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1.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(8): 702-706, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31638567

RESUMO

Objective To analyze the changes and correlation between inflammation and Klotho expression in kidney tissue of mice with acute kidney injury (AKI) induced by cisplatin, and to explore the role and possible mechanism of Klotho in AKI induced by cisplatin. Methods Eighteen male C57BL/6 mice were randomly divided into 0-day group, 1-day group and 3-day group with 6 mice in each group. The mice were killed at 0, 1 and 3 days after a single intraperitoneal injection of 25 mg/kg of cisplatin, and the serum and kidney tissues were collected. The content of serum creatinine (Scr) and blood urea nitrogen (BUN) were measured by biochemical analyzer, and the pathological changes of kidney tissues were observed by HE staining. Neutrophil gelatinase-associated lipocalin (NGAL), tumor necrosis factor α (TNF-α), NLR family pyrin domain containing 3 (NLRP3), Klotho, signal transducer and activator of transcription 3 (STAT3), phosphorylated STAT3 (p-STAT3), nuclear factor-kappa B (NF-κB), phosphorylated NF-kappa B (p-NF-κB) were detected by Western blot analysis. Spearman rank correlation test was used to analyze the correlations. Results The content of serum Scr and BUN in 1-day and 3-day groups were significantly higher than those in 0-day group, and inflammatory cell infiltration, renal tubular epithelial cell exfoliation, edema and accumulation of cell fragments were seen in 1-day and 3-day groups. In the 3-day group, the content of NGAL, TNF-α, NLRP3, p-STAT3, STAT3, p-NF-κB and NF-κB proteins in renal tissues significantly increased, and the expression of TNF-α, p-STAT3 and STAT3 increased in a time-dependent manner. The expression of Klotho decreased in a time-dependent manner in the 1-day and 3-day groups, and the expression of NGAL, TNF-α, NLRP3, p-STAT3, and p-NF-κB were significantly negatively correlated with the expression of Klotho. Conclusion The activation of STAT3/NF-κB pathway by Klotho is involved in the regulation of the occurrence and development of AKI induced by cisplatin in mice.


Assuntos
Lesão Renal Aguda , Cisplatino , Regulação da Expressão Gênica , Glucuronidase , Rim , Lesão Renal Aguda/induzido quimicamente , Lesão Renal Aguda/fisiopatologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Glucuronidase/genética , Rim/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Distribuição Aleatória
2.
Appl Microbiol Biotechnol ; 103(12): 4813-4823, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31055652

RESUMO

In this study, we aimed to shift the optimal pH of acidic ß-glucuronidase from Aspergillus oryzae Li-3 (PGUS) to the neutral region by site-directed mutagenesis, thus allowing high efficient biotransformation of glycyrrhizin (GL) into glycyrrhetinic acid (GA) under higher pH where the solubility of GL could be greatly enhanced. Based on PGUS structure analysis, five critical aspartic acid and glutamic acid residues were replaced with arginine on the surface to generate a variant 5Rs with optimal pH shifting from 4.5 to 6.5. The catalytic efficiency (kcat /Km) value of 5Rs at pH 6.5 was 10.7-fold higher than that of PGUS wild-type at pH 6.5, even 1.4-fold higher than that of wild-type at pH 4.5. Molecular dynamics simulation was performed to explore the molecular mechanism for the shifted pH profile and enhanced pH stability of 5Rs.


Assuntos
Aspergillus oryzae/metabolismo , Glucuronidase/metabolismo , Ácido Glicirrízico/metabolismo , Mutagênese Sítio-Dirigida , Ácido Aspártico/metabolismo , Biotransformação , Catálise , Glucuronidase/genética , Ácido Glutâmico/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Simulação de Dinâmica Molecular
3.
Int J Mol Sci ; 20(8)2019 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-31013989

