J Med Microbiol 55 (2006), 1009-1015; DOI: 10.1099/jmm.0.46456-0
© 2006 Society for General Microbiology
ISSN 1473-5644
Gene-expression profiles in gastric epithelial cells stimulated with spiral and coccoid Helicobacter pylori
Zhi-Fang Liu1,2,3,
,
Chun-Yan Chen4,,
Wei Tang2,
Jian-Ye Zhang3,
Yao-Qin Gong1 and
Ji-Hui Jia1,2
1 The Key Laboratory of Experimental Teratology, Ministry of Education, Shandong University, 44 WenhuaXi Road, Jinan, China
2 ,3 Department of Microbiology2 , and Department of Biochemistry3 , School of Medicine, Shandong University, 44 WenhuaXi Road, Jinan, China
4 Department of Oncology, the Second Hospital of Shandong University, 247 Beiyuan Road, Jinan, China
Correspondence
Ji-Hui Jia
jiajihui{at}sdu.edu.cn
Received 7 December 2005
Accepted 3 April 2006
Human gastric epithelial immortalized GES-1 cells were infected with spiral and coccoid Helicobacter pylori. Scanning electron microscopy was used to determine the ability of the two forms of H. pylori to adhere to GES-1 cells. GES-1 cell apoptosis induced by coccoid and spiral H. pylori was analysed using flow cytometry. A cDNA microarray for 22 000 human genes was used to identify the gene-expression differences in GES-1 cells infected with the two forms of H. pylori, and the gene expression identified by the cDNA microarray was confirmed by RT-PCR. Scanning electron microscope observation showed that both coccoid and spiral bacteria can adhere to GES-1 cells. After 4 h infection, apoptosis induction was 27.4 % for spiral-form infection and 10.2 % for coccoid-form infection. Of 268 differentially expressed genes identified by cDNA microarray, 166 showed higher expression with the spiral H. pylori infection than with the coccoid H. pylori infection. To the best of the authors' knowledge, this is the first report that GES-1 cells infected with spiral H. pylori have higher expression of cxcl10, ccl11, ccl5, gro
, TLR5, ATF3, fos, fosl2, gadd45a and myc. The cells infected with coccoid H. pylori had higher expression of survivin. The global profile of gene expression in GES-1 cells infected with coccoid and spiral H. pylori is described for the first time.
Abbreviations: IL, interleukin; RGS, regulator of G protein signalling.
These authors contributed equally to this paper. 
 |
INTRODUCTION
|
|---|
Helicobacter pylori is a prevalent Gram-negative microaerophilic bacterium that infects human gastric mucosa and causes many digestive diseases, such as chronic gastritis, peptic ulcers and gastric cancer (Nomura et al., 1991; Parsonnet et al., 1991; Graham et al., 1992). H. pylori exists in two morphological forms: an actively dividing spiral form and a non-culturable but viable coccoid form (Bode et al., 1993). Spiral H. pylori converts to the coccoid form under various unfavourable conditions, such as extended incubation (Catrenich & Maki, 1991; Reynolds & Penn, 1994), antibiotic treatment (Bode et al., 1993), nutrient deprivation (Mizoguchi et al., 1999), increased oxygen tension (Catrenich & Maki, 1991; Cellini et al., 1994), increased temperature (Shahamat et al., 1993), aerobic culture and alkaline pH (Cellini et al., 1994). Many researchers think that this morphological change may be a transitory adaptation to a particular environment as a means of species preservation, and that it may play an important role in antibiotic resistance and make the bacterium more difficult to eradicate. Also, the coccoid form might lead to difficult recovery, easy relapse and epidemic transmission (Mizoguchi et al., 1998). The spiral bacteria are the most common form found in the human stomach, but the coccoid bacteria are observed in more severely damaged regions of the gastric mucosa (Saito et al., 2003; Chan et al., 1994; Janas et al., 1995). Several reports have shown that although the coccoid form is non-culturable, it may be viable, as transcription and translation may actively take place in coccoid cells (Sisto et al., 2000; Monstein & Jonasson, 2001; Ng et al., 2003). Vijayakumari et al. (1995) have shown that the adhesion of the coccoid form to Kato III cells in vitro is similar to the interaction detected in the gastric epithelium infected with the spiral form in vivo (Hessey et al., 1990). Wang et al. (1997) have shown that both coccoid and spiral H. pylori can infect BALB/c A mice, and both forms can cause acute gastritis in BALB/c A mice. Furthermore, Nilsson et al. (2002) have reported that ATP is detectable for at least 25 days after the morphological change from the spiral form to the coccoid form. It is therefore probable that the coccoid form, like the spiral form, is viable and infective, even though it is non-culturable in vitro.
