叶酸拮抗同型半胱氨酸诱导的内皮细胞凋亡的作用机制

目的： 明确同型半胱氨酸（Hcy）对内皮细胞凋亡的影响以及叶酸的拮抗作用，阐明Bax和Bcl-2在同型半胱氨酸诱导内皮细胞凋亡及叶酸拮抗中的作用。方法: 用不同浓度的Hcy处理内皮细胞后，应用末端转移标记技术（TUNEL）以及Annexin V/PI染色加流式细胞术了解细胞凋亡状态，免疫组化方法检测Bax、Bcl-2的表达。结果: Hcy能促进细胞凋亡，叶酸具有拮抗作用。Hcy能促进细胞Bax、Bcl-2的表达，上调Bax/Bcl-2比值，叶酸能减少细胞表达Bax及Bcl-2，下调Bax/Bcl-2比值。结论: Bax、Bcl-2参与了Hcy诱导内皮细胞凋亡以及叶酸拮抗作用的过程。     

14-3-3σ调控p27抑制Rat1-Akt细胞增殖

目的： 研究腺病毒介导14-3-3·σ（Ad-14-3-3·σ）对Akt过表达Rat1-Akt细胞增殖的影响，并探讨其作用是否通过调控p27而实现。 方法： 通过5-溴-2′脱氧尿嘧啶（BrdU）实验检测Ad-14-3-3·σ对Rat1-Akt细胞增殖的影响，并通过激酶分析法和免疫荧光实验探讨Ad-14-3-3·σ对p27磷酸化水平及其在细胞内定位的影响。 结果： Ad-14-3-3σ转染的细胞BrdU阳性率（45%）低于PBS处理组（100%）或Ad-β-gal转染的对照组细胞（98%）。14-3-3σ可降低磷酸化p27的水平和减少Akt介导的p27在胞浆中的定位。 结论： 转染14-3-3σ基因能抑制Akt过表达细胞株Rat1-Akt增殖，14-3-3σ通过降低Akt激酶磷酸化p27的活性，阻断Akt介导的p27胞浆错位，从而发挥其抑制Rat1-Akt细胞增殖。     

预测蛋白质相互作用位点的计算方法研究(一)

许多重要的细胞过程如信号转导、转运、细胞运动以及多数调节机制均由蛋白-蛋白之间的相可作用介导，蛋白质之间的相互作用在物理上是通过在两个相互作用蛋白之间形成接触面的短残基序列来实现。识别蛋白-蛋白相互作用位点，以及检测相互作用氨基酸残基之间的特异性与强度特异性，是一个具有重要应用前景的课题，它的应用范围从理性的药物设计到代谢和信号转导网络的分析。虽然有不少准确度不断提高的实验技术和计算方法来检测蛋白质之间的相互作用，但很少有方法能够精确地指出参与蛋白质相互作用的特定残基及其位置，而这些信息是将相互作用数据直接应用于药物开发所必需的。随着生物信息学和计算生物学的发展，通过研究已知蛋白-蛋白相互作用位点的这些不同特征．出现了一些利用序列与结构信息顶测蛋白-蛋白相互作用位点的计算方法。本文简要介绍了近年来在顶测蛋白-蛋白的相互作用位点方面取得一定进展的计算方法，包括基于基因组信息的计算方法、基于蛋白质初级序列的计算方法以及基于蛋白复合物结构信息的计算方法。虽然这些方法在过去儿年里取得了显著的进展，但是大多数在这方面的研究仍处于起步阶段．而现在数据库的不足和实验技术的缺陷对计算预测方法的进一步发展和公平性评价也存在着较大的影响，要提高蛋白-蛋白相巨作用位点预测的鲁棒性与可靠性，仍要有很多的工作要做。（发表在这里的是第一部分）     

Are thrombocyte membranes altered in Alzheimer''s disease? A morphometric and biochemical study

Morphometric analysis of thrombocytes from patients with Alzheimer's disease, from patients with multi-infarct dementia, and from young and agematched healthy control donors, did not reveal any Alzheimer-related increase in internal membranes. Biochemical analysis showed a reduced cholesterol content of thrombocyte membrane preparations from Alzheimer patients relative to age-matched controls, but not relative to multi-infarct dementia patients. Overall distribution of protein kinase C activity (PKC) between cytosol and membrane, in resting as well as in activated thrombocytes from Alzheimer patients, was similar to that in the control groups. However, both Alzheimer and multi-infarct dementia patients had lower cytosolic levels of basal kinase and PKC activities than age-matched controls, while only Alzheimer patients had lower cytoskeletal PKC activity than controls.     

