RESUMO
Although hexagonal boron nitride (h-BN) was initially considered a less promising photocatalyst due to its large band gap and apparent chemical inertness, its unique two-dimensional lamellar structure coupled with high stability and environmental friendliness, as the second largest van der Waals material after graphene, provides a unique platform for photocatalytic innovation. This review not only highlights the intrinsic qualities of h-BN with photocatalytic potentials, such as high stability, environmental compatibility, and tunable bandgap through various modification strategies but also provides a comprehensive overview of the recent advances in h-BN-based nanomaterials for environmental and energy applications, as well as an in-depth description of the modification methods and fundamental properties for these applications. In addition, we discuss the challenges and prospects of h-BN-based nanomaterials for future photocatalysis.
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Brd4 has been intensively investigated as a promising drug target because of its implicated functions in oncogenesis, inflammation, and HIV-1 transcription. The formation of the Brd4-P-TEFb (CDK9/Cyclin T1) complex and its regulation of transcriptional elongation are critical for HIV latency reactivation and expression of many oncogenes. To further investigate the mechanism of the Brd4-P-TEFb complex in controlling elongation, mass spectrometry-based quantitative proteomics of the CDK9 interactome was performed. Upon treatment with the selective BET bromodomain inhibitor JQ1, 352 proteins were successfully identified with high confidence as CDK9-interacting proteins. Among them, increased bindings of HSP90 and CDC37 to CDK9 were particularly striking, and our data suggest that the HSP90-CDC37-P-TEFb complex is involved in controlling the dynamic equilibrium of the P-TEFb complex during BETi-induced reactivation of HIV-1 latency. Furthermore, the HSP90-CDC37-P-TEFb complex directly regulates HIV-1 transcription and relies on recruitment by heat shock factor 1 (HSF1) for binding to the HIV-1 promoter. These results advance the understanding of HSP90-CDC37-P-TEFb in HIV-1 latency reversal and enlighten the development of potential strategies to eradicate HIV-1 using a combination of targeted drugs.
Assuntos
HIV-1 , Fatores de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , HIV-1/genética , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteômica , Chaperonas Moleculares/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Transcrição GênicaRESUMO
Stable isotope chemical labeling methods have been widely used for high-throughput mass spectrometry (MS)-based quantitative proteomics in biological and clinical applications. However, the existing methods are far from meeting the requirements for high sensitivity detection. In the present study, a novel isobaric stable isotope N-phosphorylation labeling (iSIPL) strategy was developed for quantitative proteome analysis. The tryptic peptides were selectively labeled with iSIPL tag to generate the novel reporter ions containing phosphoramidate P-N bond with high intensities under lower collision energies. iSIPL strategy are suitable for peptide sequencing and quantitative analysis with high sensitivity and accuracy even for samples of limited quantity. Furthermore, iSIPL coupled with affinity purification and mass spectrometry was applied to measure the dynamics of cyclin dependent kinase 9 (CDK9) interactomes during transactivation of the HIV-1 provirus. The interaction of CDK9 with PARP13 was found to significantly decrease during Tat-induced activation of HIV-1 gene transcription, suggesting the effectiveness of iSIPL strategy in dynamic analysis of protein-protein interaction in vivo. More than that, the proposed iSIPL strategy would facilitate large-scale accurate quantitative proteomics by increasing multiplexing capability.
Assuntos
Proteoma , Espectrometria de Massas em Tandem , Proteoma/análise , Espectrometria de Massas em Tandem/métodos , Fosforilação , Peptídeos/química , Marcação por Isótopo/métodos , IsótoposRESUMO
The Siah1 and Siah2 ubiquitin ligases are implicated in diverse biological processes ranging from cellular stress responses, signaling to transcriptional regulation. A key substrate of Siah1 is ELL2, which undergoes proteolysis upon polyubiquitination. ELL2 stimulates transcriptional elongation and is a subunit of the Super Elongation Complex (SEC) essential for HIV-1 transactivation. Previously, multiple transcriptional and post-translational mechanisms are reported to control Siah's expression and activity. Here we show that the activity of Siah1/2 can also be suppressed by host cell factor 1 (HCF1), and the hitherto poorly characterized HCF2, which themselves are not degraded but can bind and block the substrate-binding domain (SBD) of Siah1/2 to prevent their autoubiquitination and trans-ubiquitination of downstream targets including ELL2. This effect stabilizes ELL2 and enhances the ELL2-SEC formation for robust HIV-1 transactivation. Thus, our study not only identifies HCF1/2 as novel activators of HIV-1 transcription through inhibiting Siah1 to stabilize ELL2, but also reveals the SBD of Siah1/2 as a previously unrecognized new target for HCF1/2 to exert this inhibition.
Assuntos
Fator C1 de Célula Hospedeira/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Chlorocebus aethiops , Células HeLa , Humanos , Proteínas Nucleares/química , Ligação Proteica , Fatores de Elongação da Transcrição/metabolismo , Ubiquitina-Proteína Ligases/química , UbiquitinaçãoRESUMO
Three-dimensional vertically aligned graphene (3DVAG) was prepared by a unidirectional freezing method, and its electrochemical performances were evaluated as electrode materials for zinc-ion hybrid supercapacitors (ZHSCs). The prepared 3DVAG has a vertically ordered channel structure with a diameter of about 20-30 µm and a length stretching about hundreds of microns. Compared with the random structure of reduced graphene oxide (3DrGO), the vertical structure of 3DVAG in a three-electrode system showed higher specific capacitance, faster ion diffusion, and better rate performance. The specific capacitance of 3DVAG reached 66.6 F·g-1 and the rate performance reached 92.2%. The constructed 3DVAG zinc-ion hybrid supercapacitor also showed excellent electrochemical performance. It showed good capacitance retention up to 94.6% after 3000 cycles at the current density of 2 A·g-1.
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The activation of CD81 [the portal of entry of hepatitis C virus (HCV)] by agonistic antibody results in phosphorylation of Ezrin via Syk kinase and is associated with inactivation of the Hippo pathway and increase in yes-associated protein (Yap1). The opposite occurs when glypican-3 or E2 protein of HCV binds to CD81. Hepatocyte-specific glypican-3 transgenic mice have decreased levels of phosphorylated (p)-Ezrin (Thr567) and Yap, increased Hippo activity, and suppressed liver regeneration. The role of Ezrin in these processes has been speculated, but not proved. We show that Ezrin has a direct role in the regulation of Hippo pathway and Yap. Forced expression of plasmids expressing mutant Ezrin (T567D) that mimics p-Ezrin (Thr567) suppressed Hippo activity and activated Yap signaling in hepatocytes in vivo and enhanced activation of pathways of ß-catenin and leucine rich repeat containing G protein-coupled receptor 4 (LGR4) and LGR5 receptors. Hepatoma cell lines JM1 and JM2 have decreased CD81 expression and Hippo activity and up-regulated p-Ezrin (T567). NSC668394, a p-Ezrin (Thr567) antagonist, significantly decreased hepatoma cell proliferation. We additionally show that p-Ezrin (T567) is controlled by epidermal growth factor receptor and MET. Ezrin phosphorylation, mediated by CD81-associated Syk kinase, is directly involved in regulation of Hippo pathway, Yap levels, and growth of normal and neoplastic hepatocytes. The finding has mechanistic and potentially therapeutic applications in hepatocyte growth biology, hepatocellular carcinoma, and HCV pathogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Hepatócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Humanos , Camundongos , FosforilaçãoRESUMO
Maintenance of the corneal refractive power and tissue transparency is essential for normal vision. Real-time characterization of changes in corneal cells during suffering stresses or wound healing may provide a way to identify novel targets, whose therapeutic manipulation can improve the outcome of this response induced by injury. Here we describe a novel user friendly and effective confocal real-time confocal microscopy attachment that monitors the effects of anisoosmotic stress on cell morphology and corneal thickness in situ. Corneal epithelial nuclei gradually became highly reflective in the isotonic group and the corneal stroma was slightly thickened as compared with that seen prior to 60 min exposure to a hypotonic solution. After 30 min of exposure to hypertonic stress, the corneal stromal cells became crenate and shriveled. The hyper-reflective area of the corneal stroma in the hypo-osmotic group was significantly larger than that in the other two groups, as demonstrated by 3D reconstruction imaging. The hypotonic fresh chlorinated pool water was observed to cause atrophy of corneal epithelial nuclei, while the isosmotic bee venom solution caused high reflection of the corneal stroma layer and corneal endothelial cell damage. With the microscopic attachment, the inward movement of corneal epithelial cells toward the denuded central region was detected in the serum-treated group. The microscopy attachment is an effective system for obtaining a more detailed understanding of the time dependent losses in the corneal cell structure and tissue architecture of full thickness corneas induced by osmotic stress or cytotoxic agents.
