Messenger RNA surveillance systems monitoring proper translation termination

Organisms have evolved an elaborate set of quality control systems to ensure the fidelity of the genetic information flow. The mRNA surveillance systems work in this context by monitoring the quality of mRNAs to ensure that they are suitable for translation. In this review, recent achievements in the investigation of mRNA surveillance pathways, including nonsense-mediated mRNA decay and nonstop-mediated mRNA surveillance pathway, will be discussed.     

Mitochondrial DNA haplogroup D4a is a marker for extreme longevity in Japan

We report results from the analysis of complete mitochondrial DNA (mtDNA) sequences from 112 Japanese semi-supercentenarians (aged above 105 years) combined with previously published data from 96 patients in each of three non-disease phenotypes: centenarians (99-105 years of age), healthy non-obese males, obese young males and four disease phenotypes, diabetics with and without angiopathy, and Alzheimer's and Parkinson's disease patients. We analyze the correlation between mitochondrial polymorphisms and the longevity phenotype using two different methods. We first use an exhaustive algorithm to identify all maximal patterns of polymorphisms shared by at least five individuals and define a significance score for enrichment of the patterns in each phenotype relative to healthy normals. Our study confirms the correlations observed in a previous study showing enrichment of a hierarchy of haplogroups in the D clade for longevity. For the extreme longevity phenotype we see a single statistically significant signal: a progressive enrichment of certain "beneficial" patterns in centenarians and semi-supercentenarians in the D4a haplogroup. We then use Principal Component Spectral Analysis of the SNP-SNP Covariance Matrix to compare the measured eigenvalues to a Null distribution of eigenvalues on Gaussian datasets to determine whether the correlations in the data (due to longevity) arises from some property of the mutations themselves or whether they are due to population structure. The conclusion is that the correlations are entirely due to population structure (phylogenetic tree). We find no signal for a functional mtDNA SNP correlated with longevity. The fact that the correlations are from the population structure suggests that hitch-hiking on autosomal events is a possible explanation for the observed correlations.     

Detection of micronucleated cells and gene expression changes in glandular stomach of mice treated with stomach-targeted carcinogens

To assess the genotoxicity of chemicals on the stomach, we developed in vivo assays that can detect micronucleus induction and gene expression changes in epithelial cells of the glandular stomach in mice. Male BALB/c mice were orally given a single dose (100 mg/kg) of N-nitroso-N-methylurea (MNU) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as stomach-targeted carcinogens. The glandular stomach was excised at 4h, 3 and 4 days after administration, and a single cell suspension of epithelial cells was prepared from the everted glandular stomach by EDTA treatment. For determination of micronucleus induction, gastric epithelial cells on days 3 and 4 after administration were fixed with 10% neutral-buffered formalin, stained with a combination of AO-DAPI, and analyzed under fluorescence microscopy. We also examined the induction of micronuclei in peripheral blood of these mice on days 2 and 3 after administration. Moreover, total RNA was extracted from gastric epithelial cells at 4h after administration, and p21 and plk2 expression was analyzed using a quantitative RT-PCR technique. 1) A significant increase of micronucleated cells was observed in the glandular stomach in mice treated with N-nitroso-N-methylurea (MNU) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) compared to mice treated with vehicle. 2) In peripheral blood, induction of micronuclei was observed in mice treated with MNU but not with MNNG. 3) p21 and plk2, which related to cell cycle arrest, were up-regulated in the glandular stomach in mice treated with MNU or MNNG compared to mice treated with vehicle. The present study showed that these assays using glandular stomach may help to evaluate the genotoxicity of chemicals after oral administration.     

The role of cysteine 116 in the active site of the antitumor enzyme L-methionine gamma-lyase from Pseudomonas putida

The cysteinyl residue at the active site of L-methionine gamma-lyase from Pseudomonas putida (MGL_Pp) is highly conserved among the heterologous MGLs. To determine the role of Cys116, we constructed 19 variants of C116X MGL_Pp by saturation mutagenesis. The Cys116 mutants possessed little catalytic activity, while their affinity for each substrate was almost the same as that of the wild type. Especially, the C116S, C116A, and C116H variants composed active site catalytic function as measured by the kinetic parameter k(cat) toward L-methionine. Furthermore, the mutagenesis of Cys116 also affected the substrate specificity of MGL_Pp at the active center. Substitution of Cys116 for His led to a marked increase in activity toward L-cysteine and a decrease in that toward L-methionine. Propargylglycine inactivated the WT MGL, C116S, and C116A mutants. Based on these results, we postulate that Cys116 plays an important role in the gamma-elimination reaction of L-methionine and in substrate recognition in the MGLs.     

