RESUMO
Leprosy, caused by Mycobacterium leprae, rarely affects children younger than 5 years. Here, we studied a multiplex leprosy family that included monozygotic twins aged 22 months suffering from paucibacillary leprosy. Whole genome sequencing identified three amino acid mutations previously associated with Crohn's disease and Parkinson's disease as candidate variants for early onset leprosy: LRRK2 N551K, R1398H and NOD2 R702W. In genome-edited macrophages, we demonstrated that cells expressing the LRRK2 mutations displayed reduced apoptosis activity following mycobacterial challenge independently of NOD2. However, employing co-immunoprecipitation and confocal microscopy we showed that LRRK2 and NOD2 proteins interacted in RAW cells and monocyte-derived macrophages, and that this interaction was substantially reduced for the NOD2 R702W mutation. Moreover, we observed a joint effect of LRRK2 and NOD2 variants on Bacillus Calmette-Guérin (BCG)-induced respiratory burst, NF-κB activation and cytokine/chemokine secretion with a strong impact for the genotypes found in the twins consistent with a role of the identified mutations in the development of early onset leprosy.
Assuntos
Predisposição Genética para Doença , Hanseníase , Criança , Humanos , Alelos , Genótipo , Hanseníase/genética , Mutação , Proteína Adaptadora de Sinalização NOD2/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genéticaRESUMO
Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) are gastrointestinal pathogens responsible for severe diarrheal illness. EHEC and EPEC form "attaching and effacing" lesions during colonization and, upon adherence, inject proteins directly into host intestinal cells via the type III secretion system (T3SS). Injected bacterial proteins have a variety of functions but generally alter host cell biology to favor survival and/or replication of the pathogen. Non-LEE-encoded effector A (NleA) is a T3SS-injected effector of EHEC, EPEC, and the related mouse pathogen Citrobacter rodentium. Studies in mouse models indicate that NleA has an important role in bacterial virulence. However, the mechanism by which NleA contributes to disease remains unknown. We have determined that the following translocation into host cells, a serine and threonine-rich region of NleA is modified by host-mediated mucin-type O-linked glycosylation. Surprisingly, this region was not present in several clinical EHEC isolates. When expressed in C. rodentium, a non-modifiable variant of NleA was indistinguishable from wildtype NleA in an acute mortality model but conferred a modest increase in persistence over the course of infection in mixed infections in C57BL/6J mice. This is the first known example of a bacterial effector being modified by host-mediated O-linked glycosylation. Our data also suggests that this modification may confer a selective disadvantage to the bacteria during in vivo infection.
Assuntos
Escherichia coli Enteropatogênica , Proteínas de Escherichia coli , Humanos , Animais , Camundongos , Fatores de Virulência/metabolismo , Células HeLa , Glicosilação , Proteínas de Escherichia coli/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Persons living with HIV (PLWH) are at increased risk of tuberculosis (TB). HIV-associated TB is often the result of recent infection with Mycobacterium tuberculosis (M. tuberculosis) followed by rapid progression to disease. Alveolar macrophages (AMs) are the first cells of the innate immune system that engage M. tuberculosis, but how HIV and antiretroviral therapy (ART) affect the anti-mycobacterial response of AMs is not known. To investigate the impact of HIV and ART on the transcriptomic and epigenetic response of AMs to M. tuberculosis, we obtained AMs by bronchoalveolar lavage from 20 PLWH receiving ART, 16 control subjects who were HIV-free (HC), and 14 subjects who received ART as preexposure prophylaxis (PrEP) to prevent HIV infection. Following in vitro challenge with M. tuberculosis, AMs from each group displayed overlapping but distinct profiles of significantly up- and downregulated genes in response to M. tuberculosis. Comparatively, AMs isolated from both PLWH and PrEP subjects presented a substantially weaker transcriptional response. In addition, AMs from HC subjects challenged with M. tuberculosis responded with pronounced chromatin accessibility changes while AMs obtained from PLWH and PrEP subjects displayed no significant changes in their chromatin state. Collectively, these results revealed a stronger adverse effect of ART than HIV on the epigenetic landscape and transcriptional responsiveness of AMs.