羟丁酸钠对缺氧缺血后新生大鼠海马CA1区Bcl-2、Bax蛋白表达的影响

目的：观察羟丁酸钠(GHB)对新生大鼠缺氧缺血性脑损伤（HIBD）后海马CA1区神经元Bcl-2、Bax蛋白表达的影响。方法：生后 7 d SD大鼠采用Rice等法，制成HIBD动物模型。新生大鼠随机分成假手术（sham）组、缺氧缺血（HI）组、GHB组。其中GHB组包括GHB50（50 mg/kg）、GHB100（100 mg/kg）、GHB200（200 mg/kg）亚组。各组在缺氧完成后 1 h、3 h、24 h、72 h 和 168 h 时点取脑切片作HE染色，用免疫组化染色观察Bcl-2、Bax蛋白的表达。结果：①光镜下HE染色结果：HI组海马CA1区锥体细胞排列紊乱，锥体细胞减少，海马带宽窄不一，可见细胞肿胀和核碎裂。GHB50组和GHB100组可减轻锥体细胞层病理改变。②免疫组化染色结果：HI组缺血缺氧后1h海马CA1区 Bcl-2、Bax表达开始增强，24 h 时达到高峰，其后逐渐减弱。在GHB50组和GHB100组可使Bcl-2表达明显高于HI组（P<0.05，P<0.01），Bax表达明显低于HI组（P<0.05，P<0.05）。结论：GHB可通过对Bcl-2、Bax表达的调控抑制新生大鼠HIBD后海马CA1区神经元损伤。     

Serous papillary carcinoma of the endometrium arising from endometrial polyps: a clinical, histological, and immunohistochemical study of 13 cases

Serous papillary carcinoma is an aggressive tumor. Point mutations in the p53 suppressor gene might explain in part the rapid growth of this malignant tumor and its unfavorable outcome. The aims of this study were to evaluate the behavior of serous papillary carcinoma developing in endometrial polyps and to assess the p53 protein overexpression. Patients included in this study were treated in our institution between 1982 and 2003. All clinical and pathological materials were examined. A p53 protein immunohistochemical analysis was performed on paraffin-embedded tissues. Thirteen serous papillary carcinomas arising from benign polyps of the endometrium were identified. The patients' age averaged 73 years. All patients were treated surgically. After an average follow-up of 22 months, 54% of the patients were dead or alive with disease. Of 10 serous papillary carcinomas, 8 (80%) for which paraffin blocks were available overexpressed the p53 protein. A serous papillary carcinoma arising from benign polyps of the endometrium remains a malignant neoplasia with an unfavorable outcome even if the primary tumor is limited to the polyp. The high rate of protein p53 overexpression suggests that a p53 gene mutation occurs early in the disease and might explain the rapid growth of the tumor.     

神经元保护蛋白TAT-Bcl-XL的体外高效表达及其功能的初步研究

目的：在原核表达系统中表达对凋亡神经元具有保护作用的重要蛋白Bcl-XL与蛋白质转导序列（PTD）的融合蛋白，并检测重组蛋白对重金属离子所诱导的细胞凋亡的保护作用。方法：通过RT-PCR的方法，用Bcl-XL特异引物从乳腺癌细胞系MCF-7细胞的总RNA中扩增出Bcl-XL基因，构建相应的原核表达载体，体外表达的TAT-Bcl-XL融合蛋白经镍亲和层析介质纯化后，通过免疫荧光方法检测其转导293T细胞的能力；并用流式细胞仪检测融合蛋白抑制细胞凋亡的能力。结果：经RT-PCR从MCF-7细胞总RNA中得到相应的TAT-Bcl-XL基因片段，并将其克隆入pCRT7/CT-TOPO载体中，重组质粒pTBTOPO转化大肠杆菌后，在SDS-PAGE和Western blot的结果中出现了与预期分子量相同的蛋白条带和阳性信号，纯化的TAT-Bcl-XL重组蛋白经免疫荧光检测，主要分布于细胞的细胞质中。流式细胞仪的检测结果显示融合表达蛋白可以有效地抑制Zn2＋离子所诱导的细胞凋亡，使细胞的存活率提高40%。结论：TAT-Bcl-XL融合蛋白在大肠杆菌中获得高效表达，初步的功能性检测表明融合蛋白具有抑制细胞凋亡的功能。     

Modulation of the inhibitory action of ATP on acetylcholine-activated current by protein phosphorylation in rat sympathetic neurons