In man, the gene-expression changes in gastric epithelial cells induced by the spiral form of H. pylori infection have been well reported (Chiou et al., 2001; Cox et al., 2001; Susanne et al., 2002). However, a comparison of the differences in gene expression between gastric epithelial cells infected with coccoid and those infected with spiral H. pylori has not hitherto been reported. The aim of this study was to assess by cDNA microarray the differences in gene expression in the gastric epithelial immortalized cell line GES-1 infected with coccoid and spiral H. pylori.
|
METHODS
|
|---|
Cell culture.
The human gastric epithelial immortalized GES-1 cells were maintained in our laboratory. The cells were routinely cultured in RPMI 1640 medium supplemented with 10 % fetal calf serum, 100 U penicillin ml–1 and 100 U streptomycin ml–1 in air with 5 % CO2 at 37 °C.
Bacterial culture and induction of the coccoid form.
The wild-type H. pylori 26695 strain was kindly provided by Dr Zhang Jianzhong of the Chinese Disease Control and Prevention Center, Changping District, Beijing. The bacterium was cultured on Columbia agar with 5 % (v/v) sheep blood under microaerobic conditions (5 % O2, 10 % CO2, 85 % N2) at 37 °C. After the bacteria had been grown for 48 h, more than 90 % of the bacteria were in the spiral form. To obtain coccoid H. pylori, the bacteria were maintained for 10 days; after this period, more than 90 % of the bacteria were coccoid.
Co-cultivation of gastric epithelial cell line GES-1 with H. pylori.
GES-1 cells were prepared by seeding 2x106 cells on plates. After overnight incubation, the medium was replaced with fresh medium without antibiotics. The spiral and coccoid H. pylori were harvested from plates by suspension in 2 ml PBS and washing twice with PBS. After centrifugation at 4000 r.p.m. for 5 min, the bacteria were resuspended in 2 ml PBS and added immediately to cell culture plates. The ratio of the number of bacteria to the number of cells was 100 : 1. Gastric epithelial cells infected with spiral or coccoid H. pylori were cultured for 4 h in air with 5 % CO2 at 37 °C.
Scanning electron microscope observation.
GES-1 cells were grown to confluence on cover glasses in six-well plates, and spiral or coccoid bacteria were added to each cover glass at a ratio of about 100 : 1. The cultures were placed in air with 5 % CO2 at 37 °C for 4 h in a total volume of 2 ml. Then, the cover glasses were removed and washed twice with PBS. The cells were fixed first with 2.5 % glutaraldehyde for >2 h, and then with 2 % osmic acid. Adherence of coccoid and spiral H. pylori to GES-1 cells was observed with a Hitachi S2520 scanning electron microscope. The number of adherent bacteria on 25 GES-1 cells was counted. The t test was used for statistical analysis.
Apoptosis assay by flow cytometry.
About 1x106 cells infected with coccoid or spiral H. pylori were harvested by trypsin digestion and centrifugation (at 1000 r.p.m. for 5 min), then washed twice with PBS. Then the cells were fixed with 70 % ethanol. The apoptosis of GES-1 cells was determined by flow cytometry.
RNA extraction.
