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1.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(1): 17-20, 2020 Jan 10.
Artigo em Chinês | MEDLINE | ID: mdl-31922588

RESUMO

OBJECTIVE: To explore the genetic basis of a pedigree affected with hereditary spherocytosis. METHODS: Peripheral blood samples were collected from 17 members of the pedigree. Genomic DNA of the proband was subjected to next generation sequencing. Candidate variant was validated by co-segregation analysis. pCAS2(c.5798+1G) and pCAS2(c.5798+1A) plasmids were constructed by homologous recombination and transfected into 293T cells. Reverse transcription PCR, TA cloning and Sanger sequencing were used to analyze the effect of candidate variant on splicing. Meanwhile, peripheral blood RNAs were extracted to analyze the effect of candidate variant on splicing in vivo. RESULTS: The proband was found to carry a c.5798+1G>A variant of the SPTB gene. The variant has co-segregated with the phenotype in the pedigree. In vitro and in vivo splicing experiments confirmed that the mutation has significantly affected the splicing, resulting in shift of reading frame and produced a premature termination codon. CONCLUSION: The novel c.5798+1G>A variant of the SPTB gene probably underlies the pathogenesis of hereditary spherocytosis in this pedigree.


Assuntos
Espectrina , Esferocitose Hereditária , Códon sem Sentido/genética , Variação Genética , Células HEK293 , Humanos , Mutação/genética , Linhagem , Plasmídeos , Processamento de RNA , Espectrina/genética , Esferocitose Hereditária/genética , Transfecção
2.
Chemosphere ; 238: 124689, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31524624

RESUMO

Pharmaceutical effluents released from industries are accountable to deteriorate the aquatic and soil environment through indirect toxic effects. Microbes are adequately been used to biodegrade pharmaceutical industry wastewater and present study was envisaged to determine biodegradation of pharmaceutical effluent by Micrococcus yunnanensis. The strain showed 42.82% COD (Chemical oxygen demand) reduction before optimization. After applying Taguchi's L8 array as an optimization technique, the biodegradation rate was enhanced by 82.95% at optimum conditions (dextrose- 0.15%, peptone 0.1%, inoculum size 4% (wv-1), rpm 200, pH 8 at 25 °C) within 6 h. The confirmation of pharmaceuticals degradation was done by 1H NMR (Nuclear magnetic resonance) studies followed by elucidation of transformation pathways of probable drugs in the effluent through Q-Tof-MS (Quadrupole Time of Flight- Mass Spectrometry). The cytotoxicity evaluation of treated and untreated wastewater was analyzed on Human Embryonic Kidney (HEK 293) cells using Alamar Blue assay, which showed significant variance.


Assuntos
Biodegradação Ambiental , Resíduos Industriais/análise , Micrococcus/metabolismo , Preparações Farmacêuticas/análise , Águas Residuárias/química , Poluentes Químicos da Água/análise , Análise da Demanda Biológica de Oxigênio , Linhagem Celular , Indústria Farmacêutica , Células HEK293 , Humanos
3.
J Clin Pathol ; 73(1): 7-13, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31422373

RESUMO

AIMS: Hereditary protein S (PS) deficiency is one of the natural anticoagulant deficiencies causing thrombophilia. We herein described a young male with recurrent deep venous thrombosis, who was diagnosed as type I PS deficiency with compound heterozygous mutations of PROS1 gene. We aimed to analyse the relationship between the genotype and phenotype detection and investigate the pathological mechanisms of PROS1 mutations causing PS deficiency. METHODS: Genetic analysis of PROS1 gene was carried out by direct sequencing. Thrombin generation potential and the inhibition function of thrombin generation by plasma PS were detected by thrombin generation test (TGT). The mRNA transcription level of mutant PS in vitro was measured by real-time PCR, while the protein level was evaluated by western blot and ELISA. Cellular distribution of the protein was further analysed by immunofluorescence. RESULTS: Compound heterozygous mutations (PROS1 c.1551_1552delinsG, p.Thr518Argfs*39 and PROS1 c.1681C>T, p.Arg561Trp) were identified in the propositus, and the former one was a novel small indel mutation. TGT results showed impaired inhibition of thrombin generation with the addition of activated protein C in his parents with certain heterozygous mutations. In vitro expression study, p.Thr518Argfs*39 mutant produced truncated protein retained in the cytoplasm, while p.Arg561Trp mutant partially affected the secretion of PS. Both mutations are located in C-terminal sex hormone-binding globulin (SHBG)-like domain of PS. CONCLUSIONS: Compound heterozygous mutations identified in the study have strong detrimental effect, causing severe type I PS deficiency in the propositus. SHBG-like domain of PS might play an important role in PS secretion system.


