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1.
BMC Ophthalmol ; 19(1): 168, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375076

RESUMO

BACKGROUND: Age-related macular degeneration (AMD) is the primary cause of blindness and severe vision loss in developed countries and is responsible for 8.7% of blindness globally. Ultraviolet radiation can induce DNA breakdown, produce reactive oxygen species, and has been implicated as a risk factor for AMD. This study investigated the effects of UVA radiation on Human retinal pigment epithelial cell (ARPE-19) growth and protein expression. METHODS: ARPE-19 cells were irradiated with a UVA lamp at different doses (5, 10, 20, 30 and 40 J/cm2) from 10 cm. Cell viability was determined by MTT assay. Visual inspection was first achieved with inverted light microscopy and then the DeadEnd™ Fluorometric TUNEL System was used to observe nuclear DNA fragmentation. Flow cytometry based-Annexin V-FITC/PI double-staining was used to further quantify cellular viability. Mitochondrial membrane potential was assessed with JC-1 staining. 2D electrophoresis maps of exposed cells were compared to nonexposed cells and gel images analyzed with PDQuest 2-D Analysis Software. Spots with greater than a 1.5-fold difference were selected for LC-MS/MS analysis and some confirmed by western blot. We further investigated whether caspase activation, apoptotic-related mitochondrial proteins, and regulators of ER stress sensors were involved in UVA-induced apoptosis. RESULTS: We detected 29 differentially expressed proteins (9 up-regulated and 20 down-regulated) in the exposed cells. Some of these proteins such as CALR, GRP78, NPM, Hsp27, PDI, ATP synthase subunit alpha, PRDX1, and GAPDH are associated with anti-proliferation, induction of apoptosis, and oxidative-stress protection. We also detected altered protein expression levels among caspases (caspase 3 and 9) and in the mitochondrial (cytosolic cytochrome C, AIF, Mcl-1, Bcl-2, Bcl-xl, Bax, Bad, and p-Bad) and ER stress-related (p-PERK, p-eIF2α, ATF4 and CHOP) apoptotic pathways. CONCLUSIONS: UVA irradiation suppressed the proliferation of ARPE-19 cells in a dose-dependent manner, caused quantitative loses in transmembrane potential (ΔΨm), and induced both early and late apoptosis.


Assuntos
Degeneração Macular/patologia , Estresse Oxidativo , Proteômica/métodos , Epitélio Pigmentado da Retina/metabolismo , Raios Ultravioleta , Apoptose , Sobrevivência Celular , Células Cultivadas , Cromatografia Líquida , Citocromos c/metabolismo , Humanos , Degeneração Macular/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/efeitos da radiação , Transdução de Sinais , Espectrometria de Massas em Tandem
2.
Eye Contact Lens ; 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31313699

RESUMO

OBJECTIVES: To investigate the changing indications for corneal transplantations in southern Taiwan from 2008 to 2018 and compare the results with those of other previous studies. METHODS: We retrospectively reviewed the records of patients who underwent corneal transplantations from January 2008 to December 2018 at Kaohsiung Veterans General Hospital. The patients' age, sex, indication for corneal transplantation, and the surgical technique were recorded and analyzed. RESULTS: In total, 336 patients (421 eyes) were included in our study from January 1, 2008, to December 31, 2018. The mean age was 63.6±15.3 years; 371 penetrating keratoplasty (88.1%), 35 deep anterior lamellar keratoplasty (8.3%), and 15 Descemet-stripping automated endothelial keratoplasty (3.6%) were performed. The leading indication for corneal transplantation was regraft (n=103, 24.5%), followed by bullous keratopathy (n=82, 19.5%), corneal scar and opacity (n=79, 18.8%), keratitis (n=59, 14.0%), corneal dystrophy (n=58, 13.8%), other indications (n=26, 6.2%), and keratoconus (n=14, 3.3%). CONCLUSION: The indications for corneal transplantations changed with time in Taiwan. Regraft was the leading indication, but there was a decreasing trend over time. The proportion of bullous keratopathy increased significantly over the past decade and is the second most common indication, similar to the most developed countries. Both corneal scar and opacity, and keratitis showed a decreasing trend of occurrence compared with a previous study in Taiwan. The proportion of keratoconus remained low, making it the least common indication for corneal transplantation.

