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Background: Direct oral anticoagulants (DOACs) are the recommended first-line therapy for ischemic stroke prevention in patients with nonvalvular atrial fibrillation (NVAF). However, the safety and effectiveness of edoxaban for this indication requires monitoring over the long term in real-world settings., Methods: ETNA-AF-Japan (trial no. UMIN000017011) was a prospective, multicenter observational study (part of postmarketing surveillance in Japan). NVAF patients due to receive edoxaban to prevent ischemic stroke were enrolled between 13 April 2015 and 30 September 2017., Results: A total of 11 569 patients were enrolled. For the 11 111 patients (female, 40.6%) whose data comprised the safety analysis set, age, body weight, creatinine clearance (CLcr), and CHADS 2 score were 74.2 ± 10.0 years, 60.0 ± 12.7 kg, 63.9 ± 25.8 mL/min, and 2.2 ± 1.3, respectively (mean ± SD). The majority (86.3%) received edoxaban in accordance with package insert information. The mean duration of treatment was 561.9 ± 261.2 days. The annual incidence (95% confidence interval) of all bleeding events and major bleeding events was 5.60% (5.25%-5.98%) and 1.02% (0.88%-1.18%), respectively. The annual incidence of ischemic stroke (excluding transient ischemic attack, TIA) or systemic embolism was 1.08% (0.93%-1.25%). Multivariate analysis showed low body weight, low CLcr, history of gastrointestinal bleeding, anemia, and use of an antiplatelet agent to be associated with major bleeding, and history of ischemic stroke or TIA, vascular disease, and antiplatelet agent use to be associated with ischemic stroke (excluding TIA) or systemic embolism., Conclusions: These results provide real-world evidence for the long-term good safety and effectiveness profile of edoxaban in Japanese NVAF patients under clinical practice., Competing Interests: TY and YK have received remuneration from Bayer Yakuhin Ltd., Boehringer Ingelheim Japan Co. Ltd., Bristol Myers Squibb, and Daiichi Sankyo Co. Ltd. for consulting and lecturing services. TY has additionally received remuneration from Ono Pharmaceutical Co. Ltd. and Toa Eiyo Ltd. for consulting and lecturing services, and research funding from Bayer Yakuhin Ltd., Bristol Myers Squibb, and Daiichi Sankyo Co. Ltd. Daiichi Sankyo Co. Ltd. employs TN and KS. The In‐House Committee of Daiichi Sankyo Co., Ltd. and the Ministry of Health, Labour and Welfare of Japan (8 November 2014) approved the study protocol.ETNA‐AF‐Japan was carried out in accordance with the Good Post‐marketing Study Practice of the MHLW. The study protocol was submitted to the MHLW after its approval by the relevant committee at Daiichi Sankyo Co. Ltd. All study participants provided informed consent. The study is registered with the UMIN Clinical Trials Registry (trial no. UMIN000017011)., (© 2021 The Authors. Journal of Arrhythmia published by John Wiley & Sons Australia, Ltd on behalf of the Japanese Heart Rhythm Society.)

Background: The safety and effectiveness of edoxaban in real-world clinical settings have not yet been elucidated thoroughly among Japanese patients with nonvalvular atrial fibrillation (NVAF). We report the one-year interim results of ETNA-AF-Japan (Edoxaban Treatment in routiNe clinical prActice in patients with nonvalvular Atrial Fibrillation: UMIN000017011), an ongoing two-year study., Methods: ETNA-AF-Japan is a prospective, real-world multicenter observational study that analyzes the long-term safety and effectiveness of edoxaban. Physicians recorded clinical characteristics, bleeding events, and clinical events of ischemic stroke and systemic embolism, among others., Results: In total, 11 569 patients with NVAF were enrolled. The demographic and clinical characteristics of the patients in the safety analysis set (n = 11 107) were a mean age of 74.2 ± 10.0 years; female sex, 40.6%; age ≥75 years, 52.4%; body weight ≤60 kg, 54.3%; creatinine clearance ≤50 mL/min, 31.2%; mean CHADS 2 score of 2.2 ± 1.3. The mean treatment period was 311.2 days (median; 366.0 days), and ~80% of patients continued edoxaban treatment. In the safety analysis, the incidence of all bleeding events was 6.32% [95% CI: 5.87, 6.79] (n = 702), and for major bleeding, it was 1.08% [0.90, 1.29] (n = 120). In the effectiveness analysis set (n = 11 059), the incidence of ischemic stroke (excluding TIA) or systemic embolism was 1.10% [0.92, 1.32] (n = 122)., Conclusions: At one-year follow-up, the results showed no major concerns about the safety and effectiveness of edoxaban in Japanese patients with NVAF in a real-world clinical setting., Competing Interests: TY received consulting and lecture fees from Daiichi Sankyo Co. Ltd., Bayer Yakuhin Ltd., Bristol Myers Squibb, Ono Pharmaceutical Co. Ltd., Toa Eiyo Ltd., and Boehringer Ingelheim Japan Co. Ltd. and research funding from Daiichi Sankyo Co. Ltd., Bayer Yakuhin Ltd., and Bristol Myers Squibb. YK received consulting and lecture fees from Daiichi Sankyo Co. Ltd., Bayer Yakuhin Ltd., Bristol Myers Squibb, and Boehringer Ingelheim Japan Co. Ltd. TN and KS are employees of Daiichi Sankyo Co. Ltd., (© 2020 The Authors. Journal of Arrhythmia published by John Wiley & Sons Australia, Ltd on behalf of the Japanese Heart Rhythm Society.)

Objective: Oral leukoplakia is keratinized lesions in the buccal mucosa, tongue, and gingiva. It is the most common oral precancerous lesion; oxidative stresses and irrelevant autophagy have been reported to be the cause of oncogenesis. p62, a cytoplasmic protein induced by oxidative stress, is an adaptor protein involved in the formation of protein aggregates and induction and inhibition of autophagy. The inhibition of autophagy induces p62 overexpression and promotes oncogenesis via the oncogenic signaling pathway. The aim of the present study was to elucidate the involvement of intracellular expression of p62 in oral leukoplakia and to address its potential clinical implementation as a biomarker to predict malignant transformation., Material and Methods: Fifty samples from subjects with confirmed oral leukoplakia were evaluated by immunohistochemical staining for the expression of p62, 8-hydroxy-2'-deoxyguanosine (8-OHdG), Ki67, and p53. Univariate and multivariate logistic regression analyses were performed to evaluate the association between p62, 8-OHdG, Ki67, and p53 and clinical characteristics, including epithelial dysplasia., Results: Significant associations were observed between p62 expression in the nucleus, p62 aggregation, and epithelial dysplasia (adjusted odds ratio [OR] = 5.75; 95% confidence interval [CI]: [1.28, 26.2]; .024 and OR = 6.16; 95% CI: [1.01, 37.4]; .048, respectively). The expression of p62 in the cytoplasm and the levels of 8-OHdG, Ki67, and p53 were not significantly associated with epithelial dysplasia. A significant relationship was found between p62 expression in the nucleus and p53 expression (OR = 3.94; 95% CI: [1.14, 13.6]; .031)., Conclusions: The results suggested that p62 expression in the nucleus and p62 aggregation can be potential markers to predict the malignant transformation of oral leukoplakia., Competing Interests: The author(s) declare that there is no potential conflict of interest relevant to the study, authorship, and publication of this article.

Objectives: Secretory carcinoma (SC, mammary analogue secretory carcinoma) is a salivary gland tumor with ETV6-NTRK3 gene fusion, and its differential diagnosis includes acinic cell carcinoma (ACC). As hemorrhage is often seen in SC, we hypothesized that magnetic resonance imaging (MRI) and immunohistochemical analyses could distinguish SC from ACC., Study Design: Retrospective study., Methods: We used ETV6-NTRK3 gene fusion analyses to reclassify 19 parotid gland tumors that had previously been diagnosed as SC or ACC, and then investigated hemorrhage in both hematoxylin-eosin (H&E)-stained sections and MRIs, and immunohistochemical expression of S-100, mammaglobin, DOG1, and α-amylase., Results: The 19 tumors were genetically reclassified into 11 (58%) SC and 8 (42%) ACC. Combined S-100 and mammaglobin were specific for SC; whereas DOG1 was specific for ACC, and α-amylase was expressed only in 4 ACC cases (50%). H&E staining showed hemorrhage with hemosiderin deposition in all SC cases, and T2-weighted MRI showed hypointense areas in all investigated SC cases, but not in ACC., Conclusion: Hemorrhage with hemosiderin deposition is frequently present in SC, and hemorrhage findings in MRI and an immunohistochemical panels for S-100, mammaglobin and DOG1 can distinguish SC from ACC., Level of Evidence: 3b.

Background: In "real-world" practice, anticoagulant therapy is indicated for patients whose clinical profiles are not addressed in randomized clinical trials. We assessed the effectiveness and safety of dabigatran versus warfarin in "real-world" Japanese patients with non-valvular atrial fibrillation (NVAF)., Methods: Among 613 NVAF patients who initiated dabigatran or warfarin therapy during the period between 2011 and 2013, 362 patients were included in the study after propensity score adjustment. The median follow-up period was 1.3 years. The effectiveness and safety outcomes were thromboembolism and major bleeding, respectively., Results: The propensity-matched hazard ratios of thromboembolism and major bleeding with dabigatran were 1.03 (95% CI: 0.12-8.04, p =0.971) and 0.15 (95% CI: 0.01-0.90, p =0.037), respectively., Conclusions: The ability of dabigatran to prevent thromboembolism is comparable to that of warfarin; however, the major bleeding rate is lower with dabigatran in "real-world" NVAF patients.

Background: Little is known about physiological anticoagulation effects via antithrombin III (AT III) and protein C/S (PC/PS) in patients using new oral anticoagulants (NOACs)., Methods: We evaluated 120 consecutive patients with non-valvular atrial fibrillation (AF) receiving NOACs. Patients were randomly divided into three groups: a dabigatran group (DG, N =40), a rivaroxaban group (RG, N =40) or an apixaban group (AG, N =40). A warfarin group (WG, N =40) was matched with NOAC groups for age, sex and type of AF during the same time period. Blood samples were obtained in pretreatment, trough and peak phases to measure the activity of physiological coagulation inhibitors, including AT III and PC/PS or thrombus formation markers such as D-dimer and thrombin-antithrombin complex (TAT)., Results: D-dimer, TAT and AT III values for the NOAC groups were equivalent in the peak and trough phases. PC/PS activity in both phases was equally maintained in the pretreatment phase in the NOAC groups, while the activity in the WG was significantly suppressed in steady state. Moreover, no differences in trends for PC/PS activity were observed among NOAC groups., Conclusions: PC/PS activity was constant in both peak and trough phases in the patients on NOACs compared with activity of those on warfarin. In addition, there was no difference in the findings among NOACs.

Background: In the new pathologic classification of lung adenocarcinoma proposed by IASLC/ATS/ERS in 2011, lepidic type adenocarcinomas are constituted by three subtypes; adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and lepidic predominant invasive adenocarcinoma (LPIA). Although these subtypes are speculated to show sequential progression from preinvasive lesion to invasive lung cancer, changes of protein expressions during these processes have not been fully studied yet. This study aims to glimpse a proteomic view of the early lepidic type lung adenocarcinomas., Methods: A total of nine formalin-fixed and paraffin-embedded (FFPE) lepidic type lung adenocarcinoma tissues were selected from our archives, three tissues each in AIS, MIA and LPIA. The tumor and peripheral non-tumor cells in these FFPE tissues were collected with laser microdissection (LMD). Using liquid chromatography-tandem mass spectrometry (MS/MS), protein compositions were compared with respect to the peptide separation profiles among tumors collected from three types of tissues, AIS, MIA and LPIA. Proteins identified were semi-quantified by spectral counting-based or identification-based protein-based approach, and statistical evaluation was performed by pairwise G-tests., Results: A total of 840 proteins were identified. Spectral counting-based semi-quantitative comparisons of all identified proteins through AIS to LPIA have revealed that the protein expression profile of LPIA was significantly differentiated from other subtypes. 70 proteins including HPX, CTTN, CDH1, EGFR, MUC1 were found as LPIA-type marker candidates, 15 protein candidates for MIA-type marker included CRABP2, LMO7, and NPEP, and 26 protein candidates for AIS-type marker included LTA4H and SOD2. The STRING gene set enrichment resulted from the protein-protein interaction (PPI) network analysis suggested that AIS was rather associated with pathways of focal adhesion, adherens junction, tight junction, that MIA had a strong association predominantly with pathways of proteoglycans in cancer and with PI3K-Akt. In contrast, LPIA was associated broadly with numerous tumor-progression pathways including ErbB, Ras, Rap1 and HIF-1 signalings., Conclusions: The proteomic profiles obtained in this study demonstrated the technical feasibility to elucidate protein candidates differentially expressed in FFPE tissues of LPIA. Our results may provide candidates of disease-oriented proteins which may be related to mechanisms of the early-stage progression of lung adenocarcinoma.

Background: Non-vitamin K antagonist oral anticoagulants (NOACs) show a favorable balance between efficacy and safety compared with warfarin for patients with non-valvular atrial fibrillation (NVAF). In "real-world" practice, however, NOAC adherence and persistence among patients are not clear. The aim of this study is to evaluate NOAC and warfarin persistence in Japanese patients with NVAF who newly started these drugs., Methods: We retrospectively studied 401 patients with NVAF who had newly started NOACs during the first 18 months after our hospital adopted their use (197 dabigatran, 107 rivaroxaban, 102 apixaban) and 200 patients with NVAF who had newly started warfarin during the same period. The endpoint was drug discontinuation for each drug., Results: During the follow-up period (up to a maximum of 24 months), 113 (28%) patients who had newly started NOACs and 33 (17%) patients who had newly started warfarin discontinued the drug. The persistence rates of patients prescribed NOACs was lower than that of patients prescribed warfarin at 3, 6, and 12 months (85% versus 93%, 79% versus 88%, and 70% versus 82%, respectively). One-tenth of patients who had newly started NOACs discontinued the drug by their own decision. Drug adverse events, worsening renal dysfunction, and patient desire were the major causes of NOAC discontinuation., Conclusions: The rate of persistence of prescribed NOACs was significantly lower than that of warfarin in Japanese patients with NVAF.

