Biomedical subjects

S K Shankar

Publications and source records attributed to S K Shankar.

At least 109 records · Page 6Linked to original sources

PubMed1993

Purification of multiple forms of cytochrome P450 from a human brain and reconstitution of catalytic activities.

The present study demonstrates the presence of multiple forms of cytochrome P450 (P450) in human brain obtained at autopsy, the purification of various isoforms to apparent homogeneity, and the monooxygenase activities in reconstituted systems. Sequential chromatography on octylamino-Sepharose 4B, DEAE-Sephacel, and DEAE-cellulose yielded four isoforms of P450 (A, B, C, and D) with specific contents of 11.0, 9.4, 12.5, and 8.3 nmol of P450/mg protein, respectively. While the forms A, B, and C were apparently homogeneous as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the P450D was not homogeneous. The apparent molecular masses of the four forms of P450 were 60,200 Da (P450A), 60,900 Da (P450B), 60,200 Da (P450C), and 61,000 Da (P450D), respectively. NADPH cytochrome P450 reductase (reductase) was also partially purified from the brain microsomes. Immunoblot analysis of the four forms of human purified P450, using antisera to purified rat liver P450 (IIB1 + IIB2), rat liver P450 (1A1 + 1A2), phenobarbital-inducible rat brain P450, human liver P450 IIE1, P450 1A2, P450 IIC, and P450 IIIA4, indicated differential immunological cross-reactivity. The monooxygenase activities of the purified human brain P450s were demonstrated with various substrates (aminopyrine, morphine, aniline, 7-ethoxycoumarin, and nifedipine) as examined in reconstituted systems consisting of purified human brain P450, purified rat brain NADPH cytochrome P450 reductase, deoxycholate, phospholipid, and NADPH.

Brain↗

PubMed1993

Catalytic activity and immunohistochemical localization of flavin-containing monooxygenase in rat kidney.

The presence of flavin-containing monooxygenase activity was examined in rat kidney microsomes using N,N-dimethylaniline and methimazole as substrates. Western immunoblot analysis using antisera to porcine liver and rabbit lung flavin-containing monooxygenase indicated immunological cross-reactivity between rat kidney, porcine liver and rabbit lung flavin-containing monooxygenase. Immunohistochemical studies using antisera to rabbit lung flavin-containing monooxygenase demonstrated localization of this enzyme in the proximal and distal convoluted tubules of the renal cortex, the collecting ducts in the renal medulla, but not the glomeruli. This observation indicates the colocalization of flavin-containing monooxygenase and cytochrome P-450 in the metabolically active and absorptive compartment of the renal parenchyma.

Aniline Compounds↗

PubMed1993

Billion-fold difference in the toxic potencies of two excitatory plant amino acids, L-BOAA and L-BMAA: biochemical and morphological studies using mouse brain slices.

Plant amino acids beta-N-oxalylamino-L-alanine (L-BOAA, present in Lathyrus sativus) and beta-N-methylamino-L-alanine (L-BMAA, present in Cycas circinalis) have been implicated in the pathogenesis of human neurological disorders lathyrism and amyotrophic lateral sclerosis-Parkinson's dementia complex of Guam (ALS-PD), respectively. In view of the conflicting reports that have emerged on the role of L-BMAA in ALS-PD, we reinvestigated the comparative toxicity of L-BMAA and L-BOAA. We report here the potent toxicity of L-BOAA as examined in an in vitro model consisting of sagittal slices of mouse brain. Incubation of sagittal slices of mouse brain with L-BOAA (1 pM) resulted in significant leakage of lactate dehydrogenase (LDH) and potassium from the slices into the medium. Under similar conditions, L-BMAA-induced LDH leakage from the slices into the medium was observed only at very high concentration of the toxin, namely 1 mM. N-Methyl-D-aspartate (NMDA) receptor antagonists ameliorated the toxic effects of L-BMAA, while non-NMDA receptor antagonists (quinoxalinediones) protected against the toxicity of L-BOAA. Incubation of slices with L-BOAA for 1 h resulted in extensive vacuolation and degeneration of neurons in the thalamus and brain stem, and to a lesser extent in the hippocampus and cerebellar nuclei. The large sized neurons appeared to be affected to a greater extent than the smaller ones. The neurons in other areas of the brain also revealed variable degree of degeneration with swelling of axons and dendrites.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1993

Changes in lactate dehydrogenase isoenzyme pattern in patients with tumors of the central nervous system.

