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1.
Acta Pharmacol Sin ; 41(3): 348-357, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31506573

RESUMO

Intestinal mucositis is a common side effect of anticancer regimens that exerts a negative impact on chemotherapy. Superoxide dismutase (SOD) is a potential therapy for mucositis but efficient product is not available because the enzyme is degraded following oral administration or induces an immune reaction after intravascular infusion. Multi-modified Stable Anti-Oxidant Enzymes® (MS-AOE®) is a new recombinant SOD with better resistance to pepsin and trypsin. We referred it as MS-SOD to distinguish from other SODs. In this study we investigated its potential to alleviate 5-FU-induced intestinal injury and the mechanisms. An intestinal mucositis model was established in C57/BL6 mice by 5-day administration of 5-FU (50 mg/kg every day, ip). MS-SOD (800 IU/10 g, ig) was given once daily for 9 days. 5-FU caused severe mucositis with intestinal morphological damage, bodyweight loss and diarrhea; MS-SOD significantly decreased the severity. 5-FU markedly increased reactive oxygen species (ROS) and inflammatory cytokines in the intestine which were ameliorated by MS-SOD. Furthermore, MS-SOD modified intestinal microbes, particularly reduced Verrucomicrobia, compared with the 5-FU group. In Caco2 cells, MS-SOD (250-1000 U/mL) dose-dependently decreased tBHP-induced ROS generation. In RAW264.7 cells, MS-SOD (500 U/mL) had no effect on LPS-induced inflammatory cytokines, but inhibited iNOS expression. These results demonstrate that MS-SOD can scavenge ROS at the initial stage of injury, thus play an indirect role in anti-inflammatory and barrier protein protection. In conclusion, MS-SOD attenuates 5-FU-induced intestinal mucositis by suppressing oxidative stress and inflammation, and influencing microbes. MS-SOD may exert beneficial effect in prevention of intestinal mucositis during chemotherapy in clinic.


Assuntos
Fluoruracila/efeitos adversos , Mucosa Intestinal/metabolismo , Superóxido Dismutase/metabolismo , Administração Oral , Animais , Fluoruracila/administração & dosagem , Fluoruracila/metabolismo , Injeções Intraperitoneais , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Superóxido Dismutase/administração & dosagem
2.
J Appl Toxicol ; 35(12): 1473-80, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25727789

RESUMO

Basic Violet 14, Direct Red 28 and Acid Red 26 are classified as carcinogenic dyes in the European textile ecology standard, despite insufficient toxicity data. In this study, the toxicity of these dyes was assessed in a zebrafish model, and the underlying toxic mechanisms were investigated. Basic Violet 14 and Direct Red 28 showed acute toxicity with a LC50 value at 60.63 and 476.84 µg ml(-1) , respectively, whereas the LC50 of Acid Red 26 was between 2500 and 2800 µg ml(-1) . Treatment with Basic Violet 14, Direct Red 28 and Acid Red 26 resulted in common developmental abnormalities including delayed yolk sac absorption and swimming bladder deflation. Hepatotoxicity was observed in zebrafish treated with Basic Violet 14, and cardiovascular toxicity was found in zebrafish treated with Acid Red 26 at concentrations higher than 2500 µg ml(-1) . Basic Violet 14 also caused significant up-regulation of GCLC gene expression in a dose-dependent manner whereas Acid Red 26 induced significant up-regulation of NKX2.5 and down-regulation of GATA4 at a high concentration in a dose-dependent manner. These results suggest that Basic Violet 14, Direct Red 28 and Acid Red 26 induce developmental and organ-specific toxicity, and oxidative stress may play a role in the hepatotoxicity of Basic Violet 14, the suppressed GATA4 expression may have a relation to the cardiovascular toxicity of Acid Red 26.


