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PubMed1996

Mutational analysis of nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of critical amino acid residues involved in exopolysaccharide alginate synthesis.

We report the utilization of site-directed and random mutagenesis procedures in the gene encoding nucleoside diphosphate kinase (ndk) from Pseudomonas aeruginosa in order to examine the role of Ndk in the production of alginate by this organism. Cellular levels of the 16-kDa form of the Ndk enzyme are greatly reduced in P. aeruginosa 8830 with a knockout mutation in the algR2 gene (8830R2::Cm); this strain is also defective in the production of the exopolysaccharide alginate. In this study, we isolated four mutations in ndk (Ala-14-->Pro [Ala14Pro], Gly21Val, His117Gln, and Ala125Arg) which resulted in the loss of Ndk biochemical activity; hyperexpression of any of these four mutant genes did not restore alginate production to 8830R2::Cm. We identified six additional amino acid residues (Ser-43, Ala-56, Ser-69, Glu-80, Gly-91, and Asp-135) whose alteration resulted in the inability of Ndk to complement alginate production. After hyperproduction in 8830R2::Cm, it was determined that each of these six mutant Ndks was biochemically active. However, in four cases, the in vivo levels of Ndk were reduced, which consequently affected the growth of 8830R2::Cm in the presence of Tween 20. Two mutant Ndk proteins which could not complement the alginate synthesis defect in 8830R2::Cm were not affected in any characteristic examined in the present study. All of the mutant Ndks characterized which were still biochemically active formed membrane complexes with Pk, resulting in GTP synthesis. Two of the four Ndk activity mutants (His117Gln and Ala125Arg) identified were capable of being truncated to 12 kDa and formed a membrane complex with Pk; however, the complexes formed were inactive for GTP synthesis. The other two Ndk activity mutants could be truncated to 12 kDa but were not detected in membrane fractions. These results further our understanding of the role of Ndk in alginate synthesis and identify amino acid residues in Ndk which have not previously been studied as critical to this process.

Alginates↗

PubMed1997

Characterization of the alginate biosynthetic gene cluster in Pseudomonas syringae pv. syringae.

Alginate, a copolymer of D-mannuronic acid and L-guluronic acid, is produced by a variety of pseudomonads, including Pseudomonas syringae. Alginate biosynthesis has been most extensively studied in P. aeruginosa, and a number of structural and regulatory genes from this species have been cloned and characterized. In the present study, an alginate-defective (Alg-) mutant of P. syringae pv. syringae FF5 was shown to contain a Tn5 insertion in algL, a gene encoding alginate lyase. A cosmid clone designated pSK2 restored alginate production to the algL mutant and was shown to contain homologs of algD, alg8, alg44, algG, algX (alg60), algL, algF, and algA. The order and arrangement of the structural gene cluster were virtually identical to those previously described for P. aeruginosa. Complementation analyses, however, indicated that the structural gene clusters in P. aeruginosa and P. syringae were not functionally interchangeable when expressed from their native promoters. A region upstream of the algD gene in P. syringae pv. syringae was shown to activate the transcription of a promoterless glucuronidase (uidA) gene and indicated that transcription initiated upstream of algD as described for P. aeruginosa. Transcription of the algD promoter from P. syringae FF5 was significantly higher at 32 degrees C than at 18 or 26 degrees C and was stimulated when copper sulfate or sodium chloride was added to the medium. Alginate gene expression was also stimulated by the addition of the nonionic solute sorbitol, indicating that osmolarity is a signal for algD expression in P. syringae FF5.

Alginates↗

PubMed2002

Mutant analysis and cellular localization of the AlgI, AlgJ, and AlgF proteins required for O acetylation of alginate in Pseudomonas aeruginosa.

Alginate is an extracellular polysaccharide produced by mucoid strains of Pseudomonas aeruginosa that are typically isolated from the pulmonary tracts of chronically infected cystic fibrosis patients. Alginate is a linear polymer of D-mannuronate and L-guluronate with O-acetyl ester linkages on the O-2 and/or O-3 position of the mannuronate residues. The presence of O-acetyl groups plays an important role in the ability of the polymer to act as a virulence factor, and the algF, algJ, and algI genes are known to be essential for the addition of O-acetyl groups to alginate. To better understand the mechanism of O acetylation of alginate, the cellular locations of the AlgI, AlgJ, and AlgF proteins were determined. For these studies, defined nonpolar algI, algJ, and algF deletion mutants of P. aeruginosa strain FRD1 were constructed, and each mutant produced alginate lacking O-acetyl groups. Expression of algI, algJ, or algF in trans in the corresponding mutant complemented each O acetylation defect. Random phoA (alkaline phosphatase [AP] gene) fusions to algF, algJ, and algI were constructed. All in-frame fusions to algF and algJ had AP activity, indicating that both AlgF and AlgJ were exported to the periplasm. Immunoblot analysis of spheroplasts and periplasmic fractions showed that AlgF was released with the periplasmic contents but that AlgJ remained with the spheroplast fraction. An N-terminal sequence analysis of AlgJ showed that its putative AlgJ signal peptide was not cleaved, suggesting that AlgJ is anchored to the cytoplasmic membrane by its uncleaved signal peptide. AP gene fusions were also used to map the membrane topology of AlgI, and the results suggest that it is an integral membrane protein with seven transmembrane domains. These results suggest that AlgI-AlgJ-AlgF may form a complex in the membrane that is the reaction center for O acetylation of alginate.

Acetylation↗

PubMed2001

Ocular hypotensive efficacy and safety of once daily carteolol alginate.

BACKGROUND/AIM: Carteolol is a beta adrenoceptor antagonist used topically to reduce intraocular pressure, typically twice daily. In an effort to provide a once daily dosing regimen, carteolol was formulated with 1% alginic acid. The objective of this study was to evaluate the efficacy and safety of carteolol alginate solution in comparison with standard carteolol solution. METHODS: This was a double masked, parallel group, multicentre study. Patients with ocular hypertension or open angle glaucoma (n=235) were randomly assigned to receive either carteolol alginate once daily [corrected] or standard carteolol solution, twice daily. The masking was maintained through the use of a vehicle in the evening for the alginate group. Patients were evaluated at baseline, 15, 60, and 120 days. RESULTS: At 0900 (presumed trough) on day 60, mean reductions in intraocular pressure (IOP) from baseline were 6.09 (SD 2.97) and 6.09 (3.18) mm Hg for the standard carteolol and alginate, respectively. At 1100 (presumed peak), mean reductions were 6.51 (2.53) and 6.47 (2.76) mm Hg, respectively. Results were similar at other times (day 15 and day 120). The most common side effect was transient stinging on instillation of drops, which did not differ significantly between groups. There were no differences of note in other ocular or systemic signs or symptoms. CONCLUSION: The new alginate formulation of carteolol 2% given once daily was as effective as standard carteolol 2% given twice daily with no meaningful differences regarding safety.

Adolescent↗

PubMed2005

Multifunctional drug delivery system using starch-alginate beads for controlled release.

Utilizing starch-containing alginate beads, a novel drug delivery system (DDS) was developed. With the starch inside, the composite bead could be dried in its original bead shape and handled in the dried state. By employing alginate multi-coating strategy on the starch-alginate beads, detained or controlled release was efficiently achieved and successfully demonstrated for a model peptide drug, L-phenylalanine. The initial latent time and release rate of the drug inside the beads were able to be controlled simply by varying the number of multi-coatings. While the latent time for the initial release was negligible for non-coated starch-alginate beads, the latent times of beads coated one, two, and four times increased to 15, 30, and 70 min, respectively. Furthermore, the alginate component of the composite beads could adsorb and remove heavy metals such as lead from the body. These multifunctional beads combined with the novel coating process will greatly benefit alginate gel-based DDS.

Alginates↗

PubMed2001

Isolation and characterizartion of alginic acid from commercially cultured Nemacystus decipiens (Itomozuku).

An alginate was isolated from commercially cultured Nemacystus decipiens which had been harvested in Yonashiro Town (Okinawa, Japan). The yield of the alginate was 1.6% (w/w of wet alga), and the uronic acid, ash and moisture contents of the alginate were 86.0%, 12.0%, and 2.3% (w/w), respectively. The molecular mass of the alginate was estimated to be about 1.5 x 10(5). The infrared spectrum and optical rotation of the alginate were in agreement with those of the standard alginate. D-Mannuronic acid and L-guluronic acid were identified by 1H- and 13C-NMR spectroscopy, the molar ratio of both sugar residues being estimated to be 0.72:1.00.

Alginates↗

PubMedDate not supplied

Nasal augmentation using injectable alginate and mesenchymal stem cells in the rabbit.

BACKGROUND: The goal of this study was to evaluate the use of the autogenous mesenchymal stem cells (MSCs) impregnated in an injectable alginate gel containing platelet-rich plasma (PRP) for nasal augmentation in rabbit model. METHODS: Bone marrow-derived MSCs were isolated and expanded from New Zealand white rabbits. At confluence, the cells were mixed with sodium alginate solution. PRP was prepared from the rabbits and it was immediately mixed into the alginate-cell mixture. The cell-PRP-alginate mix was injected into a subcutaneous nasal area. Eight weeks after injection changes in facial contour, newly formed nasal hump was analyzed and the amount of chondroitin sulfate in tissue was measured. RESULTS: Augmented nasal dorsa maintained their original shape until harvest. Immunohistochemical staining revealed that the deposited matrix was composed of type II collagen and that it was distributed abundantly and widely in the connective tissue of the tissue generated. The amount of chondroitin sulfate (main component of the proteoglycan in cartilage) produced was significantly higher when MSCs and PRP-alginate were used. CONCLUSION: Injectable PRP-alginate gel containing autologous mesenchymal stem cells may offer a useful means of facial soft tissue augmentation.

Alginates↗

PubMed1997

Alginate production by Azotobacter vinelandii.

Although all commercial alginates are today of algal origin, there is interest in the production of alginate-like polymers from bacteria. The species Azotobacter vinelandii seems to be the best candidate for the industrial production of alginate molecules characterized by a chemical composition, molecular mass and molecular mass distribution suited to a well defined application, especially required in the biotechnological, biomedical and pharmaceutical fields. The production of alginate by A. vinelandii has been to date widely investigated both in batch (mainly in the shaken flask scale) and in continuous cultures. This article summarizes current knowledge on the structure and properties of alginates and their applications and presents an overview of up-dated research on the physiology, genetics and kinetics of the production of alginate by Azotobacter vinelandii and its rheology, including the results of our recent studies.

Alginates↗

PubMed1999

Effects of sodium alginate and fish oil to reduce trichloroethylene accumulation in rats.

This study was conducted to examine the effect of dietary sodium alginate and fish oil on bodily accumulated trichloroethylene (TCE), which has been widely used as a halogenated solvent and is metabolized at a high rate. Each of three groups of rats was fed on either of diets containing cellulose-soybean oil (control), Na-alginate-soybean oil or cellulose-fish oil for 3 wk, and thereafter given a single oral dose of TCE (100 mg (0.76 mmol)/rat). TCE levels in the blood were monitored for 10 h after the administration of TCE. The peak concentrations of TCE in the blood tended to be higher in the alginate and fish oil groups as compared with those in the cellulose-soybean oil group, but not to a significant extent. TCE concentrations in the liver, kidney, brain and the three fat tissues (epididymal, perirenal and subcutaneous) were significantly lower in the alginate and fish oil groups than in the cellulose-soybean oil group. Fat tissue weights were also lower in the alginate group and fish oil group. The hepatic drug metabolizing enzymes could not account for the remarkable decreases of residual TCE contents in the alginate and fish oil groups. These findings indicate that the metabolism and excretion of TCE might be accelerated in animals with reduced fat tissue mass.

Adipose Tissue↗

PubMed2003

Biopex acquires anti-washout properties by adding sodium alginate into its liquid phase.

Biopex sets upon mixing with its liquid phase due to chelate reaction between sodium succinate and calcium phosphate, then gradually transforms to apatite. As a result of apatite formation, set Biopex shows excellent tissue responses and good osteoconductivity. However, Biopex would be washed out when the paste is exposed to body fluid before its setting reaction. Washed out cement causes an inflammatory response. In this investigation, therefore, sodium alginate was added to the liquid phase of Biopex and its effects on washout properties and transformation to apatite were studied. We found that cement remaining ratio, an index of the anti-washout property, of the Biopex increased by increasing the amounts of added sodium alginate to reach 100% when the liquid phase of Biopex contained 0.6% or more sodium alginate. Biopex gradually transformed to apatite regardless of the amount of added sodium alginate at least when the concentration of the sodium alginate in the liquid phase was 1.0% or less. It is concluded therefore, that addition of sodium alginate is very useful for Biopex to acquire the anti-washout property.

Alginates↗

PubMed2004

Effects of added sodium alginate on mechanical strength of apatite cement.

Effects of added sodium alginate on the mechanical strength of Biopex, one type of apatite cement, were investigated since sodium alginate addition is very effective for Biopex to acquire anti-washout property. Addition of sodium alginate into the liquid phase of Biopex resulted in a slower transformation to apatitic monolith. As a result, mechanical strength of set Biopex in terms of diametral tensile strength (DTS) decreased when it was hardened in an incubator kept at 37 degrees C and 100% relative humidity for 7 days. However, DTS value increased with increase in the amount of added sodium alginate when the Biopex paste was immersed in 0.9% saline at 37 degrees C for 7 days. Set Biopex with less sodium alginate also showed larger porosity. Based on these findings, we concluded that added sodium alginate was effective in increasing the mechanical strength of Biopex by inhibiting liquid penetration into its paste when it is exposed to body fluids.

Alginates↗

PubMed2004

Human monoclonal antibodies to Pseudomonas aeruginosa alginate that protect against infection by both mucoid and nonmucoid strains.

Two fully human mAbs specific for epitopes dependent on intact carboxylate groups on the C6 carbon of the mannuronic acid components of Pseudomonas aeruginosa alginate were found to promote phagocytic killing of both mucoid and nonmucoid strains as well as protection against both types of strains in a mouse model of acute pneumonia. The specificity of the mAbs for alginate was determined by ELISA and killing assays. Some strains of P. aeruginosa did not make detectable alginate in vitro, but in vivo protection against lethal pneumonia was obtained and shown to be due to rapid induction of expression of alginate in the murine lung. No protection against strains genetically unable to make alginate was achieved. These mAbs have potential to be passive therapeutic reagents for all strains of P. aeruginosa and the results document that alginate is a target for the proper type of protective Ab even when expressed at low levels on phenotypically nonmucoid strains.

Acute Disease↗

PubMed2003

A new alginate-based rapid method for determining coliforms in milk.

A new rapid method for monitoring coliforms was developed on the basis of the instant gelling effects of alginate and calcium. The effectiveness of this new method in the detection of coliforms was evaluated. Tests involving Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, total coliforms in milk, cold-injured coliforms, and total coliforms in raw milk were carried out. The bacterial samples were diluted in 0.2% peptone water containing 90 mM CaCl2 and added into test tubes containing modified purple broth base medium. Coliform concentrations were determined on the basis of the time of color change and gas production in the alginate tubes. All results obtained by the alginate method correlated strongly with those obtained by the conventional violet red bile agar (VRBA) plating method. The alginate method reduced detection time by 12 to 14 h compared with the conventional VRBA plating method. The alginate method can be applied in field studies more easily than melted-agar systems can. The results of this study indicate that the alginate method is an accurate, rapid, simple, and economical way to monitor and estimate concentrations of total coliforms in food.

Alginates↗

PubMed2000

Characterization of an encapsulation device for the production of monodisperse alginate beads for cell immobilization.

