RESUMO
Faithful transfer of parental histones to newly replicated daughter DNA strands is critical for inheritance of epigenetic states. Although replication proteins that facilitate parental histone transfer have been identified, how intact histone H3-H4 tetramers travel from the front to the back of the replication fork remains unknown. Here, we use AlphaFold-Multimer structural predictions combined with biochemical and genetic approaches to identify the Mrc1/CLASPIN subunit of the replisome as a histone chaperone. Mrc1 contains a conserved histone-binding domain that forms a brace around the H3-H4 tetramer mimicking nucleosomal DNA and H2A-H2B histones, is required for heterochromatin inheritance, and promotes parental histone recycling during replication. We further identify binding sites for the FACT histone chaperone in Swi1/TIMELESS and DNA polymerase α that are required for heterochromatin inheritance. We propose that Mrc1, in concert with FACT acting as a mobile co-chaperone, coordinates the distribution of parental histones to newly replicated DNA.
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Epigenetic inheritance of heterochromatin requires DNA-sequence-independent propagation mechanisms, coupling to RNAi, or input from DNA sequence, but how DNA contributes to inheritance is not understood. Here, we identify a DNA element (termed "maintainer") that is sufficient for epigenetic inheritance of pre-existing histone H3 lysine 9 methylation (H3K9me) and heterochromatin in Schizosaccharomyces pombe but cannot establish de novo gene silencing in wild-type cells. This maintainer is a composite DNA element with binding sites for the Atf1/Pcr1 and Deb1 transcription factors and the origin recognition complex (ORC), located within a 130-bp region, and can be converted to a silencer in cells with lower rates of H3K9me turnover, suggesting that it participates in recruiting the H3K9 methyltransferase Clr4/Suv39h. These results suggest that, in the absence of RNAi, histone H3K9me is only heritable when it can collaborate with maintainer-associated DNA-binding proteins that help recruit the enzyme responsible for its epigenetic deposition.
Assuntos
Montagem e Desmontagem da Cromatina , Metilação de DNA , DNA Fúngico/genética , Hereditariedade , Heterocromatina/genética , Sequências Reguladoras de Ácido Nucleico , Schizosaccharomyces/genética , Fatores Ativadores da Transcrição/genética , Fatores Ativadores da Transcrição/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Fúngico/metabolismo , Epigênese Genética , Regulação Fúngica da Expressão Gênica , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Proteínas/genética , Proteínas/metabolismo , Interferência de RNA , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
BACKGROUND: The pathogenesis of primary membranous nephropathy (MN) involves the antibodies against antigens on the cell surface of podocytes, with the majority of M-type phospholipase A2 receptor (PLA2R), and a profound podocyte dysfunction. The effects of anti-PLA2R antibodies directly to the podocytes remain unclear. METHODS: Anti-PLA2R antibodies from patients with PLA2R-associated MN were affinity-purified using a column coupled with recombinant human PLA2R protein. Their effects on conditionally immortalized human podocytes were assessed by apoptosis assays, cellular calcium detection, wound healing assay, and immunofluorescent staining. Proteomics analysis was performed by LC-MS/MS and on PANTHER database. RESULTS: The stimulation by anti-PLA2R antibodies could induce early-stage apoptosis of podocytes (MFI of Annexin V = 104.3 ± 19.2 vs. 36.7 ± 7.6, p = 0.004). The increase of calcium concentration in podocytes (MFI = 3309.3 ± 363.6 vs. 1776.3 ± 212.7, p = 0.015) might attribute to the endoplasmic reticulum calcium efflux. The expression of calcium/calmodulin-dependent protein kinase IV (CaMK4) was also increased (MFI = 134.4 ± 9.8 vs. 105.3 ± 10.1, p = 0.011). Proteomics results suggested that anti-PLA2R antibody treatment led to damage on cellular structure, and produced functional disorders on protein binding, actin filament binding, and microtubule motor activity. The staining of F-actin on foot process was reduced (MFI = 27.3 ± 2.8 vs. 47.5 ± 1.0, p = 0.001) and the motility and adherence capacity of podocytes were reduced (number of migrated cells = 44.7 ± 3.1 vs. 53.3 ± 4.9, p = 0.001) after incubation with anti-PLA2R antibodies. CONCLUSION: These data indicate that anti-PLA2R antibodies may directly induce podocyte damage independent of the complement system, which expands the mechanism of anti-PLA2R antibodies on MN.
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Glomerulonefrite Membranosa , Podócitos , Cromatografia Líquida , Glomerulonefrite Membranosa/patologia , Humanos , Podócitos/patologia , Receptores da Fosfolipase A2 , Espectrometria de Massas em TandemRESUMO
Millions of cis-regulatory elements are predicted to be present in the human genome, but direct evidence for their biological function is scarce. Here we report a high-throughput method, cis-regulatory element scan by tiling-deletion and sequencing (CREST-seq), for the unbiased discovery and functional assessment of cis-regulatory sequences in the genome. We used it to interrogate the 2-Mb POU5F1 locus in human embryonic stem cells, and identified 45 cis-regulatory elements. A majority of these elements have active chromatin marks, DNase hypersensitivity, and occupancy by multiple transcription factors, which confirms the utility of chromatin signatures in cis-element mapping. Notably, 17 of them are previously annotated promoters of functionally unrelated genes, and like typical enhancers, they form extensive spatial contacts with the POU5F1 promoter. These results point to the commonality of enhancer-like promoters in the human genome.
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Mapeamento Cromossômico/métodos , Testes Genéticos/métodos , Sequências Reguladoras de Ácido Nucleico/genética , Algoritmos , Células Cultivadas , Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência de DNA , Análise de Célula ÚnicaRESUMO
The eddy current displacement sensor (ECDS) is used to realize the precise detection of the rotor radial position in the magnetic suspension motor. The eccentricity between the probe axis and the measured surface normal reduces the measurement accuracy. An ECDS mathematical model is established to analyze the influence of the measured surface curvature and eccentricity on detection results. The eddy current density distribution law of the measured surface is obtained by using the finite element method (FEM). The experimental platform is set up based on the practical engineering structure, which contains two kinds structures of the single probe and the differential. The compensation method is introduced to reduce the error caused by the eccentricity. The displacement measurement error with and without compensation are tested separately. The results show that the largest full-scale error is less than 0.8% after compensation in the single probe structure, and 0.6% in the differential structure. For the engineering application, the orthogonal direction measured value is used as the eccentricity, and the compensation order of big then small is proposed. It is thus proved that the compensation method provides a guarantee for accurate feedback and control of the rotor radial position in the magnetic suspension motor system.
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Adult T-cell leukemia (ATL) is a highly aggressive T-cell malignancy induced by human T-cell leukemia virus type 1 (HTLV-1) infection. Long noncoding RNA (lncRNA) plays a critical role in the development and progression of multiple human cancers. However, the function of lncRNA in HTLV-1-induced oncogenesis has not been elucidated. In the present study, we show that the expression level of the lncRNA ANRIL was elevated in HTLV-1-infected cell lines and clinical ATL samples. E2F1 induced ANRIL transcription by enhancing its promoter activity. Knockdown of ANRIL in ATL cells repressed cellular proliferation and increased apoptosis in vitro and in vivo As a mechanism for these actions, we found that ANRIL targeted EZH2 and activated the NF-κB pathway in ATL cells. This activation was independent of the histone methyltransferase (HMT) activity of EZH2 but required the formation of an ANRIL/EZH2/p65 ternary complex. A chromatin immunoprecipitation assay revealed that ANRIL/EZH2 enhanced p65 DNA binding capability. In addition, we observed that the ANRIL/EZH2 complex repressed p21/CDKN1A transcription through H3K27 trimethylation of the p21/CDKN1A promoter. Taken together, our results implicate that the lncRNA ANRIL, by cooperating with EZH2, supports the proliferation of HTLV-1-infected cells, which is thought to be critical for oncogenesis.IMPORTANCE Human T-cell leukemia virus type 1 (HTLV-1) is the pathogen that causes adult T-cell leukemia (ATL), which is a unique malignancy of CD4+ T cells. A role for long noncoding RNA (lncRNA) in HTLV-1-mediated cellular transformation has not been described. In this study, we demonstrated that the lncRNA ANRIL was important for maintaining the proliferation of ATL cells in vitro and in vivo ANRIL was shown to activate NF-κB signaling through forming a ternary complex with EZH2 and p65. Furthermore, epigenetic inactivation of p21/CDKN1A was involved in the oncogenic function of ANRIL. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA ANRIL in ATL and provides an important clue to prevent or treat HTLV-1-associated human diseases.
