Interstitial fluid flow and cyclic strain differentially regulate cardiac fibroblast activation via AT1R and TGF-β1

Cardiac fibroblasts are exposed to both cyclic strain and interstitial fluid flow in the myocardium. The balance of these stimuli is affected by fibrotic scarring, during which the fibroblasts transition to a myofibroblast phenotype. The present study investigates the mechanisms by which cardiac fibroblasts seeded in three-dimensional (3D) collagen gels differentiate between strain and fluid flow. Neonatal cardiac fibroblast-seeded 3D collagen gels were exposed to interstitial flow and/or cyclic strain and message levels of collagens type I and III, transforming growth factor β1 (TGF-β1), and α-smooth muscle actin (α-SMA) were assessed. Flow was found to significantly increase and strain to decrease expression of myofibroblast markers. Corresponding immunofluorescence indicated that flow and strain differentially regulated α-SMA protein expression. The effect of flow was inhibited by exposure to losartan, an angiotensin II type 1 receptor (AT1R) blocker, and by introduction of shRNA constructs limiting AT1R expression. Blocking of TGF-β also inhibited the myofibroblast transition, suggesting that flow-mediated cell signaling involved both AT1R and TGF-β1. Reduced smad2 phosphorylation in response to cyclic strain suggested that TGF-β is part of the mechanism by which cardiac fibroblasts differentiate between strain-induced and flow-induced mechanical stress. Our experiments show that fluid flow and mechanical deformation have distinct effects on cardiac fibroblast phenotype. Our data suggest a mechanism in which fluid flow directly acts on AT1R and causes increased TGF-β1 expression, whereas cyclic strain reduces activation of smad proteins. These results have relevance to the pathogenesis and treatment of heart failure.     

β-Arrestin Regulation of Myosin Light Chain Phosphorylation Promotes AT1aR-mediated Cell Contraction and Migration

Over the last decade, it has been established that G-protein-coupled receptors (GPCRs) signal not only through canonical G-protein-mediated mechanisms, but also through the ubiquitous cellular scaffolds β-arrestin-1 and β-arrestin-2. Previous studies have implicated β-arrestins as regulators of actin reorganization in response to GPCR stimulation while also being required for membrane protrusion events that accompany cellular motility. One of the most critical events in the active movement of cells is the cyclic phosphorylation and activation of myosin light chain (MLC), which is required for cellular contraction and movement. We have identified the myosin light chain phosphatase Targeting Subunit (MYPT-1) as a binding partner of the β-arrestins and found that β-arrestins play a role in regulating the turnover of phosphorylated myosin light chain. In response to stimulation of the angiotensin Type 1a Receptor (AT1aR), MLC phosphorylation is induced quickly and potently. We have found that β-arrestin-2 facilitates dephosphorylation of MLC, while, in a reciprocal fashion, β-arrestin 1 limits dephosphorylation of MLC. Intriguingly, loss of either β-arrestin-1 or 2 blocks phospho-MLC turnover and causes a decrease in the contraction of cells as monitored by atomic force microscopy (AFM). Furthermore, by employing the β-arrestin biased ligand [Sar¹,Ile⁴,Ile⁸]-Ang, we demonstrate that AT1aR-mediated cellular motility involves a β-arrestin dependent component. This suggests that the reciprocal regulation of MLC phosphorylation status by β-arrestins-1 and 2 causes turnover in the phosphorylation status of MLC that is required for cell contractility and subsequent chemotaxic motility.     

Discovery of the bifunctional modulator of angiotensin II type 1 receptor (AT1R) and PPARγ derived from the AT1R antagonist,Fimasartan

Inspired by the well-known PPARγ partial agonism of angiotensin II type 1 receptor (AT1R) antagonists exemplified by an antihypertensive drug, Telmisartan, efforts to identify compounds with the dual activities have been pursued in order to control the two major metabolic disorders, hypertension and hyperglycemia simultaneously. Lead compound 18 derived from the AT1R antagonist, Fimasartan, has successfully presented the possibility to control the medical conditions by a single molecule.     

