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R Ray

Publications and source records attributed to R Ray.

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

Acetylcholine exocytosis in PC12 cells deficient in SNAP-25.

Stimulus-induced acetylcholine (ACh) exocytosis from presynaptic nerve terminals involves two important steps: fusion of ACh loaded vesicles at presynaptic release sites, followed by release into the synaptic cleft. We studied the role of the putative vesicle fusion protein SNAP-25 in this process. The nerve growth factor-differentiated PC12 cell line was used as an experimental model. A bee venom tetradecapeptide (INLKALAALAKKIL-NH2) phospholipase A2 (PLA2) activator, mastoparan, was used to induce ACh release. Treatment of PC12 cells with appropriate antisense oligonucleotides blocked SNAP-25 expression, as judged by Western blot protein analysis with a specific monoclonal antibody. Despite apparent elimination of SNAP-25, treatment of differentiated PC12 cells with mastoparan and high (80 mM) K+ induced ACh exocytosis. The results indicate that in PC12 cells, ACh exocytosis due to mastoparan plus K+ can occur in the absence of SNAP-25.

Acetylcholine↗
PubMed1997

Roles of the structure and orientation of ligands and ligand mimics inside the ligand-binding pocket of the vitamin D-binding protein.

1alpha,25-Dihydroxyvitamin D3, the vitamin D hormone, manifests its diverse biological properties by specifically binding to the vitamin D sterol-binding pockets of vitamin D-binding protein (DBP) and vitamin D receptor. In the past, several affinity, photoaffinity, and chemical modification studies have been carried out to probe the vitamin D sterol-binding pocket of DBP and to evaluate the relationship between the structure of this pocket and the functions of the protein. In the present study, we examined the steric requirements inside this pocket by considering conformational structures of various bromoacetate derivatives of 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3 and their abilities to covalently and specifically modify this pocket. We observed that, although 25-hydroxyvitamin D3 3beta-bromoacetate (25-OH-D3-3-BE), 1alpha,25-dihydroxyvitamin D3 3beta-bromoacetate [1alpha,25(OH)2D3-3-BE], 1alpha,25-dihydroxyvitamin D3 1alpha-bromoacetate [1alpha,25(OH)2D3-1-BE], and 1alpha,25-dihydroxyvitamin D3 1alpha,3beta-dibromoacetate [1alpha,25(OH)2D3-1,3-di-BE] bound DBP in a specific manner, only [3H]-25-OH-D3-3-BE and [3H]-1alpha,25(OH)2D3-3-BE affinity labeled the protein. BNPS-skatole cleavages of [3H]-25-OH-D3-3-BE- and 3H-1alpha,25(OH)2D3-3-BE-labeled DBP samples produced the same labeled peptide (N-terminal), demonstrating the specificity of labeling by these analogs. Energy-minimized conformational structures of these bromoacetate derivatives indicated significant changes in the A-ring conformations of these analogs. These structural changes were invoked to explain the inability of [3H]-1alpha,25(OH)2D3-1-BE and [3H]-1alpha,25(OH)2D3-1,3-di-BE to affinity label DBP. Overall, these studies suggested that the vitamin D sterol-binding pocket in DBP is sterically quite restrictive. This information could be potentially important in designing future vitamin D-based drugs for several diseases.

Acetates↗
PubMed1997

Transcriptional repression of p53 promoter by hepatitis C virus core protein.

Our previous results have suggested that the putative core protein of hepatitis C virus (HCV) transcriptionally regulates cellular and viral genes, inhibits cisplatin and c-myc-mediated apoptotic cell death under certain conditions, and transforms primary rat embryo fibroblast cells with a cooperative oncogene. Because HCV appears to cause hepatocellular carcinoma, we evaluated the regulatory role of the HCV core protein on p53, a well known tumor suppressor gene, by an in vitro transfection assay. HCV core protein repressed transcriptional activity of the p53 promoter when tested separately in COS7 and HeLa cells. Deletion mutational analysis of the HCV core gene indicated that the regulatory domain involved in the repression of p53 transcriptional activity is located around amino acid residues 80-122 encompassing a putative DNA binding motif and two major phosphorylation sites. Results from this study suggest that the putative core protein may have an important biological role in the promotion of cell growth by repressing p53 transcription, and this appears to be consistent with certain earlier observations about HCV core moving into the nucleus.

Animals↗
PubMed1997

Randomized trial of breast self-examination in Shanghai: methodology and preliminary results.

