Up-regulation of adipogenin, an adipocyte plasma transmembrane protein, during adipogenesis

Until now, the various proteins highly expressed in adipose tissues have been identified and characterized by traditional gene cloning techniques. However, methods of computer analysis have been developed to compare the levels of expression among various tissues, and genes whose expression levels differ significantly between tissues have been found. Among these genes, we report on the possible function of a new adipose-specific gene, showed higher expression in adipose tissue through ‘Search Expression’ on Genome Institute of Norvartis Research Foundation (GNF) SymAtlas v0.8.0. This database has generated and analyzed gene expression of each gene in diverse samples of normal tissues, organs, and cell lines. This newly discovered gene product was named adipogenin because of its role in stimulating adipocyte differentiation and development. Adipogenin mRNA was highly expressed in four different fat depots, and exclusively expressed in adipocytes isolated from adipose tissues. The level of adipogenin mRNA was up-regulated in the subcutaneous and visceral adipose tissues of mice fed a high-fat diet compared to those on the control diet. The expression of adipogenin mRNA is dramatically elevated during adipocyte differentiation of 3T3-L1 cells. Troglitazone, which up-regulated peroxisome proliferators-activated receptor γ2 (PPAR-γ2) expression, increased adipogenin mRNA expression, although this gene was down-regulated by retinoic acid. Confocal image analyses of green-fluorescent protein-adipogenin (pEGFP-adipogenin) transiently expressed in 3T3-L1 adipocytes showed that adipogenin was strictly localized to membranes and was absent from the cytosol. Moreover, small interfering RNA (siRNA) mediated a reduction of adipogenin mRNA in 3T3-L1 cells and blocked the process of adipocyte differentiation. These results indicate that adipogenin, an adipocyte-specific membrane protein, may be involved with adipogenesis, as one of the regulators of adipose tissue development.Yeon-Hee Hong and Daisuke Hishikawa contributed equally to this work     

Restraint stress delays solid gastric emptying via a central CRF and peripheral sympathetic neuron in rats

Central corticotropin-releasing factor (CRF) delays gastric emptying through the autonomic nervous system. CRF plays an important role in mediating delayed gastric emptying induced by stress. However, it is not clear whether a sympathetic or parasympathetic pathway is involved in the mechanism of central CRF-induced inhibition of solid gastric emptying. The purpose of this study was to investigate whether 1) CRF inhibits solid gastric emptying via a peripheral sympathetic pathway and 2) stress-induced inhibition of solid gastric emptying is mediated via a central CRF and peripheral sympathetic pathways. Using male Sprague-Dawley rats, CRF was injected intracisternally with or without various adrenergic-blocking agents. To investigate whether central CRF-induced inhibition of solid gastric emptying is mediated via a peripheral sympathetic pathway, rats underwent celiac ganglionectomy 1 wk before the gastric emptying study. After solid meal ingestion (90 min), gastric emptying was calculated. To investigate the role of endogenous CRF in stress-induced delayed gastric emptying, a CRF type2 receptor antagonist, astressin2-B, was intracisternally administered. Rats were subjected to a restraint stress immediately after the feeding. Intracisternal injection of CRF (0.1-1.0 microg) dose-dependently inhibited solid gastric emptying. The inhibitory effect of CRF on solid gastric emptying was significantly blocked by guanethidine, propranolol, and celiac ganglionectomy but not by phentolamine. Restraint stress significantly delayed solid gastric emptying, which was improved by astressin2-B, guanethidine, and celiac ganglionectomy. Our research suggests that restraint stress inhibits solid gastric emptying via a central CRF type2 receptor and peripheral sympathetic neural pathway in rats.     

Distinct roles of nitric oxide synthases and interstitial cells of Cajal in rectoanal relaxation

Nitric oxide (NO) relaxes the internal anal sphincter (IAS), but its enzymatic source(s) remains unknown; neuronal (nNOS) and endothelial (eNOS) NO synthase (NOS) isoforms could be involved. Also, interstitial cells of Cajal (ICC) may be involved in IAS relaxation. We studied the relative roles of nNOS, eNOS, and c-Kit-expressing ICC for IAS relaxation using genetic murine models. The basal IAS tone and the rectoanal inhibitory reflex (RAIR) were assessed in vivo by a purpose-built solid-state manometric probe and by using wild-type, nNOS-deficient (nNOS-/-), eNOS-deficient (eNOS-/-), and W/W(v) mice (lacking certain c-Kit-expressing ICC) with or without L-arginine or N(omega)-nitro-L-arginine methyl ester (L-NAME) treatment. Moreover, the basal tone and response to electrical field stimulation (EFS) were studied in organ bath using wild-type and mutant IAS. In vivo, the basal tone of eNOS-/- was higher and W/W(v) was lower than wild-type and nNOS-/- mice. L-arginine administered rectally, but not intravenously, decreased the basal tone in wild-type, nNOS-/-, and W/W(v) mice. However, neither L-arginine nor L-NAME affected basal tone in eNOS-/- mice. In vitro, L-arginine decreased basal tone in wild-type and nNOS-/- IAS but not in eNOS-/- or wild-type IAS without mucosa. The in vivo RAIR was intact in wild-type, eNOS-/-, and W/W(v) mice but absent in all nNOS-/- mice. EFS-induced IAS relaxation was also reduced in nNOS-/- IAS. Thus the basal IAS tone is largely controlled by eNOS in the mucosa, whereas the RAIR is controlled by nNOS. c-Kit-expressing ICC may not be essential for the RAIR.     

