Biomedical subjects

B Malewicz

Publications and source records attributed to B Malewicz.

11 recordsLinked to original sources

PubMed1991

Enhanced gap junction formation with LDL and apolipoprotein B.

Gap junctions are plasma membrane specializations involved in direct cell-cell communication. Intercellular communication is dependent upon the assembly of gap junction structures and would be influenced by agents which alter the assembly process. We investigated the effects of low density lipoprotein (LDL) on gap junction assembly between cultured Novikoff cells using quantitative dye transfer and freeze-fracture electron microscopic methods. We observed a concentration-dependent increase in dye transfer (maximum effect at 2.5 micrograms/ml) and a sixfold increase in the number of aggregated gap junction particles per cell. Immunoblots of Novikoff cells probed with anti-connexin43 antibody revealed no detectable increase in gap junction protein (connexin) levels. The influence of the different components of LDL on junction formation was also examined. First, we treated cells with cholesterol (0-150 microM) in serum-free BSA media and observed a decrease in junction assembly. Second, we added apolipoprotein-B (apo-B) in phosphatidyl choline vesicles to the cells and observed a concentration-dependent increase in dye transfer (maximum effect at 2.5 micrograms protein/ml) and a fivefold increase in the number of aggregated gap junction particles per cell. The addition of phosphatidyl choline vesicles without apo-B had no effect on gap junction formation. Thus, we demonstrated that gap junction assembly can be modulated by LDL and apo-B treatments.

Animals↗

PubMed1990

Lyso platelet activating factor (LysoPAF) and its enantiomer. Total synthesis and carbon-13 NMR spectroscopy.

Described is a reaction sequence for the total synthesis of lyso platelet activating factor (lysoPAF; 1-O-alkyl-sn-glycero-3-phosphocholine) and its enantiomer. The procedure is versatile and yields optically pure isomers of defined chain length. The synthesis is equally suited for the preparation of lysoPAF analogues and its enantiomers with unsaturation in the long aliphatic chain. First, rac-1(3)-O-alkylglycerol is prepared by alkylation of rac-isopropylideneglycerol with alkyl methanesulfonate followed by acid-catalyzed removal of the ketal group. The primary hydroxy group of alkylglycerol is then protected by tritylation, the secondary hydroxy group is acylated, and the protective trityl group is removed under mild acidic conditions with boric acid on silicic acid, essentially without acyl migration. Condensation of the diradylglycerol with bromoethyl dichlorophosphate in diethyl ether, hydrolysis of the resulting chloride, and nucleophilic displacement of the bromine with trimethylamine gives rac-1-O-alkyl-2-acylglycero-3-phosphocholine in good overall yield. The racemic alkylacylglycerophosphocholine is finally treated with snake venom phospholipase A2 (Ophiophagus hannah) which affords 1-O-alkyl-sn-glycero-3-phosphocholine (lysoPAF) of natural configuration in optically pure form. The "unnatural" 3-O-alkyl-2-O-acyl-sn-glycero-1-phosphocholine enantiomer, which is not susceptible to phospholipase A2 cleavage, gives 3-O-alkyl-sn-glycero-1-phosphocholine upon deacylation with methanolic sodium hydroxide. Homogeneity and structure of the intermediates and final products were ascertained by carbon-13 nuclear magnetic resonance spectroscopy on monomeric solutions.

Acylation↗

PubMed1990

Lipids in gap junction assembly and function.

Gap junctions (GJ) are important regulators of cellular function. They provide channels for the direct movement of small molecules between cells and thus control cell-to-cell transfer of metabolites and the transmission of various stimuli. Gap junctions have been shown to be involved in a multitude of cellular processes ranging from cell synchronization and neuronal function to cell differentiation and carcinogenesis. Much knowledge has been gained in recent years concerning the structure and molecular organization of GJ proteins; yet, the mechanisms that control and modulate gap junction assembly and function are still not well understood. Although it is quite apparent that the GJ proteins assemble in the lipid milieu of the plasma membrane, and that the cluster of proteins assembled in the junction do function in a lipid environment, there is a general paucity of information on the role of lipids in the gap junction assembly process and in the function of gap junctions. The present review is a comprehensive account of current knowledge on gap junction lipids. We also discuss what is known to date on the involvement of lipids in gap junction formation. Special emphasis is being placed on the potential role of membrane cholesterol in gap junction assembly and function.

Animals↗

PubMed1990

Increased gap junction assembly between cultured cells upon cholesterol supplementation.

