Exploring amino acids responsible for the temperature profile of glycoside hydrolase family 45 endoglucanase EGL3 from Humicola grisea.
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EGL3 and RCE1 are glycoside hydrolase family 45 endoglucanases isolated from Humicola grisea and Rhizopus oryzae respectively. The amino acid sequences of the two endoglucanases are homologous; on the other hand, the optimum temperature of EGL3 is higher than that of RCE1. In this study, four chimeric endoglucanases, named ER1, ER2, ER3 and ER4, in which one of four sequential amino acid regions of the EGL3 catalytic domain (CAD) was replaced by the corresponding RCE1 amino acids, were constructed to explore the region responsible for the EGL3 temperature profile. Then their temperature profiles were compared with that of the recombinant EGL3. Replacement of the N-terminal region of EGL3 with that of RCE1 caused the EGL3 temperature profile to shift to a lower temperature. These results suggest that the N-terminal amino acids of the EGL3 are responsible for the EGL3 temperature profile. (+info)
Degradation of cellulose and hemicelluloses by the brown rot fungus Piptoporus betulinus--production of extracellular enzymes and characterization of the major cellulases.
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Piptoporus betulinus is a common wood-rotting fungus parasitic for birch (Betula species). It is able to cause fast mass loss of birch wood or other lignocellulose substrates. When grown on wheat straw, P. betulinus caused 65% loss of dry mass within 98 days, and it produced endo-1,4-beta-glucanase (EG), endo-1,4-beta-xylanase, endo-1,4-beta-mannanase, 1,4-beta-glucosidase (BG), 1,4-beta-xylosidase, 1,4-beta-mannosidase and cellobiohydrolase activities. The fungus was not able to efficiently degrade crystalline cellulose. The major glycosyl hydrolases, endoglucanase EG1 and beta-glucosidase BG1, were purified. EG1 was a protein of 62 kDa with a pI of 2.6-2.8. It cleaved cellulose internally, produced cellobiose and glucose from cellulose and cellooligosaccharides, and also showed beta-xylosidase and endoxylanase activities. The K(m) for carboxymethylcellulose was 3.5 g l(-1), with the highest activity at pH 3.5 and 70 degrees C. BG1 was a protein of 36 kDa with a pI around 2.6. It was able to produce glucose from cellobiose and cellooligosaccharides, but also produced galactose, mannose and xylose from the respective oligosaccharides and showed some cellobiohydrolase activity. The K(m) for p-nitrophenyl-1,4-beta-glucoside was 1.8 mM, with the highest activity at pH 4 and 60 degrees C, and the enzyme was competitively inhibited by glucose (K(i)=5.8 mM). The fungus produced mainly beta-glucosidase and beta-mannosidase activity in its fruit bodies, while higher activities of endoglucanase, endoxylanase and beta-xylosidase were found in fungus-colonized wood. (+info)
Toll like receptor-3 ligand poly-ICLC promotes the efficacy of peripheral vaccinations with tumor antigen-derived peptide epitopes in murine CNS tumor models.
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BACKGROUND: Toll-like receptor (TLR)3 ligands serve as natural inducers of pro-inflammatory cytokines capable of promoting Type-1 adaptive immunity, and TLR3 is abundantly expressed by cells within the central nervous system (CNS). To improve the efficacy of vaccine strategies directed against CNS tumors, we evaluated whether administration of a TLR3 ligand, polyinosinic-polycytidylic (poly-IC) stabilized with poly-lysine and carboxymethylcellulose (poly-ICLC) would enhance the anti-CNS tumor effectiveness of tumor peptide-based vaccinations. METHODS: C57BL/6 mice bearing syngeneic CNS GL261 glioma or M05 melanoma received subcutaneous (s.c.) vaccinations with synthetic peptides encoding CTL epitopes--mEphA2 (671-679), hgp100 (25-33) and mTRP-2 (180-188) for GL261, or ovalbumin (OVA: 257-264) for M05. The mice also received intramuscular (i.m.) injections with poly-ICLC. RESULTS: The combination of subcutaneous (s.c.) peptide-based vaccination and i.m. poly-ICLC administration promoted systemic induction of antigen (Ag)-specific Type-1 CTLs expressing very late activation antigen (VLA)-4, which confers efficient CNS-tumor homing of vaccine-induced CTLs based on experiments with monoclonal antibody (mAb)-mediated blockade of VLA-4. In addition, the combination treatment allowed expression of IFN-gamma by CNS tumor-infiltrating CTLs, and improved the survival of tumor bearing mice in the absence of detectable autoimmunity. CONCLUSION: These data suggest that poly-ICLC, which has been previously evaluated in clinical trials, can be effectively combined with tumor Ag-specific vaccine strategies, thereby providing a greater index of therapeutic efficacy. (+info)
Carboxymethylcellulose binds to human corneal epithelial cells and is a modulator of corneal epithelial wound healing.
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PURPOSE: In this study, the ability of carboxymethylcellulose (CMC), used in artificial tear formulations, to interact with corneal-epithelial-cells (HCECs) and facilitate corneal epithelial wound healing was investigated. METHODS: HCECs were incubated with fluorescein-labeled CMC (F-CMC). CMC-epithelial binding was measured by spectrophotometry. The effect on F-CMC binding by hyaluronic acid (HA) or glucose was measured after preincubation in HA, mAb to CD44, or glucose, or mAb to GluT-1. F-CMC binding to fibronectin or collagen was measured by incubating proteins with F-CMC. The wound widths were measured 18 hours after confluent HCECs were scratch wounded. The ability of CMC to induce cell chemotaxis, proliferation, or migration was measured by quantitative assay. The efficacy of CMC in promoting epithelial wound healing was also tested in a rabbit epithelial scrape-wound model. RESULTS: CMC remained bound to the HCECs for 2 hours. Preincubation of HCECs with glucose or mAb to GluT-1, but not with HA or mAb to CD44, reduced the binding of CMC to HCECs from 43.7% to 67.2% or 10.9% to 25.3%, respectively. CMC bound significantly to fibronectin (3.1-fold) or collagen (9.3-fold) compared with the control (BSA), and such binding enhanced cell adhesion. CMC stimulated re-epithelialization of HCECs scratched in vitro and in vivo rabbit cornea epithelial scrape wounds. CMC stimulated cell migration but not proliferation. CONCLUSIONS: CMC probably binds to HCECs through interaction of its glucopyranose subunits with glucose transporters. CMC binding to the matrix proteins stimulated HCEC attachment, migration, and re-epithelialization of corneal wounds. (+info)
Histopathology of ossicular grafts and implants in chronic otitis media.
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OBJECTIVES: We describe the histopathology of ossicular grafts and implants so as to provide insight into factors that may influence functional results after surgery for chronic otitis media. METHODS: Histopathologic observations were made on 56 cases: 50 surgical specimens and 6 temporal bone cases in which the graft was sectioned in situ. RESULTS AND CONCLUSIONS: Autogenous malleus, incus, and cortical bone grafts behaved in a similar manner and maintained their morphological size, shape, and contour for extended periods of time, at least up to 30 years. These histopathologic observations support the continued use of autograft ossicular and cortical bone grafts for middle ear reconstruction. Cartilage grafts developed chondromalacia with resulting loss of stiffness and showed a tendency to undergo resorption. Synthetic prostheses made of porous plastic (Plastipore, Polycel) elicited foreign body giant cell reactions with various degrees of biodegradation of the implants. Prostheses made of hydroxyapatite and Bioglass were enveloped by a lining of connective tissue and mucosal epithelium. The Bioglass material was broken down into small fragments and partially resorbed by a host response within the middle ear. These results warrant caution in the use of prostheses made of porous plastic or Bioglass. (+info)
Antihyperglycemic effect of Cephalotaxus sinensis leaves and GLUT-4 translocation facilitating activity of its flavonoid constituents.
