利用T7和PR双启动子的新型表达载体的构建及其应用

构建具有λＰＲ与Ｔ７双启动动子的新型原核表达载体ｐＤＯＧ，以便使用同一载体研究不同诱导条件下，重组蛋白在大肠杆菌中表达，降解以及折叠的情况，从而选择最佳的诱导条件。方法采用ｐＢＶ２２０与ＰＥＴ－３表达载体作为原始材料，将ＰＥＴ－３上包括Ｔ７启动子与Ｔ７ｇ－１０释译起始序列的一段序列克隆到     

Alzheimer-Demenz und normales Altern Klinische,neuroradiologische, neurophysiologische und molekularbiologische Verlaufsdaten

Zusammenfassung 30 Patienten mit klinisch diagnostizierter Alzheimer-Demenz (AD) und 55 etwa gleichaltrige Kontrollprobanden wurden über einen 2-Jahres-Zeitraum prospektiv klinisch, neuroradiologisch und elektroenzephalographisch untersucht, um die longitudinalen Ver?nderungen auf diesen Untersuchungsebenen und ihre Zusammenh?nge zu studieren. In der Kontrollgruppe waren im Beobachtungszeitraum keine wesentlichen Ver?nderungen auf einer der Untersuchungsebenen zu verzeichnen. Bereits inital bestanden signifikante Unterschiede zwischen Patienten und Kontrollen hinsichtlich der kognitiven Leistung, der Ventrikelweite und der absoluten Theta- und Delta-Power. Innerhalb der Patientengruppe verschlechterten sich w?hrend des zweij?hrigen Beobachtungszeitraums die Werte der Blessed-Demenzskala um 7 ± 7 Punkte und im Mini-Mental-State Test um 8 ± 4 Punkte. Die Volumina der Seitenventrikel weiteten sich um mehr als 30 % des Ausgangswertes und die absolute Delta- oder Theta-Power stieg um mehr als 10 % des Ausgangswertes an. Hierdurch nahmen die Unterschiede zwischen Kontroll- und Patientengruppe nochmals zu. Wir konnten keine Zusammenh?nge zwischen Krankheitsbeginn, Alter, Apolipoprotein E4 Gendosis und Krankheitsverlauf belegen. Ein initial schlechter Wert der Patienten auf der Blessed-Skala war mit st?rkeren morphologischen und EEG-Ver?nderungen im Verlauf korreliert, w?hrend initial hohe Theta-Power eine st?rkere funktionelle und kognitive Verschlechterung innerhalb der Patientengruppe pr?dizierte.      

The neurochemistry of Alzheimer's disease

Our knowledge of the neurochemical pathology of AD has increased immensely the last years. Although it is now clear that mutations in the APP gene can cause some rare hereditary forms of AD, and that ApoE4 is a prominent risk factor for AD, we at present know little about the underlying cause of AD in the general population and the biochemical mechanisms by which the apolipoprotein E4 isoform affects AD pathogenesis. It is hoped that the near future will see a resolution of the current controversies in AD research, including: 1) whether APP mutations cause Alzheimer's disease by affecting Aβ deposition or the function of APP itself; 2) whether abnormal phosphorylation of tau is a central pathogenetic event, or whether it occurs as epiphenomena that reflect general neurodegeneration in a variety of disease processes; 3) Whether Aβ deposition in the brain is the central event in AD or whether it occurs as epiphenomena in a variety of brain disorders such as head trauma; and 4) whether altered tau phosphorylation occurs secondary to Aβ deposition or vice versa, and what the link is (if any) between the two processes.     

东部马脑炎病毒E2基因与GM-CSF共表达质粒的构建及其免疫原性初步研究

目的:利用粒细胞-巨噬细胞集落刺激因子(GM-CSF)作为基因佐剂,提高东部马脑炎病毒E2基因重组质粒的免疫效果.方法:分别扩增E2基因与GM-CSF基因,连接进入含有内部核糖体插入位点(IRES)的真核表达载体中,构建共表达质粒.Lipofectamine2000介导转染真核细胞BHK-21,反转录PCR检测E2和GM-CSF两个基因的转录,Western印迹法和间接免疫荧光法(IFA)检测细胞内E2基因表达产物的抗原性.通过肌肉注射免疫BALB/c小鼠,IFA法测定血清抗体效价,FACS测定免疫小鼠脾细胞中CD4 /CD8 淋巴细胞构成比,初步观察共表达质粒的免疫原性.结果:经酶切鉴定与序列测定证明重组共表达质粒中含有E2和GM-CSF两个基因且序列正确.转染细胞的反转录PCR可见E2和GM-CSF两个基因的转录.Western印迹结果可见转染细胞的裂解液在相对分子质量50 000位置有特异性条带.将转染细胞制成荧光抗原片,经IFA检测可见胞浆中出现特异荧光.免疫小鼠的最高血清抗体效价为1:160,但细胞免疫未观察到明显的提高.结论:构建的共表达质粒pE2-IRES-mGM-CSF具有良好的免疫原性,能有效刺激小鼠特异性体液免疫应答,提高了E2基因重组质粒诱导的血清抗体效价.     

