Effect of the potent and selective DP1 receptor antagonist, asapiprant (S-555739), in animal models of allergic rhinitis and allergic asthma.

Prostaglandin (PG) D2 elicits responses through either the DP1 and/or DP2 receptor. Experimental evidence suggests that stimulation of the DP1 receptor contributes to allergic responses, such that antagonists are considered to be directed therapies for allergic diseases. In this study, we demonstrate the activity of a novel synthetic DP1 receptor antagonist termed asapiprant (S-555739) for the DP1 receptor and other receptors in vitro, and assess the efficacy of asapiprant in several animal models of allergic diseases. We determined the affinity and selectivity of asapiprant for the DP1 receptor in binding assays. In the animal models of allergic rhinitis, changes in nasal resistance, nasal secretion, and cell infiltration in nasal mucosa were assessed after antigen challenge with and without asapiprant. Similarly, in the animal models of asthma, the effect of antigen challenge with and without asapiprant on antigen-induced bronchoconstriction, airway hyper-responsiveness, mucin production, and cell infiltration in lung were assessed. In binding studies, asapiprant exhibited high affinity and selectivity for the DP1 receptor. Significant suppression of antigen-induced nasal resistance, nasal secretion, and cell infiltration in nasal mucosa was observed with asapiprant treatment. In addition, treatment with asapiprant suppressed antigen-induced asthmatic responses, airway hyper-responsiveness, and cell infiltration and mucin production in lung. These results show that asapiprant is a potent and selective DP1 receptor antagonist, and exerts suppressive effects in the animal models of allergic diseases. Thus, asapiprant has potential as a novel therapy for allergic airway diseases.

FOXO1 activates glutamine synthetase gene in mouse skeletal muscles through a region downstream of 3'-UTR: possible contribution to ammonia detoxification.

Skeletal muscle is a reservoir of energy in the form of protein, which is degraded under catabolic conditions, resulting in the formation of amino acids and ammonia as a byproduct. The expression of FOXO1, a forkhead-type transcription factor, increases during starvation and exercise. In agreement, transgenic FOXO1-Tg mice that overexpress FOXO1 in skeletal muscle exhibit muscle atrophy. The aim of this study was to examine the role of FOXO1 in amino acid metabolism. The mRNA and protein expressions of glutamine synthetase (GS) were increased in skeletal muscle of FOXO1-Tg mice. Fasting induced FOXO1 and GS expression in wild-type mice but hardly increased GS expression in muscle-specific FOXO1 knockout (FOXO1-KO) mice. Activation of FOXO1 also increased GS mRNA and protein expression in C2C12 myoblasts. Using a transient transfection reporter assay, we observed that FOXO1 activated the GS reporter construct. Mutation of a putative FOXO1-binding consensus sequence in the downstream genomic region of GS decreased basal and FOXO1-dependent reporter activity significantly. A chromatin immunoprecipitation assay showed that FOXO1 was recruited to the 3' region of GS in C2C12 myoblasts. These results suggest that FOXO1 directly upregulates GS expression. GS is considered to mediate ammonia clearance in skeletal muscle. In agreement, an intravenous ammonia challenge increased blood ammonia concentrations to a twofold higher level in FOXO1-KO than in wild-type mice, demonstrating that the capacity for ammonia disposal correlated inversely with the expression of GS in muscle. These data indicate that FOXO1 plays a role in amino acid metabolism during protein degradation in skeletal muscle.

Detecting cause of dislocation after total hip arthroplasty by patient-specific four-dimensional motion analysis.

BACKGROUND: Dislocation is a major complication after total hip arthroplasty. Prosthesis impingement is considered to be an important cause of dislocation because impingement marks are more frequently found on retrieved cups or liners in patients who have undergone revision surgery because of dislocation (80%-94%) than in those who have undergone reoperation for other reasons (51%-56%). However, it remains a question whether impingement marks are the cause of dislocation or are instead its result. To clarify the issue, it is necessary to confirm noninvasively whether the point of impingement matches the patient's hip position when dislocation occurs. METHODS: Using four-dimensional patient-specific analysis, we recorded prosthesis impingement in 10 hips with instability after primary total hip arthroplasty when the patients reproduced the dislocation-causing motion. FINDINGS: We found prosthesis impingement to be related to at least instability in 6 of 10 hips with dislocation after primary total hip arthroplasty and in 4 of 4 hips that underwent revision surgery for recurrent dislocation. All impingements occurred between the anterior wall of the liner and the stem neck in posterior dislocation and between the posterior wall of the liner and the stem neck in anterior dislocation. Revision surgery in 1 of those 4 hips revealed 2 impingement marks on the retrieved liner that closely matched the prosthesis impingement point and the dislocation pathway of the metal head on the liner that were detected earlier during motion analysis. INTERPRETATION: Prosthesis impingement is an important factor in dislocation after total hip arthroplasty.

