Biomedical subjects

R Ray

Publications and source records attributed to R Ray.

At least 19 recordsLinked to original sources

PubMed1995

25-Hydroxy[26,27-methyl-3H]vitamin D3-3 beta-(1,2-epoxypropyl)ether: an affinity labeling reagent for human vitamin D-binding protein.

Vitamin D-binding protein (DBP) is primarily involved in the binding and transportation of vitamin D3 and its various metabolites to target organs and tissues. This is manifested by the ability of DBP to bind vitamin D3 and its metabolites with high affinity. In the present study we developed 25-hydroxyvitamin D3-3 beta-(1,2-epoxypropyl)ether (25-OH-D3-epoxide) as an affinity labeling reagent of human DBP (hDBP). Competitive radioligand binding assays of 25-OH-D3-epoxide with hDBP demonstrated that the binding affinity of this analog was similar to that of 25-hydroxyvitamin D3 (25-OH-D3). Incubation of 25-hydroxy[26(27)-3H]-vitamin D3-3 beta-(1,2-epoxypropyl)ether [[3H]25-OH-D3-epoxide] with hDBP covalently labeled the protein. When the incubation was carried out in the presence of a large excess of 25-OH-D3, labeling was removed completely. When human Cohn IV fraction, containing hDBP, was incubated with [3H]25-OH-D3-epoxide a single protein band, corresponding to hDBP, was labeled. Labeling was completely obliterated in the presence of a large amount of 25-OH-D3. However, an equivalent amount of 7-dehydrocholesterol had no effect on labeling. These results demonstrated that [3H]25-OH-D3-epoxide most probably labeled the vitamin D sterol-binding domain of hDBP.

Affinity Labels↗

PubMed1995

Hepatitis C virus core protein: synthesis, affinity purification and immunoreactivity with infected human sera.

The genomic region encoding the core (C) protein (amino acids 1-162) of hepatitis C virus (HCV) was expressed in Escherichia coli as a recombinant (re-) protein with the maltose-binding protein (MBP) using the prokaryotic expression vector pMAL-CR1. The fusion protein (C::MBP) was identified as a approx. 62-kDa polypeptide by immunoblot analysis using antiserum to MBP and HCV-infected human sera. The size of C::MBP corresponded to the calculated combined molecular mass of the approx. 20-kDa HCV C protein and the approx. 42-kDa MBP. The approx. 62-kDa C::MBP was purified using amylose resin as a matrix in affinity chromatography, and showed specific reactivity with HCV-infected human sera. These results suggest that C::MBP may serve as a source of the core antigen for immunological studies on HCV infection.

ATP-Binding Cassette Transporters↗

PubMed1995

Trp-145 is essential for the binding of 25-hydroxyvitamin D3 to human serum vitamin D-binding protein.

Chemical modification of specific amino acid residues in a protein has been a valuable tool in identifying amino acid residues that are responsible for ligand binding of a protein. In the present investigation, we targeted Trp and His residues in human serum vitamin D-binding protein (hDBP) by modifying them with specific chemical modifiers. We also evaluated the results of these modifications in the binding of 25-hydroxy[26(27)-3H]vitamin D3 ([3H]25-OH-D3) to hDBP. We observed a dose-dependent loss of binding activity by N-bromosuccinimide (specific for Trp). Similar results were observed with diethylpyrocarbonate (specific for His). Furthermore, loss of [3H]25-OH-D3-binding was protected by preincubation of hDBP samples with an excess of 25-hydroxyvitamin D3. These results strongly emphasized the importance of Trp (single residue at position 145) and 1 His residue (out of a total of 6) in the vitamin D sterol-binding by vitamin D-binding protein.

Bromosuccinimide↗

PubMed1995

Affinity purification of human plasma vitamin D-binding protein.

During the course of our studies to probe the vitamin D ligand-binding domains of vitamin D-binding protein and vitamin D receptor, we developed a synthetic procedure to modify the 3 beta-hydroxyl group of vitamin D3 and its 25-hydroxy- and 1,25-dihydroxy metabolites with a 3'-aminopropylether group. In the present study we have coupled 25-hydroxyvitamin D3-3 beta-3'-aminopropylether to an activated Sepharose matrix. Using this stable and reusable affinity matrix we have purified human vitamin D-binding protein from human plasma to homogeneity.

Binding Sites↗

PubMed1995

Sulfur mustard-induced increase in intracellular free calcium level and arachidonic acid release from cell membrane.