Total RNA was extracted from the cells with the Trizol reagent (Invitrogen), according to the manufacturer's instructions. The amount of RNA was measured by A260.
cDNA microarray.
The cDNA microarray consisted of 22 000 distinct cDNAs, which were purchased from Qiagen. The array also included cDNAs of housekeeping genes, such as ß-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), to serve as internal controls. Cy3-dCTP or Cy5-dCTP were incorporated when 50 µg total RNA was reverse-transcribed into cDNA using M-MLV reverse transcriptase and random hexamer primer (MBI). The cDNA probes from the cells co-cultured with spiral H. pylori were incorporated with Cy3, while the cDNA probes from the cells co-cultured with coccoid H. pylori were incorporated with Cy5. Different fluorescently labelled cDNA probes were mixed in 30 µl hybridization buffer (3x SSC, 0.2 % SDS, 5x Denhardt's solution and 25 % formamide) and applied to the microarray following incubation at 42 °C for 16 h. After hybridization, the slide was washed with 0.2 % SDS/2x SSC at 42 °C for 5 min, and then was washed with 0.2x SSC at room temperature for 5 min. The fluorescent images of the hybridized microarray were scanned with a ScanArray Express fluorescent laser confocal slide scanner (Packard Bioscience). Images and quantitative data of the gene-expression levels were analysed by GenePix Pro 4.0 image analysis software (Axon Instruments).
Semi-quantitative RT-PCR.
We used RT-PCR to verify the microarray data. The primers for RT-PCR are listed in Table 1
. The internal standard ß-actin was co-amplified with the specific genes. The total RNA was reverse-transcribed into cDNA using M-MLV reverse transcriptase and random hexamer primer (MBI). The cDNAs were amplified by 30 PCR cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, with a further period at 72 °C for 1 min. As a final step, extension was at 72 °C for 10 min. PCR products were separated on 1.5 % agarose gels containing 0.5 g ethidium bromide ml–1 and visualized by UV transillumination.
|
RESULTS AND DISCUSSION
|
|---|
Gastric adenocarcinoma cell lines, such as AGS, MKN28, Kato III and SGC7901, are often used to study the interaction between H. pylori and gastric mucosal epithelial cells. These cells may have mutated in the process of evolution, and as a consequence, their genetic backgrounds may display many differences from that of normal gastric mucosal epithelial cells. Thus, it is far from certain that the interactions between normal gastric mucosal epithelial cells and H. pylori are accurately reflected in these malignant cell lines. In this study, we used an immortalized fetal gastric mucosal cell line transformed with SV40. The cell line was established by Ke et al. (1994). It has a phenotype similar to that of normal stomach mucosal cells, and does not grow into tumour masses in nude mice. Therefore, it was hoped that our results could reflect more accurately the interaction of H. pylori with normal gastric mucosa. Asahi et al. (2000) and Odenbreit et al. (2001) have shown that CagA translocation can occur 30 min after infection, and that it is at a maximum in the time range of about 4–5 h (Susanne et al., 2002). Cole et al. (1997) have observed that the adhesion of coccoid and spiral H. pylori at a constant bacteria : cells ratio does not increase significantly after 2 h. Accordingly, the time point of 4 h chosen in this study seemed to be appropriate.
Adhesion of spiral and coccoid H. pylori to GES-1 cells
A scanning electron microscope was used to observe the adhesion of spiral and coccoid H. pylori to GES-1 cells. After GES-1 cells had been infected with spiral H. pylori for 4 h, we found that a number of H. pylori can adhere to the GES-1 cell surfaces (Fig. 1A). After coccoid H. pylori had infected GES-1 cells for 4 h, adhesion to GES-1 cell surface was observed with this form also (Fig. 1B). The mean adhesion index of spiral H. pylori was 28.08±9.82, while the mean adhesion index of coccoid H. pylori was 22.84±9.88. There were no significant differences in the adhesion index between the coccoid and spiral H. pylori (P>0.05).