An encapsulation device, designed on the basis of the laminar jet break-up technique, is characterized for cell immobilization with different types of alginate. The principle of operation of the completely sterilizable encapsulator, together with techniques for the continuous production of beads from 250 microm to 1 mm in diameter, with a size distribution below 5%, at a flow rate of 1-15 mL/min, is described. A modification of the device, to incorporate an electrostatic potential between the alginate droplets and an internal electrode, results in enhanced monodispersity with no adverse effects on cell viability. The maximum cell loading capacity of the beads strongly depends on the nozzle diameter as well as the cells used. For the yeast Phaffia rhodozyma, it is possible to generate 700 microm alginate beads with an initial cell concentration of 1 x 10(8) cells/mL of alginate whereas only 1 x 10(6) cells/ml could be entrapped within 400 microm beads. The alginate beads have been characterized with respect to mechanical resistance and size distribution immediately after production and as a function of storage conditions. The beads remain stable in the presence of acetic acid, hydrochloric acid, water, basic water, and sodium ions. The latter stability applies when the ratio of sodium: calcium ions is less than 1/5. Complexing agents such as sodium citrate result in the rapid solubilization of the beads due to calcium removal. The presence of cells does not affect the mechanical resistance of the beads. Finally, the mechanical resistance of alginate beads can be doubled by treatment with 5-10 kDa chitosan, resulting in reduced leaching of cells.

Acetic Acid↗

PubMed2002

Metabolism of human articular chondrocytes cultured in alginate beads. Longterm effects of interleukin 1beta and nonsteroidal antiinflammatory drugs.

OBJECTIVES: To investigate the longterm effects (12 days) of nonsteroidal antiinflammatory drugs [NSAID: aceclofenac (ACECLO), sodium diclofenac (DICLO), indomethacin (INDO), nimesulide (NIM), rofecoxib (ROFE), celecoxib (CELE), piroxicam (PIROX), and ibuprofen (IBUP)] on the metabolism of human chondrocytes cultured in alginate beads. METHODS: Enzymatically isolated osteoarthritic (OA) chondrocytes were cultured in alginate beads in a well defined culture medium for 12 days. The DNA content was measured according to a fluorimetric method and cell proliferation was determined by the incorporation of 3H-thymidine in the newly synthesized DNA. Interleukin 6 (IL-6) and IL-8, stromelysin [matrix metalloproteinase-3 (MMP-3)], and aggrecan (AGG) production were assayed by specific enzyme amplified sensitivity immunoassays, and prostaglandin E2 (PGE2) production by specific radioimmunoassay. All NSAID were tested at the mean peak plasma concentration (Cmax) obtained after oral administration of a therapeutic dose. RESULTS: In alginate beads, chondrocytes synthesized high amounts of AGG, which were largely (98%) immobilized in the alginate matrix. A large amount (43%) of the IL-8 produced was stored in the alginate beads, whereas almost all IL-6 production (94%) was released in the culture supernatant. At the therapeutic concentration, all NSAID tested fully blocked PGE2 production. ACECLO, DICLO, INDO, NIM significantly inhibited basal and IL-1beta stimulated IL-6 production; CELE and IBUP only inhibited IL-1beta stimulated IL-6 production; and ROFE and PIROX had no significant effects. No NSAID showed significant effects on basal and IL-1beta stimulated IL-8 production, except CELE and IBUP, which slightly increased basal IL-8 production. ACECLO and INDO increased AGG content by 25% in the alginate beads, while the other NSAID were without significant effect. No NSAID were able to modify the inhibitory effect of IL-1beta on AGG production. NSAID did not modify MMP-3 production. CONCLUSION: The mechanism of action of NSAID seems to be multifactorial and not limited to the inhibition of cyclooxygenases. Further, in our culture conditions, at the Cmax and by comparison with other NSAID, ACECLO and INDO show an advantageous activity profile. They fully blocked PGE2 production, inhibited IL-6 synthesis, and increased aggrecan synthesis. These effects appear advantageous for the longterm treatment of chronic joint diseases such as osteoarthritis.

