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1.
Electron. j. biotechnol ; 52: 35-44, July. 2021. tab, ilus
Article in English | LILACS | ID: biblio-1283494

ABSTRACT

BACKGROUND: Alginates are polysaccharides used in a wide range of industrial applications, with their functional properties depending on their molecular weight. In this study, alginate production and the expression of genes involved in polymerization and depolymerization in batch cultures of Azotobacter vinelandii were evaluated under controlled and noncontrolled oxygen transfer rate (OTR) conditions. RESULTS: Using an oxygen transfer rate (OTR) control system, a constant OTR (20.3 ± 1.3 mmol L 1 h 1 ) was maintained during cell growth and stationary phases. In cultures subjected to a controlled OTR, alginate concentrations were higher (5.5 ± 0.2 g L 1 ) than in cultures under noncontrolled OTR. The molecular weight of alginate decreased from 475 to 325 kDa at the beginning of the growth phase and remained constant until the end of the cultivation period. The expression level of alyA1, which encodes an alginate lyase, was more affected by OTR control than those of other genes involved in alginate biosynthesis. The decrease in alginate molecular weight can be explained by a higher relative expression level of alyA1 under the controlled OTR condition. CONCLUSIONS: This report describes the first time that alginate production and alginate lyase (alyA1) expression levels have been evaluated in A. vinelandii cultures subjected to a controlled OTR. The results show that automatic control of OTR may be a suitable strategy for improving alginate production while maintaining a constant molecular weight.


Subject(s)
Polysaccharide-Lyases/metabolism , Oxygen Transfer , Azotobacter vinelandii/metabolism , Oxygen/metabolism , Gene Expression , Polymerase Chain Reaction , Azotobacter vinelandii/genetics , Alginates/metabolism , Fermentation , Molecular Weight
2.
Braz. j. med. biol. res ; 54(6): e10423, 2021. tab, graf
Article in English | LILACS | ID: biblio-1285668

ABSTRACT

About 3000 tons of beans are not used in human food due to hardening. Several studies on bean-derived bioactive peptides have shown potential to treat some diseases, including those relying on oxidative dysfunctions. We assessed the effects of peptides extracted from hardened bean Phaseolus vulgaris (PV) on reactive oxygen species (ROS) and nitric oxide (NO) production, cytotoxic and cytoprotective effects in endothelial cells, and oxidonitrergic-dependent vasodilating effects. Extract was composed by peptide fraction <3 kDa (PV3) from hardened common bean residue. PV3 sequences were obtained and analyzed with bioinformatics. Human umbilical vein endothelial cells were treated with 10, 20, 30, and 250 µg/mL PV3. Oxidative stress was provoked by 3% H2O2. Cytotoxicity and cytoprotective effects were evaluated by MTT assay, whereas, ROS and NO were quantified using DHE and DAF-FM fluorescent probes by confocal microscopy. NO- and endothelium-dependent vasodilating effects of PV3 were assessed in isolated aortic rings. We found 35 peptides with an average mass of 1.14 kDa. There were no cell deaths with 10 and 20 μg/mL PV3. PV3 at 30 μg/mL increased cell viability, while cytotoxicity was observed only with 250 μg/mL PV3. PV3 at 10 μg/mL was able to protect cells from oxidative stress. PV3 also increased NO release without causing cell death. It also reduced relative ROS production induced by H2O2. PV3 vasodilating effects relied on endothelium-dependent NO release. PV3 obtained from low-commercial-value bean displays little cytotoxicity and exerts antioxidant effects, whereas it increases endothelial NO release.


Subject(s)
Humans , Phaseolus , Peptides/pharmacology , Endothelium , Hydrogen Peroxide , Molecular Weight , Antioxidants/pharmacology
3.
Cienc. tecnol. salud ; 8(1)2021. il 27 c
Article in Spanish | LILACS, LIGCSA, DIGIUSAC | ID: biblio-1352956

ABSTRACT

Polymeric membrane technologies demand the synthesis of new polymers to enhance their equilibrium, thermal, and transport properties. Therefore, the focus of this investigation was the evaluation of the equilibrium and thermal properties of a sulfonated fluoroblock copolymer blend membrane composed of sulfonated poly(styre-ne-isobutylene-styrene) (SIBS SO3H) and a novel sulfonated fluoroblock copolymer composed of poly(4-fluo-rostyrene) (P4FS), poly(styrene) (PS) and poly(isobutylene) (PIB). The fluoroblock copolymer was synthesized using Atom Transfer Radical Polymerization (ATRP) and cationic polymerization. First, the molecular weight and the thermal stability of the block copolymer were determined using Gel Permeation Chromatography (GPC) and Thermogravimetric Analysis (TGA). Second, the chemical composition was monitored utilizing Fourier Transform Infrared spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR) spectroscopy. The molecular weight of P4FS-b-PS was Mn ~ 36,100; this value increased 8% after the cationic polymerization. The equilibrium properties of the membrane were evaluated using the water uptake and Ion-Exchange Capacity. The degradation behavior and the thermal transitions were determined using TGA and Differential Scanning Calorimetry (DSC), respectively. This newly membrane exhibited water uptake higher than 608% related to the improvement of 36% in the ion-exchange capacity and the increment of 25.31% and 25.24% in the energy required to produce the thermal transitions induced by the addition of the sulfonated fluoroblock copolymer.


