Differential inhibition by dikegulac of dividing and stationary cells in in vitro cultures

Dikegulac sodium 2,3:4,6 di-O-isopropylidene-2-keto-L-gulonate) is a translocatable agent used for chemically pinching terminal buds to break apical dominance. It does not inhibit mature leaves. Cell culture systems were developed and used to ascertain whether or not it differentially inhibits green tissue and whether or not it differentially inhibits stationary cells at the concentrations used. A differential inhibition of dividing cells was found.Abbreviation FDA fluorescein diacetate     

Dimorphism-associated changes in amino acid transport of Candida albicans

Abstract Amino acid uptake was followed during pH-regulated dimorphism of Candida albicans . It was observed that transport activities of various amino acids differed with the morphological phenotype. The uptake rates of l-alanine , l -phenylalanine and of l -lysine were lower and those of l -methionine were higher in elongated hypha (germ tube), while the rates of glycine, l -glutamic acid and l -proline were similar in bud and hyphal phenotypes. Minimum threshold of amino acids transport activity is required at the time of phenotypic commitment in a diverging population of Candida albicans .     

Hexose uptake by Catharanthus roseus cell suspensions is inhibited by a high medium salt content

Summary The uptake of glucose and fructose from the medium by Catharanthus roseus cell suspensions was strongly inhibited by high medium salt concentration, such as found in LS (Linsmaier and Skoog 1965) medium. After inoculation into standard LS nutrient medium with less than 5 mM hexose no uptake occurred, while in low salt medium hexose was completely depleted. At a hexose concentration of 50 mM the uptake rate was higher in low salt medium than in standard medium. The lower rate of uptake at high salt concentration was not the result of a pH or osmotic effect of the salts. Probably the affinity of the hexose carrier is affected by the ion concentration of the medium. The decrease in medium salt concentration during normal batch culture probably will have a considerable effect on hexose uptake.Abbreviations LS Linsmaier and Skoog - S sucrose - N mineral nitrogen - K K₂SO₄ - F fructose     

A microdialysis study in rat brain of dihydrokainate,a glutamate uptake inhibitor

Microdialysis in neostriatum of anaesthetized rats was performed to study effects on amino acid efflux of the glutamate uptake-inhibitor dihydrokainate (DHK). Both basal and K⁺-evoked (100 mM) efflux of glutamate increased in the presence of DHK. The increase in the basal glutamate efflux occurred at lower DHK concentrations than during K⁺-depolarization (when the extracellular glutamate concentration was several-fold higher), confirming that DHK is a competitive inhibitor. The increase in basal efflux caused by DHK did not exhibit Ca²⁺-dependency, whereas ∼50% of the increase in glutamate efflux during K⁺-depolarization was Ca²⁺-dependent. The Ca²⁺-dependent efflux is related to transmitter release, whereas the Ca²⁺-independent efflux is probably due to metabolic events and/or transport of DHK into cells in exchange for glutamate. Taurine efflux in response to DHK increased both during basal conditions and K⁺-depolarization, probably secondary to the increase in glutamate concentration, whereas aspartate, GABA, glutamine and alanine effluxes did not change.     

Selection for Highly Biased Amino Acid Frequency in the TolA Cell Envelope Protein of Proteobacteria

The bacterial cell envelope protein TolA functions to maintain the integrity of the cell membrane. This protein contains high levels of alanine and lysine that are used in the formation of alpha helices, which are required for normal protein function. The neutral model of molecular evolution predicts that amino acid composition and nucleotide composition are driven by the underlying GC content, as a result of mutation bias. However, this study shows that selection has acted to maintain high levels of alanine and lysine in the TolA protein of Proteobacteria, which in turn has biased nucleotide composition in the corresponding tolA gene.     

Chromosome variation in dividing protoplasts and cell suspensions of wheat

Summary The cytology of bread wheat (Triticum aestivum) suspension lines, recycled lines (selected for high division frequency) and their dividing protoplasts, have been examined. Extensive numerical and structural chromosome variation was present in all the lines. The most frequently observed chromosome numbers were around 2n=32, indicating that considerable chromosome loss from the normal wheat complement (2n=6x=42) had occurred during selection of the lines. Chromosome aberrations also indicated loss of chromosome arms and chromosome segments. The implications of this variation for studies on transformation and for the potential regeneration of whole plants from protoplasts of bread wheat are discussed.     

Monolayer culture of parenchymal rat hepatocytes on collagen-coated microcarriers. A hepatocyte system for short- and long-term metabolic studies

Summary A method is described for the attachment to and monolayer culture of adult rat hepatocytes on collagen-coated or fibronectin-coated microbeads or both in a chemically defined serum-free medium. Protein synthesis measured by the incorporation of [³H]leucine into protein was four-fold higher in the hepatocyte microcarrier cultures than in isolated hepatocyte suspensions. The hepatocyte microcarrier cultures showed acute responsiveness to insulin of fatty acid synthesis, glucose incorporation into glycogen, and decarboxylation of [1-¹⁴C]pyruvate. Microcarrier-cultured hepatocytes have the combined advantages of monolayer culture and suspension systems. They are a potential tool for the study of long-term as well as acute effects of hormones. This work was supported by the British Diabetic Association.     

