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Biomedical subjects

S K Ray

Publications and source records attributed to S K Ray.

At least 37 records · Page 2Linked to original sources
PubMed1999

Diverse stimuli induce calpain overexpression and apoptosis in C6 glioma cells.

Calpain, a Ca2+-activated cysteine protease, has been implicated in apoptosis of immune cells. Since central nervous system (CNS) is abundant in calpain, the possible involvement of calpain in apoptosis of CNS cells needs to be investigated. We studied calpain expression in rat C6 glioma cells exposed to reactive hydroxyl radical (.OH) [formed via the Fenton reaction (Fe2++H2O2+H+-->Fe3++H2O+.OH)], interferon-gamma (IFN-gamma), and calcium ionophore (A23187). Cell death, cell cycle, calpain expression, and calpain activity were examined. Diverse stimuli induced apoptosis in C6 cells morphologically (chromatin condensation as detected by light microscopy) and biochemically [DNA fragmentation as detected by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay]. Oxidative stress arrested a population of C6 cells at the G2/M phase of cell cycle. The levels of mRNA expression of six genes were analyzed by the reverse transcriptase-polymerase chain reaction (RT-PCR). Diverse stimuli did not alter beta-actin (internal control) expression, but increased calpain expression, and the upregulated bax (pro-apoptotic)/bcl-2 (anti-apoptotic) ratio. There was no significant increase in expression of calpastatin (endogenous calpain inhibitor). Western blot analysis showed an increase in calpain content and degradation of myelin-associated glycoprotein (MAG), a calpain substrate. Pretreatment of C6 cells with calpeptin (a cell-permeable calpain inhibitor) blocked calpain overexpression, MAG degradation, and DNA fragmentation. We conclude that calpain overexpression due to.OH stress, IFN-gamma stimulation, or Ca2+ influx is involved in C6 cell death, which is attenuated by a calpain-specific inhibitor.

Animals↗
PubMed1999

Calpain activity and translational expression increased in spinal cord injury.

Calpain, a calcium-activated neutral proteinase, has been implicated in myelin and cytoskeletal protein degradation following spinal cord injury. In the present study, we examined the activity and transcriptional expression of calpain in spinal cord injury lesions via Western blotting analysis and RT-PCR, respectively. No increases in transcriptional expression of calpain or calpastatin, the endogenous inhibitor, were observed in the lesion at 1, 4, 24, and 72 h following injury. However, calpain activity (as measured by calpain-specific degradation of the endogenous substrate fodrin) was marginally increased at 4 h and significantly increased by 129.8% at 48 h compared to sham controls after injury. Calpain translational expression was localized in injured spinal cords using double immunofluorescent labeling which revealed increased calpain expression in astrocytes compared to sham controls. These results suggest that calpain produced by astrocytes located in or near spinal cord injury lesions may participate in myelin/axon degeneration following injury.

Animals↗
PubMed1999

Calpeptin and methylprednisolone inhibit apoptosis in rat spinal cord injury.

Intracellular free Ca2+ and free radicals are increased following spinal cord injury (SCI). These can activate calpain to degrade cytoskeletal proteins leading to apoptotic and necrotic cell death. Primary injury triggers a cascade of secondary injury, which spreads to rostral and caudal areas. We tested calpain involvement in apoptosis in five 1-cm segments of rat spinal cord with injury (40 g-cm) induced at T12 by weight-drop. Animals were immediately treated with calpeptin (250 micrograms/kg) and methylprednisolone (165 mg/kg) and sacrificed at 48 hr. Untreated SCI rats manifested 68-kD neurofilament protein (NFP) degradation (indicating calpain activity), and internucleosomal DNA fragmentation (indicating apoptosis). Both calpain activity and apoptosis were highest in the lesion, and decreased with increasing distance from the lesion. Treatment decreased 68-kD NFP degradation with reduction in apoptosis in all five areas. Thus, calpeptin and methylprednisolone are found to be neuroprotective in SCI.

Animals↗
PubMed1998

Calpain expression varies among different rat and bovine central nervous system regions.

