Biomedical subjects

Hirokazu Fukuda

Publications and source records attributed to Hirokazu Fukuda.

At least 19 recordsLinked to original sources

PubMed2007

Induction of abnormal nuclear shapes in two distinct modes by overexpression of serine/threonine protein phosphatase 5 in Hela cells.

Okadaic acid-sensitve serine/threonine protein phosphatase 5 (PP5) is expressed ubiquitously in various tissues and is considered to participate in many cellular processes. PP5 has a catalytic domain in the C-terminal region and three tetratricopeptide repeat (TPR) motifs in the N-terminal region, which are suspected to function as a protein-protein interaction domain. Physiological roles of PP5 are still largely unknown, although several PP5-binding proteins were reported and a few in vivo functions of PP5 were suggested. In the present study, the effects of expression of the full-length wild-type PP5 fused with EGFP (EGFP-PP5(WT)) and its phosphatase-dead mutant EGFP-PP5(H304A) were investigated. Transient expression of either EGFP-PP5(WT) or EGFP-PP5(H304A) in HeLa cells induced deformed nuclei with a 10-fold frequency compared to that of EGFP. Abnormal-shaped nuclei were also substantially increased by induced moderate expression of PP5 in tet-on HeLa cells. Many HeLa cells expressing EGFP-PP5(WT) possessed multi-nuclei separated from each other by nuclear membrane, while expression of EGFP-PP5(H304A) induced deformed nuclei which were multiple-like in shape, but not separated completely and were surrounded by one nuclear membrane. These results suggest that PP5 plays important roles at the M-phase of the cell cycle, especially in separation of chromosomes and formation of nuclear membrane.

Amino Acid Sequence↗

PubMed2006

Molecular mechanisms for maintenance of G-rich short tandem repeats capable of adopting G4 DNA structures.

Mammalian genomes contain several types of repetitive sequences. Some of these sequences are implicated in various specific cellular events, including meiotic recombination, chromosomal breaks and transcriptional regulation, and also in several human disorders. In this review, we document the formation of DNA secondary structures by the G-rich repetitive sequences that have been found in several minisatellites, telomeres and in various triplet repeats, and report their effects on in vitro DNA synthesis. d(GGCAG) repeats in the mouse minisatellite Pc-1 were demonstrated to form an intra-molecular folded-back quadruplex structure (also called a G4' structure) by NMR and CD spectrum analyses. d(TTAGGG) telomere repeats and d(CGG) triplet repeats were also shown to form G4' and other unspecified higher order structures, respectively. In vitro DNA synthesis was substantially arrested within the repeats, and this could be responsible for the preferential mutability of the G-rich repetitive sequences. Electrophoretic mobility shift assays using NIH3T3 cell extracts revealed heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and A3, which were tightly and specifically bound to d(GGCAG) and d(TTAGGG) repeats with K(d) values in the order of nM. HnRNP A1 unfolded the G4' structure formed in the d(GGCAG)(n) and d(TTAGGG)(n) repeat regions, and also resolved the higher order structure formed by d(CGG) triplet repeats. Furthermore, DNA synthesis arrest at the secondary structures of d(GGCAG) repeats, telomeres and d(CGG) triplet repeats was efficiently repressed by the addition of hnRNP A1. High expression of hnRNPs may contribute to the maintenance of G-rich repetitive sequences, including telomere repeats, and may also participate in ensuring the stability of the genome in cells with enhanced proliferation. Transcriptional regulation of genes, such as c-myc and insulin, by G4 sequences found in the promoter regions could be an intriguing field of research and help further elucidate the biological functions of the hnRNP family of proteins in human diseases.

3T3 Cells↗

PubMed2005

Entrainment in a chemical oscillator chain with a pacemaker.

Entrainment by a pacemaker was investigated experimentally and numerically in a chain of chemical oscillators using coupled discrete Belousov-Zhabotinsky reaction oscillators. The spontaneous frequency of each oscillator depended on the concentration of catalyst ions. The coupling strengths among the nearest neighbor oscillators were controlled by changing the spacing distance (d) between beads. When the coupling strength was sufficiently strong, the pacemaker entrained other oscillators in the chain. Subsequently, the trigger waves propagating from a pacemaker were observed. The range of trigger wave propagation area, i.e., the number of entrained oscillators, depended on d. Numerical simulation for the system described the experimental results well. Furthermore, photic noise maximized the strength of entrainment at an optimal noise intensity.