Assuntos
Coagulação Sanguínea/genética , Proteínas Sanguíneas/genética , Heterozigoto , Mutação , Deficiência de Proteína S/genética , Trombose Venosa/genética , Adulto , Proteínas Sanguíneas/metabolismo , Feminino , Predisposição Genética para Doença , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Deficiência de Proteína S/sangue , Deficiência de Proteína S/diagnóstico , Recidiva , Via Secretória , Índice de Gravidade de Doença , Trombina/metabolismo , Trombose Venosa/sangue , Trombose Venosa/diagnóstico
4.
Biosci Biotechnol Biochem ; 84(1): 126-133, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31538545

RESUMO

Insects must intake sterol compounds because of their inability to synthesize cholesterol de novo. In phytophagous insects, enzymatic conversion of phytosterols to cholesterol involving 24-dehydrocholesterol reductase (DHCR24) exerts to acquire cholesterol. Here, we reported the presence of two DHCR24 homologs in the silkworm Bombyx mori, BmDHCR24-1 and -2, with several transcript variants. Consistent with the data of spatial expression analyses by RT-PCR, predominant enzymatic activity of DHCR24 was observed in B. mori larval midgut whereas weak activity was observed in the other tissues examined. In addition, BmDHCR24-1 expression in HEK293 cells showed an enzymatic activity, but BmDHCR24-2 did not, although both BmDHCR24s were localized in the endoplasmic reticulum, where the mammalian DHCR24s are located to exert their enzymatic activities. The present data indicated that BmDHCR24-1 but not BmDHCR24-2 contributes to conversion of phytosterols to cholesterol mainly in the midgut of the phytophagous lepidopteran larvae.


Assuntos
Bombyx/enzimologia , Colesterol/biossíntese , Proteínas de Insetos/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Animais , Células HEK293 , Humanos , Proteínas de Insetos/genética , Larva/enzimologia , Túbulos de Malpighi/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Fitosteróis/metabolismo , Plantas/química , Plasmídeos/genética , Homologia de Sequência do Ácido Nucleico , Distribuição Tecidual , Transcrição Genética , Transfecção
5.
J Enzyme Inhib Med Chem ; 35(1): 96-108, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31690133

RESUMO

A series of analogues of Amb639752, a novel diacylglycerol kinase (DGK) inhibitor recently discovered by us via virtual screening, have been tested. The compounds were evaluated as DGK inhibitors on α, θ, and ζ isoforms, and as antagonists on serotonin receptors. From these assays emerged two novel compounds, namely 11 and 20, which with an IC50 respectively of 1.6 and 1.8 µM are the most potent inhibitors of DGKα discovered to date. Both compounds demonstrated the ability to restore apoptosis in a cellular model of X-linked lymphoproliferative disease as well as the capacity to reduce the migration of cancer cells, suggesting their potential utility in preventing metastasis. Finally, relying on experimental biological data, molecular modelling studies allow us to set a three-point pharmacophore model for DGK inhibitors.


Assuntos
Lipase Lipoproteica/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Lipase Lipoproteica/metabolismo , Linfócitos/efeitos dos fármacos , Células MCF-7 , Modelos Moleculares , Estrutura Molecular , Monócitos/efeitos dos fármacos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos
6.
J Environ Pathol Toxicol Oncol ; 38(2): 119-131, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31679275

