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.

Use of cycloheximide to study independent lipid metabolism of Chlamydia trachomatis cultivated in mouse L cells grown in serum-free medium.

A system for measuring chlamydial lipid synthesis was developed with mouse L cells grown in serum-free modified Waymouth 752/l medium in a shaker culture. Host lipid synthesis was reduced approximately 90% when cells were incubated for 24 h in medium containing cycloheximide (2 micrograms/ml). Lipid metabolism was monitored by measuring the incorporation of [3H]isoleucine into the total lipid of normal and infected cells. The results suggested that lipid synthesis of Chlamydia trachomatis lymphogranuloma venereum (LGV-404L) was not inhibited by cycloheximide treatment when the chlamydiae were grown in L cells, whereas host lipid synthesis was inhibited. Chlamydial lipid metabolism began about 6 to 12 h after infection when the noninfectious reticulate body was found and continually increased until the beginning of the appearance of intracellular infectious elementary bodies at 24 to 30 h.

Treponema innocens lipids and further description of an unusual galactolipid of Treponema hyodysenteriae.

The lipids of Treponema innocens, type strain B256, formerly considered a nonpathogenic isolate of T. hyodysenteriae, have been analyzed and compared with the lipids of T. hyodysenteriae. The lipids of T. innocens comprised 16% of the cell dry weight. Polar lipids amounted to about two-thirds of the total lipids and consisted of 61.9% phospholipids and 38.1% glycolipid. Neutral lipids consisted mainly of sterols. The phospholipids were principally phosphatidylglycerol, phosphatidylcholine, and cardiolipin. Minor amounts of lysophosphatidylcholine, sphingomoyelin, and a relatively nonpolar, unidentified phospholipid were present. The latter lipid has not been detected in T. hyodysenteriae. The glycolipid fraction of T. innocens contained a single component, monoglucosyldiglyceride, in contrast to the occurrence in T. hyodysenteriae of two components: monogallactosyldiglyceride and a less-polar glycolipid tentatively identified as acylmonogalactosyldiglyceride (the additional acyl moieties being 86.6% acetyl, 11.6% propionyl, and 1.6% n-butyryl groups). Alk-1-enyl ether analogs comprised 24.6% of the total phospholipids and glycolipid of T. innocens, or about one-third of the amount in T. hyodysenteriae. The acyl and alk-1-enyl moieties of T. innocens consisted of greater than or equal to 92% of 14:0, iso-15:0, and 16:0 chains. In contrast to T. hyodysenteriae, anteiso-15:0 moieties were not detected, and a reversed distribution of 14:0 and iso-15:0 alk-1-enyl moieties occurred in the two species.

Alk-1-enyl ether phospholipids (plasmalogens) and glycolipids of Treponema hyodysenteriae. Analysis of acyl and alk-1-enyl moieties.

The lipids of Treponema hyodysenteriae B78, the etiologic agent of swine dysentery, comprised 16.4% of the cell dry weight, and consisted of 37.4% glycolipids, 28.6% phospholipids, and 34.0% neutral lipids. Monogalactosyldiacylglycerol, a major lipid in all Treponema except Treponema pallidum, comprised 80% of the glycolipids. An unidentified galactolipid less polar than monogalactosyldiacylglycerol was also detected. Phosphatidylglycerol (19.5% of the total lipids) was the major phospholipid. Phosphatidylcholine, characteristically the major phospholipid of treponemes, comprised 6.1% of the total lipids. Cardiolipin and lysophosphatidylcholine were minor components. The alk-1-enyl ether forms of both the phospholipids (plasmalogens) and glycolipids predominated. The alk-1-enyl ether forms of monogalactosyldiacylglycerol, the unidentified galactolipid, phosphatidylglycerol, cardiolipin, and phosphatidylcholine were 88.3, 96.4, 74.8, 60.6, and 6.3%, respectively. The acyl and alk-1-enyl chains of the organism were qualitatively similar and differed dramatically from those of the medium indicating a capability for fatty acid synthesis that most Treponema do not possess. Saturated C14, C15, and C16 chains comprised more than 95% of the acyl and alk-1-enyl groups. About 25% of the chains were iso-15:0, anteiso-15:0, and other branched moieties.

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.

Unique lipid composition of Treponema pallidum (Nichols virulent strain).

The lipid composition of Treponema pallidum (Nichols virulent strain) was determined after purification of the organisms from the infected testes of corticosteroid-treated rabbits by differential centrifugation, filtration through Nuclepore membranes, and sedimentation in Hypaque density gradients. The total lipids were comprised of 32.2% neutral lipids, mainly cholesterol, and 67.8% phospholipids consisting of phosphatidylcholine (32.1%), sphingomyelin (14.8%), cardiolipin (13.0%), phosphatidylethanolamine (6.2%), phosphatidylinositol-serine (1.2%), and lysophosphatidylcholine (0.4%). Monoglycosyldiglyceride, a glycolipid comprising 25 to 50% of thetotal lipid of all Treponema previously examined, was not detected. The fatty acid composition was similar but quntitatively distinct from that of the infected testes tissue.