Novikoff hepatoma cells provide an excellent model system for the study of gap junction assembly, a process that could be influenced by lipids and other factors at numerous points. Since it is possible to alter the cellular levels of cholesterol in these cells, it was added to the cells in serum-supplemented medium and changes in gap junction assembly were evaluated. Cells were dissociated and reaggregated following exposure to a range of cholesterol concentrations for 24 h. A five- to sixfold increase in the number of aggregated gap junction particles and a 50% increase in cellular cholesterol content were observed with 20 microM added cholesterol. A 1-h exposure to added cholesterol, during cell reaggregation, resulted in a fourfold increase in the number of aggregated gap junction particles, demonstrating that the effect was rapid. The number of aggregated gap junction particles and formation plaque areas were used as measures of junction assembly and assayed by quantitative freeze-fracture and electron microscopy. Junctional permeabilities were evaluated by means of dye transfer times following the intracellular microinjection of Lucifer Yellow. Increased dye transfer was observed between cholesterol-treated cells, which suggested that the increase in assembly was accompanied by an increase in junction permeability. Cells were treated with cycloheximide (100 micrograms ml-1) and actinomycin D (10 micrograms ml-1) to determine whether protein and RNA syntheses were involved in the enhanced gap junction assembly. Cycloheximide but not actinomycin D blocked the increased junction assembly observed with added cholesterol. These results suggested that protein synthesis, but not RNA synthesis, is necessary for the increased gap junction formation observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Lysophosphatidylcholine stabilizes small unilamellar phosphatidylcholine vesicles. Phosphorus-31 NMR evidence for the "wedge" effect.

Sonication of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-sn-glycero-3-phosphocholine (lysoPC, up to approximately 30 mol %) produces small unilamellar vesicles (SUV, 250-265 A diameter). Phosphorus-31 NMR of the POPC/lysoPC vesicles gives rise to four distinct peaks for POPC and lysoPC in the outer and in the inner bilayer leaflet which can be used to localize and quantify the phospholipids in both vesicle shells. Addition of paramagnetic ions (3 mM Pr3+) enhances outside/inside chemical shift differences and allows monitoring of membrane integrity by the absence of Pr3+ in the vesicle interior. 31P NMR shows that lysoPC in these highly curved POPC/lysoPC vesicles prefers the outer bilayer leaflet. LysoPC incorporation into POPC SUV furthermore causes a substantial and concentration-dependent decrease in spin-spin relaxations (T*2) of the outside POPC phosphorus signals from 55 ms for pure POPC vesicles (v1/2, 5.8 Hz) to 29.5 ms (v1/2, 10.8 Hz) for POPC/lysoPC vesicles containing 25 mol % lysoPC. Our findings are consistent with the idea of a cone-shaped lysoPC molecule which, for geometric reasons, is preferentially accommodated in the outer bilayer leaflet. LysoPC incorporation into POPC SUV restricts POPC headgroup motion and tightens phospholipid packing, but only in the outer bilayer shell.

Lipid Bilayers↗

PubMed1988

Asymmetry of lysophosphatidylcholine/cholesterol vesicles is sensitive to cholesterol modulation.

Sonication of lysophosphatidylcholine (lysoPC; 20 mumol/mL) and cholesterol (chol) in aqueous medium produces lamellar structures over a wide range of concentrations. From 25 to 47 mol % cholesterol, electron microscopy (EM) after negative staining showed extended stacklike lamellae about 40 A thick. From 50 to 60 mol % chol, freeze-fracture EM showed homogeneous populations of small unilamellar vesicles averaging 260-310 A in diameter. Phosphorus-31 nuclear magnetic resonance was used to characterize the stacklike lamellae and to measure the distribution of the lysophospholipid between the outer and inner leaflet of the vesicles as a function of sterol concentration. We found that in lysoPC/chol dispersions containing less than equimolar amounts of cholesterol (25-47 mol %), the entire phosphorus signal (40.5 ppm) was shifted downfield by 10.5 ppm upon addition of Pr3+ (2.4 mM), consistent with the stacklike lamellar structures in which all lysoPC head groups are accessible to the ions. By contrast, addition of Pr3+ to lysoPC/chol vesicles containing equimolar or higher amounts of cholesterol (up to 60 mol %) gave rise to two phosphorus peaks. The more intense downfield signal (51.0 ppm) responsive to paramagnetic ions was assigned to lysoPC located in the outer vesicle leaflet. The upfield signal (40.5 ppm), which was not affected by the ions, was assigned to inside lysoPC. For lysoPC/chol (1:1) vesicles, an outside to inside lysophospholipid ratio (Ro/i) of 6.5 was determined. Essentially the same Ro/i value (6.7) was obtained on lysoPC/chol (1:1) vesicles which after dialysis contained only entrapped Pr3+.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol↗

PubMed1987

Phosphatidylcholine as the choline donor in sphingomyelin synthesis.