RESUMO

BACKGROUND/AIMS: LncRNAs are significant regulators in multiple cancers including hepatocellular carcinoma (HCC). Recently, lncRNA ANRIL has been reported to be elevated during multiple cancer types, exhibiting oncogenic roles. However, the exact biological mechanism of ANRIL is still poorly understood in HCC. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) assays were utilized to detect expressions of ANRIL, miR-384, and STAT3. CCK8 and EDU assays were employed to evaluate HCC cell proliferation. A flow cytometry assay was used to detect the HCC cell cycle and cell apoptosis. The scratch migration and Transwell invasion assays were performed to test cell migration and invasion, respectively. RIP and RNA pull-down assays were carried out to confirm the correlation between ANRIL and miR-384. The dual-luciferase reporter assay was used to prove the association between miR-384 and STAT3. Western blotting analysis was performed to examine protein levels of STAT3. IHC and HE staining were employed to detect Ki-67 and histopathology. RESULTS: ANRIL expression was upregulated in HCC cells, including SMCC7721, HepG2, MHCC-97H, SNU449 and HUH-7 cells, in comparison to the normal human liver cells LO2. Knockdown of ANRIL suppressed HCC cell proliferation and induced cell cycle arrest and apoptosis. HCC cell migration and invasion capacity were inhibited by inhibition of ANRIL. Bioinformatics analyses revealed that ANRIL could interact with miR-384. miR-384 was significantly decreased in HCC cells, and overexpression of miR-384 repressed HCC progression. STAT3 was predicted as a target of miR-384, and miR-384 can modulate STAT3 levels negatively in vitro. ANRIL can suppress HCC development through regulating miR-384 and STAT3 in vivo. CONCLUSION: ANRIL is involved in HCC progression by direct targeting of miR-384 and STAT3. Also, ANRIL could act as a potential candidate for HCC diagnosis, prognosis, and therapy.


Assuntos
Carcinogênese/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Fator de Transcrição STAT3/genética , Carcinoma Hepatocelular/fisiopatologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Progressão da Doença , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/fisiopatologia , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Fator de Transcrição STAT3/metabolismo
7.
Yi Chuan ; 41(10): 939-949, 2019 Oct 20.
Artigo em Chinês | MEDLINE | ID: mdl-31624056

RESUMO

Mutations in Hypoxanthine-guanine Phosphoribosyltransferase1 (HPRT1) gene can lead to metabolic disorder of hypoxanthine and guanine metabolism, and other severe symptoms such as hypophrenia, gout, and kidney stones, called the Lesch-Nyhan disease (LND). Although the mutations are widely distributed throughout the HPRT1 gene, there are some isolated hot spots. In this study, we aim to introduce two previously reported hot spots, c.508 C>T and c.151 C>T, which could lead to premature translational termination in HPRT1 gene. Through CRISPR/Cas9 mediated homology-directed repair (HDR) by using single-stranded oligo-deoxyribonucleotides (ssODN) as donor template, we obtained cell clones containing these two mutations in HEK293T or HeLa cells. Targeted mutation of c.508 C>T and c.151 C>T reached to 16.3% and 10%, respectively. We further detect HPRT1 protein levels with Western blot and enzyme activity with 6-TG in 5 different cell clones. HPRT1 protein and its enzymatic activity both was hardly detected in homozygous mutant cells, while reduced HPRT1 protein expression and enzymatic activity was detected in heterozygous mutant cells. Our study will be beneficial to those who working on generation of cell or animal models of HRPT1 mutations, and provides a basis for further investigations on the genetic mechanism of Lesch-Nyhan disease.


Assuntos
Sistemas CRISPR-Cas , Hipoxantina Fosforribosiltransferase/genética , Mutação Puntual , Células HEK293 , Células HeLa , Humanos , Síndrome de Lesch-Nyhan/genética
8.
DNA Cell Biol ; 38(11): 1374-1386, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31599655

