Oxygen free radicals induced release of lysosomal enzymes in vitro

Summary The effect of oxygen free radicals, generated by xanthine and xanthine oxidase, was studied on the release of lysosomal hydrolase from rat liver lysosomes in vitro. A lysosomal enriched subcellular fraction was prepared, using differential centrifugation technique, from the homogenate of rat liver. The biochemical purity of the lysosomal fraction was established by using the markers of different cellular organelles. Oxygen free radicals were generated in vitro by the addition of xanthine and xanthine oxidase. The release of lysosomal hydrolase (-glucuronidase) from the lysosomal fraction was measured. There was a 3 to 4 fold increase in the release of -glucuronidase activity in the presence of xanthine and xanthine oxidase when compared to that in the absence of xanthine and xanthine oxidase. In the presence of superoxide dismutase (SOD), a scavenger of oxygen free radicals, the xanthine and xanthine oxidase system was unable to induce the release of -glucuronidase activity from the lysosomes. Sonication (2 bursts for 15 sec each) and Lubrol (2 mg/10 mg lysosomal protein) treatment, which are known to cause membrane disruption, also induced the release of -glucuronidase from lysosomal fraction. This release of -glucuronidase by sonication and lubrol treatment was not prevented by SOD. These data indicate that lysosomal disruption is a consequence of oxygen free radicals, generated by xanthine and xanthine oxidase.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EGTA Ethylene Glycol Bis-(-aminoethyl ether)N,N,-N,N-tetracetic acid - Tris Tris (hydroxymethyl) aminomethane - SOD Superoxide Dismutase     

Localization of cytochrome P-450 in the colonic mucosa of 3-methylcholanthrene-pretreated and untreated rats An immunohistochemical study

Summary Immunohistochemical localization of cytochrome P-450 in the colonic mucosa of 3-methylcholanthrene-pretreated and untreated rats was studied by indirect fluorescent antibody staining technique. A polyclonal antibody for cytochrome P-450_MC purified from hepatic microsomes of 3-methylcholanthrene-pretreated rats was used for this experiment. A strong immunofluorescence was found to be localized in the cytoplasm of the surface epithelium of the mucosa in the colon of 3-methylcholanthrene-pretreated rats. A faint immunofluorescence was also observed in the epithelium of untreated rats. 7-Ethoxycoumarin O-deethylase activity of colonic microsomes was significantly enhanced by 3-methylcholanthrene-pretreatment in parallel with an increase in the intensity of immunostaining for cytochrome P-450_MC in Western blotting analysis. This is the first report on the localization of cytochrome P-450 in the colonic mucosa.     

Structural studies on cyanobacterial photosystem I

The cyanobacterial photosystem, I complex from Synechococcus sp. PCC6301 contains polypeptides of apparent M_r of 70,000, 18,000, 17,700, 16,000 and 10,000. Procedures were developed for the purification of the M_r 17,700 and 10,000 polypeptides. Amino acid analyses showed the absence of cystine and cysteine from these polypeptides. Amino-terminal sequences of 98 residues for the M_r 17,700 polypeptide and of 42 residues for the M_r 10,000 polypeptide were determined. Studies of pigment distribution within the photosystem I complex indicated that the binding of chlorophyll a and -carotene is in part dependent on the presence of these polypeptides.Abbreviations PSI photosystem I - P700 reaction center of PSI - SDS sodium dodecylsulfate - TBS tris-buffered saline - TTBS TBS containing Tween-20     

Mapping and disruption of the cybB gene coding for cytochrome b561 in Escherichia coli

Abstract The cybB gene on a plasmid encoding cytochrome b ₅₆₁ in Escherichia coli was disrupted by insertion of Km^rl determinant DNA. The cromosomal cybB gene was replaced by the inactivated cybB gene on the plasmid by homologous recombination using λ phage lysogenization and heat-induction. The replacement was confirmed by Southern and Western blotting analyses. Deficiency on the cybB gene product did not affect the growth properties of the cells, and the oxidase activities of the cells dependent on various substrates were similar to those of the parental strain. Cytochrome b ₅₆₁ is concluded to be expressed in E. coli , but may not play a major role in cell growth. In the genetic map of E. coli , the cybB gene was determined by conjugational and transductional crosses to be at 31 min between trg and terC .     

