'Modulation of the enzymatic activities of replicative helicase (DnaB) by interaction with Hp0897: a possible mechanism for helicase loading in Helicobacter pylori'.

DNA replication in Helicobacter pylori is initiated from a unique site (oriC) on its chromosome where several proteins assemble to form a functional replisome. The assembly of H. pylori replication machinery is similar to that of the model gram negative bacterium Escherichia coli except for the absence of DnaC needed to recruit the hexameric DnaB helicase at the replisome assembly site. In the absence of an obvious DnaC homologue inH. pylori, the question arises as to whether HpDnaB helicase is loaded at theHp-replication origin by itself or is assisted by other unidentified protein(s). A high-throughput yeast two-hybrid study has revealed two proteins of unknown functions (Hp0897 and Hp0340) that interact with HpDnaB. Here we demonstrate that Hp0897 interacts with HpDnaB helicase in vitro as well as in vivo Furthermore, the interaction stimulates the DNA binding activity of HpDnaB and modulates its adenosine triphosphate hydrolysis and helicase activities significantly. Prior complex formation of Hp0897 and HpDnaB enhances the binding/loading of DnaB onto DNA. Hp0897, along with HpDnaB, colocalizes with replication complex at initiation but does not move with the replisome during elongation. Together, these results suggest a possible role of Hp0897 in loading of HpDnaB at oriC.

Next-generation sequencing and de novo assembly, genome organization, and comparative genomic analyses of the genomes of two Helicobacter pylori isolates from duodenal ulcer patients in India.

The prevalence of different H. pylori genotypes in various geographical regions indicates region-specific adaptations during the course of evolution. Complete genomes of H. pylori from countries with high infection burdens, such as India, have not yet been described. Herein we present genome sequences of two H. pylori strains, NAB47 and NAD1, from India. In this report, we briefly mention the sequencing and finishing approaches, genome assembly with downstream statistics, and important features of the two draft genomes, including their phylogenetic status. We believe that these genome sequences and the comparative genomics emanating thereupon will help us to clearly understand the ancestry and biology of the Indian H. pylori genotypes, and this will be helpful in solving the so-called Indian enigma, by which high infection rates do not corroborate the minuscule number of serious outcomes observed, including gastric cancer.

Statins modulate transcriptional activity of heme-oxygenase-1 promoter in NIH 3T3 Cells.

Statins, inhibitors of HMG CoA reductase, have pleiotropic effects independent of their capacity to lower cholesterol. Heme-oxygenase-1(HO-1) plays an important role as an anti-oxidant and anti-inflammatory enzyme. In the present study, we used NIH 3T3 cells which express HO-1 to investigate the molecular mechanisms of HO-1 induction by statins. Simvastatin or fluvastatin induced a significant increase in HO-1 protein expression and mRNA levels. Both statins stimulated activity of a mouse HO-1 promoter (-1,287 to +73 bp)/luciferase reporter gene, 3.25 ± 0.23 (Mean ± S.E.M., n = 15, P < 0.001, t-test) and 3.13 ± 0.33 (Mean ± S.E.M., n = 6, P < 0.001, t-test), respectively. This effect was more pronounced in the short proximal promoter than the full promoter of HO-1. Gel retardation experiments for C/EBP and upstream stimulatory factor (USF) DNA-binding activities using simvastatin- or fluvastatin-treated cells showed significant nuclear protein-DNA complexes which were supershifted with antibodies specific for C/EBP ß and δ or USF-1 and USF-2. Point mutations of the proximal HO-1 promoter (-149 to +73 bp) for the myc/max which binds USF or the C/EBP binding sequences showed a reduction in statin-induced reporter activity whereas no role of the distal C/EBP binding elements located at -4 kb was observed. Moreover, overexpression of mutated C/EBP ß and USF factor or the siRNA for both factors supported a role of these transcription factors in statin-dependent induction of HO-1, with a clearer effect for C/EBP.

Curcumin Encapsulated into Biocompatible Co-Polymer PLGA Nanoparticle Enhanced Anti-Gastric Cancer and Anti-Helicobacter Pylori Effect.

