Biomedical subjects

M Tsujimura

Publications and source records attributed to M Tsujimura.

At least 19 recordsLinked to original sources

PubMed2001

Fe-type nitrile hydratase.

The characteristic features of Fe-type nitrile hydratase (NHase) from Rhodococcus sp. N-771 are described. Through the biochemical analyses, we have found that nitric oxide (NO) regulates the photoreactivity of this enzyme by association with the non-heme iron center and photoinduced dissociation from it. The regulation is realized by a unique structure of the catalytic non-heme iron center composed of post-translationally modified cysteine-sulfinic (Cys-SO2H) and -sulfenic acids (Cys-SOH). To understand the biogenic mechanism and the functional role of these modifications, we constructed an over-expression system of whole NHase and individual subunits in Escherichia coli. The results of the studies on several recombinant NHases have shown that the Cys-SO2H oxidation of alphaC112 is indispensable for the catalytic activity of Fe-type NHase.

Binding Sites↗

PubMed2001

Detection of serum thermolabile beta-2 macroglycoprotein (Hakata antigen) by enzyme-linked immunosorbent assay using polysaccharide produced by Aerococcus viridans.

Although a serum thermolabile beta-2 macroglycoprotein (TMG) may play a role in host defense as a lectin, little is known of its related physiological functions, mainly due to a lack of appropriate methods for tracing the functions of TMG. We identified a polysaccharide from Aerococcus viridans, PSA, which reacts with TMG, and based on this finding, we developed an enzyme-linked immunosorbent assay to trace the functions of TMG. Using ethanol precipitation and DEAE-Sepharose and Sephacryl S-400 column chromatographies, we isolated PSA from cultured medium of A. viridans, and it exhibited specific binding against TMG in blood samples. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the isolated PSA showed ladder bands that implied the existence of repeating units composed of D-glucose, N-acetyl-D-glucosamine, D-mannose, and D-xylose, as confirmed by gas chromatography-mass spectrometry. SDS-PAGE and immunochemical analysis, using rabbit anti-TMG antibody, showed that PSA specifically binds solely to intact serum TMG but not to TMG heated at 56 degrees C for 30 min, a condition under which antigenicity is lost. TMG in serum samples bound to PSA in a dose-dependent manner, and this binding was clearly suppressed by addition of PSA. These observations indicate that PSA is a useful adsorbent to TMG and can be used to develop appropriate methods for tracing the functions of TMG.

Antibodies, Bacterial↗

PubMed2001

Granulomatous tissue formation of shikon and shikonin by air pouch method.

"The extract of shikon" (SK) and shikonin play important roles in the development of granulomatous tissue formation. To reveal the augmenting effect of SK or shikonin on vascular endothelial growth factor (VEGF) production and neovascularization, we investigated murine granulomatous tissue induced by SK and shikonin, comparing them to pouches in which trehalose 6,6'-dimycolate (TDM) was injected. The development of granulomatous tissue formation was evaluated by the wet weight of pouch walls. At day 5 and 7 after SK and shikonin injection, prominent granulomatous tissue formation was detected. Histological observations on the development of granulomatous tissue showed that the pouch was formed in the submuscular connective tissue and necrotic tissue directly facing the cavity and granulomatous tissue developed in the connective tissue. At day 1, VEGF-positive neutrophils accumulated in the pouch wall. Granulomatous tissue formation and neovascularization by injection of SK or shikonin was not more prominent than TDM. However, the present results indicate that SK and shikonin induce neovascularization in granulomatous tissue.

Animals↗

PubMed2000

Arginine 56 mutation in the beta subunit of nitrile hydratase: importance of hydrogen bonding to the non-heme iron center.

