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M Nakao

Publications and source records attributed to M Nakao.

At least 91 records · Page 5Linked to original sources
PubMed2001

Epigenetics: interaction of DNA methylation and chromatin.

Epigenetic regulation is the mechanism by which gene function is selectively activated or inactivated in the cells. It provides higher-ordered and more specified genetic information, compared with the whole genome itself. Recently, a variety of regulatory proteins including DNA methyltransferases, methyl-CpG binding proteins, histone-modifying enzymes, chromatin remodeling factors, and their multimolecular complexes have been identified. These facilitate our understanding of the molecular basis for transcription, DNA replication, mutation and repair, DNA recombination, and chromosome dynamics, which are crucial for normal cell regulation. Abnormalities in the epigenetic states represent human disease phenotypes, especially developmental defects and tumorigenesis. Therefore, epigenetics will become the focus and a major target for emerging biological and medical discoveries.

Acetylation↗
PubMed2001

PU.1 is involved in the regulation of B lineage-associated and developmental stage-dependent expression of the germinal center-associated DNA primase GANP.

Germinal center-associated DNA primase (GANP) associated with MCM3 of the DNA replication complex is up-regulated selectively in germinal center B cells. We studied promoter activity of the 5' region involved in the developmental stage-dependent expression in B lineage cells by luciferase reporter assay. Selective regulation of ganp expression was observed in the -737-bp promoter region in B and plasma cell lines but was significantly low in pre-B and T cell lines. The deletion constructs displayed a gap decrease after shortening the region from -134 to -108 bp. Further narrowing suggested the involvement of the PU.1 consensus sequence at -126 bp by electrophoretic mobility shift assay. The protein component PU.1 complex is not inhibited with mutated probes at the consensus site but is inhibited with the known PU.1 probe of CD72 and with anti-PU.1 antibody. Moreover, introduction of PU.1 cDNA enhanced the reporter gene activity in a dose-dependent manner in B cells, whereas the reporter construct with the mutated PU.1 site did not respond. Anti-CD40 stimulation induced the reporter activity with a 100% increase, which is not observed with the PU.1-mutated reporter construct. These results demonstrate that the germinal center-associated DNA primase expression is partly regulated by the transcription factor PU.1 expressed in B lineage cells.

Acetyltransferases↗
PubMed2001

Malonyl-CoA:anthocyanin 5-O-glucoside-6"'-O-malonyltransferase from scarlet sage (Salvia splendens) flowers. Enzyme purification, gene cloning, expression, and characterization.

The orange to blue coloration of flowers in nature is, in most cases, provided by anthocyanins, a class of plant flavonoids, many of which are modified by malonyl group(s). However, the identity of the enzyme catalyzing the malonylation reaction remains to be established. Here, we describe for the first time the purification, characterization, and cDNA cloning of an anthocyanin malonyltransferase from scarlet sage (Salvia splendens) flowers. The purified enzyme (termed Ss5MaT1) was a monomeric 50-kDa protein catalyzing the regiospecific transfer of the malonyl group from malonyl-CoA to the 6"'-hydroxyl group of the 5-glucosyl moiety of anthocyanins. Ss5MaT1 showed a k(cat) value of 7.8 s(-1) at 30 degrees C and pH 7.0 for the malonylation of bisdemalonylsalvianin (pelargonidin 3-(6"-O-caffeyl-beta-glucopyranoside)-5-beta-glucopyranoside) and K(m) values of 101 microm and 57 microm for bisdemalonylsalvianin and malonyl-CoA, respectively. p-Coumaric acid, which mimics an aromatic acyl group linked to the 3-glucosidic moiety of an anthocyanin substrate, was a competitive inhibitor with respect to the substrate. This strongly suggests that the presence of an aromatic acyl group at the 3-glucosidic moiety of anthocyanin is important for substrate recognition by the enzyme. On the basis of the partial amino acid sequences of the purified enzyme, we isolated a cDNA encoding Ss5MaT1. Ss5MaT1 consisted of 462 amino acids and shared motifs that are commonly found among members of a versatile plant acyltransferase family, which was recently shown to include numerous homologs of unknown biochemical functions. Northern blot analysis revealed that the transcripts of Ss5MaT1 were detected in petals, sepals, bracts, and red stems, in accordance with the pigment accumulation patterns. Phylogenetic analysis suggests that the aliphatic and aromatic acylations of anthocyanins are generally catalyzed by subfamily members of the plant acyltransferase family.

Acyltransferases↗
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

Large-scale evaluation of imprinting status in the Prader-Willi syndrome region: an imprinted direct repeat cluster resembling small nucleolar RNA genes.

