Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.939
Filtrar
1.
Cell Physiol Biochem ; 54(2): 195-210, 2020 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-32083406

RESUMO

BACKGROUND/AIMS: Idiopathic pulmonary fibrosis (IPF) is a specific form of progressive and chronic interstitial lung disease of unknown cause. IPF is characterized by excessive deposition of extracellular matrix (ECM) and destructive pathological remodeling due to epithelial-to-mesenchymal transition (EMT). Eventually, lung interstitium thickens and stiffens and breathing becomes difficult. It has been well established that the transforming growth factor-ß1 (TGF-ß1)/Smad signaling pathway plays a critical role in the pathogenesis of pulmonary fibrosis. TGF-ß1-mediated activation of mitogen activated protein kinase (MAPK) family affects Smad signaling. p90RSK is a serine/threonine kinase and is activated by the extracellular signal-regulated kinase (ERK) signaling pathway. However, the roles played by p90RSK in TGF-ß1 signaling and the pathogenesis of pulmonary fibrosis remain unknown. METHODS: We investigated whether p90RSK regulates the pathogenesis of pulmonary fibrosis using in vitro and in vivo systems and Western blotting, real-time quantitative PCR, transcriptional activity assays and immunofluorescence studies. RESULTS: Pharmacological inhibition of p90RSK by FMK or inhibition of p90RSK with adenoviral vector encoding a dominant negative form of p90RSK suppressed TGF-ß1-induced ECM accumulation and EMT in lung epithelial cells and fibroblasts. Interestingly, FMK significantly inhibited TGF-ß1-induced Smad3 nuclear translocation and smad binding element-dependent transcriptional activity, but not Smad3 phosphorylation. Furthermore, in a mouse model of bleomycin-induced lung fibrosis, FMK ameliorated pulmonary fibrosis. CONCLUSION: These findings indicate that p90RSK plays critical roles in pulmonary fibrosis, which suggests it be viewed as a novel therapeutic target for the treatment of lung fibrosis.


Assuntos
Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteína Smad3/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Isoquinolinas/farmacologia , Cetonas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Piridinas/farmacologia , Pirróis/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Proteína Smad3/antagonistas & inibidores , Proteína Smad3/genética , Ativação Transcricional/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
2.
Gene ; 734: 144380, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-31978511

RESUMO

Tolypocladium guangdongense, formerly known as Cordyceps guangdongensis, is a widely cultivated fungus of the Cordyceps s.l. species that has been investigated over the last 12 years. It has the potential to be used in a number of applications in the health and pharmaceutical industries for it has shown its high nutritional and medicinal values according to previous animal studies. qRT-PCR (quantitative reverse transcription polymerase chain reaction) is extensively used to analyze the expression pattern and molecular mechanisms of functional genes under differentially experimental conditions. The expression stability of reference genes used for normalization determines the reliability of qRT-PCR results, indicating the importance of selection and validation of reference genes before gene expression analysis. In the present study, three statistical algorithms, geNorm, NormFinder and BestKeeper, were used for analyzing the expression stability of nineteen candidate reference genes (CRGs) in T. guangdongense. Investigation were carried out under differentially experimental conditions, which included differentially developmential stages (mycelia, primordia, young and mature fruiting bodies), different carbon sources, cold and heat stresses. The results showed that histone H4 and tubulin beta chain 2 (ß-tub2) were the most and least stable genes, respectively, across all the experimental samples. Moreover, analysis of individual data sets exhibited different stability and expression profiles of reference genes. The vacuolar protein sorting gene VPS was the most stable gene expressed under the differentially developmental stages and temperature stresses, whereas H4 was the most stably expressed gene under different carbon sources. Therefore, it can be proposed that VPS and H4 are the preferred reference genes for normalization of gene expression under different experimental conditions. The results of our present study will enable more accurate evaluation of gene expression in T. guangdongense using the optimal reference gene for qRT-PCR analysis.


Assuntos
Cordyceps/genética , Genes Fúngicos , Algoritmos , Cordyceps/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Histonas/genética , Padrões de Referência , Estresse Fisiológico/genética , Temperatura Ambiente , Tubulina (Proteína)/genética
4.
Int J Cancer ; 146(4): 1099-1113, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31187490