Assuntos
Compostos Azo/toxicidade , Vermelho Congo/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Corantes de Rosanilina/toxicidade , Peixe-Zebra/embriologia , Alternativas ao Uso de Animais , Animais , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/embriologia , Larva , Dose Letal Mediana , Fígado/efeitos dos fármacos , Fígado/embriologia , Fígado/ultraestrutura , Testes de Toxicidade
3.
Biochim Biophys Acta ; 1834(1): 464-9, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22819876

RESUMO

Substantial evidence suggests that transient production of reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) is an important signaling event triggered by the activation of various cell surface receptors. Major targets of H(2)O(2) include protein tyrosine phosphatases (PTPs). Oxidation of the active site Cys by H(2)O(2) abrogates PTP catalytic activity, thereby potentially furnishing a mechanism to ensure optimal tyrosine phosphorylation in response to a variety of physiological stimuli. Unfortunately, H(2)O(2) is poorly reactive in chemical terms and the second order rate constants for the H(2)O(2)-mediated PTP inactivation are ~10M(-1)s(-1), which is too slow to be compatible with the transient signaling events occurring at the physiological concentrations of H(2)O(2). We find that hydroxyl radical is produced from H(2)O(2) solutions in the absence of metal chelating agent by the Fenton reaction. We show that the hydroxyl radical is capable of inactivating the PTPs and the inactivation is active site directed, through oxidation of the catalytic Cys to sulfenic acid, which can be reduced by low molecular weight thiols. We also show that hydroxyl radical is a kinetically more efficient oxidant than H(2)O(2) for inactivating the PTPs. The second-order rate constants for the hydroxyl radical-mediated PTP inactivation are at least 2-3 orders of magnitude higher than those mediated by H(2)O(2) under the same conditions. Thus, hydroxyl radical generated in vivo may serve as a more physiologically relevant oxidizing agent for PTP inactivation. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.


Assuntos
Peróxido de Hidrogênio/química , Radical Hidroxila/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/química , Animais , Catálise , Bovinos , Humanos , Cinética , Oxirredução , Ácidos Sulfênicos/química
4.
Biochem J ; 441(2): 623-32, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-21967612

RESUMO

The formation of intracellular nitrogen-based oxidants has important physiological and pathological consequences. CK (creatine kinase), which plays a key role in intracellular energy metabolism, is a main target of low concentrations of oxidative and nitrative stresses. In the present study, the interaction between cytosolic CKs [MM-CK (muscle-type CK) and BB-CK (brain-type CK)] and MTs [metallothioneins; hMT2A (human MT-IIA) and hMT3 (human MT-III)] were characterized by both in vitro and intact-cell assays. MTs could successfully protect the cytosolic CKs against inactivation induced by low concentrations of PN (peroxynitrite) and NO both in vitro and in hMT2A-overexpressing H9c2 cells and hMT3-knockdown U-87 MG cells. Under high PN concentrations, CK formed granule-like structures, and MTs were well co-localized in these aggregated granules. Further analysis indicated that the number of cells containing the CK aggregates negatively correlated with the expression levels of MTs. In vitro experiments indicated that MTs could effectively protect CKs against aggregation during refolding, suggesting that MT might function as a chaperone to assist CK re-activation. The findings of the present study provide direct evidence of the connection between the two well-characterized intracellular systems: the precisely balanced energy homoeostasis by CKs and the oxidative-stress response system using MTs.


Assuntos
Creatina Quinase Forma BB/metabolismo , Creatina Quinase Forma MM/metabolismo , Metalotioneína/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Óxido Nítrico/farmacologia , Ácido Peroxinitroso/farmacologia , Animais , Células CHO , Creatina Quinase Forma BB/antagonistas & inibidores , Creatina Quinase Forma MM/antagonistas & inibidores , Cricetinae , Citosol/enzimologia , Humanos , Metalotioneína 3 , Camundongos , Chaperonas Moleculares/farmacologia
5.
Zhongguo Zhong Yao Za Zhi ; 38(12): 1915-8, 2013 Jun.
Artigo em Zh | MEDLINE | ID: mdl-24066583

