Homolog of FlhDC, a master regulator for flagellum synthesis: required for pathogenicity in Erwinia carotovora subsp. carotovora

 Erwinia carotovora subsp. carotovora (Ecc) is a causal agent of soft-rot diseases in a wide variety of plants. Here, we have isolated nonmotile mutants in Ecc by in vivo insertional mutagenesis using a transposon Tn5. The sequence disrupted by the Tn5 insertion in YMU1 and YMU5 mutants was highly homologous to that of flhC and flhD genes, respectively. They are involved in the initiation of the expression of flagellum-related genes in many gram-negative bacteria such as Escherichia coli and Salmonella. With electron microscopy, the flhC and the flhD homolog mutants were shown to be aflagellate. Furthermore, the virulence of these mutants was greatly reduced in Chinese cabbage and potato compared to that of the parental strain. These results suggest that flagellar formation is required for the pathogenicity of Ecc. Received: November 5, 2002 / Accepted: December 2, 2002 Acknowledgments This research was supported in part by Grant-in-Aid (12052210) and by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (13073).     

Oxidative stress inducers potentiate 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated pre-cardiac edema in larval zebrafish

We reported the involvement of oxidative stress and prostaglandins including thromboxane and prostacyclin in pre-cardiac edema (early edema) caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). While the involvement of oxidative stress in TCDD-induced toxicity has been frequently reported, the mechanism of its action is still unclear. In the present study, oxidative stress inducers including paraquat, hydrogen peroxide (H₂O₂) and rotenone augmented early edema (edema) induced by a low concentration of TCDD (0.1 ppb) at 55 hr post fertilization (hpf), while each of them alone did not cause edema. Edema caused by TCDD plus oxidative stress inducers was almost abolished by antioxidants, an antagonist for thromboxane receptor (ICI-192,605) and an agonist for prostacyclin receptor (beraprost), suggesting that the site of action of these inducers was in the regular signaling pathway after activation of aryl hydrocarbon receptor type 2 (AHR2) by TCDD. Oxidative stress inducers also enhanced edema caused by an agonist for the thromboxane receptor (U46619), and the enhancement was also inhibited by antioxidants. Sulforaphane and auranofin, activators of Nrf2 that is a master regulator of anti-oxidative response, did not affect U46619-evoked edema but almost abolished TCDD-induced edema and potentiation by paraquat in both TCDD- and U46619-induced edema. Taken together, the results suggest that oxidative stress augments pre-cardiac edema caused by TCDD via activation of thromboxane receptor-mediated signaling in developing zebrafish. As paraquat and other oxidative stress inducers used also are environmental pollutants, interaction between dioxin-like compounds and exogenous source of oxidative stress should also be considered.     

Immunostimulatory effects of natural human interferon-alpha (huIFN-α) on carps Cyprinus carpio L.

Human interferon-α (huIFN-α) is an important immunomodulatory substance used in the treatment and prevention of numerous infectious and immune-related diseases in animals. However, the immunostimulatory effects of huIFN-α in fish remain to be investigated. In the current study, the immune responses of the carp species Cyprinus carpio L. to treatment with huIFN-α were analyzed via measurement of superoxide anion production, phagocytic activity and the expression of cytokine genes including interleukin-1β, tumor necrosis factor-α and interleukin 10. Low doses of huIFN-α were administered orally once a day for 3 days, and sampling was carried out at 1, 3 and 5 days post-treatment. Our results indicate that a low dose of huIFN-α significantly increased phagocytic activity and superoxide anion production in the carp kidney. The huIFN-α-treated fish also displayed a significant upregulation in cytokine gene expression. The current study demonstrates the stimulatory effects of huIFN-α on the carp immune system and highlights the immunomodulatory role of huIFN-α in fish.     

Nitrocapsanthin and nitrofucoxanthin, respective products of capsanthin and fucoxanthin reaction with peroxynitrite

The in vitro reactivity of capsanthin (1) and fucoxanthin (2) with peroxynitrite was investigated, and the reaction products produced by scavenging with peroxynitrite were analyzed. (14'Z)-Nitrocapsanthin (3) and 12-nitrocapsanthin (4) were isolated from the products of the reaction of capsanthin with peroxynitrite. Similarly, (14Z)-15-nitrofucoxanthin (5), (11Z)-11-nitrofucoxanthin (6), and (14Z,9'Z)-15-nitrofucoxanthin (7) were obtained from the reaction of peroxynitrite reaction with fucoxanthin. Capsanthin and fucoxanthin inhibited the nitration of tyrosine by peroxynitrite. Furthermore, nitrocapsanthins (3 and 4) and nitrofucoxanthins (5 and 6) exhibited an inhibitory effect on Epstein-Barr virus early antigen activation in Raji cells and an antiproliferative effect on human pancreatic carcinoma. Moreover, nitrocapsanthins (3 and 4) inhibited carcinogensis of mouse skin tumors initiated by 7,12-dimethylbenz[a]anthracene (DMBN).     

