A study of the fatty acid metabolism of the yeast Pityrosporum ovale

The yeast Pityrosporum ovale, a skin saprophyte, will only grow if fatty acids of chain length greater than C(10) are added to the culture medium. 9-Hydroxypalmitic acid is the major product of metabolism of even-carbon-number fatty acids; 9-hydroxystearic acid is also found. The optimum pH for this conversion is pH4.5. The hydroxy fatty acids produced are found bound in a polar form in the aqueous phase of the culture medium. Growth of the organisms is facilitated by presentation of the substrate as a two-phase liquid system.     

Factors Influencing the Enhancement of the Infectivity of Poliovirus Ribonucleic Acid by Diethylaminoethyl-Dextran

The enhancement by diethylaminoethyl-dextran (DEAE-D) of the infectivity of poliovirus ribonucleic acid (RNA) for cell cultures was demonstrated by infective-center as well as by plaque assays, both in nonprimate (L) and primate cell systems (MK, HeLa, LLC-MK(2)). The sensitivity of plaque assays was greatly improved by using a tris (hydroxymethyl)aminomethane-buffered synthetic medium (basal medium Eagle) and freshly confluent cell monolayers. Enhancement of nucleic acid infectivity was directly dependent on the molecular weight of the DEAE-D. Two observations bearing on the action of DEAE-D appeared important: ribonuclease activity was reduced by DEAE-D, and cells pretreated with DEAE-D remained susceptible to infection with RNA in isotonic medium. Appreciable susceptibility of the treated cells persisted for at least 2 hr; the susceptible state could be reversed at will by an application of heparin. Enhancement of nucleic acid infectivity was independent of an effect of DEAE-D on intact virus and agar inhibitors.     

Molecular cloning and nucleotide sequence of cDNA encoding the entire precursor of rat liver medium chain acyl coenzyme A dehydrogenase

cDNA encoding the precursor of rat liver medium chain acyl-CoA dehydrogenase (EC 1.3.99.3) was cloned and sequenced. The longest cDNA insert isolated was 1866 bases in length. This cDNA encodes the entire protein of 421-amino acids including a 25-amino acid leader peptide and a 396-amino acid mature polypeptide. The identity of the medium chain acyl-CoA dehydrogenase clone was confirmed by matching the amino acid sequence predicted from the cDNA to the NH2-terminal and nine internal tryptic peptide sequences derived from pure rat liver medium chain acyl-CoA dehydrogenase. The calculated molecular masses of the precursor medium chain acyl-CoA dehydrogenase, the mature medium chain acyl-CoA dehydrogenase, and the leader peptide are 46,600, 43,700, and 2,900 daltons, respectively. The leader peptide contains five basic amino acids and only one acidic amino acid; thus, it is positively charged, overall. Cysteine residues are unevenly distributed in the mature portion of the protein; five of six are found within the NH2-terminal half of the polypeptide. Comparison of medium chain acyl-CoA dehydrogenase sequence to other flavoproteins and enzymes which act on coenzyme A ester substrates did not lead to unambiguous identification of a possible FAD-binding site nor a coenzyme A-binding domain. The sequencing of other homologous acyl-CoA dehydrogenases will be informative in this regard.     

Improved medium for clonal growth of human diploid fibroblasts at low concentrations of serum protein.

A new medium (MCDB 104) has been developed which will support clonal growth of WI-38 cells at concentrations of serum protein as low as 25 micrograms per ml (equivalent to 0.05% serum). The principal factors responsible for reduction of the protein requirement are: (a) adjustment of all nutrient concentrations in medium F12 to experimentally determined optimum values for WI-38 cells; (b) supplementation with trace elements; (c) replacement of hypoxanthine and folic acid with adenine and folinic acid; and (d) coating of the culture surface with polylysine. Individually, many of these modifications exert only a small effect on cellular growth at reduced protein concentrations, but collectively their effect has been very substantial. Other strains of fibroblast-like human diploid cells from amniotic fluid, fetal lung and newborn foreskin also will grow at reduced concentrations of serum protein in the new medium.     

