RESUMEN
Obesity contributes to the development of non-alcoholic fatty liver disease (NAFLD), which is characterized by the upregulated expression of two key inflammatory mediators: tumor necrosis factor (Tnfa) and monocyte chemotactic protein 1 (Mcp1; also known as Ccl2). However, the chromatin make-up at these genes in the liver in obese individuals has not been explored. In this study, to identify obesity-mediated epigenetic changes at Tnfa and Ccl2, we used a murine model of obesity induced by a high-fat diet (HFD) and hyperphagic (ob/ob) mice. Chromatin immunoprecipitation (ChIP) assay was used to determine the abundance of permissive histone marks, namely histone H3 lysine 9 and 18 acetylation (H3K9/K18Ac), H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 36 trimethylation (H3K36me3), in conjunction with polymerase 2 RNA (Pol2) and nuclear factor (Nf)-κB recruitment in the liver. Additionally, to correlate the liver tissue-derived ChIP measurements with a robust in vitro transcriptional response at the Tnfa and Ccl2 genes, we used lipopolysaccharide (LPS) treatment to induce an inflammatory response in Hepa1-6 cells, a cell line derived from murine hepatocytes. ChIP revealed increased H3K9/K18Ac at Tnfa and Ccl2 in the obese mice, although the differences were only statistically significant for Tnfa (p<0.05). Unexpectedly, the levels of H3K4me3 and H3K36me3 marks, as well as Pol2 and Nf-κB recruitment, did not correspond with the increased expression of these two genes in the obese mice. By contrast, the acute treatment of Hepa1-6 cells with LPS significantly increased the H3K9/K18Ac marks, as well as Pol2 and Nf-κB recruitment at both genes, while the levels of H3K4me3 and H3K36me3 marks remained unaltered. These results demonstrate that increased Tnfa and Ccl2 expression in fatty liver at the chromatin level corresponds to changes in the level of histone H3 acetylation.
Asunto(s)
Quimiocina CCL2/genética , Histonas/metabolismo , Hígado/metabolismo , Lisina/metabolismo , Obesidad/genética , Factor de Necrosis Tumoral alfa/genética , Acetilación/efectos de los fármacos , Animales , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , ADN Polimerasa II/metabolismo , Dieta Alta en Grasa/efectos adversos , Hígado Graso/etiología , Hígado Graso/genética , Hígado Graso/metabolismo , Expresión Génica , Lipopolisacáridos/farmacología , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Ratones Obesos , FN-kappa B/metabolismo , Obesidad/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The mitochondrial amidoxime reducing component mARC is the fourth mammalian molybdenum enzyme. The protein is capable of reducing N-oxygenated structures, but requires cytochrome b5 and cytochrome b5 reductase for electron transfer to catalyze such reactions. It is well accepted that the enzyme is involved in N-reductive drug metabolism such as the activation of amidoxime prodrugs. However, the endogenous function of the protein is not fully understood. Among other functions, an involvement in lipogenesis is discussed. To study the potential involvement of the protein in energy metabolism, we tested whether the mARC protein and its partners are regulated due to fasting and high fat diet in mice. We used qRT-PCR for expression studies, Western Blot analysis to study protein levels and an N-reductive biotransformation assay to gain activity data. Indeed all proteins of the N-reductive system are regulated by fasting and its activity decreases. To study the potential impact of these changes on prodrug activation in vivo, another mice experiment was conducted. Model compound benzamidoxime was injected to mice that underwent fasting and the resulting metabolite of the N-reductive reaction, benzamidine, was determined. Albeit altered in vitro activity, no changes in the metabolite concentration in vivo were detectable and we can dispel concerns that fasting alters prodrug activation in animal models. With respect to high fat diet, changes in the mARC proteins occur that result in increased N-reductive activity. With this study we provide further evidence that the endogenous function of the mARC protein is linked with lipid metabolism.
