Hydrogen peroxide-induced cellular injury is associated with increase in endogenous fluorescence from rat gastric mucosal epithelial cell culture: A new method for detecting oxidative cellular injury by fluorescence measurement

To develop a new method of detecting cellular injury caused by oxygen radicals, we studied endogenous fluorescence from the cultured cells of a rat gastric mucosal epithelial cell line. Measurement with an ultra-high sensitivity camera-image processor system under an inverted epifluorescence microscope showed that the fluorescence intensity of the cells increased time- and dose-dependently after the addition of hydrogen peroxide (H₂O₂), an oxygen radical precursor, to the medium. This increase was inhibited by the presence of catalase. Phase-contrast and fluorescence microscopy revealed that the fluorescence was emitted from granular substances in the cytoplasm of the injured cells. The spectral pattern of excitation and emission indicated that the fluorescent substances were flavins. In cell-free experiments, glutathione reductase which has flavin adenine dinucleotide (FAD) at the active site, increased in fluorescence after incubation with H₂O₂ in the presence of reduced glutathione and glutathione peroxidase. These findings indicate that FAD in the cytoplasm of cells injured by H₂O₂ increased in endogenous fluorescence according to the extent of injury, and suggest that fluorescence measurement may be a simple method in cellular toxicology to detect oxygen radical-induced injuries. (Received July 28, 1997; accepted Sept. 26, 1997)     

Impairment by activation of TRPA1 of gastric epithelial restitution in a wound model using RGM1 cell monolayer

We examined the influence of TRPA1 on the epithelial restitution using a rat gastric epithelial cell line RGM1 monolayer. RGM1 cells were inoculated in 24-well plates cultured for 24 hr, and then starved for 24 hr in a culture medium at 37 °C under 5 % CO₂ in air. After obtaining a confluent RGM1 cell monolayer, a round artificial wound of constant size was induced in the center of the cell monolayer using a pencil-type mixer with a rotating silicon tip. The repair process was monitored by quantitatively measuring the area of the epithelial wound (cell-free area). Immediately after the wound induction, cells at the edge of wound started to form lamellipodia, migrating toward the center of wound, and by so doing the cell-free area was decreased over time. The addition of icilin, the TRPA1 agonist, suppressed the recovery of the epithelial wound in a concentration-dependent manner. Likewise, another TRPA1 agonist, ally isothiocyanate, also significantly inhibited the wound repair. In addition, the recovery of the epithelial wound was potently inhibited when the ambient temperature was lowered to 17 °C, the threshold temperature where TRPA1 is known to be activated. By contrast, the wound healing was not affected by either menthol, the TRPM8 agonist, or capsaicin, the TRPV1 agonist. These results showed for the first time that the activation of TRPA1 inhibited the repair of the epithelial wound in the stomach, probably by the suppression of cell migration, and suggested the involvement of TRPA1 in the mechanism of gastric epithelial restitution. Received 8 August 2006; accepted 7 November 2006     

East Asian-type Helicobacter pylori cytotoxin-associated gene A protein has a more significant effect on growth of rat gastric mucosal cells than the Western type

BACKGROUND AND AIM: Cytotoxin-associated gene A (CagA) protein from H. pylori was reported to be injected into host gastric epithelial cells via a bacterial type IV secretion system, thereby modifying signal transduction. It is classified into two major subtypes, Western and East Asian. The present study aimed to compare the effects of East Asian-type and Western-type CagA on host cell growth. METHODS: A tetracycline (tet)-off system and cagA genes from Western and East Asian-type H. pylori (NCTC 11637 and F32) were transfected into untransformed rat gastric mucosal (RGM1) cells to establish RGM1-CagA cell lines in which CagA expression could be controlled by tetracycline. These cell lines were used to investigate the effect of CagA protein expression on cell growth with BrdU and water-soluble tetrazolium reagent (WST-8) assays. CagA expression, phosphorylation and extracellular signal-regulated kinase (ERK) activation were examined with immunoprecipitation and Western blotting analysis. RESULTS: 5-Bromo-2'deoxyuridine (BrdU) and WST-8 assays demonstrated significant increases in DNA replication and RGM1 cell growth after CagA protein expression. ERK phosphorylation was enhanced when CagA protein was expressed in RGM1-CagA cells. Moreover, the East Asian-type CagA showed a significantly greater effect on ERK activation and host cell growth than the Western type. PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor, suppressed ERK phosphorylation and the CagA protein-induced increase in RGM1-CagA cell growth. CONCLUSIONS: CagA protein expression induces an increase in RGM1-CagA cell proliferation via the mitogen-activated protein kinase (MAPK) signal pathway. The East Asian-type CagA showed a significantly greater effect on cell growth than the Western type, suggesting that the East Asian CagA-positive strain may have an important role in pathogenesis.     

