Polymerase chain reaction (PCR) amplification of IS6110 sequences to detect Mycobacterium tuberculosis complex from formalin-fixed paraffin-embedded tissues of deer (Axis axis).
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Animals↗
Biomedical subjects
S K Ray
Publications and source records attributed to S K Ray.
Activation of multiple molecular mechanisms for apoptosis in human malignant glioblastoma T98G and U87MG cells treated with sulforaphane.
Glioblastoma is the most malignant and prevalent brain tumor that still remains incurable. Recent studies reported anti-cancer effect of the broccoli-derived compound sulforaphane. We explored the mechanisms of sulforaphane-mediated apoptosis in human glioblastoma T98G and U87MG cells. Wright staining and ApopTag assay confirmed apoptosis in glioblastoma cells treated with sulforaphane. Increase in intracellular free Ca2+ was detected by fura-2 assay, suggesting activation of Ca2+-dependent pathways for apoptosis. Western blotting was used to detect changes in expression of Bax and Bcl-2 proteins resulting in increased Bax:Bcl-2 ratio that indicated a commitment of glioblastoma cells to apoptosis. Upregulation of calpain, a Ca2+-dependent cysteine protease, activated caspase-12 that in turn caused activation of caspase-9. With the increased Bax:Bcl-2 ratio, cytochrome c was released from mitochondria to cytosol for sequential activation of caspase-9 and caspase-3. Increased calpain and caspase-3 activities generated 145 kD spectrin breakdown product and 120 kD spectrin breakdown product, respectively. Activation of caspase-3 also cleaved the inhibitor-of-caspase-activated-DNase. Accumulation of apoptosis-inducing-factor in cytosol suggested caspase-independent pathway of apoptosis as well. Two of the inhibitor-of-apoptosis proteins were downregulated because of an increase in 'second mitochondrial activator of caspases/Direct inhibitor-of-apoptosis protein binding protein with low pI.' Decrease in nuclear factor kappa B and increase in inhibitor of nuclear factor kappa B alpha expression favored the process of apoptosis. Collectively, our results indicated activation of multiple molecular mechanisms for apoptosis in glioblastoma cells following treatment with sulforaphane.
Analysis of Variance↗
Inhibition of calpain and caspase-3 prevented apoptosis and preserved electrophysiological properties of voltage-gated and ligand-gated ion channels in rat primary cortical neurons exposed to glutamate.
Glutamate toxicity in traumatic brain injury, ischemia, and Huntington's disease causes cortical neuron death and dysfunction. We tested the efficacy of calpain and caspase-3 inhibitors alone and in combination to prevent neuronal death and preserve electrophysiological functions in rat primary cortical neurons following glutamate exposure. Cortical neurons exposed to 0.5 microM glutamate for 24 h committed mostly apoptotic death as determined by Wright staining and ApopTag assay. Levels of expression, formation of active forms, and activities of calpain and caspase-3 were increased following glutamate exposure. Also, in situ double labeling identified conformationally active caspase-3-p20 fragment and chromatin condensation in apoptotic neurons. Pretreatment of cortical neurons with 0.2 microM N-benzyloxylcarbonyl-Leu-Nle-aldehyde (calpain-specific inhibitor) and 100 microM N-benzyloxylcarbonyl-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-fluoromethyl ketone (caspase-3-specific inhibitor) provided strong neuroprotection. Standard patch-clamp techniques were used to measure the whole-cell currents associated with Na+ channels, N-methyl-D-aspartate receptors, and kainate receptors. The lack of a change in capacitance indicated that neurons treated with inhibitor(s) plus glutamate did not undergo apoptotic shrinkage and maintained the same size as the control neurons. Whole-cell currents associated with Na+ channels, N-methyl-D-aspartate receptors, and kainate receptors were similar in amplitude and activation/inactivation kinetics for cells untreated and treated with inhibitor(s) and glutamate. Spontaneous synaptic activity as observed by miniature end-plate currents was also similar. Prevention of glutamate-induced apoptosis by calpain and caspase-3 inhibitors preserved normal activities of crucial ion channels such as Na+ channels, N-methyl-D-aspartate receptors, and kainate receptors in neurons. Our studies strongly imply that calpain and caspase-3 inhibitors may also provide functional neuroprotection in the animal models of traumatic brain injury and neurodegenerative diseases.
