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PubMed2006

Cavernous nerve regeneration by biodegradable alginate gel sponge sheet placement without sutures.

OBJECTIVES: Alginate, a biodegradable polysaccharide, may be applied to facilitate nerve regeneration. We attempted to regenerate excised cavernous nerves by filling the gap with a biodegradable alginate gel sponge sheet without sutures. METHODS: Bilateral cavernous nerves of male Wistar rats were excised to make an approximately 2-mm gap. A piece of freeze-dried alginate sheet was then placed over the gap to cover each stump without suturing (alginate group). We also performed sham operations (sham group) and bilateral nerve excision alone (excision group). RESULTS: Erection was confirmed visually by mating behavior in 9%, 36% and 73% of alginate rats at 2, 4, and 12 weeks, respectively. In contrast, no excision rats had an erection. At 12 weeks postoperatively, electrical stimulation of the pelvic plexus increased intracavernous pressure in the sham group and alginate rats with restored erectile function. However, no increase occurred in the excision group. In a retrograde neural tracing study with FluoroGold at 12 weeks, rich FluoroGold-positive cells were observed in the sham group and alginate rats with restoration, but very few were found in the excision group. Double-labeled immunochemistry with anti-S-100 and beta-tubulin III antibodies showed that the neural gap was connected with the regenerated nerve fibers at 12 weeks. CONCLUSIONS: The results of our animal study have demonstrated that by simply filling the nerve gap using an alginate sheet, the cavernous nerve can be regenerated and erectile function may be restored.

Absorbable Implants↗

PubMed1996

Four alginate dressings in the treatment of partial thickness wounds: a comparative experimental study.

Alginate-based absorption materials for wound treatment are meeting with increasing clinical acceptance. The purpose of this study was to compare four different calcium alginate dressings (Algosteril, Comfeel Alginate, Kaltostat and Sorbsan) with respect to wound fluid retaining ability, adherence, dressing residues, epithelialisation and inflammatory cell infiltration using a standardised partial-thickness wound model in domestic pigs. Wound fluid spread laterally onto surrounding normal skin by about 40% more with Sorbsan than with the other alginate dressings after 24 h of treatment (P = 0.026). The corresponding figure after 66 h of treatment was 20% (P = 0.030). Algosteril (mean 1.7 [sem 0.3]) adhered significantly (P = 0.014) more to the wounds than Comfeel Alginate (mean 0.2 [0.2]). Kaltostat (mean 1.8 [0.3]) left significantly (P = 0.038) more dressing residues on the wound surface at dressing removal than the Comfeel Alginate dressing (mean 0.8 [0.2]). In the effect on epithelialisation or dermal inflammation there was no statistically significant difference at significance level 5% among the four alginate dressings, as assessed by light microscopy. In summary, the four alginate dressings showed significant differences in important handling characteristics and did not differ significantly in their effect on epithelialisation.

Alginates↗

PubMed2001

The effects of alginate composition on encapsulated betaTC3 cells.

The effects of alginate composition on the growth of murine insulinoma betaTC3 cells encapsulated in alginate/poly-L-lysine/alginate (APA) beads, and on the overall metabolic and secretory characteristics of the encapsulated cell system, were investigated for four different types of alginate. Two of the alginates used had a high guluronic acid content (73% in guluronic acid residues) with varying molecular weight, while the other two had a high mannuronic acid content (68% in mannuronic acid residues) with varying molecular weight. Each composition was tested using two different polymer concentrations. Our data show that betaTC3 cells encapsulated in alginates with a high guluronic acid content experienced a transient hindrance in their metabolic and secretory activity because of growth inhibition. Conversely, betaTC3 cells encapsulated in alginates with a high mannuronic acid content experienced a rapid increase in metabolic and secretory activity as a result of rapid cell growth. Our data also demonstrate that an increase in either molecular weight or concentration of high mannuronic acid alginates did not alter the behavior of the encapsulated betaTC3 cells. Conversely, an increase in molecular weight and concentration of high guluronic acid alginates prolonged the hindrance of glucose metabolism, insulin secretion and cell growth. These observations can be best interpreted by changes in the microstructure of the alginate matrix, i.e., interaction between the contiguous guluronic acid residues and the Ca2+ ions, as a result of the different compositions.

Alginates↗

PubMed2001

Hexuronyl C5-epimerases in alginate and glycosaminoglycan biosynthesis.

