Entrapment of lipid vesicles and membrane protein-lipid vesicles in gel bead pores

Phospholipid vesicles were entrapped in gel beads of Sepharose 6B and Sephacryl S-1000 during vesicle preparation by dialysis. Egg-yolk phospholipids solubilized with cholate or octyl glucoside were dialysed together with gel beads for 2.5 days in a flat dialysis bag. Some vesicles were formed in gel bead pores and vesicles of sufficient size became trapped. Red cell membrane protein-phospholipid vesicles could be immobilized in the same way. Non-trapped vesicles were carefully removed by chromatographic procedures and by centrifugation. The amount of entrapped vesicles increased with the initial lipid concentration and was dependent on the relative sizes of vesicles and gel pores. The largest amount of trapped vesicles, corresponding to 9.5 mumol of phospholipids per ml gel, was achieved when Sepharose 6B gel beads were dialysed with cholate-solubilized lipids at a concentration of 50 mM. In this case the vesicles had an average diameter of 60 nm and an internal volume of 15 microliters/ml gel. The amount of vesicles trapped in Sephacryl S-1000 gel beads upon dialysis under the same conditions was smaller: 2.2 mumol of phospholipids per ml gel. Probably most of the gel pores were too large to trap such vesicles. Larger vesicles, with an average diameter of 230 nm, were entrapped in the Sephacryl S-1000 matrix in an amount corresponding to 3.0 mumol phospholipids per ml gel upon dialysis of the gel beads and octyl glucoside-solubilized lipids at a concentration of 20 mM. The internal volume of these vesicles was 22 microliters/ml gel. The yield of immobilized phospholipids was up to 19%. The entrapped vesicles were somewhat unstable: 9% of the phospholipids were released during 9 days of storage at 4 degrees C. By the dialysis entrapment method vesicles can be immobilized in the gel beads without using hydrophobic ligands or covalent coupling.     

Temperature-dependent insertion of prolipoprotein into Escherichia coli membrane vesicles and requirements for ATP, soluble factors, and functional SecY protein for the overall translocation process.

The requirements for the translocation of prolipoprotein into membrane vesicles were examined in an in vitro system. As measured by the eventual modification and processing of the prolipoprotein to form mature lipoprotein, the overall translocation process was found to require ATP hydrolysis, the presence of some heat-labile soluble cytoplasmic translocation factors, and the function of a cytoplasmic membrane protein, SecY/PrlA. However, the initial step of complete insertion of prolipoprotein into the membrane vesicles occurred without apparent requirements of a nucleotide, cytoplasmic translocation factors, or a functional SecY/PrlA membrane protein. Immunopurified prolipoprotein spontaneously inserted into membrane vesicles at elevated temperatures and required ATP and cytoplasmic translocation factors to form mature lipoprotein. The prolipoprotein inserted most efficiently into liposomes made of negatively charged phospholipids, indicating the importance of phospholipids in protein translocation. These results suggest that ATP hydrolysis and the actions of both cytoplasmic translocation factors and a functional SecY/PrlA membrane protein occur at a step(s) after the insertion of the precursors into membrane vesicles. The initial step of spontaneous insertion of prolipoprotein into membranes is in good agreement with membrane trigger hypothesis proposed by W. Wickner (Annu. Rev. Biochem. 48:23-45, 1979) and the helical hairpin hypothesis proposed by D. M. Engleman and T. A. Steitz (Cell 23:411-422, 1981).     

Novel hybrid maturases in unstable pseudorevertants of maturaseless mutants of yeast mitochondrial DNA.

Unstable pseudorevertants of mitochondrial mutants of Saccharomyces cerevisiae lacking the maturase function encoded by the fourth intron of the cytochrome b gene (bI4) were isolated. They were found to be heteroplasmic cells owing their regained ability to respire (and grow on glycerol medium) to the presence of a rearranged (rho-) mtDNA that contains an in-frame fusion of the reading frames of the group I introns bI4 and intron 4 alpha of the coxl gene encoding subunit I of cytochrome c oxidase (aI4 alpha). The products of those gene fusions suppress the bI4 maturase deficiency still present in those heteroplasmic cells. Similar heteroplasmic pseudorevertants of a group II maturaseless mutant of the first intron of the coxI gene were characterized; they result from partial deletion of the coxI gene that fuses the reading frames of introns 1 and 2. These heteroplasms provide independent support for the existence of RNA maturases encoded by group I and group II introns. Also, since the petite/mit- heteroplasms arise spontaneously at very high frequencies they provide a system that can be used to obtain mutants unable to form or maintain heteroplasmic cells.     

