Influence of leukotriene pathway polymorphisms on clinical responses to montelukast in Japanese patients with asthma.

WHAT IS KNOWN AND OBJECTIVE: Montelukast, a cysteinyl leukotriene receptor 1 antagonist, is safe and efficacious in patients with asthma. The mechanisms underlying the significant interpatient variability in response to montelukast are not clear but are believed to be, in part, because of genetic variability. METHODS: To examine the associations between polymorphisms in candidate genes in the leukotriene pathway and outcomes in patients with asthma on montelukast for 4-8 weeks, we evaluated the changes in peak expiratory flow (PEF), forced expiratory volume in 1 s (FEV(1·0) ) and patients' subjective symptom before and after montelukast treatment. DNA was collected from 252 Japanese participants. RESULTS AND DISCUSSION: Two single-nucleotide polymorphisms (SNPs) in the ALOX5 (rs2115819) and LTA4H (rs2660845) genes were successfully typed. There was no difference between members of the general population (n = 200) and patients (n = 52) in each genotype frequency. Significant associations were found between SNP genotypes in the LTA4H gene and changes in PEF and FEV(1·0) . The PEF and FEV(1·0) responses to montelukast in the A/A genotypes (n = 4) for the LTA4H SNP were significantly higher than those in the G allele carriers (A/G+G/G) (n = 17). WHAT IS NEW AND CONCLUSION: Despite the small sample size, our results suggest that genetic variation in leukotriene pathway candidate genes contributes to variability in clinical responses to montelukast in Japanese patients with asthma.

Development of a multi-channel 320 GHz interferometer for high density plasma measurement in Heliotron J.

We report the development of a new interferometer with two stable, high-power, 320 GHz solid-state sources in Heliotron J. A heterodyne Michelson interferometer optical scheme is employed. Two solid-state oscillators are utilized as sources with a fixed frequency at 320 GHz and frequency tunable of 312-324 GHz. Quasi-optical techniques are used for beam transmission. The beam is elongated in the vertical direction with two off-axis parabolic mirrors and injected into the plasma as a sheet beam for the multi-channel measurement (>5 ch.). Passing through the plasma, the beam is reflected at a retroreflector-array installed at the vacuum chamber wall. The retroreflector-array is a bunch of retroreflector structures, which can suppress the beam refraction caused by plasma without much space inside a vacuum chamber unlike a single retroreflector and can facilitate the system design. The source, detectors, and the retroreflector-array are tested to evaluate their basic performance on a tabletop experiment.

Doppler ultrasonography-aided early diagnosis of venous thromboembolism after total knee arthroplasty.

OBJECTIVES: Postoperative deep venous thrombosis (DVT) is usually asymptomatic but can result in a fatal pulmonary embolism (PE). To assess the ability of transcranial Doppler (TCD) ultrasound apparatus to detect venous emboli in patients who had undergone total knee arthroplasty (TKA). METHODS: Forty-eight patients undergoing TKA were examined postoperatively by using compression ultrasonography, computed tomographic angiography, and TCD ultrasonography that detected high-intensity transient signals (HITS) in femoral veins. An original scoring system based on both the number of HITS and the locations of DVT was tested for its accuracy in predicting PE development. RESULTS: Twenty-three of the 48 patients had DVT postoperatively, and 8 had an asymptomatic PE. The sensitivity and specificity of the HITS assessment alone in identifying PE development were 75% and 92.5%, respectively. The scoring system, however, had a sensitivity of 100% and a specificity of 85% and the area under the receiver operating characteristic (ROC) curve (AUC) was 0.96. CONCLUSIONS: Application of a scoring system based on the detection of both DVT and HITS may be an effective and efficient method of screening for PE after knee arthroplasty.

Role of Ca2+ mobilization and Ca2+ sensitization in 8-iso-PGF 2 alpha-induced contraction in airway smooth muscle.