RESUMO

The dried Whitmania pigra is used for the treatment of cardiovascular and cerebrovascular diseases in traditional Chinese medicine. Bellamya purificata is widely distributed in the Chang Jiang River basin, it is natural diets of W. pigra. Current study was conducted to compare and analyze the nutritional ingredient in W. pigra, body fluid and flesh of B. purificata. Results showed that the contents of protein, crude fat and total sugar in W. pigra, body fluid and flesh of B. purificata were significantly different (P < 0.05). Protein content in W. pigra accounts up to 65.01%. The contents of inorganic elements and amino acid were abundant in W. pigra, body fluid and flesh of B. purificata. The content of essential amino acids in them were 32.6, 221.59, 40.78 mg x g(-1), respectively. The content of flavor amino acid in them were 27.51, 14.5, 32.03 mg x g(-1), while the coresponding content of antioxidant amino acid were 8.81, 5.91, 9.73 mg x g(-1), respectively. The individual amino acids of high content in them were Glu, Asp and Leu. Macro elements Ca, P, Mg and trace elements Zn, Si, Fe were abundant. It could be speculated that W. pigra may be a promising novel food, and the present results provide a foundation to develop artificial feed for W. Pigra.


Assuntos
Gastrópodes/química , Sanguessugas/química , Aminoácidos/análise , Animais , Medicina Tradicional Chinesa
6.
Biochem J ; 436(2): 437-45, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21426302

RESUMO

Muscle contraction requires high energy fluxes, which are supplied by MM-CK (muscle-type creatine kinase) which couples to the myofibril. However, little is known about the detailed molecular mechanisms of how MM-CK participates in and is regulated during muscle contraction. In the present study, MM-CK is found to physically interact with the slow skeletal muscle-type MyBPC1 (myosin-binding protein C1). The interaction between MyBPC1 and MM-CK depended on the creatine concentration in a dose-dependent manner, but not on ATP, ADP or phosphocreatine. The MyBPC1-CK interaction favoured acidic conditions, and the two molecules dissociated at above pH 7.5. Domain-mapping experiments indicated that MM-CK binds to the C-terminal domains of MyBPC1, which is also the binding site of myosin. The functional coupling of myosin, MyBPC1 and MM-CK is further corroborated using an ATPase activity assay in which ATP expenditure accelerates upon the association of the three proteins, and the apparent K(m) value of myosin is therefore reduced. The results of the present study suggest that MyBPC1 acts as an adaptor to connect the ATP consumer (myosin) and the regenerator (MM-CK) for efficient energy metabolism and homoeostasis.


Assuntos
Proteínas de Transporte/fisiologia , Creatina Quinase Forma MM/metabolismo , Fibras Musculares de Contração Lenta/fisiologia , Miosinas/metabolismo , Animais , Metabolismo Energético/fisiologia , Células HEK293 , Homeostase/fisiologia , Humanos , Camundongos , Fibras Musculares de Contração Lenta/enzimologia , Músculo Esquelético/citologia , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Ligação Proteica/fisiologia , Recrutamento Neurofisiológico/fisiologia
7.
Int Urol Nephrol ; 54(7): 1681-1691, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34783980

RESUMO

PURPOSE: To evaluate the effects of manganese superoxide dismutase (Mn-SOD) from thermophilic bacterium HB27 (name as Tt-SOD) on chemical cystitis. METHODS: Control and experimental rats were infused by intravesical saline or hydrochloric acid (HCl) on the first day of the experiments. Saline, sodium hyaluronate (SH) or Tt-SOD were infused intravesically once a day for three consequent days. On the fifth day, the rats were weighted and sacrificed following a pain threshold test. The bladder was harvested for histological and biochemical analyses. RESULTS: Tt-SOD could reduce the bladder index, infiltration of inflammatory cells in tissues, serum inflammatory factors and SOD levels, mRNA expression of inflammatory factors in tissues, and increase perineal mechanical pain threshold and serum MDA and ROS levels in HCl-induced chemical cystitis. Furthermore, Tt-SOD alleviated inflammation and oxidative stress by the negative regulation of the NF-κB p65 and p38 MAPK signaling pathway. CONCLUSIONS: Intravesical instillation of Tt-SOD provides protective effects against HCl-induced cystitis.


