The transient receptor potential protein homologue TRP6 is the essential component of vascular alpha(1)-adrenoceptor-activated Ca(2+)-permeable cation channel.

The Drosophila transient receptor potential protein (TRP) and its mammalian homologues are thought to be Ca(2+)-permeable cation channels activated by G protein (G(q/11))-coupled receptors and are regarded as an interesting molecular model for the Ca(2+) entry mechanisms associated with stimulated phosphoinositide turnover and store depletion. However, there is little unequivocal evidence linking mammalian TRPs with particular native functions. In this study, we have found that heterologous expression of murine TRP6 in HEK293 cells reproduces almost exactly the essential biophysical and pharmacological properties of alpha(1)-adrenoceptor-activated nonselective cation channels (alpha(1)-AR-NSCC) previously identified in rabbit portal vein smooth muscle. Such properties include activation by diacylglycerol; S-shaped current-voltage relationship; high divalent cation permeability; unitary conductance of 25 to 30 pS and augmentation by flufenamate and Ca(2+); and blockade by Cd(2+), La(3+), Gd(3+), SK&F96365, and amiloride. Reverse transcriptase-polymerase chain reaction and confocal laser scanning microscopy using TRP6-specific primers and antisera revealed that the level of TRP6 mRNA expression was remarkably high in both murine and rabbit portal vein smooth muscles as compared with other TRP subtypes, and the immunoreactivity to TRP6 protein was localized near the sarcolemmal region of single rabbit portal vein myocytes. Furthermore, treatment of primary cultured portal vein myocytes with TRP6 antisense oligonucleotides resulted in marked inhibition of TRP6 protein immunoreactivity as well as selective suppression of alpha(1)-adrenoceptor-activated, store depletion-independent cation current and Ba(2+) influx. These results strongly indicate that TRP6 is the essential component of the alpha(1)-AR-NSCC, which may serve as a store depletion-independent Ca(2+) entry pathway during increased sympathetic activity.

Phorbol 12,13-diacetate-induced contraction of the canine basilar artery: role of protein kinase C.

The pharmacological and biochemical mechanisms of contractile responses to the protein kinase C (PKC) activator phorbol-12,13-diacetate (PDA) were investigated in canine basilar arteries. In the normal medium, PDA elicited a strong, dose-related, and slow-developing sustained contraction. Among the constrictors examined, including serotonin, prostaglandin F2 alpha, and endothelin, only PDA yielded contractions in a Ca2(+)-free medium. In both media, the PDA-induced contractions were virtually inhibited by either staurosporine, H-7, or quinacrine, while neither neurotransmitter blockades nor R24571 (calmidazolium) exerted significant effects. In addition, it was shown that 8-bromocyclic GMP, but not 8-bromocyclic AMP, markedly curtailed the PDA-induced contractions. Biochemical analysis, furthermore, showed that PDA induced increased phosphorylations of 27- and 96-kDa and proteins other than the myosin light chain (MLC) 20-kDa protein. Thus, the present results open up a novel mechanism of sustained cerebral artery contractions, where PKC activation rather than Ca2+/calmodulin/MLC system plays a key role that is regulated both by phospholipase A2 and by cyclic GMP.

Application of hepatitis B core particles produced by human primary hepatocellular carcinoma (PLC/342) propagated in nude mice to the determination of anti-HBc by passive hemagglutination.

Human primary hepatocellular carcinoma (PLC/342), carried by nude mice, produces hepatitis B core particles as well as hepatitis B surface antigen particles. Core particles purified form PLC/342 tumors displayed epitopes of hepatitis B core antigen (HBcAg) but not epitopes of hepatitis B e antigen (HBeAg) on their surface, unlike core particles prepared from Dane particles, derived from plasma of asymptomatic carriers, that expressed epitopes of both HBcAg and HBeAg. Core particles obtained from PLC/342 tumors were applied to the determination of antibody to HBcAg (anti-HBc) by passive hemagglutination. The assay detected anti-HBc not only in individuals with persistent infection with hepatitis B virus and in those who had recovered from transient infection, but also in patients with acute type B hepatitis, indicating that it can detect anti-HBc of either IgG or IgM class. A liberal availability of core particles from tumors carried by nude mice, taken together with an easy applicability of the method, would make the passive hemagglutination for anti-HBc a valuable tool in clinical and epidemiological studies, especially in places where sophisticated methods are not feasible.