Background: Electrode positioning is important for the operation of implantable loop recorders (ILRs). This study aimed to investigate optimal electrode positions for the implantation of ILRs., Methods: Fifteen male subjects participated in the study and R wave amplitude data were collected from 15 areas in the left anterior chest area, with 4 variations of electrode angles and body positions., Results: The estimated minimum R wave amplitude values were greater than 0.3 mV (manufacturer-recommended value) when electrodes were angled vertically and positioned on the midclavicular line of the third and the forth intercostal spaces or on the left sternal border of the fourth and fifth intercostal space and when angled at 45° to the right on the left sternal border of the third and the forth intercostal space., Conclusions: Exploring areas around the left sternal border of the fourth intercostal space - where the R wave amplitudes are least affected by body positions - is a reliable method for choosing implantation locations for ILRs.

Purpose: To obtain safety and effectiveness data on a combined anti-HIV drug, Epzicom (abacavir 600 mg/lamivudine 300 mg), a post-marketing surveillance on Epzicom that was required by the Japanese regulatory authority was conducted between January 2005 and December 2010., Methods: A joint survey (HIV-related drug [HRD] survey) has been conducted involving manufacturers of drugs for treatment of HIV infection in Japan. Safety and effectiveness data from total 624 cases (1107.3 person-years) registered to the HRD surveys and received Epzicom were obtained. Adverse drug reactions (ADRs) were defined as adverse events (AE) of which association with Epzicom could not be 'ruled out'., Results: It was found that the incidence of ADR was 32.4% (202/624 cases) on the case basis. In addition, the frequently reported ADR included hyperlipidaemia (59 cases), hypertriglyceridaemia (21 cases), blood bilirubin increased (19 cases), gamma-glutamyltransferase increase (14 cases), blood triglyceride increase (14 cases) and rash (14 cases). Serious AEs were seen in 19 patients (30 events), including one death (no evident association with Epzicom). There were four cases (0.6%) of survey-defined 'hypersensitivity', and the incidence was 0.9% (4/445) among abacavir naïve patients; none of which was reported as serious. No case of myocardial infarction was reported. One pregnant case who delivered a normal baby by caesarean section was reported to have experienced aggravation of anaemia and nausea., Conclusions: The post-marketing surveillance indicated that the incidence of both ischaemic heart disease and hypersensitivity associated with Epzicom was considerably low, suggesting that this drug can be safely used in the Japanese population., (© 2014 The Authors. Pharmacoepidemiology and Drug Safety published by John Wiley & Sons, Ltd.)

Purpose: Abacavir is a nucleoside reverse transcriptase inhibitor indicated for human immunodeficiency virus (HIV) infection. In Japan, Ziagen® (300-mg abacavir sulfate) has been marketed since 1999. To obtain safety data on Ziagen, a mandatory postmarketing surveillance was conducted between September 1999 and September 2009., Methods: A joint survey [HIV-related Drug Surveys (HRD)] has been conducted involving manufacturers of drugs for HIV treatment in Japan. Safety data from total 643 cases (1345.7 person-years) registered to the HRD surveys and received Ziagen were obtained. Adverse drug reaction (ADR) was defined as adverse event of which association with abacavir could not be "ruled out.", Results: It was found that the overall frequency of ADR was 47.6% (306/643); the common ADRs were "hyperlipidemia," "nausea," "increased γ-glutamyltransferase level," "increased blood triglycerides," "abnormal hepatic function," and so on. Serious adverse events were reported in 65 subjects; however, none of the three fatal cases were clearly associated with Ziagen use. The survey-defined hypersensitivity has been infrequently reported in 15 subjects (2.3%). Although some studies had indicated of the association between abacavir and myocardial infarction, no ischemic heart diseases were reported in the present survey. Two of the three pregnant cases delivered normal neonates (one induced abortion)., Conclusions: During the mandatory postmarketing survey of Ziagen, there were no cases of ischemic heart diseases, and the incidence of hypersensitivity was considerably low. These indicated that abacavir can be safely used in Japanese HIV+ population. However, the safety profile of Ziagen should be continued to be monitored through pharmacovigilance., (© 2014 The Authors. Pharmacoepidemiology and Drug Safety published by John Wiley & Sons, Ltd.)

Background and Purpose: Sorbin and SH3-domain-containing-1 (SORBS1) is an important adaptor protein in insulin-signalling pathway, and its genetic polymorphism may regulate the activity of insulin resistance. We investigated the association between the SORBS1 T228A polymorphism and ischaemic stroke., Methods: Genotyping was achieved by a rapid-cycle PCR and melting curve analysis using fluorescent probes in 1049 incident cases of ischaemic stroke and 1049 age- and sex-matched control subjects recruited from the Hisayama study., Results: The allele distributions of the SORBS1 T228A polymorphism were similar amongst cases and controls. The multivariate-adjusted odds ratio (OR) of the AA genotype for ischaemic stroke was 2.897 (95% CI, 0.907-8.018) compared with the TT genotype. In terms of stroke subtype, there was a trend toward a difference in the AA genotypes for lacunar infarction, compared with the TT genotype (OR = 8.740, P = 0.0510), and combined TT and TA genotypes (OR = 8.768, P = 0.0505). The other polymorphisms genotyped were not associated with any subtypes of ischaemic stroke. T228A polymorphism of SORBS1 was not associated with the prevalence of diabetes., Conclusions: The AA genotype of SORBS1 T228A polymorphism may play a role in lacunar infarction in the Japanese population.

The C242T polymorphism of p22phox, a component of NAD(P)H oxidase, may have an impact on cardiovascular diseases; however, the association between this polymorphism and brain infarction is not fully understood. Here, we investigate the relationship between the C242T polymorphism and brain infarction in Japan. We recruited 1055 patients with brain infarction and 1055 control subjects. A chi-squared test revealed that the T-allele frequency was lower in patients with cardioembolic infarction (5.6%) than in control subjects (11.0%, P < 0.001); however, allele frequencies in patients with lacunar and atherothrombotic infarction (11.2%) were not significantly different from those in control subjects (11.0%). A multivariate-adjusted conditional logistic regression analysis also revealed no association between CT + TT genotype, and lacunar and atherothrombotic infarction (odds ratio = 0.97, 95% confidence interval: 0.72-1.32). To investigate the functional effects of the C242T polymorphism, we examined superoxide production in COS-7 cells cotransfected with Nox4 and p22phox of each genotype. The superoxide-producing activity in those cells expressing p22phox with the T allele was not significantly different from that in cells expressing p22phox with the C allele. The present results suggest that the p22phox C242T polymorphism may have a protective effect against cardioembolic infarction, but is not related to lacunar and atherothrombotic infarction in Japan.