BACKGROUND: The efficacy of breast self-examination in helping to reduce mortality from breast cancer has not been rigorously demonstrated. PURPOSE: To assess efficacy, a large, randomized trial was initiated in Shanghai, China. METHODS: From October 1989 to October 1991, 267040 current and retired female employees associated with 520 factories in the Shanghai Textile Industry Bureau were randomly assigned on the basis of factory to either a self-examination instruction group (133375 women) or a control group (133665 women). The women were born within the period from 1925 through 1958. Women in the instruction group were given intensive training in breast self-examination, including the use of silicone breast models and personalized instruction, plus two subsequent reinforcement sessions and multiple reminders to practice the technique. Women in the control group were asked to attend training sessions on the prevention of low back pain. All women have been followed for the development of breast diseases and for death from breast cancer. RESULTS: A high level of participation during the first 4-5 years of the trial was documented among women in the instruction group. Randomly sampled women in this group demonstrated greater proficiency in detecting lumps in breast models than did randomly sampled women in the control group. Approximately equal numbers of breast cancers were detected in the two groups (331 in the instruction group and 322 in the control group) through 1994, which is the last year for which case-finding efforts have been completed. The breast cancers detected in the instruction group were not diagnosed at an appreciably earlier stage or smaller size than those in the control group. More benign breast lesions were detected in the instruction group than in the control group (1457 versus 623, respectively), suggesting a higher index of suspicion for women who received training. Cumulative breast cancer mortality rates through 5 years from entry into the study were nearly equivalent for the two groups. CONCLUSIONS: Breast self-examination has not led to a reduction in mortality from breast cancer in this study cohort in the first several years since the trial began. A shift toward the diagnosis of disease at a less advanced stage in women given instruction has also not been demonstrated. Longer follow-up of participants in this trial is required before final assessment can be made of the efficacy of breast self-examination. IMPLICATIONS: At this time, there is insufficient evidence to recommend for or against the teaching of breast self-examination.

Adult↗
PubMed1997

Bacterial expression of human vitamin D-binding protein (Gc2) in functional form.

In this report, we report the first expression of human vitamin D-binding protein (hDBP), a serum protein with several functions and a multidomained structure, in Escherichia coli. The recombinant protein (reDBP) was expressed as a fusion partner of glutathione S-transferase in order to facilitate proper folding of the reDBP; E. coli-expressed DBP was found to be fully functional with respect to vitamin D sterol binding, interaction with actin, and cross-reactivity with anti-DBP antibody. Furthermore, both natural DBP and reDBP were affinity-labeled with 25-hydroxyvitamin D3-3-bromo[1-14C]acetate in a similar fashion. Availability of an expression system for hDBP in functional form provides opportunity to develop mutants and truncated DBPs to study multiple ligand-binding properties of this protein in relationship with its structure.

Actins↗
PubMed1997

Differential alterations in metabolic pattern of the six major UsnRNAs during development.

The uridylic acid rich nuclear RNAs (U1-U6 snRNAs) are involved mainly in the processing of pre-mRNA and pre-rRNA. So, any control of cell growth through pre-mRNA/pre-rRNA processing may have some regulation through altered UsnRNAs metabolism. With this idea, attempts have been made to see how the metabolism of the six major UsnRNAs' changed during the normal process of cellular proliferation associated with differentiation from pluripotent/totipotent stem cells of early embryonic stage to much more differentiated state of different cell/tissue lineages in different tissues/organs during the fetal and neonatal stages of growth. It has been seen that the levels of the six major UsnRNAs were high in day 8 embryo when the cells were mainly pluripotent/totipotent in nature, and during the progression of embryonic development the levels of these UsnRNAs gradually decreased (approximately 35-65%) up to the midgestational period (day 13) with some exception, when the organogenesis has already been started. However in the fetal life, the levels of these UsnRNAs were maximum or comparable around 18 +/- 2 days of gestation in comparison to that in day 8 embryo when the kinetics of the maturational status of the different organs were quite high. But, the levels of these UsnRNAs' became low during day 21 of fetal life or in day 0 of birth (perturation period) in all the tissues/organs except high UsnRNAs' level in spleen. In the neonatal life, around 3 +/- 1 days of birth these UsnRNAs' levels again became maximum in all the tissues/organs (except in thymus) followed by decrease up to 5/6 days, and to become steady with slight increase within one to two weeks, when the kinetics of the organ maturation reached to a steady state. In case of thymus, the levels of the U3-U6 snRNAs were high on day 0 of birth followed by decrease in their level on day 1/2 and then increased to become steady within 2-4 weeks; whereas the U1 and U2 snRNAs' levels were high on day 3 of birth and the subsequent changes were similar to that in other tissues/organs. Thus the different UsnRNAs' metabolism in the perturation period and in the early stages of neonatal life has indicated the differential cellular functions in these two stages of development. These alterations in the metabolism of these UsnRNAs might be due to the differential changes in the rate of synthesis of these UsnRNAs and/or with their differential turnover rate in the different stages of development. Also, the differential variations of these UsnRNAs' levels have been observed among the different tissues/organs at the respective stages of development indicating the differences in the UsnRNAs' metabolism among the different cell/tissue lineages. Thus, it can be concluded that the metabolism of these UsnRNAs were developmentally regulated with some cell/tissue lineage variations, which might have some role in the developmentally regulated cellular process of proliferation and differentiation, through altered RNA splicing and processing.

