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1.
Nat Commun ; 12(1): 4718, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354069

RESUMO

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.


Assuntos
Fosfatidato Fosfatase/química , Células 3T3-L1 , Adipogenia , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Sequência Conservada , Cristalografia por Raios X , Células HEK293 , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Genética
2.
Meat Sci ; 171: 108292, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32896773

RESUMO

Beef rolls for hot pot are usually stored and transported in a frozen state, and the beef color deteriorates quickly. This paper reports on an investigation into the effect of packaging method, freezing temperature and storage time on instrumental color, pH, myoglobin state, lipid oxidation (TBARS) and total volatile basic nitrogen (TVB-N) of beef rolls. It was shown that the color of beef rolls at -18 °C was better than that at -12 °C overall, and the OxyMb% and pH values were higher, while the MetMb% and TBARS were lower with storage at -18 °C. With the extension of storage time, the instrumental color, OxyMb% and pH values of beef rolls decreased. Correspondingly, the MetMb% and TBARS showed an upward trend. However, the TVB-N of all treatments did not exceed the Chinese standard during 180d of storage. The results of this paper provide a number of recommendations for the storage of frozen beef rolls to extend color-shelf life.


Assuntos
Cor , Embalagem de Alimentos/métodos , Congelamento , Produtos da Carne/análise , Animais , Bovinos , Armazenamento de Alimentos/métodos , Concentração de Íons de Hidrogênio , Mioglobina/análise , Nitrogênio/análise , Substâncias Reativas com Ácido Tiobarbitúrico/análise
3.
J Infect Dev Ctries ; 11(12): 962-966, 2018 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31626603

RESUMO

INTRODUCTION: H9N2 avian influenza viruses (AIV) can transmit in chicken flocks through direct contact and aerosols. Nevertheless, data on airborne transmission of AIV is very limited, especially under field conditions. To fill this literature gap, this study was designed to investigate airborne transmission of H9N2 AIV originating from infected chicken flocks under field conditions, with the aim to further characterize the airborne transmission of H9N2 AIV. METHODOLOGY: Oropharyngeal swabs were collected from different diseased chickens to confirm H9N2 AIV infection. All glass impingers 30 (AGI-30) were used to collect indoor, upwind and downwind air samples for three chicken houses with H9N2 AIV infected chickens. Swabs and air samples were tested for H9N2 AIV using a real-time reverse transcription polymerase chain reaction (RRT-PCR). H9N2 AIV was isolated in embryonated chicken eggs and hemagglutinin (HA) gene sequence similarity of the isolated AIV was compared. RESULTS: The results showed that indoor air samples were all RRT-PCR positive for H9N2 AIV. Downwind air samples collected between 10 m and 1.5 km away from the chicken houses were also found positive with an average load 2.62-5.21×103 RNA copies/m3. However, upwind air samples were all negative for H9N2 AIV. In addition, H9N2 AIV was isolated from swabs and indoor air samples. CONCLUSION: In summary, this study provides insights into the airborne transmission of H9N2 AIV under field conditions.

4.
DNA Repair (Amst) ; 42: 1-10, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27130982

RESUMO

DNA double-strand breaks (DSBs) are potentially lethal lesions repaired by two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). Homologous recombination preferentially reunites cognate broken ends. In contrast, non-homologous end-joining could ligate together any two ends, possibly generating dicentric or acentric fragments, leading to inviability. Here, we characterize the yeast NHEJ pathway in populations of pure G1 phase cells, where there is no possibility of repair using a homolog. We show that in G1 yeast cells, NHEJ is a highly effective repair pathway for gamma-ray induced breaks, even when many breaks are present. Pulsed-field gel analysis showed chromosome karyotypes following NHEJ repair of cells from populations with multiple breaks. The number of reciprocal translocations was surprisingly low, perhaps zero, suggesting that NHEJ preferentially re-ligates the "correct" broken ends instead of randomly-chosen ends. Although we do not know the mechanism, the preferential correct ligation is consistent with the idea that broken ends are continuously held together by protein-protein interactions or by larger scale chromatin structure.