RESUMO

Acquired chemoresistance is a critical issue for advanced bladder cancer patients during long-term treatment. Recent studies reveal that a fraction of tumor cells with enhanced tumor-initiating potential, or cancer stem-like cells (CSCs), may particularly contribute to acquired chemoresistance and recurrence. Thus, CSC characterization will be the first step towards understanding the mechanisms underlying advanced disease. Here we generated long-term patient-derived cancer cells (PDCs) from bladder cancer patient specimens in spheroid culture, which is favorable for CSC enrichment. Pathological features of bladder cancer PDCs and PDC-dependent patient-derived xenografts (PDXs) were basically similar to those of their corresponding patients' specimens. Notably, CSC marker aldehyde dehydrogenase 1A1 (ALDH1A1), a critical enzyme that synthesizes retinoic acid (RA), was abundantly expressed in PDCs. ALDH1A1 inhibitors and shRNAs repressed both PDC proliferation and spheroid formation, whereas all-trans RA could rescue ALDH1A1 shRNA-suppressed spheroid formation. ALDH inhibitor also reduced the in vivo growth of PDC-derived xenografts. ALDH1A1 knockdown study showed that tubulin beta III (TUBB3) was one of the downregulated genes in PDCs. We identified functional RA response elements in TUBB3 promoter, whose transcriptional activities were substantially activated by RA. Clinical survival database reveals that TUBB3 expression may associate with poor prognosis in bladder cancer patients. Moreover, TUBB3 knockdown was sufficient to suppress PDC proliferation and spheroid formation. Taken together, our results indicate that ALDH1A1 and its putative downstream target TUBB3 are overexpressed in bladder cancer, and those molecules could be applied to alternative diagnostic and therapeutic options for advanced disease.


Assuntos
/metabolismo , Retinal Desidrogenase/metabolismo , Tubulina (Proteína)/biossíntese , Neoplasias da Bexiga Urinária/metabolismo , /antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Progressão da Doença , Regulação para Baixo , Células HEK293 , Xenoenxertos , Humanos , Masculino , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Retinal Desidrogenase/antagonistas & inibidores , Retinal Desidrogenase/genética , Receptor alfa de Ácido Retinoico , Transdução de Sinais , Esferoides Celulares , Tretinoína , Tubulina (Proteína)/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia
5.
PLoS One ; 14(12): e0219231, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31841523

RESUMO

The deluge of sequence information in the recent times provide us with an excellent opportunity to compare organisms on a large genomic scale. In this study we have tried to decipher the variation in the gene organization and structuring of a vital bacterial gene called ftsZ which codes for an integral component of the bacterial cell division, the FtsZ protein. FtsZ is homologous to tubulin protein and has been found to be ubiquitous in eubacteria. FtsZ is showing increasing promise as a target for antibacterial drug discovery. Our study of ftsZ protein from 143 different bacterial species spanning a wider range of morphological and physiological type demonstrates that the ftsZ gene of about ninety three percent of the organisms show relatively biased codon usage profile and significant GC deviation from their genomic GC content. Comparative codon usage analysis of ftsZ and a core housekeeping gene rpoB demonstrated that codon usage pattern of ftsZ CDS is shaped by natural selection to a large extent and mimics that of a housekeeping gene. We have also detected a tendency among the different organisms to utilize a core set of codons in structuring the ftsZ coding sequence. We observed that the compositional frequency of the amino acid serine in the FtsZ protein appears to be a indicator of the bacterial lifestyle. Our meticulous analysis of the ftsZ gene linked with the corresponding FtsZ protein show that there is a bias towards the use of specific synonymous codons particularly in the helix and strand regions of the multi-domain FtsZ protein. Overall our findings suggest that in an indispensable and vital protein such as FtsZ, there is an inherent tendency to maintain form for optimized performance in spite of the extrinsic variability in coding features.


Assuntos
Proteínas de Bactérias/genética , Códon/genética , Proteínas do Citoesqueleto/genética , Sequência de Aminoácidos , Bactérias/genética , Proteínas de Bactérias/metabolismo , Composição de Bases , Simulação por Computador , Proteínas do Citoesqueleto/metabolismo , Genes Bacterianos/genética , Genômica/métodos , Fases de Leitura Aberta , Seleção Genética , Tubulina (Proteína)/genética
6.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 31(4): 404-409, 2019 Sep 23.
Artigo em Chinês | MEDLINE | ID: mdl-31612676

