Aminophospholipid glycation and its inhibitor screening system: a new role of pyridoxal 5'-phosphate as the inhibitor

Peroxidized phospholipid-mediated cytotoxity is involved in the pathophysiology of a number of diseases [i.e., the abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) found in the plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (deoxy-D-fructosyl PE, or Amadori-PE), because Amadori-PE causes oxidative stress. However, lipid glycation inhibitor has not been discovered yet because of the lack of a lipid glycation model useful for inhibitor screening. We optimized and developed a lipid glycation model considering various reaction conditions (glucose concentration, temperature, buffer type, and pH) between PE and glucose. Using the developed model, various protein glycation inhibitors (aminoguanidine, pyridoxamine, and carnosine), antioxidants (ascorbic acid, alpha-tocopherol, quercetin, and rutin), and other food compounds (L-lysine, L-cysteine, pyridoxine, pyridoxal, and pyridoxal 5'-phosphate) were evaluated for their antiglycative properties. Pyridoxal 5'-phosphate and pyridoxal (vitamin B(6) derivatives) were the most effective antiglycative compounds. These pyridoxals could easily be condensed with PE before the glucose/PE reaction occurred. Because PE-pyridoxal 5'-phosphate adduct was detectable in human red blood cells and the increased plasma Amadori-PE concentration in streptozotocin-induced diabetic rats was decreased by dietary supplementation of pyridoxal 5'-phosphate, it is likely that pyridoxal 5'-phosphate acts as a lipid glycation inhibitor in vivo, which possibly contributes to diabetes prevention.     

Molecular evidence demonstrating the basidiomycetous fungus Cryptococcus curvatus is the dominant microbial eukaryote in sediment at the Kuroshima Knoll methane seep

The Kuroshima Knoll, located in the southern Ryukyu Arc, is known to actively bubble with gas containing methane and hydrogen sulfide from numerous fissures in the large carbonate pavement. Although ecological studies regarding macrobenthos and bacteria from Kuroshima Knoll have been intensively conducted, the community structure and ecological importance of microbial eukaryotes (protists) have not yet been investigated. In the present study, we directly extracted DNA from sediment of the Kuroshima Knoll at a depth of 640 m and constructed genetic libraries of PCR-amplified eukaryotic small-subunit ribosomal DNA (SSU rDNA). Although the SSU rDNA sequences of several types of benthic foraminifers were retrieved from the surface of the sediment, all other sequences (just below the sediment surface to approximately 9 cm below sediment surface) were derived from the basidiomycetous yeast Cryptococcus curvatus. Furthermore, sequences of the internal transcribed spacer of rDNA (ITS-rDNA) retrieved from the same sediment were identical to that of C. curvatus originating from terrestrial habitats. The diversity of microbial eukaryotes in the Kuroshima Knoll sediment seems to be extremely low and significantly different from that of other marine environments previously reported.     

Possible molecular mechanisms of species recognition by barnacle larvae inferred from multi-specific sequencing analysis of proteinaceous settlement-inducing pheromone

Gregarious settlement is essential for reproduction and survival of many barnacles. A glycoprotein, settlement-inducing protein complex (SIPC) has been recognized as a signal for settlement and it is expressed in both conspecific adults and larvae. Although the settlement-inducing activities of SIPC are species-specific, the molecular-based mechanism by which larvae distinguish conspecific SIPC from the SIPC of other species is still unknown. Here, the complete primary structure of the SIPC of Megabalanus coccopoma, as well as the partial structure of the SIPCs of Balanus improvisus, Megabalanus rosa, and Elminius modestus are reported. These SIPCs contain highly variable regions that possibly modulate the affinity for the receptor, resulting in the species specificity of SIPC. In addition, the distribution patterns of potential N-glycosylation sites were seen to be different among the various species. Differences in such post-translational modifications may contribute to the species specificity of SIPC.     

Development of a glutathione production process from proteinaceous biomass resources using protease-displaying <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Glutathione is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae, and supplementation of fermentation with several amino acids can increase intracellular GSH content. More recently, however, focus has been given to protein as a resource for biofuel and fine chemical production. We demonstrate that expression of a protease on the cell surface of S. cerevisiae enables the direct use of keratin and soy protein as a source of amino acids and that these substrates enhanced intracellular GSH content. Furthermore, fermentation using soy protein also enhanced cell concentration. GSH fermentation from keratin and to a greater extent from soy protein using protease-displaying yeast yielded greater GSH productivity compared to GSH fermentation with amino acid supplementation. This protease-displaying yeast is potentially applicable to a variety of processes for the bio-production of value-added chemicals from proteinaceous biomass resources.     

Improved identification of agonist-mediated Gα(i)-specific human G-protein-coupled receptor signaling in yeast cells by flow cytometry

Flow cytometry enables comparative quantification, population analysis, and high-throughput screening of agonist-mediated G-protein-coupled receptor (GPCR) signaling in genetically engineered yeasts. By using flow cytometry, we found that transformation of yeast cells with a low plasmid number is critical both for the construction of large screening libraries and for stable signal transmission in cell ensembles. Based on these findings, we constructed an engineered yeast strain for the improved identification of signal promotion by Gα(i)-specific human GPCRs using flow cytometry.     

Characterization of a bacterial laminaribiose phosphorylase

Bacterial laminaribiose phosphorylase (LBP(bac)) was first identified and purified from cell-free extract of Paenibacillus sp. YM-1. It phosphorolyzed laminaribiose into α-glucose 1-phosphate and glucose, but did not phosphorolyze other glucobioses. It slightly phosphorolyzed laminaritriose and higher laminarioligosaccharides. The specificity of the degree of polymerization of the substrate was clearly different from that of the enzyme of Euglena gracilis (LBP(Eug)): LBP(bac) was more specific to laminaribiose than LBP(Eug). It showed acceptor specificity in reverse phosphorolysis similar to LBP(Eug). Cloning of the gene encoding LBP(bac) (lbpA) has revealed that LBP(bac) is a member of the glucoside hydrolase family 94, which includes cellobiose phosphorylase, cellodextrin phosphorylase, and N,N'-diacetylchitobiose phosphorylase. The genes that encode the components of an ATP-binding cassette sugar transporter specific to laminarioligosaccharides were identified upstream of lbpA, suggesting that the role of LBP(bac) is to utilize laminaribiose generated outside the cell. This role is different from that of LBP(Eug), which participates in the utilization of paramylon, the intracellular storage 1,3-β-glucan.     

Construction of a metagenomic library for the marine sponge Halichondria okadai

Symbionts of the marine sponge Halichondria okadai are promising as a source of natural products. Metagenomic technology is a powerful tool for accessing the genetic and biochemical potential of bacteria. Hence, we established a method of recovering bacterial-enriched metagenomic DNA by stepwise centrifugation. The metagenomic DNA was analyzed by ultrafast 454-pyrosequencing technology, and the results suggested that more than three types of bacterial DNA, Alphaproteobacteria, Actinobacteria, and Cyanobacteria, had been recovered, and that eukaryotic genes comprised only 0.02% of the metagenomic DNA. These results indicate that stepwise centrifugation and real-time quantitative PCR were effective for separating sponge cells and symbiotic bacteria, and that we constructed a bacteria-enriched metagenomic library from a marine sponge, H. okadai, selectively for the first time.     

Comparison of vildagliptin twice daily vs. sitagliptin once daily using continuous glucose monitoring (CGM): Crossover pilot study (J-VICTORIA study)

ABSTRACT: BACKGROUND: No previous studies have compared the DPP-4 inhibitors vildagliptin and sitagliptin in terms of blood glucose levels using continuous glucose monitoring (CGM) and cardiovascular parameters. METHODS: Twenty patients with type 2 diabetes mellitus were randomly allocated to groups who received vildagliptin then sitagliptin, or vice versa. Patients were hospitalized at 1 month after starting each drug, and CGM was used to determine: 1) mean (+/- standard deviation) 24-hour blood glucose level, 2) mean amplitude of glycemic excursions (MAGE), 3) fasting blood glucose level, 4) highest postprandial blood glucose level and time, 5) increase in blood glucose level after each meal, 6) area under the curve (AUC) for blood glucose level [greater than or equal to]180 mg/dL within 3 hours after each meal, and 7) area over the curve (AOC) for daily blood glucose level <70 mg/dL. Plasma glycosylated hemoglobin (HbA1c), glycoalbumin (GA), 1,5-anhydroglucitol (1,5AG), immunoreactive insulin (IRI), C-peptide immunoreactivity (CPR), brain natriuretic peptide (BNP), and plasminogen activator inhibitor-1 (PAI-1) levels, and urinary CPR levels, were measured. RESULTS: The mean 24-hour blood glucose level was significantly lower in patients taking vildagliptin than sitagliptin (142.1 +/- 35.5 vs. 153.2 +/- 37.0 mg/dL; p = 0.012). In patients taking vildagliptin, MAGE was significantly lower (110.5 +/- 33.5 vs. 129.4 +/- 45.1 mg/dL; p = 0.040), the highest blood glucose level after supper was significantly lower (206.1 +/- 40.2 vs. 223.2 +/- 43.5 mg/dL; p = 0.015), the AUC ([greater than or equal to]180 mg/dL) within 3 hours was significantly lower after breakfast (484.3 vs. 897.9 mg/min/dL; p = 0.025), and urinary CPR level was significantly higher (97.0 +/- 41.6 vs. 85.2 +/- 39.9 mug/day; p = 0.008) than in patients taking sitagliptin. There were no significant differences in plasma HbA1c, GA, 1,5AG, IRI, CPR, BNP, or PAI-1 levels between patients taking vildagliptin and sitagliptin. CONCLUSIONS: CGM showed that mean 24-hour blood glucose, MAGE, highest blood glucose level after supper, and hyperglycemia after breakfast were significantly lower in patients with type 2 diabetes mellitus taking vildagliptin than those taking sitagliptin. There were no significant differences in BNP and PAI-1 levels between patients taking vildagliptin and sitagliptin. Trial registration UMIN000007687 KEYWORDS: Vildagliptin; Sitagliptin; Continuous glucose monitoring (CGM); Brain natriuretic peptide (BNP); plasminogen activator inhibitor-1 (PAI-1).     

Impact of the genome wide supported NRGN gene on anterior cingulate morphology in schizophrenia

Background

The rs12807809 single-nucleotide polymorphism in NRGN is a genetic risk variant with genome-wide significance for schizophrenia. The frequency of the T allele of rs12807809 is higher in individuals with schizophrenia than in those without the disorder. Reduced immunoreactivity of NRGN, which is expressed exclusively in the brain, has been observed in Brodmann areas (BA) 9 and 32 of the prefrontal cortex in postmortem brains from patients with schizophrenia compared with those in controls.

Methods

Genotype effects of rs12807809 were investigated on gray matter (GM) and white matter (WM) volumes using magnetic resonance imaging (MRI) with a voxel-based morphometry (VBM) technique in a sample of 99 Japanese patients with schizophrenia and 263 healthy controls.

Results

Although significant genotype-diagnosis interaction either on GM or WM volume was not observed, there was a trend of genotype-diagnosis interaction on GM volume in the left anterior cingulate cortex (ACC). Thus, the effects of NRGN genotype on GM volume of patients with schizophrenia and healthy controls were separately investigated. In patients with schizophrenia, carriers of the risk T allele had a smaller GM volume in the left ACC (BA32) than did carriers of the non-risk C allele. Significant genotype effect on other regions of the GM or WM was not observed for either the patients or controls.

Conclusions

Our findings suggest that the genome-wide associated genetic risk variant in the NRGN gene may be related to a small GM volume in the ACC in the left hemisphere in patients with schizophrenia.     

Jacaric acid,a linolenic acid isomer with a conjugated triene system,has a strong antitumor effect in vitro and in vivo

In this study, we compared the cytotoxic effects of natural conjugated linolenic acids (CLnAs) on human adenocarcinoma cells (DLD-1) in vitro, with the goal of finding CLnA isomers with strong cytotoxic effects. The antitumor effect of the CLnA with the strongest cytotoxic effect was then examined in mice. The results showed that all CLnA isomers have strong cytotoxic effects on DLD-1 cells, with jacaric acid (JA) having the strongest effect. Examination of the mechanism of cell death showed that CLnAs induce apoptosis in DLD-1 cells via lipid peroxidation. The intracellular levels of incorporated CLnAs were measured to examine the reason for differences in cytotoxic effects. These results showed that JA was taken into cells efficiently. Collectively, these results suggest that the cytotoxic effect of CLnAs is dependent on intracellular incorporation and induction of apoptosis via lipid peroxidation. JA also had a strong preventive antitumor effect in vivo in nude mice into which DLD-1 cells were transplanted. These results suggest that JA can be used as a dietary constituent for prevention of cancer.