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1.
Phytomedicine ; 109: 154613, 2023 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-36610112

RESUMEN

BACKGROUND: Small cell lung cancer (SCLC) is an aggressive tumor with high brain metastasis (BM) potential. There has been no significant progress in the treatment of SCLC for more than 30 years. Cordycepin has shown the therapeutic potential for cancer by modulating multiple cellular signaling pathways. However, the effect and mechanism of cordycepin on anti-SCLC BM remain unknown. PURPOSE: In this study, we focused on the anti-SCLC BM effect of cordycepin in the zebrafish model and its potential mechanism. STUDY DESIGN AND METHODS: A SCLC xenograft model based on zebrafish embryos and in vitro cell migration assay were established. Cordycepin was administrated by soaking and microinjection in the zebrafish model. RNA-seq assay was performed to analyze transcriptomes of different groups. Geno Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were performed to reveal the underlying mechanism. Real-time qPCR was used to verify the effects of cordycepin on the key genes. RESULTS: Cordycepin showed lower cytotoxicity in vitro compared with cisplatin, anlotinib and etoposide, but showed comparable anti-proliferation and anti-BM effects in zebrafish SCLC xenograft model. Cordycepin showed significant anti-SCLC BM effects when administrated by both soaking and microinjection. RNA-seq demonstrated that cordycepin was involved in vitamin D metabolism, lipid transport, and proteolysis in cellular protein catabolic process pathways in SCLC BM microenvironment in zebrafish, and was involved in regulating the expressions of key genes such as cyp24a1, apoa1a, ctsl. The anti-BM effect of cordycepin in SCLC was mediated by reversing the expression of these genes. CONCLUSION: Our work is the first to describe the mechanism of cordycepin against SCLC BM from the perspective of regulating the brain microenvironment, providing new evidence for the anti-tumor effect of cordycepin.


Asunto(s)
Neoplasias Encefálicas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Animales , Humanos , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/patología , Pez Cebra , Neoplasias Pulmonares/patología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Microambiente Tumoral
2.
Bioorg Med Chem Lett ; 26(19): 4720-4723, 2016 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-27575480

RESUMEN

Strontium fructose 1,6-diphosphate (FDP-Sr) is a new strontium-containing compound. The primary aim of this study was to clarify whether the structure component of FDP-Sr, FDP could benefit the protective effect of Sr (II) against oxidative stress induced apoptosis, and meanwhile to further explore the important role of Wnt/ß-catenin signaling in the anti-apoptosis effect of FDP-Sr in response to oxidative stress induced by H2O2 in an osteoblastic MC3T3-E1 cell line. Results showed that FDP-Sr could improve the osteoblastic differentiation under oxidative stress with induced cell proliferation and improved mineralization. The inhibition effect of FDP-Sr on cell apoptosis induced by H2O2 was proved by reduced reactive oxygen species production and activated caspase3. Under oxidative stress, mRNA and protein levels of phospho-ß-catenin reduced, while ß-catenin increased in the FDP-Sr treatment cell, leaded to the up-regulations of Runx2 and OPG at both mRNA and protein levels, finally improved the differentiation of osteoblasts. By the engagement of Wnt/ß-catenin pathway's inhibitor (XAV-939), the protective effects of FDP-Sr on osteoblastic differentiation against oxidative stress were repressed along with inhibited wnt/ß-catenin signaling and reduced mRNA and protein levels of Runx2 and OPG. In conclusion, FDP-Sr was demonstrated to protect osteoblast differentiation from oxidative damage induced by H2O2 through up-regulation of Wnt/ß-catenin signaling, and FDP in FDP-Sr was able to directly improve the oxidative stress injury through its ROS scavenging ability.


