Search PubMed⌕ Search

Biomedical subjects

Kenji Yamamoto

Publications and source records attributed to Kenji Yamamoto.

At least 37 records · Page 2Linked to original sources
PubMed2006

Syndromic craniosynostosis with elbow joint contracture.

This paper reports a new type of syndromic craniosynostosis that was diagnosed by DNA analysis of the patient's fibroblast growth factor receptor (FGFR) genes. At birth, a male infant had ocular proptosis, a pseudotail, and obstructed respiration. He developed craniosynostosis, craniofacial dysmorphism, hydrocephalus, and bilateral contracture of his elbow joints. His treatment included fronto-orbital advancements and a ventriculoperitoneal shunt. Genetic analysis revealed that he was heterozygous for a missense mutation in exon 9 of the FGFR2 gene that resulted in an amino acid substitution of cysteine for serine at residue 351 (Ser351Cys). Seven cases with this mutation had previously been reported. All had severe craniosynostosis with midface hypoplasia, elbow joint contracture, developmental retardation, and early death.

Amino Acid Substitution↗
PubMed2006

In vitro and in vivo immunomodulating activities of Shoyu polysaccharides from soy sauce.

We investigated the effects of Shoyu polysaccharides (SPS) prepared from soy sauce on immune functions in mice. SPS enhanced the consumption of glucose by peritoneal macrophages in vitro. In addition, oral administration of SPS to 6-week-old male BALB/c mice for 2 weeks at a dose of 3.0 mg/day increased the capacity of peritoneal macrophages to consume glucose as compared to control mice. We observed the regulatory effect of SPS on the balance of Th1/Th2 cell responses in mice. In splenic lymphocytes stimulated with Con A in vitro, SPS significantly suppressed the production of IL-4 and enhanced that of IFN-gamma. Furthermore, in mice treated with SPS at 3.0 mg per day for 2 weeks, the balance of Th1/Th2 cell responses was shifted to predominantly Th1 cell responses. These findings suggest that SPS effectively enhanced both macrophage and lymphocyte function in vitro and in vivo, and soy sauce would be a potentially promising food for enhancing host defenses.

Animals↗
PubMed2005

Chemo-enzymatic synthesis of eel calcitonin glycosylated at two sites with the same and different carbohydrate structures.

Naturally occurring glycopeptides and glycoproteins usually contain more than one glycosylation site, and the structure of the carbohydrate attached is often different from site to site. Therefore, synthetic methods for preparing peptides and proteins that are glycosylated at multiple sites, possibly with different carbohydrate structures, are needed. Here, we report a chemo-enzymatic approach for accomplishing this. Complex-type oligosaccharides were introduced to the calcitonin derivatives that contained two N-acetyl-D-glucosamine (GlcNAc) residues at different sites by treatment with Mucor hiemalis endo-beta-N-acetylglucosaminidase. Using this enzymatic transglycosylation reaction, three glycopeptides were produced, a calcitonin derivative with the same complex-type carbohydrate at two sites, and two calcitonin derivatives each with one complex-type carbohydrate and one GlcNAc. Starting from the derivatives with one complex-type carbohydrate and one GlcNAc, a high-mannose-type oligosaccharide was successfully transferred to the remaining GlcNAc using another endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae. Thus, we were able to obtain glycopeptides containing not only two complex-type carbohydrates, but also both complex and high-mannose-type oligosaccharides in a single molecule. Using the resultant glycosylated calcitonin derivatives, the effects of di-N-glycosylation on the structure and the activity of calcitonin were studied. The effect appeared to be predictable from the results of mono-N-glycosylated calcitonin derivatives.

Amino Acid Sequence↗
PubMed2005

Polyhistidine tract expansions in HOXA1 result in intranuclear aggregation and increased cell death.

