Abstract  Moist wounds were known to heal more rapidly than dry wounds. Hydrogel wound dressings were suitable for the moist wound healing because of their hyperhydrous structure. Chitosan was a strong candidate as a base material for hydrogel wound dressings because the polymer had excellent biological properties that promoted wound healing. We previously developed physically-crosslinked chitosan cryogels, which were prepared solely by freeze-thawing of a chitosan-gluconic acid conjugate (CG) aqueous solution, for wound treatment. The CG cryogels were disinfected by immersing in 70% ethanol before applying to wounds in our previous study. In the present study, we examined the influence of autoclave sterilization (121°C, 20 min) on the characteristics of CG cryogel because complete sterilization was one of the fundamental requirements for medical devices. We found that optimum value of gluconic acid content of CG, defined as the number of the incorporated gluconic acid units per 100 glucosamine units of chitosan, was 11 for autoclaving. An increased crosslinking level of CG cryogel on autoclaving enhanced resistance of the gels to enzymatic degradation. Furthermore, the autoclaved CG cryogels retained favorable biological properties of the pre-autoclaved CG cryogels in that they showed the same hemostatic activity and efficacy in repairing full-thickness skin wounds as the pre-autoclaved CG cryogels. These results showed the great potential of autoclavable CG cryogels as a practical wound dressing.

Abstract  Heinen et al reported the use of [U-13C6]gluconolactone to trace the PPP in microorganisms (Appl. Environ. Microbiology 07/2006, P 4743). We adapted this technique for use in perfused hearts and livers from rats. The tissue was extracted in acetonitrile:water (50:50) and the supernatant applied to an ion exchange solid phase extraction cartridge. The sugar phosphates were eluted with 50 mM ammonium formate in 50% methanol-water (recovery was 90%). We used a hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems) coupled with HPLC to monitor the sugar phosphates using the enhanced resolution mode. We used hydrophilic interaction liquid chromatography to avoid ion pairing reagents and to increase the detection sensitivity. The flux through the PPP is calculated as (uptake of [U-13C6]gluconolactone)/(M6 enrichment of 6-P-gluconate). The latter can be used in tracer amounts because of low background of analytes at M6 and M5. The production of NADPH in the PPP is equal to twice its flux. The contribution of the oxidative branch of the PPP is the enrichment ratio (M5 ribose-5-P)/(M6 6-P-gluconate). Supported by NIH Roadmap grant 5 R33 DK070291.

Abstract  The aim of this trial was to assess the effect that calcium gluconate priming of 468 broilers has on the antibacterial activity of a standard dose of enrofloxacin. Hence, a series of oral pharmacokinetic studies were carried out in four groups of broilers medicated individually through an oral cannula as follows: group A, medicated only with enrofloxacin 10mg/kg; group B, receiving immediately one after the other, calcium gluconate (200mg/kg) and enrofloxacin 10mg/kg; group C, dosed first with calcium gluconate (200mg/kg) and 1h later enrofloxacin (10mg/kg); and group D, dosed first with calcium gluconate (200mg/kg) and 2h later enrofloxacin (10mg/kg). Broilers were bled at different times after the dose of enrofloxacin and antibacterial activity, measured as concentration of enrofloxacin, was measured by an agar diffusion assay. Results revealed that group D the greatest values of maximum serum concentration (Cs(max)), area under the concentration vs. time curve (AUC) and area under the moment curve (AUMC). These values were statistically higher than the corresponding ones derived from groups A, B and C (P<0.05). Taking Cs(max) and AUC values of group A as reference baseline, an increase of 24% and 50%, respectively, was obtained in group D. Group B had the lowest Cs(max), AUC, AUMC and elimination half life (T(1/2)beta) and these values were statistically different from groups A, C and D (P<0.05). The T(1/2)beta was statistically longer in groups C and D as compared with A and B, and the former groups were also different between each other (P<0.05). These results show that if calcium gluconate is first dosed to broilers and 2h later enrofloxacin is administered (as in group D), a more pronounced antibacterial activity of enrofloxacin can be obtained. A challenge of this sequential dosing scheme in a field trial may reveal its clinical value.

