Biomedical subjects

O P Mishra

Publications and source records attributed to O P Mishra.

At least 19 recordsLinked to original sources

PubMed2006

Hypoxia-induced Bax and Bcl-2 protein expression, caspase-9 activation, DNA fragmentation, and lipid peroxidation in mitochondria of the cerebral cortex of newborn piglets: the role of nitric oxide.

The present study tests the hypothesis that cerebral hypoxia results in increased ratio of Bax/Bcl-2, activation of caspase-9, lipid peroxidation, and DNA fragmentation in mitochondria of the cerebral cortex of newborn piglets and that the inhibition of nitric oxide synthase by N-nitro-L-arginine during hypoxia will prevent the events leading to mitochondrial DNA fragmentation. To test this hypothesis, six piglets, 3-5 days old, were divided into three groups: normoxic (n=5), hypoxic (n=5), and hypoxic-nitric oxide synthase (n=4). Hypoxic animals were exposed to a FiO2 of 0.6 for 60 min. Nitric oxide synthase (40 mg/kg) was infused over 60 min prior to hypoxia. Tissue hypoxia was confirmed by measuring levels of ATP and phosphocreatine. Cerebral cortical tissue mitochondria were isolated and purified using a discontinuous ficoll gradient. Mitochondrial Bax and Bcl-2 proteins were determined by Western blot. Caspase-9 activity in mitochondria was determined spectro-fluorometrically using fluorogenic substrate for caspase-9. Fluorescent compounds, an index of mitochondrial membrane lipid peroxidation, were determined spectrofluorometrically. Mitochondrial DNA was isolated and separated by electrophoresis on 1% agarose gel and stained with ethidium bromide. ATP levels (micromol/g brain) were 4.52+/-0.34 in normoxic, 1.18+/-0.29 in hypoxic (P<0.05) and 1.00+/-0.26 in hypoxic-nitric oxide synthase animals (P<0.05 vs. normoxic). Phosphocreatine levels (micromol/g brain) were 3.61+/-0.33 in normoxic, 0.70+/-0.20 in hypoxic (P<0.05 vs. normoxic) and 0.57+/-0.14 in hypoxic-nitric oxide synthase animals (P<0.05 vs. normoxic, P=NS vs. hypoxic). Bax density in mitochondrial membranes was 160+/-28 in normoxic and 324+/-65 in hypoxic (P<0.001 vs. normoxic). Bcl-2 density mitochondria was 96+/-18 in normoxic and 98+/-20 in hypoxic (P=NS vs. normoxic). Mitochondrial caspase-9 activity (nmol/mg protein/h) was 1.32+/-0.23 in normoxic and 2.25+/-0.24 in hypoxic (P<0.01 vs. normoxic). Levels of fluorescent compounds (microg of quinine sulfate/g protein) were 12.48+/-4.13 in normoxic and 37.92+/-7.62 in hypoxic (P=0.003 vs. normoxic). Densities (ODxmm2) of low molecular weight DNA fragments were 143+/-38 in normoxic, 365+/-152 in hypoxic, (P<0.05 vs. normoxic) and 163+/-25 in hypoxic-nitric oxide synthase animals (P<0.05 vs. hypoxic, P=NS vs. normoxic). The data demonstrate that hypoxia results in increased mitochondrial proapoptotic protein Bax, increased mitochondrial caspase-9 activity, increased mitochondrial lipid peroxidation, and increased fragmentation of DNA in mitochondria of the cerebral cortex of newborn piglets. The administration of a nitric oxide synthase inhibitor, nitric oxide synthase, prior to hypoxia prevented fragmentation of mitochondrial DNA, indicating that the hypoxia-induced mitochondrial DNA fragmentation is NO-mediated. We propose that NO free radicals generated during hypoxia lead to NO-mediated altered expression of Bax leading to increased ratio of pro-apoptotic/anti-apoptotic protein resulting in modification of mitochondrial membrane, and subsequently Ca2+-influx and fragmentation of mitochondrial DNA.

Adenosine Triphosphate↗

PubMed2006

Evaluation of polymerase chain reaction and adenosine deaminase assay for the diagnosis of tuberculous effusions in children.

