Cytotoxic and cytolytic activity of nonadecafluoro-n-decanoic acid on Acholeplasma laidlawii

We studied the interactions between the perfluorinated fatty acid nonadecafluoro-n-decanoic acid (NDFDA) and the cell wall-less procaryote Acholeplasma laidlawii, which were cultured in an identical medium base but with different serum supplements. When grown in mycoplasma media supplemented with PPLO serum fraction (Difco Laboratories, Detroit, Mich.), A. laidlawii was rapidly killed by low concentrations of toxicant (less than 1.0 mM). At higher concentrations (greater than 10 mM), NDFDA treatment appeared to lyse cells. A. laidlawii cells grown in horse serum-supplemented mycoplasma media were both killed and lysed at the same NDFDA concentration (greater than 10 mM). These data suggest that this perfluorinated fatty acid can be cytotoxic and cytolytic to mycoplasmas. Changes in active concentrations occurred in parallel with changes in growth medium serum supplementation, which is known to alter mycoplasma membrane composition. We propose that NDFDA interacts with the membranes of A. laidlawii cells, resulting in cell death or cell lysis or both.     

Cyclodextrins as carriers of cholesterol and fatty acids in cultivation of mycoplasmas.

The design of fully or partly defined media for mycoplasma cultivation involves the need to provide the essential lipids, cholesterol and long-chain fatty acids, in an assimilable and nontoxic form. This study introduces cyclodextrins (CDs) as carriers of these lipids, thus suggesting alternatives to serum or bovine serum albumin (BSA). The effects of beta-CD and two forms of chemically modified beta-CD, dimethyl-beta-CD (Dimeb) and hydroxypropyl-beta-CD (Hyprob), on the growth of Mycoplasma capricolum and Acholeplasma laidlawii were investigated in a basal medium as well as in serum- and BSA-supplemented media. beta-CD was found to inhibit the growth of the sterol-requiring M. capricolum in both serum and BSA media, but it stimulated the growth of the sterol-independent A. laidlawii. Inhibition by beta-CD was explained by its capacity to form a water-insoluble CD-cholesterol complex, thus rendering it unavailable to the cells. Dimeb, despite its strong complexing ability for lipids, was found to be toxic to all mycoplasma species in both liquid cultures and agar diffusion susceptibility tests. In sharp contrast to beta-CD and Dimeb, Hyprob (with a degree of substitution of 4.2) added at 5 and 10 mM to a basal medium supplemented with lipids permitted growth of M. capricolum. Comparison of growth curves in the two conventional serum and BSA media with those in two Hyprob media revealed comparable growth and growth rates.     

Nisin resistance distinguishes Mycoplasma spp. from Acholeplasma spp. and provides a basis for selective growth media

The sensitivity of 11 Mycoplasma and 5 Acholeplasma species to the bacteriocin nisin was determined. When applied on filter paper discs to lawns of acholeplasma cells, nisin (20 nmol per disc) gave 3.5- to 7.0-mm zones of growth inhibition. The inclusion of 0.2 mM nisin in agar medium reduced the number of Acholeplasma laidlawii colonies by a factor of more than 10(6), and in a salts solution, 75 microM nisin killed more than 99.9% of cells within 1 min. Under similar conditions, nisin had no significant effect upon the growth or survival of Mycoplasma species. At low concentrations (1 to 3 microM), nisin stimulated glucose oxidation by A. laidlawii and Acholeplasma oculi. However, in comparison with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a recognized protonophore and uncoupler of respiration, the maximum extent of stimulation was low, < or = 20%, compared with up to 180% for CCCP. Also, in contrast to results obtained with CCCP, at concentrations only slightly above those causing stimulation of acholeplasma oxygen uptake, nisin strongly inhibited respiration. Inhibition of oxygen uptake was greater for A. laidlawii cells grown in the absence of cholesterol, and on agar medium, growth inhibition by nisin decreased with increasing concentrations of cholesterol. Nisin resistance may be a valuable characteristic in the selection and identification of Mycoplasma spp.     

