RESUMO
As an important glycosaminoglycan hydrolase, chondroitin lyases can hydrolyze chondroitin sulfate (CS) and release disaccharides and oligosaccharides. They are further divided into chondroitin AC, ABC, and B lyases according to their spatial structure and substrate specificity. Chondroitin AC lyase can hydrolyze chondroitin sulfate A (CS-A), chondroitin sulfate C (CS-C), and hyaluronic acid (HA), making it an essential biocatalyst for the preparation of low molecular weight chondroitin sulfate, analysis of the structure of the chondroitin sulfate, treatment of spinal cord injury, and purification of heparin. This paper provides an overview of reported chondroitin AC lyases, including their properties and the challenges faced in industrial applications. Up to now, although many attempts have been adopted to improve the enzyme properties, the most important factors are still the low activity and stability. The relations between the stability of the enzyme and the spatial structure were also summarized and discussed. Also perspectives for remodeling the enzymes with protein engineering are included.
Assuntos
Sulfatos de Condroitina , Liases , Condroitina Liases/química , Condroitina Liases/metabolismo , Sulfatos de Condroitina/química , Sulfatos de Condroitina/metabolismo , Ácido Hialurônico/química , Ácido Hialurônico/metabolismo , Liases/metabolismo , Especificidade por SubstratoRESUMO
BACKGROUND: This study aimed to explore the effects of (body mass index) BMI on health related quality of life (HRQoL) among the elderly in Jiangsu, China. METHODS: A total of 10,257 community dwelling elderly (≥60 years old) were enrolled in a cross-sectional study. HRQoL was measured via the Eq-5d-3 L. Chi-square tests and one-way ANOVA analyses were used to compare the frequencies and scores of Eq-5d responses among different BMI groups (defined as "underweight", "normal weight", "overweight" and "obese"). Logistic regression analyses were conducted to examine the associations between BMI and HRQoL. RESULTS: Among the subjects, the proportion of "normal weight", "underweight", "overweight" and "obese" were 66.0, 8.3, 23.1, and 2.6%, respectively. The score of the Eq-5d index among total participants was 0.8036 and the Visual Analog Scale (VAS) score was 75.47. For both the responses frequency and scores of Eq-5d-3 L, there were significant differences among BMI groups (P < 0.001). The Logistic regression model showed that both in men and women, underweight elderly were more likely to suffer low HRQoL. The adjusted odds ratio (OR) with a 95% confidence interval (CI) for Eq-5d index/VAS was 2.03 (1.48, 2.79)/1.83 (1.34, 2.50) in men and 1.47(1.09,1.98)/1.52(1.20,1.91) in women. Overweight women more likely to have a low Eq-5d index, while overweight men were less likely to have a low Eq-5d VAS. CONCLUSION: This study shows that underweight is an explicit risk factor of low HRQoL in both the male and female elderly, while the effect of overweight on low HRQoL varies slightly by gender.
Assuntos
Índice de Massa Corporal , Nível de Saúde , Sobrepeso/epidemiologia , Qualidade de Vida , Magreza/epidemiologia , Idoso , China/epidemiologia , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Distribuição por SexoRESUMO
Enzymatic preparation of low-molecular-weight chondroitin sulfate (LMWCS) has received increasing attention. In this work, a chondroitin sulfate lyase ABC (Chon-ABC) was successfully cloned, expressed, and characterized. The Km and Vmax of the Chon-ABC were 0.54 mM and 541.3 U mg-1, respectively. The maximal activity was assayed as 500.4 U mg-1 at 37 °C in pH 8.0 phosphate buffer saline. The half-lives of the Chon-ABC were 133 d and 127 min at 4 °C and 37 °C, respectively. Enzymatic preparation of LMWCS was performed at room temperature for 30 min. The changes between the substrate and product were analyzed with mass spectrometry (MS), high-performance liquid chromatography (HPLC), gel permeation chromatography (GPC), and nuclear magnetic resonance (NMR). Overall, the Chon-ABC from Bacteroides thetaiotaomicron is competitive in large-scale enzymatic preparation of LMWCS for its high activity, stability, and substrate specificity.
