Toshiaki Nikai

Isolation and biochemical characterization of hemorrhagic toxin f from the venom of Crotalus atrox (Western Diamondback Rattlesnake)

Hemorrhagic toxin f (HT-f) was isolated from Crotalus atrox (Western Diamondback Rattlesnake) venom by a five-step purification procedure. Homogeneity was established by the formation of a single band in acrylamide gel electrophoresis, isoelectric focusing, and sodium dodecyl sulfate (SDS)-electrophoresis. HT-f has a molecular weight of 64,000 and contains 572 amino acid residues. It contains 1 mol of zinc per mol of protein. Zinc is essential for both hemorrhagic and proteolytic activities. HT-f possesses proteolytic activity hydrolyzing the Val-Asn, Gln-His, Leu-Cys, His-Leu, Ala-Leu, and Tyr-Leu bonds of oxidized insulin B chain. HT-f did not coagulate fibrinogen to fibrin, yet it did hydrolyze the gamma chain of fibrinogen without affecting either the A alpha or B beta chains. This is the first time that a hemorrhagic toxin was shown to have fibrinogenase activity. HT-f was shown to differ immunologically from other hemorrhagic toxins such as HT-a and HT-c. HT-f also possesses lethal toxicity. When zinc was removed the apo-HT-f lost its lethal toxicity. HT-f produced not only local hemorrhage in the skin and muscle, but also produced systemic hemorrhage in internal organs such as the intestine, kidney, lung, heart, and liver.

Role of nitric oxide and cyclic GMP in the dizocilpine-induced impairment of spontaneous alternation behavior in mice.

The activation of N-methyl-D-aspartate receptors induces the synthesis of nitric oxide, which activates soluble guanylate cyclase and leads to the formation of cyclic GMP in the brain. The inhibition of nitric oxide production, as well as the blockade of N-methyl-D-aspartate receptors, has been reported to prevent the induction of hippocampal long-term potentiation and learning and memory formation in vivo, although the effects of inhibitors of nitric oxide synthase are still controversial. We investigated the putative role of nitric oxide and cyclic GMP in dizocilpine-induced memory impairment in mice. The nitric oxide synthase inhibitors, NG-nitro-L-arginine methyl ester and 7-nitro indazole, as well as dizocilpine, a non-competitive N-methyl-D-aspartate receptor antagonist, dose-dependently impaired spatial working memory in mice, assessed by their spontaneous alternation behavior in a Y-maze. The inhibitory effects of both NG-nitro-L-arginine methyl ester and dizocilpine on their behavior were completely reversed by 8-bromo-cyclic GMP. Cyclic GMP levels in the cerebellum were reduced by treatment with dizocilpine. NG-Nitro-L-arginine methyl ester and 7-nitro indazole reduced cyclic GMP levels in the cerebral cortex/hippocampus and cerebellum, and the suppressive effect of NG-nitro-L-arginine methyl ester on cyclic GMP levels in the cerebral cortex/hippocampus was reversed by co-treatment with L-arginine. Cyclic AMP levels in the brain were not affected by treatment with either dizocilpine, NG-nitro-L-arginine methyl ester, or 7-nitro indazole. Neither NG-nitro-L-arginine methyl ester nor L-arginine had any effect on monoamine and acetylcholine metabolism in the brain. These results suggest that the reduction in nitric oxide/cyclic GMP production in the brain may be responsible for dizocilpine-induced impairment of spontaneous alternation behavior in a Y-maze.

