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1.
J Integr Plant Biol ; 2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33438356

RESUMO

Heterochromatin is widespread in eukaryotic genomes and has diverse impacts depending on its genomic context. Previous studies have shown that a protein complex, the ASI1-AIPP1-EDM2 (AAE) complex, participates in polyadenylation regulation of several intronic heterochromatin-containing genes. However, the genome-wide functions of AAE are still unknown. Here, we show that the ASI1 and EDM2 mostly target the common genomic regions on a genome-wide level and preferentially interacts with genetic heterochromatin. Polyadenylation [(poly(A)] sequencing reveals that AAE complex has a substantial influence on poly(A) site usage of heterochromatin-containing genes, including not only intronic heterochromatin-containing genes but also the genes showing overlap with heterochromatin. Intriguingly, AAE is also involved in the alternative splicing regulation of a number of heterochromatin-overlapping genes, such as the disease resistance gene RPP4. We provided evidence that genic heterochromatin is indispensable for the recruitment of AAE in polyadenylation and splicing regulation. In addition to conferring RNA processing regulation at genic heterochromatin-containing genes, AAE also targets some transposable elements (TEs) outside of genes (including TEs sandwiched by genes and island TEs) for epigenetic silencing. Our results reveal new functions of AAE in RNA processing and epigenetic silencing, and thus represent important advances in epigenetic regulation. This article is protected by copyright. All rights reserved.

2.
Nat Commun ; 11(1): 6212, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33277495

RESUMO

Histone 3 Lys 27 trimethylation (H3K27me3)-mediated epigenetic silencing plays a critical role in multiple biological processes. However, the H3K27me3 recognition and transcriptional repression mechanisms are only partially understood. Here, we report a mechanism for H3K27me3 recognition and transcriptional repression. Our structural and biochemical data showed that the BAH domain protein AIPP3 and the PHD proteins AIPP2 and PAIPP2 cooperate to read H3K27me3 and unmodified H3K4 histone marks, respectively, in Arabidopsis. The BAH-PHD bivalent histone reader complex silences a substantial subset of H3K27me3-enriched loci, including a number of development and stress response-related genes such as the RNA silencing effector gene ARGONAUTE 5 (AGO5). We found that the BAH-PHD module associates with CPL2, a plant-specific Pol II carboxyl terminal domain (CTD) phosphatase, to form the BAH-PHD-CPL2 complex (BPC) for transcriptional repression. The BPC complex represses transcription through CPL2-mediated CTD dephosphorylation, thereby causing inhibition of Pol II release from the transcriptional start site. Our work reveals a mechanism coupling H3K27me3 recognition with transcriptional repression through the alteration of Pol II phosphorylation states, thereby contributing to our understanding of the mechanism of H3K27me3-dependent silencing.

3.
Biotechnol Bioeng ; 117(7): 2279-2294, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32175589

RESUMO

Beyond their widespread application as genome-editing and regulatory tools, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems also play a critical role in nucleic acid detection due to their high sensitivity and specificity. Recently developed Cas family effectors have opened the door to the development of new strategies for detecting different types of nucleic acids for a variety of purposes. Precise and efficient nucleic acid detection using CRISPR-Cas systems has the potential to advance both basic and applied biological research. In this review, we summarize the CRISPR-Cas systems used for the recognition and detection of specific nucleic acids for different purposes, including the detection of genomic DNA, nongenomic DNA, RNA, and pathogenic microbe genomes. Current challenges and further applications of CRISPR-based detection methods will be discussed according to the most recent developments.

4.
Comput Struct Biotechnol J ; 17: 904-916, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31346383

RESUMO

Over the decades, the biological role of microRNAs (miRNAs) in the post-transcriptional regulation of gene expression has been discovered in many cancer types, thus initiating the tremendous expectation of their application as biomarkers in the diagnosis, prognosis, and treatment of cancer. Hence, the development of efficient miRNA detection methods in vitro is in high demand. Extensive efforts have been made based on the intrinsic properties of miRNAs, such as low expression levels, high sequence homology, and short length, to develop novel in vitro miRNA detection methods with high accuracy, low cost, practicality, and multiplexity at point-of-care settings. In this review, we mainly summarized the newly developed in vitro miRNA detection methods classified by three key elements, including biological recognition elements, additional micro-/nano-materials and signal transduction/readout elements, their current challenges and further applications are also discussed.