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Fator de Transcrição E2F1/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , RNA Longo não Codificante/genética , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células Jurkat , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Transplante de Neoplasias , Transdução de Sinais , Regulação para CimaRESUMO
BACKGROUND: Previous studies have demonstrated that oral administration of curcumin exhibited an anti-arthritic effect despite its poor bioavailability. The present study aimed to explore whether the gut-brain axis is involved in the therapeutic effect of curcumin. METHODS: The collagen-induced arthritis (CIA) rat model was induced by immunization with an emulsion of collagen II and complete Freund's adjuvant. Sympathetic and parasympathetic tones were measured by electrocardiographic recordings. Unilateral cervical vagotomy (VGX) was performed before the induction of CIA. The ChAT, AChE activities, and serum cytokine levels were determined by ELISA. The expression of the high-affinity choline transporter 1 (CHT1), ChAT, and vesicular acetylcholine transporter (VAChT) were determined by real-time PCR and immunohistochemical staining. The neuronal excitability of the vagus nerve was determined by whole-cell patch clamp recording. RESULTS: Oral administration of curcumin restored the imbalance between the sympathetic and parasympathetic tones in CIA rats and increased ChAT activity and expression of ChAT and VAChT in the gut, brain, and synovium. Additionally, VGX eliminated the effects of curcumin on arthritis and ACh biosynthesis and transport. Electrophysiological data showed that curcumin markedly increased neuronal excitability of the vagus nerve. Furthermore, selective α7 nAChR antagonists abolished the effects of curcumin on CIA. CONCLUSIONS: Our results demonstrate that curcumin attenuates CIA through the "gut-brain axis" by modulating the function of the cholinergic system. These findings provide a novel approach for mechanistic studies of anti-arthritic compounds with low oral absorption and bioavailability.
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Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Encéfalo/metabolismo , Curcumina/uso terapêutico , Trato Gastrointestinal/metabolismo , Acetilcolina/antagonistas & inibidores , Acetilcolina/metabolismo , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Artrite Experimental/patologia , Encéfalo/efeitos dos fármacos , Células Cultivadas , Colina O-Acetiltransferase , Curcumina/farmacologia , Feminino , Trato Gastrointestinal/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Antagonistas Nicotínicos/farmacologia , Gânglio Nodoso/efeitos dos fármacos , Gânglio Nodoso/metabolismo , Distribuição Aleatória , Ratos , Ratos Wistar , Vagotomia/tendências , Nervo Vago/cirurgiaRESUMO
Myeloperoxidase (MPO) is a common target antigen of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis and is recognized in one-third of patients with anti-glomerular basement membrane (GBM) disease. Our previous study identified over 60% of patients with anti-GBM disease recognizing linear peptides of MPO heavy chain. Here we tested whether aberrant glycosylation alters MPO antigenicity through exposure of neo-epitopes on MPO molecules. Atypical glycosylated MPO molecules, including all possible glycosylation types, were prepared by exoglycosidase and endoglycosidase treatments. Antibodies were detected from the sera of 40 patients with anti-GBM disease without the coexistence of MPO-ANCA. Circulating antibodies against aberrant glycosylated MPO existed in 21 of these patients. Non-glycan MPO and MPO with only N-acetylglucosamine had high frequencies of recognition (16 and 15 patients, respectively). Antibodies binding to aberrant glycosylated MPO could not be inhibited by intact MPO or GBM antigen. When applied to ethanol-fixed neutrophils from normal individuals, these antibodies yielded a typical cytoplasmic staining pattern (c-ANCA). Antigen specificity was detected in 90% of the antibodies using five peptides containing one of the five N-glycosylation sites each, mostly on N323, N355, and N391. The antibodies were restricted to IgG1 subclass, could activate complement, and induce neutrophil degranulation in vitro. Thus, aberrant glycosylated MPO exposed neo-epitopes and was recognized by half of the patients with anti-GBM disease. Their antibodies possessed pathogenic characteristics and may be associated with kidney injury.