D-valine as a selective agent for normal human and rodent epithelial cells in culture.

A nutrient medium has been developed to enable the growth of normal epithelial cells while selectively inhibiting fibroblast proliferation. In this medium, D-valine is substituted for L-valine; and only those cells containing D-amino acid oxidase can convert the D-amino acid into its essential L-enantiomer. The ability to select for cells with this enzyme has enabled us to maintain epithelial cell populations free from fibroblast overgrowth. The presence of D-amino acid oxidase has been histochemically confirmed in the epithelial cells selected from renal cell suspensions and explants. The ability to proliferate in the selective medium is transmitted to the clonal progeny of these cells. Moreover, epithelial cell proliferation of this medium indicates the presence of D-amino acid oxidase, which we have detected in tissues where it had not previously been reported-fetal human kidney, lung, and cord. Fibroblasts will not grow in the selective medium, but will proliferate normally if the product of the D-amino acid oxidase reaction is supplied.     

Regeneration of plants from leaf explants of micropropagated clonal Eucalyptus grandis

Summary Callus production along with caulogenesis was obtained from leaf explants of micropropagated clonal Eucalyptus grandis after six to twelve weeks of culture. Out of eight clones tested, six were amenable to shoot production using simple media containing naphthaleneacetic acid and either 6-benzyladenine or zeatin. Differences in growth regulator requirements for organogenesis were observed between different clones. These shoots were then elongated on a medium containing gibberellic acid and rooted using media derived from the micropropagation medium. Light conditions were also found to be important for regeneration. This protocol is the first published on regeneration from nonseedling material and it will facilitate the Agrobacterium tumefaciens -mediated transformation of selected clonal Eucalyptus grandis.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA naphthaleneacetic acid - PAR photosynthetically active radiation - PVP polyvinyl pyrrolidone - SH Schenk and Hildebrandt (1972) medium     

Phage formation in Staphylococcus muscae cultures. VIII. Effect of the protein factor and aspartic acid on virus synthesis with various bacterial strains

1. Four strains of Staphylococcus muscae have been isolated which differ in their growth rates and phage syntheses in Fildes' synthetic medium. 2. Two of the strains when singly infected cannot release phage in Fildes' synthetic medium unless a substance present in certain acid-hydrolyzed proteins is added to the medium. One of these strains also requires other substance(s) present in acid-hydrolyzed proteins in order to grow in Fildes' medium. 3. The two strains which do not require the addition of the phage-stimulating factor have been found either to synthesize this substance, or one similar to it. One of these strains will not grow in Fildes' medium unless substance(s) present in acid-hydrolyzed proteins is added to the medium. 4. The purified acid-hydrolyzed protein factor necessary for virus liberation does not affect the multiplication rate of uninfected S. muscae cells in Fildes' synthetic medium. 5. The substance is not needed for the adsorption or the invasion of the host cell by the virus. In the absence of the factor, the virus is adsorbed to the cell and "kills" it. 6. An analysis carried out by means of the one-step growth curve technique has indicated that the substance is not concerned simply with the mechanism of virus release, but is necessary for some initial stage in virus synthesis. 7. With one bacterial strain not requiring the AHPF, aspartic acid had to be present at least during the minimum latent period for the cell to form virus. 8. In the absence of aspartic acid, the virus was adsorbed to the cell and killed it, but no virus was released from singly infected bacteria. 9. If the cells were grown in a medium containing aspartic acid and then resuspended in the medium minus aspartic acid, no virus was released, although such cells contained at least two times the amount of aspartic acid necessary for the burst size in the complete medium. 10. Aspartic acid, a constituent of the virus particle, appears from an analysis of one-step growth curves to take part in the initial phase of phage synthesis. 11. The effect of amino acids on virus formation is discussed in relation to the time sequence of virus protein and desoxyribonucleic acid synthesis.     

