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Biomedical subjects

M Shirakawa

Publications and source records attributed to M Shirakawa.

At least 19 recordsLinked to original sources
PubMed2006

Distance determination in human ubiquitin by pulsed double electron-electron resonance and double quantum coherence ESR methods.

Recently, distance measurements by pulsed ESR (electron spin resonance) have been obtained using pulsed DEER (double electron-electron resonance) and DQC (double quantum coherence) in SDSL (site directed spin labeling) proteins. These methods can observe long range dipole interactions (15-80A). We applied these methods to human ubiquitin proteins. The distance between the 20th and the 35th cysteine was estimated in doubly spin labeled human ubiquitin. Pulsed DEER requires two microwave sources. However, a phase cycle is not usually required in this method. On the other hand, DQC-ESR at X-band ( approximately 9GHz) can acquire a large echo signal by using pulses of short duration and high power, but this method has an ESEEM (electron spin echo envelope modulation) problem. We used a commercial pulsed ESR spectrometer and compared these two methods.

Algorithms↗
PubMed2005

[The correlation between flow pattern during cardiopulmonary bypass and patency of the coronary artery bypass grafts].

Recently the availability of transit time flow measurement (TTFM) is reported especially in off-pump coronary artery bypass grafting (CABG). But little is known about TTFM findings in on-pump CABG. We examined the correlation between the TTFM flow pattern and the angiography findings in on-pump CABG. The subjects consisted of 52 patients who underwent on-pump CABG and angiography early after operation. In these patients, 55 internal thoracic artery (ITA), 17 gastroepiploic artery (GEA), 13 saphenous vein graft (SVG) and 41 radial artery (RA) were tested with TTFM during cardiopulmonary bypass (CPB). TTFM demonstrated a diastolic filling pattern in 53 ITA, 16 GEA, 13 SVG and 36 RA. The angiography revealed that all these grafts were perfectly patent with the exception of a GEA with a flow competition pattern. TTFM revealed an abnormal flow pattern in 2 ITA (these 2 grafts were revised during CPB and the angiography demonstrated their perfect patency), 1 GEA (to and fro pattern), 0 SVG and 5 RA (the abnormal pattern was due to graft spasm in 3 of 5, and the angiography revealed their perfect patency, however, the angiography detected stenosis in the remaining 2 grafts). The present study found that the TTFM flow pattern during CPB correlated well with the angiography findings. TTFM during CPB was useful to detect graft failure, and grafts were revised safely during CPB.

Aged↗
PubMed2005

[Advantages of side-to-side anastomosis in the assessment and revision of coronary artery bypass grafting].

With the advent of drug eluting stents in percutaneous coronary intervention, required quality level of coronary artery bypass grafting (CABG) has been elevating. To obtain nearly perfect patency of bypass grafts, intraoperative assessment and repair of abnormal grafts are helpful. We report systematic revision and safe repair technique of arterial grafts in CABG. Side-to-side instead of commonly used end-to-side anastomosis of arterial grafts is the first step in this technique. When any abnormalities are noted in intraoperative flowmetry of a graft, the distal surgical clip is removed. Free flow of the graft is measured. A coronary probe is gently inserted into the graft and the coronary artery. Vasodilators can be injected into the graft if necessary. When direct revision of the anastomosis is indicated, the graft is cut longitudinally from the distal end up to just proximal to the anastomotic site. The shape of the anastomosis can be observed directly without removing sutures. When re-anastomosis is not indicated, the distal remnant graft tissue is folded back and utilized as a patch. Thus the graft can be easily closed without narrowing.

Anastomosis, Surgical↗
PubMed2004

Electrocardiographic Q-waves as a predictor of mortality in patients with cerebral infarction.