BACKGROUND: Isoprostanes are prostaglandin (PG)-like compounds synthesized by oxidative stress, not by cyclooxygenase, and increase in bronchoalveolar lavage fluid of patients with asthma. The airway inflammation implicated in this disease may be amplified by oxidants. Although isoprostanes are useful biomarkers for oxidative stress, the action of these agents on airways has not been fully elucidated. OBJECTIVE: This study was designed to determine the intracellular mechanisms underlying the effects of oxidative stress on airway smooth muscle, focused on Ca(2+) signalling pathways involved in the effect of 8-iso-PGF(2 alpha). METHODS: Using simultaneous recording of isometric tension and F(340)/F(380) (an indicator of intracellular concentrations of Ca(2+), [Ca(2+)]i, we examined the correlation between tension and [Ca(2+)]i in response to 8-iso-PGF(2 alpha) in the fura-2 loaded tracheal smooth muscle. RESULTS: Augmented tension and F(340)/F(380) by 8-iso-PGF(2 alpha) were attenuated by ICI-192605, an antagonist of thromboxane A(2) receptors (TP receptors). Moreover, D609, an antagonist of phosphatidylcholine-specific phospholipase C, markedly reduced both the tension and F(340)/F(380) induced by 8-iso-PGF(2 alpha), whereas U73122, an antagonist of phosphatidylinositol-specific phospholipase C, modestly inhibited them by 8-iso-PGF(2 alpha). SKF96365, a non-selective antagonist of Ca(2+) channels, markedly reduced both tension and F(340)/F(380) by 8-iso-PGF(2 alpha). However, diltiazem and verapamil, voltage-dependent Ca(2+) channel inhibitors, modestly attenuated tension although their reduction of F(340)/F(380) was not different from that by SKF96365. Y-27632, an inhibitor of Rho-kinase, significantly attenuated contraction induced by 8-iso-PGF(2 alpha) without reducing F(340)/F(380), whereas GF109203X and Go6983, protein kinase C inhibitors, did not markedly antagonize them although reducing F(340)/F(380) with a potency similar to Y-27632. CONCLUSION: 8-iso-PGF(2 alpha) causes airway smooth muscle contraction via activation of TP receptors. Ca(2+) mobilization by SKF96365- and D609-sensitive Ca(2+) influx and Ca(2+) sensitization by Rho-kinase contribute to the intracellular mechanisms underlying the action of 8-iso-PGF(2 alpha). Rho-kinase may be a therapeutic target for the physiologic abnormalities induced by oxidative stress in airways.

Expression of reticulon 3 in Alzheimer's disease brain.

AIMS: Reticulon 3 (RTN3), a member of the reticulon family of proteins, interacts with the beta-secretase, beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1), and inhibits its activity to produce beta-amyloid protein. The aim of the present study was to clarify the biological role of RTN3 in the brain and its potential involvement in the neuropathology of Alzheimer's disease (AD). METHODS: We performed immunohistochemical and biochemical analyses using a specific antibody against RTN3 to investigate the expression and subcellular localization of RTN3 in control and AD brain tissue samples. RESULTS: Western blot analysis revealed no significant differences in the RTN3 levels between control and AD brains. Immunohistochemical staining showed that RTN3 immunoreactivity was predominantly localized in pyramidal neurones of the cerebral cortex. The patterns of RTN3 immunostaining were similar in control and AD cerebral cortices, and senile plaques were generally negative for RTN3. Biochemical subcellular fractionation disclosed that RTN3 colocalized with BACE1 in various fractions, including the endoplasmic reticulum and the Golgi apparatus. Double-immunofluorescence staining additionally indicated that RTN3 was localized in both endoplasmic reticulum and Golgi compartments in neurones. CONCLUSIONS: These results show that RTN3 is primarily expressed in pyramidal neurones of the human cerebral cortex and that no clear difference of RTN3 immunoreactivity is observable between control and AD brains. Our data also suggest that there is considerable colocalization of RTN3 with BACE1 at a subcellular level.

Mast cell tryptase causes homologous desensitization of beta-adrenoceptors by Ca2+ sensitization in tracheal smooth muscle.