Abstract  The microbial oxidative cellulose degradation system is attracting significant research attention after the recent discovery of lytic polysaccharide mono-oxygenases. A primary product of the oxidative and hydrolytic cellulose degradation system is cellobionic acid (CbA), the aldonic acid form of cellobiose. We previously demonstrated that the intracellular enzyme belonging to glycoside hydrolase family 94 from cellulolytic fungus and bacterium is cellobionic acid phosphorylase (CBAP), which catalyzes reversible phosphorolysis of CbA into glucose 1-phosphate and gluconic acid (GlcA). In this report, we describe the biochemical characterization and the three-dimensional structure of CBAP from the marine cellulolytic bacterium Saccharophagus degradans. Structures of ligand-free and complex forms with CbA, GlcA, and a synthetic disaccharide product from glucuronic acid were determined at resolutions of up to 1.6 Å. The active site is located near the dimer interface. At subsite +1, the carboxylate group of GlcA and CbA is recognized by Arg-609 and Lys-613. Additionally, one residue from the neighboring protomer (Gln-190) is involved in the carboxylate recognition of GlcA. A mutational analysis indicated that these residues are critical for the binding and catalysis of the aldonic and uronic acid acceptors GlcA and glucuronic acid. Structural and sequence comparisons with other glycoside hydrolase family 94 phosphorylases revealed that CBAPs have a unique subsite +1 with a distinct amino acid residue conservation pattern at this site. This study provides molecular insight into the energetically efficient metabolic pathway of oxidized sugars that links the oxidative cellulolytic pathway to the glycolytic and pentose phosphate pathways in cellulolytic microbes.

Abstract  The influence of substrate composition on the yield, nature, and composition of exopolysaccharides (EPS) produced by the food-grade strain Gluconacetobacter xylinus I-2281 was investigated during controlled cultivations on mixed substrates containing acetate and either glucose, sucrose, or fructose. Enzymatic activity analysis and acid hydrolysis revealed that two EPS, gluconacetan and levan, were produced by G. xylinus. In contrast to other acetic acid strains, no exocellulose formation has been measured. Considerable differences in metabolite yields have been observed with regard to the carbohydrate source. It was shown that glucose was inadequate for EPS production since most of this substrate (0.84 C-mol/C-mol) was oxidized into gluconic acid, 2-ketogluconic acid, and 5-ketogluconic acid. In contrast, sucrose and fructose supported a 0.35 C-mol/C-mol gluconacetan yield. In addition, growing G. xylinus on sucrose produced a 0.07 C-mol/C-mol levan yield. The composition of EPS remained unchanged during the course of the fermentations. Levan sucrase activity was found to be mainly membrane associated. In addition to levan production, an analysis of levan sucrase's activity also explained the formation of glucose oxides during fermentation on sucrose through the release of glucose. The biosynthetic pathway of gluconacetan synthesis has also been explored. Although the activity of key enzymes showed large differences to be a function of the carbon source, the ratio of their activities remained similar from one carbon source to another and corresponded to the ratio of precursor needs as deduced from the gluconacetan composition.

Abstract  Powdered sodium erythorbate (SE) and a 3:1 mixture of glucono-delta-lactone and sodium erythorbate (GDL:SE) were coated electrostatically onto the surface of meats to extend the shelf life. The total number of microorganisms after refrigerated storage was reduced an average of 2 logs, with little difference by type of powder. Coliforms, mesophiles and psychrotrophs showed equivalent reductions. The color, as measured by the a value, was better for the treated samples than the control. GDL:SE samples were redder than SE. Electrostatic coating produced better results than nonelectrostatic coating due to the increased transfer efficiency of the process. Electrostatic coating also reduced the dust that is produced when powder is coated nonelectrostatically. (c) 2005 Elsevier Ltd. All rights reserved.

Abstract  Maintenance of physiological levels of intracellular and extracellular calcium is essential for life. Increased intracellular calcium levels are involved in cell death (apoptosis and necrosis) and are associated with positive responses in the Comet assay in vitro. In addition, high calcium and vitamin D intakes were reported to induce apoptosis in adipose tissue in obese mice and to increase DNA-migration in the Comet assay. To investigate increased serum concentration of calcium as a potential confounding factor in the regulatory Comet assay in vivo, we induced mild hypercalcemia in male Wistar rats by 3-day continuous intravenous infusion of calcium gluconate and performed the Comet assay in the liver in line with regulatory guidelines. The results of the study showed that mild increases in serum calcium concentration (up to 1.4 times above the concurrent control) and increased urinary calcium concentration (up to 27.8 times above the concurrent control) results in clinical signs like mild tremor, faster respiration rate and decreased activity in a few animals. However, under the conditions of the study, no increase in the %Tail DNA in the Comet assay and no indication of liver damage as determined by histopathological means were observed. Thus, mild increases in plasma calcium did not lead to positive results in a genotoxicity assessment by the Comet assay in the rat liver. This result is important as it confirms the reliability of this assay for regulatory evaluation of safety.