View larger version (85K):
[in this window]
[in a new window]
|
Fig. 1. Adhesion of H. pylori to GES-1 cells observed by scanning electron microscopy. (A) Adhesion of spiral-form H. pylori to a GES-1 cell; (B) adhesion of coccoid-form H. pylori to a GES-1 cell. Bars, (A) 1.83 µm, (B) 2.22 µm.
|
|
cDNA microarray results
Fig. 2 shows a representative scatter plot for gene-expression levels, where cDNA probes, derived from the cells co-cultured with coccoid or spiral H. pylori, were hybridized and labelled with Cy5 and Cy3 fluorochromes, respectively. A Cy3 : Cy5 ratio between 2 and 0.5 indicated only small mRNA expression differences and was of no significance. A ratio of more than 2 or less than 0.5 indicated significant mRNA expression differences. Thus, the upper spots in Fig. 2 indicate that expression of the corresponding gene was greater in cells infected with coccoid H. pylori than in cells infected with spiral H. pylori, and the lower spots indicate the reverse. The intermediate spots indicate similar mRNA expression levels. From Fig. 2, most of the genes examined showed only small differences.
View larger version (44K):
[in this window]
[in a new window]
|
Fig. 2. Representative scatter plot of cDNA microarray analysis. The scatter plot indicates the relation between relative fluorointensities of Cy5-labelled cDNA (from coccoid-form-infected cells) and those of Cy3-labelled cDNA (from spiral-form-infected cells). For each gene, the RNA expression level in the spiral-infected cells is given on the x axis [log10(intensity of Cy3)] and the expression level of the same gene in the coccoid-infected cells is plotted on the y axis [log10(intensity ofCy5)].
|
|
Two hundred and sixty-eight genes showed more than a twofold difference in expression. One hundred and sixty-six genes in a co-culture with spiral H. pylori had a more than twofold increase in expression compared with a co-culture of coccoid H. pylori. One hundred and two genes in a co-culture with coccoid H. pylori had a more than twofold increase in expression compared with a co-culture with spiral H. pylori. Some of the differences in gene expression are listed in Table 2. Among the genes showing differential expression, the regulator of G protein signalling (RGS)2 and PCDHB8 genes showed the highest elevation in spiral H. pylori-infected cells compared with coccoid H. pylori-infected cells. Since the function of the PCDHB8 gene is not clear at present, the significance of the increase in the expression of this gene needs to be further investigated. RGS2 belongs to a family of diverse and multifunctional proteins able not only to regulate G-protein-coupled receptor signalling by binding to active G subunits and by acting as GTPase-activating proteins (GAPs), but also to act as effector antagonists by interfering with the binding of downstream effectors (Hollinger & Hepler, 2002). RGS2 is a potent regulator of Gq signalling and is also a recently identified regulatory protein for adenylate cyclase signalling (Sinnarajah et al., 2001). Recent research indicates that RGS2 is an important negative-regulatory factor in cardiac hypertrophy through complex mechanisms involving modulation of MAP kinase signalling pathways (Zou et al., 2006). However, there are few reports of the effect of RGS2 in gastric epithelial cells; further investigation is needed to determine the significance of the differential expression of this gene in cells infected with the two bacterial forms.
GES-1 cell apoptosis induced by spiral and coccoid H. pylori infection
The results of flow cytometry analysis of the apoptosis of GES-1 cells are shown in Fig. 3. After 4 h infection, spiral H. pylori infection could induce 27.4 % apoptosis of GES-1 cells (Fig. 3A), while coccoid H. pylori infection could cause only 10.2 % apoptosis (Fig. 3B). Therefore, spiral H. pylori infection induced a significantly higher level of GES-1 cell apoptosis than coccoid H. pylori infection.