Effect of Escherichia coli lipopolysaccharide on the microviscosity of liver plasma membranes and hepatocyte suspensions and monolayers

The fluorescence probe 1,6-diphenylhexa-1,3,5-triene (DPH) was used for monitoring structural perturbations induced by lipopolysaccharide (LPS) of Escherichia coli (0111:B4) in plasma membranes of rat liver. Changes in microviscosity were observed in plasma membrane preparations from control rats after treatment with LPS and in plasma membrane preparations from liver perfused with LPS. In both systems fluorescence polarization was measured from which microviscosity was calculated. This parameter increases with LPS treatment. From temperature dependence studies was inferred that LPS interaction with plasma membrane preparations induces an increase of both the polarization term (r0/r-1)-1 and flow activation energy (delta E). Addition of LPS to hepatocyte suspensions also induces an increase on microviscosity and a delay in the fall of microviscosity induced by a temperature rise in hepatocyte monolayers grown on microcover slides. These data suggest that LPS interaction can be attributed to its binding to membrane hydrophobic regions in a non-specific manner.     

The influence of some inhibitors on the formation of caffeic acid in cultures ofPerilla cell suspensions

A cell suspension culture, prepared fromPerilla frutescens var.crispa callus induced by Murashige and Skoog (1962) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 ml/l) and kinetin (0.1 mg/l), contained caffeic acid derivatives as the phenolic components. Fresh and dry weights of the cells increased exponentially for about 11 days after transfer to a fresh medium. The contents of caffeic acid and protein also reached a maximum on the 11th day, but α-amino nitrogen phenylalanine and tyrosine continued to increase in amount until the 20th to 23rd day. Caffeic acid formation in the cells was increased by lowering the concentration of 2,4-D. The administration ofl-2-aminooxy-3-phenylpropionic acid (l-AOPP), 2-aminooxyacetic acid (AOA) andN-(phosphonomethyl)glycine (glyphosate) to the cells inhibited caffeic acid formation to a large extent. An 80% inhibition of caffeic acid formation was caused by 10⁻⁴Ml-AOPP whereas phenylalanine and tyrosine contents of the cells became 7.5 and 2.3 times higher at thisl-AOPP concentration than those in the control. An 85% inhibition of caffeic acid formation was achieved at 10⁻³M glyphosate concentration, while 10⁻³M AOA inhibited caffeic acid formation by 95% and also growth rate by 80%. The influence of inhibitors on caffeic acid formation is discussed in relation to the level of α-amino nitrogen, particularly aromatic amino acids, in the cell suspension cultures.     

Characterization of a secondary uptake system for l-glutamate in Corynebacterium glutamicum

Abstract A new transport system for the uptake of l-glutamate was characterized in Corynebacterium glutamicum strain Δ glu, in which the previously described binding protein-dependent glutamate uptake system is not present. Kinetic characterization revealed a highly specific secondary transport system, dependent on sodium ions. Glutamate uptake showed Michaelis-Menten kinetics, with a K _m of 0.6 mM and a V _max of 15 nmol min⁻¹ (mg dw)⁻¹. For the co-transported sodium ions, a relatively low K _m of 3.3 mM was determined.     

Chemical Components of Cell Cultures of Arnebia euchroma

Five naphthaquinones were isolated from the petroleum ether extract of the cell cultures of Arnebia euchroma (Royle) Johnst.. Their structures were identified as deoxyshikonin, β,β-dimethylacrylshikonin, acetylshikonin, teracrylshikonin and β-hydroxyisovalerylshikonin by means of chemical methods, HPLC, IR and 1H-NMR data respectively. There was no shikonin in the cell cultures, which was obtained from the roots of A. euchroma. The contents of each compound in the cell cultures are different from those in the roots of A. euchroma.     

Effects of Free Fatty Acids on Synaptosomal Amino Acid Uptake Systems

Abstract: The Na⁺-dependent synaptosomal uptakes of proline, aspartic acid, glutamic acid, and γ-aminobutyric acid were strongly inhibited by monounsaturated fatty acids. With oleic acid, half-maximal inhibition was observed at about 15 μM. The Na⁺-independent uptakes of leucine, phenylalanine, histidine, and valine were less sensitive to inhibition by the unsaturated fatty acids. In contrast, the uptakes of all of these amino acids were unaffected by saturated fatty acids. The inhibition of proline uptake (and that of the other Na⁺-dependent amino acids) by oleic acid was overcome by the addition of serum albumin and the data presented further indicate that the previously reported stimulation of proline uptake by albumin could be related to its fatty acid binding properties.     

High Density Cultivation of Panax notoginseng Cell Cultures with Methyl Jasmonate Elicitation in a Centrifugal Impeller Bioreactor

True ginseng roots contain “active compounds” called ginsenosides. The enhanced production of useful bioactive ginsenosides by high‐density cell cultures of Panax notoginseng in a self‐developed centrifugal impeller bioreactor (CIB) was achieved by adding methyl jasmonic acid (MJA) during cultivation. The production of the major, individual ginsenosides Rg₁, Re and Rb₁ was significantly enhanced in both 3‐L and 30‐L CIBs. The production titer of Rg₁, Re and Rb₁ ginsenosides in the 30‐L CIB was improved from 42 ± 8, 42 ± 9 and 41 ± 6 mg/L without MJA elicitation, to 104 ± 6, 71 ± 5 and 95 ± 6 mg/L with MJA elicitation, respectively. The ratio of Rb/Rg was slightly improved by MJA treatment in a 3‐L CIB but no apparent difference was observed in a 30‐L CIB. This work is useful for the understanding of the effects of large‐scale production on the individual ginseng saponins produced by plant cell cultures     

Cell Cycle-Specific Requirement for Mevalonate, but Not for Cholesterol, for DNA Synthesis in Glial Primary Cultures

The requirement for the sterol biosynthetic pathway for the occurrence of DNA synthesis in glial cells and, in particular, the relative roles of cholesterol and of mevalonate have been studied. Primary cultures of developing glial cells were synchronized by reducing the content of fetal calf serum (FCS) in the culture medium from 10% to 0.1% (vol/vol) for 48 h between days 4 and 6 in culture. Reversal of the resulting quiescent state by the return of the cultures to 10% serum caused after 24 h a marked increase in DNA synthesis, and this increase was prevented by the simultaneous addition of mevinolin, a specific inhibitor of the sterol biosynthetic pathway at the 3-hydroxy-3-methylglutaryl coenzyme A reductase step, at the time of serum repletion. A dose-dependent reversal of the mevinolin inhibition of DNA synthesis occurred with simultaneous addition of mevalonate to the culture medium. The induction of DNA synthesis by serum repletion, its inhibition by mevinolin, and the reversal of the inhibition by mevalonate were unaffected by a 95% reduction in exogenous cholesterol produced by utilization of lipoprotein-poor serum (LPPS) rather than FCS. Similarly, return of quiescent cultures to 10% LPPS containing mevinolin and sufficient low-density lipoprotein (LDL) to raise the cholesterol concentration 80-fold failed to restore DNA synthesis. In addition, reversal of the mevinolin inhibition of DNA synthesis by mevalonate occurred despite the continuous presence of mevinolin if mevalonate was added as late as 12 h after serum repletion, but not if added after 16 h or more.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of Na+-Dependent, Veratridine-Sensitive Synaptosomal Amino Acid Uptakes by Serum Albumin

Abstract: Bovine serum albumin (BSA) is shown to stimulate selectively the synaptosomal uptakes of those amino acids that are dependent on external Na⁺ and that are inhibited by veratridine. Thus, the stimulation can be seen in the case of aspartic acid, glutamic acid, glycine, proline, and γ-aminobutyric acid, but not with serine and threonine. Further, studies on the interaction of veratridine, valinomycin, and BSA on the uptake of proline suggest that the primary action of the albumin is to increase the influx of proline. Such an action could result as a consequence of stabilization of the Na⁺ gradient by increased endogenous levels of ATP. Intrasynaptosomal ATP was increased in the presence of BSA but significantly decreased by veratridine.     

Sodium-Dependent Proline Uptake in the Rat Hippocampal Formation: Association with Ipsilateral-Commissural Projections of CA3 Pyramidal

Na+-dependent uptake of L-[3H]proline was measured in a crude synaptosomal preparation from the entire rat hippocampal formation or from isolated hippocampal regions. Among hippocampal regions, Na+-dependent proline uptake was significantly greater in areas CA1 and CA2-CA3-CA4 than in the fascia dentata, whereas there was no marked regional difference in the distribution of Na+-dependent gamma-[14C]aminobutyric acid ([14C]GABA) uptake. A bilateral kainic acid lesion, which destroyed most of the CA3 hippocampal pyramidal cells, reduced Na+-dependent proline uptake by an average of 41% in area CA1 and 52% in area CA2-CA3-CA4, without affecting the Na+-dependent uptake of GABA. In the fascia dentata, neither proline nor GABA uptake was significantly altered. Kinetic studies suggested that hippocampal synaptosomes take up proline by both a high-affinity (KT = 6.7 microM) and a low-affinity (KT = 290 microM) Na+-dependent process, whereas L-[14C]glutamate is taken up predominantly by a high-affinity (KT = 6.1 microM) process. A bilateral kainic acid lesion reduced the Vmax of high-affinity proline uptake by an average of 72%, the Vmax of low-affinity proline uptake by 44%, and the Vmax of high affinity glutamate uptake by 43%, without significantly changing the affinity of the transport carriers for substrate. Ipsilateral-commissural projections of CA3 hippocampal pyramidal cells appear to possess nearly as great a capacity for taking up proline as for taking up glutamate, a probable transmitter of these pathways. Therefore proline may play an important role in transmission at synapses made by the CA3-derived Schaffer collateral, commissural, and ipsilateral associational fibers.