HOXA1 gene is part of a cluster of homeotic selector genes that regulates the anteroposterior patterning of mammals during embryonic development. HOXA1 encodes two alternatively spliced mRNAs with two isoforms, A and B, the former contains the homeodomain and expressed in early embryonic development. HOXA1 contains a string of 10 histidine repeats. However, individuals heterozygous for 7, 9, 11, and 12 histidine repeat variants were present among the Japanese population, notably in some autism cases. To determine the biological implications of the different polyhistidine repeat lengths, we expressed these variants in COS-7 and a human neuroblastoma cell line (SK-N-SH). Expression of expanded variants of HOXA1 isoform A, containing 11 and 12 polyhistidine, resulted in early and great degree of protein aggregation in the nucleus. This aggregation resulted in accelerated cell death in cells expressing 11 and 12 expanded variants compared to those transfected with 7 and 10 polyhistidine variants. Furthermore, we showed that these aggregates were ubiquitinated and were inhibited by a histidine-modifying compound, DEPC. These data suggest that HOXA1 protein with polyhistidine tract expansions misfold, aggregate, and have a toxic effect on cell.

Animals↗
PubMed2005

Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo-alpha-N-acetylgalactosaminidase from Bifidobacterium longum.

We found endo-alpha-N-acetylgalactosaminidase in most bifidobacterial strains, which are predominant bacteria in the human colon. This enzyme catalyzes the liberation of galactosyl beta1,3-N-acetyl-D-galactosamine (Galbeta1,3GalNAc) alpha-linked to serine or threonine residues from mucin-type glycoproteins. The gene (engBF) encoding the enzyme has been cloned from Bifidobacterium longum JCM 1217. The protein consisted of 1,966 amino acid residues, and the central domain (590-1381 amino acid residues) exhibited 31-53% identity to hypothetical proteins of several bacteria including Clostridium perfringens and Streptococcus pneumoniae. The recombinant protein expressed in Escherichia coli liberated Galbeta1,3GalNAc disaccharide from Galbeta1,3GalNAcalpha1pNP and asialofetuin, but did not release GalNAc, Galbeta1,3(GlcNAcbeta1,6)GalNAc, GlcNAcbeta1,3GalNAc, and Galbeta1,3GlcNAc from each p-nitrophenyl (pNP) substrate, and also did not release sialo-oligosaccharides from fetuin, indicating its strict substrate specificity for the Core 1-type structure. The stereochemical course of hydrolysis was determined by (1)H NMR and was found to be retention. Site-directed mutagenesis of a total of 22 conserved Asp and Glu residues suggested that Asp-682 and Asp-789 are critical residues for the catalytic activity of the enzyme. The enzyme also exhibited transglycosylation activity toward various mono- and disaccharides and 1-alkanols, demonstrating its potential to synthesize neoglycoconjugates. This is the first report for the isolation of a gene encoding endo-alpha-N-acetylgalactosaminidase from any organisms and for the establishment of a new glycoside hydrolase family (GH family 101).

Amino Acid Sequence↗
PubMed2005

Photodegradative surfactants: photolyses of p-alkylbenzyltrimethylammonium and alkylbenzyldimethylammonium halides in aqueous solution.

A series of benzyl-containing ammonium salts, p-alkylbenzyltrimethylammonium halides (C(n)BA: 1a-c) and alkylbenzyldimethylammonium halides (C(n)AB: 2a-d), have been prepared and their photodegradabilities in aqueous solutions have been compared. The photolytic decomposition proceeded by heterolytic and homolytic cleavages of the benzyl-nitrogen bond. The conversion yields were almost the same for all surfactants, whereas the product yields were slightly dependent on the alkyl-chain length. After irradiation, C(8)BA (1b) and C(12)BA (1c) were converted to non-surfactants, whereas C(12)AB (2c) and C(16)AB (2d) still remained surface-active. Their solution properties were concomitantly changed.

Journal Article↗
PubMed2005

A role for cathepsin E in the processing of mast-cell carboxypeptidase A.