Abstract  BACKGROUND: Peripheral blood progenitor cell (PBPC) collection has become the main source of hematopoietic cells for high-dose chemotherapy with stem cell rescue and, in some protocols, for allogeneic hematopoietic transplantation. This procedure is complicated in the smallest children because of difficulties related to their weight, and there is little published experience. We have conducted a prospective study to analyze the incidence of adverse events during PBPC collection in the smallest children (< or = 10 kg).

METHODS: From January 2000 to November 2005, 257 leukapheresis were performed in our unit, and 13 of them (5%) in 12 children weighing up to 10 kg (median 9 kg, range 5.8-10.9 kg).

RESULTS: Most cases had hypovolemic signs during the procedure (usually tachycardia); six cases had hypotension, five of them with pallor and diaphoresis, and, of those, two also had nausea. In all these cases infusion of saline or plasma volume expanders resolved the clinical findings. In two cases the nausea related to hypocalcemia was resolved after calcium gluconate infusion. Changes in platelet counts were also remarkable, with a median platelet loss of 52%.

DISCUSSION: Leukapheresis with continuous-flow cell separators has frequent complications related to volume shift in the smallest children. These adverse events are mild and easily resolved with standard measures for hypovolemia, as plasma expander or normal saline infusions. However, we recommend that the procedure should only be performed by teams with extensive experience in the field.

Abstract  The most abundant plasma protein, human serum albumin (HSA), is known to undergo several conformational transitions in an acidic environment. To avoid buffer effects and correlate global and local structural changes, we developed a continuous acidification method and simultaneously monitored the protein changes by both small-angle scattering (SAXS) and fluorescence. The progressive acidification, based on the hydrolysis of glucono-δ-lactone from pH 7 to pH 2.5, highlighted a multistep unfolding involving the putative F form (pH 4) and an extended and flexible conformation (pH < 3.5). The scattering profile of the F form was extracted by component analysis and further 3D modeled. The effect of acid unfolding at this intermediate stage was assigned to the rearrangement of the three albumin domains drifting apart toward a more elongated conformation, with a partial unfolding of one of the outer domains. To test the stabilizing effect of fatty acids, here palmitic acid, we compared the acid unfolding process of albumin with and without ligand. We found that when binding the ligand, the native conformation was favored up to lower pH values. Our approach solved the problem of realizing a continuous, homogeneous, and tunable acidification with simultaneous characterization applicable to study processes triggered by a pH decrease.

Abstract  Cofactor F420, a 5-deazaflavin involved in obligatory hydride transfer, is widely distributed among archaeal methanogens and actinomycetes. Owing to the low redox potential of the cofactor, F420-dependent enzymes play a pivotal role in central catabolic pathways and xenobiotic degradation processes in these organisms. A physiologically essential deazaflavoenzyme is the F420-dependent glucose-6-phosphate dehydrogenase (FGD), which catalyzes the reaction F420 + glucose-6-phosphate → F420H2 + 6-phospho-gluconolactone. Thereby, FGDs generate the reduced F420 cofactor required for numerous F420H2-dependent reductases, involved e.g., in the bioreductive activation of the antitubercular prodrugs pretomanid and delamanid. We report here the identification, production, and characterization of three FGDs from Rhodococcus jostii RHA1 (Rh-FGDs), being the first experimental evidence of F420-dependent enzymes in this bacterium. The crystal structure of Rh-FGD1 has also been determined at 1.5 Å resolution, showing a high similarity with FGD from Mycobacterium tuberculosis (Mtb) (Mtb-FGD1). The cofactor-binding pocket and active-site catalytic residues are largely conserved in Rh-FGD1 compared with Mtb-FGD1, except for an extremely flexible insertion region capping the active site at the C-terminal end of the TIM-barrel, which also markedly differs from other structurally related proteins. The role of the three positively charged residues (Lys197, Lys258, and Arg282) constituting the binding site of the substrate phosphate moiety was experimentally corroborated by means of mutagenesis study. The biochemical and structural data presented here provide the first step towards tailoring Rh-FGD1 into a more economical biocatalyst, e.g., an F420-dependent glucose dehydrogenase that requires a cheaper cosubstrate and can better match the demands for the growing applications of F420H2-dependent reductases in industry and bioremediation.

