流式细胞计的研制

流式细胞计是近年发展起来的一种综合应用光学机械,电子学,流体动力学,激光和计算机的高技术生物医学仪器。它在生物学基础研究和临床医学研究中有广泛的应用。我们最近研制成功国内首台多参数流式细胞计,并已通过中国科学院院级鉴定。本文介绍该仪器的原理,主要结构与技术要点和使用结果等,它将有助于我国流式细胞术及其应用研究工作的开展。     

高胆固醇血症家兔红细胞在交、直流电场中的电泳行为

本文对高胆固醇血症家兔红细胞在交、直流电场中的电泳行为进行了多指标测定,结果显示,高脂组与正常组相比红细胞聚集能力增强,变形能力及膜流动性下降,表明高脂血症可能较易导致血栓形成。中药有效成分8501有对抗高脂引起的红细胞上述改变的作用,提示8501可能对血栓和动脉粥样硬化斑块形成有防治作用。     

银额果蝇的B染色体研究:1.昆明群体的Bs数目和频率

本研究发现银额果蝇昆明群体有丝分裂中期核型中存在B染色体,出现频率为69.1%。目前,在已研究过的来自各个地区的银额果蝇中,昆明群体的B染色体频率最高。B染色体数目为1-6条。该群体内单雌系间的B染色体数目不同,个体间和细胞间的B染色体数目也不同。在核型中,B染色体最小,形态稳定,点状,C-带和G-带呈阳性。     

Nuclease hypersensitivity in the beta-globin gene region of K562 cells

We have investigated chromatin structure in the beta-globin gene region of the K562 human erythroleukemic cell line by using S1 and DNase I nuclease sensitivity assays. Despite the lack of beta-globin gene expression in these cells, we find nuclease-hypersensitive sites to these enzymes in its 5' and 3' flanking regions in K562 chromatin. This result is in contrast to previous reports in which no hypersensitive sites were found in the immediate vicinity of this gene. In the 3' region, one major hypersensitive site at 0.9 kpb 3' and three minor hypersensitive sites at 0.7 kbp, 0.5 kbp 3' and 0.2 kbp 5' of the polyadenylation site were observed; these sites are very similar to those found in fetal liver and adult bone marrow cells in which the beta-globin gene is expressed. We find hypersensitive sites to both enzymes in the 5' region of the beta-globin gene: a major site 0.8 kbp 5' to the cap site, and two minor sites 1.2 and 1.5 kbp 5' to the cap site. The -0.8 kbp site is also present in plasmids containing the beta-globin gene. Our results suggest that the lack of beta-globin gene expression may be related to the lack of hypersensitivity sites in the immediate (150 bp) 5' flanking region of the beta-globin gene, as occurs in other active globin genes.     

Reduced cyclosporin accumulation in multidrug-resistant cells

Cyclosporin accumulation was reduced by 50% or more in multidrug- resistant CHRC5 CHO cells with high levels of P-glycoprotein expression compared to drug sensitive AuxB1 CHO cells. This difference could be overcome by verapamil which is known to interact with P-glycoprotein and reverse multidrug resistance. The difference in cyclosporin accumulation between sensitive and resistant cells decreased with increasing cyclosporin concentrations suggesting that cyclosporine itself regulated its own accumulation through interaction with P-glycoprotein. Indeed, cyclosporin also reversed differences in vinblastine accumulation between resistant and sensitive cell lines. Since P-glycoprotein is highly expressed in the kidney which is also a target for cyclosporin toxicity, the effects of verapamil on cyclosporin accumulation were studied in two renal cell lines, rat mesangial cells and LLCPK1, cells. Verapamil increased cyclosporin accumulation by approximately 70%. These results suggest that cellular cyclosporine accumulation is regulated at least in part by its interaction with P-glycoprotein.     

