Masamichi Hirai

Susceptibility of Helicobacter pylori isolates to metronidazole, clarithromycin and amoxycillin in vitro and in clinical treatment in Japan

Primary and acquired resistance to antibiotics is an important factor in determining the reason for treatment failure in Helicobacter pylori infection. We examined the relationship between the susceptibility of H. pylori isolates and the efficacy of chemotherapy.

Isolation of cDNAs encoding a substrate for protein kinase C: Nucleotide sequence and chromosomal mapping of the gene for a human 80K protein

An acidic phosphoprotein of Mr 80,000, the 80K protein, is a substrate for protein kinase C in fibroblasts and epidermal carcinoma cells. We purified the 80K protein from human squamous carcinoma Ca9-22 cells and fractionated it into two distinct molecular species, designated the 80K-L and 80K-H proteins. The amino acid sequences of the NH2-terminal region and cyanogen bromide-cleaved fragments of the 80K-H protein were determined and a corresponding oligonucleotide sequence was synthesized. Using this as a probe, two cDNA clones, lambda 80H-1 and lambda 80H-2, were selected from a lambda gt10 cDNA library from human A431 cells. The nucleotide sequence has an open reading frame of 1581 nucleotides encoding a protein of 527 amino acids. The deduced amino acid sequence revealed an extremely Glu-rich region. RNA blot analysis with the lambda 80H-1 cDNA clone detected two polyadenylated transcripts of 2.3 and 3.5 kb in Ca9-22 cells. Spot blot hybridization using flow-sorted human chromosomes provided evidence that the gene (G19P1) encoding 80K-H protein maps to human chromosome 19.

A proton pump inhibitor, E3810, has antibacterial activity through binding to Helicobacter pylori

Helicobacter pylori infection is causally related to atrophic gastritis, and it may also be associated with peptic ulcer and gastric carcinoma. Eradication ofH. pylori is recommended in patients with such diseases, especially in those with peptic ulcer. A new potent proton pump inhibitor, E3810, had an antibacterial effect onH. pylori, as has been reported for omeprazole and lansoprazole, two other proton pump inhibitors. The minimum inhibitory concentration of E3810 was 1.57–3.13 μg/ml, lower than that of omeprazole or lansoprazole. To clarify the mechanism of the antibacterial effect of E3810, we analyzed the characteristics of E3810 binding toH. pylori. Scatchard plot analysis of this binding showed a curvilinear profile, indicating the presence of several molecules with different affinities to E3810 onH. pylori. The binding capacity of E3810 toH. pylori was calculated to be about 2×106 sites/cell. These results suggested that E3810 has an antibacterial effect againstH. pylori and that the effect may be mediated through direct binding toH. pylori.

Expression of cathepsin E in pancreas: A possible tumor marker for pancreas, a preliminary report

Ductal cancers of the pancreas frequently express markers of gastrointestinal epithelial cells. Cathepsin E (CTSE) is a non‐secretory, intracellular, but non‐lysosomal proteinase found in the highest concentration in the superficial epithelial cells of the stomach. The aims of our study were to examine the expression of CTSE in the pancreas, to establish an assay system of CTSE and to evaluate the diagnostic usefulness of CTSE in the pancreatic juice. Eleven patients with pancreatic ductal adenocarcinoma, 10 with mucin‐producing adenoma, 3 with intraductal papillary hyperplasia and 43 with chronic pancreatitis were examined. Surgically resected pancreatic tissues were subjected to immunohistochemistry for CTSE. Pancreatic juice was collected from the patients and subjected to sandwich ELISA and Western analysis for detecting CTSE. Positive staining for CTSE was observed in pancreatic ductal adenocarcinoma by immunohistochemistry. CTSE was also expressed in mucin‐producing adenoma, intraductal papillary hyperplasia and mucinous hyperplasia. CTSE in the pancreatic juice was present in 8 of 11 patients with pancreatic ductal adenocarcinoma, 5 of 10 patients with mucin‐producing tumor, 1 of 3 patients with intraductal papillary hyperplasia and 4 of 43 patients with chronic pancreatitis. The detection frequency of CTSE in the pancreatic juice was significantly higher in the patients with pancreatic ductal adenocarcinoma than in the patients with chronic pancreatitis. Our findings suggest that the expression of CTSE is associated with the pathogenesis of pancreatic ductal adenocarcinoma, that CTSE in the pancreatic juice seems to be a useful marker for a definitive diagnosis and that CTSE may be expressed at a relatively early stage of multistep carcinogenesis in pancreatic lesions.