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Autoanticorpos/sangue , Epitopos , Glomerulonefrite/imunologia , Hemorragia/imunologia , Pneumopatias/imunologia , Peroxidase/imunologia , Peroxidase/metabolismo , Processamento de Proteína Pós-Traducional , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Estudos de Casos e Controles , Degranulação Celular , Ativação do Complemento , Glomerulonefrite/sangue , Glomerulonefrite/diagnóstico , Glomerulonefrite/enzimologia , Glicosilação , Hemorragia/sangue , Hemorragia/diagnóstico , Hemorragia/enzimologia , Humanos , Pneumopatias/sangue , Pneumopatias/diagnóstico , Pneumopatias/enzimologia , Neutrófilos/enzimologia , Neutrófilos/imunologia , Ligação ProteicaRESUMO
Norisoboldine (NOR), the main active ingredient of the dry root of Lindera aggregata, was previously proven to have substantial therapeutic effects on collagen-induced arthritis (CIA) in mice by oral administration. However, it exhibited a very poor bioavailability in normal rats. The pharmacokinetic-pharmacodynamics disconnection attracts us to explore its anti-arthritic mechanism in more detail. In this study, NOR, administered orally, markedly attenuated the pathological changes in CIA rats, which was accompanied by the down-regulation of pro-inflammatory cytokines and the up-regulation of anti-inflammatory cytokine IL-10. Pharmacokinetic studies demonstrated that the plasma concentration of NOR was moderately elevated in CIA rats compared with normal rats, but it was still far lower than the minimal effective concentration required for inhibiting the proliferation and activation of T lymphocytes in vitro. Interestingly, NOR was shown to regulate the balance between Th17 and regulatory T (Treg) cells in the intestinal lymph nodes more strikingly than in other tissues. It could increase the expression of Foxp3 mRNA in both gut and joints, and markedly up-regulate the number of integrin α4ß7 (a marker of gut source)-positive Foxp3(+) cells in the joints of CIA rats. These results suggest that the gut might be the primary action site of NOR, and NOR exerts anti-arthritis effect through regulating the balance between Th17 and Treg cells in intestinal lymph nodes and yielding a trafficking of lymphocytes (especially Treg cells) from the gut to joint. The findings of the present study also provide a plausible explanation for the anti-arthritic effects of poorly absorbed compounds like NOR.
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Alcaloides/farmacologia , Anti-Inflamatórios/farmacologia , Artrite Experimental/tratamento farmacológico , Articulações/efeitos dos fármacos , Linfonodos/efeitos dos fármacos , Nódulos Linfáticos Agregados/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Administração Oral , Alcaloides/administração & dosagem , Alcaloides/sangue , Alcaloides/farmacocinética , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/sangue , Anti-Inflamatórios/farmacocinética , Artrite Experimental/sangue , Artrite Experimental/induzido quimicamente , Artrite Experimental/imunologia , Artrite Experimental/patologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Colágeno Tipo II , Citocinas/sangue , Feminino , Fatores de Transcrição Forkhead/metabolismo , Adjuvante de Freund , Mediadores da Inflamação/sangue , Articulações/imunologia , Articulações/metabolismo , Articulações/patologia , Linfonodos/imunologia , Linfonodos/metabolismo , Mesentério , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/metabolismo , Ratos Wistar , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th17/imunologia , Células Th17/metabolismoRESUMO
Introduction: CM313 is currently under clinical investigation for treatments of multiple myeloma, systemic lupus erythematosus, and immune thrombocytopenia. We aimed to report the preclinical profile of the novel therapeutic anti-CD38 monoclonal antibody (mAb) CM313, with an emphasis on the difference with other CD38-targeting mAb. Methods: The binding of CM313 to CD38 recombinant protein across species was assessed using ELISA. The binding of CM313 to CD38-positive (CD38+) cells was detected using flow cytometry assays. CM313-induced complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and apoptosis on different CD38+ cells were assessed by LDH release assays or flow cytometry assays. The effect of CM313 on CD38 enzymatic activity was measured using fluorescence spectroscopy. CM313 immunotoxicity in human blood was assessed using flow cytometry assays, ELISA, and LDH release assays. Anti-tumor activity of CM313 was assessed in multiple mouse xenograft models. Safety profile of CM313 were evaluated in cynomolgus monkeys and human CD38 transgenic (B-hCD38) mice. Results: There exist unique sequences at complementarity-determining regions (CDR) of CM313, which facilitates its affinity to CD38 is consistently higher across a spectrum of CD38+ cell lines than daratumumab. In vitro studies showed that CM313 induces comparable killing activity than daratumumab, including ADCC, CDC, ADCP, apoptosis induced by Fc-mediated cross-linking, and effectively inhibited the enzymatic activity of CD38. However, CM313 showed more potent CDC than isatuximab. In vivo, CM313 dose-dependently inhibited xenograft tumor growth, both as a monotherapy and in combination with dexamethasone or lenalidomide. Furthermore, CM313 was well tolerated with no drug-related clinical signs or off-target risks, as evidenced by 4-week repeat-dose toxicology studies in cynomolgus monkeys and B-hCD38 mice, with the later study showing no observed adverse effect level (NOAEL) of 300mg/kg once weekly. Discussion: CM313 is a novel investigational humanized mAb with a distinct CDR sequence, showing comparable killing effects with daratumumab and stronger CDC activity than isatuximab, which supports its clinical development.
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ADP-Ribosil Ciclase 1 , Anticorpos Monoclonais , Citotoxicidade Celular Dependente de Anticorpos , Animais , Feminino , Humanos , Camundongos , ADP-Ribosil Ciclase 1/imunologia , ADP-Ribosil Ciclase 1/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antineoplásicos Imunológicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Macaca fascicularis , Glicoproteínas de Membrana , Camundongos Transgênicos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Polycomb repressive complexes 1 and 2 (PRC1 and 2) are required for heritable repression of developmental genes. The cis- and trans-acting factors that contribute to epigenetic inheritance of mammalian Polycomb repression are not fully understood. Here, we show that, in human cells, ectopically induced Polycomb silencing at initially active developmental genes, but not near ubiquitously expressed housekeeping genes, is inherited for many cell divisions. Unexpectedly, silencing is heritable in cells with mutations in the H3K27me3 binding pocket of the Embryonic Ectoderm Development (EED) subunit of PRC2, which are known to disrupt H3K27me3 recognition and lead to loss of H3K27me3. This mode of inheritance is less stable and requires intact PRC2 and recognition of H2AK119ub1 by PRC1. Our findings suggest that maintenance of Polycomb silencing is sensitive to local genomic context and can be mediated by PRC1-dependent H2AK119ub1 and PRC2 independently of H3K27me3 recognition.
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Inativação Gênica , Histonas , Proteínas do Grupo Polycomb , Ubiquitinação , Humanos , Histonas/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Proteínas do Grupo Polycomb/genética , Complexo Repressor Polycomb 2/metabolismo , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 1/genética , Genoma Humano , Epigênese Genética , MutaçãoRESUMO
BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus etiologically associated with adult T-cell leukemia (ATL). The HTLV-1 bZIP factor (HBZ), which is encoded by minus strand of provirus, is expressed in all ATL cases and supports the proliferation of ATL cells. However, the precise mechanism of growth promoting activity of HBZ is poorly understood. RESULTS: In this study, we showed that HBZ suppressed C/EBPα signaling activation induced by either Tax or C/EBPα. As mechanisms of HBZ-mediated C/EBPα inhibition, we found that HBZ physically interacted with C/EBPα and diminished its DNA binding capacity. Luciferase and immunoprecipitation assays revealed that HBZ repressed C/EBPα activation in a Smad3-dependent manner. In addition, C/EBPα was overexpressed in HTLV-1 infected cell lines and fresh ATL cases. HBZ was able to induce C/EBPα transcription by enhancing its promoter activity. Finally, HBZ selectively modulated the expression of C/EBPα target genes, leading to the impairment of C/EBPα-mediated cell growth suppression. CONCLUSION: HBZ, by suppressing C/EBPα signaling, supports the proliferation of HTLV-1 infected cells, which is thought to be critical for oncogenesis.