不同年龄白发性高血压大鼠肾脏AT1R和AT2R的表达

目的本研究观察不同月龄自发性高血压大鼠（SHR）肾脏ALR和ALR表达,初步探讨AT1R和AT2R在高血压发生、发展过程中的可能作用。方法1月龄组（S1）、2月龄组（S2）、3月龄组（S3）、6月龄组（S6）和9月龄组（墨）雄性SHR共5组,每组各6只,各组均有相应月龄匹配的Wistar-Kyoto大鼠（WKY）作对照。采用RBP-I型大鼠血压心率测定仪测量大鼠尾动脉收缩压（SBP）;放免法测定血浆血管紧张素Ⅱ（AngⅡ）;免疫组化染色结合计算机图像分析方法测定肾脏AT1R和ALR表达水平。结果（1）SHR SBP随着月龄的增加而上升,S6后趋于稳定。（2）1个月后SHR血浆AngⅡ浓度均高于S1（P〈0.05）,而S2、S3、S6和S9之间无明显差别（P〉0.05）;1个月后SHR血浆AngⅡ浓度均高于相应配对的WKY组（P〈0.05）;而WKY各月龄组均无明显差别（P〉0.05）。（3）SHR肾脏AT1R随着月份的增加而增加（P〈0.05）,且高于相应配对的WKY组（P〈0.05）。SHR肾脏ABR随着月份的增加而降低,S6明显降低（P〈0.05）,S6和S9比较无明显差别（P〉0.05）;且均低于相应配对的WKY组（P〈0.05）。WKY各月龄组AT1R和AT2R无明显差别（P〉0.05）。结论SHR肾脏AT1R表达水平比WKY高,并随着年龄的增加而递增;AT2R表达水平比WKY低,并随着年龄的增加而降低。     

AT试剂对秋茶的影响

我们于1993年对丘陵秋茶进行了AT_1和AT_3两种试剂喷洒试验,增产效果明显,现介绍如下:安徽省国营九连山茶场的试验区地势平坦,肥力中等,供试品种为祁门槠叶种,树龄20多年,单行条植。     

Cytogenetic characterization of ataxia telangiectasia (AT) heterozygotes using lymphoblastoid cell lines and chronic γ-irradiation

Summary Lymphoblastoid cell lines (LCLs) derived from two patients identified as ataxia telangiectasia (AT), two obligate AT heterozygotes and two controls (healthy subjects with no known genetic disease or relationship to AT patients) were compared with respect to the induction of chromosomal breaks by acute and chronic -irradiation. Although there was a considerable increase in the frequency of chromosomal breaks per cell in the LCLs of AT patients resulting from acute irradiation, the small increase occurring in the LCLs of the AT heterozygotes made it difficult to distinguish them from the controls. Following chronic -irradiation, however, the frequency of chromosomal breaks per cell in the LCLs of the AT heterozygotes occupied a significantly distinct position from that of the controls. These observations suggested that the use of chronic irradiation may be a better choice in the cytogenetic characterization of AT heterozygotes.     

Role of Vascular Smooth Muscle PPARγ in Regulating AT1 Receptor Signaling and Angiotensin II-Dependent Hypertension

Peroxisome proliferator activated receptor γ (PPARγ) has been reported to play a protective role in the vasculature; however, the underlying mechanisms involved are not entirely known. We previously showed that vascular smooth muscle-specific overexpression of a dominant negative human PPARγ mutation in mice (S-P467L) leads to enhanced myogenic tone and increased angiotensin-II-dependent vasoconstriction. S-P467L mice also exhibit increased arterial blood pressure. Here we tested the hypotheses that a) mesenteric smooth muscle cells isolated from S-P467L mice exhibit enhanced angiotensin-II AT₁ receptor signaling, and b) the increased arterial pressure of S-P467L mice is angiotensin-II AT₁ receptor dependent. Phosphorylation of mitogen-activated protein/extracellular signal-regulated kinase (ERK1/2) was robustly increased in mesenteric artery smooth muscle cell cultures from S-P467L in response to angiotensin-II. The increase in ERK1/2 activation by angiotensin-II was blocked by losartan, a blocker of AT₁ receptors. Angiotensin-II-induced ERK1/2 activation was also blocked by Tempol, a scavenger of reactive oxygen species, and correlated with increased Nox4 protein expression. To investigate whether endogenous renin-angiotensin system activity contributes to the elevated arterial pressure in S-P467L, non-transgenic and S-P467L mice were treated with the AT₁ receptor blocker, losartan (30 mg/kg per day), for 14-days and arterial pressure was assessed by radiotelemetry. At baseline S-P467L mice showed a significant increase of systolic arterial pressure (142.0±10.2 vs 129.1±3.0 mmHg, p<0.05). Treatment with losartan lowered systolic arterial pressure in S-P467L (132.2±6.9 mmHg) to a level similar to untreated non-transgenic mice. Losartan also lowered arterial pressure in non-transgenic (113.0±3.9 mmHg) mice, such that there was no difference in the losartan-induced depressor response between groups (−13.53±1.39 in S-P467L vs −16.16±3.14 mmHg in non-transgenic). Our results suggest that interference with PPARγ in smooth muscle: a) causes enhanced angiotensin-II AT₁ receptor-mediated ERK1/2 activation in resistance vessels, b) and may elevate arterial pressure through both angiotensin-II AT₁ receptor-dependent and -independent mechanisms.     

