平滑肌来源的IL-17C通过招募IL-17A导致动脉粥样硬化

正动脉粥样硬化发病过程中,白细胞浸润血管并通过趋化因子和细胞因子网络与血管细胞进行频繁互动。已有研究结果表明,IL-17A等炎症因子具有促进动脉粥样硬化发生的作用。然而同为IL-17家族一员的IL-17C在动脉粥样硬化过程中的作用却并不十分清楚。已有结果表明,IL-17C在动脉粥样硬化病变处表达。作者用ApoE敲除鼠的主动脉进行细胞因子分析,发现IL-17C的表     

重组人IL-17A和IL-17F的制备方法及活性测定

人IL-17A和IL-17F具有很高的同源性,在炎症性疾病、自身免疫性疾病和肿瘤中都发挥着重要的作用,是当前研究的热点.应用原核表达系统在大肠杆菌BL21(DE3)中高效表达了人IL-17A和IL-17F;经培养条件的优化,未发现可溶性目的蛋白的表达,免疫印记分析显示,重组蛋白位于包涵体中;对包涵体进行洗涤、凝胶过滤层析纯化和柱上复性,获得重折叠的可溶性蛋白;随后用SDS-PAGE对蛋白样品进行了纯度分析、采用免疫印记和质谱的方法鉴定蛋白产物成分、用ME3T3-E1和RAW264.7两个细胞株对IL-17A、IL-17F的生物学活性进行测定.结果显示,柱上复性的方法制备的谊重组蛋白具有较高的纯度和活性.建立的重组人IL-17A和IL-17F的制备方法可为相关研究中细胞因子的大量应用提供参考.     

Kin17表达促进乳腺上皮细胞MCF-10A增殖

Kin17是一个与DNA复制、DNA修复有关的蛋白质,在人类的各种组织中表达均很低.乳腺上皮细胞生长增殖的分子机制尚未阐明.为了探讨Kin17与乳腺上皮细胞增殖的关系,检测了Kin17在不同增殖状况下的MCF-10A细胞中的表达情况,并把KIN17基因插入真核表达载体pCDNA3.1-(+)中,构建重组质粒pCDNA3.1-Kin17,通过转染MCF-10A细胞,检测Kin17的表达对MCF-10A细胞的增殖、DNA复制活性及信号分子表达的影响;同时在转染Kin17特异性小干扰RNA（siRNA_Kin17）后,分析MCF-10A细胞的Kin17表达及细胞生长状况.实验结果显示,经高浓度血清刺激后,细胞中Kin17表达升高,而且生长越快的细胞,Kin17表达越强;转染重组质粒pCDNA3.1-Kin17明显提高了MCF-10A细胞中Kin17的表达,同时Kin17的上调表达促进了细胞的增殖速度与DNA复制活性,增强了cyclin D1的表达水平.当转染siRNA_Kin17时使Kin17含量下调,MCF-10A细胞生长速度的抑制不显著.实验结果表明,Kin17与乳腺上皮细胞的DNA复制及生长增殖密切相关.对Kin17在乳腺上皮细胞增殖中的作用及分子调控机制的深入探讨,将有助于揭示乳腺癌细胞快速增殖的潜在机制.     

猪白细胞介素17A基因的克隆与鉴定

猪白细胞介素17A(IL-17A)是猪白介素17家族的重要成员,在猪体内发挥重要作用的细胞因子。本研究将猪的外周血单核细胞分离后,用Con A刺激22 h后提取细胞的总RNA,RT-PCR方法扩增出猪IL-17A基因,将其PCR产物回收纯化克隆至p MD-18T载体上,转化至大肠杆菌DH5α,PCR和双酶切鉴定筛选阳性重组克隆质粒。结果显示,筛选的重组克隆质粒为阳性克隆质粒,基因片段为目的基因。克隆获得的猪IL-17A基因的序列与Gen Bank上公布的猪IL-17A基因序列同源性高,为99.4%。本研究成功克隆了IL-17A基因,有助于研究其在猪病毒性疾病中的作用。     

