CDC25A is able to induce resumption of meiosis but compromises metaphase I-metaphase II transition in mouse oocytes
Šolc, Petr; Šašková, Adéla; Baran, V.; Kubelka, Michal; Motlík, Jan
2007 - English
We have shown that CDC25A protein is expressed in GV-stage oocytes but decreases, in CDK1-dependent manner, during meiotic maturation. As compared with GV-stage only a very low level of CDC25A protein is present at metaphase I (MI) and metaphase II (MII) stages. CDC25A mRNA is stable during entire meiotic maturation. Exogenous CDC25A was sufficient to overcome cAMP-mediated GV-stage block. Using microinjection of GFP-CDC25A and GFP-CDC25B mRNAs constructs we have revealed that CDC25A is exclusively nuclear protein until nuclear envelope break down (NEBD). In contrast CDC25B localizes to cytoplasm at GV-stage oocytes and translocates to nucleus shortly before NEBD. Overexpression of GFP-CDC25A, to interfere with CDC25A degradation during meiotic maturation, resulted in MI block characterized with problems in chromosome congression and spindle formation. This MI block was accompanied with the transient reduction of both CDK1 and MAPK activities. RNAi mediated CDC25A knock-down resulted in a reduced ability to resume meiosis and to reach MII. These data demonstrate that behavior of CDC25A during female meiosis differs significantly from mitosis and CDC25A is involved in both, resumption of meiosis and also in metaphase I spindle formation as a prerequisite for correct MI-MII transition. It is evident that CDC25B is not only important CDC25 phosphatase for meiotic maturation but also CDC25A has its meiotic specific role. Práce se zabývá expresí a funkcí CDC25A v myších oocytech. Bylo zjištěno, že CDC25A protein se exprimuje v GV-oocytech, avšak jeho hladina při meiotickém zrání klesá, přičemž na konci meiosy je v metafase II oocytech pouze velmi nízká hladina CDC25A proteinu. Tento CDC25A proteinový pokles je závislý na CDK1 aktivitě, nesouvisí však se změnami hladiny Cdc25A mRNA, která zůstává konstantní. Pomocí funkčních studií (RNA interference, mikroinjekce mRNA) jsme jasně prokázali, že CDC25A je nezbytná pro znovuzahájení meiosy a tvorbu metafase I spindelu. Pokles CDC25A aktivity po znovuzahájení meiosy je klíčový pro korektní metafase I – metafase II přechod.
Keywords:
meiosis
Available at various institutes of the ASCR
CDC25A is able to induce resumption of meiosis but compromises metaphase I-metaphase II transition in mouse oocytes
We have shown that CDC25A protein is expressed in GV-stage oocytes but decreases, in CDK1-dependent manner, during meiotic maturation. As compared with GV-stage only a very low level of CDC25A protein ...
Modulation of intracellular caspase machinery using organ culture approach
Matalová, Eva; Fleischmannová, Jana; Norek, Adam; Míšek, Ivan
2007 - English
Organ explant cultures eliminate animal suffering during experiments but simultaneously allow investigations of intact organ, tissue and cell systems with preserved interactions corresponding to the situation in vivo. Moreover, the explants can be manipulated in several ways using modern micromanipulation techniques (e.g. electroporation, inhibition). We show possible usage of explant culture approaches in embryonal apoptosis research in three models – mouse limb digitalization, tooth germ morphogenesis, separation of middle ear ossicles. Data from specific pharmaceutical inhibitions focused on key apoptotic molecules – caspases - are demonstrated. Orgánové explantátové kultury minimalizují experimentální zásahy na zvířatech, ale současně umožňují studium intaktních orgánů a tkání se zachováním mezibuněčných interakcí odpovídajících systémům in vivo. Explantáty mohou být navíc modulovány celou řadou moderních mikromanipulačních technik (elektroporace, inhibice, atd.). Příspěvek ukazuje možnost využití explantátových kultur při studiu apoptotických událostí během embryogeneze, a to na modelu digitalizace myších končetin, morfogeneze zubních základů a separace středoušních kůstek. Demonstrovány jsou výsledky specifických farmakologických inhibic intracelulárních signálních drah se zaměřením na kaspázy jako klíčové molekuly realizace apoptotických signálů.