[Intestinal motility after massive small bowel resection].

To evaluate intestinal motility after 80% massive distal small bowel resection (MSBR), we continuously monitored interdigestive and postprandial bowel motility using bipolar electrodes and/or contractile strain gage force transducers in conscious beagle dogs before, and at 0-4 weeks and 8-13 months after the surgery. Fasting duodenal migrating myoelectric (or motor) complexes (MMCs) occurred at longer intervals in the short-term after 80% MSBR than in controls, and were simulated in long-term that in controls. MMCs arising from the duodenum were often migrated to the proximal jejunum, the jejunum above the anastomosis, and to the terminal ileum beyond the anastomosis. The velocity of duodenal MMC propagation was slowed in every intestinal segment in the short-term, and had not recovered even long after the operation. The duration of the postprandial period without duodenal MMCs was prolonged significantly in the short-term, and still remained longer in the long-term than in controls. These findings suggest that changes in gut motility after MSBR tend to compensate for the shorter intestine and maintain small bowel absorption early postoperatively, and adaptations of motility would occur over the long-term to increased intestinal absorption.

Natural infection with attaching and effacing Escherichia coli (O 103:H-) in chicks.

The existence of natural infection with attaching and effacing Escherichia coli (AEEC) in chicks was reported. Numerous Gram-negative bacilli were attached to the enterocyte in association with lesions characterized on a wavy appearance of the mucosal surface in the intestines of six chicks. Immunohistochemically, these bacteria reacted positively with antiserum to Escherichia coli (O 103). By electron microscopy, numerous colibacilli were seen to be closely attached to the surface membranes of the enterocytes. In regions of bacterial attachment, almost all cell microvilli were effaced, and some of the remaining ones were elongated and/or disoriented. Part of the cell membrane formed a cup invagination and pedestal-like protrusion associated with the attached bacteria. A concentration of electron-dense material was seen beneath the adherent organisms. Bacteriologically, numerous E. coli (O 103:H-) were isolated from the jejunal contents of two chicks and the E. coli did not produce verotoxin or enterotoxin. The characteristic lesions could also be induced in the cecal mucosa of young chicks experimentally inoculated with the isolated E. coli. This is the first report of natural infection in chicks with AEEC.

[Evaluation of F1 + 2/TAT ratios in Japanese patients with antiphospholipid syndrome].

The clinical manifestations of the antiphospholipid syndrome(APS) include arterial and venous thrombosis and a fetal loss, but the pathogenic mechanisms remain unclear. To clarify the mechanism of thrombogenic state in APS, we investigated the markers for thrombosis including thrombin-antithrombin complex(TAT) in patients with antiphospholipid antibodies(aPL). Prothrombin fragment 1 + 2(F1 + 2) in patients with APS and in autoimmune disease patients with aPL increased significantly compared with those obtained in autoimmune disease patients without aPL or in control subjects. However, there was not a significant difference in the TAT level of each group, suggesting that the formation of TAT was impeded in APS. To investigate which aPL is responsible for the disturbance of the TAT formation, the ratio of F1 + 2/TAT was calculated. The ratio increased in patients with lupus anticoagulant, especially with prolonged kaolin clotting time, and furthermore the ratio strongly increased in patients with IgG type-anticardiolipin antibodies(aCL). Our results suggest that IgG-aCL is associated with thrombogenic state in APS because free thrombin is present in patients' blood by impeding the formation of TAT by mainly IgG-aCL.

[Clinical early phase II study of bicalutamide (Casodex) in patients with prostatic cancer].

To investigate the efficacy and safety of bicalutamide (Casodex) with its clinically recommended dose, the randomized early phase II study was performed in 124 patients with prostatic cancer (stage C, D). The patients were given 50, 80 or 100 mg of bicalutamide orally once a day in fixed doses for 12 weeks; 122 patients were eligible for evaluation. The overall response rate was 50.0% (20/40), 61.0% (25/41) and 53.7% (22/41) in the 50 mg, 80 mg and 100 mg groups, respectively. The response rate in prostate lesion, bone and lymph node metastases was slightly higher in the 80 mg group than in the 50 mg and 100 mg groups. The proportion of patients showing a response with regard to serum PSA (CR and PR) was 84.2, 92.7 and 97.6% in the 50, 80 and 100 mg groups, respectively. The incidence of adverse reactions was 65.0, 61.0 and 61.0% in the 50, 80 and 100 mg groups, respectively, and there was no significant difference in overall safety rating in the three groups. Frequent adverse reactions were gynecomastia and breast pain. Only one patient in the 80 mg group was withdrawn due to shortness of breath. Serum concentrations of LH, testosterone and estradiol increased significantly after treatment. Bicalutamide was concluded to be effective and well tolerated in patients with prostatic cancer, and its recommended dose was 80 mg once daily.