RESUMO

The incidence of aging-related disorders may be decreased through strategies influencing the expression of longevity genes. Although numerous approaches have been suggested, no effective, safe, and easily applicable approach is yet available. Efficacy of low-dose fluvastatin and valsartan, separately or in combination, on the expression of the longevity genes in middle-aged males, was assessed. Stored blood samples from 130 apparently healthy middle-aged males treated with fluvastatin (10 mg daily), valsartan (20 mg daily), fluvastatin-valsartan combination (10 and 20 mg, respectively), and placebo (control) were analyzed. They were taken before and after 30 days of treatment and, additionally, five months after treatment discontinuation. The expression of the following longevity genes was assessed: SIRT1, PRKAA, KLOTHO, NFE2L2, mTOR, and NF-κB. Treatment with fluvastatin and valsartan in combination significantly increased the expression of SIRT1 (1.8-fold; p < 0.0001), PRKAA (1.5-fold; p = 0.262) and KLOTHO (1.7-fold; p < 0.0001), but not NFE2L2, mTOR and NF-κB. Both fluvastatin and valsartan alone significantly, but to a lesser extent, increased the expression of SIRT1, and did not influence the expression of other genes. Five months after treatment discontinuation, genes expression decreased to the basal levels. In addition, analysis with previously obtained results revealed significant correlation between SIRT1 and both increased telomerase activity and improved arterial wall characteristics. We showed that low-dose fluvastatin and valsartan, separately and in combination, substantially increase expression of SIRT1, PRKAA, and KLOTHO genes, which may be attributed to their so far unreported pleiotropic beneficial effects. This approach could be used for prevention of ageing (and longevity genes)-related disorders.


Assuntos
Fluvastatina/farmacologia , Expressão Gênica/efeitos dos fármacos , Longevidade/genética , Doenças Neurodegenerativas/prevenção & controle , Valsartana/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Adulto , Envelhecimento/genética , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Relação Dose-Resposta a Droga , Feminino , Fluvastatina/uso terapêutico , Glucuronidase/genética , Humanos , Masculino , Pessoa de Meia-Idade , Efeito Placebo , Sirtuína 1/genética , Telomerase/metabolismo , Valsartana/uso terapêutico
4.
Ren Fail ; 41(1): 175-182, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30942135

RESUMO

OBJECTIVE: To detect the combination protective effect of bone marrow mesenchymal stem cells (BMSCs) and Klotho gene on the renal ischemia-reperfusion injury (RIRI). METHODS: BMSCs isolated from rats were transfected with Klotho gene to form BMSCKl. We injected BMSCKl to allogenic rat RIRI model. After 24 h and 72 h, we detected the serum creatinine (SCr), malondialdehyde (MDA), and superoxide dismutase (SOD) in renal tissue, Hematoxylin-eosin (HE) staining, and TUNEL of renal pathology. The expression of FoxO1 and p-FoxO1 in post-hypoxia tubular epithelial cells of normal rat kidney (NRK-52E) were detected by Western blot after cocultured with BMSCKl. RESULTS: Comparing with BMSCCon group, Rats in BMSCKl group had lower SCr and MDA but higher SOD. Both HE and TUNEL score of renal tissue in BMSCKl group were lower than that of BMSCCon group. Western blot indicated that FoxO1 was upregulated, while p-FoxO1 was downregulated in post-hypoxia NRK-52E cells. CONCLUSIONS: BMSCs transfected with Klotho gene can further ameliorate RIRI. The possible mechanism may be attributed to the upregulation of SOD in NRK-52E caused by Klotho-FoxO1 axis.


Assuntos
Glucuronidase/metabolismo , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Traumatismo por Reperfusão/terapia , Animais , Engenharia Celular/métodos , Linhagem Celular , Técnicas de Cocultura , Modelos Animais de Doenças , Regulação para Baixo , Glucuronidase/genética , Humanos , Rim/irrigação sanguínea , Rim/metabolismo , Rim/patologia , Masculino , Proteínas do Tecido Nervoso/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Superóxido Dismutase/metabolismo , Transfecção , Resultado do Tratamento , Regulação para Cima
5.
Biomed Pharmacother ; 113: 108756, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30870716

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a lethal disease of unknown aetiology that largely presents in the elderly. The mechanisms related to aging such as fibroblast senescence have been strongly implicated in pathology of IPF. We have previously demonstrated the protective effects of IL-18 binding protein (IL-18BP) against bleomycin (BLM)-induced pulmonary fibrosis (PF) via inhibition of epithelial-to-mesenchymal transition. However, the role of IL-18 in fibroblast senescence in PF is still unknown. The aim of this study was to investigate the effects of IL-18 on fibroblast senescence in the development of PF. We found that SA-ß-gal positive cells, the proportion of cells in G1 phase, and expressions of p21 and p53 were increased in primary lung fibroblasts isolated from BLM-challenged mice, while the fibroblasts from IL-18BP-treated mice showed decreased senescence propensity. We further demonstrated that IL-18 was sufficient to trigger senescence of primary lung fibroblasts. The senescence-associated secretory phenotype (SASP) of fibroblasts treated with IL-18 robustly stimulated a fibrotic phenotype in pulmonary fibroblasts. Moreover, the expression of Klotho, an anti-senescence protein, was down-regulated after IL-18 treatment in primary lung fibroblasts. Overexpression of Klotho reversed the senescence and SASP induced by IL-18 in lung fibroblasts. In summary, we reported for the first time that IL-18 promoted the lung fibroblast senescence and SASP in PF through blocking Klotho pathway. Neutralize IL-18 by IL-18BP exhibited antifibrotic effects partly by suppressing lung fibroblast senescence in PF. It contributes to the growing evidence that IL-18 could be a therapeutic target for PF.


Assuntos
Fibroblastos/patologia , Glucuronidase/genética , Fibrose Pulmonar Idiopática/fisiopatologia , Interleucina-18/metabolismo , Animais , Bleomicina/toxicidade , Senescência Celular/fisiologia , Regulação para Baixo , Fibrose Pulmonar Idiopática/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo
6.
Neurobiol Aging ; 78: 18-28, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30851437

RESUMO

Alzheimer's disease (AD) is the most common type of senile dementia. The antiaging gene Klotho is reported to decline in the brain of patients and animals with AD. However, the role of Klotho in the progression of AD remains elusive. The present study explored the effects and underlying mechanism of Klotho in a mouse model of AD. The upregulation of cerebral Klotho expression was mediated by an intracerebroventricular injection of a lentiviral vector that encoded Klotho (LV-KL) in 7-month-old amyloid precursor protein/presenilin 1 transgenic mice. Three months later, LV-KL significantly induced Klotho overexpression in the brain and effectively ameliorated cognitive deficit and AD-like pathology in amyloid precursor protein/presenilin 1 mice. LV-KL induced autophagy activation and protein kinase B/mammalian target of rapamycin inhibition both in AD mice and BV2 murine microglia. These results suggest that the upregulation of Klotho expression in the brain may promote the autophagic clearance of amyloid beta and protect against cognitive deficits in AD mice. These findings highlight the preventive and therapeutic potential of Klotho for the treatment of AD.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/terapia , Encéfalo/metabolismo , Disfunção Cognitiva/genética , Disfunção Cognitiva/terapia , Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Glucuronidase/administração & dosagem , Glucuronidase/genética , Lentivirus , Peptídeos beta-Amiloides/metabolismo , Animais , Autofagia , Modelos Animais de Doenças , Vetores Genéticos/farmacologia , Glucuronidase/metabolismo , Glucuronidase/farmacologia , Humanos , Injeções Intraventriculares , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Regulação para Cima
7.
Methods Mol Biol ; 1932: 175-185, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30701500

RESUMO

microRNAs are noncoding RNAs of 20-24 nucleotides (nt) in length that act as repressors of genes and are important in key developmental processes in the entire life cycle of plants. To determine the function of a microRNA, the first step is to resolve its expression pattern; this can be achieved by in situ hybridization, RNA blot assays, or quantitative PCR. However, the study of the expression of a MIR gene is straightforward with the use of reporter proteins such as ß-D-glucuronidase (GUS), GFP, or mCherry. To do this, it is necessary to clone the promoter region of the MIR gene and place it upstream of the reporter gene; in this way the activity of the promoter will be a direct reflection of the expression of the MIR gene. Here, we indicate step by step how to make transcriptional fusion constructs from the cloning of a promoter region of a MIR gene fused to the classical reporter proteins GUS and mCherry, the latter with codon optimization for better expression in Arabidopsis thaliana. This method is particularly useful to dissect the promoter region of a MIR gene and to find its expression pattern in a tissue and developmental specific manner.


Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , MicroRNAs/genética , RNA de Plantas/genética , Proteínas Recombinantes de Fusão/genética , Clonagem Molecular , Genes de Plantas/genética , Genes Reporter/genética , Glucuronidase/genética , Regiões Promotoras Genéticas/genética , Transcrição Genética/genética
8.
Methods Mol Biol ; 1915: 103-108, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30617799

RESUMO

The DEFECTIVE KERNEL1 (DEK1) gene encodes the unique plant calpain protein, which is fundamental for cell specification, development, and growth of land plants. The ß-glucuronidase (GUS) reporter gene system has been used to characterize and localize gene expression over several decades. When cloning a promoter upstream of the uidA/GUS reporter gene, you visualize when the promoter is activating expression by monitoring enzymatic activity of GUS, by detecting ß-glucuronidase cleavage products. Here we describe a protocol for monitoring the DEK1 promoter activity in different tissues in Arabidopsis by GUS staining.


