BACKGROUND: Oocytes of large animal species isolated from small ovarian follicles (< 2 mm) are less competent to support early embryonic development after in vitro maturation and fertilization than their counterparts isolated from medium-sized and preovulatory follicles. This study aimed to assess the effect of a new maturation medium containing FGF2, LIF, and IGF1 (FLI medium) on the meiotic and developmental competence of pig cumulus-oocytes complexes (COCs) derived from the small and medium-sized follicles. METHODS: The growing oocytes were isolated from 1 to 2 (small follicle; SF) and the fully-grown ones from 3 to 6 (large follicle; LF) mm follicles and matured in a control M199 medium with gonadotropins and EGF and the FLI medium enriched by the triplet of growth factors. The matured oocytes were parthenogenetically activated and cultured to the blastocyst stage. Chromatin configuration before and during the culture and MAP kinase activity were assessed in the oocytes. Finally, the expression of cumulus cell genes previously identified as markers of oocyte quality was assessed. RESULTS: The maturation and blastocyst rates of oocytes gained from LF were significantly higher than that from SF in the control medium. In contrast, similar proportions of oocytes from LF and SF completed meiosis and developed to blastocysts when cultured in FLI. Most of the oocytes freshly isolated from SF possessed germinal vesicles with fine filaments of chromatin (GV0) or chromatin surrounding the nucleolus (GVI; 30%); the oocytes from LF were mainly in GVI (or GVII) exhibiting a few small lumps of chromatin beneath the nuclear membrane. When cultured in the FLI medium for 16 h, an acceleration of the course of maturation in oocytes both from SF and LF compared to the control medium was observed and a remarkable synchrony in the course of chromatin remodeling was noticed in oocytes from SF and LF. CONCLUSIONS: This work demonstrates that the enrichment of culture medium by FGF2, LIF, and IGF1 can enhance the meiotic and developmental competence of not only fully-grown, but also growing pig oocytes and significantly thus expanding the number of oocytes available for various assisted reproductive technology applications.

• Klíčová slova
• Chromatin configuration, Developmental potential, Follicle size, Gene expression, Growth factors, MAPK activation, Oocyte maturation,
• MeSH
• chromatin metabolismus   MeSH
• fibroblastový růstový faktor 2 * farmakologie   metabolismus   MeSH
• IVM techniky * MeSH
• meióza MeSH
• oocyty metabolismus   MeSH
• ovariální folikul MeSH
• prasata MeSH
• těhotenství MeSH
• zvířata MeSH
• Check Tag
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chromatin MeSH
• fibroblastový růstový faktor 2 * MeSH

Several peripheral membrane proteins are known to form membrane pores through multimerization. In many cases, in biochemical reconstitution experiments, a complex distribution of oligomeric states has been observed that may, in part, be irrelevant to their physiological functions. This phenomenon makes it difficult to identify the functional oligomeric states of membrane lipid interacting proteins, for example, during the formation of transient membrane pores. Using fibroblast growth factor 2 (FGF2) as an example, we present a methodology applicable to giant lipid vesicles by which functional oligomers can be distinguished from nonspecifically aggregated proteins without functionality. Two distinct populations of fibroblast growth factor 2 were identified with (i) dimers to hexamers and (ii) a broad population of higher oligomeric states of membrane-associated FGF2 oligomers significantly distorting the original unfiltered histogram of all detectable oligomeric species of FGF2. The presented statistical approach is relevant for various techniques for characterizing membrane-dependent protein oligomerization.

• MeSH
• buněčná membrána metabolismus   MeSH
• fibroblastový růstový faktor 2 * metabolismus   MeSH
• lipidy MeSH
• membránové proteiny * metabolismus   MeSH
• membrány MeSH
• multimerizace proteinu MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fibroblastový růstový faktor 2 * MeSH
• lipidy MeSH
• membránové proteiny * MeSH

