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Nat Commun ; 10(1): 1979, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040286


Hospital acquired infections (HAIs) and the emergence of antibiotic resistant strains are major threats to human health. Copper is well known for its high antimicrobial efficacy, including the ability to kill superbugs and the notorious ESKAPE group of pathogens. We sought a material that maintains the antimicrobial efficacy of copper while minimizing the downsides - cost, appearance and metallic properties - that limit application. Here we describe a copper-glass ceramic powder as an additive for antimicrobial surfaces; its mechanism is based on the controlled release of copper (I) ions (Cu1+) from cuprite nanocrystals that form in situ in the water labile phase of the biphasic glass ceramic. Latex paints containing copper-glass ceramic powder exhibit ≥99.9% reduction in S. aureus, P. aeruginosa, K. aerogenes and E. Coli colony counts when evaluated by the US EPA test method for efficacy of copper-alloy surfaces as sanitizer, approaching that of benchmark metallic copper.

Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Cerâmica/química , Cobre/química , Nanopartículas/química , Klebsiella pneumoniae/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos
J Biomol Screen ; 17(9): 1180-91, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22885730


Stem cells hold great potential in drug discovery and development. However, challenges remain to quantitatively measure the functions of stem cells and their differentiated products. Here, we applied fluorescent imaging, quantitative real-time PCR, and label-free dynamic mass redistribution (DMR) assays to characterize the differentiation process of the ReNcell VM human neural progenitor stem cell. Immunofluorescence imaging showed that after growth factor withdrawal, the neuroprogenitor stem cell was differentiated into dopaminergic neurons, astrocytes, and oligodendrocytes, thus creating a neuronal cell system. High-performance liquid chromatography analysis showed that the differentiated cell system released dopamine upon depolarization with KCl. In conjunction with quantitative real-time PCR, DMR assays using a G-protein-coupled receptor agonist library revealed that a subset of receptors, including dopamine D(1) and D(4) receptors, underwent marked alterations in both receptor expression and signaling pathway during the differentiation process. These findings suggest that DMR assays can decode the differentiation process of stem cells at the cell system level.

Imagem Molecular/métodos , Células-Tronco Neurais/metabolismo , Neurogênese , Reação em Cadeia da Polimerase em Tempo Real/métodos , Astrócitos/citologia , Astrócitos/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Dopamina/análise , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Imunofluorescência/métodos , Humanos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Cloreto de Potássio/farmacologia , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D4/metabolismo , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/fisiologia , Bibliotecas de Moléculas Pequenas/farmacologia
Nat Chem Biol ; 7(12): 909-15, 2011 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-22020552


G protein-coupled receptors (GPCRs) transmit exogenous signals to the nucleus, promoting a myriad of biological responses via multiple signaling pathways in both healthy and cancerous cells. However, little is known about the response of cytosolic metabolic pathways to GPCR-mediated signaling. Here we applied fluorescent live-cell imaging and label-free dynamic mass redistribution assays to study whether purine metabolism is associated with GPCR signaling. Through a library screen of GPCR ligands in conjunction with live-cell imaging of a metabolic multienzyme complex for de novo purine biosynthesis, the purinosome, we demonstrated that the activation of endogenous Gα(i)-coupled receptors correlates with purinosome assembly and disassembly in native HeLa cells. Given the implications of GPCRs in mitogenic signaling and of the purinosome in controlling metabolic flux via de novo purine biosynthesis, we hypothesize that regulation of purinosome assembly and disassembly may be one of the downstream events of mitogenic GPCR signaling in human cancer cells.

Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Multimerização Proteica , Purinas/biossíntese , Receptores Acoplados a Proteínas-G/metabolismo , Células HeLa , Humanos , Ligantes , Complexos Multienzimáticos/biossíntese , Purinas/metabolismo , Receptores Acoplados a Proteínas-G/agonistas , Transdução de Sinais
Oncogene ; 23(49): 8049-64, 2004 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-15377997


