Oxysterols As Indices of Oxidative Stress in Man After Paraquat Ingestion

The aim of this study is to evaluate oxidative stress in man after paraquat ingestion by analyzing 7 &#102 - and 7 &#103 -hydroperoxycholest-5-en-3 &#103 -ol (7 &#102 - and 7 &#103 -OOH) as well as oxysterols, cholesterol oxidation products, as indices of lipid peroxidation. Lung, kidney, and liver were collected at autopsy from seven patients with paraquat poisoning and seven controls matched for age and sex. We identified for the first time 7-ketocholesterol (7-keto) and 7-hydroxycholesterol (7 &#102 -OH and 7 &#103 -OH) in human kidney by LC-MS. Next, we quantified 7 &#102 -OOH and 7 &#103 -OOH by HPLC with postcolumn chemiluminescence as well as oxysterols by HPLC-UV. Both 7 &#102 -OOH and 7 &#103 -OOH detected in lung and kidney from the controls were as low as the paraquat group. In contrast, we found both 7-keto and 7 &#103 -OH in lung and 7-keto in kidney from the paraquat group were significantly higher than from the controls. This is the first report on accumulated oxysterols in lung and kidney from human paraquat poisoning. It seems to reflect greater oxidative stress in the pathology of paraquat intoxication.     

Irreversible inactivation of glutathione S-transferase-π by a low concentration of naphthoquinones

π-Class glutathione S-transferase (GST-π) was very potently inactivated by oxidants such as H₂O₂, xanthine-xanthine oxidase and naphthoquinones. Thiols and glutathione analogs including dithiothreitol, reduced gluta-thione, cysteine, cysteamine, S-methyl-SG, S-hexyl-SG and S-decyl-SG protected GST-π from the inactivation, but a substrate analog (2,4-dinitrophenol), superoxide dismutase and catalase did not, suggesting that the cysteinyl residue(s) in/nearby the glutathione binding site (G-site) may be oxidatively modified by these oxidants. Many reductants and radical scavengers including butylated hydoxytoluene, α-tocopherol, ascorbate, uric acid, mannitol, tyrosine, tryptophan, histidine, quercitrin and bilirubin had no effect on the inactivation. GST-π pretreated with cystamine was reactivated very efficiently by 50 mM DTT following incubation with 1,2-naphthoquinone, whereas cystamine-untreated GST-π was not reactivated.     

Plasma B-type Natriuretic Peptide as a Predictor of Cardiovascular Events in Subjects with Atrial Fibrillation: A Community-Based Study

Objectives

Atrial fibrillation (AF) is a significant public health issue due to its high prevalence in the general population, and is associated with an increased risk of cardiovascular (CV) events including systemic thrombo-embolism, heart failure, and coronary artery disease. The relationship between plasma B-type natriuretic peptide (BNP) and CV risk in real world AF subjects remains unknown.

Methods

The subject of the study (n = 228; mean age = 69 years) was unselected individuals with AF in a community-based population (n = 15,394; AF prevalence rate = 1.5%). The CV event free rate within each BNP tertile was estimated, and Cox regression analysis was performed to examine the relative risk of the onset of CV events among the tertiles. The prognostic ability of BNP was compared to an established risk score for embolic events (CHADS2 score). In addition, to determine the usefulness of BNP as a predictor in addition to CHADS2 score, we calculated Net Reclassification Improvement (NRI) and Integrated Discrimination Improvement (IDI) indices.

Results

During the follow-up period 58 subjects experienced CV events (52 per 1,000 person-years). The event-free ratio was significantly lower in the highest tertile (p < 0.02). After adjustment for established CV risk factors, the hazard ratio (HR) of the highest tertile was significantly higher than that of the lowest tertile (HR = 2.38; p < 0.02). The predictive abilities of plasma BNP in terms of sensitivity and specificity for general CV events were comparable to those of CHADS2 score. Adding BNP to the CHADS2 score only model improved the NRI (0.319; p < 0.05) and the IDI (0.046; p < 0.05).

Conclusion

Plasma BNP is a valuable biomarker both singly or in combination with an established scoring system for assessing general CV risk including stroke, heart failure and acute coronary syndrome in real-world AF subjects.     