Assuntos
Reparo do DNA por Junção de Extremidades , Fase G1/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Cromossomos Fúngicos/genética , Reparo do DNA por Junção de Extremidades/efeitos da radiação , Fase G1/efeitos da radiação , Raios gama/efeitos adversos , Saccharomyces cerevisiae/efeitos da radiação
5.
J Mol Signal ; 11: 1, 2016 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-27096005

RESUMO

The carboxy (C)-termini of G protein coupled receptors (GPCR) dictate essential functions. The KTXXXW motif C-terminus of Frizzleds (FZD) has been implicated in recruitment of Dishevelled (DVL). Through study of FZD4 and its associated ligand Norrin, we report that a minimum of three residues distal to the KTXXXW motif in the C-terminal tail of Frizzled-4 are essential for DVL recruitment and robust Lef/Tcf-dependent transcriptional activation in response to Norrin.

6.
Meat Sci ; 117: 50-6, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26946476

RESUMO

The objective of this study was to investigate the effects of modified atmosphere packaging (MAP) systems on shelf-life and quality of beef steaks with high marbling. Four packaging types were used including 80% O2 MAP (80% O2+20% CO2), 50% O2 MAP (50% O2+30% CO2+20% N2), carbon monoxide MAP (0.4% CO+30% CO2+69.6% N2) and vacuum packaging (VP). Steaks were displayed under simulated retail conditions at 4°C for 12days. Purge loss, pH, color stability, oxidative stability and microbial counts were monitored. Aerobically packaged steaks exhibited a bright-red color at the first 4days. However, discoloration and oxidation became major factors limiting their shelf-life to 8days. Compared with aerobic packaging, anaerobic packaging extended shelf-life of heavily marbled beef steaks, due to better color stability, together with lower oxidation and microbial populations. Among all packaging methods, CO-MAP had the best preservation for steaks, with more red color than other packaging types.


Assuntos
Tecido Adiposo/anatomia & histologia , Músculo Esquelético/anatomia & histologia , Carne Vermelha/análise , Animais , Atmosfera , Bovinos , Embalagem de Alimentos , Concentração de Íons de Hidrogênio , Água
7.
J Mol Signal ; 10: 5, 2015 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-27096003

RESUMO

Familial exudative vitreoretinopathy (FEVR) is a disease state characterized by aberrant retinal angiogenesis. Norrin-induced activation of Frizzled-4 (Fz4) has a major role in regulating beta-catenin levels in the eye that, in turn, modulate the blood retina barrier (BRB). Here we gain insight on the basis of the pathology of a FEVR implicated F328S Fz4 mutant by study. The receptor exhibits a substantially reduced ability to activate Lef/Tcf-dependent transcription. This impaired activation correlates with a decreased ability to stabilize and recruit Dishevelled-2 (Dvl2) to the cell surface. Aromaticity at position 328 of the intracellular loop 2 (iloop2) is revealed similarly as a prerequisite for Dvl2 recruitment to the Fz4. This aromaticity at 328 enables normal Norrin-induced canonical activation. The corresponding position in iloop2 of other Frizzleds likely functions in Dvl recruitment.

8.
J Mol Signal ; 9(1): 3, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24690384

RESUMO

BACKGROUND: Protein phosphorylation of G-protein-coupled receptors (GPCR) is central to the myriad of functions that these ubiquitous receptors perform in biology. Although readily addressable with the use of phospho-specific antibodies, analysis phosphorylation at the level of stoichiometry requires receptor isolation and advanced proteomics. We chose two key sites of potential phosphorylation of human beta2-adrenergic receptor (ß2AR residues S355 and S356) to ascertain the feasibility of applying targeted mass spectrometry to establishing the stoichiometry of the phosphorylation. METHOD: We stimulated HEK293 cells stably expressing Flag-tagged ß2AR-eGFP with 10 µM beta-adrenergic agonist (isoproterenol) and made use of proteomics and targeted mass spectrometry (MS) to quantify the molar ration of phosphorylation on S355 and S356 versus non-phosphorylated receptor in agonist-treated cells. RESULTS: Phosphorylation of either S355 or S356 residue occurred only for agonist-occupied ß2AR. The results demonstrated that pS356 is the dominant site of protein phosphorylation. The abundance of the p356 was 8.6-fold more than that of pS355. Calculation of the molar ratio of phosphorylated (pS355 plus pS356) versus non-phosphorylated receptor reveals that at high occupancy of the receptor only 12.4% of the ß2AR is phosphorylated at these sites. CONCLUSIONS: Application of advanced proteomics and use of the most sensitive targeted MS strategy makes possible the detection and quantification of phosphorylation of very low abundance peptide digests of ß2AR. Establishing the stoichiometry of two key sites of agonist-stimulated phosphorylation with ß2AR is an essential first-step to global analysis of the stoichiometry of GPCR phosphorylation.