Mast-cell carboxypeptidase A is stored in the secretory granule and is released, together with a range of other inflammatory mediators, upon mast-cell degranulation. Carboxypeptidase A, like all mast-cell proteases, is stored in the granule as an active enzyme (i.e. with its propeptide removed). Although the processing mechanisms for the other classes of mast-cell proteases (in particular the chymases) have been clarified to some extent, the processing of procarboxypeptidase A is poorly characterized. Here, we show that mast cells from mice lacking the aspartic protease cathepsin E display an accumulation of procarboxypeptidase A, indicating a defect in carboxypeptidase-A processing. By contrast, mast cells lacking cathepsins B, L or D have normal carboxypeptidase-A processing. Furthermore, recombinant cathepsin E was found to process recombinant procarboxypeptidase A in vitro, under conditions resembling those found in mast-cell granules. Immunohistochemical analysis revealed staining for cathepsin E in mast cells from normal mice but not in mast cells from mice lacking heparin, indicating that cathepsin E is bound to heparin proteoglycan within mast-cell granules. In accordance with this notion, affinity chromatography showed that recombinant cathepsin E bound strongly to heparin under acidic conditions (the conditions prevailing in mast-cell granules) but not at neutral pH. Moreover, mast-cell degranulation resulted in the release of cathepsin E. Taken together, our results indicate that cathepsin E is located in mast-cell secretory granules in complex with heparin proteoglycans, and that it has a role in the processing of procarboxypeptidase A into active protease.

Animals↗
PubMed2005

Synthesis of mono-glucose-branched cyclodextrins with a high inclusion ability for doxorubicin and their efficient glycosylation using Mucor hiemalis endo-beta-N-acetylglucosaminidase.

The mono-glucose-branched cyclodextrins having an appropriate spacer between the beta-cyclodextrin and a glucose moiety were synthesized from beta-cyclodextrin and arbutin. They had the significantly high association constants for doxorubicin, the anticancer agent, in the range of 10(5)-10(6)M(-1), and worked as highly reactive glycosyl acceptors for the transglycosylation reaction by endo-beta-N-acetylglucosaminidase of Mucor hiemalis to produce sialo-complex type oligosaccharide-branched cyclodextrins in the high yields of 65-67%.

Acetylglucosaminidase↗
PubMed2005

No association of FOXP2 and PTPRZ1 on 7q31 with autism from the Japanese population.

Autism is a child-onset pervasive developmental disorder, with a significant role of genetic factors in its development. Genome-wide linkage studies have suggested a 7q region as a susceptibility locus for autism. We investigated several single nucleotide polymorphisms (SNPs) of Forkhead Box P2 (FOXP2) and Protein-Tyrosine Phosphatase, Receptor-type, Zeta-1 (PTPRZ1) at the 7q region in Japanese patients with autism and healthy controls. No significant difference was observed, after correction for the multiple testing, in allele, genotype or haplotype frequencies of the SNPs of FOXP2 or PTPRZ1 between patients and controls. No evidence was thus obtained for a major role of FOXP2 or PTPRZ1 in the development of autism.

Adolescent↗
PubMed2005

The role of the cathepsin E propeptide in correct folding, maturation and sorting to the endosome.

Cathepsin E (CE) is an endosomal aspartic proteinase of the A1 family that is highly homologous to the lysosomal aspartic proteinase cathepsin D (CD). Newly synthesized CE undergoes several proteolytic processing events to yield mature CE, from which the N-terminal propeptide usually comprising 39 amino acids is removed. To define the role of the propeptide of CE in its biosynthesis and processing, we constructed two fusion proteins using chimeric DNAs encoding the CE propeptide fused to the mature CD tagged with HA at the COOH terminus (termed ED-HA) and encoding the CD propeptide fused to the mature CE (termed DE). Pulse-chase analysis revealed that wild-type CE expressed in human embryonic kidney cells is autoproteolytically processed into mature CE within a 12-h chase, whereas the chimeric DE failed to be converted into mature CE even after a 24-h chase. The DE chimera was nevertheless capable of acid-dependent autoactivation in vitro to yield a catalytically active form, although its specificity constants (kcat/Km) were considerably high but less (35%) than those of the wild-type CE. By contrast, the chimeric ED-HA expressed in HeLa cells underwent neither processing into a catalytically active enzyme nor acid-dependent autoactivation in vitro. The ED-HA protein was less stable than wt-CD-HA, as determined on pulse-chase analysis and on trypsin digestion. These data indicate that the propeptide of CE is essential for the correct folding, maturation, and targeting of this protein to its final destination.