A hydrophilic ultrafiltration membrane, regenerated cellulose, facilitates the size-selectable permeability of hydrophilic solutes in reverse micellar solution. By using an ultrafiltration membrane with a molecular weight cutoff of 3,500, we demonstrate a nonaggregating protein refolding technique based on the dialysis of reverse micellar solution. This realizes concurrent removal of denaturants, urea and 2-mercaptoethanol, and the supply of redox reagents, reduced and oxidized glutathione (GSH, GSSG), to promote renaturation of proteins. Two mg/ml ribonuclease A (RNase A) was refolded completely without any dilution and aggregation for 60 h. The refolding behavior of RNase A is strongly influenced by the ratio of GSH and GSSG. Moreover, we recovered 90% of the refolded RNase A from AOT reverse micellar solution with acetone precipitation and beta-cyclodextrin washing. These findings should facilitate the production of a continuous protein refolding membrane reactor., (2004 Wiley Periodicals, Inc.)

1. Excessive production of extracellular matrix is thought to be involved in the progression of glomerulonephritis and glomerulosclerosis. In chronic glomerulonephritis, fibronectin has been shown to accumulate in the glomeruli, accompanied by cell proliferation. 2. Glomerulonephritis was induced in rats by the injection of anti-glomerular basement membrane antibody. The rats showed proteinuria and histological alterations in the glomeruli. An increase in fibronectin levels in the culture medium of isolated nephritic glomeruli was confirmed, and was associated with the development of nephritis. Immunohistochemical staining demonstrated a marked accumulation of fibronectin in the glomeruli of nephritic rats. 3. Attenuated generation of cyclic AMP was also observed in the nephritic glomeruli treated with forskolin, prostaglandin E1 or adenosine. 4. Forskolin, prostaglandin E2 and 8-bromo-cyclic AMP markedly reduced the production of fibronectin by the nephritic glomeruli compared with controls in a dose-dependent manner. 8-Bromo-cyclic AMP suppressed the production of fibronectin by cultured mesangial cells. 5. These findings suggest that the attenuated generation of cyclic AMP in response to ligands is connected to the augmented accumulation of fibronectin in nephritic glomeruli, and may facilitate the development of methods for treating glomerulonephritis and glomerulosclerosis.

To determine the effect of arthritis on the disposition of flurbiprofen (FP) and its acyl glucuronide (FPG) as well as formation of covalent adducts with plasma protein, a pharmacokinetic study was carried out in adjuvant-induced arthritic (AA) rats. In control animals the pharmacokinetics of FP were stereoselective following intravenous bolus injection of rac-FP: (-)-(R)-FP showed higher plasma clearance (CL(tot)) and shorter mean residence time (MRT) compared to (+)-(S)-FP. The CL(tot) and clearance for the glucuronide formation (CL(glu)) of both enantiomers in AA rats were extremely increased compared to those in control rats. Increased total clearance in AA rats was due, at least in part, to a remarkable increase in the plasma unbound fraction of FP, consistent with a decrease in the plasma albumin level. The yield of covalent binding of FP to plasma protein in AA rats was less than that in controls, being consistent with the decrease in the plasma acyl glucuronide level., (Copyright 2003 Wiley-Liss, Inc.)

The susceptibility of 129/SvJ mice to infection with Strongyloides venezuelensis was compared with that of C57BL/6 mice. After a primary infection, daily egg output in faeces (EPG) from 129/SvJ mice was lower and terminated earlier than that from C57BL/6 mice. Adult worm recovery from the small intestine of 129/SvJ mice on day 7 was also lower than that of C57BL/6 mice. When the numbers of larvae recovered from the lungs were examined on days 2, 3 and 4 after a primary infection, they were comparable between the two strains. On the other hand, when an equal number of larvae recovered from the lungs of each strain on day 3 were implanted orally into homologous strain mice, the magnitude of EPG and the number of adult worms in the small intestine on day 5 after implantation were significantly lower in 129/SvJ than in C57BL/6 mice. The number of mucosal mast cells in the jejunum was not significantly different between 129/SvJ and C57BL/6 naive mice. Total chondroitin sulphate concentration in the gut washings obtained from naive mice was significantly higher in 129/SvJ (11.34 +/- 9.48) than in C57BL/6 mice (1.09 +/- 0.77, P < 0.05). These results indicate that the natural resistance of 129SvJ mice to S. venezuelensis infection is expressed at the intestine, probably due to higher concentration of chondroitin sulphate, which prevents establishment of S. venezuelensis.

We examined the contribution of Ser203 of the human beta2-adrenoceptor (beta2-AR) to the interaction with isoprenaline. The affinity of (-)-isoprenaline was reduced by substitution of an alanine for Ser203, as well as for Ser204 and Ser207. An (-)-isoprenaline derivative with only one hydroxyl group, at the meta-position, showed reduced affinity for wild-type beta2-AR and S207A-beta2-AR and even lower affinities for S203A-beta2-AR and S204A-beta2-AR. By contrast, an (-)-isoprenaline derivative with only a para-hydroxyl group showed reduced affinity for wild-type beta2-AR but the serine to alanine mutations did not cause further decreases. The EC50 value for cyclic AMP generation in response to (-)-isoprenaline was increased, by about 120 fold, for each alanine-substituted beta2-AR mutant. These results suggest that Ser203 of the human beta2-AR is important for both ligand binding and receptor activation.

Background: Malignant myoepithelioma (MME) of the salivary gland, also known as myoepithelial carcinoma, is rare and its biologic behavior has not been clarified fully., Methods: Ten cases of MME were analyzed for their clinicopathologic features and immunohistochemical characteristics, focusing on prognostic factors and tumor differentiation. In addition, six cases of benign myoepithelioma (BME) also were examined for comparison., Results: The ten patients with MME (3 men and 7 women) ranged in age from 48-81 years (mean, 61.9 years). Seven cases of MME arose in the parotid salivary gland, two in the submandibular salivary gland, and one in minor salivary glands of the soft palate. In the current series, the incidence of MME was 0.45% among 1945 cases of major salivary gland tumors. Seven cases of MME developed from a benign preexisting tumor (six in pleomorphic adenoma and one in BME). Four of nine patients with MME died of the disease and two patients developed a recurrence. It was shown that MMEs were comprised of one cell type or a combination of two cell populations; these included, in order of incidence, epithelioid, spindle, and plasmacytoid cells. Patients with MME with marked cellular pleomorphism and perineural invasion had a poor prognosis. Immunohistochemically, putative myoepithelial markers such as muscle actins, cytokeratin 14, vimentin, and calponin, and S-100 protein were expressed highly in MME. High and low molecular weight cytokeratins and epithelial membrane antigen also frequently were positive in MME. p53 expression was observed in five MME cases, four of which either recurred or were fatal. Cellular proliferative activity assessed by mitotic count and the Ki-67 labeling index was significantly higher in MME cases than in BME cases. In limited cases, such cellular proliferative activity was shown to have prognostic value. Ultrastructurally, the tumor cells displayed certain myoepithelial characteristics., Conclusions: MME is a rare salivary gland tumor showing clinicopathologic diversity and presenting with various stages of myoepithelial differentiation. Histologic aggressiveness, marked cellular pleomorphism, p53 expression, and high cell proliferative activity were found to be correlated with a poor clinical outcome.

