Formation of adducts in the reaction of glyoxal with 2'-deoxyguanosine and with calf thymus DNA

The reactions of glyoxal with 2′-deoxyguanosine and calf thymus single- and double-stranded DNA in aqueous buffered solutions at physiological conditions resulted in the formation of two previously undetected adducts in addition to the known reaction product 3-(2′-deoxy-β-d-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one (Gx-dG). The adducts were isolated and purified by reversed-phase liquid chromatography and structurally characterised by UV absorbance, mass spectrometry, ¹H and ¹³C NMR spectroscopy. The hitherto unknown adducts were identified as: 5-carboxymethyl-3-(2′-deoxy-β-d-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one (Gx₂-dG) and N²-(carboxymethyl)-9-(2′-deoxy-β-d-erythro-pentofuranosyl)-purin-6(9H)-one (Gx₁-dG). Both adducts were shown to arise from Gx-dG. Gx-dG and Gx₂-dG were found to be unstable and partly transformed to Gx₁-dG, which is a stable adduct and seems to be the end-product of the glyoxal reaction with 2′-deoxyguanosine. All adducts formed in the reaction of glyoxal with 2′-deoxyguanosine were observed in calf thymus DNA. Also in DNA, Gx₁-dG was the only stable adduct. The transformation of Gx-dG to Gx₁-dG seemed to take place in single-stranded DNA and therefore, Gx₁-dG may be a potentially reliable biomarker for glyoxal exposure and may be involved in the genotoxic properties of the compound.     

Development of porcine transgenic nuclear-transferred embryos derived from fibroblast cells transfected by the novel technique of nucleofection or standard lipofection

The aim of our study was to determine the in vitro developmental potential of porcine nuclear-transferred (NT) embryos that had been reconstructed with Tg(pWAPhGH-GFPBsd) transgene-expressing fibroblast cells. The gene construct was introduced into fibroblast cells by the novel method of nucleofection or standard lipofection. NT oocytes derived from foetal and adult dermal fibroblast cells were stimulated by either simultaneous fusion and electrical activation (Groups IA and IB) or sequential electrical and chemical activation (Groups IIA and IIB). The percentages of cloned embryos that reached the morula and blastocyst stages were 152/254 (59.8%) and 77/254 (30.3%) or 139/276 (50.4%) and 45/276 (16.3%) in Groups IA or IB, respectively. The rates of NT embryos that developed to the morula and blastocyst stages were 103/179 (57.5%) and 41/179 (22.9%) or 84/193 (43.5%) and 27/193 (14.0%) in Groups IIA and IIB, respectively. In conclusion, the in vitro developmental competences of porcine transgenic NT embryos that had been reconstructed with the Tg(pWAPhGH-GFPBsd) gene-transfected fibroblast cells were relatively high. Further, the nucleofection efficiency of all the porcine fibroblast cell lines as estimated by intra-vitam fluorescent evaluation based on the index of reporter eGFP transgene expression was nearly 100%. However, PCR analysis for transgene screening confirmed the absence of Tg(pWAPhGH-GFPBsd) fusion gene in some of the nucleofected cell lines. To our knowledge, the novel method of nucleofection is the first to transfect nuclear donor cells in the production of transgenic cloned embryos.     

Chromatographic investigation on acyl migration in betacyanins and their decarboxylated derivatives

Chemopreventive and antioxidant action of betalain pigments can differ in dependence on their stereoselective properties, therefore, it is necessary to use relevant methods for monitoring of their possible stereoisomers. Chromatographic characterisation of a group of new isomers of various 6'-O-acylated betacyanins and decarboxylated betacyanins which were generated at low concentration by intramolecular pH-dependent acyl migration was studied in aqueous solutions by HPLC separation with diode-array and mass spectrometric detection. Under alkaline conditions (pH 10.5) the rate of migration was dramatically accelerated, however, always favouring the 6'-O-position and it was much less prominent at lower pH (under 7.0). The possible products of the partial rearrangement were tentatively identified as the 3'-O- and 4'-O-acylated forms and their relative retention times were provided. In malonylated betacyanins and 17-decarboxy-betacyanins the 4'-O-forms were characterised in RP-HPLC by higher retention than the 6'-O forms, whereas the 3'-O-forms were always the most polar. In contrast, the isomerisation of hylocerenin and 17-decarboxy-hylocerenin resulted in different chromatographic profiles of the migration products. In 2-decarboxy- and 2,17-bidecarboxy-betacyanins the 3'-O- and 4'-O-acylated forms eluted always before the 6'-O-acylated betacyanins. The investigations on acyl migration in isolated 4'-O-malonyl-betanin confirmed the strong tendency of reverse acyl migration (4'-->6') and also partial 4'-->3' rearrangement which were leading to the final monoester regioisomeric distribution (%) close to 87:7:6 (6'-O-, 4'-O-, 3'-O-).     

Identification and molecular characterization of <Emphasis Type="Italic">Theileria</Emphasis> sp. infecting red deer (<Emphasis Type="Italic">Cervus elaphus</Emphasis>) in northwestern Poland

Piroplasms from Theileria genus were detected in blood and spleen of red deer Cervus elaphus culled during the months of September 2004–January 2005 in northwestern Poland. The polymerase chain reaction revealed the presence of Theileria deoxyribonucleic acid in 88% (36 of 41) of the animals examined. Molecular characterization of the parasites based on large piece of 18S ribosomal ribonucleic acid gene containing hypervariable region V4 showed 99.9% similarity to two Theileria spp. sequences: Theileria sp. 3185/02 and Theileria capreoli BAB1158. Phylogenetic analysis confirmed that the three isolates cluster together with high bootstrap support. It is supposed that those pathogens can be classified as one group characteristic for the Eurasian continent, contrary to protozoon of Theileria from the T. cervi group, which are often found on the North American continent and can also infect the representatives of Cervidae. In conclusion, this study suggested that free-living C. elaphus in northwestern Poland are a competent reservoir of Theileria sp. ZS T04 C.e. parasites, although the vector of the piroplasms is still unknown.     

Gastrointestinal tract metabolism of young turkeys fed diets supplemented with pure nystose or a fructooligosaccharide mixture

In a four-week experiment on 60 7-day-old BUT-9 male turkeys the effects of dietary fructooligosaccharides (pure nystose and a fructooligosaccharide mixture) supplemented at 1 and 2%, were studied on ileal and caecal metabolism. The control carbohydrate was cellulose, added also at 1 or 2%. Each dietary treatment consists of 10 birds kept individually. The average degree of polymerisation of the nystose and oligofructose preparation amounted to 2.9 and 4.1, respectively. The addition of nystose significantly decreased the pH value and viscosity in the ileal contents compared with the cellulose treatment. On the other hand, the oligofructose preparation increased the activity of sucrase and lactase in the ileal mucosal by 30-60% and 33-47%, respectively. Both fructan preparations similarly acidified the caecal and colonic digesta (by 0.2-0.4 pH units) as well as diminished the activity of bacterial harmful beta-glucuronidase (by 24-40%), but only nystose caused an enlargement of the caeca and effectively reduced caecal ammonia concentration, especially at a higher dose. Oligofructose supplementation at 2% caused a 3.5-fold increase of bacterial activity of alph- and beta-galactosidase, while 2% nystose resulted in 1.7 and 3 times higher alpha- and beta-glucosidases activities, respectively. Compared to oligofructose, dietary nystose increased propionic and decreased butyric fermentation in caeca. Nystose and oligofructose preparations added at 2% reduced the triacylglycerol concentration in the serum in comparison to the addition of 2% cellulose by 46 and 25%, respectively. Beside the fact that dietary levels of supplementation were of great importance, the results indicated that even small difference in the length of carbohydrate chain may cause different physiological responses.     

Decrease in sex difference in premature mortality during system transformation in Poland

In recent years, sex differences in mortality have followed diverse patterns of change in developed countries. As there is no analogous evidence from Poland, the aim of this study was to describe the pattern of change in excess male mortality among Polish inhabitants aged 35-64 during 1995-2002, when the major socioeconomic transformation occurred, and compare it with sex differences in mortality observed in the late 1980s. During the study period, excess male mortality decreased significantly, independently of age and educational level. The reduction in mortality was observed in both sexes, but its magnitude was greater in men. These changes resulted mainly from a reduction in mortality rates due to cardiovascular disease and lung cancer in males and a concomitant increase in mortality rates due to lung cancer and suicides in females. Although, in general, excess male mortality decreased, social gradients related to this phenomenon increased. Subjects (in particularly men) who had graduated from university benefited the most, their magnitude of reduction in mortality rates being the greatest. Changes in social environment during the transformation period in Poland are suggested as major determinants of these changes, but further studies are needed.     

Growth hormone therapy in boy with panhypopituitarism may induce pilomatricoma recurrence: case report

Pilomatricoma is usually a solitary subcutaneous nodule. Recurrence of the nodule after surgical excision is very rare. Pilomatricoma occurrence in patients with growth hormone (GH) deficiency has not been reported, yet. We report a 14-year-old boy with pilomatricoma and panhypopituitarism. After GH therapy had been started, we observed two relapses of previously completely excised pilomatricoma in the same location and a new pilomatricoma formation on the chin.     

Estimation of binding parameters for the protein–protein interaction using a site-directed spin labeling and EPR spectroscopy

Sensitivity of the electron paramagnetic resonance (CW EPR) to molecular tumbling provides potential means for studying processes of molecular association. It uses spin-labeled macromolecules, whose CW EPR spectra may change upon binding to other macromolecules. When a spin-labeled molecule is mixed with its liganding partner, the EPR spectrum constitutes a linear combination of spectra of the bound and unbound ligand (as seen in our example of spin-labeled cytochrome c ₂ interacting with cytochrome bc ₁ complex). In principle, the fraction of each state can be extracted by the numerical decomposition of the spectrum; however, the accuracy of such decomposition may often be compromised by the lack of the spectrum of the fully bound ligand, imposed by the equilibrium nature of molecular association. To understand how this may affect the final estimation of the binding parameters, such as stoichiometry and affinity of the binding, a series of virtual titration experiments was conducted. Our non-linear regression analysis considered a case in which only a single class of binding sites exists, and a case in which classes of both specific and non-specific binding sites co-exist. The results indicate that in both models, the error due to the unknown admixture of the unbound ligand component in the EPR spectrum causes an overestimation of the bound fraction leading to the bias in the dissociation constant. At the same time, the stoichiometry of the binding remains relatively unaffected, which overall makes the decomposition of the EPR spectrum an attractive method for studying protein–protein interactions in equilibrium. Our theoretical treatment appears to be valid for any spectroscopic techniques dealing with overlapping spectra of free and bound component.