Asunto(s)
Citocromo-B(5) Reductasa/metabolismo , Citocromos b5/metabolismo , Dieta Alta en Grasa , Ayuno , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Animales , Benzamidinas/metabolismo , Línea Celular Tumoral , Activación Enzimática , Humanos , Hiperfagia/metabolismo , Metabolismo de los Lípidos , Masculino , RatonesRESUMEN
Although mitochondrial dysfunction is implicated in the pathogenesis of obesity, the molecular mechanisms underlying obesity-related metabolic abnormalities are not well established. We performed mitochondrial quantitative proteomic and whole transcriptome analysis followed by functional annotations within liver and skeletal muscles, using fasted and non-fasted 16- and 48-week-old high-fat diet (HFD)-fed and normal diet-fed (control group) wild-type C56BL/6J mice, and hyperphagic ob/ob and db/db obese mice. Our study identified 1,675 and 704 mitochondria-associated proteins with at least two peptides in liver and muscle, respectively. Of these, 221 liver and 44 muscle proteins were differentially expressed (adjusted p values ≤ 0.05) between control and all obese mice, while overnight fasting altered expression of 107 liver and 35 muscle proteins. In the liver, we distinguished a network of 27 proteins exhibiting opposite direction of expression changes in HFD-fed and hyperphagic mice when compared to control. The network centered on cytochromes P450 3a11 (Cyp3a11) and 4a14 (Cyp4a14), and fructose-bisphosphate aldolase B (Aldob) proteins which bridged proteins cluster involved in Metabolism of xenobiotics with proteins engaged in Fatty acid metabolism and PPAR signaling pathways. Functional annotations revealed that most of the hepatic molecular alterations, which characterized both obesity and fasting, related to different aspects of energy metabolism (such as Fatty acid metabolism, Peroxisome, and PPAR signaling); however, only a limited number of functional annotations could be selected from skeletal muscle data sets. Thus, our comprehensive molecular overview revealed that both obesity and fasting states induce more pronounced mitochondrial proteome changes in the liver than in the muscles.
Asunto(s)
Ayuno/metabolismo , Mitocondrias Hepáticas/metabolismo , Mitocondrias Musculares/metabolismo , Obesidad/metabolismo , Proteínas/metabolismo , Animales , Dieta Alta en Grasa , Ácidos Grasos/metabolismo , Hiperfagia/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Músculo Esquelético/metabolismo , Proteínas/genéticaRESUMEN
Colon anastomosis is therapeutically challenging because multiple, usually undetectable factors influence a spectrum of repair mechanisms. We hypothesized that low molecular weight heparins, routinely administered perioperatively, may differentially affect gene expression related to colon healing. Twenty pairs of untreated and enoxaparin-treated rats underwent left-side hemicolectomy with a primary end-to-end anastomosis. Normal colon and anastomotic bowel segments were resected on day 0 and on days 1, 3, 5, and 7 after surgery, respectively. Serial anastomosis transverse cross-sections were evaluated microscopically and by microarray (Rat Genome 230 2.0, Affymetrix). Differentially expressed probe sets were annotated with Gene Ontology. We also examined the influence of enoxaparin on fibroblast proliferation and viability in vitro. Among the 5476 probe sets, we identified differential expression at each healing time point, yielding 79 subcategories. Most indicated genes were involved in wound healing, including multicellular organismal development, locomotory behavior, immune response, cell adhesion, inflammatory response, cell-cell signaling, blood vessel development, and tissue remodeling. Although we found no intensity differences in histological features of healing between enoxaparin-treated and control rats, treatment did induce significant expression changes during early healing. Of these changes, 83 probe sets exhibited at least twofold changes and represented different functional annotations, including inflammatory response, regulation of transcription, regulation of apoptosis, and angiogenesis. Fibroblast culture confirmed an anti-viability effect of enoxaparin. Enoxaparin affects colon wound-related gene expression profiles, but further studies will resolve whether heparin treatment is a risk factor after intestinal surgery, at least in some patients.
