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1.
Protein & Cell ; (12): 881-893, 2020.
Article in English | WPRIM | ID: wpr-880884

ABSTRACT

Cytokines are secreted by various cell types and act as critical mediators in many physiological processes, including immune response and tumor progression. Cytokines production is precisely and timely regulated by multiple mechanisms at different levels, ranging from transcriptional to post-transcriptional and posttranslational processes. Monocyte chemoattractant protein-1 induced protein 1 (MCPIP1), a potent immunosuppressive protein, was first described as a transcription factor in monocytes treated with monocyte chemoattractant protein-1 (MCP-1) and subsequently found to possess intrinsic RNase and deubiquitinase activities. MCPIP1 tightly regulates cytokines expression via various functions. Furthermore, cytokines such as interleukin 1 beta (IL-1B) and MCP-1 and inflammatory cytokines inducer lipopolysaccharide (LPS) strongly induce MCPIP1 expression. Mutually regulated MCPIP1 and cytokines form a complicated network in the tumor environment. In this review, we summarize how MCPIP1 and cytokines reciprocally interact and elucidate the effect of the network formed by these components in cancer-related immunity with aim of exploring potential clinical benefits of their mutual regulation.


Subject(s)
Chemokine CCL2/immunology , Humans , Interleukin-1beta/immunology , Neoplasm Proteins/immunology , Neoplasms/pathology , Ribonucleases/immunology , Transcription Factors/immunology
2.
Rev. Inst. Adolfo Lutz ; (77): 1-5, 2018.
Article in Portuguese | LILACS, SES-SP, SESSP-ACVSES, SESSP-IALPROD, SES-SP, SESSP-IALACERVO | ID: biblio-1103499

ABSTRACT

Em laboratório de biologia molecular existem normas para prevenir que nucleases destruam os ácidos nucleicos em análise. Rígida adesão a estas normas é primordial, principalmente em laboratórios de análises clínicas e ao se lidar com amostras com número restrito de cópias do genoma-alvo. Em contraposição, diversas nucleases têm tido importância fundamental, por exemplo, na identificação do ácido nucleico de vírus, investigação de RNA mensageiro, purificação de vírus em abordagem metagenômica, edição de genomas com o sistema CRISPR/Cas e descoberta de enzimas. O conhecimento de como nucleases podem ser tanto vilãs quanto aliadas é essencial na formação de todos que trabalham no campo de biologia molecular.


In a molecular biology laboratory there are standards to prevent nucleases from destroying the nucleic acids under analysis. Strict adherence to these standards is paramount, mainly in clinical analysis laboratories and when dealing with samples with a limited number of copies of the target genome. In contrast, several nucleases have been of fundamental importance, for example, in the identification of the type of viral nucleic acid, investigation of messenger RNA, virus purification in metagenomic approach, genome editing with the CRISPR/Cas system, and enzyme discovery. Knowledge of how nucleases can be both villains and allies is essential in the training of all working in the field of molecular biology.


Subject(s)
Ribonucleases , Viruses , Clinical Laboratory Techniques , Deoxyribonucleases , Molecular Biology
3.
Article in English | WPRIM | ID: wpr-69358

ABSTRACT

Echinostoma cinetorchis is an oriental intestinal fluke causing significant pathological damage to the small intestine. The aim of this study was to determine a full-length cDNA sequence of E. cinetorchis endoribonuclease (RNase H; EcRNH) and to elucidate its molecular biological characters. EcRNH consisted of 308 amino acids and showed low similarity to endoribonucleases of other parasites (<40%). EcRNH had an active site centered on a putative DDEED motif instead of DEDD conserved in other species. A recombinant EcRNH produced as a soluble form in Escherichia coli showed enzymatic activity to cleave the 3′-O-P bond of RNA in a DNA-RNA duplex, producing 3′-hydroxyl and 5′-phosphate. These findings may contribute to develop antisense oligonucleotides which could damage echinostomes and other flukes.


