Magainin 2 and PGLa are cationic, amphipathic antimicrobial peptides which when added as equimolar mixture exhibit a pronounced synergism in both their antibacterial and pore-forming activities. Here we show for the first time that the peptides assemble into defined supramolecular structures along the membrane interface. The resulting mesophases are quantitatively described by state-of-the art fluorescence self-quenching and correlation spectroscopies. Notably, the synergistic behavior of magainin 2 and PGLa correlates with the formation of hetero-domains and an order-of-magnitude increased membrane affinity of both peptides. Enhanced membrane association of the peptide mixture is only observed in the presence of phophatidylethanolamines but not of phosphatidylcholines, lipids that dominate bacterial and eukaryotic membranes, respectively. Thereby the increased membrane-affinity of the peptide mixtures not only explains their synergistic antimicrobial activity, but at the same time provides a new concept to increase the therapeutic window of combinatorial drugs.

• MeSH
• antibakteriální látky chemie   izolace a purifikace   farmakologie   MeSH
• buněčná membrána chemie   účinky léků   MeSH
• ethanolaminy chemie   MeSH
• fixní kombinace léků MeSH
• fluorescenční barviva chemie   MeSH
• fluorescenční spektrometrie MeSH
• fosfatidylcholiny chemie   MeSH
• fosfatidylethanolaminy chemie   MeSH
• fosfatidylglyceroly chemie   MeSH
• kationické antimikrobiální peptidy chemie   izolace a purifikace   farmakologie   MeSH
• kůže chemie   MeSH
• lipidové dvojvrstvy chemie   MeSH
• magaininy chemie   izolace a purifikace   farmakologie   MeSH
• proteiny Xenopus chemie   izolace a purifikace   farmakologie   MeSH
• sloučeniny boru chemie   MeSH
• synergismus léků MeSH
• vazba proteinů MeSH
• Xenopus laevis MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Bile acids are compounds with many functions in liver and other parts of gastrointestinal tract. Apart from long-known ability of bile acids to form micelles and help with both intestinal digestion and absorption of lipids, other (but not less important) properties of these molecules are still neglected. Among pleitropic effects of bile acids belong regulation of cholesterol homeostasis, antimicrobial properties and novel functions discovered in last decades. It seems that the notion of bile acids as compounds important mainly for their physico-chemical properties should be reevaluated and we have to put stress on the importance of bile acids as structures with hormonal functions. Moreover, the molecules of bile acids have unusual properties, which predetermine them for attractive drug designs, as vectors for delivery of active substances, production of chiral compounds, dendrons, or even photoresistant molecules for microcircuits.

• MeSH
• antivirové látky MeSH
• biologické markery MeSH
• dendrimery MeSH
• kationické antimikrobiální peptidy izolace a purifikace   MeSH
• nanočástice MeSH
• nosiče léků MeSH
• Squalus MeSH
• vztahy mezi strukturou a aktivitou MeSH
• žlučové kyseliny a soli * farmakologie   metabolismus   terapeutické užití   MeSH
• Publikační typ
• práce podpořená grantem MeSH

The present study was performed to evaluate the antibacterial activities of an antimicrobial peptide (CSpK14) and the synergies thereof with β-lactams against vancomycin-resistant Staphylococcus aureus (VRSA) and Enterococci (VRE). Our strain was isolated from fermented food (kimchi), which is 99.79 % homologous with Bacillus amyloliquefaciens subsp. plantarum FZB42(T). CSpK14 was purified to homogeneity by diammonium sulfate precipitation, concentration, dialysis, and followed by two-stage chromatographic separation, i.e., Sepharose Cl-6B and Sephadex G-25 chromatography, and had a molar mass of ~4.6 kDa via Tricine SDS-PAGE and in situ examination. It was stable at pH 6.0-11.5 and temperature up to 80 °C. In addition, it was also stable with various metal ions, solvents, and proteases. The N-terminal amino acid sequence was H-Y-D-P-G-D-D-S-G-N-T-G and did not show any significant homology with reported peptides. However, it shows some degrees of identity with alpha-2-macroglobulin and ligand-gated channel protein from different microorganisms. CSpK14 significantly reduced the minimum inhibitory concentrations (MICs) of β-lactams and had no effect on non-β-lactams against VRSA and VRE. MICs of CSpK14/oxacillin and CSpK14/ampicillin were reduced by 8- to 64-fold and 2- to 16-fold, respectively. The time killing assay between CSpK14/oxacillin (2.29-2.37 Δlog10CFU/mL at 24 h) and CSpK14/ampicillin (2.30-2.38 Δlog10CFU/mL at 24 h) being >2-fold and fractional inhibitory concentration index ˂0.5 revealed synergy. Furthermore, the biofilms formed by VRSA and VRE were reduced completely. CSpK14 was simple to purify, had low molecular mass, was stable over a wide pH range or tested chemicals, had broad inhibitory spectrum, and possessed potent synergistic antimicrobial-antibiofilm properties. CSpK14 synergistically enhanced the efficacy of β-lactams and is therefore suitable for combination therapy.

