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This study investigated the role of N6-methyladenosine (m6A) and alternative splicing (AS) in bladder cancer (BLCA). The BLCA-related RNA expression profiles and AS events were downloaded from the UCSC Xena and SpliceSeq databases, respectively. Differentially expressed AS (DEAS) was screened, and prognostic-related DEAS events were used to construct prognostic risk models based on Cox proportional hazards regression analysis. Receiver operating characteristic curves and multivariate Cox analysis were used to evaluate the predictive efficiency and independence of these models. We also constructed a protein-to-protein interaction (PPI) network and a regulation network of splicing factors (SFs) and DEAS events. In total, 225 m6A-related prognostic-related DEAS events were identified. The predictive ability of each prognostic model was good, and the alternate terminator model showed the best performance when the area under the curve was 0.793. The risk score of the model was an independent prognostic factor for BLCA. The PPI network revealed that AKT serine/threonine kinase 1, serine- and arginine-rich SF6, and serine- and arginine-rich SF2 had higher-node degrees. A complex regulator correlation was shown in the SF and DEAS networks. This study provides insights for the subsequent understanding of the role of AS events in BLCA.

Novel starch resources isolated from accessible botanical origins are of special interest to food scientists in the context of food security. In this study, Agriophyllum squarrosum starches (AS-1, AS-2, and AS-3) were isolated from three ecotypes of A. squarrosum seeds and compared with quinoa starch (QS). The mean particle diameter of AS granules ranged from 1.12 to 1.15 μm, and AS amylopectin had a significantly higher Mw than QS (p

Two separate experiments were carried out to evaluate the effects of incremental doses of 10 exogenous endo-acting α-amylase and exo-acting glucoamylase; 1LAT (bacterial α-amylase), 2AK, 3AC, 4Cs4, 5Trga, 6Afuga, 7Fvga, and 10Tg (fungal α-amylases, glucoamylases, and α-glucosidase), 8Star and 9Syn (fungal amylase-mixtures; experiment 1) and three exogenous proteases; 11P14L, 12P7L, and 13P30L (bacterial proteases; experiment 2) on in vitro dry matter digestibility (IVDMD) and in vitro starch digestibility (IVSD) of mature dent corn grain using a batch culture system. Incremental doses of the exogenous enzymes (0, 0.25, 0.50, 0.75, and 1.00 mg/g of dried substrate) were applied directly to the substrate (0.5 g of ground corn, 4 mm) in sextuplicate (experiment 1) or quadruplicate (experiment 2) within F57 filter bags, which were incubated at 39 °C in buffered rumen fluid for 7 h. Rumen fluid was collected 2–3 h after the morning feeding from three lactating dairy cows and pooled. Cows were consuming a midlactation total mixed ration (TMR; 1.60 Mcal/kg DM and 15.4%; net energy of lactation and crude protein, respectively). Three independent runs were carried out for each experiment. Data were analyzed as a randomized complete block design using run as the blocking factor. Dose was used as a fixed factor while run was considered a random factor. Linear, quadratic, and cubic orthogonal contrasts were also tested. In experiment 1, enzymes 2AK, 3AC, and 10Tg did not increase (P > 0.10) IVDMD and IVSD, whereas 0.25 mg of enzymes 1LAT, 5Trga, and 8Star increased (P < 0.01) IVDMD by 23%, 47%, and 62% and IVSD by 35%, 41%, and 58%, respectively, compared with the control. Enzymes 4Cs4, 6Afuga, 7Fvga, and 9Syn linearly increased IVDMD and IVSD (P < 0.01). Greatest increases in IVDMD (82.9%) and IVSD (85.9%) resulted with 1 mg of 6Afuga compared to control. In experiment 2, the lowest dose of exogenous proteases 11P14L and 12P7L increased (P < 0.01) IVDMD by 98% and 87% and IVSD by 57% and 64%, respectively, whereas the highest dose of 13P30L increased (P = 0.02) IVDMD by 44.8% and IVSD by 30%, relative to the control. In conclusion, IVSD and IVDMD were increased by one α-amylase, certain glucoamylases, and all proteases tested, with the glucoamylase 6Afuga in experiment 1 and the neutral protease 12P7L in experiment 2, increasing IVDMD and IVSD to the greater extents. Future in vivo studies are required to validate these findings before these enzyme additives can be recommended for improving the digestibility of mature dent corn grain.

