Significance of the caspase family in Helicobacter pylori induced gastric epithelial apoptosis.

BACKGROUND AND AIMS: H. pylori infection results in an increased epithelial apoptosis in gastritis and duodenal ulcer patients. We investigated the role and type of activation of caspases in H. pylori-induced apoptosis in gastric epithelial cells. METHODS: Differentiated human gastric cancer cells (AGS) and human gastric mucous cell primary cultures were incubated with H. pylori for 0.5-24 hours in RPMI 1640 medium, and the effects on cell viability, epithelial apoptosis, and activity of caspases were monitored. Apoptosis was analyzed by detection of DNA-fragments by Hoechst stain(R), DNA-laddering, and Histone-ELISA. Activities of caspases were determined in fluorogenic assays and by Western blotting. Cleavage of BID and release of cytochrome c were analyzed by Western blot. Significance of caspase activation was investigated by preincubation of gastric epithelial cells with cell permeable specific caspase inhibitors. RESULTS: Incubation of gastric epithelial cells with H. pylori caused a time and concentration dependent induction of DNA fragmentation (3-fold increase), cleavage of BID, release of cytochrome c and a concomittant sequential activation of caspase-9 (4-fold), caspase-8 (2-fold), caspase-6 (2-fold), and caspase-3 (6-fold). No effects on caspase-1 and -7 were observed. Activation of caspases preceded the induction of DNA fragmentation. Apoptosis could be inhibited by prior incubation with the inhibitors of caspase-3, -8, and -9, but not with that of caspase-1. CONCLUSIONS: Activation of certain caspases and activation of the mitochondrial apoptotic pathway are essential for H. pylori induced apoptosis in gastric epithelial cells.

Helicobacter pylori reduces intracellular glutathione in gastric epithelial cells.

Helicobacter pylori infection has been associated with stimulation of gastric mucosal reactive oxygen species (ROS) production, and it was postulated that ROS production is due to neutrophil infiltration and activation. The aim of this study was to investigate the direct effect of H. pylori on ROS formation in gastric epithelial cells in vitro. The human gastric cancer cell line HM02 was incubated with H. pylori for 24 hr, and the effects on cell number and the intracellular radical scavenger reduced glutathione (GSH) were assessed. H. pylori caused a concentration-dependent reduction of cellular GSH concentrations over a broad bacteria-to-cell ratio (1.4-42) in the absence of cell necrosis. The radical scavengers MnTBAP (a cell permeable superoxide dismutase) and ebselen provided protection against H. pylori-induced decrease in cellular GSH concentrations. We conclude that H. pylori directly decreases cellular GSH concentrations in gastric epithelial cells. We suggest that this effect is caused by the release of ROS by H. pylori.

Helicobacter pylori causes DNA damage in gastric epithelial cells.

Helicobacter pylori infection has been considered as a risk factor for gastric carcinoma. Strong evidence exists that reactive oxygen species (ROS) play an important role in carcinogenesis, and in vivo investigations have shown increased synthesis of ROS in the gastric mucosa of H.pylori-infected patients. In the present study the direct effects of H.pylori on ROS and DNA synthesis, induction of apoptosis and DNA repair were investigated in the gastric epithelial cell lines AGS and HM02. Incubation of gastric cells with H.pylori extract induced the synthesis of ROS, diminished the levels of reduced glutathione (GSH), induced DNA fragmentation and increased DNA synthesis in gastric cells. Poly(ADP-ribose) formation was increased in gastric cells exposed to H.pylori extract. FACS analysis of gastric cells exposed to H.pylori extract did not reveal any change in the percentage of cells in the G(2)/M phase of the cell cycle. The radical scavengers MnTBAP (a cell permeable superoxide dismutase mimic), ebselen (a GSH peroxidase mimic) and high doses of catalase completely blocked H.pylori extract-induced elevation in DNA synthesis. Our results indicate that H.pylori extract directly induces the synthesis of ROS in gastric epithelial cells and causes DNA damage.

Role of vacA and cagA in Helicobacter pylori inhibition of mucin synthesis in gastric mucous cells.

The aim of this study was to investigate the effect of Helicobacter pylori on the function of gastric mucous cells. H. pylori (10(4) to 10(7) CFU/well) was incubated with the mucin-producing gastric cell line HM02 for 12 and 24 h. Mucin synthesis and secretion were determined by the incorporation of D-N-[acetyl-(14)C]glucosamine into intracellular and released high-molecular-weight glycoproteins. cagA-positive, cytotoxin-producing and non-cytotoxin-producing H. pylori strains impaired the incorporation of D-N-[acetyl-(14)C]glucosamine into intracellular glycoproteins. Significant inhibition of mucin synthesis was noted after 12 and 24 h of cocultivation with a bacterial load of >/=10(5) bacteria (bacterium/cell ratio = 0.25). The cagA-positive, cytotoxin-producing strains (HP64, HP57, and HP87) caused significantly stronger inhibition of intracellular mucin synthesis than the cagA-positive, non-cytotoxin-producing strains (HP05, HP83, and HP84). The cagA-negative, non-cytotoxin-producing strains (HP01, HP04, and HP85) did not affect intracellular mucin synthesis. The results indicate that H. pylori directly impairs mucin synthesis in gastric mucous cells and that cytotoxic cagA-positive strains cause more profound inhibition of mucin synthesis. We suggest that the increased inhibitory effect of cagA-positive, cytotoxin-producing strains on mucin synthesis can be considered one possible factor responsible for the increased risk of developing peptic ulceration with these H. pylori strains.