BACKGROUND: The current disadvantages (high cost, toxicity, resistance) of chemotherapy for gastric cancer opted people for alternative therapy from natural source. Curcumin (natural product) possess multiple biological activities but low bio-availability limits their uses as therapeutic. The Nano-formulation of curcumin increased the bioavailability and productivity of anti-cancer and anti-bacterial properties. The present study was initiated to determine the anti-cancer and anti-bacterial effect of Nano curcumin against gastric cancer and H. pylori. METHODS: Curcumin loaded PLGA nanoparticles (CUR-NPs) was prepared by single emulsion solvent evaporation method. The MIC were determined using agar dilution method to find the anti-H. Pylori activity of Nano curcumin. The cytotoxicity of Nano curcumin was evaluated by MTT assay and the apoptotic effect (cell cycle arrest and morphology change) was shown by PI staining and microscopy. RESULTS: The MIC of nanocurcumin and curcumin for all four H. pylori strains were 8 µg/ml and 16 µg/ml respectively. The inhibition rate of gastric cancer cells after treatment with curcumin was increased from 6% to 67% for 24h, from 8% to 75% for 48h, from 10% to 83% for 72h. In case of nanocurcumin, the inhibition rate increased from 7% to 69% for 24h, 11% to 87% for 48h and 16% to 97% for 72h. The IC50 of curcumin and Nano-curcumin were 24.20 µM and 18.78 µM respectively for 72 h. The population of cells in sub-G0 population increased from 4.1% in the control group to 24.5% and 57.8% when treated with curcumin and nanocurcumin respectively. After 72h of treatment with nanocurcumin, the apoptotic cells population increased as compared to native curcumin treated cells. CONCLUSION: The Nano curcumin might be used as a potential therapeutics against gastric cancer and H. Pylori. There is need of further in vivo study in order to validate CUR-NPs activity.

Expression of a naturally occurring angiotensin AT(1) receptor cleavage fragment elicits caspase-activation and apoptosis.

Several transmembrane receptors are documented to accumulate in nuclei, some as holoreceptors and others as cleaved receptor products. Our prior studies indicate that a population of the 7-transmembrane angiotensin type-1 receptor (AT(1)R) is cleaved in a ligand-augmented manner after which the cytoplasmic, carboxy-terminal cleavage fragment (CF) traffics to the nucleus. In the present report, we determine the precise cleavage site within the AT(1)R by mass spectrometry and Edman sequencing. Cleavage occurs between Leu(305) and Gly(306) at the junction of the seventh transmembrane domain and the intracellular cytoplasmic carboxy-terminal domain. To evaluate the function of the CF distinct from the holoreceptor, we generated a construct encoding the CF as an in-frame yellow fluorescent protein fusion. The CF accumulates in nuclei and induces apoptosis in CHO-K1 cells, rat aortic smooth muscle cells (RASMCs), MCF-7 human breast adenocarcinoma cells, and H9c2 rat cardiomyoblasts. All cell types show nuclear fragmentation and disintegration, as well as evidence for phosphotidylserine displacement in the plasma membrane and activated caspases. RASMCs specifically showed a 5.2-fold increase (P < 0.001) in CF-induced active caspases compared with control and a 7.2-fold increase (P < 0.001) in cleaved caspase-3 (Asp174). Poly(ADP-ribose)polymerase was upregulated 4.8-fold (P < 0.001) in CF expressing cardiomyoblasts and colocalized with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). CF expression also induces DNA laddering, the gold-standard for apoptosis in all cell types studied. CF-induced apoptosis, therefore, appears to be a general phenomenon as it is observed in multiple cell types including smooth muscle cells and cardiomyoblasts.

The trafficking protein GABARAP binds to and enhances plasma membrane expression and function of the angiotensin II type 1 receptor.