Lactate dehydrogenase (LDH) isoenzymes were studied in biopsy samples obtained from 100 benign and malignant brain tumors. Diagnosis was confirmed by histopathology. It is observed that all tumors investigated had elevated LDH activity and showed a LDH isoenzyme pattern which is different from that of normal brain. A pronounced cathodal shift was seen in malignant tumors like medulloblastoma, grade 3-4 astrocytomas and neuroblastomas, whereas anodal pattern was seen in benign tumors like grade 1-2 astrocytomas and oligodendrogliomas. Some tumors like meningiomas showed a midzone pattern like increased LDH3. It was possible to differentiate certain tumors on the basis of LDH isoenzyme pattern like medulloblastomas into differentiated and undifferentiated; craniopharyngiomas into recurring and non-recurring ones. LDH1/LDH5 ratio was low (< 1.0) in malignant tumors and high (5.0-14.0) in benign tumors and it was useful in differentiating tumors according to the degree of malignancy and biological behavior. It is observed that both LDH isoenzyme pattern and LDH1/LDH5 ratio could be used as an adjuvant to histopathological grading of brain tumors.

Astrocytoma↗

PubMed1993

Balo's concentric sclerosis diagnosed intravitam on brain biopsy.

A 28-year-old woman presented with acute right hemiparesis evolving to bilateral hemiplegia and a mute state within 1 week on a background of paranoid psychosis of 8 years duration. CT scan revealed multiple bilateral hypodense enhancing lesions in the central white matter. Biopsy of the lesion showed lamellar alternating zones of demyelination and remyelination, typical of Balo's concentric sclerosis, an acute variant of multiple sclerosis. Immunohistochemical staining for neurofilament showed greater damage to the axis cylinders by the disease than generally perceived by silver stains. Steroid therapy produced a gratifying and sustained improvement. Follow up CT scans and psychometry revealed regression of the demyelinating lesions and improvement of the cognitive state.

Adult↗

PubMed1993

Cerebral flavin-containing monooxygenase-mediated metabolism of antidepressants in brain: immunochemical properties and immunocytochemical localization.

Flavin-containing monooxygenase (FMO)-mediated oxidation of the model substrates N,N-dimethylaniline and methimazole, and the antidepressants imipramine and fluoxetine, was determined in rat brain microsomes. No sex-related difference was observed in the activity of the FMO-mediated metabolism of the four substrates examined. The Km values for flavin-containing monooxygenase-mediated metabolism of N,N-dimethylaniline and methimazole were 2.8 and 0.8 mM, respectively, and the Km values for the oxidation of the antidepressants imipramine and fluoxetine were 20.9 and 9.8 microM, respectively. The Vmax values for oxidation of N,N-dimethylaniline, methimazole, imipramine and fluoxetine were 340, 31, 182 and 470 nmol nicotinamide adenine dinucleotide phosphate oxidized/min/mg protein, respectively. Western immunoblot analysis using antisera to purified porcine liver FMO did not reveal any immunological cross-reactivity with male or female brain microsomal protein. Antibody to rabbit lung flavin-containing monooxygenase cross-reacted with brain microsomes as examined by Western immunoblot studies. Addition of the antibody raised against rabbit lung FMO resulted in inhibition (43% inhibition) of the FMO-mediated metabolism of imipramine. Immunocytochemical examination of rat brain sections using the above antibody revealed the preferential localization of flavin-containing monooxygenase in the neuronal cell body. The flavin-containing monooxygenase-mediated metabolism of antidepressant drugs by brain microsomes is of profound pharmacological significance.

Animals↗

PubMed1993

Herpes simplex encephalitis: a diagnostic and therapeutic reapprisal.

Herpes Simplex Encephalitis (HSE) appears to be underdiagnosed in India, though viral encephalitides constitutes an important entity with significant morbidity. With an upsurge in AIDS, HSE may perhaps emerge as an important opportunistic infection in future. We discuss the clinical features and laboratory evaluation of nine cases of HSE seen in the last 12 years at our center. Diagnosis was established by brain biopsy in one, virological studies in six and at autopsy in three. Immunocytochemically viral antigens could be localized in 4 biopsied/autopsied brain tissue and in CSF cells on a cytospin preparation in one. This has facilitated rapid diagnosis in our cases. Virus isolation was successful in two. Three subjects were treated with acyclovir and all survived with variable morbidity. Four patients expired and none of them had received any specific antiviral drugs. Rapid diagnosis and early treatment with acyclovir has been highlighted.

Acyclovir↗

PubMed1992

Microsomal cytochrome P450 in human brain regions.

Cytochrome P450 (P450) levels were quantitated in microsomes from human brain regions obtained at autopsy. The reduced carbon monoxide binding spectra of cortical microsomes showed two absorption maxima at 449 and 425 nm. On solubilization of the microsomes, essentially a single peak was observed at 449 nm. The P450 levels in human brain cortical microsomes varied from 0.03 to 0.12 nmol/mg protein among the seven samples examined. The concentration of the hemeprotein present as nmol/g tissue was highest in the brain stem and cerebellum and lowest in the striatum and hippocampus.