Loss of paternal gene expression at the imprinted domain on proximal human chromosome 15 causes Prader-Willi syndrome (PWS), a complex multiple-anomaly disorder involving variable mental retardation, hyperphasia leading to obesity and infantile hypotonia with failure to thrive. Although numerous paternally expressed transcripts have been identified that reside in the candidate region, the individual contributions to the development of PWS have not been firmly established. Recent studies of mouse models carrying a cytogenetic deletion suggest that paternal deficiency of the SNRPN-IPW interval is critical for perinatal lethality of potential relevance to PWS. Here we determined the allelic expression profiles of a total of 118 cDNA clones using monochromosomal hybrids retaining either a paternal or maternal human chromosome 15. Our results demonstrated a preponderance of unusual transcripts lacking protein-coding potential that were expressed exclusively from the paternal copy of the critical interval. This interval was also found to encompass a large direct repeat (DR) cluster displaying a potentially active chromatin conformation of paternal origin, as suggested by enhanced sensitivity to nuclease digestion. Database searches revealed an unexpected organization of tandemly repeated consensus elements, all of which possessed well-defined box C and D sequences characteristic of small nucleolar RNAs (snoRNAs). Southern blot analysis further demonstrated a considerable degree of phylogenetic conservation of the DR locus in the genomes of all mammalian species tested, but not in chicken, Xenopus and Drosophila. These findings imply a potential direct contribution of the DR locus, representing a cluster of multiple snoRNA genes, to certain phenotypic features of PWS.

Base Sequence↗
PubMed2001

RUNX1/AML1: a central player in hematopoiesis.

It has been well established that a number of transcription factors play critical roles in regulating the fate of hematopoietic stem cell populations. One of them is the leukemia-associated transcription factor acute myeloid leukemia 1 (AML1; also known as runt-related transcription factor 1, or RUNX1). This gene was originally cloned from the breakpoint of the t(8;21) reciprocal chromosome translocation and was later recognized as one of the most frequent targets of leukemia-associated gene aberrations. Gene-targeting experiments revealed that transcriptionally active AML1 is essential for the establishment of definitive hematopoiesis. More specifically, this gene functions in the emergence of the hematopoietic progenitor cells from the hemogenic endothelium by budding in the aorta-gonad-mesonephros region, and its expression points to the sites with strong potential for the emergence of hematopoietic stem cells. This review discusses aspects of the biologic properties of AML1 in early hematopoietic development.

Animals↗
PubMed2001

Molecular cloning of the complement (C1r/C1s/MASP2-like serine proteases from the common carp (Cyprinus carpio).

The classical pathway of complement composed of C1, C4, and C2 is an antibody-dependent activation cascade that is present in jawed vertebrates. C1 is a Ca2+-dependent complex of C1q, C1r, and C1s, and analogous to an initiation complex of the lectin pathway of complement, which consists of the mannose-binding lectin (MBL) homologous to C1q and the MBL-associated serine proteases (MASPs) homologous to C1r and C1s. Thus divergence of Clq and MBL and that of C1r, C1s and the MASPs are considered to be crucial events in the establishment and evolution of the classical complement pathway. However, molecular information on the C1 subcomponents is very limited in lower vertebrates. Here we describe two distinct C1r/C1s/MASP2-like cDNA clones (C1r/s-A, C1r/s-B) isolated from the common carp (Cyprinus carpio). They share 83% identity at the amino acid level and have a domain structure similar to that of C1r/C1s/MASPs from other species. The serine protease domain of the carp homologues lacks the histidine loop and is encoded by a single exon containing an AGY codon for the active serine residue, as in mammalian C1r, C1s, and MASP2. Southern blot and PCR analyses indicated that the carp has at least three copies of the C1r/s-A gene and a single C1r/s-B gene. Although phylogenetic tree analysis does not definitively assign carp C1r/s-A and C1r/s-B, they might represent ancestral molecules which later diverged into C1r, C1s, and MASP2 of higher vertebrates.

Alleles↗
PubMed2001

Design of PCR primers and a gene probe for extensive detection of poly(3-hydroxybutyrate) (PHB)-degrading bacteria possessing fibronectin type III linker type-PHB depolymerases.

For rapid and sensitive detection of poly(3-hydroxybutyrate) (PHB)-degrading bacteria, a PCR primer set (PHB primers) and a gene probe (PHB probe) were designed, based on the homologous regions of six fibronectin type III linker domain-encoding sequences laid on a variety of PHB depolymerase genes listed in the GenBank. PCR using PHB primers amplified DNA fragments with the expected sizes from all the tested bacterial strains used for primer design; and all of the amplified fragments gave positive signals by Southern hybridization with the PHB probe. No amplified fragments were observed from negative controls. To evaluate the availability of the PHB primers and PHB probe, they were applied to 57 wild-type, PHB-degrading bacteria newly isolated from a variety of environments. The PHB primers amplified DNA fragments with expected sizes from 50 of the 57 wild-type strains, while the PHB probe showed positive signals against the amplified fragments from 47 strains. These results suggest that the primer and probe system established in this study can detect a considerable proportion of the potential PHB-degrading bacteria and can be applied to evaluate PHB-degradation potential in a natural environment, in combination with direct DNA extraction methods.

