重组小鼠干细胞逆转录病毒载体高效基因转染CD41+、UT7、U937和MDA-MB-435细胞

目的:研究重组小鼠干细胞逆转录病毒载体介导基因转染,探索一条高效基因转染的途径,为重组小鼠干细胞逆转录病毒载体在基因转染中的应用提供理论依据和奠定实验基础。方法:①逆转录病毒载体的构建:EC1-4(repeats1-4ofcadherin-5extracellulardomains)基因克隆产物和mutant(Ser222A)MEK1基因克隆产物,Bg1Ⅱ和EcoRⅠ限制性内切核酸酶切割后,克隆进入逆转录病毒表达载体pMSCV。②CD41⁺细胞的获取和细胞培养:从脐带血分离的CD34⁺细胞通过TPO诱导表达CD41,FACS分离CD41⁺细胞。高糖DMEM培养液培养NIH3T3和MDA-MB-435细胞,U937细胞培养在RPMI-1640培养液,UT7细胞是细胞因子依赖性细胞株,Iscove'smodifiedDulbeco's培养液中加入GM-CSF。③测定病毒滴度:逆转录病毒载体转入包装细胞293,36h后收集病毒上清液,感染NIH3T3细胞,流式细胞仪测定病毒滴度。④Westernblot:基因转染CD41⁺、UT7、U937和MDA-MB-435细胞,Westernblot检测基因产物的表达。结果:293细胞产生高滴度MEK1pMSCV病毒:3.1×10⁷,高滴度EC1-4pMSCV病毒:1.0×10⁸。用稀释8倍的病毒转染基因,重组逆转录病毒MEK1pMSCV转染白血病细胞株UT7和U973,GFP阳性细胞(转染阳性细胞)分别是60.73％、72.56％。重组逆转录病毒MEK1pMSCV转染原代培养细胞CD41⁺,GFP阳性细胞为30.57％。重组逆转录病毒EC1-4pMSCV转染人乳腺癌细胞株MDA-MB-435,GFP阳性细胞为97.54％。TPO作用CD41⁺和UT7细胞以及血清对U973细胞的作用,显示出外源mutationMEK基因的dominantnegative的效应,实验组磷酸化的MEK1减少。EC1-4基因转染的MDA-MB-435细胞表达了EC1-4基因产物。结论:重组小鼠干细胞逆转录病毒载体能高效基因转染CD41⁺、UT7、U937和MDA-MB-435细胞,转染的基因能稳定地表达。     

Mechanisms of the CYNAP phenomenon: evidence in the Bw49/Bw50 model for epitopes with different spatial orientation of antibody

The mechanism of the cytotoxic-negative, absorption-positive (CYNAP) phenomenon was studied using the model of the Bw49/Bw50 split of the BW21 antigen. Anti-Bw49 antibody bound 60% as well to Bw 50 as to Bw49 cells; however, even at a cytotoxic titer of 64 against Bw49 cells, the antibody was not cytotoxic to Bw50 Cells. At equal numbers of antibody molecules bound, the anti-Bw49 antibody activated C4 and C3, and induced lysis for Bw49 but not for the Bw50 cells. These data are consistent with a model in which different spatial orientations for shared epitopes can account for CYNAP reactivity for at least some selected Bw4/Bw6-associated splits of B locus antigens.     

