OBJECTIVES: While controlled thermal changes in subcutaneous tissue have been used to trigger apoptosis of fat cells and have been proven clinically efficacious, another mechanism of electromagnetic stress suggests that fat apoptosis could be achieved by a non-thermal manner as well. This animal model study investigates the use of a non-invasive high-intensity magnetic field device to induce apoptosis in fat cells. METHODS: Yorkshire pigs (N = 2) received one treatment (30 minutes) in the abdominal area using a High-Intensity Focused Electromagnetic (HIFEM) device. Punch biopsy samples of fat tissue and blood samples were collected at the baseline, 1 and 8 hours after the treatment. Biopsy samples were sectioned and evaluated for the levels of an apoptotic index (AI) by the TUNEL method. Statistical significance was examined using the rANOVA and Tukey's test (α 5%). Biopsy samples were also assessed for molecular biomarkers. Blood samples were evaluated to determine changes related to fat and muscle metabolism. Free fatty acids (FFA), triacylglycerol (TG), glycerol and glucose (Glu) were used as the main biomarkers of fat metabolism. Creatinine, creatinine kinase (CK), lactate dehydrogenase (LDH) and interleukin 6 (IL6) served as the main biomarkers to evaluate muscle metabolism. RESULTS: In treated pigs, a statistically significant increase in the apoptotic index (AI) (P = 1.17E-4) was observed. A significant difference was found between AI at baseline (AI = 18.75%) and 8-hours post-treatment (AI = 35.95%). Serum levels of fat and muscle metabolism indicated trends (FFA -0.32 mmol · l-1 , -28.1%; TG -0.24 mmol · l-1 , -51.8%; Glycerol -5.68 mg · l-1 , -54.8%; CK +67.58 μkat · l-1 , +227.8%; LDH +4.9 μkat · l-1 ,+35.4%) suggesting that both adipose and muscle tissue were affected by HIFEM treatment. No adverse events were noted to skin and surrounding tissue. CONCLUSIONS: Application of a high-intensity electromagnetic field in a porcine model results in adipocyte apoptosis. The analysis of serum levels suggests that HIFEM treatment influences fat and muscle metabolism. Lasers Surg. Med. 51:47-53, 2019. © 2018 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.

• MeSH
• apoptóza účinky záření   MeSH
• biologické markery krev   MeSH
• biopsie MeSH
• břišní tuk účinky záření   MeSH
• koncové značení zlomů DNA in situ MeSH
• magnetoterapie metody   MeSH
• prasata MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Apoptosis has been recognized as a type of programmed cell death connected with characteristic morphological and biochemical changes in cells. This programmed cell death plays an important role in the genesis of a number of physiological and pathological processes. Thus, it can be very important to detect the signs of apoptosis in a study of cellular metabolism. The present paper provides an overview of methods often being used for detecting DNA fragmentation as one of the most specific findings in apoptosis. To date, three routine assays have been developed for detecting DNA fragmentation: DNA ladder assay, TUNEL assay, and comet assay. All these methods differ in their principles for detecting DNA fragmentation. DNA ladder assay detects the characteristic "DNA ladder" pattern formed during internucleosomal cleavage of DNA. Terminal deoxynUcleotidyl transferase Nick-End Labeling (TUNEL) assay detects DNA strand breaks using terminal deoxynucleotidyl transferase catalyzing attachment of modified deoxynucleotides on the DNA strand breaks. Comet assay can be used for detecting nucleus breakdown producing single/double-strand DNA breaks. The aim of this review is to describe the present knowledge on these three methods, including optimized approaches, techniques, and limitations.

• MeSH
• apoptóza genetika   fyziologie   MeSH
• biotest metody   MeSH
• DNA analýza   genetika   metabolismus   MeSH
• fragmentace DNA * MeSH
• kometový test metody   MeSH
• koncové značení zlomů DNA in situ metody   MeSH
• lidé MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Human amniotic membrane (HAM) is used as an allograft in regenerative medicine or as a source of pluripotent cells for stem cell research. Various decontamination protocols and solutions are used to sterilize HAM before its application, but little is known about the toxicity of disinfectants on HAM cells. In this study, we tested two decontamination solutions, commercial (BASE·128) and laboratory decontamination solution (LDS), with an analogous content of antimycotic/antibiotics for their cytotoxic effect on HAM epithelial (EC) and mesenchymal stromal cells (MSC). HAM was processed in a standard way, placed on nitrocellulose scaffold, and decontaminated, following three protocols: (1) 6 h, 37 °C; (2) 24 h, room temperature; (3) 24 h, 4 °C. The viability of EC was assessed via trypan blue staining. The apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). The mean % (±SD) of dead EC (%DEC) from six fresh placentas was 12.9 ± 18.1. Decontamination increased %DEC compared to culture medium. Decontamination with BASE·128 for 6 h, 37 °C led to the highest EC viability (81.7%). Treatment with LDS at 24 h, 4 °C resulted in the lowest EC viability (55.9%) in the set. MSC were more affected by apoptosis than EC. Although the BASE·128 expresses lower toxicity compared to LDS, we present LDS as an alternative decontamination solution with a satisfactory preservation of cell viability. The basic formula of LDS will be optimised by enrichment with nutrient components, such as glucose or vitamins, to improve cell viability.