3.
Int J Mol Sci ; 20(15)2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31349542

RESUMO

Fungal keratitis (FK) is one of the most severe corneal infectious diseases. FK often leads to poor visual prognosis and thus requires accurate diagnosis. Conventional approaches, including clinical diagnoses, smears, and cultures, often fail to provide reliable diagnostic value. Omics approaches, such as those using genomic, metagenomic, and tear proteomic data sources, provide promising features for improving the diagnosis and monitoring the progression of FK. Genomic approaches are based mainly on detecting amplicons of ribosomal RNA genes, and internal transcribed spacers are gradually gaining popularity in clinical practices. A metagenomic approach based on 16S rRNA genes may help monitor the dynamic change of conjunctival microbiota associated with an FK event, whereas that based on shot-gun and 18S rRNA target enrichment sequencing could have the potential to diagnose FK using clinical samples. A tear proteomic approach may provide comprehensive information about ocular surface defense and injury during FK. Representative up- and down-regulated proteins during FK could also be used as biomarkers to determine the clinical course and develop a treatment strategy in different stages of FK. Consequently, a personalized tear proteomic approach will soon play a key role in FK management.

4.
PLoS One ; 14(3): e0213932, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30870514

RESUMO

Age-related macular degeneration (AMD) is an ocular disease with retinal degeneration. Retinal pigment epithelium (RPE) degeneration is mainly caused by long-term oxidative stress. Kinase activity could be either protective or detrimental to cells during oxidative stress; however, few reports have described the role of kinases in oxidative stress. In this study, high-throughput screening of kinome siRNA library revealed that erb-b2 receptor tyrosine-protein kinase 2 (ERBB2) knockdown reduced reactive oxygen species (ROS) production in ARPE-19 cells during oxidative stress. Silencing ERBB2 caused an elevation in microtubule associated protein light chain C3-II (MAP1LC3B-II/I) conversion and sequesterone (SQSTM)1 protein level. ERBB2 deprivation largely caused an increase in autophagy-regulating protease (ATG4B) expression, a protease that negatively recycles MAP1LC3-II at the fusion step between the autophagosome and lysosome, suggesting ERBB2 might modulate ATG4B for autophagy induction in oxidative stress-stimulated ARPE-19 cells. ERBB2 knockdown also caused an accumulation of nuclear factor erythroid 2-related factor 2 (NRF2) and enhanced its transcriptional activity. In addition, ERBB2 ablation or treatment with autophagy inhibitors reduced oxidative-induced cytotoxic effects in ARPE-19 cells. Furthermore, ERBB2 silencing had little or no additive effects in ATG5/7-deficient cells. Taken together, our results suggest that ERBB2 may play an important role in modulating autophagic RPE cell death during oxidative stress, and ERBB2 may be a potential target in AMD prevention.

5.
Int J Colorectal Dis ; 2018 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-30003361

RESUMO

BACKGROUND: Periodontitis is a frequently cited extraintestinal manifestation of Crohn's disease (CD). Despite a plethora of investigations and a recent meta-analysis linking CD and periodontitis, no study has estimated the risk of periodontitis among CD patients with respect to a comparison group nor has any investigation analyzed the effect of CD-specific medications on the risk of periodontitis. The present cohort study compared CD patients and matched subjects without a history of inflammatory bowel disease (IBD) to estimate the effect of CD and CD-specific pharmaceutical prescriptions on the risk of developing periodontitis by leveraging a population-based dataset in Taiwan. METHODS: We sourced 6657 CD patients and 26,628 comparison subjects without a history of IBD from the Taiwan National Health Insurance Database. Cox proportional hazards regressions were used to estimate the risk of subsequent periodontitis by CD status and pharmaceutical prescription during the follow-up period. RESULTS: After adjusting for socioeconomic status (SES), urbanicity, selected medical co-morbidities, and CD-specific pharmaceutical prescriptions, the hazard ratio (HR) for subsequent periodontitis among patients with CD was 1.36 (95% CI = 1.25-1.48) that of comparison subjects. There was not a significant difference in risk between genders or across ages. Steroids (95% CI = 0.66-0.77) appeared to confer a protective effect and Aspirin, Plavix, and Licodin were marginally protective (95% CI = 0.76-0.95). CONCLUSION: This is the first study to report an increased HR for subsequent periodontitis among CD patients when compared to matched comparison subjects without IBD. The protective effect of some pharmaceuticals may suggest that treatment of CD protects against periodontitis.

