Characterization of a midgut mucin-like glycoconjugate of Lutzomyia longipalpis with a potential role in Leishmania attachment

BACKGROUND: Leishmania parasites are transmitted by phlebotomine sand flies and a crucial step in their life-cycle is the binding to the sand fly midgut. Laboratory studies on sand fly competence to Leishmania parasites suggest that the sand flies fall into two groups: several species are termed "specific/restricted" vectors that support the development of one Leishmania species only, while the others belong to so-called "permissive" vectors susceptible to a wide range of Leishmania species. In a previous study we revealed a correlation between specificity vs permissivity of the vector and glycosylation of its midgut proteins. Lutzomyia longipalpis and other four permissive species tested possessed O-linked glycoproteins whereas none were detected in three specific vectors examined. RESULTS: We used a combination of biochemical, molecular and parasitological approaches to characterize biochemical and biological properties of O-linked glycoprotein of Lu. longipalpis. Lectin blotting and mass spectrometry revealed that this molecule with an apparent molecular weight about 45-50 kDa corresponds to a putative 19 kDa protein with unknown function detected in a midgut cDNA library of Lu. longipalpis. We produced a recombinant glycoprotein rLuloG with molecular weight around 45 kDa. Anti-rLuloG antibodies localize the native glycoprotein on epithelial midgut surface of Lu. longipalpis. Although we could not prove involvement of LuloG in Leishmania attachment by blocking the native protein with anti-rLuloG during sand fly infections, we demonstrated strong binding of rLuloG to whole surface of Leishmania promastigotes. CONCLUSIONS: We characterized a novel O-glycoprotein from sand fly Lutzomyia longipalpis. It has mucin-like properties and is localized on the luminal side of the midgut epithelium. Recombinant form of the protein binds to Leishmania parasites in vitro. We propose a role of this molecule in Leishmania attachment to sand fly midgut

Structural insight into the calcium ion modulated interdomain electron transfer in cellobiose dehydrogenase

AbstractCellobiose dehydrogenase (CDH) from wood degrading fungi represents a subclass of oxidoreductases with unique properties. Consisting of two domains exhibiting interdomain electron transfer, this is the only known flavocytochrome involved in wood degradation. High resolution structures of the separated domains were solved, but the overall architecture of the intact protein and the exact interface of the two domains is unknown. Recently, it was shown that divalent cations modulate the activity of CDH and its pH optimum and a possible mechanism involving bridging of negative charges by calcium ions was proposed. Here we provide a structural explanation of this phenomenon confirming the interaction between negatively charged surface patches and calcium ions at the domain interface

Assignment of sigma factors of RNA polymerase to promoters in Corynebacterium glutamicum

Dostalova H, Holatko J, Busche T, et al. Assignment of sigma factors of RNA polymerase to promoters in Corynebacterium glutamicum. AMB EXPRESS. 2017;7(1): 133.Corynebacterium glutamicum is an important industrial producer of various amino acids and other metabolites. The C. glutamicum genome encodes seven sigma subunits (factors) of RNA polymerase: the primary sigma factor SigA (sA), the primary-like sigma(B) and five alternative sigma factors (sigma(C), sigma(D), sigma(E), sigma(H) and sigma(M)). We have developed in vitro and in vivo methods to assign particular sigma factors to individual promoters of different classes. In vitro transcription assays and measurements of promoter activity using the overexpression of a single sigma factor gene and the transcriptional fusion of the promoter to the gfpuv reporter gene enabled us to reliably define the sigma factor dependency of promoters. To document the strengths of these methods, we tested examples of respective promoters for each C. glutamicum sigma factor. Promoters of the rshA (anti-sigma for sH) and trxB1 (thioredoxin) genes were found to be sigma(H)-dependent, whereas the promoter of the sigB gene (sigma factor sigma(B)) was sigma(E)-and sigma(H)-dependent. It was confirmed that the promoter of the cg2556 gene (iron-regulated membrane protein) is sigma(C)-dependent as suggested recently by other authors. The promoter of cmt1 (trehalose corynemycolyl transferase) was found to be clearly sigma(D)-dependent. No sigma(M)-dependent promoter was identified. The typical housekeeping promoter P2sigA (sigma factor sigma(A)) was proven to be sigma(A)-dependent but also recognized by sigma(B). Similarly, the promoter of fba (fructose-1,6-bisphosphate aldolase) was confirmed to be sigma(B)-dependent but also functional with sigma(A). The study provided demonstrations of the broad applicability of the developed methods and produced original data on the analyzed promoters

Research questions to facilitate the future development of European long-term ecosystem research infrastructures : A horizon scanning exercise

Distributed environmental research infrastructures are important to support assessments of the effects of global change on landscapes, ecosystems and society. These infrastructures need to provide continuity to address long-term change, yet be flexible enough to respond to rapid societal and technological developments that modify research priorities. We used a horizon scanning exercise to identify and prioritize emerging research questions for the future development of ecosystem and socio-ecological research infrastructures in Europe. Twenty research questions covered topics related to (i) ecosystem structures and processes, (ii) the impacts of anthropogenic drivers on ecosystems, (iii) ecosystem services and socio-ecological systems and (iv), methods and research infrastructures. Several key priorities for the development of research infrastructures emerged. Addressing complex environmental issues requires the adoption of a whole-system approach, achieved through integration of biotic, abiotic and socio-economic measurements. Interoperability among different research infrastructures needs to be improved by developing standard measurements, harmonizing methods, and establishing capacities and tools for data integration, processing, storage and analysis. Future research infrastructures should support a range of methodological approaches including observation, experiments and modelling. They should also have flexibility to respond to new requirements, for example by adjusting the spatio-temporal design of measurements. When new methods are introduced, compatibility with important long-term data series must be ensured. Finally, indicators, tools, and transdisciplinary approaches to identify, quantify and value ecosystem services across spatial scales and domains need to be advanced.Peer reviewe

Některé návrhy úprav daňové soustavy ČR

Import 20/04/2006Prezenční výpůjčkaVŠB - Technická univerzita Ostrava. Ekonomická fakulta. Katedra (154) financ