Animals↗
PubMed2005

Simultaneous multicolor detection system of the single-molecular microbial antigen with total internal reflection fluorescence microscopy.

Immunological diagnostic methods have been widely performed and showed high performance in molecular and cellular biology, molecular imaging, and medical diagnostics. We have developed novel methods for the fluorescent labeling of several antibodies coupled with fluorescent nanocrystal QDs. In this study we demonstrated that two bacterial toxins, diphtheria toxin and tetanus toxin, were detected simultaneously in the same view field of a cover slip by using directly QD-conjugated antibodies. We have succeeded in detecting bacterial toxins by counting luminescent spots on the evanescent field with using primary antibody conjugated to QDs. In addition, each bacterial toxin in the mixture can be separately detected by single excitation laser with emission band pass filters, and simultaneously in situ pathogen quantification was performed by calculating the luminescent density on the surface of the cover slip. Our results demonstrate that total internal reflection fluorescence microscopy (TIRFM) enables us to distinguish each antigen from mixed samples and can simultaneously quantitate multiple antigens by QD-conjugated antibodies . Bioconjugated QDs could have great potentialities for in practical biomedical applications to develop various high-sensitivity detection systems.

Antigens↗
PubMed2005

Essential roles of Homer-1a in homeostatic regulation of pyramidal cell excitability: a possible link to clinical benefits of electroconvulsive shock.

Homer-1a/Vesl1S, a member of the scaffold protein family Homer/Vesl, is expressed during seizure and serves to reduce seizure susceptibility. Cellular mechanisms for this feedback regulation were studied in neocortex pyramidal cells by injecting Homer-1a protein intracellularly. The injection reduced membrane excitability as demonstrated in two ways. First, the resting potential was hyperpolarized by 5-10 mV. Second, the mean frequency of spikes evoked by depolarizing current injection was decreased. This reduction of excitability was prevented by applying each of the followings: the calcium chelator BAPTA, the calcium store depletor cyclopiazonic acid (CPA), the insitol-1,4,5-trisphosphate receptor (IP(3)R) blocker heparin, the phospholipase C (PLC) inhibitor U-73122, the metabotropic glutamate receptor (mGluR) antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP), and the large-conductance calcium activated potassium channel (BK channel) antagonist charybdotoxin. The small-conductance calcium activated potassium channel (SK channel) blocker dequalinium was ineffective. These findings suggest that activation of mGluR by Homer-1a produced IP(3), which caused inositol-induced calcium release and a consequent BK channel opening, thus hyperpolarizing the injected neurons. In slices from rats subjected to electroconvulsive shock (ECS), a comparable reduction of excitability was observed without Homer-1a injection. The ECS-induced reduction of excitability was abolished by MPEP, charybdotoxin, heparin or BAPTA. Intracellular injection of anti-Homer-1a antibody was suppressive as well, but anti-Homer-1b/c antibody was not. We propose that ECS-induced Homer-1a stimulated the same pathway as did the injected Homer-1a, thereby driving a feedback regulation of excitability.

Animals↗
PubMed2005

Homer 1a enhances spike-induced calcium influx via L-type calcium channels in neocortex pyramidal cells.