Assuntos
Proteínas de Arabidopsis/genética , Calpaína/genética , Glucuronidase/genética , Regiões Promotoras Genéticas/genética , Sequência de Aminoácidos , Arabidopsis , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/isolamento & purificação , Calpaína/química , Calpaína/isolamento & purificação , Regulação da Expressão Gênica de Plantas , Genes Reporter/genética , Glucuronidase/química , Plantas Geneticamente Modificadas
9.
Virology ; 529: 1-6, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30622027

RESUMO

Vaccinia virus (VACV), like many other viruses, binds to cell surface heparan sulfate (HS) prior to infecting cells. Since HS is ubiquitously expressed extracellularly, it seemed likely that VACV-HS interaction may impede virus spread, with host heparanase, the only known mammalian endoglycosidase that can degrade HS, potentially overcoming this problem. In support of this hypothesis, we found that, compared to wild type, mice deficient in heparanase showed a 1-3 days delay in the spread of VACV to distant organs, such as ovaries, following intranasal inoculation, or to ovaries and spleen following intramuscular inoculation. These delays in spread occurred despite heparanase deficiency having no effect on VACV replication at inoculation sites. Subsequent in vitro studies revealed that heparanase treatment released VACV from HS expressing, but not HS deficient, infected cell monolayers. Collectively these data suggest that VACV relies on host heparanase to degrade HS in order to spread to distant sites.


Assuntos
Glucuronidase/metabolismo , Heparitina Sulfato/metabolismo , Vírus Vaccinia/metabolismo , Animais , Glucuronidase/genética , Heparitina Sulfato/genética , Humanos , Camundongos , Camundongos Knockout , Replicação Viral
10.
BMC Cancer ; 19(1): 9, 2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30611221

RESUMO

BACKGROUND: Dung beetle glycosaminoglycan is known to possess anti-aging activities. However, its anti-cancer mechanisms are not fully elucidated yet. The objective of this study was to evaluate the anti-cancer effect of insect-derived polymer dung beetle glycosaminoglycan (GAG) after intraperitoneally injecting it to melanoma mice induced by B16F10 cells. METHODS: To determine molecular mechanism involved in the anti-cancer effect of dung beetle GAG, its origin N-glycan under 3KD Dalton was assayed for melanoma cell cytotoxicity. Quantitative comparisons of adhesive molecule on extracellular matrix and activities of tissue inhibitor of metalloprotease 2 (TIMP-2) were also investigated. In vivo anti-cancer effect of dung beetle GAG on solid tumor size, survival time and gene-expression profiles was also assayed using B10F10 melanoma mice model. Mice with induced melanoma were then treated with Catharsius molossus (dung beetle) GAG (CaG) at 5 mg/kg for 8 weeks to investigate its anti-cancer effects compared to bumblebee (Bombus ignitus) queen glycosaminoglycan (IQG) and Huechys sanguinea glycosaminoglycan (HEG). RESULTS: These N-glycans derived from these GAG were composed of many linear heparinoid polysaccharides, polymers with hexose and N-acetylhexose. Adminstration with these GAGs increased survival time and decreased melanoma sizes in mice, in accordance with their inhibitory effects on cell growth ratio of melanoma B16F10. In addition, treatment with N-glycans derived from theses glycosaminoglycan increased activities of TIMP-2 in HMVEC cells pretreated with TNF-alpha and in melanoma cells, suggesting that they had anti-inflammatory and anticancer activities. In DNA microarray results, compared to control, CaG treated mouse group showed upregulation of 192 genes including collagen,typeI,alpha1 (Col1a1), consistent with the highly increased in vitro extracellular matrix (ECM) adhesion on collagen 1 and up-regulation of heparanase (Hpse). After treatment with CaG, a total of 152 genes were down-regulated, including nuclear RNA export factor (Nxf3) and hyaluronan proteoglycan link protein1 (Hapln1). CONCLUSIONS: Glycosaminoglycan, CaG can strengthen ECM by increasing activity of TIMP-2 and adhesion activity on collagen known to inhibit changes of ECM, leading to tumor cell invasion and progression.


