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1.
Biomed Res Int ; 2019: 1341370, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016184

RESUMO

A novel lectin (ABL) was purified from the dried fruiting bodies of Agaricus bitorquis. An efficient 3-step purification protocol involved two consecutive steps of ion exchange chromatography on Q-Sepharose and SP-Sepharose and gel filtration by FPLC on Superdex 75. ABL is a monomeric protein with the molecular mass of 27.6 kDa, which is different from other lectins from genus Agaricus. Its N-terminal amino acid sequence is EYTISIRVYQTNPKGFNRPV which is unique and sharing considerably high similarity of other mushroom lectins. The hemagglutinating activity of the lectin was inhibited by inulin. Based on hemagglutination tests, ABL prefers rabbit, human type A, and AB erythrocytes to human type B and O erythrocytes. The lectin inhibits the activity of HIV-1 reverse transcriptase and the proliferation of leukemia cell (L1210) with an IC50 value of 4.69 and 4.97 µM, respectively. Furthermore, ABL demonstrates the highest mitogenic activity with a response of 24177.7 ± 940.6 [3H-methyl] thymidine counts per minute (CPM) at a concentration of 0.91 µM.


Assuntos
Agaricales/química , Agaricus/química , Proliferação de Células/efeitos dos fármacos , Transcriptase Reversa do HIV/antagonistas & inibidores , Inulina/metabolismo , Lectinas/farmacologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Hemaglutinação/efeitos dos fármacos , Testes de Hemaglutinação/métodos , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Coelhos
2.
Sci Rep ; 9(1): 1151, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718596

RESUMO

Agaricus bisporus is in general cultivated on wheat and rice straw in China. However, millet straw is a potential alternative resource for Agaricus bisporus cultivation, but this has hardly been studied. In the present study, the feasibility of millet straw based mushroom production was analyzed by three successive trials. Mature compost demonstrated high quality with total nitrogen, pH, and C/N ratio of 2.0%, 7.5, and 18:1 respectively, which was suitable for mushroom mycelia growth. During composting, 47-50% of cellulose, 63-65% of hemicellulose, and 8-17% lignin were degraded, while 22-27% of cellulose, 14-16% of hemicellulose, and 15-21% of lignin were consumed by A. bisporus mycelia during cultivation. The highest FPUase and CMCase were observed during mushroom flushes. Endo-xylanase had the key role in hemicellulose degradation with high enzyme activity during cultivation stages. Laccase participated in lignin degradation with the highest enzyme activity in Pinning stage followed by a sharp decline at the first flush. Yield was up to 20 kg/m2, as this is similar to growth on wheat straw, this shows that millet straw is an effective resource for mushroom cultivation. Actinobacteria, Bacteroidetes, Chloroflexi, Deinococcus-Thermus, Firmicutes, and Proteobacteria were the dominant phyla, based on 16S rRNA gene sequencing during composting. The key environmental factors dominating bacterial communities of the samples were determined to be pH value, cellulose content, and hemicellulose content for prewetting and premixed phase of basic mixture (P0); moisture content for phase I (PI); and nitrogen content, lignin content, and ash content for phase II (PII), respectively.

3.
Zhongguo Gu Shang ; 31(10): 889-893, 2018 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-30373338

RESUMO

OBJECTIVE: To compare clinical effect between calcaneal locking plates and tension band with Kirschner's nail for the treatment of patellar fracture. METHODS: From December 2009 to December 2017, 58 patients with patellar fracture were divided into plate group(calcaneal locking plate) and tension band(tension band with Kirschner's nail) by surgical method. There were 29 patients in plate group, including 14 males and 15 females, aged from 18 to 72 years old with an average of (36.9±11.5) years old; while there were 29 patients in tension band group, including 17 males and 12 females, aged from 20 to 70 years old with an average of (37.7±14.4) years old. Operative time, blood loss, fracture healing time, follow-up time and postoperative complications were compared between two groups. Böstman score was applied to compared therapeutic effects at 12 months after operation. RESULTS: There was no significant differences in following-up time between plate group(18.4±2.6) months and tension band group(17.8±3.6) months. According to Böstman score at 12 months after operation, plate group was (28.5±4.6) and (25.7±4.3) in tension band group, and had statistical difference between two group(t=2.395, P=0.020). Twenty-six patients got excellent results, 3 moderate in plate group; while 14 patients got excellent results, 11 moderate and 4 poor in tension band group, and had obviously meaning between two groups(χ²=12.17 P=0.02). There were no significant differences in operative time(t=1.978, P=0.53), blood loss(t=1.740, P=0.87), fracture healing time(t=0.65, P=0.517) and postoperative complications(χ²=0.268, P=0.604) between two groups. CONCLUSIONS: Calcaneal locking plates for patellar fracture has advantages of more wide range of clinical application, more reliable fixation, and more satisfactory surgical curative method, its clinical effects is better than that of tension band with kirschner nail.