Calcium-activated neutral proteinase (calpain) is a ubiquitous, cytosolic endopeptidase which is believed to play a role in many neural functions. In the present study, we examined the transcriptional and translational expression of microcalpain (microcalpain) and millicalpain (mcalpain) isoforms and the endogenous inhibitor calpastatin in rat and bovine spinal cord, brain stem, cerebellum, and cerebral cortex tissues using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. In rat central nervous system (CNS) samples, the microcalpain and mcalpain transcriptional expression was highest in white matter-enriched areas. Calpastatin mRNA expression demonstrated no significant differences among the CNS areas. Calpain and calpastatin translational expression levels were greatest in the spinal cord. In bovine CNS, microcalpain transcriptional expression was greatest in the spinal cord, while other CNS regions showed no significant differences. Bovine mcalpain transcriptional expression was similar among various CNS regions but marginally greater in the cortex. Translational expression of bovine calpain was greatest in the brain stem, while that of calpastatin was highest in the cerebral cortex. These results indicate that calpain expression varies among different CNS regions and is often highest in white matter-enriched areas.

Animals↗
PubMed1998

A splice variant of E2-2 basic helix-loop-helix protein represses the brain-specific fibroblast growth factor 1 promoter through the binding to an imperfect E-box.

We previously demonstrated that a cis-element (-489 to -467) in the brain-specific fibroblast growth factor (FGF)-1 promoter (FGF-1.B) binds multiple nuclear factors, and this binding enhances transcriptional activity of this promoter. Here we report the isolation of three cDNA clones, VL1, VL2 and VL3, from a human brain stem cDNA expression library using four tandem repeats of the 26-base pair sequence (-492 to -467) as the probe. These cDNA clones represent the variant of bHLH protein E2-2/SEF2-1 in having 12 additional nucleotides encoding the amino acids RSRS. The glutathione S-transferase (GST) fusion proteins of VLl, VL2, and VL3 immunologically react with anti-E2-2 antibody and anti-GST-VL2 antibody. Electrophoretic mobility shift assay and methylation interference assay revealed that the GST fusion proteins specifically bind to an imperfect E-box sequence (GACCTG) present in the 26-base pair sequence. Transient expression of the full-length E2-2 without RSRS in U1240MG glioblastoma cells resulted in repression of FGF-1.B promoter activity. We further showed a significant repression of promoter activity (>40 fold) by E2-2 (lacking the amino acid sequence RSRS) when the E47 reporter construct, containing a hexameric E-box site, was used. In contrast, the E2-2 variant containing the RSRS sequence has no significant effect on either the FGF-1 promoter or E47 promoter. These results suggest that the relative abundance of the two splice variants of E2-2 in brain could be an important determinant for the expression of FGF-1.

Alternative Splicing↗
PubMed1998

Review of eye plaque dosimetry based on AAPM Task Group 43 recommendations. American Association of Physicists in Medicine.

PURPOSE: AAPM Task Group 43 recently revised the dosimetry recommendations for 125I seeds. We reviewed these guidelines and studied the effects of the recommendations on the prescription absorbed dose for patients who have undergone eye plaque therapy in our clinic. METHODS AND MATERIALS: 95 consecutive patients were chosen for this study. Absorbed doses at various points of clinical interest were computed based on conventional dose calculation algorithm (3, 4, 7) and TG-43 recommendations. For three representative plaques chosen, the seeds are approximated by isotropic point and line sources, respectively, and absorbed doses were calculated at all points on the central axis of the plaque. RESULTS: For apical heights shorter than 5 mm, treatment plans using model 6711 seeds delivered 10-13% lower absorbed doses than that calculated previously. For lesions with apical heights 5 mm or larger, the absorbed dose was 6-12% lower than prescribed. Calculations for model 6702 seeds indicated that TG-43 recommendations would produce 0-6% lower absorbed doses. Point doses calculated along the central axis of the plaque and isodose distributions at various levels showed that point source approximation of the seeds was clinically acceptable. CONCLUSIONS: TG-43 recommendations, if implemented, would result in lower absorbed doses unless the dose prescription is modified. The clinicians need to be aware of the dosimetric implications of these recommendations. The seeds may be approximated by isotropic point sources.

Brachytherapy↗
PubMed1998

"Resultant bond moment" as a newly developed electronic parameter in the design of antibacterial, antiprotozoal nitroimidazole derivatives.