Subject(s)
Amino Acids , Catalytic Domain , DNA, Complementary , Echinostoma , Endoribonucleases , Escherichia coli , Intestine, Small , Oligonucleotides, Antisense , Parasites , Ribonuclease H , Ribonucleases , RNA , Trematoda
4.
Protein & Cell ; (12): 735-749, 2017.
Article in English | WPRIM | ID: wpr-756951

ABSTRACT

Mammalian mitochondrial genome encodes a small set of tRNAs, rRNAs, and mRNAs. The RNA synthesis process has been well characterized. How the RNAs are degraded, however, is poorly understood. It was long assumed that the degradation happens in the matrix where transcription and translation machineries reside. Here we show that contrary to the assumption, mammalian mitochondrial RNA degradation occurs in the mitochondrial intermembrane space (IMS) and the IMS-localized RNASET2 is the enzyme that degrades the RNAs. This provides a new paradigm for understanding mitochondrial RNA metabolism and transport.


Subject(s)
Cell Line , Humans , Mitochondrial Membranes , Metabolism , Protein Transport , RNA , Chemistry , Metabolism , RNA Stability , RNA, Mitochondrial , Ribonucleases , Metabolism , Tumor Suppressor Proteins , Metabolism
5.
Bol. latinoam. Caribe plantas med. aromát ; 15(1): 18-28, ene. 2016. ilus, graf
Article in Spanish | LILACS | ID: biblio-907514

ABSTRACT

Con el objetivo de aislar y caracterizar parcialmente las enzimas ribonucleasas (RNasas) contenidas en el látex de Calotropis procera y Pedilanthus tithymaloides, se colectaron muestras de plantas adultas. Las proteínas solubles fueron extraídas con acetato de sodio y centrifugación a 16.000 x g durante 15 min y fraccionadas por cromatografía de intercambio iónico. Se estimó la masa molecular a través de ecuaciones de regresión lineal. Se realizaron pruebas de glicosilación. En ambas especies, las proteínas con actividad RNasa presentaron una masa molecular entre 28 y 30 kDa. No existe evidencia de proteínas glicosiladas en el látex de C. procera. En P. tithymaloides la RNasa es una proteína glicosilada.


In order to isolate and characterize partially ribonucleases (RNases) enzymes contained in the latex from Calotropis procera and Pedilanthus tithymaloides, samples were collected from mature plants. Soluble proteins were extracted with sodium acetate and centrifugation at 16,000 xg for 15 min and fractionated by ion exchange chromatography. Molecular mass was estimated by linear regression equations. Glycosylation tests were conducted. In both species, proteins with RNase activity showed a molecular mass between 28 and 30 kDa. No evidence of glycosylated proteins in latex from C. procera. In P. tithymaloides, RNase may be a glycosylated protein.


Subject(s)
Calotropis/enzymology , Euphorbiaceae/enzymology , Latex/chemistry , Ribonucleases/isolation & purification , Ribonucleases/metabolism , Calotropis/chemistry , Euphorbiaceae/chemistry , Glycosylation
6.
Article in English | WPRIM | ID: wpr-165767

ABSTRACT

IL-6 is an inflammatory cytokine and its overexpression plays an important role in osteoarthritis (OA) pathogenesis. Expression of IL-6 is regulated post-transcriptionally by MCPIP1. The 3' untranslated region (UTR) of MCPIP1 mRNA harbors a miR-139 'seed sequence', therefore we examined the post-transcriptional regulation of MCPIP1 by miR-139 and its impact on IL-6 expression in OA chondrocytes. Expression of miR-139 was found to be high in the damaged portion of the OA cartilage compared with unaffected cartilage from the same patient and was also induced by IL-1beta in OA chondrocytes. Inhibition of miR-139 decreased the expression of IL-6 mRNA by 38% and of secreted IL-6 protein by 40%. However, overexpression of miR-139 increased the expression of IL-6 mRNA by 36% and of secreted IL-6 protein by 56%. These data correlated with altered expression profile of MCPIP1 in transfected chondrocytes. Studies with a luciferase reporter construct confirmed the interactions of miR-139 with the 'seed sequence' located in the 3' UTR of MCPIP mRNA. Furthermore, miR-139 overexpression increased the catabolic gene expression but expression of anabolic markers remained unchanged. Overexpression of miR-139 also induced apoptosis in OA chondrocytes. Importantly, we also discovered that IL-6 is a potent inducer of miR-139 expression in OA chondrocytes. These findings indicate that miR-139 functions as a post-transcriptional regulator of MCPIP1 expression and enhances IL-6 expression, which further upregulates miR-139 expression in OA chondrocytes. These results support our hypothesis that miR-139-mediated downregulation of MCPIP1 promotes IL-6 expression in OA. Therefore, targeting miR-139 could be therapeutically beneficial in the management of OA.


