<em>Enterococcus faecalis</em> Infection Causes Inflammation, Intracellular Oxphos-Independent ROS Production, and DNA Damage in Human Gastric Cancer Cells

Background: Achlorhydria caused by e.g. atrophic gastritis allows for bacterial overgrowth, which induces chronic inflammation and damage to the mucosal cells of infected individuals driving gastric malignancies and cancer. Enterococcus faecalis (E. faecalis) can colonize achlohydric stomachs and we therefore wanted to study the impact of E. faecalis infection on inflammatory response, reactive oxygen species (ROS) formation, mitochondrial respiration, and mitochondrial genetic stability in gastric mucosal cells. Methods: To separate the changes induced by bacteria from those of the inflammatory cells we established an in vitro E. faecalis infection model system using the gastric carcinoma cell line MKN74. Total ROS and superoxide was measured by fluorescence microscopy. Cellular oxygen consumption was characterized non-invasively using XF24 microplate based respirometry. Gene expression was examined by microarray, and response pathways were identified by Gene Set Analysis (GSA). Selected gene transcripts were verified by quantitative real-time polymerase chain reaction (qRT-PCR). Mitochondrial mutations were determined by sequencing. Results: Infection of MKN74 cells with E. faecalis induced intracellular ROS production through a pathway independent of oxidative phosphorylation (oxphos). Furthermore, E. faecalis infection induced mitochondrial DNA instability. Following infection, genes coding for inflammatory response proteins were transcriptionally up-regulated while DNA damage repair and cell cycle control genes were down-regulated. Cell growth slowed down when infected with viable E. faecalis and responded in a dose dependent manner to E. faecalis lysate. Conclusions: Infection by E. faecalis induced an oxphos-independent intracellular ROS response and damaged the mitochondrial genome in gastric cell culture. Finally the bacteria induced an NF-kappa B inflammatory response as well as impaired DNA damage response and cell cycle control gene expression

A Comprehensive Sequence and Disease Correlation Analyses for the C-Terminal Region of CagA Protein of Helicobacter pylori

Chronic Helicobacter pylori infection is known to be associated with the development of peptic ulcer, gastric cancer and gastric lymphoma. Currently, the bacterial factors of H. pylori are reported to be important in the development of gastroduodenal diseases. CagA protein, encoded by the cagA, is the best studied virulence factor of H. pylori. The pathogenic CagA protein contains a highly polymorphic Glu-Pro-Ile-Tyr-Ala (EPIYA) repeat region in the C-terminal. This repeat region is reported to be involved in the pathogenesis of gastroduodenal diseases. The segments containing EPIYA motifs have been designated as segments A, B, C, and D; however the classification and disease relation are still unclear. This study used 560 unique CagA sequences containing 1,796 EPIYA motifs collected from public resources, including 274 Western and 286 East Asian strains with clinical data obtained from 433 entries. Fifteen types of EPIYA or EPIYA-like sequences are defined. In addition to four previously reported major segment types, several minor segment types (e.g., segment B′, B′′) and more than 30 sequence types (e.g., ABC, ABD) were defined using our classification method. We confirm that the sequences from Western and East Asian strains contain segment C and D, respectively. We also confirm that strains with two EPIYA segment C have a greater chance of developing gastric cancer than those with one segment C. Our results shed light on the relationships between the types of CagAs, the country of origin of each sequence type, and the frequency of gastric disease

Childhood drowning in South Africa: local data should inform prevention strategies

Drowning is an important cause of childhood injury, however, little is known about drowning in Africa. The aim of this study is to investigate submersion incidents in Cape Town, South Africa and provide specific prognostic factors as well as to develop age-appropriate prevention strategies. A retrospective chart review performed at the Red Cross War Memorial Children's Hospital in Cape Town, South Africa. Patients admitted because of 'drowning' or 'near-drowning' between January 2007 and April 2013 were included. 75 children were included. 63 (84 %) survived without complications, 8 (10.7 %) died and 4 (5.3 %) had permanent neurological sequelae. The median age was 2.2 years (range 0.1-12.4). 46 (60.5 %) incidents happened in or around the home, only 14 (18.7 %) were witnessed. 42 (56 %) took place in a pool (29 private, 13 public). Significant predictors of the outcome were: estimated submersion time, duration of apnea, unresponsive and dilated pupils, intubation and use of inotropes. On arrival at the ER we found these significant predictors of the outcome: CPR, a GCS < 5, hypothermia, bradycardia, asystole, as well as the PIM2-calculated mortality risk for patients admitted to the ICU. The majority of incidents were unwitnessed and occurred in or around the home. Prevention programs should be focused on adult supervision for younger children and creating awareness on the dangers of drowning in the home environment. While bathing in baths or buckets, children should never be left alone and parents should be made aware of the dangers. In our study, the majority of incidents occurred in swimming pools and limiting access to these could prevent many incidents of drowning among older children. Although children of all language groups are at risk for drowning, English- or Afrikaans-speaking children were particularly at risk for drowning in private pools while Xhosa-speaking children mostly drowned in baths or buckets. We also report multiple prognostic factors for the outcome, but none of them were absolute predictive of the outcome, indicating that each victim of submersion deserves full resuscitative treatment

Isolation of cDNA coding for an ubiquitous membrane protein deficient in high Na+, low K+ stomatocytic erythrocytes

Human red blood cells (RBCs) that are deficient in an integral membrane- associated protein (“stomatin“) of apparent molecular mass 31 Kd show a catastrophic increase in passive membrane permeability to the univalent cations Na+ and K+ and are stomatocytic in shape. We have purified this protein from normal RBC membranes and isolated a cDNA clone coding for it. The deduced protein sequence is unrelated to that of any known ion- transport-related protein. Selective solubilization studies using detergents show that while the protein is strongly associated with the phospholipid bilayer, it also binds to the cytoskeleton. The predicted polypeptide has a single trans-membranous hydrophobic segment near the N-terminus, which would locate it in the membrane; the large C-terminal domain is hydrophilic and cytoplasmic in orientation and is presumed to be responsible for the attachment to the cytoskeleton. By inference, the protein has the function of closing a latent ion channel. The messenger RNA encoding this protein is ubiquitously distributed in different human cell types and tissues and is thus presumably a widely distributed regulator of transmembrane cation fluxes. As a membrane- bound inhibitor protein of Na+ and K+ transport, it is unique among the known components of membrane-transport proteins.</jats:p

Isolation of cDNA coding for an ubiquitous membrane protein deficient in high Na+, low K+ stomatocytic erythrocytes

Abstract Human red blood cells (RBCs) that are deficient in an integral membrane- associated protein (“stomatin“) of apparent molecular mass 31 Kd show a catastrophic increase in passive membrane permeability to the univalent cations Na+ and K+ and are stomatocytic in shape. We have purified this protein from normal RBC membranes and isolated a cDNA clone coding for it. The deduced protein sequence is unrelated to that of any known ion- transport-related protein. Selective solubilization studies using detergents show that while the protein is strongly associated with the phospholipid bilayer, it also binds to the cytoskeleton. The predicted polypeptide has a single trans-membranous hydrophobic segment near the N-terminus, which would locate it in the membrane; the large C-terminal domain is hydrophilic and cytoplasmic in orientation and is presumed to be responsible for the attachment to the cytoskeleton. By inference, the protein has the function of closing a latent ion channel. The messenger RNA encoding this protein is ubiquitously distributed in different human cell types and tissues and is thus presumably a widely distributed regulator of transmembrane cation fluxes. As a membrane- bound inhibitor protein of Na+ and K+ transport, it is unique among the known components of membrane-transport proteins.</jats:p