Assuntos
Epigênese Genética , Infecções por HIV/imunologia , Macrófagos Alveolares/imunologia , Mycobacterium tuberculosis/imunologia , Adulto , Idoso , Antirretrovirais/efeitos adversos , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Macrófagos Alveolares/metabolismo , Masculino , Pessoa de Meia-Idade , Profilaxia Pré-Exposição , TranscriptomaRESUMO
Type-1 reactions (T1R) are pathological inflammatory episodes and main contributors to nerve damage in leprosy. Here, we evaluate the genewise enrichment of rare protein-altering variants in 7 genes where common variants were previously associated with T1R. We selected 474 Vietnamese leprosy patients of which 237 were T1R-affected and 237 were T1R-free matched controls. Genewise enrichment of nonsynonymous variants was tested with both kernel-based (sequence kernel association test [SKAT]) and burden methods. Of the 7 genes tested 2 showed statistical evidence of association with T1R. For the LRRK2 gene an enrichment of nonsynonymous variants was observed in T1R-free controls (PSKAT-O = 1.6 × 10-4). This genewise association was driven almost entirely by the gain-of-function variant R1628P (P = 0.004; odds ratio = 0.29). The second genewise association was found for the Parkin coding gene PRKN (formerly PARK2) where 7 rare variants were enriched in T1R-affected cases (PSKAT-O = 7.4 × 10-5). Mutations in both PRKN and LRRK2 are known causes of Parkinson's disease (PD). Hence, we evaluated to what extent such rare amino acid changes observed in T1R are shared with PD. We observed that amino acids in Parkin targeted by nonsynonymous T1R-risk mutations were also enriched for mutations implicated in PD (P = 1.5 × 10-4). Hence, neuroinflammation in PD and peripheral nerve damage due to inflammation in T1R share overlapping genetic control of pathogenicity.
Assuntos
Hanseníase , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Mutação , Doença de Parkinson , Ubiquitina-Proteína Ligases , Feminino , Humanos , Hanseníase/genética , Hanseníase/metabolismo , Hanseníase/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Masculino , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Studies have demonstrated that the solute carrier family 11 member 1 (SLC11A1) is heavily glycosylated and phosphorylated in macrophages. However, the mechanisms of SLC11A1 phosphorylation, and the effects of phosphorylation on SLC11A1 activity remain largely unknown. Here, the tyrosine phosphorylation of SLC11A1 is observed in SLC11A1-expressing U937 cells when differentiated into macrophages by phorbol myristate acetate (PMA). The phosphorylation of SLC11A1 is almost completely blocked by treatment with PP2, a selective inhibitor of Src family kinases. Furthermore, we found that SLC11A1 is a direct substrate for active c-Src kinase and siRNA-mediated knockdown of cellular Src (c-Src) expression results in a significant decrease in tyrosine phosphorylation. We found that PMA induces the interaction of SLC11A1 with c-Src kinase. We demonstrated that SLC11A1 is phosphorylated by Src family kinases at tyrosine 15 and this type of phosphorylation is required for SLC11A1-mediated modulation of NF-κB activation and nitric oxide (NO) production induced by LPS. Our results demonstrate important roles for c-Src tyrosine kinase in phosphorylation and activation of SLC11A1 in macrophages.
Assuntos
Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Diferenciação Celular , Macrófagos/citologia , Tirosina/metabolismo , Quinases da Família src/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteína Tirosina Quinase CSK , Linhagem Celular Tumoral , Humanos , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , FosforilaçãoRESUMO
Promoter deletion analysis is a useful tool for identifying important regulatory regions involved in transcriptional control of gene expression. In this approach, a series of promoter deletion fragments are fused to a reporter gene, such as chloramphenicol acetyltransferase or luciferase gene in a vector, and then transfected into cells for induction. Screening the expression level of the reporter gene using either a qualitative or a quantitative assay, allows to identify the regulatory regions of interest (e.g., cis-acting elements or enhancer) in the promoter.Luciferase genes have been widely used as reporter genes for their sensitivity and efficiency. Firefly and Renilla luciferases are two commonly used reporters, which oxidize different substrates to generate quantifiable luminescence. Therefore, the enzymatic activities of firefly and Renilla luciferases can be sequentially measured in a single sample by controlling reaction conditions. Here, we describe a dual-luciferase reporter assay, where the promoter of interest is fused to a firefly luciferase reporter and is co-transfected into cells with an internal control vector (pRL-CMV) to express Renilla luciferase. Both the Firefly and Renilla luciferases are measured using a dual-luciferase reporter assay system which improves experimental accuracy.