5.
J Biotechnol ; 296: 69-74, 2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-30885657

RESUMO

Synthetic scaffold systems, which exhibit enzyme clustering effect, have been considered as an important parallel approach for metabolic flux control and pathway enhancement. Here, we described an improved DNA-based scaffold system for synthetic tri-enzymatic pathway in Escherichia coli. With plasmid DNA serving as scaffold and exogenous enzymes fused with rationally designed transcription activator-like effectors (TALEs), our approach successfully clustered three TALE-fused enzymes and significantly increased the production of a mevalonate-producing tri-enzymatic pathway with the optimized scaffold structure and plasmid copy number. These results further suggested the scalability and robustness of the TALE-based scaffold system, and we can assume that it can be used on numerous multi-enzyme metabolic pathways due to its programmable features.


Assuntos
DNA/genética , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Efetores Semelhantes a Ativadores de Transcrição/química , DNA/química , Escherichia coli/genética , Ácido Mevalônico/química , Ácido Mevalônico/metabolismo , Plasmídeos/genética , Efetores Semelhantes a Ativadores de Transcrição/genética
6.
Microb Cell Fact ; 17(1): 120, 2018 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-30064437

RESUMO

For years, microbes have been widely applied as chassis in the construction of synthetic metabolic pathways. However, the lack of in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. In recent years, multiple methods have been applied to the construction of small metabolic clusters by spatial organization of heterologous metabolic enzymes. These methods mainly focused on using engineered molecules to bring the enzymes into close proximity via different interaction mechanisms among proteins and nucleotides and have been applied in various heterologous pathways with different degrees of success while facing numerous challenges. In this paper, we mainly reviewed some of those notable advances in designing and creating approaches to achieve spatial organization using different intermolecular interactions. Current challenges and future aspects in the further application of such approaches are also discussed in this paper.


Assuntos
Engenharia Metabólica/métodos , Redes e Vias Metabólicas
7.
ACS Synth Biol ; 7(3): 807-813, 2018 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-29486117

RESUMO

MicroRNAs have been reported as related to multiple diseases and have potential applications in diagnosis and therapeutics. However, detection of miRNAs remains improvable, given their complexity, high cost, and low sensitivity as of currently. In this study, we attempt to build a novel platform that detects miRNAs at low cost and high efficacy. This detection system contains isothermal amplification, detecting and reporting process based on rolling circle amplification, CRISPR-Cas9, and split-horseradish peroxidase techniques. It is able to detect trace amount of miRNAs from samples with mere single-base specificity. Moreover, we demonstrated that such scheme can effectively detect target miRNAs in clinical serum samples and significantly distinguish patients of non-small cell lung cancer from healthy volunteers by detecting the previously reported biomarker: circulating let-7a. As the first to use CRISPR-Cas9 in miRNA detection, this method is a promising approach capable of being applied in screening, diagnosing, and prognosticating of multiple diseases.


Assuntos
Sistemas CRISPR-Cas/genética , Custos e Análise de Custo , Técnicas Genéticas/economia , MicroRNAs/análise , MicroRNAs/economia , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/genética , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , MicroRNAs/genética , Sondas RNA/metabolismo
8.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 25(4): 992-997, 2017 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-28823257