9.
J Sci Food Agric ; 94(4): 699-706, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23881861

RESUMO

BACKGROUND: Xylanases have attracted much attention because of their potential applications. Unfortunately, the commercialization of xylanases is limited by their low catalytic activities. The aim of this study was to improve the activity of a xylanase by optimization of the expression conditions and to investigate its characterization. RESULTS: The activity of recombinant AuXyn11A (reAuXyn11A), a family 11 xylanase from Aspergillus usamii E001 expressed in Pichia pastoris GS115, reached 912.6 U mL⁻¹ under the optimized conditions, which was 2.14 times as high as that expressed using the standard protocol. After the endogenous 18-aa propeptide had been processed in P. pastoris, reAuXyn11A (188-aa mature peptide) was secreted and purified with a specific activity of 22 714 U mg⁻¹. It displayed maximum activity at pH 5 and 50 °C and was stable in the pH range 4-8 and at a temperature of 45 °C or below. Its activity was not significantly affected by most metal ions and EDTA. Xylooligosaccharides ranging from xylobiose (X2) to xylohexaose (X6) were produced from insoluble corncob xylan by reAuXyn11A. CONCLUSION: Its high specific activity and good enzymatic properties suggest that reAuXyn11A is a potential candidate for applications in industrial processes.


Assuntos
Aspergillus/enzimologia , Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Pichia/metabolismo , Dissacarídeos/metabolismo , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/isolamento & purificação , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/isolamento & purificação , Precursores Enzimáticos/metabolismo , Estabilidade Enzimática , Tecnologia de Alimentos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Temperatura Alta , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Peso Molecular , Processamento de Proteína Pós-Traducional , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Via Secretória , Especificidade por Substrato , Xilanos/metabolismo
10.
Wei Sheng Wu Xue Bao ; 53(4): 346-52, 2013 Apr 04.
Artigo em Chinês | MEDLINE | ID: mdl-23858709

RESUMO

OBJECTIVE: To reveal the correlation between thermostability of xylanase EvXyn11(TS) and its N-terminal disulfide bridge, an EvXyn11(TS)-encoding gene (Syxyn11) was synthesized and subjected to site-directed mutagenesis. METHODS: Multiple homology alignment of protein primary structures between the EvXyn11(TS) and several GH family 11 xylanases displayed that, in their N-termini, only EvXyn11(TS) contained a disulfide bridge (Cys5-Cys32), whose effect on the xylanase thermostability was predicted by molecular dynamics simulation. We constructed a gene Syxyn11(M), encoding the mutated xylanase (EvXyn11(M)) without N-terminal disulfide bridge. Then, Syxyn11 and Syxyn11(M) were expressed in Pichia pastoris GS115, and temperature and pH properties of the expressed enzymes were analyzed. RESULTS: The analytical results displayed that the temperature optimum of EvXyn11(M) was 70 degrees C, which was 15 degrees C lower than that of EvXyn11(TS). The half-life (t1/2(90)) of EvXyn11(TS) at 90 degrees C was 32 min, while the t1/2(70) of EvXyn11(M) at 70 degrees C was only 8.0 min. CONCLUSION: The important role of the N-terminal disulfide bridge on the thermostability of EvXyn11(TS) was first predicted by molecular dynamics simulation, and confirmed by site-directed mutagenesis. This work provided a novel strategy to improve thermostabilities of the mesophilic family 11 xylanases with high specific activities.