Asunto(s)
Anastomosis Quirúrgica/métodos , Heparina de Bajo-Peso-Molecular/efectos adversos , Heparina de Bajo-Peso-Molecular/uso terapéutico , Cicatrización de Heridas/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Colon/efectos de los fármacos , Colon/lesiones , Colon/metabolismo , Enoxaparina/efectos adversos , Enoxaparina/uso terapéutico , Perfilación de la Expresión Génica , Humanos , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Componente Principal , Ratas , Ratas WistarRESUMEN
Definitive identification of promoters, their cis-regulatory motifs, and their trans-acting proteins requires experimental analysis. To define the HNRNPK promoter and its cognate DNA-protein interactions, we performed a comprehensive study combining experimental approaches, including luciferase reporter gene assays, chromatin immunoprecipitations (ChIP), electrophoretic mobility shift assays (EMSA), and mass spectrometry (MS). We discovered that out of the four potential HNRNPK promoters tested, the one containing the palindromic motif TCTCGCGAGA exhibited the highest activity in a reporter system assay. Although further EMSA and MS analyses, performed to uncover the identity of the palindrome-binding transcription factor, did identify a complex of DNA-binding proteins, neither method unambiguously identified the pertinent direct trans-acting protein(s). ChIP revealed similar chromatin states at the promoters with the palindromic motif and at housekeeping gene promoters. A ChIP survey showed significantly higher recruitment of PARP1, a protein identified by MS as ubiquitously attached to DNA probes, within heterochromatin sites. Computational analyses indicated that this palindrome displays features that mark nucleosome boundaries, causing the surrounding DNA landscape to be constitutively open. Our strategy of diverse approaches facilitated the direct characterization of various molecular properties of HNRNPK promoter bearing the palindromic motif TCTCGCGAGA, despite the obstacles that accompany in vitro methods.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Regiones Promotoras Genéticas/genética , Ribonucleoproteínas/genética , Inmunoprecipitación de Cromatina/métodos , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética/métodos , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo K , Humanos , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/genética , Unión ProteicaRESUMEN
BACKGROUND: Aberrant activation of protein kinases is one of the essential oncogenic driving forces inherent to the process of tumorigenesis. The protein kinase CK2 plays an important role in diverse biological processes, including cell growth and proliferation as well as in the governing and transduction of prosurvival signals. Increased expression of CK2 is a hallmark of some cancers, hence its antiapoptotic properties may be relevant to cancer onset. Thus, the designing and synthesis of the CK2 inhibitors has become an important pursuit in the search for cancer therapies. RESULTS: Using a high-throughput microarray approach, we demonstrate that two potent inhibitors of CK2, 4,5,6,7-tetrabromo-benzimidazole (TBBz) and 2-Dimethyloamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), blocked mitogen induced mRNA expression of immediate early genes. Given the impact of these inhibitors on the process of transcription, we investigated their effects on RNA Polymerase II (RNAPII) elongation along the mitogen inducible gene, EGR1 (early growth response 1), using chromatin immunoprecipitation (ChIP) assay. ChIP analysis demonstrated that both drugs arrest RNAPII elongation. Finally, we show that CDK9 kinase activity, essential for the triggering of RNAPII elongation, was blocked by TBBz and to lesser degree by DMAT. CONCLUSIONS: Our approach revealed that small molecules derived from halogenated imidazole compounds may decrease cell proliferation, in part, by inhibiting pathways that regulate transcription elongation.
