Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Genetic linkage between lipoprotein(a) phenotype and a DNA polymorphism in the plasminogen gene

Coronary heart disease risk correlates directly with plasma concentrations of lipoprotein(a) (Lp(a)), a low-density lipoprotein-like particle distinguished by the presence of the glycoprotein apolipoprotein(a) (apo(a)), which is bound to apolipoprotein B-100 (apoB-100) by disulfide bridges. Size isoforms of apo(a) are inherited as Mendelian codominant traits and are associated with variations in the plasma concentration of lipoprotein(a). Plasminogen and apo(a) show striking protein sequence homology, and their genes both map to chromosome 6q26-27. In a large family with early coronary heart disease and high plasma concentrations of Lp(a), we found tight linkage between apo(a) size isoforms and a DNA polymorphism in the plasminogen gene; plasma concentrations of Lp(a) also appeared to be related to genetic variation at the apo(a) locus. We found free recombination between the same phenotype and alleles of the apoB DNA polymorphism. This suggests that apo(a) size isoforms and plasma lipoprotein(a) concentrations are each determined by genetic variation at the apo(a) locus.     

A new polymorphism in the factor VIII gene for prenatal diagnosis of hemophilia A.

A restriction fragment length polymorphism (RFLP) has been found in the gene for clotting factor VIII. Defects in this gene are the cause of hemophilia A. The DNA polymorphism affects an XbaI site in intron 22 of the gene. Two alleles occur in a frequency of 59 and 41 percent of the X chromosomes tested. Furthermore, about 25 percent of females who are homozygous for the previously reported BclI RFLP in the factor VIII gene are heterozygous for the XbaI polymorphism. This new RFLP thus represents a significant addition to available probes for the DNA-based prenatal diagnosis and carrier detection of this disease.     

Human lecithin-cholesterol acyltransferase gene: complete gene sequence and sites of expression.

The human lecithin-cholesterol acyltransferase (LCAT) gene has been sequenced to completion. The gene is divided into six exons spanning approximately 4,200 bp. Exon five codes for amino acids homologous to the interfacial active site of several lipases, and also codes for an amphipathic alpha-helix resembling the carboxy terminus of apolipoprotein E. Blot hybridization data suggest that there is only one LCAT gene in humans. The 1550 base LCAT mRNA can be detected in liver and HepG2 (hepatocyte) cells, but not in small intestine, spleen, pancreas, placenta or adrenal tissue.     

Rhesus monkey apolipoprotein(a). Sequence, evolution, and sites of synthesis

Human lipoprotein(a) is a low density lipoprotein-like lipoprotein whose concentration in plasma is correlated with atherosclerosis. The characteristic protein component of lipoprotein(a) is apolipoprotein(a) (apo(a)) which is disulfide-linked to apolipoprotein B-100. Sequencing of rhesus monkey apo(a) cDNA suggests that this protein, like human apo(a), is highly similar to plasminogen. Sequence data suggests that a plasminogen-like protease activity and kringle 1-, 2-, 3-, and 5-like domains are unnecessary for apo(a) function, but a highly repeated kringle four-like domain is important. Liver is the major site of apo(a) RNA synthesis; reduced amounts of message were also found in testes and brain. Co-expression with apoB-100 and plasminogen in rhesus tissues is not mandatory.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

Reconstitution of lipoprotein(a) by infusion of human low density lipoprotein into transgenic mice expressing human apolipoprotein(a).

Lipoprotein(a) (Lp(a)) is an atherosclerosis-causing lipoprotein that circulates in human plasma as a complex of low density lipoprotein (LDL) and apolipoprotein(a) (apo(a)). It is not known whether apo(a) attaches to LDL within hepatocytes prior to secretion or in plasma subsequent to secretion. Here we describe the development of a line of mice expressing the human apo(a) transgene under the control of the murine transferrin promoter. The apo(a) was secreted into the plasma, but circulated free of lipoproteins. When human (h)-LDL was injected intravenously, the circulating apo(a) rapidly associated with the lipoproteins, as determined by nondenaturing gel electrophoresis. Human HDL and mouse LDL had no such effect. When h-VLDL was injected, there was a delayed association of apo(a) with the lipoprotein fraction which suggests that apo(a) preferentially associated with a metabolic product of VLDL. The complex of apo(a) with LDL formed both in vivo and in vitro was resistant to boiling in the presence of detergents and denaturants, but was resolved upon disulfide reduction. These studies suggest that apo(a) fails to associate with mouse lipoproteins due to structural differences between human and mouse LDL, and that Lp(a) formation can occur in plasma through the association of apo(a) with circulating LDL.     

Hapten-specific T cell response to azobenzenearsonate-N-acetyl-L-tyrosine in the Lewis rat. I. Induction and suppression of delayed-type hypersensitivity and in vitro proliferative responses

Pretreatment of Lewis rats with a single i.p. injection of ABA-N-acetyl-tyrosine in incomplete Freund's adjuvant induced an unresponsiveness for delayed-type hypersensitivity to subsequent immunization with the same antigen in complete Freund's adjuvant. Complete suppression of in vitro antigen-induced proliferative responses required repeated pretreatment. Passive transfer of lymphoid cells from spleen and lymph nodes but not sera from suppressed rats induced unresponsiveness of hapten-specific T cell functions. Nylon wool-nonadherent cells and cells panned on F(ab')2 of rabbit anti-Lewis rat Ig plates suppressed the induction of DTH and in vitro antigen-stimulated proliferation. Adult thymectomy increased DTH and failed to abolish the induction of suppression.