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1.
Int J Mol Sci ; 22(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34445538

RESUMO

Decellularized tissues are biocompatible materials that engraft well, but the age of their source has not been explored for clinical translation. Advanced glycation end products (AGEs) are chemical cross-links that accrue on skeletal muscle collagen in old age, stiffening the matrix and increasing inflammation. Whether decellularized biomaterials derived from aged muscle would suffer from increased AGE collagen cross-links is unknown. We characterized gastrocnemii of 1-, 2-, and 20-month-old C57BL/6J mice before and after decellularization to determine age-dependent changes to collagen stiffness and AGE cross-linking. Total and soluble collagen was measured to assess if age-dependent increases in collagen and cross-linking persisted in decellularized muscle matrix (DMM). Stiffness of aged DMM was determined using atomic force microscopy. AGE levels and the effect of an AGE cross-link breaker, ALT-711, were tested in DMM samples. Our results show that age-dependent increases in collagen amount, cross-linking, and general stiffness were observed in DMM. Notably, we measured increased AGE-specific cross-links within old muscle, and observed that old DMM retained AGE cross-links using ALT-711 to reduce AGE levels. In conclusion, deleterious age-dependent modifications to collagen are present in DMM from old muscle, implying that age matters when sourcing skeletal muscle extracellular matrix as a biomaterial.


Assuntos
Envelhecimento/metabolismo , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Músculo Esquelético/metabolismo , Envelhecimento/patologia , Animais , Matriz Extracelular/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia
2.
Nat Methods ; 18(7): 771-774, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34168373

RESUMO

We develop an automatic method for synaptic partner identification in insect brains and use it to predict synaptic partners in a whole-brain electron microscopy dataset of the fruit fly. The predictions can be used to infer a connectivity graph with high accuracy, thus allowing fast identification of neural pathways. To facilitate circuit reconstruction using our results, we develop CIRCUITMAP, a user interface add-on for the circuit annotation tool CATMAID.


Assuntos
Encéfalo/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Sinapses/fisiologia , Animais , Encéfalo/citologia , Bases de Dados Factuais , Drosophila melanogaster , Microscopia Eletrônica , Vias Neurais
3.
Cell ; 184(3): 759-774.e18, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33400916

RESUMO

To investigate circuit mechanisms underlying locomotor behavior, we used serial-section electron microscopy (EM) to acquire a synapse-resolution dataset containing the ventral nerve cord (VNC) of an adult female Drosophila melanogaster. To generate this dataset, we developed GridTape, a technology that combines automated serial-section collection with automated high-throughput transmission EM. Using this dataset, we studied neuronal networks that control leg and wing movements by reconstructing all 507 motor neurons that control the limbs. We show that a specific class of leg sensory neurons synapses directly onto motor neurons with the largest-caliber axons on both sides of the body, representing a unique pathway for fast limb control. We provide open access to the dataset and reconstructions registered to a standard atlas to permit matching of cells between EM and light microscopy data. We also provide GridTape instrumentation designs and software to make large-scale EM more accessible and affordable to the scientific community.


Assuntos
Envelhecimento/fisiologia , Drosophila melanogaster/ultraestrutura , Microscopia Eletrônica de Transmissão , Neurônios Motores/ultraestrutura , Células Receptoras Sensoriais/ultraestrutura , Animais , Automação , Conectoma , Extremidades/inervação , Nervos Periféricos/ultraestrutura , Sinapses/ultraestrutura
4.
Nat Neurosci ; 23(12): 1637-1643, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32929244