RESUMO

OBJECTIVE: To investigate the expression of some genes in Pomacea canaliculata infected with Angiostrongylus cantonensis, so as to provide insight into the preliminary understanding of the interactions between Angiostrongylus cantonensis and its intermediate host Pomacea canaliculata. METHODS: P. canaliculata was fed with rat faces containing the first-stage larvae of A. cantonensis. Three to five P. canaliculata was sampled 1, 10 days and 20 days after feeding, and the hemolymph, hepatopancreas, kidney, intestinal tract, head-foot and gill tissues were collected, while uninfected P. canaliculata served as controls. Total RNA was extracted from various tissues of P. canaliculata at different time points post-infection, and transcribed reversely into cDNA. Based on previous transcriptome sequencing results, 10 genes associated with immune defense, signal transduction, cell growth and metabolism, stress response were selected, and the gene expression was determined in the hemolymph tissues of P. canaliculata 1, 10 days and 20 days post-infection with A. cantonensis using real-time fluorescent quantitative PCR assay, and the α-tubulin gene expression was quantified in the hepatopancreas, kidney, head-foot, intestinal tract and gill tissues of P. canaliculata infected with A. cantonensis. RESULTS: Higher CELA1 gene expression was detected in the infection group than in the control group 1 (t = 12.32, P < 0.05), 10 days (t = 23.51, P < 0.05) and 20 days post-infection (t = 34.92, P < 0.05), and the CELA1 expression increased with the time of infection. The GST gene expression was (7.26 ± 1.80) times higher in the infection group than in the control group 1 day post-infection, and was significantly lower in the infection group than in the control group 10 days (t = 23.89, P < 0.05) and 20 days post-infection (t = 19.83, P < 0.05). Higher ferritin gene expression was found in the infection group than in the control group 10 days post-infection (t = 32.76, P < 0.05), and higher CRT gene expression was seen in the infection group than in the control group 1 (t = 7.23, P < 0.05), 10 days (t = 5.78, P < 0.05) and 20 days post-infection (t = 6.32, P < 0.05). In addition, the greatest α-tubulin gene expression was observed in the the hepatopancreatic tissues of P. canaliculata (F = 17.58, P < 0.05), and the α-tubulin gene expression altered in various tissues of P. canaliculata post-infection with A. cantonensis, with the most remarkable reduction of α - tubulin gene expression seen in the hepatopancreatic tissues (P < 0.05). CONCLUSIONS: Following A. cantonensis infection in P. canaliculata, the expression of multiple genes is altered, and the expression of α-tubulin gene is inhibited in multiple tissues. The findings provide a basis for the further elucidation of the interactions between P. canaliculata and A. cantonensis.


Assuntos
Angiostrongylus cantonensis , Gastrópodes , Interações Hospedeiro-Parasita , Infecções por Strongylida , Tubulina (Proteína) , Angiostrongylus cantonensis/fisiologia , Animais , Gastrópodes/genética , Gastrópodes/parasitologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Interações Hospedeiro-Parasita/genética , Larva , Ratos , Tubulina (Proteína)/genética
7.
Zhongguo Dang Dai Er Ke Za Zhi ; 21(10): 972-976, 2019 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-31642429

RESUMO

OBJECTIVE: To study the types and characteristics of TUBB1 mutation in children with congenital hypothyroidism (CH) and thyroid dysgenesis (TD) in Shandong, China. METHODS: Mutations of the whole coding region of the TUBB1 gene were analyzed for 289 children with CH and TD in Shandong. Whole-genome DNA was extracted from peripheral blood leukocytes. PCR multiplication was performed for the whole coding region of the TUBB1 gene. Sanger sequencing was performed for the PCR products, and a biological information analysis was performed. RESULTS: Among the 289 children with CH and TD, 4 (1.4%) were found to have a c.952C>T(p.R318W) heterozygous mutation in the TUBB1 gene, resulting in the change of tryptophan into arginine at codon 318 of TUBB1 protein. This mutation was evaluated as "potentially pathogenic" based on the classification criteria and guidelines for genetic variation by American College of Medical Genetics and Genomics. CONCLUSIONS: A novel mutation is detected in the exon of the TUBB1 gene in children with CH and TD in Shandong, suggesting that the TUBB1 gene may be a candidate pathogenic gene for CH children with TD.


Assuntos
Hipotireoidismo Congênito , Disgenesia da Tireoide , Tubulina (Proteína)/genética , Criança , China , Hipotireoidismo Congênito/genética , Análise Mutacional de DNA , Humanos , Mutação , Disgenesia da Tireoide/genética
8.
Phytopathology ; 109(12): 2096-2106, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31559903