The scaffold protein family Homer/Vesl serves to couple surface receptors or channels with endoplasmic calcium release channels. Homer 1a/Vesl-1S is regarded as regulating such coupling in an activity-dependent manner. The present calcium photometry and electrophysiological measurement revealed that Homer 1a up-regulates voltage-dependent calcium channels (VDCCs), depending on inositol-1,4,5-trisphosphate (IP3) receptors (IP3Rs). In rat neocortex pyramidal cells, intracellular injection by diffusion from the patch pipette (referred to as 'infusion') of Homer 1a protein enhanced spike-induced calcium increase, depending on both the protein concentration and spike frequency. Induction of this enhancement was disrupted by blockers of key molecules of the mGluR-IP3 signalling pathway, including metabotropic glutamate receptors (mGluRs), phospholipase C and IP3Rs. However, infusion of IP3 failed to mimic the effect of Homer 1a, suggesting requirement for a second Homer 1a-mediated signalling as well as the mGluR-IP3 signalling. In contrast to the induction, maintenance of this enhancement was independent of the mGluR-IP3 signalling, taking the form of augmented calcium influx via L-type VDCCs. Presumably due to the VDCC up-regulation, threshold currents for calcium spikes were reduced. Given that Homer 1a induction is thought to down-regulate neural excitability and hence somatic spike firing, this facilitation of calcium spikes concomitant with such attenuated firing may well have a critical impact on bi-directional synaptic plasticity.

Animals↗
PubMed2005

A functional virulence complex composed of gingipains, adhesins, and lipopolysaccharide shows high affinity to host cells and matrix proteins and escapes recognition by host immune systems.

Arg-gingipain (Rgp) and Lys-gingipain (Kgp) are Porphyromonas gingivalis cysteine proteinases implicated as major virulence factors in pathologies of periodontitis. We purified a 660-kDa cell-associated gingipain complex existing as a homodimer of two catalytically active monomers which comprises their catalytic and adhesin domains. Electron microscopy revealed that the complex was composed of a globular particle with a 10-nm external diameter possessing one or two electron-dense hole-like structures. Two-dimensional gel electrophoresis and immunoblot analyses revealed the association of lipopolysaccharide (LPS) with the catalytic domains and a hemagglutinin domain, Hgp44, of Rgp and Kgp in the complex. The complex significantly degraded human type I collagen and elastin and strongly disrupted viability of human gingival fibroblasts and umbilical vein endotherial cells with an efficiency which was higher than that of the monomeric gingipains. The native complex produced only a small amount of nitrogen dioxide, tumor necrosis factor alpha, and interleukin-6 by macrophages, whereas the heat-denatured complex resulted in increased production. Inhibition of the proteolytic activities of the gingipain complex did not up-regulate the cytokine production, indicating that the functional domains in LPS are structurally masked by the complex proteins. These results indicate the importance of the complex in evasion of host defense mechanisms as well as in host tissue breakdown.

Adhesins, Bacterial↗
PubMed2005

Novel bifidobacterial glycosidases acting on sugar chains of mucin glycoproteins.

Bifidobacterium bifidum was found to produce a specific 1,2-alpha-L-fucosidase. Its gene (afc A) has been cloned and the DNA sequence was determined. The Afc A protein consisting of 1959 amino acid residues with a predicted molecular mass of 205 kDa can be divided into three domains; the N-terminal function-unknown domain (576 aa), the catalytic domain (898 aa), and the C-terminal bacterial Ig-like domain (485 aa). The recombinant catalytic domain specifically hydrolyzed the terminal alpha-(1-->2)-fucosidic linkages of various oligosaccharides and sugar chains of glycoproteins. The primary structure of the catalytic domain exhibited no similarity to those of any glycoside hydrolases but showed similarity to those of several hypothetical proteins in a database, which resulted in establishment of a novel glycoside hydrolase family (GH family 95). Several bifidobacteria were found to produce a specific endo-alpha-N-acetylgalactosaminidase, which is the endoglycosidase liberating the O-glycosidically linked galactosyl beta1-->3 N-acetylgalactosamine disaccharide from mucin glycoprotein. The molecular cloning of endo-alpha-N-acetylgalactosaminidase was carried out on Bifidobacterium longum based on the information in the database. The gene was found to comprise 1966 amino acid residues with a predicted molecular mass of 210 kDa. The recombinant protein released galactosyl beta1-->3 N-acetylgalactosamine disaccharide from natural glycoproteins. This enzyme of B. longum is believed to be involved in the catabolism of oligosaccharide of intestinal mucin glycoproteins. Both 1,2-alpha-L-fucosidase and endo-alpha-N-acetylgalactosaminidase are novel and specific enzymes acting on oligosaccharides that exist mainly in mucin glycoproteins. Thus, it is reasonable to conclude that bifidobacteria produce these enzymes to preferentially utilize the oligosaccharides present in the intestinal ecosystem.