Nisin resistance distinguishes Mycoplasma spp. from Acholeplasma spp. and provides a basis for selective growth media.

The sensitivity of 11 Mycoplasma and 5 Acholeplasma species to the bacteriocin nisin was determined. When applied on filter paper discs to lawns of acholeplasma cells, nisin (20 nmol per disc) gave 3.5- to 7.0-mm zones of growth inhibition. The inclusion of 0.2 mM nisin in agar medium reduced the number of Acholeplasma laidlawii colonies by a factor of more than 10(6), and in a salts solution, 75 microM nisin killed more than 99.9% of cells within 1 min. Under similar conditions, nisin had no significant effect upon the growth or survival of Mycoplasma species. At low concentrations (1 to 3 microM), nisin stimulated glucose oxidation by A. laidlawii and Acholeplasma oculi. However, in comparison with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a recognized protonophore and uncoupler of respiration, the maximum extent of stimulation was low, < or = 20%, compared with up to 180% for CCCP. Also, in contrast to results obtained with CCCP, at concentrations only slightly above those causing stimulation of acholeplasma oxygen uptake, nisin strongly inhibited respiration. Inhibition of oxygen uptake was greater for A. laidlawii cells grown in the absence of cholesterol, and on agar medium, growth inhibition by nisin decreased with increasing concentrations of cholesterol. Nisin resistance may be a valuable characteristic in the selection and identification of Mycoplasma spp.     

Radiation studies of Acholeplasma laidlawii: the role of membrane composition

Acholeplasma laidlawii, a mycoplasma, is unable to synthesize unsaturated fatty acids but it will incorporate them into its plasma membrane if they are supplied exogeneously. Thus the fatty acid composition of the cell membrane can be defined by growing the organism in media containing specific fatty acids. We obtained cells with predominantly one type of unsaturated fatty acid (either oleic, linoleic or linolenic acid) or cells with only saturated fatty acid in the cell membrane. The cells were irradiated with 7 MeV electrons and the effect of membrane fatty acid composition on cell survival was examined. At 200 Gy/min and 0.5 degrees C (melting ice) there was little difference in the radiation sensitivities of the cells grown in unsaturated fatty acids either in aerated or anoxic radiation conditions. However, the cells containing saturated fatty acids irradiated in anoxic conditions were markedly more sensitive than the cells containing unsaturated fatty acids. At 200 Gy/min and 37 degrees C the two types of cells were of similar sensitivity both in aerated and anoxic radiation conditions. At 5 Gy/min at 0.5 degrees C the cells containing linolenic acid (18:3) were less sensitive than those containing solely saturated fatty acids. However, at 5 Gy/min at 37 degrees C there was no difference in sensitivity between these two types of cell. Our results strongly argue against the involvement of lipid peroxidation as a molecular change leading to cell death.     

Identification of albumin-bound fatty acids as the major factor in serum-induced lipid accumulation by cultured cells