BACKGROUND: Recent studies have revealed that in asthma, mast cells infiltrate to the smooth muscle layer and release tryptase, an enzymatic activator of protease-activated receptor 2 (PAR2). This phenomenon, mast cell myositis, is proposed as a new feature of asthma. However, little is known about the involvement of mast cell myositis in the pathophysiology of asthma. OBJECTIVE: This study was designed to determine whether mast cell degranulation has any functional impact on beta-adrenoceptors via PAR2 in airway smooth muscle. Moreover, we focused on Ca(2+) signalling as a mechanism underlying alteration of smooth muscle tone and responsiveness. METHODS: Isometric tension and F(340)/F(380), an indicator of the concentration of intracellular Ca(2+) ([Ca(2+)](i)), were simultaneously measured using fura-2-loaded tissues isolated from guinea-pig tracheal smooth muscle. RESULTS: Tryptase (1-100 nm) caused tension with elevated F(340)/F(380), and after exposure to tryptase for 15 min the inhibitory effect of isoprenaline (ISO) against methacholine was attenuated without elevating F(340)/F(380) in a concentration-dependent manner. Tryptase (<1 nm) had a modest effect on tension, but prolonged treatment (

Fabrication of lead-free piezoelectric (Na(0.5)K(0.5))NbO(3) ceramics by a modified solid-state reaction method.

Sodium potassium niobate, (Na(0.5)K(0.5))NbO(3), fine powder has been successfully synthesized at the low temperature of 550 degrees C through a modified solid-state reaction method, in which urea [CO(NH(2))(2)] plays an important role. High-density (Na(0.5)K(0.5))NbO(3) ceramics could be obtained by conventional sintering of the synthesized (Na(0.5)K(0.5))NbO(3) fine powder with the addition of 0.03 mol% Co(3)O(4) as a sintering additive. The crystal structure, microstructure, and dielectric and piezoelectric properties were characterized. The (Na(0.5)K(0.5))NbO(3) ceramic showed a comparatively saturated P-E hysteresis loop. The (Na(0.5)K(0.5))NbO(3) ceramic also displayed piezoelectricity with a piezoelectric constant d(33) of 126 pC/N and a planar electromechanical coupling factor k(p) of 33%.

Beta-adrenergic agonists regulate KCa channels in airway smooth muscle by cAMP-dependent and -independent mechanisms.

Stimulation of calcium-activated potassium (KCa) channels in airway smooth muscle cells by phosphorylation-dependent and membrane-delimited, G protein actions has been reported (Kume, H. A. Takai, H. Tokuno, and T. Tomita. 1989. Nature [Lond.]. 341:152-154; Kume, H., M. P. Graziano, and M. I. Kotlikoff. 1992. Proc. Natl. Acad. Sci. USA. 89:11051-11055). We show that beta-adrenergic receptor/channel coupling is not affected by inhibition of endogenous ATP, and that activation of KCa channels is stimulated by both alpha S and cAMP-dependent protein kinase (PKA). PKA stimulated channel activity in a dose-dependent fashion with an EC50 of 0.12 U/ml and maximum stimulation of 7.38 +/- 2.04-fold. Application of alpha S to patches near maximally stimulated by PKA significantly increased channel activity to 15.1 +/- 3.65-fold above baseline, providing further evidence for dual regulatory mechanisms and suggesting that the stimulatory actions are independent. Analysis of channel open-time kinetics indicated that isoproterenol and alpha S stimulation of channel activity primarily increased the proportion of longer duration events, whereas PKA stimulation had little effect on the proportion of short and long duration events, but resulted in a significant increase in the duration of the long open-state. cAMP formation during equivalent relaxation of precontracted muscle strips by isoproterenol and forskolin resulted in significantly less cAMP formation by isoproterenol than by forskolin, suggesting that the degree of activation of PKA is not the only determinant of tissue relaxation. We conclude that beta-adrenergic stimulation of KCa channel activity and relaxation of tone in airway smooth muscle occurs, in part, by means independent of cyclic AMP formation.