Proteins that bind to the intracellular expanses, particularly cytoplasmic tail regions, of heptahelical integral membrane receptors are of particular interest in that they can mediate or modulate trafficking or intracellular signaling. In an effort to distinguish new proteins that might promote angiotensin II type 1 (AT(1)) receptor intracellular events, we screened a yeast 2-hybrid mouse brain library with the rat AT(1A) receptor (AT(1)R) carboxyl terminus and identified GABARAP, a protein involved in intracellular trafficking of the GABA(A) receptor, as a binding partner for the AT(1)R. Interaction of GABARAP with the AT(1)R carboxyl terminus was further substantiated using GST pull-down assays, and binding of the full-length tagged AT(1)R to GABARAP was verified using coimmunoprecipitation. Bioluminescence resonance energy transfer assays further confirmed specific interaction of GABARAP with AT(1)R. Moreover, GABARAP clearly increased the steady-state level of plasma membrane-associated AT(1)R in PC-12 cells. Cotransfection of GABARAP with an AT(1)R fluorescent fusion protein increased PC-12 cell surface expression of the AT(1)R more than 6-fold when standardized to the level of intracellular expression. Furthermore, GABARAP overexpression in CHO-K1 cells engineered to express AT(1)R increased angiotensin II binding sites 3.7-fold and angiotensin II-induced phospho-extracellular signal-regulated kinase 1/2 and cellular proliferation significantly over levels obtained with AT(1)R overexpression alone. In addition, small interfering RNA-mediated knockdown of GABARAP reduced the steady-state levels of the AT(1)R fluorescent fusion protein by 43% and its cell surface expression by 84%. Immunoblot analyses confirmed the quantitative image data. We conclude that GABARAP binds to and promotes trafficking of the AT(1)R to the plasma membrane.

Novel virulence factor dupA of Helicobacter pylori as an important risk determinant for disease manifestation: An overview.

Helicobacter pylori (H. pylori) is a microaerophilic, Gram-negative, human gastric pathogen found usually in the mucous lining of stomach. It infects more than 50% of the world's population and leads to gastroduodenal diseases. The outcome of disease depends on mainly three factors: Host genetics, environment and bacterial factors. Among these, bacterial virulence factors such as cagA, vacA are well known for their role in disease outcomes. However, based on the global epidemiological results, none of the bacterial virulence (gene) factors was found to be associated with particular diseases like duodenal ulcer (DU) in all populations. Hence, substantial importance has been provided for research in strain-specific genes outside the cag pathogenicity island, especially genes located within the plasticity regions. dupA found within the plasticity regions was first demonstrated in 2005 and was proposed for duodenal ulcer development and reduced risk of gastric cancer in certain geographical regions. Due to the discrepancies in report from different parts of the world in DU development related to H. pylori virulence factor, dupA became an interesting area of research in elucidating the role of this gene in the disease progression. In this review, we shed light on the detailed information available on the polymorphisms in dupA and their clinical relevance. We have critically appraised several pertinent studies on dupA and discussed their merits and shortcomings. This review also highlights dupA gene as an important biomarker for DU in certain populations.

Measurement of membrane-bound human heme oxygenase-1 activity using a chemically defined assay system.

Heme oxygenase (HO) catalyzes heme degradation in a reaction requiring NADPH-cytochrome P450 reductase (CPR). Although most studies with HO used a soluble 30-kDa form, lacking the C-terminal membrane-binding region, recent reports show that the catalytic behavior of this enzyme is very different if this domain is retained; the overall activity was elevated 5-fold, and the K(m) for CPR decreased approximately 50-fold. The goal of these studies was to accurately measure HO activity using a coupled assay containing purified biliverdin reductase (BVR). This allows measurement of bilirubin formation after incorporation of full-length CPR and heme oxygenase-1 (HO-1) into a membrane environment. When rat liver cytosol was used as the source of partially purified BVR, the reaction remained linear for 2 to 3 min; however, the reaction was only linear for 10 to 30 s when an equivalent amount of purified, human BVR (hBVR) was used. This lack of linearity was not observed with soluble HO-1. Optimal formation of bilirubin was achieved with concentrations of bovine serum albumin (0.25 mg/ml) and hBVR (0.025-0.05 microM), but neither supplement increased the time that the reaction remained linear. Various concentrations of superoxide dismutase had no effect on the reaction; however, when catalase was included, the reactions were linear for at least 4 to 5 min, even at high CPR levels. These results not only show that HO-1-generated hydrogen peroxide leads to a decrease in HO-1 activity but also provide for a chemically defined system to be used to examine the function of full-length HO-1 in a membrane environment.