Arginine 56 in the beta subunit (betaArg56) of the iron-containing nitrile hydratase (NHase), one of the strongly conserved residues within the NHase family, is known to form hydrogen bonds to the sulfinyl (-SO2H) and sulfenyl (-SOH) groups of the post-translationally modified cysteine residues in the catalytic center. BetaArg56 was substituted by tyrosine, glutamate or lysine, respectively, and the respective mutant enzymes generated by reconstitution were characterized. The betaR56K mutant complex exhibited about 1% of the enzymatic activity of native NHase, while the others were totally inactive. The kinetic analysis of the betaR56K mutant complex exhibited a drastic decrease in turnover number and decreases in kinetic constants for substrate and inhibitors as compared to the native NHase. Changes in UV-visible absorption and light-induced Fourier transform infrared difference spectra suggest that betaArg56 is involved in the positioning of the -SO2H and -SOH groups of the modified Cys residues in the catalytic center so as to fine tune the electronic state of the iron center suitable for catalysis. Thus, betaArg56 is essential for catalysis.

Arginine↗

PubMed2000

An angiotensin system in the anterior hypothalamic area anterior is involved in the maintenance of hypertension in spontaneously hypertensive rats.

An overactive brain renin-angiotensin system is one of the factors contributing to the pathogenesis of hypertension in spontaneously hypertensive rats (SHR). We examined brain sites where enhanced activity of an angiotensin system is responsible for the pathogenesis of hypertension in SHR. The angiotensin receptor antagonist, losartan was injected into tissues around rostral parts of the third ventricle in conscious rats. Losartan (0.22 nmol) injected into the anterior hypothalamic area, anterior (AHA) produced a depressor response in SHR but not in Wistar Kyoto rats (WKY). Angiotensin II (0.091-0.91 pmol) injected into the AHA produced a pressor response in both WKY and SHR, and the pressor response to angiotensin II was greater in SHR than that of WKY. Carbachol (3 pmol) injected into the AHA also produced a pressor response in WKY and SHR, and the pressor response to carbachol was almost the same in both strains of rats. Release of angiotensin peptides in the AHA was greater in SHR than that of WKY. These findings suggest that an angiotensin system in the AHA is enhanced and this enhancement of angiotensin system is involved in the maintenance of hypertension in SHR. Both increased pressor reactivity to angiotensin II and increased release of angiotensin peptides in the AHA appear to be related to this enhancement of angiotensin system in SHR.

Angiotensin II↗

PubMed2000

Blockade of angiotensin receptors in the anterior hypothalamic preoptic area lowers blood pressure in DOCA-salt hypertensive rats.

It has been established that deoxycorticosterone acetate (DOCA)-salt hypertensive rats have an overactive brain angiotensin-system. The purpose of the present study was to identify the brain sites showing enhanced angiotensin-system activity responsible for the pathogenesis of hypertension in DOCA-salt hypertensive rats. The angiotensin receptor antagonist, losartan, was injected into brain ventricles or into tissues around the rostral parts of the third ventricle in conscious DOCA-salt hypertensive rats. Losartan (1 microg) injection into the lateral ventricle or into the rostral parts of the third ventricle produced a depressor response, whereas the agent did not affect blood pressure when injected into the caudal parts of the third ventricle or into the fourth ventricle. Losartan (0.1 microg) injection into the anterior hypothalamic preoptic area, anterior (AHA) produced a depressor response. Angiotensin II (0.1-1 ng) injection into the AHA produced a pressor response in sham-operated and DOCA-salt hypertensive rats, and the pressor response to angiotensin II (1 ng) was greater in DOCA-salt hypertensive rats than that in sham-operated rats. Release of angiotensin peptides in the AHA was greater in DOCA-salt hypertensive rats than that in sham-operated rats. These findings suggest that the angiotensin-system in the AHA is enhanced, and that this enhancement is involved in the maintenance of hypertension in DOCA-salt hypertensive rats. Both increased pressor reactivity to angiotensin II and increased release of angiotensin peptides in the AHA appear to be related to this enhancement of the angiotensin-system in DOCA-salt hypertensive rats.

Angiotensin II↗

PubMed2000

The self-choice elaboration effects on incidental memory of Japanese historical facts.