Assuntos
Calcâneo , Fraturas Ósseas , Adolescente , Adulto , Idoso , Placas Ósseas , Estudos de Casos e Controles , Feminino , Fixação Interna de Fraturas , Fraturas Ósseas/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Adulto Jovem
4.
Biochimie ; 148: 46-54, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29501482

RESUMO

A novel extracellular laccase was purified from fermentation broth of the white rot fungus Trametes sp. F1635 by a three-step protocol including two consecutive ion-exchange chromatography steps on DEAE-Sepharose and SP-Sepharose, and a final gel-filtration on Superdex 75. The purified laccase (TsL) was a monomeric protein with the molecular mass of 64.8 kDa. It demonstrated high oxidation activity of 4.00 × 104 U/mg towards ABTS. Its N-terminal amino acid sequence was AIGPVADLTIINNAV which was unique and sharing high similarity of other fungal laccases. TsL was a yellow laccase based on absorption spectrum analysis. It demonstrated an acidic pH optimum of 2.6 and temperature optimum of 50 °C towards ABTS. The Km and Vmax values towards ABTS were estimated to 18.58 µM and 1.35 µmol/min, respectively. TsL manifested effective decolorization activity towards eriochrome black T (EBT), remazol brilliant blue R (RBBR), malachite green (MG), and eriochrome black T (EBT) (over 60%). Violuric acid (VA) and acetosyringone (AS) were the optimal mediators for the laccase in dye decolorization. Results suggest that TsL demonstrates great potential for dye decolorization and water treatment.


Assuntos
Corantes/metabolismo , Espaço Extracelular/enzimologia , Lacase/metabolismo , Trametes/citologia , Sequência de Aminoácidos , Cor , Fermentação , Concentração de Íons de Hidrogênio , Cinética , Lacase/química , Peso Molecular , Temperatura , Trametes/enzimologia
5.
Int J Biol Macromol ; 95: 920-927, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27793681

RESUMO

A novel laccase was purified from fermentation broth of the white rot fungus Cerrena unicolor strain GSM-01 following three ion-exchange chromatography steps and one gel-filtration step. The purified enzyme was determined to be a monomeric protein of 63.2kDa and demonstrated high oxidation activity of 2.05×104U/mg towards ABTS. Its cDNA, gene, and amino acid sequences were obtained. It possessed high sequence similarity with that of other laccases but different enzymatic properties. It manifested optimal pH and temperature of 2.6 and 45°C, respectively. Fe3+ and Fe2+ were the most efficient inhibitors towards Cerrena unicolor laccase (CUL), while Mn2+ can slightly enhance the laccase activity of 3.8-10.5%. The Km and Vmax of CUL were estimated to 302.7µM and 13.6µMm-1, respectively. CUL was effective in the decolorization of bromothymol blue, evans blue, methyl orange, and malachite green with decolorization efficiencies of 50%-85%. It possesses potential application in textile and environmental industries.


Assuntos
Corantes/metabolismo , Espaço Extracelular/enzimologia , Lacase/genética , Lacase/metabolismo , Polyporaceae/citologia , Sequência de Aminoácidos , Clonagem Molecular , Cor , Espaço Extracelular/genética , Concentração de Íons de Hidrogênio , Cinética , Lacase/química , Lacase/isolamento & purificação , Peso Molecular , Polyporaceae/enzimologia , Especificidade por Substrato , Temperatura
6.
Nutrients ; 8(10)2016 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-27690088

RESUMO

To investigate the antitumor effect of anthocyanins extracted from Chinese bayberry fruit (Myrica rubra Sieb. et Zucc.), a nude mouse tumor xenograft model was established. Treatments with C3G (cyanidin-3-glucoside, an anthocyanin) significantly suppressed the growth of SGC-7901 tumor xenografts in a dose-dependent manner. Immunohistochemical staining showed a significant increase in p21 expression, indicating that the cell cycle of tumor xenografts was inhibited. qPCR screening showed that C3G treatment up-regulated the expression of the KLF6 gene, which is an important tumor suppressor gene inactivated in many human cancers. Western blot showed that C3G treatments markedly increased KLF6 and p21 protein levels, inhibited CDK4 and Cyclin D1 expression, but did not notably change the expression of p53. These results indicated that KLF6 up-regulates p21 in a p53-independent manner and significantly reduces tumor proliferation. This study provides important information for the possible mechanism of C3G-induced antitumor activity against gastric adenocarcinoma in vivo.

7.
Int J Biol Macromol ; 93(Pt A): 837-842, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27642125

RESUMO

A novel laccase was isolated from fermentation broth of the mycorrhizal fungus Leucoagaricus naucinus LAC-04 by using a protocol that comprising ion-exchange chromatography steps on DEAE-cellulose, SP-Sepharose, and Q-Sepharose, and finally gel filtration by fast protein liquid chromatography on Superdex 75. The laccase (LNL) was purified with a purification fold of 21.19 and a recovery rate of 19.8%. It is a monomeric protein with a molecular mass of 56kDa. LNL lacks absorption around 600nm, which indicates that the purified laccase is a yellow laccases. LNL demonstrates an optimal pH of 2.2 and an optimal temperature range of 30-60°C using ABTS as the substrate. It is inhibited in the presence of EDTA and metal ions including Cd2+, Co2+, Cu2+. The Km of the laccase towards ABTS is estimated to 50.12µM at pH 2.2 and 30°C. Moreover, the purified laccase manifests effective decolorizing activity towards azo, heterocyclic, and aromatic dyes including Bromothymol Blue, Eriochrome Black T, Evans Bue, Fuchsin Basic, and Remazol Brilliant Blue R.


Assuntos
Agaricales/enzimologia , Corantes/química , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Lacase/química , Lacase/isolamento & purificação , Estabilidade Enzimática , Concentração de Íons de Hidrogênio
8.
Indian J Biochem Biophys ; 50(3): 196-201, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23898482

RESUMO

A 15 kDa ribonuclease (RNase) was purified from dried fruiting bodies of the wild edible mushroom Armillaria luteo-virens. The simple 4-step purification protocol involved ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on SP-Sepharose and a final gel filtration by FPLC on Superdex-75. The RNase was unadsorbed on Affi-gel blue gel, but adsorbed on DEAE-cellulose and SP-Sepharose. The N-terminal amino acid sequence of purified RNase was AGVQYKLTILLV, which showed low sequence homology to those of previously reported RNases. The optimal pH and temperature of the enzyme were very close to 4.0 and 70 degrees C, respectively. The enzyme showed considerably high ribonucleolytic activity and broad specificity towards polyhomoribonucleotides, with a specificity of poly(U) > poly(C) > poly (G) > poly(A). The ribonucleolytic activities towards poly(U), poly(C), poly(G) and poly(A) were 279.5, 184.1, 69.9 and 52.3 U/mg, respectively.


Assuntos
Agaricales/enzimologia , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Ribonucleases/química , Ribonucleases/isolamento & purificação , Animais , Ativação Enzimática , Estabilidade Enzimática , Especificidade por Substrato
9.
Biomed Res Int ; 2013: 540239, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23586045

RESUMO

A monomeric phytase with a molecular mass of 14 kDa was acquired from fresh fruiting bodies of the shiitake mushroom Lentinus edodes. The isolation procedure involved chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, Affi-gel blue gel, and a final fast protein liquid chromatography-gel filtration on Superdex 75. The purified phytase demonstrated the unique N-terminal amino acid sequence DPKRTDQVN, which exhibited no sequence similarity with those of other phytases previously reported. It expressed its maximal activity at pH 5.0 and 37 °C. Phytase activity manifested less than 20% change in activity over the pH range of 3.0-9.0, considerable thermostability with more than 60% residual activity at 70 °C, and about 40% residual activity at 95°C. It displayed a wide substrate specificity on a variety of phosphorylated compounds with the following ranking: ATP > fructose-6-phosphate > AMP > glucose-6-phosphate > ADP > sodium phytate > ß -glycerophosphate. The phytase activity was moderately stimulated by Ca(2+), but inhibited by Al(3+), Mn(2+), Zn(2+), and Cu(2+) at a tested concentration of 5 mM.


