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1.
Membranes (Basel) ; 11(10)2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34677524

RESUMO

By a sol-gel method, a BiFeO3 (BFO) capacitor is fabricated and connected with the control thin film transistor (TFT). Compared with a control thin-film transistor, the proposed BFO TFT achieves 56% drive current enhancement and 7-28% subthreshold swing (SS) reduction. Moreover, the effect of the proposed BiFeO3 capacitor on IDS-VGS hysteresis in the BFO TFT is 0.1-0.2 V. Because dVint/dVGS > 1 is obtained at a wide range of VGS, it reveals that the incomplete dipole flipping is a major mechanism to obtain improved SS and a small hysteresis effect in the BFO TFT. Experimental results indicate that sol-gel BFO TFT is a potential candidate for digital application.

2.
Cancers (Basel) ; 13(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070078

RESUMO

Radiotherapy, a common component in cancer treatment, can induce adverse effects including fibrosis in co-irradiated tissues. We previously showed that differential DNA methylation at an enhancer of diacylglycerol kinase alpha (DGKA) in normal dermal fibroblasts is associated with radiation-induced fibrosis. After irradiation, the transcription factor EGR1 is induced and binds to the hypomethylated enhancer, leading to increased DGKA and pro-fibrotic marker expression. We now modulated this DGKA induction by targeted epigenomic and genomic editing of the DGKA enhancer and administering epigenetic drugs. Targeted DNA demethylation of the DGKA enhancer in HEK293T cells resulted in enrichment of enhancer-related histone activation marks and radiation-induced DGKA expression. Mutations of the EGR1-binding motifs decreased radiation-induced DGKA expression in BJ fibroblasts and caused dysregulation of multiple fibrosis-related pathways. EZH2 inhibitors (GSK126, EPZ6438) did not change radiation-induced DGKA increase. Bromodomain inhibitors (CBP30, JQ1) suppressed radiation-induced DGKA and pro-fibrotic marker expression. Similar drug effects were observed in donor-derived fibroblasts with low DNA methylation. Overall, epigenomic manipulation of DGKA expression may offer novel options for a personalized treatment to prevent or attenuate radiotherapy-induced fibrosis.

3.
Nat Methods ; 16(5): 401-404, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30988467

RESUMO

Profiling of both the genome and the transcriptome promises a comprehensive, functional readout of a tissue sample, yet analytical approaches are required to translate the increased data dimensionality, heterogeneity and complexity into patient benefits. We developed a statistical approach called Texomer ( https://github.com/KChen-lab/Texomer ) that performs allele-specific, tumor-deconvoluted transcriptome-exome integration of autologous bulk whole-exome and transcriptome sequencing data. Texomer results in substantially improved accuracy in sample categorization and functional variant prioritization.


Assuntos
Perfilação da Expressão Gênica/métodos , Genoma Humano , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Transcriptoma/genética , Alelos , DNA de Neoplasias/genética , Exoma/genética , Humanos , Mutação , Polimorfismo de Nucleotídeo Único
4.
PeerJ ; 6: e5852, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30397550

RESUMO

Background: The need for read-based phasing arises with advances in sequencing technologies. The minimum error correction (MEC) approach is the primary trend to resolve haplotypes by reducing conflicts in a single nucleotide polymorphism-fragment matrix. However, it is frequently observed that the solution with the optimal MEC might not be the real haplotypes, due to the fact that MEC methods consider all positions together and sometimes the conflicts in noisy regions might mislead the selection of corrections. To tackle this problem, we present a hierarchical assembly-based method designed to progressively resolve local conflicts. Results: This study presents HAHap, a new phasing algorithm based on hierarchical assembly. HAHap leverages high-confident variant pairs to build haplotypes progressively. The phasing results by HAHap on both real and simulated data, compared to other MEC-based methods, revealed better phasing error rates for constructing haplotypes using short reads from whole-genome sequencing. We compared the number of error corrections (ECs) on real data with other methods, and it reveals the ability of HAHap to predict haplotypes with a lower number of ECs. We also used simulated data to investigate the behavior of HAHap under different sequencing conditions, highlighting the applicability of HAHap in certain situations.

