Abstract  We herein present a three-in-one nanoplatform for sensing, self-assembly, and cascade catalysis, enabled by cyclodextrin modified gold nanoparticles (CD@AuNPs). Monodisperse AuNPs 15-20 nm in diameter are fabricated in an eco-friendly way by the proposed one-step colloidal synthesis method using CD as both reducing agents and stabilizers. First, the as-prepared AuNPs are employed as not only scaffolds but energy acceptors for turn-on fluorescent sensing based on guest replacement reaction. Then, the macrocyclic supramolecule functionalized AuNPs can be controllably assembled and form well-defined one- and two-dimensional architectures using tetrakis(4-carboxyphenyl)porphyrin as mediator. Finally, in addition to conventional host-guest interaction based properties, the CD@AuNPs possess unpredictable catalytic activity and exhibit mimicking properties of both glucose oxidase and horseradish peroxidase simultaneously. Especially, the cascade reaction (glucose is first catalytically oxidized and generates gluconic acid and H2O2; then the enzymatic H2O2 and preadded TMB (3,3',5,5'-tetramethylbenzidine) are further catalyzed into H2O and oxTMB, respectively) is well-achieved using the AuNPs as the sole catalyst. By employing a joint experimental-theoretical study, we reveal that the unique catalytic properties of the CD@AuNPs probably derive from the special topological structures of CD molecules and the resulting electron transfer effect from the AuNP surface to the appended CD molecules.

Abstract  Previous studies have suggested that oral zinc supplementation can help reduce the duration of the common cold; however, the use of intranasal (IN) zinc is strongly associated with anosmia, or the loss of the sense of smell, in humans. Prior studies from this lab showed that upregulation of metallothioneins (MT) is a rapid and robust response to zinc gluconate (ZG). Therefore, we assessed the role of MT in the recovery of nasal epithelial damage resulting from IN zinc administration. The main studies in this investigation used a high dose of ZG (170mM) to ensure ablation of the olfactory mucosa, so that the progression of histological and functional recovery could be assessed. In vivo studies using wild-type, MT1/2 knockout mice (MT KO), and heterozygotes administered ZG by IN instillation showed profound loss of the olfactory mucosa in the nasal cavity. Recovery was monitored, and a lower percentage of the MT KO mice were able to smell 28 d after treatment; however, no significant difference was observed in the rate of cell proliferation in the basal layer of the olfactory epithelium between MT KO and wild-type mice. A lower concentration of ZG (33mM), equivalent to that found in homeopathic IN ZG preparations, also caused olfactory epithelial toxicity in mice. These studies suggest that the use of zinc in drug formulations intended for IN administration in humans must be carefully evaluated for their potential to cause olfactory functional deficits.

Abstract  Glucose oxidase (GOx) is an enzyme produced by Aspergillus, Penicillium and other fungi species. It catalyzes the oxidation of β-d-glucose (by the molecular oxygen or other molecules, like quinones, in a higher oxidation state) to form d-glucono-1,5-lactone, which hydrolyses spontaneously to produce gluconic acid. A coproduct of this enzymatic reaction is hydrogen peroxide (H₂O₂). GOx has found several commercial applications in chemical and pharmaceutical industries including novel biosensors that use the immobilized enzyme on different nanomaterials and/or polymers such as polyethylenimine (PEI). The problem of GOx immobilization on PEI is retaining the enzyme native activity despite its immobilization onto the polymer surface. Therefore, the molecular dynamic (MD) study of the PEI ligand (C14N8_07_B22) and the GOx enzyme (3QVR) was performed to examine the final complex PEI-GOx stabilization and the affinity of the PEI ligand to the docking sites of the GOx enzyme. The docking procedure showed two places/regions of major interaction of the protein with the polymer PEI: (LIG1) of -5.8 kcal/mol and (LIG2) of -4.5 kcal/mol located inside the enzyme and on its surface, respectively. The values of enthalpy for the PEI-enzyme complex, located inside of the protein (LIG1) and on its surface (LIG2) were computed. Docking also discovered domains of the GOx protein that exhibit no interactions with the ligand or have even repulsive characteristics. The structural data clearly indicate some differences in the ligand PEI behavior bound at the two places/regions of glucose oxidase.