Aβ40(L17A/F19A) mutant diminishes the aggregation and neurotoxicity of Aβ40

Aggregated β-amyloid peptides (Aβ) are neurotoxic and responsible for neuronal death both in vitro and in vivo. From the structural point of view, Aβ self-aggregation involves a conformational change in the peptide. Here, we investigated the relationship between conformational changes and amino acid residues of Aβ₄₀. Urea unfolding in combination with NMR spectroscopy was applied to probe the stabilization of Aβ₄₀ conformation. L17 and F19 residues were found more sensitive to environmental changes than the other residues. Replacement of these two residues with alanine could stabilize the conformation of Aβ₄₀. Further analysis indicated that the Aβ₄₀(L17A/F19A) mutant could diminish the aggregation and reduce the neurotoxicity. These results suggest that L17 and F19 are the critical residues responsible for conformational changes which may trigger neurotoxic cascade of Aβ₄₀.     

Identifying a novel mutation of CYP17A1 gene from five Chinese 17α-hydroxylase/17, 20-lyase deficiency patients

Mutations of CYP17A1 gene could cause complete or partial, combined or isolated 17α-hydroxylase/17,20-lyase enzyme deficiencies (17OHD). We intended to investigate the CYP17A1 mutation in five unrelated patients and analyze its possible influence on phenotype of an atypical 17OHD patient presented with micropenis, hypertension and intermittent hypokalemia. Steroid hormones were assayed in these patients. A novel missense mutation (c.1169C>G, p. Thr390Arg) located in exon 7 was detected in one of the patients. Homozygous c. 985_987delinsAA, p. Tyr329fs mutation was found in two patients, while compound heterozygous mutations (c. 985_987delinsAA, p. Tyr329fs/c. 932–939 del, p. Val311fs and c. 287G>A, p. Arg96Gln/c. 985_987delinsAA, p. Tyr329fs) were found in two other patients, respectively. Then, steric model analysis of CYP17A1 showed that the novel mutation T390R changed the local structure as well as the electrostatic potential of the nearby beta sheet. Finally, site-directed mutagenesis and in vitro expression were used to analyze the activity of novel mutant CYP17A1. It indicated the T390R mutant retained part of enzyme activity, which was consistent to the clinical features. In conclusion, we identified a novel missense mutation of CYP17A1 gene from a patient with micropenis, hypertension and intermittent hypokalemia, which varied from other four patients. It also expanded our understanding of genotype–phenotype correlation of the disease.     

基于MOD17A3的陕西省植被NPP变化特征

利用2000-2006年MOD17A3数据集的年均NPP数据和GIS技术定量分析了陕西省植被NPP的时空变化特征,结果表明:陕西省年NPP变化范围为340 ～434 g C·m-2·a-1,平均值为383 g C·m-2·a-1;年均NPP分布全省呈现北低南高,关中、陕南呈现西高东低的趋势.长城沿线风沙区年平均NPP在0～200gC·m-2·a-1,黄土高原丘陵沟壑区年平均NPP在200～ 300 g C·m-2·a-1,中部林区年平均NPP在400～500 g C·m-2·a-1,渭北旱作农业区年平均NPP在300～400 g C·m-2·a-1,关中灌溉农作区年平均NPP大部在400～500 g C·m-2·a-1,秦巴山地林区年平均NPP ＞400 gC·m-2·a-1.与2000年相比,2006年陕西省年NPP大部分地区是增加的,年NPP增加的面积占国土面积的90.5％.陕西省NPP线性变化趋势以增加为主,NPP变化百分率增加10％以上的面积所占陕西省国土面积的比例为50.6％;植被NPP的变化百分率＞10％的植被主要分布在延安市以北地区,说明通过实施退耕还林等生态建设工程,这些地区植被状况得到较好的改善.     