5.
Methods Mol Biol ; 1757: 557-577, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29761470

RESUMO

The i5k Workspace@NAL is a genome database tailored toward newly sequenced arthropod genomes and their research communities. With 56 arthropod genomes and counting, the i5k Workspace strives to facilitate public data access, visualization, and community curation across arthropod species. Any researcher with an arthropod genome project who would like to take advantage of the i5k Workspace facilities is encouraged to submit their data. In this chapter, we explain how to use the i5k Workspace@NAL to submit, find, and improve arthropod genomics data.


Assuntos
Artrópodes/genética , Bases de Dados Genéticas , Genoma , Genômica , Animais , Biologia Computacional/métodos , Genômica/métodos , Anotação de Sequência Molecular , Software , Interface Usuário-Computador , Navegador
6.
Environ Sci Technol ; 52(10): 6009-6022, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29634279

RESUMO

Hyalella azteca is a cryptic species complex of epibenthic amphipods of interest to ecotoxicology and evolutionary biology. It is the primary crustacean used in North America for sediment toxicity testing and an emerging model for molecular ecotoxicology. To provide molecular resources for sediment quality assessments and evolutionary studies, we sequenced, assembled, and annotated the genome of the H. azteca U.S. Lab Strain. The genome quality and completeness is comparable with other ecotoxicological model species. Through targeted investigation and use of gene expression data sets of H. azteca exposed to pesticides, metals, and other emerging contaminants, we annotated and characterized the major gene families involved in sequestration, detoxification, oxidative stress, and toxicant response. Our results revealed gene loss related to light sensing, but a large expansion in chemoreceptors, likely underlying sensory shifts necessary in their low light habitats. Gene family expansions were also noted for cytochrome P450 genes, cuticle proteins, ion transporters, and include recent gene duplications in the metal sequestration protein, metallothionein. Mapping of differentially expressed transcripts to the genome significantly increased the ability to functionally annotate toxicant responsive genes. The H. azteca genome will greatly facilitate development of genomic tools for environmental assessments and promote an understanding of how evolution shapes toxicological pathways with implications for environmental and human health.


Assuntos
Anfípodes , Poluentes Químicos da Água , Animais , Ecotoxicologia , Sedimentos Geológicos , América do Norte , Testes de Toxicidade
7.
Oncotarget ; 7(37): 59845-59859, 2016 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-27542222

RESUMO

Recent reports demonstrate that the expression of protein kinase C alpha (PKCα) in triple-negative breast cancer (TNBC) correlates with decreased survival outcomes. However, off-target effects of targeting PKCα and limited understanding of the signaling mechanisms upstream of PKCα have hampered previous efforts to manipulate this ubiquitous gene. This study shows that the expression of both myeloid zinc finger 1 (MZF-1) and Ets-like protein-1 (Elk-1) correlates with PKCα expression in TNBC. We found that the acidic domain of MZF-1 and the heparin-binding domain of Elk-1 facilitate the heterodimeric interaction between the two genes before the complex formation binds to the PKCα promoter. Blocking the formation of the heterodimer by transfection of MZF-160-72 or Elk-1145-157 peptide fragments at the MZF-1 / Elk-1 interface decreases DNA-binding activity of the MZF-1 / Elk-1 complex at the PKCα promoter. Subsequently, PKCα expression, migration, tumorigenicity, and the epithelial-mesenchymal transition potential of TNBC cells decrease. These subsequent effects are reversed by transfection with full-length PKCα, confirming that the MZF-1/Elk-1 heterodimer is a mediator of PKCα in TNBC cells. These data suggest that the next therapeutic strategy in treating PKCα-related cancer will be developed from blocking MZF-1/Elk-1 interaction through their binding domain.