Assuntos
Dissulfetos/metabolismo , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Sequência de Aminoácidos , Endo-1,4-beta-Xilanases/genética , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/métodos , Pichia/genética , Pichia/metabolismo , Engenharia de Proteínas/métodos , Alinhamento de Sequência , Temperatura
11.
PLoS One ; 8(5): e64766, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23741390

RESUMO

The AuMan5A, an acidophilic glycoside hydrolase (GH) family 5 ß-mannanase derived from Aspergillus usamii YL-01-78, consists of an only catalytic domain (CD). To perfect enzymatic properties of the AuMan5A, a family 1 carbohydrate-binding module (CBM) of the Trichoderma reesei cellobiohydrolase I (TrCBH I), having the lowest binding free energy with cellobiose, was selected by in silico design, and fused into its C-terminus forming a fusion ß-mannanase, designated as AuMan5A-CBM. Then, its encoding gene, Auman5A-cbm, was constructed as it was designed theoretically, and expressed in Pichia pastoris GS115. SDS-PAGE analysis displayed that both recombinant AuMan5A-CBM (reAuMan5A-CBM) and AuMan5A (reAuMan5A) were secreted into the cultured media with apparent molecular masses of 57.3 and 49.8 kDa, respectively. The temperature optimum of the reAuMan5A-CBM was 75°C, being 5°C higher than that of the reAuMan5A. They were stable at temperatures of 68 and 60°C, respectively. Compared with reAuMan5A, the reAuMan5A-CBM showed an obvious decrease in K m and a slight alteration in V max. In addition, the fusion of a CBM of the TrCBH I into the AuMan5A contributed to its cellulose-binding capacity.


Assuntos
Aspergillus/química , Celulose 1,4-beta-Celobiosidase/química , Celulose/química , Proteínas Recombinantes de Fusão/química , beta-Manosidase/química , Sequência de Aminoácidos , Sequência de Bases , Carboidratos/química , Celulose/metabolismo , Celulose 1,4-beta-Celobiosidase/genética , Celulose 1,4-beta-Celobiosidase/metabolismo , Ativação Enzimática , Estabilidade Enzimática , Evolução Molecular , Expressão Gênica , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Filogenia , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Temperatura , beta-Manosidase/genética , beta-Manosidase/metabolismo
12.
J Ind Microbiol Biotechnol ; 40(1): 75-83, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23053346

RESUMO

A cDNA gene (Auxyn10A), which encodes a mesophilic family 10 xylanase from Aspergillus usamii E001 (abbreviated to AuXyn10A), was amplified and inserted into the XhoI and NotI sites of pPIC9K(M) vector constructed from a parent pPIC9K. The recombinant expression vector, designated pPIC9K(M)-Auxyn10A, was transformed into Pichia pastoris GS115. All P. pastoris transformants were spread on a MD plate, and then inoculated on geneticin G418-containing YPD plates for screening multiple copies of integration of the Auxyn10A. One transformant expressing the highest recombinant AuXyn10A (reAuXyn10A) activity of 368.6 U/ml, numbered as P. pastoris GSX10A4-14, was selected by flask expression test. SDS-PAGE assay demonstrated that the reAuXyn10A was extracellularly expressed with an apparent M.W. of 39.8 kDa. The purified reAuXyn10A displayed the maximum activity at pH 5.5 and 50 °C. It was highly stable at a broad pH range of 4.5-8.5, and at a temperature of 45 °C. Its activity was not significantly affected by EDTA and several metal ions except Mn(2+), which caused a strong inhibition. The K(m) and V(max), towards birchwood xylan at pH 5.5 and 50 °C, were 2.25 mg/ml and 6,267 U/mg, respectively. TLC analysis verified that the AuXyn10A is an endo-ß-1,4-D-xylanase, which yielded a major product of xylotriose and a small amount of xylose, xylotetraose, and xylopentose from birchwood xylan, but no xylobiose.


Assuntos
Aspergillus/enzimologia , Endo-1,4-beta-Xilanases/metabolismo , Aspergillus/genética , Clonagem Molecular , DNA Complementar , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Oligossacarídeos/química , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Xilanos/metabolismo
13.
Biotechnol Bioeng ; 110(4): 1028-38, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23097144