Asunto(s)
Formicinas/química , Estrés Oxidativo/efectos de los fármacos , Ribonucleótidos/química , Transducción de Señal , Proteínas Wnt/metabolismo , Células 3T3 , Animales , Ratones
3.
Biotechnol Biofuels ; 9: 103, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27175216

RESUMEN

BACKGROUND: Water-forming NADH oxidase can oxidize cytosolic NADH to NAD(+), thus relieving cytosolic NADH accumulation in Saccharomyces cerevisiae. Previous studies of the enzyme were conducted under aerobic conditions, as O2 is the recognized electron acceptor of the enzyme. In order to extend its use in industrial production and to study its effect on anaerobes, the effects of overexpression of this oxidase in S. cerevisiae BY4741 and Clostridium acetobutylicum 428 (Cac-428) under anaerobic conditions were evaluated. RESULTS: Glucose was exhausted in the NADH oxidase-overexpressing S. cerevisiae strain (Sce-NOX) culture after 26 h, while 43.51 ± 2.18 g/L residual glucose was left in the control strain (Sce-CON) culture at this time point. After 30 h of fermentation, the concentration of ethanol produced by Sce-NOX reached 36.28 ± 1.81 g/L, an increase of 56.38 % as compared to Sce-CON (23.20 ± 1.16 g/L), while the byproduct glycerol was remarkably decreased in the culture of Sce-NOX. In the case of the C. acetobutylicum strain (Cac-NOX) overexpressing NADH oxidase, glucose consumption, cell growth rate, and the production of acetone-butanol-ethanol (ABE) all decreased, while the concentrations of acetic acid and butyric acid increased as compared to the control strain (Cac-CON). During fermentation of Cac-CON and Cac-NOX in 100-mL screw-capped bottles, the concentrations of ABE increased with increasing headspace. Additionally, several alternative electron acceptors in C. acetobutylicum fermentation were tested. Nitroblue tetrazolium and 2,6-dichloroindophenol were lethiferous to both Cac-CON and Cac-NOX. Methylene blue could relieve the effect caused by the overexpression of the NADH oxidase on the metabolic network of C. acetobutylicum strains, while cytochrome c aggravated the effect. CONCLUSIONS: The water-forming NADH oxidase could regulate the metabolism of both the S. cerevisiae and the C. acetobutylicum strains in anaerobic conditions. Thus, the recombinant S. cerevisiae strain might be useful in industrial production. Besides the recognized electron acceptor O2, methylene blue and/or the structural analogs may be the alternative elector acceptor of the NADH oxidase in anaerobic conditions.

4.
J Mater Chem B ; 3(32): 6626-6633, 2015 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-32397689

RESUMEN

An efficient and biocompatible NADH regeneration system to promote ADH-catalysed oxidation reactions is reported. Carbon nanomaterials facilitate enhanced enzyme attachment within their hierarchical nano-structure. According to enzyme protein molecular computer simulation analysis, different electron transfer efficiencies on CNTs or GNSs enzyme-catalyzed electrodes result in different electric charge distributions around ADH, which affects its molecular spatial arrangement and three-dimensional conformation. The nanostructure enhances the enzyme-electrode interaction and electron transfer rate. 1.4- and 1.9-fold higher current density are reached on CNTs and GNSs, respectively, versus the carbon cloth control for the bio-electrochemical NADH regeneration. Maximum NADH production rates are 2.11 and 3.01 times higher than that on the unmodified carbon cloth control. The use of the efficient carbon nanomaterial electrochemical reactor leads to a highly conductive three-dimensional cathode for the improvement of bio-electrochemical NADH regeneration, making the nanomaterial an extremely efficient material from an engineering perspective as well.

