端粒单链结合蛋白hPOT1核定位结构域的鉴定

目的：鉴定端粒单链结合蛋白hPOT1核定位结构域。方法：通过hPOT1的不同长度缺失突变体与EGFP绿色荧光蛋白融合，转染细胞后用荧光显微镜观察融合蛋白在细胞内的定位。结果：确定了hPOT进核序列是定位于第355—375位氨基酸之间的富含碱性氨基酸序列，其中R^377，R^373影响hPOT1的细胞核内定位。结论：鉴定出hPOT1的核定位结构域，该结构域影响了hPOT1不同形式剪接体蛋白的细胞内分布。为进一步研究hPOT1在端粒末端调控中的作用提供了线索。     

人肝再生增强因子的cDNA克隆与序列分析

利用大鼠肝再生增强因子核苷酸序列，从人胎肝ｃＤＮＡ文库中筛选到人肝再生增强因子ｃＤＮＡ克隆，该克隆含有１．５ｋｂ的外源插入片段，核苷酸序列测定表明含３７５ｂｐ的读码框架，能编码１２５个氨基酸的蛋白质；与大鼠肝再生增强因子相比，核苷酸序列测定表明含３７５ｂｐ的读码框架，能编码１２５个氨基酸的蛋白质；与大鼠肝再生增强因子相比，核到序列同源性达８７％，氨基酸序列同源性达８４．８％。     

三氧化二砷反式激活基因AsTP2的克隆化及生物信息学分析

目的三氧化二砷反式激活靶基因(AsTP2)的克隆化研究。方法对构建的三氧化二砷差异表达的肝HepG2细胞cDNA消减文库进行筛选，利用RT-PCR技术获得新基因AstTP2的编码序列，结合生物信息学对其氨基酸序列、染色体定位和基因功能进行分析比较。结果AsTP2基因编码区为1119nt，编码产物为372aa。经核苷酸序列数据库(GenBank)和蛋白质一级结构序列数据库(SwissProt)同源序列的搜寻，发现未知功能的同源蛋白，说明克隆的AsTP2基因属于未知功能新基因，GenBank注册号为AY744366。该基因在三氧化二砷诱导的HepG2细胞中表达上调。结论克隆了一条新的三氧化二砷反式激活靶基因AsTP2，为进一步研究三氧化二砷的反式激活作用和揭示砷致癌的生物学机制提供新线索。     

12例继发于桥脑出血的肥大性下橄榄核变性的MRI表现

目的 探讨桥脑出血继发肥大性下橄榄核变性(HOD)的MRI表现.方法 回顾性分析12例桥脑出血后继发HOD的MRI表现,12例均行MRI常规序列和扩散加权成像(DWI)、磁敏感加权成像(SWI)或梯度回波(T2*WI)序列扫描,2例行扩散张量成像(DTI)扫描.结果 MRI表现为同侧或双侧下橄榄核体积增大或无明显变化,T1WI呈等或稍低信号,T2WI呈稍高或高信号,液体衰减反转恢复序列(FLAIR)呈等或稍高信号,DWI呈等或稍高信号,ADC图呈等或稍高信号,SWI或T2*WI呈等或稍高信号,其对原发出血病灶显示最好,DTI示病变侧纤维束稀少,3例并发双侧桥臂变性.结论 HOD多继发于桥脑背盖部出血,有特定的发病部位和较为特征的MRI表现,结合其原发病变可对HOD作出正确诊断.     

神经元核内包涵体病的临床和MRI特征分析(附6例报告)

目的分析6例成人型神经元核内包涵体病患者的临床及MRI特征,提高临床对成人型神经元核内包涵体病的影像认识。方法选取6例经皮肤活检确诊的成人型神经元核内包涵体病的临床、病理及影像资料,并复习国内外相关文献。结果6例成人型神经元核内包涵体病患者临床症状不一,但头颅MRI检查显示明显对称性脑白质病变,T₂WI和T₂flair序列出现对称性高信号,T₁WI低信号,DWI均可见皮质、髓质交界区高信号,以双侧额叶明显,DWI高信号在双侧额叶与顶叶交界区呈断崖样缺失;2例双侧基底节区出现斑片状T₂WI高信号;1例小脑半球中部蚓部旁(蚓旁区)出现T₂flair高信号;1例胼胝体区出现T₂flair高信号。结论成人型神经元核内包涵体病的MRI表现具有一定特征,头颅MRI检查特别是DWI序列对成人型神经元核内包涵体病生前诊断具有重要价值。     

