Changes in Seroprevalence of Helicobacter pylori Infection over 20 Years in Jinju, Korea, from Newborns to the Elderly.

BACKGROUND: The objective of this study was to examine changes in the prevalence of cytotoxic-associated gene A (CagA) positive Helicobacter pylori infection in Jinju, Korea, over the last 20 years. METHODS: Three cross-sectional analyses were conducted concurrently. A total of 1,305 serum samples were collected from 1994-1995, 2004-2005, and 2014-2015, respectively. The presence of immunoglobulin (Ig) G, IgA, and IgM antibodies against H. pylori CagA protein was examined by western blotting. RESULTS: Overall, seropositivity for anti-CagA IgG antibody was significantly decreased from 63.2% to 42.5% over the last 20 years (P < 0.001). Anti-CagA IgG seropositivities in children and young adults aged 10-29 years decreased from 1994 (60.0%-85.0%) to 2015 (12.5%-28.9%). The age when plateau of increasing IgG seropositivity was reached in each study period shifted from the 15-19 year-old group in 1994-1995 (85.0%) to the 40-49 year-old group in 2014-2015 (82.5%). Overall seropositive rates of anti-CagA IgA and IgM antibodies did not change significantly either over the last 20 years. CONCLUSION: H. pylori infection rate in children and young adults declined over 20 years in Jinju, probably due to improved sanitation, housing, or economy.

Helicobacter pylori Antigens Inducing Early Immune Response in Infants.

To identify the Helicobacter pylori antigens operating during early infection in sera from infected infants using proteomics and immunoblot analysis. Two-dimensional (2D) large and small gel electrophoresis was performed using H. pylori strain 51. We performed 2D immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) antibody immunoblotting using small gels on sera collected at the Gyeongsang National University Hospital from 4-11-month-old infants confirmed with H. pylori infection by pre-embedding immunoelectron microscopy. Immunoblot spots appearing to represent early infection markers in infant sera were compared to those of the large 2D gel for H. pylori strain 51. Corresponding spots were analyzed by matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS). The peptide fingerprints obtained were searched in the National Center for Biotechnology Information (NCBI) database. Eight infant patients were confirmed with H. pylori infection based on urease tests, histopathologic examinations, and pre-embedding immunoelectron microscopy. One infant showed a 2D IgM immunoblot pattern that seemed to represent early infection. Immunoblot spots were compared with those from whole-cell extracts of H. pylori strain 51 and 18 spots were excised, digested in gel, and analyzed by MALDI-TOF-MS. Of the 10 peptide fingerprints obtained, the H. pylori proteins flagellin A (FlaA), urease ß subunit (UreB), pyruvate ferredoxin oxidoreductase (POR), and translation elongation factor Ts (EF-Ts) were identified and appeared to be active during the early infection periods. These results might aid identification of serological markers for the serodiagnosis of early H. pylori infection in infants.

Dermabacter jinjuensis sp. nov., a novel species of the genus Dermabacter isolated from a clinical specimen.

A Gram-stain-positive, catalase-positive, facultatively anaerobic, non-motile, coryneform bacterium, designated strain 32T, was isolated from a closed pus sample from a patient having finger necrosis in Korea. Strain 32T was considered as representing a novel species according to its initial identification by matrix-assisted laser desorption/ionization-time-of-flight MS. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 32T belonged to the genus Dermabacter and was closely related to Dermabacter hominis DSM 7083T (=ATCC 49369T) (98.34 % similarity). Optimal growth was observed at 30-40 °C and pH 7. Growth occurred in the presence of 0-6 % (w/v) NaCl. Menaquinones MK-8, MK-7 and MK-9 were the major respiratory quinones. The major polar lipids were phosphatidylethanolamine, phosphatidylcholine, glycolipid and two unknown lipids. The major cellular fatty acids were anteiso-C17 : 0, anteiso-C15 : 0, iso-C16 : 0 and iso-C15 : 0. The DNA G+C content of strain 32T was 62.58 mol%, and the mean level of DNA-DNA relatedness between strain 32T and D. hominis ATCC 49369T was 49±1.6 %. Based on the phenotypic and genotypic characteristics, strain 32T is confirmed to represent a novel species of the genus Dermabacter, for which the name Dermabacter jinjuensis sp. nov. is proposed. The type strain is 32T (=NCCP 16133T=DSM 101003T).