RESUMO

Imaging neuronal networks provides a foundation for understanding the nervous system, but resolving dense nanometer-scale structures over large volumes remains challenging for light microscopy (LM) and electron microscopy (EM). Here we show that X-ray holographic nano-tomography (XNH) can image millimeter-scale volumes with sub-100-nm resolution, enabling reconstruction of dense wiring in Drosophila melanogaster and mouse nervous tissue. We performed correlative XNH and EM to reconstruct hundreds of cortical pyramidal cells and show that more superficial cells receive stronger synaptic inhibition on their apical dendrites. By combining multiple XNH scans, we imaged an adult Drosophila leg with sufficient resolution to comprehensively catalog mechanosensory neurons and trace individual motor axons from muscles to the central nervous system. To accelerate neuronal reconstructions, we trained a convolutional neural network to automatically segment neurons from XNH volumes. Thus, XNH bridges a key gap between LM and EM, providing a new avenue for neural circuit discovery.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Neurônios/ultraestrutura , Animais , Axônios/fisiologia , Axônios/ultraestrutura , Córtex Cerebral/citologia , Córtex Cerebral/fisiologia , Córtex Cerebral/ultraestrutura , Dendritos/fisiologia , Dendritos/ultraestrutura , Drosophila melanogaster , Feminino , Holografia , Imageamento Tridimensional , Aprendizado de Máquina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/fisiologia , Neurônios Motores/ultraestrutura , Músculo Esquelético/inervação , Músculo Esquelético/ultraestrutura , Nanotecnologia , Redes Neurais de Computação , Células Piramidais/ultraestrutura , Células Receptoras Sensoriais/fisiologia , Células Receptoras Sensoriais/ultraestrutura , Tomografia
5.
Biochemistry ; 58(33): 3527-3536, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31386347

RESUMO

CPAF (chlamydial protease-like activity factor) is a Chlamydia trachomatis protease that is translocated into the host cytosol during infection. CPAF activity results in dampened host inflammation signaling, cytoskeletal remodeling, and suppressed neutrophil activation. Although CPAF is an emerging antivirulence target, its catalytic mechanism has been unexplored to date. Steady state kinetic parameters were obtained for recombinant CPAF with vimentin-derived peptide substrates using a high-performance liquid chromatography-based discontinuous assay (kcat = 45 ± 0.6 s-1; kcat/Km = 0.37 ± 0.02 µM-1 s-1) or a new fluorescence-based continuous assay (kcat = 23 ± 0.7 s-1; kcat/Km = 0.29 ± 0.03 µM-1 s-1). Residues H105, S499, E558, and newly identified D103 were found to be indispensable for autoproteolytic processing by mutagenesis, while participation of C500 was ruled out despite its proximity to the S499 nucleophile. Pre-steady state kinetics indicated a burst kinetic profile, with fast acylation (kacyl = 110 ± 2 s-1) followed by slower, partially rate-limiting deacylation (kdeacyl = 57 ± 1 s-1). Both kcat- and kcat/Km-pH profiles showed single acidic limb ionizations with pKa values of 6.2 ± 0.1 and 6.5 ± 0.1, respectively. A forward solvent deuterium kinetic isotope effect of 2.6 ± 0.1 was observed for D2Okcatapp, but a unity effect was found for D2Okcat/Kmapp. The kcat proton inventory was linear, indicating transfer of a single proton in the rate-determining transition state, most likely from H105. Collectively, these data provide support for the classification of CPAF as a serine protease and provide a mechanistic foundation for the future design of inhibitors.


Assuntos
Chlamydia trachomatis/enzimologia , Endopeptidases/metabolismo , Serina Proteases/metabolismo , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cinética , Proteólise , Fatores de Virulência
6.
J Cancer ; 8(15): 2950-2958, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28928886