The Upper Cave at Zhoukoudian and the origins of the Mongoloids

The best evidence for identifying the inhabitants of northeast Asia in the terminal Pleistocene or early Holocene periods is provided by the human burials from the Upper Cave at Zhoukoudian, and in particular the “Old Man”. Apart from the Minatogawa finds on Okinawa, all Late Pleistocene human remains from East Asia that are reasonably well published are poorly preserved and often have equivocal dates. Since the time the Upper Cave was excavated it has been supposed that the human remains were the link between “Peking Man” and the modern Mongoloid complex. We have carried out multivariate analyses of the “Old Man” cranium employing new measurements of Weidenreich's cast of the skull and comparative data from Howells' survey of modern human groups. Despite our expectations the analyses have not shown the “Old Man” to be closely linked with the Mongoloids. Considering all the evidence available we conclude that the common belief of a close biological relationship between the people buried in the Upper Cave and the modern Mongoloids is not yet adequately demonstrated. Of crucial importance in interpreting the Upper Cave burials is their antiquity, which is still commonly thought to be in the order of at least 18 000 years. We believe that recent C-14 dates of about 11 000 years, determined from animal bones, indicate the earliest possible date for the burials. The time span between the Upper Cave burials and the earliest known modern Mongoloids in north China is in the order of about 4–5000 years. It is possible that a major population shift has occurred in north China between the terminal Pleistocene and the mid Holocene, when farming first appears. If this is so, the Upper Cave people may not have been closely allied to the Mongoloid groups that now inhabit East Asia and the Americas.     

Structure and carcinogenicity of Dibenzo(c,g)carbazole derivatives

The carcinogenicity of several groups of carcinogens is evoked with particular reference to Dibenzo(c,g)carbazole derivatives. The activity of these derivatives is discussed with respect to their species and organ specificity. The enzymatic equipment is decisive as to whether the compounds formed can react with DNA or are simply detoxified and eliminated. All these carcinogens are complete carcinogens, i.e. they have the property of both initiation and promotion.     

Structural and functional domains of cellobiohydrolase I from trichoderma reesei

Limited proteolysis (papain) of the cellobiohydrolase I (CBH I, 65 kDa) from Trichoderma reesei led to the seperation of two functional domains: a core protein (55 kDa) containing the active site, and a C-terminal glycopeptide (10 kDa) implicated in binding to the insoluble matrix (cellulose). The quaternary structures of the intact CBH I and its core in solution are now compared by small angle X-ray scattering (SAXS) measurements. The molecular parameters derived for the core (Rg=2.09 nm, D_max=6.5 nm) and for the intact enzyme (Rg=4.27 nm, D_max=18 nm) indicate very different shapes. The resulting models show a tadpole-like structure for the intact enzyme where the isotropic part coincides with the core protein and the flexible tail part should be identified with the C-terminal glycopeptide. Thus in this enzyme, functional differentiation is reflected in structural peculiarities.Abbreviations SAXS small angle X-ray scattering - SDS-PAGE SDS-polyacrylamide gel electrophoresis - IEF-PAG polyacrylamide gel isoelectric focusing; cellobiohydrolase (CBH, 1,4--glucan cellobio hydrolase (E.C.3.2.1.91)) - D_max maximum diameter - Rg radius of gyration     

Formation of the Neurotransmitter Glycine from the Anticonvulsant Milacemide Is Mediated by Brain Monoamine Oxidase B

