RESUMO
BACKGROUND: Hepatitis B infection is a health problem that affects more than 400 million people all over the world. We aimed to evaluate the usability of prolidase enzyme that plays an important role in collagen synthesis. Prolidase levels increase in hepatic damage, which can be used as diagnostic parameters in the progressions to chronic hepatitis B (CHB) infection by evaluating it in different clinical forms of hepatitis B infection. METHODS: A total of 69 patients who presented to our clinic with chronic hepatitis B (CHB) infection, 72 patients with inactive hepatitis B infection (IHB), and 45 healthy volunteers were included into this study. Alanine transaminase (ALT), Aspartate aminotransferase (AST) and prolidase levels of patients were measured. Hepatic biopsy was performed in patients with CHB infection. Prolidase levels were evaluated in three different groups, and its correlations with fibrosis were investigated. RESULTS: Prolidase was different between all groups (P < 0.001). Prolidase level was found to be higher in CHB and IHB compared to the control group. There was no correlation between this enzyme, fibrosis, and histological activity index. CONCLUSION: In this present study, it is shown that prolidase levels increase in hepatitis B infection. It may be used as a biochemical marker in the chronic hepatitis B.
Assuntos
Dipeptidases/sangue , Hepatite B Crônica/enzimologia , Fígado/enzimologia , Adulto , Alanina Transaminase/sangue , Aspartato Aminotransferases/sangue , Biomarcadores/sangue , Colágeno/metabolismo , Progressão da Doença , Feminino , Hepatite B Crônica/sangue , Hepatite B Crônica/diagnóstico , Humanos , Cirrose Hepática/diagnóstico , Masculino , Adulto JovemRESUMO
BACKGROUND: The aim of this study was to evaluate oxidative stress and to determine the activity of paraoxonase and arylesterase in patients with osteomyelitis compared to healthy controls. METHOD: In total, 30 patients diagnosed with osteomyelitis and 30 healthy volunteers were enrolled in the study. Paraoxonase and arylesterase activities were measured spectrophotometrically. Serum lipid hydroperoxide (LOOH) concentrations were measured by ferrous oxidation with xylenol orange (FOX) assay as markers of oxidative stress. RESULTS: Serum paraoxonase and arylesterase activities were significantly lower in patients with osteomyelitis compared to control individuals (all p < 0.05). Serum LOOH concentrations were significantly higher in patients with osteomyelitis than those in controls (p < 0.05). Arylesterase activity was inversely correlated with triglyceride (r =- 0.49; p = 0.005) and cholesterol concentrations (r =- 0.41; p = 0.025). CONCLUSION: In light of the findings obtained from the present study, it may be assumed that decreased activity of serum paraoxonase and increased concentrations of LOOH observed in osteomyelitis patients appear to be related to the increased oxidative stress and inflammatory conditions present in these patients, and may cause a much more severe status of the disease.
Assuntos
Arildialquilfosfatase/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Osteomielite/enzimologia , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Adulto JovemRESUMO
The immediate early (IE) proteins of human cytomegalovirus (hCMV) have diverse roles in directing viral and host cell transcription. Among these is the ability of IE2 to induce transcription of the IL1B gene that codes for IL-1beta in monocytes. This function is partially explained by interaction between IE2 and the host cell transcription factor Spi-1/PU.1 (Spi-1). We now show that maximal IE2 function also depends on productive interactions localizing to two C/EBP sites on the IL1B promoter suggesting either bi- or tri-molecular interactions between IE2, Spi-1 and C/EBPbeta at two different locations on the promoter. The IE2 interaction region on Spi-1 was previously mapped to the DNA-binding ETS domain and overlaps the region of Spi-1 that interacts with the transcription factor C/EBPbeta, a factor known to be critical for the induction of IL1B in response to Toll/IL-1 receptor (TIR) family signal transduction. The Spi-1 interacting region of IE2 maps to amino acids 315-328, a sequence that also interacts with the bZIP domain of C/EBPbeta. An expression vector coding for amino acids 291-364 of IE2 can suppress LPS induction of a co-transfected IL1B enhancer-promoter fragment in a monocyte cell line. This inhibition is likely the result of competition between Spi-1 and C/EBPbeta, thus blunting gene induction.