Abstract  NAD(P)-dependent glucose-1-dehydrogenase (GDH) has been used for glucose determination and NAD(P)H production in bioreactors. Thermostable glucose dehydrogenase exhibits potential advantage for its application in biological processes. The function of the putative GDH gene (ST1704, 360-encoding amino acids) annotated from the total genome analysis of a thermoacidophilic archeaon Sulfolobus tokodaii strain 7 was investigated to develop more effective application of GDH. The gene encoding S. tokodaii GDH was cloned and the activity was expressed in Escherichia coli, which did not originally possess GDH. This shows that the gene (ST1704) codes the sequence of GDH. The enzyme was effectively purified from the recombinant E. coli with three steps containing a heat treatment and two successive chromatographies. The native enzyme (molecular mass: 160 kDa) is composed of a tetrameric structure with a type of subunit (41 kDa). The enzyme utilized both NAD and NADP as the coenzyme. The maximum activity for glucose oxidation in the presence of NAD was observed around pH 9 and 75 degreesC in the presence of 20 mM Mg2+. The enzyme showed broad substrate specificity: several monosaccarides such as 6-deoxy-D-glucose, 2-amino-2-deoxy-D-glucose and D-Xylose were oxidized as well as D-glucose as the electron donor. D-Mannose, D-ribose and glucose-6-phosphate were inert as the donor. The enzyme showed high thermostability: remarkable loss of activity was not observed up to 80 degreesC by incubation for 15 min at pH 8.0. In addition, the enzyme was stable in a wide pH range of 5.0-10.5 by incubation at 37 degreesC. From the steady-state kinetic analysis, the enzyme reaction of D-glucose oxidation proceeds via a sequential ordered Bi-Bi mechanism: NAD and D-glucose bind to the enzyme in this order and then D-glucono-1,5-lactone and NADH are released from the enzyme in this order. The amino acid sequence alignment showed that S. tokodaii GDH exhibited high homology with the Sulfolobus solfataricus hypothetical glucose dehydrogenase and a Thermoplasma acidophilum one. (C) 2003 Elsevier B.V. All rights reserved.

Abstract  Background: Primary hypoparathyroidism (PH) is an endocrine disorder characterized by decreased production and/or release of parathyroid hormone (PTH). Dogs and cats are rarely affected, and typical clinical signs include an abrupt onset of neurological and neuromuscular signs. The diagnosis is based on the history, clinical signs, and laboratory findings of hypocalcaemia, as well as the exclusion of other causes of tetany. Treatment involves stabilization of serum calcium with specific therapy. This study aimed to report three cases of PH: two canine cases and the first feline case in Brazil.

Cases: 1) A 8-year-old male Yorkshire terrier was brought to the clinic with a history of tetanic crisis. The owner reported that the animal had been previously diagnosed with epilepsy and since treated with phenobarbital. The physical examination revealed hyperthermia, tachypnea, tachycardia, salivation, and ataxia with hyperextension of the anterior and posterior limbs. Emergency treatment included intravenous (IV) administration of calcium gluconate, and the animal showed clinical improvement within an hour. Overall, the complete blood count was within the normal range, but the calcium and PTH levels were below the normal ranges. Therefore, PH was confirmed. Calcium carbonate and vitamin D3 supplementation were prescribed, and the treatment with phenobarbital was suspended. During the following year, the dog suffered three episodes of hypocalcaemia, even with appropriate administration of medication by the owner. In the last crisis, the animal died, probably because of acute renal failure (ARF). 2) A 7-year-old male Pinscher was presented for veterinary care with a history of 15 days of tetanic and seizure crises. The physical examination revealed hyperthermia, seizure activity, and hyperextension of the anterior and posterior limbs. Levels of total calcium and PTH were below the normal ranges. The animal showed clinical improvement with IV administration of calcium gluconate. Therefore, PH was diagnosed from the clinical signs, response to emergency treatment, and values of calcium and PTH. Calcium carbonate and vitamin D3 supplementation were prescribed. The owner had trouble purchasing calcitriol, and the animal died before the follow-up appointment. 3) A 14-year-old female mixed-breed cat was brought to the clinic with a history of anorexia and vomiting. From the results of the complete blood count, serum biochemistry, and electrocardiogram, the cat was diagnosed initially with hypertrophic cardiomyopathy with hepatic lipidosis due to prolonged anorexia. However, the serum ionized calcium and PTH levels were below normal ranges. Thus, on the basis of the clinical symptoms and serum calcium and PTH levels, the animal was diagnosed with PH. Treatment consisted of ongoing supplementation with vitamin D3 and calcium carbonate, and the existing treatment for heart disease was continued. The treatment did not result in any clinical improvement, and the animal was euthanized.

