Cadmium-induced apoptosis of primary epithelial lung cells: Involvement of Bax and p53, but not of oxidative stress

The lung is a target organ for cadmium (Cd) toxicity. Apoptosis induced by cadmium acetate (CdAc) was studied in alveolar type 2 cells and Clara cells isolated from rat lung. Relatively low concentrations of CdAc (1–10 μmol/L) induced apoptosis after exposure for 20 h. Type 2 cells were more sensitive than Clara cells to Cd-induced apoptosis and loss of cell viability. On exposure to 10 μmol/L CdAc, the levels of the apoptosis-modulating proteins p53 and Bax were increased at 2 h and 5–12 h, respectively. The expression of p53 preceded the expression of Bax and the apoptotic process. The exposure to 10 μmol/L CdAc did not significantly increase the formation of cellular reactive oxygen species (ROS). However, after exposure to a high concentration of CdAc (100 μmol/L), a 30% increase of the ROS level was observed. No significant nitric oxide production was measured following CdAc exposure. Catalase, superoxide dismutase, dimethyl sulfoxide, or tetramethylthiourea did not protect against Cd-induced apoptosis. In conclusion, the results show that Clara cells and type 2 cells are sensitive to Cd-induced apoptosis. Increased levels of p53 and Bax are suggested to be involved in the apoptosis. The apoptosis did not appear to be mediated by oxidative pathways. This revised version was published online in August 2006 with corrections to the Cover Date.     

Plasmid-borne AmpC beta-lactamases

Historically, it was thought that ampC genes encoding class C beta-lactamases were located solely on the chromosome but, within the last 12 years, an increasing number of ampC genes have been found on plasmids. These have mostly been acquired by ampC-deficient pathogenic bacteria, which consequently are supplied with new and additional resistance phenotypes. This review discusses the phylogenetic origin of the plasmid-encoded AmpC beta-lactamases, their occurrence, and mode of spread, as well as their hydrolytic properties.     

Silver staining of proteins on electroblotting membranes and intensification of silver staining of proteins separated by polyacrylamide gel electrophoresis

A fast and convenient method for silver staining of proteins on electroblotting membranes was developed based on Gallyas' histochemical intensifier and applied to human endothelial cell proteins separated by one- and two-dimensional electrophoresis and electroblotted to polyvinyl difluoride membranes. The method allowed detection of proteins on membranes with a sensitivity equal to the sensitivity of the most sensitive silver-staining protocols for electrophoresis gels. Also, the method was compatible with preceding immunostaining on the same membrane. Furthermore, an intensifying method for proteins in silver-stained SDS-PAGE gels was developed based on Gallyas' histochemical intensifier. This method was applied to proteins separated by one- and two-dimensional gel electrophoresis and visualized by one of several silver-staining methods. Maximal intensification was achieved for the less sensitive but fast acidic silver-staining protocols, but even for the very sensitive alkaline protocols a significant increase in signal to noise ratio was obtained. In particular, negatively stained or invisible proteins on the silver-stained gels were found to be visualized by the Gallyas stain. Proteins from silver-stained and Gallyas-stained gels were identified by mass spectrometry, and the intensification procedure was fully compatible with mass spectrometry.     

Gliadin T cell epitope selection by tissue transglutaminase in celiac disease. Role of enzyme specificity and pH influence on the transamidation versus deamidation process

Tissue transglutaminase (TG2) can modify proteins by transamidation or deamidation of specific glutamine residues. TG2 has a major role in the pathogenesis of celiac disease as it is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides that are recognized by CD4(+), DQ2-restricted T cells from the celiac lesions. Capillary electrophoresis with fluorescence-labeled gliadin peptides was used to separate and quantify deamidated and transamidated products. In a competition assay, the affinity of TG2 to a set of overlapping gamma-gliadin peptides was measured and compared with their recognition by celiac lesion T cells. Peptides differed considerably in their competition efficiency. Those peptides recognized by intestinal T cell lines showed marked competition indicating them as excellent substrates for TG2. The enzyme fine specificity of TG2 was characterized by synthetic peptide libraries and mass spectrometry. Residues in positions -1, +1, +2, and +3 relative to the targeted glutamine residue influenced the enzyme activity, and proline in position +2 had a particularly positive effect. The characterized sequence specificity of TG2 explained the variation between peptides as TG2 substrates indicating that the enzyme is involved in the selection of gluten T cell epitopes. The enzyme is mainly localized extracellularly in the small intestine where primary amines as substrates for the competing transamidation reaction are present. The deamidation could possibly take place in this compartment as an excess of primary amines did not completely inhibit deamidation of gluten peptides at pH 7.3. However, lowering of the pH decreased the reaction rate of the TG2-catalyzed transamidation, whereas the rate of the deamidation reaction was considerably increased. This suggests that the deamidation of gluten peptides by TG2 more likely takes place in slightly acidic environments.     