肝豆状核变性的脑部MRI表现(附5例报告)

目的 评价MRI对诊断肝豆状核变性（HLD）的意义。方法 5例由临床神经学与生化学证实的HLD患均经脑部T1加权和T2加权自旋回波序列磁共振成像（MRI）。1例患还经Gd-DTPA增强扫描。记录了每个序列形态学特征和MRI信号特征。结果 在T2加权像上，所有异常病灶均呈高信号，例如，全部病例两侧豆状核和3例的两侧尾状核均呈对称性分布的高信号，2例的中脑见对称性分布的腔隙性高信号，1例右侧颞叶深部白质内见局限性高信号，而在T1加权像上，所有异常病灶均呈低信号，1例经Gd-DTPA增强扫描后，病灶无强化。脑萎缩改变见于3例，表现为脑沟、脑裂、脑池加宽，及脑室系统扩张。尾状核轻度萎缩见于1例。结论 肝豆状核变性的特征性MRI表现是其诊断的重要依据，尤其是T2加权SE序列应推荐为诊断HLD的首选方法。     

肝豆状核变性的MRI表现及临床诊断价值

目的 探讨肝豆状核变性(WD)的MRI表现及临床诊断价值。方法 选取28例WD患者的MRI资料进行分析，主要病变位于神经系统和肝脏，患者均行3.0T MRI检查。结果 28例WD患者中脑部MRI发现异常信号16例，评分为(8.12±4.92)分，改良Young量表评分(19.27±6.13)分，相关系数为0.69,差异有统计学意义(P<0.05);其余12例为肝脏病变，其中8例患者肝脏T₁WI呈多发高信号小结节，T₂WI及T₂WI脂肪抑制序列则表现为多发低信号结节，1例多发结节表现周围更高信号的间隔包围，呈相对“蜂窝状”的特征，MRI增强扫描结节未见明显异常强化。结论 通过对高场强MRI脑型、肝型WD的信号特点分析，有助于临床早期准确的诊断，为WD的随访及疗效评估提供依据。     

乙型肝炎病毒e抗原在肝细胞内作用蛋白AK026018亚细胞定位的研究

目的：确定肝细胞内乙型肝炎病毒e抗原(HBeAg)作用蛋白AK026018的亚细胞定位，初步研究该蛋白的生物学功能。方法：应用逆转录聚合酶链反应(RT-PCR)技术，从HepG2细胞中扩增编码AK026018蛋白的全基因，构建真核表达载体pEGFP-AK，转染HepG2、293T细胞系，在激光共聚焦荧光显微镜下与转染空载体pEGFP-N1的细胞比较观察。结果：经限制性内切酶分析和DNA序列测定鉴定构建的重组表达载体正确。通过激光共聚焦荧光显微镜观察，转染了重组栽体pEGFP-AK的细胞绿色荧光信号较集中分布于胞浆中。而空载体pEGFP-N1转染的细胞绿色荧光信号在整个细胞中均匀分布。结论：成功分离了AK026018全基因序列并构建了真核表达载体，该基因表达产物亚细胞定位于细胞浆中。     

肝豆状核变性脑部MRI与临床相关性探讨

目的 :探讨肝豆状核变性脑部 MRI改变及其与临床的关系。方法 :对 9例经临床及化验证实的肝豆状核变性患者的脑部 MRI表现进行了分析。结果 :发病部位依次为壳核 10 0 % ;苍白球 88.8% ;红核 77.7% ;黑质 77.7% ;丘脑 6 6 .6 % ;齿状核 33.3% ;桥脑 2 2 .2 %。脑部 MRI主要改变为铜沉积所致的继发性长 T1 低信号和长 T2 高信号 ,由铜沉积顺磁作用直接引起的短 T2 低信号出现较少。结论 :肝豆状核变性脑部改变以继发为主 ,MRI表现与临床症状具有一定相关性意义。 FSEIR是显示病灶的最佳序列     