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Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/antagonistas & inibidores , Proliferação de Células , Interações Hospedeiro-Patógeno , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Linfócitos T/virologia , Proteínas Virais/metabolismo , Adulto , Idoso , Linhagem Celular , DNA/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Proteínas dos RetroviridaeRESUMO
The rixosome and PRC1 silencing complexes are associated with deSUMOylating and deubiquitinating enzymes, SENP3 and USP7, respectively. How deSUMOylation and deubiquitylation contribute to rixosome- and Polycomb-mediated silencing is not fully understood. Here, we show that the enzymatic activities of SENP3 and USP7 are required for silencing of Polycomb target genes. SENP3 deSUMOylates several rixosome subunits, and this activity is required for association of the rixosome with PRC1. USP7 associates with canonical PRC1 (cPRC1) and deubiquitinates the chromodomain subunits CBX2 and CBX4, and inhibition of USP activity results in disassembly of cPRC1. Finally, both SENP3 and USP7 are required for Polycomb- and rixosome-dependent silencing at an ectopic reporter locus. These findings demonstrate that SUMOylation and ubiquitination regulate the assembly and activities of the rixosome and Polycomb complexes and raise the possibility that these modifications provide regulatory mechanisms that may be utilized during development or in response to environmental challenges.
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Núcleo Celular , Complexo Repressor Polycomb 1 , Peptidase 7 Específica de Ubiquitina/metabolismo , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Ubiquitinação , Núcleo Celular/metabolismoRESUMO
We characterized the tissue repair response after penetrating traumatic brain injury (pTBI) in this study. Seventy specific pathogen-free Kunming mice were randomly divided into the following groups: normal control, 1, 3, 7, 15, 21, and 30 days after pTBI. Hematoxylin and eosin (H&E) staining, immunohistochemistry, and immunofluorescence were performed to examine and monitor brain tissue morphology, and the distribution and expression of lymphatic-specific markers lymphatic vessel endothelial receptor-1 (LYVE-1), hematopoietic precursor cluster of differentiation 34 (CD34) antigen, and Prospero-related homeobox-1 (PROX1) protein. H&E staining revealed that damaged and necrotic tissues observed on day 1 at and around the injury site disappeared on day 7, and there was gradual shrinkage and disappearance of the lesion on day 30, suggesting a clearance mechanism. We explored the possibility of lymphangiogenesis causing this clearance as part of the post-injury response. Notably, expression of lymphangiogenesis markers LYVE-1, CD34, and PROX1 was detected in damaged mouse brain tissue but not in normal tissue. Moreover, new lymphatic cells and colocalization of LYVE-1/CD34 and LYVE-1/PROX1 were also observed. Our findings of the formation of new lymphatic cells following pTBI provide preliminary insights into a post-injury clearance mechanism in the brain. Although we showed that lymphatic cells are implicated in brain tissue repair, further research is required to clarify the origin of these cells.
Assuntos
Encéfalo/metabolismo , Encéfalo/patologia , Traumatismos Cranianos Penetrantes/imunologia , Animais , Antígenos CD34/análise , Antígenos CD34/imunologia , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Encéfalo/imunologia , Lesões Encefálicas Traumáticas/imunologia , Diferenciação Celular , China , Endotélio Vascular/metabolismo , Feminino , Proteínas de Homeodomínio/análise , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Linfangiogênese/fisiologia , Vasos Linfáticos/patologia , Masculino , Proteínas de Membrana Transportadoras/análise , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Necrose , Proteínas Supressoras de Tumor/análise , Proteínas Supressoras de Tumor/metabolismoRESUMO
Our previous studies demonstrated that oral administration of madecassoside could markedly attenuate collagen-induced arthritis in rats, a rodent model of rheumatoid arthritis. As the autonomic nervous system is critically involved in the modulation of peripheral inflammation and immune response, the present study aims to explore the possible involvement of adrenergic and cholinergic nerves in the effect of madecassoside on rheumatoid arthritis. Arthritis was induced by chicken collagen in rats, and madecassoside was orally administered daily for two weeks from day 14 after the primary immunization. The antagonists of adrenoceptor and cholinergic receptors were co-administered with madecassoside, respectively. Unilateral cervical vagotomy was performed four days before the arthritis induction. The results showed that madecassoside (30â¯mg/kg) treatment markedly ameliorated arthritis symptoms in rats, mainly evidenced by the reduction of paw swelling and arthritis index scores. Co-administration of madecassoside with atropine (an antagonist of the muscarinic acetylcholine receptor) or hexamethonium (an antagonist of the nicotinic acetylcholine receptor) markedly diminished the therapeutic effects of madecassoside in arthritis. However, co-administration with phentolamine (an antagonist of the α-adrenoceptor) or propranolol (an antagonist of the ß-adrenoceptor) did not alter the effect of madecassoside on arthritis. Furthermore, unilateral cervical vagotomy significantly reduced the anti-arthritis efficacy of madecassoside, including the amelioration of clinical symptoms, as well as the inhibition of the production of pro-inflammatory cytokines except T lymphocytes-related cytokines. These findings suggest that madecassoside exerts inhibitory effects on collagen-induced arthritis through, at least partially, the peripheral cholinergic system.