Assuntos
Fatores de Transcrição Kruppel-Like/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteína Quinase C-alfa/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Proteínas Elk-1 do Domínio ets/metabolismo , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Feminino , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fragmentos de Peptídeos/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Domínios Proteicos/genética , Proteína Quinase C-alfa/genética , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Proteínas Elk-1 do Domínio ets/genética
8.
Mol Med Rep ; 14(2): 1636-42, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27357025

RESUMO

AXL receptor tyrosine kinase is overexpressed in triple-negative breast cancer (TNBC), and has a function in cancer progression and metastases. However, the mechanism underlying AXL gene regulation in TNBC remains unknown. In this study, the involvement of protein kinase C α (PKCα) in the expression of AXL was investigated in human TNBC cells. The microarray data from other studies showed that PKCα is significantly correlated with AXL expression in TNBC cell lines. Tissue array analysis also confirmed their correlation in TNBC. The PKCα inhibitor Go6976 was used to treat MDA­MB­231 and Hs578T TNBC cells, which resulted in decreased expression of AXL and epithelia-mesenchymal transition-related gene vimentin, and decreased cell proliferation. An MZF­1 acidic domain fragment (MZF-1 peptide), which was designed to downregulate PKCα expression, was transfected into the cells and resulted in inhibition of AXL expression. This effect was reversed by co­treatment with the constitutive form of PKCα. Moreover, the downregulation of PKCα was also confirmed by treatment with TAT­fused MZF­1 peptide. Thus, the current study proposes that AXL may be correlated with PKCα­dependent TNBC cells, and could be modulated by MZF­1 peptides.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteína Quinase C-alfa/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Adulto , Idoso , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Fatores de Transcrição Kruppel-Like/química , Fatores de Transcrição Kruppel-Like/metabolismo , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Fragmentos de Peptídeos/farmacologia , Proteína Quinase C-alfa/genética , Proteoma , Proteômica/métodos , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
9.
J Econ Entomol ; 109(3): 1378-1386, 2016 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-27106222

RESUMO

Pesticide resistance poses many challenges for pest control, particularly for destructive pests such as diamondback moths ( Plutella xylostella ). Organophosphates have been used in the field since the 1950s, leading to selection for resistance-related gene variants and the development of resistance to new insecticides in the diamondback moth. Identifying actual and potential genes involved in resistance could offer solutions for control. This study established resistant diamondback moth strains from two different collections using mevinphos. Two sets of transcriptome sequencing (RNA-Seq) data were generated for pairs of mevinphos-resistant versus susceptible (wild-type) strains. One susceptible strain containing 14 giga base pairs was assembled into a reference-based assembly using published scaffold sequences as reference. Differential expression data between resistant and susceptible strains revealed 944 transcripts (803 with annotations) showing upregulation and 427 transcripts (150 with annotations) showing downregulation. Around 6.8% of the differential expression transcripts (65) could be categorized as associated with well-known resistance mechanisms such as penetration, detoxification, and behavior response; of these 65 transcripts, 38 showed upregulation, and 12 relating to penetration were upregulated when the transcripts of 19 cytochrome P450s, 2 zeta-class glutathione S-transferases, and 4 ATP-binding cassette transporters showed upregulation. In addition, 11 groups of transcripts related to olfactory perception appeared to be downregulated in trade-off situations. Quantitative polymerase chain reaction expression results were consistent with RNA-Seq data. Possible roles of these differentially expressed genes in resistance mechanisms are discussed in this study.

10.
BMC Genomics ; 17: 220, 2016 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-26969372

RESUMO

BACKGROUND: Recent advances in sequencing technology have opened a new era in RNA studies. Novel types of RNAs such as long non-coding RNAs (lncRNAs) have been discovered by transcriptomic sequencing and some lncRNAs have been found to play essential roles in biological processes. However, only limited information is available for lncRNAs in Drosophila melanogaster, an important model organism. Therefore, the characterization of lncRNAs and identification of new lncRNAs in D. melanogaster is an important area of research. Moreover, there is an increasing interest in the use of ChIP-seq data (H3K4me3, H3K36me3 and Pol II) to detect signatures of active transcription for reported lncRNAs. RESULTS: We have developed a computational approach to identify new lncRNAs from two tissue-specific RNA-seq datasets using the poly(A)-enriched and the ribo-zero method, respectively. In our results, we identified 462 novel lncRNA transcripts, which we combined with 4137 previously published lncRNA transcripts into a curated dataset. We then utilized 61 RNA-seq and 32 ChIP-seq datasets to improve the annotation of the curated lncRNAs with regards to transcriptional direction, exon regions, classification, expression in the brain, possession of a poly(A) tail, and presence of conventional chromatin signatures. Furthermore, we used 30 time-course RNA-seq datasets and 32 ChIP-seq datasets to investigate whether the lncRNAs reported by RNA-seq have active transcription signatures. The results showed that more than half of the reported lncRNAs did not have chromatin signatures related to active transcription. To clarify this issue, we conducted RT-qPCR experiments and found that ~95.24% of the selected lncRNAs were truly transcribed, regardless of whether they were associated with active chromatin signatures or not. CONCLUSIONS: In this study, we discovered a large number of novel lncRNAs, which suggests that many remain to be identified in D. melanogaster. For the lncRNAs that are known, we improved their characterization by integrating a large number of sequencing datasets (93 sets in total) from multiple sources (lncRNAs, RNA-seq and ChIP-seq). The RT-qPCR experiments demonstrated that RNA-seq is a reliable platform to discover lncRNAs. This set of curated lncRNAs with improved annotations can serve as an important resource for investigating the function of lncRNAs in D. melanogaster.


