Biomedical subjects

E Ohtsuka

Publications and source records attributed to E Ohtsuka.

At least 19 recordsLinked to original sources

PubMed2005

Real-time PCR assays based on distinct genomic regions for cytomegalovirus reactivation following hematopoietic stem cell transplantation.

Real-time PCR has many advantages compared with antigenemia and qualitative PCR assays for detecting cytomegalovirus (CMV) infection in patients following SCT. However, the procedure used in each report was not standardized. This study compares the CMV load detected by real-time PCR assays amplifying distinct genomic regions. Real-time PCR assays based on US17, UL65, immediate early protein (IE) and glycoprotein B(gB) were selected and comparisons were made between each genomic region, and with antigenemia and nested PCR (IE region) in 18 SCT patients. The CMV load detected by real-time PCR using all combinations of primers targeting distinct genomic regions and by antigenemia assays correlated well. However, US17 and UL65-PCR could detect CMV earlier than gB-PCR, antigenemia and nested PCR assays. In longitudinal analysis, gB-PCR demonstrated a trend for showing a lower viral load in some patients than US17-, UL65- and IE-PCR. Moreover, the results suggest that a cutoff level of 500 copies/ml might be used to decide when to initiate treatment. We propose that monitoring should be carried out using real-time PCR assays targeting the US17 region and that a CMV load of 500 copies/ml could be used as a cutoff value for initiating treatment in patients following SCT, receiving immunoglobulin prophylaxis.

Adult↗

PubMed2003

Prevalence of Helicobacter pylori in NSAID users with gastric ulcer.

OBJECTIVE: Regarding the interaction of Helicobacter pylori and non-steroidal anti-inflammatory drugs (NSAIDs), we cannot accept unanimous conclusions in inducing gastric ulcer. We therefore evaluated the role of Helicobacter pylori and NSAIDs in inducing gastric ulcer. METHODS: Dyspeptic patients receiving NSAIDs underwent endoscopic examination. Gastric ulcer formation and H. pylori status were investigated. Biopsy specimens from the antrum and lower body of the stomach were prepared for the rapid urease test and pathological evaluation. Anti-H. pylori antibody was measured by enzyme-linked immunosorbent assay. RESULTS: Two hundred and twenty-six patients receiving NSAIDs (220 chronic and six on-demand users) underwent gastrofibrescopic examination. There were 110 patients with gastric ulcer and 111 non-ulcer patients with gastritis. The remaining five patients had neither. NSAID users with gastric ulcer showed a low prevalence of H. pylori compared with those without them [55/110 (50.0%) vs 79/111 (71.2%), P < 0.01]. The same tendency was seen when patients receiving low-dose aspirin and those with rheumatoid arthritis were analysed separately [13/29 (44.8%) vs 50/62 (80.6%), P < 0.01, and 11/33 (33.3%) vs 16/26 (61.5%), P < 0.06 with Yates' correction, respectively]. CONCLUSION: Helicobacter pylori infection appeared to be a risk factor for developing gastritis, but we found no evidence that it increases gastric ulcer formation in NSAID users with dyspepsia.

Aged↗

PubMed2002

Successful bone marrow transplantation from an unrelated donor in a patient with adult T cell leukemia.

We report a 51-year-old male with adult T cell leukemia (ATL) who received a BMT from an HLA-identical unrelated donor. The ATL proved refractory to chemotherapy, and he underwent BMT conditioned with CY/TBI. Complications of encephalitis of unknown origin were successfully treated with steroid therapy and the patient has been in CR for 16 months after BMT. Human T cell leukemia virus type 1 proviral DNA loads were reduced to undetectable levels in PBMC sampled 12 months after BMT. This encouraging result suggests that BMT from an unrelated donor should be considered for ATL even if the disease is refractory to chemotherapy.

Bone Marrow Transplantation↗

PubMed2001

Roles of mast cells and histamine in mosquito bite-induced allergic itch-associated responses in mice.