Unique T Cells with Unconventional Cytokine Profiles Induced in the Livers of Mice during Schistosoma mansoni Infection

During infection with Schistosoma, serious hepatic disorders are induced in the host. The liver possesses unique immune systems composed of specialized cells that differ from those of other immune competent organs or tissues. Host immune responses change dramatically during Schistosoma mansoni infection; in the early phase, Th1-related responses are induced, whereas during the late phase Th2 reactions dominate. Here, we describe unique T cell populations induced in the liver of mice during the period between Th1- and Th2-phases, which we term the transition phase. During this phase, varieties of immune cells including T lymphocytes increase in the liver. Subsets of CD4⁺ T cells exhibit unique cytokine production profiles, simultaneously producing both IFN-γ and IL-13 or both IFN-γ and IL-4. Furthermore, cells triply positive for IFN-γ, IL-13 and IL-4 also expand in the S. mansoni-infected liver. The induction of these unique cell populations does not occur in the spleen, indicating it is a phenomenon specific to the liver. In single hepatic CD4⁺ T cells showing the unique cytokine profiles, both T-bet and GATA-3 are expressed. Thus, our studies show that S. mansoni infection triggers the induction of hepatic T cell subsets with unique cytokine profiles.     

Flavonoids isolated from the leaves of Melicope triphylla and their extracellular-superoxide dismutase-inducing activity

Two novel flavonoids, named meliflavones A (1) and B (2), were isolated from the leaves of Melicope triphylla (Lam.) Merr., along with thirteen known compounds (3–15). Four of the polymethoxyflavonoids bearing a prenyloxy (3-methylbut-2-enyloxy) function (1, 3–5) induced the expression of extracellular-superoxide dismutase (EC-SOD) in a human leukemic U937 cell-based assay.     

Interference in DNA Replication Can Cause Mitotic Chromosomal Breakage Unassociated with Double-Strand Breaks

Morphological analysis of mitotic chromosomes is used to detect mutagenic chemical compounds and to estimate the dose of ionizing radiation to be administered. It has long been believed that chromosomal breaks are always associated with double-strand breaks (DSBs). We here provide compelling evidence against this canonical theory. We employed a genetic approach using two cell lines, chicken DT40 and human Nalm-6. We measured the number of chromosomal breaks induced by three replication-blocking agents (aphidicolin, 5-fluorouracil, and hydroxyurea) in DSB-repair-proficient wild-type cells and cells deficient in both homologous recombination and nonhomologous end-joining (the two major DSB-repair pathways). Exposure of cells to the three replication-blocking agents for at least two cell cycles resulted in comparable numbers of chromosomal breaks for RAD54^−/−/KU70^−/− DT40 clones and wild-type cells. Likewise, the numbers of chromosomal breaks induced in RAD54^−/−/LIG4^−/− Nalm-6 clones and wild-type cells were also comparable. These data indicate that the replication-blocking agents can cause chromosomal breaks unassociated with DSBs. In contrast with DSB-repair-deficient cells, chicken DT40 cells deficient in PIF1 or ATRIP, which molecules contribute to the completion of DNA replication, displayed higher numbers of mitotic chromosomal breaks induced by aphidicolin than did wild-type cells, suggesting that single-strand gaps left unreplicated may result in mitotic chromosomal breaks.     

Genetic Diversity and Distribution of the Ciguatera-Causing Dinoflagellate Gambierdiscus spp. (Dinophyceae) in Coastal Areas of Japan

Background

The marine epiphytic dinoflagellate genus Gambierdiscus produce toxins that cause ciguatera fish poisoning (CFP): one of the most significant seafood-borne illnesses associated with fish consumption worldwide. So far, occurrences of CFP incidents in Japan have been mainly reported in subtropical areas. A previous phylogeographic study of Japanese Gambierdiscus revealed the existence of two distinct phylotypes: Gambierdiscus sp. type 1 from subtropical and Gambierdiscus sp. type 2 from temperate areas. However, details of the genetic diversity and distribution for Japanese Gambierdiscus are still unclear, because a comprehensive investigation has not been conducted yet.

Methods/Principal Finding

A total of 248 strains were examined from samples mainly collected from western and southern coastal areas of Japan during 2006–2011. The SSU rDNA, the LSU rDNA D8–D10 and the ITS region were selected as genetic markers and phylogenetic analyses were conducted. The genetic diversity of Japanese Gambierdiscus was high since five species/phylotypes were detected: including two reported phylotypes (Gambierdiscus sp. type 1 and Gambierdiscus sp. type 2), two species of Gambierdiscus (G. australes and G. cf. yasumotoi) and a hitherto unreported phylotype Gambierdiscus sp. type 3. The distributions of type 3 and G. cf. yasumotoi were restricted to the temperate and the subtropical area, respectively. On the other hand, type 1, type 2 and G. australes occurred from the subtropical to the temperate area, with a tendency that type 1 and G. australes were dominant in the subtropical area, whereas type 2 was dominant in the temperate area. By using mouse bioassay, type 1, type 3 and G. australes exhibited mouse toxicities.

