Expression of the previously reported defensin of the tick Dermacentor marginatus (defDM) was analysed in different organs by RT-PCR. mRNA of the defDM gene was detected in the hemolymph, midgut and salivary glands. Moreover defDM was isolated from the tick hemolymph using RP-HPLC and its sequence was determined by mass spectrometry and Edman degradation. Synthetic peptide was used for determining biological activities. The results showed an anti-Gram-positive bacterial role for the defensin. As D. marginatus ticks appear not to be vectors of the Lyme disease agent of the complex Borrelia burgdorferi sensu lato, we tested the influence of defDM on Borrelia afzelii. There is a very clear borrelicidal activity of the defensin, which is concentration dependent and suggests a possible role in the clearing of Borrelia ingested by D. marginatus ticks.

• MeSH
• antibakteriální látky izolace a purifikace   farmakologie   MeSH
• antifungální látky izolace a purifikace   farmakologie   MeSH
• antivirové látky izolace a purifikace   farmakologie   MeSH
• Borrelia burgdorferi komplex účinky léků   MeSH
• defensiny izolace a purifikace   farmakologie   MeSH
• Dermacentor chemie   MeSH
• mikrobiální testy citlivosti MeSH
• proteiny členovců izolace a purifikace   farmakologie   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Recently, we identified a new insect defensin, named lucifensin that is secreted/excreted by the blowfly Lucilia sericata larvae into a wound as a disinfectant during the medicinal process known as maggot therapy. Here, we report the total chemical synthesis of this peptide of 40 amino acid residues and three intramolecular disulfide bridges by using three different protocols. Oxidative folding of linear peptide yielded a peptide with a pattern of disulfide bridges identical to that of native lucifensin. The synthetic lucifensin was active against Gram-positive bacteria and was not hemolytic. We synthesized three lucifensin analogues that are cyclized through one native disulfide bridge in different positions and having the remaining four cysteines substituted by alanine. Only the analogue cyclized through a Cys16-Cys36 disulfide bridge showed weak antimicrobial activity. Truncating lucifensin at the N-terminal by ten amino acid residues resulted in a drop in antimicrobial activity. Linear lucifensin having all six cysteine residues alkylated was inactive. Circular dichroism spectra measured in the presence of α-helix-promoting compounds showed different patterns for lucifensin and its analogues. Transmission electron microscopy revealed that Bacillus subtilis treatment with lucifensin induced significant changes in its envelope.

• MeSH
• antiinfekční látky chemická syntéza   chemie   MeSH
• cirkulární dichroismus MeSH
• defensiny chemická syntéza   chemie   genetika   MeSH
• disulfidy chemie   MeSH
• larva chemie   MeSH
• sbalování proteinů MeSH
• sekundární struktura proteinů MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Phlebotomus papatasi is the vector of Leishmania major, causing cutaneous leishmaniasis in the Old World. We investigated whether P. papatasi immunity genes were expressed toward L. major, commensal gut microbes, or a combination of both. We focused on sand fly transcription factors dorsal and relish and antimicrobial peptides (AMPs) attacin and defensin and assessed their relative gene expression by qPCR. Sand fly larvae were fed food with different bacterial loads. Relish and AMPs gene expressions were higher in L3 and early L4 larval instars, while bacteria 16S rRNA increased in late L4 larval instar, all fed rich-microbe food compared to the control group fed autoclaved food. Sand fly females were treated with an antibiotic cocktail to deplete gut bacteria and were experimentally infected by Leishmania. Compared to non-infected females, dorsal and defensin were upregulated at early and late infection stages, respectively. An earlier increase of defensin was observed in infected females when bacteria recolonized the gut after the removal of antibiotics. Interestingly, this defensin gene expression occurred specifically in midguts but not in other tissues of females and larvae. A gut-specific defensin gene upregulated by L. major infection, in combination with gut-bacteria, is a promising molecular target for parasite control strategies.