Assuntos
Matriz Extracelular/efeitos dos fármacos , Glicosaminoglicanos/farmacologia , Melanoma Experimental/tratamento farmacológico , Inibidor Tecidual de Metaloproteinase-2/genética , Animais , Proliferação de Células/efeitos dos fármacos , Besouros/química , Colágeno Tipo I/genética , Matriz Extracelular/genética , Proteínas da Matriz Extracelular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucuronidase/genética , Glicosaminoglicanos/química , Humanos , Camundongos , Invasividade Neoplásica/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteoglicanas/genética
11.
Mech Ageing Dev ; 178: 33-40, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30633899

RESUMO

Klotho gene polymorphisms have been implicated in healthy aging, but inconsistences in findings from previous case-control studies have raised concerns regarding the associations between KLOTHO gene polymorphisms and susceptibility to aging-related diseases and longevity. Hence, this meta-analysis was performed. We assessed the associations between two polymorphisms (G-395 A/rs1207568 and F352 V/rs9536314) and five parameters (urolithiasis, cognitive impairment, cardiovascular disease, cancer, and longevity) by calculating pooled odds ratios with 95% confidence intervals. According to the pooled results, the G allele of the G-395 A polymorphism conferred a significantly higher risk of urolithiasis; G-395 A was related to the susceptibility to cardiovascular disease under allele, dominant, and recessive models. There was no significant association between the G-395 A polymorphism and cognitive impairment among the elderly. The F allele of the F352 V polymorphism protected against breast and ovarian cancer susceptibility. Interestingly, based on the results of the subgroup analysis, the F352 V polymorphism was associated with the overall risk of neoplasms in BRCA1 mutation carriers but not in BRCA2 mutation carriers. Moreover, the F allele played a protective role in determining human longevity. In conclusion, Klotho G-395 A polymorphisms were associated with urolithiasis and cardiovascular disease but not with cognitive impairment. Additionally, Klotho F352 V polymorphisms were associated with cancers and longevity.


Assuntos
Glucuronidase/fisiologia , Envelhecimento Saudável/genética , Longevidade/genética , Polimorfismo Genético/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Doenças Cardiovasculares/genética , Estudos de Casos e Controles , Disfunção Cognitiva/genética , Feminino , Glucuronidase/genética , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/genética , Urolitíase/genética
12.
Semin Nephrol ; 39(1): 57-75, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30606408

RESUMO

Acute kidney injury (AKI) is associated with many of the same mineral metabolite abnormalities that are observed in chronic kidney disease. These include increased circulating levels of the osteocyte-derived, vitamin D-regulating hormone, fibroblast growth factor 23 (FGF23), and decreased renal expression of klotho, the co-receptor for FGF23. Recent data have indicated that increased FGF23 and decreased klotho levels in the blood and urine could serve as novel predictive biomarkers of incident AKI, or as novel prognostic biomarkers of adverse outcomes in patients with established AKI. In addition, because FGF23 and klotho exert numerous classic as well as off-target effects on a variety of organ systems, targeting their dysregulation in AKI may represent a unique opportunity for therapeutic intervention. We review the pathophysiology, kinetics, and regulation of FGF23 and klotho in animal and human studies of AKI, and we discuss the challenges and opportunities involved in targeting FGF23 and klotho therapeutically.


Assuntos
Lesão Renal Aguda/sangue , Fatores de Crescimento de Fibroblastos/metabolismo , Glucuronidase/metabolismo , Rim/patologia , Lesão Renal Aguda/etiologia , Lesão Renal Aguda/terapia , Animais , Biomarcadores/sangue , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Estado Terminal , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/sangue , Fatores de Crescimento de Fibroblastos/fisiologia , Fibrose , Glucuronidase/genética , Glucuronidase/fisiologia , Glucuronidase/uso terapêutico , Humanos , Prognóstico , Insuficiência Renal Crônica/sangue , Índice de Gravidade de Doença , Transdução de Sinais
13.
Food Funct ; 10(1): 325-332, 2019 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-30574980

RESUMO

Theanine (γ-glutamylethylamide), an amino acid in tea, is a putative neuroprotective and antioxidant compound capable of improving lifespan and cognitive function. Because we previously reported cognitive dysfunction in klotho mutant mice via down-regulation of janus kinase 2 (JAK2) and signal transducer and activator of transcription3 (STAT3), M1 muscarinic cholinergic receptor (M1 mAChR), and ERK signaling, we, therefore, investigated whether self-administration of theanine affects memory dysfunction in response to klotho gene depletion in mice, and whether theanine modulates the JAK2/STAT3, M1 mAChR, and ERK signaling network. Theanine significantly attenuated memory impairments in klotho mutant mice. Moreover, theanine self-administration significantly attenuated inhibitions of JAK2/STAT3 phosphorylation, M1 mAChR expression, and ERK1/2 phosphorylation in the hippocampus of klotho mutant mice. Consistently, AG490, a JAK2/STAT3 inhibitor, dicyclomine, an M1 mAChR antagonist, or U0126, an ERK1/2 inhibitor, significantly counteracted theanine-induced attenuation of memory impairment induced by klotho gene depletion in mice. Our study suggests that theanine attenuates memory impairments in a genetic aging model via up-regulation of JAK2/STAT3, M1 mAChR, and ERK signaling.


Assuntos
Glucuronidase/deficiência , Glutamatos/administração & dosagem , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/genética , Animais , Feminino , Glucuronidase/genética , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Memória/efeitos dos fármacos , Transtornos da Memória/metabolismo , Camundongos , Camundongos Knockout , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo
14.
J Pharm Biomed Anal ; 162: 91-100, 2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30227357

RESUMO

Metal ions can be enzyme cofactors and can directly influence the kinetics of biochemical reactions that also influence the biological production and quality attributes of therapeutic proteins, such as glycan formation and distribution. However, the concentrations of metals in commercially available chemically defined media can range from 1 to 25,000 ppb. Because such concentration changes can impact cell growth, manufacturing yield and product quality the alteration/fluctuation in media composition should be well controlled to maintain product quality. Here, we describe a platform of analytical methods to determine the composition of several metals in different sample matrices using an advanced automated Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). These methods, validated to ICH Q2R1 regulatory validation parameters, were successfully applied to- (a) screen cell culture media; (b) determine changes in the metal concentration during cell growth in spinner flasks, and, (c) determine effect on the glycosylation pattern and homogeneity of an IgG3:κ produced from a murine-hybridoma cell line in bench-top parallel bioreactors due to a spike in copper and iron concentration. Our results show that maintenance of metal content in the cell culture media is critical for product consistency of the IgG3:κ produced.


Assuntos
Anticorpos Monoclonais/biossíntese , Cobre/metabolismo , Meios de Cultura/metabolismo , Glucuronidase/biossíntese , Imunoglobulina G/biossíntese , Cadeias kappa de Imunoglobulina/biossíntese , Ferro/metabolismo , Espectrometria de Massas/métodos , Animais , Anticorpos Monoclonais/genética , Reatores Biológicos , Células CHO , Proliferação de Células , Cricetulus , Glucuronidase/genética , Glicosilação , Hibridomas , Imunoglobulina G/genética , Cadeias kappa de Imunoglobulina/genética , Espectrometria de Massas/normas , Camundongos , Controle de Qualidade , Reprodutibilidade dos Testes , Fatores de Tempo , Transfecção
15.
Ann Anat ; 221: 27-37, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30240906

RESUMO

In a rat model of the andropause we aimed to examine the influence of daidzein, soy isoflavone, on the structure and function of parathyroid glands (PTG) and the expression levels of some of the crucial regulators of Ca2+ and Pi homeostasis in the kidney, and to compare these effects with the effects of estradiol, serving as a positive control. Middle-aged (16-month-old) male Wistar rats were divided into the following groups: sham-operated (SO), orchidectomized (Orx), orchidectomized and estradiol-treated (Orx+E; 0.625mg/kg b.w./day, s.c.) as well as orchidectomized and daidzein-treated (Orx+D; 30mg/kg b.w./day, s.c.) group. Every treated group had a corresponding control group. PTH serum concentration was decreased in Orx+E and Orx+D groups by 10% and 21% (p<0.05) respectively, in comparison with the Orx. PTG volume was decreased in Orx+E group by 16% (p<0.05), when compared to the Orx. In Orx+E group expression of NaPi 2a was lower (p<0.05), while NaPi 2a abundance in Orx+D animals was increased (p<0.05), when compared to Orx. Expression of PTH1R was increased (p<0.05) in Orx+E group, while in Orx+D animals the same parameter was decreased (p<0.05), in comparison with Orx. Klotho expression was elevated (p<0.05) in Orx+D rats, in regard to Orx. Orx+D induced reduction in Ca2+/creatinine and Pi/creatinine ratio in urine by 32% and 16% (p<0.05) respectively, in comparison with Orx. In conclusion, presented results indicate the more coherent beneficial effects of daidzein compared to estradiol, on disturbed Ca2+ and Pi homeostasis, and presumably on bone health, in the aging male rats.


Assuntos
Andropausa , Modelos Animais de Doenças , Glucuronidase/efeitos dos fármacos , Isoflavonas/farmacologia , Fitoestrógenos/farmacologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/efeitos dos fármacos , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Glucuronidase/genética , Glucuronidase/metabolismo , Masculino , Orquiectomia , Ratos , Ratos Wistar , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismo , Regulação para Cima
16.
Mol Med Rep ; 19(3): 1803-1808, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30592280

RESUMO

Aldosterone has an important role in the progression of renal fibrosis. In the present study, the concentration of aldosterone and klotho (KL) in the serum of patients with chronic kidney disease (CKD) were analyzed. A negative correlation was observed between aldosterone and KL, suggesting that KL may serve a protective role in CKD. Subsequently, an aldosterone­induced CKD mouse model was established using a single nephrectomy and subcutaneous osmotic pump with aldosterone and 1% high­salt drinking water. It was demonstrated that fibronectin 1 (Fn1) expression levels were higher in high aldosterone mice, whereas KL expression levels were low. In addition, the results demonstrated that histone deacetylase 1 (HDAC1) protein expression levels were upregulated in the renal distal convoluted tubules of high aldosterone mice, whereas acetylated H3K9 (H3K9Ac) was significantly downregulated. To determine the transcriptional activation status, chromatin immunoprecipitation polymerase chain reaction (PCR) was used to validate binding of H3K9Ac to the KL gene promoter site. It was revealed that the binding product of the KL promoter could be PCR­amplified at the H3K9Ac site from wild­type and low aldosterone mice; however, amplification of the binding product was not observed in high aldosterone mice. In conclusion, aldosterone significantly inhibited H3K9 acetylation by upregulating HDAC1 protein expression levels in the renal distal convoluted tubule cells, resulting in its inability to bind to the KL promoter, loss of transcription of the KL gene and increased expression of the renal fibrosis gene, Fn1.


Assuntos
Glucuronidase/genética , Histona Desacetilase 1/metabolismo , Rim/metabolismo , Rim/patologia , Transcrição Genética , Acetilação , Adulto , Idoso , Idoso de 80 Anos ou mais , Aldosterona , Animais , Regulação para Baixo , Feminino , Fibronectinas/metabolismo , Fibrose , Glucuronidase/metabolismo , Histonas/metabolismo , Humanos , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Lisina/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Insuficiência Renal Crônica/genética , Regulação para Cima/genética
17.
BMC Cardiovasc Disord ; 18(1): 237, 2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30547758

RESUMO

BACKGROUND: Klotho, possibly an age-regulating protein, is considered an important factor contributing to the lifespan and pathophysiology of hypertension and coronary artery disease (CAD). The present study was carried out aiming to investigate the association of Klotho-rs564481 (C1818T) gene polymorphism with hypertension and CAD. METHODS: A total of 286 CAD-suspicious subjects were entered into this case-control study. The polymorphism was investigated in hypertensive patients with no CAD (H-Tens, n = 60); hypertensive patients with CAD (CAD + H-Tens, n = 95); CAD patients with no hypertension (CAD, n = 61); and non-hypertensive non-CAD subjects, which were regarded as the control group (Ctrl, n = 70). Genotype and allele frequencies were assessed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: A significant difference was found in allele frequency of Klotho C1818T among the four research groups (P = 0.03). It was also found that wild-type homozygote subjects were negatively associated with hypertension as compared to heterozygote ones (OR = 0.07 [95% CI: 0.008-0.69] P = 0.02). Moreover, in the subgroups older than 57 years old, dominant genetic model demonstrated a negative association with CAD combined with hypertension (OR = 0.31 [95% CI: 0.10-0.95] P = 0.04). CONCLUSIONS: In conclusion, Klotho C1818T variant may be associated with a decreased risk of hypertension. Moreover, aging enhanced positive effects of the Klotho polymorphism on CAD combined with hypertension, indicating the possibility that the KLOTHO gene might play a part in the age-related occurrence of CAD combined with hypertension.


Assuntos
Doença da Artéria Coronariana/genética , Glucuronidase/genética , Hipertensão/genética , Polimorfismo Genético , Adulto , Fatores Etários , Idoso , Pressão Sanguínea/genética , Estudos de Casos e Controles , Angiografia Coronária , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/epidemiologia , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Heterozigoto , Homozigoto , Humanos , Hipertensão/diagnóstico , Hipertensão/epidemiologia , Hipertensão/fisiopatologia , Irã (Geográfico)/epidemiologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Fatores de Risco
18.
J Agric Food Chem ; 66(43): 11380-11389, 2018 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-30296070

RESUMO

In this study, computation-aided design on the basis of structural analysis was employed to rationally identify a highly dynamic C-terminal region that regulates the stability, expression level, and activity of a GH2 fungal glucuronidase from Aspergillus oryzae Li-3 (PGUS). Then, four mutants with a precisely truncated C-terminal region in different lengths were constructed; among them, mutant D591-604 with a 3.8-fold increase in half-life at 65 °C and a 6.8 kJ/mol increase in Gibbs free energy showed obviously improved kinetic and thermodynamic stability in comparison to PGUS. Mutants D590-604 and D591-604 both showed approximately 2.4-fold increases in the catalytic efficiency kcat/ Km and 1.8-fold increases in the expression level. Additionally, the expression level of PGUS was doubled through a C-terminal region swap with bacterial GUS from E. coli (EGUS). Finally, the robust PGUS mutants D590-604 and D591-604 were applied in the preparation of glycyrrhetinic acid with 4.0- and 4.4-fold increases in concentration through glycyrrhizin hydrolysis by a fed-batch process.


