin-stimulated ceramide elevation, however, did not result from sphingomyelin hydrolysis, but rather from de novo synthesis via activation of the enzyme ceramide synthase. An obligatory role for ceramide synthase was defined, since its natural specific inhibitor, fumonisin B1, blocked daunorubicin-induced ceramide elevation and apoptosis. These studies demonstrate that ceramide synthase activity can be regulated in eukaryotes and constitute definitive evidence for a requirement for ceramide elevation in the induction of apoptosis.

Amidohydrolases↗
PubMed1995

Overexpression of SIS2, which contains an extremely acidic region, increases the expression of SWI4, CLN1 and CLN2 in sit4 mutants.

The Saccharomyces cerevisiae SIS2 gene was identified by its ability, when present on a high copy number plasmid, to increase dramatically the growth rate of sit4 mutants. SIT4 encodes a type 2A-related protein phosphatase that is required in late G1 for normal G1 cyclin expression and for bud initiation. Overexpression of SIS2, which contains an extremely acidic carboxyl terminal region, stimulated the rate of CLN1, CLN2, SWI4 and CLB5 expression in sit4 mutants. Also, overexpression of SIS2 in a CLN1 cln2 cln3 strain stimulated the growth rate and the rate of CLN1 and CLB5 RNA accumulation during late G1. The SIS2 protein fractionated with nuclei and was released from the nuclear fraction by treatment with either DNase I or micrococcal nuclease, but not by RNase A. This result, combined with the finding that overexpression of SIS2 is extremely to a strain containing lower than normal levels of histones H2A and H2B, suggests that SIS2 might function to stimulate transcription via an interaction with chromatin.

Amino Acid Sequence↗
PubMed1995

Embryo-associated immunosuppressor factor is produced at the maternal-fetal interface in human pregnancy.

PROBLEM: To study whether embryo associated immunosuppressor factor (EASF) is synthesized at the maternal-fetal interface. METHOD: Anti-EASF monoclonal antibody H5D12 was used to identify EASF. Paraffin-embedded sections were prepared from placental and fetal tissues and immunohistochemistry was done by the avidin-biotin-peroxidase technique. EASF was affinity purified using H5D12-Sepharose 4B from culture media of placental villi and analyzed for immunosuppressive activity (by Concanavalin A-induced lymphocyte proliferation assay) and molecular weight identity (by metabolic labeling studies with 35S-methionine followed by immunoprecipitation and SDS-PAGE). RESULTS: Immunohistochemical studies demonstrates intense immunostaining of villous syncytiotrophoblast and cytotrophoblast cells of first trimester placental tissues. Hoffbauer cells and decidual cells stained positive. The same cells in second and third trimester placental tissues stained weakly. However, the endothelium and smooth muscle cells of fetal blood vessels, fetal ovarian stroma and primordial follicles, kidney epithelium, cerebral neurons, and glial cells all stained negative. The affinity-purified EASF from the conditioned media of placental villi (less than 12 wk gestational age) was identified as a 37-kDa molecule with immunosuppressive activity. Metabolic labeling studies revealed that placental villi from early gestational age secretes a major factor of 37-kDa and minor factors of 41-kDa and 47-kDa molecular weight. CONCLUSIONS: Monoclonal antibody H5D12 identifies a factor that is produced by the pre-implantation embryo and also synthesized by decidua and trophoblast cells.

Chaperonin 10↗
PubMed1995

Phospholipase A2 activation in human neutrophils requires influx of extracellular Ca2+ and leukotriene B4.

We have demonstrated that phospolipase A2 (PLA2) activation in human neutrophils requires both the influx of extracellular Ca2+ and leukotriene B4 (LTB4). Surprisingly, the eicosanoids (LTB4 and its omega-oxidation products) formed were quantitatively very similar in both thapsigargin (Thap)- and A-23187-stimulated neutrophils. In contrast, Thap had very little effect on the activation of PLA2 when 5-lipoxygenase (5-LO) was blocked by BW755C or MK-886, whereas A-23187 caused a substantial activation. The lack of PLA2 activation in Thap-stimulated neutrophils results from the inhibition of LTB4 formation in the presence of 5-LO inhibitors. It appears that A-23187 activates both LTB4-dependent and -independent PLA2, whereas Thap activates LTB4-dependent PLA2. Experiments with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid demonstrated that activation of Thap-sensitive PLA2 and 5-LO requires the influx of Ca2+. Neither the transient elevation of cytosolic Ca2+ from intracellular stores nor the sustained Ca2+ influx alone without LTB4 appears sufficient to cause the activation of LTB4-dependent PLA2. We suggest that the activation of LTB4-dependent PLA2 involves 1) a sustained elevation of cytosolic Ca2+ coupled to the influx of extracellular Ca2+ and 2) a coupling between LTB4 and its receptor. We conclude that LTB4-dependent PLA2 plays an important role in the poststimulatory formation of lipid mediators such as prostaglandins, leukotrienes, and platelet-activating factor.

