Measurement of protease activity of live Uronema marinun (Ciliata: Scuticociliatida) by fluorescence polarization

The proteolytic activity of live Uronema marinum was analyzed by a fluorescence polarization (FP) technique. Protease activity was measured as a decrease in the FP value using fluorescein isothiocynate (FITC)-casein as a protein substrate. A time-dependent decrease in FP occurred in plate wells containing live U. marinum. Supplementation with the cysteine protease inhibitor E-64 had no significant inhibitory effect on the decrease in FP at any of the concentrations used. In contrast, supplementation with 1,10-phenanthroline resulted in complete inhibition of proteolysis for 30 min at 1 mM and for 1 h at 2 and 5 mM. Effective inhibition of the proteolytic activity of live U. marinum by 1,10-phenanthroline indicated that metalloproteases are the main proteases excreted by U. marinum. As U. marinum has a high potential for systemically invading and destroying fish tissues, the metalloproteases excreted by live U. marinum are likely to be involved in the invasion of host tissues and the pathogenicity of the parasite.     

TRPC5 as a candidate for the nonselective cation channel activated by muscarinic stimulation in murine stomach

We investigated which transient receptor potential (TRP) channel is responsible for the nonselective cation channel (NSCC) activated by carbachol (CCh) in murine stomach with RT-PCR and the electrophysiological method. All seven types of TRP mRNA were detected in murine stomach with RT-PCR. When each TRP channel was expressed, the current-voltage relationship of mTRP5 was most similar to that recorded in murine gastric myocytes. mTRP5 showed a conductance order of Cs(+) > K(+) > Na(+), similar to that in the murine stomach. With 0.2 mM GTPgammaS in the pipette solution, the current was activated transiently in both NSCC in the murine stomach and the expressed mTRP5. Both NSCC activated by CCh in murine stomach and mTRP5 were inhibited by intracellularly applied anti-G(q/11) antibody, PLC inhibitor U-73122, IICR inhibitor 2-aminoethoxydiphenylborate, and nonspecific cation channel blockers La(3+) and flufenamate. There were two other unique properties. Both the native NSCC and mTRP5 were activated by 1-oleoyl-2-acetyl-sn-glycerol. Without the activation of NSCC by CCh, the NSCC in murine stomach was constitutively active like mTRP5. From the above results, we suggest that mTRP5 might be a candidate for the NSCC activated by ACh or CCh in murine stomach.     

Effect of dietary pectin on the production of immunoglobulins and cytokines by mesenteric lymph node lymphocytes in mouse colitis induced with dextran sulfate sodium

The present study explores the dietary effect of pectin on the MLN lymphocyte functions of mice with dextran sulfate sodium (DS)-induced colitis. We found that the immunoglobulin (Ig)A level in mesenteric lymph node (MLN) lymphocytes was high, while the IgE level was lower, in mice fed with pectin than in those fed with cellulose. Interestingly, the fecal IgA concentration of the pectin-fed mice was significantly higher than that of the cellulose-fed mice. The concentrations of interferon-gamma and interleukin (IL)-2 treated with concanavalin A (ConA) were significantly higher in the pectin-fed group than in the cellulose-fed group. Although dietary pectin did not affect the IL-4 and IL-10 levels, the activation-induced IL-4 and IL-10 secretion was lower in MLN cells of the pectin-fed mice than of the cellulose-fed mice following DS-induced colitis. Based on these findings, we propose that the effect of dietary pectin on mice with DS-induced colitis is mediated by the manipulation of Th1 cells. Furthermore, the inhibitory effect of IL-4 and IL-10 by dietary pectin may play an important role in promoting a change in Th1/Th2 balance toward Th1-dominant immunity.     

Hox genes from the earthworm Perionyx excavatus

The Hox genes of the oligochaete, Perionyx excavatus, were surveyed using PCR and phylogenetic analysis. We were able to identify 11 different Hox gene fragments. Comparative and phylogenetic analyses revealed that this oligochaete would have at least five Hox genes of the anterior group, including three copies of labial-type, five of the central group and one of the posterior group. This is the first report regarding sequence information and phylogenetic analysis of Hox genes in the earthworm.     

Synthesis of methyl beta-D-fructoside catalyzed by levansucrase from Rahnella aquatilis

Methyl beta-D-fructoside(MF) was formed from sucrose and methanol by a transfructosylation reaction using recombinant levansucrase from Rahnella aquatilis. The increase in the yield of MF formation was achieved by increasing methanol concentration. The enzyme stability at higher concentrations of methanol was maintained by lowering the reaction temperature. The optimum temperature and sucrose concentration for MF formation was 10 degrees C and 50 gL(-1) respectively and the yield of MF was 70%.     

On-line sample preparation of paraquat in human serum samples using high-performance liquid chromatography with column switching

A new ion-pair high-performance liquid chromatographic method with column-switching has been developed for the determination of paraquat in human serum samples. The diluted serum sample was injected onto a precolumn packed with LiChroprep RP-8 (25-40 μm) and polar serum components were washed out by 3% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 5 mM sodium octanesulfonate. After valve switching to inject position, concentrated compounds were eluted in the back-flush mode and separated on an Inertsil ODS-2 column with 17% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 10 mM sodium octanesulfonate. The total analysis time per sample was about 30 min and mean recovery was 98.5±2.8% with a linear range of 0.1–100 μg/ml. This method has been successfully applied to serum samples from incidents by paraquat poisoning.     

miR-185 Plays an Anti-Hypertrophic Role in the Heart via Multiple Targets in the Calcium-Signaling Pathways

MicroRNA (miRNA) is an endogenous non-coding RNA species that either inhibits RNA translation or promotes degradation of target mRNAs. miRNAs often regulate cellular signaling by targeting multiple genes within the pathways. In the present study, using Gene Set Analysis, a useful bioinformatics tool to identify miRNAs with multiple target genes in the same pathways, we identified miR-185 as a key candidate regulator of cardiac hypertrophy. Using a mouse model, we found that miR-185 was significantly down-regulated in myocardial cells during cardiac hypertrophy induced by transverse aortic constriction. To confirm that miR-185 is an anti-hypertrophic miRNA, genetic manipulation studies such as overexpression and knock-down of miR-185 in neonatal rat ventricular myocytes were conducted. The results showed that up-regulation of miR-185 led to anti-hypertrophic effects, while down-regulation led to pro-hypertrophic effects, suggesting that miR-185 has an anti-hypertrophic role in the heart. Our study further identified Camk2d, Ncx1, and Nfatc3 as direct targets of miR-185. The activity of Nuclear Factor of Activated T-cell (NFAT) and calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ) was negatively regulated by miR-185 as assessed by NFAT-luciferase activity and western blotting. The expression of phospho-phospholamban (Thr-17), a marker of CaMKIIδ activity, was also significantly reduced by miR-185. In conclusion, miR-185 effectively blocked cardiac hypertrophy signaling through multiple targets, rendering it a potential drug target for diseases such as heart failure.     

Modulation of Sweet Taste by Umami Compounds via Sweet Taste Receptor Subunit hT1R2

Although the five basic taste qualities—sweet, sour, bitter, salty and umami—can be recognized by the respective gustatory system, interactions between these taste qualities are often experienced when food is consumed. Specifically, the umami taste has been investigated in terms of whether it enhances or reduces the other taste modalities. These studies, however, are based on individual perception and not on a molecular level. In this study we investigated umami-sweet taste interactions using umami compounds including monosodium glutamate (MSG), 5’-mononucleotides and glutamyl-dipeptides, glutamate-glutamate (Glu-Glu) and glutamate-aspartic acid (Glu-Asp), in human sweet taste receptor hT1R2/hT1R3-expressing cells. The sensitivity of sucrose to hT1R2/hT1R3 was significantly attenuated by MSG and umami active peptides but not by umami active nucleotides. Inhibition of sweet receptor activation by MSG and glutamyl peptides is obvious when sweet receptors are activated by sweeteners that target the extracellular domain (ECD) of T1R2, such as sucrose and acesulfame K, but not by cyclamate, which interact with the T1R3 transmembrane domain (TMD). Application of umami compounds with lactisole, inhibitory drugs that target T1R3, exerted a more severe inhibitory effect. The inhibition was also observed with F778A sweet receptor mutant, which have the defect in function of T1R3 TMD. These results suggest that umami peptides affect sweet taste receptors and this interaction prevents sweet receptor agonists from binding to the T1R2 ECD in an allosteric manner, not to the T1R3. This is the first report to define the interaction between umami and sweet taste receptors.