Phage T4 lysozyme is a well folded and highly soluble protein that is widely used as an insertion tag to improve solubility and crystallization properties of poorly behaved recombinant proteins. It has been used in the fusion protein strategy to facilitate crystallization of various proteins including multiple G protein-coupled receptors, lipid kinases, or sterol binding proteins. Here, we present a structural and biochemical characterization of its novel, metal ions-binding mutant (mbT4L). We demonstrate that mbT4L can be used as a purification tag in the immobilized-metal affinity chromatography and that, in many respects, it is superior to the conventional hexahistidine tag. In addition, structural characterization of mbT4L suggests that mbT4L can be used as a purification tag compatible with X-ray crystallography.
Amyloidózy tvoří různorodou skupinu onemocnění charakterizovanou tvorbou patologického bílkovinného materiálu s následným ukládáním v tkáních a orgánech. Příčinou hereditární amyloidózy je patologická germinální mutace v genu kódujícím některý z amyloidogenních prekurzorových proteinů. Hereditární amyloidóza patří mezi onemocnění s autozomálně dominantní dědičností a značnou fenotypovou variabilitou a penetrancí. Vznik hereditární amyloidózy je v současné době spojován s mutacemi proteinu transthyretinu, apolipoproteinu AI a AII, fibrinogenu, gelsolinu, lysozymu a cystatinu C. Světová incidence hereditární amyloidózy je velice variabilní s dobře patrnými rozdíly mezi endemickými a neendemickými oblastmi výskytu. Incidence v rámci České republiky je neznámá. Hereditární amyloidózy patří mezi raritní onemocnění a povědomí o nich mezi lékaři je nízké, diagnostika často velmi komplikovaná. Kvůli dědičnému charakteru onemocnění, patří důkladně odebraná rodinná anamnéza mezi stěžejní diagnostické aspekty. Toto souhrnné sdělení je zaměřeno na popis sedmi nejčastějších dosud známých typů hereditárních amyloidóz, jejich genetický podklad, klinický obraz a možnosti léčby.
Amyloidosis represents a heterogeneous group of diseases characterized by the deposition of pathological material of protein nature in target tissues and organs. Hereditary amyloidosis is a disease with autosomal dominant inheritance and considerable phenotypic variability and penetrance. It is caused by a germline mutation in the gene encoding amyloidogenic precursor protein. Development of hereditary amyloidosis is currently associated with mutations in the following amyloidogenic precursor proteins: transthyretin, apolipoprotein AI and AII, fibrinogen, gelsolin, lysozyme, cystatin C. Worldwide incidence of hereditary amyloidosis is highly variable with clearly visible differences between endemic areas and non-endemic areas. The incidence of hereditary amyloidosis in the Czech Republic remains unknown. As hereditary amyloidosis is a rare disease and awareness among physicians is low, diagnosis is often very difficult. Due to the hereditary nature of the disease, carefully acquired family medical history is a key from a diagnostic aspect. This review described the seven most common types of hereditary amyloidosis, their genetic background, clinical features and treatment options.
- MeSH
- amyloidóza * etiologie genetika klasifikace metabolismus patologie MeSH
- apolipoprotein A-I genetika MeSH
- apolipoprotein A-II genetika MeSH
- bodová mutace MeSH
- cystatiny genetika MeSH
- fibrinogen genetika MeSH
- gelsolin genetika MeSH
- kardiovaskulární nemoci etiologie genetika MeSH
- klinický obraz nemoci MeSH
- lidé MeSH
- muramidasa genetika MeSH
- prealbumin genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Survival of earthworms in the environment depends on their ability to recognize and eliminate potential pathogens. This work is aimed to compare the innate defense mechanisms of two closely related earthworm species, Eisenia andrei and Eisenia fetida, that inhabit substantially different ecological niches. While E. andrei lives in a compost and manure, E. fetida can be found in the litter layer in forests. Therefore, the influence of environment-specific microbiota on the immune response of both species was followed. Firstly, a reliable method to discern between E. andrei and E. fetida based on species-specific primers for cytochrome c oxidase I (COI) and stringent PCR conditions was developed. Secondly, to analyze the immunological profile in both earthworm species, the activity and expression of lysozyme, pattern recognition protein CCF, and antimicrobial proteins with hemolytic function, fetidin and lysenins, have been assessed. Whereas, CCF and lysozyme showed only slight differences in the expression and activity, fetidin/lysenins expression as well as the hemolytic activity was considerably higher in E. andrei as compared to E. fetida. The expression of fetidin/lysenins in E. fetida was not affected upon the challenge with compost microbiota, suggesting more substantial changes in the regulation of the gene expression. Genomic DNA analyses revealed significantly higher level of fetidin/lysenins (determined using universal primer pairs) in E. andrei compared to E. fetida. It can be hypothesized that E. andrei colonizing compost as a new habitat acquired an evolutionary selection advantage resulting in a higher expression of antimicrobial proteins.