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A method for specific immobilization of whole-cell with covalent bonds was developed through a click reaction between alkyne and azide groups. In this approach, magnetic nanoparticle Fe3O4@SiO2-NH2-alkyne was synthesized with Fe3O4 core preparation, SiO2 coating, and alkyne functionalization on the surface. The azides were successfully integrated onto the cell surface of the recombinant E. coli harboring glycerol dehydrogenase, which was employed as the model cell. The highest immobilization yield of 83% and activity recovery of 94% were obtained under the conditions of 0.67 mg mg-1 cell-support ratio, pH 6.0, temperature 45 °C, and 20 mM Cu2+ concentration. The immobilized cell showed good reusability, which remained over 50% of initial activity after 10 cycles of utilization. Its activity was 9.7-fold higher than that of the free cell at the condition of pH 8.0 and each optimal temperature. Furthermore, the immobilized cell showed significantly higher activity, operational stability, and reusability.
Assuntos
Enzimas Imobilizadas , Nanopartículas de Magnetita , Azidas , Química Click , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Polissacarídeos , Dióxido de SilícioRESUMO
Chondroitin AC lyase can efficiently hydrolyze chondroitin sulfate (CS) to low molecule weight chondroitin sulfate, which has been widely used in clinical therapy, including anti-tumor, anti-oxidation, hypolipidemic, and anti-inflammatory. In this work, a novel chondroitin AC lyase from Pedobacter xixiisoli (PxchonAC) was cloned and overexpressed in Escherichia coli BL21 (DE3). The characterization of PxchonAC showed that it has specific activities on chondroitin sulfate A, Chondroitin sulfate C and hyaluronic acid with 428.77, 270.57, and 136.06 U mg-1, respectively. The Km and Vmax of PxchonAC were 0.61 mg mL-1 and 670.18 U mg-1 using chondroitin sulfate A as the substrate. The enzyme had a half-life of roughly 660 min at 37 °C in the presence of Ca2+ and remained a residual activity of 54 % after incubated at 4 °C for 25 days. Molecular docking revealed that Asn123, His223, Tyr232, Arg286, Arg290, Asn372, and Glu374 were mainly involved in the substrate binding. The enzymatic hydrolysis product was analyzed by gel permeation chromatography, demonstrating PxchonAC could hydrolyze CS efficiently.
Assuntos
Oligossacarídeos , Sequência de Aminoácidos , Condroitina Liases/genética , Condroitina Liases/metabolismo , Clonagem Molecular , Humanos , Simulação de Acoplamento Molecular , PedobacterRESUMO
Sea cucumbers are one of many marine echinoderm animals that contain valuable nutrients and medicinal compounds. The bioactive substances in sea cucumbers make them have promising biological and pharmacological properties, including antioxidant, anti-bacterial, and anti-tumor effects. In this study, sea cucumber intestinal peptide (SCIP) is a small molecular oligopeptide (<1,000 Da) extracted from sea cucumber intestines hydrolyzed by alkaline protease. The analysis of amino acid composition showed that hydrophobic amino acids and branched-chain amino acids were rich in SCIP. Nowadays, although increasing studies have revealed the biological functions of the sea cucumber active substances, there are few studies on the function of SCIP. Furthermore, due to the anti-cancer activity being an essential characteristic of sea cucumber active substances, we also investigated the anti-cancer potential and the underlying mechanism of SCIP in vivo and in vitro. The results indicate that SCIP inhibits the growth of MCF-7 tumor cells in zebrafish and increases the apoptosis of human breast cancer MCF-7 cells. Further mechanism studies confirm that SCIP promotes the expression of apoptosis-related proteins and thus promotes the breast cancer cells (MCF-7) apoptosis via inhibition of PI3K/AKT signal transduction pathway.
RESUMO
L-Tagatose, a promising building block in the production of many value-added chemicals, is generally produced by chemical routes with a low yield, which may not meet the increasing demands. Synthesis of l-tagatose by enzymatic oxidation of d-galactitol has not been applied on an industrial scale because of the high cofactor costs and the lack of efficient cofactor regeneration methods. In this work, an efficient and environmentally friendly enzymatic method containing a galactitol dehydrogenase for d-galactitol oxidation and a water-forming NADH oxidase for regeneration of NAD+ was first designed and used for l-tagatose production. Supplied with only 3 mM NAD+, subsequent reaction optimization facilitated the efficient transformation of 100 mM of d-galactitol into l-tagatose with a yield of 90.2 % after 12 h (obtained productivity: 7.61 mM.h-1). Compared with the current chemical and biocatalytic methods, the strategy developed avoids by-product formation and achieves the highest yield of l-tagatose with low costs. It is expected to become a cleaner and more promising route for industrial biosynthesis of l-tagatose.