Bacteria↗
PubMed2001

Immune-relevant (including acute phase) genes identified in the livers of rainbow trout, Oncorhynchus mykiss, by means of suppression subtractive hybridization.

To develop tools for analysis of the acute phase response, we used suppression subtractive hybridization of cDNAs from the livers of trout in an unchallenged state and in the course of a response to injection with a Vibrio bacterin emulsified in Freund's Incomplete Adjuvant. The resulting cDNA library contains 300-600bp long fragments of 25 or more immune-relevant genes. Fifteen were previously unreported for salmonids, and 12 were not known from any fish species. Known acute phase proteins include serum amyloid A, transferrin and precerebellin-like protein; trout C-polysaccharide-binding protein 1 is probably also an acute phase protein. Components of both the complement system (n=5) and the clotting system (n=3), as well as lectins, various binding proteins, a putative antibacterial peptide, a chemotaxin, an anti-oxidant enzyme, as well as some likely cell-surface receptors and metabolic and lysosomal enzymes are represented in the library. One clone closely resembles a group of Toll-like receptors, including the human IL-1 receptor. Three cDNAs appear to represent complete open reading frames.

Acute-Phase Proteins↗
PubMed2001

Organization of the NKEF gene and its expression in the common carp (Cyprinus carpio).

Natural killer enhancing factor (NKEF) is a member of the newly defined peroxiredoxin (Prx) family. Its functions are to enhance the cytotoxic capacity of natural killer cells and to prevent DNA and protein from being damaged by oxidative stress in the presence of thiol compounds. However, little is known about the structure and function of NKEF in lower vertebrates. We have recently cloned a cDNA encoding NKEF from the common carp (Cyprinus carpio) by use of suppression subtractive hybridization (SSH). In the present study, we used PCR to obtain a genomic DNA which covers the entire coding region of carp NKEF. In the 3363bp-long genomic sequence, six exons and five introns were identified. The carp NKEF gene has splice donor/acceptor site sequences at the boundaries of exons and introns, and contains two Val-Cys-Pro (VCP) motifs. The exon/intron organization of the carp NKEF gene shows complete conservation with other members of the Prx family. Genomic Southern blotting analyses suggest that carp has multiple copies of the NKEF gene. RT-PCR analyses reveal that carp NKEF has very different expression levels not only in tissues but also from individuals.

Amino Acid Sequence↗
PubMed2001

Functional analyses of MeCP2 mutations associated with Rett syndrome using transient expression systems.

Rett syndrome, an X-linked neurodevelopmental disorder, is a major cause of mental retardation in females. Recent genetic analyses have revealed that mutations in the methyl-CpG-binding protein gene encoding MeCP2 are associated with Rett syndrome. In this study, we used transient expression systems to investigate the functional significance of mutations seen in patients with Rett syndrome. Missense mutations in the methyl-CpG-binding domain were analyzed by the transfection in mouse L929 cells and Drosophila SL2 cells. The L929 cells were utilized to investigate the effects of mutations on the affinity for heterochromatin, where methylated CpG dinucleotides are extremely enriched. The SL2 cells were utilized to analyze their effects on transcriptional repression activities. R106W and F155S mutations led to the substantial impairment of MeCP2 functions, showing the loss of accumulation of the mutated protein to mouse heterochromatin and the reduction of the transcriptional repressive activity in Drosophila SL2 cells. Intriguingly, the R133C mutant retained the functionality equivalent to MeCP2 in these analyses. On the other hand, the T158M mutation exhibited the intermediate level of the impairment of functions in both analyses. Thus, these functional assays are useful to evaluate the consequences of mutation in the methyl-CpG-binding domain of MeCP2 and provide an insight into the relationship between the genotype and the severity of Rett syndrome.

Animals↗
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

DNA methylation is a critical cell-intrinsic determinant of astrocyte differentiation in the fetal brain.

Astrocyte differentiation, which occurs late in brain development, is largely dependent on the activation of a transcription factor, STAT3. We show that astrocytes, as judged by glial fibrillary acidic protein (GFAP) expression, never emerge from neuroepithelial cells on embryonic day (E) 11.5 even when STAT3 is activated, in contrast to E14.5 neuroepithelial cells. A CpG dinucleotide within a STAT3 binding element in the GFAP promoter is highly methylated in E11.5 neuroepithelial cells, but is demethylated in cells responsive to the STAT3 activation signal to express GFAP. This CpG methylation leads to inaccessibility of STAT3 to the binding element. We suggest that methylation of a cell type-specific gene promoter is a pivotal event in regulating lineage specification in the developing brain.

