淫羊藿苷可缓解肾阳虚小鼠睾酮及性腺雄激素受体基因的表达下调

本文主要研究肾阳虚(kidney yang deficiency)小鼠血清睾酮及性腺雄激素受体基因的表达,旨在揭示淫羊藿苷(icariin)对肾阳虚症状的影响．雄性小鼠随机分为6组,除正常组注射生理盐水外,其余组注射氢化可的松15 d;再分别给大、中、小剂量组淫羊藿苷,阳性组甲基睾酮,正常组和模型对照组蒸馏水,灌胃15 d．放射免疫法测血清中睾酮含量; RT-PCR和免疫组织化学方法检测雄激素受体基因在性腺组织中mRNA和蛋白质的表达情况．对照组小鼠平均体重最轻,与正常组比较差异显著(P＜0.05)．对照组小鼠血清睾酮的含量显著低于正常组(P＜0.05);大、中剂量给药组,阳性组睾酮与正常组相比差异不显著(P＞0.05);性腺组织中,对照组雄激素受体和mRNA比正常组表达量低,差异显著(P＜0.05);中、小剂量给药组,阳性组雄激素受体和mRNA与正常组相比差异不显著(P＞0.05). 结果表明,肾阳虚小鼠血清睾酮含量,性腺雄激素受体mRNA和蛋白质的表达与正常组相比均有所下降,淫羊藿苷能够抑制其下降,缓解肾阳虚症状．     

Dynamic SUMOylation Is Linked to the Activity Cycles of Androgen Receptor in the Cell Nucleus

Despite of the progress in the molecular etiology of prostate cancer, the androgen receptor (AR) remains the major druggable target for the advanced disease. In addition to hormonal ligands, AR activity is regulated by posttranslational modifications. Here, we show that androgen induces SUMO-2 and SUMO-3 (SUMO-2/3) modification (SUMOylation) of the endogenous AR in prostate cancer cells, which is also reflected in the chromatin-bound receptor. Although only a small percentage of AR is SUMOylated at the steady state, AR SUMOylation sites have an impact on the receptor''s stability, intranuclear mobility, and chromatin interactions and on expression of its target genes. Interestingly, short-term proteotoxic and cell stress, such as hyperthermia, that detaches the AR from the chromatin triggers accumulation of the SUMO-2/3-modified AR pool which concentrates into the nuclear matrix compartment. Alleviation of the stress allows rapid reversal of the SUMO-2/3 modifications and the AR to return to the chromatin. In sum, these results suggest that the androgen-induced SUMOylation is linked to the activity cycles of the holo-AR in the nucleus and chromatin binding, whereas the stress-induced SUMO-2/3 modifications sustain the solubility of the AR and protect it from proteotoxic insults in the nucleus.     

Desensitization of the Neurokinin 1 Receptor Is Mediated by the Receptor Carboxy-Terminal Region, but Is Not Caused by Receptor Internalization

Abstract: The carboxy-terminal cytoplasmic regions of the rat neurokinin 1 (substance P) and neurokinin 2 (neurokinin A) receptors have been exchanged to determine if this region of the neurokinin 1 receptor is involved in its desensitization. When expressed at similar levels in stably transfected Chinese hamster ovary (CHO) cell lines, receptors containing the carboxy-terminal region of the neurokinin 1 receptor desensitized significantly more (as measured by reduction of the inositol 1,4,5-trisphosphate response) when preexposed for 1 min to 1 µ M neurokinin, indicating a role for the carboxy-terminal region of the neurokinin 1 receptor in its desensitization. Measurement of receptor internalization using radiolabeled neurokinins (0.3 n M ) indicated that ∼75–80% of the receptors were internalized in each cell line after 10 min at 37°C, with no observable correlation between neurokinin receptor desensitization and internalization. Measurement of loss of receptor surface sites for cell lines CHO NK1 and CHO NK1NK2 following exposure to 1 µ M substance P also indicated no obvious relationship between the percent desensitization and percent of receptors internalized. Also, two inhibitors of neurokinin 1 receptor internalization, phenylarsine oxide and hyperosmolar sucrose, did not inhibit neurokinin 1 receptor desensitization. The protein kinase inhibitors Ro 31-8220, staurosporine, and Zn²⁺ had no effect on neurokinin 1 receptor desensitization, indicating that the kinases affected by these agents are not rate-limiting in neurokinin 1 receptor desensitization in this system.     