Assuntos
Clonagem Molecular/métodos , Genes Reporter , Luciferases de Vaga-Lume/genética , Luciferases de Renilla/genética , Regiões Promotoras Genéticas , Extratos Celulares/química , Ensaios Enzimáticos , Deleção de Genes , Células HL-60 , Humanos , Luciferases de Vaga-Lume/biossíntese , Luciferases de Renilla/biossíntese , Plasmídeos/genética , TransfecçãoRESUMO
BACKGROUND: It is mandatory to confirm the absence of mutations in the KRAS gene before treating metastatic colorectal cancers with epidermal growth factor receptor inhibitors, and similar regulations are being considered for non-small cell lung carcinomas (NSCLC) and other tumor types. Routine diagnosis of KRAS mutations in NSCLC is challenging because of compromised quantity and quality of biological material. Although there are several methods available for detecting mutations in KRAS, there is little comparative data regarding their analytical performance, economic merits, and workflow parameters. METHODS: We compared the specificity, sensitivity, cost, and working time of five methods using 131 frozen NSCLC tissue samples. We extracted genomic DNA from the samples and compared the performance of Sanger cycle sequencing, Pyrosequencing, High-resolution melting analysis (HRM), and the Conformité Européenne (CE)-marked TheraScreen DxS and K-ras StripAssay kits. RESULTS AND CONCLUSIONS: Our results demonstrate that TheraScreen DxS and the StripAssay, in that order, were most effective at diagnosing mutations in KRAS. However, there were still unsatisfactory disagreements between them for 6.1% of all samples tested. Despite this, our findings are likely to assist molecular biologists in making rational decisions when selecting a reliable, efficient, and cost-effective method for detecting KRAS mutations in heterogeneous clinical tumor samples.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Colorretais , Proteínas Proto-Oncogênicas/genética , Análise de Sequência de DNA , Proteínas ras/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/secundário , Análise Custo-Benefício , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Humanos , Mutação , Proteínas Proto-Oncogênicas p21(ras) , Sensibilidade e Especificidade , Análise de Sequência de DNA/economia , Análise de Sequência de DNA/métodosRESUMO
The solute carrier family 11 member 1 (SLC11A1) gene is strictly regulated and exclusively expressed in myeloid lineage cells. However, little is known about the transcriptional regulation of the SLC11A1 gene during myeloid development. In this study, we used HL-60 cells as a model to investigate the regulatory elements/factors involved in the transactivation of the SLC11A1 gene during phorbol 12-myristate 13-acetate (PMA)-induced macrophage differentiation of HL-60 cells. Promoter deletion analysis showed that a 7-base AP-1-like element (TGACTCT) was critical for the responsiveness of the SLC11A1 promoter to PMA. Stimulation by PMA induced the binding of ATF-3 and the recruitment of two components of the SWI/SNF complex, BRG1 and ß-actin, to this element in an ATF-3-dependent manner. RNAi-mediated depletion of ATF-3 or BRG1 markedly decreased SLC11A1 gene expression and its promoter activity induced by PMA. Luciferase reporter experiments demonstrated that ATF-3 cooperated with BRG1 and ß-actin to activate the SLC11A1 promoter. Furthermore, we showed that PMA can induce the proximal (GT/AC)(n) repeat sequence to convert to the Z-DNA structure in the SLC11A1 gene promoter, and depletion of BRG1 resulted in a significant decrease of Z-DNA formation. Our results demonstrated that recruitment of the SWI/SNF complex initiated Z-DNA formation and subsequently helped to transactivate the SLC11A1 gene.
