This article describes the characterization and application of collagenase-based chitosan nanofiber membranes with rat burns. Electrospun chitosan nanofibers were functionalized with clostridial collagenase using carbodiimide chemistry. The immobilized collagenase was characterized by enzyme activity, kinetic constants, and dry storage stability measurements using a Pz-peptide substrate. The apparent kinetic constants KM and Vmax of immobilized collagenase showed a high affinity for the peptide substrate compared to the free enzyme. Drying of chitosan membranes with immobilized collagenase ensured 98 % stability of enzyme activity after rehydration. The effect of collagenase immobilized on chitosan nanofibers on the burn of the rat model was compared with a control treatment with chitosan nanofibers. The healing of the wound with both materials was terminated after 30 days at the same time, although the collagenase wound healed more rapidly during healing. The scar area size after the application of collagenase-containing chitosan nanofiber membranes was 31.6 % smaller than when only chitosan nanofibers were used.

• MeSH
• chitosan terapeutické užití   MeSH
• Clostridium histolyticum MeSH
• enzymy imobilizované MeSH
• hojení ran * účinky léků   MeSH
• krysa rodu rattus MeSH
• kůže zranění   MeSH
• mikrobiální kolagenasa * metabolismus   terapeutické užití   MeSH
• nanovlákna terapeutické užití   MeSH
• pilotní projekty MeSH
• rány a poranění farmakoterapie   patologie   MeSH
• stabilita enzymů MeSH
• výsledek terapie MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH

Polycyklické aromatické uhlovodíky (PAU) představují skupinu významných kontaminantů pracovního i životního prostředí. Při expozici PAU v pracovním prostředí se vedle inhalační expoziční cesty může významně uplatňovat i expozice dermální. Stávající experimentální údaje o intenzitě a rychlosti penetrace látek do systémové cirkulace jsou zatím omezené. Předkládaný článek se zabývá metodickou a interpretační problematikou transepidermální absorpce PAU in vitro. Byla sledována intenzita (Flux) a rychlost (Lag time) penetrace naftalenu, fenanthrenu, pyrenu a benzo[a]pyrenu přes epidermální membránu z prasečího boltce. Experiment byl prováděn ve vertikálních statických difuzních komůrkách dle Franze (n = 32) a jako rozpouštědlo byl použit isopropyl-myristát. Parametr Flux (nmol/cm2/hod) dosáhl hodnot 95,7 ± 45,5 u naftalenu, 19,5 ± 8,7 u fenanthrenu, 4,38 ± 1,98 u pyrenu a 0,21 ± 0,08 u benzo[a]pyrenu. Parametr Lag time (hod) hodnot 0,26 ± 0,17 u naftalenu, 2,12 ± 0,41 u fenanthrenu, 3,25 ± 0,50 u pyrenu a 11,2 ± 4,08 u benzo[a]pyrenu. Hodnota parametru Flux klesala s molární hmotností PAU, zatímco hodnota parametru Lag time s molární hmotností PAU stoupala. Množství PAU, které za daný časový úsek penetrovalo přes epidermální membránu, se pohybovalo mezi 0,24 % (benzo[a]pyren) až 0,84 % (fenanthren) aplikované dávky. Penetrace PAU přes epidermální membránu vykazovala, v porovnání s experimenty na plné kůži, nižší stupeň variability dat. Z výsledků vyplývá, že použití epidermální membrány by mohlo zpřesňovat jak odhad vnitřní dávky PAU po dermální expozici, tak i odhad souvisejícího zdravotního rizika v rámci konzervativního expozičního scénáře. Experimenty s epidermální membránou jsou však časově i experimentálně náročné, bez možnosti objektivní kontroly integrity epidermální membrány, což může vést k finanční náročnosti testování, ztrátám vzorků a v neposlední řadě i k nárůstu rozdílů hodnot dat, získaných v různých laboratořích.