AIM: To evaluate and compare the utility of polymerase chain reaction (PCR) for the diagnosis of tuberculous effusions in children. METHODS: PCR, adenosine deaminase (ADA) activity and absolute lymphocyte count (ALC) were evaluated in the fluid of 31 tuberculous (20 pleural, 8 ascites and 3 pericardial) and 24 non-tuberculous (10 transudtative ascites, 8 empyema thoracis, 3 malignant pleural and 3 pyopericardium) effusions. RESULTS: Fluid PCR for Mycobacterium tuberculosis was positive in 74% of tuberculous effusions, whereas it was falsely positive in 13% of the non-tuberculous group. The mean fluid ADA and ALC values were significantly higher in tuberculous effusions than in non-tuberculous effusions (p<0.001). The sensitivity and specificity of PCR, ADA (> or =38 IU/l) and ALC (> or =275/mm3) were 74% and 88%, 81% and 75%, and 90% and 83%, respectively, in diagnosing tuberculous effusions. The sensitivity of PCR, ADA and ALC was 100%, 100% and 88%, respectively, for confirmed tuberculous effusions. When the two tests were combined (either/or positive), the sensitivity increased (90-100%) at the expense of specificity. When both the tests were positive, then the specificity markedly increased (92-96%), but sensitivity of the tests decreased. CONCLUSION: Fluid PCR alone should not be relied on as a single test; rather, combined analysis with either ADA or ALC could be more useful in the diagnosis of tuberculous effusions in children.

Adenosine Deaminase↗

PubMed2006

[The role played by calcium in neuronal injury following neonatal hypoxia or convulsions].

AIM: Calcium plays a complex and pivotal role both in neuronal development and function, and in hypoxia/ ischemia-induced cell death. In this paper, we review current concepts of calcium function emphasizing the neonatal period. DEVELOPMENT: Calcium enters the neuron through glutamate receptors (NMDA and AMPA) located on the neuronal membrane. After hypoxia or seizures, there is a conformational change of the receptors, with increased flow of calcium into the cytoplasm. Cytoplasmatic calcium triggers activation of several free-radical generation pathways, including the nitric oxide pathway, with a deleterious effect upon the neuron. Calcium then enters the neuronal nucleus, through specific receptors on the nuclear membrane. In our experience, hypoxia and neonatal seizures create nuclear membrane dysfunction, increasing the nitric-oxide-dependent flow of calcium into the nucleus. Nuclear calcium increase is critical for genetic transcription, pro-apoptotic gene activation and a cascade of biochemical and molecular events that lead to an increase of caspases and apoptotic neuronal death. CONCLUSIONS: Calcium has a crucial role in neuronal damage after neonatal hypoxia or seizures. A better knowledge of the pathogenic mechanisms that lead to neuronal damage after neonatal hypoxia or seizures will assist in future development of efficacious neuroprotective therapies.

Calcium↗

PubMed2006

Nitric oxide-mediated mechanism of neuronal nitric oxide synthase and inducible nitric oxide synthase expression during hypoxia in the cerebral cortex of newborn piglets.

Previously, we have shown that hypoxia results in increased generation of nitric oxide free radicals in the cerebral cortex of newborn piglets that may be due to up-regulation of nitric oxide synthases, neuronal nitric oxide synthase and inducible nitric oxide synthase. The present study tests the hypothesis that hypoxia results in increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase in the cerebral cortex of newborn piglets and that the increased expression is nitric oxide-mediated. Newborn piglets, 2-4 days old, were divided to normoxic (n=4), hypoxic (n=4) and hypoxic-treated with 7-nitro-indazole-sodium salt, a selective neuronal nitric oxide synthase inhibitor (hypoxic-7-nitro-indazole-sodium salt, n=6, 1 mg/kg, 60 min prior to hypoxia). Piglets were anesthetized, ventilated and exposed to an FiO2 of 0.21 or 0.07 for 60 min. Cerebral tissue hypoxia was documented biochemically by determining ATP and phosphocreatine. The expression of neuronal nitric oxide synthase and inducible nitric oxide synthase was determined by Western blot using specific antibodies for neuronal nitric oxide synthase and inducible nitric oxide synthase. Protein bands were detected by enhanced chemiluminescence, analyzed by imaging densitometry and the protein band density expressed as absorbance (OD x mm(2)). The density of neuronal nitric oxide synthase in the normoxic, hypoxic and hypoxic-7-nitro-indazole-sodium salt groups was: 41.56+/-4.27 in normoxic, 61.82+/-3.57 in hypoxic (P<0.05) and 47.80+/-1.56 in hypoxic-7-nitro-indazole-sodium salt groups (P=NS vs normoxic), respectively. Similarly, the density of inducible nitric oxide synthase in the normoxic, hypoxic and hypoxic-7-nitro-indazole-sodium salt groups was: 105.21+/-9.09, 157.71+/-13.33 (P<0.05 vx normoxic), 117.84+/-10.32 (p=NS vx normoxic), respectively. The data show that hypoxia results in increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase proteins in the cerebral cortex of newborn piglets and that the hypoxia-induced increased expression is prevented by the administration of 7-nitro-indazole-sodium salt. Furthermore, the neuronal nitric oxide synthase inhibition prevented the inducible nitric oxide synthase expression for a period of 7 days after hypoxia. Since administration of 7-nitro-indazole-sodium salt prevents nitric oxide generation by inhibiting neuronal nitric oxide synthase, we conclude that the hypoxia-induced increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase is mediated by neuronal nitric oxide synthase derived nitric oxide. We speculate that during hypoxia nitric oxide-mediated up-regulation of nitric oxide synthases will continue the perpetual cycle of nitric oxide generation-->NOS up-regulation-->nitric oxide generation resulting in hypoxic neuronal death.

Adenosine Triphosphate↗

PubMed2006

Miltefosine in children with visceral leishmaniasis: a prospective, multicentric, cross-sectional study.

OBJECTIVE: Miltefosine, an alkyl phospholipid has been found effective against visceral leishmaniasis (VL) in adults in various studies. The authors safety, tolerance and efficacy of Miltefosine and compared with available gold standard anti-Ieishmanial drug, Amphotericin B, a parenteral formulation in children with VL. METHODS: All consecutive children aged 1 yr to 14 yr, presented with fever, splenomegaly and positive LD body in splenic smear examination, admitted in pediatric ward of Nalanda Medical college and Child care center between 1st July 03 to 30th June 05 were taken for study. Patients were randomized into four groups. Group-l and 2 patients were given Miltefosine in dose of 2.5 mg/Kg day o.d. or b.i.d. per orally to a maxiIpum of 100 mg and group 3 and 4 Amphotericin B at a dose of 1 mg/Kg/day (total: 15 mg/Kg). All patients were followed at completion of therapy, 3 months and 6 months for clinical response, splenic size and parasitologically. RESULTS: Out of 125 children, 44 were in group-I, 20 in group-2, 38 in group-3 and 23 in group- 4, 124 patients had parasitological cure with relapse in one patient of group 1 during follow up. One patient in-group II had no response with first course but became parasitologically negative with 2nd course of Miltefosine. In-group I, one patient had persistent splenomegaly and found to have associated portal hypertension. Final cure rate with Miltefosine and Amphotericin B was 93.2%, 95%, 92.1% and 91.3% in-group 1, 2, 3 and 4 respectively, which are statistically insignificant. Majority of patients had pancytopenia. Eievated". AL T (>3 times of normal) were seen in 28, 11, 19 and 13 patients of group 1, 2, 3 and group 4 respectively which returned to normal in subsequent follow up. Raised BUN was observed more in patients who got Amphotericin B i.e. 65.42% and 73.91 % in-group 3 and 4 respectively. GI side effects i.e. diarrhea and vomiting were observed in 26 and 23 patients in-group 1 and 2 respectively. CONCLUSION: Miltefosine is safe, well tolerable, and highly effective and has same efficacy as Amphotericin B in newly diagnosed and SAG resistant children with visceral leishmaniasis.

Amphotericin B↗

PubMed2005

Hypoxia modifies nuclear calcium uptake pathways in the cerebral cortex of the guinea-pig fetus.

Nuclear Ca2+ signals are thought to play a critical role in the initiation and progression of programmed cell death. The present study tests the hypothesis that hypoxia alters nuclear Ca2+ transport pathways and leads to an increase in nuclear Ca(2+)-influx in cerebral cortical neuronal nuclei. To test this hypothesis the effect of tissue hypoxia on high affinity Ca(2+)-ATPase activity and the binding characteristics of inositol 1,4,5-triphosphate (IP3) and inositol 1,3,4,5-tetrakisphosphate (IP4) receptors were studied in neuronal nuclei from the cerebral cortex of guinea-pig fetuses. Results show increased high-affinity Ca(2+)-ATPase activity (nmol/mg protein/h) in the hypoxic group 969.7+/-79 as compared with 602.4+/-90.9 in the normoxic group, P<0.05. The number of IP3 receptors (Bmax, fmol/mg protein) increased from 61+/-21 in the normoxic group to 164+/-49 in the hypoxic group, P<0.05. K(d) values did not change following hypoxia. In contrast, IP4 receptor Bmax (fmol/mg protein) and K(d) (nM) values increased from 360+/-32 in the normoxic group to 626+/-136 in the hypoxic group (P<0.001) and, from 26+/-1 in the normoxic group to 61+/-9 in the hypoxic group (P<0.001), respectively. 45Ca(2+)-influx (pmol/mg protein) significantly increased from 6.3+/-1.9 in the normoxic group to 10.9+/-1.1 the hypoxic group (P<0.001). The data show that hypoxia modifies nuclear Ca2+ transport pathways and results in increased nuclear Ca(2+)-influx. We speculate that hypoxia increases nuclear Ca2+ uptake from the cytoplasm to the nucleoplasm, resulting in increased transcription of proapoptotic genes and subsequent activation of programmed cell death pathways.