Usefulness of morphological characteristics for the differentiation of benign from malignant solitary pulmonary lesions using HRCT.

The aim of this study was to analyze different characteristics on high-resolution computed tomography (HRCT) that help differentiate benign solitary pulmonary lesions (BSPLs) from malignant solitary pulmonary lesions (MSPLs). High-resolution computed tomography was performed on 104 consecutive patients with SPLs. The whole lesion was examined with a slice thickness of 1 mm and a 12-cm field of view. All lesions were surgically excised within 24 h of the CT examination. Satellite nodules, cavitations, and necrosis were found only in MSPLs. Useful characteristics for the differentiation of BSPLs from MSPLs were the presence of spicules (p < 0.00005), spicules extending to the visceral pleura (p < 0.0005), the vessel sign (p < 0.0005), pleural retraction (p < 0.001), circumscribed pleural thickening (p < 0.001), the bronchus sign (p < 0.005), the presence of ground-glass attenuation adjacent to the SPL (p < 0.01), the density of the lesion (p < 0.05), and the length of spicules (p < 0.05). Using the significant characteristics p < 0.01 for the identification of MSPLs, a sensitivity of 91.4 % and a specificity of 56.5 % (accuracy of 83.7 %) was found. A precise morphological assessment of the periphery of the pulmonary lesion is necessary. The HRCT technique is useful in differentiation of BSPLs from MSPLs. However, metastases strongly resembled benign lesions in terms of size and edge type, and chronic inflammatory pseudotumors as a group mimic MSPLs.

The Helicobacter pylori fatty acid cis-9,10-methyleneoctadecanoic acid stimulates protein kinase C and increases DNA synthesis of gastric HM02 cells.

Protein kinase C (PKC) has been implicated in the control of epithelial proliferative activity and in the process of malignant transformation. Helicobacter pylori (H.p.) infection is associated with increased gastric epithelial cell proliferation and has been linked with gastric carcinoma. In the present study, we report that the H.p. fatty acid cis-9,10-methyleneoctadecanoic acid (MOA) directly activates PKC (Ka 3.3 microM). The effect of MOA upon PKC activation was Ca2+ dependent but did not require phosphatidylserine as phospholipid cofactor. MOA increased the stimulatory effect of phosphatidylserine at low Ca2+ (1 microM) concentrations. These findings indicate that MOA interacts at the phospholipid- and the diacylglycerol-binding domain to elicit PKC activation. Treatment of gastric mucous cells HM02 caused translocation of PKC from the cytosol to the nuclear, mitochondrial and membrane fraction. Furthermore, MOA stimulated [3H]thymidine incorporation into the DNA of HM02 cells. Our results show that the H.p. fatty acid MOA activates PKC and increases DNA synthesis in gastric epithelial cells.

Antioxidative protection in a high-melatonin organism: the dinoflagellate Gonyaulax polyedra is rescued from lethal oxidative stress by strongly elevated, but physiologically possible concentrations of melatonin.

Lethal oxidative stress was investigated in the dinoflagellate Gonyaulax polyedra by measuring the dying-peak of bioluminescence during circadian phases of low physiological light emission, low bioluminescence capacity, and low sensitivity to stimulatory agents. Measurements were carried out in constant darkness after transfer of cells from light at CT 6 (circadian time, 0600 hr). H2O2 (0.08 mM), when administered 1 hr after transfer of cells, led to a multifold, long-lasting enhancement of light emission, which is typical for lethal cell damage. At the circadian phases of investigation, melatonin did not substantially stimulate bioluminescence up to concentrations of 0.5 mM. At this concentration, addition of melatonin prevented the dying-peak and reduced bioluminescence to almost basal values. The high concentration of melatonin applied is not unphysiological in Gonyaulax, because the indoleamine can increase to levels of several millimolar, e.g., in response to temperature signals. These protective effects of melatonin seem to be caused mainly by the direct action of melatonin as an antioxidant, because the major enzymes of antioxidative protection were not stimulated by melatonin, although some of them responded to H2O2. The activities of neither superoxide dismutase, hemoperoxidase/catalase, glutathione peroxidase, nor haloperoxidase were enhanced under the influence of melatonin; glutathione S-transferase activity increased only slightly.