Polycyclic aromatic hydrocarbons (PAHs) are a group of significant contaminants in the occupational and living environment. In addition to the inhalation route of exposure in the occupational environment, there is also significant the dermal route of exposure. Existing experimental data on the intensity and rate of penetration of these substances into systemic circulation are still limited. The presented paper is focused on methodological and interpretative issues of trans-epidermal absorption of PAHs in vitro. In this study, we assessed the intensity (Flux) and rate (Lag time) of penetration of naphthalene, phenanthrene, pyrene and benzo[a]pyrene through the epidermal membrane derived from a pig ear. The experiment was performed using static vertical Franz diffusion cells (n = 32) and isopropyl myristate was used as a solvent. Flux (nmol/cm2/hour) reached 95.7 ± 45.5 in the case of naphthalene, 19.5 ± 8.7 of phenanthrene, 4.38 ± 1.98 of pyrene and 0.21 ± 0.08 of benzo[a]pyrene. Lag time (hour) was 0.26 ± 0.17 in the case of naphthalene, 2.12 ± 0.41 of phenanthrene, 3.25 ± 0.50 of pyrene and 11.2 ± 4.08 of benzo[a]pyrene. The Flux value decreased with the molecular weight of PAHs, while the Lag time increased with the molecular weight of PAHs. The amount of PAHs that penetrated through the epidermal membrane in a given time period ranged between 0.24% (benzo[a]pyrene) and 0.84% (phenanthrene) of the applied dose. The penetration of PAHs through the epidermal membrane showed a lower degree of data variability compared to full thickness skin experiments. The results suggest that the use of the epidermal membrane could define more accurately both the estimation of the internal dose of PAHs after dermal exposure and the estimation of the associated health risk within a conservative exposure scenario. However, experiments with using the epidermal membrane are time consuming and experimentally demanding, with no option of an objective control of the integrity of the epidermal membrane, which can lead to costly testing, loss of samples and, finally, an increase in the differences in data values obtained in different laboratories.

• MeSH
• epidermis anatomie a histologie   patofyziologie   MeSH
• interpretace statistických dat MeSH
• kožní absorpce * fyziologie   MeSH
• modely u zvířat MeSH
• polycyklické aromatické uhlovodíky * škodlivé účinky   MeSH
• prasata MeSH
• techniky in vitro * metody   statistika a číselné údaje   MeSH
• ušní boltec MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH

OBJECTIVE: This study aimed to investigate whether out-of-hospital cardiac arrest (OHCA) may induce severe DNA damage measured using comet assay in successfully resuscitated humans and to evaluate a short-term prognostic role. METHODS: In this prospective, controlled, blinded study (1/2013-1/2014), 41 patients (age, 63±14 years) successfully resuscitated from non traumatic OHCA and 10 healthy controls (age, 53±17 years) were enrolled. DNA damage [double-strand breaks (DSBs) and single-strand breaks (SSBs)] was measured using comet assay in peripheral lymphocytes sampled at admission. Clinical data were recorded (according to Utstein style). A good short-term prognosis was defined as survival for 30 days. RESULTS: Among the patients, there were 71% (29/41) short-term survivors. After OHCA, DNA damage (DSBs and SSBs) was higher (11.0±7.6% and 0.79±2.41% in tail) among patients than among controls (1.96±1.63% and 0.02±0.03% in tail), and it was more apparent for DSBs (p<0.001 and p=0.085). There was no difference in the DNA damage between patients with cardiac and non-cardiac etiology, or between survivors and nonsurvivors. Among Utstein style parameters, ventricular fibrillation, asystole, and early electrical defibrillation influenced DSBs; none of the factors influenced SSBs. Factors influencing survival were SSBs, ventricular fibrillation, length of cardiopulmonary resuscitation by professionals ≤15 min, cardiogenic shock, and postanoxic encephalopathy. In contrast to DSBs [area under the curve (AUC)=0.520], SSBs seem to have a potential in prognostication (AUC=0.639). CONCLUSION: This study for the first time demonstrates revelation of DNA damage using comet assay in patients successfully resuscitated from OHCA. Whether DNA damage measured using comet assay may be a prognostic marker remains unknown, although our data may encourage some suggestions.