5.
Biotechnol Biofuels ; 7(1): 5, 2014 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-24401161

RESUMEN

BACKGROUND: Clostridium acetobutylicum can propagate on fibrous matrices and form biofilms that have improved butanol tolerance and a high fermentation rate and can be repeatedly used. Previously, a novel macroporous resin, KA-I, was synthesized in our laboratory and was demonstrated to be a good adsorbent with high selectivity and capacity for butanol recovery from a model solution. Based on these results, we aimed to develop a process integrating a biofilm reactor with simultaneous product recovery using the KA-I resin to maximize the production efficiency of biobutanol. RESULTS: KA-I showed great affinity for butanol and butyrate and could selectively enhance acetoin production at the expense of acetone during the fermentation. The biofilm reactor exhibited high productivity with considerably low broth turbidity during repeated batch fermentations. By maintaining the butanol level above 6.5 g/L in the biofilm reactor, butyrate adsorption by the KA-I resin was effectively reduced. Co-adsorption of acetone by the resin improved the fermentation performance. By redox modulation with methyl viologen (MV), the butanol-acetone ratio and the total product yield increased. An equivalent solvent titer of 96.5 to 130.7 g/L was achieved with a productivity of 1.0 to 1.5 g · L-1 · h-1. The solvent concentration and productivity increased by 4 to 6-fold and 3 to 5-fold, respectively, compared to traditional batch fermentation using planktonic culture. CONCLUSIONS: Compared to the conventional process, the integrated process dramatically improved the productivity and reduced the energy consumption as well as water usage in biobutanol production. While genetic engineering focuses on strain improvement to enhance butanol production, process development can fully exploit the productivity of a strain and maximize the production efficiency.

6.
Acta Pharmacol Sin ; 33(4): 479-89, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22426695

RESUMEN

AIM: To evaluate the protective effects of strontium fructose 1,6-diphosphate (FDP-Sr), a novel strontium salt that combined fructose 1,6-diphosphate (FDP) with strontium, on bone in an ovariectomy-induced model of bone loss. METHODS: Eighty female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated. Three months later, the rats were assigned to six groups (10 for each): sham-operated, OVX control, OVX+FDP-Sr (110, 220, or 440 mg/kg), or OVX+strontium ranelate (SR, 180 mg/kg). Drugs were administered orally for 3 months. When the treatment was terminated, the following parameters were assessed: bone mineral density (BMD), the biomechanical properties of the femur and lumbar vertebrae, trabecular histomorphology, serum phosphorus, calcium, bone-specific alkaline phosphatase (B-ALP), tartrate-resistant acid phosphatase 5b (TRACP5b), N-telopeptide of type I collagen (NTx) and a series of markers for oxidative stress. Receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) levels in serum were measured using ELISA and their gene expression levels in the bone were measured using R-T PCR. RESULTS: Treatment with FDP-Sr (220 or 440 mg/kg) or SR (180 mg/kg) significantly increased the BMD and improved the bone microarchitecture and bone strength in OVX rats. The treatments also decreased in the levels of H(2)O(2) and MDA, restored the CAT level in serum and bone marrow, increased the serum B-ALP and decreased NTx and TRACP 5b in OVX rats. Treatment with FDP-Sr decreased the RANKL level, and increased the OPG level in serum in a dose-dependent manner. It also significantly down-regulated the RANKL expression and up-regulated OPG expression in bone marrow. CONCLUSION: FDP-Sr may be an effectve treatment for postmenopausal osteoporosis that acts, in part, via a decrease in osteoclastogenesis through the OPG\RANKL\RANK pathway.


Asunto(s)
Fructosadifosfatos/uso terapéutico , Factores Inmunológicos/uso terapéutico , Osteoporosis Posmenopáusica/prevención & control , Animales , Densidad Ósea/efectos de los fármacos , Huesos/efectos de los fármacos , Huesos/patología , Femenino , Fructosadifosfatos/farmacología , Humanos , Factores Inmunológicos/farmacología , Factor I del Crecimiento Similar a la Insulina/análisis , Osteoporosis Posmenopáusica/sangre , Osteoporosis Posmenopáusica/patología , Osteoprotegerina/sangre , Estrés Oxidativo/efectos de los fármacos , Ligando RANK/sangre , Ratas , Ratas Sprague-Dawley , Receptor Activador del Factor Nuclear kappa-B/sangre , Transducción de Señal
7.
Bioresour Technol ; 102(2): 1704-8, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20801644