6.
Cornea ; 37(12): 1542-1550, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30044251

RESUMO

PURPOSE: To elucidate the bioburden of a lens care system in patients with contact lens (CL)-related keratitis. METHODS: In this prospective case-control study, the CL storage case was used as the target of bioburden assessment. Participants were CL wearers with (n = 26) or without (n = 24) keratitis in southern Taiwan. The case fluid sample was consecutively collected and assessed using a dot hybridization assay (DHA) for bioburden assessment. The DHA was composed of 3 universal bacterial probes for detecting all bacteria, genus-specific probes for Pseudomonas and Acinetobacter, and 1 probe for Acanthamoeba. The receiver operating characteristic curve was used to analyze the differentiating performance of a probe. RESULTS: The storage case bioburden was significantly higher in those with CL-related keratitis than in control subjects; patients with CL-related keratitis had stronger standardized signals in all universal bacterial probes. Moreover, in the cases of these patients, the bioburden was significantly heavier in confirmed infectious keratitis than in presumed noninfectious keratitis. Receiver operating characteristic analysis showed that stronger signals in all 3 universal bacterial probes and the Pseudomonas probe might suggest that the wearers have infectious keratitis. Only the storage case of the Acanthamoeba keratitis case showed positive detection by the Acanthamoeba probe. CONCLUSIONS: A heavier bioburden in the lens storage case was associated with a higher risk of CL-related keratitis and infectious keratitis. Inappropriate maintenance of the CL will lead to microbial contamination and transfer the pathogen onto the ocular surface causing keratitis accordingly. The DHA assessment for the lens storage case might provide an alternative way to differentiate infectious from noninfectious CL-related keratitis.


Assuntos
Biofilmes , Lentes de Contato/parasitologia , DNA Bacteriano/análise , DNA de Protozoário/análise , Contaminação de Equipamentos , Infecções Oculares Parasitárias/parasitologia , Embalagem de Produtos/instrumentação , Ceratite por Acanthamoeba , Adulto , Estudos de Casos e Controles , Soluções para Lentes de Contato/efeitos adversos , Infecções Oculares Parasitárias/epidemiologia , Infecções Oculares Parasitárias/metabolismo , Feminino , Humanos , Incidência , Masculino , Reação em Cadeia da Polimerase , Estudos Prospectivos , Taiwan/epidemiologia , Adulto Jovem
7.
PLoS One ; 12(3): e0174736, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28358857

RESUMO

Indocyanine green (ICG) and brilliant blue G (BBG) are commonly used vital dyes to remove internal limiting membrane (ILM) in vitreoretinal surgery. The vital dyes have shown cytotoxic effects in ocular cells. Autophagy is a stress responsive pathway for either protecting cells or promoting cell death. However, the role of autophagy in ocular cells in response to the vital dyes remains unknown. In this study, we found that ICG and BBG reduced cell viability in both human retinal pigment epithelial ARPE-19 and mouse photoreceptor 661W cells. ICG and BBG induced lipidated GFP-LC3-II and LC3-II in ARPE-19 and 661W cells. Combination treatment with the autophagy inhibitor chloroquine indicated that ICG and BBG reduced autophagic flux in ARPE-19 cells, whereas the vital dyes induced autophagic flux in 661W cells. Moreover, genetic and pharmacological ablation of autophagy enhanced vital dyes-induced cytotoxicity in ocular cells. Dietary supplements, including resveratrol, lutein, and CoQ10, induced autophagy and diminished the cytotoxic effects of ICG and BBG in ocular cells. These results suggest that autophagy may protect ARPE-19 and 661W cells from vital dyes-induced damage.


Assuntos
Autofagia/efeitos dos fármacos , Corantes/efeitos adversos , Verde de Indocianina/efeitos adversos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Corantes de Rosanilina/efeitos adversos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cloroquina , Humanos , Luteína/administração & dosagem , Camundongos , Substâncias Protetoras/administração & dosagem , Resveratrol , Epitélio Pigmentado da Retina/fisiopatologia , Epitélio Pigmentado da Retina/cirurgia , Estilbenos/administração & dosagem , Ubiquinona/administração & dosagem , Ubiquinona/análogos & derivados , Vitrectomia/efeitos adversos
8.
Medicine (Baltimore) ; 95(40): e5072, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27749578