The subjects performed an orienting task involving 3 conditions, followed by unexpected tests which included free recall, name-matching and name-selec tion. Conditions were designed to force self-generated elaboration, self-choice elaboration, and experimenter-provided elaboration. In the self-generated elaboration condition, subjects were presented target sentences, e.g., Nobunaga ODA burned down ENRYAKUJI Temple, and asked to answer an elaborative interrogation, e.g., Why did Nobunaga ODA burn down ENRYAKUJI Temple? about each sentence. In the self-choice elaboration condition, subjects selected one of the alternative answers to an elaborative interrogation about each sentence. In the experimenter provided elaboration condition, subjects were presented an answer which they rated for congruity as the correct answer to the elaborative interrogation. In the free recall test, self generated elaboration led to better performance than the other two conditions for which no difference was observed. However, in the name-matching and name-selection tests, scores were better for self choice elaboration and self-generated elaboration than for experimenter-provided elaboration. These results were interpreted as demonstrating that self choice elaboration, in addition to self-generated elaboration, led to effective encoding in memory.

Adolescent↗

PubMed1999

Functional expression of nitrile hydratase in Escherichia coli: requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine.

The nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated on nitrosylation of the non-heme iron center and activated on photo-dissociation of nitric oxide (NO). The nitrile hydratase operon consists of six genes encoding NHase regulator 2, NHase regulator 1, amidase, NHase alpha subunit, NHase beta subunit and NHase activator. We overproduced the NHase in Escherichia coli using a T7 expression system. The NHase was functionally expressed in E. coli only when the NHase activator encoded downstream of the beta subunit gene was co-expressed and the transformant was grown at 30 degrees C or less. A ligand cysteine, alphaCys112, of the recombinant NHase was also post-translationally modified to a cysteine-sulfinic acid similar to for the native NHase. Although another modification of alphaCys114 could not be identified because of the instability under acidic conditions, the recombinant NHase could be reversibly inactivated by nitric oxide.

Amino Acid Sequence↗

PubMed1998

Novel non-heme iron center of nitrile hydratase with a claw setting of oxygen atoms.

The iron-containing nitrile hydratase (NHase) is a photoreactive enzyme that is inactivated in the dark because of persistent association with NO and activated by photo-dissociation of NO. The crystal structure at 1.7 A resolution and mass spectrometry revealed the structure of the non-heme iron catalytic center in the nitrosylated state. Two Cys residues coordinated to the iron were post-translationally modified to Cys-sulfenic and -sulfinic acids. Together with another oxygen atom of the Ser ligand, these modifications induced a claw setting of oxygen atoms capturing an NO molecule. This unprecedented structure is likely to enable the photo-regulation of NHase and will provide an excellent model for designing photo-controllable chelate complexes and, ultimately, proteins.

Bacterial Proteins↗

PubMed1997

Structure of the photoreactive iron center of the nitrile hydratase from Rhodococcus sp. N-771. Evidence of a novel post-translational modification in the cysteine ligand.

Nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated by nitrosylation of the non-heme iron center and activated by photodissociation of nitric oxide (NO). To obtain structural information on the iron center, we isolated peptide complexes containing the iron center by proteolysis. When the tryptic digest of the alpha subunit isolated from the inactive form was analyzed by reversed-phase high performance liquid chromatography, the absorbance characteristic of the nitrosylated iron center was observed in the peptide fragment, Asn105-Val-Ile-Val-Cys-Ser-Leu-Cys-Ser-Cys-Thr-Ala-Trp-Pro-Ile-Leu - Gly-Leu-Pro-Pro-Thr-Trp-Tyr-Lys128. The peptide contained 0.79 mol of iron/mol of molecule as well as endogenous NO. Subsequently, by digesting the peptide with thermolysin, carboxypeptidase Y, and leucine aminopeptidase M, we found that the minimum peptide segment required for the nitrosylated iron center is the 11 amino acid residues from alphaIle107 to alphaTrp117. Furthermore, by using mass spectrometry, protein sequence, and amino acid composition analyses, we have shown that the 112th Cys residue of the alpha subunit is post-translationally oxidized to a cysteine-sulfinic acid (Cys-SO2H) in the NHase. These results indicate that the NHase from Rhodococcus sp. N-771 has a novel non-heme iron enzyme containing a cysteine-sulfinic acid in the iron center. Possible ligand residues of the iron center are discussed.

