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Biomedical subjects

H Nakai

Publications and source records attributed to H Nakai.

At least 19 recordsLinked to original sources
PubMed2003

Dysferlin mutations in Japanese Miyoshi myopathy: relationship to phenotype.

OBJECTIVE: To study dysferlin gene mutations and genotype-phenotype correlations in Japanese patients with Miyoshi myopathy (MM). BACKGROUND: MM is an autosomal recessive distal muscular dystrophy that arises from mutations in the dysferlin gene. This gene is also mutated in families with limb girdle muscular dystrophy 2B. METHODS: The authors examined 25 Japanese patients with MM. Genomic DNA was extracted from the peripheral lymphocytes of the patients. The PCR products of each of 55 exons were screened by single strand conformation polymorphism or direct sequencing from the PCR fragments. RESULTS: The authors identified 16 different mutations in 20 patients with MM; 10 were novel. Mutations in Japanese patients are distributed along the entire length of the gene. CONCLUSIONS: Four mutations (C1939G, G3370T, 3746delG, and 4870delT) are relatively more prevalent in this population, accounting for 60% of the mutations in this study. This study revealed that the G3370T mutation was associated with milder forms of MM and the G3510A mutation was associated with a more severe form.

Adult↗
PubMed2001

Escherichia coli PriA helicase: fork binding orients the helicase to unwind the lagging strand side of arrested replication forks.

Escherichia coli PriA is a primosome assembly protein with 3' to 5' helicase activity whose apparent function is to promote resumption of DNA synthesis following replication-fork arrest. Here, we describe how initiation of helicase activity on DNA forks is influenced by both fork structure and by single-strand DNA-binding protein. PriA could recognize and unwind forked substrates where one or both arms were primarily duplex, and PriA required a small (two bases or larger) single-stranded gap at the fork in order to initiate unwinding. The helicase was most active on substrates with a duplex lagging-strand arm and a single-stranded leading-strand arm. On this substrate, PriA was capable of translocating on either the leading or lagging strands to unwind the duplex ahead of the fork or the lagging-strand duplex, respectively. Fork-specific binding apparently orients the helicase domain to unwind the lagging-strand duplex. Binding of single-strand-binding protein to forked templates could inhibit unwinding of the duplex ahead of the fork but not unwinding of the lagging-strand duplex or translocation on the lagging-strand template. While single-strand-binding protein could inhibit binding of PriA to the minimal, unforked DNA substrates, it could not inhibit PriA binding to forked substrates. In the cell, single-strand-binding protein and fork structure may direct PriA helicase to translocate along the lagging-strand template of forked structures such that the primosome is specifically assembled on that DNA strand.

Base Sequence↗
PubMed2001

ABO-incompatible living-donor kidney transplantation in children.

BACKGROUND: Due to a severe shortage of suitable cadaveric allografts for children awaiting kidney transplants, we have performed a series of ABO-incompatible living kidney transplantations (LKT) at our institution. METHODS: Between July 1989 and March 2000, 16 pediatric patients (3 female, 13 male) underwent ABO-incompatible LKT. The mean age at transplantation was 10.9+/-4.3 years (range 5.1-15.0 years). The donor to recipient ABO blood antigen incompatibility was as follows: A1-->O, 5 patients; B-->O, 6 patients; A1B-->B, 2 patients; and A1B -->B, A1-->B, or B-->A1, 1 patient each. The median pretransplantation anti-A1 titers of eight A-incompatible recipients were 1:128 (IgM, range 1:16 to 1:512) and 1:32 (IgG, range 1:2 to 1:128). Median anti-B titers of seven B-incompatible recipients were 1:32 (IgM, range 1:4 to 1:128) and 1:8 (IgG, range 1:2 to 1:64). All patients received three or four sessions of plasmapheresis (PP) and/or immunoadsorption (IA) to remove the anti-A and/or anti-B antibodies before transplantation. Immunosuppression initially consisted of cyclosporine, methylprednisolone, cyclophosphamide, and antilymphocyte globulin. Splenectomy was performed on all recipients at the time of transplantation. RESULTS: The patients were followed for 6 to 122 months with a mean follow-up of 63 months. All 16 recipients who underwent ABO-incompatible LKT achieved a pretransplant isoagglutinin titer less than 1:8 with 3-4 sessions of PP/IA treatment. Of 16 patients, 10 patients had rebound increase in their IgM and/or IgG anti-A/B titers to greater than 1:64 or predepletion levels within 10 days posttransplantation. In addition, nine patients developed renal dysfunction in association with the rebound increase in their anti-A/B. One patient lost his graft because of uncontrolled delayed hyperacute rejection, whereas eight other recipients recovered completely with pulse steroids and PP/IA therapy. After the third week posttransplant, there was no correlation between the occurrence of AR and their isoagglutinin titers. Moreover, no antibody-mediated rejection was observed, even in recipients with continued high titer anti-A and/or anti-B antibodies. Patient survival is 100% to date. The actuarial 1-year and 5-year graft survival rates are 87% and 85%, respectively. No fatal infectious complications occurred despite the combination of splenectomy and immunosuppressive drugs. CONCLUSIONS: We have demonstrated that with adequate pre- and posttransplant management, successful kidney transplantation across the ABO barrier is possible in the pediatric population. "Accommodation" of the allografts occurred within 2 weeks of transplantation. Subsequently, the long-term graft outcome of ABO-incompatible LKT was comparable to that of ABO-compatible LKT.