Subject(s)
3' Untranslated Regions , Aged , Apoptosis , Chondrocytes/metabolism , Down-Regulation , Female , Gene Expression Regulation , Humans , Interleukin-6/genetics , Male , MicroRNAs/genetics , Middle Aged , Osteoarthritis/genetics , RNA, Messenger/genetics , Ribonucleases/genetics , Transcription Factors/genetics , Up-Regulation
7.
Journal of Breast Cancer ; : 235-241, 2015.
Article in English | WPRIM | ID: wpr-112054

ABSTRACT

PURPOSE: Circular RNAs (circRNAs), a novel class of RNAs, perform important functions in biological processes. However, the role of circRNAs in the mammary gland remains unknown. The present study is aimed at identifying and characterizing the circRNAs expressed in the mammary gland of lactating rats. METHODS: Deep sequencing of RNase R-enriched rat lactating mammary gland samples was performed and circRNAs were predicted using a previously reported computational pipeline. Gene ontology terms of circRNA-producing genes were also analyzed. RESULTS: A total of 6,824 and 4,523 circRNAs were identified from rat mammary glands at two different lactation stages. Numerous circRNAs were specifically expressed at different lactation stages, and only 1,314 circRNAs were detected at both lactation stages. The majority of the candidate circRNAs map to noncoding intronic and intergenic regions. The results demonstrate a circular preference or specificity of some genes. DAVID analysis revealed an enrichment of protein kinases and related proteins among the set of genes encoding circRNAs. Interestingly, four protein-coding genes (Rev3l, IGSF11, MAML2, and LPP) that also transcribe high levels of circRNAs have been reported to be involved in cancer. CONCLUSION: Our findings provide the basis for comparison between breast cancer profiles and for selecting representative circRNA candidates for future functional characterization in breast development and breast cancer.


Subject(s)
Animals , Biological Phenomena , Breast , Breast Neoplasms , DNA, Intergenic , Female , Gene Ontology , High-Throughput Nucleotide Sequencing , Introns , Lactation , Mammary Glands, Human , Phosphotransferases , Protein Kinases , Rats , Ribonucleases , RNA , RNA, Untranslated , Sensitivity and Specificity
8.
Chinese Journal of Biotechnology ; (12): 1632-1642, 2015.
Article in Chinese | WPRIM | ID: wpr-240548

ABSTRACT

Ranpirnase (onconase, ONC) is a new drug, with weak RNase activity and strong cytotoxicity to various tumor cells in vitro and in vivo. This study is to obtain recombination onconase (rONC) with high bioactivity. Based on the codon preference of Pichia pastoris, we designed and synthesized the gene according to cDNA sequences of ONC and the α mating factor's prepeptide. We screened positive clones after transforming the recombination plasmids into P. pastoris X-33, GSS115 and SMD1168. We screened the best combination of seven different vectors and host strains. Moreover, we optimized culture condition in shake flasks and 10 L bioreactor, and purified rONC from the supernatant after inducing it with 0.25% methanol by aqueous two-phase extraction coupling G50 molecular exclusion method. The highest rONC production was 13 mg/L in pPICZα-A/X-33/ONC combination under the condition of pH 5.5 and 23 degrees C in shake flasks for 7 d; and that the highest rONC production was 180 mg/L when the induction is performed in the lower basic salt medium with pH 5.5 in the 10 L bioreactor for 7 d. The yield of rONC is more than 90% at a purity of above 95%. rONC can kill various tumor cells in vitro. The expression and purification of rONC would be useful for further investigation of this new drug.