Batch fermentation of whey ultrafiltrate by Lactobacillus helveticus for lactic acid production

Summary Cheese whey ultrafiltrate (WU) was used as the carbon source for the production of lactic acid by batch fermentation with Lactobacillus helveticus strain milano. The fermentation was conducted in a 400 ml fermentor at an agitation rate of 200 rpm and under conditions of controlled temperature (42° C) and pH. In the whey ultrafiltrate-corn steep liquor (WU-CSL) medium, the optimal pH for fermentation was 5.9. Inoculum propagated in skim milk (SM) medium or in lactose synthetic (LS) medium resulted in the best performance in fermentation (in terms of growth, lactic acid production, lactic acid yield and maximum productivity of lactic acid), as compared to that propagated in glucose synthetic (GS) medium. The yeast extract ultrafiltrate (YEU) used as the nitrogen/growth factor source in the WU medium at 1.5% (w/v) gave the highest maximum productivity of lactic acid of 2.70 g/l-h, as compared to the CSL and the tryptone ultrafiltrate (TU). L. helveticus is more advantageous than Streptococcus thermophilus and Lactobacillus delbrueckii for the production of lactic acid from WU. The L. helveticus process will provide an alternative solution to the phage contamination in dairy industries using Lactobacillus bulgaricus.     

Improved medium for clonal growth of human diploid fibroblasts at low concentrations of serum protein

Summary A new medium (MCDB 104) has been developed which will support clonal growth of WI-38 cells at concentrations of serum protein as low as 25 μg per ml (equivalent to 0.05% serum). The principal factors responsible for reduction of the protein requirement are: (a) adjustment of all nutrient concentrations in medium F12 to experimentally determined optimum values for WI-38 cells; (b) supplementation with trace elements; (c) replacement of hypoxanthine and folic acid with adenine and folinic acid; and (d) coating of the culture surface with polylysine. Individually, many of these modifications exert only a small effect on cellular growth at reduced protein concentrations, but collectively their effect has been very substantial. Other strains of fibroblast-like human diploid cells from amniotic fluid, fetal lung and newborn foreskin also will grow at reduced concentrations of serum protein in the new medium. This work was supported by Grant No. AG00310 from the National Institute on Aging, and by Contract No. 223-74-1156 from the Bureau of Biologics, U.S. Food and Drug Administration.     

Release of free fatty acids from Ehrlich ascites tumor cells

Ehrlich ascites tumor cells release free fatty acids (FFA) during in vitro incubation in media that contain albumin. The released FFA are derived by lipolysis from endogenous lipid esters. Addition of glucose to the incubation medium greatly decreases the quantity of fatty acid released by the cells. Cyanide, which inhibits endogenous lipid oxidation but not lipolysis, increases the quantity of fatty acid released to media containing albumin and causes free fatty acid to accumulate in the cells in the absence of exogenous albumin. The release of fatty acid, either preformed or derived by lipolysis during prolonged incubations, occurs under conditions of net fatty acid uptake from the incubation medium. Net release of fatty acid from the cell occurs only when fatty acid-extracted albumin is present in the extracellular medium; extrapolation of the data suggests that net release will not occur under physiological conditions. It is postulated that free fatty acid uptake and release are independent processes, the direction of net fatty acid movement being determined by the relationship between cellular free fatty acid concentration (regulating efflux) and the molar ratio of free fatty acid to albumin in the extracellular medium (regulating uptake).     

Micropropagation and seed cryopreservation of the critically endangered species Tennessee yellow-eye grass, Xyris tennesseensis Kral

Xyris tennesseensis is a critically endangered species native to the southeastern USA. A micropropagation protocol was developed which may assist in the safeguarding and augmentation of dwindling natural populations of this ecologically and medically valuable plant. Four different batches of seeds were sterilized using hydrogen peroxide and germinated in vitro on modified one third-strength Murashige and Skoog medium. Shoot multiplication from seedling tissue was obtained using modified one third-strength Murashige and Skoog medium containing 1?mg/l kinetin and 0.1?C0.5?mg/l ??-naphthaleneacetic acid. Optimal shoot size and sustainable multiplication rates of three to five per 2-mo subculture occurred on medium containing 0.3?C0.4?mg/l ??-naphthaleneacetic acid. Shoots rooted successfully when placed on growth regulator-free medium for 10?d followed by transfer to greenhouse soil under high humidity. Use of seed cryopreservation resulted in significant increases in germination compared to control treatments with average germination rates of 97%. Shoot tip cultures from soil-grown plants of X. tennesseensis and Xyris spathifolia were also developed using the above protocols. Plant tissue culture tools will assist in the multiplication, long-term storage, and conservation of these rare and valuable plants as well as provide a template for the micropropagation of other Xyris species.     