• MeSH
• ampicilin farmakologie   MeSH
• antibakteriální látky biosyntéza   izolace a purifikace   farmakologie   MeSH
• Bacillus amyloliquefaciens klasifikace   imunologie   metabolismus   MeSH
• biofilmy účinky léků   růst a vývoj   MeSH
• chromatografie iontoměničová MeSH
• enterokoky rezistentní vůči vankomycinu účinky léků   růst a vývoj   MeSH
• fylogeneze MeSH
• kationické antimikrobiální peptidy biosyntéza   izolace a purifikace   farmakologie   MeSH
• kombinovaná farmakoterapie MeSH
• mikrobiální testy citlivosti MeSH
• oxacilin farmakologie   MeSH
• rezistence na vankomycin účinky léků   MeSH
• sekvence aminokyselin MeSH
• stabilita proteinů MeSH
• Staphylococcus aureus účinky léků   růst a vývoj   MeSH
• synergismus léků MeSH
• Publikační typ
• časopisecké články MeSH

Venoms of hymenopteran insects have attracted considerable interest as a source of cationic antimicrobial peptides (AMPs). In the venom of the solitary bee Hylaeus signatus (Hymenoptera: Colletidae), we identified a new hexadecapeptide of sequence Gly-Ile-Met-Ser-Ser-Leu-Met-Lys-Lys-Leu-Ala-Ala-His-Ile-Ala-Lys-NH2. Named HYL, it belongs to the category of α-helical amphipathic AMPs. HYL exhibited weak antimicrobial activity against several strains of pathogenic bacteria and moderate activity against Candida albicans, but its hemolytic activity against human red blood cells was low. We prepared a set of HYL analogues to evaluate the effects of structural modifications on its biological activity and to increase its potency against pathogenic bacteria. This produced several analogues exhibiting significantly greater activity compared to HYL against strains of both Staphylococcus aureus and Pseudomonas aeruginosa even as their hemolytic activity remained low. Studying synergism of HYL peptides and conventional antibiotics showed the peptides act synergistically and preferentially in combination with rifampicin. Fluorescent dye propidium iodide uptake showed the tested peptides were able to facilitate entrance of antibiotics into the cytoplasm by permeabilization of the outer and inner bacterial cell membrane of P. aeruginosa. Transmission electron microscopy revealed that treatment of P. aeruginosa with one of the HYL analogues caused total disintegration of bacterial cells. NMR spectroscopy was used to elucidate the structure-activity relationship for the effect of amino acid residue substitution in HYL.

• MeSH
• antibakteriální látky farmakologie   MeSH
• kationické antimikrobiální peptidy chemie   izolace a purifikace   farmakologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• molekulární struktura MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• Pseudomonas aeruginosa účinky léků   MeSH
• Staphylococcus aureus účinky léků   MeSH
• včelí jedy farmakologie   MeSH
• včely chemie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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

Antimicrobial peptides (AMP) which kill microbes by a fundamentally different mechanism of action than do traditional antibiotics are a new category of compounds capable of fighting resistant pathogens. The cationic AMP isolated from insects comprise a significant group among those 2000 peptides already registered in The Antimicrobial Peptide Database. The novel AMP include lucifensins – the insect defensins isolated from medicinal larvae of flies which are routinely used in maggot therapy and AMP ranking among -helical amphipathic peptides isolated from the venom of stinging hymenopterans. The AMP isolated from the venom show strong antimicrobial activities and low or moderate toxicity to eukaryotic cells. Systematic structure modification of these naturally occurring AMP resulted in their analogs with enhanced antimicrobial activities.

