Strenuous exercise induces delayed-onset muscle damage including oxidative damage of cellular components. Oxidative stress to muscle cells impairs glucose uptake via disturbance of insulin signaling pathway. We investigated glucose uptake and insulin signaling in relation to oxidative protein modification in muscle after acute strenuous exercise. ICR mice were divided into sedentary and exercise groups. Mice in the exercise group performed downhill running exercise at 30 m/min for 30 min. At 24 hr after exercise, metabolic performance and insulin-signaling proteins in muscle tissues were examined. In whole body indirect calorimetry, carbohydrate utilization was decreased in the exercised mice along with reduction of the respiratory exchange ratio compared to the rested control mice. Insulin-stimulated uptake of 2-deoxy-[3H]glucose in damaged muscle was decreased after acute exercise. Tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and phosphatidyl-3-kinase/Akt signaling were impaired by exercise, leading to inhibition of the membrane translocation of glucose transporter 4. We also found that acute exercise caused 4-hydroxy-nonenal modification of IRS-1 along with elevation of oxidative stress in muscle tissue. Impairment of insulin-induced glucose uptake into damaged muscle after strenuous exercise would be related to disturbance of insulin signal transduction by oxidative modification of IRS-1.

• MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• glukosa metabolismus   MeSH
• inzulin metabolismus   MeSH
• kondiční příprava zvířat MeSH
• kosterní svaly metabolismus   MeSH
• myši inbrední ICR MeSH
• myši MeSH
• oxidace-redukce MeSH
• oxidační stres MeSH
• přenašeč glukosy typ 4 metabolismus   MeSH
• proteiny insulinového receptorového substrátu metabolismus   MeSH
• protoonkogenní proteiny c-akt metabolismus   MeSH
• signální transdukce MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

BACKGROUND: The implantable cardioverter-defibrillator (ICD) is the standard therapy to prevent sudden cardiac death in patients with coronary artery disease and unstable ventricular tachyarrhythmias. The prospective multinational SMS (Substrate Modification Study) was designed to assess whether prophylactic ablation of the arrhythmogenic substrate reduces or prevents the recurrence of ventricular tachycardia/ventricular fibrillation in such patients. METHODS AND RESULTS: Of 111 patients included in an intention-to-treat analysis, 54 were randomly assigned catheter ablation plus ICD implantation (ablation group: 68±8 years; 47 men), whereas 57 were assigned ICD implantation without catheter ablation (ICD-only group: 66±8 years; 46 men). Primary study end point was the time to first recurrence of ventricular tachycardia/ventricular fibrillation. ICD episodes were assessed and verified by an independent board. Patients were followed up for 2.3±1.1 years. The primary end point was reached by 25 ablation patients and 26 ICD-only patients. Two-year event-free survival was estimated at 49.0% (95% confidence interval, 33.3%-62.9%) in the former and 52.4% (36.7%-65.9%) in the latter groups. Comparison of episode incidence revealed no significant difference in the primary end point (P=0.84). In an Andersen-Gill regression model with multiple end point recurrences, the difference between the study arms significantly favored catheter ablation for both the primary end point and all but one of the predefined subgroups of detected arrhythmia events. CONCLUSIONS: SMS failed to meet the primary end point of time to first ventricular tachycardia/ventricular fibrillation recurrence. However, catheter ablation did reduce the total number of ICD interventions during the duration of follow-up. CLINICAL TRIAL REGISTRATION: https://clinicaltrials.gov. Unique identifier: NCT00170287.

• MeSH
• analýza přežití MeSH
• defibrilátory implantabilní * MeSH
• dospělí MeSH
• fibrilace komor diagnóza   mortalita   chirurgie   MeSH
• hodnocení rizik MeSH
• Kaplanův-Meierův odhad MeSH
• katetrizační ablace metody   mortalita   MeSH
• komorová tachykardie diagnóza   mortalita   chirurgie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mapování potenciálů tělesného povrchu MeSH
• náhlá srdeční smrt prevence a kontrola   MeSH
• nemoci koronárních tepen diagnóza   mortalita   chirurgie   MeSH
• neparametrická statistika MeSH
• prognóza MeSH
• proporcionální rizikové modely MeSH
• senioři MeSH
• stupeň závažnosti nemoci MeSH
• výsledek terapie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• randomizované kontrolované studie MeSH
• srovnávací studie MeSH

A simple, versatile, protein-repulsive, substrate-independent biomimetic surface modification is presented that is based on the creation of a PEO brush on a polydopamine anchoring layer and its capacity for selective follow-up modifications with various ligands using a copper-catalyzed alkyne-azide cycloaddition reaction. The desired surface concentration of peptide biomimetic ligands can be controlled by adjusting the peptide concentration in the reaction mixture, then measuring the activity of (125)I-radiolabeled peptides that are immobilized on the substrates. The performance of the prepared substrates is tested in cell cultures with MEF cells and a human ECC line.