Nitric oxide stimulates heme oxygenase-1 gene transcription via the Nrf2/ARE complex to promote vascular smooth muscle cell survival.

OBJECTIVE: Previous studies from our laboratory and others found that NO is a potent inducer of heme oxygenase-1 (HO-1) gene transcription in vascular smooth muscle cells (SMC), however, the mechanism responsible for the induction of HO-1 gene expression has not been elucidated. In the present study, we determined the signaling pathway responsible for the induction of HO-1 and its biological significance. METHODS: Cultured rat aortic SMC were exposed to nitrosative stress by treating cells with various NO donors or with inflammatory cytokines. RESULTS: Nitrosative stress stimulated an increase in HO-1 mRNA expression and promoter activity in vascular SMC. However, mutation of the antioxidant response element (ARE) in the HO-1 promoter or overexpression of a dominant-negative mutant of NF-E2-related factor-2 (Nrf2) abrogated the activation by NO. Electromobility shift assays using an ARE probe detected a complex that was significantly increased in intensity by NO. In addition, the migration of this complex was retarded by using an antibody directed against Nrf2. NO also increased Nrf2 mRNA expression, total and nuclear Nrf2 levels, and the binding of Nrf2 to the HO-1 promoter. Finally, treatment of SMC with NO stimulated apoptosis that was increased by HO-1 inhibition. CONCLUSIONS: These results demonstrate that nitrosative stress induces HO-1 gene transcription through the activation of the Nrf2/ARE complex to counteract NO-induced apoptosis of vascular SMC. The capacity of nitrosative stress to activate Nrf2 and stimulate HO-1 gene transcription may represent a critical adaptive response to maintain cell viability at sites of vascular inflammation and atherosclerosis.

Heme oxygenase-1 gene enhancer manifests silencing activity in a chromatin environment prior to oxidative stress.

The expression of heme oxygenase-1 (HO-1) is regulated by E1 and E2 enhancers, both of which contain multiple Maf recognition elements (MAREs). In living cells, MAREs are bound by Bach1/MafK heterodimers, hence maintaining a quiescent state of the HO-1 gene (hmox-1). However, in transient transfection assays, they act as transcriptional enhancers. Therefore MAREs may manifest their function only in a chromatin environment. By using NIH3T3 cell pools stably transfected with EGFP reporter genes driven by the wild-type or mutated E2 enhancer, we demonstrate that the E2 MAREs function as transcriptional silencers depending on the binding of Bach1/MafK heterodimer in vivo only in a chromatin environment. After cadmium treatment, they switched into transcriptional enhancers. Surprisingly, single MARE site did not exhibit such function. Furthermore, by using DNase I hypersensitivity assay, we demonstrate that simple chromatin condensations were not involved in the Bach1-mediated repression. We conclude that, in a chromatin environment, the E2 MAREs function as transcriptional silencers depending on binding of Bach1/MafK heterodimer.

Helicobacter pylori strains harboring babA2 from Indian sub population are associated with increased virulence in ex vivo study.