The objective was to evaluate the effect of adding an exogenous glucoamylase (GA) enzyme from the fungus Trichoderma reesei on in vitro fermentation, growth performance, and carcass characteristics of feedlot steers fed a dry-rolled corn (DRC)-based diet. Experiment 1 evaluated three levels of added enzyme (0, 0.24, and 0.72 GA enzyme units) and two corn particle sizes (CPS; 2 and 4 mm) in a factorial arrangement using a 7 h in vitro batch culture fermentation. Addition of GA increased (P < 0.01) in vitro dry matter disappearance by 13% and decreased final pH (P < 0.01). Molar proportion of propionate increased with GA inclusion (P < 0.01). A smaller CPS increased (P < 0.01) in vitro dry matter disappearance and total volatile fatty acid and decreased final pH (P < 0.01). A smaller CPS also decreased (P < 0.01) the molar proportion of acetate and increased (P < 0.01) the molar proportion of butyrate. In experiment 2, Angus × Simmental steers (n = 105; initial body weight [BW] = 329 ± 38 kg) were used to evaluate the inclusion of an exogenous GA on growth performance and carcass characteristics. Steers were fed a basal diet consisting of 60% DRC, 17.5% modified distillers grains with solubles, 12.5% corn silage, and 10% dry supplement on a dry matter basis for 136 d. Steers were blocked by weight and allotted to pens. Pens were randomly assigned to one of three treatments (5 pens/treatment): diet with no GA (CON), low inclusion of GA (122 enzyme units/kg dry matter [DM]; LGA), or high inclusion of GA (183 enzyme units/kg DM; HGA). Inclusion of GA did not affect (P ≥ 0.23) final BW, dry matter intake (DMI), or average daily gain (ADG) for the 136-d feeding period. Feed conversion was affected (P = 0.02) by treatment with steers fed HGA having ~8% greater G:F compared with LGA and CON. Treatment did not affect (P = 0.32) fecal starch. Inclusion of GA did not affect (P ≥ 0.19) carcass traits including hot carcass weight, 12th rib fat thickness, yield grade, longissimus muscle area, or marbling score. Overall, results suggest inclusion of exogenous GA enzyme increased in vitro dry matter disappearance in batch culture and improved feed conversion in steers fed 183 enzyme units/kg DM during the finishing phase.

The objective was to evaluate the effect of adding an exogenous glucoamylase (GA) enzyme to a finishing diet on in vitro digestion and performance of feedlot cattle. Experiment 1 evaluated 3 levels of added enzyme (0, 0.24, and 0.72 GA enzyme units) and 2 corn particle sizes (2 and 4 mm) in a 7 h in vitro batch culture fermentation. No interactions were observed (P > 0.10). Addition of GA increased (P < 0.01) in vitro dry matter disappearance by 9%. A smaller corn particle increased (P < 0.01) in vitro dry matter disappearance by 11%. In Experiment 2, Angus × Simmental steers (N = 105; BW = 340 ± 39 kg) were blocked by weight and assigned to 15 pens. Pens were randomly assigned to one of 3 treatments: control (CON) diet with no added enzymes, low inclusion of GA (122 enzyme units/kg DM; LGA), or high inclusion of GA (183 enzyme units/kg DM; HGA). Steers were fed a basal diet consisting of 60% dry rolled corn, 17.5% modified distillers grains with solubles, 12.5% corn silage, and 10% of a corn-based supplement on a DM basis for 139 d. Inclusion of GA did not affect (P ≥ 0.24) final BW, DMI, or ADG overall for the 136 d feeding period. Steers fed HGA had increased (P = 0.02) G:F compared with LGA and CON with least square means of 0.213, 0.194, and 0.197, respectively. Inclusion of GA did not affect (P ≥ 0.19) carcass traits including HCW, 12th rib fat thickness, yield grade, longissimus muscle area, or marbling score. Overall, results suggest inclusion of exogenous glucoamylase enzyme increased in vitro dry matter disappearance in batch culture and improved feed conversion of the finishing diet fed to feedlot steers at 0.381 g enzyme protein/kg DM.

The objective was to investigate the effects of 500 and 2000 FTU/kg phytase inclusions in maize–based diets with appropriately reduced nutrient specifications in comparison to a positive control diet. Diets were offered to Ross 308 broiler chickens from 7 to 28 days post-hatch and growth performance, nutrient utilisation parameters, sodium, starch and protein (N) digestibility coefficients were obtained in four small intestinal segments and IP6 phytate degradation coefficients were determined in the gizzard and in four small intestinal segments. The transition from positive control to negative control diets compromised weight gains, FCR and toe ash by 10.3%, 6.57% and 11.5%, respectively. However, 2000 FTU/kg phytase supplementation completely restored these parameters. At 500 FTU/kg phytase significantly increased starch digestibility coefficients by 12.7% (0.879 versus 0.780) in the distal jejunum and by 4.41% (0.947 versus 0.907) in the proximal ileum and significantly increased starch disappearance rates in all four small intestinal segments. Significant increases in protein (N) digestibility and disappearance rates were limited to the proximal ileum where 500 FTU/kg phytase increased protein (N) digestibility coefficients by 6.08% (0.785 versus 0.740). Phytase, at both inclusions, significantly increased the recovery of sodium in the three anterior small intestinal segments. Interestingly, sodium digestibility coefficients were correlated (P = 0.051 −

A series of MnO

The quinone-dependent alcohol dehydrogenase (PQQ-ADH, E.C. 1.1.5.2) from the Gram-negative bacterium Pseudogluconobacter saccharoketogenes IFO 14464 oxidizes primary alcohols (e.g. ethanol, butanol), secondary alcohols (monosaccharides), as well as aldehydes, polysaccharides, and cyclodextrins. The recombinant protein, expressed in Pichia pastoris, was crystallized, and three-dimensional (3D) structures of the native form, with PQQ and a Ca2+ ion, and of the enzyme in complex with a Zn2+ ion and a bound substrate mimic were determined at 1.72 angstrom and 1.84 angstrom resolution, respectively. PQQ-ADH displays an eight-bladed beta-propeller fold, characteristic of Type I quinone-dependent methanol dehydrogenases. However, three of the four ligands of the Ca2+ ion differ from those of related dehydrogenases and they come from different parts of the polypeptide chain. These differences result in a more open, easily accessible active site, which explains why PQQ-ADH can oxidize a broad range of substrates. The bound substrate mimic suggests Asp333 as the catalytic base. Remarkably, no vicinal disulfide bridge is present near the PQQ, which in other PQQ-dependent alcohol dehydrogenases has been proposed to be necessary for electron transfer. Instead an associated cytochrome c can approach the PQQ for direct electron transfer.