光学方法二维成像前庭核神经元的突触传导

为探索前庭核神经元传导的时空二维特征，用吸光性电压敏感染料RH155染色新生小鼠的脑干切片20min，电刺激前庭神经，用光学记录膜电位技术成像前庭核的神经电活动。结果显示，电刺激前庭神经后光学记录显示前庭核出现神经电兴奋，在部分实验中观察到同侧及对侧前庭核的兴奋传导；前庭核神经兴奋有激发延迟和高峰延迟；所记录的光学信号具有光吸收波长特性，表明光学记录的可靠性；光学信号包括尖峰状快反应信号和持续较长时间的慢反应信号，所有兴奋信号被20μmol/L河豚毒(特异性钠通道阻断剂)不可逆性消除，表明快反应信号来源于钠通道介导的动作电位，慢反应信号被无钙液可逆性阻断，表明慢反应信号可能为兴奋性突触后电位。研究表明，光学记录膜电位方法可以在神经细胞群的水平上直观观察脑干前庭核神经电活动的时空二维方式及其兴奋性突触传递过程。     

人体19种氨基酸单体的核磁共振波谱特征信息表达

目的:随着生物医学迅猛的发展,更多的新技术新方法被引入实验室,并逐步应用于人体超微结构与功能的研究,其中多肽、氨基酸单体的生物活性研究愈加受到有关课题的重视。并且多学科的渗透和多技术的交叉已形成新的模式,鉴定其结构的性质分析手段亦跃升到借助物理化学等高新技术,通过物质本身官能团的多组信息进行特征分析。方法:根据构成蛋白质骨架的主要原素——氢具有产生磁矩、有自旋能力的特性,采用Nuclear magnetic resonance—NMR核磁共振(氢)波谱技术,对L-CLY等19种氨基酸进行了研究。结果:15种氨基酸的波谱特征表达与国外相关研究十分吻合,并且首次获得L.D—His和L.D—Trp4种氨基酸的结构信息。结论:本组研究设立HMR分析条件:脉宽PW=5.8,增益RG=26,驰豫=0.2.采样NS=36,谱宽SW=4000Hz,偏置01=3800条。实验证明能满足分析需要。     

Mobilisation of structural proteins during exercise

In general, the mobilisation of structural proteins is necessary for enzyme synthesis and for renewing cellular structures with amino acids and precursors of nucleotic acids. However, during exercise the adaptive synthesis of proteins occurs only in the liver to some extent. In muscle tissue most protein synthesis is suppressed, although the synthesis of certain proteins in muscle remains unchanged or even increases. The general suppression of protein synthesis in muscle leaves much of the free amino acid pool unused. The breakdown of tissue proteins may also increase in various tissues, but there is no convincing evidence for proteolysis of contractile proteins in active muscle. As a result of these processes, an increased pool of available free amino acids is created. The main use of free amino acids is connected with the energy requirement of muscular activity, through the oxidation of branched-chain amino acids and the use of alanine in gluconeogenesis. In active muscles the output of alanine is increased. It is based on usage of pyruvate, which is produced in increased amounts due to intensified glycogenolysis and glycolysis, and of amino groups, which are liberated in oxidation of branched-chain amino acids. In the liver, alanine is consumed. The carbon skeleton of alanine is required for gluconeogenesis and the liberated amino groups are used in ureagenesis. The branched-chain amino acids are transported from the liver to active muscle for their oxidation. The increases in the free amino acid pool, in the rate of the glucose-alanine cycle, and in the use of amino acids in the liver are stimulated by an increased level of glucocorticoids and a decreased level of insulin during exercise. During recovery after exercise the use of amino acids for adaptive protein synthesis is intensified. This coincides with a persistently high rate of protein breakdown, constituting an increased rate of protein turnover. During recovery, the production of 3-methylhistidine by previously active muscles increases. It results in an increase in urinary output of 3-methylhistidine after exercise. Immediately after exercise the level of free 3-methylhistidine is elevated in the intestine for only a short time and the fact that it does not contribute significantly to the delayed increase in the excretion of 3-methylhistidine excretion after exercise must be considered as a sign of increased turnover of contractile proteins, helping to restore a good contractile function.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ingestion of casein and whey proteins result in muscle anabolism after resistance exercise

