去糖基化突变体肾素元的分子特点

应用野生体和去糖基化突变体肾素元ｃＤＮＡ，转染ＧＨ４细胞，经＾３５Ｓ代谢标记表达产物，免疫沉淀，ＳＤＳ．ＰＡＧＥ电泳检测观察去糖基后肾素元结构与功能的变化。发现去糖基化后的肾素元突变体，除半衰期缩短外，其分子粘度明显进高，染细胞清除作为代谢标记去糖基化肾素元的时间延长。     

非糖基化尿激酶原突变体的研究

目的:构建一种非糖基化、抗凝血酶激活的尿激酶原突变体,在CHO-dhfr-(二氢叶酸还原酶缺陷型-中国仓鼠卵巢)细胞中表达,收取无血清培养上清,并鉴定其活性。方法:使用PCR扩增的方法,将302位天冬酰胺(Asn302)突变为丙氨酸(Ala302),去掉糖基化位点;将156位精氨酸(Arg156)突变为赖氨酸(Lys156),去掉凝血酶作用位点。将突变体基因转染到CHO-dhfr-细胞中。收取无血清培养上清,并用纤维蛋白板溶解圈法鉴定活性。结果:PCR产物经琼脂糖电泳鉴定分子量略大于1200道尔顿(dalton),与理论分子量1296道尔顿符合。转染成功的细胞株用于扩大培养。纤维蛋白板溶解圈法测定上清活性为295.58IU/ml。结论:非糖基化、抗凝血酶的单链尿激酶型纤溶酶原激活剂突变体构建成功。转染后细胞生长良好,表达水平较高。     

糖基化终产物的结构及其检测方法的研究进展

糖基化终产物（advancedglycationendproducts,AGEs）是还原糖（如葡萄糖）与蛋白质、氨基酸等的游离氨基端通过一系列复杂的非酶促反应形成的结构多样的不可逆聚合物[1],它们在糖尿病慢性并发症发生发展中的作用正越来越引起广泛关注[2]。检测体内AGEs对临床进一步研究糖尿病慢性并发症的发生、评估并发症的程度、监测长期血糖水平以及指导糖尿病治疗等将具有十分重要的意义[3,4]。然而由于AGEs的结构复杂多样,至今尚缺乏标准的商品化AGEs检测手段可供使用。我们就AGEs的结构及其有关检测方法方面的研究进展做一综述。一、AGEs…     

肾素抑制剂治疗高血压的研究进展

随着对高血压病治疗研究的深入,以肾素为靶点的肾素抑制剂在高血压病中的应用越来越受到人们的重视。肾素抑制剂作用于肾素-血管紧张素系统(RAS)的限速步骤,理论上较ACEI和ARB具有潜在的优势,为高血压病的治疗提供了新的思路。     

云南省居民的肾素和醛固酮水平

目的 了解云南省居民肾素和醛固酮水平。方法 收集“2020年中国居民心血管病及其危险因素监测”项目中云南省入选的调查对象9 996人,调查对象于清晨空腹采集立位血液标本,应用全自动化学发光免疫分析仪(LIAISON°XL,type2210)检测立位血浆醛固酮浓度(PAC)、直接肾素浓度(DRC),计算醛固酮与肾素比值(ARR);对PAC、DRC、ARR使用百分位数法计算双侧的参考范围,分别计算2.5、97.5百分位数(P_2.5,P_97.5)及5、95百分位数(P₅,P₉₅)的参考值,并分析年龄、性别、体重指数和海拔等与PAC、DRC、ARR的关系。结果 调查对象立位PAC、DRC、ARR检测数值均不服从正态分布,三项指标拟定参考范围分别为,PAC(P_2.5,P_97.5):(2.77,28.80)ng/dL;DRC(P₅,P₉₅):(3.14,82.78)mU/L;ARR(P_2.5,P     

异常糖基化IgA1分子与IgA肾病

上世纪60年代,应用免疫组化技术,Berger与Hinglais[1]首次描述了IgA、IgG在肾小球系膜区共同沉积的肾小球疾病,并将其命名为"IgA肾病(IgAN)"(又名Berger病).     

糖尿病大鼠阴茎组织糖基化终产物含量的变化和糖基化终产物受体的表达情况

目的研究糖尿病阴茎组织糖基化终产物(AGEs)含量的变化和糖基化终产物受体(RAGE)的表达情况。方法用葡萄糖和血红蛋白(Hb)在体外孵育形成Hb-AGEs,免疫新西兰兔,制备AGEs抗血清,建立竞争性ELISA法,测定大鼠阴茎组织AGEs含量。应用RT-PCR研究RAGEmRNA表达。结果各组糖尿病大鼠未经治疗时阴茎组织AGEs水平均较对照(NC)组明显升高(P<0.01),8周时糖尿病用胰岛素治疗(DMI)组及糖尿病用胰岛素和二氨基酚嗪(DAP)治疗(DMID)组的AGEs水平较糖尿病未治疗(DM)组降低(P<0.05～0.01),至16周DMID组上述指标较DMI组明显降低(P<0.01)。RAGEmRNA在DM组大鼠阴茎组织的表达明显高于NC组(P<0.01)。结论糖尿病大鼠阴茎组织AGEs水平明显升高,RAGEmRNA高表达,胰岛素和DAP治疗可降低AGEs水平。     

