Enzymic arrangement and allosteric regulation of the aromatic amino acid pathway in Neisseria gonorrhoeae

The pathway construction and allosteric regulation of phenylalanine and tyrosine biosynthesis was examined in Neisseria gonorrhoeae. A single 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase enzyme sensitive to feedback inhibition by l-phenylalanine was found. Chorismate mutase and prephenate dehydratase appear to co-exist as catalytic components of a bifunctional enzyme, known to be present in related genera. The latter enzyme activities were both feedback inhibited by l-phenylalanine. Prephenate dehydratase was strongly activated by l-tyrosine. NAD⁺-linked prephenate dehydrogenase and arogenate dehydrogenase activities coeluted following ion-exchange chromatography, suggesting their identity as catalytic properties of a single broad-specificity cyclohexadienyl dehydrogenase. Each dehydrogenase activity was inhibited by 4-hydroxyphenylpyruvate, but not by l-tyrosine. Two aromatic aminotransferases were resolved, one preferring the l-phenylalanine:2-ketoglutarate substrate combination and the other preferring the l-tyrosine: 2-ketoglutarate substrate combination. Each aminotransferase was also able to transaminate prephenate. The overall picture of regulation is one in which l-tyrosine modulates l-phenylalanine synthesis via activation of prephenate dehydratase. l-Phenylalanine in turn regulates early-pathway flow through inhibition of DAHP synthase. The recent phylogenetic positioning of N. gonorrhoeae makes it a key reference organism for emerging interpretations about aromatic-pathway evolution.     

The periplasmic nitrate reductase of Rhodobacter capsulatus; purification,characterisation and distinction from a single reductase for trimethylamine-N-oxide,dimethylsulphoxide and chlorate

The periplasmic dissimilatory nitrate reductase from Rhodobacter capsulatus N22DNAR⁺ has been purified. It comprises a single type of polypeptide chain with subunit molecular weight 90,000 and does not contain heme. Chlorate is not an alternative substrate. A molybdenum cofactor, of the pterin type found in both nitrate reductases and molybdoenzymes from various sources, is present in nitrate reductase from R. capsulatus at an approximate stoichiometry of 1 molecule per polypeptide chain. This is the first report of the occurrence of the cofactor in a periplasmic enzyme. Trimethylamine-N-oxide reductase activity was fractionated by ion exchange chromatography of periplasmic proteins. The fractionated material was active towards dimethylsulphoxide, chlorate and methionine sulphoxide, but not nitrate. A catalytic polypeptide of molecular weight 46,000 was identified by staining for trimethylamine-N-oxide reductase activity after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The same polypeptide also stained for dimethylsulphoxide reductase activity which indicates that trimethylamine-N-oxide and dimethylsulphoxide share a common reductase.Abbreviations DMSO dimethylsulphoxide - LDS lithium dodecyl sulphate - MVH reduced methylviologen - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - TMAO trimethylamine-N-oxide     

Kinetic measurement of bacterial adherence to eukaryotic cells

We developed a kinetic assay using a monolayer of differentiated respiratory epithelium in culture to assess bacterial adherence. Mean residence time of bacteria in the tissue culture chamber was estimated from a model-independent (moment) analysis of the rate of bacterial washout from perfused Rose chambers. Results with this method compared favorably with visual assessment of adherence and double radiolabel method with H. influenzae. Adherence was assessed with low inoculae of H. influenzae, P. cepacia and P. aeruginosa avoiding cytotoxic effects seen when large inoculae are added to eukaryotic cells. This method will provide a means of assessing adherence of pathogenic respiratory bacteria to their cellular target at low inoculae.     

Induction of hepatic drug metabolizing enzymes by DL-methionine in rats

A single intraperitoneal injection of DL-methionine (500 mg/kg body wt.) to adult male Wistar rats was shown to significantly induce all the components of the hepatic microsomal mixed function oxidase system such as NADPH cytochrome C reductase activity, cytochromes P-450 and b₅, as well as activities of drug metabolizing enzymes such as aminopyrine demethylase and uridine 5′ -diphosphate-glucuronosyltransferase. Combined administration of nicotinamide (250 mg/kg body wt.) and DL-methionine (500 mg/kg body wt.) was shown to bring about an additional increase (25-30%) in the activities of these enzymes as compared to their induction on independent administration of the two endobiotics. In rats bearing Yoshida sarcoma (ascites) tumour as well as in normal rats injected with serum from tumour bearing animals, the decreased activities of hepatic mixed function oxidases could be restored to their normal levels by administration of DL-methionine (500 mg/kg body wt.) to these rats. Whereas actinomycin D (1 mg/kg body wt.) had no effect on the increased incorporation of [¹⁴C] labelled leucine into microsomal proteins following administration of nicotinamide, the enhanced incorporation of the label following DL-methionine administration was completely inhibited by the same dose of actinomycin D. Administration of cycloheximide (0·5 mg/kg body wt.) to rats could completely inhibit the increased incorporation of [¹⁴C] leucine into hepatic microsomal proteins following independent administration of nicotinamide and DL-methionine. Similar inhibitory pattern with actinomycin D and cycloheximide was also demonstrated in case of induction of NADPH cytochromeC reductase activity by both these endobiotics.     

Antagonistic potential of Gliocladium virens and Trichoderma longibrachiatum to phytopathogenic fungi

Three isolates of Gliocladium virens (G1, G2 and G3) and two of Trichoderma longibrachiatum (T1 and T2) were screened against isolates of three soilborne plant pathogens namely Rhizoctonia solani, Sclerotium rolfsii and Pythium aphanidermatum. G. virens exhibited stronger hyperparasitism and wider biological spectrum than T. longibrachiatum. Further, similarities as well as variation was observed in the ability of the various isolates to invade the test pathogens in dual culture. For the hyperparasites, acidic pH range (5.0 to 5.5) favoured both growth and spore germination. The hyperparasites made direct contact with the pathogens followed by varied modes of attack invariably leading to cell disruption. Antagonists, G1 and G3 revealed strong antibiosis while T2 showed moderate effect. All the isolates produced enhanced levels of lytic enzymes adaptively and there were marked differences among them. However, no correlation was observed between these attributes and the hyperparasitic potential of the various isolates in dual culture. The relevance and the role of enzymes and toxic metabolite(s) in the antagonism of G. virens and T. longibrachiatum to these pathogens are discussed.     

Degradation of 2-chloroethanol by wild type and mutants of Pseudomonas putida US2

A strain of Pseudomonas putida was isolated that was able to degrade 2-chloroethanol. The degradation proceeded via 2-chloroacetaldehyde and chloroacetate to glycolate. In crude extracts the enzymes for this degradation pathway could be detected. All enzymes proved to be inducible. The dehalogenase that catalyzed the dehalogenation of chloroacetate to glycolate was further characterized. It consisted of a single polypeptide chain with a molecular mass of 28 kDa. After induction the dehalogenase was expressed at a high level. In a mutant resistant to high concentrations of 2-chloroethanol the dehalogenase was no longer expressed. The mechanism of resistance seemed to be due to the inability to convert chloroacetate and export of this compound out of the cell.Non-standard abbreviations CEO 2-chloroethanol - DCPIP 2,6-dichlorophenolindophenol - FPLC fast protein liquid chromatography - PAGE polyacrylamide gelelectrophoresis - PES phenazine ethosulfate - PMS phenazine methosulfate - PQQ pyrroloquinoline quinone     

Intercellular structure in a many-celled magnetotactic prokaryote

A many-called magnetotactic prokaryote obtained from brackish water was observed to possess intercellular connections at points of contact between the outer membranes of constituent cells. Each aggregate organism consisted of 10 to 30 individual Gram-negative cells containing material with the appearance of poly--hydroxybutyrate and magnetosomes of unusual arrangement, structure and composition. The aggregate, which possessed prokaryotic-type flagella arranged at the outwards surfaces of each cell, showed motility indicative of co-ordination between individual component cells. These results suggest that this organism could be a multicellular prokaryote.     

Degradation of 3-chlorobiphenyl by in vivo constructed hybrid pseudomonads

Abstract 3-Chlorobiphenyl-degrading bacteria were obtained from the mating between Pseudomonas putida strain BN10 and Pseudomonas sp. strain B13. Strains such as BN210 resulted from the transfer of the genes coding the enzyme sequence for the degradation of chlorocatechols from B13 into BN10, whereas B13 derivatives such as B131 have acquired the biphenyl degradation sequence from BN10. During growth of the hybrid strains on 3-chlorobiphenyl 90% chloride was released. Activities of phenylcatechol 2,3-dioxygenase, benzoate dioxygenase, catechol 1,2-dioxygenase, chloromuconate cyloisomerase and 4-carboxymethyl-enebut-2-en-4-olide hydrolase were found in 3-chlorobiphenyl-grown cells. The hybrid strains were found to convert some congeners of the Aroclor 1221 mixture such as mono- and dichloro-substituted biphenyls.     

Soils contain two different activities for oxidation of hydrogen

Abstract Hydrogen oxidation rates were measured in a neutral compost soil and an acidic sandy loam at H₂ mixing ratios of 0.01 to 5000 ppmv. The kinetics were biphasic showing two different K _m values for H₂, one at about 10–40 nM dissolved H₂, the other at about 1.2–1.4 μM H₂. The low- K _m activity was less sensitive to chloroform fumigation than the high- K _m activity. If sterile soil was amended with Paracoccus denitrificans or a H₂-oxidizing strain isolated from compost soil, it exhibited only a high- K _m (0.7–0.9 μM) activity. It also failed to utilize H₂ mixing ratios below a threshold of 1.6–3.0 ppmv H₂ (160–300 mPa). A similar result was obtained when fresh soil samples were suspended in water, and H₂ oxidation was determined from the decrease of dissolved H₂. However, H₂ was again utilized to mixing ratios lower than 0.05 ppmv, if the supernatant of the soil suspension or the settled soil particles were dried onto sterile soil or purified quarz sand. Obviously, soils contain two different activities for oxidation of H₂: (1) a high- K _m, high-threshold activity which apparently is due to aerobic H₂-oxidizing bacteria, and (2) a low- K _m, low-threshold activity whose origin is unknown but presumably is due to soil enzymes.     

大鼠隔—海马通路损伤对海马内递质含量及酶活力的影响

单侧切断大鼠海马缴和部分穹窿可使海马部分去神经。损伤后7d,海马内胆碱能系统中乙酰胆碱(ACh)含量下降72.5%,胆碱乙酰基转移酶(ChAT)活力下降45.7%,胆碱酯酶活力下降52.2%,在单胺能系统中,去甲肾上腺素(NA)含量下降16.3%,多巴胺(DA)含量下降31.3%,5-羟色胺含量下降30.3%。在损伤过程中,海马内氨基酸含量没有改变。实验结果表明,海马缴和穹窿是脑内胆碱能和单胺能传入神经到达海马靶区的部分共同通路。