Dose-different effects of orexin-A on the renal sympathetic nerve and blood pressure in urethane-anesthetized rats

Previous studies have demonstrated that central injection of orexin-A affects renal sympathetic nerve activity (RSNA) and blood pressure (BP) in both anesthetized and unanesthetized rats. In the present study, we examined, using urethane-anesthetized rats, the dose-dependent effects of intravenous (iv) or intralateral cerebral ventricular (LCV) injection of various doses of orexin-A on RSNA and BP. We found that injection of a low dose of orexin-A (10 ng iv or 0.01 ng LCV) suppressed RSNA and BP significantly. Conversely, a high dose (1000 ng iv or 10 ng LCV) of orexin-A elevated both RSNA and BP significantly. Pretreatment with either iv or LCV injection of thioperamide, a histaminergic H(3)-receptor antagonist, eliminated the effects of a low dose of orexin-A on both RSNA and BP. Both iv and LCV injection of diphenhydramine, a histaminergic H(1)-receptor antagonist, abolished the effects of a high dose of orexin-A on RSNA and BP. Furthermore, bilateral lesions of the hypothalamic suprachiasmatic nucleus (SCN) abolished the effects of both low and high doses of orexin-A on RSNA and BP. These findings suggest that orexin-A affects RSNA and BP in a dose-dependent manner and that the SCN and histaminergic nerve may be involved in the dose-different effects of orexin-A in rats.     

A Dominant Mutation in the Light-Oxygen and Voltage2 Domain Vicinity Impairs Phototropin1 Signaling in Tomato

In higher plants, blue light (BL) phototropism is primarily controlled by the phototropins, which are also involved in stomatal movement and chloroplast relocation. These photoresponses are mediated by two phototropins, phot1 and phot2. Phot1 mediates responses with higher sensitivity than phot2, and phot2 specifically mediates chloroplast avoidance and dark positioning responses. Here, we report the isolation and characterization of a Nonphototropic seedling1 (Nps1) mutant of tomato (Solanum lycopersicum). The mutant is impaired in low-fluence BL responses, including chloroplast accumulation and stomatal opening. Genetic analyses show that the mutant locus is dominant negative in nature. In dark-grown seedlings of the Nps1 mutant, phot1 protein accumulates at a highly reduced level relative to the wild type and lacks BL-induced autophosphorylation. The mutant harbors a single glycine-1484-to-alanine transition in the Hinge1 region of a phot1 homolog, resulting in an arginine-to-histidine substitution (R495H) in a highly conserved A′α helix proximal to the light-oxygen and voltage2 domain of the translated gene product. Significantly, the R495H substitution occurring in the Hinge1 region of PHOT1 abolishes its regulatory activity in Nps1 seedlings, thereby highlighting the functional significance of the A′α helix region in phototropic signaling of tomato.Being sessile in nature, plants have developed diverse sets of sensory mechanisms, integrating external cues such as light, water, and temperature to adapt their growth and development to the ambient environment. Plants have evolved a cohort of photoreceptors such as red/far-red light-sensing phytochromes (Chen and Chory, 2011), UV-A/blue light (BL)-sensing phototropins (Christie, 2007; Holland et al., 2009; Suetsugu and Wada, 2013), cryptochromes (Yu et al., 2010; Liu et al., 2011), Zeitlupe (ZTL)/Flavin-binding, Kelch repeat, F-box protein1/light-oxygen and voltage (LOV)-kelch protein2 members of the ZTL/ADAGIO putative family of photoreceptors (Suetsugu and Wada, 2013), and UV-B light-sensing UV-B resistance8 (Heijde and Ulm, 2012), enabling them to sense nearly the full range of the solar spectrum. One of the most visually obvious photoresponses of flowering plants involves the growth and orientation of organs toward or away from light, particularly during the early stages of growth and the establishment of seedlings (Iino, 1990) and during gap-filling situations in dense canopy conditions (Ballaré, 1999) for optimizing photosynthesis and interspecies/intraspecies competition. Several studies involving the relative effectiveness of different wavelengths of the solar spectrum as well as monitoring of lateral differences in light intensity revealed that the directional growth of plants is specifically mediated by the UV-A/blue region of the visible spectrum. Molecular genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants inhibited in hypocotyl curvature toward BL revealed that, among the UV-A light-/BL-specific photoreceptors, the phototropins perceive ambient light as a cue for directional growth (Liscum and Briggs, 1995; Kagawa et al., 2001).Phototropins have been identified in several plant species, ranging from the green alga Chlamydomonas reinhardtii to higher plants (Briggs et al., 2001). To date, two members of the phototropins have been reported from higher plants, phot1 and phot2, which share sequence homology (Sakai et al., 2001). Physiological analyses with Arabidopsis mutants lacking phot1 and phot2 have revealed that, in addition to regulating the hypocotyl curvature of seedlings toward BL (Huala et al., 1997; Christie et al., 1998), phototropins also regulate a diverse range of responses in flowering plants (Christie and Murphy, 2013; Hohm et al., 2013). These responses include chloroplast movements (Sakai et al., 2001), nuclear positioning (Iwabuchi et al., 2007), stomatal opening (Kinoshita et al., 2001), sun tracking (Inoue et al., 2008b), leaf expansion (Ohgishi et al., 2004), leaf movements (Inoue et al., 2005), leaf photomorphogenesis (Kozuka et al., 2011), leaf flattening (Sakamoto and Briggs, 2002), and the rapid inhibition of the growth of etiolated hypocotyls (Folta and Spalding, 2001).While both phot1 and phot2 overlap in function in regulating phototropism, chloroplast accumulation, leaf expansion, and stomatal opening, they also exhibit differential photosensitivity to BL, where phot1 is more sensitive to low-fluence BL than phot2. Both phot1 and phot2 redundantly regulate the chloroplast accumulation toward low-fluence BL, and phot2 exclusively regulates the chloroplast avoidance from high-fluence BL (Jarillo et al., 2001; Kagawa et al., 2001), while phot1 solely mediates the rapid inhibition of the elongation of etiolated hypocotyls (Folta and Spalding, 2001). Analysis of mutants downstream of blue light perception by phototropins revealed that the phototropin signaling branches out at an early step, and phot1 and phot2 trigger distinct photoresponses recruiting multiple signaling partners (Christie and Murphy, 2013; Hohm et al., 2013).Molecular characterizations have shown that phototropins are plasma membrane-associated Ser/Thr kinases containing a photosensory domain (Briggs and Christie, 2002) in the N-terminal region composed of two LOV domains (LOV1 and LOV2) and the kinase domain at the C-terminal end. The LOV1 and LOV2 domains bind the FMN as chromophore and are responsible for BL sensing by phototropin. Although phototropins characteristically possess two LOV domains, the photoregulation of phototropin activity is predominantly mediated by LOV2 (Christie, 2007). The exposure to BL also causes adduct formation between the FMN and the Cys residue in LOV domains and leads to the phosphorylation of phototropin, which is believed to be the primary step in the transmission of phototropic signals (Christie et al., 1998; Sakai et al., 2000). To decipher the functions of different domains of phototropins, many different substitution mutants of phototropins have been generated, which have enabled the elucidation of the functional significance of the different domains (Matsuoka and Tokutomi, 2005; Jones et al., 2007; Kong et al., 2007; Inoue et al., 2008a). Inoue et al. (2008a) showed that the BL-induced autophosphorylation of Ser-851 in the C-terminal kinase domain of phototropin is the primary step for initiating stomatal opening, phototropism, chloroplast accumulation, and leaf flattening. Mutational studies also revealed that the photosensory N-terminal domain of phototropin acts as a kinase inhibitor, where the LOV2 domain inhibits the activity of kinase domain by binding to it, and BL exposure is required for the dissociation of the LOV2 domain, enabling phosphorylation of the kinase domain (Matsuoka and Tokutomi, 2005; Jones et al., 2007).While our current understanding of phototropism has been greatly facilitated by the isolation of phototropins and their signaling mutants, the phot mutants identified to date are loss-of-function alleles. The lack of dominant-negative alleles or alleles with increased sensitivity to phototropic stimulus has hindered exploration into the roles of different domains of phot proteins in regulating phototropic signaling. In addition, the dearth of mutants defective in phototropin or phototropin-mediated responses has been a major bottleneck in furthering our understanding of the function of phototropins in crop species. Although phototropin homologs have been identified from a variety of crop species, including oat (Avena sativa; Zacherl et al., 1998), rice (Oryza sativa; Kanegae et al., 2000), and tomato (Solanum lycopersicum; Sharma et al., 2007; Sharma and Sharma, 2007), only the coleoptile phototropism1 mutant of rice has been isolated, which is defective in BL phototropism (Haga et al., 2005).Here, we report that in a mutant screen for nonphototropic seedlings under continuous BL, we recovered a strong dominant-negative mutation of phot1. The dominant-negative mutations are useful to elucidate redundant functions, as mutant protein in addition to suppressing its own functions can also suppress the function of its partners. The characterization of this new phot1 mutant revealed that the dominant activity is caused by the substitution of an Arg residue located in the A′α helix in the Hinge1 region between the LOV1 and LOV2 domains. Our study shows the functional importance of the A′α helix (Halavaty and Moffat, 2007) in regulating phot1-mediated signaling in tomato.     

In Vitro Susceptibility of Chlamydia pecorum to Macrolides,Tetracyclines, Quinolones and β-Lactam

The in vitro susceptibility of Chlamydia pecorum to two macrolides (clarithromycin and erythromycin), two tetracyclines (doxycycline and minocycline), two quinolones (ofloxacin and ciprofloxacin) and one β-lactam (ampicillin) was determined. The MICs were 0.004 to 0.008 μg/ml for clarithromycin, 0.008 to 0.031 μg/ml for doxycycline and minocycline, 0.063 to 0.125 μg/ml for erythromycin, 0.25 to 0.5 μg/ml for ofloxacin and 0.25 to 1.0 μg/ml for ciprofloxacin. The MIC for ampicillin was greater than 1,024 μg/ml. The results show clarithromycin and doxycycline are the two most effective drugs against C. pecorum.     

