N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM): an alternative label for fluorescence tracing.

N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM), a fluorescent thiol reagent, has a high molar extinction coefficient and a high quantum yield when it reacts with protein sulphhydryl bonds. DACM proved to be effective as a fluorescent probe when used for labelling rabbit immunoglobulin G anti-human gamma-chain (IgG). DACM-labelled rabbit immunoglobulin G anti-human gamma-chain conjugates (DACM-IgG) can be purified efficiently by Sephadex G-50 gel chromatography. The immunological properties of the IgG are not altered appreciably by conjugation. As a label DACM has advantages with regard to ease of preparation, specific immunofluorescence and strong photoresistance.

Fluorescent staining of microfilaments with heavy meromyosin labeled with N-(7-dimethylamino-4-methylcoumarinyl) maleimide.

For fluorescent staining of microfilaments in cells, heavy meromyosin (HMM) or subfragment-1 (S-1) was labeled with a novel thiol-directed fluorescent dye, N-(7-dimethylamino-4-methylcoumarinyl) maleimide (DACM), instead of the usual dyes, such as fluorescein-isothiocyanate (FITC). DACM-labeled HMM or S-1 gave characteristic fluorescence patterns to a variety of cell types similar to those reported with the use of FITC-labeled HMM or S-1 or with immunofluorescence techniques using anti-actin antibody. The fluorescence of DACM was fairly photoresistant as compared with FITC, so that HMM or S-1 required only 1 mol of the dye per myosin head. Consequently, F-actin need not be used to preserve the actin binding activity of the myosin fragments when labeling with the dye.

Multiplicity of mitochondrial inner membrane antigens from beef heart reacting with antimitochondrial antibodies in sera of patients with primary biliary cirrhosis.

Mitochondrial inner membrane proteins extracted from beef heart tissue were examined for reactivity to antimitochondrial antibody (AMA) present in sera of patients with primary biliary cirrhosis (PBC) by an immunoblotting technique. Four proteins, which reacted with AMA, had molecular weights of 70 kDa, 54 kDa, 51 kDa and 45 kDa, as defined by their RF in SDS-PAGE gel. There was no correlation between the number of specificities and the titers of AMA as determined by immunofluorescence analysis. The 70-kDa protein was dissociated into a 36-kDa protein by trypsin digestion which still reacted with AMA. The reactivity to AMA of the 54-, 51- and 45-kDa proteins was abolished by trypsin digestion.

Rabeprazole, amoxycillin and low- or high-dose clarithromycin for cure of Helicobacter pylori infection.

BACKGROUND: Rabeprazole sodium is a proton pump inhibitor. AIM: To evaluate the efficacy and safety of 1-week triple therapy with rabeprazole, amoxycillin and clarithromycin for the eradication of Helicobacter pylori. METHODS: A total of 100 subjects with H. pylori were randomly divided into two groups of 1-week triple therapy with rabeprazole 10 mg b.d., amoxycillin 750 mg b.d. and either clarithromycin 200 mg b.d. (RAC400, n=50) or clarithromycin 400 mg b. d. (RAC800, n=50). Endoscopic examination with four biopsies (two specimens from the antrum and two from the gastric body) was performed. The status of H. pylori infection was determined using culture and histology (Giemsa stain) of the biopsy specimens. Sensitivity to clarithromycin was determined using the E-test: MIC > 8 g/mL was considered to be resistant, whereas MIC < 2 g/mL was considered to be sensitive. Cure was defined as no evidence of H. pylori infection 1 month after completion of treatment. RESULTS: There were no significant differences in the clinical characteristics of the two groups. Eradication rates (intention-to-treat and per protocol, respectively) were: RAC400: 86% (95% CI: 76-95%) and 89% (95% CI: 80-97%); RAC800: 94% (95% CI: 87-100%) and 97% (95% CI: 94-100%). There was no significant difference between the eradication rates of either regimen. Three subjects with failed eradication in the RAC400 group were all infected with a clarithromycin-resistant strain before beginning the therapy. Haemorrhagic colitis was the only severe adverse event, which was observed in one patient in the RAC800 group. CONCLUSION: One-week triple therapy with rabeprazole, amoxycillin and low-dose clarithromycin is effective for the eradication of H. pylori infection.

Comparison of ranitidine and lansoprazole in short-term low-dose triple therapy for Helicobacter pylori infection.

