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K Shiga

Publications and source records attributed to K Shiga.

At least 55 records · Page 3Linked to original sources
PubMed1996

Three-dimensional structure of porcine kidney D-amino acid oxidase at 3.0 A resolution.

The X-ray crystallographic structure of porcine kidney D-amino acid oxidase, which had been expressed in Escherichia coli transformed with a vector containing DAO cDNA, was determined by the isomorphous replacement method for the complex form with benzoate. The known amino acid sequence, FAD and benzoate were fitted to an electron density map of 3.0 A resolution with an R-factor of 21.0%. The overall dimeric structure exhibits an elongated ellipsoidal framework. The prosthetic group, FAD, was found to be in an extended conformation, the isoalloxazine ring being buried in the protein core. The ADP moiety of FAD was located in the typical beta alpha beta dinucleotide binding motif, with the alpha-helix dipole stabilizing the pyrophosphate negative charge. The substrate analog, benzoate, is located on the re-face of the isoalloxazine ring, while the si-face is blocked by hydrophobic residues. The carboxylate group of benzoate is ion-paired with the Arg283 side chain and is within interacting distance with the hydroxy moiety of Tyr228. The phenol ring of Tyr224 is located just above the benzene ring of benzoate, implying the importance of this residue for catalysis. There is no positive charge or alpha-helix dipole near N(1) of flavin. Hydrogen bonds were observed at C(2) = O, N(3)-H, C(4) = O, and N(5) of the flavin ring.

Animals↗
PubMed1996

In vitro refolding and unfolding of subunits of electron-transferring flavoprotein: characterization of the folding intermediates and the effects of FAD and AMP on the folding reaction.

Electron-transferring flavoprotein (ETF) from pig kidney is composed of two subunits (alpha and beta, molecular weights of 33,000 and 29,000) and two small molecules, FAD and AMP. In this study, in vitro refolding and unfolding of the subunits of ETF were carried out with urea as the denaturing reagent. The refolding reaction of alpha and beta was revealed to proceed kinetically in two steps: D in equilibrium with I-->N, where D,I, and N denote the denatured, intermediate, and native forms, respectively. The features of the I forms of alpha and beta, described below, are consistent with the concept of the so-called "molten globule state," which is frequently observed in protein refolding. (i) The conversion between D and I was very rapid. (ii) The I form showed as much secondary structure as the N form as judged from the far-UV circular dichroism. (iii) The solvent accessibility of the I form, estimated by the analysis of equilibrium unfolding experiments, was intermediate between those of the D and N forms. (iv) The standard free energy of the I form is almost the same as that of the D form. The refolding reaction progressed more slowly and the environment of the tryptophan chromophore was changed more drastically in beta refolding that in alpha refolding. We previously reported that the reconstitution of holoETF from denatured subunits is speeded up by increasing the AMP concentration. In this study, the effects of AMP, FAD, and the other subunit on the single subunit folding were examined, but no effect was detected. This result suggests that AMP plays a role in a later process, namely, assembly of the four components (refolded alpha and beta, FAD, and AMP).

Adenosine Monophosphate↗
PubMed1996

Preparation of poly(D,L-lactide) and copoly(lactide-glycolide) microspheres of uniform size.

In an attempt to prepare monodisperse poly(D,L-lactide) and copoly(lactide-glycolide) microspheres, a novel emulsification technique (membrane emulsification) was employed and the preparation conditions which might affect the monodispersity were evaluated. With this technique nearly monodisperse poly(D,L-lactide) and copoly(lactide-glycolide) microspheres were successfully prepared and their sizes were controllable only by making use of microporous glass membranes of different pore sizes. However, in the present system of emulsion (methylene chloride/water) the surfactant used was limited to ionic ones and the amount of polymers available for the formation of microspheres was inevitably too small in concentration to entrap sufficient amounts of drug. As for the drug release, the effect of particle size was not appreciable but the method of solvent removal gave a great influence; the solvent extraction method showed a more drug-sustaining effect than did the solvent evaporation method. The present results suggest the possibility of making drug-loaded and biodegradable monodisperse microspheres.