ABO Blood-Group System↗
PubMed2001

Conformational dynamics of a transposition repressor in modulating DNA binding.

The repressor of bacteriophage Mu functions in the establishment and maintenance of lysogeny by binding to Mu operator DNA to shut down transposition. A domain at its N terminus functions in DNA binding, and temperature-sensitive mutations in this domain can be suppressed by truncations at the C terminus. To understand the role of the C-terminal tail in DNA binding, a fluorescent probe was attached to the C terminus to examine its environment and its movement with respect to the DNA binding domain. The emission spectrum of this probe indicated that the C terminus was in a relatively hydrophobic environment, comparable to the environment of the probe attached within the DNA-binding domain. Fluorescence of two tryptophan residues located within the DNA-binding domain was quenched by the probe attached to the C terminus, indicating that the C terminus is in close proximity to this domain. Addition of DNA, even when it did not contain operator DNA, reduced quenching of tryptophan fluorescence, indicating that the tail moves away from the DNA-binding domain as it interacts with DNA. The presence of the tail also produced a trypsin hypersensitive site within the DNA-binding domain; mutant repressors with an altered or truncated C terminus were relatively resistant to cleavage at this site. Interaction of the wild-type repressor with DNA greatly reduced cleavage at the site. A repressor with a temperature-sensitive mutation in the DNA-binding domain was especially sensitive to cleavage by trypsin even in the presence of DNA, and the C-terminal tail failed to move in the presence of DNA at elevated temperatures. These results indicate that the tail sterically inhibits DNA binding and that it moves during establishment of repression. Such conformational changes are likely to be involved in communication between repressor protomers for cooperative DNA binding.

Amino Acid Sequence↗
PubMed2001

Mouse Atp6f, the gene encoding the 23-kDa proteolipid of vacuolar proton translocating ATPase.

The 23-kDa proteolipid subunit of mouse vacuolar-type proton-translocating ATPase (V-ATPase) was predicted to be a hydrophobic polypeptide of 205 amino acid residues with five putative transmembrane segments. It exhibits sequence similarity to Vma16p of Saccharomyces cerevisiae and vha-4 of Caenorhabdittis elegans (83 and 84%, respectively). Southern blot analysis indicated that the proteolipid is encoded by a single gene, Atp6f, in the mouse genome. Atp6f was mapped to approximately 55 cM on chromosome 4, and its genomic organization is similar to that of the human gene: 8 exons separated by 7 introns, with boundaries matching the GT-AG rule. RNA blotting demonstrated that Atp6f is transcribed as 1.0- and 1.8-kb mRNAs in multiple tissues to varying degrees. The major transcription initiation sites are at -13 and -58 bp upstream of the translation initiation codon. The epitope-tagged 23-kDa protoelipid was localized in endomembrane organelles in CHO cells, as expected for a component of a vacuolar-type proton pump.

Alternative Splicing↗
PubMed2001

Design and synthesis of a highly selective EP2-receptor agonist.

EP2-receptor selective agonist 3 was identified by the structural hybridization of butaprost 1a and PGE(2) 2a. Based on this information, a chemically more stabilized 4 was discovered as another highly selective EP2-receptor agonist, iv administration of which to anesthetized rats suppressed uterine motility, while PGE(2) 2a stimulated uterine motility.

Alprostadil↗
PubMed2001

Handoff from recombinase to replisome: insights from transposition.