• MeSH
• analýza přežití MeSH
• dospělí MeSH
• dvojitá slepá metoda MeSH
• elektrická defibrilace MeSH
• kardiopulmonální resuscitace MeSH
• kometový test MeSH
• lidé středního věku MeSH
• lidé MeSH
• lymfocyty metabolismus   MeSH
• poškození DNA * MeSH
• prognóza MeSH
• prospektivní studie MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• senzitivita a specificita MeSH
• studie případů a kontrol MeSH
• zástava srdce mimo nemocnici mortalita   terapie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• Geografické názvy
• Turecko MeSH

AIMS: Goeckerman therapy is based on combined exposure to UV radiation (UVA, UVB) and crude coal tar (PAHs). Some indicators suggest a genotoxic hazard, however, the level of genotoxic risk of the therapy has not yet been investigated sufficiently. This study aims to assesss the genotoxic risk. METHODS: The studied group consisted of patients with chronic stable plaque psoriasis treated by Goeckerman therapy (n = 29). Heparin-treated peripheral blood samples were collected one day before the first treatment and immediately after the last procedure. The lymphocytes were isolated from the blood. The level of genotoxicity was evaluated using an alkaline version of the Comet assay which detects DNA single strand breaks (DNA-SSBs), a neutral version of the Comet assay which detects DNA double strand breaks (DNA-DSBs), and using chromosomal aberrations. RESULTS: The level of DNA-SSBs increased insignificantly (median; Q1-Q3): 1.4 (0.4; 0.1-1.4) vs. 2.5 (0.6; 0.3-2.7) %tDNA (P = 0.11) and the level of DNA-DSBs increased significantly: 7.8 (6.5; 3.4-10.5) vs. 20.7 (19.3; 14.2-24.6) % DNA (P < 0.001). The total number of aberrated cells (P < 0.001) and structurally aberrated cells (P < 0.001) increased significantly. CONCLUSION: The elevated levels of the DNA-DSBs and the chromosomal aberrations in the peripheral lymphocytes indicated a genotoxic hazard. However, the elevated level of the chromosomal abnormalities was below the upper level of the reference range for healthy Czech adults. While, the genotoxic risk appears to be low, Goeckerman treatment represents a further contribution to the lifetime load of genotoxic factors.

• MeSH
• chromozomální aberace účinky léků   účinky záření   MeSH
• chronická nemoc MeSH
• dehet uhelný škodlivé účinky   MeSH
• dospělí MeSH
• dvouřetězcové zlomy DNA účinky léků   účinky záření   MeSH
• keratolytika škodlivé účinky   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lymfocyty * MeSH
• psoriáza terapie   MeSH
• senioři MeSH
• terapie ultrafialovými paprsky škodlivé účinky   MeSH
• ultrafialové záření škodlivé účinky   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

In the present study, we investigated the anticancer action of the trithiolato arene ruthenium complex, [(η-p-MeC6H4Pr)2Ru2(μ-S-p-C6H4OH)3]Cl, named diruthenium-2, both in vitro and in vivo. The mechanism of antiproliferative, cytotoxic, and DNA-damaging activity, and the effect on expressions of cell cycle regulatory proteins were investigated using a WST-1-based proliferation assay, lactate dehydrogenase leakage assay, comet assay, flow cytometry, and western blot analysis. In-vivo anticancer activity was evaluated using Ehrlich tumor-bearing NMRI mice. Diruthenium-2 inhibited the growth of all cancer cell lines used, the most sensitive being gastric (AGS), breast cancer (BT-549, MCF-7, MDA-MB-231), and leukemic (HL-60, MOLT-4) cells. In MCF-7 cells, it caused a G1/S cell cycle arrest, along with an increase in the expression of protein p21 and cyclin B1. We also observed increased levels of MRN complex proteins, which, together with the results from the comet assay, indicate the formation of DNA double-strand breaks. In tumor-bearing mice, diruthenium-2 at doses of 3 and 5 mg/kg inhibits the growth of solid Ehrlich tumor, although weaker than cisplatin. However, it did not prolong the post-therapeutic survival. Our results suggest the in-vitro potential of diruthenium-2 should be further evaluated in studies using other in-vivo models.