Immunomodulatory effects of alpha melanocyte stimulating hormone on common carp (Cyprinus carpio L.)

The immunomodulatory effects of phagocytic cells in common carp Cyprinus carpio L., by alpha melanocyte stimulating hormone (alpha-MSH) was analyzed in vitro. Carp head kidney leucocytes were cultured in RPMI 1640 medium containing 0.05, 0.1, 1 and 10 nM alpha-MSH and the production of superoxide anion was measured via the reduction of nitroblue tetrazolium (NBT) in vitro. Macrophages incubated with alpha-MSH showed an increase in the production of superoxide anion in comparison to control macrophages cultured without hormone. Phagocytic cells treated with alpha-MSH also displayed increased phagocytosis. Furthermore, carp lymphocytes treated with alpha-MSH increased the mitogenic responses to phytohaemoagglutinin (PHA-P). These results show that alpha-MSH directly influences fish immune responses.     

Development of an efficient method for recovery of Puumala and Puumala-related viruses by inoculation of Mongolian gerbils

Puumala (PUU) virus and PUU-related viruses are difficult to isolate in cell culture. To determine whether animal inoculation would be a better alternative for virus recovery, the Sotkamo strain of PUU virus was inoculated into several animal species. Newborn Mongolian gerbils (MGs), mice, and rats were infected with the Sotkamo strain by intracerebral (ic), intraperitoneal (ip), and subcutaneous (sc) inoculation. Antibodies to PUU appeared in MGs at 30 days post-infection (dpi), and in mice and rats at 15 dpi. Interestingly, virus appeared at 7 dpi in lung and brain of MGs inoculated via ic and ip routes. Virus was detected in all tested tissues of MGs at 15 dpi, with a peak level of 1.36 x 10 (5) focus forming units (FFU)/g in brain tissue. The virus titer declined with the onset of the antibody response and became undetectable by 75 dpi, when the antibody titer reached the maximum level. The appearance of the virus in mice and rats was delayed as compared to MGs, and the virus titer was apparently lower, at approximately 4 to 8 x 10(3) FFU/g, at 15 dpi. In addition, lung homogenates of antibody-positive Clethrionomys (C.) rufocanus (captured in Tobetsu, Hokkaido, Japan) were inoculated into MGs by the ic route. PUU-related viral RNA was detected at 16 dpi in the brains of MG inoculated with the lung homogenate, and antibodies were detected at 45 dpi. These findings indicate that newborn MG inoculation is an efficient method to recover PUU and PUU-related viruses.     

Examination of the onset of stable crack growth under fracture toughness testing of paper

Successive temperature distribution images around the notch tips during fracture toughness testing of paper were obtained by means of an infrared thermography system. Analysis of the images gave the critical times when the temperature significantly rose at the notch tip and when the distance between two maximum temperature spots started to decrease during the testing. Other successive microscopic images around the notch tips showed the relation between crack opening and displacement and the transitional point of the relation. The onset of stable or unstable crack growth as indicated in these critical times and the point agree with each other. For the specimen with a small width, an unstable crack starts to grow at the maximum load point without the stable crack growth period. On the other hand, a stable crack grows before the maximum load point unless the specimen width is small.The period of the stable crack growth increases with an increase in width. Differing from the methods based on thermal images to determine the onset of crack growth, the microscopic method is applicable at a wide range of strain rates and is thus suitable for quasistatic fracture toughness testing.This work was presented in part at the 8th annual meeting of the Society of Packaging Science and Technology, Japan, Tokyo, June 1999     

Ion channel mRNA distribution and expression in the sinoatrial node and right atrium of dogs and monkeys

There are limited data on the gene expression profiles of ion channels in the sinoatrial node (SAN) of dogs and monkeys. In this study, the messenger RNA (mRNA) expression profiles of various ion channels in the SAN of naïve dogs and monkeys were examined using RNAscope^®in situ hybridization and compared with those in the surrounding right atrium (RA) of each species. Regional-specific Cav1.3 and HCN4 expression was observed in the SAN of dogs and monkeys. Additionally, HCN1 in dogs was only expressed in the SAN. The expression profiles of Cav3.1 and Cav3.2 in the SAN and RA were completely different between dogs and monkeys. Dog hearts only expressed Cav3.2; however, Cav3.1 was detected only in monkeys, and the expression score in the SAN was slightly higher than that in the RA. Although Kir3.1 and NCX1 in dogs were equally expressed in both the SAN and RA, the expression scores of these genes in the SAN of monkeys were slightly higher than those in the RA. The Kir3.4 expression score in the SAN of dogs and monkeys was also slightly higher than that in the RA. The mRNA expression scores of Kv11.1/ERG and KvLQT1 were equally observed in both the SAN and RA of dogs and monkeys. HCN2 was not detected in dogs and monkeys. In summary, we used RNAscope to demonstrate the SAN-specific gene expression patterns of ion channels, which may be useful in explaining the effect of pacemaking and/or hemodynamic effects in nonclinical studies.