Molecular species composition of membrane phosphatidylcholine influences the rate of cholesterol efflux from human erythrocytes and vesicles of erythrocyte lipid

The efflux of [3H]cholesterol from prelabelled human erythrocytes having modified phosphatidylcholine compositions was measured during 24-h incubations in the presence of unlabelled acceptor liposomes composed of equimolar amounts of egg phosphatidylcholine and cholesterol. The cells were modified by replacement of part of the native phosphatidylcholine with either dipalmitoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine or dilinoleoylphosphatidylcholine catalyzed by phosphatidylcholine-specific transfer protein from bovine liver. The results indicated that the efflux of [3H]cholesterol was faster from erythrocytes in which the dipalmitoylphosphatidylcholine content was increased from 7 to 25% of the total, than from cells enriched in palmitoyloleoylphosphatidylcholine or dioleoylphosphatidylcholine. Incorporation of dilinoleoylphosphatidylcholine to a level of 13% of the total phosphatidylcholine slowed the rate of efflux of [3H]sterol. The phosphatidylcholine replacements produced no significant differences in cholesterol/phospholipid ratio before or after 24 h of incubation with the acceptor egg phosphatidylcholine-cholesterol vesicles. Using vesicles prepared from erythrocyte lipid, modified to reflect the changes in the phosphatidylcholine composition induced in the whole cells, the same influence of composition on the rate of cholesterol exchange was evident. Enhancement of the dipalmitoylphosphatidylcholine content from 7 to 25% of the total phosphatidylcholine pool increased the rate of [3H]cholesterol efflux, while the addition of the same amount of dilinoleoylphosphatidylcholine slowed it compared to controls. The magnitude of the effect was comparable in intact cells and erythrocyte lipid vesicles enriched in dipalmitoylphosphatidylcholine, while the influence of dilinoleoylphosphatidylcholine was more marked in the intact cells. These results demonstrate that changes in the molecular species composition of the phosphatidylcholine pool can influence the rate of exchange of cholesterol but not necessarily the cellular content of sterol in the human erythrocyte. The influence of this phospholipid appears to be expressed independently of the presence of membrane protein or an underlying cytoskeleton.     

Lipid molecular shape affects erythrocyte morphology: a study involving replacement of native phosphatidylcholine with different species followed by treatment of cells with sphingomyelinase C or phospholipase A2

In a previous report it was shown that the replacement of native erythrocyte phosphatidylcholine (PC) with different PC species which have defined acyl chain compositions can lead to morphological changes (Kuypers, F.A., W. Berendsen, B. Roelofsen, J. A. F. Op den Kamp, and L.L.M. van Deenen, 1984, J. Cell Biol., 99:2260-2267). It was proposed that differences in molecular shape between the introduced PC species and normal erythrocyte PC caused the membrane to bend outwards or inwards, depending on the shape of the PC exchanged. To support this proposal, two requirements would have to be fulfilled: the exchange reaction would take place only with the outer lipid monolayer of the erythrocyte, and the extent of lipid transbilayer movement would be restricted. If this theory is correct, any treatment causing unilateral changes in lipid molecular shape should lead to predictable morphological changes. Since this hypothesis is a refinement of the coupled bilayer hypothesis, but so far lacks experimental support, we have sought other means to change lipid molecular shape unilaterally. Shape changes of human erythrocytes were induced by the replacement of native PC by various PC species using a phosphatidylcholine-specific transfer protein: by hydrolysis of phospholipids in intact cells using sphingomyelinase C or phospholipase A2, and by the combination of both procedures. The morphological changes were predictable; additive when both treatments were applied, and explicable on the basis of the geometry of the lipid molecules involved. The results strongly support the notion that lipid molecular shape affects erythrocyte morphology.     