Calcimycin↗
PubMed1995

Effects of phenamil on potassium and calcium channels of guinea pig ventricular myocytes.

Phenamil, a phenyl-substituted amiloride derivative and a potent inhibitor of epithelial-type sodium channels, produces significant prolongation of the action potential in isolated ventricular preparations, a tissue in which epithelial-type sodium channels have not been reported. Experiments were therefore carried out to examine the possible electrophysiological effects of phenamil on whole-cell ionic currents recorded in voltage-clamped guinea pig ventricular myocytes. At higher concentrations than those required to inhibit the epithelial sodium channels, phenamil (10-60 microM as compared to nanomoles for epithelial sodium channels) significantly increased the action potential duration in ventricular myocytes. It also induced a slow progressive depolarization which was followed eventually by oscillatory potentials near -35 mV. Higher concentrations of phenamil accelerated these changes. These effects were partially reversible. Voltage-clamp experiments using slow voltage ramps from -140 mV to +60 mV or step protocols from -120 mV to +30 mV revealed a prominent (approximately 70% at -120 mV) inhibitory effect of phenamil (50 microM) on the inwardly rectifying potassium current. Phenamil (50 microM) had little effect on the delayed rectifier potassium currents recorded by using long (5 sec) voltage steps from -40 mV to +60 mV. In the absence of K+, calcium current (L-type) was not affected by phenamil at concentrations up to 100 microM. Our data support the concept that phenamil-induced action potential prolongation in guinea pig cardiac myocytes may be produced by inhibition of inwardly rectifying potassium current.

Action Potentials↗
PubMed1994

H5D12 monoclonal antibody identifies a 24-kD immunosuppressor factor produced by human chorio carcinoma cells.

PROBLEM: The purpose of this study was to identify and characterize embryo-associated immunosuppressor factor (EASF) secreted by chorio carcinoma cells with the help of EASF-specific monoclonal antibody H5D12 (raised against EASF purified from embryo growth media of in vitro fertilized human ova). METHOD: Paraffin-embedded slides of human chorio carcinoma as well as control cell lines were prepared, and immunohistochemistry was done by the avidin-biotin-peroxidase technique. EASF was affinity purified using H5D12-Sepharose 4B from culture media of cell lines and analyzed for immunosuppressive activity (by Concanavalin-A-induced lymphocyte proliferation assay) and molecular weight identity (by metabolic-labelling studies with 35S-methionine followed by immunoprecipitation and SDS-PAGE). RESULTS: H5D12 showed intense immunostaining of BeWo chorio carcinoma cells. Biosynthetic labeling studies identifies this factor as a 24-kD molecule, and EASF bioassay indicates that this factor possesses immunosuppressive activity. No such immunosuppressive activity or similar molecules were identified when control cell lines were analyzed. CONCLUSIONS: Monoclonal antibody H5D12 recognizes a 24-kD factor with immunosuppressive activity that is secreted by chorio carcinoma cells, which suggests that this is a unique factor and may be one of the key regulators of reproductive functions.

Antibodies, Monoclonal↗
PubMed1994

Serum lipid profile determines platelet reactivity to native and modified LDL-cholesterol in humans.

The effects of thrombin (0.2 U/ml) and native (n-LDL), malondialdehyde-modified (MDA-LDL) and auto-oxidized (ox-LDL) low-density lipoproteins (20 micrograms of protein/ml) on platelet activation were evaluated in seven hyperlipidemic patients and compared to seven controls (fasting serum cholesterol 8.49 +/- 0.5 and 4.61 +/- 0.4 mM, respectively). Basal and thrombin-induced increases in platelet intracellular free calcium ion concentration ([Ca2+]i; fura-2) were similar in hyperlipidemic patients and controls (45 +/- 5 vs 42 +/- 3 and 635 +/- 51 vs 599 +/- 69 mM, respectively). n-LDL, MDA-LDL and ox-LDL increased basal [Ca2+]i (16, 36 and 81 percent, p < 0.01 between LDL-types), increases were consistently smaller in patients. There was an inverse relationship between LDL-induced responses and fasting serum LDL cholesterol as well as LDL/HDL ratio. In conclusion, modified LDL activated platelets to a greater extent than n-LDL, suggesting different types of LDL-receptors. Their agonistic effect was inversely related to the fasting serum lipid profile, suggesting that blunting of platelet responses to LDL could represent a protective mechanism in hyperlipidemic patients.

Adult↗
PubMed1993

Pyrazine compounds and the measurement of cytosolic Ca2+.