Amino Acid Sequence↗

PubMed1997

[A case of reactive arthritis fallen after shigellosis infection].

A 25-year-old woman was admitted for general arthralgia in July, 1989. Reactive arthritis with arthralgia after Shigellosis was diagnosed by sex, localization of arthralgia and positive for HLA-B 27. Within 3 weeks after starting diclifenac sodium 75 mg/day, the arthralgia remitted. It has been reported that patients who are positive for HLA-B 27 have a more severe acute or chronic sacroiliitis, and our case may support this report.

Adult↗

PubMed1997

[The relation between IgG subclasses and clinical manifestations in patients with active systemic lupus erythematosus].

The levels of IgG subclasses were determined in patients with active systemic lupus erythematosus (SLE). The levels of all IgG subclasses in these patients were higher than normal controls. However, there were variations in the increase of IgG subclasses, especially IgG 1 or IgG 2. As concerning the relation to the clinical findings, the levels of IgG 1 significantly increased in patients with renal involvements and those of IgG 3 increased in patients with low complements. Thus, it was suggested that some IgG subclass related to some clinical findings.

Humans↗

PubMed1996

Resonance Raman evidence that photodissociation of nitric oxide from the non-heme iron center activates nitrile hydratase from Rhodococcus sp. N-771.

Nitrile hydratase (NHase) from Rhodococcus sp. N-771, which contains a non-heme iron center in the catalytic site, has been known to be activated by light illumination. Recently, endogenous nitric oxide (NO) was found in this enzyme by FTIR spectroscopy [Noguchi et al. (1995) FEBS Lett. 358, 9-12]. In order to directly detect the bonding between NO and the iron atom and the reaction of NO upon photoactivation, resonance Raman spectra of the NHase were measured with 413 nm excitation at 85 K. Two prominent bands at 592 and 570 cm-1 were observed in the inactive from, and both of them were completely lost upon photoactivation. Upon subsequent introduction of 15NO, the active NHase was converted to the inactive form again and the above two bands were restored with downshifts by 10 and 12 cm-1, respectively. Also, the excitation profiles of these bands in the 350-500 nm region mostly followed the absorption spectrum arising from the iron center. From these isotopic shifts and the excitation profiles, the two Raman bands were assigned to the Fe-NO stretching and bending vibrations that are probably coupled with each other. The results provided solid evidence that NO is bound to the non-heme iron in the inactive NHase and its photodissociation activates the enzyme.

Binding Sites↗

PubMed1996

Photoreactive nitrile hydratase: the photoreaction site is located on the alpha subunit.

Nitrile hydratase (NHase) from Rhodococcus sp. N-771 exists in active and inactive forms. The inactive NHase is immediately activated by light irradiation and changes to the active form. To characterize the photoreactive center, the inactive NHase was denatured by 6 M urea, and two kinds of subunits (alpha and beta) were separated and purified by anion-exchange chromatography. In a manner similar to the native NHase, the isolated alpha subunit showed two absorption peaks at 280 and 370 nm, which were diminished by light irradiation. However, irradiation failed to elicit the appearance of absorption peaks at around 400 nm and at 710 nm, which were characteristic of the activated enzyme. The beta subunit seemed not to possess any photoreactive chromophore because its absorption spectrum was not altered by light irradiation. Neither of the subunits showed NHase activity before and after light irradiation, but the inactive NHase was reconstituted by incubating the two subunits together in the dark at 4 degrees C for 1 h. Light irradiation of the beta subunit did not affect subsequent complex formation or NHase activity. However, the irradiated alpha subunit could not assemble with the beta subunit, and no activity was recovered. These results demonstrate that the chromophore(s) responsible for the photoactivation of NHase are entirely located on the alpha subunit, and imply that light irradiation induces conformational change of the alpha subunit.

