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Biomedical subjects

K Kakinuma

Publications and source records attributed to K Kakinuma.

At least 37 records · Page 2Linked to original sources
PubMed1999

[Development of the quantification method of TTV-DNA and clinical application].

We developed a method to measure the quantity of TTV-DNA. This measurement is based on the principle of the real time PCR method using the TaqMan probe. By measuring the change of the fluorescent intensity caused by FRET, we could detect the amount of TTV-DNA. This method has the characteristics that the possibility of the contamination is very rare when it is compared with the usual PCR method, because the reaction system contains UNG and dUTP. This quantification method is useful for the future research of TTV to study the relationship between this virus and diseases.

Biomarkers↗
PubMed1999

[Virological response to interferon therapy in patients dually infected with TT virus and hepatitis C virus].

In 30 chronic hepatitis C patients co-infected with TTV, TTV DNA in the sera immediately after cessation of IFN and 6 months after the end of IFN was examined. Sustained loss of TTV DNA was observed in 12 of 30 (40.0%) patients. In 7 of 30 (23.3%) patients, TTV DNA re-appeared 6 months after becoming undetectable at the end of IFN therapy. In the remaining 11 patients (36.7%), TTV DNA remained detectable during the entire follow-up period. The ALT values correlated only with the presence of HCV RNA regardless of the effect of IFN on TTV replication. This study indicates that IFN therapy is effective against TTV.

Adult↗
PubMed1999

[Detection rate of TTV-DNA in healthy medical workers].

We examined the positive rate in healthy medical workers about TT virus (TTV) which was a new hepatitis virus reported in 1997. The healthy medical workers showed a positive rate of 24%. Because there was no significant difference of the positive rate between the medical workers and general healthy persons we could not conclude that the positive rate was influenced by their profession. Generally it is known that a positive rate changes according to PCR primers used for the measurement. As for TTV as well, it is reported that a positive rate is greatly dependent on the selected region of primer. Therefore, we must pay attention to the evaluation of the positive rate for each assay system, especially using different primers.

Adult↗
PubMed1998

A novel ether core lipid with H-shaped C80-isoprenoid hydrocarbon chain from the hyperthermophilic methanogen Methanothermus fervidus.

A new ether lipid core (designated as FU) was found in Methanothermus fervidus total lipid. Comparison with caldarchaeol showed lower mobility of FU on TLC and smaller molecular weight (m/z 1298) by 2 mass units on FAB-MS. Treatment of FU with HI followed by displacement with silver acetate afforded long chain alcohol acetate (ROAc), which was further saponified with mild alkali to its free alcohol (ROH). ROH is the long chain alcohol prepared from FU. The molecular weights of ROAc and ROH were shown by MS to be 1354 and 1186, respectively. These results suggested that the molecular formula of ROH was C80H162O4, and ROH had four hydroxyl groups, and one molecule of ROH was bound with two molecules of glycerol by four ether linkages. Because FU was not oxidized by NaIO4 and specific rotation [alpha]D of FU coincided with that of caldarchaeol, it seems that the ether linkages of FU are formed with hydroxyl groups of the sn-2 and sn-3 positions of each glycerol moiety. The structure of FU was suggested to be a modified caldarchaeol in which two hydrocarbon chains are bridged with a covalent bond. Although a few points remain to be elucidated before the final conclusion can be reached on the structure of FU due to difficulty in complete structure determination done even with every approach currently available, the most possible position of the bridge in FU hydrocarbon was proposed from the data of EI-MS of ROAc and 1H-NMR of FU. The hydrocarbon chain looks like H-shaped C80 isoprenoid.

Ether↗
PubMed1998

A novel mutation at a probable heme-binding ligand in neutrophil cytochrome b558 in atypical X-linked chronic granulomatous disease.

A membrane-bound cytochrome b558, a heterodimer consisting of gp91-phox and p22-phox, is a critical component of the superoxide (O2-)-generating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. Chronic granulomatous disease (CGD) is characterized by recurrent bacterial infection caused by a defect of the oxidase. Both subunits are absent from phagocytes in typical X-linked recessive CGD patients who are primarily defective in gp91-phox. We report here an atypical case of X-linked CGD in which neutrophils showed a complete absence of O2--forming NADPH oxidase activity, but a small amount (about 10% of control) of both subunits was detected by immunoblot analysis. Spectrophotometric studies of the neutrophils with a recently developed sensitive method gave no evidence for the heme spectrum in the cytochrome b558, of this CGD. Reverse transcription/polymerase chain reaction and sequence analysis revealed a C to T transition replacing histidine at amino acid position 101 (His101) by tyrosine in gp91-phox. These results provide evidence that His101 of gp91-phox is the one of the heme-binding ligands of cytochrome b558.

