Helicobacter pylori induces mitochondrial DNA mutation and reactive oxygen species level in AGS cells.

To investigate the role of ROS in the helicobacter pylori (Hp) induced mtDNA mutations, AGS cells were treated by extracts of Hp11638 or Hp11638M. The ROS levels, cytochrome C reductions, and intracellular ATP levels were measured. The coding region and the D-Loop region were amplified and sequenced. Results showed the ROS levels, cytochrome C reduction and mtDNA mutations were markedly increased and cell viability decreased after treatment with both Hp extracts, and 616 mutations were detected in D-Loop region and 3 heteroplasmic point mutations in the Cytb gene. No mutations were found in the coding region. The mutation rates of mtDNA D-Loop region were positively correlated with the ROS levels and negatively to the ATP levels.

[Mitochondrial DNA mutations in gastric endothelial cells induced by extract of helicobacter pylori in vitro].

OBJECTIVE: To investigate the relationship between the helicobacter pylori (HP) infection and the genetic instability of mitochondrial DNA (mtDNA) in human gastric adenocarcinoma epithelial cells (AGS). METHODS: After treated with extracts of HP11638 (CagA+, VacA+) or Hp11638 mutant strain (CagA+, VacA-), AGS cells were collected, and mitochondrial DNA was extracted and Cox-I, Cox-II, Cox-III, ATPase6, ATPase8 and Cytb genes and the D-Loop region were amplified by PCR and then sequenced. RESULTS: The mutation rates of the mtDNA in AGS cells were correlated with the extracts of the two HP strains in a concentration- and time-dependent manner. But the mtDNA mutation rate in AGS cells treated with the HP11638 extract was higher than that treated with the Hp11638 mutant extract. Total of 616 mutations in D-Loop region were detected, including 489 point mutations, 81 insertions and 46 deletions. Among them, 70.9% (437/616) belonged to GC to AT and AT to GC transition. Seventeen out of 20 (85%) AGS cells treated with extract of HP had mutations in 303PolyC, 16184PolyC and 514CA regions of mtDNA D-Loop. No mutation was detected in Cox-I, Cox-II, Cox-III, ATPase6 and ATPase8 genes, three point mutations were found in the Cytb gene. CONCLUSION: HP can cause the accumulation of mutations in mtDNA, in particular, in the D-Loop region, and the VacA participated in the process.

[Gastrointestinal dysfunction in acute severe mountain sickness and its relation with multiple organ dysfunction syndrome].

OBJECTIVE: To investigate the relationship between gastrointestinal dysfunction (GD) and multiple organ dysfunction syndrome (MODS) in acute severe mountain sickness (ASMS), including high altitude pulmonary edema (HAPE), and high altitude cerebral edema (HACE), by a retrospective study of medical records and prospective study of hospitalized patients. METHODS: In retrospective study, the clinical data of 3 184 inpatients of General Hospital of Tibetan Military Command suffering from ASMS in the past 50 years (from June, 1958 to June, 2007) were collected. Statistical analysis was performed to study the relationship between GD and MODS in these patients. For the prospective study, 10 admitted patients of ASMS were included. Gastroscopic examination was performed for the ASMS patients, and gastric and duodenal mucosa was scrutinized. At the same time, 30 g of glutamine (Gln) capsule was orally ingested each day for 3 days after the first day of admission. Ten healthy volunteers were included in the control group, and received the same treatment. The levels of serum diamine oxidase (DAO), malonic dialdehyde (MDA), endotoxin and lactulose/mannitol (L/M) ratio were detected before and after treatment in two groups. RESULTS: First, 49.8% of the patients with ASMS were complicated with GD, with 1.5% of fairy stool, and 1.0% with occult blood in stool. In 83 cases of ASMS complicated with MODS, 21.7% (18 cases) appeared GD, and the score of GD was 5.5 in the total score of all organ injury. Second, endoscopic examination showed extensive edema and localized hemorrhage in gastrointestinal mucous membrane, with dotted and patched erosion in gastric antrum and fundus. The pre-treatment DAO, MDA, and endotoxin were higher in the observation group than those in the control group (all P<0.01). After 3 days of Gln capsule treatment, DAO, MDA, and endotoxin were significantly decreased in the observation group (P<0.05 or P<0.01). The pre-treatment L/M ratio in observation group was significantly higher than that in healthy control group (150.69+/-19.91 vs. 117.91+/-17.78, P<0.01). The L/M ratio was significantly decreased after the treatment, as it decreased to 129.37+/-19.75 (P<0.05). However, no significant change in the healthy control group was observed. CONCLUSION: GD plays a major role in the pathogenesis of MODS in ASMS patients.

[Mutations in the D-loop region of mitochondrial DNA and the ROS level in the tissue of hepatocellular carcinoma].

To explore the relationship between ROS level and mutations in D-Loop region of mtDNA, mutations in the D-Loop region of mtDNA and the ROS level in primary hepatocarcinoma tissues were studied. We amplified the D-Loop region of mtDNA of 20 hepatocarcinomas and their adjacent tissue by PCR and then sequencing. ROS in tissue was measured by flow cytometry. mtDNA mutations were detected in 40% (8 of 20) tumor samples. 53 point mutations were detected in eight tumour samples, including 2 insertions, 11 deletions and 40 point mutations. 75% point mutations were T-C and C-T transition. They were four microsatellites among the mutations. Mutations in the adjacent tissues were always companied with mutations in tumour tissues. The mutation frequency in tumour tissues was higher than that in adjacent tissue. There was a larger unidentified deletion. The ROS level in hepatocarcinoma tissue was much higher than control (P<0.01). Meanwhile, we found the ROS level in hepatocarcinoma tissues with mutated mtDNA D-Loop was higher than that hepatocarcinoma tissue normal mtDNA D-Loop, and the ROS level in hepatocarcinoma adjacent tissue with mutated mtDNA D-Loop was higher than that in hepatocarcinoma adjacent tissue with normal mtDNA D-Loop. It was concluded that the D-Loop region of mitochondrial DNA was a highly polymorphoric and mutable region and mutation rate was relatively high in patients with hepaticellular carcinoma, and the abnormal ROS level might be the point mutation in the mitochondrial DNA and hepatocarcinogenesis related to ROS.

Two functional loci in the promoter of EPAS1 gene involved in high-altitude adaptation of Tibetans.

EPAS1 involves in the hypoxic response and is suggested to be responsible for the genetic adaptation of high-altitude hypoxia in Tibetans. However, the detailed molecular mechanism remains unknown. In this study, a single nucleotide polymorphism rs56721780:G>C and an insertion/deletion (indel) polymorphism -742 indel in the promoter region showed divergence between Tibetans and non-Tibetan lowlanders. rs56721780:G>C regulated the transcription of EPAS1 by IKAROS family zinc finger 1 (IKZF1), which was identified as a new transcriptional repressor for EPAS1 gene. It demonstrated that the C allele of rs56721780:G>C decreased the binding of IKZF1, leading to the attenuated transcriptional repression of EPAS1 gene. The insertion at -742 indel provided a new binding site for Sp1 and was related to the activation of EPAS1 promoter. Further functional analysis revealed that lysyl oxidase (LOX) gene, which was reported to be responsible for extracellular matrix protein cross-linking of amnion previously, was a direct target of EPAS1. The CC genotype at rs56721780:G>C and the insertion genotype at -742 indel were found associated with higher EPAS1 and LOX expression levels in amnion, as well as higher birth weight of Tibetan newborns, suggesting that EPAS1 gene might play important roles in the development of amnion, fetus growth and high-altitude adaptation of Tibetans.