Overexpression and activation of the murine double minute 2 (MDM2) or nuclear factor activated T cells 1 (NFAT1) oncoproteins frequently occur in pancreatic cancer. Most MDM2 inhibitors under development target MDM2-p53 binding have little no effect on cancers without functional p53, including Some available compounds indirectly inhibit NFAT1 activity by interfering with calcineurin activity, but there are currently specific against NFAT1. Here we performed a high-throughput virtual...

Background— Intravenous prostacyclin is approved for treating pulmonary arterial hypertension (PAH), but it has a short half-life and must be delivered systemically via an indwelling intravenous catheter. We hypothesize that localized jugular vein delivery of prostacyclin-producing cells may provide sustained therapeutic effects without the limitations systemic delivery. Methods Results— generated vector expressing human cyclooxygenase isoform 1 synthase fusion protein produces from...

Abstract Promoting the paracrine effects of human mesenchymal stem cell (hMSC) therapy may contribute to improvements in patient outcomes. Here we develop an innovative strategy enhance hMSCs. In a mouse hindlimb ischaemia model, examine hMSCs which novel triple-catalytic enzyme is introduced stably produce prostacyclin (PGI 2 -hMSCs). We show that PGI -hMSCs facilitate perfusion recovery and running capability as compared with control or iloprost (a stable analogue). Transplanted do not...

Thromboxane A2 receptor (TP receptor), a prostanoid receptor, belongs to the G protein-coupled family, composed of three intracellular loops and extracellular connecting seven transmembrane helices. The highly conserved domains receptors were found in second loop (eLP2), which was proposed be involved ligand recognition. 3D structure eLP2 would help further explain binding mechanism. Analysis human TP model generated from molecular modeling based on bacteriorhodopsin crystallographic...

Cyclooxygenase isoform-2 (COX-2) and microsomal prostaglandin E2 synthase-1 (mPGES-1) are inducible enzymes that become up-regulated in inflammation some cancers. It has been demonstrated their coupling reaction of converting arachidonic acid (AA) into (PG) (PGE2) is responsible for Understanding reactions at the molecular cellular levels a key step toward uncovering pathological processes inflammation. In this paper, we describe structure-based enzyme engineering which produced novel hybrid...

Salicylic acid (SA), an endogenous signaling molecule of plants, possesses anti-inflammatory and anti-neoplastic actions in human. Its derivative, aspirin, is the most commonly used analgesic drug. Aspirin sodium salicylate (salicylates) have been reported to multiple pharmacological actions. However, it unclear whether they bind a cellular protein. Here, we report for first time purification from human fibroblasts approximately 78 kDa binding protein with sequence identity immunoglobulin...

The second extracellular loop (eLP2) of the thromboxane A(2) receptor (TP) had been proposed to be involved in ligand binding. Through two-dimensional (1)H NMR experiments, overall three-dimensional structure a constrained synthetic peptide mimicking eLP2 determined by our group (Ruan, K.-H., So, S.-P., Wu, J., Li, D., Huang, A., and Kung, J. (2001) Biochemistry 40, 275-280). To further identify residues binding, TP antagonist, SQ29,548 was used interact with peptide. High resolution NOESY,...

Prostacyclin (PGI2), a vascular protector with vasodilation and antithrombotic properties, is synthesized by coupling reactions of cyclooxygenase (COX, the first enzyme) PGI2 synthase (PGIS, second using arachidonic acid (AA) as an initial substrate. The COX product, prostaglandin H2 (PGH2) also command substrate for other prostanoid enzymes that produce distinct eicosanoids, such thromboxane A2 (TXA2). actions TXA2 to cause vasoconstriction platelet aggregation oppose vasodilatory...

Abstract Background Conventionally the active ingredients in herbal extracts are separated into individual components, by fractionation, desalting, and followed high-performance liquid chromatography (HPLC). In this study we have tried to directly screen water-soluble fractions of herbs with potential before purification or extraction. We propose that mimicking prostaglandin E 1 (PGE ) 2 can be identified non-purified fraction. PGE is a potent anti-inflammatory molecule used for treating...