Adrenalectomy-Induced Increase of Brain Protein Synthesis Is Antagonized by Corticosterone Replacements in Free-Moving Rats

Abstract: The autoradiographic method with l [³⁵S]-methionine was used to determine whether changes in glucocorticoid circulating levels were associated with changes in local rates of protein synthesis in rat brain. Chronic bilateral adrenalectomy induced an increase of me-thionine incorporation rates into proteins in 60 of the 62 brain regions examined (mean effect, +50%). This effect was confirmed biochemically and quantified by correcting for the relative contribution of methionine derived from protein degradation to the precursor pool for protein synthesis in the whole brain. Acute or chronic administration of corticosterone, at doses that normalize basal levels of adrenocorticotrophic hormone, reversed or prevented the adrenalectomy-induced increase of protein synthesis in most regions. However, in nearly all the regions studied (59 of 62), acute corticosterone administration to sham-operated rats did not change the apparent rate of protein synthesis. These results demonstrate that glucocorticoids exert a generalized inhibitory action on brain protein synthesis, because the stimulatory and persistent effect of adrenalectomy on protein synthesis was antagonized by corticosterone replacements at physiological doses. Thus, the regulation of overall brain protein synthesis by glucocorticoids emphasizes the role of neuroendocrine events on long-term neurochemical processes.     

Regulation of Androgen Receptor Activity by Transient Interactions of Its Transactivation Domain with General Transcription Regulators

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靶向抗肿瘤蛋白iRGD-CDD的原核表达及生物活性鉴定

目的: 原核表达并纯化具有特异性成纤维细胞激活蛋白(FAPα)酶切位点的靶向抗肿瘤GP-CDD-iRGD融合蛋白,利用FAPα的酶切功能切除融合标签,检测其对FAPα阳性肿瘤细胞株的毒性。方法: 设计并合成GP-CDD-iRGD基因,插入pGEX-4T3 载体,构建重组表达质粒,转化至BL21大肠杆菌感受态细胞中,IPTG诱导表达,SDS-PAGE分析重组融合蛋白的表达,Western blot检测融合蛋白的表达,经GST亲和柱纯化融合蛋白后,通过体外细胞毒性试验(MTT法)和细胞凋亡实验评价该融合蛋白的靶向抗肿瘤活性。结果: 成功构建重组原核表达质粒pGEX-GP-CDD-iRGD,可溶性表达相对分子量约为36 kDa的融合蛋白GST-GP-CDD-iRGD,纯化后蛋白纯度约为90%,经MTT实验测定其对FAPα阳性4T1细胞株的ED50约为18.5μmol/L,流式细胞术检测到其对FAPα阳性4T1细胞株具有选择性毒性作用,早期凋亡比例达到约28%。结论: 原核表达的重组融合蛋白GP-CDD-iRGD对FAPα阴性4T1细胞株未显示毒性,而对FAPα阳性4T1细胞株具有显著的促凋亡作用,为进一步研究其在体内的靶向抗肿瘤活性提供了依据。     

非典型性肺炎病毒S蛋白受体结合域的可溶性表达

试验目的是获得S蛋白受体结合域基因,并获得其高效表达,为SARS病毒受体的进一步研究奠定一定的基础。首先通过P（取方法获得了S蛋白起始密码和SalⅠ限制性内切酶之间包含受体结合区的片段,然后将该基因定向克隆到pET-22b原核表达载体,构建了per—22b—S1重组质粒,转化大肠杆菌并诱导目的蛋白的表达,经SDS—PAGE没有发现明显的目的蛋白带。利用载体和S1基因上的NeoⅠ酶切位点,将S1基因的信号肽序列和部分疏水序列切掉后,构建pET—22b—SNS重组质粒。pET—22b—SNS重组质粒仍然包含受体结合域序列,并且阅读框没有改变。将pET—22b—SNS转化大肠杆菌,发现明显的目的蛋白带。Westem blot结果表明表达蛋白为SARS病毒S1蛋白的一部分。     

A Time-Dependent Increase in Glial Fibrillary Acidic Protein Expression and Glutamine Synthetase Activity in Long-Term Subculture of the GL15 Glioma Cell Line

1. Astrocytes are the most numerous cellular elements in the central nervous tissue, where they play a critical role in physiological and pathological events. The biological signals regulating astrocyte growth and differentiation are relevant for both physiology and pathology, but they are still little understood.2. Using a poorly differentiated glioma cell line, GL15, we investigated whether, in long-term subculture, this could upregulate the expression of glial fibrillary acidic protein (GFAP), as described in some rodent astrocyte cell lines. Under the same culture conditions, we investigated glutamine synthetase (GS) activity, growth-associated protein (GAP)-43 expression, and expression of several neutrotrophic factors.3. A dramatic increase in GFAP expression was evidenced by Western blotting during progressive in vitro growth of GL15 cells. GS specific activity was also upregulated in long-term culture. The time spent in vitro by GL15 cells did not affect GAP-43 and neutrophic factor BDNF and NT3 expression as revealed by RT-PCR analysis.4. Our results suggest that, in GL15, GFAP and GS genes may have common or integrated regulatory mechanisms elicited at the cell confluency which could be relevant for both astrocyte physiology and astrocyte pathology. These mechanisms are not involved in GAP-43 and neutrophic factor BDNF and NT3 expression.     

