YH12852, a potent and highly selective 5-HT4 receptor agonist, significantly improves both upper and lower gastrointestinal motility in a guinea pig model of postoperative ileus.

BACKGROUND: Postoperative ileus (POI) is a transient gastrointestinal (GI) dysmotility that commonly develops after abdominal surgery. YH12852, a novel, potent and highly selective 5-hydroxytryptamine 4 (5-HT4 ) receptor agonist, has been shown to improve both upper and lower GI motility in various animal studies and may have applications for the treatment of POI. Here, we investigated the effects and mechanism of action of YH12852 in a guinea pig model of POI to explore its therapeutic potential. METHODS: The guinea pig model of POI was created by laparotomy, evisceration, and gentle manipulation of the cecum for 60 seconds, followed by closure with sutures under anesthesia. Group 1 received an oral administration of vehicle or YH12852 (1, 3, 10 or 30 mg/kg) only, while POI Group 2 was intraperitoneally pretreated with vehicle or 5-HT4 receptor antagonist GR113808 (10 mg/kg) prior to oral dosing of vehicle or YH12852 (3 or 10 mg/kg). Upper GI transit was evaluated by assessing the migration of a charcoal mixture in the small intestine, while lower GI transit was assessed via measurement of fecal pellet output (FPO). KEY RESULTS: YH12852 significantly accelerated upper and lower GI transit at the doses of 3, 10, and 30 mg/kg and reached its maximal effect at 10 mg/kg. These effects were significantly blocked by pretreatment of GR113808 10 mg/kg. CONCLUSION AND INFERENCES: Oral administration of YH12852 significantly accelerates and restores delayed upper and lower GI transit in a guinea pig model of POI. This drug may serve as a useful candidate for the treatment of postoperative ileus.

Low central venous pressure with milrinone during living donor hepatectomy.

Maintaining a low central venous pressure (CVP) has been frequently used in liver resections to reduce blood loss. However, decreased preload carries potential risks such as hemodynamic instability. We hypothesized that a low CVP with milrinone would provide a better surgical environment and hemodynamic stability during living donor hepatectomy. Thirty-eight healthy adult liver donors were randomized to receive either milrinone (milrinone group, n = 19) or normal saline (control group, n = 19) infusion during liver resection. The surgical field was assessed using a four-point scale. Intraoperative vital signs, blood loss, the use of vasopressors and diuretics and postoperative laboratory data were compared between groups. The milrinone group showed a superior surgical field (p < 0.001) and less blood loss (142 +/- 129 mL vs. 378 +/- 167 mL, p < 0.001). Vital signs were well maintained in both groups but the milrinone group required smaller amounts of vasopressors and less-frequent diuretics to maintain a low CVP. The milrinone group also showed a more rapid recovery pattern after surgery. Milrinone-induced low CVP improves the surgical field with less blood loss during living donor hepatectomy and also has favorable effects on intraoperative hemodynamics and postoperative recovery.

Pinusolide and 15-methoxypinusolidic acid attenuate the neurotoxic effect of staurosporine in primary cultures of rat cortical cells.

BACKGROUND AND PURPOSE: Apoptosis is a fundamental process required for neuronal development but also occurs in most of the common neurodegenerative disorders. In an attempt to obtain an anti-apoptotic neuroprotective compound from natural products, we isolated the diterpenoids, pinusolide and 15-MPA, from B. orientalis and investigated their neuroprotective activity against staurosporine (STS) -induced neuronal apoptosis. In addition, we determined the anti-apoptotic mechanism of these compounds in rat cortical cells. EXPERIMENTAL APPROACH: Primary cultures of rat cortical cells injured by STS were used as an in vitro assay system. Cells were pretreated with pinusolide or 15-MPA before exposure to STS. Anti-apoptotic activities were evaluated by the measurement of cytoplasmic condensation and nuclear fragmentation. The levels of cellular peroxide, malondialdehyde (MDA) and [Ca(2+)]i, as well as the activities of superoxide dismutase (SOD) and caspase-3/7, were measured. KEY RESULTS: Pinusolide and 15-MPA, at a concentration of 5.0 ìM, reduced the condensed nuclei and rise in [Ca(2+)]i that accompanies apoptosis induced by 100 nM STS. Pinusolide and 15-MPA also protected the cellular activity of SOD, an antioxidative enzyme reduced by STS insult. Furthermore, the overproduction of reactive oxygen species and lipid peroxidation induced by STS was significantly reduced in pinusolide and 15-MPA treated cells. In addition, pinusolide and 15-MPA inhibited STS-induced caspase-3/7 activation. CONCLUSIONS AND IMPLICATIONS: These results show that pinusolide and 15-MPA protect neuronal cells from STS-induced apoptosis, probably by preventing the increase in [Ca(2+)]i and cellular oxidation caused by STS, and indicate that they could be used to treat neurodegenerative diseases.

Structural analysis of multifunctional peptide motifs in human bifunctional tRNA synthetase: identification of RNA-binding residues and functional implications for tandem repeats.