View larger version (24K):
[in this window]
[in a new window]
|
Fig. 3. GES-1 cell apoptosis induced by coccoid and spiral H.pylori infection. The left peak represents the apoptosis rate, the middle peak G0/G1-phase cells. DNA content on the x axis represents the logarithm of fluorescence intensity. (A) Cell apoptosis induced by spiral H. pylori infection; the apoptosis rate was 27.4 %. (B) Cell apoptosis induced by coccoid H.pylori infection; the apoptosis rate was 10.2 %.
|
|
The cDNA microarray showed gene-expression differences in apoptosis-related genes, such as the increased expression of proapoptotic genes gadd45a, myc and fos, and the reduced expression of the antiapoptosis gene survivin in spiral H. pylori-infected GES-1 cells (Table 2
). The RT-PCR confirmed the expression differences of gadd45a, fos and survivin (Fig. 3B). These gene-expression differences may contribute to the high apoptosis rate in cells infected with spiral H. pylori. Gastric mucosal integrity is maintained by a balance between the rate of cell loss and the rate of epithelial cell regeneration (Xia & Talley, 2001). Thus, under normal circumstances, the rate of cell loss by apoptosis is matched by the rate of new cell production by proliferation (Hall et al., 1994). However, spiral H. pylori infection may induce apoptosis in gastric epithelial cells and destroy the balance, resulting in an increase in cell proliferation as a host response to apoptosis. This will result in increased proliferation of the gastric mucosa (Shirin et al., 1999; Yamaguchi et al., 2000; Xia & Talley, 2001). Once spiral H. pylori converts to the coccoid form, the gastric epithelial cells maintain the high proliferation rate. However, the coccoid form can induce a lower degree of apoptosis and can thus lead to the augmentation of cells, which may contribute to gastric cancer after H. pylori infection.
Immune reaction gene-expression differences between spiral and coccoid H. pylori-infected GES-1 cells
Among these differences in gene expression, we found an increased expression of several cytokines and chemokines, such as interleukin (IL)-8, IL-17, IL-18, IL-26, IL-17E, ccl11, ccl5, cxcl10 and gro in spiral H. pylori-infected GES-1 cells (Table 2). RT-PCR analysis confirmed the cDNA microarray results for genes IL-8, ccl11, ccl5, cxcl10 and gro (Fig. 4A). Cole et al. (1997) and Osaki et al. (2002) have shown that coccoid H. pylori have a reduced ability to induce IL-8 secretion compared with spiral H. pylori in AGS and KATO cells. The present study has also shown that the gene-expression level of IL-8 in GES-1 cells infected with coccoid H. pylori is lower than that of cells infected with spiral H. pylori. This is in keeping with the results of Cole et al. (1997). IL-8, gro and cxcl10 belong to the C-X-C chemokines, which preferentially recruit neutrophils. On the other hand, ccl5 and ccl11 belong to the C-C chemokines, which mainly attract lymphocytes and monocytes (Miller & Krangel, 1992; Baggiolini, 1993). Thus, the cells infected with spiral H. pylori have significantly greater mRNA expression of both C-X-C and C-C chemokines than those infected with coccoid H. pylori. Shimoyama et al. (1998) have shown that enhanced chemokine responses can result in a histologically more intense gastritis. Therefore, infection with spiral H. pylori may cause a more pronounced inflammatory response and lead to a more severe gastritis. This may increase the chance of the conversion of the spiral to the coccoid form of H. pylori. The coccoid form may exist in the host in a ‘silent’ state (Cole et al., 1997) and induce a weaker inflammatory response. This is probably a transitory adaptation to a particular environment and contributes to species preservation. Once the environment changes, the coccoid form can also convert to the spiral form (Bode et al., 1993), which in turn can lead to chronic infection and difficulty in the eradication of the pathogen.