Keywords:
caspase machinery
Available at various institutes of the ASCR
Modulation of intracellular caspase machinery using organ culture approach
Organ explant cultures eliminate animal suffering during experiments but simultaneously allow investigations of intact organ, tissue and cell systems with preserved interactions corresponding to the ...
Aurora-A is involved in resumption of meiosis and spindle formation in mouse oocytes
Šašková, Adéla; Šolc, Petr; Baran, V.; Kubelka, Michal; Motlík, Jan
2007 - English
We study the role of Aurora-A during meiotic maturation of mouse oocytes. Total Aurora-A is present in the nucleus in GV-stage oocytes (G2 equivalent). Additionally, active Aurora-A is localized entirely to the centrosome (MTOC) shorly before germinal vesicle breakdown (GVBD). Compared to somatic cells, where active Aurora-A is at the centrosomes and the spindle poles, active Aurora-A is strictly localized on MTOCs at metaphase I in oocytes. We show that activation of centrosomal Aurora-A is independent on PI3K-PKB and CDK1 signaling pathways. This was proved by cultivation of oocytes in presence of roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor). Treated oocytes show high phosphorylation of Aurora-A on T288 and centrosome amplification despite the presence of intact nuclear envelope. Silencing of Aurora-A by RNA interference induces incorrect spindle assembly. Oocytes are arrested in prometaphase I and unable to reach metaphase II. After microinjection of eGFP-Aurora-A mRNA into GV-stage oocytes, overexpression of Aurora-A leads to distortion of MI spindle organization as well. Our results indicate that Aurora-A is the key centrosomal player in meiotic maturation, essential for proper spindle formation and metaphase I - metaphase II transition. Práce se zabývá úlohou Aurora-A kinázy během meiotického zrání in vitro. Pomocí farmakologické inhibice PI3K, PKB a CDK1 bylo prokázáno, že Aurora-A, se aktivuje velmi časně při znovuzahájení meiosy, a toto aktivace je nezávislá na PI3K-PKB a CDK1 signalizaci. Dále bylo prokázáno (RNA interference, mikroinjekce mRNA), že Aurora-A je nutná pro korektní výstavbu metafáze I spindelu a pro metafase I – metafase II přechod.
Keywords:
meiosis
Available at various institutes of the ASCR
Aurora-A is involved in resumption of meiosis and spindle formation in mouse oocytes
We study the role of Aurora-A during meiotic maturation of mouse oocytes. Total Aurora-A is present in the nucleus in GV-stage oocytes (G2 equivalent). Additionally, active Aurora-A is localized ...
CDC25A is able to induce resumption of meiosis but compromises metaphase I-metaphase II transition in mouse oocytes
Šolc, Petr; Šašková, Adéla; Baran, V.; Kubelka, Michal; Motlík, Jan
2007 - English
We have shown that CDC25A protein is expressed in GV-stage oocytes but decreases, in CDK1-dependent manner, during meiotic maturation. As compared with GV-stage only a very low level of CDC25A protein is present at metaphase I (MI) and metaphase II (MII) stages. CDC25A mRNA is stable during entire meiotic maturation. Exogenous CDC25A was sufficient to overcome cAMP-mediated GV-stage block. Using microinjection of GFP-CDC25A and GFP-CDC25B mRNAs constructs we have revealed that CDC25A is exclusively nuclear protein until nuclear envelope break down (NEBD). In contrast CDC25B localizes to cytoplasm at GV-stage oocytes and translocates to nucleus shortly before NEBD. Overexpression of GFP-CDC25A, to interfere with CDC25A degradation during meiotic maturation, resulted in MI block characterized with problems in chromosome congression and spindle formation. This MI block was accompanied with the transient reduction of both CDK1 and MAPK activities. RNAi mediated CDC25A knock-down resulted in a reduced ability to resume meiosis and to reach MII. These data demonstrate that behavior of CDC25A during female meiosis differs significantly from mitosis and CDC25A is involved in both, resumption of meiosis and also in metaphase I spindle formation as a prerequisite for correct MI-MII transition. It is evident that CDC25B is not only important CDC25 phosphatase for meiotic maturation but also CDC25A has its meiotic specific role. Práce se zabývá expresí a funkcí CDC25A v myších oocytech. Bylo zjištěno, že CDC25A protein se exprimuje v GV-oocytech, avšak jeho hladina při meiotickém zrání klesá, přičemž na konci meiosy je v metafase II oocytech pouze velmi nízká hladina CDC25A proteinu. Tento CDC25A proteinový pokles je závislý na CDK1 aktivitě, nesouvisí však se změnami hladiny Cdc25A mRNA, která zůstává konstantní. Pomocí funkčních studií (RNA interference, mikroinjekce mRNA) jsme jasně prokázali, že CDC25A je nezbytná pro znovuzahájení meiosy a tvorbu metafase I spindelu. Pokles CDC25A aktivity po znovuzahájení meiosy je klíčový pro korektní metafase I – metafase II přechod.
Keywords:
meiosis
Available at various institutes of the ASCR
CDC25A is able to induce resumption of meiosis but compromises metaphase I-metaphase II transition in mouse oocytes
We have shown that CDC25A protein is expressed in GV-stage oocytes but decreases, in CDK1-dependent manner, during meiotic maturation. As compared with GV-stage only a very low level of CDC25A protein ...
Protein fractionation and relative quantitation using PF 2D and ITRAQ for biomarker quest
Gadher, S. J.; Skalníková, Helena; Halada, Petr; Řehulka, Pavel; Chmelík, Josef; Kovářová, Hana
2007 - English
ProteomeLab PF 2D System - Protein Fractionation in 2 Dimensions (Beckman Coulter, Fullerton, CA, USA) has been developed to fractionate complex protein mixtures by chromatofocusing in the first dimension followed by high-resolution non-porous silica reversed phase chromatography (RP LC) in the second dimension. Despite the high-resolution power of ProteomeLab PF 2D, UV-based quantitation could be compromised due to possible co-elution of several proteins into one fraction. Hence, we present an optimized protocol for application of isobaric tags for relative and absolute quantitation (iTRAQ) and MALDI-TOF/TOF mass spectrometry to obtain quantitative data from peptides derived by tryptic digestions of intact proteins fractionated by ProteomeLab PF 2D technique. To demonstrate the feasibility of such an approach, protein expression patterns obtained from the ProteomeLab PF 2D fractionation of human T-lymphoblastic leukemia CEM cell line were utilised.
Keywords:
protein fractionation; proteomics
Available at various institutes of the ASCR
Protein fractionation and relative quantitation using PF 2D and ITRAQ for biomarker quest
ProteomeLab PF 2D System - Protein Fractionation in 2 Dimensions (Beckman Coulter, Fullerton, CA, USA) has been developed to fractionate complex protein mixtures by chromatofocusing in the first ...
Quest for protein biomarkers of neural stem cells differentiation
Skalníková, Helena; Halada, Petr; Vodička, Petr; Motlík, Jan; Horing, O.; Jensen, O. N.; Gadher, S. J.; Pelech, S.; Kovářová, Hana
2007 - English
Understanding neurogenesis and neural cell differentiation presents a unique challenge for treatment of nervous system disorders. To gain more insight about molecular mechanism of differentiation of neural cells, we applied different proteomic approaches using classical 2-DE followed by MS and antibody microarrays. Based on 2-DE, profile of constituent proteins of neural stem cells and their differentiated progenies was estabilished at first and then the protein species that are significantly up or down regulated during the differentiation were selected. Differentiation of neural cells was accompanied by changes in the expression of proteins involved in DNA and RNA binding, mRNA processing and transport, stress responses, iron storage and redox regulation. Immunoblot verified changes of hnRNP A1, hnRNP A2/B1, RafB, heme-oxygenasy 2, GRK2 proteins and alphaB-crystallin (S45), CDK1/2 (Y15), PKC mu (S738+S742), proline-rich Akt substrate (T246) phosphorylations during differentiation.
Keywords:
neurogenesis; cell diferentiation
Available at various institutes of the ASCR
Quest for protein biomarkers of neural stem cells differentiation
Understanding neurogenesis and neural cell differentiation presents a unique challenge for treatment of nervous system disorders. To gain more insight about molecular mechanism of differentiation of ...