Characterization of a novel cell damage model induced by acid and pepsin using rat gastric epithelial cells: Protective effect of sucralfate

We have established a new model for rat gastric epithelial cell (RGM1) damage caused by both acid and pepsin. Exposure of RGM1 to an acidified medium (pH 3.5–5.0) for 10-50 min decreased cell viability in a time- and pH-dependent manner. Pepsin (0.5–1.0 mg/mL) at pH 4.5 potentiated cell damage in a concentration-dependent manner. Based on these results, two types of cell damage models caused by incubation of cells at pH 4.0 and with pepsin (0.75 mg/mL) at pH 4.5 for 30 min, respectively, were established. The intracellular pH (pH) gradually decreased with a decrease in medium pH and an increase in exposure time. At pH ≤ 4.0, pH_i reached approximately pH 6.3. Pepsin at pH 4.5 caused a further reduction in pH_i compared with the acidified medium alone. Pepsin pre-incubated with pepstatin did not induce any cell damage. Pretreatment with sucralfate (0.1 -3 mg/mL) for 2 h significantly prevented cell damage caused at both pH 4.0 and with pepsin at pH 4.5 in a concentration-dependent manner. Sucralfate (3 mg/mL) significantly prevented the reduction in pH_i at pH 4.0 or with pepsin at pH 4.5. 16,16-Dimethyl prostaglandin E₂ (30 μg/mL) had no effect on either cell damage or pH_i. These cell damage models involving RGM1 are useful for studying the mechanism underlying cell damage and for the screening of cytoprotective drugs.     

Interleukin-1β Inhibits Growth Factor-Stimulated Restoration of Wounded Rat Gastric Epithelial Cell Monolayers

We examined the effect of interleukin-1(IL-1) on spontaneous and enhanced restoration(cell migration and proliferation) using an in vitrowound model comprising a confluent monolayer of ratgastric epithelial RGM1 cells. Repair of an artificialwound in a cell monolayer was found to be time- andconcentration-dependent when the cells were incubatedwith epidermal growth factor (EGF) or transforming growth factor (TGF)- alone for up to 24hr. The growth factors also stimulated DNA synthesissignificantly for 24 hr in a concentration-relatedmanner. IL-1 had no effect on wound restoration in the absence of the growth factors. However, itmarkedly inhibited the restoration enhanced by EGF andTGF-, the inhibition being about 60% and 70%,respectively. In addition, IL-1 significantly reduced the DNA synthesis stimulated by thegrowth factors. The EGF- and TGF--enhancedrestoration was reduced by about 30% by mitomycin C,which potently inhibited the stimulated DNA synthesis.Mitomycin C had no effect on the spontaneous restoration.Even when treated with mitomycin C, the inhibitoryeffect of IL-1 on the enhanced wound repair wasstill observed; however, the extent of the inhibition was decreased. These results indicate thatIL-1 inhibits the migration as well as theproliferation of gastric epithelial cells enhanced byEGF and TGF-, resulting in a failure of woundhealing.     

