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
- inhibitory proteasomu MeSH
- kathepsin B metabolismus MeSH
- kosterní svaly metabolismus účinky léků MeSH
- krysa rodu rattus MeSH
- peptidy farmakologie MeSH
- potkani Wistar MeSH
- proteasomový endopeptidasový komplex 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
The aim of our study was to evaluate the differences in protein and amino acid metabolism after subcutaneous turpentine administration in the soleus muscle (SOL), predominantly composed of red fibres, and the extensor digitorum longus muscle (EDL) composed of white fibres. Young rats (40-60 g) were injected subcutaneously with 0.2 ml of turpentine oil/100 g body weight (inflammation) or with the same volume of saline solution (control). Twenty-four hours later SOL and EDL were dissected and incubated in modified Krebs-Heinseleit buffer to estimate total and myofibrillar proteolysis, chymotrypsin-like activity of proteasome (CHTLA), leucine oxidation, protein synthesis and amino acid release into the medium. The data obtained demonstrate that in intact rats, all parameters measured except protein synthesis are significantly higher in SOL than in EDL. In turpentine treated animals, CHTLA increased and protein synthesis decreased significantly more in EDL. Release of leucine was inhibited significantly more in SOL. We conclude that turpentine-induced inflammation affects more CHTLA, protein synthesis and leucine release in EDL compared to SOL.
- MeSH
- financování organizované MeSH
- krysa rodu rattus MeSH
- leucin metabolismus MeSH
- potkani Wistar MeSH
- proteasomový endopeptidasový komplex metabolismus MeSH
- svalová vlákna typu I účinky léků MeSH
- svalová vlákna typu II účinky léků MeSH
- terpentýn farmakologie MeSH
- zánět chemicky indukované MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
Beta-hydroxy-beta-metylbutyrát (HMB) je metabolitem leucinu, jenž vykazuje antikatabolické účinky a příznivě ovlivňuje imunitní systém. Mechanismus účinku nebyl dosud zcela objasněn. HMB může sloužit jako zdroj pro syntézu cholesterolu, zřejmě zasahuje také do ubikvitin-proteazomového proteolytického systému. HMB je užíván jako potravinový doplněk při silovém tréninku, zejména pro nárůst síly a množství netukové tkáně. V posledních letech se také testuje jako součást terapie kachexie různé etiologie (např. nádory, AIDS).
Beta-hydroxy-beta-methylbutyrate (HMB) is the leucine metabolite, which shows anti-catabolic effect and beneficially affects the immune system. The mechanism of action is still not fully understood. HMB may serve as the source for cholesterol synthesis and it probably affects the ubiquitin-proteasome proteolytic system. HMB is used as a dietary supplement during resistance-training, especially for increase of power and non-fat body mass. It has been also tested in recent years as a part of cachexia treatment (cancer, AIDS, etc.).
- MeSH
- arginin terapeutické užití MeSH
- glutamin terapeutické užití MeSH
- kachexie MeSH
- klinické zkoušky jako téma MeSH
- kosterní svaly MeSH
- lidé MeSH
- potravní doplňky MeSH
- proteasomový endopeptidasový komplex účinky léků MeSH
- valeráty farmakokinetika farmakologie terapeutické užití MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
BACKGROUND/AIMS: Growth hormone (GH) could have the potential to improve protein metabolism in sepsis but glutamine deficiency has been reported after GH treatment. The aim was to investigate the effects of glutamine deficiency in sepsis with and without GH treatment on protein and amino acid metabolism. METHODS: Cecal ligation and puncture (CLP) was used as a model of sepsis. Serious glutamine deficiency was induced by administration of glutamine synthetase inhibitor, methionine sulfoximine (MSO). Young Wistar rats were divided into 5 groups: control; CLP; CLP+MSO; CLP+GH, and CLP+MSO+GH. Parameters of protein metabolism were measured on incubated soleus and extensor digitorum longus muscles: [1-14C]leucine was used to estimate protein synthesis and leucine oxidation, tyrosine release was used to evaluate protein breakdown. Amino acid concentrations in plasma, skeletal muscle and incubation media were measured by HPLC. RESULTS/CONCLUSIONS: A reduced muscle glutamine concentration after MSO treatment is not associated with changes in the rates of protein synthesis or breakdown. MSO treatment decreased glutamine release from skeletal muscle and plasma glutamine concentration. Severe glutamine deficiency in GH-treated septic rats resulted in increased release of branched-chain amino acids from skeletal muscle. Copyright 2006 S. Karger AG, Basel.
- MeSH
- aminokyseliny metabolismus MeSH
- financování organizované MeSH
- glutamin analýza metabolismus nedostatek MeSH
- kosterní svaly metabolismus MeSH
- krysa rodu rattus MeSH
- náhodné rozdělení MeSH
- potkani Wistar MeSH
- růstový hormon farmakologie MeSH
- sepse metabolismus patofyziologie MeSH
- svalové proteiny metabolismus MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- ženské pohlaví MeSH
- zvířata MeSH
Proteasome inhibitors are novel therapeutic agents which may be used in treatment of cancer and other severe disorders. We studied the effect of proteasome inhibitor MG-132 on protein and amino acid metabolism. In MG-132-treated rats we observed a significant decrease in proteasome-dependent proteolysis in skeletal muscle and an increase in whole-body protein turnover (i.e., increase in whole-body proteolysis and protein synthesis). Proteasome-dependent proteolysis was activated in the liver and kidney, protein synthesis increased in skeletal muscle, liver, and kidney. Insignificant changes were found in jejunum and colon. MG-132 administration induced a significant increase in concentration of several amino acids in blood plasma and their decrease in jejunum and colon. We conclude that administration of MG-132 affects both protein anabolic and protein catabolic pathways via the direct effect on proteasome-dependent proteolysis and indirect effect on proteolysis and protein synthesis via unidentified mediators.
- MeSH
- aminokyseliny metabolismus MeSH
- financování organizované MeSH
- inhibitory proteasomu MeSH
- krysa rodu rattus MeSH
- leupeptiny farmakologie MeSH
- metabolická clearance účinky léků MeSH
- orgánová specificita MeSH
- potkani Wistar MeSH
- proteasomový endopeptidasový komplex metabolismus MeSH
- proteom metabolismus MeSH
- tkáňová distribuce MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