RESUMO

A mesophilic xylanase from Aspergillus oryzae CICC40186 (abbreviated to AoXyn11A) belongs to glycoside hydrolase family 11. The thermostability of AoXyn11A was significantly improved by substituting its N-terminus with the corresponding region of a hyperthermostable family 11 xylanase, EvXyn11(TS) . The suitable N-terminus of AoXyn11A to be replaced was selected by the comparison of B-factors between AoXyn11A and EvXyn11(TS) , which were generated and calculated after a 15 ns molecular dynamic (MD) simulation process. Then, the predicted hybrid xylanase (designated AEx11A) was modeled, and subjected to a 2 ns MD simulation process for calculating its total energy value. The N-terminus substitution was confirmed by comparing the total energy value of AEx11A with that of AoXyn11A. Based on the in silico design, the AEx11A was constructed and expressed in Pichia pastoris GS115. After 72 h of methanol induction, the recombinant AEx11A (reAEx11A) activity reached 82.2 U/mL. The apparent temperature optimum of reAEx11A was 80°C, much higher than that of reAoXyn11A. Its half-life was 197-fold longer than that of reAoXyn11A at 70°C. Compared with reAoXyn11A, the reAEx11A displayed a slight alteration in K(m) but a decrease in V(max).


Assuntos
Aspergillus oryzae/enzimologia , Endo-1,4-beta-Xilanases/metabolismo , Engenharia Genética , Substituição de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , DNA Complementar/genética , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Estabilidade Enzimática , Genes Fúngicos , Temperatura Alta , Cinética , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
14.
Appl Biochem Biotechnol ; 167(8): 2198-211, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22689149

RESUMO

A full-length complementary DNA (cDNA) of Auxyn11D, a gene that encodes a novel endo-ß-1,4-D-xylanase of Aspergillus usamii E001 (abbreviated to AuXyn11D), was obtained using 3'- and 5'-rapid amplification of cDNA ends (RACE) methods. The cDNA sequence is 855 bp in length, containing 5'- and 3'-untranslated regions and a 696-bp open reading frame (ORF) that encodes a 32-aa signal peptide and a 199-aa mature peptide (namely AuXyn11D). Multiple homology alignment of amino acid sequences verified that AuXyn11D belongs to glycoside hydrolase family 11. Moreover, a mature peptide-encoding cDNA fragment of Auxyn11D was cloned and expressed in Pichia pastoris GS115. One P. pastoris transformant expressing the highest recombinant AuXyn11D (reAuXyn11D) activity of 15.0 U/mL, labeled as P. pastoris GSAuXyn4-16, was chosen by shake flask test. SDS-PAGE assay demonstrated that the reAuXyn11D, a glycosylated protein with an apparent molecular mass of 32.0 kDa, was secreted into the medium. The purified reAuXyn11D displayed the highest activity at pH 4.5 and 55 °C. It was stable at a pH range of 3.5-6.5 and at a temperature of 50 °C or below. Its activity was not significantly affected by most of metal ions tested and EDTA, but increased by Ca(2+) and inhibited by Mn(2+). The K(m) and V(max) of the reAuXyn11D towards birchwood xylan were 6.32 mg/mL and 391.6 U/mg, respectively.


Assuntos
Aspergillus/enzimologia , Clonagem Molecular , Endo-1,4-beta-Xilanases/genética , Proteínas Fúngicas/genética , Expressão Gênica , Pichia/genética , Sequência de Aminoácidos , Aspergillus/química , Aspergillus/genética , Sequência de Bases , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular , Pichia/metabolismo , Alinhamento de Sequência
15.
Sheng Wu Gong Cheng Xue Bao ; 28(12): 1441-9, 2012 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-23593868

RESUMO

A mesophilic xylanase from Aspergillus oryzae, abbreviated to AoXyn11A, belongs to glycoside hydrolase family 11. Using AoXyn11A as the parent, the thermotolerant hybrid xylanase, we constructed AEx11A by substituting its N-terminus with the corresponding region of a hyperthermostable family 11 xylanase, EvXyn11(TS). AoXyn11A- and AEx11A-encoding genes were expressed in Pichia pastoris GS115 separately, and effects of temperatures on expressed products were determined and compared. The optimum temperature (T(opt)) of AEx11A was 75 degrees C and its half-life at 70 degrees C (t1/2(70)) was 197 min, improved as compared with those (T(opt) = 50 degrees C, t1/2(70) = 1.0 min) of AoXyn11A. Homology modeling of the AEx11A's structure and comparison between structures of AEx11A and AoXyn11A revealed that one disulfide bridge (Cys5-Cys32) was introduced into AEx11A resulted from N-terminus substitution. To explore the effect of the disulfide bridge on the thermostability of AEx11A, it was removed from AEx11A by site-directed mutagenesis (C5T). Analytical results show that the T(opt) of the mutant AEx11A (AEx11A(C5T)) dropped to 60 degrees C from 75 degrees C of AEx11A, and its t1/2(70) and t1/2(80) also decreased to 3.0 and 1.0 min from 197 and 25 min.