Comparison of a Variable-Number Tandem-Repeat (VNTR) Method for Typing Mycobacterium avium with Mycobacterial Interspersed Repetitive-Unit-VNTR and IS1245 Restriction Fragment Length Polymorphism Typing

ABSTRACT Mycobacterium avium complex (MAC) infections are increasing annually in various countries, including Japan, but the route of transmission and pathophysiology of the infection remain unclear. Currently, a variable-number tandem-repeat (VNTR) typing method using the Mycobacterium avium tandem repeat (MATR) loci (MATR-VNTR) is employed in Japan for epidemiological studies using clinical isolates of M. avium. In this study, the usefulness of this MATR-VNTR typing method was compared with that of the IS1245-restriction fragment length polymorphism (IS1245-RFLP) typing method and a mycobacterial interspersed repetitive-unit (MIRU)-VNTR typing method reported previously (V. C. Thibault, M. Grayon, M. L. Boschiroli, C. Hubbans, P. Overduin, K. Stevenson, M. C. Gutierrez, P. Supply, and F. Biet, J. Clin. Microbiol. 45:2404-2410, 2007). Seventy clinical isolates identified as M. avium from human immunodeficiency virus-negative patients with MAC infections were used. MATR-VNTR typing using 15 loci distinguished 56 patterns of different allele profiles, yielding a Hunter-Gaston discriminatory index (HGDI) of 0.990. However, IS1245-RFLP and MIRU-VNTR typing yielded HGDIs of 0.960 and 0.949, respectively, indicating that MATR-VNTR has an excellent discriminatory power compared with MIRU-VNTR and IS1245-RFLP typing. Moreover, concomitant use of the MATR-VNTR method and IS1245-RFLP typing increased the HGDI to 0.999. MATR-VNTR typing is inexpensive and easy to perform and could thus be useful in establishing a digital multifacility database that will greatly contribute to the clarification of the transmission route and pathophysiology of M. avium infections.

Brain dysfunction associated with an induction of nitric oxide synthase following an intracerebral injection of lipopolysaccharide in rats

We investigated the pathophysiological role of nitric oxide synthesized by inducible nitric oxide synthase in the brain, by injecting lipopolysaccharide directly into the rat cerebral cortex/hippocampus. The levels of nitric oxide metabolites, nitrite and nitrate, began to increase in a dose-dependent manner with a 3-h lag, and reached approximately seven-fold the basal levels 8 h after the direct injection of lipopolysaccharide (5 microg). The lipopolysaccharide-induced increase in nitrite and nitrate levels was inhibited by treatment with the specific inducible nitric oxide synthase inhibitor aminoguanidine. The protein synthesis inhibitor cycloheximide delayed the onset of the increase in nitric oxide metabolite levels, and reduced the peak levels. Lipopolysaccharide increased Ca2+-independent, but not Ca2+-dependent, nitric oxide synthase activity in the brain. Intense nicotinamide adenine dinucleotide phosphate-diaphorase activity was observed in round cells in the vicinity of the site of injection of lipopolysaccharide 8 h after the injection. Neuronal death was observed seven days after the injection of lipopolysaccharide. Spatial memory, as assessed by performance in a water maze task and spontaneous alternation behavior in a Y-maze, was significantly impaired in rats which had had previous bilateral injections of lipopolysaccharide into the hippocampus. The lipopolysaccharide-induced neuronal death and spatial memory impairments were prevented by aminoguanidine. These results suggest that direct injection of lipopolysaccharide into the brain causes an induction of inducible nitric oxide synthase in vivo. Furthermore, it is suggested that nitric oxide produced by inducible nitric oxide synthase is responsible for the lipopolysaccharide-induced brain dysfunction.

Involvement of Salmonella Pathogenicity Island 2 in the Up-Regulation of Interleukin-10 Expression in Macrophages: Role of Protein Kinase A Signal Pathway

