DNA nanotechnology is a rapidly growing research area, where DNA may be used for wide range of applications such as construction of nanodevices serving for large scale of diverse purposes. Likewise a panel of various purified fluorescent proteins is investigated for their ability to emit their typical fluorescence spectra under influence of particular excitation. Hence these proteins may form ideal donor molecules for assembly of fluorescence resonance emission transfer (FRET) constructions. To extend the application possibilities of fluorescent proteins, while using DNA nanotechnology, we developed nanoconstruction comprising green fluorescent protein (GFP) bound onto surface of surface active nanomaghemite and functionalized with gold nanoparticles. We took advantage of natural affinity between gold and thiol moieties, which were modified to bind DNA fragment. Finally we enclosed doxorubicin into fullerene cages. Doxorubicin intercalated in DNA fragment bound on the particles and thus we were able to connect these parts together. Because GFP behaved as a donor and doxorubicin as an acceptor using excitation wavelength for GFP (395 nm) in emission wavelength of doxorubicin (590 nm) FRET was observed. This nanoconstruction may serve as a double-labeled transporter of doxorubicin guided by force of external magnetic force owing to the presence of nanomaghemite. Further nanomaghemite offers the possibility of using this technology for thermotherapy.

• Klíčová slova
• Fullerene, Intercalation, Maghemite nanoparticles, Surface modification, Thiol modified DNA,
• MeSH
• DNA chemie   MeSH
• doxorubicin chemie   MeSH
• fullereny MeSH
• luminescentní proteiny chemie   MeSH
• magnetické nanočástice chemie   MeSH
• nanotechnologie metody   MeSH
• rezonanční přenos fluorescenční energie metody   MeSH
• zelené fluorescenční proteiny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• doxorubicin MeSH
• fullereny MeSH
• luminescentní proteiny MeSH
• magnetické nanočástice MeSH
• zelené fluorescenční proteiny MeSH

Transgenic mice expressing green fluorescent protein (GFP) are useful in transplantation experiments. When we used ubiquitin-GFP (UBC-GFP) transgenic mice to study the availability of niches for transplanted hematopoietic stem and progenitor cells, the results were strikingly different from the corresponding experiments that used congenic mice polymorphic in the CD45 antigen. Analysis of these unexpected results revealed that the hematopoiesis of UBC-GFP mice was outcompeted by the hematopoiesis of wild-type (WT) mice. Importantly, UBC-GFP mice engrafted the transplanted bone marrow of WT mice without conditioning. There was a significant bias toward lymphopoiesis in the WT branch of chimeric UBC-GFP/WT hematopoiesis. A fraction of immature Sca-1+ cells in the spleen of UBC-GFP mice expressed GFP at a very high level. The chimeric hematopoiesis was stable in the long term and also after transplantation to secondary recipient mice. The article thus identifies a specific defect in the hematopoiesis of UBC-GFP transgenic mice that compromises the lymphoid-primed hematopoietic stem cells in the bone marrow and spleen. Stem Cells 2018;36:1237-1248.

• Klíčová slova
• Bone marrow transplantation, Cell competition, Extramedullary hematopoiesis, Green fluorescent protein, Hematopoiesis, Lymphopoiesis, Stem cell niche, Stem cells, Transgenic mouse,
• MeSH
• chiméra MeSH
• hematopoetické kmenové buňky metabolismus   MeSH
• hematopoéza MeSH
• kostní dřeň metabolismus   MeSH
• lymfocyty metabolismus   MeSH
• lymfopoéza MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• slezina metabolismus   MeSH
• splenektomie MeSH
• thymus metabolismus   MeSH
• transplantace hematopoetických kmenových buněk * MeSH
• ubikvitin metabolismus   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ubikvitin MeSH
• zelené fluorescenční proteiny MeSH

The Lewis lung tumor has been extensively studied in both syngeneic and allogeneic mouse models. However, its metastatic potential and mechanism are poorly understood. The aim of the present study was to develop a highly metastatic lymph-node targeting, imageable model of the Lewis lung carcinoma in a syngeneic host. We report here a syngeneic model of the Lewis lung carcinoma in which the carcinoma cells are labeled with green fluorescent protein (GFP). The tumor cells were transplanted in the dorsal side of the ear of C57-B16 mice in order to give the tumor cells access to the lymphatic system. This model of the Lewis lung carcinoma extensively metastasized to numerous lymph nodes throughout the body of the animal as well as visceral organs, as visualized by fluorescence microscopy using the bright GFP signal. Twenty-one different metastatic sites, including lymph nodes throughout the body, were identified among the cohort of transplanted animals. The data demonstrate a predilection of the Lewis lung carcinoma for lymphatic pathways for metastasis throughout the animal body. The concomitant macrometastases to the visceral organs observed in this model may be remetastasis from the lymph nodes. This model of the Lewis lung carcinoma should be very useful in defining cellular trafficking and targeting mechanisms of metastasis, in particular those involving lymphatic pathways.

