RESUMO
Exploring the potential lead compounds for Alzheimer's disease (AD) remains one of the challenging tasks. Here, we report that the plant extract conophylline (CNP) impeded amyloidogenesis by preferentially inhibiting BACE1 translation via the 5' untranslated region (5'UTR) and rescued cognitive decline in an animal model of APP/PS1 mice. ADP-ribosylation factor-like protein 6-interacting protein 1 (ARL6IP1) was then found to mediate the effect of CNP on BACE1 translation, amyloidogenesis, glial activation, and cognitive function. Through analysis of the 5'UTR-targetd RNA-binding proteins by RNA pulldown combined with LC-MS/MS, we found that FMR1 autosomal homolog 1 (FXR1) interacted with ARL6IP1 and mediated CNP-induced reduction of BACE1 by regulating the 5'UTR activity. Without altering the protein levels of ARL6IP1 and FXR1, CNP treatment promoted ARL6IP1 interaction with FXR1 and inhibited FXR1 binding to the 5'UTR both in vitro and in vivo. Collectively, CNP exhibited a therapeutic potential for AD via ARL6IP1. Through pharmacological manipulation, we uncovered a dynamic interaction between FXR1 and the 5'UTR in translational control of BACE1, adding to the understanding of the pathophysiology of AD.
Assuntos
Doença de Alzheimer , Animais , Camundongos , Regiões 5' não Traduzidas , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Cromatografia Líquida , Proteína do X Frágil da Deficiência Intelectual/genética , Biossíntese de Proteínas , Espectrometria de Massas em TandemRESUMO
Mitochondrial Tu translation elongation factor (TUFM or EF-Tu) is part of the mitochondrial translation machinery. It is reported that TUFM expression is reduced in the brain of Alzheimer's disease (AD), suggesting that TUFM might play a role in the pathophysiology. In this study, we found that TUFM protein level was decreased in the hippocampus and cortex especially in the aged APP/PS1 mice, an animal model of AD. In HEK cells that stably express full-length human amyloid-ß precursor protein (HEK-APP), TUFM knockdown or overexpression increased or reduced the protein levels of ß-amyloid protein (Aß) and ß-amyloid converting enzyme 1 (BACE1), respectively. TUFM-mediated reduction of BACE1 was attenuated by translation inhibitor cycloheximide (CHX) or α-[2-[4-(3,4-Dichlorophenyl)-2-thiazolyl]hydrazinylidene]-2-nitro-benzenepropanoic acid (4EGI1), and in cells overexpressing BACE1 constructs deleting the 5' untranslated region (5'UTR). TUFM silencing increased the half-life of BACE1 mRNA, suggesting that RNA stability was affected by TUFM. In support, transcription inhibitor Actinomycin D (ActD) and silencing of nuclear factor κB (NFκB) failed to abolish TUFM-mediated regulation of BACE1 protein and mRNA. We further found that the mitochondria-targeted antioxidant TEMPO diminished the effects of TUFM on BACE1, suggesting that reactive oxygen species (ROS) played an important role. Indeed, cellular ROS levels were affected by TUFM knockdown or overexpression, and TUFM-mediated regulation of apoptosis and Tau phosphorylation at selective sites was attenuated by TEMPO. Collectively, TUFM protein levels were decreased in APP/PS1 mice. TUFM is involved in AD pathology by regulating BACE1 translation, apoptosis, and Tau phosphorylation, in which ROS plays an important role.
Assuntos
Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Modelos Animais de Doenças , Mitocôndrias/patologia , Fator Tu de Elongação de Peptídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/fisiologia , Animais , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Fator Tu de Elongação de Peptídeos/genética , Fosforilação , Presenilina-1/fisiologiaRESUMO
Thioredoxin-2 (TXN2) is a mitochondrial protein and represents one of the intrinsic antioxidant enzymes. It has long been recognized that mitochondrial dysfunction and oxidative stress contribute to the pathogenesis of Alzheimer's disease (AD). We hypothesized that mitochondrial TXN2 might play a role in AD-like pathology. In this study, we found that in SH-SY5Y and HEK cells stably express full-length human amyloid-ß precursor protein (HEK-APP), TXN2 silencing or over-expression selectively increased or decreased the transcription of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), respectively, without altering the protein levels of others enzymes involved in the catalytic processing of APP. As a result, ß-amyloid protein (Aß) levels were significantly decreased by TXN2. In addition, in cells treated with 3-nitropropionic acid (3-NP) that is known to increase reactive oxygen species (ROS) and promote mitochondrial dysfunction, TXN2 silencing resulted in further enhancement of BACE1 protein levels, suggesting a role of TXN2 in ROS removal. The downstream signaling might involve NFκB, as TXN2 reduced the phosphorylation of p65 and IκBα; and p65 knockdown significantly attenuated TXN2-mediated regulation of BACE1. Concomitantly, the levels of cellular ROS, apoptosis-related proteins and cell viability were altered by TXN2 silencing or over-expression. In APPswe/PS1E9 mice, an animal model of AD, the cortical and hippocampal TXN2 protein levels were decreased at 12 months but not at 6 months, suggesting an age-dependent decline. Collectively, TXN2 regulated BACE1 expression and amyloidogenesis via cellular ROS and NFκB signaling. TXN2 might serve as a potential target especially for early intervention of AD.
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Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Proteínas Mitocondriais/metabolismo , Tiorredoxinas/metabolismo , Doença de Alzheimer/metabolismo , Animais , Encéfalo/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Mitocôndrias/metabolismo , Transdução de Sinais/fisiologiaRESUMO
MMP13 (matrix metallopeptidase 13) plays a key role in bone metabolism and cancer development, but has no known functions in Alzheimer's disease. In this study, we used high-throughput small molecule screening in SH-SY5Y cells that stably expressed a luciferase reporter gene driven by the BACE1 (ß-site amyloid precursor protein cleaving enzyme 1) promoter, which included a portion of the 5' untranslated region (5'UTR). We identified that CL82198, a selective inhibitor of MMP13, decreased BACE1 protein levels in cultured neuronal cells. This effect was dependent on PI3K (phosphatidylinositide 3-kinase) signalling, and was unrelated to BACE1 gene transcription and protein degradation. Further, we found that eukaryotic translation initiation factor 4B (eIF4B) played a key role, as the mutation of eIF4B at serine 422 (S422R) or deletion of the BACE1 5'UTR attenuated MMP13-mediated BACE1 regulation. In APPswe/PS1E9 mice, an animal model of Alzheimer's disease, hippocampal Mmp13 knockdown or intraperitoneal CL82198 administration reduced BACE1 protein levels and the related amyloid-ß precursor protein processing, amyloid-ß load and eIF4B phosphorylation, whereas spatial and associative learning and memory performances were improved. Collectively, MMP13 inhibition/CL82198 treatment exhibited therapeutic potential for Alzheimer's disease, via the translational regulation of BACE1.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Benzofuranos/uso terapêutico , Disfunção Cognitiva/tratamento farmacológico , Metaloproteinase 13 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/uso terapêutico , Morfolinas/uso terapêutico , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Células Cultivadas , Fatores de Iniciação em Eucariotos/genética , Técnicas de Silenciamento de Genes , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Oligopeptídeos/genética , Fosfatidilinositol 3-Quinases/metabolismo , RatosRESUMO
ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10) is the α-secretase that is involved in APP (ß-amyloid precursor protein) processing. Enhancement of the nonamyloidogenic APP pathway by ADAM10 provides therapeutic potential for Alzheimer's disease (AD). By using high-throughput screening that targeted ADAM10, we determined that apicidin-an inhibitor of HDACs (histone deacetylases)-significantly increased mRNA and protein levels of ADAM10 in SH-SY5Y cells. A luciferase assay revealed that the nucleotides -444 to -300 in the ADAM10 promoter were sufficient to mediate this effect. In addition, knockdown of USF1 (upstream transcription factor 1) and HDAC2/3 prevented apicidin regulation of ADAM10. Moreover, USF1 acetylation was increased by apicidin, which enhanced the association of USF1 with HDAC2/3 and with the ADAM10 promoter. We further found that apicidin did not affect the phosphorylation of ERK or USF1; however, ERK inhibitor U0126 blocked the effect of apicidin on ADAM10. Finally, apicidin increased the level of α-site C-terminal fragment from APP and reduced the production of ß-amyloid peptide 1-42. Collectively, our study provides evidence that ADAM10 expression can be regulated by HDAC2/3 inhibitor apicidin via USF1-dependent mechanisms in which ERK signaling plays an important role. Thus, HDAC regulation of ADAM10 might shed new light on the understanding of AD pathology.-Hu, X.-T., Zhu, B.-L., Zhao, L.-G., Wang, J.-W., Liu, L., Lai, Y.-J., He, L., Deng, X.-J., Chen, G.-J. Histone deacetylase inhibitor apicidin increases expression of the α-secretase ADAM10 through transcription factor USF1-mediated mechanisms.
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Proteína ADAM10/genética , Secretases da Proteína Precursora do Amiloide/genética , Inibidores de Histona Desacetilases/farmacologia , Proteínas de Membrana/genética , Peptídeos Cíclicos/farmacologia , Fatores Estimuladores Upstream/metabolismo , Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ativação Transcricional/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Fatores Estimuladores Upstream/genéticaRESUMO
BACKGROUND: A decreased plasma level of soluble form of the receptor for advanced glycation end products (sRAGE) in patients with Alzheimer's disease (AD) has been reported. However, no evidence has shown whether the sRAGE plasma level of AD patients may differentiate from other types of dementia. METHODS: Our study assessed sRAGE concentrations in the following 121 individuals in Chongqing area: 36 patients with AD, 12 with vascular dementia (VaD), 14 with mixed dementia (MD), 24 with other dementia (OD) including Parkinson's disease dementia, frontotemporal dementia, paralytic dementia and 35 cognitively normal controls. The total plasma level of sRAGE was determined using sandwich ELISA method. RESULTS: sRAGE concentration in AD is significantly decreased compared with healthy controls. However, the receiver operating characteristic curve analysis of sRAGE between the AD and the control shows a low diagnostic accuracy. CONCLUSIONS: Our results demonstrate that sRAGE may assist the diagnosis of AD from normal individuals, but cannot differentiate AD from VaD, MD or OD.
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Doença de Alzheimer/sangue , Produtos Finais de Glicação Avançada/sangue , Atividades Cotidianas , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Demência Vascular/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Entrevista Psiquiátrica Padronizada , Pessoa de Meia-Idade , Curva ROC , Análise de RegressãoRESUMO
It is long been suggested that one-carbon metabolism (OCM) is associated with Alzheimer's disease (AD), whereas the potential mechanisms remain poorly understood. Taking advantage of chemical biology, that mitochondrial serine hydroxymethyltransferase (SHMT2) directly regulated the translation of ADAM metallopeptidase domain 10 (ADAM10), a therapeutic target for AD is reported. That the small-molecule kenpaullone (KEN) promoted ADAM10 translation via the 5' untranslated region (5'UTR) and improved cognitive functions in APP/PS1 mice is found. SHMT2, which is identified as a target gene of KEN and the 5'UTR-interacting RNA binding protein (RBP), mediated KEN-induced ADAM10 translation in vitro and in vivo. SHMT2 controls AD signaling pathways through binding to a large number of RNAs and enhances the 5'UTR activity of ADAM10 by direct interaction with GAGGG motif, whereas this motif affected ribosomal scanning of eukaryotic initiation factor 2 (eIF2) in the 5'UTR. Together, KEN exhibits therapeutic potential for AD by linking OCM with RNA processing, in which the metabolic enzyme SHMT2 "moonlighted" as RBP by binding to GAGGG motif and promoting the 5'UTR-dependent ADAM10 translation initiation.
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Doença de Alzheimer , Glicina Hidroximetiltransferase , Animais , Camundongos , Regiões 5' não Traduzidas , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Glicina Hidroximetiltransferase/genética , RNA Mensageiro/genéticaRESUMO
Two new species of Antrodia, A. aridula and A. variispora, are described from western China. Phylogeny based on a six-gene dataset (ITS + nLSU + nSSU + mtSSU + TEF1 + RPB2) demonstrates that samples of the two species form two independent lineages within the clade of Antrodia s.s. and are different in morphology from the existing species of Antrodia. Antrodia aridula is characterized by its annual and resupinate basidiocarps with angular to irregular pores of 2-3 mm each and oblong ellipsoid to cylindrical basidiospores measuring 9-12 × 4.2-5.3 µm, growing on gymnosperm wood in a dry environment. Antrodia variispora is characterized by its annual and resupinate basidiocarps with sinuous or dentate pores with a size of 1-1.5 mm each and oblong ellipsoid, fusiform, pyriform, or cylindrical basidiospores measuring 11.5-16 × 4.5-5.5 µm, growing on the wood of Picea. The differences between the new species and morphologically similar species are discussed in this article.