1. We have reported that Ba2+ causes endothelium-dependent relaxation of canine coronary arteries through NO synthesis in Ca2+-free and depolarizing solution. To determine the cellular mechanisms by which the endothelium-dependent relaxation occurs, we used fura-2 fluorometry (F350 and F390; excitation wavelengths, 350 and 390 nm, respectively) and estimated the intracellular Ba2+ concentration in endothelial and vascular smooth muscle cells. 2. Ba2+ (10(-3) M) increased the fura-2 ratio (F350/F390) recorded from a combined preparation of smooth muscle and endothelium (0.445+/-0.073, n = 4) and contracted the arteries in the presence of 80 mM K+ (0.22+/-0.06 g, n = 4). 3. Diltiazem (3 x l0(-6) M) blocks Ba2+ entry into vascular smooth muscle cells via L-type Ca2+ channels. In this condition, Ba2+ increased the fura-2 ratio in endothelial cells (0.141+/-0.014, n = 5) and relaxed the underlying smooth muscle (0.08+/-0.01 g, n = 5) by a mechanism which was sensitive to 10(-4) M NG-methyl-L-arginine (L-NMMA). 4. Ba2+-induced relaxation was not attenuated with repeated application and was elicited even after endothelium-dependent relaxations in response to 10(-6) M bradykinin were abolished due to tachyphylaxis. Neither 10(-2) M caffeine nor 10(-6) M thapsigargin had effect upon Ba2+-induced relaxation. 5. To further rule out changes in intracellular Ca2+ as a mechanism of Ba2+-induced relaxation, fura-2 fluorescence was measured at the isosbestic wavelengths for Ca2+ (360 nm) and Ba2+ (370 nm) in endothelium-intact arteries. Ba2+ altered F360, but not F370, suggesting little or no contribution of intracellular Ca2+ to the phenomenon of Ba2+-induced relaxation. 6. These results suggest that the Ba2+-induced relaxation is due to its direct activation of endothelial NO synthesis without mobilization of intracellular Ca2+.

Background: Basal cell adenocarcinoma (BCAC) of the salivary gland is a rare tumor and recently described entity. Eleven cases of BCAC are presented here and compared with basal cell adenoma (BCA) through assessment of cell proliferative activity, apoptosis, and expression of p53, bcl-2, and epidermal growth factor receptor (EGFR) because these two tumors show close similarity in some cytologic and architectural characteristics., Methods: Formalin fixed, paraffin embedded sections of 11 cases of BCAC and 9 cases of BCA, selected from the authors' files of 1851 primary tumors of the major salivary gland, were examined using immunostaining for Ki-67 (MIB-1), p53, bcl-2, and EGFR. In addition, apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling., Results: The incidence of BCAC was 0.6% among patients with major salivary gland tumors in the current series. Nine cases of BCAC arose in the parotid gland and two were of submandibular gland origin. Approximately 50% of the patients had recurrences, but no patient developed metastases or died of disease. Vascular involvement (75%), perineural invasion (36%), and necrosis (45%) were common features. Cell proliferative activity, including mitotic count, Ki-67 labeling index (LI), and apoptotic index were significantly higher in BCAC than BCA. More than four mitotic figures per ten high-power fields or a Ki-67 LI > 5% appeared to be limited to cases of BCAC. Considering those cases expressing p53 or EGFR in > 10% of tumor cells as positive, 6 of the 11 BCAC cases were positive for p53 and 3 were positive for EGFR. In contrast, all BCA cases were negative for p53 and EGFR. Although all cases of BCA were strongly positive for bcl-2 (> 50% of tumor cells), 3 of 11 cases of BCAC were completely negative., Conclusions: BCAC is a rare salivary gland tumor with a relatively high recurrence rate. Examination of cell proliferation, apoptosis, and expression of p53, bcl-2, and EGFR were found to be useful in distinguishing malignant basal cell tumors from their benign counterparts arising in the salivary gland.

Background: In organ transplantation, patients with peripheral blood mononuclear cells (PBMCs) that exhibit resistance to cyclosporine (INN, ciclosporin) or glucocorticoids in vitro are refractory to therapy based on these drugs in vivo. However, detection or distribution of the resistant patients with immunologic disorders remains to be documented., Methods: Drug sensitivity tests were performed with PBMCs from four subject groups: 69 healthy subjects, 100 patients with chronic renal failure, 38 patients with nephrosis, and 51 patients with psoriasis. The values for the concentration that produces 50% lymphocyte-mitosis inhibition (IC50) of the drugs on PBMC blastogenesis were estimated, and individual variations or group differences in the IC50 values were examined., Results: The median cyclosporine IC50 values of the four subject groups were similar, but large individual deviations in the IC50 values were observed. Individual differences in prednisolone IC50 values were spread from 1 to 3500 ng/ml. When compared with healthy subjects, a significantly large number of the patients with chronic renal failure group exhibited low responses to prednisolone (p < 0.04). In contrast, no significant difference in the methylprednisolone IC50 was observed among the groups. Normal upper thresholds for IC50 values of these drugs were estimated from the mean + 2 standard deviations (SD) of the IC50 values of healthy PBMCs, and the patients with IC50 values above these levels were considered to be resistant. The incidence of resistant patients with nephrosis or psoriasis was similar to that of healthy subjects; however, the incidence of cyclosporine- or prednisolone-resistant subjects with chronic renal failure was significantly higher (p < 0.04). Significant correlations between PBMC sensitivity to cyclosporine in vitro and clinical efficacy of the drug in vivo were observed in renal transplant recipients and in patients with psoriasis., Conclusions: A large subset of patients with chronic renal failure showed PBMC resistance to cyclosporine and prednisolone. Hyperresistant patients have a high risk of being refractory to immunosuppressive therapy with one of these drugs. Alternative treatment should be considered according to the individual drug-sensitivity data.

