A组轮状病毒SA11VP6基因的克隆和表达

从ＳＡ１１ＶＰ６基因全序列克隆开始,设计一对两端带有酶切位点的引物,逆转录ＰＣＲ扩增出ＶＰ６全基因ＣＤＮＡ。经酶切后插入ＰＵＣ１９,构建了ＶＰ６全基因克隆ＰＲＡ６。再经酶切后插入痘苗病毒载休质凿ＰＪＳＡ１１７５中。利用Ｌｉｐｏｆｅｃｔｉｎ导入ＴＫ１４３细胞,利用ＴＫ基因和Ｌａｃ基因作为重组病毒的筛选标记。表达产物用单克隆抗体ＥＬＩＳＡ法检测,发现细胞培养上清和细胞裂解液都是阳性。Ｗｅｓｔｅｒｎ ｂ     

Action of Cystine in the Cytotoxic Response of Escherichia Coli Cells Exposed to Hydrogen Peroxide

Cystine markedly enhanced the cytotoxic response of Escherichia coli cells to concentrations of hydrogen peroxide resulting in mode one killing, but displayed little effect in mode two killed cells. The effect of cystine was concentration-dependent over a range of 5-50 μM and did not further increase at higher levels. Cystine had similar effects in other bacterial systems.

In order to sensitize the cells to the oxidative injury, the amino acid must be present during exposure to the oxidant since no enhancement of the cytotoxic response can be observed in cystine pre-loaded cells. In addition, no further enhancement of cytotoxicity could be detected when cystine was added before and left during challenge with the oxidant. The enhancing effect of cystine on oxidative injury of E. coli cells appears to be directly mediated by the amino acid and in fact cysteic acid, the most likely oxidation product, had no effect on the killing of bacterial cells elicited by hydrogen peroxide. Other disulfide compounds such as oxidized glutathione, cystamine and dithionitrobenzoic acid only slightly increased the susceptibility of bacteria to the oxidant. The effect of the disulfides was not concentration-dependent over a range of 200-800 μM and was statistically significant only for cystamine.

Taken together, these results indicate that cystine markedly increases the cytotoxic response of bacteria to hydrogen peroxide and suggest that the amino acid might impair the cellular defence machinery against hydrogen peroxide. This effect may involve a thiol-disulfide exchange reaction at the cell membrane level.     

Bioconversion of substituted naphthalenes to the corresponding salicylic acids

The Pseudomonas fluorescens N3 was isolated from soil for its ability to utilize naphthalene as a carbon source. The strain transforms 2,3-dimethyl-, 2-methoxy-, 1- and 2-ethylnaphthalenes to the corresponding salicylic acids competitively with chemical synthesis. The identification of 2-hydroxy-2-carboxy-7-ethylchromane by biotransformation of 2-ethylnaphthalene, contributes to elucidating the steps involved in the catabolic pathways of naphthalenes to salicylaldehydes. Correspondence to: F. Pelizzoni     

The proliferating cell marker monoclonal antibody Ki-67 recognizes specific antigens associated with the nuclear envelope of the early Drosophila embryo

Summary— Immunofluorescence and immunoelectron microscopy indicated that the antibody raised against the nuclear antigen Ki-67 of mammalian cells recognized antigenic determinants of early Drosophila embryos, localized on the outside of the nuclear envelope. Hence, the nuclear envelope of Drosophila appears to share a similar epitope with the chromosome scaffold of mitotic mammalian cells. With the progression of mitosis the antigen persisted around the mitotic spindle region and was also found in the pole regions at metaphase and anaphase. The antibody also stained the equatorial regions of the spindles from anaphase to late telophase. The antibody may therefore be used as a biochemical marker of the nuclear envelope for studying nuclear membrane biogenesis and behavior during the mitotic divisions of the Drosophila embryo.     

Compartmentalization of SHC, GRB2 and mSOS, and hyperphosphorylation of Raf-1 by EGF but not insulin in liver parenchyma.

