RESUMO
Phospholipase D (PLD) is a critical component of intracellular signal transduction and has been implicated in many important biological processes. It has been observed that there are abnormalities in PLD expression in many human cancers, and PLD is thus recognized as a potential diagnostic biomarker as well as a target for drug discovery. We report for the first time a phospholipid-modified nanoprobe for ratiometric upconversion fluorescence (UCF) sensing and bioimaging of PLD activity. The nanoprobe can be synthesized by a facile one-step self-assembly of a phospholipid monolayer composed of poly(ethylene glycol) (PEG)ylated phospholipid and rhodamine B-labeled phospholipid on the surface of upconversion nanoparticles (UCNPs) NaYF4: 20%Yb, 2%Er. The fluorescence resonance energy transfer (FRET) process from the UCF emission at 540 nm of the UCNPs to the absorbance of the rhodamine B occurs in the nanoprobe. The PLD-mediated hydrolysis of the phosphodiester bond makes rhodamine B apart from the UCNP surface, leading to the inhibition of FRET. Using the unaffected UCF emission at 655 nm as an internal standard, the nanoprobe can be used for ratiometric UCF detection of PLD activity with high sensitivity and selectivity. The PLD activity in cell lysates is also determined by the nanoprobe, confirming that PLD activity in a breast cancer cell is at least 7-fold higher than in normal cell. Moreover, the nanoprobe has been successfully applied to monitoring PLD activity in living cells by UCF bioimaging. The results reveal that the nanoprobe provides a simple, sensitive, and robust platform for point-of-care diagnostics and drug screening in biomedical applications.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Molecular/métodos , Nanopartículas/química , Fosfolipase D/análise , Fosfolipídeos/química , Linhagem Celular/enzimologia , Linhagem Celular Tumoral/enzimologia , Fluorescência , Humanos , Hidrólise , Imagem Molecular/instrumentação , Polietilenoglicóis/química , Rodaminas/química , Soluções , Itérbio/química , Ítrio/químicaRESUMO
The majority of caspases, cysteine-dependent aspartate-directed proteases, being in their activated state are involved in regulation of apoptosis by cleaving protein substrates harboring specific target motifs. Basically all biochemical and morphological changes in an apoptotic cell, including cell shrinkage, chromatin condensation, DNA fragmentation, and plasma membrane blebbing, are consequence of caspase-mediated proteolysis. Thus, uncovering activities of unique caspases are key determinants of the apoptotic process. This chapter describes a set of experimental protocols available for characterization, quantification and inhibition of caspase activities in mammalian cell cultures, including immunoblotting, usage of synthetic substrates, flow cytometry, and microscopic techniques.
Assuntos
Caspases/isolamento & purificação , Caspases/metabolismo , Biologia Molecular/métodos , Animais , Caspases/genética , Linhagem Celular/enzimologia , Ativação Enzimática/genética , Citometria de Fluxo , Humanos , Mamíferos/genética , Transdução de SinaisRESUMO
A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz's L15 medium and incubated at 32(°)C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10-14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.
Assuntos
Abelhas/citologia , Linhagem Celular , Animais , Linhagem Celular/enzimologia , Diploide , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Embrião não Mamífero/citologia , Subunidades Proteicas/metabolismoRESUMO
Mitochondrial targeted manganese superoxide dismutase is a major antioxidant enzyme, the levels of which modulate the response of cells, tissues and organs to ionizing irradiation. We developed a Tet-regulated MnSOD mouse (MnSOD(tet)) to examine the detailed relationship between cellular MnSOD concentration and radioresistance and carried out in vitro studies using bone marrow culture derived stromal cell lines (mesenchymal stem cells). Homozygous MnSOD(tet/tet) cells had low levels of MnSOD, reduced viability and proliferation, increased radiosensitivity, elevated overall antioxidant stores, and defects in cell proliferation and DNA strand-break repair. Doxycycline (doxy) treatment of MnSOD(tet/tet) cells increased MnSOD levels and radioresistance from ñ of 2.79 ± 1.04 to 8.69 ± 1.09 (P = 0.0060) and normalized other biologic parameters. In contrast, MnSOD(tet/tet) cells showed minimal difference in baseline and radiation induced mRNA and protein levels of TGF-ß, Nrf2 and NF-κB and radiation induced cell cycle arrest was not dependent upon MnSOD level. These novel MnSOD(tet/tet) mouse derived cells should be valuable for elucidating several parameters of the oxidative stress response to ionizing radiation.