RESUMO

This study was conducted using TagSNPs to systematically explore the relationship between ARID5B polymorphisms and the occurrence, clinical characterization, and prognosis of acute myeloid leukemia (AML). A total of 569 unrelated AML patients and 410 healthy individuals from West China were recruited, and ARID5B TagSNPs were genotyped using iMLDR® (improved multiplex ligation detection reaction). It was found that the association of ARID5B polymorphisms with AML was most significant in acute promyelocytic leukemia (APL), and exclusively in males, the mutant alleles of rs6415872, rs2393726, rs7073837, rs10821936, and rs7089424 were found to increase the risk of developing APL in men, the odds ratio (OR) were 1.36, 1.74, 1.45, 1.53, and 1.56 (all p < 0.05), respectively. Haplotype analysis revealed that haplotype [AACCG] increased the risk of male APL with an OR of 1.53 (95% confidence interval: 1.10-2.14, p = 0.012). Besides, there was a strong positive additive interaction between rs6415872 and rs10821936, rs7089424, respectively, and cases attributed to the interaction of rs6415872, rs10821936, and rs7089424 accounted for 100%. Furthermore, ARID5B single nucleotide polymorphisms were found associated with clinical features of AML, and rs6415872 was shown to be an independent prognosis factor in APL patients. Besides, dual luciferase report assay showed that rs6415872 may affect the binding activity of PPARG with ARID5B. ARID5B polymorphisms contribute to male APL risk, clinical feature, and prognosis, suggesting the importance of ARDI5B in AML pathogenesis and development, and the gender and subtype preference may prompt some specific mechanisms of ARID5B. Besides, ARID5B polymorphisms might be a potential prognosis biomarker.


Assuntos
Proteínas de Ligação a DNA/genética , Leucemia Promielocítica Aguda/diagnóstico , Leucemia Promielocítica Aguda/genética , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/genética , Adulto , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Genótipo , Células HEK293 , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/epidemiologia , Leucemia Mieloide Aguda/genética , Leucemia Promielocítica Aguda/epidemiologia , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Prognóstico , Caracteres Sexuais , Análise de Sobrevida
9.
Zhonghua Gan Zang Bing Za Zhi ; 27(9): 687-692, 2019 Sep 20.
Artigo em Chinês | MEDLINE | ID: mdl-31594093

RESUMO

Objective: To construct the recombinant adenoviral containing fructose 1, 6-biphosphatase 1 (FBP1), and to investigate whether FBP1 has effect on autophagy and proliferation in liver cancer cells (HepG2). Methods: FBP1 cDNA sequence was amplified by PCR and cloned in adenovirus vector pAdTrack-TO4, and then recombinant adenovirus plasmid pAdTrack-FBP1 was constructed. The recombinant adenovirus plasmid was transfected into HEK293 cells by Lipofectamine 3000. High-titer of recombinant adenovirus AdFBP1 was obtained by packaging and amplification. HepG2 cells were infected with recombinant adenovirus AdFBP1, and the Mock and AdGFP group were set at the same time. Western blot and confocal laser scanning microscopy were used to observe the effect of FBP1 on the level of autophagy in hepatocellular carcinoma cells, and the effect of FBP1on the proliferation was observed by MTS and colony formation assay. A t-test and one-way ANOVA were used to compare the mean between group. Results: A high-titer recombinant adenovirus FBP1 was successfully constructed. Western blot and confocal laser scanning microscopy showed that the level of autophagy in AdFBP1 group was significantly lower than that in AdGFP group. Western blot results showed that LC3-II protein expression level in AdGFP was 1.10 ± 0.10 and 0.30 ± 0.01 in AdFBP1 group, F = 90.36, P < 0.01. Confocal laser scanning microscopy analysis showed that the average number of autophages in AdGFP was 28.33 ± 1.53 and 12.33 ± 1.53 in AdFBP1group, F = 97.40, P < 0.01. In addition, the results of colony formation assay and MTS assay showed that the proliferation of liver cancer cells in the AdFBP1 group was significantly inhibited compared with the AdGFP group. The results of colony formation showed that the cell clones in the AdGFP group was 65.66 ± 2.57 and 34.00 ± 2.00 in AdFBP1 group, F = 141.50, P < 0.01. MTS results showed that the absorbance of AdGFP group at 96h was 39.13 ± 2.21 and 30.61 ± 3.33 in AdFBP1 group, F = 7.80, P < 0.05. Conclusion: FBP1 inhibited the autophagy and proliferation in liver cancer cells (HepG2).