PURPOSE: Determination of the anabolic response to exercise and nutrition is important for individuals who may benefit from increased muscle mass. Intake of free amino acids after resistance exercise stimulates net muscle protein synthesis. The response of muscle protein balance to intact protein ingestion after exercise has not been studied. This study was designed to examine the acute response of muscle protein balance to ingestion of two different intact proteins after resistance exercise. METHODS: Healthy volunteers were randomly assigned to one of three groups. Each group consumed one of three drinks: placebo (PL; N = 7), 20 g of casein (CS; N = 7), or whey proteins (WH; N = 9). Volunteers consumed the drink 1 h after the conclusion of a leg extension exercise bout. Leucine and phenylalanine concentrations were measured in femoral arteriovenous samples to determine balance across the leg. RESULTS: Arterial amino acid concentrations were elevated by protein ingestion, but the pattern of appearance was different for CS and WH. Net amino acid balance switched from negative to positive after ingestion of both proteins. Peak leucine net balance over time was greater for WH (347 +/- 50 nmol.min(-1).100 mL(-1) leg) than CS (133 +/- 45 nmol.min(-1).100 mL(-1) leg), but peak phenylalanine balance was similar for CS and WH. Ingestion of both CS and WH stimulated a significantly larger net phenylalanine uptake after resistance exercise, compared with the PL (PL -5 +/- 15 mg, CS 84 +/- 10 mg, WH 62 +/- 18 mg). Amino acid uptake relative to amount ingested was similar for both CS and WH (approximately 10-15%). CONCLUSIONS: Acute ingestion of both WH and CS after exercise resulted in similar increases in muscle protein net balance, resulting in net muscle protein synthesis despite different patterns of blood amino acid responses.     

The shape of DNA elution dose-response curves under non-denaturing conditions: the contribution of the degree of chromatin condensation.

We have re-examined the effect of detergent type, pH and temperature of lysis on the shape of the DNA elution dose response under non-denaturing conditions using plateau-phase CHO cells. Results practically identical to those previously published (Okayasu and Iliakis, 1989) were obtained, with a 1 h incubation at 60 degrees C during lysis with sodium-N-laurylsarcosine (NLS) resulting in almost linear dose-response curves. We also examined chromatin decondensation as a contributing factor in the linearization observed in the elution dose-response curve under the above conditions. When nuclei with condensed chromatin were prepared from irradiated cells, applied on the filter and lysed with NLS at room temperature, a shoulder-type elution dose-response curve was obtained only slightly higher than that of cells lysed under the same conditions. However, when nuclei prepared from irradiated cells were applied on the filter after relaxation of chromatin by incubation in low ionic strength buffer and lysed with NLS at room temperature, an almost linear dose-response curve was obtained similar to that of cells lysed with NLS at 60 degrees C. Lysis with NLS at 60 degrees C of nuclei with relaxed chromatin did not further modify the DNA elution dose-response curve. Based on these results we propose that the linearization of the DNA elution dose-response curve observed after chromatin decondensation reflects a reduction in the degree of chromatin compactness in the nuclear complexes that leads to a relatively uniform distribution of the DNA on the filter and reduces trapping of elutable material in the compact nuclear structures otherwise present. Since high radiation doses dissolve compact nuclear structures, trapping of elutable material is expected to be highest at low doses of radiation, leading to the observed reduction in the fraction of DNA eluted per Gy at low versus high radiation doses and thus to the observed shoulder. Furthermore, we propose that the linearization of the DNA elution dose-response curves observed in cells lysed in NLS at 60 degrees C may also be due to a decondensation of the nuclear complexes on the filter as a result of the combined action of detergent and high temperature. The notion of a correlation between DNA elution dose response and cell radiosensitivity was examined in two human (SQ20B, SCC61) and two Chinese hamster (V-79, irs-2) cell lines with widely different radiosensitivities.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heat Shock (45°C) Results in an Increase of Nuclear Matrix Protein Mass in HeLa Cells

Summary

The nuclear matrix from HeLa cells heated at 45°C was isolated to determine the effect of thermal shock on its composition and structure. The matrix from unheated cells contained about 10 per cent of total cell protein and was observed to be a spherical particle with a diameter ranging from 3 to 5 μm with the major constituent polypeptides having molecular weights of 45, 47, 55, 57, 59 and 65 kilodaltons. The nuclear-matrix protein mass increased linearly with increasing exposure time at 45°C with no observable change in its size or shape. The additional proteins were observed in general to have molecular weights greater than 45 kilodaltons, with marked increases in polypeptides of 28·5, 38·5, 60, 66, 75, 81, 88, 100 and 115 kilodaltons. An exponential relationship was observed between heat-induced cytotoxicity and the nuclear matrix protein mass increase. A 15 per cent increase in matrix protein mass was sustained prior to the onset of cytotoxicity, while a 35 per cent increase in matrix protein content was associated with a 63 per cent probability of cell killing. The results indicate that redistribution of cell protein or alterations in the mass or structure of the nuclear matrix may be involved in heat-induced cytotoxicity.     

