Altered mouse leukemia L1210 thymidylate synthase, associated with cell resistance to 5-fluoro-dUrd, is not mutated but rather reflects posttranslational modification

Thymidylate synthase purified from 5-fluoro-dUrd-resistant mouse leukemia L1210 cells (TSr) was less sensitive to slow-binding inhibition by 5-fluoro-dUMP than the enzyme from the parental cells (TSp), both enzyme forms differing also in sensitivity to several other dump analogues, apparent molecular weights of monomer and dimer, and temperature dependence of the catalyzed reaction. Direct sequencing of products obtained from RT-PCR, performed on total RNA isolated from the parental and 5-fluoro-dUrd-resistant cells, proved both nucleotide sequences to be identical to the mouse thymidylate synthase coding sequence published earlier (NCBI protein database access no. NP_067263). This suggests that the altered properties of TSr are caused by a factor different than protein mutation, presumably posttranslational modification. As a possibility of rat thymidylate synthase phosphorylation has been recently demonstrated (Samsonoff et al. (1997) J Biol Chem 272: 13281), the mouse enzyme amino-acid sequence was analysed, revealing several potential phosphorylation sites. In order to test possible influence of the protein phosphorylation state on enzymatic properties, endogenous TSp and TSr were purified in the presence of inhibitors of phosphatases. Although both enzyme forms were phosphorylated, as shown by electrophoretical separation followed by phosphoprotein detection, the extent of phosphorylation was apparently similar. However, the same two purified enzyme preparations, compared to the corresponding preparations purified in the absence of phosphatase inhibitors, showed certain properties, including sensitivity to the slow-binding inhibition by FdUMP, altered. Thus properties dependence on phosphorylation was indicated.     

Body mass and measurements of the European bison during postnatal development

Body mass of the European bisonBison bonasus (Linnaeus, 1758) was determined for 152 animals (86 males, 66 females) from captive breeding centre in Białowieża and for 492 (248, 244) animals from the free-ranging herd in the Białowieża Forest. Body measurements were taken for a total of 472 bison (204, 268). Age-related changes in body mass and measurements of 375 bison (165, 210) were analysed. Bison body mass was significantly correlated with age. Captive bison were heavier than those from a free-ranging herd. Measurements showed the changes in bison body structure in the postnatal development. All measurements were highly correlated with age and mass of the animals and increased most intensively during the first year of life. Sex-related differences in body mass and measurements were most pronounced in bison ≥ 3 years old. Physical development of females ends at the age of 5 years, and that of males at the age of 7 years.     

Effect of maximal-intensity exercise on systemic nitro-oxidative stress in men and women

Objectives: The aim of this study was to test the hypotheses: (1) there is a negative correlation between protein and lipid oxidative damage following maximal-intensity exercise, and oxygen uptake and work intensity (%VO_2max) at the respiratory compensation point (RCP) in women and men; (2) nitro-oxidative stress following maximal-intensity exercise results from the intensification of anaerobic processes and muscle fibre micro-damage.

Methods: Study participants comprised 20 women (21.34±1.57 years) and 20 men (21.97±1.41 years) who performed a treadmill incremental test (IT); VO_2max: 45.08?±?0.91 and 57.38?±?1.22?mL?kg^?1?min^?1 for women and men, respectively. The oxidized low-density lipoprotein (ox-LDL), 3-nitrotyrosine (3-NT) concentration and creatine kinase (CK) as well as lactate dehydrogenase (LDH) activity were measured in the blood serum, and total antioxidative capacity (TAC) and lactate concentration (Lac) were determined in blood plasma before and after IT.

Results: After the IT, increases in ox-LDL, 3-NT, CK, and LDH were seen in both groups (P?P?P?Conclusions: The gain of ox-LDL and 3-NT following maximal-intensity exercise is independent of VO_2max, oxygen consumption and exercise intensity at RCP. This increase of ox-LDL and 3-NT is indicative of similar lipid and protein damage in women and men. A significant increase in TAC in women following maximal-intensity exercise is the result of muscle fibre micro-injuries.     

Plant–herbivore interactions: Combined effect of groundwater level,root vole grazing,and sedge silicification