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.

Radiolabeling of Treponema pallidum (Nichols virulent strain) in vitro with precursors for protein and RNA biosynthesis.

We observed uptake of [U-14C]serine, U-14C-labeled amino acid hydrolysates, and [2-14C]uracil by virulent Treponema pallidum in vitro for at least 96 h. No uptake of [2-14C]thymine, [1-14C]pyruvate, [U-14C]pyruvate, and [2-14C]uridine was detected. Treponemal protein and RNA biosynthetic activity was identified by erythromycin inhibition of amino acid and uracil uptake. Radioactivity due to uptake of radiolabeled amino acids by residual testicular cells in the cultures remained at background levels regardless of the presence or absence of cycloheximide. Accumulation of the radiolabeled substrates by T. pallidum proceeded at a linear rate for 48 to 96 h during incubation in vitro. The longevity of substrate uptake using the system of incubation described will facilitate future studies on the metabolism of the microorganism to help determine essential growth factors and environmental conditions for multiplication of T. pallidum in vitro.

Contrast of Glycogenesis and protein synthesis in monkey kidney cells and HeLa cells infected with Chlamydia trachomatis lymphogranuloma venereum.

Glycogen metabolism of monkey kidney (LLC-MK-2) cells and HeLa 229 cells infected with a Chlamydia trachomatis lymphogranuloma venereum 440 L (LGV) was studied. The growth cycle of LGV in both host cells was similar; however, a greater number of infectious organism developed intracellularly and were released into the medium during LGV infection of HeLa 229 cells than MK-2 cells. A rapid infection accompanied by a high rate of glycogen synthesis and a short period of accumulation was found in GeLa 229 cells infected with LGV. LGV infected MK-2 cells started to accumulate glycogen about the same time as HeLa 229 cells; however, the rate of glycogen synthesis was lower and the period of accumulation was longer. The LGV agent grew in cycloheximide-treated cells in the absence of host cell protein synthesis. Protein synthesis associated with LGV throughout the developmental cycle was similar in both cell types and could be abolished by chloramphenicol. The continued synthesis of glycogen in the presence of cycloheximide suggested that the synthesis of glycogen was directed by the organism in both MK-2 cells and HeLa 229 cells.

Effects of fatty acids on motility retention by Treponema pallidum in vitro.

Treponema pallidum (Nichols virulent strain) was incubated under 75% N2 + 20% H2 + 5% CO2 in prereduced serum-free modified Eagle-Richter medium supplemented with different concentrations of various long-chain fatty acids complexed with fatty acid-free bovine serum albumin. Motility retention was greater in medium with oleic acid containing 15 rather than 2 mg of albumin per ml. Palmitic, stearic, oleic, or linoleic acid alone caused rapid loss of motility at concentrations as low as 5 microgram/ml. Elaidic acid (92 microgram/ml) alone had no effect on motility. Various combinations of saturated plus unsaturated fatty acids did not inhibit motility retention or were less inhibitory than either of the individual fatty acid components. The combination of palmitic plus oleic acids was least toxic. Rapid loss of motility occurred with pairs of unsaturated or saturated fatty acids, or with Tween 40, 60, or 80, alone or combined. Autoxidation of oleic acid resulted in decreased toxicity for T. pallidum but increased toxicity for baby hamster kidney cells.

Unsustained multiplication of treponema pallidum (nichols virulent strain) in vitro in the presence of oxygen.

Treponema pallidum (Nichols virulent strain) was incubated with or without oxygen using a modified medium supplemented with reduced glutathione and a variety of nutrients (PRNF10-B). Two- to fourfold increases in treponemal numbers were observed in cultures without mammalian cells within 96 h of incubation under 5 to 6% oxygen. Treponemal motility and multiplication were maintained more satisfactorily in cultures that were diluted and transferred daily, using an equal volume of fresh medium. Treponemes incubated without oxygen did not significantly increase in number. Virulent microorganisms were detected for at least 96 h in the cell-free system. In the presence of 3 to 4% oxygen, two- to fivefold increases in treponemal numbers were observed in the supernatant fluids of cultures containing human prepuce cells after 48 to 120 h at 35 degrees C. Without oxygen, treponemal numbers rarely approached a threefold increase. Virulent treponemes were detected by the rabbit skin lesion test after at least 120 h in vitro. Regardless of the system of incubation, increases in treponemal numbers could not be sustained for longer than 120 h, and treponemal virulence decreased as a function of time in vitro.