Assuntos
6-Fitase/química , 6-Fitase/isolamento & purificação , Cogumelos Shiitake/enzimologia , Sequência de Aminoácidos , Cromatografia , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Peso Molecular , Especificidade por Substrato , Temperatura
10.
J Basic Microbiol ; 53(10): 868-75, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23322529

RESUMO

A monomeric acid phosphatase (ACP) with a molecular mass of 72.5 kDa was purified from fresh fruiting bodies of cultured Schizophyllum commune mushroom. The isolation procedure entailed ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. It demonstrated a unique N-terminal amino acid sequence of NAPWAQIDEV, which exhibited 60% amino acid identity to that of S. commune hypothetical histidine ACP based on its genome sequence, but less than 30% amino acid identity to that of other fungal ACPs previously reported. The ACP exhibited an optimum temperature at 50 °C, an optimum pH at pH 4.6, and was considerably stable at a pH range of 4.0 to 9.0, and a temperature range of 20-40 °C. The Km of the purified enzyme for ρ-nitrophenyl phosphate (ρNPP) was 0.248 mM and the Vmax was 9.093 × 10(-3) µM/min. ACP activity was strongly inhibited by Al(3+) and Fe(3+) , but enhanced by Co(2+) , Mg(2+) , and Ca(2+) at a concentration of 0.5 mM.


Assuntos
Fosfatase Ácida/isolamento & purificação , Fosfatase Ácida/metabolismo , Carpóforos/enzimologia , Schizophyllum/enzimologia , Fosfatase Ácida/química , Sequência de Aminoácidos , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Carpóforos/fisiologia , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Peso Molecular , Especificidade por Substrato , Temperatura
11.
J Biomed Biotechnol ; 2012: 736472, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23093860

RESUMO

A novel 68 kDa laccase was purified from the mycorrhizal fungus Agaricus placomyces by utilizing a procedure that comprised three successive steps of ion exchange chromatography and gel filtration as the final step. The monomeric enzyme exhibited the N-terminal amino acid sequence of DVIGPQAQVTLANQD, which showed only a low extent of homology to sequences of other fungal laccases. The optimal temperature for A. placomyces laccase was 30°C, and optimal pH values for laccase activity towards the substrates 2,7'-azinobis[3-ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS) and hydroquinone were 5.2 and 6.8, respectively. The laccase displayed, at 30°C and pH 5.2, K(m) values of 0.392 mM towards hydroquinone and 0.775 mM towards ABTS. It potently suppressed proliferation of MCF 7 human breast cancer cells and Hep G2 hepatoma cells and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity with an IC(50) of 1.8 µM, 1.7 µM, and 1.25 µM, respectively, signifying that it is an antipathogenic protein.


Assuntos
Agaricus/enzimologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Lacase/administração & dosagem , Lacase/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Inibidores da Transcriptase Reversa/química , Proliferação de Células/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/farmacologia , Células Hep G2 , Humanos , Células MCF-7 , Neoplasias Experimentais/patologia
12.
Zhonghua Nan Ke Xue ; 14(8): 701-3, 2008 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-18817341

RESUMO

OBJECTIVE: To detect the levels of nitric oxide synthetase (NOS) and carbon monoxide (CO) in the penile corpus cavernous of adult male Wistar rats with high homocysteine (Hhcy) and to explore the relationship of NOS and CO levels with erectile dysfunction. METHODS: Twenty Wistar rats were equally and randomly divided into a control and an Hhcy group and fed on normal diet and normal diet with 3.0% methionine respectively. Four weeks later, the levels of NOS and CO in the penile corpus cavernous were detected by ultraviolet spectrophotometry and that of serum homocysteine by the cycle enzyme method. RESULTS: Compared with the control group, the levels of NOS and CO in the penile corpus cavernous were significantly lower in the Hhcy group, (6.45 +/- 1.12) nmol/(g x min) vs (10.77 +/- 0.60) nmol/(g x min) and (10.60 +/- 0.92) micromol/L vs (13.36 +/- 0.44) micromol/L, while that of homocysteine was significantly higher, (22.32 +/- 1.65) micromol/L) vs (4.90 +/- 1.73) micromol/L. CONCLUSION: Four-week diet with methionine can cause Hhcy and significantly decreased levels of NOS and CO in the penile corpus cavernous in Wistar rats. Hhcy is an independent risk factor of erectile dysfunction.


Assuntos
Monóxido de Carbono/metabolismo , Hiper-Homocisteinemia/fisiopatologia , Óxido Nítrico Sintase/metabolismo , Pênis/metabolismo , Animais , Homocisteína/sangue , Hiper-Homocisteinemia/sangue , Masculino , Ratos , Ratos Wistar , Espectrofotometria Ultravioleta
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