Immature capsids of the Betaretrovirus, Mason-Pfizer Monkey virus (M-PMV), are assembled in the pericentriolar region of the cell, and are then transported to the plasma membrane for budding. Although several studies, utilizing mutagenesis, biochemistry, and immunofluorescence, have defined the role of some viral and host cells factors involved in these processes, they have the disadvantage of population analysis, rather than analyzing individual capsid movement in real time. In this study, we created an M-PMV vector in which the enhanced green fluorescent protein, eGFP, was fused to the carboxyl-terminus of the M-PMV Gag polyprotein, to create a Gag-GFP fusion that could be visualized in live cells. In order to express this fusion protein in the context of an M-PMV proviral backbone, it was necessary to codon-optimize gag, optimize the Kozak sequence preceding the initiating methionine, and mutate an internal methionine codon to one for alanine (M100A) to prevent internal initiation of translation. Co-expression of this pSARM-Gag-GFP-M100A vector with a WT M-PMV provirus resulted in efficient assembly and release of capsids. Results from fixed-cell immunofluorescence and pulse-chase analyses of wild type and mutant Gag-GFP constructs demonstrated comparable intracellular localization and release of capsids to untagged counterparts. Real-time, live-cell visualization and analysis of the GFP-tagged capsids provided strong evidence for a role for microtubules in the intracellular transport of M-PMV capsids. Thus, this M-PMV Gag-GFP vector is a useful tool for identifying novel virus-cell interactions involved in intracellular M-PMV capsid transport in a dynamic, real-time system.

• MeSH
• biologický transport MeSH
• buněčná membrána metabolismus   MeSH
• fluorescenční barviva metabolismus   MeSH
• genetické vektory genetika   MeSH
• genové produkty gag genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• kapsida metabolismus   MeSH
• kinetika MeSH
• lidé MeSH
• Masonův-Pfizerův opičí virus genetika   metabolismus   fyziologie   MeSH
• mikrotubuly metabolismus   virologie   MeSH
• molekulární zobrazování MeSH
• pohyb MeSH
• proviry genetika   metabolismus   fyziologie   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• sestavení viru MeSH
• transport proteinů MeSH
• viabilita buněk MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• fluorescenční barviva MeSH
• genové produkty gag MeSH
• rekombinantní fúzní proteiny MeSH
• zelené fluorescenční proteiny MeSH

Tetraspanins are integral membrane proteins that function as organizers of multimolecular complexes and modulate function of associated proteins. Mammalian genomes encode approximately 30 different members of this family and remotely related eukaryotic species also contain conserved tetraspanin homologs. Tetraspanins are involved in a number of fundamental processes such as regulation of cell migration, fusion, immunity and signaling. Moreover, they are implied in numerous pathological states including mental disorders, infectious diseases or cancer. Despite the great interest in tetraspanins, the structural and biochemical basis of their activity is still largely unknown. A major bottleneck lies in the difficulty of obtaining stable and homogeneous protein samples in large quantities. Here we report expression screening of 15 members of the human tetraspanin superfamily and successful protocols for the production in S. cerevisiae of a subset of tetraspanins involved in human cancer development. We have demonstrated the subcellular localization of overexpressed tetraspanin-green fluorescent protein fusion proteins in S. cerevisiae and found that despite being mislocalized, the fusion proteins are not degraded. The recombinantly produced tetraspanins are dispersed within the endoplasmic reticulum membranes or localized in granule-like structures in yeast cells. The recombinantly produced tetraspanins can be extracted from the membrane fraction and purified with detergents or the poly (styrene-co-maleic acid) polymer technique for use in further biochemical or biophysical studies.

• MeSH
• glykosylace MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• subcelulární frakce MeSH
• tetraspaniny genetika   metabolismus   MeSH
• transport proteinů MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• rekombinantní fúzní proteiny MeSH
• tetraspaniny MeSH
• zelené fluorescenční proteiny MeSH

Embryonic stem cells (ESCs), with their dual capacity to self-renew and differentiate, are commonly used to study differentiation, epigenetic regulation, lineage choices, and more. Using non-directed retroviral integration of a YFP/Cherry exon into mouse ESCs, we generated a library of over 200 endogenously tagged fluorescent fusion proteins and present several proof-of-concept applications of this library. We show the utility of this library to track proteins in living cells; screen for pluripotency-related factors; identify heterogeneously expressing proteins; measure the dynamics of endogenously labeled proteins; track proteins recruited to sites of DNA damage; pull down tagged fluorescent fusion proteins using anti-Cherry antibodies; and test for interaction partners. Thus, this library can be used in a variety of different directions, either exploiting the fluorescent tag for imaging-based techniques or utilizing the fluorescent fusion protein for biochemical pull-down assays, including immunoprecipitation, co-immunoprecipitation, chromatin immunoprecipitation, and more.