La tecnología de membranas poliméricas requiere de la síntesis de nuevos polímeros que mejoren sus propiedades de equilibrio, térmicas y de transporte. Esta investigación tuvo como objetivo determinar las pro-piedades de equilibrio y térmicas de una membrana compuesta de poli(estireno-isobutileno-estireno) sulfonado (SIBS SO3H) y un fluoropolímero en bloque sulfonado compuesto de poli(4-fluorostireno) (P4FS), poli(estireno) (PS) y poli(isobutileno) (PIB). El fluoropolímero en bloque se sintetizó utilizando la técnica de polimerización radical por transferencia atómica (ATRP por sus siglas en inglés) y polimerización catiónica. El peso molecular y la estabilidad térmica del fluoropolímero en bloque fueron determinadas por medio de Cromatografía de Permeación en Gel (GPC) y un análisis termogravimétrico (TGA). La composición química se monitorizó utilizando espectroscopía infrarroja por transformada de Fourier (FTIR) y espectroscopía de Resonancia Magnética Nuclear (RMN). El peso molecular de P4FS-b-PS fue Mn ~ 36,100; este valor aumentó un 8% después de la polimerización catiónica. Las propiedades de equilibrio de la membrana fueron evaluadas por medio de la absorción de agua y la capacidad de intercambio iónico. El comportamiento de degradación y las transiciones térmicas se determinaron utilizando TGA y Calorimetría Diferencial de Barrido (DSC). Esta nueva membrana exhibió una absorción de agua mayor del 608% relacionada con la mejora del 36% en la capacidad de intercambio iónico y el incremento en 25.31% y 25.24% en la energía requerida para producir las transiciones termales inducidas por la adición del fluoropolímero sulfonado en bloque.


Subject(s)
Polymers/analysis , Membranes, Artificial , Polystyrenes/analysis , Absorption , Polymerization , Ion Exchange , Molecular Weight
4.
Article in Chinese | WPRIM | ID: wpr-880177

ABSTRACT

OBJECTIVE@#To investigate the therapeutic effect of spleen low molecular weight extracts on epileptics hydrochloride-induced leukopenia in mice and explore its mechanism.@*METHODS@#The model of leukopenia in mice was established by the injection of epirubicin hydrochloride (10 mg/kg). After the injection of chemotherapeutic drugs, leukocytopenia mice were treated with different doses of spleen low molecular weight extract, Ganoderma oral solution and recombinant granulocyte colony stimulating factor (rhG-CSF). The general survival status indicators such as body weight, coat color and athletic ability of mice in each group were recorded; the tail vein blood of mice in each group was collected and the white blood cell count in them was calculated; bone marrow of mice was taken and bone marrow smears were observed.@*RESULTS@#In the model group, the weight of the mice gradually decreased in the later period, their coat became dark and rough, and the ability to exercise decreased, while the mice in the treatment groups showed different degrees of improvement in their survival status except for the mice treated by rhG-CSF. There was no significant fluctuation in the white blood cell count of the blank control mice. After injection of epirubicin, the white blood cell count of peripheral blood in the model mice and treated mice were decreased. The white blood cell count was lower in the mice treated with high-dose low molecular weight extract and rhG-CSF than that in other experimental groups. Bone marrow smear showed that the proportion of bone marrow nucleated cells in the mice treated with the low molecular weight extract of the spleen was significantly higher than that of model mice (P<0.05).@*CONCLUSION@#The low molecular weight spleen extracts can significantly improve the hematopoietic state of mouse bone marrow, promote the proliferation of inhibited bone marrow cells, and thus has the effect of treating leukopenia in mice.


Subject(s)
Animals , Epirubicin , Granulocyte Colony-Stimulating Factor , Leukocyte Count , Leukopenia/drug therapy , Mice , Molecular Weight , Plant Extracts , Recombinant Proteins , Spleen
5.
Chinese Journal of Biotechnology ; (12): 2260-2276, 2020.
Article in Chinese | WPRIM | ID: wpr-878484

ABSTRACT

Dissolving pulp consists of high purity cellulose and is widely used to as raw materials for the production of regenerated cellulose fiber, cellulose ester and cellulose ether. The characteristic of dissolving pulp affects greatly the production and processing performance of subsequent products. The α-cellulose content, hemicellulose content, pulp viscosity, ash, transition metal ion content, fiber morphology, molecular weight distribution of cellulose and the reactivity are the important properties. Because of its green, mild and high efficiency, the application of enzymes in improving the properties of dissolving pulp has a promising application prospect and has been researched significantly. In this review, the main properties of dissolving pulp are presented first, followed by a recommendation of the enzymes to improve these properties. The application and current research of cellulase and xylanase in improving the properties of dissolving pulp are emphasized. The main problems and the future research areas in improving the properties of dissolving pulp by enzymes are revealed. Finally, the technology prospects in this field are proposed.


