Lipopolysaccharide augments expression and secretion of vascular endothelial growth factor in rat ventricular myocytes

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is highly expressed in the myocardium under various stimuli including hypoxia and ischemia. On the other hand, lipopolysaccharide (LPS) causes systemic inflammatory response syndrome (SIRS), which consists of systemic pathophysiological changes related to vascular hyperpermeability. To test the hypothesis that VEGF is one of the important mediators of SIRS, we examined effects of LPS on the VEGF expression and secretion in cultured neonatal rat ventricular myocytes. LPS (10 microg/ml) rapidly (within 1 h) augmented the levels of VEGF mRNA in these cells. Pharmacological inhibition of nucleic factor-kappaB or tyrosine kinases did not affect the LPS-induced augmentation of VEGF mRNA expression, while these treatments markedly suppressed the up-regulation of inducible nitric oxide synthase (iNOS) expression by LPS. The VEGF concentrations in the conditioned media were also significantly increased by the LPS treatment of 6 h. In conclusion, LPS augments VEGF expression and secretion in rat ventricular myocytes, suggesting that VEGF may be involved in pathogenesis of SIRS. LPS may induce VEGF mRNA through the signaling pathways that are distinct from those responsible for the iNOS induction.     

Cobalt-substituted Fe-type nitrile hydratase of Rhodococcus sp. N-771

When the genes encoding alpha and beta subunits of Fe-type nitrile hydratase (NHase) from Rhodococcus sp. N-771 were expressed in Escherichia coli in Co-supplemented medium without co-expression of the NHase activator, the NHase specifically incorporated not Fe but Co ion into the catalytic center. The produced Co-substituted enzyme exhibited rather weak NHase activity, initially. However, the activity gradually increased by the incubation with an oxidizing agent, potassium hexacyanoferrate. The oxidizing agent is likely to activate the Co-substituent by oxidizing the Co atom to a low-spin Co(3+) state and/or modification of alphaCys-112 to a cysteine-sulfinic acid. It is suggested that the NHase activator not only supports the insertion of an Fe ion into the NHase protein but also activates the enzyme via the oxidation of its iron center.     

Saccharide production from methanol by transposon 5 mutants derived from the extracellular polysaccharide-producing bacterium Methylobacillus sp. strain 12S

A CH₃OH-utilizing bacterium that has the ability to produce extracellular polysaccharide (EPS) was isolated from a soil sample, and was identified as the obligate methylotroph Methylobacillus sp. strain 12S on the basis of its 16S rDNA sequence and growth-substrate specificity. The EPS produced by strain 12S was purified and the sugar composition was analysed by GC-MS and HPLC to reveal that the EPS was a heteropolymer composed of glucosyl, galactosyl, and mannosyl residues in the molar ratio 3:1:1. In order to produce mono- and/or oligosaccharides by single-step fermentation from CH₃OH, stain 12S was mutagenized by transposon 5. Among eleven EPS-deficient mutants, three strains were found to accumulate significant amounts of reducing sugars in the media. The amounts of the reducing sugars produced by the mutants (>ca. 700 mg glucose equivalent/l) were >11–22 times higher than those produced by the wild-type strain (<ca. 60 mg glucose equivalent/l). The GC-MS analysis showed that all the mutants accumulated glucose, erythrose, threose and a disaccharide-like compound in the media. Received: 25 August 1999 / Received revision: 15 March 2000 / Accepted: 24 March 2000     

Isolation and characterization of the genes encoding a novel oxygenase component of angular dioxygenase from the gram-positive dibenzofuran-degrader Terrabacter sp. strain DBF63

A gram-positive bacterium Terrabacter sp. strain DBF63 is able to degrade dibenzofuran (DF) via initial dioxygenation by a novel angular dioxygenase. The dbfA1 and dbfA2 genes, which encode the large and small subunits of the dibenzofuran 4,4a-dioxygenase (DFDO), respectively, were isolated by a polymerase chain reaction-based method. DbfA1 and DbfA2 showed moderate homology to the large and small subunits of other ring-hydroxylating dioxygenases (less than 40%), respectively, and some motifs such as the Fe(II) binding site and the [2Fe-2S] cluster ligands were conserved in DbfA1. DFDO activity was confirmed in Escherichia coli cells containing the cloned dbfA1 and dbfA2 genes with the complementation of nonspecific ferredoxin and ferredoxin reductase component of E. coli. Under this condition, these cells exhibited angular dioxygenation of DF and dibenzo-p-dioxin, and monooxygenation of fluorene, but not angular dioxygenation of carbazole, xanthene, and phenoxathiin. Phylogenetic analysis revealed that DbfA1 formed a branch with recently reported large subunits of polycyclic aromatic hydrocarbon (PAH) dioxygenase from gram-positive bacteria but did not cluster with that of other angular dioxygenases, i.e., DxnA1 from Sphingomonas sp. strain RW1 [Armengaud, J., Happe, B., and Timmis, K. N. J. Bacteriol. 180, 3954-3966, 1998] and CarAa from Pseudomonas sp. strain CA10 [Sato, S., Nam, J.-W., Kasuga, K., Nojiri, H., Yamane, H., and Omori, T. J. Bacteriol. 179, 4850-4858, 1997].     