Nineteen nitroimidazole derivatives have been reported to be effective antibacterial against one strain each of Bacteroides fragilis and Clostridium perfringens. The negative logarithms of minimum inhibitory concentrations (MICs) of these nitroimidazoles were fitted by a biostatistical relation involving a newly designed structural descriptor, the Resultant Bond Moment (RBM). RBM was developed on the basis of the chemical attribute of bond moments of heteroatoms. RBM reflects the electronic characteristic of the molecule. Apart from this, steric, electronic, and hydrophobic parameters were also utilised in the search of a best fit regression equation. On the basis of these equations new investigative nitroimidazole derivatives have been predicted concerning their antibacterial and antiprotozoal efficacy.

Anti-Bacterial Agents↗
PubMed1998

A report of diphtheria surveillance from a rural medical college hospital.

A 5-year sentinel surveillance of diphtheria from 1989 to 1993 was undertaken at a rural medical college hospital. No significant change in the number of diphtheria cases was observed in spite of sustained high level of diphtheria, pertussis, tetanus vaccine-3 doses (DPT3) coverage. Most of the diphtheria cases occurred during July to November. Age distribution of diphtheria cases showed that more than 75% occurred above 2 years age (except in 1989) and around 65% cases above 3 years age. The age shift in diphtheria signified success of primary diphtheria immunisation, as well as indicated the lack of coverage with booster doses at appropriate ages. Because of high coverage with primary diphtheria immunisation there was decrease in circulating toxigenic C diphtheriae resulting in less natural boosting of antibody titre. Thus, in absence of booster immunisation, the older children and adults were more vulnerable to diphtheria. The findings of the study justified the need of emphasising importance of booster diphtheria immunisation at appropriate ages for effective control of diphtheria.

Adult↗
PubMed1997

Prevalence of canine demodicosis in Orissa (India).

Examination of the records of 12 hospitals from the five districts of Orissa showed that 50,987 dogs were presented for treatment during the period of 5 years covering 1987-1988 to 1991-1992. Demodicosis was detected in 1697 (3%) cases. There was no difference in the occurrence of the disease in male (51%) and female (49%) dogs. The disease was recorded in 60%, 23% and 17% of dogs up to 1 year, 1-2 years and above 2 years of age respectively, suggesting that young dogs up to 1 year of age are more susceptible. Further study revealed that Tibetan apso are more susceptible (47%) than Doberman (23%), Alsatian (18%) and mongrels (12%). A total of 912 dogs with natural dermatitis were examined during the 5-year period and 35% of the dogs were found to have demodicosis. Out of these, 208 dogs (65%) had localised lesions and 111 (35%) had generalised lesions. The study revealed that dogs up to 1 year of age are more frequently affected (60%) than dogs of 1-2 years (23%) and above 2 years of age (17%). There was no difference between the susceptibility of male (49%) and female (51%) dogs. The prevalence was high in Tibetan apso (41%) as compared to Doberman (26%), Alsatian (16%) and mongrels (17%).

Age Factors↗
PubMed1997

Transcriptional activation of fibroblast growth factor 1.B promoter is mediated through an 18-base pair cis-acting element.

Four different transcripts encoding fibroblast growth factor 1 (FGF-1, also known as aFGF) have been previously identified in our laboratory. Among them, FGF-1.B is the major transcript expressed specifically in the neuronal cells in brain tissue. Using the transient transfection experiment in a glioblastoma cell line, U1240MG, that expresses 1.B, we previously identified two regulatory regions (RR1 and RR2) in the brain-specific promoter, FGF-1.B. In the present study, we showed that the minimal region required for the DNA-protein interaction in RR2 resides in an 18-base pair (-484 to -467) sequence, by using DNase I footprinting and methylation interference studies and electrophoretic mobility shift assays. This minimal cis-acting element was found to be sufficient in enhancing the reporter activity driven by the heterologous herpes simplex virus thymidine kinase promoter in the 1.B-positive U1240MG cell line. This enhancing effect, however, was not detected in a glioblastoma cell line, U1242MG, which is negative for 1.B expression. By electrophoretic mobility shift assays, we also identified a specific DNA-protein complex, namely complex I, which is specific for 1.B-positive cell lines and human brain tissue. By in situ UV cross-linking experiment, we further showed that complex I contains two major DNA-binding proteins of apparent molecular masses of 37 and 98 kDa. Our results suggest that the formation of complex I, resulting from the heterodimerization of a 37-kDa protein (1.B-specific) and a 98-kDa protein (ubiquitous) may likely be a prerequisite for the enhanced expression of 1.B transcript in neuronal cells.

