Ghrelin Augments the Expressions and Secretions of Proinflammatory Adipokines,VEGF120 and MCP‐1, in Differentiated 3T3‐L1 Adipocytes

Ghrelin is a physiological‐active peptide with growth hormone‐releasing activity, orexigenic activity, etc. In addition, the recent study has also suggested that ghrelin possesses the pathophysiological abilities related with type 2 diabetes. However, the ghrelin‐direct‐effects implicated in type 2 diabetes on peripheral tissues have been still unclear, whereas its actions on the central nervous system (CNS) appear to induce the development of diabetes. Thus, to assess its peripheral effects correlated with diabetes, we investigated the regulatory mechanisms about adipokines, which play a central role in inducing peripheral insulin resistance, secreted from mature 3T3‐L1 adipocytes stimulated with ghrelin in vitro . The stimulation with 50 nmol/L ghrelin for 24 h resulted in the significant 1.9‐fold increase on vascular endothelial growth factor‐120 (VEGF₁₂₀) releases (p < 0.01) and the 1.7‐fold on monocyte chemoattractant protein‐1 (MCP‐1) (p < 0.01) from 3T3‐L1 adipocytes, respectively, while ghrelin failed to enhance tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), IL‐6, IL‐10 and adiponectin secretions. In addition, Akt phosphorylation on Ser473 and c‐Jun NH₂‐terminal protein kinase (JNK) phosphorylation on Thr183/Tyr185 were markedly enhanced 1.4‐fold (p < 0.01) and 1.6‐fold (p < 0.01) in the ghrelin‐stimulated adipocytes, respectively. Furthermore, the treatment with LY294002 (50 μmol/L) and Wortmannin (10nmol/L), inhibitors of phosphatidylinositol 3‐kinase (PI3K), significantly decreased the amplified VEGF₁₂₀ secretion by 29% (p < 0.01) and 28% (p < 0.01) relative to the cells stimulated by ghrelin alone, respectively, whereas these inhibitors had no effects on increased MCP‐1 release. On the other hand, JNK inhibitor SP600125 (10 μmol/L) clearly reduced the increased MCP‐1, but not VEGF₁₂₀, release by 35% relative to the only ghrelin‐stimulated cells (p < 0.01). In conclusion, ghrelin can enhance the secretions of proinflammatory adipokines, VEGF₁₂₀ and MCP‐1, but fails to affect IL‐10 and adiponectin which are considered to be anti‐inflammatory adipokines. Moreover, this augmented VEGF₁₂₀ release is invited through the activation of PI3K pathways and the MCP‐1 is through JNK pathways. Consequently, our results strongly suggest that ghrelin can induce the development of diabetes via its direct‐action in peripheral tissues as well as via in CNS. J. Cell. Physiol. 230: 199–209, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Identification of a flavin‐containing S‐oxygenating monooxygenase involved in alliin biosynthesis in garlic

S‐Alk(en)yl‐l ‐cysteine sulfoxides are cysteine‐derived secondary metabolites highly accumulated in the genus Allium. Despite pharmaceutical importance, the enzymes that contribute to the biosynthesis of S‐alk‐(en)yl‐l ‐cysteine sulfoxides in Allium plants remain largely unknown. Here, we report the identification of a flavin‐containing monooxygenase, AsFMO1, in garlic (Allium sativum), which is responsible for the S‐oxygenation reaction in the biosynthesis of S‐allyl‐l ‐cysteine sulfoxide (alliin). Recombinant AsFMO1 protein catalyzed the stereoselective S‐oxygenation of S‐allyl‐l ‐cysteine to nearly exclusively yield (R_CS_S)‐S‐allylcysteine sulfoxide, which has identical stereochemistry to the major natural form of alliin in garlic. The S‐oxygenation reaction catalyzed by AsFMO1 was dependent on the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD), consistent with other known flavin‐containing monooxygenases. AsFMO1 preferred S‐allyl‐l ‐cysteine to γ‐glutamyl‐S‐allyl‐l ‐cysteine as the S‐oxygenation substrate, suggesting that in garlic, the S‐oxygenation of alliin biosynthetic intermediates primarily occurs after deglutamylation. The transient expression of green fluorescent protein (GFP) fusion proteins indicated that AsFMO1 is localized in the cytosol. AsFMO1 mRNA was accumulated in storage leaves of pre‐emergent nearly sprouting bulbs, and in various tissues of sprouted bulbs with green foliage leaves. Taken together, our results suggest that AsFMO1 functions as an S‐allyl‐l ‐cysteine S‐oxygenase, and contributes to the production of alliin both through the conversion of stored γ‐glutamyl‐S‐allyl‐l ‐cysteine to alliin in storage leaves during sprouting and through the de novo biosynthesis of alliin in green foliage leaves.     