Subject(s)
Antineoplastic Agents , Metabolism , Bioreactors , Cell Line, Tumor , Codon , DNA, Complementary , Genetic Vectors , Humans , Pichia , Metabolism , Recombinant Proteins , Ribonucleases
9.
Chinese Journal of Virology ; (6): 629-633, 2015.
Article in Chinese | WPRIM | ID: wpr-296237

ABSTRACT

To prepare virus-like particles containing FluA/B mRNA as RNA standard and control in Influenza RNA detection, the genes coding the coat protein and maturase of E. coli bacteriophage MS2 were amplified and cloned into D-pET32a vector. Then we inserted 6 histidines to MS2 coat protein by QuikChange Site-Directed Mutagenesis Kit to construct the universal expressing vector D-pET32a-CP-His. In addition, the partial gene fragments of FluA and FluB were cloned to the down-stream of expressing vector. The recombinant plasmid D-pET32a-CP-His-FluA/B was transformed to BL21 with induction by IPTG. The virus-like particles were purified by Ni+ chromatography. The virus-like particles can be detected by RT-PCR, but not PCR. They can be conserved stably for at least 3 months at both 4 degrees C and -20 degrees C. His-tagged virus-like particles are more stable and easier to purification. It can be used as RNA standard and control in Influenza virus RNA detection.


Subject(s)
Escherichia coli , Genetics , Metabolism , Influenza A virus , Genetics , Metabolism , Influenza B virus , Genetics , Metabolism , RNA, Messenger , Genetics , Metabolism , RNA, Viral , Genetics , Metabolism , Recombinant Fusion Proteins , Genetics , Metabolism , Ribonucleases , Chemistry , Virion , Genetics , Metabolism
10.
Genomics & Informatics ; : 70-75, 2015.
Article in English | WPRIM | ID: wpr-216096

ABSTRACT

MicroRNAs (miRNAs) have been demonstrated to play an important role in carcinogenesis. Previous studies revealed that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. In this study, we measured the plasma expression levels of three miRNAs (miR-21, miR-27a, and miR-155) to investigate the usefulness of miRNAs for gastric cancer detection. We initially examined plasma miRNA expression levels in a screening cohort consisting of 15 patients with gastric cancer and 15 healthy controls from Korean population, using TaqMan quantitative real-time polymerase chain reaction. We observed that the expression level of miR-27a was significantly higher in patients with gastric cancer than in healthy controls, whereas the miR-21 and miR-155a expression levels were not significantly higher in the patients with gastric cancer. Therefore, we further validated the miR-27a expression level in 73 paired gastric cancer tissues and in a validation plasma cohort from 35 patients with gastric cancer and 35 healthy controls. In both the gastric cancer tissues and the validation plasma cohort, the miR-27a expression levels were significantly higher in patients with gastric cancer. Receiver-operator characteristic (ROC) analysis of the validation cohort, revealed an area under the ROC curve value of 0.70 with 75% sensitivity and 56% specificity in discriminating gastric cancer. Thus, the miR-27a expression level in plasma could be a useful biomarker for the diagnosis and/or prognosis of gastric cancer.


Subject(s)
Carcinogenesis , Cohort Studies , Diagnosis , Humans , Mass Screening , MicroRNAs , Plasma , Prognosis , Real-Time Polymerase Chain Reaction , Ribonucleases , ROC Curve , Sensitivity and Specificity , Stomach Neoplasms
11.
Acta cir. bras ; 29(8): 485-492, 08/2014. graf
Article in English | LILACS | ID: lil-719186

ABSTRACT

PURPOSE: To obtain a decellularized tracheal scaffold associating traditional approaches with the novel light-emitting diode (LED) proposal. METHODS: This study was performed with New Zealand adult rabbits weighing 3.0 - 4.0 kg. Different protocols (22) were used combining physical (agitation and LED irradiation), chemical (SDS and Triton X-100 detergents), and enzymatic methods (DNase and RNase). RESULTS: Generally, the cells surrounding soft tissues were successfully removed, but none protocol removed cells from the tracheal cartilage. However, longer protocols were more effective. The cost-benefits relation of the enzymatic processes was not favorable. It was possible to find out that the cartilaginous tissue submitted to the irradiation with LED 630nm and 475 nm showed an increased number of gaps without cells, but several cells were observed to be still present. CONCLUSION: The light-emitting diode is a promising tool for decellularization of soft tissues. .