Binding Sites↗

PubMed1995

Human parvovirus B19 infection in blood donors.

Using an immunodiffusion assay, we tested all of the blood units donated at the Fukuoka Red Cross Blood Center from June 1991 to July 1994 for B19 antigen. Over this 3-year trial period, we detected 16 viremic cases out of approximately 560,000 blood donors. Interestingly, most of the viremic donors (15 out of 16) were detected between February 1992 and January 1993, which coincided with a local erythema infectiosum epidemic in the Fukuoka area (December 1991 to August 1992). In particular, we detected 4 cases of viremia in March 1992, which was the peak of the erythema infectiosum epidemic. The incidence of B19 viremia in this peak period was approximately 1/4,000. The viremic donors ranged in age from 17 to 45 years, and most (11/16) were between 31 and 39 years old. By ELISA, using virus particles purified from viremic donor plasma as antigen, we analyzed the prevalence of B19-specific antibody among blood donors. The antibody-positive rate was approximately 40% in donors 16-30 years old, gradually increased in middle age, and reached a peak of 92% in donors more than 61 years old.

Adolescent↗

PubMed1995

Antiscorbutic activity of 6-bromo-6-deoxy-L-ascorbic acid in the guinea pig.

The antiscorbutic activity of 6-bromo-6-deoxy-L-ascorbic acid (I) in guinea pigs was compared to that of ascorbic acid (AsA). The growth test results in guinea pigs did not show any significant difference between the body weight increase in the respective 2 groups given compound (I) and that in the group given AsA. Scorbutic guinea pigs were found to completely recover from the disease after administration of the compound (I). There were no significant differences in the plasma alkaline phosphatase activity and the hydroxyproline content in the tibia between the group given compound (I) and that given AsA. The total vitamin C contents in the various tissues were similar in the 2 groups given compound (I) and the C-deficient group but they were lower than the levels for the group given AsA. It was shown that antiscorbutic activity was characteristic of compound (I) and it was almost equal to the effect of AsA.

Alkaline Phosphatase↗

PubMed1993

Essential role of the Arg112 residue of cytochrome P450cam for electron transfer from reduced putidaredoxin.

Cytochrome P450cam (CYP101) of Pseudomonas putida PpG1 in which Arg112 is substituted by Cys was isolated by in vitro random mutagenesis of the camC gene DNA coding for P450cam. The absorption spectra of the purified mutant enzyme were similar to those of the wild type enzyme, but its substrate-dependent NADH oxidation activity in the presence of putidaredoxin (Pd) and putidaredoxin reductase (PdR) was extremely low. The rate constant of electron transfer from reduced Pd to the heme of the mutant P450cam, measured on an anaerobic stopped flow apparatus, was 1/400 of that of the wild type enzyme and the dissociation constant of the mutant P450cam for oxidized Pd was several fold higher than that of the wild type enzyme. A considerable decrease in mid-point potential of the mutant enzyme was also noted. We conclude that Arg112, which is located on the surface of the P450cam molecule and hydrogen-bonded to one of the heme propionate chains, plays an essential role in the electron transfer from Pd.

Arginine↗

PubMed1993

Synthesis and antiscorbutic activity of vitamin C analogue: L-threo-hex-2-enaro-1,4-lactone ethyl ester in the guinea pig.

L-threo-hex-2-enaro-1,4-lactone ethyl ester (II) was synthesized by the modified Fisher's esterification and its sodium salt was obtained almost quantitatively. Confirmation of the compound was made by elementary analysis, as well as IR, UV, MS and NMR spectra. The antiscorbutic activity was compared to that of ascorbic acid and the result showed that (II) did not have Vitamin C activity. The results of this investigation indicate that a close relationship exists between the chemical structure of the C-6 position of ascorbic acid analogues and the development of vitamin C activity.

Alkaline Phosphatase↗