Factors responsible for the high lipogenic activity of rabbit serum were investigated using an assay procedure based on the gravimetric determination of the 24 hr increase in cell lipid. Cellular synthesis of fatty acids was inhibited by the presence of serum in the assay medium. Approximately 90% of the increase in cell lipid produced by serum fractions was due to triglyceride accumulation. Fractionation of rabbit serum by precipitation with ammonium sulfate or by ultracentrifugation in high density medium, both indicated that three-quarters of its lipogenic activity was associated with albumin. The lipoproteins prepared by ultracentrifugation also exhibited about one-half the activity of whole serum. The lipogenic activity of albumin was confirmed by the high potency of the albumin isolated in a nearly pure form from proteins of d>1.21 by precipitation with trichloroacetic acid and extraction with ethanol. As judged from chemical and isotopic analysis, neither the lipid content nor the lipid composition of the albumin was appreciably altered during its isolation. Of the albumin-bound lipids, only the free fatty acids, as determined by DEAE column chromatography, were present in an amount sufficient to account for the observed increase in cell triglycerides. In control experiments with horse serum of low lipogenic activity, the proteins of d>1.21 also possessed low activity in conjunction with a low content of free fatty acid. However, the albumin isolated from the latter preparation exhibited the high lipogenic activity of rabbit serum albumin. Chemical and isotopic analysis of the recovered horse serum albumin revealed that its free fatty acid content was the same as that of rabbit serum albumin. These results indicated that the isolation of horse serum albumin was attended by a substantial increase in its free fatty acid content. When the rabbit serum and horse serum content of media were adjusted to provide equivalent concentrations of albumin-bound fatty acids, the rabbit liver cells grown on the former media accumulated more lipid than cells grown on the latter media. This difference was shown to be due to the higher concentration of albumin per micro mole of fatty acid in horse serum as compared with rabbit serum. Consequently, the albumin to fatty acid ratio also controls the lipogenic activity of a serum. A linear relationship is presented which relates the cell lipid content to the molar ratio of albumin to free fatty acids and to the absolute concentration of free fatty acids in the medium.     

Ammonia production and amino acid metabolism by rat renal papillary epithelial cells in culture

A significant percentage of excreted ammonium is added to tubular fluid along the medullary collecting duct. However, it is not clear whether this ammonia is produced in the cortex and delivered into the medulla or is produced directly by medullary cells. To address this issue, rat epithelial cells derived from the renal papilla were grown in continuous culture and their ability to generate ammonia was examined. When grown in Dulbecco's modified Eagle's medium with 4 mM glutamine, these cells produced ammonia at a rate of approximately 27 nmol/10(6) cells/h. When these cells were grown in minimum essential medium without glutamine, ammonia production fell to 7 nmol/10(6) cells/ h. Increasing the glutamine concentrations of minimum essential medium to 4 mM increased ammonia production to slightly greater than 30 nmol/10(6) cells/ h. Increasing the media concentration of glutamate, glycine, or asparagine resulted in no significant increase in ammoniagenesis. Analysis of media amino acid concentration revealed that glutamine was the main amino acid consumed while alanine was the predominant amino acid produced. The glutaminase activity of these cells appears to be primarily phosphate-dependent, similar to that observed in vitro in papillary tubules. Alterations of K+ or H+ ion concentration did not alter ammoniagenesis, but addition of 2.5 mM ammonium chloride significantly reduced net ammonia production. It is concluded that rat papillary epithelial cells have the intrinsic ability to utilize glutamine to generate ammonia and alanine. In vivo ammonia produced locally in the medulla may contribute to final urinary ammonium excretion.     

Infrared spectroscopic study of the gel to liquid-crystal phase transition in live Acholeplasma laidlawii cells

The temperature dependences of the infrared spectra of deuterium-labeled plasma membranes of live Acholeplasma laidlawii B cells and of the isolated plasma membranes demonstrate that the profiles of the gel to liquid-crystal phase transitions are very different. At temperatures within the range of the phase transition, the live mycoplasma is able to keep the "fluidity" of its plasma membrane at a much higher value than that of the isolated plasma membrane at the same temperature. The difference is particularly pronounced at and around the temperature of growth. Live Acholeplasma laidlawii, grown at 37 degrees C on a fatty acid depleted medium supplemented with myristic acid (C14:0), pentadecanoic acid (C15:0), or palmitic acid (C16:0), are highly "fluid"; i.e., at the temperature of growth, the fractional population of the liquid-crystalline phase is 95-100% at 37 degrees C, whereas in the case of the isolated plasma membranes the fractional population of the liquid-crystalline phase at 37 degrees C is only 58% (C14:0), 36% (C15:0), or 38% (C16:0).     