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The objectives of this study were to investigate the antihyperglycemic effect of Cephalotaxus sinensis leaves and to identify the active components. The antihyperglycemic effect of various fractions (FA, FB, FC, FD) of the 80% ethanol extract of the leaves was evaluated in streptozotocin (STZ)-induced diabetic rats. Among the tested fractions, FC was the most active. FC (0.48 g/kg) given orally for 10 d reduced significantly (p<0.001) the blood glucose of STZ-induced diabetic rats. The food and water intakes of FC (0.48 g/kg)-treated diabetic rats were reduced significantly (p<0.001) when compared to the 0.5% carboxymethyl cellulose (CMC)-treated diabetic rats. The activity-guided fractionation of the ethanol extract of C. sinensis leaves furnished three flavonoid compounds, apigenin-5-O-[alpha-L-rhamnopyranosyl-(1-->4)-6-O-beta-D-acetylglucopyranoside] (1), apigenin (2), and apigenin-5-O-[alpha-L-rhamnopyranosyl-(1-->4)-6-O-beta-D-glucopyranoside] (3). The elevation of GLUT-4 protein level in membrane preparations from mice adipocytes was detected by Western blot analysis after adipocytes were pre-incubated with FC (0.1, 1, 10 mg/ml), apigenin (0.1, 2 mg/ml) and apigenin-5-O-[alpha-L-rhamnopyranosyl-(1-->4)-6-O-beta-D-acetylglucopyranoside] (0.1, 2 mg/ml), respectively. Phytochemical investigation and HPLC-DAD analysis of FC indicated that the flavonoids were the major constituents in this fraction. These results suggest that the fraction from C. sinensis leaves is a promising drug for the treatment of diabetes, and that the flavonoids from this plant are the active constituents. (+info)
Effect of hydrophilic swellable polymers on dissolution enhancement of carbamazepine solid dispersions studied using response surface methodology.
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The objective of this work was to study dissolution enhancement efficiency and solid dispersion formation ability of hydrophilic swellable polymers such as sodium carboxymethyl cellulose (Na-CMC), sodium starch glycolate (SSG), pregelatinized starch (PGS), and hydroxypropylmethyl cellulose (HPMC) with carbamazepine using 3(2) full factorial design for each of the polymers. Solid dispersions of carbamazepine were prepared using solvent evaporation method with around 70% solvent recovery. The independent variables were the amount of polymer and organic solvent. The dependent variables assessed were percentage drug dissolved at various time points and dispersion efficiency (ie, in terms of particle size of solid dispersion). Solid dispersions were evaluated for percentage drug dissolved, wettability, differential scanning calorimetry, scanning electron microscopy, and angle of repose. Multiple linear regression of results obtained led to equations, which generated contour plots to relate the dependent variables. Similarity factor and mean dissolution time were used to compare dissolution patterns obtained in distilled water and simulated gastric fluid United States Pharmacopeia (USP) XXVI of pH 1.2. Maximum drug dissolution was obtained with polymer order Na-CMC>SSG>PGS>HPMC. Particle size of drug was reduced ~10-15, 3-5, 5-7, and 10-25 times in Na-CMC, SSG, PGS, and HPMC solid dispersions, respectively; whereas wettability of solid dispersions was found in the order of Na-CMC>HPMC>PGS>SSG. Angle of repose was found to be in the range of 29 degrees to 35 degrees for all solid dispersions, which shows good flowability characteristics. HPMC showed increase in drug dissolution up to an optimized level; however, further increase in its concentration decreased drug dissolution. (+info)
Synthesis of enzymatically-gellable carboxymethylcellulose for biomedical applications.
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We synthesized a carboxymethylcellulose with phenol moieties by covalently incorporating tyramine into carboxymethylcellulose using aqueous-phase carbodiimide activation chemistry. The resulting hydrogel was obtained from an aqueous solution of the conjugate via the horseradish peroxidase-catalyzed oxidation reaction of phenols by consuming H(2)O(2), where the gelation speed depended on the concentrations of enzyme and H(2)O(2). The viability of the mammalian cells enclosed within the hydrogel prepared from 1.5% (w/v) conjugate solution containing 5 units/ml horseradish peroxidase and 1 mM H(2)O(2), was 80% after 24 h. These results demonstrate that this carboxymethylcellulose with phenol moieties has potential for biomedical applications including tissue-engineering. (+info)