RESUMEN

The enzymatic hydrolysate of spent yeast cells was evaluated as a nitrogen source for succinic acid production by Actinobacillus succinogenes NJ113, using corn fiber hydrolysate as a carbon source. When spent yeast cell hydrolysate was used directly as a nitrogen source, a maximum succinic acid concentration of 35.5 g/l was obtained from a glucose concentration of 50 g/l, with a glucose utilization of 95.2%. Supplementation with individual vitamins showed that biotin was the most likely factor to be limiting for succinic acid production with spent yeast cell hydrolysate. After supplementing spent yeast cell hydrolysate and 90 g/l of glucose with 150 µg/l of biotin, cell growth increased 32.5%, glucose utilization increased 37.6%, and succinic acid concentration was enhanced 49.0%. As a result, when biotin-supplemented spent yeast cell hydrolysate was used with corn fiber hydrolysate, a succinic acid yield of 67.7% was obtained from 70.3 g/l of total sugar concentration, with a productivity of 0.63 g/(l h). Our results suggest that biotin-supplemented spent yeast cell hydrolysate may be an alternative nitrogen source for the efficient production of succinic acid by A. succinogenes NJ113, using renewable resources.


Asunto(s)
Actinobacillus/metabolismo , Fibras de la Dieta/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Ácido Succínico/síntesis química , Residuos/análisis , Zea mays/metabolismo , Actinobacillus/efectos de los fármacos , Biotina/farmacología , Carbono/farmacología , Fermentación/efectos de los fármacos , Glucosa/farmacología , Hidrólisis/efectos de los fármacos , Nitrógeno/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Vitaminas/farmacología , Zea mays/efectos de los fármacos
8.
Bioprocess Biosyst Eng ; 34(4): 411-8, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21103890

RESUMEN

Escherichia coli AFP111, a pflB, ldhA, ptsG triple mutant of E. coli W1485, can be recovered for additional succinate production in fresh medium after two-stage fermentation (an aerobic growth stage followed by an anaerobic production stage). However, the specific productivity is lower than that of two-stage fermentation. In this study, three strategies were compared for reusing the cells. It was found when cells were aerobically cultivated at the end of two-stage fermentation without supplementing any carbon source, metabolites (mainly succinate and acetate) could be consumed. As a result, enzyme activities involved in the reductive arm of tricarboxylic acid cycle and the glyoxylate shunt were enhanced, yielding a succinate specific productivity above g⁻¹(DCW)h⁻¹ and a mass yield above 0.90 g g⁻¹ in the subsequent anaerobic fermentation. In addition, the intracellular NADH of cells subjected to aerobic cultivation with metabolites increased by more than 3.6 times and the ratio of NADH to NAD+ increased from 0.4 to 1.3, which were both favorable for driving the TCA branch to succinate.


Asunto(s)
Biotecnología/métodos , Escherichia coli/metabolismo , Ácido Succínico/química , Aerobiosis , Anaerobiosis , Técnicas de Química Analítica/métodos , Ciclo del Ácido Cítrico , Medios de Cultivo , Fermentación , Modelos Químicos , NAD/química , Factores de Tiempo
9.
Bioprocess Biosyst Eng ; 34(1): 95-102, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20640447

RESUMEN

Batch fermentative production of welan gum by Alcaligenes sp. CGMCC2428 was investigated under various oxygen supply conditions using regulating agitation speed. Based on a three kinetic parameters analysis that includes specific cell growth rate (µ), specific glucose consumption rate (q (s)), and specific welan formation rate (q (p)), a two-stage agitation speed control strategy was proposed to achieve high concentration, high yield, and high viscosity of welan. During the first 22 h, the agitation speed in 7.5 L fermenter was controlled at 800 rpm to maintain high µ for cell growth. The agitation was then reduced step-wise to 600 rpm to maintain a changing profile with stable dissolved oxygen levels and obtain high qp for high welan accumulation. Finally, the maximum concentration of welan was reached at 26.3 ± 0.89 g L(-1) with a yield of 0.53 ± 0.003 g g(-1) and the welan gum viscosity of 3.05 ± 0.10 Pa s, which increased by an average of 15.4, 15.2, and 20.1% over the best results controlled by constant agitation speeds.


