Containing Carboxymethyl Cellulose Research Articles

Stabilization or Oxidation of Nanoscale Zerovalent Iron at Environmentally Relevant Exposure Changes Bioavailability and Toxicity in Medaka Fish

Nanoscale zerovalent iron (nZVI)-based nanotechnologies are increasingly being used for environmental remediation; however, the fate and ecotoxicologic effects of nZVI remain unclear. Larvae of medaka fish (Oryzias latipes) underwent 3-14 days' aqueous exposure to thoroughly characterized solutions containing carboxymethyl cellulose (CMC)-stabilized nZVI, bare nZVI, nanoscale iron oxide (nFe(3)O(4)) or ferrous ion [Fe(II)(aq)] at μg/L-mg/L levels to assess the causal toxic effect(s) of iron nanoparticles (NPs). Acute larval mortality was decreased in the order of Fe(II)(aq) > CMC-nZVI > nZVI > nFe(3)O(4). CMC-nZVI (100 mg/L) increased hypoxia and reactive oxygen species (ROS) and Fe(II)(aq) production, thus increasing mortality and oxidative stress response as compared with unstabilized nZVI. Additionally, nFe(3)O(4) and nZVI were more bioavailable than suspended CMC-nZVI or Fe(II)(aq). Antioxidant activities were significantly altered by induced intracellular ROS levels in larvae with subchronic exposure to nFe(3)O(4) or Fe(II)(aq) at environmentally relevant concentrations (0.5-5 mg/L). We report on different organizational biomarkers used for rapidly assessing the lethal and sublethal toxicity of nZVI and its stabilized or oxidized products. The toxicity results implicate a potential ecotoxicological fate and impact of nZVI on the aquatic environment.

Toxicity assessments of nanoscale zerovalent iron and its oxidation products in medaka (Oryzias latipes) fish

Iron-based nanotechnologies are increasingly used for environmental remediation; however, toxicologic impacts of iron nanoparticles on the aquatic ecosystem remain poorly understood. We treated larvae of medaka fish (Oryzias latipes) with thoroughly characterized solutions containing carboxymethyl cellulose (CMC)-stabilized nanoscale zerovalent iron (nZVI), aged nanoscale iron oxides (nFe-oxides) or ferrous ion (Fe[II]) for 12–14days’ aqueous exposure to assess the causal toxic effect(s) of iron NPs on the fish. With the CMC-nZVI solution, the dissolved oxygen level decreased, and a burst of reactive oxygen species (ROS) was generated as Fe(II) oxidized to ferric ion (Fe[III]); with the other two iron solutions, these parameters did not significantly change. CMC-nZVI and Fe(II) solutions caused acute lethally and sublethally toxic effects in medaka larvae, with nFe-oxide-containing solutions causing the least toxic effects. We discuss modes of toxic action of iron NPs and chronic toxic effects in terms of hypoxia, Fe(II) toxicity and ROS-mediated oxidative damage.

Cellulolytic activity and virulence of Ophiostoma ulmi and O. novo‐ulmi isolates

SummaryThe cellulolytic activity (exoglucanase, endoglucanase and β‐glucosidase) of Ophiostoma ulmi (four isolates), O. novo‐ulmi (19 isolates) and ‘fast‐waxy’ (five isolates) was determined in growth media containing carboxymethylcellulose (CMC) and cellulose powder. Differences in enzyme activities were observed among isolates, irrespective of the species and substrate used. Inoculation experiments on Ulmus minor with randomly selected isolates of O. ulmi (two isolates), O. novo‐ulmi (five isolates) and ‘fast‐waxy’ (two isolates) were also performed. Disease was assessed as the percentage of leaves showing yellowing and browning. Ophiostoma novo‐ulmi and ‘fast‐waxy’ isolates exhibited a great variability in their capacities to cause the disease. In the presence of CMC, a significant correlation between the activity of exoglucanase and β‐glucosidase in vitro and virulence was found.

