CGDEase, a Pseudomonas fluorescens protein of the PLC/APase superfamily with CDP-ethanolamine and (dihexanoyl)glycerophosphoethanolamine hydrolase activity induced by osmoprotectants under phosphate-deficient conditions

A novel enzyme, induced by choline, ethanolamine, glycine betaine or dimethylglycine, was released at low temperature and phosphate from Pseudomonas fluorescens (CECT 7229) suspensions at low cell densities. It is a CDP-ethanolamine pyrophosphatase/(dihexanoyl)glycerophosphoethanolamine phosphodiesterase (CGDEase) less active on choline derivatives, and inactive on long-chain phospholipids, CDP-glycerol and other NDP-X compounds. The reaction pattern was typical of phospholipase C (PLC), as either phosphoethanolamine or phosphocholine was produced. Peptide-mass analyses, gene cloning and expression provided a molecular identity for CGDEase. Bioinformatic studies assigned it to the PLC branch of the phospholipase C/acid phosphatase (PLC/APase) superfamily, revealed an irregular phylogenetic distribution of close CGDEase relatives, and suggested their genes are not in operons or conserved contexts. A theoretical CGDEase structure was supported by mutagenesis of two predicted active-site residues, which yielded essentially inactive mutants. Biological relevance is supported by comparisons with CGDEase relatives, induction by osmoprotectants (not by osmotic stress itself) and repression by micromolar phosphate. The low bacterial density requirement was related to phosphate liberation from lysed bacteria in denser populations, rather than to a classical quorum-sensing effect. The results fit better a CGDEase role in phosphate scavenging than in osmoprotection.     

A combined metabolic/polymerization kinetic model on the microbial production of poly(3-hydroxybutyrate)

In the present work, an integrated dynamic metabolic/polymerization kinetic model is developed for the prediction of the intracellular accumulation profile and the molecular weight distribution of poly(3-hydroxybutyrate) (P(3HB) or PHB) produced in microbial cultures. The model integrates two different length/time scales by combining a polymerization kinetic model with a metabolic one. The bridging point between the two models is the concentration of the monomer unit (i.e. 3-hydroxybutyryl-CoA) produced during the central aerobic carbon metabolism. The predictive capabilities of the proposed model are assessed by the comparison of the calculated biopolymer concentration and number average molecular weight with available experimental data obtained from batch and fed-batch cultures of Alcaligenes eutrophus and Alcaligenes latus. The accuracy of the proposed model was found to be satisfactory, setting this model a valuable tool for the design of the process operating profile for the production of different polymer grades with desired molecular properties.     

Amphiphilic model conetworks based on cross-linked star copolymers of benzyl methacrylate and 2-(dimethylamino)ethyl methacrylate: synthesis, characterization, and DNA adsorption studies

Six amphiphilic model conetworks of a new structure, that of cross-linked "in-out" star copolymers, were synthesized by the group transfer polymerization (GTP) of the hydrophobic monomer benzyl methacrylate (BzMA) and the ionizable hydrophilic monomer 2-(dimethylamino)ethyl methacrylate (DMAEMA) in a one-pot preparation. The synthesis took place in tetrahydrofuran (THF) using tetrabutylammonium bibenzoate (TBABB) as the catalyst, 1-methoxy-1-(trimethylsiloxy)-2-methyl-propene (MTS) as the initiator, and ethylene glycol dimethacrylate (EGDMA) as the cross-linker. Three heteroarm star-, two star block-, one statistical copolymer star-, and one homopolymer star-based networks were prepared. The synthesis of these star-based networks involved four to six steps, including the preparation of the linear (co)polymers, the "arm-first" and the "in-out" star copolymers, and finally the network. The precursors and the extractables were characterized using gel permeation chromatography (GPC) and proton nuclear magnetic resonance (1H NMR) spectroscopy. The degrees of swelling (DSs) of all the networks were measured in THF, while the aqueous DSs were measured as a function of pH. The DSs at low pH were higher than those at neutral or high pH because of the protonation of the DMAEMA units and were found to be dependent on the structure of the network. The DSs in THF were higher than those in neutral water and were independent of the structure. Finally, DNA adsorption studies onto the networks indicated that the DNA binding was governed by electrostatics.     

ADP-ribose pyrophosphatase-I partially purified from livers of rats overdosed with acetaminophen reveals enzyme inhibition in vivo reverted in vitro by dithiothreitol