[Diagnosis of malignant thyroid tumor using high-frequency ultrasonography with piezoelectric transducer, soft X-rays and aspiration biopsy cytology].

Diagnosis of thyroid cancer has been based mainly on palpation and also soft X-rays because of its calcification. As the gland itself is very small and Aspiration Biopsy Cytology (ABC) has had a high level of diagnostic accuracy the clinical significance of ultrasonography did not become apparent for a long time. However, recently it has become possible to carry out detailed study and to detect suspect histological structures, following the remarkable progress which has been made in ultrasonography, using a high-frequency piezoelectric transducer. A single mechanical arch scanner, with a piezoelectric 7.5-MHz transducer (SAL-25A Toshiba Corp.) was employed for examination using the water bath method. The rates of correct diagnosis for each of the above methods among 847 nodular goiters including 184 malignant tumors which were microscopically examined were shown to be: ultrasonography 89.7%, soft X-rays 66.9% and ABC 86.4%. Ultrasonography was capable to revealing a few cases which could not be shown by ABC or soft X-rays. Using these three methods jointly, there was 1 false positive and no false negative cases. This study revealed that combined diagnosis with ultrasonography using a high-frequency piezoelectric transducer improved the accuracy of examination of malignant thyroid tumors.

[Establishment of permanent atrial fibrillation and time course of the amplitude of the f wave].

Atrial fibrillation is one of the arrhythmias that increase with increasing age. In this study we compared transition intervals from sinus rhythm to permanent atrial fibrillation and the time course of the f wave amplitude immediately after the transition between 32 younger (less than 65 years) and 44 elderly patients (greater than or equal to 65 years) in whom transition from sinus rhythm to permanent atrial fibrillation was confirmed on serial ECG recordings. Each group was classified into three categories according their underlying diseases: hypertensive heart disease, valvular disease, and lone atrial fibrillation. In patients with hypertensive heart disease or lone atrial fibrillation, there was no significant difference in the transition intervals between the younger and the elderly groups. In both groups the transition intervals were significantly (p less than 0.05) longer in patients with lone atrial fibrillation than those in patients with hypertensive heart disease (44.6 vs. 12.5 months in younger and 26.8 vs. 12.9 months in elderly). A significant positive correlation (r = 0.58, p less than 0.01) was observed between the final P wave and the initial f wave amplitude on establishment of permanent atrial fibrillation in all patients. In the younger group, the initial f wave amplitude of patients with valvular disease (0.27 +/- 0.04 mV, mean +/- SE) was significantly larger than those of patients with hypertensive heart disease (0.15 +/- 0.03 mV, p less than 0.05) and of patients with lone atrial fibrillation (0.16 +/- 0.01 mV, p less than 0.05). The f wave amplitude of valvular disease was significantly decreased after 1 year (0.22 +/- 0.03 mV, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

[A case of malignant mesothelioma of the pleura and the peritoneum detected by sudden onset of back pain and pleural effusion and ascites].

A case of malignant mesothelioma of the pleura and the peritoneum is reported. In April 1996, a 40-year-old men noticed sudden onset of back pain. Radiographic examinations and MRI revealed pleural effusions, ascites, ringed enhanced tumorous lesions in the right posterior diaphragm along the abdominal aorta, and marked thickening of the right diaphragm with moderate signal intensity. On thoracoscopic surgery, there were white small nodules on the intercostal parietal pleura. Tumor cells of a tubulopapillary pattern had large rounded nuclei and eosinophilic cytoplasms in a partially glandlike arrangement. Cytoplasms of tumor cells stained for alcian blue disappeared after hyaluronidase digestion. Immunohistochemical examinations showed positive staining for keratin but negative for CEA. Electron micrographs showed numerous long thin microvilli, desmosomes and intermediate tonofilaments. From these findings, malignant mesothelioma was diagnosed. The malignant mesothelioma cells of the pleura in this case were considered to disseminate the peritoneum directly through the diaphragm or its lymphatic canals. MRI and thoracoscopic surgery were useful for the demonstration of the pleural disseminations and abdominal invasions.

