丙型肝炎病毒H株包膜蛋白在昆虫细胞中的表达及意义

目的　获得丙型肝炎病毒 (HCV) H株包膜糖蛋白的重组杆状病毒 ,在昆虫细胞中稳定表达包膜糖蛋白 E1和 E2 ,并初步应用于丙型肝炎患者血清抗体的检测。方法　用 PCR方法自 HCV H株扩增出包膜蛋白 E基因 ,构建重组杆状病毒 ,并在昆虫细胞中稳定表达包膜糖蛋白 E1和 E2。免疫荧光及 Western blot方法鉴定表达产物。用表达的 E1和 E2蛋白与 35例丙型肝炎患者血清反应。结果　昆虫细胞中表达的 E蛋白呈现 3种分子大小 ,E1的相对分子量为 2 1× 10 3和 33× 10 3 ,E2的相对分子量为 6 0× 10 3。在 35份感染者血清中 ,有 4例 E1抗体阳性 ,其中 1例检出 E2抗体。在 9例 PCR检测 HCV RNA阳性而抗 HCV检测阴性的血清中 ,有 3例血清与表达的 E蛋白反应。结论　 HCV E蛋白基因可在昆虫细胞中稳定表达 ,表达的 E1和 E2蛋白可用于 HCV患者的血清学诊断     

HUMAN MONOCYTES MAINTAINED IN CULTURE ACQUIRE FUNCTIONAL RESPONSIVENESS TO PLATELET-ACTIVATING FACTOR THAT IS INDEPENDENT OF INCREASES IN PROTEIN TYROSINE PHOSPHORYLATION

1. Acute (day 0) stimulation with platelet-activating factor (PAF) did not elicit superoxide anion (02^-) generation from adherent monocytes. However, by day 2 of culture, PAF induced an increase in O2^- generation that was inhibited by pretreat-ment with the PAF receptor antagonist WEB 2086. 2. The lack of effect of PAF on O2^- generation was not due to the absence of receptors, as PAF stimulated an increase in tyrosine phosphorylation and intracellular calcium ([Ca²⁺]_i) on both days 0 and 2 of culture. 3. Pretreatment with the protein tyrosine kinase inhibitor methyl 2 ,5-dihydroxycinnamate inhibited PAF-induced tyrosine phosphorylation; however, this inhibitor failed to inhibit PAF-induced O₂^- generation. In contrast, pretreatment with the protein kinase C inhibitor staurosporine had no effect on PAF-induced tyrosine phosphorylation, but did inhibit PAF-induced O₂^- generation. 4. These results indicate that monocytes maintained in culture acquire a functional response to PAF through a mechanism that appears to be independent of PAF receptor expression, coupling to increases in [Ca²⁺]_i or tyrosine phosphorylation.     

急性脑血管病患者血清髓鞘碱性蛋白含量研究

用酶联免疫吸附法对３０例急性脑血管病（ＣＶＤ）患者及３２位正常人血清髓鞘碱性蛋白（ＭＢＰ）含量进行检测。结果表明：急性ＣＶＤ组患者血清ＭＢＰ含量显著高于正常人组（Ｐ＜０．０１）；血清ＭＢＰ含量与急性ＣＶＤ的严重程度相关。提示检测血清ＭＢＰ含量对急性ＣＶＤ诊断及预后判断有重要价值     

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.     

唾液蛋白与脾肾两虚、气血不足的关系

实验表明,脾肾两虚、气血不足患者唾液蛋白明显下降,与正常人相比差异明显(P<0.01);脾肾两虚患者唾液蛋白与血浆白蛋白及总蛋白量成正相关性,气血不足患者唾液蛋白与血色素、红细胞量亦成正相关性。     

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…     

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.