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BACKGROUND: Accumulation of hyperphosphorylated Tau (pTau) contributes to the formation of neurofibrillary tangles in Alzheimer's disease (AD), and targeting Tau/pTau metabolism has emerged as a therapeutic approach. We have previously reported that mitochondrial 3-hydroxy-3-methylglutaryl-COA synthase 2 (HMGCS2) is involved in AD by promoting autophagic clearance of amyloid-ß protein precursor via ketone body-associated mechanism, whether HMGCS2 may also regulate Tau metabolism remains elusive. OBJECTIVE: The present study was to investigate the role of HMGCS2 in Tau/p degradation. METHODS: The protein levels of Tau and pTau including pT217 and pT181, as well as autophagic markers LAMP1 and LC3-II were assessed by western blotting. The differentially regulated genes by HMGCS2 were analyzed by RNA sequencing. Autophagosomes were assessed by transmission electron microscopy. RESULTS: HMGCS2 significantly decreased Tau/pTau levels, which was paralleled by enhanced formation of autophagic vacuoles and prevented by autophagic regulators chloroquine, bafilomycin A1, 3-methyladenine, and rapamycin. Moreover, HMGCS2-induced alterations of LAMP1/LC3-II and Tau/pTau levels were mimicked by ketone body acetoacetate or ß-hydroxybutyrate. Further RNA-sequencing identified ankyrin repeat domain 24 (ANKRD24) as a target gene of HMGCS2, and silencing of ANKRD24 reduced LAMP1/LC3-II levels, which was accompanied by the altered formation of autophagic vacuoles, and diminished the effect of HMGCS2 on Tau/pTau. CONCLUSION: HMGCS2 promoted autophagic clearance of Tau/pTau, in which ketone body and ANKRD24 played an important role.
Assuntos
Doença de Alzheimer , Proteínas tau , Humanos , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Corpos Cetônicos , Sirolimo/farmacologia , Autofagia/fisiologia , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismoRESUMO
Although several features of apoptosis and autophagy have been reported in the larval organs of Lepidoptera during metamorphosis, solid experimental evidence for autophagy is still lacking. Moreover, the role of the two processes and the nature of their relationship are still cryptic. In this study, we perform a cellular, biochemical and molecular analysis of the degeneration process that occurs in the larval midgut of Bombyx mori during larval-adult transformation, with the aim to analyze autophagy and apoptosis in cells that die under physiological conditions. We demonstrate that larval midgut degradation is due to the concerted action of the two mechanisms, which occur at different times and have different functions. Autophagy is activated from the wandering stage and reaches a high level of activity during the spinning and prepupal stages, as demonstrated by specific autophagic markers. Our data show that the process of autophagy can recycle molecules from the degenerating cells and supply nutrients to the animal during the non-feeding period. Apoptosis intervenes later. In fact, although genes encoding caspases are transcribed at the end of the larval period, the activity of these proteases is not appreciable until the second day of spinning and apoptotic features are observable from prepupal phase. The abundance of apoptotic features during the pupal phase, when the majority of the cells die, indicates that apoptosis is actually responsible for cell death and for the disappearance of larval midgut cells.
Assuntos
Apoptose/fisiologia , Autofagia/fisiologia , Bombyx/crescimento & desenvolvimento , Metamorfose Biológica/fisiologia , Animais , Autofagia/genética , Bombyx/citologia , Bombyx/metabolismo , Caspases/metabolismo , Sistema Digestório/anatomia & histologia , Sistema Digestório/metabolismo , Larva/citologia , Larva/metabolismo , Larva/ultraestrutura , Pupa/metabolismoRESUMO
BACKGROUND: To explore the value of quantitative magnetic resonance imaging (i.e., T2 map technique) in the diagnosis of crush injury in rabbit hind limb muscles. MATERIALS AND METHODS: We established a rabbit hind limb crush injury model and performed examinations on magnetic resonance imaging (MRI) (T1WI, T2WI, and T2 map), muscle pathology, serum level of muscular troponin (sTnI), and urine myoglobin (Myo) at 1, 3, 7, 14, and 30 d after injury to investigate the correlation of MRI, library examination, and histopathology. RESULTS: T2WI of the injured muscle showed high signal intensity at 1, 3, and 7 d after crush injury and the T2 value continued to rise. The pathologic findings of the muscle included swollen and ruptured cells, expanded extra-cellular space, inflammatory reactions, and fine muscle fiber regeneration. The serum sTnI and urine Myo were high. At 14 d, these indices returned to normal gradually. The T2WI changes and T2 value were positively associated with the pathological changes of the muscles, serum sTnI and urine Myo. However, the signal intensity of T1WI did not vary significantly at different time points. CONCLUSION: T2WI and T2 value from T2 mapping are very useful methods of choice to evaluate the distribution and extension of the affected muscles. The high signal intensity on T2WI of the affected muscles after crush injury may be due to an increased extracellular space, local inflammation, and incomplete muscle fiber regeneration.
Assuntos
Membro Posterior/lesões , Imageamento por Ressonância Magnética , Músculo Esquelético/lesões , Músculo Esquelético/patologia , Pressão/efeitos adversos , Lesões dos Tecidos Moles/etiologia , Lesões dos Tecidos Moles/patologia , Animais , Biomarcadores/metabolismo , Membro Posterior/metabolismo , Masculino , Modelos Animais , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/metabolismo , Mioglobinúria/urina , Coelhos , Sensibilidade e Especificidade , Troponina/sangueRESUMO
Sulfuretin is a flavonoid that protects cell from damage induced by reactive oxygen species and inflammation. In this study, we investigated the role of sulfuretin in the processing of amyloid precursor protein (APP), in association with the two catalytic enzymes the α-secretase a disintegrin and metalloproteinase (ADAM10), and the beta-site APP cleaving enzyme 1 (BACE1) that play important roles in the generation of ß amyloid protein (Aß) in Alzheimer's disease (AD). We found that sulfuretin increased the levels of the immature but not the mature form of ADAM10 protein. The enhanced ADAM10 transcription by sulfuretin was mediated by the nucleotides -444 to -300 in the promoter region, and was attenuated by silencing or mutation of transcription factor retinoid X receptor (RXR) and by GW6471, a specific inhibitor of peroxisome proliferator-activated receptor α (PPAR-α). We further found that sulfuretin preferentially increased protein levels of the immature form of APP (im-APP) but significantly reduced those of BACE1, sAPPß and ß-CTF, whereas Aß1-42 levels were slightly increased. Finally, the effect of sulfuretin on BACE1 and im-APP was selectively attenuated by the translation inhibitor cycloheximide and by lysosomal inhibitor chloroquine, respectively. Taken together, (1) RXR/PPAR-α signaling was involved in sulfuretin-mediated ADAM10 transcription. (2) Alteration of Aß protein level by sulfuretin was not consistent with that of ADAM10 and BACE1 protein levels, but was consistent with the elevated level of im-APP protein, suggesting that im-APP, an isoform mainly localized to trans-Golgi network, plays an important role in Aß generation.
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OBJECTIVES: Tumor necrosis factor-alpha (TNF-alpha) may play an important role in host's immune response to mycobacterium tuberculosis (M. tuberculosis) infection. This study was to investigate the association of TNF-alpha gene polymorphism with pulmonary tuberculosis (TB) among patients with coal worker's pneumoconiosis (CWP). METHODS: A case-control study was conducted in 113 patients with confirmed CWP complicated with pulmonary TB and 113 non-TB controls with CWP. They were matched in gender, age, job, and stage of pneumoconiosis. All participants were interviewed with questionnaires and their blood specimens were collected for genetic determination with informed consent. The TNF-alpha gene polymorphism was determined with polymerase chain reaction of restriction fragment length polymorphism (PCR-RFLP). Frequency of genotypes was assessed for Hardy-Weinberg equilibrium by chi-square test or Fisher's exact probability. Factors influencing the association of individual susceptibility with pulmonary TB were evaluated with logistic regression analysis. Gene-environment interaction was evaluated by a multiplicative model with combined OR. All data were analyzed using SAS version 8.2 software. RESULTS: No significant difference in frequency of the TNF-alpha-308 genotype was found between CWP complicated with pulmonary TB and non-TB controls (chi2 = 5.44, P = 0.07). But difference in frequency of the TNF-alpha-308 A allele was identified between them (chi2 = 5.14, P = 0.02). No significant difference in frequencies of the TNF-alpha-238 genotype and allele (P = 0.23 and P = 0.09, respectively) was found between cases and controls either, with combined (GG and AA) OR of 3.96 (95% confidence interval of 1.30-12.09) at the -308 locus of the TNF-alpha gene, as compared to combination of the TNF-alpha-238 GG and TNF-alpha-308 GG genotypes. Multivariate-adjusted odds ratio of the TNF-alpha-238 GG and TNF-alpha-308 GA genotypes was 1.98 (95% CI of 1.06-3.71) for risk for pulmonary TB in patients with CWP. There was a synergic interaction between the TNF-alpha-308 GG genotype and body mass index (OR = 4.92), as well as an interaction between the TNF-alpha-308 GG genotype and history of BCG immunization or history of TB exposure. And, the interaction of the TNF-alpha-238 GG genotype and history of BCG immunization or TB exposure with risk for pulmonary TB in them was also indicated. CONCLUSIONS: TNF-alpha-308 A allele is associated with an elevated risk for pulmonary TB, whereas TNF-alpha-238 A allele was otherwise.
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Antracose/complicações , Tuberculose Pulmonar/genética , Fator de Necrose Tumoral alfa/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Exposição Ambiental , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Regiões Promotoras GenéticasRESUMO
The effects of seed size and drought stress on the growth and physiological characteristics of Quercus wutaishanica seedlings were investigated under shading conditions of a pot experiment in greenhouse. There were four treatments, including 80% field water content (FWC), 60% FWC, 40% FWC, and 20% FWC [CK, light drought stress (LDS), medium drought stress (MDS), and high drought stress (HDS), respectively]. The results showed that leaf area per plant, total dry mass, and root-shoot ratio of Q. wutaishanica seedlings regenerated from large seeds (3.05±0.38 g) were significantly higher than those from small seeds (1.46±0.27 g) in all four treatments. Shoot height, basal stem diameter, leaf number, specific leaf area, relative growth rate, and net assimilation rate of the seedlings from large seeds were higher than those of seedlings from small seeds under the treatments of LDS, MDS and HDS. Activities of peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) in large-seeded seedlings were higher than those of small-seeded seedlings under all treatments, while the contents of MDA, soluble protein, free proline, and total chlorophyll of large-seeded seedlings were higher than those of small-seeded seedlings only under some drought stress treatments. All growth parameters except the root-shoot ratio decreased with the increases of drought stress. The HDS treatment resulted in 19.4% and 20.0% decline in total dry mass of large- and small-seeded seedlings respectively, compared with those of CK. With increasing drought stress, the activities of POD, CAT, and SOD decreased after an initial increase. POD activity of large- and small-seeded seedling under MDS treatment was 126.7% and 142.1% higher than CK, while CAT was 170.0% and 151.9% higher than CK, respectively. However, the MDA content of seedlings from large and small seeds under HDS treatment was 86.5% and 68.9% higher than that of CK, respectively. The contents of soluble protein, free proline, and total chlorophyll rose at first and then fell with increasing drought stress, and soluble protein content in large- and small-seeded seedlings experienced MDS enhanced 320.7% and 352.7%, respectively. Those results indicated that large-seeded seedlings of Q. wutaishanica had stronger drought tolerance than small-seeded seedlings due to their growth and physiology advantages. Large-seeded seedlings with stronger resistance to drought stress should be applied to artificial regeneration of the degraded secondary Q. wutaishanica plantations.
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Quercus , Plântula , Clorofila , Secas , Sementes , Estresse FisiológicoRESUMO
Vps34 (vacuolar protein-sorting 34) plays important role in autophagy and endosomal trafficking. These processes are closely associated protein ubiquitination and degradation. We have hypothesized that Vps34 ubiquitination status would also control its degradation. Here, we report that our results did not support this assumption. In cells transiently transfected with ubiquitin (UB) constructs contained different lysine residues (Ks), Vps34 ubiquitination could occur regardless of the presence of any Ks in UB. However, Vps34 protein levels were not significantly altered in cells transiently transfected with these UB mutants. We further found that Vps34 protein was altered by pharmacological manipulation of E2/E3 activity; yet this effect was not significantly affected by UB overexpression. In vivo experiments revealed that in APP/PS1 mice, an animal model of Alzheimer's disease (AD), although ubiquitination of Vps34 was significantly reduced, Vps34 protein levels remained unchanged. Vps34 indeed was subjected to proteasomal or lysosomal degradation, as prolonged treatment of proteasomal inhibitor MG132 or lysosomal inhibitor chloroquine elevated Vps34 protein levels. We conclude that unlike most of other proteins, Vps34 ubiquitination is not closely associated with its degradation.
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BACKGROUND: Through the whole life of eukaryotes, autophagy plays an important role in various biological events including development, differentiation and determination of lifespan. A full set of genes and their encoded proteins of this evolutionarily conserved pathway have been identified in many eukaryotic organisms from yeast to mammals. However, this pathway in the insect model organism, the silkworm Bombyx mori, remains poorly investigated. RESULTS: Based on the autophagy pathway in several model organisms and a series of bioinformatic analyses, we have found more than 20 autophagy-related genes from the current database of the silkworm Bombyx mori. These genes could be further classified into the signal transduction pathway and two ubiquitin-like pathways. Using the mRNA extracted from the silkgland, we cloned the full length cDNA fragments of some key genes via reverse transcription PCR and 3' rapid amplification of cDNA ends (RACE). In addition, we found that the transcription levels of two indicator genes BmATG8 and BmATG12 in the silkgland tend to be increased from 1st to 8th day of the fifth instar larvae. CONCLUSION: Bioinformatics in combination with RT-PCR enable us to remodel a preliminary pathway of autophagy in the silkworm. Amplification and cloning of most autophagy-related genes from the silkgland indicated autophagy is indeed an activated process. Furthermore, the time-course transcriptional profiles of BmATG8 and BmATG12 revealed that both genes are up-regulated along the maturation of the silkgland during the fifth instar. These findings suggest that the autophagy should play an important role in Bombyx mori silkgland.
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Autofagia , Bombyx/citologia , Bombyx/genética , Clonagem Molecular , Proteínas de Insetos/genética , Sequência de Aminoácidos , Animais , Bombyx/química , Bombyx/metabolismo , Regulação da Expressão Gênica , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Dados de Sequência Molecular , Alinhamento de Sequência , Transdução de SinaisRESUMO
Drosomycin (Drs) encoding an inducible 44-residue antifungal peptide is clustered with six additional genes, Dro1, Dro2, Dro3, Dro4, Dro5, and Dro6, forming a multigene family on the 3L chromosome arm in Drosophila melanogaster. To get further insight into the regulation of each member of the drosomycin gene family, here we investigated gene expression patterns of this family by either microbe-free injury or microbial challenges using real time RT-PCR. The results indicated that among the seven drosomycin genes, Drs, Dro2, Dro3, Dro4, and Dro5 showed constitutive expressions. Three out of five, Dro2, Dro3, and Dro5, were able to be upregulated by simple injury. Interestingly, Drs is an only gene strongly upregulated when Drosophila was infected with microbes. In contrast to these five genes, Dro1 and Dro6 were not transcribed at all in either noninfected or infected flies. Furthermore, by 5' rapid amplification of cDNA ends, two transcription start sites were identified in Drs and Dro2, and one in Dro3, Dro4, and Dro5. In addition, NF-kappaB binding sites were found in promoter regions of Drs, Dro2, Dro3, and Dro5, indicating the importance of NF-kappaB binding sites for the inducibility of drosomycin genes. Based on the analyses of flanking sequences of each gene in D. melanogaster and phylogenetic relationship of drosomycins in D. melanogaster species-group, we concluded that gene duplications were involved in the formation of the drosomycin gene family. The possible evolutionary fates of drosomycin genes were discussed according to the combining analysis of gene expression pattern, gene structure, and functional divergence of these genes.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Evolução Molecular , Regulação da Expressão Gênica , Variação Genética , Família Multigênica/genética , Animais , Sítios de Ligação , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/metabolismo , Perfilação da Expressão Gênica , Genes de Insetos , Filogenia , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
Tuber indicum is one of the most renowned commercialized fungi in China. Mycorrhizal investigations, however, have been carried out mainly with exotic trees. Up to now there is no detailed description of morphology of the mycorrhizae formed with the indigenous hosts of T. indicum. Containerized seedlings of two indigenous hosts of the fungus in southwestern China, Pinus armandii and Castanea mollissima, were inoculated with aqueous spore suspension of T. indicum in two kinds of substrates. Mycorrhizae began to form 4 months after inoculation and were harvested at 9 months. The contributing fungus of the mycorrhizae was confirmed to be T. indicum by morphological and ITS-rDNA sequence analyses. The morphology of emanating hyphae and epidermoid-like mantle appearance was similar to the mycorrhizae obtained with some European trees. The high morphological variation and the similarity to that of Tuber melanosporum makes it difficult to distinguish the mycorrhizae of the two species by morphology alone. The synthesis of mycorrhizae of T. indicum with its indigenous hosts will be of great significance for planned cultivation of the Asian black truffles.
Assuntos
Ascomicetos/crescimento & desenvolvimento , Fagaceae/microbiologia , Micorrizas/crescimento & desenvolvimento , Pinus/microbiologia , Ascomicetos/citologia , Ascomicetos/genética , Ascomicetos/isolamento & purificação , Micorrizas/citologia , Micorrizas/genética , Micorrizas/isolamento & purificaçãoRESUMO
Some lepidopteran lysozymes have been reported to display activity against Gram-positive and Gram-negative bacteria, in contrast to most lysozymes that are active only against Gram-positive bacteria. OstrinLysC, a c-type lysozyme, was purified from the Asian corn borer, Ostrinia furnacalis Guenée (Lepidoptera: Pyralidae), and shows activity against Gram-positive and Gram-negative bacteria. The NH2-terminal amino acid sequence was determined by Edman degradation and used in a homology cloning strategy. The gene coding for OstrinLysC contains three exons and two introns. The expression profile of the OstrinlysC gene was examined by quantitative real-time PCR. Following injection of the larvae with bacteria, the OstrinlysC gene is strongly up-regulated in immune tissues. Transcripts were also detected in gut tissue. After feeding the larvae with bacteria, OstrinlysC transcripts increased in immune tissues. A very low level of transcript abundance was also detected in gut tissue. These results suggested that the OstrinlysC gene is involved in immune responses. The three dimensional structure of OstrinLysC was predicted. Based on comparison of the 3-D structure of OstrinLysC with that of silkworm lysozyme and chicken lysozyme, we hypothesize that the positive charge-rich surface and the short loop-2, which is close to the cluster of hydrophobic residues, may play important roles in the interaction with the outer membrane of Gram-negative bacterial cell walls.
Assuntos
Mariposas/enzimologia , Mariposas/genética , Muramidase/genética , Muramidase/metabolismo , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Regulação da Expressão Gênica , Hemolinfa/química , Modelos Moleculares , Dados de Sequência Molecular , Muramidase/química , Muramidase/farmacologia , Filogenia , Estrutura Terciária de Proteína , Alinhamento de Sequência , Staphylococcus aureus/efeitos dos fármacosRESUMO
The etiology of Alzheimer's disease (AD) has been intensively studied. However, little is known about the molecular alterations in early-stage and late-stage AD. Hence, we performed RNA sequencing and assessed differentially expressed genes (DEGs) in the hippocampus of 18-month and 7-month-old APP/PS1 mice. Moreover, the DEGs induced by treatment with nicotine, the nicotinic acetylcholine receptor agonist that is known to improve cognition in AD, were also analyzed in old and young APP/PS1 mice. When comparing old APP/PS1 mice with their younger littermates, we found an upregulation in genes associated with calcium overload, immune response, cancer, and synaptic function; the transcripts of 14 calcium ion channel subtypes were significantly increased in aged mice. In contrast, the downregulated genes in aged mice were associated with ribosomal components, mitochondrial respiratory chain complex, and metabolism. Through comparison with DEGs in normal aging from previous reports, we found that changes in calcium channel genes remained one of the prominent features in aged APP/PS1 mice. Nicotine treatment also induced changes in gene expression. Indeed, nicotine augmented glycerolipid metabolism, but inhibited PI3K and MAPK signaling in young mice. In contrast, nicotine affected genes associated with cell senescence and death in old mice. Our study suggests a potential network connection between calcium overload and cellular signaling, in which additional nicotinic activation might not be beneficial in late-stage AD.