Study of the eta c(1 (1)S0) state of charmonium formed in p-barp annihilations and a search for the eta c'(2 (1)S0).
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Journal Article↗
Biomedical subjects
R Ray
Publications and source records attributed to R Ray.
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↗
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↗
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↗
Metabolism of 3H-1 alpha,25-dihydroxyvitamin D3 in cultured human keratinocytes.
In the present investigation we studied the metabolism of 1 alpha,25-dihydroxy-[1 beta-3H]vitamin D3 (3H-1,25(OH)2D3) in culture-grown human keratinocytes (CHK). Our results showed that the cellular uptake of 3H-1,25(OH)2D3, upon incubation with CHK, occurred very rapidly; and it paralleled a decrease in the concentration of 3H-1,25(OH)2D3 in the medium. The amount of 3H-calcitroic acid, on the other hand, increased slowly in the medium, while the concentration of 3H-calcitroic acid in the cell remained undetectable during the whole period of incubation. When the cells were preincubated with 1,25(OH)2D3 (10(-8)M), conversion of 3H-1,25(OH)2D3 to 3H-calcitroic acid increased almost twofold, indicating that 1,25(OH)2D3 catalyzed its own catabolism.
Calcitriol↗
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↗
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↗
Aminopropylation of vitamin D hormone (1 alpha,25-dihydroxyvitamin D3), its biological precursors, and other steroidal alcohols: an anchoring moiety for affinity studies of sterols.
In this communication we describe a simple two-step procedure for the conversion of several steroidal alcohols to their aminopropyl ether derivatives. To demonstrate the usefulness of this procedure we synthesized a second-generation photoaffinity labeling analog of 1 alpha,25-dihydroxyvitamin D3, and a 25-hydroxyvitamin D3 derivative containing a long and chemically stable tether. Utilities of these aminopropyl ether derivatives of steroids in various affinity studies of receptor proteins are discussed.
Alcohols↗
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↗
Transcriptional regulation of cellular and viral promoters by the hepatitis C virus core protein.
The genomic region encoding the hepatitis C virus (HCV) core protein was cloned into a mammalian expression vector to study its role on the transcriptional regulation of cellular proto-oncogene and viral promoters. Using a transient transfection assay in human hepatocellular carcinoma (HepG2) cells, we demonstrate that the HCV core protein activates the human c-myc, Rous sarcoma virus long terminal repeat (LTR), and simian virus 40 (SV40) early promoters; and suppresses the c-fos promoter and human immunodeficiency virus type 1 (HIV-1) LTR activity. The transcriptional regulation of cellular proto-oncogenes by the HCV core protein suggests possible involvement of the core protein in the deregulation of normal hepatocyte growth and hepatocarcinogenesis.
3T3 Cells↗
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↗
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↗
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↗
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↗
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↗
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↗
Search for antiproton decay at the Fermilab antiproton accumulator.
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Journal Article↗
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↗