Several of the pyrazine derivatives are widely used for inhibiting sodium flux via Na+/Ca2+ exchange or Na+/H+ exchangers or through the epithelial cation channels. These processes can profoundly affect cytosolic Ca2+. We found that the widely used fluorescent probes fura-2 and indo-1 could not be used to measure the effect of pyrazine analogs on the cytosolic free calcium ([Ca2+]i) of YAC-1 lymphoma cells treated with the pore-forming protein cytolysin/perforin. We show that the excitation spectra of pyrazine derivatives that specifically inhibit Na+/Ca2+ exchange [5-(N-4-chlorobenzyl)-2',4'-dimethylbenzamil], Na+/H+ exchange [5-(N-ethyl-N-isopropyl)-amiloride], and Na+ channels (phenamil) overlap with those of fura-2 and indo-1. In the presence of Ca2+, fluorescence readings for fura-2 plus drug are greater than those of fura-2 alone with the typically used 340- and 380-nm excitation light wavelengths; F380 readings were more affected than F340 readings. The effect was drug dose dependent. Hence, calculations that use F340 readings in the presence of pyrazine derivatives will result in overestimates of [Ca2+]i, while those that use the corresponding ratio readings, R340/380, will result in underestimates of [Ca2+]i. We found that the luminescent intracellular Ca2+ indicator aequorin could be used successfully with pyrazine derivatives, and that the ability of these compounds to enhance cytolysin/perforin-mediated increases in [Ca2+]i corresponded to their previously reported ability to inhibit Na+/Ca2+ exchange in pituitary cell plasma membrane vesicles. YAC-1 lymphoma cells are easy to culture and handle and may be a useful model for the studies of the Na+/Ca2+ exchanger in situ.

Aequorin↗
PubMed1993

Role of sodium in antigen-induced contraction of tracheal smooth muscle in dogs.

We examined the role of Na+ influx in the airway response to antigen (ragweed pollen extract) in sensitized dogs, using amiloride analogs to block Na(+)-dependent processes. In in vivo studies, respiratory resistance was measured in amiloride treated and untreated groups. The resistance increased by 9.3 cmH2O.L-1.sec in response to ragweed aerosol in the untreated group, but increased only by 5.2 cmH2O.L-1.sec in the treated group. In in vitro studies, isometric tension was measured in ragweed pollen sensitized tracheal strips. Tissues were treated with amiloride or its derivatives (50 microM) for specifically blocking Na+ channels (phenamil), Na(+)-H+ exchanger [5-(N-methyl-N-guanidinocarbonyl methyl)-amiloride] or Na(+)-Ca2+ exchanger [5-(4-chlorobenzyl)-2',4'-dimethylbenzamil]. In untreated strips, tension increased in response to ragweed by 1.9 +/- 0.5 mN/mg. The increase was reduced by phenamil (95.2 +/- 2.5%; P < 0.01) and amiloride (41.7 +/- 13.1%; P < 0.01), but not by the other two agents. Furthermore, phenamil also inhibited histamine-induced tension response and histamine-induced 22Na+ uptake of the muscle. We conclude that antigen-induced airway response is attenuated by blocking Na+ influx in smooth muscle.

Amiloride↗
PubMed1993

Production and characterization of monoclonal antibodies to embryo-associated immunosuppressor factor (EASF) produced by human pre-implantation embryo.

Balb/c mice were immunized with pre-implantation embryo-associated immunosuppressor factor (EASF) (purified from embryo growth media of in vitro fertilized human ova). Hybridoma clones were produced by fusing their spleen cells with NS1 and P3X653 myeloma cell lines. The presence of specific anti-EASF monoclonal antibodies (mAb) in the hybridoma culture supernatants were tested by enzyme-linked immunosorbent assay. A total of 15 hybridoma clones were selected, and their products were purified and characterized. Each mAb bound specifically to its antigen in a dose-dependent manner. The affinity-purified EASF from embryo growth media demonstrated immunosuppressive activity on Concanavalin A-induced lymphocytes and the presence of 14 kDa, 24 kDa and 37 kDa factors. No such activity or similar molecules were identified when control growth media were analyzed. This clearly demonstrates that these mAb are indeed EASF-specific and are able to recognize biologically active immunosuppressive components in embryo growth media. These mAbs are presently being tested for the development of EASF-specific assay system.

Animals↗
PubMed1993

Inhibition of immunopurified DNA polymerase-alpha from PA-3 prostate tumor cells by platinum (II) antitumor drugs.

Cisplatin (cis-diamminedichloroplatinum(II); cis-DDP) is used as an effective drug for treatment of a variety of cancers, such as carcinomas of bladder, ovarian, and testicular origin. Immunopurified DNA polymerase-alpha from rat prostate tumor PA-3 cells was inhibited (50%) in the presence of cis-DDP (165 microM) and PtCl2(en) (cis-dichloroethylenediamine platinum (II); DEDAP) (75 microM) and remained uninhibited in the presence of trans-DDP. Immunopurified DNA polymerase-alpha was preincubated with cis-DDP and separated from unreacted cis-DDP by gel filtration chromatography. The platinated DNA polymerase-alpha was unable to initiate the DNA chain extension reaction. N-ethylmaleimide (1 mM), a thiol group modifier, also inhibited (95%) the DNA polymerase-alpha catalyzed reaction in vitro. Possible disruption of a zinc-finger motif of the DNA polymerase-alpha polypeptide chain by replacement of zinc is suggested.

Animals↗