Characterization of angiotensin II-receptor subtypes in podocytes

Glomerular podocytes play a key role in maintaining the integrity of the glomerular filtration barrier. This function may be regulated by angiotensin II (Ang II) through activation of cell-surface receptors. Although studies suggest that podocytes express receptors for Ang II, the Ang II binding site has not been characterized with radioligand binding techniques. We therefore used iodine 125-labeled Ang II to monitor Ang II-receptor density during differentiation of a mouse podocyte cell line. Scatchard analyses of equilibrium binding data revealed a single class of high-affinity binding sites (dissociation constant approximately 3 nmol/L) in both differentiated and nondifferentiated cells. During differentiation, the density of Ang II-receptor sites increased roughly 15-fold in differentiated podocytes (maximal density of specific binding sites 881 fmol/mg protein) compared with that in nondifferentiated cells (52 fmol/mg protein; P<.005). Glomerular podocytes expressed messenger RNA for AT1A, AT1B, and AT2 receptor subtypes, and competitive binding studies found that differentiated podocytes expressed mostly AT1 receptors (approximately 75%) with lesser amounts of AT2 (approximately 25%). Up-regulation of Ang II-receptor number was associated with increased Ang II-receptor responsiveness, as evidenced by enhanced Ang II-stimulated inositol phosphate (IP) generation and incorporation of tritiated thymidine. Both [3H]thymidine incorporation and IP generation were mediated by AT1-receptor activation. These data suggest that glomerular podocytes express a high-affinity binding site for Ang II with pharmacologic characteristics of both AT1 and AT2 receptors. This receptor site is up-regulated during podocyte differentiation, and receptor activation induces both IP generation and DNA synthesis by AT1-dependent mechanisms. We speculate that activation of podocyte Ang II receptors contributes to glomerular damage in disease states.     

Isothiafludine,a novel non-nucleoside compound,inhibits hepatitis B virus replication through blocking pregenomic RNA encapsidation

Aim： To investigate the action of isothiafludine （NZ-4）, a derivative of bis-heterocycle tandem pairs from the natural product leucamide A, on the replication cycle of hepatitis B virus （HBV） in vitro and in vivo. Methods： HBV replication cycle was monitored in HepG2.2.15 cells using qPCR, qRT-PCR, and Southern and Northern blotting. HBV protein expression and capsid assembly were detected using Western blotting and native agarose gel electrophoresis analysis. The interaction of pregenomic RNA （pgRNA） and the core protein was investigated by RNA immunoprecipitation. To evaluate the anti-HBV effect of NZ-4 in vivo, DHBV-infected ducks were orally administered NZ-4 （25, 50 or 100 mg.k~l{1-1） for 15 d. Results： NZ-4 suppressed intracellular HBV replication in HepG2.2.15 cells with an IC~o value of 1.33 pmol/L, whereas the compound inhibited the cell viability with an IC5o value of 50.4 pmol/L. Furthermore, NZ-4 was active against the replication of various drug- resistant HBV mutants, including 3TC/ETV-dual-resistant and ADV-resistant HBV mutants. NZ-4 （5, 10, and 20 pmol/L） concentration- dependently reduced the encapsidated HBV pgRNA, resulting in the assembly of replication-deficient capsids in HepG2.2.15 cells. Oral administration of NZ-4 dose-dependently inhibited DHBV DNA replication in the DHBV-infected ducks. Conclusion： NZ-4 inhibits HBV replication by interfering with the interaction between pgRNA and HBcAg in the capsid assembly process thus increasing the replication-deficient HBV capsids. Such mechanism of action might provide a new therapeutic strategy to combat H BV infection.     

New clues to identify proteins correlated with Attractin

In our previous study, the age‐dependent testis vacuolisation and sperm dysfunction were found in Attractin (Atrn)‐deficient mice, Atrn^mg‐3J, which is a null or nearly null allele. To explore the potential mechanism involved in these pathological changes, Attractin knock‐down in mouse Sertoli cells TM4 (psiAtrn‐TM4) was successfully established by stable RNA interference. The TM4 transfected by psiRNA‐hH1 (psiRNA‐TM4) was the control group, in which the expression of Atrn was not affected. The proteomic changes among the psiAtrn‐TM4, primary cultures of Sertoli cells of Atrn^mg‐3J (mu‐Sc) and control cells (psiRNA‐TM4) were compared by two‐dimensional gel electrophoresis. Fifteen differentially expressed protein spots of those cells were identified by matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry and the NCBI proteins database. Except the decreased expression of superoxide dismutase (SOD), there were several novel proteins associated with Atrn function, including downregulated ATP synthase, peroxiredoxin 2 and upregulated caspase 6, ketohexokinase, etc., in psiAtrn‐TM4 and mu‐Sc. These data suggest that these differentially expressed proteins may be associated with the function of Atrn in Sertoli cells, thus providing a new clue to interpret the mechanism of testis degeneration in Atrn mutants.     