Regulatory mutations affecting the synthesis of cellulase in Pseudomonas fluorescens

Pseudomonas fluorescens , was cultured in basal medium containing carboxymethyl-cellulose (CMC) as inducer and glucose or glycerol as carbon and energy sources. Ethylmethanesulphonate (EMS) was used to mutagenize the wild-type organism to produce mutants. The isolated mutants were screened for the isolation of catabolite repression resistant mutants in the presence of 1% (w/v) glucose as carbon source. A total of fifty mutants were isolated. All the mutants produced cellulase in the presence of CMC as an inducer with specific activity of 0.057, 0.088 and 0.074 units/mg protein for the wild-type, catabolite repression resistant mutant4 (CRRmt 4 ) and catabolite repression resistant mutant24 (CRRmt 24 ), respectively. It was observed that addition of glucose or glycerol as carbon and energy sources to the culture medium resulted into considerable reduction in the cellulolytic activity. However, glycerol appeared to be a better carbon and energy source than glucose which inhibited enzyme expression in most of the strains used in this study. It was also observed that potent cellulase production occurred at the exponential growth phase of the organism. The isolated mutants were grouped into three classes based on their induction ratios namely; unimproved mutants, catabolite repression resistant mutants and mutants with highest induction ratio but sensitive to catabolite repression in the presence of high glucose concentration. The overall results obtained showed that cellulolytic activity in P. fluorescens was regulated by catabolite repression. Key words: Pseudomonas fluorescens , ethylmethanesulphonate, mutants, cellulose, catabolite repression, induction ratio. African Journal of Biotechnology Vol. 4 (8), pp. 838-843

Regulatory mutations affecting the synthesis of cellulase in Pseudomonas fluorescens

Pseudomonas fluorescens , was cultured in basal medium containing carboxymethyl-cellulose (CMC) as inducer and glucose or glycerol as carbon and energy sources. Ethylmethanesulphonate (EMS) was used to mutagenize the wild-type organism to produce mutants. The isolated mutants were screened for the isolation of catabolite repression resistant mutants in the presence of 1% (w/v) glucose as carbon source. A total of fifty mutants were isolated. All the mutants produced cellulase in the presence of CMC as an inducer with specific activity of 0.057, 0.088 and 0.074 units/mg protein for the wild-type, catabolite repression resistant mutant4 (CRRmt 4 ) and catabolite repression resistant mutant24 (CRRmt 24 ), respectively. It was observed that addition of glucose or glycerol as carbon and energy sources to the culture medium resulted into considerable reduction in the cellulolytic activity. However, glycerol appeared to be a better carbon and energy source than glucose which inhibited enzyme expression in most of the strains used in this study. It was also observed that potent cellulase production occurred at the exponential growth phase of the organism. The isolated mutants were grouped into three classes based on their induction ratios namely; unimproved mutants, catabolite repression resistant mutants and mutants with highest induction ratio but sensitive to catabolite repression in the presence of high glucose concentration. The overall results obtained showed that cellulolytic activity in P. fluorescens was regulated by catabolite repression. Key words: Pseudomonas fluorescens , ethylmethanesulphonate, mutants, cellulose, catabolite repression, induction ratio. African Journal of Biotechnology Vol. 4 (8), pp. 838-843

Effect of Solid State Transition on the Physical Stability of Suspensions Containing Bupivacaine Lipid Microparticles

Bupivacaine lipid microparticles were prepared and evaluated as a parenteral sustained-release dosage form for postoperative pain management. Bupivacaine free base was incorporated into a molten tristearin matrix and lipid microparticles were subsequently formed from this molten mixture by a spray-congealing process. A 3% injectable bupivacaine lipid microparticle suspension was prepared by dispersing 30% bupivacaine lipid microparticles in an aqueous medium containing carboxymethylcellulose (CMC), mannitol, and Tween 80. Upon room temperature storage, the fluid suspension gradually changed into a nonflowing semisolid (gelation) as a result of crystal growth of bupivacaine. However, suspensions prepared with bupivacaine lipid microparticles that were previously annealed at an elevated temperature remained fluid upon long-term storage. Differential scanning calorimetry (DSC), x-ray powder diffraction (XRPD), and isoperibol solution calorimetry were used to investigate the changes in the solid-state properties of tristearin and bupivacaine in the lipid microparticles before and after the heat treatment. The DSC and XRPD results indicate that after 24 hours of heating at 40°C, tristearin was completely converted from the unstable α form to the stable β form. Using the isoperibol solution calorimetric method, bupivacaine was found to transform into a more stable form after the lipid microparticles were heated at 60°C for 24 hours. The generation of the unstable solid forms of tristearin and bupivacaine was attributed to the resolidification of both components from the molten mixture during the spray-congealing process.

Pre-clinical investigation of the efficacy of an artificial tear solution containing hydroxypropyl-guar as a gelling agent

Purpose. Pre-clinical studies of a new artificial tear product (SystaneTM Lubricating Eye Drops Alcon Laboratories, Inc., Fort Worth, TX) containing the novel gelling agent hydroxypropyl-guar (HP-guar) and two demulcents, polyethylene glycol 400 (PEG) and propylene glycol (PG) were conducted to determine the ability of the product to protect ocular surface epithelial cells from desiccation in vivo and in vitro, and to promote recovery of the damaged corneal epithelial barrier in vivo. Other leading artificial tear products were also evaluated as comparators to determine the relative effectiveness of different polymer systems. Methods. Damage due to desiccation was assessed by measuring corneal uptake of methylene blue compared to untreated corneas. Corneas of anesthetized rabbits were treated with the new artificial tear product and subjected to desiccation by holding the eyelids open for 2 hours with a speculum. Control eyes were subjected to desiccation without application of a tear formulation. To measure recovery of the corneal epithelium from damage, corneas of anethesthetized rabbits were exposed to 0.01% benzalkonium chloride (BAC) for 5 minutes to increase epithelial permeability. The corneas were exposed to the new artificial tear for 1.5 hours followed by measurement of uptake of 5,6 carboxyfluorescein (CF). In the desiccation and CF uptake experiments, the new tear product was also compared to a tear product formulation without HP-guar and to a commercially available artificial tear containing carboxymethyl cellulose (CMC) and Purite®. In a third set of experiments, immortalized human corneal epithelial cells and Chang conjunctival cells in culture were exposed to the PEG/PG/HP-guar tear product, the control formulation without HP-guar, a tear formulation preserved with BAC, or the artificial tear containing CMC/Purite® for 15 min. The tear formulation was removed and the cells were exposed to desiccating conditions in a laboratory safety hood for 10 or 30 min. Cell viability was determined using the MTT assay. Results. The in vivo desiccation model, showed that the new tear product, SystaneTM, offered complete protection of the cornea from desiccation (methylene blue uptake not different than naïve control). Following exposure to 0.01% BAC, the new artificial tear product also provided an environment in which the corneal epithelium recovered completely from damage (CF uptake not different than normal, untreated cornea). This level of protection was not observed when corneas were treated with other formulations. Results from the in vitro desiccation procedure indicated that viability of corneal epithelial and Chang cells treated with the PEG/PG/HP-guar product was significantly greater than viability of cells treated with the tear product without HP-guar or the tear products containing BAC or CMC/Purite®. Conclusions. The tear product containing HP-guar, PEG 400 and propylene glycol satisfies several pre-clinical criteria for an appropriate artificial tear formulation. It gives long-term desiccation protection of the intact cornea and also epithelial cells in culture and has no apparent deleterious affects on cells. It also provides conditions in which a damaged corneal epithelium can recover normal barrier function. The combination of ingredients in the formulation appears to provide an effective mucomimetic artificial tear product. These pre-clinical data suggest that the product will be effective in providing superior relief for the dry eye sufferer.

