Geranyl modification on the tryptophan residue of ComXRO-E-2 pheromone by a cell-free system

ComX pheromone is an isoprenoidal oligopeptide containing a modified tryptophan residue, which stimulates natural genetic competence in the gram-positive bacterium Bacillus. Since posttranslational prenylation on the tryptophan residue has not been reported except in ComX pheromone, the universality of this modification has not yet been elucidated. In this paper, we established a cell-free system, whereby the tryptophan residue in peptides is modified with a geranyl group by modifying enzyme ComQ. In addition, we investigated enzymatic reaction conditions using an in vitro enzyme reaction system. This is the first report of in vitro geranylation on the tryptophan residue. This system is potentially a useful tool for elucidating the universality of prenylation on the tryptophan residue.     

Soymorphin-5, a soy-derived μ-opioid peptide, decreases glucose and triglyceride levels through activating adiponectin and PPARα systems in diabetic KKAy mice

Soymorphin-5 (YPFVV) derived from soybean β-conglycinin β-subunit is a μ-opioid agonist peptide having anxiolytic-like activity. Here, we show that soymorphin-5 improves glucose and lipid metabolism after long-term oral administration to KKAy mice, a type 2 diabetes model animal. Soymorphin-5 inhibited hyperglycemia without an increase in plasma insulin levels in KKAy mice. Soymorphin-5 also decreased plasma and liver triglyceride (TG) levels and liver weight, suggesting that soymorphin-5 improved lipid metabolism. Soymorphin-5 increased plasma adiponectin concentration and liver mRNA expression of AdipoR2, a subtype of adiponectin receptor that is involved in stimulating the peroxisome proliferator-activated receptor (PPAR)α pathway and fatty acid β-oxidation. The expressions of the mRNA of PPARα and its target genes acyl-CoA oxidase, carnitine palmitoyltransferase 1 A, and uncoupling protein-2, in the liver were also increased after oral administration of soymorphin-5. Furthermore, des-Tyr-soymorphin-5 (PFVV) without μ-opioid and anxiolytic-like activities did not decrease blood glucose levels in KKAy mice. These results suggest that μ-opioid peptide soymorphin-5 improves glucose and lipid metabolism via activation of the adiponectin and PPARα system and subsequent increases of β-oxidation and energy expenditure in KKAy mice.     

BDNF-Dependent Accumulation of Palmitoleic Acid in CNS Neurons

Although it is known that brain-derived neurotrophic factor (BDNF) plays a critical role in neuronal survival and differentiation, its effect on lipid homeostasis is poorly understood. To understand them, we here investigated the effect of BDNF on the fatty acid composition of primary neurons. A detailed analysis of the fatty acid composition of BDNF-stimulated primary neurons revealed that BDNF treatment led to a significant and selective increase in intracellular palmitoleic acid (PLO) levels. Correspondingly, BDNF induced the expression of the enzyme responsible for PLO synthesis [stearoyl-CoA desaturase-1]. In addition, this increase was suppressed by K252a, an inhibitor for tropomyosin-related kinase (Trk) receptors, indicating that BDNF-dependent increase in the PLO was mediated through the activation of TrkB. Further, PLO in culture media was reduced by BDNF treatment. This result suggested that BDNF suppressed extracellular release of PLO. Taken together, these data indicate that BDNF increases intracellular PLO both by activating its biosynthesis and by suppressing its extracellular release.     

Reduction of glucose uptake through inhibition of hexose transporters and enhancement of their endocytosis by methylglyoxal in Saccharomyces cerevisiae

Diabetes mellitus is characterized by an impairment of glucose uptake even though blood glucose levels are increased. Methylglyoxal is derived from glycolysis and has been implicated in the development of diabetes mellitus, because methylglyoxal levels in blood and tissues are higher in diabetic patients than in healthy individuals. However, it remains to be elucidated whether such factors are a cause, or consequence, of diabetes. Here, we show that methylglyoxal inhibits the activity of mammalian glucose transporters using recombinant Saccharomyces cerevisiae cells genetically lacking all hexose transporters but carrying cDNA for human GLUT1 or rat GLUT4. We found that methylglyoxal inhibits yeast hexose transporters also. Glucose uptake was reduced in a stepwise manner following treatment with methylglyoxal, i.e. a rapid reduction within 5 min, followed by a slow and gradual reduction. The rapid reduction was due to the inhibitory effect of methylglyoxal on hexose transporters, whereas the slow and gradual reduction seemed due to endocytosis, which leads to a decrease in the amount of hexose transporters on the plasma membrane. We found that Rsp5, a HECT-type ubiquitin ligase, is responsible for the ubiquitination of hexose transporters. Intriguingly, Plc1 (phospholipase C) negatively regulated the endocytosis of hexose transporters in an Rsp5-dependent manner, although the methylglyoxal-induced endocytosis of hexose transporters occurred irrespective of Plc1. Meanwhile, the internalization of hexose transporters following treatment with methylglyoxal was delayed in a mutant defective in protein kinase C.     

