???????????????????ε???????????

??????????Щ?????????????????????????????????(H2)??????????????????????о??飬???????о?????????????????????????????Concanavalin A (ConA)?????С?????????????????????????????????????????????????????????????????????AST??ALT??TNF-????IFN-???????????????????????????????ConA??????????????????ò??????????????????????????????????????????????????????ConA??????????????????????????????????????????????????ConA??????????????????TNF-????IFN-?á???Щ?????????????????????????????????ConA?????????????????

Kajiya M, Sato K, Silva MJ, Ouhara K, Do PM, Shanmugam KT, Kawai T. Hydrogen from intestinal bacteria is protective for Concanavalin A-induced hepatitis. Biochem Biophys Res Commun. 2009 Aug 21;386(2):316-21.

????

????(H2)???????ж??????????????????С??????е??????Ч???????[1]?????????о????????????????????????????????????????????????У????????????????????????????[2]??[3]??[4]????????????????????????????????????????????????????Ч??????????????????????????????????????????????????????б?????????????????á?

?????????????????????о??У??????????????????????????????й???????[1]??[2]??[3]??[4]???????????Щ??????????????(E. coli)???????????????????????[5]???????????????????????[6]????????????????????????????????????????????????????????????????????????о??漰??????????????????????????????????????

????????A (ConA) ??????????????????????????????????T????????????????????????????????????????????????????????????????????????????????????????[7]??[8]??????ConA??????????????????????????????????????????????????????????[9]?????????????SCID???ConA???????????????????????T?????ConA???????????е?????????[10]??[11]??????ConA???????????????????[11]???????????????????????????????????????????????ConA?鵼??T???????????????д???????????????????????????????? (TNF-??) ???????? (IFN-??)????Щ????????????T????????????????ConA???????????????а????????[12]??[13]??[14]??

???Concanavalin A???????????С?????????о??????(1)????????????????????????????????(2)?????????????????????????????

????????

????

8??10?????????C57BL/6jС??????????滷???У??????????12С????????????о???????????????Forsyth IACUC???????

????????GFP?????

???о?????????W3110????ATCC 27325????????????????hypF????????PMD23?????????1??????????????HypF???????????????????????????????????????????95% [15]??[16]??????紩????????????????????????а?????????????Ampr?????????pGFPuv-???壨Clontech, Mountain View, CA???????????????????????W3110gfp+??Ampr+/GFP+/HypF+???????PMD23gfp+??Ampr+/GFP+/HypF−????????а????????100 ??g/ml????Luria?CBertani??LB??????????????

?????????

???????????????Unisense A/S, Aarhus, Denmark??????Hayashida????[3]?????????????С???????в???????????H2??????С??????CO2???????????????????????????????25-G????????????·???С????????????????????????????????????????С????????б??????????壨25??C??781??M??37??C??721??M???????????????????????????????δ??????????????0??M ???????????????????????????????????????????磬???????????????????0.7??M/min????

????????????

??????????????壨Airgas, Salem, NH???????????????У?????????????????780??M??25??C???????????????????????????????б?????????????pH 7.6????????????????λ??ORP??-511 mV????

????????A??????????????

??鷽??-A????1?????﹩????п?????????????????8 mg/ml???????????1.6 mg/ml??????????????????????????3????2?????????????????鶯??????????????????????3????????С???????ConA??Sigma, St. Louis, MO, 15 mg/kg????????????????????????0??2??10С????????е?ALT??AST??

??鷽??-B????1?????﹩????п?????????????????4 mg/ml??????????0.8 mg/ml????????????0.1 mg/ml?????????????3????2????????????????????????????ú??а????????0.1 mg/ml?????????3????????С?????ConA??15 mg/kg??????????????????a????Щ??ConA????12С????????0??3С?????????????????780??M??pH 7.6??????1 ml [????]??n = 5/?飩??b????Щ??????????????1 ml [????]??n = 5/?飩??ConA?????????????????а?????????????鷽??-B??????????2A?С?

?1. ?????????????????????????????????С???ConA?????????????????????A????????????????????????????е??????????????????У????n=3??????B????????????????δ??????С??????????????????????20 mg/10 ml LB?????????n=3??????37??C?·???1С???12С?????????????????е?????????C??D????ConA??15 mg/kg????????????????????????????????8 mg/ml???????????1.6 mg/ml????С?????????ЩС???????????????????3???????2?????????????????????????е?ALT??C????AST??D??????????????????С??????????????????*p<0.05??**p<0.01???????????????t???飩??