• MeSH
• biomimetika * MeSH
• cyklizace MeSH
• kultivované buňky MeSH
• lidé MeSH
• povrchové vlastnosti MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A new route for coating various substrates with antifouling polymer layers was developed. It consisted in deposition of an amino-rich adhesion layer by means of RF magnetron sputtering of Nylon 6,6 followed by the well-controlled, surface-initiated atom transfer radical polymerization of antifouling polymer brushes initiated by bromoisobutyrate covalently attached to amino groups present in the adhesion layer. Polymer brushes of hydroxy- and methoxy-capped oligoethyleneglycol methacrylate and carboxybetaine acrylamide were grafted from bromoisobutyrate initiator attached to a 15 nm thick amino-rich adhesion layer deposited on gold, silicon, polypropylene, and titanium-aluminum-vanadium alloy surfaces. Well-controlled polymerization kinetics made it possible to control the thickness of the brushes at a nanometer scale. Zero fouling from single protein solutions and a reduction of more than 90% in the fouling from blood plasma observed on the uncoated surfaces was achieved. The feasibility of functionalization with bioactive compounds was tested by covalent attachment of streptavidin onto poly(oligoethylene glycol methacrylate) brush and subsequent immobilization of model antibodies and oligonucleotides. The procedure is nondestructive and does not require any chemical preactivation or the presence of reactive groups on the substrate surface. Contrary to current antifouling modifications, the developed coating can be built on various classes of substrates and preserves its antifouling properties even in undiluted blood plasma. The new technique might be used for fabrication of biotechnological and biomedical devices with tailor-made functions that will not be impaired by fouling from ambient biological media.

• MeSH
• mikroskopie atomárních sil MeSH
• povrchové vlastnosti MeSH
• proteiny chemie   MeSH
• spektrální analýza metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Rhomboid proteases are increasingly being explored as potential drug targets, but their potent and specific inhibitors are not available, and strategies for inhibitor development are hampered by the lack of widely usable and easily modifiable in vitro activity assays. Here we address this bottleneck and report on the development of new fluorogenic transmembrane peptide substrates, which are cleaved by several unrelated rhomboid proteases, can be used both in detergent micelles and in liposomes, and contain red-shifted fluorophores that are suitable for high-throughput screening of compound libraries. We show that nearly the entire transmembrane domain of the substrate is important for efficient cleavage, implying that it extensively interacts with the enzyme. Importantly, we demonstrate that in the detergent micelle system, commonly used for the enzymatic analyses of intramembrane proteolysis, the cleavage rate strongly depends on detergent concentration, because the reaction proceeds only in the micelles. Furthermore, we show that the catalytic efficiency and selectivity toward a rhomboid substrate can be dramatically improved by targeted modification of the sequence of its P5 to P1 region. The fluorogenic substrates that we describe and their sequence variants should find wide use in the detection of activity and development of inhibitors of rhomboid proteases.

• MeSH
• fluorescenční barviva chemie   MeSH
• kinetika MeSH
• liposomy MeSH
• peptidy metabolismus   MeSH
• proteasy metabolismus   MeSH
• rezonanční přenos fluorescenční energie MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH

Chondroitin sulfate proteoglycans inhibit regeneration, neuroprotection, and plasticity following spinal cord injury. The development of a second-generation chondroitinase ABC enzyme, capable of being secreted from mammalian cells (mChABC), has facilitated the functional recovery of animals following severe spinal trauma. The genetically modified enzyme has been shown to efficiently break down the inhibitory extracellular matrix surrounding cells at the site of injury, while facilitating cellular integration and axonal growth. However, the activity profile of the enzyme in relation to the original bacterial chondroitinase (bChABC) has not been determined. Here, we characterize the activity profile of mChABC and compare it to bChABC, both enzymes having been maintained under physiologically relevant conditions for the duration of the experiment. We show that this genetically modified enzyme can be secreted reliably and robustly in high yields from a mammalian cell line. The modifications made to the cDNA of the enzyme have not altered the functional activity of mChABC compared to bChABC, ensuring that it has optimal activity on chondroitin sulfate-A, with an optimal pH at 8.0 and temperature at 37 °C. However, mChABC shows superior thermostability compared to bChABC, ensuring that the recombinant enzyme operates with enhanced activity over a variety of physiologically relevant substrates and temperatures compared to the widely used bacterial alternative without substantially altering its kinetic output. The determination that mChABC can function with greater robustness under physiological conditions than bChABC is an important step in the further development of this auspicious treatment strategy toward a clinical application.