OBJECTIVE: To determine whether abnormal EKG findings could be predictive factors for death after a stroke event. METHODS: Patients with acute cerebral infarction who were 35 to 98 years old during April 1996 through November 2000 were analyzed (n = 216). A standard 12-lead EKG was recorded for each patient after acute cerebral infarction. The authors prospectively investigated the association between abnormal EKG findings and the risk for death after stroke over a 1-year period. RESULTS: Using multivariate Cox proportional hazards models, the authors found age (hazard ratio of mortality per year 1.10, 95% CI 1.06 to 1.15, p < 0.001), sex (female; hazard ratio of mortality 3.42, 95% CI 1.43 to 8.19, p = 0.006), and the presence of Q-waves in more than two leads (hazard ratio of mortality 2.75, 95% CI 1.23 to 6.14, p = 0.013) were independently associated with death after stroke. CONCLUSION: The presence of Q-waves in more than two leads could be a predictive factor for death after acute cerebral infarction.

Adult↗
PubMed2004

Activation of WNT family expression and signaling in squamous cell carcinomas of the oral cavity.

The WNT family activates an oncogenic signaling mediated through beta-catenin and is up-regulated in a variety of malignant neoplasms. The signaling translocates beta-catenin into the nucleus and stimulates carcinoma cells in the epithelial-mesenchymal transition (EMT). However, WNT expression and signaling in oral carcinomas have not been examined. The present study focused on unveiling the involvement of WNTs in oral carcinomas, and showed that carcinoma cells express 11 of 19 WNT family members by reverse-transcription/PCR. WNT-expressing carcinoma cells exhibited increased beta-catenin levels in the cytoplasmic pool and translocation to the nucleus. The activation state of signaling correlated with the expression of membrane-type 1 matrix metalloproteinase, which degrades territorial matrices in carcinoma invasion. Immunohistochemistry disclosed that WNT3 expression and nuclear localization of beta-catenin were predominant in carcinoma cells at the invasive front. These results suggest that enhanced WNT expression and signaling accelerate the progression of carcinomas via activating EMTs and local invasiveness.

Carcinoma, Squamous Cell↗
PubMed2001

Solution structure of the methyl-CpG binding domain of human MBD1 in complex with methylated DNA.

In vertebrates, the biological consequences of DNA methylation are often mediated by protein factors containing conserved methyl-CpG binding domains (MBDs). Mutations in the MBD protein MeCP2 cause the neurodevelopmental disease Rett syndrome. We report here the solution structure of the MBD of the human methylation-dependent transcriptional regulator MBD1 bound to methylated DNA. DNA binding causes a loop in MBD1 to fold into a major and novel DNA binding interface. Recognition of the methyl groups and CG sequence at the methylation site is due to five highly conserved residues that form a hydrophobic patch. The structure indicates how MBD may access nucleosomal DNA without encountering steric interference from core histones, and provides a basis to interpret mutations linked to Rett syndrome in MeCP2.

Acetylation↗
PubMed2001

Solution structure and dynamic character of the histidine-containing phosphotransfer domain of anaerobic sensor kinase ArcB from Escherichia coli.

An Escherichia coli sensor kinase, ArcB, transfers a phosphoryl group to a partner response regulator in response to anaerobic conditions. Multidimensional NMR techniques were applied to determine the solution structure of the histidine-containing phosphotransfer signaling domain of ArcB (HPt(ArcB)), which has a phosphorylation site, His717. The backbone dynamics were also investigated by analyses of the (15)N relaxation data and amide hydrogen exchange rates. Furthermore, the protonation states of the histidine imidazole rings were characterized by means of (1)H and (15)N chemical shifts at various pHs. The determined solution structure of HPt(ArcB) contains five helices and forms a four-helix bundle motif like other HPt domains. The obtained order parameters, S (2), [(1)H]-(15)N heteronuclear NOE values, and chemical exchange parameters, R(ex), showed that the alpha-helical regions of HPt(ArcB) are rigid on both picosecond to nanosecond and microsecond to millisecond time scales. On the other hand, helix D, which contains His717, exhibited low protection factors of less than 4000, indicating the presence of fluctuations on a slower time scale in helix D. These results suggest that HPt(ArcB) may undergo a small conformational change in helix D upon phosphorylation. It was also shown that the imidazole ring of His717 has a pK(a) value of 6.76, which is similar to that of a solvent-exposed histidine imidazole ring, and that a pair of deprotonated neutral tautomers are rapidly exchanged with each other. This is consistent with the solution structure of HPt(ArcB), in which the imidazole ring of His717 is exposed to the solvent.