Differential involvement of cell cycle reactivation between striatal and cortical neurons in cell death induced by 3-nitropropionic acid

Recent evidence suggests that unscheduled cell cycle activity leads to neuronal cell death. 3-Nitropropionic acid (3-NP) is an irreversible inhibitor of succinate dehydrogenase and induces cell death in both striatum and cerebral cortex. Here we analyzed the involvement of aberrant cell cycle progression in 3-NP-induced cell death in these brain regions. 3-NP reduced the level of cyclin-dependent kinase inhibitor p27 in striatum but not in cerebral cortex. 3-NP also induced phosphorylation of retinoblastoma protein, a marker of cell cycle progression at late G(1) phase, only in striatum. Pharmacological experiments revealed that cyclin-dependent kinase activity and N-methyl-d-aspartate (NMDA) receptor were cooperatively involved in cell death by 3-NP in striatal neurons, whereas only NMDA receptor was involved in 3-NP-induced neurotoxicity in cortical neurons. Death of striatal neurons was preceded by elevation of somatic Ca(2+) and activation of calpain, a Ca(2+)-dependent protease. Both striatal p27 down-regulation and cell death provoked by 3-NP were dependent on calpain activity. Moreover, transfection of p27 small interfering RNA reduced striatal cell viability. In cortical neurons, however, there was no change in somatic Ca(2+) and calpain activity by 3-NP, and calpain inhibitors were not protective. These results suggest that 3-NP induces aberrant cell cycle progression and neuronal cell death via p27 down-regulation by calpain in striatum but not in the cerebral cortex. This is the first report for differential involvement of cell cycle reactivation in different brain regions and lightens the mechanism for region-selective vulnerability in human disease, including Huntington disease.     

Quality control of photosystem II: reactive oxygen species are responsible for the damage to photosystem II under moderate heat stress

Moderate heat stress (40 degrees C for 30 min) on spinach thylakoid membranes induced cleavage of the reaction center-binding D1 protein of photosystem II, aggregation of the D1 protein with the neighboring polypeptides D2 and CP43, and release of three extrinsic proteins, PsbO, -P, and -Q. These heat-induced events were suppressed under anaerobic conditions or by the addition of sodium ascorbate, a general scavenger of reactive oxygen species. In accordance with this, singlet oxygen and hydroxyl radicals were detected in spinach photosystem II membranes incubated at 40 degrees C for 30 min with electron paramagnetic resonance spin-trapping spectroscopy. The moderate heat stress also induced significant lipid peroxidation under aerobic conditions. We suggest that the reactive oxygen species are generated by heat-induced inactivation of a water-oxidizing manganese complex and through lipid peroxidation. Although occurring in the dark, the damages caused by the moderate heat stress to photosystem II are quite similar to those induced by excessive illumination where reactive oxygen species are involved.     

A novel regulatory function of selenocysteine lyase, a unique catalyst to modulate major urinary protein

Mouse selenocysteine lyase (SCL) catalyzes the decomposition of -selenocysteine into -alanine and selenium with pyridoxal 5′-phosphate as a coenzyme. When using SCL as bait in a yeast two-hybrid screening method, major urinary protein I (MUP-I) was identified as a protein that interacts with SCL. This interaction was confirmed with an in vitro binding assay. MUP-I is known as a pheromone-binding protein that accommodates volatile effectors to affect the physiology and behavior of mice. We found that the binding of 2-naphthol to MUP-I was significantly inhibited by SCL, suggesting that SCL regulates the binding capacity of MUP-I.     

Phylogenetic Relationships Among JC Virus Strains in Japanese/Koreans and Native Americans Speaking Amerind or Na-Dene

Abstract Many genetic studies using human mtDNA or the Y chromosome have been conducted to elucidate the relationships among the three Native American groups speaking Amerind, Na-Dene, and Eskimo-Aleut. Human polyomavirus JC (JCV) may also help to gain insights into this issue. JCV isolates are classified into more than 10 geographically distinct genotypes (designated subtypes here), which were generated by splits in the three superclusters, Types A, B, and C. A particular subtype of JCV (named MY) belonging to Type B is spread in both Japanese/Koreans and Native Americans speaking Amerind or Na-Dene. In this study, we evaluated the phylogenetic relationships among MY isolates worldwide, using the whole-genome approach, with which a highly reliable phylogeny of JCV isolates can be reconstructed. Thirty-six complete sequences belonging to MY (10 from Japanese/Koreans, 24 from Native Americans, and 2 from others), together with 54 belonging to other subtypes around the world, were aligned and subjected to phylogenetic analysis using the neighbor-joining and maximum-likelihood methods. In the resultant phylogenetic trees, the MY sequences diverged into two Japanese/Korean and five Native American clades with high bootstrap probabilities. Two of the Native American clades contained isolates mainly from Na-Denes and the others contained isolates mainly from Amerinds. The Na-Dene clades were not clustered together, nor were the Amerind clades. In contrast, the two Japanese/Korean clades were clustered at a high bootstrap probability. We concluded that there is no distinction between Amerinds and Na-Denes in terms of indigenous JCVs, although they are linguistically distinguished from each other.