Adult↗

PubMed1992

Neuroanatomy of Cysticercus cellulosae (Cestoda) as revealed by acetylcholinesterase and nonspecific esterase histochemistry.

The nervous system of Cysticercus cellulosae, the metacestode stage of the tapeworm Taenia solium, was delineated using histochemical methods for the localization of the enzyme markers; nonspecific esterase and acetylcholinesterase. The main features of the nervous system include a pair of cerebral ganglia, a circumcerebral nerve ring, a rostellar nerve ring, and anterior and posterior nerves and their branches. The posterior nerves form a subtegumental network in the strobila and the bladder wall. A nerve network around excretory tubules could also be demonstrated, suggesting neuronal control of excretion in the metacestode. No sheath was observed around the nervous system. The morphological features described suggest "cephalization" of the nervous system in this parasite.

Acetylcholinesterase↗

PubMed1991

Effect of chronic ethanol ingestion on phosphate content of neurofilament proteins and neurofilament associated protein phosphatase in rat spinal cord.

Rats were trained to drink alcohol solution by gradually increasing the ethanol content [2.5-15% (v/v)] in drinking water. After 11 months of alcohol (15% v/v) ingestion, animals were guillotined and the spinal cords were used for the preparation of neurofilaments (NF). NF triplet proteins were separated by SDS-PAGE and the phosphate contents of individual components were estimated. Results indicated a significant increase in phosphate content of 200 KD protein in alcohol fed rats (30.19 +/- 4.12 mol of phosphate/mole of protein: p less than 0.001) compared to control group (18.42 +/- 3.91 mol of phosphate/mole of protein). No significant change in the phosphate content of 150KD and 68KD components of NF were seen in experimental group. Further, the studies on NF associated protein phosphatase activity indicated a significant decrease in phosphatase activity among the alcohol fed rats (14.10 +/- 2.5 mU; p less than 0.001) against NF rich fraction as a substrate, as compared to control (20.15 +/- 2.15 mU). While the observed decrease in NF associated protein phosphatase would possibly explain the increase in phosphate content of NF proteins in alcohol fed rats, the precise mechanism of decrease in enzyme activity remains to be elucidated. Nevertheless, the change seen in phosphate content and NF associated protein phosphatase activity as a result of ethanol ingestion would possibly form the biochemical basis of some of the neuropathological changes seen in alcoholics.

Alcoholism↗

PubMed1991

A simple and inexpensive slicer for preparation of brain slices.

A simple and inexpensive slicer has been developed for the preparation of slices of mouse or rat brain. The instrument consists of razor blades, separated by an 0.5 mm thick polyethylene sheet (1 x 1 cm), mounted on metal screws through a hole in the center of the polyethylene sheet. Using this slicer, 6-8 uniform slices of 500 microns thickness were obtained from mouse or rat brain. These brain slices were incubated in a medium consisting of artificial cerebrospinal fluid for 1 h at 37 degrees C under an oxygen atmosphere and the activities of various subcellular marker enzymes were assayed. The slice weights and the activities of the enzymes did not vary significantly in different batches of slices. Morphological evaluation of the slices revealed well-preserved neurons. Histochemical staining for mitochondrial enzymes revealed intense staining of neuronal cells and lighter staining of the white matter in all the regions examined. These slices could serve as a useful in vitro model for studying brain function and the effect of various toxicants on the brain.

Animals↗

PubMed1991

Acute ataxic neuropathy: a clinical, electrophysiological and morphological study.

Sensory ataxia as the chief manifestation of acute neuropathy is rather rare. Of the 224 cases of acute polyneuropathy seen over 6 years (1984-1990) only 10 patients (M:F 3:7) had disabling ataxia as the presenting feature. Their ages ranged from 14-61 years. Antecedent febrile illness was present in 6 patients and the peak deficit evolved over 2-25 days. Severe ataxia, paresthesia, distal areflexia and predominant joint sense loss were common to all, motor weakness was either absent or insignificant. CSF was acellular and revealed elevated protein in 3 subjects. All patients had electrophysiological evidence of severe sensory neuropathy with mild or no motor neuropathy. Sural nerve biopsy in one patient showed loss of large, as well as small, diameter myelinated fibres, secondary demyelination, but no evidence of inflammation. At follow up marginal to moderate improvement in ataxia was noted in only 5 patients. Absence of ophthalmoplegia and motor weakness, poor prognosis and characteristic electrophysiological and histopathological observations suggest that acute ataxic neuropathy may be a distinct entity.

Adolescent↗

PubMed1991

Creutzfeldt-Jakob disease in India (1971-1990).

Thirty cases including 20 definite and 10 probable cases of Creutzfeldt-Jakob disease (CJD) seen in India between 1971 and 1990 are reported. Demographic analysis has shown similarities to the previously published reports from other parts of the world. Though 21 (70%) of cases were from two centers--Bombay and Bangalore-, suggesting clustering, this seems to be more apparent than real. One subject worked in the medical field, where possibility of iatrogenic transmission could not be ruled out. None of the cases had positive family history of CJD. There is no epidemiological data of CJD from India so far and hence this report is one such pilot study.

