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BACKGROUND: Patients with decompensated cirrhosis face poor prognosis and increased mortality risk. Rifaximin, a non-absorbable antibiotic, has been shown to have beneficial effects in preventing complications and improving survival in these patients. However, the underlying mechanisms of rifaximin's effects remain unclear. METHODS: We obtained fecal samples from decompensated cirrhotic patients undergoing rifaximin treatment and controls, both at baseline and after 6 months of treatment. Shotgun metagenome sequencing profiled the gut microbiome, and untargeted metabolomics analyzed fecal metabolites. Linear discriminant and partial least squares discrimination analyses were used to identify differing species and metabolites between rifaximin-treated patients and controls. RESULTS: Forty-two patients were enrolled and divided into two groups (26 patients in the rifaximin group and 16 patients in the control group). The gut microbiome's beta diversity changed in the rifaximin group but remained unaffected in the control group. We observed 44 species with reduced abundance in the rifaximin group, including Streptococcus_salivarius, Streptococcus_vestibularis, Haemophilus_parainfluenzae, etc. compared to only four in the control group. Additionally, six species were enriched in the rifaximin group, including Eubacterium_sp._CAG:248, Prevotella_sp._CAG:604, etc., and 14 in the control group. Furthermore, rifaximin modulated different microbial functions compared to the control. Seventeen microbiome-related metabolites were altered due to rifaximin, while six were altered in the control group. CONCLUSION: Our study revealed distinct microbiome-metabolite networks regulated by rifaximin intervention in patients with decompensated cirrhosis. These findings suggest that targeting these specific metabolites or related bacteria might be a potential therapeutic strategy for decompensated cirrhosis.
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Cirrose Hepática , Metagenoma , Humanos , Rifaximina/uso terapêutico , Cirrose Hepática/complicações , Resultado do Tratamento , Antibacterianos/uso terapêuticoRESUMO
Because of its high economic value and potential for adaptation to subtropical climates, Indian jujube (Zizyphus mauritiana Lam.) is one of the most important fruit crops introduced into south of Guizhou Province, China. In December 2020, approximately 10 to 15% of the harvested jujube (Z. mauritiana Lam. Wuqian) showed fruit rot symptoms after storage at 4°C for 10-15 days in Luodian county (25°34'N, 106°82'E). Symptoms of brown, circular, watery lesions were observed on the jujube fruits. Small pieces (c.a. 5 mm) at the margins of rot tissue were incubated on PDA plates at 25°C in darkness after surface sterilization in 1.5% NaClO for 45 s followed with triple washes using sterile distilled water. Two monoconidial isolates were obtained after incubation and identical colony morphologies were observed with olive grey, cottony aerial mycelium which became darker after 10 days growth. The colony reverse began white but turned brown with age. Conidia, produced in orange masses, were mainly cylindrical with the size of 9.2-16.8 µm (average 13.7 µm) × 3.8-6.2 µm (average 4.6 µm) (n = 50), typical of Colletotrichum spp. (Vieira et al. 2014). For further identification, DNA of these two isolates were extracted and were used for multi-locus genotyping. Five loci, including the ITS region, partial sequences of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), ß-tubulin (BTU) and chitin synthase (CHS) genes, were amplified and sequenced with primers of ITS1/ITS4, GDF1/GDR1, ACT512F/ ACT783R, Bt2a/Bt2b and CHS79F/CHS354R, respectively. No differences was found between the isolates at any of the loci and one sequence for each locus was deposited in the Genebank database under accessions OL376803, OL404925, OL404926, OL404927 and OL404928, respectively. Blastn results indicated that the ITS, GAPDH, ACT, BTU and CHS sequences of the jujube isolates shared 100%, 98.56%, 96.62%, 99.48% and 99.33% similarity with those of ex-type strain ICMP 18581 of C. fructicola (GenBank Accession Nos. JX010165, JX010033, JX009501, JX010405 and JX009866). Phylogenetic analysis including published ITS, GAPDH, ACT, BTU and CHS data for C. fructicola and other Colletotrichum species was performed using MEGA 6.0. Based on morphological and molecular data, the jujube isolates were identified as C. fructicola. Pathogenicity was determined for both isolates on jujube fruits cultivar "Wuqian". Fruit surface was sterilized with 75% ethanol, air dried, and wounded with a needle by piercing into 2 mm depth. Ten microliters of a spore suspension (1 × 106 spores/ml) or sterilized water were applied to one of two wounds on the same fruit. There were six replicate inoculations for each isolate and the whole experiment was repeated twice. Treated fruit were maintained in a growth chamber with 80% relative humidity at 25°C. Symptoms of fruit rots, identical the original observations, developed around the infection sites at 3 days post inoculation. These began as light brown, circular lesions, which got darker with orange spore masses after 7 days and both isolates caused identical symptoms. However, the wounds inoculated with water remained asymptomatic. C. fructicola was successfully reisolated from the infected areas to fulfill Koch's postulates. To the best of our knowledge, this is the first report of jujube fruit rot caused by C. fructicola in China, which may become an emerging problem considering the area expansion of Z. mauritiana cultivation and transportation of its fruit. Funding: Funding was provided by Science and Technology Foundation of Guizhou Province (Guizhou Science Base [2020]1Y104), Talent Development Program of Guizhou Province (Qian Jiaohe KY [2021]080), Innovation and Entrepreneurship Training Program of Guizhou University (Guo Chuangzi [2020]017). Reference: (1) Vieira, W., et al. 2014. Fungal Divers. 67(1): 181-202.