ABSTRACT Salmonellae are facultative intracellular bacteria capable of surviving within macrophages. Salmonella pathogenicity island 2 (SPI-2) is required for growth within macrophages and for virulence in mice. In this study, we show the involvement of SPI-2 in a signal transduction pathway that induces cytokine expression in Salmonella-infected macrophages. High levels of interleukin-10 (IL-10) mRNA were induced in macrophages by infection with wild-type salmonellae compared to a strain carrying a mutation in the spiC gene, which is encoded within SPI-2. The two strains had the same effect on the expression of proinflammatory cytokines such as IL-1α, IL-6, and tumor necrosis factor alpha. IL-10 expression was dose dependently blocked by treatment of infected macrophages with the protein kinase A (PKA) inhibitor H-89, while IL-10 expression was increased by the PKA activator dibutyryl cyclic AMP. Cyclic AMP-dependent PKA activity was higher in macrophages infected with wild-type salmonellae compared to the spiC mutant, and Ser132 phosphorylation of cyclic AMP response element-binding protein (CREB), which is an important mediator of PKA activation, correlated with the levels of PKA activity. Taken together, these results indicate that salmonellae cause an SPI-2-dependent increase in PKA activity that leads to CREB phosphorylation, resulting in up-regulation of IL-10 expression in Salmonella-infected macrophages. Suppression of IL-10 expression by an antisense oligonucleotide did not affect the growth of wild-type salmonellae within macrophages, whereas growth was dose dependently inhibited by H-89, suggesting that the PKA signaling pathway plays a significant role in intramacrophage Salmonella survival.

Biochemical characterization of hemorrhagic toxins with fibrinogenase activity isolated from Crotalus ruber ruber venom

Three hemorrhagic toxins with proteolytic activity were isolated from the venom of Crotalus ruber ruber (red rattlesnake). Molecular weights of HT-1, HT-2, and HT-3 were 60,000, 25,000, and 25,500, respectively. Although HT-3 was a basic protein, HT-1 and HT-2 were slightly acidic proteins. Total amino acid residues were 482,207, and 221 for HT-1, HT-2, and HT-3, respectively. Protease activity of all the toxins was inhibited in the presence of EDTA or o-phenanthroline, suggesting that the toxins are metalloproteins. Analyses for various metals by inductively coupled plasma-atomic emission spectrometry indicated that sodium, potassium, zinc, and calcium atoms were present in significant quantities. With all three toxins, there was roughly 1 mol of zinc to 1 mol of protein; the results for calcium were not consistent. All three hemorrhagic toxins degraded the A alpha chain of fibrinogen, while HT-1 also degraded the B beta chain. Although fibrinogen was degraded by the three toxins, no clots were observed, indicating that the proteolytic specificities of the three toxins were different from those of thrombin. The hemorrhagic toxins increased creatine kinase activity in mice serum, indicating muscle damage, which was substantiated by histological examination.

Isolation and characterization of hemorrhagic factors a and b from the venom of the Chinese habu snake (Trimeresurus mucrosquamatus)

Hemorrhagic factors a and b were isolated from Trimeresurus mucrosquamatus venom by Sephadex G-100, CM-Sephadex C-50 and DEAE-Sephacel column chromatographies. The hemorrhagic factors were homogeneous, as established by a single band on acrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Molecular weights of 15 000 and 27 000 were found for hemorrhagic factors a and b, respectively. Factor a possesses proteolytic activity hydrolyzing the His(10)-Leu(11), Tyr(16)-Leu(17) and Arg(22)-Gly(23) bonds of oxidized insulin B chain, whereas, factor b hydrolyzed only the Ala(14)-Leu(15) bond. Hemorrhagic activity of these hemorrhagic factors was inhibited by ethylenediaminetetraacetic acid, 1,10-phenanthroline or p-chloromercuribenzoate, but not by soybean trypsin inhibitor or diisopropyl fluorophosphate. The hemorrhagic factors were injected into the skin of the back of albino rabbits, and the minimum hemorrhagic dose of factors a and b was 1.7 and 2.3 micrograms, respectively. These purified hemorrhagic factors were not lethal at 15 micrograms/g in mice. Factor a hydrolyzed the B beta chain of fibrinogen, while factor b hydrolyzed the A alpha chain. Hemorrhagic factor a was shown to differ immunologically from factor b. Factors a and b produced systemic hemorrhage in internal organs such as the heart and stomach of mice. Moreover, factor b produced hemorrhage in the liver.