Detection of Staphylococcus hyicus exfoliative toxin genes by dot blot hybridization and multiplex polymerase chain reaction

We designed a novel DNA probe and novel PCR primer sets for detecting the genes coding for Staphylococcus hyicus (S. hyicus) exfoliative toxin (ET). In dot blot hybridization, the novel DNA probe hybridized with chromosomal DNA of ExhA-, ExhB-, ExhC-, ExhD-, and SHETA-producing strains. This probe also hybridized with the plasmid DNA of a SHETB-producing strain. In Southern blot hybridization, the probe hybridized with a 1.5 kb HindIII fragment of chromosomal DNA from a SHETA-producing strain. The above fragment was cloned into E. coli and the nucleotide sequence of the SHETA gene determined, this gene proved to have almost the same homology (99.6%) as the ExhB gene. It was therefore thought that SHETA is a subtype of ExhB. In multiplex PCR using five primer sets, each gene gave a band distinguishable from the others. This multiplex PCR system has high specificity among the well-known S. hyicus ET genes. Of the 69 known ET-producing S. hyicus strains, 38, 19, 10, 2 and 1 strains have exhB, exhD exhA, shetb and exhC genes, respectively.     

Efficient establishment of primary fibroblast cultures from the hawksbill sea turtle (Eretmochelys imbricata)

The hawksbill sea turtle (Eretmochelys imbricata) is a critically endangered species at a risk of extinction. Preservation of the genomic and cellular information of endangered animals is important for future genetic and biological studies. Here, we report the efficient establishment of primary fibroblast cultures from skin tissue of the hawksbill sea turtle. We succeeded in establishing 19 primary cultures from 20 hawksbill sea turtle individuals (a success rate of 95%). These cells exhibited a fibroblast-like morphology and grew optimally at a temperature of 26°C, but experienced a loss of viability when cultured at 37°C. Chromosomal analysis using the primary cells derived here revealed that hawksbill sea turtles have a 2n?=?56 karyotype. Furthermore, we showed that our primary cell cultures are free of several fish-related viruses, and this finding is important for preservation purposes. To our knowledge, this report is the first to describe primary cell cultures established from normal tissues of the hawksbill sea turtle. The results will contribute to the preservation of biodiversity, especially for the sea turtles that are critically endangered owing to human activities.     

Bestrophin genes are expressed in Xenopus development

The Bestrophin-1/VMD2 gene has been implicated in Best disease, a juvenile-onset vitelliform macular dystrophy. The Bestrophin proteins have anion channel activity, and the four mammalian members share sequence homologies in multiple transmembrane domains and an RFP-tripeptide motif. The expression patterns and functions of the Bestrophin genes in retinal pigment epithelium have been studied intensively, whereas little is known about their roles in vertebrate embryogenesis. This study examined the roles of four Xenopus tropicalis homologs of BEST genes. The xtBest genes showed spatially and temporally distinct expression. xtBest-2 was the only maternally expressed Best gene, and both xtBest-2 and the Xenopus laevis Best-2 gene were expressed at the edge of the blastopore lip including the organizer. Ectopic expression of xBest-2 caused defects in dorsal axis formation and in mesodermal gene expression during gastrulation. These results suggest a new role of the Bestrophin family genes in early vertebrate embryogenesis.     

International guide to prescribing psychotropic medication for the management of problem behaviours in adults with intellectual disabilities

Psychotropic medications are used regularly to manage problem behaviours among people with intellectual disabilities. This causes concern because often these medications are used out of their licensed indications in this context. The WPA Section on Psychiatry of Intellectual Disability has recently developed an evidence and consensus-based international guide for practitioners for the use of psychotropic medications for problem behaviours among adults with intellectual disabilities. This guide advises on assessment of behaviours, producing a formulation, initiation of treatment, assessment of out-come and adverse effects, follow-up arrangements, and possibility of discontinuation of treatment.     

Cytokine profiles in chickens infected with virulent and avirulent Marek's disease viruses: Interferon-gamma is a key factor in the protection of Marek's disease by vaccination

Marek's disease has been controlled by vaccination with avirulent strains of MDV. However, the protection mechanism following vaccination is not fully understood. In this study immune responses of PBMC and splenocytes derived from vaccinated chickens challenged with virulent MDV were examined using real-time PCR and ELISA. Higher levels of IFN-γ induction were observed in chickens vaccinated during the latent phase of infection with virulent MDV than in similarly challenged, unvaccinated chickens. Furthermore, the mean expression of IFNGR2 and IFN regulatory factor-3 mRNAs was significantly higher in vaccinated than in unvaccinated chickens. These results show that IFN-γ could be one of the important factors in prevention of MD by vaccination and is effective during the latent phase of the infection.     

How costly are ejaculates for Japanese macaques?

Much sexual selection theory is based on the idea that ejaculate is cheap. Since further details are unknown our aim was to determine the energy that primate males require for ejaculate production. We addressed this problem by measuring the energy content (in kJ) of ejaculates from Japanese macaques (Macaca fuscata) using standard bomb calorimetry. Then, we estimated the relative amount of energy that individuals require for ejaculate production by relating the net energy content of ejaculates to males daily basal metabolic rate (BMR). Fresh macaque ejaculate contains 3.0 kJ ml^–1. Assuming a mean volume of 2.7 ml an average macaque ejaculate contains 8.1 kJ. Depending on the individuals body mass (6–13 kg) and the number and volume of the ejaculates, macaque males are assumed to use between at least 0.8% and at most 6.0% of their BMR for ejaculate production per day during the breeding season. Even when regarding only the minimal energy investment of 0.8% of daily BMR for ejaculate production, clearly ejaculates come with some cost for primate males.