Asunto(s)
Antineoplásicos/farmacología , Bencimidazoles/farmacología , Quinasa de la Caseína II/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , ARN Polimerasa II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo , Proliferación Celular , Inmunoprecipitación de Cromatina , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/genética , Quinasa 9 Dependiente de la Ciclina/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Perfilación de la Expresión Génica , Halogenación , Células HeLa , Humanos , Transcripción GenéticaRESUMEN
A product of the Helicobacter pylori hp0596 gene (Tip-alpha) is a highly immunogenic homodimeric protein, unique for this bacterium. Cell fractionation experiments indicate that Tip-alpha is anchored to the inner membrane. In contrast, the three-dimensional model of the protein suggests that Tip-alpha is soluble or, at least, largely exposed to the solvent. hp0596 gene knockout resulted in a significant decrease in the level of H. pylori colonization as measured by real-time PCR assay. In addition, the Tip-alpha recombinant protein was determined to stimulate macrophage to produce IL-1alpha and TNF-alpha. Both results imply that Tip-alpha is rather loosely connected to the inner membrane and potentially released during infection.
Asunto(s)
Proteínas Bacterianas/inmunología , Mucosa Gástrica/microbiología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/patogenicidad , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Infecciones por Helicobacter/inmunología , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Activación de Macrófagos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Organismos Libres de Patógenos Específicos , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Human mitochondrial polynucleotide phosphorylase (hPNPase) is an exoribonuclease localized in mitochondria. The exact physiological function of this enzyme is unknown. Recent studies have revealed the existence of a relationship between induction of hPNPase mRNA and both cellular senescence and growth arrest of melanoma cells following beta-interferon treatment. The aim of this study was to verify whether the augmented hPNPase mRNA level results in increase of the protein level. In several cell lines established from five metastatic melanoma patients we did not find any such correlation. However, an elevated level of hPNPase protein was observed in interferon-induced HeLa and Jurkat cells. This increase was correlated with a slight shortening of poly(A) tails of mitochondrial ND3 transcript.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Interferón beta/metabolismo , Melanoma/metabolismo , Mitocondrias/enzimología , Polirribonucleótido Nucleotidiltransferasa/biosíntesis , ARN Mensajero/metabolismo , Regulación hacia Arriba , Secuencia de Bases , Línea Celular Tumoral , Exorribonucleasas/metabolismo , Células HeLa , Humanos , Células Jurkat , Datos de Secuencia MolecularRESUMEN
The heterogeneous nuclear ribonucleoprotein K is an ancient RNA/DNA-binding protein that is involved in multiple processes that compose gene expression. The pleiotropic action of K protein reflects its ability to interact with different classes of factors, interactions that are regulated by extracellular signals. We used affinity purification and MS to better define the repertoire of K protein partners. We identified a large number of new K protein partners, some typically found in subcellular compartments, such as plasma membrane, where K protein has not previously been seen. Electron microscopy showed K protein in the nucleus, cytoplasm, mitochondria, and in vicinity of plasma membrane. These observations greatly expanded the view of the landscape of K protein-protein interaction and provide new opportunities to explore signal transduction and gene expression in several subcellular compartments.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo K/química , Proteómica/métodos , Animales , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Inmunohistoquímica , Neoplasias Hepáticas/patología , Espectrometría de Masas/métodos , Mapeo de Interacción de Proteínas , Ratas , Transducción de Señal , Fracciones SubcelularesRESUMEN
Dsb proteins control the formation and rearrangement of disulfide bonds during the folding of membrane and exported proteins. Here we examined the role of DsbI protein in Helicobacter pylori pathogenesis and demonstrated that a dsbI mutant impaired in disulfide bond formation revealed a greatly reduced ability to colonize mice gastric mucosa.
