The skin is known to be the largest organ in human organism creating interface with outer environment. The skin provides protective barrier against pathogens, physical and chemical insults, and against uncontrolled loss of water. The barrier function was primarily attributed to the stratum corneum (SC) but recent studies confirmed that epidermal tight junctions (TJs) also play important role in maintaining barrier properties of the skin. Independent observations indicate that barrier function and its recovery is impaired in aged skin. However, trans-epidermal water loss (TEWL) values remains rather unchanged in elderly population. UV radiation as major factor of photoageing impairs TJ proteins, but TJs have great self-regenerative potential. Since it may be possible that TJs can compensate TEWL in elderly due to its regenerative and compensatory capabilities, important question remains to be answered: how are TJs regulated during skin ageing? This review provides an insight into TJs functioning as epidermal barrier and summarizes current knowledge about the impact of ageing on the barrier function of the skin and epidermal TJs.
- MeSH
- epidermis metabolismus patologie účinky záření MeSH
- kožní absorpce MeSH
- lidé MeSH
- permeabilita MeSH
- perspiratio insensibilis MeSH
- stárnutí kůže * účinky záření MeSH
- stárnutí metabolismus patologie MeSH
- těsný spoj metabolismus patologie účinky záření MeSH
- ultrafialové záření škodlivé účinky MeSH
- věkové faktory MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Hyaluronan-based tissue substitutes are promising materials in cartilage reconstruction surgery. Herein, the chondrogenesis of human mesenchymal stem cells (MSC) in a hydrogel based on a tyramine derivative of hyaluronan crosslinked by hydrogen peroxidase (HA-TA) was evaluated. Human MSC seeded in the scaffold were incubated in standard chondrogenic medium and medium enriched with bone morphogenetic protein-6 (BMP6). Cell viability, the gene expression of selected markers (collagen type II, aggrecan, SOX9, collagen type X, and osteopontin), and the histological characteristics were examined during three weeks of in vitro cultivation. The tissue reaction of both unseeded and MSC seeded HA-TA scaffolds were tested in vivo after subcutaneous application in rats for 12 weeks. The data showed that cells resisted the process of crosslinking and remained viable for the whole time while exhibiting changes in cell organization. Human MSC cultivated in HA-TA hydrogel expressed genes of both chondrogenic and osteogenic differentiation and the addition of BMP6 revealed a tendency to potentiate both processes. Histological analysis of HA-TA in vivo implants did not reveal a chronic inflammatory reaction. In both cases, in vivo HA-TA implants were continuously degraded and MSC-seeded hydrogels tended to form clusters similar to in vitro samples. In conclusion, MSC chondrogenic differentiation may proceed in a HA-TA scaffold that is biocompatible. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3523-3530, 2014.
- MeSH
- buněčná diferenciace * účinky léků genetika MeSH
- chondrogeneze * účinky léků genetika MeSH
- experimentální implantáty MeSH
- imobilizované buňky cytologie účinky léků metabolismus MeSH
- krysa rodu rattus MeSH
- kyselina hyaluronová farmakologie MeSH
- lidé MeSH
- mezenchymální kmenové buňky cytologie účinky léků metabolismus MeSH
- PEG-DMA hydrogel farmakologie MeSH
- peroxidasa metabolismus MeSH
- reagencia zkříženě vázaná farmakologie MeSH
- regulace genové exprese účinky léků MeSH
- subkutánní tkáň účinky léků MeSH
- tyramin farmakologie MeSH
- viabilita buněk účinky léků MeSH
- zobrazování trojrozměrné MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Skin exposure to ultraviolet (UV) light evokes a complex stress response in keratinocytes. Keratin filament organization provides structural stability and mechanical integrity of keratinocytes. Involucrin is a transglutaminase substrate protein contributing to the formation of insoluble cornified envelopes. However, a more complex role for keratins and involucrin has been proposed, including the regulation of cell stress response. The aim was to evaluate modulations of keratin 1, 10 and involucrin expression in HaCaT in the light of the complex response of these cells to UV-B radiation, including effects on c-Jun and matrix metalloproteinase 1 (MMP-1) gene expression and production of interleukin (IL) 6 and 8. A UV-B (300±5 nm) dose of 10 mJ/cm(2) was selected since this dose resulted in a partial decrease in cell viability in contrast to higher UV-B doses, which induced complete cell death 48 h after treatment. The UV-B radiation induced significant expression of keratin 1 and 10 and decreased expression of involucrin. This was accompanied by increased expression of c-Jun and MMP-1 and IL-6 and IL-8 production. The data suggest that the expression of keratin 1, 10 and involucrin is modulated in HaCaT keratinocytes as a part of the complex stress response to UV radiation.
- Publikační typ
- časopisecké články MeSH
Endotoxin administration is frequently used as a model of systemic inflammatory response which is considered the important pathogenetic factor in muscle wasting development in severe illness, such as sepsis, cancer, injury, AIDS and others. The main purpose of this study was determining the effect of various doses of endotoxin on protein and amino acid metabolism in two types of rat skeletal muscle. Sepsis was induced by intraperitoneal administration of endotoxin in a dose of 1, 3 and 5 mg/kg body weight (bw); control animals received a corresponding volume of the saline solution. After 24 h, extensor digitorum longus (EDL) and soleus (SOL) muscles were isolated and used for determination of total and myofibrillar proteolysis, protein synthesis, activity of cathepsins B and L, chymotrypsin-like activity of proteasome and amino acid release. The endotoxemia induced the body weight loss, the rise of total cholesterol and triglyceride plasma concentration and the protein catabolic state in skeletal muscle, which was caused by a higher increase in protein breakdown (due to activation of the proteasome system) than protein synthesis. The more significant effect of endotoxin was seen in EDL than SOL. The dose of 5 mg of endotoxin/kg bw induced the most significant changes in parameters of the protein and amino acid metabolism measured and could be therefore considered appropriate for studies of protein catabolism in young rat skeletal muscle at 24 h after endotoxin treatment.
- MeSH
- endotoxiny farmakologie MeSH
- kathepsin B metabolismus MeSH
- kosterní svaly metabolismus MeSH
- krysa rodu rattus MeSH
- myofibrily metabolismus MeSH
- potkani Wistar MeSH
- proteasomový endopeptidasový komplex metabolismus MeSH
- sepse metabolismus MeSH
- svalové proteiny metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Exposure to ultraviolet (UV) irradiation has detrimental effects on skin accompanied by the increased metabolism of hyaluronan (HA), a linear polysaccharide important for the normal physiological functions of skin. In this study, the modulation of human keratinocyte response to UVB irradiation by HA (970 kDa) was investigated. Immortalized human keratinocytes (HaCaT) were irradiated by a single dose of UVB and immediately treated with HA for 6 and 24 h. The irradiation induced a significant decrease in the gene expression of CD44 and toll-like receptor 2 6 h after irradiation. The expressions of other HA receptors, including toll-like receptor 4 and the receptor for HA-mediated motility, were not detected in either the control or UVB-irradiated or HA-treated HaCaT cells. UVB irradiation induced a significant decrease in the gene expression of HA synthase-2 and hyaluronidase-2 6 h after irradiation. The expressions of HA synthase-3 and hyaluronidase-3 were not significantly modulated by UV irradiation. Interestingly, HA treatment did not significantly modulate any of these effects. In contrast, HA significantly suppressed UVB-induced pro-inflammatory cytokine release including interleukin-6 and interleukin-8. Similarly, HA treatment reduced the UVB-mediated production of transforming growth factor β1. HA treatment also significantly reduced the UV irradiation-mediated release of soluble CD44 into the media. Finally, HA partially, but significantly, suppressed the UVB-induced decrease in cell viability. Data indicate that HA had significant protective effects for HaCaT cells against UVB irradiation.
- MeSH
- antigeny CD44 biosyntéza genetika MeSH
- exprese genu MeSH
- glukuronosyltransferasa biosyntéza genetika MeSH
- hyaluronoglukosaminidasa biosyntéza genetika MeSH
- interleukin-6 biosyntéza MeSH
- interleukin-8 biosyntéza MeSH
- keratinocyty účinky léků metabolismus účinky záření MeSH
- kůže účinky léků metabolismus účinky záření MeSH
- kyselina hyaluronová metabolismus farmakologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- toll-like receptor 2 biosyntéza genetika MeSH
- transformující růstový faktor beta biosyntéza MeSH
- ultrafialové záření MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVES: Determination of cell numbers is a crucial step in studies focused on cytokinetics and cell toxicity. The impedance-based analysis employing electronic sensor array system xCELLigence System allowing label-free dynamic monitoring of relative viable adherent cell amounts was compared with the most utilized methods for relative quantification of viable cell numbers based on a determination of cellular metabolism. DESIGN: Colorimetric assay based on reduction of tetrazolium salt (MTT) by mitochondrial enzymes and chemiluminiscent assay based on intracellular adenosine triphosphate (ATP) determination were compared with the impedance-based system. Cell morphology was compared by microscopic evaluation. Normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF), together with 3T3 mouse fibroblast and HaCaT keratinocyte cell lines were employed. RESULTS: The progress of cell growth curves obtained by different methods during 72 hours reflected cell type and cell seeding densities. The impedance-based method was found to be applicable for the determination of the cell proliferation of 3T3 fibroblasts, HaCaT and NHDF, since the comparison of this method with ATP and MTT determinations showed a comparable results. In contrast, the proliferation of NHEK measured by the impedance-based method did not correlate with other methodological approaches. This could be accounted to the specific morphological appearance of these cells. CONCLUSION: The study shows the impedance-based detection of viable adherent cells is a valuable approach for cytokinetics and pharmacological studies. However, the specific morphological characteristics of cell lines have to be considered employing this method for determination of cell proliferation without using other reference methods.
