Gluco-oligomers initially formed by the reuteransucrase enzyme of Lactobacillus reuteri 121 incubated with sucrose and malto-oligosaccharides

<p>The probiotic bacterium Lactobacillus reuteri 121 produces a complex, branched (1 -> 4, 1 -> 6)-alpha-d-glucan as extracellular polysaccharide (reuteran) from sucrose (Suc), using a single glucansucrase/glucosyltransferase (GTFA) enzyme (reuteransucrase). To gain insight into the reaction/product specificity of the GTFA enzyme and the mechanism of reuteran formation, incubations with Suc and/or a series of malto-oligosaccharides (MOSs) (degree of polymerization (DP2-DP6)) were followed in time. The structures of the initially formed products, isolated via high-performance anion-exchange chromatography, were analyzed by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and 1D/2D H-1/C-13 NMR spectroscopy. Incubations with Suc only, acting as both donor and acceptor, resulted in elongation of Suc with glucose (Glc) units via alternating (alpha 1 -> 4) and (alpha 1 -> 6) linkages, yielding linear gluco-oligosaccharides up to at least DP similar to 12. Simultaneously with the ensemble of oligosaccharides, polymeric material was formed early on, suggesting that alternan fragments longer than DP similar to 12 have higher affinity with the GTFA enzyme and are quickly extended, yielding high-molecular-mass branched reuteran (4 x 10(7) Da). MOSs (DP2-DP6) in the absence of Suc turned out to be poor substrates. Incubations of GTFA with Suc plus MOSs as substrates resulted in preferential elongation of MOSs (acceptors) with Glc units from Suc (donor). This apparently reflects the higher affinity of GTFA for MOSs compared with Suc. In accordance with the GTFA specificity, most prominent products were oligosaccharides with an (alpha 1 -> 4)/(alpha 1 -> 6) alternating structure.</p>

Phenylbutazone in the horse: a review

Phenylbutazone is an acidic, lipophilic, nonsteroidal anti-inflammatory drug (NSAID). It is extensively metabolized in the horse. The metabolites so far identified, oxyphenbutazone, y-hydroxyphenylbutazone and y-hydroxyoxyphenbutazone. account for some 25-30% of administered dose over 24 h. The plasma half-life of phenylbutazone and termination of its pharmacological action are determined primarily by its rate of hepatic metabolism. Phenylbutazone acts by inhibiting the cyclooxygenase enzyme system, which is responsible for synthesis of prostanoids such as PGE?. It appears to act on prostaglalidin-H synthase and prostacyclin synthase, after conversion by prostaglandin-H synthase to reactive intermediates. It markedly reduces prostanoid-dependent swelling, edema, erythema, and hypersensitivity to pain in inflamed tissues. Its principal use in the horse is for treatment of soft tissue inflammation. Phenylbutazone is highly bound (\u3e 98%) to plasma protein. After i.v. injection, blood levels decline with an elimination half-life of 3-10 h. The plasma kinetics of phenylbutazone may be dose dependent, with the plasma half-life increasing as the drug dosage level increases. Plasma residues of the drug at 24 h after a single i.v. dose of 2 g/450 kg average about 0.9 pg/ml, but considerable variation occurs. If dosing is repeated, the plasma residue accumulates to give mean residual blood levels of approxiniately 4.5 pg/ml on Day 5 after 4 days of dosing. Approximately similar blood levels are found after a combination of oral and i.v. dosing. Experiments on large numbers of horses in training have been undertaken to ascertain the population distributions of residual blood levels after such dosing schedules. Absorption of phenylbutazone from the gastrointestinal tract is influenced by the dose administered and the relationship of dosing to feeding. Access to hay can delay the time of peak plasma concentration to 18 h or longer. Under optimal conditions, the bioavailability of oral phenylbutazone is probably in the region of 70%. Paste preparations may be more slowly absorbed than other preparations and yield higher residual plasma levels at 24 h after dosing, but further controlled studies are required. Phenylbutazone is easily detected in the plasma and urine of horses but concentrations in saliva are low. It is quantitated for forensic purposes by HPLC. The variability of this method between laboratories is about k 25%. Increasing urinary pH increases the urinary concentration of phenylbutazone and its metabolites up to 200-fold. However, urinary pH has little effect on the plasma half-life of phenylbutazone, which is determined mainly by hepatic metabolism and possibly by biliary secretion. Phenylbutazone has a narrow therapeutic index in the horse. If the administered dose is greater than recommended by the manufacturer, toxic effects may be produced, especially if high dose administration is maintained for more than a few days. Signs of toxicity include anorexia, depression, oral and GI ulcers, plasma protein losing enteropathy, and death from shock. Other side-effects include toxic neutropenia, hepatotoxicity and renal papillary necrosis; the latter may occur if access to water is restricted. If phenylbutazone is withdrawn in the early stages of toxicity, the prognosis is good. Late withdrawal is associated with delayed recovery. Death may occur up to 50 days after withdrawal of the drug. This toxicity can be antagonized by administration of prostaglandins

Structural analysis of rebaudioside A derivatives obtained by Lactobacillus reuteri 180 glucansucrase-catalyzed trans-α-glucosylation

The wild-type Gtf180-ΔN glucansucrase enzyme from Lactobacillus reuteri 180 was found to catalyze the α-glucosylation of the steviol glycoside rebaudioside A, using sucrose as glucosyl donor in a transglucosylation process. Structural analysis of the formed products by MALDI-TOF mass spectrometry, methylation analysis and NMR spectroscopy showed that rebaudioside A is specifically α-d-glucosylated at the steviol C-19 β-d-glucosyl moiety (55% conversion). The main product is a mono-(α1 → 6)-glucosylated derivative (RebA-G1). A series of minor products, up to the incorporation of eight glucose residues, comprise elongations of RebA-G1 with mainly alternating (α1 → 3)- and (α1 → 6)-linked glucopyranose residues. These studies were carried out in the context of a program directed to the improvement of the taste of steviol glycosides via enzymatic modification of their naturally occurring carbohydrate moieties

Enzymatic depolymerization of alginate by two novel thermostable alginate lyases from Rhodothermus marinus

Alginate (alginic acid) is a linear polysaccharide, wherein (1→4)-linked β-D-mannuronic acid and its C5 epimer, α-L-guluronic acid, are arranged in varying sequences. Alginate lyases catalyze the depolymerization of alginate, thereby cleaving the (1→4) glycosidic linkages between the monomers by a β-elimination mechanism, to yield unsaturated 4-deoxy-L-erythro-hex-4-enopyranosyluronic acid (Δ) at the non-reducing end of resulting oligosaccharides (α-L-erythro configuration) or, depending on the enzyme, the unsaturated monosaccharide itself. In solution, the released free unsaturated monomer product is further hydrated in a spontaneous (keto-enol tautomerization) process to form two cyclic stereoisomers. In this study, two alginate lyase genes, designated alyRm3 and alyRm4, from the marine thermophilic bacterium Rhodothermus marinus (strain MAT378), were cloned and expressed in Escherichia coli. The recombinant enzymes were characterized, and their substrate specificity and product structures determined. AlyRm3 (PL39) and AlyRm4 (PL17) are among the most thermophilic and thermostable alginate lyases described to date with temperature optimum of activity at ∼75 and 81°C, respectively. The pH optimum of activity of AlyRm3 is ∼5.5 and AlyRm4 at pH 6.5. Detailed NMR analysis of the incubation products demonstrated that AlyRm3 is an endolytic lyase, while AlyRm4 is an exolytic lyase, cleaving monomers from the non-reducing end of oligo/poly-alginates

Trans-α-glucosylation of stevioside by the mutant glucansucrase enzyme Gtf180-ΔN-Q1140E improves its taste profile

The adverse health effects of sucrose overconsumption, typical for diets in developed countries, necessitate use of low-calorie sweeteners. Following approval by the European Commission (2011), steviol glycosides are increasingly used as high-intensity sweeteners in food. Stevioside is the most prevalent steviol glycoside in Stevia rebaudiana plant leaves, but it has found limited applications in food products due to its lingering bitterness. Enzymatic glucosylation is a strategy to reduce stevioside bitterness, but reported glucosylation reactions suffer from low productivities. Here we present the optimized and efficient alpha-glucosylation of stevioside using the mutant glucansucrase Gtf180-Delta N-Q1140E and sucrose as donor substrate. Structures of novel products were elucidated by NMR spectroscopy, mass spectrometry and methylation analysis; stevioside was mainly glucosylated at the steviol C-19 glucosyl moiety. Sensory analysis of the alpha-glucosylated stevioside products by a trained panel revealed a significant reduction in bitterness compared to stevioside, resulting in significant improvement of edulcorant/organoleptic properties

High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains—selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH4Cl/glucose–glycerol–methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man8–15GlcNAc2). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Manup-to-30GlcNAc2). The latter finding made the X-33 strain not very suitable for generating 15N-labeled material. Therefore, 15N-phCG was expressed in the GS115 strain using the new optimized protocol. The 15N-enrichment was evaluated by 15N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that 15N-phCG/GS115 had the same folding as urinary hCG. Furthermore, 15N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays

The optimal imaging window for dysplastic colorectal polyp detection using c-Met targeted fluorescence molecular endoscopy

Fluorescence molecular endoscopy (FME) is an emerging technique that has the potential to improve the 22% colorectal polyp detection miss-rate. We determined the optimal dose-to-imaging interval and safety of FME using EMI-137, a c-Met-targeted fluorescent peptide, in a population at high risk for colorectal cancer. Methods: We performed in vivo FME and quantification of fluorescence by multidiameter single-fiber reflectance/single-fiber fluorescence spectroscopy in 15 patients with a dysplastic colorectal adenoma. EMI-137 was intravenously administered (0.13 mg/kg) at a 1-, 2- or 3-h dose-to-imaging interval (n = 3 patients per cohort). Two cohorts were expanded to 6 patients on the basis of target-to-background ratios. Fluorescence was correlated to histopathology and c-Met expression. EMI-137 binding specificity was assessed by fluorescence microscopy and in vitro experiments. Results: FME using EMI-137 appeared to be safe and well tolerated. All dose-to-imaging intervals showed significantly higher fluorescence in the colorectal lesions than in surrounding tissue, with a target-to-background ratio of 1.53, 1.66, and 1.74 for the 1-, 2-, and 3-h cohorts, respectively, and a mean intrinsic fluorescence of 0.035 vs. 0.023 mm-1 (P < 0.0003), 0.034 vs. 0.021 mm-1 (P < 0.0001), and 0.033 vs. 0.019 mm-1 (P < 0.0001), respectively. Fluorescence correlated with histopathology on a macroscopic and microscopic level, with significant c-Met overexpression in dysplastic mucosa. In vitro, a dose-dependent specific binding was confirmed. Conclusion: FME using EMI-137 appeared to be safe and feasible within a 1- to 3-h dose-to-imaging interval. No clinically significant differences were observed among the cohorts, although a 1-h dose-to-imaging interval was preferred from a clinical perspective. Future studies will investigate EMI-137 for improved colorectal polyp detection during screening colonoscopies

First-in-human administration of a live-attenuated RSV vaccine lacking the G-protein assessing safety, tolerability, shedding and immunogenicity: a randomized controlled trial

Background: Human respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in early infancy and in elderly. A pediatric vaccine against RSV would not only prevent morbidity and mortality amongst infants and young children but could also reduce transmission to elderly. The RSVDG vaccine consists of a live-attenuated RSV that lacks the G attachment protein. RSVDG is severely impaired in binding to host cells and exhibits reduced infectivity in preclinical studies. Intranasal immunization of cotton rats with RSVDG vaccine protected against replication of wildtype RSV, without inducing enhanced disease.Methods: We performed a first-in-human trial with primary objective to evaluate safety and shedding of RSV Delta G (6.5 log(10) CCID50) after intranasal administration. Healthy adults aged between 18 and 50, with RSV neutralizing serum titers below 9.6 log(2), received a single dose of either vaccine or placebo (n = 48, ratio 3:1). In addition to safety and tolerability, nasal viral load, and systemic and humoral immune responses were assessed at selected time points until 4 weeks after immunization.Results: Intranasal administration of RSV Delta G was well tolerated with no findings of clinical concern. No infectious virus was detected in nasal wash samples. Similar to other live-attenuated RSV vaccines, neutralizing antibody response following inoculation was limited in seropositive adults.Conclusions: A single dose of 6.5 log(10) CCID50 of RSV Delta G was safe and well-tolerated in seropositive healthy adults. RSV Delta G was sufficiently attenuated but there were no signs of induction of antibodies. Safety and immunogenicity can now be explored in children and eventually in seronegative infants. (C) 2020 The Author(s). Published by Elsevier Ltd.Host-parasite interactio