We investigated itch-associated responses (scratching) to mosquito bites and the role of histamine and mast cells in mosquito-induced itching in mice. Although the first bites of mosquito Aedes albopictus did not increase scratching, repeated bites increased scratching. The response was not diminished even after an interval of 2 months. Similarly, repeated intradermal (i.d.) injections of salivary gland extract (SGE) from Aedes albopictus increased scratching after SGE injection itself and mosquito bites. The scratching peaked within 10 min and almost subsided by 60 min. The opioid antagonist naloxone (1 mg/kg, s.c.) inhibited scratching following SGE injection. Although the non-sedative H1-histamine-receptor antagonist terfenadine (30 mg/kg, p.o.) significantly suppressed scratching induced by histamine (100 nmol/site, i.d.) in either naive or mosquito-sensitized mice, it did not affect mosquito-induced scratching in mosquito-sensitized mice. Repeated injections of SGE increased scratching in mast cell-deficient (WBB6F1-W/Wv) mice as well as in normal (WBB6F1-+/+) littermates. Repeated exposure to mosquito bites roughly doubled serum concentrations of total IgE and IgG1, but not IgG2a. Repeated injections of SGE markedly increased plasma extravasation induced by mosquito bites and such an increase was almost completely suppressed by terfenadine (30 mg/kg, p.o.). The results show the presence of histamine-mediated and histamine-independent mechanisms in cutaneous itching and suggest that histamine probably released from mast cells does not play an important role in itching in immediate allergic reaction. Our murine model of mosquito itching may be useful for studying the mechanisms of immediate allergic itching.

Aedes↗

PubMed2000

A role for MHR1, a gene required for mitochondrial genetic recombination, in the repair of damage spontaneously introduced in yeast mtDNA.

A nuclear recessive mutant in Saccharomyces cerevisiae, mhr1-1, is defective in mitochondrial genetic recombination at 30 degrees C and shows extensive vegetative petite induction by UV irradiation at 30 degrees C or when cultivated at a higher temperature (37 degrees C). It has been postulated that mitochondrial DNA (mtDNA) is oxidatively damaged by by-products of oxidative respiration. Since genetic recombination plays a critical role in DNA repair in various organisms, we tested the possibility that MHR1 plays a role in the repair of oxidatively damaged mtDNA using an enzyme assay. mtDNA isolated from cells grown under standard (aerobic) conditions contained a much higher level of DNA lesions compared with mtDNA isolated from anaerobically grown cells. Soon after a temperature shift from 30 to 37 degrees C the number of mtDNA lesions increased 2-fold in mhr1-1 mutant cells but not in MHR1 cells. Malonic acid, which decreased the oxidative stress in mitochondria, partially suppressed both petite induction and the temperature-induced increase in the amount of mtDNA damage in mhr1-1 cells at 37 degrees C. Thus, functional mitochondria require active MHR1, which keeps the extent of spontaneous oxidative damage in mtDNA within a tolerable level. These observations are consistent with MHR1 having a possible role in mtDNA repair.

Aerobiosis↗

PubMed2000

No genetic association between alpha-2 macroglobulin I1000V polymorphism and Japanese sporadic Alzheimer's disease.

Alpha-2 macroglobulin (A2M) is a serum pan-protease inhibitor that is related with the pathogenesis of Alzheimer's disease (AD) through its ability to mediate amyloid beta degradation. Recently, it has been reported that the I1000V polymorphism in A2M gene might increase the risk of AD. In the present study, we investigated this mutation in 95 healthy controls and in 111 sporadic AD cases by polymerase chain reaction-restriction fragment length polymorphism method in order to study this hypothesis in the Japanese AD population. Allelic frequencies with the I1000V polymorphism in the gene were 7.4 and 6.8% in the control and AD groups, respectively. Our results failed to demonstrate an association between this polymorphism and Japanese sporadic AD, and the A2M I1000V mutation does not seem to be a risk factor per se for sporadic AD.

Aged↗

PubMed2000

Construction of new ribozymes requiring short regulator oligonucleotides as a cofactor.

A hairpin loop and an oligonucleotide bound to the loop form one-half of the pseudoknot structure. We have designed an allosteric hammerhead ribozyme, which is activated by the introduction of this motif by using a short complementary oligonucleotide as a cofactor. Stem II of the hammerhead ribozyme was substituted with a non-self-complementary loop sequence (loop II) to abolish the cleavage activity. The new ribozyme had almost no cleavage activity of the target RNA. However, it exhibited the cleavage activity in the presence of a cofactor oligoribonucleotide, which is complementary to loop II of the ribozyme. The activity is assumed to be derived from the formation of a pseudo-stem structure between the cofactor oligonucleotide and loop II. The structure including the loop may be similar to the pseudo-half-knot structure. The activation efficiencies of the cofactor oligonucleotides were decreased as the lengths of the oligonucleotides increased, and the ribozyme with a longer loop II was more active than that with a short loop II. Oligoribonucleotides with 3'-dangling purine bases served as efficient cofactors of the ribozyme, and a 2'-O-methyloligonucleotide enhanced the cleavage activity of the ribozyme most efficiently, by as much as about 750-fold as compared with that in the absence of the oligonucleotide. Cofactor oligonucleotides with a cytidine base at the 3'-end also activated a ribozyme with the G10.1.G11.1 mutation, which eliminates the cleavage activity in the wild-type. The binding sites of the oligonucleotide were identified by photo-crosslinking experiments and were found to be the predicted sites in the loop. This is the first report of a design aimed at positively controlling the activity of ribozymes by employing a structural motif. This method can be applied to control the activities of other functional RNAs with hairpin loops.

