Apidaecins represent an important group of antimicrobial peptides occurring in honey bee hemolymph, where they play an important role as key components of humoral immunity. The present study demonstrates the development of a highly sensitive assay for apidaecin 1 isoforms quantification in the hemolymph or body parts from honey bee individuals. The analytical protocol comprises apidaecins 1 purification and enrichment steps by weak cation-exchange chromatography (WCX) in laboratory-made WCX-Tip microcolumns combined with a desalting step on a reversed-phase sorbent (C8) carried in StageTips. Apidaecin-enriched fraction was analyzed by a reversed-phase based nanoliquid chromatography (C4) separation coupled with high-resolution mass spectrometry. The method performance was validated in its specificity, linearity (0-5pmol), recovery (∼45%), precision (<10% at 0.1pmol), limit of detection (∼50fmol), limit of quantification (0.1pmol) and sample stability. The method was successfully applied to analyze the content of apidaecin 1 isoforms in the following samples: hemolymph - 13.0ng/μL (95% confidence interval of 7.5-18.6ng/μL), thoraxes - 36.2ng/unit (95% CI of 18.9-53.6ng/unit) and heads - 12.9ng/unit (95% CI of 9.1-16.7ng/unit). Freshly emerged bees had apidaecin 1 isoforms levels below the limit of detection. Thus it was possible to use them as a competitive matrix for calibration standards to prevent losses of highly basic apidaecins. This new protocol for apidaecin 1 isoforms quantification represents a promising tool to study the role of apidaecins in honey bee immunity and can be considered as a proof-of-concept for the development of sensitive quantification methods for basic antimicrobial peptides in various organisms.
- MeSH
- hmotnostní spektrometrie metody MeSH
- kalibrace MeSH
- kationické antimikrobiální peptidy izolace a purifikace MeSH
- kationty chemie MeSH
- limita detekce MeSH
- protein - isoformy izolace a purifikace MeSH
- včely chemie MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Biochemical characteristics of patatin proteins purified by ion-exchange and affinity chromatography from tubers of 20 potato cultivars were studied to evaluate their genotype differences with respect to utility groups, table potato cultivars (TPCs) and processing potato cultivars (PPCs). Both groups of cultivars showed similar values of protein content in dry matter (3.98-7.39%) and of patatin relative abundance (5.40-35.40%). Three mass levels (∼40.6, 41.8, and 42.9 kDa) of purified patatins were found by MALDI-TOF MS within all cultivars. Differences among mass levels corresponding with the mass of sugar antenna (∼1.2 kDa) confirmed the previous concept of different glycosylation extentsin patatin proteins. It was showed that the individual types of patatin varying in their masses occur in the patatin family in a ratio specific for each of the cultivars, with the lowest mass type being the major one. Electrophoretic analyses demonstrated wide cultivar variability in number of patatin forms. Especially 2D-PAGE showed 17-23 detected protein spots independently on the utility group. Specific lipid acyl hydrolase (LAH) activity of purified patatins from the individual tested cultivars varied between 0.92 and 5.46 μmol/(min mg). Patatin samples within most of the TPCs exhibited higher values of specific LAH activity than samples of PPCs. It may be supposed that individual patatin forms do not have similar physiological roles.