RESUMO

BACKGROUND: Secondhand smoke (SHS) exposure is deleterious to pregnant women and their unborn children. The prevalence of SHS exposure among pregnant women is particularly high in many Asian countries where approximately half of the male population smokes. We aim to investigate the efficacy of an intervention based on an expanded Health Belief Model (HBM) incorporating self-efficacy to educate and empower pregnant women to reduce their SHS exposure. METHODS: We conducted a 3-arm randomized controlled trial (N = 50 in each arm) comparing the effectiveness of group-based and individual-based interventions with a treatment-as-usual group. A questionnaire tapping into constructs of the expanded HBM was administered at baseline and 1- and 2-month follow-ups. Exhaled carbon monoxide was used to determine SHS exposure (>=6 ppm). ANOVA was used to compare HBM construct scores, self-efficacy for rejecting SHS exposure, and SHS rejection behavior among the 3 groups at baseline and the 1- and 2-month follow-ups, while logistic regression analysis was used to compare the risk of exposure to SHS at each follow-up. RESULTS: The group-based intervention significantly improved health beliefs, self-efficacy, and self-reported rejection behaviors. The individual-based intervention effect was limited to some health belief constructs and SHS rejection behaviors. Both group- and individual-based interventions showed significant reductions in SHS exposure 2 months after the intervention (P < 0.0001). CONCLUSION: Group-based educational interventions based on the HBM are particularly effective in training pregnant women to avoid and refuse exposure to SHS. Policy makers should consider offering group-delivered programs to educate and empower pregnant women to reduce their SHS exposure.


Assuntos
Aconselhamento/métodos , Grupos Focais , Resultado da Gravidez/epidemiologia , Autorrelato , Poluição por Fumaça de Tabaco/prevenção & controle , Adulto , Feminino , Humanos , Gravidez , Prevalência , Inquéritos e Questionários , Taiwan/epidemiologia , Poluição por Fumaça de Tabaco/efeitos adversos , Poluição por Fumaça de Tabaco/estatística & dados numéricos
9.
Taiwan J Ophthalmol ; 5(2): 99-102, 2015 Apr-Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29018677

RESUMO

This article reports the clinical course and treatment of ocular adnexal lymphoma based on a retrospective review of five cases with a histologically approved ocular adnexal lymphoma at Kaohsiung Veterans General Hospital over 10 years. Extranodal B-cell lymphoma in the orbit, lacrimal gland, eyelid, or conjunctiva was found in these patients. Four of them were female, and they were aged 45-64 years. All patients were also consulted with hematologists for possible systemic involvement and therapeutic plan. The patient with retrobulbar and orbital apex involvement received systemic chemotherapy. The patient with lacrimal gland involvement experienced tumor recurrence after local excision, and therefore received adjuvant radiotherapy. The remaining three patients had localized lymphoma on the eyelid or bulbar conjunctiva, and they all showed no recurrence after surgical excision. The incidence of ocular adnexal lymphoma has risen worldwide over the last few decades. Although most cases are confined to ocular adnexal, some may also be associated with disseminated lymphoma. Accurate diagnosis and staging is mandatory for appropriate treatment. Generally speaking, localized and low-grade ocular adnexal lymphoma involved eyelid or conjunctiva seem to have good outcome after surgical excision only. Systemic chemotherapy should be considered in patients with advanced disease or systemic manifestations, and radiotherapy also offers a good choice for lacrimal gland lymphoma.

10.
J Ocul Pharmacol Ther ; 26(3): 231-6, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20565308

RESUMO

PURPOSE: Although the exact pathogenesis of age-related macular degeneration (AMD) is not clear, most studies indicate a role for retinal pigment epithelial (RPE) cell damage and death caused by oxidative stress. The purpose of this study was to examine the potential protective effects of lutein, zeaxanthin, meclofenamic acid, and resveratrol on the acrolein-induced oxidative stress in human RPE cells. METHODS: Cultured human RPE R-50 cells were treated with acrolein at different concentrations and treatment times. The protective effects of lutein (100 microM), zeaxanthin (100 microM), meclofenamic acid (30 microM), and resveratrol (10 microM) were investigated by pretreatment with the above agents before toxicant exposure in acute toxicity models and cotreatment with the toxicant in chronic toxicity models. The synergistic effects of acrolein and hydrogen peroxide exposure were also studied. Fluorescent latex beads were used to assess the phagocytic function of the cells. RESULTS: Acrolein inhibited the phagocytic function of human RPE R-50 cells, and the inhibitory effects were time dependent. Pretreatment with lutein, zeaxanthin, meclofenamic acid, or resveratrol alleviated the inhibition of phagocytosis in the acute acrolein and combined acrolein/hydrogen peroxide toxicity models. Synergistic effects were seen between zeaxanthin and resveratrol or meclofenamic acid. Cotreatment with lutein, zeaxanthin, meclofenamic acid, or resveratrol showed a protective effect against the damage caused by 7-day acrolein exposure followed by hydrogen peroxide treatment. CONCLUSIONS: Our results indicated an inhibitory effect of compounds found in cigarette smoke on human RPE phagocytosis, and lutein, zeaxanthin, meclofenamic acid, and resveratrol each offered protection against this inhibition. Therefore, red wine polyphenol, resveratrol, might ameliorate acrolein-induced or age-related RPE degeneration, such as AMD.