Lung Cancer Cells Often Express High Levels of Protein Kinase C Activity

We analyzed protein kinase C (PKC) activity in twenty‐two tumor cell lines derived from lung, pancreas, stomach, tongue and vulva, and found that lung cancer cells often (9 out of 13) exhibit significantly higher PKC activity than other types of cancer cells. The PKC in these lung cancer cells was separated into one major and one minor peaks by a Mono Q column chromatography. The PKC in the major peak had an absolute requirement for Ca2±, phosphatidylserine and 12‐O‐tetradeca‐noylphorbol‐13‐acetate (TPA), as expected. However, the PKC in the minor peak did not require TPA for its activation. Hydroxyapatite column chromatography revealed that the PKC in the major peak is type III. These results indicate that in lung cancer cells type III PKC activity is often elevated compared to other types of cancer cells. The growth of many lung cancer cell lines was inhibited by TPA.

Cytotoxin and urease activities of Helicobacter pylori isolates from Japanese patients with atrophic gastritis or duodenal ulcer

The vacuolating cytotoxin and urease secreted by Helicobacter pylori are thought to be virulent factors. Because vacuolation is potentiated by the presence of ammonium ion, which is produced by urease in vitro, it is of interest to examine whether cytotoxin and urease work reciprocally in the development of atrophic gastritis or duodenal ulcer. In the present study, patients (all H. pyloripositive) were divided into four groups: mild atrophic gastritis (group 1; nine patients), severe atrophic gastritis (group 2; 36 patients), duodenal ulcer with mild atrophic gastritis (group 3; 19 patients) and duodenal ulcer with severe atrophic gastritis (group 4; 12 patients). Cytotoxin production and urease activity of H. pylori isolated from these patients were analysed. Cytotoxin production was observed in four of nine (44.4%), 28 of 36 (77.8%), 11 of 19 (57.9%) and eight of 12 (66.7%) isolates from groups 1, 2, 3 and 4, respectively. Cytotoxin‐producing H. pylori isolates were found significantly more in patients with severe atrophy than in patients with mild atrophy (P= 0.048). The mean of relative activity of cytotoxin in H. pylori isolate was 1. 6. ± 2. 3, 7. 9. ± 7. 4, 5. 8. ± 6. 0 and 9. 0 ± 9. 1 in groups 1, 2, 3 and 4, respectively. Helicobacter pylori isolates from severe atrophy or duodenal ulcer patients in groups 2 or 4 possessed significantly higher activity than those from non‐ulcer patients in group 1 (P= 0.017 and 0.030, respectively). The mean of urease activity was 8. 6 ± 4. 6, 10. 0 ± 5. 9, 10. 0 ± 8. 5 and 11. 2 ± 7. 7 IU/mg in groups 1, 2, 3 and 4, respectively. These differences indicated no statistical significance. In each H. pylori isolate, the production of cytotoxin and urease were independent, which indicated that there was no reciprocal effect between them in vivo. Thus, cytotoxin‐producing H. pylori isolates were more prevalent in patients with severe atrophic gastritis and the cytotoxin activities of H. pylori isolates from the patients with severe atrophic gastritis or duodenal ulcer were much higher than those from the patients with mild atrophic gastritis, which suggested that vacuolating cytotoxin may be a disease‐inducing factor.