Correlation between the presence of a fifty-megadalton plasmid in Salmonella dublin and virulence for mice.

All of the Salmonella dublin organisms isolated from cattle in Japan (20 strains) and in Europe (2 strains) harbored one plasmid of approximately 50 megadaltons (Mdal). The biochemical reactions of a derivative strain (5230P-) cured of the 50-Mdal plasmid were those of its parental strain 5230P+. However, the strain 5230P- proved to be 100- to 1,000-fold less virulent for mice compared with that of the strain 5230P+, suggesting that the virulent phenotypes of S. dublin may be mediated by the 50-Mdal plasmid.

A comparison of three methods used for treatment of ranula.

PURPOSE: This report compares the treatment results for oral and plunging ranulas. PATIENTS AND METHODS: Twenty-seven patients were retrospectively examined concerning treatment methods and recurrence on the basis of their medical records. The surgical specimens were also examined microscopically. RESULTS: Four patients had excision of the ranula only, 22 had marsupialization, and nine had removal of the sublingual gland combined with the excision of the ranula. These treatments showed a 25.0%, 36.4% and 0% recurrence rate, respectively. Histological observation revealed no epithelial lining in any of the examined specimens. CONCLUSIONS: Removal of the sublingual gland combined with the ranula was the most reliable method among the above three used.

Prompt decrease of circulating hepatitis C virus in patients with chronic hepatitis C after treatment with interferon.

Patients with chronic hepatitis C were treated with interferon (IFN) and followed for hepatitis C virus (HCV) RNA and antibody to HCV (anti-HCV) in serum. The response was correlated with decrease in serum levels of HCV RNA, as well as HCV genotypes and liver histopathology. Response to IFN, estimated by clearance of HCV RNA and normalization of aminotransferase levels at 6 months after the withdrawal of IFN, was observed in 11 (31%) of 35 patients infected with HCV of genotype II/1b, 13 (72%) of 18 with genotype III/2a, and 2 (33%) of 6 with genotype IV/2b; a single patient with genotype I/1a responded while the one doubly infected with HCV of genotypes II/1b and IV/2b did not. Response was seen in 10 (71%) of 14 patients with chronic persistent hepatitis, 14 (39%) of 36 with chronic active hepatitis 2A, and 3 (27%) of 11 with 2B. Response was achieved less often in patients with high than low pretreatment levels of HCV RNA. HCV RNA dropped sharply on a day after the start of IFN, and continued to decrease during the 2 weeks, irrespective of the response to IFN or HCV genotypes. In contrast, anti-HCV decreased more gradually and only in responders to IFN. These results support the rapid development of an IFN-mediated antiviral effect on HCV, and support therapeutic effects of IFN dependent on histopathology of liver as well as HCV RNA titers and genotypes.

Functional roles of cyclic guanosine 3',5'-monophosphate analogue in cerebral vasodilation.

1. Vasodilating effects of cyclic nucleotides in cerebral vasculature were examined using membrane permeable cyclic nucleotide analogues, 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP). 2. In isolated canine basilar artery (CBA), 8-Br-cGMP but not 8-Br-cAMP, significantly inhibited Ca(2+)-induced and agonist [serotonin(5-HT), prostaglandin(PG)F2 alpha or endothelin]-induced contraction, in a concentration-dependent manner. 3. When Ca2+ was depleted from intracellular store sites by pretreatment with A23187, 8-Br-cGMP but not 8-Br-cAMP strongly attenuated contractions induced by Ca(2+)-influx. 4. Neither 8-Br-cGMP nor 8-Br-cAMP modified contraction induced by caffeine which elicits Ca2+ release from intracellular Ca2+ store. 5. 8-Br-cGMP lowered the high K(+)-induced sustained [Ca2+]i elevation. 6. These results suggest that, at least in CBA, cGMP exerts its inhibitory effect on the contraction induced by influx of Ca2+, by reducing the level of [Ca2+]i and reducing [Ca2+]i sensitivity of the contractile machinery.