- MeSH
- druhová specificita MeSH
- glykosylace MeSH
- hlízy rostlin chemie MeSH
- karboxylesterhydrolasy chemie izolace a purifikace metabolismus MeSH
- molekulová hmotnost MeSH
- protein - isoformy chemie izolace a purifikace metabolismus MeSH
- rostlinné proteiny analýza chemie izolace a purifikace metabolismus MeSH
- Solanum tuberosum chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
This study concerned the occurrence of fecal bacteria with plasmid-mediated quinolone resistance (PMQR) genes in rooks (Corvus frugilegus, medium-sized corvid birds) wintering in continental Europe during winter 2010/2011. Samples of fresh rook feces were taken by cotton swabs at nine roosting places in eight European countries. Samples were transported to one laboratory and placed in buffered peptone water (BPW). The samples from BPW were enriched and subcultivated onto MacConkey agar (MCA) supplemented with ciprofloxacin (0.06 mg/L) to isolate fluoroquinolone-resistant bacteria. DNA was isolated from smears of bacterial colonies growing on MCA and tested by PCR for PMQR genes aac(6')-Ib, qepA, qnrA, qnrB, qnrC, qnrD, qnrS, and oqxAB. All the PCR products were further analyzed by sequencing. Ciprofloxacin-resistant bacteria were isolated from 37% (392 positive/1,073 examined) of samples. Frequencies of samples with ciprofloxacin-resistant isolates ranged significantly from 3% to 92% in different countries. The qnrS1 gene was found in 154 samples and qnrS2 in 2 samples. The gene aac(6')-Ib-cr was found in 16 samples. Thirteen samples were positive for qnrB genes in variants qnrB6 (one sample), qnrB18 (one), qnrB19 (one), qnrB29 (one), and qnrB49 (new variant) (one). Both the qnrD and oqxAB genes were detected in six samples. The genes qnrA, qnrC, and qepA were not found. Wintering omnivorous rooks in Europe were commonly colonized by bacteria supposedly Enterobacteriaceae with PMQR genes. Rooks may disseminate these epidemiologically important bacteria over long distances and pose a risk for environmental contamination.
- MeSH
- antibakteriální látky farmakologie MeSH
- Enterobacteriaceae genetika izolace a purifikace MeSH
- enterobakteriální infekce epidemiologie mikrobiologie veterinární MeSH
- feces mikrobiologie MeSH
- fluorochinolony farmakologie MeSH
- mikrobiální testy citlivosti MeSH
- nemoci ptáků epidemiologie mikrobiologie MeSH
- plazmidy klasifikace genetika izolace a purifikace MeSH
- polymerázová řetězová reakce veterinární MeSH
- protein - isoformy klasifikace genetika izolace a purifikace MeSH
- proteiny z Escherichia coli klasifikace genetika izolace a purifikace MeSH
- vrány mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
Plant seed oil bodies, subcellular lipoprotein inclusions providing storage reserves, are composed of a neutral lipid core surrounded by a phospholipid monolayer with several integrated proteins that play a significant role in stabilization of the particles and probably also in lipid mobilization. Oil bodies' proteins are generally very hydrophobic, due to the long uncharged sequences anchoring them into the lipid core, which makes them extremely difficult to handle and to digest successfully. Although oil bodies have been intensively studied during last decades, not all their proteins have been identified yet. To overcome the problems connected with their identification, a method based on SDS-PAGE, in-gel digestion and LC-MS/MS analysis was used. Digestion was carried out with trypsin and chymotrypsin, single or in combination, which increased significantly the number of identified peptides, namely the hydrophobic ones. Thanks to this methodology it was possible to achieve an extensive coverage of proteins studied, to analyze their N-terminal modifications and moreover, to detect four new oil bodies' protein isoforms, which demonstrates the complexity of oil bodies' protein composition.
- MeSH
- Arabidopsis chemie metabolismus MeSH
- chromatografie kapalinová MeSH
- chymotrypsin chemie MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- hmotnostní spektrometrie MeSH
- molekulární sekvence - údaje MeSH
- oleje rostlin chemie MeSH
- peptidové fragmenty analýza chemie MeSH
- protein - isoformy chemie klasifikace izolace a purifikace MeSH
- proteiny huseníčku chemie izolace a purifikace metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční analýza proteinů MeSH
- semena rostlinná chemie metabolismus MeSH
- trypsin chemie MeSH
- vakuoly chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
Ačkoliv příčiny Alzheimerovy nemoci ještě nejsou přesně známé, zdá se, že klíčovou roli v patogenezi onemocnění hrají peptidy amyloidu fi. Toxicita peptidů je úzce spjatá s jejich schopností oligomerizovat a agregovat nebo tvořit komplexy s jinými endogenními molekulami (např. receptory, transportéry, enzymy, oxysteroly apod.). Mechanizmy fyziologických interakcí peptidů amyloidu β a jejich přechod k patologickým efektům však ještě nebyly detailněji objasněny. Experimentální výsledky signalizují, že fyziologické akce lze pravděpodobně přisoudit monomemí rozpustné L-izoformé peptidů a že jsou zprostředkovány stereospecifickou vazbou k příslušné molekule. Syntetické D-izoformy nebo reverzní peptidy jsou zde neúčinné. Naproti tomu toxické účinky agregovaných L- i D-izoforem peptidů amyloidu β na membránu si jsou velice podobné. Ve studii jsou porovnány komplexy peptidů amyloidu β s R- a S-oxysteroly. Diskutováno je také možné použití přirozeně se vyskytujících enantiomerů oxysterolů a syntetických D-peptidů při léčení Alzheimerovy nemoci.
