The fifth residue of the coat protein of turnip mosaic virus is responsible for long-distance movement in a local-lesion host and aphid transmissibility in a systemic host.

HC-Pro and CP genes of a potyvirus facilitate cell-to-cell movement and are involved in the systemic movement of the viruses. The interaction between HC-Pro and CP is mandatory for aphid transmission. Two turnip mosaic virus (TuMV) isolates, RC4 and YC5, were collected from calla lily plants in Taiwan. The virus derived from the infectious clone pYC5 cannot move systemically in Chenopodium quinoa plants and lacks aphid transmissibility in Nicotiana benthamiana plants, like the initially isolated virus. Sequence analysis revealed that two amino acids P5 and A206, of YC5 CP uniquely differ from RC4 and other TuMV strains. Recombination assay and site-directed mutagenesis revealed that the fifth residue of leucine (L) at the N-terminal region of CP (TuMV-RC4), instead of proline (P) (TuMV-YC5), is critical to permit the systemic spread in C. quinoa plants. Moreover, the single substitution mutant YC5-CPP5L became aphid transmissible similar to RC4. Fluorescence microscopy revealed that YC5-GFP was restricted in the petioles of inoculated leaves, while YC5-CPP5L-GFP translocated through the petioles of inoculated leaves, main stem, and the petioles of upper uninoculated leaves of C. quinoa plants. In addition, YC5-GUS was blocked at the basal part of the petiole connecting to the main stem of the inoculated C. quinoa plants, while YC5-CPP5L-GFP translocated to the upper leaves. Thus, a single amino acid, the residue L5 at the N-terminal region right before the 6DAG8 motif, is critical for the systemic translocation ability of TuMV in a local-lesion host and for aphid transmissibility in a systemic host.

Rhodium-Catalyzed Domino Hydroformylation/Double-Cyclization Reaction of Arylacetylenecarboxamides: Diastereoselectivity Studies and Application in the Synthesis of 1-Azabicyclo[x.y.0]alkanes.

A domino method for the rapid syntheses of 1-azabicyclo[x.y.0]alkane scaffolds, such as indolizidines, quinolizidines, decahydropyridoazepines, and their derivatives, has been developed. This strategy involved a rhodium-catalyzed hydroformylation of allyl-, 3-butenyl-, or homoallyl amides, followed by two spontaneous ring closures under mild conditions. The reaction scope and diastereoselectivity were fully explored by changing the substitution pattern on the amide and by altering the length of the carbon chain. This method was applied to the syntheses of natural alkaloids tashiromine and epilupinine. The clear differences between the reactivities of two isomeric amide substrates, 3-butenamide 1 j and homoallyl amide 1 i, could be attributed to better HOMO-LUMO overlap in the transition states that were derived from butenamides during cyclization. This explanation was supported by DFT calculations.

Calpain activity and Toll-like receptor 4 expression in platelet regulate haemostatic situation in patients undergoing cardiac surgery and coagulation in mice.

Human platelets express Toll-like receptors (TLR) 4. However, the mechanism by which TLR4 directly affects platelet aggregation and blood coagulation remains to be explored. Therefore, in this study, we evaluated the platelet TLR4 expression in patients who underwent CABG surgery; we explored the correlation between platelet TLR4 expression and the early outcomes in hospital of patients. Additionally, C57BL/6 and C57BL/6-Tlr(LPS-/-) mice were used to explore the roles of platelet TLR4 in coagulation by platelet aggregometry and rotation thromboelastometry. In conclusion, our results highlight the important roles of TLR4 in blood coagulation and platelet function. Of clinical relevance, we also explored novel roles for platelet TLR4 that are associated with early outcomes in cardiac surgery.

The role of calpain-myosin 9-Rab7b pathway in mediating the expression of Toll-like receptor 4 in platelets: a novel mechanism involved in α-granules trafficking.

