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3.
J Photochem Photobiol B ; 189: 66-73, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30312922

RESUMO

In current years, the development of efficient green methods for synthesis of metal oxide nanomaterials has attracted a great attention to the researchers since the plant-mediated synthesis is a cost-effective and a good alternative to chemical and physical methods. An efficient and eco-friendly route has been developed for the green synthesis of CuO nanoparticles (NPs) by Madhuca longifolia plant extract which acts as a non-toxic reducing agent. X-ray diffraction studies reveal the good crystallinity of the synthesized NPs and FTIR spectra confirm the synthesis of these NPs. UV-visible absorption spectra showed that the NPs have been reached at different nano scale level depending on their synthesis procedures. TEM images indicate that as-synthesized CuO NPs are spherical in shape with their different size ranges and they show different band gap values which is confirmed by Tauc's formula. The NPs exhibit good photoluminescence property depending on their particle size and they also show excellent photocatalytic activity towards the degradation of methylene blue (MB) in presence of visible light irradiation which will be a promising material for waste water treatment. The synthesized CuO NPs show good antibacterial activity against bacterial strains namely E. coli BL21 DE3 Gram-negative, S. aureus Gram-positive and B.subtilis Gram-positive and the results have been compared against Ampicillin and Tetracycline.


Assuntos
Química Verde/métodos , Madhuca/metabolismo , Nanopartículas Metálicas/química , Extratos Vegetais/química , Purificação da Água/métodos , Antibacterianos , Bactérias/efeitos dos fármacos , Cobre , Oxirredução , Águas Residuárias
4.
PLoS One ; 9(8): e104148, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25089703

RESUMO

The scavenger receptor MARCO mediates macrophage recognition and clearance of pathogens and their polyanionic ligands. However, recent studies demonstrate MARCO expression and function in dendritic cells, suggesting MARCO might serve to bridge innate and adaptive immunity. To gain additional insight into the role of MARCO in dendritic cell activation and function, we profiled transcriptomes of mouse splenic dendritic cells obtained from MARCO deficient mice and their wild type counterparts under resting and activating conditions. In silico analysis uncovered major alterations in gene expression in MARCO deficient dendritic cells resulting in dramatic alterations in key dendritic cell-specific pathways and functions. Specifically, changes in CD209, FCGR4 and Complement factors can have major consequences on DC-mediated innate responses. Notably, these perturbations were magnified following activation with the TLR-4 agonist lipopolysaccharide. To validate our in silico data, we challenged DC's with various agonists that recognize all mouse TLRs and assessed expression of a set of immune and inflammatory marker genes. This approach identified a differential contribution of MARCO to TLR activation and validated a major role for MARCO in mounting an inflammatory response. Together, our data demonstrate that MARCO differentially affects TLR-induced DC activation and suggest targeting of MARCO could lead to different outcomes that depend on the inflammatory context encountered by DC.


Assuntos
Células Dendríticas/imunologia , Imunidade Inata , Inflamação/imunologia , Receptores Imunológicos/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Moléculas de Adesão Celular/metabolismo , Fator I do Complemento/metabolismo , Simulação por Computador , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Regulação da Expressão Gênica/imunologia , Inflamação/metabolismo , Lectinas Tipo C/metabolismo , Ligantes , Lipopolissacarídeos/administração & dosagem , Macrófagos/imunologia , Camundongos , Receptores de Superfície Celular/metabolismo , Receptores de IgG/metabolismo , Receptores Imunológicos/metabolismo , Receptores Depuradores/imunologia , Receptor 4 Toll-Like/agonistas
5.
Bioorg Med Chem Lett ; 23(9): 2614-8, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23540646

RESUMO

A series of compounds containing an α,ß-unsaturated carbonyl moiety, such as chalcones and coumarins were designed, synthesized and tested in a variety of assays to assess their potential as anti-Alzheimer's disease (AD) agents. The investigations included the inhibition of cholinesterases (AChE, BuChE), the inhibition of amyloid beta (Aß) self-assembly and the disassembly of preformed Aß oligomers. Several compounds showed excellent potential as multifunctional compounds for AD. Docking studies for 16 that performed well in all the assays gave a clear interpretation of various interactions in the gorge of AChE. Based on the results, the long-chain coumarin scaffold appears to be a promising structural template for further AD drug development.