The sugar residues in most polysaccharides are incorporated as their corresponding monomers during polymerization. Here we summarize the three known exceptions to this rule, involving the biosynthesis of alginate, and the glycosaminoglycans, heparin/heparan sulfate and dermatan sulfate. Alginate is synthesized by brown seaweeds and certain bacteria, while glycosaminoglycans are produced by most animal species. In all cases one of the incorporated sugar monomers are being C5-epimerized at the polymer level, from D-mannuronic acid to L-guluronic acid in alginate, and from D-glucuronic acid to L-iduronic acid in glycosaminoglycans. Alginate epimerization modulates the mechanical properties of seaweed tissues, whereas in bacteria it seems to serve a wide range of purposes. The conformational flexibility of iduronic acid units in glycosaminoglycans promotes apposition to, and thus functional interactions with a variety of proteins at cell surfaces and in the extracellular matrix. In the bacterium Azotobacter vinelandii the alginates are being epimerized at the cell surface or in the extracellular environment by a family of evolutionary strongly related modular type and Ca(2+)-dependent epimerases (AlgE1-7). Each of these enzymes introduces a specific distribution pattern of guluronic acid residues along the polymer chains, explaining the wide structural variability observed in alginates isolated from nature. Glycosaminoglycans are synthesized in the Golgi system, through a series of reactions that include the C5-epimerization reaction along with extensive sulfation of the polymers. The single, Ca(2+)-independent, epimerase in heparin/heparan sulfate biosynthesis and the Ca(2+)-dependent dermatan sulfate epimerase(s) also generate variable epimerization patterns, depending on other polymer-modification reactions. The alginate and heparin epimerases appear unrelated at the amino acid sequence level, and have probably evolved through independent evolutionary pathways; however, hydrophobic cluster analysis indicates limited similarity. Seaweed alginates are widely used in industry, while heparin is well established in the clinic as an anticoagulant.

Alginates↗

PubMedDate not supplied

Difference in concentration dependence of relaxation critical exponent N for alginate solutions at sol-gel transition induced by calcium cations.

The sol-gel transition in aqueous alginate solutions induced by chelation with calcium cations from in situ release has been investigated with viscoelastic methods. Two alginate samples having different molecular weights (MW) were used over the concentration C(Alg) of 2 approximately 6 wt % with different mole ratio f of Ca2+ to the alginate repeat unit. The gel point f(gel) and relaxation critical exponent n were determined according to Winter's criterion, the later agrees well with that obtained from the relaxation modulus. The results indicate that the power law is valid for the dynamic relaxation at the gel point and the critical gel possesses the self-similarity in structure. With increasing C(Alg), f(gel) for the alginate with lower MW decreases dramatically and n is almost constant of about 0.71. In contrast, f(gel) for the higher MW alginate with is almost a constant and n decreases from 0.72 then levels off at 0.37 with increasing C(Alg), indicating that the concentration dependence of n varies with MW of alginate in the starting solution. The fractal dimension d(f) estimated from n suggests a denser structure in the critical gel of higher MW alginate. Either n or d(f) has been found to follow one curve for the two samples if plotted against the number of cross-link junctions per polymer chain, which is proportional to the alginate MW.

Alginates↗

PubMed1990

The use of 13C-n.m.r. spectroscopy to monitor alginate biosynthesis in mucoid Pseudomonas aeruginosa.

The biosynthesis of alginate by a mucoid strain of Pseudomonas aeruginosa, isolated from a cystic-fibrosis patient, was monitored by using 13C-n.m.r. spectroscopy of bacterial cultures incubated with 1-13C- or 2-13C-enriched fructose. When 1-13C- or 2-13C-enriched fructose was used as the precursor of alginate, enrichment with 13C in the constituent uronic acid monomers of the polysaccharide could only be detected in C-1 or C-2 respectively, indicating that alginate is synthesized in Ps. aeruginosa directly from fructose, with the hexose molecule being retained intact; this rules out the involvement of C3 intermediates, which occurs when glucose is the alginate precursor. The absence of detectable poly-L-gluluronate block sequences from the alginate of Ps. aeruginosa was confirmed, and it was shown that there is no modification of the arrangement of the constituent uronic acids between polymerization to form alginate and the appearance of the mature alginate in the extracellular medium. The 13C-n.m.r. data also provided independent evidence for acetylation on D-mannuronate residues and for the ratio of D-mannuronate to L-guluronate residues in newly synthesized alginate, which had previously been determined only for material secreted from bacteria into the extracellular medium.