Bacteriophage Mu replicates as a transposable element, exploiting host enzymes to promote initiation of DNA synthesis. The phage-encoded transposase MuA, assembled into an oligomeric transpososome, promotes transfer of Mu ends to target DNA, creating a fork at each end, and then remains tightly bound to both forks. In the transition to DNA synthesis, the molecular chaperone ClpX acts first to weaken the transpososome's interaction with DNA, apparently activating its function as a molecular matchmaker. This activated transpososome promotes formation of a new nucleoprotein complex (prereplisome) by yet unidentified host factors [Mu replication factors (MRF alpha 2)], which displace the transpososome in an ATP-dependent reaction. Primosome assembly proteins PriA, PriB, DnaT, and the DnaB--DnaC complex then promote the binding of the replicative helicase DnaB on the lagging strand template of the Mu fork. PriA helicase plays an important role in opening the DNA duplex for DnaB binding, which leads to assembly of DNA polymerase III holoenzyme to form the replisome. The MRF alpha 2 transition factors, assembled into a prereplisome, not only protect the fork from action by nonspecific host enzymes but also appear to aid in replisome assembly by helping to activate PriA's helicase activity. They consist of at least two separable components, one heat stable and the other heat labile. Although the MRF alpha 2 components are apparently not encoded by currently known homologous recombination genes such as recA, recF, recO, and recR, they may fulfill an important function in assembling replisomes on arrested replication forks and products of homologous strand exchange.

Adenosine Triphosphatases↗
PubMed2001

Development of orally active nonpeptidic inhibitors of human neutrophil elastase.

5-Amino-2-phenylpyrimidin-6-ones, some of their desamino derivatives, and miscellaneous derivatives were synthesized and biologically evaluated on both in vitro activity and oral activity in an acute hemorrhagic assay. These compounds contained an alpha-keto-1,3,4-oxadiazole moiety to bind covalently to the Ser-195 hydroxy group of human neutrophil elastase (HNE). Among those tested, compounds 11a-c,e,i-l(F), 11d,e,k(H), 21d,e,k(F), and 21d,e(H) showed a good oral profile. RS-Mixture 3(H) was selected for clinical evaluation based on its oral potency, duration of action, enzyme selectivity, safety profile, and ease of synthesis. Structure-activity relationships (SARs) are discussed.

Administration, Oral↗
PubMed2001

In hemophilia A and autoantibody inhibitor patients: the factor VIII A2 domain and light chain are most immunogenic.

Factor VIII (fVIII) is a protein cofactor essential for blood coagulation, and it binds in the factor Xase complex to factors IXa, X, and phospholipid. In about 30% of severe hemophilia A patients, treatment with fVIII leads to production of anti-fVIII antibodies. Anti-fVIII autoantibodies also rarely appear in normal individuals. Those antibodies that inactivate fVIII (inhibitors) prevent optimal fVIII therapy. Inhibitor epitopes were previously localized to the fVIII A2, A3, and C2 domains and to an acidic amino acid region between A1 and A2. Such anti-fVIII antibodies interfere with fVIII binding to components of the factor Xase complex and prevent blood coagulation. When total anti-fVIII titers were determined for each fVIII domain in 43 inhibitor plasmas by immunoprecipitation (IP) and inhibitor neutralization assays, the anti-light chain (LCh) antibody titer was highest, anti-A2 was intermediate, and anti-A1 and anti-B were low. The relative immunogenicity of the fVIII domains in hemophilic and autoantibody inhibitor patients was similar.

Antibodies, Monoclonal↗
PubMed2001

The crystal structure of the complex of non-peptidic inhibitor of human neutrophil elastase ONO-6818 and porcine pancreatic elastase.

The crystal structure of a new inhibitor of human neutrophil elastase (HNE), N-[2-[5-(tert-butyl)-1,3,4-oxadiazol-2-yl]-(IRS)-1-(methylethyl)-2-oxoethyl]-2-(5-amino-6-oxo-2-phenyl-6H-pyrimidin-1-ly)acetamide (ONO-6818, 1) complexed to porcine pancreatic elastase (PPE) has been determined at 1.86 A resolution. Analytical results provided evidence of a 1:1 complex in which the electrophilic ketone of 1 covalently bound to O gamma of Ser195 at the active site of PPE. The role of the unique electron-withdrawing ketone of 1 has been elucidated.

Animals↗
PubMed2001

Design and synthesis of new orally active inhibitors of human neutrophil elastase.