Assuntos
Dissulfetos/química , Endo-1,4-beta-Xilanases/genética , Estabilidade Enzimática/genética , Mutagênese Sítio-Dirigida/métodos , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Substituição de Aminoácidos , Aspergillus oryzae/enzimologia , Sequência de Bases , Dissulfetos/metabolismo , Endo-1,4-beta-Xilanases/biossíntese , Endo-1,4-beta-Xilanases/química , Dados de Sequência Molecular , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
16.
J Mol Signal ; 6: 8, 2011 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-21831305

RESUMO

BACKGROUND: The family of A-kinase-anchoring proteins, AKAPs, constitutes a group of molecular scaffolds that act to catalyze dynamic interactions of protein kinase A, protein kinase C, tyrosine kinases, G-protein-coupled receptors and ion channels. AKAP5 (MW ~47 kDa) and AKAP12 (MW ~191 kDa) homo-oligomerize, but whether or not such AKAPs can hetero-oligomerize into supermolecular scaffolds of increased complexity is unknown. RESULTS: Affinity chromatography using immobilized AKAPs as "bait" demonstrates unequivocally that AKAP5 and AKAP12 do form minimally hetero-dimers. Steric-exclusion chromatography of AKAP5 and AKAP12 mixtures revealed the existence of very large, supermolecular complexes containing both AKAPs. Docking of AKAP5 to AKAP12 was increased 4-fold by beta-adrenergic agonist stimulation. Overexpression of AKAP12 was found to potentiate AKAP5-mediated Erk1/2 activation in response to stimulation with beta-adrenergic agonist. CONCLUSION: AKAP5 and AKAP12 are capable of forming hetero-oligomeric supermolecular complexes that influence AKAP locale and function.

17.
J Mol Signal ; 6: 3, 2011 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-21554706

RESUMO

BACKGROUND: A-kinase-anchoring proteins, AKAPs, constitute a family of scaffolds that play an essential role in catalyzing the spatial-temporal, dynamic interactions of protein kinase A, protein kinase C, tyrosine kinases, G-protein-coupled receptors and ion channels. We studied AKAP5 (AKAP79; MW ~47 kDa) and AKAP12 (gravin, SSECKS; MW ~191 kDa) to probe if these AKAP scaffolds oligomerize. RESULTS: In gel analysis and sodium-dodecyl sulfate denaturation, AKAP12 behaved with a MW of a homo-dimer. Only in the presence of the chaotropic agent 8 M urea did gel analysis reveal a monomeric form of AKAP12. By separation by steric-exclusion chromatography, AKAP12 migrates with MW of ~840 kDa, suggestive of higher-order complexes such as a tetramer. Interestingly, the N-(1-840) and C-(840-1782) terminal regions of AKAP12 themselves retained the ability to form dimers, suggesting that the structural basis for the dimerization is not restricted to a single "domain" found within the molecule. In either sodium dodecyl sulfate or urea, AKAP5 displayed a relative mobility of a monomer, but by co-immunoprecipitation in native state was shown to oligomerize. When subjected to steric-exclusion chromatography, AKAP5 forms higher-order complexes with MW ~220 kDa, suggestive of tetrameric assemblies. CONCLUSION: Both AKAP5 and AKAP12 display the capacity to form supermolecular homo-oligomeric structures that likely influence the localization and function of these molecular scaffolds.

18.
Meat Sci ; 88(3): 597-601, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21376477

RESUMO

This study investigated the effect of pre-rigor rapid chilling (RC) on the rate of pH and temperature decline, shear force values and ultrastructure of M. longissimus from beef carcasses exposed to electrical stimulation (ES). Chinese bull carcasses were electrically stimulated, and the alternate sides of the carcasses either were chilled conventionally (CC, 0-4°C, air speed 0.5 m/s for 24 h) or they underwent RC (-14±1°C, air speed 3 m/s for 2 h and then cooled under CC conditions until 24 h post-mortem). The results showed that RC increased the rate of temperature decline (P<0.05) and decreased the rate of pH decline (P<0.05). There were no significant differences in the shear force value and sarcomere length of M. longissimus between the two treatments (P>0.05). The results of this study indicate that RC has no detrimental impact on the tenderness of beef carcasses exposed to ES.