Increased systemic glucose tolerance with increased muscle glucose uptake in transgenic mice overexpressing RXRγ in skeletal muscle.

BACKGROUND: Retinoid X receptor (RXR) γ is a nuclear receptor-type transcription factor expressed mostly in skeletal muscle, and regulated by nutritional conditions. Previously, we established transgenic mice overexpressing RXRγ in skeletal muscle (RXRγ mice), which showed lower blood glucose than the control mice. Here we investigated their glucose metabolism. METHODOLOGY/PRINCIPAL FINDINGS: RXRγ mice were subjected to glucose and insulin tolerance tests, and glucose transporter expression levels, hyperinsulinemic-euglycemic clamp and glucose uptake were analyzed. Microarray and bioinformatics analyses were done. The glucose tolerance test revealed higher glucose disposal in RXRγ mice than in control mice, but insulin tolerance test revealed no difference in the insulin-induced hypoglycemic response. In the hyperinsulinemic-euglycemic clamp study, the basal glucose disposal rate was higher in RXRγ mice than in control mice, indicating an insulin-independent increase in glucose uptake. There was no difference in the rate of glucose infusion needed to maintain euglycemia (glucose infusion rate) between the RXRγ and control mice, which is consistent with the result of the insulin tolerance test. Skeletal muscle from RXRγ mice showed increased Glut1 expression, with increased glucose uptake, in an insulin-independent manner. Moreover, we performed in vivo luciferase reporter analysis using Glut1 promoter (Glut1-Luc). Combination of RXRγ and PPARδ resulted in an increase in Glut1-Luc activity in skeletal muscle in vivo. Microarray data showed that RXRγ overexpression increased a diverse set of genes, including glucose metabolism genes, whose promoter contained putative PPAR-binding motifs. CONCLUSIONS/SIGNIFICANCE: Systemic glucose metabolism was increased in transgenic mice overexpressing RXRγ. The enhanced glucose tolerance in RXRγ mice may be mediated at least in part by increased Glut1 in skeletal muscle. These results show the importance of skeletal muscle gene regulation in systemic glucose metabolism. Increasing RXRγ expression may be a novel therapeutic strategy against type 2 diabetes.

Histological evaluation of artifacts in tongue tissue produced by the CO2 laser and the electrotome.

OBJECTIVE: The purpose of this study was to investigate the clinical usefulness of laser surgery in pathologic diagnosis following excisional biopsy of human oral mucosa by CO(2) laser and electrotome. BACKGROUND DATA: When performing pathologic diagnosis and microscopic analysis of specimens excised by the CO(2) laser, there have been concerns about thermal denaturation of the excisional margin that may prevent diagnosis. MATERIALS AND METHODS: Thirty tongue tissue samples from humans that were surgically resected using a CO(2) laser (continuous wave mode for 10 cases and pulse wave mode for 10 cases) and an electrotome (10 cases) were formalin-fixed and paraffin-embedded. These preserved specimens were then cut into thick sections and subjected to hematoxylin and eosin staining followed by microscopic assessment. RESULTS: Despite the differences between the CO(2) laser and electrotome methods, similar thermal denaturation, such as carbonization, vacuolar degeneration, and elongation of nuclei, were observed at the excisional margins for both methods. Use of the CO(2) laser, particularly in pulse wave mode, reduced the amount of thermal denaturation significantly (p < 0.01) compared to the electrotome. CONCLUSION: These results indicate that the CO(2) laser is better than the electrotome as a means of making excisions for performing pathologic diagnosis.

2-Arylimino-5,6-dihydro-4H-1,3-thiazines as a new class of cannabinoid receptor agonists. Part 3: Synthesis and activity of isosteric analogs.

Structure-activity relationships and efforts to optimize the pharmacokinetic profile of isosteric analogs of 2-arylimino-5,6-dihydro-4H-1,3-thiazines as cannabinoid receptor agonists are described. Among those examined, compound 25 showed potent affinity for cannabinoid receptor 1 (CB1) and receptor 2 (CB2). This compound displayed oral bioavailability and analgesic activity.

Suppression of immunoglobulin production by a novel dihydroorotate dehydrogenase inhibitor, S-2678.