• Publikační typ
• časopisecké články MeSH

Studies of the human defensins have been hampered by the lack of a simple expression system allowing for rapid production of functional peptide forms. Here, we describe a Saccharomyces cerevisiae AH22 expression system that meets that condition. The 42 amino acid form of human beta-defensin-1 was expressed under the control of the ADH1 promoter. The optimum conditions for expression were determined and the stable maintenance of the pVT103L-hBD-1 chimeric vector in the yeast population was confirmed. Expressed hBD-1 was secreted into the medium (approximately 55 microg l(-1)) and purified using cation-exchange chromatography. Isolated defensin exhibited strong bactericidal effect on Escherichia coli ML-35p. We conclude that the expression system described here will be a useful tool where readily prepared and active forms of the human defensins are needed.

• MeSH
• antibakteriální látky biosyntéza   farmakologie   izolace a purifikace   MeSH
• beta-defensiny biosyntéza   farmakologie   genetika   chemie   MeSH
• Escherichia coli cytologie   účinky léků   MeSH
• genetické vektory genetika   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• proliferace buněk účinky léků   MeSH
• rekombinantní proteiny biosyntéza   farmakologie   chemie   izolace a purifikace   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• sekvence aminokyselin MeSH
• Check Tag
• lidé MeSH

The hard-bodied tick Ixodes ricinus (castor bean tick) is the most common tick species in Europe. I. ricinus is a vector of the causative agents of diseases that affect humans and animals including tick-borne encephalitis, borreliosis, tick-borne fever and babesiosis. The innate immune system provides ticks with quite an efficient defence against some pathogenic microorganisms in the event of their penetration into the tick body or through the blood meal. Antimicrobial peptides (AMPs) constitute an important feature of the tick immune system. Defensins are a well-known class of AMPs. Members of the defensin family of proteins have been reported in several tick species. So far, only two defensins had been identified from I. ricinus. In this study, we report the identification of six novel putative defensins from I. ricinus at the genomic and transcriptional levels. At the genomic level they show differences with one being intronless, while others contain two introns. The expression pattern of these molecules in the salivary glands, midgut, ovary, Malpighian tubules, haemolymph and the tick cell line IRE/CTVM19 was determined. Some of them are tissue specific while others seem to be ubiquitous. Molecular and phylogenetic analyses show that these novel members of the I. ricinus defensin family differ phylogenetically and structurally; nevertheless, the cysteine pattern is highly conserved among the family members. Finally, antimicrobial-peptide prediction tools were used to predict putative antimicrobial activity of our defensins. They show putative antimicrobial activity mainly against Gram-positive bacteria. This study displays the diversity of the defensin family in the tick I. ricinus.

• MeSH
• buněčné linie MeSH
• defensiny genetika   metabolismus   MeSH
• fylogeneze MeSH
• klíště genetika   MeSH
• klonování DNA MeSH
• morčata MeSH
• proteiny členovců genetika   metabolismus   MeSH
• sekvenční analýza DNA MeSH
• transkriptom MeSH
• zvířata MeSH
• Check Tag
• morčata MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Infekce kloubních náhrad jsou považovány za jednu z nejzávažnějších komplikací v endoprotetice. Diagnóza těchto infekcí je obtížná. V sou-časnosti neexistuje jediný samostatný test, který by byl považován za zlatý standard a byl dostatečně spolehlivý a přesný, s odpovídající senzi-tivitou a specificitou, vyžadována je kombinace více vyšetřovacích postupů. Účelem tohoto článku je posoudit současné poznatky o užitečnosti biomarkerů stanovovaných v krvi a synoviální tekutině, které by mohly pomoci při diagnostice infekce kloubní náhrady se zaměřením na synoviální alfa-defenzin.

Joint replacement infections are considered to be one of the most serious complications in endoprosthesis. The diagnosis of these infections is challenging. There is no individual gold-standard test that has robust sensitivity and specificity, and a combination of investigations is required. The purpose of this article is to review the current evidence on the utility of blood and synovial fluid biomarkers to help aid in the diagnosis of joint replacement infection with focusing on synovial fluid alpha-defensin.