Journal Article↗

PubMed2005

Unfolding of higher DNA structures formed by the d(CGG) triplet repeat by UP1 protein.

Fragile X syndrome is caused by expansion of a d(CGG) triplet repeat in the 5'-untranslated region of the first exon of the FMR1 gene resulting in silencing of the gene. The d(CGG) repeat has been reported to form hairpin and quadruplex structures in vitro, and formation of these higher structures could be responsible for its unstable expansion in the syndrome, although molecular mechanisms underlying the repeat expansion still remain elusive. We have previously proved that UP1, a proteolytic product of hnRNP A1, unfolds the intramolecular quadruplex structures of d(GGCAG)5 and d(TTAGGG)4 and abrogates the arrest of DNA synthesis at d(GGG)n sites. Here, we demonstrate that the d(CGG) repeat forms a peculiar DNA structure, which deviates from the canonical B-form structure. In addition, UP1 was demonstrated by CD spectrum analysis to unfold this characteristic higher structure of the d(CGG) repeat and to abrogate the arrest of DNA synthesis at the site. This ability of UP1 suggests that unfolding of unusual DNA structures of a triplet repeat is required for DNA synthesis processes.

Circular Dichroism↗

PubMed2005

Up-regulation of hnRNP A1 gene in sporadic human colorectal cancers.

We have previously reported that the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a major hnRNP, binds to G-rich repetitive sequences and quadruplex (G4') structures in DNA, including the 5'-TTAGGG-3' telomere repeat and 5'-GGCAG-3' short-tandem-repeat. DNA synthesis arrest at the (GGG) sites within these repeats in vitro was retrieved by the addition of the hnRNP A1 protein or its N-terminal proteolytic product, UP1, in a dose-dependent manner. Therefore, functional perturbation of hnRNP A1 may abrogate the genomic stability of telomere repeats and other G-rich sequences, independent of its major role in transcriptional and translational regulation. In the present study, we conducted genetic and expression analysis of the hnRNP A1 gene in sporadic human colorectal cancers to clarify its possible involvement in human carcinogenesis. Of 30 lesions, one harbored a mutation at the -11 position from the translation initiation site, but none in the coding region. A single nucleotide polymorphism, an A or G-allele, was found in the 5' upstream promoter region of the gene. Quantitative gene expression analysis revealed that 60% (18/30) of cases showed over-expression of hnRNP A1 in cancer tissues by 2-fold or greater, compared to their normal colon tissues, with values of 78, 64 and 40% for clinicopathological stages II, III and IV, respectively. Although the biological consequences of hnRNP A1 overexpression in colorectal cancers remain to be clarified, it could contribute to maintenance of telomere repeats in cancer cells with enhanced cell proliferation. Alternatively, since the variations in the stoichiometry of hnRNP family proteins are considered to affect cell-specific gene expression, quantitative alteration of hnRNP A1 could result in facilitation of transformation of colon epithelial cells as a consequence of transcriptional and translational perturbation.

Adult↗

PubMed2004

LRP130, a single-stranded DNA/RNA-binding protein, localizes at the outer nuclear and endoplasmic reticulum membrane, and interacts with mRNA in vivo.