Conclusions/Significance

This study revealed a surprising diversity of Japanese Gambierdiscus and the distribution of five species/phylotypes displayed clear geographical patterns in Japanese coastal areas. The SSU rDNA and the LSU rDNA D8–D10 as genetic markers are recommended for further use.     

Changes in bacterial glycolipids as an index of intestinal lactobacilli and epithelial glycolipids in the digestive tracts of mice after administration of penicillin and streptomycin

The major lipid constituent of symbiotic gram-positive bacteria in animals are phosphatidylglycerol, cardiolipin and dihexaosyl diglycerides (DH-DG), whose hydrophobic structures are characteristic of the environments, and the carbohydrate structures of DH-DGs are bacterial species-characteristic. Immunization of rabbits with intestinal lactobacilli generated antibodies against DH-DGs and their modified structures, among which Galα1-6-substituted DH-DG, i.e., Lactobacillus tetrahexaosyl diglyceride (LacTetH-DG), reacted with antibodies more intensely than DH-DG. Whereas, from the 16S-rRNA sequence, the intestinal lactobacilli in murine digestive tracts were revealed to be L. johnsonii, in which LacTetH-DG is present at the concentration of 2.2 ng per 1?×?10⁶ cells. To obtain more accurate estimates of intestinal lactobacilli in several regions of the digestive tract of mice, LacTetH-DG was detected by TLC-immunostaining with anti-Lactobacillus antisera, being found in the stomach, cecum and colon of normal breeding mice, 1.0?×?10⁹, 3.5?×?10⁹ and 7.4?×?10⁹ cells, respectively. Administration of penicillin and streptomycin for 6 days resulted in a reduction in the number of intestinal lactobacilli, the levels being 0 %, 30 % and 4 % of the control ones in the stomach, cecum and colon, respectively, which was associated with the accumulation of the contents in the tracts from the stomach to the cecum and with diarrhea. In addition, a reduced amount of fucosyl GA1 (FGA1) and a compensatory increase in GA1 due to the reduced activity of α1,2-fucosyltransferase in the small intestine and the enhanced discharge of FGA1 into the contents occurred in mice, probably due to the altered population of bacteria caused by administration of penicillin and streptomycin.     

Generation of Rhesus Macaque-Tropic HIV-1 Clones That Are Resistant to Major Anti-HIV-1 Restriction Factors

Human immunodeficiency virus type 1 (HIV-1) replication in macaque cells is restricted mainly by antiviral cellular APOBEC3, TRIM5α/TRIM5CypA, and tetherin proteins. For basic and clinical HIV-1/AIDS studies, efforts to construct macaque-tropic HIV-1 (HIV-1mt) have been made by us and others. Although rhesus macaques are commonly and successfully used as infection models, no HIV-1 derivatives suitable for in vivo rhesus research are available to date. In this study, to obtain novel HIV-1mt clones that are resistant to major restriction factors, we altered Gag and Vpu of our best HIV-1mt clone described previously. First, by sequence- and structure-guided mutagenesis, three amino acid residues in Gag-capsid (CA) (M94L/R98S/G114Q) were found to be responsible for viral growth enhancement in a macaque cell line. Results of in vitro TRIM5α susceptibility testing of HIV-1mt carrying these substitutions correlated well with the increased viral replication potential in macaque peripheral blood mononuclear cells (PBMCs) with different TRIM5 alleles, suggesting that the three amino acids in HIV-1mt CA are involved in the interaction with TRIM5α. Second, we replaced the transmembrane domain of Vpu of this clone with the corresponding region of simian immunodeficiency virus SIVgsn166 Vpu. The resultant clone, MN4/LSDQgtu, was able to antagonize macaque but not human tetherin, and its Vpu effectively functioned during viral replication in a macaque cell line. Notably, MN4/LSDQgtu grew comparably to SIVmac239 and much better than any of our other HIV-1mt clones in rhesus macaque PBMCs. In sum, MN4/LSDQgtu is the first HIV-1 derivative that exhibits resistance to the major restriction factors in rhesus macaque cells.