RESUMO

Ethaboxam is a ß-tubulin inhibitor registered for the control of oomycete pathogens. The current study was established to determine the ethaboxam sensitivity of the plant pathogen Phytophthora sojae and investigate the potential for the emergence of fungicide resistance. The effective concentration for 50% inhibition (EC50) of 112 Phytophthora sojae isolates exhibited a unimodal distribution with a mean EC50 for ethaboxam of 0.033 µg/ml. Establishing this baseline sensitivity provided critical data for monitoring changes in ethaboxam-sensitivity in field populations. The potential for fungicide resistance was investigated using adaptation on ethaboxam-amended V8 agar, which resulted in the isolation of 20 resistant mutants. An assessment of the biological characteristics of the mutants including mycelial growth, sporulation, germination rate and pathogenicity indicated that the resistance risk in Phytophthora sojae was low to medium with no cross-resistance between ethaboxam and cymoxanil, metalaxyl, flumorph, and oxathiapiprolin being detected. However, positive cross-resistance was found between ethaboxam and zoxamide for Q8L and I258V but negative cross-resistance for C165Y. Further investigation revealed that the ethaboxam-resistant mutants had point mutations at amino acids Q8L, C165Y, or I258V of their ß-tubulin protein sequences. CRISPR/Cas9-mediated transformation experiments confirmed that the Q8L, C165Y, or I258V mutations could confer ethaboxam resistance in Phytophthora sojae and that the C165Y mutation induces high levels of resistance. Taken together, the results of the study provide essential data for monitoring the emergence of resistance and resistance management strategies for ethaboxam, as well as for improving the design of novel ß-tubulin inhibitors for future development.


Assuntos
Resistência a Medicamentos , Phytophthora , Mutação Puntual , Tiazóis , Tiofenos , Tubulina (Proteína) , Resistência a Medicamentos/genética , Phytophthora/efeitos dos fármacos , Phytophthora/genética , Doenças das Plantas/parasitologia , Soja/parasitologia , Tiazóis/farmacologia , Tiofenos/farmacologia , Tubulina (Proteína)/genética
9.
Exp Parasitol ; 206: 107754, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31473211

RESUMO

Dermatophagoides farinae is an important source of indoor allergens that shows strong tolerance to external temperatures. However, the regularity and mechanism of tolerance are still unclear. Based on our previous RNA-seq and annotation of D. farinae under temperature stress, it is planned to identify differentially expressed genes (DEGs) involved in the temperature stress response by quantitative real-time PCR (qRT-PCR). However, the lack of reference genes directly limited the detection and confirmation of DEGs. Accordingly, in this study, we have selected six candidates as reference genes in D. farinae: 60S RP L11, 60S RP L21, α tubulin, GAPDH, Der f Mal f 6, and calreticulin, and evaluated their expression stabilities as affected by heat and cold stresses, using geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder methods. Then the expression level of 15 DEGs were detected and verified. geNorm analysis showed that α tubulin and calreticulin were the most stable reference genes under heat stress and cold stress of D. farinae. Similar evaluation results were obtained by NormFinder and BestKeeper, in which 60S RP L21 and α tubulin were the most stable reference genes. By comparative ΔCt method and a comprehensive evaluation of RefFinder, α tubulin was identified as the most ideal reference gene of D. farinae under heat and cold stresses. Furthermore, qRT-PCR detection results of 15 DEGs were almost identical to the RNA-seq results, indicating that α tubulin is stable as a reference gene. This study provided technical support for DEGs expression studies in D. farinae using qRT-PCR.


Assuntos
Calreticulina/genética , Dermatophagoides farinae/genética , Temperatura Ambiente , Tubulina (Proteína)/genética , Animais , Antígenos de Dermatophagoides/genética , Primers do DNA/química , Dermatophagoides farinae/fisiologia , Feminino , Amplificação de Genes , Perfilação da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Anotação de Sequência Molecular , RNA/química , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Ribossômicas/genética , Análise de Sequência de RNA , Transcriptoma/genética , Temperatura de Transição , Sequenciamento Completo do Exoma
10.
BMC Oral Health ; 19(1): 207, 2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31484557

RESUMO

BACKGROUND: The oral flagellated protozoan Trichomonas tenax has been associated with patients with periodontal disease. However, no recent studies have been conducted on the prevalence of T. tenax in Chile. The aim of this study was to determine the presence of T. tenax in patients with periodontal disease, admitted to the Dental Clinic of the University of Antofagasta, Chile, through Polymerase Chain Reaction (PCR) amplification of the beta-tubulin gene. METHODS: An observational, cross-sectional study was conducted on 50 patients diagnosed with periodontal disease, 20 of them with gingivitis and 30 with periodontitis. T. tenax was identified by PCR amplification of the beta-tubulin gene. Associations between the protozoan and periodontal disease or the presence of risk factors to establish T. tenax infection were determined using the chi-square test and binary logistic regression analysis. RESULTS: T. tenax was present in 28 out of 50 (56%) of patients with periodontal disease, and was more prevalent when associated with periodontitis (21 out of 30; 70%) than dental plaque-induced gingivitis (7 out of 20; 35%). Non-statistically-significant associations were observed between the presence of T. tenax and age, gender, smoking habit or diabetes. Statistically significant associations were observed between the presence of T. tenax and periodontal disease, and between T. tenax and the Periodontal Screening and Recording (PSR) index. CONCLUSION: T. tenax showed a high presence in patients with progressive states of periodontal diseases. Consequently, T. tenax detection is strongly recommended in patients with periodontal disease diagnosis and with a PSR index greater than 3.