Adolescent↗
PubMed1998

Proliferative potential of recurrent intracranial meningiomas as evaluated by labelling indices of BUdR and Ki-67, and tumour doubling time.

This study was designed to provide the reciprocal relationship among labelling indices of 5-bromodeoxyuridine (BUdR LI), Ki-67 (Ki-LI), and tumour doubling time (Td) of recurrent meningiomas. In our series of 182 primary intracranial meningiomas, 46 cases recurred. The average of BUdR LI and Ki LI for nonrecurrent meningiomas were 0.77 +/- 0.13% and 4.71 +/- 1.96%, respectively. Recurrent meningiomas had significantly higher LIs at the first operation: BUdR LI was 3.77 +/- 1.22% and Ki LI was 14.78 +/- 3.17%. The recurrent ratio significantly increased with the degrees of each LI. And the linear regression analysis has demonstrated a significant correlation between BUdR and Ki LI. Td was calculated accurately by NIH, a computer software. Td showed a significant inverse correlation with each of the labelling indices. Consequently, BUdR, Ki LIs and Td of individual tumours correlate mutually well. Of the 46 recurrent cases, 4 received radiation after the operation. Td of the irradiated meningiomas tended to be longer than expected for their higher level of BUdR and Ki LIs before radiation therapy. Thus, it was shown that the radiation therapy delays the regrowth of meningiomas.

Adult↗
PubMed1998

Spectrophotometric determination of neutrophil cytochrome b558 of chronic granulomatous disease.

BACKGROUND: Chronic granulomatous disease (CGD) is an inherited disease characterized clinically by severe recurrent bacterial infections from infancy. This disease is a disorder of the formation of superoxide (O2-) by the neutrophil NADPH oxidase system, mostly due to defects in cytochrome b558 (cyt b558), which is one of the oxidase components. Diagnosis of CGD has been performed by the assay of the O2- forming activity, immunological determination of defects in the oxidase components, and or spectrophotometry of cyt b558. However, spectrophotometric analysis of the b-type heme is difficult with small amounts of blood from infant CGD patients, as the limited amounts of neutrophils are contaminated with a relatively high ratio of hemoglobin (Hb) that interferes with the heme spectrum of cyt b558. This report presents an accurate method for the spectrophotometric analysis of cyt b558 in a small amount of CGD neutrophils that were treated with CO gas in a safe procedure instead of the previously reported CO-bubbling method. METHODS AND RESULTS: The difference of the reduced minus oxidized cyt b558 spectrum was measured under no interference from oxy Hb at the alpha and beta bands and differentiated as d[delta A]/d lambda (lambda = wavelength) to obtain further evidence for the defects of the cyt b558 heme spectrum. The interference from CO-insensitive met Hb was eliminated by subtracting the absorption peak at the Soret (gamma) band of the contaminating met Hb, which was estimated from the CO-treated and untreated spectra of the same, hemolyzed sample. CONCLUSIONS: This spectrophotometric method is feasible for the determination of abnormality and heme content of cyt b558 with a small amount of CGD neutrophils in 10-20 mL of blood even in the presence of contaminating Hb.

Cytochrome b Group↗
PubMed1998

Substrate specificity of 2-deoxy-scyllo-inosose synthase, the starter enzyme for 2-deoxystreptamine biosynthesis, toward deoxyglucose-6-phosphates and proposed mechanism.

A crucial enzyme in the biosynthesis of the 2-deoxystreptamine aglycon of clinically important aminocyclitol antibiotics is 2-deoxy-scyllo-inosose synthase (DOIS), which is responsible for the initial carbocycle formation of 2-deoxy-scyllo-inosose (1) from D-glucose-6-phosphate (G-6-P) (2). To get more insight into the mechanism and substrate specificity, deoxy-D-glucose-6-phosphates (deoxy-G-6-P) were chemically synthesized and subjected to the reaction with DOIS. The enzyme appeared to use 2-deoxy- and 3-deoxy-G-6-P as substrates, both of which were converted into the corresponding dideoxy-scyllo-inosose products, but 4-deoxy-G-6-P failed in cyclization by DOIS. These results clearly support the proposed reaction mechanism involving the initial oxidation at C-4 of the G-6-P substrate. Another implication is the potential use of DOIS for the preparation of useful dideoxyinososes.

