Biomedical subjects

H Ide

Publications and source records attributed to H Ide.

At least 19 recordsLinked to original sources

PubMed1992

A novel, sensitive, and specific assay for abasic sites, the most commonly produced DNA lesion.

Free radicals produce a wide spectrum of damages; among these are DNA base damages and abasic (AP) sites. Although several methods have been used to detect and quantify AP sites, they either are relatively laborious or require the use of radioactivity. A novel reagent for detecting abasic sites in DNA was prepared by reacting O-(carboxymethyl)hydroxylamine with biotin hydrazide in the presence of carbodiimide. This reagent, called Aldehyde Reactive Probe (ARP), specifically tagged AP sites in DNA with biotin residues. The number of biotin-tagged AP sites was then determined colorimetrically by an ELISA-like assay using avidin/biotin complex conjugated to horseradish peroxidase as the indicator enzyme. With heat/acid-depurinated calf thymus or bacteriophage f1 DNA, ARP detected femtomoles of AP sites in DNA. Using this assay, DNA damages generated in calf thymus, phi X174 RF, and f1 single-stranded DNA, X-irradiated in phosphate buffer, were easily detectable at 10 rad (0.1 Gy). Furthermore, ARP sites were detectable in DNA isolated from heat-inactivated X-irradiated (10 Gy) and methyl methanesulfonate (MMS)-treated (5 microM) Escherichia coli cells. The rate of production of ARP sites was proportional to the X-ray dose as well as to the concentration of MMS. Thus, the sensitivity and simplicity of the ARP assay should provide a potentially powerful method for the quantitation of AP sites or other DNA lesions containing an aldehyde group.

Animals↗

PubMed1992

Purification of serine hydroxymethyltransferase from Bacillus stearothermophilus with ion-exchange high-performance liquid chromatography.

The gene of serine hydroxymethyltransferase (SHMT) of a thermophilic bacterium Bacillus stearothermophilus was expressed in Escherichia coli, and SHMT was successfully purified from the crude extract of E. coli in two steps while maintaining the enzymatic activity. The purification steps involved ammonium sulphate precipitation followed by high-performance liquid chromatographic separation using the anion-exchange column Fractogel EMD DEAE-650(S). In addition to the DEAE column, three other types of anion- and cation-exchange columns were also studied for their ability to separate SHMT, and the performance of the four columns were compared.

Ammonium Sulfate↗

PubMed1992

Properties of a monoclonal antibody for the detection of abasic sites, a common DNA lesion.

The abasic site is one of the most frequent changes occurring in DNA and has been shown to be lethal and mutagenic. An abasic site in DNA can be tagged by reaction with O-4-nitrobenzylhydroxylamine (NBHA), resulting in the formation of an oxime linkage between the abasic site and the NBHA moiety. In order to measure NBHA-tagged abasic sites, a monoclonal antibody was elicited against a 5'-phosphodeoxyribosyl O-4-nitrobenzyl hydroxylamine-BSA conjugate. The antibody was specific for the NBHA residue as demonstrated by hapten inhibition, with IC50 values for 5'-phosphodeoxyribosyl-NBHA, deoxyribosyl-NBHA, ribosyl-NBHA and NBHA of 0.3 microM, 5 microM, 5 microM and 7 microM, respectively. Other haptens examined, including benzylhydroxylamine, 5'-phosphodeoxyribosyl-, deoxyribosyl-, and ribosyl-benzylhydroxylamine, showed no inhibition even at 1 mM. The antibody showed high specificity for NBHA-modified AP sites in DNA and exhibited no cross reactivity with normal DNA bases, otherwise-modified DNA bases or unmodified AP sites. Using a direct ELISA assay, the antibody detected 1 AP site (after NBHA-modification) per 10,000 base-pairs or approximately 10 femtomoles of AP sites in DNA. DNA lesions were detectable in 60Co gamma-irradiated DNA at a dose as low as 10 rad (0.1 Gy) and the production of antibody detectable sites was proportional to the gamma-ray dose. Since NBHA reacts with lesions containing an aldehyde group, the simplicity and sensitivity of the antibody assay should provide a useful method for the quantitation of AP sites or other DNA lesions containing an aldehyde group.

Animals↗

PubMed1992

Early diagnosis of tuberculosis by fibreoptic bronchoscopy.