• MeSH
• amnion cytologie   MeSH
• apoptóza MeSH
• dekontaminace metody   MeSH
• koncové značení zlomů DNA in situ MeSH
• kryoprezervace metody   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mezenchymální kmenové buňky cytologie   MeSH
• počet buněk MeSH
• těhotenství MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

This study examined the impact of cow body condition on the quality of bovine preimplantation embryos. The embryos (n = 107) were flushed from dairy cows and classified according to a five-point scale body condition score (BCS2 n = 17; BCS3 n = 31; BCS4 n = 11) on the 7th day after insemination and then analyzed for development, dead cell index (DCI), cell number and actin cytoskeleton quality. The highest embryo recovery rate (P < 0.05) was recorded in the BCS3 group and the lowest in the BCS4 group. More transferable (morula, blastocyst) embryos were obtained from the BCS4 cows (79%), compared with the BCS2 (64%) or BCS3 (63%) animals. However, cell numbers were higher in the BCS2 and BCS3 groups (P < 0.05) compared with the BCS4 embryos. Conversely, the DCI was lowest in the BCS2 (3.88%; P < 0.05) and highest in the BCS4 (6.56%) embryos. The proportion of embryos with the best actin quality (grade I) was higher in the BCS2 and BCS3 cows compared with the BCS4 group. Almost 25% of all embryos showed fragmented morphology and a higher DCI (5.65%) than normal morulas (1.76%). More fragmented embryos were revealed in the BCS2 (28.6%) and BCS4 (31.25%) groups, and less (19.15%) in the BCS3 group. The cell numbers in such embryos were lower in the BCS4 (22.57) than in the BCS2 (46.25) or BCS3 (42.4) groups. In conclusion, the body condition of dairy cows affects the quality of preimplantation embryos. A BCS over 3.0 resulted in a higher incidence of poor (fragmented) embryos.

• MeSH
• apoptóza MeSH
• blastocysta cytologie   metabolismus   MeSH
• buněčné dělení MeSH
• fertilizace in vitro metody   MeSH
• koncové značení zlomů DNA in situ MeSH
• konfokální mikroskopie MeSH
• mikrofilamenta metabolismus   MeSH
• mlékárenství MeSH
• morula cytologie   metabolismus   MeSH
• počet buněk MeSH
• přenos embrya metody   MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Abnormal aggregation of Tau in glial cells has been reported in Alzheimer's disease (AD) and other tauopathies; however, the pathological significance of these aggregates remains unsolved to date. In this study, we evaluated whether full-length Tau (Tau441) and its aspartic acid421-truncated Tau variant (Tau421) produce alterations in the normal organization of the cytoskeleton and plasma membrane (PM) when transiently expressed in cultured C6-glial cells. Forty-eight hours post-transfection, abnormal microtubule bundling was observed in the majority of the cells, which expressed either Tau441 or Tau421. Moreover, both variants of Tau produced extensive PM blebbing associated with cortical redistribution of filamentous actin (F-Actin). These effects were reverted when Tau-expressing cells were incubated with drugs that depolymerize F-Actin. In addition, when glial cells showing Tau-induced PM blebbing were incubated with inhibitors of the Rho-associated protein kinase (ROCK) signaling pathway, both formation of abnormal PM blebs and F-Actin remodeling were avoided. All of these effects were initiated upstream by abnormal Tau-induced microtubule bundling, which may release the microtubule-bound guanine nucleotide exchange factor-H1 (GEF-H1) into the cytoplasm in order to activate its major effector RhoA-GTPase. These results may represent a new mechanism of Tau toxicity in which Tau-induced microtubule bundling produces activation of the Rho-GTPase-ROCK pathway that in turn mediates the remodeling of cortical Actin and PM blebbing. In AD and other tauopathies, these Tau-induced abnormalities may occur and contribute to the impairment of glial activity.

• MeSH
• aktiny účinky léků   metabolismus   MeSH
• buněčná membrána účinky léků   metabolismus   patologie   MeSH
• buněčné linie MeSH
• cytoplazma metabolismus   MeSH
• elektroforéza MeSH
• fluorescenční protilátková technika MeSH
• kinázy asociované s rho metabolismus   MeSH
• koncové značení zlomů DNA in situ MeSH
• konfokální mikroskopie MeSH
• krysa rodu rattus MeSH
• neuroglie účinky léků   metabolismus   patologie   MeSH
• proteiny tau genetika   metabolismus   MeSH
• rhoA protein vázající GTP metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• transfekce MeSH
• tubulin metabolismus   MeSH
• výměnné faktory guaninnukleotidů metabolismus   MeSH
• western blotting MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Apoptosis is an active energy-consuming mechanism of cell death, which may contribute to heart failure in patients with dilated cardiomyopathy. Dilated cardiomyopathy is a common clinical outcome of many prolonged cardiac insults, and therefore is considered as the most prevalent form of cardiomyopathy. Loss of heart mass is highly correlated with the heart failure and mortality, thus the purpose of this study was to define the apoptotic index in patients with dilated cardiomyopathy. Apoptosis was detected by the TUNEL method in 30 patients. Biopsies were obtained from the left ventricle, and at least three specimens were taken. TUNEL-positive cardiomyocytes were found in 26 of 30 cases (86.7 %) and the mean apoptotic index for the entire specimen series was 5.41 ± 1.70 %. The analysis showed that patients with dilated cardiomyopathy had significantly higher apoptotic index (P < 0.001) than healthy subjects. One subject (man, 41 years old) had a markedly elevated apoptotic index of 52.2 %. In the remaining subjects, the percentage of cardiomyocyte death ranged from 0 % to 15.5 %. The high percentage of apoptosis found in our study may be in accordance with the clinically manifested cardiac failure in patients with dilated cardiomyopathy since in most patients we recorded the left ventricular ejection fraction values below 30 %.

• MeSH
• apoptóza * MeSH
• biopsie MeSH
• dilatační kardiomyopatie patologie   patofyziologie   MeSH
• dospělí MeSH
• kardiomyocyty patologie   MeSH
• koncové značení zlomů DNA in situ MeSH
• lidé středního věku MeSH
• lidé MeSH
• myokard patologie   MeSH
• tepový objem MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Shock waves can cause significant cytotoxic effects in tumor cells and tissues both in vitro and in vivo. However, understanding the mechanisms of shock wave interaction with tissues is limited. We have studied in vivo effects of focused shock waves induced in the syngeneic sarcoma tumor model using the TUNEL assay, immunohistochemical detection of caspase-3 and hematoxylin-eosin staining. Shock waves were produced by a multichannel pulsed-electrohydraulic discharge generator with a cylindrical ceramic-coated electrode. In tumors treated with shock waves, a large area of damaged tissue was detected which was clearly differentiated from intact tissue. Localization and a cone-shaped region of tissue damage visualized by TUNEL reaction apparently correlated with the conical shape and direction of shock wave propagation determined by high-speed shadowgraphy. A strong TUNEL reaction of nuclei and nucleus fragments in tissue exposed to shock waves suggested apoptosis in this destroyed tumor area. However, specificity of the TUNEL technique to apoptotic cells is ambiguous and other apoptotic markers (caspase-3) that we used in our study did not confirmed this observation. Thus, the generated fragments of nuclei gave rise to a false TUNEL reaction not associated with apoptosis. Mechanical stress from high overpressure shock wave was likely the dominant pathway of tumor damage.

• MeSH
• design vybavení MeSH
• elektrostimulační terapie přístrojové vybavení   metody   MeSH
• eosin MeSH
• experimentální nádory patologie   terapie   MeSH
• fluorescence MeSH
• hematoxylin MeSH
• imunohistochemie metody   MeSH
• kaspasa 3 metabolismus   MeSH
• koncové značení zlomů DNA in situ MeSH
• potkani inbrední LEW MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Apoptosis in hair follicles often is studied under pathological conditions; little is known about apoptotic mechanisms during normal hair follicle formation and maintenance. We investigated proteins of intrinsic apoptotic pathway, Bim and Puma, during hair follicle development and the first catagen stage using immunofluorescence to describe their expression patterns and to correlate them with apoptosis as determined by TUNEL assay. Both proteins were found in developing follicles. Bim and Puma overlapped apoptosis only partially during physiological apoptotic stage and they were present in non-apoptotic parts of the follicles. Our findings suggest that these primary apoptotic molecules participate in postnatal development and maintenance of hair follicles.

