The hatching of common carp (Cyprinus carpio L.) embryos in response to exposure to different concentrations of cryoprotectant at low temperatures

The hatching performance of embryos of the common carp (Cyprinus carpio L.) was examined after 1, 7, 14, 21, or 28 days of storage at -8, -6, -4, -2, 0, 2, or 4 degrees C with different concentrations of methanol (0.5-7.0 M in 0.5 M steps) or varying concentrations of methanol in 0.1 M sucrose or trehalose. Preserved embryos failed to hatch after storage at -8 and -6 degrees C, regardless of the duration of storage or the concentrations tested. Likewise, there was no hatching out above 5.0 M concentration of methanol, even with the addition of sucrose or trehalose. After storage at 2 or 4 degrees C, the hatching rate was higher with mixtures of methanol (1.5 M) and trehalose (0.1 M) than with methanol plus sucrose or methanol alone. At 4 degrees C, the solution containing 1.5 M methanol supplemented with trehalose gave the highest hatching response of embryos stored for 14 days. Comparison of hatching after 24h of storage at the effective temperatures (-4, -2, 0, 2, and 4 degrees C) revealed that low concentrations of methanol were effective at high temperatures and high concentrations at sub-zero temperatures. The combination of 0.1 M trehalose with 1.5 M methanol gave the highest percentage hatching out both at 4 and 2 degrees C. At 0 degrees C, the highest percentage hatching occurred with 0.1 M trehalose plus 2.5 M methanol and at -2 and 4 degrees C, the best results were with 0.1 M trehalose plus 3.0 M methanol.     

Immunoreactivity of iNOS in porcine uterus after infusions of Escherichia coli endotoxin

The aim of this study was to investigate the distribution of inducible isoform of nitric oxide synthase (iNOS) in the porcine uterus after infusions of Escherichia coli endotoxin (lipopolysaccharide, LPS). In the group I (treated; n=6), 1 mg of LPS was infused into both the left and right uterine horn starting from the 4th to the 10th day of the estrous cycle, twice a day. In the group II (control; n=6), saline was infused into the uterus. The uterine horns were collected on the 14th day of the estrous cycle. Cryostat sections from the paraformaldehyde fixed tissues were stained immunohistochemically to estimate the distribution of iNOS. The luminal and glandular epithelium was stained more intensely for iNOS in the LPS-treated gilts than in the control animals. After LPS infusions, iNOS staining in vascular endothelial cells was also more intense than that observed in the controls. The present study has revealed that infusions of LPS into the porcine uterus result in an increase in the intensity of iNOS staining in some structures of this organ and supports our earlier data that NO can mediate an inflammatory effect of LPS in the uterus.     

Environmental impact of heated mining waters on clitellate (Annelida: Clitellata) assemblages

Mining is a relatively highly monitored industry. While chemical pollutants (toxic ions, radionuclides, etc.) have mostly been eliminated from mining waters, other types of environmental pollution (temperature regime alterations, high concentrations of various anions, etc.) can affect benthic invertebrates. In this study, we focused on the effect of mining water effluent on the diversity and density of aquatic Clitellata. Four sampling sites were selected. Three sites in a natural stream (the Nedvědi?ka River, Czech Republic), one upstream and two downstream from the mining effluent, and one site on the mining waters were sampled monthly during 2008–2009. Environmental variables were recorded in and samples were collected from two types of habitats — riffles and pools. The response of clitellate assemblages was evaluated using principal component analysis and generalised estimating equations. The results indicated that the mining effluent caused partial species exchange and had negative effects on clitellate taxa richness and abundance. These responses were specific to both the habitat (riffle/pool) and species sampled. In each of the different taxa studied, we observed one of four typical clitellate responses: (a) elimination of stenotherm species; (b) reduction of clitellate species followed by quick recovery; (c) neutral response; or (d) positive influence. We found that aquatic clitellates, which are considered to be eurytopic with broad ecological valences, are also sensitive to even slight environmental pollution.     

O-GlcNAcase: Promiscuous Hexosaminidase or Key Regulator of O-GlcNAc Signaling?

O-GlcNAc signaling is regulated by an opposing pair of enzymes: O-GlcNAc transferase installs and O-GlcNAcase (OGA) removes the modification from proteins. The dynamics and regulation of this process are only beginning to be understood as the physiological functions of both enzymes are being probed using genetic and pharmacological approaches. This minireview charts the discovery and functional and structural analysis of OGA and summarizes the insights gained from recent studies using OGA inhibition, gene knock-out, and overexpression. We identify several areas of “known unknowns” that would benefit from future research, such as the enigmatic C-terminal domain of OGA.     

Application of Arabidopsis tissue-specific CRUC promoter in the Cre/loxP self-excision strategy for generation of marker-free oilseed rape: potential advantages and drawbacks

The aim of the present study was to explore the usability of Arabidopsis cruciferin C (CRUC) promoter in the Cre/loxP self-excision strategy with the minimal rate of an ectopic expression of the cre recombinase. For this, a plant transformation vector containing the gus reporter gene driven by the light-sensitive Lhca3.St1 instead of double dCaMV 35S promoter and one pair loxP sites flanking the cre and the nptII genes was prepared. Agrobacterium-mediated transformations of three economically important oilseed rape (Brassica napus L.) cultivars Campino, Hunter and Topas as well as tobacco (Nicotiana tabacum L.) as a reference system were performed. The integration of the T-DNA into genome of all Brassica cultivars was accompanied by DNA deletions/rearrangements on the both T-DNA ends. The disruption of the Cre/loxP recombination system in oilseed rape was observed. On the contrary, no such events were detected in tobacco. The applied strategy did restrict an ectopic CRUC activity to some extent and a set of transgenic tobacco T₀ plants without premature excision event was obtained. Our data showed that a choice of the promoter can limit undesired activation of the Cre/loxP cassette.     

Agrobacterium tumefaciens-mediated transformation of bush monkey-flower (Mimulus aurantiacus Curtis) with a new reporter gene ZsGreen

A successful in vitro Agrobacterium-mediated transformation protocol was developed for Mimulus aurantiacus, a model species for ecological and evolutionary genetics and a promising ornamental plant. Three binary vectors were tested, each containing the hptII selectable marker gene and one of the reporter genes: gusA, EGFP or ZsGreen, all of them under CaMV 35S promoter. Genetic transformation was achieved through 4 days of co-cultivation of leaf, petiole and hypocotyl explants with Agrobacterium tumefaciens strain LBA 4404. Explants produced transformed callus tissue on solid modified Murashige and Skoog medium supplemented with 1 mg L^?1 6-benzylaminopurine, 0.5 mg L^?1 1-naphthaleneacetic acid, 30 g L^?1 sucrose and 20 or 50 mg L^?1 hygromycin B. All three reporter genes were expressed in callus tissue but the intensity of expression gradually decreased during further plant development. The new reporter gene ZsGreen proved suitable for plant transformation experiments since very intense and bright fluorescence was detected. Out of 1,760 co-cultured explants, 110 plants were regenerated and all of them were found to be PCR positive for the selection and/or reporter genes. Chemiluminescent Southern blot analysis revealed that 91 % of the regenerated plants (100 T₀ plants) contained T-DNA integrated in their genome. Transformation efficiency varied from 1.4 to 23.3 % for hypocotyl and petiole explants, respectively. Integration of some backbone sequences in plant genomes was confirmed in 75.3 % of T₀ plants. Using this protocol, stable transformants expressing selectable marker gene hptII and one of the reporter genes (gusA, ZsGreen or EGFP) were obtained in 4–5 months.