Total Knee Arthroplasty Is Superior to Open Wedge High Tibial Osteotomy in Terms of Pain Relief for Patients With Osteoarthritis.

BACKGROUND: Globally, total knee arthroplasty (TKA) is widely performed on patients with osteoarthritis. Meanwhile, open wedge high tibial osteotomy (OWHTO) has garnered attention in our country as a joint-preserving procedure. This study aimed to retrospectively compare the postoperative clinical outcomes of TKA and OWHTO for patients with osteoarthritis. METHODS: We selected 94 patients (106 knees) who underwent OWHTO or TKA between 2013 and 2018, had complete clinical data, and were followed up for >2 years. Patients were classified into 2 groups depending on the procedure (TKA: n = 49; OWHTO: n = 45). Patients in the A (= arthroplasty) group were significantly older, with a worse range of motion (ROM) than those in the O (osteotomy) group. There were no significant differences regarding sex and body mass index between groups. Operative time, perioperative blood loss, knee ROM, and Japanese Knee Injury and Osteoarthritis Outcome Score (J-KOOS) were compared between the groups. RESULTS: Significant differences were found between the A and O groups regarding operative time (120 ± 27.2 vs 80.3 ± 23.3 minutes), perioperative blood loss (505.4 ± 271.8 vs 322.6 ± 196.1 mL), knee ROM (flexion; 123.4 ± 16.3° vs 133.7 ± 12.8°), and J-KOOS for pain (87.4 ± 12.5 vs 78.1 ± 15.2 points) and symptoms (86.6 ± 12.3 vs 79.1 ± 13.3 points). There were no significant differences regarding other J-KOOS subscales. CONCLUSIONS: OWHTO involved shorter operative times and less blood loss. However, the O group reported less pain relief. The A group represents an older, likely less active patient population. Therefore, OWHTO is a possible joint-preserving treatment options in younger active patients who may not be interested in arthroplasty.

Intra-articular administration of EP2 enhances the articular cartilage repair in a rabbit model.

We have reported the usefulness of chondrocyte sheets on articular cartilage repair in animal experiments. Here, we investigated the regenerative effects of EP2 signalling with or without chondrocyte sheets. Forty-five rabbits were used, with six rabbits in each of the six groups and nine rabbits for chondrocytes and synovial cells harvesting to fabricate triple-layered chondrocyte sheets: osteochondral defect only (control, Group A), EP2 agonist (Group B), EP2 antagonist (Group C), chondrocyte sheets (Group D), EP2 agonist and chondrocyte sheets (Group E), and EP2 antagonist and chondrocyte sheets (Group F). After surgery, the weight distribution ratio was measured as an indicator of pain alleviation. Injections of the EP2 agonist or EP2 antagonist were given from 4 weeks after surgery. The rabbits were sacrificed at 12 weeks, and the repaired tissues were evaluated for histology. The weight distribution ratio and International Cartilage Repair Society grading were as follows: Group A: 40.5% ± 0.2%, 14.8 ± 0.5; Group B: 43.4% ± 0.7%, 25.4 ± 0.8; Group C: 38.7% ± 0.7%, 13.7 ± 0.3; Group D: 48.6% ± 0.6%, 40.2 ± 0.5; Group E: 49.1% ± 0.3%, 40.5 ± 0.4; and Group F; 46.8% ± 0.4%, 38.7 ± 0.5. Significant differences in histology and pain alleviation were observed between groups except between Groups A and C, between Groups D and E, and between Groups D and F. These findings show that the intra-articular administration of an EP2 agonist achieved pain alleviation and tissue repair. However, no synergistic effect with chondrocyte sheets was observed.

Platelet-activated serum might have a therapeutic effect on damaged articular cartilage.

Platelet-activated serum (PAS) was collected from rabbits. This contains high concentrations of growth factors, including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)-BB, and transforming growth factor-beta (TGF-ß). PAS was injected into the knee joints of Japanese White rabbits subjected to anterior cruciate ligament transection (ACL-T) to investigate its therapeutic effects on articular cartilage. The effect of Avastin (an anti-VEGF monoclonal antibody) on VEGF expression was also investigated. The levels of VEGF, PDGF-BB, and TGF-ß in PAS, platelet-rich plasma (PRP) and autologous serum from untreated rabbits were analysed by enzyme-linked immunosorbent assays. The samples (n = 24 rabbits) were divided into control (C), PAS (S), Avastin (A) and PAS + Avastin (S + A) treatment groups. Intra-articular injections were administered weekly for 7 weeks after ACL-T, during which the weight distribution ratios of the damaged limbs were evaluated. Histological evaluation was performed 12 weeks after ACL-T using Mankin score. The VEGF, PDGF-BB and TGF-ß expression levels were significantly higher (P < 0.05) in the PAS than in the PRP or autologous serum samples. The weight distribution ratios of damaged limbs improved significantly after ACL-T in all treatment groups (P < 0.05). The proximal medial, distal medial and lateral aspects of joints in the treatment groups showed significant differences in Mankin scores compared with controls (P < 0.05). The damaged limb weight distribution ratios, Mankin scores and articular cartilage structure did not differ significantly among the three treatment groups, which all showed significant improvements in structure compared with controls. Copyright © 2017 John Wiley & Sons, Ltd.

The effects of using vitrified chondrocyte sheets on pain alleviation and articular cartilage repair.

The effect of using vitrified-thawed chondrocyte sheets on articular cartilage repair was examined because the methods for storing chondrocyte sheets are essential for allogeneic chondrocyte sheet transplantation. Six Japanese white rabbits were used as sources of articular chondrocytes and synovial cells. Chondrocytes were harvested from the femur, and synovial cells were harvested from inside the knee joints. After coculture of the chondrocytes with synovial cells, triple-layered chondrocyte sheets were fabricated. Eighteen rabbits were used, with six rabbits in each of three groups: osteochondral defect only (control, group A); chondrocyte sheets (group B); and vitrified-thawed chondrocyte sheets (group C). An osteochondral defect was created on the femur. After transplantation, the weight distribution ratio of the undamaged and damaged limbs was measured as a pain-alleviating effect. The rabbits were euthanized at 12 weeks, and the transplanted tissues were evaluated for histology (Safranin O staining and immunostaining) using the International Cartilage Repair Society grading system. For both evaluations, significant differences were observed between groups A and B, and between groups A and C (p < 0.05). No significant differences were observed between groups B and C. Thus, pain-alleviating effects and tissue repair were achieved using vitrified-thawed chondrocyte sheets. Copyright © 2017 John Wiley & Sons, Ltd.

Effects of a cell-free method using collagen vitrigel incorporating TGF-ß1 on articular cartilage repair in a rabbit osteochondral defect model.

We studied the ability of collagen vitrigel material to repair cartilage in vivo when used alone or with transforming growth factor-ß (TGF-ß). We measured the time course and quantity of TGF-ß1 released from the collagen vitrigel in vitro to quantify the controlled release of TGF-ß1. Over 14 days, 0.91 ng of TGF-ß was released from the collagen vitrigel. Osteochondral defects were made in the femoral trochlear groove in 36 Japanese white rabbits, which were divided into three groups: untreated group (group A), collagen vitrigel-implanted group (group B), and TGF-ß1-incorporated collagen vitrigel-implanted group (group C). The weight distribution ratio between the affected and unaffected limbs served as an indicator of pain. Animals were sacrificed at 4 and 12 weeks after surgery, and their tissues were assessed histologically. The weight distribution ratio increased in all groups and did not differ significantly between groups at 12 weeks. Group A needed 6 weeks to attain maximum improvement, and groups B and C showed near-maximum improvement at 4 and 2 weeks, respectively. The International Cartilage Repair Society II score improved significantly in group C relative to the other groups. These findings suggest that sustained, slow release of TGF-ß caused early pain mitigation and cartilage repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2592-2602, 2017.

Usefulness of using laser-induced photoacoustic measurement and 3.0 Tesla MRI to assess knee cartilage damage: a comparison study.

BACKGROUND: T2 mapping is an MRI method particularly reflective of the collagen arrangement in the cartilage, and diffusion tensor (DT) imaging captures the diffusion of water molecules. Laser-induced photoacoustic measurement (LIPA) makes it possible to assess not only the thickness of the cartilage layer but also its viscoelastic properties. By assessing cartilage damage assessment using LIPA and 3.0 Tesla MRI (T2 mapping and DT imaging), this study investigates the usefulness of the various methods. METHODS: The International Cartilage Repair Society (ICRS) classification was used to classify 29 bone cartilage pieces excised during surgical procedures. At the same time, LIPA was performed at sites matching the area of cartilage damage. MRI was performed preoperatively to measure the T2 and the apparent diffusion coefficient. In addition, tissue sections for histological assessment using the Mankin score were prepared for each ICRS grade, and the results with the various methods were compared. RESULTS: With DT imaging, significant differences were observed in all grades (P < 0.01). With T2 mapping, significant differences were observed in all grades except for grade 1 versus grade 2 (P < 0.01). With LIPA, significant differences were observed in ICRS grade 1 versus grade 3 (P < 0.05), grade 1 versus grade 4 (P < 0.01), grade 2 versus grade 4 (P < 0.01), and grade 3 versus grade 4 (P < 0.05). With the Mankin score, significant differences were observed in ICRS grade 1 versus grade 3 (P < 0.01), grade 1 versus grade 4 (P < 0.01), grade 2 versus grade 4 (P < 0.01), and grade 3 versus grade 4 (P < 0.01). Correlations were observed in all combinations of ICRS grade with DT imaging, T2 mapping, LIPA, and Mankin score. Correlations were observed between the degree of histological degeneration and DT imaging, T2 mapping, and ICRS grade, but LIPA had a weaker correlation than MRI. CONCLUSIONS: In the assessment of knee osteoarthritis, there are instances where it is difficult to assess the damaged cartilage site with MRI alone, and we believe that it is desirable to use a combination of LIPA and MRI.

Bevacizumab, an anti-vascular endothelial growth factor antibody, inhibits osteoarthritis.

INTRODUCTION: Angiogenesis is an important factor in the development of osteoarthritis (OA). We investigated the efficacy of bevacizumab, an antibody against vascular endothelial growth factor and an inhibitor of angiogenesis, in the treatment of OA using a rabbit model of anterior cruciate ligament transection. METHODS: First, we evaluated the response of gene expression and histology of the normal joint to bevacizumab treatment. Next, in a rabbit model of OA induced by anterior cruciate ligament transection, we used macroscopic and histological evaluations and real-time polymerase chain reaction (PCR) to examine the responses to intravenous (systemic) administration of bevacizumab (OAB IV group). We also investigated the efficacy of intra-articular (local) administration of bevacizumab in OA-induced rabbits (OAB IA group). RESULTS: Histologically, bevacizumab had no negative effect in normal joints. Bevacizumab did not increase the expression of genes for catabolic factors in the synovium, subchondral bone, or articular cartilage, but it increased the expression of collagen type 2 in the articular cartilage. Macroscopically and histologically, the OAB IV group exhibited a reduction in articular cartilage degeneration and less osteophyte formation and synovitis compared with the control group (no bevacizumab; OA group). Real-time PCR showed significantly lower expression of catabolic factors in the synovium in the OAB IV group compared with the OA group. In articular cartilage, expression levels of aggrecan, collagen type 2, and chondromodulin-1 were higher in the OAB IV group than in the OA group. Histological evaluation and assessment of pain behaviour showed a superior effect in the OAB IA group compared with the OAB IV group 12 weeks after administration of bevacizumab, even though the total dosage given to the OAB IA group was half that received by the OAB IV group. CONCLUSIONS: Considering the dosage and potential adverse effects of bevacizumab, the local administration of bevacizumab is a more advantageous approach than systemic administration. Our results suggest that intra-articular bevacizumab may offer a new therapeutic approach for patients with post-traumatic OA.

Efficient screening for astaxanthin-overproducing mutants of the yeast Xanthophyllomyces dendrorhous by flow cytometry.

Astaxanthin possesses higher antioxidant activity than other carotenoids and, for this and other reasons, has great commercial potential for use in the aquaculture, pharmaceutical, and food industries. The basidiomycetous yeast Xanthophyllomyces dendrorhous is one of the best natural producers of astaxanthin, but wild-type cells accumulate only a small amount of astaxanthin. In this study, we developed an efficient flow cytometry method to screen for astaxanthin-overproducing mutants of X. dendrorhous. We first examined the relationship between cellular astaxanthin content and the intensity of fluorescence emitted from the cell. Although the fluorescence emission maximum of astaxanthin dissolved in acetone occurred at 570 nm, intracellular astaxanthin content correlated better with emission at around 675 nm in different X. dendrorhous strains. Using this emission wavelength, we screened cells mutagenized with ethyl methanesulfonate and successfully isolated mutants that produced 1.5-3.8-fold more astaxanthin than parent cells. This method enabled us to obtain overproducers five times more efficient than conventional screening from plate culture.

Cloning, sequence analysis, and expression in Escherichia coli of gene encoding N-Benzyl-3-pyrrolidinol dehydrogenase from Geotrichum capitatum.

The gene encoding N-benzyl-3-pyrrolidinol dehydrogenase (DDBJ/EMBL/GenBank accession no. AB294179), a useful biocatalyst for producing (S)-N-benzyl-3-pyrrolidinol, was cloned from the genomic DNA of Geotrichum capitatum JCM 3908. The gene contained an open reading frame consisting of 1023 nucleotides corresponding to 340 amino acid residues. The subunit molecular weight was calculated to be 39,000. The predicted amino acid sequence did not have significant similarity to those of N-benzyl-3-pyrrolidinone reductases reported previously. From 30 mM N-benzyl-3-pyrrolidinone, (S)-N-benzyl-3-pyrrolidinol was obtained with a yield >99.9% and an enantiomeric excess >99.9% in 1-h and 2-h reactions without NADH addition by the resting cells of Escherichia coli HB 101 strains harboring the expression plasmids pSG-POBS and pSF-POBS that possess the glucose dehydrogenase gene and formate dehydrogenase gene as an NADH-reproducing system, respectively, besides the N-benzyl-3-pyrrolidinol dehydrogenase gene. N-Benzyl-3-pyrrolidinol dehydrogenase activity (0.56 U/mg) was observed in E. coli (pSG-POBS), which was 17-fold the specific activity observed in G. capitatum JCM 3908.

Purification and characterization of 2-aminoacetophenone reductase of newly isolated Burkholderia sp. YT.

We found that a newly isolated Burkholderia sp. produced (R)-2-amino-1-phenylethanol from 2-aminoacetophenone, showing the high stereospecificity. NADPH-dependent 2-aminoacetophenone reductase purified to homogeneity was a dimer with a molecular mass of 65,000. The purified enzyme did not reduce acetophenone and 1-phenyl-1-propanone. The purified enzyme converted 2-aminoacetophenone to only (R)-2-amino-1-phenylethanol.

Functional analysis of genes encoding putative oxidoreductases in Aspergillus oryzae, which are similar to fungal fructosyl-amino acid oxidase.

We found 11 genes (FAO1-11) encoding putative oxidoreductases in the Aspergillus oryzae genome, which are similar to fungal fructosyl-amino acid oxidases. The cDNAs corresponding to the genes were cloned and expressed in Escherichia coli. rFao2 had fructosyl-amino acid oxidase activity, whereas rFao1 did not show any enzyme activity, even though the deduced amino acid sequence of Fao1 is identical to that of one of the fructosyl-amino acid oxidase isozymes from Aspergillus oryzae. rFao7 and rFao8 showed oxidase activity toward sarcosine, L-pipecolate, and L-proline. rFao10 was active toward only sarcosine, of the substrates tested. The functions of the other proteins were also predicted from a phylogenetic analysis.

Screening of carbon dioxide-requiring extreme oligotrophs from soil.

We screened soil samples for CO(2)-requiring extreme oligotrophs similar to Rhodococcus erythropolis N9T-4, which can grow on a basal salt agar medium without an organic carbon source. From 387 soil samples, three isolates were obtained and identified as Streptomyces spp. by 16S rDNA analysis. The isolates required gaseous CO(2) for growth and grew on a basal salt medium solidified by silica gel. These results suggest that such CO(2)-requiring oligotrophs occur widely in nature.

An extremely oligotrophic bacterium, Rhodococcus erythropolis N9T-4, isolated from crude oil.

Rhodococcus erythropolis N9T-4, which was isolated from crude oil, showed extremely oligotrophic growth and formed its colonies on a minimal salt medium solidified using agar or silica gel without any additional carbon source. N9T-4 did not grow under CO(2)-limiting conditions but could grow on a medium containing NaHCO(3) under the same conditions, suggesting that the oligotrophic growth of N9T-4 depends on CO(2). Proteomic analysis of N9T-4 revealed that two proteins, with molecular masses of 45 and 55 kDa, were highly induced under the oligotrophic conditions. The primary structures of these proteins exhibited striking similarities to those of methanol: N,N'-dimethyl-4-nitrosoaniline oxidoreductase and an aldehyde dehydrogenase from Rhodococcus sp. These enzyme activities were three times higher under oligotrophic conditions than under n-tetradecane-containing heterotrophic conditions, and gene disruption for the aldehyde dehydrogenase caused a lack of growth on the minimal salt medium. Furthermore, 3-hexulose 6-phosphate synthase and phospho-3-hexuloisomerase activities, which are key enzymes in the ribulose monophosphate pathway in methylotrophic bacteria, were detected specifically in the cell extract of oligotrophically grown N9T-4. These results suggest that CO(2) fixation involves methanol (formaldehyde) metabolism in the oligotrophic growth of R. erythropolis N9T-4.

Production of optically active 1,2,4-butanetriol from corresponding racemate by Microbial stereoinversion.

Sterigmatomyces elviae DSM 70852 produced 12 g/l (S)-1,2,4-butanetriol (enantiomeric excess >99.9%) from 20 g/l racemate in 82 h. From the results of the inversion of an (R)-isomer to an (S)-isomer and GC-MS, it was suggested that (R)-1,2,4-butanetriol is oxidized to 1,4-dihydroxy-2-butanone, which is reduced to an (S)-isomer.

Purification and characterization of alcohol dehydrogenase reducing N-benzyl-3-pyrrolidinone from Geotrichum capitatum.

(S)-N-Benzyl-3-pyrrolidinol is widely used in the synthesis of pharmaceuticals as a chiral building block. We produced 30 mM (S)-N-benzyl-3-pyrrolidinol (enantiometric excess > 99.9%) from the corresponding ketone N-benzyl-3-pyrrolidinone with more than 99.9% yield in 28 h of the resting-cell reaction of Geotrichum capitatum JCM 3908. NAD(+)-dependent alcohol dehydrogenase reducing N-benzyl-3-pyrrolidinone from G. capitatum JCM 3908 was purified to homogeneity by ammonium sulfate fractionation and a series of DEAE-Toyopearl, Butyl-Toyopearl, Superdex 200, and Hydroxyapatite column chromatographies. The results of SDS-PAGE and HPLC showed the enzyme to be a dimer with a molecular mass of 78 kDa. The purified enzyme produced (S)-N-benzyl-3-pyrrolidinol (e.e.>99.9%) from N-benzyl-3-pyrrolidinone. The enzyme reduced 2,3-butanedione, 2-hexanone, cyclohexanone, propionaldehyde, n-butylaldehyde, n-hexylaldehyde, n-octylaldehyde, n-valeraldehyde, and benzylacetone more effectively than it did N-benzyl-3-pyrrolidinone. No activity was detected towards N-benzyl-2-pyrrolidinone or 2-pyrrolidinone. The activity towards (R)-N-benzyl-3-pyrrolidinol was not detected under the assay conditions employed. The oxidizing activity of the enzyme was higher towards 2-propanol, 2-butanol, 2-pentanol, 2-hexanol, 3-hexanol, and 1-phenyl-2-propanol than towards (S)-N-benzyl-3-pyrrolidinol. The K(m) values for N-benzyl-3-pyrrolidinone reduction and (S)-N-benzyl-3-pyrrolidinol oxidation were 0.13 and 8.47 mM, respectively. To our knowledge, this is the first time that an N-benzyl-3-pyrrolidinol/N-benzyl-3-pyrrolidinone oxidoreductase was purified from a eukaryote; moreover, this is the first report of (S)-N-benzyl-3-pyrrolidinol dehydrogenase activity in microorganisms. This enzyme showed features different from those of known prokaryotic N-benzyl-3-pyrrolidinone reductases. This enzyme will be very useful for the production of chiral compounds.

Purification, characterization, and gene cloning of glycerol dehydrogenase from Hansenula ofunaensis, and its expression for production of optically active diol.

Optically active alcohol is an important building block as a versatile chiral synthon for the asymmetric synthesis of pharmaceuticals and agrochemicals. We purified and characterized glycerol dehydrogenase from Hansenula ofunaensis and prepared optically active 1,2-octanediol using a recombinant Escherichia coli harboring the glycerol dehydrogenase gene. The deduced amino acid sequence was investigated for identities with those of other alcohol dehydrogenases in the NCBI databank. The identification of the unknown product of a resting-cell reaction was performed by GC-MS. In the deduced amino acid sequence composed of 376 residues, the NAD(H) binding pattern and cysteine residues that correspond to the cysteine ligands at the zinc atom were conserved as they are in alcohol dehydrogenases from other origins. Glycerol dehydrogenase from Hansenula polymorpha DL-1 (Pichia angusta, DDBJ/EMBL/GenBank accession no. BAD32688) had the highest identity to our enzyme, showing 73% identity. Our glycerol dehydrogenase catalyzed the NAD(+)-dependent oxidation of long-chain secondary alcohols such as 1,2-pentanediol, 1,2-hexanediol, 1,2-heptanediol, and 1,2-octanediol. Activities toward 2,4-pentanediol and 2,5-hexanediol were hardly detected. From these results, it was confirmed that our enzyme requires two hydroxyl groups on adjacent carbon atoms for oxidation. 2,3-Pentanedione, 2,3-hexanedione, and 3,4-hexanedione were significantly reduced. The transformants oxidized only (R)-1,2-octanediol in 50 mM racemate (R:S=52:48), and produced (S)-1,2-octanediol (24 mM, <99.9% e.e.) after 24 h of incubation. The reaction product was suggested to be 1-hydroxy-2-octanone by GC-MS, which showed secondary hydroxyl groups oxidized. Glycerol dehydrogenase from H. ofunaensis could be useful for the production of long-chain optically active secondary alcohols.

Bacterial communities in petroleum oil in stockpiles.

Bacterial communities in crude-oil samples from Japanese oil stockpiles were investigated by 16S rRNA gene cloning, followed by denaturing gradient gel electrophoresis (DGGE) analysis. 16S rRNA genes were successfully amplified by PCR after isooctane treatment from three kinds of crude-oil sample collected at four oil stockpiles in Japan. DGGE profiles showed that bacteria related to Ochrobactrum anthropi, Burkholderia cepacia, Stenotrophomonas maltophilia, Propionibacterium acnes, and Brevundimonas diminuta were frequently detected in most crude-oil samples. The bacterial communities differed in the sampling time and layer. Among the predominant bacteria detected in the crude oil, only three species were found for bacteria isolated on agar plates and were related to Burkholderia, Stenotrophomonas, and Propionibacterium, while Ochrobactrum sp. could not be isolated although this species seemed to be the most abundant bacterium in crude oil from the DGGE profiles. Using an archaea-specific primer set, methanogens were found in crude-oil sludge but not in crude-oil samples, indicating that methanogens might be involved in sludge formation in oil stockpiles.

Fructosyl-amino acid oxidases of Aspergillus oryzae are induced by the reaction product, glucosone.

Aspergillus oryzae has two fructosyl-amino acid oxidase (FAOD) isozymes (AoFao1 and AoFao2), which are different in the substrate specificities. Northern blot analysis showed both FAO genes were induced by autoclave-browned medium containing l-lysine or l-valine. Studies with a mutant, that had a disrupted AoFAO2 gene, revealed that the expression of AoFAO1 by fructosyl l-valine depended on the expression of AoFAO2. Both genes were also induced by one of the FAOD-reaction products, glucosone. In contrast, other alpha-dicarbonyl compounds, which display a similar structure to that of glucosone were not able to induce the genes expression. These results imply that glucosone may contribute to the expression of FAO genes.

Occurrence of fructosyl-amino acid oxidase-reactive compounds in fungal cells.

Fructosyl-amino acid oxidase (FAOD)-reactive fraction (FRY) was found in commercial yeast extract. FRY showed very hydrophilic property and was adsorbed to phenylboronate silica gel, indicating that it contained the Amadori compound. TLC and amino acid analyses revealed that glucosone, lysine, and arginine were produced from FRY after incubation with FAOD. TOF-MS analysis confirmed that FRY is a mixture of fructosyl lysine and fructosyl arginine. These compounds were also detected in mycelial extract of an FAOD-producer, Aspergillus terreus GP1, grown on the minimum medium, suggesting that a glycation reaction occurs in fungal cells and that FAOD acts toward the resultant Amadori compounds.

Functional analysis of fructosyl-amino acid oxidases of Aspergillus oryzae.

Three active fractions of fructosyl-amino acid oxidase (FAOD-Ao1, -Ao2a, and -Ao2b) were isolated from Aspergillus oryzae strain RIB40. N-terminal and internal amino acid sequences of FAOD-Ao2a corresponded to those of FAOD-Ao2b, suggesting that these two isozymes were derived from the same protein. FAOD-Ao1 and -Ao2 were different in substrate specificity and subunit assembly; FAOD-Ao2 was active toward N(epsilon)-fructosyl N(alpha)-Z-lysine and fructosyl valine (Fru-Val), whereas FAOD-Ao1 was not active toward Fru-Val. The genes encoding the FAOD isozymes (i.e., FAOAo1 and FAOAo2) were cloned by PCR with an FAOD-specific primer set. The deduced amino acid sequences revealed that FAOD-Ao1 was 50% identical to FAOD-Ao2, and each isozyme had a peroxisome-targeting signal-1, indicating their localization in peroxisomes. The genes was expressed in Escherichia coli and rFaoAo2 showed the same characteristics as FAOD-Ao2, whereas rFaoAo1 was not active. FAOAo2 disruptant was obtained by using ptrA as a selective marker. Wild-type strain grew on the medium containing Fru-Val as the sole carbon and nitrogen sources, but strain Delta faoAo2 did not grow. Addition of glucose or (NH(4))(2)SO(4) to the Fru-Val medium did not affect the assimilation of Fru-Val by wild-type, indicating glucose and ammonium repressions did not occur in the expression of the FAOAo2 gene. Furthermore, conidia of the wild-type strain did not germinate on the medium containing Fru-Val and NaNO(2) as the sole carbon and nitrogen sources, respectively, suggesting that Fru-Val may also repress gene expression of nitrite reductase. These results indicated that FAOD is needed for utilization of fructosyl-amino acids as nitrogen sources in A. oryzae.