三个与agropine合成相关的基因在大肠杆菌微细胞中的表达

pTiAch 5 TR区与在植物中积累agropine相关的三个基因片段已导入pINIIA或pACYC184,在大肠杆菌微细胞中表达出杂合蛋白。三个基因片段的pINIIA重组质粒的表达得到稳定蛋白、不稳定蛋白和在两个相表达出相似蛋白的三种结果。基因2'HindⅢ片段上的UGAA序列是在两个相表达相似蛋白的原因。从产生的杂合蛋白分子量和读码方式看,pTiAch 5中基因2'和基因1'的结构与pTi 15955近似,但由基因0'片段产生的杂合蛋白分子量比根据pTi15955 DNA序列数据推测的稍大。从表达强度和微细胞操作看,这个系统还不适应从大肠杆菌中方便地纯化杂合蛋白的需要。     

Establishment of the human hepatocellular carcinoma cell line and its characteristics

c-Hc-4 has been established and maintained for more than seven years. The hepatocellular carcinoma originated in 45-year old man with liver cirrhosis. The cell grew in vitro forming a sheet of monolayered cells and firmly attaching to the inner surface of cultured flasks. Morphologically they showed epithelial-like pattern. The doubling time was about 20 hours. Their modal chromosome number was 58. Serial heterologous transplantation in nude mice was successful. The histological finding was almost the same patterns as those in the primary tumor. The cultured cells produced alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA).     

A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae

A strain of Vibrio cholerae, which had been engineered to express high levels of the non-toxic B subunit (EtxB) of Escherichia coli heat-labile enterotoxin, was subjected to transposon (TnphoA) mutagenesis. Two chromosomal TnphoA insertion mutations of the strain were isolated that showed a severe defect in the amount of EtxB produced. The loci disrupted by TnphoA in the two mutant derivatives were cloned and sequenced, and this revealed that the transposon had inserted at different sites in the same gene. The open reading frame of the gene predicts a 200-amino-acid exported protein, with a Cys-X-X-Cys motif characteristic of thioredoxin, protein disulphide isomerase, and DsbA (a periplasmic protein required for disulphide bond formation in E. coli). The V. cholerae protein exhibited 40% identity with the DsbA protein of E. coli, including 90% identity in the region of the active-site motif. Introduction of a plasmid encoding E. coli DsbA into the V. cholerae TnphoA derivatives was found to restore enterotoxin formation, whilst expression of Etx or EtxB in a dsbA mutant of E. coli confirmed that DsbA is required for enterotoxin formation in E. coli. These results suggest that, since each EtxB subunit contains a single intramolecular disulphide bond, a transient intermolecular interaction with DsbA occurs during toxin subunit folding which catalyses formation of the disulphide in vivo.     

Immunocytochemical determination of estrogen and progesterone receptors in human endometrial adenocarcinoma cells (Ishikawa cells).

The regulation of both estrogen and progesterone receptor levels in human endometrial adenocarcinoma cells of the Ishikawa line was investigated immunocytochemically by using monoclonal antibodies. Positive staining for estrogen and progesterone receptors was observed in the nuclei of Ishikawa cells. Intercellular heterogeneity in receptor content was evident from the presence of receptor-positive or -negative cells and from differences in staining intensity of positive cells. Quantitative analysis was performed by scoring the staining intensity and the proportion of positively stained cells. The time and dose-dependent stimulatory effect of estradiol added to culture media on progesterone receptor levels was studied by applying both immunocytochemical and biochemical methods. Estradiol at 10 nM (optimal concentration) increased the intensity score for PR from an initial value of 10.1 to 78.3 after 72 h incubation, and the proportion of the positive staining cells from 6.7 to 42.7%. Promegestone (R5020) was effective at 1 microM concentration in decreasing the intensity score for ER from 31.1 to 14.6 after 72 h exposure and the proportion of positive cells from 19.0 to 11.4%.     

活性氧对巨噬细胞呼吸爆发影响及云芝多糖的保护作用

用化学发光法观察到叔丁基氢过氧化物对培养的小鼠腹腔巨噬细胞呼吸爆发有强烈的抑制作用。云芝多糖经腹腔注射后,能增强巨噬细胞呼吸爆发功能对叔丁基氢过氧化物损伤的抵抗力。云芝多糖处理的巨噬细胞谷胱甘肽过氧化物酶基础活力显著提高,在叔丁基氢过氧化物作用下,云芝多糖处理的巨噬细胞仍有较高的谷胱甘肽过氧化物酶活力。说明巨噬细胞的免疫功能与谷胱甘肽过氧化物酶活力有关,非特异性免疫多糖可提高细胞抗氧化能力,减轻活性氧损伤作用。     

Blue Light-Induced Phototropic Response and the Intracellular Photoreceptive Site in Adiantum Protonemata

Blue light-induced phototropism in Adiantum protonemata wasinvestigated with microbeam irradiation. Brief irradiation withblue light effectively induced a phototropic response when itwas applied to a half-side of the apical 200d µm regionof a protonema. The phototropic response was partly reversedby the subsequent far-red light irradiation but the full reversalof the response was not observed even when the fluence of far-redlight was increased. In the far-red reversible part of the response,blue/far-red photoreversibility was repeatedly observed. Thus,both phytochrome and a blue light-absorbing pigment (other thanphytochrome) seem to be involved in the blue light-induced phototropicresponse in Adiantum protonemata. Furthermore, detailed studiesof the far-red light effect on the fluence-response curve forblue lightinduced phototropism revealed that the concomitantmediation by the two receptors was limited to the response inthe relatively higher fluence range of blue light and that theblue light-absorbing pigment alone was responsible in the lowerfluence range. In the higher fluence range, the response mediatedby the blue light-absorbing pigment became saturated and thephytochrome response became evident, indicating a differencein the sensitivities of the two receptor pigments to blue light. When various regions of half-sides of protonemata were irradiatedwith a blue microbeam of 10 µm width, irradiation at theapical 5–25 µm region was most effective both forphytochrome- and blue light-absorbing pigment-mediated response,indicating that the site of blue light perception is almostidentical for each response. (Received July 14, 1986; Accepted September 26, 1986)     

Isolation and characterization of the human ornithine transcarbamylase gene: structure of the 5'-end region

We isolated over 20 phage clones carrying the ornithine transcarbamylase (OTC) [EC 2.1.3.3] gene, from two independently constructed human genomic DNA libraries, using as probes either a rat OTC cDNA or several nuclear DNA fragments derived from some of these isolated clones. These clones, classified into 10 different groups, overlapped and spanned a region of more than 85 kilobase pairs of the human genomic DNA. Restriction mapping and Southern blot analyses demonstrated that one of the clones covers the 5'-end region of the OTC gene. We sequenced the 5'-end region of the OTC gene and found that it covered 665 base pairs of the 5'-flanking region, the complete first exon and a part of the first intron (150 base pairs). In the 5'-flanking region, there were two pairs of putative CAAT and TATA boxes and one enhancer core-like sequence, GTGGAAAG. The first exon contained a coding region for most of the OTC presequence, i.e. 26 out of 32 amino acid residues of the presequence, including the initiation methionine.