Effects of forskolin analogs, phosphodiesterase inhibitors and 8-bromo cyclic AMP on plasma exudations induced with bradykinin and prostaglandin E1 in rat skin

The effects of forskolin analogs, phosphodiesterase inhibitors and 8-bromo cyclic AMP on plasma exudations induced with bradykinin and prostaglandin E1 in rat skin were investigated using [125I]bovine serum albumin (125I-BSA). Forskolin, forskolin 7-ethyl carbonate and 7-desacetylforskolin, which are potent activators of adenylate cyclase, greatly potentiated the bradykinin-induced plasma exudation and inhibited the prostaglandin E1-induced response. On the other hand, 14,15-dihydroforskolin and 1,9-dideoxyforskolin, which are weak or inactive as activators of adenylate cyclase, did not have any significant effect on bradykinin and prostaglandin E1-induced plasma exudations. The phosphodiesterase inhibitors, ZK 62711, dipyridamole, HL 725, and 3-isobutyl-1-methylxanthine potentiated the bradykinin-induced plasma exudation and inhibited the prostaglandin E1-induced response. Papaverine had biphasic effects on the bradykinin-response and slight inhibitory effects on the prostaglandin E1-response. 8-Bromo cyclic AMP in the doses of 0.01 to 1 microgram potentiated the bradykinin-induced plasma exudation, but had no effect at doses of 10 and 100 micrograms. 8-Bromo cyclic AMP at all doses significantly inhibited the prostaglandin E1-induced response. The results suggest that the effects of forskolin and its analogs on plasma exudations induced with bradykinin and prostaglandin E1 in rat skin derive from activation of cyclic AMP-generating systems.     

Reduction of Hg2+ with reduced mammalian cytochrome c by cytochrome c oxidase purified from a mercury-resistant acidithiobacillus ferrooxidans strain, MON-1

Acidithiobacillus ferrooxidans AP19-3, ATCC 23270, and MON-1 are mercury-sensitive, moderately mercury-resistant, and highly mercury-resistant strains respectively. It is known that 2,3,5,6-tetramethyl-p-phenylendiamine (TMPD) and reduced cytochrome c are used as electron donors specific for cytochrome c oxidase. Resting cells of strain MON-1 had TMPD oxidase activity and volatilized metal mercury with TMPD as an electron donor. Cytochrome c oxidase purified from strain MON-1 reduced mercuric ions to metalic mercury with reduced mammalian cytochrome c as well as TMPD. These mercury volatilization activities with reduced cytochrome c and TMPD were completely inhibited by 1 mM NaCN. These results indicate that cytochrome c oxidase is involved in mercury reduction in A. ferrooxidans cells. The cytochrome c oxidase activities of strains AP19-3 and ATCC 23270 were completely inhibited by 1 muM and 5 muM of mercuric chloride respectively. In contrast, the activity of strain MON-1 was inhibited 33% by 5 muM, and 70% by 10 muM of mercuric chloride, suggesting that the levels of mercury resistance in A. ferrooxidans strains correspond well with the levels of mercury resistance of cytochrome c oxidase.     

Composition of myofiber types in the vocalis muscles involved in rapid closure of the glottis in Japanese macaques

Myofiber types of the medial thyroarytenoid (vocalis) muscle, lateral thyroarytenoid muscle, and cricothyroid muscle of the Japanese macaque were examined with enzyme-histochemical methods. For comparison, the semitendinosus muscle of the Japanese macaque and the thyroarytenoid (vocalis) muscle of cattle, sheep, and pig were examined with the same methods. The vocalis muscle of the Japanese macaque was composed exclusively of fast-twitch/oxidative/glycolytic (FOG) myofibers; it differed from the lateral thyroarytenoid, cricothyroid, and semitendinosus muscles of the Japanese macaque and from the vocalis muscles of the other animals, which consisted of slow-twitch/oxidative, FOG-, and fast-twitch/glycolytic myofibers or type IIC myofibers. The histochemical properties of the vocalis muscle of the Japanese macaque show that the vocalis muscle has a capacity to close the glottis rapidly and completely.     