Assuntos
Drosophila melanogaster/genética , RNA Longo não Codificante/genética , Animais , Cromatina/genética , Imunoprecipitação da Cromatina , Anotação de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA
11.
Anticancer Res ; 34(11): 6467-72, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25368247

RESUMO

UNLABELLED: Aim/Materials and Methods: In order to develop better drugs against non-small cell lung cancer (NSCLC), we screened a variety of compounds and treated the human lung adenocarcinoma cell line A549 with different drug concentrations. We then examined the cell viability using the MTT assay. RESULTS: Data show that a new candidate drug, acriflavine (ACF), suppresses the viability of A549 cells in a concentration- and time-dependent manner. Flow cytometry analysis revealed that ACF significantly caused cell growth arrest in the G2/M phase on A549 cells. Moreover, ACF decreased Bcl-2 expression and increased Bax expression. The content of cleaved poly(ADP-ribose)polymerase-1 (PARP-1) and caspase-3 are significantly increased. These findings suggest that ACF is cytotoxic against A549 cells and suppresses A549 cells growth through the caspase-3 activation pathway. In the in vivo test, nude mice bearing A549 cells xenografts by intravenous injection were randomly assigned into two groups: control and experimental group. Treatment was initiated 10 days after implantation and intraperitoneal injection of 0.9% normal saline or 2 mg/kg of ACF was continued daily for five weeks. ACF treatment significantly decreased tumor size and tumor spots on lung surface of tumor-bearing mice. CONCLUSION: ACF can inhibit cell growth in A549 cells. Our results may assist on the delineation of the mechanism(s) leading to NSCLC cell growth inhibition and provide a new antitumor strategy against NSCLC.


Assuntos
Acriflavina/farmacologia , Adenocarcinoma/tratamento farmacológico , Anti-Infecciosos Locais/farmacologia , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Adenocarcinoma/patologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/patologia , Citometria de Fluxo , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Anticancer Res ; 34(7): 3549-56, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24982368

RESUMO

Patients suffering from advanced hepatocellular carcinoma can generally be treated only by targeted therapy to achieve a survival rate that lasts a few months more than that achieved with conventional therapy. To develop better drugs against hepatocellular carcinoma, we screened a variety of compounds and treated four human hepatocellular carcinoma (HCC) cell lines with different drug concentrations. We then examined cell viability using the MTT assay. Results show that a new candidate drug, acriflavine (ACF), suppresses the viability of HCC cell lines in a dose-dependent manner. Flow cytometry analysis reveals that ACF significantly induces the accumulation of a Sub-G1 population of Mahlavu cells. Moreover, ACF decreases Bcl-2 expression and caspase-3 activation. The content of cleaved poly-(ADP-ribose)polymerase-1 (PARP-1) is significantly increased. These findings suggest that ACF suppresses HCC cell growth through the caspase-3 activation pathway. Compared to clinically-approved drugs, the IC50 of ACF (1 µM) is nearly ten-fold lower than that of sorafenib (13 µM). In the in vivo test, nude mice received Mahlavu cell xenografts subcutaneously and were randomly assigned into two groups: control and experimental groups. Treatment was initiated 3 days after implantation and intraperitoneal injection of 0.9 % normal saline or 2 mg/Kg of ACF was continued daily for five weeks. Tumors were palpable in vehicle-treated mice by day 3 and grew to approximately 2000 mm3 by the end of the experiment, whereas mice treated with ACF experience tumor growth to approximately 500 mm3. We, thus, suggest that ACF can inhibit cell growth in HCC cells. Our results may assist the delineation of the mechanism(s) leading to HCC cell growth inhibition and provide a new target therapy capable to prolong the survival rate of patients in advanced stage.