Allosteric Regulation↗

PubMed2000

Crystal structure of the 64M-2 antibody Fab fragment in complex with a DNA dT(6-4)T photoproduct formed by ultraviolet radiation.

DNA photoproducts with (6-4) pyrimidine-pyrimidone adducts formed by ultraviolet radiation are implicated in mutagenesis and cancer, particularly skin cancer. The crystal structure of the Fab fragment of the murine 64M-2 antibody specific to DNA T(6-4)T photoproducts is determined as a complex with dT(6-4)T, a (6-4) pyrimidine-pyrimidone photodimer of dTpT, at 2.4 A resolution to a crystallographic R-factor of 0.199 and an R(free) value of 0.279. The 64M-2 Fab molecule is in an extended arrangement with an elbow angle of 174 degrees, and its five complementarity-determining regions, except L2, are involved in the ligand binding. The bound dT(6-4)T ligand adopting a ring structure with (6-4) linked 5' thymine-3' pyrimidone bases is fully accommodated in an antigen-binding pocket of about 15 Ax10 A. The 5'-thymine and 3'-pyrimidone bases are in half-chair and planar conformations, respectively, and are nearly perpendicular to each other. The 5'-thymine base is hydrogen-bonded to Arg95H and Ser96H, and is in van der Waals contact with Tyr100iH. The 3'-pyrimidone base is hydrogen-bonded to His35H, and is in contact with Trp33H. Three water molecules are located at the interface between the bases and the Fab residues. Hydrogen bonds involving these water molecules also contribute to Fab recognition of the dT(6-4)T bases. The sugar-phosphate backbone connecting the bases is surrounded by residues His27dL, Tyr32L, Ser92L, Trp33H, and Ser58H, but is not hydrogen-bonded to these residues.

Animals↗

PubMed2000

DNA binding mode of the Fab fragment of a monoclonal antibody specific for cyclobutane pyrimidine dimer.

Monoclonal antibodies specific for the cyclobutane pyrimidine dimer (CPD) are widely used for detection and quantification of DNA photolesions. However, the mechanisms of antigen binding by anti-CPD antibodies are little understood. Here we report NMR analyses of antigen recognition by TDM-2, which is a mouse monoclonal antibody specific for the cis - syn -cyclobutane thymine dimer (T[ c, s ]T). (31)P NMR and surface plasmon resonance data indicated that the epitope recognized by TDM-2 comprises hexadeoxynucleotides centered on the CPD. Chemical shift perturbations observed for TDM-2 Fab upon binding to d(T[ c, s ]T) and d(TAT[ c, s ]TAT) were examined in order to identify the binding sites for these antigen analogs. It was revealed that d(T[ c, s ]T) binds to the central part of the antibody-combining site, while the CPD-flanking nucleotides bind to the positively charged area of the V(H)domain via electrostatic interactions. By applying a novel NMR method utilizing a pair of spin-labeled DNA analogs, the orientation of DNA with respect to the antigen-binding site was determined: CPD-containing oligonucleotides bind to TDM-2 in a crooked form, draping the 3'-side of the nucleotides onto the H1 and H3 segments, with the 5'-side on the H2 and L3 segments. These data provide valuable information for antibody engineering of TDM-2.

Animals↗

PubMed2000

Allogeneic bone marrow transplantation-related transmission of human T lymphotropic virus type I (HTLV-I).

We report here the first case report of bone marrow transplantation (BMT)-related transmission of human T lymphotropic virus type-I (HTLV-I). Antibodies against HTLV-I-associated antigens (anti-HTLV-I) were detected in the serum from the BMT recipient 12 days post BMT. IgG against gag core proteins (anti-p19 and anti-p24) appeared earlier than IgM against gag and env proteins (anti-p19, anti-p24 and anti-gp46) during seroconversion. The data presented here differs from blood transfusion-related seroconversion. This phenomenon may be due to the engraftment of anti-HTLV-I producing cells from the donor.

Adolescent↗

PubMed2000

Two-terpyridine.Cu(II) complexes-containing antisense systems for rapid and highly site-specific RNA cleavage.

In an approach toward artificial ribonucleases, novel RNA cleaving systems were constructed that contained two terpyridine.Cu(II) residues. The first antisense system used tandem Cu(II) complex--2'-O-methyloligonucleotide 5'- and 3'-conjugates to cleave an RNA substrate. The second system, which will be described in a future paper, contained two contiguous Cu(II) complex residues at an internal site of a 2'-O-methyloligonucleotide. We found that the first system rapidly cleaved RNA with high site-specificity. Based on these results, we expect the second system to also show efficient RNA cleavage.

Base Sequence↗

PubMed2000

Functional domain-assembly in hairpin ribozymes.

Functional structures of hairpin ribozymes have been investigated by constructing various chemically modified molecules. Domain-exchange and linker insertion experiments were performed to find active conformations of the RNA enzyme showing cleavage activity. Our experiments and other evidence suggest that the active structure has a bent conformation, and that domain-interactions are essential for the cleavage activity.

Nepovirus↗

PubMed2000

[Leukocytopenia and thrombocytopenia preceded by human parvovirus B19 infection: report of three adult cases].

In Saiki City, Oita, Japan, erythema infectiosum in children has been prevalent from June, 1999 to the time of writing (January, 2000). We present three adult cases of parvovirus B19-associated leukocytopenia and thrombocytopenia that developed during this epidemic. Between June and November, 1999, a 32-year-old woman, a 38-year-old woman, and a 63-year-old man were referred to our hospital for treatment of leukocytopenia and thrombocytopenia. All complained of common cold-like symptoms. Their WBC counts (percentage of neutrophils) were 1,000/microliter (70%), 1,900/microliter (40%) and 1,680/microliter (40%), their hemoglobin levels 9.4 g/dl, 9.8 g/dl and 14.9 g/dl, and their platelet counts 10.8 x 10(4)/microliter, 6.9 x 10(4)/microliter and 4.5 x 10(4)/microliter, respectively. The diagnosis of parvovirus B19 infection was documented by the presence of B19-specific IgM antibodies or serum positivity for viral DNA. In two cases, the leukocytopenia and thrombocytopenia resolved gradually. In the other case, leukocytopenia, thrombocytopenia and B19 infection persisted for more than two months. These cases suggest that parvovirus B19 may be a common cause of leukocytopenia and thrombocytopenia even in adult patients without hematological disorders (erythropoietic stress), and that testing for parvovirus infection is justified in such patients, even if anemia is slight, especially when erythema infectiosum is prevalent.

Adult↗

PubMed1999

Investigation of the recognition of an important uridine in an internal loop of a hairpin ribozyme prepared using post-synthetically modified oligonucleotides.

We introduced 4-thio- ((4S)U), 2-thio- ((2S)U), 4- O -methyluridine ((4Me)U) and cytidine substitutions for U+2, which is an important base for cleavage in a substrate RNA. Oligonucleotides containing 4-thio- and 4- O -methyluridine were prepared by a new convenient post-synthetic modification method using a 4- O - p -nitrophenyl-uridine derivative. A hairpin ribozyme cleaved the substrate RNA with either C+2, (4S)U+2 or (4Me)U+2 at approximately 14-, 6- and 4-fold lower rates, respectively, than that of the natural substrate. In contrast, the substrate with a (2S)U+2 was cleaved with the same activity as the natural substrate. These results suggest that the O4 of U+2 is involved in hydrogen bonding at loop A, but the O2 of U+2 is not recognized by the active residues. Circular dichroism data of the ribozymes containing (4S)U+2 and (2S)U+2, as well as the susceptibility of the thiocarbonyl group to hydrogen peroxide, suggest that a conformational change of U+2 occurs during the domain docking in the cleavage reaction. We propose here the conformational change of U+2 from the ground state to the active molecule during the reaction.

Circular Dichroism↗

PubMed1999

Genetic association between alpha-2 macroglobulin and Japanese sporadic Alzheimer's disease.

Alpha-2 macroglobulin (encoded by the gene A2M) is a serum pan-protease inhibitor that may be related with the pathogenesis of Alzheimer's disease (AD) because of its ability to mediate amyloid beta degradation. Recently, several groups have reported that the five-nucleotides deletion in A2M gene at the 5' splice site of exon 18 might increase risk for AD. In the present study, therefore, this mutation was studied in 69 healthy controls and 55 sporadic AD cases by polymerase chain reaction- restriction fragment length polymorphism method. The allelic frequencies with the deletion (A2M-2) are 9.4 and 6.4% in the control and AD groups, respectively. There is no significant difference in the A2M-2 frequencies between the controls and sporadic AD cases. This is the first report to study the frequencies of A2M-2 in Japanese AD cases, suggesting its no genetic association with sporadic AD.