Alginates↗

PubMed2002

Transforming growth factor-beta in calcium alginate beads for the treatment of articular cartilage defects in the rabbit.

PURPOSE: Articular cartilage has only limited capability for intrinsic repair. The use of growth factors has been suggested to improve the repair of cartilage after injury. Reliable delivery systems for these agents are needed. In this study we tested calcium alginate for the delivery of TGF-beta in the treatment of osteochondral defects in the rabbit knee. TYPE OF STUDY: Randomized trial animal study and basic science study. METHODS: In vitro, to establish the kinetics of TGF-beta release from the alginate, 125I- labeled TGF-beta was suspended in 1.2% sodium alginate at concentrations of 1 microg/mL and 10 microg/mL. Beads were formed from 50 microL aliquots and placed into standard culture medium by immersion in calcium chloride solution and incubated at 37 degrees C. A gamma counter was used to measure the amount of TGF-beta that was released into the medium at various time points. In vivo, osteochondral defects were created in the trochlear grooves of 32 New Zealand White rabbits. Defects were treated with plain alginate or with alginate containing TGF-beta at 20 ng/mL or 2,000 ng/mL. Untreated defects served as a control. Animals were killed after 6 and 12 weeks. Knee joints were evaluated grossly with a 12-point grading scale. Histologic sections of the repair tissue were stained with Safranin O and evaluated using a 24-point grading scale by 2 independent blinded observers. Mean scores and standard deviations were calculated. P values were determined using the Student t test. RESULTS: The TGF-beta was released at a surprisingly slow but steady rate. Release rates extrapolated from the gamma counter measurements were 0.25% per hour and 0.33% per hour, for the 1 microg/mL and 10 microg/mL beads, respectively. Gross analysis scores at 6 and 12 weeks resulted in higher scores for both TGF-beta groups without reaching statistical significance. The lower TGF-beta concentration reached the highest scores, whereas the higher concentration (2,000 ng/mL) resulted in increased osteophyte formation. Histologic analysis at 6 weeks resulted in average scores ranging from 14.5 for empty defects and 18.1 for alginate-treated defects, to 20.0 and 20.3 for the 2,000 ng/mL and 20 ng/mL TGF-beta groups, respectively (P <.05). At 12 weeks, histologic scores ranged from 14.9 for empty and 14.5 for alginate to 20.1 and 20.5 for the 2,000 ng/mL and 20 ng/mL TGF-beta groups, respectively (P <.05). These results indicate a significant improvement of the quality of the repair tissue at 6 and 12 weeks with TFG-beta treatment, especially at the lower concentration. CONCLUSIONS: The use of alginate allows the controlled delivery of TGF-beta selectively to the site of injury, potentially avoiding systemic side effects. Furthermore, treatment with TGF-beta appears to improve the repair of articular cartilage defects. Longer-term studies are needed to assess whether the benefits of the TGF-beta treatment can be sustained.

Alginates↗

PubMed2003

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques.

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged inmonolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.

Alginates↗

PubMed2001

Preparation and physical characterization of alginate microparticles using air atomization method.

Alginate microparticles were prepared using an air atomization method and varying formulation and processing conditions. Thereafter, the size and surface morphology of alginate microparticles were characterized. The trapping efficiencies of the ketoconazole, acetaminophen, vitamin C, and Bifidobacteria bifidum as model core materials were then determined. The air atomization process produced free-flowing and small-size microparticles after the freeze-drying process. The size distribution and surface morphology varied depending on the concentration of wall-forming materials and processing conditions. Generally, the geometric mean size increased as the concentration of alginate and poly-1-lysine and the delivery rate increased, but the air pressure decreased. Most of all, the ratio of delivery rate of alginate solution and air pressure could affect the size and surface morphology of alginate microparticles. However, the geometric mean size of alginate poly-1-lysine microparticles reproducibly ranged from about 80 to 130 microm. The microparticles were irregularly spherical or elliptical. The trapping efficiencies of ketoconazole, acetaminophen, vitamin C, and bifidobacteria were determined to be 71.5%, 60.1%, 1.6%, and 31%, respectively, when alginate concentration (1.5%), poly-1-lysine concentration (0.02%), air pressure (0.75 bar), delivery rate (8 ml/min), and spraying distance (45 cm) were applied. The current microencapsulation process using the air atomization method provides an alternative to entrapping small molecules and macromolecules without using harmful organic solvents. In addition, the small-size and free-flowing alginate microparticles containing active substances can be used as an intermediate in pharmaceutical applications.