Assuntos
Proteínas de Bactérias , Cistite , Superóxido Dismutase , Animais , Proteínas de Bactérias/uso terapêutico , Cistite/induzido quimicamente , Cistite/terapia , Ácido Clorídrico/efeitos adversos , Inflamação/metabolismo , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/uso terapêutico , Bexiga Urinária/patologia
8.
J Biomol Struct Dyn ; 38(10): 2884-2897, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31345118

RESUMO

In this study, we aimed to identify critical factors associated with superoxide dismutase 2 (SOD2) in human keratinocytes through gene and protein expression profiling approaches. After recombinant SOD2 was exogenously added to culture media, we conducted serial OMICS studies, which included RNA sequencing analysis, integrated antibody-chip arrays, and the implementation of bioinformatics algorithms, in order to reveal genes and proteins that are possibly associated with SOD2 in keratinocytes. These approaches identified several novel genes and proteins in keratinocytes that are associated with exogenous SOD2. These novel genes included DCT, which was up-regulated, and CD38, GPR151, HCK, KIT, and AFP, which were down-regulated. Among them, CD38 and KIT were also predicted as hub proteins in PPI mappings. By integrating the datasets obtained from these complementary high-throughput OMICS studies and utilizing the strengths of each method, we obtained new insights into the functional role of externally added SOD2 in skin cells and into several critical genes that are thought to play important roles in SOD2-associated skin function. The approach used here could help contribute to our clinical understanding of SOD2-associated applications and may be broadly applicable to a wider range of diseases. AbbreviationsSOD2superoxide dismutase 2DAVIDthe database for annotation, visualization and integrated discoveryKEGGKyoto Encyclopedia of Genes and GenomesPPIprotein-protein interactionsHTSHigh-throughput screeningCommunicated by Ramaswamy H. Sarma.


Assuntos
Biologia Computacional , Superóxido Dismutase , Humanos , Queratinócitos , Análise de Sequência de RNA , Superóxido Dismutase/genética
9.
Mol Cell Biol ; 26(1): 199-208, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16354691

RESUMO

Kaiso is a BTB domain protein that associates with the signaling molecule p120-catenin and binds to the methylated sequence mCGmCG or the nonmethylated sequence CTGCNA to modulate transcription. In Xenopus laevis, xKaiso deficiency leads to embryonic death accompanied by premature gene activation in blastulae and upregulation of the xWnt11 gene. Kaiso has also been proposed to play an essential role in mammalian synapse-specific transcription. We disrupted the Kaiso gene in mice to assess its role in mammalian development. Kaiso-null mice were viable and fertile, with no detectable abnormalities of development or gene expression. However, when crossed with tumor-susceptible Apc(Min/+) mice, Kaiso-null mice showed a delayed onset of intestinal tumorigenesis. Kaiso was found to be upregulated in murine intestinal tumors and is expressed in human colon cancers. Our data suggest that Kaiso plays a role in intestinal cancer and may therefore represent a potential target for therapeutic intervention.


Assuntos
Neoplasias Intestinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Marcação de Genes , Neoplasias Intestinais/metabolismo , Camundongos , Camundongos Mutantes , Neurônios/citologia , Fenótipo , Ativação Transcricional , Regulação para Cima
10.
J Enzyme Inhib Med Chem ; 24(1): 234-40, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18608781

RESUMO

The present work focused on the kinetics of the inhibitory effects of the leaf extract of Siberian Crabapple, named Shan jingzi in China, on chicken liver fatty acid synthase. The results showed that this extract had much stronger inhibitory ability on fatty acid synthase than that from green teas described in many previous reports. The inhibitory ability of this extract is closely related to the extracting solvent, and the time of extraction was also an important influencing factor. The inhibitory types of this extract on diffeerent substrates of chicken liver fatty acid synthase, acetyl-CoA, malonyl-CoA and NADPH, were found to be noncompetitive, uncompetitive and mixed, respectively. The studies here shed a new light on the exploration for inhibitors of fatty acid synthase.


