Host blood proteins, represented mainly by hemoglobin and serum albumin, serve as the ultimate source of amino acids needed for de novo protein synthesis during tick development and reproduction. While uptake and processing of hemoglobin by tick gut cells have been studied in detail, molecular mechanisms of host serum albumin degradation remain unknown. In this work, we have used artificial membrane feeding of Ixodes ricinus females on a hemoglobin-free diet in order to characterize the proteolytic machinery involved in albuminolysis. Morphological comparisons of ticks fed on whole blood (BF) and serum (SF) at microscopic and ultrastructural levels showed that albumin and hemoglobin have different trafficking routes in tick gut cells. Analysis in vitro with selective inhibitors demonstrated that albumin is degraded at an acidic pH by a network of cysteine and aspartic peptidases with predominant involvement of cysteine cathepsins having endo- and exopeptidase activities. The cleavage map of albumin and the roles of individual peptidases in albumin degradation were determined. These results indicate that the albuminolytic pathway is controlled by the same proteolytic system that is responsible for hemoglobinolysis. This was further supported by the overall similarity of gut peptidase profiles in SF and BF ticks at the transcriptional and enzymatic activity levels. In conclusion, our work provides evidence that although hemoglobin and albumin are transported differentially during heterophagy they are digested by a common multienzyme proteolytic network. This central digestive system, critical for successful blood feeding in tick females, thus represents a valuable target for novel anti-tick interventions.

• Klíčová slova
• Albumin digestion, Gut, Proteolysis, Tick,
• MeSH
• aspartátové proteasy metabolismus   MeSH
• cysteinové proteasy metabolismus   MeSH
• hemoglobiny metabolismus   MeSH
• klíště enzymologie   MeSH
• koncentrace vodíkových iontů MeSH
• proteolýza * MeSH
• sérový albumin metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aspartátové proteasy MeSH
• cysteinové proteasy MeSH
• hemoglobiny MeSH
• sérový albumin MeSH

Liquid chromatography coupled with mass spectrometry is widely used in the field of proteomic analysis after off-line protein digestion. On-line digestion with chromatographic column connected in a series with immobilized enzymatic reactor is not often used approach. In this work we investigated the impact of chromatographic conditions on the protein digestion efficiency. The investigation of trypsin reactor activity was performed by on-line digestion of N-α-benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA), followed by separation of the digests on the mixed-mode column. Two trypsin column reactors with the different trypsin coverage on the bridged ethylene hybrid particles were evaluated. To ensure optimal trypsin activity, the separation temperature was set at 37.0 °C and the pH of the mobile phase buffer was maintained at 8.5. The on-line digestion itself ongoing during the initial state of gradient was carried out at a low flow rate using a mobile phase that was free of organic modifiers. Proteins such as cytochrome C, enolase, and myoglobin were successfully digested on-line without prior reduction or alkylation, and the resulting peptides were separated using a mixed-mode column. Additionally, proteins that contain multiple cysteines, such as α-lactalbumin, albumin, β-lactoglobulin A, and conalbumin, were also successfully digested on-line (after reduction and alkylation). Moreover, trypsin immobilized enzymatic reactors were utilized for over 300 injections without any noticeable loss of digestion activity.

• Klíčová slova
• Immobilized enzymatic reactor, Mass spectrometry, Mixed-mode column, On-line protein digestion, Trypsin digestion,
• MeSH
• alkylace MeSH
• enzymy imobilizované MeSH
• laktalbumin * MeSH
• proteolýza MeSH
• proteomika * MeSH
• trypsin MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• enzymy imobilizované MeSH
• laktalbumin * MeSH
• trypsin MeSH

The ingestion of chromogenic or fluorescent substrates for protease detection enables the visualization of digestive processes in mites in vivo due to their transparent bodies. The substrates for protease detection were offered to Lepidoglyphus destructor, and the resulting signals were observed in specimens under a compound microscope. The protease activity was successfully localized using chromogenic substrates (azoalbumin, AAPpNA, SAAPFpNA, elastin-orcein, SA(3) pNA, ZRRpNA, ArgpNA, and MAAPMpNA) and fluorescent substrates (casein-fluorescein, albumin-fluorescein, AAPAMC, BAAMC, ZRRAMC, ArgAMC, and AGPPPAMC). No activity was detected using the keratin azure and BApNA substrates. In the mesodeum, trypsin-like activity generated by hydrolysis of the BApNA substrate was not observed, but the BAAMC substrate allowed the visualization of trypsin-like activity in food boli in the posterior mesodeum. The results indicate that cathepsins B, D, and G and cathepsin H or aminopeptidase-like activities are present in the midgut of L. destructor. Among these activities, cathepsin D-like activity was identified for the first time in the gut of L. destructor. All proteases mentioned are produced in the mesodeal lumen and form the food bolus together with ingested food, afterward passing through the gut to be defecated. The method used enables the visualization of protease activities in the gut of transparent animals.