Assuntos
Citomegalovirus/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Interleucina-1beta/genética , Transativadores/metabolismo , Transcrição Gênica , Proteína beta Intensificadora de Ligação a CCAAT/química , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular , Citomegalovirus/imunologia , Células HeLa , Humanos , Proteínas Imediatamente Precoces/química , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Peptídeos/química , Peptídeos/imunologia , Peptídeos/metabolismo , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/química , TransfecçãoRESUMO
Rapid induction of transcription is known to be mediated by factors which bind DNA following post-translational modification. We report here that non-tyrosine phosphorylated (NTP)-Stat1 is involved in a cooperative interaction with Spi-1/PU.1 and IRF8 to form a pre-associated, poised complex for IL1B gene induction. A double point mutation at a putative STAT binding site, which overlaps this composite Spi-1 x IRF8 site located in the LPS and IL-1 response element (LILRE), inhibited human IL1B LPS-dependent reporter activity to about 10 percent of the control wild type vector. Chromatin immunoprecipitation revealed stimulation-independent constitutive binding of IRF8, Spi-1 and NTP-Stat1 at the LILRE, while binding of C/EBP beta was activated at an adjacent C/EBP beta site after LPS stimulation. In contrast to Stat1, IRF8 was tyrosine phosphorylated following LPS treatment. Supporting the involvement of NTP-Stat1, LPS-induced IL1B reporter activity in monocytes was enhanced by ectopic expression of NTP-Stat1 Y701F. In contrast, co-expression of a Y211F IRF8 mutein functioned as a dominant-negative inhibitor of LPS-induced IL1B reporter activity. In vitro DNA binding using extracts from LPS-treated monocytes confirmed that the LILRE enhancer constitutively binds a trimolecular complex containing IRF8, Spi-1 and NTP-Stat1. Binding studies using in vitro-expressed proteins revealed that NTP-Stat1 enhanced the binding of Spi-1 and IRF8 to the LILRE. Co-expression of TRAF6, an LPS surrogate, with Spi-1 and IRF8 enhanced IL1B reporter activity in HEK293R cells, which was dramatically reduced when Y211F IRF8 was co-expressed. These results suggest that the rapid transcriptional induction of an important inflammatory gene is dependent upon constitutive cooperative binding of a Spi-1 x IRF8 x NTP-Stat1 complex to the LILRE, which primes the gene for immediate induction following IRF8 phosphorylation. Phosphorylation of chromatin pre-associated factors like IRF8 may be an important strategy for the rapid transcriptional activation of genes involved in innate immunity.
Assuntos
Fatores Reguladores de Interferon/metabolismo , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Lipopolissacarídeos/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição STAT1/fisiologia , Transativadores/metabolismo , Ativação Transcricional/imunologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Fatores Reguladores de Interferon/fisiologia , Camundongos , Fosforilação , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas/fisiologia , Elementos de Resposta , Fator de Transcrição STAT1/metabolismo , Transativadores/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Ativação Transcricional/genética , Tirosina/metabolismoRESUMO
MicroRNAs (miRNAs) are 18- to 22-nucleotide-long, single-stranded, noncoding RNAs that regulate important biological processes including differentiation, proliferation, and response to cellular stressors such as hypoxia, nutrient depletion, and traversion of the cell cycle by controlling protein expression within the cell. Many investigators have profiled cancer tissue and serum miRNAs to identify potential therapeutic targets, understand the pathways involved in tumorigenesis, and identify diagnostic tumor signatures. In the setting of pancreatic cancer, obtaining pancreatic tissue is invasive and impractical for early diagnosis. Several groups have profiled miRNAs that are present in the blood as a means to diagnose tumor progression and predict prognosis/survival or drug resistance. Several miRNA signatures found in pancreatic tissue and the peripheral blood, as well as the pathways that are associated with pancreatic cancer, are reviewed here in detail. Three miRNA biomarkers (miR-21, miR-155, and miR-200) have been repetitively identified in both pancreatic cancer tissue and patients' blood. Those miRNAs regulate and are regulated by the central genetic and epigenetic changes observed in pancreatic cancer including p53, transforming growth factor ß, p16(INK4A), BRCA1/2, and Kras. These miRNAs are involved in DNA repair, cell cycle, and cell invasion and also play important roles in promoting metastases.
