Oxidative damage of the gastric mucosa in Helicobacter pylori positive chronic atrophic and nonatrophic gastritis, before and after eradication

Background. Helicobacter pylori is the main cause of gastritis and a primary carcinogen. The aim of this study was to assess oxidative damage in mucosal compartments of gastric mucosa in H. pylori positive and negative atrophic and nonatrophic gastritis. Materials and methods. Five groups of 10 patients each were identified according to H. pylori positive or negative chronic atrophic (Hp‐CAG and CAG, respectively) and nonatrophic gastritis (Hp‐CG and CG, respectively), and H. pylori negative normal mucosa (controls). Oxidative damage was evaluated by nitrotyrosine immunohistochemistry in the whole mucosa and in each compartment at baseline and at 2 and 12 months after eradication. Types of intestinal metaplasia were classified by histochemistry. Results. Total nitrotyrosine levels appeared significantly higher in H. pylori positive than in negative patients, and in Hp‐CAG than in Hp‐CG (p < .001); no differences were found between H. pylori negative gastritis and normal mucosa. Nitrotyrosine were found in foveolae and intestinal metaplasia only in Hp‐CAG. At 12 months after H. pylori eradication, total nitrotyrosine levels showed a trend toward a decrease in Hp‐CG and decreased significantly in Hp‐CAG (p = .002), disappearing from the foveolae (p = .002), but remaining unchanged in intestinal metaplasia. Type I and II of intestinal metaplasia were present with the same prevalence in Hp‐CAG and CAG, and did not change after H. pylori eradication. Conclusions. Oxidative damage of the gastric mucosa increases from Hp‐CG to Hp‐CAG, involving the foveolae and intestinal metaplasia. H. pylori eradication induces a complete healing of foveolae but not of intestinal metaplasia, reducing the overall oxidative damage in the mucosa.     

Staphylococcus aureus - induced tumor necrosis factor - related apoptosis - inducing ligand expression mediates apoptosis and caspase-8 activation in infected osteoblasts

Background

Bacterial mercury resistance is based on enzymatic reduction of ionic mercury to elemental mercury and has recently been demonstrated to be applicable for industrial wastewater clean-up. The long-term monitoring of such biocatalyser systems requires a cultivation independent functional community profiling method targeting the key enzyme of the process, themerAgene coding for the mercuric reductase. We report on the development of a profiling method formerAand its application to monitor changes in the functional diversity of the biofilm community of a technical scale biocatalyzer over 8 months of on-site operation.

Results

Based on an alignment of 30merAsequences from Gram negative bacteria, conserved primers were designed for amplification ofmerAfragments with an optimized PCR protocol. The resulting amplicons of approximately 280 bp were separated by thermogradient gelelectrophoresis (TGGE), resulting in strain specific fingerprints for mercury resistant Gram negative isolates with differentmerAsequences. ThemerAprofiling of the biofilm community from a technical biocatalyzer showed persistence of some and loss of other inoculum strains as well as the appearance of new bands, resulting in an overall increase of the functional diversity of the biofilm community. One predominant new band of themerAcommunity profile was also detected in a biocatalyzer effluent isolate, which was identified asPseudomonas aeruginosa. The isolated strain showed lower mercury reduction rates in liquid culture than the inoculum strains but was apparently highly competitive in the biofilm environment of the biocatalyzer where moderate mercury levels were prevailing.

Conclusions

ThemerAprofiling technique allowed to monitor the ongoing selection for better adapted strains during the operation of a biocatalyzer and to direct their subsequent isolation. In such a way, a predominant mercury reducingPs. aeruginosastrain was identified by its unique mercuric reductase gene.     

