• Publikační typ
• abstrakt z konference MeSH

The degradation of maternal proteins is one of the most important events during early development, and it is presumed to be essential for embryonic genome activation (EGA), but the precise mechanism is still not known. It is thought that a large proportion of the degradation of maternal proteins is mediated by the ubiquitin-proteolytic system. In this study we focused on the expression of the Skp1-Cullin1-F-box (SCF) complex, a modular RING-type E3 ubiquitin-ligase, during bovine preimplantation development. The complex consists of three invariable components--Cul1, Skp1, Rbx1 and F-box protein, which determines the substrate specificity. The protein level and mRNA expression of all three invariable members were determined. Cul1 and Skp1 mRNA synthesis was activated at early embryonic stages, at the 4c and early 8c stage, respectively, which suggests that these transcripts are necessary for preparing the embryo for EGA. CUL1 protein level increased from MII to the morula stage, with a significant difference between MII and L8c, and between MII and the morula. The CUL1 protein was localized primarily to nuclei and to a lesser extent to the cytoplasm, with a lower signal in the inner cell mass (ICM) compared to the trophectoderm (TE) at the blastocyst stage. The level of SKP1 protein significantly increased from MII oocytes to 4c embryos, but then significantly decreased again. The localization of the SKP1 protein was analysed throughout the cell and similarly to CUL1 at the blastocyst stage, the staining was less intensive in the ICM. There were no statistical differences in RBX1 protein level and localization. The active SCF-complex, which is determined by the interaction of Cul1 and Skp1, was found throughout the whole embryo during preimplantation development, but there was a difference at the blastocyst stage, which exhibits a much stronger signal in the TE than in the ICM. These results suggest that all these genes could play an important role during preimplantation development. This paper reveals comprehensive expression profile, the basic but important knowledge necessary for further studying.

• MeSH
• blastocysta metabolismus   ultrastruktura   MeSH
• embryonální vývoj genetika   MeSH
• F-box proteiny genetika   metabolismus   MeSH
• fertilizace in vitro MeSH
• genetická transkripce MeSH
• kulinové proteiny genetika   metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• oocyty cytologie   růst a vývoj   metabolismus   MeSH
• proteinligasy komplexu SCF genetika   metabolismus   MeSH
• proteiny asociované s kinázou S-fáze genetika   metabolismus   MeSH
• signální transdukce MeSH
• skot MeSH
• spermie cytologie   metabolismus   MeSH
• substrátová specifita MeSH
• vývojová regulace genové exprese MeSH
• zinkové prsty genetika   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Proper timing of degradation of maternal protein reserves is important for early embryonic development. The major modification that triggers proteins to degradation is ubiquitination, mediated by ubiquitin-proteolytic system. We focus here on Skp 1-Cul 1-F-box complex (SCF-complex), E3 ubiquitin-ligase, a part of ubiquitin-proteolytic system, which transfer ubiquitin to the substrate protein. We describe in this chapter the methods for the characterization of the expression profile of mRNA and protein of invariant members of SCF-complex and for the definition of SCF-complex activity.

• MeSH
• aktivace transkripce MeSH
• embryonální vývoj * MeSH
• proteinligasy komplexu SCF metabolismus   MeSH
• proteolýza MeSH
• skot MeSH
• stanovení celkové genové exprese MeSH
• ubikvitinligasy metabolismus   MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Claspin is an adaptor protein that facilitates the ataxia telangiectasia and Rad3-related (ATR)-mediated phosphorylation and activation of Chk1, a key effector kinase in the DNA damage response. Efficient termination of Chk1 signaling in mitosis and during checkpoint recovery requires SCF(betaTrCP)-dependent destruction of Claspin. Here, we identify the deubiquitylating enzyme ubiquitin-specific protease 7 (USP7) as a novel regulator of Claspin stability. Claspin and USP7 interact in vivo, and USP7 is required to maintain steady-state levels of Claspin. Furthermore, USP7-mediated deubiquitylation markedly prolongs the half-life of Claspin, which in turn increases the magnitude and duration of Chk1 phosphorylation in response to genotoxic stress. Finally, we find that in addition to the M phase-specific, SCF(betaTrCP)-mediated degradation, Claspin is destabilized by the anaphase-promoting complex (APC) and thus remains unstable in G1. Importantly, we demonstrate that USP7 specifically opposes the SCF(betaTrCP)- but not APC(Cdh1)-mediated degradation of Claspin. Thus, Claspin turnover is controlled by multiple ubiquitylation and deubiquitylation activities, which together provide a flexible means to regulate the ATR-Chk1 pathway.