Modulation by protein phosphorylation of the relation between acetylcholine (ACh)-activated current (I _ACh) and adenosine triphosphate-(ATP)-activated current (I _ATP) was investigated with the whole-cell voltage-clamp technique in rat sympathetic neurons. During simultaneous activation by 100 M ATP of an inward current, the current evoked by 100 M ACh was reduced to 60–70% of that in the absence of ATP. Effects of compounds that are known to modulate protein phosphorylation were tested by including them in the intracellular solution. The reduction ofI _ACh by ATP was not observed when K252a (1 M), a non-selective protein kinase inhibitor, adenosine 5-O-(3-thiotriphosphate) (ATP[S], 1 mM) or,-methylene ATP (1 mM) were included in the intracellular solution. Activators of protein kinases, adenosine 3,5-cyclic monophosphate (cAMP, 100 M), guanosine 3,5-cyclic monophosphate (cGMP, 100 M), phorbol 12-myristate 13-acetate (PMA, 1 M), also abolished the reduction by ATP ofI _ACh. The effects of okadaic acid, a protein phosphatase inhibitor, were paradoxical: okadaic acid (2 M) itself abolished the reduction by ATP ofI _ACh but it antagonized the abolishment by cAMP or cGMP of the reduction ofI _ACh. Okadaic acid did not affect the disappearance of the reduction ofI _ACh by ATP in the presence of intracellular PMA. The results suggest that the interaction betweenI _ACh andI _ATP is regulated by protein phosphorylation/dephosphorylation. Possible mechanisms underlying the effects of these modulators of protein phosphorylation are discussed.     

Isolation,characterization, phosphorylation and site of synthesis of Spinacia chloroplast ribosomal proteins

Summary We have characterized the ribosomal proteins from Spinacia chloroplasts using two-dimensional gel electrophoresis. The 30S and 50S subunits contain 23–25 and 36 ribosomal proteins, respectively. In contrast to prokaryotic ribosomes, chloroplast ribosomes contain at least one (and possibly two) phosphorylated ribosomal proteins. Isolated chloroplasts synthesize in the presence of (³⁵S) labeled methionine and cysteine at least seven 30S and thirteen 50S ribosomal proteins which are assembled into (pre)ribosomes. This suggests that about one third of the chloroplast ribosomal proteins is encoded by the chloroplast DNA itself. The identity of several labeled proteins in the two-dimensional gel electrophoretic patterns which did not comigrate with stained chloroplast ribosomal proteins is discussed.Abbreviations CBB Coomassie Brilliant Blue - CHI cycloheximide - cp chloroplast - DTT dithiotreitol - EDTA ethylene diamine tetraacetate - EGTA ethylene glycol-bis (-amino ethyl ether) N,N-tetraacetic acid - kD kilodalton - LHCP light harvesting chlorophyll a/b protein - PMSF phenyl methyl sulfonyl fluoride - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS sodiumdodecylsulphate     

Elucidation of the protein kinase C-dependent apoptosis pathway in distinct subsets of T lymphocytes in MRL-lpr/lpr mice

MRL-lpr mice are severely impaired in the Fas pathway of apoptosis induction. We here evaluate another pathway of apoptosis induction in MRL-lpr mice which is protein kinase C (PKC) dependent. Despite the defect of the Fas pathway, apoptosis developed during culture in vitro in splenic T lymphocytes from MRL-lpr mice more extensively than in T lymphocytes from MRL-+/+ mice. Apoptosis induction in the former cells was then found to be greatly promoted by PKC inhibitor H-7, and partially prevented by PKC activator phorbol 12-myristate 13-acetate (PMA). High sensitivity to H-7, but not to PKA inhibitor HA 1004, of these cells for apoptosis induction was confirmed by detailed time course and dose-dependency experiments of the drug effect. Population analysis showed that both CD4⁺ T lymphocytes and CD8⁺ T lymphocytes from MRL-lpr mice were highly sensitive to H-7, whereas CD8⁺ T lymphocytes, but not CD4⁺ T lymphocytes, from MRL-+/+ mice were susceptible to the reagent. Interestingly, B220⁺Thy-1⁺CD4^?CD8^? T lymphocytes from MRL-lpr mice were most sensitive to H-7 for apoptosis induction. Correspondingly, the membrane-translocated activated PKC-α level in splenic T lymphocytes from MRL-lpr was more extensively up-regulated by PMA than in splenic T lymphocytes from MRL-+/+. These results suggest that some signal consistently activates PKC in MRL-lpr T lymphocytes, and this event is needed for survival of these cells. On the other hand, CD4⁺CD8⁺ thymocytes were deleted by apoptosis in culture with PMA, whether these thymocytes were from MRL-lpr mice or MRL-+/+ mice. This finding suggested that the apoptosis induction pathway linked to PKC activation is intact in CD4⁺ CD8⁺ thymocytes from the Fas-defective MRL-lpr mice. We conclude from these results that the PKC-dependent signal pathways for either cell death or cell activation are intact or even accelerated in lpr mice, which could both compensate for the loss of the Fas pathway and promote the generation of autoreactive T lymphocytes.