Alpha-amylase from Bacillus licheniformis mutants near to the catalytic site: effects on hydrolytic and transglycosylation activity

The alpha-amylase from Bacillus licheniformis is the most widely used enzyme in the starch industry owing to its hyperthermostability, converting starch to medium-sized oligosaccharides. Based on sequence alignment of homologous amylases, we found a semi-conserved sequence pattern near the active site between transglycosidic and hydrolytic amylases, which suggested that hydrophobicity may play a role in modifying the transglycosylation/hydrolysis ratio. Based on this analysis, we replaced residue Val286 by Phe and Tyr in Bacillus licheniformis alpha-amylase. Surprisingly, the two resultant mutant enzymes, Val286Phe and Val286Tyr, showed two different behaviors. Val286Tyr mutant was 5-fold more active for hydrolysis of starch than the wild-type enzyme. In contrast, the Val286Phe mutant, differing only by one hydroxyl group, was 3-fold less hydrolytic than the wild-type enzyme and apparently had a higher transglycosylation/hydrolysis ratio. These results are discussed in terms of affinity of subsites, hydrophobicity and electrostatic environment in the active site. The engineered enzyme reported here may represent an attractive alternative for the starch transformation industries as it affords direct and substantial material savings and requires no process modifications.     

Kinetic analysis of artificial peptide self-replication. Part I: the homochiral case

Computational kinetic analysis of a lately discovered homochiral peptide self-replicator is presented. A 6-step kinetic model was designed that addresses the main reactions and hydrophobic interactions involved in this template-directed, autocatalytic system and that gave rise to excellent fitting of 4 previously published independent experimental series. The model sheds light on the mechanistic principle of the reaction system and illustrates directly a number of dynamic properties such as the observed autocatalytic efficiency. It was found that the dynamics are basically governed by two reversible hydrophobic interactions: between the template and a peptide fragment and between two template species. The later association was determined to be considerably more favored, which leads to the predominant presence of the catalytically inactive template dimer in the reaction system. Our results show that the involvement of a template trimer is not necessary to obtain the observed fittings.     

Peroxynitrite flux-mediated LDL oxidation is inhibited by manganese porphyrins in the presence of uric acid

We have studied the role of three Mn(III)porphyrins differing in charge, alkyl substituent length and reactivity, on LDL exposed to low fluxes of peroxynitrite (PN) in the presence of uric acid. Mn(III)porphyrins (5 microM, MnTE-2-PyP(5+), MnTnOct-2-PyP(5+), and MnTCPP(3-)) plus uric acid (300 microM) inhibited cholesteryl ester hydroperoxide formation, changes in REM as well as spared alpha- and gamma-tocopherol. MnTnOct-2-PyP(5+), the more lipophilic compound, was the most effective in protecting LDL lipids, while MnTCPP(3-) exerted the lesser protection. Mn(III)porphyrins react fast with PN ( approximately 10(5)-10(7) M(-1) s(-1)) to yield a O=Mn(IV) complex. The stoichiometry of uric acid consumption was approximately 1.7 moles per mol of PN, in agreement with reactions with both the O=Mn(IV) complex and nitrogen dioxide. A shift from an anti- to a pro-oxidant action of the Mn(III)porphyrin was observed after uric acid was significantly consumed, supporting competition reactions between LDL targets and uric acid for the O=Mn(IV) complex. Overall, the data is consistent with the catalytic reduction of PN in a cycle that involves a one electron oxidation of Mn(III) to Mn(IV) by PN followed by the reduction back to Mn(III) by uric acid. These antioxidant effects should predominate under in vivo conditions having plasma uric acid concentration range between 150 and 500 microM.     

Generalized microsporidiosis caused by Encephalitozoon sp. in a pediatric patient with Bruton's disease

We present the case of a four-year-old boy with a history of repeated upper respiratory tract infections and pyoderma. He presented fever, seizures, inability to talk, loss of swallowing, fine tremor in the upper extremities; positive bilateral Babinski reflex and quadriparesis. The diagnosis of Bruton's disease and generalized microporidiosis was based on immunologic analysis, smear tests with chromotrope R2 stain and indirect immunofluorescense with monoclonal 3B6 antibody for Encephalitozoon species in samples of spinal fluid, bronchial and paranasal sinus aspirates and stool, which were all positive. The patient was treated with albendazol during 72 days; he left the hospital in a good condition, walking, talking and able to swallow. His laboratory test controls were negative; he is followed up in the outpatient department.     