A simple cultural method for the presumptive detection of the yeasts Brettanomyces/Dekkera in wines

AIMS: The development of a simple and reliable procedure, compatible with routine use in wineries, for the presumptive detection of Brettanomyces/Dekkera from wine and wine-environment samples. METHODS AND RESULTS: The method of detection of these yeasts employs a selective enrichment medium. The medium contains glucose (10 g l(-1)) as carbon and energy source, cycloheximide (20 mg l(-1)) to prevent growth of Saccharomyces, chloramphenicol (200 mg l(-1)) to prevent growth of bacteria and p-coumaric acid (20 mg l(-1)) as the precursor for the production of 4-ethyl-phenol. After the inoculation with wine, the medium is monitored by visual inspection of turbidity and by periodic olfactive analysis. Contaminated wines will develop visible turbidity in the medium and will produce the 4-ethyl-phenol off-odour, which can be easily detected by smelling. CONCLUSIONS: A selective enrichment liquid medium was developed to differentially promote the growth and activity of Brettanomyces/Dekkera. The method is simple to execute, employing a simple-to-prepare medium and a periodic olfactive detection. SIGNIFICANCE AND IMPACT OF THE STUDY: The characteristics of the procedure make it particularly applicable in a wine-making environment thus presenting important advantages to the wine industry.     

Comparison of the nutritional value of low and medium tannin sorghum grains for pigs

The effect of sorghum grain of low (0.32%) and medium (0.94%) tannic acid content was studied in a growth experiment on Large White pigs. Medium tannin sorghum in pig diets had no significant adverse effect on growth, back fat thickness or feed conversion efficiency, but it was associated with a trend towards lower growth rate. All-sorghum diets, with either low or medium tannin, resulted in a significantly inferior feed conversion ratio compared to the wheat control. It was concluded that sorghum grain with up to about 0.94% tannic acid can be used as the main or sole grain in grower pig diets without the risk of any significant adverse effect on growth or back fat thickness.The production of medium tannin sorghum grain in bird-infested areas will depend on the extent to which pig producers would be prepared to use it in pig diets.     

Rapid clonal propagation of Saccharum officinarum L. Vars. CO-6907 and CO-86249 and to assess the genetic uniformity through molecular markers

Abstract

An efficient protocol was developed for in vitro clonal propagation of Saccharum officinarum Vars. CO-6907 and CO-86249 through axillary meristem culture. Maximum meristem elongation was achieved on Murashige and Skoog's (MS) medium supplemented with 0.5 mg/L 6-benzyladenine (BA) and 0.5 mg/L kinetin (Kn) within 15 days of culture. Multiple shoots were induced from meristems on MS basal medium supplemented with 1.0 mg/L BA, 0.5 mg/L Kn, 0.25 mg/L 1-napthaleneacetic acid (NAA) and 3% (w/v) sucrose. Addition of 0.1–0.25 mg/L gibberellic acid into the multiplication medium found the better shoot elongation. Repeated subculture on multiplication medium induces higher rate of shoot multiplication. The root induction from excised microshoots was achieved on half-strength MS medium supplemented with 1.0–2.0 mg/L NAA or indole-3-butyric acid and 6% (w/v) sucrose. While either decreasing or increasing of sucrose concentration in the rooting medium, the percentage of rooting was reduced. Maximum percentage of rooting was achieved on medium having 2.0 mg/L NAA with 6% (w/v) sucrose. About 80% of micropropagated plantlets were hardened in the greenhouse and successfully established in the soil. Random Amplified Polymorphic DNA marker was used to detect the variability among the micropropagated plants developed through in vitro. The results showed that there was no polymorphism among the micropropagated plants. This study will help for propagation of quality planting material of high-yielding variety of sugarcane for commercialization.     