Assuntos
Predisposição Genética para Doença/genética , MicroRNAs/genética , Neoplasias Pancreáticas/genética , Diagnóstico Precoce , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/terapia , PrognósticoRESUMO
Interleukin-1ß and Tumor Necrosis Factor α play related, but distinct, roles in immunity and disease. Our study revealed major mechanistic distinctions in the Toll-like receptor (TLR) signaling-dependent induction for the rapidly expressed genes (IL1B and TNF) coding for these two cytokines. Prior to induction, TNF exhibited pre-bound TATA Binding Protein (TBP) and paused RNA Polymerase II (Pol II), hallmarks of poised immediate-early (IE) genes. In contrast, unstimulated IL1B displayed very low levels of both TBP and paused Pol II, requiring the lineage-specific Spi-1/PU.1 (Spi1) transcription factor as an anchor for induction-dependent interaction with two TLR-activated transcription factors, C/EBPß and NF-κB. Activation and DNA binding of these two pre-expressed factors resulted in de novo recruitment of TBP and Pol II to IL1B in concert with a permissive state for elongation mediated by the recruitment of elongation factor P-TEFb. This Spi1-dependent mechanism for IL1B transcription, which is unique for a rapidly-induced/poised IE gene, was more dependent upon P-TEFb than was the case for the TNF gene. Furthermore, the dependence on phosphoinositide 3-kinase for P-TEFb recruitment to IL1B paralleled a greater sensitivity to the metabolic state of the cell and a lower sensitivity to the phenomenon of endotoxin tolerance than was evident for TNF. Such differences in induction mechanisms argue against the prevailing paradigm that all IE genes possess paused Pol II and may further delineate the specific roles played by each of these rapidly expressed immune modulators.
Assuntos
Genes Precoces/genética , Interleucina-1beta/genética , Ativação Transcricional , Fator de Necrose Tumoral alfa/genética , Animais , Transporte Biológico/efeitos dos fármacos , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Ontologia Genética , Loci Gênicos/genética , Glucose/metabolismo , Células HEK293 , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Monócitos/metabolismo , Nucleossomos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , RNA Polimerase II/química , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacosRESUMO
Endogenous damage associated molecular pattern molecules (DAMPs) released from necrotic, damaged or stressed cells are associated with an inflammatory response. Whether the microRNA (miR) expression signature of this response is different from that of a pathogen associated molecular pattern (PAMP)-stimulated inflammatory response is unknown. We report here that miR-34c and miR-214 are significantly expressed in fresh human peripheral blood mononuclear cells (PBMCs) exposed to DAMP-containing freeze-thaw lysates, or to conditioned media from serum-starved and glucose-deprived cells (p<6×10(-4) and p<3.7×10(-3)), respectively. Interestingly, only miR-34c expression was differentially expressed in PBMCs exposed to freeze-thaw lysates or conditioned media from wildtype High Mobility Group B1 (HMGB1(+/+)) mouse embryonic fibroblast (MEF) cells, when compared to cultures exposed to lysates or conditioned media from HMGB1(-/-) MEFs. miR-155 expression in these cultures was negligible, but was significantly expressed in PBMCs stimulated with Lipopolysaccahride (LPS) or most other Toll-like receptor (TLR) ligands, making it the prototypic "PAMPmiR". Exposure to a damaged human colorectal carcinoma cell line lysate (HCT116) similarly resulted in increased miR-34c and miR-214 levels. When PBMCs were pre-transfected with anti-miR-34c and then exposed to lysate, expression levels of IKKγ mRNA, a putative target of miR-34c, increased, while protein levels of IKKγ in cultures transfected with a pre-miR-34c were abrogated. Levels of miR-34c expression (as well as pro-inflammatory cytokines, IL-1ß and TNFα) decreased when PBMC cultures were briefly pre-incubated with the K(+) channel (inflammasome) inhibitor, glybenclamide, suggesting that inflammasome activation is upstream of miR-34c expression in response to DAMPs. Our findings demonstrate that a specific microRNA expression signature is associated with the inflammatory response to damaged/injured cells and carries implications for many acute and chronic inflammatory disorders.
Assuntos
Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , MicroRNAs/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/farmacologia , Humanos , Inflamassomos/metabolismo , Lipopolissacarídeos/farmacologia , CamundongosRESUMO
Tumor necrosis factor alpha converting enzyme (TACE) is a sheddase overexpressed in cancers that generates cancer cell growth and survival factors, and is implicated in carcinogenesis and tumor growth. This indicates that TACE could be a potentially important cancer biomarker. Unexpectedly, TACE expression in cancer tissues does not correlate with cancer stage or invasiveness. Although TACE sheddase activity is a more direct and potentially better indicator of TACE biology and might be a better cancer biomarker than TACE expression, it has not been studied in cancer tissues. In the present study, we developed a reliable specific assay for quantification of TACE sheddase activity, investigated TACE activity and TACE protein expression in head and neck cancer (HNC) tissues, and examined the correlation of the results with HNC clinical stages and likelihood to recur. We found that HNC cell lines and tissues contained remarkably higher quantities of TACE activity and TACE protein than normal keratinocytes or oral mucosa. siRNA silencing of TACE resulted in the inhibition of release of the tumorogenic factors amphiregulin and transforming growth factor alpha, and tumor protective factors tumor necrosis factor receptors from HNC cells. Importantly, TACE activity, but not TACE protein expression, was significantly higher in large, T3/T4, primary tumors relative to small, T1/T2, primary tumors, and especially in primary tumors likely to recur relative to those unlikely to recur. These data show that increased TACE activity in cancer is biologically and clinically relevant, and indicate that TACE activity could be a significant biomarker of cancer prognosis.
Assuntos
Proteínas ADAM/metabolismo , Neoplasias de Cabeça e Pescoço/enzimologia , Recidiva Local de Neoplasia/enzimologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/genética , Proteína ADAM17 , Adulto , Idoso , Idoso de 80 Anos ou mais , Anfirregulina , Western Blotting , Células Cultivadas , Família de Proteínas EGF , Ensaio de Imunoadsorção Enzimática , Feminino , Glicoproteínas/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Técnicas Imunoenzimáticas , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Queratinócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Mucosa Bucal/metabolismo , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores do Fator de Necrose Tumoral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Fator de Crescimento Transformador alfa/metabolismoRESUMO
Prior studies have identified molecules involved in IL-1 signaling that transmit the extracellular stimulus to downstream kinase molecules causing altered transcriptional activity. Many of these investigations have relied heavily on ligand induced protein-protein interactions detected by immuno-coprecipitation to map the cascade of events from receptor binding to activation of downstream signaling intermediates. Direct protein interactions have not been commonly reported. An in vitro study was undertaken to better define the direct protein-protein interactions involved in IL-1 signaling. Results indicate that IRAK2 is capable of direct association with either IL-1R(I) or IL-1R(AcP). IRAK2 is also capable of associating directly with MyD88 by distinct regions. Finally, IRAK2 interactions with TRAF6 were mapped and demonstrate differences from more proximal signaling intermediates. A model is presented that reflects the specific molecular interactions responsible for recruiting signaling intermediates to the IL-1 receptor cytoplasmic domains.