The effects of phytase supplementation on growth performance, nutrient utilisation, starch and protein digestive dynamics in broiler chickens offered maize-, sorghum- and wheat-based diets were determined in a previous study (Liu et al., 2014). Responses to phytase were most pronounced in maize-based diets, which suggest that more phytate was degraded in these diets. Relevant retained samples of grain, diets and digesta from four small intestinal segments were retrospectively analysed for concentrations of phytate, sodium and starch pasting properties to investigate the hypothesis that phytate in maize-based diets was more completely degraded by exogenous phytase. Exogenous phytase significantly (P

Ochratoxin, with ochratoxin A as the dominant form, is one of the five major mycotoxins most harmful to humans and animals. It is produced by Aspergillus and Penicillium species and occurs in a wide range of agricultural products. Detoxification of contaminated food is a challenging health issue. In the present paper we report the identification, characterization and crystal structure (at 2.2 angstrom) of a novel microbial ochratoxinase from Aspergillus niger. A putative amidase gene encoding a 480 amino acid polypeptide was cloned and homologously expressed in A. niger. The recombinant protein is N-terminally truncated, thermostable, has optimal activity at pH similar to 6 and 66 degrees C, and is more efficient in ochratoxin A hydrolysis than carboxypeptidase A and Y, the two previously known enzymes capable of degrading this mycotoxin. The subunit of the homo-octameric enzyme folds into a two-domain structure characteristic of a metal dependent amidohydrolase, with a twisted TIM (triosephosphateisomerase)-barrel and a smaller beta-sandwich domain. The active site contains an aspartate residue for acid base catalysis, and a carboxylated lysine and four histidine residues for binding of a binuclear metal centre.

α-Glucosidase is in the glycoside hydrolase family 13 (13AG) and 31 (31AG). Only 31AGs can hydrate the D-glucal double bond to form α-2-deoxyglucose. Because 1,5-anhydrofructose (AF), having a 2-OH group, mimics the oxocarbenium ion transition state, AF may be a substrate for α-glucosidases. α-Glucosidase-catalyzed hydration produced α-glucose from AF, which plateaued with time. Combined reaction with α-1,4-glucan lyase and 13AG eliminated the plateau. Aspergillus niger α-glucosidase (31AG), which is stable in organic solvent, produced ethyl α-glucoside from AF in 80% ethanol. The findings indicate that α-glucosidases catalyze trans-addition. This is the first report of α-glucosidase-associated glucose formation from AF, possibly contributing to the salvage pathway of unutilized AF.

Phytase (EC 3.1.3.-) hydrolyzes phytate (IP(6)) present in cereals and grains to release inorganic phosphate (P(i)), thereby making it bioavailable. The most commonly used method to assay phytase, developed nearly a century ago, measures the P(i) liberated from IP(6). This traditional endpoint assay is time-consuming and well known for its cumbersomeness in addition to requiring extra caution for handling the toxic regents used. This article reports a simple, fast, and nontoxic kinetic method adaptable for high throughput for assaying phytase using IP(6)-lysozyme as a substrate. The assay is based on the principle that IP(6) forms stable turbid complexes with positively charged lysozyme in a wide pH range, and hydrolysis of the IP(6) in the complex is accompanied by a decrease in turbidity monitored at 600 nm. The turbidity decrease correlates well to the released P(i) from IP(6). This kinetic method was found to be useful in assaying histidine acid phytases, including 3- and 6-phytases, a class representing all commercial phytases, and alkaline β-propeller phytase from Bacillus sp. The influences of temperature, pH, phosphate, and other salts on the kinetic assay were examined. All salts, including NaCl, CaCl(2), and phosphate, showed a concentration-dependent interference.

Red and near-infrared (NIR) organic light-emitting devices (OLEDs) were fabricated based on Tetra (2-Isopropyl-5-methylphenoxyl) substituted metal-free phthalocyanine (Tetra-H2Pc). The devices were fabricated by vacuum deposition and spin coating methods. The electroluminescence (EL) intensity at about 910 nm in the devices based on Tetra-H2Pc was increased by about 14 times compared with the intensity at about 930 nm in the devices based on unsubstituted metal-free phthalocyanine (H2Pc) in the same device structures. The improvement in the EL intensity was attributed to the large steric hindrance of non-peripheral phenoxyl substituent of Tetra-H2Pc. It was found that the NIR EL spectra of the doped devices exhibited a strong dependence on the concentration of Tetra-H2Pc. The emission at 740 nm was from Tetra-H2Pc monomer, while the emissions around 910 nm and 870 nm were from excimer or higher aggregated species.

A novel copper phthalocyanine bearing phenoxy-substituents was prepared and characterized by MS and Elemental analysis. Its UV/Vis absorption and photoluminescence (PL) spectra were investigated. Organic light-emitting devices (OLEDs) were demonstrated by employing this copper phthalocyanine doped into 4, 4′-N,N′-dicarbazole-biphenyl (CBP). Room-temperature electrophosphorescence was observed at about 1.1 μm due to transitions from the first excited triplet state to the singlet ground state (T 1–S0) of this CuPc. The intensity of NIR emission at lower doping concentrations (about 10 wt %) was extremely high compared with devices doped with fluorescent dyes. The results indicated that direct charge trapping appears to be the dominant mechanism.