Angiotensinogen and angiotensin-converting enzyme mRNA decrease and AT1 receptor mRNA and protein increase in epididymal fat tissue accompany age-induced elevation of adiposity and reductions in expression of GLUT4 and peroxisome proliferator-activated receptor (PPARγ)

Elevated adiposity is one of the accompanying features of increased age in humans and animals. Angiotensin II (Ang II) is considered as growth promoting peptide to be involved in hypertrophic enlargement of adipose tissue. However, systemic renin-angiotensin system (RAS) seems to decrease with increased age of rats. Local adipose tissue RAS might be independent of the systemic one. Therefore we performed a comprehensive study using rats with increased age from 9 to 26 weeks and evaluated angiotensinogen, angiotensin-converting enzyme (ACE) and AT(1) receptor mRNA in epididymal adipose tissue by RT-PCR. In addition, we determined AT(1) receptor protein by Western blotting and Ang II binding. These RAS parameters were correlated with expression of selected adiposity-dependent proteins such as leptin, adiponectin, insulin-dependent glucose transporter (GLUT4) and PPARgamma. Angiotensinogen and ACE expression decreased with increased age and adiposity. On the contrary, AT(1) receptor mRNA and protein was significantly elevated in 26-week-old rats though the Ang II binding was not different between 9 and 26-week-old animals. These results suggest dynamic adaptation of local adipose tissue RAS components to increased age and adiposity most likely by decreasing local Ang II formation which is thereafter compensated by increased expression of AT(1) receptor. However, this increase in AT(1) receptor mRNA and protein is not reflected in increased receptor binding. We believe that this complex regulation of adipose tissue RAS slows down the negative age and adiposity related changes in adipose tissue leptin, adiponectin, GLUT4 and PPARgamma.     

心梗后心肌重构过程中AT1A，AT2受体表达的变化

为探讨AT1,AT2受体在心肌重构演变过程中的作用,本实验应用免疫组化,电镜技术和图像分析方法,观察了大鼠心梗后心肌重构过程中非醒,AT1,AT2受体表达的动态变化,结果显示,心梗术后3d,电镜显示非梗塞区心肌细胞肌原纤维横纹消失,线粒体肿胀,成纤维细胞增多,免疫组化显示AT1A受体在非梗塞区心肌组织表达明显升高（P＜0．001）,AT2受体表达无明显变化（P＞0．05）,心梗术后14天,可见心肌细胞肌原纤维模纹,心肌细胞间胶原纤维明显增多。同时AT1A受本在心肌的表达比心梗术后3天时减弱,但仍高于对照组（P＜0．05）,AT2受体表达明显增加（P＜0．001）,结果提示：心梗后非梗塞区心肌AT1A,AT2受体表达先后上调,可能参与介导心肌重构过程。     

The differentiation of bone marrow cells to functional T lymphocytes following implantation of thymus grafts and thymic stroma in nude and AT×BM mice

Cardiac allografts were used to compare the immunologic capacity of nude mice and adult, thymectomized, lethally irradiated, bone marrow-reconstituted (AT × BM) mice. Neither nude nor AT × BM mice were able to reject cardiac allografts of any party. However, both rejected grafts of any party following implantation of neonatal thymus or thymus from 3-week-old syngeneic mice. Irradiated syngeneic thymus grafts (800 R) were equally effective in restoring host responsiveness against allografts. In contrast, allogeneic thymus grafts restored the capacity to reject second-party heart grafts only in AT × BM mice. Second-party grafts persisted indefinitely when placed on nude mice implanted with an allogeneic, unirradiated thymus graft. Third-party grafts transplanted 17 weeks after reconstitution, however, were rejected. Irradiated nude mice given normal littermate bone marrow and simultaneously grafted with second-party thymus and heart allografts also failed to reject their second-party heart grafts. The difference in ultimate capacity to respond between AT × BM and nude mice suggests that a maturational defect exists in the nude mouse enviroment which impedes development of precursor T lymphocytes.     

