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1.
Am J Hum Genet ; 109(8): 1436-1457, 2022 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-35907405

RESUMO

ADGRL1 (latrophilin 1), a well-characterized adhesion G protein-coupled receptor, has been implicated in synaptic development, maturation, and activity. However, the role of ADGRL1 in human disease has been elusive. Here, we describe ten individuals with variable neurodevelopmental features including developmental delay, intellectual disability, attention deficit hyperactivity and autism spectrum disorders, and epilepsy, all heterozygous for variants in ADGRL1. In vitro, human ADGRL1 variants expressed in neuroblastoma cells showed faulty ligand-induced regulation of intracellular Ca2+ influx, consistent with haploinsufficiency. In vivo, Adgrl1 was knocked out in mice and studied on two genetic backgrounds. On a non-permissive background, mice carrying a heterozygous Adgrl1 null allele exhibited neurological and developmental abnormalities, while homozygous mice were non-viable. On a permissive background, knockout animals were also born at sub-Mendelian ratios, but many Adgrl1 null mice survived gestation and reached adulthood. Adgrl1-/- mice demonstrated stereotypic behaviors, sexual dysfunction, bimodal extremes of locomotion, augmented startle reflex, and attenuated pre-pulse inhibition, which responded to risperidone. Ex vivo synaptic preparations displayed increased spontaneous exocytosis of dopamine, acetylcholine, and glutamate, but Adgrl1-/- neurons formed synapses in vitro poorly. Overall, our findings demonstrate that ADGRL1 haploinsufficiency leads to consistent developmental, neurological, and behavioral abnormalities in mice and humans.


Assuntos
Transtorno do Espectro Autista , Deficiência Intelectual , Transtornos do Neurodesenvolvimento , Receptores Acoplados a Proteínas G , Receptores de Peptídeos , Adulto , Animais , Transtorno do Espectro Autista/genética , Modelos Animais de Doenças , Haploinsuficiência/genética , Humanos , Deficiência Intelectual/genética , Camundongos , Camundongos Knockout , Transtornos do Neurodesenvolvimento/genética
2.
Differentiation ; 89(1-2): 11-21, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25578479

RESUMO

The nuclear lamina, comprised of the A and B-type lamins, is important in maintaining nuclear shape and in regulating key nuclear functions such as chromatin organization and transcription. Deletion of the A-type lamins results in genome instability and many cancers show altered levels of A-type lamin expression. Loss of function mutations in the mouse Lmna gene result in early postnatal lethality, usually within 3-5 weeks of birth making an analysis of the role of lamins in carcinogenesis difficult. To circumvent early lethality, and determine the role of the A-type lamins in specific tissues in older mice we derived a conditional allele of Lmna(FL/FL) (floxed). Lmna(FL/FL) was specifically deleted in the gastrointestinal (GI) epithelium by crossing the Lmna(FL/FL) mice with Villin-Cre mice. Mice lacking Lmna in the GI are overtly normal with no effects on overall growth, longevity or GI morphology. On a GI specific sensitized (Apc(Min/+)) background, polyp numbers are unchanged, but polyp size is slightly increased, and only in the duodenum. Our findings reveal that although A-type lamins are dispensable in the postnatal GI epithelium, loss of Lmna under malignant conditions may, to a limited extent, enhance polyp size indicating that A-type lamins may regulate cell proliferation in the transformed GI epithelium.