• MeSH
• alfa-defensiny * analýza   MeSH
• antibakteriální látky terapeutické užití   MeSH
• artrocentéza MeSH
• artroplastiky kloubů MeSH
• biologické markery krev   MeSH
• infekce spojené s protézou * diagnóza   MeSH
• lidé MeSH
• náhrada kyčelního kloubu škodlivé účinky   MeSH
• synoviální tekutina chemie   MeSH
• totální endoprotéza kolene škodlivé účinky   MeSH
• Check Tag
• lidé MeSH

Defensiny jsou antimikrobiální a imunomodulační peptidy, které jsou důležitou součástí přirozené imunity. Autoři stručně charakterizují přirozenou imunitu, chemickou stavbu a funkci defensinů. Zabývají se především lidskými defensiny na respiračních sliznicích a jejich účinkem na bakterie, a to hlavně na grampozitivní bakterii Staphylococcus aureus. Defensiny mají přímý destrukční účinek na bakteriální stěnu, ale mají i imunomodulační účinek. Lidské defensiny dělíme podle struktury na alfa a beta 1, beta 2, beta 3 a beta 4. Alfa jsou v granulocytech, ve sliznici urogenitálního a intestinálního traktu. Beta defensiny jsou ve všech epiteliálních tkáních. V epitelu dýchacích cest jsou beta defensiny 1 produkovány konstitučně a beta defensiny 2 a 3 jsou indukovány jako odpověď na infekční agens. Některé bakterie mohou modifikovat svoji stěnu tak, aby byly méně citlivé na defensiny. U bakterie Staphylococcus aureus byly zjištěny geny Dlt a MprF, které jsou zodpovědné za modifikaci kyseliny teichoové a phosphatidylglycerolu v membráně takovým způsobem, že se sníží atraktivita bakterie pro pozitivně nabité defensiny. Poruchy těchto genů způsobují zvýšenou senzitivitu této bakterie na defensiny. Nové poznatky o antimikrobiálních peptidech nám umožňují lépe rozumět infekčním onemocněním a nabízejí nové perspektivy v terapii těchto onemocnění.

Defensins are antimicrobial and immunomodulation peptides, which are the important part of natural immunity. The authors briefly characterize natural immunity, chemical structure and function of defensins. The review deals preferentially with human defensins on respiratory mucous membranes and their effects on bacteria, mainly on gram-negative Staphylococcus aureus. Defensins exert a direct destructive effect on bacterial wall, but they possess an immunomodulation effect as well. Human defensins are divided according to their structure to alpha, beta 1, beta 2, beta 3 and beta 4. Alpha defensins are in granulocytes, in mucosa of urogenital and intestinal tract. Beta defensins are in all epithelial tissues. In the epithelium of respiratory pathways beta defensins 1 are produced constitutively and beta defensins 2 and 3 are induced as a response to infection agents. Some bacteria can modify their wall and thereby become less sensitive to defensins. In the bacteria Staphylococcus aureus the genes Dlt and Mrpf were found responsible for modification of teichoic acid and phosphatidylglycerol in the membrane in such a way that it decreases attractiveness of the bacteria for the positively charged defensins. Defects in these genes caused increased sensitivity of this bacterium to defensins. New knowledge of antibacterial peptides helps us to understand better infectious diseases and offers new perspectives in the therapy of these diseases.