• MeSH
• fluorescenční mikroskopie MeSH
• karcinom plic Lewisové sekundární   MeSH
• luminescentní proteiny * MeSH
• lymfatické metastázy MeSH
• lymfatické uzliny patologie   MeSH
• modely nemocí na zvířatech * MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• Retroviridae genetika   MeSH
• transplantace nádorů MeSH
• zelené fluorescenční proteiny * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• luminescentní proteiny * MeSH
• zelené fluorescenční proteiny * MeSH

The chick-embryo model has been an important tool to study tumor growth, metastasis, and angiogenesis. However, an imageable model with a genetic fluorescent tag in the growing and spreading cancer cells that is stable over time has not been developed. We report here the development of such an imageable fluorescent chick-embryo metastatic cancer model with the use of green fluorescent protein (GFP). Lewis lung carcinoma cells, stably expressing GFP, were injected on the 12th day of incubation in the chick embryo. GFP-Lewis lung carcinoma metastases were visualized by fluorescence, after seven days additional incubation, in the brain, heart, and sternum of the developing chick embryo, with the most frequent site being the brain. The combination of streptokinase and gemcitabine was evaluated in this GFP metastatic model. Twelve-day-old chick embryos were injected intravenously with GFP-Lewis lung cancer cells, along with these two agents either alone or in combination. The streptokinase-gemcitabine combination inhibited metastases at all sites. The effective dose of gemcitabine was found to be 10 mg/kg and streptokinase 2000 IU per embryo. The data in this report suggest that this new stably fluorescent imageable metastatic-cancer chick-embryo model will enable rapid screening of new antimetastatic agents.

• MeSH
• deoxycytidin aplikace a dávkování   analogy a deriváty   MeSH
• gemcitabin MeSH
• karcinom plic Lewisové farmakoterapie   MeSH
• kuřecí embryo MeSH
• léky antitumorózní - screeningové testy metody   MeSH
• metastázy nádorů prevence a kontrola   MeSH
• modely nemocí na zvířatech * MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• streptokinasa aplikace a dávkování   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• deoxycytidin MeSH
• gemcitabin MeSH
• streptokinasa MeSH
• zelené fluorescenční proteiny MeSH

Catalytic properties and high adsorption affinity of nucleic acids and proteins to silver amalgam electrode surface make this kind of electrified interface perspective for bioanalytical and biomedical applications. For the first time, a basal-plane pyrolytic graphite electrode (bPGE) has been used as a substrate for electrodeposition of silver amalgam particles (AgAPs). Optimization of the resulting composition, surface morphology and electrochemical properties of the AgAPs was done by scanning electron microscopy with energy disperse X-ray spectroscopy, image processing software and voltammetric detection of electrochemically reducible model organic nitro compound, 4-nitrophenol. Spectro-electrochemical applicability of bPGE-AgAP has been demonstrated by electrolysis of 4-nitrophenol. Simultaneous UV-Vis-chronoamperometry provided information on the number of exchange electrons and the reduction rate constants. Preferential adsorption of the fluorescently labelled calf thymus DNA and the green fluorescent protein (GFP) on the surface of AgAPs was observed by fluorescence microscopy. In contrast to previously studied indium-tin oxide and vapour-deposited gold decorated by AgAPs, herein the presented bPGE-AgAP has provided sufficiently wide negative potential window allowing direct electroanalysis of non-labelled DNA and GFP using intrinsic electrochemical signals independently of the fluorescent labelling. The bPGE-AgAP can thus be expected to find application opportunities in protein electrochemistry, (bio)sensor development or in-situ spectro-electrochemical studies.

