Search PubMed⌕ Search

Biomedical subjects

O Gotoh

Publications and source records attributed to O Gotoh.

At least 37 records · Page 2Linked to original sources
PubMed1997

Structural characterization of the gene and corresponding cDNA for the cytochrome P450rm from Rhodotorula minuta which catalyzes formation of isobutene and 4-hydroxylation of benzoate.

Cytochrome P450rm was previously isolated from the basidiomycete yeast Rhodotorula minuta as a bifunctional enzyme with isobutene-forming and benzoate 4-hydroxylase activities. We cloned the gene and corresponding cDNA for P450rm in order to characterize the enzyme in the context of fungal phylogeny and physiology. From the cDNA sequence, P450rm was deduced to have 527 amino acids with a calculated molecular weight of 59136. P450rm shared 48% amino acid sequence identity with CYP53A1 from Aspergillus niger, indicating that the gene belongs to a novel subfamily of CYP53, CYP53B. However, the organization of the P450rm gene, which has eight exons and seven introns, differed completely to that of CYP53A1. Northern analysis demonstrated that the level of P450rm mRNA expression increased when L-phenylalanine was used as sole carbon source. These results suggest that P450rm has been well conserved during the evolution of fungi as a benzoate 4-hydroxylase in the dissimilation pathway starting from L-phenylalanine

Amino Acid Sequence↗
PubMed1997

Transcriptional activation domain of human BTEB2, a GC box-binding factor.

BTEB2 is a GC box-binding transcription factor containing proline-rich and zinc finger domains as transactivation and DNA binding domains, respectively. We have identified a small region in the proline-rich domain indispensable for its transcription-enhancing activity by transfection experiments using various expression plasmids with point mutations or small deletions in the domain. This region comprising about 10 amino acids was relatively hydrophobic and rich in proline and alanine residues. BTEB2 purified from a baculovirus expression system could enhance transcription, depending on the presence of GC boxes in the promoter region of templates in an in vitro transcription assay. BTEB2 deleted of the hydrophobic region, however, lost the transcription-enhancing activity, in confirmation of the above results. Basic transcription factors which possibly interact with BTEB2 were examined. Initiation factors, TFIIB, TFIIE beta, and TFIIF beta as well as the TATA box-binding protein (TBP) were found to interact with BTEB2 when analyzed by in vitro binding experiments, although these interactions could not be attributed to the proline-rich domain. We discussed factors which interact with and transmit the transcriptional activity of the BTEB2 activation domain to basic transcriptional machinery.

Amino Acid Sequence↗
PubMed1997

Structural and evolutionary studies on sterol 14-demethylase P450 (CYP51), the most conserved P450 monooxygenase: I. Structural analyses of the gene and multiple sizes of mRNA.

The structure of rat CYP51 gene encoding sterol 14-demethylase was examined. The CYP51 gene spanned about 18 kb and contained 10 exons. The copy number of CYP51 in the rat genome was determined to be one. In addition, one CYP51 processed (intron-less) pseudogene covering the coding and ca. 600-bp 3'-noncoding sequences of CYP51 cDNA was found in the rat genome. Multiple transcription initiation sites were predicted by primer extension and 5'-RACE methods using poly(A)+ RNA from liver, ovary, and testis, and the major ones were located at 126 and 123 nucleotides upstream from the initiation ATG codon. The primer extension also showed several minor sites around the major ones. In addition to these sites, other minor initiation sites were also predicted at around 330 and 460 nucleotides upstream from the initiation ATG codon. No TATA box was found in the putative promoter region, but multiple GC boxes were found around the cap sites, supporting the previously inferred housekeeping nature of CYP51 gene and the existence of the multiple transcription initiation sites. A few consensus transcription regulatory elements such as CRE were found in the 5'-flanking region. Four polyadenylation signals were found in the 3'-noncoding region by the 3'-RACE method. Three of them were used to generate 3.1-, 2.7-, and 2.3-kb mRNAs in liver and ovary. The remaining one was used only in testis to generate 1.9-kb mRNA having an unusually short trailer sequence, suggesting a specific regulatory mechanism for generating CYP51 mRNA in testis different from that in liver and ovary.

