Biochemical studies on cell fusion. I. Lipid composition of fusion- resistant cells

A series of stable cell mutants of mouse fibroblasts were previously isolated (Roos, D. S. and R. L. Davidson, 1980, Somatic Cell Genet., 6:381-390) that exhibit varying degrees of resistance to the fusion-inducing effect of polyethylene glycol (PEG), but are morphologically similar to the parental cells from which they were derived. Biochemical analysis of these mutant cell lines has revealed differences in whole cell lipid composition which are directly correlated with their susceptibility to fusion. Fusion-resistant cells contain elevated levels of neutral lipids, particularly triglycerides and an unusual ether-linked lipid, O-alkyl, diacylglycerol. This ether lipid is increased approximately 35-fold over parental cells in the most highly PEG-resistant cell line. Fusion-resistant cells also contain more highly saturated fatty acyl chains (ratio of saturated to polyunsaturated fatty acids [S/P ratio] approximately 4:1) than the parental line (S/P ratio approximately 1:1). Cells which are intermediate in their resistance to PEG have ether lipid and fatty acid composition which is intermediate between the parental cells and the most fusion-resistant mutants. In a related communication (Roos, D. S. and P. W. Choppin, 1985, J. Cell. Biol., 100:1591-1598) evidence is presented that alteration of lipid content can predictably control the fusion response of these cells.     

Biochemical, immunological, and cell genetic studies in glycogenosis type II.

Fibroblasts from patients with the adult, juvenile, and infantile form of glycogenosis type II (Pompe disease) were cultured under standardized conditions, and the activity of acid alpha-glucosidase (E.C.3.2.1.20) towards glycogen, maltose, and 4-methylumbelliferyl-alpha-D-glucopyranoside was measured. Glycogen levels in muscle biopsies and in cultured fibroblasts from patients were determined. Residual enzyme activities varying from 7%-22% were detected in fibroblasts from patients with the adult form but not from patients with the infantile form of glycogenosis II. An inverse correlation was found between the severity of the clinical manifestation and the degree of residual enzyme activity in the fibroblasts. The kinetic and electrophoretic properties of acid alpha-glucosidase in fibroblasts from the adult patients and from control individuals were similar. Immunological studies suggested that the decrease of acid alpha-glucosidase activity is caused by a mutation that affects the production or degradation of the enzyme rather than its catalytic activity. Complementation studies were carried out by fusing fibroblasts from patients with the adult, juvenile, and infantile form of glycogenosis II, but neither conventional assays on multikaryons nor enzyme assays on single binuclear heterokaryons gave any evidence for genetic heterogeneity among these forms.     

Long-term live cell microscopy studies of lipid droplet fusion dynamics in adipocytes

During the adipogenic differentiation process of mesenchymal stem cells, lipid droplets (LDs) grow slowly by transferring lipids between each other. Recent findings hint at the possibility that a fusion pore is involved. In this study, we analyze lipid transfer data obtained in long-term label-free microscopy studies in the framework of a Hagen-Poiseuille model. The data obtained show a LD fusion process in which the lipid transfer directionality depends on the size difference between LDs, whereas the respective rates depend on the size difference and additionally on the diameter of the smaller LDs. For the data analysis, the viscosity of the transferred material has to be known. We demonstrate that a viscosity-dependent molecular rotor dye can be used to measure LD viscosities in live cells. On this basis, we calculate the diameter of a putative lipid transfer channel which appears to have a direct dependence on the diameter of the smaller of the two participating LDs.     

Biochemical studies on bovine adenovirus type 3. II. Incomplete virus.

Incomplete virus of oncogenic bovine adenovirus type 3 (BAV3) was highly purified and its biological activity was studied. The production of incomplete virus was found to increase with a high multiplicity of infection and with a large amount of arginine in the growth medium. On infection of contact-inhibited mouse cells, incomplete virus induced cellular DNA synthesis and focus formation. Moreover, this virus was oncogenic to newborn hamsters. On infection of calf kidney cells, a permissive cell line, viral early and late RNA, viral DNA, and almost all the viral late proteins were produced, but no mature progeny virus was detected. It is, therefore, suggested that incomplete virus of BAV3 may be unable to synthesize a protein(s) (perhaps a kind of maturation protein[s]) essential for assembly of viral macromolecules for maturation.     

Biochemical studies on the lipid content ofTilapia nilotica andSparus auratus

1. Seasonal variations of total lipids, free fatty acids, triglycerides, phospholipids and cholesterol content of the freshwater fishTilapia nilotica and the marine fishSparus auratus were investigated.2. Male fish ofS. auratus showed higher muscular and hepatic total lipids and hepatic free fatty acids than those ofT. nilotica (P < 0.05).3. The mean differences in gonadal male lipids of the two species were not significant.Tilapia nilotica female fish showed a significantly higher content of hepatic free fatty acids, phospholipids and cholesterol (P < 0.01, 0.01, 0.05respectively) and gonadal total lipids, triglycerides, and cholesterol (P < 0.05) than those ofS. auratus females.4. In contrastS. auratus females exhibited higher muscular total lipids, triglycerides, phospholipids and cholesterol content (P < 0.01, 0.05, 0.02, 0.05, respectively) and gonadal phospholipids (P < 0.05) than those of theT. nilotica females.5. In general hepatic and gonadal lipids of freshwater fishT. nilotica were higher than those of the marine fishS. auratus, and in contrast the marine fish contained higher muscular lipids than the freshwater fish.     