Assuntos
Autofagia , Proliferação de Células , Frutose-Bifosfatase/metabolismo , Neoplasias Hepáticas/patologia , Adenoviridae , Vetores Genéticos , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/enzimologia , Transfecção
10.
Anticancer Res ; 39(10): 5427-5436, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570437

RESUMO

BACKGROUND/AIM: Renal cell carcinoma (RCC) is one of the most common tumor diseases in adults, and new specific biomarkers are urgently needed to define diagnosis and prognosis of patients with RCC as well as monitor the outcome of therapeutic interventions. The enzyme nicotinamide N-methyltransferase (NNMT) is believed to represent such a marker molecule in RCC therapy. MATERIALS AND METHODS: NNMT expression was examined by western blotting in samples from patients with RCC and in RCC cell lines. Effects of NNMT on cell growth and metabolism were assessed using the Hoechst 33342 reagent assay and Vita-Orange cell viability assay. Incubation experiments were performed to study the influence of methionine and interleukin-6 (IL6) on expression of NNMT. RESULTS: In patient samples, NNMT was up-regulated depending on the stage of progression. Investigations in an RCC cell culture model showed that after modulation of NNMT expression, cellular metabolism, but not cell growth was affected. This regulatory function was also dependent on the presence of the NNMT precursor substrate methionine and IL6. CONCLUSION: The metabolism-regulatory activity of NNMT depends on the precursor substrate methionine and the presence of IL6. The function of methionine appears to be dependent on the stage of progression, since in individual RCC cell lines, opposing effects on metabolism were demonstrated. This, in turn, reflects the thoroughly complex situation in the clinic.


Assuntos
Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Metionina/metabolismo , Nicotinamida N-Metiltransferase/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Progressão da Doença , Células HEK293 , Humanos , Interleucina-6/metabolismo , Prognóstico , Regulação para Cima/fisiologia
11.
Nat Cell Biol ; 21(10): 1219-1233, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31576058

RESUMO

Protein trafficking requires coat complexes that couple recognition of sorting motifs in transmembrane cargoes with biogenesis of transport carriers. The mechanisms of cargo transport through the endosomal network are poorly understood. Here, we identify a sorting motif for endosomal recycling of cargoes, including the cation-independent mannose-6-phosphate receptor and semaphorin 4C, by the membrane tubulating BAR domain-containing sorting nexins SNX5 and SNX6. Crystal structures establish that this motif folds into a ß-hairpin, which binds a site in the SNX5/SNX6 phox homology domains. Over sixty cargoes share this motif and require SNX5/SNX6 for their recycling. These include cargoes involved in neuronal migration and a Drosophila snx6 mutant displays defects in axonal guidance. These studies identify a sorting motif and provide molecular insight into an evolutionary conserved coat complex, the 'Endosomal SNX-BAR sorting complex for promoting exit 1' (ESCPE-1), which couples sorting motif recognition to the BAR-domain-mediated biogenesis of cargo-enriched tubulo-vesicular transport carriers.


Assuntos
Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Nexinas de Classificação/química , Nexinas de Classificação/metabolismo , Motivos de Aminoácidos/genética , Animais , Drosophila melanogaster , Células HEK293 , Células HeLa , Humanos , Domínios Proteicos/genética , Transporte Proteico/fisiologia , Receptor IGF Tipo 2/química , Receptor IGF Tipo 2/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , Nexinas de Classificação/genética
12.
Cell Physiol Biochem ; 53(4): 687-700, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31577078

RESUMO

BACKGROUND/AIMS: Apelin and its G protein-coupled receptor APJ (gene symbol Aplnr) are strongly expressed in magnocellular vasopressinergic neurons suggesting that the apelin/APJ system plays a key role at the central level in regulating salt and water balance by counteracting the antiduretic action of vasopressin (AVP). Likewise, recent studies revealed that apelin exerts opposite effects to those of vasopressin induced on water reabsorption via a direct action on the kidney collecting duct. However, the underlying mechanisms of the peripheral action of apelin are not clearly understood. Here, we thus investigated the role of the apelin/APJ system in the regulation of water balance in the kidney, and more specifically its involvement in modulating the function of aquaporin-2 (AQP2) in the collecting duct. METHODS: Mouse cortical collecting duct cells (mpkCCD) were incubated in the presence of dDAVP and treated with or without apelin-13. Changes in AQP2 expression and localization were determined by immunoblotting and confocal immunofluorescence staining. RESULTS: Herein, we showed that the APJ was present in mpkCCD cells. Treatment of mpkCCD with apelin-13 reduced the cAMP production and antagonized the AVP-induced increase in AQP2 mRNA and protein expressions. Immunofluorescent experiments also revealed that the AVP-induced apical cell surface expression of AQP2, and notably its phosphorylated isoform AQP2-pS269, was considerably reduced following apelin-13 application to mpkCCD cells. CONCLUSION: Our data reinforce the aquaretic role of the apelin/APJ system in the fine regulation of body fluid homeostasis at the kidney level and its physiological opposite action to the antiduretic activity of AVP.