BACKGROUND: The babA2 gene along with the cagA and vacA of Helicobacter pylori has been considered as a risk factor for the disease outcome in certain populations. This study was aimed to understand the role of babA2 of H. pylori with the background of cagA and vacA in disease manifestations in Indian sub population. METHODS: A total of 114 H. pylori strains isolated from duodenal ulcer (DU) (n = 53) and non-ulcer dyspepsia (NUD) patients (n = 61) were screened for the prevalence of these virulence markers by PCR. The comparative study of IL-8 production and apoptosis were done by co-culturing the AGS cell line with H. pylori strains with different genotypes. Adherence assay was performed with babA2 positive and negative strains. Two isogenic mutants of babA2 were constructed and the aforesaid comparative studies were carried out. RESULTS: PCR results indicated that 90.6 % (48/53), 82 % (50/61) and 73.6 % (39/53) strains from DU patients were positive for cagA, vacA, and babA2, respectively. Whereas the prevalence of these genes in NUD subjects were 70.5 % (43/61); 69.8 % (37/53), and 65.6 % (39/61), respectively. Although adherence to AGS cells was comparable among strains with babA2 positive and negative genotypes, but the triple positive strains could induce highest degree of IL-8 production and apoptosis, followed by the cagA (-)/vacA (-)/babA2 (+) strains and triple negative strains, respectively. The wild type strains showed significantly higher IL-8 induction as well as apoptosis in ex vivo than its isogenic mutant of babA2. CONCLUSION: PCR study demonstrated that there was no significant association between the distribution of babA2 genotype or of triple positive strains and disease outcome in this sub population. The adherence assay showed that there was no significant difference in the extent of adherence to AGS cells among babA2 positive and negative strains. But the ex vivo study indicated that the triple positive or even the babA2 only positive strains are involved in increased virulence. The wild type strains also exhibited increased virulence compared to the babA2 mutant strains. This inconsistency demonstrated that bacterial genotype along with host genetic polymorphisms or other factors play important role in determining the clinical manifestation of H. pylori infections.

Low prevalence of clarithromycin-resistant Helicobacter pylori isolates with A2143G point mutation in the 23S rRNA gene in North India.

Resistance of Helicobacter pylori to clarithromycin is associated with a single base substitution in the 23S rRNA gene. In this study, clarithromycin-resistant H. pylori isolates were analysed for the presence of 23S rRNA gene mutations. H. pylori were isolated from 68 patients suffering from various gastroduodenal diseases in North India. Minimum inhibitory concentrations (MICs) were determined by the agar dilution method, and point mutations in clarithromycin-resistant strains were identified by PCR-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing. Clarithromycin resistance was observed in 11.8% (8/68) of the H. pylori isolates in North India. The A2143G point mutation in the 23S rRNA gene was found in 87.5% (7/8) of the clarithromycin-resistant strains, and the A2142G mutation in association with the T2182C mutation was found in 12.5% (1/8). In conclusion, the continued high prevalence of clarithromycin-sensitive H. pylori strains (88.2%) observed in this study allows the use of the triple-therapy regimen for the treatment of H. pylori infection in this region. Surveillance studies need to be conducted at regular intervals for clarithromycin resistance in the population. To our knowledge, this is the first study in India to report that point mutations at position A2143G and at A2142G in association with T2182C are associated with clarithromycin resistance, confirming reports from other parts of the world.

Helicobacter pylori plasticity region genes are associated with the gastroduodenal diseases manifestation in India.

BACKGROUND: Almost all Helicobacter pylori infected person develop gastritis and severe gastritis is supposed to be the denominator of peptic ulcer diseases, which may lead to gastric cancer. However, it is still an enigma why few strains are associated with ulcer formation, while others are not related with any disease outcome. Although a number of putative virulence factors have been reported for H. pylori, there are contradictory results regarding their connotation with diseases. Recently, there has been a significant attention in strain-specific genes outside the cag pathogenicity island, especially genes within plasticity regions. Studies demonstrated that certain genes in this region may play important roles in the pathogenesis of H. pylori-associated diseases. The aim of this study was to assess the role of selected genes (jhp0940, jhp0945, jhp0947 and jhp0949) in the plasticity region in relation to risk of H. pylori-related diseases in Indian population. METHODS: A total of 113 H. pylori strains isolated from duodenal ulcer (DU) (n = 61) and non-ulcer dyspepsia (NUD) subjects (n = 52) were screened by PCR and Dot-Blot to determine the presence of these genes. The comparative study of IL-8 production and apoptosis were also done by co-culturing the AGS cells with H. pylori strains of different genotype. RESULTS: PCR and Dot-Blot results indicated that the prevalence rates of jhp0940, jhp0945, jhp0947 and jhp0949 in the H. pylori strains were 9.8, 47.5, 50.8, 40.9 % and 17.3, 28.8, 26.9, 19.2 % isolated from DU and NUD, respectively. IL-8 production and apoptotic cell death were significantly higher in H. pylori strains containing jhp0945, jhp0947 and jhp0949 than the strains lacking those genes. Results indicated that the prevalence of jhp0945, jhp0947 and jhp0949 are associated with increased risk of severe diseases in India. CONCLUSION: Our study showed that presence of jhp0945, jhp0947 and jhp0949 were significantly associated with symptomatic expressions along with the increased virulence during in vitro study whereas jhp0940 seems to be negatively associated with the disease. These results suggest that jhp0945, jhp0947 and jhp0949 could be useful prognostic markers for the development of duodenal ulcer in India.

