Reconstituted U1 small nuclear ribonucleoprotein complex restores 5' splice site cleavage activity

Functional reconstitution of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was performed using in vitro transcribed U1 snRNA. Hela cell nuclear extract was depleted of its constituent snRNPs by centrifugation at 100,000 X g. The supernatant was devoid of snRNAs and lacked cleavage activity in splicing reactions using in vitro transcribed beta-globin pre-mRNA as substrate. The resulting pellet which contained the snRNAs, retained 5' splice site cleavage activity in a similar splicing reaction. Supplementation of the inactive supernatant fraction with in vitro transcribed U1 snRNA, partially restored 5' splice site cleavage activity thereby demonstrating the specific requirement of U1 snRNP in the initial stage of pre-mRNA splicing.     

Isolation of the cDNA for human prostaglandin H synthase

Prostaglandin H Synthase (PGHS, cyclooxygenase) is a 67 kd protein which catalyzes the first step in prostaglandin synthesis. The primary amino acid sequence and the molecular mechanisms regulating expression are unknown. We report here isolation of a cDNA clone for the enzyme from human vascular endothelial cells for use in such studies. High titre, polyclonal antiserum against PGHS was developed in rabbits. The antiserum was monospecific, reacted with cyclooxygenase on Western blots at a limiting dilution of 1:500,000 and immunoprecipitated cyclooxygenase synthesized by in vitro translation of PGHS messenger RNA. It was used to screen a lambda gt11 cDNA expression library from human endothelial cells. Three positive clones were isolated. Following plaque purification, one clone reacted strongly with two other polyclonal antisera independently raised against highly purified cyclooxygenase and the aspirin-acetylated enzyme. Western blot analysis confirmed production of a large approximately 180 kd fusion protein of cyclooxygenase and beta-galactosidase. The cDNA insert of approximately 2.2 kilo base pairs was excised and subcloned into plasmid pUC8. A 24 nucleotide DNA probe, synthesized according to the amino acid sequence of the aspirin-acetylation site of cyclooxygenase, hybridized strongly with the 2.2 kbp cDNA insert. It is concluded that the 2.2 kbp cDNA insert represents a cDNA clone for human cyclooxygenase, which also expresses the aspirin-acetylation site. This is the first reported isolation of the cDNA for this enzyme, and will facilitate further studies on the primary sequence and on the regulation of the enzyme at the molecular level.     

Differential recovery of prostacyclin synthesis in cultured vascular endothelial vs. smooth muscle cells after inactivation of cyclooxygenase with aspirin

Conflicting findings from clinical trials on the use of aspirin in preventing myocardial infarction emphasize the importance of understanding the effects of aspirin on vascular cells. Cultured vascular endothelial cells and smooth muscle cells of human, rat and bovine origin synthesized prostacyclin, a key component in vascular homeostasis, when superfused with 14C arachidonic acid. Prostacyclin synthesis was inactivated following brief treatment with aspirin, which irreversibly acetylates cyclooxygenase. Marked differences were observed between endothelial and smooth muscle cells in the recovery of cyclooxygenase after aspirin treatment. Smooth muscle cells recovered within 3 hours by a process that required serum factors replaceable by epidermal growth factor (EGF) and TGF-beta. Recovery in both smooth muscle and endothelial cells was blocked by cycloheximide but not by actinomycin-D. Endothelial cell recovery occurred much more slowly, requiring up to 24 hours and was not dependent on serum factors or EGF. Furthermore, it was suppressed by growth inducing agents such as endothelial cell growth factor (ECGF) and was enhanced by conditions favoring growth arrest and cellular differentiation. Regulation of expression and recovery of cyclooxygenase following inactivation by aspirin thus differs considerably in the endothelial and smooth muscle compartments of the vasculature.     