Phase I clinical trial of a recombinant canarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigen and the B7.1 co-stimulatory molecule

 The generation of cytotoxic effector T cells requires delivery of two signals, one derived from a specific antigenic epitope and one from a costimulatory molecule. A phase I clinical trial was conducted with a non-replicating canarypoxvirus (ALVAC) constructed to express both human carcinoembryonic antigen (CEA) and the B7.1 costimulatory molecule. This was the first study in cancer patients to determine if the delivery of costimulation with a tumor vaccine was feasible and improved immune responses. Three cohorts of six patients, each with advanced CEA-expressing adenocarcinomas, were treated with increasing doses of an ALVAC-CEA-B7.1 vaccine (4.5 × 10⁶, 4.5 × 10⁷, and 4.5 × 10⁸ plaque-forming units, PFU). Patients were vaccinated by intramuscular injection every 4 weeks for 3 months and monitored for side-effects, tumor growth and anti-CEA immune responses. ALVAC-CEA- B7.1 at doses up to 4.5 × 10⁸ PFU was given without evidence of significant toxicity or autoimmune reactions. Three patients experienced clinically stable disease that correlated with increasing CEA-specific precursor T cells, as shown by in vitro interferon-γ enzyme-linked immunoassay spot tests (ELISPOT). These three patients underwent repeated vaccination resulting in augmented CEA-specific T cell responses. This study represents the first use of costimulation to enhance antitumor vaccines in cancer patients. This approach resulted in CEA-specific immunity associated with stable diseases in three patients. This study also demonstrated that CEA-specific T cell responses could be sustained by repeated vaccinations. Although the number of patients was small, the addition of B7.1 to virus-based vaccines may improve immunological and stable diseases to vaccination against tumor-associated antigens with tolerable toxicity. Received: 6 May 2000 / Accepted: 13 July 2000     

Molecular evidence for selection on female color polymorphism in the damselfly Ischnura graellsii

The significance of female color polymorphism in Odonata remains controversial despite many field studies. The importance of random factors (founder effects, genetic drift and migration) versus selective forces for the maintenance of this polymorphism is still discussed. In this study, we specifically test whether the female color polymorphism of Ischnura graellsii (Odonata, Coenagrionidae) is under selection in the wild. We compared the degree of genetic differentiation based on RAPD markers (assumed to be neutral) with the degree of differentiation based on color alleles. Weir and Cockerham's theta values showed a significant degree of population differentiation for both sets of loci (RAPD and color alleles) but the estimated degree of population differentiation (theta) was significantly greater for the set of RAPD loci. This result shows that some sort of selection contributes to the maintenance of similar color morph frequencies across the studied populations. Our results combined with those of previous field studies suggest that at least in some I. graellsii populations, density-dependent mechanisms might help to prevent the loss of this polymorphism but cannot explain the similarity in morph frequencies among populations.     

Hemin is kinetically trapped in cytochrome b(5) from rat outer mitochondrial membrane

Cytochrome b(5) from the outer mitochondrial membrane of rat liver (OM cyt b(5)) is substantially more stable to thermal and chemical denaturation than cytochrome b(5) from the endoplasmic reticulum of bovine liver (microsomal, or Mc cyt b(5)). In contrast, the corresponding apoproteins have similar stability, suggesting stronger interactions between hemin and the polypeptide in OM cyt b(5). Whereas complete transfer of hemin from bovine Mc cyt b(5) to apomyoglobin at pH 5.2 takes less than 1 h, hemin transfer from OM cyt b(5) is unmeasurably slow. Coupled with the previously reported 1:1 ratio of hemin orientational isomers in OM cyt b(5), this finding suggests that the cofactor is kinetically trapped under physiologically relevant conditions. This conclusion is confirmed by (1)H NMR studies which show that the hemin isomeric ratio changes when the protein is incubated for several hours at 68 degrees C. Interestingly, the orientational isomer favored in OM cyt b(5) is the form less favored in all other known cytochromes b(5).