Molecular aspects of hypodontia
Fleischmannová, Jana; Krejčí, P.; Matalová, Eva; Míšek, Ivan
2007 - English
Numeric dental anomalies are the most common craniofacial congenital malformations in humans. More than 20 % of human population miss one or more third molars, approximately 5 % of population display agenesis of another tooth. In this contribution molecular events underlying tooth formation (more than 200 genes have been identified so far) and defects in tooth germ formation are correlated. Both syndromic forms of hypodonia (e.g. Rieger syndrome, anhidrotic ectodermal dysplasia) and non-syndromic forms (related to pax9, msx1, axin2) were investigated. Abnormality v počtu zubů jsou nejčastější kraniofaciální malformací v lidské populaci. Více než 20 % lidí nemá vyvinutý jeden nebo více třetích molárů a zhruba 5 % populace vykazuje agenezi dalších zubů. Příspěvek se zabývá korelací molekulárních kroků (zatím je známo asi 200 genů, které provázejí embryonální vývoj zubů) s výskytem defektů formování zubních základů. Sledovány jsou jak syndromické formy hypodoncií (např. Riegerův syndrom, anhidrotická ektodermální dysplazie), tak nesyndromické, se kterými jsou spojovány především klíčové regulační geny pax9, msx1, axin2.
Keywords:
hypodontia
Available at various institutes of the ASCR
Molecular aspects of hypodontia
Numeric dental anomalies are the most common craniofacial congenital malformations in humans. More than 20 % of human population miss one or more third molars, approximately 5 % of population display ...
Aurora-A is involved in resumption of meiosis and spindle formation in mouse oocytes
Šašková, Adéla; Šolc, Petr; Baran, V.; Kubelka, Michal; Motlík, Jan
2007 - English
We study the role of Aurora-A during meiotic maturation of mouse oocytes. Total Aurora-A is present in the nucleus in GV-stage oocytes (G2 equivalent). Additionally, active Aurora-A is localized entirely to the centrosome (MTOC) shorly before germinal vesicle breakdown (GVBD). Compared to somatic cells, where active Aurora-A is at the centrosomes and the spindle poles, active Aurora-A is strictly localized on MTOCs at metaphase I in oocytes. We show that activation of centrosomal Aurora-A is independent on PI3K-PKB and CDK1 signaling pathways. This was proved by cultivation of oocytes in presence of roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor). Treated oocytes show high phosphorylation of Aurora-A on T288 and centrosome amplification despite the presence of intact nuclear envelope. Silencing of Aurora-A by RNA interference induces incorrect spindle assembly. Oocytes are arrested in prometaphase I and unable to reach metaphase II. After microinjection of eGFP-Aurora-A mRNA into GV-stage oocytes, overexpression of Aurora-A leads to distortion of MI spindle organization as well. Our results indicate that Aurora-A is the key centrosomal player in meiotic maturation, essential for proper spindle formation and metaphase I - metaphase II transition. Práce se zabývá úlohou Aurora-A kinázy během meiotického zrání in vitro. Pomocí farmakologické inhibice PI3K, PKB a CDK1 bylo prokázáno, že Aurora-A, se aktivuje velmi časně při znovuzahájení meiosy, a toto aktivace je nezávislá na PI3K-PKB a CDK1 signalizaci. Dále bylo prokázáno (RNA interference, mikroinjekce mRNA), že Aurora-A je nutná pro korektní výstavbu metafáze I spindelu a pro metafase I – metafase II přechod.
Keywords:
meiosis
Available at various institutes of the ASCR
Aurora-A is involved in resumption of meiosis and spindle formation in mouse oocytes
We study the role of Aurora-A during meiotic maturation of mouse oocytes. Total Aurora-A is present in the nucleus in GV-stage oocytes (G2 equivalent). Additionally, active Aurora-A is localized ...
