A photoluminescent and electrochemiluminescent probe based on an iridium(III) complex with a boronic acid-functionalised ancillary ligand for the selective detection of mercury(II) ions.

Exposure to mercury(II) ions (Hg2+) can cause various diseases such as Minamata disease, acrodynia, Alzheimer's disease, and Hunter-Russell syndrome, and even organ damage. Therefore, real-time and accurate monitoring of Hg2+ in environmental samples is crucial. In this study, we report a photoluminescent (PL) and electrochemiluminescent (ECL) probe based on a cyclometalated Ir(III) complex for the selective detection of Hg2+. The introduction of a reaction site, o-aminomethylphenylboronic acid, on the ancillary ligands allowed a prompt transmetalation reaction to take place between Hg2+ and boronic acid. This reaction resulted in significant decreases of the PL and ECL signals due to the photo-induced electron transfer from the Ir(III) complex to the Hg2+ ions. The probe was applied to the selective detection of Hg2+, and the signal changes revealed a linear correlation with Hg2+ concentrations in the range of 0-10 µM (LOD = 0.72 µM for PL, 8.03 nM for ECL). The designed probe allowed the successful quantification of Hg2+ in tap water samples, which proves its potential for the selective detection of Hg2+ in environmental samples.

Electrochemiluminescence of dimethylaminonaphthalene-oxazaborine donor-acceptor luminophores.

Donor-acceptor (D-A) type molecules with a skeleton consisting of a dimethylaminonaphthalene donor and an oxazaborine acceptor were designed as efficient electrochemiluminescence (ECL) luminophores with tunable intramolecular charge transfer (ICT). The D-A ECL luminophores demonstrated that the ICT characteristics play a critical role in the electrochemistry and ECL of luminophores in the presence of tri-n-propylamine, which was rationalised experimentally and computationally. Furthemore, dual-peaked ECL-potential behaviours of the luminophores were rationalised using two competitive pathway ECL mechanisms, elucidated through the use of spooling ECL spectroscopy.

Highly Selective Electrochemiluminescence Chemosensor for Sulfide Enabled by Hierarchical Reactivity.

Hydrogen sulfide (H2S) is a well-known toxic gas with the odor of rotten eggs. Several reaction-based electrochemiluminescence (ECL) chemosensors for H2S have been developed; however, no homogeneous ECL probe with high selectivity toward H2S in aqueous media has been reported. Herein, we report an iridium(III) complex-based ECL chemodosimetric probe employing two 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) groups known as a photo-induced electron transfer quencher and a reaction site for the selective detection of H2S; the detection mechanism involves H2S being clearly distinguished from biothiols based on the different cleavage rates of the two NBD groups and extremely weak ECL interferences caused by reaction by-products. The probe was rationally designed to improve selectivity toward H2S within the ECL analysis platform by enabling the removal of nonspecific background signals observed via fluorescence analysis. This analytical system exhibited remarkable selectivity toward H2S, a rapid reaction rate, and high sensitivity (LOD = 57 nM) compared to conventional fluorescence methods. Furthermore, the probe could successfully quantify H2S in tap water samples and commercial ammonium sulfide solutions, which demonstrates the effectiveness of this probe in field monitoring.

Conventional open-tray impression versus intraoral digital scan for implant-level complete-arch impression.

STATEMENT OF PROBLEM: The best-fit method is frequently used to evaluate the accuracy of different implant impression techniques. However, the method includes inherent superimposition errors, which may accumulate and become more exaggerated in complete-arch impressions. PURPOSE: The purpose of this in vitro study was to evaluate and compare the trueness and precision of conventional open-tray impressions and intraoral digital scans at the implant level in an edentulous maxillary model with 6 implant replicas without superimposition. MATERIAL AND METHODS: A master model was fabricated using epoxy resin by duplicating a maxillary edentulous cast that had 6 implant replicas in the right first molar, right first premolar, right lateral incisor, left lateral incisor, left first premolar, and left first molar positions. The conventional open-tray, splinted-coping impression technique was used to fabricate 10 definitive casts (group CI). Intraoral digital scans were performed, after which scan bodies were connected to each implant replica to fabricate 10 digital models (group IOS). For the master model and group CI, a computerized coordinate-measuring machine was used to determine the 3D spatial orientation of the implant replicas. For group IOS, the scan bodies were converted to implant replicas using a digital library, and an inspection software program was used to measure the implant replicas. To compare the accuracies of different impression techniques, a 3D part coordinate system was set to compute the centroid and projection angles of each implant replica. The changes in the centroid coordinates (linear displacement: Δx, Δy, Δz, and ΔD; ΔD=Δx2+Δy2+Δz2) and projection angles onto XY and ZX planes (angular displacement: ΔθXY and ΔθZX) were statistically compared (α=.05). RESULTS: Group CI gave more accurate trueness values than group IOS for overall Δx (P<.001), Δy (P =.029), Δz (P<.001), and ΔD (P<.001). Furthermore, group CI had more accurate precision values for Δx, Δy, and Δz. Group IOS exhibited a statistically greater angular displacement in the ZX plane (P=.002), but the difference was only 0.24 degrees. No differences were found between the 2 groups for the angular displacement in the XY plane (P=.529). CONCLUSIONS: Conventional open-tray impressions produced significantly smaller linear displacements than the digital scan obtained using an intraoral scanner at the implant level in a complete-arch model.

Electrogenerated Chemiluminescent Chemodosimeter Based on a Cyclometalated Iridium(III) Complex for Sensitive Detection of Thiophenol.

Thiophenol is the simplest aromatic thiol that is utilized for various applications in industry and agriculture. However, it should be used with care because thiophenol is readily absorbed into the human body by inhalation and ingestion, which leads to serious internal injuries. Thus, there is an urgent need for real-time and accurate monitoring of thiophenol. Despite remarkable advantages of electrogenerated chemiluminescence (ECL) analysis, ECL thiophenol probes have never been reported. Herein, a new strategy for the rapid detection of thiophenol by use of an ECL turn-on chemodosimeter based on a cyclometalated Ir(III) complex is described. This analytical system showed superior sensitivity [limit of detection (LOD) value, 3.8 nM] in comparison to the conventional fluorescence method. In addition, our system exhibited remarkable selectivity and reaction rate toward thiophenol over other analytes. Moreover, it was successfully applied to quantify thiophenol in real water samples, providing a new proof-of-concept for field monitoring based on ECL.

Biotransformation of polyunsaturated fatty acids to bioactive hepoxilins and trioxilins by microbial enzymes.

Hepoxilins (HXs) and trioxilins (TrXs) are involved in physiological processes such as inflammation, insulin secretion and pain perception in human. They are metabolites of polyunsaturated fatty acids (PUFAs), including arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid, formed by 12-lipoxygenase (LOX) and epoxide hydrolase (EH) expressed by mammalian cells. Here, we identify ten types of HXs and TrXs, produced by the prokaryote Myxococcus xanthus, of which six types are new, namely, HXB5, HXD3, HXE3, TrXB5, TrXD3 and TrXE3. We succeed in the biotransformation of PUFAs into eight types of HXs (>35% conversion) and TrXs (>10% conversion) by expressing M. xanthus 12-LOX or 11-LOX with or without EH in Escherichia coli. We determine 11-hydroxy-eicosatetraenoic acid, HXB3, HXB4, HXD3, TrXB3 and TrXD3 as potential peroxisome proliferator-activated receptor-γ partial agonists. These findings may facilitate physiological studies and drug development based on lipid mediators.

Structure-based prediction and identification of 4-epimerization activity of phosphate sugars in class II aldolases.

Sugar 4-epimerization reactions are important for the production of rare sugars and their derivatives, which have various potential industrial applications. For example, the production of tagatose, a functional sweetener, from fructose by sugar 4-epimerization is currently constrained because a fructose 4-epimerase does not exist in nature. We found that class II D-fructose-1,6-bisphosphate aldolase (FbaA) catalyzed the 4-epimerization of D-fructose-6-phosphate (F6P) to D-tagatose-6-phosphate (T6P) based on the prediction via structural comparisons with epimerase and molecular docking and the identification of the condensed products of C3 sugars. In vivo, the 4-epimerization activity of FbaA is normally repressed. This can be explained by our results showing the catalytic efficiency of D-fructose-6-phosphate kinase for F6P phosphorylation was significantly higher than that of FbaA for F6P epimerization. Here, we identified the epimerization reactions and the responsible catalytic residues through observation of the reactions of FbaA and L-rhamnulose-1-phosphate aldolases (RhaD) variants with substituted catalytic residues using different substrates. Moreover, we obtained detailed potential epimerization reaction mechanism of FbaA and a general epimerization mechanism of the class II aldolases L-fuculose-1-phosphate aldolase, RhaD, and FbaA. Thus, class II aldolases can be used as 4-epimerases for the stereo-selective synthesis of valuable carbohydrates.

High-yield production of pure tagatose from fructose by a three-step enzymatic cascade reaction.

OBJECTIVE: To produce tagatose from fructose with a high conversion rate and to establish a high-yield purification method of tagatose from the reaction mixture. RESULTS: Fructose at 1 M (180 g l-1) was converted to 0.8 M (144 g l-1) tagatose by a three-step enzymatic cascade reaction, involving hexokinase, plus ATP, fructose-1,6-biphosphate aldolase, phytase, over 16 h with a productivity of 9 g l-1 h-1. No byproducts were detected. Tagatose was recrystallized from ethanol to a purity of 99.9% and a yield of 96.3%. Overall, tagatose at 99.9% purity was obtained from fructose with a yield of 77%. CONCLUSION: This is the first biotechnological production of tagatose from fructose and the first application of solvent recrystallization for the purification of rare sugars.

Complete genome sequence of Stenotrophomonas sp. KACC 91585, an efficient bacterium for unsaturated fatty acid hydration.

Hydroxy fatty acids (HFAs) such as 10-hydroxystearic acid (10-HSA) and 10-hydroxy-12(Z)-octadecenoic acid (10-HOD), which are similar to ricinoleic acid, are important starting materials and intermediates for the industrial manufacture of many commodities. Stenotrophomonas sp. KACC 91585, which was isolated from lake sediment, is an efficient bacterium for unsaturated fatty acid hydration that produces 10-HSA and 10-HOD from oleic acid and linoleic acid, respectively, with high conversion rates. The complete genome of this strain is 4,541,729bp with 63.83% GC content and devoid of plasmids. Sets of genes involved in the fatty acid biosynthesis and modification as well as modified lipids were identified in the genome, and these genes were concerned with HFA production. This genome sequence provides molecular information and elucidation for HFA production, and will be used as an efficient biocatalyst source for the biotechnological production of HFA.

D-Allulose Production from D-Fructose by Permeabilized Recombinant Cells of Corynebacterium glutamicum Cells Expressing D-Allulose 3-Epimerase Flavonifractor plautii.

A d-allulose 3-epimerase from Flavonifractor plautii was cloned and expressed in Escherichia coli and Corynebacterium glutamicum. The maximum activity of the enzyme purified from recombinant E. coli cells was observed at pH 7.0, 65°C, and 1 mM Co2+ with a half-life of 40 min at 65°C, Km of 162 mM, and kcat of 25280 1/s. For increased d-allulose production, recombinant C. glutamicum cells were permeabilized via combined treatments with 20 mg/L penicillin and 10% (v/v) toluene. Under optimized conditions, 10 g/L permeabilized cells produced 235 g/L d-allulose from 750 g/L d-fructose after 40 min, with a conversion rate of 31% (w/w) and volumetric productivity of 353 g/L/h, which were 1.4- and 2.1-fold higher than those obtained for nonpermeabilized cells, respectively.

Alternative Biotransformation of Retinal to Retinoic Acid or Retinol by an Aldehyde Dehydrogenase from Bacillus cereus.

UNLABELLED: A novel bacterial aldehyde dehydrogenase (ALDH) that converts retinal to retinoic acid was first identified in Bacillus cereus The amino acid sequence of ALDH from B. cereus (BcALDH) was more closely related to mammalian ALDHs than to bacterial ALDHs. This enzyme converted not only small aldehydes to carboxylic acids but also the large aldehyde all-trans-retinal to all-trans-retinoic acid with NAD(P)(+) We newly found that BcALDH and human ALDH (ALDH1A1) could reduce all-trans-retinal to all-trans-retinol with NADPH. The catalytic residues in BcALDH were Glu266 and Cys300, and the cofactor-binding residues were Glu194 and Glu457. The E266A and C300A variants showed no oxidation activity. The E194S and E457V variants showed 15- and 7.5-fold higher catalytic efficiency (kcat/Km) for the reduction of all-trans-retinal than the wild-type enzyme, respectively. The wild-type, E194S variant, and E457V variant enzymes with NAD(+) converted 400 µM all-trans-retinal to 210 µM all-trans-retinoic acid at the same amount for 240 min, while with NADPH, they converted 400 µM all-trans-retinal to 20, 90, and 40 µM all-trans-retinol, respectively. These results indicate that BcALDH and its variants are efficient biocatalysts not only in the conversion of retinal to retinoic acid but also in its conversion to retinol with a cofactor switch and that retinol production can be increased by the variant enzymes. Therefore, BcALDH is a novel bacterial enzyme for the alternative production of retinoic acid and retinol. IMPORTANCE: Although mammalian ALDHs have catalyzed the conversion of retinal to retinoic acid with NAD(P)(+) as a cofactor, a bacterial ALDH involved in the conversion is first characterized. The biotransformation of all-trans-retinal to all-trans-retinoic acid by BcALDH and human ALDH was altered to the biotransformation to all-trans-retinol by a cofactor switch using NADPH. Moreover, the production of all-trans-retinal to all-trans-retinol was changed by mutations at positions 194 and 457 in BcALDH. The alternative biotransformation of retinoids was first performed in the present study. These results will contribute to the biotechnological production of retinoids, including retinoic acid and retinol.

Selective Production of 9R-Hydroxy-10E,12Z,15Z-Octadecatrienoic Acid from α-Linolenic Acid in Perilla Seed Oil Hydrolyzate by a Lipoxygenase from Nostoc Sp. SAG 25.82.

Hydroxy fatty acids (HFAs) derived from omega-3 polyunsaturated fatty acids have been known as versatile bioactive molecules. However, its practical production from omega-3 or omega-3 rich oil has not been well established. In the present study, the stereo-selective enzymatic production of 9R-hydroxy-10E,12Z,15Z-octadecatrienoic acid (9R-HOTE) from α-linolenic acid (ALA) in perilla seed oil (PO) hydrolyzate was achieved using purified recombinant 9R-lipoxygenase (9R-LOX) from Nostoc sp. SAG 25.82. The specific activity of the enzyme followed the order linoleic acid (LA) > ALA > γ-linolenic acid (GLA). A total of 75% fatty acids (ALA and LA) were used as a substrate for 9R-LOX from commercial PO by hydrolysis of Candida rugosa lipase. The optimal reaction conditions for the production of 9R-HOTE from ALA using 9R-LOX were pH 8.5, 15°C, 5% (v/v) acetone, 0.2% (w/v) Tween 80, 40 g/L ALA, and 1 g/L enzyme. Under these conditions, 9R-LOX produced 37.6 g/L 9R-HOTE from 40 g/L ALA for 1 h, with a conversion yield of 94% and a productivity of 37.6 g/L/h; and the enzyme produced 34 g/L 9R-HOTE from 40 g/L ALA in PO hydrolyzate for 1 h, with a conversion yields of 85% and a productivity of 34 g/L/h. The enzyme also converted 9R-hydroxy-10E,12Z-octadecadienoic acid (9R-HODE) from 40 g/L LA for 1.0 h, with a conversion yield of 95% and a productivity of 38.4 g/L. This is the highest productivity of HFA from both ALA and ALA-rich vegetable oil using LOX ever reported. Therefore, our result suggests an efficient method for the production of 9R-HFAs from LA and ALA in vegetable oil using recombinant LOX in biotechnology.

Biochemical properties of retinoid-converting enzymes and biotechnological production of retinoids.

Retinoids are a class of compounds that are forms of vitamin A and include retinal, retinol, retinoic acid, and retinyl ester. Retinal is involved in visual cycle, retinol has anti-infective, anticancer, antioxidant, and anti-wrinkle functions, and retinoic acid is used to treat acne and cancer. Retinol, retinoic acid, and retinyl ester are used in cosmetic and pharmaceutical industries. In this article, the biochemical properties and active sites and reaction mechanisms of retinoid-converting enzymes in animals and bacteria, including retinol dehydrogenase, alcohol dehydrogenase, aldo-keto reductase, and aldehyde dehydrogenase, are reviewed. The production of retinoids, using retinoid-producing enzymes and metabolically engineered cells, is also described. Uncharacterized bacterial proteins are suggested as retinoid-converting enzymes, and the production of retinoids using metabolically engineered cells is proposed as a feasible method.

Production of 13S-hydroxy-9(Z)-octadecenoic acid from linoleic acid by whole recombinant cells expressing linoleate 13-hydratase from Lactobacillus acidophilus.

Linoleate 13-hydratase from Lactobacillus acidophilus LMG 11470 converted linoleic acid to hydroxyl fatty acid, which was identified as 13S-hydroxy-9(Z)-octadecenoic acid (13-HOD) by GC-MS and NMR. The expression of linoleate 13-hydratase gene in Escherichia coli was maximized by using pACYC plasmid and super optimal broth with catabolite repression (SOC) medium containing 40mM Mg(2+). To optimize induction conditions, recombinant cells were cultivated at 37°C, 1mM isopropyl-ß-d-thiogalactopyranoside was added at 2h, and the culture was further incubated at 16°C for 18h. Recombinant cells expressing linoleate 13-hydratase from L. acidophilus were obtained under the optimized expression conditions and used for 13-HOD production from linoleic acid. The optimal reaction conditions were pH 6.0, 40°C, 0.25% (v/v) Tween 40, 25gl(-1) cells, and 100gl(-1) linoleic acid, and under these conditions, whole recombinant cells produced 79gl(-1) 13-HOD for 3h with a conversion yield of 79% (w/w), a volumetric productivity of 26.3gl(-1)h(-1), and a specific productivity of 1.05g g-cells(-1)h(-1). To the best of our knowledge, the recombinant cells produced hydroxy fatty acid with the highest concentration and productivity reported so far.

Unveiling of novel regio-selective fatty acid double bond hydratases from Lactobacillus acidophilus involved in the selective oxyfunctionalization of mono- and di-hydroxy fatty acids.

The aim of this study is the first time demonstration of cis-12 regio-selective linoleate double-bond hydratase. Hydroxylation of fatty acids, abundant feedstock in nature, is an emerging alternative route for many petroleum replaceable products thorough hydroxy fatty acids, carboxylic acids, and lactones. However, chemical route for selective hydroxylation is still quite challenging owing to low selectivity and many environmental concerns. Hydroxylation of fatty acids by hydroxy fatty acid forming enzymes is an important route for selective biocatalytic oxyfunctionalization of fatty acids. Therefore, novel fatty acid hydroxylation enzymes should be discovered. The two hydratase genes of Lactobacillus acidophilus were identified by genomic analysis, and the expressed two recombinant hydratases were identified as cis-9 and cis-12 double-bond selective linoleate hydratases by in vitro functional validation, including the identification of products and the determination of regio-selectivity, substrate specificity, and kinetic parameters. The two different linoleate hydratases were the involved enzymes in the 10,13-dihydroxyoctadecanoic acid biosynthesis. Linoleate 13-hydratase (LHT-13) selectively converted 10 mM linoleic acid to 13S-hydroxy-9(Z)-octadecenoic acid with high titer (8.1 mM) and yield (81%). Our study will expand knowledge for microbial fatty acid-hydroxylation enzymes and facilitate the designed production of the regio-selective hydroxy fatty acids for useful chemicals from polyunsaturated fatty acid feedstocks.

Critical appraisal of implant impression accuracies: A systematic review.

STATEMENT OF PROBLEM: Different assessment methods have been used to measure the accuracy of implant impression techniques; therefore, the readers should understand the benefits and limitations of each assessment method used. PURPOSE: The purpose of this systematic review was to classify the implant impression studies by the assessment methods and techniques used and to understand the characteristics of each assessment method. The results of published studies were also analyzed to draw meaningful conclusions about the accuracy of the implant impressions. MATERIAL AND METHODS: An electronic search of the MEDLINE/PubMed database was performed in February 2013 using specific search terms and predetermined criteria to identify and assess laboratory studies of the accuracy of implant impression techniques. A final list of articles deemed to be of interest was comprehensively reviewed by 2 reviewers to ensure that these were suitable for the purpose of this review. The results of the current review were also compared with results from a previous systematic review. RESULTS: A total of 56 studies met the inclusion criteria for this review. Thirty-seven studies measured the amount of linear distortion, and 17 studies compared the angular change to assess the accuracy. Most linear or angular distortions were only measured in 2 dimensions, and 3-dimensional analysis was rare. More than 80% of the studies compared nonsplinting versus splinting, direct versus indirect techniques, and different impression materials. CONCLUSIONS: In recent publications, the direct or splint technique showed more accurate results than the indirect or nonsplinted technique. In contrast to external connection implants, inconsistent results were reported for internal connection implants.

Microbial synthesis of plant oxylipins from γ-linolenic acid through designed biotransformation pathways.

Secondary metabolites of plants are often difficult to synthesize in high yields because of the large complexity of the biosynthetic pathways and challenges encountered in the functional expression of the required biosynthetic enzymes in microbial cells. In this study, the biosynthesis of plant oxylipins--a family of oxygenated unsaturated carboxylic acids--was explored to enable a high-yield production through a designed microbial synthetic system harboring a set of microbial enzymes (i.e., fatty acid double-bond hydratases, alcohol dehydrogenases, Baeyer-Villiger monooxygenases, and esterases) to produce a variety of unsaturated carboxylic acids from γ-linolenic acid. The whole cell system of the recombinant Escherichia coli efficiently produced (6Z,9Z)-12-hydroxydodeca-6,9-dienoic acid (7), (Z)-9-hydroxynon-6-enoic acid (15), (Z)-dec-4-enedioic acid (17), and (6Z,9Z)-13-hydroxyoctadeca-6,9-dienoic acid (2). This study demonstrated that various secondary metabolites of plants can be produced by implementing artificial biosynthetic pathways into whole-cell biocatalysis.

Efficacy, tolerability, and safety of oxybutynin chloride in pediatric neurogenic bladder with spinal dysraphism: a retrospective, multicenter, observational study.

PURPOSE: Anticholinergics are a key element in treating neurogenic detrusor overactivity, but only limited data are available in the pediatric population, thus limiting the application to children even for oxybutynin chloride (OC), a prototype drug. This retrospective study was designed to provide data regarding the efficacy, tolerability, and safety of OC in the pediatric population (0-15 years old) with spinal dysraphism (SD). MATERIALS AND METHODS: Records relevant to OC use for neurogenic bladder were gathered and scrutinized from four specialized clinics for pediatric urology. The primary efficacy outcomes were maximal cystometric capacity (MCC) and end filling pressure (EFP). Data on tolerability, compliance, and adverse events (AEs) were also analyzed. RESULTS: Of the 121 patient records analyzed, 41 patients (34%) received OC at less than 5 years of age. The range of prescribed doses varied from 3 to 24 mg/d. The median treatment duration was 19 months (range, 0.3-111 months). Significant improvement of both primary efficacy outcomes was noted following OC treatment. MCC increased about 8% even after adjustment for age-related increases in MCC. Likewise, mean EFP was reduced from 33 to 21 cm H2O. More than 80% of patients showed compliance above 70%, and approximately 50% of patients used OC for more than 1 year. No serious AEs were reported; constipation and facial flushing consisted of the major AEs. CONCLUSIONS: OC is safe and efficacious in treating pediatric neurogenic bladder associated with SD. The drug is also tolerable and the safety profile suggests that adjustment of dosage for age may not be strictly observed.