Assuntos
Linhagem Celular/efeitos da radiação , Raios gama/efeitos adversos , Tolerância a Radiação/genética , Células Estromais/efeitos da radiação , Superóxido Dismutase/fisiologia , Animais , Medula Óssea , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular/enzimologia , Células Clonais/enzimologia , Células Clonais/efeitos da radiação , Ensaio de Unidades Formadoras de Colônias , Quebras de DNA/efeitos da radiação , Doxiciclina/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/efeitos da radiação , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/enzimologia , Mutagênese Sítio-Dirigida , Fator 2 Relacionado a NF-E2/biossíntese , Fator 2 Relacionado a NF-E2/genética , NF-kappa B/biossíntese , NF-kappa B/genética , Plasmídeos/genética , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Células Estromais/enzimologia , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genética , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genéticaRESUMO
The bioactivation of pro-toxicants is the biological process through which some chemicals are metabolized into reactive metabolites. Therefore, in vitro toxicological evaluation should ideally be conducted in cell systems retaining adequate metabolic competency and relevant to the route of exposure. The respiratory tract is the primary route of exposure to inhaled pro-toxicants and lung-derived BEAS-2B cell line has been considered as a potentially suitable model for in vitro toxicology testing. However, its metabolic activity has not been characterized. We performed a gene expression analysis for 41 metabolism-related genes and compared the profile with liver- and lung-derived cell lines (HepaRG, HepG2 and A549). To confirm that mRNA expression was associated with the corresponding enzyme activity, we used a series of metabolic substrates of CYPs (CYP1A1/1B1, CYP1A2, CYP2A6/2A13 and CYP2E1) known to bioactivate inhaled pro-toxicants. CYP activities were compared between BEAS-2B, HepaRG, HepG2, and A549 cells and published literature on primary bronchial epithelium cells (HBEC). We found that in contrast to HBEC, BEAS-2B and A549 have limited CYP activity which was in agreement with their CYP gene expression profile. Control cell lines such as HepG2 and HepaRG were metabolically active for the tested CYPs. We recommend that similar strategies can be used to select suitable cell systems in the context of pro-toxicant assessment.
Assuntos
Linhagem Celular Tumoral/enzimologia , Linhagem Celular/enzimologia , Sistema Enzimático do Citocromo P-450/genética , Regulação Enzimológica da Expressão Gênica , Sobrevivência Celular/efeitos dos fármacos , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Perfilação da Expressão Gênica , Humanos , Pulmão/citologia , Dibenzodioxinas Policloradas/toxicidade , Reação em Cadeia da Polimerase , Testes de ToxicidadeRESUMO
Rohu gill cell line (LRG) was established from gill tissue of Indian major carp (Labeo rohita), a freshwater fish cultivated in India. The cell line was maintained in Leibovitz's L-15 supplemented with 10 % foetal bovine serum (FBS). This cell line has been sub-cultured more than 85 passages over a period of 2 years. The LRG cell line consists of both epithelial and fibroblastic-like cells. The cells were able to grow at a wide range of temperatures from 22 to 32 °C, the optimum temperature being 28 °C. The growth rate of gill cells increased as the FBS proportion increased from 2 to 20 % at 28 °C. The plating efficiency was also high (34.37 %). The viability of the LRG cell line was 70-80 % after 6 months of storage in liquid nitrogen. The karyotype analysis revealed a diploid count of 50 chromosomes. The gill cells of rohu were successfully transfected with pEGFP-N1. Amplification of mitochondrial Cox1 gene using primers specific to L. rohita confirmed the origin of this cell line from L. rohita. The cytotoxicity of malathion was assessed in LRG cell line using multiple endpoints such as 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Neutral Red assay, Alamar Blue assay and Coomassie Blue protein assay. Acute toxicity assay on fish was conducted by exposing L. rohita for 96 h to malathion under static conditions. Statistical analysis revealed good correlation with r (2) = 0.946-0.990 for all combinations between endpoints employed. Linear correlations between each in vitro effective concentration 50 and the in vivo lethal concentration 50 data were highly significant.
Assuntos
Técnicas de Cultura de Células/métodos , Linhagem Celular/citologia , Cyprinidae/fisiologia , Brânquias/citologia , Testes de Toxicidade/métodos , Animais , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/enzimologia , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genes Mitocondriais/efeitos dos fármacos , Genes Mitocondriais/genética , Brânquias/efeitos dos fármacos , Brânquias/enzimologia , Inseticidas/toxicidade , Malation/toxicidade , Reprodutibilidade dos Testes , Poluentes da Água/toxicidade , Poluentes Químicos da Água/toxicidadeRESUMO
Autosomal-dominant missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a common genetic cause of PD (Parkinson's disease). LRRK2 is a multidomain protein with kinase and GTPase activities. Dominant mutations are found in the domains that have these two enzyme activities, including the common G2019S mutation that increases kinase activity 2-3-fold. However, there is also a genetic variant in some populations, G2385R, that lies in a C-terminal WD40 domain of LRRK2 and acts as a risk factor for PD. In the present study we show that the G2385R mutation causes a partial loss of the kinase function of LRRK2 and deletion of the C-terminus completely abolishes kinase activity. This effect is strong enough to overcome the kinase-activating effects of the G2019S mutation in the kinase domain. Hsp90 (heat-shock protein of 90 kDa) has an increased affinity for the G2385R variant compared with WT (wild-type) LRRK2, and inhibition of the chaperone binding combined with proteasome inhibition leads to association of mutant LRRK2 with high molecular mass native fractions that probably represent proteasome degradation pathways. The loss-of-function of G2385R correlates with several cellular phenotypes that have been proposed to be kinase-dependent. These results suggest that the C-terminus of LRRK2 plays an important role in maintaining enzymatic function of the protein and that G2385R may be associated with PD in a way that is different from kinase-activating mutations. These results may be important in understanding the differing mechanism(s) by which mutations in LRRK2 act and may also have implications for therapeutic strategies for PD.
Assuntos
Mutação , Doença de Parkinson/enzimologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Substituição de Aminoácidos , Linhagem Celular/enzimologia , Linhagem Celular/ultraestrutura , Cromatografia em Gel , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Microssomos/metabolismo , Mutação de Sentido Incorreto , Neuritos/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Fosforilação , Proteínas Serina-Treonina Quinases/químicaRESUMO
OBJECTIVE: To investigate hydrogen sulfide and its synthases, cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE), in human prostatic tissue and cells. METHODS: CBS and CSE in human prostatic tissue and cells were located using immunostaining. Western blot and a sulfur-sensitive electrode were used to evaluate the expression levels and catalytic activity of CBS and CSE. We analyzed the association between dihydrotestosterone-added or hormone-reduced medium-induced CBS/CSE protein levels with androgen receptor levels in prostate cancer lines. All experiments were repeated ≥3 times. RESULTS: Endogenous hydrogen sulfide and its synthases existed in various areas of human prostatic tissue and cells. Cell activity and CBS/CSE protein levels were greatest in the androgen-dependent prostate cancer cell LNCaP among all cells and downregulated by dihydrotestosterone. CONCLUSION: Hydrogen sulfide and its synthases in human prostatic tissue and cells were modulated by dihydrotestosterone, which could suggest a potential therapy for prostatic disease.
Assuntos
Cistationina beta-Sintase/análise , Cistationina gama-Liase/análise , Sulfeto de Hidrogênio/análise , Próstata/química , Adenocarcinoma/química , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Androgênios , Linhagem Celular/química , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/enzimologia , Linhagem Celular Tumoral/química , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/enzimologia , Meios de Cultura/farmacologia , Cistationina beta-Sintase/biossíntese , Cistationina beta-Sintase/genética , Cistationina gama-Liase/biossíntese , Cistationina gama-Liase/genética , Di-Hidrotestosterona/farmacologia , Regulação para Baixo/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Células Epiteliais/química , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Hormônio-Dependentes/química , Neoplasias Hormônio-Dependentes/enzimologia , Neoplasias Hormônio-Dependentes/patologia , Próstata/citologia , Próstata/enzimologia , Neoplasias da Próstata/química , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Receptores Androgênicos/análise , Receptores Androgênicos/biossíntese , Receptores Androgênicos/genética , Células Estromais/química , Células Estromais/efeitos dos fármacos , Células Estromais/enzimologiaRESUMO
ADAMTS-13 is the Von Willebrand factor (vWF) cleaving protease, responsible for the cleavage and down-regulation of the pro-thrombotic properties of ultra large VWF multimers. It is expressed predominantly by the hepatic stellate cells of the liver, but is also found to be expressed in other tissues, including brain. Reduced ADAMTS-13 is associated with a variety of thrombotic microangiopathies. Since the cellular origin and regulation of ADAMTS-13 expression in the brain is unknown, we aimed to investigate this in four different central nervous system (CNS)-derived cell lines, SHSY-5Y (human neuroblastoma), U373 (human astroglioma), CHME-3 (human foetal microglia) and hCMEC/D3 (adult human brain endothelial cells). All cell lines expressed ADAMTS-13 mRNA constitutively with neuroblastoma cells showing the highest expression. Interleukin (IL)-1ß down-regulated ADAMTS-13 mRNA expression in astroglioma cells and microglial cells whereas TNF and IL-6 treatment showed no significant differences in ADAMTS-13 mRNA expression in any cell line tested. ADAMTS-13 protein expression was reduced in a dose-dependent manner only in astroglioma cells following stimulation by IL-1ß. The ability of IL-1ß to significantly reduce ADAMTS-13 mRNA expression in human microglia and astroglioma cells suggests a role in the haemostasis of the local microenvironment under inflammatory conditions. This is the first report of ADAMTS-13 expression in cells of the CNS; however, its function remains to be determined.
Assuntos
Proteínas ADAM/genética , Astrócitos/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Interleucina-1beta/farmacologia , Microglia/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Proteínas ADAM/biossíntese , Proteína ADAMTS13 , Adulto , Astrócitos/enzimologia , Astrócitos/patologia , Astrocitoma/enzimologia , Astrocitoma/patologia , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Linhagem Celular/enzimologia , Linhagem Celular Tumoral/enzimologia , Indução Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hemostasia/fisiologia , Humanos , Técnicas In Vitro , Interleucina-6/farmacologia , Microglia/enzimologia , Microglia/patologia , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/biossíntese , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Neurônios/enzimologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The inability to produce recombinant glycoproteins with authentic N-glycans is a limitation of many heterologous protein expression systems. In the baculovirus-insect cell system, this limitation has been addressed by glycoengineering insect cell lines with mammalian genes encoding protein N-glycosylation functions ("glycogenes") under the transcriptional control of constitutive promoters. However, a potential problem with this approach is that the metabolic load imposed by the expression of multiple transgenes could adversely impact the growth and/or stability of glycoengineered insect cell lines. Thus, we created a new transgenic insect cell line (SfSWT-5) with an inducibly mammalianized protein N-glycosylation pathway. Expression of all six glycogenes was induced when uninfected SfSWT-5 cells were cultured in growth medium containing doxycycline. Higher levels of expression and induction were observed when SfSWT-5 cells were cultured with doxycycline and infected with a baculovirus. Interestingly, there were no major differences in the short-term growth properties of SfSWT-5 cells cultured with or without doxycycline. Furthermore, there were no major differences in the phenotypic stability of these cells after continuous culture for over 300 passages with or without doxycycline. Baculovirus-infected Sf9 and SfSWT-5 cells produced about the same amounts of a model recombinant glycoprotein, but only the latter sialylated this product and sialylation was more pronounced when the cells were treated with doxycycline. In summary, this is the first report of a lower eukaryotic system with an inducibly mammalianized protein N-glycosylation pathway and the first to examine how the presumed metabolic load imposed by multiple transgene expression impacts insect cell growth and stability.
Assuntos
Linhagem Celular/metabolismo , Engenharia Genética , Glicosiltransferases/biossíntese , Oxo-Ácido-Liases/biossíntese , Spodoptera/citologia , Animais , Baculoviridae/genética , Bovinos , Linhagem Celular/enzimologia , Proliferação de Células , Clonagem Molecular , Regulação da Expressão Gênica , Vetores Genéticos , Glicoproteínas/biossíntese , Glicoproteínas/metabolismo , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Humanos , Redes e Vias Metabólicas , Camundongos , Oxo-Ácido-Liases/genética , Oxo-Ácido-Liases/metabolismo , Fenótipo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Oxidative stress plays a pivotal role in uncontrolled neuro-inflammation leading to many neurological diseases including Alzheimer's. One of the major antioxidant enzymes known to prevent deleterious effects due to oxidative stress is Cu,Zn-superoxide dismutase (SOD). In this study, we examined the regulatory function of SOD on the LPS-induced signaling pathways leading to NF-kappaB activation, expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in BV-2 cells using cell-permeable SOD. Treatment of BV-2 cells with cell-permeable SOD led to a decrease in LPS-induced reactive oxygen species (ROS) generation and significantly inhibited protein and mRNA levels of iNOS and COX-2 upregulated by LPS. Production of NO and PGE2 in LPS stimulated BV-2 cells was significantly abrogated by pretreatment with a cell-permeable SOD fusion protein. Furthermore, cell-permeable SOD inhibited LPS-induced NF-kappaB DNA-binding activity and activation of MAP kinases including ERK, JNK, and p38 in BV-2 cells. These data indicate that SOD has a regulatory function for LPS-induced NF-kappaB activation leading to expression of iNOS and COX-2 in BV-2 cells and suggest that cell-permeable SOD is a feasible therapeutic agent for regulation of ROS-related neurological diseases.
Assuntos
Ciclo-Oxigenase 2/biossíntese , Microglia/enzimologia , Óxido Nítrico Sintase Tipo II/biossíntese , Superóxido Dismutase/fisiologia , Animais , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/enzimologia , Permeabilidade da Membrana Celular , Ciclo-Oxigenase 2/genética , Dinoprostona/biossíntese , Regulação para Baixo/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , HIV-1/genética , Humanos , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Microglia/efeitos dos fármacos , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Superóxido Dismutase/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/fisiologiaRESUMO
The cellular actions of ouabain are complex and involve different pathways, depending on the cell type and experimental conditions. Several studies have reported that Madin-Darby canine kidney (MDCK) cellular sensitivity to ouabain is not related to Na-K-ATPase inhibition, and others showed that some cell types, such as Ma104, are resistant to ouabain toxicity albeit their Na-K-ATPase isoforms possess high affinity for this glycoside. We describe here that the effects of ouabain and ouabagenin also diverge in MDCK and Ma104 cells, being MDCK cells more resistant to ouabagenin, while Ma104 cells are resistant to both molecules. This feature seems to correlate with induction of cell signaling, since ouabain, but not ouabagenin, induced an intense and sustained increase in tyrosine phosphorylation levels in MDCK cells. Moreover, ouabain-induced phosphorylation in Ma104 cells was approximately half than that observed in MDCK cells. The proportion between alpha and beta subunits of Na-K-ATPase was similar in MDCK cells, though Ma104 cells presented more alpha subunits, located mainly at the cytoplasm. Furthermore, a fluorescent ouabain-analog labeled mainly the cytoplasm of Ma104 cells, the opposite of that seen in MDCK cells, corroborating the results using anti-Na-K-ATPase antibodies. Hence, the results suggest that ouabain and ouabagenin differ in terms of Na-K-ATPase inhibition and cell signaling activation in MDCK cells. Additionally, MDCK and Ma104 cell lines respond differently to ouabain, perhaps due to an intrinsic ability of this glycoside to selectively reach the cytoplasm of Ma104 cells.
Assuntos
Rim/efeitos dos fármacos , Ouabaína/análogos & derivados , Ouabaína/toxicidade , Animais , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/enzimologia , Linhagem Celular/metabolismo , Cães , Relação Dose-Resposta a Droga , Haplorrinos , Rim/enzimologia , Rim/metabolismo , Microscopia de Fluorescência , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
The design, synthesis and structure-activity relationships of a novel series of N-phenyl-substituted pyrrole, 1,2-pyrazole and 1,2,3-triazole acid analogs as PPAR ligands are outlined. The triazole acid analogs 3f and 4f were identified as potent dual PPARalpha/gamma agonists both in binding and functional assays in vitro. The 3-oxybenzyl triazole acetic acid analog 3f showed excellent glucose and triglyceride lowering in diabetic db/db mice.
Assuntos
Azóis/síntese química , Desenho de Fármacos , PPAR alfa/agonistas , PPAR gama/agonistas , Animais , Azóis/farmacologia , Linhagem Celular/enzimologia , Cristalografia por Raios X , Feminino , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Transgênicos , PPAR alfa/metabolismo , PPAR gama/metabolismo , Relação Estrutura-AtividadeRESUMO
In trabecular bone fracture repair in vivo, osteogenesis occurs through endochondral ossification under hypoxic conditions, or through woven bone deposition in the vicinity of blood vessels. In vitro osteogenesis assays are routinely used to test osteoblastic responses to drugs, hormones, and biomaterials for bone and cartilage repair applications. These cell culture models recapitulate events that occur in woven bone synthesis, and are carried out using primary osteoblasts, osteoblast precursors such as bone marrow-derived mesenchymal stromal cells (BMSCs), or various osteoblast cell lines. With time in culture, cell differentiation is typically assessed by examining levels of alkaline phosphatase activity (an early osteoblast marker) and by evaluating the assembly of a collagen (type I)-containing fibrillar extracellular matrix that mineralizes. In this review, we have made a comparative analysis of published osteogenic assays using calvarial cells, calvaria-derived cell lines, and bone marrow stromal cells. In all of these cell types, alkaline phosphatase activity shows similar progression over time using a variety of osteogenic and mineralizing media conditions; however, levels of alkaline phosphatase activity are not proportional to observed mineralization levels.
Assuntos
Minerais/metabolismo , Osteoblastos/fisiologia , Osteogênese , Fosfatase Alcalina/metabolismo , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/enzimologia , Células da Medula Óssea/fisiologia , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/enzimologia , Linhagem Celular/fisiologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/enzimologia , Células Cultivadas/fisiologia , Colágeno Tipo I/metabolismo , Meios de Cultura/farmacologia , Matriz Extracelular/metabolismo , Humanos , Técnicas In Vitro , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Camundongos , Especificidade de Órgãos , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Coelhos , Ratos , Crânio/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/enzimologia , Células Estromais/fisiologiaRESUMO
The recepteur d'origine nantais (RON) is a receptor tyrosine kinase (RTK) in the scatter factor family, which includes the c-Met receptor. RON exhibits increased expression in a significant number of human breast cancer tissues as well as in many established breast cancer cell lines. Recent studies have indicated that in addition to ligand-dependent signaling events, RON also promotes signals in the absence of its only known ligand, MSP, when expressed in epithelial cells. In this study, we found that when expressed in MCF-10A breast epithelial cells, RON exhibits both MSP-dependent and MSP-independent signaling, which lead to distinct biological outcomes. In the absence of MSP, RON signaling promotes cell survival, increased cell spreading and enhanced migration in response to other growth factors. However, both RON-mediated proliferation and migration require the addition of MSP in MCF-10A cells. Both MSP-dependent and MSP-independent signaling by RON are mediated in part by Src family kinases. These data suggest that RON has two alternative modes of signaling that can contribute to oncogenic behavior in normal breast epithelial cells.
Assuntos
Mama/citologia , Transformação Celular Neoplásica , Células Epiteliais/enzimologia , Fator de Crescimento de Hepatócito/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Transdução de Sinais/fisiologia , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular/citologia , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/enzimologia , Linhagem Celular Transformada/citologia , Linhagem Celular Transformada/efeitos dos fármacos , Linhagem Celular Transformada/enzimologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Forma Celular/efeitos dos fármacos , Forma Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Camundongos , Células NIH 3T3/citologia , Células NIH 3T3/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/fisiologia , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/fisiologiaRESUMO
Removal of apoptotic cells is an essential process for normal development and tissue maintenance. Importantly, apoptotic cells stimulate their phagocytosis by macrophages while actively suppressing inflammatory responses. Growth arrest specific gene 6 (Gas6) is involved in this process, bridging phosphatidylserine residues on the surface of apoptotic cells to the Axl/Mer family of tyrosine kinases which stimulate phagocytosis. Animals with mutations or loss of these receptors exhibit phenotypes reflective of impaired phagocytosis and a hyperactive immune response. We report that Gas6 induces phagocytosis in microglia through a novel non-classical phagocytic mechanism. Gas6 stimulates a type-II-related phagocytic response, but requires Vav phosphorylation and Rac activation, distinguishing it from the classical type II mechanism. Importantly, Gas6 suppressed lipopolysaccharide-induced expression of the inflammatory molecules IL-1beta and iNOS. Gas6 inhibited iNOS expression through suppression of promoter activity. The present data provide direct evidence for the role of Gas6 receptors in mediating an anti-inflammatory response to ligands found on apoptotic cells with the simultaneous stimulation of phagocytosis. These data provide a mechanistic explanation for the phenotype observed in animals lacking Axl/Mer receptors.
Assuntos
Inflamação/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Microglia/efeitos dos fármacos , Proteínas Oncogênicas/fisiologia , Fagocitose/efeitos dos fármacos , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Encéfalo/metabolismo , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/enzimologia , Linhagem Celular/fisiologia , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Lipopolissacarídeos/toxicidade , Camundongos , Microglia/enzimologia , Microglia/fisiologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Fagocitose/fisiologia , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-vav/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Transcrição Gênica/efeitos dos fármacos , c-Mer Tirosina Quinase , Proteínas rac de Ligação ao GTP/metabolismo , Receptor Tirosina Quinase AxlRESUMO
Brain ischemia and the following reperfusion are important causes for brain damage and leading causes of brain morbidity and human mortality. Numerous observations exist describing the neuronal damage during ischemia/reperfusion, but the outcome of such conditions towards glial cells still remains to be elucidated. Microglia are resident macrophages in the brain. In this study, we investigated the anoxia/reoxygenation caused damage to a microglial cell line via determination of energy metabolism, free radical production by dichlorofluorescein fluorescence and nitric oxide production by Griess reagent. Consequences of oxidant production were determined by measurements of protein oxidation and lipid peroxidation, as well. By using site-specific antioxidants and inhibitors of various oxidant-producing pathways, we identified major sources of free radical production in the postanoxic microglial cells. The protective influences of these compounds were tested by measurements of cell viability and apoptosis. Although, numerous free radical generating systems may contribute to the postanoxic microglial cell damage, the xanthine oxidase- and the cyclooxygenase-mediated oxidant production seems to be of major importance.
Assuntos
Ciclo-Oxigenase 1/fisiologia , Ciclo-Oxigenase 2/fisiologia , Proteínas de Membrana/fisiologia , Microglia/enzimologia , Xantina Oxidase/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Hipóxia Celular/fisiologia , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/enzimologia , Inibidores de Ciclo-Oxigenase/farmacologia , Metabolismo Energético/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Etilenodiaminas/toxicidade , Fluoresceínas/metabolismo , Radicais Livres , Indometacina/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Microglia/efeitos dos fármacos , Oxidantes/metabolismo , Oxirredução , Estresse Oxidativo , Oxigênio/farmacologia , Oxipurinol/farmacologia , Carbonilação Proteica/efeitos dos fármacos , Reperfusão , Sulfanilamidas/toxicidade , Xantina Oxidase/antagonistas & inibidoresRESUMO
The fate of human hematopoietic stem cells (HSCs)/progenitor cells (HPCs) is influenced by bone marrow (BM) stromal cells. To investigate the role of stromal cells in the hematopoietic support, we have transduced human fetal BM stromal cells (FBMSCs) with a human telomerase catalytic subunit (hTERT). One of the resultant cell lines was identified as osteoblasts, because it contained mineral deposits and constitutively expressed osteogenic genes osteocalcin, osteopontin, collagen type I, osteoblast marker alkaline phosphatase, but not marrow stromal cell marker STRO-1 and CD105. The hTERT-transduced fetal BM-derived osteoblastic cells (FBMOB-hTERT) can actively maintain the capacity of self-renewal and multipotency of HSCs/HPCs at least partly through transcriptional up-regulation of hematopoietic growth factors such as stem cell growth factors (SCFs) and Wnt-5A during interaction with HSCs/HPCs. The enhanced transcription of SCFs and Wnt-5A appears to be mediated by CD29 signaling. Moreover, the FBMOB-hTERT cells seem superior to primary FBMSCs in supporting hematopoiesis, because they are more potent than primary FBMSCs in supporting the ex vivo expansion and long-term culture initiating cells activity of HSCs. The FBMOB-hTERT cell line has been maintained in vitro more than 125 population doublings without tumorigenicity. The results indicate that the FBMOB-hTERT is useful for the study of molecular mechanisms by which osteoblasts support hematopoiesis.
Assuntos
Células da Medula Óssea/citologia , Linhagem Celular/citologia , Hematopoese , Osteoblastos/citologia , Telomerase/genética , Animais , Células da Medula Óssea/enzimologia , Domínio Catalítico , Linhagem Celular/enzimologia , Feto/citologia , Hematopoese/genética , Fatores de Crescimento de Células Hematopoéticas/genética , Fatores de Crescimento de Células Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/enzimologia , Humanos , Camundongos , Osteoblastos/enzimologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Células Estromais/citologia , Células Estromais/enzimologia , Transdução Genética , Regulação para Cima , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt-5a , Quinases da Família srcRESUMO
To simplify the titration of infectious varicella-zoster virus (VZV), we generated a reporter cell line that produced luciferase in a dose-dependent manner upon infection with cell-free VZV. A few VZV-infected cells were detectable by coculturing with the cell line. We demonstrated the usefulness of the cell line for antiviral studies.
Assuntos
Antivirais/farmacologia , Linhagem Celular/enzimologia , Linhagem Celular/virologia , Herpesvirus Humano 3/genética , Testes de Sensibilidade Microbiana/métodos , Sequência de Bases , Genes Reporter/genética , Herpesvirus Humano 3/efeitos dos fármacos , Luciferases/biossíntese , Luciferases/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Regiões Promotoras GenéticasRESUMO
We have previously demonstrated that the serine/threonine protein phosphatase-1 (PP-1) plays an important role in promoting cell survival. However, the molecular mechanisms by which PP-1 promotes survival remain largely unknown. In the present study, we provide evidence to show that PP-1 can directly dephosphorylate a master regulator of apoptosis, p53, to negatively modulate its transcriptional and apoptotic activities, and thus to promote cell survival. As a transcriptional factor, the function of p53 can be greatly regulated by phosphorylation and dephosphorylation. While the kinases responsible for phosphorylation of the 17 serine/threonine sites have been identified, the dephosphorylation of these sites remains largely unknown. In the present study, we demonstrate that PP-1 can dephosphorylate p53 at Ser-15 and Ser-37 through co-immunoprecipitation, in vitro and in vivo dephosphorylation assays, overexpression and silence of the gene encoding the catalytic subunit for PP-1. We further show that mutations mimicking constitutive dephosphorylation or phosphorylation of p53 at these sites attenuate or enhance its transcriptional activity, respectively. As a result of the changed p53 activity, expression of the downstream apoptosis-related genes such as bcl-2 and bax is accordingly altered and the apoptotic events are either largely abrogated or enhanced. Thus, our results demonstrate that PP-1 directly dephosphorylates p53, and dephosphorylation of p53 has as important impact on its functions as phosphorylation does. In addition, our results reveal that one of the molecular mechanisms by which PP-1 promotes cell survival is to dephosphorylate p53, and thus negatively regulate p53-dependent death pathway.