Salmonella enterica serovar Typhimurium infection induces cyclooxygenase 2 expression in macrophages: involvement of Salmonella pathogenicity island 2.

ABSTRACT Salmonella pathogenicity island 2 (SPI-2) is required for intramacrophage survival and systemic infection in mice. We have recently reported that Salmonella enterica causes activation of the protein kinase A (PKA) signaling pathway in a manner dependent on SPI-2, resulting in the upregulation of interleukin-10 expression in macrophages (K. Uchiya et al., Infect. Immun. 72:1964-1973, 2004). We show in the present study the involvement of SPI-2 in a signal transduction pathway that induces the expression of cyclooxygenase 2 (COX-2), an inducible enzyme involved in the synthesis of prostanoids. High levels of prostaglandin E2 (PGE2) and prostacyclin (PGI2), which are known to activate the PKA signaling pathway via their receptors, were induced in J774 macrophages infected with wild-type Salmonella compared to a strain carrying a mutation in the spiC gene, located within SPI-2. The increased production of both prostanoids was dependent on COX-2. COX-2 expression was dose dependently blocked by treatment with a specific inhibitor of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway, and the phosphorylation level of ERK1/2 was higher in macrophages infected with wild-type Salmonella compared to the spiC mutant. Taken together, these results indicate that Salmonella causes an SPI-2-dependent ERK1/2 activation that leads to increased COX-2 expression, resulting in the upregulation of PGE2 and PGI2 production in macrophages. A COX-2 inhibitor inhibited not only Salmonella-induced activation of the PKA signaling pathway but also growth of wild-type Salmonella within macrophages, suggesting that Salmonella utilizes the COX-2 pathway to survive within macrophages and that the mechanism involves activation of the PKA signaling pathway.

Hemorrhagic activity and muscle damaging effect of Pseudomonas aeruginosa metalloproteinase (elastase).

Pseudomonas aeruginosa elastase, a Bacillus subtilis thermolysin-like zinc-proteinase was examined for hemorrhagic activity and its effect on muscle and endothelial cells. Subcutaneous and intramuscular injections of elastase into mice caused severe hemorrhage with an acute increase of creatine phosphokinase activity in serum. The elastase also possessed fibrinogenolytic and fibrinolytic activities. The Aalpha and Bbeta chains of fibrinogen were completely hydrolyzed as demonstrated by their electrophoretic disappearance on SDS polyacrylamide gels. The pathological study indicates that elastase induces changes in the structure of the vascular wall and causes leakage of the plasma component and red and white blood cells into the extravascular tissue. This is further supported by results showing injury to cultured endothelial cells and macrophages. These data indicate that P. aeruginosa elastase directly affects endothelial cells and destroys the basement membrane of blood vessels to cause hemorrhage. Since fibrinogenolytic activity is an additional component of this elastase and this activity induces the hemorrhagic tendency, the damage in tissues could become increasingly severe.

Purification of a proteinase (Ac5-proteinase) and characterization of hemorrhagic toxins from the venom of the hundred-pace snake (Agkistrodon acutus)

Ac5-Proteinase (15.2 mg) was isolated from Agkistrodon acutus venom (1 g) by column chromatography on Sephadex G-75, CM-Sephadex C-50 and CM-Cellulose. Ac5-Proteinase was homogeneous by disc electrophoresis on polyacrylamide gel at pH 4.3 and also by SDS-disc polyacrylamide gel electrophoresis. Ac1-, Ac2-, Ac3- and Ac5-proteinases possessed lethal and hemorrhagic activities, but Ac4-proteinase had no lethal activity. These activities were inhibited completely by ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline or cysteine. The molecular weights of Ac1-, Ac2-, Ac3-, Ac4- and Ac5-proteinases were approximately 24,500, 25,000, 57,000, 33,000 and 24,000, respectively. Ac1-, Ac2-, Ac4- and Ac5-proteinases did not contain any carbohydrates, but Ac3-proteinase contained 0.1% carbohydrate by weight.