Toxicoproteomic analysis of a mouse model of nonsteroidal anti-inflammatory drug-induced gastric ulcers

Nonsteroidal anti-inflammatory drugs (NSAIDs) are valuable agents; however, their use has been limited by their association with mucosal damage in the upper gastrointestinal tract. NSAIDs inhibit cyclooxygenase and consequently block the synthesis of prostaglandins, which have cytoprotective effects in gastric mucosa; these effects on prostaglandins have been thought to be major cause of NSAID-induced ulceration. However, studies indicate that additional NSAID-related mechanisms are involved in formation of gastric lesions. Here, we used a toxicoproteomic approach to understand cellular processes that are affected by NSAIDs in mouse stomach tissue during ulcer formation. We used fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS)-which consists of fluorogenic derivatization, separation and fluorescence detection by LC, and identification by LC-tandem mass spectrometry-in this proteomic analysis of pyrolic stomach from control and diclofenac (Dic)-treated mice. FD-LC-MS/MS results were highly sensitive; 10 differentially expressed proteins were identified, and all 10 were more highly expressed in Dic-treated mice than in control mice. Specifically, expression levels of 78 kDa glucose-regulated protein (GRP78), heat shock protein beta-1 (HSP27), and gastrin were more than 3-fold higher in Dic-treated mice than in control mice. This study represents a first step to ascertain the precise actors of early NSAID-induced ulceration.     

Population Genetics of an Expanding Family of Mobile Genetic Elements

A model of an expanding family of dispersed repetitive DNA was studied. Based on the previous result of the model of duplicative transposition, an approximate solution to give allelism and identify coefficients as functions of time was obtained, and theoretical predictions were verified by Monte Carlo experiments. The results show that, even if the copy number per genome increases very rapidly, allelism and identity coefficients may take a long time to reach equilibrium. The changes of allelism and allelic identity are similar to that of homozygosity at an ordinary single locus, whereas that of nonallelic identity can be much slower, particularly when the copy number per genome is large. Thus, many existing families of highly repetitive sequences may represent nonequilibrium states for nonallelic identity. The present model may be extended to include other evolutionary forces such as gene conversion or the recurrent insertion from normal gene copies.     

GSE Is a Maternal Factor Involved in Active DNA Demethylation in Zygotes

After fertilization, the sperm and oocyte genomes undergo extensive epigenetic reprogramming to form a totipotent zygote. The dynamic epigenetic changes during early embryo development primarily involve DNA methylation and demethylation. We have previously identified Gse (gonad-specific expression gene) to be expressed specifically in germ cells and early embryos. Its encoded protein GSE is predominantly localized in the nuclei of cells from the zygote to blastocyst stages, suggesting possible roles in the epigenetic changes occurring during early embryo development. Here, we report the involvement of GSE in epigenetic reprogramming of the paternal genome during mouse zygote development. Preferential binding of GSE to the paternal chromatin was observed from pronuclear stage 2 (PN2) onward. A knockdown of GSE by antisense RNA in oocytes produced no apparent effect on the first and second cell cycles in preimplantation embryos, but caused a significant reduction in the loss of 5-methylcytosine (5mC) and the accumulation of 5-hydroxymethylcytosine (5hmC) in the paternal pronucleus. Furthermore, DNA methylation levels in CpG sites of LINE1 transposable elements, Lemd1, Nanog and the upstream regulatory region of the Oct4 (also known as Pou5f1) gene were clearly increased in GSE-knockdown zygotes at mid-pronuclear stages (PN3-4), but the imprinted H19-differential methylated region was not affected. Importantly, DNA immunoprecipitation of 5mC and 5hmC also indicates that knockdown of GSE in zygotes resulted in a significant reduction of the conversion of 5mC to 5hmC on LINE1. Therefore, our results suggest an important role of maternal GSE for mediating active DNA demethylation in the zygote.     