Biocompatible Materials↗
PubMed1996

[Cheiro-oral syndrome following a cortical brain infarction in the precentral gyrus and at the base of the central sulcus. A case report].

We report a patient who manifested cheiro-oral syndrome following a cortical brain infarction without involvement of postcentral gyrus. A 67-year-old man was admitted to our hospital because of sudden paresthesia in his right face and right thumb and index finger. His cranial magnetic resonance imaging disclosed that the infarction was located in the left precentral sulcus and at the base of the central sulcus, while the left postcentral gyrus was not involved. His sensory symptom was attributed to the lesion of the Brodmann's area 3a, which is located at the base of the central sulcus. We postulate that the lesions responsible for cortical cheiro-oral syndrome are dispersed more widely than those for thalamic or pontine ones, since the somatotopy of face and hand in the cortex is distributed more extensively than that in the thalamus or pons.

Aged↗
PubMed1995

Functional expression of two forms of rat acyl-CoA oxidase and their substrate specificities.

Using the reverse transcription of RNA followed by the polymerase chain reaction, we cloned the cDNAs for the rat acyl-CoA oxidases I and II, which are produced by alternative splicing from a single gene, and developed a system for their expression in Escherichia coli. The homogeneous preparations of these enzymes, without proteolytic procession, showed oxidase activity with acyl-CoAs having various acyl-chain lengths. The two types of the enzyme exhibited different substrate specificities with respect to the acyl-chain length, acyl-CoA oxidase I showing the optimum activity at shorter chain-length relative to acyl-CoA oxidase II.

Acyl-CoA Oxidase↗
PubMed1995

Substrate activating mechanism of short-chain acyl-CoA, medium-chain acyl-CoA, long-chain acyl-CoA, and isovaleryl-CoA dehydrogenases from bovine liver: a resonance Raman study on the 3-ketoacyl-CoA complexes.

We have shown previously that acetoacetyl-CoA bound to medium-chain acyl-CoA dehydrogenase from pig kidney is transformed into an enolate form, O = C(3)-C(2)H = C(1)-O-, and that the interaction between the C(4a) = N(5) moiety of flavin and the O = C(3)-C(2)H = C(1)-O- moiety of acetoacetyl-CoA is important for the charge-transfer interaction [Nishina, Y. et al. (1992) J. Biochem. 111, 699-706]. In this study, we examined four kinds of acyl-CoA dehydrogenases [short-chain acyl-CoA (SCAD), medium-chain acyl-CoA (MCAD), long-chain acyl-CoA (LCAD), and isovaleryl-CoA (IVD) dehydrogenases] from bovine liver. The Raman spectra of non-labeled and isotopically labeled acetoacetyl-CoA in keto-form revealed that the 1,716-cm-1 and 1,650-cm-1 bands were derived from the C(3) = O and the C(1) = O stretching mode, respectively. In the charge-transfer complexes of acetoacetyl-CoA with the four kinds of dehydrogenases, the resonance Raman (RR) bands corresponding to the C(3) = O and the C(1) = O of acetoacetyl-CoA were observed at around 1,643-1,622 and 1,506-1,476 cm-1, respectively, indicating that acetoacetyl-CoA was transformed into the enolate form as the result of the complexation with the enzymes. Further, in RR spectra with excitation at 632.8 nm, within the charge-transfer band of the complexes of acetoacetyl-CoA with the four acyl-CoA dehydrogenases, both bands associated with the C(4a) = N(5) moiety of oxidized flavin and the O = C(3)-C(2)H = C(1)-O- moiety of acetoacetyl-CoA were enhanced, but the benzene portion of oxidized flavin was not. These results indicate that the substrate activating mechanism is common to all four kinds of dehydrogenases, i.e., the interaction between the C(1) = O of acetoacetyl-CoA and the positively polarized atoms of the enzymes located in close proximity to the oxygen atom of C(1) = O is important, and the C(4a) = N(5) moiety of flavin participates in the interaction. Some kinds of 3-ketoacyl-CoAs were tested instead of acetoacetyl-CoA and essentially similar results were obtained. The positions of the bands derived from the C(1)-O- moiety of 3-ketoacyl-CoAs were different by ca. 30 cm-1 in two groups, i.e., ca. 1,475 cm-1 for SCAD and MCAD and ca. 1,505 cm-1 for LCAD and IVD, that is, RR spectra can classify the four dehydrogenases into two groups.

Acyl-CoA Dehydrogenase↗
PubMed1995

Structural modulation of 2-enoyl-CoA bound to reduced acyl-CoA dehydrogenases: a resonance Raman study of a catalytic intermediate.

A catalytic intermediate, the so-called "purple complex," of acyl-CoA dehydrogenase is produced on its reaction with the substrate, acyl-CoA. The purple complex is a charge-transfer complex between the reduced enzyme and the product, enoyl-CoA. Resonance Raman spectra of the purple complexes of three acyl-CoA dehydrogenases [short-chain acyl-CoA (SCAD), medium-chain acyl-CoA (MCAD), and isovaleryl-CoA (IVD) dehydrogenases] were measured with excitation at 632.8 nm within charge-transfer absorption bands. The 1,577 cm-1 band of the SCAD purple complex formed in the reaction with butyryl-CoA is mainly associated with the C(1) = O stretching of crotonyl-CoA, judging from the isotopic frequency shifts upon 13C or 18O substitution of butyryl-CoA. The 1,627 cm-1 band of the C(1) = O moiety of crotonyl-CoA in solution shifted downward by 50 cm-1 on complexation with reduced SCAD. This large frequency shift indicates a substantial interaction between C(1) = O and the enzyme, and is further evidence for an appreciable contribution of a polarized form of the C(1) = O moiety in the enzyme-bound enoyl-CoA. This frequency shift can be explained by the hydrogen bond of C(1) = O. The 1,577 cm-1 band of the MCAD purple complex remained constant, regardless of the acyl carbon-chain length (from C4 to C16 of the substrate, acyl-CoA); the alky chain scarcely affected the interaction of the C(1) = O moiety in the active site.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl Coenzyme A↗
PubMed1995

Structures of charge-transfer complexes of flavoenzyme D-amino acid oxidase: a study by resonance Raman spectroscopy and extended Hückel molecular orbital method.

The structure of the ligand in a charge-transfer complex of oxidized D-amino acid oxidase (DAO) formed upon aerobic addition of chloropyruvate and ammonium sulfate was determined by resonance Raman spectroscopy. The ligand in the complex is in the enamine form, 2-amino-3-chloracrylate, not in the imine form, 2-imino-3-chloropropionate. This conclusion is consistent with our hypothesis that the ligands in the charge-transfer complexes of oxidized DAO and reduced DAO are generally in the enamine and imine forms, respectively. The calculation of HOMO and LUMO of some enamine and imine forms by the extended Hückel molecular orbital method indicated that the enamine form is a better electron donor than the imine form and the imine form is a better electron acceptor than the enamine form. These results, as well as the information about the structure of enzyme-bound ligand, support the following ideas. (i) In the charge-transfer complex of reduced DAO, the reduced flavin is an electron donor. (ii) In the charge-transfer complex of reduced DAO, the reduced flavin is an electron donor and the ligand is an electron acceptor. Resonance Raman studies on the charge-transfer complexes of oxidized DAO and those of reduced DAO, and calculated results of HOMO and LUMO for some enamine and imine forms, corroborated the structure of the stacking form between the flavin ring and the ligand in the active site of DAO.

Ammonium Sulfate↗
PubMed1995

[Clinical evaluation of 123I-metaiodobenzylguanidine myocardial scintigram in patients with valvular heart disease].

Myocardial sympathetic nerve function can be evaluated by 123I-metaiodobenzylguanidine (MIBG) scintigraphy. Congestive heart failure is closely related to myocardial sympathetic nerve function. This study evaluated the severity of congestive heart failure in 30 patients with valvular heart disease [aortic regurgitation (AR): n = 20, mean age = 70 +/- 13 years; mitral regurgitation (MR): n = 10, mean age = 61 +/- 18 years], who had chronic heart failure by MIBG myocardial scintigraphy. Single photon emission computed tomography (SPECT) and anterior planar myocardial images were obtained 15 minutes (initial images) and 4 hours (delayed images) after injection of MIBG (111MBq). Defect score was determined by the delayed SPECT images visually as a semi-quantitative index. Myocardial MIBG uptake was quantified by the heart to upper mediastinum uptake ratio on the delayed anterior planar images (H/M) and mean cardiac MIBG washout rate during 4 hours was calculated from the bull's eye display data (clearance). These indices were compared with the NYHA class and echocardiographic findings of the patients. MIBG regional defect in the delayed image was most frequently seen in the inferoposterior wall, and defect score and clearance were significantly higher and the H/M ratio was significantly lower in NYHA class III patients than in class II patients. In patients with AR, clearance significantly correlated with left ventricular end-systolic dimension. In patients with MR, both the H/M ratio and clearance significantly correlated with left atrial dimension. Defect score, H/M, and clearance were closely related to the severity of AR and MR. These results indicate that MIBG scan can be used to assess the severity of valvular heart disease.

3-Iodobenzylguanidine↗
PubMed1994

Epidermal growth factor receptor overexpression in esophageal carcinoma. An immunohistochemical study correlated with clinicopathologic findings and DNA amplification.

BACKGROUND: Many studies have reported the increased expression of epidermal growth factor receptor (EGFR) in various human malignancies and its association with the biologic behavior of the tumors. METHODS: We performed an immunohistochemical analysis of the EGFR in 217 cases of human esophageal squamous cell carcinoma, 161 lymph node metastases and 23 foci of squamous dysplasias. The findings were correlated with clinicopathologic features, including the clinical outcome. Southern blot analysis was performed in 42 cases for the detection of DNA amplification of the EGFR gene and subsequently was correlated with EGFR immunoreactivity. RESULTS: Epidermal growth factor receptor overexpression was detected in 71% of primary tumors and 88% of lymph node metastases, as compared to nonpathologic adjacent esophageal epithelium. Statistically significant correlations were observed between EGFR overexpression and sex, age, histologic type, and the presence of invasion. Tumor staining was classified into two patterns, homogeneous and heterogeneous, based on the distribution of EGFR-positive cells. The immunostaining patterns of primary tumors had a statistically significant correlation with histologic type, the presence of adventitial invasion, histologic stage and lymph node metastasis. There was a tendency toward a worse prognosis for those patients with EGFR overexpression in the primary tumor. Greater than 90% of the foci of squamous dysplasia demonstrated homogeneous EGFR overexpression. DNA amplification of the EGFR was observed in 21% of primary tumors, and all demonstrated immunohistochemical overexpression. CONCLUSIONS: Immunohistochemical overexpression of the EGFR, which was more frequent than EGFR DNA amplification, appears to play an important role in biologic behavior of human esophageal squamous cell carcinomas.

Blotting, Southern↗
PubMed1994

Preparation of separated alpha and beta subunits of electron-transferring flavoprotein in unfolded forms and their restoration to the native holoprotein form.

Electron-transferring flavoprotein (ETF) isolated from pig kidney is a heterodimer containing one FAD and one AMP [Sato, K. et al. (1993) J. Biochem. 114, 215-222]. This paper presents a method for separation of the alpha and beta subunits on a preparative scale. The subunits were separated by cation-exchange chromatography in the presence of 6 M urea at pH 7.6. The subunits were obtained in unfolded forms with denaturant. The unfolded subunits were restored to the heterodimeric form having FAD and AMP with high yield after incubation with FAD and AMP under non-denaturing conditions. The far-UV CD spectrum, the fluorescence and absorption spectra of the bound FAD, and the enzyme activity of the reconstituted ETF were all identical to those of the ETF isolated from pig kidney. Thus, an experimental system was established for the reconstitution of ETF from the four components, alpha, beta, FAD, and AMP. Analyses of the absorption spectra of the separated subunits suggested that the contents of aromatic residues were one tryptophan and six tyrosines in alpha and one tryptophan and one tyrosine in beta. The possibility that one subunit binds with FAD or AMP in the absence of the other subunit was examined by measuring the flavin and protein fluorescence spectra, but no spectral changes reflecting such binding were detected. This result suggests that the coexistence of both the subunits is necessary for the binding of FAD and AMP with the protein.

Adenosine Monophosphate↗
PubMed1993

13C- and 15N-NMR studies on medium-chain acyl-CoA dehydrogenase reconstituted with 13C- and 15N-enriched flavin adenine dinucleotide.

The 13C- and 15N-NMR spectra of porcine kidney medium-chain acyl-CoA dehydrogenase (MCAD) reconstituted with 13C- and 15N-enriched FADs were measured. The positions of selective enrichment were C(2), C(4), C(4 alpha), C(10 alpha), N(1), N(3), and N(5) of the isoalloxazine nucleus of FAD. The NMR signals of the labeled atoms were observed as broad but distinct peaks in each NMR spectrum. The chemical shift values of the 2-, 4-, 4 alpha-, and 10 alpha-13C for the oxidized form of MCAD were 159.5, 166.8, 141.1, and 155.5 ppm, respectively, relative to the methyl resonance of 3-(trimethylsilyl)propionic acid-d4, while those of 1-, 3-, and 5-15N for the oxidized form were 183.6, 161.1, and 334.7 ppm, relative to liquid ammonia, respectively. The upfield shift of 2-13C of MCAD relative to that of FMN in the aqueous medium and its downfield shift relative to that of tetraacetylriboflavin in an apolar medium imply that a weaker hydrogen bond exists between C(2) = O and apoMCAD or a water molecule than that of free FMN with a water molecule. That the 4-13C resonance was observed downfield-shifted relative to that of free FMN in aqueous solution suggests a strong hydrogen bond between C(4) = O and apoMCAD. The chemical shift for 4 alpha-13C in oxidized MCAD is considerably downfield-shifted from that of FMN or any other flavoprotein observed thus far, indicating a unique environment around this position in MCAD. The 1-15N resonance of MCAD was most upfield-shifted among the flavoproteins observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl Coenzyme A↗
PubMed1993

Electron-transferring flavoprotein has an AMP-binding site in addition to the FAD-binding site.

Mammalian electron-transferring flavoprotein (ETF) has been reported to consist of two non-identical subunits and one FAD. The present paper shows that ETF purified from pig kidney contains one more molecule, an AMP. ETF was denatured by guanidine hydrochloride and ultrafiltered for the purpose of removing proteins. The filtrate was analyzed by reverse-phase chromatography. Two peaks appeared on the chromatogram: they were identified as FAD and AMP, and their molar amounts were identical, indicating that ETF contains one AMP molecule. ApoETF, which was prepared by KBr treatment of ETF, also contains one AMP molecule. ApoETF, which was prepared by KBr treatment of ETF, also contain one AMP molecule. These results clearly demonstrate that ETF has an AMP-binding site in addition to the FAD-binding site. AMP-free apoETF was prepared by guanidine treatment of ETF. Mixing AMP-free apoETF, FAD, and AMP produced reconstituted ETF, which showed the same properties as native ETF. Mixing AMP-free apoETF and FAD produced AMP-free ETF, regardless of the coexistence of ATP or ADP: the AMP-binding site cannot bind FAD, ADP, or ATP. The enzymatic activity of the AMP-free ETF for electron transfer from substrate-reduced medium-chain acyl-CoA dehydrogenase to 2,6-dichlorophenolindophenol was identical to that of native ETF. This indicates that the AMP contained in holoETF has no apparent influence on this enzymatic activity. A role of AMP recognized in this study is that AMP facilitates the formation of holoETF from AMP-free apoETF, FAD, and AMP.

Adenosine Monophosphate↗
PubMed1993

[A case of acute coronary syndrome followed by 201Tl, 123I-BMIPP and 123I-MIBG myocardial imagings before and after PTCA].