Study of Completed Archaeal Genomes and Proteomes： Hypothesis of Strong Mutational AT Pressure Existed in Their Common Predecessor

The number of completely sequenced archaeal genomes has been sufficient for a large-scale bioinformatic study.We have conducted analyses for each coding region from 36 archaeal genomes using the original CGS algorithm by calculating the total GC content(G+C),GC content in first,second and third codon positions as well as in fourfold and twofold degenerated sites from third codon positions,levels of arginine codon usage(Arg2:AGA/G;Arg4:CGX),levels of amino acid usage and the entropy of amino acid content distribution.In archaeal genomes with strong GC pressure,arginine is coded preferably by GC-rich Arg4 codons,whereas in most of archaeal genomes with G+C0.6,arginine is coded preferably by AT-rich Arg2 codons.In the genome of Haloquadratum walsbyi,which is closely related to GC-rich archaea,GC content has decreased mostly in third codon positions,while Arg4Arg2 bias still persists.Proteomes of archaeal species carry characteristic amino acid biases:levels of isoleucine and lysine are elevated,while levels of alanine,histidine,glutamine and cytosine are relatively decreased.Numerous genomic and proteomic biases observed can be explained by the hypothesis of previously existed strong mutational AT pressure in the common predecessor of all archaea.     

Structure and Interactions of Myosin-binding Protein C Domain C0: CARDIAC-SPECIFIC REGULATION OF MYOSIN AT ITS NECK?*

Myosin-binding protein C (MyBP-C) is a multidomain protein present in the thick filaments of striated muscles and is involved in both sarcomere formation and contraction regulation. The latter function is believed to be located at the N terminus, which is close to the motor domain of myosin. The cardiac isoform of MyBP-C is linked to hypertrophic cardiomyopathy. Here, we use NMR spectroscopy and biophysical and biochemical assays to study the three-dimensional structure and interactions of the cardiac-specific Ig-like domain C0, a part of cardiac MyBP-C of which little is known. The structure confirmed that C0 is a member of the IgI class of proteins, showing many of the characteristic features of this fold. Moreover, we identify a novel interaction between C0 and the regulatory light chain of myosin, thus placing the N terminus of the protein in proximity to the motor domain of myosin. This novel interaction is disrupted by several cardiomyopathy-linked mutations in the MYBPC3 gene. These results provide new insights into how cardiac MyBP-C incorporates in the sarcomere and how it can contribute to the regulation of muscle contraction.     

A Structural Transition in d(AT) n ·d(AT) n Inserts within Superhelical DNA

Abstract

We have constructed plasmids carrying d(AT)_n·d(AT)_n inserts of different lengths. Two- dimensional gel electrophoresis patterns show that an increase in the negative superhelicity of these DNAs brings about a structural transition within the inserts, resulting in a reduction of the superhelical stress. However, this reduction corresponds to the expected values neither for cruciform nor for the Z form. Those DNA topoisomers in which the structural transition had occurred proved to be specifically recognizable by single-strand-specific endonuclease SI, with the cleavage site situated at the centre of the insert. These data, as well as kinetic studies, suggest that the cloned d(AT)_n·d(AT) _n sequences adopt a cruciform rather than the Z-form structure. We discuss plausible reasons of the discrepancy between the observed superhelical stress release and that expected for the transition of the insert to the cruciform state.     

人A1AT蛋白的生物信息学分析

通过生物信息学预测分析程序发现,人A1AT(alpha-1 antitrypsin)蛋白是由418个氨基酸组成的外分泌蛋白质,其等电点为5.37。同源性分析推断,在其第73~93位和第351~372位氨基酸间存在两个重要的功能区域。此外,A1AT蛋白二级结构中存在13个较大的α螺旋区域,占总蛋白质的42.58%。高级结构的分析发现,RCL结构域的存在对A1AT的结构和功能具有重要的影响。GO和KEGG分析发现,A1AT参与机体抗炎症反应、凝血反应以及细胞的迁移、侵袭、增殖过程。分析所得的A1AT生物信息学数据为其在疾病诊断和治疗方面的研究提供了重要的理论数据。     

The B°AT1 amino acid transporter from rat kidney reconstituted in liposomes: kinetics and inactivation by methylmercury