Amino Acid Sequence↗
PubMed2005

A new selective substrate for cathepsin E based on the cleavage site sequence of alpha2-macroglobulin.

Cathepsin E is an intracellular aspartic proteinase of the pepsin family predominantly expressed in cells of the immune system and believed to contribute to homeostasis by participating in host defense mechanisms. Studies on its enzymatic properties, however, have been limited by a lack of sensitive and selective substrates. For a better understanding of the importance of this enzyme in vivo, we designed and synthesized a highly sensitive peptide substrate for cathepsin E based on the sequence of the specific cleavage site of alpha2-macroglobulin. The substrate constructed, MOCAc-Gly-Ser-Pro-Ala-Phe-Leu-Ala-Lys(Dnp)-D-Arg-NH2 [where MOCAc is (7-methoxycoumarin-4-yl)acetyl and Dnp is dinitrophenyl], derived from the cleavage site sequence of human alpha2-macroglobulin, was the most sensitive and selective for cathepsin E, with k(cat)/K(m) values of 8-11 microM(-1) s(-1), whereas it was resistant to hydrolysis by the analogous aspartic proteinases cathepsin D and pepsin, as well as the lysosomal cysteine proteinases cathepsins B, L, and H. The assay allows the detection of a few fmol of cathepsin E, even in the presence of plasma and cell lysate, and gives accurate results over a wide enzyme concentration range. This substrate might represent a useful tool for monitoring and accurately quantifying cathepsin E, even in crude enzyme preparations.

Animals↗
PubMed2005

Treatment options in the management of ovarian cancer.

The standard regimen used as primary chemotherapy of ovarian cancer is combination chemotherapy using paclitaxel and carboplatin. The main objective of first-line chemotherapy is to induce complete response. Although most cases respond to the initial chemotherapy, many cases relapse within 3 years. Such relapsed and persistent cases become resistant to first-line chemotherapy and require second-line chemotherapy. Objectives of such a second-line chemotherapy are to obtain disease palliation to cease disease progression. Meanwhile, consolidation or maintenance chemotherapy may be added to prevent or inhibit disease relapse for patients with advanced disease after induction of complete remission by a primary chemotherapy. When the unresectable tumour is presumed by primary surgery, neoadjuvant chemotherapy may be selected. Recently, conventional cytotoxic anticancer drugs containing paclitaxel have been shown to be capable of inhibiting angiogenesis. The notion of 'redefining' chemotherapeutic drugs has been recognised; thus, continuous low-dose chemotherapy -- so-called metronomic chemotherapy -- has been approved as a new concept. Many new molecular-targeted therapies became available for clinical cancer therapy. The explosion of new molecular targets and the development and application of many powerful technologies should accelerate the discovery of innovative molecular therapeutics. Understanding the molecular mechanisms will help to clarify the pathways in ovarian cancer development and help to identify new therapeutic and diagnostic targets. These are exciting times for new drug development and the treatment of cancer. Cautious optimism should prevail for all investigators involved in translating these exciting new biological findings into new pharmacological agents for treatment of cancer.

Angiogenesis Inhibitors↗