Bovine colostrum or milk as a serum substitute for the cultivation of a mouse hybridoma

A mouse-mouse hybridoma was grown in serum-free medium supplemented with bovine milk or colostrum. Bovine colostrum supported growth of the hybridoma whereas bovine milk alone did not support cellular proliferation. For growth in medium supplemented with colostrum, the maximum cell concentration achieved was 1.4 x 10(6) cells/mL in 2.2% colostrum, which is 44% of that obtained in 9% serum. When cells were grown in media containing milk and low amounts of serum (<1%) the maximum cell concentration in 2.2% milk with 0.4% serum was 2 x 10(6) cells/ml, whereas it was only 0.2 x 10(6) cells/ml and 1.3 x 10(6) cells/ml in 2.2% milk alone and 0.4% serum alone, respectively. Similar behavior was observed for growth in media containing colostrum and low amounts of serum. The monoclonal antibody production in media containing combinations of serum and milk or colostrum was comparable to that obtained in media with higher serum concentrations. Experiments performed with conditioned media suggest that the rapid decrease in viability, after the maximum cell concentration has been reached, is partially due to the presence of some inhibitory components generated during the cell culture rather than due to depletion of some serum components.     

Synthesis of saturated long chain fatty acids from sodium acetate-1-C14 by Mycoplasma

Three strains of Mycoplasma, M. laidlawii A and B, and Mycoplasma sp. A60549, were grown in broth containing sodium acetate-1-C(14). The methyl esters of the phospholipid fatty acids of harvested radioactive cells were prepared and identified by comparison of their mobilities to known radioactive fatty acid methyl esters by use of a modified reversed-phase partition-thin layer chromatographic technique. No radioactive methyl oleate or methyl linoleate was detected. Compounds migrating as radioactive methyl myristate, stearate, palmitate, and, with less certainty, laurate and octanoate were detected. The qualitative findings for all three organisms appeared similar. M. laidlawii B synthesized a radioactive substance, presumably a saturated fatty acid detected as the methyl ester derivative, which migrated in a position intermediate to methyl myristate-1-C(14) and methyl palmitate-1-C(14). This work indicates that M. laidlawii A and B and Mycoplasma sp. A60549 are capable, in a complex medium containing fatty acids, of synthesizing saturated but not unsaturated fatty acids entirely or in part from acetate.     

Osmolar Concentration and Fixation of Mycoplasmas

Broth cultures of Acholeplasma laidlawii were fixed with various concentrations of cacodylate-buffered glutaraldehyde. The shape and ultrastructure of the organisms varied with the osmolar concentration of the fixative. When the fixation mixture was hypertonic to the culture medium, ultrathin sections suggested that the cells had shrunk. Phosphate buffer, sodium chloride, or sucrose at comparable osmolaities had the same effect as sodium cacodylate. Glutaraldehyde itself also contributed to the osmotic effects of the fixation mixture but to a lesser extent than salts or sucrose, to which the cell membrane is impermeable. The osmolar concentration of the fixation mixture seemed of greater importance than pH in determining morphology. The mycoplasma was still susceptible to damage by high concentrations of cacodylate after fixation with 2.5% glutaraldehyde. The best procedure was to fix and wash the organism under conditions isotonic with the growth medium. These conditions were also satisfactory for a filamentous mycoplasma, Mycoplasma orale.     

Regulation of palmitic acid synthesis in cultured glial cells: effects of lipid on fatty acid synthetase, acetyl-CoA carboxylase, fatty acid and sterol synthesis.