Maintenance of neuronal positions in organized ganglia by SAX-7, a Caenorhabditis elegans homologue of L1

The L1 family of cell adhesion molecules is predominantly expressed in the nervous system. Mutations in human L1 cause neuronal diseases such as HSAS, MASA, and SPG1. Here we show that sax-7 gene encodes an L1 homologue in Caenorhabditis elegans. In sax-7 mutants, the organization of ganglia and positioning of neurons are abnormal in the adult stage, but these abnormalities are not observed in early larval stage. Misplacement of neurons in sax-7 mutants is triggered by mechanical force linked to body movement. Short and long forms of SAX-7 exhibited strong and weak homophilic adhesion activities in in vitro aggregation assay, respectively, which correlated with their different activities in vivo. SAX-7 was localized on plasma membranes of neurons in vivo. Expression of SAX-7 only in a single neuron in sax-7 mutants cell-autonomously restored its normal neuronal position. Expression of SAX-7 in two different head neurons in sax-7 mutants led to the forced attachment of these neurons. We propose that both homophilic and heterophilic interactions of SAX-7 are essential for maintenance of neuronal positions in organized ganglia.     

Responses of the silkworm tyramine receptor to 2-phenylethylamines and 5-phenyloxazoles

Tyramine (TA), a biogenic amine, attenuates intracellular cAMP production by acting on its receptor in insects. Several non-biogenic amines were examined for their actions on native and heterologously expressed silkworm TA receptors. 5-(4-Hydroxyphenyl)oxazole, which showed an attenuating effect on cAMP production in silkworm-head membranes, did not attenuate forskolin-stimulated cAMP production in HEK-293 cells expressing the silkworm TA receptor, although the compound bound to the cloned receptor. 2-Phenylethylamines (2-PEAs), which showed positive and negative effects on cAMP production in silkworm-head membranes, inhibited [3H]TA binding to the cloned TA receptor. 2-Chloro-2-(4-chlorophenyl)ethylamine was the most potent inhibitor of [3H]TA binding among the 2-PEAs tested, with an IC50 of 30.4 nM. This compound acted as an antagonist and abolished TA-attenuation of forskolin-stimulated cAMP production in the cloned TA receptor. The discrepancy in the effects of the non-biogenic amines on the native and cloned TA receptors remains to be further examined. A newly synthesized 2-PEA, 2-chloro-2-(4-hydroxyphenyl)ethylamine, attenuated forskolin-stimulated cAMP production in the cloned TA receptor, indicating that the para-hydroxy group is important for the agonist action.     

Visualization of conformational distribution of short to medium size segments in globular proteins and identification of local structural motifs

Analysis of the conformational distribution of polypeptide segments in a conformational space is the first step for understanding a principle of structural diversity of proteins. Here, we present a statistical analysis of protein local structures based on interatomic C(alpha) distances. Using principal component analysis (PCA) on the intrasegment C(alpha)-C(alpha) atomic distances, the conformational space of protein segments, which we call the protein segment universe, has been visualized, and three essential coordinate axes, suitable for describing the universe, have been identified. Three essential axes specified radius of gyration, structural symmetry, and separation of hairpin structures from other structures. Among the segments of arbitrary length, 6-22 residues long, the conservation of those axes was uncovered. Further application of PCA to the two largest clusters in the universe revealed local structural motifs. Although some of motifs have already been reported, we identified a possibly novel strand motif. We also showed that a capping box, which is one of the helix capping motifs, was separated into independent subclusters based on the C(alpha) geometry. Implications of the strand motif, which may play a role for protein-protein interaction, are discussed. The currently proposed method is useful for not only mapping the immense universe of protein structures but also identification of structural motifs.     

The effect of long-range interactions on the secondary structure formation of proteins

The influence of long-range residue interactions on defining secondary structure in a protein has long been discussed and is often cited as the current limitation to accurate secondary structure prediction. There are several experimental examples where a local sequence alone is not sufficient to determine its secondary structure, but a comprehensive survey on a large data set has not yet been done. Interestingly, some earlier studies denied the negative effect of long-range interactions on secondary structure prediction accuracy. Here, we have introduced the residue contact order (RCO), which directly indicates the separation of contacting residues in terms of the position in the sequence, and examined the relationship between the RCO and the prediction accuracy. A large data set of 2777 nonhomologous proteins was used in our analysis. Unlike previous studies, we do find that prediction accuracy drops as residues have contacts with more distant residues. Moreover, this negative correlation between the RCO and the prediction accuracy was found not only for beta-strands, but also for alpha-helices. The prediction accuracy of beta-strands is lower if residues have a high RCO or a low RCO, which corresponds to the situation that a beta-sheet is formed by beta-strands from different chains in a protein complex. The reason why the current study draws the opposite conclusion from the previous studies is examined. The implication for protein folding is also discussed.     

Searching for protein-protein interaction sites and docking by the methods of molecular dynamics, grid scoring, and the pairwise interaction potential of amino acid residues

In CAPRI Rounds 1 and 2, we assumed that because there are many ionic charges that weaken electrostatic interaction forces in living cells, the hydrophobic interaction force might be important entropically. As a result of Rounds 1 and 2, the predictions for binding sites and geometric centers were acceptable, but those of the binding axes were poor, because only the largest benzene cluster was used for generating the initial docking structures. These were generated by fitting of benzene clusters formed on the surface of receptor and ligand. In CAPRI Rounds 3-5, the grid-scoring sum on the protein-protein interaction surface and the pairwise potential of the amino acid residues, which were indicated as coming easily into the protein-protein interaction regions, were used as the calculation methods, along with the smaller benzene clusters that participated in benzene cluster fitting. Good predicted models were obtained for Targets 11 and 12. When the modeled receptor proteins were superimposed on the experimental structures, the smallest ligand root-mean-square deviation (RMSD) values corresponding to the RMSD between the model and experimental structures were 6.2 A and 7.3 A, respectively.