Antioxidant defense mechanisms of cereal aphids based on ascorbate and ascorbate peroxidase

Effects of plant o-dihydroxyphenols on ascorbate (ASA) content and ascorbate peroxidase (APOX) activity in the tissues of the grain aphid Sitobion avenae and the bird cherry-oat aphid Rhopalosiphum padi were studied. Among the aphid morphs, the highest ASA content and APOX activity were noted for larvae and the lowest for wingless apterae. When exposed to o-dihydroxyphenols, aphids of both species contained significantly lower concentrations of ASA and higher APOX activity than the controls. Among the studied compounds, caffeic acid had the strongest effect on ASA-based antioxidant responses in that caffeic acid caused a 5-fold decrease of ASA in aphid tissues. The influences of the plant o-dihydroxyphenols on antioxidant defense mechanisms within the cereal aphid species are discussed.     

Regional distribution of heterozygous 657del5 mutation carriers of theNBS1 gene in Wielkopolska province (Poland)

The homozygous 657del5 mutation, called Slavic mutation, of the NBS1 gene, causes the Nijmegen Breakage Syndrome (NBS). This syndrome is connected with a high incidence of malignancies in early childhood. A high frequency of NBS heterozygotes was found among patients with melanoma, breast, ovary and prostate cancer. The aim of our research was to determine the frequency of 657del5 mutation of the NBS1 gene in the population of Wielkopolska province. For this purpose, we analysed blood samples from anonymous Guthrie cards. In a group of 2090 newborns from the whole province, we found 16 heterozygous mutation carriers. The frequency of 1/131 is higher than 1/190 reported for populations from other regions in Poland. We observed differential regional distribution of heterozygous 657del5 mutation carriers within the province: among 464 samples from the eastern part of Wielkopolska we found 6 carriers (1/77), in contrast to the southern part without any carrier among 625 samples analysed. The high mean frequency of heterozygous 657del5 mutation (1/131) in Wielkopolska province may be associated with cancer incidence in this region.     

Does DNA methylation pattern mark generative development in winter rape?

In this paper we report on changes in DNA methylation pattern in rape apices and leaves during transition from vegetative to reproductive stage due to grafting and/or vernalization. Grafted plants of winter rape (Brassica napus L., var. "Górczański") (stock from vernalized, scion from non-vernalized plants) were used together with vernalized non-grafted plants. In addition, methylation status was determined also in spring rape (var. "M?ochowski") grown under normal and low temperature. The methylation-sensitive amplification polymorphism (MSAP) method with EcoRI/MspI and EcoRII/HpaII restriction enzymes was employed. The majority (ca. 68%) of analyzed loci (566 in winter and 551 in spring rape) were monomorphic, i.e. did not undergo methylation. Both cultivars showed a similar degree of methylation. 188 loci in winter and 176 in spring cultivars expressed changes in the methylation pattern. All differentially amplified fragments resulted from either full methylation of an internal cytosine or from hemi-methylation of an external cytosine. A pair-wise comparison showed that a similar number of loci underwent development-related methylation changes in apices of the winter and spring rape. The majority (80%) of changes were demethylation events in generative (vernalized) apices of the winter cultivar. However, an increased number of demethylated loci was detected in vernalized apices in comparison with generative, non-vernalized ones. In apices of vegetative and generative grafted plants the same number of demethylation events was observed. Overall, 10 MSAP loci were detected that expressed methylation changes in vernalized apices only; among them 7 loci underwent demethylation after vernalization and remained methylated in both vegetative and generative non-vernalized stage. Only 1 locus was demethylated in generative non-vernalized apices. Thus, most of demethylation events can be ascribed to vernalization and not to the generative stage. In leaves of winter rape methylation and demethylation events occurred with similar frequency, while in the spring cultivar more demethylation events were detected. The results show that during vernalization and transition to the generative stage different sets of genes are activated.     

Hypoglycemic potency of novel trivalent chromium in hyperglycemic insulin-deficient rats

Two sources of chromium III, "chromium 454" and "chromium picolinate," were tested in insulin-deficient Streptozocin-treated diabetic rats. This model was selected in order to evaluate the possibility of any hypoglycemic potency of chromium in a relative absence of blood insulin concentration. Three weeks of the treatment with CRC454 and CrP resulted in a 38% and 11% reduction of blood glucose levels, respectively. Body weight gains were equally improved by both treatments. Blood levels of CK, ALT and AST were significantly reduced by CRC454 and CrP. These results might suggest that any hypoglycemic effect of trivalent chromium under insulin-deficient conditions could be largely dependent upon the type of chromium agent and associated characteristics such as solubility and bioavalibility. In contrast, improvement of body weight gains and blood levels of CK, AST and ALT seems to be less dependent on the type of chromium compound under these experimental conditions. In conclusion, CRC454 showed significant reduction of hyperglycemia under insulin-deficient conditions.     

Identification of a new subfamily of HNH nucleases and experimental characterization of a representative member, HphI restriction endonuclease

The restriction endonuclease (REase) R. HphI is a Type IIS enzyme that recognizes the asymmetric target DNA sequence 5'-GGTGA-3' and in the presence of Mg(2+) hydrolyzes phosphodiester bonds in both strands of the DNA at a distance of 8 nucleotides towards the 3' side of the target, producing a 1 nucleotide 3'-staggered cut in an unspecified sequence at this position. REases are typically ORFans that exhibit little similarity to each other and to any proteins in the database. However, bioinformatics analyses revealed that R.HphI is a member of a relatively big sequence family with a conserved C-terminal domain and a variable N-terminal domain. We predict that the C-terminal domains of proteins from this family correspond to the nuclease domain of the HNH superfamily rather than to the most common PD-(D/E)XK superfamily of nucleases. We constructed a three-dimensional model of the R.HphI catalytic domain and validated our predictions by site-directed mutagenesis and studies of DNA-binding and catalytic activities of the mutant proteins. We also analyzed the genomic neighborhood of R.HphI homologs and found that putative nucleases accompanied by a DNA methyltransferase (i.e. predicted REases) do not form a single group on a phylogenetic tree, but are dispersed among free-standing putative nucleases. This suggests that nucleases from the HNH superfamily were independently recruited to become REases in the context of RM systems multiple times in the evolution and that members of the HNH superfamily may be much more frequent among the so far unassigned REase sequences than previously thought.     

Helicobacter pylori cagA Gene Polymorphism Affects the Total Antioxidant Capacity of Human Saliva

Background:  We aimed to evaluate the total antioxidant capacity (TAC) of saliva in healthy Helicobacter pylori -positive and negative saliva individuals.
Materials and Methods:  A total of 102 human saliva samples were checked for the presence of H. pylori DNA ( ureA and cagA gene fragments).
TAC of saliva was estimated by ABTS radical cation (ABTS^{• +} ) decolorization assay.
Results:  PCR analysis revealed that 36 subjects were ureA-/cagA- , 24 were ureA+/cagA- and 42 were ureA+/cagA+ . Smoking habits had no evident effect on H. pylori infection.
We found that TAC of the ureA-/cagA- material, after 10 seconds reaction reflecting fast-reacting antioxidants, was significantly higher than of ureA+/cagA- and ureA+/cagA+ samples (p < .01 and p < .001, respectively). Similar results were obtained for reaction time of 3 minutes measuring slow-reacting antioxidants (p < .001). We also estimated ureA+/cagA- and ureA+/cagA+ samples alone and reported a statistically significant decrease in the TAC_3min value of ureA+/cagA+ compared with ureA+/cagA- samples (p < .05).
Conclusions:  Our data demonstrated that altered redox equilibrium may be associated with more frequent occurrence of H. pylori in the saliva samples.     

Interaction of selected divalent metal ions with human ataxin-3 Q36

The mode of interaction of ataxin-3 Q36 (AT-3 Q36) with selected endogenous and exogenous metal ions, namely, Zn²⁺, Cu²⁺, Ni²⁺, and Cd²⁺, was examined. Metal-ion-induced structural changes of the protein were monitored by fluorescence as well as Fourier transform Raman spectroscopy. We found that the cations tested lead to a decrease in α-helical content and a concurrent increase in β-sheet as well as undefined (β-turn and random-coil) structures. The most evident effect was observed for copper and nickel cations. After titration with these cations, the AT3 Q36 secondary structure content (27% α-helices in the presence of either ion, 31 and 27% β-sheets for Cu²⁺ and Ni²⁺, respectively) was similar to that observed for the aggregated form of the protein (27% α-helices, 36% β-sheets). Using the 1-anilinonaphthalene-8-sulfonate hydrophobic fluorescence probe, we showed that the presence of the metal ions tested led to the formation of solvent-exposed hydrophobic patches of AT-3 Q36, and that such an effect decreased with increasing ionic radius.