Action of polyethylene glycol on the fusion of human erythrocyte membranes

Summary Factors affecting the polyethylene glycol (PEG)-induced membrane fusion were examined. Human erythrocyte membrane ghosts, cytoskeleton-free vesicles budded from erythrocytes, mechanically disrupted erythrocyte vesicles, and recombinant vesicles from glycophorin and egg phosphatidylcholine were used as models. Fusion was monitored by darkfield light microscopy and by freeze-fracture electron microscopy. Osmotic swelling was found necessary for fusion between membrane ghosts following PEG treatment. The sample with the highest fusion percentage was sealed ghosts incubated in hypotonic media after at least 5 min of treatment in <25% PEG. At similar osmolarity, glycerol, dextran and PEG produced progressively more pronounced intramembranous particle (IMP) patching, correlating with their increasing fusion percentages. The patching of IMP preceded cell-cell contact, and occurred without direct PEG-protein interaction. The presence of cytoskeletal elements in small vesicles had no significant effect on fusion, nor on the aggregation of intramembranous particle (IMP) upon PEG treatment. Disrupting the membrane by lysolecithin, dimethylsulfoxide, retinol or mild sonication resulted in the fragmentation of ghosts without an increase in fusion percentage. The purity of the commercial PEG used had no apparent effect on fusion. We concluded that the key steps in PEG-induced fusion of cell membrane are the creation of IMP-free zones, and the osmotic swelling of cells after the formation of bilayer contacts during the PEG treatment. Cell cytoskeleton affects PEG-induced fusion only to the extent of affecting IMP patching.     

A comparative study of the tapetum, retina and skull of the ferret, dog and cat

Results of this investigation indicate that the ferret (Mustela putorius) closely resembles the dog (Canis familiaris) and should be a useful research animal alternative. The tapetum lucidum is a common structure present in the eyes of dogs, cats (Felis catus) and other nocturnal animals. Our study showed that this structure was present in ferret eyes. The color or reflection of the ferret and dog tapetum was remarkably reduced by the general fixation with glutaraldehyde. However, this color fading phenomenon was not observed in the cat tapetum. These observations led to this comparative study on several morphological, histochemical and biochemical parameters on mature ferrets, dogs and cats including: (1) the number of center tapetum cell layers, (2) thickness of center tapetum, (3) presence of a microtubule-like structure in each tapetal rod, (4) presence of electron-dense cores in tapetal rods after prolonged fixation in glutaraldehyde, (5) retention of reflection or color of tapetum after prolonged glutaraldehyde fixation, (6) zygomatic bones of eye orbits, (7) zinc content in tapetum, (8) cysteine in the tapetum, (9) cysteine sulfinic acid decarboxylase in liver, (10) thickness of retina from center tapetum, (11) anterior view of skull configuration, and (12) lateral view of skull configuration (jaw and teeth). Among these 12 parameters, ferret and dog were similar in the first nine points; ferret and cat were similar to each other only in the last two points. There was no difference in retinal thickness among these three animals.     

Mg~(2+)影响嵌有线粒体H~+-ATP酶脂酶体的表面电荷密度和流动性

<正> 我们实验室曾报道,用胆酸盐透析法将猪心线粒体H~+-ATP酶嵌入大豆磷脂脂质体形成脂酶体时,透析液中Mg~(2+)的存在会降低脂酶体的膜脂流动性,并明显提高重建H~+-ATP酶的活性以及对寡霉素或DCCD的敏感性,因而推论Mg~(2+)的作用很可能是通过改变膜脂的物理状态,形成了维持H~+-ATP酶较高活性的合适构象。但共确切的作用机制仍     

光呼吸代谢物乙醇酸、乙醛酸和草酸对烟草叶片硝酸还原的影响

乙醇酸、乙醛酸和草酸能明显促进烟草(Nicotiana rustica)叶片在黑暗中的硝酸还原,光呼吸抑制剂a-羟基吡啶甲烷磺酸能消除前二者的促进作用而不能完全消除草酸的作用。草酸+NAD~+能显著促进离体的硝酸还原。烟叶提取液加入草酸和NAD~+后生成NADH和CO_2认为活体内由乙醛酸氧化生成的草酸是经脱氢生成NADH供硝酸还原之用。未能证明在烟叶内存在乙醇酸脱氨酶,因此排除由乙醇酸直接脱氢以还原硝酸的可能。     

玉米黄早4雄性不育系、保持系过氧化物酶同工酶的比较研究

在玉米黄早4雄性不育系、保持系的10个组织(叶、根、茎、芽鞘、胚轴、苞叶、穗轴、花丝、雌蕊、花药)中共检测出18种正、负极向过氧化物酶,其中有7个组织不育系与保持系间过氧化物酶没有差异,只有在3个组织中(叶、茎、花药)不育系与保持系间的过氧化物酶存在差异,说明玉米黄早4雄性不育系及保持系的过氧化物酶可能由细胞核基因编码。不育系与保持系个别组织内过氧化物酶存在差异,可能是由于核内编码过氧化物酶的基因表达异常所引起,而这种表达异常,可能是与不育系中不育细胞质基因调控核基因的表达有关。     

Proteases from human immunodeficiency virus and avian myeloblastosis virus show distinct specificities in hydrolysis of multidomain protein substrates.