Subject(s)
Animals , Rabbits , Light , Tissue Scaffolds , Tissue Engineering/methods , Trachea/ultrastructure , Deoxyribonucleases/metabolism , Detergents/pharmacology , Extracellular Matrix/ultrastructure , Ribonucleases/metabolism , Trachea/drug effects , Trachea/enzymology
12.
Indian J Biochem Biophys ; 2013 Jun; 50(3): 196-201
Article in English | IMSEAR | ID: sea-147302

ABSTRACT

A 15 kDa ribonuclease (RNase) was purified from dried fruiting bodies of the wild edible mushroom Armillaria luteo-virens. The simple 4-step purification protocol involved ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on SP-Sepharose and a final gel filtration by FPLC on Superdex-75. The RNase was unadsorbed on Affi-gel blue gel, but adsorbed on DEAE-cellulose and SP-Sepharose. The N-terminal amino acid sequence of purified RNase was AGVQYKLTILLV, which showed low sequence homology to those of previously reported RNases. The optimal pH and temperature of the enzyme were very close to 4.0 and 70°C, respectively. The enzyme showed considerably high ribonucleolytic activity and broad specificity towards polyhomoribonucleotides, with a specificity of poly(U)>poly(C)>poly (G)>poly(A). The ribonucleolytic activities towards poly(U), poly(C), poly(G) and poly(A) were 279.5, 184.1, 69.9 and 52.3 U/mg, respectively.


Subject(s)
Agaricales/enzymology , Animals , Enzyme Activation , Enzyme Stability , Fungal Proteins/chemistry , Fungal Proteins/isolation & purification , Ribonucleases/chemistry , Ribonucleases/isolation & purification , Substrate Specificity
13.
Acta Physiologica Sinica ; (6): 616-622, 2013.
Article in Chinese | WPRIM | ID: wpr-297530

ABSTRACT

The aim of the present study is to investigate whether monocyte chemotactic protein-1 (MCP-1)-induced vascular smooth muscle cell (VSMC) proliferation is mediated via monocyte chemotactic protein-1 induced protein-1 (MCPIP1). MCPIP1 expressions in cultured VSMC were detected by real-time PCR and Western blot following MCP-1 incubation. After MCPIP1 was silenced by siRNA, cell number was counted by hemocytometer, VSMC activity was analyzed by CCK-8 kit, percentage of DNA synthesis was detected by EdU kit, percentage of S phase cell numbers were measured by flow cytometry, and c-fos mRNA expression induced by MCP-1 or platelet-derived growth factor (PDGF) was detected by real-time PCR. The results showed MCP-1 increased MCPIP1 mRNA and up-regulated MCPIP1 protein expression in dose- and time-dependent manners. Cell counts, cellular activity, the percentage of DNA synthesis, and the percentage of S phase cell numbers were remarkably decreased in MCPIP1 siRNA group, compared with those in MCP-1 group. The enhancing effect of MCP-1 or PDGF on c-fos mRNA expression was inhibited by MCPIP1 siRNA. These results suggest that MCP-1-induced VSMC proliferation is mediated via MCPIP1, and the underlying mechanism may involve c-fos expression up-regulation.


Subject(s)
Cell Proliferation , Cells, Cultured , Chemokine CCL2 , Pharmacology , Humans , Muscle, Smooth, Vascular , Cell Biology , Myocytes, Smooth Muscle , Cell Biology , Platelet-Derived Growth Factor , Pharmacology , Real-Time Polymerase Chain Reaction , Ribonucleases , Metabolism , Transcription Factors , Metabolism , Up-Regulation
14.
Article in Chinese | WPRIM | ID: wpr-254534

ABSTRACT

<p><b>OBJECTIVE</b>To assess the association of RNASET2 gene polymorphisms and haplotypes with Graves disease (GD) in Han Chinese population from coastal regions of Shandong Province.</p><p><b>METHODS</b>A total of 471 GD patients and 472 controls were enrolled. Genotypes of single nucleotide polymorphisms (SNPs) in RNASET2 gene were determined with a Taqman probe on a Fluidigm EPl platform. Haplotypes and their frequencies were analyzed with a SHEsis online software.</p><p><b>RESULTS</b>There was a significant difference in allele frequencies of rs3777722, rs3777723 and rs9355610 between the GD patients and the controls (P=0.018; P=0.028; P=0.021).Allele frequencies of rs3777722 and rs9355610 were significantly lower in GD than in the controls (P=0.018, P=0.021). Haplotypes A-A-C-A and A-A-T-A were significantly more common in the control group compared with the GD group (P=0.046, OR=0.448, 95%CI:0.200-1.006; P=0.049, OR=0.823, 95%CI:0.678-0.999). The frequency of C-G-C-G haplotype was significantly higher in GD patient group than the control group (P=0.018).</p><p><b>CONCLUSION</b>RNASET2 gene polymorphisms and haplotypes are associated with GD in Han population from coastal areas of Shandong Province. rs3777722 and rs9355610 may contribute to the risk for GD.</p>


Subject(s)
Adolescent , Adult , Aged , Aged, 80 and over , Asians , Genetics , Child , Female , Gene Frequency , Genetic Predisposition to Disease , Graves Disease , Genetics , Haplotypes , Humans , Male , Middle Aged , Polymorphism, Single Nucleotide , Ribonucleases , Genetics , Tumor Suppressor Proteins , Genetics , Young Adult
15.
Article in English | WPRIM | ID: wpr-161071

ABSTRACT

The traditional light microscopy has limitations for precise growth assays of malaria parasites in culture or for assessment of new compounds for antimalarial activity; the speed and high reproducibility of flow cytometry can overcome these limitations. A flow cytometric method using PicoGreen, a DNA-binding fluorochrome, was developed with optimal precision suitable for performing growth assays of low-parasitemia field isolates. In addition, intra- and inter-person reproducibility of the flow cytometric and the microscopic method were compared in order to quantitatively demonstrate the improved precision. RNase treatment contributed to the precision of the flow cytometric measurements by enhancing the signal-to-noise ratios. Coefficients of variation of the method were smaller than 10% for 0.1% or higher parasitemia samples. The intra- and inter-person coefficients of variation of the flow cytometric method were three to six times smaller than those of the microscopic method. The flow cytometric method developed in this study yielded substantially more precise results than the microscopic method, allowing determination of parasitemia levels of 0.1% or higher, with coefficients of variation smaller than 10%. Thus, the PicoGreen method could be a reliable high sensitivity assay for analysis of low parasitemia samples and might be applied to a high throughput system testing antimalarial drug activity.


Subject(s)
Flow Cytometry , Fluorescent Dyes/chemistry , Humans , Microscopy , Organic Chemicals/chemistry , Parasitemia/diagnosis , Plasmodium falciparum/isolation & purification , Reproducibility of Results , Ribonucleases/metabolism , Signal-To-Noise Ratio
16.
Protein & Cell ; (12): 903-910, 2012.
Article in English | WPRIM | ID: wpr-757849

ABSTRACT

MCP-1-induced protein-1 (MCPIP1) is a newly identified protein that is crucial to immune regulation. Mice lacking MCPIP1 gene suffer from severe immune disorders, and most of them cannot survive longer than 12 weeks. Considerable progress has been made in revealing the mechanism underlying the immune regulatory function of MCPIP1. MCPIP1 can act as an RNase to promote the mRNA degradation of some inflammatory cytokines, such as IL-6 and IL-1. Pre-microRNAs are also confirmed to be the substrate of MCPIP1 RNase. The structure of MCPIP1 N-terminal conserved domain shows a PilT N-terminus-like RNase structure, further supporting the notion that MCPIP1 has RNase activity. MCPIP1 can also deubiquitinate TNF receptor-associated factor family proteins, which are known to mediate immune and inflammatory responses. In this review, we summarize recent progress on the immune regulatory role of MCPIP1 and discuss the mechanisms underlying its function.


Subject(s)
Amino Acid Sequence , Animals , Humans , Immunity , Molecular Sequence Data , Ribonucleases , Metabolism , Transcription Factors , Chemistry , Metabolism , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins , Metabolism , Ubiquitination
17.
Annals of Dermatology ; : 393-397, 2012.
Article in English | WPRIM | ID: wpr-162701