Animals↗

PubMed2004

Immune status with empyema thoracis.

OBJECTIVE: To evaluate the humoral and cell mediated immune status of children with empyema thoracis. METHODS: Serum IgG, IgA, IgM, Complement C3 assay and cell mediated immunity (CMI) tests were performed in 33 patients of empyema thoracis, and 14 healthy age matched controls. RESULTS: The mean serum IgG and IgA levels in empyema thoracis and its subgroups were significantly raised as compared to controls. The overall values of IgG and IgA were 104% (p<0.001) and 114% (p<0.01) of normal mean, respectively. The mean serum IgM and complement C3 levels did not differ significantly in both the groups. The frequency of negative skin reaction to purified protein derivative (PPD) was significantly higher in children with empyema thoracis as compared to controls (p<0.05). The mean absolute lymphocyte count (ALC) was significantly decreased and serum adenosine deaminase (ADA) activity was significantly raised in empyema thoracis in comparison to controls. The overall ALC was 76.1% (p<0.01) and serum ADA activity was 169.4% (p<0.001) of normal mean, respectively. No significant differences were observed in the mean levels of immunoglobulins, complement C3 and CMI tests between pyothorax and pyopneumothorax and pleural fluid culture positive and negative cases. CONCLUSIONS: Thus, both humoral and cell mediated immunity were affected in empyema thoracis patients. However, CMI demonstrated more pronounced change in comparison to humoral immunity.

Adenosine Deaminase↗

PubMed2004

Nitric oxide-mediated activation of extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) during hypoxia in cerebral cortical nuclei of newborn piglets.

Previous studies have shown that mitogen-activated protein kinases, such as extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK), mediate signal transduction from cell surface receptors to the nucleus and phosphorylate anti-apoptotic proteins thereby regulating programmed cell death. The present study tests the hypotheses that hypoxia activates ERK and JNK in neuronal nuclei of newborn piglets and the hypoxia-induced activation of ERK and JNK is mediated by nitric oxide (NO). Activated ERK and JNK were assessed by determining phosphorylated ERK and JNK using immunoblotting in six normoxic (Nx) and 10 hypoxic (Hx) and five N-nitro-L-arginine (a NOS inhibitor, 40 mg/kg,) -pretreated hypoxic (N-nitro-L-arginine [NNLA]-Hx) 3-5 day old piglets. Hypoxia was induced by decreasing inspired oxygen from 21% to 7% for 60 min. Cerebral tissue hypoxia was documented biochemically by determining the tissue levels of ATP and phosphocreatine (PCr). Cortical neuronal nuclei were isolated and the nuclear protein was analyzed for activated ERK and JNK using anti-phosphorylated ERK and JNK antibodies. Protein bands were detected using enhanced chemiluminescence method and analyzed by imaging densitometry. Protein density was expressed as absorbance ODxmm(2). ATP levels were 4.57+/-0.45 micromoles/g brain in the Nx group, 1.29+/-0.23 micromoles/g brain in the Hx group (P<0.05 vs Nx) and 1.50+/-0.14 micromoles/g brain in the NNLA-Hx group (P<0.05 vs Nx). PCr levels were 3.77+/-0.36 micromoles/g brain in the Nx group, 0.77+/-0.13 micromoles/g brain in the hypoxic group (P<0.05) and 1.02+/-0.24 in the NNLA-Hx group (P<0.05 vs Nx). Density of phosphorylated ERK protein was 170.5+/-53.7 ODxmm(2) in the Nx group as compared with 419.6+/-63.9 ODxmm(2) in the hypoxic group (P<0.001 vs Nx) and 270.0+/-28.7 in the NNLA-Hx group (P<0.002 vs Hx). Density of phosphorylated JNK protein was 172.8+/-42.8 ODxmm(2) in the normoxic group as compared with 364.6+/-60.1 ODxmm(2) in the Hx group (P<0.002) and 254.8+/-24.8 in the NNLA-Hx group (P<0.002 vs Hx). The data demonstrate increased phosphorylation of ERK and JNK during hypoxia indicating that hypoxia results in activation of ERK and JNK in neuronal nuclei of newborn piglets. The administration of NNLA, a NOS inhibitor, prevented the hypoxia-induced phosphorylation of ERK and JNK indicating that the hypoxia-induced activation of ERK and JNK in the cerebral cortical nuclei of newborn piglets is NO-mediated