Antibodies to peptide determinants in transforming growth factor beta and their applications

Polyclonal antibodies have been raised to a series of synthetic peptides which correspond to essentially all regions of the transforming growth factor beta 1 (TGF-beta 1) molecule. All antisera were evaluated for their abilities to react with TGF-beta 1 and TGF-beta 2 in either the native or reduced form in enzyme-linked immunosorbent assays, Western blots, and immunoprecipitation assays. While all antisera demonstrated some ability to recognize TGF-beta 1 in these systems, there was limited cross-reactivity with TGF-beta 2, suggesting that substantial sequence or conformational differences exist between the two growth factors. On Western blots 5-10 ng of purified human platelet TGF-beta 1 could be detected when probed with affinity-purified peptide antisera generated against peptides corresponding to residues 48-77, 50-75, and 78-109 of the 112 amino acid TGF-beta 1 monomer. Antisera raised against peptides 50-75 and 78-109 were most effective in immunoprecipitating reduced and native 125I-TGF-beta 1, respectively. The antisera also were tested for their effectiveness in blocking the binding of 125I-TGF-beta 1 to its receptor. Anti-peptide 78-109 and anti-peptide 50-75 blocked 80% and 40% of the binding, respectively, while antibodies against amino-terminal peptides were without effect. These data suggest that the carboxyl-terminal region of TGF-beta 1 may play a significant role in the binding of the native ligand to its receptor.     

Altered plasma membrane ultrastructure in multidrug-resistant cells

Multidrug resistance is mediated by P-glycoprotein, an integral plasma membrane component which is thought to function as a drug export pump. This model can explain drug resistance, but fails to account for the broader pleiotropy of the multidrug resistance phenotype. We report here a freeze-fracture study revealing increases in the densities of protoplasmic face intramembrane particles in multidrug-resistant Chinese hamster ovary (CHO) and human leukemic cells. The intramembrane particle density in a CHO cell revertant which had lost the characteristics of the multidrug resistance phenotype was indistinguishable from that of the drug-sensitive parental cell line. This demonstration of a global multidrug resistance-linked change in plasma membrane architecture may have significant implications for understanding the variety of concurrent membrane-related changes which are not easily explained by the current model for multidrug resistance.     

A surface component on GH3 pituitary cells that recognizes transforming growth factor-beta, activin, and inhibin

We have examined the ability of various forms of activin and inhibin, which are structurally related to transforming growth factor-beta (TGF-beta), to interact with various types of cell surface TGF-beta binding sites. Activin AB, inhibin A, and inhibin B were unable to compete with 125I-TGF-beta 1 for binding to the TGF-beta receptor types I, II, or III that coexist in human skin fibroblasts, rat liver epithelial cells, and mink lung epithelial cells. In contrast, activins and inhibins effectively competed for TGF-beta 1 binding to GH3 rat pituitary tumor cells. Binding of TGF-beta 1 to GH3 cells was mediated by about 2700 sites/cell with a Kd = 90 pM. Affinity labeling of these GH3 binding sites by cross-linking to 125I-TGF-beta 1 yielded 70-74-kDa labeled complexes distinct from previously identified TGF-beta binding components. Labeling of these 70-74-kDa components with 125I-TGF-beta 1 was inhibited by TGF-beta 1, TGF-beta 2, activin AB, and inhibin B at concentrations in the high picomolar to low nanomolar range, but it was not significantly affected by other polypeptide hormones and growth factors tested. The 70-74-kDa labeled GH3 components represent a novel type of cell surface TGF-beta binding protein that is unique in its ability to recognize various other members of the TGF-beta family of bioactive polypeptides.     

Cell-surface expression of a membrane-anchored form of the human chorionic gonadotropin alpha subunit

We carried out experiments designed to generate a novel cell-surface protein from a small glycosylated secretory protein. DNA encoding the entire precursor of human chorionic gonadotropin (hCG, alpha subunit) was fused precisely to DNA encoding the transmembrane and cytoplasmic domains of the vesicular stomatitis virus glycoprotein. When expressed in animal cells this DNA encoded the 92-amino acid hCG-alpha subunit anchored in cellular membranes by an extension composed of the 49 carboxyl-terminal amino acids of vesicular stomatitis virus glycoprotein. This hybrid protein was transported efficiently to the plasma membrane of animal cells. The two asparagine-linked glycans on the anchored form of hCG-alpha were large and heterogeneous when compared to those on the secretory form. Experiments employing in vitro mutagenesis and the glycosylation inhibitor tunicamycin established that the presence of at least one of the two asparagine-linked glycans was required for expression of the anchored molecule on the cell surface. However, as reported previously, secretion of hCG-alpha occurred in the absence of glycosylation. Also, mutations eliminating the second glycosylation site (at amino acid 78) in both the anchored or secreted forms apparently led to partial denaturation or a conformational change interfering with transport of the protein.