FGF2 is a cell survival factor involved in tumor-induced angiogenesis that is secreted through an unconventional secretory pathway based upon direct protein translocation across the plasma membrane. Here, we demonstrate that both PI(4,5)P2-dependent FGF2 recruitment at the inner plasma membrane leaflet and FGF2 membrane translocation into the extracellular space are positively modulated by cholesterol in living cells. We further revealed cholesterol to enhance FGF2 binding to PI(4,5)P2-containing lipid bilayers. Based on extensive atomistic molecular dynamics (MD) simulations and membrane tension experiments, we proposed cholesterol to modulate FGF2 binding to PI(4,5)P2 by (i) increasing head group visibility of PI(4,5)P2 on the membrane surface, (ii) increasing avidity by cholesterol-induced clustering of PI(4,5)P2 molecules triggering FGF2 oligomerization, and (iii) increasing membrane tension facilitating the formation of lipidic membrane pores. Our findings have general implications for phosphoinositide-dependent protein recruitment to membranes and explain the highly selective targeting of FGF2 toward the plasma membrane, the subcellular site of FGF2 membrane translocation during unconventional secretion of FGF2.

• MeSH
• buněčná membrána metabolismus   MeSH
• cholesterol * metabolismus   MeSH
• fibroblastový růstový faktor 2 * metabolismus   MeSH
• fosfatidylinositol-4,5-difosfát * metabolismus   MeSH
• lipidové dvojvrstvy * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholesterol * MeSH
• fibroblastový růstový faktor 2 * MeSH
• fosfatidylinositol-4,5-difosfát * MeSH
• lipidové dvojvrstvy * MeSH

In-membrane oligomerization is decisive for the function (or dysfunction) of many proteins. Techniques were developed to characterize membrane-inserted oligomers and the hereby obtained oligomerization states were intuitively related to the function of these proteins. However, in many cases, it is unclear whether the obtained oligomerization states are functionally relevant or are merely the consequence of nonspecific aggregation. Using fibroblast growth factor 2 (FGF2) as a model system, we addressed this methodological challenge. FGF2 oligomerizes in a PI(4,5)P2-dependent manner at the inner plasma membrane leaflet. This process results in membrane insertion and the formation of a lipidic membrane pore, the key intermediate in unconventional secretion of FGF2. To tackle the problem of discriminating functional oligomers from irrelevant aggregates, we present a statistical single molecule and single vesicle assay determining the brightness of individually diffusing in-membrane oligomers and correlating their oligomerization state with membrane pore formation. Importantly, time-dependent membrane pore formation was analyzed with an ensemble of single vesicles providing detailed statistics. Our findings demonstrate that quantifying oligomeric states alone does not allow for a deep understanding of the structure-function relationship of membrane-inserted oligomers.

• MeSH
• buněčná membrána chemie   metabolismus   MeSH
• fibroblastový růstový faktor 2 chemie   metabolismus   MeSH
• fluorescenční spektrometrie MeSH
• kvarterní struktura proteinů MeSH
• multimerizace proteinu * MeSH
• permeabilita MeSH
• poréznost MeSH
• unilamelární lipozómy chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fibroblastový růstový faktor 2 MeSH
• unilamelární lipozómy MeSH

Fibroblast growth factor (FGF) signaling is crucial for mammary gland development. Although multiple roles for FGF signaling in the epithelium have been described, the function of FGF signaling in mammary stroma has not been elucidated. In this study, we investigated FGF signaling in mammary fibroblasts. We found that murine mammary fibroblasts express FGF receptors FGFR1 and FGFR2 and respond to FGF ligands. In particular, FGF2 and FGF9 induce sustained ERK1/2 signaling and promote fibroblast proliferation and migration in 2D cultures. Intriguingly, only FGF2 induces fibroblast migration in 3D extracellular matrix (ECM) through regulation of actomyosin cytoskeleton and promotes force-mediated collagen remodeling by mammary fibroblasts. Moreover, FGF2 regulates production of ECM proteins by mammary fibroblasts, including collagens, fibronectin, osteopontin and matrix metalloproteinases. Finally, using organotypic 3D co-cultures we show that FGF2 and FGF9 signaling in mammary fibroblasts enhances fibroblast-induced branching of mammary epithelium by modulating paracrine signaling, and that knockdown of Fgfr1 and Fgfr2 in mammary fibroblasts reduces branching of mammary epithelium. Our results demonstrate a pleiotropic role for FGF signaling in mammary fibroblasts, with implications for regulation of mammary stromal functions and epithelial branching morphogenesis.