Acetaminophen↗

PubMed2003

Chitosan-alginate multilayer beads for gastric passage and controlled intestinal release of protein.

Chitosan-alginate beads loaded with a model protein, bovine serum albumin (BSA) were investigated to explore the temporary protection of protein against acidic and enzymatic degradation during gastric passage. Optimum conditions were established for preparation of homogenous, spherical, and smooth chitosan-alginate beads loaded with BSA. Multilayer beads were prepared by additional treatment with either chitosan or alginate or both. The presence of chitosan in the coagulation bath during bead preparation resulted in increased entrapment of BSA. During incubation in simulated gastric fluid (SGF pH 1.2), the beads showed swelling and started to float but did not show any sign of erosion. Inclusion of pepsin in the gastric fluid did not show a further effect on the properties of the beads. Release studies were done in simulated gastric fluid (SGF pH 1.2) and subsequently in simulated intestinal fluid (SIF pH 7.5) to mimic the physiological gastrointestinal conditions. After transfer to intestinal fluid, the beads were found to erode, burst, and release the protein. Microscopic and macroscopic observations confirmed that the release of protein was brought about by the burst of beads. Chitosan-reinforced calcium-alginate beads showed delay in the release of BSA. The multilayer beads disintegrated very slowly. The enzymes pepsin and pancreatin did not change the characteristics of BSA-loaded chitosan-alginate beads. Single layer chitosan-alginate beads released 80-90% of the model protein within 12h while multilayer beads released only 40-50% in the same period of time. The release from chitosan-alginate beads and multilayer beads in SIF was further delayed without prior incubation in SGF. It is concluded that alginate beads reinforced with chitosan offer an excellent perspective for controlled gastrointestinal passage of protein drugs.

Alginates↗

PubMed2004

Oral controlled release formulation for highly water-soluble drugs: drug--sodium alginate--xanthan gum--zinc acetate matrix.

An oral controlled release formulation matrix for highly water-soluble drugs was designed and developed to achieve a 24-hour release profile. Using ranitidine HCl as a model drug, sodium alginate formulation matrices containing xanthan gum or zinc acetate or both were investigated. The caplets for these formulations were prepared by direct compression and the in vitro release tests were carried out in simulated intestinal fluid (SIF, pH 7.5) and simulated gastric fluid (SGF, pH 1.2). The release of the drug in the sodium alginate formulation containing only xanthan gum completed within 12 hours in the SIF, while the drug release in the sodium alginate formulation containing only zinc acetate finished almost within 2 hours in the same medium. Only the sodium alginate formulation containing both xanthan gum and zinc acetate achieved a 24-hour release profile, either in the SIF or in the pH change medium. In the latter case, the caplet released in the SGF for 2 hours was immediately transferred into the SIF to continue the release test. The results showed that the presence of both xanthan gum and zinc acetate in sodium alginate matrix played a key role in controlling the drug release for 24 hours. The helical structure and high viscosity of xanthan gum might prevent zinc ions from diffusing out of the ranitidine HCl--sodium alginate--xanthan gum--zinc acetate matrix so that zinc ions could react with sodium alginate to form zinc alginate precipitate with a cross-linking structure. The cross-linking structure might control a highly water-soluble drug to release for 24 hours. Evaluation of the release data showed the release mechanism for the novel formulation might be attributed to the diffusion of the drug.

Administration, Oral↗

PubMed2001

Intervertebral disc cells exhibit differences in gene expression in alginate and monolayer culture.