Animals↗

PubMed2004

Effect of neuronal nitric oxide synthase inhibition on CA2+/calmodulin kinase kinase and CA2+/calmodulin kinase IV activity during hypoxia in cortical nuclei of newborn piglets.

The present study tests the hypothesis that cerebral tissue hypoxia results in increased Ca(2+)/calmodulin (CaM) kinase kinase activity and that the administration of nitric oxide synthase inhibitors (N-nitro-l-arginine [NNLA], or 7-nitroindazole sodium [7-NINA]) prior to the onset of hypoxia will prevent the hypoxia-induced increase in the enzyme activity. To test this hypothesis, CaM kinase kinase and CaM kinase IV activities were determined in normoxic, hypoxic, NNLA-treated hypoxic, and 7-NINA-treated hypoxic piglets. Hypoxia was induced (FiO(2)=0.05-0.08x1 h) and confirmed biochemically by tissue levels of ATP and phosphocreatine. CaM kinase kinase activity was determined in a medium containing protein kinase and phosphatase inhibitors, calmodulin, and a specifically designed CaM kinase kinase target peptide. CaM kinase IV activity was determined by (33)P-incorporation into syntide-2 in a buffer containing protein kinase and phosphatase inhibitors. Compared with normoxic animals, ATP and phosphocreatine levels were significantly lower in all hypoxic piglets whether or not pretreated with nitric oxide synthase inhibitors. There was a significant difference among CaM kinase kinase activity (pmol/mg protein/min) in normoxic (76.84+/-14.1), hypoxic (138.86+/-18.2, P<0.05 vs normoxia), NNLA-pretreated hypoxic (91.34+/-19.3; P=NS vs normoxia, P<0.05 vs hypoxia) and 7-NINA-pretreated hypoxic animals (100.12+/-23.3; P=NS vs normoxia, P<0.05 vs hypoxia). There was a significant difference among CaM kinase IV activity (pmol/mg protein/min) in normoxia (1270.80+/-126.1), hypoxia (2680.80+/-136.7; P<0.05 vs normoxia), NNLA-pretreated hypoxia (1666.00+/-154.8; P<0.05 vs normoxia, P<0.05 vs hypoxia), and 7-NINA-pretreated hypoxic (1712.9+/-231.5; P=NS vs normoxia, P<0.05 vs hypoxia). We conclude that the hypoxia-induced increase in CaM kinase kinase and CaM kinase IV activity is mediated by neuronal NOS-derived NO.

Adenosine Triphosphate↗

PubMed2004

Effect of hypoxia on protein phosphatase 2A activity, subcellular distribution and expression in cerebral cortex of newborn piglets.

Protein phosphatase (PP) 2A (PP2A), a major serine/threonine phosphatase highly active in the brain, is known to regulate programmed cell death by different mechanisms including downregulation of Ca++/calmodulin-dependent kinase IV (CaMK IV). Previous studies have shown that CaMK IV activity is increased following cerebral hypoxia. In the present study, we tested the hypothesis that PP2A activity and expression in neuronal nuclei are decreased following hypoxia in newborn piglets. PP and PP2A activities were determined in cerebral subcellular fractions spectrophotometrically using a serine phosphopeptide in the presence or absence of microcystine. The activity of CaMK IV in neuronal nuclei was determined by 33P-incorporation into syntide 2 in the presence or absence of either 1 mM EGTA or 0.8 mM CaCl2 and 1 mM calmodulin. The expressions of PP2A and CaMK IV were measured using Western blot. Following hypoxia, nuclear Ca++-dependent kinase IV activity increased two-fold (P<0.001), whereas PP2A and PP activities significantly decreased (P<0.05) in the neuronal nuclei and membranes but not in the cytosol (P=NS). The distribution of the activity of PP2A was 60% in the cytosol, 35% in membranes and 5% in the neuronal nuclei. The expression of PP2A protein showed a 14% increase and for CaMK IV protein a 100% increase during hypoxia. We propose that due to the decreased activity of PP and PP2A following hypoxia in the neuronal nuclei there is a shift in the balance of the phosphorylation/dephosphorylation system toward increased phosphorylated state thereby increasing activity of the nuclear CaMK IV, modulator of programmed cell death. Since there is only slight increase in the PP2A protein expression, we conclude that the changes observed in the activity of PP2A are due to hypoxia-induced modification of the enzyme itself. We also provide evidence that PP2A is a potential regulator of CaMK IV during hypoxia.

