Variation in tolerance to drought among Scandinavian populations of <Emphasis Type="Italic">Arabidopsis lyrata</Emphasis>

The ability to cope with water limitation influences plant distributions, and several plant traits have been interpreted as adaptations to drought stress. In Scandinavia, the perennial herb Arabidopsis lyrata occurs in open habitats that differ widely in climate and water availability in summer, suggesting differential selection on drought-related traits. We conducted two greenhouse experiments to examine differentiation in drought response traits among six Scandinavian populations, and to determine whether leaf trichomes confer protection against drought. We quantified tolerance to drought as fitness (survival and biomass of survivors) when exposed to drought relative to fitness under non-drought conditions. Two Swedish populations from shores along the Bothnian Bay had higher tolerance to drought than four riverbed populations from Norway. Under conditions of drought, the shore populations experienced less leaf damage compared to the riverbed populations, and their survival and biomass were less reduced relative to non-drought conditions. Across populations, tolerance to drought was positively related to leaf mass per area and negatively related to flowering propensity and proportion roots, but not related to plant size at the initiation of the drought treatment. In populations polymorphic for trichome production, trichome-producing plants were more tolerant to drought than glabrous plants. The results suggest that both leaf morphology and life-history traits contribute to differential drought response in natural populations of A. lyrata, and that this system offers excellent opportunities for examining the adaptive value and genetic basis of drought-related traits.     

Immunohistochemical detection of tankyrase 2 in human breast tumors and normal renal tissue

Tankyrase, which functions at telomeres and other cellular compartments, is thought to be a positive regulator of telomerase; its isoenzyme tankyrase 2 has been cloned as a putative cancer antigen. This pilot immunohistochemical study was designed to examine whether tumors overexpress tankyrase 2. An antibody was generated by using synthetic peptide specific for tankyrase 2 and was tested by Western blot and immunocytochemically; no cross-reaction with isoenzyme 1 was revealed. Among tissue sections, two tumors of 18 specimens were positive for tankyrase 2. Others were negative or contained barely detectable protein. The surrounding normal tissues were negative. Tankyrase 2 was also revealed in epithelial cells of a limited number of normal renal tubules, whereas other renal tissues were negative. These data suggest that tankyrase 2 is not expressed ubiquitously in human tissues. To determine whether the up-regulation of tankyrase 2 is associated with tissue regeneration and cell proliferation, we compared the activity and concentration of the enzyme in a model human embryonic kidney cell line 293 arrested by serum deprivation and restimulated with serum. The serum-starved quiescent cell culture exhibited detectable protein as did the proliferating cells; enzyme activity dramatically increased in the latter. We conclude that pathologic overexpression of tankyrase 2 in some tumors may be a result of the cancer-related adaptation of the malignant cells dependent on tankyrase activity. Under normal conditions, the protein might be up-regulated during cell differentiation and also posttranslationally in proliferating cells. This study was supported by the Russian Foundation for Basic Research (grant no. 03-04-48835, principal investigator A.N. Kuimov)     

Beyond gastric acid reduction: proton pump inhibitors induce heme oxygenase-1 in gastric and endothelial cells

Proton pump inhibitors (PPIs) have been demonstrated to prevent gastric mucosal injury by mechanisms independent of acid inhibition. Here we demonstrate that both omeprazole and lansoprazole protect human gastric epithelial and endothelial cells against oxidative stress. This effect was abrogated in the presence of the heme oxygenase-1 (HO-1) inhibitor ZnBG. Exposure to either PPI resulted in a strong induction of HO-1 expression on mRNA and protein level, and led to an increased activity of this enzyme. Expression of cyclooxygenase isoforms 1 and 2 remained unaffected, and COX-inhibitors did not antagonize HO-1 induction by PPIs. Our results suggest that the antioxidant defense protein HO-1 is a target of PPIs in both endothelial and gastric epithelial cells. HO-1 induction might account for the gastroprotective effects of PPIs independently of acid inhibition, especially in NSAID gastropathy. Moreover, our findings provide additional perspectives for a possible but yet unexplored use of PPIs in vasoprotection.     

Streptococcal pyrogenic exotoxin B cleaves properdin and inhibits complement-mediated opsonophagocytosis

Streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, is an important virulence factor in group A streptococcal (GAS) infection. The reduction of phagocytic activity by SPE B may help prevent bacteria from being ingested. In this study, we investigated the mechanism SPE B uses to enable bacteria to resist opsonophagocytosis. Using Western blotting and an affinity column immobilized with SPE B, we found that both SPE B and C192S, an SPE B mutant lacking protease activity, bound to serum properdin, and that SPE B, but not C192S, degraded serum properdin. Further study showed that SPE B-treated, but not C192S-treated, serum blocked the alternative complement pathway. Reconstitution of properdin into SPE B-treated serum unblocked the alternative pathway. GAS opsonized with SPE B-treated serum was more resistant to neutrophil killing than GAS opsonized with C192S-treated or normal serum. These results suggest that a novel SPE B mechanism, one which degrades serum properdin, enables GAS to resist opsonophagocytosis.     

A crystallographic and spectroscopic study on the effect of X-ray radiation on the crystal structure of Melanocarpus albomyces laccase

Laccases (p-diphenol dioxygen oxidoreductases) belong to the family of blue multicopper oxidases, which catalyse the four-electron reduction of dioxygen to water concomitantly through the oxidation of substrate molecules. Blue multicopper oxidases have four coppers, a copper (T1) forming a mononuclear site and a cluster of three coppers (T2, T3, and T3') forming a trinuclear site. Because X-rays are known to liberate electrons during data collection and may thus affect the oxidation state of metals, we have investigated the effect of X-ray radiation upon the crystal structure of a recombinant laccase from Melanocarpus albomyces through the use of crystallography and crystal absorption spectroscopy. Two data sets with different strategies, a low and a high-dose data set, were collected at synchrotron. We have observed earlier that the trinuclear site had an elongated electron density amidst coppers, suggesting dioxygen binding. The low-dose synchrotron structure showed similar elongated electron density, but the high-dose X-ray radiation removed the bulk of this density. Therefore, X-ray radiation could alter the active site of laccase from M. albomyces. Absorption spectra of the crystals (320, 420, and 590nm) during X-ray radiation were measured at a home laboratory. Spectra clearly showed how that the band at 590nm had vanished, resulting from the T1 copper being reduced, during the long X-ray measurements. The crystal colour changed from blue to colourless. Absorptions at 320 and 420nm seemed to be rather permanent. The absorption at 320nm is due to the T3 coppers and it is proposed that absorption at 420nm is due to the T2 copper when dioxygen or a reaction intermediate is close to this copper.     

Nonhost resistance of barley is successfully manifested against Magnaporthe grisea and a closely related Pennisetum-infecting lineage but is overcome by Magnaporthe oryzae

Magnaporthe oryzae is a major pathogen of rice (Oryza sativa L.) but is also able to infect other grasses, including barley (Hordeum vulgare L.). Here, we report a study using Magnaporthe isolates collected from other host plant species to evaluate their capacity to infect barley. A nonhost type of resistance was detected in barley against isolates derived from genera Pennisetum (fontaingrass) or Digitaria (crabgrass), but no resistance occurred in response to isolates from rice, genus Eleusine (goosegrass), wheat (Triticum aestivum L.), or maize (Zea mays L.), respectively. Restriction of pathogen growth in the nonhost interaction was investigated microscopically and compared with compatible interactions. Real-time polymerase chain reaction was used to quantify fungal biomass in both types of interaction. The phylogenetic relationship among the Magnaporthe isolates used in this study was investigated by inferring gene trees for fragments of three genes, actin, calmodulin, and beta-tubulin. Based on phylogenetic analysis, we could distinguish different species that were strictly correlated with the ability of the isolates to infect barley. We demonstrated that investigating specific host interaction phenotypes for a range of pathogen isolates can accurately highlight genetic diversity within a pathogen population.     

Sequence stability of the T-DNA – plant junctions in tissue culture in Arabidopsis transgenic lines

The stability of the inserted transgenes and particularly the junction regions of transgenic events is a concern of food labeling, traceability and post release monitoring, as these regions are used for development of event-specific DNA-based detection methods. During the standard agricultural breeding practices, the transgenic lines can be exposed to completely different conditions than those in the laboratory environment. Some of these conditions have the potential to affect the stability of the transgenic locus and the surrounding DNA. As tissue culture is recognized as a stressful and mutagenic factor, we have analyzed the effect of this process on the stability of the junction regions at nucleotide level in five Arabidopsis thaliana transgenic lines in comparison with the respective integration loci in ColO and C24 ecotypes. No indication of any kind of alteration at nucleotide level of the junctions was found. The relevance of the stability of the plant–T-DNA junction regions for application of the DNA-based methods in commercial transgenic plants is discussed.     

Structures of the core oligosaccharide and O-units in the R- and SR-type lipopolysaccharides of reference strains of Pseudomonas aeruginosa O-serogroups

Highly phosphorylated core oligosaccharides and those substituted with one O-antigen repeating unit were obtained by mild acid degradation or strong alkaline hydrolysis of lipopolysaccharide samples from 23 reference strains representing all Pseudomonas aeruginosa O-serogroups. Studies by high-resolution electrospray ionization mass spectrometry and two-dimensional NMR spectroscopy revealed both conserved and variable structural features of the lipopolysaccharides of various O-serogroups. The upstream terminal saccharide of the O-antigen, which contributes most to the immunospecificity of the bacteria, was defined in 11 from a total of 13 O-serogroups. The data obtained link together the known biosynthesis pathways, genetics and serology of the P. aeruginosa lipopolysaccharide.     

The role of enzyme dynamics and tunnelling in catalysing hydride transfer: studies of distal mutants of dihydrofolate reductase

Residues M42 and G121 of Escherichia coli dihydrofolate reductase (ecDHFR) are on opposite sides of the catalytic centre (15 and 19 A away from it, respectively). Theoretical studies have suggested that these distal residues might be part of a dynamics network coupled to the reaction catalysed at the active site. The ecDHFR mutant G121V has been extensively studied and appeared to have a significant effect on rate, but only a mild effect on the nature of H-transfer. The present work examines the effect of M42W on the physical nature of the catalysed hydride transfer step. Intrinsic kinetic isotope effects (KIEs), their temperature dependence and activation parameters were studied. The findings presented here are in accordance with the environmentally coupled hydrogen tunnelling. In contrast to the wild-type (WT), fluctuations of the donor-acceptor distance were required, leading to a significant temperature dependence of KIEs and deflated intercepts. A comparison of M42W and G121V to the WT enzyme revealed that the reduced rates, the inflated primary KIEs and their temperature dependences resulted from an imperfect potential surface pre-arrangement relative to the WT enzyme. Apparently, the coupling of the enzyme's dynamics to the reaction coordinate was altered by the mutation, supporting the models in which dynamics of the whole protein is coupled to its catalysed chemistry.