• Klíčová slova
• lucifensin,
• MeSH
• antibakteriální látky * izolace a purifikace   MeSH
• antiinfekční látky izolace a purifikace   MeSH
• defensiny * farmakokinetika   farmakologie   chemie   izolace a purifikace   klasifikace   terapeutické užití   MeSH
• farmaceutické databáze MeSH
• histatiny aplikace a dávkování   farmakokinetika   terapeutické užití   MeSH
• Hymenoptera * chemie   imunologie   MeSH
• kationické antimikrobiální peptidy * farmakokinetika   farmakologie   chemie   izolace a purifikace   terapeutické užití   MeSH
• lidé MeSH
• magaininy aplikace a dávkování   farmakokinetika   terapeutické užití   MeSH
• mikrobiální testy citlivosti MeSH
• molekulární struktura MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

The peptide named codesane (COD), consisting of 18 amino acid residues and isolated from the venom of wild bee Colletes daviesanus (Hymenoptera : Colletidae), falls into the category of cationic α-helical amphipathic antimicrobial peptides. In our investigations, synthetic COD exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria and Candida albicans but also noticeable hemolytic activity. COD and its analogs (collectively referred to as CODs) were studied for the mechanism of their action. The interaction of CODs with liposomes led to significant leakage of calcein entrapped in bacterial membrane-mimicking large unilamellar vesicles made preferentially from anionic phospholipids while no calcein leakage was observed from zwitterionic liposomes mimicking membranes of erythrocytes. The preference of CODs for anionic phospholipids was also established by the blue shift in the tryptophan emission spectra maxima when the interactions of tryptophan-containing COD analogs with liposomes were examined. Those results were in agreement with the antimicrobial and hemolytic activities of CODs. Moreover, we found that the studied peptides permeated both the outer and inner cytoplasmic membranes of Escherichia coli. This was determined by measuring changes in the fluorescence of probe N-phenyl-1-naphthylamine and detecting cytoplasmic β-galactosidase released during the interaction of peptides with E. coli cells. Transmission electron microscopy revealed that treatment of E. coli with one of the COD analogs caused leakage of bacterial content mainly from the septal areas of the cells.

• MeSH
• antibakteriální látky chemická syntéza   izolace a purifikace   farmakologie   MeSH
• Escherichia coli účinky léků   metabolismus   ultrastruktura   MeSH
• fluorescence MeSH
• fosfolipidy metabolismus   MeSH
• hemolýza účinky léků   MeSH
• hydrofobní a hydrofilní interakce MeSH
• kationické antimikrobiální peptidy genetika   izolace a purifikace   farmakologie   MeSH
• liposomy metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• permeabilita buněčné membrány účinky léků   MeSH
• racionální návrh léčiv MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• transmisní elektronová mikroskopie MeSH
• tryptofan chemie   MeSH
• včelí jedy chemie   genetika   izolace a purifikace   farmakologie   MeSH
• včely genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A novel antimicrobial peptide, designated macropin (MAC-1) with sequence Gly-Phe-Gly-Met-Ala-Leu-Lys-Leu-Leu-Lys-Lys-Val-Leu-NH2 , was isolated from the venom of the solitary bee Macropis fulvipes. MAC-1 exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria, antifungal activity, and moderate hemolytic activity against human red blood cells. A series of macropin analogs were prepared to further evaluate the effect of structural alterations on antimicrobial and hemolytic activities and stability in human serum. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were significantly increased while their toxicity against human red blood cells was decreased. The activity enhancement is related to the introduction of either l- or d-lysine in selected positions. Furthermore, all-d analog and analogs with d-amino acid residues introduced at the N-terminal part of the peptide chain exhibited better serum stability than did natural macropin. Data obtained by CD spectroscopy suggest a propensity of the peptide to adopt an amphipathic α-helical secondary structure in the presence of trifluoroethanol or membrane-mimicking sodium dodecyl sulfate. In addition, the study elucidates the structure-activity relationship for the effect of d-amino acid substitutions in MAC-1 using NMR spectroscopy.