Although causes of Alzheimer disease are not known yet in detail, it appears that amyloid ß peptides can play a key role in the pathogenesis of the dementia. The toxicity of peptides is closely associated with their ability to oligomerize and aggregate or to create complexes with various endogenous molecules (e.g., with receptors, carriers, enzymes, oxysterols, etc.). However, mechanisms of their physiological interactions and the shift to pathological effects have not been yet elucidated. The experimental results indicate that the physiological actions of peptides are probably associated with the monomeric soluble L-isoform and mediated by stereospecific binding to corresponding molecule. Synthetic D-isoforms or reverse peptides are not effective in this respect. On the other hand, the toxic actions of aggregated L- and D-isoforms on membranes are very similar. In the study, the complexes of amyloid ß peptides and of R-/S-oxysterols are compared. Possible applications of naturally occurring enantiomers of oxysterols and of synthetic D-peptides in patients with Alzheimer disease are also discussed.
- MeSH
- Alzheimerova nemoc diagnóza enzymologie etiologie MeSH
- amyloid izolace a purifikace metabolismus škodlivé účinky MeSH
- amyloidní beta-protein izolace a purifikace metabolismus škodlivé účinky MeSH
- biomedicínský výzkum MeSH
- cholesterol izolace a purifikace metabolismus škodlivé účinky MeSH
- financování organizované MeSH
- hydroxycholesteroly metabolismus MeSH
- lidé MeSH
- medicína založená na důkazech trendy MeSH
- peptidy metabolismus terapeutické užití MeSH
- protein - isoformy izolace a purifikace MeSH
- Check Tag
- lidé MeSH
Rettův syndrom (RTT) je vážné X-vázané neurologické onemocnění, postihující především dívky. Patří mezi poruchy autistického spektra a je charakterizován zejména regresem psychomotorického vývoje, ztrátou řeči, mikrocefalií, stereotypními pohyby rukou a záchvaty. Většina případů RTT je způsobena de novo mutacemi v genu pro metyl-CpG-vazebný protein 2 (MECP2) a jen velmi ojediněle se jedná o familiární výskyt. Produkt tohoto genu, MeCP2 protein, sehrává důležitou roli v chromatinové remodelaci, regulaci genové exprese a také se účastní modulace RNA sestřihu. Některé případy atypického RTT mohou být způsobeny mutacemi v dalších genech, např. CDKL5, FOXG1 nebo NTNG1. Tento přehledový článek uvádí souhrn současných poznatků o Rettovu syndromu, klinickém obrazu pacientů v jednotlivých stadiích, molekulární podstatě, diagnostických kritériích, terapeutických přístupech a o možnostech DNA diagnostiky.
Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder affecting almost exclusively girls. It belongs to the family of autistic spectrum disorders, and it is characterized by psychomotor regression, loss of acquired speech, microcephaly, repetitive stereotypic hand movements, and seizures. Most of RTT cases are caused by de novo mutations in the gene for the methyl-CpG-binding protein 2 (MECP2), and familial cases are extremely rare. The MECP2 gene product plays an important role in chromatin remodeling, regulation of gene expression and is also involved in RNA splicing. Some atypical RTT cases are caused by mutations in other genes, such as CDKL5, FOXG1 or NTNG1. In this paper we give an overview of RTT, its clinical aspects, molecular basis, diagnostic criteria, medical management and DNA diagnosis.
- MeSH
- diagnostické techniky molekulární metody využití MeSH
- farmakoterapie metody škodlivé účinky využití MeSH
- financování organizované MeSH
- genetická heterogenita MeSH
- geny vázané na chromozom X genetika MeSH
- klinický obraz nemoci MeSH
- lidé MeSH
- mentální retardace diagnóza etiologie genetika MeSH
- místa sestřihu RNA genetika MeSH
- mutace genetika MeSH
- neurologické manifestace MeSH
- polymerázová řetězová reakce využití MeSH
- progrese nemoci MeSH
- protein - isoformy genetika izolace a purifikace MeSH
- protein 2 vázající methyl-CpG izolace a purifikace MeSH
- Rettův syndrom diagnóza etiologie terapie MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- přehledy MeSH
The human p73 protein is essential for normal morphogenesis and maintenance of neural tissue. Recently, several TP73 transcripts have been revealed in medulloblastoma (MB), the most common malignant brain tumor in children. Here, we performed immunohistochemical analysis on 29 MB specimens using anti-p73alpha and anti-DeltaNp73 antibodies. Real-time PCR quantification was performed to assess TAp73 and DeltaNp73 transcripts in a subset of 13 MB samples. Normal cerebellar tissues and RNA were used for comparison. Pilot clinical-pathological correlations were also provided. We report significant differences for TAp73 and DeltaNp73 mRNA expression between tumor tissues and reference (P = 0.013, P = 0.028). Immunohistochemically, 52 and 29% MB samples were positive for p73alpha and DeltaNp73, respectively. p73alpha expression was found to be in both the nucleus and cytoplasm, whereas DeltaNp73 was localized predominantly in the cytoplasm. In normal cerebellum, positive staining for p73alpha and DeltaNp73 was observed in the Purkinje cells of newborns, not adult samples, which supports the developmental role of TP73 during organogenesis of the human cerebellum. Survival analysis has shown negative relationship of DeltaNp73-immunoreactivity with overall survival (OS) and event free survival (EFS) (P = 0.026 and P = 0.127, respectively). For p73alpha-positive cases, the negative trend in OS (P = 0.149) and EFS (P = 0.216) was also apparent. Our results indicate the involvement of p73 protein in MB tumorigenesis and define TP73 as a potential prognostic and therapeutic target for medulloblastom.
- MeSH
- dítě MeSH
- DNA vazebné proteiny genetika imunologie metabolismus MeSH
- dospělí MeSH
- financování organizované MeSH
- imunohistochemie MeSH
- lidé MeSH
- meduloblastom genetika imunologie metabolismus MeSH
- messenger RNA analýza metabolismus MeSH
- míra přežití MeSH
- mladiství MeSH
- mozek metabolismus patofyziologie patologie MeSH
- nádorová transformace buněk genetika metabolismus MeSH
- nádorové biomarkery analýza genetika metabolismus MeSH
- nádorové supresorové proteiny genetika imunologie metabolismus MeSH
- nádory mozku genetika metabolismus patofyziologie MeSH
- předškolní dítě MeSH
- prognóza MeSH
- protein - isoformy genetika izolace a purifikace metabolismus MeSH
- regulace genové exprese u nádorů genetika MeSH
- retrospektivní studie MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
The levels of two major serum amyloid A precursor isoforms, SAA1 and SAA2, which are associated with high-density lipoproteins (HDL) are increased during inflammation. The hydrophobic character and the small size difference--corresponding to just 0.8 kDa--between these two members of the SAA family hinder their separation and purification on a large scale by conventional methods. In the current work, both mouse SAA proteins were purified from HDL-SAA and acute-phase serum within 10 h in a one-step procedure using the high-resolution, continuous-elution preparative gel electrophoresis Prep-Cell system in combination with Tris/Glycine SDS-PAGE. Moreover, applying the Tris/Tricine system on the Prep-Cell resulted not only in purification of the SAA proteins, but also in their separation within 16 h. The SAA isoforms were freed from SDS using a Centricon concentrator and were identified using monoclonal antibodies. Optical density profile plots of gel protein or Western blot bands in combination with a colorimetric spectrophotometric protein assay showed that the recovery of the isoforms ranged from 38% to 60%. These results show that the preparative gel electrophoresis system Prep-Cell is a suitable device for separating SAA1 and SAA2 proteins in a simplified, convenient, and fast procedure, which can be applied on a small or large scale.
- MeSH
- barvení stříbrem metody MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- finanční podpora výzkumu jako téma MeSH
- hydrofobní a hydrofilní interakce MeSH
- myši MeSH
- protein - isoformy izolace a purifikace MeSH
- proteinové prekurzory izolace a purifikace MeSH
- sérový amyloid A izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
