Masafumi Nakao

Helicobacter pylori infection induces gastric cancer in Mongolian gerbils

BACKGROUND & AIMS Although epidemiological studies have indicated that Helicobacter pylori infection plays a crucial role in gastric carcinogenesis in humans, there is no direct proof that H. pylori is actually associated with gastric carcinogenesis. The purpose of this study was to elucidate the relationship between H. pylori infection and gastric carcinogenesis using an animal model of long-term H. pylori infection. METHODS Mongolian gerbils were orally inoculated with H. pylori, and the sequential morphological changes in the stomach were examined for up to 62 weeks. RESULTS H. pylori was constantly detected in all infected animals throughout the study. At the 26th week, severe active chronic gastritis, ulcers, and intestinal metaplasia could be observed in infected animals. By the end of the study, adenocarcinoma had developed in the pyloric region of 37% of the infected animals. All tumors consisted of well-differentiated intestinal-type epithelium, and their development seemed to be closely related to intestinal metaplasia. CONCLUSIONS We have successfully demonstrated that long-term infection with H. pylori induces adenocarcinoma in Mongolian gerbils. The observations are thus highly suggestive of the involvement of H. pylori infection in gastric carcinogenesis in humans.

Lansoprazole, a novel benzimidazole proton pump inhibitor, and its related compounds have selective activity against Helicobacter pylori.

The activities of various types of antiulcer agents against Helicobacter pylori (formerly called Campylobacter pylori) strains were determined by an agar dilution method. Among the compounds tested, two benzimidazole proton pump inhibitors, lansoprazole (AG-1749) and omeprazole, were found to have significant activities against this organism. The activity of lansoprazole was comparable to that of bismuth citrate, with MICs ranging from 3.13 to 12.5 micrograms/ml, and fourfold more potent than that of omeprazole. A major metabolite and two acid-converted rearrangement products of lansoprazole also exhibited good activities comparable or superior to that of the parent compound. Exposure to lansoprazole of H. pylori growing in a liquid medium led to an extensive loss of viability without a reduction in culture turbidity and produced an aberrant bacterial morphology characterized by the irregular constriction of cells and the collapse of cell surface structures. The antibacterial activity of lansoprazole and its related compounds was selective against H. pylori; common aerobic and anaerobic bacteria and Campylobacter jejuni were not inhibited by 100 micrograms/ml. Images

Growth Inhibitory and Bactericidal Activities of Lansoprazole Compared with Those of Omeprazole and Pantoprazole against Helicobacter pylori

Helicobacter pylori plays a role in the pathogenesis of both duodenal and gastric ulcers. The aim of this study was to evaluate the effect of the proton pump inhibitor (PPI), lansoprazole, commonly used in eradication regimens, on growth, bactericidal activity and morphology of H. pylori in vitro in comparison with other PPIs.

In Vitro and In Vivo Antibacterial Activities of TAK-083, an Agent for Treatment of Helicobacter pylori Infection

ABSTRACT The antibacterial activity of TAK-083 was tested against 54 clinical isolates of Helicobacter pylori and was compared with those of amoxicillin, clarithromycin, and metronidazole. The growth-inhibitory activity of TAK-083 was more potent than that of amoxicillin, clarithromycin, or metronidazole (the MICs at which 90% of the strains are inhibited were 0.031, 0.125, 64, and 8 μg/ml, respectively). The antibacterial activity of TAK-083 was highly selective against H. pylori; there was a >30-fold difference between the concentration of TAK-083 required to inhibit the growth of H. pylori and that required to inhibit the growth of common aerobic and anaerobic bacteria. Exposure ofH. pylori strains to TAK-083 at the MIC or at a greater concentration resulted in an extensive loss of viability. When four H. pylori strains were successively subcultured in the medium containing subinhibitory concentrations of TAK-083, no significant change in the MICs of this compound was observed. TAK-083 strongly inhibited the formation of tryptophanyl-tRNA in H. pylori while exhibiting little effect on the same system in eukaryotes. TAK-083 was efficacious in the treatment of gastric infection caused by H. pylori in Mongolian gerbils. The results presented here indicate that TAK-083 is a promising candidate for the treatment of H. pylori infection.

Role of disulfide bonds in folding and secretion of human lysozyme in saccharomycescerevisiae

We examined folding and secretion of human lysozyme using four mutants each lacking two cysteines expressed in a yeast secretion system. Our results have revealed that the formation of the disulfide bond Cys6/Cys128 in human lysozyme is a prerequisite for correct folding in vivo in yeast. Substitution of Ala for Cys77 and Cys95 gave eight-fold greater secretion of a molecule with almost the same specific activity as that of the native enzyme. Substitutions of the other cysteines gave molecules that were secreted at a lower rate and had lower specific activities than the native enzyme. These are the first findings that the individual disulfide bonds of human lysozyme have different functions in folding and secretion in vivo.

Intracellular degradation of recombinant proteins in relation to their location in Escherichia coli cells

Abstract Recombinant interferon-αA (rIFN-αA) accumulated in Escherichia coli 294 was rapidly degraded in vivo when glucose or oxygen in the medium was limited, whereas recombinant interferon-γ (rIFN-γ) and recombinant interleukin-2 (rIL-2) were stable under the same conditions. The degradation in vitro of rIL-2 by E. coli proteases was as rapid as that of rIFN-αA. On the other hand, rIFN-γ maintained the same activity after proteolysis in vitro. Western blot analysis of the reaction mixture, however, showed that the rIFN-γ molecule was converted into a smaller protein of about 15 kDa by losing the COOH-terminal portion of the peptide. Electron microscopic observations showed that rIFN-γ and rIL-2 accumulated and formed inclusion bodies. No clear inclusion bodies were found in the cells accumulating rIFN-αA, which is rapidly degraded in vivo. These results indicate that intracellular location and protein structure are related to the stability in vivo of recombinant proteins. rIFN-αA was also degraded rapidly in a lon− mutant of the same organism, suggesting that protease La does not participate in this degradation process.