Critical Role of H2O2 Generated by NOX4 during Cellular Response under Glucose Deprivation

Glucose is the most efficient energy source, and various cancer cells depend on glycolysis for energy production. For maintenance of survival and proliferation, glucose sensing and adaptation to poor nutritional circumstances must be well organized in cancer cells. While the glucose sensing machinery has been well studied in yeasts, the molecular mechanism of glucose sensing in mammalian cells remains to be elucidated. We have reported glucose deprivation rapidly induces AKT phosphorylation through PI3K activation. We assumed that regulation of AKT is relevant to glucose sensing and further investigated the underlying mechanisms. In this study, AKT phosphorylation under glucose deprivation was inhibited by galactose and fructose, but induced by 2-deoxyglucose (2-DG). Both 2-DG treatment and glucose deprivation were found to induce AKT phosphorylation in HepG2 cells. These findings suggested that glucose transporter may not be involved in the sensing of glucose and induction of AKT phosphorylation, and that downstream metabolic events may have important roles. A variety of metabolic stresses reportedly induce the production of reactive oxygen species (ROS). In the present study, glucose deprivation was found to induce intracellular hydrogen peroxide (H₂O₂) production in HepG2 cells. N-acetylcysteine (NAC), an antioxidant reagent, reduced both the increase in cellular H₂O₂ levels and AKT phosphorylation induced by glucose deprivation. These results strongly suggest that the glucose deprivation-induced increase of H₂O₂ in the cells mediated the AKT phosphorylation. RNA interference of NOX4, but not of NOX5, completely suppressed the glucose deprivation-induced AKT phosphorylation as well as increase of the intracellular levels of ROS, whereas exogenous H₂O₂ could still induce AKT phosphorylation in the NOX4-knockdown cells. In this study, we demonstrated that the ROS generated by NOX4 are involved in the intracellular adaptive responses by recognizing metabolic flux.     

Small-molecular inhibitors of Ca2+-induced mitochondrial permeability transition (MPT) derived from muscle relaxant dantrolene

A structure consisting of substituted hydantoin linked to a 5-(halophenyl)furan-2-yl group via an amide bond was identified as a promising scaffold for development of low-molecular-weight therapeutic agents to treat vascular dysfunction, including ischemia/reperfusion injury. Among the compounds synthesized, 5-(3,5-dichlorophenyl)-N-{2,4-dioxo-3-[(pyridin-3-yl)methyl]imidazolidin-1-yl}-2-furamide (17) possessed the most potent inhibitory activity against Ca²⁺-induced mitochondrial swelling. The structural development, synthesis and structure–activity relationship of these compounds are described.     

Fire ants: What do rural and urban areas show us about occurrence, diversity,and ancestral state reconstruction?

In South America, Solenopsis saevissima and S. invicta are the most common fire ants. Nests are founded in areas under anthropic interference like urban or rural areas, but S. invicta is found preferentially in those with the greatest anthropic interference. However, we do not know the rates at which they exist in anthropized areas next to high density of native vegetation. Areas with 60 to 90% of native Atlantic Forest were selected to verify the occurrence of both species in rural and urban areas. We investigated the molecular diversity and applied the reconstruction of the ancestral state analysis for each species. A total of 186 nests were analyzed and we found that the two species had the same proportion in the urban area. However, S. saevissima had a higher rate of prevalence in the rural area, in addition to having a greater number of haplotypes and ancestry associated with this type of habitat for the region. S. invicta had the same number of haplotypes in both rural and urban regions, and less haplotypic diversity. We conclude that S. saevissima is a species typically associated with rural areas and S. invicta, although present, is not dominant in urban areas. Keywords: Biodiversity, haplotype diversity, DNA barcoding, mtDNA     

Kibizu concentrated liquid suppresses the accumulation of lipid droplets in 3T3-L1 cells

Adipocyte size is closely related to the occurrence of diabetes, metabolic syndrome, and insulin resistance. Thus, researchers are searching for active substances that function to reduce adipocyte size. In the present study, we focused on sugar cane vinegar, Kibizu, and evaluated the function of Kibizu to reduce adipocyte size by using an in vitro model system, because people in Amami Oshima famous for longevity regularly consume Kibizu. Results showed that Kibizu treatment significantly reduced the size and number of lipid droplets in 3T3-L1 cells, relative to treatment with Kurozu, another traditional vinegar. Results of an extraction experiment suggest that the active components in Kibizu are lipophilic and hydrophobic. In addition, an in vivo experiment on rats treated with Kibizu showed that the active components were contained in large vein blood. Results of an additional in vivo experiment suggest that metabolites generated by Kibizu-treated rats are primarily contained or modified specifically in the large vein blood.