Assuntos
Aquaporina 2/metabolismo , Desamino Arginina Vasopressina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Transporte Proteico/efeitos dos fármacos , Animais , Receptores de Apelina/metabolismo , Aquaporina 2/genética , Linhagem Celular , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos
13.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 50(4): 540-545, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31642232

RESUMO

OBJECTIVE: To explore the anti-virus effect of AY358935 gene cloned by our research team on vesicular stomatitis virus (VSV), and studytheanti-virus mechanism. METHODS: HEK293 cells were stably transfected by the AY358935 gene recombinant plasmid pcDNA3.1-AY358935 or pcDNA3.1 blank plasmid respectively. Then VSV was added into the cell wells to infect the above cells at the multiplicity of infection (MOI) of 0.001. The virus titers in the liquid supernatant of the above three groups of cells were detected on different time, and the mortality of cells of each group was tested with trypan blue exclusion test at 24 h post VSV infection. Total RNA was extracted from the cells that stably transfected with target gene for the whole genome-wide cDNA microarray analysis. RESULTS: ① Virus titer:The virus titer in the liquid supernatant of pcDNA-3.1-AY358935 transfection cells group was obviously lower than those in pcDNA-3.1 transfection cell group and blank control cell group at 12 h post infection. The virus titerin the liquid supernatant of three groups were (7.16±2.33)×105 PFU/mL, (6.25±2.05)×106 PFU/mL and (7.75±2.54)×106 PFU/mL respectively at 18 h post infection. At that time, the virus titerin the liquid supernatant of pcDNA3.1-AY358935 group was nearly 10 times lower than those of other two groups (P < 0.01). ②Mortality of cells:The cell mortality of pcDNA3.1-AY358935 group, pcDNA3.1 group and blank group were (35.00±6.68)%, (78.33±15.03)% and (83.34±14.98)% respectively at 24 h post infection.The cell mortality of pcDNA3.1-AY358935 group was significantly decreased comparing with other two groups (P < 0.01). ③Result of genes chip analysis: compared with pcDNA3.1 group, 30 cell genes were up-regulated by more than 3 times in pcDNA3.1-AY358935 group. Among them, the proportion of interferon-activating gene, interferon-effect gene, cytokine and chemokine was 27%, 17%, and 20%, respectively. CONCLUSION: AY358935 gene hasan anti-VSV effect, and its anti-virus mechanism may involve the interferon-associated natural immune response.


Assuntos
Proteínas de Transporte/genética , Estomatite Vesicular/imunologia , Animais , Citocinas , Células HEK293 , Humanos , Interferons , Plasmídeos , Transfecção , Vesiculovirus
14.
Cell Biochem Biophys ; 77(4): 367-377, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31659617

RESUMO

Cisplatin is a widely used anti-cancer drug. However, cisplatin is limited in clinical treatment because of its severe nephrotoxicity. This study reported whether O-GSP can antagonize the cisplatin-induced cytotoxicity in HEK293 cells through inducing HO-1 protein expression. We previously demonstrated O-GSP can increase the survival rate of HEK293 and have protective effect on HEK293 cells. Herein, We found that O-GSP can antagonize cisplatin nephrotoxicity through regulating the expression of HO-1. O-GSP promotes the translocation of Nrf2 in the nucleus, and activates the ERKN JNK pathway and p38 MAPK pathway. Interestingly, p38 MAPK plays a major role in HO-1 expression induced by O-GSP. And O-GSP can modulate the decrease of Nrf2 and HO-1 expression induced by cisplatin, and improve the cisplatin-induced activity and apoptosis rate of cells by stimulating the expression of HO-1. However, the protective effects of O-GSP are inhibited by ZnPP IX. Collectively, the results indicated that O-GSP induced the expression of HO-1 through p38MAPK and Nrf2 pathway in HEK293 cells.