The mechanism of action of the alkylating agent bis-(2-chloroethyl)sulfide (sulfur mustard, SM) was studied using the in vitro mouse neuroblastoma-rat glioma hybrid NG108-15 clonal cell line model. Following 0.3 mM SM exposure, cell viability remained high (> 80% of untreated control) up to 9 hr and then declined steadily to about 40% of control after 20-24 hr. During the early period of SM exposure, when there was no significant cell viability loss, the following effects were observed. The cellular glutathione level decreased 20% after 1 hr and 34% after 6 hr. Between 2 and 6 hr, there was a time-dependent increase (about 10 to 30%) in intracellular free calcium (Ca2+), which was localized to the limiting membrane of swollen endoplasmic reticula and mitochondria, to euchromatin areas of the nucleus, and to areas of the cytosol and plasma membrane. Moreover, there was also a time-dependent increase in the release of isotopically labeled arachidonic acid ([3H]AA) from cellular membranes. Increase in [3H]AA release was 28% at 3 hr and about 60-80% between 6 and 9 hr. This increase in [3H]AA release was inhibited by quinacrine (20 microM), which is a phospholipase (PLA2) inhibitor. At 16 hr after SM exposure, there was a large increase (about 200% of control) in [3H]AA release, which was coincident with a 50% loss of cell viability. These results suggest a Ca(2+)-mediated toxic mechanism of SM via PLA2 activation and arachidonate release.

Animals↗

PubMed1995

In situ hybridization detection of short viral amplicon sequences within cultured cells and body fluids after the in situ polymerase chain reaction.

Using single primer pairs, intracellular gene sequences of cytomegalovirus (CMV-Towne's strain) and alpha-tubulin were amplified (in situ PCR) from cells in human body fluids and in suspensions. Visualization of CMV amplificants was carried out by in situ hybridization (ISH), using both a biotinylated double-stranded DNA probe and a radiolabelled oligonucleotide probe. Visualization of alpha-tubulin amplificants was achieved using both radiolabelled single-stranded cRNA and oligonucleotide probes. Liberated amplificants were also identified by bands of expected size by gel electrophoresis. The specificity of the PCR products was confirmed by Southern blot analysis. Intracellular amplification was identified both in unfixed cells and, optimally, after brief alcohol fixation, whilst maintaining relative isotonicity in all working solutions. For CMV, enhanced signal was observed in cells (cultured fibroblasts or urine sediment) undergoing in situ PCR using either biotinylated or radiolabelled probes compared with controls undergoing ISH alone. For alpha-tubulin, radiolabelled riboprobes and oligoprobes only produced signals within cells (human peripheral lymphocytes, ascitic fluid and bladder washings from routine cytological specimens) after in situ PCR, but not after ISH alone. Morphological evaluation was superior with biotinylated probes, and minimal back-diffusion effect was found compared with radiolabelled probes. Up to 80% of cells survived thermal cycling. In situ PCR detected short sequence (100 bp) foreign DNA and low copy number genomic DNA, and was superior to ISH alone. In contrast to radiolabelled probes, very small CMV amplificants could be detected without a significant 'back-diffusion' effect when using the large biotinylated probe in this model system.

Animals↗

PubMed1995

Unidimensionality of alcohol dependence syndrome.

To evaluate the coherence of Alcohol Dependence Syndrome (ADS) as defined by DSM III-R criteria, 482 males were diagnosed as having ADS from a representative general population survey. Nine DSM III-R criteria were factor analyzed to assess unidimensionality. Unlike our earlier report on the same issue, a single-factor model, rather than a two-factor model, supports the coherence of the dependence syndrome explaining the relationship among the nine criteria for community data. When combining criteria into cumulative scales, they formed good approximation of Guttman scales and further supported unidimensionality. Withdrawal and withdrawal avoidance had maximum loading (0.9) and thus can be considered as highly central criteria in defining the syndrome, while preoccupation measured the most severe level of dependence (z = 0.21).

Adolescent↗

PubMed1995

N-methyl-D-aspartate- or glutamate-mediated toxicity in cultured rat cortical neurons is antagonized by FPL 15896AR.