AIM: To evaluate the efficacy and safety of two 1-week low-dose triple-therapy drug regimens involving antisecretory drugs for Helicobacter pylori infection. 99 patients with H. pylori infection were treated with either lansoprazole or ranitidine used together with clarithromycin and metronidazole. METHODS: The drug combination and administration periods in the proton pump inhibitor group were lansoprazole 30 mg o.m., clarithromycin 200 mg b.d. and metronidazole 250 mg b.d., all given for 7 days (LCM group). The ranitidine group received ranitidine 150 mg b.d., clarithromycin 200 mg b.d. and metronidazole 250 mg b.d. also for 7 days (RCM group). The presence or absence of H. pylori was determined from gastric biopsy specimens taken from both the antrum and the body, by smear, culture and tissue section (Giemsa stain). Cure was defined as failure to find evidence of H. pylori infection 4 weeks after antimicrobial therapy had ended. RESULTS: The cure of H. pylori infection was 88% in the LCM group (44 of 50; 95% confidence interval (CI) = 79-97%) and 92% in the RCM group (45 of 49; 95% CI = 84-99%). The incidence of adverse events was 16% and 18% for the two groups, respectively. CONCLUSIONS: No significant differences in cure rate and safety profiles were noted between the two regimens, suggesting that moderate acid inhibition using an H2-blocker is sufficient to achieve optimal H. pylori eradication.

The value of LYM-1 cells for examining vacuole formation and loss of cell viability induced by culture supernates of Helicobacter pylori.

Some strains of Helicobacter pylori are known to produce an extracellular cytotoxin that causes vacuolation in cultured mammalian cells. Screening for such strains makes use of HeLa cells which may not be sensitive enough to detect minimal changes. The aim of this study was to develop a more sensitive cell line. Vacuole formation was examined in HeLa cells, as well as four other cell lines established in this laboratory by ammonium chloride induction. Among five cell lines tested, LYM-1 cells were most sensitive for the detection of intracellular vacuolation with this agent. Loss of cell viability of LYM-1 and HeLa cells induced by H. pylori culture supernates was also examined: LYM-1 were more sensitive than HeLa cells. Cell death was not always accompanied by vacuole formation. This suggests that the mechanism whereby cell death occurs must be different from that for vacuole formation. LYM-1 cells may be useful when measuring vacuole formation and cell death of the cultured cells induced by culture supernates of clinical isolates of H. pylori.

Efficacy of clarithromycin in eradicating Helicobacter pylori.

We reviewed reports on the efficacy of clarithromycin in eradicating Helicobacter pylori. Reports on dual and 1-week triple therapy that included clarithromycin were analyzed to assess the eradication rates of H. pylori infection. Dual therapy with clarithromycin and a proton pump inhibitor achieved eradication rates of 38%-83%, and 1-week triple therapy with omeprazole, clarithromycin, and other antibiotics achieved eradication rates, of 80%-96%. The incidence of side effects with all regimens was low. Clarithromycin is useful for treatment of H. pylori infection. One-week triple therapy that includes clarithromycin and a proton pump inhibitor effectively eradicates H. pylori.

Endoscopic topical therapy for the treatment of Helicobacter pylori infection.

We modified a novel topical therapeutic method for the treatment of Helicobacter pylori infection to increase its effectiveness and tolerability. Sixty-six patients (with nonulcer dyspepsia, inactive ulcer, or active ulcer) were given lansoprazole (30 mg, h.s.) and pronase (18,000 tyrosine units, b.i.d.) orally for 2 days before the topical therapy. One hundred milliliters of 7% sodium bicarbonate solution containing bismuth subnitrate, amoxicillin, metronidazole (at two different regimens), and pronase was instilled into the stomach through an endoscope. A double-lumen tube with a balloon at the tip was inserted into the duodenum along with the endoscope. The balloon was inflated with 25 ml of air and was lodged postbulbarly. The solution was kept in the stomach for 2 h, and the patient's position was changed every 15 min from the sitting to the supine, prone, and right lateral position, each position being maintained twice, to expose the entire gastric mucosa. The solution was aspirated at the end of the procedure. H. pylori infection was cured in 16/22 (72.7%) of patients with nonulcer dyspepsia, in 21/26 (80.7%) of patients with inactive ulcer, and in 1/18 (5.6%) patients with active ulcer. H. pylori eradication was confirmed 4 weeks after the therapeutic procedure by smear, culture, and histology of antral and corpus biopsy specimens. Side effects (loose stools) were observed in two patients only, and one patient had loss of appetite. These effects were transient. This endoscopic topical therapy for H. pylori infection is a safe, effective, and well tolerated procedure. With further modifications of the drug regimens and the method itself, this procedure could be of interest as anti-H. pylori therapy.

