Volume 5, Issue 6

2DG causes cytotoxicity in cancer cells by disrupting thiol metabolism while Doxorubicin (DOX) induces cytotoxicity in tumor cells by generating reactive oxygen species (ROS). Here we examined the combined cytotoxic action of 2DG and DOX in rapidly dividing T47D breast cancer cells vs. slowly growing MCF-7 breast cancer cells. T47D cells exposed to the combination of 2DG/DOX significantly decreased cell survival compared to controls, while 2DG/DOX had no effect on MCF-7 cells. 2DG/DOX also disrupted the oxidant status of T47D treated cells, decreased intracellular total glutathione and increased glutathione disulfide (%GSSG) compared to MCF-7 cells. Lipid peroxidation increased in T47D cells treated with 2DG and/or DOX, but not in MCF-7 cells. T47D cells were significantly protected by NAC, indicating that the combined treatment exerts its action by increasing ROS production and disrupting antioxidant stores. When we inhibited glutathione synthesis with BSO, T47D cells became more sensitive to 2DG/DOX-induced cytotoxicity, but NAC significantly reversed this cytotoxic effect. Finally, 2DG/DOX, and BSO significantly increased the %GSSG in T47D cells, an effect which was also reversed by NAC. Our results suggest that exposure of rapidly dividing breast cancer cells to 2DG/DOX enhances cytotoxicity via oxidative stress and via disruptions to thiol metabolism.

Retinoic X receptor alpha (RXRA), a member of nuclear receptor superfamily, plays a key role in development, metabolism, glucose homeostasis, and intestinal cholesterol balance. The aim of this study was to examine an association of RXR alpha introne 5 A(39526)AA polymorphism and ABO blood groups with chronic heart failure (CHF) in the Czech population. A total of 238 patients with chronic heart failure and a control group of 246 subjects were included in the study. The RXR alpha gene polymorphism and ABO blood groups were detected by PCR and RFLP methods. Significant differences in distributions of RXRA A(39526)AA alleles and genotypes between CHF patients and controls were observed (Pg=0.03, Pa=0.02). The RXRA gene polymorphism differences of within blood group A between CHF patients and controls were highly significant in genotype distributions (Pg=0.002) and in allele frequency comparisons (Pa=0.0001). The prevalence of AA allele in CHF patients with A blood group was four-fold lower than in controls with the same blood group (OR=0.24; Pcorr=0.0001). A highly significant association of RXRA introne single-nucleotide insertion polymorphism and A blood group in chronic heart failure was observed. Our results suggest close linkage between RXRA A(39526)AA polymorphism and ABO blood groups.

Stressful experience during the early postnatal period may influence processes associated with neurogenesis (i.e. proliferation, cell death, appearance of astrocytes or cell differentiation) in the neonatal rat rostral migratory stream (RMS). To induce stress, pups were subjected to maternal deprivation daily for three hours, starting from the first postnatal day till the seventh postnatal day. Immunohistochemical methods were used to visualize proliferating cells and astrocytes; dying cells and nitrergic cells were visualized using histochemical staining. Quantitative analysis showed that maternal deprivation decreased the number of proliferating cells and significantly increased the number of dying cells in the RMS. Maternal deprivation did not influence the appearance of astrocytes in the RMS, but caused premature differentiation of nitrergic cells. In control rats, nitrergic cells can be observed in the RMS as early as the tenth postnatal day. In maternally deprived pups, these cells were detected as early as the seventh postnatal day. The observed earlier appearance of nitrergic cells in the RMS was associated with altered proliferation and increased cell dying and this observation supports the hypothesis that nitric oxide has an anti-proliferative role in the RMS. Our study demonstrates that maternal deprivation represents a stressful condition with a profound impact on early postnatal neurogenesis.

The bulk of large plant genomes consists of retrotransposons. Retrotransposons are able to integrate into a multitude of loci in the genome, and can thereby generate insertional polymorphism between individuals, as well as mutations and new characteristics. Retrotransposons are largely quiescent during development, but become more active in response to biotic and abiotic stresses, which cause them to produce larger pools of transcripts. The present study assesses the structural dynamics and putative transcriptional activation of BARE-1 and WIS 2-1A retrotransposons in ethyl methanesulfonate (EMS)-induced hexaploid wheat and Triticale sphaerococcum mutants, via an RT-PCR approach that utilized the retrotransposon based markers SSAP, IRAP and REMAP. Our results demonstrate a polymorphic pattern distribution of BARE-1/WIS 2-1A members, and identify new insertions. The WIS 2-1A retrotransposon members have a modulated transcriptional profile, which strongly suggests that EMS treatment encourages their activation Retrotransposon-based methods are efficient for fingerprinting and genetic polymorphism studies of sphaerococcum mutant forms, and can illuminate the genome dynamics affecting the genes responsible for the sphaerococcum phenotype.

