CAM assay Dotaz Zobrazit nápovědu
Cancer cells facilitate tumor growth by creating favorable tumor micro-environments (TME), altering homeostasis and immune response in the extracellular matrix (ECM) of surrounding tissue. A potential factor that contributes to TME generation and ECM remodeling is the cytoskeleton-associated human death-associated protein kinase 1 (DAPK1). Increased tumor cell motility and de-adhesion (thus, promoting metastasis), as well as upregulated plasminogen-signaling, are shown when functionally analyzing the DAPK1 ko-related proteome. However, the systematic investigation of how tumor cells actively modulate the ECM at the tissue level is experimentally challenging since animal models do not allow direct experimental access while artificial in vitro scaffolds cannot simulate the entire complexity of tissue systems. Here, we used the chorioallantoic membrane (CAM) assay as a natural, collagen-rich tissue model in combination with all-optical experimental access by multiphoton microscopy (MPM) to study the ECM remodeling potential of colorectal tumor cells with and without DAPK1 in situ and even in vivo. This approach demonstrates the suitability of the CAM assay in combination with multiphoton microscopy for studying collagen remodeling during tumor growth. Our results indicate the high ECM remodeling potential of DAPK1 ko tumor cells at the tissue level and support our findings from proteomics.
- Publikační typ
- časopisecké články MeSH
Antimicrobial peptides are currently considered as promising antiviral compounds. Current assays to evaluate the effectivity of peptides against enveloped viruses based on liposomes or hemolysis are encumbered by the artificial nature of liposomes or distinctive membrane composition of used erythrocytes. We propose a novel assay system based on enzymatic Ebola virus-like particles containing sensitive luciferase reporter. The assay was validated with several cationic and anionic peptides and compared with lentivirus inactivation and hemolytic assays. The assay is sensitive and easy to perform in standard biosafety level laboratory with potential for high-throughput screens. The use of virus-like particles in the assay provides a system as closely related to the native viruses as possible eliminating some issues associated with other more artificial set ups. We have identified CAM-W (KWKLWKKIEKWGQGIGAVLKWLTTWL) as a peptide with the greatest antiviral activity against infectious lentiviral vectors and filoviral virus-like particles.
- MeSH
- anionty MeSH
- antivirové látky farmakologie MeSH
- hemoragická horečka Ebola prevence a kontrola virologie MeSH
- kationické antimikrobiální peptidy farmakologie MeSH
- Lentivirus účinky léků genetika MeSH
- lidé MeSH
- liposomy chemie MeSH
- peptidy farmakologie MeSH
- virus Ebola účinky léků patogenita MeSH
- VLP vakcíny MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- autofagie * fyziologie MeSH
- biotest metody normy MeSH
- lidé MeSH
- počítačová simulace MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
- směrnice MeSH
The transient receptor potential channel TRPC6 is a non-selective cation channel which modulates the calcium level in eukaryotic cells (including sensory receptor cells) in response to external signals. Calmodulin (CaM) is a ubiquitously expressed Ca(2+) binding protein that is an important mediator of Ca(2+)-dependent regulation of the TRPC6 channel. One CaM binding site was identified within the C-tail of TRPC6. The aim of this study is to map in detail the CaM and inositol (1,4,5)-triphosphate receptor binding (CIRB) domain in the C-terminal region of mouse TRPC6 that is capable of interacting with CaM using in vitro binding assays. Besides the set of positively charged amino acid residues Arg852, Lys856, Arg864, Lys859/Arg860, a hydrophobic Ile857, at the position 1 in 1-5-10 motif, was located and the effect of replacing it with a neutral residue was tested using fluorescence anisotropy measurement. Participation of Ile857 could indicate a strong role of this conserved CaM binding motif.