终末糖基化产物在糖尿病勃起功能研究中的进展

<正>勃起功能障碍(ED)是指在有性交意愿的情况下阴茎持续至少3个月不能达到和(或)维持充分的勃起状态而获得满意的性生活~([1])。DM中ED患者为正常人群的3倍,且ED发生较正常人群早10~15年~([2~4])。DM阴茎组织中终末糖基化产物(AGEs)含量较正常人明显增多~([5]),应用AGEs抑制剂能改善DM大鼠勃起功能~([6])。本文对AGEs在DMED的发生、发展中     

Human Prorenin: Pathophysiology and Clinical Implications

ABSTRACT

Renin and prorenin, the latter after conversion to renin, are usually measured by indirect RIA using antibodies against angiotensin I. They can now also be measured by direct RIA using monoclonal antibodies reacting with total immunoreactive renin (renin plus prorenin) or with renin alone. Results of measurements in renal and peripheral venous plasma indicate that normally a large proportion of prorenin in plasma is of renal origin and they support the concept of separate pathways for prorenin and renin secretion by the JG-cells. Acute stimulation causes a prompt increase of plasma renin without any change in prorenin. During chronic stimulation both renin and prorenin are increased, in such a way that the ratio between the two is higher the stronger the stimulus. Thus, with acute stimulation only the release of stored renin appears to be increased (regulated pathway), whereas during chronic stimulation the synthesis and secretion of prorenin are also (constitutive pathway).

During pregnancy, in the early luteal phase of the menstrual cycle and after ovarian hyperstimulation with gonadotropins, a normal or somewhat elevated plasma level of renin is associated with a disproportionally high level of prorenin. This is an indication of extrarenal production of prorenin and in these conditions the ovary, probably corpus luteum, seems to be the main source. In most patients with renin-producing tumors plasma prorenin is also disproportionally high. In diabetes mellitus complicated by micro-angiopathy plasma prorenin is also elevated whereas renin is normal or even low. In diabetics with end-stage nephropathy we found no significant veno-arterial difference in prorenin across the kidneys, despite high circulating prorenin and a very low blood flow through these kidneys, suggesting that also in these patients part of the increased prorenin in plasma is of extrarenal origin.

Thus, measurements of prorenin in plasma in various pathological conditions may contribute to a better understanding of the physiological role of the renal and extrarenal renin-angiotensin systems.     

Ovarian Prorenin

We review here recent evidence that the ovaries synthesize and secrete prorenin and we explore the possible reasons why prorenin, and not active renin, is formed almost exclusively i n this extra-renal site. Very high concentrations of prorenin are present in the human ovary in the fluid inside mature follicles. This ovarian prorenin appears to be secreted into the circulation since plasma prorenin increases in normal women for two to three days at mid-menstrual cycle, at the time of ovulation. No change in plasma active renin occurs at this time. Plasma prorenin increases much more at mid-cycle in women whose ovaries have been hyperstimulated with gonadotropins. Their mid-cycle increment in plasma prorenin (after hCG) is directly related to the number of ovarian follicles. Plasma prorenin also increases markedly (10-fold) in pregnant women within two weeks after conception, in parallel with the rise in endogenous hCG. The ovaries are the apparent source of the increase in plasma prorenin during pregnancy since no such increase occurred in a woman with ovarian failure who conceived after receiving a donor egg. These results suggest that the ovaries synthesize and secrete prorenin in response to stimulation by gonadotropic hormones. Future studies will investigate the potential role of ovarian prorenin in human reproductive function. W e postulate the existence of a prorenin receptor which activates prorenin and, in consequence, activates a local renin-angiotensin system. The functioning of this system may be regulated by changes in prorenin and its receptor.     