• MeSH
• dietní proteiny metabolismus   MeSH
• gastrointestinální trakt anatomie a histologie   enzymologie   MeSH
• proteasy metabolismus   MeSH
• roztoči anatomie a histologie   fyziologie   MeSH
• trávení * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dietní proteiny MeSH
• proteasy MeSH

The aim of this study was to evaluate the influence of model (alcohol, sugar, salt, protein and acid) and real foods and beverages on the viability of probiotics during incubation and artificial digestion. Viability of monocultures Lactobacillus acidophilus CCM4833 and Bifidobacterium breve CCM7825T, and a commercial mixture of 9 probiotic bacterial strains, was tested by cultivation assay and flow cytometry. In model foods, the best viability was determined in the presence of 0.2 g/L glucose, 10% albumin and 10% ethanol. As the most suitable real food for probiotic survival, complex protein and carbohydrate substrates were found, such as beef broth, potato salad with pork, chicken with rice, chocolate spread, porridge and yoghurt. The best liquid was milk and meat broth, followed by Coca-Cola, beer and coffee. Viability of probiotics was higher when consumed with meals than with beverages only. Addition of prebiotics increased the viability of probiotics, especially in presence of instant and fast foods. Generally, the highest viability of probiotics during artificial digestion was observed in mixed culture in the presence of protein, sugar and fat, or their combination. The increase of cell viability observed in such foods during model digestion may further contribute to the positive effect of probiotics on human health.

• Klíčová slova
• cell viability, food matrices, food stress, model digestion, probiotics,
• Publikační typ
• časopisecké články MeSH

Non-enzymatic posttranslational modifications of bovine serum albumin (BSA) by various oxo-compounds (glucose, ribose, glyoxal and glutardialdehyde) have been investigated using high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE). Both of these methods used mass spectrometric (MS) detection. Three enzymes (trypsin, pepsin, proteinase K) were used to digest glycated BSA. The extent of modification depended on the selected oxo-compound. Reactivity increased progressively from glucose to glutardialdehyde (glucose

• MeSH
• elektroforéza kapilární metody   MeSH
• hmotnostní spektrometrie metody   MeSH
• posttranslační úpravy proteinů MeSH
• sérový albumin hovězí chemie   MeSH
• skot MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• sérový albumin hovězí MeSH

BACKGROUND: The endothelial glycocalyx (EG) plays a vital role in the physiology and pathophysiology of human microcirculation. Having relevant EG damage model would be important tool for testing new interventions aiming at EG protection and recovery. We describe the first in vivo EG damage model in pig. OBJECTIVE: To investigate the course of animal EG damage induced by specific enzymes. MATERIAL AND METHODS: Four anesthetized piglets received enzymes: 1g hyaluronidase and 25 IU heparanase I intravenously. Blood and urine samples were collected at baseline and 20/40/60/80/100/120 min for detecting markers of endothelial and EG function. Sublingual microcirculation and EG thickness were assessed by Side-stream Dark Field (SDF) imaging and Perfused Boundary Region (PBR) respectively. EG of the mesentery artery was visualized in fluorescent microscopy. RESULTS: Biochemical marker of EG damage syndecan-1 showed temporary increase with return to baseline and was reflected by PBR values. Albumin levels suggested brief period of capillary leakage (decrease in the serum, increase in the urine) with a trend to normalization. Urine glycosaminoglycans peaked at 120 minutes. Microcirculatory perfusion parameter showed significant alteration. Diffusion parameters were altered with no statistical significance. CONCLUSION: EG damage induced by specific enzymes was reflected by temporary changes of biochemical makers together with alteration of microcirculation and changes in fluorescent microscopy of EG layer. Our results support to further validate presented model of EG damage on a larger number of animals.

• Klíčová slova
• Microcirculation, albuminuria, endothelial glycocalyx, heparanase, hyaluronidase,
• MeSH
• glykokalyx * MeSH
• kapiláry MeSH
• mikrocirkulace MeSH
• pilotní projekty MeSH
• prasata MeSH
• trávení MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The major objective of proteomics is to identify and examine the large numbers of proteins extracted from complex biological systems. This is generally achieved by combining various techniques of protein separation with a mass spectrometric analysis of proteins that are digested enzymatically. Recently, several alternatives to this standard protocol have been developed for efficient and fast protein digestion. One option is the use of modified trypsin instead of native trypsin for the in-gel digestion of proteins. Microwave, ultrasonic-assisted protein enzymatic digestion and proteolysis accelerated by infrared radiation are other suitable alternatives. The application of the variable performance of the fast enzymatic digestion of proteins by using different techniques is reported here. The advantage of these methods is to have the ability to detect proteins in a shorter span of time. For example, using alternative protein digestion takes only minutes, in contrast to the several hours required by conventional methods. To demonstrate the suitability of this fast procedure, the digestion of carbonic anhydrase, bovine serum albumin, lysozyme and proteins extracted from plants (Hordeum vulgare, Arabidopsis thaliana) were used. Considering that the required reaction time for the conventional method is much longer, these applied methodic approaches tend to give in-gel digestion a much higher efficiency rating. This study examines the fast, efficient and low-cost proteolytic strategies for the digestion process, and for protein identification based on the use of ultrasound and infrared technology. In addition, comparisons of the applied techniques were studied. Several differences were found, suggesting the potential use of proteolysis accelerated by infrared radiation.