The virally encoded proteases from human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) have been compared relative to their ability to hydrolyze a variant of the three-domain Pseudomonas exotoxin, PE66. This exotoxin derivative, missing domain I and referred to as LysPE40, is made up of a 13-kilodalton NH2-terminal translocation domain II connected by a segment of 40 amino acids to enzyme domain III of the toxin, a 23-kilodalton ADP-ribosyltransferase. HIV protease hydrolyzes two peptide bonds in LysPE40, a Leu-Leu bond in the interdomain region and a Leu-Ala bond in a nonstructured region three residues in from the NH2-terminus. Neither of these sites is cleaved by the AMV enzyme; hydrolysis occurs, instead, at an Asp-Val bond in another part of the interdomain segment and at a Leu-Thr bond in the NH2-terminal region of domain II. Synthetic peptides corresponding to these cleavage sites are hydrolyzed by the individual proteases with the same specificity displayed toward the protein substrate. Peptide substrates for one protease are neither substrates nor competitive inhibitors for the other. A potent inhibitor of HIV type 1 protease was more than 3 orders of magnitude less active toward the AMV enzyme. These results suggest that although the crystallographic models of Rous sarcoma virus protease (an enzyme nearly identical to the AMV enzyme) and HIV type 1 protease show a high degree of similarity, there exist structural differences between these retroviral proteases that are clearly reflected by their kinetic properties.     

Concerted generation of Ig isotype diversity in human fetal bone marrow

The human fetal bone marrow B cell compartment of 14- to 21-wk gestational age was examined phenotypically and with respect to Ig H chain commitment and diversity. A dramatic expansion of fetal marrow B cell pools at 16- to 18-wk gestational age characterizes a rapid and concerted chain of differentiation events. Transiently up to 1/4 of nucleated marrow cells are CD20+/CD21+ cells which begin to express surface Ig other than IgM. Limiting dilution analysis of EBV-infected marrow cells delineated a virtually exclusive commitment to IgM production until 15 wk and the absolute and relative number of these cells were small (approximately 5% of comparable adult values). In parallel to the rapid increase in total B cell pools size, cells committed and able to secrete any of the five Ig isotypes are generated by 16-wk gestational age and by 18 wk the frequencies of these cells rapidly reach levels typical for adult peripheral tissue such as blood or lymph node. Fetal L chain diversity always anticipated that observed in adult serum. In addition to rising pool sizes and diverse IgH expression, EBV transformability is a major variable during this period of B cell development with up to 2/3 of B lineage cells transformable, about half of which are pre-B cells. By 21-wk gestational age transformable pre-B cells have disappeared and (as in adult tissue) approximately 10 to 20% of CD20+ cells are transformable. The rapid, concerted expression of full H chain diversity during a narrow period in fetal development is unique to marrow and implies a lymphopoietic process in a privileged site rather than an immunologic differentiation event. During this event, the relative proportions between the different IgH classes expressed, resembled that found in adult tissue, perhaps suggesting that B cell inherent programming rather than only antigenic forces determine heavy chain choice. The staggered expression, early in postnatal life, of IgH regions 3' of the C mu locus may reflect regulatory functions rather than inherent immaturity of the B lineage.     

Identification and nucleotide sequence of the Leptospira biflexa serovar patoc trpE and trpG genes.

Leptospira biflexa is a representative of an evolutionarily distinct group of eubacteria. In order to better understand the genetic organization and gene regulatory mechanisms of this species, we have chosen to study the genes required for tryptophan biosynthesis in this bacterium. The nucleotide sequence of the region of the L. biflexa serovar patoc chromosome encoding the trpE and trpG genes has been determined. Four open reading frames (ORFs) were identified in this region, but only three ORFs were translated into proteins when the cloned genes were introduced into Escherichia coli. Analysis of the predicted amino acid sequences of the proteins encoded by the ORFs allowed us to identify the trpE and trpG genes of L. biflexa. Enzyme assays confirmed the identity of these two ORFs. Anthranilate synthase from L. biflexa was found to be subject to feedback inhibition by tryptophan. Codon usage analysis showed that there was a bias in L. biflexa towards the use of codons rich in A and T, as would be expected from its G + C content of 37%. Comparison of the amino acid sequences of the trpE gene product and the trpG gene product with corresponding gene products from other bacteria showed regions of highly conserved sequence.     

Induction of anti-EBNA-1 protein by 12-O-tetradecanoylphorbol-13-acetate treatment of human lymphoblastoid cells.

Binding of the Epstein-Barr virus (EBV) nuclear antigen (EBNA-1) to BamHI-C DNA was studied by affinity column chromatography followed by immunoblotting with human serum specific for EBNA-1. Two species of EBNA-1 (68 and 70 kilodaltons) were identified in nuclear extracts of the EBV-positive Burkitt's lymphoma cell line Raji and not in nuclear extracts of the EBV-negative Burkitt's lymphoma cell line BJAB. Both EBNA-1s bound specifically to the region required for EBV plasmid DNA maintenance (oriP) located in the BamHI-C fragment. Upon treatment with 12-O-tetradecanoylphorbol-13-acetate, which activates latent EBV genome in Raji cells, the 68-kilodalton EBNA-1 was uncoupled from binding to EBV oriP. Nuclear extracts from 12-O-tetradecanoylphorbol-13-acetate-treated BJAB cells also uncoupled the binding of both EBNA-1s to oriP. DNA-cellulose column chromatography identified two protein species which competed for and uncoupled the binding of EBNA-1 to oriP. The two cellular competitors we called anti-EBNA-1 proteins had molecular masses of 60 and 40 kilodaltons, respectively. They were not found in nuclear extracts of BJAB cells not activated by 12-O-tetradecanoylphorbol-13-acetate.