Enhanced Anthocyanin Production from Grape Callus in an Air-Lift Type Bioreactor Using a Viscous Additive-Supplemented Medium

An N-medium containing carboxymethyl cellulose (CMC) was applied to an air-lift type bioreactor culture of grape (Vitis vinifera cv. Bailey alicant A.) callus, and anthocyanin production was investigated. Grape callus grew well at an air flow rate of 80 ml/min and anthocyanin production was significantly increased in the N-medium, reaching 17 mg/l after 7 d of culture. The anthocyanin content of the N-medium was about two times higher than that of the conventional medium without CMC. The effect of air flow rate was also investigated within the range from 40 to 160 ml/min. A twofold increase in anthocyanin content was obtained at all the air flow rates tested in the N-medium. The distribution of grape callus size obtained after 7 d of the bioreactor culture was investigated. The average callus size was 490 mum which was 1.6 times larger than that obtained in the conventional medium. It was found that large calli with a relatively high anthocyanin pigment content were formed in the bioreactor culture using the N-medium. The fluid dynamics in the bioreactor was also investigated at three points (top, middle and bottom) in the bioreactor by laser doppler velocimetry. The average axial velocity of the circulated medium was 0.4 times lower than that of the conventional medium while their average radial velocities were almost the same (zero). The standard deviation of radial velocity fluctuation in the N-medium was also 0.4 times less than that in the conventional medium. These results suggest that turbulent flow occurred in the bioreactor culture using the conventional medium and the degree of turbulent flow decreased significantly when 0.8% CMC was added to the medium to prepare the N-medium. A change of the flow pattern is considered to be the cause of the decrease in hydrodynamic stress, resulting in enhanced pigment production due to the enlargement of the callus.

Enhanced anthocyanin production from grape callus in an Air-Lift type bioreactor using a viscous additive-supplemented medium

An N-medium containing carboxymethyl cellulose (CMC) was applied to an air-lift type bioreactor culture of grape ( Vitis vinifera cv. Bailey alicant A.) callus, and anthocyanin production was investigated. Grape callus grew well at an air flow rate of 80 ml/min and anthocyanin production was significantly increased in the N-medium, reaching 17 mg/ l after 7 d of culture. The anthocyanin content of the N-medium was about two times higher than that of the conventional medium without CMC. The effect of air flow rate was also investigated within the range from 40 to 160 ml/min. A twofold increase in anthocyanin content was obtained at all the air flow rates tested in the N-medium. The distribution of grape callus size obtained after 7 d of the bioreactor culture was investigated. The average callus size was 490 μm which was 1.6 times larger than that obtained in the conventional medium. It was found that large calli with a relatively high anthocyanin pigment content were formed in the bioreactor culture using the N-medium. The fluid dynamics in the bioreactor was also investigated at three points (top, middle and bottom) in the bioreactor by laser doppler velocimetry. The average axial velocity of the circulated medium was 0.4 times lower than that of the conventional medium while their average radial velocities were almost the same (zero). The standard deviation of radial velocity fluctuation in the N-medium was also 0.4 times less than that in the conventional medium. These results suggest that turbulent flow occurred in the bioreactor culture using the conventional medium and the degree of turbulent flow decreased significantly when 0.8% CMC was added to the medium to prepare the N-medium. A change of the flow pattern is considered to be the cause of the decrease in hydrodynamic stress, resulting in enhanced pigment production due to the enlargement of the callus.

Purification and characterization of wall-localized cellulase from maize coleoptiles

Wall-localized cellulase was partially purified from freeze-dried maize coleoptiles by a combination of DEAE-Sepharose, Superdex-200 gel filtration and Hydroxyapatite column chromatography. Activity was measured by both reducing sugar assay and dot assay on agarose gel containing carboxymethylcellulose(CMC). In situ activity staining on a nondenaturing gel overlaid on agarose gel containing CMC turned out to be a quite reliable method to detect cellulase activity. The molecular mass of partially-purified cellulase was determined to be about 53 kD based on SDS-PAGE, and the N-terminal amino acid sequence of this cellulase was NH2-AGAKGANXLGGLXRA. The enzyme hydrolyzed CMC with an optimal pH of 4.5 and optimal temperature of 40°C. It also catalyzed carboxymethylcellulose with aK m of 2.02 mg/mL and aV max of 160 ng/h/mL The β-1,4-glucosyl linkages of CMC, fibrous cellulose and lichenan were cleaved specifically by this enzyme. Reducing reagents such as cysteine-HCI, dithiothreitol and glutathione strongly enhanced the activity, suggesting that SH-groups of the enzyme were protected from oxidation. N-ethylmaleimide which is a sulfhydryl-reacting reagent did not seem to inhibit the activity, indicating that cysteine residues were not located near the active site of the enzyme. These results will be valuable in understanding the structure of wall-localized cellulase in maize coleoptiles and in predicting its possible function in the cell wall.

Simple and rapid method for the isolation of Aspergillus niger mutants with enhanced cellulase and xylanase activity

An agar plate isolation technique was developed for screening the mutants of Aspergillus niger. Fungal growth in solid media containing carboxymethylcellulose(CMC) was selected within 2–3 days. The mutants selected by the new technique showed a remarkable enhancement of CMCase, FPase, β-glucosidase, xylanase, and β-xylosidase activities. These results clearly show that the new isolation technique is a practical method for the selection of better strains.

PREPARATION AND THERMAL PROPERTIES OF POROUS COMPOSITES OF NYLON-6 AND CARBOXYMETHYLCELLULOSE-RESIN

Two kinds of porous nylon-6 membranes containing or not containing carboxymethylcellulose (CMC) resins were prepared from a methanol solution of nylon-6 and calcium chloride. The sorption effects of calcium chloride on the thermal properties of these membranes were investigated by DSC, together with the observation of morphologies. From the DSC curves, it was revealed that calcium chloride disturbed the reorganization of nylon-6 crystals during the heating process. The mixing of CMC resins gave the porosities with specific shapes to the nyloxi-6/CMC-resin composites.

Solid Media Containing Carboxymethylcellulose to Detect Cx Cellulase Activity of Micro-organisms

Solid media containing carboxymethyl cellulose (CMC) were developed to detect CX cellulose-producing micro-organisms. Hydrolysis of CMC was seen as a clear zone around colonies after flooding plates with 1% aqueous hexadecyltrimethyl-ammonium bromide. Tests with ten bacterial and four fungal species showed that the degree of substitution (DS) of the CMC affects both growth and enzyme production. Most of the organisms produced more CX cellulase on CMC with a DS of 0-9, but CMC with a DS of 0-4 was better for one fungus. A qualitative measure of cellulase production may be obtained by calculating the ratio of zone size to colony diameter. Solid media containing CMC provided a more rapid assay of CX cellulose production than a medium containing native cellulose.

In vitro stimulation of antibody formation by peritoneal cells. I. Plaque technique of high sensitivity enabling access to the cells.

An improved method for the short-term culture of mouse peritoneal cells in a medium containing carboxymethylcellulose (CMC), sheep erythrocytes (SRBC), and guinea pig complement is described. It involves preparation of microcultures, of thickness 12-15 micro and volume 3.6 microl, under paraffin oil. With such cultures, peritoneal cells from normal, unimmunized young male CBA mice give about 3000 hemolytic plaques per million cells cultured, this figure being attained within 24 hr. The plaque detection method is about four times as sensitive as the Jerne technique. A method is described whereby such plaque-forming cells (PFC) can be transferred, by micromanipulation, to fresh monolayer cultures containing SRBC, CMC, and complement. In this fashion, the secretory capacity and susceptibility to inhibitors of peritoneal PFC can be tested in detail. Using this technique, evidence is presented that the hemolytic substance responsible for plaque formation is actually secreted by the cell at the center of the plaque, and is not a complement component but probably an antibody. Studies on the time of plaque appearance after cell transfer, and the subsequent growth rate of the zone of hemolysis, have been performed. They speak against the idea that the PFC is either a reservoir of cytophilic antibody or a "background" PFC. Rather they suggest that active antibody secretion is induced in the cell at some defined time point in culture. Detailed kinetics of the rate of appearance of plaques in peritoneal cell cultures revealed an exponential phase lasting from about 3 to about 13 hr with a doubling time of 2 hr. The reasons for this are not known. A greatly heightened reactivity was shown in peritoneal cells of mice that had been pregnant several times. Cultures of such cells showed more rapid plaque appearance and a peak activity about 20 times higher than with cells from young male mice. Cultures in which 1 cell in 10 formed a plaque were not infrequent. A series of experiments on germ-free mice showed reactivity similar to that of conventional mice from the same strain and source. The significance of the findings for cellular immunology are discussed.