ABSTRACT

BACKGROUND: Acne inversa is a chronic, suppurative relapsing inflammatory skin disease that primarily affects the axillae, perineum and inframammary regions. Evidence suggests that the innate immune system is involved in the pathogenesis of acne inversa. OBJECTIVE: To investigate the role of the innate immune system in acne inversa. METHODS: Skin biopsies were obtained from inflammatory skin lesions (n=17) and from non-lesional skin (intraindividual control, n=17) of patients with acne inversa. Additional skin lesions were taken from patients with chronic venous leg ulcers (interindividual control, n=5). Quantitative real-time reverse transcription-polymerase chain reaction was used to determine the mRNA levels of antimicrobial peptides and proteins (AMPs), including human beta-defensin (hBD)-1, hBD-2 and hBD-3, LL-37 (cathelicidin) and Ribonuclease 7 (RNase 7). mRNA levels were also determined for inflammatory and anti-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha), matrix metalloproteinase-1 (MMP1), interleukin (IL)-1beta, IL-6, IL-8 and IL-10. RESULTS: The mRNA levels of hBD-2, LL-37, IL-1beta, IL-6, IL-8, IL-10 and MMP1 were significantly higher in acne inversa lesions compared to non-lesional skin (p<0.05). A significant positive correlation expression was observed between hBD-2 mRNA expression and LL-37 (rho=0.53, p=0.03), and between hBD-2 and RNAse 7 (rho=0.68, p=0.006). When compared to the chronic venous leg ulcer lesions, acne inversa lesions showed a significantly higher expression of RNase 7 mRNA, while IL-1 beta, IL-6, IL-8, TNF-alpha and MMP1 mRNA expression was significantly higher in the chronic venous leg ulcer lesions (p<0.05). CONCLUSION: The AMP, cytokine milieu and tissue proteases in acne inversa lesions differ significantly from non-lesional skin and chronic venous leg ulcers. The positively correlating up-regulation of AMPs in acne inversa indicates an important role of the innate immune system in the pathogenesis of this disorder.


Subject(s)
Acne Vulgaris , Axilla , Biopsy , Cytokines , Hidradenitis Suppurativa , Humans , Immune System , Interleukin-10 , Interleukin-1beta , Interleukin-6 , Interleukin-8 , Interleukins , Leg Ulcer , Matrix Metalloproteinase 1 , Peptide Hydrolases , Peptides , Perineum , Proteins , Ribonucleases , RNA, Messenger , Skin , Skin Diseases , Tumor Necrosis Factor-alpha , Up-Regulation
18.
Article in English | WPRIM | ID: wpr-33242

ABSTRACT

BACKGROUND: MicroRNAs (miRNAs) have demonstrated their potential as biomarkers for lung cancer diagnosis. In recent years, miRNAs have been found in body fluids such as serum, plasma, urine and saliva. Circulating miRNAs are highly stable and resistant to RNase activity along with, extreme pH and temperatures in serum and plasma. In this study, we investigated serum miRNA profiles that can be used as a diagnostic biomarker of non-small cell lung cancer (NSCLC). METHODS: We compared the expression profile of miRNAs in the plasma of patients diagnosed with lung cancer using an miRNA microarray. The data from this assay were validated by quantitative real-time PCR (qRT-PCR). RESULTS: Six miRNAs were overexpressed and three miRNAs were underexpressed in both tissue and serum from squamous cell carcinoma (SCC) patients. Sixteen miRNAs were overexpressed and twenty two miRNAs were underexpressed in both tissue and serum from adenocarcinoma (AC) patients. Of the four miRNAs chosen for qRT-PCR analysis, the expression of miR-23a was consistent with microarray results from AC patients. Receiver operating characteristic (ROC) curve analyses were done and revealed that the level of serum miR-23a was a potential marker for discriminating AC patients from chronic obstructive pulmonary disease (COPD) patients. CONCLUSION: Although a small number of patients were examined, the results from our study suggest that serum miR-23a can be used in the diagnosis of AC.


Subject(s)
Adenocarcinoma , Biomarkers , Body Fluids , Carcinoma, Non-Small-Cell Lung , Carcinoma, Squamous Cell , Gene Expression Profiling , Humans , Hydrogen-Ion Concentration , Lung , Lung Neoplasms , MicroRNAs , Plasma , Pulmonary Disease, Chronic Obstructive , Real-Time Polymerase Chain Reaction , Ribonucleases , ROC Curve , Saliva
19.
Article in English | WPRIM | ID: wpr-94348