• Klíčová slova
• DNA damage, GFP, differentiation, embryonic stem cells, fluorescence, imaging, live imaging, microscopy, pluripotency, protein dynamics,
• MeSH
• buněčná diferenciace genetika   MeSH
• exprese genu * MeSH
• genetická heterogenita MeSH
• genová knihovna MeSH
• myší embryonální kmenové buňky cytologie   metabolismus   MeSH
• myši MeSH
• poškození DNA MeSH
• rekombinantní fúzní proteiny genetika   MeSH
• reportérové geny * MeSH
• transportní proteiny MeSH
• vazba proteinů MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• rekombinantní fúzní proteiny MeSH
• transportní proteiny MeSH

D6 is a promoter/enhancer of the mDach1 gene that is involved in the development of the neocortex and hippocampus. It is expressed by proliferating neural stem/progenitor cells (NSPCs) of the cortex at early stages of neurogenesis. The differentiation potential of NSPCs isolated from embryonic day 12 mouse embryos, in which the expression of green fluorescent protein (GFP) is driven by the D6 promoter/enhancer, has been studied in vitro and after transplantation into the intact adult rat brain as well as into the site of a photochemical lesion. The electrophysiological properties of D6/GFP-derived cells were studied using the whole-cell patch-clamp technique, and immunohistochemical analyses were carried out. D6/GFP-derived neurospheres expressed markers of radial glia and gave rise predominantly to immature neurons and GFAP-positive cells during in vitro differentiation. One week after transplantation into the intact brain or into the site of a photochemical lesion, transplanted cells expressed only neuronal markers. D6/GFP-derived neurons were characterised by the expression of tetrodotoxin-sensitive Na(+)-currents and K (A)- and K (DR) currents sensitive to 4-aminopyridine. They were able to fire repetitive action potentials and responded to the application of GABA. Our results indicate that after transplantation into the site of a photochemical lesion, D6/GFP-derived NSPCs survive and differentiate into neurons, and their membrane properties are comparable to those transplanted into the non-injured cortex. Therefore, region-specific D6/GFP-derived NSPCs represent a promising tool for studying neurogenesis and cell replacement in a damaged cellular environment.

• MeSH
• bikukulin metabolismus   MeSH
• biologické markery metabolismus   MeSH
• blokátory draslíkových kanálů metabolismus   MeSH
• buněčná diferenciace fyziologie   MeSH
• embryo savčí * anatomie a histologie   fyziologie   MeSH
• GABA antagonisté metabolismus   MeSH
• GABA metabolismus   MeSH
• kmenové buňky cytologie   fyziologie   MeSH
• krysa rodu Rattus MeSH
• metoda terčíkového zámku MeSH
• myši MeSH
• neurony cytologie   fyziologie   MeSH
• promotorové oblasti (genetika) MeSH
• rekombinantní fúzní proteiny * genetika   metabolismus   MeSH
• telencefalon * cytologie   patologie   fyziologie   MeSH
• transplantace kmenových buněk * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bikukulin MeSH
• biologické markery MeSH
• blokátory draslíkových kanálů MeSH
• GABA antagonisté MeSH
• GABA MeSH
• rekombinantní fúzní proteiny * MeSH

Cell reprogramming requires efficient delivery of reprogramming transcription factors into the cell nucleus. Here, we compared the robustness and workload of two protein delivery methods that avoid the risk of genomic integration. The first method is based on fusion of the protein of interest to a protein transduction domain (PTD) for delivery across the membranes of target cells. The second method relies on de novo synthesis of the protein of interest inside the target cells utilizing synthetic mRNA (syn-mRNA) as a template. We established a Cre/lox reporter system in three different cell types derived from human (PANC-1, HEK293) and rat (BRIN-BD11) tissues and used Cre recombinase to model a protein of interest. The system allowed constitutive expression of red fluorescence protein (RFP), while green fluorescence protein (GFP) was expressed only after the genomic action of Cre recombinase. The efficiency of protein delivery into cell nuclei was quantified as the frequency of GFP+ cells in the total cell number. The PTD method showed good efficiency only in BRIN-BD11 cells (68%), whereas it failed in PANC-1 and HEK293 cells. By contrast, the syn-mRNA method was highly effective in all three cell types (29-71%). We conclude that using synthetic mRNA is a more robust and less labor-intensive approach than using the PTD-fusion alternative.