Helicobacter pylori HP0425 Targets the Nucleus with DNase I-Like Activity.

BACKGROUND AND AIMS: Nuclear targeting of bacterial proteins has a significant impact on host cell pathology. Helicobacter pylori have many nuclear targeting proteins that translocate into the nucleus of host cells. H. pylori HP0425, annotated as hypothetical, has a nuclear localization signal (NLS) sequence, but its function has not been demonstrated. The aim of this experiment was to address the nuclear translocation of HP0425 and determine the effect of HP0425 pathology on host cells. MATERIALS AND METHODS: To investigate the nuclear localization of HP0425, it was expressed in AGS and MKN-1 cells as a GFP fusion protein (pEGFP-HP0425), and its localization was analyzed by confocal microscopy. Recombinant HP0425 (rHP0425) protein was overproduced as a GST fusion protein in Escherichia coli and purified by glutathione-affinity column chromatography. Purified rHP0425 was examined for cytotoxicity and DNase activity. RESULTS: The pEGFP-HP0425 fluorescence was expressed in the nucleus and cytosol fraction of cells, while it was localized in the cytoplasm in the negative control. This protein exhibited DNase activity under various conditions, with the highest DNase activity in the presence of manganese. In addition, the rHP0425 protein efficiently decreased cell viability in a concentration-dependent manner. CONCLUSIONS: These results suggest that HP0425 carrying a nuclear localization signal sequence translocates into the nucleus of host cells and degrades genomic DNA by DNase I-like enzymatic activity, which is a new pathogenic strategy of H. pylori in the host.

Correlations between the CagA Antigen and Serum Levels of Anti-Helicobacter pylori IgG and IgA in Children.

We tested correlations between anti-Helicobacter pylori IgG and IgA levels and the urease test, anti-CagA protein antibody, degree of gastritis, and age. In total, 509 children (0-15 years) were enrolled. Subjects were stratified as 0-4 years (n = 132), 5-9 years (n = 274), and 10-15 years (n = 103) and subjected to the urease test, histopathology, ELISA, and western blot using whole-cell lysates of H. pylori strain 51. The positivity rate in the urease test (P = 0.003), the degree of chronic gastritis (P = 0.021), and H. pylori infiltration (P < 0.001) increased with age. The median titer for anti-H. pylori IgG was 732.5 IU/mL at 0-4 years, 689.0 IU/mL at 5-9 years, and 966.0 IU/mL at 10-15 years (P < 0.001); the median titer for anti-H. pylori IgA was 61.0 IU/mL at 0-4 years, 63.5 IU/mL at 5-9 years, and 75.0 IU/mL at 10-15 years (P < 0.001). The CagA-positivity rate was 26.5% at 0-4 years, 36.5% at 5-9 years, and 46.6% at 10-15 years for IgG (P = 0.036), and 11.3% at 0-4 years, 18.6% at 5-9 years, and 23.3% at 10-15 years for IgA (P < 0.001). Anti-H. pylori IgG and IgA titers increased with the urease test grade, chronic gastritis degree, active gastritis, and H. pylori infiltration. Presence of CagA-positivity is well correlated with a high urease test grade and high anti-H. pylori IgG/IgA levels.

Immunohistochemical Expressions of MUC2, MUC5AC, and MUC6 in Normal, Helicobacter pylori Infected and Metaplastic Gastric Mucosa of Children and Adolescents.