The neuroprotective action of (S)-alpha-phenyl-2-pyridineethanamine dihydrochloride (FPL 15896AR), a novel noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, was examined in primary rat cortical neuronal cultures. Exposure of cortical cultures to NMDA (50 microM) or glutamate (50 microM) for 15 min resulted in the death of 85-95% of the neurons during the next 24 h. This neurotoxicity was completely eliminated by adding FPL 15896AR (50 microM) to the cultures during the time of NMDA or glutamate exposure. Neuroprotective concentrations of FPL 15896AR also inhibited other acute effects of NMDA. FPL 15896AR (50 microM) prevented the loss of membrane-associated protein kinase C activity that developed by 4 h after transient exposure to 50 microM NMDA or 50 microM glutamate. FPL 15896AR also reduced by approximately 35% the magnitude of NMDA-triggered increases in intracellular free Ca2+ concentration in the cortical cultures. These data indicate that NMDA-mediated toxicity in cultured cortical neurons can be blocked by the NMDA antagonist FPL 15896AR.

Animals↗

PubMed1995

Characterization of a live, attenuated human parainfluenza type 3 virus candidate vaccine strain.

Characterization of a temperature-sensitive and live, attenuated human parainfluenza type 3 virus strain (cp45) grown at a permissive temperature (32 degrees C) suggested that the virus efficiently multiplies in cell lines and retains antigenic and functional properties of the envelope glycoproteins. When grown at a nonpermissive temperature (39.5 degrees C), the cp45 virus exhibited poor replication; however, shifting to a permissive temperature allowed virus growth. Although at a nonpermissive temperature virus polypeptide synthesis was significantly reduced, the hemagglutinin-neuraminidase and fusion glycoproteins were transported to cell surfaces and retained their characteristic biologic activities. Studies on mRNA synthesis from the P protein gene suggested a poor transcriptional activity of the cp45 virus at a nonpermissive temperature. Results from this study indicate that the temperature sensitivity of cp45 virus is related to altered transcriptional activity and a marked reduction in virus polypeptide synthesis.

Antibodies, Monoclonal↗

PubMed1995

Immune responses to plasmid DNA encoding the hepatitis C virus core protein.

Hepatitis C virus (HCV) is a major causative agent of parenterally transmitted non-A, non-B hepatitis. The genomic region encoding the virion-associated core protein is relatively conserved among HCV strains. To generate a DNA vaccine capable of expressing the HCV core protein, the genomic region encoding amino acid residues 1 to 191 of the HCV-1 strain was amplified and cloned into an eukaryotic expression vector. Intramuscular inoculation of recombinant plasmid DNA into BALB/c mice (H-2d) generated core-specific antibody responses, lymphoproliferative responses, and cytotoxic T-lymphocyte activity. Our results suggest that the HCV core polynucleotide warrants further investigation as a potential vaccine against HCV infection.

3T3 Cells↗

PubMed1995

Controlling America's health care costs via health care futures.

This article examines the new health care futures contract to be released by the Chicago Board of Trade sometime during calendar year 1995. Health care futures--futures contracts on health care insurance--represent a new class of financial products never before available to financial practitioners. The innovation allows health care underwriters to effectively freeze their health care costs. Besides helping to control a cost that currently consumes one seventh of our national income, health care futures also allow underwriters to plan far more effectively.

Actuarial Analysis↗

PubMed1994

Changes in UsnRNA biosynthesis during rat liver regeneration.

Partial hepatectomy (P.H.) induces a partially synchronized growth response of liver under normal regulation of growth. In this phase changes in cellular morphology, radial distribution pattern of cells and other biological as well as major biochemical changes are well documented. Here, we have shown that the cellular content of UsnRNAs altered during this proliferative phase as well. The level of spliceosomal UsnRNAs (U1, U2, U4-U6) gradually decreased by 30-50% upto 48 hrs of P.H. followed by gradual increase to reach the normal level within one month of P.H. The U3 snRNA level on the other hand, was nearly equal to that in normal liver at 48 hrs of P.H. but in 24 and 72 hrs of P.H. its level was high (4 fold) in contrast to that in other UsnRNAs. Thus, it is clear from our data that the level of all the six UsnRNAs decreased during 48 hrs of P.H. compared to that after first 24 hrs. This has been correlated in the kinetics of UsnRNAs' synthesis (in terms of labelling) in isolated hepatocytes, where the rate of labelling of all the six UsnRNAs increased 20-30% in 24 hrs regenerating hepatocytes (R.H.) followed by sharp decrease by 30-50% within next 24 hrs, compared to that in the normal hepatocytes. But from 72 hrs onwards in R.H. the rate of labelling of all the six UsnRNAs again increased by 30-50% (compared to that in normal hepatocytes) followed by decrease of their labelling-rate to reach the normal level in R.H. within one month of P.H.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Changes in UsnRNA biosynthesis during rat liver regeneration.