Animals↗
PubMed1997

Prolonged pyrexia in a diabetic due to systemic aspergillosis.

Immuno-compromised patients are susceptible to a variety of opportunistic infections. Systemic aspergillosis is one such common infection in neutropenic subjects. A case of primary cutaneous aspergillosis with fungimia in a diabetic is reported.

Aspergillosis↗
PubMed1997

Use it or lose it.

Explore the source record for details and available documents.

Journal Article↗
PubMed1996

Suppression of apoptotic cell death by hepatitis C virus core protein.

We have previously demonstrated the role of hepatitis C virus (HCV) core protein in the transcriptional regulation of cellular and unrelated viral promoters. Furthermore, the core protein in cooperation with H-ras oncogene transforms primary rat embryo fibroblast cells to the tumorigenic phenotype. In the present study, the functional role of HCV core protein was investigated to determine its potential to inhibit the onset of apoptotic cell death. Expression of HCV core protein inhibited cisplatin mediated apoptosis in human cervical epithelial cells, and apoptosis induced by the overexpression of c-myc in Chinese hamster ovarian cells. Results from these studies suggest that the core protein may have a biological implication in the pathogenesis of HCV infection.

Animals↗
PubMed1996

Effects of the vitamin D3 analog 1 alpha, 25-dihydroxyvitamin D3-3 beta-bromoacetate on rat osteosarcoma cells: comparison with 1 alpha, 25-dihydroxyvitamin D3.

The actions of the hormonal form of vitamin D, 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25-(OH)2 D3], are mediated by both genomic and nongenomic mechanisms. Several vitamin D synthetic analogs have been developed in order to identify and characterize the site(s) of action of 1 alpha, 25-(OH)2D3 in many cell types including osteoblastic cells. We have compared the effects of 1 alpha, 25-(OH)2D3 and a novel 1 alpha, 25-(OH)2D3 bromoester analog (1,25-(OH)2-BE) that covalently binds to vitamin D receptors. Rat osteosarcoma cells that possess (ROS 17/2.8) or lack (ROS 24/1) the classic intracellular vitamin D receptor were studied to investigate genomic and nongenomic actions. In ROS 17/2.8 cells plated at low density, the two vitamin D compounds (1 x 10(-8) M) caused increased cell proliferation, as assessed by DNA synthesis and total cell counts. Northern blot analysis revealed that the mitogenic effect of both agents was accompanied by an increase in steady-state osteocalcin mRNA levels, but neither agent altered alkaline phosphatase mRNA levels in ROS 17/2.8 cells. ROS 17/2.8 cells responded to 1,25-(OH)2-BE but not the natural ligand with a significant increase in osteocalcin secretion after 72, 96, 120, and 144 hr of treatment. Treatment of ROS 17/2.8 cells with the bromoester analog also resulted in a significant decrease in alkaline phosphatase-specific activity. To compare the nongenomic effects of 1 alpha, 25-(OH)2D3 and 1,25-(OH)2-BE intracellular calcium was measured in ROS 24/1 cells loaded with the fluorescent calcium indicator Quin 2. At 2 x 10(-8) M, both 1 alpha,25-(OH)2D3 and 1, 25-(OH)2-BE increased intracellular calcium within 5 min. Both the genomic and nongenomic actions of 1,25-(OH)2-BE are similar to those of 1 alpha,25-(OH)2D3, and since 1,25-(OH)2-BE has more potent effects on osteoblast function than the naturally occurring ligand due to more stable binding, this novel vitamin D analog may be useful in elucidating the structure and function of cellular vitamin D receptors.

Animals↗
PubMed1996

Effect of anthracycline antitumor antibiotics (adriamycin and nogalamycin) and cycloheximide on the biosynthesis and processing of major UsnRNAs.

