The cell cycle-regulated protein human GTSE-1 controls DNA damage-induced apoptosis by affecting p53 function

GTSE-1 (G2 and S phase-expressed-1) protein is specifically expressed during S and G2 phases of the cell cycle. It is mainly localized to the microtubules and when overexpressed delays the G2 to M transition. Here we report that human GTSE-1 (hGTSE-1) protein can negatively regulate p53 transactivation function, protein levels, and p53-dependent apoptosis. We identified a physical interaction between the C-terminal regulatory domain of p53 and the C-terminal region of hGTSE-1 that is necessary and sufficient to down-regulate p53 activity. Furthermore, we provide evidence that hGTSE-1 is able to control p53 function in a cell cycle-dependent fashion. hGTSE-1 knock-down by small interfering RNA resulted in a S/G2-specific increase of p53 levels as well as cell sensitization to DNA damage-induced apoptosis during these phases of the cell cycle. Altogether, this work suggests a physiological role of hGTSE-1 in apoptosis control after DNA damage during S and G2 phases through regulation of p53 function.     

Lipid peroxidation in skeletal muscle of obese as compared to endurance-trained humans: a case of good vs. bad lipids?

Intra-myocellular triglycerides (IMTG) accumulate in the muscle of obese and endurance-trained (ET) humans and are considered a pathogenic factor in the development of insulin resistance, in the former. We postulate that this paradox may be associated with the peroxidation status of the IMTG. IMTG content was the same in the obese and ET subjects. The lipid peroxidation/IMTG ratio was 4.2-fold higher in the obese subjects. Hence, obesity results in an increased level of IMTG peroxidation while ET has a protective effect on IMTG peroxidation. This suggests a link between the lipid peroxidation/IMTG ratio and insulin resistance.     

An ultracentrifugation analysis of two hundred fish genomes

The goal of this study was to provide a comprehensive view of the compositional characteristics of fish genomes. We therefore expanded the number of fish species that we had explored so far in their DNAs by analytical ultracentrifugation in CsCl density gradient from 122 to 201. This study included representatives from three out of nine orders of Elasmobranchs (sharks and rays), both orders of dipnoan lungfishes, and both orders of chondrosteans (sturgeons and bichirs). We also studied 19 out of 38 teleostean orders, which represent all but four (minor) superorders of the subdivision Teleostei, a group comprising about 23,600 species (96% of all extant fishes). This leaves for further studies two subclasses, Holocephali (chimaeras), and Coelacanthimorpha (gombessas). In spite of this substantial increase in the number of species and orders analysed, all average properties (the modal buoyant density, rho(0), the average buoyant density, , the CsCl profile asymmetry, A, and the compositional heterogeneity, H), and all their ranges were unchanged compared to a previous study [J. Mol. Evol. 31 (1990) 265]. This suggests that, in all likelihood, the properties reported in the present paper can be considered as generally valid for all fish genomes.     

Comparative analysis of the human dystrophin and utrophin gene structures

We present analysis of intronic sequences in the human DMD and UTRN genes. In both genes accumulation of repeated elements could account for intron expansion. Out-of-frame rod-domain exons have stronger splice sites and are separated by significantly longer introns as compared to in-frame exons. These features are unique for the two homologs and not shared by other spectrin superfamily genes.     

Interactions between Zn and Cu in LEC rats, an animal model of Wilson's disease

The effect of oral Zn treatment was studied in the liver and kidneys of 26 male Long-Evans Cinnamon (LEC) rats (mutant animals, 5 weeks old) in relation to both the interaction between Zn and Cu and the localisation and concentration of metallothionein (MT). Rats receiving 80 mg zinc acetate daily by gavage and control rats receiving no treatment were killed after 1 or 2 weeks. By immunohistochemical and analytical chemical techniques we revealed that treated rats had higher levels of MT in the hepatic and renal cells compared to untreated ones. Tissue Zn concentrations were significantly higher in treated rats compared to untreated whereas Cu concentrations decreased in the liver and kidneys as indicated by analytical chemical analyses. MT levels also decreased with treatment period. A histochemical procedure, obtained using autofluorescence of Cu-metallothioneins, confirms these findings: after 2 weeks, the signal decreased in both the liver and kidney sections. This gives a greater understanding of the mechanism of Cu metabolism in the two tissues considered. These results suggest that Zn acts both to compete for absorption on the luminal side of the intestinal epithelium and to induce the synthesis of MT.     

Modification of the enantioselectivity of two homologous thermophilic carboxylesterases from<Emphasis Type="Italic"> Alicyclobacillus acidocaldarius</Emphasis> and<Emphasis Type="Italic"> Archaeoglobus fulgidus</Emphasis> by random mutagenesis and screening

The esterase genes est2 from Alicyclobacillus acidocaldarius and AF1716 from Archaeoglobus fulgidus were subjected to error-prone PCR in an effort to increase the low enantioselectivity of the corresponding enzymes EST2 and AFEST, respectively. The model substrate ( RS)- p-nitrophenyl-2-chloropropionate was chosen to produce ( S)-2-chloropropionic acid, an important intermediate in the synthesis of some optically pure compounds, such as the herbicide mecoprop. In the case of EST2, a single mutant, Leu212Pro, was obtained showing a slightly enhanced preference toward the ( S) substrate; in the case of AFEST, a double mutant, Leu101Ile/Asp117Gly, was obtained showing an increased preference in the opposite direction. The 3-D structures of the EST2 and AFEST enzymes were analyzed by molecular modeling to determine the effects of the mutations. Mutations were positioned differently in the structures, but in both cases caused small modifications around the active site and in the oxyanion loop.     