Assuntos
Hexoses/biossíntese , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Desidrogenase do Álcool de Açúcar/metabolismo , Hexoses/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Molecular , TemperaturaRESUMO
This study aimed to explore the correlation of kinesin family member 2A (KIF2A) expression with disease risk, clinical characteristics, and prognosis of acute myeloid leukemia (AML), and investigate the effect of KIF2A knockdown on AML cell activities in vitro. Bone marrow samples were collected from 176 AML patients and 40 healthy donors, and KIF2A expression was measured by real-time quantitative polymerase chain reaction. Treatment response, event-free survival (EFS), and overall survival (OS) were assessed in AML patients. In vitro, KIF2A expression in AML cell lines and CD34+ cells (from healthy donors) was measured, and the effect of KIF2A knockdown on AML cell proliferation and apoptosis in HL-60 and KG-1 cells was detected. KIF2A expression was greater in AML patients compared to healthy donors, and receiver operating characteristic curve indicated that KIF2A expression predicted increased AML risk (area under curve: 0.793 (95%CI: 0.724-0.826)). In AML patients, KIF2A expression positively correlated with white blood cells, monosomal karyotype, and high risk stratification. Furthermore, no correlation of KIF2A expression with complete remission or hematopoietic stem cell transplantation was found. Kaplan-Meier curves showed that KIF2A expression was negatively correlated with EFS and OS. In vitro experiments showed that KIF2A was overexpressed in AML cell lines (KG-1, HL-60, ME-1, and HT-93) compared to CD34+ cells, moreover, cell proliferation was reduced but apoptosis was increased by KIF2A knockdown in HL-60 and KG-1 cells. In conclusion, KIF2A showed potential to be a biomarker and treatment target in AML.
Assuntos
Cinesinas/genética , Leucemia Mieloide Aguda , Adulto , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Feminino , Técnicas de Silenciamento de Genes , Células HL-60 , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Taxa de SobrevidaRESUMO
As one of the most extensively studied glycosaminoglycan lyases, heparinase I has been used in producing low or ultra-low molecular weight heparin. Its' important applications are to neutralize the heparin in human blood and analyze heparin structure in the clinic. However, the low productivity and activity of the enzyme have greatly hindered its applications. In this study, a novel Hep-I from Bacteroides cellulosilyticus (BcHep-I) was successfully cloned and heterologously expressed in E. coli BL21 (DE3) as a soluble protein. The molecular mass and isoelectric point (pI) of the enzyme are 44.42 kDa and 9.02, respectively. And the characterization of BcHep-I after purified with Ni-NTA affinity chromatography suggested that it is a mesophilic enzyme. BcHep-I can be activated by 1 mM Ca2+, Mg2+, and Mn2+, while severely inhibited by Zn2+, Co2+, and EDTA. The specific activity of the enzyme was 738.3 U·mg-1 which is the highest activity ever reported. The Km and Vmax were calculated as 0.17 mg·mL-1 and 740.58 U·mg-1, respectively. Besides, the half-life of 300 min at 30°C showed BcHep-I has practical applications. Homology modeling and substrate docking revealed that Gln15, Lys74, Arg76, Lys104, Arg149, Gln208, Tyr336, Tyr342, and Lys338 were mainly involved in the substrate binding of Hep-I, and 11 hydrogen bonds were formed between heparin and the enzyme. These results indicated that BcHep-I with high activity has great potential applications in the industrial production of heparin, especially in the clinic to neutralize heparin.
