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991.
Ewa Kaniewska Alicja Sielicka Padmini Sarathchandra Iwona Pelikant-Małecka Mariola Olkowicz Ewa M. Słomińska 《Nucleosides, nucleotides & nucleic acids》2014,33(4-6):305-312
Extracellular nucleotides control mechanisms such as thrombosis or inflammation that are important in several pathologies, including heart valve disease and calcification. Ectonucleoside triphosphate diphosphohydrolase 1 (eNTPD1, CD39) and ecto-5′-nucleotidase (e5NT, CD73) are ectoenzymes that convert adenosine triphosphate to adenosine diphosphate, adenosine monophosphate and finally to adenosine. Changes in activities of these enzymes influence extracellular nucleotide concentrations and therefore could be involved in valve pathology. This study aimed to analyze type of cells, specific area, level of expression and biochemical function of CD39 and CD73 in pig aortic valves. Samples were collected from aortic valves of domestic pigs. Histological sections were cut from paraffin embedded tissue blocks. Following incubation with primary antibody against CD39 or CD73, washing and secondary goat anti-rabbit secondary antibodies, slides were viewed with NanoZoomer scanner. Substantial expression CD39 and CD73 was observed in two main types of valve cells: endothelial and valve interstitial cells. Subsequently, biochemical function of CD39 and CD73 was evaluated in cells cultured from pig aortic valve. Breakdown of extracellular nucleotides added to cell medium was analyzed with high performance liquid chromatography. In the interstitial cells, the CD73 products formation was much faster than in endothelium, while for the CD39 activity this relation was opposite. Expression and high concentration of CD39 and CD73 products in endothelium are expected, but presence of CD73 in valve interstitial cells is a surprise. We conclude that CD39 and CD73 and their enzymatic activities that convert extracellular nucleotides are highly expressed and could have special function in the valve. 相似文献
992.
Thomas Tilling Ewa Wladykowski Antonio Virgilio Failla Pia Houdek Johanna M. Brandner Ingrid Moll 《Histochemistry and cell biology》2014,141(4):407-421
Merkel cells, the neurosecretory cells of skin, are essential for light-touch responses and may probably fulfill additional functions. Whether these cells derive from an epidermal or a neural lineage has been a matter of dispute for a long time. In mice, recent studies have clearly demonstrated an epidermal origin of Merkel cells. Given the differences in Merkel cell distribution between human and murine skin, it is, however, unclear whether the same holds true for human Merkel cells. We therefore attempted to gain insight into the human Merkel cell lineage by co-immunodetection of the Merkel cell marker protein cytokeratin 20 (CK20) with various proteins known to be expressed either in epidermal or in neural stem cells of the skin. Neither Sox10 nor Pax3, both established markers of the neural crest lineage, exhibited any cell co-labeling with CK20. By contrast, β1 integrin, known to be enriched in epidermal stem cells, was found in nearly 70 % of interfollicular epidermal and 25 % of follicular Merkel cells. Moreover, LRIG1, also enriched in epidermal stem cells, displayed significant co-immunolabeling with CK20 as well (approximately 20 % in the interfollicular epidermis and 7 % in the hair follicle, respectively). Further epidermal markers were detected in sporadic Merkel cells. Cells co-expressing CK20 with epidermal markers may represent a transitory state between stem cells and differentiated cells. β1 integrin is probably also synthesized by a large subset of mature Merkel cells. Summarizing, our data suggest that human Merkel cells may originate from epidermal rather than neural progenitors. 相似文献
993.
Ewa Grabowska Urszula Wronska Milena Denkiewicz Malgorzata Jaszczur Aleksandra Respondek Malgorzata Alabrudzinska Catherine Suski Karolina Makiela‐Dzbenska Piotr Jonczyk Iwona J. Fijalkowska 《Molecular microbiology》2014,92(4):659-680
The role of replicative DNA polymerases in ensuring genome stability is intensively studied, but the role of other components of the replisome is still not fully understood. One of such component is the GINS complex (comprising the Psf1, Psf2, Psf3 and Sld5 subunits), which participates in both initiation and elongation of DNA replication. Until now, the understanding of the physiological role of GINS mostly originated from biochemical studies. In this article, we present genetic evidence for an essential role of GINS in the maintenance of replication fidelity in Saccharomyces cerevisiae. In our studies we employed the psf1‐1 allele (Takayama et al., 2003) and a novel psf1‐100 allele isolated in our laboratory. Analysis of the levels and specificity of mutations in the psf1 strains indicates that the destabilization of the GINS complex or its impaired interaction with DNA polymerase epsilon increases the level of spontaneous mutagenesis and the participation of the error‐prone DNA polymerase zeta. Additionally, a synergistic mutator effect was found for the defects in Psf1p and in the proofreading activity of Pol epsilon, suggesting that proper functioning of GINS is crucial for facilitating error‐free processing of terminal mismatches created by Pol epsilon. 相似文献
994.
