Kiyohisa Uchida

Disruption of microtubules as an early sign of irreversible ischemic injury. Immunohistochemical study of in situ canine hearts.

Structural disruption of the cytoskeleton may be involved in irreversible ischemic injury. In the present study, ischemic changes in microtubules during various periods of myocardial ischemia were studied with an immunohistochemical technique in open-chest dogs. In intact myocardium, microtubules were stained as a filamentous network throughout cytoplasm and a circular network around the nucleus, which disappeared with colchicine treatment. In brief ischemia of less than 15 minutes, microtubule patterns were unaltered. After 20 minutes, however, characteristic microtubule stains were partly lost in patchy lesions. As an increase in ischemic period, lesions of loss of microtubule stains were increased in number and size. After 120 minutes of reperfusion following 60 minutes of ischemia, the lesions with intact actin filaments but with disrupted microtubules were replaced by the severely injured cells in which the regular myofibrillar registrations were distinctly disrupted. After 24 hours of reperfusion following 40 minutes of occlusion of the left circumflex artery, the percent area of disrupted microtubules at 40 minutes of ischemia was replaced by that of irreversibly injured lesions in the posterior papillary muscle. These results indicate that disruption of microtubules during ischemia heralds irreversible ischemic injury. However, in in vitro study, the myocardium incubated in hypoxic solution for 60-120 minutes demonstrated earlier disruption of the microtubules than the vinculin. Electron microscopic study also showed minimal irreversible changes in the lesions with disrupted microtubules. Thus, taken together, we conclude that microtubules that support the structural integration of myofibrils and other organelles are disrupted in severe myocardial ischemia before the irreversible injury, promoting the irreversible change after reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered Bile Acid Metabolism in Nonobese, Spontaneously Diabetic (NOD) Mice

Cholesterol and bile acid metabolism was examined in nonobese, spontaneously diabetic (NOD) female mice before and after the development of diabetes. After the development of glucosuria, the plasma and liver cholesterol levels, gallbladder bile weight after 5-h fasting, biliary cholesterol, phospholipid and bile acid concentrations, the lithogenic index, the pool size of bile acids, and fecal sterol excretion markedly increased, but fecal bile acid excretion and fractional turnover rates for the cholic acid and chenodeoxycholic acid groups decreased. The distribution percentage of bile acids in the small intestine did not change, but it increased in the gallbladder and decreased in the large intestine. One striking finding was a change in the bile acid composition: increases were recorded in cholic and deoxycholic acids while decreases occurred in bile acids derived from chenodeoxycholic acid, such as β-muricholic and ursodeoxycholic acids in the bile and α-muricholic, β-muricholic, ω-muricholic, and hyodeoxychollc acids in the feces. Therefore, the cholic acid group/chenodeoxycholic acid group (CA/CDCA) ratio increased in the bile, feces, and small and large intestines after the development of diabetes. These changes were very similar to those observed in alloxan-treated mice, suggesting that the changes found in NOD mice are caused by insulin deficiency.

Effect on ergosterol biosynthesis of a fungicide, SSF-109, in Botrytis cinerea

Abstract Treatment of Botrytis cinerea with a novel fungicide SSF-109, (dl)-cis-1-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)cyclo-heptanol (0.45μgml−1), gave five 14α-methyl sterols, 24-methylene-24(25)-dihydrolanosterol, 24-methylene-24(25)-dihydrolanosten-3-one, obtusifoliol, obtusifolione, and 14α-methylfecosterol, together with ergosterol and ergosta-5,8,22-trien-3β-ol. SSF-109 was found to inhibit the biosynthesis of ergosterol at the 14α-demethylation step.

Bile Acid Metabolism in Benign Recurrent Intrahepatic Cholestasis: Comparative Studies on the Icteric and Anicteric Phases of a Single Case

Bile acid compositions in the serum, urine, bile, and feces were examined in a typical case of benign recurrent intrahepatic cholestasis for a period of 3 yr. The serum cholesterol level remained almost constant. The serum and urinary levels of total bile acids increased markedly during the icteric phase but returned to normal toward the anicteric phase. Daily fecal excretion of bile acids in the anicteric phase was about three times the normal value. Bile acids in the bile and feces, and in the serum and urine, mainly consisted of chenodeoxycholic and cholic acids, with little deoxycholic acid even in the anicteric phase. Scarcely any coprostanol was found in the feces. These observations suggest that metabolism by intestinal bacteria was altered in this patient. The ratio of cholic acid/chenodeoxycholic acid in the serum was above 2.0 in the icteric phase but was reduced to 0.9 in the anicteric phase. At least three unusual bile acids, designated as peak 7a, 11, and 13, were detected in the feces. The amount of bile acid in peak 7a reached about 10% of the total in the anicteric phase.

Bile acid metabolism in partially hepatectomized rats

The bile flow and the bile acid secretion, calculated on liver weight basis, increased 12 H and 24 H after 60-70% hepatectomy and returned to the initial levels thereafter. The biliary phospholipid secretion much more increased than bile acids, but the cholesterol secretion decreased. Bile acid composition changed with an increase of the cholic acid group and a decrease of the chenodeoxycholic acid group in both bile and feces. These changes almost disappeared on Day 14. The pool size of bile acid decreased maximally on Day 4 to about 40% of the initial, but the distribution of bile acids in the enterohepatic circulation was not changed. The fecal cholesterol and coprostanol markedly decreased on Day 2 but gradually returned to the initial levels according to the recovery of diet intake. The fecal bile acids decreased on Day 2, increased on Day 4, and returned to the normal range after Day 7. In conclusion, the regenerating liver secretes more bile, bile acids and phospholipids, and less cholesterol than the normal liver. Cholic acid predominates in the bile acids. These changes restored to the initial levels by about one week after the operation.