Adult↗

PubMed1990

Rat brain cytochromes P-450: catalytic, immunochemical properties and inducibility of multiple forms.

Cytochrome P-450 (P-450) and associated mono-oxygenase activities were estimated in male and female rat brain microsomes. The P-450 concentration in male rat brain was one-tenth the corresponding hepatic levels, which is considerably higher than earlier reports. A distinct sex-related difference was observed in the levels of total P-450 and mono-oxygenase activities known to be mediated by P-450b,e; the female brain levels were 60% of those in the males. Immunoinhibition and immunoblot studies using antisera to P-450b,e and P-450c,d indicated the presence of multiple forms of P-450, immunologically similar to P-450b,e, P-450c and P-450d in the rat brain. Prior treatment with phenobarbital resulted in two-fold increase of total P-450 and selective induction of aminopyrine N-demethylase (APD) and morphine N-demethylase (MND) activities. Administration of 3-methylcholanthrene, selectively induced the levels of ethoxycoumarin O-deethylase (ECD) and arylhydrocarbon hydroxylase, although the levels of total P-450 were not increased. 3-Methylcholanthrene induction was also accompanied by a shift in the absorption maximum of the reduced carbon monoxide difference spectrum from 452 to 448 nm. Immunocytochemical localization using antibodies to P-450b,e indicated the presence of P-450 predominantly in the neuronal cell bodies and to a lesser extent in the fibre tracts in cerebral cortex, cerebellum, thalamus, hypothalamus, hippocampus and brainstem. These studies indicate that the brain contains significant amounts of P-450, which exists in multiple forms and can be selectively induced by prior exposure to phenobarbital or 3-methylcholanthrene.

Animals↗

PubMed1990

NADPH cytochrome P-450 reductase in rat, mouse and human brain.

NADPH cytochrome P-450 reductase (P-450 reductase), an essential component of the cytochrome P-450 mono-oxygenase system, has been estimated in rat and mouse brain, and seven human brains obtained at autopsy. The ratio of cytochrome P-450 to P-450 reductase is lower in the rat and mouse brains (2.5-4.0) as compared to the respective livers (10.0-11.0). The rat and mouse brain P-450 reductase were immunologically similar to the rat liver P-450 reductase as examined by immunochemical inhibition, Ouchterlony double diffusion and immunoblot. The antisera to rat liver P-450 reductase inhibited rat brain aminopyrine N-demethylase activity to the same extent as NADPH cytochrome c reductase, suggesting that the level of P-450 reductase controls the rate of this cytochrome P-450 mediated activity. The human brain NADPH cytochrome c reductase exhibited regional variation, maximal activity being observed in the brain stem region. Immunochemical inhibition and immunoblot studies revealed immunological cross-reactivity between rat liver reductase and human brain medulla, while none was observed in cortex or cerebellum. Immunocytochemical studies on human brain medulla using antisera to rat liver P-450 reductase indicated localization of the P-450 reductase in neuronal cell body.

Adolescent↗

PubMed1990

Vertebro-basilar insufficiency due to carotid stenosis.

Two patients with internal carotid disease and normal/hypoplastic vertebro-basilar system presenting with symptoms of vertebrobasilar insufficiency are presented. The role of associated anomalies of the circle of Willis or major vessels in the pathogenesis of symptoms is highlighted.

Carotid Artery Diseases↗

PubMed1990

Simultaneous cerebral arterial and venous thrombosis.

Simultaneous thrombotic occlusion of cerebral arterial and venous systems in the same individual is rare. Three patients of stroke in whom autopsy revealed co-existing arterial and venous infarctions, major arterial occlusion (vertebral 1, internal carotid 1) and dural sinus and cortical vein thrombosis are reported. Role of infarcted brain, haemorrheological factors and alcohol in the pathogenesis of this rare event is highlighted.

Adult↗

PubMed1989

Xenobiotic metabolism in human brain--presence of cytochrome P-450 and associated mono-oxygenases.

The cytochromes P-450, a family of heme proteins, play an important role in the oxidation of drugs and carcinogens, as well as endogenous substrates. We report the presence of cytochrome P-450 and associated mono-oxygenase activity in human brain regions and their selective enrichment in the brainstem. Immunocytochemical studies on human medulla with antibodies raised to phenobarbital-inducible rat liver cytochrome P-450 indicate that the enzyme is primarily localized in the neuronal cell bodies and to a lesser extent in the axons. These observations indicate that the human brain could be involved in metabolism of xenobiotics and endogenous compounds, mediated through cytochrome P-450.

Adult↗