Assuntos
Ácido Graxo Sintases/antagonistas & inibidores , Malus/química , Extratos Vegetais/isolamento & purificação , Animais , Galinhas , Cinética , Extratos Vegetais/farmacologia , Folhas de Planta/química , Solventes
12.
J Biomol Struct Dyn ; 24(4): 359-68, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17206851

RESUMO

The monomeric state of creatine kinase (CK) was stably captured at the equilibrium state by employing cysteine residue modifications in the presence of a denaturant, and at a partially folded state. The partially folded monomeric CK (PF-CK) was aggregated with kinetic order, which was mainly caused by the hydrophobic surface interactions between the CK subunits. The artificial chaperone, described as a SDS-cyclodextrin, was applied to prevent aggregation as well as to refold the PF-CK: SDS treatment onto the monomeric CK can significantly block aggregation and can be successfully refolded in the solutions containing cyclodextrins and DTT. Three types of cyclodextrins such as alpha-, beta-, and gamma-cyclodextrins were applied to strip SDS from the enzyme molecule, and each kinetic course was measured. The intrinsic fluorescence changes showed that reactivation occurred and this accompanied the conformational changes. The size exclusion chromatography detected the variously trapped monomeric CKs such as the thiol residue modified PF-CK, the SDS-binding PF-CK, the cyclodextrin treated PF-CK, and the DTT treated SDS-binding PF-CK. Our study demonstrated monomer CK aggregation for the first time; we also demonstrated the complex reassociation of CK during refolding with the aid of the SDS-cyclodextrin, and these pathways followed first-order kinetics.


Assuntos
Creatina Quinase/química , Creatina Quinase/metabolismo , Ciclodextrinas/farmacologia , Cisteína , Dodecilsulfato de Sódio/farmacologia , Animais , Ativação Enzimática , Cinética , Músculo Esquelético/enzimologia , Dobramento de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Coelhos
13.
Int J Biol Macromol ; 41(4): 439-46, 2007 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-17673285

RESUMO

The dimeric native state of creatine kinase (CK) was aggregated at conspicuous levels during cysteine modification at the active site with using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) under a high enzyme concentration. Measuring the ANS-binding fluorescence revealed that the hydrophobic surface of CK was increased by cysteine modification due to the flexible active site, and this resulted in insoluble aggregation, probably via non-specific hydrophobic interactions. To determine whether the aggregates can be refolded, 3M guanidine hydrochloride (GdnHCl) was used to dissolve the aggregates into the denatured form. Refolding of the solubilized enzyme sample was then conducted, accompanied by deprivation of DTNB from the CK in the presence of DTT. As a result, CK was reactivated by up to 40% with partial recovery of the tertiary (78%) and secondary structures (77%). To further elucidate its kinetic refolding pathway, both time interval measurements and a continuous substrate reaction were performed. The results showed that the refolding behavior was similar to the manner of normal CK folding with respect to the following two-phase kinetic courses. Additionally, the rate constants for the dimerization of the unfolded CK were dependent on the enzyme concentration and this was irrespective to the DTT concentrations, suggesting the rate-limiting steps of CK reassociation. The present study will expand our insight into the flexibility of the enzyme active site, which might act as a risk factor for inducing the unfavorable aggregation and partial refolding pathway of CK, as well as inducing an intermediate-like state recovery from aggregation.


Assuntos
Creatina Quinase/química , Creatina Quinase/metabolismo , Cisteína/química , Dobramento de Proteína , Naftalenossulfonato de Anilina/metabolismo , Animais , Sítios de Ligação , Cromatografia em Gel , Dicroísmo Circular , Creatina Quinase/análise , Dimerização , Ácido Ditionitrobenzoico/farmacologia , Ditiotreitol/farmacologia , Relação Dose-Resposta a Droga , Corantes Fluorescentes/metabolismo , Guanidina/farmacologia , Cinética , Modelos Biológicos , Músculos/enzimologia , Desnaturação Proteica/efeitos dos fármacos , Renaturação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Coelhos , Espectrofotometria Ultravioleta
14.
Int J Biol Macromol ; 40(5): 437-43, 2007 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-17173966