Assuntos
Antineoplásicos/farmacologia , Biflavonoides/farmacologia , Catequina/farmacologia , Cisplatino/farmacologia , Heme Oxigenase-1/metabolismo , Proantocianidinas/farmacologia , Regulação para Cima/efeitos dos fármacos , Vitis/química , Apoptose/efeitos dos fármacos , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Fator 2 Relacionado a NF-E2/metabolismo , Sementes/química , Sementes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vitis/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
Photochem Photobiol Sci ; 18(11): 2740-2747, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31573014

RESUMO

Cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) is associated with memory formation and controls cell survival and proliferation via regulation of downstream gene expression in tumorigenesis. As a transcription factor, CREB binds to cAMP response elements. Phosphorylation of CREB triggers transcriptional activation of CREB downstream genes following the interaction of the kinase-inducible domain (KID) of CREB with the KID interaction domain (KIX) of CREB-binding protein. Nevertheless, because of the lack of single-cell analytical techniques, little is known about spatiotemporal regulation of CREB phosphorylation. To analyze CREB activation in single living cells, we developed genetically encoded bioluminescent sensors using luciferase-fragment complementation: the sensors are designed based on KID-KIX interaction with a single-molecule format. The luminescence intensity of the sensor, designated as CREX (a sensor of CREB activation based on KID(CREB)-KIX interaction), increased by phosphorylation of CREB. Moreover, the luminescence intensity of CREX was sufficient to detect CREB activation in live-cell bioluminescence imaging for single-cell analysis because of the higher sensitivity. CREX sensor is expected to contribute to elucidation of the spatiotemporal regulation of CREB phosphorylation by applying single-cell analysis.


Assuntos
Proteína de Ligação a CREB/análise , Medições Luminescentes/métodos , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Colforsina/química , Células HEK293 , Humanos , Luciferases/química , Luciferases/metabolismo , Fosforilação , Ligação Proteica , Domínios Proteicos/genética , Análise de Célula Única , Imagem com Lapso de Tempo
16.
Chem Commun (Camb) ; 55(88): 13291-13294, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31626249

RESUMO

O-GlcNAc transferase (OGT) glycosylates numerous proteins and is implicated in many diseases. To date, most OGT inhibitors lack either sufficient potency or characterized specificity in cells. We report the first targeted covalent inhibitor that predominantly reacts with OGT but does not affect other functionally similar enzymes. This study provides a new strategy to interrogate cellular OGT functions and to investigate other glycosyltransferases.


Assuntos
Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Inibidores Enzimáticos/química , Células HEK293 , Humanos , Células MCF-7 , Modelos Moleculares , N-Acetilglucosaminiltransferases/metabolismo
17.
Med Oncol ; 36(11): 95, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31637536

RESUMO

Ovarian cancer is one of the most lethal gynecological cancers; owning to its late detection and chemoresistance, understanding the pathogenesis of this malignant tumor is much critical. Previous studies have reported that ubiquitin-specific peptidase 39 (USP39) is generally overexpressed in a variety of cancers, including hepatocellular carcinoma, gastric cancer and so forth. Furthermore, USP39 is proved to be associated with the proliferation of malignant tumors. However, the function and mechanism of USP39 in ovarian cancer have not been elucidated. In the present study, we observed that USP39 was frequently overexpressed in human ovarian cancer and was highly correlated with TNM stage. Suppression of USP39 markedly inhibited the growth and migration of ovarian cancer cell lines HO-8910 and SKOV3 and induced cell cycle G2/M arrest. Moreover, knockdown of USP39 inhibited ovarian tumor growth in a xenograft model. In addition, our findings indicated that cell cycle arrest induced by USP39 knockdown might be involved in p53/p21 signaling pathway. Furthermore, we found that the depletion of USP39 inhibited the migration of ovarian cancer cells via blocking epithelial-mesenchymal transition. Taken together, these results suggest that USP39 may play vital roles in the genesis and progression and may serve as a potential biomarker for diagnosis and therapeutic target of ovarian cancer.