Milacemide (2-n-pentylaminoacetamide) is a secondary monoamine that in the brain is converted to glycinamide and glycine. This oxidative reaction was suspected to involve the reaction of monoamine oxidase (MAO). Using mitochondrial preparations from tissues that contain MAO-A and -B (rat brain and liver), MAO-A (human placenta), and MAO-B (human platelet and bovine adrenal chromaffin cell), it has been established that mitochondria containing MAO-B rather than MAO-A oxidize (H2O2 production and glycinamide formation) milacemide. The apparent Km (30-90 microM) for milacemide oxidation by mitochondrial MAO-B preparations is significantly lower than that for milacemide oxidation by mitochondrial MAO-A (approximately 1,300 microM). In vitro MAO-B (l-deprenyl and AGN 1135) rather than MAO-A (clorgyline) selectively inhibited the oxidation of milacemide. These in vitro data are matched by ex vivo experiments where milacemide oxidation was compared to oxidation of serotonin (MAO-A) and beta-phenylethylamine (MAO-B) by brain mitochondria prepared from rats pretreated with clorgyline (0.5-10 mg/kg) and l-deprenyl (0.5-10 mg/kg). Furthermore, in vivo experiment demonstrated that l-deprenyl selectively increased the urinary excretion of [14C]milacemide and the total radioactivity with a concomitant decrease of [14C]glycinamide. Such changes were not observed after clorgyline treatment, but were evident only at doses beyond clorgyline selectivity. The present data therefore demonstrate that milacemide is a substrate for brain MAO-B, and its conversion to glycinamide, further transformed to the inhibitory neurotransmitter, glycine, mediated by this enzyme may contribute to its pharmacological activities.     

Monoamine Oxidase-Inhibiting Properties of SR 95191, a New Pyridazine Derivative, in the Rat: Evidence for Selective and Reversible Inhibition of Monoamine Oxidase Type A In Vivo but Not In Vitro

In rodents, SR 95191 [3-(2-morpholinoethylamino)-4-cyano-6-phenylpyridazine] has been shown to be active in animal models of depression. The profile of activity of SR 95191 suggests that the compound is a selective and short-acting type A monoamine oxidase (MAO) inhibitor (MAOI) in vivo. In the present study, the interaction of SR 95191 with MAO-A and MAO-B activity was further examined in vivo and in vitro. In brain, liver, and duodenum of pretreated rats, SR 95191 selectively inhibited MAO-A (ED50 = 3-5 mg/kg, p.o.), whereas MAO-B was only weakly inhibited for doses as high as 300 mg/kg, p.o. In vivo, SR 95191 (1-100 mg/kg, p.o.) antagonized, in a dose-dependent fashion, the irreversible inhibition of brain and liver MAO-A induced by phenelzine. Finally, dopamine and 5-hydroxytryptamine depleted from their striatal stores by tetrabenazine were able to displace SR 95191 from the active site of MAO-A. However, ex vivo, kinetic studies showed that the inhibitory effect of SR 95191 (1-10 mg/kg) towards MAO-A was noncompetitive and was unchanged after dilution or dialysis. In vitro, the inhibition of brain MAO-A, but not MAO-B, by SR 95191 was time dependent, with a 19-fold decrease in the IC50 values being observed over a 30-min incubation period (140 to 7.5 microM). At this time, the SR 95191-induced inhibition of MAO-A was not removed by repeated washings. When the reaction was started by adding the homogenate without prior preincubation with SR 95191, the inhibition of brain MAO-A was fully competitive (Ki = 68 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural Features of Human Monoamine Oxidase A Elucidated from cDNA and Peptide Sequences

Monoamine oxidase (MAO), an important enzyme for the degradation of amine neurotransmitters, has been implicated in neuropsychiatric illness. The amino acid sequence for one form of the enzyme, MAO-A, has been deduced from human cDNA clones and verified against proteolytic peptides. The covalent binding site for the flavin adenine dinucleotide (FAD) cofactor is near the C-terminal region. The presence of features characteristic of the ADP-binding fold suggests that the N-terminal region is also involved in the binding of FAD. These cDNAs should facilitate the study of the structure, function, and intracellular targeting of MAO, as well as the analysis of its expression in normal and pathological states.