• MeSH
• adaptorové proteiny signální transdukční * metabolismus   MeSH
• anafázi podporující komplex MeSH
• buněčné linie MeSH
• fosforylace MeSH
• G1 fáze MeSH
• komplexy ubikvitinligas * metabolismus   fyziologie   MeSH
• lidé MeSH
• poškození DNA MeSH
• proteinkinasy metabolismus   MeSH
• proteinligasy komplexu SCF * metabolismus   fyziologie   MeSH
• substrátová specifita MeSH
• thiolesterasa ubikvitinu * metabolismus   fyziologie   MeSH
• ubikvitinace fyziologie   MeSH
• Check Tag
• lidé MeSH

We have investigated amino acid concentrations and protein metabolism in musculus extensor digitorum longus (EDL, fast-twitch, white muscle) and musculus soleus (SOL, slow-twitch, red muscle) of rats sacrificed in the fed state or after one day of starvation. Fractional protein synthesis rates (FRPS) were measured using the flooding dose method (L-[3,4,5-3H]phenylalanine). Activities of two major proteolytic systems in muscle (the ubiquitin-proteasome and lysosomal) were examined by measurement of chymotrypsin like activity of proteasome (CTLA), expression of ubiquitin ligases atrogin-1 and muscle-ring-finger-1 (MuRF-1), and cathepsin B and L activities. Intramuscular concentrations of the most of non-essential amino acids, FRPS, CTLA and cathepsin B and L activities were in postprandial state higher in SOL when compared with EDL. The differences in atrogin-1 and MuRF-1 expression were insignificant. Starvation decreased concentrations of a number of amino acids and increased concentrations of valine, leucine, and isoleucine in blood plasma. Starvation also decreased intramuscular concentrations of a number of amino acids differently in EDL and SOL, decreased protein synthesis (by 31 % in SOL and 47 % in EDL), and increased expression of atrogin-1 and MuRF-1 in EDL. The effect of starvation on CTLA and cathepsin B and L activities was insignificant. It is concluded that slow-twitch (red) muscles have higher rates of protein turnover and may adapt better to brief starvation when compared to fast-twitch (white) muscles. This phenomenon may play a role in more pronounced atrophy of white muscles in aging and muscle wasting disorders.

• MeSH
• aminokyseliny krev   metabolismus   MeSH
• časové faktory MeSH
• fyziologická adaptace MeSH
• lyzozomy metabolismus   MeSH
• postprandiální období * MeSH
• potkani Wistar MeSH
• potravinová deprivace * MeSH
• proteasomový endopeptidasový komplex metabolismus   MeSH
• proteinligasy komplexu SCF genetika   metabolismus   MeSH
• proteolýza MeSH
• svalová vlákna typu I metabolismus   MeSH
• svalová vlákna typu II metabolismus   MeSH
• svalové proteiny biosyntéza   genetika   metabolismus   MeSH
• TRIM protein genetika   metabolismus   MeSH
• ubikvitinace MeSH
• ubikvitinligasy genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

The mechanism of maternal protein degradation during preimplantation development has not been clarified yet. It is thought that a lot of maternal proteins are degraded by the ubiquitin-proteasome system. In this study, we focused on the role of the SCF (Skp1-Cullin-F-box) complexes during early bovine embryogenesis. We inhibited them using MLN4924, an inhibitor of SCF complex ligases controlled by neddylation. Oocytes maturated in MLN4924 could be fertilized, but we found no cumulus cell expansion and a high number of polyspermy after in vitro fertilization. We also found a statistically significant deterioration of development after MLN4924 treatment. After treatment with MLN4924 from the four-cell to late eight-cell stage, we found a statistically significant delay in their development; some of the treated embryos were, however, able to reach the blastocyst stage later. We found reduced levels of mRNA of EGA markers PAPOLA and U2AF1A, which can be related to this developmental delay. The cultivation with MLN4924 caused a significant increase in protein levels in MLN4924-treated oocytes and embryos; no such change was found in cumulus cells. To detect the proteins affected by MLN4924 treatment, we performed a Western blot analysis of selected proteins (SMAD4, ribosomal protein S6, centromeric protein E, P27, NFKB inhibitor alpha, RNA-binding motif protein 19). No statistically significant increase in protein levels was detected in either treated embryos or oocytes. In summary, our study shows that SCF ligases are necessary for the correct maturation of oocytes, cumulus cell expansion, fertilization, and early preimplantation development of cattle.

• MeSH
• blastocysta cytologie   účinky léků   fyziologie   MeSH
• časové faktory MeSH
• cyklopentany farmakologie   MeSH
• embryo savčí MeSH
• embryonální vývoj účinky léků   MeSH
• IVM techniky metody   veterinární   MeSH
• kultivované buňky MeSH
• multiproteinové komplexy antagonisté a inhibitory   metabolismus   MeSH
• oocyty cytologie   účinky léků   fyziologie   MeSH
• oogeneze účinky léků   MeSH
• proteinligasy komplexu SCF antagonisté a inhibitory   metabolismus   fyziologie   MeSH
• pyrimidiny farmakologie   MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• buněčná membrána metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• buněčný cyklus * MeSH
• cyklin E genetika   metabolismus   MeSH
• cyklin-dependentní kinasa 2 MeSH
• cyklin-dependentní kinasy * metabolismus   MeSH
• cysteinové endopeptidasy metabolismus   MeSH
• exprese genu MeSH
• F-box proteiny * MeSH
• fosforylace MeSH
• kinasy CDC2-CDC28 * MeSH
• lidé MeSH
• multienzymové komplexy metabolismus   MeSH
• mutace MeSH
• nádory prsu genetika   MeSH
• nádory vaječníků genetika   MeSH
• nádory etiologie   MeSH
• peptidsynthasy * metabolismus   MeSH
• proteasomový endopeptidasový komplex MeSH
• protein-serin-threoninkinasy * metabolismus   MeSH
• proteinligasy komplexu SCF MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• ubikvitinligasy * MeSH
• ubikvitiny * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH

F-box proteins represent the substrate-specificity determinants of the SCF ubiquitin ligase complex. We previously reported that the F-box protein Grr1p is one of the proteins involved in the transmission of glucose-generated signal for proteolysis of the galactose transporter Gal2p and fructose-1,6-bisphosphatase. In this study, we show that the other components of SCF(Grr1), including Skp1, Rbx1p, and the ubiquitin-conjugating enzyme Cdc34, are also necessary for glucose-induced Gal2p degradation. This suggests that transmission of the glucose signal involves an SCF(Grr1)-mediated ubiquitination step. However, almost superimposable ubiquitination patterns of Gal2p observed in wild-type and grr1Delta mutant cells imply that Gal2p is not the primary target of SCF(Grr1) ubiquitin ligase. In addition, we demonstrate here that glucose-induced Gal2p proteolysis is a cell-cycle-independent event.

• MeSH
• buněčný cyklus fyziologie   MeSH
• financování organizované MeSH
• mapování interakce mezi proteiny MeSH
• proteinligasy komplexu SCF metabolismus   MeSH
• proteiny přenášející monosacharidy metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Saccharomyces cerevisiae cytologie   metabolismus   MeSH
• ubikvitinligasy metabolismus   MeSH

PURPOSE: The current study aimed to (1) examine the sociodemographic, clinical and psychological factors related to subjective cognitive functioning (SCF); (2) analyze the complex mutual interconnections between SCFs; and (3) address patients' perspectives on SCF and supportive care. METHOD: A heterogeneous sample of oncological patients (N = 566) was recruited.Items inquiring about the senses, attention, memory, spatial functions, decision-making and speech were administered. A network of subjectively perceived changes in cognitive functions was estimated while three open-ended questions addressed patients' perspectives on SCF. RESULTS: Within the network, deficits in spatial perception, attention focus and problem-solving ability had the highest strength index while the deficits related to the senses were the least influential. CONCLUSION: The findings indicate that psychological-based interventions focused on higher cognitive functions could improve patients' quality of life. The presence of supportive care and available information could strengthen SCF intervention and prevention for patients with cancer.

• MeSH
• dospělí MeSH
• kognice * MeSH
• kvalita života psychologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádory * psychologie   terapie   MeSH
• senioři nad 80 let MeSH
• senioři 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

Reliable and simple methods are required for detection of low concentrations of cytokines and some other proteins in complex biological fluids. This is especially important when monitoring the immune responses under various physiological and pathophysiological conditions in vivo or following production of these compounds in in vitro systems. Cytokines and other immunologically active molecules are being predominantly detected by enzyme-linked immunosorbent assays (ELISA) and newly also by immuno-polymerase chain reactions (iPCR). New simplified variants of iPCR have recently been described where antibodies are connected with multiple DNA templates through gold nanoparticles (Au-NPs) to form a new class of detection reagents. In this study we compared functionalized Au-NP-based iPCR (Nano-iPCR) with standard ELISA and iPCR for the detection of interleukin (IL)-3 and stem cell factor (SCF). The same immunoreagents (IL-3- and SCF-specific polyclonal antibodies and their biotinylated forms) were used throughout the assays. The obtained data indicate that both Nano-iPCR and iPCR are superior in sensitivity and detection range than ELISA. Furthermore, Nano-iPCR is easier to perform than the other two methods. Nano-iPCR was used for monitoring changes in concentration of free SCF during growth of mast cells in SCF-conditioned media. The results show that growing cultures gradually reduce the amount of SCF in supernatant to 25% after 5 days. The combined data indicate that Nano-iPCR assays may be preferable for rapid detection of low concentrations of cytokines in complex biological fluids.

• MeSH
• cytokiny analýza   imunologie   MeSH
• ELISA MeSH
• imunoanalýza metody   statistika a číselné údaje   MeSH
• indikátory a reagencie MeSH
• interleukin-3 analýza   imunologie   MeSH
• kovové nanočástice MeSH
• kultivované buňky MeSH
• mastocyty imunologie   MeSH
• myši MeSH
• nanotechnologie MeSH
• polymerázová řetězová reakce metody   statistika a číselné údaje   MeSH
• protilátky MeSH
• růstový faktor kmenových buněk analýza   imunologie   MeSH
• senzitivita a specificita MeSH
• zlato MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH