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.     

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)     

Specific binding of three neurotoxins with phospholipase A2 activity to synaptosomal membrane preparations from the guinea pig brain

Snake presynaptic toxins such as crotoxin, β-bungarotoxin and taipoxin block neuromuscular transmission through inhibiting the release of acetylcholine by their phospholipase A₂ activities. On the other hand, many other phospholipase A₂s show little neurotoxicity. It is likely that the difference lies in whether high affinity binding to nerve cell membranes exists or not. To test this idea, crotoxin, β-bungarotoxin and taipoxin were first radioactively labeled with Na(¹²⁵I) without loss of their neurotoxicity. Using the radioactive toxins we have found that each of the three showed specific binding to synaptosomal membranes from guinea pig brain. In contrast, we could not detect specific binding of a non-neurotoxic pancreatic phospholipase A₂. Crotoxin and taipoxin, but not β-bungarotoxin, also bound specifically to membrane preparation from other tissues. The binding of each toxin was not greatly affected by the other two toxins. The photoaffinity labeling technique has been used to obtain further information about the components which bind crotoxin. For this purpose, (¹²⁵I) crotoxin was derivatized with N-hydroxysuccinimidyl-4-azidobenzoate. Autoradiographic analysis of the membranes following photoirradiation in the presence of the modified crotoxin revealed that an 85K dalton component was preferentially covalently conjugated with the crotoxin analogue in a specific manner.     

Efficient expression of an Epstein-Barr nuclear antigen in Drosophila cells transfected with Epstein-Barr virus DNA.

In a search for exogenous promoters which function in cultured Drosophila cells, we have co-transfected a D. melanogaster cell line with an Epstein-Barr virus (EBV) cosmid clone which encodes the Epstein-Barr nuclear antigen (EBNA-1). Here we report that Drosophila cells containing stably integrated copies of EBNA-1 encoding DNA synthesise a polypeptide of mol. wt. identical to that of authentic EBNA-1, which is detectable with EBNA-positive but not EBNA-negative human serum. As in EBV-transformed lymphoblastoid cells, this neo-antigen is associated with the nucleus of transfected cells suggesting that cellular localisation signals which operate in mammalian cells are also recognised in insect cells.     

Regulated expression of a Drosophila melanogaster heat shock locus after stable integration in a Drosophila hydei cell line.

DNA-mediated cotransformation has been used to transfer a Drosophila melanogaster heat shock locus into cultured Drosophila hydei cells by use of the copia-based selectable vector pCV2gpt and of pMH10A, a cloned 87A7 heat shock locus encoding a mutant heat shock protein (hsp). Transformed lines contain between 50 and 200 copies of both plasmids, each separately organized as a head-to-tail concatemer which is stably maintained in the transformed lines. Exposure of the cotransformants to heat shock temperatures induces the regulated expression of the hsp RNA and the mutant hsp in all the lines analyzed.