OBJECTIVES: The aim of this study was to investigate expression of gastric mucins in children and adolescents and to assess their relations with age and Helicobacter pylori (H. pylori) infection. METHODS: Gastric biopsies were collected from 259 pediatric and adulthood patients with gastrointestinal symptoms among all of patients undergone gastroduodenoscopy from 1990 to 2004 at Gyeongsang National University hospital and assorted based on H. pylori infection, age, and intestinal metaplasia as follows; H. pylori infection before 5 years of age or not, H. pylori infection between 5 and 9 years of age or not, H. pylori infection between 10 and 14 years of age or not, H. pylori infection between 20 and 29 years of age or not and intestinal metaplasia between 21 and 35 years of age. Total 810 tissue slides from the subjects were examined regarding expressions of Mucin2 (MUC2), Mucin5AC (MUC5AC), and Mucin6 (MUC6) in nine groups using immunohistochemical stains. A semiquantitative approach was used to score the staining extent of tissue slide. RESULTS: Increased expressions of MUC2, MUC5AC, and MUC6 were noted in intestinal metaplasia compared with subjects infected with H. pylori between 20 and 29 years. Gastric expressions of MUC5AC were decreased in older than 5 years with H. pylori compared with in older than 5 years without H. pylori (p < .001). Expressions of MUC2 and MUC6 did not change significantly by H. pylori status. Some nuclear expressions of MUC2 and MUC6 were noted in children without intestinal metaplasia. CONCLUSIONS: MUC5AC might be affected by chronic H. pylori infection. In addition to biomarkers for intestinal metaplasia or prognostic factors for gastric cancer in adults, MUC2 and MUC6 in children might have an another role, based on ectopic gastric nuclear expressions of MUC2 and MUC6 in children without intestinal metaplasia.

Helicobacter pylori γ-glutamyl transpeptidase-induced Ca(2+) release via PLC-IP3 receptors in AGS cells.

In our previous study, γ-glutamyl transpeptidase (GGT) isolated from Helicobacter pylori induced apoptosis of AGS cells. Here, we investigate Ca(2+) effects on GGT-induced apoptosis. The GGT transiently and significantly increased intracellular Ca(2+) concentration ([Ca(2+)]i) in AGS cells in a dose-dependent manner (P < 0.05). The GGT-induced Ca(2+) increase resulted from Ca(2+) influx and release through the phospholipase C - inositol 1,4,5-trisphosphate (PLC-IP3) pathway. The GGT-induced apoptosis was significantly reduced by treatment with U73122 (a PLC inhibitor) and xestospongin (an IP3 receptor antagonist) (P < 0.05). These results indicate that GGT could induce apoptosis of AGS cells by high levels of [Ca(2+)]i.

Correlation between positive rate and number of biopsy samples on urease test in childhood Helicobacter pylori infection.

To identify the correlation between the number of gastric biopsy samples and the positive rate, we compared the results of urease test using one and three biopsy samples from each 255 children who underwent gastroduodenoscopy at Gyeongsang National University Hospital. The children were divided into three age groups: 0-4, 5-9, and 10-15 yr. The gastric endoscopic biopsies were subjected to the urease test. That is, one and three gastric antral biopsy samples were collected from the same child. The results of urease test were classified into three grades: Grade 0 (no change), 1 (6-24 hr), 2 (1-6 hr), and 3 (<1 hr). The positive rate of urease test was increased by the age with no respect to the number of gastric biopsy samples (one biopsy P = 0.001, three biopsy P < 0.001). The positive rate of the urease test was higher on three biopsy samples as compared with one biopsy sample (P < 0.001). The difference between one and three biopsy samples was higher in the children aged 0-9 yr. Our results indicate that the urease test might be a more accurate diagnostic modality when it is performed on three or more biopsy samples in children.

Relationship between headache and mucosal mast cells in pediatric Helicobacter pylori-negative functional dyspepsia.