Impact of Prior Platinum-Based Therapy on Patients Receiving Salvage Systemic Treatment for Advanced Urothelial Carcinoma.

BACKGROUND: Trials of salvage therapy for advanced urothelial carcinoma have required prior platinum-based therapy. This practice requires scrutiny because non-platinum-based first-line therapy may be offered to cisplatin-ineligible patients. PATIENTS AND METHODS: Data of patients receiving salvage systemic chemotherapy were collected. Data on prior first-line platinum exposure were required in addition to treatment-free interval, hemoglobin, Eastern Cooperative Oncology Group performance status, albumin, and liver metastasis status. Cox proportional hazard regression was used to evaluate their association with overall survival (OS) after accounting for salvage single-agent or combination chemotherapy. RESULTS: Data were obtained from 455 patients previously exposed to platinum-based therapy and 37 not exposed to platinum. In the group exposed to prior platinum therapy, salvage therapy consisted of a single-agent taxane (n = 184) or a taxane-containing combination chemotherapy (n = 271). In the group not exposed to prior platinum therapy, salvage therapy consisted of taxane or vinflunine (n = 20), 5-fluorouracil (n = 1), taxane-containing combination chemotherapy (n = 12), carboplatin-based combinations (n = 2), and cisplatin-based combinations (n = 2). The median OS for the prior platinum therapy group was 7.8 months (95% confidence interval, 7.0, 8.1), and for the group that had not received prior platinum therapy was 9.0 months (95% confidence interval, 6.0, 11.0; P = .50). In the multivariable analysis, prior platinum therapy versus no prior platinum exposure did not confer an independent impact on OS (hazard ratio, 1.10; 95% confidence interval, 0.75, 1.64; P = .62). CONCLUSION: Prior platinum- versus non-platinum-based chemotherapy did not have a prognostic impact on OS after accounting for major prognostic factors in patients receiving salvage systemic chemotherapy for advanced urothelial carcinoma. Lack of prior platinum therapy should not disqualify patients from inclusion onto trials of salvage therapy.

VHL gene inactivation in an endolymphatic sac tumor associated with von Hippel-Lindau disease.

The authors present a case of endolymphatic sac tumor, a rare adenomatous tumor of the temporal bone, in a patient with von Hippel-Lindau (VHL) disease. Sequencing and microsatellite analysis of DNA samples from the tumor showed a 1-base pair deletion in exon 1 of the VHL gene and loss of heterozygosity at chromosome 3p25.5, the locus for the VHL gene. The results indicate that VHL gene inactivation contributed to the oncogenesis of endolymphatic sac tumor and provide molecular genetic proof that this tumor is associated with VHL disease.

Muscarinic regulation of membrane ion channels in airway smooth muscle cells.

We have demonstrated that stimulation of airway smooth muscle by muscarinic agonists results in a coordinated modulation of two membrane ion channel proteins. Both channels are modulated in a similar way, although their effects on open-channel probability are opposite. The voltage-dependence of channel activity is shifted to more positive potentials in the case of KCa, and to more negative potentials in the case of the voltage-dependent calcium channels. Similarly, KCa channel dwell-time kinetics are shifted to short open lifetimes, whereas the long open state is favored for the large-amplitude voltage-dependent calcium channel. Although little is known about the molecular coupling of calcium channels, muscarinic inhibition of KCa channels is mediated through a pertussis toxin-sensitive guanine nucleotide binding protein.

Phosphorylation and spatiotemporal distribution of KW8 (NDRF/NeuroD2), a NeuroD family basic helix-loop-helix protein.