View larger version (35K):
[in this window]
[in a new window]
|
Fig. 4. RT-PCR analysis of mRNA expression in spiral or coccoid H. pylori-infected GES-1 cells. (A) RT-PCR analysis of the expression of the immune-reaction genes IL-8, ccl11, ccl5, cxcl10 and gro. (B) RT-PCR analysis of the expression of the apoptosis-related genes gadd45a, fos and survivin
|
|
The cDNA microarray results also showed an increase in the expression of fos and fosL2 in spiral H. pylori-infected cells. The fos- and fosl2-encoded C-fos and Fra-2 transcription factors can combine with other proteins to form transcription-factor complexes that are involved in various processes, such as cell proliferation, apoptosis, survival of tumour cells, invasion, growth and angiogenesis (Eferl & Wagner, 2003). In the present study, we also found gene-expression differences for other transcription factors, signal transduction factors and cell adhesion molecules between infections with the two H. pylori morphological types (Table 2).
In conclusion, to the best of our knowledge, this is the first study to report the global assessment of gene-expression differences by cDNA microarray in gastric epithelial cells infected with spiral or coccoid forms of H. pylori. Of the 22 000 genes, 268 showed a twofold gene-expression difference in Cy5 : Cy3 ratio in gastric epithelial cells induced by spiral and coccoid H. pylori infection, which was regarded as differential expression. These results reveal the differences between the two H. pylori morphological types in inducing changes in GES-1 cell gene expression. Also, these results provide basic data and a research direction for further investigating the interaction of coccoid and spiral H. pylori and gastric epithelial cells.
|
ACKNOWLEDGEMENTS
|
|---|
This work was supported by the National Natural Science Foundation of China (no. 30270079), the Scientific Foundation of Innovative Research Team in Shandong University, the Natural Science Foundation of Shandong Province, People's Republic of China (no. Y2004C03), and the Science Foundation of Shandong Province, People's Republic of China (no. 2005GG3202087). We gratefully acknowledge the invaluable help of Dr Jeremy Ganz for critically reading this manuscript.
|
REFERENCES
|
|---|
Asahi, M., Azuma, T., Ito, S. & 9 other authors (2000). Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells. J Exp Med 191, 593–602.[Abstract/Free Full Text]
Baggiolini, M. (1993). Chemotactic and inflammatory cytokines – CXC and CC proteins. In The Chemokines, pp. 1–10. Edited by I. J. D. Lindley. New York: Plenum.
Bode, G., Mauch, F. & Malfertheiner, P. (1993). The coccoid forms of Helicobacter pylori. Criteria for their viability. Epidemiol Infect 111, 483–490.[Medline]
Catrenich, C. E. & Maki, K. M. (1991). Characterization of the morphologic conversion of Helicobacter pylori from bacillary to coccoid forms. Scand J Gastroenterol Suppl 181, 58–64.
Cellini, L., Allocati, N., Di Campli, E. & Dainelli, B. (1994). Helicobacter pylori: a fickle germ. Microbiol Immunol 38, 25–30.[Medline]
Chan, W. Y., Hui, P. K., Leung, K. M., Chow, J., Kwok, F. & Ng, C. S. (1994). Coccoid forms of Helicobacter pylori in the human stomach. Am J Clin Pathol 102, 503–507.[Medline]
Chiou, C. C., Chan, C. C., Sheu, D. L., Chen, K. T., Li, Y. S. & Chan, F. C. (2001). Helicobacter pylori infection induced alteration of gene expression in human gastric cells. Gut 48, 598–604.[Abstract/Free Full Text]