- MeSH
- Bacteria klasifikace genetika imunologie MeSH
- biologické toxiny genetika imunologie MeSH
- cytotoxicita imunologická genetika imunologie MeSH
- druhová specificita MeSH
- ekosystém * MeSH
- exprese genu MeSH
- hemolýza genetika imunologie MeSH
- hnůj mikrobiologie parazitologie MeSH
- molekulární sekvence - údaje MeSH
- muramidasa genetika imunologie MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- Oligochaeta klasifikace genetika imunologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- přirozená imunita genetika imunologie MeSH
- proteiny genetika imunologie MeSH
- půdní mikrobiologie MeSH
- respirační komplex IV genetika MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- sekvenční homologie nukleových kyselin MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Caenorhabditis elegans has been increasingly used to study the innate immunity and for the screening of microbe/host-specific pathogenic factors. Staphylococcus aureus-mediated infections with live C. elegans were performed on solid (full-lawn) and liquid assays. S. aureus required 90 ± 10 h for the complete killing of C. elegans, but the infection was started only after 32 h of exposure with 20% inoculum of S. aureus. The short time exposure studies revealed that, in 20% of inoculum, continuous exposure to the pathogen was required for the killing of nematode. In 100% of inoculum, only 8 h of exposure was sufficient to kill the C. elegans. To evaluate kinetically at the innate immune level, the regulation of representative candidate antimicrobial genes was investigated. Both semi-quantitative reverse transcriptase polymerase chain reaction (PCR) and real-time PCR analyses indicated the regulation of candidate immune regulatory genes of lysozyme (lys-7), cysteine protease (cpr-2), and C-type lectin (clec-60 and clec-87) family members during the course of S. aureus infections, indicating the possible contribution of the above players during the host immune response against S. aureus exposures.
- MeSH
- bakteriální léková rezistence MeSH
- Caenorhabditis elegans genetika imunologie metabolismus MeSH
- cysteinové proteasy genetika imunologie metabolismus MeSH
- exprese genu MeSH
- interakce hostitele a patogenu MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lektiny typu C genetika imunologie metabolismus MeSH
- muramidasa genetika imunologie metabolismus MeSH
- počet mikrobiálních kolonií MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- přirozená imunita MeSH
- proteiny Caenorhabditis elegans genetika imunologie metabolismus MeSH
- stafylokokové infekce genetika imunologie mikrobiologie MeSH
- Staphylococcus aureus fyziologie MeSH
- virulence MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lysozyme is a widely distributed antimicrobial protein having specificity for cleaving the beta-(1,4)-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (GlcNAc) of peptidoglycan of the bacterial cell walls and thus efficiently contributes to protection against infections caused mainly by Gram-positive bacteria. In the present study, we assembled a full-length cDNA of a novel invertebrate-type lysozyme from Eisenia andrei earthworm (EALys) by RT-PCR and RACE system. The primary structure of EALys shares high homology with other invertebrate lysozymes; however the highest, 72% identity, was shown for the destabilase I isolated from medicinal leech. Recombinant EALys expressed in Escherichia coli exhibited the lysozyme and isopeptidase activity. Moreover, real-time PCR revealed increased levels of lysozyme mRNA in coelomocytes of E. andrei after the challenge with both Gram-positive and Gram-negative bacteria.
- MeSH
- Bacillus subtilis imunologie patogenita MeSH
- bakteriální adheze MeSH
- chitinasy metabolismus MeSH
- Echinodermata genetika MeSH
- endopeptidasy metabolismus MeSH
- Escherichia coli genetika imunologie patogenita MeSH
- glukosamin analogy a deriváty imunologie metabolismus MeSH
- grampozitivní bakteriální infekce imunologie MeSH
- hydrolýza MeSH
- infekce vyvolané Escherichia coli imunologie MeSH
- interakce hostitele a patogenu MeSH
- klonování DNA MeSH
- kyseliny muramové imunologie metabolismus MeSH
- lyasy štěpící vazby C-N metabolismus MeSH
- muramidasa genetika imunologie metabolismus MeSH
- Oligochaeta enzymologie genetika imunologie MeSH
- pijavka lékařská genetika MeSH
- sekvenční homologie MeSH
- virulence MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