Some representatives of the bivalve family Sphaeriidae are assumed to be polyploid. In this study, 11 sphaeriid species (nine of the genus Pisidium, one of Musculium, and one of Sphaerium) inhabiting central Europe were studied karyologically, 10 of them for the first time. Analysis revealed high chromosome numbers (from 140 to 240). To elucidate the origin of high chromosome numbers, DNA contents were measured by flow cytometry in 5 of the studied species and, for comparison, in S. corneum and S. nucleus, which are known to be diploid (2n=30). Species with high chromosome counts yielded very similar DNA contents that are not higher than in the related species with low diploid numbers. This finding contradicts a possible origin of these species by recent polyploidization or hybridization of related species. Chromosome complements of the investigated species with high chromosome numbers differ from those with low 2n in their small chromosome size and the high proportion of subtelo- or acrocentric chromosomes. This indicates their possible origin either by an ancient polyplodization event followed by chromosomal rearrangements or by multiple chromosome fissions.

The influence of magnetic fields on hatching and chromatin state of brine shrimp, Artemia sp., was investigated. Dry Artemia cysts were exposed to a magnetic field of intensity 25 mT for 10 min. The magnetic field was applied in different variants: constant field, rotating field of different directions (right-handed and left-handed) and different magnet polarization. The effect of ultra wideband pulse radiation and microwave radiation was also investigated. The energy density on the surface of object exposed to ultra wideband pulse radiation was 10−2, 10−3, 10−4, 10−5 and 10−6 W/cm2, the power of microwave radiation was 10−4 and 10−5 W/cm2, exposure time - 10 s. After incubation of the cysts for 48 hours in sea water the hatching percentage of Artemia from exposed cysts was higher than in controls. The number of heterochromatin granules was significantly higher in the nauplia (newborn larvae of Artemia) developed from cysts that had been exposed to magnetic and electromagnetic fields. The data obtained demonstrate an increase in percentage hatching of Artemia cysts after treatment with magnetic and electromagnetic fields and chromatin condensation in nauplia. We have also shown different effects of right-handed and left-handed rotating magnetic fields on these processes.

Recombinant human growth hormone (rhGH) has been widely used in many medical applications. Large scale production of rhGH is important for a wider application of this molecule in the field of proteomics. This investigation reports a modified Agrobacterium-mediated method for transfer and expression of human growth hormone gene in the popular edible mushroom Pleurotus eryngii. A binary vector pCAMBIA1304 containing the hGH2 gene was constructed and introduced into Pleurotus eryngii via Agrobacterium tumefaciens-mediated transformation. Integration of hGH2 into mushroom genome was detected by PCR and expression was confirmed by Western blot assay. The successful expression of the human growth hormone gene in mushroom suggests that the proposed modified transformation system is probably useful for the production of transgenic mushrooms.

The antioxidative activity of the extract from Conyza canadensis in plasma treated with peroxynitrite (ONOO−) (0.1 mM) was studied. C. canadensis is known to possess a broad set of pharmacological effects because of content of various antioxidants, antiplatelet and anticoagulant compounds. The aim of our study was to assess if this extract protects plasma proteins against oxidative/nitrative damages induced by ONOO−. The plasma components are continuously exposed to reactive oxygen/nitrogen species action. Peroxynitrite evokes oxidative stress and induces undesirable effects in biological systems and causes damage to biomolecules. The extract from Conyza (50–2500 mg/ml) caused a dose-dependent reduction of protein nitration by 90%. The oxidation of plasma proteins was diminished by about 75%. ONOO− oxidized the plasma thiol groups and this process was inhibited by tested extract. The level of reduced protein thiols was increased thrice at the lowest concentration of extract (50 mg/ml). The highest concentration of extract decreased twice the level of protein thiols in reduced forms and increased the homocysteine level about 4.5 times. The obtained results demonstrated that the extract from Conyza possesses antioxidative properties in vitro, protects plasma proteins against toxicity induced by peroxynitrite and has modulating effects on thiol/disulfide redox status.

The methanol extracts of four Parmeliaceae lichens (Hypogymnia physodes, Evernia prunastri, Flavoparmelia caperata and Parmelia sulcata) were screened for antioxidant properties and total phenol content. The H. physodes extract was the most effective at reducing iron(III) and scavenging 1,1-diphenyl-2-picrylhydrazyl radicals, while the P. sulcata extract was the most effective in reducing molybdenum(VI) in an acidic medium. The E. prunastri and H. physodes extracts contained more Folin-Ciocalteu reagent reactive substances than the F. caperata and P. sulcata extracts. Significant activity of the H. physodes extract in DPPH and reducing Fe(III) assays suggest that this lichen can be considered as a potential source of antioxidants.

In the crude ethanol extracts obtained from the rhizome and roots of Rheum palmatum L., Rheum undulatum L. and Rheum rhaponticum L. growing in Poland concentration of polyphenols ranged from 46.11 to 76.45 mg/g. Concentration of tannins ranged from 7.07% to 8.67%, while anthracene derivatives and anthraquinones varied by species - R. palmatum measured 36.3 and 34 mg/g, while R. undulatum or R. rhaponticum did not exceed 20.4 and 18.1 or 19.8 mg/g and 16.6 mg/g, respectively. Using a broth microdilution method it was found that all of the Rheum spp. extracts were more active against reference strains of Gram-positive bacteria (Staphylococcus spp.) than against those of Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis). The strongest inhibitory effect against Staphylococcus spp. was exerted by R. undulatum extract with MIC = 125–250 μg/mL. The moderate in vitro antibacterial activity of R. undulatum suggests that this plant, often used in the European cuisine to improve flavour, may be also important and useful as an alternative or auxiliary medicine remedy in the treatment of uncomplicated superficial infections caused especially by clinically important staphylococci, potentially pathogenic S. aureus or opportunistic S. epidermidis.

The incidence of candidiasis among immunocompromised patients and emergence of antimycotics resistant strains has increased significantly. The aims of this study were: to examine the in vitro activity of antimycotics and biocides against Candida clinical isolates; to detect cross-resistance of fungi to these preparations and to estimate whether disinfectants applied in hospital areas are active against clinical Candida isolates. In vitro susceptibility of 102 Candida isolates to eight antimycotics was examined by Etest and ATB Fungus. Sensitivity of these strains to four disinfectants and an antiseptic agent was tested according to EN 1275:2005. Amphotericin B, caspofungin and 5-fluorocytosine were the most effective antimycotics against all Candida isolates. Resistance to itraconazole and fluconazole was observed among C. krusei and C. glabrata. The MICs (Minimal Inhibitory Concentrations) for ketoconazole, voriconazole and posaconazole against Candida albicans ranged: 0.003 - >32 μg/ml and one strain was resistant to three agents tested. All analysed Candida strains were sensitive to biocides containing either chlorine, aldehyde, alcohol mixtures, glucoprotamin or chlorhexidine gluconate with isopropanol. Sensitivity to these agents was observed at concentrations lower than those concentrations recommended by manufacturers to achieve proper biocidal activity to those preparations. Our data suggest that these disinfectants can be effectively applied in clinical wards to prevent nosocomial Candida infections.

Uropathogenic strains of E. coli isolated from urine of patients with urinary tract infections were tested for antibiotic sensitivity using bio-Merieux kits and ATB-UR 5 expression system. The virulence of strains was evaluated by serum bactericidal assay, macrophage “killing” and bacterial adhesive tests. Survival capability of strains was assessed under starvation in saline. The results showed that quinolone-resistant uropathogenic strains of E. coli exhibit significantly reduced adhesive potential but relatively high resistance to serum and macrophage bactericidity. In contrast to laboratory strains, the quinolone-resistant uropathogenic clinical isolate demonstrated increased viability during starvation in saline. Our study suggests that quinolone-resistant uropathogenic strains are highly adaptable clones of E. coli, which can exhibit compensatory viability potential under unfavorable conditions. The clinical occurrence of such phenotypes is likely to contribute to the survival, persistence and spread strategy of resistant bacteria.

The investigation was carried out to ascertain the cause of mass mortalities in European perch (Perca fluviatilis L.) in North Lithuanian rivers in 2008 that reached a maximum in mid-autumn at a water temperature of 6°C. Marked changes were detected in morphophysiological parameters (spleen-, gill-, liver- and heart-somatic indices), which corresponded to changes in haematological parameters (leukocyte count was significantly elevated or reduced) of the infected fish. The viable bacterial counts in the gill, liver and kidney of infected live fish samples ranged from 2.3±0.3 × 103 to 6.3×00B1;0.4 × 103 c.f.u./g. The BOX-PCR fingerprinting technique was used for characterization and identification of the isolated bacterial strains from liver and kidney of infected perch. The bacterial isolates were identified by sequence analysis of the 16S rRNA gene. Pathogenic Aeromonas salmonicida subsp. salmonicida has been identified as a possible causative agent of the observed mortality in European perch.

The effects of six basal media on in vitro shoot proliferation of the greek grapevines Vitis vinifera L. cv. ‘Malagouzia’ and ‘Xinomavro’ were investigated. Galzy and Zlenco proved to be the most effective for ‘Malagouzia’ and ‘Xinomavro’, respectively. If only BA was present in the medium, shoot development was poor and the plantlets were chlorotic. When the medium was supplemented with BA and NAA, growth was enhanced. The best ratio (in μM) of growth regulators was 0.5/0.3 for ‘Malagouzia’, and 0.1/0.03 for ‘Xinomavro’, which resulted in the highest number of microshoots per explant and greatest proliferation rate. The development of ‘Malagouzia’ and ‘Xinomavro’ explants at 21±2 and 26±2°C was also investigated, revealing the higher temperature to be more effective. Regarding rooting, 0.5 μM IBA improved root formation at 26°C for ‘Malagouzia’ and 0.5 μM IBA at 21°C for ‘Xinomavro’. Moreover, 0.5 μM IBA resulted in a higher rooting percentage (>95%) and proved to be more beneficial for the overall morphological appearance of the plantlets of ‘Malagouzia’. After acclimatization, survival of microshoots cultivated in media with IBA was higher than those in NAA.

Rhodiola rosea is an endangered medicinal plant used for cancer, cardiovascular, and nervous system diseases therapy. Due to its limited distribution and sustainability alternative methods for production of its valuable substances are under investigation. Using in vitro techniques apical and rhizome buds, leaf nodes, stem and radix segments from wild plants and in vitro seedlings were plated on 24 modified Murashige and Skoog (1962) media. Decontamination of plant material was successful only in 21% of the schemes. The best shoot induction was obtained from seedling explants on media containing 2 mg/l zeatin or N6-benzylaminopurine, each. Their reduction stimulated shoot formation in the next passages (multiplication rate up to 5). Efficient rooting was induced on half-strength MS with 2 mg/l Indole-3-butyric acid and stimulated by adding 0.2 mg/l Indolyl-3-acetic acid. Regenerants rooted in perlite, peat, and soil (1:1:2), adapted in greenhouse, and transplanted in the mountains survived (70%) and developed like the wild plants. Salidroside content of these plants after one or two years was high (0.64 and 0.61% in rhizomes and 0.62 and 0.53% in roots, respectively). This is the first established efficient scheme for micropropagation of Bulgarian R. rosea allowing habitats restoration, germplasm conservation, and potential application of biotechnology for production of valuable substances.

Interest in the biochemical composition of Viburnum opulus fruit has intensified due to the food industry’s demand for natural vitamins, pigments and other substances that enhance the value of different foods. The present study was conducted to determine the agro-biological and biochemical variability of V. opulus and to select the genotypes that could best serve as sources of health promoting substances. Twelve selected genotypes were evaluated. ‘Leningradskaya Otbornaya’, V. opulus var. americanum, ‘Zarnitsa’, and local clone P2 were determined to be the best genotypes for growth in commercial plantations. Fruits of the local clone P3 were characterised by large amounts of total phenolics, ascorbic acid, and reducing sugars. V. opulus var. sargentii and V. opulus var. americanum contained exceptionally large amounts of total phenolics, 1460.0 and 1400.0 mg/100 g, respectively. The amount of ascorbic acid varied from 12.4 to 41.4 mg/100 g, the amount of carotenoids varied from 1.4 to 2.8 mg/100 g, the amount of anthocyanins varied from 23.2 to 44.6 mg/100 g, and the amount of total phenolics varied from 753.0 to 1460.0 mg/100 g. The presence of these large amounts of biologically active compounds enables their use as potent antioxidants. The data describing agro-biological characteristics, biochemical components, and health promoting activities of V. opulus fruits will increase the understanding of this plant and facilitate its use in the food and pharmaceuticals industry.