RESUMO

Objective: Describe for the first time the clinical, epidemiological features of vulvar cancer in southwest China. Identify risk factors and provide reference for the prevention of vulvar cancer. Method: We retrospectively analyzed 885 patients admitted to the West China Second University Hospital for vulvar diseases between 2006 and 2016. Vulvar cancer patients with previously diagnosed vulvar nonneoplastic epithelial disorders (n=132) were analyzed and compared to those without prior history of vulvar nonneoplastic epithelial disorders (n=219). Comparisons were also made among cancer patients and non-cancer patients with vulvar nonneoplastic epithelial disorders (n=288) and vulvar squamous intraepithelial lesions (n=246). The risk factors leading to vulvar cancer for the patients with vulvar nonneoplastic epithelial disorder were analyzed by univariate analysis. Furthermore, differences of the epidemiological features of vulvar nonneoplastic epithelial disorders, vulvar squamous intraepithelial lesion and vulvar cancer were identified. Results: According to the univariate analysis, age, first coital age, educational level, smoking, history of vaginal atrophy, HPV infection, lesion sites of the upper vulva and histo-pathological changes are strongly positively correlated with vulvar cancer. By comparing the features of vulvar cancer with those of the vulvar nonneoplastic epithelial disorder and vulvar squamous intraepithelial lesion, we found that on average patients with vulvar cancer had the highest age (ranged from 50 to 59), the lowest first coital age and the highest number of pregnancies and births. The incidences of vulvar nonneoplastic epithelial disorder and vulvar cancer were 1/1000 and 2.5/100,000 respectively with an increasing trend during last 10 years. Conclusion: Age, first coital age, educational level, smoking, atrophic vagina history, HPV infection, lesion sites of the upper vulva and histo-pathological changes are the risk factors that lead to vulvar cancer. Vulvar nonneoplastic epithelial disorder, vulvar squamous intraepithelial lesion and vulvar cancer each has distinct epidemiological features. Prompt surgical intervention and subsequent treatments are the key to a better outcome of vulvar cancer.

7.
J Obstet Gynaecol Res ; 43(4): 768-774, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28418206

RESUMO

Anti-N-methyl-d-aspartate receptor (anti-NMDA-R) encephalitis is an autoimmune disorder that was first described by Dr Vitaliani in 2005. In 2007, Dalmau et al. found anti-NMDA-R antibody expressed both in the hippocampus and prefrontal nerve cell membrane, finally proposing the diagnosis of autoimmune anti-NMDA-R encephalitis. Most of the patients are female (91%), with ages ranging from 4 to 76 years. The average age is 23 years, a birth peak age, although anti-NMDA-R encephalitis is rare during pregnancy. The disorder is characterized by prominent psychosis, dyskinesias, seizures, autonomic disturbance, and central hypoventilation. We report a 24-year-old woman hospitalized at 28 gestational weeks with acute-onset psychosis. Over the course of 3 weeks, her mental status worsened until she fell into a coma. Both serum and cerebrospinal fluid anti-NMDA-R antibodies were found to be positive. At cesarean section, a healthy baby boy was born and a wedge-shaped bilateral ovarian resection was performed. Treatment with corticosteroids, intravenous immunoglobulin, and plasmapheresis can lead to improved outcomes for both mother and baby.


Assuntos
Encefalite Antirreceptor de N-Metil-D-Aspartato/diagnóstico , Coma/diagnóstico , Complicações na Gravidez/diagnóstico , Adulto , Encefalite Antirreceptor de N-Metil-D-Aspartato/complicações , Coma/etiologia , Feminino , Humanos , Gravidez , Adulto Jovem
12.
BMC Genomics ; 16: 853, 2015 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-26499117

RESUMO

BACKGROUND: Exposure to dichlorvos (DDVP), an organophosphorus pesticide, is known to result in neurotoxicity as well as other metabolic perturbations. However, the molecular causes of DDVP toxicity are poorly understood, especially in cells other than neurons and muscle cells. To obtain a better understanding of the process of non-neuronal DDVP toxicity, we exposed zebrafish to different concentrations of DDVP, and investigated the resulting changes in liver histology and gene transcription. RESULTS: Functional enrichment analysis of genes affected by DDVP exposure identified a number of processes involved in energy utilization and stress response in the liver. The abundance of transcripts for proteins involved in glucose metabolism was profoundly affected, suggesting that carbon flux might be diverted toward the pentose phosphate pathway to compensate for an elevated demand for energy and reducing equivalents for detoxification. Strikingly, many transcripts for molecules involved in ß-oxidation and fatty acid synthesis were down-regulated. We found increases in message levels for molecules involved in reactive oxygen species responses as well as ubiquitination, proteasomal degradation, and autophagy. To ensure that the effects of DDVP on energy metabolism were not simply a consequence of poor feeding because of neuromuscular impairment, we fasted fish for 29 or 50 h and analyzed liver gene expression in them. The patterns of gene expression for energy metabolism in fasted and DDVP-exposed fish were markedly different. CONCLUSION: We observed coordinated changes in the expression of a large number of genes involved in energy metabolism and responses to oxidative stress. These results argue that an appreciable part of the effect of DDVP is on energy metabolism and is regulated at the message level. Although we observed some evidence of neuromuscular impairment in exposed fish that may have resulted in reduced feeding, the alterations in gene expression in exposed fish cannot readily be explained by nutrient deprivation.


Assuntos
Diclorvós/toxicidade , Metabolismo Energético/efeitos dos fármacos , Inseticidas/toxicidade , Fígado/efeitos dos fármacos , Fígado/metabolismo , Peixe-Zebra/metabolismo , Animais , Apoptose/genética , Metabolismo dos Carboidratos/genética , Colinesterases/metabolismo , Análise por Conglomerados , Metabolismo Energético/genética , Ativação Enzimática/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Fígado/patologia , Modelos Biológicos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Resposta a Proteínas não Dobradas , Peixe-Zebra/genética
13.
J Proteome Res ; 13(8): 3583-95, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24978939

RESUMO

The toxicity of dichlorvos (DDVP), an organophosphate (OP) pesticide, classically results from modification of the serine in the active sites of cholinesterases. However, DDVP also forms adducts on unrelated targets such as transferrin and albumin, suggesting that DDVP could cause perturbations in cellular processes by modifying noncholinesterase targets. Here we identify novel DDVP-modified targets in lysed human hepatocyte-like cells (HepaRG) using a direct liquid chromatography-mass spectrometry (LC-MS) assay of cell lysates incubated with DDVP or using a competitive pull-down experiments with a biotin-linked organophosphorus compound (10-fluoroethoxyphosphinyl-N-biotinamidopentyldecanamide; FP-biotin), which competes with DDVP for similar binding sites. We show that DDVP forms adducts to several proteins important for the cellular metabolic pathways and differentiation, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin. We validated the results using purified proteins and enzymatic assays. The study not only identified novel DDVP-modified targets but also suggested that the modification directly inhibits the enzymes. The current approach provides information for future hypothesis-based studies to understand the underlying mechanism of toxicity of DDVP in non-neuronal tissues. The MS data have been deposited to the ProteomeXchange with identifier PXD001107.


Assuntos
Adutos de DNA/efeitos dos fármacos , Diclorvós/toxicidade , Hepatócitos/efeitos dos fármacos , Inseticidas/toxicidade , Actinas/metabolismo , Biotina/análogos & derivados , Linhagem Celular , Cromatografia Líquida , Diclorvós/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Inseticidas/metabolismo , Espectrometria de Massas , Compostos Organofosforados
14.
Exp Ther Med ; 3(6): 1026-1032, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22970012

RESUMO

Esophageal carcinoma is the sixth most common cause of cancer-related mortality in the world. Senescence and apoptosis are assumed to be two main mechanisms that inhibit age-related carcinogenesis. p14(ARF), p15(INK4b) and p16(INK4a), which are known to induce senescence by regulating G(1) cell cycle arrest, have been identified as senescence markers. However, the mechanism by which senescence and apoptosis causes neoplasia in esophageal squamous cell carcinoma (ESCC) has not been identified. In this study, 20 cases of normal esophageal tissues, 11 cases of esophageal intraepithelial dysplasia (EID) and 60 cases of ESCC were obtained and pathologically diagnosed. Immunohistochemical staining was performed to assess the expression of p14(ARF), p15(INK4b), p16(INK4a), skp2, bcl-2 and ki-67. The senescence markers p14(ARF) and p16(INK4a) were found to be expressed in 15 and 10% of the normal tissues, 82 and 73% of the EID cases and 100 and 88% of the ESCC cases, respectively. The expression of p15(INK4b) was low in normal tissues, while 92% of the ESCC specimens were diffusely and markedly stained, involving the basal, middle and upper portion of the epithelium. The nuclear expression markers ki-67 and skp2 were highly expressed in ESCC tissues (100 and 72%, respectively). bcl-2 was expressed weakly in normal tissues (10%) and demonstrated various staining patterns in carcinoma specimens (strong in 60%, negative in 40%). MI was 0.09% in normal tissues and 0.95% in the ESCC specimens. Apart from the increased proliferation in esophageal carcinogenesis, as indicated in the ki-67 and skp2 indices, there was an increased expression of senescence-associated molecular markers in the ESCC specimens, which indicates that the senescence pathway may be activated and become a part of cancer development. Of greatest interest to us was that, when compared with clinical information, the expression of the senescence markers was markedly high in the poorly differentiated specimens with lymph node metastasis, indicating that senescence markers may have diagnostic potential in clinical settings.

15.
Int J Biol Markers ; 27(4): e305-13, 2012 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-23280128

RESUMO

Inflammatory breast cancer (IBC) accounts for a small fraction but aggressive form of epithelial breast cancer. Although the role of thrombin in cancer is beginning to be unfolded, its impact on the biology of IBC remains unknown. The purpose of this study was to establish the role of thrombin on the invasiveness of IBC cells. The IBC SUM149 cell line was treated with thrombin in the absence or presence of the epidermal growth factor receptor (EGFR) inhibitor erlotinib and protease-activated receptor 1 (PAR1) inhibitor. The effects of pharmacological inhibitors on the ability of thrombin to stimulate the growth rate and invasiveness were examined. We found that the inhibition of putative cellular targets of thrombin action suppresses both the growth and invasiveness of SUM149 cells in a concentration-dependent manner. In addition, thrombin-mediated increased invasion of SUM149 cells was routed through EGFR phosphorylation, and in turn, stimulation of the p21-activated kinase (Pak1) activity in a EGFR-sensitive manner. Interestingly, thrombin-mediated activation of the Pak1 pathway stimulation was blocked by erlotinib and PAR1 inhibitor. For proof-of-principle studies, we found immunohistochemical evidence of Pak1 activation as well as expression of PAR1 in IBC. Thrombin utilizes EGFR to relay signals promoting SUM149 cell growth and invasion via the Pak1 pathway. The study provides the rationale for future therapeutic approaches in mitigating the invasive nature of IBC by targeting Pak1 and/or EGFR.


Assuntos
Receptores ErbB/metabolismo , Neoplasias Inflamatórias Mamárias/metabolismo , Neoplasias Inflamatórias Mamárias/patologia , Receptor PAR-1/metabolismo , Trombina/farmacologia , Quinases Ativadas por p21/metabolismo , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/patologia , Ativação Enzimática/efeitos dos fármacos , Receptores ErbB/genética , Feminino , Humanos , Neoplasias Inflamatórias Mamárias/genética , Microscopia Confocal , Invasividade Neoplásica , Fosforilação/efeitos dos fármacos , Receptor PAR-1/genética , Transdução de Sinais/efeitos dos fármacos
16.
J Biol Chem ; 285(43): 32787-32792, 2010 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-20702415

RESUMO

Although metastasis tumor antigen 1 (MTA1) contributes to the responsiveness of macrophages to LPS, the underlying mechanism remains unknown. Here, we investigated the role of MTA1 in the regulation of expression and function of MyD88, a proximal component of NF-κB signaling. We discovered that MTA1 targets MyD88 and that MyD88 is a NF-κB-responsive gene in LPS-stimulated macrophages. We found that MTA1 is required for MyD88-dependent stimulation of NF-κB signaling and expression of proinflammatory cytokines such as IL-1ß, MIP2, and TNF-α as MTA1 depletion leads to a substantial reduction in the expression of NF-κB target genes. In addition, LPS-mediated stimulation of MyD88 transcription was accompanied by an enhanced recruitment of MTA1, RNA polymerase II, and p65RelA complex to the NF-κB consensus sites in the MyD88 promoter. Interestingly, the recruitment of both MTA1 and MyD88 expression is effectively blocked by NF-κB inhibitor parthenolide. Selective knockdown of MyD88 by a dominant negative mutant of MyD88 or selective siRNA also impairs the ability of LPS to stimulate the NF-κB target genes. These findings reveal an inherent coregulatory role of MTA1 upon the expression of MyD88 and suggest that MTA1 regulation of MyD88 may constitute at least one of the mechanisms by which MTA1 stimulates LPS-induced NF-κB signaling in stimulated macrophages.


Assuntos
Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Linhagem Celular , Citocinas/biossíntese , Citocinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Mediadores da Inflamação/metabolismo , Macrófagos Peritoneais/citologia , Camundongos , Fator 88 de Diferenciação Mieloide/genética , Proteínas Repressoras , Elementos de Resposta/fisiologia , Sesquiterpenos/farmacologia , Transdução de Sinais/genética , Transativadores , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fatores de Transcrição/genética , Transcrição Genética/efeitos dos fármacos , Transcrição Genética/genética
17.
Int J Gynecol Cancer ; 20(6): 918-25, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20683396

RESUMO

OBJECTIVES: Tumor infiltrating macrophages play an important role in tumor progression. Macrophage chemoattractant protein-1 (MCP-1) is one of the major chemokines responsible for inducing macrophage migration. Our objective was to investigate chemotherapy-induced modulation of MCP-1 in ovarian cancer by investigating macrophage infiltration, tumor vascularity, and MCP-1 expression after chemotherapy exposure. METHODS: MA-148 ovarian cancer cells were treated with paclitaxel (43 pg/mL) and carboplatin (5 microg/mL) alone or in combination. Reverse transcription-polymerase chain reaction determined MCP-1 transcript levels and enzyme-linked immunosorbent assay evaluated MCP-1 protein production at multiple time points. The effect of kinase inhibitors on MCP-1 expression was investigated. In vivo MCP-1 production was examined in tumor-bearing mice and immunohistochemistry with fluorescein isothiocyanate conjugated anti-mouse F4/80 antibody, phycoerythrin-anti-CD31, and terminal deoxynucleotide transferase dUTP nick-end labeling assay were performed. RESULTS: Macrophage chemoattractant protein-1 transcript levels were up-regulated in MA-148 after treatment with paclitaxel and carboplatin individually and in combination. The greatest elevation was seen with combination therapy: 2.5-fold increase in the MCP-1 protein levels from baseline (P = 0.011) with the mitogen-activated protein kinase and janus kinases/signal transducers and activators of transcription pathways appearing to be involved in the regulation of MCP-1 production. In vivo mouse studies confirmed increased MCP-1 production after chemotherapy; however, there was no significant difference in macrophage, apoptosis, or vessel density. CONCLUSIONS: Macrophage chemoattractant protein-1 is up-regulated in ovarian cancer after chemotherapy in vitro and in vivo. Whether MCP-1 production is increased because of a stress-induced response or a scavenger response promoting macrophage infiltration remains unknown. Chemotherapy induction of MCP-1 in ovarian cancer suggests this chemokine plays an important role in the immune response occurring after chemotherapy exposure.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Quimiocina CCL2/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Animais , Carboplatina/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Quimiocina CCL2/biossíntese , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imuno-Histoquímica , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/análise , Neoplasias Ovarianas/patologia , Paclitaxel/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Regulação para Cima
18.
J Biol Chem ; 285(31): 23590-7, 2010 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-20519513

RESUMO

The MTA1 coregulator (metastatic tumor antigen 1), a component of the nucleosome remodeling and deacetylase (NuRD) complex, has been intimately linked with human cancer, but its role in inflammatory responses remains unknown. Here, we discovered that MTA1 is a target of inflammation, and stimulation of macrophages with Escherichia coli lipopolysaccharide (LPS) stimulates MTA1 transcription via the NF-kappaB pathway. Unexpectedly, we found that MTA1 depletion in LPS-stimulated macrophages impairs NF-kappaB signaling and expression of inflammatory molecules. MTA1 itself acts as a transcriptional coactivator of inflammatory cytokines in LPS-stimulated macrophages, and in contrast, it acts as a corepressor in resting primary macrophages as its depletion induced cytokine expression. LPS stimulates S-nitrosylation of histone deacetylase 2 (HDAC2) and interferes with its binding to MTA1, which, in turn, resulted in the loss of corepressor behavior of MTA1.HDAC complex in activated macrophages. Consequently, the net levels of inflammatory cytokines in LPS-stimulated macrophages from MTA1(-/-) mice were high compared with wild-type mice. Accordingly, MTA1(-/-) mice were much more susceptible than control mice to septic shock induced by LPS, revealing that MTA1 protects mice from deregulated host inflammatory response. These findings reveal a previously unrecognized, critical homeostatic role of MTA1, both as a target and as a component of the NF-kappaB circuitry, in the regulation of inflammatory responses.


Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica , NF-kappa B/metabolismo , Nucleossomos/metabolismo , Animais , Clonagem Molecular , Escherichia coli/metabolismo , Histona Desacetilase 2/metabolismo , Homeostase , Inflamação , Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Transgênicos , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras , Transativadores , Fatores de Transcrição/metabolismo
19.
Mol Cell Biochem ; 338(1-2): 255-61, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20054616

RESUMO

Sprouty1 (Spry1) is a conserved antagonist of FGF signaling. The goal of this study was to further explore the downstream mechanisms governing Spry1 inhibition of endothelial cell proliferation. Up-regulation of Spry1 in HUVECs inhibited tube formation on Matrigel (n = 6, P < 0.001). This was associated with decreased proliferation as measured by BrdU incorporation (n = 6, P < 0.001) and increased protein expression of the cyclin-dependent kinase inhibitor 1A (CDKN1A), p21 and cyclin-dependent kinase inhibitor 1B (CDKN1B), p27. A transcriptional analysis using a targeted human angiogenesis array following up-regulation of Spry1 demonstrated a >2-fold increase in an anti-angiogenic factor, serpin peptidase inhibitor, clad F (Serpinf1), and a >2-fold decrease in pro-angiogenic factors fms-related tyrosine kinase 1 (FLT1), angiopoietin2 (Ang-2), and placental growth factor (PGF) (n = 2). To define upstream mechanisms that may regulate endogenous Spry1, we performed a search for responsive elements upstream of the promoter region. This search resulted in the identification of multiple degenerate hypoxia responsive elements. Exposure to hypoxia resulted in a significant increase in Spry1 expression (n = 8, P < 0.01). These findings shed new light on downstream signaling pathways associated with Spry1 anti-proliferative responses, and provide new evidence that hypoxia stimulates Spry1 expression.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteínas de Membrana/metabolismo , Neovascularização Fisiológica , Fosfoproteínas/metabolismo , Proliferação de Células , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Humanos , Hipóxia/metabolismo , Proteínas de Membrana/genética , Fosfoproteínas/genética , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima
20.
J Biol Chem ; 285(10): 6980-6, 2010 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-20022949

RESUMO

Nitric oxide has been implicated in the pathogenesis of inflammatory disorders, including hepatitis B virus-associated hepatocellular carcinoma. Transactivator protein HBx, a major regulator of cellular responses of hepatitis B virus, is known to induce the expression of MTA1 (metastasis-associated protein 1) coregulator via NF-kappaB signaling in hepatic cells. However, the underlying mechanism of HBx regulation of the inducible nitric-oxide synthase (iNOS) pathway remains unknown. Here we provide evidence that MTA1 is a positive regulator of iNOS transcription and plays a mechanistic role in HBx stimulation of iNOS expression and activity. We found that the HBx-MTA1 complex is recruited onto the human iNOS promoter in an NF-kappaB-dependent manner. Pharmacological inhibition of the NF-kappaB signaling prevented the ability of HBx to stimulate the transcription, the expression, and the activity of iNOS; nevertheless, these effects could be substantially rescued by MTA1 dysregulation. We further discovered that HBx-mediated stimulation of MTA1 is paralleled by the suppression of miR-661, a member of the small noncoding RNAs, recently shown to target MTA1. We observed that miR-661 controls of MTA1 expression contributed to the expression and activity of iNOS in HBx-expressing HepG2 cells. Accordingly, depletion of MTA1 by either miR-661 or siRNA in HBx-expressing cells severely impaired the ability of HBx to modulate the endogenous levels of iNOS and nitrite production. Together, these findings reveal an inherent role of MTA1 in HBx regulation of iNOS expression and consequently its function in the liver cancer cells.


Assuntos
Óxido Nítrico Sintase Tipo II/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/fisiologia , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virologia , Camundongos , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Sesquiterpenos/farmacologia , Transativadores/genética , Fatores de Transcrição/genética , Proteínas Virais Reguladoras e Acessórias
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