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AIM: This study aimed to investigate the effects of endometrial stromal cells (ESC)-derived interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1 on macrophage polarization in endometriosis. METHODS: Macrophage polarization was measured in eutopic endometrium of control participants ('normal endometrium'), eutopic endometrium of patients with endometriosis ('eutopic endometrium') and ectopic endometrium of endometriosis patients ('ectopic endometrium') by immunohistochemical staining. Expression of IL-6 and MCP-1 were measured in the eutopic and ectopic endometrium through enzyme-linked immunosorbent assays. Expression of CD163 was measured in human acute monocytic leukemia (THP-1) cell-derived macrophages that were treated with conditional medium induced by tumor necrosis factor (TNF)-α, TNF-α + anti-IL-6 or TNF-α + anti-MCP-1 via flow cytometry. RESULTS: The ratio of CD163+/CD68+ macrophages in the normal endometrium was higher than that in the eutopic endometrium, while differences between the eutopic and ectopic endometrium were not statistically significant. IL-6 and MCP-1 exhibited enhanced expression in the ectopic endometrium group and decreased expression in the eutopic endometrium group. TNF-α could promote the expression of ESC-derived IL-6 and MCP-1. Intervention with TNF-α-induced conditioned medium resulted in the upregulation of CD163 in THP-1 cells, while conditional medium induced with IL-6 and MCP-1 neutralizing antibodies decreased the proportion of CD163+ macrophages significantly. CONCLUSION: In endometriosis patients, the macrophages of the eutopic endometrium polarize toward M1 compared with the normal endometrium, and those of the ectopic endometrium were mainly M2-polarized. Under the action of TNF-α, ESC-derived IL-6 and MCP-1 could stimulate peritoneal macrophages toward M2-polarization, which could modulate endometriosis.
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Quimiocina CCL2/metabolismo , Endometriose/imunologia , Endométrio/imunologia , Interleucina-6/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Endométrio/citologia , Endométrio/metabolismo , Feminino , Humanos , Macrófagos/metabolismo , Cultura Primária de Células , Receptores de Superfície Celular/metabolismo , Células Estromais/metabolismo , Fator de Necrose Tumoral alfaRESUMO
A central component of the cellular stress response is p21(WAF1/CIP1), which regulates cell proliferation, survival, and differentiation. Inflammation and cell stress often up-regulate p21 posttranscriptionally by regulatory mechanisms that are poorly understood. ZO-1-associated nucleic acid binding protein (ZONAB)/DbpA is a Y-box transcription factor that is regulated by components of intercellular junctions that are affected by cytokines and tissue damage. We therefore asked whether ZONAB activation is part of the cellular stress response. Here, we demonstrate that ZONAB promotes cell survival in response to proinflammatory, hyperosmotic, and cytotoxic stress and that stress-induced ZONAB activation involves the Rho regulator GEF-H1. Unexpectedly, stress-induced ZONAB activation does not stimulate ZONAB's activity as a transcription factor but leads to the posttranscriptional up-regulation of p21 protein and mRNA. Up-regulation is mediated by ZONAB binding to specific sites in the 3'-untranslated region of the p21 mRNA, resulting in mRNA stabilization and enhanced translation. Binding of ZONAB to mRNA is activated by GEF-H1 via Rho stimulation and also mediates Ras-induced p21 expression. We thus identify a unique type of stress and Rho signaling activated pathway that drives mRNA stabilization and translation and links the cellular stress response to p21 expression and cell survival.
