Regulation of renal ouabain-resistant Na+-ATPase by leptin, nitric oxide, reactive oxygen species, and cyclic nucleotides: implications for obesity-associated hypertension

This study examined the effect of leptin on renal ouabain-resistant Na(+)-ATPase, which drives the reabsorption of about 10% of sodium transported in the proximal tubule. Chronic leptin administration (0.25 mg/kg s.c. twice daily for seven days) increased Na(+)-ATPase activity by 62.9%. This effect was prevented by the coadministration of superoxide dismutase mimetic, tempol, or the NADPH oxidase inhibitor, apocynin (2 mM in the drinking water). Acutely administered NO donors decreased Na(+)-ATPase activity. This effect was abolished by soluble guanylate cyclase inhibitor, ODQ, but not by protein kinase G inhibitors. Exogenous cGMP reduced Na(+)-ATPase activity, but its synthetic analogues, 8-bromo-cGMP and 8-pCPT-cGMP, were ineffective. The inhibitory effect of NO donors and cGMP was abolished by EHNA, an inhibitor of cGMP-stimulated phosphodiesterase (PDE2). Exogenous cAMP analogue and dibutyryl-cAMP increased Na(+)-ATPase activity and abolished the inhibitory effect of cGMP. Finally, the administration of superoxide-generating mixture (xanthine oxidase+hypoxanthine) increased Na(+)-ATPase activity. The results suggest that nitric oxide decreases renal Na(+)-ATPase activity by stimulating cGMP, which in turn activates PDE2 and decreases cAMP concentration. Increased production of reactive oxygen species may lead to the elevation of Na(+)-ATPase activity by scavenging NO and limiting its inhibitory effect. Chronic hyperleptinemia is associated with increased Na(+)-ATPase activity due to excessive oxidative stress.     

The Effects of Rosiglitazone,Metformin, and Diet with Exercise in Nonalcoholic Fatty Liver Disease

Our aım was to evaluate effects of metformin, rosiglitazone, and diet with exercise in nonalcoholic fatty liver disease. Forty-seven patients (mean age, 44±10 years; 17 female) whose ALT levels had been high for at least 6 months and with hepatosteatosis detected by liver biopsy and/or USG were enrolled in this study. Of these, 12 were treated with 850 mg/day metformin (group 1), 11 with 4 mg/day rosiglitazone (group 2), and 24 with diet and exercise (group 3) for 1 year. ALT normalization at months 6 and 12 was accepted as treatment response. Liver biopsy was performed in all patients in groups 1 and 2 before treatment and 12 patients (4 in group 1, 8 in group 2) after treatment; but in group 3 it was performed only in patients who approved this procedure (12 patients). Body mass index did not change in groups 1 and 2, but it decreased significantly in group 3 (30±3 to 28±2 kg/m²) at month 12. Treatment response rate was 33.3, 54.5, and 54.2% in groups 1, 2, and 3, respectively, at month 6. This rate was 22.2, 37.5, and 41.2 in groups 1, 2, and 3, respectively, at month 12. Rate of steatosis and stage of fibrosis did not change after treatment. Diet with exercise seems to be superior to metformin and rosiglitazone. Decreasing treatment response at month 12 compared to month 6 may be due to fluctuations of ALT levels. Treatment response should be evalulated histologically.     

抗原特异TCR Vα6-pIRES2-EGFP和TCRV β13-pIRES2-EGFP真核表达载体的构建及共转染研究

目的 构建携带弥漫大B型非霍奇金淋巴瘤(DLBL)相关抗原特异性的TCR Vα和TCR Vβ基因片段的真核表达载体TCR Vα-pIRES2.EGFP和TCR Vβ-plRES2-EGFP,将其共同转染Raji和Jurkat细胞.方法 前期研究从1例DLBL患者外周血T细胞中发现克隆性增殖T细胞受体(TCR)Vα6和Vβ13亚家族T细胞,在此基础上利用RT-PCR扩增TCR Va6和TCR Vβ13基因全长序列后,分别将其定向克隆入带有绿色荧光蛋白(EGFP)的真核表达载体pIRES2-EGFP,酶切和核酸序列测定分析方法鉴定重组质粒TCR Vα6-pIRKS2-EGFP和TCR Vβ13-pIRES2-EGFP的正确性;利用核转染技术(nueleofector)将其分别或共同转染Raji细胞和Jurkat细胞,24 h后利用激光共聚焦显微镜观察EGFP的瞬时表达情况,48h后利用实时定量PCR检测TCR Vα6和TCR Vβ13基因的表达情况,Western blot检测EGFP蛋白的表达情况.结果 获得来自DLBL患者的TCR Vα6和TCR Vβ13基因全长序列,酶切分析和核酸序列测定证实TCR Vα6-pIRES2-EGFP和TCR Vβ13-pIRES2-EGFP重组质粒构建正确;转染24 h后,激光共聚焦显微镜下可观察到EGFP的表达,40%以上细胞发出绿色荧光,单独转染和共转染组荧光产生情况相似;实时定量PCR在单独转染和共转染组均町检测到TCR Vα6和TCR Vβ13基因的表达,共转染组2基因的表达水平稍低于单独转染组;Western blot检测在单独转染和共转染组均显示EGFP蛋白的表达,2组的蛋白杂交带强度相似.结论 成功构建了DLBL特异性的TCR Vα6-pIRES2-EGFP和TCR Vβ13-pIRES2-EGFP真核表达质粒,两者可同时转染到细胞中,并实现了体外共表达.     