Chromatography, High Pressure Liquid↗
PubMed1998

Overproduction and substrate specificity of 3-isopropylmalate dehydrogenase from Thiobacillus ferrooxidans.

We constructed an overexpression system in Escherichia coli of the leuB gene coding for 3-isopropylmalate dehydrogenase in Thiobacillus ferrooxidans. E. coli harboring the plasmid we constructed, pKK leuB1, produced 17-fold the enzyme protein of the expression system previously used for purification. The substrate specificity of the enzyme was analyzed with synthetic (2R, 3S)-3-alkylmalates. The 3-isopropylmalate dehydrogenase of Thiobacillus ferrooxidans had broad specificity toward the alkylmalates.

3-Isopropylmalate Dehydrogenase↗
PubMed1998

Structure and biosynthesis of FD-594; a new antitumor antibiotic.

The structure of a novel antitumor antibiotics FD-594 (1), produced by Streptomyces sp. TA-0256, was determined to have a glycosylated pyrano[4',3':6,7]naphtho[1,2-b]-xanthene skeleton by means of spectral data. The biosynthetic studies of the chromophore of 1 was also carried out by feeding experiments with [1-13C]-, [2-13C]-, and [1,2-13C2]sodium acetate. The labeling pattern was determined by 13C NMR including 2D INADEQUATE experiments, which allowed us to elucidate that the chromophore of 1 is derived from 14 acetate, followed by the loss of one carbon atom.

Antibiotics, Antineoplastic↗
PubMed1997

Saxitoxin as a toxic principle of a freshwater puffer, Tetraodon fangi, in Thailand.

Saxitoxin was identified in a freshwater puffer, Tetraodon fangi, which caused food poisoning in Thailand. Tetrodotoxin, a puffer toxin, was not detected in the species by the HPLC-fluorometric analysis, showing that tetrodotoxin is absent or under any detectable level. The result of this study shows that saxitoxin can be a major toxin in puffer.

Animals↗
PubMed1997

Microspectrophotometry of nitric oxide-dependent changes in hemoglobin in single red blood cells incubated with stimulated macrophages.

A highly sensitive microspectrophotometer was developed to measure spectral changes of oxyhemoglobin (oxy Hb) in single red blood cells (RBC) incubated with stimulated macrophages as a model of nitric oxide (NO) dependent cytotoxicity. Our microspectrophotometer, using a modified acousto-optic tunable filter (AOTF) and a 2-dimensional CCD array, allows fast spectrophotometric data acquisition. Human RBC treated with various concentrations of NO showed spectral changes due to the conversion of oxy Hb to methemoglobin (met Hb), in which the change in absorption differences at alpha (557 590 nm) and beta (542-525 nm) bands showed a linear relationship with the concentration of NO up to 100 microM. In contrast to highly diffusible NO, nitrite ions (NO2-) seem to enter RBC very slowly, resulting in negligible formation of met Hb in the presence of 5 mM glucose even during a prolonged incubation period. RBC were incubated with murine macrophages with and without lipopolysaccharide (LPS) in the presence of glucose for 24 and 40 h and subjected to the microspectrophotometric assay. The RBC incubated with LPS-stimulated macrophages showed significant changes in the spectrum due to NO-dependent conversion of oxy Hb to met Hb, which corresponded to the spectral changes of RBC treated with a several times higher concentration of NO than that in the culture medium. The trapping efficiency was calculated from the amounts of the NO released from macrophages and of the met Hb formed in the RBC, which gave a high efficiency (43%). The results suggest that RBC trap NO directly by cell cell interaction with macrophages. This spectrophotometric system is available for use with just a few drops of samples to study NO-specific cytotoxicity as a model of RBC without the use of any chemical reagent, in parallel with microscopic observations on changes of the cellular morphology under physiological conditions, such as membrane damage leading to hemolysis, adherence, and phagocytosis.

Animals↗
PubMed1997

Kinetic isotope effect and reaction mechanism of 2-deoxy-scyllo-inosose synthase derived from butirosin-producing Bacillus circulans.