ABSTRACT

OBJECTIVES: In the present study, three kinds of tissues cells (pulp, gingiva, and periodontal ligament) were investigated if those cells express MMP and TIMP when they were stimulated with neuropeptides (substance P, CGRP) or proinflammatory cytokine, TNF-alpha. MATERIALS AND METHODS: The cells cultured from human dental pulp (PF), gingiva (GF) and periodontal ligament were (PDLF) stimulated with Mock, SP, TNF-alpha, and CGRP for 24 hrs and 48 hrs. for an RNase protection assay and Enzyme Linked Immunosorbent Assay. Cells (PF, GF and PDLF) seeded in 100 mm culture dish were stimulated with SP (10(-5), 10(-8) M) or only with medium (Mock stimulation) for 4hrs and for 24 hrs for RNase Protection Assay, and they were stimulated with CGRP (10(-5) M) and TNF-alpha (2 ng/mL) for 24 hrs and with various concentraion of TNF-alpha (2, 10, and 100 ng/mL) for Rnase Protection Assay with a human MMP-1 probe set including MMP 1, 2, 8, 7, 8, 9, 12, and TIMP 2, 3. In addition, cells (PF, GF and PDLF) were stimulated with Mock and various concentraion of TNF-alpha(2, 10, and 100 ng/mL) for 24 hrs and with TNF-alpha(10 ng/mL) for 48 hrs, and the supernatents from the cells were collected for Enzyme Linked Immunosorbent Assay (ELISA) for MMP-1 and MMP-13. RESULTS: The expression of MMPs in PF, GF, PDLF after stimulation with SP and CGRP were not changed compared with Mock stimulation for 4 hrs and 24 hrs. The expression of MMP-1, -12, -13 24 hrs after stimulation with TNF-alpha were upregulated, however the expression of TIMP-3 in PF, GF, PDLF after stimulation with TNF-alpha were downregulated. TNF-alpha (2 ng/mL, 10 ng/mL, 100 ng/mL) increased MMP-1 and MMP-12 expression in PF dose dependently for 24 hrs. CONCLUSIONS: TNF-alpha in the area of inflammation may play an important role in regulating the remodeling of dentin, cementum, and alveolar bone.


Subject(s)
Dental Cementum , Dental Pulp , Dentin , Enzyme-Linked Immunosorbent Assay , Gingiva , Humans , Inflammation , Matrix Metalloproteinases , Neuropeptides , Periodontal Ligament , Ribonucleases , Seeds , Substance P , Tissue Inhibitor of Metalloproteinase-3 , Tumor Necrosis Factor-alpha
20.
Protein & Cell ; (12): 755-763, 2011.
Article in English | WPRIM | ID: wpr-757044

ABSTRACT

The human CCR4-NOT deadenylase complex consists of at least nine enzymatic and non-enzymatic subunits. Accumulating evidence suggests that the non-enzymatic subunits are involved in the regulation of mRNA deadenylation, although their precise roles remain to be established. In this study, we addressed the function of the CNOT1 subunit by depleting its expression in HeLa cells. Flow cytometric analysis revealed that the sub G(1) fraction was increased in CNOT1-depleted cells. Virtually, the same level of the sub G1 fraction was seen when cells were treated with a mixture of siRNAs targeted against all enzymatic subunits, suggesting that CNOT1 depletion induces apoptosis by destroying the CCR4-NOT-associated deadenylase activity. Further analysis revealed that CNOT1 depletion leads to a reduction in the amount of other CCR4-NOT subunits. Importantly, the specific activity of the CNOT6L immunoprecipitates-associated deadenylase from CNOT1-depleted cells was less than that from control cells. The formation of P-bodies, where mRNA decay is reported to take place, was largely suppressed in CNOT1-depleted cells. Therefore, CNOT1 has an important role in exhibiting enzymatic activity of the CCR4-NOT complex, and thus is critical in control of mRNA deadenylation and mRNA decay. We further showed that CNOT1 depletion enhanced CHOP mRNA levels and activated caspase-4, which is associated with endoplasmic reticulum ER stress-induced apoptosis. Taken together, CNOT1 depletion structurally and functionally deteriorates the CCR4-NOTcomplex and induces stabilization of mRNAs, which results in the increment of translation causing ER stress-mediated apoptosis. We conclude that CNOT1 contributes to cell viability by securing the activity of the CCR4-NOT deadenylase.


Subject(s)
Apoptosis , Caspases, Initiator , Genetics , Metabolism , Cell Survival , Endoplasmic Reticulum , Enzyme Activation , Flow Cytometry , HEK293 Cells , HeLa Cells , Humans , Protein Subunits , Genetics , Metabolism , RNA Stability , RNA, Messenger , RNA, Small Interfering , Genetics , Metabolism , Ribonucleases , Metabolism , Stress, Physiological , Transcription Factor CHOP , Genetics , Metabolism , Transcription Factors , Genetics , Metabolism , Transfection
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