In the present study, anthracycline antitumor antibiotics (e.g. adriamycin and nogalamycin), the potent RNA synthesis inhibitors and cycloheximide, the protein synthesis inhibitor, have been used to understand the events of biosynthesis and processing of major UsnRNAs (U1-U6). The anthracyclines inhibit the UsnRNAs biosynthesis (in terms of labelling) differentially in a dose dependent manner. The inhibitory effect of adriamycin and nogalamycin reached plateau at a concentration of 2.5 micrograms/ 10(6) cells/ml and 0.1 microgram/10(6) cells/ml respectively and indicates that nogalamycin is more inhibitory than adriamycin. The inhibition of the UsnRNAs synthesis (in terms of labelling) became maximum within 30 min of incubation and remained unaltered even after 2 h. Thus, it shows that the anthracyclines preferentially inhibit the initiation of the UsnRNA genes' transcription as it has been seen in cases of other large RNAs' synthesis by some other laboratories. The higher inhibitory effect of the anthracyclines on the biosynthesis of U5 and U6 compared to other UsnRNAs indicates the presence of more binding sites on the U5 and U6 snRNA genes. In presence of the anthracyclines, there was high retention of cytoplasmic major pre-UsnRNAs/ UsnRNAs which indicates that the elongation of the UsnRNA synthesis is probably impaired along with initiation; because for the proper processing of the pre-UsnRNAs, formation of the correct secondary structure of that pre-UsnRNA is necessary. Cycloheximide showed some differential effect on the pol II transcribed UsnRNAs (U1-U5) biosynthesis (in terms of labelling) however it has no effect on the pol III transcribed U6 snRNA. It implies that in the pol II transcribed UsnRNAs, some transacting labile factors, either activator or inhibitor, are involved. Whereas, the processing of the UsnRNAs (either pol II or pol III transcribed) was affected more or less in a similar fashion in presence of cycloheximide, indicating the involvement of some transacting labile factors in this event.

Animals↗
PubMed1996

Affinity labeling of rat serum vitamin D binding protein.

Vitamin D binding protein (DBP) plays an essential role in the vitamin D hormone endocrine system in sequestering vitamin D3 and its metabolites with high affinity, and transporting them to various target organs and tissues. In the present investigation, 25-hydroxyvitamin D3-3 beta-(1,2-epoxypropyl)ether (25-OH-D3-epoxide) and 25-hydroxyvitamin D3-3 beta-bromoacetate (25-OH-D3-BE), synthetic analogs of 25-hydroxyvitamin D3 (25-OH-D3), were developed as affinity alkylating reagents for the covalent modification of the 25-OH-D3-binding site in rat vitamin D binding protein (rDBP). Competitive radioligand binding assays of 25-OH-D3-BE and 25-OH-D3-epoxide with affinity-purified rDBP demonstrated that these analogs displaced 25-hydroxy[26(27)-3H]vitamin D3 (3H-25-OH-D3), specifically bound to rDBP, in a dose-dependent fashion. Incubation of rDBP samples with radiolabeled versions of these analogs, i.e., 3H-25-OH-D3-epoxide and 3H-25-OH-D3-BE, resulted in the covalent labeling of rDBP. This labeling was largely prevented when incubations were carried out in the presence of an excess of 25-OH-D3, the natural ligand for rDBP. Labeling-specificity by these analogs was further demonstrated by the covalent labeling, inhibited by coincubation with a large excess of 25-OH-D3, of a single protein band, upon incubating rat serum Cohn IV fraction with 3H-25-OH-D3-epoxide and 3H-25-OH-D3-BE. Collectively, these results strongly suggested that 3H-25-OH-D3-epoxide and 3H-25-OH-D3-BE covalently modified the 25-OH-D3-binding site in rDBP. The reagents described in this report could be important in mapping the 25-OH-D3-binding pocket in rDBP.

Affinity Labels↗
PubMed1996

1 alpha,25-(OH)2-vitamin D3 analogs with minimal in vivo calcemic activity can stimulate significant transepithelial calcium transport and mRNA expression in vitro.