Abstract  Glucose oxidase and catalase were immobilized on poly(diethylaminoethyl methacrylate-g-ethylene glycol) gels by copolymerization of the constituent monomers and the functionalized enzyme solutions. The hydrogels were prepared in the form of discs and microparticles. The amount and the activity of enzymes immobilized in the matrix were determined. The hydrogels were tested for their response to glucose by exposing microparticles to varying concentrations of glucose. The generation of gluconic acid as a result of the reaction of glucose with oxygen was investigated as a function of polymer parameters, such as crosslinking ratio and enzyme loading. Pulsatile variation of the glucose concentration was used to confirm the glucose-dependent swelling properties of these hydrogels.

Abstract  BACKGROUND: Several diseases affect bone healing and physiology. Many drugs that are commonly used in orthopaedics as "analgesics" or anti-inflammatory agents impair bone healing. Stressful conditions are associated with decreased serum osteocalcin concentration. High endorphin levels alter calcium metabolism, blocking the membrane channels by which calcium normally enters cells. The consequent decrease of intracellular calcium impairs the activities of calcium-related enzymes. Naloxone is a pure opioid antagonist. Morphine-induced osteocalcin inhibition was abolished when osteoblasts were incubated with naloxone. Naloxone restored the altered cellular and tissue physiology by removing beta-endorphins from specific receptors. However, this is only possible if the circulating Ca concentration is adequate. The aim of the present study was to evaluate the efficacy of parenteral naloxone administration in inducing fast mineralization and callus remodelling in a group of sheep with a standardised bone lesion.

METHODS: Twenty ewes were randomly assigned to 4 treatment groups. Group A acted as control, group B received a solution of calcium gluconate, group C a solution of naloxone, and group D a solution of calcium gluconate and naloxone. A transverse hole was drilled in the left metacarpus, including both cortices, then parenteral treatment was administered intramuscularly, daily for four weeks. Healing was evaluated by weekly radiographic examination for eight weeks. For quantitative evaluation, the ratio of the radiographic bone density between the drill area and the adjacent cortical bone was calculated. After eight weeks the sheep were slaughtered and a sample of bone was collected for histopathology

RESULTS: Group D showed a higher radiographic ratio than the other groups. Sheep not treated with naloxone showed a persistently lower ratio in the lateral than the medial cortex (P < 0.01). Histopathology of bone samples showed more caverns and fewer osteoblasts in group D than in the other groups (P </= 0.001).

CONCLUSION: A low-dose parenteral regimen of naloxone enhances mineralization and remodelling of the callus in healing cortical defects in sheep, especially if associated with calcium gluconate.

Abstract  Zinc is both an essential and potentially toxic metal. It is widely believed that oral zinc supplementation can reduce the effects of the common cold; however, there is strong clinical evidence that intranasal (IN) zinc gluconate (ZG) gel treatment for this purpose causes anosmia, or the loss of the sense of smell, in humans. Using the rat olfactory neuron cell line, Odora, we investigated the molecular mechanism by which zinc exposure exerts its toxic effects on olfactory neurons. Following treatment of Odora cells with 100 and 200μM ZG for 0-24h, RNA-seq and in silico analyses revealed up-regulation of pathways associated with zinc metal response, oxidative stress, and ATP production. We observed that Odora cells recovered from zinc-induced oxidative stress, but ATP depletion persisted with longer exposure to ZG. ZG exposure increased levels of NLRP3 and IL-1β protein levels in a time-dependent manner, suggesting that zinc exposure may cause an inflammasome-mediated cell death, pyroptosis, in olfactory neurons.

Abstract  A new caffeoylgluconic acid derivative, trans-caffeoyl-6-O-D-gluconic acid methyl ester (1), together with two known compounds named trans-caffeoyl-6-O-D-glucono-γ-lactone (2) and trans-caffeoyl-6-O-D-gluconic acid (3), was isolated from the nearly ripe fruits of Evodia rutaecarpa (Juss.) Benth.. These compounds were isolated by various separation methods associated with the UPLC-Q-TOF-MS technique. Their structures were elucidated on the basis of extensive spectroscopic methods.