Surgical outcome of urethroplasty using penile circular fasciocutaneous flap for anterior urethral stricture.

PURPOSE: Penile circular fasciocutaneous flap urethroplasty is a useful technique for a long anterior urethral stricture due to the flap's hairless nature and ample length. We investigated the surgical outcomes of urethroplasty for a complex anterior urethral stricture, performed using a penile circular fasciocutaneous flap. MATERIALS AND METHODS: Between 2008 and 2013, we performed a retrospective review of 29 patients who underwent urethroplasty using a penile circular fasciocutaneous flap and had at least 6 months of follow-up. A total of 20 cases utilized only a fasciocutaneous flap, while 9 cases combined a fasciocutaneous flap with other surgery. Success was defined as no requirement of additional urethral instrumentation. RESULTS: The overall success rate was 68.9% (20 out of 29 cases) at a median follow-up of 19 months. Furthermore, fasciocutaneous flap urethroplasty rendered the actual stricture-free rate of 79.3%. The location of recurrence was mostly at the junction of the flap. Among 9 surgical failures, 5 cases were treated successfully by using an additional surgical procedure. Fistula repair was needed in 1 case 4 months later. Further, periodic urethral dilation was performed in the remaining 3 cases. The failure rate was significantly higher in patients with suprapubic cystostomy than in patients without suprapubic cystostomy. The most common complication was post-micturition dribbling. CONCLUSIONS: Penile circular fasciocutaneous flap urethroplasty is a useful method for the reconstruction of a long anterior urethral stricture. A sufficient healthy margin should be acquired for better surgical results due to the fact that most recurrence occurs at the junction of the flap.

Molecular characterization of an aldo-keto reductase from Marivirga tractuosa that converts retinal to retinol.

A recombinant aldo-keto reductase (AKR) from Marivirga tractuosa was purified with a specific activity of 0.32unitml(-1) for all-trans-retinal with a 72kDa dimer. The enzyme had substrate specificity for aldehydes but not for alcohols, carbonyls, or monosaccharides. The enzyme turnover was the highest for benzaldehyde (kcat=446min(-1)), whereas the affinity and catalytic efficiency were the highest for all-trans-retinal (Km=48µM, kcat/Km=427mM(-1)min(-1)) among the tested substrates. The optimal reaction conditions for the production of all-trans-retinol from all-trans-retinal by M. tractuosa AKR were pH 7.5, 30°C, 5% (v/v) methanol, 1% (w/v) hydroquinone, 10mM NADPH, 1710mgl(-1) all-trans-retinal, and 3unitml(-1) enzyme. Under these optimized conditions, the enzyme produced 1090mgml(-1) all-trans-retinol, with a conversion yield of 64% (w/w) and a volumetric productivity of 818mgl(-1)h(-1). AKR from M. tractuosa showed no activity for all-trans-retinol using NADP(+) as a cofactor, whereas human AKR exhibited activity. When the cofactor-binding residues (Ala158, Lys212, and Gln270) of M. tractuosa AKR were changed to the corresponding residues of human AKR (Ser160, Pro212, and Glu272), the A158S and Q270E variants exhibited activity for all-trans-retinol. Thus, amino acids at positions 158 and 270 of M. tractuosa AKR are determinant residues of the activity for all-trans-retinol.