Amino Acid Sequence↗
PubMed1997

Structural and evolutionary studies on sterol 14-demethylase P450 (CYP51), the most conserved P450 monooxygenase: II. Evolutionary analysis of protein and gene structures.

Phylogenetic analyses based on protein sequence data indicated that sterol 14-demethylase P450 (CYP51) and bacterial CYP51-like protein were joined into a distinctive evolutionary cluster, CYP51 cluster, within the CYP protein superfamily. The most probable branch topology of the CYP51 phylogenetic tree was (bacteria, (plants, (fungi, mammals))), which is comparable to the phylogeny of major kingdoms of living matter, suggesting that CYP51 has been conserved from the era of prokaryotic evolution. This may be strong evidence supporting the prokaryotic origin of P450. Structure of flanking regions and the number and insertion sites of introns are quite different between mammalian and fungal CYP51s. This fact indicates that different mechanisms are operative in evolution of protein sequences and gene structures. CYP51 is the first example violating the well-documented rule that the basic structure of a gene, including intron insertion sites, is well conserved in each P450 family. One CYP51 processed a pseudogene was found in rat genome. Nonsynonymous nucleotide divergence observed between the pseudogene and CYP51 cDNA was less than one-fifth of the synonymous divergence. This unusually low rate of nonsynonymous nucleotide changes in the pseudogene suggests that it may be derived from another CYP51, which might have been active for a significant duration in the past.

Amino Acid Sequence↗
PubMed1996

Significant improvement in accuracy of multiple protein sequence alignments by iterative refinement as assessed by reference to structural alignments.

The relative performances of four strategies for aligning a large number of protein sequences were assessed by referring to corresponding structural alignments of 54 independent families. Multiple sequence alignment of a family was constructed by a given method from the sequences of known structures and their homologues, and the subset consisting of the sequences of known structures was extracted from the whole alignment and compared with the structural counterpart in a residue-to-residue fashion. Gap-opening and -extension penalties were optimized for each family and method. Each of the four multiple alignment methods gave significantly more accurate alignments than the conventional pairwise method. In addition, a clear difference in performance was detected among three of the four multiple alignment methods examined. The currently most popular progressive method ranked worst among the four, and the randomized iterative strategy that optimizes the sum-of-pairs score ranked next worst. The two best-performing strategies, one of which was newly developed, both pursue an optimal weighted sum-of-pairs score, where the pair weights were introduced to correct for uneven representations of subgroups in a family. The new method uses doubly nested iterations to make alignment, phylogenetic tree and pair weights mutually consistent. Most importantly, the improvement in accuracy of alignments obtained by these iterative methods over pairwise or progressive method tends to increase with decreasing average sequence identity, implying that iterative refinement is more effective for the generally difficult alignment of remotely related sequences. Four well-known amino acid substitution matrices were also tested in combination with the various methods. However, the effects of substitution matrices were found to be minor in the framework of multiple alignment, and the same order of relative performance of the alignment methods was observed with any of the matrices.

Algorithms↗
PubMed1996

cDNA cloning of a murine homologue of Drosophila single-minded, its mRNA expression in mouse development, and chromosome localization.

