Protease-activated receptor-2-mediated Ca2+ signaling in guinea pig tracheal epithelial cells

The protease-activated receptor-2 (PAR-2), a G protein-coupled receptor activated by trypsin, contributes to the pathogenesis of inflammatory disease including asthma. Here, we examined the mechanisms by which stimulation of PAR-2 induces an increase in intracellular Ca2+ concentration ([Ca2+]i) in guinea pig tracheal epithelial cells. Trypsin (0.01-3 units/ml) dose-dependently induced a transient increase in [Ca2+]i, the increase being blocked by soybean trypsin inhibitor (SBTI 1 microM). An increase in [Ca2+]i was also induced by an agonist peptide for PAR-2 (SLIGRL-NH2, 0.001-10 microM) but not by thrombin (3 units/ml, an activator for PAR-1, PAR-3 or PAR-4). Repeated or cross stimulation of trypsin or SLIGRL-NH2 caused marked desensitization of the [Ca2+]i response. These responses of [Ca2+]i to trypsin and SLIGRL-NH2 were attenuated by a phospholipase C inhibitor, U-73122, and a Ca2+-ATPase inhibitor, thapsigargin (100 nM), while removal of Ca2+ and a L-type Ca2+-channel blocker, verapamil, were without significant effects. Further, trypsin was without effect on the rate of fura 2 quenching by Mn2+ entry as an indicator of Ca2+ influx. Thus, stimulation of PAR-2 appears to increase [Ca2+]i through the mobilization of Ca2+ from intracellular stores probably via phospholipase Cbeta-linked generation of a second messenger.     

DNA Methylation Profile Distinguishes Clear Cell Sarcoma of the Kidney from Other Pediatric Renal Tumors

A number of specific, distinct neoplastic entities occur in the pediatric kidney, including Wilms’ tumor, clear cell sarcoma of the kidney (CCSK), congenital mesoblastic nephroma (CMN), rhabdoid tumor of the kidney (RTK), and the Ewing’s sarcoma family of tumors (ESFT). By employing DNA methylation profiling using Illumina Infinium HumanMethylation27, we analyzed the epigenetic characteristics of the sarcomas including CCSK, RTK, and ESFT in comparison with those of the non-neoplastic kidney (NK), and these tumors exhibited distinct DNA methylation profiles in a tumor-type-specific manner. CCSK is the most frequently hypermethylated, but least frequently hypomethylated, at CpG sites among these sarcomas, and exhibited 490 hypermethylated and 46 hypomethylated CpG sites in compared with NK. We further validated the results by MassARRAY, and revealed that a combination of four genes was sufficient for the DNA methylation profile-based differentiation of these tumors by clustering analysis. Furthermore, THBS1 CpG sites were found to be specifically hypermethylated in CCSK and, thus, the DNA methylation status of these THBS1 sites alone was sufficient for the distinction of CCSK from other pediatric renal tumors, including Wilms’ tumor and CMN. Moreover, combined bisulfite restriction analysis could be applied for the detection of hypermethylation of a THBS1 CpG site. Besides the biological significance in the pathogenesis, the DNA methylation profile should be useful for the differential diagnosis of pediatric renal tumors.     

Outbreeding Depression Caused by Intraspecific Hybridization Between Local and Nonlocal Genotypes in Abies sachalinensis

To evaluate the effects of intraspecific hybridization of local and nonlocal genotypes on growth traits of progeny in Abies sachalinensis, we performed reciprocal crossing between nonlocal trees in a high‐elevation zone (1,100–1,200 m asl) and local trees in a low‐elevation zone (530 m asl) in Hokkaido, northern Japan, in 1979 and established a common garden experiment using local × local (female × male ), local × nonlocal , nonlocal × local , and nonlocal × nonlocal progeny in the low‐elevation zone in 1986. Survival, height, diameter at breast height (dbh), needle nitrogen content, specific leaf area (SLA), and needle area per shoot diameter (NA) of 25‐year‐old progeny were measured in 2005. The survival rate was consistently high (>85% on average). Reductions in height and dbh were apparent in F1 hybrids compared with local × local progeny. Furthermore, outbreeding depression was significant in height growth of nonlocal × local F1 hybrids and in dbh of both F1 hybrids. Reductions in growth traits may be related to morphological needle traits, such as the low values of SLA and NA. Elevation guidelines for A. sachalinensis seed zones are discussed to ensure the long‐term viability of both restored and native populations.     