Assuntos
Transformação Celular Neoplásica/genética , Instabilidade Genômica , Pólipos Intestinais/genética , Lamina Tipo A/genética , Animais , Proliferação de Células/genética , Epitélio/crescimento & desenvolvimento , Epitélio/patologia , Trato Gastrointestinal/crescimento & desenvolvimento , Trato Gastrointestinal/patologia , Pólipos Intestinais/patologia , Lamina Tipo A/metabolismo , Camundongos , Especificidade de Órgãos
3.
Dev Cell ; 59(2): 175-186.e8, 2024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38159568

RESUMO

Ectodermal appendages, such as the mammary gland (MG), are thought to have evolved from hair-associated apocrine glands to serve the function of milk secretion. Through the directed differentiation of mouse embryonic stem cells (mESCs), here, we report the generation of multilineage ESC-derived mammary organoids (MEMOs). We adapted the skin organoid model, inducing the dermal mesenchyme to transform into mammary-specific mesenchyme via the sequential activation of Bone Morphogenetic Protein 4 (BMP4) and Parathyroid Hormone-related Protein (PTHrP) and inhibition of hedgehog (HH) signaling. Using single-cell RNA sequencing, we identified gene expression profiles that demonstrate the presence of mammary-specific epithelial cells, fibroblasts, and adipocytes. MEMOs undergo ductal morphogenesis in Matrigel and can reconstitute the MG in vivo. Further, we demonstrate that the loss of function in placode regulators LEF1 and TBX3 in mESCs results in impaired skin and MEMO generation. In summary, our MEMO model is a robust tool for studying the development of ectodermal appendages, and it provides a foundation for regenerative medicine and disease modeling.


Assuntos
Proteínas Hedgehog , Células-Tronco Embrionárias Murinas , Camundongos , Animais , Proteínas Hedgehog/metabolismo , Glândulas Mamárias Animais , Células Epiteliais , Diferenciação Celular , Organoides
4.
J Cell Sci ; 123(Pt 22): 3944-55, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-20980386

RESUMO

During the initial stage of neuromuscular junction (NMJ) formation, nerve-derived agrin cooperates with muscle-autonomous mechanisms in the organization and stabilization of a plaque-like postsynaptic specialization at the site of nerve-muscle contact. Subsequent NMJ maturation to the characteristic pretzel-like appearance requires extensive structural reorganization. We found that the progress of plaque-to-pretzel maturation is regulated by agrin. Excessive cleavage of agrin via transgenic overexpression of an agrin-cleaving protease, neurotrypsin, in motoneurons resulted in excessive reorganizational activity of the NMJs, leading to rapid dispersal of the synaptic specialization. By contrast, expression of cleavage-resistant agrin in motoneurons slowed down NMJ remodeling and delayed NMJ maturation. Neurotrypsin, which is the sole agrin-cleaving protease in the CNS, was excluded as the physiological agrin-cleaving protease at the NMJ, because NMJ maturation was normal in neurotrypsin-deficient mice. Together, our analyses characterize agrin cleavage at its proteolytic α- and ß-sites by an as-yet-unspecified protease as a regulatory access for relieving the agrin-dependent constraint on endplate reorganization during NMJ maturation.


Assuntos
Agrina/metabolismo , Junção Neuromuscular/metabolismo , Serina Endopeptidases/metabolismo , Animais , Linhagem Celular , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Fibras Nervosas/metabolismo , Serina Endopeptidases/biossíntese , Medula Espinal/citologia , Transmissão Sináptica/fisiologia
5.
Neuron ; 49(6): 823-32, 2006 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-16543131

RESUMO

Myelin, the insulating layers of membrane wrapped around axons by oligodendrocytes, is essential for normal impulse conduction. It forms during late stages of fetal development but continues into early adult life. Myelination correlates with cognitive development and can be regulated by impulse activity through unknown molecular mechanisms. Astrocytes do not form myelin, but these nonneuronal cells can promote myelination in ways that are not understood. Here, we identify a link between myelination, astrocytes, and electrical impulse activity in axons that is mediated by the cytokine leukemia inhibitory factor (LIF). These findings show that LIF is released by astrocytes in response to ATP liberated from axons firing action potentials, and LIF promotes myelination by mature oligodendrocytes. This activity-dependent mechanism promoting myelination could regulate myelination according to functional activity or environmental experience and may offer new approaches to treating demyelinating diseases.


