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).     

Secretion of inflammatory mediators by isolated rat Kupffer cells: the effect of octreotide

Aims: We studied the production of inflammatory mediators by rat KC and the possible in vitro effect of the somatostatin analogue octreotide. Methods: Primary KC cultures were incubated with LPS added alone or with different concentrations of octreotide. The production of TNF, IL-6, IL-10, IL-12 and IL-13 was assessed in culture supernatants by ELISA and that of nitric oxide (NO) by a modification of the Griess reaction. Results: Isolated KC produced a basal amount of TNF, IL-6, IL-12, IL-13, and NO but not IL-10. LPS-stimulated KC secreted significantly increased amounts of TNF (P<0.001), IL-6 (P<0.01), IL-10 (P<0.001), IL-12 (P<0.01), and NO (P<0.001) whereas IL-13 production remained constant. Octreotide reduced IL-12 (P<0.05) and increased IL-13 (P<0.05) production by unstimulated KC. Furthermore, octreotide suppressed TNF production (P<0.05), without modifying TNF mRNA expression and decreased iNOS expression and NO (P≈0.05) production by LPS-activated KC. These effects were reversed with Wortmannin pre-treatment suggesting that octreotide may act via interference with phosphatidylinositol 3-kinase pathways. Conclusions: These data demonstrate that KC is a source of multiple inflammatory mediators, indicating a critical role in liver inflammatory disorders. Octreotide modulates inflammatory mediator production by isolated KC, suggesting that it might have immunoregulatory and anti-inflammatory effects in liver diseases.     

Xenobiotic conjugation systems in deer compared with cattle and rat

The ability of cattle and deer liver to catalyse xenobiotic conjugation reactions was investigated and compared with that of the rat. Marked differences in the activity of these enzymes were noted between the domestic animals and rats. Hepatic microsomal epoxide hydrolase activity in cattle and deer, determined using benzo[a]pyrene 4,5-oxide as substrate, was nearly twice that of the rat. In contrast, glutathione S-transferase activity in hepatic cytosol, determined with 1-chloro-2,4-dinitrobenzene as substrate, was significantly lower in the cattle and deer. When 1,2-dichloro-4-nitrobenzene served as the accepting substrate, no activity was detectable in the cattle and deer. Similarly, glutathione reductase activity and total glutathione levels were markedly lower in the cattle and deer compared with the rat. Cytosolic sulfotransferase activity, monitored using 2-naphthol as substrate, was higher in cattle compared with the rat. Finally, microsomal UDP-glucuronosyl transferase activity, determined using 1-napththol as substrate, did not differ significantly among the three species.     

Mutagenic activity of the glutathione S-transferase substrate 1-chloro-2,4-dinitrobenzene (CDNB) in the Salmonella mutagenicity assay

The mutagenicity of the commonly used glutathione S-transferase substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) was investigated in the Salmonella mutagenicity assay. CDNB induced a concentration-dependent mutagenic response in Salmonella typhimurium strain TA98. Incorporation of an activation system derived from Aroclor 1254-induced rats did not influence mutagenic response. Under the same conditions DCNB failed to display mutagenic activity. The mutagenic activity of CDNB was attenuated in bacterial strains under-expressing nitroreductase or O-acetylase activity but, in contrast, it was exaggerated in an O-acetylase over-expressing strain. It is inferred that CDNB exhibits a mutagenic response following reduction of the nitro-group to the hydroxylamine, which is further acetylated to form the acetoxy derivative that presumably breaks down spontaneously to generate the nitrenium ion, the likely ultimate mutagen.