BACKGROUND: Although many patients with functional dyspepsia experience headache concurrently with dyspeptic symptoms, studies suggesting mechanisms underlying this phenomenon are limited. Herein, we explore the relationship between gastrointestinal inflammatory cells and presence of headache associated with dyspeptic symptoms in children with HELICOBACTER PYLORI -negative functional dyspepsia. METHODS: Fifty-six patients with H. PYLORI -negative functional dyspepsia underwent upper endoscopy with biopsy to investigate recurrent epigastric pain or discomfort. Patients were divided into two groups according to self-reported presence of headache associated with dyspeptic symptoms. Inflammatory cells including mast cells, and enteroendocrine cells in the gastroduodenal mucosa were evaluated. Associations between headache presence and cellular changes in the gastroduodenal mucosa were examined. RESULTS: Headache was not associated with the grade of lymphocytes, neutrophil infiltration, or enteroendocrine cell density in the gastroduedenal mucosa. However, headache was significantly associated with high mast cell density in the body (27.81 ± 8.71 vs. 20.30 ± 8.16, P < 0.01) and duodenum (23.16 ± 10.40 vs. 14.84 ± 5.88, P < 0.01). CONCLUSIONS: Presence of headache associated with dyspeptic symptoms is strongly related to mucosal mast cell density in pediatric patients with H. PYLORI -negative functional dyspepsia. Thus, our results may help clinicians understand and treat headache during dyspeptic symptoms in such pediatric patients.

Anthocyanins from black soybean inhibit Helicobacter pylori-induced inflammation in human gastric epithelial AGS cells.

Infection with Helicobacter pylori leads to gastritis, peptic ulcers and gastric cancer. Moreover, when the gastric mucosa is exposed to H. pylori, gastric mucosal inflammatory cytokine interleukin-8 (Il-8) and reactive oxygen species increase. Anthocyanins have anti-oxidative, antibacterial and anti-inflammatory properties. However, the effect of anthocyanins in H. pylori-infected cells is not yet clear. In this study, therefore, the effect of anthocyanins on H. pylori-infected human gastric epithelial cells was examined. AGS cells were pretreated with anthocyanins for 24 hrs followed by H. pylori 26695 infection for up to 24 hrs. Cell viability and ROS production were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 2',7'-dichlorofluorescein diacetate assay, respectively. Western blot analyses and RT-PCR were performed to assess gene and protein expression, respectively. IL-8 secretion in AGS cells was measured by ELISA. It was found that anthocyanins decrease H. pylori-induced ROS enhancement. Anthocyanins also inhibited phosphorylation of mitogen-activated protein kinases, translocation of nuclear factor-kappa B and Iκßα degradation. Furthermore anthocyanins inhibited H. pylori-induced inducible nitric oxide synthases and cyclooxygenase-2 mRNA expression and inhibited IL-8 production by 45.8%. Based on the above findings, anthocyanins might have an anti-inflammatory effect in H. pylori-infected gastric epithelial cells.

Changing pattern of antibiotic resistance of Helicobacter pylori in children during 20 years in Jinju, South Korea.

BACKGROUND: The antimicrobial resistance capability of Helicobacter pylori is one of the critical factors in the failure to treat this pathogen. The purpose of this study was to investigate the changing pattern of primary antibiotic resistance rates in children in the southern central part of South Korea from 1990 to 2009. METHODS: H. pylori strains were isolated from children who had undergone upper endoscopy at Gyeongsang National University Hospital, including 58 children from 1990-1994 and 33 children from 2005-2009. The susceptibility of H. pylori strains to erythromycin, clarithromycin, azithromycin, amoxicillin, tetracycline, metronidazole, furazolidone, levofloxacin, ciprofloxacin, moxifloxacin, and rifabutin was tested using the serial twofold agar dilution method. RESULTS: The resistance rate to erythromycin increased significantly from 13.8% in 1990-1994 to 33.3% in 2005-2009 (P = 0.032). Clarithromycin resistance increased from 6.9% to 18.2%. Metronidazole resistance decreased from 32.8% to 27.3%. The minimum inhibitory concentration of azithromycin and erythromycin showed definite shifts to higher concentrations in 2005-2009 compared with the strains sampled in 1990-1994 (P = 0.021 and P = 0.025, respectively). The frequency of both macrolide- and metronidazole-resistant strains was 13.8% in 1990-1994 and 15.2% in 2005-2009. No associations were detected between multidrug-resistant strains and the two study periods. CONCLUSIONS: The antibiotic resistance rates of H. pylori in Jinju had a different pattern to other regions. The antibiotic resistance rates of H. pylori showed geographic variation, and local data should be provided as a guideline for treating H. pylori infection.