STUDY DESIGN: The mRNA levels of aggrecan and collagen were quantified in intervertebral disc cells cultured under three conditions: primary alginate culture, monolayer culture, and re-encapsulation in alginate after monolayer culture. OBJECTIVES: To compare the phenotype of intervertebral disc cells under different culture conditions and to investigate the reversibility of cell phenotype after re-encapsulation in alginate after monolayer culture. SUMMARY OF BACKGROUND DATA: The intervertebral disc contains heterogeneous populations of cells that vary with anatomic region. These cells possess significant differences in phenotype that can be preserved in vitro, although the effect of culture conditions on the phenotype of these cells is poorly understood. METHODS: The intervertebral disc cells of 4-5-month-old pigs were isolated enzymatically from three anatomic zones: anulus fibrosus (AF), transition zone (TZ), and nucleus pulposus (NP). Gene expression levels of aggrecan and collagen Types I and II were measured using a quantitative reverse transcriptase--polymerase chain reaction. RESULTS: Gene expression levels of anulus fibrosus and transition zone cells were shifted in monolayer compared with alginate, although the shift was partially reversed when re-encapsulated in alginate. However, NP cells appeared to be insensitive to culture conditions. Furthermore, characteristic patterns of gene expression among AF, TZ, and NP cells in primary alginate culture did not exist in monolayer culture, but they were also observed after re-encapsulation in alginate. CONCLUSION: The findings of this study suggest that anulus fibrosus and transition zone cells undergo a reversible shift in phenotype when cultured in monolayer compared with alginate. These differences suggest that the culture system exerts a strong influence on cell phenotype and may play a role in the response of these cells to biophysical and biochemical stimuli in vitro.

Aggrecans↗

PubMed1992

Effects of growth temperature on alginate synthesis and enzymes in Pseudomonas aeruginosa variants.

Spontaneous variation of the level of alginate synthesis in Pseudomonas aeruginosa was associated with changes in the activity of all four enzymes leading to synthesis of GDP-mannuronic acid, the activated precursor for polymerization. For the high-alginate-producing variant 8821M, alginate yield and properties, as well as the levels of alginate enzymes, were dependent on growth temperature. In contrast, levels of alginate and enzymes in the mucoid parent strain 8821 were very low and near temperature-independent. The difference in the specific activity of GDP-mannose dehydrogenase (GMD), encoded by the algD gene, between the two strains was associated with the alginate biosynthetic ability and with the degree of activation of the algD promoter, measured using the algD-xylE transcription fusion on plasmid pVD2X. Maximal activity of the four enzymes was observed in strain 8821M grown at 30 degrees C, a temperature below the optimum for growth (35 degrees C). The effect of temperature on GMD activity could not be explained by the regulation of the algD promoter by temperature, since expression of pVDZX appeared to be more active at 35 degrees C, when the decrease of pVD2X copy number with increasing temperature was taken into account. The involvement of enzymes that catalyse steps downstream from the formation of the activated precursor should also be considered, as suggested by differences in the molecular mass of alginates synthesized by the two strains at various temperatures. Acetyl content of alginates increased as temperature decreased and strain 8821M produced the highest levels of acetylated polymers. The degree of acetylation appeared to be related to growth rate and could reflect acetyl-CoA availability.

Acetylation↗

PubMed1994

GDP-mannose dehydrogenase is the key regulatory enzyme in alginate biosynthesis in Pseudomonas aeruginosa: evidence from metabolite studies.

The Pseudomonas aeruginosa enzyme GDP-mannose dehydrogenase (GMD) is encoded by the algD gene, and previous genetic studies have indicated that it is a key regulatory and committal step in the biosynthesis of the polysaccharide alginate. In the present study the algD gene has been cloned into the broad-host-range expression vector pMMB66EH and GMD overexpressed in mucoid and genetically-related non-mucoid strains of P. aeruginosa. The metabolic approach of P. J. Tatnell, N. J. Russell & P. Gacesa (1993), J Gen Microbiol 139, 119-127, has been used to investigate the subsequent effect of GMD overexpression on the intracellular concentrations of the key metabolites GDP-mannose and GDP-mannuronate, which have been related to GMD activity and total alginate production. The overexpression of algD in mucoid and non-mucoid strains resulted in elevated GMD activities compared to wild-type strains; there was a concomitant reduction in GDP-mannose concentrations and greatly increased GDP-mannuronate concentrations. However, significantly, alginate biosynthesis was detected only in mucoid strains and GMD overexpression resulted in only a marginal increase in exopolysaccharide production. The GDP-mannuronate concentrations in mucoid strains which overexpressed GMD were always significantly greater than those of GDP-mannose, indicating that GMD was no longer the major kinetic control point in the biosynthesis of alginate by these genetically-manipulated strains. The small but significant increase in alginate production by such strains together with the increased GDP-mannuronate concentrations is interpreted as meaning that a later enzyme of the alginate pathway has become the major kinetic control point and now determines the extent of alginate production. This study has provided direct metabolic evidence that GMD is the key regulatory enzyme in alginate biosynthesis in P. aeruginosa.