Assuntos
Astrócitos/efeitos da radiação , Comunicação Celular/fisiologia , Estimulação Elétrica/métodos , Proteínas da Mielina/metabolismo , Oligodendroglia/fisiologia , Potenciais de Ação/fisiologia , Potenciais de Ação/efeitos da radiação , Adenosina/farmacologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Anestésicos Locais/farmacologia , Animais , Anticorpos/farmacologia , Astrócitos/fisiologia , Axônios/efeitos dos fármacos , Axônios/metabolismo , Axônios/efeitos da radiação , Compostos Azo , Western Blotting/métodos , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/efeitos da radiação , Contagem de Células/métodos , Células Cultivadas , Córtex Cerebral/citologia , Técnicas de Cocultura/métodos , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Proteínas de Drosophila/metabolismo , Interações Medicamentosas , Embrião de Mamíferos , Ensaio de Imunoadsorção Enzimática/métodos , Gânglios Espinais/citologia , Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica/métodos , Interleucina-6/imunologia , Interleucina-6/metabolismo , Fator Inibidor de Leucemia , Camundongos , Modelos Biológicos , Proteína Básica da Mielina/metabolismo , Glicoproteína Associada a Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito , Naftalenos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Antígenos O/metabolismo , RNA Mensageiro/biossíntese , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Células-Tronco , Tetrodotoxina/farmacologia , Tionucleotídeos/farmacologia
6.
Cancer Res ; 80(8): 1630-1643, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-31911550

RESUMO

Pancreatic cancer is a disease with limited therapeutic options. Resistance to chemotherapies poses a significant clinical challenge for patients with pancreatic cancer and contributes to a high rate of recurrence. Oncogenic KRAS, a critical driver of pancreatic cancer, promotes metabolic reprogramming and upregulates NRF2, a master regulator of the antioxidant network. Here, we show that NRF2 contributed to chemoresistance and was associated with a poor prognosis in patients with pancreatic cancer. NRF2 activation metabolically rewired and elevated pathways involved in glutamine metabolism. This curbed chemoresistance in KRAS-mutant pancreatic cancers. In addition, manipulating glutamine metabolism restrained the assembly of stress granules, an indicator of chemoresistance. Glutaminase inhibitors sensitized chemoresistant pancreatic cancer cells to gemcitabine, thereby improving the effectiveness of chemotherapy. This therapeutic approach holds promise as a novel therapy for patients with pancreatic cancer harboring KRAS mutation. SIGNIFICANCE: These findings illuminate the mechanistic features of KRAS-mediated chemoresistance and provide a rationale for exploiting metabolic reprogramming in pancreatic cancer cells to confer therapeutic opportunities that could be translated into clinical trials. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/8/1630/F1.large.jpg.


Assuntos
Resistencia a Medicamentos Antineoplásicos/fisiologia , Glutamina/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Antimetabólitos Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/mortalidade , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Glutaminase/antagonistas & inibidores , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Mutação , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidade , Prognóstico , Distribuição Aleatória , Análise Serial de Tecidos , Regulação para Cima , Gencitabina
7.
J Virol ; 82(12): 5860-8, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18400857

RESUMO

The human nuclear envelope proteins emerin and lamina-associated polypeptide 2alpha (LAP2alpha) have been proposed to aid in the early replication steps of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). However, whether these factors are essential for HIV-1 or MLV infection has been questioned. Prior studies in which conflicting results were obtained were highly dependent on RNA interference-mediated gene silencing. To shed light on these contradictory results, we examined whether HIV-1 or MLV could infect primary cells from mice deficient for emerin, LAP2alpha, or both emerin and LAP2alpha. We observed HIV-1 and MLV infectivity in mouse embryonic fibroblasts (MEFs) from emerin knockout, LAP2alpha knockout, or emerin and LAP2alpha double knockout mice to be comparable in infectivity to wild-type littermate-derived MEFs, indicating that both emerin and LAP2alpha were dispensable for HIV-1 and MLV infection of dividing, primary mouse cells. Because emerin has been suggested to be important for infection of human macrophages by HIV-1, we also examined HIV-1 transduction of macrophages from wild-type mice or knockout mice, but again we did not observe a difference in susceptibility. These findings prompted us to reexamine the role of human emerin in supporting HIV-1 and MLV infection. Notably, both viruses efficiently infected human cells expressing high levels of dominant-negative emerin. We thus conclude that emerin and LAP2alpha are not required for the early replication of HIV-1 and MLV in mouse or human cells.


Assuntos
Proteínas de Ligação a DNA/genética , HIV-1/fisiologia , Proteínas de Membrana/genética , Proteínas Nucleares/genética , Infecções por Retroviridae/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Humanos , Rim/citologia , Vírus da Leucemia Murina/patogenicidade , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Células NIH 3T3 , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína
8.
FASEB J ; 22(6): 1861-73, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18230682

RESUMO

The synaptic serine protease neurotrypsin is considered to be essential for the establishment and maintenance of cognitive brain functions, because humans lacking functional neurotrypsin suffer from severe mental retardation. Neurotrypsin cleaves agrin at two homologous sites, liberating a 90-kDa and a C-terminal 22-kDa fragment from the N-terminal moiety of agrin. Morphological analyses indicate that neurotrypsin is contained in presynaptic terminals and externalized in association with synaptic activity, while agrin is localized to the extracellular space at or in the vicinity of the synapse. Here, we present a detailed biochemical analysis of neurotrypsin-mediated agrin cleavage in the murine brain. In brain homogenates, we found that neurotrypsin exclusively cleaves glycanated variants of agrin. Studies with isolated synaptosomes obtained by subcellular fractionation from brains of wild-type and neurotrypsin-overexpressing mice revealed that neurotrypsin-dependent cleavage of agrin was concentrated at synapses, where the most heavily glycanated variants of agrin predominate. Because agrin has been shown to play an important role in the formation and the maintenance of excitatory synapses in the central nervous system, its local cleavage at the synapse implicates the neurotrypsin/agrin system in the regulation of adaptive reorganizations of the synaptic circuitry in the context of cognitive functions, such as learning and memory.


Assuntos
Agrina/metabolismo , Fragmentos de Peptídeos/metabolismo , Serina Endopeptidases/metabolismo , Sinapses/metabolismo , Agrina/química , Animais , Química Encefálica , Cognição , Camundongos , Fragmentos de Peptídeos/química , Polissacarídeos/análise , Serina Endopeptidases/análise , Sinapses/química
9.
Methods Mol Biol ; 512: 159-67, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19347277

RESUMO

Despite the current availability of an impressive in vitro assay battery developed to quantitatively analyze the broad panel of small compounds and macromolecules that possess the inflammatory potential, little methodology exists nowadays that affords a researcher or clinician to quantify the ultimate output on the level of cell signaling response caused by inflammatory pathway stimulation. As a matter of fact, majority of analytical tools measure bona fide inflammatory substances (e.g., cytokines or chemokines) by their direct binding to secondary reagents such as specific antibodies or other selectively affine substrates with the final readout generated via quantification of the resulting complexes. Although specific and highly reproducible, this approach provides no discrimination between biologically active versus inactive input analyte nor does it address the differential biological potential for the questioned substances related to their in vivo stability and biodistribution. In a search for alternative solutions, a novel strategy is emerging that employs cell-based methods of inflammatory substance measurements allowing to detect and quantify the downstream effects of analyte's activity translated in terms of inflammatory pathways stimulation. In addition, application of cell based assays simultaneously permits entry level evaluation of compound toxicity and endows with a powerful approach to perform high-throughput screenings of, e.g., small molecule libraries in a quest for novel compounds capable of influencing the inflammation process.