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Anti-Inflamatórios/uso terapêutico , Artrite Experimental/tratamento farmacológico , Triterpenos/uso terapêutico , Nervo Vago , Administração Oral , Animais , Artrite Experimental/imunologia , Artrite Experimental/patologia , Citocinas/sangue , Citocinas/imunologia , Feminino , Articulação do Joelho/efeitos dos fármacos , Articulação do Joelho/patologia , Ratos Wistar , VagotomiaRESUMO
In a substantial number of patients with crescentic glomerulonephritis, both anti-glomerular basement membrane (GBM) antibodies and anti-neutrophil cytoplasmic antibodies (ANCA) are detected simultaneously. ANCA is presumed to be the initial event but the mechanism is unknown. In the present study, we investigated the antibodies against linear epitopes on Goodpasture autoantigen in sera from patients with ANCA-associated vasculitis, aiming to reveal the mechanisms of the coexistence of the two kinds of autoantibodies. Thirty-one patients with ANCA-associated vasculitis were enrolled in this study. Twenty-four overlapping linear peptides were synthesized across the whole sequence of Goodpasture autoantigen. Serum antibodies against linear peptides were detected by ELISA and their associations with clinical features were further analyzed. Twenty-five out of the thirty-one (80.6%) sera from patients with ANCA-associated vasculitis possessed antibodies against linear peptides on Goodpasture autoantigen. These antibodies could be detected in 50% of patients with normal renal function (Scr ≤ 133 µmol/L), 70% of patients with moderate renal dysfunction (133 µmol/L < Scr ≤ 600 µmol/L), and 94% of patients with renal failure (Scr > 600 µmol/L) (P = 0.032). The highest recognition frequencies were found for peptides P4 (51.6%), P14 (54.8%), and P24 (54.8%), which contained the sequences that constitute the conformational epitopes of EA (P4) and EB (P14) recognized by anti-GBM antibodies. The level of anti-P4 antibodies was positively correlated with the percentage of crescents in glomeruli (r = 0.764, P = 0.027). Patients with anti-P24 antibodies had a significantly higher prevalence of renal dysfunction on diagnosis (88.2 vs. 42.9%, P = 0.018). Antibodies against linear epitopes on Goodpasture autoantigen could be detected in sera of patients with ANCA-associated vasculitis, which might mediate the production of antibodies towards the conformational epitopes on Goodpasture autoantigen, namely, the anti-GBM antibodies.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/sangue , Anticorpos Anticitoplasma de Neutrófilos/sangue , Autoanticorpos/sangue , Autoantígenos/química , Colágeno Tipo IV/química , Epitopos/imunologia , Adulto , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/complicações , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Especificidade de Anticorpos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Insuficiência Renal/epidemiologiaRESUMO
Chemotherapy as a routine method for clinical treatment of cancer has disadvantages such as significant toxicity and strong resistance. In order to improve the efficacy of the drugs and reduce the by-effects, we tried to combine static magnetic field (SMF) with cisplatin or adriamycin. The growth of Hepa1-6 cells treated with the static magnetic field (SMF) combined with cisplatin or adriamycin was significantly inhibited, as detected with MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) test. Combined treatment group cells underwent significant morphological changes as observed by HE (Hematoxylin and eosin) staining under optical microscope. Cell cycle analysis indicated that SMF increased the ratio of cells arrested in G2/M phase caused by cisplatin, and when treated with SMF combined with adriamycin, cells were almost arrested in G1 and G2/M phase. SCGE test showed that SMF can enhance the ability of cisplatin or adriamycin to promote cell DNA damage. Atomic force microscope observation found that the combination of antitumor drugs and magnetic field treatment induced larger and deeper holes on the cell membrane, and surface structure damage is serious. The combination of antitumor drugs and magnetic field technology effectively inhibits the growth of tumor cells, and reduces drug doses. The results implicate this method as potential cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Doxorrubicina/farmacologia , Campos Magnéticos , Ciclo Celular , Linhagem Celular Tumoral/efeitos dos fármacos , Dano ao DNA , HumanosRESUMO