The mitochondrial permeability transition pore complex (PTPC) is involved in the control of the mitochondrial membrane permeabilization during apoptosis, necrosis and autophagy. Indeed, the adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC), two major components of PTPC, are the targets of a variety of proapoptotic inducers. Using co-immunoprecipitation and proteomic analysis, we identified some of the interacting partners of ANT in several normal tissues and human cancer cell lines. During chemotherapy-induced apoptosis, some of these interactions were constant (e.g. ANT-VDAC), whereas others changed strongly concomitantly with the dissipation of the mitochondrial transmembrane potential and until nuclear degradation occurred (e.g. Bax, Bcl-2, subunits of the respiratory chain, a subunit of the phosphatase PP2A, phospholipase PLC beta 4 and IP3 receptor). In addition, a glutathione-S-transferase (GST) interacts with ANT in normal tissue, in colon carcinoma cells and in vitro. This interaction is lost during apoptosis induction, suggesting that GST behaves as an endogenous repressor of PTPC and ANT pore opening. Thus, ANT is connected to mitochondrial proteins as well as to proteins from other organelles such as the endoplasmic reticulum forming a dynamic polyprotein complex. Changes within this ANT interactome coordinate the lethal response of cells to apoptosis induction.

Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Canais Iônicos/fisiologia , Translocases Mitocondriais de ADP e ATP/fisiologia , Animais , Linhagem Celular Tumoral , Glutationa/fisiologia , Glutationa Transferase/fisiologia , Humanos , Cinética , Proteínas de Transporte da Membrana Mitocondrial , Proteômica , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Ratos , Proteína X Associada a bcl-2
Cancer Res ; 63(2): 541-6, 2003 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-12543814


Bcl-2 is a prosurvival factor that reportedly prevents the nonspecific permeabilization of mitochondrial membranes, yet enhances specific ADP/ATP exchange by these organelles. Here, we show that Bcl-2 enhances the ADP/ATP exchange in proteoliposomes containing the purified adenine nucleotide translocase (ANT) in isolated mitochondria and mitoplasts, as well as in intact cells in which mitochondrial matrix ATP was monitored continuously using a specific luciferase-based assay system. Conversely, Bax, which displaces Bcl-2 from ANT in apoptotic cells, inhibits ADP/ATP exchange through a direct action on ANT. The Bax-mediated inhibition of ADP/ATP exchange can be separated from Bax-stimulated formation of nonspecific pores by ANT. Chemotherapy-induced apoptosis caused an inhibition of ANT activity, which preceded the loss of the mitochondrial transmembrane potential and could be prevented by overexpression of Bcl-2. These data are compatible with a model of mitochondrial apoptosis regulation in which ANT interacts with either Bax or Bcl-2, which both influence ANT function in opposing manners. Bcl-2 would maintain the translocase activity at high levels, whereas Bax would inhibit the translocase function of ANT.

Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Células HeLa , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Transfecção , Proteína X Associada a bcl-2
Ann N Y Acad Sci ; 1010: 126-42, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15033708


The permeability transition pore complex (PTPC), a mitochondrial polyprotein complex, has been previously described to be involved in the control of mitochondrial membrane permeabilization (MMP) during chemotherapy-induced apoptosis. PTPC may contain proteins from both mitochondrial membranes [e.g., voltage-dependent anion channel (VDAC), PRAX-1, peripheral benzodiazepine receptor (PBR), adenine nucleotide translocator (ANT)], from cytosol (e.g., hexokinase II, glycerol kinase), from matrix [e.g., cyclophilin D (CypD)], and from intermembrane space (e.g., creatine kinase). PTPC may also interact with tumor suppressor proteins (i.e., Bax and Bid), oncoprotein homologues of Bcl-2 and some viral proteins, which can regulate apoptosis induced by pore opening. ANT and VDAC are the target of numerous pro-apoptotic MMP inducers. However, the precise composition of PTPC as well as the respective role of each PTPC component represent major issues in the understanding MMP process. Using several experimental strategies that combine co-immunoprecipitation, proteomics, and functional tests with proteoliposomes, we and others have been able to characterize some of the intra/inter-PTPC protein interactions leading to a better understanding of the process of MMP. In addition, this approach could identify new putative members and regulators of PTPC pro-apoptotic function and new targets of viral protein involved in the modulation of apoptosis during infection.

Apoptose/fisiologia , Proteínas Virais/fisiologia , Animais , Linhagem Celular Tumoral , Humanos , Membranas Intracelulares/fisiologia , Canais Iônicos/fisiologia , Mitocôndrias/patologia , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , Permeabilidade , Porinas/fisiologia , Canais de Ânion Dependentes de Voltagem