Therapeutic activity of plant-derived alkaloid conophylline on metabolic syndrome and neurodegenerative disease models

Increasing metabolic syndromes including type-2 diabetes mellitus, obesity, and steatohepatitis are serious problems in most countries in the world. Neurodegenerative diseases such as Alzheimer, Parkinson’s, and Huntington’s diseases are increasing in many countries. However, therapy for these diseases is not sufficient yet. Thus, effective chemotherapy for these diseases is being expected. Conophylline is an alkaloid isolated from the leaves of Ervatamia microphylla and related plants. It was found to induce beta-cell differentiation in the precursor pancreatic cells. Oral administration of this compound ameliorated type-2 diabetes mellitus model in mice and rats. Later, fibrosis of the pancreatic islets was found to be greatly reduced by conophylline in the pancreatic islets. It also inhibited chemically induced liver cirrhosis. Further study indicated that conophylline inhibited non-alcoholic steatohepatitis in the model mice. On the one hand, loss of autophagy often causes protein aggregation to give neural cell death. Conophylline was found to activate autophagy in cultured neural cells. Activation of autophagy ameliorated cellular models of Parkinson’s and Huntington’s diseases. Thus, conophylline is likely to be useful for the development of chemotherapy for metabolic and neurodegenerative diseases.     

Defective repair of radiation-induced DNA damage is complemented by a CHORI-230-65K18 BAC clone on rat chromosome 4

The Long Evans cinnamon (LEC) rat is highly susceptible to X-irradiation due to defective DNA repair and is thus a model for hepatocellular carcinogenesis. We constructed a bacterial artificial chromosome (BAC) contig of rat chromosome 4 completely covering the region associated with radiation susceptibility. We used transient and stable transfections to demonstrate that defective DNA repair in LEC cells is fully complemented by a 200-kb BAC, CHORI-230-65K18. Further analysis showed that the region associated with radiation susceptibility is located in a 128,543-bp region of 65K18 that includes the known gene Rpn1. However, neither knockdown nor overexpression of Rpn1 indicated that this gene is associated with radiation susceptibility. We also mapped three ESTs (TC523872, TC533727, and CB607546) in the 128,543-bp region, suggesting that 65K18 contains an unknown gene associated with X-ray susceptibility in the LEC rat.     

Crystal structure of Thermus thermophilus Delta1-pyrroline-5-carboxylate dehydrogenase

Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDh) plays an important role in the metabolic pathway from proline to glutamate. It irreversibly catalyzes the oxidation of glutamate-gamma-semialdehyde, the product of the non-enzymatic hydrolysis of Delta(1)-pyrroline-5-carboxylate, into glutamate with the reduction of NAD(+) into NADH. We have confirmed the P5CDh activity of the Thermus thermophilus protein TT0033 (TtP5CDh), and determined the crystal structure of the enzyme in the ligand-free form at 1.4 A resolution. To investigate the structural basis of TtP5CDh function, the TtP5CDh structures with NAD(+), with NADH, and with its product glutamate were determined at 1.8 A, 1.9 A, and 1.4 A resolution, respectively. The solved structures suggest an overall view of the P5CDh catalytic mechanism and provide insights into the P5CDh deficiencies in the case of the human type II hyperprolinemia.     

Crystal Structure of an Exo-1,5-α-l-arabinofuranosidase from Streptomyces avermitilis Provides Insights into the Mechanism of Substrate Discrimination between Exo- and Endo-type Enzymes in Glycoside Hydrolase Family 43

Exo-1,5-α-l-arabinofuranosidases belonging to glycoside hydrolase family 43 have strict substrate specificity. These enzymes hydrolyze only the α-1,5-linkages of linear arabinan and arabino-oligosaccharides in an exo-acting manner. The enzyme from Streptomyces avermitilis contains a core catalytic domain belonging to glycoside hydrolase family 43 and a C-terminal arabinan binding module belonging to carbohydrate binding module family 42. We determined the crystal structure of intact exo-1,5-α-l-arabinofuranosidase. The catalytic module is composed of a 5-bladed β-propeller topologically identical to the other family 43 enzymes. The arabinan binding module had three similar subdomains assembled against one another around a pseudo-3-fold axis, forming a β-trefoil-fold. A sugar complex structure with α-1,5-l-arabinofuranotriose revealed three subsites in the catalytic domain, and a sugar complex structure with α-l-arabinofuranosyl azide revealed three arabinose-binding sites in the carbohydrate binding module. A mutagenesis study revealed that substrate specificity was regulated by residues Asn-159, Tyr-192, and Leu-289 located at the aglycon side of the substrate-binding pocket. The exo-acting manner of the enzyme was attributed to the strict pocket structure of subsite −1, formed by the flexible loop region Tyr-281–Arg-294 and the side chain of Tyr-40, which occupied the positions corresponding to the catalytic glycon cleft of GH43 endo-acting enzymes.     

Platelet derived growth factor regulates ABCA1 expression in vascular smooth muscle cells

The ATP-binding cassette transporter A1 (ABCA1) regulates lipid efflux from peripheral cells to High-density lipoprotein. The platelet-derived growth factor (PDGF) is a potent mitogen that enables vascular smooth muscle cells to participate in atherosclerosis. In this report, we showed that PDGF suppressed endogenous expression of ABCA1 in cultured vascular smooth muscle cells. Exposure of CRL-208 cells to PDGF elicited a rapid phosphorylation of a kinase downstream from PI3-K, Akt. The constitutively active form of both p110, a subunit of PI3-K, and Akt inhibited activity of the ABCA1 promoter. In conclusion, PI3-K-Akt pathways participate in PDGF-suppression of ABCA1 expression.