Toll-like receptors (TLRs) plays a critical role in innate immunity. In 2004, Aslam R. and Shiraki R. first determined that murine and human platelets express functional TLRs. Additionally, Andonegui G. demonstrated that platelets express TLR4, which contributes to thrombocytopenia. However, the underlying mechanisms of TLR4 expression by platelets have been rarely explored until now. The aim of this study was to identify the mechanism of TLR4 expression underlying thrombin treatment. The human washed platelets were used in this study. According to flowcytometry and western blot analysis, the surface levels of TLR4 were significantly enhanced in thrombin-activated human platelets and decreased by TMB-8, calpeptin, and U73122, but not Y27632 (a Rho-associated protein kinase ROCK inhibitor) indicating that thrombin-mediated TLR4 expression was modulated by PAR/PLC pathway, calcium and calpain. Co-immunoprecipitation (co-IP) assay demonstrated that the interaction between TLR4 and myosin-9 (a substrate of calpain) was regulated by calpain; cleavage of myosin-9 enhanced TLR4 expression in thrombin treated platelets. Transmission electron microscope data indicated that human platelets used α-granules to control TLR4 expression; the co-IP experiment suggested that myosin-9 did not coordinate with Rab7b to negatively regulate TLR4 trafficking in thrombin treated platelets. In summary, phospholipase Cγ-calpain-myosin 9-Rab7b axis was responsible for the mechanism underlying the regulation of TLR4 containing α-granules trafficking in thrombin-stimulated platelets, which was involved in coagulation.

GroEL1, a heat shock protein 60 of Chlamydia pneumoniae, impairs neovascularization by decreasing endothelial progenitor cell function.

The number and function of endothelial progenitor cells (EPCs) are sensitive to hyperglycemia, hypertension, and smoking in humans, which are also associated with the development of atherosclerosis. GroEL1 from Chlamydia pneumoniae has been found in atherosclerotic lesions and is related to atherosclerotic pathogenesis. However, the actual effects of GroEL1 on EPC function are unclear. In this study, we investigate the EPC function in GroEL1-administered hind limb-ischemic C57BL/B6 and C57BL/10ScNJ (a toll-like receptor 4 (TLR4) mutation) mice and human EPCs. In mice, laser Doppler imaging, flow cytometry, and immunohistochemistry were used to evaluate the degree of neo-vasculogenesis, circulating level of EPCs, and expression of CD34, vWF, and endothelial nitric oxide synthase (eNOS) in vessels. Blood flow in the ischemic limb was significantly impaired in C57BL/B6 but not C57BL/10ScNJ mice treated with GroEL1. Circulating EPCs were also decreased after GroEL1 administration in C57BL/B6 mice. Additionally, GroEL1 inhibited the expression of CD34 and eNOS in C57BL/B6 ischemic muscle. In vitro, GroEL1 impaired the capacity of differentiation, mobilization, tube formation, and migration of EPCs. GroEL1 increased senescence, which was mediated by caspases, p38 MAPK, and ERK1/2 signaling in EPCs. Furthermore, GroEL1 decreased integrin and E-selectin expression and induced inflammatory responses in EPCs. In conclusion, these findings suggest that TLR4 and impaired NO-related mechanisms could contribute to the reduced number and functional activity of EPCs in the presence of GroEL1 from C. pneumoniae.

Domino Rh-catalyzed hydroformylation-double cyclization of o-amino cinnamyl derivatives: applications to the formal total syntheses of physostigmine and physovenine.

A parallel, versatile and efficient route to synthesis of pyrrolidinoindoline and tetrahydrofuranoindoline alkaloids from cinnamyl derivatives has been developed, featuring a domino Rh-catalyzed hydroformylation-double cyclization sequence. This method can be applied to the syntheses of anti-Alzheimer drugs such as physostigmine and physovenine.

TNF-α-decreased thrombomodulin expression in monocytes is inhibited by propofol through regulation of tristetraprolin and human antigen R activities.

Thrombomodulin (TM) is expressed on the surface of monocytes and is a key regulator of actual immune capacity. Propofol is an anesthetic agent that exerts anti-inflammatory effects. The objective of this study was to determine whether propofol could modulate TM in TNF-α-stimulated monocytes. THP-1 cells and male New Zealand rabbits were used in this study. The results showed that TNF-α decreases the TM expression by mediating posttranscriptional modification, and this inhibition may be repressed by treatment with propofol. Immunofluorescence, immunoprecipitation, and pull-down assays were used to demonstrate that Rac1-dependent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, Cdc42, and p38 mitogen-activated protein kinase activation, as well as tristetraprolin (TTP) expression, all contributed to the downregulation of TM in TNF-α-treated cells. Propofol reversed the effects of TNF-α on TM downregulation. Propofol mediated the expression of intracellular TTP and the distribution of cytosolic human antigen R (HuR) and changed their interactions with the 3'-untranslated region of TM mRNA regulating by Cdc42 and Rac1. In addition, the animal study showed that propofol regulates TM, TTP, and HuR expression on monocytes in TNF-α-treated rabbits. In conclusion, the inhibition of TM expression in TNF-α-treated monocytes was mediated by the activation of NADPH oxidase and the expression of TTP. Propofol may inhibit the downregulation of TM by mediating NADPH oxidase and TTP inactivation and through the activation of HuR in vitro and in vivo. Utilizing TTP and HuR to control TM expression may be a promising approach for controlling systemic inflammation, and propofol may possess potential implications for the clinical immunity of monocytes after anesthesia or surgery.