Assuntos
Bacteroides/enzimologia , Heparina Liase/genética , Heparina Liase/metabolismo , Heparina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroides/genética , Sítios de Ligação , Cálcio/metabolismo , Clonagem Molecular , Ativação Enzimática , Heparina Liase/química , Ligação de Hidrogênio , Magnésio/metabolismo , Manganês/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação ProteicaRESUMO
This study aimed to investigate the correlation of long noncoding RNA zinc finger antisense 1 (lncRNA ZFAS1) expression with disease risk, disease severity and inflammatory cytokines levels in lumbar disc degeneration (LDD) patients.83 LDD patients underwent surgery and 28 traumatized, non-LDD patients underwent lumbar disc surgery (controls) were consecutively enrolled in this case-control study. Lumbar disc tissue was obtained during surgery and herniated nucleus pulposus (HNP) was isolated to detect lncRNA ZFAS1 expression and inflammatory cytokines mRNA levels by RT-qPCR, and determine protein levels of inflammatory cytokines by western blot.HNP lncRNA ZFAS1 expression in LDD patients was up-regulated compared with controls (Pâ<â.001), and receiver operating characteristic (ROC) curve showed lncRNA ZFAS1 expression disclosed a good predictive value for LDD risk with area under curve (AUC) 0.753 (95% CI 0.646-0.859). And after adjustment by age, gender and body mass index (BMI), lncRNA ZFAS1 (Pâ=â.017) remained to be an independent predictive factor for higher LDD risk. In addition, lncRNA ZFAS1 expression was positively associated with Modified Pfirrmann Grade (Pâ=â.015). As to inflammatory cytokines, lncRNA ZFAS1 expression was observed to be positively correlated with TNF-α (Pâ=â.002), IL-1ß (Pâ=â.007) and IL-6 (Pâ=â.015) mRNAs expressions while reversely associated with IL-10 mRNA level (Pâ=â.014); and lncRNA ZFAS1 expression was also positively correlated with protein levels of TNF-α (Pâ=â.038) and IL-6 (Pâ=â.027) while reversely associated with IL-10 protein expression (Pâ=â.039).lncRNA ZFAS1 expression associates with increased risk, elevated disease severity and higher inflammatory cytokines levels in LDD patients.
Assuntos
Citocinas/metabolismo , Degeneração do Disco Intervertebral/metabolismo , Deslocamento do Disco Intervertebral/metabolismo , Disco Intervertebral/metabolismo , Vértebras Lombares , RNA Antissenso/metabolismo , RNA Longo não Codificante/metabolismo , Adulto , Biomarcadores/análise , Western Blotting , Estudos de Casos e Controles , Citocinas/análise , Feminino , Humanos , Interleucina-10/análise , Interleucina-10/metabolismo , Interleucina-1beta/análise , Interleucina-1beta/metabolismo , Interleucina-6/análise , Interleucina-6/metabolismo , Disco Intervertebral/química , Masculino , Pessoa de Meia-Idade , RNA Longo não Codificante/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Índice de Gravidade de Doença , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/metabolismoRESUMO
This study aimed to explore the correlation of kinesin family member 2A (KIF2A) expression with disease risk, clinical characteristics, and prognosis of acute myeloid leukemia (AML), and investigate the effect of KIF2A knockdown on AML cell activities in vitro. Bone marrow samples were collected from 176 AML patients and 40 healthy donors, and KIF2A expression was measured by real-time quantitative polymerase chain reaction. Treatment response, event-free survival (EFS), and overall survival (OS) were assessed in AML patients. In vitro, KIF2A expression in AML cell lines and CD34+ cells (from healthy donors) was measured, and the effect of KIF2A knockdown on AML cell proliferation and apoptosis in HL-60 and KG-1 cells was detected. KIF2A expression was greater in AML patients compared to healthy donors, and receiver operating characteristic curve indicated that KIF2A expression predicted increased AML risk (area under curve: 0.793 (95%CI: 0.724-0.826)). In AML patients, KIF2A expression positively correlated with white blood cells, monosomal karyotype, and high risk stratification. Furthermore, no correlation of KIF2A expression with complete remission or hematopoietic stem cell transplantation was found. Kaplan-Meier curves showed that KIF2A expression was negatively correlated with EFS and OS. In vitro experiments showed that KIF2A was overexpressed in AML cell lines (KG-1, HL-60, ME-1, and HT-93) compared to CD34+ cells, moreover, cell proliferation was reduced but apoptosis was increased by KIF2A knockdown in HL-60 and KG-1 cells. In conclusion, KIF2A showed potential to be a biomarker and treatment target in AML.