?2. ????????????????????????????C57BL/6jС??ConA????????????????A????鷽??B????????????????????????????ConA????0??2??6С??????С???ALT??C????AST??D????TNF-????E????IFN-???F???????????????????????????????????C??D??E??F???е?????????α?????????????????????n=5????*p<0.05??**p<0.01???????????????t???飩??

??鷽??C????1?????﹩?????????B??????????????????????????????3????2????????????????????????????ú??а????????1 mg/ml?????????3????????С?????ConA??15 mg/kg??????????????????a????Щ??E. coli W3110gfp+?????????n=5????b????Щ??PMD23gfp+?????????n=5??????????E. coli???????????????????α????????10^9???/100 ??l??5%?????????????????/?? [????]???????Popperι?????ConA????2????С??????ConA?????????????????а?????????????????鷽??C???????????3A?С?

?3. ?ò????????????????????????????????????????????????????????????C57BL/6С????ConA?????????????A????鷽??C?????????????????????????B??????а????????100 ??g/ml????LB??????????12С????????????PMD23??Ampr+/GFP+/HypF−????W3110??Ampr+/GFP+/HypF+??????????????????ConA????0??2??6С??????С???ALT??B????AST??C????TNF-????D????IFN-???E???????????????????????????????????????????????n=5????∗p<0.05??∗∗p<0.01?????t?????????????

??????????????????????????????

??????????е??????????????ALT?????????????????????AST????????????????????????ü??????в??????????????????У?Biotron Diagnostics, Hemet, CA?????????????????????飨ELISA??????У?PeproTech, Rocky Hill, NJ?????д??????????TNF-????IFN-????????

????????????????

??ConA????10С???????С?????????????????????????????????????????H&E??????

????????????????????????????????????

???Histopaque??Sigma????????????????C57BL/6jС???????з?????????????????96????У???????????2??10^5/????ú???10% FBS??l-???????????????RPMI????????????????????????????????175??350??700 ??M?????????96????е??????ConA??1 ??g/ml?????24С???????ConA???????????????????????ELISA???TNF-????IFN-?á???96????е???????????????[3H]?????0.5 ??Ci????????48С????????????????16С???????????????????????????????????????е???????cpm????

???

??????????У?????????????????????????????[17]???о?????????????С??θ??????е??????????20-80 ??M?????????????????Km????20??????[6]??[18]????????Щ??????????踹????????????????????????????????????????????裬С????3???????????????????????????????????????2?????????????????????????????????????????????????????????????????????Ч????????δ??????С??1.6 ?? 0.5 ?? Log10^9 CFU/g?????????????С??7.0 ?? 6.1 ?? Log10^7 CFU/g?????1A????????????е?????????????м???????????????????С?????????????????????????м??????????????????????????????????????????????С???????????????в????????е??????????????????????????С?????????????????????????????????δ??????С??????????????????????????????????1B??????Щ??????????????????????????仯??????С????????λ??????????????????????????????????????????????е????????????????????

???????????????в?????????????????????????С???ConA????????????????????ConA??15 mg/kg???????????????????????δ??????С?????????鷽??-A????ALT??AST???????????????????????δ??????С?????????в????1C??D???????????????δ???????????2С????????????ALT??AST?????????????????С?????????????????0С?????????????????????1C??D????????????????????????????????δ??????С????????????????????С????????????????????????????1?????????????????????????С???ConA???????????????????仰?????п?????????????????????????????ConA?????????????

??????????????????????????????????ConA????????????????????俹???????????С?????????????????????????????С???ConA???????????????????????????裬?????????????С?????????????????????????????????鷽??-B????????????????????????????????????????????????????????????С????ConA????6С???????????????ALT??AST???2B??C??????????????????T???????????????????TNF-????IFN-?????????????????С?????????????????????????????2D??E????

????????????????????????ConA????????????????????????????С??????????????????????????????????1?????????????????W3110gfp+??2??????????????????PMD23gfp+??????ConA????????3A????鷽??-C???3B??W3110gfp+??PMD23gfp+?????????????????????а???????????????С???У????????а????????????????????С?????л????GFP+???????????????????????????????W3110gfp+???????С??? small intestine??large intestine??cecum ?? liver ?м????????????????????PMD23gfp+???????С??????Щ?????б??????????????????????????2??????Я??PMD23gfp+??????С??????ConA????6С???????????ALT??AST????W3110????С??????????????3C??D?????????TNF-????IFN-???????Я??W3110gfp+??С?????????????Я??PMD23gfp+??????С???3E??F??????????????鷽??-A??-B??-C?????????????????????????????????ConA???????????????з????????á?

