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

Flavin-containing monooxygenase mediated metabolism of psychoactive drugs by human brain microsomes

Flavin-containing monooxygenases (FMO) catalyze the oxidation of certain xenobiotics and drugs which contain a nucleophilic heteroatom. Here we report the first assessment of human brain flavin-containing monooxygenase from tissues obtained at autopsy from seven traffic accident victims. Human brain microsomes catalyzed the S-oxidation or N-oxidation of model substrates methimazole and N,N-dimethylaniline, respectively. The psychoactive drugs chlorpromazine, imipramine and fluoxetine, were also metabolized by human brain FMO. 'Western' immunoblot analyses revealed immunological cross-reactivity of the human brain FMO with rabbit pulmonary FMO. Immunocytochemistry further revealed the localization of the FMO predominantly in the neuronal cell bodies in the magnocellular reticular nuclei, colliculi and substantia nigra. Human brain clearly contains an active FMO system, and it is conceivable that such enzyme(s) are significantly involved in the local metabolism and modulation of pharmacological effects of psychoactive drugs

Exceptional molecular and coreceptor-requirement properties of molecular clones isolated from an Human Immunodeficiency Virus Type-1 subtype C infection

<p>Abstract</p> <p>Background</p> <p>The pathogenic significance of coreceptor switch in the viral infection of HIV-1 is not completely understood. This situation is more complex in subtype C infection where coreceptor switch is either absent or extremely rare. To gain insights into the mechanisms that underlie coreceptor requirement of subtype C, we screened several primary viral isolates and identified a clinical sample that demonstrated a potential to grow on standard T-cell lines with no detectable CCR5 expression. The subject was diagnosed with HIV-1 associated dementia in the absence of opportunistic infections of the brain. To isolate molecular clones from this virus, we devised a novel strategy based on anchor primers that target a sequence in the reverse transcriptase, highly conserved among diverse subtypes of HIV-1.</p> <p>Results</p> <p>Using this strategy, we isolated 8 full-length molecular clones from the donor. Two of the eight molecular clones, 03In94_D17 and 03In94_D24, (D17 and D24) generated replication-competent viruses. Phylogenetic analysis of the full-length viral sequences revealed that both clones were non-recombinant subtype C viruses. They contain intact open reading frames in all the viral proteins. Both the viral clones are endowed with several unique molecular and biological properties. The viral promoter of the clones is characterized by the presence of four NF-kB binding elements, a feature rarely seen in the subtype C HIV-1 LTR. Interestingly, we identified the coexistence of two different forms of Rev, a truncated form common to subtype C and a full-length form less common for this subtype, in both proviral and plasma virus compartments. An exceptional property of the viruses, atypical of subtype C, is their ability to use a wide range of coreceptors including CCR5, CXCR4, and several others tested. Sequence analysis of Env of D17 and D24 clones identified differences within the variable loops providing important clues for the expanded coreceptor use. The V1, V2 and V4 loops in both of the molecular clones are longer due to the insertion of several amino acid residues that generated potential N-linked glycosylation sites.</p> <p>Conclusion</p> <p>The exceptional biological and molecular properties of these clones make them invaluable tools to understand the unique pathogenic characteristics of subtype C.</p

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

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. Immunocytochernical studies on human brain medulla using antisera to rat liver P-450 reductase indicated localization of the P-450 reductase in neuronal cell body

Billionfold 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 β-N-oxalylamino-L-alanine (L-BOAA, present in Lathyrus sativus) and β-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. Thus, the present study demonstrates the potent toxicity of L-BOAA and elucidates for the first time, the billion-fold difference in the concentration of L-BOAA and L-BMAA required to elicit similar toxic response in vitro, using mouse brain slices. The study also demonstrates the selective vulnerability of certain regions of the brain to toxic insult by L-BOAA

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 (1×1cm), mounted on metal screws through a hole in the center of the polyethylene sheet. Using this slicer, 6-8 uniform slices of 500 μm 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°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

Thiol oxidation and loss of mitochondrial complex I precede excitatory amino acid-mediated neurodegeneration

Human ingestion of “chickling peas ” from the plant Lathyrus sativus, which contains an excitatory amino acid, L-BOAA (L-�-N-oxalylamino-L-alanine), leads to a progressive corticospinal neurodegenerative disorder, neurolathyrism. Exposure to L-BOAA, but not its optical enantiomer D-BOAA, causes mitochondrial dysfunction as evidenced by loss of complex I activity in vitro in male mouse brain slices and in vivo in selected regions of mouse CNS (lumbosacral cord and motor cortex). Loss of complex I activity in lumbosacral cord after L-BOAA administration to mice was accompanied by concurrent loss of glutathione. The inhibited complex I activity in mitochondria isolated from lumbosacral cord of animals treated with L-BOAA rebounded after incubation with the thiol-reducing agent dithiothreitol, indicating that oxidation of protein thiols to disulfides was responsible for enzyme inhibition. The inhibition of comple

Correction to: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway

Upon publication of the original article [1], it was noticed that there is an error in Fig. 10, the dialog box in panel (b) was missing. The correct Fig. 10 is shown below

An idiopathic epilepsy syndrome linked to 3q13.3-q21 and missense mutations in the extracellular calcium sensing receptor gene

Objective: To identify the disease locus in a three-generation south Indian family having several of its members affected with idiopathic epilepsy. Methods: Genome-wide parametric linkage analysis was performed with 382 autosomal markers. Mutational analysis of the positional candidate genes in linked interval was performed by direct sequencing of genomic DNA from the proband in the family. Expression analysis in human adult brain was performed by Western blotting. Results: A novel epilepsy genetic locus on chromosome 3q13.3-q21 was identified by linkage analysis. This locus comprises about 12 megabases of the genomic interval, with its proximal and distal genetic boundaries defined by microsatellite markers, D3S3675 and D3S1551, respectively. In this interval, we found a novel, patient-specific, missense variant, Arg898Gln, at the extracellular calcium sensing receptor (CASR), a gene belonging to the G-protein-coupled receptor family. CASR expression was detected in the temporal lobe, frontal lobe, parietal lobe, cerebellum, and hippocampus. Four additional, potentially pathogenic, missense CASR variants, Glu354Ala, Ile686Val, Ala988Val, and Ala988Gly, were observed in five individuals affected with idiopathic generalized epilepsy. Interpretation: A novel idiopathic epilepsy locus has been mapped on chromosome 3q13.3-q21, as evident by presence of significant genetic linkage. Identification of novel, rare missense CASR variants at evolutionary-conserved residues in epilepsy patients and CASR expression in various subregions of human brain raises an interesting possibility of involvement of CASR in pathophysiology of epileptic disorders