• Publikační typ
• časopisecké články MeSH

UNLABELLED: Protein turnover is essential in all living organisms for the maintenance of normal cell physiology. In eukaryotes, most cellular protein turnover involves the ubiquitin-proteasome pathway, in which proteins tagged with ubiquitin are targeted to the proteasome for degradation. In contrast, most bacteria lack a proteasome but harbor proteases for protein turnover. However, some actinobacteria, such as mycobacteria, possess a proteasome in addition to these proteases. A prokaryotic ubiquitination-like tagging process in mycobacteria was described and was named pupylation: proteins are tagged with Pup (prokaryotic ubiquitin-like protein) and directed to the proteasome for degradation. We report pupylation in another actinobacterium, Streptomyces coelicolor. Both the morphology and life cycle of Streptomyces species are complex (formation of a substrate and aerial mycelium followed by sporulation), and these bacteria are prolific producers of secondary metabolites with important medicinal and agricultural applications. The genes encoding the pupylation system in S. coelicolor are expressed at various stages of development. We demonstrated that pupylation targets numerous proteins and identified 20 of them. Furthermore, we established that abolition of pupylation has substantial effects on morphological and metabolic differentiation and on resistance to oxidative stress. In contrast, in most cases, a proteasome-deficient mutant showed only modest perturbations under the same conditions. Thus, the phenotype of the pup mutant does not appear to be due solely to defective proteasomal degradation. Presumably, pupylation has roles in addition to directing proteins to the proteasome. IMPORTANCE: Streptomyces spp. are filamentous and sporulating actinobacteria, remarkable for their morphological and metabolic differentiation. They produce numerous bioactive compounds, including antifungal, antibiotic, and antitumor compounds. There is therefore considerable interest in understanding the mechanisms by which Streptomyces species regulate their complex physiology and production of bioactive compounds. We studied the role in Streptomyces of pupylation, a posttranslational modification that tags proteins that are then directed to the proteasome for degradation. We demonstrated that the absence of pupylation had large effects on morphological differentiation, antibiotic production, and resistance to oxidative stress in S. coelicolor. The phenotypes of pupylation and proteasome-defective mutants differed and suggest that pupylation acts in a proteasome-independent manner in addition to its role in proteasomal degradation.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• delece genu MeSH
• molekulární sekvence - údaje MeSH
• posttranslační úpravy proteinů MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• Streptomyces coelicolor genetika   růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

While polymers are widely utilized materials in the biomedical industry, they are rarely used in an unmodified state. Some kind of a surface treatment is often necessary to achieve properties suitable for specific applications. There are multiple methods of surface treatment, each with their own pros and cons, such as plasma and laser treatment, UV lamp modification, etching, grafting, metallization, ion sputtering and others. An appropriate treatment can change the physico-chemical properties of the surface of a polymer in a way that makes it attractive for a variety of biological compounds, or, on the contrary, makes the polymer exhibit antibacterial or cytotoxic properties, thus making the polymer usable in a variety of biomedical applications. This review examines four popular methods of polymer surface modification: laser treatment, ion implantation, plasma treatment and nanoparticle grafting. Surface treatment-induced changes of the physico-chemical properties, morphology, chemical composition and biocompatibility of a variety of polymer substrates are studied. Relevant biological methods are used to determine the influence of various surface treatments and grafting processes on the biocompatibility of the new surfaces-mammalian cell adhesion and proliferation is studied as well as other potential applications of the surface-treated polymer substrates in the biomedical industry.

• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

• MeSH
• anaerobní bakterie enzymologie   genetika   klasifikace   MeSH
• bachor mikrobiologie   MeSH
• bakteriální chromozomy fyziologie   MeSH
• finanční podpora výzkumu jako téma MeSH
• molekulární evoluce MeSH
• restrikční enzymy analýza   fyziologie   MeSH
• substrátová specifita MeSH

Methanosarcina barkeri (DSM 800) is a metabolically versatile methanogen and shows distinct metabolic status under different substrate regimes. However, the mechanisms underlying distinct transcriptional profiles under different substrate regimes remain elusive. In this study, based on transcriptional analysis, the growth performances and gene expressions of M. barkeri fed on acetate, H2 + CO2, and methanol, respectively, were investigated. M. barkeri showed higher growth performances under methanol, followed by H2 + CO2 and acetate, which corresponded well with the variations of gene expressions. The α diversity (evenness) of gene expressions was highest under the acetate regime, followed by H2 + CO2 and methanol, and significantly and negatively correlated with growth performances. The gene co-expression analysis showed that "Energy production and conversion," "Coenzyme transport and metabolism," and "Translation, ribosomal structure, and biogenesis" showed deterministic cooperation patterns of intra- and inter-functional classes. However, "Posttranslational modification, protein turnover, chaperones" showed exclusion with other functional classes. The gene expressions and especially the relationships among them potentially drove the shifts of metabolic status under different substrate regimes. Consequently, this study revealed the diversity-related ecological strategies that a high α diversity probably provided more fitness and tolerance under natural environments and oppositely a low α diversity strengthened some specific physiological functions, as well as the co-responses of gene expressions to different substrate regimes.

• MeSH
• archeální RNA genetika   MeSH
• Euryarchaeota genetika   metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• kultivační média chemie   MeSH
• kyselina octová chemie   MeSH
• methanol chemie   MeSH
• Methanosarcina barkeri genetika   metabolismus   MeSH
• oxid uhličitý chemie   MeSH
• regulace genové exprese u archeí MeSH
• sekvenční analýza RNA MeSH
• substrátová specifita MeSH
• transkriptom * MeSH
• vodík chemie   MeSH
• Publikační typ
• časopisecké články MeSH