Assuntos
Anti-Inflamatórios/farmacologia , Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Inflamação/induzido quimicamente , Fator de Necrose Tumoral alfa/farmacologia , Anti-Inflamatórios/análise , Células HeLa , Humanos , Preparações Farmacêuticas , Fator de Necrose Tumoral alfa/análise
12.
Adv Drug Deliv Rev ; 136-137: 82-96, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30273617

RESUMO

Nanotechnology provides many solutions to improve conventional drug delivery and has a unique niche in the areas related to the specific targeting of the immune system, such as immunotherapies and vaccines. Preclinical studies in this field rely heavily on the combination of in vitro and in vivo methods to assess the safety and efficacy of nanotechnology platforms, nanoparticle-formulated drugs, and vaccines. While certain types of toxicities can be evaluated in vitro and good in vitro-in vivo correlation has been demonstrated for such tests, animal studies are still needed to address complex biological questions and, therefore, provide a unique contribution to establishing nanoparticle safety and efficacy profiles. The genetic, metabolic, mechanistic, and phenotypic diversity of currently available animal models often complicates both the animal choice and the interpretation of the results. This review summarizes current knowledge about differences in the immune system function and immunological responses of animals commonly used in preclinical studies of nanomaterials. We discuss challenges, highlight current gaps, and propose recommendations for animal model selection to streamline preclinical analysis of nanotechnology formulations.


Assuntos
Sistema Imunitário/inervação , Modelos Animais , Nanoestruturas/química , Nanotecnologia , Animais , Sistema Imunitário/imunologia
13.
Elife ; 72018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30457553

RESUMO

A presynaptic adhesion G-protein-coupled receptor, latrophilin-1, and a postsynaptic transmembrane protein, Lasso/teneurin-2, are implicated in trans-synaptic interaction that contributes to synapse formation. Surprisingly, during neuronal development, a substantial proportion of Lasso is released into the intercellular space by regulated proteolysis, potentially precluding its function in synaptogenesis. We found that released Lasso binds to cell-surface latrophilin-1 on axonal growth cones. Using microfluidic devices to create stable gradients of soluble Lasso, we show that it induces axonal attraction, without increasing neurite outgrowth. Using latrophilin-1 knockout in mice, we demonstrate that latrophilin-1 is required for this effect. After binding latrophilin-1, Lasso causes downstream signaling, which leads to an increase in cytosolic calcium and enhanced exocytosis, processes that are known to mediate growth cone steering. These findings reveal a novel mechanism of axonal pathfinding, whereby latrophilin-1 and Lasso mediate both short-range interaction that supports synaptogenesis, and long-range signaling that induces axonal attraction.


Assuntos
Cones de Crescimento/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Sinapses/fisiologia , Animais , Linhagem Celular , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteólise
14.
Curr Cancer Drug Targets ; 5(5): 325-44, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16101381

RESUMO

Chronic inflammation has long been suggested to constitute a risk factor for a variety of epithelial cancers such as malignancies of prostate, cervix, esophagus, stomach, liver, colon, pancreas, and bladder. An inflammatory response is typically accompanied by generation of free radicals, stimulation of cytokines, chemokines, growth and angiogenic factors. Free radicals, capable of both directly damaging DNA and affecting the DNA repair machinery, enhance genetic instability of affected cells, thus contributing to the first stage of neoplastic transformation also known as "initiation". Cytokines and growth factors can further promote tumor growth by stimulating cell proliferation, adhesion, vascularization, and metastatic potential of later stage tumors. Nuclear factor kappa B (NF-kappaB) is a family of ubiquitously expressed transcription factors that are widely believed to trigger both the onset and the resolution of inflammation. NF-kappaB also governs the expression of genes encoding proteins essential in control of stress response, maintenance of intercellular communications, and regulation of cellular proliferation and apoptosis. Recent data have expanded the concept of inflammation as a critical component in carcinogenesis suggesting new anti-inflammatory therapies for a complementary approach in treating a variety of tumor types. These observations highlighted the NF-kappaB pathway as an attractive avenue for drug discovery and development. The present review will outline recent advances in our understanding of NF-kappaB function in the inflammatory processes and its input in tumor initiation/promotion, as well as summarize the development of animal and cell culture models for validating drug candidates with NF-kappaB-modulating activities, and applications of the latter in cancer therapy.


Assuntos
Antineoplásicos/uso terapêutico , Inflamação/etiologia , NF-kappa B/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/etiologia , Animais , Antineoplásicos/farmacologia , Citocinas/metabolismo , Desenho de Fármacos , Radicais Livres/metabolismo , Regulação da Expressão Gênica , Humanos , Inflamação/metabolismo , Inflamação/fisiopatologia , Glicoproteínas de Membrana/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Neoplasias/patologia , Receptores de Superfície Celular/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like , Fator de Necrose Tumoral alfa/metabolismo
15.
Nat Med ; 21(11): 1337-43, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26479921

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive neoplasm characterized by a marked fibro-inflammatory microenvironment, the presence of which can promote both cancer induction and growth. Therefore, selective manipulation of local cytokines is an attractive, although unrealized, therapeutic approach. T cells possess a unique mechanism of p38 mitogen-activated protein kinase (MAPK) activation downstream of T cell receptor (TCR) engagement through the phosphorylation of Tyr323 (pY323). This alternative p38 activation pathway is required for pro-inflammatory cytokine production. Here we show in human PDAC that a high percentage of infiltrating pY323(+) T cells was associated with large numbers of tumor necrosis factor (TNF)-α- and interleukin (IL)-17-producing CD4(+) tumor-infiltrating lymphocytes (TILs) and aggressive disease. The growth of mouse pancreatic tumors was inhibited by genetic ablation of the alternative p38 pathway, and transfer of wild-type CD4(+) T cells, but not those lacking the alternative pathway, enhanced tumor growth in T cell-deficient mice. Notably, a plasma membrane-permeable peptide derived from GADD45-α, the naturally occurring inhibitor of p38 pY323(+) (ref. 7), reduced CD4(+) TIL production of TNF-α, IL-17A, IL-10 and secondary cytokines, halted growth of implanted tumors and inhibited progression of spontaneous KRAS-driven adenocarcinoma in mice. Thus, TCR-mediated activation of CD4(+) TILs results in alternative p38 activation and production of protumorigenic factors and can be targeted for therapeutic benefit.


Assuntos
Carcinoma Ductal Pancreático/imunologia , Citocinas/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Pancreáticas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Animais , Linfócitos T CD4-Positivos , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Progressão da Doença , Humanos , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-17/imunologia , Interleucina-17/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosforilação , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética
16.
J Immunol Methods ; 281(1-2): 51-63, 2003 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-14580881

RESUMO

The control of NF-kappaB activation is a proven therapeutic strategy in the treatment of multiple inflammatory disorders. Drug discovery and development for such a therapy demands a battery of assays to reliably demonstrate both clinical effectiveness and biological safety of prospective medications. Unlike traditional in vitro biochemical analyses, cell-based assays more closely mimic the actual in vivo physiologic environment, addressing simultaneously biological activity and toxicity issues. A novel assay system, based solely on the drug resistance of a genetically engineered cell line, has been developed to provide rapid quantitative evaluation of the (anti)-inflammatory potential of test substances. The assay principle is based on the ability of bona fide inflammatory agents to activate the transcription factor NF-kappaB in cultured cells. In our model, expression of a dual drug resistance marker, driven by an NF-kappaB-dependent minimal promoter, provides a selective and highly sensitive scheme with a quantitative readout to detect biochemical agents with pro-or anti-inflammatory properties. The novel cell-based system is inexpensive, simple to perform (requiring only basic cell culture skills), accurate, and provides sensitivity comparable to that of the electrophoretic mobility shift assay and quantitative ELISA. In addition, the dual selection capability of the model provides a powerful tool to discover novel molecular components of the NF-kappaB signal transduction pathway.


Assuntos
Anti-Inflamatórios/farmacologia , Inflamação/induzido quimicamente , NF-kappa B/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Células HeLa , Humanos , Lipopolissacarídeos/farmacologia , Puromicina/farmacologia , Retroviridae/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/análise
19.
PLoS One ; 8(2): e57960, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23469119

RESUMO

Cell fate commitment of spinal progenitor neurons is initiated by long-range, midline-derived, morphogens that regulate an array of transcription factors that, in turn, act sequentially or in parallel to control neuronal differentiation. Included among these are transcription factors that regulate the expression of receptors for guidance cues, thereby determining axonal trajectories. The Ig/FNIII superfamily molecules TAG1/Axonin1/CNTN2 (TAG1) and Neurofascin (Nfasc) are co-expressed in numerous neuronal cell types in the CNS and PNS - for example motor, DRG and interneurons - both promote neurite outgrowth and both are required for the architecture and function of nodes of Ranvier. The genes encoding TAG1 and Nfasc are adjacent in the genome, an arrangement which is evolutionarily conserved. To study the transcriptional network that governs TAG1 and Nfasc expression in spinal motor and commissural neurons, we set out to identify cis elements that regulate their expression. Two evolutionarily conserved DNA modules, one located between the Nfasc and TAG1 genes and the second directly 5' to the first exon and encompassing the first intron of TAG1, were identified that direct complementary expression to the CNS and PNS, respectively, of the embryonic hindbrain and spinal cord. Sequential deletions and point mutations of the CNS enhancer element revealed a 130bp element containing three conserved E-boxes required for motor neuron expression. In combination, these two elements appear to recapitulate a major part of the pattern of TAG1 expression in the embryonic nervous system.


Assuntos
Contactina 2/genética , Gânglios Sensitivos/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Sequências Reguladoras de Ácido Nucleico/genética , Medula Espinal/embriologia , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Moléculas de Adesão Celular/metabolismo , Embrião de Galinha , Sequência Conservada , Elementos E-Box/genética , Evolução Molecular , Gânglios Sensitivos/citologia , Gânglios Sensitivos/metabolismo , Redes Reguladoras de Genes/genética , Humanos , Camundongos , Dados de Sequência Molecular , Neurônios Motores/metabolismo , Mutagênese , Fatores de Crescimento Neural/metabolismo , Especificidade de Órgãos , Ratos , Rombencéfalo/citologia , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismo , Raízes Nervosas Espinhais/metabolismo
20.
Nanomedicine (Lond) ; 6(7): 1175-88, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21675859

RESUMO

AIMS: While numerous studies have reported on nanoparticle uptake by phagocytic cells, the mechanisms of this uptake are poorly understood. A metastudy of research focusing on biological particulate matter has postulated that nanoparticles cannot be phagocytosed and therefore must enter cells via pinocytosis. The purpose of this study was to identify the route(s) of uptake of gold nanoparticles in vitro and to determine if these route(s) depend on particle size. MATERIALS & METHODS: The parent RAW264.7 cell line and its derivatives, transduced with a virus carrying siRNA to macrophage scavenger receptor A, were used as model phagocytes. Citrate-stabilized gold colloids were used as model nanoparticles. We used chemical inhibitors known to interfere with specific routes of particulate uptake. We developed multifocal light microscopy methods including multifocal stack analysis with NIH ImageJ software to analyze cell uptake. RESULTS: Irrespective of size, gold nanoparticles are internalized by macrophages via multiple routes, including both phagocytosis and pinocytosis. If either route was blocked, the particles entered cells via the other route. CONCLUSION: Gold nanoparticles with hydrodynamic sizes below 100 nm can be phagocytosed. Phagocytosis of anionic gold colloids by RAW264.7 cells is mediated by macrophage scavenger receptor A.


Assuntos
Coloide de Ouro/metabolismo , Macrófagos/citologia , Receptores Depuradores/metabolismo , Receptores Depuradores Classe A/metabolismo , Animais , Linhagem Celular , Macrófagos/metabolismo , Camundongos , Fagocitose , Pinocitose
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