Abstract— C6 glial cells in culture were utilized to study the regulation of the important lipogenic enzymes, fatty acid synthetase and acetyl-CoA carboxylase, and the synthesis of fatty acids and sterols. Regulation of these phenomena by lipid was demonstrated by the following observations. First, removal of serum from the culture medium was accompanied over the next five days by 2–3-fold increases in the lipogenic enzymatic activities and in 5–15-fold increases in rates of incorporation of acetate into fatty acids and sterols. Second, cells grown in delipidated serum exhibited approx 2-fold higher levels of activity of the lipogenic enzymes and 5–10-fold higher rates of synthesis of fatty acids and sterols than cells grown in normal calf serum. Third, cells grown in serum-free medium supplemented with concentrations of fatty acid comparable to those present in medium supplemented with serum exhibited activities of fatty acid synthetase comparable to those exhibited by cells grown in the serum-supplemented medium. The mechanism of these effects on fatty acid synthetase was shown by immunochemical techniques to involve alterations in content rather than in catalytic efficiency of the enzyme. The changes in content of the synthetase were caused by alterations in enzyme synthesis. In view of morphological and biochemical data suggesting that C6 cells are related to differentiating cells with properties of both astrocytes and oligodendroglia, the present data may indicate that regulation of palmitic acid synthesis by fatty acid or a product thereof occurs in brain during development.     

Alteration of the fatty acid composition of membrane phospholipids in mouse lymphoid cells

A simple method is described for introducing exogenous fatty acids into the membrane phospholipids of the murine leukemia cell EL-4, and into the membrane phospholipids of resting mouse lymphocytes. The method involves culturing of the cells with free or methylated fatty acids at concentrations up to 50 microgram/ml. The presence of serum in the culture medium does not interfere with fatty acid uptake, but does increase the growth rate and viability of the cells. Membrane lipid composition returns to normal after the cells are grown in medium without exogenous fatty acid. Fractionation of the cell membranes confirmed that exogenous fatty acids were incorporated into the phospholipids of the plasma membrane.     

Changes in serum influence the fatty acid composition of established cell lines

Summary The fatty acid composition of different kinds of commercially available serum used to supplement cell culture media differs widely. As compared with fetal bovine serum, horse and bovine calf serum have a very high content of linoleic acid (18:2) and are low in arachidonic acid (20:4). (Fatty acids are abbreviated as number of carbon atoms: number of double bonds). Swine serum contains substantial amounts of both 18:2 and 20:4. Only fetal bovine serum contains more than 1% docosahexaenoic acid (22:6). Considerable differences in fatty acid composition occur when cells are grown in media containing any of these different serum supplements. The 18:2 and 20:4 content of 3T3 mouse fibroblast phospholipids is highest when the medium contains horse serum, intermediate with bovine calf serum, and lowest with swine or fetal bovine serum. Likewise, the highest phospholipid 18:2 content in Madin-Darby canine kidney cells (MDCK) occurs when the medium contains horse serum. With MDCK cells, however, growth in swine serum produces the highest 20:4 content. The 3T3 cell phospholipids accumulate more than 1% 22:6 only when the medium contains fetal bovine serum, whereas in no case do the MDCK cell phospholipids accumulate appreciable amounts of 22:6. The fact that the cellular fattyacid composition is likely to change should be taken into account when changes are contemplated in the serum used to grow established cell lines. These studies were supported by Arteriosclerosis Specialized Center of Research Grant HL 14,230 from the National Heart, Lung, and Blood Institute, National Institutes of Health.     

Polyethylene glycol-dependent transfection of Acholeplasma laidlawii with mycoplasma virus L2 DNA

Phenol-extracted DNA from mycoplasma virus L2 was able to transfect Acholeplasma laidlawii in the presence of polyethylene glycol. Transfection was sensitive to DNase and was most efficient with 36% (wt/vol) polyethylene glycol 8000 and cells in logarithmic growth. Virus production by the transfected cells was similar to that of the cells infected by intact virus. L2 DNA transfected A. laidlawii with a single-hit dose-response curve, reaching saturation at high DNA concentrations. Optimum transfection frequencies were about 10(-7) transfectants per L2 DNA molecule and 10(-4) transfectants per CFU. When DNA was present in saturating amounts, the number of transfectants increased linearly with the number of CFU present in the transfection mixture, suggesting that DNA uptake does not occur by a mechanism involving cell fusion. The cleavage of the superhelical mycoplasma virus L2 genome with restriction endonucleases that cleave the DNA molecule once reduced the transfection frequency. Host cell modification and restriction of transfecting L2 DNA were similar to those for infecting L2 virions.     