To identify new orally active inhibitors, further modification of 1 (ONO-6818) was performed. Peptidic derivatives 4b, 4c and 4n showed more potent inhibitory activity than nonpeptidic derivatives 3a-c. As a result, a series of peptidic inhibitors, 4a-s and 5a-v, were discovered. Among these N-aryl derivatives 5a-g, 5i, 5m and 5o-v showed oral activity. Their oral activity showed good correlation with their metabolic stability. Compounds 5h and 5j-l, which were extremely metabolically unstable in hamster plasma, did not show oral activity. Oral activity was considered to be determined by a combination of at least two factors: oral absorption and metabolic stability.

Administration, Oral↗
PubMed2001

No evidence for AT2R gene derangement in human urinary tract anomalies.

BACKGROUND: It has been recently found that mice, especially males, with a disrupted angiotensin type 2 receptor (AT2R) gene, which is located on the X-chromosome, often have a range of congenital anomalies of the kidney and urinary tract (CAKUT), including renal hypoplasia, and that Caucasian male patients with ureteropelvic junction stenosis (UPJ) and multicystic dysplastic kidneys frequently have A-G transition in intron 1 of the AT2R gene. We have previously found that renal hypoplasia is remarkably predominant in Japanese boys. METHODS: We investigated sex ratios for the frequency of each CAKUT. The frequency of the A-G transition between the controls and 66 Japanese boys with CAKUT were compared. There was renal hypoplasia in 16, UPJ in 17, vesicoureteral in 20, and other anomalies in 13. We also investigated whether any mutations in AT2R genes were detectable in patients with renal hypoplasia. RESULTS: In contrast to mice with a disruption of the AT2R gene, the male-to-female ratios in human patients proved to be considerably variable: 16 for renal hypoplasia, 2.1 for UPJ, 0.8 for vesicoureteral, and 1.2 for others. The frequency of the A-G transition was not different between the control population and the patients with CAKUT [31 of 102 (30%) vs. 23 of 66 (35%), respectively]. A sequencing study disclosed no mutations in nine boys with renal hypoplasia. CONCLUSIONS: These findings indicate that the AT2R gene may not play a major role in the development of renal hypoplasia and other CAKUT in humans, at least in the Japanese population.

Alleles↗
PubMed2001

Tissue engineering research in oral implant surgery.

In this article, we introduce some of the more extensively evaluated technologies using concepts of tissue engineering. We report on hard tissue engineering and soft tissue engineering and their utility for dental implant therapy. For hard tissue engineering, we evaluated human recombinant bone morphogenetic protein-2 and marrow mesenchymal stem cells using a model of sinus augmentation procedure in rabbit. We also describe distraction osteogenesis as another category for hard tissue engineering. In addition, we evaluate soft tissue management using cultured epithelial grafting for soft tissue engineering. The results of our tissue regeneration materials and methods in this study are positive. When the tissue engineering materials are used in clinics in the future, implant surgery could be the leading field.

Absorbable Implants↗
PubMed2001

Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo.

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Although the vector genomes are found both as extrachromosomes and as chromosomally integrated forms in hepatocytes, the relative proportion of each has not yet been clearly established. Using an in vivo assay based on the induction of hepatocellular regeneration via a surgical two-thirds partial hepatectomy, we have determined the proportion of integrated and extrachromosomal rAAV genomes in mouse livers and their relative contribution to stable gene expression in vivo. Plasma human coagulation factor IX (hF.IX) levels in mice originating from a chromosomally integrated hF.IX-expressing transposon vector remained unchanged with hepatectomy. This was in sharp contrast to what was observed when a surgical partial hepatectomy was performed in mice 6 weeks to 12 months after portal vein injection of a series of hF.IX-expressing rAAV vectors. At doses of 2.4 x 10(11) to 3.0 x 10(11) vector genomes per mouse (n = 12), hF.IX levels and the average number of stably transduced vector genomes per cell decreased by 92 and 86%, respectively, after hepatectomy. In a separate study, one of three mice injected with a higher dose of rAAV had a higher proportion (67%) of integrated genomes, the significance of which is not known. Nevertheless, in general, these results indicate that, in most cases, no more than approximately 10% of stably transduced genomes integrated into host chromosomes in vivo. Additionally, the results demonstrate that extrachromosomal, not integrated, genomes are the major form of rAAV in the liver and are the primary source of rAAV-mediated gene expression. This small fraction of integrated genomes greatly decreases the potential risk of vector-related insertional mutagenesis associated with all integrating vectors but also raises uncertainties as to whether rAAV-mediated hepatic gene expression can persist lifelong after a single vector administration.

Animals↗