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Biomedical subjects

M Suyama

Publications and source records attributed to M Suyama.

At least 19 recordsLinked to original sources
PubMed2001

Site-directed removal of N-glycosylation sites in BST-1/CD157: effects on molecular and functional heterogeneity.

Cyclic ADP ribose (cADPR) is a novel second messenger that releases calcium from intracellular calcium stores, but works independently of inositol 1,4,5-trisphosphate. In mammals ADP-ribosyl cyclase function is found in two membrane proteins, CD38 and bone marrow stromal cell antigen 1 (BST-1)/CD157. These enzymes are exposed extracellularly and also possess cADPR hydrolase activity, but an intracellular soluble ADP-ribosyl cyclase has been reported in human T-cells. Previously, a soluble form of BST-1/CD157 (sBST-1), which lacked the glycosylphosphatidylinositol-anchored portion, was expressed by a baculovirus-insect-cell system. In this study, we have purified the sBST-1, and it migrated as two major bands by SDS/PAGE, suggesting that it is post-translationally modified. BST-1 contains four putative N-glycosylation sites. Tunicamycin treatment reduced sBST-1 expression in the culture medium, indicating that N-glycosylation is essential for secretion. Site-directed mutagenesis was performed to generate sBST-1 mutants (N1-N4), each preserving a single N-glycosylation site. N1, N3 and N4 were well secreted into the medium, and were each detected as a single band. Although N3 and N4 retained the ADP-ribosyl cyclase activity, the cADPR-hydrolase activity was retained only in N4. We conclude that N-glycosylation of sBST-1 facilitates the folding of the nascent polypeptide chain into a conformation that is conductive for intracellular transport and enzymic activity. Furthermore a crystal has been obtained using the N4 mutant, but not the wild-type sBST-1. Thus the artificial engineering of N-glycosylation sites could be an effective method to generate homogeneous material for structural studies.

ADP-ribosyl Cyclase↗
PubMed2001

Evolution of prokaryotic gene order: genome rearrangements in closely related species.

Conservation of gene order in prokaryotes has become important in predicting protein function because, over the evolutionary timescale, genomes are shuffled so that local gene-order conservation reflects the functional constraints within the protein. Here, we compare closely related genomes to identify the rate with which gene order is disrupted and to infer the genes involved in the genome rearrangement.

Evolution, Molecular↗
PubMed2001

Leakage of energy to the body surface during defibrillation shock by an implantable cardioverter-defibrillator (ICD) system--experimental evaluation during defibrillation shocks through the right ventricular lead and the subcutaneous active-can in canines.

The leakage of electrical current to the body surface during defibrillation shock delivery by an implantable cardioverter-defibrillator (ICD) device (the Medtronic Jewel Plus PCD system) was evaluated in 5 dogs. The defibrillation shocks were delivered between the active-can implanted in the left subclavicular region and the endocardial lead placed in the right ventricle at the energy levels of 1, 2, 8, 12, 24 and 34 J. During each delivery, the electrical current leakage from the body surface was measured by electrodes connected to a circuit at 4 recording positions: (A) parallel-subcutaneous (the electrodes were fixed in the subcutaneous tissue of the left shoulder and the right lower chest, and the direction of the electrode vector was parallel to the direction of the defibrillation energy flow); (B) cross-subcutaneous (the electrodes were fixed in the subcutaneous tissue of the right shoulder and the left lower chest, and the vector of the electrodes was roughly perpendicular to the direction of the energy flow); (C) parallel-surface (the electrodes were fixed with ECG paste on the shaved skin surface at the left shoulder and the right lower chest); and (D) surface grounded (the electrodes were fixed on the shaved skin surface at the left shoulder and the left foot, which was grounded). The circuit resistance was set at a variable level (100-5,000 ohms) in accordance with the resistance measured through each canine body. Leakage energies were measured in 750 defibrillation shocks with each circuit resistance in 5 dogs. The leakage energy increased in accordance with the increase of the delivered energy and the decrease of the circuit resistance in all 4 recording positions. When the circuit resistance was set at 1,000 ohms, the leakage energy during shock delivery at 34 J was 32+/-17 mJ at position A, 5+/-9 mJ at B, 10+/-9 mJ at C, and 4+/-3 mJ at D (p=0.042). The peak current was highest at position A and was 87+/-22 mA with a circuit resistance of 1,000 ohms. The power of the leakage energy depended on the delivered energy and the impedance between the electrodes. The angle between the alignment of the recording electrodes and the direction of the energy flow was another important factor in determining the leakage energy. Although the peak current of the leakage energy reached the level of macro shock, the highest leakage energy from the body surface was considerably less because of the short duration of the shock delivery.