Differential effects of physiological concentrations of retinoic acid in vitro on chondrogenesis and myogenesis in chick craniofacial mesenchyme

Recent studies have shown that in the developing limb bud retinoic acid is a skeletal morphogen at physiological levels, but a potent teratogen at higher levels. Retinoic acid has also been shown to be teratogenic during facial development, but very low levels may have an as yet unspecified role in normal development. In the present study the effects of retinoic acid on chondrogenesis and myogenesis by craniofacial cells grown in micromass cell culture were investigated. Retinoic acid, at concentrations of 0.01-100 ng/ml, was supplied to cells derived from day-4 (H.H stage 23/24) chick embryo mandibular, maxillary and frontonasal processes, grown in micromass cultures for 4 days in both serum-containing and defined media. Based on Alcian-blue-staining, concentrations of retinoic acid of 0.1-1 ng/ml were found to enhance chondrogenesis by mandibular cells grown in defined medium, while greater concentrations up to 100 ng/ml inhibited chondrogenesis. By contrast, chondrogenesis was generally retarded by all concentrations of retinoic acid applied to frontonasal cells grown in defined medium and when applied to both mandibular and frontonasal cells when grown in serum-containing medium. Cells from stage-23/24 maxillae did not display any significant chondrogenic activity in either medium under these culture conditions. Unlike chondrogenesis, myogenesis in mandibular, frontonasal and maxillary cultures was greater in defined than serum-containing medium, based on the appearance of immunologically detectable muscle myosin, and was reduced considerably less in defined medium by all concentrations of retinoic acid tested. In the presence of serum however, myogenesis was retarded with increasing concentrations of retinoic acid beyond 1 ng/ml in micromass cultures from all three facial regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mycoplasmal infection of insect cell cultures

Twenty-five cell cultures of three insect orders from eight laboratories were tested for mycoplasmal infection. Acholeplasma laidlawii was detected in one culture, an incidence of 4.0%. A. laidlawii, Mycoplasma orale, M. arginini, but not M. hyorhinis, could establish infections of drosophila Dm-1 cell cultures at 25 degrees C. In prospective studies, drosophila Dm-1 cultures were intentionally infected with broth-propagated A. laidlawii and M. hyorhinis. M. hyorhinis did not grow and was eliminated from the Dm-1 cultures during consecutive passages. A. laidlawii grew without obvious cytopathic effects during six weekly passages; titers of over 10(7) CFU/ml were recorded at Passages 2 and 5 (p2 and p5). Minimal cell culture infectious doses were also determined during these studies. 0.1 milliliter cell samples were inoculated into Leighton tubes containing either fresh M1A culture medium or 3T6 indicator cells in McCoy's 5a medium. After 4 d of incubation at 25 and 37 degrees C, respectively, the cover slips were stained by DNA fluorochrome Hoechst 33258 (A. laidlawii) or by specific fluorescein-conjugated antiserum (M. hyorhinis). At p2 with both mycoplasma species, the procedure using M1A medium and incubation at 25 degrees C without 3T6 cells was inferior to indicator cells. In five of six experiments at least a two-log higher titer of mycoplasmas was needed to be detected with M1A and 25 degrees C. At p5 no difference could be found. Uridine phosphorylase assays of Dm-1 cultures infected with A. laidlawii, M. hyorhinis, M. orale, and M. arginini gave clearly positive results only with A. laidlawii. The ratio of incorporated uridine to incorporated uracil method yielded false positives with two drosophila cell lines. Suggestions for assay of mycoplasmas in invertebrate cell cultures are given.     

