Immunoelectron microscopic studies on centromere-kinetochore complexes detached from chromosomes

The centromere-kinetochore complexes of Chinese hamster ovary (CHO) cells were detached and separated from the condensed chromatin by treatment with hydroxyurea and caffeine. By labelling the complex for immunoelectron microscopy (immuno-EM) with a mixture of antibodies against centromere proteins (anti-CENP-A,-B, -C) in some cells, we could demonstrate complete detachment of the complexes. No remnants were left at the bulk of condensed chromatin in these cells. In some mitotic cells complex and chromatin were found side by side. It could be shown that the fine structure of the separated material of the complex differs significantly from that of the rest of chromatin. The complex consists of proteins and DNA. This leads us to suppose that the organization of chromatin in the centromere-kinetochore complex is different.     

Antimutagenic activities of naturally occurring polyamines in Chinese hamster ovary cells in vitro

Spermine and spermidine, ubiquitous polyamines present in bacteria and animal cells, are also involved in cell growth. Since they interact with the double helix, they can stabilize the DNA molecule. Recent evidence of the antimutagenic and anticarcinogenic capacity of spermine has focused attention on the mechanism(s) by which such agents can protect cells from induced damages. In the present paper we show the ability of spermine and spermidine to decrease the level of sister chromatid exchanges induced in Chinese hamster ovary cells cultivated in vitro, by treating them with Psoralen + UVA irradiation (able to induce mainly monoadducts and DNA cross-links). Two different mechanisms of polyamine action can be invoked to explain the preservative activity of this class of agents.     

Differential effects of luminol,nickel, and arsenite on the rejoining of ultraviolet light and alkylation-induced DNA breaks

When Chinese hamster ovary cells were treated with ultraviolet (UV) light or methyl methanesulfonate (MMS), a large number of DNA strand breaks could be detected by alkaline elution. These strand breaks gradually disappeared if the treated cells were allowed to recover in a drug-free medium. The presence of nickel or arsenite during the recovery incubation retarded the disappearance of UV-induced strand breaks, whereas the disappearance of MMS-induced strand breaks was retarded by the presence of arsenite or of luminol, a new inhibitor for poly(ADP-ribose) synthetase. Luminol, however, had no apparent effect on the repair of UV-induced DNA strand breaks, and nickel had no effect on the repair of MMS-induced DNA strand breaks. When UV- or MMS-treated cells were incubated in cytosine arabinofuranoside (AraC) plus hydroxyurea (HU), a large amount of low molecular weight DNA was detected by alkaline sucrose sedimentation. The molecular weight of these DNAs increased if the cells were further incubated in a drug-free medium. This rejoining of breaks in cells pretreated with UV plus AraC and HU was inhibited by nickel and by arsenite, but not by luminol. The rejoining of breaks in cells pretreated with MMS plus AraC and HU was inhibited by luminol and by arsenite, but not by nickel. These results suggest that different enzymes may be used in DNA resynthesis and/or ligation during the repairing of UV- and MMS-induced DNA strand breaks, and that nickel, luminol, and arsenite may have differential inhibitory effects on these enzymes. © 1994 Wiley-Liss, Inc.     

Heterologous Expression of Rat Testis GABAA Receptor β3t Splicing Variant in CHO Cells

Objective To characterize a possible retention function of unique sequence in the 5'end of rat testis GABAA receptor β3t splicing variantMethods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and two eukaryotic expression recombinant plasmids of pEGFP-N1 and pEGFP-C1 were constructed respectively by fusing green fluorescent protein to the N or C-terminus of β3t isoform. The recombinant plasmids were transfected into CHO cells by calcium phosphate co-precipitation method. Fluorescence microscope and laser confocal microscope were used to analyze localization of β3t in the transfected cells. ConA-Texas-Red was used to label cell ER and the localization of rat testis β3t splicing variant in CHO cells was determined.Results When rat testis β3t splicing variant was expressed in CHO cells, two expression patterns were delineated, the distributions of uniform and mainly discrete intracellular compartments respectively. The chimera product failed to be translocated into the cell surface when expressed in CHO cells; whereas the β3 subunit of rat brain was incorporated into the plasma membrane.Conclusion The inability of β3t to target into the ER may be a consequence of the unique 25 specific amino acid segments in the N terminus.     

Genetic toxicology testing of Di(2-ethylhexyl) terephthalate

Di(2-ethylhexyl) terephthalate (DEHT) is a commercially produced chemical (Kodaflex® DOTP) that is used as a general purpose, low-volatility plasticizer for polyvinyl chloride and other polymeric materials. Less than 30 million kilograms of DEHT are produced annually. DEHT is isomeric with di(2-ethylhexyl) phthalate (DEHP), a nongenotoxic rodent carcinogen whose mode of action has been suggested to derive from its ability to produce hepatocellular proliferation and/or hepatic peroxisome proliferation. Thus it is important to know the behavior of DEHT in genotoxicity assays in order to compare it with that of DEHP and other phthalate ester plasticizers. It is known from previously published studies that rats fed DEHT in the diet at 2,000 mg/kg produce urine that is negative in the Ames Salmonella bacterial mutagenicity assay in the presence and absence of induced rat liver S-9 and in the presence and obsence of β-glucuronidase/aryl sulfatase. Reported here are the results of direct testing of DEHT in the Ames plate incorporation assay, the Chinese hamster ovary/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) in vitro mammalian cell mutagenicity assay, and an in vitro chromosome aberrations assay using CHO cells. The results for mono(ethylhexyl) terephthalate (MEHT), a metabolite of DEHT, in the Ames Salmonella bacterial mutagenicity assay are also presented. All test results for both DEHT and MEHT were found to be negative, and it is therefore concluded that DEHT, like its isomeric relative DEHP, is not genotoxic. © 1994 Wiley-Liss, Inc.     

抗人角蛋白全IgG基因真核表达载体的构建及表达

目的：构建抗角蛋白抗体基因的真核表达载体，并在CHO(dhfr^-)细胞中表达。方法：从含抗角蛋白抗体基因的原核Fab表达载体中，扩增VH及VL基因。以回收的PCR产物为模板，用重叠PCR扩增带有前导序列的VH、VL基因。经XbaⅠ/BamHⅠ和XhoⅠ/HindⅢ酶切后，分别插入真核表达载体pWD中，构建重组载体pWDkH。经PCR和测序鉴定正确后，用lipofectAMINE2000转染CHO(dhfr^-)细胞。取培养上清检测抗人角蛋白全IgG的表达。结果：构建了抗人角蛋白基因的真核表达载体pWDkH，并在CHO(dhfr^-)细胞中表达：结论：在CHO(dhfr^-)细胞中成功地表达了具有抗原结合活性的抗人角蛋白IgG，为其临床应用奠定了基础。     

血浆纤维蛋白原与冠心病患者胆固醇、血液流变学的相关性及药物干预的研究

目的：研究血浆纤维蛋白原（ｆｉｂｒｉｎｏｇｅｎ,ＦＧ）与冠心病（ＣＨＤ）患者胆固醇、血液流变学的相关性及腹蛇抗栓酶（ＡＨＶＡＴ）的治疗效果。方法：观察６６ 例ＣＨＤ 患者血浆ＦＧ、胆固醇（ＣＨＯ）及血浆粘度、低切变血粘度、红细胞聚集指数等血液流变学指标与６０ 例正常人对照。将６６例ＣＨＤ患者随机分为ＡＨＶＡＴ治疗组３２ 例及肝素治疗组３４ 例,观察治疗前后上述指标的变化。结果：ＣＨＤ组血浆ＦＧ、ＣＨＯ、血浆粘度、低切变血粘度、红细胞聚集指数均明显高于正常对照组（Ｐ＜ ０．０１）。ＣＨＤ组患者血浆ＦＧ 水平与ＣＨＯ、血浆粘度、低切变血粘度、红细胞聚集指数呈直线相关（Ｐ＜ ０．０５, Ｐ＜ ０．０１）。用ＡＨＶＡＴ治疗后上述指标明显下降（Ｐ＜ ０．０５）。结论：血浆ＦＧ 升高与ＣＨＯ 升高有关,血浆ＦＧ 升高可导致ＣＨＤ患者血浆粘度、低切变血粘度及红细胞聚集指数的升高,用ＡＨＶＡＴ 治疗可降低ＣＨＤ患者血浆ＦＧ及ＣＨＯ水平,从多方面改善血液流变学指标     