Changes in anti-group a rotavirus antibody seroprevalence and levels in the Western Gyeongnam province of Korea over 16 years.

To observe how anti-group A rotavirus antibody seropositivity rates and levels have changed in the western region of Gyeongnam Province, 2,030 serum samples collected at four collection periods (1989-1990, 1994-1995, 1999-2000, and 2004-2005) were tested by Enzyme-Linked Immunosorbent Assay for IgG, and IgA antibodies reacting to recombinant VP6 protein. The seroprevalences exhibit no regular patterns over a 16-yr period. For all four collection periods, the anti-rVP6 IgG levels rose steadily during the first 5 months of life, after which they remained high. However, the 2-9 yr and 10-39 yr groups had significantly higher IgG levels in 1999-2000 and 2004-2005, respectively, than in the other collection periods. The 1-5 mo, 40- ≥ 60 yr, and 4-29 yr groups had significantly higher IgA levels in 1989-1990, 1999-2000, and 2004-2005, respectively. The 4 yr (25.0%), 5-9 yr (18.8%), 10-14 yr (41.1%), 20-29 yr (35.0%), and 30-39 yr (20.0%) groups in 2004-2005 had significant higher IgA seropositivity rate compared to the other three collection periods. These observations suggest that in the western region of Gyeongnam Province since the late 1990s, rotavirus reinfection has occurred more frequently than previously, with all ages being at risk.

Effects of Helicobacter pylori γ-glutamyltranspeptidase on apoptosis and inflammation in human biliary cells.

BACKGROUND: Several studies have reported the presence of H. pylori in individuals with hepatobiliary diseases, but in vitro and in vivo studies are still needed. Here, we determined the effects of H. pylori γ-glutamyltranspeptidase (GGT) on the induction of apoptosis and IL-8 production in a human cholangiocarcinoma cell line (KKU-100 cells). METHODS: Cell viability and DNA synthesis were examined by MTT and BrdU assays, respectively. RT-PCR and western blot analysis were performed to assess gene and protein expression, respectively. IL-8 secretion in KKU-100 cells was measured by ELISA. RESULTS: Exposure to the H. pylori ggt (+) strain decreased KKU-100 cell survival and DNA synthesis when compared with cells exposed to the H. pylori ggt mutant strain. Treatment with recombinant H. pylori GGT (rHP-GGT) dramatically decreased cell survival and DNA synthesis, and stimulated apoptosis; these features corresponded to an increased level of iNOS gene expression in KKU-100 cells treated with rHP-GGT. RT-PCR and western blot analyses revealed that rHP-GGT treatment enhanced the expression of pro-apoptotic molecules (Bax, Caspase-9, and Caspase-3) and down-regulated the expression of anti-apoptotic molecules (Bcl-2 and Bcl-xL). The extrinsic-mediated apoptosis molecules, including Fas and activated Caspase-8, were not expressed after treatment with rHP-GGT. Furthermore, rHP-GGT significantly stimulated IL-8 secretion in KKU-100 cells. CONCLUSION: Our data indicate that H. pylori GGT might be involved in the development of cancer in hepatobiliary cells by altering cell kinetics and promoting inflammation.

Antigenic Determinant of Helicobacter pylori FlaA for Developing Serological Diagnostic Methods in Children.