Ewa Burchacka Michal Zdzalik Justyna‐Stec Niemczyk Katarzyna Pustelny Grzegorz Popowicz Benedykt Wladyka Adam Dubin Jan Potempa Marcin Sienczyk Grzegorz Dubin Jozef Oleksyszyn 《Protein science : a publication of the Protein Society》2014,23(2):179-189
Staphylococcus aureus is responsible for a variety of human infections, including life-threatening, systemic conditions. Secreted proteome, including a range of proteases, constitutes the major virulence factor of the bacterium. However, the functions of individual enzymes, in particular SplA protease, remain poorly characterized. Here, we report development of specific inhibitors of SplA protease. The design, synthesis, and activity of a series of α-aminoalkylphosphonate diaryl esters and their peptidyl derivatives are described. Potent inhibitors of SplA are reported, which may facilitate future investigation of physiological function of the protease. The binding modes of the high-affinity compounds Cbz-PheP-(OC6H4−4-SO2CH3)2 and Suc-Val-Pro-PheP-(OC6H5)2 are revealed by high-resolution crystal structures of complexes with the protease. Surprisingly, the binding mode of both compounds deviates from previously characterized canonical interaction of α-aminoalkylphosphonate peptidyl derivatives and family S1 serine proteases. 相似文献
995.
Anna D. Grabowska Ewa Wywia? Stanislaw Dunin-Horkawicz Anna M. ?asica Marc M. S. M. W?sten Anna Nagy-Staroń Renata Godlewska Katarzyna Bocian-Ostrzycka Katarzyna Pieńkowska Pawe? ?aniewski Janusz M. Bujnicki Jos P. M. van Putten E. Katarzyna Jagusztyn-Krynicka 《PloS one》2014,9(9)
Background
Bacterial Dsb enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between their cysteine residues. The Dsb protein network has been well characterized in cells of the model microorganism Escherichia coli. To gain insight into the functioning of the Dsb system in epsilon-Proteobacteria, where it plays an important role in the colonization process, we studied two homologs of the main Escherichia coli Dsb oxidase (EcDsbA) that are present in the cells of the enteric pathogen Campylobacter jejuni, the most frequently reported bacterial cause of human enteritis in the world.Methods and Results
Phylogenetic analysis suggests the horizontal transfer of the epsilon-Proteobacterial DsbAs from a common ancestor to gamma-Proteobacteria, which then gave rise to the DsbL lineage. Phenotype and enzymatic assays suggest that the two C. jejuni DsbAs play different roles in bacterial cells and have divergent substrate spectra. CjDsbA1 is essential for the motility and autoagglutination phenotypes, while CjDsbA2 has no impact on those processes. CjDsbA1 plays a critical role in the oxidative folding that ensures the activity of alkaline phosphatase CjPhoX, whereas CjDsbA2 is crucial for the activity of arylsulfotransferase CjAstA, encoded within the dsbA2-dsbB-astA operon.Conclusions
Our results show that CjDsbA1 is the primary thiol-oxidoreductase affecting life processes associated with bacterial spread and host colonization, as well as ensuring the oxidative folding of particular protein substrates. In contrast, CjDsbA2 activity does not affect the same processes and so far its oxidative folding activity has been demonstrated for one substrate, arylsulfotransferase CjAstA. The results suggest the cooperation between CjDsbA2 and CjDsbB. In the case of the CjDsbA1, this cooperation is not exclusive and there is probably another protein to be identified in C. jejuni cells that acts to re-oxidize CjDsbA1. Altogether the data presented here constitute the considerable insight to the Epsilonproteobacterial Dsb systems, which have been poorly understood so far. 相似文献996.
Olga Alster Anna Bielak-Zmijewska Grazyna Mosieniak Maria Moreno-Villanueva Wioleta Dudka-Ruszkowska Aleksandra Wojtala Monika Kusio-Kobia?ka Zbigniew Korwek Alexander Burkle Katarzyna Piwocka Jan K. Siwicki Ewa Sikora 《PloS one》2014,9(8)
Nibrin plays an important role in the DNA damage response (DDR) and DNA repair. DDR is a crucial signaling pathway in apoptosis and senescence. To verify whether truncated nibrin (p70), causing Nijmegen Breakage Syndrome (NBS), is involved in DDR and cell fate upon DNA damage, we used two (S4 and S3R) spontaneously immortalized T cell lines from NBS patients, with the founding mutation and a control cell line (L5). S4 and S3R cells have the same level of p70 nibrin, however p70 from S4 cells was able to form more complexes with ATM and BRCA1. Doxorubicin-induced DDR followed by cell senescence could only be observed in L5 and S4 cells, but not in the S3R ones. Furthermore the S3R cells only underwent cell death, but not senescence after doxorubicin treatment. In contrary to doxorubicin treatment, cells from all three cell lines were able to activate the DDR pathway after being exposed to γ-radiation. Downregulation of nibrin in normal human vascular smooth muscle cells (VSMCs) did not prevent the activation of DDR and induction of senescence. Our results indicate that a substantially reduced level of nibrin or its truncated p70 form is sufficient to induce DNA-damage dependent senescence in VSMCs and S4 cells, respectively. In doxorubicin-treated S3R cells DDR activation was severely impaired, thus preventing the induction of senescence. 相似文献
997.