Assuntos
Proteínas de Transporte de Cátions/biossíntese , Diferenciação Celular/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , DNA Forma Z/metabolismo , Macrófagos/metabolismo , Elementos de Resposta/fisiologia , Fatores de Transcrição/metabolismo , Ativação Transcricional/fisiologia , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Carcinógenos/farmacologia , Proteínas de Transporte de Cátions/genética , Diferenciação Celular/efeitos dos fármacos , Proteínas Cromossômicas não Histona/genética , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Forma Z/genética , Células HL-60 , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/genética , Ativação Transcricional/efeitos dos fármacosRESUMO
Secretory leukocyte protease inhibitor (SLPI) is an anti-inflammatory protein that is observed at high levels in asthma patients. Resiquimod, a TLR7/8 ligand, is protective against acute and chronic asthma, and it increases SLPI expression of macrophages in vitro. However, the protective role played by SLPI and the interactions between the SLPI and resiquimod pathways in the immune response occurring in allergic asthma have not been fully elucidated. To evaluate the role of SLPI in the development of asthma phenotypes and the effect of resiquimod treatment on SLPI, we assessed airway resistance and inflammatory parameters in the lungs of OVA-induced asthmatic SLPI transgenic and knockout mice and in mice treated with resiquimod. Compared with wild-type mice, allergic SLPI transgenic mice showed a decrease in lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), and plasma IgE levels (p < 0.001). Allergic SLPI knockout mice displayed phenotype changes significantly more severe compared with wild-type mice. These phenotypes included lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), cytokine levels in the lungs (p < 0.05), and plasma IgE levels (p < 0.001). Treatment of asthmatic transgenic mice with resiquimod increased the expression of SLPI and decreased inflammation in the lungs; resiquimod treatment was still effective in asthmatic SLPI knockout mice. Taken together, our study showed that the expression of SLPI protects against allergic asthma phenotypes, and treatment by resiquimod is independent of SLPI expression, displayed through the use of transgenic and knockout SLPI mice.
Assuntos
Alérgenos/administração & dosagem , Asma/enzimologia , Asma/imunologia , Inibidor Secretado de Peptidases Leucocitárias/fisiologia , Doença Aguda , Animais , Asma/patologia , Hiper-Reatividade Brônquica/enzimologia , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/fisiopatologia , Movimento Celular/genética , Movimento Celular/imunologia , Modelos Animais de Doenças , Inflamação/enzimologia , Inflamação/genética , Inflamação/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Inibidor Secretado de Peptidases Leucocitárias/biossíntese , Inibidor Secretado de Peptidases Leucocitárias/deficiênciaRESUMO
BACKGROUND: Secreted protein, acidic and rich in cysteine (SPARC) is a matricellular protein that mediates cell-matrix interactions. It has been shown, depending on the type of cancer, to possess either pro- or anti-tumorigenic properties. The transcriptional regulation of the SPARC gene expression has not been fully elucidated and the effects of anti-cancer drugs on this process have not been explored. RESULTS: In the present study, we demonstrated that chromatin remodeling factor Brg-1 is recruited to the proximal SPARC promoter region (-130/-56) through an interaction with transcription factor Sp1. We identified Brg-1 as a critical regulator for the constitutive expression levels of SPARC mRNA and protein in mammary carcinoma cell lines and for SPARC secretion into culture media. Furthermore, we found that Brg-1 cooperates with Sp1 to enhance SPARC promoter activity. Interestingly, fenretinide [N-4(hydroxyphenyl) retinamide, 4-HPR], a synthetic retinoid with anti-cancer properties, was found to up-regulate the transcription, expression and secretion of SPARC via induction of the Brg-1 in a dose-dependent manner. Finally, our results demonstrated that fenretinide-induced expression of SPARC contributes significantly to a decreased invasion of mammary carcinoma cells. CONCLUSIONS: Overall, our results reveal a novel cooperative role of Brg-1 and Sp1 in mediating the constitutive and fenretinide-induced expression of SPARC, and provide new insights for the understanding of the anti-cancer effects of fenretinide.
Assuntos
DNA Helicases/fisiologia , Fenretinida/farmacologia , Neoplasias Mamárias Experimentais/patologia , Proteínas Nucleares/fisiologia , Osteonectina/genética , Fator de Transcrição Sp1/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , DNA , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/fisiopatologia , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Células Tumorais CultivadasRESUMO
Studies have shown that nuclear translocation of actin occurs under certain conditions of cellular stress; however, the functional significance of actin import remains unclear. Here, we demonstrate that during the phorbol 12-myristate 13-acetate (PMA)-induced differentiation of HL-60 cells toward macrophages, beta-actin translocates from the cytoplasm to the nucleus and that this process is dramatically inhibited by pretreatment with p38 mitogen-activated protein kinase inhibitors. Using chromatin immunoprecipitation-on-chip assays, the genome-wide maps of beta-actin binding to gene promoters in response to PMA treatment is analyzed in HL-60 cells. A gene ontology-based analysis shows that the identified genes belong to a broad spectrum of functional categories such as cell growth and differentiation, signal transduction, response to external stimulus, ion channel activity, and immune response. We also demonstrate a correlation between beta-actin occupancy and the recruitment of RNA polymerase II at six selected target genes, and beta-actin knockdown decreases the mRNA expression levels of these target genes induced by PMA. We further show that nuclear beta-actin is required for PMA-induced transactivation of one target gene, solute carrier family 11 member 1, which is important for macrophage activation. Our data provide novel evidence that nuclear accumulation of beta-actin is involved in transcriptional regulation during macrophage-like differentiation of HL-60 cells.
Assuntos
Actinas/metabolismo , Transporte Ativo do Núcleo Celular , Regulação da Expressão Gênica , Macrófagos/metabolismo , Transcrição Gênica , Diferenciação Celular , Células HL-60 , Humanos , Hibridização In Situ , Monócitos/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Ativação TranscricionalRESUMO
Cutaneous wound healing is a complex process, which is heavily dependent on successful inflammatory action. Mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MAPKAPK-2 or MK2), a major substrate of p38 MAPK, has been shown to be a major player in multiple inflammatory diseases, but its role in cutaneous wound healing has not yet been explored. In this study, by comparing excisional wounds made on the backs of MK2 knockout (KO) and MK2 wild-type (WT) mice, we found that the kinetics of wound healing are significantly affected by the absence of MK2 (P=0.010 to P<0.001). Histological examination showed a higher level of acanthosis of the migrating wound keratinocyte layer as well as a higher level of collagen deposition in the granulation tissue of the wounds from MK2 WT mice compared with those from MK2 KO mice. Interestingly, although MK2 did not influence macrophage and neutrophil infiltration of the wounds, the expression of many cytokines and chemokines was significantly affected at different days post wounding. Furthermore, the delayed healing rate of wounds in MK2 KO mice can be significantly improved by passive transfer of macrophages with intact MK2. Overall, these results show a critical role for MK2 gene expression in macrophages participating in the process of cutaneous wound healing.
Assuntos
Dermatite/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Cicatrização/fisiologia , Animais , Colágeno/genética , Colágeno/metabolismo , Citocinas/metabolismo , Dermatite/imunologia , Dermatite/patologia , Feminino , Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Queratinócitos/fisiologia , Macrófagos/imunologia , Macrófagos/transplante , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica/fisiologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/metabolismoRESUMO
The models on direct N2O emissions from rice paddies under different water regimes developed by the authors were validated against field measurements in China reported in 2005-2007 and in other regions. In flooding rice paddies (F), N2O emission predicted by the model was consistent with previous reports in other regions. Under the water regime of flooding-midseason drainage-reflooding (F-D-F), the model developed in this study was comparable to that established by using worldwide database. The models also well fitted N2O emissions from rice paddies under the water regime of flooding-midseason drainage-reflooding-moisture but without waterlogging (F-D-F-M) in China. Consistency of rice production data derived from the database of this study with those reported in previous studies suggests that the model input data of rice production had high reliability. The input data showed that water management and nitrogen input regimes have greatly changed in rice paddies since the 1950s. During the 1950s-1970s, about 20%-25% of the rice paddy was continuous water logging, and 75%-80% under the water regime of F-D-F. Since the 1980s, about 12%-16%, 77% and 7%-12% of paddy fields were under the water regimes of F, F-D-F and F-D-F-M, respectively. Total N input during the rice growing season averaged 87.49 kg x hm(-2) in the 1950s and 224.64 kg x hm(-2) in the 1990s. Chemical N input during the rice growing season has increased from 37.4 kg x hm(-2) in the 1950s to 198.8 kg x hm(-2) in the 1990s, accounting for 43% and 88% of the seasonal total N inputs, respectively. Manure N input was applied at stable rate, ranging from 45.2 kg x hm(-2) to 48.2 kg x hm(-2) during the 1950s-1970s, but thereafter it decreased over time. The contribution of manure N to total N inputs has decreased from 52% in the 1950s to 9% in the 1990s. Crop residue N retained during the rice growing season has increased from 4.9 kg x hm(-2) in the 1950s to 6.3 kg x hm(-2) in the 1980s. A high spatial variation of nitrogen inputs during the rice growing season was found in the 1950s-1970s, while it was not pronounced in the 1980s-1990s. Overall, the results of this study suggest that the models could be used to quantify direct N2O emissions from rice paddies under various water regimes in China.
Assuntos
Agricultura/métodos , Poluentes Atmosféricos/análise , Modelos Teóricos , Óxido Nitroso/análise , Oryza/crescimento & desenvolvimento , China , Monitoramento Ambiental , Fertilizantes/análise , Inundações , Nitrogênio/análise , Oryza/metabolismo , Estações do Ano , Solo/análiseRESUMO
Based on statistical analysis of field N2O measurements in rice paddies in China, the models on direct N2O emissions under different water regimes were established. After successes in model validation and input data verification, the models were used to quantify changes in direct N2O emissions from paddy fields during the rice growing season in mainland China between the 1950s and the 1990s. Due to increases in rice planting area and nitrogen input and changes in water regime, the models predicted that seasonal N2O-N emissions from rice paddies have increased from 9.55 Gg each year in the 1950s to 32.26 Gg N2O-N in the 1990s, which was accompanied by the increase in rice yield over the period 1950s-1990s. During the period 1950s-1990s, seasonal N2O-N emissions from rice paddies have increased, on average, at a rate of 6.74 Gg per decade. Seasonal N2O fluxes in rice paddies were estimated to be 0.32 kg x hm(-2) in the 1950s and 1.00 kg x hm(-2) in the 1990s, which accounted for 0.37% and 0.46% of the seasonal total N inputs, respectively. The uncertainties in N2O estimate were estimated to be 59.8% in the 1950s and 37.5% in the 1990s. Seasonal N2O emissions from rice paddies in the region of middle and lower Yangtze River contributed 51% -56% to its national total. In the 1990s, N2O emissions during the rice growing season accounted for 8%-11% of the reported annual total of N2O emissions from croplands in China, suggesting that paddy rice development could have contributed to mitigating agricultural N2O emissions in the past decades. However, seasonal N2O emissions would be increased given that saving-water irrigation and nitrogen inputs are increasingly adopted in rice paddies in China.