Partial hepatectomy (P.H.) induces a partially synchronized growth response of liver under normal regulation of growth. In this phase changes in cellular morphology, radial distribution pattern of cells and other biological as well as major biochemical changes are well documented [24]. Here, we have shown that the cellular content of UsnRNAs altered during this proliferative phase as well. The level of spliceosomal UsnRNAs (U1, U2, U4-U6) gradually decreased by 30-50% upto 48 hrs of P.H. followed by gradual increase to reach the normal level within one month of P.H. The U3 snRNA level on the other hand, was nearly equal to that in normal liver at 48 hrs of P.H. but in 24 and 72 hrs of P.H. its level was high (4 fold) in contrast to that in other UsnRNAs. Thus, it is clear from our data that the level of all the six UsnRNAs decreased during 48 hrs of P.H. compared to that after first 24 hrs. This has been correlated in the kinetics of UsnRNAs' synthesis (in terms of labelling) in isolated hepatocytes, where the rate of labelling of all the six UsnRNAs increased 20-30% in 24 hrs regenerating hepatocytes (R.H.) followed by sharp decrease by 30-50% within next 24 hrs, compared to that in the normal hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Binding characteristics of a membrane receptor that recognizes 1 alpha,25-dihydroxyvitamin D3 and its epimer, 1 beta,25-dihydroxyvitamin D3.

The steroid hormone 1 alpha,25-dihydroxyvitamin D3 has been shown to exert rapid effects (15 s to 5 min) in osteoblasts. These effects occur in osteoblast-like cells lacking the nuclear vitamin D receptor, ROS 24/1, suggesting that a separate signalling system mediates the rapid actions. These non-genomic actions include rapid activation of phospholipase C and opening of calcium channels, pointing to a membrane localization of this signalling system. Previous studies have shown that the 1 beta epimer of 1 alpha,25-dihydroxyvitamin D3 can block these rapid actions, indicating that the 1 beta epimer may bind to the receptor responsible for the rapid actions in a competitive manner. We have assessed the displacement of 3H-1 alpha,25-dihydroxyvitamin D3 by vitamin D compounds, as well as the apparent dissociation constant of 1 alpha,25-dihydroxyvitamin D3 and its 1 beta epimer for the membrane receptor in membrane preparations from ROS 24/1 cells. Increasing concentrations of 1 alpha,25-dihydroxyvitamin D3, 7.25 nM to 725 nM, displaced 3H-1 alpha,25-dihydroxyvitamin D3 from the membranes with 725 nM of the hormone displacing 40-49% of the radioactivity. Similarly, 1 beta,25-dihydroxyvitamin D3, 7.25 nM and 72.5 nM, displaced 1 alpha,25-dihydroxyvitamin D3 binding while 25-hydroxyvitamin D3, 72.5 nM and 725 nM, did not. The apparent dissociation constant (KD) for 1 alpha,25-dihydroxyvitamin D3 was determined from displacement of 3H-1 alpha,25-dihydroxyvitamin D3 yielding a value of 8.1 x 10(-7) M by Scatchard analysis. The KD for the 1 beta epimer determined from displacement of 3H-1 beta,25-dihydroxyvitamin D3 was 4.8 x 10(-7) M.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Inhibition of bioenergetics alters intracellular calcium, membrane composition, and fluidity in a neuronal cell line.