Assuntos
Chalconas/química , Inibidores da Colinesterase/síntese química , Cumarínicos/química , Desenho de Fármacos , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Sítios de Ligação , Domínio Catalítico , Chalconas/síntese química , Chalconas/uso terapêutico , Inibidores da Colinesterase/química , Inibidores da Colinesterase/uso terapêutico , Cumarínicos/síntese química , Cumarínicos/uso terapêutico , Humanos , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade
6.
Biochemistry ; 52(7): 1137-48, 2013 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-23346953

RESUMO

The design and application of an effective, new class of multifunctional small molecule inhibitors of amyloid self-assembly are described. Several compounds based on the diaryl hydrazone scaffold were designed. Forty-four substituted derivatives of this core structure were synthesized using a variety of benzaldehydes and phenylhydrazines and characterized. The inhibitor candidates were evaluated in multiple assays, including the inhibition of amyloid ß (Aß) fibrillogenesis and oligomer formation and the reverse processes, the disassembly of preformed fibrils and oligomers. Because the structure of the hydrazone-based inhibitors mimics the redox features of the antioxidant resveratrol, the radical scavenging effect of the compounds was evaluated by colorimetric assays against 2,2-diphenyl-1-picrylhydrazyl and superoxide radicals. The hydrazone scaffold was active in all of the different assays. The structure-activity relationship revealed that the substituents on the aromatic rings had a considerable effect on the overall activity of the compounds. The inhibitors showed strong activity in fibrillogenesis inhibition and disassembly, and even greater potency in the inhibition of oligomer formation and oligomer disassembly. Supporting the quantitative fluorometric and colorimetric assays, size exclusion chromatographic studies indicated that the best compounds practically eliminated or substantially inhibited the formation of soluble, aggregated Aß species, as well. Atomic force microscopy was also applied to monitor the morphology of Aß deposits. The compounds also possessed the predicted antioxidant properties; approximately 30% of the synthesized compounds showed a radical scavenging effect equal to or better than that of resveratrol or ascorbic acid.


Assuntos
Amiloide/antagonistas & inibidores , Antioxidantes/farmacologia , Hidrazonas/química , Hidrazonas/farmacologia , Relação Estrutura-Atividade , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Antioxidantes/química , Compostos de Bifenilo/química , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Hidrazonas/síntese química , Microscopia de Força Atômica , Estrutura Molecular , Picratos/química , Superóxidos/química
7.
Infect Immun ; 81(2): 402-10, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23166161

RESUMO

TRIF is an adaptor molecule important in transducing signals from intracellularly signaling Toll-like receptor 3 (TLR3) and TLR4. Recently, TLR2 was found to signal from intracellular compartments. Using a synthetic ligand for TLR2/1 heterodimers, as well as Borrelia burgdorferi, which is a strong activator of TLR2/1, we found that TLR2 signaling can utilize TRIF. Unlike TRIF signaling by other TLRs, TLR2-mediated TRIF signaling is dependent on the presence of another adaptor molecule, MyD88. However, unlike MyD88 deficiency, TRIF deficiency does not result in diminished control of infection with B. burgdorferi in a murine model of disease. This appears to be due to the effects of MyD88 on phagocytosis via scavenger receptors, such as MARCO, which are not affected by the loss of TRIF. In mice, TRIF deficiency did have an effect on the production of inflammatory cytokines, suggesting that regulation of inflammatory cytokines and control of bacterial growth may be uncoupled, in part through transduction of TLR2 signaling through TRIF.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/imunologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Borrelia burgdorferi/imunologia , Inflamação/imunologia , Doença de Lyme/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Animais , Inflamação/metabolismo , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Ligantes , Doença de Lyme/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Fagocitose/imunologia , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Transdução de Sinais/imunologia
8.
Curr Opin Clin Nutr Metab Care ; 15(4): 323-9, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22669047

