Relationship between dopamine-stimulated phospholipid methylation and the single-carbon folate pathway.

In a previous study we demonstrated the ability of dopamine (DA) to stimulate phospholipid methylation (PLM) via a novel mechanism involving the D4 dopamine receptor (D4R) in which single-carbon folates appeared to be the primary source of methyl groups. To further understand the relationship between D4R-mediated PLM and folate metabolism, we examined the effect of several folate pathway interventions on the level of basal and DA-stimulated incorporation of [14C]-labeled formate into phospholipids in cultured SH-SY5Y neuroblastoma cells. These interventions included: (i) Overexpression of methenyltetrahydrofolate synthetase (MTHFS). (ii) Treatment with 5-formylTHF. (iii) Treatment with the MTHFS inhibitor 5-formyltetrahydrohomofolic acid (5-formylTHHF). (iv) Growth in nucleoside-free media. 31P-NMR was also used to follow DA-induced changes in cell phospholipid composition. MTHFS overexpression and 5-formylTHHF treatment, both of which lower 5-methylTHF levels, each reduced basal PLM and its stimulation by DA. In contrast, 5-formylTHF, which increases 5-methylTHF, caused a dose-dependent increase in both basal and DA-stimulated PLM. Growth in nucleoside-free media caused time-dependent changes in PLM, which were due to the absence of purine nucleosides. While basal PLM was maintained at a reduced level, DA-stimulated PLM was initially increased followed by a later decrease. Together, these findings indicate a close functional relationship between single-carbon folate metabolism and DA-stimulated PLM, consistent with a role for 5-methylTHF as the methyl donor for the D4R-mediated process.

Increased synthesis of phosphocholine is required for UV-induced AP-1 activation.

Exposure of mammalian cells to UV irradiation stimulates phosphatidylcholine hydrolysis and activates the transcription factor AP-1. Since phosphocholine (PCho), a phospholipid metabolite, is a potential regulator of mitogenesis and carcinogenesis, we examined the effect of UV exposure on the formation of PCho and the possible mediatory role of PCho in UVB-and UVC-induced activation of AP-1 in mouse JB6 epidermal cells. We found that both UVB and UVC irradiation resulted in increased PCho levels. Hemicholinium-3 (HC-3), an inhibitor of choline kinase, strongly inhibited UV-induced AP-1 activity. By contrast, relatively low levels of PCho (80 microM) or choline (20 microM) nearly doubled UV-induced AP-1 activity, while higher (2-20 mM) concentrations of PCho alone stimulated AP-1 activity 6-8-fold. Importantly, HC-3 inhibited only the stimulatory effect of choline, but not of PCho, on AP-1 activity. Of the mitogen-activated protein (MAP) kinases involved in the regulation of AP-1 activity, UVC stimulated the MAP kinase family ERK-1/ERK-2, JNK as well as p38 kinase activity. These UVC effects were all inhibited by HC-3. With UVB, by contrast, only the activation of ERK-1/ERK-2 was inhibited by HC-3. The data suggest that increased formation of PCho is required for UV-induced activation of AP-1 by an ERK-1/ERK-2-dependent mechanism.

Phosphorylation of ethanolamine, methylethanolamine, and dimethylethanolamine by overexpressed ethanolamine kinase in NIH 3T3 cells decreases the co-mitogenic effects of ethanolamines and promotes cell survival.

Ethanolamine (Etn), as well as its N-methyl (MeEtn) and N,N-dimethyl (Me2Etn) analogues, were recently shown to potentiate the stimulatory effect of insulin on DNA synthesis in serum-starved NIH 3T3 fibroblasts. In the present work we assessed the impact of the co-mitogenic effects of Etn and its methyl analogues on cell proliferation and cell survival, and examined whether the cell growth regulatory effects of these ethanolamines involve an Etn-kinase-mediated phosphorylation step. For this purpose, NIH 3T3 sublines highly overexpressing Drosophila Etn kinase and an appropriate vector control line were utilized and the effects of Etn, MeEtn, Me2Etn, methylamine (MeNH2), and dimethylamine (Me2NH) were studied. 31P-NMR analysis of the water-soluble cell metabolites revealed that both MeEtn and Me2Etn, but not choline, are excellent substrates for the expressed Etn kinase. The methylated ethanolamines (MeEtn and Me2Etn) and methylamines (MeNH2, Me2NH) were used as Etn models that can or cannot be phosphorylated, respectively. In serum-starved vector control cells, both MeNH2 (1 mM) and Me2NH (1 mM) were more effective than Etn in enhancing insulin-induced DNA synthesis, and both were almost as effective as MeEtn and Me2Etn. However, in the Etn kinase overexpressor cells the potentiating effects of Etn, MeEtn and Me2Etn, but not those of MeNH2 and Me2NH, were significantly reduced. Moreover, in the overexpressor cells, lower concentrations of Etn (50-200 microM) inhibited the combined mitogenic effects of Me2NH (1 mM) and insulin. These data are consistent with a mechanism in which the phosphorylated and non-phosphorylated ethanolamines are negative and positive regulators of insulin-induced mitogenesis, respectively. After incubating the cells for 13 days in serum-free medium in 96-well microplates, there was a steady decrease in cell numbers in both cell lines. However, between 6-13 days, 0.1-1 mM MeEtn and, particularly, Me2Etn provided significant protection against cell death in the Etn kinase overexpressor cells. In vector control cells, only Me2Etn in combination with insulin had similar effects on cell survival. The data suggest that phosphorylated ethanolamines may function as promoters of cell survival.