RESUMO

OBJECTIVE: To investigate the clinical characteristics of 31 acute myeloid leukemia (AML) patients with chromosome 21 aberrations. METHODS: Karyotypes of 168 newly diagnosed AML patients in Second Xiangya Hospital from Jan 2014 to July 2016 were reviewed for the presence of chromosome 21 aberrations (accounting for 18.45%). Clinical manifestation, as well as prognostic gene mutations distribution and immune classification were analyzed. RESULTS: Out of 168 AML newly diagnosed patients, 31 cases with chromosome 21 aberrations including t(8;21) accounting for 67.74% (21/31), and trisomy 21 (16.13%,5/31), 2 variants were found as t(1; 21) and t(1; 21; 8); 77 cases had normal karyotype, and 60 cases possessed other chromosomes aberrations. Statistically significant differences did not exist among age, sex and white blood cell count (P>0.05). However, the 21 cases in chromosome aberrations group were predisposed to lower hemoglobin and platelet count(P<0.05). 5 cases of Trisomy 21 were characterized by M5 2 cases, M1 one case, M2 one case M4 one case. And the rate of C-kit/D816V mutation was higher in t(8;21) aberrations group when 7 prognostic genes including FLT3/ITD, C-kit/D816V, NPM1, DNMT3A, TET2 were analyzed, and the immune classification of t(8; 21) aberration group inclined to CD19+, CD34+ but CD33-, CD64-. And trisomy 21 displayed a trend to CD34+ and CD7+. CONCLUSION: Chromosome 21 is easily involved in acute myeloid leukemia. The patients with involvement of this aberration have characteristic clinic changes.


Assuntos
Cromossomos Humanos Par 21 , Leucemia Mieloide Aguda , Aberrações Cromossômicas , Humanos , Cariotipagem , Mutação , Prognóstico , Tirosina Quinase 3 Semelhante a fms
9.
J Membr Biol ; 250(3): 315-325, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28597209

RESUMO

Tarantula toxins compose an important class of spider toxins that target ion channels, and some are known to interact with lipid membranes. In this study, we focus on a tarantula toxin, Jingzhaotoxin-III (JZTx-III) that specifically targets the cardiac voltage-gated sodium channel Na[Formula: see text]1.5 and is suspected to be able to interact with lipid membranes. Here, we use an all-atom model and long-term molecular dynamics simulations to investigate the interactions between JZTx-III and lipid membranes of different compositions. Trajectory analyses show that JZTx-III has no substantial interaction with the neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids, but binds to membranes containing negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG). The most intriguing observations in our simulation are the different interactions between the toxin and the membrane in the mixed and pure POPG membrane systems. The POPC/POPG mixed membrane undergoes a phase transition to a rippled phase upon binding of the toxin, while the pure POPG membrane has no apparent change. Moreover, the binding of JZTx-III to both of the mixture and the pure POPG membrane systems induce small conformational changes. The sequence alignment shows that JZTx-III may not partition into the lipid bilayer due to the mutations of a C-terminal hydrophobic residue and some charged residues that affect toxin orientation. Taken together, JZTx-III and lipid membranes have unique effects on each other that may facilitate the specific binding of JZTx-III to Na[Formula: see text]1.5. This computational study also enriches our understanding of the potential complex interactions between spider toxins and lipid membranes.


Assuntos
Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Simulação de Dinâmica Molecular , Venenos de Aranha/química , Potenciais da Membrana , Fosfatidilcolinas/química
10.
Chin Med J (Engl) ; 128(22): 3050-4, 2015 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-26608985