Animals↗
PubMed2001

Comparative analysis of colonization of Helicobacter pylori and glycolipids receptor density in Mongolian gerbils and mice.

The Mongolian gerbil has been used as an excellent experimental animal model for studying Helicobacter pylori infection because it can stably colonize and induce severe chronic gastritis, ulceration, and cancer-simulating human diseases in this animal. In contrast, H. pylori can only induce mild inflammation in many mouse models. The aim in this study is to clarify the difference of induction of pathological lesions in the two animal models. SPF ICR mice and Mongolian gerbils were inoculated with a clinically isolated strain of H. pylori. Six weeks after inoculation, bacteria colonizing the stomach were counted. Immunohistochemical staining and biochemical analyses of three putative receptor glycolipids were performed with monoclonal antibodies to the respective glycolipids. Significantly higher numbers of H. pylori were recovered from the stomachs of Mongolian gerbils than mice (5.77 +/- 0.46 log CFU vs 4.17 +/- 0.55 log CFU, P < 0.01). Immunohistochemical studies showed that sulfatide expression in the gastric mucosa of Mongolian gerbils was much stronger than that in mice, whereas the expression of Lewis(b) glycolipid and GM3 were almost equal. Quantitative analysis of each glycolipid by thin-layer chromatography confirmed the results of immunohistochemical study, showing 4.1 times higher sulfatide content in the Mongolian gerbil stomach. The content of both Lewis(b) and GM3 was almost equivalent in these two animals. In conclusions, higher levels of sulfatide expression, a putative adhesion receptor, in the gastric mucosa of Mongolian gerbils may allow abundant colonization by H. pylori, resulting in the development of gastric lesions in this animal model.

Animals↗
PubMed2001

Congestion of superior mesenteric veins and small bowel mucosal injury after endoscopic treatment of esophageal varices in patients with portal hypertension.

We investigated treatment-induced changes in venous return from the small bowel and small bowel intestinal mucosal injury induced by the treatment of esophageal varices in patients with portal hypertension. A total of 14 patients (age 59.8+/-9.5 years, five women and 9 men) who received prophylactic treatment of esophageal varices between December 1998 and March 1999 were investigated. Diamine oxidase (DAO) activity was measured before and after treatment. Changes in blood flow of the portal and superior mesenteric veins were investigated by Doppler ultrasonography in six patients. A significant decrease in DAO activity was observed three days after treatment (11.5+/-1.6 units/liter prior to treatment versus 8.6+/-1.6 units/liter three days after treatment; P < 0.001). Decreases in superior mesenteric and portal venous flow velocity were observed in four and three patients, respectively. In two patients with an increase in the cross-sectional area of the superior mesenteric vein with delayed venous return, a marked decrease in DAO activity was observed three days after treatment. In patients with portal hypertension, rapid reduction of pooling of portal flow caused by the treatment of esophageal varices can induce transient congestion of the mesenteric venous system which can produce some small bowel mucosal injury.

Amine Oxidase (Copper-Containing)↗
PubMed2001

A novel maternally expressed gene, ATP10C, encodes a putative aminophospholipid translocase associated with Angelman syndrome.

Lack of a maternal contribution to the genome at the imprinted domain on proximal chromosome 15 causes Angelman syndrome (AS) associated with neurobehavioral anomalies that include severe mental retardation, ataxia and epilepsy. Although AS patients have infrequent mutations in the gene encoding an E6-AP ubiquitin ligase required for long-term synaptic potentiation (LTP), most cases are attributed to de novo maternal deletions of 15q11-q13. We report here that a novel maternally expressed gene, ATP10C, maps within the most common interval of deletion and that ATP10C expression is virtually absent from AS patients with imprinting mutations, as well as from patients with maternal deletions of 15q11-q13.

Adenosine Triphosphatases↗
PubMed2001

International prognostic scoring system and TP53 mutations are independent prognostic indicators for patients with myelodysplastic syndrome.

We applied the International Prognostic Scoring System (IPSS) to our series of 118 patients with myelodysplastic syndrome (MDS) to determine its validity, and also used univariate and multivariate analyses to evaluate the prognostic significance of TP53 configurations. Sixteen patients with the mutation had a strikingly worse prognosis and the multivariate analysis demonstrated that this alteration was the most significant factor. The prognostic comparison between patients with and without the mutation within each IPSS subgroup showed a significant difference in the intermediate subgroups. A combination of clinical manifestations and genetic configurations provided us with more accurate prognostic information in MDS patients.

Adolescent↗