Alk-1-enylacyl, alkylacyl, and diacyl subclasses of native ethanolamine and choline glycerophospholipids can be quantified directly by phosphorus-31 NMR in solution.

We show that phosphorus-31 nuclear magnetic resonance spectroscopy can be used to distinguish and to quantify the alk-1-enylacyl, alkylacyl, and diacyl glycerophosphoethanolamine (GPE) subclasses, and the respective glycerophosphocholine (GPC) subclasses, in their native form without prior degradation or derivatization, provided the phospholipids are observed in the nonaggregated state. Monomeric phospholipid distribution is ascertained by recording the spectra, after removal of metal ions, on CDCl3/CD3OD/D2O (50:50:15, by vol) solutions. The utility of this approach is exemplified for the ethanolamine glycerophospholipids (EPL) from bovine brain and the choline glycerophospholipids (CPL) from bovine heart. Sharp and well-resolved resonances are obtained for alkylacylGPE (+0.395 ppm; re 1% H3PO4), alkenylacylGPE (+0.353 ppm), and diacylGPE (+0.315 ppm), and for alkylacylGPC (-0.383 ppm), alkenyl-acylGPC (-0.436 ppm) and diacylGPC (-0.451 ppm). Integrated peak areas are shown to closely correlate with dose. Accurate quantitation of EPL and CPL subclasses at submicromolar levels can further be facilitated by use of synthetic dialkylGPE (+0.602 ppm) and dialkylGPC (-0.196 ppm) as internal standards. The method is simple, rapid, sensitive and reproducible, and permits the complete resolution and direct quantitation of all ethanolamine and choline glycerophospholipid subclasses quite independent of fatty chain length and degree of unsaturation.

Lipid differentiation in MP26 junction enriched membranes of bovine lens fiber cells.

The present study was undertaken to address the question whether lipid differentiation occurs in junctional domains which could imply a functional requirement for specific lipids in junctional structures. Junction enriched membranes were isolated from bovine lens fiber cells using Tris and urea treatment, and the presence of junctional structures was ascertained by electron microscopy. Enrichment in major intrinsic protein (MIP, MP26) was monitored by SDS polyacrylamide gel electrophoresis. Junctional lipids were extracted by a modified Folch procedure, to quantitatively recover cholesterol, and lipid classes were analyzed. While 99.5% of total lens protein was solubilized in the course of junction isolation, 43.9% of cell phospholipids (PL) and 64.1% of cell cholesterol (Chol) were conserved. Cholesterol was by far the predominant lipid in the junction enriched lens fiber cell membranes (833 nmol/mg protein) and was more abundant than all phospholipids combined (682 nmol/mg protein). In isolating the junctional membranes, cholesterol levels increased 144-fold, and average phospholipid levels increased 99-fold, which resulted in an increase in Chol/PL ratio from 0.84 to 1.22. Different phospholipids showed substantially different degrees of enrichment with highest enrichments seen for the phosphatidylethanolamine fraction (152-fold) and sphingomyelin (101-fold). Thus, the phospholipids of the junction enriched membranes consisted mainly of ethanolamine glycerophospholipids (37.3%) and sphingomyelin (28.6%), with lesser amounts of choline glycerophospholipids (23.5%) and phosphatidylserine (9.2%) present. Our data suggest that the MP26 junction enriched membranes of bovine lens fiber cells contain differentiated lipid domains, and that cholesterol, ethanolamine glycerophospholipids and sphingomyelin are the prevalent boundary lipids of the major intrinsic protein in these domains.

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.

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.

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.

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)

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.

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)

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)

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.

Potentiation of antiviral activity of acyclovir by polyene macrolide antibiotics.