The early diagnosis of Helicobacter pylori infection is important for gastric cancer prevention and treatment. Although endoscopic biopsy is widely used for H. pylori diagnosis, an accurate biopsy cannot be performed until a lesion becomes clear, especially in pediatric patients. Therefore, it is necessary to develop convenient and accurate methods for early diagnosis. FlaA, an essential factor for H. pylori survival, shows high antigenicity and can be used as a diagnostic marker. We attempted to identify effective antigens containing epitopes of high diagnostic value in FlaA. Full-sized FlaA was divided into several fragments and cloned, and its antigenicity was investigated using Western blotting. The FlaA fragment of 1345-1395 bp had strong immunogenicity. ELISA was performed with serum samples from children by using the 1345-1395 bp recombinant antigen fragment. IgG reactivity showed 90.0% sensitivity and 90.5% specificity, and IgM reactivity showed 100% sensitivity and specificity. The FlaA fragment of 1345-1395 bp discovered in the present study has antigenicity and is of high value as a candidate antigen for serological diagnosis. The FlaA 1345-1395 bp epitope can be used as a diagnostic marker for H. pylori infection, thereby controlling various gastric diseases such as gastric cancer and peptic ulcers caused by H. pylori.

Role of cagA-positive Helicobacter pylori on cell proliferation, apoptosis, and inflammation in biliary cells.

BACKGROUND AND AIMS: The pathogenesis of Helicobacter pylori in the human hepatobiliary system has not been clearly elucidated. We compared the effects of H. pylori cagA(+) and cagA(-) mutant strains on cell proliferation, apoptosis, and inflammation in a cholangiocarcinoma (CCA) cell line (KKU-100). METHODS: MTT and BrdU were used to determine cell viability and DNA synthesis, respectively. The results were further investigated by RT-PCR and Western-blot analysis. The production of interleukin-8 (IL-8) was measured by ELISA assay. RESULTS: At low H. pylori inocula (cell-bacteria ratio of 1:1), the H. pylori cagA(+) strain showed a significant stimulation in KKU-100 cell growth (109 ± 1.79%) and DNA synthesis (131 ± 3.39%) than did the H. pylori cagA(-) strain (95 ± 3.06% and 120 ± 2.32%, respectively), through activation of the anti-apoptotic bcl-2 gene, MAP kinase and NF- κB cascade. By contrast, at high H. pylori inocula (cell-bacteria ratio of 1:200), the H. pylori cagA(+) strain showed a significant reduction in KKU-100 cell survival (49 ± 2.47%) and DNA synthesis (49 ± 1.14%) than did the H. pylori cagA(-) strain (60 ± 1.30% and 75 ± 4.00%, respectively), by increased iNOS, p53 and bax, while decreased bcl-2. Additionally, caspase-8 and -3 protein were activated. The H. pylori cagA (+) strain had significantly stronger effect on IL-8 production than did the cagA(-) strain. CONCLUSIONS: These results suggest that the H. pylori cagA(+) strain may play an important role in the development of biliary cancer by disturbing cell proliferation, apoptosis, and promoting cell inflammation in the CCA cell line.

Development of a surface plasmon resonance-based immunosensor for the rapid detection of cardiac troponin I.

The concentration of cardiac troponin I (cTnI) in blood is an important marker for heart muscle cell damage. A surface plasmon resonance (SPR)-based immunosensor was devised for the rapid and specific detection of cTnI. It was constructed by crosslinking a monoclonal antibody P-II-13, which was generated against a loop region (aa 84-94) of cTnI protein as an epitope peptide, onto a chemically modified thin gold film. The performance of the sensor was examined with respect to the SPR signal intensity versus cTnI concentration. The signal intensity was directly correlated with the cTnI concentration in the range of 0-160 µg/l. The sensor signal was saturated when the concentration of cTnI approached 660 µg/l with the SPR intensity of 172 RU. The lower detection limit of the sensor was 68 ng/l cTnI, which was comparable to ELISA-based commercial cTnI detection systems.

