Continuous activation of the immune system inside a tissue can lead to remodelling of the tissue structure and creation of a specific microenvironment, such as during the tumour development. Chronic inflammation is a central player in stimulating changes that alter the tissue stroma and can lead to fibrotic evolution. In the colon mucosa, regulatory mechanisms, including TGF-β1, avoid damaging inflammation in front of the continuous challenge by the intestinal microbiome. Inducing either DSS colitis or AOM colorectal carcinogenesis in AVN-Wistar rats, we evaluated at one month after the end of each treatment whether immunological changes and remodelling of the collagen scaffold were already in development. At this time point, we found in both models a general downregulation of pro-inflammatory cytokines and even of TGF-β1, but not of IL-6. Moreover, we demonstrated by multi-photon microscopy the simultaneously presence of pro-fibrotic remodelling of the collagen scaffold, with measurable changes in comparison to the control mucosa. The scaffold was significantly modified depending on the type of induced stimulation. These results suggest that at one month after the end of the DSS or AOM inductions, a smouldering inflammation is present in both induced conditions, since the pro-inflammatory cytokines still exceed, in proportion, the local homeostatic regulation of which TGF-β1 is a part (inflammatory threshold). Such an inflammation appears sufficient to sustain remodelling of the collagen scaffold that may be taken as a possible pathological marker for revealing pre-neoplastic inflammation.
- Publikační typ
- časopisecké články MeSH
Nanodiamonds (ND) serve as RNA carriers with potential for in vivo application. ND coatings and their administration strategy significantly change their fate, toxicity, and effectivity within a multicellular system. Our goal was to develop multiple ND coating for effective RNA delivery in vivo. Our final complex (NDA135b) consisted of ND, polymer, antisense RNA, and transferrin. We aimed (i) to assess if a tumor-specific coating promotes NDA135b tumor accumulation and effective inhibition of oncogenic microRNA-135b and (ii) to outline off-targets and immune cell interactions. First, we tested NDA135b toxicity and effectivity in tumorospheres co-cultured with immune cells ex vivo. We found NDA135b to target tumor cells, but it binds also to granulocytes. Then, we followed with NDA135b intravenous and intratumoral applications in tumor-bearing animals in vivo. Application of NDA135b in vivo led to the effective knockdown of microRNA-135b in tumor tissue regardless administration. Only intravenous application resulted in NDA135b circulation in peripheral blood and urine and the decreased granularity of splenocytes. Our data show that localized intratumoral application of NDA135b represents a suitable and safe approach for in vivo application of nanodiamond-based constructs. Systemic intravenous application led to an interaction of NDA135b with bio-interface, and needs further examination regarding its safety.
- Publikační typ
- časopisecké články MeSH
Glucose oxidase (GOX) is a homodimeric glycoprotein with tightly bound one molecule of FAD cofactor per monomer of the protein. GOX has numerous applications, but the preparation of biotechnologically interesting GOX sensors requires a removal of the native FAD cofactor. This process often leads to unwanted irreversible deflavination and, as a consequence, to the low enzyme recovery. Molecular mechanisms of reversible reflavination are poorly understood; our current knowledge is based only on empiric rules, which is clearly insufficient for further development. To develop conceptual understanding of flavin-binding competent states, we studied the effect of deflavination protocols on conformational properties of GOX. After deflavination, the apoform assembles into soluble oligomers with nearly native-like holoform secondary structure but largely destabilized tertiary structure presumambly due to the packing density defects around the vacant flavin binding site. The reflavination is cooperative but not fully efficient; after the binding the flavin cofactor, the protein directly disassembles into native homodimers while the fraction of oligomers remains irreversibly inactivated. Importantly, the effect of Hofmeister salts on the conformational properties of GOX and reflavination efficiency indicates that the native-like residual tertiary structure in the molten-globule states favorably supports the reflavination and minimizes the inactivated oligomers. We interpret our results by combining the ligand-induced changes in quaternary structure with salt-sensitive, non-equilibrated conformational selection model. In summary, our work provides the very first steps toward molecular understanding the complexity of the GOX reflavination mechanism.
- MeSH
- Aspergillus niger enzymologie MeSH
- biokatalýza MeSH
- cirkulární dichroismus MeSH
- diferenciální skenovací kalorimetrie MeSH
- flavinadenindinukleotid chemie metabolismus MeSH
- glukosaoxidasa chemie metabolismus MeSH
- multimerizace proteinu MeSH
- protein - isoformy chemie metabolismus MeSH
- sekundární struktura proteinů MeSH
- spektrofotometrie ultrafialová MeSH
- stabilita proteinů MeSH
- teplota MeSH
- terciární struktura proteinů MeSH
- Publikační typ
- časopisecké články MeSH
Yeasts, historically considered to be single-cell organisms, are able to activate different differentiation processes. Individual yeast cells can change their life-styles by processes of phenotypic switching such as the switch from yeast-shaped cells to filamentous cells (pseudohyphae or true hyphae) and the transition among opaque, white and gray cell-types. Yeasts can also create organized multicellular structures such as colonies and biofilms, and the latter are often observed as contaminants on surfaces in industry and medical care and are formed during infections of the human body. Multicellular structures are formed mostly of stationary-phase or slow-growing cells that diversify into specific cell subpopulations that have unique metabolic properties and can fulfill specific tasks. In addition to the development of multiple protective mechanisms, processes of metabolic reprogramming that reflect a changed environment help differentiated individual cells and/or community cell constituents to survive harmful environmental attacks and/or to escape the host immune system. This review aims to provide an overview of differentiation processes so far identified in individual yeast cells as well as in multicellular communities of yeast pathogens of the Candida and Cryptococcus spp. and the Candida albicans close relative, Saccharomyces cerevisiae. Molecular mechanisms and extracellular signals potentially involved in differentiation processes are also briefly mentioned.
Adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) of the whooping cough agent Bordetella pertussis penetrates phagocytes expressing the integrin complement receptor 3 (CR3, CD11b/CD18, α(M)β(2) or Mac-1). CyaA translocates its adenylate cyclase (AC) enzyme domain into cell cytosol and catalyzes unregulated conversion of ATP to cAMP, thereby subverting cellular signaling. In parallel, CyaA forms small cation-selective membrane pores that permeabilize cells for potassium efflux, contributing to cytotoxicity of CyaA and eventually provoking colloid-osmotic cell lysis. To investigate whether the single-pass α-helical transmembrane segments of CR3 subunits CD11b and CD18 do directly participate in AC domain translocation and/or pore formation by the toxin, we expressed in CHO cells variants of CR3 that contained artificial transmembrane segments, or lacked the transmembrane segment(s) at all. The results demonstrate that the transmembrane segments of CR3 are not directly involved in the cytotoxic activities of CyaA but serve for maintaining CR3 in a conformation that is required for efficient toxin binding and action.
- MeSH
- adenosintrifosfát chemie MeSH
- adenylátcyklasový toxin metabolismus MeSH
- AMP cyklický biosyntéza MeSH
- antigeny CD11b genetika metabolismus MeSH
- antigeny CD18 genetika metabolismus MeSH
- biologický transport fyziologie MeSH
- Bordetella pertussis metabolismus MeSH
- buněčné linie MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- fagocyty metabolismus MeSH
- lidé MeSH
- makrofágový antigen 1 biosyntéza genetika metabolismus MeSH
- signální transdukce fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