Assuntos
Temperatura Baixa , Manipulação de Alimentos/métodos , Carne/análise , Músculo Esquelético/química , Animais , Bovinos , Fenômenos Químicos , China , Temperatura Baixa/efeitos adversos , Técnicas Eletroquímicas , Concentração de Íons de Hidrogênio , Masculino , Microscopia Eletrônica de Transmissão , Músculo Esquelético/ultraestrutura , Controle de Qualidade , Sarcômeros/ultraestrutura , Resistência ao Cisalhamento , Fatores de Tempo
19.
Cells Tissues Organs ; 185(1-3): 162-74, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17587822

RESUMO

Epithelial-mesenchymal transitions (EMTs) occur in organogenesis throughout embryonic development and are recapitulated during epithelial tissue injury and in carcinoma progression. EMTs are regulated by complex, precisely orchestrated cell signaling and gene expression networks, with the participation of key developmental pathways. Here we review context-dependent modules of gene regulation by hairy/enhancer-of-split-related (H/E(spl)) repressors downstream of transforming growth factor-beta (TGF-beta)/Smad and Notch signals in EMT and in other phenotype transitions such as differentiation and cancer. Based on multiple models of disease-related EMT, we propose that Polycomb group epigenetic silencers and histone-lysine methyl-transferases EZH1 and EZH2 are candidate targets of H/E(spl)-mediated transcriptional repression, in a process accompanied by replacement of modified core histone H3 with de novo synthesized histone variant H3.3B. Finally, we discuss the potential significance of this scenario for EMT in the light of recent findings on gene regulation by histone modifications and chromatin structure changes.


Assuntos
Cromatina/química , Células Epiteliais/citologia , Mesoderma/citologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular , Células Epiteliais/metabolismo , Humanos , Córtex Renal/citologia , Mesoderma/metabolismo , Modelos Biológicos , Fator de Crescimento Transformador beta/genética
20.
J Biol Chem ; 279(16): 15968-74, 2004 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-14761967

RESUMO

Despite growing evidence for a mitochondrial localization of nitric oxide (NO) synthase and a broadening spectrum of NO actions on mitochondrial respiration and apoptosis, the basis for interaction between the enzyme and the organelle remain obscure. Here we investigated mitochondrial localization of endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells and human embryonic kidney cells transfected or infected with eNOS expression vectors. Copurification of eNOS with mitochondria was observed in both human umbilical vein endothelial cells and eNOS-expressing human embryonic kidney cells. Immunodetectable eNOS was cleaved from mitochondria by proteinase K treatment, suggesting eNOS association with the outer mitochondrial membrane. Localization of eNOS to a proteinase K-cleavable site on the cytoplasmic face of the outer membrane was confirmed by immunogold labeling of non-permeabilized mitochondria. Markers for mitochondrial subfractions ruled out the possibility of eNOS association with an intramitochondrial site or inverted mitochondrial particles. Denaturation of eNOS did not attenuate association with mitochondria. Mutant eNOS lacking a pentabasic amino acid sequence within the autoinhibitory domain (residues 628-632 of the bovine eNOS) showed dramatically reduced binding to the mitochondrial but not to the plasma membrane, which was associated with increased oxygen consumption. Collectively, these findings argue in favor of eNOS localization to the outer mitochondrial membrane in endothelial cells and identify elements of a novel anchoring mechanism.


Assuntos
Mitocôndrias/enzimologia , Óxido Nítrico Sintase/metabolismo , Transporte Biológico , Linhagem Celular , Endopeptidase K/metabolismo , Humanos , Membranas Intracelulares/enzimologia , Membranas Intracelulares/ultraestrutura , Microscopia Imunoeletrônica , Mitocôndrias/ultraestrutura , Mutação , Óxido Nítrico Sintase/ultraestrutura , Óxido Nítrico Sintase Tipo III
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