Characterization of iron-sulfur clusters in rat liver submitochondrial particles by electron paramagnetic resonance spectroscopy. Alterations produced by chronic ethanol consumption

Iron-sulfur clusters present in rat liver submitochondrial particles were characterized by ESR at temperatures between 30 and 5.5 K combined with potentiometric titrations. The spectral and thermodynamic characteristics of the iron-sulfur clusters were generally similar to those previously reported for pigeon or bovine heart submitochondrial particles. Clusters N-1a, N-1b, N-2, N-3 and N-4 of NADH dehydrogenase had midpoint oxidation-reduction potentials at pH 7.5 of ?425, ?265, ?85, ?240 and ?260 mV, respectively. Clusters S-1 and S-3 of succinate dehydrogenase had midpoint potentials of 0 and +65 mV, respectively. The iron-sulfur cluster of electron-transferring flavoprotein-ubiquinone oxidoreductase exhibited the g_z signal at g = 2.08 and had a midpoint potential of +30 mV. This signal was relatively prominent in rat liver compared to pigeon or bovine heart.Submitochondrial particles from rats chronically treated with ethanol (36% of total calories, 40 days) showed decreases of 20–30% in amplitudes of signals due to clusters N-2, N-3 and N-4 compared to those from pair-fed control rats. Signals from clusters N-1b, S-1, S-3 and electron-transferring flavoprotein-ubiquinone oxidoreductase were unaffected. Microwave power-saturation behavior was similar for both submitochondrial particle preparations, suggesting that the lower signal amplitudes reflected a lower content of these particular clusters. NADH dehydrogenase activity was significantly decreased (46%), whilst succinate dehydrogenase activity was elevated (25%), following chronic ethanol consumption. The results indicate that chronic ethanol treatment leads to an alteration of the structure and function of the NADH dehydrogenase segment of the electron transfer chain. This alteration is one of the factors contributing to the lower respiration rates observed following chronic ethanol administration.     

Simulating Evolution by Gene Duplication

By considering the recent finding that unequal crossing over and other molecular interactions are contributing to the evolution of multigene families, a model of the origin of repetitive genes was studied by Monte Carlo simulations. Starting from a single gene copy, how genetic systems evolve was examined under unequal crossing over, random drift and natural selection. Both beneficial and deteriorating mutations were incorporated, and the latter were assumed to occur ten times more frequently than the former. Positive natural selection favors those chromosomes with more beneficial mutations in redundant copies than others in the population, but accumulation of deteriorating mutations (pseudogenes) have no effect on fitness so long as there remains a functional gene. The results imply the following: Positive natural selection is needed in order to acquire gene families with new functions. Without it, too many pseudogenes accumulate before attaining a functional gene family. There is a large fluctuation in the outcome even if parameters are the same. When unequal crossing over occurs more frequently, the system evolves more rapidly. It was also shown, under realistic values of parameters, that the genetic load for acquiring a new gene is not as large as J.B.S. Haldane suggested, but not so small as in a model in which a system for selection started from already redundant genes.     

Evolutionary History of the GABA Transporter (GAT) Group Revealed by Marine Invertebrate GAT-1

The GABA transporter (GAT) group is one of the major subgroups in the solute career 6 (SLC6) family of transmembrane proteins. The GAT group, which has been well studied in mammals, has 6 known members, i.e., a taurine transporter (TAUT), four GABA transporters (GAT-1, -2, -3, - 4), and a creatine transporter (CT1), which have important roles in maintaining physiological homeostasis. However, the GAT group has not been extensively investigated in invertebrates; only TAUT has been reported in marine invertebrates such as bivalves and krills, and GAT-1 has been reported in several insect species and nematodes. Thus, it is unknown how transporters in the GAT group arose during the course of animal evolution. In this study, we cloned GAT-1 cDNAs from the deep-sea mussel, Bathymodiolus septemdierum, and the Antarctic krill, Euphausia superba, whose TAUT cDNA has already been cloned. To understand the evolutionary history of the GAT group, we conducted phylogenetic and synteny analyses on the GAT group transporters of vertebrates and invertebrates. Our findings suggest that transporters of the GAT group evolved through the following processes. First, GAT-1 and CT1 arose by tandem duplication of an ancestral transporter gene before the divergence of Deuterostomia and Protostomia; next, the TAUT gene arose and GAT-3 was formed by the tandem duplication of the TAUT gene; and finally, GAT-2 and GAT-4 evolved from a GAT-3 gene by chromosomal duplication in the ancestral vertebrates. Based on synteny and phylogenetic evidence, the present naming of the GAT group members does not accurately reflect the evolutionary relationships.     

A simple method forin situ freezing of anchorage-dependent cells including rat liver parenchymal cells

We developed a simple method for freezing anchorage-dependent cells, including primary cultured rat liver parenchymal cells, without detaching the cells from the culture dish. The method consists of preculture of the cells to confluence, changing the growth medium to a conventional freezing medium, packaging in a container, and storage at –80°C. After thawing and changing the freezing medium to regular growth medium, cell growth was nearly identical to that of cells freshly seeded into a new dish.