A β-4-(2-isopropyl-5-methylphenoxylphthalocyaninato)titanium(IV) oxide (TiOPc) was prepared and characterized by MS, 1H NMR, and elemental analysis. Cyclic voltammograms show that this TiOPc has two quasi-reversible reduction couples and two quasi-reversible to irreversible oxidations processes. The first reductions are two-electron processes, confirmed by spectroelectrochemistry to be due to TiIVPc2−/TiIIIPc3− redox processes. The second reductions are two-electron processes during which TiIIIPc3− was reduced to TiIIIPc5− species. Spectroelectrochemistry showed that oxidation occurs at the ring during the first oxidation. However, spectroelectrochemistry also showed that upon the second oxidation, the molecule decomposes. Chronocoulometry confirmed transfer of two electrons at the first and second reduction steps.

We demonstrate near-ultraviolet frequency two-dimensional organic-photonic-crystal laser using resonator structures that offer two-dimensional distributed feedback at room temperature. The gain medium is N,N′-di-1-naphthyl-N, N′-diphenylbenzidine (NPB) which is deposited on lithographically patterned silica structure. We observe surface emitting lasing action with the central beam normal to the surface and the far-field emission characteristics of this laser. The devices operate in the blue spectral region, and exhibit very low threshold energies for oscillation (0.3 kW/cm2). The far field is also shown to contain information about the quality of the photonic crystal. This emission pattern offers a convenient and powerful way to evaluate the nature of laser action in such resonators. © 2007 Wiley Periodicals, Inc. Microwave Opt Technol Lett 50: 382–385, 2008; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mop.23102

A novel, solvothermal method for directly growing crystals of the β-form of copper phthalocyanine that involved using quinoline as solvent and cooling the reaction mixture to room temperature is proposed. Needle-like, single crystals of CuPc up to 10 mm in length were prepared using 1,3-diiminoisoindoline and Cu(AcO)2·2H2O in the quinoline solvent. The influence of the temperatures, growth time and solvent volumes on crystal yield were determined.

Microthecin (2-hydroxy-2-(hydroxymethyl)-2H-pyran-3(6H)-one), which has anti-microbial activity, is one of the end products of an alternative glycogen and starch degrading pathway, the anhydrofructose pathway. It is formed from 1,5-anhydro-d-fructose (AF), a product of α-1,4-glucan lyase (EC 4.2.2.13) from glycogen and starch by aldos-2-ulose dehydratase (AUDH; EC 4. 2.1.110). In the current study, the yield and purity of microthecin was examined with respect to pH and buffers using AUDH purified from the fungus Phanerochaete chrysosporium. It was found that AUDH had a Km of 5.4 and 4.9 mM towards AF and its natural substrate glucosone, respectively, while its Vmax with AF was 5.5 times higher than that with glucosone. Higher molar conversion of 90% was obtained in a reactor with pH controlled around 5.0 and 24°C with de-ionized water as the solvent. Microthecin was found to be most stable in de-ionized water at a pH around 7.0 and stable in freeze-dried form. Under acidic conditions microthecin was degra...

The synthesis of a zinc phthalocyanine (Pc) bearing four phenoxyl substituents was described and its molecular structure was fully characterized by 1 H NMR, MS, elemental analysis, UV–vis and IR, which were consistent with the proposed structure. This Pc possesses excellent solubility in polar organic solvents, such as dichloromethane and THF, and doesn't readily aggregate to the dimer. TOF-MS and elemental analysis prove its excellent purity. The compound's electrochemical redox reactions in THF occurred in the Pc ring and were quasi-reversible single-electron processes. The electrochemical reaction mechanism was proposed.

The use of distiller dried grains with solubles (DDGS) as alternative to conventional animal feed for non-ruminants is challenged by the high content of non-starch polysaccharides and varying protein quality. In this study the enzymatic degradation of corn- and wheat DDGS was evaluated, in vitro, by use of four xylanases from two different glycoside hydrolase families, GH10 and GH11, along with protease and phytase. Wheat DDGS showed the highest degree of enzymatic degradation due to a lower degree of cell wall complexity compared with that of corn DDGS. For corn DDGS,the combination of xylanase and protease yielded the highest degree of enzymatic degradation, indicating close association of arabinoxylan and protein within the cell wall matrix. Collectively, the GH10xylanase degraded DDGS more efficiently than the GH11 xylanases, due to the complexity of the DDGS substrate and the substrate affinity of the GH10xylanase. The current in vitro results indicate a high potential of xylanase in combination with protease to efficiently degrade DDGS and promote nutrient release in diets for non-ruminant animals.

A new method of growing single crystal for β-form copper phthalocyanine (CuPc) is presented in this paper. Melted anthracene was used as solvent of CuPc. The method, vaporizing the solvent using an automatic exaltation machine, was employed to grow CuPc single crystals. The needle-like single crystals of CuPc up to 11.6 mm in length were obtained by applying this method. The influences of different temperatures, exaltation speeds and concentrations on the single crystals growth were also discussed. The method was called exaltation–evaporation growth method.