1. We have studied the difference in receptor binding activity between partial and full beta2-adrenoceptor agonists and the abilities of the agonists to interact with Ser204 and Ser207 in the fifth transmembrane region of the beta2-adrenoceptor, amino acid residues that are important for activation of the beta2-adrenoceptor. 2. In the binding study with [125I]-iodocyanopindolol, the Ki values of (+/-)-salbutamol, (+/-)-salmeterol, TA-2005 and (-)-isoprenaline for the beta2-adrenoceptor expressed in COS-7 cell membranes were 3340, 21.0, 12.0 and 904 nM, respectively. The beta1/beta2 selectivity of these agonists was in the order of (+/-)-salmeterol (332 fold) > TA-2005 (52.8) > (+/-)-salbutamol (6.8) > (-)-isoprenaline (1.1), and the beta3-/beta2-adrenoceptor selectivity of these agonists was in the order of TA-2005 (150 fold) > (+/-)-salmeterol (88.6) > (+/-)-salbutamol (10.4) > (-)-isoprenaline (3.2). 3. The maximal activation of adenylyl cyclase by stimulation of the beta1-, beta2- and beta3-adrenoceptors by TA-2005 was 32, 100 and 100% of that by (-)-isoprenaline, respectively, indicating that TA-2005 is a full agonist at the beta2- and beta3-adrenoceptors and a partial agonist at the beta1-adrenoceptor. (+/-)-Salbutamol and (+/-)-salmeterol were partial agonists at both beta1- (8% and 9% of (-)-isoprenaline) and beta2- (83% and 74% of (-)-isoprenaline) adrenoceptors. 4. The affinities of full agonists, TA-2005 and (-)-isoprenaline, were markedly decreased by substitution of Ala for Ser204 (S204A) of the beta2-adrenoceptor, whereas this substitution slightly reduced the affinities of partial agonists, (+/-)-salbutamol and (+/-)-salmeterol. Although the affinities of full agonists for the S207A-beta2-adrenoceptor were decreased, those of partial agonists for the S207A-beta2-adrenoceptor were essentially the same as for the wild type receptor. 5. The constitutively active mutant (L266S, L272A) of the beta2-adrenoceptor had an increased affinity for all four agonists. The affinities of full agonists were decreased by substitution of Ser204 of the constitutively active mutant, whereas the degree of decrease was smaller than that caused by the substitution of the wild type receptor. Although the affinities of (+/-)-salbutamol and (+/-)-salmeterol for the S207A-beta2-adrenoceptor were essentially the same as those for the wild type beta2-adrenoceptor, the affinities of (+/-)-salbutamol and (+/-)-salmeterol for the constitutively active beta2-adrenoceptor were decreased by substitution of Ser207. 6. These results suggest that Ser204 and Ser207 of the wild type and constitutively active beta2-adrenoceptors differentially interacted with beta2-selective agonists.

1. We tested the hypothesis that activation of large conductance calcium-activated potassium channels is involved in dilator responses of the basilar artery to acetylcholine in vivo. Using a cranial window in anaesthetized rats, we examined responses of the basilar artery to acetylcholine. 2. Topical application of acetylcholine (10(-6) and 10(-5) M) increased diameter of the basilar artery from 238 +/- 7 microns to 268 +/- 7 and 288 +/- 7 microns, respectively (P < 0.05 vs. baseline diameter). Iberiotoxin (10(-8) M), an inhibitor of large conductance calcium-activated potassium channels, did not affect baseline diameter of the basilar artery. In the presence of 10(-8) M iberiotoxin, 10(-6) and 10(-5) M acetylcholine increased diameter of the basilar artery from 239 +/- 7 microns to 246 +/- 7 and 261 +/- 7 microns, respectively. Thus, iberiotoxin attenuated acetylcholine-induced dilatation of the basilar artery (P < 0.05). 3. Sodium nitroprusside (10(-7) and 10(-6) M) increased diameter of the basilar artery from 242 +/- 9 microns to 310 +/- 12 and 374 +/- 13 microns, respectively (P < 0.05 vs. baseline diameter). In the presence of iberiotoxin (10(-8) M), sodium nitroprusside (10(-7) and 10(-6) M) increased diameter of the basilar artery from 243 +/- 6 microns to 259 +/- 9 and 311 +/- 12 microns, respectively. Thus, iberiotoxin attenuated dilator responses of the basilar artery to sodium nitroprusside (P < 0.05). 4. Iberiotoxin partly inhibited dilator responses of the basilar artery to forskolin, a direct activator of adenylate cyclase, but did not affect vasodilatation produced by levcromakalim, a potassium channel opener. 5. These results suggest that dilator responses of the basilar artery to acetylcholine and sodium nitroprusside are mediated, in part, by activation of large conductance calcium-activated potassium channels. Because both acetylcholine and sodium nitroprusside have been shown to activate guanylate cyclase via nitric oxide, activation of large conductance calcium-activated potassium channels may be one of the major mechanisms by which cyclic GMP causes dilatation of the basilar artery in vivo.

Background: Undifferentiated carcinoma with lymphoid stroma (UCLS) arising in the salivary gland is a rare tumor, except for its geographic concentration among Eskimo and southern Chinese populations. Five cases of salivary gland UCLS in Japanese patients are presented here and compared with benign lymphoepithelial lesions (BLELs) with regard to their association with Epstein-Barr virus (EBV), p53 expression, and cell proliferative activity., Methods: Formalin fixed, paraffin embedded sections of 5 cases of UCLS selected from 1676 patients with primary tumors of the major salivary gland and 7 cases of BLEL were examined using in situ hybridization for EBV-encoded RNAs (EBERs), along with immunostaining for p53 and Ki-67 (MIB-1)., Results: The incidence of UCLS was 0.3% among patients with major salivary gland tumors. EBER hybridization signals were strongly expressed in most of the neoplastic cells in all five cases of UCLS, whereas there was no signal in BLEL and surrounding nontumorous salivary gland tissues. All cases of UCLS showed strongly positive immunostaining for p53 in the tumor cell nuclei. By contrast, only one case of BLEL was positively stained for p53 in dysplastic epimyoepithelial cells, which showed focal immunostaining. Cell proliferative activity assessed using the Ki-67 labeling index was significantly higher in tumor cells of UCLS than in epimyoepithelial cells of BLEL., Conclusions: UCLS of the salivary gland is an extremely rare tumor in Japanese patients characterized by consistent association with EBV. Distinction between BLEL and UCLS is possible by examining for evidence of EBV infection, expression of p53, and cell proliferative activity.

1. The relative proportions of beta 1- and beta 2-adrenoceptors were determined by radioligand binding studies in three different rat myocardial preparations: membranes prepared from rat ventricle (ventricular membranes), membranes prepared from rat isolated ventricular myocytes (myocyte membranes), and myocytes isolated from rat ventricle (myocytes). 2. Competition experiments using CGP 20712A or ICI 118,551 with [125I]-iodocyanopindolol ([125I]-ICYP) revealed high- and low-affinity binding sites in ventricular membranes. The concentration at which each beta-antagonist occupied 100% of its high-affinity binding sites was 300 nM for CGP 20712A (beta 1-adrenoceptor) and 50 nM for ICI 118,551 (beta 2-adrenoceptor). 3. The density of high-affinity (beta 1-adrenoceptor) and low-affinity (beta 2-adrenoceptor) binding sites for CGP 20712A was measured by a saturation experiment using [125I]-ICYP in the presence and absence of 300 nM CGP 20712A. In ventricular membranes, the proportions of high-affinity and low-affinity binding sites for CGP 20712A were 73% and 27%, respectively, whereas in myocyte membranes, the corresponding figures were 90% and 10%, respectively. The density of low-affinity binding sites for CGP 20712A in ventricular membranes, defined as [125I]-ICYP-specific binding in the presence of 300 nM CGP 20712A, was decreased by addition of 50 nM ICI 118,551, whereas that in myocyte membranes was not affected. 4. In myocytes, specific binding of [125I]-ICYP and [3H]-CGP 12177 was not detected by saturation experiments performed in the presence of 300 nM CGP 20712A. 5 In myocytes, the activation of adenylate cyclase caused by beta2-adrenoceptors was not detected in the presence of 10 nM, 100 nM or 1000 nM CGP 20712A, which selectively antagonized beta1-adrenoceptors.Furthermore, the concentration-response curve for isoprenaline-stimulated cyclic AMP accumulation was not shifted by 10 nm or 100 nM ICI 118,551, which selectively antagonized beta2-adrenoceptors, but was shifted to the right by 1000 nM ICI 118,551.6 These results indicate that beta2-adrenoceptors are not present on rat ventricular myocytes and that beta2-adrenoceptor stimulation does not cause any detectable production of cyclic AMP. We conclude that only beta1-adrenoceptors exist on rat ventricular myocytes.

1. The effects of elevation of extracellular K+ concentration ([K+]o) on the negative inotropic potencies of three representative calcium antagonists, diltiazem, verapamil and nifedipine, were investigated in guinea-pig papillary muscle preparations. 2. The negative inotropic effect of diltiazem was potentiated 110 fold when [K+]o was raised from 2.7 mM to 12.7 mM. The effect of verapamil was also potentiated to a lesser extent, but that of nifedipine was not affected. 3. Resting membrane potentials in ventricular muscles were about -80 mV and -60 mV in 2.7 mM K+ and 12.7 mM K+, respectively. 4. To clarify the mechanism responsible for the differential potentiation of the negative inotropic effects, the blocking actions of the three calcium antagonists on the L-type Ca2+ channel current (ICa(L)) were compared at the holding potentials of -80 mV and -60 mV by the whole-cell patch-clamp technique. 5. The use-dependent blocking effect of diltiazem on ICa(L) was enhanced markedly by the change in the holding potential from -80 mV to -60 mV. The effect of verapamil was also enhanced to a lesser extent but that of nifedipine was not affected in this range of depolarization. 6. The differential effects of the [K+]o elevation on the negative inotropic potencies of the three calcium antagonists are explained by the differences in voltage-dependency of their use-dependent blocking effects on ICa(L). 7. The properties of diltiazem and verapamil observed in this study may contribute to their protective effects on the ischaemic myocardium, without affecting the normal myocardium.

(-)-Isoproterenol (ISO), used as bronchodilator, and (-)-(R)-1-(3,4-dihydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino]ethanol (T-0509), a new cardiotonic agent, were determined in rat plasma (only 25 microL) using a fully automated catecholamine (CA) analyser that includes in-line pre-extraction of CAs, coupled with an HPLC-ethylenediamine condensation reaction and a peroxyoxalate chemiluminescence detection system (HPLC-ED-PO-CL). The chromatographic conditions were: precolumn, SERUMOUT-CEX; buffer for delivering CAs in the precolumn, 10 mM potassium phosphate buffer (pH 7.5)/ethanol (92 + 8, v/v) (1 mL/min); precolumn clean-up solution, 4% phosphoric acid/acetonitrile (50 + 50, v/v) (1 mL/min); analytical column, TSKgel ODS-80Ts, 250 x 4.6 mm i.d.; eluent, 75 mM potassium acetate buffer (pH 3.2)/50 mM potassium phosphate buffer (pH 3.2)/acetonitrile (83.6 + 4.4 + 12, v/v/v for ISO and 76 + 4 + 20 v/v/v for T-0509) (1 mL/min); fluorogenic reagent solution, 105 mM ED and 175 mM imidazole in acetonitrile/ethanol (90 + 10, v/v) (0.25 mL/min); chemiluminogenic reaction solution, 0.25 mM TDPO, 150 mM hydrogen peroxide and 110 mM TFA in dioxane/ethyl acetate (50 + 50, v/v) (1.4 mL/min). Colterol, (+/-)-1-(3,4-dihydroxyphenyl)-2-[(1,1-dimethyl)ethylamino]ethanol, (+/-)-N-t-butylnorepinephrine was used as an internal standard (IS) for ISO, and (+/-)-1-(3,4-dihydroxyphenyl)-2-[(3,4,5- trimethoxyphenethyl)amino]ethanol, (+/-)-(3,4,5-trimethoxyphenethyl- aminomethyl)-3,4-dihydroxybenzylalcohol (T-1583) for T-0509. The detection limits for ISO and T-0509 were 1.3 and 0.9 fmol on injection, respectively.

A calcium antagonist, diltiazem, was infused continuously into Sprague-Dawley rats through the left femoral vein at four different flow rates. The mean arterial blood pressure and concentrations of plasma norepinephrine (NE) were measured in each single rat (n = 5) and the correlations between them were studied. Blood (150 microL) was collected 13 times during the infusion. Plasma NE was determined by HPLC-ethylenediamine condensation reaction-peroxyoxalate chemiluminescence detection system (HPLC-ED-PO-CL). In four cases from 5 rats, the blood pressure reduction caused by diltiazem was inversely correlated to logarithm of plasma NE concentration. The relation was expressed as Y = -alogX+m. The coefficients of correlation were -0.9506, -0.9293, -0.9341 and -0.8675, respectively. The correlation for the last rat was worse (r = -0.0799). The good correlation would imply that the sympathetic nervous system released NE to maintain blood pressure up to the normal level, responding to the sympathetic nervous system released NE to maintain blood pressure up to the normal level, responding to the blood pressure reduction caused by diltiazem. The present experiment proved the feasibility of the determination method of NE utilizing HPLC-ED-PO-CL detection in applying to the individual rats.