Unadapted and adapted to starvation Acholeplasma laidlawii cells induce different responses of Oryza sativa, as determined by proteome analysis

For the first time, we studied the phytopathogenicity toward Oryza sativa L. of unadapted and adapted to unfavorable environment (starvation) cells of Acholeplasma laidlawii PG8--ubiquitous mycoplasma found in the soil, waste waters, tissues of the highest eukaryotes and being the basic contaminant of cell cultures and a causative agent of phytomycoplasmoses. The features of morphology, ultrastructural organization and proteomes of unadapted and adapted cells of the mycoplasma and infected plants were presented. Using 2D-DIGE and MS, 43 proteins of O. sativa L. that were differentially expressed in the leaves of plants cultivated in media with A. laidlawii PG8 were identified. The qualitative and quantitative responses of the plant proteome toward adapted and unadapted mycoplasma cells differed. That may be explained by differences in the virulence of the corresponding bacterial cells. Using 2D-DIGE and MS, 82 proteins that were differentially expressed in adapted and unadapted mycoplasma cells were detected. In adapted cells of the mycoplasma, in comparison with unadapted ones, a significant increase in the expression of PNPase--a global regulator of virulence in phytopathogenic bacteria occurred; there was also decreased expression of 40 proteins including 14 involved in bacterial virulence and the expression of 31 proteins including 5 involved in virulence was not detected. We propose that differences in the phytopathogenicity of adapted and unadapted A. laidlawii PG8 cells may be related to features of their proteomes and membrane vesicles.     

NUTRITIONAL REQUIREMENTS FOR OVULE FORMATION IN EXCISED PISTILS OF NIGELLA

The nutritional requirements for ovule formation in Nigella saliva L. were investigated by growing excised pistils on defined media. Pistils grown on a medium containing the minerals of Murashige and Skoog produced significantly more ovules than on a medium containing the minerals of Bilderback. When the nitrogen, sulfate, and phosphate of the Bilderback medium were adjusted to levels comparable to those of the Murashige and Skoog medium, a similar number of ovules was formed. The effect of different forms and concentrations of nitrogen on ovule formation and pistil growth was investigated. High concentrations of nitrate (40 mil) favored pistil growth and ovule formation, but comparable levels of ammonium were toxic. When ammonium at concentrations above 10 mM was added to nitrate media, ovule formation was inhibited. A medium containing low concentrations of ammonium (10 mM) and nitrate (5 mM) supported more ovule formation and pistil growth in young pistils than a low-nitrate (5 mM) medium without ammonium. However, ovule formation on a medium containing 10 mM ammonium and 5 mM nitrate was significantly less than on a medium containing only 15 mM nitrate. Low concentrations of organic nitrogen in the form of α-alanine (1 mM) and γ-aminobutyric acid (5 mM) supported ovule formation and pistil growth similar to a high nitrate medium.     

The turkey myogenic satellite cell: optimization of in vitro proliferation and differentiation

Myogenic satellite cells were isolated from the pectoralis major muscle of young growing tom turkeys. These cells were capable of proliferating and forming large multinucleated myotubes in vitro. Of 36 media-sera combinations evaluated, McCoy's 5A medium containing 15% chicken serum (CS) promoted the greatest level of proliferation and subsequent myotube formation when cells were induced to differentiate (P less than 0.05). Myotube formation was maximized following exposure of cultures to Dulbecco's Modified Eagle's Medium (DMEM) containing 1% horse serum (HS; DMEM-1% HS) for 4 days. Satellite cells grown under these conditions generally resulted in cultures containing greater than 90% fused nuclei. Cells plated in the presence of DMEM-10% HS resulted in greater attachment and larger cultures (and consequently a greater fused nuclei number) when transferred to growth media than similarly grown cultures plated in McCoy's 5A medium-10% CS, regardless of substrata tested (P less than 0.05). The greatest proliferation and myotube formation was seen in cultures grown in gelatin-coated wells. Proliferation was maximized in McCoy's 5A medium containing 18% CS, although this was not significantly different than the proliferation with media containing 15% CS (P greater than 0.05). Our results (1) document that the postnatal myogenic satellite cell can be isolated from the turkey in sufficient quantities for biological studies and (2) identify culture conditions which optimize proliferation and differentiation of these cells in vitro.