Alginates↗

PubMed1987

Construction and characterization of Pseudomonas aeruginosa algB mutants: role of algB in high-level production of alginate.

The algB gene, which is involved in the production of alginate in Pseudomonas aeruginosa, was localized to approximately 2.2 kilobases of DNA from strain FRD by using transposon Tn501 insertion mutagenesis, subcloning, and complementation techniques. The previously reported alg-50(Ts) mutation, which confers the phenotype of temperature-sensitive alginate production, was here designated as an algB allele. A transduction-mediated gene replacement technique was used for site-directed mutagenesis to isolate and characterize algB::Tn501 mutants of P. aeruginosa FRD. Although algB::Tn501 mutants had a nonmucoid phenotype (indicating an alginate deficiency), they still produced about 1 to 5% of wild-type levels of alginate in most growth media and up to 16% in very rich media. The algB::Tn501 mutations had no apparent effect on growth rate or growth requirements. Using another gene replacement technique called excision marker rescue, we constructed a chromosomal algB deletion (delta algB) mutant of P. aeruginosa FRD. The delta algB mutant also produced low levels of alginate as did the algB::Tn501 mutants. The alginate produced by algB::Tn501 mutants resembled wild-type alginate by all criteria studied: molecular weight, acetylation, and proportion of mannuronic and guluronic acids. Thus, the algB gene product is apparently involved in the high-level production of alginate by P. aeruginosa and is not directly involved in the pathway leading to its biosynthesis. Chromosomal mapping of an algB::Tn501 insertion showed linkage to the trp-2 marker on the FRD chromosome as does the algB50(Ts) mutation. The excision marker rescue technique was also used to place the algB::Tn501 marker on the chromosome of characterized strains of P. aeruginosa PAO. The algB::Tn501 mutation mapped near 21 min on the PAO chromosome.

Alginates↗

PubMed1997

Posttranslational control of the algT (algU)-encoded sigma22 for expression of the alginate regulon in Pseudomonas aeruginosa and localization of its antagonist proteins MucA and MucB (AlgN).

Pseudomonas aeruginosa strains associated with cystic fibrosis are often mucoid due to the copious production of alginate, an exopolysaccharide and virulence factor. Alginate gene expression is transcriptionally controlled by a gene cluster at 68 min on the chromosome: algT (algU)-mucA-mucB (algN)-mucC (algM)-mucD (algY). The algT gene encodes a 22-kDa alternative sigma factor (sigma22) that autoregulates its own promoter (PalgT) as well as the promoters of algR, algB, and algD. The other genes in the algT cluster appear to regulate the expression or activity of sigma22. The goal of this study was to better understand the functional interactions between sigma22 and its antagonist regulators during alginate production. Nonmucoid strain PAO1 was made to overproduce alginate (indicating high algD promoter activity) through increasing sigma22 in the cell by introducing a plasmid clone containing algT from mucA22(Def) strain FRD1. However, the bacterial cells remained nonmucoid if the transcriptionally coupled mucB on the clone remained intact. This suggested that a stoichiometric relationship between sigma22 and MucB may be required to control sigma factor activity. When the transcription and translational initiation of algT were measured with lacZ fusions, alginate production correlated with only about a 1.2- to 1.7-fold increase in algT-lacZ activity, respectively. An algR-lacZ transcriptional fusion showed a 2.8-fold increase in transcription with alginate production under the same conditions. A Western blot analysis of total cell extracts showed that sigma22 was approximately 10-fold higher in strains that overproduced alginate, even though algT expression increased less than 2-fold. This suggested that a post-transcriptional mechanism may exist to destabilize sigma22 in order to control certain sigma22-dependent promoters like algD. By Western blotting and phoA fusion analyses, the MucB antagonist of sigma22 was found to localize to the periplasm of the cell. Similar experiments suggest that MucA localizes to the inner membrane via one transmembrane domain with amino- and carboxy-terminal domains in the cytoplasm and periplasm, respectively. These data were used to propose a model in which MucB-MucA-sigma22 interact via an inner membrane complex that controls the stability of sigma22 protein in order to control alginate biosynthesis.