Discussion: The two dogs presented with the classic symptoms reported in the literature. However, the cat with the same pathology had different clinical signs. In the three cases, the diagnosis was confirmed according to the procedure described in the literature, ie, clinical symptoms, serum calcium and PTH levels below normal ranges, and response to treatment. The stabilization of the serum calcium concentration with calcium and vitamin D3 supplementation corroborates the literature. None of the study patients survived: the first dog probably died because of ARF due to excessive crises; the second dog died because of the advanced stage of the disease and the difficulty in purchasing the drugs prescribed; the cat was euthanized because it did not show any response to treatment, perhaps because it had more than one disease concurrently.

Abstract  Two independent isolates of a Gram-stain-positive, facultatively anaerobic, motile, rod-shaped bacterium were recovered from cheonggukjang, a Korean fermented soybean paste food product. Preliminary sequencing analysis of the 16S rRNA gene indicated that these strains were related most closely to Bacillus sonorensis KCTC-13918T and Bacillus licheniformis DSM 13T. In phenotypic characterization, the novel strains were found to grow between 15 and 55 °C and to tolerate up to 8 % (w/v) NaCl. Furthermore, the strains grew in media of pH 5-10 (optimal growth at pH 7.0). The predominant cellular fatty acids were anteiso-C15 : 0 and iso-C15 : 0.The isoprenoid quinone was menaquinone 7 (MK-7). The cell-wall peptidoglycan contained meso-diaminopimelic acid. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unknown glycolipid. Draft genomes of the two strains were determined and in silico DNA-DNA hybridizations with their nearest neighbour (B. sonorensis KCTC-13918T) revealed 29.9 % relatedness for both strains. Phylogenomic analysis of the genomes was conducted with the core genome (799 genes) of all strains in the Bacillus subtilis group and the two strains formed a distinct monophyletic cluster. In addition, the strains differed from the two most closely related species in that they did not metabolize maltose, d-galactose, d-sorbitol or d-gluconic acid. The DNA G+C content was 45.9 mol%. Based upon the consensus of phylogenetic and phenotypic analyses, these strains represent a novel species of the genus Bacillus, for which the name Bacillus glycinifermentans sp. nov. is proposed. The type strain is GO-13T ( = KACC 18425T = NRRL B-65291T).

Abstract  Substrate versatility of Pseudomonas taetrolens was evaluated for the first time in a co-fermentation system combining cheese whey and glucose, glycerol or lactose as co-substrates. Results showed that P. taetrolens displayed different production patterns depending on the co-substrate supplied. Whereas the presence of glucose led to a simultaneous co-production of lactobionic (78g/L) and gluconic acid (8.8g/L), lactose feeding stimulated the overproduction of lactobionic acid from whey with a high specific productivity (1.4g/gh) and yield (100%). Co-substrate supply of glycerol conversely led to reduced lactobionic acid yield (82%) but higher cell densities (1.8g/L), channelling the carbon source towards cell growth and maintenance. Higher carbon availability impaired the metabolic activity as well as membrane integrity, whereas lactose feeding improved the cellular functionality of P. taetrolens. Insights into these mixed carbon source strategies open up the possibility of co-producing lactobionic and gluconic acid into an integrated single-cell biorefinery.

Abstract  The myo-inositol catabolism pathway of Bacillus subtilis has not been fully characterized but was proposed to involve step-wise multiple reactions that finally yielded acetyl-CoA and dihydroxyacetone phosphate. It is known that the iolABCDEFGHIJ operon is responsible for the catabolism of inositol. IolG catalyses the first step of myo-inositol catabolism, the dehydrogenation of myo-inositol, producing 2-keto-myo-inositol (inosose). The second step was thought to be the dehydration of inosose. Genetic and biochemical analyses of the iol genes led to the identification of iolE, encoding the enzyme for the second step of inositol catabolism, inosose dehydratase. The reaction product of inosose dehydratase was identified as D-2,3-diketo-4-deoxy-epi-inositol.