Purification and enzymatic characterization of CMP-sialic acid: beta-galactosyl1----3-N-acetylgalactosaminide alpha 2----3-sialyltransferase from human placenta

A CMP-NeuAc:Gal beta 1----3GalNAc-R alpha 2----3-sialyltransferase has been purified over 20,000-fold from a Triton X-100 extract of human placenta by affinity chromatography on concanavalin A-Sepharose and CDP-hexanolamine-Sepharose in a yield of 10%. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions revealed that the enzyme consists of a major polypeptide species with a molecular weight of 41,000 and some minor forms with molecular weights of 40,000, 43,000, and 65,000, respectively, which can be resolved partially by gel filtration on Sephadex G-100. Isoelectric focusing revealed that the enzyme occurs in a major and a minor charged form with pI values of 5.0-5.5 and 6.0, respectively. Acceptor specificity studies indicated that the enzyme catalyzes the incorporation of sialic acid from CMP-NeuAc into glycoproteins, glycolipids, and oligosaccharides which possess a terminal Gal beta----3GalNAc unit. Analysis of the structure of the product chain by high-pressure liquid chromatography and thin layer chromatography as well as methylation analysis revealed that a NeuAc alpha 2----3Gal beta 1----3GalNAc sequence is elaborated. The best glycoprotein acceptors are antifreeze glycoprotein and porcine submaxillary asialo/afucomucin. The disaccharide Gal beta 1----3GalNAc-Thr shows values for Km and V which are close to those of the latter glycoprotein. Lactose as well as oligosaccharides in which galactose is linked beta 1----3 or beta 1----4 to N-acetylglucosamine are less efficient acceptors. Of the glycolipids tested only gangliosides GM1 and GD1b served as an acceptor. The enzyme does not show an absolute aglycon specificity, and attaches sialic acid regardless the anomeric configuration of the N-acetylgalactosaminyl residue in the accepting Gal beta 1----3GalNAc unit. By use of specific acceptor substrates it could be demonstrated that the purified enzyme is free from other known sialyltransferase activities. Studies with rabbit antibodies raised against a partially purified sialyltransferase preparation indicated that the enzyme is immunologically unrelated to a Gal beta 1----4GlcNAc-R alpha 2----3-sialyltransferase, which previously had been identified in human placenta (Van den Eijnden, D.H., and Schiphorst, W. E. C. M. (1981) J. Biol. Chem. 256, 3159-3162). Initial-rate kinetic studies suggest that the sialyltransferase operates through a mechanism involving a ternary complex of enzyme, sugar donor, and acceptor. This is the first report on the extensive purification and characterization of a sialyltransferase from a human tissue.     

Presence of markers for virulence in the unique short region or repeat region or both of pseudorabies hybrid viruses.

The unique short region and part of the repeat region of virulent pseudorabies virus strain NIA-3 was replaced by the corresponding region of the avirulent NIA-4 strain by transfection with subgenomic DNA fragments. The resulting hybrid virus showed a reduced virulence in both mice and pigs. Therefore, important markers for virulence are located in the unique short or repeat region or both of pseudorabies virus. We provide evidence that the terminally located repeat is not required for the generation of progeny with intact pseudorabies virus genomes. Apparently, the terminal repeat is regenerated from the internal repeat.     

Purification and Characterization of the Pea Chloroplast Pyruvate Dehydrogenase Complex : A Source of Acetyl-CoA and NADH for Fatty Acid Biosynthesis

The pyruvate dehydrogenase complex has been purified 76-fold, to a specific activity of 0.6 μmoles per minute per milligram protein, beginning with isolated pea (Pisum sativum L. var Little Marvel) chloroplasts. Purification was accomplished by rate zonal sedimentation, polyethyleneglycol precipitation, and ethyl-agarose affinity chromatography. Characterization of the substrates as pyruvate, NAD⁺, and coenzyme-A and the products as NADH, CO₂, and acetyl-CoA, in a 1:1:1 stoichiometry unequivocally established that activity was the result of the pyruvate dehydrogenase complex. Immunochemical analysis demonstrated significant differences in structure and organization between the chloroplast pyruvate dehydrogenase complex and the more thoroughly characterized mitochondrial complex. Chloroplast complex has a higher magnesium requirement and a more alkaline pH optimum than mitochondrial complex, and these properties are consistent with light-mediated regulation in vivo. The chloroplast pyruvate dehydrogenase complex is not, however, regulated by ATP-dependent inactivation. The properties and subcellular localization of the chloroplast pyruvate dehydrogenase complex are consistent with its role of providing acetyl-CoA and NADH for fatty acid synthesis.     