998.
Jeremi K. Ochab Jacek Tyburczyk Ewa Beldzik Dante R. Chialvo Aleksandra Domagalik Magdalena Fafrowicz Ewa Gudowska-Nowak Tadeusz Marek Maciej A. Nowak Halszka Oginska Jerzy Szwed 《PloS one》2014,9(9)
The timing and dynamics of many diverse behaviors of mammals, e.g., patterns of animal foraging or human communication in social networks exhibit complex self-similar properties reproducible over multiple time scales. In this paper, we analyze spontaneous locomotor activity of healthy individuals recorded in two different conditions: during a week of regular sleep and a week of chronic partial sleep deprivation. After separating activity from rest with a pre-defined activity threshold, we have detected distinct statistical features of duration times of these two states. The cumulative distributions of activity periods follow a stretched exponential shape, and remain similar for both control and sleep deprived individuals. In contrast, rest periods, which follow power-law statistics over two orders of magnitude, have significantly distinct distributions for these two groups and the difference emerges already after the first night of shortened sleep. We have found steeper distributions for sleep deprived individuals, which indicates fewer long rest periods and more turbulent behavior. This separation of power-law exponents is the main result of our investigations, and might constitute an objective measure demonstrating the severity of sleep deprivation and the effects of sleep disorders. 相似文献
999.
Michaela Zorn-Kruppa Pia Houdek Ewa Wladykowski Maria Engelke Melinda Bartok Karsten R. Mewes Ingrid Moll Johanna M. Brandner 《PloS one》2014,9(12)
The depth of injury (DOI) is a mechanistic correlate to the ocular irritation response. Attempts to quantitatively determine the DOI in alternative tests have been limited to ex
vivo animal eyes by fluorescent staining for biomarkers of cell death and viability in histological cross sections. It was the purpose of this study to assess whether DOI could also be measured by means of cell viability detected by the MTT assay using 3-dimensional (3D) reconstructed models of cornea and conjunctiva. The formazan-free area of metabolically inactive cells in the tissue after topical substance application is used as the visible correlate of the DOI. Areas of metabolically active or inactive cells are quantitatively analyzed on cryosection images with ImageJ software analysis tools. By incorporating the total tissue thickness, the relative MTT-DOI (rMTT-DOI) was calculated. Using the rMTT-DOI and human reconstructed cornea equivalents, we developed a prediction model based on suitable viability cut-off values. We tested 25 chemicals that cover the whole range of eye irritation potential based on the globally harmonized system of classification and labelling of chemicals (GHS). Principally, the MTT-DOI test method allows distinguishing between the cytotoxic effects of the different chemicals in accordance with all 3 GHS categories for eye irritation. Although the prediction model is slightly over-predictive with respect to non-irritants, it promises to be highly valuable to discriminate between severe irritants (Cat. 1), and mild to moderate irritants (Cat. 2). We also tested 3D conjunctiva models with the aim to specifically address conjunctiva-damaging substances. Using the MTT-DOI method in this model delivers comparable results as the cornea model, but does not add additional information. However, the MTT-DOI method using reconstructed cornea models already provided good predictability that was superior to the already existing established in
vitro/ex
vivo methods. 相似文献
1000.
Radioactive coenzyme Q(10) ([(3)H]CoQ) was synthesized in a way that the metabolites produced retained the radioactivity. Administration of the lipid to rats intraperitoneally resulted in an efficient uptake into the circulation, with high concentrations found in spleen, liver, and white blood cells; lower concentrations in adrenals, ovaries, thymus, and heart; and practically no uptake in kidney, muscle, and brain. In liver homogenate most [(3)H]CoQ appeared in the organelles, but it was also present in the cytosol and transport vesicles. Mitochondria, purified on a metrizamide gradient, had a very low concentration of [(3)H]CoQ, which was mainly present in the lysosomes. All organs that took up the labeled lipid also contained water-soluble metabolites. The majority of metabolites excreted through the kidney and appeared in the urine. Some metabolites were also present in the feces, which further contained nonmetabolized [(3)H]CoQ, excreted through the bile. The major metabolites were purified from the urine, and the mass spectrometric fragmentation showed that these compounds, containing the ring with a short side chain, are phosphorylated. Thus, the results demonstrate that CoQ is metabolized in all tissues, the metabolites are phosphorylated in the cells, transported in the blood to the kidney, and excreted into the urine. 相似文献