Asunto(s)
Alcaligenes/metabolismo , Microbiología Industrial/métodos , Polisacáridos Bacterianos/biosíntesis , Alcaligenes/crecimiento & desarrollo , Reactores Biológicos , Medios de Cultivo , Fermentación , Glucosa/metabolismo , Cinética , Oxígeno/metabolismo , Polisacáridos Bacterianos/análisis
10.
Bioprocess Biosyst Eng ; 33(9): 1077-85, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20589397

RESUMEN

Propionic acid was produced in a multi-point fibrous-bed (MFB) bioreactor by Propionibacterium freudenreichii CCTCC M207015. The MFB bioreactor, comprising spiral cotton fiber packed in a modified 7.5-l bioreactor, was effective for cell-immobilized propionic acid production compared with conventional free cell fermentation. Batch fermentations at various glucose concentrations were investigated in the MFB bioreactor. Based on analysis of the time course of production, a fed-batch strategy was applied for propionic acid production. The maximum propionic acid concentration was 67.05 g l(-1) after 496 h of fermentation, and the proportion of propionic acid to total organic acids was approximately 78.28% (w/w). The MFB bioreactor exhibited excellent production stability during batch fermentation and the propionic acid productivity remained high after 78 days of fermentation.


Asunto(s)
Reactores Biológicos , Fermentación , Microbiología Industrial/métodos , Propionatos/química , Propionibacterium/metabolismo , Biotecnología/métodos , Diseño de Equipo , Concentración de Iones de Hidrógeno , Microscopía Electrónica de Rastreo/métodos , Espectrofotometría/métodos , Factores de Tiempo
11.
Biotechnol Lett ; 32(10): 1413-8, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20495946

RESUMEN

Escherichia coli AFP111 cells recovered from spent two-stage fermentation broth were investigated for additional production of succinic acid under anaerobic conditions. Recovered cells produced succinic acid in an aqueous environment with no nutrient supplementation except for glucose and MgCO(3). In addition, initial glucose concentration and cell density had a significant influence on succinic acid mass yield and productivity. Although the final concentration of succinic acid from recovered cells was lower than from two-stage fermentation, an average succinic acid mass yield of 0.85 g/g was achieved with an average productivity of 1.81 g/l h after three rounds of recycling, which was comparable to two-stage fermentation. These results suggested that recovered cells might be reused for the efficient production of succinic acid.


Asunto(s)
Escherichia coli/genética , Escherichia coli/metabolismo , Redes y Vías Metabólicas/genética , Ácido Succínico/metabolismo , Anaerobiosis , Fermentación , Ingeniería Genética , Glucosa/metabolismo , Magnesio/metabolismo
12.
Bioprocess Biosyst Eng ; 33(8): 911-20, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20204662

RESUMEN

The effects of redox potential used as a control parameter on the process of succinic acid production in batch cultures of Actinobacillus succinogenes NJ113 have been investigated. In batch fermentation, cell growth and metabolite distribution were changed with redox potential levels in the range of -100 to -450 mV. From the results, the ORP level of -350 mV was preferable, which resulted in high succinic acid yield (1.28 mol mol(-1)), high succinic acid productivity (1.18 g L(-1) h(-1)) and high mole ratio of succinic acid to acetic acid (2.02). The mechanism of redox potential regulation was discussed by metabolic flux analysis and the ratio of NADH/NAD+. We expected that redox potential can be used as a valuable parameter to monitor and control much more anaerobic fermentation production.


Asunto(s)
Actinobacillus/metabolismo , Ácido Succínico/metabolismo , Reactores Biológicos , Fermentación , Oxidación-Reducción
13.
Bioorg Med Chem Lett ; 20(8): 2461-4, 2010 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-20346668

RESUMEN

A series of novel 2-arylimino-3-aryl-thiazolidine-4-ones was designed, synthesized and tested for in vitro antibiofilm activity against Staphylococcus epidermidis. Among them tested, some compounds with carboxylic acid groups showed good antibiofilm activity. The antibiofilm concentration of 1x was 6.25 microM. The structure-activity relationships revealed that incorporation of 2-phenylfuran moiety could greatly enhance antibiofilm activity of thiazolidine-4-one.


Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Biopelículas , Diseño de Fármacos , Tiazoles/química , Tiazoles/farmacología , Antibacterianos/síntesis química , Staphylococcus epidermidis/efectos de los fármacos , Tiazoles/síntesis química
14.
Bioresour Technol ; 101(9): 3159-63, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20064709

RESUMEN

Cyclic adenosine monophosphate (cAMP) was synthesized through the purine salvage synthesis pathway by Arthrobacter A302. Results showed that hypoxanthine was the best of the precursors, and the cAMP concentration reached 4.06 g/L. For inhibition of the glycolytic pathway, sodium fluoride was found the optimal effector, which was further studied on cAMP production. With the addition of 0.4 g/L of sodium fluoride, the maximal cAMP concentration reached 11.04 g/L, and the concentrations of lactic acid, alpha-ketoglutarate and citric acid were decreased by 77%, 86% and 76%, respectively. Meanwhile, the specific activities of glyceraldehyde 3-phosphate dehydrogenase, phosphofructokinase and pyruvate kinase were decreased by 66%, 61%, and 46%, respectively. By contrast the activity of 6-phosphoglucose dehydrogenase was increased by 100%, which demonstrated the redistribution of metabolic flux. This is the first study to reveal the regulatory mechanisms of different effectors on cAMP production among the EMP pathway, HMP pathway and TCA cycle.


Asunto(s)
Arthrobacter/metabolismo , AMP Cíclico/biosíntesis , Arthrobacter/efectos de los fármacos , Arthrobacter/enzimología , Arthrobacter/crecimiento & desarrollo , Ácidos Carboxílicos/metabolismo , Fermentación/efectos de los fármacos , Glucólisis/efectos de los fármacos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Fluoruro de Sodio/farmacología
15.
Bioprocess Biosyst Eng ; 33(5): 565-71, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19768471

RESUMEN

A new yeast, isolated from natural osmophilic sources, produces D-arabitol as the main metabolic product from glucose. According to 18S rRNA analysis, the NH-9 strain belongs to the genus Kodamaea. The optimal culture conditions for inducing production of D-arabitol were 37 degrees C, neutral pH, 220 rpm shaking, and 5% inoculum. The yeast produced 81.2 +/- 0.67 g L(-1) D-arabitol from 200 g L(-1) D-glucose in 72 h with a yield of 0.406 g g(-1) glucose and volumetric productivity Q(P) of 1.128 g L(-1) h(-1). Semi-continuous repeated-batch fermentation was performed in shaker-flasks to enhance the process of D-arabitol production by Kodamaea ohmeri NH-9 from D-glucose. Under repeated-batch culture conditions, the highest volumetric productivity was 1.380 g L(-1) h(-1).


Asunto(s)
Saccharomycetales/crecimiento & desarrollo , Alcoholes del Azúcar/metabolismo , Glucosa/metabolismo , ARN de Hongos/genética , ARN Ribosómico 18S/genética , Saccharomycetales/genética , Saccharomycetales/aislamiento & purificación , Saccharomycetales/metabolismo
16.
Appl Biochem Biotechnol ; 160(4): 1017-31, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19455429

RESUMEN

An organic solvent-stable protease from Pseudomonas aeruginosa PT121 was purified in a single step with 55% recovery by hydrophobic interaction chromatography on a Phenyl Sepharose High Performance matrix. The purified protease was homogenous on SDS-PAGE and had an estimated molecular mass of 33 kDa. The optimal pH and temperature conditions for enzyme activity were 8.0 and 60 degrees C, respectively. The enzyme was classified as a metalloprotease based on its strong inhibition by EDTA and 1,10-phenanthroline and exhibited good stability across a broad pH range (6.0-11.0). The protease was quite stable in the presence of various water-miscible organic solvents. This is a unique property of the protease which makes it an ideal choice for application in aqueous-organic phase organic synthesis including peptides synthesis. The synthetic activity of the protease was tested using N-carbobenzoxy-L-asparagine (Z-Asp) and L-phenylalaninamide (Phe-NH2) as substrate in the presence of various water-miscible organic solvents for aspartame precursor synthesis. The highest yield was obtained in the presence of 50% DMSO (91%). The synthesis rate in the presence of DMSO was also much higher than the rates in the other tested organic solvents, and the initial rates of Z-Asp-Phe-NH2 synthesis in mixtures of various water-miscible organic solvents, with the exception of ethanol, correlated with the yields of Z-Asp-Phe-NH2. Furthermore, the PT121 protease was able to use various carboxyl components (Z-AA) and Phe-NH2 as substrates to catalyze the syntheses of the dipeptides, indicating that this protease has a broad specificity for carboxylic acid residue.