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Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Células Epiteliais/fisiologia , Proteínas de Choque Térmico/metabolismo , Estabilidade de RNA/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Apoptose/fisiologia , Células CACO-2 , Sobrevivência Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Cães , Células Epiteliais/citologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/fisiopatologia , Rim/citologia , Proteínas de Membrana/metabolismo , Necrose/metabolismo , Fosfoproteínas/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , RNA Mensageiro/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Estresse Mecânico , Junções Íntimas/fisiologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteína da Zônula de Oclusão-1RESUMO
PURPOSE: To evaluate the clinical efficacy of letrozole on ovulation induction and hormone replacement therapy (HRT) during endometrial preparation for frozen-thawed embryo transfer (FET). METHODS: We analyzed totally 1,230 cycles of patients that underwent FET from October 2010 to September 2012. Seven hundred and thirteen cycles of patients with ovulation disorders that underwent FET were randomly assigned to two groups by case control study. 359 cycles received letrozole ovulation induction and 354 cycles received HRT during endometrial preparation for FET, respectively. In the corresponding period, 517 cycles of patients with normal ovulation in the natural cycle group for FET endometrial preparation served as controls. Reproduction-related clinical outcomes of patients in the three groups were compared. RESULTS: The embryo implantation rate of patients in letrozole group (30.4 %) was significantly higher than the HRT group (22.8 %, P < 0.05). The clinical pregnancy rate of patients in the letrozole group (53.2 %) was significantly higher than the HRT group (44.4 %, P < 0.05), while no significant difference was observed between the letrozole and natural cycle groups (51.3 %, P > 0.05). Estradiol levels on the day of human chorionic gonadotropin administration in the letrozole group were significantly lower than those in the natural cycle group (280.32 ± 125.39 pg/ml and 351.06 ± 123.03 pg/ml, respectively; P < 0.05). The live birth rate of patients in letrozole group (44.6 %) was significantly higher than the HRT group (32.5 %, P < 0.05), while abortion rate (12.0 %) was significantly lower than the HRT group (21.0 %, P < 0.05). There were no significant differences in number of mature follicles, endometrial thickness, duration of follicle growth between the letrozole and the natural cycle groups, and there were no significant differences in twin birth rate and ectopic pregnancy rate among the three groups (all P values >0.05). CONCLUSIONS: Ovulation induction with letrozole during endometrial preparation for FET has a higher rate of pregnancy success and a lower abortion rate than HRT. Letrozole treatment exhibits clinical progression and outcomes similar to those patients undergoing a natural cycle or normal ovulation cycle. Therefore, letrozole treatment may be an effective option in endometrial preparation for FET in patients with ovulation disorders or irregular menstruation.
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Inibidores da Aromatase/farmacologia , Transferência Embrionária/métodos , Nitrilas/farmacologia , Indução da Ovulação/métodos , Triazóis/farmacologia , Aborto Espontâneo/epidemiologia , Adulto , Coeficiente de Natalidade , Estudos de Casos e Controles , China/epidemiologia , Criopreservação , Implantação do Embrião , Feminino , Terapia de Reposição Hormonal , Humanos , Letrozol , Folículo Ovariano/efeitos dos fármacos , Gravidez , Resultado da Gravidez , Taxa de Gravidez , Gravidez Ectópica/epidemiologiaRESUMO
A simple approach for copper-promoted S-arylation reactions utilizing triarylbismuths or triarylantimonys as arylating reagents has been described. These reactions can be performed under mild conditions and exhibit remarkable functional group tolerance and chemoselectivity. The corresponding 2-arylthiopyridine 1-oxide derivatives and arylthioanilines/phenols have been successfully synthesized, achieving good to excellent yields across over 49 examples.
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Non-alcoholic steatohepatitis (NASH) is the progressive stage of non-alcoholic fatty liver disease (NAFLD). The non-absorbable antibiotic rifaximin has been used for treatment of irritable bowel syndrome, traveling diarrhea, and hepatic encephalopathy, but the efficacy of rifaximin in NASH patients remains controversial. This study investigated the effects and underlying mechanisms of rifaximin treatment in mice with methionine and choline deficient (MCD) diet-induced NASH. We found that rifaximin greatly ameliorated hepatic steatosis, lobular inflammation, and fibrogenesis in MCD-fed mice. Bacterial 16S rRNA sequencing revealed that the gut microbiome was significantly altered in MCD-fed mice. Rifaximin treatment enriched 13 amplicon sequence variants (ASVs) belonging to the groups Muribaculaceae, Parabacteroides, Coriobacteriaceae_UCG-002, uncultured Oscillospiraceae, Dubosiella, Rikenellaceae_RC9_gut_group, Mucispirillum, and uncultured Desulfovibrionaceae. However, rifaximin treatment also reduced seven ASVs in the groups Aerococcus, Oscillospiraceae, uncultured Ruminococcaceae, Bilophila, Muribaculaceae, Helicobacter, and Alistipes in MCD-fed mice. Bile acid-targeted metabolomic analysis indicated that the MCD diet resulted in accumulation of primary bile acids and deoxycholic acid (DCA) in the ileum. Rifaximin delivery reduced DCA levels in MCD-fed mice. Correlation analysis further showed that DCA levels were associated with differentially abundant ASVs modulated by rifaximin. In conclusion, rifaximin may ameliorate NASH by decreasing ileal DCA through alteration of the gut microbiome in MCD-fed mice. Rifaximin treatment may therefore be a promising approach for NASH therapy in humans.
