Characterization of PBZ1, a probenazole-inducible gene, in suspension-cultured rice cells

Probenazole (PBZ) induces non-race specific resistance in rice plants against rice blast fungus and PBZ1 was identified as a PBZ-inducible gene from rice. The induction of PBZ1 expression in suspension-cultured rice cells was investigated. Northern blot analysis indicated that PBZ1 was induced by PBZ in a dose-dependent manner. Enzyme-linked immunosorbent assay (ELISA) showed a dose and time-dependent accumulation of PBZ1 protein. Both mRNA and protein analysis showed that PBZ1 was not induced by salicylic acid or an active metabolite, 1,2-benzisothiazole-1,1-dioxide.     

Molecular characterization of L-amino acid oxidase from Agkistrodon halys blomhoffii with special reference to platelet aggregation

L-Amino acid oxidase (LAO, EC 1.4.3.2) is widely distributed in snake venom, and induces apoptosis in vascular endothelial cells, causing prolonged bleeding from vessel walls at bite sites. The effect of snake venom LAOs on platelet function is controversial. Further, we have little information on their structural characterization. We purified M (mamushi)-LAO, a single-chain glycoprotein with a molecular mass of 60 kDa and a pI of 4.9, from Agkistrodon halys blomhoffii (Japanese mamushi) venom, and determined the N-terminal and several internal amino acid sequences of this enzyme. Molecular cloning based on these data was conducted to elucidate its full-length cDNA structure (2192 nucleotides), which includes a putative 18 amino acid residue signal peptide and a 504 residue mature subunit. The predicted M-LAO translation product shares 87.3% identity with that of Crotalus adamanteus (Southeastern diamondback rattlesnake) LAO. M-LAO, up to a final concentration of 2.6 microM, inhibited both agonist- and shear stress-induced platelet aggregation (SIPA) dose-dependently. In agonist-induced platelet aggregation, M-LAO predominantly inhibited the second aggregation, but with a marginal inhibition of the first. In SIPA, the inhibition was more dramatic under low-shear stress than high-shear stress, and was enhanced by the presence of L-leucine, a substrate of this enzyme. Catalase, a H2O2 scavenger, totally quenched such enhancement. These results suggest that M-LAO inhibits the interaction between activated platelet integrin alphaIIb/beta3 and fibrinogen through the continuous generation of H2O2, and may contribute to prolonged bleeding from the vessels at snake bite sites.     

Characterization of a major form of human isatin reductase and the reduced metabolite.

Isatin, an endogenous indole, has been shown to inhibit monoamine oxidase, and exhibit various pharmacological actions. However, the metabolism of isatin in humans remains unknown. We have found high isatin reductase activity in the 105,000 g supernatants of human liver and kidney homogenates, and have purified and characterized a major form of the enzyme in the two tissues. The hepatic and renal enzymes showed the same properties, including an M(r) of 31 kDa, substrate specificity for carbonyl compounds and inhibitor sensitivity, which were also identical to those of recombinant human carbonyl reductase. The identity of the isatin reductase with carbonyl reductase was immunologically demonstrated with an antibody against the recombinant carbonyl reductase. About 90% of the soluble isatin reductase activity in the liver and kidney was immunoprecipitated by the antibody. The Km (10 microm) and k(cat)/K(m) (1.7 s(-1) x microm(-1)) values for isatin at pH 7.0 were comparable to those for phenanthrenequinone, the best xenobiotic substrate of carbonyl reductase. The reduced product of isatin was chemically identified with 3-hydroxy-2-oxoindole, which is also excreted in human urine. The inhibitory potency of the reduced product for monoamine oxidase A and B was significantly lower than that of isatin. The results indicate that the novel metabolic pathway of isatin in humans is mediated mainly by carbonyl reductase, which may play a critical role in controlling the biological activity of isatin.     

Cloning and overproduction of the rpoZ gene encoding an RNA polymerase omega subunit from a deep-sea piezophilic Shewanella violacea strain DSS12.

We have cloned the rpoZ gene, encoding RNA polymerase omega protein, by PCR approach from the deep-sea piezophilic and psychrophilic bacterium, Shewanella violacea strain DSS12. The cloned gene, 285bp in length, was found to encode a protein consisting of 94 amino acid residues with a molecular mass of 10,327 Da. Significant homology was evident comparing the RpoZ protein of S. violacea with that of Shewanella oneidensis (69% identity), Vibrio cholerae (65% identity), Escherichia coli K-12 (64% identity) and Haemophilus influenzae (61% identity). From the Northern blot analysis, S. violacea rpoZ gene was expressed constitutively under pressure conditions of 0.1, 30 and 50MPa. We constructed expression plasmid to overproduce the RpoZ protein and transformed into E. coli JM109 as a host of overproduction. Upon induction, the recombinant protein encoded by plasmid pQrpoZ was overexpressed and purified using Ni2+ affinity column.     

Reconstitution and characterization of NtrC protein in a deep-sea piezophilic bacterium, Shewanella violacea strain DSS12

NtrC protein of piezophilic Shewanella violacea was overexpressed and purified, to confirm the protein-DNA interaction. An electrophoretic mobility shift assay demonstrated that the NtrC recognizes the sequence for NtrC binding within the region upstream of the glnA operon. Western blot analysis also showed that the NtrC is expressed at a higher level under high-pressure conditions than under atmospheric pressure conditions.     