1. Previous studies, demonstrated that endothelium-dependent relaxations which are resistant to nitro-L-arginine (an inhibitor of nitric oxide synthase) are accompanied by membrane hyperpolarization in the porcine coronary artery. The present experiments were designed to characterize further this type of endothelium-dependent relaxation in response to bradykinin by measuring isometric force in isolated rings of that artery. The experiments were performed in the presence of indomethacin to rule out vasoactive prostanoids. 2. Bradykinin induced comparable endothelium-dependent relaxations of proximal and distal rings of porcine coronary arteries contracted with prostaglandin F2 alpha in the presence of nitro-L-arginine. 3. Bradykinin and SIN 1 (a donor of nitric oxide) reduced contractions induced by prostaglandin F2 alpha in an additive fashion in the presence of nitro-L-arginine. 4. Bradykinin (in the presence of nitro-L-arginine) relaxed the tissues contracted with tetraethylammonium, prostaglandin F2 alpha, phorbol 12, 13-diacetate or endothelin, with similar pD2 values. 5. The time course of the relaxations induced by bradykinin (in the presence of nitro-L-arginine) and UK14304 (an alpha 2-adrenoceptor agonist, in the absence of the inhibitor of nitric oxide synthase) were comparable. 6. These results suggest that, in the porcine coronary artery, nitro-L-arginine-resistant relaxations (a) are distributed similarly in the proximal and distal parts of the artery, (b) contribute to inhibition of vascular smooth muscle with nitric oxide in an additive fashion, (c) occur during contractions induced by various contractile agents and (d) do not precede those mediated by nitric oxide.

1. The effects of the calmodulin antagonists, calmidazolium and fendiline were investigated on endothelium-dependent hyperpolarization in the canine coronary artery. The membrane potential of vascular smooth muscle cells was measured with the microelectrode technique. 2. Smooth muscle cells of the canine coronary artery had a resting membrane potential of -50 mV. Bradykinin and the Ca(2+)-ionophore, A23187, induced concentration- and endothelium-dependent hyperpolarization. The hyperpolarization induced by a supramaximal concentration of bradykinin (10(-6) M) reached approximately 20 mV. 3. Calmidazolium (10(-5) M) and fendiline (10(-4) M) inhibited hyperpolarization induced by bradykinin and A23187. By contrast, calmidazolium did not affect the hyperpolarization induced by lemakalim, an opener of ATP-sensitive K(+)-channels. 4. These observations suggest that calmodulin is involved in the generation of endothelium-dependent membrane hyperpolarization of vascular smooth muscle.

1. The role of calmodulin in endothelium-dependent relaxations in the canine coronary artery, was investigated by use of the inhibitor of calmodulin, calmidazolium. 2. The endothelium-dependent relaxations to adenosine diphosphate (ADP) and nebivolol, a beta-adrenoceptor antagonist, in control solution, and to bradykinin in high potassium solution (to inhibit endothelium-dependent hyperpolarization), were abolished by nitro-L-arginine (30 microM), an inhibitor of nitro oxide-synthase. Calmidazolium (10 microM) did not inhibit these relaxations. 3. Calmidazolium did not affect the endothelium-independent relaxations to SIN-1, an exogenous donor of nitric oxide (NO). 4. The relaxations to bradykinin and to the calcium ionophore A23187 in control solution were inhibited to a small extent by calmidazolium (10 microM). 5. Bradykinin and A23187 induced relaxations in the presence of nitro-L-arginine (30 microM) that were abolished by calmidazolium (10 microM) but not affected by glibenclamide (10 microM), an inhibitor of ATP-sensitive K+ channels. 6. The endothelium-independent relaxations to lemakalim, an ATP-sensitive K+ channel opener, were not affected by calmidazolium (10 microM) but were inhibited by glibenclamide (10 microM). 7. These results suggest that calmidazolium does not inhibit the endothelium-dependent relaxations due to endothelium-derived NO in the canine coronary artery but inhibits either the production of endothelium-derived hyperpolarizing factor (EDHF) from endothelial cells or its effects on vascular smooth muscle cells. Furthermore these results suggest that EDHF contributes to endothelium-dependent relaxations in the canine coronary artery.

1. The pharmacological actions of T-0509, a 3-hydroxy derivative of denopamine, were studied in various guinea-pig tissues; these effects were compared with those of isoprenaline, denopamine and xamoterol. 2. The intrinsic activities of the positive inotropic actions of T-0509, denopamine and xamoterol compared with isoprenaline (= 100%) in the papillary muscle were 99%, 83% and 28%, respectively, while their relative potencies (EC50 agonist EC50 isoprenaline) were 0.23, 33 and 1.4, respectively. The intrinsic activities of T-0509, denopamine and xamoterol as positive chronotropic agents in the right atria were 98%, 69% and 48%, respectively, and their equipotent concentrations (isoprenaline = 1) were 0.24, 50 and 4, respectively. 3. The positive chronotropic actions of T-0509 and denopamine were antagonized by bisoprolol (3 x 10(-8) M), but not by ICI 118,551 (3 x 10(-8) M). 4. The intrinsic activity of T-0509 in histamine-contracted tracheae was similar to that of isoprenaline, but its equipotent concentration was 38; the effects of both agents were antagonized by ICI 118,551 (3 x 10(-8) M), but not by bisoprolol (3 x 10(-8) M). Denopamine and xamoterol did not show any agonist activity on guinea-pig trachea. 5. Denopamine and xamoterol antagonized the positive chronotropic (pA2, denopamine: 6.98, xamoterol: 7.75) and tracheal relaxant (pA2, denopamine: 5.39, xamoterol: 6.25) effects of isoprenaline. 6. Isoprenaline, T-0509 and denopamine, but not xamoterol, contracted the guinea-pig aorta in a decreasing order in the presence of propranolol (10(-6) M).7. Based on the above studies, T-0509 appears to be a highly selective betaI-adrenoceptor agonist with full agonist properties, while denopamine and xamoterol appear to be selective, but partial betaI-adrenoceptor agonists.

A 26-year-old man with erythroleukemia was found to have circulating micromegakaryocytes . Megakaryocytic features were defined by morphologic and cytochemical studies using light and electron microscopy with platelet-megakaryocyte peroxidase staining. This appears to be the first reported instance of erythroleukemia with circulating micromegakaryocytes .

In circular smooth muscle cells of the guinea-pig stomach (antrum), diltiazem (10(-6)-10(-5)M) blocked the overshooting spike potential generated either spontaneously or by electrical stimulation in the presence of 2 mM tetraethylammonium chloride, but did not block the slow wave and the abortive spike potential. The membrane was depolarized by high concentrations of diltiazem (more than 3 X 10(-6)M), and this depolarization was associated with an increase in the membrane resistance. The interval between slow waves was shortened to about 0.90 times the control (14.7s) by 10(-5)M diltiazem. Transmural nerve stimulation evoked an inhibitory junction potential (i.j.p.) and enhanced the subsequently generated slow wave. Tetrodotoxin (3 X 10(-7)M) blocked both responses but atropine (10(-6)M) blocked only the latter. Diltiazem (more than 10(-6)M) increased the amplitude of the i.j.p. and depressed the enhancement of the slow wave produced by transmural nerve stimulation, presumably due to depolarization of the membrane. The latency for the i.j.p. remained the same in the presence of diltiazem (10(-5)M). It is concluded that in the guinea-pig stomach, diltiazem blocks Ca-influx during the generation of the overshooting spike potential, but not the Ca-influx related to generation of the abortive spike potential or the slow wave. The cholinergic excitatory and the non-adrenergic, non-cholinergic inhibitory transmission may not be much affected by diltiazem.