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Tight junctions are heteromeric protein complexes that act as signaling centers by mediating the bidirectional transmission of information between the environment and the cell interior to control paracellular permeability and differentiation. Insight into tight junction-associated signaling mechanisms is of fundamental importance for our understanding of the physiology of epithelia and endothelia in health and disease.
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Comunicação Celular , Células Endoteliais/enzimologia , Células Epiteliais/enzimologia , Transdução de Sinais , Junções Íntimas/enzimologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Comunicação Celular/genética , Diferenciação Celular , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Complexos Multiproteicos , Permeabilidade , Transdução de Sinais/genéticaRESUMO
Epithelial tight junctions recruit different types of signalling proteins that regulate cell proliferation and differentiation. Little is known about how such proteins interact functionally and biochemically with each other. Here, we focus on the Y-box transcription factor ZONAB (zonula occludens 1-associated nucleic-acid-binding protein)/DbpA (DNA-binding protein A) and the Rho GTPase activator guanine nucleotide exchange factor (GEF)-H1/Lbc's first cousin, which are two tight-junction-associated signalling proteins that regulate proliferation. Our data show that the two proteins interact and that ZONAB activity is Rho-dependent. Overexpression of GEF-H1 induces accumulation of ZONAB in the nucleus and activates transcription. Microtubule-affinity regulating kinase/partition-defective-1, another type of GEF-H1-associated signalling protein, remains in the cytoplasm and partially co-localizes with the exchange factor. GEF-H1 and ZONAB are required for expression of endogenous cyclin D1, a crucial RhoA signalling target gene, and GEF-H1-stimulated cyclin D1 promoter activity requires ZONAB. Our data thus indicate that GEF-H1 and ZONAB form a signalling module that mediates Rho-regulated cyclin D1 promoter activation and expression.
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Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Proteínas de Ligação a DNA/genética , Cães , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Ligação Proteica , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais , Junções Íntimas/metabolismo , Fatores de Transcrição/genéticaRESUMO
OBJECTIVE: This study investigated the alterations in macrophage polarization in patients with endometriosis as well as the underlying molecular mechanisms. METHODS: Peritoneal washings, serum samples, and endometrial tissues were collected from endometriosis patients and control subjects. Endometrial stromal cells (ESCs) were isolated from endometrial tissue, and conditioned medium was prepared by treating ESCs with or without various concentrations of interleukin- (IL-) 6, estrogen, or progestin. The frequencies of CD86+ and CD163+ cells and expression levels of these markers as well as the cytokines IL-12 and IL-10 were measured in THP-1- (human monocytic leukemia cell) derived macrophages. RESULTS: There was a decrease in the percentage of CD86+ macrophages in the peritoneal wash solution of patients with endometriosis. Ectopic endometrial homogenates could promote M1 to M2 macrophage polarization in response to lipopolysaccharide (LPS), as evidenced by the increased percentage of CD163+ macrophages and increased IL-10 expression as well as a decreased percentage of CD86+ cells and lower IL-12 expression. In contrast, addition of serum from women with endometriosis to THP-1 cells resulted in the polarization of macrophages towards both M1 and M2 phenotypes. Upregulation of Smad2/Smad3 in macrophages upon exposure to eutopic and ectopic endometrial homogenates as well as serum of women with endometriosis was observed, and blockage of Smad2/Smad3 with their inhibitor SB431542 could reverse the macrophage polarization from M1 to M2. Conditioned medium induced by IL-6, but neither estrogen nor progestin, could facilitate M2 polarization. Neutralization of IL-6 diminished macrophage M2 polarization in endometriosis. CONCLUSION: This study provides detailed evidence supporting alterations in M1 to M2 macrophage polarization that may contribute to the initiation as well as progression of endometriosis.