Unfavorable hip stress distribution after Legg–Calvé–Perthes syndrome: A 25‐year follow‐up of 135 hips

To study the effect of hip and pelvis geometry on development of the hip after Perthes disease, we determined the resultant hip force and contact hip stress distribution in a population of 135 adult hips of patients who had been treated for Perthes disease in childhood. Contra‐lateral hips with no record of disease were taken as the control population. Biomechanical parameters were determined by mathematical models for resultant hip force in one‐legged stance and for contact hip stress, which use as an input the geometrical parameters assessed from anteroposterior radiographs. The mathematical model for stress was upgraded to account for the deviation of the femoral head shape from spherical. No differences were found in resultant hip force and in peak contact hip stress between the hips that were in childhood subject to Perthes disease and the control population, but a considerable (148%) and significant (p < 0.001) difference was found in the contact hip stress gradient index, expressing an unfavorable, steep decrease of contact stress at the lateral acetabular rim. This finding indicates an increased risk of early coxarthritis in hips subject to Perthes disease. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:8–16, 2014.     

Outcomes of Long Tapered Hydroxyapatite-Coated Stems in Revision Total Hip Arthroplasty

The purpose of this study was to evaluate the outcome of femoral component revisions using a long tapered HA coated femoral revision stem. Between 2001 and 2008, 55 femoral component revisions were performed using this stem. Forty-one patients were available for follow up evaluation at average of 59 months. The clinical results were evaluated using the HHS and serial radiographs were evaluated for loosening. The mean HHS was 71 (range 22–100). Three hips required revision of KAR stem (1 aseptic loosening, 1 infection, 1 limb length discrepancy). Only one prosthesis demonstrated radiographic evidence of subsidence. Our study suggests that long tapered HA coated revision femoral components can provide stable fixation and in-growth in cases where there is good proximal femoral bone stock and favorable canal geometry.     

Different patterns of cerebellar abnormality and hypomyelination between POLR3A and POLR3B mutations

Background

Mutations of POLR3A and POLR3B have been reported to cause several allelic hypomyelinating disorders, including hypomyelination with hypogonadotropic hypogonadism and hypodontia (4H syndrome). Patients and methods: To clarify the difference in MRI between the two genotypes, we reviewed MRI in three patients with POLR3B mutations, and three with POLR3A mutations. Results: Though small cerebellar hemispheres and vermis are common MRI findings with both types of mutations, MRI in patients with POLR3B mutations revealed smaller cerebellar structures, especially vermis, than those in POLR3A mutations. MRI also showed milder hypomyelination in patients with POLR3B mutations than those with POLR3A mutations, which might explain milder clinical manifestations. Conclusions: MRI findings are distinct between patients with POLR3A and 3B mutations, and can provide important clues for the diagnosis, as these patients sometimes have no clinical symptoms suggesting 4H syndrome.     

从运动神经元的TDP-43蛋白表达和ADAR2活性探讨肌萎缩侧索硬化症的发病机制

肌萎缩性侧索硬化症(ALS)是以上下两级运动神经元进行性丢失为特征的神经系统变性疾病,是运动神经元病(MND)中最常见的类型。运动神经元中的病理性TDP-43是ALS的病理特征。此外,散发性ALS运动神经元中作用于RNA的次黄嘌呤腺苷脱氢酶(ADAR2)减少、具有未经编辑Q/R位点的谷氨酸受体2(GluR2)表达增加,α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体(AMPAR)属性受影响,Ca2+通透性增加,Ca2+流入胞质增加导致神经元死亡。ALS运动神经元死亡包含病理性TDP-43和ADAR2活性下降,两种病理变化可能在细胞死亡中存在一定联系。ADAR2 mRNA是TDP-43蛋白的靶RNA,TDP-43蛋白在ADAR2的表达中起调节作用。近年来,研究者探讨ALS的基因治疗可能性:动物实验结果提示,外周静脉给予9型腺相关病毒载体(AAV9),可上调鼠运动神经元ADAR2,引发外源性ADAR2在中枢神经元表达,从而有效防治运动功能障碍。运动神经元的获救可能与TDP-43基因的正常表达有关。AAV9介导的ADAR2基因植入可能为ALS的基因治疗提供新的前景。     

Beclin 1 基因对恶性胶质瘤细胞 U87 自噬活性和增殖的影响简

目的研究BeclinJ基因对U87胶质瘤细胞自噬和增殖的影响。方法实验分5组：空白对照组．pSUPER—Bec组（表达BeclinsiRNA）与其阴性对照组（pSUPER—non组）,pcDNA3．1-Bec组（表达BeclinJ基因质粒）与其阴性对照组（pcDNA3．1-non组）。分别转染U87细胞,采用免疫印迹检测Beclin1、LC3和p62蛋白在各组的表达;用氚标胸腺嘧啶脱氧核苷（3H-TdR）检测各组细胞增殖率。结果转染后48h,pSUPER—Bec组Beclin1、LC3B—11蛋白表达下降,p62蛋白表达升高。pcDNA3．1-Bec组Beclin1、LC3B-Ⅱ蛋白表达升高,p62蛋白表达下降。与空白对照组或pcDNA3．1-non组相比．pcDNA3．1-Bec组细胞增殖速度降低（P〈0．05）,其他各组细胞增殖速度无明显差异。结论恶性胶质瘤细胞中自噬相关基因Beclinj表达下降,过表达Beclinl蛋白能增强细胞的自噬活性,抑制细胞的增殖活性。