The inhibition of protein C anticoagulant activity by anti-ß2-glycoprotein I (ß2GPI) antibodies isolated from patients with antiphospholipid syndrome by chromatography methods.

Abstract Antiphospholipid antibodies (aPL) are associated with an increased risk of thrombosis; however, the mechanism remains unknown. Recent studies have focused on the impediment of protein C anticoagulant activity by anti-ß2-glycoprotein I (ß2GPI) antibodies (aß2GPI Ab). We purified IgG fractions containing a high concentration of aß2GPI Ab from patients with antiphospholipid syndrome (APS) and then investigated the effect of purified aß2GPI Ab on the activity of activated protein C (APC). Using a three-step chromatography method (DEAE-sepharose column, phosphatidylserine polyacrylamide gel column dependent on the presence of ß2GPI, and protein G column chromatography), we successfully isolated anti-ß2GPI IgG from nine patients with APS. Seven of nine samples inhibited APC activity in a concentration-dependent manner only in the presence of ß2GPI, as observed by a chromogenic assay that was able to determine thrombin activity even in the presence of APC. The extent of APC inhibition by these fractions appeared to be related to aß2GPI Ab titers of the purified IgG. However, the inhibitory effect of IgG from patients was not detected in the absence of ß2GPI. IgG purified from three normal subjects did not affect APC activity. Herein, we show a useful method for the isolation of IgG containing a high concentration of aß2GPI Ab. Moreover, the present findings indicate that inhibition by aß2GPI Ab on APC anticoagulant activity could explain one of the mechanisms for the thrombotic state in APS.

[A study of drinking habits of rural middle and old aged residents in Ibaraki Prefecture--relationship between problem drinking, depression and personality (Part 1). On male subjects].

Although there has been considerable research conducted regarding the relationship between alcohol dependency and clinical depression, there have been few reports which have investigated the relationship between alcohol consumption and depression in the general populace of residents of rural communities in Japan. We investigated this correlation in a rural community located in Ibaraki Prefecture, the findings of which are described in this report. The subjects of this investigation were residents of the southern part of the town of K in Ibaraki Prefecture age 40 and over, all of whom had participated in a medical examination program. With respect to the men surveyed, the proportions of problem drinkers and severe problem drinkers were 10.4% and 25.7%, respectively. According to the results we obtained with respect to correlation with personality, problem drinkers were found most frequently in shut-in personality types (37.5%). In regard to the correlation with depression, 7% of the problem drinkers and severe problem drinkers exhibited an SDS score of 50 or higher. In addition, the non-drinking group demonstrated a significantly higher degree of depression in comparison to the normal drinker and severe problem drinker groups.

[A study of drinking habits of rural middle and old aged residents in Ibaraki Prefecture--relationship between problem drinking, depression and personality (Part 2). On female subjects].

In looking at the findings of the women subjects of this investigation, alcohol consumption decreased rapidly in younger women with the borderline being roughly at the age of 50 years. This is consistent with general trends in Japan. In terms of the correlation with personality, the proportion of drinkers in those groups consisting of syntonic, self-revelation, nervous and persistent personality types was high at 34-37%. 3 out of the 7 women who were severe problem drinkers were of the persistent personality type. As such, it is believed that depression contributes to problem drinking in middle-aged and elderly women residing in rural communities. According to the results of this investigation focused on women residing in rural communities, 30% of the severe problem drinkers were also suffering from depression. As this figure closely resembles the proportion noted in clinical investigations, it was suggested that in the process of alcoholism becoming involved with health care institutions, there are other contributing factors besides clinical depression.

A huge immature cervical teratoma in a newborn: report of a case.

A neonate with a large cervical mass was transferred to our hospital at 4 days of age. A computed tomography scan showed a contrast-enhanced solid mass with multiple cystic elements and fine calcification. Ultrasonography also revealed a predominantly solid mass with calcification, containing multiple cysts. These studies suggested a teratoma, but could not rule out a hemangioma. The tumor was removed on the 12th day of life. A pathological study revealed an immature teratoma that demonstrated fetal type cartilage and an immature neural tube. The operative complete removal of a cervical teratoma in neonates is recommended as soon as possible. The management of a pediatric cervical teratoma should also be similar to that of a sacrococcygeal teratoma. The incidence of cervical teratoma in all pediatric teratomas ranges from 2.3%-9.3% in the West, and from 1.6%-8.3% in Japan.