Effects of Atrial Natriuretic Factor Infusion on Plasma Prorenin Levels in Hypertensive Males

To evaluate the influence of atrial natriuretic factor (ANF) infusion on circulating prorenin, 20 essential hypertensive males, aged between 40 and 60 years, were studied. After 2 weeks under normal sodium intake (120 mmol NaCl per day), patients were randomly assigned to receive either ANF (0.01 fmol/Kg/min) (n.12 patients) or its vehicle (50 mL of isotonic Saline) (n.8 patients) over a period of 60 minutes. Blood samples for plasma renin activity (PRA), prorenin and aldosterone (PAC) were taken at time -60, 0, 20, 40, 60, 120, 180, 240 minutes (infusion time: from 0 to 60 minutes). PRA and PAC decreased during the ANF infusion (PRA: from 0.33±0.05 ng/L/s at time 0 to 0.10±0.06 ng/L/s at 60 minutes, p<0.0001; PAC: from 389.2±99.8 pmol/L at time 0 to 148.7±44.3 pmol/L at 60 minutes, p<0.0001), while returned immediately to baseline levels after the infusion was stopped (PRA: 0.37±0.11 ng/L/s at 180 minutes, PAC: 251.6±72.1 pmol/L at time 180 minutes). On the contrary, plasma prorenin increased during ANF infusion (from 1.66±0.58 ng/L/s at time 0 to 2.44±0.72 ng/L/s at 60 minutes, p<0.05), and returned to baseline levels after the end of the infusion (1.86±0.83 ng/L/s at 180 minutes). These data indicate that ANF infusion may alter only the circulating levels of active renin, without affecting plasma prorenin secretion.     

Effect of Angiotensinogen Concentration on Renin Activity Following Nephrectomy and Adrenalectomy: Implications in the Activation of Plasma Prorenin

We have studied the effects of bilateral nephrectomy and adrenalectomy on angiotensinogen concentration, plasma renin activity, and total plasma renin activity obtained after trypsin activation in rats and compared with controls. Our results show that the plasma angiotensinogen concentration of nephrectomized (NX) rats is 5-fold higher than that of adrenalectomized (AX) rats. On the other hand, plasma angiotensinogen concentration of AX rats was about 2.5-fold lower than that of control rats. While NX rat plasma possess no renin activity, its mixing (1:1, v/v) with AX plasma results in 2-fold increase in renin activity over that observed for AX plasma alone. These results suggest that the apparent increase in renin activity upon mixing these two plasmas is at least partly due to an overall increase in angiotensinogen concentration in the mixture. To show that it is not due to activation of NX plasma prorenin by a convertase from AX plasma, prorenin-free NX rat plasma was prepared by using an anti-renin immunoaffinity column. When this prorenin-free NX plasma was mixed (1:1, v/v) with AX, again a 2-fold increase in renin activity was observed which is attributed to the overall increase in plasma angiotensinogen in the mixture. It is concluded that rat plasma prorenin is probably not activated within the circulation by a prorenin convertase from the rat kidney.     

Characterization of epitopes on the 25 kD protein of the macrogametes/zygotes of Plasmodium falciparum

A sexual stage-specific protein of Plasmodium falciparum with a Mr of 25,000 is one of the target antigens of transmission-blocking antibodies. The contributions of tertiary structure and post-translational modifications (glycosylation and acylation) to the structure of the epitopes on this protein were the subject of detailed investigations. After modification of the three-dimensional structure and modification or cleavage of carbohydrate groups and linked fatty acids, the immunological reactivity was investigated by three different techniques: (i) immunoprecipitation of radiolabelled proteins, (ii) enzyme-linked immunosorbent assay (ELISA), and (iii) Western blotting. The results of the experiments indicate that the immunological reactivity of the major epitopes on the 25 kD protein, including the epitope involved in transmission-blocking immunity, are dependent on the tertiary structure of the protein and on the presence of linked fatty acids, but not on the presence or absence of carbohydrate groups.     

Molecular Characterization of Human Papillomavirus Type 159 (HPV159)

Human papillomavirus type 159 (HPV159) was identified in an anal swab sample and preliminarily genetically characterized by our group in 2012. Here we present a detailed molecular in silico analysis that showed that the HPV159 viral genome is 7443 bp in length and divided into five early and two late genes, with conserved functional domains and motifs, and a non-coding long control region (LCR) with significant regulatory sequences that allow the virus to complete its life cycle and infect novel host cells. HPV159, clustering into the cutaneotropic Betapapillomavirus (Beta-PV) genus, is phylogenetically most similar to HPV9, forming an individual phylogenetic group in the viral species Beta-2. After testing a large representative collection of clinical samples with HPV159 type-specific RT-PCR, in addition to the anal canal from which the first HPV159 isolate was obtained, HPV159 was further detected in other muco-cutaneous (4/181, 2.2%), mucosal (22/764, 2.9%), and cutaneous (14/554, 2.5%) clinical samples, suggesting its extensive tissue tropism. However, because very low HPV159 viral loads were estimated in the majority of positive samples, it seemed that HPV159 mainly caused clinically insignificant infections of the skin and mucosa. Using newly developed, highly sensitive HPV159-specific nested PCRs, two additional HPV159 LCR viral variants were identified. Nevertheless, all HPV159 mutations were demonstrated outside important functional domains of the LCR, suggesting that the HPV159 viral variants were most probably not pathogenically different. This complete molecular characterization of HPV159 enhances our knowledge of the genome characteristics, tissue tropism, and phylogenetic diversity of Beta-PVs that infect humans.