The bacterium Sinorhizobium morelense S-30.7.5 was isolated by a microbial screening using the sugar 1,5-anhydro- d -fructose (AF) as the sole carbon source. This strain metabolized AF by a novel pathway involving its reduction to 1,5-anhydro- d -mannitol (AM) and the further conversion of AM to d -mannose by C-1 oxygenation. Growth studies showed that the AF metabolizing capability is not confined to S. morelense S-30.7.5 but is a more common feature among the Rhizobiaceae . The AF reducing enzyme was purified and characterized as a new NADPH-dependent monomeric reductase (AFR, EC 1.1.1.-) of 35.1 kDa. It catalyzed the stereoselective reduction of AF to AM and also the conversion of a number of 2-keto aldoses (osones) to the corresponding manno-configurated aldoses. In contrast, common aldoses and ketoses, as well as nonsugar aldehydes and ketones, were not reduced. A database search using the N-terminal AFR sequence retrieved a putative 35-kDa oxidoreductase encoded by the open reading frame Smc04400 localized on the chromosome of Sinorhizobium meliloti 1021. Based on sequence information for this locus, the afr gene was cloned from S. morelense S-30.7.5 and overexpressed in Escherichia coli . In addition to the oxidoreductase of S. meliloti 1021, AFR showed high sequence similarities to putative oxidoreductases of Mesorhizobium loti , Brucella suis , and B. melitensis but not to any oxidoreductase with known functions. AFR could be assigned to the GFO/IDH/MocA family on the basis of highly conserved common structural features. His 6 -tagged AFR was used to demonstrate the utility of this enzyme for AF analysis and synthesis of AM, as well as related derivatives.

The anhydrofructose pathway describes the degradation of glycogen and starch to metabolites via 1,5-anhydro-D-fructose (1,5AnFru). Enzymes that form 1,5AnFru, ascopyrone P (APP), and ascopyrone M (APM) have been reported from our laboratory earlier. In the present study, APM formed from 1,5AnFru was found to be the intermediate to the antimicrobial microthecin. The microthecin forming enzyme from the fungus Phanerochaete chrysosporium proved to be aldos-2-ulose dehydratase (AUDH, EC 4.2.1.-), which was purified and characterized for its enzymatic and catalytic properties. The purified AUDH showing a molecular mass of 97.4 kDa on SDS-PAGE was partially sequenced. Total 332 amino acid residues in length were obtained, representing some 37% of the AUDH protein. The obtained amino acid sequences showed no homology to known proteins but to an unannotated DNA sequence in Scaffold 62 of the published genome of the fungus. The alignment revealed three introns of the identified AUDH gene (Audh; ph.chr), thus the first gene coding for a neutral sugar dehydratase is identified. AUDH was found to be a bi-functional enzyme, being able to dehydrate 1,5AnFru to APM and further isomerizing the APM formed to microthecin. The optimal pH for the formation of APM and microthecin was pH 5.8 and 6.8, respectively. AUDH showed 5 fold higher activity toward 1,5AnFru than toward its analogue glucosone, when tested at concentrations from 0.6 mM to 0.2 M. Based on the characteristic UV absorbance of microthecin (230 nm) and APM (262 nm) assay methods were developed for the microthecin forming enzymes.

1,5-Anhydro-D-fructose (AF) is a monosaccharide occurring in edible morels, red seaweeds and certain mammalian tissues. It can be formed directly from starch and glycogen in vivo by alpha-1,4-glucan lyase (EC 4.2.2.13). In this study, the toxicity, absorption and metabolism of AF using bacteria, mammalian cells, rat and mouse models were examined. In Ames test, AF showed no genotoxicity using five strains of the bacterium Salmonella typhimurium TA 98, 100, 102, 1535 and 1537. AF caused no mammalian gene mutation as tested with mouse lymphoma L5178Y cells. AF did not cause toxic symptoms in rats when it was administered as a single oral dose of 5 g/kg and observed over a 14-day period. Furthermore, at necropsy, no signs of abnormality were detected. Daily intraperitoneal (ip) administration of 2 g/kg AF to mice did not induce adverse effects throughout a 28-day period. Radioactive tracing experiments using 14 C-labeled AF indicated that AF was efficiently absorbed since the major portion of radioactive material was recovered in urine. Further work using unlabeled AF indicated that the cyclic polyol 1,5-anhydro-D-sorbitol (AS) increased dramatically in both blood and urine upon AF administration at 1 g/kg ip, suggesting the existence of an efficient reduction mechanism from AF to AS, which was then excreted in urine. In conclusion, these studies indicate that AF had low or no toxicity and showed no mutagenicity.

Ferulic acid dehydrodimers (DFA) and dehydrotrimers (TriFA) ester-linked to plant cell wall polymers may cross-link not only cell wall polysaccharides but also other cell wall components including proteins and lignin, thus enhancing the rigidity and potentially affecting the enzymatic degradation of the plant cell wall. Corn, wheat, and mixed-cereal distiller’s dried grains with solubles (DDGS) were investigated for composition of DFAs and TriFAs by reversed phase high-performance liquid chromatography with ultraviolet detection. Corn DDGS contained 5.3 and 5.9 times higher contents of total DFAs than wheat and mixed-cereal DDGS, respectively. Furthermore, the contents of total TriFAs were 5.7 and 6.3 times higher in corn DDGS than in wheat and mixed-cereal DDGS, respectively. In addition, both corn grains and corresponding DDGS had similar profiles of individual DFAs and TriFAs, indicating that ferulic acid cross-links in the corn cell wall are presumably not modified during fermentation and DDGS processing.