• Klíčová slova
• Branching morphogenesis, Collagen, Extracellular matrix, Fibroblast, Fibroblast growth factor, Mammary gland, Mouse, Stroma,
• MeSH
• fibroblastový růstový faktor 2 metabolismus   MeSH
• fibroblastový růstový faktor 9 metabolismus   MeSH
• fibroblasty cytologie   metabolismus   MeSH
• MAP kinasový signální systém * MeSH
• mléčné žlázy zvířat cytologie   embryologie   MeSH
• myši inbrední ICR MeSH
• myši MeSH
• parakrinní signalizace * MeSH
• receptor fibroblastových růstových faktorů, typ 1 metabolismus   MeSH
• receptor fibroblastových růstových faktorů, typ 2 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Fgf9 protein, mouse MeSH Prohlížeč
• Fgfr1 protein, mouse MeSH Prohlížeč
• Fgfr2 protein, mouse MeSH Prohlížeč
• fibroblastový růstový faktor 2 MeSH
• fibroblastový růstový faktor 9 MeSH
• receptor fibroblastových růstových faktorů, typ 1 MeSH
• receptor fibroblastových růstových faktorů, typ 2 MeSH

Melanoma is one of the most aggressive cancers which has very low response rate and survival rate. Melanoma cells are known to be inherently resistant to the chemotherapy which results in poor outcomes and even failure of the therapy. For this reason, a better understanding of underlying mechanism of melanoma pathogenesis and resistance is required, so that more efficient and novel therapeutic strategies can be developed. Survivin is a protein which is overexpressed in melanoma cells and is known to impart resistance to them against apoptosis. Also, melanoma cells overexpress Vascular Endothelial Growth Factor (VEGF) and basic Fibroblast Growth Factor (bFGF) angiogenic growth factors which lead to aggressive angiogenesis in melanoma cells thereby making the treatment more challenging. This hypothesis presents a combinatorial approach against melanoma where an anti-survivin agent and an anti-angiogenic agent are combined with a chemotherapeutic drug and loaded in surface functionalized liposomes in order to target specific mechanisms of melanoma, thus overcoming its resistance. Thus, the study aims to overcome the resistance of melanoma cells by developing a wise combination of drugs and achieve a higher response rate in resistant melanoma model, which is usually not achieved with the existing treatment modalities.

• MeSH
• antigeny CD44 metabolismus   MeSH
• antitumorózní látky chemie   MeSH
• dakarbazin terapeutické užití   MeSH
• fibroblastový růstový faktor 2 metabolismus   MeSH
• inhibitory angiogeneze chemie   MeSH
• inhibitory apoptózy chemie   MeSH
• lidé MeSH
• liposomy chemie   MeSH
• melanom metabolismus   terapie   MeSH
• nádory kůže terapie   MeSH
• nanočástice chemie   MeSH
• patologická angiogeneze MeSH
• proliferace buněk MeSH
• survivin MeSH
• teoretické modely MeSH
• vaskulární endoteliální růstový faktor A metabolismus   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny CD44 MeSH
• antitumorózní látky MeSH
• BIRC5 protein, human MeSH Prohlížeč
• CD44 protein, human MeSH Prohlížeč
• dakarbazin MeSH
• fibroblastový růstový faktor 2 MeSH
• inhibitory angiogeneze MeSH
• inhibitory apoptózy MeSH
• liposomy MeSH
• survivin MeSH
• vaskulární endoteliální růstový faktor A MeSH