Animals↗

PubMed2004

Effect of hypoxia on the expression and activity of mitogen-activated protein (MAP) kinase-phosphatase-1 (MKP-1) and MKP-3 in neuronal nuclei of newborn piglets: the role of nitric oxide.

Mitogen-activated protein kinase-1 (MAPK-1) and MAPK-3 regulate survival and programmed cell death of neurons under stress conditions. The activity of MAPK-1 and MAPK-3 is regulated by dual specificity phosphatases: MKP-1 and MKP-3. In previous studies, we have shown that cerebral hypoxia results in increased activation of MAPK-1 and MAPK-3. Furthermore, we have shown that the hypoxia-induced activation of MAPK is nitric oxide (NO)-mediated. The present study tested the hypothesis that hypoxia results in altered expression and activity of MKP-1 and MKP-3 in neuronal nuclei and the administration of 7-nitro-indazole (7-NINA; 1 mg/kg, 60 min prior to hypoxia), a selective nNOS inhibitor, will prevent the hypoxia-induced alteration in the expression and activity of MKP-1 and MKP-3. To test this hypothesis expression and activity of MKP-1 and MKP-3 were determined in neuronal nuclei of normoxic (Nx; n=5), hypoxic (Hx; n=5) and 7-NINA-pretreated-hypoxic (7-NINA-Hx; n=5). Hypoxia was achieved by exposing the animals to an FiO2 of 0.07 for 60 min. Cerebral tissue hypoxia was documented biochemically by determining ATP and phosphocreatine levels. Neuronal nuclei were isolated using discontinuous sucrose gradient centrifugation and purified. Nuclear proteins were analyzed by Western blot using specific antibodies for MKP-1 and MKP-3 (Santa Cruz, CA, USA). The protein band density was determined by imaging densitometry and expressed as OD x mm2. The density of MKP-1 was 61.57+/-5.68, 155.86+/-44.02 and 69.88+/-25.54 in the Nx, Hx and 7-NINA-Hx groups, respectively (P<0.05, ANOVA). Similarly, the density of MKP-3 was 66.46+/-5.88, 172.04+/-33.10 and 116.88+/-14.66 in the Nx, Hx and 7-NINA-Hx groups, respectively (P<0.05, ANOVA). The data show an increased expression of MKP-1 and MKP-3 during hypoxia in neuronal nuclei of newborn piglets and the administration of 7-NINA, an nNOS inhibitor, prevented the hypoxia-induced increased expression of MKP-1 and MKP-3. The activity of MKP-1 (pmol/min) was 176.17+/-16.95 in Nx, 97.56+/-10.64 in Hx and 130+/-14.42 in the 7-NINA-Hx groups, respectively (P<0.05, ANOVA). Similarly the activity of MKP-3 was 104.11+/-12.17 in Nx, 36.29+/-16.88 in Hx and 77.89+/-20.18 in the 7-NINA groups, respectively (P<0.05, ANOVA). The results demonstrate that cerebral hypoxia results in increased expression of MKP-1 and MKP-3 expression that was prevented by the administration of 7-NINA. In contrast, hypoxia resulted in decreased activity of MKP-1 and MKP-3 that was prevented by the administration of a nNOS inhibitor. We conclude that hypoxia-induced decrease in MKP-1 and MKP-3 activity is not due to altered expression but due to NO-mediated modification of the cysteine residue at the active site of these dual specificity phosphatases, a mechanism of their inactivation that leads to activation of MAP kinases.

Adenosine Triphosphate↗

PubMed2004

Serum immunoglobulin E in idiopathic nephrotic syndrome.