A synthesis of 7α-hydroxyandrost-4-ene-3, 17-dione

The first convenient chemical synthesis of 7α-hydroxyandrost-4-ene-3,17-dione is reported. Androsta-4,6-diene-3,17-dione was converted into its 6α,7α-epoxy-derivative; reduction of the epoxide with aluminium amalgam gave 7α-hydroxyandrost-4-ene-3,17-dione. This reducing agent is more efficient than chromous acetate for the purpose.     

A progesterone receptor affinity chromatography reagent: 17α-Hexynyl nortestosterone sepharose

Several affinity chromatography reagents have been proposed for purification of progesterone receptor (PgR), and significant results have been achieved with some of these. None, however, have approached the results achieved in affinity chromatography of estrogen receptor. We have therefore synthesized a number of new 19-nortestosterone derivatives capable of chemically stable linkage with Sepharose beads, and have identified one with very high PgR affinity for further study. We first synthesized the epoxides of 17α-allyl nortestosterone, by analogy with the estradiol derivatization of Greene and Jensen. The relative affinity of these epoxides for PgR from T47D human breast cancer cells, however, was only around 5% that of R5020, and affinity beads prepared from them bound very little PgR. We then reacted appropriately protected 17α-ethynyl-nortestosterone with a series of diiodo alkanes, and found that 17α-(6'-iodohex-1'-ynyl)nortestosterone had an affinity of 22% relative to R5020, equal to the affinity of progesterone itself. Reaction with Thiopropyl-Sepharose 6B yielded hexynyl-nortestosterone-Sepharose beads with a ligand density of about 7 micromoles/ml beads. One-hundred μl of these beads adsorbed 71% of the PgR present in 1 ml ofcytosol from T47D cells. This adsorption was inhibited by 10 μM progesterone but not Cortisol, indicating the specificity of the binding. Comparisions with NADAC and Sterogel, other affinity beads used for PgR purification, show that the former takes up much less receptor, while the latter takes up and releases similar amounts of receptor but more extraneous protein, and is less stable. We therefore believe that hexynyl-nortestosterone-Sepharose, having a high density of a high affinity ligand, and having chemically and biochemically stable covalent bonds, should be a good reagent for affinity purification of PgR.     

NMR structure of the water soluble Aβ17–34 peptide

Alzheimer''s disease is the most common neurodegenerative disorder in the world. Its most significant symptoms are memory loss and decrease in cognition. Alzheimer''s disease is characterized by aggregation of two proteins in the brain namely Aβ (amyloid β) and tau. Recent evidence suggests that the interaction of soluble Aβ with nAChR (nicotinic acetylcholine receptors) contributes to disease progression. In this study, we determine the NMR structure of an Aβ_17–34 peptide solubilized by the addition of two glutamic acids at each terminus. Our results indicate that the Aβ peptide adopts an α-helical structure for residues 19–26 and 28–33. The α-helical structure is broken around residues S26, N27 and K28, which form a kink in the helical conformation. This α-helix was not described earlier in an aqueous solution without organic solvents, and at physiological conditions (pH 7). These data are in agreement with Aβ adopting an α-helical conformation in the membrane before polymerizing into amyloid β-sheets and provide insight into the intermediate state of Aβ in Alzheimer''s disease.     

L17A/F19A Substitutions Augment the α-Helicity of β-Amyloid Peptide Discordant Segment

β-amyloid peptide (Aβ) aggregation has been thought to be associated with the pathogenesis of Alzheimer’s disease. Recently, we showed that L17A/F19A substitutions may increase the structural stability of wild-type and Arctic-type Aβ₄₀ and decrease the rates of structural conversion and fibril formation. However, the underlying mechanism for the increase of structural stability as a result of the alanine substitutions remained elusive. In this study, we apply nuclear magnetic resonance and circular dichroism spectroscopies to characterize the Aβ₄₀ structure, demonstrating that L17A/F19A substitutions can augment the α-helicity of the residues located in the α/β-discordant segment (resides 15 to 23) of both wild-type and Arctic-type Aβ₄₀. These results provide a structural basis to link the α-helicity of the α/β-discordant segment with the conformational conversion propensity of Aβ.     