PDGF受体结合域与乙肝病毒核心抗原的融合表达

化学合成血小板源性生长因子受体结合域１３肽基因,并与乙肝病毒核心抗原基因５′端融合,序列分析表明化学合成的１３肽基因及融合后基因的阅读框架正确．将融合基因亚克隆于ｔａｃ启动子控制的ｐＥＴ３ａ表达质粒中并于大肠杆菌中表达．表达产物经ＥＬＩＳＡ、ＷｅｓｔｒｅｎＢｌｏｔ鉴定表明,融合蛋白已被表达,其单位分子量与推算值一致．电镜观察证明所表达的融合蛋白能形成颗粒．     

Understanding the Origins of Loss of Protein Function by Analyzing the Effects of Thousands of Variants on Activity and Abundance

Understanding and predicting how amino acid substitutions affect proteins are keys to our basic understanding of protein function and evolution. Amino acid changes may affect protein function in a number of ways including direct perturbations of activity or indirect effects on protein folding and stability. We have analyzed 6,749 experimentally determined variant effects from multiplexed assays on abundance and activity in two proteins (NUDT15 and PTEN) to quantify these effects and find that a third of the variants cause loss of function, and about half of loss-of-function variants also have low cellular abundance. We analyze the structural and mechanistic origins of loss of function and use the experimental data to find residues important for enzymatic activity. We performed computational analyses of protein stability and evolutionary conservation and show how we may predict positions where variants cause loss of activity or abundance. In this way, our results link thermodynamic stability and evolutionary conservation to experimental studies of different properties of protein fitness landscapes.     

人源类溶菌酶蛋白6在毕赤酵母中的重组表达及活性分析

利用毕赤酵母(Pichia pastoris)重组表达人源类溶菌酶蛋白6(human lysozyme-like protein6,h Lyzl6),对其酶学性质进行分析。根据毕赤酵母密码子偏爱性设计并人工合成h Lyzl6基因,将其连接至含有乙醇氧化酶启动子(AOX1)的p PIC9K质粒构建重组表达载体p PIC9K-hlyzl6;重组表达载体经线性化后电转化入毕赤酵母GS115感受态细胞,经G418筛选获得高拷贝重组菌株后进行甲醇诱导表达。经甲醇诱导72 h后发酵液上清中酶活性达到最高值,发酵液上清经SDSPAGE检测在14.8 k Da处有重组h Lyzl6蛋白条带,分子量符合预期,通过甲壳素亲和层析可对其进行纯化;采用比浊法测定h Lyzl6酶学活性,结果表明h Lyzl6对溶壁微球菌(Micrococcus lysodeikticus)有较好的杀灭作用,最适反应温度为40℃,最适p H为5.5,其酶活力为54 700U/mg,Cu2+对其活性有明显抑制,EC50为30.2799 mg/L。采用基因工程方法首次在毕赤酵母GS115成功表达了重组h Lyzl6,证实其在体外具有杀菌活性,初步揭示h Lyzl6在男性生殖系统先天性免疫中发挥了一定作用,为进一步研究h Lyzl6的功能和应用开发奠定了基础。     

SDH-SV2C-L4融合蛋白的表达及山梨糖脱氢酶活性检测

目的:构建SDH-SV2C-L4融合蛋白表达载体,在大肠杆菌中表达具有山梨糖脱氢酶(SDH)活性的融合蛋白。方法:将C亚型突触囊泡蛋白2大突环L4(SV2C-L4)基因与SDH基因以GGGS柔性接头连接,在大肠杆菌DH5α中表达;用NBT染色和DCIP脱色的方法检测融合蛋白的SDH活性。结果:DNA测序及SDS-PAGE结果显示构建了融合蛋白表达载体,并表达了SDH-SV2C-L4融合蛋白,相对分子质量约80×103;DCIP脱色及NBT染色均检测到融合蛋白的SDH活性。结论:与SV2C-L4融合的SDH仍具有活性,为下一步SV2C-L4活性检测方法的建立及SDH与SV2C-L4的其他相关研究奠定了基础。     

Interplay of Protein Disorder in Retinoic Acid Receptor Heterodimer and Its Corepressor Regulates Gene Expression

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