Ligand-induced trafficking of the sphingosine-1-phosphate receptor EDG-1

The endothelial-derived G-protein-coupled receptor EDG-1 is a high-affinity receptor for the bioactive lipid mediator sphingosine-1-phosphate (SPP). In the present study, we constructed the EDG-1-green fluorescent protein (GFP) chimera to examine the dynamics and subcellular localization of SPP-EDG-1 interaction. SPP binds to EDG-1-GFP and transduces intracellular signals in a manner indistinguishable from that seen with the wild-type receptor. Human embryonic kidney 293 cells stably transfected with the EDG-1-GFP cDNA expressed the receptor primarily on the plasma membrane. Exogenous SPP treatment, in a dose-dependent manner, induced receptor translocation to perinuclear vesicles with a tau1/2 of approximately 15 min. The EDG-1-GFP-containing vesicles are distinct from mitochondria but colocalize in part with endocytic vesicles and lysosomes. Neither the low-affinity agonist lysophosphatidic acid nor other sphingolipids, ceramide, ceramide-1-phosphate, or sphingosylphosphorylcholine, influenced receptor trafficking. Receptor internalization was completely inhibited by truncation of the C terminus. After SPP washout, EDG-1-GFP recycles back to the plasma membrane with a tau1/2 of approximately 30 min. We conclude that the high-affinity ligand SPP specifically induces the reversible trafficking of EDG-1 via the endosomal pathway and that the C-terminal intracellular domain of the receptor is critical for this process.     

Large-scale search of SNPs for type 2 DM susceptibility genes in a Japanese population

The etiology of type 2 diabetes (DM) is polygenic. We investigated here genes and polymorphisms that associate with DM in the Japanese population. Single-nucleotide polymorphisms (SNPs) of 398 derived from 120 candidate genes were examined for association with DM in a population-based case-control study. The study group consisted of 148 cases and 227 controls recruited from Funagata, Japan. No evident subpopulation structure was detected for the tested population. The association tests were conducted with standard allele positivity tables (chi(2) tests) between SNP genotype frequency and case-control status. The independent association of the SNPs from serum triglyceride levels and body mass index was examined by multiple logistic regression analysis. A value of P<0.01 was accepted as statistically significant. Six genes (met proto-oncogene, ATP-binding cassette transporter A1, fatty acid binding protein 2, LDL receptor defect C complementing, aldolase B, and sulfonylurea receptor) were shown to be associated with DM.     

Immunosuppressive and anti-angiogenic sphingosine 1-phosphate receptor-1 agonists induce ubiquitinylation and proteasomal degradation of the receptor

Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator, regulates lymphocyte trafficking, vascular permeability, and angiogenesis by activation of the S1P1 receptor. This receptor is activated by FTY720-P, a phosphorylated derivative of the immunosuppressant and vasoactive compound FTY720. However, in contrast to the natural ligand S1P, FTY720-P appears to act as a functional antagonist, even though the mechanisms involved are poorly understood. In this study, we investigated the fate of endogenously expressed S1P1 receptor in agonist-activated human umbilical vein endothelial cells and human embryonic kidney 293 cells expressing green fluorescent protein-tagged S1P1. We show that FTY720-P is more potent than S1P at inducing receptor degradation. Pretreatment with an antagonist of S1P1, VPC 44116, prevented receptor internalization and degradation. FTY720-P did not induce degradation of internalization-deficient S1P1 receptor mutants. Further, small interfering RNA-mediated down-regulation of G protein-coupled receptor kinase-2 and beta-arrestins abolished FTY720-P-induced S1P1 receptor degradation. These data suggest that agonist-induced phosphorylation of S1P1 and subsequent endocytosis are required for FTY720-P-induced degradation of the receptor. S1P1 degradation is blocked by MG132, a proteasomal inhibitor. Indeed, FTY720-P strongly induced polyubiquitinylation of S1P1 receptor, whereas S1P at concentrations that induced complete internalization was not as efficient, suggesting that receptor internalization is required but not sufficient for ubiquitinylation and degradation. We propose that the ability of FTY720-P to target the S1P1 receptor to the ubiquitinylation and proteasomal degradation pathway may at least in part underlie its immunosuppressive and anti-angiogenic properties.     