Assuntos
Agricultura/métodos , Poluentes Atmosféricos/análise , Modelos Teóricos , Óxido Nitroso/análise , Oryza/crescimento & desenvolvimento , China , Monitoramento Ambiental , Oryza/metabolismo , Estações do Ano , Solo/análiseRESUMO
Toll-like receptor ligands (TLRLs) produced by various pathogens activate mitogen-activated protein kinases (MAPKs). While the dependence on p38 MAPK activation for the induction of inflammatory genes by the TLR4L, lipopolysaccharide (LPS), has been well documented, the importance of the p38 pathway in gene regulation by other TLRLs is less well understood. We have focused our analysis on two TLRLs with therapeutic potential, imidazoquinoline S28463 (TLR7L) and CpG DNA (TLR9L), to explore in detail their effects on the regulation of gene expression in macrophages. Here we report that activation of the p38 MAPK/MK2 pathway is crucial for both S28463- and CpG-induced cytokine and chemokine production. We show that the stability of TNF mRNA induced by CpG DNA and S28463 is not dependent on the p38 MAPK/MK2 pathway, in contrast to LPS-induced TNF mRNA. Using a GFP reporter construct under the control of the 3' untranslated region of the TNF gene, we demonstrate that S28463 and CpG DNA-induced MK2 signalling regulates TNF mRNA primarily at the translational level, whereas LPS-induced MK2 signalling regulates both the stability and translational efficiency of TNF mRNA. Overall, these data provide insight into distinct molecular mechanisms of gene expression regulation by different Toll-like receptor ligands.
Assuntos
Aminoquinolinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Fator de Necrose Tumoral alfa/genética , Animais , Quimiocinas/genética , Quimiocinas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular , Ligantes , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/biossíntese , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
The solute carrier family 11 member 1 (SLC11A1, formerly NRAMP1) gene is associated with infectious and autoimmune diseases and plays an important role in macrophage activation. Human SLC11A1 mRNA contains an AU-rich element (ARE) within the 3' untranslated region; however, its role in the regulation of SLC11A1 gene expression has not been elucidated. Here we analyze the expression of SLC11A1 in human monocytes and HL-60 cells and then use HL-60 cells as a model to determine whether RNA-binding protein HuR is associated with the ARE and involved in SLC11A1 mRNA turnover. Our results demonstrate a binding of HuR to the SLC11A1 ARE in phorbol myristate acetate (PMA)-differentiated cells dramatically increased compared to that in undifferentiated cells. Interestingly, PMA-induced accumulation of cytoplasmic HuR occurs in parallel with an increase in the binding of HuR to SLC11A1 ARE and with an increase in the SLC11A1 mRNA level. This suggests that HuR's cytoplasmic localization plays an important role in the regulation of SLC11A1 expression. We also observe that down-regulation of HuR expression by RNA interference (RNAi) results in a decrease in SLC11A1 expression which can be restored by the addition of recombinant HuR protein to the RNAi-treated cells. Finally, we show that HuR overexpression in HL-60 cells significantly increases the SLC11A1 mRNA stability. Taken together, our data demonstrate that HuR is a key mediator of posttranscriptional regulation and expression of the SLC11A1 gene.
Assuntos
Antígenos de Superfície/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Regulação da Expressão Gênica , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas/metabolismo , Antígenos de Superfície/análise , Antígenos de Superfície/genética , Sequência de Bases , Diferenciação Celular , Citoplasma/química , Citoplasma/metabolismo , DNA Recombinante/genética , Regulação para Baixo , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Células HL-60 , Humanos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Dados de Sequência Molecular , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/genética , Acetato de Tetradecanoilforbol/farmacologia , TransfecçãoRESUMO
Tuberculosis(TB) remains one of the major problems in global health. Macrophage (MPhi) apoptosis, induced by Mycobacterium tuberculosis (Mtb), is a cornerstone of effective innate microbial defense mechanism. Elucidation of the complex apoptosis-related gene expression may facilitate understanding the mechanism and regulation of macrophage apoptosis in response to Mtb, and contribute to developing novel measures to counter TB. DNA microarray containing 19,200 gene or gene fragments was used to compare the macrophage cell line U937 gene expression response to the clinical and laboratory Mtb infection. Northern blotting and RT-PCR were used to confirm the microarray results. Mtb H37Rv infection were found to downregulate the bcl-2, vitamin D receptor, interferon regulatory factor 3, cytochrome c oxidase, gene expression by 2-, 3-, 3-, 2.5-fold, respectively, while the clinical strain infection leads to upregulate the SOD2, SOD3, serine protease, toll-like receptor 2, signal transducer and activator (STAT1), hypoxia-inducible factor 22, 2.9-, 2.5-, 2.5-, 2.2-, 2.4-, 5.9-fold respectively. The findings suggest that the clinical strain infection tends to override the macrophage apoptosis by which the host attempt to limit the growth of the invader. The research on the complex factors network involved in the interaction will benefit the vaccine and novel drug target development.