Increased Risk of Severe Gastric Symptoms by Virulence Factors vacAs1c, alpA, babA2, and hopZ in Helicobacter pylori Infection.

Two virulence factors of Helicobacter pylori, cagA and vacA, have been known to play a role in the development of severe gastric symptoms. However, they are not always associated with peptic ulcer or gastric cancer. To predict the disease outcome more accurately, it is necessary to understand the risk of severe symptoms linked to other virulence factors. Several other virulence factors of H. pylori have also been reported to be associated with disease outcomes, although there are many controversial descriptions. H. pylori isolates from Koreans may be useful in evaluating the relevance of other virulence factors to clinical symptoms of gastric diseases because the majority of Koreans are infected by toxigenic strains of H. pylori bearing cagA and vacA. In this study, a total of 116 H. pylori strains from Korean patients with chronic gastritis, peptic ulcers, and gastric cancers were genotyped. The presence of virulence factors vacAs1c, alpA, babA2, hopZ, and the extremely strong vacuolating toxin was found to contribute significantly to the development of severe gastric symptoms. The genotype combination vacAs1c/alpA/babA2 was the most predictable determinant for the development of severe symptoms, and the presence of babA2 was found to be the most critical factor. This study provides important information on the virulence factors that contribute to the development of severe gastric symptoms and will assist in predicting clinical disease outcomes due to H. pylori infection.

A thin-layer liquid culture technique for the growth of Helicobacter pylori.

BACKGROUND AND AIMS: Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H. pylori in liquid media in order to get affluent growth and a simple approach for examining bacterial properties. We introduce here a thin-layer liquid culture technique for the growth of H. pylori. METHODS: A thin-layer liquid culture system was established by adding liquid media to a 90-mm diameter Petri dish. Optimal conditions for bacterial growth were investigated and then viability, growth curve, and released proteins were examined. RESULTS: Maximal growth of H. pylori was obtained by adding 3 mL of brucella broth supplemented with 10% horse to a Petri dish. H. pylori grew in both DMEM and RPMI-1640 supplemented with 10% fetal bovine serum and 0.5% yeast extract. Serum-free RPMI-1640 supported the growth of H. pylori when supplemented with dimethyl-beta-cyclodextrin (200 microg/mL) and 1% yeast extract. Under optimal growth, H. pylori grew exponentially for 28 hours, reaching a density of 3.4 OD(600) with a generation time of 3.3 hours. After 24 hours, cultures at a cell density of 1.0 OD(600) contained 1.3 +/- 0.1 x 10(9 )CFU/mL. gamma-Glutamyl transpeptidase, nuclease, superoxide dismutase, and urease were not detected in culture supernatants at 24 hours in thin-layer liquid culture, but were present at 48 hours, whereas alcohol dehydrogenase, alkylhydroperoxide reductase, catalase, and vacuolating cytotoxin were detected at 24 hours. CONCLUSIONS: Thin-layer liquid culture technique is feasible, and can serve as a versatile liquid culture technique for investigating bacterial properties of H. pylori.

Crystallization and preliminary crystallographic analysis of decameric and monomeric forms of C49S mutant thioredoxin-dependent AhpC from Helicobacter pylori.

Cys49Ser mutant Helicobacter pylori alkyl hydroperoxide reductase (C49S HpAhpC) was purified under reducing conditions in monomeric and decameric forms. The monomeric form was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.25 A resolution and belonged to space group C2, with unit-cell parameters a = 245.8, b = 140.7, c = 189.5 A, beta = 127 degrees , and contained 20 molecules in the asymmetric unit. A crystal of the decameric form was obtained by the microbatch crystallization method and diffracted to 2.8 A resolution. It belonged to space group C222, with unit-cell parameters a = 257.5, b = 417.5, c = 95.6 A. The structure of the monomeric form of C49S HpAhpC has been solved by the molecular-replacement method.