Asunto(s)
Infecciones por Helicobacter/microbiología , Helicobacter pylori/patogenicidad , Proteína Disulfuro Isomerasas/metabolismo , Factores de Virulencia/genética , Virulencia/genética , Animales , Modelos Animales de Enfermedad , Mucosa Gástrica/microbiología , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Ratones , Ratones Endogámicos C57BL , Oxidación-Reducción , Proteína Disulfuro Isomerasas/genéticaRESUMEN
Heterogeneous nuclear ribonucleoprotein K (hnRNP K protein) is an RNA/DNA-binding protein that acts in several compartments, including mitochondria. It integrates cellular signaling cascades with multiple processes of gene expression mechanisms. Our studies demonstrate that: (1) insulin activates the import of hnRNP K protein into mitochondria in vitro and in vivo; (2) overexpression of hnRNP K protein modulates insulin-activated mitochondrial gene expression; and (3) insulin treatment stimulates binding of hnRNP K protein to mitochondrial DNA. Based on these and our previously reported results we conclude that hnRNP K protein may be a mediator of mitochondrial response to insulin.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo K/fisiología , Insulina/farmacología , Mitocondrias/efectos de los fármacos , Animales , ADN Mitocondrial , Regulación de la Expresión Génica , Humanos , Mitocondrias/fisiología , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/análisis , RatasRESUMEN
hnRNP K protein, which localizes to the nucleus, cytoplasm and mitochondria, is involved in the various cellular processes that compose gene expression. We used a SAGE-based assay to profile RNAs associated with hnRNP K protein in rat mitochondria. RNA was isolated from mitoplasts obtained from highly purified and RNase-treated mitochondria. Total RNA and RNA associated with hnRNP K protein were then used as input material for generating two SAGE libraries. Mitochondrion-derived tags isolated from the total mitoplast RNA library represented 86.3%, while those isolated from the library constructed from RNA associated with hnRNP K protein represented only 28.2% of selected tags. Thus, an unexpected number of nuclear-encoded RNAs were purified from mitochondria. Many of these transcripts were co-purified with hnRNP K protein, and high levels of nuclear-encoded RNAs co-immunoprecipitating with K protein corresponded to elevated hnRNP K protein levels of the organelle. The most abundant RNAs that were co-purified with hnRNP K protein represented transcripts originating from satellite I DNA. While satellite I RNA levels were higher in the nucleus and cytoplasm than in mitochondria, the most abundant binding of satellite I transcripts to hnRNP K protein was found in mitochondria. The role of satellite I RNA in mitochondria remains to be elucidated.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo K/química , Mitocondrias/metabolismo , Satélite de ARN/genética , Animales , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Biblioteca de Genes , Técnicas Genéticas , Humanos , Proteínas de la Membrana/química , Unión Proteica , Ratas , Receptor de Insulina/metabolismo , Canales de Translocación SECRESUMEN
Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) is an RNA/DNA-binding protein involved in many processes that regulate gene expression. K protein's pleiotropic action reflects the diversity of its molecular interactions. Many of these interactions have been shown to be regulated by phosphorylation. K protein contains more than seventy potential phosphorylation sites. We used an integrated approach of mass spectrometry and computer analysis to explore patterns of K protein phosphorylation. We found that in vitro a single kinase can phosphorylate K protein on multiple sites spanning the entire length of the protein, including residues contained within the RNA/DNA-binding domains. 2-D gel electrophoresis of K protein purified from cells identified 5-8 spots. Mass spectrometry of K protein isolated from proliferating cells and from cells under oxidative stress revealed the same pattern of phosphopeptides. The structural implications of phosphorylation are discussed.
Asunto(s)
Caseína Quinasas/química , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Secuencia de Aminoácidos , Animales , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Conformación Proteica , RatasRESUMEN
Insulin is the most potent anabolic hormone. The greatest sensitivity to insulin is exhibited by muscle, liver and adipose cells. To study links between insulin and mitochondrial function over the course of cellular quiescence and differentiation, we quantified mitochondrial RNA and DNA in L6 myoblasts and HTC-IR hepatocytes cultured under low-serum conditions in the presence of insulin. The expression of the whole set of mitochondrial genes was determined using reverse transcriptase (RT)-real time PCR. Cell proliferation was assayed by the incorporation of (3)H-thymidine and myoblast differentiation was analyzed by morphological and biochemical markers of myogenesis. Low growth factor concentration in medium decreased proliferation of both cell types and induced differentiation of myoblasts. The expression of all mitochondrial genes decreased in quiescent hepatocytes whereas it increased in quiescent differentiated myotubes, as compared with proliferating cells, similarly to reflecting the expression of the insulin receptor gene, both in myoblasts and hepatocytes. The kinetics of mitochondrial RNA levels were similar to the expression patterns of two nuclear genes, subunit e of mitochondrial ATP-synthase and uncoupling protein-2; however, they did not reflect changes in mitochondrial DNA content. Insulin accelerated myogenesis and expression of both mitochondrial and nuclear genes in differentiated myotubes but not in quiescent hepatocytes. Our studies prove that myogenesis may require the orchestrated transcriptional activation of both mitochondrial and nuclear genes and provide additional evidence confirming the regulatory impact of insulin on the function of muscle mitochondria.