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Coristoma/imunologia , Endometriose/imunologia , Endométrio/imunologia , Macrófagos/imunologia , Soro/imunologia , Células Estromais/fisiologia , Adulto , Diferenciação Celular , Técnicas de Cocultura , Citocinas/metabolismo , Feminino , Humanos , Imunomodulação , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Células THP-1RESUMO
Bradykinin and interleukin-1beta (IL-1beta) induce cyclooxygenase 2 (COX-2) in human airway smooth muscle cells. Here we extended our study to explore the gene transcriptional regulation. By transfection with various COX-2 promoter reporter constructs, we found that the bp -327-to-+59 promoter region was essential for COX-2 gene transcription by bradykinin and IL-1beta and that the cyclic AMP response element (CRE) was critical in bradykinin-induced transcription, whereas nuclear factor IL-6 and CRE and, to a lesser extent, nuclear factor-kappaB (NF-kappaB) were involved in IL-1beta-induced transcription. An electrophoretic mobility shift assay revealed that both bradykinin and IL-1beta elicited CRE-binding protein-1 (CREB-1) binding, and IL-1beta also elicited CCAAT/enhancer-binding protein beta and NF-kappaB binding to their respective elements in the COX-2 promoter. These transcription factors were associated with the COX-2 promoter, which was dynamically linked to different patterns of histone H4 acetylation by bradykinin and IL-1beta, as demonstrated by chromatin immunoprecipitation. We also revealed that endogenous prostaglandin E(2) was critical in bradykinin-induced COX-2 transcription initiation and involved in IL-1beta-induced COX-2 transcription at a latter stage. Our result provide the first evidence that COX-2 transcriptional regulation by different stimuli involves different promoter elements and transcription factors and is associated with chromatin remodeling after selective histone H4 acetylation in a stimulus-specific manner.
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Bradicinina/farmacologia , Interleucina-1/farmacologia , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintases/genética , Acetilação , Transporte Ativo do Núcleo Celular , Sequência de Bases , Sítios de Ligação/genética , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Ciclo-Oxigenase 2 , DNA/genética , DNA/metabolismo , Dinoprostona/metabolismo , Histonas/metabolismo , Humanos , Técnicas In Vitro , Proteínas de Membrana , Modelos Biológicos , Dados de Sequência Molecular , Músculo Liso/efeitos dos fármacos , Músculo Liso/enzimologia , Regiões Promotoras Genéticas , Músculos Respiratórios/efeitos dos fármacos , Músculos Respiratórios/enzimologia , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
We previously reported that proinflammatory mediator bradykinin (BK) induces cyclooxygenase (COX)-2 expression in human airway smooth muscle (HASM), but the mechanism is unknown in any biological system. Here, we studied the role of specific protein kinase C (PKC) isozyme(s) in COX-2 expression. Among the eight PKC isozymes present in HASM cells, the Ca2+-independent PKC-delta and -epsilon and the Ca2+-dependent PKC-alpha and -betaI were translocated to the nucleus upon BK stimulation. BK-induced COX-2 expression and prostaglandin E2 (PGE2) accumulation were mimicked by the direct PKC activator phorbol 12-myristate 13-acetate (PMA) and inhibited by the broad spectrum PKC inhibitor bisindolylmaleimide I. However, the selective Ca2+-dependent PKC isozyme inhibitor Go 6976 had no effect. Furthermore, the membrane-permeable calcium chelator BAPTA-AM had no effect on BK-induced COX-2 expression and COX activity despite its inhibition of PGE2 accumulation, suggesting the involvement of Ca2+-independent PKC isozymes. Rottlerin, a PKC-delta inhibitor, also had no effect, likely implicating PKC-epsilon. BK-stimulated transcriptional activation of a COX-2 promoter reporter construct was enhanced by overexpression of wild-type PKC-epsilon and abolished by a dominant negative PKC-epsilon, but it was not affected by wild-type or dominant negative PKC-alpha or -delta. Collectively, our results demonstrate that PKC-e mediates BK-induced COX-2 expression in HASM cells.
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Bradicinina/farmacologia , Isoenzimas/biossíntese , Isoenzimas/fisiologia , Pulmão/enzimologia , Músculo Liso/enzimologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Proteína Quinase C/fisiologia , Células Cultivadas , Ciclo-Oxigenase 2 , Dinoprostona/biossíntese , Humanos , Isoenzimas/genética , Pulmão/citologia , Proteínas de Membrana , Modelos Biológicos , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Mutação , Prostaglandina-Endoperóxido Sintases/genética , Proteína Quinase C/genética , Proteína Quinase C-épsilon , RNA Mensageiro/biossíntese , Ativação TranscricionalRESUMO
OBJECTIVE: To investigate the inhibition of HPV18E6 gene in HeLa cell transfected with plasmid expressing human papilloma virus 18 E6 (HPV18E6) short hairpin RNA (shRNA). METHODS: We synthesized two HPV18E6 shRNA frames and sub-cloned them into pSUPER which can express shRNA in mammalian cells to construct pE6-1shRNA and pE6-2shRNA which were mutant in E6 shRNA frame. The pE6-1shRNA, pE6-2shRNA and pcDNA3.1 were co-transfected into HeLa cells by cationic liposome respectively and the positive transfectants were selected by G418. The HPV18E6 mRNA and protein expression level was detected by semi-quantitative RT-PCR and streptavidin-peroxidase conjugated method (SP) to assay the inhibitory effects of pE6shRNA. RESULTS: We successfully constructed several new HeLa cell lines transfected with pE6-1shRNA and pE6-2shRNA. In the HeLa cells without transfection and the HeLa cells transfected with pE6-1shRNA plasmid, the HPV18E6 mRNA levels were 1.14 +/- 0.45, 0.76 +/- 0.28 respectively, and the difference of HPV18E6 mRNA levels was significant (P < 0.05). The inhibition efficiency of HPV18E6 gene mRNA was 33.3% and the HPV18E6 protein levels were declined after transfection with pE6-1shRNA. In the HeLa cells transfected with pE6-2shRNA and pSUPER plasmids, HPV18E6 mRNA and protein expression levels were not different from those in wild HeLa cells. CONCLUSIONS: The pE6-1shRNA plasmid can inhibit HPV18E6 expression in HeLa cells, which is persistent, specific and heritable.
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Proteínas de Ligação a DNA/genética , Proteínas Oncogênicas Virais/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Células HeLa , Humanos , Imuno-Histoquímica , Proteínas Oncogênicas Virais/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologiaRESUMO
A series of the new ruthenium(II) complexes with different number of aldehyde groups have been synthesized and characterized for the simple and selective sensing of homocysteine (Hcy) and cysteine (Cys). The reaction of these ruthenium(II) complexes with Hcy and Cys afforded thiazinane or thiazolidine derivatives which resulted in the obvious changes in the UV-visible spectra and strong enhancement of the luminescence intensity of the system. The luminescence enhancement of [Ru(dmb)(2)(L2)](2+) (dmb: 4,4'-dimethyl-2,2'-bipyridine) showed a good linearity in the concentration of 4.2-350 µM and 6-385 µM with the detection limits of 0.3 µM and 1 µM for Hcy and Cys, respectively. The absorption and emission bands from metal-to-ligand charge transfer transition in the visible region and the large Stokes shift of the ruthenium(II) complex chromophore made it suitable for biological applications.
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Cisteína/química , Homocisteína/química , Compostos Organometálicos/química , Rutênio/química , Cátions Bivalentes , Medições Luminescentes , Compostos Organometálicos/síntese química , Espectrofotometria UltravioletaRESUMO
A colorimetric and luminescent bifunctional Ru(II) complex-modified gold nano-probe for sensing ctDNA was developed. A new water-soluble Ru(II) complex containing an aromatic α-diimine ligand with an extended π system was found to be emissive at about 588nm in water and the emission intensity of the complex was enhanced about 3.4-fold upon addition of calf thymus DNA (ctDNA) in aqueous buffer with no color changes. The detection limit was 70nM with a 1.2% relative standard deviation (RSD, n=5). The Ru(II) complex chromophore was used to modify 8nm MHA carboxylate-functionalized gold nanoparticles to produce a colorimetric and luminescent bifunctional probe for sensing ctDNA in aqueous buffer at room temperature. The obvious change in visible color and enhancement of emission intensity of the functionalized gold-Ru(II) complex colloids upon addition of ctDNA was due to the electron or energy transfer between Ru(II) chromophore and the Au-NPs. The limit of detection was 1.0nM for ctDNA with a 4.5% RSD (n=5). With such a high sensitivity, the bifunctional Ru(II) complex-modified gold nano-probe will be potentially suitable for the DNA sensing in bioanalytical application.