Subject(s)
Cellulase , Molecular Weight , Viscosity , Wood
6.
Article in English | WPRIM | ID: wpr-762175

ABSTRACT

PURPOSE: Occupational asthma may be induced by high- or low-molecular weight allergens (HMWA or LMWA, respectively). The study was conducted to compare the pattern of bronchial response in 200 HMWA-induced asthmatics (n = 130) and LMWA-induced asthmatics (n = 70). METHODS: The study participants underwent a single-blind, placebo-controlled specific inhalation challenge (SIC) with workplace allergens, accompanied by evaluation of non-specific bronchial hyperresponsiveness (NSBHR) with methacholine before and after the SIC. RESULTS: A single early bronchial response more frequently occurred in HMWA-induced asthmatics than in LMWA-induced asthmatics (86.2% vs. 20%). An isolated late bronchial response or atypical patterns were more frequently observed in LMWA-induced asthmatics than in LMWA-induced asthmatics (45.7% vs. 3.8% or 34.3% vs. 10%, respectively). Baseline NSBHR before SIC was more often detected in LMWA-induced asthmatics than in HMWA-induced asthmatics (81.4% vs. 54.6%), and the median value of the provocation concentration of methacholine was relevantly lower in these patients before and after SIC. A significant 3-fold increase in NSBHR after SIC was observed more often in LMWA-induced asthmatics than in HMWA-induced asthmatics (82.8% vs. 66.1%). In addition, compared to LMWA-induced asthmatics, HMWA-induced asthmatics were older, were more frequently active smokers, showed lower level of NSBHR, and more frequently continued their work in harmful occupational exposure. CONCLUSIONS: The results of this study suggest that HMWA-induced asthmatics may have milder clinical courses and that there is a possibility of job continuation despite asthma exacerbation requiring medical surveillance.


Subject(s)
Allergens , Asthma , Asthma, Occupational , Bronchial Hyperreactivity , Humans , Immunoglobulin E , Inhalation , Methacholine Chloride , Molecular Weight , Occupational Exposure , Prognosis
7.
Acta ortop. mex ; 33(3): 166-168, may.-jun. 2019.
Article in Spanish | LILACS | ID: biblio-1248656

ABSTRACT

Resumen: Introducción: La osteoartrosis es actualmente la quinta causa de discapacidad en el adulto. Su prevalencia aumenta a partir de los 40 años. La viscosuplementación es un tratamiento para las etapas iniciales de la osteoartrosis de rodilla. Objetivo: Comparar la eficacia y la calidad de vida en pacientes con gonartrosis sintomática tratados con ácido hialurónico de alto y mediano peso molecular. Material y métodos: Estudio analítico, retrospectivo, transversal, observacional de expedientes clínicos de pacientes con diagnóstico de gonartrosis grado II y grado III, de Octubre de 2017 a Junio de 2018, tratados con viscosuplementación con ácido hialurónico de alto o de mediano peso molecular; se analizó la calidad de vida y la escala de WOMAC de manera previa a la primera aplicación y con seguimiento a los dos, cuatro y seis meses. Resultados: 194 pacientes, con edades entre 30 y 87 años (promedio 61.85; DE = 11.3); 134 mujeres (69.1%) y 60 hombres (30.9%). En ambos grupos la evolución fue hacia la mejoría, con disminución en el puntaje WOMAC, sin diferencia significativa entre grupos. El puntaje de WOMAC tuvo un aumento mínimo pasados seis meses de la aplicación en ambos grupos, lo que se interpreta como disminución del efecto terapéutico. Dos pacientes tuvieron reacciones adversas mínimas localizadas, las cuales se resolvieron con medidas generales. Conclusión: El ácido hialurónico en ambas presentaciones, de alto y de bajo peso molecular, es efectivo en el tratamiento de la gonartrosis sintomática sin diferencia con el peso molecular.


Abstract: Introduction: OA is currently the fifth leading cause of adult disability, its prevalence increases from age 40. Viscosupplementation is a treatment for the early stages of knee OA. Objective: To compare efficacy and quality of life in patients with symptomatic gonartrosis, treated with high and low molecular weight hyaluronic acid. Material and methods: Analytical, retrospective, cross-sectional, observational study of clinical records of patients diagnosed with grade II and grade III gonarthrosis, October 2017 to June 2018, treated with viscosupplementation with high molecular weight hyaluronic acid or low molecular weight; WOMAC quality of life and scale were analyzed prior to the first application and tracked at two, four and six months. Results: 194 patients, aged between 30 to 87 years (promedio 61.85; SD = 11.3); 134 women (69.1%), 60 men (30.9%). In both groups the evolution was towards improvement, with a decrease in the WOMAC score, with no significant difference between groups. The WOMAC score had a minimum increase after 6 months of application in both groups, which is interpreted as decreasing the therapeutic effect. Two patients had localized minimal adverse reactions which were resolved with general measures. Conclusion: Hyaluronic acid in both presentations, high and low molecular weight, are effective in the treatment of symptomatic goonarthrosis without difference to molecular weight.