• MeSH
• HL-60 buňky MeSH
• komplexní sloučeniny chemie   farmakologie   MeSH
• leukemie farmakoterapie   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory prsu farmakoterapie   MeSH
• nádory žaludku farmakoterapie   MeSH
• protinádorové látky chemie   farmakologie   MeSH
• ruthenium chemie   farmakologie   MeSH
• screeningové testy protinádorových léčiv MeSH
• xenogenní modely - testy protinádorové aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

PURPOSE: This study evaluates early changes in human mesenchymal stem cells (MSC) isolated from dental pulp and periodontal ligament after γ-irradiation and the effect of ataxia-telangiectasia mutated (ATM) inhibition. METHODS: MSC were irradiated with 2 and 20 Gy by (60)Co. For ATM inhibition, specific inhibitor KU55933 was used. DNA damage was measured by Comet assay and γH2AX detection. Cell cycle distribution and proteins responding to DNA damage were analyzed 2-72 h after the irradiation. RESULTS: The irradiation of MSC causes an increase in γH2AX; the phosphorylation was ATM-dependent. Irradiation activates ATM kinase, and the level of p53 protein is increased due to its phosphorylation on serine15. While this phosphorylation of p53 is ATM-dependent in MSC, the increase in p53 was not prevented by ATM inhibition. A similar trend was observed for Chk1 and Chk2. The increase in p21 is greater without ATM inhibition. ATM inhibition also does not fully abrogate the accumulation of irradiated MSC in the G2-phase of the cell-cycle. CONCLUSIONS: In irradiated MSC, double-strand breaks are tagged quickly by γH2AX in an ATM-dependent manner. Although phosphorylations of p53(ser15), Chk1(ser345) and Chk2(thr68) are ATM-dependent, the overall amount of these proteins increases when ATM is inhibited. In both types of MSC, ATM-independent mechanisms for cell-cycle arrest in the G2-phase are triggered.

• MeSH
• dávka záření MeSH
• DNA vazebné proteiny antagonisté a inhibitory   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mezenchymální kmenové buňky cytologie   fyziologie   účinky záření   MeSH
• nádorové supresorové proteiny antagonisté a inhibitory   MeSH
• periodontální vaz cytologie   fyziologie   účinky záření   MeSH
• protein-serin-threoninkinasy antagonisté a inhibitory   MeSH
• proteiny buněčného cyklu antagonisté a inhibitory   MeSH
• vztah dávky záření a odpovědi MeSH
• záření gama MeSH
• zubní dřeň cytologie   fyziologie   účinky záření   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

In recent years, α-tomatine has been studied for its anticancer activity. In the present study, we focused on the cytotoxic effect of α-tomatine in the MCF-7 human breast adenocarcinoma cell line, its mechanism of action, biotransformation and stability in the culture medium. We observed an inhibition of cell proliferation and viability at concentrations of 6 and 9 µM but then a recovery of cells occurred. The recovery was not caused by the biotransformation of α-tomatine in MCF-7 cells, but by a substantial decrease in the concentration of α-tomatine in the culture medium due to its binding with cholesterol. Regarding the mechanism of action of α-tomatine, we observed no DNA damage, no changes in the levels of the proteins p53 and p21(WAF1/Cip1), and no apoptosis (neither activated caspase-8 and -9, nor sub-G1 peak, or morphological signs). We found a loss of ATP in α-tomatine-treated cells. These results support the conclusion that α-tomatine does not induce apoptosis in the MCF-7 cell line.

• MeSH
• apoptóza účinky léků   MeSH
• buněčný cyklus účinky léků   MeSH
• cholesterol metabolismus   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• nádory prsu farmakoterapie   metabolismus   patologie   MeSH
• proliferace buněk účinky léků   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• tomatin aplikace a dávkování   analogy a deriváty   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The aim of the study was to develop a diet which causes stable hyperglycaemia and development of diabetes in female Zucker diabetic fatty (ZDF) rats. We also examined whether worsened wound healing is caused only by hyperglycaemia or whether it is caused by more factors. Four types of special diets with a different content of fat were fed to eight groups of 3–7 (fa/fa or fa/+) rats. The following diets were used: H1 (24.6% fat), H2 (33.2%), C13004 (25.6%), and St1 (3.4%). We detected significant diet-dependent changes of weight and concentration of glucose in animals with leptin mutation (fa/fa). All examined indicators were significantly (P < 0.001) higher in (fa/fa) animals compared to the fa/+ ones no matter what diet they ate. All diets with high-fat content caused increased glycaemia, but only the diet with 24.6% fat caused a significant (P < 0.01) increase of glycaemia. Our results have proved that this diet is the most suitable to invoke and keep hyperglycaemia. The diet with 25.6% fat is suitable to invoke stable slightly increased glycaemia (10 mmol/l) and hyperinsulinaemia. On the other hand, the diet with 33.2% fat is unsuitable. We did not observe a significant influence of diet on wound healing. We developed a new diet more suitable for induction of stable hyperglycaemia in female ZDF rats than commercially available mixtures. Our study is the first to present recommendations for adjusting a high-fat diet to produce stable hyperglycaemia and hyperinsulinaemia in the rat model.