• MeSH
• alfa-defensiny genetika   imunologie   klasifikace   MeSH
• beta-defensiny genetika   imunologie   klasifikace   MeSH
• defensiny imunologie   klasifikace   terapeutické užití   MeSH
• financování organizované MeSH
• imunologické faktory genetika   imunologie   MeSH
• kationické antimikrobiální peptidy genetika   imunologie   MeSH
• lidé MeSH
• rezistence dýchacích cest fyziologie   genetika   imunologie   MeSH
• Staphylococcus aureus genetika   imunologie   MeSH
• Check Tag
• lidé MeSH

BACKGROUND: There have been conflicting reports in the literature on association of gene copy number with disease, including CCL3L1 and HIV susceptibility, and β-defensins and Crohn's disease. Quantification of precise gene copy numbers is important in order to define any association of gene copy number with disease. At present, real-time quantitative PCR (QPCR) is the most commonly used method to determine gene copy number, however the Paralogue Ratio Test (PRT) is being used in more and more laboratories. FINDINGS: In this study we compare a Pyrosequencing-based Paralogue Ratio Test (PPRT) for determining beta-defensin gene copy number with two currently used methods for gene copy number determination, QPCR and triplex PRT by typing five different cohorts (UK, Danish, Portuguese, Ghanaian and Czech) of DNA from a total of 576 healthy individuals. We found a systematic measurement bias between DNA cohorts revealed by QPCR, but not by the PRT-based methods. Using PRT, copy number ranged from 2 to 9 copies, with a modal copy number of 4 in all populations. CONCLUSIONS: QPCR is very sensitive to quality of the template DNA, generating systematic biases that could produce false-positive or negative disease associations. Both triplex PRT and PPRT do not show this systematic bias, and type copy number within the correct range, although triplex PRT appears to be a more precise and accurate method to type beta-defensin copy number.

• MeSH
• beta-defensiny genetika   MeSH
• genetická predispozice k nemoci MeSH
• genom lidský genetika   MeSH
• genová dávka * MeSH
• kohortové studie MeSH
• lidé MeSH
• mapování chromozomů metody   MeSH
• molekulární sekvence - údaje MeSH
• populace MeSH
• populační genetika metody   MeSH
• sekvence nukleotidů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Geografické názvy
• Česká republika MeSH
• Dánsko MeSH
• Ghana MeSH
• Portugalsko MeSH
• Spojené království MeSH

• MeSH
• defensiny genetika   MeSH
• exprese genu fyziologie   MeSH
• finanční podpora výzkumu jako téma MeSH
• hmyzí proteiny genetika   MeSH
• včely genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH

BACKGROUND: The immune system of ticks is stimulated to produce many pharmacologically active molecules during feeding and especially during pathogen invasion. The family of cationic peptides - defensins - represents a specific group of antimicrobial compounds with six conserved cysteine residues in a molecule. RESULTS: Two isoforms of the defensin gene (def1 and def2) were identified in the European tick Ixodes ricinus. Expression of both genes was induced in different tick organs by a blood feeding or pathogen injection. We have tested the ability of synthetic peptides def1 and def2 to inhibit the growth or directly kill several pathogens. The antimicrobial activities (expressed as minimal inhibition concentration and minimal bactericidal concentration values) against Gram positive bacteria were confirmed, while Gram negative bacteria, yeast, Tick Borne Encephalitis and West Nile Viruses were shown to be insensitive. In addition to antimicrobial activities, the hemolysis effect of def1 and def2 on human erythrocytes was also established. CONCLUSIONS: Although there is nothing known about the realistic concentration of defensins in I. ricinus tick body, these results suggest that defensins play an important role in defence against different pathogens. Moreover this is a first report of a one amino acid substitution in a defensins molecule and its impact on antimicrobial activity.

• MeSH
• anatomické struktury zvířat imunologie   MeSH
• antiinfekční látky izolace a purifikace   farmakologie   MeSH
• defensiny genetika   imunologie   izolace a purifikace   MeSH
• erytrocyty účinky léků   MeSH
• gramnegativní bakterie účinky léků   MeSH
• grampozitivní bakterie účinky léků   MeSH
• klíště genetika   imunologie   MeSH
• kvasinky účinky léků   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• morčata MeSH
• protein - isoformy genetika   imunologie   izolace a purifikace   MeSH
• stanovení celkové genové exprese MeSH
• viry účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• morčata MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH