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1.
Biochimie ; 165: 131-140, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31356846

RESUMO

Swainsonine is a major toxic ingredients of locoweed plants, ingestion of these plants may cause locoism in livestock characterized by extensive cellular vacuolar degeneration of multiple tissues. However, so far, the mechanisms responsible for vacuolar degeneration induced by SW are not known. In this study, we investigated the role of autophagy in SW-induced TCMK-1 cells using Western blotting, transmission electron microscopy, immunofluorescent microscopy and qRT-PCR. The results showed that SW treatment increased the levels of LC3-II. The co-localization of LC3-II and lysosomal protein LAMP-2 results suggested that SW treatment does not interfere with fusion between autophagosome and lysosome. TEM results indicated that SW induced aggregation of the lysosome around the autophagosome. In addition, SW treatment suppressed p-PI3K, p-Akt, p-mTOR, p-p70S6K and p-4EBP1 level. In conclusion, SW induced autophagy via pI3K/AKT/mTOR signaling pathway and revealed the role of autophagy in causing the SW toxicity characterized by the vacuolar degeneration.

2.
Appl Microbiol Biotechnol ; 103(17): 6919-6932, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31332488

RESUMO

Filamentous fungi play an important role in human health and industrial/agricultural production. With the increasing number of full genomes available for fungal species, the study of filamentous fungi has brought about a wider range of genetic manipulation opportunities. However, the utilization of traditional methods to study fungi is time consuming and laborious. Recent rapid progress and wide application of a versatile genome editing technology, i.e., the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-related nuclease 9) system, has revolutionized biological research and has many innovative applications in a wide range of fields showing great promise in research and application of filamentous fungi. In this review, we introduce the CRISPR/Cas9 genome editing technology focusing on its application in research of filamentous fungi and we discuss the general considerations of genome editing using CRISPR/Cas9 system illustrating vector construction, multiple editing strategies, technical consideration of different sizes of homology arms on genome editing efficiency, off-target effects, and different transformation methodologies. In addition, we discuss the challenges encountered using CRISPR/Cas9 technology and give the perspectives of future applications of CRISPR/Cas9 technology for basic research and practical application of filamentous fungi.

3.
J Trace Elem Med Biol ; 55: 15-19, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31345353

RESUMO

The trace element strontium has a significant impact on cartilage metabolism. However, the direct effects of strontium on alkaline phosphatase (ALP), a marker of bone growth, and bone morphogenetic protein-4 (BMP-4), which plays a key role in the regulation of bone and cartilage development, are not entirely clear. In order to understand the mechanisms involved in these processes, the chondrocytes were isolated from Wistar rat articular cartilage by enzymatic digestion and cultured under standard conditions. They were then treated with strontium at 0.5, 1.0, 2.0, 5.0, 20.0 and 100.0 µg/mL for 72 h. The mRNA abundance and protein expression levels of ALP and BMP-4 were measured using real-time polymerase chain reaction (real-time PCR) and Western blot analysis. The results showed that the levels of expression of ALP and BMP-4 in chondrocytes increased as the concentration of strontium increased relative to the control group, and the difference became significant at 1.0 µg/mL strontium (P<0.05). These results indicated that strontium could be involved in cartilage development via regulating ALP and BMP-4 expression.

4.
Nat Immunol ; 20(7): 879-889, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31182807

RESUMO

CD8+ T cells and natural killer (NK) cells are central cellular components of immune responses against pathogens and cancer, which rely on interleukin (IL)-15 for homeostasis. Here we show that IL-15 also mediates homeostatic priming of CD8+ T cells for antigen-stimulated activation, which is controlled by a deubiquitinase, Otub1. IL-15 mediates membrane recruitment of Otub1, which inhibits ubiquitin-dependent activation of AKT, a kinase that is pivotal for T cell activation and metabolism. Otub1 deficiency in mice causes aberrant responses of CD8+ T cells to IL-15, rendering naive CD8+ T cells hypersensitive to antigen stimulation characterized by enhanced metabolic reprograming and effector functions. Otub1 also controls the maturation and activation of NK cells. Deletion of Otub1 profoundly enhances anticancer immunity by unleashing the activity of CD8+ T cells and NK cells. These findings suggest that Otub1 controls the activation of CD8+ T cells and NK cells by functioning as a checkpoint of IL-15-mediated priming.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Cisteína Endopeptidases/metabolismo , Interleucina-15/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Cisteína Endopeptidases/deficiência , Enzimas Desubiquitinantes/metabolismo , Modelos Animais de Doenças , Metabolismo Energético , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interleucina-15/genética , Melanoma Experimental , Camundongos , Camundongos Transgênicos , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Interleucina-15/metabolismo , Tolerância a Antígenos Próprios/genética , Tolerância a Antígenos Próprios/imunologia , Transdução de Sinais , Especificidade do Receptor de Antígeno de Linfócitos T , Ubiquitinação
5.
Environ Toxicol Pharmacol ; 71: 103214, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31252312

RESUMO

Lipololysaccharides (LPS) can disrupt the gut barrier. How dose LPS affects the immune performance of mesenteric lymph nodes? The results showed the hematological parameters significantly changed after LPS treatment. The length of intestinal villus was shortened and the depth of crypts was deepened, especially on the ileum. After LPS treatment 6 h, 12 h, the number of CD3+ T cells and CD4/CD8 in the mesenteric lymph nodes of ileum were reduced significantly; the levels of IFN-γ, TNF-ɑ and IL-2 were significantly decreased, and the levels of IL-6 and IL-10 were significantly increased in the ileum. The content of sIgA in the ileum was significantly decreased after LPS treatment 3 h, 6 h and was increased after LPS treatment 12 h. LPS through mesenteric lymph nodes, which induces the immune function reduced and the ileum injured obviously after treatment 6 h. Furthermore, the performance of intestinal immune performance was the lowest after LPS treatment 6 h.

6.
Org Lett ; 21(13): 5051-5054, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31199154

RESUMO

Ochrocephalamines B-D (1-3), composed of fused quinolizidine and octahydroquinoline rings, were isolated from Oxytropis ochrocephala Bunge. Ochrocephalamine B (1) has a unique bridged tetracyclic ring skeleton fused with a lactam ring. The structures of 1-3 were elucidated using spectroscopic and computational approaches. Ochrocephalamine C (2) and D (3) demonstrated potent anti-HBV activities and are more potent against the secretion of HBeAg than that of HBsAg.

7.
Nature ; 569(7758): 718-722, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31118511

RESUMO

Nucleic acids from bacteria or viruses induce potent immune responses in infected cells1-4. The detection of pathogen-derived nucleic acids is a central strategy by which the host senses infection and initiates protective immune responses5,6. Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA sensor7,8. It catalyses the synthesis of cyclic GMP-AMP (cGAMP)9-12, which stimulates the induction of type I interferons through the STING-TBK1-IRF-3 signalling axis13-15. STING oligomerizes after binding of cGAMP, leading to the recruitment and activation of the TBK1 kinase8,16. The IRF-3 transcription factor is then recruited to the signalling complex and activated by TBK18,17-20. Phosphorylated IRF-3 translocates to the nucleus and initiates the expression of type I interferons21. However, the precise mechanisms that govern activation of STING by cGAMP and subsequent activation of TBK1 by STING remain unclear. Here we show that a conserved PLPLRT/SD motif within the C-terminal tail of STING mediates the recruitment and activation of TBK1. Crystal structures of TBK1 bound to STING reveal that the PLPLRT/SD motif binds to the dimer interface of TBK1. Cell-based studies confirm that the direct interaction between TBK1 and STING is essential for induction of IFNß after cGAMP stimulation. Moreover, we show that full-length STING oligomerizes after it binds cGAMP, and highlight this as an essential step in the activation of STING-mediated signalling. These findings provide a structural basis for the development of STING agonists and antagonists for the treatment of cancer and autoimmune disorders.

8.
Res Vet Sci ; 124: 317-320, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31030119

RESUMO

Aconitine, a major aconitum alkaloid, is well known for its high toxicity that induces severe arrhythmias and neurological symptoms. One mechanism of aconitine-induced toxic responses is the induction of apoptosis. Apoptosis and autophagy are interconnected processes and the two pathways share critical components. In this study, we investigated the role of autophagy in aconitine-induced toxicity using mouse model. 120 mice were randomly divided into 4 experimental groups (normal saline), low dose group (0.14 µmol/L), medium dose group (0.28 µmol/L) and high dose group (0.56 µmol/ L). 30 mice in each group were administered with aconitine (lavage) for 30 days. The livers were collected for analysis of autophagy-related proteins by Western blotting. The expression of LC3II/LC3I ratio and Beclin 1 were found to increase and then decrease with the highest expression at 10 days and the p62 showed a time-dependent decreases. Autophagy is regulated by the mTOR pathway, we further analyzed the effects of aconitine on this pathway and found aconitine inhibited, phosphorylation of p-PI3K, p-Akt and p-mTOR. The p-p70s6k and p-4EBP1 which are downstream of mTOR were concomitantly decreased. These results suggest that aconitine induce autophagy in mouse liver. The PI3K/Akt/mTOR signaling pathway is involved in the regulation of aconitine-induced autophagy in the liver of mice.


Assuntos
Aconitina/toxicidade , Autofagia/genética , Fígado/efeitos dos fármacos , Transdução de Sinais/genética , Animais , Relação Dose-Resposta a Droga , Feminino , Fígado/fisiologia , Masculino , Camundongos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Agonistas do Canal de Sódio Disparado por Voltagem/toxicidade
9.
Immunity ; 50(3): 591-599.e6, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30893587

RESUMO

Immune suppression is a crucial component of immunoregulation and a subgroup of nucleotide-binding domain (NBD), leucine-rich repeat (LRR)-containing proteins (NLRs) attenuate innate immunity. How this inhibitory function is controlled is unknown. A key question is whether microbial ligands can regulate this inhibition. NLRC3 is a negative regulator that attenuates type I interferon (IFN-I) response by sequestering and attenuating stimulator of interferon genes (STING) activation. Here, we report that NLRC3 binds viral DNA and other nucleic acids through its LRR domain. DNA binding to NLRC3 increases its ATPase activity, and ATP-binding by NLRC3 diminishes its interaction with STING, thus licensing an IFN-I response. This work uncovers a mechanism wherein viral nucleic acid binding releases an inhibitory innate receptor from its target.


Assuntos
DNA Viral/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interferon Tipo I/metabolismo , Proteínas de Membrana/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Imunidade Inata/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Ácidos Nucleicos/metabolismo , Ligação Proteica/imunologia
10.
BMC Microbiol ; 19(1): 35, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30744547

RESUMO

BACKGROUND: The indolizidine alkaloid-swainsonine is produced by an endophytic fungus Alternaria oxytropis, which was isolated from locoweeds. Swainsonine has many biological activities such as anti-tumorigenic, anti-viral and bacteriostatic. However, the full complement of metabolites produced by Alternaria oxytropis is not known. This study is a chemical analysis of Alternaria oxytropis metabolites, which not only unravels the potential compounds from the fermentation broth but also in which solvent are they extracted, facilitating industrial application. RESULTS: Alternaria oxytropis isolated from Oxytropis gansuensis was cultured in Czapek's medium for 30d to collect the fermentation broth. The fermentation broth is treated with methanol and then evaporated to dryness to obtain a concentrate of the fermentation broth. The concentrate is added with water for the subsequent fractional extraction with petroleum ether, chloroform, ethyl acetate and n-butanol. Different fractions of the extract were eluted by wet packing and dry loading. The obtained eluate was combined by TLC to detect the same fraction, and then characterized by GC-MS and LC-MS. The results of GC-MS showed that 105 different compounds existed in the petroleum ether, chloroform, and ethyl acetate phases of Alternaria oxytropis fermentation broth. Moreover, the results of LC-MS indicated that the fermentation broth of Alternaria oxytropis contained five alkaloids, 2-hydroxy-indolizidine, retronecine, lentiginosine, swainsonine and swainsonine N-oxide. CONCLUSIONS: In addition to swainsonine and swainsonine N-oxide, 2-hydroxy-indolizidine, retronecine and lentiginosine were identified as the secondary metabolites of Alternaria oxytropis. Other compounds were also detected including 5,6-dihydroergosterol, eburicol, lanosterol, and L-phenylalanyl-L-proline lactam, which have potential applications as drugs.

11.
Immunity ; 50(1): 51-63.e5, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30635239

RESUMO

Interferon-inducible human oligoadenylate synthetase-like (OASL) and its mouse ortholog, Oasl2, enhance RNA-sensor RIG-I-mediated type I interferon (IFN) induction and inhibit RNA virus replication. Here, we show that OASL and Oasl2 have the opposite effect in the context of DNA virus infection. In Oasl2-/- mice and OASL-deficient human cells, DNA viruses such as vaccinia, herpes simplex, and adenovirus induced increased IFN production, which resulted in reduced virus replication and pathology. Correspondingly, ectopic expression of OASL in human cells inhibited IFN induction through the cGAS-STING DNA-sensing pathway. cGAS was necessary for the reduced DNA virus replication observed in OASL-deficient cells. OASL directly and specifically bound to cGAS independently of double-stranded DNA, resulting in a non-competitive inhibition of the second messenger cyclic GMP-AMP production. Our findings define distinct mechanisms by which OASL differentially regulates host IFN responses during RNA and DNA virus infection and identify OASL as a negative-feedback regulator of cGAS.


Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Infecções por Vírus de DNA/imunologia , Vírus de DNA/fisiologia , Infecções por Vírus de RNA/imunologia , Vírus de RNA/imunologia , 2',5'-Oligoadenilato Sintetase/genética , Animais , AMP Cíclico/metabolismo , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nucleotidiltransferases/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais , Células THP-1 , Replicação Viral
12.
Biol Trace Elem Res ; 2018 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-30414002

RESUMO

Strontium (Sr) can reduce cartilage degeneration and stimulate cartilage matrix formation. Angiogenesis plays a developmental role in chondrogenesis, and was stimulated by growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2). However, the direct influence of Sr on VEGF and FGF2 expressions in chondrocytes is not entirely clear. The objective of this study was to investigate the effects of different Sr concentrations on VEGF and FGF2 expressions in rat chondrocytes in vitro. Chondrocytes were isolated from Wistar rat articular by enzymatic digestion. As a Sr source, strontium chloride hexahydrate (SrCl2·6H2O) was added to the culture solution at final concentrations of 0, 0.5, 1.0, 2.0, 5.0, 20.0, and 100.0 µg/mL. After 72 h of continuous culture, mRNA abundance and protein expression levels of VEGF and FGF2 in the chondrocytes were determined by real-time polymerase chain reaction (real-time PCR) and Western blot, respectively. The results showed that VEGF and FGF2 expressions were dose-dependently elevated with Sr concentration in chondrocytes. The mRNA abundance and protein expression levels of VEGF were extremely significantly higher than those in the control group (P < 0.01) at 1.0 µg/mL Sr treatment. For FGF2, there were markedly significant differences in mRNA and protein expression from control group (P < 0.01) when the Sr-treated concentration exceeded 5.0 µg/mL and 20.0 µg/mL, respectively. These results indicated that Sr might involve in the cartilage angiogenesis via regulating expression of VEGF and FGF2z.

13.
Cell ; 175(2): 488-501.e22, 2018 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-30270045

RESUMO

Detection of viruses by innate immune sensors induces protective antiviral immunity. The viral DNA sensor cyclic GMP-AMP synthase (cGAS) is necessary for detection of HIV by human dendritic cells and macrophages. However, synthesis of HIV DNA during infection is not sufficient for immune activation. The capsid protein, which associates with viral DNA, has a pivotal role in enabling cGAS-mediated immune activation. We now find that NONO is an essential sensor of the HIV capsid in the nucleus. NONO protein directly binds capsid with higher affinity for weakly pathogenic HIV-2 than highly pathogenic HIV-1. Upon infection, NONO is essential for cGAS activation by HIV and cGAS association with HIV DNA in the nucleus. NONO recognizes a conserved region in HIV capsid with limited tolerance for escape mutations. Detection of nuclear viral capsid by NONO to promote DNA sensing by cGAS reveals an innate strategy to achieve distinction of viruses from self in the nucleus.