The effect of inhibited bioenergetics and ATP depletion on membrane composition and fluidity was examined in cultured neuroblastoma-glioma hybrid NG108-15 cells. Sodium cyanide (CN) and 2-deoxyglucose (2-DG) were used to block, oxidative phosphorylation and anaerobic glycolysis, respectively. Endoplasmic reticulum (ER) Ca(2+)-pump activity measured by 45Ca2+ uptake was > 92% inhibited in intact cells incubated with CN (1 mM) and 2-DG (20 mM) for 30 min. In addition, exposure of cells to CN and 2-DG caused a 134% increased release of isotopically labeled arachidonic acid (3H-AA) or arachidonate-derived metabolites from membranes. Removal of Ca2+ from the incubation medium ablated the CN/2-DG induced release of 3H-AA or its metabolites. Membrane fluidity of intact cells was measured by electron spin resonance spectroscopy using the spin label 12-doxyl stearic acid. The mean rotational correlation time (tau c) of the spin label increased 49% in CN/2-DG exposed cells compared to controls, indicating a decrease in membrane fluidity. These results show that depletion of cellular ATP results in inhibition of the ER Ca(2+)-pump, loss of AA from membranes, and decreased membrane fluidity. We propose that impaired bioenergetics can increase intracellular Ca2+ as a result of Ca(2+)-pump inhibition and thereby activate Ca(2+)-dependent phospholipases causing membrane effects. Since neurons derive energy predominantly from oxidative metabolism, ATP depletion during brain hypoxia may initiate a similar cytotoxic mechanism.

Adenosine Triphosphate↗

PubMed1994

Dementia in elderly patients: can the 3R mental stimulation programme improve mental status?

The aim of this study was to determine whether the 3R mental stimulation programme can improve the mental status score (MSS) of a group of patients with dementia attending a day care centre. Fifteen patients with dementia attending a day care centre were exposed to the 3R stimulation programme over eight weeks while 15 controls were not. All patients from both groups were subjected to pre- and post-exposure questionnaires modified from the Mental Status Questionnaire of Kahn et al. A mental status score (MSS) was thus calculated on both occasions. All patients in the exposed group showed an improved mental status score, with the mean 'pre-exposure' and 'post-exposure' scores of 4.4 and 7.3, respectively (p < 0.001; paired t test). In contrast, the mental status scores of 12 control patients had deteriorated and mean 'pre-exposure' and 'post-exposure' scores were 4.1 and 3.4, respectively (p < 0.05; paired t test). Using multiple covariance analysis, the adjusted 'post-exposure' mean mental status score was 7.1 for the exposed and 3.6 for the controlled group (p < 0.001). The 3R programme appears to be effective for short-term mental stimulation of demented elderly people.

Aged↗

PubMed1994

Seroreactivity and genomic amplification profile of hepatitis C virus from patients with chronic liver disease in India.

Patients with chronic liver disease were screened initially for antibodies to the hepatitis C virus using commercially available assay systems. Positive sera were tested for amplification of the 5' noncoding region, and putative structural and nonstructural genomic regions of hepatitis C virus by reverse transcription-polymerase chain reaction. Eight out of 22 patients who were seropositive showed presence of viral RNA by amplification from genomic regions. Sera from four subjects showed amplification of the 5' noncoding region, a different serum showed amplification of the capsid region, and three other sera showed amplification of the nonstructural genomic region of the virus. An amplification profile of the RNA-positive samples suggests genomic sequence divergence of hepatitis C virus circulating among Indian patients.

Base Sequence↗

PubMed1994

Peptide immunogen mimicry of putative E1 glycoprotein-specific epitopes in hepatitis C virus.

Hepatitis C virus (HCV) accounts for most cases of acute and chronic non-A and non-B hepatitis with serious consequences that may lead to hepatocellular carcinoma. The putative envelope glycoproteins (E1 and E2) of HCV probably play a role in the pathophysiology of the virus. In order to map the immunodominant domains of the E1 glycoprotein, two epitopes from amino acid residues 210 to 223 (P1) and 315 to 327 (P2) were predicted from the HCV sequence. Immunization of mice with the synthetic peptides conjugated to bovine serum albumin induced an antibody response, and the antisera immunoprecipitated the E1 glycoprotein (approximately 33 kDa) of HCV expressed by recombinant vaccinia virus. A panel of HCV-infected human sera was also tested with the synthetic peptides by enzyme-linked immunosorbent assay for epitope-specific responses. Of 38 infected serum samples, 35 (92.1%) demonstrated a spectrum of reactivity to the P2 peptide. On the other hand, only 17 of 38 (44.7%) serum samples were reactive to the P1 peptide. Strains of HCV exhibit a striking genomic diversity. The predicted P1 epitope showed localization in the sequence-variable region, and the P2 epitope localized in a highly conserved domain. Results from this study suggest that the E1 glycoprotein of HCV contains at least two potential antigenic epitopes. Synthetic peptides corresponding to these epitopes and antisera to these peptides may serve as the monospecific immunological reagents to further determine the role of E1 glycoprotein in HCV infection.

Amino Acid Sequence↗