Radical scavenging activity of spring mountain herbs in the Shikoku mountain area and identification of antiradical constituents by simple HPLC detection and LC-MS methods

The functionality of spring mountain herbs, which were collected in the Kajigamori mountain area of Shikoku area in Japan, was investigated in the course of our studies for utilizing local plant resources. The radical scavenging activity of the extracts from seventeen herbs was measured. Among these herbs, two extracts from Polystichym ovato-paleaceum (Japanese name: Tsuyanashiinode) and Sambucus racemosa subsp. sieboldiana (Japanese name: Niwatoko) showed potent DPPH radical scavenging activity. The material evidence for the potent activity of the extracts was studied by a combination of our developed method for detecting antiradical compounds, LC-MS/MS, and enzymatic hydrolysis.     

Cytotoxic prodigiosin family pigments from Pseudoalteromonas sp. 1020R isolated from the Pacific coast of Japan

Pseudoalteromonas sp. 1020R, isolated from the Pacific coast of Japan, produces prodigiosin family pigments. Structural analysis indicated that these are prodigiosin (2-methyl-3-pentyl-prodiginine) and three other prodigiosin congeners which differ only in the lengths of the alkyl side chains. These compounds exhibited different extents of cytotoxicity against U937 leukemia cells, and cell death was accompanied by typical features of apoptosis.     

Secreted Nucleobindin-2 Inhibits 3T3-L1 Adipocyte Differentiation

Nucleobindin-2 is a 420 amino acid EF-hand Ca2+ binding protein that can be further processed to generate an 82 amino terminal peptide termed Nesfatin-1. To examine the function of secreted Nucleobindin-2 in adipocyte differentiation, cultured 3T3-L1 cells were incubated with either 0 or 100 nM of GST, GST-Nucleobindin-2, prior to and during the initiation of adipocyte differentiation. Nucleobindin-2 treatment decreased neutral lipid accumulation (Oil-Red O staining) and expression of several marker genes for adipocyte differentiation (PPARγ, aP2, and adipsin). When Nucleobindin- 2 was constitutively secreted into cultured medium, cAMP content and insulin stimulated CREB phosphorylation were significantly reduced. On the other hand, intracellularly overexpressed Nucleobindin-2 failed to affect cAMP content and CREB phosphorylation. Taken together, these data indicate that secreted Nucleobindin-2 is a suppressor of adipocyte differentiation through inhibition of cAMP production and insulin signal.     

Molecular determinants that convert hormone sensitive lipase into gibberellin receptor

Gibberellins (GAs) are tetracyclic, diterpenoid plant hormones, essential for many developmental processes in higher plants. Plants perceive GA through a nuclear-localized GA receptor, GA INSENSITIVE DWARF1 (GID1). From sequence similarity, it is suggested that GID1 evolved from a hormone-sensitive lipase (HSL), and recent x-ray crystallography of the GA-GID1 complex has given insights into how GID1 recognizes GA. Analyses of the GA signaling pathway in several plant species further suggest that the GID1-mediated GA signaling pathway emerged in the vascular plant lineage and since then regulation of GA recognition specificity seems to have been fine tuned to strictly regulate the on-off GA signal.     

Ca2+ ion transport through channels formed by α-hemolysin analyzed using a microwell array on a Si substrate

For the functional analysis of ion channel activity, an artificial lipid bilayer suspended over microwells was formed that ruptured giant unilamellar vesicles on a Si substrate. Ca(2+) ion indicators (fluo-4) were confined in the microwells by sealing the microwells with a lipid bilayer. An overhang formed at the microwells prevented the lipid membrane from falling into them and allowed the stable confinement of the fluorescent probes. The transport of Ca(2+) ions through the channels formed by α-hemolysin inserted in a lipid membrane was analyzed by employing the fluorescence intensity change of fluo-4 in the microwells. The microwell volume was very small (1-100 fl), so a highly sensitive monitor could be realized. The detection limit is several tens of ions/s/μm(2), and this is much smaller than the ion current in a standard electrophysiological measurement. Smaller microwells will make it possible to mimic a local ion concentration change in the cells, although the signal to noise ratio must be further improved for the functional analysis of a single channel. We demonstrated that a microwell array with confined fluorescent probes sealed by a lipid bilayer could constitute a basic component of a highly sensitive biosensor array that works with functional membrane proteins. This array will allow us to realize high throughput and parallel testing devices.