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Y Shiro

Publications and source records attributed to Y Shiro.

At least 55 records · Page 3Linked to original sources
PubMed1999

Dynamic light-scattering and preliminary crystallographic studies of the sensor domain of the haem-based oxygen sensor FixL from Rhizobium meliloti.

FixL is a transmitter protein in a two-component system which acts as an oxygen sensor when the symbiotic Rhizobia resides in root nodules of host plants. The oxygen-sensor domain of Rhizobium meliloti FixL (RmFixLH) was purified by His-tag affinity and isoelectronic focusing chromatographies, without the use of gel-filtration chromatography. Dynamic light-scattering measurements of RmFixLH thus obtained revealed it to be monodispersive and present as a homodimer in solution. A single crystal of RmFixLH in the met (Fe3+) form was grown in 100 mM acetic acid/NaOH buffer at pH 4.6 in the presence of 200 mM ammonium acetate, using 40%(w/v) PEG 4000 as a precipitant. A crystal of the ferrous CO form of RmFixLH was also prepared by reduction of the met crystal with Na2S2O4 in an atmosphere of CO. The crystals (0.2 x 0.05 x 0.01 mm) belong to the monoclinic system (C2) with unit-cell parameters a = 60.94, b = 37.44, c = 54.14 A, beta = 115.29 degrees and diffract X-rays to 1.7 A resolution at station BL44B2 of SPring-8, Japan. Bijvoet difference Patterson maps show a clear peak corresponding to the haem iron in RmFixLH.

Bacterial Proteins↗
PubMed1999

[Huntington's disease: clinical and molecular genetics].

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms. An unstable CAG trinucleotide repeat expansion within the first exon of the responsible gene "IT15", encoding huntingtin, was identified. The clinical phenotype strongly correlates with the number of CAG repeat. The mutant huntingtin is expressed ubiquitously as same as the wild-type huntingtin, suggesting a toxic gain of function of the mutant huntingtin. The identification of huntingtin-interacting proteins and intranuclear aggregates containing a fragment of mutant huntingtin provide new insights into the pathophysiological mechanisms underlying HD. Moreover, the progress in transgenic animal models for HD will be critical for understanding the development of HD and for the testing of new therapeutics.

Animals↗
PubMed1998

Site-directed mutagenesis of the conserved threonine (Thr243) of the distal helix of fungal cytochrome P450nor.

Cytochrome P450nor (P450nor) is a heme enzyme which catalyzes NO reduction in denitrifying fungi. Threonine 243 (Thr243) of P450nor, which corresponds to the conserved threonine of monooxygenase cytochrome P450s, was replaced by 18 different amino acids via site-directed mutagenesis. The mutation did not seriously affect the optical absorption and the CD spectral properties of the enzyme in several oxidation, ligation, or spin states or the association rate constant for association of NO with the ferric iron, suggesting subtle and local structural changes in the heme environment on Thr243 mutation. However, the NO reduction activity was dramatically altered by Thr243 mutation, depending on the properties of the replaced amino acids. The catalytic activity, as measured by N2O formation and NADH consumption, was considerably retained on substitution of Asn, Ser, and Gly for Thr243, while it was profoundly decreased or lost on substitution with other amino acids. Kinetic analysis of the reaction of the enzymes with NO and NADH indicated that the decrease in the enzymatic activity upon Thr243 mutation mainly results from a decrease in the rate of reduction of the ferric-NO complex with NADH. On the basis of these enzymatic, kinetic, and spectroscopic results, as well as on the basis of the crystal data for native P450nor [Park, S.-Y., et al. (1997) Nat. Struct. Biol. 4, 827-832], the role of the conserved threonine at the 243 position in the NO reduction reaction by P450nor is discussed. We also discuss structural similarities or differences in the vicinity of the conserved threonine between P450nor and other monooxygenase P450s.

Catalysis↗
PubMed1998

Identification of the hydrophobic amino acid residues required for heme assembly in the rhizobial oxygen sensor protein FixL.

