Transfer of chirality by dithiophosphate ligands and chiral discrimination in the stereoselective formation of square-planar Ni(II) complexes

In the formation reaction of Ni(2+) with the chiral racemic ligand, (R)(R)bdtp(-)/(S)(S)bdtp(-), bdtp(-) = [SSPOCH)CH(3))CH(CH(3))O](-), cyclo- O,O'-[1,2-dimethylethylene] dithiophosphato ion, the meso-complex Ni[(R)(R)(lambda)bdtp][(S)(S)(delta)-bdtp] is stereoselectively produced. The meso-complex was compared with the enantiopure crystals of (+)(589)Ni[(R)(R)(lambda)bdtp](2) or (-)(589)Ni[(S)(S)(delta)bdtp](2), as well as racemic crystals, rac-(+/-)Ni[bdtp](2), which were prepared from the solution containing the two enantiomers in a 1:1 ratio. Dissociation constants in solutions indicate different stability of the meso and enantiopure complexes depending on the solvent, whereas a more efficient crystal packing, weak H-bonding, and nonbonding interactions contribute to stabilization of the meso-species over the racemic one. Molecular structures show that the outer five-membered ligand ring adopts the half-chair conformation C(2) with either the lambda or the delta chirality and the methyl groups are in equatorial (e) positions. Enantiopure ligands of (+)(589)Ni[(R)(R)(lambda)bdtp](2) and (-)(589)Ni[(S)(S)(delta)bdtp](2) induce chirality into the symmetric SSNiSS chromophore with slightly helical distortion. Thus, their CD spectra exhibit weak negative or positive Cotton effects at 662 nm. CD spectra in L(+)- and D(-)diethyltartrate of the meso-complex and racemic crystal, rac-(+/-)Ni[bdtp](2), exhibit different weak Cotton effects of opposite sign. Complexes dissociate in methanol; rac-(+/-)Ni[bdtp](2) in methanol undergoes a crystallization-induced second-order asymmetric transformation which finally yields crystals of the meso-Ni[(R)(R)(lambda)bdtp][(S)(S)(delta)bdtp] complex.     

Neuromedin U has a novel anorexigenic effect independent of the leptin signaling pathway

Neuromedin U (NMU) is a hypothalamic neuropeptide that regulates body weight and composition. Here we show that mice lacking the gene encoding NMU (Nmu(-/-) mice) develop obesity. Nmu(-/-) mice showed increased body weight and adiposity, hyperphagia, and decreased locomotor activity and energy expenditure. Obese Nmu(-/-) mice developed hyperleptinemia, hyperinsulinemia, late-onset hyperglycemia and hyperlipidemia. Notably, however, treatment with exogenous leptin was effective in reducing body weight in obese Nmu(-/-) mice. In addition, central leptin administration did not affect NMU gene expression in the hypothalamus of rats. These results indicate that NMU plays an important role in the regulation of feeding behavior and energy metabolism independent of the leptin signaling pathway. These characteristic functions of NMU may provide new insight for understanding the pathophysiological basis of obesity.     

Identification of neuron-specific promoters in Ciona intestinalis

We isolated 5' flanking regions of four genes, Ci-Galphai1, Ci-arr, Ci-vAChTP, and Ci-vGAT, each of which is expressed in distinct sets of neurons in the central nervous system of the ascidian Ciona intestinalis, and we examined their function by introducing green fluorescent protein (GFP)-fusion constructs into Ciona embryos. The reporter gene driven by the 5' flanking region of Ci-Galphai1, Ci-arr, and Ci-vAChTP recapitulated the endogenous gene expression patterns, while that of Ci-vGAT can drive GFP expression in particular subsets of neurons expressing the endogenous gene. Deletion analysis revealed that the Ci-Galphai1 promoter consists of multiple regulatory modules controlling the expression in different types of cells. The GFP fluorescence enabled visualization of cell bodies and axons of different sets of neurons in ascidian larvae. These promoters can be a powerful tool for studying molecular mechanisms of neuronal development as well as neuron networks and functions in ascidians.     

Quercetin-Induced Melanogenesis in a Reconstituted Three-Dimensional Human Epidermal Model

Quercetin (3,5,7,3',4'-pentahydroxyflavone) is one of the most abundant natural flavonoids. It is present in various common vegetables and fruits. In this report, we examined the effect of quercetin on melanogenesis using a three-dimensional reconstituted human epidermal culture model, MelanoDerm, which is a new commercially-available cultured human epidermis containing functional melanocytes. Treatment with 10 microM quercetin induced an increase of tyrosinase activity in cultured epidermis after 3-5 days in time-dependent manner. In the quercetin-treated epidermis, furthermore, melanin content and tyrosinase expression were markedly increased, as shown by immunohistochemistry after a 7-day culture period. Ultrastructural studies clearly indicated an accumulation of mature melanosomes (stages III and IV) inside the basal layer of the cultured epidermis after the quercetin treatment. In addition, the dendrites of melanocytes extended further towards the adjacent keratinocytes after quercetin treatment. These results suggest that quercetin has an effect on maturation of melanosomes and that quercetin has the potential to induced melanogenesis in human epidermis.     