• MeSH
• antibakteriální látky chemická syntéza   chemie   farmakologie   MeSH
• antifungální látky chemická syntéza   chemie   farmakologie   MeSH
• Bacillus subtilis účinky léků   MeSH
• erytrocyty účinky léků   MeSH
• Escherichia coli účinky léků   MeSH
• kationické antimikrobiální peptidy chemie   izolace a purifikace   farmakologie   MeSH
• Micrococcus luteus účinky léků   MeSH
• mikrobiální testy citlivosti MeSH
• molekulární modely MeSH
• Pseudomonas aeruginosa účinky léků   MeSH
• Staphylococcus aureus účinky léků   MeSH
• včelí jedy chemie   izolace a purifikace   farmakologie   MeSH
• včely chemie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Antimicrobial peptides are considered as promising supplements to or substitutes for conventional antibiotics due to their different mechanisms of action. Biological activities of antimicrobial peptides are influenced by their conformations. The solution conformations of 18 amino acid residues peptide melectin (MEP) and its synthetic analogues including C-terminal and N-terminal fragments were studied using electronic and vibrational circular dichroism spectroscopies.

• MeSH
• cirkulární dichroismus * MeSH
• kationické antimikrobiální peptidy * imunologie   izolace a purifikace   terapeutické užití   MeSH
• molekulární konformace * MeSH
• spektroskopie infračervená s Fourierovou transformací metody   přístrojové vybavení   využití   MeSH
• včelí jedy MeSH
• Publikační typ
• práce podpořená grantem MeSH

Úvod. Peptidický hormon hepcidin je hlavním faktorem ovlivňujícím homeostázu železa (Fe). Zajišťuje komunikaci mezi místy střádání Fe (hepatocyty a makrofágy) a místy, kde se Fe vstřebává (enterocyty), spotřebovává (erytroidní buňky) nebo recykluje a uvolňuje do krevního oběhu (makrofágy). Syntéza hepcidinu je regulována signály reagujícími na zánět, aktivitu erytropoézy, hladinu Fe, jeho zásoby v organismu a tenzi kyslíku. Zvýšení hladiny hepcidinu vede k zadržení Fe v enterocytech a makrofázích a snížení jeho hladiny v plazmě. Cíl práce. Zhodnotit hladiny hepcidinu a jejich diagnostický přínos u dětských pacientů s vybranými typy anémií: Diamondovou-Blackfanovou anémií (DBA), deficitem pyruvátkinázy (PK), sideropenickou anémií (IDA) a anémií doprovázející střevní záněty (IBD). Pacienti a metody. Hladina hepcidinu byla vyšetřena metodou proteomické analýzy u 33 dětí – 17 chlapců a 16 dívek (6 pacientů s DBA, 5 pacientů s deficitem PK, 10 pacientů se IDA a 12 pacientů s IBD) ve věku 6 měsíců až 18 let. Hladiny byly srovnávány se souborem 16 zdravých dětí vyšetřených před plánovanými chirurgickými výkony. Výsledky. U pacientů s těžkou formou DBA jsou hladiny hepcidinu signifikantně vyšší než u kontrol (p < 0,0005) i přes vysoké hladiny erytropoetinu, což je zcela rozdílný nález ve srovnání s pacienty s talasemií. Příčinou je pravděpodobně absence „erytroidního regulátoru“ při těžké redukci erytropoézy. U pacientů s deficitem PK je hladina hepcidinu naopak signifikantně nižší (p < 0,02), což teoreticky odpovídá akceleraci erytropoézy. Pro tuto teorii svědčí i zvýšená hladina potenciálního erytroidního regulátoru produkce hepcidinu: GDF15. Nízká hladina hepcidinu může přispívat k prohloubení přetížení Fe u těchto pacientů. U pacientů se IDA byly nalezeny signifikantně nižší hladiny (p < 0,01) hepcidinu, které jsou pravděpodobně výrazem zvýšené poptávky organismu po Fe. Mezi hladinami hepcidinu u pacientů s IBD a kontrolami nebyl překvapivě zjištěn signifikantní rozdíl. U dětských pacientů s IBD pravděpodobně převažuje skutečný deficit Fe nad klasickým obrazem anémie chronických chorob. Závěr. Stanovení hladin hepcidinu může zpřesnit diagnostiku celé řady anémií informací o aktuálním stavu metabolismu železa. Může poskytnout nejen důležitou informaci o aktuálním deficitu železa pro potřeby erytropoézy, stupni přetížení železem, ale i o aktuální schopnosti enterocytů vstřebávat Fe ze střevního lumen. Může být vodítkem při rozhodnutí o indikacích perorální a parenterální aplikace železa. Znalost hladiny hepcidinu může při korelaci s hladinami ostatních proteinů účastnících se regulace metabolismu Fe přinést velmi důležité poznatky o dosud nejasných aspektech homeostázy Fe v organismu.