Alkyne-mediated domino hydroformylation/double cyclization: mechanistic insight and synthesis of (±)-tashiromine.

A novel domino reaction, alkyne-mediated domino hydroformylation/double cyclization, has been developed for rapid preparation of indolizidine type alkaloids. DFT calculations were applied for rationales of reactivity and selectivity. A concise synthesis of tashiromine as the application of the methodology is also reported.

Ginkgo biloba extract suppresses endotoxin-mediated monocyte activation by inhibiting nitric oxide- and tristetraprolin-mediated toll-like receptor 4 expression.

Monocytes expressing toll-like receptor 4 (TLR4) play a major role in regulating the innate immune response and are involved in systemic inflammation. Previous studies have shown that Ginkgo biloba extract (GBE) may act as a therapeutic agent for some cardiovascular and neurological disorders. The objective of this study was to determine whether GBE could modulate immunity in human cells. The monocytic cell line THP-1 was used. Enzyme-linked immunosorbent assay results showed that lipopolysaccharide (LPS) induces the expression of monocyte chemotactic protein-1 (MIP-1), tumor necrosis factor-α, stromal cell-derived factor-1, and MIP-1α, and this induction may be repressed by GBE treatment due to TLR4 blockade. The Griess reagent assay and western blot analysis showed that GBE-mediated inhibition of TLR4 expression was associated with the activation of mitogen-activated protein kinase and production of nitric oxide (NO). Actinomycin D chase experiments demonstrated that GBE decreased the TLR4 mRNA stability in cells. Confocal microscopy and real-time polymerase chain reaction showed that GBE induced the expression of intracellular tristetraprolin (TTP). Transfection with TTP siRNA reversed the effects of GBE in naïve or TLR4-overexpressing cells. Treatment with SNAP (an NO donor) may increase intracellular TTP expression in cells. Immunoprecipitation analysis showed that GBE mediates TTP activation and increases the interaction of TTP with the 3' untranslated region (UTR) of TLR4 mRNA by regulating NO production. Our findings indicate that GBE could decrease the sensitivity of monocytes to LPS. Utilizing TTP to control TLR4 expression may be a promising approach for controlling systemic inflammation, and GBE may have potential applications in the clinical treatment of immune diseases.

Preliminary surgical results of single-incision transumbilical laparoscopic bariatric surgery.

BACKGROUND: Recently, single-incision laparoscopic surgery (SILS) has been used for bariatric procedures, and this surgery is considered a type of minimally invasive surgery. When SILS is performed via the transumbilical route, the resultant abdominal wound is hidden and the cosmetic outcome is better. However, because of the small angle of manipulation and difficulty in liver traction, this technique is not used to perform complex bariatric surgery. In this prospective study, we used our novel technique, which involves the use of a liver-suspension tape and umbilicoplasty of an omega-shaped incision (omega umbilicoplasty), to perform laparoscopic bariatric surgery via the single-incision transumbilical (SITU) approach. We then assessed the safety and effectiveness of our surgical technique. METHODS: We started performing and developing this technique from December 2008. Until July 2009, 40 consecutive patients underwent 40 bariatric procedures: two adjustable gastric band placements, six sleeve gastrectomies, and 32 Roux-en-Y gastric bypass operations, including five cases where concomitant cholecystectomy was performed. RESULTS: The mean operation time was 93.4 min and the mean duration of postoperative hospitalization was 1.15 days. No perioperative or postoperative complications or deaths occurred. Most patients were very satisfied with the cosmetic outcomes. CONCLUSION: Our technique can be safely and effectively used for SITU laparoscopic bariatric surgery. This technique will soon be used for advanced abdominal surgeries besides bariatric ones.

Synthesis of 3-alkoxymethylcoumarin from 3-cyanochromene via a novel intermediate 2-phenylimino-3-alkoxymethylchromene.