• MeSH
• elektroforéza v polyakrylamidovém gelu metody   MeSH
• molekulární sekvence - údaje MeSH
• peptidové fragmenty analýza   chemie   metabolismus   MeSH
• proteiny analýza   chemie   metabolismus   MeSH
• proteomika metody   MeSH
• rostlinné proteiny MeSH
• sekvence aminokyselin MeSH
• skot MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• trypsin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• peptidové fragmenty MeSH
• proteiny MeSH
• rostlinné proteiny MeSH
• trypsin MeSH

• MeSH
• albuminy * MeSH
• gastrektomie MeSH
• gastrointestinální nemoci diagnóza   dietoterapie   metabolismus   MeSH
• lidé MeSH
• radioizotopy chromu * MeSH
• trávicí systém metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• Názvy látek
• albuminy * MeSH
• radioizotopy chromu * MeSH

Carnivorous plants have evolved in nutrient-poor wetland habitats. They capture arthropod prey, which is an additional source of plant growth limiting nutrients. One of them is nitrogen, which occurs in the form of chitin and proteins in prey carcasses. In this study, the nutritional value of chitin and protein and their digestion traits in the carnivorous sundew Drosera capensis L. were estimated using stable nitrogen isotope abundance. Plants fed on chitin derived 49% of the leaf nitrogen from chitin, while those fed on the protein bovine serum albumin (BSA) derived 70% of its leaf nitrogen from this. Moreover, leaf nitrogen content doubled in protein-fed in comparison to chitin-fed plants indicating that the proteins were digested more effectively in comparison to chitin and resulted in significantly higher chlorophyll contents. The surplus chlorophyll and absorbed nitrogen from the protein digestion were incorporated into photosynthetic proteins - the light harvesting antennae of photosystem II. The incorporation of insect nitrogen into the plant photosynthetic apparatus may explain the increased rate of photosynthesis and plant growth after feeding. This general response in many genera of carnivorous plants has been reported in many previous studies.

• Klíčová slova
• Carnivorous plant, Chitin, Chlorophyll, Drosera, Nitrogen uptake, Photosynthesis, Plant chitinase,
• MeSH
• biologické pigmenty metabolismus   MeSH
• biomasa MeSH
• chitin farmakologie   MeSH
• Drosera účinky léků   metabolismus   MeSH
• dusík farmakologie   MeSH
• izotopy dusíku MeSH
• listy rostlin účinky léků   metabolismus   MeSH
• sérový albumin hovězí farmakologie   MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biologické pigmenty MeSH
• chitin MeSH
• dusík MeSH
• izotopy dusíku MeSH
• sérový albumin hovězí MeSH

In this work, magnetosomes produced by microorganisms were chosen as a suitable magnetic carrier for covalent immobilization of thermostable trypsin conjugates with an expected applicability for efficient and rapid digestion of proteins at elevated temperatures. First, a biogenic magnetite was isolated from Magnetospirillum gryphiswaldense and its free surface was coated with the natural polysaccharide chitosan containing free amino and hydroxy groups. Prior to covalent immobilization, bovine trypsin was modified by conjugating with α-, β- and γ-cyclodextrin. Modified trypsin was bound to the magnetic carriers via amino groups using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling reagents. The magnetic biomaterial was characterized by magnetometric analysis and electron microscopy. With regard to their biochemical properties, the immobilized trypsin conjugates showed an increased resistance to elevated temperatures, eliminated autolysis, had an unchanged pH optimum and a significant storage stability and reusability. Considering these parameters, the presented enzymatic system exhibits properties that are superior to those of trypsin forms obtained by other frequently used approaches. The proteolytic performance was demonstrated during in-solution digestion of model proteins (horseradish peroxidase, bovine serum albumin and hen egg white lysozyme) followed by mass spectrometry. It is shown that both magnetic immobilization and chemical modification enhance the characteristics of trypsin making it a promising tool for protein digestion.

• MeSH
• cyklodextriny chemie   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• enzymy imobilizované chemie   metabolismus   MeSH
• magnetické nanočástice chemie   MeSH
• Magnetospirillum chemie   metabolismus   MeSH
• opakované použití vybavení MeSH
• oxid železnato-železitý chemie   izolace a purifikace   metabolismus   MeSH
• proteiny chemie   metabolismus   MeSH
• skot MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• stabilita enzymů MeSH
• teplota MeSH
• trypsin chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyklodextriny MeSH
• enzymy imobilizované MeSH
• magnetické nanočástice MeSH
• oxid železnato-železitý MeSH
• proteiny MeSH
• trypsin MeSH