Assuntos
Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteína Supressora de Tumor p53/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Transição Epitelial-Mesenquimal , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular , Técnicas de Silenciamento de Genes , Células HEK293 , Xenoenxertos , Humanos , Imuno-Histoquímica , Pontos de Checagem da Fase M do Ciclo Celular , Camundongos , Camundongos Nus , Estadiamento de Neoplasias , Neoplasias Ovarianas/enzimologia , Transdução de Sinais , Proteases Específicas de Ubiquitina/biossíntese , Proteases Específicas de Ubiquitina/genética
18.
Nat Cell Biol ; 21(10): 1273-1285, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548606

RESUMO

Chromosome translocation is a major cause of the onset and progression of diverse types of cancers. However, the mechanisms underlying this process remain poorly understood. Here, we identified a non-homologous end-joining protein, IFFO1, which structurally forms a heterotetramer with XRCC4. IFFO1 is recruited to the sites of DNA damage by XRCC4 and promotes the repair of DNA double-strand breaks in a parallel pathway with XLF. Interestingly, IFFO1 interacts with lamin A/C, forming an interior nucleoskeleton. Inactivating IFFO1 or its interaction with XRCC4 or lamin A/C leads to increases in both the mobility of broken ends and the frequency of chromosome translocation. Importantly, the destruction of this nucleoskeleton accounts for the elevated frequency of chromosome translocation in many types of cancer cells. Our results reveal that the lamin A/C-IFFO1-constituted nucleoskeleton prevents chromosome translocation by immobilizing broken DNA ends during tumorigenesis.


Assuntos
Carcinogênese/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Lamina Tipo A/metabolismo , Translocação Genética , Animais , Carcinoma/genética , Cromossomos Humanos , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Proteínas de Filamentos Intermediários/genética , Camundongos , Matriz Nuclear/metabolismo , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Associadas à Matriz Nuclear/fisiologia
19.
Nat Cell Biol ; 21(10): 1179-1190, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548608

RESUMO

Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors, as is exemplified by reprogramming somatic cells to pluripotent stem cells through the expression of OCT4, KLF4, SOX2 and cMYC. How transcription factors orchestrate the complex molecular changes around their target gene loci remains incompletely understood. Here, using KLF4 as a paradigm, we provide a transcription-factor-centric view of chromatin reorganization and its association with three-dimensional enhancer rewiring and transcriptional changes during the reprogramming of mouse embryonic fibroblasts to pluripotent stem cells. Inducible depletion of KLF factors in PSCs caused a genome-wide decrease in enhancer connectivity, whereas disruption of individual KLF4 binding sites within pluripotent-stem-cell-specific enhancers was sufficient to impair enhancer-promoter contacts and reduce the expression of associated genes. Our study provides an integrative view of the complex activities of a lineage-specifying transcription factor and offers novel insights into the nature of the molecular events that follow transcription factor binding.


Assuntos
Reprogramação Celular/genética , Montagem e Desmontagem da Cromatina/genética , Elementos Facilitadores Genéticos , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Células-Tronco Pluripotentes/metabolismo
20.
Nat Cell Biol ; 21(10): 1206-1218, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548609

RESUMO

Cholesterol activates the master growth regulator, mTORC1 kinase, by promoting its recruitment to the surface of lysosomes by the Rag guanosine triphosphatases (GTPases). The mechanisms that regulate lysosomal cholesterol content to enable mTORC1 signalling are unknown. Here, we show that oxysterol binding protein (OSBP) and its anchors at the endoplasmic reticulum (ER), VAPA and VAPB, deliver cholesterol across ER-lysosome contacts to activate mTORC1. In cells lacking OSBP, but not other VAP-interacting cholesterol carriers, the recruitment of mTORC1 by the Rag GTPases is inhibited owing to impaired transport of cholesterol to lysosomes. By contrast, OSBP-mediated cholesterol trafficking drives constitutive mTORC1 activation in a disease model caused by the loss of the lysosomal cholesterol transporter, Niemann-Pick C1 (NPC1). Chemical and genetic inactivation of OSBP suppresses aberrant mTORC1 signalling and restores autophagic function in cellular models of Niemann-Pick type C (NPC). Thus, ER-lysosome contacts are signalling hubs that enable cholesterol sensing by mTORC1, and targeting the sterol-transfer activity of these signalling hubs could be beneficial in patients with NPC.


Assuntos
Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Doenças de Niemann-Pick/metabolismo , Receptores de Esteroides/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Receptores de Esteroides/genética , Transdução de Sinais , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
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