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Biomedical subjects

E Ohtsuka

Publications and source records attributed to E Ohtsuka.

At least 55 records · Page 3Linked to original sources
PubMed1998

Specificities and rates of binding of anti-(6-4) photoproduct antibody fragments to synthetic thymine photoproducts.

Pyrimidine (6-4) pyrimidone photoproducts are some of the major DNA photolesions induced by ultraviolet (UV) light. A monoclonal antibody (64M5) specific to a (6-4) photoproduct has been established and the corresponding single-chain antibody (64M5scFv) has been prepared. In this study, we characterized the ligand selectivities of 64M5 and 64M5scFv using synthetic octadeoxynucleotides containing either a central cis-syn cyclobutane thymine dimer (T[c,s]T), the (6-4) photoproduct of TpT (T[6-4]T), or its Dewar isomer (T[Dewar]T) by means of enzyme-linked immunosorbent assays (ELISA). Both 64M5 and 64M5scFv recognized T[6-4]T, but not the other photoproducts. We synthesized several biotinylated oligonucleotides of different lengths containing (T[6-4]T) to analyze the effects of the antigen size on the binding rates of an antigen binding fragment (64M5Fab) and 64M5scFv by means of surface plasmon resonance. The association rate constants for oligonucleotides of different sizes containing T[6-4]T as to 64M5Fab were found to be almost the same (1.9-5.6 x 10(5) M(-1) x s(-1)), while the dissociation rate constant for the largest oligonucleotide (d8mer, 8.0 x 10(-5) s(-1)) was significantly smaller than that for the d2mer (4.2 x 10(-2) s(-1)). These results indicate that 64M5Fab recognized the d2mer as the epitope and that the binding affinity for T[6-4]T depended on the flanking oligonucleotides. The dissociation rate constants for 64M5scFv as to the antigen analogs were almost the same as those for the various T[6-4]T-oligonucleotides as to 64M5Fab, suggesting that the conformations of these antibody binding regions are pretty similar to each other.

Antibody Specificity↗
PubMed1998

Modification and application of self-trimming hairpin ribozymes to targeting a transcribed RNA in vitro.

The three-domain hairpin ribozyme was improved by increasing the number of linker bases connecting domain I' with domain II from six to seven, and a new ribozyme was designed to release a trans-acting ribozyme that is able to bind with target RNAs by three hybridizing arms. The trans-cleavage activities of the trimmed ribozyme were used to cleave a long target RNA, with 85 bases transcribed from a synthetic gene encoding induced nitric oxide synthase (iNOS). Although the ribozyme with three-hybridizing arms did not efficiently cleave the long target RNA, the overall activity, from self-trimming to the trans-cleavage reaction of the target RNA, was higher than that of the ribozyme with two hybridizing arms.

Base Sequence↗
PubMed1998

The role of surface lysines in pyrimidine (6-4) pyrimidone photoproduct binding by a high-affinity antibody.

The roles of four VH surface lysine side-chains in antibody recognition of DNA containing a central pyrimidine (6-4) pyrimidone photoproduct was investigated. Three of the four lysines (at positions H62, H64 and H66) are strongly conserved; however, the presence of lysine at position H68 was highly unusual. Each lysine was replaced individually by alanine; in addition, a quadruple mutant was also created. Studies of the binding kinetics revealed that these lysine residues mainly influenced the association phase. Our results suggest that these side chains help to guide the DNA polyanion to the antigen binding pocket by electrostatic effects.

Antibody Affinity↗
PubMed1998

[Lymphoma associated hemophagocytic syndrome with plasmacytosis in the bone marrow and hypergammaglobulinemia].

A 72-year-old woman was admitted to our hospital because of fever, anemia and thrombocytopenia in March 1997. Laboratory findings showed elevated serum LDH levels and polyclonal gammopathy. Bone marrow aspiration samples revealed hemophagocytosis and plasmacytosis. Although serum interleukin-6 was elevated, serum interferon-lambda and tumor necrosis factor-alpha were below detectable limits. Magnetic resonance images disclosed a tumor in the patient's pelvic cavity. The tumor was resected and diagnosed as non-Hodgkin's lymphoma. The patient was treated with combination chemotherapy and has remained in complete remission. Also, histiocyte and plasma cell counts in the bone marrow fell significantly and the serum interleukin-6 level returned to the normal range. We reasoned that lymphoma cells may have induced plasmacytosis in the bone marrow and polyclonal gammopathy accompanied by hemophagocytic syndrome.

Aged↗
PubMed1997

Antigen structural requirements for recognition by a cyclobutane thymine dimer-specific monoclonal antibody.

A monoclonal antibody (TDM-2) specific to a UV-induced cyclobutane pyrimidine dimer (T[cis-syn]T) has previously been established; however,the immunization had used UV-irradiated calf-thymus DNA containing a heterogeneous mixture of photoproduct sites. We investigated here the structural requirements of antigen recognition by the antibody using chemically synthesized antigen analogs. TDM-2 bound with cis-syn,but not trans-syn thymine dimer,and could bind strongly with four nucleotide analogs in which the cis-syn pyrimidine dimer was located in the center. Antigen analogs containing abasic linkers at the 5'- or 3'-side of the cis-syn cyclobutane pyrimidine dimer were synthesized and tested for binding to TDM-2. The results indicated that TDM-2 recognizes not only the cyclobutane ring but also both the 5'- and 3'-side nucleosides of the cyclobutane dimer. Furthermore,it was proved that either the 5'- or 3'-side phosphate group at a cyclobutane dimer site was absolutely required for the affinity to TDM-2. The antibody showed a strong binding to single stranded DNA but indicated little binding to double stranded DNA.

Animals↗
PubMed1997

A new type of hairpin ribozyme consisting of three domains.

We have constructed a new hairpin ribozyme with three stem-loop domains. In the ribozyme, another domain (domain I') was connected to the 3'-end of domain II of the parent hairpin ribozyme, and the new ribozyme can be trimmed after transcription from the DNA template using T7 RNA polymerase. Since a mutant ribozyme containing a substitution at the essential base in domain I' lacked the 3'-trimming reaction, the autoprocessing activity was proved to be derived from the catalytic reaction, similar to the wild-type ribozyme. Furthermore, the structure of the cleavage site from the self-trimming reaction was identified as a 2',3'-cyclic phosphate, which is the same as that of the wild-type. The processed ribozyme was designed to cleave an external substrate RNA derived from the mRNA of the human inducible nitric oxide synthase and was proved to cleave at the expected, unique site. The hairpin ribozyme containing the three-domains exhibited the 3'-self-trimming activity even in a runoff transcription reaction from the plasmid harboring the ribozyme gene with the three domains. The new type of hairpin ribozyme thus obtained has three stem-loop domains and is able to act as a catalytic RNA for both cis and trans cleavage. These ribozymes are of interest from the point of the structure-function relationship of the hairpin ribozyme and provide an important insight into over understanding of the role of the domain-domain interaction in the catalytic activity.

Base Sequence↗
PubMed1997

A recurrent translocation, t(16;21)(q24;q22), associated with acute myelogenous leukemia: identification by fluorescence in situ hybridization.

Chromosome fluorescence in situ hybridization (FISH) analyses were performed on bone marrow cells in 3 adult patients with MDS or AML with a (16;21)(q24;q22) translocation. FISH analyses with AML1 probes at 21q22 proved in all 3 patients splitting of the AML1 gene at a region spanning exons 5 and 6 and the translocation of its 5' segment to distal 16q. Chromosome painting FISH analysis in patient 1 proved the translocation of the distal 21q segment to 16q, but it failed to prove the presumed translocation of the distal 16q segment to 21q, most likely because of its small size.

Adult↗
PubMed1997

Solution structure of an RNA.2'-O-methylated RNA hybrid duplex containing an RNA.DNA hybrid segment at the center.

The solution structure of an RNA.2'-O-methylated RNA hybrid duplex containing an RNA.DNA hybrid segment at its center, (ggagaugac).(GmUmCmATCTCmCm), where lowercase letters, capital letters, and capital letters with the subscript m are RNA, DNA, and 2'-O-methylated RNA residues, respectively, was determined by observing the NMR spectra and performing the full relaxation matrix refinement. The 2'-O-methylation gives several characteristic features to oligoribonucleotides. In addition, this hybrid duplex is cleaved at a specific position by Escherichia coli ribonuclease HI, and so the role of the tertiary structure during the substrate recognition by the enzyme is of interest. The NOE connectivities among the proton resonances revealed that the duplex was a right handed helix. The 2'-O-methylated RNA segments had a typical C3'-endo conformation, and the 2'-O-methyl groups were directed to the minor groove of this duplex, taking the torsion angles phi (C1'-C2'-02'-CH3) that were all gauche(+). The DNA residues in the central RNA.DNA hybrid duplex formed the C3'-endo conformation, except for the middle thymine residue. No remarkable structural discontinuities were observed around the junction sites at either the 5'- or 3'-end of the DNA. The overall structure was close to the typical A-form duplex.

Crystallography, X-Ray↗
PubMed1997

Flavin adenine dinucleotide as a chromophore of the Xenopus (6-4)photolyase.

Two types of enzyme utilizing light from the blue and near-UV spectral range (320-520 nm) are known to have related primary structures: DNA photolyase, which repairs UV-induced DNA damage in a light-dependent manner, and the blue light photoreceptor of plants, which mediates light-dependent regulation of seedling development. Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)photoproducts] are the two major photoproducts produced in DNA by UV irradiation. Two types of photolyases have been identified, one specific for CPDs (CPD photolyase) and another specific for (6-4)photoproducts [(6-4)photolyase]. (6-4)Photolyase activity was first found in Drosophila melanogaster and to date this gene has been cloned only from this organism. The deduced amino acid sequence of the cloned gene shows that (6-4)photolyase is a member of the CPD photolyase/blue light photoreceptor family. Both CPD photolyase and blue light photoreceptor are flavoproteins and bound flavin adenine dinucleotides (FADs) are essential for their catalytic activity. Here we report isolation of a Xenopus laevis(6-4)photolyase gene and show that the (6-4)photolyase binds non- covalently to stoichiometric amounts of FAD. This is the first indication of FAD as the chromophore of (6-4)photolyase.

Animals↗
PubMed1997

Behçet's disease associated with myelodysplastic syndromes. A case report and a review of the literature.

BACKGROUND: Behçet's disease has rarely been reported in association with myelodysplastic syndromes (MDS). Increased production of reactive oxygen species (ROS) by neutrophils has a primary role in the pathogenesis of Behçet's disease. However, decreased production of ROS by neutrophils has frequently been reported in patients with MDS. The current study was undertaken to determine the role of ROS production in a patient with Behçet's disease and MDS. METHODS: A patient with MDS with trisomy 8 who developed Behçet's disease is described and a review of the literature of patients with Behçet's disease in MDS is presented. The production of ROS by neutrophils was investigated by luminol-enhanced chemiluminescence (CL) assay. RESULTS: Based on a review of the literature, 10 cases of Behçet's disease associated with MDS have been reported to date. Nine patients had undergone cytogenetic analysis of bone marrow cells, 7 of whom (78%) had trisomy 8. Neutrophils taken from the authors' patient during the active phase of Behçet's disease demonstrated an increased CL response. Moreover, serum from this patient increased the CL emission of neutrophils from healthy volunteers. CONCLUSIONS: These data suggest that trisomy 8 predisposes to Behçet's disease in patients with MDS. Furthermore, an increased ROS production by neutrophils may be associated with the diverse clinical findings in this disease. In this study, neutrophils were activated directly by serum factors.

Adult↗
PubMed1997

Construction of hairpin ribozymes with a three-way junction.

Hairpin ribozymes with high cleavage activities were designed. An extra sequence was introduced at the 3'-end of the hairpin ribozyme to increase the binding to the substrate RNA, as compared to the wild-type hairpin ribozyme. A three-way junction (TWJ) was formed between the newly designed ribozyme and the substrate RNA. The complex with a solid TWJ showed less RNA cleavage activity than the wild-type hairpin ribozyme. However, the ribozyme with a TWJ with five unpaired bases or propandiol phosphate linkers had higher cleavage activity than the parent ribozyme without the TWJ. When a cis-cleavage system, in which the 5'-end of the substrate RNA was conjugated to the 3'-end of the ribozyme, was employed, the complex with the TWJ containing unpaired bases was also cleaved faster than the complex with the solid TWJ. This suggested that these differences in the cleavage activities were derived from the confirmation, and this was proven by nondenaturing gel electrophoresis. The TWJ hairpin ribozyme containing unpaired bases is able to bind strongly with substrate RNAs and to cleave them efficiently. Since the three-way ribozyme presented here is more active than the wild-type ribozyme, this type of ribozyme can serve as a more efficient tool to control RNA activities in vitro and in vivo.

Base Sequence↗
PubMed1997

Identification of specific amino acid residues of adenovirus 12 E1A involved in transformation and p300 binding.

The early region 1A (E1A) gene of highly oncogenic adenovirus type 12 (Ad12) was analyzed for transforming activity and protein binding using specific mutations. The Ad12 E1A proteins were found to bind p300 protein mainly within the CR1 region, although mutations that affect both p300 binding and transformation were identified in both the CR1 and the N-terminal region. The most critical mutation dlf89 located in the CR1 region was further dissected by point mutations and the results identified 68S as the most critical for transformation and 67E as the most critical for p300 binding. Specific mutations that retain p300 binding but impair transcriptional repression of a viral enhancer were also identified in both the N-terminal and CR1 regions.