Abstract  Previously, we identified the uncharacterized predicted membrane protein PA2663 of Pseudomonas aeruginosa PAO1 as a virulence factor using a poplar tree model; PA2663 was induced in the poplar rhizosphere and, upon inactivation, it caused 20-fold lower biofilm formation (Attila et al., Microb Biotechnol, 2008). Here, we confirmed that PA2663 is related to biofilm formation by restoring the wild-type phenotype by complementing the PA2663 mutation in trans and investigated the genetic basis of its influence on biofilm formation through whole-transcriptome and -phenotype studies. Upon inactivating PA2663 by transposon insertion, the psl operon that encodes a galactose- and mannose-rich exopolysaccharide was highly repressed (verified by RT-PCR). The inactivation of PA2663 also repressed 13 pyoverdine genes, which eliminated the production of the virulence factor pyoverdine in P. aeruginosa. The inactivation of PA2663 also affected other quorum-sensing-related phenotypes in that it repressed the Pseudomonas quinolone signal (PQS) genes, which abolished PQS production, and repressed lasB, which decreased elastase activity sevenfold. Genes were also induced for motility and attachment (PA0499, PA0993, PA2130, and PA4549) and for small molecule transport (PA0326, PA1541, PA1632, PA1971, PA2214, PA2215, PA2678, and PA3407). Phenotype arrays also showed that PA2663 represses growth on D: -gluconic acid, D: -mannitol, and N-phthaloyl-L: -glutamic acid. Hence, the PA2663 gene product increases biofilm formation by increasing the psl-operon-derived exopolysaccharides and increases pyoverdine synthesis, PQS production, and elastase activity while reducing swarming and swimming motility. We speculate that PA2663 performs these myriad functions as a novel membrane sensor.

Abstract  Spatial organization of multiple enzymes at specific positions for a controlled reaction cascade has attracted wide attention in recent years. Here, we report the construction of a biomimetic enzyme cascade organized on DNA triangle prism (TP) nanostructures to enable the efficient catalytic production of nitric oxide (NO) on a single microbead. Two enzymes, glucose oxidase (GOx) and horseradish peroxidase (HRP), were assembled at adjacent locations on a DNA TP nanostructure by using DNA-binding protein adaptors with small interenzyme distances. In the cascade, the first enzyme, GOx, converts glucose into gluconic acid in the presence of oxygen. The produced H2 O2 intermediate is rapidly transported to the second enzyme, HRP, which oxides hydroxyurea into NO and other nitroxyl species. The pH near the surface of the negatively charged DNA nanostructures is believed to be lower than that in the bulk solution; this creates an optimal pH environment for the anchored enzymes, which results in higher yields of the NO product. Furthermore, the multienzyme system was immobilized on a microbead mediated by a DNA adaptor, and this enabled the efficient catalytic generation of gas molecules in the microreactor. Therefore, this work provides an alternative route for the biomimetic generation of NO through enzyme cascades. In particular, the dynamic binding capability of the DNA sequence enabled the positions of the protein enzyme and the DNA nanostructure to be reversed, which allowed the cascade catalysis to be modulated.

Abstract  Lipopolysaccharide (LPS), which can cause acute airway inflammatory reactions, constitutes one of the most common substances to establish acute lung injury (ALI) models in mice. Studies suggest that calcium gluconate offers the possibility of suppressing the immune response, and this study was intended to explore the effects of calcium gluconate on LPS-induced ALI in mice. Mice inhaled with LPS were intraperitoneally injected with calcium gluconate (12.5, 25, 50 mg/kg). IL-1β, IL-6 and TNF-α levels in bronchoalveolar lavage fluid (BALF) were determined by ELISA. The expression of signaling proteins, phosphorylation extracellular regulated protein kinases (p-ERK), was detected using Western Blot in lung tissues. In our study, the release of inflammatory cytokines IL-1β, IL-6 and TNF-α in BALF increased after inhalation of LPS. Post-treatment with calcium gluconate inhibited LPS-induced airway inflammatory injury and the release of inflammatory cytokines. In addition, LPS promoted the expression of signaling protein p-ERK while calcium gluconate was capable of reversing this change. Overall, calcium gluconate inhibits LPS-induced ALI in mice, which may take effects through the inhibition of ERK phosphorylation.