A combination of the RT-PCR method and subsequent screening of the cDNA library of mouse skeletal muscle with the cDNA isolated by RT-PCR used as a probe led to isolation of cDNAs encoding a polypeptide (mSim) with bHLH and PAS domains which show high similarity to the corresponding regions of Drosophila Sim, a master regulator in neurogenesis. Experiments using a GST-fusion protein demonstrated that mSim heterodimerizes with Arnt (Ah receptor nuclear translocator), even more efficiently than AhR (Ah receptor) does with Arnt. RNA blot analysis using RNAs from various tissues of mice indicated that mSim transcript is expressed in several limited tissues such as muscle, kidney and lung of adult animals. Distribution of mSim mRNA was always accompanied with that of Arnt. All the results suggest a regulatory role of mSim in partnership with Arnt. Chromosomal location of the mSim gene was determined by fluorescent in situ hybridization to be localized on the C3.3-C4 band of mouse chromosome 16 which is syntenic with the human chromosome 21q22 carrying the Down syndrome critical region, where a gene highly homologous to the Drosophila sim was localized. Whole mount in situ hybridization using a unique part of mSim cDNA sequence showed that mSim mRNA was expressed in the ventral diencephalon, branchial arches and limbs. These findings will provide an approach to the cause of the Down syndrome as well as the elucidation of the functional roles of mSim in animal development.

Age Factors↗
PubMed1996

Sterol 14-demethylase P450 (P45014DM*) is one of the most ancient and conserved P450 species.

To determine the orthology of sterol 14-demethylase (P45014DM), the only known P450 enzyme distributed widely in eukaryotes with a conserved metabolic role, the full-length amino acid sequences of rat and human P45014DMs were determined from the cloned cDNA sequences, and compared with those of the corresponding fungal proteins (CYP51). The amino acid identity value between given pairs of P45014DMs ranged from 93% (human/rat) to 39% (human or rat/Saccharomyces cerevisiae). All the P45014DMs formed a single cluster in a phylogenetic tree constructed from representative P450 protein sequences currently available. The nearest neighbors to the P45014DM cluster in the phylogenetic tree were CYP7 (cholesterol 7 alpha-hydroxylase) and CYP8 (prostacyclin synthase), and the divergence point of fungal and mammalian P45014DMs was clearly more recent than that of P45014DM and CYP7/CYP8. These lines of evidence show that fungal and mammalian P45014DMs are really orthologous. This is the first example of orthologous P450s occurring in distinct kingdoms. P45014DM may be an ancient P450 which arose before the divergence of major eukaryotic branches and has been conserved throughout evolution. The amino acid identity value (93%) between human and rat P45014DMs was comparable to those observed for some housekeeping enzymes. In addition, a processed pseudogene of P45014DM was found in a rat genomic DNA library, suggesting the expression of P45014DM in germ line cells. These facts suggest that P45014DM may be a housekeeping enzyme essential for the viability of mammals.

Amino Acid Sequence↗
PubMed1996

Structure of the gene encoding the human differentiation-stimulating factor/leukemia inhibitory factor receptor.

The human gene encoding the differentiation-stimulating factor (D-factor)/leukemia inhibitory factor (LIF) receptor was cloned and its structure was analyzed. The gene spans more than 70 kilobases and contains 20 exons. The D-factor/LIF receptor can be subdivided into several regions; cytokine receptor homologous domain 1, an Ig-like domain, cytokine receptor homologous domain 2, three fibronectin type III domains, a transmembrane domain and a cytoplasmic region. Each domain of the receptor is encoded by a set of exons. There is a TATA sequence upstream of the transcription initiation site. One unit of the Alu sequence is present in the 5' flanking region. An NF-IL6 site is located 31 bases downstream of the transcription initiation site.

Base Sequence↗
PubMed1996

Glasgow Coma Scale in the prediction of outcome after early aneurysm surgery.