- MeSH
- adenosintrifosfát metabolismus MeSH
- buněčné linie MeSH
- buňky 3T3 MeSH
- časové faktory MeSH
- elektrická impedance MeSH
- fibroblasty cytologie MeSH
- keratinocyty cytologie MeSH
- kolorimetrie MeSH
- kultivované buňky MeSH
- lidé MeSH
- luminiscenční měření MeSH
- mitochondrie enzymologie metabolismus MeSH
- myši MeSH
- oxidace-redukce MeSH
- počet buněk metody MeSH
- proliferace buněk MeSH
- škára cytologie MeSH
- tetrazoliové soli metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
The proteasome inhibitors are used as research tools to study of the ATP-dependent ubiquitin-proteasome system. Some of them are at present undergoing clinical trials to be used as therapeutic agents for cancer or inflammation. These diseases are often accompanied by muscle wasting. We herein demonstrate findings about new proteasome inhibitors, belactosin A and C, and their direct effect on protein metabolism in rat skeletal muscle. M. soleus (SOL) and m. extensor digitorum longus (EDL) were dissected from both legs of male rats (40-60 g) and incubated in a buffer containing belactosin A or C (30 microM) or no inhibitor. The release of amino acids into the medium was estimated using high performance liquid chromatography to calculate total and myofibrillar proteolysis. Chymotrypsin-like activity (CTLA) of proteasome and cathepsin B, L activity were determined by fluorometric assay. Protein synthesis and leucine oxidation were detected using specific activity of L-[1-14C] leucine added to medium. Inhibited and control muscles from the same rat were compared using paired t-test. The results indicate that after incubation with both belactosin A and C total proteolysis and CTLA of proteasome decreased while cathepsin B, L activity did not change in both SOL and EDL. Leucine oxidation was significantly enhanced in SOL, protein synthesis decreased in EDL. Myofibrillar proteolysis was reduced in both muscles in the presence of belactosin A only. In summary, belactosin A and C affected basic parameters of protein metabolism in rat skeletal muscle. The response was both muscle- and belactosin-type-dependent.
- MeSH
- aminokyseliny metabolismus MeSH
- chymotrypsin antagonisté a inhibitory metabolismus MeSH
- kathepsin B metabolismus MeSH
- kosterní svaly metabolismus účinky léků MeSH
- krysa rodu rattus MeSH
- peptidy farmakologie MeSH
- potkani Wistar MeSH
- proteasomový endopeptidasový komplex antagonisté a inhibitory MeSH
- svalové proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Beta-hydroxy-beta-methylbutyrate (HMB) is a leucine metabolite with protein anabolic effect. The aim of the study was to examine the role of exogenous HMB on leucine and protein metabolism in whole body and selected tissues. Rats were administered by HMB (0.1 g/kg b.w.) or by saline. The parameters of whole-body protein metabolism were evaluated 24 h later using L-[1-14C]leucine and L-[3,4,5-3H]phenylalanine. Changes in proteasome dependent proteolysis and protein synthesis were determined according the "chymotrypsin-like" enzyme activity and labeled leucine and phenylalanine incorporation into the protein. A decrease in leucine clearance and whole-body protein turnover (i.e., a decrease in whole-body proteolysis and protein synthesis) was observed in HMB treated rats. Proteasome-dependent proteolysis decreased significantly in skeletal muscle, changes in heart, liver, jejunum, colon, kidney, and spleen were insignificant. Decrease in protein synthesis was observed in the heart, colon, kidney, and spleen, while an increase was observed in the liver. There were no significant changes in leucine oxidation. We conclude that protein anabolic effect of HMB in skeletal muscle is related to inhibition of proteolysis in proteasome. Alterations in protein synthesis in visceral tissues may affect several important functions and the metabolic status of the whole body.
- MeSH
- financování organizované MeSH
- játra metabolismus účinky léků MeSH
- kosterní svaly metabolismus účinky léků MeSH
- krysa rodu rattus MeSH
- ledviny metabolismus účinky léků MeSH
- myokard metabolismus MeSH
- N-acetyltransferasa aminokyselin metabolismus MeSH
- potkani Wistar MeSH
- proteiny metabolismus MeSH
- slezina metabolismus účinky léků MeSH
- srdce účinky léků MeSH
- střeva metabolismus účinky léků MeSH
- valeráty farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- MeSH
- financování organizované MeSH
- Publikační typ
- abstrakty MeSH