?????????????????T???????TNF-????IFN-?????ConA????????????е?????????[12][13][14]????????????????????????ConA?????T?????TNF-????IFN-??????????????ConA??????????????????MNL????????????????????????????С????4?????????????????????????ConA???MNL???????????????????????????????MNL????????4A???????TNF-????IFN-?????????????4B??C???????????????????????????????MNL?????????????IFN-?????????????????????о?????????????????????ConA?鵼??T??????????????????????TNF-????IFN-???????????

?4. ??????ConA???????????????????????Ч?????C57BL/6С??????з?????????????????????96??????????????????????????????????175??350??700??M??????1С???????????????ConA??1??g/ml????????·??24С????????ELISA??????????????????????48С??????[3H]???????????????????????????????????A????????????????TNF-????IFN-????????B??C???????????????SI?????????????????????cpm??δ?????????cpm???????????????????????????????????????????????��∗p < 0.05, ∗∗p < 0.01????????????t???飩??

????

????????????????????????????????????????????о??б????????????[19][20]??????????????????鵼??????????????????????в???????Щ?о??????????С?????????С??????????????????????????????????????????????????????????????[21]?????????????????????????????????????????Concanavalin A?????С???????п???????????о?????????????????鵼????????????????????????????????????鷽??A??B??C?????????????????ConA???????????????з??????????????????????????????????????????????????????????

????????????????????????????????Ч???????????????????????????????????????????????????????????????????????????е???????1??/???????????????????????????2????/??????????????????????????????????θ???????????????????о??г?????????????????????????????????????????????????????????????????θ?е????????????????籨??????????????????Helicobacter hepaticus[6]?????????????裬???????θ???????в???????????????????????????????

?????????????о??????????????Ч??????????????????????????????????????????????????????[1][2][3][4]?????в?????????????????????????????????????????????????о????????????????????????????????????????H2????????????ConA?????????????????????????????????TNF-????IFN-?á?????????????ROS????????????NF-kB????·??????TNF-??????[22]????????????????NADPH-???????NOX???????????NADPH????ROS[23]??????????????????????????????ROS?????????????????淴????????????鵼???ConA?????????????TNF-????IFN-?????????????漰???????????Ч????

????????о??????????????????????????????????Concanavalin A???????????????????????????????????????б????????????????????????????????????д??????????????????????????????????????????????鶯?????????????????????????????????б???????????Ψ??????????????????????????????????е??????????????????????????????????????????????????????????翹???????????????????????????????????????????????????

????????

????????????С??????????????????????????????鲻???С???????????

- (A) δ??????????????????С??

- (B) ?????Con A???10С????????????С??

- (E) ?????Con A???10С???????????С??

??Щ????????????????????????-????(HE)??????????200????

??????鲹?????????????????????????????????????????????????????????????С??????????????????????????????????

- (1) ????????????W3110gfp+??

- (2) ???????????PMD23gfp+????鷽??C????

?????????е????????????Day-1???????????????????????n = 3?????????*p < 0.05?????????????????????????????t???飩??

Bacterial strains.  A wild type Escherichia coli strain W3110 (ATCC 27325) and a hypF deletion derivative, strain PMD23, that is defective in all three hydrogenase activities of the bacterium were used in this study. Strain PMD23 was constructed using the methods described by Datsenko and Wanner (1). For construction of strain PMD23, two PCR primers that also contain 50 bases long hypF DNA flanking the ends of the region to be deleted were used to amplify a kanamycin resistance gene cassette in plasmid pKD4 (ATGGCAAAAAACACATC TTGCGGT GTCCAAC TGCGTATTCGTGGCAAAGTGTGTAGGCTGGAGCTGCTTC and CACCCGCCGGTAAACTCTGTGGAAAGAGCAATGTGAAATCAGCGAGATAACATATGAATATCCTCCTTAGT; underlined, hypF sequence). The resulting PCR product was introduced into E. coli strain BW25113 (1) by electroporation and kanamycin-resistant transformants were selected on rich medium with kanamycin (50 mg/L) and tested for fermentative H2 production (2). All kanamycin-resistant transformants tested were defective for H2 production. Using phage P1 (3) the ??hypF-FRT-kanR-FRT mutation was transduced to wild type E. coli strain W3110 (strain PMD22). The kanamycin gene cassette in strain PMD22 was removed as described by Datsenko and Wanner (1) that left one FRT sequence at the site of deletion. The resulting kanamycin-sensitive strain, PMD23 carries a deletion of 2088 bp of the 2253 bp long hypF gene.

References

1. Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U. S. A. 97:6640-6645.

2. Lee, J. H., P. Patel, P. Sankar, and K. T. Shanmugam. 1985. Isolation and characterization of mutant strains of Escherichia coli altered in H2 metabolism. J. Bacteriol. 162:344-352.

3. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor laboratory, Cold Spring Harbor, NY.