Abstract  INTRODUCTION: Micronutrient deficiencies are one of the most important public health issues worldwide and iron (Fe) deficiency anemia is the most prevalent micronutrient deficiency. Iron deficiency often coexists with calcium deficiency and iron and calcium supplementation often overlap. This has led to investigations into the interaction between these two minerals, and whether calcium may inhibit iron absorption in the gut.

OBJECTIVE: To determine the effect of various calcium salts on non-heme iron bioavailability in fasted women of childbearing age.

METHODS: A randomized and single blinded trial was conducted on 27 women of childbearing age (35-45 years old) divided into 2 groups (n1 = 13 and n2 = 14, respectively). On four different days, after an overnight fast, they received 5 mg of Fe as FeSO4 (labeled with 55Fe or 59Fe) with 800 mg of elemental calcium in the form of either calcium chloride, calcium gluconate, calcium citrate, calcium carbonate, calcium lactate, calcium sulfate or calcium phosphate. Calcium chloride was used as the control salt in both groups. Iron was labeled with the radioisotopes 59Fe or 55Fe, and the absorption of iron was measured by erythrocyte incorporation of radioactive Fe RESULTS: 800 mg of elemental calcium as calcium citrate produced a significant decrease in non-heme iron bioavailability (repeated measures ANOVA, F = 3.79, p = 0.018).

CONCLUSION: Of the various calcium salts tested, calcium citrate was the only salt that decreased non-heme iron bioavailability relative to the calcium chloride control when taken on an empty stomach. These results suggest that inhibition of non-heme iron absorption in fasted individuals is dependent upon the calcium salt in question and not solely dependent on the presence of calcium.

Abstract  This study aimed to evaluate the effects of different concentration methods (nanofiltration and evaporation) and heat treatments on the gel properties of milk protein concentrate (MPC). The MPC gels were produced using glucono-δ-lactone (GDL) as an acidifier with different preheat treatments (30 min at 80°C and 5 min at 92°C). We then evaluated the effect of preheat treatments on MPC gel properties, including storage modulus (G'), loss tangent (tan δ), firmness, whey separation, and microstructure. The results indicated that without preheating, evaporation (EP)-MPC had higher G' and firmness, and lower tan δ and whey separation than nanofiltration (NF)-MPC. These results suggest that EP-MPC produced a better acid-induced gel than NF-MPC when no preheat treatments were performed. After preheating, however, except for a very small difference in the final G' (EP-MPC was higher), the 2 MPC did not differ significantly in firmness, final tan δ, or whey separation. Additionally, compared with the gel of unheated MPC, both preheat-treated gels (NF-MPC and EP-MPC) achieved increased G' and firmness and decreased tan δ and whey separation. The preheat-treated MPC also displayed a more flexible-stranded network. These findings demonstrate that, given a suitable heating treatment, NF-MPC compares favorably with EP-MPC in achieving desired gel properties.

Abstract  Floral nectar plays important roles in the interaction between animal-pollinated plants and pollinators. Its components include water, sugars, amino acids, vitamins, and proteins. Growing empirical evidence shows that most of the proteins secreted in nectar (nectarines) are enzymes that can tailor nectar chemistry for their animal mutualists or reduce the growth of microorganisms in nectar. However, to date, the function of many nectarines remains unknown, and very few plant species have had their nectar proteome thoroughly investigated. Mucuna sempervirens (Fabaceae) is a perennial woody vine native to China. Nectarines from this species were separated using two-dimensional gel electrophoresis, and analyzed using mass spectrometry. A L-gulonolactone oxidase like protein (MsGulLO) was detected, and the full length cDNA was cloned: it codes for a protein of 573 amino acids with a predicted signal peptide. MsGulLO has high similarity to L-gulonolactone oxidase 5 (AtGulLO5) in Arabidopsis thaliana, which was suggested to be involved in the pathway of ascorbate biosynthesis; however, both MsGulLO and AtGulLO5 are divergent from animal L-gulonolactone oxidases. MsGulLO was expressed mainly in flowers, and especially in nectary before blooming. However, cloning and gene expression analysis showed that L-galactonolactone dehydrogenase (MsGLDH), a vital enzyme in plant ascorbate biosynthesis, was expressed in all of flowers, roots, stems, and especially leaves. MsGulLO was purified to near homogeneity from raw MS nectar by gel filtration chromatography. The enzyme was determined to be a neutral monomeric protein with an apparent molecular mass of 70 kDa. MsGulLO is not a flavin-containing protein, and has neither L-galactonolactone dehydrogenase activity, nor the L-gulonolactone activity that is usual in animal GulLOs. However, it has weak oxidase activity with the following substrates: L-gulono-1,4-lactone, L -galactono-1,4-lactone, D-gluconic acid-δ-lactone, glucose, and fructose. MsGulLO is suggested to function in hydrogen peroxide generation in nectar but not in plant ascorbate biosynthesis.

Abstract  The traditional way of producing wine is through the use of Saccharomyces cerevisiae in order to convert glucose and fructose into alcohol. In the case of red wines, after this alcoholic fermentation lactic bacteria Oenococus oeni is used to stabilize wine from a microbiological point of view by converting malic acid into lactic acid that it is not a microbiological substract. The yeast species Schizosaccharomyces pombe was traditionally considered spoilage yeast. Nevertheless, during the last decade it started to be used due to its unique malic acid deacidification ability to reduce the harsh acidity of wines from northern Europe, by converting malic acid to ethanol and CO2 without producing lactic acid as lactic bacteria does. Additionally, during the last years, S. pombe has started to be used to solve the problems of modern winemaking industry such as increasing food quality or food safety. Some of those new uses, different from its traditional malic acid deacidification, are: high autolytic polysaccharides release, gluconic acid reduction, urease activity that make impossible ethyl carbamate (toxic compound) formation, high pyruvic acid production, that is related to color improvement, and removing lactic bacteria subtracts while avoiding biogenic amines (toxic compounds such as histamine) formation.

Abstract  Bacterial response to environmental stimuli is essential for survival. In response to fluctuating environmental conditions, the physiological status of bacteria can change due to the actions of transcriptional regulatory machinery. The synthesis and accumulation of polyhydroxyalkanoates (PHAs) are one of the survival strategies in harsh environments. In this study, we used transcriptome analysis of Pseudomonas putida KT2440 to gain a genome-wide view of the mechanisms of environmental-friendly biopolymers accumulation under nitrogen-limiting conditions during conversion of metabolically different carbon sources (sodium gluconate and oleic acid). Transcriptomic data revealed that phaG expression is associated with medium-chain-length-PHAs' synthesis not only on sodium gluconate but also on oleic acid, suggesting that PhaG may play a role in this process, as well. Moreover, genes involved in the β-oxidation pathway were induced in the PHAs production phase when sodium gluconate was supplied as the only carbon and energy source. The transition from exponential growth to stationary phase caused a significant expression of genes involved in nitrogen metabolism, energy supply, and transport system. In this study, several molecular mechanisms, which drive mcl-PHAs synthesis, have been investigated. The identified genes may provide valuable information to improve the efficiency of this bioprocess and make it more economically feasible.

Abstract  The synthesis of a range of analogues of the migrastatin macrolide core has been achieved from tri-O-acetyl-D-glucal in order to facilitate structure-activity studies. Efficient macrolactone formation was achieved in the presence of a reactive olefin, by increasing steric hindrance in the olefin environment. Acyclic analogues of migrastatin, structurally related to dorrigocin A, have also been prepared from D-glucal. The dorrigocin A analogues were prepared using the combination of the cross metathesis of ethyl 6-heptenoate with a glycal derivative and a subsequent allylic rearrangement-alkene isomerisation reaction (Perlin reaction). A synthetic route is thus provided that will enable dorrigocin A analogues to be prepared in parallel to migrastatin analogues in the search for novel anti-cancer and anti-arthritic therapeutics. Biological evaluation of one migrastatin and one dorrigocin A sugar derived analogue show that they inhibit proliferation and serum-induced migration of tumour and synovial cells at higher concentrations than evodiamine. Dorrigocin A analogues displayed similar potency to analogues of the migrastatin core.

Abstract  Microwave-assisted organic synthesis (MAOS) of D-gluconic acid can be efficiently done by oxidation of D- glucose with bromine water, upon irradiation with microwave ( MW). It was also used for the conversion of D-gluconic acid to ethyl D-gluconate, D-glucono-1,4- and 1,5-lactones, gluconyl hydrazide, and gluconyl phenylhydrazide in yields comparable to those obtained by conventional methods, but in much shorter times. A convenient microwave- mediated condensation of D- gluconic acid with o-phenylenediamines provided the respective acyclonucleoside benzimidazole in short time and good yield.

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Abstract  This study examining factors contributing to the production or elimination of domoic acid (DA) in cultures of Pseudo-nitzschia multiseries showed that in axenic cultures doubling the silicate concentrations increased growth, but not DA levels. DA concentration for axenic cultures was increased by the addition of gluconolactone (GlcA), especially in cultures with increased silicon. In non-axenic cultures, there were similar increases in growth with increased silicon, but a reduction of DA production in the presence of GlcA. Detailed examinations confirmed these findings and also showed that in non-axenic cultures, glucose alone resulted in a reduction of DA while a combination of glucose with gluconolactone resulted in a complete elimination of DA. Subsequent trials with axenic P. multiseries cultures showed that additions of DA or DA plus glucose introduced at the early stationary growth phase and incubated for 5 d had no impact on DA concentrations. In contrast, a 6 d incubation of the associated bacteria separated from the non-axenic diatom cultures showed reductions of added DA concentrations ranging from 46 to 72 %, depending upon co-additives. The diatom does not use extracellular DA present in surrounding culture medium whereas bacteria associated with the diatom can utilize DA readily. Reductions in the production of DA by aging P. multiseries cultures appear to be the result of changing balances over time among bacteria associated with the diatom. These data coupled with results from other studies indicate that the amount of DA measured in P. multiseries cultures is a result of competitive interaction, i.e. a function of the diatom's production rate versus the extra-cellular utilization of DA by associated bacteria.

Abstract  Red blood cell (RBC)-derived adenosine triphosphate (ATP) has been proposed as an integral component in the regulation of oxygen supply to skeletal muscle. In ex vivo settings RBCs have been shown to release ATP in response to a number of stimuli, including stimulation of adrenergic receptors. Further evidence suggested that ATP release from RBCs was dependent on activation of adenylate cyclase (AC)/cyclic adenosine monophosphate (cAMP)-dependent pathways and involved the pannexin 1 (Panx1) channel. Here we show that RBCs express Panx1 and confirm its absence in Panx1 knockout (-/-) RBCs. However, Panx1-/- mice lack any decrease in exercise performance, challenging the assumptions that Panx1 plays an essential role in increased blood perfusion to exercising skeletal muscle and therefore in ATP release from RBCs. We therefore tested the role of Panx1 in ATP release from RBCs ex vivo in RBC suspensions. We found that stimulation with hypotonic potassium gluconate buffer resulted in a significant increase in ATP in the supernatant, but this was highly correlated with RBC lysis. Next, we treated RBCs with a stable cAMP analog, which did not induce ATP release from wild-type or Panx1-/- mice. Similarly, multiple pharmacological treatments activating AC in RBCs increased intracellular cAMP levels (as measured via mass spectrometry) but did not induce ATP release. The data presented here question the importance of Panx1 for exercise performance and dispute the general assumption that ATP release from RBCs via Panx1 is regulated via cAMP.

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  Synthetic methods were elaborated for d-glucals attached to oxadiazoles by a C-C bond. Introduction of the double bond was effected by either DBU induced elimination of PhCOOH from the O-perbenzoylated glucopyranosyl precursors or Zn/N-methylimidazole mediated reductive elimination from the 1-bromoglucopyranosyl starting compounds. Alternatively, heterocyclizations of 2-deoxy-d-arabino-hex-1-enopyranosyl cyanide were also carried out. Test compounds were obtained by Zemplén debenzoylation, however, none of them showed significant inhibition of rabbit muscle glycogen phosphorylase b.