14.
BMC Vet Res ; 14(1): 236, 2018 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-30103741

RESUMO

BACKGROUND: During peripartum period, dairy cows are highly susceptible to energy metabolism disorders such as fatty liver and ketosis. Angiopoietin-like protein 4 (ANGPTL4) and fibroblast growth factor 21 (FGF21), known as hepatokines, play important roles in lipid metabolism. The purposes of our study were to evaluate variations of serum ANGPTL4 and FGF21 concentrations in periparturient dairy cows and changes in these serum analyte concentrations of energy-related metabolic disorders in early lactation dairy cows. This study was divided into two experiments. Experiment I: Blood parameters were measured in healthy periparturient Holstein cows from 4 wk antepartum to 4 wk postpartum (n = 219). In this experiment, weekly blood samples were obtained from 4 wk before the expected calving date through 4 wk after calving. Experiment II: Blood parameters were measured in healthy cows (n = 30) and cows with clinical ketosis (n = 29) and fatty liver (n = 25) within the first 4 wk of lactation. In the present study, all blood samples were collected from the coccygeal vein in the early morning before feeding. RESULTS: Serum ANGPTL4 and FGF21 concentrations peaked at parturition, and declined rapidly over the following 2 wk Serum ANGPTL4 and FGF21 concentrations were positively correlated with serum non-esterified fatty acids (NEFA) concentration (r = 0.856, P = 003; r = 0.848, P = 0.004, respectively). Cows with clinical ketosis and fatty liver had significantly higher serum ANGPTL4 and FGF21 concentrations than healthy cows (P < 0.01). CONCLUSION: Serum ANGPTL4 and FGF21 concentrations were elevated during peripartum period, suggesting that energy balance changes that were associated with parturition contributed significantly to these effects. Although FGF21 and ANGPTL4 could play important roles in the adaptation of energy metabolism, they may be involved in the pathological processes of energy metabolism disorders of dairy cows in the peripartum period.


Assuntos
Proteína 4 Semelhante a Angiopoietina/sangue , Doenças dos Bovinos/sangue , Fatores de Crescimento de Fibroblastos/sangue , Doenças Metabólicas/veterinária , Ácido 3-Hidroxibutírico/sangue , Ácido 3-Hidroxibutírico/metabolismo , Animais , Bovinos , Doenças dos Bovinos/metabolismo , Ácidos Graxos não Esterificados/sangue , Ácidos Graxos não Esterificados/metabolismo , Feminino , Doenças Metabólicas/sangue , Doenças Metabólicas/metabolismo , Parto/sangue , Parto/metabolismo , Período Pós-Parto/sangue , Período Pós-Parto/metabolismo , Gravidez/sangue
15.
J Dairy Sci ; 101(9): 8513-8523, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29960773

RESUMO

Ketosis causes serious economic losses for the modern dairy industry because it is a highly prevalent metabolic disease among cows in high-producing herds during the transition period. Due to some striking similarities between diabetes in humans and ketosis in dairy cows, there is potential for the use of methylglyoxal (MGO)-commonly used in human diabetics-as a biomarker in dairy cattle. However, currently no data are available about the presence of MGO in the serum of dairy cattle or about the characteristics of its production or its potential contribution in the pathogenesis of ketosis. To determine the potential origin and pathway of formation of MGO, cows in different metabolic conditions [i.e., non-subclinically ketotic dairy cows in early lactation (n = 7), subclinically ketotic dairy cows in early lactation (n = 8), overconditioned dry cows (BCS >4.25, n = 6), and nonlactating heifers (n = 6)] were selected. Serum MGO concentrations were determined and correlated with indicators of the glucose and lipid metabolism and with haptoglobin (Hp) as an inflammatory marker. The serum MGO concentrations in subclinically ketotic cows (712.60 ± 278.77 nmol/L) were significantly greater than in nonlactating heifers (113.35 ± 38.90 nmol/L), overconditioned dry cows (259.71 ± 117.97 nmol/L), and non-subclinically ketotic cows (347.83 ± 63.56 nmol/L). In serum of lactating cows, concentrations of glucose and fructosamine were lower than in heifers and were negatively correlated with MGO concentrations. Even so, concentrations of metabolic and inflammatory markers such as dihydroxyacetone phosphate, nonesterified fatty acids, ß-hydroxybutyrate, acetone, and Hp were remarkably higher in subclinically ketotic cows compared with nonlactating heifers; these metabolites were also positively correlated with MGO. In human diabetics elevated MGO concentrations are stated to originate from both hyperglycemia and the enhanced lipid metabolism, whereas higher MGO concentrations in subclinically ketotic cows were not associated with hyperglycemia. Therefore, our data suggest MGO in dairy cows to be a metabolite produced from the metabolization of acetone within the lipid metabolization pathway and from the metabolization of dihydroxyacetone phosphate. Furthermore, the highly positive correlation between MGO and Hp suggests that this reactive compound might be involved in the proinflammatory state of subclinical ketosis in dairy cows. However, more research is needed to determine the potential use of MGO as a biomarker for metabolic failure in dairy cows.

