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PubMed1997

Fumarase C activity is elevated in response to iron deprivation and in mucoid, alginate-producing Pseudomonas aeruginosa: cloning and characterization of fumC and purification of native fumC.

We report the discovery of fumC, encoding a fumarase, upstream of the sodA gene, encoding manganese superoxide dismutase, in Pseudomonas aeruginosa. The fumC open reading frame, which terminates 485 bp upstream of sodA, contains 1,374 bp that encode 458 amino acids. A second 444-bp open reading frame located between fumC and sodA, called orfX, showed no homology with any genes or proteins in database searches. A fumarase activity stain revealed that P. aeruginosa possesses at least two and possibly three fumarases. Total fumarase activity was at least approximately 1.6-fold greater in mucoid, alginate-producing bacteria than in nonmucoid bacteria and decreased 84 to 95% during the first 5 h of aerobic growth, followed by a rapid rise to maximum activity in stationary phase. Bacteria exposed to the iron chelator 2,2'-dipyridyl, but not ferric chloride, demonstrated an increase in fumarase activity. Mucoid bacteria produced approximately twofold-higher levels of the siderophores pyoverdin and pyochelin than nonmucoid bacteria. Northern blot analysis revealed a transcript that included fumC, orfX, and sodA, the amount of which was increased in response to iron deprivation. A P. aeruginosa fumC mutant produced only approximately 40% the alginate of wild-type bacteria. Interestingly, a sodA mutant possessed an alginate-stable phenotype, a trait that is typically unstable in vitro. These data suggest that mucoid bacteria either are in an iron-starved state relative to nonmucoid bacteria or simply require more iron for the process of alginate biosynthesis. In addition, the iron-regulated, tricarboxylic acid cycle enzyme fumarase C is essential for optimal alginate production by P. aeruginosa.

Alginates↗

PubMed2004

AlgX is a periplasmic protein required for alginate biosynthesis in Pseudomonas aeruginosa.

Alginate, an exopolysaccharide produced by Pseudomonas aeruginosa, provides the bacterium with a selective advantage that makes it difficult to eradicate from the lungs of cystic fibrosis (CF) patients. Previous studies identified a gene, algX, within the alginate biosynthetic gene cluster on the P. aeruginosa chromosome. By probing cell fractions with anti-AlgX antibodies in a Western blot, AlgX was localized within the periplasm. Consistent with these results is the presence of a 26-amino-acid signal sequence. To examine the requirement for AlgX in alginate biosynthesis, part of algX in P. aeruginosa strain FRD1::pJLS3 was replaced with a nonpolar gentamicin resistance cassette. The resulting algXDelta::Gm mutant was verified by PCR and Western blot analysis and was phenotypically nonmucoid (non-alginate producing). The algXDelta::Gm mutant was restored to the mucoid phenotype with wild-type P. aeruginosa algX provided on a plasmid. The algXDelta::Gm mutant was found to secrete dialyzable oligouronic acids of various lengths. Mass spectroscopy and Dionex chromatography indicated that the dialyzable uronic acids are mainly mannuronic acid dimers resulting from alginate lyase (AlgL) degradation of polymannuronic acid. These studies suggest that AlgX is part of a protein scaffold that surrounds and protects newly formed polymers from AlgL degradation as they are transported within the periplasm for further modification and eventual transport out of the cell.

Alginates↗

PubMed1992

Role of alginate in infection with mucoid Pseudomonas aeruginosa in cystic fibrosis.

BACKGROUND: Chronic bronchopulmonary infection with mucoid, alginate producing Pseudomonas aeruginosa occurs characteristically in patients with cystic fibrosis. Alginate may be a virulence factor for P aeruginosa infection in such patients. METHODS: Forced vital capacity (FVC), nutritional state and the antibody response to P aeruginosa were determined at regular intervals from three years before chronic P aeruginosa infection to 10 years afterwards in 73 patients with cystic fibrosis. All patients were treated intensively with antipseudomonal chemotherapy during the study period. RESULTS: FVC was reduced in all patients who subsequently developed P aeruginosa infection before they acquired the infection, indicating significant pre-existing lung damage when compared with patients who remained free of P aeruginosa. Lung function and nutritional state remained unchanged after 10 years of infection, except in the patients who died of P aeruginosa lung infection. The FVC and height and weight of patients infected with nonmucoid strains of P aeruginosa were similar to those of uninfected patients. Patients infected with mucoid strains had poorer lung function and nutritional state for the first five years after infection compared with patients with nonmucoid strains. Such infection was also associated with greater IgG and IgA antibody responses to P aeruginosa standard antigen compared with nonmucoid infection. Concentrations of antibody to alginate were similar in patients with non-mucoid and mucoid infection. Noticeably increased concentrations of IgA antibodies to P aeruginosa standard antigen were observed early after the onset of infection in patients who subsequently died. CONCLUSION: Alginate producing P aeruginosa infection is associated with a hyperimmune response and poor clinical condition, suggesting that alginate production is a virulence factor in such infections in patients with cystic fibrosis.

Adolescent↗

PubMed1980

Inhibition of the intestinal absorption of iron by sodium alginate and guar gum in rats.

Na-alginate as well as guar gum inhibit the absorption of a 59Fe-labelled iron dose (360 nmol) from tied-off jejunal segments of either normal or iron-deficient rats. In order to inhibit the absorption of the iron dose by half as compared with normal rats to which ionized iron was administered 1.2--8 mg of guar gum and 8-30 mg Na-alginate was necessary. In iron-deficient rats the highest dose dose of Na-alginate tested, 100 mg, inhibited the absorption of iron by about 20%; the highest dose of guar gum, 30 mg, inhibited the amount of iron absorbed by about 25%. An artificial diet containing 10% of either guar gum and Na-alginate fed for 3 days inhibited the absorption of iron in normal but not in iron-deficient rats. Also, in these experiments guar gum proved to be more effective than Na-alginate.

Alginates↗

PubMed2006

Formulation, characterization and release studies of alginate microspheres encapsulated with tetanus toxoid.

Alginate is a safe, non-immunogenic and inexpensive natural polymer with high mucoadhesive properties. Alginate microspheres can be used as a delivery system for antigens to mucosal surfaces. In the present study alginate microspheres were prepared by an emulsification technique. The effects of sonication time, concentration of alginate, emulsifier and calcium chloride, and also the volume of calcium solution, were evaluated on mean size, size range, surface roughness and porosity, sphericity and clumping of microspheres using an optical microscope and particle size analyzer. The most desirable conditions were 90 s sonication, 3% alginate solution, 2% surfactant and 60 ml of 0.33% CaCl2 in octanol. The resulting microspheres had a mean size of 1.34 +/- 0.3 microm and size range of 0.3 +/- 2.0 microm, with no surface roughness and porosity, low clumping and high sphericity. The encapsulation efficiency was about 47.7%. All batches showed nearly the same release profiles with a low burst release. The stability of the model antigen (tetanus toxoid (TT)) extracted from microspheres was confirmed by SDS-PAGE; and the antigenicity of TT was studied by ELISA and found to be 91 +/- 5% of the original TT. It can be concluded that, with regard to the size and morphological characteristics of the prepared microspheres and their ability in preserving the antigenicity of the encapsulated TT, they could be used as a delivery system for mucosal delivery of TT.

1-Octanol↗

PubMed1984

Pulmonary hypersensitivity in the alginate industry.

Salts of alginic acid are complex polymerised polysaccharides which are chemically extracted from seaweed. Workers in the alginate industry are exposed to dust from dried milled seaweed and pure alginate compounds. In this survey of one of the two factories in Britain producing alginates, we found evidence of pulmonary hypersensitivity to seaweed dust in seven per cent of the total work force, and evidence of precipitating antibody to sodium alginate and seaweed extracts in the serum of 4.5 per cent of the work force. Challenge testing of a number of employees with symptoms showed a dual response with immediate airways obstruction, and a later loss of lung volume, with associated impairment of transfer factor.

Alginates↗

PubMed2005

Investigation of a new microcapsule membrane combining alginate, chitosan, polyethylene glycol and poly-L-lysine for cell transplantation applications.

Microencapsulation of living cells may serve as an alternative therapy for patients requiring organ transplants. One of the limiting factors in the progress of such therapy is attaining a biocompatible and mechanically stable polymer. The current study investigates the potential of a novel membrane combining alginate, chitosan, polyethylene glycol (PEG) and poly-L-lysine (PLL) with the objective of proposing a membrane suitable for cell entrapment that may overcome some of the shortcomings of the widely studied alginate-poly-L-lysine-alginate (APA) capsules. The novel microcapsule was formulated using a 1.5% alginate solution coated with 0.05% chitosan, 0.1% PEG and 0.05% poly-L-lysine with a final layer of 0.1% alginate. Microcapsules having a diameter of 450 +/- 30 microm were prepared. Upon citrate treatment, the membrane remained intact and retained its spherical structure. The membrane was able to support liver cell proliferation and the encapsulated cells were capable of secreting proteins. The study demonstrated that the new membrane can be used for cell entrapment. However, further investigations are needed to assess its potential for long term transplantation and usage in the development of bioartificial organs.

Albumins↗

PubMedDate not supplied

Correction of bilirubin conjugation in the Gunn rat using hepatocytes immobilized in alginate gel beads as an extracorporeal bioartificial liver.

A new extracorporeal bioartificial liver using alginate-entrapped hepatocytes was developed and evaluated for its ability to correct the lack of bilirubin conjugation in the Gunn rat. Hepatocytes were harvested from Sprague-Dawley rats by the two-step collagenase perfusion method and then immobilized in Ca(++)-alginate beads. The ability of immobilized hepatocytes to conjugate bilirubin was investigated in vitro by comparison with hepatocyte monolayer cultures. The bioartificial liver consisted of a cylindric bioreactor containing either alginate beads with hepatocytes (test group) or alginate beads alone (control group). Gunn rats were connected to this bioreactor via an extracorporeal circulation and bile fractions were collected at hourly intervals. Both bilirubin monoconjugates and bilirubin diconjugates were measured in the bile by high pressure liquid chromatography. Hepatocyte viability in alginate beads was determined prior to and at the end of each experiment and found to be unchanged (75%). In the test group, the concentration of bilirubin conjugates increase rapidly, attaining median values of 72.26 microM and 92.59 microM for mono and diconjugated bilirubin respectively, during a 3 h period of extracorporeal circulation. In the control group, the levels of either conjugate did not exceed 0.87 microM throughout the experiments. Statistical analysis showed a significant difference between the two groups (p < 0.0023). These results suggest that the bioartificial liver used in this study represents an effective method for the temporary correction of the Gunn rat's genetic defect. Such a system might be of therapeutic interest in acute liver failure.

Alginates↗

PubMed2003

Investigations on neomycin production with immobilized cells of Streptomyces marinensis NUV-5 in calcium alginate matrix.

The purpose of this investigation was to study the effect of Streptomyces marinensis NUV-5 cells immobilized in calcium alginate for the production of neomycin. The effect of various parameters, such as the effect of alginate concentration (1%, 2%, 3%, 4%, and 5% wt/vol), the effect of cation (CaCl2, BaCl2, and SrCl2), the concentration of cation (0.01M, 0.125M, 0.25M, 0.375M, and 0.5M), the curing times (1, 6, 11, 16, and 21 hours), and the diameter of the bead (1.48, 2.16, 3.24, 4.46, and 5.44 mm), on neomycin production and bead stability were studied. The effect of maltose (4%, 3%, 2%, and 1% wt/vol) and sodium glutamate (0.6%, 0.3%, 0.15%, and 0.075% wt/vol) concentration on neomycin production was also studied. Better neomycin production was achieved with optimized parameters, such as alginate at 2% wt/vol, 0.25M CaCl2, 1-hour curing time, and 3.24 mm bead diameter. Effective neomycin production was achieved with 3% wt/vol maltose and 0.6% wt/vol sodium glutamate concentration. The repeated batch fermentations were conducted (every 96 hours) using the optimized alginate beads, employing the production medium with 3% wt/vol maltose and 0.6% wt/vol sodium glutamate along with mineral salts solution. The increase in antibiotic production was observed up to the 5th cycle, and later gradual decrease in antibiotic production was observed. Comparison of the total antibiotic production with free cells and immobilized cells was also done. An enhanced antibiotic productivity of 32% was achieved with immobilized cells over the conventional free-cell fermentation, while 108% more productivity was achieved over the washed free-cell fermentation. From these results it is concluded that the immobilized cells of S marinensis NUV-5 in calcium alginate are more efficient for the production of neomycin with repeated batch fermentation.

Alginates↗

PubMed2005

Papain entrapment in alginate beads for stability improvement and site-specific delivery: physicochemical characterization and factorial optimization using neural network modeling.

This work examines the influence of various process parameters (like sodium alginate concentration, calcium chloride concentration, and hardening time) on papain entrapped in ionotropically cross-linked alginate beads for stability improvement and site-specific delivery to the small intestine using neural network modeling. A 3(3) full-factorial design and feed-forward neural network with multilayer perceptron was used to investigate the effect of process variables on percentage of entrapment, time required for 50% and 90% of the enzyme release, particle size, and angle of repose. Topographical characterization was conducted by scanning electron microscopy, and entrapment was confirmed by Fourier transform infrared spectroscopy and differential scanning calorimetry. Times required for 50% (T(50)) and 90% (T(90)) of enzyme release were increased in all 3 of the process variables. Percentage entrapment and particle size were found to be directly proportional to sodium alginate concentration and inversely proportional to calcium chloride concentration and hardening time, whereas angle of repose and degree of cross-linking showed exactly opposite proportionality. Beads with >90% entrapment and T(50) of <10 minutes could be obtained at the low levels of all 3 of the process variables. The inability of beads to dissolve in acidic environment, with complete dissolution in buffer of pH >or=6.8, showed the suitability of beads to release papain into the small intestine. The shelf-life of the capsules prepared using the papain-loaded alginate beads was found to be 3.60 years compared with 1.01 years of the marketed formulation. It can be inferred from the above results that the proposed methodology can be used to prepare papain-loaded alginate beads for stability improvement and site-specific delivery.

Alginates↗

PubMed2006

Incorporation of proteins within alginate fibre-based scaffolds using a post-fabrication entrapment method.

In this study, a physical entrapment process was explored for the incorporation of proteins within preformed fibrous alginates and the release profile was tuned by varying the processing parameters. The entrapment process was carried out in a series of aqueous solutions at room temperature and involved pre-swelling of the fibrous alginate within a Na(+)-rich solution, followed by exposure to the protein of choice and entrapping it by re-establishing cross-links of alginate with BaCl2. Entrapment and release of fluorescein isothiocyanate-labelled bovine serum albumin (FITC-BSA), a model protein, was studied. It was found that a sustained release of the incorporated protein in cell culture medium for about 6 days was achieved. The main factors determining the release profile included the NaCl/CaCl2 ratio in the pre-swelling solution, protein concentration, and the exposure time. To retard protein release, alginate fibres with entrapped FITC-BSA were processed together with poly(D, L-lactide) (PDLLA) into porous alginate fibre/PDLLA composites using supercritical CO2. In this manner, release of the protein for up to 3 months was achieved.

3T3 Cells↗

PubMed1996

Application of alginate gel as a vehicle for liposomes. I. Factors affecting the loading of drug-containing liposomes and drug release.

To explore the feasibility of alginate gel as a vehicle for liposomes, we investigated the effects of various factors associated with the loading of drug-containing liposomes into the gel beads. The loading process includes (I) mixing of liposomes and alginate solution, (2) calcium induced gelation of alginates, (3) the time-dependent contraction of a gel body squeezing out interior water, and (4) possible leakage or release of a drug entrapped in liposomes in a series of each of theses processes. These effects were examined in terms of the leakage of a marker 5(6)-carboxyfluorescein (CF) from liposomes of egg phosphatidylcholine (EPC) and EPC/cholesterol (EPC/Cho) and liposome (phosphorus) release from curing and fully-cured gel beads whose initial polymer concentrations were 4 and 2%. Major findings were: (1) Alginate induced the leakage of a water-soluble drug incorporated in the liposomes as a function of the polymer concentration and the mixing time. (2) Calcium ions also stimulated the leakage of the drug. EPC/Cho liposomes were several times more resistant to the leakage of CF than were EPC liposomes. (3) The liposomes were well loaded without any loss in the gel bead despite the squeezing outflow of water and the bead contraction during gel curing. (4) Such curing caused leakage of the drug from the EPC liposomes in the very early stage while no effect was observed in the EPC/Cho liposomes. (5) In the gel-eroding medium (pH 7.4 Tris-HCl, 37 degrees C), the total drug release was controlled by the erosion rate of the bead body. Immediately after the bead erosion, EPC liposomes retained about 60% of the drug in the 2% bead and only about 20% in the 4% bead, whereas EPC/Cho liposomes retained more than 85% regardless of the initial alginate concentration. The results provide valuable information for the design and applicability of the gel-loaded liposome delivery system.

Alginates↗

PubMed2003

1H-NMR study of Na alginates extracted from Sargassum spp. in relation to metal biosorption.

The use of a number of species of marine brown algae in the implementation of bioremediation strategies for toxic heavy metals is being considered and evaluated. The biosorption capacity of these algae for heavy metals resides mainly in a group of linear polysaccharides known as alginates that occur as a gel in the algal thallus. The potential for selective metal binding by the biomass of two species of Sargassum was evaluated by 1H-NMR (nuclear magnetic resonance) following a high temperature, alkaline extraction and purification of their alginate polysaccharide. The alkaline extraction protocol applied to Sargassum fluitans and Sargassum siliquosum yielded alginate samples of low viscosity, suitable for direct acquisition of well-resolved spectra. Estimates of both the ratio of beta-D-mannopyranuronosyl (M) and alpha-L-gulopyranuronosyl (G) residues along the polymer chain and the frequencies of occurrence of diad uronic acid residue pairs were obtained. Guluronic acid (G) was the major component in all extracts and the GG diads accounted for more than 49% of the polymer diads. Whereas the performance of Sargassum spp. in the metal biosorption process is a function of both its alginate content and composition, the occurrence of "G-blocks" in both purified alginates and in the raw brown seaweed is critical because it results in a well-established selectivity for divalent ions, potentially increasing the commercial effectiveness of targeted biosorption as a means of remediation.

Alginates↗

PubMedDate not supplied

The effect of capsule composition on the biocompatibility of alginate-poly-l-lysine capsules.

The encapsulation of islets of Langerhans in alginate-poly-l-lysine has been proposed as a method for the immunoprotection of transplanted islets. Although several capsule compositions have been reported, there has been no published study concerning the effect of capsule composition on the severity of the foreign body reaction. Empty capsules were prepared from high mannuronic acid alginate and were coated with: (1) poly-l-lysine alone, (2) poly-l-lysine plus high guluronic acid alginate, or (3) poly-l-lysine plus high mannuronic acid alginate. The capsules were placed in the renal subcapsular space or the peritoneal cavity, and retrieved after three weeks of histological examination. The recipients were WAG/01a, nude (athymic), diabetic BB, and non-diabetes prone BB rats. The severity of reaction to the capsules was determined by measuring the thickness of the pericapsular cell infiltrate or by a scoring system. The severity of the reaction to the capsules was strain-dependent in both the renal and peritoneal sites, with the BB and nude rats displaying the most severe responses. The degree of response was not affected by capsule composition in the renal subcapsular space, but in the peritoneum, the high mannuronic acid alginate capsules provoked the weakest response, and this type of capsule will be used for future transplantation work. The infiltrating cells were characterised by immunohistochemistry and electron microscopy and found to be mostly fibroblasts and macrophages.

Alginates↗

PubMedDate not supplied

Emulsification preparation of calcium alginate beads in the presence of sequesterant.

A biocompatible emulsification method has been developed for microencapsulation of live cells and enzymes within a calcium alginate matrix. Fabrication of alginate beads was achieved by premixing a sequestering agent (sodium polyphosphate) and the calcium source (calcium sulphate) with the hydrogel monomer prior to the introduction to the oil phase. The competition between the sequesterant and sodium alginate in binding the available calcium ions results in a slowing down of the rate of polymerization and thereby lead to a successful calcium alginate bead formation. The mean diameter of the fabricated beads may be easily controlled by employing soy bean lecithin as an emulsifier. The polymerization time in this process may vary between 3 and 35 min depending on the ratio of sequesterant to that of calcium source at constant sodium alginate concentration. This preparation method avoids the use of pH extremes at all times and therefore is particularly suitable for encapsulating pH-sensitive cells and enzymes.