Prostacyclin (PGI2) is a key vascular protector, metabolized from endogenous arachidonic acid (AA). Its actions are mediated through the PGI2 receptor (IP) and nuclear receptor, peroxisome proliferator-activated γ (PPARγ). Here, we found that involved in regulating cellular microRNA (miRNA) expression its receptors mouse adipose tissue-derived primary culture cell line expressing novel hybrid enzyme gene (COX-1-10aa-PGIS), cyclooxygenase-1 (COX-1) synthase (PGIS) linked with 10-amino linker....

The three-dimensional structure of a synthetic peptide corresponding to the N-terminal membrane anchor domain (residues 1—25) prostaglandin I2 synthase (also known as cytochrome P450 8A1), an eicosanoid-synthesizing microsomal P450, has been determined by two-dimensional 1H NMR spectroscopy in trifluoroethanol and dodecylphosphocholine which mimic hydrophobic environment. A combination experiments, including NOESY, TOCSY double-quantum-filtered COSY, was used obtain complete assignments for...

To overcome the difficulty of characterizing structures extracellular loops (eLPs) G protein‐coupled receptors (GPCRs) other than rhodopsin, we have explored a strategy to generate three‐dimensional structural model for GPCR, thromboxane A 2 receptor. This structure was completed by assembly NMR computation‐guided constrained peptides that mimicked and connected conserved seven transmembrane domains. The structure‐based reveals features eLPs, in which second loop (eLP ) disulfide bond...

It remains a challenge to achieve the stable and long-term expression (in human cell lines) of previously engineered hybrid enzyme [triple-catalytic (Trip-cat) enzyme-2; Ruan KH, Deng H & So SP (2006) Biochemistry45, 14003-14011], which links cyclo-oxygenase isoform-2 (COX-2) prostacyclin (PGI(2)) synthase (PGIS) for direct conversion arachidonic acid into PGI(2) through enzyme's Trip-cat functions. The upregulation biosynthesis vascular protector, PGI(2), in cells is an ideal model...

Prostacyclin (PGI2) is a potent vasodilator and important mediator of vascular homeostasis; however, its clinical use limited because short (<2-min) half-life. Thus, we hypothesize that the engineered endothelial progenitor cells (EPCs) constitutively secrete high levels PGI2 may overcome this limitation therapy. A cDNA encoding COX-1-10aa-PGIS, which links human cyclooxygenase-1 (COX-1) to prostacyclin synthase (PGIS), was delivered via nucleofection into outgrowth EPCs derived from rat...

A 3-D model of human thromboxane A2 synthase (TXAS) was constructed using a homology modeling approach based on information from the 2.0 Å crystal structure hemoprotein domains cytochrome P450BM-3 and P450cam. is bacterial fatty acid monooxygenase resembling eukaryotic microsomal P450s in primary function. TXAS shares 26.4% residue identity 48.4% similarity with domain. The score between much higher than that Alignment domain or P450cam determined through sequence searches. main-chain...

Human thromboxane A2 synthase (TXAS) exhibits spectral characteristics of cytochrome P450 but lacks monooxygenase activity. Its distinctive amino acid sequence makes TXAS the sole member family 5 in superfamily. To better understand structure-function relationship this unusual P450, we have recently constructed a three-dimensional model for using P450BM-3 as template (Ruan, K.-H., Milfeld, K., Kulmacz, R. J., and Wu, K. (1994) Protein Eng. 7, 1345-1551) identified potential active site...

A retroviral vector (BAG) was used to transfer human prostaglandin H synthase (PGHS-1) gene into a endothelial cell line for enhancement of PGI2 synthesis. Cells infected with BAG containing PGHS-1 cDNA in the sense orientation relative promoter (PGHS(S)) expressed 30-fold increase mRNA but, due reading frame shift, did not show an PGHS protein or synthesis, while those reverse viral (PGHS(R)), produced > 10-fold over control (169 +/- 22 vs 14.8 1.2 amol/micrograms RNA) concordant (5.82 1.07...