RESUMO

PURPOSE OF REVIEW: Epigenetic modifications are heritable alterations of the genome, which can govern gene expression without altering the DNA sequence. The purpose of this review is to render an overview of the possible mechanisms of epigenetic regulation of gene expression in response to environmental pollutants leading to cardiovascular diseases (CVD). RECENT FINDINGS: An era of cataloging epigenetic marks of the various diseased states has recently commenced, including those within the genes responsible for atherosclerosis, ischemia, hypertension and heart failure. From varied study approaches directed either toward the general understanding of the key pathway regulatory genes, or sampling population cohorts for global and gene-specific changes, it has been possible to identify several epigenetic signatures of environmental exposure relevant to CVD. Signatures of epigenetic dysregulation can be detected in peripheral blood samples, even within a few hours of environmental exposure. However, the field now faces the demand for thorough, systematic, rationalized approaches to establish the relation of exposure-driven epigenetic changes to clinical outcomes, by using sophisticated and reliable research designs and tools. SUMMARY: An understanding of chromatin remodelling in response to environmental stimuli conducive to CVD is emerging, with the promise of novel diagnostic and therapeutic candidates.


Assuntos
Doenças Cardiovasculares/genética , Exposição Ambiental/efeitos adversos , Poluentes Ambientais/efeitos adversos , Epigênese Genética , Regulação da Expressão Gênica , Interação Gene-Ambiente , Cromatina , Epigenômica , Humanos
9.
Am J Respir Cell Mol Biol ; 45(5): 1036-44, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21562316

RESUMO

Lung macrophages use the scavenger receptor MARCO to bind and ingest bacteria, particulate matter, and post cellular debris. We investigated the role of MARCO in influenza A virus (IAV) pneumonia. In contrast to higher susceptibility to bacterial infection, MARCO(-/-) mice had lower morbidity and mortality from influenza pneumonia than wild-type (WT) mice. The early course of influenza in MARCO(-/-) lungs was marked by an enhanced but transient neutrophilic inflammatory response and significantly lower viral replication compared with the WT mice. At later time points, no significant differences in lung histopathology or absolute numbers of T lymphocyte influx were evident. Uptake of IAV by WT and MARCO(-/-) bronchoalveolar lavage macrophages in vitro was similar. By LPS coadministration, we demonstrated that rapid neutrophil and monocyte influx during the onset of influenza suppressed viral replication, indicating a protective role of early inflammation. We hypothesized that the presence of increased basal proinflammatory post cellular debris in the absence of scavenging function lowered the inflammatory response threshold to IAV in MARCO(-/-) mice. Indeed, MARCO(-/-) mice showed increased accumulation of proinflammatory oxidized lipoproteins in the bronchoalveolar lavage early in the infection process, which are the potential mediators of the observed enhanced inflammation. These results indicate that MARCO suppresses a protective early inflammatory response to influenza, which modulates viral clearance and delays recovery.


Assuntos
Macrófagos Alveolares/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Pneumonia Viral/metabolismo , Receptores Imunológicos/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/química , Vírus da Influenza A/metabolismo , Vírus da Influenza A/fisiologia , Lipopolissacarídeos/toxicidade , Lipoproteínas/análise , Lipoproteínas/metabolismo , Macrófagos Alveolares/patologia , Macrófagos Alveolares/virologia , Masculino , Camundongos , Neutrófilos/metabolismo , Neutrófilos/patologia , Neutrófilos/virologia , Infecções por Orthomyxoviridae/patologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Receptores Imunológicos/genética , Replicação Viral
10.
Am J Respir Cell Mol Biol ; 45(1): 111-9, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20870897