A review of mechanistic studies on aromatase (CYP19) and 17α-hydroxylase-17,20-lyase (CYP17)

In the conventional P-450 dependent hydroxylation reaction, the Fe(III) resting state of the enzyme, by a single electron transfer, is reduced to Fe(II), which reacts with O(2) to produce a Fe(III)-O-O intermediate. The latter following the transfer of another electron furnishes a ferric-peroxyanion, Fe(III)-O-O(-), which after protonation leads to the fission of the O-O bond resulting in the formation of Fe(V)O, the key player in the hydroxylation process. Certain members of the P-450 family, including CYP17 and CYP19, catalyze, at the same active site, not only the hydroxylation process but also an acyl-carbon bond cleavage reaction which has been interpreted to involve the nucleophilic attack of the ferric-peroxyanion, Fe(III)-O-O(-), on the acyl carbon to furnish a tetrahedral intermediate which fragments, leading to acyl-carbon cleavage. Evidence is presented to show that in the case of CYP17 the attack of Fe(III)-O-O(-) on the target carbon is promoted by cytochrome b(5), which acts as a conformational regulator of CYP17. It is this regulation of CYP17 that provides a safety mechanism which ensures that during corticoid biosynthesis, which involves 17α-hydroxylation by CYP17, androgen formation is avoided. Finally, a brief account is presented of the inhibitors, of the two enzymes, which have been designed on the basis of their mechanism of action. Article from the Special issue on 'Targeted Inhibitors'.     

17β—雌二醇下调血管平滑肌内皮素A型受体的表达

为进一步探讨雌激素对心血管的保护作用,实验在双侧卵巢去势大鼠模型和培养的血管平滑肌细胞（VSMCs)上,观察17β－雌二醇（E2）对血管反应性及VSMCs增殖的影响,以RT－PCR和Western blot检测内皮素受体（ETAR）的表达,结果显示：去势雌性大鼠血管对内皮素（ET－1）的反应性明显增高,ETAR特异性受体阻断剂BQ123能完全阻断ET－1对VSMCs增殖的影响,E2能明显抑制ET－1对VSMCs增殖的作用,RT－PCR结果显示E2能抑制ETAR mRNA的表达,Western blot进一步证实E2能抑制ETAR蛋白表达,E2受体阻断剂Tamoxifen能部分抑制ET－1对VSMCs的增殖及ETAR的mRNA和蛋白 的表达。以上结果提示;ET－1促VSMCs增殖的效应主要是由ETAR介导的,雌激素能通过下调ETAR来抑制ET－1对VSMCs 促增殖的作用和血管对ET－1的反应,且此作用与雌激素受体有关。     

生防工程菌株BS-17A稳定性与抑菌活性初步研究

目的:BS-17是内生细菌,对番茄灰霉病菌、叶霉病菌和枯萎病菌具有显著抑菌活性,为了跟踪研究野生型菌株BS-17在番茄根围和叶围的定殖情况,构建了1株带有黄绿荧光蛋白基因标记的生防菌株BS-17A.方法:采用NYD连续培养的方法和平皿抑菌试验的方法对工程菌株的遗传稳定性和抑菌活性进行了初步研究.结果:该工程菌在无选择压力培养基中连续培养50h,质粒遗传稳定性为94%,对番茄灰霉病菌Botrytis cinerea、叶霉病菌Cladosporiumfulvum和枯萎病菌Fusarium oxysporum的抑制作用与野生菌无显著差异,平皿抑菌率分别为85.5%、86.5%和89.8%.结论:该工程菌具有较强的遗传稳定性和抑菌活性.     