OBJECTIVE: A prospective study was performed to analyze whether the Glasgow Coma Scale (GCS) was useful in predicting the outcome after early surgical intervention for aneurysmal subarachnoid hemorrhage (SAH). METHODS: In a consecutive series of 765 patients who underwent surgery for aneurysms within 7 days after SAH, the level of consciousness was assessed by the GCS just before surgery and the outcome was graded by the Glasgow Outcome Scale 6 months after surgery. RESULTS: The patient distribution in accordance with the GCS sum scores in descending order from 15 to 3 was as follows: 334, 140, 58, 27, 20, 26, 27, 19, 26, 17, 20, 27, and 24 patients, respectively. In general, the higher the preoperative GCS score was, the better the surgical outcome was. The overall surgical result was significantly correlated with the preoperative GCS score (rs = 0.615, P < 0.001). With respect to the levels that distinguish the outcome along the GCS axis, a significant difference in the outcome was observed only between the GCS scores of 15 and 14 (P < 0.001, Wilcoxon test). CONCLUSION: The GCS proved useful in the preoperative evaluation of patients with SAH, in terms of outcome prediction. It is suggested that the SAH scale proposed by the World Federation of Neurosurgical Societies be reexamined, because differences in outcomes were not clear between the GCS scores of 13 and 12 or between those of 7 and 6, in which Grades III and IV and Grades IV and V are differentiated in the scale, respectively.

Adult↗
PubMed1996

P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature.

We provide here a list of 481 P450 genes and 22 pseudogenes, plus all accession numbers that have been reported as of October 18, 1995. These genes have been described in 85 eukaryote (including vertebrates, invertebrates, fungi, and plants) and 20 prokaryote species. Of 74 gene families so far described, 14 families exist in all mammals examined to date. These 14 families comprise 26 mammalian subfamilies, of which 20 and 15 have been mapped in the human genome and the mouse genome, respectively. Each subfamily usually represents a cluster of tightly linked genes widely scattered throughout the genome, but there are exceptions. Interestingly, the CYP51 family has been found in mammals, filamentous fungi and yeast, and plants-attesting to the fact that this P450 gene family is very ancient. One functional CYP51 gene and two processed pseudogenes, which are the first examples of intronless pseudogenes within the P450 superfamily, have been mapped to three different human chromosomes. This revision supersedes the four previous updates in which a nomenclature system, based on divergent evolution of the superfamily, has been described. For the gene, we recommend that the italicized root symbol "CYP' for human ("Cyp' for mouse and Drosophila), representing "cytochrome P450', be followed by an Arabic number denoting the family, a letter designating the subfamily (when two or more exist), and an Arabic numeral representing the individual gene within the subfamily. A hyphen is no longer recommended in mouse gene nomenclature. "P' ("ps' in mouse and Drosophila) after the gene number denotes a pseudogene; "X' after the gene number means its use has been discontinued. If a gene is the sole member of a family, the subfamily letter and gene number would be helpful but need not be included. The human nomenclature system should be used for all species other than mouse and Drosophila. The cDNAs, mRNAs and enzymes in all species (including mouse) should include all capital letters, and without italics or hyphens. This nomenclature system is similar to that proposed in our previous updates.

Alleles↗
PubMed1996

Two new members of the murine Sim gene family are transcriptional repressors and show different expression patterns during mouse embryogenesis.

From a cDNA library of mouse skeletal muscle, we have isolated mouse Sim1 (mSim1) cDNA encoding a polypeptide of 765 amino acids with striking amino acid identify in basic helix-loop-helix (89% identify) and PAS (89 % identify) domains to previously identified mSim2, although the carboxy-terminal third of the molecule did not show any similarity to mSim2 or Drosophila Sim (dSim). Yeast two-hybrid analysis and coimmunoprecipitation experiments demonstrated that both of the mSim gene products interacted with Arnt even more efficiently than AhR, a natural partner of Arnt, suggesting a functional cooperativity with Arnt. In sharp contrast with dSim having transcriptional-enhancing activity in the carboxy-terminal region, the two mSims possessed a repressive activity toward Arnt in the heterodimer complex. This is the first example of bHLH-PAS proteins with transrepressor activity, although some genetic data suggest that dSim plays a repressive role in gene expression (Z. Chang, D. Price, S. Bockheim, M. J. Boedigheimer, R. Smith, and A. Laughon, Dev. Biol. 160:315-322, 1993; D. M. Mellerick and M. Nirenberg, Dev. Biol. 171:306-316, 1995). Whole-mount in situ hybridization showed restricted and characteristic expression patterns of the two mSim mRNAs in various tissues and organs during embryogenesis, such as those for the somite, the nephrogenic cord, and the mesencephalon (for mSim1) and those for the diencephalon, branchial arches, and limbs (for mSim2). From sequence similarity and chromosomal localization, it is concluded that mSim2 is an ortholog of hSim2, which is proposed to be a candidate gene responsible for Down's syndrome. The sites of mSim2 expression showed an overlap with the affected regions of the syndrome, further strengthening involvement of mSim2 in Down's syndrome.