KW8, a NeuroD family basic helix-loop-helix protein, was initially cloned during the course of screening for the genes related to long term potentiation in rat hippocampal slice. Its homologue NDRF/NeuroD was also reported. In this report its phosphorylation and spatiotemporal distribution was studied. KW8 was expressed not only during embryonic and neonatal periods but also in adults. In adult, KW8 was expressed only in brain tissues, such as the cerebral cortex, hippocampus and cerebellum. Immunohistological studies revealed that KW8 was localized in the nuclei of neurons. On immunoblotting of rat brain tissue, COS-1 cells and Neuro2A cells overexpressing KW8, this protein was detected as several diffuse bands. Alkaline phosphatase treatment reduced the molecular weights of these bands. Metabolic labeling with 32Pi in COS-1 cells confirmed that the KW8 protein was phosphorylated in vivo. Some of the physiological functions of KW8 might be regulated by this phosphorylation. In yeast, the GAL4 fusion protein containing the C-terminal region of KW8 activated transcription of the reporter gene, suggesting that KW8 had transcriptional activity.

Norbin, a neurite-outgrowth-related protein, is a cytosolic protein localized in the somatodendritic region of neurons and distributed prominently in dendritic outgrowth in Purkinje cells.

Distribution of norbin protein in rats was characterized by immunohistochemical study. It was distributed not only in whole brain but also in peripheral nervous system. The protein was localized in the somata, except for nuclei, and dendrites of neurons. Subcellular fractionation revealed that norbin is a cytosolic protein. Prominent distribution was observed in dendrites of dendritic outgrowth in Purkinje cells. Norbin should play a role in somatodendritic functions of neurons.

A partial connectin cDNA encoding a novel type of RSP motifs isolated from chicken embryonic skeletal muscle.

A partial cDNA encoding 811 amino acids of connectin (titin), a giant elastic protein of muscle (3,000 kDa), was cloned from a chicken embryonic skeletal muscle cDNA library using antibodies to muscle connectin. The encoded product was the C terminal segment of connectin. The predicted sequences consisted of 5 type II motifs (immunoglobulin C2 type) separated by 5 interdomain insertions. One interdomain insertion had significant homology (RSP) to KSP repeats found in human cardiac C-terminal connectin and another had a high sequence homology to porin (67.7%; 31 amino acids).

Localization of three fragments of connectin in chicken breast muscle sarcomeres.

Connectin (titin) links the Z line to the myosin filament in sarcomeres of vertebrate skeletal muscle. An 800 kDa fragment of alpha-connectin runs from the Z line up to the N2 line region in the I band, and the following beta-connectin portion runs up to the edge of the M line on the myosin filament in chicken breast muscle sarcomeres. Immunoelectron microscopy showed that a 400 kDa fragment following the 800 kDa fragment reaches the edge of the myosin filament and, thereafter a 1,700 kDa fragment runs to the M line on the myosin filament in chicken breast muscle sarcomeres. When stretched, the epitopes to anti-400 kDa fragment antibodies outside the myosin filament moved toward the inside of the I band.

Adrenomedullin and proadrenomedullin N-terminal 20 peptide induce histamine release from rat peritoneal mast cell.

Adrenomedullin (ADM)-induced histamine release from rat peritoneal mast cells was investigated. We compared the ability of full-length ADM to induce histamine release to the fragments ADM-(1-25) and ADM-(22-52), as well as proadrenomedullin N-terminal 20 peptide (PAMP). ADM (10(-8) to 10(-5) M) and PAMP (10(-8) to 10(-5) M) dose-dependently increased histamine release from peritoneal mast cell preparations. The effect of ADM-(1-25) was similar to ADM, whereas ADM-(22-52) did not show any effects. These data suggest the relative importance of the ADM C-terminal fragment, which contains a six-membered ring structure. Histamine release, induced by ADM, was significantly and dose-dependently inhibited by the addition of ADM-(22-52) (10(-5) M), Ca(2+) (0.5 to 2.0 mM), and benzalkonium chloride (3 to 7 microM), a selective inhibitor of Gi type G proteins. In contrast, PAMP (10(-5) M)-induced histamine release was not inhibited by Ca(2+). These results suggest that ADM induce histamine release via a putative ADM receptor in a manner sensitive to Gi-protein function and extracellular Ca(2+) concentration, and that PAMP might produce its effect by a different mechanism than ADM.