IL-17A基因单核苷酸位点多态性与胃癌预后的相关性研究

探讨白介素17A基因多态性与胃癌预后的关系。129例研究对象纳入生存分析,分成死亡和存活两组,用基因测序的方法检测血液样本IL-17A基因SNP位点rs3748067、rs17880588基因型分布。rs3748067位点有3种基因型T/T、C/T、C/C,rs17880588位点有2种基因型A/G、G/G。比较存活组和死亡组之间的基因型分布频率和单点等位基因分布频率,发现rs3748067的基因型C/T在死亡组的分布频率较存活组高,基因型T/T和C/C在死亡组的分布频率低于存活组,两组之间分布频率差异有统计学意义(P0.05)。杂合型C/T基因型在存活组分布低于死亡组(OR=2.051,CI=0.016~1.420),该位点基因型杂合可能为胃癌预后的一种危险因素。rs17880588的两种基因型A/G、G/G在存活组和死亡组的分布频率差异无统计学意义(P0.05)。结论:IL-17A基因rs3748067位点SNP与胃癌预后可能有相关性。     

IL-17A在呼吸道慢性炎症性疾病中的研究进展

白介素-17A(IL-17A)是一种促炎因子,是IL-17细胞因子家族中的一员。该家族的细胞因子分别被命名为IL-17A至IL-17F,IL-17A是该家族中最具代表性的成员,它能够促进炎症细胞释放多种趋化因子、细胞因子和抗菌肽等,诱导中性粒细胞的聚集和增殖,是连接固有免疫和适应性免疫的桥梁。IL-17A的家族细胞因子在很多肺部过敏性、自身免疫性甚至肿瘤性疾病的发生发展和宿主防御中发挥着关键作用,在哮喘、慢性阻塞性肺病(COPD)、囊性纤维化(CF)、结节病、支气管扩张等呼吸道慢性炎症性疾病中均存在异常表达,虽然在多种疾病中未能阐明IL-17A影响疾病发展的具体作用机制,但已证明其水平与疾病的发展存在关联,这不仅为研究相关疾病的发病机制提供了新的切入点,也为其新型治疗手段的研究提供了新的思路。本文就IL-17A在呼吸道慢性炎症性疾病中的研究进展进行综述。     

IL17A impairs blood–testis barrier integrity and induces testicular inflammation

Experimental autoimmune orchitis is a useful model for studying testicular inflammation and germ/immune cell interactions. Th17 cells and their hallmark cytokine IL17A were reported to be involved in the development of autoimmune orchitis. The aim of the present work is to investigate the pathogenic role of IL17A in rat testis. In vitro experiments were performed in order to analyze effects of IL17A on Sertoli cell tight junctions. The addition of IL17A to normal rat Sertoli cell cultures induced a significant decline in transepithelial electrical resistance and a reduction of occludin expression and redistribution of occludin and claudin 11, altering the Sertoli cell tight junction barrier. Intratesticular injection of 1 μg of recombinant rat IL17A to Sprague–Dawley rats induced increased blood–testis barrier permeability, as shown by the presence of biotin tracer in the seminiferous tubule adluminal compartment, and delocalization of occludin and claudin 11. Results showed that IL17A induced focal inflammatory cell infiltration in the interstitium and germ cell sloughing in adjacent seminiferous tubules. Moreover, an increase in TUNEL+ apoptotic germ cells was also observed. Inflammatory ED1+ macrophages were the main population infiltrating the interstitium following IL17A injection. This correlated with an increase in mRNA expression of the monocyte chemoattractant protein Ccl2, its receptor Ccr2 and the vascular cell adhesion molecule Vcam1. Overall results suggest a relevant role of IL17A in the development of testicular inflammation, facilitating the recruitment of immune cells to the testicular interstitium and inducing impairment of blood–testis barrier function.