Adult↗

PubMed1999

Conformational multiplicity of the antibody combining site of a monoclonal antibody specific for a (6-4) photoproduct.

The antigen binding site of monoclonal antibody 64M5, which possesses a high degree of affinity for DNA containing pyrimidine (6-4) pyrimidone photoproducts, were investigated by use of stable-isotope-assisted NMR spectroscopy. A variety of 64M5 Fab fragments specifically labeled with 13C and 15N at backbone amide groups were prepared. Extensive assignments of amide resonances originating from the variable region of 64M5 were made by using 2D-HN(CO) measurements along with recombination of the heavy and light chains of 64M5. On the basis of chemical shift changes of the amide resonances caused upon addition of d(T[6-4]T) and d(GTAT[6-4]TATG), the binding sites of 64M5 Fab for the (6-4) photodimer and for the oligodeoxynucleotides flanking it were identified. It was revealed that the L1 and L3 segments, which are responsible for the binding to (6-4) photodimer, exhibit conformational multiplicities in the absence of antigens, and take different conformations between the d(T[6-4]T) and d(GTAT[6-4]TATG)-bound forms. On the basis of spectral comparison with another Fab fragment with a similarity in the amino acid sequence of the VL domain of 64M5, we suggest that the conformational multiplicities observed in the present study is caused by a substitution of an amino acid residue at the position of a key residue in L3 canonical structure, which leads to a preferable effect on the antigen binding, and by a specific combination of L1 and L3 canonical structures.

Amino Acid Sequence↗

PubMed1999

Human MMH (OGG1) type 1a protein is a major enzyme for repair of 8-hydroxyguanine lesions in human cells.

8-Hydroxyguanine (8-OH-G) is the site of a frequent mutagenic lesion of DNA, produced by oxidative damage. MutM of E. coli and OGG1 of Saccharomyces cervisiae are known to possess 8-OH-G glycosylase activity and apurinic (AP) site lyase activity to repair 8-OH-G lesions. Recently, cDNA clones of human OGG1 homologues (hMMH) of four isoforms (type 1a, type 1b, type 1c, and type 2) were isolated. However, it is unknown whether expression of endogenous hMMH proteins actually occurs in mammalian cells. Here using hMMH type 1a-specific antibody and cells overexpressing tag-fused hMMH type 1a, we show the expression of hMMH type 1a protein in many types of human cells and show that endogenous hMMH type 1a protein has 8-OH-G glycosylase/AP lyase activity. Furthermore, we show that upon depletion of hMMH type 1a protein in a whole cell extract by its antibody, most of the AP lyase activity is lost, indicating that hMMH type 1a protein is a major enzyme for repair of 8-OH-G lesions in human cells.

Antibodies↗

PubMed1999

Probing the interaction between a high-affinity single-chain Fv and a pyrimidine (6-4) pyrimidone photodimer by site-directed mutagenesis.

We have used site-directed mutagenesis to uncover the origin of the binding affinity differences exhibited by a series of monoclonal antibodies that recognize pyrimidine (6-4) pyrimidone photoproducts in the context of single- or double-stranded DNA. In this study, we have focused on two antibodies-64M3 and 64M5-that share the same binding specificity but differ in their affinities. We used single-chain Fv (scFv) derivatives for these studies since they can be easily expressed in Escherichia coli. To facilitate this, we also developed a simple, on-column refolding procedure for scFvs that is rapid and does not require high dilution. We took several precautions to ensure that the scFvs faithfully reflected the behavior of the parent monoclonal antibodies. Results obtained from chimeric scFvs constructed from 64M3 and 64M5 suggested that the higher affinity of the 64M5 antibody was mainly due to its VL region. Loop-grafting studies in which VH CDR loops of 64M3 were individually transplanted into 64M5 were consistent with this hypothesis. Since the VL sequences of 64M3 and 64M5 differ at only three positions (L30, L50, and L90), alanine-scanning mutagenesis was used to assess the importance of these three residues in DNA binding by 64M5. These studies highlighted the importance of all three VL CDR loops; furthermore, they suggested that photoproduct binding involved conformational changes within the VL region.

Alanine↗