Fetal hepatic and umbilical uptakes of glucogenic substrates during a glucagon-somatostatin infusion

To test the hypothesis that fetal hepatic glutamate output diverts the products of hepatic amino acid metabolism from hepatic gluconeogenesis, ovine fetal hepatic and umbilical uptakes of glucose and glucogenic substrates were measured before and during fetal glucagon-somatostatin (GS) infusion and during the combined infusion of GS, alanine, glutamine, and arginine. Before the infusions, hepatic uptake of lactate, alanine, glutamine, arginine, and other substrates was accompanied by hepatic output of pyruvate, aspartate, serine, glutamate, and ornithine. The GS infusion induced hepatic output of 1.00 +/- 0.07 mol glucose carbon/mol O(2) uptake, an equivalent reduction in hepatic output of pyruvate and glutamate carbon, a decrease in umbilical glucose uptake and placental uptake of fetal glutamate, an increase in hepatic alanine and arginine clearances, and a decrease in umbilical alanine, glutamine, and arginine uptakes. The latter result suggests that glucagon inhibits umbilical amino acid uptake. We conclude that fetal hepatic pyruvate and glutamate output is part of an adaptation to placental function that requires the fetal liver to maintain both a high rate of catabolism of glucogenic substrates and a low rate of gluconeogenesis.     

Giardia cysts in wastewater treatment plants in Italy

Reductions in annual rainfall in some regions and increased human consumption have caused a shortage of water resources at the global level. The recycling of treated wastewaters has been suggested for certain domestic, industrial, and agricultural activities. The importance of microbiological and parasitological criteria for recycled water has been repeatedly emphasized. Among water-borne pathogens, protozoa of the genera Giardia and Cryptosporidium are known to be highly resistant to water treatment procedures and to cause outbreaks through contaminated raw or treated water. We conducted an investigation in four wastewater treatment plants in Italy by sampling wastewater at each stage of the treatment process over the course of 1 year. The presence of the parasites was assessed by immunofluorescence with monoclonal antibodies. While Cryptosporidium oocysts were rarely observed, Giardia cysts were detected in all samples throughout the year, with peaks observed in autumn and winter. The overall removal efficiency of cysts in the treatment plants ranged from 87.0 to 98.4%. The removal efficiency in the number of cysts was significantly higher when the secondary treatment consisted of active oxidation with O(2) and sedimentation instead of activated sludge and sedimentation (94.5% versus 72.1 to 88.0%; P = 0.05, analysis of variance). To characterize the cysts at the molecular level, the beta-giardin gene was PCR amplified, and the products were sequenced or analyzed by restriction. Cysts were typed as assemblage A or B, both of which are human pathogens, stressing the potential risk associated with the reuse of wastewater.     

Metallothionein-1 and metallothionein-2 gene expression and localisation of apoptotic cells in Zn-treated LEC rat liver

The aims of the present work were to determine the effect of long-term treatment with zinc (Zn) on metallothionein (MT) concentrations and to study the levels of both MT-1 and MT-2 mRNAs in Long-Evans Cinnamon (LEC) rat liver. We also identified apoptotic cells comparing two cytochemical techniques. Thirteen rats received 50 mg zinc acetate daily by gavage, 13 rats received no treatment, and both groups were killed after 60 days. Finally four rats were killed 35 days after birth (T(0)). The results demonstrate that the Zn-treated group had higher levels of MT than both the untreated and basal ones. Quantification of mRNA indicates that the level of the Zn-treated group was significantly higher than the untreated group. Confocal fluorescent staining with monoclonal antibody (Mab) against single-strand DNA localised the hepatic cells that had chromatin condensation and nuclear fragmentation typical of apoptosis, especially in the untreated group sections. The intensity and quantity of fluorescence decreased in both the treated and basal groups. The higher sensitivity of Mab staining compared to TUNEL, which revealed both apoptotic and necrotic cells, reflects the different action mechanism of the two techniques. These findings confirm, in LEC rats, the important role of Zn in cellular protection in relation to MT expression and apoptotic processes as cellular responses to DNA damage by free radicals.     

L-Propionyl-carnitine as superoxide scavenger, antioxidant, and DNA cleavage protector

L-Propionylcarnitine, a propionyl ester of L-carnitine, increases the intracellular pool of L-carnitine. It exhibits a high affinity for the enzyme carnitine acetyltransferase (CAT) and, thus, is readily converted into propionyl-coenzyme A and free carnitine. It has been reported that L-propionylcarnitine possesses a protective action against heart ischemia–reperfusion injury; however, the antioxidant mechanism is not yet clear. L-Propionylcarnitine might reduce the hydroxyl radical production in the Fenton system, by chelating the iron required for the generation of hydroxyl radicals. To obtain a better insight into the antiradical mechanism of L-propionylcarnitine, the present research analyzed the superoxide scavenging capacity of L-propionylcarnitine and its effect on linoleic acid peroxidation. In addition, the effect of L-propionylcarnitine against DNA cleavage was estimated using pBR322 plasmid. We found that L-propionylcarnitine showed a dose-dependent free-radical scavenging activity. In fact, it was able to scavenge superoxide anion, to inhibit the lipoperoxidation of linoleic acid, and to protect pBR322 DNA from cleavage induced by H₂O₂ UV-photolysis. This revised version was published online in July 2006 with corrections to the Cover Date.