Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis and non-competitive inhibition by the small molecule “crystallization chaperone” 5′-O-tritylinosine (KIN59)

Thymidine phosphorylase (TP) is a catabolic enzyme in thymidine metabolism that is frequently upregulated in many solid tumors. Elevated TP levels are associated with tumor angiogenesis, metastasis and poor prognosis. Therefore, the use of TP inhibitors might offer a promising strategy for cancer treatment. The tritylated inosine derivative 5′-O-tritylinosine (previously designated KIN59) is a non-competitive inhibitor of TP which was previously found to be instrumental for the crystallization of human TP. A combination of computational studies including normal mode analysis, automated ligand docking and molecular dynamics simulations were performed to define a plausible binding site for 5′-O-tritylinosine on human TP. A cavity in which 5′-O-tritylinosine could fit was identified in the vicinity of the Gly405-Val419 loop at a distance of about 11 Å from the substrate-binding site. In the X-ray crystal structure, this pocket is characterized by an intricate hydrogen-bonding network in which Asp203 was found to play an important role to afford the loop stabilization that is required for efficient enzyme catalysis. Site-directed mutagenesis of this amino acid residue afforded a mutant enzyme with a severely compromised catalytic efficiency (V_max/K_m of mutant enzyme ∼50-fold lower than for wild-type TP) and pronounced resistance to the inhibitory effect of 5′-O-tritylinosine. In contrast, the D203A mutant enzyme kept full sensitivity to the competitive inhibitors 6-aminothymine and 6-amino-5-bromouracil, which is in line with the kinetic properties of these inhibitors. Our findings reveal the existence of a previously unrecognized site in TP that can be targeted by small molecules to inhibit the catalytic activity of TP.     

Effect of valsartan on erythropoietin and hemoglobin levels in stage III-IV chronic kidney disease patients

Angiotensin-converting enzyme inhibitors (ACEIs) were accepted as a potential cause of inadequate epoetin response in chronic kidney disease (CKD) patients. We aimed to determine the effects of valsartan, an angiotensin receptor blocker (ARB), on serum ertyhropoietin levels and on certain biochemical and haematological parameters in hypertensive CKD patients. Twenty-two stage III-IV CKD patients (mean age; 56.8 +/- 8.9 years, 12 male 10 female) were included in the study. Before initiating the treatment, current anti-hypertensive treatments (if any) were discontinued, and blood samples were collected after a washout period of 3 weeks. Valsartan 80 mg/day was started, and additional anti-hypertensive agents were given according to study protocol if needed. One way Anova and paired t-tests were used for statistical comparisons. Serum blood urea nitrogen (BUN), creatinine, uric acid, potassium, haemoglobin and erythropoietin values were measured, and glomerular filtration rates were calculated before and 3, 6 and 90 days after valsartan treatment, a significant reduction in EPO level was observed at 3rd (19.6 +/- 24.0 vs. 13.8 +/- 8.5, p = 0.010), 6th (12.1 +/- 7.6, p = 0.009), and 90th days (8.3 +/- 5.4, p = 0.007). When pre-treatment values were compared with 90th day results, no significant change was observed in terms of hgb, htc, serum BUN, creatinine, uric acid, potassium, and GFR values. In conclusion, valsartan, an ARB, did not decrease haemoglobin levels in stage III-IV CKD patients despite significant reduction in serum erythropoietinlevels, so ARBs may be preferred to ACEIs in CKD patients when indicated.     

Plasma membrane potential of lymphocytes from ataxia telangiectasia patients

The plasma membrane potential of lymphocytes prepared from ataxia telangiectasia (AT) patients and normal subjects was assessed using the optical indicator bis-(3-phenyl-5-oxoisoxazol-4-yl) pentamethineoxonol (oxonol-V). AT lymphocytes had a potential of -46 +/- 9 mV and normal lymphocytes had a potential of -63 +/- 4 mV. The intracellular cation content (Na+ and K+) of AT and normal lymphocytes was similar. AT and normal lymphocytes were both depolarized by extracellular K+ and to a similar extent. This study indicates that one feature characterizing ataxia telangiectasia is a modification of the ability of the lymphocyte cell membrane to sustain a normal membrane potential.     

Valsartan and coronary haemodynamics in early post-myocardial infarction in rats

Angiotensin II AT1 receptor blockade (AT1-) has been shown to prolong survival in post-myocardial infarction (MI) heart failure in rats. In this study, we investigated whether an early AT1-induced improvement in coronary vasodilatation reserve (CVR) might be involved in this beneficial effect. Wistar rats with MI were treated daily and orally for 6 weeks with valsartan, 5 (MI-V5) or 50 mg/kg (MI-V50). MI-controls and sham-operated rats (S-controls) received no treatment. Subsequently, systemic and coronary haemodynamics (at baseline and at maximal vasodilatation, CVR fluospheres) were investigated in the conscious state, and cardiac remodelling (hypertrophy and fibrosis) was assessed. As compared to MI-controls. valsartan (5 mg/kg), had no effect on systemic haemodynamics or myocardial hypertrophy and fibrosis development, gave slightly improved basal left and right ventricular coronary flow and resistance values, but decreased left and right CVR values. Valsartan (50 mg/kg), decreased blood pressure (-11%) and left ventricular end diastolic pressure (-32%), limited the development of cardiac hypertrophy (19%) but not that of fibrosis, slightly improved basal left ventricular flow and resistance values but only the right ventricular CVR value was increased. We conclude that in rats with post-MI. an early AT1-induced improvement in coronary haemodynamics is not responsible for the long-term survival prolongation observed. Furthermore. that cardiac hypertrophy was prevented whereas fibrosis was not, suggests that the latter is a pivotal determinant of CVR.     

Angiotensin receptors in Dupuytren’s disease: a target for pharmacological treatment?