[(Dihydroindenyl)oxy]alkonic acid inhibits the cystic fibrosis transmembrane conductance regulator.

We investigated the effects of [(dihydroindenyl)oxy]alkaonic acid (DIOA) on the Cl(-) secretion in Calu-3 human airway epithelial cells that exclusively express the cystic fibrosis transmembrane conductance regulator (CFTR) as an apical Cl(-) channel. The 5'-nitro-2-(3-phenylpropylamino) benzoate (NPPB)-sensitive short-circuit current (I(sc)) and apical conductance were markedly reduced by DIOA (100 microM) in the presence and absence of isoproterenol (10 nM). Replacement of the butyl group in DIOA with a methyl group attenuated the inhibitory effects. The ED(50) of DIOA (17.0+/-1.0 microM) was almost equivalent to that of NPPB (15.6+/-2.1 microM). In conclusion, DIOA inhibits CFTR as strongly as NPPB does.

Intradermal application of nociceptin increases vascular permeability in rats: the possible involvement of histamine release from mast cells.

Intradermal application of nociceptin was used to investigate its in vivo effect on the inflammatory response in rats. Intradermal nociceptin (5 pmol/site-5 nmol/site) increased vascular permeability in a dose-dependent manner. The increased vascular permeability by nociceptin (5 nmol/site) was dose-dependently inhibited by the histamine H1 receptor antagonist pyrilamine (50 pmol/site-5 nmol/site). In rat peritoneal mast-cell preparation, nociceptin (10(-8)-10(-4) M) dose-dependently stimulated histamine release. The effect of nociceptin (10(-5) M) occurred rapidly (within 30 s) and was inhibited by pertussis toxin, Ca2+, but was not sensitive to naloxone, a classical opioid receptor antagonist. These characteristics are in agreement with features of the opioid-receptor-like 1 (ORL1) receptor, a non-classical opioid receptor linked to a pertussis toxin-sensitive G protein. Taken together, these data suggest that nociceptin, likely acting via the ORL1 receptor at the site of inflammation, might be critical for the enhancement of the inflammatory response by stimulating histamine release from mast cells.

Surgical treatment of renal cell carcinoma associated with Budd-Chiari syndrome: report of four cases and review of the literature.

AIMS: Renal cell carcinoma is sometimes associated with inferior vena caval tumour thrombus, but occlusion of hepatic veins by the tumour thrombus causing liver dysfunction, the so-called Budd Chiari syndrome, is relatively uncommon. There are only a few reports in the literature which discuss this condition. METHODS: Four cases admitted to our hospital over a 7-year period and eight cases reported in detail in the English and the Japanese literature were included in this study. They are classified into two groups: mild/silent, without liver failure, and severe, with liver failure. RESULTS: Five patients were classified as mild/silent and seven as severe. Clinical manifestations were mild in the former cases and acute in the latter. Surgery was performed in four of the former cases but only in one case of the latter cases. CONCLUSIONS: In mild cases, surgical treatment seems to avoid imminent hepatic failure effectively and should be performed as soon as possible. In such cases Budd Chiari syndrome in itself does not affect the prognosis. In severe cases, however, surgical treatment is very difficult and risky due to the existing hepatic failure.

The presenilin 2 loop domain interacts with the mu-calpain C-terminal region.

Presenilin 2 (PS2) is a gene responsible for the early-onset familial Alzheimer's disease (AD). PS2 mutations are considered to be closely related to the pathogenesis of AD. We screened for proteins that interact with PS2 to understand its pathological and physiological functions. Using the PS2 loop domain as the bait, the yeast two-hybrid system was used for screening, and mu-calpain was identified as a PS2 binding protein. In COS-1 cells, the interaction of PS2 with mu-calpain was confirmed by immunoprecipitation. These results suggested that PS2 and mu-calpain interact with each other, and might regulate each other's functions.