Lipid vesicle-cell interactions. II. Induction of cell fusion

The ability of lipid vesicles of simple composition (lecithin, lysolecithin, and stearylamine) to induce cells of various types to fuse has been investigated. One in every three or four cells in monolayer cultures can be induced to fuse with a vesicle dose of about 100 per cell. At such dosages and for exposures of 15 min to 1 h, vesicles have essentially no effect on cell viability. Under anaerobic conditions, these cells lyse rather than fuse. Avian erythrocytes are readily fused with lipid vesicles in the presence of dextran. Fusion indices increase linearly with the zeta potential of the vesicles (increasing stearylamine content), indicating that contact between vesicle and cell membrane is required. Fusion indices increase sublinearly with increasing lysolecithin content. Divalent cations increase fusion indices at high vesicle doses. The data presented are consistent with the hypothesis that cell fusion occurs via simultaneous fusion of a vesicle with two adhering cell membranes.     

Biochemical studies on sporulation in blue-green algae. II. Factors affecting glycogen accumulation

Inorganic nitrogens sources like nitrate, nitrite enhanced sporulation and glycogen accumulation in Anabaena sp. but ammonium chloride neither influenced sporulation nor glycogen accumulation. Acetate and citrate also stimulated early sporulation and glycogen level was higher over nitrogen free control. Nitrogen and carbon sources in combination proved to be useful in inducing early sporulation and increased content of glycogen. Phosphate and calcium also affected glycogen accumulation significantly, although, the sporulation was found to be of the same order as in nitrogen free medium. Sulphate initiated early sporulation, the mechanism of which is not known.     

Effect of lipid composition on the "membrane response" induced by a fusion peptide

To understand the initial stages of membrane destabilization induced by viral proteins, the factors important for binding of fusion peptides to cell membranes must be identified. In this study, effects of lipid composition on the mode of peptides' binding to membranes are explored via molecular dynamics (MD) simulations of the peptide E5, a water-soluble analogue of influenza hemagglutinin fusion peptide, in two full-atom hydrated lipid bilayers composed of dimyristoyl- and dipalmitoylphosphatidylcholine (DMPC and DPPC, respectively). The results show that, although the peptide has a common folding motif in both systems, it possesses different modes of binding. The peptide inserts obliquely into the DMPC membrane mainly with its N-terminal alpha helix, while in DPPC, the helix lies on the lipid/water interface, almost parallel to the membrane surface. The peptide seriously affects structural and dynamical parameters of surrounding lipids. Thus, it induces local thinning of both bilayers and disordering of acyl chains of lipids in close proximity to the binding site. The "membrane response" significantly depends upon lipid composition: distortions of DMPC bilayer are more pronounced than those in DPPC. Implications of the observed effects to molecular events on initial stages of membrane destabilization induced by fusion peptides are discussed.     

Membrane mobility agents. II. Active promoters of cell fusion.

The membrane mobility agent, A2C, actively promotes the fusion of hen erythrocytes under conditions similar to those used by Lucy et al. for glyceryl monooleate.     

Biochemical studies on rat liver Golgi apparatus. II. Further characterization of isolated Golgi fraction.

Although the preparation of rat liver Golgi apparatus isolated by our method contains appreciable activities of NADH- and NADPH-cytochrome c reductases and glucose-6-phosphatase, these enzymes as well as thiamine pyrophosphatase of the extensively fragmented Golgi fraction are partitioned in aqueous polymer two-phase systems quite differently from those associated with microsomes. Similarly, the partition patterns of acid phosphatase and 5'-nucleotidase of the Golgi fragments differ from those of homogenized lysosomes and plasma membrane, respectively. It is concluded that most, if not all, of these marker enzymes in the Golgi fraction cannot be ascribed to contamination by the non-Golgi organelles. In sucrose density gradient centrifugation the NADH- and NADPH-cytochrome c reductase activities of the Golgi fraction behave identically with galactosyltransferase but differently from the reductase activities of microsomes, again indicating that the reductases are inherently associated with the Golgi apparatus. NADPH-cytochrome c reductase of the Golgi preparation is immunologically identical with that of microsomes. The marker enzymes mentioned above and galactosyltransferase behave differently from one another when the Golgi fragments are subjected to partitioning in aqueous polymer two-phase systems, suggesting that these enzymes are not uniformly distributed in the Golgi apparatus structure.