Prodrugs of CL316243: a selective beta3-adrenergic receptor agonist for treating obesity and diabetes.

CL316243 is a highly selective and potent beta3-adrenergic receptor agonist, and has been shown in rodent models to be an effective agent for treating obesity and Type II diabetes. To improve the oral absorption and pharmacokinetic profiles of CL316243, a number of prodrugs have been synthesized and evaluated. Several ester-type prodrugs show significant improvements in oral bioavailability in both rodent and primate models.     

Discovery of SMP-304, a novel benzylpiperidine derivative with serotonin transporter inhibitory activity and 5-HT1A weak partial agonistic activity showing the antidepressant-like effect

We report the discovery of a novel benzylpiperidine derivative with serotonin transporter (SERT) inhibitory activity and 5-HT_1A receptor weak partial agonistic activity showing the antidepressant-like effect. The 3-methoxyphenyl group and the phenethyl group of compound 1, which has weak SERT binding activity, but potent 5-HT_1A binding activity, were optimized, leading to compound 35 with potent and balanced dual SERT and 5-HT_1A binding activity, but also potent CYP2D6 inhibitory activity. Replacement of the methoxy group in the left part of compound 35 with a larger alkoxy group, such as ethoxy, isopropoxy or methoxy-ethoxy group ameliorated CYP2D6 inhibition, giving SMP-304 as a candidate. SMP-304 with serotonin uptake inhibitory activity and 5-HT_1A weak partial agonistic activity, which could work as a 5-HT_1A antagonist, displayed faster onset of antidepressant-like effect than a representative SSRI paroxetine in an animal model.     

Chronic elevated calcium blocks AMPK-induced GLUT-4 expression in skeletal muscle

Muscle contraction stimulates glucose transport independent of insulin. Glucose uptake into muscle cells is positively related to skeletal muscle-specific glucose transporter (GLUT-4) expression. Therefore, our objective was to determine the effects of the contraction-mediated signals, calcium and AMP-activated protein kinase (AMPK), on glucose uptake and GLUT-4 expression under acute and chronic conditions. To accomplish this, we used pharmacological agents, cell culture, and pigs possessing genetic mutations for increased cytosolic calcium and constitutively active AMPK. In C2C12 myotubes, caffeine, a sarcoplasmic reticulum calcium-releasing agent, had a biphasic effect on GLUT-4 expression and glucose uptake. Low-concentration (1.25 to 2 mM) or short-term (4 h) caffeine treatment together with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR), had an additive effect on GLUT-4 expression. However, high-concentration (2.5 to 5 mM) or long-term (4 to 30 h) caffeine treatment decreased AMPK-induced GLUT-4 expression without affecting cell viability. The negative effect of caffeine on AICAR-induced GLUT-4 expression was reduced by dantrolene, which desensitizes the ryanodine receptor. Consistent with cell culture data, increases in GLUT-4 mRNA and protein expression induced by AMPK were blunted in pigs possessing genetic mutations for both increased cytosolic calcium and constitutively active AMPK. Altogether, these data suggest that chronic exposure to elevated cytosolic calcium concentration blocks AMPK-induced GLUT-4 expression in skeletal muscle.     

The role of ether-a-go-go-related gene K(+) channels in glucocorticoid inhibition of adrenocorticotropin release by rat pituitary cells

The present study investigated the role of K(+) channels in the inhibitory effect of glucocorticoid on adrenocorticotropin (ACTH) release induced by corticotropin-releasing hormone (CRH) using cultured rat anterior pituitary cells. Apamin and charybdotoxin (CTX) did not have a significant effect on ACTH release induced by CRH (1 nM). Tetraethylammonium (TEA), a broad spectrum K(+) channel blocker, increased the ACTH response to CRH only at the highest concentration (10 mM). The exposure to 100 nM corticosterone for 60 min inhibited the CRH-induced ACTH release. Neither TEA, apamin, nor CTX affected the inhibitory effect of corticosterone. In contrast, astemizole (Ast) and E-4031, ether-a-go-go-related gene (erg) K(+) channel blockers, abolished the inhibitory effect of corticosterone on CRH-induced ACTH release (1.25+/-0.10 vs. 1.45+/-0.11 ng/well at 10 microM Ast, p>0.05, 1.71+/-0.16 vs. 1.91+/-0.32 ng/well at 10 microM E-4031, p>0.05, vehicle vs. corticosterone). RT-PCR demonstrated all three subtypes of rat-erg mRNAs in the pituitary and corticosterone increased only erg1 mRNA up to 2.47+/-0.54 fold. In conclusion, erg K(+) channels were up-regulated by glucocorticoid, and have indispensable roles in delayed glucocorticoid inhibition of CRH-induced ACTH release by rat pituitary cells.     

Ants on plants: a meta-analysis of the role of ants as plant biotic defenses

We reviewed the evidence on the role of ants as plant biotic defenses, by conducting meta-analyses for the effects of experimental removal of ants on plant herbivory and fitness with data pooled from 81 studies. Effects reviewed were plant herbivory, herbivore abundance, hemipteran abundance, predator abundance, plant biomass and reproduction in studies where ants were experimentally removed (n = 273 independent comparisons). Ant removal exhibited strong effects on herbivory rates, as plants without ants suffered almost twice as much damage and exhibited 50% more herbivores than plants with ants. Ants also influenced several parameters of plant fitness, as plants without ants suffered a reduction in biomass (−23.7%), leaf production (−51.8%), and reproduction (−24.3%). Effects were much stronger in tropical regions compared to temperate ones. Tropical plants suffered almost threefold higher herbivore damage than plants from temperate regions and exhibited three times more herbivores. Ant removal in tropical plants resulted in a decrease in plant fitness of about 59%, whereas in temperate plants this reduction was not statistically significant. Ant removal effects were also more important in obligate ant–plants (=myrmecophytes) compared to plants exhibiting facultative relationships with hemiptera or those plants with extrafloral nectaries and food bodies. When only tropical plants were considered and the strength of the association between ants and plants taken into account, plants with obligate association with ants exhibited almost four times higher herbivory compared to plants with facultative associations with ants, but similar reductions in plant reproduction. The removal of a single ant species increased plant herbivory by almost three times compared to the removal of several ant species. Altogether, these results suggest that ants do act as plant biotic defenses, but the effects of their presence are more pronounced in tropical systems, especially in myrmecophytic plants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. N. P. de U. Barbosa, L. Diniz, Y. Oki and F. Pezzini contributed equally to this work and are listed in alphabetical order.     

Mobilization and Acquisition of Sparingly Soluble P-Sources by Brassica Cultivars under P-Starved Environment I. Differential Growth Response,P-Efficiency Characteristics and P-Remobilization

Phosphorus （P） starvation is highly notorious for limiting plant growth around the globe. To combat P-starvation, plants constantly sense the changes in their environment, and elicit an elegant myriad of plastic responses and rescue strategies to enhance P-solublization and acquisition from bound soil P-forms. Relative growth responses, P-solublization and P- acquisition ability of 14 diverse Brassica cultivars grown with sparingly soluble P-sources （Rock-P （RP） and Ca3（PO4）2 （TCP）） were evaluated in a solution culture experiment. Cultivars showed considerable genetic diversity in terms of biomass accumulation, concentration and contents of P and Ca in shoots and roots, P-stress factor （PSF） and P use efficiency. Cultivars showed variable P-stress tolerance, and cultivars depicting low PSF and high P-efficiency values were better adaptable to P-starvation. In experiment 2, after initial feeding on optimum nutrition for 12 d after transplanting （DAT）, class-I （low P-tolerant （Oscar and Con-II）） and class-II （low P-sensitive （Gold Rush and RL-18）） cultivars were exposed to P-free environment for 25 d. All of the cultivars remobilized P from above ground parts to their roots during growth in P-free environment, the magnitude of which was variable in tested cultivars. P-concentrations （[P]s） at 37 DAT were higher in developing compared with developed leaves. Translocation of absorbed P from metabolically inactive to active sites in P- stressed plants may have helped class-I cultivars to establish a better rooting system, which provided a basis for enhanced P-utilization efficiency （PUE） and tolerance against P-stress. By supplying TCP and RP spatially separated from other nutrients in split root study, class-I cultivars were still able to mobilize RP and TCP more efficiently compared with class-II cultivars. To compare the growth behavior under P-stress, cultivars were grown in pots for 41 d after sowing, using a soil low in P （NaHCO3-extractable P = 3.97 mg/kg, M     

Mobilization and Acquisition of Sparingly Soluble P-Sources by Brassica Cultivars under P-Starved Environment II. Rhizospheric pH changes,Redesigned Root Architecture and Pi-Uptake Kinetics

Non-mycorrhizal Brassica does not produce specialized root structures such as cluster or dauciform roots but is an effective user of P compared with other crops. In addition to P-uptake, utilization and remobilization activity, acquisition of orthophosphate （Pi） from extracellular sparingly P-sources or unavailable bound P-forms can be enhanced by biochemical rescue mechanisms such copious H＋-efflux and/or carboxylates exudation into rhizosphere by roots via plasmalemma H＋ ATPase and anion channels triggered by P-starvation. To visualize the dissolution of sparingly soluble Ca-phosphate （Ca-P）, newly formed Ca-P was suspended in agar containing other essential nutrients. With NH4＋ applied as the N source, the precipitate dissolved in the root vicinity can be ascribed to rhizosphere acidification, whereas no dissolution occurred with nitrate nutrition. To observe in situ rhizospheric pH changes, images were recorded after embedding the roots in agar containing bromocresol purple as a pH indicator. P-tolerant cultivar showed a greater decrease in pH than the sensitive cultivar in the culture media （the appearance of typical patterns of various colors of pH indicator in the root vicinity）, and at stress P-level this acidification was more prominent. In experiment 2, low P-tolerant class-I cultivars （Oscar and Con-II） showed a greater decrease in solution media pH than low P-sensitive class-II （Gold Rush and RL-18） cultivars, and P-contents of the cultivars was inversely related to decrease in culture media pH. To elucidate P-stress- induced remodeling and redesigning in a root architectural system, cultivars were grown in rhizoboxes in experiment 3. The elongation rates of primary roots increased as P-supply increased, but the elongation rates of the branched zones of primary roots decreased. The length of the lateral roots and topological index values increased when cultivars were exposed to a P-stress environment. To elucidate Pi-uptake kinetics, parameters related to P influx： maximal     

The HECT Domain of the Ubiquitin Ligase Rsp5 Contributes to Substrate Recognition

Ubiquitin modification of endosomal membrane proteins is a signal for active inclusion into the Multivesicular Body (MVB) pathway, resulting in lysosomal degradation. However, the endosome represents a dynamic site of protein sorting with a majority of proteins destined for recycling, rather than MVB targeting. Substrate recognition by ubiquitin ligases is therefore highly regulated. We have investigated substrate recognition by the Nedd4 ortholog Rsp5 as a model for understanding ligase-substrate interactions. Rsp5 interacts directly with its substrate Cps1 via a novel interaction mode. Perturbation of this mode of interaction revealed a compensatory role for the Rsp5 adaptor Bsd2. These results highlight the ability of Rsp5 to interact with substrates via multiple modalities, suggesting additional mechanisms of regulating this interaction and relevant outcomes.     

Function of α subunit of heterotrimeric G protein in brassinosteroid response of rice plants

The α subunit of heterotrimeric G-proteins (Gα) is involved in a broad range of aspects of the brassinosteroid (BR) response, such as the enhancement of lamina bending. However, it has been suggested from epistatic analysis of d1 and d61, which are mutants deficient for Gα and the BR receptor BRI1, that Gα and BRI1 may function via distinct pathways in many cases. In this study, we investigated further the genetic interaction between Gα and BRI1. We report the analysis of transformants of T65d1 and T65d1/d61-7 into which were introduced a constitutively active form of Gα, Q223L. The application of 24-epi-brassinolide (24-epiBL) to T65d1 expressing Q223L still resulted in elongation of the coleoptile and, in fact, it was enhanced over the wild-type plant (WT) level in a concentration dependent manner. In T65d1/d61-7 expressing Q223L, the seed size was enlarged over that of d61-7 due to activation of Gα. These results suggest that Q223L is able to augment the BR response in response to 24-epiBL and also that Q223L functions independently of BRI1 in the process of determining seed morphology, given that Q223L was functional in the BRI1-deficient mutant, d61-7.Key words: brassinosteroid, BRASSINOSTEROID INSENSITIVE1 (BRI1), genetic interaction, G-protein α subunit, rice plants, seed morphology, transgenic plants