A novel fluorometric ultramicro determination of serum leucine aminopeptidase using a coumarine derivative.

A simple and rapid rate assay of serum leucine aminopeptidase is described, using a novel fluorogenic substrate, 7-L-leucyl-4-methylcoumarinylamide. The reaction is initiated by adding 10 microliter of serum, and the fluorescence development for 1 min due to the 7-amino-4-methylcoumarin liberated at 37 degrees C is followed directly on a recorder. The proposed method is proved to be free from error due to the adsorption of the substrate dye to serum albumin and to be applicable to hyperbilirubinemic sera by simple correction. The values obtained by this method showed good correlation with those obtained by the conventional method of Goldbarg and Rutenberg (Goldbarg, J.A. and Rutenberg, A.M. (1958) Cancer 11, 283-291).

Determination of endotoxin using fluorescent probe.

Bacterial endotoxin induces gel-formation of amebocyte lysate. This gelation test, the limulus test, was first described by Levin and Bang [1]. The limulus test has been widely used for the detection of endotoxin, not only in fundamental bacteriological research but also in clinical diseases [2]. Although the technique of the limulus test is simple, the endotoxin level can not be determined quantitatively. We developed a method to determine the endotoxin in physiological saline solution and glucose solutions using a fluorescent probe, fluorescamine.

Dual therapy with lansoprazole and clarithromycin for eradication of Helicobacter pylori.

AIM: To evaluate the eradication of Helicobacter pylori by therapy with a combination of 60 mg lansoprazole and 800 mg clarithromycin. PATIENTS AND METHODS: In an open therapeutic trial, 30 H. pylori-positive patients with active ulcer disease took 30 mg lansoprazole twice a day and 400 mg clarithromycin twice a day for the first 2 weeks, followed by 30 mg lansoprazole once a day for 4-6 weeks. Endoscopy was performed both before and at the end of therapy, and 4 weeks after the end of the therapy. H. pylori was detected by using a combination of smear, culture and tissue sections. RESULTS: Complete pain relief occurred within 3 days in all patients and all ulcers were healed by the end of the therapy. The H. pylori clearance rate was 83.3% and the eradication rate was 73.3%. A minor side effect (metallic taste) was reported by only one patient (3.3%). CONCLUSIONS: Therapy with a combination of 60 mg lansoprazole and 800 mg clarithromycin is efficacious in the eradication of H. pylori and has the advantage of a low incidence of side effects and quick pain relief for patients with active ulcers.

[A case of hepatocellular carcinoma (HCC) with lung metastasis which responded to chemotherapy with a single use of 1-hexylcarbamoyl-5-fluorouracil (HCFU)].

To our knowledge, there has been only one report pertaining to the efficacy of HCFU for treatment of metastatic lung lesion of HCC, so we reported a case of HCC with lung metastasis which responded to chemotherapy with a single use of HCFU. A 64-year-old male was diagnosed as having HCC with lung metastasis by biochemical examination, abdominal CT, hepatic arteriogram and chest X-P. He had been treated previously with gamma-interferon and mitoxantrone, which were assessed as NC and PD, respectively. Two months after last chemotherapy, HCFU was administrated at a dose of 400 mg/body everyday for 8 months. After 4 weeks metastatic lung lesions showed remarkable regression (47% decrease) and disappeared completely 9 weeks later. The size of primary liver tumor gradually decreased during therapy and revealed marked improvement (85.7% decrease) after about 2 months of this therapy. During these periods serum levels of alpha-fetoprotein dropped from 430 ng/ml to less than 10 ng/ml. He is presently still alive and the duration of the PR attained to 35 weeks. As side effects, hypoproteinemia, anorexia and hot sensation were observed.

[A case of recurrent gastric cancer successfully treated with a combination of cisplatin and carmofur].

A 51-year-old man with recurrent gastric cancer was treated by combined administration of Cisplatin and Carmofur. The target sites were the abdominal lymph nodes and the area of invasion to the stomach. Cisplatin (50 mg/body/day) was given for 3 days, while Carmofur (400-800 mg/body/day) was administered daily in 1 course. After 1 course of administration, the target tumor was reduced in size and the therapy was continued. A complete response was confirmed by upper gastrointestinal roentgenography, endoscopy and echography after 8 courses of Cisplatin administration. The patient has survived for 2 years 6 months in a state of CR. This case suggests that a combination therapy of Cisplatin and Fluoropyrimidine derivatives might be effective for gastric cancer.