Assuntos
Gengivite/microbiologia , Doenças Periodontais/microbiologia , Tricomoníase/diagnóstico , Trichomonas/isolamento & purificação , Tubulina (Proteína)/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Chile/epidemiologia , Estudos Transversais , Clínicas Odontológicas , Feminino , Amplificação de Genes , Gengivite/diagnóstico , Gengivite/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Periodontais/diagnóstico , Doenças Periodontais/epidemiologia , Reação em Cadeia da Polimerase , Prevalência , Universidades
11.
PLoS Negl Trop Dis ; 13(8): e0007471, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31369562

RESUMO

BACKGROUND: Preventive chemotherapy (PC) with benzimidazole drugs is the backbone of soil-transmitted helminth (STH) control programs. Over the past decade, drug coverage has increased and with it, the possibility of developing anthelmintic resistance. It is therefore of utmost importance to monitor drug efficacy. Currently, a variety of novel diagnostic methods are available, but it remains unclear whether they can be used to monitor drug efficacy. In this study, we compared the efficacy of albendazole (ALB) measured by different diagnostic methods in a head-to-head comparison to the recommended single Kato-Katz. METHODS: An ALB efficacy trial was performed in 3 different STH-endemic countries (Ethiopia, Lao PDR and Tanzania), each with a different PC-history. During these trials, stool samples were evaluated with Kato-Katz (single and duplicate), Mini-FLOTAC, FECPAKG2, and qPCR. The reduction rate in mean eggs per gram of stool (ERR) and mean genome equivalents / ml of DNA extract (GERR) were calculated to estimate drug efficacy. PRINCIPAL FINDINGS AND CONCLUSIONS: The results of the efficacy trials showed that none of the evaluated diagnostic methods could provide reduction rates that were equivalent to a single Kato-Katz for all STH. However, despite differences in clinical sensitivity and egg counts, they agreed in classifying efficacy according to World Health Organization (WHO) guidelines. This demonstrates that diagnostic methods for assessing drug efficacy should be validated with their intended-use in mind and that other factors like user-friendliness and costs will likely be important factors in driving the choice of diagnostics. In addition, ALB efficacy against STH infections was lower in sites with a longer history of PC. Yet, further research is needed to identify factors that contribute to this finding and to verify whether reduced efficacy can be associated with mutations in the ß-tubulin gene that have previously been linked to anthelmintic resistance. TRIAL REGISTRATION: ClinicalTrials.gov NCT03465488.


Assuntos
Albendazol/uso terapêutico , Anti-Helmínticos/uso terapêutico , Helmintíase/diagnóstico , Helmintíase/tratamento farmacológico , Solo/parasitologia , Administração Oral , Albendazol/administração & dosagem , Animais , Brasil , Criança , Testes Diagnósticos de Rotina/métodos , Etiópia , Fezes/parasitologia , Feminino , Helmintos/genética , Humanos , Laos , Masculino , Contagem de Ovos de Parasitas/métodos , Sensibilidade e Especificidade , Tanzânia , Tubulina (Proteína)/genética , Organização Mundial da Saúde
12.
PLoS Genet ; 15(8): e1008243, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31386652

RESUMO

Tubulin genes encode a series of homologous proteins used to construct microtubules which are essential for multiple cellular processes. Neural development is particularly reliant on functional microtubule structures. Tubulin genes comprise a large family of genes with very high sequence similarity between multiple family members. Human genetics has demonstrated that a large spectrum of cortical malformations are associated with de novo heterozygous mutations in tubulin genes. However, the absolute requirement for many of these genes in development and disease has not been previously tested in genetic loss of function models. Here we directly test the requirement for Tuba1a, Tubb2a and Tubb2b in the mouse by deleting each gene individually using CRISPR-Cas9 genome editing. We show that loss of Tubb2a or Tubb2b does not impair survival but does lead to relatively mild cortical malformation phenotypes. In contrast, loss of Tuba1a is perinatal lethal and leads to significant forebrain dysmorphology. We also present a novel mouse ENU allele of Tuba1a with phenotypes similar to the null allele. This demonstrates the requirements for each of the tubulin genes and levels of functional redundancy are quite different throughout the gene family. The ability of the mouse to survive in the absence of some tubulin genes known to cause disease in humans suggests future intervention strategies for these devastating tubulinopathy diseases.


