Language：English   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science  

Perilymphatic fistula is defined as an abnormal communication between the perilymph-filled space and the middle ear, or cranial spaces. The manifestations include a broad spectrum of neuro-otological symptoms such as hearing loss, vertigo/dizziness, disequilibrium, aural fullness, tinnitus, and cognitive dysfunction. By sealing the fistula, perilymphatic fistula is a surgically correctable disease. Also, appropriate recognition and treatment of perilymphatic fistula can improve a patient’s condition and hence the quality of life. However, the difficulty in making a definitive diagnosis due to the lack of an appropriate biomarker to detect perilymph leakage has caused a long-standing debate regarding its management. We have reported a clinical test for the diagnosis of perilymphatic fistula by detecting a perilymph specific protein, Cochlin-tomoprotein, as a diagnostic marker using a western blot. The aim of this study is to establish an ELISA-based human Cochlin-tomoprotein detection test and to evaluate its diagnostic accuracy in clinical subjects. The results of ELISA showed good dilution reproducibility. The mean concentration was 49.7±9.4 of 10 perilymph samples. The ROC curve in differentiating the perilymph leakage condition from the normal middle ear was significant (P &lt
0.001) with an area under the curve (AUC) of 0.918 (95% CI 0.824–0.100). We defined the diagnostic criteria as follows: CTP&lt
0.4 negative
0.4≦CTP&lt
0.8 intermediate
0.8≦CTP(ng/ml) positive in the clinical usage of the hCTP ELISA, and sensitivity and specificity were 86.4% and 100%, respectively. We further tested the expression specificity of the Cochlin-tomoprotein by testing blood and CSF samples. The concentration was below the detection limit (0.2 ng/ml) in 38 of the 40 blood, and 14 of the 19 CSF samples. We report the accuracy of this test for the diagnosis of perilymphatic fistula. Using ELISA, we can improve the throughput of the test. Furthermore, it is useful for a large-scale study to characterize the clinical picture and delineate the management of this medical condition.

DOI： 10.1371/journal.pone.0191498

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Objective: To investigate the positive rate for the Cochlin tomo-protein (CTP: an inner ear-specific protein) detection test among patients with inner ear-related clinical manifestations and evaluate the clinical characteristics of definite perilymphatic fistula (PLF).
Methods: We have performed an ELISA-based CTP detection test using middle ear lavage (MEL) samples from 497 cases of suspected PLF enrolled from 70 clinical centers nationwide between 2014 and 2015. In addition to the CTP-positive rate, audio-vestibular symptoms were compared between CTP-positive and -negative cases.
Results: 8-50% of patients in category 1 (trauma, middle and inner ear disease cases), and about 20% of those in categories 2, 3 and 4 (external origin antecedent events, internal origin antecedent events, and without antecedent event, respectively) were positive for CTP. In category 1 cases, the earlier tested samples showed a higher CTP-positive rate, whereas no differences were observed in categories 2, 3 or 4. The characteristic clinical features in the earlier tested cases were nystagmus and fistula sign in CTP test-positive cases in category 1, and streaming water-like tinnitus in those in categories 2, 3 and 4.
Conclusion: The present study clarified that CTP detection test-positive patients exist at considerable rates among patients with inner ear-related manifestations.

DOI： 10.1080/00016489.2017.1300940

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

The molecular mechanisms underlying age-related hearing loss are unknown, and currently, there is no treatment for this condition. Recent studies have shown that microRNAs (miRNAs) and age-related diseases are intimately linked, suggesting that some miRNAs may present attractive therapeutic targets. In this study, we obtained 8 human temporal bones from 8 elderly subjects at brain autopsy in order to investigate the expression profile of miRNAs in the inner ear with miRNA arrays. A mean of 478 different miRNAs were expressed in the samples, of which 348 were commonly expressed in all 8 samples. Of these, levels of 16 miRNAs significantly differed between young elderly and old elderly subjects. miRNAs, which play important roles in inner ear development, were detected in all samples, i.e., in both young and old elderly subjects, whether with or without hearing loss. Our results suggest that these miRNAs play important roles not only in development, but also in the maintenance of inner ear homeostasis. (C) 2017 S. Karger AG, Basel

DOI： 10.1159/000479724

Web of Science

PubMed

researchmap

Language：English   Publisher：MEDICAL ASSOC NIPPON MEDICAL SCH  

DOI： 10.1272/jnms.83.140

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Mammalian xanthine oxidoreductase can exist in both dehydrogenase and oxidase forms. Conversion between the two is implicated in such diverse processes as lactation, anti-bacterial activity, reperfusion injury and a growing number of diseases. We have constructed a variant of the rat liver enzyme that lacks the carboxy-terminal amino acids 1316-1331; it appears to assume an intermediate form, exhibiting a mixture of dehydrogenase and oxidase activities. The purified variant protein retained similar to 50-70% of oxidase activity even after prolonged dithiothreitol treatment, supporting a previous prediction that the C-terminal region plays a role in the dehydrogenase to oxidase conversion. In the crystal structure of the protein variant, most of the enzyme stays in an oxidase conformation. After 15min of incubation with a high concentration of NADH, however, the corresponding X-ray structures showed a dehydrogenase-type conformation. On the other hand, disulfide formation between Cys535 and Cys992, which can clearly be seen in the electron density map of the crystal structure of the variant after removal of dithiothreitol, goes in parallel with the complete conversion to oxidase, resulting in structural changes identical to those observed upon proteolytic cleavage of the linker peptide. These results indicate that the dehydrogenase-oxidase transformation occurs rather readily and the insertion of the C-terminal peptide into the active site cavity of its subunit stabilizes the dehydrogenase form. We propose that the intermediate form can be generated (e.g. in endothelial cells) upon interaction of the C-terminal peptide portion of the enzyme with other proteins or the cell membrane.
DatabaseCoordinate sets and structure factors for the four crystal structures reported in the present study have been deposited in the Protein Data Bank under the identification numbers , , , and .

DOI： 10.1111/febs.13277

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (international conference proceedings)  

researchmap

Language：English   Publishing type：Research paper (international conference proceedings)  

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Xanthinuria due to xanthine dehydrogenase (XDH) deficiency is a rare genetic disorder characterized by hypouricemia and the accumulation of xanthine in the urine. We have identified an Afghan girl whose xanthinuria could be classified as type I xanthinuria based on an allopurinol loading test Three mutations were identified in the XDH gene, 141insG, C2729T (T910M) and C3886T (R1296W). Site-directed mutagenesis followed by expression analysis in Escherichia coli revealed that not only the frame shift mutation 141insG impairs XDH activity, but also the missense mutation C2729T, while C3886T resulted in major residual activity of about 50% of the wild type. In this report, a case of xanthinuria type I with mutations of XDH was identified and characterized by expression studies. (C) 2012 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.cca.2012.08.011

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：INFORMA HEALTHCARE  

Conclusions: The changes in the cochlin isoforms in the perilymph may provide important insights to the understanding of cochlin function and the pathogenesis of related inner ear diseases. Objectives: Cochlin is involved in various pathologies of the inner ear. Altered levels of cochlin isoforms in developing inner ear tissue were reported previously. The purpose of this study was to elucidate the cochlin isoform expression in the perilymph of rats during postnatal development in relation to Coch gene mRNA expression. Methods: We studied the cochlin isoforms in the rat perilymph during postnatal development by Western blot analysis. Real-time PCR was also performed to elucidate the expression level of Coch mRNA in the developing inner ear of rats. Results: Western blot analysis showed that the expression of p63s in the perilymph was highest on the 12th day after birth (DAB12), the earliest age at which we could identify the perilymphatic space microscopically, and it decreased gradually as the cochlea developed. On the other hand, the expression of Cochlin-tomoprotein (CTP) was lowest on DAB12 and increased gradually up to DAB24. COCH mRNA was detected from DAB3 and gradually increased to DAB15, and then gradually decreased to DAB70.

DOI： 10.3109/00016489.2012.687456

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS AS  

Conclusions: We have cloned guinea pig Coch cDNA and the sequence information will be useful for future molecular study combined with physiological experiments. Proper Coch gene expression appears to be dependent on the unique extracellular micro-environment of the inner ear in vivo. These results provide insight into the Coch gene expression and its regulation. Objective: To characterize the guinea pig Coch gene, we performed molecular cloning and expression analysis in the inner ear and cultured fibrocytes of the spiral ligament. Methods: The Coch cDNA was isolated using RACE. Cochlin isofoms were studied by Western blot using three different types of mammalian inner ear. The cochlear fibrocytes were cultured and characterized by immunostaining. Coch gene expression in the fibrocytes was investigated and the influence of cytokine stimulation was evaluated. Results: The full-length 1991 bp Coch cDNA that encodes a 553 amino acid protein was isolated. The sequence had significant homology with other mammals, and the sizes of the Cochlin isoforms were identical. In the cultured fibrocytes, Coch mRNA was expressed in a very small amount and the isoform production was different, compared with the results in vivo. Cytokine stimulation did not alter the level of mRNA expression or isoform formation.

DOI： 10.3109/00016480903493766

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

Proteomic analysis of inner ear proteins revealed unique properties of cochlin, encoded by the COCH gene. We detected 3 cochlin isoforms, p63s, p44s and p40s, in the inner ear tissue and a short 16-kDa isoform, cochlin-tomoprotein (CTP), in the perilymph. The role of the cochlin isoforms has not been elucidated. To improve our understanding of the mechanism of cochlin isoform expression, we investigated rat cochlin mRNA expression in the inner ear and other organs. We performed RNA-ligation-mediated amplification of cDNA ends (RLM-RACE) using RNA isolated from the inner ear and spleen of rats, which are known to express abundant cochlin mRNA. We also examined the expression profile of full-length cochlin mRNA by nested RT-PCR in the cerebrum, cerebellum/brain stem, eye, inner ear, thyroid gland, thymus gland, lung, heart, liver, spleen, adrenal gland, kidney and blood. We verified CTP expression in rat perilymph by Western blot. By RLM-RACE, alternately spliced variants of cochlin mRNA with 3 different lengths were detected (2442, 2008 and 724 bp). The two longer mRNAs encode full-length cochlin with different polyadenylation signals in the 3'-untranslated region, which are expressed both in the ear and spleen. The short variant encodes the limulus factor C, cochlin, late gestation lung protein (LCCL) domain and the N-terminal sequence of the von Willebrand factor A (vWFA1) domain, and this variant was detected only in the ear. All 3 variants have the same transcriptional start site. By RT-PCR, we found that full-length cochlin was expressed in all organs examined, with a splice variant in the heart. By Western blot, we detected short isoforms (11-17 kDa) in the perilymph. Cochlin isoform formation is regulated, at least in part, by alternative splicing at the transcriptional level. The short mRNA was detected only in the inner ear, and this variant may provide a clue to understanding the formation and function of cochlin isoforms. Copyright (C) 2009 S. Karger AG, Basel

DOI： 10.1159/000231634

Web of Science

PubMed

researchmap

Language：English   Publisher：BENTHAM SCIENCE PUBL LTD  

Cysteine (cysteinyl residue) modifications in proteins result in diversity in protein functions. The reaction specificity of a protein with a modified cysteine residue is determined by the overall conditions of the protein, including the spatial position of the cysteine residue, electrostatic interactions between cysteine residue and other charged residues, spatial interactions between the cysteine residue and a chemical compound, electrophilicity of the chemical compound, and the pH of the solution. In cysteine-dependant enzymes, each specific type of cysteine modification characterizes the catalytic mechanism of the enzyme. Recently, the catalytic mechanisms of peroxiredoxins and cysteine proteases, which contain a cysteine residue(s) in their catalytic sites, have been elucidated. In the catalytic process of peroxiredoxins, a sulfenyl intermediate is formed by oxidation of the catalytic cysteine residue. On the other hand, in cysteine proteases, the catalytic cysteine residue reacts with the carboxyl carbon of a peptide substrate to form an intermediate complex via S-alkylation. In this review, we introduce the most current information on the applications of cysteine thiol chemistry for in vitro glycoprotein synthesis. Recently, a glycoprotein (monocyte chemotactic protein-3), containing an intact human complex-type sialyloligosaccharide has been chemically synthesized. The procedure used for this could have applications in the development of new protein-based drugs, including antineoplastic drugs and antibiotics. It can also potentially be applied for improving the half-life and reducing the toxicity of these drugs, and for preventing the development of multidrug resistance.

DOI： 10.2174/092986709789760643

Web of Science

PubMed

researchmap

DOI： 10.2174/092986709789712880

PubMed

researchmap

DOI： 10.1093/jb/mvn111

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Rat heme-binding protein 23 (HBP23)/peroxiredoxin (Prx I) belongs to the 2-Cys peroxiredoxin type I family and exhibits peroxidase activity coupled with reduced thioredoxin (Trx) as an electron donor. We analyzed the dimer-oligomer interconversion of wild-type and mutant HBP23/Prx I by gel filtration and found that the C52S and C173S mutants existed mostly as decamers, whereas the wild type was a mixture of various forms, favoring the decamer at higher protein concentration and lower ionic salt concentration and in the presence of dithiothreitol. The C83S mutant was predominantly dimeric, in agreement with a previous crystallographic analysis (Hirotsu, S., Abe, Y., Okada, K., Nagahara, N., Hori, H., Nishino, T., and Hakoshima, T.(1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12333-12338). X-ray diffraction analysis of the decameric C52S mutant revealed a toroidal structure (diameter, similar to 130 angstrom; inside diameter, similar to 55 angstrom; thickness, similar to 45 angstrom). In contrast to human Prx I, which was recently reported to exist predominantly as the decamer with Cys(83)-Cys(83) disulfide bonds at all dimer-dimer interfaces, rat HBP23/Prx I has a Cys(83)-Cys(83) disulfide bond at only one dimer-dimer interface (S-S separation of similar to 2.1 angstrom), whereas the interactions at the other interfaces (mean S-S separation of 3.6 angstrom) appear to involve hydrophobic and van der Waals forces. This finding is consistent with gel filtration analyses showing that the protein readily interconverts between dimer and oligomeric forms. The C83S mutant exhibited similar peroxidase activity to the wild type, which is exclusively dimeric, in the Trx/Trx reductase system. Athigher concentrations, where the protein was mostly decameric, less efficient attack of reduced Trx was observed in a [C-14] iodoacetamide incorporation experiment. We suggest that the dimer-decamer interconversion may have a regulatory role.

DOI： 10.1074/jbc.M705753200

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Reactive oxygen species are generated by various systems, including NADPH oxidases, xanthine oxidoreductase (XOR) and mitochondrial respiratory enzymes, and contribute to many physiological and pathological phenomena. Mammalian xanthine dehydrogenase (XDH) can be converted to xanthine oxidase (XO), which produces both superoxide anion and hydrogen peroxide in a molar ratio of about 1:3, depending upon the conditions. Here, we present a mutant of rat XOR that displays mainly XO activity with a superoxide:hydrogen peroxide production ratio of about 6:1. In the mutant, tryptophan 335, which is a component of the amino acid cluster crucial for switching from the XDH to the XO conformation, was replaced with alanine, and phenylalanine 336, which modulates FAD's redox potential through stacking interactions with the flavin cofactor, was changed to leucine. When the mutant was expressed in Sf9 cells, it was obtained in the XO form, and dithiothreitol treatment only partially restored the pyridine nucleotide-binding capacity. The crystal structure of the dithiothreitol-treated mutant at 2.3 angstrom resolution showed the enzyme's two subunits to be quite similar, but not identical: the cluster involved in conformation-switching was completely disrupted in one subunit, but remained partly associated in the other one. The chain trace of the active site loop in this mutant is very similar to that of the bovine XO form. These results are consistent with the idea that the XDH and XO forms of the mutant are in an equilibrium that greatly favours the XO form, but the equilibrium is partly shifted towards the XDH form upon incubation with dithiothreitol.

DOI： 10.1093/jb/mvm054

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

Xanthine oxidase (oxidoreductase; XOR) and aldehyde oxidase (AO) are similar in protein structure and prosthetic group composition, but differ in substrate preference. Here we show that mutation of two amino acid residues in the active site of human XOR for purine substrates results in conversion of the substrate preference to AO type. Human XOR and its Glu803-to-valine (E803V) and Arg881-to-methionine (R881M) mutants were expressed in an Escherichia coli system. The E803V mutation almost completely abrogated the activity towards hypoxanthine as a substrate, but very weak activity towards xanthine remained. On the other hand, the R881M mutant lacked activity towards xanthine, but retained slight activity towards hypoxanthine. Both mutants, however, exhibited significant aldehyde oxidase activity. The crystal structure of E803V mutant of human XOR was determined at 2.6 angstrom resolution. The overall molybdopterin domain structure of this mutant closely resembles that of bovine milk XOR; amino acid residues in the active centre pocket are situated at very similar positions and in similar orientations, except that Glu803 was replaced by valine, indicating that the decrease in activity towards purine substrate is not due to large conformational change in the mutant enzyme. Unlike wild-type XOR, the mutants were not subject to time-dependent inhibition by allopurinol.

DOI： 10.1093/jb/mvm053

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Rat 3-mercaptopyruvate sulfurtransferase (MST) contains three exposed cysteines as follows: a catalytic site cysteine, Cys(247), in the active site and Cys(154) and Cys(263) on the surface of MST. The corresponding cysteine to Cys(263) is conserved in mammalian MSTs, and Cys(154) is a unique cysteine. MST has monomer-dimer equilibrium with the assistance of oxidants and reductants. The monomer to dimer ratio is maintained at similar to 92:8 in 0.2M potassium phosphate buffer containing no reductants under air-saturated conditions; the dimer might be symmetrical via an intersubunit disulfide bond between Cys(154) and Cys(154) and between Cys(263) and Cys(263), or asymmetrical via an intersubunit disulfide bond between Cys(154) and Cys(263). Escherichia coli reduced thioredoxin (Trx) cleaved the intersubunit disulfide bond to activate MST to 2.3- and 4.9-fold the levels of activation of dithiothreitol (DTT)-treated and DTT-untreated MST, respectively. Rat Trx also activated MST. On the other hand, reduced glutathione did not affect MST activity. E. coli C35S Trx, in which Cys(35) was replaced with Ser, formed some adducts with MST and activated MST after treatment with DTT. Thus, Cys(32) of E. coli Trx reacted with the redox-active cysteines, Cys(154) and Cys(263), by forming an intersubunit disulfide bond and a sulfenyl Cys(247). A consecutively formed disulfide bond between Trx and MST must be cleaved for the activation. E. coli C32S Trx, however, did not activate MST. Reduced Trx turns on a redox switch for the enzymatic activation of MST, which contributes to the maintenance of cellular redox homeostasis.

DOI： 10.1074/jbc.M605931200

Web of Science

PubMed

researchmap

DOI： 10.1016/j.placenta.2006.10.005

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Peroxiredoxin-LL (Prxll) and glutathione peroxidase-l (GPxl) are regulators of the redox system that is one of the most crucial supporting systems in neurons. This system is an antioxidant enzyme defense system and is synchronously linked to other important cell supporting systems. To clarify the common self-survival mechanism of the residual motor neurons affected by amyotrophic lateral sclerosis (ALS), we examined motor neurons from 40 patients with sporadic ALS (SALS) and 5 patients with superoxide dismutase 1 (SOD1)-mutated familial ALS (FALS) from two different families (frame-shift 126 mutation and A4 V) as well as four different strains of the SOD1-mutated ALS models (H46R/G93A rats and G1H/G1L-G93A mice). We investigated the immunohistochemical expression of Prxll/GPxl in motor neurons from the viewpoint of the redox system. In normal subjects, Prxll/GPxl immunoreactivity in the anterior horns of the normal spinal cords of humans, rats and mice was primarily identified in the neurons: cytoplasmic staining was observed in almost all of the motor neurons. Histologically, the number of spinal motor neurons in ALS decreased with disease progression. Immunohistochemically, the number of neurons negative for Prxll/GPxl increased with ALS disease progression. Some residual motor neurons coexpressing Prxll/GPxl were, however, observed throughout the clinical courses in some cases of SALS patients, SOD1-mutated FALS patients, and ALS animal models. In particular, motor neurons overexpressing Prxll/GPxl, i.e., neurons showing redox system up-regulation, were commonly evident during the clinical courses in ALS. For patients with SALS, motor neurons overexpressing Prxll/GPx1 were present mainly within approximately 3 years after disease onset, and these overexpressing neurons thereafter decreased in number dramatically as the disease progressed. For SOD1-mutated FALS patients, like in SALS patients, certain residual motor neurons without inclusions also overexpressed Prxll/GPxl in the short-term-surviving FALS patients. In the ALS animal models, as in the human diseases, certain residual motor neurons showed overexpression of Prxll/GPxl during their clinical courses. At the terminal stage of ALS, however, a disruption of this common Prxll/GPx1-overexpression mechanism in neurons was observed. These findings lead us to the conclusion that the residual ALS neurons showing redox system up-regulation would be less susceptible to ALS stress and protect themselves from ALS neuronal death, whereas the breakdown of this redox system at the advanced disease stage accelerates neuronal degeneration and/or the process of neuronal death.

DOI： 10.1007/s00401-005-1019-3

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Mammalian xanthine dehydrogenase can be converted to xanthine oxidase by modification of cysteine residues or by proteolysis of the enzyme polypeptide chain. Here we present evidence that the Cys(535) and Cys(992) residues of rat liver enzyme are indeed involved in the rapid conversion from the dehydrogenase to the oxidase. The purified mutants C535A and/or C992R were significantly resistant to conversion by incubation with 4,4'-dithiodipyridine, whereas the recombinant wildtype enzyme converted readily to the oxidase type, indicating that these residues are responsible for the rapid conversion. The C535A/C992R mutant, however, converted very slowly during prolonged incubation with 4,4'-dithiodipyridine, and this slow conversion was blocked by the addition of NADH, suggesting that another cysteine couple located near the NAD(+) binding site is responsible for the slower conversion. On the other hand, the C535A/C992R/C1316S and C535A/C992R/C1324S mutants were completely resistant to conversion, even on prolonged incubation with 4,4'-dithiodipyridine, indicating that Cys(1316) and Cys(1324) are responsible for the slow conversion. The crystal structure of the C535A/C992R/C1324S mutant was determined in its demolybdo form, confirming its dehydrogenase conformation.

DOI： 10.1074/jbc.M501830200

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

In mammals, xanthine oxidoreductase is synthesized as a dehydrogenase (XDH) but can be readily converted to its oxidase form (XO) either by proteolysis or modification of cysteine residues. The crystal structures of bovine milk XDH and XO demonstrated that atoms in the highly charged active-site loop (Gln-423-Lys-433) around the FAD cofactor underwent large dislocations during the conversion, blocking the approach of the NAD+ substrate to FAD in the XO form as well as changing the electrostatic environment around FAD. Here we identify a unique cluster of amino acids that plays a dual role by forming the core of a relay system for the XDH/XO transition and by gating a solvent channel leading toward the FAD ring. A more detailed structural comparison and site-directed mutagenesis analysis experiments showed that Phe-549, Arg-335, Trp-336, and Arg-427 sit at the center of a relay system that transmits modifications of the linker peptide by cysteine oxidation or proteolytic cleavage to the active-site loop (Gln-423-Lys-433). The tight interactions of these residues are crucial in the stabilization of the XDH conformation and for keeping the solvent channel closed. Both oxidative and proteolytic generation of XO effectively leads to the removal of Phe-549 from the cluster causing a reorientation of the bulky side chain of Trp-336, which then in turn forces a dislocation of Arg-427, an amino acid located in the active-site loop. The conformational change also opens the gate for the solvent channel, making it easier for oxygen to reach the reduced FAD in XO.

DOI： 10.1073/pnas.1431485100

Web of Science

PubMed

researchmap

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press Inc.  

Drosophila ma-l gene was suggested to encode an enzyme for sulfuration of the desulfo molybdenum cofactor for xanthine dehydrogenase (XDH) and aldehyde oxidase (AO). The human molybdenum cofactor sulfurase (HMCS) gene, the human ma-l homologue, is therefore a candidate gene responsible for classical xanthinuria type II, which involves both XDH and AO deficiencies. However, HMCS has not been identified as yet. In this study, we cloned the HMCS gene from a cDNA library prepared from liver. In two independent patients with classical xanthinuria type II, we identified a C to T base substitution at nucleotide 1255 in the HMCS gene that should cause a CGA (Arg) to TGA (Ter) nonsense substitution at codon 419. A classical xanthinuria type I patient and healthy volunteers lacked this mutation. These results indicate that a functional defect of the HMCS gene is responsible for classical xanthinuria type II, and that HMCS protein functions to provide a sulfur atom for the molybdenum cofactor of XDH and AO. © 2001 Academic Press.

DOI： 10.1006/bbrc.2001.4719

Scopus

PubMed

researchmap

Language：English   Publisher：The Japanese Biochemical Society  

Phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA) were found to inhibit strongly the citrulline formation activity of neuronal nitric oxide synthase (nNOS; EC 1.14.13.39). Such inhibition was not observed with any other phospholipid examined. A kinetic analysis of purified nNOS showed no significant change in apparent K(m) for L-Arg or NADPH caused by these inhibitory phospholipids. Electron paramagnetic resonance analysis revealed no significant spectral perturbation of the ferriheme or flavin semiquinone upon the addition of PIP2. On the other hand, a lower enhancement of the NADPH diaphorase activity by Ca2+-calmodulin was observed in the presence of PIP2 and PA, and the citrulline formation activity was protected from phospholipid inhibition by preincubation with Ca2+-calmodulin. Moreover, trypsin digestion analysis showed that the cleavage site within the calmodulin-binding site of nNOS was specifically protected from trypsin by the addition of PIP2 and PA. These results strongly suggest that PIP2 and PA inhibit the citrulline formation activity of nNOS by blocking the interaction of the enzyme with Ca2+-calmodulin.

DOI： 10.1093/oxfordjournals.jbchem.a022523

Scopus

PubMed

researchmap

Language：Japanese   Publisher：(一社)日本救急医学会  

J-GLOBAL

researchmap

Language：Japanese   Publisher：(一社)日本Shock学会  

J-GLOBAL

researchmap

Language：Japanese   Publisher：日本医科大学医学会  

DOI： 10.1272/manms.7.169

CiNii Books

researchmap

Language：Japanese   Publisher：The Medical Association of Nippon Medical School  

The protein expression system in bacteria is widely used to overproduce recombinant proteins. Many established expression vectors are on the market. The <i>Escherichia coli</i> expression system is extremely useful for biochemical and biophysical analysis. Here, we describe the results of the expression of mammalian protein in <i>E. coli</i> and an experiment on the structural analysis of recombinant protein.<br>

DOI： 10.1272/manms.7.169

researchmap

Language：Japanese   Publisher：一般社団法人 日本痛風・核酸代謝学会  

DOI： 10.6032/gnam.34.47

CiNii Books

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：Japanese   Publisher：一般社団法人 日本痛風・核酸代謝学会  

DOI： 10.6032/gnam.33.214

CiNii Books

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

J-GLOBAL

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese   Publisher：日本産科婦人科学会  

CiNii Books

researchmap

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：W B SAUNDERS CO LTD  

Web of Science

researchmap

Language：Japanese  

CiNii Books

researchmap

Language：Japanese   Publisher：(一社)日本痛風・核酸代謝学会  

researchmap

Language：Japanese   Publisher：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.44.S28_4

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese  

CiNii Books

researchmap

Language：Japanese   Publisher：(一社)日本痛風・核酸代謝学会  

researchmap

Language：Japanese   Publisher：(株)日本臨床社  

researchmap

Language：Japanese  

J-GLOBAL

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

researchmap

Language：Japanese   Publisher：(一社)日本痛風・核酸代謝学会  

モリブデンコファクターのモリブデンに硫黄原子を配位すると考えられる酵素Human Molybdenum cofactor sulfurase(HMCS)をクローニングし,この酵素がキサンチン尿症タイプIIの責任遺伝子であることを証明するため,本症の2例において解析を試みた.その結果,HMCSが本症の責任遺伝子である可能性が強く示唆された

researchmap

Language：Japanese   Publisher：(公社)日本生化学会  

J-GLOBAL

researchmap

Language：Japanese  

CiNii Books

researchmap

Language：Japanese   Publisher：(一社)日本痛風・核酸代謝学会  

researchmap

Language：Japanese   Publisher：(一社)日本痛風・核酸代謝学会  

researchmap

J-GLOBAL

researchmap

Language：Japanese   Publisher：日本生物工学会  

CiNii Books

researchmap