Rhizobial FixL is a novel heme protein, which senses environmental oxygen tension and directs signal transduction via protein phosphotransfer. To identify the essential residues for heme assembly in Rhizobium meliloti FixL, we individually replaced the 18 invariant hydrophobic amino acid residues (F, I, L, and V) in the heme-containing domain with alanine and histidine. Spectroscopic measurements of the soluble fractions from fixL recombinant Escherichia coli revealed that V152, F162, F170, I172, L185, F226, L230, and F243 as well as the proximal ligand H194 were indispensable for heme assembly. Autoxidation rates of purified I209H, I210A, and I210H were 65-fold, 15-fold, and 15-fold, respectively, faster than that of the wild type, although they retained heme in the protein. The absorption peak in the Soret region of the ferric I209H or I210H was red-shifted, suggesting that the ferric heme is a hexa-coordinate form in these mutants.

Amino Acid Sequence↗
PubMed1998

The effects of heme modification on reactivity, ligand binding properties and iron-coordination structures of cytochrome P450nor.

Artificial cytochrome P450nors (nitric oxide reductase) were prepared by replacing the native protoheme with various 2,4-substituted hemes: meso-, deutero-, and diacetyldeutero-hemes. For these samples, the ratio of low spin/high spin states of the ferric resting enzyme were varied, indicating that the coordination of the water molecule at the iron sixth site was affected by the electron withdrawing capacities of the heme 2,4-substituents. The binding of the water molecule reduces the rate of binding of nitric oxide (NO) to the ferric iron. In addition, the reduction reaction of the ferric-NO complex with NADH, which constitutes the second step in the NO reduction, was facilitated by the electron withdrawing capacity of 2,4-substituents. Consequently, proto- (native-) P450nor exhibited the highest overall enzymatic activity (NO reduction activity), while the enzymes containing diacetyl-, deutero-, and meso-hemes had considerably lower activities, since the NO reduction activity is determined by a balance of the reaction rates of the above two steps. The optical absorption spectra of the ferric-NO and the ferrous-CO complexes of the reconstituted enzymes show that the electron density on the heme in both states was modulated by the substituent groups. However, the resonance Raman spectral measurements showed that the Fe-NO and N-O stretching frequencies in the ferric-NO complex were insensitive to the electron density of the heme while the Fe-CO and C-O stretching frequencies in the ferrous-CO complex were sensitively varied by the electron withdrawing capacity of the 2,4-substituent. The differences are discussed in terms of the difference in the iron-ligand bond characters between the ferric-NO and the ferrous-CO complexes.

Binding Sites↗
PubMed1998

Myoglobin mutants giving the largest geminate yield in CO rebinding in the nanosecond time domain.

We have measured the rebinding of carbon monoxide (CO) to some distal mutants of myoglobin (Mb) in the time range from 10(-8) to 10(-1) s by flash photolysis, in which the photodissociated CO rebinds to the heme iron without escaping to the solvent water from the protein matrix. We have found that the double mutants [His64-->Val/Val68-->Thr (H64V/V68T) and His64-->Val/Val68-->Ser (H64V/V68S)] have an extremely large geminate yield (70-80%) in water at 5 degreesC, in contrast to the 7% of the geminate yield of wild-type Mb. The CO geminate yields for these two mutants are the largest in those of Mb mutants reported so far, showing that the two mutants have a unique heme environment that favors CO geminate rebinding. Comparing the crystal structures and 1H-NMR and vibrational spectral data of H64V/V68T and H64V/V68S with those of other mutants, we discuss factors that may control the nanosecond geminate CO rebinding and CO migration in the protein matrix.

Carbon Monoxide↗
PubMed1998

Reversible pitch perception deficit due to carbamazepine.

Carbamazepine (CBZ) is a drug frequently used to treat variety of neurological diseases or symptoms. Among its adverse effects, auditory disturbance is described rarely. In the present report, we describe an 18-year-old girl who noted false lowering of perceived pitch after starting CBZ treatment for epilepsy, and review the literature reporting CBZ-associated abnormal pitch perception.

Adolescent↗
PubMed1997

Crystallization, preliminary diffraction and electron paramagnetic resonance studies of a single crystal of cytochrome P450nor.