DNA fragmentation factor 45 knockout mice exhibit longer memory retention in the novel object recognition task compared to wild-type mice

Apoptosis is an important process in the development and function of the central nervous system (CNS). To study the role of DNA fragmentation factor 45 (DFF45/ICAD) in CNS function, we previously generated DFF45 knockout mice. We found that whereas they exhibit apparently normal CNS development, DFF45 knockout mice exhibit an increased number of granule cells in the dentate gyrus and enhanced spatial learning and memory compared to wild-type mice in a Morris water maze test. In this study, we examined the performance of the DFF45 knockout mice in a novel object recognition task to measure short-term nonspatial memory that is believed to depend on the hippocampal formation. Both wild-type and DFF45 knockout mice exhibited novel object recognition 1 h posttraining. However, whereas wild-type mice no longer did so, DFF45 knockout mice were still able to differentiate the novel versus the familiar object 3 h posttraining. The longer memory retention in DFF45 knockout mice did not last up to 24 h as neither wild-type nor DFF45 knockout mice demonstrated novel object recognition 24 h posttraining. These results suggest that a lack of DFF45 facilitates hippocampus-dependent nonspatial memory, as well as hippocampus-dependent spatial memory.     

Laboratory compliance with federal government and professional society recommendations

Quality assurance issues have assumed growing importance in the cytology laboratory. The 1988 Clinical Laboratories Improvement Amendment (CLIA '88) (United States Department of Health and Human Services, Federal Register: U.S. Government Printing Office 1990;55:9495) regulates the patient identifiers and clinical data on the requisition form but does not mandate physician compliance to provide the information. We investigated the use of patient identifiers and clinical data by laboratories as specimen acceptance/rejection criteria. We surveyed 81 board certified cytopathologists and 235 randomly selected cytology laboratories for acceptance criteria of cytology specimens and received responses from 104. Approximately two thirds of all responding laboratories had specific criteria for rejecting specimens on the basis of inadequate identification or clinical data. While the vast majority required the specimens to be identified with patient name, collection date, and specimen source, a minority of laboratories required clinical information such as LMP, prior atypical cytologic/histologic specimens, and history of previous therapy. Little correlation was found between practice setting and the use of rejection criteria. Diagn Cytopathol 1994; 11:85–92. © 1994 Wiley-Liss, Inc.     

Standard skin prick testing and sensitization to inhalant allergens across Europe--a survey from the GALEN network

Skin prick testing (SPT) is the standard method for diagnosing allergic sensitization but is to some extent performed differently in clinical centres across Europe. There would be advantages in harmonizing the standard panels of allergens used in different European countries, both for clinical purposes and for research, especially with increasing mobility within Europe and current trends in botany and agriculture. As well as improving diagnostic accuracy, this would allow better comparison of research findings in European allergy centres. We have compared the different SPT procedures operating in 29 allergy centres within the Global Allergy and Asthma European Network (GA(2)LEN). Standard SPT is performed similarly in all centres, e.g. using commercial extracts, evaluation after 15-20 min exposure with positive results defined as a wheal >3 mm diameter. The perennial allergens included in the standard SPT panel of inhalant allergens are largely similar (e.g. cat: pricked in all centres; dog: 26 of 29 centres and Dermatophagoides pteronyssinus: 28 of 29 centres) but the choice of pollen allergens vary considerably, reflecting different exposure and sensitization rates for regional inhalant allergens. This overview may serve as reference for the practising doctor and suggests a GA(2)LEN Pan-European core SPT panel.     

Apoptosis and apoptosis-associated perturbations of peripheral blood lymphocytes during HIV infection: comparison between AIDS patients and asymptomatic long-term non-progressors

This study was designed to compare the degree of lymphocyte apoptosis and Fas-Fas ligand (FasL) expression in AIDS patients and long-term non-progressors (LTNPs) and correlate these parameters with apoptosis-associated perturbations in lymphocyte function. LTNPs had a lower frequency of apoptotic CD4+ and CD8+ T cells compared with subjects with AIDS. This correlated with a lower frequency of cells expressing Fas and FasL. The frequency of selected lymphocyte populations exhibiting a disrupted mitochondrial transmembrane potential (DeltaPsim) and increased superoxide generation was lower in LTNPs than in patients with AIDS; these abnormalities were associated with lower levels of caspase-1 activation in LTNPs. The results indicate a significantly reduced level of apoptosis and apoptosis-associated parameters in LTNPs than in patients developing AIDS. Based on these findings, a crucial role for mitochondria can be predicted in the process of lymphocyte apoptosis during the evolution of AIDS.     