Subject(s)
Humans , Male , Female , Adult , Aged , Aged, 80 and over , Osteoarthritis, Knee/therapy , Viscosupplementation , Quality of Life , Cross-Sectional Studies , Retrospective Studies , Treatment Outcome , Hyaluronic Acid , Injections, Intra-Articular , Middle Aged , Molecular Weight
8.
J. appl. oral sci ; 27: e20180663, 2019. tab, graf
Article in English | LILACS, BBO | ID: biblio-1040224

ABSTRACT

Abstract Objective: To investigate the use of polymethyl methacrylate (PMMA) electrospun fiber mats containing different amounts of polyethylene oxide (PEO) as a doxycycline delivery system and to test antibacterial activity against an oral pathogen. Methodology: PMMA powders or PEO (mol wt 200 Kd) (10,20,30% w/w/) were dissolved in N, N-dimethylformamide (DMF) to obtain a final polymer concentration of 15% in DMF (w/v). 2% Doxycycline monohydrate was added to the solutions and submitted to vortex mixing. The solution was transferred to a plastic syringe and fit into a nanofiber electrospinning unit. The parameters applied were: voltage at 17.2 kV; distance of 20 cm between the needle tip and the collector plate; target speed at 2 m/min; and transverse speed at 1cm/min. Syringe pump speed was 0.15 mm/min. The drug release analysis was performed by removing aliquots of the drug-containing solution (in PBS) at specific periods. Doxycycline release was quantified using RP-HPLC. Fiber mats from all groups had their antibacterial action tested against S. mutans based on inhibition halos formed around the specimens. The experiments were performed in triplicate. Gravimetric analysis at specific periods was performed to determine any polymer loss. Morphological characterization of the electrospun fibers was completed under an optical microscope followed by SEM analysis. Results: The addition of PEO to the PMMA fibers did not affect the appearance and diameter of fibers. However, increasing the %PEO caused higher doxycycline release in the first 24 h. Fibers containing 30% PEO showed statistically significant higher release when compared with the other groups. Doxycycline released from the fibers containing 20% or 30% of PEO showed effective against S. mutans. Conclusion: The incorporation of PEO at 20% and 30% into PMMA fiber mat resulted in effective drug release systems, with detected antibacterial activity against S. mutans.


Subject(s)
Polyethylene Glycols/pharmacokinetics , Doxycycline/pharmacokinetics , Polymethyl Methacrylate/pharmacokinetics , Nanofibers/chemistry , Anti-Bacterial Agents/pharmacokinetics , Polyethylene Glycols/chemistry , Streptococcus mutans/drug effects , Time Factors , Water/chemistry , Microscopy, Electron, Scanning , Reproducibility of Results , Analysis of Variance , Chromatography, High Pressure Liquid/methods , Doxycycline/chemistry , Polymethyl Methacrylate/chemistry , Immersion , Anti-Bacterial Agents/chemistry , Molecular Weight
9.
Article in Chinese | WPRIM | ID: wpr-773138

ABSTRACT

In this study,the protein in different Cordyceps samples,which include fresh sample( S1),22 ℃ drying sample( S2),37 ℃ drying sample( S3) and 60 ℃ drying sample( S4),were analyzed by sodium dodecylsupinate-polyacrylamide gel electrophoresis( SDS-PAGE) and two-dimensional electrophoresis( 2-DE). The total protein contents in Cordyceps samples were from 1. 655-4. 493 mg·g~(-1) and the protein contents in fresh sample was the highest. The results of SDS-PAGE showed that the mainly ranges of protein molecular weight of Cordyces samples were 10-100 kDa and the numbers of protein bands were 28 to 41,the fresh sample had the maximum number of protein bands. The 2-DE profiles were analyzed by PDQuest software. The resulted indicated that 488-876 protein spots were detected in different Cordyceps samples and the isoelectric point( pI) was distributed between 4. 5 and 6. 5,the protein molecular weight was distributed in 10-20 kDa and 25-100 kDa,the fresh sample had the maximum number of protein spots. Therefore,the drying process could decrease contents and species of protein in Cordyceps,and the different drying conditions had different effects on protein. These results provide a reference for improving the drying process of Cordyceps.