Stabilities of N-acetyl-β-d-glucosaminidase (NAG) isoenzymes in urine: advantage of NAG isoenzyme B measurement in clinical applications

N-Acetyl-beta-D-glucosaminidase (NAG) is a widely used urinary enzyme for the assessment of renal diseases. We studied the stabilities of NAG isoenzymes in urine at 37 degrees C by enzyme assay and ELISA using a model simulating in vivo conditions. The stabilities were found to be affected by the pH. Under mild acidic condition (about pH 6), there was no significant loss of enzymatic activity of NAG isoenzyme A, enzymatic activity of NAG isoenzyme B and immunological activity of NAG isoenzyme B even after 8 h incubation. In contrast, under alkaline condition (about pH 8), the enzymatic activity of NAG isoenzyme A was rapidly lost, whereas both enzymatic and immunological activities of NAG isoenzyme B were maintained at more than 80% of their initial values. Also, we found that the ratios of endogenous NAG isoenzyme B to total NAG were elevated in alkaline urine samples. These results indicate that NAG isoenzyme A, which is a major isoenzyme in normal urine (pH 5-7), seems to be inactivated in alkaline urine. Our results suggest that for alkaline urine, NAG isoenzyme B should be measured to avoid misinterpretation of total NAG enzymatic activities.

Effects of β-lactam antibiotics on intestinal microflora and bile acid metabolism in rats

Wistar male rats were treated for six days with broad spectrum β-lactam antibiotics, latamoxef, and cefotaxime. On the seventh day, the number of fecal anaerobic microbes decreased, total fecal bile acids decreased, and bile acid pools increased. Secondary bile acids such as β-hyocholic, hyodeoxycholic, lithocholic, and deoxycholic acids decreased in the feces while the primary bile acids, cholic, β-muricholic, and chenodeoxycholic acids, became predominant. Coprostanol, a microbial metabolite of cholesterol, also disappeared from the feces during the treatment. The cecum enlarged to almost twice the size of that in control rats, whereas the liver weight was not significantly changed. After treatment was stopped, the number of fecal microbes returned to the initial counts within a week, but restoration of bile acid and cholesterol metabolism required at least three weeks.

Composition of cecal bile acids in ex-germfree mice inoculated with human intestinal bacteria

Germfree (GF) mice were orally inoculated with human fecal suspension or various components of human fecal microbiota. Three weeks after the inoculation, cecal bile acid composition of these mice was examined. More than 80% of total bile acids was deconjugated in the cecal contents of ex-GF mice associated with human fecal dilutions of 10−2 or 10−6, or anaerobic growth from a dilution of 10−6. In these ex-GF mice, deoxycholic acid accounted for about 20% of total bile acids. In the cecal contents of ex-GF mice associated only with clostridia, unconjugated bile acids made up less than 40% of total bile acids, about half of those in other ex-GF groups. However, the percentage of deoxycholic acid in these mice was the same as that in the other groups. These results indicate that dominant anaerobic bacterial combination is efficient for deconjugation of primary bile acids, and that clostridia in the human feces may play an important role in 7α-dehydroxylation of unconjugated primary bile acids in the intestine.

Effect of sex hormones on hypoprothrombinemia induced by N-Methyltetrazolethiol in rats

N-Methyltetrazolethiol (NMTT) increased prothrombin time (PT) and decreased plasma factor VII and prothrombin levels only in vitamin K-deficient male rats. In female rats identical treatment with NMTT did not produce hypoprothrombinemia. Conventional and germ-free rats displayed no significant difference in the manifestation of hypoprothrombinemia, but the increase of PT in NMTT-treated vitamin K-deficient rats was greater in the germ-free males. Estradiol administration or castration of male rats retarded manifestation of vitamin K deficient syndromes such as increases of PT and activated partial thromboplastin time (APTT), decreases of plasma factor VII and prothrombin levels, and increases of plasma and liver descarboxyprothrombin (PIVKA) levels, and testosterone injection to the castrated rats restored these changes. In female rats testosterone treatment or castration enhanced the manifestation of hypoprothrombinemia and estradiol treatment to the castrated females retarded it. Gamma-glutamyl-carboxylase activity was increased by vitamin K-deficiency but not inhibited by testosterone or NMTT. These data suggest that estrogen protects the rat against manifestation of hypoprothrombinemia even with NMTT treatment, while androgen enhances vitamin K deficiency, and supplementation of vitamin K prevents its deficiency in NMTT-treated rats.

Age-related changes of bile acid metabolism in rats

Cholesterol and bile acid leves were examined in young (8 weeks), middle-aged (12 months) and old (24 months) germ-free male rats, and young (8 weeks) and middle-aged (12 months) conventional male rats. The plasma cholesterol levels were higher in the aged rats, being more marked in the conventional rats. The liver cholesterol levels also increased with age and the increases were almost identical for both groups. No age-related changes were found in the biliary bile acid secretion, the pool size and distribution of bile acids in the bile, small intestine and large intestine, nor in the turnover frequency of bile acids, but the pool size in the young and middle-aged germ-free rats was much larger than that in the conventional rats. The turnover frequency was less in the germ-free rats. The bile acid synthesis presumed from the fecal bile acid excretion decreased in the aged germ-free rats but not in the conventional rats. A most remarkable age-related change was found in the bile acid composition; cholic acid increased and beta-muricholic acid derived from chenodeoxycholic acid in the rat decreased by aging, resulting in an increase of the CA/CDCA ratio (bile acids belonging to the cholic acid group/bile acids to the chenodeoxycholic acid group) in the bile, feces and pool. These results suggest that cholic acid synthesis increases while chenodeoxycholic acid synthesis is impaired by aging in rats.