非酒精性脂肪肝大鼠脂质代谢及病理变化的动态观察

目的通过高脂饮食建立NAFLD大鼠模型,连续监测4～16周模型动物肝功能、脂质代谢、胰岛素抵抗及肝细胞凋亡在NAFLD进展过程中的变化情况及相互关系,为该模型在脂肪肝发病机制、脂肪肝治疗药物评价等方面的应用提供参考依据。方法 SD大鼠50只,除正常对照组外,其余动物饲喂高脂饲料,分别检测4,8,12,16周大鼠血清GLU、CHO、TG、HDL、LDL、GPT、GOT及胰岛素水平,肝脏组织切片进行病理学及细胞凋亡观察,进一步分析大鼠肝功能、脂质代谢、胰岛素抵抗及肝细胞凋亡对肝组织病理改变的影响。结果模型组大鼠4周后就出现肝功能损伤,脂质代谢紊乱、胰岛素抵抗,肝细胞凋亡8 W后明显增加,肝细胞脂变及炎症为肝组织病理变化的主要特征,且造模时间越长,病变程度越严重。结论经过高脂饲料的喂养,SD大鼠在4～16周内可形成病变程度逐步加重的NAFLD模型,肝功能损伤,脂质代谢紊乱及肝细胞凋亡是引起非酒精性脂肪肝中脂肪变性和炎症的重要因素,该模型可应用于脂肪肝治疗药物评价等方面。     

几种旱生灌木种子萌发对干旱胁迫的响应

以不同渗透势的PEG(6 0 0 0 )溶液 (- 0 .3～ - 2 .7MPa)为模拟干旱胁迫条件 ,研究了柠条 (Caraganakorshinskii)、花棒 (Hedysarumscoparium)和白沙蒿 (Artemisiasphaerocephala)种子发芽、幼苗生长和累积吸水率对干旱胁迫的响应 ,讨论了参试种子发芽特性、初生根长度与幼苗建植成活率的关系 .结果表明 ,干旱胁迫下柠条发芽率最高 ,其次为花棒 ,白沙蒿最低 . - 0 .3～ - 0 .6MPa渗透势下白沙蒿发芽 10d的初生根长度显著大于花棒和柠条 (P <0 .0 5 ) ,三者的测量值依次为 7.9、4 .5和 3.1cm .干旱胁迫条件下参试种子发芽率与种子 72h累积吸水率均呈极显著的正相关 (P <0 .0 1) .     

Characterization of a K26Q site-directed mutant of human parathyroid hormone expressed in yeast

Human parathyroid hormone (hPTH) is susceptible to proteolytical cleavage both in humans and when expressed as a secretory product in Escherichia coli (H?gseth, A., Blingsmo, O. R., Saether, O., Gautvik, V. T., Holmgren, E., Hartmanis, M., Josephson, S., Gabrielsen, O. S., Gordeladze, J. O., Alestr?m, P., and Gautvik, K. M. (1990) J. Biol. Chem. 265, 7338-7344) and Saccharomyces cerevisiae (Gabrielsen, O. S., Reppe, S., Saether, O., Blingsmo, O. R., Sletten, K., Gordeladze, J. O., H?gset, A., Gautvik, V. T., Alestr?m, P., Oyen, T. B., and Gautvik, K. M. (1990) Gene (Amst.) 90, 255-262). In the latter system, one major site of cleavage was identified (Arg25-Lys26 decreased Lys27). To produce hPTH resistant to this proteolytic processing, a point mutation changing Lys26 to Gln was introduced, and the modified gene expressed in S. cerevisiae as a fusion protein with the alpha-factor leader sequence. The resulting major form of hPTH secreted to the growth medium was of full length showing that the mutation had eliminated internal processing. Consequently, the yield of the mutant hormone was significantly higher than obtained with the natural peptide. Using improved purification procedures, a significantly higher purity was also obtained. The secreted mutant hPTH-(1-84,Q26) had the correct size, full immunological reactivity with two different hPTH antisera, correct amino acid composition and N-terminal sequence, and correct mass as determined by mass spectrometry. Furthermore, the introduced mutation did not reduce the biological activity of the hormone as judged from its action in three biological assay systems: 1) a hormone-sensitive osteoblast adenylate cyclase assay; 2) an in vivo calcium mobilizing assay in rats; and 3) an in vitro bone resorption assay.     

Mutations in radial spoke head genes and ultrastructural cilia defects in East-European cohort of primary ciliary dyskinesia patients

Primary ciliary dyskinesia (PCD) is a rare (1/20,000), multisystem disease with a complex phenotype caused by the impaired motility of cilia/flagella, usually related to ultrastructural defects of these organelles. Mutations in genes encoding radial spoke head (RSPH) proteins, elements of the ciliary ultrastructure, have been recently described. However, the relative involvement of RSPH genes in PCD pathogenesis remained unknown, due to a small number of PCD families examined for mutations in these genes. The purpose of this study was to estimate the involvement of RSPH4A and RSPH9 in PCD pathogenesis among East Europeans (West Slavs), and to shed more light on ultrastructural ciliary defects caused by mutations in these genes. The coding sequences of RSPH4A and RSPH9 were screened in PCD patients from 184 families, using single strand conformational polymorphism analysis and sequencing. Two previously described (Q109X; R490X) and two new RSPH4A mutations (W356X; IVS3_2-5del), in/around exons 1 and 3, were identified; no mutations were found in RSPH9. We estimate that mutations in RSPH4A, but not in RSPH9, are responsible for 2-3% of cases in the East European PCD population (4% in PCD families without situs inversus; 11% in families preselected for microtubular defects). Analysis of the SNP-haplotype background provided insight into the ancestry of repetitively found mutations (Q109X; R490X; IVS3_2-5del), but further studies involving other PCD cohorts are required to elucidate whether these mutations are specific for Slavic people or spread among other European populations. Ultrastructural defects associated with the mutations were analyzed in the transmission electron microscope images; almost half of the ciliary cross-sections examined in patients with RSPH4A mutations had the microtubule transposition phenotype (9+0 and 8+1 pattern). While microtubule transposition was a prevalent ultrastructural defect in cilia from patients with RSPH4A mutations, similar defects were also observed in PCD patients with mutations in other genes.     

Mechanisms of attenuation of abdominal sepsis induced acute lung injury by ascorbic acid

Bacterial infections of the lungs and abdomen are among the most common causes of sepsis. Abdominal peritonitis often results in acute lung injury (ALI). Recent reports demonstrate a potential benefit of parenteral vitamin C [ascorbic acid (AscA)] in the pathogenesis of sepsis. Therefore we examined the mechanisms of vitamin C supplementation in the setting of abdominal peritonitis-mediated ALI. We hypothesized that vitamin C supplementation would protect lungs by restoring alveolar epithelial barrier integrity and preventing sepsis-associated coagulopathy. Male C57BL/6 mice were intraperitoneally injected with a fecal stem solution to induce abdominal peritonitis (FIP) 30 min prior to receiving either AscA (200 mg/kg) or dehydroascorbic acid (200 mg/kg). Variables examined included survival, extent of ALI, pulmonary inflammatory markers (myeloperoxidase, chemokines), bronchoalveolar epithelial permeability, alveolar fluid clearance, epithelial ion channel, and pump expression (aquaporin 5, cystic fibrosis transmembrane conductance regulator, epithelial sodium channel, and Na(+)-K(+)-ATPase), tight junction protein expression (claudins, occludins, zona occludens), cytoskeletal rearrangements (F-actin polymerization), and coagulation parameters (thromboelastography, pro- and anticoagulants, fibrinolysis mediators) of septic blood. FIP-mediated ALI was characterized by compromised lung epithelial permeability, reduced alveolar fluid clearance, pulmonary inflammation and neutrophil sequestration, coagulation abnormalities, and increased mortality. Parenteral vitamin C infusion protected mice from the deleterious consequences of sepsis by multiple mechanisms, including attenuation of the proinflammatory response, enhancement of epithelial barrier function, increasing alveolar fluid clearance, and prevention of sepsis-associated coagulation abnormalities. Parenteral vitamin C may potentially have a role in the management of sepsis and ALI associated with sepsis.     

Altered oxido-reductive state in the diabetic heart: loss of cardioprotection due to protein disulfide isomerase

Diabetes is associated with an increased risk of heart failure, in part explained by endoplasmic reticulum stress and apoptosis. Protein disulfide isomerase (PDI) prevents stressed cardiomyocytes apoptosis. We hypothesized that diabetes impairs PDI function by an alteration in its oxido-reductive state. Myocardial biopsies harvested from the anterolateral left ventricular wall from diabetic (n = 7) and nondiabetic (n = 8) patients were used to assess PDI expression and cardiomyocyte death. A mouse model of diabetes (streptozotocin injection, 130 mg/mL) was used to study PDI expression and its redox state after ischemia/reperfusion injury induced by 30-min occlusion of the left anterior coronary artery followed by reperfusion. Transthoracic echocardiography was performed to assess cardiac remodeling after 1 wk. Western blot analysis was used to analyze PDI expression, and methoxy-polyethyleneglycol-maleimide was used to assess its redox state. Dehydroascorbate (DHA) administration was used to restore the PDI redox state. Diabetic patients had a greater number of transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells than nondiabetic patients despite a greater myocardial PDI expression suggesting altered PDI function. Diabetic mice had a worse postinfarction remodeling associated with an altered PDI redox state. DHA treatment restored functional PDI redox state and ameliorated post-myocardial infarction remodeling. An increase in PDI levels with a paradoxical decrease of its active form occurs in the diabetic heart after ischemia and may explain the lack of protective effects of PDI in diabetes. Restoration of PDI redox state prevents adverse remodeling. The potential significance of these findings deserves to be validated in a clinical setting.     

未净化Z：ZCLA长爪沙鼠封闭群繁育及遗传稳定性分析

目的分析未净化Z：ZCLA长爪沙鼠封闭群繁育和生长指标,并利用直接测序法分析其遗传稳定性。方法随机选择40对Z：ZCLA长爪沙鼠,记录其繁殖胎次、产子数等指标,分析其仔鼠的生长发育情况。遗传稳定性分析选择Z：ZCLA长爪沙鼠非同胞、非亲代个体33只,提取肝脏基因组DNA,PCR扩增D-Loop序列,产物纯化后双向测序,测序结果与Z：ZCLA长爪沙鼠标准序列比对。结果 Z：ZCLA长爪沙鼠每胎产仔7只,胎间隔多在20～60 d间,雄性体重高于雌性。遗传稳定性分析检测发现33只Z：ZCLA长爪沙鼠序列与标准序列完全一致。结论 Z：ZCLA长爪沙鼠群体内未发现遗传多态性,说明该群体具有较好的遗传稳定性。