宫颈癌细胞放射损伤后IER5蛋白表达及其相互作用蛋白的筛选和验证

目的 了解IER5蛋白经放射损伤后的表达变化,并筛选IER5蛋白在放射损伤后可能参与DNA损伤修复通路中的相互作用蛋白。方法 HeLa细胞经4 Gy γ射线照射后在不同时间点收集细胞,提取总蛋白质和细胞核蛋白行Western blot检验;构建3×Flag标签融合表达的IER5蛋白表达载体,转染人肾上皮细胞293T并照射,收集细胞行免疫沉淀富集IER5的结合蛋白,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离免疫共沉淀复合物,考马斯亮蓝染色观察差异条带并切胶酶解后行质谱分析,对可能相互作用蛋白结果进行Western blot验证。结果 IER5蛋白照后4 h表达逐渐增加,12 h至高峰,持续至48 h;细胞核内IER5蛋白表达也有增加趋势;质谱分析数据表明,照射组检测出374个蛋白,对照组检测出256个蛋白,两组比较差异蛋白41个,照射组中包括与DNA结合、代谢、损伤修复相关的10个蛋白;其中,与DNA损伤修复密切相关的1型聚ADP核糖基化聚合酶（PARP1）得到Western blot验证。结论 IER5是放射损伤相关蛋白,其可能参与DNA损伤修复通路。     

Incorporation of Tritium into Organic Compounds of Brain

We have re-examined the effect of detergent type, pH and temperature of lysis on the shape of the DNA elution dose response under non-denaturing conditions using plateau-phase CHO cells. Results practically identical to those previously published (Okayasu and Iliakis, 1989) were obtained, with a 1 h incubation at 60°C during lysis with sodium-N-laurylsarcosine (NLS) resulting in almost linear dose-response curves. We also examined chromatin decondensation as a contributing factor in the linearization observed in the elution dose-response curve under the above conditions. When nuclei with condensed chromatin were prepared from irradiated cells, applied on the filter and lysed with NLS at room temperature, a shoulder-type elution dose-response curve was obtained only slightly higher than that of cells lysed under the same conditions. However, when nuclei prepared from irradiated cells were applied on the filter after relaxation of chromatin by incubation in low ionic strength buffer and lysed with NLS at room temperature, an almost linear dose-response curve was obtained similar to that of cells lysed with NLS at 60°C. Lysis with NLS at 60°C of nuclei with relaxed chromatin did not further modify the DNA elution dose-response curve. Based on these results we propose that the linearization of the DNA elution dose-response curve observed after chromatin decondensation reflects a reduction in the degree of chromatin compactness in the nuclear complexes that leads to a relatively uniform distribution of the DNA on the filter and reduces trapping of elutable material in the compact nuclear structures otherwise present. Since high radiation doses dissolve compact nuclear structures, trapping of elutable material is expected to be highest at low doses of radiation, leading to the observed reduction in the fraction of DNA eluted per Gy at low versus high radiation doses and thus to the observed shoulder. Furthermore, we propose that the linearization of the DNA elution dose-response curves observed in cells lysed in NLS at 60°C may also be due to a decondensation of the nuclear complexes on the filter as a result of the combined action of detergent and high temperature. The notion of a correlation between DNA elution dose response and cell radiosensitivity was examined in two human (SQ20B, SCC61) and two Chinese hamster (V-79, irs-2) cell lines with widely different radiosensitivities. We found that radiation-sensitive and radiation-resistant cell lines give similar DNA elution dose-response curves, suggesting that elution dose response and cell radiosensitivity are not necessarily coupled.     

Radiation-induced increase in the release of amino acids by isolated, perfused skeletal muscle

Local exposure of the hindquarter of the rat to 15 Gy of gamma-radiation resulted, 4-6 h after irradiation, in an increased release of amino acids by the isolated, perfused hindquarter preparation, 70 per cent of which is skeletal muscle. This increase in release involves not only alanine and glutamine which are synthesized to a large extent de novo in muscle, but also those amino acids which are not metabolized by muscle and, therefore, released in proportion to their occurrence in muscle proteins. Because metabolic parameters and content of energy-rich phosphate compounds in muscle remain unchanged, it is unlikely that general cellular damage is the underlying cause of the radiation-induced increase in amino acid release. The findings strongly favour the hypothesis that the increased availability of amino acids results from enhanced protein breakdown in skeletal muscle which has its onset shortly after irradiation. This radiation-induced disturbance in protein metabolism might be one of the pathogenetic factors in the aetiology of radiation myopathy.