Serum immunoglobulin E (IgE) and absolute eosinophil count (AEC) were assayed in 48 children with idiopathic nephrotic syndrome and 20 controls. The mean serum IgE (p < 0.001) and AEC (p < 0.05) were significantly raised in active nephrotic syndrome compared with controls. The mean values of IgE and AEC in nephrotic patients were 228.6 and 164.5 per cent of the normal mean, respectively. The serum IgE and AEC were significantly abnormal in all the subgroups (first attack, infrequent, and frequent relapsers), except AEC in infrequent relapses, when compared with the controls. No significant differences in the parameters were observed among the different subgroups of cases. In remission, the mean IgE and AEC were unchanged in comparison with their corresponding values at diagnosis. Thus, the levels are raised not only in active but also in remission, reflecting abnormal immune status even in the disease-free state.

Case-Control Studies↗

PubMed2003

Bacterial antigen detection test in meningitis.

OBJECTIVE: To evaluate the role of bacterial antigen detection test in cerebrospinal fluid (CSF) for a rapid etiological diagnosis of bacterial meningitis. METHODS: The study included 36 cases of bacterial meningitis and 14 controls. Latex particle agglutination test (LPA test) for detection of bacterial antigen was done in the CSF using slidex meningitis kit (Biomeriux, France). RESULTS: Using LPA test, an etiological diagnosis could be made in 83% cases of bacterial meningitis. In contrast, CSF Gram stain and culture showed 36% and 6% positivity, respectively. The sensitivity and specificity of LPA test were 83% and 100%, respectively. The common etiological organisms were S. pneumoniae, H. influenzae type b and N. meningitidis A. S. pneumoniae was encountered in all age groups while H. influenzae type b was found only below one year of age. CONCLUSIONS: LPA test is a rapid and superior diagnostic tool as compared to CSF Gram stain and culture. The study recommends LPA test as an adjunct laboratory test for rapid etiological diagnosis of bacterial meningitis for prompt institution of proper antibiotics.

Adolescent↗

PubMed2003

Serum immunoglobulin E in early childhood wheezing.

OBJECTIVE: To study the serum Ig E levels in patients presenting with wheezing under the age of 24 months. METHODS: The study was conducted on 38 cases and 35 controls. The children presenting with first attack of wheezing were clinically considered as bronchiolitis and those with recurrent attacks with positive history of atopy in the patient or in their first degree relatives were considered as bronchial asthma. The Ig E estimation was done by immune assay method. RESULTS: The mean serum Ig E was significantly higher in asthma patients (p < 0.001). It was also higher in bronchiolitis patients but the difference was not statistically significant. However, 26 per cent of these patients had values above 1 SD of the control mean. These patients were considered as potential cases of asthma. CONCLUSION: The determination of serum Ig E may be of value in identifying those children presenting with first wheezing attack, who may develop asthma in future. Only a long-term follow-up of these patients can give an answer to the postulation that raised Ig E level at the time of first viral lower respiratory tract infection is a potential marker for the development of recurrent wheezing in later life.

Asthma↗

PubMed2003

Hypoxia-induced modification of poly (ADP-ribose) polymerase and dna polymerase beta activity in cerebral cortical nuclei of newborn piglets: role of nitric oxide.

Previous studies have shown that poly (ADP-ribose) polymerase (PARP) and DNA polymerase beta, nuclear enzymes, are associated with cell replication and DNA repair. The present study tests the hypothesis that hypoxia results in increased PARP and DNA polymerase activity in cerebral cortical neuronal nuclei to repair the hypoxia-induced damage to genomic DNA. Studies were conducted in 13 anesthetized and ventilated newborn piglets (age 3-5 days) divided into normoxic (n=5) and hypoxic (n=8) groups. Hypoxia was induced by decreasing inspired oxygen from 21% to 7% for 60 min. Cerebral tissue hypoxia was documented biochemically by determining the tissue levels of ATP and phosphocreatine (PCr). Following isolation of the cortical neuronal nuclei, the activity of PARP and DNA polymerase beta was determined. During hypoxia, the tissue ATP level decreased by 73% from 4.12+/-0.67 micromol/g brain to 1.12+/-0.34 micromol/g brain, and PCr decreased by 78% from 4.14+/-0.68-0.90+/-0.20 micromol/g brain. In hypoxic neuronal nuclei, PARP activity significantly increased from 5.88+/-0.51 pmol NAD/mg protein/h in normoxic nuclei to 10.04+/-2.02 (P=0.001). PARP activity inversely correlated with tissue ATP (r=0.78) and PCr levels (r=0.81). Administration of N-nitro-L-arginine prior to hypoxia decreased the hypoxia-induced increase in PARP activity by 67%. Endogenous DNA polymerase beta activity increased from 0.96+/-0.13 in normoxic nuclei to 1.39+/-0.18 nmol/mg protein/h in hypoxic nuclei (P<0.005). DNA polymerase beta activity in the presence of exogenous template increased from 1.54+/-0.14 in the normoxic to 2.42+/-0.26 nmol/mg protein/h in the hypoxic group (P<0.005). DNA polymerase beta activity in the presence or absence of template inversely correlated with the tissue ATP (r=0.95 and 0.84, respectively) and PCr levels (r=0.93 and 0.77, respectively). These results demonstrate that the activity of PARP and DNA polymerase beta enzymes increase with the increase in degree of cerebral tissue hypoxia. Furthermore, the results demonstrate a direct correlation between the PARP and the DNA polymerase beta activity. We conclude that tissue hypoxia results in increased PARP and DNA polymerase beta activities indicating activation of DNA repair mechanisms that may result in potential neuronal recovery following hypoxia and the hypoxia-induced increase in PARP activity is NO-mediated.