Cole, S. P., Cirillo, D., Kagnoff, M. F., Guiney, D. G. & Eckmann, L. (1997). Coccoid and spiral Helicobacter pylori differ in their abilities to adhere to gastric epithelial cells and induce interleukin-8 secretion. Infect Immun 65, 843–846.[Abstract]
Cox, J. M., Clayton, C. L., Tomita, T., Wallace, D. M., Robinson, P. A. & Crabtree, J. E. (2001). cDNA array analysis of cag pathogenicity island-associated Helicobacter pylori epithelial cell response genes. Infect Immun 69, 6970–6980.[Abstract/Free Full Text]
Eferl, R. & Wagner, E. F. (2003). AP-1: a double-edged sword in tumorigenesis. Nat Rev Cancer 3, 859–868.[CrossRef][Medline]
Graham, D. Y., Lew, G. M., Klein, P. D., Evans, D. J., Saeed, Z. A. & Malaty, H. M. (1992). Effect of treatment of Helicobacter pylori infection on the long-term recurrence of gastric or duodenal ulcer: a randomized controlled study. Ann Intern Med 116, 705–708.[Medline]
Hall, P. A., Coates, P. J., Ansari, A. & Hopwood, D. (1994). Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis. J Cell Sci 107, 3569–3577.[Abstract]
Hessey, S. J., Spencer, J., Wyatt, J. I., Sobala, G., Rathbone, B. J., Axon, A. T. & Dixon, M. F. (1990). Bacterial adhesion and disease activity in Helicobacter associated chronic gastritis. Gut 31, 134–138.[Abstract/Free Full Text]
Hollinger, S. & Hepler, J. R. (2002). Cellular regulation of RGS proteins: modulators and integrators of G protein signaling. Pharmacol Rev 54, 527–559.[Abstract/Free Full Text]
Janas, B., Czkwianianc, E., Bak-Romaniszyn, L., Bartel, H., Tosik, D. & Planeta-Malecka, I. (1995). Electron microscopic study of association between coccoid forms of Helicobacter pylori and gastric epithelial cells. Am J Gastroenterol 90, 1829–1833.[Medline]
Ke, Y., Ning, T. & Wang, B. (1994). Establishment and characterization of a SV40 transformed human fetal gastric epithelial cell line-GES-1. Zhonghua Zhong Liu Za Zhi 16, 7–10 (in Chinese).[Medline]
Miller, M. D. & Krangel, M. S. (1992). Biology and biochemistry of the chemokines: a family of chemotactic and inflammatory cytokines. CRC Crit Rev Immunol 12, 17–46.
Mizoguchi, H., Fujioka, T., Kishi, K., Nishizono, A., Kodama, R. & Nasu, M. (1998). Diversity in protein synthesis and viability of Helicobacter pylori coccoid forms in response to various stimuli. Infect Immun 66, 5555–5560.[Abstract/Free Full Text]
Mizoguchi, H., Fujioka, T. & Nasu, M. (1999). Evidence for viability of coccoid forms of Helicobacter pylori. J Gastroenterol 34 (Suppl. 11), 32–36.[Medline]
Monstein, H. J. & Jonasson, J. (2001). Differential virulence-gene mRNA expression in coccoid forms of Helicobacter pylori. Biochem Biophys Res Commun 285, 530–536.[CrossRef][Medline]
Ng, B. L., Quak, S. H., Aw, M., Goh, K. T. & Ho, B. (2003). Immune responses to differentiated forms of Helicobacter pylori in children with epigastric pain. Clin Diagn Lab Immunol 10, 866–869.[Medline]
Nilsson, H. O., Blom, J., Abu-Al-Soud, W., Ljungh, A. A., Andersen, L. P. & Wadstrom, T. (2002). Effect of cold starvation, acid stress, and nutrients on metabolic activity of Helicobacter pylori. Appl Environ Microbiol 68, 11–19.[Abstract/Free Full Text]
Nomura, A., Stemmermann, G. N., Chyou, P. H., Kato, I., Perezperez, G. I. & Blaser, M. J. (1991). Helicobacter pylori infection and gastric carcinoma among Japanese Americans in Hawaii. N Engl J Med 325, 1132–1136.[Abstract]
