Age-related Changes in Bovine α-crystallin and High-molecular-weight Protein

The high-molecular-weight (HMW) protein from the lens is composed mostly of α-crystallin in a highly aggregated state. Bovine HMW protein was carefully separated from α-crystallin by size-exclusion chromatography. α-Crystallin has chaperone-like ability whereby it stabilizes other proteins under conditions of stress (e.g. heat). Comparison of bovine HMW protein and α-crystallin shows that the HMW protein has a markedly reduced chaperone ability compared to α-crystallin. However, in contrast to the results of other workers, we observe no alteration with age in the ability of α-crystallin to act as a chaperone. Using electrospray ionisation mass spectrometry, changes in the phosphorylation of the α-crystallin subunits with age have been quantified. Phosphorylation of α-crystallin occurs early in life but does not alter in proportion after about three years of age. In addition, phosphorylation of the A subunit of α-crystallin has little effect on its chaperone ability. As is found in the artificially prepared HMW complex of α- and γ-crystallin, NMR spectroscopy shows that in the naturally occurring HMW protein, the short C-terminal extension of the α_Bsubunit has lost its flexibility whereas the α_Asubunit extension is still flexible. Post-translational modifications therefore seem to have little effect on the chaperone action of α-crystallin, but alterations in the quaternary structure of α-crystallin via incorporation into the HMW aggregate, lead to major changes in the chaperone ability of the protein. The results are consistent with the notion that one of the contributing factors to cataract formation in the lens is the depletion of α-crystallin with age as it is converted into the HMW protein.     

从包涵体中纯化重组人幽门螺杆菌热休克蛋白A亚单位

目的：建立一种有效从包涵体中纯化重组人幽门螺杆菌热休克蛋白A亚单位HspA的方法。方法：重组基因工程大肠杆菌发酵后，表达的Hp r HspA包涵体经洗涤，变性，复性，采用Q Sepharose High Performance阴离子交换层析和Superdex75凝胶过滤层析分离纯化，使用SDS－PAGE和HPLC检测纯度，选用ELISA和动物实验对纯化蛋白的免疫学活性和生物学活性进行鉴定。结果：Hp的HspA包涵经洗涤和溶解后，Hp的HspA的纯度＞60％，包涵体溶解液经阴离子交换层析和凝胶过滤层析后纯度超过95％，Hp的HspA纯品经检测具有良好的免疫学活性和生物学活性。结论：本研究建立的分离纯化方法可从包涵体中获得高纯度的重组HspA蛋白，为进一步的动物实验研究奠定了基础。     

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.     

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。