Assuntos
Antioxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Estilbenos/farmacologia , Acroleína/administração & dosagem , Acroleína/toxicidade , Células Cultivadas , Citoproteção/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Peróxido de Hidrogênio/toxicidade , Luteína/farmacologia , Ácido Meclofenâmico/farmacologia , Fagocitose/efeitos dos fármacos , Resveratrol , Epitélio Pigmentado da Retina/citologia , Fumar/efeitos adversos , Xantofilas/farmacologia , Zeaxantinas
11.
Proc Natl Acad Sci U S A ; 102(44): 15960-4, 2005 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16247010

RESUMO

Dyskeratosis congenita is a rare inherited disorder characterized by abnormal skin manifestations. Morbidity and mortality from this disease is usually due to bone marrow failure, but idiopathic pulmonary fibrosis and an increased cancer predisposition also occur. Families with autosomal dominant dyskeratosis congenita display anticipation and have mutations in the telomerase RNA gene. We identified a three-generation pedigree with autosomal dominant dyskeratosis congenita, anticipation, and telomere shortening. We show that a null mutation in motif D of the reverse transcriptase domain of the protein component of telomerase, hTERT, is associated with this phenotype. This mutation leads to haploinsufficiency of telomerase, and telomere shortening occurs despite the presence of telomerase. This finding emphasizes the importance of telomere maintenance and telomerase dosage for maintaining tissue proliferative capacity and has relevance for understanding mechanisms of age-related changes.


Assuntos
Antecipação Genética , Proteínas de Ligação a DNA/genética , Disceratose Congênita/genética , Genes Dominantes/genética , Haplótipos , Telomerase/genética , Adolescente , Adulto , Proliferação de Células , Criança , Pré-Escolar , Proteínas de Ligação a DNA/deficiência , Disceratose Congênita/etiologia , Saúde da Família , Feminino , Dosagem de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Fenótipo , Telomerase/deficiência , Telômero/ultraestrutura
12.
Kaohsiung J Med Sci ; 21(6): 286-90, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16035573

RESUMO

A 30-year-old healthy female presented with a 1-year history of chronic mucous discharge, tearing, and irritation in the left eye. Slit-lamp examination revealed severe papillary and follicular reaction surrounding a movable subconjunctival mass on the left upper tarsal conjunctiva. Incision and curettage were performed to establish the diagnosis. Multiple peculiar black mulberry nodules were obtained. The clumps of septate hyphae seen with periodic acid-Schiff stain were characteristic of fungus ball (aspergilloma). The patient's symptoms improved significantly after surgery without any antifungal therapy. Although rarely reported, aspergillus is a common fungus in the conjunctiva that may seed into the subconjunctiva. We present this case to remind ophthalmologists of such a rare cause of recalcitrant conjunctival inflammation in immunocompetent patients.


Assuntos
Aspergilose/patologia , Doenças da Túnica Conjuntiva/patologia , Infecções Oculares Fúngicas/patologia , Adulto , Feminino , Humanos
13.
Proc Natl Acad Sci U S A ; 102(23): 8080-5; discussion 8077-9, 2005 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-15849264

RESUMO

Telomerase is essential for maintaining telomere length and chromosome stability in stem cells, germline cells, and cancer cells. The telomerase ribonucleoprotein complex consists of two essential components, a catalytic protein component and an RNA molecule that provides the template for telomeric repeat synthesis. A pseudoknot structure in the human telomerase RNA is conserved in all vertebrates and is essential for telomerase activity. It has been proposed that this highly conserved structure functions as a dynamic structure with conformational interchange between the pseudoknot and a hairpin with intraloop base pairings. To examine the structural and functional requirements of the pseudoknot structure, we made mutations in the proposed base-paired regions in the pseudoknot. Although mutations that disrupted the pseudoknot P3 helix abolished activity as predicted, mutations that disrupted the intraloop hairpin base pairings did not reduce telomerase activity, indicating that the intraloop hairpin is not required for telomerase function. This functional study thus provides evidence against the previous proposed molecular-switch model of telomerase pseudoknot function and supports a static pseudoknot structure. The mutational analysis further suggests that telomerase RNA can function independent of the proposed intermolecular pairings between pseudoknot regions on two RNA molecules.