RESUMO

Osmolytes are a series of different kinds of small molecules that can maintain the correct conformation of protein by acting as molecular chaperons. In this study, the protective effects of four compatible osmolytes, i.e., proline, sucrose, DMSO and glycerol, were studied during arginine kinase (EC 2.7.3.3) unfolding and aggregation. The results showed that all the osmolytes applied in this study obviously prevented AK unfolding and inactivation that was due to a GdnHCl denaturant by reducing the inactivation rate constants (k(i)), increasing the transition free energy changes (DeltaDeltaG(i)) and increasing the value for the midpoint of denaturation (C(m)). Furthermore, the osmolytes remarkably prevented AK aggregation in a concentration-dependent manner during AK refolding. Our results strongly indicated that osmolytes were not only metabolism substrates, but they were also important compounds with significant physiological protective functions for proteins, especially in some extremely harsh environments.


Assuntos
Arginina Quinase/química , Arginina Quinase/metabolismo , Dimetil Sulfóxido/farmacologia , Glicerol/farmacologia , Prolina/farmacologia , Dobramento de Proteína , Sacarose/farmacologia , Naftalenossulfonato de Anilina , Animais , Ativação Enzimática/efeitos dos fármacos , Fluorescência , Gafanhotos/enzimologia , Guanidina/farmacologia , Cinética , Desnaturação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína/efeitos dos fármacos , Espectrometria de Fluorescência
15.
Mol Med Rep ; 16(6): 7939-7948, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28983596

RESUMO

CDGSH iron sulfur domain 2 (CISD2) has been found to be important in carcinogenesis. However, the role of CISD2 in glioma remains to be elucidated. The present study aimed to investigate the role of CISD2 in glioma using the reverse transcription­quantitative polymerase chain reaction, western blotting, co­immunoprecipitation assay, immunofluorescence staining and other methods. The results demonstrated that the mRNA and protein levels of CISD2 were found to be upregulated in glioma tissues, compared with the levels in matched normal tissues. Clinical data analysis showed that the level of CISD2 was negatively correlated with the survival rates of patients with glioma. In addition, high levels of CISD2 were associated with advanced clinical stage, relapse, vascular invasion and increased tumor size. The inhibition of CISD2 suppressed the proliferation and survival of glioma cells in vitro and in vivo. Mechanistically, it was found that small interfering RNA­induced knock down of CISD2 inhibited the proliferation of glioma cells through activating beclin­1­mediated autophagy. The results also revealed that CISD2 was a target of microRNA (miR)­449a. Together, the results of the present study demonstrated that CISD2 was increased in glioma samples and was associated with poor prognosis and aggressive tumor behavior. The miR­449a/CISD2/beclin­1­mediated autophagy regulatory network contributed to the proliferation of glioma cells. Targeting this pathway may be a promising strategy for glioma therapy.


Assuntos
Autofagia/genética , Proteína Beclina-1/genética , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/genética , Interferência de RNA , Adulto , Idoso , Animais , Proteína Beclina-1/metabolismo , Biomarcadores Tumorais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Modelos Animais de Doenças , Feminino , Inativação Gênica , Glioma/mortalidade , Glioma/patologia , Xenoenxertos , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Pessoa de Meia-Idade , Modelos Biológicos , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Carga Tumoral
16.
Biomed Pharmacother ; 93: 230-237, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28645007

