Endothelin receptor antagonist reverses decreased NO system in the kidney in vivo during exercise

Vascular endothelial cells produce endothelin (ET)-1, a potent vasoconstrictor peptide, and nitric oxide (NO), a potent vasodilator substance. There are interactions between ET-1 and NO. Exercise results in a marked decrease in renal blood flow. We previously reported that exercise causes an increase of ET-1 production in the kidney, whereas production of NO in the kidney is decreased. Furthermore, we recently revealed that the magnitude of decrease in blood flow to the kidney during exercise was significantly attenuated by the administration of the endothelin-A (ET(A)) receptor antagonist, strongly suggesting that endogenously increased ET-1 participates in the decrease of blood flow in the kidney during exercise. Because it was demonstrated that ET-1 depresses NO synthase (NOS) activity of cultured cells in vitro, we hypothesized that an increase of ET-1 production in kidney during exercise contributes to a decrease of NO production in kidney in vivo. We studied whether administration of the ET(A) receptor antagonist attenuates the decreases of NOS activity and NO production in the kidney during exercise. Rats performed treadmill running for 30 min after pretreatment with an ET(A) receptor antagonist (TA-0201, 0.5 mg/kg; TA-0201-treated exercise group) or vehicle (vehicle-treated exercise group). Control rats remained at rest (vehicle-treated sedentary group). Blood flow in the kidney was decreased by this exercise, but the magnitude of the decrease after pretreatment with TA-0201 was significantly smaller than that after pretreatment with vehicle. NOS activity in kidney was significantly lower in the vehicle-treated exercise group than in the vehicle-treated sedentary group, whereas that in the TA-0201-treated exercise group was significantly higher than that in the vehicle-treated exercise group. Expressions of endothelial NOS protein and NOx, the stable end product of NO, i.e., nitrite/nitrate, concentration in the kidney were significantly lower in the vehicle-treated exercise group than in the vehicle-treated sedentary group, whereas those in the TA-0201-treated exercise group were significantly higher than those in the vehicle-treated exercise group. The data suggest that increased ET-1 production in the kidney during exercise contributes to the decreases of NOS activity and NO production. Therefore, the present study provides a possibility that the exercise-induced increase in production of ET-1 in the kidney causes a decrease in blood flow in the kidney through two pathways, i.e., vasoconstrictive action and the action of attenuating NO production.     

A purified agonist-activated G-protein coupled receptor: truncated octopus Acid Metarhodopsin

G-protein coupled receptors (GPCRs) mediate responses to many types of extracellular signals. So far, bovine rhodopsin, the inactive form of a GPCR, is the only member of the family whose three dimensional structure has been determined. It would be desirable to determine the structure of the active form of a GPCR. In this paper, we report the large scale preparation of a stable, homogenous species, truncated octopus rhodopsin (t-rhodopsin) in which proteolysis has removed the proline-rich C-terminal; this species retains the spectral properties and the ability for light-induced G-protein activation of unproteolyzed octopus rhodopsin. Moreover, starting from this species we can prepare a pure, active form of pigment, octopus t-Acid Metarhodopsin which has an all-trans-retinal as its agonist. Photoisomerization of t-Acid Metarhodopsin leads back to the inactive form, t-rhodopsin with the inverse agonist 11-cis-retinal. Octopus t-Acid Metarhodopsin can activate an endogenous octopus G-protein in the dark and this activity is reduced by irradiation with orange light which photoregenerates t-Acid Metarhodopsin back to the initial species, t-rhodopsin.     

GID2, an F-box subunit of the SCF E3 complex, specifically interacts with phosphorylated SLR1 protein and regulates the gibberellin-dependent degradation of SLR1 in rice

The phytohormone gibberellin (GA) controls growth and development in plants. Previously, we identified a rice F-box protein, gibberellin-insensitive dwarf2 (GID2), which is essential for GA-mediated DELLA protein degradation. In this study, we analyzed the biological and molecular biological properties of GID2. Expression of GID2 preferentially occurred in rice organs actively synthesizing GA. Domain analysis of GID2 revealed that the C-terminal regions were essential for the GID2 function, but not the N-terminal region. Yeast two-hybrid assay and immunoprecipitation experiments demonstrated that GID2 is a component of the SCF complex through an interaction with a rice ASK1 homolog, OsSkp15. Furthermore, an in vitro pull-down assay revealed that GID2 specifically interacted with the phosphorylated Slender Rice 1 (SLR1). Taken these results together, we conclude that the phosphorylated SLR1 is caught by the SCFGID2 complex through an interacting affinity between GID2 and phosphorylated SLR1, triggering the ubiquitin-mediated degradation of SLR1.     

Circadian-associated rice pseudo response regulators (OsPRRs): insight into the control of flowering time

A small family of plant proteins, designated PSEUDO RESPONSE REGULATORS (PRRs), is crucial for a better understanding of the molecular link between circadian rhythm and photoperiodic control of flowering time in the dicotyledonous model plant Arabidopsis thaliana. Recently, we showed that the monocotyledonous model plant Oryza sativa also has homologous members of the OsPRR family (Oryza sativa PRR). In the previous experiments with rice, we mainly characterized a japonica variety (Nipponbare). By employing an indica variety (Kasalath), in this study we further characterized OsPRRs with reference to the photoperiod sensitivity Hd (Heading date) QTL (quantitative trait loci) implicated in the control of flowering time in rice. The circadian-controlled and sequential expression profiles of the five OsPRR genes were observed not only for Nipponbare but also for Kasalath. Then each of these OsPRR genes was mapped on the rice chromosomes. Among these OsPRR genes, OsPRR37 was mapped very closely to Hd2-QTL, which was identified as the major locus that enhances the photoperiod sensitivity of flowering in Nipponbare. Furthermore, we found that Kasalath has a severe mutational lesion in the OsPRR37 coding sequence.     

Mutation analysis of the histidine residues in the glycylglycine endopeptidase ALE-1

A novel staphylolytic enzyme, ALE-1, is a glycylglycine endopeptidase produced by Staphylococcus capitis EPK1. ALE-1 possesses seven histidines. Chemical modification studies using diethylpyrocarbonate and iodoacetic acid suggested that a histidine or tyrosine residue(s) in the molecule is important for the organism's staphylolytic activity. All of the histidine residues, one tyrosine, and one aspartic acid residue in the N-terminally truncated ALE-1 (DeltaN-term ALE-1) were systematically altered by site-directed mutagenesis, and the enzyme activities and metal contents of the variants were measured. Our studies indicated that His-150, His-200, His-231, His-233, and Asp-154 are essential for the enzyme activity of DeltaN-term ALE-1. Except for His-150 and Asp-154, all of these amino acids were located within the 38-amino-acid region conserved among 11 proteins, including 5 staphylolytic endopeptidases. Inductively coupled plasma-mass spectrometric analysis of DeltaN-term ALE-1 revealed that it contains one atom of zinc per molecule. Measurement of the zinc content of the mutant DeltaN-term ALE-1 suggested that His-150 and -233 are important for zinc binding; their loss in these variant enzymes coincided with the loss of staphylolytic activity. These results strongly suggest that ALE-1 is a novel member of zinc metalloproteases.     

Differential activities of plant polyphenols on the binding and internalization of cholera toxin in vero cells

Plant polyphenols, RG-tannin, and applephenon had been reported to inhibit cholera toxin (CT) ADP-ribosyltransferase activity and CT-induced fluid accumulation in mouse ileal loops. A high molecular weight fraction of hop bract extract (HBT) also inhibited CT ADP-ribosyltransferase activity. We report here the effect of those polyphenols on the binding and entry of CT into Vero cells. Binding of CT to Vero cells or to ganglioside GM1, a CT receptor, was inhibited in a concentration-dependent manner by HBT and applephenon but not RG-tannin. These observations were confirmed by fluorescence microscopy using Cy3-labeled CT. Following toxin binding to cells, applephenon, HBT, and RG-tannin suppressed its internalization. HBT or applephenon precipitated CT, CTA, and CTB from solution, creating aggregates larger than 250 kDa. In contrast, RG-tannin precipitated CT poorly; it formed complexes with CT, CTA, or CTB, which were demonstrated with sucrose density gradient centrifugation and molecular weight exclusion filters. In agreement, CTA blocked the inhibition of CT internalization by RG-tannin. These data suggest that some plant polyphenols, similar to applephenon and HBT, bind CT, forming large aggregates in solution or, perhaps, on the cell surface and thereby suppress CT binding and internalization. In contrast, RG-tannin binding to CT did not interfere with its binding to Vero cells or GM1, but it did inhibit internalization.     

Zymographic characterization of bacteriolytic enzymes produced by oral streptococci

Zymographic analysis was performed to know the bacteriolytic enzyme profiles of 4% SDS extracts of oral streptococci, Streptococcus mutans, S. sobrinus, S. sanguis, S. mitis and S. salivarius. We investigated the five strains in each species and found that the profile was very similar among strains of the same species except for S. salivarius(the profile was classified into two types). On the other hand, the profile was considerably different among species. Two major bacteriolytic enzymes of S. mutans showing molecular mass of 80 and 100 kDa were found using SDS-boiled S. mutans or S. sobrinus cells as substrate. These bacteriolytic activities were less apparent in the gel containing S. mitis or S. salivarius, and also not detectable in the gel containing S. sanguis. S. sobrinus extract showed only one bacteriolytic band (78 kDa) as strong activity using S. sobrinus cells as substrate. S. sanguis extract showed no bacteriolytic bands using any streptococcal cells. Extracts of either S. mitis or S. salivarius showed weak activity by using respective strains as substrate.     

Dissection of the phosphorylation of rice DELLA protein, SLENDER RICE1

DELLA proteins are repressors of gibberellin signaling in plants. Our previous studies have indicated that gibberellin signaling is derepressed by SCF(GID2)-mediated proteolysis of the DELLA protein, SLENDER RICE1 (SLR1), in rice. In addition, the gibberellin-dependent increase of phosphorylated SLR1 in the loss-of-function gid2 mutant suggests that the SCF(GID2)-mediated degradation of SLR1 might be initiated by gibberellin-dependent phosphorylation. To confirm the role of phosphorylation of SLR1 in its gibberellin-dependent degradation, we revealed that SLR1 is phosphorylated on an N-terminal serine residue(s) within the DELLA/TVHYNP and polyS/T/V domain. However, gibberellin-induced phosphorylation in these regions was not observed in the gid2 mutant following the constitutive expression of SLR1 under the control of the rice actin1 promoter. Treatment with gibberellin induced both the phosphorylated and non-phosphorylated forms of SLR1 with similar induction kinetics in gid2 mutant cells. Both the phosphorylated and non-phosphorylated SLR1 proteins were degraded by gibberellin treatment with a similar half-life in the rice callus cells, and both proteins interacted with recombinant glutathione S-transferase (GST)-GID2. These results demonstrate that the phosphorylation of SLR1 is independent of its degradation and is dispensable for the interaction of SLR1 with the GID2/F-box protein.