• MeSH
• buněčné jádro metabolismus   MeSH
• buněčné klony MeSH
• DNA metabolismus   MeSH
• genetické inženýrství MeSH
• HEK293 buňky MeSH
• integrasy metabolismus   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• proteinové domény MeSH
• průtoková cytometrie MeSH
• rekombinantní fúzní proteiny chemie   metabolismus   MeSH
• reprodukovatelnost výsledků MeSH
• technika přenosu genů * MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Cre recombinase MeSH Prohlížeč
• DNA MeSH
• integrasy MeSH
• messenger RNA MeSH
• rekombinantní fúzní proteiny MeSH
• zelené fluorescenční proteiny MeSH

Nicotinamide phosphoribosyltransferase (NAMPT) is located in both the nucleus and cytoplasm and has multiple biological functions including catalyzing the rate-limiting step in NAD synthesis. Moreover, up-regulated NAMPT expression has been observed in many cancers. However, the determinants and regulation of NAMPT's nuclear transport are not known. Here, we constructed a GFP-NAMPT fusion protein to study NAMPT's subcellular trafficking. We observed that in unsynchronized 3T3-L1 preadipocytes, 25% of cells had higher GFP-NAMPT fluorescence in the cytoplasm, and 62% had higher GFP-NAMPT fluorescence in the nucleus. In HepG2 hepatocytes, 6% of cells had higher GFP-NAMPT fluorescence in the cytoplasm, and 84% had higher GFP-NAMPT fluorescence in the nucleus. In both 3T3-L1 and HepG2 cells, GFP-NAMPT was excluded from the nucleus immediately after mitosis and migrated back into it as the cell cycle progressed. In HepG2 cells, endogenous, untagged NAMPT displayed similar changes with the cell cycle, and in nonmitotic cells, GFP-NAMPT accumulated in the nucleus. Similarly, genotoxic, oxidative, or dicarbonyl stress also caused nuclear NAMPT localization. These interventions also increased poly(ADP-ribosyl) polymerase and sirtuin activity, suggesting an increased cellular demand for NAD. We identified a nuclear localization signal in NAMPT and amino acid substitution in this sequence (424RSKK to ASGA), which did not affect its enzymatic activity, blocked nuclear NAMPT transport, slowed cell growth, and increased histone H3 acetylation. These results suggest that NAMPT is transported into the nucleus where it presumably increases NAD synthesis required for cell proliferation. We conclude that specific inhibition of NAMPT transport into the nucleus might be a potential avenue for managing cancer.

• Klíčová slova
• GFP fusion, NAMPT, cancer, epigenetics, nicotinamide adenine dinucleotide (NAD), nuclear localization, pre–B cell colony enhancing factor (PBEF), sirtuin, visfatin,
• MeSH
• akrylamidy farmakologie   MeSH
• aktivní transport - buněčné jádro MeSH
• buněčné jádro metabolismus   MeSH
• buňky 3T3-L1 MeSH
• buňky Hep G2 MeSH
• cytoplazma metabolismus   MeSH
• histony metabolismus   MeSH
• kontrolní body buněčného cyklu MeSH
• lidé MeSH
• mutageneze cílená MeSH
• myši MeSH
• NAD metabolismus   MeSH
• nikotinamidfosforibosyltransferasa chemie   genetika   metabolismus   MeSH
• oxidační stres MeSH
• piperidiny farmakologie   MeSH
• poly(ADP-ribosa)polymerasy metabolismus   MeSH
• proliferace buněk MeSH
• rekombinantní fúzní proteiny chemie   genetika   metabolismus   MeSH
• sirtuiny metabolismus   MeSH
• viabilita buněk účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• akrylamidy MeSH
• histony MeSH
• N-(4-(1-benzoylpiperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide MeSH Prohlížeč
• NAD MeSH
• nikotinamidfosforibosyltransferasa MeSH
• piperidiny MeSH
• poly(ADP-ribosa)polymerasy MeSH
• rekombinantní fúzní proteiny MeSH
• sirtuiny MeSH

The HsdR subunit of the type I restriction-modification system EcoR124I is responsible for the translocation as well as the restriction activity of the whole complex consisting of the HsdR, HsdM and HsdS subunits, and while crystal structures are available for the wild type and several mutants, the C-terminal domain comprising approximately 150 residues was not resolved in any of these structures. Here, three fusion constructs with the GFP variant pHluorin developed to overexpress, purify and crystallize the C-terminal domain of HsdR are reported. The shortest of the three encompassed HsdR residues 887-1038 and yielded crystals that belonged to the orthorhombic space group C2221, with unit-cell parameters a = 83.42, b = 176.58, c = 126.03 Å, α = β = γ = 90.00° and two molecules in the asymmetric unit (VM = 2.55 Å(3) Da(-1), solvent content 50.47%). X-ray diffraction data were collected to a resolution of 2.45 Å.

• Klíčová slova
• EcoR124I, Escherichia coli, GFP, HsdR, pHluorin, restriction-modification system,
• MeSH
• difrakce rentgenového záření MeSH
• Escherichia coli chemie   enzymologie   genetika   MeSH
• exprese genu MeSH
• klonování DNA MeSH
• krystalizace MeSH
• krystalografie rentgenová MeSH
• plazmidy chemie   metabolismus   MeSH
• podjednotky proteinů chemie   genetika   metabolismus   MeSH
• proteiny z Escherichia coli chemie   genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny chemie   genetika   metabolismus   MeSH
• restrikční endonukleasy typu I chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• zelené fluorescenční proteiny chemie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• endodeoxyribonuclease EcoR124I MeSH Prohlížeč
• HsdR protein, E coli MeSH Prohlížeč
• PHluorin MeSH Prohlížeč
• podjednotky proteinů MeSH
• proteiny z Escherichia coli MeSH
• rekombinantní fúzní proteiny MeSH
• restrikční endonukleasy typu I MeSH
• zelené fluorescenční proteiny MeSH

Despite the urgent need for assays to visualize insulin secretion there is to date no reliable method available for measuring insulin release from single cells. To address this need, we developed a genetically encoded reporter termed RINS1 based on proinsulin superfolder GFP (sfGFP) and mCherry fusions for monitoring insulin secretion. RINS1 expression in MIN6 β cells resulted in proper processing yielding single-labeled insulin species. Unexpectedly, glucose or drug stimulation of insulin secretion in β cells led to the preferential release of the insulin-sfGFP construct, while the mCherry-fused C-peptide remained trapped in exocytic granules. This physical separation was used to monitor glucose-stimulated insulin secretion ratiometrically by total internal reflection fluorescence microscopy in single MIN6 and primary mouse β cells. Further, RINS1 enabled parallel monitoring of pulsatile insulin release in tolbutamide-treated β cells, demonstrating the potential of RINS1 for investigations of antidiabetic drug candidates at the single-cell level.

• Klíčová slova
• TIRF, biosensor, calcium, fluorescence, glucose, granule, insulin, mCherry, oscillation, potassium channel, superfolder GFP, tolbutamide,
• MeSH
• beta-buňky cytologie   účinky léků   metabolismus   MeSH
• biosenzitivní techniky MeSH
• buněčné linie MeSH
• červený fluorescenční protein MeSH
• fluorescenční mikroskopie MeSH
• glukosa farmakologie   MeSH
• hypoglykemika farmakologie   MeSH
• inzulin metabolismus   MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• myši MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• reportérové geny MeSH
• sekrece inzulinu MeSH
• tolbutamid farmakologie   MeSH
• vápník metabolismus   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glukosa MeSH
• hypoglykemika MeSH
• inzulin MeSH
• luminescentní proteiny MeSH
• rekombinantní fúzní proteiny MeSH
• tolbutamid MeSH
• vápník MeSH
• zelené fluorescenční proteiny MeSH