We carried out a retrospective study of the methods used to achieve an early diagnosis of 67 patients with pulmonary tuberculosis treated at our institute between 1984 and 1989. Sputum bacteriology was positive in 56 of the 67 patients, 22 were positive on microscopical examination of smears and on culture and 34 on culture alone. The 11 patients with negative sputum bacteriology were all diagnosed by fibreoptic bronchoscopy. In addition, 21 of the 34 smear-negative/culture-positive patients were examined by fibreoptic bronchoscopy and the initial diagnosis was made in 7 of these. Thus the initial diagnosis was made by sputum bacteriology in 49 cases and by fibreoptic bronchoscopy in 18 cases. The median number of days between obtaining a specimen and starting therapy was 7 days for sputum microscopy, 41 days for sputum culture, 7 days for microscopic examination of bronchoscopy specimens, 51 days for culture of the same and 19 days for biopsy. Fibreoptic bronchoscopy is therefore useful for the diagnosis of cases of tuberculosis in which tubercle bacilli are not detected in sputum and, in some instances, for an earlier diagnosis of smear-negative/culture-positive patients.

Adult↗

PubMed1992

Effect of naloxone on focal cerebral ischemia in cats.

We have examined the effects of a single injection of naloxone (5 mg/kg, i.v.) in cats with cerebral ischemia produced by transorbital occlusion of the middle cerebral artery (MCA). Cerebral blood flow (CBF) was measured and the cerebral metabolic rate of oxygen consumption (CMRO2) was estimated based on measurements of arteriovenous (A-V) oxygen difference. Six cats were treated with naloxone 30 minutes after occlusion and 8 were treated 2 hours after occlusion. In 6 control animals, naloxone produced a 10-15% increase in mean arterial blood pressure (MABP), CBF and CMRO2 lasting 30 minutes. MCA occlusion reduced CBF by 70-75% in the ipsilateral MCA territory and by 15% in the contralateral hemisphere. Naloxone increased CBF by 3.5-6% in the ischemic region and 10-22% in the contralateral hemisphere in both treatment groups to the same extent as seen in control animals. There was no significant change in A-V oxygen difference and the estimated increase in CMRO2 of non-ischemic regions of both treatment groups was similar to that of control animals. These effects were transient and lasted 15-60 minutes. We have concluded that naloxone caused a transient increase in cerebral metabolism which equals or exceeds the corresponding increase in CBF. Therefore, naloxone would not be beneficial, and may be detrimental in the treatment of cerebral ischemia.

Animals↗

PubMed1992

Significance of the serum CA125 level in recurrent ovarian cancer.

Thirty-four recurrent cases of 89 patients with ovarian cancer treated in our department between 1985 and 1989 were examined for changes in serum CA125 level. 1) Upon confirmation of recurrence, 17 patients were positive for CA125 and 15 were negative. 2) According to the histopathological type, the rate of CA125 positivity in patients with recurrence was high for serous adenocarcinoma, suggesting that determination of CA125 is useful for detection of recurrence. In contrast, all patients with mucinous adenocarcinoma or endometrioid adenocarcinoma were negative for CA125. 3) The CA125 positivity rate upon confirmation of recurrence was 9% in patients whose CA125 was less than 1,000 U/ml on initial examination, suggesting that close management of such patients is necessary. 4) Elevation of CA125 by 3 steps or more within the normal range (less than 35 U/ml) was useful for predicting recurrence. 5) The cut-off level of CA125 during follow-up should be set at 16 U/ml. 6) It was difficult to evaluate remission with only the serum CA125 level. It is impossible to avoid second look operation at present. These results indicate that pretreatment values and changes of the parameter within the normal range (less than 35 U/ml) have to be considered when using CA125 as a marker of tumor recurrence.

Adenocarcinoma↗

PubMed1992

[A case of Candida albicans endocarditis with impaired lung function].

A 53-year-old male was admitted to the hospital because of Candida albicans endocarditis. He had had a thoracoplasty due to pulmonary tuberculosis and showed severe restructive lung function. In 1987 and '89, trachiostomy was made because of respiratory failure. The patient was well until nine months earlier, when he consulted a physician because of fever. The investigations failed in finding the cause of the fever. He was administered antituberculosis agents and antiinflammatory drugs but had a fever every day. Two months before admission, a cardiac ultrasonographic study showed evident vegetations with mitral regurgitation. From the above course and examinations, a diagnosis of Candida albicans endocarditis was made. Infusions of CEZ, TOB, PIPC and miconazole for more than one month was ineffective. In November, 1990, he was referred to our medical center for the purpose of operation. A blood culture proved Candida albicans infection. An intravenous administration of fluconazole 400 mg/day was begun. However, there was pulmonary bleeding probably due to heparin used for prevention of atrial thrombosis and he developed fever, hypoexemia, ventricular tachycardia, and hyponatremia. He underwent mitral-valve replacement with a SJM valve. Culture of the vegetated mitral valve again proved Candida albicans. After operation, hypoexemia, ventricular tachycardia, hyponatremia were improved gradually. However he had an eosinophilia, eruption, and dyspnea. We suspected a drug eruption of fluconazole. Lymphocyte stimulating test of fluconazole proved positive. After the episode, he had no symptoms and was discharged.

Candidiasis↗

PubMed1992

Inflammatory cytokines and enzymes in synovial fluid of patients with rheumatoid arthritis and other arthritides.

Cytokines and lysosomal enzymes, which are produced by inflammatory cells, play a role in inflammation. We have found that synovial fluid (SF) in rheumatoid and septic arthritis contained a large number of white blood cells (WBCs) and high levels of cytokines and enzymes, while in contrast the SF of osteoarthritis and traumatic arthritis did not contain significant amounts. Measurements of WBCs, cytokines and enzymes in SF are useful for evaluating clinical disease activity. Assays for WBCs and enzymes are simple and rapid when compared to those for cytokines.

Adult↗

PubMed1992

Effect of leukotriene antagonist on experimental delayed cerebral vasospasm.

Experimental delayed cerebral vasospasm was produced in 16 adult mongrel dogs by the "two-hemorrhage" method of intracisternal injections of autologous arterial blood. Group 1 was a control group. Group 2 was a treatment group that received an intravenous injection of ONO-1078, a novel potent leukotriene antagonist, once a day for 7 days just after the first cisternal injection of the blood. Angiography was performed on Days 0 and 7, and the cerebrospinal fluid levels of leukotriene C4 (LTC4) were measured on Days 0, 3, and 7. The cisternal levels of LTC4 increased after subarachnoid hemorrhage in both groups. But the cerebrospinal fluid levels of LTC4 in the treatment group were significantly lower than those in the control group (P less than 0.05). The angiographic vasospasm after subarachnoid hemorrhage was partially prevented with the treatment of intravenous injections of ONO-1078 (P less than 0.001). These results suggest that LTC4 may play a role in the pathogenesis of delayed cerebral vasospasm, directly or indirectly, and ONO-1078 may have a therapeutic effect on the prevention of the development of delayed cerebral vasospasm.

Animals↗

PubMed1992

An artificial blood vessel with an endothelial-cell monolayer.

An artificial blood vessel with an endothelial-cell monolayer was used as an arterial substitute in rats. Endothelial cells were isolated from the aorta of a Wistar rat by the digestion method. The cell identification was established by the cobblestone appearance of a confluent cell monolayer, by an expression of factor VIII-related antigen, and by the presence of Weibel-Palade bodies. The luminal surface of the thin-walled polytetrafluoroethylene (PTFE) graft (4 mm in diameter and 10 mm in length) was coated with an endothelial-cell monolayer for 7 days in vitro. An interpositional graft was placed using the endothelial cell-coated PTFE prosthesis on the right common carotid artery in seven rats. A total of 10 rats received an interpositional graft with the noncoated PTFE prosthesis as a control. The patency rate at 1 month after implantation was significantly higher in the coated group than in the control group. The vascular prosthesis with an endothelial-cell monolayer is a promising technique to inhibit the development of thrombosis.

Animals↗

PubMed1992

Morphogenetic roles of retinoic acid.

We integrated our information on morphogenetic roles of retinoic acid, focusing on development of chick limb. Retinoic acid has been considered to be a putative morphogen released from the ZPA. However, since exogenous retinoic acid induces expression of RAR beta, but not grafted ZPA, the ZPA is unlikely to produce retinoic acid. From the result that retinoic acid-treated cells induce digit duplication, retinoic acid converts anterior cells into ZPA cells. We found that retinoic acid also induces expression of homeobox genes indirectly and that bFGF enhances expression of the homeobox genes. Thus, we considered that retinoic acid and growth factors cooperate with each other to activate homeobox genes. A morphogenetic role of retinoic acid is to coordinate developmental stage of each cells by controlling expression of homeobox genes.

Animals↗

PubMed1992

Interaction of antisense DNA with nucleic acids/proteins.

In order to study interaction of various types of labeled antisense DNAs were prepared. Fluorescein and 2,2,6,6-tetramethypiperidine-N-oxyl were the label molecules, which were introduced to 5'-end of oligonucleotides and their analogs. Interactions of labeled antisense DNAs with nucleic acids or proteins such as HSA, HIG and TF, were studied by UV, fluorescence depolarization spectroscopy, and ESR spectroscopy. Hybrid formation of antisense DNAs with oligonucleotides in solution could be monitored by the increase in fluorescence anisotropy (r) and by intensity change in ESR spectra. When phosphorothioate type antisense molecules anchoring fluorescein (F-OPT) were mixed with proteins, r drastically increased, whereas ODN slightly increased. These results suggest that OPTs have much more affinity for proteins than ODNs.

Blood Proteins↗

PubMed1992

Syntheses and properties of fluorescent labeled oligonucleotides containing deoxyethenoadenosine at 5' end.

The fluorescent labeled oligodeoxyribonucleotides which contain deoxyethenoadenosione (d epsilon A) at their 5' end were prepared by treating CPG bound oligonucleotides with 5'-DMTr-deoxyethenoadenosine-3'-H-phosphonate. The hybrid formation of d epsilon A-oligonucleotide with its complementary DNA was studied by fluorescence spectroscopy. The fluorescence of d epsilon A in a single strand was largely quenched by stacking interaction with the base at 3' position. When d epsilon A-oligonucleotides hybridized with their complementary strands, relative fluorescence quantum yields (Qrel) against d epsilon A changed in specific manners. These results suggest that d epsilon A-oligonucleotides are applicable to study the local structure of DNA in solution.

Base Sequence↗

PubMed1992

Separation and characterization of diastereoisomeric oligonucleotide.

For developing the antisense method, two types of oligonucleotides suitable for antisense molecules were explored for their essential properties. One is oligonucleoside phosphorothioate, and all their possible R/S diastereoisomers were separated and purified by reversed phase liquid chromatography. Isolated diastereoisomers have been investigated for the correlation between their configuration and hybridization manner. Separation, R/S characterization and spectroscopical properties of these oligonucleotides will be discussed.

Chromatography, High Pressure Liquid↗

PubMed1992

Effects of base damages on DNA replication--mechanism of preferential purine nucleotide insertion opposite abasic site in template DNA.

DNA polymerase preferentially inserts purine nucleotides opposite non-instructive lesions such as abasic sites during DNA replication. In order to elucidate the mechanism of the preferential insertion, a DNA template containing a model abasic site and primers containing 4 different nucleotides (A,G,C,T) at primer terminus were synthesized. The stability of the primer terminus nucleotide placed opposite the abasic site was evaluated on the basis of its sensitivity to 3'-5' exonuclease associated with DNA polymerase.

DNA↗

PubMed1992

NMR studies on solution structure of single-stranded oligonucleotides causing line broadening.

Unusual line broadening of 1H-NMR lines attributable to the proton (8H) of guanine residues was observed for all tetradeoxyoligonucleotides tested here which have a specific base sequence of dGXXG (X = A or T). For the same samples, line broadening was also obtained in the 31P-NMR spectra. These broadened signals did not become sharp up to 60 degrees C. This unusual spectral phenomenon has been attributed by 2D-NMR and differential NOE to the compact solution structure of the oligonucleotides.

Magnetic Resonance Spectroscopy↗

PubMed1992

Sequence-specific DNA recognition by basic peptides.

A chiral template with C2 symmetry has been used for modeling a dimeric interface of DNA binding protein. An oligopeptide derived from the basic region of MyoD, a recently described "helix-loop-helix" class of DNA binding protein, has been tethered to the template. Among the four models which differ in chirality and polarity with respect to the arrangement of two subunits, only one dimer model with right-handed and C-terminus to C-terminus arrangement of the peptide subunits binds DNA containing native MyoD binding sequence.

DNA↗

PubMed1992

Monomer-dimer equilibrium of nitroso-aromatic spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid, sodium salt (DBNBS), in aqueous solution.

3,5-Dibromo-4-nitrosobenzenesulfonic acid, sodium salt (DBNBS), which is widely used as a spin trap in biological systems, was allowed to form the monomer-dimer equilibrium in aqueous solution, and its equilibrium constant (Kc) was determined by NMR measurement and visible spectrum analysis. The Kc values obtained from the two different methods were virtually identical and evaluated as 1.3 x 10(-3) mol dm-3 at 25 degrees C. Further, the molar extinction coefficient of DBNBS monomer (epsilon 760) was determined to be 34 dm3mol-1cm-1. The present results indicate that only a minor portion (ca. 20%) of dissolved DBNBS (10 mmol dm-3) is practically capable of spin trapping under the conditions most widely used in biological studies.

Benzenesulfonates↗