Assuntos
Conformação de Ácido Nucleico , RNA/química , RNA/metabolismo , Telomerase/química , Telomerase/metabolismo , Sequência de Bases , Humanos , Espectroscopia de Ressonância Magnética , Mutação/genética , RNA/genética , Relação Estrutura-Atividade , Telomerase/genética
16.
Genes Dev ; 17(22): 2747-52, 2003 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-14630939

RESUMO

Telomerase uses a short template sequence in its intrinsic RNA component to synthesize telomere repeats. Disruption of the helix P1b in human telomerase RNA or alteration of its distance from the template resulted in telomerase copying residues past the normal template boundary both in vivo and in vitro. Therefore, helix P1b is important for template boundary definition in human telomerase. Mouse telomerase RNA lacks helix P1b, and the boundary is established at 2 nt downstream of the 5'-end. The divergent structure of boundary definition elements in mammals, yeast, and ciliates suggests diverse mechanisms for template boundary definition in telomerase.


Assuntos
RNA/química , RNA/metabolismo , Telomerase , Telomerase/química , Telomerase/metabolismo , Moldes Genéticos , Animais , Pareamento de Bases , Sequência de Bases , Proteínas de Ligação a DNA , Humanos , Kluyveromyces/enzimologia , Kluyveromyces/genética , Camundongos , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , RNA/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Telomerase/genética , Telômero/genética , Telômero/metabolismo , Tetrahymena thermophila/enzimologia , Tetrahymena thermophila/genética
17.
EMBO J ; 22(2): 304-14, 2003 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-12514136

RESUMO

Telomerase contains two essential components: an RNA molecule that templates telomeric repeat synthesis and a catalytic protein component. Human telomerase is processive, while the mouse enzyme has much lower processivity. We have identified nucleotide determinants in the telomerase RNA that are responsible for this difference in processivity. Mutations adjacent to the template region of human and mouse telomerase RNA significantly altered telomerase processivity both in vitro and in vivo. We also identified functionally important nucleotides in the pseudoknot domain of telomerase RNA that potentially mediate the incompatibility between human TERT and mouse telomerase RNA. These experiments identify essential residues of the telomerase RNA that regulate telomerase activity and processivity.


Assuntos
Nucleotídeos/metabolismo , RNA/genética , RNA/metabolismo , Telomerase/genética , Telomerase/metabolismo , Animais , Domínio Catalítico , Proteínas de Ligação a DNA , Humanos , Camundongos , Modelos Genéticos , Mutação , Conformação de Ácido Nucleico , Especificidade da Espécie , Moldes Genéticos
18.
Nucleic Acids Res ; 30(2): 592-7, 2002 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-11788723

RESUMO

Telomerase is an enzyme that maintains telomere length by adding telomeric sequence repeats onto chromosome ends. The telomerase ribonucleoprotein complex consists of two essential components, a reverse transcriptase and an RNA molecule that provides the template for telomeric repeat synthesis. A common secondary structure of vertebrate telomerase RNA has been proposed based on a phylogenetic comparative analysis of 35 sequences. Here we report the identification of an additional essential base-paired region in the CR4-CR5 domain of mammalian telomerase RNA, termed P6.1. Mouse telomerase RNAs with mutations that disrupted base pairings in the P6.1 helix were unable to reconstitute telomerase activity in vivo. In contrast, an RNA mutant with compensatory mutations that restored base pairings in the P6.1 helix restored telomerase activity. In an in vitro reconstitution system stable base pairing of the P6.1 stem was required for the RNA-protein interaction between the CR4-CR5 domain and the telomerase reverse transcriptase (TERT) protein. Interestingly, two RNA mutations, one that extends the P6.1 stem and one that alters the conserved nucleotides of the L6.1 loop, allowed RNA-protein binding but significantly impaired telomerase activity. These data establish the presence of the P6.1 stem-loop and its importance for the assembly and enzymatic activity of the mammalian telomerase complex.


Assuntos
Conformação de Ácido Nucleico , RNA/química , RNA/metabolismo , Telomerase/química , Telomerase/metabolismo , Animais , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a DNA , Fibroblastos , Mamíferos/genética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Filogenia , Ligação Proteica , RNA/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Telomerase/genética , Transfecção
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