Odenbreit, S., Gebert, B., Puls, J., Fischer, W. & Haas, R. (2001). Interaction of Helicobacter pylori with professional phagocytes: role of the Cag pathogenicity island and translocation, phosphorylation and processing of CagA. Cell Microbiol 3, 21–31.[CrossRef][Medline]
Osaki, T., Yamaguchi, H., Taguchi, H., Fukada, M., Kawakami, H., Hirano, H. & Kamiya, S. (2002). Interleukin-8 induction and adhesion to the coccoid form of Helicobacter pylori. J Med Microbiol 51, 295–299.[Abstract/Free Full Text]
Parsonnet, J., Friedman, G. D., Vandersteen, D. P., Chang, Y., Vogelman, J. H. & Orenteich, N. (1991). Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med 325, 1127–1131.[Abstract]
Reynolds, D. J. & Penn, C. W. (1994). Characteristics of Helicobacter pylori growth in a defined medium and determination of its amino acid requirements. Microbiology 140, 2649–2656.[Abstract]
Saito, N., Konishi, K., Sato, F., Kato, M., Takeda, H., Sugiyama, T. & Asaka, M. (2003). Plural transformation-processes from spiral to coccoid Helicobacter pylori and its viability. J Infect 46, 49–55.[CrossRef][Medline]
Shahamat, M., Mai, U., Paszko-Kolva, C., Kessel, M. & Colwell, R. R. (1993). Use of autoradiography to assess viability of Helicobacter pylori in water. Appl Environ Microbiol 59, 1231–1235.[Abstract/Free Full Text]
Shimoyama, T., Everett, S. M., Dixon, M. F., Axon, A. T. & Crabtree, J. E. (1998). Chemokine mRNA expression in gastric mucosa is associated with Helicobacter pylori cagA positivity and severity of gastritis. J Clin Pathol 51, 765–770.[Abstract]
Shirin, H., Sordillo, E. M., Oh, S. H., Yamamoto, H., Delohery, T., Weinstein, I. B. & Moss, S. F. (1999). Helicobacter pylori inhibits the G1 to S transition in AGS gastric epithelial cells. Cancer Res 59, 2277–2281.[Abstract/Free Full Text]
Sinnarajah, S., Dessauer, C. W., Srikumar D. & 7 other authors (2001). RGS2 regulates signal transduction in olfactory neurons by attenuating activation of adenylyl cyclase III. Nature 409, 1051–1055.[CrossRef][Medline]
Sisto, F., Brenciaglia, M. I., Scaltrito, M. M. & Dubini, F. (2000). Helicobacter pylori: ureA, cagA and vacA expression during conversion to the coccoid form. Int J Antimicrob Agents 15, 277–282.[CrossRef][Medline]
Susanne, B., Athanasios, M., Manfred, R. & Alexander, M. H. (2002). Gene expression profiling in AGS cells stimulated with Helicobacter pylori isogenic strains (cagA positive or cagA negative). Infect Immun 70, 988–992.[Abstract/Free Full Text]
Vijayakumari, S., Khin, M. M., Jiang, B. & Ho, B. (1995). The pathogenic role of the coccoid form of Helicobacter pylori. Cytobios 82, 251–260.[Medline]
Wang, X., Sturegard, E., Rupar, R., Nilsson, H. O., Aleljung, P. A., Carlen, B., Willen, R. & Wadstrom, T. (1997). Infection of BALB/c A mice by spiral and coccoid forms of Helicobacter pylori. J Med Microbiol 46, 657–663.[Abstract]
Xia, H. H. & Talley, N. J. (2001). Apoptosis in gastric epithelium induced by Helicobacter pylori infection: implications in gastric carcinogenesis. Am J Gastroenterol 96, 16–26.[Medline]
Yamaguchi, T., Nakajima, N., Kuwayama, H., Ito, Y., Iwasaki, A. & Arakawa, Y. (2000). Gastric epithelial cell proliferation and apoptosis in Helicobacter pylori-infected mice. Aliment Pharmacol Ther 14 (Suppl. 1), 68–73.[Medline]
Zou, M. X., Roy, A. A., Zhao, Q., Kirshenbaum, L. A., Karmazyn, M. & Chidiac, P. (2006). RGS2 is upregulated by and attenuates the hypertrophic effect of 1-adrenergic activation in cultured ventricular myocytes. Cell Signal (in press).
TOP
INTRODUCTION
METHODS
