IN VITRO ANALYSIS OF τ PHOSPHORYLATION SITES AND ITS BIOLOGICAL ACTIVITY

INTRODUCTIONThemicrotubule－associatedprotein蚲ishyperphos－phorylatedandglycosylatedinAlzheimerdisease（AD），andtheseabnormalmodificationsformedthebasisofprogressivelyretrogradeneurofibrillarydegenerationseeninADbrainandtherebythedementia（１，２）．ADab－normallyphosphorylated蚲notonlyismicrotubuleas－semblyincompetent，butalsoinhibitsassemblyanddis－assemblesthepreassembledmicrotubulesinvitro（３）．Inthetangle－bearingneuronsinADbrain，thenormalcytoskeletonisdisruptedandreplacedwi…     

Agilent 2100 Bioanalyzer在基因差异表达研究中的应用

目的观察Agilent 2100 Bioanalyzer 芯片分析系统(以下简称Bioanalyzer)在基因差异表达研究中的应用。方法应用限制性显示技术分别从正常和热休克处理后的酿酒酵母细胞中分离出cDNA片段,然后再用Bioanalyzer和传统的琼脂糖凝胶电泳技术对RD-PCR产物进行检测分析。结果Bioanalyzer能更快速、敏感地分离和显示差异表达的基因片段,并且通过对差异片段进行定量比较,发现了数个表达有明显差异的基因片段。结论Bioanalyzer在基因差异表达研究中具有重要的应用价值。     

Ubiquitin and heat shock protein expression in amyotrophic lateral sclerosis

The expression of two heat shock proteins, HSP72 and p57, in addition to ubiquitin, has been studied immunocytochemically in nine amyotrophic lateral sclerosis (ALS) cases and 10 age-matched controls. HSP72 and p57 antibodies did not identify the characteristic ubiquitin-immunoreactive inclusions present in anterior horn cells in ALS spinal cord. Antibodies to HSP72, but not to p57 or ubiquitin, strongly labelled structures corresponding to polyglucosan bodies in spinal grey matter. Such immunoreactive profiles were more abundant in ALS cases, although they were also present in control material. They were sometimes identified by haematoxylin and eosin and periodic acid Schiff reaction, but were not labeled by phosphotungstic acid haematoxylin or by antibodies to glial fibrillary acidic protein. Although ubiquitin, HSP72 and p57 are stress-induced proteins, they are expressed differently and might therefore have different significance in neuronal degeneration.     

肥胖患者血清C-反应蛋白水平变化与代谢综合征的关系

目的　比较肥胖患者急性期标志 -血清C 反应蛋白 (CRP)水平的变化并探讨与代谢综合征的关系。方法　选择 15 3例肥胖患者和 4 7例正常体重者 ,测定血清CRP水平 ,同时检测体重指数 (BMI)、腰围 (W )、腰臀比 (WHR)、血压、糖脂代谢参数、空腹胰岛素 (FIN)、胰岛素原 (PI)、胰岛素原 /胰岛素比值 (P/I) ,并对导致血清CRP改变的因素进行相关分析研究。结果　 ( 1)肥胖组血清CRP高于体重正常组 (P <0 .0 0 1) ,单纯肥胖组与肥胖伴糖代谢异常组之间无明显差异 (P =0 .4 13 ) ;( 2 )肥胖组相关分析显示血清CRP与W、WHR、SBP、TG、PI、HOMA IR、P/I呈正相关 ;( 3 )矫正年龄和BMI后 ,血清CRP与SBP、WHR和RI仍存在相关性 ,与其他变量的相关性消失。结论　 1、肥胖患者血清CRP水平明显高于非肥胖者 ,与SBP、TG、HOMA IR呈正相关 ,提示肥胖患者血清CRP水平升高与代谢综合征、胰岛素抵抗有密切关系 ;( 2 )血清CRP水平与P/I也呈正相关 ,提示血清CRP升高与 β 细胞功能缺陷可能也有一定关系 ;( 3 )BMI、WHR、SBP和PI是血清CRP升高的主要相关因素     

乳酸林格氏液、高渗盐水和全血对失血性休克犬血液动力学的影响

以失血性休克犬为研究对象，比较乳酸林格氏液（LR）、高渗盐水（HS）和全血（WB）对其血液动力学的影响。经右颈外静脉插入Swan-Ganz飘浮导管，分别在全身氧供（DO2）恢复至休克前水平时，以及液体复苏后5、10、15、30、60和120min时测量动物的各项血液动力学指标。结果显示，HS仅需要11．83ml/kg的液体量，在4．97min时即可使休克犬的DO2恢复至休克前的水平，而LR组和WB组则分别需要52．08ml/kg和23．33ml/kg的液体量，在20．83min和9．33min时才能使休克犬的DO2恢复至休克前的水平。3组动物在DO2恢复至休克前水平时其血液动力学指标均能恢复至休克前的水平。提示高渗盐水比乳酸林格氏液和全血更适合失血性休克患者的早期紧急液体复苏治疗。     

胞嘧啶脱氨酶的原核表达和多克隆抗体的制备

目的：在原核系统中表达并纯化大肠杆菌胞嘧啶脱氨酶（cytosine deaminase,CD)，制备鼠抗大肠杆菌CD多克隆抗体，方法：亚克隆CD基因到原核表达载体pMAL-c2和pBV222中，并转化入大肠杆菌DH5α内，诱导表达并纯化MBP-CD和6his-CD融合蛋白，用纯化的MBP-CD融合蛋白免疫小鼠制备多克隆抗体。结果：通过重组质粒的酶切筛选出重组阳性克隆，成功地表达和纯化出MBP-CD和6his-CD融合蛋白，用纯化的MBP-CD成功制备了鼠抗CD多克隆抗体，并用6his-CD和GST-CD重组蛋白进行Western印迹分析，证实了抗体的正确性，结论：应用多克隆抗体可以检测体内外CD基因的表达，为临床前和临床上深入开展CD基因的生物治疗研究提供重要的实验材料。     

Folding and function of the myelin proteins from primary sequence data.

To explain how the myelin proteins are involved in the organization and function of the myelin sheath requires knowing their molecular structures. Except for P2 basic protein of PNS myelin, however, their structures are not yet known. As an aid to predicting their molecular folding and possible functions, we have developed a FORTRAN program to analyze the primary sequence data for proteins, and have applied this to the myelin proteins in particular. In this program, propensities for the secondary structure conformations as well as physical-chemical parameters are assigned to the amino acids and the pattern of these parameters is examined by calculating their average values, autocorrelation functions and Fourier transforms. To compare two proteins, their sequences are aligned using a unitary scoring matrix, and homologies are searched by plotting a two-dimensional map of the correlation coefficients. Comparison of the corresponding myelin basic proteins (MBP) and P0 glycoproteins (P0) for rodent and shark showed that the conserved residues included most of the amino acids which were predicted to form the alpha or beta conformations, while the altered residues were mainly in the hydrophilic and turn or coil regions. In both rodent and shark the putative extracellular domain of P0 glycoprotein displayed consecutive peaks of beta propensity similar to that for the immunoglobulins, while the cytoplasmic domain showed alpha-beta-alpha folding. To trace the immunoglobulin fold along the P0 sequence, we compared the beta propensity curve of P0 with that of the immunoglobulin M603, whose three-dimensional structure has been determined. We propose that the flat beta-sheets of P0 are orientated parallel to the membrane surface to facilitate their homotypic interaction in the extracellular space. An extra beta-fold in the extracellular domain of shark P0 compared with rodent P0 was found, and this may result in a greater attraction between the apposed extracellular surfaces and may account for a smaller extracellular space as measured by x-ray diffraction. A computer search of the myelin protein sequences for functional motifs revealed sites for N-glycosylation, phosphorylation, nucleotide binding, and certain enzyme activities. We note especially that there are potential nucleotide binding sites in proteolipid protein (PLP), MBP and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). This is consistent with the experimental observations that PLP acts like an ionophore or proton channel when reconstituted into planar lipid bilayers, MBP binds GTP, and CNP catalyzes in vitro the hydrolysis of 2',3'-nucleotides into corresponding 2'-nucleotides.