Aggrecans↗

PubMed2004

[Heterotopic chondrogenesis of human adipose tissue-derived stromal cells loading on alginate gel].

OBJECTIVE: To isolate and chondro-inductive culture of human adipose tissue-derived stromal cells and to study their heterotopic chondrogenesis by loading them on alginate gel. METHODS: Liposuction human adipose tissues were minced and digested with collagenase type I. The obtained stromal cells were primarily cultured in BGJb medium for ten days. Secondary harvested cells were cultured in DMEM-F12 medium supplemented with 10%FBS, 6.25 mg/L insulin, 10 mg/L TGF-beta1, 50 mg/L of freshly prepared L-ascorbate for 14 days. After in vitro assay of chondrogenic phenotypes, the cells at density of 10(10)/L were mixed with 1.2% alginate sodium and 102 mmol/L CaCl(2). The cross-linking cell-alginate gel were injected into four BALB/C athymic mice subcutaneously (1 ml for each mouse). Meanwhile, the auto-controls were set by injecting equal dose of simple alginate gel and pure cells in two opposite buttocks of the same mouse subcutaneously. Two mice were sacrificed at fourth and eighth week postoperatively and all samples were removed, fixed, embedded in paraffin and cut into sections of 5 micro m thick. HE staining, Alcian blue and modified Masson's trichrome staining were employed to observe chondrogenesis histologically. RESULTS: Alcian blue and immunocytochemical staining revealed chondroitin sulfate and collagen II in cell matrix after having been chondro-inductive cultured for 14 days. At intervals of fourth and eighth week, heterotopic chondrogenesis is (cartilage formed) within cell-alginate injected sites were found in all mice but negatively in auto-controls. Histologically the hypertrophic chondrocytes were among cartilage matrix in different staining. All alginate gel and solitory cells absorbed within two to three weeks postoperatively in auto-controls. CONCLUSION: It seems that stromal cells derived from human adipose tissue presents a potential for chondrogenic differentiation.

Adipose Tissue↗

PubMed1990

Comparisons of different types and concentrations of alginates for encapsulation of Lagenidium giganteum (Oomycetes: Lagenidiales), a fungal pathogen of mosquito larvae.

Six different types of alginates used to encapsulate Lagenidium giganteum gave similar levels of fungal infection in Culex quinquefasciatus larvae. Initial infection levels when the capsules were immersed in water after 6 days of storage (15 and 25 degrees C) were 100% for all types of alginate and after 42 days of storage was 62-100%, depending on the type of alginate. Infectivity was 24-100% after the encapsulated fungus were left in water for 7 days and after 15 days was 0 to 26%, depending on the alginate. When 2 of the alginates were tested at different concentrations to give high, medium and low viscosity solutions, the fungus encapsulated using lower concentration alginate solutions usually gave the highest level of infectivity.

Alginates↗

PubMed1978

[Fundamental studies of elastic impression materials (part III). Viscoelastic properties from start of mix for alginate impression materials (author's transl)].

The viscoelastic properties during setting of commercial alginate impression materials vary considerably from one product to another. In order to obtain an acculate impression, it is important to understand these differences in viscoelastic properties. The purpose of this investigation is to develop the method of measuring viscoelastic properties during setting and to determine precise setting characteristic of commercial alginate impression materials. The materials used in this investigation were mixed according to the manufacturer's recommendation at a temperature of 23 +/- 0.5 degrees C and transferred after 60 sec from start of mixing to the testing instrument in an air cabinet at 32 +/- 0.5 degrees C and allowed to set. The mixing time for materials was 30 seconds. The apparatus used in this investigation is the oscillating rheometer modifiied by Inoue and Wilson (1977). The working times of impression materials procured at retail in alginate paste were shorter than those of alginate Impression materials in powder. Alginate, Jeltrate (regular), Xantalgin (normal) and Zelgan had long working time. In case of these materials, the variation of elastic modulus during the time of working was small markedly. All other materials used in this investigation, with the exception of Alginate, indicated a considerable increase torsional shear modulus after the setting time determined by the method of British standard.

Alginates↗