LRP130 (also known as a LRPPRC) is an RNA and single-stranded DNA-binding protein, and recently identified as a candidate gene responsible for the Leigh syndrome, a French-Canadian type cytochrome c oxidase deficiency. However, the biological function of LRP130 still remains largely unresolved. In the present study, we found that the C-terminal half of the mouse LRP130 located within a 120 amino acid sequence (a.a. 845-964) binds to synthetic RNA homopolymers, poly(G), poly(U), and poly(C), as well as r(CUGCC)(6). Assessment of the subcellular localization indicated both nuclear/endoplasmic reticulum (ER) and mitochondrial fractions to be positive. To further analyze the subcellular localization of LRP130, a nuclear/ER fraction was fractionated into the nucleoplasm (NP) and nuclear envelope (NE)/ER, and the latter was further separated into outer nuclear membrane (ONM)/ER and inner nuclear membrane (INM) by treatment with Triton X-100. LRP130 was detectable in all three fractions, and the distribution pattern was in good accordance with that known for ONM/ER proteins. Interestingly, immunostaining of HeLa cells demonstrated nuclear rim staining of LRP130, specifically at the outside of the NE and also at ER, and association of LRP130 with poly(A)(+) RNA was restricted only to the ONM/ER fraction. Overexpression of full-length mouse LRP130 fused with EGFP resulted in nuclear accumulation of poly(A)(+) RNA in HeLa cells. Taking all these results together, it is suggested that LRP130, a novel type of RNA-binding protein, associates with mRNA/mRNP complexes at the outside of NE and ER, and plays a role in control of mRNA metabolisms.

Animals↗

PubMed2004

Circadian rhythm formation in plant seedling: global synchronization and bifurcation as a coupled nonlinear oscillator system.

Circadian rhythm formation is studied in seedlings after germination measuring their respiratory metabolism. The circadian rhythm is clearly observed at about 170h (the onset time t(CR-ON)) after germination of seeds in natural conditions in a dark incubator. There are no clear cyclic signals in gas exchange before t(CR-ON). Application of external triggers (temperature shocks) near the onset of the rhythm in seedling growth strongly affects formation processes. The onset is shifted earlier up to 50h by application of perturbations. This fact may suggest that the circadian rhythms appear via subcritical bifurcation.

Adaptation, Physiological↗

PubMed2004

[Postintubation granuloma of the larynx].

A 50-year-old woman underwent tonsillectomy under general anesthesia. She developed progressive hoarseness two month after the tonsillectomy, and a large granuloma of the larynx was found. Surgical removal of the granuloma was performed by laryngo-microsurgery. We should keep in mind that postintubation granuloma of the larynx might develop after tracheal intubation, and careful airway manipulation is needed to avoid this potential complication.

Anesthesia, General↗

PubMed2003

Destruction of quadruplex by proteins, and its biological implications in replication and telomere maintenance.

The minisatellite DNA Pc-1 consists of tandem repeats of d(GGCAG). We previously reported that a d(GGCAG)n strand folds into an intramolecular quadruplex under physiological conditions and that during replication the progression of DNA polymerase is blocked by the quadruplex in vitro. Therefore, the formation of the quadruplex was supposed to be responsible for the hypermutable features of Pc-1. Then, we have identified proteins that bind to Pc-1, one of which is hnRNP A1. Here, we have demonstrated that hnRNP A1 destroys the quadruplex of Pc-1 on binding and abrogates the arrest of DNA polymerase at the repeat. Thus, hnRNP A1 functions as if it is a chaperon to assist Pc-1 DNA to form the proper folding suitable for replication. We have also found that hnRNP A1 and a related protein, hnRNP D, destroy the quadruplex of telomere DNA, which suggests the involvement of these proteins in telomere maintenance as DNA chaperons.

DNA Replication↗

PubMed2003

[Anesthetic management for infected abdominal aneurysm].

A 48-year-old man was scheduled for repair of mycotic abdominal aortic aneurysm. Extraanatomic bypass was performed due to infection of the aneurysm. General anesthesia combined with continuous epidural anesthesia was performed. In spite of severe blood loss, hemodynamic state was stable during anesthesia and postoperative period. He was managed in the intensive care unit without any complication. In this anesthetic management, it is very important to maintain adequate cardiac output and circulating blood volume to prevent visceral ischemia.

Anesthesia, Epidural↗

PubMed2003

[Heparin resistance during cardiopulmonary bypass].