A quantitative method for thein vitro study of sounds produced by prosthetic aortic heart valves Part II: an experimental,comparative study of the sounds produced by a normal and simulated-abnormal Starr-Edwards series 2400 aortic prosthesis

A comparative study was made of the sounds produced by a normal Starr-Edwards 2400 aortic valve prosthesis with those produced by the same valve but having a simulated overgrowth at the apex of the struts. Comparisons were made over the entire cardiac cycle for time and amplitude, power-density spectra, power-distribution spectra, power-distribution surfaces associated with individual valves, and three-dimensional power-distribution-difference surface. Power-density spectra were compared for portions of the cycle corresponding to the opening, systolic, and closing sounds of the valve. Physical parameters of an acoustical model were estimated from the power-density spectra. The results showed that each comparison gave information pertinent to the simulated malfunction. Opening. systolic and closing sounds, respectively, were different for each valve. The opening sound of the abnormal valve displayed a much lower frequency. Systolic sounds for the two valves were similar in frequency, but the normal valve produced more total power for this sound. The closing sound of the abnormal valve occurred later than that of the normal valve. These differences were more clearly seen when viewed in the frequency domain.     

A Direct Comparison of the Skin Conductance and Skin Resistance Methods

The purpose of the present study was a direct comparison between simultaneous recordings of skin conductance and skin resistance. Sixty male students received a series of 30 white noise stimuli, while measures were taken continuously from four sites on the palmar surfaces of the fingers. Evaluations were made for response amplitudes, recovery, and for an approximate area measure. Magnitude of reactions and reliabilities were compared using ANOVA procedures. Behavioral concordances were estimated as correlations with the subjects' ratings of stimulus intensities. Conductance and resistance measures do not differ in amplitude, in area, or in strength of their reliabilities and behavioral concordances. No differences in any respect are found between sites. Skin conductance yields significantly (p < .01) shorter recovery times than skin resistance, which is discussed in terms of membrane permeability change.     

人血清免疫球蛋白工作标准的制备与标定

本文报导混合浓缩人血清工作标准(89-1)的制备和用WHO相应参考标准血清标定出其中五类免疫球蛋白(Igs)及IgG4亚类含量。该制剂主要用于体外测定IgG、IgA、IgM的工作标准,也可供作体外测定IgD、IgE、IgG4的工作标准。混合前每份血清均无乙型肝炎表面抗原、转氨酶正常。定量分装为每安瓿0.5ml,经冷冻干燥后,4℃保存。参照国际参考标准血清67/97及67/37,分别以免疫单扩散法标定89-1中IgG、IgA、ISM含量;以酶联免疫吸附试验(ELISA)标定IgD、IgG4和IgE含量。其中IgG、IgG4、IgA、IgM含量,应用本室制备的相应单克隆抗体(McAb)标定,而IgD和IgE应用多克隆抗体(PcAb)进行标定。并用五剂量法及四剂量法分别计算Igs含量。     

A comparison of three methods for titration of poliovirus vaccines

Two methods for in vitro endpoint titration of poliovirus — the roller tube and the microtitration assay — were compared with each other and with the plaque assay, using secondary vervet monkey kidney cells and Vero cells as indicators. The roller tube method is the most reliable under difficult working conditions, but is otherwise cumbersome and expensive. The microtitre method is the most economical and the plaque assay the most sensitive. By suspending freshly trypsinized indicator cells with the virus dilutions before planting, it was possible to simplify the microtitre method considerably. The sensitivity of the plaque assay was improved for Vero cells by absorbing the virus onto freshly planted monolayers. The method was scaled down to a semi-micro level by using 24-well cell culture trays. The slower rate of plaque development under a low calcium overlay medium facilitated a more accurate plaque count.