DNA↗
PubMed1997

Studies on 'zinc deficiency syndrome' in black bengal goats (Capra hircus) fed with fodder (Andropogon gayanus) grown on soil treated with an excess of calcium and phosphorus fertilizer.

Overliming and excessive application of superphosphate caused a zinc deficiency in the soil and so reduced the uptake of zinc by fodder plants. Bucks reared on such fodder had significantly (p < 0.01) less zinc in their hair compared with controls and suffered from 'conditioned zinc deficiency syndrome' with a significant (p < 0.01) loss of body weight, stunted growth, alopecia, lethargy, abnormal (kyphotic) gait, anorexia, digestive and respiratory problems. Oral supplementation with zinc sulphate very rapidly improved these conditions to near normality. Histological examination of samples of skin and testis from the zinc-deficient bucks revealed formation of excessive keratin, retention of nuclei in the stratum corneum and reduction in the width of the stratum granulosum in the skin, while samples of testis indicated degenerative changes, including atrophy of the seminiferous tubules, hyperplasia of the germinal epithelium and thickening of the walls of blood vessels.

Alopecia↗
PubMed1997

Amphibian FGF-1 is structurally and functionally similar to but antigenically distinguishable from its mammalian counterpart.

Recent studies have shown that fibroblast growth factors (FGF) play an important role in the diverse cellular mechanisms involved with vertebrate development. One system which has received a great deal of attention is the developing limb in part because of the extensive epithelial-mesenchymal interactions that take place during this process. Because it closely parallels the developmental process of the limb and is a model for wound repair, the phenomenon of amphibian limb regeneration has been used to investigate the role of FGF in these processes. We have recently reported on the cloning and functional characterization of an FGF receptor (FGFR) isolated from amphibian regenerative tissue. In this report, we describe the isolation and characterization of an FGF-1 molecule from the newt, Notophthalmus viridescens. Amino acid sequence comparisons indicate that the newt FGF-1 exhibits between 79 to 83% identity with FGF-1 from mammalian and avian species. The full length cDNA of the newt FGF-1 was cloned into a prokaryotic expression vector and purified from E. coli. Although the newt FGF-1 shares a high degree of primary amino acid sequence similarity with other FGF-1 molecules, the recombinant protein was not detected in a Western blot analysis using a polyclonal antibody directed against mammalian FGF-1. Despite the antigenic divergence, the newt FGF-1 was capable of binding to NIH/3T3 and Chinese hamster ovary cells overexpressing mammalian and amphibian FGFRs with dissociation constants comparable to those reported for mammalian FGF-1. Newt FGF-1 could also be cross-linked to receptors on the surface of NIH/3T3 cells. In addition, it elicits a mitogenic response in NIH/3T3 cells indistinguishable from human recombinant FGF-1.

3T3 Cells↗
PubMed1996

Pathogenic autoantibodies are routinely generated during the response to foreign antigen: a paradigm for autoimmune disease.

The immune system's ability to distinguish self and nonself is essential for both host defense against foreign agents and protection of self-antigens from autoimmune destruction. Such discrimination is complicated by extensive structural homology shared between foreign and self antigens. One hypothesis to explain the development of an autoimmune response is that some B cells activated by foreign antigen acquire, through somatic mutation, specificity for both the eliciting foreign antigen and self antigen. If such clones arise frequently, there must be a mechanism for their elimination. We have analyzed the extent of autoreactivity arising in a nonautoimmune host during the response to a foreign antigen. To overcome the process of apoptosis in primary B cells that might routinely eliminate autoreactive clones, we generated B-cell hybridomas from spleen cells of immunized mice by using a fusion partner constitutively expressing bcl-2. Multiple lines were obtained that recognize simultaneously the hapten phosphorylcholine and the self antigen double-stranded DNA. This dual specificity was not present early but was detected by day 10 after immunization. Some of these cross-reactive antibodies deposit in kidneys in a pattern similar to what is seen in autoimmune disease. These results demonstrate that autoantibodies arise at a high frequency as part of a response to foreign antigen. It has previously been shown that autoreactivity is regulated by central deletion; these data demonstrate a need for negative selection in peripheral lymphoid organs also, to regulate autoantibodies acquiring their self-specificity by somatic mutation.

Animals↗
PubMed1995

Functional characterization of the brain-specific FGF-1 promoter, FGF-1.B.