Pronounced conversion of the metal-specific activity of superoxide dismutase from Porphyromonas gingivalis by the mutation of a single amino acid (Gly155Thr) located apart from the active site

Glycine 155, which is located approximately 10 A from the active metal sites, is mostly conserved in aligned amino acid sequences of manganese-specific superoxide dismutases (Mn-SODs) and cambialistic SOD (showing the same activity with Fe and Mn) from Porphyromonas gingivalis, but is substituted for threonine in most Fe-SODs. Since Thr155 is located between Trp123 and Trp125, and Trp123 is one member of the metal-surrounding aromatic amino acids, there is a possibility that the conversion of this amino acid may cause a conversion of the metal-specific activity of cambialistic P. gingivalis SOD. To clarify this possibility, we have prepared a mutant of the P. gingivalis SOD with conversion of Gly155 to Thr. The ratios of the specific activities of Fe- to Mn-reconstituted enzyme, which are measured by the xanthine oxidase/cytochrome c method, increased from 0.6 in the wild-type to 11.2 in the mutant SODs, indicating the conversion of the metal-specific activity of the enzyme from a cambialistic type to an Fe-specific type. The visible absorption spectra of the Fe- and Mn-reconstituted mutant SODs closely resembled those of Fe-specific SOD. Furthermore, the EPR spectra of the Fe- and Mn-reconstituted mutant SODs also closely resembled those of Fe-specific SOD. Three-dimensional structures of the Fe-reconstituted wild-type SOD and Mn-reconstituted mutant SOD have been determined at 1.6 A resolution. Both structures have identical conformations, orientations of residues involved in metal binding, and hydrogen bond networks, while the side chain of Trp123 is moved further toward the metal-binding site than in wild-type SOD. A possible contribution of the structural differences to the conversion of the metal-specific activity through rearrangement of the hydrogen bond network among Trp123, Gln70, Tyr35, and the metal-coordinated solvent is discussed.     

Volatilization of mercury by an iron oxidation enzyme system in a highly mercury-resistant Acidithiobacillus ferrooxidans strain MON-1

A highly mercury-resistant strain Acidithiobacillus ferrooxidans MON-1, was isolated from a culture of a moderately mercury-resistant strain, A. ferrooxidans SUG 2-2 (previously described as Thiobacillus ferrooxidans SUG 2-2), by successive cultivation and isolation of the latter strain in a Fe2+ medium with increased amounts of Hg2+ from 6 microM to 20 microM. The original stain SUG 2-2 grew in a Fe2+ medium containing 6 microM Hg2+ with a lag time of 22 days, but could not grow in a Fe2+ medium containing 10 microM Hg2+. In contrast, strain MON-1 could grow in a Fe2+ medium containing 20 microM Hg2+ with a lag time of 2 days and the ability of strain MON-1 to grow rapidly in a Fe2+ medium containing 20 microM Hg2+ was maintained stably after the strain was cultured many times in a Fe2+ medium without Hg2+. A similar level of NADPH-dependent mercury reductase activity was observed in cell extracts from strains SUG 2-2 and MON-1. By contrast, the amounts of mercury volatilized for 3 h from the reaction mixture containing 7 microM Hg2+ using a Fe(2+)-dependent mercury volatilization enzyme system were 5.6 nmol for SUG 2-2 and 67.5 nmol for MON-1, respectively, indicating that a marked increase of Fe(2+)-dependent mercury volatilization activity conferred on strain MON-1 the ability to grow rapidly in a Fe2+ medium containing 20 microM Hg2+. Iron oxidizing activities, 2,3,5,6-tetramethyl-p-phenylenediamine (TMPD) oxidizing activities and cytochrome c oxidase activities of strains SUG 2-2 and MON-1 were 26.3 and 41.9 microl O2 uptake/mg/min, 15.6 and 25.0 microl O2 uptake/mg/min, and 2.1 and 6.1 mU/mg, respectively. These results indicate that among components of the iron oxidation enzyme system, especially cytochrome c oxidase activity, increased by the acquisition of further mercury resistance in strain MON-1. Mercury volatilized by the Fe(2+)-dependent mercury volatilization enzyme system of strain MON-1 was strongly inhibited by 1.0 mM sodium cyanide, but was not by 50 nM rotenone, 5 microM 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HQNO), 0.5 microM antimycin A, or 0.5 microM myxothiazol, indicating that cytochrome c oxidase plays a crucial role in mercury volatilization of strain MON-1 in the presence of Fe2+.     