The potentiation of the antiviral activity of acyclovir [9-[(2-hydroxyethoxy)methyl]guanine] by polyene macrolide antibiotics has been studied as a function of the macrolide structure. The 12 polyenes chosen for this study represented the major structural groups of these antibiotics and induced in mammalian cells repairable membrane alterations or irreversible cell damage. The potentiating activity of the polyene macrolides was determined based on the differential decrease of in vitro production of infectious virions in the presence of acyclovir alone or in combination with the polyene. Pseudorabies virus, a representative herpesvirus susceptible to acyclovir, was replicated in BHK-21 cells grown in serum-free medium to avoid the interference of serum factors in the polyene macrolide-cell interaction. The potentiation activity of the polyene antibiotics was concentration dependent. The enhancement of the antiviral activity of acyclovir was observed at polyene concentrations which had no direct effect on pseudorabies virus replication in BHK-21 cells. The optimal potentiating concentrations of polyenes were 2 to 15 times lower than that inducing 50% of potassium efflux from BHK-21 cells. The highest potentiating activity was observed for the methyl ester of the trimethylammonium derivative of aureofacin B, which reduced the pseudorabies virus titer by two orders of magnitude. Potentiation by polyene macrolides appeared to coincide with the K+-dependent membrane repair process. The acyclovir potentiating activity was associated with polyene macrolide antibiotics having a large and rigid macrolide ring (amphotericin B and aureofacin). Polyene antibiotics with small and rigid (pimaricin and filipin) or large but flexible (nystatin A1 and lienomycin) macrolide rings showed no potentiation of the antiviral effect of acyclovir.

Combined effect of acyclovir and amphotericin B on the replication of pseudorabies virus in BHK-21 cells.

Acyclovir, known as an antiherpetic agent, showed an inhibitory effect on the propagation of pseudorabies virus in BHK-21 cells. The antiviral effect of acyclovir was observed by plaque reduction, as well as by the inhibition of the virus-stimulated uptake of thymidine by BHK-21 cells. Amphotericin B potentiated the antiviral activity of acyclovir. The optimal concentrations of polyene antibiotic expressing the potentiating effect were lower than required for the induction of K+ leakage from the cells. There was no evident amphotericin B-induced stimulation of thymidine incorporation into infected BHK-21 cells. The model presented may be useful to study the potentiation phenomenon of polyene macrolide antibiotics.

Differential selective toxicity of DMS-aureofacin components.

Trimethylammonium methyl esters (DMS) of polyene macrolides are products of the methylation of native polyene antibiotics with dimethyl sulfate. We isolated individual components of the DMS-aureofacin complex and characterized their toxicity and activity to induce permeability changes in cell membranes. The DMS-aureofacin complex contained five components readily separated by thin-layer chromatography on polygram cellulose plates. DMS-Aureofacin A, a major component of the complex (90%), showed poor selective toxicity between yeast cells and mammalian cells grown in culture. DMS-Aureofacin B (6%) and DMS-aureofacin E (4%) exhibited very high biological activities and differed qualitatively in selective toxicity. DMS-Aureofacin B was much more active for mammalian cells than for yeast cells. In contrast, DMS-aureofacin E was much more active for yeast cells than for mammalian cells. DMS-Aureofacin C and D were present in the complex in only minute quantities which did not permit their biological characterization.

Repair of membrane alterations induced in baby hamster kidney cells by polyene macrolide antibiotics.

We studied the correlation between chemical characteristics of 13 polyene macrolide antibiotics and the ability to repair the membrane permeability changes induced by polyenes in BHK-21 cells grown in shaker culture. It had been demonstrated that large-macrolide-ring polyenes with rigid molecules (heptaenes) induced specific membrane permeability pathways which were repaired by the eucaryotic cells under the proper conditions. The influence of environmental conditions on the repair process was examined. Aureofacin trimethylammonium methyl ester derivative was used as a selected representative of polyene macrolides inducing specific pathways. The factors influencing the repair process, monitored by measuring the ability of BHK-21 cells to control K+ membrane transport, were examined during and after cell contact with the antibiotic. We found that the repair process was dependent upon the temperature, the concentration of the antibiotic, time of its contact with cells, potassium concentration in the medium, and availability of an energy source. The repair process occurred in the presence of cycloheximide, which inhibited protein synthesis in BHK-21 cells. Results showed that the repair process plays an important role in mammalian cell recovery from the toxic effects of polyenes.

Dissociation between the induction of potassium efflux and cytostatic activity of polyene macrolides in mammalian cells.

The paper contains data on the induction of K+ efflux and viability of baby hamster kidney (BHK-21) cells after their treatment with macrolide antibiotics inducing specific pores in membrane. New water-soluble semisynthetic derivatives of amphotericin B and aureofacin (N-glycosyl and trimethylammonium methyl ester derivatives) as well as the parent compounds were used to compare the concentration of antibiotics inducing permeabilizing and cytostatic effects. We found that a two- to eight-times-higher concentration of polyene antibiotic was required to observe a cytostatic effect than for release of 50% of the cellular potassium (K50 concentration) from BHK-21 cells. These differences were larger for water-soluble derivatives than for the parent compounds. The amount of intracellular potassium in treated cells incubated under optimal growth conditions was higher than in cells which had been further washed with K+-free maintenance medium. The membrane permeability changes induced by low concentrations of specific polyenes were observed to be reversible. BHK-21 cells were able to repair polyene-induced membrane permeability within 3 to 12 h under optimal growth conditions, after cell treatment with K50 concentration of specific macrolide antibiotics. The repair phenomenon is postulated as an explanation for the dissociation observed between permeabilizing and cytostatic effect of specific polyenes in BHK-21 cells.

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.