In vitro stimulation of antibody formation by peritoneal cells. II. Cell interactions and effects of immunochemical or metabolic inhibitors.

Peritoneal cells (PC) from normal, unimmunized mice were placed in ultra-thin monolayer cultures containing carboxymethylcellulose (CMC), sheep red blood cells (SRBC), and complement, and tested for the appearance of plaques of lysis. The behavior of PC from young male mice and from female mice that had given birth to several litters (retired breeder mice) was studied. It was found that cells from spleen, mesenteric lymph node, thymus, bone marrow, thoracic duct lymph, or Peyer's patches could not form plaques in the CMC microcultures. Also, various combinations of these cells did not lead to plaque formation. When cells from any of these sources were mixed with PC, there was either no effect or an actual inhibition of plaque formation, the plaque counts being lower than would have been expected from the number of PC present in the mixture. Optimal plaque formation by peritoneal cells was found to be dependent on an optimal cell concentration, this optimum being around 5 x 10(6)/ml for young male mice and 0.5 x 10(6)/ml for retired breeders. Inhibition of plaque formation was found with either supra- or suboptimal cell concentrations. The inhibition by excess cell concentration may have been a simple nutritional or nonspecific overcrowding effect, as it could also be induced by an addition of an excess of spleen or lymph node cells. The failure of more dilute PC preparations to give adequate numbers of plaques appeared to be more specific, as plaque numbers could not be restored to normal by addition of spleen cells. The suggestion was that some cell to cell interaction between PC was involved. This dependence on cell concentration was not seen with immunized spleen PFC. Plaque appearance could be specifically and reversibly suppressed by placing PC in a medium containing rabbit anti-mouse IgM serum. Anti-IgG serum had no such effect. These experiments strengthened our view, expressed in the accompanying paper, that plaque formation was due to the formation of IgM, hemolytic antibody to SRBC by the PC. Metabolic inhibitors were incorporated into monolayer cultures and had different effects with the different types of PFC used. In the case of spleen cells from mice actively immunized against SRBC 4 days before killing, actinomycin D had no effect on plaque counts and puromycin reduced plaque numbers by a factor of 2. In the case of PC from young male mice, actinomycin D in concentrations above 0.01 microg/ml caused reductions down to < 2% of control values in plaque counts, and puromycin (10 microg/ml) had a similar effect. The PC from retired breeder mice occupied an intermediate position between the two cases just discussed. A compartment of cells, equal to about one-fifth of the total normal PFC compartment, was identified as resistant to high concentrations of either actinomycin D or puromycin, being similar in these respects to PFC from spleens of intentionally preimmunized mice. The mitotic poison, Colcemid, did not affect plaque counts in any situation tested. The theoretical implications of these results are briefly discussed.

Induction of Cellulase Biosynthesis by Cellobiose Octaacetate in &lt;i&gt;Aspergillus humicola&lt;/i&gt;

Aspergillus humicola, one of the major cellulase-producing fungi, was used in this study for carboxymethylcellulase (CMCase) production using Winstead's basal broth supplanted with cellobiose octaacetate (COA), a synthetic carbon source. Under all conditions, the enzyme biosynthesis was remarkably increased when the inducer COA was added to the production medium containing carboxymethylcellulose (CMC). Maximum enzyme production (1.62 U/ml) was achieved in COA-containing at 37°C. The enzyme production was highest at initial pH 5.5 and after 7 days incubation. The enzyme exhibited maximum activity at 40°C with a reaction pH 5.5. CMCase activity was inhibited by its own substrate CMC at concentration higher than 1.0%. The study clearly demonstrated that COA is a good inducer for extracellular CMCase production by the fungus. Keywords: Aspergillus humicola, Carboxymethylcellulase (CMCase), Carboxymethylcellulose (CMC), Cellobiose octaacetate (COA)DOI: http://dx.doi.org/10.3329/bjm.v23i2.889 Bangladesh J Microbiol, Volume 23, Number 2, December 2006, pp 174-176