Subject(s)
Cordyceps , Chemistry , Desiccation , Methods , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Fungal Proteins , Molecular Weight
10.
Chinese Journal of Biotechnology ; (12): 159-168, 2019.
Article in Chinese | WPRIM | ID: wpr-771390

ABSTRACT

Two proteins of similar molecular weight (named as ASPR-C-1 and ASPR-C-2) from the crude drug of Angelica sinensis were purified and characterized by 80% ammonium sulfate precipitation, Sephadex G-50 gel filtration chromatography, and DEAE-Sepharose anion exchange chromatography. The molecular weight of ASPR-C-1 and ASPR-C-2 on SDS-PAGE was 17.33 kDa and 17.18 kDa, respectively. They were mainly monomeric in solution, but partially formed dimers and they were glycoproteins with glycosyl content of 2.6% and 8.2%, respectively. Both ASPR-C-1 and ASPR-C-2 were identified to be members of pathogenesis-related 10 family of proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and have ribonuclease activities with the specific activity of 73.60 U/mg and 146.76 U/mg, respectively. The optimum pH of the two isoforms was similar, at about 5.6, while their optimum temperatures were different. The optimum temperature of ASPR-C-1 was 50 ℃, and that of ASPR-C-2 was 60 ℃. Both isoforms presented highest thermal stability at 60 ℃. However, ASPR-C-2 was more thermotolerant than ASPR-C-1. The latter was rapidly inactivated and retained only about 20% residual activity while the former still maintained about 80% of its original activity at a higher treatment temperature (80 to 100 ℃). In addition, Fe²⁺ had an activating effect on the ribonuclease activities of two isoforms while Ca²⁺, Mg²⁺, Zn²⁺, Mn²⁺, Ag⁺, Cu²⁺, EDTA (Elhylene diamine tetraacetic acid), dithiothreitol and sodium dodecylsulphate showed different degrees of inhibition of the enzyme activities. Our findings provide a foundation for further research on the biological function of PR-10 protein from Angelica sinensis.


Subject(s)
Angelica sinensis , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Hydrogen-Ion Concentration , Kinetics , Molecular Weight , Protein Isoforms , Temperature
11.
Article in English | WPRIM | ID: wpr-758954

ABSTRACT

The intra-articular use of hyaluronic acid (HA) for the treatment of synovitis and osteoarthritis is still controversial. As a consequence, corticosteroids remain the most frequently employed therapeutic agents, despite their potential systemic and local deleterious effects. This study examined the anti-inflammatory, antioxidant, and chondroprotective activities of low and high molecular weight hyaluronic acid (LMW-HA and HMW-HA) on lipopolysaccharide (LPS)-induced synovitis in horses compared to triamcinolone acetonide (TA). LPS was injected in the metacarpophalangeal joints, which were treated intra-articularly with either TA (as control) or LMW-HA or HMW-HA. Joint clinical evaluation and synovial fluid (SF) analysis were performed at 0, 8, 24, and 48 h. The white blood cell counts (WBC), prostaglandin E2 (PGE2), interleukin (IL)-1, IL-6, IL-10, tumor necrosis factor-α, chondroitin sulfate (CS) and HA concentrations, oxidative burst, and HA molecular weights were measured. TA reduced the lameness, swelling, and PGE2 release but increased the SF CS concentrations enormously at 24h and 48h, and decreased the SF HA modal molecular weight. These results indicate the breakdown of articular cartilage aggrecan and SF HA. In contrast, LMW-HA and HMW-HA were less effective in reducing the inflammation symptoms, but preserved the joints because only a modest increase in CS occurred at 24 h, decreasing at 48 h, and the SF HA was maintained. The HA-treatment also had anti-inflammatory actions, and LMW-HA was the most effective in reducing the release of cytokine. In summary, the HA treatment inhibited efficiently the digestion of cartilage proteoglycans and SF HA breakdown.


Subject(s)
Adrenal Cortex Hormones , Aggrecans , Cartilage , Cartilage, Articular , Chondroitin Sulfates , Digestion , Dinoprostone , Horses , Hyaluronic Acid , Inflammation , Interleukin-10 , Interleukin-6 , Interleukins , Joints , Leukocyte Count , Metacarpophalangeal Joint , Molecular Weight , Necrosis , Osteoarthritis , Proteoglycans , Respiratory Burst , Synovial Fluid , Synovitis , Triamcinolone , Triamcinolone Acetonide
12.
Article in English | WPRIM | ID: wpr-758949

ABSTRACT

Chronic kidney disease is considered to be most common in geriatric domestic cats. It has been reported that the feline viral rhinotracheitis, calicivirus, and panleukopenia (FVRCP) vaccine prepared from the Crandell-Rees feline kidney (CRFK) cell line can induce cross-reactions of antibodies with feline kidney tissues. As an anti-cat kidney antibody was not available commercially for this study of autoantibody in cats, the purpose of this study was to produce anti-cat kidney antibody in rabbits for further study of autoantibody in cats after FVRCP vaccination. Kidney proteins from cadaveric cats were extracted and immunized into rabbits using Montanide as the adjuvant. Based on enzyme-linked immunosorbent assay measurement, all immunized rabbits produced high levels of anti-cat kidney antibodies and some began to produce antibodies as early as 2 weeks after immunization. Immunofluorescence staining of rabbit sera showed kidney-bound antibodies in glomerulus, Bowman's capsule, apical surface of the proximal convoluted tubule, peritubular surface, and interstitial cells. Western blot analysis of cat kidney proteins revealed molecular weights (M.W.) of 72, 55, 47, and 31 kDa, while binding to the CRFK cell proteins was observed at M.W. of 43 and 26 kDa. The antibody that recognized the 47 kDa protein was similarly detected in cats with autoantibody presence after FVRCP vaccination. The kidney-bound antibody profile at different time points and its patterns in rabbits could be used as a model for the study of autoantibody to cat kidney in feline chronic kidney diseases.


Subject(s)
Animals , Antibodies , Blotting, Western , Bowman Capsule , Cadaver , Cats , Cell Line , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Immunization , Kidney , Molecular Weight , Rabbits , Renal Insufficiency, Chronic , Vaccination , Vaccines
13.
Chinese Journal of Biotechnology ; (12): 816-826, 2019.
Article in Chinese | WPRIM | ID: wpr-771328

ABSTRACT

A new method to express oligomerized feruloyl esterase (FAE) in Pichia pastoris GS115 to improve the catalytic efficiency was developed. It was realized by fusing the foldon domain at the C-terminus of FAE, and the fusion protein was purified by histidine tag. Fusion of the feruloyl esterase with the foldon domain resulted spontaneously forming a trimer FAE to improve the catalytic performance. The oligomerized FAE and monomeric FAE were obtained by purification. The apparent molecular weight of the oligomerized FAE was about 110 kDa, while the monomeric FAE about 40 kDa, and the optimum temperature of the oligomerized FAE was 50 °C, which is the same as the monomeric one. The optimal pH of the oligomerized FAE is 5.0, while the optimal pH of the monomer FAE is 6.0. When compared with the monomeric ones, the catalytic efficiency (kcat/Km) of the oligomerized FAE increased 7.57-folds. The catalytic constant (kcat) of the oligomerized FAE increased 3.42-folds. The oligomerized FAE induced by foldon have advantages in the catalytic performances, which represents a simple and effective enzyme-engineering tool. The method proposed here for improving the catalytic efficiency of FAE would have great potentials for improving the catalytic efficiency of other enzymes.


Subject(s)
Carboxylic Ester Hydrolases , Metabolism , Catalysis , Molecular Weight , Pichia , Genetics , Metabolism , Polymerization , Protein Engineering , Substrate Specificity
14.
Article in English | WPRIM | ID: wpr-719490

ABSTRACT

PURPOSE: The aim of the present study was to develop a serodiagnostic test for differentiation infected from vaccinated animal (DIVA) strategy accompanying the marker vaccine lacking an immunodominant epitope (IDE) of nucleoprotein of Newcastle disease virus (NDV). MATERIALS AND METHODS: Recombinant epitope-repeat protein (rERP) gene encoding eight repeats of the IDE sequence (ETQFLDLMRAVANSMR) by tetra-glycine linker was synthesized. Recombinant baculovirus carrying the rERP gene was generated to express the rERP in insect cells. Specificity and sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) employing the rERP was evaluated. RESULTS: The rERP with molecular weight of 20 kDa was successfully expressed by the recombinant baculovirus in an insect-baculovirus system. The rERP was antigenically functional as demonstrated by Western blotting. An indirect ELISA employing the rERP was developed and its specificity and sensitivity was determined. The ELISA test allowed discrimination of NDV infected sera from epitope deletion virus vaccinated sera. CONCLUSION: The preliminary results represent rERP ELISA as a promising DIVA diagnostic tool.


Subject(s)
Animals , Baculoviridae , Blotting, Western , Discrimination, Psychological , Enzyme-Linked Immunosorbent Assay , Insecta , Molecular Weight , Newcastle disease virus , Newcastle Disease , Nucleoproteins , Sensitivity and Specificity
15.
Article in English | LILACS, VETINDEX | ID: biblio-1002500

ABSTRACT

Studies on toad poison are relevant since they are considered a good source of toxins that act on different biological systems. Among the molecules found in the toad poison, it can be highlighted the cardiotonic heterosides, which have a known mechanism that inhibit Na+/K+-ATPase enzyme. However, these poisons have many other molecules that may have important biological actions. Therefore, this work evaluated the action of the low molecular weight components from Rhinella schneideri toad poison on Na+/K+-ATPase and their anticonvulsive and / or neurotoxic effects, in order to detect molecules with actions of biotechnological interest. Methods: Rhinella schneideri toad (male and female) poison was collected by pressuring their parotoid glands and immediately dried and stored at -20 °C. The poison was dialysed and the water containing the low molecular mass molecules (< 8 kDa) that permeate the dialysis membrane was collected, frozen and lyophilized, resulting in the sample used in the assays, named low molecular weight fraction (LMWF). Na+/K+ ATPase was isolated from rabbit kidneys and enzyme activity assays performed by the quantification of phosphate released due to enzyme activity in the presence of LMWF (1.0; 10; 50 and 100 µg/mL) from Rhinella schneideri poison. Evaluation of the L-Glutamate (L-Glu) excitatory amino acid uptake in brain-cortical synaptosomes of Wistar rats was performed using [3H]L-glutamate and different concentration of LMWF (10-5 to 10 µg/µL). Anticonvulsant assays were performed using pentylenetetrazole (PTZ) and N-methyl-D-aspartate (NMDA) to induce seizures in Wistar rats (n= 6), which were cannulated in the lateral ventricle and treated with different concentration of LMWF (0.25; 0.5; 1.0; 2.0; 3.0 and 4.0 µg/µL) 15 min prior to the injection of the seizure agent. Results: LMWF induced a concentration-dependent inhibition of Na+/K+-ATPase (IC50% = 107.5 μg/mL). The poison induces an increased uptake of the amino acid L-glutamate in brain-cortical synaptosomes of Wistar rats. This increase in the L-glutamate uptake was observed mainly at the lowest concentrations tested (10-5 to 10-2 µg/µL). In addition, this fraction showed a very relevant central neuroprotection on seizures induced by PTZ and NMDA. Conclusions: LMWF from Rhinella schneideri poison has low molecular weight compounds, which were able to inhibit Na+/K+-ATPase activity, increase the L-glutamate uptake and reduced seizures induced by PTZ and NMDA. These results showed that LMWF is a rich source of components with biological functions of high medical and scientific interest.(AU)


Subject(s)
Animals , Poisons , Synaptosomes , Bufo rana , Neuroprotection , Anticonvulsants , Glutamic Acid , Molecular Weight
16.
Braz. j. microbiol ; 49(3): 452-462, July-Sept. 2018. tab, graf
Article in English | LILACS | ID: biblio-951792

ABSTRACT

Abstract Exopolysaccharide (EPS) biopolymers produced by microorganisms play a crucial role in the environment such as health and bio-nanotechnology sectors, gelling agents in food and cosmetic industries in addition to bio-flocculants in the environmental sector as they are degradable, nontoxic. This study focuses on the improvement of EPS production through manipulation of different culture and environmental conditions using response surface methodology (RSM). Plackett-Burman design indicated that; molasses, yeast extract and incubation temperature are the most effective parameters. Box-Behnken RSM indicated that; the optimum concentration for each parameter was 12% (w/v) for molasses, 6 g/L yeast extract and 30 °C for incubation temperature. The most potent bacterial isolate was identified as Bacillus velezensis KY498625. After production, EPS was extracted, purified using DEAE-cellulose, identified using Fourier transform infrared (FTIR), gel permeation chromatography (GPC) and gas chromatography-mass spectroscopy (GC-MS). The result indicated that; it has molecular weight 1.14 × 105 D consisting of glucose, mannose and galactose.


Subject(s)
Polysaccharides, Bacterial/metabolism , Bacillus/metabolism , Polysaccharides, Bacterial/isolation & purification , Polysaccharides, Bacterial/chemistry , Bacillus/chemistry , Industrial Microbiology , Spectroscopy, Fourier Transform Infrared , Culture Media/metabolism , Culture Media/chemistry , Molecular Weight
17.
Braz. j. microbiol ; 49(2): 414-421, Apr.-June 2018. tab, graf
Article in English | LILACS | ID: biblio-889234

ABSTRACT

Abstract Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935 bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35 kDa exhibited highest expression at 0.5 mM concentration of IPTG. Expressed recombinant protein of 35 kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35 kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80 µg and 200 µg.


Subject(s)
Antifungal Agents/pharmacology , Chitinases/pharmacology , Hordeum/enzymology , Recombinant Proteins/metabolism , Antifungal Agents/chemistry , Antifungal Agents/isolation & purification , Blotting, Western , Chitinases/chemistry , Chitinases/genetics , Chitinases/isolation & purification , Chromatography, Affinity , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Hordeum/genetics , Molecular Weight , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Sequence Homology, Amino Acid
18.
Electron. j. biotechnol ; 33: 29-35, May. 2018. tab, graf, ilus
Article in English | LILACS | ID: biblio-1022834

ABSTRACT

Background: P64k is a Neisseria meningitidis high molecular weight protein present in meningococcal vaccine preparations. The lpdA gene, which encodes for this protein, was cloned in Escherichia coli and the P64k recombinant protein was expressed in E. coli K12 GC366 cells under the control of a tryptophan promoter. P64k was expressed as an intracellular soluble protein about 28% of the total cellular protein. Several scale-up criteria of fermentation processes were studied to obtain the recombinant P64k protein at the pilot production scale. Results: The best operational conditions at a larger scale production of P64k recombinant protein were studied and compared using the four following criteria: Constant Reynold's number (Re constant), Constant impeller tip speed (n di constant), Constant power consumption per unit liquid volume (P/V constant) and Constant volumetric oxygen transfer coefficients (KLa/k constant). The highest production of the recombinant protein was achieved based on the constant KLa/k scale-up fermentation criterion, calculating the aeration rate (Q) and the impeller agitation speed (n) by iterative process, keeping constant the KLa/k value from bench scale. The P64k protein total production at the 50 l culture scale was 546 mg l -1 in comparison with the 284 mg l -1 obtained at 1.5 l bench scale. Conclusions: The methodology described herein, for the KLa/k scale-up fermentation criterion, allowed us to obtain the P64k protein at 50 l scale. A fermentation process for the production of P64k protein from N. meningitidis was established, a protein to be used in future vaccine formulations in humans.