Free ADP-ribose reacts nonenzymatically with proteins and can lead to intracellular damage. The low-Km ADP-ribose pyrophosphatase-I (ADPRibase-I) is well suited to control free ADP-ribose and nonenzymatic ADP-ribosylation. In vitro, the acetaminophen metabolite N-acetyl-p-benzoquinoneimine (NAPQI) decreases ADPRibase-I Vmax and increases Km, effects not reverted by dithiothreitol (DTT) and attributed to enzyme arylation. The present study was conducted to test whether acetaminophen overdose affected ADPRibase-I in vivo. Rats pretreated with 3-methylcholanthrene and L-buthionine-[S,R]-sulfoximine to potentiate acetaminophen toxicity received an intraperitoneal dose of either acetaminophen (800 mg/ kg; n = 5) or vehicle (n = 3). ADPRibase-I partially purified from acetaminophen-overdosed rats showed a decreased Vmax (0.32+/-0.09 versus 0.60+/-0.03 mU/mg of liver protein; p<0.01) not reverted by DTT and an increased Km for ADP-ribose (1.39+/-0.31 versus 0.67+/-0.05 microM; p<0.01) that, contrary to the in vitro NAPQI effect, was reverted by DTT. Incubation of partially purified ADPRibase-I from normal rat liver with oxidized glutathione elicited a time- and dose-dependent, DTT-reverted increase of Km, without change of Vmax. The results indicate that the activity of ADPRibase-I can be regulated by thiol exchange and that the increase of Km, elicited by acetaminophen overdosage was related to the oxidative stress caused by the drug. It remains to be seen whether an increase of free ADP-ribose concomitant to ADPRibase-I inhibition could contribute to the hepatotoxicity of acetaminophen.     

Origin, diffusion, and differentiation of Y-chromosome haplogroups E and J: inferences on the neolithization of Europe and later migratory events in the Mediterranean area

The phylogeography of Y-chromosome haplogroups E (Hg E) and J (Hg J) was investigated in >2400 subjects from 29 populations, mainly from Europe and the Mediterranean area but also from Africa and Asia. The observed 501 Hg E and 445 Hg J samples were subtyped using 36 binary markers and eight microsatellite loci. Spatial patterns reveal that (1). the two sister clades, J-M267 and J-M172, are distributed differentially within the Near East, North Africa, and Europe; (2). J-M267 was spread by two temporally distinct migratory episodes, the most recent one probably associated with the diffusion of Arab people; (3). E-M81 is typical of Berbers, and its presence in Iberia and Sicily is due to recent gene flow from North Africa; (4). J-M172(xM12) distribution is consistent with a Levantine/Anatolian dispersal route to southeastern Europe and may reflect the spread of Anatolian farmers; and (5). E-M78 (for which microsatellite data suggest an eastern African origin) and, to a lesser extent, J-M12(M102) lineages would trace the subsequent diffusion of people from the southern Balkans to the west. A 7%-22% contribution of Y chromosomes from Greece to southern Italy was estimated by admixture analysis.     

Optknock: a bilevel programming framework for identifying gene knockout strategies for microbial strain optimization

The advent of genome-scale models of metabolism has laid the foundation for the development of computational procedures for suggesting genetic manipulations that lead to overproduction. In this work, the computational OptKnock framework is introduced for suggesting gene deletion strategies leading to the overproduction of chemicals or biochemicals in E. coli. This is accomplished by ensuring that a drain towards growth resources (i.e., carbon, redox potential, and energy) must be accompanied, due to stoichiometry, by the production of a desired product. Computational results for gene deletions for succinate, lactate, and 1,3-propanediol (PDO) production are in good agreement with mutant strains published in the literature. While some of the suggested deletion strategies are straightforward and involve eliminating competing reaction pathways, many others suggest complex and nonintuitive mechanisms of compensating for the removed functionalities. Finally, the OptKnock procedure, by coupling biomass formation with chemical production, hints at a growth selection/adaptation system for indirectly evolving overproducing mutants.     

Amifostine protects lymphocytes during radiotherapy and stimulates expansion of the CD95/Fas and CD31 expressing T-cells,in breast cancer patients

A large body of experimental research supports the anti-neoplastic activity of cellular and humoral immunity. Disease and therapy-related immune suppression may be important on the treatment outcome or on the subsequent course of the malignant disease. The aim of the study was to investigate the efficacy of amifostine in preventing the immunological toxicity of post-operative radiotherapy (RT) in breast cancer patients. Using flow-cytometry, we examined comparatively the peripheral blood lymphocytic subpopulations in breast cancer patients undergoing conventional post-operative RT versus a hypofractionated accelerated RT scheme combined with amifostine (HypoARC) administration. Despite the higher radiation dose intensity delivered in the HypoARC group, a significant protection of CD4, CD8, CD19 and CD56 subtypes by amifostine was noted. We further focused on two interesting CD4/CD8 subpopulations involved in cellular apoptosis and trans-endothelial migration, namely the CD95/Fas and CD31 positive lymphocytes. Amifostine protected and induced expansion of these subtypes, which could contribute to the maintenance of a high burden of tumor infiltrating lymphocytes during therapy. It is suggested that amifostine effectively protects lymphocytes against RT, which may enhance the efficacy of the latter. The clinical impact of the CD95(+) and CD31(+) T-cell immunological modulation induced by amifostine requires further investigation.     

Production of a thermostable extracellular lipase by Kluyveromyces marxianus

Kluyveromyces marxianus was grown in submerged culture in a complex medium with several potential inducers of lipolytic activity (triacylglycerols, fatty acids). The highest extracellular lipolytic enzyme production (about 80 U ml^–1 in 3 d) was obtained when the medium was supplemented with 2 g urea l^–1 plus 5 g tributyrin l^–1. Addition of surfactants (1 g l^–1) did not improve production. The lipase had a high thermal stability in aqueous solution (73% residual activity after 9 d at 50 °C, 16 min half-life time at 100 °C). It was also stable at acidic pH and showed good tolerance to organic solvents (70% residual activity after 2 d in n-hexane of cyclohexane).