Background: Attempts at the pharmacological treatment of Dupuytren’s disease have so far been unsuccessful, and the disease is not yet fully understood on a cellular level. The Renin-Angiotensin System has long been understood to play a circulating hormonal role. However, there is much evidence showing Angiotensin II to play a local role in wound healing and fibrosis, with ACE inhibitors being widely used as an anti-fibrotic agent in renal and cardiac disease.

Methods: This study was designed to investigate the presence of Angiotensin II receptors 1 (AT1) and 2 (AT2) in Dupuytren’s tissue to form a basis for further study into the pharmacological treatment of this condition. Tissue was harvested from 11 patients undergoing surgery for Dupuytren’s disease. Each specimen was processed into frozen sections and immunostaining was employed to identify AT1 and AT2 receptors.

Results: Immunostaining for AT1 receptors was mildly positive in one patient and negative in all the remaining patients. However, all specimens stained extensively for AT2 receptors. This suggests that the expression of AT2 receptors is more prominent than AT1 receptors in Dupuytren’s disease.

Conclusion: These findings have opened a new avenue for future research involving ACE inhibitors, AT2 agonists, and AT2 antagonists in Dupuytren’s disease.     

Thrombosis in congenital deficiencies of AT III, protein C or protein S: a study of 44 children

Abstract. Congenital deficiency in coagulation inhibitors is a cause of hereditary thrombotic disease. The severity of symptoms is variable and depends on the type of deficit. In this paper, 44 children suffering from deep venous thrombosis, with a mean age of 5 years, were studied. A search for Lupus anticoagulant (LA) and coagulation inhibitor deficiency showed: 3/44 cases (6.8%) had protein S deficiency, 2/44 cases (4.5%) had protein C deficiency, 1/44 cases (2.3%) had deficiencies in both protein C and S; no cases of AT III deficiency and LA was positive in 2/44 cases (4.5%). Only 1 case of APC resistance out of 13 studied was found. Four family studies were performed and confirmed the congenital origin of the disorder.     

ACE DD genotype: an independent predisposition factor to venous thromboembolism

BACKGROUND: The renin angiotensin system affects haemostasis through different mechanisms; data on the possible role of angiotensin-converting enzyme I/D polymorphism in the pathogenesis of deep venous thrombosis are conflicting, and no information is available regarding the A1166C polymorphism of the angiotensin type 1 receptor gene. In order to investigate this issue, angiotensin-converting enzyme and AT1R polymorphisms were genotyped in 336 consecutive venous thromboembolism patients and 378 controls. MATERIALS AND METHODS: Haemostasis-related risk factors have been evaluated by routine tests. Factor V Leiden, Factor II (G20210A), angiotensin-converting enzyme (I/D), and angiotensin type 1 receptor (A1166C) polymorphisms have been identified by molecular analysis. RESULTS: We documented a significant association between angiotensin-converting enzyme DD genotype and venous thromboembolism (OR=2.19 95%CI 1.51-3.17 adjusted for acquired and haemostasis-related risk factors, P<0.0001); in patients with haemostasis-related risk factors, angiotensin-converting enzyme DD genotype modified the risk of venous thromboembolism in hyperhomocysteinaemic and Factor V Leiden patients and was associated with the risk of recurrent venous thromboembolism (OR=1.83 95%CI 1.06-3.17 P=0.03). In patients without haemostasis-related risk factors the angiotensin-converting enzyme DD genotype was still an independent predictor of venous thromboembolism (OR=3.29 95%CI 2.17-4.98 adjusted for acquired risk factors, P<0.0001). No significant association between the angiotensin type 1 receptor CC genotype and venous thromboembolism was found. CONCLUSIONS: This study shows that angiotensin-converting enzyme DD genotype represents a susceptibility marker of thrombosis in subjects apparently without predisposing factors and traditional thrombophilic alterations, and increases the risk of venous thromboembolism in subjects in whom a thrombogenic condition occurs. Moreover, angiotensin-converting enzyme DD genotype may be considered a new predisposing factor to venous thromboembolism recurrence.     

衔接蛋白2敲除对AT1受体介导的AngⅡ内吞的影响

目的 通过RNAi沉默AP2-μ2亚单位的表达,观察其是否影响AT1R介导的AngⅡ内吞.方法 通过设计构建的AP2-μ2 RNAi表达载体,转染H9C2细胞,通过Western印迹验证沉默效果;激光共聚焦显微镜观察沉默AP2-μ2基因对AT1R介导的AngⅡ内吞的影响.结果 蛋白质水平检测siRNA重组质粒对AP2-μ2蛋白表达的影响,可见pSH1Si-AP2 plasmid-1表达质粒可明显抑制AP2-μ2蛋白表达.给予AngⅡ不同时间点,可见其在15 min时受体内吞至胞内数量最多.重组质粒沉默AP2-μ2亚单位后,外源加入AngⅡ后,可明显抑制AT1R介导的AngⅡ内吞. 结论 AT1R介导AngⅡ内吞的发生,与其他GPCR一样,主要通过网格蛋白依赖的内吞方式进行,并且需要AP2蛋白参与.     

β-抑制蛋白在AT_1受体信号通路中的作用及意义

血管紧张素Ⅱ(AngⅡ)是肾素-血管紧张素系统的重要活性物质,广泛参与心血管活动的调节。AngⅡ通过与其受体结合,诱导激活复杂的胞内信号通路,是其参与多种病理生理过程的基础。β-抑制蛋白(β-arrestins)是一类负反馈调节G蛋白偶联受体的多功能蛋白,广泛存在心血管系统,是近期的研究热点。该文主要综述β-抑制蛋白在AngⅡ诱导的AngⅡ一型受体(AT1受体)信号通路中的作用及意义。