Assuntos
Acriflavina/farmacologia , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Ciclo Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto
13.
J Clin Invest ; 122(5): 1881-94, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22466651

RESUMO

Dysregulation of canonical Wnt signaling is thought to play a role in colon carcinogenesis. ß-Catenin, a key mediator of the pathway, is stabilized upon Wnt activation and accumulates in the nucleus, where it can interact with the transcription factor T cell factor (TCF) to transactivate gene expression. Normal colonic epithelia express a truncated TCF-1 form, called dnTCF-1, that lacks the critical ß-catenin-binding domain and behaves as a transcriptional suppressor. How the cell maintains a balance between the two forms of TCF-1 is unclear. Here, we show that ERM-binding phosphoprotein 50 (EBP50) modulates the interaction between ß-catenin and TCF-1. We observed EBP50 localization to the nucleus of human colorectal carcinoma cell lines at low cell culture densities and human primary colorectal tumors that manifested a poor clinical outcome. In contrast, EBP50 was primarily membranous in confluent cell lines. Aberrantly located EBP50 stabilized conventional ß-catenin/TCF-1 complexes and connected ß-catenin to dnTCF-1 to form a ternary molecular complex that enhanced Wnt/ß-catenin signaling events, including the transcription of downstream oncogenes such as c-Myc and cyclin D1. Genome-wide analysis of the EBP50 occupancy pattern revealed consensus binding motifs bearing similarity to Wnt-responsive element. Conventional chromatin immunoprecipitation assays confirmed that EBP50 bound to genomic regions highly enriched with TCF/LEF binding motifs. Knockdown of EBP50 in human colorectal carcinoma cell lines compromised cell cycle progression, anchorage-independent growth, and tumorigenesis in nude mice. We therefore suggest that nuclear EBP50 facilitates colon tumorigenesis by modulating the interaction between ß-catenin and TCF-1.


Assuntos
Transporte Ativo do Núcleo Celular , Carcinoma/metabolismo , Neoplasias Colorretais/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Fator 1 de Transcrição de Linfócitos T/metabolismo , beta Catenina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Sequência de Bases , Sítios de Ligação , Carcinoma/patologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Transformação Celular Neoplásica , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Transplante de Neoplasias , Domínios PDZ , Fosfoproteínas/química , Fosfoproteínas/genética , Regiões Promotoras Genéticas , Ligação Proteica , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genética , Fator 1 de Transcrição de Linfócitos T/química
14.
Eur J Oral Sci ; 118(2): 151-8, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20487004

RESUMO

Osteoarthritis (OA) sometimes occurs as a consequence of repeated microtrauma involved in parafunction, which may lead to microfracture in the subchondral bone. The aim of this in vitro study was to evaluate the effects of subchondral osteoblasts in loading with repeated excessive mechanical stress on the metabolism of overlying chondrocytes. A high-magnitude cyclic tensile stress of 15 kPa (30 cycles min(-1)) was applied to the cultured osteoblasts obtained from porcine mandibular condyles. The chondrocytes in alginate beads were then co-cultured with mechanically stressed or unstressed osteoblasts. Chondrocytes co-cultured with unstressed osteoblasts showed a phenotypic shift to hypertrophic chondrocytes, characterized by decreased expression of type II collagen, aggrecan, Sry-related HMG box (SOX-9), and cartilage oligomeric matrix protein (COMP) genes and increased expression of type X collagen and bone sialoprotein (BSP) genes, suggesting that the co-culture may change the chondrocyte differentiation to some extent. These changes were more distinct in chondrocytes co-cultured with excessively mechanically stressed osteoblasts. After co-culture with stressed osteoblasts, the expressions of matrix metalloproteinase (MMP)1, MMP3 and MMP13 genes were also enhanced and the synthesis of DNA, proteoglycan and collagen were significantly decreased in chondrocytes. These results demonstrate that alterations in cartilage metabolism can be induced by stressed osteoblasts, indicating a possible explanation for the onset and progression of OA.


