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Mapping of a transcription promoter located inside the priA gene of the Bacillus subtilis chromosome

100%
Hinc K., Iwanicki A., Seror S., Obuchowski M.
Acta Biochimica Polonica
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2006
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vol. 53
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issue 3
497-505
EN
The genome sequence of the Gram-positive soil bacterium Bacillus subtilis was completed in 1997 (Kunst et al., 1998) and the results included the identification of a putative transcription unit encompassing the yloI to yloS genes. Within this region of the B. subtilis chromosome 11 putative open reading frames were found with a wide diversity of probable functions. In this work we have analyzed transcription in the region of the priA-cpgA genes and we have mapped a promoter which is located inside the priA gene and its activity directs transcription of the def-yloM genes. Moreover, this transcript can be extended at low level to the prpC-priK-cpgA genes. Analysis of the sequence in proximity of the transcription start site revealed a sequence suitable for the housekeeping σA subunit of RNA polymerase. Analysis of the β-glactosidase activity of transcription fusions revealed that the identified promoter is active at low level and its activity is increased during late exponential phase of growth.
2
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The choice of the anchoring protein influences the interaction of recombinant Bacillus spores with the immune system

76%
Piekarska A., Pełka P., Peszyńska-Sularz G., Negri A., Hinc K., Obuchowski M., Iwanicki A.
Acta Biochimica Polonica
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2017
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vol. 64
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issue 2
239-244
EN
The technology of display of heterologous proteins on the surface of Bacillus subtilis spores enables use of these structures as carriers of antigens for mucosal vaccination. Currently, there are no technical possibilities to predict whether a designed fusion will be efficiently displayed on the spore surface and how such recombinant spores will interact with cells of the immune system. In this study, we compared four variants of B. subtilis spores presenting a fragment of a FliD protein from Clostridium difficile in fusion with CotB, CotC, CotG or CotZ spore coat proteins. We show that these spores promote their own phagocytosis and activate both, the J774 macrophages and JAWSII dendritic cells of murine cell lines. Moreover, we used these spores for mucosal immunization of mice. We conclude that the observed effects vary with the type of displayed FliD-spore coat protein fusion and seem to be mostly independent of its abundance and localization in the spore coat structure.
3
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Helicobacter pylori antigens, acetylsalicylic acid, LDL and 7-ketocholesterol - their potential role in destabilizing the gastric epithelial cell barrier. An in vitro model of Kato III cells

76%
Gajewski A., Mnich E., Szymański K., Hinc K., Obuchowski M., Moran A., Chmiela M.
Acta Biochimica Polonica
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2016
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vol. 63
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issue 1
145-152
EN
Colonization of gastric tissue in humans by H. pylori Gram-negative bacteria initiates gastric and duodenal ulcers and even gastric cancers. Infections promote inflammation and damage to gastric epithelium which might be followed by the impairment of its barrier function. The role of H. pylori components in these processes has not been specified. H. pylori cytotoxicity may potentially increase in the milieu of anti-inflammatory drugs including acetylsalicylic acid (ASA). The lipid transport-associated molecule such as low density lipoprotein (LDL), which is a classic risk factor of coronary heart disease (CHD) and 7-ketocholesterol (7-kCh) a product of cholesterol oxidation, which may occur during the oxidative stress in LDL could also be considered as pro-inflammatory. The aim of this study was to evaluate the cytotoxicity of H. pylori antigens, ASA, LDL and 7-kCh towards Kato III gastric epithelial cells, on the basis of the cell ability to reduce tetrazolium salt (MTT) and morphology of cell nuclei assessed by 4',6-diamidino-2-phenylindole (DAPI) staining. Kato III cells were stimulated for 24 h, at 37°C and 5% CO2, with H. pylori antigens: cytotoxin associated gene A (CagA) protein, the urease A subunit (UreA), lipopolysaccharide (LPS) and ASA, LDL or 7-kCh. H. pylori LPS, ASA, LDL and 7-kCh, but not H. pylori glycine acid extract (GE), demonstrated cytotoxicity against Kato III cells, which was related to a diminished percentage of MTT reducing cells and to an increased cell population with the signs of DNA damage. The results suggest that damage to gastric epithelial cells can be induced independently by H. pylori antigens, ASA and endogenous lipid transport-associated molecules. During H. pylori infection in vivo, especially in CHD patients, synergistic or antagonistic interactions between these factors might possibly influence the disease course. Further study is necessary to explain these potential effects.
4
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Immunoregulation of antigen presenting and secretory functions of monocytic cells by Helicobacter pylori antigens in relation to impairment of lymphocyte expansion

64%
Mnich E., Gajewski A., Rudnicka K., Gonciarz W., Stawerski P., Hinc K., Obuchowski M., Chmiela M.
Acta Biochimica Polonica
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2015
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vol. 62
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issue 4
641-650
EN
The role of Helicobacter pylori (H. pylori) antigens in driving a specific immune response against the bacteria causing gastroduodenal disorders is poorly understood. Using a guinea pig model mimicking the natural history of H. pylori infection, we evaluated the effectiveness of immature and mature macrophages in promoting the blastogenesis of splenocytes from H. pylori infected and uninfected animals, in response to H. pylori antigens: glycine acid extract (GE), cytotoxin associated gene A protein (CagA), urease A (UreA) and lipopolysaccharide (LPS). Lymphocyte expansion was assessed in 72 h cell cultures, containing: immature or mature macrophages derived from bone marrow monocytes, unstimulated or stimulated with H. pylori antigens for 2 h. The proliferation was expressed as a ratio of [3H]-thymidine incorporation into DNA of antigen-stimulated to unstimulated cells and the DNA damage was determined by DAPI cell staining. TGF-β and IFN-γ were assessed immunoenzymatically in cell culture supernatants. Lymphocytes of control and H. pylori-infected animals proliferated intensively in response to phytohaemagglutinin (PHA) and in co-cultures with immature or mature macrophages treated with CagA or UreA (significantly) and GE (slightly) exluding the cultures containing H. pylori or E. coli LPS. This lymphocyte growth inhibition was related to DNA damage of monocytic cells in response to H. pylori or E. coli LPS and secretion of regulatory TGF-β, but not proinflammatory IFN-γ. Impaired homeostasis of monocytic cell function related to DNA damage and TGF-β release, in response to H. pylori LPS may lead to the suppression of adaptive immune response against the bacteria and development of chronic infection.
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