Cells defective in sphingolipids biosynthesis express low amounts of muscle nicotinic acetylcholine receptor

The properties of the nicotinic acetylcholine receptor (AChR) are modulated by its lipid microenvironment. Studies of such modulation are hampered by the cell's homeostatic mechanisms that impede sustained modification of membrane lipid composition. We have devised a novel strategy to circumvent this problem and study the effect of changes in plasma membrane lipid composition on the functional properties of AChR. This approach is based on the stable transfection of AChR subunit cDNAs into cells defective in a specific lipid metabolic pathway. In the present work we illustrate this new strategy with the successful transfection of a temperature-sensitive Chinese hamster ovary (CHO) cell line, SPB-1, with the genes corresponding to the four adult mouse AChR subunits. The new clone, SPB-1/SPH, carries a mutation of the gene coding for serine palmitoyl transferase, the enzyme that catalyses the first step in sphingomyelin (Sph) biosynthesis. This defect causes a decrease of Sph de novo synthesis at non-permissive temperatures. The IC50 for inhibition of alpha-BTX binding with the agonist carbamoylcholine exhibited values of 3.6 and 2.7 microm in the wild-type and Sph-deficient cell lines, respectively. The corresponding IC50 values for the competitive antagonist D-tubocurarine (D-TC) were 2.8 and 3.4 microm, respectively. No differences in single-channel properties were observed between wild-type and mutant cell lines grown at the non-permissive, lipid defect-expressing temperature using the patch-clamp technique. Both cells exhibited two open times with mean values of 0.35 +/- 0.05 and 1.78 +/- 0.2 ms at 12 degrees C. Taken together, these results suggest that the AChR is expressed as the complete heteroligomer. However, only 10-20% of the total AChR synthesized reached the surface membrane in the mutant cell line and exhibited a higher metabolic turnover, with a half-life about 50% shorter than the wild-type cells. When control CHO-K1/A5 cells were treated with fumonisin B1, an inhibitor of sphingosine (sphinganine) N-acetyltransferase (ceramide synthase), a 45.5% decrease in cell surface AChR expression was observed. The results suggest that sphingomyelin deficiency conditions AChR targeting to the plasma membrane.     

重组人类杀菌肽基因在CHO细胞中的表达

目的 检测重组人类杀菌肽基因（BPI）在真核细胞中的表达.方法 采用脂质体转染法将本室构建的pCDNA3.1-BP1500及pCDNA3.1-BPI600重组子导入中国仓鼠卵巢上皮细胞（CHO）,G418筛选出带有重组子的细胞集落,RT-pCR检测BPI在CHO细胞集落中的表达.结果 转染后G418抗性CHO细胞中可检测到BPImRNA的表达.结论 pCDNA3.1-BPI重组子被成功转染至CHO,并得到有效表达.     

转染VMAT2基因的中国仓鼠卵巢细胞对毒物抵抗作用的研究

目的:研究毒物鱼藤酮和1-甲基-4-苯基吡啶离子(MPP+)对野生型及转染囊泡单胺转运蛋白(VMAT2)基因的中国仓鼠卵巢细胞(CHO)的毒性作用。方法:将不同浓度的MPP+、鱼藤酮与野生型CHO细胞(WT-CHO)和转染VMAT2基因的CHO细胞(VMAT2-CHO)共同培养,采用细胞MTT比色法和形态学观察,探讨毒物对细胞的毒性作用,通过激光共聚焦显微镜观察毒物对2种细胞的细胞内钙浓度([Ca2+]i)的影响。结果:VMAT2-CHO在一定时间(72 h)内对MPP+(0.2-2.0 mmol/L)和鱼藤酮(0.05-1.0μmol/L)的毒性有抵抗作用(P<0.05)。结论:VMAT2可以抵抗一定浓度的MPP+和鱼藤酮引起的神经细胞毒性作用。