This study was conducted to evaluate the efficacy of a novel Buttiauxella phytase to pigs fed P-deficient, corn-soybean meal diets. One hundred and twenty crossbred piglets (9.53 ± 0.84 kg) were allocated to one of five treatments which consisted of four low P diets (0.61 % Ca and 0.46 % total P) supplemented with 0, 500, 1,000, or 20,000 FTU/kg phytase as well as a positive control diet (0.77 % Ca and 0.62 % total P). Each treatment had six replicated pens with four pigs per pen. Pigs were fed the experimental diets for 28 days. Phytase supplementation linearly improved (P

There is a steadily increasing need to expand sustainable resources, and carbohydrates are anticipated to play an important role in this respect, both for bulk and fine chemical preparation. The enzyme α-(1→4)-glucan lyase degrades starch to 1,5-anhydro- d -fructose. This compound, which has three different functional properties, a prochiral center together with a permanent pyran ring, renders it a potential chiral building block for the synthesis of valuable and potentially biologically active compounds. 1,5-Anhydro- d -fructose is found in natural materials as a degradation product of α-(1→4)-glucans. The occurrence of lyases and the metabolism of 1,5-anhydro- d -fructose are reviewed in the biological part of this article. In the chemical part, the elucidated structure of 1,5-anhydro- d -fructose will be presented together with simple stereoselective conversions into hydroxy/amino 1,5-anhydro hexitols and a nojirimycin analogue. Synthesis of 6- O -acylated derivatives of 1,5-anhydro- d -fructose substituted with long fatty acid residues is carried out using commercially available enzymes. Those reactions lead to compounds with potential emulsifying properties. The use of protected derivatives of 1,5-anhydro- d -fructose for the synthesis of natural products is likewise reviewed. The potential utilization of this chemical building block is far from being exhausted. Since 1,5-anhydro- d -fructose now is accessible in larger amounts through a simple-enzyme catalyzed degradation of starch by α-(1→4)-glucan lyase, the application of 1,5-anhydro- d -fructose may be considered a valuable contribution to the utilization of carbohydrates as the most abundant resource of sustainable raw materials.

The current work reports on isolation and physico-chemical characterization of floridean starch from three species of agarophytic macro red algae. As determined by 1H-NMR spectroscopy, the average chain length and degree of branching frequency of this starch were 18 and 4.8, respectively. According to its amylopectin chain length distribution obtained by Dionex analysis, the crystalline polymorph of floridean starch from the red alga Gracilariopsis lemaneiformis was deduced to be C-type and this was further supported from its X-ray crystallographic pattern. Enzymatic analysis of its glucose 6-phosphate content showed that floridean starch had a low level of covalently linked phosphate (1 nmol per milligram starch) and this was further confirmed by 31P-NMR. The absorbance peak of floridean starch with iodine occurred at 527—530 nm and the blue value was low (0.1), indicating the absence of amylose, which was confirmed by differential scanning calorimetry (DSC). Floridean starch exhibited low gelatinization temperature, low viscosity, high clarity and little or no retrogradation upon repetitive freeze-thaw cycles, as studied by DSC and rapid viscosity analysis (RVA). These results are discussed in light of the functional properties and the structure of floridean starch.

Corn-, wheat- and mixed cereal Distillers' Dried Grains with Solubles (DDGS) were investigated for compositional variability among DDGS origins, ethanol plants, and the relationship between corn and corresponding DDGS. A total of 138 DDGS samples were analyzed by use of Near Infrared Reflectance Spectroscopy for common constituents, while 63 DDGS samples along with 11 corn samples were characterized for their non-starch polysaccharide (NSP) content. The results indicated that the compositional profile of DDGS reflected the nutrient content of the parent grain but with a greater content of remaining nutrients (e.g. protein, fat, fibre and minerals) after fermentation of starch to ethanol. Corn DDGS differentiated from wheat DDGS by a greater content of fat (P≤0.006), insoluble-NSP (P

Purification of α-1,4-glucan lyase from red algae and fungi has previously been reported. The α-glucan lyase converts α-glucans to 1,5- anhydro-d-fructose. In this study, an endophyte was detected between the algal cells of Gracilariopsis sp., but not penetrating the cell walls. Histological staining was consistent with the endophyte being fungal and immunohistochemistry revealed that it possessed an α-1,4-glucan lyase differing from that of the host. The endophyte α-glucan lyase was recognized by antisera against both fungal and red algal α-glucan lyases, whereas the α-glucan lyase found in the algal tissue was recognized only by the antisera against algal α-glucan lyase. The lyases occur independently in the host and in the endophyte, and were sometimes detected simultaneously. The endophyte was only observed in Gracilariopsis sp. collected in China; no endophyte could be detected in Gracilariopsis sp. from California or in Gracilaria chilensis.

α-1,4-グルカンリアーゼ(EC4.2.2.13)は、マルトース、マルトオリゴ糖、澱粉およびグリコーゲンのようなα-1,4-グルコシド結合からなるグルコースオリゴマーやポリマーに作用して、1,5-アンヒドロ-D-フラクトースを生成する酵素である。分子量は約120kDa、作用至適pHは4.0-6.5であり、シングルポリペプタイドからなっている。α-1,4-グルカンリアーゼはα-1,4-グルコシド結合に対し高い特異性を有しており、α-1,6-グルコシド結合に対しては、殆ど作用しない。また、本酵素はエキソ型の酵素作用を示し、非還元末端側から還元末端あるいは枝別れ箇所まで順次グルコース単位で切断し、1,5-アンヒドロ-D-フラクトースを生成する。つまり、マルトースを作用基質とした場合、グルコースと1,5-アンヒドロ-D-フラクトースが等モル量生成してくることになる。一方、アミロペクチンやグリコーゲンに作用させた場合は、1,5-アンヒドロ-D-フラクトースとα-1,6-グルコシド結合部分で分枝構造を多く含むリミットデキストリンを生成する。著者らは、現在までに、カビや藻類からα-1,4-グルカンリアーゼを精製し、その遺伝子のクローン化もおこなってきた。1,5-アンヒドロ-D-フラクトースおよびその代謝産物である1,5-アンヒドロ-D-グルシトールが、大腸菌やほ乳類において見出されている。この事実は、大腸菌やほ乳類ではもう一つのグリコーゲン分解経路が機能している可能性を示唆するものである。

We demonstrate the near-infrared (NIR) organic light-emitting devices (OLEDs) based on copper hexadecafluorophthalocyanine (CuPcF16 doped into 2,2,2"-(1,3,5-benzenetriyl)tris-[1-phenyl-1H-benzimidazole] (TPBI). The device structure is ITO/ NPB/ TPBI:CuPcF16/BCP/Alq3/Al. Room-temperature electroluminescence is observed at about 1106 nm due to transitions from the first excited triplet state to the ground state (T1-S0) of CuPcF16. The result indicates that Forster and Dexter energy transfers play a minor role in these devices, while the direct charge trapping is the dominant mechanism. The absorption spectra of CuPcF16 solution in pyridine and vacuum sublimed films on quartz have also been investigated.

Regioselective acylation of 1,5-anhydro- d -fructose was performed with dodecanoic acid to give 1,5-anhydro-6- O -dodecanoyl- d -fructose, chemically in 50% yield and enzymatically in quantitative yield. Quantitative conversions were also obtained using hexadecanoic and octadecanoic acids as acyl donors.

In the past few years a novel enzyme alpha-1,4-glucan lyase (EC 4.2. 2.13), which releases 1,5-anhydrofructose from starch and glycogen, has been cloned and characterized from red algae and fungi. Accumulated evidence indicates that the lytic degradation of starch and glycogen also occurs in other organisms. The present review focuses on the biochemical and molecular aspects of eight known alpha-1,4-glucan lyases and their genes from red algae and fungi. While the amino acid sequence identity is 75-80% among the alpha-1, 4-glucan lyases from each of the taxonomic groups, the identity between the algal and fungal alpha-1,4-glucan lyases is only 25-28%. Notably database searches disclosed that the alpha-1,4-glucan lyases have a clear identity of 23-28% with alpha-glucosidases of glycoside hydrolase family 31, thus for the first time linking enzymes from the class of hydrolases with that of lyases. The alignment of lyases and alpha-glucosidases revealed seven well-conserved regions, three of which have been reported to be involved in catalysis and substrate binding in alpha-glucosidases. The shared substrate and inhibitor specificity and sequence similarity of alpha-1,4-glucan lyases with alpha-glucosidases suggest that related structural elements are involved in the two different catalytic mechanisms.

We here report genes encoding a newly discovered class of starch- and glycogen-degrading enzyme, α-1,4-glucan lyase (EC 4.2.2.13), which degrades starch and glycogen to 1,5-anhydro-D-fructose. Two lyases were purified and partially sequenced from the macrofungi Morchella costata and M. vulgaris. The obtained lyase amino acid sequences were used to generate PCR primers, which were further used to probe the fungal genomic libraries. Two lyase genes (Agll1;Mo.cos and Agll1;Mo.vul) from the two fungi were fully sequenced and found to contain a coding region of 3201 bp and 3213 bp, respectively. A total of 13 small introns were found in each of the two genes with identical positions. The two lyase genes share 86% identity at the amino acid level. They encode mature lyases with 1066 and 1070 amino acids, respectively. The deduced molecular masses of 121 530 and 121 971 Da agree with the values found for the two purified lyases. A structure analysis of the promoter regions of the lyase genes revealed a number of putative regulatory DNA elements, such as the AREA and CREA sites, which are related to nitrogen and carbon metabolism, respectively, and the CCAAT/CAAT boxes, which are related to basal expression of genes. A third lyase gene (Agll1;Pe.ost) from the fungus Peziza ostracoderma was partially sequenced to 557 bp. The amino acid sequence deduced from this nucleotide fragment shares 76% identity with the M. costata lyase. Heterologous expression of the M. costata lyase gene was achieved intracellularly in Pichia pastoris and Aspergillus niger.

Acetylation of 1,5-anhydro-D-fructose under acidic conditions gave two crystalline acetylated dimeric forms, which by X-ray analysis were shown to be diastereomeric spiroketals formed between C-2 and C-2/C-3. The structures of the compounds differed only at the configuration at C-2. Acetylation or benzoylation of 1,5-anhydro-D-fructose in pyridine yielded 3,6-di-O-acetyl-1,5-anhydro-4-deoxy-D-glycero-hex-3-enos-2-ulopyra-nos or crystalline 1,5-anhydro-3,6-di-O-benzoyl-4-deoxy-D-glycero-hex-3-enos-2-ulo-pyranose. 1. Presented at the 9th European Carbohydrate Symposium, Utrecht, Netherlands, July 6-1 1, 1997, Poster A138.

