The Ubiquitin-Proteasome 26s Pathway in Liver Cell Protein Turnover: Effect of Ethanol and Drugs

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Samuel W. French and R. J. Mayer. The presentations were (1) The ubiquitin-proteasome 26s pathway in liver cell protein turnover: Effect of alcohol and drugs, by Samuel W. French and F. Bardag-Gorce; (2) The role of CYP2E1 phosphorylation and degradation pathway in the induction of the enzyme, by Magnus Ingelman-Sundberg; (3) Role of proteasome in the proteolysis of oxidized proteins in experimental chronic alcoholism, by Helen Rouach; (4) Alcohol, proteolysis and liver cancer, by R. J. Mayer; (5) Effect of ethanol feeding on the ATP-ubiquitin-proteasome pathway in the liver cell, by F. Bardag-Gorce; (6) Novel mechanisms and targets for intracellular transport of CYP2E1, by E. Neve; and (7) Gankyrin, an oncoprotein commonly over expressed in hepatoma, by H. Higashitsuji.     

卡托普利对高血压冠状动脉壁肥厚和储备力下降的预防作用

目的研究卡托普利对高血压冠状动脉壁肥厚和储备力下降的预防作用。方法４ｗ大鼠设３组：分别为自发性高血压大鼠（ＳＨＲ）组、ＳＨＲ口服卡托普利组（ＳＨＲ＋Ｃ）和正常血压大鼠（ＷＫＹ）组,饲养１２ｗ。结果ＳＨＲ＋Ｃ组较ＳＨＲ组,收缩压、冠状动脉壁横截面积、横截面积与内径比及中层血管平滑肌细胞宽度显著下降,最大冠状动脉流量增加,与ＷＫＹ组无显著差异。结论卡托普利能完全预防ＳＨＲ冠状动脉壁肥厚和储备力下降。     

参三七皂苷Rg1预适应对5-氮胞苷诱导大鼠骨髓间充质干细胞向心肌细胞转化的干预

目的:用药物预适应方法进行干细胞诱导已有报道,本实验观察中药参三七皂苷Rg1对5-氮胞苷诱导大鼠骨髓间充质干细胞向心肌细胞转化中的作用。方法:实验于2003-01/05在南京医科大学药理教研室完成。①实验材料:清洁级SD大鼠8只。参三七皂苷Rg18mg,批号20021017,由云南省长春花生物制剂公司提供,加入不含胎牛血清的IMDM培养液10mL,调配成10-4mol/L溶液,4℃保存。5-氮胞苷(Sigma公司,批号021209)。②实验方法:贴壁法体外培养大鼠骨髓间充质干细胞。设立4组:空白对照组常规培养后进行无血清处理,每3d换液1次;5-氮胞苷单用组单纯以10μmol/L的5-氮胞苷进行处理,其终浓度为1×10-8moL/L,连续诱导15d;5-氮胞苷 参三七皂苷Rg1预适应组分别加入0.1,1μmol/L参三七皂苷Rg1培养液处理24h,再各以10μmol/L的5-氮胞苷进行诱导,其终浓度为1×10-8moL/L,连续诱导15d。③实验评估:取第2代骨髓间充质干细胞,绘制生长曲线并计算群体倍增时间。观察诱导后骨髓间充质干细胞的生长形态学特征和细胞超微结构变化。激光共聚焦显微镜测定细胞表面积变化和细胞内钙离子浓度。结果:①5-氮胞苷诱导后骨髓间充质干细胞的生长形态学特征和细胞超微结构变化:骨髓间质干细胞胞体逐渐增大并伸出细长突起,在突起末端出现分支,部分相邻细胞的突起连接成网,形态学上表现出向心肌细胞方向转化的特征。其超微结构呈梭形,有明显的肌丝,细胞核呈单椭圆形,位于细胞中央,间质干细胞形似心肌细胞。②参三七皂苷Rg1预适应对5-氮胞苷诱导的骨髓间充质干细胞增殖特性的影响:与5-氮胞苷单用组比较,5-氮胞苷 参三七皂苷Rg1预适应组从第3天开始细胞数明显增加,细胞生长曲线均无明显的生长平台期,达到高峰后细胞数开始减少。③参三七皂苷Rg1预适应对5-氮胞苷诱导的骨髓间充质干细胞表面积的影响:与空白对照组骨髓间充质干细胞表面积比较,5-氮胞苷单用组明显降低,0.1,1μmol/L参三七皂苷Rg1预适应则能显著升高5-氮胞苷诱导的骨髓间充质干细胞表面积(P<0.01)。④参三七皂苷Rg1对5-氮胞苷诱导的骨髓间充质干细胞内游离钙水平的影响:与空白对照组比较,5-氮胞苷诱导4周后骨髓间充质干细胞内游离Ca2 相对荧光强度均明显升高(t=6.72,P<0.01),且5-氮胞苷 1μmol/L参三七皂苷Rg1预适应组升高幅度大于5-氮胞苷单用组(t=3.13,P<0.05)。结论:①参三七皂苷Rg1预适应在体外可显著刺激5-氮胞苷诱导的鼠骨髓间充质干细胞向心肌细胞转化和增殖,改善细胞形态,刺激细胞内钙离子增加。②参三七皂苷Rg1与5-氮胞苷对骨髓间充质干细胞向心肌细胞定向分化产生协同效应。     