Peroxidasin Is Secreted and Incorporated into the Extracellular Matrix of Myofibroblasts and Fibrotic Kidney

Mammalian peroxidases are heme-containing enzymes that serve diverse biological roles, such as host defense and hormone biosynthesis. A mammalian homolog of Drosophila peroxidasin belongs to the peroxidase family; however, its function is currently unknown. In this study, we show that peroxidasin is present in the endoplasmic reticulum of human primary pulmonary and dermal fibroblasts, and the expression of this protein is increased during transforming growth factor-β1-induced myofibroblast differentiation. Myofibroblasts secrete peroxidasin into the extracellular space where it becomes organized into a fibril-like network and colocalizes with fibronectin, thus helping to form the extracellular matrix. We also demonstrate that peroxidasin expression is increased in a murine model of kidney fibrosis and that peroxidasin localizes to the peritubular space in fibrotic kidneys. In addition, we show that this novel pathway of extracellular matrix formation is unlikely mediated by the peroxidase activity of the protein. Our data indicate that peroxidasin secretion represents a previously unknown pathway in extracellular matrix formation with a potentially important role in the physiological and pathological fibrogenic response.Peroxidases are heme-containing enzymes with highly conserved structure, serving diverse functions in the plant and animal kingdom.¹ Peroxidases catalyze the oxidation of various substrates in the presence of H₂O₂. Mammalian peroxidases have an important role in several physiological processes including host defense and hormone biosynthesis. The family of mammalian peroxidases consists of myeloperoxidase, eosinophil peroxidase, lactoperoxidase, thyroid peroxidase, and the mammalian peroxidasin. Myeloperoxidase, eosinophil peroxidase, and lactoperoxidase have antimicrobial activity and serve in the first line of host defense, while thyroid peroxidase has an essential role in the biosynthesis of thyroid hormones.^2,3,4 The function of the mammalian peroxidasin is currently unknown. Peroxidases in plants and in lower animal species frequently participate in extracellular matrix (ECM) formation. In the presence of H₂O₂, peroxidases enzymatically cross-link extracellular proteins through tyrosine residues.⁵ ECM stabilization by dityrosine bridges is well-documented during sea urchin fertilization, where secreted ovoperoxidase is responsible for the formation of cross-links.⁶ Dityrosine formation is also involved in the stabilization of C. elegans cuticle, where dual oxidases, carrying both NADPH oxidase and peroxidase-like domains, provide hydrogen peroxide for the crosslinking reaction.⁷Peroxidasin (PXDN), a unique form of peroxidase was first identified in Drosophila melanogaster.⁸ Beside containing a peroxidase domain, which is highly homologous to other animal peroxidases, peroxidasin also contains protein domains characteristic for proteins of the ECM. Drosophila PXDN was found to be expressed in several stages of development, but the exact function remained unknown.⁸ Little is still known about the mammalian PXDN protein. A human homolog of Drosophila PXDN was originally identified as a p53-responsive gene product from a colon cancer cell line, but it was not characterized in detail.⁹ An independent cloning effort, using subtractive hybridization also led to the identification of the mammalian PXDN gene, which was originally named melanoma gene 50, based on the expression in melanoma samples.¹⁰ This latter study has characterized PXDN as a possible potent melanoma-associated antigen, but it did not examine the possible physiological role of the protein.Here we demonstrate that peroxidasin is expressed by human primary cells, including fibroblasts of different origin, where the protein is localized to the endoplasmic reticulum. On stimulation by transforming growth factor (TGF)-β1, differentiating myofibroblasts show increased expression of peroxidasin. The protein becomes secreted to the extracellular space where it is organized into a fibril-like network. We also show that this pathway of ECM formation is probably not mediated by the peroxidase activity of the protein. Our results suggest that beside the secretion of well-known constituents of the ECM, PXDN secretion by myofibroblasts is a novel way of ECM modification in wound repair and tissue fibrosis.     