201TlCl (Tl), 123I-beta-methyl-p-iodophenyl pentadecanoic acid (BMIPP) and 123I-metaiodobenzylguanidine (MIBG) images were applied before and after PTCA to a patient with acute coronary syndrome who had repeated ischemic attacks on light effort. A decreased tracer uptake was noted at the mid and apical anteroseptal regions in each image before PTCA. MIBG uptake was most decreased and Tl uptake was least decreased, but Tl uptake at exercise decreased as severely as MIBG uptake at rest. After PTCA an uptake was improved in each image. Both before and after PTCA, the area with decreased BMIPP uptake corresponded to the region with deteriorated wall motion obtained by echocardiography. It was suspected that the decreased MIBG uptake correlates with "area at risk" and that the decreased BMIPP uptake correlates with abnormal wall motion. Taken together those findings, it was indicated that an application of these cardiac scintigraphy is useful for the evaluation of comprehending metabolic abnormality and recovering process of ischemic myocardium.

3-Iodobenzylguanidine↗
PubMed1993

[The distribution and kinetics of 123I-MIBG in normal human hearts].

123I-metaiodobenzylguanidine (MIBG) myocardial scintigraphy was performed in twelve normal human subjects, and the distribution and clearance of MIBG were estimated. In PLANAR studies, heart to mediastinum (H/M) and lung to mediastinum activity ratio (L/M) were 2.68 +/- 0.45 and 1.82 +/- 0.23 in initial images, and 2.75 +/- 0.44 and 1.55 +/- 0.13 in delayed images. The clearance of MIBG in the lung was more rapid than that in the heart, 41.6 +/- 4.1 vs. 29.8 +/- 3.2 (%), therefore MIBG cardiac profiles were more clarified in delayed images. In SPECT studies, the regional relative uptake (RRU) in anterior, septal, inferior and lateral wall were 100.0 +/- 0, 93.9 +/- 6.9, 85.0 +/- 10.0 and 104.9 +/- 8.3 (%) in initial images, and 100.0 +/- 0, 96.8 +/- 9.6, 79.4 +/- 8.3 and 99.1 +/- 7.9 (%) in delayed images, respectively. The RRU in inferior wall was significantly lower than those in the other walls. The regional clearance rate (RCR) in these four walls were 25.1 +/- 4.6, 22.5 +/- 8.2, 29.7 +/- 8.3, 29.3 +/- 3.4 (%), respectively. The RCR in inferior and lateral wall were significantly larger than those in anterior and septal walls. The RRU in basal, mid and apical portion were 100.0 +/- 0, 104.5 +/- 3.3, 98.9 +/- 12.1 (%) in initial images, and 100.0 +/- 0, 103.9 +/- 4.5, 96.8 +/- 15.2 (%) in delayed images, respectively. The RRU in mid portion was significantly higher than that in basal portion.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Iodobenzylguanidine↗
PubMed1993

[Effect of verapamil on myocardial ischemia in patients with hypertrophic cardiomyopathy: evaluation by exercise 201Tl SPECT].

Effect of verapamil on myocardial ischemia in patients with hypertrophic cardiomyopathy (HCM) was evaluated by exercise stress myocardial 201Tl SPECT (EX-Tl). EX-Tl were performed before and after 8.8 weeks of oral verapamil (240 mg/day) in 12 patients with HCM who showed transient 201Tl perfusion defects under control conditions. 201Tl perfusion defect was visually scored and judged for 4 grades as normal (0), mild defect (1), moderate defect (2), and severe defect (3). Transient Dilation Index (TDI) was calculated as an index of subendocardial ischemia. Improvements of defect score were demonstrated in 10 patients after administration of verapamil. Two patients showed no change of defect score. Mean defect score decreased significantly from 5.50 to 3.03 (p < 0.001). Although 11 of 12 patients showed abnormal TDI under control conditions, 10 of them revealed improvements of TDI and 7 of those 10 patients disclosed normal TDI after verapamil. Mean TDI decreased from 1.263 to 1.090 significantly (p < 0.01). In conclusion, verapamil may improve myocardial ischemia in patients with HCM.

Adult↗
PubMed1992

The primary structure of rat rig/ribosomal protein S15 gene.

We have isolated rat rig/ribosomal protein S15 gene from a DNA library derived from a rat insulinoma and determined the complete nucleotide sequence. The rat rig/S15 gene is composed of four exons and three introns spanning 2 kbp and exhibits distinctive structural features unique for a ribosomal protein gene.

Amino Acid Sequence↗