Alginates↗

PubMed1998

Deletion of algK in mucoid Pseudomonas aeruginosa blocks alginate polymer formation and results in uronic acid secretion.

Chronic pulmonary infection with Pseudomonas aeruginosa is a common and serious problem in patients with cystic fibrosis (CF). The P. aeruginosa isolates from these patients typically have a mucoid colony morphology due to overproduction of the exopolysaccharide alginate, which contributes to the persistence of the organisms in the CF lung. Most of the alginate biosynthetic genes are clustered in the algD operon, located at 34 min on the chromosome. Alginate biosynthesis begins with the formation of an activated monomer, GDP-mannuronate, which is known to occur via the products of the algA, algC, and algD genes. Polymannuronate forms in the periplasm, but the gene products involved in mannuronate translocation across the inner membrane and its polymerization are not known. One locus of the operon which remained uncharacterized was a new gene called algK between alg44 and algE. We sequenced algK from the mucoid CF isolate FRD1 and expressed it in Escherichia coli, which revealed a polypeptide of the predicted size (52 kDa). The sequence of AlgK showed an apparent signal peptide characteristic of a lipoprotein. AlgK-PhoA fusion proteins were constructed and shown to be active, indicating that AlgK has a periplasmic subcellular localization. To test the phenotype of an AlgK-mutant, the algK coding sequence was replaced with a nonpolar gentamicin resistance cassette to avoid polar effects on genes downstream of algK that are essential for polymer formation. The algKdelta mutant was nonmucoid, demonstrating that AlgK was required for alginate production. Also, AlgK- mutants demonstrated a small-colony phenotype on L agar, suggesting that the loss of AlgK also caused a growth defect. The mutant phenotypes were complemented by a plasmid expressing algK in trans. When the algKdelta mutation was placed in an algJ::Tn501 background, where algA was not expressed due to polar transposon effects, the growth defect was not observed. AlgK- mutants appeared to accumulate a toxic extracellular product, and we hypothesized that this could be an unpolymerized alginate precursor. High levels of low-molecular-weight uronic acid were produced by the AlgK- mutant. When AlgK- culture supernatants were subjected to dialysis, high levels of uronic acids diffused out of the dialysis sac, and no uronic acids were detectable after extensive dialysis. In contrast, the mucoid wild-type strain produced only polymerized uronic acids (i.e., alginate), whereas the algKdelta algJ::Tn501 mutant produced no uronic acids. Thus, the alginate pathway in an AlgK- mutant was blocked after transport but at a step before polymerization, suggesting that AlgK plays an important role in the polymerization of mannuronate to alginate.

Alginates↗

PubMed1996

Alginate, the slime exopolysaccharide of Pseudomonas aeruginosa, binds human leukocyte elastase, retards inhibition by alpha 1-proteinase inhibitor, and accelerates inhibition by secretory leukoprotease inhibitor.

The interaction of alginate from Pseudomonas aeruginosa ATCC 39324 with human leukocyte elastase was studied by determining the effects of the polysaccharide on the amidolytic activity of the enzyme toward a range of synthetic peptide substrates of different length. The data support a model in which each elastase molecule interacts with a total of 19 uronic acid units on the alginate, primarily through electrostatic forces. Binding of alginate results in occlusion of distal subsites, most likely S4 and S5, of the enzyme's extended substrate-binding domain. As a result, alginate alone appears to be a weak inhibitor of the hydrolysis of long synthetic peptide substrates and [14C]elastin by elastase. Alginate also has effects on the antielastase function of naturally occurring protease inhibitors in the lung: It reduces the association rate of elastase and alpha 1-proteinase inhibitor, whereas it increases the association rate of elastase and secretory leukoprotease inhibitor. In the presence of 36 micrograms/ml alginate, the median concentration found in sputum from cystic fibrosis patients infected with mucoid strains of P. aeruginosa, the second-order rate constant for inhibition of elastase by secretory leukoprotease inhibitor is 2.6-fold greater than that for alpha 1-proteinase inhibitor. Alginate has only a minor effect on the antielastase activities of elafin and a recombinant form of the isolated C-terminal domain of secretory leukoprotease inhibitor. Based on these findings, alginate may be an important factor in determining the local distribution of leukocyte elastase and perturbing the overall protease-antiprotease balance in the infected lungs of cystic fibrosis patients.

Alginates↗