Rat liver parenchyma harbors equal numbers of epidermal growth factor (EGF) and insulin receptors. Following administration of a saturating dose of EGF (10 micrograms/100 g body weight), there was a rapid (t1/2 approximately 1.1 min) internalization of receptor coincident with its tyrosine phosphorylation at residue 1173 and receptor recruitment of the adaptor protein SHC, its tyrosine phosphorylation and its association with GRB2 and the Ras guanine nucleotide exchange factor, mSOS, largely in endosomes. This led to a cytosolic pool of a complex of tyrosine-phosphorylated SHC, GRB2 and mSOS. It was demonstrated that these constituents were linked to Ras activation by the characteristic decrease in Raf-1 mobility on SDS-PAGE, which was maintained for 60 min after a single bolus of administered EGF. While insulin administration (15 micrograms/100 g body weight) led to insulin receptor beta-subunit tyrosine phosphorylation and internalization, there was little detectable tyrosine phosphorylation of SHC, recruitment of GRB2, association of a complex with mSOS or any detectable change in the mobility of Raf-1. Therefore, in normal physiological target cells in vivo, distinct signaling pathways are realized after EGF or insulin receptor activation, with regulation of this specificity most probably occurring at the locus of the endosome.     

An oxygenation-sensitive dye binding to Carcinus maenas hemocyanin

The interaction of 4',6-diamidino-2-phenylindole (DAPI) with Carcinus maenas hemocyanin has been investigated by steady state fluorescence, dynamic fluorescence and circular dichroism measurements. The dye binds to apohemocyanin (without copper) as well as to oxygenated hemocyanin and to deoxygenated hemocyanin with very similar affinities (kd approximately equal to 1 microM ) and number of binding sites (one per subunit). In contrast, the fluorescence quantum yield enhancement of DAPI bound to oxygenated hemocyanin is nearly 60% lower than that observed for deoxygenated and apo forms. The decrease of fluorescence of the dye bound to deoxygenated hemocyanin is a sigmoidal function of the oxygen partial pressure, specular to that observed by following the absorbance of the copper-oxygen charge transfer band at 340 nm. This result provides preliminary evidence that DAPI may be used as a functional probe to monitor the cooperative binding of oxygen to the protein. The higher fluorescence quantum yield of DAPI bound to either apohemocyanin or deoxygenated protein is characterized by a single fluorescence decay with lifetime of about 3 ns, while with the oxygenated protein two components of about 1 ns and 3.0 ns are observed. This result is interpreted assuming the existence of two rotamers of DAPI in solution (Szabo et al. Photochem. Photobiol. 44 (1986) 143-150) both able to interact with oxygenated hemocyanin but only one to deoxygenated and apo forms. We conclude that the different fluorescence behaviour of the dye induced by the presence of oxygen bound to the protein is probably due to a structural change of hemocyanin in cooperative interaction with oxygen. Furthermore, the interaction is confirmed by the induced negative ellipticity of DAPI bound to apohemocyanin and deoxy-hemocyanin and by the increase of fluorescence anisotropy of DAPI bound to all forms of protein investigated.     

The effects of nutrient addition and pH manipulation in bag experiments on the phytoplankton of a small acidic lake in West Virginia,USA

In situ bag experiments were performed during summer and autumn in a small acidic lake, Tibbs Run Lake, West Virginia, USA. The objective was to evaluate phytoplankton responses to pH manipulation and nutrient addition. Increasing the pH from below 4.5 to over 6.3 resulted in great declines in phytoplankton biovolume. There was also a succession from dinoflagellates (Peridinium inconspicuum to small chlorophytes. The trend was more rapid where phosphorus (P) additions were made along with pH enhancement. During summer, P limitation was indicated, while nitrogen (N) appeared to limit production in autumn. In both seasons, nutrient additions greatly altered the phytoplankton composition in high pH treatments, but had no discernable effects at (the natural) low pH. A low pH, P addition treatment in autumn was the single exception. When N was subsequently added, phytoplankton composition changed dramatically, probably because the proceeding P additions caused severe secondary N-limitation. In general, however, the results supported the view that phytoplankton compositional responses to nutrient additions are suppressed in low pH, relative to high pH lake water.     

Thermodynamic basis of site-specific cooperativity

Cooperative phenomena in biological macromolecules arise from the interaction of many distinct subsystems, such as structural domains or binding sites. Cooperative properties of the system as a whole, like protein folding or allosteric transitions, are subject to the restrictions imposed by thermodynamic stability. These restrictions, however, do not apply in the case of individual subsystems open to interactions with the rest of the macromolecule. The site-specific properties of such subsystems can be understood in general thermodynamic terms from those of a multicomponent system under particular conditions. The analogy provides a thermodynamic basis for site-specific Cooperativity. © 1994 John Wiley & Sons, Inc.