The gross anatomical features, namely stem, blade and petiole and the micromorphological features, trichome, pollen and nutlet of Salvia vermifolia Hedge & Hub.-Mor. (Sect. Aethiopis Bentham, Lamiaceae) were examined using light microscopy (LM) and scanning electron microscopy (SEM). S. vermifolia is a perennial endemic herb growing on igneous and serpentine slopes in Sivas province, Central Anatolia, Turkey. The results obtained from anatomical studies show that the stem is made up of 4–8-layers of collenchyma cells and 1-3-layers chlorenchyma cells. The blade is amphistomatic, bifacial, with 2-3-layered palisade cells on the adaxial. The petiole has three large central bundles and six small subsidiary bundles. Peltate glandular, capitate glandular and non-glandular trichomes are present in S. vermifolia. The peltate glandular trichomes are present in abundance on the calyx and corolla, and capitate glandular trichomes are predominant on the calyx, inflorescence axis, pedicel and stem. The acicular non-glandular trichomes are mainly situated on the calyx, corolla, inflorescence axis and pedicels whereas the floccose non-glandular trichomes are common on both sides of the leaf, inflorescence axis and petiole. The pollen grains are hexacolpate to octacolpate, medium to large (P, polar axis=45.29–62.49 μm; E, equatorial axis=45.32-62.38 μm), oblate-spheroidal to prolate-spheroidal (P/E=0.91–1.02) and their exine ornamentation is bireticulate. The nutlets are 2.70–2.90 mm long and 1.90–2.13 mm wide, brown, ovoid-oblong in their outline, rounded-trigonous in transverse section, and their surface is glabrous and colliculate.

The aim of this study was to investigate natural serpentine and non-serpentine populations of Teucrium polium aggr. and to document the differences in their morphological traits, as well as estimate which characters are most likely contributing to differentiation of the populations. Nine populations distributed both on and off serpentine soils were investigated, and 12 different morphological features of 270 individuals were studied by univariate and multivariate statistical analyses. The results did not demonstrate clear delimitation of the serpentine from the non-serpentine populations. Using these ecotypes as classification factors in the discriminant analysis, it can be stated that stem height, leaf length in the fifth leaf pair, internode length between the fourth and the fifth leaf pairs, and corolla tube length are features with the greatest discriminant ability. The character variation was generally higher for the vegetative features, while reproductive features failed to demonstrate clear differences. The similarity groupings indicated by the cluster analysis were synchronous with groups distinguished by both the discriminant and PCA analyses. Significant differences were observed between sets of vegetative characters in all populations investigated.

The study on the external morphology of the non-native black bullhead (Ameiurus melas) was carried out on a population from Slovakia, based on triple regression and geometrical analysis. The breakpoints distribution in distance-based morphometric characters indicated that black bullhead reached its definitive phenotype early in ontogeny. Ontogenetic changes in external morphology occurred continuously throughout the whole size-range of the sample examined, and inter-individual morphological variation was very low. Such uniformity may reflect the possible founder effect and/or little phenotypic plasticity of this non-native population. No sexual dimorphism was observed. This is the first detailed study on external morphology of the species, and as such it contributes to the assessment of phenotypic plasticity and/or overall morphological variability of black bullhead’s invasive populations.

Describing the sex ratio, age structure of the population and ontogenetic variability of Red fox, Vulpes vulpes (Canidae, Carnivora) skull parameters, this study is based on 416 male and 289 female skulls collected in the Czech Republic. The skulls analysed came from feral individuals, that were shot by hunters. The male to female ratio was 1:0.69 regarding the whole population. Individuals younger than one year prevailed in the population (54% males, 48% females were in their first year of life). Four growth patterns of skull dimensions were described. The first group included mainly skull length dimensions (e.g. condylobasal length). They grew rapidly until the sixth month of life, becoming stabilised afterwards. The second group comprised parameters that were stable throughout the life (e.g. cheek tooth rows). Measurements representing the third growth pattern showed continual growth (mainly width dimensions, e.g. zygomatic breadth). Conversely, smaller dimensions of postorbital breadth were observed after the sixth month of life. Postorbital breadth represented the fourth growth pattern. It was concluded, that male and female Red foxes had similar ontogenetic skull development, even though there were some differences, e.g. in jugular breadth, which increased after the age of six months in males unlike in females.