Adenosine Triphosphate↗

PubMed2003

Hypoxia-induced caspase-3 activation and DNA fragmentation in cortical neurons of newborn piglets: role of nitric oxide.

Hypoxia results in generation of nitric oxide (NO) free radicals, activation of caspase-3, and genomic DNA fragmentation. The present study tests the hypothesis that hypoxia-induced caspase-3 activation and DNA fragmentation are nitric oxide mediated. Studies were conducted in newborn piglets, divided into normoxic (n = 5), hypoxic (n = 5), and hypoxic-7-NINA (n = 6). Hypoxic-7-NINA group received the neuronal nitric oxide synthase inhibitor, 7-Nitroindazole (7-NINA). Caspase-3 activity was determined spectrofluorometrically using enzyme-specific substrates. Sections from the neocortex were stained with an antiserum recognizing active caspase-3. Purified DNA was separated by gel electrophoresis. Administration of 7-NINA resulted in decreased immunoreactivity of caspase-3 (mean LI: 20.2%) as compared to the untreated hypoxia group (mean LI: 57.5%) (P < 0.05). 7-NINA attenuated caspase-3 enzymatic activity as well in comparison to the untreated hypoxia group (P < 0.05). Furthermore, multiple low molecular weight bands corresponding to DNA fragments were present in the hypoxic but not in the normoxic or hypoxic-7-NINA groups. Inhibition of nNOS abates the hypoxia-induced increase in active caspase-3 immunoreactivity, as well as enzymatic activity in cortical neurons, and DNA fragmentation in brain homogenates. We conclude that the coordinate increase of capase-3 activity and fragmentation of nuclear DNA in the hypoxic newborn piglet brain are NO mediated.

Adenosine Triphosphate↗

PubMed2003

Cerebrospinal fluid lysozyme level for the diagnosis of tuberculous meningitis in children.

Lysozyme activity was assayed in the cerebrospinal fluid (CSF) of 32 tuberculous meningitis (TBM), 17 bacterial meningitis, 10 partially treated bacterial meningitis, 18 encephalitis and 18 control subjects. The mean CSF lysozyme activity was significantly raised (p < 0.001) in TBM patients compared with other study groups. A cut-off CSF lysozyme level of > or = 26 U/l had a sensitivity and specificity of 93.7 and 84.1 per cent, respectively for the diagnosis of TBM. Overall, it was found to be a better test than any other single test and thus can be used for rapid and early diagnosis of TBM in children.

Case-Control Studies↗

PubMed2003

Helicobacter pylori infection and recurrent abdominal pain in children.

The association of Helicobacter pylori infection with recurrent abdominal pain (RAP) was studied in 65 children aged between 3 and 12 years. Cases with a specific cause for abdominal pain were excluded from the study. All the patients were subjected to upper gastrointestinal endoscopy and antral biopsy. The biopsy specimens were subjected to rapid urease test, histopathological examination, and culture to document the presence of H. pylori in the gastric mucosa. All the patients were put on H. pylori eradication drugs for 10 days. About 69 per cent of the children had histopathological evidence of antral inflammation. Helicobacter pylori detection rate was 77 per cent. About 83 per cent of patients had complete symptomatic relief when followed over a period of 6 months. The study concludes a possible association of H. pylori with recurrent abdominal pain in children and all these patients should be considered for H. pylori eradication.

Abdominal Pain↗