Amides↗
PubMed2001

Regulation of transcription and chromatin by methyl-CpG binding protein MBD1.

DNA methylation is important for epigenetic regulation of genome, and it is interpreted by specific protein factors that contain a highly conserved methyl-CpG binding domain (MBD). There are at present five mammalian MBD family proteins: MBD1, MBD2, MBD3, MBD4 and MeCP2. In the family of methyl-CpG binding proteins, MBD1 is characterized by the presence of MBD, two or three cysteine-rich CXXC motifs, and the C-terminal transcriptional repression domain (TRD). In addition, MBD1 has at least five isoforms due to alternative splicing events, resulting in the existence of CXXC1, CXXC2, and CXXC3 in MBD1 isoform v1 (MBD1v1) and MBD1v2, and CXXC1 and CXXC2 in MBD1v3 and MBD1v4. MBD1v1 represses transcription preferentially from both unmethylated and hypomethylated promoters, while MBD1v3 inhibits hypermethylated but not unmethylated promoter activities. The MBD and CXXC3 sequences are responsible for the ability to bind methylated and unmethylated DNAs, respectively. MBD1 is also found to be a chromosomal protein that forms many foci within the nucleus. These findings suggest that MBD1 is a unique transcriptional regulator depending on the density of methyl-CpG pairs.

Animals↗
PubMed2001

Anti-HIV-1 activity of an antisense phosphorothioate oligonucleotide bearing imidazole and primary amine groups.

We have previously shown that RNA cleaving reagents with imidazole and primary amine groups on the 5'-end of antisense oligodeoxyribonucleotides could site-specifically cleave CpA as the target sequence of the substrate tRNA in vitro. In this study, a RNA cleaving reagent, composed of imidazole and primary amine groups on an antisense phosphorothioate oligonucleotide (Im-anti-s-ODN), was synthesized and evaluated for anti-HIV-1 activity in MT-4 cells. The sequence of the Im-anti-s-ODN was designed to be complementary to the HIV-1 gag-mRNA and to bind adjacent to the CpA cleavage site position. Im-anti-s-ODN encapsulated with the transfection reagent, DMRIE-C, had higher anti-HIV-1 activity than the unmodified antisense phosphorothioate oligonucleotide (anti-s-ODN) at a 2 microM concentration. Furthermore, the Im-anti-ODN encapsulated with DMRIE-C conferred sequence-specific inhibition.

Amines↗
PubMed2001

Conformation of a peptide ligand bound to its G-protein coupled receptor.

Many peptide hormones elicit a wide array of physiological effects by binding to G-protein coupled receptors. We have determined the conformation of pituitary adenylate cyclase activating polypeptide, PACAP(1--21)NH(2), bound to a PACAP-specific receptor by NMR spectroscopy. Residues 3--7 form a unique beta-coil structure that is preceded by an N-terminal extended tail. This beta-coil creates a patch of hydrophobic residues that is important for receptor binding. In contrast, the C-terminal region (residues 8--21) forms an alpha-helix, similar to that in the micelle-bound PACAP. Thus, the conformational difference between PACAP in the receptor-bound and the micelle-bound states is limited to the N-terminal seven residues. This observation is consistent with the two-step ligand transportation model in which PACAP first binds to the membrane nonspecifically and then diffuses two-dimensionally in search of its receptor; a conformational change at the N-terminal region then allows specific interactions between the ligand and the receptor.