Surveillance of triazole susceptibility of colonizing yeasts in patients with haematological malignancies

The species distribution and antifungal susceptibility of yeasts isolated from colonization cultures, and the contribution of previous fluconazole prophylaxis to the emergence of resistance, was prospectively studied in 87 neutropenic patients treated for haematological malignancies. Minimal inhibition concentrations (MICs) to fluconazole, itraconazole and voriconazole for 123 yeast isolates (65 Candida albicans, 22 Candida glabrata, 15 Saccharomyces cerevisiae, 5 Candida krusei, 5 Candida norvegensis, 11 other Candida spp.) were determined by E-test. Fluconazole MICs for Candida species and S. cerevisiae isolated from patients given prophylaxis were > or =2 mg/l in 24 of 40 (60%) isolates compared with 26 of 83 (31%) isolates from patients in the non-prophylaxis group (p =0.002). Among the non-albicans yeast species reduced susceptibility was common against itraconazole but was rarely found against voriconazole. Cross resistance between fluconazole and itraconazole was observed in 14 of all isolates, 4 of them also showing reduced susceptibility to voriconazole (MICs 4-8 mg/l). Fluconazole prophylaxis may contribute to emergence of less susceptible yeast strains also in patients with haematological malignancies not treated with allogeneic bone marrow transplantation.     

Strong Signature of Natural Selection within an FHIT Intron Implicated in Prostate Cancer Risk

Previously, a candidate gene linkage approach on brother pairs affected with prostate cancer identified a locus of prostate cancer susceptibility at D3S1234 within the fragile histidine triad gene (FHIT), a tumor suppressor that induces apoptosis. Subsequent association tests on 16 SNPs spanning approximately 381 kb surrounding D3S1234 in Americans of European descent revealed significant evidence of association for a single SNP within intron 5 of FHIT. In the current study, re-sequencing and genotyping within a 28.5 kb region surrounding this SNP further delineated the association with prostate cancer risk to a 15 kb region. Multiple SNPs in sequences under evolutionary constraint within intron 5 of FHIT defined several related haplotypes with an increased risk of prostate cancer in European-Americans. Strong associations were detected for a risk haplotype defined by SNPs 138543, 142413, and 152494 in all cases (Pearson's χ²=12.34, df 1, P=0.00045) and for the homozygous risk haplotype defined by SNPs 144716, 142413, and 148444 in cases that shared 2 alleles identical by descent with their affected brothers (Pearson's χ²=11.50, df 1, P=0.00070). In addition to highly conserved sequences encompassing SNPs 148444 and 152413, population studies revealed strong signatures of natural selection for a 1 kb window covering the SNP 144716 in two human populations, the European American (π=0.0072, Tajima's D=3.31, 14 SNPs) and the Japanese (π=0.0049, Fay & Wu's H=8.05, 14 SNPs), as well as in chimpanzees (Fay & Wu's H=8.62, 12 SNPs). These results strongly support the involvement of the FHIT intronic region in an increased risk of prostate cancer.     

Pharmacological interventions for improved colonic anastomotic healing: A meta-analysis

AIM: To identify pharmaceuticals for the prophylaxis of anastomotic leakage (AL), we systematically reviewed studies on anastomosis repair after colorectal surgery.METHODS: We searched PubMed and EMBASE for articles published between January 1975 and December 2012. We included studies in English with the primary purpose of promoting healing of anastomoses made in the colon or rectum under uncomplicated conditions. We excluded studies on adverse events from interventions, nutritional interventions or in situ physical supporting biomaterials. The primary outcome was biomechanical strength or AL. We performed meta-analyses on therapeutic agents investigated by three or more independent research groups using the same outcome. The DerSimonian-Laird method for random effects was applied with P < 0.05.RESULTS: Of the 56 different therapeutic agents assessed, 7 met our inclusion criteria for the meta-analysis. The prostacyclin analog iloprost increased the weighted mean of the early bursting pressure of colonic anastomoses in male rats by 60 mmHg (95%CI: 30-89) vs the controls, and the immunosuppressant tacrolimus increased this value by 29 mmHg (95%CI: 4-53) vs the controls. Erythropoietin showed an enhancement of bursting pressure by 45 mmHg (95%CI: 14-76). The anabolic compound growth hormone augmented the anastomotic strength by 21 mmHg (95%CI: 7-35), possibly via the up-regulation of insulin-like growth factor-1, as this growth factor increased the bursting pressure by 61 mmHg (95%CI: 43-79) via increased collagen deposition. Hyperbaric oxygen therapy increased the bursting pressure by 24 mmHg (95%CI: 13-34). Broad-spectrum matrix metalloproteinase inhibitors increased the bursting pressure by 48 mmHg (95%CI: 31-66) on postoperative days 3-4. In the only human study, the AL incidence was not significantly reduced in the 103 colorectal patients treated with aprotinin (11.7%) compared with the 113 placebo-treated patients (9.7%).CONCLUSION: This systematic review identified only one randomized clinical trial and seven therapeutic agents from pre-clinical models that could be explored further for the prophylaxis of AL after colorectal surgery.     

Mutations in CYP1B1 cause primary congenital glaucoma by reduction of either activity or abundance of the enzyme

Primary congenital glaucoma (PCG) is an autosomal recessive disorder caused predominantly by mutations in the CYP1B1 gene. A total of five frequent single nucleotide polymorphisms (SNPs) have been identified in the coding sequence of CYP1B1: rs10012C>G (p.R48G), rs1056827G>T (p.A119S), rs1056836C>G (p.V432L), rs1056837C>T (p.D449D), and rs1800440A>G (p.N453S). We performed a functional characterization of four common CYP1B1 variants presenting different coding SNP haplotypes (RAVDN, GSLDN, RALDS, and RALDN) and five CYP1B1 mutations reported for PCG patients: c.182G>A (p.G61E), c.608A>G (p.N203S), c.1033_1035del (p.L343del), c.241 T>A (p.Y81N), and c.685G>A (p.E229 K). Each mutation was embedded in its corresponding background SNP haplotype. The common variants revealed variation in enzymatic activity; among them, RAVDN showed the highest activity. Mutants p.G61E, p.N203S, and p.L343del each revealed a residual activity (<10%) of their respective haplotype. The microsomal CYP1B1 abundance relative to total protein also showed variation in common variants and a significant reduction in p.L343del, p.Y81N, and p.E229 K. The free energy of folding (DeltaDeltaG) values suggest that the lower stability of the mutants is one key property leading to the experimentally observed lower protein abundance. Our new measure of relative enzymatic activity (U/mg total protein), which combines activity and abundance values, was significantly lower for all five mutations compared to the corresponding background haplotype. We classified p.Y81N and p.E229 K not as mutations but as hypomorphic alleles, since their relative activity values are intermediate between bona fide mutations and the common variant with the lowest activity (RALDS). We propose that CYP1B1 mutations can act by either reducing enzymatic activity (p.G61E and p.N203S), reducing the abundance of the enzyme (p.Y81N and p.E229 K), or both (p.L343del).