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Biomedical subjects

M Shirakawa

Publications and source records attributed to M Shirakawa.

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

Obturator hernia: the usefulness of computed tomography in diagnosis.

BACKGROUND: Delay in diagnosis and treatment of the rare obturator hernia often leads to high resection and mortality rates. Reviews of the clinical courses and the reliability of computed tomography (CT) in early diagnosis are reported. METHODS: Seventeen patients with obturator hernias were treated between April 1983 and December 1994. Except for one man, all were emaciated women who had undergone an average of 4.7 live deliveries. The mean age was 79.9 years. RESULTS: All patients had small bowel obstruction symptoms. Howship-Romberg sign was present in 11 patients. Twelve cases were correctly diagnosed before operation. After the introduction of CT in the diagnosis of suspected hernia cases, preoperative diagnosis was made on 9 of the 10 patients (90%) and CT was performed on 8 patients with 100% accuracy. CONCLUSIONS: Definite and early diagnosis is possible with the awareness of the clinical courses of this hernia and by performing CT on suspected cases.

Aged↗
PubMed1996

Implications of the zinc-finger motif found in the DNA-binding domain of the human XPA protein.

BACKGROUND: The XPA (xeroderma pigmentosum group A) protein specifically recognizes the UV-or chemically damaged DNA lesions, and triggers the nucleotide excision repair process. This XPA protein contains the functional domain which is crucial to the recognition of damaged DNA. Its primary structure suggests that this DNA binding domain may contain a zinc-finger motif. To gain a more detailed insight into this zinc-finger motif, we have measured the 113Cd-NMR spectra of the DNA binding domains derived from the wild-type and mutant XPA proteins. RESULTS: 113Cd-NMR analysis, combined with atomic absorption and site-directed mutagenesis, revealed that the DNA binding domain contains one zinc ion, coordinated with four cysteine residues (Cys105, Cys108, Cys126, and Cys129), that is essential for correct protein folding in vivo and in vitro. CONCLUSIONS: The four ligand cysteine residues form a Cys-X2-Cys-X17-Cys-X2-Cys motif, which is reminiscent of the (Cys)4 type zinc-finger motif found in numerous transcriptional regulatory proteins. However, the secondary structure prediction and the 3D-1D compatibility analysis demonstrate that there is no structural similarity in the vicinity of the zinc-finger motif between the XPA protein and other zinc-finger containing proteins. We conclude that the XPA protein contains a new type of zinc-finger motif.

Amino Acid Sequence↗
PubMed1996

Surgical treatment of abdominal aortic aneurysms in octogenarians.

Due to the increase in life span and the decrease in mortality associated with aortic surgery, many geriatric patients now undergo surgery for abdominal aortic aneurysms (AAAs). Therefore, we studied our surgical results for AAAs in an elderly population to assess the value of such operations. Twenty-six patients aged 80 years or older underwent surgery during an 11-year period from 1984 to 1994 at our institutions, and their outcomes were compared with those of 212 younger patients. The ratio of ruptured to non-ruptured AAAs was significantly higher in the older patients (aged 80 years or older) than in the younger patients (aged 79 years or younger), and aneurysm size in cases of non-rupture was greater in older patients than in younger patients. For octogenarians with non-ruptured AAAs, the survival rate was 85.7% at 5 years, compared with 43.6% at 3 years for those with ruptured AAAs, and these figures were not significantly different from those in younger patients. The present findings support the value of our active surgical approach for octogenarians with AAAs. We believe that an aggressive approach for octogenarians will decrease the incidence of ruptured AAAs and contribute to better patient survival.

Adult↗
PubMed1996

Cloning, sequencing, and expression of a beta-amylase gene from Bacillus cereus var. mycoides and characterization of its products.

The cloned gene was composed of 1638 bp for coding plus promoter like and SD-like sequences ahead of it. The deduced amino acid sequence had high similarity with known beta-amylases. The N-terminal sequence of the cloned beta-amylase seemed to be a signal peptide. The gene was introduced into Bacillus subtilis 1A289 using pHY300PLK as a vector and the expressed protein was recovered from the culture media. The enzyme fraction produced was divided into two components upon the DEAE column chromatography. The amino acid sequence of one fraction (FrI) was the same as the mature enzyme, and the other (FrII) lacked the N-terminal amino acid residue (Ala) of the mature enzyme. The kinetic parameters of the hydrolysis catalyzed by the enzyme component FrI were measured, and the subsite affinities of the enzyme were evaluated. In conclusion, it was shown that the recombinant enzyme was the same as the mature enzyme functionally and proteochemically.

Amino Acid Sequence↗
PubMed1995

Three-dimensional dimer structure of the lambda-Cro repressor in solution as determined by heteronuclear multidimensional NMR.

The 1H, 15N and 13C magnetic resonances of the lambda-Cro repressor have been assigned almost completely, mainly through the use of heteronuclear multidimensional NMR methods. Inter-subunit NOEs were distinguished by means of heteronuclear spectral editing technique (13C double half filter technique). Based on the distance and dihedral angle constraints derived from the NMR data, the three-dimensional solution structure of the lambda-Cro repressor in the dimeric form has been calculated by the simulated annealing method. The input for the structure calculations consisted of 1H-1H distance constraints, of which 1536 were intra-subunit and 40 were inter-subunit, and dihedral angle, phi, constraints, which numbered 92. The average root-mean-square deviation (RMSD) for all backbone heavy- atoms of the 20 calculated structures for residues 3 to 59 of the total of 66 amino acid residues in both subunits was 1.57 Angstrum, while the average RMSD for each subunit in the same residue range was 0.66 Angstrum. The subunit is composed of three alpha-helices, residues 7 to 13, 16 to 23 and 27 to 36, and a three-stranded anti-parallel beta-sheet composed of residues 3 to 6, 40 to 44 and 50 to 55. The solution structure of the subunit is essentially the same as that in the crystalline form, but the structure of the dimer form in solution differs from that of the dimer unit in the crystalline form. It is suggested that the solution dimer structure is distorted to fit the recognition helices in the major grooves of DNA on complex formation.

Amino Acid Sequence↗
PubMed1995

Solution structure of the activator contact domain of the RNA polymerase alpha subunit.

The structure of the carboxyl-terminal domain of the Escherichia coli RNA polymerase alpha subunit (alpha CTD), which is regarded as the contact site for transcription activator proteins and for the promoter UP element, was determined by nuclear magnetic resonance spectroscopy. Its compact structure of four helices and two long arms enclosing its hydrophobic core shows a folding topology distinct from those of other DNA-binding proteins. The UP element binding site was found on the surface comprising helix 1, the amino-terminal end of helix 4, and the preceding loop. Mutation experiments indicated that the contact sites for transcription activator proteins are also on the same surface.

Base Sequence↗
PubMed1995

Secondary structure and folding topology of the DNA binding domain of interferon regulatory factor 2, as revealed by NMR spectroscopy.

The secondary structure elements of the DNA-binding domain of mouse interferon regulatory factor 2 [IRF-2(113)] were determined by heteronuclear multidimensional NMR spectroscopy. The sequential NOE connectivities, amide proton exchange rates, and 3JHN alpha coupling constants indicated the presence of three alpha-helical regions and four short beta-strands connected through relatively long loops. The long range NOEs indicated the four strands form an antiparallel beta-sheet and the three alpha-helices form a bundle on the sheet. The arrangement of the secondary structure elements and the overall folding topology resemble those of the DNA binding domains of bacterial activator CAP, heat shock transcription factors, and fork-head proteins, although there is no sequence homology among them.

Amino Acid Sequence↗
PubMed1995

Structure of the Oct-3 POU-homeodomain in solution, as determined by triple resonance heteronuclear multidimensional NMR spectroscopy.

The POU-homeodomain (POUH) forms the bipartite DNA-binding POU domain in association with the POU-specific domain. The 1H, 15N, and 13C magnetic resonances of the 67-amino acid long POUH of mouse Oct-3 have almost completely been assigned, mainly through the combined use of three-dimensional triple resonance NMR methods. Based on the distance and dihedral angle constraints derived from the NMR data, the solution structure of the POUH domain has been calculated by the ab initio simulated annealing method. The average RMS deviation for all backbone heavy atoms of the 20 best calculated structures for residues 9-53 of the total 67 amino acid residues is 0.44 A. The POUH domain consists of three alpha-helices (helix-I, 10-20; helix-II, 28-38; and helix-III, 42-53), and helices-II and -III form a helix-turn-helix motif. In comparison with other classical homeodomains, the folding of the three helices is quite similar. However, the length of helix-III is fairly short. In the complex of the Oct-1 POU domain with an octamer site (Klemm JD, et al., 1994, Cell 77:21-32), the corresponding region is involved in helix-III. The structural difference between these two cases will be discussed.

Amino Acid Sequence↗
PubMed1995

Effects of basic fibroblast growth factor on proliferation, the expression of osteonectin (SPARC) and alkaline phosphatase, and calcification in cultures of human pulp cells.