Abstract  The purpose of this work was to evaluate the effectiveness of a new Membrane Electrochemical Reactor (MER) for the production of gluconic acid by glucose oxidase (GOD) catalysed glucose oxidation. The GOD was confined against the electrode surface with a dialysis membrane. The role of the electrochemical step was to eliminate by oxidation the hydrogen peroxide that appeared as a by-product of the reaction and strongly inhibited and/or inactivated GOD. The dialysis MER gave a transformation ratio of 30% with an initial glucose concentration of around 300 mM. This result is significantly better than the maximum of 10% obtained when hydrogen peroxide was eliminated by addition of a large excess of catalase in solution, as is generally done. The D-MER also revealed unexpected properties of the enzyme kinetics, such as an oscillatory behaviour, which were discussed.

Abstract  Lipase-mediated regioselective biotransformations such as hydrolysis and alcoholysis of 3,4,6-tri-O-acetyl-D-glucal, 1 have been studied in organic solvent, tetrahydrofuran (THF) and two different ionic liquids, namely 1-butyl-3-methylimidazolium hexafluorophosphate, [bmim]PF6 and 1-butyl-3-methylimidazolium tetrafluoroborate, [bmim]BF4. The influence of different reaction media on the rates and regioselectivity of enzyme catalysis has been demonstrated. A marked regioselectivity towards the formation of 4,6-di-O-acetyl-D-glucal, 2 was observed in [bmim]PF6 with 84% product formation after 6 h with 98% selectivity in hydrolysis and 48% after 8 h with 98% selectivity in alcoholysis. (C) 2004 Elsevier B.V. All rights reserved.

Abstract  Klebsiella pneumoniae produces many economically important chemicals. Using glucose as a carbon source, the main metabolic product in K. pneumoniae is 2,3-butanediol. Gluconic acid is an intermediate of the glucose oxidation pathway. In the current study, a metabolic engineering strategy was used to develop a gluconic acid-producing K. pneumoniae strain. Deletion of gad, resulting in loss of gluconate dehydrogenase activity, led to the accumulation of gluconic acid in the culture broth. Gluconic acid accumulation by K. pneumoniae Δgad was an acid-dependent aerobic process, with accumulation observed at pH 5.5 or lower, and at higher levels of oxygen supplementation. Under all other conditions tested, 2,3-butanediol was the main metabolic product of the process. In fed batch fermentation, a final concentration of 422 g/L gluconic acid was produced by K. pneumoniae Δgad, and the conversion ratio of glucose to gluconic acid reached 1 g/g. The K. pneumoniae Δgad described in this study is the first genetically modified strain used for gluconic acid production, and this optimized method for gluconic acid production may have important industrial applications. Gluconic acid is an intermediate of this glucose oxidation pathway. Deletion of gad, resulting in loss of gluconate dehydrogenase activity, led to the accumulation of gluconic acid in the culture broth. In fed batch fermentation, a final concentration of 422 g/L gluconic acid was produced by the K. pneumoniae Δgad strain, and the conversion ratio of glucose to gluconic acid reached 1 g/g.

Abstract  BACKGROUND: Phosphorus is an essential macronutrient for the growth of plants. However, in most soils a large portion of phosphorus becomes insoluble and therefore, unavailable to plants. Knowledge on biodiversity of phosphate-solubilizing fluorescent pseudomonads is essential to understand their ecological role and their utilization in sustainable agriculture.

RESULTS: Of 443 fluorescent pseudomonad strains tested, 80 strains (18%) showed positive for the solubilization of tri-calcium phosphate (Ca3(PO4)2) by the formation of visible dissolution halos on Pikovskaya's agar. These phosphate solubilizing strains showed high variability in utilizing various carbon sources. Numerical taxonomy of the phosphate solubilizing strains based on their carbon source utilization profiles resulted into three major phenons at a 0.76 similarity coefficient level. Genotypic analyses of strains by BOX (bacterial repetitive BOX element)-polymerase chain reaction (PCR) resulted into three distinct genomic clusters and 26 distinct BOX profiles at a 80% similarity level. On the basis of phenotypic characterization and 16S rRNA gene phylogenetic analyses strains were identified as Pseudomonas aeruginosa, P. mosselii, P. monteilii, P. plecoglossicida, P. putida, P. fulva and P. fluorescens. These phosphate solubilizing strains also showed the production of plant growth promoting enzymes, hormones and exhibited antagonism against phytopathogenic fungi that attack on various crops. Gene specific primers have identified the putative antibiotic producing strains. These putative strains were grown in fermentation media and production of antibiotics was confirmed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC).

CONCLUSION: Present study revealed a high degree of functional and genetic diversity among the phosphate solubilizing fluorescent pseudomonad bacteria. Due to their innate potential of producing an array of plant growth promoting enzymes, hormones and antifungal metabolites these phosphate solubilizing strains are considered to play a vital role in plant growth promotion, disease suppression and subsequent enhancement of yield.

Abstract  BACKGROUND: Phosphorus deficiency is a major constraint to crop production due to rapid binding of the applied phosphorus into fixed forms not available to the plants. Microbial solubilization of inorganic phosphates has been attributed mainly to the production of organic acids. Phosphate-solubilizing microorganisms enhance plant growth under conditions of poor phosphorus availability by solubilizing insoluble phosphates in the soil. This paper describes the production of organic acids during inorganic phosphate solubilization and influence on plant growth as a function of phosphate solubilization by fluorescent Pseudomonas.

RESULTS: Nineteen phosphate-solubilizing fluorescent Pseudomonas strains of P. fluorescens, P. poae, P. trivialis, and Pseudomonas spp. produced gluconic acid, oxalic acid, 2-ketogluconic acid, lactic acid, succinic acid, formic acid, citric acid and malic acid in the culture filtrates during the solubilization of tricalcium phosphate, Mussoorie rock phosphate, Udaipur rock phosphate and North Carolina rock phosphate. The strains differed quantitatively and qualitatively in the production of organic acids during solubilization of phosphate substrates. Cluster analysis based on organic acid profiling revealed inter-species and intra-species variation in organic acids produced by Pseudomonas strains. The phosphate-solubilizing bacterial treatments P. trivialis BIHB 745, P. trivialis BIHB 747, Pseudomonas sp. BIHB 756 and P. poae BIHB 808 resulted in significantly higher or statistically at par growth and total N, P and K content over single super phosphate treatment in maize. These treatments also significantly affected pH, organic matter, and N, P, and K content of the soil.

CONCLUSION: The results implied that organic acid production by Pseudomonas strains is independent of their genetic relatedness and each strain has its own ability of producing organic acids during the solubilization of inorganic phosphates. Significant difference in plant growth promotion by efficient phosphate-solubilizing Pseudomonas strains point at the need for selecting potential strains in plant growth promotion experiments in conjunction with various phosphate substrates for their targeted application as bioinoculants.

Abstract  A series of biodegradable linear-hyperbranched barbell-like poly(ethylene glycol) (PEG)-supported poly(lactic-ran-glycolic acid) (PLGA) copolymers were synthesized with PEG, d,l-lactic acid aqueous solution, glycolic acid and gluconic acid (Glu) under bulk conditions. The branching density of the hyperbranched section was varied by controlling the molar ratio of Glu to hydroxyl-terminal groups of PEG ([Glu]/[OH]=1, 3.5, 6.0, 8.5). Chemical structures of these copolymers were confirmed using NMR spectroscopy. The molecular weights were determined using H-1 NMR group analysis and gel permeation chromatography, both results being consistent with one another. The results of hydrolytic degradation indicate that these copolymers can degrade completely in no more than three weeks. The thermal properties were evaluated using differential scanning calorimetry and thermogravimetric analysis. The results indicate that the glass transition temperatures and melt temperatures of these copolymers are not above 50 degrees C. The self-assembly behavior of the copolymers on hydrophilic surfaces was also investigated. The morphology of self-assembly films made of the copolymers was observed using atomic force microscopy, and the results indicate that these copolymers exhibit more inhomogeneous and rough structural orientated films on a silicon wafer substrate with increasing branching densities. Due to the favorable biodegradability and biocompatibility of the PLGA and PEG, the results suggest new possibilities for these novel structural amphiphilic linear-hyperbranched barbell-like copolymers as potential biomaterials. (c) 2013 Society of Chemical Industry

Abstract  Vascular endothelial growth factor receptor-2 (VEGFR-2), which is a key determinant of the angiogenecic response, is decreased in diabetic mice under oxidative stress. β-transduction repeat-containing protein (β-TrCP) has been reported to participate in VEGFR-2 degradation in thyroid cancer cells. Additionally, glycogen synthase kinase-3β (GSK‑3β) acts as a mediator in the β-TrCP-induced degradation of several proteins. However, the role played by β-TrCP and GSK‑3β in the degradation of VEGFR-2 in endothelial cells where hyperglycemia had been induced was not fully understood. In the present study, we aimed to analyze the mechanisms of VEGFR-2 degradation by studying excess reactive oxygen species (ROS) induced by hyperglycemia or glucose oxidase (GO). Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of glucose (6.6, 19.8 and 33 mM), mannitol (33 mM) and GO (1 U/ml). Angiogenic function, ROS levels, the co-location of VEGFR-2 and β-TrCP were evaluated. Cells were collected for RT-qPCR and western blot analysis. We noted that angiogenesis was impaired upon increasing the glucose concentration. When HUVECs were in a hyperglycemic state, ROS production increased, comparable to exposure to GO; GO catalyzes oxidation of glucose into H2O2 and D-glucono-δ-lactone. Phosphorylated VEGFR-2 was reduced by hyperglycemia while total VEGFR-2 was almost unaltered. However, VEGFR-2 was reduced when directly exposed to ROS, with resultant co-location of β-TrCP and VEGFR-2. Through a co-immunoprecipitation assay, we noted that ubiquitinated VEGFR-2 was significantly augmented by excess ROS. Decreased VEGFR-2 caused by ROS was ameliorated by β-TrCP siRNA, proteasome inhibitor MG132 and GSK‑3β activity inhibitor (lithium chloride and SB216763). We suggest that redundant ROS reduces VEGFR-2 through β-TrCP-mediated VEGFR-2 degradation, which is postulated to be regulated by GSK-3β.

Abstract  Diabetes mellitus is an epidemic disease that it has became a worldwide public health problem. Thus, blood glucose monitoring has attracted extensive attention. Here, we report a nanosensor based on inner filter effect (IFE) between upconversion nanoparticles (UCNPs) and squaric acid (SQA)-iron(III) for the highly sensitive and selective detection of glucose levels in human serum. In this assay, GOx-catalyzed oxidization of glucose produces gluconic acid and hydrogen peroxide (H2O2). The latter can catalytically oxidize iron(II) to iron(III) which can rapidly (<1min) coordinate with the SQA to produce (SQA)-iron(III). The absorption band of (SQA)-iron(III) largely covered the emission band of UCNPs, resulting the fluorescence emission of UCNPs was effectively quenched. Therefore, the glucose can be monitored based on the formation of SQA-iron(III). Under the optimal condition, the fluorescence quenching efficiency shows a good linear response to glucose concentration in the ranges of 7-340μmol/L with a detection limit of 2.3μmol/L. The developed method has been further applied to monitor glucose levels in human serum with satisfactory results. Compared with other fluorescence methods, current method displayed high sensitivity and signal-to-noise ratio. Meanwhile, this nanosystem is also generalizable and can be easily expanded to the detection of various H2O2-involved analytes.

Abstract  Poly(acrylic acid) (PAA) brushes coated onto silica nanoparticles have been widely utilized in bioassays due to their abilities of providing favorable microenvironments and ensuring good biological activities for biomolecules. However, traditional PAA brushes are synthesized by reversible addition-fragmentation chain transfer polymerization. Hence, it is generally difficult to control and characterize the molecular weight of the PAA brushes, which may depress the reproducibility and bring more uncertain results. Herein, atom transfer radical polymerization method is employed to synthesize β-cyclodextrin-cored PAA with uniform and controllable molecular weight. After loading on the surfaces of adamantane-functionalized silica nanoparticles via host-guest interactions, glucose oxidase and probe single strand DNA (ssDNA) are further immobilized on the as-prepared nanoparticles. Meanwhile, capture ssDNA is functionalized on amino modified magnetic beads. In the presence of ssDNA sequence of Hepatitis B Virus (HBV) containing completely matched sequence of both probe and capture ssDNA, a bioconjugate is formed and can be separated by an external magnet. The isolated glucose oxidase can further catalyze glucose into gluconic acid and H2O2, and then reduce HAuCl4 on Au seeds. By monitoring the absorption intensity change of the Au NPs at 530nm, the proposed biosensor with novel signal amplification probes can be used to detect DNA sequence of HBV with high sensitivity and selectivity in both buffer and serum samples. This developed strategy has presented a new way to construct silica nanoparticles coated by PAA brushes for the fields of clinical diagnosis and other life sciences.