RESUMO

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disease characterized by recurrent epistaxis, mucocutaneous telangiectasia, and arteriovenous malformations. The efficacy of traditional treatments for HHT is very limited. The aim of this study was to investigate the therapeutic role of thalidomide in HHT patients and the effect in FLI-EGFP transgenic zebrafish model. METHODS: HHT was diagnosed according to Shovlin criteria. Five HHT patients were treated with thalidomide (100 mg/d). The Epistaxis Severity Score (ESS), telangiectasia spots, and hepatic computed tomography angiography (CTA) were used to assess the clinical efficacy of thalidomide. The Fli-EGFP zebrafish model was investigated for the effect of thalidomide on angiogenesis. Dynamic real-time polymerase chain reaction assay, ELISA and Western blotting from patient's peripheral blood mononuclear cells and plasma were used to detect the expression of transforming growth factor beta 3 (TGF-ß3) messenger RNA (mRNA) and vascular endothelial growth factor (VEGF) protein before and after 6 months of thalidomide treatment. RESULTS: The average ESS before and after thalidomide were 6.966 ± 3.093 and 1.799 ± 0.627, respectively (P = 0.009). The "telangiectatic spot" on the tongue almost vanished; CTA examination of case 2 indicated a smaller proximal hepatic artery and decreased or ceased hepatic artery collateral circulation. The Fli-EGFP zebrafish model manifested discontinuous vessel development and vascular occlusion (7 of 10 fishes), and the TGF-ß3 mRNA expression of five patients was lower after thalidomide therapy. The plasma VEGF protein expression was down-regulated in HHT patients. CONCLUSIONS: Thalidomide reverses telangiectasia and controls nosebleeds by down-regulating the expression of TGF-ß3 and VEGF in HHT patients. It also leads to vascular remodeling in the zebrafish model.


Assuntos
Proteínas de Fluorescência Verde/metabolismo , Telangiectasia Hemorrágica Hereditária/tratamento farmacológico , Talidomida/uso terapêutico , Animais , Animais Geneticamente Modificados , Feminino , Proteínas de Fluorescência Verde/genética , Humanos , Pessoa de Meia-Idade , Telangiectasia Hemorrágica Hereditária/metabolismo , Fator de Crescimento Transformador beta3/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra
11.
Plant Physiol Biochem ; 79: 19-24, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24675567

RESUMO

CP43 is a chlorophyll a (Chl a) and ß-carotene (ß-Car) binding protein encoded by psbC gene. In this study, psbC gene isolated from Spinach was expressed in Escherichia coli in soluble state. After lysis of the cells, the apoproteins purified by nickel affinity chromatography were examined by SDS-PAGE and Western-blot. Next, reconstitution experiment was carried out in vitro and the formation of stable pigment-protein complex was analyzed by partially denaturing electrophoresis. After purifying reconstituted CP43 (rCP43) from free pigments (FPs) by sucrose gradient ultracentrifugation and subsequently ion exchange chromatography (IEC), the eluate was analyzed by partially denaturing electrophoresis to confirm stability of the reconstructed complex. Finally, analyses of spectroscopic character of the eluate revealed that in vitro reconstitution was achieved and FPs were completely removed from the pigment-protein complex. Comparison between the absorption spectra of the rCP43 and native CP43 (nCP43) showed the lack of peaks between 450 and 500 nm, illustrating that the ß-Car was stripped off rCP43. In brief, it is feasible to obtain a reconstituted protein binding Chl a only, indicating that the occupancy of the ß-Car site has small impact on the stabilization of CP43. However, ß-Car shows strong interaction with Chl a, inducing the hyperchromic effect in blue region of spectrum and the blue shift of the 438.5 nm and 673.5 nm absorption band to 437 nm and 671 nm respectively. To some extent, our research is suggestive that ß-Car, coupled loosely with CP43, contributes to the precise orientation of Chl a in vivo.


Assuntos
Clorofila/metabolismo , Escherichia coli/metabolismo , Proteínas de Plantas/metabolismo , Spinacia oleracea/metabolismo , Clorofila A , Cromatografia por Troca Iônica , Escherichia coli/genética , Proteínas de Plantas/genética , Spinacia oleracea/genética
12.
Artigo em Inglês | MEDLINE | ID: mdl-20934373