Adenovirus E1A Proteins↗
PubMed1997

Hemolytic uremic syndrome following autologous peripheral blood stem cell transplantation in a patient with malignant lymphoma.

Hemolytic uremic syndrome (HUS) has been reported in patients receiving bone marrow transplantation. However, only a few cases of HUS following autologous peripheral blood stem cell transplantation (PBSCT) have been reported. We present a case of HUS developing after autologous PBSCT in a 40-year-old man with non-Hodgkin's lymphoma. It appears that the chemotherapeutic agents administered during the conditioning regimen for PBSCT may have played an important role in the development of HUS in our patient. In the present case, the combination therapy of vincristine, methylprednisolone, and ticlopidine hydrochloride was effective.

Adult↗
PubMed1997

[Diffuse alveolar hemorrhage associated with idiopathic interstitial pneumonia syndrome after allogeneic bone marrow transplantation].

A 24-year-old male with myelodysplastic syndrome (refractory anemia) was treated with allogeneic bone marrow transplantation (BMT) from HLA-identical brother on September 9, 1994, after conditioning with busulfan and cyclophosphamide. From early in April 1995, the patient complained of cough and fever. Chest X-ray showed interstitial infiltrates involving hilar and lower lung fields. Although pulmonary infiltrates were resolved with methylprednisolone pulse therapy, on May 19, the patient rapidly deteriorated with dyspnea. Diffuse alveolar infiltrates appeared on chest X-ray and he died of respiratory failure. At autopsy, diffuse alveolar hemorrhage accompanied with thickening of the alveolar walls and interstitial infiltration of lymphocytes was demonstrated. There was no evidence of bleeding in other organs. This is a rare case of diffuse alveolar hemorrhage in the late stage of allogeneic BMT. This report should lead to an evaluation of this disease by the collection of similar cases.

Adult↗
PubMed1997

Mechanistic studies on the neighboring base damage induced by KMnO4 oxidation of 8-oxoguanine in DNA.

New types of DNA substrates containing an 8-oxoguanine residue (8-oxo-G) were prepared in order to examine the mechanisms for the neighboring base damage initiated by KMnO4 oxidation of the 8-oxo-G. The results obtained from the reactions suggested that the damage at remote sites in the single strands can be explained by an electronic interaction (redox reaction) between an oxidized 8-oxo-G species and the base (to be damaged), which are close each other in a loop structure. For the inefficient damage observed in duplex substrates, electron transfer through stacked bases might be involved.

Base Sequence↗
PubMed1997

Preliminary studies for interaction of a cyclobutane thymine dimer region with its cognate antibody.

A monoclonal antibody (TDM-2) specific to a UV-induced cyclobutane pyrimidine dimer has previously been established. We investigated here the structural requirements of antigen recognition by the antibody using chemically synthesized antigen analogs. TDM-2 bound to the cis-syn, but not the trans-syn thymine dimer, and recognized not only the cyclobutane ring but also the 5'- or 3'-side phosphate groups flanking the cyclobutane dimer site. The antibody showed strong binding to photodimer-containing single stranded DNA but indicated little binding to double stranded DNA.

Antibodies, Monoclonal↗
PubMed1996

Interaction of the basic protrusion of Escherichia coli ribonuclease HI with its substrate.

In order to determine the actual distance between the active site and the substrate binding site, termed the basic protrusion, of Escherichia coli ribonuclease HI, synthetic oligonucleotide duplexes with gradually extended overhangs were used, in which the enzymatic cleavage was restricted to a single site with 2'-O-methylnucleosides. The affinity of the enzyme for each substrate was determined by kinetic analysis. It was found that the affinity increased markedly when one nucleotide was attached to the 3' end of the DNA strand of the nine-base-pair hybrid duplex and then increased slightly as the DNA strand was extended further, whereas elongation of the strand in the other direction caused no change. When a mutant enzyme, in which three lysine residues in the basic protrusion were altered to alanine, was used, no increase in the kcat/K(m) value was observed. The results indicate that, for the productive binding, the axis from the 3' to the 5' end of the RNA strand of the substrate duplex must be oriented in agreement with the direction from the active site to the basic protrusion of the enzyme. The distance between the active site and the basic protrusion in the enzyme-substrate complex was shorter than that anticipated in modeling studies. A dynamic structure refinement, referred to as the normal mode analysis, was carried out in order to simulate the fluctuations of the basic protrusion.

Escherichia coli↗