Basic fibroblast growth factor (bFGF) may be involved in the development and repair of dentine and pulp because bFGF, its related peptides, and FGF receptors are expressed in dental mesenchymal cells. In this study, we examined the effects of bFGF on DNA synthesis, osteonectin/SPARC levels, alkaline phosphatase (ALPase) activity, their mRNA levels, and calcium levels in cultures of human pulp cells. Pulp cells were isolated from three healthy upper wisdom teeth of three patients and maintained separately. These cells produced SPARC, ALPase, and calcified nodules and there was a close correlation between the SPARC-synthetic activity of the cell lines and their levels of ALPase and calcification. The levels of SPARC, ALPase and calcium deposits in the three pulp cell cultures were 10-250 times those of human foreskin fibroblasts. Western blots showed that the pulp cells produced 38-kDa SPARC. Northern blots showed that the pulp cells expressed flg (FGF receptor type 1) transcripts throughout all culture stages, irrespective of the presence or absence of bFGF. The addition of bFGF to the pulp cultures suppressed the increases in ALPase activity, SPARC synthesis, and their mRNA levels, although it increased the incorporation of [3H]thymidine into DNA > 10-fold. The effects of bFGF on ALPase activity and SPARC synthesis were reversible. Furthermore, bFGF abolished the calcification of the extracellular matrix; the calcium content of bFGF-free cultures. These findings suggest that bFGF is a potent mitogen for human pulp cells and that it inhibits the expression of the odontoblast phenotype by the cells at least partly at pretranslational levels.

Alkaline Phosphatase↗
PubMed1995

Characteristics of multimodal co-aggregation between Fusobacterium nucleatum and streptococci.

The co-aggregation characteristics between Fusobacterium nucleatum and streptococci were examined to clarify the adherence factors participating in the co-aggregation. Nineteen strains of F. nucleatum were classified into 8 groups according to co-aggregation titer and inhibition by L-arginine, L-lysine and N-acetyl-D-galactosamine (or lactose). The inhibition activity was, however, very different from strain to strain. With two fusobacterial strains, two inhibitors, which were both inhibition negative on their own, completely inhibited the co-aggregation when used together in a mixture. In some co-aggregation pairs, the protease treatment of F. nucleatum inactivated one of the adherence factors, and resulted in the change of inhibition characteristics. These results indicate the multimodal co-aggregation of F. nucleatum with streptococci mediated by L-arginine-sensitive, L-lysine-sensitive, N-acetyl-D-galactosamine-sensitive and in some resistant factors, and that the adherence factor or factors participating the co-aggregation change according to the co-aggregation partners.

Acetylgalactosamine↗
PubMed1994

Pathway-specific regulation of the synthesis of anticoagulantly active heparan sulfate.

L cells and endothelial cells synthesize a heparan sulfate (HS) subpopulation, HSact, that exhibits anticoagulant activity due to a specific monosaccharide sequence; the remaining heparan sulfate, HSinact, lacks this region of defined structure and is anticoagulantly inactive. HSact biosynthesis was examined in these two cell types by stably expressing epitope-tagged rat ryudocan (ryudocan12CA5), which possesses three glycosaminoglycan (GAG) acceptor sites. Both HSact and HSinact were present on ryudocan12CA5 isolated from L cells and endothelial cells; thus, a core protein with a unique primary sequence initiates the synthesis of both GAGs. The expression in L cells of ryudocan12CA5 variants containing a single functional GAG acceptor site demonstrated that each of the three acceptor regions initiates the synthesis of both types of GAGs to a similar extent. Most importantly, in both cell types total HSact generation declined as a function of ryudocan12CA5 overexpression even though HSinact production increased linearly as a function of this variable. This discordant relationship is a general property of the biosynthetic machinery since in both cell types HSact production was reduced to an equal extent on protein cores of either exogenous or endogenous origins. The suppression of HSact generation was also observed with a secreted form of core protein lacking transmembrane and cytoplasmic domains or by a GAG acceptor site mutated form of core protein incapable of augmenting GAG synthesis. These results suggest that elevated intracellular levels of core protein saturate the capacity of a critical component of the HSact biosynthetic machinery. This critical component is not a member of the common set of biosynthetic enzymes involved in the production of HSact and HSinact since no structural changes were observed in either GAG during overexpression of core protein. Based upon the above data, we conclude that increased intracellular levels of ryudocan probably act by saturating the capacity of components which regulate HSact production by coordinating the function of biosynthetic enzymes.

Amino Acid Sequence↗
PubMed1994

Lysophosphatidylcholine from white muscle of bonito Euthynnus pelamis (Linnaeus): involvement of phospholipase A1 activity for its production.

A fairly large amount of lysophosphatidylcholine (LPC) was detected in the fresh muscle of bonito Euthynnus pelamis (Linnaeus). The major fatty acid esterified in LPC were highly unsaturated fatty acids, such as docosahexaenoic and eicosapentaenoic acids, and the form was mainly composed of 1-lyso-2-acyl-sn-glycero-3-phosphocholine (1-LPC). The content of this species continued to increase during 4 months of frozen storage and then decreased. Phospholipase A1 activity detected in the bonito muscle was supposed to be responsible for the accumulation of LPC.

Animals↗
PubMed1994

Characterization of ryudocan glycosaminoglycan acceptor sites.

The specificity of the glycosaminoglycan (GAG) acceptor sites of ryudocan was examined by stably expressing epitope-tagged ryudocan cDNA constructs in mouse L cells, which normally produce this proteoglycan. Immunopurified ryudocan was glycanated with both heparan sulfate (HS) and chondroitin sulfate (CS). The attachment of GAGs to ryudocan was prevented by creating Ser-->Thr mutations in all possible combinations at positions 44, 65, and 67. The resulting ryudocan of exogenous origin was immunopurified and evaluated with regard to attached GAG chains. The data reveal that ryudocan possesses three functional GAG attachment sites, that the sites are always occupied with GAG chains, and that each site is capable of bearing either HS or CS. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of GAG lyase digests of intact ryudocan reveal the production of the following multiple isoforms: pure HS-ryudocan, various HS/CS-hybrids, and pure CS-ryudocan. The data suggest that the occupancy bias of each site for HS or CS is slight and that each site functions in a relatively independent fashion. The GAG lyase analysis of partially purified L cell proteoglycans shows two pure CS-homoglycans with core proteins of M(r) = 130,000 and 52,000, respectively. A similar analysis of immunopurified L cell glypican demonstrates that this species only exists as a pure HS-homoglycan. The production of pure homoglycans by this clonal cell line strongly suggests that the functional promiscuity of GAG attachment sites of ryudocan must be encoded in the core protein structure. This property of ryudocan is not peculiar to L cells, as ryudocan synthesized by early passage human endothelial cells also bears both HS and CS. The production of multiple isoforms of ryudocan may serve to expand the functional versatility of this cell surface component and allow it to participate in many different biologic processes.

Amino Acid Sequence↗
PubMed1994

The XPA protein is a zinc metalloprotein with an ability to recognize various kinds of DNA damage.

The XPA (xeroderma pigmentosum group A) gene encodes a protein of 273 amino acids with a zinc finger motif. The human XPA cDNA was placed in an Escherichia coli expression vector for the synthesis of the recombinant XPA protein. The molecular weight of the wild-type protein was about 40 kDa in SDS-PAGE. Microinjection of the wild-type protein specifically restored the defect of UV-induced unscheduled DNA synthesis in XP-A cells. Thus, the bacterially expressed XPA protein retains biochemical properties identical to those of natural sources. The wild-type protein binds preferentially to UV-, cis-diamminedichloroplatinum(II) (cisplatin)- or osmium tetroxide (OsO4)-damaged DNA as assayed by retention on nitrocellulose filters. In addition, the data from atomic absorption and UV-CD spectra revealed that the wild-type protein is a zinc metalloprotein with secondary structure. Furthermore, the mutant protein, of which the cysteine-103 residue in the zinc finger motif was replaced with serine, has a vastly different protein conformation resulting in a loss of XP-A correcting and DNA-binding activities. These findings indicate that the XPA protein is a zinc-binding protein with affinity for various DNA damages, and a cysteine residue in the C4-type zinc finger motif is indispensable for normal protein conformation.

Amino Acids↗
PubMed1994

Inhomogeneity of cellular activation time and Vmax in normal myocardial tissue under electrical field stimulation.

To clarify the mechanism underlying the ectopic excitation after countershock, the cellular activation processes of cardiac tissue with a low-potential-gradient electric field (LPEF) were investigated in experiments using guinea pig papillary muscles and in computer simulation. Action potential upstrokes in papillary muscles during longitudinal propagation (LP) or transverse propagation (TP) were different from those of nonpropagating ones in single ventricular cells in terms of lower maximum upstroke velocity (Vmax) (LP, 231 V/s; TP, 309 V/s) and the presence of a linear ascending segment in the phase-plane plot. High Vmax (409 V/s) close to the single cell (512 V/s) was obtained in the muscle at the collision of LP (LC). Field stimulation of the muscles with LPEF < 5 V/cm caused inhomogeneous excitation suggesting multiple wave fronts, which collide with each other, and a wide spatial dispersion of Vmax (132-388 V/s). Phase-plane plots of action potential with lower Vmax were similar to LP or TP, whereas those with higher Vmax were similar to LC. In the two-dimensional discrete sheet composed of 51 x 51 elements of modified Beeler-Reuter model, the inhomogeneous excitation induced by LPEF is mimicked by setting a random variation of stimulus onset in each element. LPEF may induce inhomogeneous excitation with multiple wave fronts through a complex electrotonic interaction. This would provide a basis for the genesis of ectopic focal excitation.

Action Potentials↗