Characterization of the hrpF pathogenicity peninsula of Xanthomonas oryzae pv. oryzae

The hrp gene cluster of Xanthomonas spp. contains genes for the assembly and function of a type III secretion system (TTSS). The hrpF genes reside in a region between hpaB and the right end of the hrp cluster. The region of the hrpF gene of Xanthomonas oryzae pv. oryzae is bounded by two IS elements and also contains a homolog of hpaF of X. campestris pv. vesicatoria and two newly identified genes, hpa3 and hpa4. A comparison of the hrp gene clusters of different species of Xanthomonas revealed that the hrpF region is a constant yet more variable peninsula of the hrp pathogenicity island. Mutations in hpaF, hpa3, and hpa4 had no effect on virulence, whereas hrpF mutants were severely reduced in virulence on susceptible rice cultivars. The hrpF genes from X. campestris pv. vesicatoria, X. campestris pv. campestris, and X. axonopodis pv. citri each were capable of restoring virulence to the hrpF mutant of X. oryzae pv. oryzae. Correspondingly, none of the Xanthomonas pathovars with hrpF from X. oryzae pv. oryzae elicited a hypersensitive reaction in their respective hosts. Therefore, no evidence was found for hrpF as a host-specialization factor. In contrast to the loss of Bs3-dependent reactions by hrpF mutants of X. campestris pv. vesicatoria, hrpF mutants of X. oryzae pv. oryzae with either avrXa10 or avrXa7 elicited hypersensitive reactions in rice cultivars with the corresponding R genes. A double hrpFxoo-hpa1 mutant also elicited an Xa10-dependent resistance reaction. Thus, loss of hrpF, hpal, or both may reduce delivery or effectiveness of type III effectors. However, the mutations did not completely prevent the delivery of effectors from X. oryzae pv. oryzae into the host cells.     

Purification and biochemical characterization of the F1-ATPase from Acidithiobacillus ferrooxidans NASF-1 and analysis of the atp operon

ATPase was purified 51-fold from a chemoautotrophic, obligately acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans NASF-1. The purified ATPase showed the typical subunit pattern of the F1-ATPase on a polyacrylamide gel containing sodium dodecyl sulfate, with 5 subunits of apparent molecular masses of 55, 50, 33, 20, and 18 kDa. The enzyme hydrolyzed ATP, GTP, and ITP, but neither UTP nor ADP. The K(m) value for ATP was 1.8 mM. ATPase activity was optimum at pH 8.5 at 45 degrees C, and was activated by sulfite. Azide strongly inhibited the enzyme activity, whereas the enzyme was relatively resistant to vanadate, nitrate, and N,N'-dicyclohexylcarbodiimide. The genes encoding the subunits for the F1F(O)-ATPase from A. ferrooxidans NASF-1 were cloned as three overlapping fragments by PCR cloning and sequenced. The molecular masses of the alpha, beta, gamma, delta, and epsilon subunits of the F1 portion were deduced from the amino acid sequences to be 55.5, 50.5, 33.1, 19.2, and 15.1 kDa, respectively.     

Identification of Thiobacillus ferrooxidans strains based on restriction fragment length polymorphism analysis of 16S rDNA.

The 16S rDNA sequences from ten strains of Thiobacillus ferrooxidans were amplified by PCR. The products were compared by performing restriction fragment length polymorphism (RFLP) analysis with restriction endonucleases Alu I, Hap II, Hha I, and Hae III. The RFLP patterns revealed that T. ferrooxidans could be distinguished from other iron- or sulphur-oxidizing bacteria such as T. thiooxidans NB1-3, T. caldus GO-1, Leptospirillum ferrooxidans and the marine iron-oxidizing bacterium strain KU2-11. The RFLP patterns obtained with Alu I, Hap II, and Hae III were the same for nine strains of T. ferrooxidans except for strain ATCC 13661. The RFLP patterns for strains NASF-1 and ATCC 13661 with Hha I were distinct from those for other T. ferrooxidans strains. The 16S rDNA sequence of T. ferrooxidans NASF-1 possessed an additional restriction site for Hha I. These results show that iron-oxidizing bacteria isolated from natural environments were rapidly identified as T. ferrooxidans by the method combining RFLP analysis with physiological analysis.     

Aerobic Hydrogen Accumulation by a Nitrogen-Fixing Cyanobacterium, Anabaena sp

Hydrogen evolution by a nitrogen-fixing cyanobacterium, Anabaena sp. strain N-7363, was tested in order to develop a water biophotolysis system under aerobic conditions. A culture of the strain supplemented with carbon dioxide under an air atmosphere evolved hydrogen and oxygen gas, which reached final concentrations of 9.7 and 69.8%, respectively, after 12 days of incubation. Hydrogen uptake activity was not observed during incubation, and nitrogenase was thought to be the sole enzyme responsible for the hydrogen evolution.