Gene expression profile during the life cycle of the urochordate Ciona intestinalis

Recent whole-genome studies and in-depth expressed sequence tag (EST) analyses have identified most of the developmentally relevant genes in the urochordate, Ciona intestinalis. In this study, we made use of a large-scale oligo-DNA microarray to further investigate and identify genes with specific or correlated expression profiles, and we report global gene expression profiles for about 66% of all the C. intestinalis genes that are expressed during its life cycle. We succeeded in categorizing the data set into 5 large clusters and 49 sub-clusters based on the expression profile of each gene. This revealed the higher order of gene expression profiles during the developmental and aging stages. Furthermore, a combined analysis of microarray data with the EST database revealed the gene groups that were expressed at a specific stage or in a specific organ of the adult. This study provides insights into the complex structure of ascidian gene expression, identifies co-expressed gene groups and marker genes and makes predictions for the biological roles of many uncharacterized genes. This large-scale oligo-DNA microarray for C. intestinalis should facilitate the understanding of global gene expression and gene networks during the development and aging of a basal chordate.     

Generation of reactive oxygen species during mouse hepatic microsomal metabolism of cannabidiol and cannabidiol hydroxy-quinone

We investigated whether cannabidiol (CBD) and cannabidiol hydroxy-quinone (CBDHQ) generate reactive oxygen species (ROS) during metabolism with mouse hepatic microsomes. CBD and CBDHQ (91.5 microM) significantly suppressed lipid peroxidation in the mouse hepatic microsomes. CBDHQ also significantly decreased NADH-cytochrome b5 reductase (fp1) activity by 25% of the control activity in the hepatic microsomes, and tended to increase NADPH-cytochrome c (P450) reductase (fp2) activity. CBDHQ also significantly inhibited superoxide dismutase and catalase activities in mouse hepatic 105,000 xg supernatant. Moreover, CBDHQ significantly increased glutathione reductase activity and significantly inhibited NAD(P)H-quinone reductase activity. CBD exhibited similar effects on these enzymes, except that cannabinoid significantly inhibited glutathione reductase activity in mouse hepatic 105,000 xg supernatant. These results suggest that CBDHQ is easily converted to the semiquinone form rather than the hydroquinone form. It was also suggested that CBDHQ and CBD were capable of generating ROS as superoxide anion radicals during their metabolism with mouse hepatic microsomes or with purified fp2 by electron spin resonance spin trapping methods with 5,5-dimethyl-1-pyrroline-N-oxide. The present results suggest that CBDHQ formed during hepatic microsomal metabolism of CBD is capable of generating ROS and inducing cell toxicity.     

Recycle Use of Sphingomonas sp. CDH-7 Cells for Continuous Degradation of Carbazole in the Presence of MgCl2

Carbazole (CA) is a heterocyclic nitrogen compound contained in the crude petroleum oil and recalcitrant to removal through the refinery processes. For development of the efficient CA-degradation bioprocess, conditions for the recycle use of Sphingomonas sp. CDH-7 resting cells were examined. When the resting cells (O.D.₆₆₀ 3.3) were shaken in 50 mM K₂HPO₄-KH₂PO₄ buffer (pH 7.0) containing CA 1000 mg/L, CA 880 mg/L was degraded within 3 h, but thereafter the activity decreased markedly. However, the activity was found to be restored to the initial level after the shaking treatment for 3 h in CA-free medium solution or in the buffer containing 20 mM MgCl₂. Although the CA-degradation activity of CDH-7 resting cells was lost after 3 h of shaking in the buffer containing 100 mM EDTA, it was restored through the shaking treatment for 3 h in the buffer containing 20 mM MgCl₂. When CA was periodically added eight times at a concentration of 100 mg/L (0.599 mM) to the reaction mixture containing the resting cells, CA 778 mg/L (4.66 mM) was continuously degraded within 35 h by the recycle use of resting cells, with the restoration treatment after each CA-degradation reaction by the resting cells. Received: 28 June 2001 / Accepted: 30 July 2001     

Isolation and chemical characterization of two structurally and functionally distinct forms of botrocetin, the platelet coagglutinin isolated from the venom of Bothrops jararaca

Two distinct forms of botrocetin, the von Willebrand factor (vWF)-dependent platelet coagglutinin isolated from the venom of the snake Bothrops jararaca, were purified and characterized structurally and functionally. The apparent molecular mass of the one-chain botrocetin was 28 kDa before and 32 kDa after reduction of disulfide bonds, while that of the two-chain botrocetin was 27 kDa before and 15/14.5 kDa after reduction. Amino acid composition of the two species revealed a similar high content of potentially acidic residues (greater than 60 Asx and Glx residues/molecule) but significant differences in the content of Cys and Phe residues. The NH2-terminal sequence of the one-chain botrocetin was Ile-Ile/Val-Ser-Pro-Pro-Val-Cys-Gly-Asn-Glu-. Two constituent polypeptides of the two-chain botrocetin showed similar but different NH2-terminal sequences, distinct from that of the one-chain species: (alpha) Asp-Cys-Pro-Ser-Gly-Trp-Ser-Ser-Tyr-Glu- and (beta) Asp-Cys-Pro-Pro-Asp-Trp-Ser-Ser-Tyr-Glu-. The carbohydrate content of both species was less than 2% of the total mass, and the pI was 4.0-4.1 for the one-chain species, and 4.6, 5.3-5.4, and 7.7-7.8 for the two-chain species. No free sulfhydryl group was detected in each species. Both types of botrocetin were resistant to proteolysis at neutral pH. Incubation of 125I-labeled one-chain botrocetin with the crude venom solution resulted in no detectable structural change. On a weight basis, the two-chain botrocetin was 34 times more active than the one-chain form in promoting vWF binding to platelets.(ABSTRACT TRUNCATED AT 250 WORDS)