Functional aspects of the postovulatory follicle in the ovary of the African catfish,Clarias gariepinus,after induced ovulation

Summary In captive African catfish, Clarias gariepinus, ovulation was induced with human chorionic gonadotropin (HCG) 4 I.U./g body weight to study the function of postovulatory follicles (POFs). Ultrastructural and enzyme-histochemical data indicate that, apart from special theca cells, the granulosa of relative young POFs (i.e., from 16 h and 28 h after HCG-injection) is capable of producing steroids. Possible functions of the synthesized steroids are discussed. Histological comparison of POFs from stripped and from unstripped fish, as well as histochemical investigation of the contents of ovulated ova and granulosa of POFs at 48 h after HCG-injection, showed that the latter structure is involved in phagocytosis of the disintegrating ovulated eggs. The polysaccharide-lipid-protein material, initially taken up by heterophagolysosomes of the granulosa cells, subsequently undergoes fatty degeneration. The granulosa cells of the POFs showed strong acid phosphatase activity and abundant granular endoplasmic reticulum from 16 h after HCG-injection onward; heterophagolysosomes appeared at 32 h. These results indicate that after ovulation the phagocytotic function of the granulosa develops progressively. Autophagolysosomes, responsible for the final disintegration of POFs, become increasingly evident in the granulosa cells with increasing time after ovulation.     

In vitro culture of intramolluscan stages of the avian schistosome Trichobilharzia ocellata

Several different procedures for in vitro cultivation of intramolluscan stages of the avian schistosome Trichobilharzia ocellata were tried. A medium was found and culture conditions were established that not only supported in vitro transformation of miracidia into mother sporocysts, but also resulted in substantial subsequent growth; moreover, some degree of germinative development appeared to occur as well. Cerebral ganglia from uninfected adult snails of the intermediate host species, Lymnaea stagnalis, could produce factors promoting in vitro development of young mother sporocysts. Results are compared with data from the literature and it is concluded that greater success in in vitro culturing of young mother sporocysts of T. ocellata can be achieved than has hitherto been reported for other schistosome species. The same culture procedures were less successful when applied to other intramolluscan stages of T. ocellata, but can be used for in vitro maintenance of these stages. The procedures described here will be a useful tool in the study of schistosome-snail interactions in T. ocellata-L. stagnalis and possibly in other systems as well.     

Human c-fms proto-oncogene: comparative analysis with an abnormal allele.

The organization of the human c-fms proto-oncogene has been determined and compared with an abnormal allele. The human v-fms homologous genetic sequences are dispersed discontinuously and colinearly with the viral oncogene over a DNA region of ca. 32 kilobase pairs. The abnormal c-fms locus contains a small deletion in its 3' portion. DNA sequencing analysis indicated that it was 426 base pairs in size and located in close proximity to a putative c-fms exon.     

Human lens γ-crystallin sequences are located in the p12-qter region of chromosome 2

Summary The human -crystallin genes constitute a multigene family whose members are only expressed in the eye lens. The chromosomal location of these sequences has been determined by screening a panel of human/rodent hybrid cell lines containing overlapping subsets of human chromosomes for the presence of human -crystallin sequences. By correlating these genomic hybridization data with the chromosomal constitution of the somatic cell hybrids, all human -crystallin sequences could be assigned to chromosome 2. The use of human/hamster cell hybrids derived from human Burkitt lymphoma cells carrying a reciprocal translocation between human chromosomes 2 and 8, allowed a further localization of the sequences to the region 2p12-qter.     

Isolation of microorganisms on 3-butyn-1-ol and other acetylenic compounds

The microbial potential to degrade acetylenic compounds (alkynes) was investigated, and several fungi and bacteria were isolated on 2-propyn-1-ol, 3-butyn-1-ol, propynoic acid, and 2-butyne-1,4-diol. The results indicate that a wide variety of microorganisms may degrade alkynes in nature.