Formation of the 37KD Protein-Acetaldehyde Adduct in Liver During Alcohol Treatment is Dependent on Alcohol Dehydrogenase Activity

Protein-acetaldehyde adducts (protein-AAs) are formed in vivo during chronic alcohol ingestion. These protein-AAs reported thus far include a 37KD protein-AA in liver cytosol, cytP450IIE 1-AA in hepatic microsomes, hemoglobin-AA, and serum protein-AAs. It has been postulated that acetaldehyde or perhaps a reactive acetaldehyde radical generated by the microsomal ethanol oxidizing system (MEOS or cytP450IIE1) explains the formation of the cytP450IIE1-AA. The source of acetaldehyde responsible for the formation of the cytosolic 37KD protein-AA has not been determined. In this report, we have examined the effects of pyrazole (an ADH inhibitor) and cyanamide (an aldehyde dehydrogenase inhibitor) on the formation of the 37KD liver protein-AA in vivo and in vitro. It was found that feeding rats with an alcohol-containing liquid diet supplemented with cyanamide enhanced while a diet supplemented with pyrazole completely abolished the formation of the 37KD liver protein-AA. The liver of rats fed the pyrazole supplemented alcohol-containing diet showed significantly higher content of cytP450IIE1 than that of rats fed the diet containing alcohol alone. On the other hand, feeding the cyanamide supplemented alcohol-containing liquid diet did not further enhance the content of cytP450IIE1. Similarly, adding cyanamide to the culture medium enhanced while adding 4-methylpyrazole inhibited the production of the 37KD protein-AA by cultured hepatocytes even though the combination of alcohol and 4-methylpyrazole increased the content of cytP450IIE1 2-fold over that in control cells. These results demonstrate that the formation of the 37KD liver Protein-AA is dependent on ADH and not on MEOS.(ABSTRACT TRUNCATED AT 250 WORDS)     

刺槐素在SH-SY5Y细胞氧-葡萄糖剥夺和小鼠脑缺血再灌注中的神经保护作用

目的 探讨刺槐素在SH-SY5Y细胞氧-葡萄糖剥夺和小鼠脑缺血再灌注中的神经保护作用.方法 培养神经母细胞瘤细胞SH-SY5Y,分为正常培养组、氧-葡萄糖剥夺(oxygen-glucose deprivation,OGD)组以及刺槐素小剂量组(1μmol/L)、中剂量组(5μmol/L)和大剂量组(10 μmol/L),复氧24 h后应用噻唑蓝染色法测定细胞存活率,乳酸脱氢酶(lactate dehydrogenase,LDH)法测定LDH漏出率.60只C57小鼠随机分为假手术组、脑缺血再灌注组以及小剂量组、中剂量组和大剂量刺槐素组,每组12只.采用线栓法制作大脑中动脉闭塞模型,缺血再灌注时给予刺槐素腹腔注射(小、中、大剂量组分别为6.25、12.5和25 mg/kg).再灌注后24 h时进行神经功能评分,应用2,3,5-氯化三苯基四氮唑染色检测脑梗死体积.结果 体外实验显示,1 μmol/L、5μmol/L和10 μmol/L刺槐素组SH-SY5Y细胞存活率分别为(90.34±6.87)％(P=0.000)、(85.47 ±2.24)％ (P=0.001)和(81.79±1.77)％(P=0.008),均显著高于OGD组的(70.62± 8.89)％.1μmol/L、5μm/L和10 μm/L刺槐素组SH-SY5Y细胞LDH漏出率分别为(159.11 ±13.11)％ (P=0.021)、(155.12±24.72)％(P=0.011)和(160.92 ±7.83)％ (P =0.027),均显著低于OGD组的(180.35±10.60)％.体内实验显示,大剂量刺槐素组神经功能评分显著低于与脑缺血再灌注组[(1.67±0.85)分对(2.50±0.55)分;P=0.018].小剂量、中剂量和大剂量刺槐素组梗死体积分别为(24.14±7.10)mm3、(17.18±3.19)mm3和(12.86±1.88)mm3,均显著小于脑缺血再灌注组的[(48.81±9.48) mm3](P均=0.000).结论 体外和体内实验均显示,刺槐素具有神经保护作用.     

Effects of Protein Size on the Rate of Import of the Precursors of Aldehyde Dehydrogenase and Ornithine Transcarbamylase into Rat Liver Mitochondria

It is known that a signal peptide is required for the import of a protein into mitochondrial matrix. It is also known that a signal peptide can be attached to any protein and allow it to be imported. We recently reported that the rate of import of rat liver mitochondrial aldehyde dehydrogenase precursor was slower than that of ornithine transcarbamylase precursor (Wang TTY, Farrés J, and Weiner H. Arch Biochem Biophys 272, 440–449, 1989). It was not known if the difference in the rate of import was related to the fact that the mature portion of aldehyde dehydrogenase is larger (500 amino acids compared with 322 amino acids) or because the signal peptides were different. We further showed that treatment of the mitochondria with alcohols caused an inhibition of the import of the precursor of aldehyde dehydrogenase but not that of ornithine transcarbamylase. In the present study we constructed chimeric proteins that contained the signal peptide from one precursor protein and the mature portion from the other. We found that the rate of import was related to the overall size of the precursor protein. Consistent with this observation was finding that a truncated aldehyde dehydrogenase precursor, which contained 317 amino acids, was imported more rapidly than was the authentic precursor. Consistent with this finding was the fact that butanol caused the inhibition of only the large precursor proteins. Thus, it appears that size of the protein being imported is a major determinant of the rate at which a precursor protein is imported into mitochondria.     

高纯度诊断用乳酸脱氢酶的制备和酶促反应

设计并优化了植物乳杆茵发酵制备高纯度诊断用乳酸脱氢酶的技术路线．从培育的厌氧型植物乳杆茵中粗提乳酸脱氢酶制备液，经过DEAE Sepharose F．F．离子交换层析、Phenyl Sepharose6 F．F．疏水层析及Sephadex G-25 Fine凝胶过滤等方法进行分离纯化，比酶活达1096．8U／mg，纯度达88％，纯化倍数达到21．4倍，酶活力回收率为53．9％；高效液相色谱和SDS-PAGE电泳结果显示制得乳酸脱氢酶相对分子质量约为80000，含有两个亚基，每个亚基分子量约为39000．     

左旋四氢巴马汀对大鼠和小鼠脑缺血再灌注损伤的保护作用

目的：观察左旋四氛巴马汀（THP）对脑缺血再灌注损伤的保护作用；方法：采用大鼠和小鼠脑缺血再灌注模型。结果：在大鼠和小鼠脑缺血再灌注模型上，THP30，60mg/kg,iv及60,120mg/kgiv，可显著提高下脑组织乳酸脱氢酶（LDH）及超氧化物歧化酶(SOD)活性、明显降低丙二醛（MDA）含量。在大鼠局灶性脑缺血2h再灌注24h模型上，THP30，60mg/kg,iv可明显缩小梗死灶面积，并显著抑制脑缺血组织中LDH和SOD活性降低，减少MDA过量生成。结论：THP对大鼠和小鼠脑缺血再灌注损伤有显著保护作用。其机制与其抗氧自由基有关。     

Effects of Helicobacter pylori Infection and Gastritis on Gastric Alcohol Dehydrogenase Activity

Sex and age differences in gastric alcohol dehydrogenase activity in relation to abnormalities of gastric histology and Helicobacter pylori infection were determined in 63 patients (32 men and 31 women) undergoing upper endoscopy for gastrointestinal symptoms. No sex difference was found in gastric alcohol dehydrogenase activity. Males older than 50 years had lower enzyme activity than younger males. Patients with H. pylori and/or moderate to severe chronic and acute inflammation and epithelial mucin depletion had lower alcohol dehydrogenase activity in the antrum, but not in the fundus. H. pylori was found more frequently in the older male patients. Antral alcohol dehydrogenase was most decreased in older patients of both sexes with H. pylori infection. In conclusion, H. pylori infection and/or chronic active gastritis are important causes of low gastric alcohol dehydrogenase activity.     

硒和蛋氨酸缺乏对大鼠心肌琥珀酸脱氨酶活性的影响

本文介绍了硒和蛋氨酸缺乏对大鼠心肌琥珀酸脱氢酶(SDH)活性的影响。实验结果表明,单纯低硒或低蛋氨酸合成饲料均能引起大鼠心肌SDH活性明显下降,而低硒低蛋氨酸的复合作用则可导致该酶活性的更进一步降低。本文尚结合克山病病因进行了探讨。     

Comparative study of the absorption,elimination and acute hepatotoxic action of ethanol in guinea pigs and rats

In fed guinea pigs, an oral dose of 6.4 g/kg of ethanol given as a 40% solution (v/v) produced a maximal blood alcohol level of 6.8±0.3 mg/ml, whereas in fed rats, blood alcohol levels after the same dose did not exceed 2.1±0.2 mg/ml. Maximal blood alcohol levels in fasted animals after an oral load of 4.8 g/kg of ethanol were 6.3±0.2 mg/ml in guinea pigs and 3.7±0.3 mg/ml for rats. However, i.v. injected ethanol (1 g/kg) was eliminated at the same rate in both species (275 mg per kg · h), and ADH activity of the liver related to body weight was by 20% greater in guinea pigs than in rats.Therefore, absorption of ethanol occurs at a much slower rate in rats than in guinea pigs. This is possibly due to the fact that high ethanol concentrations strongly delay emptying of the rat stomach. Lowering the ethanol concentration accelerates absorption rate in the rat. However, even after gavage of a 10% solution peak levels of blood alcohol were still lower by 36% in rats than in guinea pigs.In guinea pigs, increased serum activities of GOT, GPT, and GLDH occurred after an oral dose of 4.8 g/kg or 6.4 g/kg of ethanol, respectively. SGOT already increased after 1.6 g/kg of ethanol p.o. After 6.4 g/kg of ethanol given to rats serum transaminase levels increased only slightly, and GLDH activity not at all. Vacuolar degeneration was the morphological substrate of ethanol-induced liver damage in guinea pigs and rats. In guinea-pigs, it occurred already after 1.6 g/kg of ethanol, whereas in rats only after 6.4 g/kg.In conclusion, the guinea pig seems to be better suited for research on alcohol toxicity than the rat.     

Vermiconversion of wastewater sludge from textile mill mixed with anaerobically digested biogas plant slurry employing Eisenia foetida

Vermicomposting is commonly used for the management of organic wastes. We have investigated the potential of an epigeic earthworm, Eisenia foetida, to transform solid textile mill sludge (STMS) spiked with anaerobically digested biogas plant slurry (BPS) into vermicompost to evaluate the feasibility of vermicomposting in industries for waste management. The growth and reproduction of E. foetida was monitored in a range of different feed mixtures for 15 weeks in laboratory under controlled experimental conditions. E. foetida did not survive in fresh STMS. But worms grew and reproduced in STMS spiked with BPS feed mixtures. A greater percentage of STMS in feed mixture affected biomass gain and cocoon production by earthworms. The maximum growth was recorded in 100% BPS. The net weight gain by E. foetida in 100% BPS was two-four-fold higher than STMS-containing feed mixtures. After 15 weeks, maximum cocoons (78) were counted in 100% BPS and minimum (26) in 60% BPS+40% STMS feed. Vermicomposting resulted in pH shift toward acidic, significant reduction in C:N ratio, and increase in nitrogen, phosphorus, and potassium contents. Microbial activity measured as dehydrogenase activity increased with time up to day 75 but decreased on day 90, indicating the exhaustion of feed and decrease in microbial activity. These experiments demonstrate that vermicomposting can be an alternate technology for the recycling and environmentally safe disposal/management of textile mill sludge using an epigeic earthworm, E. foetida, if mixed with anaerobically digested BPS in appropriate ratios.