Sinomenine (SIN) has long been used as a therapeutic agent of rheumatoid arthritis (RA) in China. However, the discrepancy between low oral bioavailability and higher minimal effective concentration made its action mode mysterious. The present study aimed to gain insight into the mechanisms by which SIN suppressed collagen-induced arthritis (CIA) in rats in view of Th17 and regulatory T (Treg) cell balance. SIN was orally administered, and the clinical symptoms of CIA rats were monitored; inflammatory cytokines levels in serum were measured by ELISA; pharmacokinetic studies were performed in normal and CIA rats; Th17 and Treg cell frequencies were analyzed by flow cytometry. The data showed that SIN treatment resulted in a dramatic decrease of arthritis scores and paw volume of CIA rats, which was accompanied by down-regulation of IL-17A and up-regulation of IL-10 in rat serum. The frequency of Treg cells was increased and the frequency of Th17 cells was decreased in the gut lymphoid tissues of SIN-treated rats. Immunohistochemistry assay demonstrated that more α4ß7-positive cells were detained in joint tissues after SIN treatment. Moreover, the anti-arthritis efficacy of SIN disappeared when it was given by intraperitoneal injection, further confirming the action of SIN was gut-dependent. In conclusion, SIN exerts anti-RA action probably through modulating the frequencies of Treg cells and Th17 cells in intestinal lymph nodes and yielding a trafficking of lymphocytes (especially Treg cells) from gut to joint.
Assuntos
Anti-Inflamatórios/farmacologia , Artrite Experimental/tratamento farmacológico , Morfinanos/farmacologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Artrite Experimental/induzido quimicamente , Artrite Reumatoide/tratamento farmacológico , Diferenciação Celular/imunologia , Colágeno , Trato Gastrointestinal/citologia , Trato Gastrointestinal/imunologia , Interleucina-10/biossíntese , Interleucina-17/biossíntese , Linfonodos/citologia , Linfonodos/imunologia , Ratos , Ratos Wistar , Linfócitos T Reguladores/citologia , Células Th17/citologiaRESUMO
(E)-3-(4-chlorophenyl)-N-(7-hydroxy-6-methoxy-2-oxo-2H-chromen-3-yl) acrylamide (SC-III3), a newly synthesized derivative of scopoletin, was previously shown to reduce the viability of HepG2 cells and tumor growth of HepG2 xenograft mouse model. It induces the death of HepG2 cells by a way irrelevant to apoptosis and necrosis. To shed light on the cytotoxic mechanisms of SC-III3, the present study addresses whether and how it can induce autophagic cell death. When HepG2 cells were incubated with various concentrations of SC-III3, autophagic vacuoles could be observed by transmission electron microscopy and monodansylcadaverine staining. Increased expressions of LC3-II to LC3-I and Beclin-1, required for autophagosome formation, were accompanied. These characteristics integrally indicated that SC-III3 could initiate autophagy in HepG2 cells. N-acetyl-l-cysteine (NAC), a ROS scavenger, could reverse SC-III3-caused ROS accumulation, but it did not affect SC-III3-induced autophagy, suggesting that ROS was not involved in SC-III3-mediated autophagy in HepG2 cells. SC-III3 significantly depressed mitochondrial function, as evidenced by disruption of mitochondrial transmembrane potential and loss of the mitochondrial cristae structure, as well as decrease of Cox-I, Cox-III, Cox-IV, and ATP levels. The autophagy and activation of AMPK-TSC2-mTOR-p70s6k pathways induced by SC-III3 in HepG2 cells could be efficiently blocked by pre-treatments of compound C (an inhibitor of AMPK). Moreover, addition of extracellular ATP to the cell culture media could reverse SC-III3-caused activation of AMPK-TSC2-mTOR-p70s6k pathway, autophagy and cell viability decrease in HepG2 cells. Collectively, SC-III3 leads to autophagy through inducing mitochondrial dysfunction, depleting ATP, and activating AMPK-mTOR pathway, which thus reflects the cytotoxic effect of SC-III3 in HepG2 cells.