Characterization of Nrf2 activation and heme oxygenase-1 expression in NIH3T3 cells exposed to aqueous extracts of cigarette smoke.

Cigarette smoke (CS) is a complex chemical mixture estimated to be composed of up to 5000 different chemicals, many of which are prooxidant. Here we show that, at least in vitro, the cellular response designed to combat oxidative stress resulting from CS exposure is primarily controlled by the transcription factor Nrf2, a principal inducer of antioxidant and phase II-related genes. The prominent role of Nrf2 in the cellular response to CS is substantiated by the following observations: In NIH3T3 cells exposed to aqueous extracts of CS (i) Nrf2 is strongly stabilized and becomes detectable in nuclear extracts. (ii) Nuclear localization of Nrf2 coincides with increased DNA binding of a putative Nrf2/MafK heterodimer to its cognate cis-regulatory site, i.e., the antioxidant-responsive element (ARE). (iii) Studies on the regulatory elements of the oxidative stress-inducible gene heme oxygenase-1 (hmox1) using various hmox1 promoter/luciferase reporter constructs revealed that the strong CS-dependent expression of this gene is primarily governed by the distal enhancers 1 ("E1") and 2 ("E2"), which both contain three canonical ARE-like stress-responsive elements (StREs). Notably, depletion of Nrf2 levels caused by RNA interference significantly compromised CS-induced hmox1 promoter activation, based on the distinct Nrf2 sensitivity exhibited by E1 and E2. Finally, (iv) siRNA-dependent knock-down of Nrf2 completely abrogated CS-induced expression of phase II-related genes. Taken together, these results confirm the outstanding role of Nrf2 both in sensing (oxidant) stress and in orchestrating an efficient transcriptional response aimed at resolving the stressing conditions.

NF-kappaB plays a key role in hypoxia-inducible factor-1-regulated erythropoietin gene expression.

OBJECTIVE: The aim of this study was to further define the signal transduction pathways leading to hypoxia-inducible factor-1 (HIF-1) erythropoietin (EPO) gene expression. MATERIALS AND METHODS: Human hepatocellular carcinoma cells (Hep3B) were exposed to hypoxia (1% oxygen) and examined for mRNA expression, as well as gene transactivation with RT-PCR and luciferase reporter gene assays, respectively. RESULTS: Treatment with LY294002 (a selective pharmacological inhibitor of phosphatidylinositol 3-kinase) significantly inhibited EPO protein and mRNA expression in Hep3B cells exposed to hypoxia for 24 hours, while treatment with PD098059 or SB203580 (selective pharmacological inhibitors of the MEK and p38 mitogen-activated protein kinase pathways, respectively) had no significant effects. The activity of AKT, a downstream target of PI3K, was increased by hypoxia and was also inhibited by LY294002. Genetic inhibition of AKT resulted in significant inhibition of NF-kappaB and HIF-1-mediated transactivation, as well as EPO gene expression, in response to hypoxia. Overexpression of constitutively active AKT resulted in increased NF-kappaB and HIF-1 transactivation. The selective inhibitor of NF-kappaB, pyrrolidine dithiocarbamate (PDTC), significantly blocked HIF-1 protein expression. Inhibition of NF-kappaB with a superrepressor dominant negative IkappaBalpha genetic construct also significantly blocked NF-kappaB and HIF-1 transactivation, as well as EPO gene expression. CONCLUSION: We propose a key role for NF-kappaB in EPO gene regulation in response to hypoxia.