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Técnicas Biossensoriais/métodos , DNA/análise , Nanopartículas Metálicas , Animais , Bovinos , Colorimetria , Ouro , Luminescência , Compostos Organometálicos , RutênioRESUMO
Recent studies have shown that a lack of eosinophils in asthmatic airway smooth muscle (ASM) bundles in contrast to the large number of mast cells is a key feature of asthma. We hypothesized that this is caused by beta-tryptase, the predominant mast cell-specific protease, abrogating the eosinophil chemotactic activities of ASM cell-derived eosinophil chemoattractants such as eotaxin and RANTES. We studied the effect of beta-tryptase on the immunoreactivities of human ASM cell-derived and recombinant eotaxin and other recombinant chemokines that are known to be produced by human ASM cells. We report in this study that purified beta-tryptase markedly reduced the immunoreactivity of human ASM cell-derived and recombinant eotaxin, but had no effect on eotaxin mRNA expression. The effect was mimicked by recombinant human beta-tryptase in the presence of heparin and was reversed by heat inactivation and the protease inhibitor leupeptin, suggesting that the proteolytic activity of tryptase is required. beta-Tryptase also exerted similar effects on recombinant RANTES, but not on the other chemokines and cytokines that were screened. Furthermore, a chemotaxis assay revealed that recombinant eotaxin and RANTES induced eosinophil migration concentration-dependently, which was abrogated by pretreatment of these chemokines with beta-tryptase. Another mast cell protease chymase also markedly reduced the immunoreactivity of eotaxin, but had no effect on RANTES and other chemokines and did not affect the influence of beta-tryptase on RANTES. These findings suggest that mast cell beta-tryptase selectively cleaves ASM-derived eotaxin and RANTES and abrogates their chemotactic activities, thus providing an explanation for the eosinophil paucity in asthmatic ASM bundles.
Assuntos
Quimiocina CCL5/metabolismo , Quimiocinas CC/metabolismo , Quimiotaxia , Eosinófilos/citologia , Mastócitos/enzimologia , Serina Endopeptidases/metabolismo , Asma/imunologia , Asma/metabolismo , Asma/patologia , Células Cultivadas , Quimiocina CCL11 , Quimiocina CCL5/imunologia , Quimiocinas CC/genética , Eosinófilos/imunologia , Eosinófilos/metabolismo , Regulação da Expressão Gênica , Humanos , Leupeptinas/metabolismo , RNA Mensageiro/genética , Proteínas Recombinantes/metabolismo , TriptasesRESUMO
Eotaxin is a potent eosinophil chemoattractant implicated in various allergic inflammatory conditions including asthma, but relatively little is known about its regulation. Human airway smooth muscle cells are an important source of eotaxin in the airway. We have previously demonstrated that beta(2)-adrenoceptor agonists (beta(2)-agonists) and glucocorticoids additively inhibit eotaxin production in human airway smooth muscle cells, but the underlying mechanisms are unclear. Here, we studied the molecular mechanisms of their actions and interactions on eotaxin gene transcription. TNF-alpha-induced eotaxin gene transcription was mediated mainly by the transcription factor NF-kappaB (p65/p50) as analyzed by luciferase reporter gene assay, Western blotting, EMSA, and electrophoretic mobility supershift assay. Chromatin immunoprecipitation assay demonstrated that TNF-alpha also induced selective histone H4 acetylation on lysines 5 and 12 at the eotaxin promoter site and p65 binding to the eotaxin promoter, resulting in eotaxin gene transcription. The inhibition of eotaxin production by beta(2)-agonists and glucocorticoids was transcriptional and not due to altered NF-kappaB nuclear translocation or in vitro promoter binding capability, but due to their inhibition of TNF-alpha-induced histone H4 acetylation and p65 in vivo binding to the promoter. Additive inhibition was achieved when the two groups of drugs were combined. Our findings reveal a novel mechanism by which beta(2)-agonists, like glucocorticoids, regulate NF-kappaB-mediated inflammatory gene expression through inhibition of histone acetylation. This provides one explanation for the benefits that result when these agents are combined to treat asthma, and may have important implications in a wide range of inflammatory diseases.