• MeSH
• apoptóza fyziologie   MeSH
• barvení a značení metody   MeSH
• fluorescenční protilátková technika metody   MeSH
• koncové značení zlomů DNA in situ metody   MeSH
• membránové proteiny metabolismus   MeSH
• myši MeSH
• nádorové supresorové proteiny metabolismus   MeSH
• proteiny regulující apoptózu metabolismus   MeSH
• protoonkogenní proteiny metabolismus   MeSH
• vlasový folikul cytologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

This study tries to elucidate the anti-proliferative and apoptotic effects of methanolic lichen extracts from Cladonia rangiformis and Cladonia convolute in MCF-7 human breast cancer cells. Lichen extracts (0-2 mg/ml) were added to MCF-7 cells for 24 h. Cell viability was tested using 3-(4,5-dimethylthiazol- 2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. Cell proliferation was observed using bromodeoxyuridine (BrdU) labelling and proliferating cell nuclear antigen (PCNA) by immunocytochemistry. The TUNEL method was used for cell death detection. The effective dose (ED50) values of methanolic extracts from C. rangiformis and C. convolute were found to be 0.905 and 0.977 mg/ml, respectively. Treatment with C. rangiformis methanolic extract (0.2-0.8 mg/ml) dose-dependently inhibited proliferation of MCF-7 cells as detected by BrdU incorporation. The inhibition was started in 0.2 mg/ml concentration of C. convoluta methanolic extract. The percent of PCNA immunopositive cells showed a decrease in MCF-7 cells treated with two lichen extracts compared to control MCF-7. Both methanolic extracts showed a significant increase in percentage of apoptosis-positive cells. These results indicate that methanolic lichen extracts from C. rangiformis and C. convolute inhibited proliferation of MCF-7 cells and caused apoptosis in MCF-7 cells. The lichens may be novel natural agents for treating breast cancer disease.

• MeSH
• adenokarcinom farmakoterapie   MeSH
• antitumorózní látky farmakologie   terapeutické užití   MeSH
• apoptóza účinky léků   MeSH
• fytoterapie MeSH
• imunohistochemie MeSH
• koncové značení zlomů DNA in situ MeSH
• lidé MeSH
• lišejníky * MeSH
• methanol MeSH
• MFC-7 buňky MeSH
• nádory prsu farmakoterapie   MeSH
• proliferace buněk účinky léků   MeSH
• rostlinné extrakty farmakologie   terapeutické užití   MeSH
• viabilita buněk účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

Eruption requires synchrony of the tooth with the surrounding tissues, particularly the bone. One important step during eruption is remodelling of the alveolar bone at the base of the tooth and along the roots. Expression of BMP6 was reported to be increased in the basal half of the dental follicle prior to eruption and inhibition of BMP6 affected bone formation at the base of the alveolar crypt. The aim of this study was to further investigate BMP6 protein in relation to tooth eruption and the corresponding bone remodelling using temporospatial correlations of BMP6 localization with morphogenetic events (proliferation, differentiation, apoptosis and bone apposition/resorption), other BMPs (BMP2 and BMP7) and three-dimensional images of tooth-bone development. BMP6 expression pattern was mapped in the mandibular molar teeth and related structures around eruption. Localization of BMP6 dominated in osteoblasts, in regions of bone formation within the alveolar crypt. These findings positively correlated with proliferation at the tooth base region, osteocalcin expression in the osteoblasts/osteocytes and BMP2 and BMP7 presence in the alveolar bone surrounding the tooth. Osteoclast activity and apoptotic elimination in the root region gradually decreased before eruption and totally ceased at eruption stages. Generally, BMP6 positively correlated with BMP2, BMP7 and osteocalcin-positive osteoblasts, and areas of bone remodelling. Moreover, BMP6 was found in the periodontium and cementoblasts. BMP6 expression in the alveolar bone accompanied tooth eruption. Notably, the expression pattern of BMP6 in the bone did not differ around individual molar teeth at the same stage of development. The expression of BMP6 in periodontal ligaments may contribute to interaction between the tooth and bone during the eruption and anchoring process.

• MeSH
• diazoniové sloučeniny MeSH
• imunohistochemie MeSH
• koncové značení zlomů DNA in situ MeSH
• kostní morfogenetický protein 6 metabolismus   MeSH
• methylová zeleň MeSH
• moláry fyziologie   MeSH
• myši MeSH
• osteoblasty metabolismus   MeSH
• osteokalcin metabolismus   MeSH
• periodontální vaz metabolismus   MeSH
• processus alveolaris fyziologie   MeSH
• prořezávání zubů fyziologie   MeSH
• remodelace kosti genetika   fyziologie   MeSH
• vývojová regulace genové exprese fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH