RESUMO
Current cell-based bone tissue regeneration strategies cannot cover large bone defects. K-carrageenan is a highly hydrophilic and biocompatible seaweed-derived sulfated polysaccharide, that has been proposed as a promising candidate for tissue engineering applications. Whether κ-carrageenan can be used to enhance bone regeneration is still unclear. In this study, we aimed to investigate whether κ-carrageenan has osteogenic potential by testing its effect on pre-osteoblast proliferation and osteogenic differentiation in vitro. Treatment with κ-carrageenan (0.5 and 2 mg/mL) increased both MC3T3-E1 pre-osteoblast adhesion and spreading at 1 h. K-carrageenan (0.125-2 mg/mL) dose-dependently increased pre-osteoblast proliferation and metabolic activity, with a maximum effect at 2 mg/mL at day three. K-carrageenan (0.5 and 2 mg/mL) increased osteogenic differentiation, as shown by enhanced alkaline phosphatase activity (1.8-fold increase at 2 mg/mL) at day four, and matrix mineralization (6.2-fold increase at 2 mg/mL) at day 21. K-carrageenan enhanced osteogenic gene expression (Opn, Dmp1, and Mepe) at day 14 and 21. In conclusion, κ-carrageenan promoted MC3T3-E1 pre-osteoblast adhesion and spreading, metabolic activity, proliferation, and osteogenic differentiation, suggesting that κ-carrageenan is a potential osteogenic inductive factor for clinical application to enhance bone regeneration.
Assuntos
Regeneração Óssea/fisiologia , Carragenina/farmacologia , Osteogênese/efeitos dos fármacos , Animais , Regeneração Óssea/efeitos dos fármacos , Carragenina/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/fisiologia , Engenharia Tecidual/métodosRESUMO
Changes of the network structure in agarose gels during storage were studied by measuring diffusion coefficient (D) of probe polymers. PGSTE 1H NMR was used to measure the D of pullulan added as a probe to show an increase with storage suggesting the formation of coarse network with thicker bundles of agarose aggregates. The hydrodynamic mesh size was estimated from the degree of restriction in the diffusion shedding light on the change of microscopic environment in the agarose gel during storage. From the gel electrophoresis, D of DNA was calculated from the friction on the DNA movement under the electric field to show an increase of D with storage suggesting the change of network structure in the aged agarose gels. The results provided a better understanding of the aging mechanism in agarose gels with the conformational changes which should be commonly observed in polysaccharides.
Assuntos
Géis/química , Espectroscopia de Prótons por Ressonância Magnética/métodos , Sefarose/química , DNA/química , Difusão , Armazenamento de Medicamentos , Eletroforese em Gel de Ágar/métodos , Glucanos/química , Hidrodinâmica , Solventes/química , ViscosidadeRESUMO
With reference to eq. (5) in the original article, the authors have neglected to specify the description of some terms in the text. They correct the mistake in the present erratum.
RESUMO
Agarose samples of low (Ag1) and high (Ag2) O -methyl content on position 6 of the galactose residue were studied in H2O and D2O. Differential scanning calorimetry, turbidity and rheological measurements showed a [Formula: see text] 2 ° C shift in the coil-to-helix transition temperature, indicating higher helix stability in D2O. The differential scanning calorimetry data could be superimposed using a temperature shift factor, suggesting similar extents of helix aggregation in both solvents. Small angle X-ray scattering of H2O and D2O gels were essentially identical, indicating no change in the small scale ( [Formula: see text] 0.05-20 nm) network structure on isotopic exchange. Larger ([Formula: see text] 1 µm) scale heterogeneities were more pronounced in deuterium gels. The 1HT2 relaxation times were measured at different H/D ratios. These relaxation times were analyzed using a model assuming regular solution mixing of H2O, HDO and D2O between the solvent and gel phases. The fit results suggested that H2O has higher affinity for the agarose network than HDO and D2O. The difference, however, was much larger for the Ag2 sample. This finding implies that the higher hydrophobic effect observed in D2O affects the hydration state much more strongly for the more hydrophobic (and more polarizable) agarose sample Ag2. As a consequence, Ag2 (but not Ag1) gels retained more H2O than D2O. In contrast, the bulk rheology of either hydrogel was not affected by the isotopic exchange.
RESUMO
A new family of binomial-like inversion sequences, named jump-and-return sandwiches (JRS), has been developed by inserting a binomial-like sequence into a standard jump-and-return sequence, discovered through use of a stochastic Genetic Algorithm optimisation. Compared to currently used binomial-like inversion sequences (e.g., 3-9-19 and W5), the new sequences afford wider inversion bands and narrower non-inversion bands with an equal number of pulses. As an example, two jump-and-return sandwich 10-pulse sequences achieved 95% inversion at offsets corresponding to 9.4% and 10.3% of the non-inversion band spacing, compared to 14.7% for the binomial-like W5 inversion sequence, i.e., they afforded non-inversion bands about two thirds the width of the W5 non-inversion band.
RESUMO
An α-l-arabinofuranosidase (Abf) encoding gene was obtained via genomic mining from a Ruminococcus albus strain. The specific activity of this GH 51 Abf was 73.3 U/mg at pH 6.0 and 50 °C. The modification of Abf, aimed at improving thermostability, was performed through different strategies. Structure-based rational design using the PoPMuSiC and the Enzyme Thermal Stability System (ETSS) predicted thermal stability of Abf and enhanced the half-life of thermal inactivation (t1/2) at 50 °C for K208W more than 11.1 times versus the wild-type (WT). Sequence-based rational design was also conducted by substituting histidine with lysine at various sites. Among eight mutants, the t1/2 at 50 °C of H337K was prolonged by 5.0-fold, and the specific activity of this mutant was increased to 121.8 U/mg. In addition, the mutant H337K was utilized with some enzymes to extract pectin from apple pomace. The enzymatically produced pectin got less moisture and ash, milder pH, and higher viscosity than its acid-extracted counterpart, indicating that Abf has an application prospect in pectin production.
Assuntos
Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Ruminococcus/enzimologia , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Glicosídeo Hidrolases/genética , Meia-Vida , Modelos Moleculares , Mutagênese Sítio-Dirigida , Pectinas/metabolismo , Conformação Proteica , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
We report on the effect of probe size and diffusion time of poly(ethylene) oxide in agarose gels. Time-dependence of the diffusion coefficient, reflecting anomalous diffusion, was observed for poly(ethylene) oxide chains with hydrodynamic radii exceeding about 20nm at an agarose concentration of 2%. The main conclusion is that the pore distribution includes pores that are only several nm across, in agreement with scattering reports in the literature. Interpretation of the diffusion coefficient dependence on the probe size based on a model of entangled rigid rods yielded a rod length of 72nm.
Assuntos
Difusão , Polietilenoglicóis/química , Sefarose/química , Géis , Cinética , Modelos Químicos , ReologiaRESUMO
An arabinanase gene was cloned by overlap-PCR from Penicillium sp. Y702 and expressed in Pichia pastoris. The recombinant enzyme was named AbnC702 with 20 U/mg of endo-arabinanase activity toward linear α-1,5-L-arabinan. The optimal pH and temperature of AbnC702 were 5.0 and 50 °C, respectively. The recombinant AbnC702 was highly stable at pH 5.0-7.0 and 50 °C. It could retain about 72.3 % of maximum specific activity at pH 5.0 after incubation for 2.5 h, which indicated AbnC702 was an acid-adapted enzyme. The K m and V max values were 24.8 ± 4.7 mg/ml and 88.5 ± 5.6 U/mg, respectively. A three-dimensional structure of AbnC702 was made by homology modeling, and the counting of acidic/basic amino residues within the region of 10 Å around the active site, as well the hydrogen bonds within the area of 5 Å around the active site, might theoretically interpret the acid adaptability of AbnC702. Analysis of hydrolysis products by thin layer chromatography (TLC) combined with high-performance liquid chromatography (HPLC) verified that the recombinant AbnC702 was an endo-1,5-α-L-arabinanase, which yielded arabinobiose and arabinotriose as major products. AbnC702 was applied in pectin extraction from apple pomace with synergistic action of α-L-arabinofuranosidase.
Assuntos
Ácidos/farmacologia , Glicosídeo Hidrolases/metabolismo , Pectinas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Malus/química , Modelos Moleculares , Alinhamento de Sequência , Especificidade por Substrato/efeitos dos fármacosRESUMO
The temperature dependencies of the (1)H T2 and diffusion coefficient (D) of a mixed solution of kappa-carrageenan and iota-carrageenan were measured by NMR. Rheological and NMR measurements suggested an exponential formation of rigid aggregates of kappa-carrageenan and a gradual formation of fine aggregates of iota-carrageenan during two step increases of G'. The results also suggested that longer carrageenan chains are preferentially involved in aggregation, thus resulting in a decrease in the average Mw of solute carrageenans. The results of diffusion measurements for poly(ethylene oxide) (PEO) suggested that kappa-carrageenan formed thick aggregates that decreased hindrance to PEO diffusion by decreasing the solute kappa-carrageenan concentration in the voids of the aggregated chains, and that iota-carrageenan formed fine aggregates that decreased the solute iota-carrageenan concentration less. DPEO in a mixed solution of kappa-carrageenan and iota-carrageenan suggested two possibilities for the microscopic network structure: an interpenetrating network structure, or micro-phase separation.
Assuntos
Carragenina/química , Difusão , Géis , Espectroscopia de Ressonância Magnética , Polietilenoglicóis/química , TemperaturaRESUMO
In the process of gene mining for novel α-L-arabinofuranosidases (AFs), the gene Celf_3321 from Cellulomonas fimi ATCC 484 encodes an AF, termed as AbfCelf, with potent activity, 19.4 U/mg under the optimum condition, pH 6.0 and 40 °C. AbfCelf can hydrolyze α-1,5-linked oligosaccharides, sugar beet arabinan, linear 1,5-α-arabinan, and wheat flour arabinoxylan, which is partly different from some previously well-characterized GH 51 AFs. The traditional substrate-specificity analysis for AFs is labor-consuming and money costing, because the substrates include over 30 kinds of various 4-nitrophenol (PNP)-glycosides, oligosaccharides, and polysaccharides. Hence, a preliminary structure and mechanism based method was applied for substrate-specificity analysis. The binding energy (ΔG, kcal/mol) obtained by docking suggested the reaction possibility and coincided with the experimental results. AbfA crystal 1QW9 was used to test the rationality of docking method in simulating the interaction between enzyme and substrate, as well the credibility of the substrate-specificity analysis method in silico.
Assuntos
Proteínas de Bactérias/química , Cellulomonas/enzimologia , Glicosídeo Hidrolases/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cellulomonas/química , Cellulomonas/genética , Clonagem Molecular , Biologia Computacional , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , Xilanos/química , Xilanos/metabolismoRESUMO
AbnZ1, with optimal pH of 6.0 and optimal temperature of 40 °C, is a cold-adapted endo-1,5-α-L-arabinanase encoded by the gene abnZ1 from Paenibacillus polymyxa Z6. The specific activity of AbnZ1 remained 54.1% of maximum at 5 °C. To apply AbnZ1 in acidic conditions, three basic hsitidine (His) residues, His(48), His(218), and His(297), around the catalytic domain were selected as mutation sites, which were replaced with Asp, Glu, Arg, and Lys, respectively, to yield 12 mutants, H48D/E/R/K, H218D/E/R/K, and H297D/E/R/K. The optimum pH of mutant H218D shifted toward the acidic direction by 0.5 unit, and the relative activity was enhanced from 20.4 to 55.7% at pH 5.0. Furthermore, the specific activity of H218D in optimal conditions was 82.6 U/mg versus that of wild type, 73.4 U/mg, and the K(m) decreased from 11.9 to 7.1 mg/mL. This work provided an arabinanase candidate for juice clarification and pectin extraction.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Paenibacillus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Bebidas/análise , Clonagem Molecular , Temperatura Baixa , Estabilidade Enzimática , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Paenibacillus/química , Paenibacillus/genética , Alinhamento de SequênciaRESUMO
We measured stress relaxation, volume of exuded water, and spatial distribution of stress in agar gels under large deformation. Gels with smaller sample size and lower concentration exuded water faster and had shorter stress relaxation time. Gels with the storage time of 3 days exuded more water and had shorter stress relaxation time than gels with the storage time of 1 day, and this tendency was remarkable for low-concentration gels. Examination of the spatial distribution of stress in a cylindrical gel under large deformation showed that the outer part of the gel had smaller stress than the inner part at an early stage, and the area with small stress gradually extended into the inner part. This result indicates that the inhomogeneity of water content caused by water exudation from the gel surface induces the stress distribution in the gel.
Assuntos
Ágar/química , Água/química , Força Compressiva , Elasticidade , Géis , Reologia , Solventes/química , Estresse Mecânico , ViscosidadeRESUMO
Changes in the molecular mobility of κ-carrageenan were observed by the pulsed field gradient stimulated echo (PGSTE) and Carr-Purcell-Meiboom-Gill (CPMG) methods for elucidating the molecular aspect of the sol-to-gel transition. The echo signal intensity of κ-carrageenan without a gradient, Ikap(0), decreased steeply near the sol-to-gel temperature (Tsg), suggesting that κ-carrageenan chains formed aggregates and a network structure. Below Tsg, the spin-spin relaxation time T2 and the diffusion coefficient of κ-carrageenan (Dkap) increased with decreasing temperature, indicating that the solute κ-carrageenan chains have a lower molecular weight Mw than chains involved in the aggregation. The diffusion coefficient of pullulan (Dpul) added as a probe molecule in κ-carrageenan solutions was measured, and the characteristic hydrodynamic screening length, ξ, was then estimated from the degree of diffusion restriction. Below a certain temperature, Dkap reached a higher value than that of Dpul, suggesting that the Mw of solute κ-carrageenan became lower than that of pullulan. GPC measurements confirmed the presence of κ-carrageenan chains with a lower Mw than that of pullulan. A simple physical model of the structural change in κ-carrageenan solution was proposed with a bimodal distribution of κ-carrageenan with higher and lower Mw than the pullulan probe. The higher Mw chains form the gel network restricting the probe's diffusion, and the lower Mw chains increase the effective viscosity. The concentration of the high Mw solute κ-carrageenan chains in 1%, 2% and 4% κ-carrageenan solutions was estimated from Ikap(0) and the total κ-carrageenan concentration, and the relation with pullulan diffusion was studied.
Assuntos
Carragenina/química , Cromatografia em Gel , Espectroscopia de Ressonância Magnética , SoluçõesRESUMO
Many biopolymers assume ordered structure in solution due to specific intermolecular interactions, and subsequently aggregate to form fibrous network structures, which play important structural and functional roles both in biomedical tissues and in biopolymeric applied materials. In this study, the pulsed-field-gradient stimulated echo (PGSTE) (1)H NMR method was utilized to elucidate the gelation mechanism and to determine the network structure of agarose. The echo signal intensity of agarose decreased with the formation of aggregated bundles, and therefore, it was used to determine the concentration of the solute agarose (c(sol)) in the gel. The diffusion coefficient of a dendrimer, added to the gel as a probe molecule, increased concomitantly with the formation of the network of aggregated bundles, suggesting apparent dilution of solute agarose in the network interspaces. The hydrodynamic mesh size (ξ) of the network was estimated from the degree of retardation of the diffusion. The dependence of ξ on c(sol) was interpreted using a simple model, where the hydrodynamic interaction of the probe molecule with a solute chain or an aggregated bundle of chains is same. Our theoretically predicted lines fitted well on the experimentally obtained plots, thus validating the use of this model.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Transição de Fase , Sefarose/química , Biopolímeros/química , Difusão , Géis/química , Hidrodinâmica , Estrutura Molecular , Soluções/química , Temperatura de TransiçãoRESUMO
We propose a new interferometer concept that can realize electron-density distribution measurement with high spatial and moderate temporal resolution. The image non-radiative dielectric guide antenna can probe a wide measurement area simultaneously. We fabricated the antenna with an electromagnetic simulator and confirmed that the simulated and measured radiation patterns are consistent with each other. In addition, we found that the antenna shows the required characteristics such as scanning characteristics, which depend on the input frequency.
RESUMO
The purpose of this study was to examine the suitability of cryoprotectant agent (CPA) impregnation protocols for the embryos of Japanese whiting (Sillago japonica), a small-sized, easy-to-rear, and prolific marine fish which may constitute a suitable experimental material for the development of cryopreservation methods for fish embryos. Our immediate goals were to assess the toxicity and permeability of various CPAs to whiting embryos of different developmental stages. Exposure of gastrula, somites, tail elongation, and pre-hatching embryos to 10%, 15%, and 20% solutions of propylene glycol (PG), methanol (MeOH), dimethyl sulfoxide (Me2SO), dimethylformamide (DFA), ethylene glycol (EG), and glycerol (Gly) in artificial sea water (ASW; 33 psu) for 20 min revealed that CPA toxicity for whiting embryos increased in the order of PGAssuntos
Criopreservação
, Crioprotetores/farmacocinética
, Crioprotetores/toxicidade
, Permeabilidade
, Animais
, Criopreservação/métodos
, Criopreservação/veterinária
, Dimetil Sulfóxido/farmacocinética
, Dimetil Sulfóxido/toxicidade
, Dimetilformamida/farmacocinética
, Dimetilformamida/toxicidade
, Embrião não Mamífero/efeitos dos fármacos
, Desenvolvimento Embrionário/efeitos dos fármacos
, Etilenoglicol/farmacocinética
, Etilenoglicol/toxicidade
, Feminino
, Peixes
, Gástrula/efeitos dos fármacos
, Glicerol/farmacocinética
, Glicerol/toxicidade
, Espectroscopia de Ressonância Magnética
, Metanol/farmacocinética
, Metanol/toxicidade
, Permeabilidade/efeitos dos fármacos
, Propilenoglicol/farmacocinética
, Propilenoglicol/toxicidade
, Soluções/química
, Somitos/efeitos dos fármacos
RESUMO
The effects of droplet size and emulsifiers on oxidative stability of polyunsaturated TAG in oil-in-water (o/w) emulsions with droplet sizes of 0.806 +/- 0.0690, 3.28 +/- 0.0660, or 10.7 +/- 0.106 microm (mean +/- SD) were investigated. Hydroperoxide contents in the emulsion with a mean droplet size of 0.831 microm were significantly lower than those in the emulsion with a mean droplet size of 12.8 microm for up to 120 h of oxidation time. Residual oxygen contents in the headspace air of the vials containing an o/w emulsion with a mean droplet size of 0.831 microm were lower compared with those of the emulsion with a mean droplet size of 12.8 microm. Hexanal developed from soybean oil TAG o/w emulsions with smaller droplet size showed significantly lower residual oxygen contents than those of the larger droplet size emulsions. Consequently, oxidative stability of TAG in o/w emulsions could be controlled by the size of oil droplet even though the origins of TAG were different. Spin-spin relaxation time of protons of acyl residues on TAG in o/w emulsions measured by H NMR suggested that motional frequency of some acyl residues was shorter in o/w emulsions with a smaller droplet size. The effect of the wedge associated with hydrophobic acyl residues of emulsifiers was proposed as a possible mechanism to explain differences in oxidative stability between o/w emulsions with different droplet sizes.
Assuntos
Emulsões/química , Óleos/química , Água/química , Óleo de Fígado de Bacalhau/química , Estabilidade de Medicamentos , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Tamanho da Partícula , Peróxidos/síntese químicaRESUMO
The binding effect of divalent cation Cu(2+) on the gelation process with a coil-helix transition in Cu(2+)/gellan aqueous solutions has been successfully elucidated by EPR, CD, and viscoelasticity measurements. Generally, Na-type gellan gum in aqueous solution can make gel when accompanied by an intrinsic coil-helix formation induced by hydrogen bonding between chains without any additional cations at T(ch)(-)(in) ( approximately 29 degrees C) with cooling temperature. An extrinsic coil-helix transition, induced by additional divalent cations in advance of the intrinsic sol-gel transition of gellan gum, is separately detected by CD measurement. The extrinsic coil-helix transition temperatures T(ch)(-)(ex) (>47 degrees C), which increased with the Cu(2+) concentration added, were nearly identical to the sol-gel transition temperature, T(sg), determined by the viscoelasticity measurement. Judging from the molar ellipticity by CD measurement and quantitative analysis of EPR spectra, it was elucidated that the helix forming process via divalent cations is composed of two steps ascribed to the different origins, i.e., a chemical binding effect via Cu(2+) ions in the initial stage and hydrogen bonds subsequently. Finally, we propose the coil-helix and the sol-gel transition mechanism initiated by the binding effect with the divalent cation, in which the partial chelate formation can cause local formation of helices and junction zones in the vicinity of the chelates at the initial stage of the process and stabilize the helices and the junction zones. On the other hand, the stabilized helices and junction zones can induce further formation and further stabilization of the Cu(2+)-gellan chelates. The mutual stabilization promotes the formation of three-dimensional network structure at the higher temperature than the intrinsic temperature for network formation.