16.
Nature ; 557(7706): 516-521, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29769717

RESUMO

Chromatin remodelling factors (CHRs) typically function to alter chromatin structure. CHRs also reside in ribonucleoprotein complexes, but little is known about their RNA-related functions. Here we show that CHR2 (also known as BRM), the ATPase subunit of the large switch/sucrose non-fermentable (SWI/SNF) complex, is a partner of the Microprocessor component Serrate (SE). CHR2 promotes the transcription of primary microRNA precursors (pri-miRNAs) while repressing miRNA accumulation in vivo. Direct interaction with SE is required for post-transcriptional inhibition of miRNA accumulation by CHR2 but not for its transcriptional activity. CHR2 can directly bind to and unwind pri-miRNAs and inhibit their processing, and this inhibition requires the remodelling and helicase activity of CHR2 in vitro and in vivo. Furthermore, the secondary structures of pri-miRNAs differed between wild-type Arabidopsis thaliana and chr2 mutants. We conclude that CHR2 accesses pri-miRNAs through SE and remodels their secondary structures, preventing downstream processing by DCL1 and HYL1. Our study uncovers pri-miRNAs as a substrate of CHR2, and an additional regulatory layer upstream of Microprocessor activity to control miRNA accumulation.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , MicroRNAs/biossíntese , Proteínas de Ligação a RNA/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , MicroRNAs/metabolismo , Ligação Proteica , Dobramento de RNA , Processamento Pós-Transcricional do RNA , Transcrição Genética
17.
Biophys J ; 114(6): 1313-1320, 2018 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-29590589

RESUMO

Many intrinsically disordered proteins (IDPs) form fuzzy complexes upon binding to their targets. Although many IDPs are weakly bound in fuzzy complexes, some IDPs form high-affinity complexes. One example is the nonstructural protein 1 (NS1) of the 1918 Spanish influenza A virus, which hijacks cellular CRKII through the strong binding affinity (Kd ∼10 nM) of its proline-rich motif (PRMNS1) to the N-terminal Src-homology 3 domain of CRKII. However, its molecular mechanism remains elusive. Here, we examine the interplay between structural disorder of a bound PRMNS1 and its long-range electrostatic interactions. Using x-ray crystallography and NMR spectroscopy, we found that PRMNS1 retains substantial conformational flexibility in the bound state. Moreover, molecular dynamics simulations showed that structural disorder of the bound PRMNS1 increases the number of electrostatic interactions and decreases the mean distances between the positively charged residues in PRMNS1 and the acidic residues in the N-terminal Src-homology 3 domain. These results are analyzed using a polyelectrostatic model. Our results provide an insight into the molecular recognition mechanism for a high-affinity fuzzy complex.

18.
J Ethnopharmacol ; 213: 376-383, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29102763

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Sophora alopecuroides L., a traditional Chinese herb, has been widely used to treat numerous diseases throughout China. Quinolizidine alkaloids were identified as active components in Sophora alopecuroides L., and Sophoridine (SRI) is the major component in the Quinolizidine alkaloids. AIM OF THE STUDY: To investigate the toxic effects of SRI in rat liver BRL-3A cells and to explore potential ROS-related mechanisms. MATERIALS AND METHODS: Cell viability, cytotoxicity, apoptosis, intracellular generation of ROS, GSH/GSSG ratio and levels of proteins in mitochondria apoptosis pathway were analyzed. RESULTS: Our data indicated that SRI could suppress BRL-3A cells viability in a concentration- and time-dependent manner and increase cytotoxicity, ROS accumulation and cell apoptosis in a concentration-dependent manner. Expressions and activities of apoptotic related proteins were upregulated, whereas expression of Bcl-2 was downregulated after treatment. Furthermore, level of H2O2 was increased, whereas level of Superoxide was not changed after treatment. Moreover, the antioxidant N-acetylcysteine reversed SRI-induced apoptosis and ROS accumulation. CONCLUSION: Our data suggest that SRI promotes rat liver BRL-3A cells apoptosis by increasing intracellular ROS accumulation.