RESUMO

The central objective was to identify the role of the PI3K-Akt activation pathway on the neuroprotection of δ-opioid receptor agonist (DADLE) against cerebral ischemia-reperfusion (I/R) injury in a rat model. Fifty-five male Sprague-Dawley (SD) rats were included to establish a middle cerebral artery occlusion (MCAO) model which were then divided into the sham, MCAO, LY294002 (MCAO+DADLE+LY294002 [inhibitor of PI3K-Akt pathway]), DADLE (MCAO+DADLE) and DMSO (MCAO+DADLE+DMSO [dimethyl sulphoxide]) groups. The cerebral infarction (CI) volume and nerve cell apoptosis was determined using TTC and TUNEL staining. Quantitative real-time polymerase chain reaction (qRT-PCR), western blotting and immunohistochemistry staining were applied for the expressions of Bad, Bax, Bcl-2 and cleaved caspase-3. The MCAO group showed higher CI volume, nerve cell apoptosis and cleaved caspase-3 expressions than the DADLE and DMSO groups, which were also higher in the LY294002 group than the DADLE group. Compared with the MCAO group, the mRNA and protein expressions of PI3K and Bcl-2, and the protein expressions of p-Akt and p-Bad were elevated, while the mRNA and protein expressions of Bax were decreased in the DADLE and DMSO groups. Decreased mRNA and protein expressions of PI3K and Bcl-2, reduced protein expressions of p-Akt and p-Bad and elevated mRNA and protein expressions of Bax exhibited in the LY294002 group than the DADLE group. These results indicate that activation of PI3K-Akt pathway promotes the neuroprotection of DADLE against cerebral I/R injury in a rat model by decreasing nerve cells apoptosis.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Opioides delta/agonistas , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Isquemia Encefálica/metabolismo , Caspase 3/metabolismo , Ciclina D1/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/metabolismo , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismo
17.
Sci Rep ; 5: 8560, 2015 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-25708299

RESUMO

PP2C family phosphatases (the type 2C family of protein phosphatases; or metal-dependent phosphatase, PPM) constitute an important class of signaling enzymes that regulate many fundamental life activities. All PP2C family members have a conserved binuclear metal ion active center that is essential for their catalysis. However, the catalytic role of each metal ion during catalysis remains elusive. In this study, we discovered that mutations in the structurally buried D38 residue of PP2Cα (PPM1A) redefined the water-mediated hydrogen network in the active site and selectively disrupted M2 metal ion binding. Using the D38A and D38K mutations of PP2Cα as specific tools in combination with enzymology analysis, our results demonstrated that the M2 metal ion determines the rate-limiting step of substrate hydrolysis, participates in dianion substrate binding and stabilizes the leaving group after P-O bond cleavage. The newly characterized catalytic role of the M2 metal ion in this family not only provides insight into how the binuclear metal centers of the PP2C phosphatases are organized for efficient catalysis but also helps increase our understanding of the function and substrate specificity of PP2C family members.


Assuntos
Metais/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Íons/química , Cinética , Manganês/química , Manganês/metabolismo , Metais/química , Simulação de Dinâmica Molecular , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Proteína Fosfatase 2C , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Transdução de Sinais
18.
Int J Biochem Cell Biol ; 36(3): 447-59, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14687923

RESUMO

The thermostability of an enzyme that exhibits phytase and acid phosphatase activities was studied. Kinetics of inactivation and unfolding during thermal denaturation of the enzyme were compared. The loss of phytase activity on thermal denaturation is most suggestive of a reversible process. As for acid phosphatase activities, an interesting phenomenon was observed; there are two phases in thermal inactivation: when the temperature was between 45 and 50 degrees C, the thermal inactivation could be characterized as an irreversible inactivation which had some residual activity and when the temperature was above 55 degrees C, the thermal inactivation could be characterized as an irreversible process which had no residual activity. The microscopic rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method [Adv. Enzymol. Relat. Areas Mol. Biol. 61 (1988) 381]. Fluorescence analyses indicate that when the enzyme was treated at temperatures below 60 degrees C for 60 min, the conformation of the enzyme had no detectable change; when the temperatures were above 60 degrees C, some fluorescence red-shift could be observed with a decrease in emission intensity. The inactivation rates (k(+0)) of free enzymes were faster than those of conformational changes during thermal denaturation at the same temperature. The rapid inactivation and slow conformational changes of phytase during thermal denaturation suggest that inactivation occurs before significant conformational changes of the enzyme, and the active site of this enzyme is situated in a relatively fragile region which makes the active site more flexible than the molecule as a whole.