Plantlets from somatic callus tissue of the East Indian Rosewood (Dalbergia latifolia Roxb.)

Callus-mediated shoot bud formation was demonstrated in Dalbergia latifolia Roxb. (East Indian Rosewood). Cultures were raised from shoot explants of six year-old plants on Murashige and Skoog (MS) medium supplemented with naphthaleneacetic acid (NAA) and benzyladenine (BA). A sequential treatment of callus with increasing BA levels and decreasing NAA ensured shoot bud induction. Rooting of shoots was achieved by a three-step culture procedure involving 1) White's(W) liquid medium containing indoleacetic acid (IAA), naphthaleneacetic acid and indolebutyric acid (IBA), 2) half-strength MS agar-solidified medium with charcoal (0.25%) and 3) half-strength MS liquid medium.Abbreviations BA Benzyladenine - IAA Indoleacetic acid - IBA Indolebutyric acid - MS Murashige and Skoog - NAA a-naphthaleneacetic acid - PVP Polyvinylpyrrolidone - W White's medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

Stereospecific distribution of plamitic acid in the triacylglycerols of rat adipocytes. Effects of varying the composition of the substrate fatty acid in vitro

The effects of inclusion of different fatty acids in the medium on the rate of esterification of palmitic acid and its stereospecific distribution among the three positions of the triacyl-sn-glycerols by preparations of rat adipocytes in vitro have been determined. Myristic acid, stearic acid, oleic acid and linoleic acid were used as diluents and the concentration of the combined unesterified fatty acids in the medium was held constant; only the proportion of palmitic acid was varied. The amount of palmitic acid esterified was always linearly related to its relative concentration in the medium and was not significantly affected by the nature of the diluent fatty acid chosen. Constant relative proportions were recovered in triacylglycerols and in intermediates in each instance. The amount of palmitic acid esterified to each of the positions of the triacyl-sn-glycerols was linearly dependent on the relative proportion in the medium but the nature of the relationship was markedly influenced by which fatty acid was present. When stearic acid was present, simple relationships were found over the whole range tested. When either myristic acid, oleic acid or linoleic acid was present, abrupt changes in the manner of esterification of palmitic acid were observed in position sn-1 when the relative concentrations of palmitic acid and the diluent reached critical values, which differed with each fatty acid. In position sn-2 when oleic acid or linoleic acid was present, a similar change was observed, and in position sn-3 it was obtained with myristic acid as diluent. The results are discussed in terms of changes in the relative affinities of the acyltransferases for palmitic acid. Palmitic acid was esterified into various molecular species in proportions that indicated acylation with non-correlative specificity at higher relative concentrations but not at lower.     

Rapid and repetitive plant regeneration in sweetpotato via somatic embryogenesis

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and petiole expiants has been developed in sweetpotato [Ipomoea batatas L. (Lam.)]. The optimal somatic embryogenic response was obtained in the genotype PI 318846-3 with a two-step protocol: (1) stage I-incubation of expiants in the dark for 2 weeks on Murashige Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5 mg/l) and 6-benzylaminopurine (0.25 mg/l) and, (2) stage II-culture in the light on MS medium with abscisic acid (ABA) (2.5 mg/l). The addition of ABA was critical for enhanced production of somatic embryos. Secondary somatic embryos were produced from the primary embryos cultured on MS medium with 2,4-D at 0.2 mg/l. The somatic embryos were converted into normal plantlets when cultured on basal MS medium. Upon transfer to soil, plants grew well and appeared normal with no mortality. The system of somatic embryogenesis described here will facilitate tissue culture, germplasm conservation and gene transfer research of sweetpotato due to its rapidity (6 to 10 weeks), prolific plant production by direct embryogenesis, ease of secondary somatic embryo production and reproducibility.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine, 2,4-D-2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - KIN kinetin - MS medium of Murashige and Skoog (1962) - NAA 1-naph-thaleneacetic acid - PIC picolinic acid - TDZ thidiazuron     

Permanent Mounting of Unstained and Aceto-Orcein Stained Cells in the Water-Soluble Medium,Abopon

For cellular morphology, mammalian cells were grown on cover slips in Leigh ton tubes, fixed in 1% osmic acid vapor for 2 min, decolorized with 30% H₂O₂ in 5% ammonium oxalate solution (1:7) for 2 min, then washed thoroughly, and finally mounted in a water-soluble medium consisting of a saturated solution of Abopon in 0.2 M phosphate buffer, pH 7.0. For chromosomal analysis of similarly cultured cells, aceto-orcein preparations were made by conventional methods, with the following minor modifications: following pretreatment with colchicine and hypotonic expansion, the cells on the cover slips were fixed in acetic-alcohol (1:3), air dried, incubated at 37° C for 15 min in 2% orcein in 45% acetic acid, rinsed in 45% acetic acid, washed several times in distilled water, and finally mounted in Abopon mounting medium. Both kinds of preparations were allowed to harden for 24 hr before being handled. Such slides will keep for years at room temperature. Studies requiring frequent comparisons of cellular and chromosomal morphology of cultured cells can thus be extended over long periods of time.     

Effect of potassium chloride on the uptake and storage of phosphate by Saccharomyces mellis

The inorganic and polyphosphate pools of Saccharomyces mellis, grown in a medium containing excess phosphate, remain associated with the cells when the cells are suspended in a saline medium. If the cells are incubated in a medium containing 2 m KCl, the cells are altered in some manner which permits most of the orthophosphate and approximately one-third of the polyphosphate to be subsequently eluted by osmotic shock. At lower salt concentrations, beta-mercaptoethanol enhances this salt effect but is inactive by itself in this respect. At equivalent ionic strengths, the sodium salt of ethylenediaminetetraacetic acid behaves exactly like KCl or any other monovalent ionic compound in altering the cell to susceptibility to osmotic shock. No special effect of this anion at either high or low concentration could be detected. Resting cells are refractory to being altered in this manner by salts if an energy source, such as glucose, is included in the reaction mixture. Cells which are depleted of phosphate reserves will immediately incorporate phosphate when suspended in a medium containing inorganic phosphate and an energy source. These cells exhibit the phenomenon of "überkompensation." In resting cells, the inclusion of KCl in the reaction mixture prevents the conversion of orthophosphate into polyphosphate and, also, gradually decreases the ability of the organism even to assimilate orthophosphate. This effect is reversible, however, since the cells will incorporate phosphate in a normal manner if the cells are transferred to a non-salinized medium, or if a nitrogen source is included in the salinized reaction mixture so that the cells are now in a medium adequate for growth.     

Relationship between pH and medium dissolved solids in terms of growth and metabolism of lactobacilli and Saccharomyces cerevisiae during ethanol production

The specific growth rates of four species of lactobacilli decreased linearly with increases in the concentration of dissolved solids (sugars) in liquid growth medium. This was most likely due to the osmotic stress exerted by the sugars on the bacteria. The reduction in growth rates corresponded to decreased lactic acid production. Medium pH was another factor studied. As the medium pH decreased from 5.5 to 4.0, there was a reduction in the specific growth rate of lactobacilli and a corresponding decrease in the lactic acid produced. In contrast, medium pH did not have any significant effect on the specific growth rate of yeast at any particular concentration of dissolved solids in the medium. However, medium pH had a significant (P < 0.001) effect on ethanol production. A medium pH of 5.5 resulted in maximal ethanol production in all media with different concentrations of dissolved solids. When the data were analyzed as a 4 (pH levels) by 4 (concentrations of dissolved solids) factorial experiment, there was no synergistic effect (P > 0.2923) observed between pH of the medium and concentration of dissolved solids of the medium in reducing bacterial growth and metabolism. The data suggest that reduction of initial medium pH to 4.0 for the control of lactobacilli during ethanol production is not a good practice as there is a reduction (P < 0.001) in the ethanol produced by the yeast at pH 4.0. Setting the mash (medium) with > or =30% (wt/vol) dissolved solids at a pH of 5.0 to 5.5 will minimize the effects of bacterial contamination and maximize ethanol production by yeast.