BACKGROUND: The introduction of HIV proteinase inhibitors (PIs) as anti-AIDS drugs resulted in decreased mortality and prolonged life expectancy of HIV-positive patients. However, rapid selection of drug-resistant HIV variants is a common complication in patients undergoing highly active anti-retroviral therapy (HAART). Thus, monitoring of clinical resistance development is indispensable for rational pharmacotherapy. OBJECTIVE: We present a non-infectious cell-based assay for drug resistance quantification of HIV proteinase (PR) - an important target of HAART. STUDY DESIGN: Previously, we showed [Lindsten K, Uhlikova T, Konvalinka J, Masucci MG, Dantuma NP. Cell-based fluorescence assay for human immunodeficiency virus type 1 protease activity. Antimicrob Agents Chemother 2001;45:2616-22] that the expression of a fusion protein (GFP-PR), comprised of HIV-1 proteinase wild-type artificial precursor (PR) and green fluorescent protein (GFP), in transiently transfected tissue culture cells depends on the presence of PR-specific inhibitors (PIs). Here we show that in the GFP-PR reporter the HIV wild-type PR can be replaced by a drug-resistant HIV PR mutant, yielding a simple and biologically relevant tool for the quantitative analysis of drug-resistant HIV PR mutants susceptibility to HIV proteinase inhibitors. RESULTS: We cloned a set of GFP-PR reporters, some of which possess a simple, well-defined drug-resistant PR mutant (G48V L90M, V82A, A71V V82T I84V, D30N, K45I); another four complex PR mutants were obtained from patients undergoing HAART. The results were compared with genotyping and enzyme kinetics data. Furthermore, we designed a single inhibitor concentration experiment setup for easy evaluation of drug resistance profiles for mutants of interest. The resistance profiles clearly demonstrate the importance of succession of individual drugs during the treatment for drug resistance development. CONCLUSION: We show that the GFP-PR assay might serve as a non-infectious, rapid, cheap, and reliable alternative to the currently used phenotypic assays.

• MeSH
• biotest metody   MeSH
• fenotyp * MeSH
• fluorescenční mikroskopie MeSH
• fluorometrie MeSH
• HeLa buňky MeSH
• HIV-proteasa účinky léků   genetika   metabolismus   MeSH
• hodnotící studie jako téma MeSH
• inhibitory HIV-proteasy farmakologie   MeSH
• kinetika MeSH
• klonování DNA MeSH
• lidé MeSH
• monoklonální protilátky metabolismus   MeSH
• mutace * MeSH
• plazmidy MeSH
• průtoková cytometrie MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• reportérové geny MeSH
• spektrofotometrie MeSH
• transfekce MeSH
• virová léková rezistence MeSH
• virové geny MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• western blotting MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• HIV-proteasa MeSH
• inhibitory HIV-proteasy MeSH
• monoklonální protilátky MeSH
• rekombinantní fúzní proteiny MeSH
• zelené fluorescenční proteiny MeSH

It was proposed that Ato1p, Ato2p and Ato3p have a role in ammonia production by Saccharomyces cerevisiae colonies (Palkova et al., Mol Biol Cell 13: 3901-3914, 2002). In this study, we show that all three Ato proteins localise to the plasma membrane and their appearance correlates with the beginning of ammonia release. The expression of ATO genes is controlled by ammonia. All three Ato-GFP proteins associate with detergent-resistant membranes; two of them, Ato1p-GFP and Ato3p-GFP, localise to patches visible under the fluorescence microscope. In contrast with Ato3p-GFP which forms stable patches, the formation of those of Ato1p-GFP is pH dependent. Ato1p-GFP patches form at pH above 6 and they disappear at pH 5 or lower. Both changes, Ato1p-GFP clustering and patches spreading are reversible. The Ato1p-GFP spreading at low pH is independent on endocytosis. These data suggest that besides the ammonia induction of Ato protein synthesis, pH may rapidly regulate Ato1p function.

• MeSH
• amoniak metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• detergenty metabolismus   MeSH
• kompartmentace buňky * MeSH
• koncentrace vodíkových iontů MeSH
• kvartérní amoniové sloučeniny metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• membránové transportní proteiny metabolismus   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Saccharomyces cerevisiae cytologie   metabolismus   MeSH
• transport proteinů MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amoniak MeSH
• ATO1 protein, S cerevisiae MeSH Prohlížeč
• ATO2 protein, S cerevisiae MeSH Prohlížeč
• ATO3 protein, S cerevisiae MeSH Prohlížeč
• detergenty MeSH
• kvartérní amoniové sloučeniny MeSH
• membránové proteiny MeSH
• membránové transportní proteiny MeSH
• rekombinantní fúzní proteiny MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• zelené fluorescenční proteiny MeSH