Expression of alternatively spliced human FGF-1 (or aFGF) transcripts is regulated in a tissue-specific manner via multiple promoters. To identify the cis-regulatory elements in the brain-specific FGF-1.B promoter, we constructed a series of promoter deletions fused to the luciferase reporter gene and transfected into an FGF-1.B positive glioblastoma cell line, U1240MG, and a 1.B negative cell line, U1242MG. Results of transient transfections indicate three elements that are involved in the positive regulation of FGF-1.B expression. The core promoter is located in a 40-base pair region (between -92 and -49), and two regulatory regions (RR-1 and RR-2) are located within the 540-base pair region 5' to the major transcription start site (defined as +1). Electrophoretic mobility shift assays and footprinting analysis have identified sequence-specific binding sites in RR-1 and RR-2. Mutants of RR-2 abolished binding to nuclear proteins and showed diminished luciferase reporter activity. The effects seen are specific for the U1240MG cell line, supporting a role for RR-2 in the tissue-specific regulation of FGF-1.B. Southwestern analysis using an oligonucleotide probe derived from RR-2 (nucleotides -489 to -467) further identified a 37-kDa protein that is present in nuclear extracts from U1240MG and brain but not from U1242MG.

Base Sequence↗
PubMed1995

Potentiation of antiproliferative effects of monoclonal antibody Lym-1 and immunoconjugate Lym-1-gelonin on human Burkitt's lymphoma cells with gamma-interferon and tumor necrosis factor.

A type I ribosome inactivating protein, gelonin, was linked to Lym-1, a murine monoclonal antibody reactive with a polymorphic determinant of class II HLA-DR histocompatibility leukocyte antigen (HLA) on human lymphoma cells, via a disulfide linkage using the heterobifunctional cross-linking agent, N-succinimidyl-3-(2-pyridyldithio) propionate. This immunotoxin was purified from unreacted gelonin and unconjugated Lym-1 by fast protein liquid chromatography using sephacryl S-300 gel filtration and blue sepharose affinity gradient separation. Binding of Lym-1-gelonin immunoconjugate to human Raji Burkitt's lymphoma cells was demonstrated by indirect immunofluorescence using flow cytometry. Lym-1-gelonin was very active in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium salt and sulforhodamine B in vitro cytotoxicity assays against the Raji lymphoma cell line and confirmed the fact that monoclonal antibody Lym-1 internalizes into human lymphoma cells. A weaker cytostatic antiproliferative effect was also noted for unconjugated Lym-1. gamma-interferon augmented the antiproliferative effects of Lym-1-gelonin conjugate and unconjugated Lym-1, by having a direct cytotoxic effect on the Raji cells. Tumor necrosis factor-alpha also enhanced the antiproliferative effect of unconjugated Lym-1, but did not significantly augment the cytotoxic activity of the Lym-1-gelonin conjugate. These results suggest that anti-HLA class II monoclonal antibodies may be useful in constructing immunotoxins for the treatment of human lymphomas and leukemias expressing HLA class II antigens, and that unconjugated anti-HLA class II monoclonal antibodies may be therapeutically useful in conjunction with recombinant cytokines, especially gamma-interferon.

Animals↗
PubMed1995

Determination of differential scatter-air ratios (dSAR) for three-dimensional scatter integration.

Scatter dose may be calculated by summing the scatter contribution from individual volume elements. These contributions may be represented by differential scatter-air ratios (dSAR). Determination of dSAR from measured data is only approximately correct for second and higher orders of scatter and yields values often limited to one significant figure. Monte Carlo calculation, on the other hand, is time intensive, requires some knowledge of the beam's x-ray spectrum, and mastering the complexities of a program such as EGS4. Total scatter dose at a point may be determined by measuring depth dose or tissue-air ratios and partitioning the dose into its primary and scatter components. Scatter may be represented by scatter-air ratios, which can be characterized by the sum of first, second, and higher orders of scatter. The first scatter dose may be computed exactly by summing the first scatter contribution from individual elements, determined from the first principle. Separation of dSAR into primary attenuation and depth-independent terms allows the latter to be precomputed once for a given energy and stored in tabular form. Second scatter may be treated in a similar manner. The higher orders of scatter are computed by subtracting the sum of calculated first and second scatter doses from the total scatter dose. Elements close to and approximately 1 cm above the point of calculation contribute most heavily to the first scatter dose. Compared to the first scatter dose, the second scatter dose contribution is lower, particularly for elements close to the point of calculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Humans↗