The neutral amino acid transporter B°-like from rat kidney, previously reconstituted in liposomes, was identified as B°AT1 by a specific antibody. Collectrin was present in the brush-border extract but not in functionally active proteoliposomes, indicating that it was not required for the transport function. Neutral amino acids behaved as competitive inhibitors of the glutamine transport mediated by B°AT1 with half saturation constants ranging from 0.13 to 4.74mM. The intraliposomal half saturation constant for glutamine was 2.0mM. By a bisubstrate kinetic analysis of the glutamine-Na(+) cotransport, a random simultaneous mechanism was found. Methylmercury and HgCl(2) inhibited the transporter; the inhibition was reversed by dithioerythritol, Cys and, at a lower extent, N-acetylcysteine but not by S-carboxymethylcysteine. The IC(50) of the transporter for methylmercury and HgCl(2) was 1.88 and 1.75μM, respectively. The reagents behaved as non-competitive inhibitors toward both glutamine and Na(+) and no protection by glutamine or Na(+) was found for the two inhibitors.     

AT₂receptors recruit c-Src, SHP-1 and FAK upon activation by Ang II in PND15 rat hindbrain

The functional role of AT(2) receptors is unclear and it activates unconventional signaling pathways, which in general do not involve a classical activation of a G-protein. In the present study, we aimed to investigate the transduction mechanism of AT(2) Ang II receptors in PND15 rat hindbrain membrane preparations, which represents a physiological developmental condition. To determine whether Ang II AT(2) receptors induced association to SHP-1 in rat hindbrain, co-immunoprecipitation assays were performed. Stimulation of Ang II AT(2) receptors induced both a transient tyr-phosphorylation and activation of SHP-1. The possible participation of c-Src in Ang II-mediated SHP-1 activation, we demonstrated by recruitment of c-Src in immunocomplexes obtained with anti AT(2) or anti-SHP-1 antibodies. The association of SHP-1 to c-Src was inhibited by PD123319 and the c-Src inhibitor PP2. Similarly, SHP-1 activity determined in AT(2)-immunocomplexes was inhibited by PD123319 and the c-Src inhibitor PP2. Following stimulation with Ang II, AT(2) receptors recruit c-Src, which was responsible for SHP-1 tyr-phosphorylation and activation. Since AT(2) receptors are involved in neuron migration, we tested the presence of FAK in immunocomplexes. Surprisingly, AT(2)-immunocomplexes contained mainly the 85kDa fragment of FAK. Besides, p125FAK associated to SHP-1. In summary, we demonstrated the presence of an active signal transduction mechanism in PND15 rat hindbrain, a developmental stage critical for cerebellar development. In this model, we showed a complex containing AT(2)/SHP-1/c-Src/p85FAK, suggesting a potential role of Ang II AT(2) receptors in cerebellar development and neuronal differentiation.     

Design, synthesis and biological activity of 6-substituted carbamoyl benzimidazoles as new nonpeptidic angiotensin II AT₁ receptor antagonists

A series of 6-substituted carbamoyl benzimidazoles were designed and synthesised as new nonpeptidic angiotensin II AT(1) receptor antagonists. The preliminary pharmacological evaluation revealed a nanomolar AT(1) receptor binding affinity for all compounds in the series, and a potent antagonistic activity in an isolated rabbit aortic strip functional assay for compounds 6f, 6g, 6h and 6k was also demonstrated. Furthermore, evaluation in spontaneous hypertensive rats and a preliminary toxicity evaluation showed that compound 6g is an orally active AT(1) receptor antagonist with low toxicity.     

Alpha-1-antitrypsin (α1AT) phenotypes and PiM subtypes in Italy. Evidence of considerable geographic variability

Summary Genetic typing of ₁AT was performed in 3751 individuals from Italian towns. The following was observed: (a) The pathologic phenotypes (SZ, MZ, ZZ) appeared to decrease progressively from northern to southern Italy; (b) the distribution of the PiM suballeles showed considerable geographic variability, but the suballele, M ₂ was more frequently encountered in southern Italy; and (c) in the large cities of southern Italy, the frequency of the deficiency more closely resembled that found in northern Italy than that of the remaining populations of the south.     

AT—AC内含子及其剪接机理的研究进展

ＡＴ┐ＡＣ内含子及其剪接机理的研究进展滕胜明镇寰（杭州大学生命科学学院,杭州３１００１２）关键词ＡＴ－ＡＣ内含子剪接机理最近发现了一种新类型的内含子,它在ｍＲＮＡ前体中存在的比例不到０．１％,其剪接位点高度保守的双核苷酸为ＡＴ和ＡＣ（ＡＴ－ＡＣ内含子...