The adherence of bladder uroepithelial cells, subsequent expression, and regulation of type 1 fimbrial genes (key mediator of attachment) in clinical multidrug-resistant uropathogenic Escherichia coli (MDR-UPECs) isolated from individuals with asymptomatic bacteriuria (ABU) remain unexplored till date. Therefore, this study aimed to investigate the underlying molecular mechanisms associated with the adherence of clinical MDR-ABU-UPECs to human a uroepithelial cell line (HTB-4), both in the absence and presence of D-Mannose. These investigations focused on phase variation, expression, and regulation of type 1 fimbriae and were compared to a prototype ABU-strain (E. coli 83972) and symptomatic MDR-UPECs. Discordant to the ABU prototype strain, MDR-ABU-UPECs exhibited remarkable adhesive capacity that was significantly reduced after D-mannose exposure, fairly like the MDR symptomatic UPECs. The type 1 fimbrial phase variation, determined by the fim switch analysis, asserted the statistically significant incidence of "both OFF and ON" orientation among the adherent MDR-ABU-UPECs with a significant reduction in phase-ON colonies post-D-mannose exposure, akin to the symptomatic ones. This was indicative of an operative and alternating type 1 fimbrial phase switch. The q-PCR assay revealed a coordinated action of the regulatory factors; H-NS, IHF, and Lrp on the expression of FimB and FimE recombinases, which further controlled the function of fimH and fimA genes in ABU-UPECs, similar to symptomatic strains. Therefore, this study is the first of its kind to provide an insight into the regulatory crosstalk of different cellular factors guiding the adhesion of ABU-UPECs to the host. Additionally, it also advocated for the need to accurately characterize ABU-UPECs.
- MeSH
- adheziny Escherichia coli genetika metabolismus MeSH
- bakteriální adheze * MeSH
- bakteriální fimbrie * genetika metabolismus MeSH
- bakteriurie mikrobiologie MeSH
- buněčné linie MeSH
- epitelové buňky * mikrobiologie MeSH
- infekce vyvolané Escherichia coli * mikrobiologie MeSH
- lidé MeSH
- mannosa metabolismus farmakologie MeSH
- mnohočetná bakteriální léková rezistence * genetika MeSH
- proteiny fimbrií * genetika metabolismus MeSH
- proteiny z Escherichia coli genetika metabolismus MeSH
- regulace genové exprese u bakterií MeSH
- uropatogenní Escherichia coli * genetika účinky léků izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Pollen germination as a crucial process in plant development strongly depends on the accessibility of carbon as energy source. Carbohydrates, however, function not only as a primary energy source, but also as important signaling components. In a comprehensive study, we analyzed various aspects of the impact of 32 different sugars on in vitro germination of Arabidopsis pollen comprising about 150 variations of individual sugars and combinations. Twenty-six structurally different mono-, di- and oligosaccharides, and sugar analogs were initially tested for their ability to support pollen germination. Whereas several di- and oligosaccharides supported pollen germination, hexoses such as glucose, fructose and mannose did not support and even considerably inhibited pollen germination when added to germination-supporting medium. Complementary experiments using glucose analogs with varying functional features, the hexokinase inhibitor mannoheptulose and the glucose-insensitive hexokinase-deficient Arabidopsis mutant gin2-1 suggested that mannose- and glucose-mediated inhibition of sucrose-supported pollen germination depends partially on hexokinase signaling. The results suggest that, in addition to their role as energy source, sugars act as signaling molecules differentially regulating the complex process of pollen germination depending on their structural properties. Thus, a sugar-dependent multilayer regulation of Arabidopsis pollen germination is supported, which makes this approach a valuable experimental system for future studies addressing sugar sensing and signaling.
- MeSH
- Arabidopsis účinky léků fyziologie MeSH
- hexosy metabolismus farmakologie MeSH
- klíčení účinky léků fyziologie MeSH
- mannosa metabolismus farmakologie MeSH
- metabolismus sacharidů * MeSH
- oligosacharidy chemie metabolismus farmakologie MeSH
- pyl metabolismus fyziologie MeSH
- sacharidy MeSH
- sacharosa metabolismus farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
A plant selection system based on the phosphomannose isomerase gene (pmi) as a selectable marker is often used to avoid selection using antibiotic resistance. Nevertheless, pmi gene is endogenous in several plant species and therefore difficult to use in such cases. Here we evaluated and compared Agrobacterium-mediated transformation of Linum usitatissimum breeding line AGT-952 (without endogenous pmi gene) and Nicotiana tabacum var. WSC-38 (with endogenous pmi gene). Transformation was evaluated for vectors bearing transgenes that have the potential to be involved in improved phytoremediation of contaminated environment. Tobacco regenerants selection resulted in 6.8% transformation efficiency when using a medium supplemented with 30 g/L mannose with stepwise decrease of the sucrose concentration. Similar transformation efficiency (5.3%) was achieved in transformation of flax. Relatively low selection efficiency was achieved (12.5% and 34.8%, respectively). The final detection of efficient pmi selection was conducted using PCR and the non-endogenous genes; pmi transgene for flax and todC2 transgene for tobacco plants.