Amino Acid Chloromethyl Ketones↗
Estrogen prevents glutamate-induced apoptosis in C6 glioma cells by a receptor-mediated mechanism.
Estrogen-mediated neuroprotection is well established; however, no single mechanism of action for this effect has yet been established. As glial cells are integral for both the intact and injured nervous system, we hypothesized that estrogen-mediated neuroprotection may partly be attributed to attenuation of glial cell apoptosis, allowing them to protect neurons following injury. To assess the protective effects of estrogen on glia, C6 rat glioma cells were treated for 24 h with 500 microM glutamate. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and apoptosis was confirmed by cell morphology and DNA fragmentation. Pretreatment with 10 nM 17beta-estradiol (estrogen) increased cell viability and attenuated apoptosis. Treatment with the stereoisomer 17alpha-estradiol, or estrogen plus estrogen receptor antagonist ICI 182,780, was significantly less effective, indicating that cytoprotection was receptor-mediated. Estrogen treatment upregulated expression of estrogen receptor alpha. Cell impermeable bovine serum albumin-conjugated estrogen was also protective, indicating activation of estrogen receptors on the cell membrane. Intracellular free [Ca2+] was increased after glutamate treatment. This increase was attenuated in cells pretreated with estrogen. Glutamate increased the activity of pro-apoptotic proteases, such as calpain and caspase-3, and these protease activities were significantly attenuated by estrogen. The mechanism by which estrogen decreased intracellular Ca2+ was examined by assaying cell viability after using inhibitors that either blocked extracellular Ca2+ influx or prevented the release of intracellular Ca2+ stores. While several inhibitors increased cell viability in glutamate-treated cells, none were as protective as estrogen, and estrogen co-treatment significantly increased cell viability. These findings indicate that estrogen-mediated cytoprotection may be related to effects on Ca2+ entry but that these effects are not limited to any one of these Ca2+ entry points alone.
Animals↗
Relatively low levels of calpain expression in juvenile rat correlate with less neuronal apoptosis after spinal cord injury.
Approximately 5% of spinal cord injuries in the US occur in patients younger than 16 years. These young patients have an increased mortality within the 24 h after trauma but have a greater capacity for functional recovery than adults, suggesting age-related differences in injury tolerance. Unfortunately, the response of the developing cord to secondary injury has not been thoroughly investigated. Calpain, a Ca(2+)-dependent protease, has been implicated in the pathogenesis of spinal cord injury (SCI) in rats. Our current investigation revealed that following SCI, calpain upregulation was qualitatively less in the 21-day-old rats than in adult rats, as shown by immunofluorescent labeling. Decreased levels of TUNEL+ neurons were also noted in juvenile rat spinal cord, indicating that the developing cord may have an increased resistance to injury.
Age Factors↗
Dental fluorosis in bovine of Nayagarh district of Orissa.
Chronic fluoride toxicity in the form of dental fluorosis was observed in cattle from nine (9) villages under two (2) blocks of Nayagarh district of Orissa. Out of 1117 cattle, 221 (18.09%) showed the signs of dental fluorosis. In all affected villages, the prevalence of dental fluorosis in calves (< 1 year age) was greater than adults. There was significant difference in prevalence in respect to age. The commonly observed signs of dental fluorosis were brown discoloration, mottling, attrition or uneven wearing of teeth with or without pitting. None of the affected animals showed characteristic signs of osteofluorosis. The mean serum and urine fluoride concentration of affected animals were significantly (P < 0.05) higher than those of control animals. Fluoride levels (mean) of ground water and surface water in two blocks were 1.30 +/- 0.16 ppm, 0. 66 +/- 0.08 ppm and 1.12 +/- 0.19 ppm, 0. 48 +/- 0.05 ppm respectively. The fluoride content of grass samples of affected and control (non-endemic) area was comparable. There was a highly positive correlation (r = + 0.664) between prevalence of dental fluorosis and fluoride content of ground water. It was concluded that fluoride intake through the water especially ground water contributed to the development of fluorosis in cattle.
Age Factors↗
Molecular mechanisms in the pathogenesis of traumatic brain injury.
Traumatic brain injury (TBI) is a serious neurodisorder commonly caused by car accidents, sports related events or violence. Preventive measures are highly recommended to reduce the risk and number of TBI cases. The primary injury to the brain initiates a secondary injury process that spreads via multiple molecular mechanisms in the pathogenesis of TBI. The events leading to both neurodegeneration and functional recovery after TBI are generalized into four categories: (i) primary injury that disrupts brain tissues; (ii) secondary injury that causes pathophysiology in the brain; (iii) inflammatory response that adds to neurodegeneration; and (iv) repair-regeneration that may contribute to neuronal repair and regeneration to some extent following TBI. Destructive multiple mediators of the secondary injury process ultimately dominate over a few intrinsic protective measures, leading to activation of cysteine proteases such as calpain and caspase-3 that cleave key cellular substrates and cause cell death. Experimental studies in rodent models of TBI suggest that treatment with calpain inhibitors (e.g., AK295, SJA6017) and neurotrophic factors (e.g., NGF, BDNF) can prevent neuronal death and dysfunction in TBI. Currently, there is still no precise therapeutic strategy for the prevention of pathogenesis and neurodegeneration following TBI in humans. The search continues to explore new therapeutic targets and development of promising drugs for the treatment of TBI.
Brain Injuries↗
Inhibition of calpain-mediated apoptosis by E-64 d-reduced immediate early gene (IEG) expression and reactive astrogliosis in the lesion and penumbra following spinal cord injury in rats.
Upregulation of calpain, a Ca(2+)-activated cysteine protease, has been implicated in apoptosis and tissue degeneration in spinal cord injury (SCI) that over time spreads from the site of injury to the surrounding regions. We examined calpain content and activity, regulation of immediate early genes (IEGs) such as c-jun and c-fos, reactive astrogliosis as the expression of glial fibrillary acidic protein (GFAP), and apoptosis-related features such as caspase-3 mRNA expression and internucleosomal DNA fragmentation in 1-cm long spinal cord segments (S1, distant rostral; S2, adjacent rostral; S3, lesion or injury; S4, adjacent caudal; and S5, distant caudal) following SCI in rats. Calpain content and production of 150 kD calpain-cleaved alpha-fodrin fragment, expression of IEGs, reactive astrogliosis, and apoptotic features were highly increased in the lesion (S3), moderately in adjacent areas (S2 and S4), and slightly in distant areas (S1 and S5) in SCI rats when compared to sham animals. Administration of the calpain-specific inhibitor E-64-d (1 mg/kg) to SCI rats continuously for 24 h inhibited calpain activity and other factors contributing to apoptosis in the lesion and surrounding areas, indicating that calpain played a key role in the pathophysiology of SCI. The results obtained from this animal model of SCI suggest that calpain inhibitor can provide neuroprotection in patients with SCI.
Animals↗
Epidemiology of undernutrition.
OBJECTIVE: The present study was undertaken to find out the magnitude of the problem of under nutrition among the children under 5 years of age and also to identify the important factors influencing the nutritional status of the children. METHODS: 30 cluster sampling technique had been applied in the study. A total of 600 children below five years of age were covered. Twenty under five children from each cluster were chosen for the study which was carried out during January to February '97. As per IAP criteria a total of 60.29% children were undernourished and 3.92% were severely undernourished. According to NCHS standard 46.57% & 6.86% children had weight below-2SD and -3SD respectively. RESULTS: A statistically significant relationship was found between the different age groups and nutritional status of under 5 children. Severe degree of malnutrition had highest prevalence under two years of age. The influence of variables like age, sex, religion, literacy status of parents and morbidity of the children were significantly associated with malnutrition. CONCLUSION: Practice of exclusive breast feeding, introduction of timely complementary feeding, education for maintaining personal hygiene, proper implementation of UIP immunization, periodic deworming, standard case management of diarrhoea and ARI as well as continuation of feeding during illness may reduce malnutrition of under-five children.
Age Distribution↗
Cell death in spinal cord injury (SCI) requires de novo protein synthesis. Calpain inhibitor E-64-d provides neuroprotection in SCI lesion and penumbra.
Degradation of cytoskeletal proteins by calpain, a Ca(2+)-dependent cysteine protease, may promote neuronal apoptosis in the lesion and surrounding areas following spinal cord injury (SCI). Clinically relevant moderate (40 g-cm force) SCI in rats was induced at T12 by a standardized weight-drop method. Internucleosomal DNA fragmentation or apoptosis in the lesion was inhibited by 24-h treatment of SCI rats with cycloheximide (1 mg/kg), indicating a requirement for de novo protein synthesis in this process. To prove an involvement of calpain activity in mediation of apoptosis in SCI, we treated SCI rats with a cell-permeable calpain inhibitor E-64-d (1 mg/kg). Following 24-h treatment, a 5-cm-long spinal cord section centered at the lesion was collected, and divided equally into five segments (1 cm each) to determine calpain activity, as shown by degradation of the 68-kD neurofilament protein (NFP), and apoptosis as indicated by internucleosomal DNA fragmentation. Neurodegeneration propagated from the site of injury to neighboring rostral and caudal regions. Both calpain activity and apoptosis were readily detectable in the lesion, and moderately so in neighboring areas of untreated SCI rats, whereas these were almost undetectable in E-64-d-treated SCI rats, and absent in sham animals. Results indicate that apoptosis in the SCI lesion and penumbra is prominently associated with calpain activity and is inhibited by the calpain inhibitor E-64-d providing neuroprotective benefit.
Animals↗
Reproductive health needs and care seeking behaviour of pavement dwellers of Calcutta.
An unabated growth of street dwellers in the city of Calcutta is reported to be due to twin reasons like, migration of rural poor people as well as uncontrolled fertility among these poor settlers of the city. A community-based study on reproductive health, fertility and related care seeking behaviour was studied among a sample of women of child bearing age living on streets of Calcutta. Besides, the quite common conditions like leucorrhoea (28.5%), menstrual irregularities (12.3%), infertility (2.5%) and STDs (1.3%) were also reported. But most of these illnesses (three-fourth) were uncared for, and the remaining one-fourth sought treatment from govemment institutions, private agencies or even from untrained practitioners (quacks). The reproductive behaviour of street dwelling women was characterised by early marriage, teenage pregnancies, and scarce use of contraceptives (32%) as well as frequent abortions (2.8%). Very few pregnant women received adequate antenatal care (3.8%). Coverage of tetanus toxoid immunisation (68.5%) and proper iron and folic acid supplementation (16.7%) were also poor. Whereas, antenatal care was received mostly from government health institutions (71%), home delivery (ie, on street) was a common practice and conducted mostly by untrained birth attendants (51.8%).
Adolescent↗
E-64-d prevents both calpain upregulation and apoptosis in the lesion and penumbra following spinal cord injury in rats.
Calpain, a Ca(2+)-dependent cysteine protease, has been implicated in cytoskeletal protein degradation and neurodegeneration in the lesion and adjacent areas following spinal cord injury (SCI). To attenuate apoptosis or programmed cell death (PCD) in SCI, we treated injured rats with E-64-d, a cell permeable and selective inhibitor of calpain. SCI was induced on T12 by the weight-drop (40 g-cm force) method. Within 15 min, E-64-d (1 mg/kg) in 1.5% DMSO was administered i.v. to the SCI rats. Following 24 h treatment, a 5-cm long spinal cord section with the lesion in the center was collected. The spinal cord section was divided equally into five 1-cm segments (S1: distant rostral, S2: near rostral, S3: lesion or injury, S4: near caudal and S5: distant caudal) for analysis. Determination of mRNA levels by reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that ratios of bax/bcl-2 and calpain/calpastatin were increased in spinal cord segments from injured rats compared to controls. Degradation of the 68-kD neurofilament protein and internucleosomal DNA fragmentation were also increased. All of these changes were maximally increased in the lesion and gradually decreased in the adjacent areas of SCI rats, while largely undetectable in E-64-d treated rats and absent in sham controls. The results indicate that apoptosis in rat SCI appears to be associated with calpain activity which can be attenuated by the calpain inhibitor E-64-d.
Animals↗
Oxidative stress and Ca2+ influx upregulate calpain and induce apoptosis in PC12 cells.
Calpain, a Ca2+-dependent cysteine protease, has previously been implicated in apoptosis or programmed cell death (PCD) in immune cells. Although oxidative stress and intracellular free Ca2+ are involved in neurodegenerative diseases, the mechanism of neuronal cell death in the central nervous system (CNS) due to these agents has not yet been defined. To explore a possible role for calpain in neuronal PCD under oxidative stress and Ca2+ influx, we examined the effects of H2O2 and A23187 on PC12 cells. Treatments caused PCD (light microscopy and TUNEL assay) with altered mRNA expression (RT-PCR) of bax (pro-apoptotic) and bcl-2 (anti-apoptotic) genes, resulting in a high bax/bcl-2 ratio. Control cells expressed 1.3-fold more microcalpain (requiring microM Ca2+) than mcalpain (requiring mM Ca2+). Expression of mcalpain was significantly increased following exposure to oxidative stress and Ca2+ influx. The mRNA levels of calpastatin (endogenous calpain inhibitor) and beta-actin (house-keeping) genes were not changed. Western analysis indicated degradation of 68 kDa neurofilament protein (NFP), a calpain substrate. Pretreatment of cells with MDL28170 (a cell permeable and selective inhibitor of calpain) prevented increase in bax/bcl-2 ratio, upregulation of calpain, degradation of 68 kDa NFP, and occurrence of PCD. These results suggest a role for calpain in PCD of PC12 cells due to oxidative stress and Ca2+ influx.
Animals↗
Combined TUNEL and double immunofluorescent labeling for detection of apoptotic mononuclear phagocytes in autoimmune demyelinating disease.
Apoptosis is usually associated with genomic DNA fragmentation which can be detected in situ by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay. We describe a combined TUNEL and double immunofluorescent labeling technique to determine the fate of inflammatory infiltrates and resident glial cells in the central nervous system following the onset of an autoimmune demyelinating disease such as experimental allergic encephalomyelitis (EAE) in rats. Anti-digoxigenin (anti-DIG) antibody conjugated with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) emitting blue fluorescence was used to detect apoptotic cell DNA, which was already labeled by modified TUNEL using alkali-stable DIG-11-dUTP. Anti-mouse IgG secondary antibody conjugated with Texas Red emitting red fluorescence was used to detect anti-rat CD11b primary antibody (clone OX-42) directed to the surface antigen of mononuclear phagocytes including microglia. Using this technique, we detected apoptotic mononuclear phagocytes (co-labeled with blue and red fluorescences) in the spinal cord sections of rats with EAE.
Animals↗
Increased calpain expression is associated with apoptosis in rat spinal cord injury: calpain inhibitor provides neuroprotection.