Assuntos
6-Fitase/química , Fosfatase Ácida/química , Conformação Proteica , 6-Fitase/antagonistas & inibidores , 6-Fitase/metabolismo , Fosfatase Ácida/antagonistas & inibidores , Fosfatase Ácida/metabolismo , Compostos de Anilina/química , Ração Animal/normas , Aditivos Alimentares/normas , Temperatura Alta , Cinética , Compostos Organofosforados/química , Fósforo/química , Desnaturação Proteica , Espectrometria de Fluorescência
19.
Int J Biol Macromol ; 31(1-3): 97-102, 2002 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-12559432

RESUMO

The inactivation and conformational changes of porcine heart lactate dehydrogenase (LDH) have been studied in sodium dodecyl sulfate (SDS) solutions. Increasing SDS concentration led to a quick and concentration-dependent inhibition of the enzyme, with complete inactivation within 5 min in the presence of 1.0 mM SDS. Meanwhile, fluorescence emission and circular dichroism spectra were used to follow the conformational changes of the enzyme during this process, concurrently showing that SDS less than 1.0 mM induced only limited conformational changes to LDH. The above results are in accordance with the suggestion by Tsou (Trends Biochem. Sci. 11 (1986) 427; Science 262 (1993) 380) that the active site usually be more flexible than the enzyme molecule as a whole. Furthermore, the results of polyacrylamide gel electrophoresis (PAGE) implied that unfolding intermediates were presented in the above process. When the SDS concentration used to treat LDH was increased, the bands of native enzyme on native PAGE faded and finally almost disappeared. Meanwhile, multiple bands with lower mobility but no activity emerged behind and enhanced correspondingly. Fast protein liquid chromatography indicated that dissociation occurred during the course of denaturation. The reasons for the above phenomena have been discussed. It was suggested that SDS, binding to LDH to form different LDH-SDS complexes, conferred an array of different unfolding states over the enzyme, and in turn resulted in the formation of the multiple bands on the native PAGE.


Assuntos
L-Lactato Desidrogenase/química , Miocárdio/enzimologia , Dodecilsulfato de Sódio/química , Animais , Cromatografia Líquida , Dicroísmo Circular , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Conformação Proteica , Dodecilsulfato de Sódio/farmacologia , Espectrometria de Fluorescência , Suínos , Fatores de Tempo
20.
Huan Jing Ke Xue ; 35(6): 2287-93, 2014 Jun.
Artigo em Zh | MEDLINE | ID: mdl-25158508

RESUMO

Luminescent bacteria have attracted more and more attention in recent years as an effective mean for biological toxicity of water environment monitoring. First of all, fluorescent protein Frex was correctly expressed in Escherichia coli, and then the effect of toxic substances on microbial metabolism in the water was monitored through the determination of the changes in the fluorescence intensity in bacteria caused by the change of NADH level in the bacteria. Then the effects of culture temperature, inducing time and the final concentration of inductor isopropyl beta-D-thiogalactopyranoside (IPTG) on the expression level and fluorescent activity of the fusion protein Frex were studied. The recombinant fluorescent bacteria was then applied in the initial detection of toxic substances in water environment. Four international standard substances of biological toxicity test including HgCl2, 3,5-dichlorophenol, potassium dichromate, and zinc sulfate heptahydrate were chosen to conduct experimental assay. The results suggested that all of these substances can cause a rapid decrease in the fluorescence of the bacteria. This test method has advantages of rapid reaction and high sensitivity. Meanwhile, the optimization of the conditions for the biological toxicity test lays foundation for subsequent application, and expands the application scope of luminescent bacteria in other aspects.


Assuntos
Escherichia coli , Proteínas Luminescentes/química , Proteínas Recombinantes de Fusão/química , Testes de Toxicidade/métodos , Poluentes Químicos da Água/análise , Bioensaio , Clorofenóis/análise , Fluorescência , Isopropiltiogalactosídeo/química , Cloreto de Mercúrio/análise , Dicromato de Potássio/análise , Temperatura , Sulfato de Zinco/análise
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