Charge dependence of Fe(II)-catalyzed DNA cleavage.

The effect of charge of the Fe(II) reagent used to induce DNA strand cleavage reactions in the presence of a source of reducing equivalents is investigated using two oligonucleotide models. The first consists of the two strands dA20 and dT20, and an equimolar complex between them. The second is a short four-arm branched DNA complex composed of four 16-mer strands. In the former case, cleavage of the 1:1 complex by three reagents with different formal charge, Fe(II).EDTA2-, Fe(II).EDDA and Fe2+, is comparable in rate to that of the individual dT20 and the dA20 strands. While the three reagents show similar cleavage rates for the duplex and single stranded molecules, they give distinctive cutting patterns in the DNA tetramer, consistent with the presence of a site of excess negative charge at the branch point. Scission induced by Fe(II).EDTA2- shows lower reactivity at the branch site relative to duplex controls, whereas Fe(II)2+ shows enhanced reactivity. Formally neutral Fe(II).EDDA shows weak loss of cutting reactivity at the branch. The position of attack by Fe(II)2+ in the branched tetramer is shifted with respect to those of Fe(II).EDTA2- or Fe(II).EDDA; a slower migrating species is also detected in the scission of dA20.dT20 duplex by Fe(II) reaction. These results suggest that the Fe(II)2+ reaction proceeds by a different mechanism from the other agents. The difference in cutting profiles induced by the neutral and negatively charged chelated complexes is consistent with a local electrostatic repulsion of a negatively charged source of radicals, not a positively charged one.     

Studies on theStellaria longipes complex (Caryophyllaceae): Isozyme variability and the relationship betweenStellaria longipes andS. longifolia

Genetic variation within and the relationship betweenStellaria longipes Goldie andS. longifolia Muhl. were studied. Ten enzyme systems were assessed in eight natural populations ofS. longipes (25 loci) and three ofS. longifolia (20 loci) using starch and polyacrylamide gel electrophoresis. Patterns of population differentiation corresponded to geographic distance. There was no evidence that polyploidS. longipes had greater electrophoretic variability than diploidS. longipes. The isozyme data confirmed extensive population differentiation in these species and, within that context, a relatively close relationship betweenS. longipes andS. longifolia. It was postulated that diploids of these two species might be the progenitors of tetraploidS. longipes.     

椭圆斜羽叶的解剖研究及斜羽叶的系统分类位置

本文根据对椭圆斜羽叶(Plagiozamites oblongifolius)钙质石化化石的研究,认为其主要特征如下:羽状复叶,叶肉无海绵组织和栅栏组织分化。羽片叶脉维管束外韧式,木质部外始式,与现代苏铁属叶脉特征相近。羽轴维管束为“U”形,外始式木质部、梯状纹孔管胞,与现代苏铁属的羽轴维管束相似。根据上述特征,目前可将斜羽叶属归入原始苏铁类植物。但是,如果斜羽叶的生殖器官同 Noeggerathia 一样,为异孢型孢子囊穗,则斜羽叶可能为一种与苏铁类起源有关的原裸子植物。     

血管平滑肌细胞的钙动力学

血管平滑肌细胞外的Ca~(2+)通过多种通道进入细胞内。Ca~(2+)通道的本质是镶嵌在膜脂质双分子层中的糖蛋白,神经介质和药物可影响Ca~(2+)通道的功能。靠近胞膜的肌质网和胞膜内侧面的高亲和性Ca~(2+)结合位点是血管平滑肌细胞内储存和释放Ca~(2+)的主要部位。胞浆[Ca~(2+)]增高后在钙调蛋白的介导下引起血管收缩。高血压等血管性疾病的发生与其平滑肌细胞的钙动力学异常有关。