Asunto(s)
ADN Mitocondrial/metabolismo , Hipoglucemiantes/farmacología , Insulina/farmacología , Mitocondrias Musculares/genética , Desarrollo de Músculos/fisiología , Mioblastos/efectos de los fármacos , ARN/metabolismo , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Expresión Génica , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Canales Iónicos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Proteínas de Transporte de Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Mioblastos/metabolismo , ARN Mitocondrial , Ratas , Proteína Desacopladora 2RESUMEN
Immunologically mediated protection against H. pylori infection is an attractive alternative to antibiotic treatment. We compared the efficacy of conventional protein vaccination with that of genetic vaccination against experimental infection with H. pylori in mice. For oral immunization, we used the recombinant peptide of an antigenic fragment of UreB (rUreB) or H. pylori-whole cell lysate antigens, and for genetic immunization, we used recombinant pcDNA and pSec plasmids inserted with the fragment of ureB or DNA of the H. pylori genomic library. Mice were challenged with the mouse stomach-adapted H. pylori Sidney Strain. The detection of gastric bacterial colonization was performed by real-time PCR of a 26-kDa Helicobacter-specific gene, and the presence of serum H. pylori-specific antibodies was determined using direct ELISA assay. The most effective treatment appeared to be oral vaccination with rUreB and either intramuscular or intradermal vaccination with DNA of the H. pylori genomic library. Intradermal genetic vaccination with genomic library DNA significantly increased the IgG antibody response. Our study revealed acceptable efficacies of genetic vaccination with DNA of the H. pylori genomic library.
Asunto(s)
Biblioteca Genómica , Infecciones por Helicobacter/inmunología , Helicobacter pylori/metabolismo , Estómago/microbiología , Vacunas de ADN/inmunología , Animales , Mucosa Gástrica/metabolismo , Infecciones por Helicobacter/terapia , Helicobacter pylori/inmunología , Ratones , Ratones Endogámicos C57BL , Plásmidos , Estómago/inmunología , Vacunación , Vacunas de ADN/administración & dosificación , Vacunas de ADN/microbiologíaRESUMEN
Accurate diagnosis of Helicobacter pylori infection is important in both clinical practice and clinical research. Molecular methods are highly specific and sensitive, and various PCR-based tests have been developed to detect H. pylori in gastric biopsy specimens. We optimized a sensitive and specific quantitative SYBR Green I real-time PCR assay for detection of H. pylori based on amplification of the fragment of a 26-kDa Helicobacter species-specific antigen gene that allows for detection of 5 bacterial cells per PCR sample. Under the assay conditions, SYBR Green I real-time PCR is highly reproducible with a precise log-linear relation in the range of six orders of magnitude of bacterial DNA concentrations. For accurate comparison of H. pylori infection in different tissue samples, the amount of total host DNA in each sample is normalized by TaqMan real-time PCR of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) pseudogenes. The developed method was validated in prophilactically immunized and experimentally infected mice and revealed a level of H. pylori gastric colonisation that was below the limit of detection for a rapid urease test. This new method established for a quantitative analysis of H. pylori in the host's stomach may be useful in experimental studies evaluating new anti-H. pylori drugs and vaccines.