Human bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) contains three tandem repeats linking the two catalytic domains. These repeated motifs have been shown to be involved in protein-protein and protein-nucleic acid interactions. The single copy of the homologous motifs has also been found in several different aminoacyl-tRNA synthetases. The solution structure of repeat 1 (EPRS-R1) and the secondary structure of the whole appended domain containing three repeated motifs in EPRS (EPRS-R123) was determined by nuclear magnetic resonance (NMR) spectroscopy. EPRS-R1 consists of two helices (residues 679-699 and 702-721) arranged in a helix-turn-helix, which is similar to other RNA binding proteins and the j-domain of DnaJ, and EPRS-R123 is composed of three helix-turn-helix motifs linked by an unstructured loop. When tRNA is bound to the appended domain, chemical shifts of several residues in each repeat are perturbed. However, the perturbed residues in each repeat are not the same although they are in the same binding surface, suggesting that each repeat in the appended domain is dynamically arranged to maximize contacts with tRNA. The affinity of tRNA to the three-repeated motif was much higher than to the single motif. These results indicate that each of the repeated motifs has a weak intrinsic affinity for tRNA, but the repetition of the motifs may be required to enhance binding affinity. Thus, the results of this work gave information on the RNA-binding mode of the multifunctional peptide motif attached to different ARSs and the functional reason for the repetition of this motif.

Fas- and tumor necrosis factor-mediated apoptosis uses the same binding surface of FADD to trigger signal transduction. A typical model for convergent signal transduction.

FADD is known to function as a common signaling conduit in Fas- and tumor necrosis factor (TNF)-mediated apoptosis. The convergent death signals from the Fas receptor and TNF receptor 1 are transferred to FADD by death domain interactions triggering the same cellular event, caspase-8 activation. In this work, we investigated whether the same binding surface of FADD is used for both signaling pathways by using FADD death domain mutants. Mutations in helices alpha2 and alpha3 of the FADD death domain, the interacting surface with the Fas death domain, affected TNF-mediated apoptosis to various extents. This indicated that TNF-mediated apoptosis uses the same binding surface of the FADD death domain as Fas-mediated apoptosis. The binding specificity is not the same, however. Some mutations affected the binding affinity of the Fas death domain for the FADD death domain, but did not influence TNF-mediated apoptosis and vice versa. Interestingly, all mutants tested that affected TNF-mediated apoptosis have structural perturbations, implying that the structural integrity, involving helices alpha2 and alpha3 in particular, is critical in TNF-mediated apoptosis. Our results suggest that different signaling molecules use a similar structural interaction to trigger the same cellular event, such as caspase-8 recruitment, which could be typical in convergent signal transduction.

Radical cyclization of beta-aminoacrylates: synthesis of (-)-indolizidine 223AB.

[reaction: see text] (-)-Indolizidine 223AB was synthesized via radical cyclization of the beta-aminoacrylate derivative of a trans-2,5-disubstituted pyrrolidine. The trans-2,5-disubstituted pyrrolidine substrate was prepared by radical cyclization of a Ses-protected beta-aminoacrylate.

Comparison of in vivo fate and immunogenicity of hepatitis B surface antigen incorporated in cationic and neutral liposomes.

To compare cationic liposomes (CatL) and neutral liposomes (NeuL), as a vaccine carrier, the in vivo fate and immunogenicity of hepatitis B surface antigen (HBsAg), incorporated in CatL and NeuL, were investigated. CatL, composed of phosphatidyl choline (PC) and stearyl amine (SA) with a molar ratio of 9:1, showed a 2.5-fold higher incorporating efficiency of HBsAg than NeuL composed of PC alone. Most of HBsAg incorporated in both liposomes existed in an antibody-available form on the outer surface of liposomes. After intramuscular injection to rats, HBsAg in CatL resided at the injection site for a longer period than that in NeuL with terminal half lives of 52.5 and 42.9 h, respectively. However, HBsAg in NeuL was more efficiently taken up by the lymphatic organs and spleen than that in CatL. Furthermore, the group treated with HBsAg in NeuL showed earlier sero-conversion with higher anti-HBsAg titre than the group treated with HBsAg in CatL. Sero-conversion rates (SCRs) in both CatL- and NeuL-treated animals were 100% after every injection carried out, except the primary injection of CatL. These results demonstrate that CatL can enhance the retention of incorporated antigen at the injection site, compared with NeuL. However, the production of antibody by HBsAg in NeuL is more effective than that by HBsAg in CatL, probably due to the higher lymphatic targeting ability of NeuL. Thus, NeuL appears to be a better carrier for HBsAg than CatL.

The solution structure of FADD death domain. Structural basis of death domain interactions of Fas and FADD.

A signal of Fas-mediated apoptosis is transferred through an adaptor protein Fas-associated death domain protein (FADD) by interactions between the death domains of Fas and FADD. To understand the signal transduction mechanism of Fas-mediated apoptosis, we solved the solution structure of a murine FADD death domain. It consists of six helices arranged in a similar fold to the other death domains. The interactions between the death domains of Fas and FADD analyzed by site-directed mutagenesis indicate that charged residues in helices alpha2 and alpha3 are involved in death domain interactions, and the interacting helices appear to interact in anti-parallel pattern, alpha2 of FADD with alpha3 of Fas and vice versa.

A multifunctional repeated motif is present in human bifunctional tRNA synthetase.

Tandem repeats located in the human bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) have been found in many different eukaryotic tRNA synthetases and were previously shown to interact with another distinct repeated motifs in human isoleucyl-tRNA synthetase. Nuclear magnetic resonance and differential scanning calorimetry analyses of an isolated EPRS repeat showed that it consists of a helix-turn-helix with a melting temperature of 59 degrees C. Specific interaction of the EPRS repeats with those of isoleucyl-tRNA synthetase was confirmed by in vitro binding assays and shown to have a dissociation constant of approximately 2.9 microM. The EPRS repeats also showed the binding activity to the N-terminal motif of arginyl-tRNA synthetase as well as to various nucleic acids, including tRNA. Results of the present work suggest that the region comprising the repeated motifs of EPRS provides potential sites for interactions with various biological molecules and thus plays diverse roles in the cell.