Assuntos
Malformações do Desenvolvimento Cortical/genética , Neurogênese/genética , Córtex Sensório-Motor/embriologia , Tubulina (Proteína)/genética , Alelos , Animais , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Embrião de Mamíferos , Etilnitrosoureia/toxicidade , Feminino , Deleção de Genes , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Malformações do Desenvolvimento Cortical/mortalidade , Malformações do Desenvolvimento Cortical/patologia , Camundongos , Camundongos Transgênicos , Microtúbulos/genética , Modelos Animais , Mutagênese/efeitos dos fármacos , Córtex Sensório-Motor/anormalidades , Especificidade da Espécie , Tubulina (Proteína)/metabolismo
13.
Int J Mol Sci ; 20(16)2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31412591

RESUMO

Recent studies have reported that TUBB3 overexpression is involved in docetaxel (DTX) resistance in prostate cancer (PCa). The aim of this study was to clarify the role of TUBB3 in DTX and cabazitaxel (CBZ) resistance, and cross-resistance between DTX and CBZ in PCa. We analyzed the effect of TUBB3 knockdown on DTX and CBZ resistance and examined the interaction between TUBB3 and PTEN. We also investigated the role of phosphoinositide 3-kinases (PI3K) inhibitor (LY294002) in DTX and CBZ resistance. TUBB3 expression was upregulated in DTX-resistant and CBZ-resistant cells. TUBB3 knockdown re-sensitized DTX-resistant cells to DTX and CBZ-resistant cells to CBZ. Additionally, TUBB3 knockdown re-sensitized DTX-resistant cell lines to CBZ, indicating that TUBB3 mediates cross-resistance between DTX and CBZ. Knockdown of TUBB3 enhanced PTEN expression, and PTEN knockout enhanced TUBB3 expression. LY294002 suppressed TUBB3 expression in DTX-resistant and CBZ-resistant cell lines. LY294002 re-sensitized DTX-resistant cell lines to DTX and CBZ-resistant cell lines to CBZ. These results suggest that TUBB3 is involved in DTX resistance and CBZ resistance. A combination of LY294002/DTX and that of LY294002/CBZ could be potential strategies for PCa treatment.


Assuntos
Antineoplásicos/farmacologia , Docetaxel/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias da Próstata/genética , Taxoides/farmacologia , Tubulina (Proteína)/genética , Linhagem Celular Tumoral , Cromonas/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Masculino , Morfolinas/farmacologia , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Ligação Proteica , Tubulina (Proteína)/metabolismo
15.
EMBO J ; 38(15): e100984, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31368598

RESUMO

Microtubules are cytoskeletal filaments essential for many cellular processes, including establishment and maintenance of polarity, intracellular transport, division and migration. In most metazoan cells, the number and length of microtubules are highly variable, while they can be precisely defined in some protozoan organisms. However, in either case the significance of these two key parameters for cells is not known. Here, we quantitatively studied the impact of modulating microtubule number and length in Plasmodium, the protozoan parasite causing malaria. Using a gene deletion and replacement strategy targeting one out of two α-tubulin genes, we show that chromosome segregation proceeds in the oocysts even in the absence of microtubules. However, fewer and shorter microtubules severely impaired the formation, motility and infectivity of Plasmodium sporozoites, the forms transmitted by the mosquito, which usually contain 16 microtubules. We found that α-tubulin expression levels directly determined the number of microtubules, suggesting a high nucleation barrier as supported by a mathematical model. Infectious sporozoites were only formed in parasite lines featuring at least 10 microtubules, while parasites with 9 or fewer microtubules failed to transmit.


Assuntos
Malária/parasitologia , Plasmodium/patogenicidade , Tubulina (Proteína)/genética , Animais , Deleção de Genes , Camundongos , Modelos Teóricos , Plasmodium/genética , Plasmodium/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Esporozoítos/genética , Esporozoítos/crescimento & desenvolvimento , Esporozoítos/patogenicidade , Tubulina (Proteína)/metabolismo
16.
Plant Dis ; 103(10): 2536-2540, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31424998