- MeSH
- Agrobacterium genetika MeSH
- bakteriální transformace genetika MeSH
- biodegradace MeSH
- geneticky modifikované rostliny genetika MeSH
- kultivační média chemie MeSH
- len účinky léků genetika MeSH
- mannosa-6-fosfátisomerasa genetika MeSH
- mannosa metabolismus farmakologie MeSH
- selekce (genetika) MeSH
- tabák účinky léků genetika MeSH
- Publikační typ
- časopisecké články MeSH
Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the interaction between a variably glycosylated envelope glycoprotein (gp120) and host-cell receptors. Approximately half of the molecular mass of gp120 is contributed by N-glycans, which serve as potential epitopes and may shield gp120 from immune recognition. The role of gp120 glycans in the host immune response to HIV-1 has not been comprehensively studied at the molecular level. We developed a new approach to characterize cell-specific gp120 glycosylation, the regulation of glycosylation, and the effect of variable glycosylation on antibody reactivity. A model oligomeric gp120 was expressed in different cell types, including cell lines that represent host-infected cells or cells used to produce gp120 for vaccination purposes. N-Glycosylation of gp120 varied, depending on the cell type used for its expression and the metabolic manipulation during expression. The resultant glycosylation included changes in the ratio of high-mannose to complex N-glycans, terminal decoration, and branching. Differential glycosylation of gp120 affected envelope recognition by polyclonal antibodies from the sera of HIV-1-infected subjects. These results indicate that gp120 glycans contribute to antibody reactivity and should be considered in HIV-1 vaccine design.
- MeSH
- AIDS imunologie MeSH
- buněčné linie MeSH
- buňky Hep G2 metabolismus MeSH
- ELISA metody MeSH
- glykosidhydrolasy metabolismus MeSH
- glykosylace MeSH
- HIV obalový protein gp120 genetika imunologie izolace a purifikace metabolismus MeSH
- HIV séropozitivita imunologie metabolismus MeSH
- HIV-1 imunologie metabolismus MeSH
- hmotnostní spektrometrie MeSH
- Jurkat buňky metabolismus MeSH
- komplementární DNA genetika MeSH
- lektin vázající mannosu genetika MeSH
- lidé MeSH
- mannosa metabolismus MeSH
- nádorové buněčné linie MeSH
- oligosacharidy chemie izolace a purifikace MeSH
- plazmidy MeSH
- polysacharidy chemie izolace a purifikace MeSH
- protilátky imunologie MeSH
- specificita protilátek MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
The purple pigmented bacterium Chromobacterium violaceum is a dominant component of tropical soil microbiota that can cause rare but fatal septicaemia in humans. Its sequenced genome provides insight into the abundant potential of this organism for biotechnological and pharmaceutical applications and allowed an ORF encoding a protein that is 60% identical to the fucose binding lectin (PA-IIL) from Pseudomonas aeruginosa and the mannose binding lectin (RS-IIL) from Ralstonia solanacearum to be identified. The lectin, CV-IIL, has recently been purified from C. violaceum [Zinger-Yosovich, K., Sudakevitz, D., Imberty, A., Garber, N. C., and Gilboa-Garber, N. (2006) Microbiology 152, 457-463] and has been confirmed to be a tetramer with subunit size of 11.86 kDa and a binding preference for fucose. We describe here the cloning of CV-IIL and its expression as a recombinant protein. A complete structure-function characterization has been made in an effort to analyze the specificity and affinity of CV-IIL for fucose and mannose. Crystal structures of CV-IIL complexes with monosaccharides have yielded the molecular basis of the specificity. Each monomer contains two close calcium cations that mediate the binding of the monosaccharides, which occurs in different orientations for fucose and mannose. The thermodynamics of binding has been analyzed by titration microcalorimetry, giving dissociation constants of 1.7 and 19 microM for alpha-methyl fucoside and alpha-methyl mannoside, respectively. Further analysis demonstrated a strongly favorable entropy term that is unusual in carbohydrate binding. A comparison with both PA-IIL and RS-IIL, which have binding preferences for fucose and mannose, respectively, yielded insights into the monosaccharide specificity of this important class of soluble bacterial lectins.
- MeSH
- bakteriální proteiny genetika chemie izolace a purifikace MeSH
- Chromobacterium chemie metabolismus MeSH
- entropie MeSH
- financování organizované MeSH
- fukosa metabolismus MeSH
- krystalizace MeSH
- lektin vázající mannosu genetika chemie izolace a purifikace MeSH
- lektiny genetika chemie izolace a purifikace MeSH
- mannosa metabolismus MeSH
- molekulární modely MeSH
- rekombinantní proteiny metabolismus MeSH
- rozpustnost MeSH
- sekundární struktura proteinů MeSH
- senzitivita a specificita MeSH
- statická elektřina MeSH
- vápník chemie MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- vodíková vazba MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- srovnávací studie MeSH
- MeSH
- aerobióza MeSH
- aktivní transport MeSH
- anaerobióza MeSH
- buněčná stěna metabolismus MeSH
- glukosa metabolismus MeSH
- hexokinasa metabolismus MeSH
- mannosa metabolismus MeSH
- matematika MeSH
- metody MeSH
- oxidativní fosforylace MeSH
- rotace MeSH
- Saccharomyces cerevisiae enzymologie metabolismus růst a vývoj MeSH
- Saccharomyces enzymologie metabolismus růst a vývoj MeSH
- stereoizomerie MeSH
- světlo MeSH
- xylosa metabolismus MeSH