We discovered a novel dihydroorotate dehydrogenase (DHO-DH) inhibitor, S-2678 ([2-fluoro-2',5'-dimethyl-4'-[6-(3-methyl-2-butenyloxy) pyridin-3-yl] biphenyl-4-yl]-(3-methyl-2-butenyl) amine). Its inhibitory activity against DHO-DH was more potent than that of A77 1726, an active metabolite of the anti-rheumatic drug leflunomide. S-2678 suppressed immunoglobulin production in mouse B cells and human peripheral blood mononuclear cells in vitro, with little or no inhibition of cell proliferation, probably through inhibition of class switch recombination in the immunoglobulin heavy chain loci in B cells. In vivo antibody production induced by systemic immunization with ovalbumin was dramatically suppressed by oral administration of S-2678, without any toxicological signs. However, S-2678 did not affect T-cell activation in vitro, and cytokine production induced by intravenous anti-CD3 antibody in mice. S-2678 did not affect host defense in a mouse model of Candida infection, whereas leflunomide severely impaired it. In conclusion, S-2678 selectively acts on B cells, resulting in antibody production, which suggests that it is useful for the treatment of humoral immunity-related diseases.

2-Arylimino-5,6-dihydro-4H-1,3-thiazines as a new class of cannabinoid receptor agonists. Part 2: orally bioavailable compounds.

Structure-activity relationships and efforts to optimize the pharmacokinetic profile of a class of 2-arylimino-5,6-dihydro-4H-1,3-thiazines as cannabinoid receptor agonists are described. Among the compounds examined, compound 14 showed potent affinity and high selectivity for CB2, and compound 23 showed potent affinities against CB1 and CB2. These compounds displayed oral bioavailability.

Synergistic effect of PGD2 via prostanoid DP receptor on TNF-alpha-induced production of MCP-1 and IL-8 in human monocytic THP-1 cells.

Prostaglandin (PG) D2, a major cyclooxygenase metabolite generated predominantly from immunologically stimulated mast cells, is thought to contribute to the pathogenesis of allergic diseases via the two PGD2 receptors, prostanoid DP receptor and chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2). Monocytes are known to express the prostanoid DP receptor, however, the role of it in inflammatory responses is still unclear. In the present study, to clarify the functional roles of prostanoid DP receptor on monocytes, we examined the effect of PGD2 on the production of monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-8 from a human monocytic cell line, THP-1. Single activation of prostanoid DP receptor hardly produced any cytokines or chemokines. However, activation with PGD2 in the presence of tumor necrosis factor (TNF)-alpha mediated significant production of MCP-1 and IL-8, but not the other cytokines and chemokines, in comparison to single stimulation with TNF-alpha. In addition, the selective prostanoid DP receptor antagonist, pinagladin ((Z)-7-[(1R,2R,3S,5S)-2-(benzothiophen-3-ylcarbonylamide)-10-norpinan-3-yl]hept-5-enoic acid) inhibited the production of MCP-1 and IL-8 upon combined stimulation with PGD2 and TNF-alpha. The synergistic production of MCP-1 and IL-8 by PGD2 was mimicked by dibutyryl cAMP (db-cAMP) and was inhibited by a protein kinase A (PKA) inhibitor. Our findings suggest that activation of the prostanoid DP receptor on THP-1 cells enhances TNF-alpha-induced MCP-1 and IL-8 production via the cAMP/PKA signaling pathway.

Entomogenous fungi that produce 2,6-pyridine dicarboxylic acid (dipicolinic acid).

An inhibitor of the prophenoloxidase activation using extract from a silkworm pupa was isolated from a culture filtrate of Cordyceps militaris and identified as dipicolinic acid (DPA). The production of DPA in Clavicipitaceae fungi was examined. Entomogenous fungi that produce DPA were integrated into one group by a phylogenetic analysis based on 18S rDNA. It is suggested that the group acquired an ability to produce DPA during its evolution from plant pathogenic fungi to entomogenous fungi.

Extracellular trypsin-like proteases produced by Cordyceps militaris.

A trypsin-like protease, P-1-1, was purified from the culture supernatant of the fungus Cordyceps militaris by (NH(4))(2)SO(4) precipitation, chromatography on DEAE Bio-Gel Agarose, TSKgel CM-5PW, and gel-filtration with HiLoad 26/60 Superdex 75 pg, and its properties were examined. Purified P-1-1 showed a single band by SDS-PAGE and was estimated to have a molecular mass of 23,405 by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The optimum pH of the enzyme was between 8.5 and 12.0. It was inhibited strongly by leupeptin and diisopropyl fluorophosphate (DFP), and definitely did by N(alpha)-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), phenylmethanesulfonyl fluoride (PMSF) and chymostatin. The carbonyl group sides of Arg and Lys were confirmed as the sites of cleavage by the enzyme toward cecropin B. These results indicate that P-1-1 is a trypsin-type serine protease. The N-terminal amino acid sequence of P-1-1 showed a high homology with those of trypsins or chymotrypsin derived from Diptera insects.