Animals↗
PubMed2000

Re-annotating the Mycoplasma pneumoniae genome sequence: adding value, function and reading frames.

Four years after the original sequence submission, we have re-annotated the genome of Mycoplasma pneumoniae to incorporate novel data. The total number of ORFss has been increased from 677 to 688 (10 new proteins were predicted in intergenic regions, two further were newly identified by mass spectrometry and one protein ORF was dismissed) and the number of RNAs from 39 to 42 genes. For 19 of the now 35 tRNAs and for six other functional RNAs the exact genome positions were re-annotated and two new tRNA(Leu) and a small 200 nt RNA were identified. Sixteen protein reading frames were extended and eight shortened. For each ORF a consistent annotation vocabulary has been introduced. Annotation reasoning, annotation categories and comparisons to other published data on M.pneumoniae functional assignments are given. Experimental evidence includes 2-dimensional gel electrophoresis in combination with mass spectrometry as well as gene expression data from this study. Compared to the original annotation, we increased the number of proteins with predicted functional features from 349 to 458. The increase includes 36 new predictions and 73 protein assignments confirmed by the published literature. Furthermore, there are 23 reductions and 30 additions with respect to the previous annotation. mRNA expression data support transcription of 184 of the functionally unassigned reading frames.

Amino Acid Sequence↗
PubMed2000

HUGE: a database for human large proteins identified in the Kazusa cDNA sequencing project.

HUGE is a database for human large proteins newly identified in the Kazusa cDNA project, the aim of which is to predict the primary structure of proteins from the sequences of human large cDNAs (>4 kb). In particular, cDNA clones capable of coding for large proteins (>50 kDa) are the current targets of the project. HUGE contains >1100 cDNA sequences and detailed information obtained through analysis of the sequences of cDNAs and the predicted proteins. Besides an increase in the number of cDNA entries, the amount of experimental data for expression profiling has been largely increased and data on chromosomal locations have been newly added. All of the protein-coding regions were examined by GeneMark analysis, and the results of a motif/domain search of each predicted protein sequence against the Pfam database have been newly added. HUGE is available through the WWW at http://www.kazusa.or.jp/huge

Animals↗
PubMed2000

Prediction of structural domains of TAP reveals details of its interaction with p15 and nucleoporins.

Vertebrate TAP is a nuclear mRNA export factor homologous to yeast Mex67p. The middle domain of TAP binds directly to p15, a protein related to the nuclear transport factor 2 (NTF2), whereas its C-terminal domain interacts with various nucleoporins, the components of the nuclear pore complex (NPC). Here, we report that the middle domain of TAP is also similar to NTF2, as well as to regions in Ras-GAP SH3 domain binding protein (G3BP) and some plant protein kinases. Based on the known three-dimensional structure of NTF2 homodimer, a heterodimerization model of TAP and p15 could be inferred. This model was confirmed by site-directed mutagenesis of residues located at the dimer interface. Furthermore, the C-terminus of TAP was found to contain a ubiquitin-associated (UBA) domain. By site-directed mutagenesis we show that a conserved loop in this domain plays an essential role in mediating TAP-nucleoporin interaction.