Introduction. The peptide hormone hepcidin is the principal factor regulating iron homeostasis. It ensures communication between sites where iron is stored (hepatocytes and macrophages) and sites where it is absorbed (enterocytes), utilised (erythroid cells) or recycled and released into the bloodstream (macrophages). Hepcidin synthesis is regulated by signals responding to inflammation, erythropoietic activity, iron level, iron stores in the organism and oxygen tension. An increase in hepcidin levels leads to iron retention in enterocytes and macrophages and to a fall in iron plasma levels. Objective. To assess hepcidin levels and their diagnostic contribution in paediatric patients with selected types of anaemia: Diamond-Blackfan anaemia (DBA), pyruvate kinase deficiency (PK), iron deficiency anaemia (IDA) and anaemia in inflammatory bowel disease (IBD). Patients and Methods. Hepcidin levels were assessed in 33 children using the proteomic analysis method – 17 boys and 16 girls (6 patients with DBA, 5 patients with PK deficiency, 10 patients with IDA and 12 patients with IBD) aged 6 months–18 years. Hepcidin levels were compared to those in a cohort of 16 healthy children examined prior to the planned surgical interventions. Results. Hepcidin levels in patients with severe forms of DBA are significantly higher than those in the controls (p<0.0005) despite high erythropoietin levels. The high hepcidin levels in DBA patients represent a completely different finding, compared to patients with thalassemia. This is likely to be caused by the absence of an “erythroid regulator” associated with severely reduced erythropoiesis. On the contrary, hepcidin levels in patients with PK deficiency are significantly lower (p<0.02), which theoretically corresponds to erythropoiesis acceleration. This theory is also supported by the increased level of the potential erythroid regulator of hepcidin production: GDF15. Low hepcidin levels may contribute to greater iron overload in these patients. In patients with IDA, significantly lower hepcidin levels (p<0.01) are found; they are likely to express the organismęs increased iron-demands. Surprisingly, no significant difference between hepcidin levels in patients with IBD and the controls was observed. In paediatric patients with IBD, true iron deficiency probably prevails over the characteristic presentation of anaemia of chronic disease. Conclusion. Determination of hepcidin levels may help in the more accurate diagnosis of a whole range of anaemias by providing information on the current status of iron metabolism. It may provide important information not only regarding the current deficiency of iron required for erythropoiesis and the degree of iron overload, but also regarding the current capacity of enterocytes to absorb iron from the intestinal lumen. It may be useful as a guide for making decisions regarding the indication of oral and parenteral iron application. Hepcidin levels, in correlation with those of other proteins involved in the regulation of iron metabolism, may bring very important insights into aspects of iron homeostasis that are as yet unclear.

• Klíčová slova
• proteomická analýza, sideropenická anémie, anémie chronických chorob, nespecifické střevní záněty, deficit pyruvátkinázy,
• MeSH
• anemie diagnóza   etiologie   klasifikace   MeSH
• chronická nemoc MeSH
• diagnostické techniky molekulární metody   využití   MeSH
• Diamondova-Blackfanova anemie diagnóza   etiologie   MeSH
• dítě MeSH
• financování organizované MeSH
• hypochromní anemie diagnóza   etiologie   MeSH
• idiopatické střevní záněty diagnóza   MeSH
• kationické antimikrobiální peptidy izolace a purifikace   krev   MeSH
• lidé MeSH
• poruchy metabolismu železa diagnóza   etiologie   MeSH
• proteomika metody   trendy   MeSH
• pyruvátkinasa izolace a purifikace   nedostatek   MeSH
• statistika jako téma MeSH
• studie případů a kontrol MeSH
• výsledky a postupy - zhodnocení (zdravotní péče) MeSH
• Check Tag
• dítě MeSH
• lidé MeSH