A 74-year-old man was scheduled for coronary artery bypass graft surgery with cardiopulmonary bypass. After intravenous heparin (200 U.kg-1), the activated clotting time (ACT) increased from 124 to 436 sec. However, it decreased to 128 sec immediately after cardiopulmonary bypass. The bypass was discontinued because the addition of heparin (200 U.kg-1) proved heparin resistance. The coronary artery bypass procedure was completed uneventfully without cardiopulmonary bypass. Several recent articles have reported that heparin resistance was corrected with antithrombin III concentrates, fresh frozen plasma, or argatroban. In this case, these drugs could not be used because the mechanism of heparin resistance remains uncertain. Thus, the off-pump technique is useful for unknown heparin resistance.

Aged↗

PubMed2003

[A case of pregnant woman with severe obsessive-compulsive disorder successfully treated by modified-electroconvulsive therapy].

Obsessive-compulsive disorder (OCD) is rare psychiatric disorder during pregnancy, and is often therapy-resistant. We report a 36-year-old pregnant woman with severe obsessive-compulsive disorder successfully treated by modified-electroconvulsive therapy. During the pregnancy, severe mysophobia and compulsive washing appeared, so severely that she was unable to lie down, endangering the life of the fetus. Since the pharmacotherapy was ineffective, modified electroconvulsive therapy (m-ECT) was performed in cooperation with the obstetrician and the anesthesiologist, along with monitored cardiotocography throughout the procedure. During the second therapy late deceleration on the fetal cardiotocogram occurred, but rapid intravenous administration of ritodrine led to the cessation of abnormal uterine contraction. Two courses of m-ECT markedly diminished her symptoms, and she delivered a healthy infant without complications. M-ECT can be an alternative treatment for pregnant patients with OCD.

Adult↗

PubMed2003

[Anesthetic management with ATP for endovascular thoracic aortic repair].

A 78-year-old man with thoracic aortic aneurysm was scheduled for endovascular stent-graft surgery without cardiopulmonary bypass. He had a history of myocardial infarction five months before and echocardiography revealed ejection fraction of 37%. Deployment of the stent-graft was performed during third degree atrioventricular (A-V) block of 32 seconds produced by adenosine 5'-triphosphate (ATP) 30 mg. There were no complications during this procedure. ATP is a convenient and suitable agent to produce transient A-V block for the precise deployment of the stent-graft in these patients with severe cardiopulmonary complications.

Adenosine Triphosphate↗

PubMed2003

[Retroperitoneal emphysema during total hip replacement].

A 55-year-old woman with rheumatic arthritis was scheduled for total hip replacement. The procedure was performed under combined spinal and epidural anesthesia. Intraoperatively, the part of acetabular roof was cracked by reaming the region but she did not complain of any symptom. However, during pulse irrigation she complained of abdominal pain. After the operation, abdominal distension was noticed and retroperitoneal emphysema was disclosed. She was managed in a ward without problems postoperatively.

Anesthesia, Epidural↗

PubMed2002

Unfolding of quadruplex structure in the G-rich strand of the minisatellite repeat by the binding protein UP1.

The mouse hypervariable minisatellite (MN) Pc-1 consists of tandem repeats of d(GGCAG) and flanked sequences. We have previously demonstrated that single-stranded d(GGCAG)(n) folds into the intramolecular folded-back quadruplex structure under physiological conditions. Because DNA polymerase progression in vitro is blocked at the repeat, the characteristic intramolecular quadruplex structure of the repeat, at least in part, could be responsible for the hypermutable feature of Pc-1 and other MNs with similar repetitive units. On the other hand, we have isolated six MN Pc-1 binding proteins (MNBPs) from nuclear extracts of NIH 3T3 cells. Here, we describe one of those MNBPs, MNBP-B, that binds to the single-stranded d(GGCAG)(n). Amino acid sequences of seven proteolytic peptide fragments of MNBP-B were determined, and the cDNA clones were isolated. MNBP-B was proven identical to the single-stranded DNA-binding protein, UP1. Recombinant UP1 bound to single-stranded d(GGCAG)(n) and other G-rich repetitive sequences, such as d(GTCAGG)(n) and d(GTTAGG)(n). In addition, UP1 was demonstrated by CD spectrum analysis to unfold the intramolecular quadruplex structure of d(GGCAG)(5) and d(TTAGGG)(4) and to abrogate the arrest of DNA synthesis at the d(GGG)(n) site. This ability of UP1 suggests that unfolding of quadruplex DNA is required for DNA synthesis processes.