Assuntos
Córnea/efeitos dos fármacos , Córnea/diagnóstico por imagem , Estresse Fisiológico , Animais , Sistemas Computacionais , Soluções Hipotônicas/farmacologia , Soluções Isotônicas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Pressão Osmótica/fisiologia , Solução Salina Hipertônica/farmacologiaRESUMO
Fiber-shaped supercapacitors (FSCs) are promising energy storage devices for portable and wearable electronics due to their miniaturized size, flexibility, and knittability. Despite the significant progress in this area, it is still a challenge to develop large capacitance and high energy density FSCs for practical applications. In this work, a hybrid fiber composed of reduced graphene oxide and polyaniline nanoparticles (r-PANI-GOF) is synthesized viain situsynthesis of polyaniline nanoparticles both on the surface and inside of graphene fibers. The areal specific capacitance of a single r-PANI-GOF electrode is as large as 1755 mF cm-2in the three-electrode system. The r-PANI-GOF hybrid fibers were also used as electrodes for making an all-solid-state FSCs. This whole device has a specific areal capacitance of up to 481 mF cm-2and a high areal energy density of 42.76µWh cm-2. The hybrid fiber electrodes with a high capacitance, and excellent flexibility may become new candidates for the development of fiber-shaped high-performance energy storage devices.
RESUMO
The general transcription factor P-TEFb, a master regulator of RNA polymerase (Pol) II elongation, phosphorylates the C-terminal domain (CTD) of Pol II and negative elongation factors to release Pol II from promoter-proximal pausing. We show here that P-TEFb surprisingly inhibits the myoblast differentiation into myotubes, and that P-TEFb and its two positive complexes are eliminated in this process. In contrast, DYRK1A, another CTD kinase known to control transcription of a subset of genes important for development and tissue homeostasis, is found to activate transcription of key myogenic genes. We show that active DYRK1A exists in a complex with the WD40-repeat protein DCAF7 that stabilizes and tethers DYRK1A to Pol II, so that DYRK1A-DCAF7 can co-migrate with and phosphorylate Pol II along the myogenic gene loci. Thus, DCAF7 modulates the kinase signaling output of DYRK1A on Pol II to stimulate myogenic transcription after active P-TEFb function is shut off.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Desenvolvimento Muscular/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Transcrição Gênica , Animais , Diferenciação Celular/genética , Ciclina T/genética , Quinase 9 Dependente de Ciclina/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Proteínas Nucleares/genética , RNA Polimerase II/química , RNA Polimerase II/genética , Canais de Translocação SEC/genética , Fatores de Transcrição/genética , Quinases DyrkRESUMO
This study describes the synthesis and vacuole-inducing activity of 5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-indole-2-carbohydrazide derivatives, including five potent derivatives 12c, 12 g, 12i, 12n, and 12A that exhibit excellent vacuole-inducing activity. Remarkably, 12A effectively induces methuosis in tested cancer cells but not human normal cells. In addition, 12A exhibits high pan-cytotoxicity against different cancer cell lines but is hardly toxic to normal cells. It is found that the 12A-induced vacuoles are derived from macropinosomes but not autophagosomes. The 12A-induced cytoplasmic vacuoles may originate from the endoplasmic reticulum (ER) and be accompanied by ER stress. The MAPK/JNK signalling pathway is involved in the 12A-induced methuotic cell death. Moreover, 12A exhibits significant inhibition of tumour growth in the MDA-MB-231 xenograft mouse model. The excellent potency and selectivity of 12A prompt us to select it as a good lead compound for further development of methuosis inducers and investigation of the molecular and cellular mechanisms underlying methuosis.
Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , Hidrazinas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Hidrazinas/síntese química , Hidrazinas/química , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
To discover new anticancer agents, two series of thiosemicarboxamide derivatives were synthesized and evaluated for their antiproliferative activity against human cancer cells inâ vitro. Most target compounds (especially 3f, 3g, and 3h) exhibit potent antiproliferative activity against HeLa cells. Importantly, compound 3h, bearing a 4-methylphenyl substituent at N position of thiourea moiety, has significant and broad-spectrum inhibitory activities against cancer cells (HepG2, HeLa, MDA-MB231, A875, and H460 cells) with low IC50 values (<5.0â µM) and shows low toxicity to normal LO2 and MRC-5 cells. Further studies show that compound 3h exerts high inhibitory activity in cancer cells by inducing the G2/M-phase arrest of cancer cells. Collectively, this study presents compound 3h as a new entity for the development of cell cycle arrest inducers for the treatment of cancer.
Assuntos
Antineoplásicos/farmacologia , Compostos de Sulfidrila/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Compostos de Sulfidrila/síntese química , Compostos de Sulfidrila/químicaRESUMO
Polyacrylonitrile (PAN)-based porous carbon was prepared by different methods of activation with PAN polymer microsphere as precursor. The morphology, structure and electrical properties for supercapacitor of the porous carbon were investigated. It was found that the morphology of PAN nanospheres tended to be destroyed in the process of one-step activation (activation and carbonization were carried out simultaneously, and could only be retained when the amount of activating agent KOH was small). While the spherical morphology could be well reserved during the two-step activation method (carbonization and activation sequentially). The specific surface area and pore volume increased first and then decreased, with the increase in activation holding time for both one-step and two-step activation methods. The specific surface area reached the maximum value with 2430 m2 g-1 for the one-step activation method and 2830 m2 g-1 for the two-step activation method. Additionally, their mass-specific capacitances were 178.8 F g-1 and 160.2 F g-1, respectively, under the current density of 1 A g-1. After 2000 cycles, the specific capacitance retentions were 92.9% and 91.3%.
RESUMO
In continuation of our previous work on the investigation of CDK9 inhibitors bearing indole moiety for the discovery of novel anticancer agents, novel methylenehydrazine-1-carboxamide derivatives with (5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-indole scaffold were designed, synthesized, and evaluated for the CDK9 inhibitory activity and anticancer activity. Biological activity results demonstrated that most of these derivatives possessed good inhibitory on the kinase activity of CDK9 such as blocking its phosphorylation function and inhibiting HIV-1 transcription. Compound 12i was found to be the most potent CDK9 inhibitor and exhibited excellent anticancer activity against HepG2, A375, MCF-7, and A549, but low toxic on normal cells including HaCaT and MCF-10A. Further studies revealed that as a result of CDK9 inhibition and subsequent inhibition of phosphorylation at Serine 2 of the RNAPII CTD, the representative compound 12i dose-dependently increased cleaved PARP level, exerting its antiproliferative effect through induction of apoptosis in cancer cells. Finally, the molecular docking analysis implied that 12i had a good binding affinity with CDK9. In summary, 12i is a potent CDK9 inhibitor and can be considered as a good lead-candidate for developing potential anticancer drugs.