RESUMO

Fusarium head blight, also called scab, is caused by Fusarium graminearum and is one of the most important destructive diseases of wheat. The frequency of carbendazim resistance in 1,132 isolates of F. graminearum recovered from fields in different regions of Henan Province in 2016, 2017, and 2018 was determined. A total of 31 F. graminearum isolates resistant to carbendazim were detected, including 30 moderately resistant isolates and one highly resistant isolate. The frequency of resistance of F. graminearum isolates to carbendazim was 2.7%. The range of effective concentration (EC50) values of 1,101 sensitive isolates and 30 moderately resistant isolates was 0.08 to 0.98 µg ml-1 and 2.73 to 13.28 µg ml-1, respectively. The mean ± SD EC50 value was 0.55 ± 0.13 µg ml-1 and 5.61 ± 2.58 µg ml-1, respectively. The EC50 value of the highly resistant isolate was 21.12 µg ml-1. Point mutation types of the carbendazim-resistant isolates were characterized by cloning the ß2-tubulin gene of 31 resistant isolates. Three point mutation types at amino acids F167Y, E198Q, and E198L in the ß2-tubulin gene of resistant isolates were identified. Among 31 resistant isolates, the frequency of point mutation types in F167Y, E198Q, and E198L of the ß2-tubulin gene was 71.0, 25.8, and 3.2%, respectively. The data indicate that F. graminearum has developed resistance to carbendazim in Henan Province, and single point mutations at amino acid F167Y were the predominant type of mutation detected.


Assuntos
Benzimidazóis , Carbamatos , Farmacorresistência Fúngica , Fusarium , Triticum , Benzimidazóis/farmacologia , Carbamatos/farmacologia , Farmacorresistência Fúngica/genética , Fungicidas Industriais , Fusarium/efeitos dos fármacos , Fusarium/genética , Genes de Plantas/genética , Mutação Puntual , Triticum/microbiologia , Tubulina (Proteína)/genética
17.
Artigo em Inglês | MEDLINE | ID: mdl-31425900

RESUMO

Anthelmintic resistant gastrointestinal helminths have become a major cause of poor health in sheep and goats. Sensitive and specific molecular markers are needed to monitor the genotypic frequency of resistance in field parasite populations. Gastrointestinal nematode resistance to benzimidazole is caused by a mutation in one of three positions within the isotype 1 ß-tubulin gene. In the absence of markers for resistance to the other broad spectrum anthelmintic classes, these provide a relevant study example. Determination of the prevalence of these single nucleotide polymorphisms in field nematode populations can be impractical using conventional molecular methods to examine individual parasites; which can be laborious and lack sensitivity in determining low levels of resistance in parasite populations. Here, we report the development of a novel method based on an Illumina MiSeq deep amplicon sequencing platform to sequence the isotype 1 ß-tubulin locus of the small ruminant gastrointestinal nematode, Teladorsagia circumcincta, and determine the frequency of the benzimidazole resistance mutations. We validated the method by assessing sequence representation bias, comparing the results of Illumina MiSeq and pyrosequencing, and applying the method to populations containing known proportions of resistant and susceptible larvae. We applied the method to field samples collected from ewes and lambs on over a period of one year on three farms, each highlighting different aspects of sheep management and approaches to parasite control. The results show opportunities to build hypotheses with reference to selection pressures leading to differences in resistance allele frequencies between sampling dates, farms and ewes or lambs, and to consider the impact of their genetic fixation or otherwise. This study provides proof of concept of a practical, accurate, sensitive and scalable method to determine frequency of anthelmintic resistance mutations in gastrointestinal nematodes in field studies and as a management tool for livestock farmers.


Assuntos
Anti-Helmínticos/farmacologia , Benzimidazóis/farmacologia , Resistência a Medicamentos , Trato Gastrointestinal/parasitologia , Análise de Sequência de DNA/métodos , Doenças dos Ovinos/parasitologia , Infecções por Strongylida/veterinária , Estrongilídios/efeitos dos fármacos , Estrongilídios/genética , Animais , Frequência do Gene/efeitos dos fármacos , Proteínas de Helminto/genética , Filogenia , Ovinos , Estrongilídios/classificação , Estrongilídios/isolamento & purificação , Infecções por Strongylida/parasitologia , Tubulina (Proteína)/genética
18.
Colloids Surf B Biointerfaces ; 182: 110324, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31288132

RESUMO

Graphene exhibits excellent mechanical strength, electrical conductivity and good biocompatibility, which make it a suitable candidate as a neural interfacing material in regenerative medicine and tissue engineering. Graphene is reported to promote both of neural stem cells (NSCs) proliferation and differentiation. However, the transcriptomes of 2D graphene-regulated NSC differentiation have not yet been investigated. To identify candidate genes, on which graphene may affect, we used next-generation RNA sequencing to analyze the transcriptome of NSCs differentiated for 21 days on a graphene substrate. These NSCs displayed highly enriched and differentially expressed genes compared with traditional cell culture in vitro. Of these, we identified motor protein genes that might regulate NSC differentiation, including cytoplasmic dynein and axonemal dynein genes, Ccdc108, Dnah5, and Dnah11. Furthermore, we analyzed the cell signaling pathway genes that might regulate NSC differentiation, and we constructed a protein-protein interaction network for the genes that are differentially expressed in NSCs on graphene compared to commercial tissue culture polystyrene substrates. We have identified genes potentially regulating the differentiation and migration of NSCs on graphene substrates, and our findings provide mechanistic evidence for the biological activities of graphene, especially in view of graphene-stem cell interactions.