Assuntos
Condrócitos/metabolismo , Osteoblastos/fisiologia , Agrecanas/análise , Fosfatase Alcalina/análise , Animais , Fenômenos Biomecânicos , Cartilagem Articular/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Colágeno/análise , Colágeno Tipo II/análise , Colágeno Tipo X/análise , DNA/análise , Proteínas da Matriz Extracelular/análise , Glicoproteínas/análise , Hipertrofia , Sialoproteína de Ligação à Integrina , Côndilo Mandibular/citologia , Proteínas Matrilinas , Metaloproteinase 1 da Matriz/análise , Metaloproteinase 13 da Matriz/análise , Metaloproteinase 3 da Matriz/análise , Fenótipo , Proteoglicanas/análise , Fatores de Transcrição SOX9/análise , Sialoglicoproteínas/análise , Estresse Mecânico , Suínos , Fator de Crescimento Transformador beta/análise
15.
Ann Biomed Eng ; 37(7): 1358-67, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19381811

RESUMO

Osteoarthritis (OA) in the temporomandibular joint (TMJ) is a degenerative disease caused by excessive external loading. Recently, it was reported that the damage in the mineralized subchondral bone caused by traumatic impact-loading is responsible for the initiation and progression of cartilage degeneration. Thus far, we have hypothesized that cytokines released from damaged subchondral bone from impact-loading affect the cartilage catabolism under pathological conditions. An impactor of 200 gw was dropped onto the top of a porcine mandibular condyle. After organ culture for 2 days, we investigated the association between the subchondral bone and cartilage using histological and biochemical experiments. The impact-loading induced the expression of IL-1beta immunohistochemically and prominently up-regulated IL-1alpha and IL-1beta mRNA levels in subchondral bone. We confirmed a significant decrease in type II collagen and aggrecan mRNA expressions in chondrocytes by co-culture with osteoblasts after impact-loading, and significant increase in mRNA and protein expressions of IL-1beta in subchondral osteoblasts from impact-loaded subchondral bone. The mRNA expressions of type II collagen, aggrecan, and type X collagen in chondrocytes were decreased significantly by the co-culture with osteoblasts pre-treated by IL-1beta, -6, and TNF-alpha. Among them, osteoblasts pre-treated by IL-1beta affected chondrocytes most strongly. It was also shown that IL-1beta-treated osteoblasts enhanced the MMP-1 mRNA level most markedly in chondrocytes among the four cytokines. These results suggest that the TMJ subjected to impact-loading can increase directly IL-1beta synthesis in the subchondral region, subsequently altering the metabolism of adjacent cartilage and may eventually resulting in the onset and progression of TMJ-OA.


Assuntos
Cartilagem/metabolismo , Cartilagem/fisiopatologia , Citocinas/metabolismo , Interleucina-1beta/metabolismo , Mandíbula/fisiopatologia , Ferimentos não Penetrantes/fisiopatologia , Animais , Técnicas de Cultura de Órgãos/métodos , Suínos , Suporte de Carga
16.
Ann Biomed Eng ; 36(5): 793-800, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18278554

