Relationship between Glycolysis and Exopolysaccharide Biosynthesis in Lactococcus lactis

The relationships between glucose metabolism and exopolysaccharide (EPS) production in a Lactococcus lactis strain containing the EPS gene cluster (Eps⁺) and in nonproducer strain MG5267 (Eps⁻) were characterized. The concentrations of relevant phosphorylated intermediates in EPS and cell wall biosynthetic pathways or glycolysis were determined by ³¹P nuclear magnetic resonance. The concentrations of two EPS precursors, UDP-glucose and UDP-galactose, were significantly lower in the Eps⁺ strain than in the Eps⁻ strain. The precursors of the peptidoglycan pathway, UDP-N-acetylglucosamine and UDP-N-acetylmuramoyl-pentapeptide, were the major UDP-sugar derivatives detected in the two strains examined, but the concentration of the latter was greater in the Eps⁺ strain, indicating that there is competition between EPS synthesis and cell growth. An intermediate in biosynthesis of histidine and nucleotides, 5-phosphorylribose 1-pyrophosphate, accumulated at concentrations in the millimolar range, showing that the pentose phosphate pathway was operating. Fructose 1,6-bisphosphate and glucose 6-phosphate were the prominent glycolytic intermediates during exponential growth of both strains, whereas in the stationary phase the main metabolites were 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvate. The activities of relevant enzymes, such as phosphoglucose isomerase, α-phosphoglucomutase, and UDP-glucose pyrophosphorylase, were identical in the two strains. ¹³C enrichment on the sugar moieties of pure EPS showed that glucose 6-phosphate is the key metabolite at the branch point between glycolysis and EPS biosynthesis and ruled out involvement of the triose phosphate pool. This study provided clues for ways to enhance EPS production by genetic manipulation.     

Untersuchungen zum Vorkommen von Pythium spp. im Boden

Investigations on the occurrence of Pythium spp. in soil: I. The isolation of Pythium spp., their distinction to macroscopically characteristics and their determination The aim of the present paper consisted in detection of Pythium spp. directly in the soil. This was possible by using a selective medium and by crumbling smallest particles of agar-covered soil on its surface. On the basis of simple morphologically criteria (growth patterns) this method allows to decide concerning the presence of high and less pathogen or apathogen Pythium spp. in a soil sample within 48 hours. About 700 isolates have been cultivated from hyphal tips, determinated and about 230 tested for pathogenicity to sugar beet seedlings in vitro. Most of the Pythia pathogen to sugar beet belong to P. ultimum Trow followed by P. paroecandrum Drechsler and P. debaryanum sensu Drechsler non Hesse. The taxonomically characteristics are demonstrated by figures of the three species.     

Functional Properties of Rare Missense Variants of Human CDH13 Found in Adult Attention Deficit/Hyperactivity Disorder (ADHD) Patients

The CDH13 gene codes for T-cadherin, a GPI-anchored protein with cell adhesion properties that is highly expressed in the brain and cardiovascular system. Previous studies have suggested that CDH13 may be a promising candidate gene for Attention Deficit/Hyperactivity Disorder (ADHD). The aims of this study were to identify, functionally characterize, and estimate the frequency of coding CDH13 variants in adult ADHD patients and controls. We performed sequencing of the CDH13 gene in 169 Norwegian adult ADHD patients and 63 controls and genotyping of the identified variants in 641 patients and 668 controls. Native and green fluorescent protein tagged wild type and variant CDH13 proteins were expressed and studied in CHO and HEK293 cells, respectively. Sequencing identified seven rare missense CDH13 variants, one of which was novel. By genotyping, we found a cumulative frequency of these rare variants of 2.9% in controls and 3.2% in ADHD patients, implying that much larger samples are needed to obtain adequate power to study the genetic association between ADHD and rare CDH13 variants. Protein expression and localization studies in CHO cells and HEK293 cells showed that the wild type and mutant proteins were processed according to the canonical processing of GPI-anchored proteins. Although some of the mutations were predicted to severely affect protein secondary structure and stability, no significant differences were observed between the expression levels and distribution of the wild type and mutant proteins in either HEK293 or CHO cells. This is the first study where the frequency of coding CDH13 variants in patients and controls is reported and also where the functional properties of these variants are examined. Further investigations are needed to conclude whether CDH13 is involved in the pathogenesis of ADHD or other conditions.     

Pharmacological Implications of A Multiplicity of Adenosine Actions in the CNS

Abstract

Adenosine (50 nM - 50 μM) in brain extracellular space acts on two major classes of receptors present on virtually every cell. Specificity of action may be achieved by altering brain adenosine levels and by using partial agonists and/or drugs that affect more than one biochemical target.     

Regulation by a β-adrenergic receptor of a Ca2+-independent adenosine 3′,5′-(cyclic)monophosphate phosphodiesterase in C6 glioma cells

The hormonal control of cyclic nucleotide phosphodiesterase (EC 3.1.4.17) activity has been studied by using as a model the isoproterenol stimulation of cyclic AMP phosphodiesterase activity in C6 glioma cells. A 2-fold increase in cyclic AMP phosphodiesterase specific activity was observed in homogenates of isoproterenol-treated cells relative to control. This increase reached a maximum 3 h after addition of isoproterenol, was selective for cyclic AMP hydrolysis, was reproduced by incubation with 8-Br cyclic AMP but not with 8-Br cyclic GMP and was limited to the soluble enzyme activity. The presence of 0.1 mM EGTA did not alter the magnitude of the increase in phosphodiesterase activity. Moreover, the calmodulin content in the cell extracts was not changed after isoproterernol. DEASE-Sephacel chromatography of the 100 000×g supernatant resolved two peaks of phosphodiesterase activity. The first peak hydrolyzed both cyclic nucleotides and was activated by Ca²⁺ and purified calmodulin. The second peak was specific for cyclic AMP but it was Ca²⁺- and calmodulin-insensitive. Isoproterenol selectively increased the specific activity of the second peak. Kinetic analysis of the cyclic AMP hydrolysis by the induced enzyme reveled a non-linear Hofstee plot with apparent K_m values of 2–5 μM. Cyclic GMP was not hydrolyzed by this enzyme in the absence or presence of calmodulin and failed to affect the kinetics of the hydrolysis of cyclic AMP. Gel filtration chromatography of the induced DEASE-Sephacel peak resolved a single peak of enzyme activity with an apparent molecular weight of 54 000.     

Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis

Applied Microbiology and Biotechnology - Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate...     

Usnic acid,as a biotic factor,changes the ploidy level in mosses

Lichens and mosses often share the same environmental conditions where they compete for substrate and other essential factors. Lichens use secondary metabolites as allelochemicals to repel surrounding plants and potential rivals. In mosses, endoreduplication leads to the occurrence of various ploidy levels in the same individual and has been suggested as an adaptation to abiotic stresses. Here, we show that also biotic factors such as usnic acid, an allelochemical produced by lichens, directly influenced the level of ploidy in mosses. Application of usnic acid changed the nuclei proportion and significantly enhanced the endoreduplication index in two moss species, Physcomitrella patens and Pohlia drummondii. These investigations add a new aspect on secondary metabolites of lichens which count as biotic factors and affect ploidy levels in mosses.