• MeSH
• diabetes mellitus MeSH
• dieta * klasifikace   MeSH
• dietní tuky * MeSH
• hmotnostní přírůstek MeSH
• hojení ran MeSH
• hyperglykemie * MeSH
• inzulin krev   MeSH
• krevní glukóza * MeSH
• krysa rodu rattus MeSH
• leptin genetika   MeSH
• matrixová metaloproteinasa 3 genetika   MeSH
• modely nemocí na zvířatech MeSH
• mutace MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

The aim of our work was to evaluate mechanisms leading to radiosensitization of MOLT-4 leukemia cells following valproic acid (VA) treatment. Cells were pretreated with 2 mM VA for 24 h followed by irradiation with a dose of 0.5 or 1 Gy. The effect of both noxae, alone and combined, was detected 1 and 24 hours after the irradiation. Induction of apoptosis was evaluated by a flow cytometry. The extent of DNA damage was further determined by phosphorylation of histone H2AX using confocal microscopy. Changes in protein expression were identified by SDS-PAGE/immunoblotting. Two-millimolar VA increased apoptosis induction after irradiation as well as phosphorylation of H2AX and provokes an increase in the level of p53 and its phosphorylation at Ser392 in 4 h post-irradiation. Likewise, p21 protein reached its maximal expression in 4 h after the irradiation of VA-treated cells. Twenty four hours later, only the p53 phosphorylated at Ser15 was detected. At the same time, the protein mdm2 (negative regulator of p53) was maximally activated. The 24-hour treatment of MOLT-4 leukemia cells with 2 mM VA results in radiosensitizing, increases apoptosis induction, H2AX phosphorylation, and also p53 and p21 activation.

• MeSH
• apoptóza účinky léků   účinky záření   MeSH
• fosforylace účinky léků   účinky záření   MeSH
• histony genetika   metabolismus   MeSH
• inhibitory enzymů farmakologie   MeSH
• kyselina valproová farmakologie   MeSH
• leukemie metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• oprava DNA účinky léků   MeSH
• poškození DNA účinky léků   účinky záření   MeSH
• protoonkogenní proteiny c-mdm2 genetika   metabolismus   MeSH
• radiosenzibilizující látky farmakologie   MeSH
• regulace genové exprese účinky léků   účinky záření   MeSH
• rho proteiny vázající GTP genetika   metabolismus   MeSH
• záření gama MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cellular response to ionizing radiation-induced damage depends on the cell type and the ability to repair DNA damage. Some types of cells undergo apoptosis, whereas others induce a permanent cell cycle arrest and do not proliferate. Our study demonstrates two types of response of embryonic diploid fibroblasts WI-38 to ionizing radiation. In the WI-38 cells p53 is activated, protein p21 increases, but the cells are arrested in G2 phase of cell cycle. Some of the cells die by apoptosis, but in remaining viable cells p16 increases, senescence associated DNA-damage foci occur, and senescence-associated beta-galactosidase activity increases, which indicate stress-induced premature senescence.

• MeSH
• apoptóza genetika   účinky záření   MeSH
• buněčné linie MeSH
• diploidie MeSH
• embryonální kmenové buňky fyziologie   účinky záření   MeSH
• fibroblasty fyziologie   účinky záření   MeSH
• ionizující záření MeSH
• lidé MeSH
• poškození DNA genetika   účinky záření   MeSH
• stárnutí buněk genetika   účinky záření   MeSH
• viabilita buněk genetika   účinky záření   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• srovnávací studie MeSH