1,5-Anhydro- d - arabino -hex-2-ulose (1,5-anhydro- d -fructose, 1,5AnFru), produced by α-1,4-glucan lyase (EC 4.2.2.13) acting on starch, glycogen, or related d -glucose oligo- and polysaccharides as substrate, reacts with alkaline 3,5-dinitrosalicylic acid reagent (DNS) at room temperature (22 °C) within 10 min. The absorbance of the reaction mixture at 550 nm at the end of the reaction was proportional to a 1,5AnFru content in the range of 0.5 to 16 μmol (80 μg to 2.6 mg) mL −1 . 1,5AnFru determined by this colorimetric, one test tube one reagent method, was in good agreement with that found by 1 H-NMR spectroscopy and HPLC. The DNS method is also specific as other reducing sugars, such as glucose, maltose maltosaccharides, starch and glycogen do not give a colour with DNS at 22 °C; therefore, they do not interfere in the determination. The DNS method is applicable to lyase assay for both cell-free extracts and purified enzyme. Methods for reducing sugar analyses, based on the reduction of ferric and cupric ions, were examined for 1,5AnFru and they proved to be quantitative but in contrast to the DNS method, they were not specific. Instead of assaying 1,5AnFru, the activity of α-1,4-glucan lyase was analysed enzymatically by quantifying glucose or 4-nitrophenol released using maltose and 4-nitrophenyl α-maltopentaoside as substrate, respectively.

α-1,4-Glucan lyase (EC 4.2.2.13) from the red seaweed Gracilariopsis lemaneiformis cleaves α-1,4-glucosidic linkages in glycogen, starch, and malto-oligosaccharides, yielding the keto-monosaccharide 1,5-anhydro-D-fructose. The enzyme belongs to glycoside hydrolase family 31 (GH31) but degrades starch via an elimination reaction instead of hydrolysis. The crystal structure shows that the enzyme, like GH31 hydrolases, contains a (β/α)8-barrel catalytic domain with B and B' subdomains, an N-terminal domain N, and the C-terminal domains C and D. The N-terminal domain N of the lyase was found to bind a trisaccharide. Complexes of the enzyme with acarbose and 1-dexoynojirimycin and two different covalent glycosyl-enzyme intermediates obtained with fluorinated sugar analogues show that, like GH31 hydrolases, the aspartic acid residues Asp(553) and Asp(665) are the catalytic nucleophile and acid, respectively. However, as a unique feature, the catalytic nucleophile is in a position to act also as a base that abstracts a proton from the C2 carbon atom of the covalently bound subsite -1 glucosyl residue, thus explaining the unique lyase activity of the enzyme. One Glu to Val mutation in the active site of the homologous α-glucosidase from Sulfolobus solfataricus resulted in a shift from hydrolytic to lyase activity, demonstrating that a subtle amino acid difference can promote lyase activity in a GH31 hydrolase.

The alpha-1,4-glucan lyase (EC 4.2.2.-), purified from the red alga Gracilariopsis lemaneiformis, is a single polypeptide with a molecular mass of 116,654 Da as determined by matrix-assisted laser-desorption mass spectrometry. It degraded maltose, maltosaccharides, amylose, amylopectin and glycogen, forming 1,5-anhydro-D-fructose from the non-reducing end groups. The substrate specificity, mode of action, and cleavage mechanism of the enzyme were studied by using various naturally occurring and synthesized substrates. This enzyme was highly specific for the alpha-1,4-D-glucosidic bond. When a linear alpha-1,4-glucan was used as substrate, the enzyme split the substrate from the non-reducing end and released 1,5-anhydro-D-fructose successively until only one glucose unit was left. When a branched pentasaccharide of 6(2)-alpha-maltosylmaltotriose, obtained from glycogen by alpha-amylase limitation, was used as substrate, the glucose group in the 4-position of the 4,6-branched residue was not cleaved off. Using maltoheptaose as substrate and following the reaction with HPLC and 1H-NMR spectroscopy, it was found that the action mode of the lyase followed a multichain attack mechanism. 1H- and 13C-NMR spectroscopic studies on unlabelled and labelled amylose (1-2H, 2-2H, 1-13C) as substrates indicated that the lyase cleaved the C-(1')-O(4) bond forming a double bond between C-1' and C-2', thus forming the enol form of 1,5-anhydro-D-fructose. It also indicated that the catalytic process of the lyase involved proton exchanges among C-1, C-2, C-3 and the solvent.

The enzyme aldos-2-ulose dehydratase/isomerase (AUDH) participates in carbohydrate secondary metabolism, catalyzing the conversion of glucosone and 1,5- d- anhydrofructose to the secondary metabolites cortalcerone and microthecin, respectively. AUDH is a homo-dimeric enzyme with subunits of 900 amino acids. The subunit consists of a seven-bladed β-propeller domain, two cupin folds and a C-terminal lectin domain. AUDH contains a structural Zn 2+ and Mg 2+ located in loop regions and two zinc ions at the bottom of two putative active-site clefts in the propeller and the cupin domain, respectively. Catalysis is dependent on these two zinc ions, as their specific removal led to loss of enzymatic activity. The structure of the Zn 2 + -depleted enzyme is very similar to that of native AUDH, and structural changes upon metal removal as the cause for the catalytic deficiencies can be excluded. The complex with the reaction intermediate ascopyrone M shows binding of this compound at two different sites, with direct coordination to Zn 2+ in the propeller domain and as second sphere ligand of the metal ion in the cupin domain. These observations suggest that the two reactions of AUDH might be catalyzed in two different active sites, about 60 A apart. The dehydration reaction most likely follows an elimination mechanism, where Zn 2+ acts as a Lewis acid polarizing the C2 keto group of 1,5- d- anhydrofructose. Abstraction of the proton at the C3 carbon atom and protonation of the leaving group, the C4 hydroxyl moiety, could potentially be catalyzed by the side chain of the suitably positioned residue His155.