RESUMO

It is well known that mechanical loading influences the endochondral bone formation essential for the growth and development of longitudinal bones. The question was, however, asked whether the effect of mechanical loading on the chondrocyte metabolism is dependent on the loading frequency. This study was aimed at evaluating the effect of tensile loadings with various frequencies on the proliferation of growth plate chondrocytes and extracellular matrix synthesis. The chondrocytes obtained from rib growth plate cartilage of 4-week-old male Wistar strain rats were cultured by day 4 and day 11 and used as proliferating and matrix-forming chondrocytes, respectively. Intermittent tensile stresses with different frequencies were applied to each stage chondrocyte. DNA syntheses were examined by measuring the incorporation of [(3)H]thymidine into the cells. Furthermore, the rates of collagen and proteoglycan syntheses were determined by measuring the incorporation of [2,3-(3)H]proline and [(35)S]sulfate into the cells, respectively. At the proliferating stage, intermittent tensions with the frequencies of 30 cycles/min and 150 cycles/min significantly (p < 0.05) upregulated the syntheses of DNA, which indicates the promotion of chondrocyte proliferation. At the matrix-forming stage, collagen, and proteoglycan syntheses also enhanced with increase of the loading frequency. In particular, the intermittent tension with the frequencies of 30 cycles/min and 150 cycles/min increased significantly (p < 0.05 or p < 0.01) both the collagen and proteoglycan syntheses. These results suggest that the proliferation and differentiation of growth plate chondrocytes are regulated by the mechanical loading and that the chondrocyte metabolism enhanced with increase of loading frequency. These may give more insight into the possible mechanism leading to endochondral bone formation.


Assuntos
Condrócitos/fisiologia , Colágeno/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/fisiologia , Lâmina de Crescimento/fisiologia , Mecanotransdução Celular/fisiologia , Proteoglicanas/metabolismo , Animais , Células Cultivadas , Lâmina de Crescimento/citologia , Masculino , Ratos , Ratos Wistar , Estresse Mecânico , Resistência à Tração/fisiologia , Suporte de Carga/fisiologia
17.
Arch Oral Biol ; 53(4): 330-6, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18160062

RESUMO

Hyaluronan (HA) exists in various living tissues as one of the major matrix macromolecules, and is well known to play an integral role in cell differentiation and proliferation. The present study was conducted to elucidate whether or not the proliferation of periodontal ligament (PDL) cells are affected specifically by the degradation of HA by hyaluronidasze (HAase). Human PDL fibroblasts were isolated and cultured with and without 15-150U/ml bovine testicular HAase from 1 to 11 days after seeding. The cells were also cultured with anti-CD44 antibody of 2 microg/ml. For the control against the anti-CD44 antibody treatment, 2 microg/ml IgG was used. The HA-dependent pericellular matrix was visualized by particle-exclusion assay. The number of cells was counted by MTT assay during the proliferation. The mRNA levels of HA synthases (HASs), HAases (HYALs) and CD44s were examined by a quantitative real-time PCR analysis. The cell proliferation was inhibited by the treatment with HAase and anti-CD44 antibody in cultured PDL fibroblasts. HASs mRNAs were down-regulated, whereas HYALs mRNAs were up-regulated significantly by the treatment with HAase and anti-CD44 antibody. The CD44s mRNA level exhibited no significant changes. These results suggest that HA may contribute to modulate the proliferation of cultured human PDL cells through a CD44-mediated mechanism.


Assuntos
Hialuronoglucosaminidase/farmacologia , Ligamento Periodontal/efeitos dos fármacos , Anticorpos Monoclonais/metabolismo , Ligação Competitiva , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucuronosiltransferase/biossíntese , Glucuronosiltransferase/genética , Humanos , Receptores de Hialuronatos/biossíntese , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/imunologia , Hialuronan Sintases , Hialuronoglucosaminidase/biossíntese , Hialuronoglucosaminidase/genética , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética
18.
Mol Biol Cell ; 18(5): 1710-22, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17332505

RESUMO

Podocalyxin/Gp135 was recently demonstrated to participate in the formation of a preapical complex to set up initial polarity in MDCK cells, a function presumably depending on the apical targeting of Gp135. We show that correct apical sorting of Gp135 depends on a bipartite signal composed of an extracellular O-glycosylation-rich region and the intracellular PDZ domain-binding motif. The function of this PDZ-binding motif could be substituted with a fusion construct of Gp135 with Ezrin-binding phosphoprotein 50 (EBP50). In accordance with this observation, EBP50 binds to newly synthesized Gp135 at the Golgi apparatus and facilitates oligomerization and sorting of Gp135 into a clustering complex. A defective connection between Gp135 and EBP50 or EBP50 knockdown results in a delayed exit from the detergent-resistant microdomain, failure of oligomerization, and basolateral missorting of Gp135. Furthermore, the basolaterally missorted EBP50-binding defective mutant of Gp135 was rapidly retrieved via a PKC-dependent mechanism. According to these findings, we propose a model by which a highly negative charged transmembrane protein could be packed into an apical sorting platform with the aid of its cytoplasmic partner EBP50.


Assuntos
Sialoglicoproteínas/metabolismo , Motivos de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Polaridade Celular , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Cães , Endocitose , Glicosilação , Microdomínios da Membrana/metabolismo , Modelos Biológicos , Mutação , Proteína Quinase C/metabolismo , Sinais Direcionadores de Proteínas/genética , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , Sialoglicoproteínas/química , Sialoglicoproteínas/genética , Transdução de Sinais
19.
Histochem Cell Biol ; 127(4): 399-414, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17180683

RESUMO

Podocalyxin (PC) was initially identified as a major sialoprotein on the apical surface of glomerular podocytes to perform the filtration barrier function. Later, it was reported to be expressed in endothelial cells, megakaryotes/platelets, and hemangioblasts, the common progenitor cells of the hematopoietic and endothelial cells. Recently, increasing numbers of reports have indicated that PC is not merely a molecule restricted at renal glomerulus, angiogenic or hematopoietic system. To further elucidate the expression pattern and address the possible physiological role of PC in adult mammals, we conducted an extensive study by immunohistochemistry and immunofluorescence staining on various tissues of healthy adult beagle dogs. By combinatory usage of two different anti-podocalyxin antibodies recognizing distinct epitopes in PC, we have demonstrated that (1) PC is expressed in renal tubules, mesothelium, myocardium, striated muscles in tongue, esophagus and extraocular region, myoepithelial cells in esophagus and salivary glands, neurons, and ependyma, etc.; (2) there are at least three forms of PC proteins, depending upon the accessibility of two different PC antibodies, expressed in different organs/systems; and (3) a particular form of PC is distributed in a vesicle-like compartment in certain organs/systems, such as the central nervous system.


Assuntos
Biomarcadores/análise , Glomérulos Renais/química , Podócitos/química , Sialoglicoproteínas/análise , Animais , Western Blotting , Células da Medula Óssea/química , Linhagem Celular , Linhagem Celular Tumoral , Sistema Digestório/química , Cães , Sistema Endócrino/química , Olho/química , Feminino , Genitália Feminina/química , Genitália Masculina/química , Humanos , Sistema Imunitário/química , Imuno-Histoquímica , Glomérulos Renais/citologia , Masculino , Miocárdio/química , Sistema Nervoso/química , Podócitos/citologia , Sistema Urinário/química
20.
J Am Soc Nephrol ; 16(6): 1612-22, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15814834

RESUMO

GP135 is an apical membrane protein expressed in polarized MDCK epithelial cells. When cultured in three-dimensional collagen gel, MDCK cells form branching tubules in response to hepatocyte growth factor stimulation in a manner that simulates the embryonic renal development. During this process, GP135 displays transient loss of membranous localization but reappears at the cell surface when nascent lumen emerges from the developing tubules. Despite being used for decades as the canonical hallmark of apical surface, the molecular identity and the significance of the dynamic expression of GP135 during the tubulogenic process remain elusive. For exploring the function of GP135, the full-length cDNA encoding GP135 was obtained. Sequence alignments and features analysis confirm GP135 as a canine homolog of podocalyxin, confirming the finding of an earlier independent study. Immunohistochemical assays on canine kidney sections identified both glomerular and tubular distribution of GP135 along the nephron. Mutant MDCK cells expressing siRNA targeted at two regions of GP135 show defects in hepatocyte growth factor-induced tubulogenesis. Re-expression of full-length and an O-linked glycosylation abbreviated construct of GP135 could recapitulate the tubulogenesis process lacking in siRNA knockdown cells; however, a deletion construct devoid of the cytoplasmic domain failed to rescue the phenotype. In summary, the data identify the MDCK apical domain marker GP135 as a tubular form of podocalyxin and provide evidence for its importance in renal tubulogenesis.


Assuntos
Túbulos Renais/fisiologia , Sialoglicoproteínas/fisiologia , Animais , Linhagem Celular , Clonagem Molecular , DNA Complementar , Cães , Células Epiteliais , Glicoproteínas de Membrana/fisiologia , Camundongos
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