Several 1 alpha,25-(OH)2-vitamin D3 (1 alpha,25-(OH)2-D3) analogs have significant antiproliferative effects in vitro but do not elevate serum calcium in vivo. We tested whether the lack of a calcemic response of a vitamin D analog in vivo is due to its inability to stimulate intestinal calcium absorption by examining the effect of several such compounds on transepithelial calcium transport in the human colonic carcinoma cell line Caco-2. The relative stimulations of calcium transport by the four A-ring diastereomers of 1 alpha, 25-(OH)2-D3 (1 alpha,3 beta) and a 3 beta-bromoacetate analog (1 alpha,3 beta-BrAc) of the vitamin following 48-h treatment of cells at 10 nM were 1 alpha,3 beta (=100%), 1 alpha,3 alpha (+45.2%), 1 beta,3 beta (-15.6%), 1 beta,3 alpha (+6.5%), and 1 alpha,3 beta-BrAc (+50.6%). This was similar to the reported affinity of these compounds for the vitamin D receptor (VDR) and suggests that VDR binding predicts calcium transport. In contrast, three noncalcemic, sidechain- or D-ring-modified analogs of vitamin D, 1 alpha,25-(OH)2-16-ene-D3, 1 alpha,25-(OH)2-16-ene-23-yne-D3, and 1 alpha,25,28-(OH)3-D2 (at 10 nM for 48 h), showed a different relationship between VDR affinity (150, 60, and 63% of 1 alpha, 25-(OH)2-D3, respectively) and calcium transport (74.1, 126, and 10%, respectively). Elevated calcium transport was accompanied by higher 24-hydroxylase and calbindin D9k mRNA levels. The data demonstrate that although some vitamin D compounds cannot stimulate calcium transport due to an inability to interact with the VDR (e.g., 1 beta isomers), other factors, e.g., differential cellular metabolism, may account for variations in biological response in vivo to various vitamin D analogs.

Biological Transport↗
PubMed1996

Affinity labeling of the 1 alpha,25-dihydroxyvitamin D3 receptor.

Genomic actions of the calciotropic hormone 1 alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) involves a multistep process that is triggered by the highly specific binding of 1,25(OH)2D3 to 1 alpha, 25-dihydroxyvitamin D3 receptor, VDR. In order to study this key step in the cascade, we synthesized 1 alpha,25-dihydroxy[26(27)-3H]vitamin D3-3-deoxy-3 beta-bromoacetate (1,25(OH)2[3H]D3-BE) and 1 alpha,25-dihydroxyvitaminD3-3 beta-[1-14C]bromoacetate(1,25(OH)2D3-[14C]BE) binding-site directed analogs of 1,25(OH)2D3, and affinity-labeled baculovirus-expressed recombinant human VDR (with 1,25(OH)2[3H]D3-BE), and naturally occurring VDRs in cytosols from calf thymus homogenate and rat osteosarcoma (ROS 17/2.8) cells (with 1,25(OH)2D3-[14C]BE). In each case, specificity of labeling was demonstrated by the drastic reduction in labeling when the incubation was carried out in the presence of an excess of nonradioactive 1 alpha,25(OH)2D3. These results strongly suggested that 1,25(OH)2[3H]D3-BE and 1,25(OH)2D3-[14C]BE covalently modified the 1,25(OH)2D3-binding sites in baculovirus-expressed recombinant human VDR and naturally occurring calf thymus VDR and rat osteosarcoma VDR, respectively.

Affinity Labels↗
PubMed1996

Direct in situ reverse transcriptase polymerase chain reaction for detection of measles virus.

New methods are described for combined intracellular reverse transcription (RT) and polymerase chain reaction (PCR) using single primer pairs, with direct incorporation of digoxigenin-11-dUTP into amplificants (direct in situ RT/PCR). Routinely used fixatives and minimal pre-treatments were employed. Target sequences of measles virus nucleocapsid (N) and phosphoprotein genes were detected within measles virus infected Vero cells, both in suspension and in formalin-fixed sections, that had been treated by in situ reverse transcription and 30 cycles of direct in situ PCR. Uninfected cells, omission of Taq polymerase, and irrelevant primers were used as controls. Distribution of measles virus within infected cells was determined by in situ hybridisation and immunocytochemistry for measles virus N gene and protein, respectively. Confirmation of amplification within sections was by gel electrophoresis, Southern blotting and sequencing of extracted amplicons. In the majority of cases, measles-infected cells exhibited intense cytoplasmic signal after direct in situ PCR; this was not seen in uninfected cells or infected cells reacted either with irrelevant primers or without Taq polymerase. Unfixed cells in suspension required nested reaction. Measles-specific in situ hybridisation and immunocytochemistry gave an identical signal distribution in sections. Nuclear artifact occurred in some sections and was unpredictable, although it was greatest either in areas of cellular damage, following DNase predigestion, or with vigorous protease pre-treatment. In situ RT-PCR is feasible for measles virus in acutely infected cells both in sections and in suspension. Further work is required to improve the procedure and to eliminate artefactual nuclear signal.

Animals↗