3T3 Cells↗

PubMed2002

LRP130, a protein containing nine pentatricopeptide repeat motifs, interacts with a single-stranded cytosine-rich sequence of mouse hypervariable minisatellite Pc-1.

Recently, we have identified and purified minisatellite DNA binding proteins (MNBPs) that bind to the mouse hypervariable minisatellite Pc-1, from NIH3T3 cells. This study describes the isolation and characterization of a mouse leucine-rich protein (mLRP130) as one of the MNBPs that binds to the C-rich strand of Pc-1. The mLRP130 cDNA was demonstrated to encode a polypeptide of 1306 amino-acid residues with a deduced molecular mass of 137 kDa, and the mLRP130 mRNA is detected in various organs, including heart, brain, liver, skeletal muscle, kidneys and testes. The mLRP130 protein has nine copies of pentatricopeptide repeat (PPR) motifs that are considered to serve as protein-protein interactions. Two forms of the mLRP130 protein were detected in NIH3T3 cells with an approximate molecular mass of 140 kDa (mLRP130) and 100 kDa (mLRP130der), and were detected mainly in nuclear and cytoplasmic fractions, respectively. Immunofluorescence microscopic analysis demonstrated dominant localization of mLRP130 at the perinuclear region, and also in the nucleus and cytoplasm with dot- or squiggle-like staining. The immunoprecipitated mLRP130 bound to the single-stranded d(CTGCC)8, but not to its complementary G-rich strand of d(GGCAG)8 or double-stranded form. Possible biological roles of mLRP130 are discussed in association with the stability of minisatellite DNA sequences.

3T3 Cells↗

PubMed2002

Skin disinfection before epidural catheterization: comparative study of povidone-iodine versus chlorhexidine ethanol.

BACKGROUND: Chlorhexidine is better than povidone-iodine for skin preparation before intravascular device insertion or blood culture collection, but it is not known whether chlorhexidine is superior in reducing the colonization of continuous epidural catheters. METHODS: Patients requiring an epidural catheter for postoperative analgesia were randomly assigned to receive skin preparation with 0.5% chlorhexidine ethanol (CE group) or 10% povidone-iodine (PI group) before catheter insertion. Using surgical aseptic techniques, catheters were inserted into either the lumbar or the thoracic epidural space. Gloves used at catheter insertion, swabs of insertion site skin and the catheter tip at catheter removal were qualitatively cultured. RESULTS: Of 70 randomly assigned patients, 62 were evaluable. The clinical characteristics of the patients and the risk factors for infection were similar in the two groups. Catheters were kept in place for 49 +/- 7 h (mean +/- SD). Seven cultures from gloves yielded microorganisms. In 1 case, the leak test of gloves was positive. Fifteen cultures of catheter insertion sites yielded microorganisms: 7/28 (25%) in the PI group and 8/34 (24%) in the CE group. Six cultures of catheter tips yielded microorganisms: 3/28 (11%) in the PI group and 3/34 (9%) in the CE group. As for the incidence of isolation of bacteria, no difference was seen between the two groups. In none of these 62 cases was any inflammatory reaction seen in the skin locally at catheter removal. CONCLUSIONS: The effect of 0.5% chlorhexidine ethanol is not different from that of 10% povidone-iodine in reducing catheter colonization associated with short-term epidural catheter placement.

Adult↗

PubMed2002

[Granulomatous polyp in the bronchus caused by anticoagulant therapy].

A 73-year-old woman was scheduled for right lower lobectomy. Her trachea was intubated with a left double-lumen endobronchial tube (Sheribronch, 35 Fr). There was no bleeding at the trachea or the bronchus during the operation. She received intravenous heparin (4000 IU.day-1) for anticoagulant therapy one hour after operation. On the first post-operative day, the mucosal bleeding in the left main bronchus was confirmed, which developed a granulomatous polyp on the 17th post-operative day. The anticoagulant therapy may have worsened slight mucosal damage caused by double lumen tube. It is important to select suitable tube size especially in patients who will receive anticoagulant therapy.

Aged↗