RESUMO
Due to the pronounced effect of the confined environment on the photochemical properties of 4-hydroxybenzylidene imidazolinone (HBI), a GFP-related chromophore, imidazolidinone and imidazothiazolone analogues have been studied as fluorescent probes. Their photoisomerization and their thermal reversion were studied under 365-nm-irradiation, resulting in observation of an enthalpy-entropy compensation effect. Theoretical studies were carried out to shed light on the thermal reversion mechanism. Moreover, photophysical studies of benzylidene imidazothiazolone in the presence of dsDNA revealed fluorescence enhancement. The prepared compounds could be considered as a valuable tool for the detailed investigation of physicochemical, biochemical, or biological systems.
Assuntos
Corantes Fluorescentes , Proteínas de Fluorescência Verde/química , Fluorescência , Termodinâmica , EntropiaRESUMO
We describe an engineered violet fluorescent protein from the lancelet Branchiostoma floridae (bfVFP). This is the first example of a GFP-like fluorescent protein with a stable fluorescent chromophore lacking an imidazolinone ring; instead, it consists of oxidized tyrosine 68 flanked by glycine 67 and alanine 69. bfVFP contains the simplest chromophore reported in fluorescent proteins and was generated from the yellow protein lanFP10A2 by two synergetic mutations, S148H and C166I. The chromophore structure was confirmed crystallographically and by high-resolution mass spectrometry. The photophysical characteristics of bfVFP (323/430 nm, quantum yield 0.33, and Ec 14,300 M-1 cm-1 ) make it potentially useful for multicolor experiments to expand the excitation range of available FP biomarkers and Förster resonance energy transfer with blue and cyan fluorescent protein acceptors.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Tirosina , Alanina , Proteínas de Fluorescência Verde/química , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Mutação , Tirosina/químicaRESUMO
A highly sensitive and specific Hg-whole-cell biosensor was developed from a non-selective variant of the Au sensor GolS and its regulatory pathway. The performance of this analytical tool was validated under laboratory and field-like conditions. This biosensor can be easily applied in cost-effective and portable semiquantitative devices to report Hg contamination in water.
Assuntos
Técnicas Biossensoriais/métodos , Ouro/química , Mercúrio/análise , Salmonella/metabolismo , Poluentes Químicos da Água/análise , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Água Doce/análise , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ligantes , Limite de Detecção , Espectrometria de FluorescênciaRESUMO
BACKGROUND: The use of biomaterials has been expanded to improve the characteristics of vaccines. Recently we have identified that the peptide PH(1-110) from polyhedrin self-aggregates and incorporates foreign proteins to form particles. We have proposed that this peptide can be used as an antigen carrying system for vaccines. However, the immune response generated by the antigen fused to the peptide has not been fully characterized. In addition, the adjuvant effect and thermostability of the particles has not been evaluated. RESULTS: In the present study we demonstrate the use of a system developed to generate nano and microparticles carrying as a fusion protein peptides or proteins of interest to be used as vaccines. These particles are purified easily by centrifugation. Immunization of animals with the particles in the absence of adjuvant result in a robust and long-lasting immune response. Proteins contained inside the particles are maintained for over 1 year at ambient temperature, preserving their immunological properties. CONCLUSION: The rapid and efficient production of the particles in addition to the robust immune response they generate position this system as an excellent method for the rapid response against emerging diseases. The thermostability conferred by the particle system facilitates the distribution of the vaccines in developing countries or areas with no electricity.
Assuntos
Antígenos/imunologia , Imunoglobulinas/metabolismo , Proteínas de Matriz de Corpos de Inclusão/química , Peptídeos/química , Vacinas/imunologia , Animais , Antígenos/química , Estabilidade de Medicamentos , Feminino , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/imunologia , Imunização , Camundongos , Nanopartículas , Tamanho da Partícula , Agregados Proteicos , Proteínas Recombinantes de Fusão/imunologia , Termodinâmica , Vacinas/químicaRESUMO
BACKGROUND: Establishing lung lymphatic drainage is thought to be important for successful lung transplantation. To date, there has been a complete absence of knowledge of how lymphatic connections are reestablished after lung transplant, despite evidence suggesting that this does indeed occur. The present study aimed to elucidate whether and how lymphatic anastomosis occurs after lung transplant. METHODS: An orthotopic murine model of lung transplant using lymphatic reporter mice and whole mount immunohistochemistry was used to evaluate the lymphatic vasculature and donor-host connections after lung transplantation. RESULTS: Immunohistochemistry of transplanted lungs demonstrated robust lymphatic vessels, and functional assays demonstrated lymphatic drainage in the transplanted lung that was comparable with that in native lungs. Lymphatic vessels in the donor lung exhibited active sprouting toward the host at the anastomosis within the first 3 days after lung transplantation, with more numerous and complex lymphatic sprouting developing thereafter. Donor lymphatic vessels were numerous at the site of anastomosis by day 14 after lung transplantation and formed physical connections with host lymphatic vessels, demonstrating a mechanism by which lymphatic drainage is reestablished in the transplanted lung. CONCLUSIONS: Lymphatic drainage after lung transplantation is established by active sprouting of donor lymphatic vessels towards the host and the formation of donor-host lymphatic connections at the level of the transplant anastomosis.
Assuntos
Aloenxertos/fisiologia , Transplante de Pulmão , Pulmão/fisiologia , Linfangiogênese/fisiologia , Vasos Linfáticos/fisiologia , Aloenxertos/diagnóstico por imagem , Animais , Corantes Fluorescentes/química , Genes Reporter/genética , Sobrevivência de Enxerto/fisiologia , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Pulmão/diagnóstico por imagem , Vasos Linfáticos/diagnóstico por imagem , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Modelos AnimaisRESUMO
We present a multicolor fluorescence microscope system, under a selective plane illumination microscopy (SPIM) configuration, using three continuous wave-lasers and a single-channel-detection camera. The laser intensities are modulated with three time-delayed pulse trains that operate synchronously at one third of the camera frame rate, allowing a sequential excitation and an image acquisition of up to three different biomarkers. The feasibility of this imaging acquisition mode is demonstrated by acquiring single-plane multicolor images of living hyphae of Neurospora crassa. This allows visualizing simultaneously the localization and dynamics of different cellular components involved in apical growth in living hyphae. The configuration presented represents a noncommercial, cost-effective alternative microscopy system for the rapid and simultaneous acquisition of multifluorescent images and can be potentially useful for three-dimensional imaging of large biological samples.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Neurospora crassa/metabolismo , Biomarcadores/metabolismo , Cor , Desenho de Equipamento , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/química , Lasers , Luz , Proteínas Luminescentes/química , Rodaminas/química , Proteína Vermelha FluorescenteRESUMO
Type 2 diabetes mellitus (T2DM) is characterized by the inability of the insulin-producing ß-cells to overcome insulin resistance. We previously identified an imprinted region on chromosome 14, the DLK1-MEG3 locus, as being downregulated in islets from humans with T2DM. In this study, using targeted epigenetic modifiers, we prove that increased methylation at the promoter of Meg3 in mouse ßTC6 ß-cells results in decreased transcription of the maternal transcripts associated with this locus. As a result, the sensitivity of ß-cells to cytokine-mediated oxidative stress was increased. Additionally, we demonstrate that an evolutionarily conserved intronic region at the MEG3 locus can function as an enhancer in ßTC6 ß-cells. Using circular chromosome conformation capture followed by high-throughput sequencing, we demonstrate that the promoter of MEG3 physically interacts with this novel enhancer and other putative regulatory elements in this imprinted region in human islets. Remarkably, this enhancer is bound in an allele-specific manner by the transcription factors FOXA2, PDX1, and NKX2.2. Overall, these data suggest that the intronic MEG3 enhancer plays an important role in the regulation of allele-specific expression at the imprinted DLK1-MEG3 locus in human ß-cells, which in turn impacts the sensitivity of ß-cells to cytokine-mediated oxidative stress.
Assuntos
Metilação de DNA , Diabetes Mellitus Tipo 2/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas de Membrana/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Linhagem Celular , Citocinas/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/química , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Diabetes Mellitus Tipo 2/patologia , Elementos Facilitadores Genéticos , Epigênese Genética , Loci Gênicos , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Ilhotas Pancreáticas/patologia , Região de Controle de Locus Gênico , Proteínas de Membrana/genética , Camundongos , Mutação , Proteínas Nucleares , Estresse Oxidativo/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Bancos de Tecidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Image correlation analysis has evolved to become a valuable method of analysis of the diffusional motion of molecules in every points of a live cell. Here we compare the iMSD and the 2D-pCF approaches that provide complementary information. The iMSD method provides the law of diffusion and it requires spatial averaging over a small region of the cell. The 2D-pCF does not require spatial averaging and it gives information about obstacles for diffusion at pixel resolution. We show the analysis of the same set of data by the two methods to emphasize that both methods could be needed to have a comprehensive understanding of the molecular diffusional flow in a live cell.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia Intravital/métodos , Microscopia de Fluorescência/métodos , Modelos Químicos , Algoritmos , Animais , Células CHO , Cricetulus , Difusão , Receptores ErbB/química , Receptores ErbB/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Microscopia Intravital/instrumentação , Microscopia de Fluorescência/instrumentaçãoRESUMO
Site-specific, genetic incorporation of unnatural amino acids (UAAs) into proteins in living cells using engineered orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs is a powerful tool for studying and manipulating protein structure and function. To date, UAA incorporation systems have been developed for several bacterial and eukaryotic model hosts. Due to the importance of Streptomyces as prolific producers of bioactive natural products and as model hosts for natural product biosynthesis and bioengineering studies, we have developed systems for the incorporation of the UAAs p-iodo-L-phenylalanine (pIPhe) and p-azido-L-phenylalanine (pAzPhe) into green fluorescent protein (GFP) in Streptomyces venezuelae ATCC 15439. Here, we describe the procedure for using this system to site-specifically incorporate pIPhe or pAzPhe into proteins of interest in S. venezuelae. The modular design of plasmids harboring UAA incorporation systems enables use of other aaRS or aaRS/tRNA pairs for the incorporation of other UAAs; and the vector backbone used allows the system to be transferred to diverse Streptomyces species via both protoplast transformation and conjugation.
Assuntos
Aminoácidos/genética , Proteínas de Bactérias/genética , Engenharia de Proteínas , Streptomyces/genética , Aminoácidos/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Ordem dos Genes , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Plasmídeos/genética , Streptomyces/metabolismo , TransfecçãoRESUMO
Cellular functions emerge from the collective action of a large number of different proteins. Understanding how these protein networks operate requires monitoring their components in intact cells. Due to intercellular and intracellular molecular variability, it is important to monitor simultaneously multiple components at high spatiotemporal resolution. However, inherent trade-offs narrow the boundaries of achievable multiplexed imaging. Pushing these boundaries is essential for a better understanding of cellular processes. Here the motivations, challenges and approaches for multiplexed imaging of intracellular protein networks are discussed. © 2016 International Society for Advancement of Cytometry.