Assuntos
Apoptose , Perfilação da Expressão Gênica , Macrófagos/metabolismo , Mycobacterium tuberculosis/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Humanos , Células U937RESUMO
OBJECTIVE: To verify whether it is effective to treat open bite cases with tip-forward bend. METHODS: Three-dimensional finite element (TDFE) models of the lower left central incisor and first molar were set up by means of CT. Stress distribution in root, PDL and alveolar bone, and the tendency of the tooth movement were obtained by calculation under different orthodontic forces. RESULTS: (1) The molar model revealed that the tensile stress concentration was at the distal cervix and the compressive stress concentration at mesial cervix. (2) The incisor model showed that the tensile stress was concentrated at apical tip and the compressive stress concentration was at the lingual side of the cervix. (3) The incisor had the tendency to elongate and move lingually. The molar tended to tip mesially and buccally. CONCLUSIONS: Arch wire with tip forward bend depends on the elongation and lingual movement of anterior teeth to treat open bite, but the anchorage molar will incline mesially further, which is not consistent with the mechanism to treat open bite.
Assuntos
Análise do Estresse Dentário , Diastema/terapia , Desenho de Aparelho Ortodôntico , Ortodontia Corretiva/métodos , Fenômenos Biomecânicos , Análise de Elementos Finitos , Humanos , Incisivo/fisiopatologia , Dente Molar/fisiopatologia , Ligamento Periodontal/fisiologiaRESUMO
OBJECTIVE: To evaluate the cytocompatibility of nanophase hydroxyapatite ceramics in vitro. METHODS: Hydroxyapatite (HA) was prepared via wet method. The grain size of the hydroxyapatite in the study was determined by scanning electron microscope and atomic force microscope with image analysis software. Primary osteoblast culture was established from rat calvaria. Cell adherence and proliferation on nanophase hydroxyapatite ceramics and conventional hydroxyapatite ceramics were examined at 1, 3, 5, 7 days. Morphology of the cells was observed by microscope. RESULTS: The average grain size of the nanophase and conventional HA was 55 nm and 780 nm, respectively. Throughout 7 days period, osteoblast proliferation on the HA was similar to that on tissue culture borosilicate glass controls, osteoblasts could attach, spread and proliferate on HA. However, compared to conventional ceramics, osteoblast proliferation on nanophase HA was significantly better after 1, 3, 5 and 7 days. CONCLUSION: Cytocompatibility of nanophase HA was significantly better than conventional ceramics.
Assuntos
Materiais Biocompatíveis , Cerâmica , Durapatita , Osteoblastos/citologia , Animais , Adesão Celular , Proliferação de Células , Células Cultivadas , Nanoestruturas , RatosRESUMO
OBJECTIVE: To investigate the effect of M. tuberculosis infection on actin in host-cells. METHODS: The form and distribution of fibrous actin and changes of actin expression were observed by confocal microscopy and Western blot analysis in macrophages infected with either M. tuberculosis H(37)R(a) or H(37)R(v) at 6 h, 12 h and 24 h after infection. Non-infected macrophages or macrophages treated with dead M. tuberculosis H(37)R(v) served as controls. RESULTS: F-actin aggregated and the actin expression was suppressed in macrophages infected with H(37)R(a) or H(37)R(v), and the effect appeared earlier in cells infected by the virulent H(37)R(v) strain than in cells infected by the avirulent H(37)R(a) strain. In macrophages treated with the dead H(37)R(v), actin was not affected. CONCLUSION: The results suggest that in the process of infection, M. tuberculosis evades the bactericidal mechanisms possibly by secretion of certain proteins or factors which affect the host-cell actin.