Amino Acid Sequence↗
PubMed1996

cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt).

We isolated mouse cDNA clones (Arnt2) that are highly similar to but distinct from the aryl hydrocarbon receptor (AhR) nuclear translocator (Arnt). The composite cDNA covered a 2,443-bp sequence consisting of a putative 2,136-bp open reading frame encoding a polypeptide of 712 amino acids. The predicted Arnt2 polypeptide carries a characteristic basic helix-loop-helix (bHLH)/PAS motif in its N-terminal region with close similarity (81% identity) to that of mouse Arnt and has an overall sequence identity of 57% with Arnt. Biochemical properties and interaction of Arnt2 with other bHLH/PAS proteins were investigated by coimmunoprecipitation assays, gel mobility shift assays, and the yeast two-hybrid system. Arnt2 interacted with AhR and mouse Sim as efficiently as Arnt, and the Arnt2-AhR complex recognized and bound specifically the xenobiotic responsive element (XRE) sequence. Expression of Arnt2 successfully rescued XRE-driven reporter gene activity in the Arnt-defective c4 mutant of Hepa-1 cells. RNA blot analysis revealed that expression of Arnt2 mRNA was restricted to the brains and kidneys of adult mice, while Arnt mRNA was expressed ubiquitously. In addition, whole-mount in situ hybridization of 9.5-day mouse embryos showed that Arnt2 mRNA was expressed in the dorsal neural tube and branchial arch 1, while Arnt transcripts were detected broadly in various tissues of mesodermal and endodermal origins. These results suggest that Arnt2 may play different roles from Arnt both in adult mice and in developing embryos. Finally, sequence comparison of the currently known bHLH/PAS proteins indicates a division into two phylogenetic groups: the Arnt group, containing Arnt, Arnt2, and Per, and the AhR group, consisting of AhR, Sim, and Hif-1alpha.

Amino Acid Sequence↗
PubMed1995

Cloning and gene mapping of the mouse homologue of the CBFA2T1 gene associated with human acute myeloid leukemia.

The human CBFA2T1 (also known as MTG8) gene, on chromosome 8, has been identified through its involvement in the t(8;21) chromosomal translocation, frequently found in acute myeloid leukemia. We report here the isolation and characterization of the mouse homologue of the CBFA2T1 gene, Cbfa2t1h. Nucleotide sequence analysis of Cbfa2t1h cDNA clones revealed an open reading frame encoding a protein of 577 amino acids with an extremely high degree of amino acid identity (99.3%) to the human protein. The nucleotide sequence is also highly conserved between mouse and human in the 5'- and 3'-untranslated regions (87.0, 92.0, and 93.7% identities for 5'-untranslated, coding, 3'-untranslated regions, respectively). The 3'-untranslated region of Cbfa2t1h contains a (CA)n dinucleotide repeat, and the polymerase chain reaction amplification of the (CA)n repeat region revealed fragment length polymorphism among mouse strains. Using this polymorphism, we have mapped Cbfa2t1h to mouse chromosome 4 close to the centromere using SMXA recombinant inbred strains and 106 intersubspecific backcross progenies of the (DBA/2 x Mae) x Mae cross. The chromosomal location was also confirmed by fluorescence in situ hybridization.

Acute Disease↗
PubMed1995

A weighting system and algorithm for aligning many phylogenetically related sequences.

Most multiple sequence alignment programs explicitly or implicity try to optimize some score associated with the resulting alignment. Although the sum-of-pairs score is currently most widely used, it is inappropriate when the phylogenetic relationships among the sequences to be aligned are not evenly distributed, since the contributions of densely populated groups dominate those of minor members. This paper proposes an iterative multiple sequence alignment method which optimizes a weighted sum-of-pairs score, in which the weights given to individual sequence pairs are adjusted to compensate for the biased contributions. A simple method that rapidly calculates such a set of weights for a given phylogenetic tree is presented. The multiple sequence alignment is refined through partitioning and realignment restricted to the edges of the tree. Under this restriction, profile-based fast and rigorous group-to-group alignment is achieved at each iteration, rendering the overall computational cost virtually identical to that using an unweighted score. Consistency of nearly 90% was attained between structural and sequence alignments of multiple divergent globins, confirming the effectiveness of this strategy in improving the quality of multiple sequence alignment.

Algorithms↗
PubMed1995

[Japan coma scale in the prediction of outcome after early surgery for aneurysmal subarachnoid hemorrhage].

The value of the 10-grade Japan Coma Scale (JCS) as a means of evaluating patients undergoing early aneurysm surgery was assessed in terms of its relationship to surgical outcome. The 13-grade Glasgow Coma Scale (GCS) was also assessed, and comparisons were made between the two. A series of 765 patients who underwent aneurysm surgery within 7 days after hemorrhage were evaluated both by JCS and GCS immediately prior to surgery, and outcome was assessed by the Glasgow Outcome Scale (GOS) 6 months postoperatively. Both the JCS and GCS scores were significantly correlated with surgical outcome (JCS vs GOS: rs = 0.586, GCS vs GOS: rs = 0.615, p < 0.001). There was no significant difference between the correlation coefficients for outcome of the JCS and GCS. In general, the better the JCS or GCS score was, the better the surgical outcome was, however, outcome among those with a JCS score of 3 was poorer than among those with a score of 10, 20, or 30. The results of this study indicate that both JCS and GCS are useful in predicting surgical outcome, but the GCS may be better than the JCS because of the problem with the score of 3 in the latter.

Acute Disease↗
PubMed1994

Molecular cloning of the human AH receptor gene promoter.

A lambda phage clone containing a promoter region of the human Ah receptor gene was isolated. This clone spanned 13.8 kb and contained the 1st exon, the sequence of which completely matched the reported Ah receptor cDNA. Using RNase protection assay and primer extension analysis, the transcription initiation sites were determined to be 643 and 615 bp upstream of the translational initiation codon ATG. This promoter did not contain a TATA box, while multiple GC boxes were present close to the determined transcription initiation sites. Comparison of the 5' flank sequence of the human Ah receptor with its murine equivalent showed several well conserved regions, containing binding sites for known transcription factors, such as Sp1. The promoter activity was confirmed by transient transfection of chimeric constructs of the Ah receptor gene and reporter gene luciferase into hepatoma HepG2 cells.

Animals↗
PubMed1994

Effect of YM737, a new glutathione analogue, on ischemic brain edema.

We investigated the effect of YM737, a monoester of glutathione (GSH), on brain edema and GSH content after occlusion of a middle cerebral artery (MCA) in the rat. The drug possesses stronger radical scavenging activity than GSH itself, and is more effectively transported into cells. Hemispheric water, sodium, and potassium contents were determined at 2.5 and 24 hours after MCA occlusion. The animals received either YM737 or GSH immediately after occlusion. Cerebral GSH content was measured by HPLC after 2.5 hours of ischemia. The increases in water and sodium contents at 2.5 and 24 hours after MCA occlusion were significantly suppressed by YM737. GSH content decreased by 53% in the caudate, and by 22% in the cortex after ischemia. YM737 significantly ameliorated the GSH decrease in the caudate, while administration of GSH showed little effects on the ischemia-induced changes in water, sodium, and GSH contents. The result suggests the role of free radicals in the pathogenesis of ischemic brain edema.

Animals↗