Assuntos
Aspergillus oryzae/enzimologia , Proteínas Fúngicas/metabolismo , Glucuronidase/metabolismo , Engenharia de Proteínas , Biotransformação , Biologia Computacional , Proteínas Fúngicas/genética , Glucuronidase/genética , Ácido Glicirretínico/metabolismo , Ácido Glicirrízico/metabolismo , Microbiologia Industrial , Simulação de Dinâmica Molecular , Proteínas Mutantes/metabolismo , Estrutura Terciária de Proteína
19.
World J Microbiol Biotechnol ; 34(10): 148, 2018 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-30218324

RESUMO

Morchella importuna is a worldwide distributed edible mushroom with high ecological and economic values, but the molecular and genetic research about this mushroom has been hindered due to lack of an efficient transformation method. Here, we report for the first time the successful transformation of M. importuna by using a hypervirulent Agrobacterium tumefaciens strain bearing the constructed binary plasmid p1391-U-GUS. The selectable markers used were the genes for hygromycin resistance under the control of the polyubquitin promoter from M. importuna. The reporter genes were those for enhanced green fluorescent protein (EGFP) and the ß-Glucuronidase (GUS) under the control of glyceraldehyde-3-phosphate dehydrogenase promoter and polyubquitin promoter respectively. The presence of the reporter gene EGFP in the transformants was confirmed by the fluorescence and confocal microscope and molecular analysis and that of the reporter gene GUS was verified by enzyme activity and molecular analysis. The analysis results of both reporter genes indicated that Agrobacterium-mediated transformation was successfully performed in M. importuna.


Assuntos
Agaricales/genética , Agrobacterium tumefaciens/genética , Ascomicetos/genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Biologia Molecular/métodos , Poliubiquitina/genética , Regiões Promotoras Genéticas/genética , Transformação Genética , Agaricales/metabolismo , Ascomicetos/citologia , Ascomicetos/metabolismo , Cinamatos/farmacologia , DNA Bacteriano/genética , Regulação Fúngica da Expressão Gênica , Genes Reporter , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Glucuronidase/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Plasmídeos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
20.
Pregnancy Hypertens ; 13: 95-99, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30177080

RESUMO

Preeclampsia (PE) is a serious disorder of human pregnancy and always is accompanied with multi-organ disorder, which severely threatens the health of both the mothers and the offspring. The oxidative stress and genetic factors involves in the development of PE. The Klotho encodes Klotho protein that is capable of increasing resistance to oxidative stress. Thus, we designed this case-control study to investigate the association between Klotho polymorphisms and the susceptibility to PE in Chinese Han women. Two single nucleotide polymorphisms (SNPs) (rs1207568 and rs564481) in Klotho were selected to be genotyped in 1002 PE patients and 1384 normal controls with TaqMan allelic discrimination real-time PCR technology. There were no significant differences in genotypic or allelic frequencies at both polymorphic sites between PE patients and controls (rs1207568: χ2 = 2.386, p = 0.303 by genotype, χ2 = 2.357, p = 0.125, OR = 1.127, 95%CI 0.968-1.312 by allele; rs564481: χ2 = 1.195, p = 0.550 by genotype, χ2 = 0.018, p = 0.894, OR = 1.010, 95%CI 0.875-1.165 by allele). Furthermore, we divided the cases into mild vs severe and early-onset vs late-onset subgroups and then analyzed the relationships between these subgroups and the control group respectively. As a consequence, no significant differences were found for both SNPs in each case. These results suggested that the genetic variants of rs1207568 and rs564481 in Klotho may not play a pivotal role in the pathogenesis of PE in Chinese Han women.


Assuntos
Predisposição Genética para Doença , Glucuronidase/genética , Pré-Eclâmpsia/genética , Diagnóstico Pré-Natal , Adulto , Grupo com Ancestrais do Continente Asiático , Estudos de Casos e Controles , China , Feminino , Humanos , Polimorfismo de Nucleotídeo Único , Pré-Eclâmpsia/diagnóstico , Gravidez , Fatores de Risco
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