Sphingomyelin synthesis was studied in cultured Novikoff rat hepatoma cells by following transfer of [14C]choline label into sphingomyelin (SPH). The study was facilitated by the fact that prelabeling of the cells with [methyl-14C]choline resulted in rapid accumulation of essentially all the label (approximately 95%) in phosphatidylcholine (PC). The redistribution of PC label during a 15-hr chase was dependent upon the extracellular choline concentration. Under conditions of free choline diffusion (500 microM choline), loss of label from PC was most pronounced, and the percentage of total radioactivity that became trapped in the extracellular water-soluble choline pool was an order of magnitude greater than in low choline medium (27 microM choline). Despite the significant loss of water-soluble label from the cells in high choline medium, SPH labeling proceeded at essentially the same rate at either choline concentration. During the label chase in 500 microM choline, the specific radioactivity of PC decreased, but the specific radioactivity of SPH continued to increase for 9-12 hr until it reached the specific radioactivity of PC. In the presence of 300 microM neophenoxine (NPO), transfer of label from PC into SPH was stimulated. NPO also decreased the specific radioactivity of PC to about the same extent as that of SPH was increased. Because transfer of choline label from PC to SPH was not affected by loss or dilution of water-soluble precursors, and because the specific radioactivity of PC and SPH, in the absence or presence of NPO, responded in a characteristic precursor product fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Fetal rhesus monkey lung cells can be grown in serum-free medium for the replication of dengue-2 vaccine virus.

Serum-free media were developed to grow diploid fetal rhesus monkey lung (DBS-FRhL-2) cells and to propagate dengue-type 2 virus vaccine strain PR-159 (dengue-2 vaccine virus). Vitamins, amino acids, growth factors, hormones and other organic compounds, and inorganic salts were substituted for fetal bovine serum. The composition of the medium that was optimal for growth of DBS-FRhL-2 cells differed from medium optimal for the propagation of dengue-2 vaccine virus. Insulin, epidermal growth factor, fibroblast growth factor, and platelet-derived growth factor were required for DBS-FRhL-2 cell proliferation in serum-free medium but were inhibitory for virus propagation. Adenosine, cytidine, guanosine, uridine, and thymidine, each at 0.01 mM concentration, were necessary as medium supplements to obtain a high yield of dengue-2 vaccine virus in DBS-FRhL-2 cells under serum-free conditions. DBS-FRhL-2 cells grown in serum-free medium produced dengue-2 vaccine virus with yields similar to those of cells grown in the presence of serum. Dengue-2 vaccine virus obtained under serum-free conditions retained its phenotypic markers such as temperature sensitivity and small plaque size.

Animals↗

PubMed1979

Energy dependence and reversibility of membrane alterations induced by polyene macrolide antibiotics in Chlorella vulgaris.

The requirement of metabolic energy for the interaction of polyene macrolide antibiotics with eukaryotic organisms remains a controversial subject (for review see ref. 1) It has been claimed that the lethal binding of these antibiotics to the sterol target component of the hydrophobic core of the membrane, in accordance with the model of de Kruijff and Demel, is an energy-dependent process. When energy production is reduced by removal of all metabolisable substrates or by adding metabolic inhibitors, polyene binding and antifungal effects are also reduced. Metabolic energy may be required to maintain binding site accessibility or to move antibiotic molecules to the active site. The interaction is also restricted at low temperatures, possibly because of the reduced thermal mobilities of the groups concerned with antibiotic uptake. However, it should be emphasised that the interaction of polyene macrolides with artificial lipid membranes is a purely physicochemical process, although the type of permeability pathways induced are similar to those observed in natural membranes. Using Chlorella vulgaris as a model organism, we demonstrate here that the interaction of polyene macrolides with sensitive cells and the induction of lethal membrane permeability changes are energy-dependent processes or purely physicochemical phenomena, depending on the structure of the antibiotic used.

Anti-Bacterial Agents↗

PubMed1979

Development of dengue virus plaques under serum-free overlay medium.

An improved plaque assay for dengue virus was developed utilizing baby hamster kidney (BHK-21) cells initially grown in shaker culture. Different media preparations were tested for uniform and fast formation of BHK-21 cell sheets. Several overlay formulas were tested to develop a rapid plaque assay in 6- and 24-well plastic plates. The best results were obtained utilizing Eagle minimal essential medium (pH 7.2 to 7.4) supplemented with 1 mg of NaHCO3 per ml and 5% newborn calf serum for the formation of cell monolayers after 8 to 24 h of incubation at 37 degrees C. Serum-free Eagle minimal essential medium supplemented with 1% methylcellulose and buffered with 10 mM N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (pH 7.4 to 7.6) was used as an overlay medium. This system allowed for plaque formation after 3 days of incubation of dengue type 2 virus and after 4 days for dengue type 1 and 4 viruses.

Animals↗

PubMed1979

Cultivation of dengue virus type 2 in baby hamster kidney cells in serum-free medium.

Propagation of dengue virus type 2 (New Guinea C strain) was performed using a shaker culture of baby hamster kidney cells (BHK-21) cultivated in serum-free modified Waymouth medium. Maximum virus titer varied from 10(8.3) to 10(8.8) plaque forming units/ml after incubation of BHK-21 cells in suspension culture at 37 dgrees C for 40-48 hours post-infection.

Animals↗