The interaction of magnesium ions with teichoic acid.

The binding of Mg2+ to the wall teichoic acid of Lactobacillus buchneri N.C.I.B. 8007 was measured by equilibrium dialysis at controlled ionic concentration and pH. In an aqueous solution containing 10mM-NaCl at pH 5.0 one Mg2+ ion was bound for every two phosphate groups of the teichoic acid, with an apparent association constant, Kassoc. = 2.7 x 10(3) M-1. On lowering the pH below the pKa of the phosphate groups the amount of bound Mg2+ decreased concomitantly with decreasing ionization of the phosphate groups. Both the amount of Mg2+ bound to the teichoic acid and the apparent association constants were similar in the presence of 10 mM concentrations of NaCl or KCl but decreased markedly in the presence of 10 mM-CaCl2 because of competition between Ca2+ and Mg2+ for the binding sites. A similar effect was found when the concentration of NaCl was increased from 0 to 50 mM. The results are discussed in relation to the function of teichoic acid in the walls of Gram-positive bacteria.     

Insulin release from a cloned hamster B-cell line (HIT-T15). The effects of glucose, amino acids, sulphonylureas and colchicine

Insulin release from statically incubated HIT-T15 cells was maximally stimulated by glucose, L-arginine and L-leucine. L-arginine stimulated insulin release in the absence of glucose. Glucose induced insulin release was potentiated by the addition of L-leucine, L-arginine and the two in combination. Both glibenclamide and chlorpropamide stimulated insulin release from HIT-T15 cells. Glibenclamide was the more potent and equivalent in insulinotrophic action to 7.5 mmol/l glucose. Only chlorpropamide significantly potentiated glucose induced insulin release. Perifused HIT-T15 cells produced a reproducible biphasic insulin response to glucose challenge which was characterised by a pronounced and sustained first phase and a reduced second phase. The stimulation of phase I by glibenclamide alone and the inhibition of phase II of glucose induced insulin release by colchicine suggested the presence of a readily available pool of insulin granules which was not rapidly restored by insulin biosynthesis and granule margination.     

Resolution of pea legumin subunits by high-performance liquid chromatography

Pea legumin was dissociated into its component subunits by 6 M urea: these were subsequently fractionated by FPLC using a combination of Mono P, Mono Q, and Mono S columns. The resolution and speed of separation were greatly improved in comparison with previous fractionations. Twelve discrete fractions were obtained and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six "normal" legumin subunits (Mr 60,000) were identified as well as some "large" (Mr 66,000) and "small" (Mr 44,000) subunits. A few polypeptides of unknown origin were also observed. Four subunits were purified to homogeneity as adjudged by electrophoresis and HPLC and in sufficient yields to permit further studies. Anomalous electrophoretic behavior of the legumin subunits was also observed.     

Induced recombination between duplicated neo genes stably integrated in the genome of CHO cells

Homologous recombination between 2 truncated neo genes stably integrated in the genome of Chinese hamster ovary (CHO) cells was studied. A vector containing a functional gpt gene and 2 tandemly arranged G418 resistance (neo) gene fragments with about 400 bp of sequence homology was transfected into CHO cells. Clonal cell lines were established from transfected cultures and the spontaneous frequency of G418-resistant revertants was found to range between 1 x 10(-4) and 5 x 10(-4). The ability of the alkylating agents MMS and HN2 to induce recombination of the transfected neo genes was studied in 2 of the cell lines. After treatment with MMS at doses that reduced survival to 10% of the control these cell lines showed a dose-dependent increase in the frequency of G418-resistant revertants. No effect was observed after treatment with HN2. All G418-resistant subclones contained a new restriction fragment indicating that a whole neo gene had been formed by rearrangement in pairs of truncated neo genes. Hence, this system can be used to study molecular mechanisms and chemical inducibility of homologous recombination in mammalian cells.     

Effects of acute and chronic exercise on sarcolemmal MCT1 and MCT4 contents in human skeletal muscles: current status

Two lactate/proton cotransporter isoforms (monocarboxylate transporters, MCT1 and MCT4) are present in the plasma (sarcolemmal) membranes of skeletal muscle. Both isoforms are symports and are involved in both muscle pH and lactate regulation. Accordingly, sarcolemmal MCT isoform expression may play an important role in exercise performance. Acute exercise alters human MCT content, within the first 24 h from the onset of exercise. The regulation of MCT protein expression is complex after acute exercise, since there is not a simple concordance between changes in mRNA abundance and protein levels. In general, exercise produces greater increases in MCT1 than in MCT4 content. Chronic exercise also affects MCT1 and MCT4 content, regardless of the initial fitness of subjects. On the basis of cross-sectional studies, intensity would appear to be the most important factor regulating exercise-induced changes in MCT content. Regulation of skeletal muscle MCT1 and MCT4 content by a variety of stimuli inducing an elevation of lactate level (exercise, hypoxia, nutrition, metabolic perturbations) has been demonstrated. Dissociation between the regulation of MCT content and lactate transport activity has been reported in a number of studies, and changes in MCT content are more common in response to contractile activity, whereas changes in lactate transport capacity typically occur in response to changes in metabolic pathways. Muscle MCT expression is involved in, but is not the sole determinant of, muscle H(+) and lactate anion exchange during physical activity.     

Bi-directional modulation of fast inhibitory synaptic transmission by leptin

The hormone leptin has widespread actions in the CNS. Indeed, leptin markedly influences hippocampal excitatory synaptic transmission and synaptic plasticity. However, the effects of leptin on fast inhibitory synaptic transmission in the hippocampus have not been evaluated. Here, we show that leptin modulates GABA_A receptor-mediated synaptic transmission onto hippocampal CA1 pyramidal cells. Leptin promotes a rapid and reversible increase in the amplitude of evoked GABA_A receptor-mediated inhibitory synaptic currents (IPSCs); an effect that was paralleled by increases in the frequency and amplitude of miniature IPSCs, but with no change in paired pulse ratio or coefficient of variation, suggesting a post-synaptic expression mechanism. Following washout of leptin, a persistent depression (inhibitory long-lasting depression) of evoked IPSCs was observed. Whole-cell dialysis or bath application of inhibitors of phosphoinositide 3 (PI 3)-kinase or Akt prevented leptin-induced enhancement of IPSCs indicating involvement of a post-synaptic PI 3-kinase/Akt-dependent pathway. In contrast, blockade of PI 3-kinase or Akt activity failed to alter the ability of leptin to induce inhibitory long-lasting depression, suggesting that this process is independent of PI 3-kinase/Akt. In conclusion these data indicate that the hormone leptin bi-directionally modulates GABA_A receptor-mediated synaptic transmission in the hippocampus. These findings have important implications for the role of this hormone in regulating hippocampal pyramidal neuron excitability.     

Litterfall and forest floor dynamics in Eucalyptus pilularis forests

Abstract Calculations relating the input of litterfall to litter or forest floor mass in forests generally assume that the forest floor reaches an equilibrium state. Based on this assumption, a decomposition factor (k) can be calculated. In the present paper, this basic assumption is questioned and the implications considered. Data on litterfall and forest floor from blackbutt (Eucalyptus pilularis) regrowth forests and plantations were collated from publications and the authors' studies to evaluate both assumptions and relationships. Blackbutt grows over a wide environmental range but its main distribution is in mild temperate to subtropical conditions. Data were from single‐plot studies, sequential studies and chronosequences in both plantations and native regrowth forests. Stands ranged in age from 3 years to maturity in the case of pure, or almost pure blackbutt stands. The forest floor biomass increased up to 12.3 tha^?1 at 33 years of age with no evidence of steady state. Litterfall increased up to 7.8 t ha^?1 year^?1 and was correlated with crown biomass. Regrowth stands were relatively undisturbed and more than 20 years of age, and litterfall ranged from 4.1 to 11.6 tha^?1 year^?1 and was correlated with stand basal area. Forest floor mass in regrowth forests was variable between the different aged stands but did not exceed 18 tha^?1, and there was no evidence that steady state was achieved. The forest floor mass was related to, and approximately 1.7 times the input of litterfall. Although the assumption of steady state was not valid, a k' factor was estimated that related input to forest floor mass and this was relatively constant across all stands and correlated with generalized environmental data. Although assumptions of forest floor equilibrium cannot be supported for E. pilularis, it still should be possible to predict forest floor mass and decomposition from stand conditions and general environmental data.     

Fast-Growing, Aerobic, Heterotrophic Bacteria from the Rhizosphere of Young Sugar Beet Plants

Fast-growing, aerobic, heterotrophic bacteria from the root surface of young sugar beet plants were inventoried. Isolation of the most abundant bacteria from the root surface of each of 1,100 plants between the second and tenth leaf stage yielded 5,600 isolates. These plants originated from different fields in Belgium and Spain. All isolates were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cellular proteins. Comparison of protein fingerprints allowed us to inventory the bacteria of individual plants of different fields or leaf stages and to analyze the composition and variability of the rhizobacterial population of young sugar beet plants. Each field harbored a specific population of bacteria which showed a highly hierarchic structure. A small number of bacteria occurring frequently at high densities dominated in each field. The major bacteria were identified as Pseudomonas fluorescens, Xanthomonas maltophilia, Pseudomonas paucimobilis, and Phyllobacterium sp. The former three species showed a high genetic variability as they were represented by different protein fingerprint types on the same or different fields or leaf stages. Twinspan analysis and relative abundance plots showed that the structure and composition of the bacterial populations varied strongly over time. Pseudomonads were typically early colonizers which were later replaced by X. maltophilia or Phyllobacterium sp.