Cytochrome P450nor (P450nor) is a heme-containing nitric oxide reductase from the denitrifying fungus, Fusarium oxysporum. This enzyme catalyzes the reduction of NO to N2O. In the present study, we report results from preliminary crystallographic and electron paramagnetic resonance (EPR) analysis of a single crystal of P450nor. The crystal was grown in 100 mM MES buffer at pH 5.6 using PEG 4000 as a precipitant. It belongs to the orthorhombic system with cell dimensions of a = 54.99 A, b = 82.66 A, c = 87.21 A, and the space group is P2(1)2(1)2(1). The crystal diffracts synchrotron radiation at higher than 2.0 A resolution, and therefore it is suitable for X-ray crystal structure analysis at atomic resolution. Bijvoet and dispersive anomalous difference Patterson maps show a clear peak corresponding to the heme iron. The structure solution is currently underway by means of MIR and MAD techniques. EPR analysis determined the orientation of the heme within the P450nor crystal.

Crystallography, X-Ray↗
PubMed1997

Functional and structural comparison of nitric oxide reductases from denitrifying fungi Cylindrocarpon tonkinense and Fusarium oxysporum.

Two isozymes of nitric oxide reductase (Nor) from the denitrifying fungus Cylindrocarpon tonkinense (c.Nor1 and c.Nor2) are the heme-enzyme cytochrome P-450's (Usuda et al. (1995) Appl. Environ. Microbiol. 61, 883-889). However, they catalyze the NO reduction to N2O, but not the monooxygenation reaction using O2. We kinetically and spectrophotometrically studied the reactions of the two Nor's with NO and electron donor, NAD(P)H, using flash-photolysis and stopped-flow rapid scan methods. The enzyme in the Fe3+ state can bind NO to yield the Fe3+ NO complex. When the resultant Fe3+ NO complex reacted with the electron donor, it was converted to the Fe3+ enzyme via a transient formation of the characteristic intermediate (I). The spectroscopic results were essentially the same as those of the Nor from another denitrifying fungus Fusarium oxysporum (f.Nor), which we previously reported (Shiro et al. (1995) J. Biol. Chem. 270, 1617-1623), suggesting that these fungal Nor's catalyze the NO reduction by the same mechanism. Most probably, the Fe3+ NO complex of the Nor is reduced with two-electrons directly transferred from NAD(P)H to yield the intermediate I, and then the I reacts with another NO to generate N2O and the Fe3+ enzyme. However, the kinetic measurements showed that the reaction rate constant of each step was variable depending on the combination of the Nor and the electron donor; i.e., c.Nor1 + NADH, c.Nor2 + NADPH, c.Nor2 + NADH and f.Nor + NADH. In particular, the rate constant for the electron transfer step from the electron donor to the Fe3+ NO enzyme is dramatically different among these systems. On the other hand, we also measured paramagnetically shifted 1H-NMR spectra of c.Nor2 and f.Nor in the ferrous (reduced) state, where the iron-bound Cys beta-CH2 signal was observed at the same position (approximately 270 ppm) for c.Nor2 and f.Nor, indicating that the Cys thiolate (S-) coordinates to the heme iron in the same fashion in the Nor's. However, the heme peripheral proton signals were subtly but significantly different in their positions between the two enzymes. On the basis of these kinetic and spectroscopic data, we suggested that the Fe-S- binding character is not essential for the NO reduction reactivity, but that the subtle difference in interaction of their hemes with the surroundings is possibly responsible for the difference in the Nor reactivity, especially in the electron transfer step from NAD(P)H to the Fe3+ NO moiety.

Basidiomycota↗
PubMed1997

Crystal structure of nitric oxide reductase from denitrifying fungus Fusarium oxysporum.

Structures of nitric oxide reductase (NOR) in the ferric resting and the ferrous CO states have been solved at 2.0 A resolution. These structures provide significant new insights into how NO is reduced in biological systems. The haem distal pocket is open to solvent, implicating this region as a possible NADH binding site. In combination with mutagenesis results, a hydrogen-bonding network from the water molecule adjacent to the iron ligand to the protein surface of the distal pocket through the hydroxyl group of Ser 286 and the carboxyl group of Asp 393 can be assigned to a pathway for proton delivery during the NO reduction reaction.

Amino Acid Sequence↗
PubMed1997

Remarkable functional aspects of myoglobin induced by diazaheme prosthetic group.

The iron complex of beta,delta-diazamesoporphyrin III, a molecular hybrid of porphyrin and phthalocyanine, was incorporated into apomyoglobin to investigate novel biological aspects of myoglobin. The reconstituted ferric protein forms an internal hemichrome with the iron-bound distal histidine. The reduced ferrous protein has extraordinarily high affinities for O2 and CO. The ferrous myoglobin is capable of strong binding with pyridine, imidazole, cyanide, and azide, and reacts moderately with ammonia. The NO complex exhibited 5-coordinate to 6-coordinate transition over 150 min. The instability of 5-coordinate NO heme is consistent with a high affinity of imidazole to the ferrous heme. The kinetic analyses of the ferrous derivatives suggest the importance of the pi orbitals in neutral ligands as well as the negative charges in anionic ligands. A high affinity of imidazole to ferrous diazaheme accounts for the internal hemochrome formation in ferrous myoglobin containing phthalocyanines.

Acids↗
PubMed1997

Polymorphism of dopamine receptors and transporter genes in neuropsychiatric diseases.

The advances made in studies on the molecular biology of the dopamine transporter (DAT) and receptors (D1, D2, D3, D4 and D5) are reviewed, and their pharmacological characteristics and the relationship between polymorphic variations of these human proteins and neuropsychiatric disorders are discussed. Data from four studies on the variants of the D2 receptor gene (Ser311-->Cys). BalI polymorphism in D3, 48-base repeat polymorphism in D4, and 40-base repeat polymorphism in the DAT gene in patients with schizophrenia, mood disorder, neurological disease and controls are reported.

Carrier Proteins↗
PubMed1997

[A man with systemic lupus erythematosus presenting with spastic paraplegia].

We report a 38-year-old man systemic lupus erythematosus who presented with an acute onset of paraplegia and urinary retention. The man had a 12-year history of nodular cutaneous mucinosis and arthralgia. In 1994, he was admitted to our hospital with a sudden onset of weakness and numbness of the right leg followed by an emergence of similar symptoms in the left leg. His elder sister had died at 16 years of age after suffering from systemic lupus erythematosus for 6 years. On examination, the patient had skin rash on his chest, back, head, forehead, and extremities. The neurological examination revealed that his tongue deviated to the right on protrusion. The muscle power was reduced to 2-3/5 in the right leg and to 4/5 in the left leg. The sensory disturbance was noted in the lower extremities with predominant involvement of the right leg. Reflexes were increased in the right biceps, triceps, both patellas, and Achilles tendons. Babinski sign was noted bilaterally. Urinary retention and constipation were also noted. The results of the blood cell count and hepatic and renal function tests were normal. Serum levels of C-reactive protein and complements (C3, C4, CH50) were also normal. Serological examinations showed increased anti-DNA antibody (14 U/ml, [normal, < 6]). Antinuclear antibody was positive at a titer of 1:1380. CSF study showed an increased protein concentration of 83 mg/dl and an IgG level of 14 mg/dl with a normal number of cells. MR images revealed a T1-low, T2-high signal lesion at the upper part of the left ventral medulla. MR images of the brain and spinal cord were normal. The patient was diagnosed as having SLE. High-dose intravenous methylprednisolone (1 g/day) pulse treatment that was started 25 days after the onset of neurological symptoms, produced partial relief. Our case presented with paraplegia with a focal lesion in the left upper ventral part of the medulla on MR images. The incidence of male SLE is low, and paraplegia is a rare complication of SLE. Thus, the medullary lesion in SLE observed in our case appears to be rare. SLE should be considered as a cause of acute onset paraplegia or myelopathy.

Adult↗
PubMed1997

[A case of idiopathic orthostatic hypotension with selective involvement of postganglionic noradrenergic neurons].

A 44-year-old man had a 30-year history of orthostatic hypotension and diarrhea. One month before admission, he suddenly lost consciousness by defecation, and was hospitalized. He became alert within two days, but he could not sit up due to severe orthostatic hypotension. At that point, he was transferred to our hospital. On admission, talipes and microdactylia were noted. Neurological examination revealed brisk patellar tendon reflexes. Anhidrosis or impotence was not present. Analyses of blood and urine yielded normal results. Cardiological examination revealed no abnormality that could be responsible for the hypotension. MR images of the brain were also normal. However, single photon emission tomography revealed diffuse hypoperfusion of the brain. A head-up tilt (50 degrees) test induced a remarkable fall in systolic blood pressure from 149 (heart rate; 65/min) to 64 mmHg (83). Immersion of hand in ice-cold water failed to increase blood pressure. Heart rate variation and cystometry results, which represent the function of parasympathetic nerves, were normal. Warming of his body caused normal sweating. Intravenous injection of low doses of norepinephrine and methoxamine increased blood pressure while isoproterenol remarkably increased heart rate, suggesting that both alpha- and beta-receptors developed supersensitivity. Instillation of 5% cocaine and 5% tyramine into the conjunctival sac failed to cause pupillary dilation. Clinical findings and pharmacological challenge test results suggested that the main lesion of his autonomic nervous system was selectively confined to the postganglionic sympathetic nerves and noradrenergic (not cholinergic) neurons. The autonomic failure of our patient can be classified as idiopathic orthostatic hypotension. However, most patients with idiopathic orthostatic hypotension or pure autonomic failure complain of anhidrosis and impotence, which were not noted in our patient. These symptomatic differences may be the result of the highly selective involvement of noradrenergic neurons in the postganglionic sympathetic system in our patient.

Adult↗
PubMed1996

Levodopoa but not bromocriptine induces AP-1 and creb DNA-binding activity in the dopamine-depleted striatum of the rat.

To elucidate the neurochemical mechanism that underlies the effect of anti-parkinsonian agents on motor activities in the dopamine-depleted striatum, we evaluated AP-I and CREB DNA-binding activity in the striatum of 6-hydroxydopamine-lesioned rats by use of a gel mobility-shift assay. Rats with a unilateral lesion of the nigrostriatal dopamine pathway were injected i.p. with either SKF38393 (a DI receptor agonist), bromocriptine (a D2 receptor agonist), or levodopa (a Dl/D2 receptor agonist). Each treatment increased the number of rotational responses of rats contralateral to the lesioned side. However, only SKF38393 and levodopa increased AP-I and CREB DNA-binding activity in the dopamine-depleted striatum. As with the expression of c-fos, supersensitive DI receptors may play a key role in the enhanced induction of AP-I and CREB DNA-binding activity in the dopamine-depleted striatum. Supershift analysis revealed that c-Fos; and Jun family proteins are the main components for AP-1 induced in the dopamine-depleted striaturn by SKF38393 or levodopa.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1996

Synergistic effect of D1 and D2 dopamine receptors on AP-1 DNA-binding activity in the striatum and globus pallidus of the rat with a unilateral 6-OHDA lesion of the medical forebrain bundle.

The synergistic effect of D1 and D2 dopamine receptors on transcription factor AP-1 was studied in the striatum and globus pallidus of rats with unilateral 6-OHDA lesions of the medial forebrain bundle. Contralateral rotational behavior in response to a challenge dose of D1 agonist with and without D2 agonist was determined by behavioral observation, and AP-1 induction was studied by a gel mobility-shift assay. Single administration of vehicle and of a low dose of the D1 agonist (SKF38393, 0.5 mg/kg, i.p.) failed to induce rotational behavior, while the D2 agonists bromocriptine (2.5 mg/kg, i.p.) and quinpirole (1 mg/kg. i.p.) induced low rate rotations. High dose of SKF38393, 10 mg/kg, i.p., and low dose D1 and D2 agonists administered together induced a higher rate of rotation. The gel mobility-shift assay also suggested a synergistic interaction between D1 and D2 receptors on AP-1 induction in both the striatum and globus pallidus ipsilateral to the 6-OHDA lesioned nigrostriatal pathway. However, the mode of AP-1 induction via each dopamine receptor subtype appeared to differ between these brain structures. Thus, in the striatum of the lesioned side, single administration of a high dose of D1 agonist, and combined administration of D1 agonist with either of the D2 agonists resulted in AP-1 induction, while in the globus pallidus, AP-1 binding was induced by the D2 agonist bromocriptine and combined administration of a low dose D1 agonist with D2 agonists, but not by D1 agonist alone, even at a high dose. These results demonstrate that a D1/D2 dopamine receptor synergism is involved is the induction of AP-1 both in the striatum and globus pallidus of the rat with ipsilateral dopamine depletion. The induction of AP-1 via each receptor subtype appears, however, to be differently regulated in these two structures.

Animals↗