Association of Intact dupA (dupA1) rather than dupA1 cluster with duodenal ulcer in Indian population.

BACKGROUND: The duodenal ulcer promoting gene (dupA) and dupA cluster in Helicobacter pylori have been described as a risk factor for duodenal ulcer development in some populations. Polymorphic gene dupA can be divided into two groups, intact dupA1 (long or short type based on the presence or absence of 615-bp extra sequences at the 5' region) having complete reading frame and other truncated dupA2 having frame-shift mutation. This study was aimed to elucidate the role of dupA of H. pylori and their clusters in the disease manifestation of Indian population. METHODS: A total of 170 H. pylori strains were screened for the presence of dupA, dupA alleles and dupA cluster by PCR and sequencing. Pro-inflammatory cytokine (IL-8) with different dupA variant H. pylori stimulated gastric epithelial cells (AGS cells) was measured by ELISA. RESULTS: A total of 50 strains (29.4%) were positive for dupA among the tested 170 strains. The prevalence of dupA1 in duodenal ulcer (DU) and non-ulcer dyspepsia (NUD) populations was found to be 25.5% (25/98) and 11.1% (8/72), respectively and 16.4% (28/170) of the tested strains had dupA1, cagA and vacAs1m1 positive. The distribution of long and short type dupA1 has not been significantly associated with the disease outcome. The dupA cluster analysis showed that 10.2% (10/98) and 8.3% (6/72) strains were positive among DU and NUD, respectively. IL-8 production was significantly higher in dupA1(+) , cagA (+), vacA (+) (902.5 ± 79.01 pg/mL) than dupA2 (+) , cagA (+) , vacA (+) (536.0 ± 100.4 pg/mL, P = 0.008) and dupA (-), cagA (+), vacA (+) (549.7 ± 104.1 pg/mL, P = 0.009). Phylogenetic analysis of dupA indicated that the Indian H. pylori strains clustered with East Asian strains but distinct from Western strains. This is the first known genetic element of Indian H. pylori that is genetically closer to the East Asian strains but differed from the Western strains. CONCLUSIONS: The intact dupA1 was significantly associated with DU than NUD (P = 0.029) but the dupA1 cluster has no role in the disease manifestation at India (P = 0.79). Thus, dupA1 can be considered as a biomarker for DU patients in India.

Regulation of heme oxygenase-1 gene transcription: recent advances and highlights from the International Conference (Uppsala, 2003) on Heme Oxygenase.

Recent investigations into the regulation of heme oxygenase-1 gene (hmox-1) transcription have exposed mechanisms of increasing diversity and complexity worthy of a gene whose expression is modulated by a seemingly endless array of physiological, pathophysiological, and nonphysiological agents and conditions. For instance, contrary to initial and prevalent assumptions that inducer-dependent gene stimulation is mediated principally by the positive action of transcription activators, it now appears that such induction may occur secondarily to deactivation of the repressor protein, Bach1. As a further complication, heme and cadmium, two potent inducers of the hmox-1 gene, inhibit Bach1 function by different mechanisms-by inhibition of DNA binding or promotion of nuclear export, respectively. Bach1 also plays a role in signal-dependent hmox-1 gene repression, an increasingly appreciated phenomenon that is manifested in a species- and cell-specific manner. Although extreme concentrations of the heme oxygenase-1 protein resulting from the opposing phenomena of gene activation and repression have physiological consequences, even minor modulation in the level of this enzyme, as elicited by variations in the length of a dinucleotide repeat region within the human hmox-1 promoter, may be of clinical relevance. Finally, mechanistic diversity is also apparent in the type and combination of protein kinase-dependent, signal transduction pathways used during hmox-1 gene activation.

Activation of the mouse heme oxygenase-1 gene by 15-deoxy-Delta(12,14)-prostaglandin J(2) is mediated by the stress response elements and transcription factor Nrf2.

The mechanism of heme oxygenase-1 (ho-1) gene activation by 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) was examined. 15d-PGJ(2) stimulated expression of HO-1 mRNA and protein and of a mouse ho-1 gene promoter/luciferase fusion construct (HO15luc) in a dose-dependent manner in mouse hepatoma (Hepa) cells. HO15luc expression was not effected by troglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligand, but induction by 15d-PGJ(2) was abrogated by the antioxidant N-acetylcysteine. The primary 15d-PGJ(2) responsive sequences were localized to a 5' distal enhancer (E1) and identified as the stress-response element, previously shown to mediate ho-1 activation by several agents, including heme and heavy metals. Treatment of Hepa cells with 15d-PGJ(2) stimulated stress-response element-binding activity as judged by electrophoretic mobility shift assays. Antibody "supershift" experiments identified NF-E2 related factor 2 (Nrf2), but not Fos, Jun, or activating transcription factor/cyclic AMP response element binding protein transcription factors, within the 15d-PGJ(2)-induced complexes. Similarly, a dominant-negative mutant of Nrf2, but not of c-Jun or c-Fos, abrogated 15d-PGJ(2)-stimulated E1 transcription activity. Finally, prior induction of HO-1 in RAW264.7 mouse macrophages by 15d-PGJ(2) attenuated cell death caused by diesel exhaust particle extracts. These results demonstrate that induction of mouse HO-1 expression by 15d-PGJ(2) is independent of PPAR-gamma but dependent on oxidative stress, is regulated by the oxidative stress-activated transcription factor Nrf2, and provides cytoprotective activity.

Sensitization of apoptotically-resistant breast carcinoma cells to TNF and TRAIL by inhibition of p38 mitogen-activated protein kinase signaling.

The mitogen-activated protein kinase (MAPK) cascade is a critical component in the regulation of cell survival and proliferation decisions. In breast carcinoma cells, activation of the p38-MAPK member of this family occurs in response to pro-inflammatory cytokines and cellular stress. The involvement of p38-MAPK in the activation of the transcription factor, NF-kappaB, suggests a potential role and mechanism for regulation of cell survival and drug resistance. Generation of the resistant MCF-7 variant (MCF-7TN-R) was achieved by prolonged exposure of MCF-7N cells to increasing concentrations of TNF. Differences in MAPK activation and function in the MCF-7 cell variants were determined. The role of the p38-MAPK pathway in regulation of resistance was determined using pharmacological (SB 203580) or molecular [Dominant Inhibitory (DI)-p38] inhibition. The effect of p38 inhibition on NF-kappaB transcriptional activation was analyzed. As compared to the sensitive MCF-7N parent cell line, the MCF-7TN-R cell line displayed significant resistance to TNF- and TRAIL-induced cell death. Analysis of the expression and phosphorylation of members of the MAPK family revealed an increased basal activation of p38 in the MCF-7TN-R variant. The p38-mediated phosphorylation and transcriptional activity were suppressed by pharmacologic inhibition with SB 230580. Treatment of MCF-7TN-R cells with SB partially restored sensitivity to TNF-induced cell death. In addition, use of a DI-p38 construct with or without the addition to TNF induced cell death, thus restoring TNF-sensitivity to these cells. The ability of p38 inhibition to restore apoptotic sensitivity was correlated with suppression of the TNF-induced cell survival pathway, NF-kappaB. The increased activation of p38-MAPK in MCF-7TN-R cells demonstrates that this signaling pathway through activation of NF-kappaB is an important route for control of resistance to cell death in breast carcinoma. Molecular and pharmacological inhibition of p38-MAPK signaling may represent a mechanism for sensitizing cancer cells to chemotherapeutic regimens and restoration of apoptotic signaling.