Effects of hypothermic hypoxia on anaerobic energy metabolism in isolated anuran livers

Many lower vertebrates (reptilian and amphibian species) are capable of surviving natural episodes of hypoxia and hypothermia. It is by specific metabolic adaptations that anurans are able to tolerate prolonged exposure to harsh environmental stresses. In this study, it was hypothesized that livers from an aquatic frog would possess an inherent metabolic ability to sustain high levels of ATP in an isolated organ system, providing insight into a metabolic system that is well-adapted for low temperature in vitro organ storage. Frogs of the species, R. pipiens were acclimated at 20 °C and at 5 °C. Livers were preserved using a clinical preservation solution after flushing. Livers from 20 °C-acclimated frogs were stored at 20 °C and 5 °C and livers from 5 °C-acclimated frogs were stored at 5 °C. The results indicated that hepatic adenylate status was maintained for 96 h during 5 °C storage, but not longer than 4–10 h during 20 °C storage. In livers from 5 °C-acclimated animals subjected to 5 °C storage, ATP was maintained at 100% throughout the 96-h period. Warm acclimation (20 °C) and 20 °C storage resulted in poorer maintenance of ATP; energy charge values dropped to 0.50 within 2 h and by 24 h, only 24% of control ATP remained. Lactate levels remained less than 25 μ mol/g dry weight in all 5 °C-stored livers; 20 °C-stored livers exhibited greater accumulation of this anaerobic end-product (lactate reached 45–50 μ mol/g by 10 h). The data imply that hepatic adenylate status is largely dependent on exposure to hypothermic hypoxia and although small amounts of ATP were accounted for by anaerobic glycolysis, there must have been either a substantial reduction in cellular energy-utilization or an efficient use of low oxygen tensions. Accepted: 24 August 1998     

MicroRNA‐351 eases insulin resistance and liver gluconeogenesis via the PI3K/AKT pathway by inhibiting FLOT2 in mice of gestational diabetes mellitus

Gestational diabetes mellitus (GDM) is known as different degree glucose intolerance that is initially identified during pregnancy. MicroRNAs (miRs) may be a potential candidate for treatment of GDM. Herein, we suggested that miR‐351 could be an inhibitor in the progression of GDM via the phosphoinositide 3‐kinase/protein kinase B (PI3K/AKT) pathway. Microarray analysis was used to identify differentially expressed genes and predict miRs regulating flotillin 2 (FLOT2). Target relationship between miR‐351 and FLOT2 was verified. Gestational diabetes mellitus mice were treated with a series of mimic, inhibitor and small interfering RNA to explore the effect of miR‐351 on insulin resistance (IR), cell apoptosis in pancreatic tissues and liver gluconeogenesis through evaluating GDM‐related biochemical indexes, as well as expression of miR‐351, FLOT2, PI3K/AKT pathway‐, IR‐ and liver gluconeogenesis‐related genes. MiR‐351 and FLOT2 were reported to be involved in GDM. FLOT2 was the target gene of miR‐351. Gestational diabetes mellitus mice exhibited IR and liver gluconeogenesis, up‐regulated FLOT2, activated PI3K/AKT pathway and down‐regulated miR‐351 in liver tissues. Additionally, miR‐351 overexpression and FLOT2 silencing decreased the levels of FLOT2, phosphoenolpyruvate carboxykinase, glucose‐6‐phosphatase, fasting blood glucose, fasting insulin, total cholesterol, triglyceride, glyeosylated haemoglobin and homeostasis model of assessment for IR index (HOMA‐IR), extent of PI3K and AKT phosphorylation, yet increased the levels of HOMA for islet β‐cell function, HOMA for insulin sensitivity index and glucose transporter 2 expression, indicating reduced cell apoptosis in pancreatic tissues and alleviated IR and liver gluconeogenesis. Our results reveal that up‐regulation of miR‐351 protects against IR and liver gluconeogenesis by repressing the PI3K/AKT pathway through regulating FLOT2 in GDM mice, which identifies miR‐351 as a potential therapeutic target for the clinical management of GDM.     