Review: Diagnosis of Helicobacter pylori infection

A number of diagnostic tests have been developed for the detection of H. pylori. Diagnostic techniques can be divided into invasive and noninvasive methods. The invasive methods require upper gastrointestinal endoscopy and involve culture of gastric biopsy specimens, examination of stained biopsies and detection of urease activity in the biopsies themselves. In addition, we have developed endoscopic diagnosis of H. pylori infection in gastric mucosa using phenol red dye‐spraying. The noninvasive methods include urea breath test and serological techniques. Although there has been considerable improvement in the techniques, a combination of at least two different techniques should be used in order to optimize the diagnostic yield. We recommend the use of one rapid test in the combination. The rapid urease test, cytology and the phenol red dye‐spraying endoscopy give results available before the patient leaves the endoscopy suite.

Lansoprazole treatment of Helicobacter pylori-positive peptic ulcers

Forty-nine patients with Helicobacter pylori (Hp)-positive gastric ulcer (GU) and 39 patients with Hp-positive duodenal ulcer (DU) were studied. Before the trial and every 3 or 4 weeks, phenol red dye spraying endoscopy, the rapid urease test, biopsy specimen histology, and culture were performed to assess the ulcer stage and to detect Hp. Patients were divided into three groups: group I received lansoprazole 30 mg/d; Group II received dual therapy of lansoprazole 30 mg/day and amoxicillin (AMPC) 1 g/day or clarithromycin (CAM) 400 mg/day, and Group III received combination therapy of lansoprazole 30 mg/day, AMPC 1 g/day, or CAM 400 mg/day, and metronidazole 500 mg/day. Patients with GU received lansoprazole for 8 weeks and patients with DU received lansoprazole for 6 weeks. The other agents were administered for 2 weeks at the beginning of the trial. There were no differences in ulcer healing among the three treatment groups in patients with GU or DU, but there were significant differences in the eradication of Hp. No side effects were observed in any of the patients. We conclude that combination therapy is likely to be most effective and is harmless for Hp-persistent patients with peptic ulcer

Reduced tyrosine phosphorylation and non-responsiveness to EGF-mediated cytotoxicity in EGF receptor-hyperproducing UCVA-1 cells

UCVA-1 cells, derived from human pancreas adenocarcinoma, have a high number of epidermal growth factor (EGF) receptors (1.0 x 10(6) per cell) but their growth is not inhibited by EGF, unlike other EGF receptor-hyperproducing tumour cells. In UCVA-1 cells EGF activates neither the phosphatidylinositol turnover nor protein kinase C. EGF, however, enhances the phosphorylation of EGF receptors at specific tyrosine residues, indicating that the EGF receptor kinase is active and subject to autophosphorylation. Downmodulation of EGF receptors by 12-O-tetradecanoylphorbol 13-acetate (TPA) is also observed. Using an anti-phosphotyrosine antibody several phosphoproteins, including EGF receptors, were immunoprecipitated from UCVA-1 cell lysates, whereas more than 20 phosphoproteins were detected in other EGF receptor-hyperproducing tumour cells (NA), indicating that tyrosine-phosphorylation of endogenous substrates by EGF receptor kinase is significantly reduced in UCVA-1 cells. Thus, non-responsiveness of UCVA-1 cells to EGF is correlated with the reduced tyrosine phosphorylation.

Protein kinase C activity and protein phosphorylation in mouse eggs

The treatment of mouse eggs with phorbol esters and diacylglycerol inhibits sperm peretration and results in biochemical modification of the zona pellucida. In this report, we have demonstrated the presence of protein kinase C (PKC) activity in mouse eggs as determined by 12-O-tetradecanoyl phorbol-13-acetate (TPA) dependent in vivo and in vitro protein phosphorylation in mouse eggs. When mouse eggs were radiolabeled with [32P]phosphate and treated with TPA, two specific proteins, 70 and 20 kDa, were phosphorylated. The 70-kDa protein was also phosphorylated in vitro by endogenous PKC. In addition, we have shown that exogenous PKC induced the in vitro phosphorylation of 70-, 55-, and 20-kDa proteins in egg extract. The 70-kDa protein was also phosphorylated in vitro after treatment of the cytosol fraction of mouse eggs with TPA, suggesting that this protein might be a specific substrate for PKC and that it is located in the cytosol. These results demonstrate that mouse eggs contain PKC activity and suggest that PKC-catalyzed protein phosphorylation of specific proteins might be involved in the regulation of egg-induced modification of the zona pellucida.