Alginates↗

PubMedDate not supplied

Ionotropic gelation: encapsulation of indomethacin in calcium alginate gel discs.

Ionotropic gelation by divalent metal interaction was employed as an approach to design a modified release multiple-unit oral drug-delivery system. This process was achieved by crosslinking an indomethacin-sodium alginate dispersion with calcium ions to induce the spontaneous formation of indomethacin-calcium alginate gel discs. A significant part in the validation of the integrity of the system, involved a preformulatory stage for the optimization of the curing conditions and potency determination of the gel discs. A three-phase approach was developed to establish the critical curing parameters. Since curing involved crosslinking of the sodium alginate with calcium ions, an optimal concentration of calcium chloride (phase one) and crosslinking reaction-time (phase two) had to be determined. Furthermore, the third phase involved the optimization of the air-drying time of the gel discs. In phases one and two, stabilization of in vitro drug-release characteristics was used as the marker of optimal crosslinking efficiency. Phase three was based on achieving fully dried gel discs by drying to constant weight at 21 degrees C under an extractor. The study revealed that optimal crosslinking efficiency was achieved in 1%w/v calcium chloride solution for 24 h and air-dried at 21 degrees C under an extractor for 48 h. The three solvent/solution systems investigated for their ability to liberate completely the drug from the matrix system were methanol, sodium citrate (1%w/v) and phosphate buffer pH 6.2. Phosphate buffer provided optimal drug removal, in addition to its ability to induce swelling of the calcium alginate gel discs. Furthermore, drug loading also increased with the use of increasing concentrations of sodium alginate in the formulations.

Administration, Oral↗

PubMedDate not supplied

Effect of calcium alginate dressings on partial-thickness wounds in swine.

The epithelization of partial-thickness wounds (PTW) has been shown to be enhanced with the use of moisture-retentive dressings (MRD), and not with nonmoisture-retentive dressings (NMRD) like gauze. This study was designed to explore this effect using two different alginate products with and without MRDs. We evaluated these alginate dressings under a polyurethane film dressing (FMRD) and under an NMRD gauze. They were compared to hydrocolloid moisture-retentive dressings (HMRD). Twelve PTW measuring 22 x 22 x 0.5 mm were made on the dorsum of six swine with a Castroviejo dermatome. Return of the epithelial barrier function was measured with an EP1 ServoMed evaporimeter. On postoperative day 4, the first alginate product under the NMRD and under the FMRD had a significantly slower healing rate than the HMRDs. By postoperative day 7, the second alginate product under the NMRD had a poorer rate of epidermal wound healing than the HMRD. Our results indicate that these alginate products have a satisfactory partial-thickness wound healing capability when used under MRDs. When used under MRDs, the return of the epithelial barrier function is delayed, indicating that these dressings should not be used on dry wounds or under gauze dressings.

Alginates↗

PubMed1995

Culture of chondrocytes in alginate and collagen carrier gels.

In this in vitro study, we compared the potential of collagen and alginate gels as carriers for chondrocyte transplantation and we studied the influence of demineralized bone matrix (DBM) on chondrocytes in the gels. Chondrocytes were assessed for cell viability, phenotype (histology), proliferation rate and sulfate incorporation. Collagen gels showed a significant increase in cell numbers, but the chondrocytes dedifferentiated into fibroblast-like cells from day 6 onwards. In alginate gels, initial cell loss was found, but the cells maintained their typical chondrocyte phenotype. Although the total quantity of proteoglycans initially synthesized per cell in collagen gel was significantly higher, expressed per cell, the quantity in alginate gel eventually surpassed collagen. No effects of culturing chondrocytes in combination with DBM could be demonstrated on cell proliferation and sulfate incorporation. The collagen and alginate gels have different advantages as carriers for chondrocyte transplantation. The high proliferation rate of chondrocytes in collagen gel may be an advantage, but the preservation of the chondrocyte phenotype and the gradually increasing proteoglycan synthesis in alginate gel is a promising method for creating a hyaline cartilage implant in vitro.

Alginates↗