Assuntos
Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Indóis/química , Simulação de Acoplamento Molecular/métodos , Pirimidinas/síntese química , Pirimidinas/uso terapêutico , Desenho de Fármacos , Humanos , Estrutura Molecular , Pirimidinas/farmacologia , Relação Estrutura-AtividadeRESUMO
In the effort to develop novel quinoline derivatives for the treatment of liver cancer, we synthesized a series of N'-Substituted methylene-4-(quinoline-4-amino) benzoylhydrazides and evaluated their biological activities as anticancer agents. Compounds 5h and 5j were found to be the potent antiproliferative agents against HepG2 cell line with an IC50 value of 12.6 ± 0.1 µM and 27.3 ± 1.7 µM, respectively. The most effective compound 5h also exhibited potent cytotoxicity against SMMC-7721 and Huh7 cells with IC50 values of 9.6 ± 0.7 µM and 6.3 ± 0.2 µM, respectively. Inspiringly, both 5h and 5j exhibited lower cytotoxic property in normal cells than hepatic carcinoma cells. Compounds 5h and 5j could down-regulate mRNA level of c-Myc and expression level of c-Myc. Meanwhile, they decreased expression level of anti-apoptotic protein Bcl-2 and increased expression levels of pro-apoptotic protein Bax and cleaved PARP with reference to tubulin. So various assays including cell colony formation, cell cycle distribution, as well as cell apoptosis and migration were performed to understand their antitumor role. It was confirmed that 5h and 5j inhibited the growth of HepG2 cells due to their anti-survival effect, induction of cell cycle arrest and cell apoptosis, and inhibition of cell migration. These results demonstrated that 5h might be as potential lead compounds to develop anticancer agents for the treatment of hepatocellular carcinoma.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Hidrazinas/química , Hidrazinas/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Antineoplásicos/síntese química , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células Hep G2 , Humanos , Hidrazinas/síntese química , Neoplasias Hepáticas/metabolismo , Quinolinas/síntese química , Quinolinas/química , Quinolinas/farmacologiaRESUMO
Incomplete tear film spreading and eyelid closure can cause defective renewal of the ocular surface and air exposure-induced epithelial keratopathy (EK). In this study, we characterized the role of autophagy in mediating the ocular surface changes leading to EK. Human corneal epithelial cells (HCECs) and C57BL/6 mice were employed as EK models, respectively. Transmission electron microscopy (TEM) evaluated changes in HCECs after air exposure. Each of these models was treated with either an autophagy inhibitor [chloroquine (CQ) or 3-methyladenine (3-MA)] or activator [Rapamycin (Rapa)]. Immunohistochemistry assessed autophagy-related proteins, LC3 and p62 expression levels. Western blotting confirmed the expression levels of the autophagy-related proteins [Beclin1 and mammalian target of rapamycin (mTOR)], the endoplasmic reticulum (ER) stress-related proteins (PERK, eIF2α and CHOP) and the PI3K/Akt/mTOR signalling pathway-related proteins. Real-time quantitative PCR (qRT-PCR) determined IL-1ß, IL-6 and MMP9 gene expression levels. The TUNEL assay detected apoptotic cells. TEM identified autophagic vacuoles in both EK models. Increased LC3 puncta formation and decreased p62 immunofluorescent staining and Western blotting confirmed autophagy induction. CQ treatment increased TUNEL positive staining in HCECs, while Rapa had an opposite effect. Similarly, CQ injection enhanced air exposure-induced apoptosis and inflammation in the mouse corneal epithelium, which was inhibited by Rapa treatment. Furthermore, the phosphorylation status of PERK and eIF2α and CHOP expression increased in both EK models indicating that ER stress-induced autophagy promoted cell survival. Taken together, air exposure-induced autophagy is indispensable for the maintenance of corneal epithelial physiology and cell survival.
Assuntos
Autofagia/fisiologia , Ceratite/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Autofagia/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Cloroquina/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Humanos , Ceratite/tratamento farmacológico , Ceratite/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator de Transcrição CHOP/metabolismoRESUMO
Glypican (GPC)-3 is overexpressed in hepatocellular carcinomas (HCCs). GPC3 binds to CD81. Forced expression of CD81 in a GPC3-expressing HCC cell line caused activation of Hippo, a decrease in ezrin phosphorylation, and a decrease in yes-associated protein (YAP). CD81 is also associated with hepatitis C virus (HCV) entry into hepatocytes. Activation of CD81 by agonistic antibody causes activation of tyrosine-protein kinase SYK (SYK) and phosphorylation of ezrin, a regulator of the Hippo pathway. In cultures of normal hepatocytes, CD81 agonistic antibody led to enhanced phosphorylation of ezrin and an increase in nuclear YAP. HCV E2 protein mimicked GPC3 and led to enhanced Hippo activity and decreased YAP in cultured normal human hepatocytes. HCC tissue microarray revealed a lack of expression of CD81 in most HCCs, rendering them insusceptible to HCV infection. Activation of CD81 by agonistic antibody suppressed the Hippo pathway and increased nuclear YAP. HCV mimicked GPC3, causing Hippo activation and a decrease in YAP. HCV is thus likely to enhance hepatic neoplasia by acting as a promoter of growth of early CD81-negative neoplastic hepatocytes, which are resistant to HCV infection, and thus have a proliferative advantage to clonally expand as they participate in compensatory regeneration for the required maintenance of 100% of liver weight (hepatostat).
Assuntos
Biomimética , Carcinoma Hepatocelular/patologia , Glipicanas/metabolismo , Hepatite C/complicações , Hepatócitos/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Tetraspanina 28/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/virologia , Proliferação de Células , Glipicanas/genética , Hepacivirus , Hepatite C/virologia , Hepatócitos/metabolismo , Via de Sinalização Hippo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Tetraspanina 28/genética , Células Tumorais CultivadasRESUMO
Pterygium postoperative granuloma (PPG) is one of the common complications of pterygium surgery. In order to provide the structural features of PPG, and to further explore its pathogenetic mechanism, we analyzed clinical and pathological characteristics of 12 PPG cases. New blood vessels were observed under a slit lamp in PPG and peripheral conjunctival tissues. In vivo confocal imaging showed that there was extensive neovascularization in the stroma, accompanied by infiltration of dendritic cells and inflammatory cells. Dense fibrous structures were observed in some PPG tissues. H&E staining results confirmed neovascularization and inflammatory cells in PPG tissues. In addition, H&E staining exhibited epithelioid tissue covering some PPG tissues. The immunofluorescence results demonstrated that the PPG epithelium was negative for K19, K10 and Muc5AC. Compared with the normal conjunctiva and pterygium, the expression of collagen IV in PPG basement membrane decreased, the expression of pan-cytokeratin (PCK), claudin 4 and E-cadherin in PPG epithelium was significantly lower, while the expression of vimentin, α-SMA and Snail was significantly increased. Therefore, our results suggest that the expression of epithelial keratin markers and goblet cell specific mucin marker is downregulated in the PPG tissues, and it likely is associated with the occurrence of EMT in granulomatous tissues.
Assuntos
Doenças da Túnica Conjuntiva/patologia , Células Epiteliais/patologia , Granuloma/patologia , Complicações Pós-Operatórias , Pterígio/cirurgia , Adulto , Biomarcadores/metabolismo , Doenças da Túnica Conjuntiva/etiologia , Doenças da Túnica Conjuntiva/metabolismo , Neovascularização da Córnea/patologia , Substância Própria/irrigação sanguínea , Regulação para Baixo , Feminino , Fibrose , Granuloma/etiologia , Granuloma/metabolismo , Humanos , Masculino , Microscopia Confocal , Pessoa de Meia-IdadeRESUMO
Recruitment of the P-TEFb kinase by HIV-1 Tat to the viral promoter triggers the phosphorylation and escape of RNA polymerase II from promoter-proximal pausing. It is unclear, however, if Tat recruits additional host factors that further stimulate HIV-1 transcription. Using a sequential affinity-purification scheme, we have identified human transcription factors/coactivators AFF4, ENL, AF9, and elongation factor ELL2 as components of the Tat-P-TEFb complex. Through the bridging functions of Tat and AFF4, P-TEFb and ELL2 combine to form a bifunctional elongation complex that greatly activates HIV-1 transcription. Without Tat, AFF4 can mediate the ELL2-P-TEFb interaction, albeit inefficiently. Tat overcomes this limitation by bringing more ELL2 to P-TEFb and stabilizing ELL2 in a process that requires active P-TEFb. The ability of Tat to enable two different classes of elongation factors to cooperate and coordinate their actions on the same polymerase enzyme explains why Tat is such a powerful activator of HIV-1 transcription.
Assuntos
HIV-1/genética , RNA Viral/biossíntese , Proteínas Repressoras/metabolismo , Ativação Transcricional , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Sítios de Ligação , Linhagem Celular , Quinase 9 Dependente de Ciclina/metabolismo , HIV-1/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Complexos Multiproteicos , Proteínas Nucleares/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Interferência de RNA , Proteínas Repressoras/genética , Fatores de Elongação da Transcrição/metabolismo , Transfecção , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
The AF4/FMR2 proteins AFF1 and AFF4 act as a scaffold to assemble the Super Elongation Complex (SEC) that strongly activates transcriptional elongation of HIV-1 and cellular genes. Although they can dimerize, it is unclear whether the dimers exist and function within a SEC in vivo. Furthermore, it is unknown whether AFF1 and AFF4 function similarly in mediating SEC-dependent activation of diverse genes. Providing answers to these questions, our current study shows that AFF1 and AFF4 reside in separate SECs that display largely distinct gene target specificities. While the AFF1-SEC is more potent in supporting HIV-1 transactivation by the viral Tat protein, the AFF4-SEC is more important for HSP70 induction upon heat shock. The functional difference between AFF1 and AFF4 in Tat-transactivation has been traced to a single amino acid variation between the two proteins, which causes them to enhance the affinity of Tat for P-TEFb, a key SEC component, with different efficiency. Finally, genome-wide analysis confirms that the genes regulated by AFF1-SEC and AFF4-SEC are largely non-overlapping and perform distinct functions. Thus, the SEC represents a family of related complexes that exist to increase the regulatory diversity and gene control options during transactivation of diverse cellular and viral genes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , HIV-1/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Ativação Transcricional , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Proteínas de Choque Térmico HSP70/biossíntese , Células HeLa , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Mutação Puntual , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Elongação da Transcrição Genética , Fatores de Elongação da TranscriçãoRESUMO
The positive transcription elongation factor b (P-TEFb) stimulates RNA polymerase elongation by inducing the transition of promoter proximally paused polymerase II into a productively elongating state. P-TEFb itself is regulated by reversible association with various transcription factors/cofactors to form several multisubunit complexes [e.g., the 7SK small nuclear ribonucleoprotein particle (7SK snRNP), the super elongation complexes (SECs), and the bromodomain protein 4 (Brd4)-P-TEFb complex] that constitute a P-TEFb network controlling cellular and HIV transcription. These complexes have been thought to share no components other than the core P-TEFb subunits cyclin-dependent kinase 9 (CDK9) and cyclin T (CycT, T1, T2a, and T2b). Here we show that the AF4/FMR2 family member 1 (AFF1) is bound to CDK9-CycT and is present in all major P-TEFb complexes and that the tripartite CDK9-CycT-AFF1 complex is transferred as a single unit within the P-TEFb network. By increasing the affinity of the HIV-encoded transactivating (Tat) protein for CycT1, AFF1 facilitates Tat's extraction of P-TEFb from 7SK snRNP and the formation of Tat-SECs for HIV transcription. Our data identify AFF1 as a ubiquitous P-TEFb partner and demonstrate that full Tat transactivation requires the complete SEC.