Cardiac atrophy is the most common complication of prolonged application of the left ventricle (LV) assist device (LVAD) in patients with advanced heart failure (HF). Our aim was to evaluate the course of unloading-induced cardiac atrophy in rats with failing hearts, and to examine if increased isovolumic loading obtained by intraventricular implantation of an especially designed spring expander would attenuate this process. Heterotopic abdominal heart transplantation (HTx) was used as a rat model of heart unloading. HF was induced by volume overload achieved by creation of the aorto-caval fistula (ACF). The degree of cardiac atrophy was assessed as the weight ratio of the heterotopically transplanted heart (HW) to the control heart. Isovolumic loading was increased by intraventricular implantation of a stainless steel three-branch spring expander. The course of cardiac atrophy was evaluated on days 7, 14, 21, and 28 after HTx Seven days unloading by HTx in failing hearts sufficed to substantially decrease the HW (-59 ± 3%), the decrease progressed when measured on days 14, 21, and 28 after HTx Implantation of the spring expander significantly reduced the decreases in whole HW at all the time points (-39 ± 3 compared with -59 ± 3, -52 ± 2 compared with -69 ± 3, -51 ± 2 compared with -71 ± 2, and -44 ± 2 compared with -71 ± 3%, respectively; P<0.05 in each case). We conclude that the enhanced isovolumic heart loading obtained by implantation of the spring expander attenuates the development of unloading-induced cardiac atrophy in the failing rat heart.

• Klíčová slova
• Cardiac atrophy, heart failure, heterotopic heart transplantation, mechanical heart unloading, spring expander,
• MeSH
• aorta chirurgie   MeSH
• atriální natriuretický faktor genetika   metabolismus   MeSH
• atrofie metabolismus   patofyziologie   prevence a kontrola   chirurgie   MeSH
• biologické markery metabolismus   MeSH
• design vybavení MeSH
• experimentální implantáty MeSH
• exprese genu MeSH
• fibroblastový růstový faktor 2 genetika   metabolismus   MeSH
• heterotopická transplantace MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• píštěle MeSH
• potkani inbrední LEW MeSH
• přenašeč glukosy typ 1 genetika   metabolismus   MeSH
• sarkoplazmatická Ca2+-ATPáza genetika   metabolismus   MeSH
• srdce patofyziologie   MeSH
• srdeční komory patofyziologie   chirurgie   MeSH
• srdeční selhání metabolismus   patofyziologie   chirurgie   terapie   MeSH
• tkáňové expandéry * MeSH
• transplantace srdce * MeSH
• vena cava superior chirurgie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Atp2a1 protein, rat MeSH Prohlížeč
• atriální natriuretický faktor MeSH
• biologické markery MeSH
• fibroblastový růstový faktor 2 MeSH
• přenašeč glukosy typ 1 MeSH
• sarkoplazmatická Ca2+-ATPáza MeSH
• Slc2a1 protein, rat MeSH Prohlížeč

Fibroblast growth factors (FGFs) serve numerous regulatory functions in complex organisms, and their corresponding therapeutic potential is of growing interest to academics and industrial researchers alike. However, applications of these proteins are limited due to their low stability. Here we tackle this problem using a generalizable computer-assisted protein engineering strategy to create a unique modified FGF2 with nine mutations displaying unprecedented stability and uncompromised biological function. The data from the characterization of stabilized FGF2 showed a remarkable prediction potential of in silico methods and provided insight into the unfolding mechanism of the protein. The molecule holds a considerable promise for stem cell research and medical or pharmaceutical applications.

• Klíčová slova
• computational design, embryonic stem cells, fibroblast growth factor, focused directed evolution, protein engineering, protein stability,
• MeSH
• bodová mutace MeSH
• design s pomocí počítače * MeSH
• embryonální kmenové buňky cytologie   metabolismus   MeSH
• fibroblastový růstový faktor 2 chemie   genetika   metabolismus   MeSH
• lidé MeSH
• počítačová simulace MeSH
• proteinové inženýrství * MeSH
• řízená evoluce molekul MeSH
• sbalování proteinů MeSH
• sekvence aminokyselin MeSH
• stabilita proteinů * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fibroblastový růstový faktor 2 MeSH

Human pluripotent stem cells (hPSC) require signaling provided by fibroblast growth factor (FGF) receptors. This can be initiated by the recombinant FGF2 ligand supplied exogenously, but hPSC further support their niche by secretion of endogenous FGF2. In this study, we describe a role of tyrosine kinase expressed in hepatocellular carcinoma (TEC) kinase in this process. We show that TEC-mediated FGF2 secretion is essential for hPSC self-renewal, and its lack mediates specific differentiation. Following both short hairpin RNA- and small interfering RNA-mediated TEC knockdown, hPSC secretes less FGF2. This impairs hPSC proliferation that can be rescued by increasing amounts of recombinant FGF2. TEC downregulation further leads to a lower expression of the pluripotency markers, an improved priming towards neuroectodermal lineage, and a failure to develop cardiac mesoderm. Our data thus demonstrate that TEC is yet another regulator of FGF2-mediated hPSC pluripotency and differentiation. Stem Cells 2017;35:2050-2059.