RESUMO

Airway smooth muscle (ASM) cells have been reported to contribute to the inflammation of asthma. Because the thiazolidinediones (TZDs) exert anti-inflammatory effects, we examined the effects of troglitazone and rosiglitazone on the release of inflammatory moieties from cultured human ASM cells. Troglitazone dose-dependently reduced the IL-1ß-induced release of IL-6 and vascular endothelial growth factor, the TNF-α-induced release of eotaxin and regulated on activation, normal T expressed and secreted (RANTES), and the IL-4-induced release of eotaxin. Rosiglitazone also inhibited the TNF-α-stimulated release of RANTES. Although TZDs are known to activate peroxisome proliferator-activated receptor-γ (PPARγ), these anti-inflammatory effects were not affected by a specific PPARγ inhibitor (GW 9662) or by the knockdown of PPARγ using short hairpin RNA. Troglitazone and rosiglitazone each caused the activation of adenosine monophosphate-activated protein kinase (AMPK), as detected by Western blotting using a phospho-AMPK antibody. The anti-inflammatory effects of TZDs were largely mimicked by the AMPK activators, 5-amino-4-imidazolecarboxamide ribose (AICAR) and metformin. However, the AMPK inhibitors, Ara A and Compound C, were not effective in preventing the anti-inflammatory effects of troglitazone or rosiglitzone, suggesting that the effects of these TZDs are likely not mediated through the activation of AMPK. These data indicate that TZDs inhibit the release of a variety of inflammatory mediators from human ASM cells, suggesting that they may be useful in the treatment of asthma, and the data also indicate that the effects of TZDs are not mediated by PPARγ or AMPK.


Assuntos
Anti-Inflamatórios/farmacologia , Cromanos/farmacologia , Miócitos de Músculo Liso/metabolismo , Sistema Respiratório/metabolismo , Tiazolidinedionas/farmacologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Anilidas/farmacologia , Antimetabólitos/farmacologia , Asma/tratamento farmacológico , Asma/genética , Asma/metabolismo , Asma/patologia , Células Cultivadas , Citocinas/biossíntese , Citocinas/genética , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Hipoglicemiantes/farmacologia , Mediadores da Inflamação/metabolismo , Metformina/farmacologia , Miócitos de Músculo Liso/patologia , PPAR gama/antagonistas & inibidores , PPAR gama/genética , PPAR gama/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Sistema Respiratório/patologia , Ribonucleotídeos/farmacologia , Rosiglitazona , Troglitazona , Vidarabina/farmacologia
11.
Am J Pathol ; 176(3): 1484-93, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20093488

RESUMO

Inflammation may play a role in the etiology of both degenerative and rheumatic cardiac valve diseases. We report here that mice deficient in tristetraprolin (TTP), a protein with known anti-inflammatory functions, develop severe left-sided cardiac valvulitis. TTP is an mRNA binding protein that inhibits inflammation by destabilizing the mRNA encoding tumor necrosis factor alpha (TNF). This leads in turn to a TNF-excess syndrome characterized by systemic inflammation. Evaluation of hearts from TTP-/- mice demonstrated gross thickening of the mitral and aortic but not the tricuspid or pulmonary valves, accompanied by inflammatory cell infiltrates. To determine whether TNF played a role in the development of this valvulitis, we examined mice deficient in both TNF receptors and in TTP; four of five of these mice exhibited no histological evidence of valvulitis, but one mouse had aortic valve leaflet thickening with a cellular infiltrate. Four additional mice had no external evidence of valvular thickening. Cardiac valves of transgenic mice expressing human TNF developed mild aortic valve leaflet edema without evidence of hypercellularity. Thus, TTP deficiency in mice leads to left-sided cardiac valvulitis with prominent inflammatory cell involvement, due, at least in part, to excess TNF. These findings support the potential involvement of TNF and inflammation in the development of cardiac valve disease in man.


Assuntos
Doenças das Valvas Cardíacas/metabolismo , Doenças das Valvas Cardíacas/patologia , Tristetraprolina/deficiência , Fator de Necrose Tumoral alfa/metabolismo , Animais , Regulação da Expressão Gênica , Doenças das Valvas Cardíacas/diagnóstico por imagem , Valvas Cardíacas/metabolismo , Valvas Cardíacas/patologia , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/deficiência , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Tristetraprolina/metabolismo , Fator de Necrose Tumoral alfa/genética , Ultrassonografia
12.
Biotechnol Prog ; 24(2): 326-33, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18302406

RESUMO

Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) family proteins are anti-inflammatory. They bind and destabilize some AU-rich element-containing mRNAs such as tumor necrosis factor mRNA. In this study, recombinant ZFP36L1/TIS11B (a TTP homologue) was overexpressed in E. coli, purified, and used for polyclonal antibody production in rabbits. The antiserum recognized nanograms of the antigen on immunoblots. This antiserum and another antiserum developed against recombinant mouse TTP were used to detect ZFP36L1 and TTP in mouse 3T3-L1 adipocytes and RAW264.7 macrophages. Immunoblotting showed that ZFP36L1 was stably expressed with a size corresponding to the lower mass size of ZFP36L1 expressed in transfected human embryonic kidney 293 cells, but TTP was induced by cinnamon extract and not by lipopolysaccharide (LPS) in adipocytes. In contrast, ZFP36L1 was undetectable, but TTP was strongly induced in LPS-stimulated RAW cells. Quantitative real-time polymerase chain reaction confirmed the higher levels of ZFP36L1 mRNA in adipocytes and TTP mRNA in RAW cells. Low levels of ZFP36L1 expression were also confirmed by Northern blotting in mouse embryonic fibroblasts. These results demonstrate that ZFP36L1 antiserum is useful in the detection of this protein and that TTP and ZFP36L1 are differentially expressed and regulated at the mRNA and protein levels in mouse adipocytes and macrophages.


Assuntos
Anticorpos/imunologia , Escherichia coli/genética , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Células 3T3 , Adipócitos/metabolismo , Animais , Fator 1 de Resposta a Butirato , Linhagem Celular , Primers do DNA , DNA Complementar/biossíntese , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Macrófagos/metabolismo , Camundongos , Proteínas Nucleares/biossíntese , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNA/biossíntese , Coelhos , Proteínas Recombinantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tristetraprolina/biossíntese , Tristetraprolina/genética
13.
Glycoconj J ; 23(9): 675-86, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17115274

RESUMO

Various species of Mycobacteria produce a major cell wall-associated lipoglycan, called Lipoarabinomannan (LAM), which is involved in the virulence of Mycobacterial species. In this study, we tried to establish the role of the increased IL-10 secretion under Arabinosylated-LAM (Ara-LAM) treatment, the LAM that induces apoptosis in host macrophages or PBMC. We have studied the survival and apoptotic factors by western blotting, and estimated nitrite generation by Griess reaction, quantified iNOS mRNA by semi-quantitative RT-PCR, and ultimately the fate of the cells were studied by Flow Cytometric Analysis of AnnexinV-FITC binding. As per our observations, neutralization of released IL-10 in C57BL/6 peritoneal macrophages prior to Ara-LAM treatment, as well as macrophages from IL-10 knockout (KO) mice treated with Ara-LAM, showed significant down regulation of pro-apoptotic factors and up regulation of survival factors. These effects were strikingly similar to those when peritoneal macrophages were subjected to TNF-alpha and IL-12 neutralization followed by Ara-LAM-treatment. However, under similar conditions virulent Mannosylated-LAM (from Mycobacterium tuberculosis) treatment of macrophages clearly depicts the importance of IL-10 in the maintenance of pathogenesis, proving its usual immunosuppressive role. Thus, from our detailed investigations we point out an unusual pro-inflammatory action of IL-10 in Ara-LAM treated macrophages, where it behaves in a similar manner as the known Th1 cytokines TNF- alpha and IL-12.


Assuntos
Interleucina-10/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/imunologia , Animais , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Macrófagos Peritoneais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/análise , Nitritos/análise , Nitritos/metabolismo , RNA Mensageiro/análise
14.
Biochemistry ; 44(15): 5923-30, 2005 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-15823052

RESUMO

While the hydrophobic driving force is thought to be a major contributor to protein stability, it is difficult to experimentally dissect out its contribution to the overall free energy of folding. We have made large to small substitutions of buried hydrophobic residues at positions 8 and 13 in the peptide/protein complex, RNase-S, and have characterized the structures by X-ray crystallography. The thermodynamics of association of these mutant S peptides with S protein was measured in the presence of different concentrations of methanol and ethanol. The reduction in the strength of the hydrophobic driving force in the presence of these organic solvents was estimated from surface-tension data as well as from the dependence of the DeltaC(p) of protein/peptide binding on the alcohol concentration. The data indicated a decrease in the strength of the hydrophobic driving force of about 30-40% over a 0-30% range of the alcohol concentration. We observe that large to small substitutions destabilize the protein. However, the amount of destabilization, relative to the wild type, is independent of the alcohol concentration over the range of alcohol concentrations studied. The data clearly indicate that decreased stability of the mutants is primarily due to the loss of packing interactions rather than a reduced hydrophobic driving force and suggest a value of the hydrophobic driving force of less than 18 cal mol(-)(1) A(2).


Assuntos
Ribonucleases/química , Ribonucleases/genética , Animais , Bovinos , Dicroísmo Circular , Cristalografia por Raios X , Estabilidade Enzimática , Interações Hidrofóbicas e Hidrofílicas , Técnicas In Vitro , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Eletricidade Estática , Termodinâmica
15.
Biol Reprod ; 73(2): 297-307, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15814898

RESUMO

Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger (TZF) proteins can bind directly to AU-rich elements (ARE) in mRNA, causing deadenylation and destabilization of the transcripts to which they bind. We describe here a novel fourth mammalian member of the TTP protein family, designated ZFP36L3, which could also bind directly to ARE-containing RNAs and could promote the deadenylation and degradation of ARE-containing target RNAs. Zfp36l3 transcript expression was detected only in placenta and extraembryonic tissues in the mouse. It was expressed throughout development in the placenta and was particularly highly expressed in the cells of the labyrinthine layer of the trophoblastic placenta. Unlike the other family members, the expression of a ZFP36L3-green fluorescent protein fusion protein was entirely cytoplasmic when expressed in 293 cells, even in the presence of the CRM1-dependent nuclear export inhibitor leptomycin B. Zfp36l3 was located on the mouse X chromosome; a similar predicted gene was present on the rat X chromosome, but there was no evidence for a similar gene in humans. ZFP36L3 may thus be a rodent-specific or even murine-specific member of the TTP protein family. Its presumed role in placental physiology may be unique to rodents or murine rodents, but this role may be subsumed by other family members in nonrodents.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Imediatamente Precoces/genética , Placenta/fisiologia , Dedos de Zinco/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Proteínas Imediatamente Precoces/biossíntese , Proteínas Imediatamente Precoces/metabolismo , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Placenta/metabolismo , Gravidez , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Sequências de Repetição em Tandem , Transfecção , Tristetraprolina , Fator de Necrose Tumoral alfa/metabolismo , Cromossomo X/genética
16.
Mol Cell Biol ; 24(14): 6445-55, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15226444

RESUMO

The mouse gene Zfp36L1 encodes zinc finger protein 36-like 1 (Zfp36L1), a member of the tristetraprolin (TTP) family of tandem CCCH finger proteins. TTP can bind to AU-rich elements within the 3'-untranslated regions of the mRNAs encoding tumor necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), leading to accelerated mRNA degradation. TTP knockout mice exhibit an inflammatory phenotype that is largely due to increased TNF secretion. Zfp36L1 has activities similar to those of TTP in cellular RNA destabilization assays and in cell-free RNA binding and deadenylation assays, suggesting that it may play roles similar to those of TTP in mammalian physiology. To address this question we disrupted Zfp36L1 in mice. All knockout embryos died in utero, most by approximately embryonic day 11 (E11). Failure of chorioallantoic fusion occurred in about two-thirds of cases. Even when fusion occurred, by E10.5 the affected placentas exhibited decreased cell division and relative atrophy of the trophoblast layers. Although knockout embryos exhibited neural tube abnormalities and increased apoptosis within the neural tube and also generalized runting, these and other findings may have been due to deficient placental function. Embryonic expression of Zfp36L1 at E8.0 was greatest in the allantois, consistent with a potential role in chorioallantoic fusion. Fibroblasts derived from knockout embryos had apparently normal levels of fully polyadenylated compared to deadenylated GM-CSF mRNA and normal rates of turnover of this mRNA species, both sensitive markers of TTP deficiency in cells. We postulate that lack of Zfp36L1 expression during mid-gestation results in the abnormal stabilization of one or more mRNAs whose encoded proteins lead directly or indirectly to abnormal placentation and fetal death.


Assuntos
Alantoide/metabolismo , Córion/metabolismo , Proteínas de Ligação a DNA , Embrião de Mamíferos/fisiologia , Proteínas Imediatamente Precoces/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Dedos de Zinco , Alantoide/anatomia & histologia , Alantoide/patologia , Animais , Fator 1 de Resposta a Butirato , Células Cultivadas , Córion/anatomia & histologia , Córion/patologia , Embrião de Mamíferos/anatomia & histologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Marcação de Genes , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Família Multigênica , Proteínas Nucleares , Fenótipo , Placenta/citologia , Placenta/metabolismo , Placenta/patologia , Estabilidade de RNA , RNA Mensageiro/metabolismo , Distribuição Tecidual , Tristetraprolina
17.
Infect Immun ; 70(12): 6828-38, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12438359

RESUMO

In vitro infection of murine peritoneal macrophages with the protozoan Leishmania donovani has been found to alter the signaling parameters of the host. The present study indicates that the enhancement of intracellular ceramide level in macrophages after infection is a major event relating to macrophage dysfunction. We have previously demonstrated that increased ceramide synthesis in host macrophages was involved in the dephosphorylation of extracellular signal-regulated kinase (ERK). In the present study, we further show that downregulation of ERK by ceramide was found to be associated with the inhibition of activated protein 1 (AP-1) and NF-kappaB transactivation. Pharmacological inhibition of ceramide synthesis by Fumonisin B1 restored the induction of AP-1 and NF-kappaB DNA-binding activities in infected BALB/c macrophages. On the contrary, in the case of macrophages from the leishmaniasis-resistant C.D2 mice, L. donovani failed to induce sustained ceramide synthesis. Enhanced mitogen-activated protein kinase phosphorylation, AP-1 and NF-kappaB DNA-binding activity, and the generation of nitric oxide (NO) were observed in L. donovani-infected C.D2 macrophages. ERK activation was necessary for the activation of transcription factors AP-1 and NF-kappaB, NO generation, and restriction of the parasite burden in the resistant murine host macrophages. Hence, the induction of ceramide synthesis in host macrophages appears to be instrumental and one of the turning points leading to silencing of the macrophage antileishmanial responses.


Assuntos
Ceramidas/metabolismo , Leishmania donovani/patogenicidade , Sistema de Sinalização das MAP Quinases , Macrófagos Peritoneais/parasitologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Células Cultivadas , Ceramidas/farmacologia , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/fisiopatologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , NF-kappa B/metabolismo , Fator de Transcrição AP-1/metabolismo
18.
J Infect Dis ; 185(12): 1704-8, 2002 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-12085314

RESUMO

This study explored the role of the proinflammatory chemokines macrophage inflammatory protein (MIP)-1alpha and macrophage chemoattractant protein (MCP)-1 for development of antileishmanial activity. There was substantial inhibition in nitrite generation in Leishmania donovani-infected macrophages. A marked elevation of nitrite generation and induction of inducible nitric oxide (NO) synthase (iNOS) mRNA was found in chemokine-primed parasite-infected macrophages. Tumor necrosis factor-alpha, which is the priming signal for NO production, was also up-regulated under similar experimental conditions. The priming with chemokine inhibited the multiplication of L. donovani amastigotes within the intramacrophageal milieu. The antileishmanial effect of chemokines was almost completely abrogated when the macrophages were preincubated with l-N-monomethyl arginine, the specific inhibitor of iNOS. The results of this investigation suggest that the CC chemokines MIP-1alpha and MCP-1 orchestrate an antileishmanial armamentarium via the induction of an NO-mediated regulatory mechanism to control the intracellular growth and multiplication of the Leishmania protozoan.


Assuntos
Quimiocina CCL2/fisiologia , Leishmania donovani , Proteínas Inflamatórias de Macrófagos/fisiologia , Macrófagos Peritoneais/metabolismo , Óxido Nítrico/biossíntese , Animais , Quimiocina CCL3 , Quimiocina CCL4 , Indução Enzimática , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Nitritos/metabolismo , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima , ômega-N-Metilarginina/farmacologia
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