Helicobacter pylori induces proinflammatory cytokines and major histocompatibility complex class II antigen in mouse gastric epithelial cells

Although Helicobacter pylori has been reported to stimulate the release of various cytokines from gastric tissue, it remains unknown whether normal and nontumorous gastric epithelial cells produce these cytokines. Therefore, in this study, we used a normal mouse gastric surface mucous cell line (GSM06) to determine whether gastric epithelial cells produce proinflammatory cytokines in response to H. pylori. The expression of MHC class II antigen was also examined, to investigate whether gastric epithelial cells participate in the immune response to H. pylori. In the study, GSM06 cells were incubated with H. pylori or its lipopolysaccharide (LPS). Proinflammatory cytokines were detected by Northern and Western blot analysis. The expression of MHC class II antigen was examined by fluorescence activated cell sorter (FACS) analysis. Genetic expression of proinflammatory cytokines such as interleukin-1alpha, tumor necrosis factor-alpha, and cytokine-induced neutrophil chemoattractant-2beta was enhanced by both intact and sonicated H. pylori, but not by H. pylori LPS. The expression of MHC class II antigen was induced by H. pylori more strongly than by interferon-gamma. We conclude that H. pylori induces the expression of proinflammatory cytokines and MHC class II antigen in gastric epithelial cells. Gastric epithelial cells may act as antigen-presenting cells and participate in the immune response to H. pylori infection.

Establishment of a model of penicillin-resistant Streptococcus pneumoniae pneumonia in healthy CBA/J mice

Examination of strain differences in the susceptibility of mice to experimental respiratory tract infection with penicillin-resistant Streptococcus pneumoniae TUM19 revealed that a fatal infection model could be induced in immunocompetent CBA/J mice, but not in C3H/HeN, C57BL/6 or ICR mice. After intranasal instillation of c. 10(6) cfu of S. pneumoniae, the bacterial counts in the lungs of CBA/J mice increased from 10(5) to 10(7) cfu after 3-5 days, and gradually increased thereafter. The challenge organisms localised mainly in the lungs until 14 days after infection. Mice began to die c. 7 days after infection, and by 3 weeks most of the mice had died. Histopathologically, infiltration of neutrophils and lymphocytes around bronchi was observed from 1 day after infection, and fibrin deposition was seen in alveolar and bronchial spaces from 5 days. This model may be useful for investigating therapy of respiratory tract infection caused by penicillin-resistant S. pneumoniae because its pathological features resemble those observed in the human disease.

Cefmenoxime (SCE-1365), a novel broad-spectrum cephalosporin: in vitro and in vivo antibacterial activities.

The activity of cefmenoxime (SCE-1365), 7 beta-[2-(2-aminothiazol-4-yl)-(Z)-2-methoxyiminoacetamido]-3-[(1-methyl-1H-tetrazol-5-yl)thiomethyl]ceph-3-em-4-carboxylic acid, was compared with that of other cephalosporins. Cefmenoxime exhibited high activity against a wide variety of gram-positive and gram-negative bacteria. The in vitro activity of cefmenoxime against Streptococcus pyogenes, Haemophilus influenzae, and Enterobacteriaceae, including indole-positive Proteus, Serratia marcescens, Enterobacter cloacae, and Citrobacter freundii, was 10 to 1,000 times greater than that of several other cephalosporins. Against Pseudomonas aeruginosa, cefmenoxime showed activity two to four times that of sulbenicillin and carbenicillin but less than that of cefsulodin. Variation in pH, addition of horse serum, and type of growth medium had definite effects on the activity of cefmenoxime, and the inoculum size affected the activity against bacterial species. In Escherichia coli cefmenoxime showed marked affinity for penicillin-binding protein 3 (PBP-3), followed by PBP-1 (1A and 1B). This affinity profile was well correlated with its filamentous cell-forming activity under extremely low drug concentrations and with its bactericidal activity against microorganisms. The high in vitro activity of cefmenoxime was reflected in the degree of protection observed in mice infected intraperitoneally with a wide variety of gram-positive and gram-negative bacteria. Furthermore, cefmenoxime showed good therapeutic activity against infection models in mice such as respiratory tract infection caused by Klebsiella pneumoniae and urinary tract infection caused by Proteus mirabilis. Images

Cloning and characterization of two novel aldo‐keto reductases (AKR1C12 and AKR1C13) from mouse stomach

In contrast to hepatic hydrosteroid dehydrogenases (HSDs) of the aldo‐keto reductase family (AKR1C), little is known about a stomach one. From a mouse stomach cDNA library, we isolated two clones encoding proteins of 323 amino acid residues. They exhibited 93.2% amino acid sequence identity and 64–68% with any known HSDs. Recombinant proteins expressed in Escherichia coli reduced 9,10‐phenanthraquinone with NAD(P)H as cofactor. The mRNAs were exclusively expressed in stomach, liver and ileum. The present study demonstrates that these proteins are new members of the HSD subfamily and they are named AKR1C12 and AKR1C13. Immunohistochemical analysis suggests that they are involved in detoxification of xenobiotics in the stomach.