• Klíčová slova
• Cardiac differentiation, Embryonic stem cells, Fibroblast growth factor, Fibroblast growth factor 2, Neural differentiation, Pluripotent stem cells, Tyrosine kinase expressed in hepatocellular carcinoma,
• MeSH
• biologické markery metabolismus   MeSH
• buněčné linie MeSH
• buněčný rodokmen * účinky léků   MeSH
• down regulace účinky léků   MeSH
• fibroblastový růstový faktor 2 metabolismus   MeSH
• lidé MeSH
• pluripotentní kmenové buňky cytologie   enzymologie   MeSH
• proliferace buněk účinky léků   MeSH
• rekombinantní proteiny farmakologie   MeSH
• tyrosinkinasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biologické markery MeSH
• fibroblastový růstový faktor 2 MeSH
• rekombinantní proteiny MeSH
• Tec protein-tyrosine kinase MeSH Prohlížeč
• tyrosinkinasy MeSH

Ventral root avulsion (VRA) triggers a strong glial reaction which contributes to neuronal loss, as well as to synaptic detachment. To overcome the degenerative effects of VRA, treatments with neurotrophic factors and stem cells have been proposed. Thus, we investigated neuroprotection elicited by human embryonic stem cells (hESC), modified to overexpress a human fibroblast growth factor 2 (FGF-2), on motoneurons subjected to VRA. Lewis rats were submitted to VRA (L4-L6) and hESC/FGF-2 were applied to the injury site using a fibrin scaffold. The spinal cords were processed to evaluate neuronal survival, synaptic stability, and glial reactivity two weeks post lesion. Then, qRT-PCR was used to assess gene expression of β2-microglobulin (β2m), TNFα, IL1β, IL6 and IL10 in the spinal cord in vivo and FGF2 mRNA levels in hESC in vitro. The results indicate that hESC overexpressing FGF2 significantly rescued avulsed motoneurons, preserving synaptic covering and reducing astroglial reactivity. The cells were also shown to express BDNF and GDNF at the site of injury. Additionally, engraftment of hESC led to a significant reduction in mRNA levels of TNFα at the spinal cord ventral horn, indicating their immunomodulatory properties. Overall, the present data suggest that hESC overexpressing FGF2 are neuroprotective and can shift gene expression towards an anti-inflammatory environment.

• Klíčová slova
• Embryonic stem cells, Fibroblast growth factor 2, Motoneurons, Neuroprotection, Neurotrophic factors, Ventral root avulsion,
• MeSH
• doxycyklin terapeutické užití   MeSH
• fibrinová tkáňová adheziva toxicita   MeSH
• fibroblastový růstový faktor 2 genetika   metabolismus   MeSH
• genetické vektory fyziologie   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• lidské embryonální kmenové buňky metabolismus   transplantace   MeSH
• míšní kořeny patologie   MeSH
• modely nemocí na zvířatech MeSH
• motorické neurony metabolismus   patologie   MeSH
• neuroglie účinky léků   metabolismus   MeSH
• pohyb buněk MeSH
• potkani inbrední LEW MeSH
• proteiny nervové tkáně metabolismus   MeSH
• radikulopatie chemicky indukované   chirurgie   MeSH
• regulace genové exprese účinky léků   genetika   MeSH
• tkáňová adheziva toxicita   MeSH
• viabilita buněk účinky léků   genetika   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• doxycyklin MeSH
• fibrinová tkáňová adheziva MeSH
• fibroblastový růstový faktor 2 MeSH
• proteiny nervové tkáně MeSH
• tkáňová adheziva MeSH