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Quimiocinas CC/genética , Glucocorticoides/farmacologia , Histonas/efeitos dos fármacos , Histonas/metabolismo , Acetilação , Albuterol/análogos & derivados , Albuterol/farmacologia , Androstadienos/farmacologia , Sequência de Bases , Linhagem Celular , Quimiocina CCL11 , DNA/genética , DNA/metabolismo , Dexametasona/farmacologia , Fluticasona , Histonas/química , Humanos , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Mutação , NF-kappa B/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/metabolismo , Fator de Transcrição STAT6 , Xinafoato de Salmeterol , Transativadores/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transfecção , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Chemokine-mediated inflammatory cell infiltration is a hallmark of asthma. We recently demonstrated that glucocorticoids and beta(2)-agonists additively or synergistically suppress tumor necrosis factor-alpha (TNFalpha)-induced production of chemokines eotaxin and interleukin-8 (IL-8), respectively, in human airway smooth muscle (HASM) cells, which may partly explain their combined benefits in asthma. Peroxisome proliferator-activated receptors (PPARs) also modulate inflammatory gene expression. We reported here that the PPARgamma agonists 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) and troglitazone, but not PPARalpha agonist WY-14643, inhibited TNFalpha-induced production of eotaxin and monocyte chemotactic protein-1 (MCP-1) but not IL-8. Eotaxin inhibition was transcriptional and additively enhanced by the glucocorticoid fluticasone and the beta(2)-agonist salmeterol, whereas MCP-1 inhibition was post-transcriptional and additively and synergistically enhanced by fluticasone and salmeterol, respectively. Coimmunoprecipitation revealed that 15d-PGJ(2) induced a protein-protein interaction between PPARgamma and the glucocorticoid receptor (GR) in TNFalpha-treated HASM cells, which was enhanced by fluticasone and salmeterol. 15d-PGJ(2), fluticasone, and salmeterol all inhibited TNFalpha-induced histone H4 acetylation at the eotaxin promoter and NF-kappaB p65 binding to the eotaxin promoter and induced PPARgamma and GR association with the eotaxin promoter, as analyzed by chromatin immunoprecipitation assay. Our data suggest that chemokine expression in HASM cells is differentially regulated by PPARgamma agonists and that the interaction between PPARgamma and GR may be responsible for the additive and synergistic inhibition of chemokine expression by PPARgamma agonists, glucocorticoids, and beta(2)-agonists, particularly the chromatin-dependent suppression of eotaxin gene transcription. The interaction may have wide applications and may provide a potential target for pharmacological and molecular intervention.
Assuntos
Albuterol/análogos & derivados , Regulação da Expressão Gênica , Glucocorticoides/metabolismo , PPAR gama/agonistas , Prostaglandina D2/análogos & derivados , Receptores de Glucocorticoides/metabolismo , Agonistas Adrenérgicos beta/metabolismo , Albuterol/farmacologia , Androstadienos/farmacologia , Western Blotting , Linhagem Celular , Quimiocina CCL11 , Quimiocina CCL2/biossíntese , Quimiocinas/metabolismo , Quimiocinas CC/biossíntese , Quimiocinas CC/metabolismo , Cromanos/farmacologia , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Fluticasona , Humanos , Imunoprecipitação , Interleucina-8/biossíntese , PPAR alfa/agonistas , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Prostaglandina D2/farmacologia , Ligação Proteica , Pirimidinas/farmacologia , RNA/química , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Xinafoato de Salmeterol , Tiazolidinedionas/farmacologia , Transcrição Gênica , Transfecção , Troglitazona , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to modulate cyclooxygenase (COX)-2 expression, but the mechanisms involved are controversial and may be cell specific. We show in this study that indomethacin (Indo), flurbiprofen (Flur), and the selective COX-2 inhibitor NS-398 induced COX-2 expression and markedly enhanced IL-1beta-induced COX-2 expression in human airway smooth muscle (HASM) cells. These effects were not reversed by exogenous PGE(2), suggesting that they are prostanoid-independent. Indeed, PGE(2) also induced and enhanced IL-1beta-induced COX-2 expression. Peroxisome proliferator-activated receptor (PPAR) alpha and PPARgamma (not PPARbeta) were expressed in HASM cells. PPARgamma activators ciglitizone (Cig) and 15-Deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), but not the PPARalpha activator WY-14643, mimicked the effect of NSAIDs on COX-2 expression. Treatment with Flur, NS-398, Cig, and 15d-PGJ(2) alone, but not Indo and WY-14643, elevated COX activity; however, neither enhanced IL-1beta-induced COX activity. Pretreatment with dexamethasone suppressed COX-2 expression, PGE(2) release, and COX activity induced by NS-398, Cig, IL-1beta, alone or in combination. Unlike IL-1beta, NS-398 and Cig did not cause NF-kappaB (p65) nuclear translocation, nor did they further enhance IL-1beta-induced NF-kappaB translocation, but they stimulated PPARgamma translocation. Indo, NS-398, Flur, and 15d-PGJ(2), but not WY-14643, induced transcriptional activity of a COX-2 reporter construct containing the peroxisome proliferator response element (PPRE) on their own and enhanced the effect of IL-1beta, but had no effect on a COX-2 reporter construct lacking the PPRE. The results suggest that COX-2 expression by NSAIDs is biologically functional, prostanoid-independent, and involves PPARgamma activation, and provide the first direct evidence that the PPRE in the promoter is required for NSAID-induced COX-2 expression.