Design and synthesis of 4,5-disubstituted-thiophene-2-amidines as potent urokinase inhibitors

A study of the S1 binding of lead 5-methylthiothiophene amidine 3, an inhibitor of urokinase-type plasminogen activator, was undertaken by the introduction of a variety of substituents at the thiophene 5-position. The 5-alkyl substituted and unsubstituted thiophenes were prepared using organolithium chemistry. Heteroatom substituents were introduced at the 5-position using a novel displacement reaction of 5-methylsulfonylthiophenes and the corresponding oxygen or sulfur anions. Small alkyl group substitution at the 5-position provided inhibitors equipotent with but possessing improved solubility.     

Salmonellae in avian wildlife in Norway from 1969 to 2000

Postmortem records of wild-living birds in Norway with laboratory-confirmed findings of salmonella infection were summarized for the period from 1969 to 2000. Salmonella spp. were isolated from 470 birds belonging to 26 species. The salmonella-positive birds included 441 small passerines, 15 gulls, 5 waterfowl, 4 birds of prey, 3 doves, and 2 crows. The bullfinch (Pyrrhula pyrrhula) was by far the most frequently recorded species (54% of the cases). Salmonella enterica serover Typhimurium was recovered from all cases except from one hooded crow (Corvus corone), which yielded serovar Paratyphi-B var. Java. Variant O:4,12 comprised 96% (451 cases) of all serovar Typhimurium isolates, including all the passerines, while variant O:4,5,12 accounted for the remaining 4% (18 cases). The occurrence of salmonellae in small passerines showed a distinct seasonality, with a peak in February and March. Plasmid profile analysis of 346 isolates of serovar Typhimurium O:4,12 detected six profiles, of which two comprised 66 and 28% of the isolates, respectively. Phage typing of 52 randomly selected isolates of serovar Typhimurium O:4,12 from passerines detected four types: DT 40 (54%), U277 (35%), DT 99 (6%), and DT 110 (4%).     

Mechanisms and rates of bacterial colonization of sinking aggregates

Quantifying the rate at which bacteria colonize aggregates is a key to understanding microbial turnover of aggregates. We used encounter models based on random walk and advection-diffusion considerations to predict colonization rates from the bacteria's motility patterns (swimming speed, tumbling frequency, and turn angles) and the hydrodynamic environment (stationary versus sinking aggregates). We then experimentally tested the models with 10 strains of bacteria isolated from marine particles: two strains were nonmotile; the rest were swimming at 20 to 60 microm s(-1) with different tumbling frequency (0 to 2 s(-1)). The rates at which these bacteria colonized artificial aggregates (stationary and sinking) largely agreed with model predictions. We report several findings. (i) Motile bacteria rapidly colonize aggregates, whereas nonmotile bacteria do not. (ii) Flow enhances colonization rates. (iii) Tumbling strains colonize aggregates enriched with organic substrates faster than unenriched aggregates, while a nontumbling strain did not. (iv) Once on the aggregates, the bacteria may detach and typical residence time is about 3 h. Thus, there is a rapid exchange between attached and free bacteria. (v) With the motility patterns observed, freely swimming bacteria will encounter an aggregate in <1 day at typical upper-ocean aggregate concentrations. This is faster than even starving bacteria burn up their reserves, and bacteria may therefore rely solely on aggregates for food. (vi) The net result of colonization and detachment leads to a predicted equilibrium abundance of attached bacteria as a function of aggregate size, which is markedly different from field observations. This discrepancy suggests that inter- and intraspecific interactions among bacteria and between bacteria and their predators may be more important than colonization in governing the population dynamics of bacteria on natural aggregates.     

Engineering of phytase for improved activity at low pH

For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles of Aspergillus fumigatus and consensus phytases. Decreasing the negative surface charge of the A. fumigatus Q27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH of A. niger NRRL 3135 phytase, A. niger pH 2.5 acid phosphatase, and Peniophora lycii phytase. Site-directed mutagenesis confirmed that, in A. fumigatus wild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues of A. niger phytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in both A. fumigatus and consensus phytase backbones), as well as the S140Y D141G double mutation (in A. fumigatus phytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.