Amino Acid Sequence↗
PubMed2000

Three-dimensional structural views of damaged-DNA recognition: T4 endonuclease V, E. coli Vsr protein, and human nucleotide excision repair factor XPA.

Genetic information is frequently disturbed by introduction of modified or mismatch bases into duplex DNA, and hence all organisms contain DNA repair systems to restore normal genetic information by removing such damaged bases or nucleotides and replacing them by correct ones. The understanding of this repair mechanism is a central subject in cell biology. This review focuses on the three-dimensional structural views of damaged DNA recognition by three proteins. The first protein is T4 endonuclease V (T4 endo V), which catalyzes the first reaction step of the excision repair pathway to remove pyrimidine-dimers (PD) produced within duplex DNA by UV irradiation. The crystal structure of this enzyme complexed with DNA containing a thymidine-dimer provided the first direct view of DNA lesion recognition by a repair enzyme, indicating that the DNA kink coupled with base flipping-out is important for damaged DNA recognition. The second is very short patch repair (Vsr) endonuclease, which recognizes a TG mismatch within the five base pair consensus sequence. The crystal structure of this enzyme in complex with duplex DNA containing a TG mismatch revealed a novel mismatch base pair recognition scheme, where three aromatic residues intercalate from the major groove into the DNA to strikingly deform the base pair stacking but the base flipping-out does not occur. The third is human nucleotide excision repair (NER) factor XPA, which is a major component of a large protein complex. This protein has been shown to bind preferentially to UV- or chemical carcinogen-damaged DNA. The solution structure of the XPA central domain, essential for the interaction of damaged DNA, was determined by NMR. This domain was found to be divided mainly into a (Cys)4-type zinc-finger motif subdomain for replication protein A (RPA) recognition and the carboxyl terminal subdomain responsible for DNA binding.

Amino Acid Motifs↗
PubMed2000

Solution structure of the chitin-binding domain of Bacillus circulans WL-12 chitinase A1.

The three-dimensional structure of the chitin-binding domain (ChBD) of chitinase A1 (ChiA1) from a Gram-positive bacterium, Bacillus circulans WL-12, was determined by means of multidimensional heteronuclear NMR methods. ChiA1 is a glycosidase that hydrolyzes chitin and is composed of an N-terminal catalytic domain, two fibronectin type III-like domains, and C-terminal ChBD(ChiA1) (45 residues, Ala(655)-Gln(699)), which binds specifically to insoluble chitin. ChBD(ChiA1) has a compact and globular structure with the topology of a twisted beta-sandwich. This domain contains two antiparallel beta-sheets, one composed of three strands and the other of two strands. The core region formed by the hydrophobic and aromatic residues makes the overall structure rigid and compact. The overall topology of ChBD(ChiA1) is similar to that of the cellulose-binding domain (CBD) of Erwinia chrysanthemi endoglucanase Z (CBD(EGZ)). However, ChBD(ChiA1) lacks the three aromatic residues aligned linearly and exposed to the solvent, which probably interact with cellulose in CBDs. Therefore, the binding mechanism of a group of ChBDs including ChBD(ChiA1) may be different from that proposed for CBDs.

Amino Acid Sequence↗
PubMed2000

Expression and characterization of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12.

Chitinase A1 from Bacillus circulans WL-12 comprises an N-terminal catalytic domain, two fibronectin type III-like domains, and a C-terminal chitin-binding domain (ChBD). In order to study the biochemical properties and structure of the ChBD, ChBD(ChiA1) was produced in Escherichia coli using a pET expression system and purified by chitin affinity column chromatography. Purified ChBD(ChiA1) specifically bound to various forms of insoluble chitin but not to other polysaccharides, including chitosan, cellulose, and starch. Interaction of soluble chitinous substrates with ChBD(ChiA1) was not detected by means of nuclear magnetic resonance and isothermal titration calorimetry. In addition, the presence of soluble substrates did not interfere with the binding of ChBD(ChiA1) to regenerated chitin. These observations suggest that ChBD(ChiA1) recognizes a structure which is present in insoluble or crystalline chitin but not in chito-oligosaccharides or in soluble derivatives of chitin. ChBD(ChiA1) exhibited binding activity over a wide range of pHs, and the binding activity was enhanced at pHs near its pI and by the presence of NaCl, suggesting that the binding of ChBD(ChiA1) is mediated mainly by hydrophobic interactions. Hydrolysis of beta-chitin microcrystals by intact chitinase A1 and by a deletion derivative lacking the ChBD suggested that the ChBD is not absolutely required for hydrolysis of beta-chitin microcrystals but greatly enhances the efficiency of degradation.

Amino Acid Sequence↗
PubMed2000

Mechanism of transcriptional regulation by methyl-CpG binding protein MBD1.

MBD1 is a mammalian protein that binds symmetrically methylated CpG sequences and regulates gene expression in association with DNA methylation. This protein possesses a conserved sequence, named methyl-CpG binding domain (MBD), among a family of methyl-CpG binding proteins that mediate the biological consequences of the methylation. In addition, MBD1 has at least five isoforms due to alternative splicing events, resulting in the presence of CXXC1, CXXC2, and CXXC3 in MBD1 isoforms v1 (MBD1v1) and MBD1v2, and CXXC1 and CXXC2 in MBD1v3 and -v4. In the present study, we have investigated the significance of MBD, CXXC, and the C-terminal transcriptional repression domain (TRD) in MBD1. A bacterially expressed MBD binds efficiently to densely methylated rather than to sparsely methylated DNAs. In both methylation-deficient Drosophila melanogaster SL2 cells and mammalian CHO-K1 cells, MBD1v1 represses transcription preferentially from both unmethylated and sparsely methylated promoters, while MBD1v3 inhibits densely methylated but not unmethylated promoter activities. The CXXC3 sequence in MBD1v1 is responsible for the ability to bind unmethylated promoter. Furthermore, we have constructed mutant-type MBD1s in which the functionally important residues Arg22, Arg30, Asp32, Tyr34, Arg44, Ser45, and Tyr52 are changed to alanine to investigate the correlation between the structure and function of the MBD in MBD1. Excepting those for Ser45 and Tyr52, none of the recombinant MBD mutants bound to the densely methylated or unmethylated DNAs, and green fluorescent protein-fused MBD1 mutants did not localize properly in the nucleus. All the MBD1v1 and -v3 mutants lost the activity of methylation-dependent gene repression. Based on these findings we have concluded that MBD1 acts as a transcriptional regulator depending on the density of methyl-CpG pairs through the cooperation of MBD, CXXC, and TRD sequences.

Amino Acid Motifs↗
PubMed1999

Solution structure of the methyl-CpG-binding domain of the methylation-dependent transcriptional repressor MBD1.

CpG methylation in vertebrates is important for gene silencing, alterations in chromatin structure and genomic stability, and differences in the DNA-methylation status are correlated with imprinting phenomena, carcinogenesis and embryonic development. Methylation signals are interpreted by protein factors that contain shared methyl-CpG-binding domains (MBDs). We have determined the solution structure of the MBD of the human methylation-dependent transcriptional repressor MBD1 by multi-dimensional heteronuclear NMR spectroscopy. It folds into an alpha/beta-sandwich structure with characteristic loops. Basic residues conserved in the MBD family are largely confined to one face of this fold and a flexible loop, which together form a large positively charged surface. Site-directed mutagenesis and chemical shift changes upon complexing with a methylated DNA facilitated identification of this surface as the DNA interaction site. In addition to three basic residues, conserved Tyr34 and Asp32 were shown to be important for the DNA binding.

Amino Acid Sequence↗