Calpain content was investigated in the lesion of rat spinal cord at 1, 4, 24, and 72 h following injury induced by the weight-drop (40 g-cm force) technique. Calpain content was increased in the lesion, and was highest at 24 h following injury. microCalpain mRNA level in the lesion was increased by 58.4% (p = 0.0135) at 24 h following trauma, compared to sham. Alterations in mRNA expression in the lesion increased bax/bcl-2 ratio by 20.8% (p = 0.0395) at this time point, indicating a commitment to apoptosis. Therapeutic effect of the calpain inhibitor E-64-d (1 mg/kg) was studied in SCI rats following administration for 24 h. Internucleosomal DNA fragmentation (apoptosis) was observed in SCI rats, but not in sham or E-64-d treated rats. These results indicate a new information that E-64-d has the therapeutic potential for inhibiting apoptosis in SCI.
Animals↗
Mutants of Xanthomonas oryzae pv. oryzae deficient in general secretory pathway are virulence deficient and unable to secrete xylanase.
Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight, a serious disease of rice. A virulence- and xylanase-deficient mutant of Xoo was isolated following ethyl methane sulfonate (EMS) mutagenesis. A cosmid clone that restored virulence and xylanase secretion was obtained from a genomic library by functional complementation. Transposon mutagenesis and marker exchange studies revealed genes on the cloned DNA that were required for xylanase production and virulence. Sequence analysis with transposon-specific primers revealed that these genes were homologues of xps F and xps D, which encode components of a protein secretion system in Xanthomonas campestris pv. campestris. Enzyme assays showed xylanase accumulation in the periplasmic space and cytoplasm of the xps F mutant and the complementing clone restored transport to the extracellular space.
Amino Acid Sequence↗
Calpain upregulation in spinal cords of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a heroin analogue, is a neurotoxin that undergoes in vivo oxidation by monoamine oxidase-B (MAO-B) to 1-methyl-4-phenylpyridinium ion (MPP+) which preferentially exerts its toxic effects on the dopaminergic neurons of the substantia nigra in brain. Spinal interneuronal pathways are also likely to be affected in the course of MPP+ neurotoxicity. The primary effect of MPP+ is mediated by irreversible inhibition of mitochondrial complex I, releasing free radicals. MPP+ may also activate N-methyl-D-aspartate (NMDA) receptors, increasing the cytosolic concentration of free Ca2+. Intracellular free radicals indirectly and free Ca2+ directly can activate Ca2+-dependent proteases such as calpain. We investigated involvement of calpain in spinal cord degeneration due to neurotoxin by subjecting male C57BL/6N mice (17 months old) to MPTP administration (12.5 mg/kg for 0.5 h; 25 mg/kg for 0.25 h; and 50 mg/kg for 0.25, 0.5, 1, 2, and 24 h). RT-PCR and Western blot analysis were performed using the thoracic segment of spinal cords from control and MPTP-administered mice. The administration of MPTP caused calpain upregulation at the mRNA and protein levels to various extents, compared to control mice. Calpain activity was measured by 68 kDa neurofilament protein (NFP) degradation, which was increased in MPTP-induced PD mice. These results suggest that calpain may play a role in spinal cord degeneration in mice with MPTP-induced PD.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
A study of dietary pattern, household food security and nutritional profile of under-five children of a community of West Bengal.
A cross-sectional study was carried out in a tribal community of West Bengal to study the dietary pattern, household food security, utilisation of services and nutrition profile of under-five children. It was observed that average calorie consumption was 2,236 with 48% food insecured families. Cereals, starchy food and green leafy vegetables consumption was higher than the recommended daily allowance while pulses (scarcely supplied in fair price shops), milk, oil and sugar were less than recommended daily allowance. Nearly 11% kcal were coming from alcohol consumption. Public distribution system should supply all essential items with an improved quality on a regular basis and supply during lean season should be ensured. Prevalence of malnutrition in the children under-five years of age was 80.90% and 9.26% were suffering from severe grades. More severely malnourished children were observed in the age group of 12-23 months, amongst female children, in the families where mothers were working and also in the families where numbers of sibling were 2 or more. Services available under Integrated Child Development Services Scheme were utilised by 47.3% children.
Breast Feeding↗