Genetic structure of populations of the tench Tinca tinca (Linnaeus, 1758)
Lajbner, Zdeněk; Linhart, Otomar; Kotlík, Petr
2007 - English
The tench is an important fish for aquaculture, which is undergoing intensive domestication, but only few studies have examined genetic structure of its populations. We present first results of a phylogeographic study based on DNA sequence data for four nuclear genes and one mitochondrial DNA gene from throughout the tench geographical distribution, including some of the known transfers outside its native Eurasian range. The multiple gene dataset revealed a strong phylogeographic partitioning between the western and eastern parts of the species range with a wide zone of overlap in Europe. Tench in European aquaculture largely represent mixtures of the two evolutionary lineages. We will discuss the likely historical processes underlying these findings. We suggest that human-mediated dispersal may have played an important role in shaping the present phylogeographic pattern. Ačkoliv lín patří mezi hospodářsky významné druhy ryb a v současnosti zažívá intenzivní domestikaci, pouze několik málo studií se doposud zabývalo genetickou strukturou jeho populací. Předkládáme zde výsledky fylogeografické studie založené na datovém souboru souboru sekvencí DNA pro čtyři jaderné a jeden mitochondriální gen. Do studie byli zahrnuty vzorky z celého areálu rozšíření druhu včetně několika oblastí, kde byl lín vysazen člověkem. Naše analýzy ukázaly hluboké fylogeografické rozdělení populací lína na západní a východní evoluční linii s širokou zónou překryvu v Evropě. Chovné linie z Evropy jsou tvořeny směsí těchto evolučních linií. Budeme diskutovat pravděpodobné historické procesy, které vedly ke zjištěné situaci. Domníváme se, že ve formování současného fylogeografického vzoru hrál podstatnou roli tok genů zprostředkovaný člověkem.
Keywords:
Tinca tinca
Available at various institutes of the ASCR
Genetic structure of populations of the tench Tinca tinca (Linnaeus, 1758)
The tench is an important fish for aquaculture, which is undergoing intensive domestication, but only few studies have examined genetic structure of its populations. We present first results of a ...
In vitro characterization of the spinal cord progenitor cells
Vitásková, Martina; Klíma, Jiří; Vodička, Petr; Motlík, Jan
2006 - English
We performed in vitro studies with the spinal cord of mice and miniature pig. Employing papain dissociation system we isolated neural progenitor cells and cultured them in DMEM/F12 HEPES, Ala-Glu, gentamicin, heparin, B27 supplement without vitamin A, N2 supplement, EGF (Epidermal Growth Factor) and bFGF (Basic Fibroblast Growth Factor). We added retinoic acid and 10% fetal bovine serum into cultivation media to induce cell differentiation. Using immunocytochemistry we confirmed cell capability to differentiate into neurons (βIII-tubulin, Map 2A - Microtubule Associated Protein), astroglia (GFAP – Glial Fibrillary Acidic Protein), and oligodendroglia (CNPase - 2',3'-cyclic nucleotide 3'-phosphodiesterase). Cells can keep non-differentiated status for a longer period of time when cultured in low adhesion culturing flask or by addition of LIF (Leukemia Inhibitory Factor) to the cultivation media. Nervové progenitorové buňky jsme izolovali z míšní tkáně myši respektive miniaturního prasete za pomoci disociačního systému s papainem. Buňky jsme kultivovali v mediu s DMEM/F12 HEPES, Ala-Glu, gentamicinem, heparinem, suplementem B27 bez vitaminu A, N2 suplementem, EGF (epidermální růstový faktor) a bFGF (základní růstový faktor fibroblastů). K navození diferenciace jsme do kultivačního media přidávali kyselinu retinovou a 10% fetální sérum. Imunocytochemicky jsme potvrdili schopnost buněk diferencovat se v neurony (βIII-tubulin, Map 2A – protein asociovaný s mikrotubuly), astroglii (GFAP – kyselý fibrilární protein glie) a oligodendroglii (CNPase - 2',3'-cyklická nucleotid 3'-fosfodiesteráza). Buňky si ponechávají svůj nediferencovaný stav po delší dobu, pokud jsou kultivovány v kultivačních lahvích s nízkou adhezivitou a nebo přidáním LIF (faktor inhibující leukemii) do kultivačního media.
Keywords:
spinal cord
Available at various institutes of the ASCR
In vitro characterization of the spinal cord progenitor cells
We performed in vitro studies with the spinal cord of mice and miniature pig. Employing papain dissociation system we isolated neural progenitor cells and cultured them in DMEM/F12 HEPES, Ala-Glu, ...
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