Fasciola hepatica: A proteolytic digestive enzyme

A proteolytic enzyme in the gut exudate of the common liver fluke has been purified. The enzyme is specific for globin with a pH optimum of 3.9–4.0 and breaks the protein down to peptides and a small percentage of free amino acids. Collagenase activity at pH 7.5 copurifies with the main globinolytic enzyme. The enzyme has a molecular weight of 12,000 and does not appear to be antigenic in fluke-infected animals.     

Characterization by photoaffinity labeling of a steroid binding protein in rat liver plasma membrane

Summary The mechanism of steroid uptake by the cell remains controversial. [³H]R5020 was utilized to characterize by photoaffinity labeling the steroid binding site in plasma membrane. This binding was saturable, reversible and had one type of binding site (K _d = 33 ± 4 nm, B _max = 32 ± 2 pmol/mg). [³H]R5020 could be prevented from binding by a variety of steroids (cortisol, progesterone, deoxycorticosterone, and levonorgestrel); estradiol did not have affinity for this binding site. The kinetics of R5020 photoactivation was time dependent and saturable. SDS-PAGE showed a specific band which corresponded to a 53-kDa peptide. The sucrose density gradient analysis has revealed the existence of a protein with a sedimentation coefficient of 3.6 ± 0.2 S. This polypeptide shows different characteristics than cytosolic steroid receptor or serum steroid binding proteins. This binding protein could correspond to the steroid binding site previously found in the plasma membrane.This work was supported by grants PB85-0461 from the Comisión Asesora de Investigatión Científica y Técnica and PGV-8612 from the Departamento de Educatión, Universidades e Investigation del Gobierno Vasco. We thank Roussel-Uclaf (France) for the nonradioactive RU-steroids kindly provided.     

非酒精性脂肪性肝病的研究进展

非酒精性脂肪性肝病(NAFLD)在西方国家较为常见,近年来在我国的发病呈上升趋势,且发展逐渐低龄化。非酒精性脂肪性肝病患者可能因持续性肝损伤而导致纤维化进展,可与慢性病毒性肝炎和酒精性肝病一样发展到终末期肝硬化,并出现肝硬化严重并发症,也有可能发展成肝癌,最终需要肝移植治疗。它严重危害人类的健康,影响人类的生活及生存质量。多因素的发病机制使其愈来愈被人们所重视,研究和了解非酒精性脂肪性肝病的流行病学、发病机制、诊断及治疗方法,对人类非常重要,如果在疾病的早期,也就是单纯性脂肪肝阶段就对疾病进行干预,这样可以取得很好的治疗效果,NAFLD是人类在本世纪需要面对的疾病之一,因此研究它的发病机制及治疗方法是非常必要的。     

Impact of CYP3A5 and MDR-1 gene polymorphisms on the dose and level of tacrolimus among living-donor liver transplanted patients: single center experience

Aim of work: To assess the impact of Cytochrome P450 3A5 (CYP3A5) and multidrug resistance-1 gene (MDR-1) single nucleotide polymorphisms on the dose and blood level of tacrolimus among liver transplanted patients.

Patients and methods: We enrolled a prospective study of 41 liver transplanted patients. Dose-adjusted trough blood concentration (C/D ratio) was calculated. Polymerase chain reaction-restriction fragment length polymorphism followed by sequencing was done for genotyping of CYP3A5*3 (6986A?>?G).

Results: At 1 week, 1 and 3 months C/D ratio were significantly lower in CYP3A5 expressers *1/*1 patients compared to non-expressers *3/*3.

Conclusion: CYP3A5 (6986A?>?G) genotype, rather than MDR-1 (2677G?>?A/T) variant, has an impact on tacrolimus pharmacokinetics.     

DNA methylation of loci within ABCG1 and PHOSPHO1 in blood DNA is associated with future type 2 diabetes risk

Identification of subjects with a high risk of developing type 2 diabetes (T2D) is fundamental for prevention of the disease. Consequently, it is essential to search for new biomarkers that can improve the prediction of T2D. The aim of this study was to examine whether 5 DNA methylation loci in blood DNA (ABCG1, PHOSPHO1, SOCS3, SREBF1, and TXNIP), recently reported to be associated with T2D, might predict future T2D in subjects from the Botnia prospective study. We also tested if these CpG sites exhibit altered DNA methylation in human pancreatic islets, liver, adipose tissue, and skeletal muscle from diabetic vs. non-diabetic subjects. DNA methylation at the ABCG1 locus cg06500161 in blood DNA was associated with an increased risk for future T2D (OR = 1.09, 95% CI = 1.02–1.16, P-value = 0.007, Q-value = 0.018), while DNA methylation at the PHOSPHO1 locus cg02650017 in blood DNA was associated with a decreased risk for future T2D (OR = 0.85, 95% CI = 0.75–0.95, P-value = 0.006, Q-value = 0.018) after adjustment for age, gender, fasting glucose, and family relation. Furthermore, the level of DNA methylation at the ABCG1 locus cg06500161 in blood DNA correlated positively with BMI, HbA1c, fasting insulin, and triglyceride levels, and was increased in adipose tissue and blood from the diabetic twin among monozygotic twin pairs discordant for T2D. DNA methylation at the PHOSPHO1 locus cg02650017 in blood correlated positively with HDL levels, and was decreased in skeletal muscle from diabetic vs. non-diabetic monozygotic twins. DNA methylation of cg18181703 (SOCS3), cg11024682 (SREBF1), and cg19693031 (TXNIP) was not associated with future T2D risk in subjects from the Botnia prospective study.     

溶瘤腺病毒ZD55对类肝癌干细胞的抑制效应

溶瘤腺病毒能够靶向和杀死癌症干细胞,被认为是一种很有前景的抗癌药物.已有研究表明,溶瘤腺病毒ZD55能够靶向肝癌,并且表现出明显的细胞毒性效应.然而,其对肝癌干细胞是否具有同样地杀伤效力仍需进一步探讨.利用悬浮培养富集类肝癌干细胞,并验证其肝癌干细胞的特征.进一步通过MTT、结晶紫染色、Hoechst染色、Western blot和流式细胞术等检测ZD55对类肝癌干细胞的细胞存活率、凋亡诱导和病理效应等.结果发现,悬浮培养的类肝癌干细胞具有自我更新和分化能力、高表达干细胞相关转录因子(如NANOG和OCT4)、处于静息状态和具有耐药性等特性,溶瘤腺病毒处理后表现出明显的细胞毒性效应和杀伤特性,类肝癌干细胞的最低生存率仅为26.7%.ZD55能够非常明显地诱导类肝癌干细胞凋亡,其凋亡率最高达到60%.因此,ZD55可能会成为靶向肝癌干细胞的一种很有前景的治疗药物,对肝癌的临床治疗具有一定的应用价值.     

Action mechanism of tachyplesin I and effects of PEGylation

PEGylation of protein and peptide drugs is frequently used to improve in vivo efficacy. We investigated the action mechanism of tachyplesin I, a membrane-acting cyclic antimicrobial peptide from Tachypleus tridentatus and the effects of PEGylation on the mechanism. The PEGylated peptide induced the leakage of calcein from egg yolk l-α-phosphatidylglycerol/egg yolk l-α-phosphatidylcholine large unilamellar vesicles similarly to the parent peptide. Both peptides induced lipid flip-flop coupled to leakage and was translocated into the inner leaflet of the bilayer, indicating that tachyplesin I forms a toroidal pore and that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. Despite their similar activities against model membranes, the peptides showed very different biological activities. The cytotoxicity of tachyplesin I was greatly reduced by PEGylation, although the antimicrobial activity was significantly weakened. We investigated the enhancement of the permeability of inner membranes induced by the peptides. Our results suggested that outer membranes and peptidoglycan layers play an inhibitory role in the permeation of the PEG moiety. Furthermore, a reduction in DNA binding by PEGylation may also contribute to the weak activity of the PEGylated peptide.