费氏艾美尔球虫（E.ferrisi）某些生物学特性的研究

采用从自然感染球虫的小鼠粪便中分离的单卵囊接种小鼠获得成功，观察了此种球虫的卵囊形态，孢子化时间，潜隐期和寄生部位等，确定此球虫为费氏艾美尔球虫。为进行费氏艾美尔球虫病的流行病学调查和防治提供了依据。     

烧伤后红细胞损伤的机制——过氧化脂质、维生素E与溶血的关系

本实验发现大鼠体表面积20％Ⅲ°烧伤后,皮肤内产生了大量丙二醛(MDA),第二天达最高,第七天有第二高峰,血浆和红细胞(RBC)MDA第三天达最高,血浆和RBC维生素E(VE)于伤后第二天后迅速下降,RBC溶血第三天最甚。对皮肤MDA、血浆MDA、RBCMDA、血浆VE、BBC VE、1％H_2O_2溶血进行相关分析后发现在不同时相,有不同的相关关系,但基本遵循烧伤皮肤MDA增加、血浆MDA增加、RBC MDA增加、血浆和RBC VE降低,溶血增加的规律。文中讨论了RBC损伤的机制。     

Proteolytic Processing Mechanisms in the Biosynthesis of Neuroendocrine Peptides: The Subtilisin-like Proprotein Convertases

The recent discovery of a novel family of precursor processing endoproteases has greatly accelerated progress in understanding the complex mechanisms underlying the maturation of prohormones, neuropeptides, and many other precursor-derived proteins. At least six members of this family have been found thus far in mammalian species, several having alternatively spliced isoforms, and related enzymes have been identified in many invertebrates, including molluscs, insects, nematodes, and coelenterates. The proprotein convertases are all dependent on calcium for activity and all possess highly conserved subtilisin-like domains with the characteristic catalytic triad of this serine protease (ordered Asp, His, and Ser along the polypeptide chain). Two members of this family, PC2(SPC2) and PC1/PC3(SPC3), appear to play a preeminent role in neuroendocrine precursor processing. Both convertases are expressed only in the brain and in the extended neuroendocrine system, while another important family member—furin/PACE (SPC1)—is expressed more ubiquitously, in almost all tissues, and at high levels in liver. SPC2 and SPC3 exhibit acidic pH optima and other properties which enhance their activity in the acidic, calcium-enriched environment of the dense-core secretory granules of the regulated pathway in neuroendocrine cells, while furin has a neutral pH optimum and is localized predominantly to the trans Golgi network where it is retained by a C-terminal transmembrane domain. Furin processes a wide variety of precursors in the constitutive pathway, such as those of growth factors, receptors, coagulation factors, and viral glycoproteins. Recent findings on the processing of proopiomelanocortin, proinsulin, proglucagon, and several other neuroendocrine precursors by SPC2 and SPC3 are discussed, along with information on the structure, properties, evolution, developmental expression, and regulation or the convertases. An inherited defect in the fat/fat mouse which affects the processing of proinsulin, and probably also many other prohormones, due to a point mutation in carboxypeptidase E has recently been identified and has begun to provide new insights into the functional integration of the individual processing steps.     

Conditions of Alzheimer-type dementia in the old-old and oldest-old patients with special reference to extreme conditions of brain aging

A neuropathological study on 1540 consecutive autopsy brains ranging from 60 to 107 years of age revealed the following points. (1) Of the of the demented cases of the plaque-predominant type, 93% were complicated with multiple tiny cortical infarcts. They showed a tendency for dementia to develop before or after the appearance or worsening of a systemic disorder such as cardiovascular disease, respiratory infection and cancer. However, there was no case showing Alzheimer-type dementia (ATD). (2) The plaque-predominant type might be an extreme condition of brain aging in terms of senile plaques (SP). It is likely that although the pathological appearance of SP alone is not responsible for dementia, its coexistence with multiple cortical infarcts could be the cause of dementia. Therefore, this type should be distinguished from ATD. (3) Primary hippocampal degeneration could also be an extreme condition of brain aging in terms of neurofibrillary tangles. This condition was different pathologically from the hippocampal lesion in ATD. (4) Several characteristics of old-old and oldest-old patients were clarified.     

维生素E对培养心肌细胞脂质过氧化损伤的保护

应用氢过氧化枯烯引发体外培养的乳鼠心肌细胞脂质过氧化损伤过程，观察了添加维生素Ｅ（２００ｍｇ／Ｌ的α－生育酚）的影响。结果显示：维生素Ｅ能明显减轻细胞脂质过氧化物的蓄积、细胞内肌酸激酶漏出和ＤＮＡ断裂，保护细胞膜流动性、肌球蛋白轻链肌酸激酶和头部ＡＴＰ酶的活性；但对￣３Ｈ－ＴｄＲ掺入的影响不显著。表明维生素Ｅ具有抵抗脂质过氧化所致心肌细胞损伤的作用。     

房室间隔缺损的形态学诊断——二维超声心动图与心血管造影,手术的比较

为了评估二维超声心动图（２ＤＥ）对房室间隔缺损（ＡＶＳＤ）的诊断和手术方法选择价值，对８７例ＡＶＳＤ的２ＤＥ，心血管造影及手术结果进行回顾对照研究，病例年龄自３月至１１月（平均３．９岁）。部分型ＡＶＳＤ４６例，过渡型１２例，完全型２９例（Ａ型２１例，Ｂ型４例，Ｃ型４例）２ＤＥ诊断与手术诊断符合率在部分型ＡＶＳＤ中为９７％，在过渡型及完全型ＡＶＳＤ中为８４％，２ＤＥ诊断与手术诊断不符合大多为过渡型与