Larval Gnathostoma spinigerum Detected in Asian Swamp Eels,Monopterus albus,Purchased from a Local Market in Yangon,Myanmar

The present study was performed to determine the infection status of swamp eels with Gnathostoma sp. larvae in Myanmar. We purchased total 37 Asian swamp eels, Monopterus albus, from a local market in Yangon in June and December 2013 and 2014. All collected eels were transferred with ice to our laboratory and each of them was examined by the artificial digestion technique. A total of 401 larval gnathostomes (1-96 larvae/eel) were detected in 33 (89.2%) swamp eels. Most of the larvae (n=383; 95.5%) were found in the muscle. The remaining 18 larvae were detected in the viscera. The advanced third-stage larvae (AdL₃) were 2.3-4.4 mm long and 0.25-0.425 mm wide. The characteristic head bulb (0.093 × 0.221 mm in average size) with 4 rows of hooklets, muscular long esophagus (1.025 mm), and 2 pairs of cervical sacs (0.574 mm) were observed by light microscopy. The average number of hooklets in the 1st, 2nd, 3rd, and 4th rows was 41, 45, 48, and 51, respectively. As scanning electron microscopic findings, the characteristic 4-5 rows of hooklets on the head bulb, a cervical papilla, tegumental spines regularly arranged in the transverse striations, and an anus were well observed. Based on these morphological characters, they were identified as the AdL₃ of Gnathostoma spinigerum. By the present study, it has been confirmed for the first time that Asian swamp eels, M. albus, from Yangon, Myanmar are heavily infected with G. spinigerum larvae.     

Genomic insights into mediator lipidomics

G protein-coupled receptors (GPCR) are used ubiquitously and widely for signal transduction across the plasma membrane. The ligands for GPCRs are structurally diverse and include peptides, odorants, photon, ions and lipids. It is thought that GPCRs evolved by gene duplication and mutational events that diversified the ligand binding and signaling properties, thereby resulting in paralogues in various organisms. Genomic sequencing efforts of various organisms indicate that GPCRs evolved very early in evolution; for example, unicellular eukaryotes use GPCRs for mating, differentiation and sporulation responses and prokarotes utilize these receptors for phototransduction, as exemplified by the bacteriorhodopsin, a photon sensor. Many GPCRs fall into subfamilies, usually determined by structural similarity to their ligands. Bioactive lipids such as lysophospholipids, eicosanoids, ether lipids and endocannabinoids, which are produced widely in evolution, also signal through GPCRs. Thus, distinct subfamilies of bioactive lipid GPCRs, such as prostanoid receptors, lysophosphatidic, sphingosine 1-phosphate, leukotrienes, hydroxy fatty acids, endocannabinoids and ether lipids exist in the mammalian genome. With the increasing availability of genomic information throughout the phylogenetic tree, orthologues of bioactive lipid receptors are found in the genomes of vertebrates and chordates but not in worms, flies or other lower organisms. This is in contrast to GPCRs for biogenic amines and polypeptide growth factors, which are conserved in invertebrates as well. Thus, it appears that with the evolution of chordates, lipids may have acquired novel roles in cell-cell communication events via GPCRs. This hypothesis will be discussed using the prostanoid and lysophospholipid signaling systems. Since such bioactive lipids play critical roles in immune, vascular and nervous systems, this suggests that lipid metabolite signaling via the GPCRs co-evolved with the development of sophisticated vascular, immune and nervous systems in chordates and vertebrates.     

A novel thermophilic pectate lyase containing two catalytic modules of Clostridium stercorarium

The Clostridium stercorarium F-9 pel9A gene encodes a pectate lyase Pel9A consisting of 1,240 amino acids with a molecular weight of 135,171. The mature form of Pel9A is a modular enzyme composed of two family-9 catalytic modules of polysaccharide lyases, CM9-1 and CM9-2, in order from the N terminus. Pel9A showed an overall sequence similarity to the hypothetical pectate lyase PelX of Bacillus halodurans (sequence identity 53%), and CM9-2 showed moderate sequence similarities to some pectate lyases of family 9. Sequence identity between CM9-1 and CM9-2 was 21.3%. The full-length Pel9A lacking the N-terminal signal peptide was expressed, purified, and characterized. The enzyme required Ca(2+) ion for its enzyme activity and showed high activity toward polygalacturonic acid but lower activity toward pectin, indicating that Pel9A is a pectate lyase. Immunological analysis using an antiserum raised against the purified enzyme indicated that Pel9A is constitutively synthesized by C. stercorarium F-9.