Asunto(s)
Metaloproteasas/metabolismo , Pseudomonas aeruginosa/enzimología , Dimetilsulfóxido/farmacología , Dipéptidos/síntesis química , Estabilidad de Enzimas , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/efectos de los fármacos , Metaloproteasas/aislamiento & purificación , Solventes/farmacología , Especificidad por Sustrato , Tensoactivos/farmacología , Temperatura
17.
J Pharm Pharmacol ; 61(2): 229-36, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19178771

RESUMEN

OBJECTIVES: Male hypogonadism is frequently associated with testopathy in patients with type 2 diabetes and in middle-aged males. We hypothesized that abnormal matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in testis have large roles to play in male hypogonadism. It has been found in diabetic rats that a novel compound, strontium fructose 1,6-diphosphate (FDP-Sr), with extra high energy supply, could reverse male hypogonadism by normalizing MMP-9 and TIMPs in the testis. We investigated whether FDP-Sr could be promising in treating diabetic testopathy. METHODS: Adult male Sprague-Dawley rats were administered a single dose of streptozocin (65 mg/kg, i.p.) to induce diabetes. The diabetic rats were treated with FDP-Sr in three doses or testosterone propionate in the final four weeks during the eight-week study. KEY FINDINGS: Serum testosterone, activity of marker enzymes, and mRNA of MMPs and TIMPs and protein of MMP-9 in the testis were detected. After eight weeks, the activity of acid phosphatase, lactate dehydrogenase, succinate dehydrogenase and g-glutamyl transpeptidase in testis were significantly decreased (P < 0.01), accompanied by down-regulated mRNA and activity of MMP-2 and MMP-9 (P < 0.01) and upregulated mRNA of TIMP-1 and TIMP-2. Downregulated MMP-9 protein and degenerative changes in histology were predominant in diabetic testis. CONCLUSIONS: FDP-Sr or testosterone propionate significantly normalized expression and activity of the MMPs-TIMPs system to attenuate changes in serum testosterone, marker enzymes and histology in testis. Effects of FDP-Sr were dose-dependent and comparable with those of testosterone propionate. By supplying extra energy, FDP-Sr could be promising in treating diabetic testopathy by normalizing abnormal MMP-9 and its endogenous inhibitors in testes.


Asunto(s)
Fructosadifosfatos/farmacología , Hipogonadismo/tratamiento farmacológico , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz/efectos de los fármacos , Estreptozocina/toxicidad , Fosfatasa Ácida/antagonistas & inhibidores , Fosfatasa Ácida/sangre , Animales , Complicaciones de la Diabetes/tratamiento farmacológico , Diabetes Mellitus Tipo 2/complicaciones , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Expresión Génica , Hiperglucemia/inducido químicamente , Hipogonadismo/complicaciones , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/sangre , Masculino , Metaloproteinasas de la Matriz/clasificación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Estreptozocina/administración & dosificación , Succinato Deshidrogenasa/antagonistas & inhibidores , Succinato Deshidrogenasa/sangre , Testículo/efectos de los fármacos , Testículo/enzimología , Testículo/ultraestructura , Testosterona/antagonistas & inhibidores , Testosterona/sangre , Inhibidores Tisulares de Metaloproteinasas/metabolismo , gamma-Glutamiltransferasa/antagonistas & inhibidores , gamma-Glutamiltransferasa/sangre
18.
J Microbiol Biotechnol ; 19(12): 1497-505, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20075610