In this paper a concise, efficient, and environmentally benign method for the synthesis of 3-alkoxymethylcoumarin is described. From the reaction of 3-cyanochromene with an alkoxide and arylamine in THF, (Z)-2-phenylimino-3-alkoxymethylchromene was obtained as a novel intermediate via an isomerization of the double bond, a 1,2-addition of alkoxide, a Michael-type addition of aniline, an another isomerization of double bond and an elimination of ammonia. Subsequently, the intermediate was converted into the desired coumarin by treatment with 15% HCl in THF in good yield.

TNF-alpha inhibits toll-like receptor 4 expression on monocytic cells via tristetraprolin during cardiopulmonary bypass.

Toll-like receptor 4 (TLR4) plays a major role in regulating the innate immune response, which is related to postoperative complications. Although inflammatory capacity and TNF-alpha synthesis were altered on monocytes after cardiopulmonary bypass (CPB), whether the CPB and the CPB-induced TNF-alpha affect TLR4 expression on monocytes have not yet clarified. We speculate that the changing of TNF-alpha level during CPB may be involved in monocytic TLR4 expression. As previous report, our enzyme-linked immunosorbent assay showed that CPB elevated the plasma level of TNF-alpha, whereas off-pump cardiac surgery does not. Flow cytometry reported decreased levels of monocytic TLR4 in patients undergoing CPB but not undergoing off-pump cardiac surgery. To elucidate whether the CPB-induced TNF-alpha is related to TLR4 down-regulation, we used human monocytic THP-1 cells. Actinomycin D chase experiments demonstrated that TNF-alpha decreased TLR4 expression and TLR4 mRNA stability on THP-1. Confocal microscopy and real-time polymerase chain reaction showed that TNF-alpha induced intracellular tristetraprolin (TTP) expression. Transfection with TTP siRNA reversed the down-regulation of TLR4 in TNF-alpha-stimulated THP-1. Treatment with ERK1/2 inhibitor and SAPK/JNK inhibitor decreased TNF-alpha-induced TTP expression. Immunoprecipitation and Western blot analysis showed that the TNF-alpha-mediated activation of TTP might be inhibited by p38 mitogen-activated protein kinase inhibitor and by PD98059. We also demonstrated in clinical samples with confocal microscopy and flow cytometry that CPB led to an elevation of TTP in monocytes. In conclusion, CPB and TNF-alpha decrease TLR4 expression on monocytes; TTP expression and mitogen-activated protein kinase-signaling pathways play critical roles in CPB- and TNF-alpha-mediated decreases of TLR4 on monocytes. Our results suggest that using TTP to control cytokine message decay rate may be a promising approach for controlling system inflammation and preventing post-CPB complications.

Role of human hepatic cytochrome P-450s in territrem A metabolism.

The ability of human liver microsomal preparations (HLM1, 2, 3, and 5), microsomes from human lymphoblasts expressing different cytochrome P-450 (CYP450) isoforms, and CYP3A4 cDNA-transfected V79 Chinese hamster cells to metabolize territrem A (TRA) was studied. The only metabolite generated by any of these preparations was 6beta-hydroxymethyl-6beta-demethylterritrem A (MA(1)). MA(1) formation was observed with all four human liver microsomal samples. Of the eight microsomal preparations from human lymphoblasts expressing different cytochrome P-450 enzymes (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) examined, only those expressing CYP2C9, CYP2D6, or CYP3A4 metabolized TRA, with that expressing CYP3A4 being the most active. No TRA metabolites were formed by control V79MZ cells, but MA(1) was formed by CYP3A4 cDNA-transfected V79 Chinese hamster cells. In order to investigate which CYP450 isoforms were involved in MA(1) formation in the human liver microsomal preparations, the effects of six isoform-specific chemical inhibitors (furafylline, sulfaphenazole, omeprazole, quinidine, ketaconazole, and diethyldithiocarbamate) and anti-3A4, anti-2C9, and anti-2D6 antibodies on TRA metabolism by HLM2 and HLM5 were examined. MA(1) formation was markedly inhibited by ketaconazole, with quinidine and sulfaphenazole having less of an effect. Anti-CYP3A4 antibody markedly inhibited MA(1) formation, while antibodies against CYP2C9 or CYP2D6 had little effect. The amount of MA(1) formed using different HLM preparations was related to the 6beta-testosterone hydroxylase activity and CYP3A4 protein content of the preparations. These results suggest that CYP3A4 is the major enzyme involved in TRA metabolism by human liver microsomes, with CYP2C9 and CYP2D6 playing a minor role.