[A case report of esophageal intramural pseudodiverticulosis].

A 76 year old man with esophageal intramural pseudodiverticulosis occurring in reflux esophagitis is presented. This unusual condition characterized by multiple small outpouchings in the esophageal wall and associated with a high incidence of benign esophageal stricture. Our case demonstrated typical radiological feature by barium swallow.

Reactivities and clinical relevance of antimitochondrial antibodies to four mitochondrial inner membrane proteins in sera of patients with primary biliary cirrhosis.

Antimitochondrial antibodies are characteristically detected in sera of patients with primary biliary cirrhosis. The antigens to which the antimitochondrial antibodies in primary biliary cirrhosis sera react have been located in the mitochondrial inner membrane. We have reported on four mitochondrial inner membrane proteins, extracted from beef heart, which reacted with antimitochondrial antibodies of primary biliary cirrhosis. These four proteins had molecular weights of 70, 54, 51 and 45 kd. Forty-six of the 50 PBC sera tested were positive with rat kidney and stomach specimens by the indirect immunofluorescent method. All sera of 50 primary biliary cirrhosis patients were positive with at least one and as many as four of the mitochondrial proteins extracted from beef heart using immunoblotting. Forty-seven primary biliary cirrhosis sera (94%) had IgG antibodies (antimitochondrial antibodies) to the antigen proteins, 43 patients (86%) IgM antimitochondrial antibodies and 38 patients (76%) IgA antimitochondrial antibodies. Seventy per cent of patients had antimitochondrial antibodies in all three immunoglobulin classes. In a few primary biliary cirrhosis sera, antimitochondrial antibodies were represented by only one class of immunoglobulin. The major antigenic proteins of mitochondrial inner membrane to which the antimitochondrial antibodies react were the 70 and 51 kd proteins. All primary biliary cirrhosis sera containing antibodies to the 54 and/or 45 kd proteins reacted with 70 kd as well. Patients with an elevation of serum bilirubin over 2 mg per dl were often found to produce antimitochondrial antibodies reactive with the 54 kd in addition to the 70 kd protein. No significant association was found between histological classifications and reactive patterns of antimitochondrial antibodies.

Isolation of tryptic fragment of antigen from mitochondrial inner membrane proteins reacting with antimitochondrial antibody in sera of patients with primary biliary cirrhosis.

Most of the sera from patients with primary biliary cirrhosis contains antimitochondrial antibodies, which react with four proteins of the mitochondrial inner membrane. We reported in a previous paper that when beef heart mitochondrial inner membrane proteins were digested by trypsin, a new reactive 36 kDa fragment with antimitochondrial antibody was obtained. This 36 kDa fragment derives from original 70 kDa protein because the monoclonal antibody specific to 70 kDa protein reacts with the 36 kDa band equivalent to 70 kDa band. The 36 kDa fragment was purified using an affinity column conjugated with an immunoglobulin-rich fraction of primary biliary cirrhosis serum containing antimitochondrial antibody, preparative electrophoresis and high-performance liquid chromatography using a reverse phase column. The final preparation showed a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis. Its amino acid composition is in good agreement with that of the subunit binding domain of the pyruvate dehydrogenase complex E2 from bovine heart.

CagA and cytotoxicity of Helicobacter pylori are not markers of peptic ulcer in Japanese patients.

BACKGROUND: The infection with cagA-positive Helicobacter pylori strains is reported to be associated with peptic ulcer disease in developed countries, but it is controversial in Asia. To investigate the relationship between the virulence factors of H. pylori and peptic ulcer disease in Japan, we compared these between ulcer and nonulcer patients. MATERIALS AND METHODS: Seventy-four strains of clinically isolated H. pylori obtained from 22 gastric ulcer (GU), 23 duodenal ulcer (DU), and 29 chronic gastritis (CG) patients were studied. The presence of vacA and cagA gene was examined by polymerase chain reaction method using two different primer sets. We evaluated the proliferation-inhibiting and lethal cytotoxicity of culture supernatants using the alamarBlue assay. RESULTS: The vacA gene was identified in all strains by the original primers. S1 strains were found in 90.9% (20/22) from GU, 95.7% (22/23) from DU, and 96.6% (28/29) from CG patients. The prevalence of cagA gene determined by the first, and second primers was 90.9% (20/22), 90.9% (20/22) in strains from GU, 87.0% (20/23), 91.3% (21/23) from DU, and 86.2% (25/29), 89.7% (26/29) from CG patients, respectively. The supernatant showed cytolethal effect in 95.5% (21/22) of strains from GU, in 100% (23/23) from DU, and in 93.1% (27/29) from CG patients. There was no significant difference in the prevalence of the virulence factors between H. pylori strains isolated from patients with peptic ulcers and those with chronic gastritis. CONCLUSIONS: These results indicate that cagA gene status and the proliferation-inhibiting and lethal cytotoxicity of supernatant are not reliable markers of ulcerogenicity of H. pylori in Japanese patients.