RESUMEN

The thermophile Bacillus fordii MH602 was screened for stereospecifically hydrolyzing DL-5-substituted hydantoins to L-alpha-amino acids. Since the reaction at higher temperature, the advantageous for enhancement of substrate solubility and for racemization of DL-5-substituted hydantoins during the conversion were achieved. The hydantoin metabolism gene cluster from thermophile was firstly reported in this paper. The genes involved in hydantoin utilization (hyu) were isolated on an 8.2 kb DNA fragment by Restriction Site-dependent PCR, and six ORFs were identified by DNA sequence analysis. The hyu gene cluster contained four genes with novel cluster organization characteristics: the hydantoinase gene hyuH, putative transport protein hyuP, hyperprotein hyuHP, and L-carbamoylase gene hyuC. The hyuH and hyuC genes were heterogeneously expressed in E. coli. The results indicated that hyuH and hyuC are involved in the conversion of DL-5-substituted hydantoins to an N-carbamyl intermediate that is subsequently converted to L-alpha-amino acids. Hydantoinase and carbamoylase from B. fordii MH602 comparing respectively with reported hydantoinase and carbamoylase showed the highest identities of 71% and 39%. The novel cluster organization characteristics and the difference of the key enzymes between thermopile B. fordii MH602 and other mesophiles were presumed to be related to the evolutionary origins of concerned metabolism.


Asunto(s)
Aminoácidos/biosíntesis , Bacillus , Hidantoínas/metabolismo , Familia de Multigenes , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Aminoácidos/química , Bacillus/genética , Bacillus/metabolismo , Proteínas Portadoras/genética , Clonación Molecular , ADN Bacteriano/análisis , ADN Bacteriano/genética , Genes Bacterianos , Calor , Hidantoínas/química , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Análisis de Secuencia de ADN , Especificidad por Sustrato
19.
Bioresour Technol ; 100(2): 919-24, 2009 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18778935

RESUMEN

Response surface methodology was employed to optimize medium composition for the production of cyclic adenosine 3',5'-monophosphate (cAMP) with Microbacterium sp. no. 205. A fractional factorial design (2(11-7)) was applied to evaluate the effects of different components in the medium. K(2)HPO(4), MgSO(4) and NaF were found to significantly influence on the cAMP production. The steepest ascent method was used to access the optimal region of the medium composition. The concentrations of the three factors were optimized subsequently using central composite design and response surface methodology. The optimal medium composition to achieve the optimal cAMP production was determined (g/L): K(2)HPO(4), 12.78; MgSO(4), 3.53 and NaF, 0.18. The corresponding cAMP concentration was 8.50 g/L, which was about 1.8-fold increase compared with that using the original medium. Validation experiments were also carried out to prove the adequacy and the accuracy of the model obtained. The cAMP fermentation in 5L fermenter reached 9.87 g/L.


Asunto(s)
Actinomycetales/metabolismo , Reactores Biológicos/microbiología , Técnicas de Cultivo de Célula/métodos , Medios de Cultivo/química , Medios de Cultivo/metabolismo , AMP Cíclico/metabolismo , Modelos Biológicos , Actinomycetales/clasificación , Algoritmos , Técnicas Químicas Combinatorias/métodos , Simulación por Computador , Especificidad de la Especie
20.
Acta Crystallogr Sect E Struct Rep Online ; 65(Pt 4): m453-4, 2009 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-21582389

RESUMEN

In the mol-ecule of the title complex, [Ni(C(30)H(29)N(3)O(5))], the Ni atom is coordinated in a distorted square-planar geometry by three N and one O atoms. The aromatic rings are oriented at dihedral angles of 29.01 (3), 79.73 (3) and 83.37 (3)°. The remaining rings adopt envelope conformations with the C and N atoms at the flap positions. In the crystal structure, inter-molecular O-H⋯O hydrogen bonds link the mol-ecules into chains along the b axis. There is also a weak C-H⋯π inter-action.

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