1. Accumulation of silica (Si) by plants can be driven by (1) herbivory pressure (and therefore plant–herbivore interactions), (2) geohydrological cycles, or (3) a combination of (1) and (2), with (1–3) possibly affecting Si concentration with a 1‐year delay.
2. To identify the relative significance of (1–3), we analyzed the concentration of Si in fibrous tussock sedge (Carex appropinquata), the population density of the root vole (Microtus oeconomus), and the groundwater level, over 11 years.
3. The largest influence of autumn Si concentration in leaves (Si_leaf) was on the level of the current‐year groundwater table, which was positive and accounted for 13.3% of its variance. The previous year''s vole population density was weakly positively correlated with Si_leaf, and it alone explained 9.5% of its variance.
4. The only variable found to have a positive, significant effect on autumn Si concentration in rhizomes (Si_rhiz) was the current‐year spring water level, explaining as much as 60.9% of its variance.
5. We conclude that the changes in Si concentration in fibrous tussock sedge are predominantly driven by hydrology, with vole population dynamics being secondary.
6. Our results provide only partial support for the existence of plant–herbivore interactions, as we did not detect the significant effects of Si tussock concentration on the vole density dynamics. This was mainly due to the low level of silicification of sedges, which was insufficient to impinge herbivores.
7. Future studies on plant–herbivore interactions should therefore aim at disentangling whether anti‐herbivore protection is dependent on threshold values of herbivore population dynamics. Furthermore, studies on Si accumulation should focus on the effect of water‐mediated Si availability.

     

Changes in wheat plastid membrane properties induced by cadmium and selenium in presence/absence of 2,4-dichlorophenoxyacetic acid

The aim of the work was to recognize the effect of cadmium (Cd) and selenium (Se) onto properties of plastid lipid membranes. Plastids were isolated from wheat calli cultured during 2 weeks on Murashige–Skoog media with presence/absence of 2,4-dichlorophenoxyacetic acid. Plastids obtained in presence of 2,4-D represented an earlier developmental stage in comparison to those, got in absence of 2,4-D, which reached a pre-chloroplast stage. The studied metals were introduced to culture media separately (2 μM Na₂SeO₄ or 800 μM CdCl₂) or together (Se + Cd). The changes of following properties of plastid envelope membrane caused by both metals were measured: composition of main lipid fractions, their fatty acid saturation, membrane fluidity, lipid peroxidation and membrane zeta potential. Results of experiments led to the conclusion that galactolipid component plays a predominant role in modification of plastid membrane properties responding to Cd and Se addition. It was shown that galactolipid protecting reaction to Cd toxic action can consists in increased plastid envelope membrane stiffness. The presence of hormone (2,4-D) and Se did not counterbalance Cd toxic effects (at least under concentration level applied in the experiments). Se applied separately can probably stimulate plastid/chloroplast transformation in wheat cells by increasing a galactolipid unsaturation degree. The zeta potentials seem to be important physicochemical parameter in determination of properties of membranes exposed to metal stress conditions.     

Regulierung des Blutdruckes beim Rothirsch (Cervus elaphus L.)

European Journal of Wildlife Research - Drei Hirsche unterschiedlichen Alters und Gewichtes wurden mit verschiedenen Narkotika betäube, die Höhe des Blutdruckes ermittelt und die...     

Thio-functionalized carbohydrate thiosemicarbazones and evaluation of their anticancer activity

Thiosemicarbazides and their analogs have shown potential medical applications as antiviral, antibacterial and anticancer drugs. We designed, synthesized and evaluated in vitro anticancer activity against ovarian (A2780), cervix (HeLa), colon (LoVo), breast (MCF-7) and brain (MO59J) human cancer cell lines of seven novel compounds –S-glycosylated thiosemicarbazones. We assessed the cyto- and genotoxic properties of all novel compounds using a variety of methods including comet assay, XTT assay, various fluorescent assays and toxicology PathwayFinder expression array. We tried to evaluate their possible mechanism of action with particular attention to induction of DNA damage and repair, apoptosis, oxidative stress analysis and cellular response in terms of changes in gene expression. The most sensitive cell line was human ovarian cancer. The results revealed that the major activity against A2780 cancer cell line displayed by our compounds is induction of DNA damage. This effect is not associated with apoptosis or oxidative stress induction and the resulting damage will not lead to cell cycle arrest. We also observed up-expression of heat shock related genes and NQO1 gene in response to our compounds. The second effect seems to be specific to glycosylated S-bond compounds as we observed it earlier. Upregulation of heat shock protein encoding genes suggest that our compounds induce stressful conditions. The nature of this phenomena (heat shock, pH shift or hypoxia) needs further study.     

Exposure to carbaryl leads to ultrastructural changes and alters activity of antioxidant enzymes in Spodoptera exigua (Lepidoptera: Noctuidae)

Abstract. The toxic effect of carbamate insecticide carbaryl on Spodoptera exigua (Lepidoptera: Noctuidae) larvae was examined. Insects appeared to be highly sensitive to it; numerous malfunctions and malformations were observed. The first ultrastructural alterations, including biological membrane swelling, nuclear condensation, and disintegration, and abnormalities around the mitochondria were observed even before massive mortality was noted. Scanning electron microscopy revealed changes in cuticle. Carbaryl depleted the wet weight of fat body mass and changed (in most of the cases elevated) the activity of antioxidant enzymes. The majority of these malformations are rather non-specific and are common among animals exposed to xenobiotics. However, they all suggest serious disruptions in metabolism, in addition to indicating that reactive oxygen species may play an important role in the toxic effect of carbaryl.