Subject(s)
Bacterial Outer Membrane Proteins/biosynthesis , Recombinant Proteins/biosynthesis , Escherichia coli/metabolism , Neisseria meningitidis/metabolism , Tryptophan , Meningococcal Vaccines , Fermentation , Molecular Weight
19.
J. appl. oral sci ; 26: e20170004, 2018. tab, graf
Article in English | LILACS, BBO | ID: biblio-893706

ABSTRACT

ABSTRACT Among the many graft materials that have been used for the treatment of bone defects in oral and maxillofacial regions is xenograft. To improve osteoconductive effects of xenografts, they have been combined with various biocompatible materials, such as hyaluronic acid and bone morphogenetic protein. Objective: To determine bone-healing capacity of high molecular weight hyaluronic acid (HA) combined with xenograft in rabbit calvarial bone defects. Material and methods: Ten adult male New Zealand rabbits (mean weight 3 kg) were included in the study. Three 6-mm-diameter bicortical cranial defects were created on calvarial bone of all rabbits. These defects were filled as follows: a) xenograft; b) HA+xenograft; c) autograft. One month after the first operation, rabbits were sacrificed. Specimens were evaluated histomorphometrically. Results: Considering multiple comparisons, differences regarding new bone were statistically significant between all groups (p<0.05). The volume of residual graft was significantly decreased in HA group compared to xenograft group (p=0.035). Marrow space, trabecular thickness (TbTh), trabecular width (TbWi), trabecular separation (TbSp), and number of node: number of terminus (NNd:NTm) in the autograft group were significantly better than xenograft and HA groups (p<0.05). However, regarding marrow space, TbTh, TbWi, TbSp, and NNd:NTm values, xenograft and HA groups showed similar results and the difference were not significant (p>0.05). Conclusion: These results support that high molecular weight hyaluronic acid could contribute to the healing of xenograft by improving the percentage of new bone formation and reducing the percentage of residual graft. However, HA did not significantly affect the quality of newly formed bone assessed by microarchitectural parameters.


Subject(s)
Humans , Animals , Male , Skull/transplantation , Wound Healing/drug effects , Bone Regeneration/drug effects , Heterografts/drug effects , Hyaluronic Acid/pharmacology , Rabbits , Skull/drug effects , Biocompatible Materials/pharmacology , Reproducibility of Results , Bone Transplantation/methods , Treatment Outcome , Disease Models, Animal , Autografts/drug effects , Cancellous Bone/drug effects , Hyaluronic Acid/chemistry , Molecular Weight
20.
Braz. j. med. biol. res ; 51(2): e6738, 2018. tab, graf
Article in English | LILACS | ID: biblio-889025

ABSTRACT

Adiponectin (APN), an adipose tissue-released adipokine with demonstrated anti-inflammatory and anti-atherogenic properties, is encoded by a gene whose polymorphisms are associated with presence of coronary artery disease (CAD). Serum APN levels are inversely related with presence and complexity of CAD. Within this context, we sought to compare levels of total APN and its high molecular weight form (HMW APN) according to clinical presentation and extent of CAD in patients undergoing elective cardiac catheterization. From March 2008 to June 2010, clinical data and blood samples for APN and HMW APN measurements were collected from 415 subjects undergoing cardiac catheterization at two tertiary centers. CAD extent was estimated by the number of coronary arteries with significant stenosis (≥70% obstruction in a major coronary artery) and by Duke Jeopardy Score (DJS). Serum APN levels were similar between groups with stable or unstable CAD (APN 9.20±5.88 vs 9.47±6.23 μg/mL, P=0.738, and HMW APN 5.31±3.72 vs 5.91±4.16 μg/mL, P=0.255), even after stratification by the number of arteries involved (single-vessel vs multivessel disease: APN 9.39±5.76 vs 9.26±6.27 μg/mL, P=0.871; HMW APN 5.29±3.79 vs 5.83±4.04 μg/mL, P=0.306) and DJS score (APN, P=0.718; HMW APN, P=0.276). We conclude that APN and HMW APN serum levels are similar across clinical presentations and different extents of CAD, despite being significantly lower in the presence of obstructive CAD.


Subject(s)
Humans , Male , Female , Middle Aged , Coronary Artery Disease/blood , Coronary Angiography , Adiponectin/blood , Prognosis , Coronary Artery Disease/pathology , Enzyme-Linked Immunosorbent Assay , Biomarkers/blood , Elective Surgical Procedures , Molecular Weight
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