Genetic characterization and evolutionary implications of a car gene cluster in the carbazole degrader Pseudomonas sp. strain CA10

The nucleotide sequences of the 27,939-bp-long upstream and 9,448-bp-long downstream regions of the carAaAaBaBbCAc(ORF7)Ad genes of carbazole-degrading Pseudomonas sp. strain CA10 were determined. Thirty-two open reading frames (ORFs) were identified, and the car gene cluster was consequently revealed to consist of 10 genes (carAaAaBaBbCAcAdDFE) encoding the enzymes for the three-step conversion of carbazole to anthranilate and the degradation of 2-hydroxypenta-2,4-dienoate. The high identities (68 to 83%) with the enzymes involved in 3-(3-hydroxyphenyl)propionic acid degradation were observed only for CarFE. This observation, together with the fact that two ORFs are inserted between carD and carFE, makes it quite likely that the carFE genes were recruited from another locus. In the 21-kb region upstream from carAa, aromatic-ring-hydroxylating dioxygenase genes (ORF26, ORF27, and ORF28) were found. Inductive expression in carbazole-grown cells and the results of homology searching indicate that these genes encode the anthranilate 1,2-dioxygenase involved in carbazole degradation. Therefore, these ORFs were designated antABC. Four homologous insertion sequences, IS5car1 to IS5car4, were identified in the neighboring regions of car and ant genes. IS5car2 and IS5car3 constituted the putative composite transposon containing antABC. One-ended transposition of IS5car2 together with the 5' portion of antA into the region immediately upstream of carAa had resulted in the formation of IS5car1 and ORF9. In addition to the insertion sequence-dependent recombination, gene duplications and presumed gene fusion were observed. In conclusion, through the above gene rearrangement, the novel genetic structure of the car gene cluster has been constructed. In addition, it was also revealed that the car and ant gene clusters are located on the megaplasmid pCAR1.     

A Toxic Protein Causative of Silver-leaf Disease Symptoms on Apple Trees

The electrophoretically homogeneous protein obtained from the culture filtrate of Stereum purpureum causes silver-leaf symptoms when injected into apple trees at a dosage of about 25 μg per tree. This preparation has a molecular weight of 32,000, and shows not only an endo-PG (polygalacturonase) activity, but also macerating action in several plant tissues. Toxic effects on apple trees qualitatively make parallel alteration in the endo-PG activity. From the chemical properties tested, it is suggested that the preparation differs from phytolysin reported as a toxic material inductive to silver-leaf symptoms on plum trees.     

Acetoacetate Decarboxylase and a Peptide with Similar Activity Produced by Bacillus polymyxa A-57

Acetoacetate decarboxylase is a valuable tool for clinical analysis of ketone bodies in human plasma. After screening many microorganisms, we found Bacillus polymyxa A-57 to be a new enzyme source with a good yield of acetoacetate decarboxylase. After purifying the intracellular enzyme by three-stage column chromatography, we identified it as a single protein by SDS-polyacrylamide gel electrophoresis. The enzyme had a molecular weight of approximately 280,000 and consisted of 10 (±2) identical subunits. It had an optimum pH of 5.9 and was stable up to about 60°C for 30min. The apparent Km and V_max values for lithium acetoacetate were 0.94 mm and 296 μmiol/min/mg, respectively. In addition, the decarboxylase activity was found in the broth. After purification, we found that it was due to an active peptide which we named A-57-9, which we identified as the antibiotic polymyxin M₁. However, the V_max value (0.25 μmol/min/mg) of the peptide was very much lower and the Km value (400 mm) was higher than those of real acetoacetate decarboxylase.     

Bolevenine, a toxic protein from the Japanese toadstool Boletus venenatus

A toxic protein, called bolevenine, was isolated from the toxic mushroom Boletus venenatus based on its lethal effects on mice. On SDS-PAGE, in either the presence or absence of 2-mercaptoethanol, this protein showed a single band of approximately 12 kDa. In contrast, based on gel filtration and MALDI-TOFMS, its relative molecular mass was estimated to be approximately 30 kDa and approximately 33 kDa, respectively, indicating that the protein consists of three identical subunits. This toxin exhibited its lethal activity following injection at 10mg/kg into mice. The N-terminal amino acid sequence was determined up to 18, and found to be similar to the previously reported bolesatine, a toxic compound isolated from Boletus satanas.