Atmospheric mercury in Changbai Mountain area, northeastern China II. The distribution of reactive gaseous mercury and particulate mercury and mercury deposition fluxes

Reactive gaseous mercury (RGM) and particulate mercury (Hgp) concentrations in ambient air from a remote site at Changbai Mountain area in northeastern China were intermittently monitored from August 2005 to July 2006 totaling 93 days representing fall, winter-spring and summer season, respectively. Rainwater and snow samples were collected during a whole year, and total mercury (THg) in rain samples were used to calculate wet depositional flux. A throughfall method and a model method were used to estimate dry depositional flux. Results showed mean concentrations of RGM and Hgp are 65 and 77 pg m⁻³. Compared to background concentrations of atmospheric mercury species in Northern Hemisphere, RGM and Hgp are significantly elevated in Changbai area. Large values for standard deviation indicated fast reactivity and a low residence time for these mercury species. Seasonal variability is also important, with lower mercury levels in summer compared to other seasons, which is attributed to scavenging by rainfall and low local mercury emissions in summer. THg concentrations ranged from 11.5 to 15.9 ng L⁻¹ in rainwater samples and 14.9-18.6 ng L⁻¹ in throughfall samples. Wet depositional flux in Changbai area is calculated to be 8.4 μg m⁻² a⁻¹, and dry deposition flux is estimated to be 16.5 μg m⁻² a⁻¹ according to a throughfall method and 20.2 μg m⁻² a⁻¹ using a model method.     

Revisiting the evolution of gonadotropin-releasing hormones and their receptors in vertebrates: secrets hidden in genomes

Gonadotropin-releasing hormone (GnRH) and its G protein-coupled receptor, GnRHR, play a pivotal role in the control of reproduction in vertebrates. To date, many GnRH and GnRHR genes have been identified in a large variety of vertebrate species using conventional biochemical and molecular biological tools in combination with bioinformatic tools. Phylogenetic approaches, primarily based on amino acid sequence identity, make it possible to classify these multiple GnRHs and GnRHRs into several lineages. Four vertebrate GnRH lineages GnRH1, GnRH2, GnRH3, and GnRH4 (for lamprey) are well established. Four vertebrate GnRHR lineages have also been proposed—three for nonmammalian GnRHRs and mammalian GnRHR2 as well as one for mammalian GnRHR1. However, these phylogenetic analyses cannot fully explain the evolutionary origins of each lineage and the relationships among the lineages. Rapid and vast accumulation of genome sequence information for many vertebrate species, together with advances in bioinformatic tools, has allowed large-scale genome comparison to explore the origin and relationship of gene families of interest. The present review discusses the evolutionary mechanism of vertebrate GnRHs and GnRHRs based on extensive genome comparison. In this article, we focus only on vertebrate genomes because of the difficulty in comparing invertebrate and vertebrate genomes due to their marked divergence.     

Drug susceptibiity testing of nontuberculous mycobacteria by broth microdilution method

Introduction-Incidence of Nontuberculous mycobacteria (NTM) has been increasing in past few years. Treatment of NTM differs from Mycobacterium tuberculosis. For proper treatment, it's important to carry out Drug Susceptibility Testing of NTM. Method of DST for NTM is different from MTB and is not available in most laboratories.Method-We performed DST on 122 isolates of NTM. Amikacin, Ciprofloxacin, Trimethoprim, Doxycycline, Moxifloxacin, Clarithromycin, Minocycline and Cefoxitin were used for Rapid Growing Mycobacteria (RGM) and Rifampicin, Clarithromycin, Ethambutol, Isoniazid and Moxifloxacin for Slow Growing Mycobacteria (SGM). M. avium Complex (MAC) was tested against Clarithromycin. Minimum inhibitor concentration was calculated as recommended by standard Clinical and Laboratory Standards Institute (CLSI) and Resazurin Microtitre Assay (REMA).Result-Most of Rapid Growing Mycobacteria were sensitive to Amikacin (76.1%) and Moxifloxacin (46.47%) while Slow Growing Mycobacteria showed only 33.3% sensitivity to Rifampicin and Moxifloxacin and 42% to Clarithromycin. M. avium-intracellulare complex showed 45–50% sensitivity to Clarithromycin. Overall, 98% concordance (Kappa = 0.98; almost perfect; 95% CI = 0.966 to 0.996) was seen between standard and REMA method of DST of NTM.Conclusion-Rapid growers showed good sensitivity to Amikacin and Moxifloxacin, while only one third SGM showed sensitivity to Rifampicin, Moxifloxacin and Clarithromycin. For proper management of NTM of eastern Rajasthan its important to know the DST profile in our area to initiate empirical therapy till the results of specific patient are available. REMA was found to give excellent concordance with standard method.