Assuntos
Alcaloides/farmacologia , Apoptose/efeitos dos fármacos , Fígado/citologia , Fígado/efeitos dos fármacos , Quinolizinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Alcaloides/antagonistas & inibidores , Animais , Proteínas Reguladoras de Apoptose/biossíntese , Contagem de Células , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação para Baixo , Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Fígado/metabolismo , Mitocôndrias/efeitos dos fármacos , Quinolizinas/antagonistas & inibidores , Ratos , Proteína X Associada a bcl-2/biossíntese
19.
Biol Trace Elem Res ; 184(2): 450-455, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29170863

RESUMO

The transforming growth factors ß1 (TGF-ß1) and TGF-ß2, as two distinct homodimers of TGF-ß superfamily, involve in chondrocyte growth and differentiation. Emerging evidence has implied that strontium (Sr) plays an important role in the bone formation and resorption, and has strong effects on stimulating human cartilage matrix formation in vitro. However, the direct effects of Sr on TGF-ß1 and TGF-ß2 expressions in chondrocytes are not entirely clear. The purpose of this study was to evaluate the influence of different Sr concentrations on the expression of TGF-ß1 and TGF-ß2 in rat chondrocytes in vitro. Chondrocytes were isolated from Wistar rat articular by enzymatic digestion. Strontium chloride hexahydrate (SrCl2·6H2O) was used as a Sr source in this study. Sr was added to the culture solution at final concentrations of 0, 0.5, 1.0, 2.0, 5.0, 20.0, and 100 µg/mL. After 72 h of continuous culture, TGF-ß1 and TGF-ß2 mRNA abundance and protein expression levels in the chondrocytes were determined by real-time polymerase chain reaction (real-time PCR) and Western blot, respectively. The results showed that TGF-ß1 and TGF-ß2 expressions in chondrocytes increased dose-dependently with Sr concentration. The mRNA abundance of TGF-ß1 and TGF-ß2 were markedly higher than those observed for control (P < 0.01) when the Sr-treated concentration exceeded 1.0 and 5.0 µg/mL, respectively. The TGF-ß1 and TGF-ß2 protein expression levels were extremely significantly higher than those in the control group (P < 0.01) at above 5.0 µg/mL Sr-treatment. These results indicated that Sr could involve in the chondrocytes metabolism via regulating TGF-ß1 and TGF-ß2 signalling.


Assuntos
Expressão Gênica/efeitos dos fármacos , Estrôncio/farmacologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta2/genética , Animais , Western Blotting , Células Cultivadas , Relação Dose-Resposta a Droga , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta2/metabolismo
20.
Antioxid Redox Signal ; 28(5): 385-400, 2018 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-28027652

RESUMO

AIMS: Many men endure immunosuppressive or anticancer treatments that contain alkylating agents before the age of sexual maturity, especially the increasing number of preadolescent males who undergo busulfan treatment for myeloablative conditioning before hematopoietic stem cell transplantation. Before sperm production, there are no sperm available for cryopreservation. Thus, it is necessary to identify a solution to ameliorate the busulfan-induced damage of spermatogonial stem cells (SSCs). RESULTS: In this study, we demonstrated that melatonin relieved the previously described SSC loss and apoptosis in mouse testes. Melatonin increased the expression of manganese superoxide dismutase (MnSOD), which regulated the production of busulfan-induced reactive oxygen species (ROS). Moreover, melatonin promoted sirtuin type 1 (SIRT1) expression. SIRT1 participated in the deacetylation of p53, which promotes p53 ubiquitin degradation. Decreased concentrations of deacetylated p53 resulted in spermatogonial cell resistance to apoptosis. Acute T cell leukemia cell assay demonstrated that melatonin does not affect busulfan-induced cancer cell apoptosis and ROS. INNOVATION: The current evidence suggests that melatonin may alleviate the side effects of alkylating drugs, such as busulfan. CONCLUSION: Melatonin promoted MnSOD and SIRT1 expression, which successfully ameliorated busulfan-induced SSC apoptosis caused by high concentrations of ROS and p53. Antioxid. Redox Signal. 28, 385-400.


Assuntos
Melatonina/administração & dosagem , Sirtuína 1/genética , Superóxido Dismutase/genética , Testículo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Bussulfano/efeitos adversos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Masculino , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Espermatogônias/efeitos dos fármacos , Espermatogônias/crescimento & desenvolvimento , Espermatogônias/patologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/patologia , Testículo/crescimento & desenvolvimento , Testículo/patologia , Proteína Supressora de Tumor p53/genética , Ubiquitina/genética
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