Differential effects of statins on endogenous H2S formation in perivascular adipose tissue

Hydrogen sulfide (H₂S) is a new gasotransmitter synthesized enzymatically from l-cysteine in cytosol and is oxidized in mitochondria. In the cardiovascular system, H₂S regulates vascular tone, inhibits atherogenesis, and protects against myocardial ischemia–reperfusion injury. We examined the effect of statins on vascular H₂S production. Male Wistar rats received pravastatin (40 mg/kg/day) or atorvastatin (20 mg/kg/day) for 3 weeks and then H₂S formation was measured in aortic media, periaortic adipose tissue (PAAT) and the liver. Only atorvastatin increased H₂S production in PAAT whereas both statins stimulated its formation in the liver. Neither statin affected H₂S production in aortic media. H₂S formation in post-mitochondrial supernatant was higher than in mitochondria-containing supernatant and was not influenced by statins in any tissue. In addition, oxidation of exogenous H₂S in isolated liver mitochondria was slower in statin-treated than in control rats. These data indicate that statins increase net H₂S production by inhibiting its mitochondrial oxidation. Statins had no effect on the activity of H₂S-metabolizing enzyme, sulfide:quinone oxidoreductase, measured at saturating coenzyme Q concentration. Both statins reduced CoQ₉ concentration in plasma and liver, but only atorvastatin decreased CoQ₉ in PAAT. Atorvastatin attenuated phenylephrine-induced contraction of PAAT+ but not of PAAT? aortic rings. Effects of atorvastatin on net H₂S production, mitochondrial H₂S oxidation and aortic contractility were abolished by supplementation of exogenous CoQ₉. In conclusion, lipophilic atorvastatin, but not hydrophilic pravastatin, increases net H₂S production in perivascular adipose tissue by inhibiting its mitochondrial oxidation. This effect is mediated by statin-induced CoQ₉ deficiency and results in the augmentation of anticontractile effect of perivascular adipose tissue.     

Spatial and temporal distribution of Ki-67 proliferation marker, Bcl-2 and Bax proteins in the developing human tooth

Objective

To investigate the spatial and temporal expression of proliferation Ki-67 marker, pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins during early development of the human tooth.

Materials and methods

Histological sections of eight human conceptuses, 5–10 postovulatory weeks old, were used for immunolocalization for Ki-67, Bax and Bcl-2 markers. Quantification was performed by calculating the fraction of Ki-67 positive cells, expressed as a mean ± SD, and analysed by Mann–Whitney test, Kruskal–Wallis and Dunn's post hoc test.

Results

In 6th–7th developmental weeks, the tooth germ and dental crest contained 37% of proliferating cells, which increased to 40% in the 8th week, and then decreased to 15% in the 10th week, whilst the proliferation in the ectomesenchyme subsequently dropped from 37% to 23%. Epithelial parts of the enamel organ displayed similar proliferation activity (31–36%), dental crest 10%, whilst enamel knot showed no proliferating activity. The tooth ectomesenchyme contained more proliferating cells (50%) than the jaw ectomesenchyme (35%), and both dropped to 28% in the 10th week. Ectomesenchyme between the tooth germs contained 23%, whilst the jaw ectomesenchyme contained 15% of proliferating cells. Bcl-2 expression had following pattern: strong in proliferating cells, moderate in tooth germs and dental crest, and weak in the ectomesenchyme. Bax co-expressed with Bcl-2 in the tooth germ and dental crest. In the reticulum and inner enamel epithelium Bcl-2 had prevalent expression, whilst Bax prevailed in the outer enamel epithelium and tooth ectomesenchyme.

Conclusions

Proliferating cells most likely influence growth of the tooth germ, Bcl-2 affects proliferation and differentiation of specific cell lineages, whilst Bax influences process of cell death.     

处方药转换非处方药评价中有关药品不良反应检索的问题探讨

通过对我国处方药转换非处方药评价工作中的药品不良反应检索进行系统分析,从检索申请人、检索范围、检索报告质量三个角度较全面地审视了处方药转换非处方药评价工作中药品不良反应检索存在的一些问题。针对现存主要问题,借鉴美国的相关经验,提出在处方药转换非处方药评价过程中,有关药品不良反应检索的改进对策和建议。