The efficacy and safety of one-week triple therapy with lansoprazole, clarithromycin, and metronidazole for the treatment of Helicobacter pylori infection in Japanese patients.

BACKGROUND: The aim of this study was to evaluate the efficacy and tolerability of 1-week, low-dose triple therapy with lansoprazole, clarithromycin, and metronidazole (LCM) for the cure of H. pylori infection and to establish the adequate dosage of a new triple therapy for Japanese patients. MATERIALS AND METHODS: One hundred four H. pylori-positive Japanese patients were assigned alternatively to one of two groups: one to receive either 30 mg lansoprazole once in the morning, 200 mg clarithromycin twice daily, and 250 mg metronidazole twice daily for 1 week (LCM1; n = 52); the other to receive 30 mg lansoprazole once in the morning, 200 mg clarithromycin twice daily, and 500 mg metronidazole twice daily for 1 week (LCM2; n = 52). H. pylori infection was assessed by smear, culture, and histological assessment (Giemsa stain) performed before and 4 weeks after cessation of the therapy. RESULTS: The overall cure rates of H. pylori infection were 92.3% (48 of 52; 95% confidence interval (CI), 85% to 100%) in LCM1 and 92.3% (48 of 52; 95% CI, 85% to 100%) in LCM2. The cure rates in the patients without prior treatment were 95.7% (44 of 46; 95% CI, 89%-100%) in LCM1 and 95.7% (45 of 47; 95% CI, 89%-100%) in LCM2. Minor side effects were observed in 7.7% of LCM1 and 9.6% of LCM2, respectively. CONCLUSION: The LCM1 regimen consisting of 30 mg lansoprazole once daily, 200 mg clarithromycin twice daily, and 250 mg metronidazole twice daily (the regular doses in ordinary use in Japan) is a highly effective and safe regimen for Japanese patients. LCM1 as a new triple therapy is a promising regimen for the first-line treatment of H. pylori infection in Japanese patients.

[A superficial carcinoma arising from a solitary hepatic cyst].

Reported is a rare superficial papillary adenocarcinoma arising from a congenital cyst, along with a survey of pertinent cases in the world literature. A 43-year-old male with a huge hepatic cyst was admitted to hospital for further examination. Since the cystic drainage done for alcoholic fixation was seromucinous initially but later contaminated by bile, an operation was initiated for dome resection and the closure of the biliary communication. At the time of the deroofing, however, small granulomas were found. And those were confirmed as being malignant by frozen section. Thus, an extended right lobectomy was performed for therapy. From its history and nature, this was considered to be a cancer arising in a congenital hepatic cyst.

Efficacy of lansoprazole in eradication of Helicobacter pylori.

Fifty-eight Helicobacter pylori-positive ulcer patients received omeprazole 20 mg (n = 15), or lansoprazole 30 mg (n = 23), lansoprazole 60 mg (n = 13), or E3810 20 mg (n = 7) q.d. Another 63 H. pylori-positive ulcer patients received lansoprazole and clarithromycin for 2 weeks. Patients received lansoprazole 30 mg and clarithromycin 400 mg (group 1, n = 22), lansoprazole 30 mg and clarithromycin 800 mg (group 2, n = 12), or lansoprazole 60 mg and clarithromycin 800 mg (group 3, n = 29). Neither proton pump inhibitor (PPI) was capable of eradication by monotherapy, but the clearance rates in the lansoprazole group were 60.9 and 69.2%, which were higher than those for omeprazole (p < 0.05). In the dual therapy, eradication rates were 50, 50, and 72.4% in groups 1, 2, and 3, respectively. Minor side effects were observed in one case each in groups 1 and 3. Lansoprazole monotherapy proved more efficacious than omeprazole monotherapy, but it was unable to eradicate H. pylori. Dual therapy with lansoprazole 60 mg and clarithromycin 800 mg was an efficacious and safe regimen for H. pylori eradication in this study.