- MeSH
- fluorescenční polarizace MeSH
- kalmodulin metabolismus MeSH
- kationtové kanály TRPC chemie genetika metabolismus MeSH
- klonování DNA MeSH
- molekulární modely MeSH
- mutageneze cílená MeSH
- myši MeSH
- retardační test MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A series of four 2‑amino‑3‑cyano‑4‑(3/4‑pyridyl)‑4H‑benzo[h]chromenes 2a-d and their dichlorido(p‑cymene)ruthenium(II) complexes 3a-d were tested for antiproliferative, vascular-disruptive, anti-angiogenic and DNA-binding activity. The coordination of the 4‑pyridyl‑4H‑naphthopyrans 2 to ruthenium led to complexes with pleiotropic effects. Unlike the free ligands 2a-d, their ruthenium complexes 3a-d showed a significant affinity for DNA as demonstrated by electrophoretic mobility shift assays (EMSA) and ethidium bromide assays. Binding of 3a-d to calf thymus DNA proceeded about 10-times faster compared with cisplatin. Treatment of HT-29 colon carcinoma, 518A2 melanoma and MCF-7Topo breast cancer cells with 3a and 3b caused an accumulation of cells in the G2/M phase and an increase of the fraction of mitotic cells in the case of HT-29, due to alterations of the microtubule cytoskeleton as shown by immunofluorescence staining. Complexes 3b-c showed a dual effect on the vascular system. They suppressed angiogenesis in zebrafish embryos and they destroyed the vasculature of the chorioallantoic membrane (CAM) in fertilized chicken eggs. They also inhibited the vasculogenic mimicry, typical of U-87 glioblastoma cells in tube formation assays.
- MeSH
- antitumorózní látky chemie farmakologie MeSH
- buňky HT-29 MeSH
- chorioalantoická membrána účinky léků MeSH
- cisplatina farmakologie MeSH
- dánio pruhované MeSH
- DNA chemie MeSH
- komplexní sloučeniny chemie MeSH
- lidé MeSH
- MFC-7 buňky MeSH
- nádorové buněčné linie MeSH
- retardační test MeSH
- ruthenium chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- adenokarcinom plic diagnóza genetika MeSH
- anaplastická lymfomová kináza genetika MeSH
- fúzní onkogenní proteiny genetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- membránové proteiny genetika MeSH
- myši SCID MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory plic diagnóza genetika MeSH
- proteiny nervové tkáně genetika MeSH
- proteiny vázající kalmodulin genetika MeSH
- staging nádorů MeSH
- xenogenní modely - testy antitumorózní aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
Purpose: The chick chorioallantoic membrane (CAM) assay can provide an alternative versatile, cost-effective, and ethically less controversial in vivo model for reliable screening of drugs. In the presented work, we demonstrate that CAM assay (in ovo and ex ovo) can be simply employed to delineate the effects of cisplatin (CDDP) and ellipticine (Elli) on neuroblastoma (Nbl) cells in terms of their growth and metastatic potential. Methods: The Nbl UKF-NB-4 cell line was established from recurrent bone marrow metastases of high-risk Nbl (stage IV, MYCN amplification, 7q21 gain). Ex ovo and in ovo CAM assays were optimized to evaluate the antimetastatic activity of CDDP and Elli. Immunohistochemistry, qRT-PCR, and DNA isolation were performed. Results: Ex ovo CAM assay was employed to study whether CDDP and Elli exhibit any inhibitory effects on growth of Nbl xenograft in ex ovo CAM assay. Under the optimal conditions, Elli and CDDP exhibited significant inhibition of the size of the primary tumor. To study the efficiency of CDDP and Elli to inhibit primary Nbl tumor growth, intravasation, and extravasation in the organs, we adapted the in ovo CAM assay protocol. In in ovo CAM assay, both studied compounds (CDDP and Elli) exhibited significant (p < 0.001) inhibitory activity against extravasation to all investigated organs including distal CAM. Conclusions: Taken together, CAM assay could be a helpful and highly efficient in vivo approach for high-throughput screening of libraries of compounds with expected anticancer activities.
- Publikační typ
- časopisecké články MeSH
AGR2 is an oncogenic endoplasmic reticulum (ER)-resident protein disulfide isomerase. AGR2 protein has a relatively unique property for a chaperone in that it can bind sequence-specifically to a specific peptide motif (TTIYY). A synthetic TTIYY-containing peptide column was used to affinity-purify AGR2 from crude lysates highlighting peptide selectivity in complex mixtures. Hydrogen-deuterium exchange mass spectrometry localized the dominant region in AGR2 that interacts with the TTIYY peptide to within a structural loop from amino acids 131-135 (VDPSL). A peptide binding site consensus of Tx[IL][YF][YF] was developed for AGR2 by measuring its activity against a mutant peptide library. Screening the human proteome for proteins harboring this motif revealed an enrichment in transmembrane proteins and we focused on validating EpCAM as a potential AGR2-interacting protein. AGR2 and EpCAM proteins formed a dose-dependent protein-protein interaction in vitro Proximity ligation assays demonstrated that endogenous AGR2 and EpCAM protein associate in cells. Introducing a single alanine mutation in EpCAM at Tyr251 attenuated its binding to AGR2 in vitro and in cells. Hydrogen-deuterium exchange mass spectrometry was used to identify a stable binding site for AGR2 on EpCAM, adjacent to the TLIYY motif and surrounding EpCAM's detergent binding site. These data define a dominant site on AGR2 that mediates its specific peptide-binding function. EpCAM forms a model client protein for AGR2 to study how an ER-resident chaperone can dock specifically to a peptide motif and regulate the trafficking a protein destined for the secretory pathway.
- MeSH
- adhezní molekula epiteliálních buněk genetika metabolismus MeSH
- lidé MeSH
- MFC-7 buňky MeSH
- peptidy metabolismus MeSH
- proteiny genetika metabolismus MeSH
- protoonkogenní proteiny c-mdm2 metabolismus MeSH
- rekombinantní proteiny metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Using scaffolds with appropriate porosity represents a potential approach for repair of critical-size bone defects. Vascularization is essential for bone formation and healing. This study investigates methods for monitoring angiogenesis within porous biopolymer scaffolds on the basis of polyhydroxybutyrate (PHB)/chitosan. We use the chick and quail chorioallantoic membrane (CAM) assay as an in vivo model focused on the formation of new blood vessels inside the implant structure. Chemical properties of the surface in biopolymer scaffold matrix were characterized as well as the tissue reaction of the CAM. Implantation of a piece of polymer scaffold results in vascular reaction, documented visually and by ultrasound biomicroscopy. Histological analysis shows myofibroblast reaction (smooth muscle actin-positive cells) without excessive collagen deposition. Cell invasion is observed inside the implant, and QH1 marker, detecting hemangioblasts and endothelial cells of quail origin, confirms the presence of vascular network. The CAM assay is a rapid and easy way to test biocompatibility and vasculogenic potential of new candidate scaffolds for bone tissue bioengineering with respect to the 3R´ s.
- MeSH
- biokompatibilní materiály MeSH
- chorioalantoická membrána krevní zásobení fyziologie MeSH
- fyziologická neovaskularizace fyziologie MeSH
- kosti a kostní tkáň * MeSH
- křepelky a křepelovití MeSH
- kuřecí embryo MeSH
- regenerace kostí fyziologie MeSH
- tkáňové inženýrství * MeSH
- tkáňové podpůrné struktury * MeSH
- zvířata MeSH
- Check Tag
- kuřecí embryo MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
This study illustrates the synthesis of functionalized carbon quantum dots (CQDs) by the one-pot pyrolysis method. The functionalization agent used in CQD synthesis was poly l- lysine (PLL). Various physicochemical techniques were employed to confirm the successful formation of PLLCQD including High resolution transmission electron microscopy (HR-TEM), UV-Vis spectroscopy, fluorescence spectroscopy; Atomic force microscopy (AFM), X-ray Photoelectron Spectroscopy (XPS) and X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. The size of PLLCQD was confirmed by HRTEM and AFM. The synthesized PLLCQD shows bright blue fluorescence and has a quantum yield of 19.35%. The highest emission band was observed at 471nm when excited to 370nm. The prepared PLLCQD exhibited excellent antibacterial activity against Escherichia coli and Staphylococcus aureus with inhibition zone 7-20 mm. The concentrations of 0.9 to 0.1gmL-1 were studied to determine minimum inhibitory concentration (MIC) by the agar well diffusion assay method. MIC of 0.2gml -1 concentration of PLLCQD is achieved. The anti-angiogenic activity of PLLCQD was determined using (Chick Chorioallantoic Membrane) CAM assay. CAM assay is a reliable in -vivo model to study angiogenesis also; many stimulators and inhibitors have been examined by this method. This study proves higher antibacterial efficiency of PLLCQD over non functionalized CQD. PLLCQD was successfully employed in bio-imaging of the bacterial cell through fluorescence microscopy. Further, PLLCQD displayed cytotoxic effect on endothelial cells and inhibited blood vessel formation in the CAM model.