2'-benzyloxychalcone derivatives stimulate glucose uptake in 3T3-L1 adipocytes

By a cell-based glucose uptake screening assay, a chalcone derivative, 3-nitro-2'-benzyloxychalcone (compound 1) was identified. Compound 1 stimulated glucose uptake and potentiated insulin-stimulated glucose uptake in a concentration-dependent manner in 3T3-L1 adipocytes. When cells were treated with various concentrations of insulin in the presence of compound 1, marked enhancement of insulin-stimulated glucose uptake was observed at each concentration, suggesting that the compound might function as an insulin sensitizer. Preliminary study on the structure-activity relationships revealed that two aromatic benzene rings tolerated several substituents, but substitution by acidic or highly polar groups abolished the activity. Among several chalcone derivatives, 4-chloro-2'-benzyloxychalcone (compound 8) showed the highest level of activity. Compound 8-stimulated glucose uptake was almost completely inhibited by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). These results suggest that the action of chalcone derivatives is mediated via a pathway involving PI3K.     

Targeted disruption of the neurochondrin/norbin gene results in embryonic lethality

Neurochondrin/norbin is a cytoplasmic protein involved in dendrite outgrowth. The expression of the gene has been restricted to neural, bone, and chondral tissues. To identify the functions of the gene in vivo, we have generated mice with a disrupted mutation in the neurochondrin/norbin gene. Histological analysis of heterozygous mutant mice indicates the possibility of specific functions of neurochondrin/norbin in chondrocyte differentiation. We defined the expression patterns of neurochondrin/norbin-lacZ fusion protein in the central nervous system. In the developing olfactory bulb, beta-galactosidase activity was detected in the mantle layer at 12.5 dpc and the strongest activity was detected in the presumptive mitral or tufted cell layer at 15.5 dpc. beta-Galactosidase activity was also detected in the lateral choroid plexus. In homozygous (-/-) mutant mice, the disruption of the neurochondrin/norbin gene leads to early embryonic death between 3.5 and 6.5 dpc. This result indicates that neurochondrin/norbin gene function is essential for the early embryogenesis.     

Metabolism of deuterium-labeled jasmonic acid and OPC 8:0 in the potato plant (Solanum tuberosum L.)

The metabolism of deuterium-labeled (+/-)-jasmonicacid and 3-oxo-2-[(Z)pent-2'-enyl]-cyclopentan-1- octanoic acid in potato (Solanum tuberosum L.) was examined by using cultures of potato single-node stems. Deuterium-labeled (+/-)-jasmonic acid and 3-oxo-2-[(Z) pent-2'-enyl]cyclopentan-1-octanoic acid, which had been prepared from commercially available methyl (+/-)-jasmonate, were fed to the cultures, and the metabolites were extracted from the plants and analyzed by a liquid chromatography-selected ion monitoring system. The metabolism of deuterium-labeled (+/-)-jasmonic acid and 3-oxo-2-[(Z)-2'-pentenyl]cyclopentan-1-octanoic acid to 5' and 4'-O-glucopyranosyloxyjasmoic acids was strongly suggested.     

Isolation and identification of sodium 2-propenyl thiosulfate from boiled garlic (Allium sativum) that oxidizes canine erythrocytes

Sodium 2-propenyl thiosulfate was identified in boiled garlic (Allium sativum). When canine erythrocytes were incubated with sodium 2-propenyl thiosulfate, the methemoglobin concentration and Heinz body percentage in erythrocytes were both increased, indicating that the compound induced oxidative damage in canine erythrocytes. It seems that this compound is one of the causative agents of garlic-induced hemolysis in dogs.     

Poly-L-glutamine forms cation channels: relevance to the pathogenesis of the polyglutamine diseases

We report that long-chain poly-L-glutamine forms cation-selective channels when incorporated into artificial planar lipid bilayer membranes. The channel was permeable to alkali cations and H(+) ions and virtually impermeable to anions; the selectivity sequence based on the single-channel conductance was H(+) > Cs(+) > K(+) > Na(+). The cation channel was characterized by long-lived open states (often lasting for several minutes to tens of minutes) interrupted by brief closings. The appearance of the channel depended critically on the length of polyglutamine chains; ion channels were observed with 40-residue stretches, whereas no significant conductance changes were detected with 29-residue tracts. The channel-forming threshold length of poly-L-glutamine was thus between 29 and 40 residues. A molecular mechanics calculation suggests a mu-helix (. Biophys. J. 69:1130-1141) as a candidate molecular structure of the channel. The channel-forming nature of long-chain poly-L-glutamine may provide a clue to the elucidation of the pathogenetic mechanism of the polyglutamine diseases, a group of inherited neurodegenerative disorders including Huntington's disease.     

Stable supply of large amounts of human Fab from the inclusion bodies in E. coli

Recombinant human Fab-H chain and L chain were separately expressed as inclusion body using Escherichia coli. After solubilization of Fab-H chain and L chain by the reduction and S-alkyldisulphidation in 8 M urea, about 100 mg of purified Fab-H chain and about 160 mg of L chain could be obtained from 1 l of each culture by ion-exchange chromatogram in the presence of 8 M urea. Combination of the lyophilized Fab-H chain and L chain could be efficiently folded to native human Fab by using the stepwise dialysis method and the human Fab was purified with cation-exchange chromatogram. In the folding procedure, it was found that cysteamine and cystamine with positive charge were effective to improve the folding yield of human Fab. Moreover, from comparison of folding yield in the presence of ten kinds of additives, it was suggested that taurine was effective to improve the folding of human Fab. Consequently, we could obtain about 60 mg of folded human Fab from 1 l of each culture under the optimum conditions.