Amino Acid Sequence↗
PubMed2000

TAP (NXF1) belongs to a multigene family of putative RNA export factors with a conserved modular architecture.

Vertebrate TAP (also called NXF1) and its yeast orthologue, Mex67p, have been implicated in the export of mRNAs from the nucleus. The TAP protein includes a noncanonical RNP-type RNA binding domain, four leucine-rich repeats, an NTF2-like domain that allows heterodimerization with p15 (also called NXT1), and a ubiquitin-associated domain that mediates the interaction with nucleoporins. Here we show that TAP belongs to an evolutionarily conserved family of proteins that has more than one member in higher eukaryotes. Not only the overall domain organization but also residues important for p15 and nucleoporin interaction are conserved in most family members. We characterize two of four human TAP homologues and show that one of them, NXF2, binds RNA, localizes to the nuclear envelope, and exhibits RNA export activity. NXF3, which does not bind RNA or localize to the nuclear rim, has no RNA export activity. Database searches revealed that although only one p15 (nxt) gene is present in the Drosophila melanogaster and Caenorhabditis elegans genomes, there is at least one additional p15 homologue (p15-2 [also called NXT2]) encoded by the human genome. Both human p15 homologues bind TAP, NXF2, and NXF3. Together, our results indicate that the TAP-p15 mRNA export pathway has diversified in higher eukaryotes compared to yeast, perhaps reflecting a greater substrate complexity.

Active Transport, Cell Nucleus↗
PubMed2000

High frequency of clonally related tumors in cases of multiple synchronous lung cancers as revealed by molecular diagnosis.

In patients with multiple synchronous lung tumors, discrimination of multicentric lung cancers from intrapulmonary metastasis is important for treatment decision, but this is sometimes difficult. The aim of this study was to retrospectively distinguish multicentric lung cancers from intrapulmonary metastases in 14 such cases by loss of heterozygosity (LOH) and p53 mutational status. DNA was extracted from microdissected tumor cells in paraffin-embedded archival tissue, and 3p14.2, 3p21, 3p25, 9p21, and 18q21.1 were investigated for LOH. Exons 5-8 of the p53 gene were examined for mutations by the PCR, followed by single-strand conformation polymorphism analysis and DNA sequencing. For cases with the same LOH pattern, we calculated a clonality index, the probability of the given LOH pattern when these tumors were hypothesized to be independent in origin. Eleven of 14 cases (79%) were thus diagnosed as having pulmonary metastasis and only one case as having genuinely multicentric lung cancers. Two cases presented difficulty in diagnosis. In several cases, the LOH patterns conflicted with p53 mutation patterns, suggesting that clonal evolution is directly affected by certain genetic changes. The combination of p53 with LOH helped increase both the sensitivity and specificity of the assay.

Adult↗
PubMed1999

Prediction of the coding sequences of unidentified human genes. XIII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.

As a part of our cDNA project for deducing the coding sequence of unidentified human genes, we newly determined the sequences of 100 cDNA clones from a set of size-fractionated human brain cDNA libraries, and predicted the coding sequences of the corresponding genes, named KIAA0919 to KIAA1018. The sequencing of these clones revealed that the average sizes of the inserts and corresponding open reading frames were 4.9 kb and 2.6 kb (882 amino acid residues), respectively. A computer search of the sequences against the public databases indicated that predicted coding sequences of 87 genes contained sequences similar to known genes, 53% of which (46 genes) were categorized as proteins relating to cell signaling/communication, cell structure/motility and nucleic acid management. The chromosomal locations of the genes were determined by using human-rodent hybrid panels unless their mapping data were already available in the public databases. The expression profiles of all the genes among 10 human tissues, 8 brain regions (amygdala, corpus callosum, cerebellum, caudate nucleus, hippocampus, substania nigra, subthalamic nucleus, and thalamus), spinal cord, fetal brain and fetal liver were also examined by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.

Animals↗
PubMed1999

HUGE: a database for human large proteins identified by Kazusa cDNA sequencing project.

HUGE is a database for human large proteins newly identified by Kazusa cDNA project, which aims to predict protein primary structures from sequences of human large cDNAs (>4 kb). In particular, cDNA clones capable of coding for large proteins (>50 kDa) are current targets of the project. More than 700 sequences of human cDNAs (average size, 5.1 kb) have been determined to date and deposited in the public databases. Notable information implied from the cDNAs and the predicted protein sequences can be obtained through HUGE via the World Wide Web at URL http://www.kazusa.or.jp/huge

Cloning, Molecular↗
PubMed1998

Prediction of the coding sequences of unidentified human genes. XII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.

In this paper, we report the sequences of 100 cDNA clones newly determined from a set of size-fractionated human brain cDNA libraries and predict the coding sequences of the corresponding genes, named KIAA0819 to KIAA0918. These cDNA clones were selected on the basis of their coding potentials of large proteins (50 kDa and more) by using in vitro transcription/translation assays. The sequence data showed that the average sizes of the inserts and corresponding open reading frames are 4.4 kb and 2.5 kb (831 amino acid residues), respectively. Homology and motif/domain searches against the public databases indicated that the predicted coding sequences of 83 genes were similar to those of known genes, 59% of which (49 genes) were categorized as coding for proteins functionally related to cell signaling/communication, cell structure/motility and nucleic acid management. The chromosomal locations and the expression profiles of all the genes were also examined. For 54 clones including brain-specific ones, the mRNA levels were further examined among 8 brain regions (amygdala, corpus callosum, cerebellum, caudate nucleus, hippocampus, substantia nigra, subthalamic nucleus, and thalamus), spinal cord, and fetal brain.

Amino Acid Sequence↗
PubMed1998

Prediction of the coding sequences of unidentified human genes. XI. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.

In our series of projects for accumulating sequence information on the coding sequences of unidentified human genes, we have newly determined the sequences of 100 cDNA clones from a set of size-fractionated human brain cDNA libraries, and predicted the coding sequences of the corresponding genes, named KIAA0711 to KIAA0810. These cDNA clones were selected according to their coding potentials of large proteins (50 kDa and more) in vitro. The average sizes of the inserts and corresponding open reading frames were 4.3 kb and 2.6 kb (869 amino acid residues), respectively. Sequence analyses against the public databases indicated that the predicted coding sequences of 78 genes were similar to those of known genes, 64% of which (50 genes) were categorized as proteins functionally related to cell signaling/communication, cell structure/motility and nucleic acid management. As additional information concerning genes characterized in this study, the chromosomal locations of the clones were determined by using human-rodent hybrid panels and the expression profiles among 10 human tissues were examined by reverse transcription-coupled polymerase chain reaction which was substantially improved by enzyme-linked immunosorbent assay.

Brain Chemistry↗
PubMed1998

Clinical implications of p53 autoantibodies in the sera of patients with non-small-cell lung cancer.

BACKGROUND: The presence of autoantibodies to p53 protein has been associated with the presence of p53 (also known as TP53) gene mutations in primary tumors and with poor prognosis. This study was undertaken to determine the clinical significance of p53 autoantibodies in patients with non-small-cell lung cancer (NSCLC). METHODS: We studied 188 consecutive patients with NSCLC who underwent pulmonary resection and for whom preoperative serum was available. The presence of p53 autoantibodies, detected by use of two amino-terminal and two carboxy-terminal peptides (20-30 mers) as antigens and an enzyme-linked immunosorbent assay, was related to various clinicopathologic parameters and to overexpression of p53 protein in the primary tumor. For 22 patients who had p53 autoantibodies before surgery, we also examined sera taken during postoperative follow-up. Reported P values are two-sided. RESULTS: Autoantibodies to p53 protein were detected in 38 patients. Patients with squamous cell carcinoma, those with more advanced disease (stage III-IV), and those with tumors that overexpressed p53 had a significantly higher incidence of p53 autoantibodies (P = .05,.0079, and .02, respectively). In all but one of the patients with postoperative serum samples, the antibody titer declined after surgery; however, there was no relationship between clinical course and this change in antibody titer. In addition, there was no relationship between the presence of p53 autoantibodies and overall survival in 171 patients who underwent potentially curative resection (P = .28); however, 13 patients with autoantibodies to amino-terminal peptides had a worse overall survival (P = .02). CONCLUSIONS: In NSCLC, the incidence of p53 autoantibodies is associated with histologic type, stage, and p53 overexpression--but not with patient survival. Our data do not support the clinical utility of p53 autoantibodies as diagnostic or prognostic markers in patients with NSCLC.

Adult↗
PubMed1998

Prediction of the coding sequences of unidentified human genes. X. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro.

As an extension of our cDNA analysis for deducing the coding sequences of unidentified human genes, we have newly determined the sequences of 100 cDNA clones from a set of size-fractionated human brain cDNA libraries, and predicted the coding sequences of the corresponding genes, named KIAA0611 to KIAA0710. In vitro transcription-coupled translation assay was applied as the first screening to select cDNA clones which produce proteins with apparent molecular mass of 50 kDa and over. One hundred unidentified cDNA clones thus selected were then subjected to sequencing of entire inserts. The average size of the inserts and corresponding open reading frames was 4.9 kb and 2.8 kb (922 amino acid residues), respectively. Computer search of the sequences against the public databases indicated that predicted coding sequences of 87 genes were similar to those of known genes, 62% of which (54 genes) were categorized as proteins related to cell signaling/communication, cell structure/motility and nucleic acid management. The expression profiles in 10 human tissues of all the clones characterized in this study were examined by reverse transcription-coupled polymerase chain reaction and the chromosomal locations of the clones were determined by using human-rodent hybrid panels.

Brain Chemistry↗
PubMed1998

A method for comparing circular genomes from gene locations: application to mitochondrial genomes.

MOTIVATION: Data on the entire structures of organelle and bacterial genomes, most of which are known to be circular, have accumulated at a rapid pace. This information enables us to utilize the locations of homologous gene pairs for measuring the dissimilarity between complete genomic structures. RESULTS: A macroscopic distance is presented for comparing circular genomes from their overall structures, on the basis of the locations of two pairs of homologous genes on the compared genomes. The novel aspect of our method is that the comparison between the genomes automatically reveals a relationship based on the information on all gene locations, by incorporating the mobility of each gene, which includes not only the gene order, but also the relative location between gene pairs. The plausibility of the newly defined distances is evaluated by means of 44 mitochondrial genomes. The genome distance shows high performance for quantitatively describing the differences between the gene organizations of the genomes. AVAILABILITY: Since the programs implementing these calculations require well-arranged gene organization data, they have not been released yet. However, one of the authors will analyze circular genomes upon request. Data on the gene organizations may be submitted electronically to the address below.

Animals↗
PubMed1998

Imaging of small pancreatic ductal adenocarcinoma.

Symptoms and laboratory studies provide only limited assistance in the diagnosis of small pancreatic carcinomas. Ultrasound and computed tomography are best suited for screening small pancreatic carcinomas because of their ease and accuracy. When findings of ultrasound and computed tomography suggest small pancreatic carcinomas, MR cholangiopancreatography and endoscopic ultrasound should be indicated. Both techniques can show very small tumors. Follow-up of 77 patients with pancreatic carcinoma in whom the tumor was resected showed a 100% 5-year survival rate of patients with tumor limited to the duct epithelium. The majority of these tumors were <1 cm. These tumors are considered early pancreatic carcinoma.

Adenocarcinoma↗