Assuntos
Dineínas do Axonema/genética , Regulação da Expressão Gênica no Desenvolvimento , Grafite/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Transcriptoma , Animais , Dineínas do Axonema/metabolismo , Diferenciação Celular , Biologia Computacional/métodos , Embrião de Mamíferos , Perfilação da Expressão Gênica , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Grafite/química , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Poliestirenos/química , Poliestirenos/farmacologia , Cultura Primária de Células , Mapeamento de Interação de Proteínas , Transdução de Sinais , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
19.
Cells ; 8(7)2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269740

RESUMO

Mutations in tubulin genes are responsible for a large spectrum of brain malformations secondary to abnormal neuronal migration, organization, differentiation and axon guidance and maintenance. Motor impairment, intellectual disability and epilepsy are the main clinical symptoms. In the present study 15 patients from a personal cohort and 75 from 21 published studies carrying mutations in TUBA1A, TUBB2B and TUBB3 tubulin genes were evaluated with the aim to define a clinical and electrophysiological associated pattern. Epilepsy shows a wide range of severity without a specific pattern. Mutations in TUBA1A (60%) and TUBB2B (74%) and TUBB3 (25%) genes are associated with epilepsy. The accurate analysis of the Electroencephalogram (EEG) pattern in wakefulness and sleep in our series allows us to detect significant abnormalities of the background activity in 100% of patients. The involvement of white matter and of the inter-hemispheric connection structures typically observed in tubulinopathies is evidenced by the high percentage of asynchronisms in the organization of sleep activity recorded. In addition to asymmetries of the background activity, excess of slowing, low amplitude and Magnetic Resonance (MR) imaging confirm the presence of extensive brain malformations involving subcortical and midline structures. In conclusion, epilepsy in tubulinopathies when present has a favorable evolution over time suggesting a not particularly aggressive therapeutic approach.


Assuntos
Encéfalo/anormalidades , Epilepsia/genética , Malformações do Desenvolvimento Cortical/genética , Tubulina (Proteína)/genética , Adolescente , Adulto , Encéfalo/diagnóstico por imagem , Encéfalo/crescimento & desenvolvimento , Criança , Pré-Escolar , Eletroencefalografia , Epilepsia/diagnóstico , Epilepsia/epidemiologia , Feminino , Humanos , Imagem por Ressonância Magnética , Masculino , Malformações do Desenvolvimento Cortical/complicações , Pessoa de Meia-Idade , Mutação , Índice de Gravidade de Doença , Fatores de Tempo , Adulto Jovem
20.
Int J Mol Sci ; 20(14)2019 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-31315202

RESUMO

Platelets are anuclear cells with a short lifespan that play an essential role in many pathophysiological processes, including haemostasis, inflammation, infection, vascular integrity, and metastasis. Billions of platelets are produced daily from megakaryocytes (platelet precursors). Despite this high production, the number of circulating platelets is stable and, under resting conditions, they maintain their typical discoid shape thanks to cytoskeleton proteins. The activation of platelets is associated with dynamic and rapid changes in the cytoskeleton. Two cytoskeletal polymer systems exist in megakaryocytes and platelets: actin filaments and microtubules, based on actin, and α- and ß-tubulin heterodimers, respectively. Herein, we will focus on platelet-specific tubulins and their alterations and role of the microtubules skeleton in platelet formation (thrombopoiesis). During this process, microtubules mediate elongation of the megakaryocyte extensions (proplatelet) and granule trafficking from megakaryocytes to nascent platelets. In platelets, microtubules form a subcortical ring, the so-called marginal band, which confers the typical platelet discoid shape and is also responsible for changes in platelet morphology upon activation. Molecular alterations in the gene encoding ß1 tubulin and microtubules post-translational modifications may result in quantitative or qualitative changes in tubulin, leading to altered cytoskeleton reorganization that may induce changes in the platelet number (thrombocytopenia), morphology or function. Consequently, ß1-tubulin modifications may participate in pathological and physiological processes, such as development.


Assuntos
Plaquetas/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Variação Genética , Humanos , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/química , Tubulina (Proteína)/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA