Angiogenic activity of sesamin through the activation of multiple signal pathways

The natural product sesamin has been known to act as a potent antioxidant and prevent endothelial dysfunction. We here found that sesamin increased in vitro angiogenic processes, such as endothelial cell proliferation, migration, and tube formation, as well as neovascularization in an animal model. This compound elicited the activation of multiple angiogenic signal modulators, such as ERK, Akt, endothelial nitric oxide synthase (eNOS), NO production, FAK, and p38 MAPK, but not Src. The MEK inhibitor PD98059 and the PI3K inhibitor Wortmannin specifically inhibited sesamin-induced activation of the ERK and Akt/eNOS pathways. These inhibitors reduced angiogenic events, with high specificity for MEK/ERK-dependent cell proliferation and migration and PI3K/Akt-mediated tube formation. Moreover, inhibition of p38 MAPK effectively inhibited sesamin-induced cell migration. The angiogenic activity of sesamin was not associated with VEGF expression. Furthermore, this compound did not induce vascular permeability and upregulated ICAM-1 and VCAM-1 expression, which are hallmarks of vascular inflammation. These results suggest that sesamin stimulates angiogenesis in vitro and in vivo through the activation of MEK/ERK-, PI3K/Akt/eNOS-, p125^FAK-, and p38 MAPK-dependent pathways, without increasing vascular inflammation, and may be used for treating ischemic diseases and tissue regeneration.     

The PPLA motif of glycogen synthase kinase 3beta is required for interaction with Fe65

Glycogen synthase kinase 3beta (GSK 3 beta) is a serine/ threonine kinase that phosphorylates substrates such as beta-catenin and is involved in a variety of biological processes, including embryonic development, metabolism, tumorigenesis, and cell death. Here, we present evidence that human GSK 3beta is associated with Fe65, which has the characteristics of an adaptor protein, possessing a WW domain, and two phosphotyrosine interaction domains, PID1 and PID2. The GSK 3beta catalytic domain also contains a putative WW domain binding motif ((371)PPLA(374)), and we observed, using a pull down approach and co-immuno-precipitation, that it interacts physically with Fe65 via this motif. In addition, we detected co-localization of GSK 3beta and Fe65 by confocal microscopy, and this co-localization was disrupted by mutation of the putative WW domain binding motif of GSK 3beta.Finally, in transient transfection assays interaction of GSK 3 beta (wt) with Fe65 induced substantial cell apoptosis, whereas interaction with the GSK 3beta AALA mutant ((371)AALA(374)) did not, and we noted that phosphorylation of the Tyr 216 residue of the GSK 3beta AALA mutant was significantly reduced compared to that of GSK 3beta wild type. Thus, our observations indicate that GSK 3beta binds to Fe65 through its (371)PPLA(374) motif and that this interaction regulates apoptosis and phosphorylation of Tyr 216 of GSK 3beta.     

Role of CAGE, a novel cancer/testis antigen, in various cellular processes, including tumorigenesis, cytolytic T lymphocyte induction, and cell motility

A cancer-associated antigen gene (CAGE) was identified by serological analysis of a recombinant cDNA expression library (SEREX). The gene was identified by screening cDNA expression libraries of human testis and gastric cancer cell lines with sera from patients with gastric cancer. CAGE was found to contain a D-E-A-D box domain and encodes a putative protein of 630 amino acids with possible helicase activity. The CAGE gene is widely expressed in various cancer tissues and cancer cell lines. Demethylation plays a role in the activation of CAGE in certain cancer cell lines where the gene is not expressed. The functional roles of CAGE in tumorigenesis, the molecular mechanisms of CAGE expression, and cell motility are also discussed.     

Evaluation of 2- and 3-stage combinations of vertical and horizontal flow constructed wetlands for treating greenhouse wastewater

The adsorption characteristics of various filter media and treatment efficiency of small pilot-scale constructed wetlands (CWs) were investigated in order to design optimum CWs for treating greenhouse wastewater. Calcite was the best filter medium for the adsorption of ammonium nitrogen and phosphorus under various temperature and pH conditions. However, removal efficiency of calcite for total nitrogen (T-N) removal was low due primarily to high nitrate levels. Thus, several hybrid CWs (containing calcite as filter media) consisting of combinations of vertical flow (VF) and horizontal flow (HF) beds were evaluated for improving efficiency for T-N removal. Both 2- and 3-stage combinations of the VF and HF beds were tested. The optimum hybrid CWs was demonstrated to be a 3-stage combination of horizontal flow (HF)–vertical flow (VF)–horizontal flow (HF), which provided suitable conditions for both nitrification and denitrification, which improved removal of T-N in wastewater containing nitrate. In the HF–VF–HF 3-stage hybrid CWs, the reduction in chemical oxygen demand (COD), T-N, and total phosphorus (T-P) in the effluent were 95.1, 68.4 and 94.3%, respectively. The removal of COD, T-N and T-P in 3-stage HF–VF–HF CWs was rapid in order of VF (second stage) ≫ HF (first stage) ≫ HF (third stage), VF (second stage) ≫ HF (third stage) > HF (first stage) and VF (second stage) ≫ HF (first stage) ≫ HF (third stage), respectively.     

Pyruvate dehydrogenase kinase-4 deficiency lowers blood glucose and improves glucose tolerance in diet-induced obese mice

The effect of pyruvate dehydrogenase kinase-4 (PDK4) deficiency on glucose homeostasis was studied in mice fed a high-fat diet. Expression of PDK4 was greatly increased in skeletal muscle and diaphragm but not liver and kidney of wild-type mice fed the high-fat diet. Wild-type and PDK4(-/-) mice consumed similar amounts of the diet and became equally obese. Insulin resistance developed in both groups. Nevertheless, fasting blood glucose levels were lower, glucose tolerance was slightly improved, and insulin sensitivity was slightly greater in the PDK4(-/-) mice compared with wild-type mice. When the mice were killed in the fed state, the actual activity of the pyruvate dehydrogenase complex (PDC) was higher in the skeletal muscle and diaphragm but not in the liver and kidney of PDK4(-/-) mice compared with wild-type mice. When the mice were killed after overnight fasting, the actual PDC activity was higher only in the kidney of PDK4(-/-) mice compared with wild-type mice. The concentrations of gluconeogenic substrates were lower in the blood of PDK4(-/-) mice compared with wild-type mice, consistent with reduced formation in peripheral tissues. Diaphragms isolated from PDK4(-/-) mice oxidized glucose faster and fatty acids slower than diaphragms from wild-type mice. Fatty acid oxidation inhibited glucose oxidation by diaphragms from wild-type but not PDK4(-/-) mice. NEFA, ketone bodies, and branched-chain amino acids were elevated more in PDK4(-/-) mice, consistent with slower rates of oxidation. These findings show that PDK4 deficiency lowers blood glucose and slightly improves glucose tolerance and insulin sensitivity in mice with diet-induced obesity.     

Hormone interactions to Leu-rich repeats in the gonadotropin receptors. III. Photoaffinity labeling of human chorionic gonadotropin with receptor Leu-rich repeat 4 peptide

Human chorionic gonadotropin (hCG) binds to the extracellular N-terminal domain, exodomain, of its receptor, and the resulting hCG-exodomain complex is thought to modulate the membrane associated domain, endodomain, of the receptor to generate hormone signal. The bulk of the exodomain is speculated to assume a crescent structure consisting of eight to nine Leu-rich repeats (LRRs), which may provide the hormone contact sites. Unfortunately, little experimental evidence is available for the precise hormone contact points in the exodomain and the endodomain. The two preceding articles (Song, Y., Ji, I., Beauchamp, J., Isaacs, N., and Ji, T. (2001) J. Biol. Chem. 276, 3426-3435; Song, Y., Ji, I., Beauchamp, J., Isaacs, N., and Ji, T. (2001) J. Biol. Chem. 276, 3436-3442) show that putative LRR2 and LRR4 are crucial for hormone binding. In particular, the N-terminal region of LRR4 assumes the hydrophobic core of the LRR4 loop, whereas the C-terminal region is crucial for signal generation. However, it is unclear whether LRR4 interacts hCG and the endodomain and how it might be involved in signal generation. In this article, our affinity labeling results present the first evidence that the N-terminal region of LRR4 interacts with hCG, preferentially the hCGalpha subunit and that the hCG/LRR4 complex interacts with exoloop 2 of the endodomain. This interaction offers a mechanism to generate hormone signal.     

Mechanisms responsible for regulation of branched-chain amino acid catabolism

The branched-chain amino acids (BCAAs) are essential amino acids and therefore must be continuously available for protein synthesis. However, BCAAs are toxic at high concentrations as evidenced by maple syrup urine disease (MSUD), which explains why animals have such an efficient oxidative mechanism for their disposal. Nevertheless, it is clear that leucine is special among the BCAAs. Leucine promotes global protein synthesis by signaling an increase in translation, promotes insulin release, and inhibits autophagic protein degradation. However, leucine's effects are self-limiting because leucine promotes its own disposal by an oxidative pathway, thereby terminating its positive effects on body protein accretion. A strong case can therefore be made that the proper leucine concentration in the various compartments of the body is critically important for maintaining body protein levels beyond simply the need of this essential amino acid for protein synthesis. The goal of the work of this laboratory is to establish the importance of regulation of the branched chain alpha-ketoacid dehydrogenase complex (BCKDC) to growth and maintenance of body protein. We hypothesize that proper regulation of the activity state of BCKDC by way of its kinase (BDK) and its phosphatase (BDP) is critically important for body growth, tissue repair, and maintenance of body protein. We believe that growth and protection of body protein during illness and stress will be improved by therapeutic control of BCKDC activity. We also believe that it is possible that the negative effects of some drugs (PPAR alpha ligands) and dietary supplements (medium chain fatty acids) on growth and body protein maintenance can be countered by therapeutic control of BCDKC activity.     

Trans-activation of mutant follicle-stimulating hormone receptors selectively generates only one of two hormone signals

Previously, we reported that a liganded LH receptor (LHR) is capable of activating itself (cis-activation) and other nonliganded LHRs to induce cAMP (trans-activation). Trans-activation of the LHR raises two crucial questions. Is trans-activation unique to LHR or common to other G protein-coupled receptors? Does trans-activation stimulate phospholipase Cbeta as it does adenylyl cyclase? To address these questions, two types of novel FSH receptors (FSHRs) were constructed, one defective in hormone binding and the other defective in signal generation. The FSHR, a G protein-coupled receptor, comprises two major domains, the N-terminal extracellular exodomain that binds the hormone and the membrane-associated endodomain that generates the hormone signals. For signal defective receptors, the exodomain was attached to glycosyl phosphatidylinositol (ExoGPI) or the transmembrane domain of CD8 immune receptor (ExoCD). ExoGPI and ExoCD can trans-activate another nonliganded FSH. Surprisingly, the trans-activation generates a signal to activate either adenylyl cyclase or phospholipase Cbeta, but not both. These results indicate that trans-activation in these mutant receptors is selective and limited in signal generation, thus providing new approaches to investigating the generation of different hormone signals and a novel means to selectively generate a particular hormone signal. Our data also suggest that the FSHR's exodomain could not trans-activate LHR.