[Differentiation of solitary pulmonary coin lesions by high-resolution computerized tomography]. / Differenzierung von solitären Lungenrundherden durch die hochauflösende Computertomographie.

PURPOSE: The aim of this prospective study was not to describe individual morphological findings in benign and malignant solitary intrapulmonary nodules; it was instead to examine in a critical manner the indications for differentiation found in the literature in order to facilitate safe differential diagnosis of benign and malignant nodules. PATIENTS AND METHODS: A total of 64 solitary pulmonary nodules were examined with high-resolution computed tomography and correlated with histological findings. Only lesions that had been removed by surgery were used. No lesion was excluded on the grounds of size. RESULTS: Useful characteristics for the differentiation of benign from malignant pulmonary nodules were: diameter and density of the lesion, air inclusion, unsharp and dystelectatic margin, the presence of spicules, length of spicules, spicules extending to the visceral pleura, pleural tail sign and cirumscribed pleural thickening. CONCLUSION: For the differentiation of benign and malignant solitary pulmonary nodules meticulous assessment of the margin of the nodule is necessary. Using the criteria mentioned, a sensitivity of 85% and a specifity of 78% can be achieved for the identification of malignant pulmonary nodules. Since it was not possible to differentiate between benign and malignant nodules with certainty using imaging methods, the chance of patient survival could only be promoted by early surgery.

Identification of characteristics for malignancy of solitary pulmonary nodules using high-resolution computed tomography.

The aim of this prospective study was to apply the described single pathological appearance and possibilities of differentiating signs from other studies and summarize them concerning their differential diagnostic importance to improve differential diagnostic strategies concerning the dignity of solitary pulmonary nodules. 55 consecutive patients with solitary pulmonary nodules were examinated using high-resolution computed tomography (HRCT) before surgery. Thereafter HRCT-diagnosis was proven by histological assessment. Only lesions which were removed by surgery were used. No lesion was excluded by size. Necrotic areas, cavitation, satellite lesions and circumscribed pleural thickening were only found in the malignant nodules. Discrimination between benign and malignant lesions was possible by: mean diameter (P<.01), mean density (P<.01) and air inclusion (P<.05), by air bronchogram/bronchiologram (P<.05), indistinct/fogged (P<.05) and dystelectatic (P<.01) margin, the presence (P<.01) and length (P<.01) of spiculae, spiculae extended into the pleura visceralis (P<.05) and pleural distension (P<.01). A single sign can be seen in either benign or malignant nodules, but if considered together with other signs it may have a different meaning. HRCT can enable a differentiation of BSPN from MSPN in the majority of cases. As imaging methods could not get a nearly complete certainty about the dignity the chance of survival of patients could be preserved exclusively by an early surgery.

Interspecific variation in sugar and amino acid transport by the avian cecum.

Previous studies of cecal sugar and amino acid transport in the domestic chicken led to a widely held generalization that the avian cecum is unimportant as a site of nutrient transport. In fact, we found that the uptake capacity of the cecum for hexose sugars and amino acids is substantial in some species of birds. Cecal transport of glucose was measurable in all five study species (Canada goose, sage grouse, domestic chicken, red-necked phalarope, and rock dove), approached or exceeded intestinal levels in the grouse and phalarope, and accounted for between 0.1% (rock dove) and 49% (sage grouse) of the whole gut's integrated uptake capacity. Proline uptake averaged higher in the proximal portion of the cecum than in any region of the small intestine for all species but the goose. The ceca contributed between 2% (rock dove) and 25% (sage grouse) of the gut's integrated uptake capacity for proline. Similar ranges were found for fructose, lysine, leucine, and aspartate. Future studies should be undertaken to search for phylogenetic and ecological correlates of the interspecific variation in cecal transport and to determine how nutrient transport integrates with other functions of the avian cecum.

Adipocyte size and number in dietary obesity resistant and susceptible rats.

Fat depot cellularity was assessed in overfed dietary obesity resistant S 5B/Pl (S) and susceptible Osborne-Mendel (OM) rats. Cell number and lipid per cell were determined for three fat depots in both 24- and 105-day-old rats. Between these two ages, fat cell number doubled in inguinal fat depots of S rats fed high- or low-fat diets and OM rats fed a low-fat diet, but quadrupled in OM rats fed a high-fat diet. The size of adipocytes in this depot was influenced by strain but not by diet. Compared to normal weight S rats, 24-day-old overfed S rats had twice as many adipocytes in the perirenal-retroperitoneal fat depot. Overfed OM rats had 3 times perirenal-retroperitoneal fat depot. Overfed OM rats had 3 times as many. In OM rats fed the high-fat diet, there was a 16-fold increase in adipocyte number between 24 and 105 days of age. Overfeeding caused a slight increase in perirenal-retroperitoneal adipocyte size in 24-day-old rats but had little influence on cell size in 105-day-old rats.