• Klíčová slova
• 4-nitrophenol, DNA, Electrodeposition, Protein analysis, Silver amalgam particles, Spectroelectrochemistry,
• MeSH
• adsorpce MeSH
• DNA analýza   MeSH
• elektrochemické techniky metody   MeSH
• mikroskopie elektronová rastrovací MeSH
• nitrofenoly analýza   MeSH
• pokovování galvanické * MeSH
• stříbro chemie   MeSH
• zelené fluorescenční proteiny analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 4-nitrophenol MeSH Prohlížeč
• DNA MeSH
• nitrofenoly MeSH
• stříbro MeSH
• zelené fluorescenční proteiny MeSH

Tagging cells of experimental organisms with genetic markers is commonly used in biomedical research. Insertion of artificial gene constructs can be highly beneficial for research as long as this tagging is functionally neutral and does not alter the tissue function. The transgenic UBC-GFP mouse has been recently found to be questionable in this respect, due to a latent stem cell defect compromising its lymphopoiesis and significantly influencing the results of competitive transplantation assays. In this study, we show that the stem cell defect present in UBC-GFP mice negatively affects T-lymphopoiesis significantly more than B-lymphopoiesis. The production of granulocytes is not negatively affected. The defect in T-lymphopoiesis causes a low total number of white blood cells in the peripheral blood of UBC-GFP mice which, together with the lower lymphoid/myeloid ratio in nucleated blood cells, is the only abnormal phenotype in untreated UBCGFP mice to have been found to date. The defective lymphopoiesis in UBC-GFP mice can be repaired by transplantation of congenic wild-type bone marrow cells, which then compensate for the insufficient production of T cells. Interestingly, the wild-type branch of haematopoiesis in chimaeric UBC-GFP/wild-type mice was more active in lymphopoiesis, and particularly towards production of T cells, compared to the lymphopoiesis in normal wild-type donors.

• MeSH
• kmenové buňky metabolismus   patologie   MeSH
• lymfopoéza * MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• myši MeSH
• T-lymfocyty metabolismus   patologie   MeSH
• ubikvitin genetika   metabolismus   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ubikvitin MeSH
• zelené fluorescenční proteiny MeSH

Our goal was to find an optimal tissue clearing protocol for whole-mount imaging of embryonic and adult hearts and whole embryos of transgenic mice that would preserve green fluorescent protein GFP fluorescence and permit comparison of different currently available 3D imaging modalities. We tested various published organic solvent- or water-based clearing protocols intended to preserve GFP fluorescence in central nervous system: tetrahydrofuran dehydration and dibenzylether protocol (DBE), SCALE, CLARITY, and CUBIC and evaluated their ability to render hearts and whole embryos transparent. DBE clearing protocol did not preserve GFP fluorescence; in addition, DBE caused considerable tissue-shrinking artifacts compared to the gold standard BABB protocol. The CLARITY method considerably improved tissue transparency at later stages, but also decreased GFP fluorescence intensity. The SCALE clearing resulted in sufficient tissue transparency up to ED12.5; at later stages the useful depth of imaging was limited by tissue light scattering. The best method for the cardiac specimens proved to be the CUBIC protocol, which preserved GFP fluorescence well, and cleared the specimens sufficiently even at the adult stages. In addition, CUBIC decolorized the blood and myocardium by removing tissue iron. Good 3D renderings of whole fetal hearts and embryos were obtained with optical projection tomography and selective plane illumination microscopy, although at resolutions lower than with a confocal microscope. Comparison of five tissue clearing protocols and three imaging methods for study of GFP mouse embryos and hearts shows that the optimal method depends on stage and level of detail required.

• Klíčová slova
• Confocal microscopy, Embryo, Green fluorescent protein (GFP), Heart, Optical projection tomography, Tissue transparency,
• MeSH
• myši transgenní MeSH
• myši MeSH
• srdce embryologie   MeSH
• zelené fluorescenční proteiny analýza   biosyntéza   genetika   MeSH
• zobrazování trojrozměrné metody   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• zelené fluorescenční proteiny MeSH

The effect of the type of leaf tissue selected for the study of green fluorescent protein (GFP) fluorescence intensity was investigated here using the T(1) generation of transgenic tobacco expressing the m-gfp5-ER gene. The fluorescence of GFP was detected by fluorescence binocular microscope coupled with the CCD camera and quantified by means of image analyses using the Lucia((R)) software. Mean brightness values from various leaf tissues were compared. First, an original data revealing the significant differences in the fluorescence intensity between the abaxial and adaxial surfaces are given. Stronger signal was detected on the abaxial side. Subsequently, the effect of the tissue location within the leaf surface was investigated and higher fluorescence was detected on the samples detached from leaf tips. Finally, the effect of the physiological age of leaves was studied using the in vitro clonally propagated plants. Leaves from the analogous positions within the plant body of three clones were investigated. The decrease in the fluorescence towards the plant top (youngest leaves) was observed in all studied plants. Surprisingly, the variability of the fluorescence within the clones of studied genotype was high enough to conclude, that the fluorescence of each individual is unique and affected by particular genotype and environment. Our study showed that the origin of leaf tissue selected for the GFP quantification is crucial and that the fluctuations in the fluorescence intensity should be taken into account when comparing the GFP fluorescence patterns of different plants. Moreover, the degree of fluorescence variability seems to be individually affected.

• MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fluorescenční mikroskopie MeSH
• geneticky modifikované rostliny genetika   metabolismus   MeSH
• listy rostlin genetika   metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• polymerázová řetězová reakce MeSH
• tabák genetika   metabolismus   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• zelené fluorescenční proteiny MeSH

Different types of lipid- and polymer-based vectors have been developed to deliver proteins into cells, but these methods showed relatively poor efficiency. Recently, a group of short, highly basic peptides known as cell-penetrating peptides (CPPs) were used to carry polypeptides and proteins into cells. In this study, expression and purification of GFP protein was performed using the prokaryotic pET expression system. We used two amphipathic CPPs (Pep-1 and CADY-2) as a novel delivery system to transfer the GFP protein into cells. The morphological features of the CPP/GFP complexes were studied by scanning electron microscopy (SEM), Zetasizer, and SDS-PAGE. The efficiency of GFP transfection using Pep-1 and CADY-2 peptides and TurboFect reagent was compared with FITC-antibody protein control delivered by these transfection vehicles in the HEK-293T cell line. SEM data confirmed formation of discrete nanoparticles with a diameter of below 300 nm. Moreover, formation of the complexes was detected using SDS-PAGE as two individual bands, indicating non-covalent interaction. The size and homogeneity of Pep-1/GFP and CADY-2/GFP complexes were dependent on the ratio of peptide/cargo formulations, and responsible for their biological efficiency. The cells transfected by Pep-1/GFP and CADY-2/GFP complexes at a molar ratio of 20 : 1 demonstrated spreading green regions using fluorescent microscopy. Flow cytometry results showed that the transfection efficiency of Pep-based nanoparticles was similar to CADY-based nanoparticles and comparable with TurboFect-protein complexes. These data open an efficient way for future therapeutic purposes.

• MeSH
• biologický transport MeSH
• exprese genu MeSH
• HEK293 buňky MeSH
• klonování DNA MeSH
• lidé MeSH
• mikroskopie elektronová rastrovací MeSH
• nanočástice chemie   metabolismus   MeSH
• penetrační peptidy metabolismus   MeSH
• plazmidy genetika   MeSH
• průtoková cytometrie MeSH
• rekombinantní proteiny genetika   izolace a purifikace   metabolismus   ultrastruktura   MeSH
• transfekce MeSH
• western blotting MeSH
• zelené fluorescenční proteiny genetika   izolace a purifikace   metabolismus   ultrastruktura   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• penetrační peptidy MeSH
• rekombinantní proteiny MeSH
• zelené fluorescenční proteiny MeSH

A constitutive expression vector pHY300-Flgfp was constructed to test the function of promoter F1 subcloned from a rice epiphyte Bacillus brevis strain DX01. The DX01 cells harboring plasmid pHY300-F1gfp were detected to produce bright green fluorescence. Subsequently, the gfp-tagged B. brevis strain was released into the soil and its survival was investigated by PCR and the detection of green fluorescence. The spatial location of in situ gfp-tagged bacterial cells on the root surface of rice seedlings was visualized. All these results indicated that green fluorescent protein is an ideal molecular marker for the detection of the activities of promoter F1, and it is also a reliable probe to monitor specific B. brevis bacteria in the environment.

• MeSH
• Bacillus genetika   fyziologie   MeSH
• barvení a značení metody   MeSH
• DNA bakterií analýza   MeSH
• exprese genu * MeSH
• fluorescence MeSH
• genetické vektory MeSH
• kořeny rostlin mikrobiologie   MeSH
• plazmidy MeSH
• polymerázová řetězová reakce MeSH
• promotorové oblasti (genetika) * MeSH
• půdní mikrobiologie * MeSH
• reportérové geny MeSH
• rýže (rod) mikrobiologie   MeSH
• semenáček mikrobiologie   MeSH
• zelené fluorescenční proteiny analýza   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• zelené fluorescenční proteiny MeSH