RESUMO

Previously, the mechanism of the thermal unfolding of Pin1 (on-line measurements) was studied, revealing that Pin1 has a relatively high thermal stability. However, it is still questionable whether the unfolding of Pin1 is reversible. In the present work, intrinsic tryptophan fluorescence, ANS fluorescence, RLS, FTIR and CD spectroscopies are used to evaluate the reversibility of the thermal unfolding of Pin1. Intrinsic tryptophan fluorescence studies indicate that structural changes around tryptophan motifs in Pin1 are possibly reversible after heat treatment (even above 98°C), for no significant change in the intensity or λ(max) of the spectra was observed. ANS fluorescence measurements indicate the irreversible exposure of the hydrophobic clusters in Pin1 after heat treatment at 98°C, with increase in the fluorescence intensity and blue shift in λmax. Also, RLS signals of the Pin1-ANS system increased after heat treatment, possibly implying both the unfolding and the aggregation of Pin1. In addition, FTIR and CD results confirmed the irreversible unfolding of the secondary structure in Pin1 after heat treatment above 90°C, showing decreases in both α-helix and ß-sheet. In summary, the present work mainly suggests that heat treatment, especially above 90°C, has an important impact on the structural stability of Pin1, and the structural unfolding induced by heat was proved to be irreversible.


Assuntos
Temperatura Alta , Peptidilprolil Isomerase/química , Naftalenossulfonato de Anilina/química , Dicroísmo Circular , Interações Hidrofóbicas e Hidrofílicas , Luz , Estrutura Secundária de Proteína , Desdobramento de Proteína , Espalhamento de Radiação , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier , Triptofano
13.
Gene Regul Syst Bio ; 4: 135-48, 2010 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-21245947

RESUMO

The protein phosphatase-2A (PP-2A), one of the major phosphatases in eukaryotes, is a heterotrimer, consisting of a scaffold A subunit, a catalytic C subunit and a regulatory B subunit. Previous studies have shown that besides regulating specific PP-2A activity, various B subunits encoded by more than 16 different genes, may have other functions. To explore the possible roles of the regulatory subunits of PP-2A in vertebrate development, we have cloned the PR55/B family regulatory subunits: ß and δ, analyzed their tissue specific and developmental expression patterns in Goldfish ( Carassius auratus). Our results revealed that the full-length cDNA for PR55/Bß consists of 1940 bp with an open reading frame of 1332 nucleotides coding for a deduced protein of 443 amino acids. The full length PR55/Bδ cDNA is 2163 bp containing an open reading frame of 1347 nucleotides encoding a deduced protein of 448 amino acids. The two isoforms of PR55/B display high levels of sequence identity with their counterparts in other species. The PR55/Bß mRNA and protein are detected in brain and heart. In contrast, the PR55/Bδ is expressed in all 9 tissues examined at both mRNA and protein levels. During development of goldfish, the mRNAs for PR55/Bß and PR55/Bδ show distinct patterns. At the protein level, PR55/Bδ is expressed at all developmental stages examined, suggesting its important role in regulating goldfish development. Expression of the PR55/Bδ anti-sense RNA leads to significant downregulation of PR55/Bδ proteins and caused severe abnormality in goldfish trunk and eye development. Together, our results suggested that PR55/Bδ plays an important role in governing normal trunk and eye formation during goldfish development.

14.
Gene Regul Syst Bio ; 3: 115-29, 2009 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-19838339

RESUMO

SG2NA is a member of the striatin protein family. In human and mouse, the SG2NA gene encodes two major protein isoforms: SG2NA alpha and SG2NA beta. The functions of these proteins, except for acting as the regulatory subunits for PP-2A, remain largely unknown. To explore the possible functions of SG2NA in lower vertebrates, we have isolated two SG2NA cDNAs from goldfish, Carassius auratus. Our results reveal that the first cDNA contains an ORF of 2118 bp encoding a deduced protein with 705 amino acids, and the second one 2148 bp coding for a deduced protein of 715 amino acids. Comparative analysis reveals that both isoforms belong to the alpha-type, and are named SG2NA alpha and SG2NA alpha(+). RT-PCR and western blot analysis reveal that the SG2NA gene is differentially expressed in 9 tissues examined. During goldfish development, while the SG2NA mRNAs remain relatively constant in the first 3 stages and then become decreased and fluctuated from gastrula to larval hatching, the SG2NA proteins are fluctuated, displaying a peak every 3 to 4 stages. Each later peak is higher than the earlier one and the protein expression level becomes maximal at hatching stage. Together, our results reveal that SG2NA may play an important role during goldfish development and also in homeostasis of most adult tissues.

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