The mechanism of 2-deoxy-scyllo-inosose synthase reaction, a carbocycle formation step from D-glucose-6-phosphate in the biosynthesis of the 2-deoxystreptamine aglycon of clinically important aminocyclitol antibiotics, was investigated with a partially purified enzyme from butirosin-producing Bacillus circulans SANK 72073, Nonlabeled and double-labeled D-[4-2H, 3-15O]glucose-6-phosphate were used for cross-over experiment, and the oxime-TMS ether derivative of the 2-deoxy-scyllo-inosose product was analyzed by GC-MS. The deuterium label at C-4 of the substrate appeared to be retained at C-6 of the inosose product without scrambling of the double-labeled isotopes. Since the transient reduction of NAD+ cofactor was proved to be essential in the 2-deoxy-scyllo-inosose reaction, the hydride abstraction and returning appeared to take place within the same glucose molecule. The observed kinetic isotope effect was estimated to be kH/kD = 2.4. These results strongly suggest that this carbocycle formation is catalyzed by a single 2-deoxy-scyllo-inosose synthase enzyme with catalytic requirement of NAD+, the mechanism of which appears to be resembled closely to the 2-deoxy-scyllo-inosose synthase in the Streptomyces fradiae.

Anti-Bacterial Agents↗
PubMed1996

Targeting chemotherapy for malignant brain tumor using thermosensitive liposome and localized hyperthermia.

Thermosensitive liposomes are microscopic vesicles that can contain drugs and release them effectively in response to hyperthermia. To deliver an antitumor drug specifically to brain tumor, the authors used thermosensitive liposomes containing cis-diamminedichloroplatinum (CDDP) in conjunction with localized brain heating. The authors then investigated the antitumor effect on rat malignant glioma. Rous sarcoma virus-induced malignant glioma cells were transplanted into the brains of Fisher rats. Ten days after tumor inoculation, the rats were assigned to one of six treatment groups: control, free CDDP, hyperthermia, free CDDP + hyperthermia, liposomes containing CDDP (CDDP-liposome), and CDDP-liposome + hyperthermia. Liposomes containing CDDP or free CDDP were injected via the tail vein. Brain tumor heating was administered by means of a radiofrequency antenna designed at our institute. The rats treated with CDDP-liposome + hyperthermia had the longest survival time and the tumor CDDP level of this group was the highest when compared to the other groups. Histopathological examination showed that tumor cells were necrotized but surrounding normal brain tissue remained undamaged. On the basis of these findings we suggest that the combination of thermosensitive liposome and localized hyperthermia may better focus antitumor drugs to the tumor, providing a significantly greater antitumor effect.

Animals↗
PubMed1995

Spectroscopic identification of the heme axial ligation of cytochrome b558 in the NADPH oxidase of porcine neutrophils.

The combination of electron paramagnetic resonance (EPR), near-infrared magnetic circular dichroism (NIR-MCD) and resonance Raman (RR) spectroscopies at cryogenic temperatures has been used to identify the axial heme ligation of the low spin cytochrome b558 component of NADPH oxidase from porcine blood neutrophils. The EPR and NIR-MCD results indicate the presence of two distinct forms in frozen solution; one with a low field g-value at 3.23 and porphyrin(pi)-to-Fe(III) charge transfer maximum at 1660 nm and the other a low field g-value at 3.00 and porphyrin(pi)-to-Fe(III) charge transfer maximum at 1510 nm. On the basis of these properties and the RR studies, both are attributed to forms of cytochrome b558 with bis-histidine axial ligation. The origin of the observed heterogeneity, the location and identity of the specific histidines involved in ligating the heme, and the role of the heme prosthetic group in O2- production are discussed in light of these results.

Animals↗
PubMed1995

Electron spin resonance studies on neutrophil cytochrome b558. Evidence that low-spin heme iron is essential for O2-. generating activity.

Cytochrome b558 purified from pig neutrophils was studied to characterize the spin state of the heme iron in relation to its O2-. generating activity. ESR spectra of cytochrome b558 either from resting or stimulated neutrophils showed a low-spin hemoprotein with g1,2,3 of 3.2, 2.1, and 1.3 (estimated). At physiological pH, the oxidized cytochrome b558 is in a purely low-spin state. On lowering or raising pH from 7, the spin state changes to high-spin. The ESR spectrum of high-spin cytochrome b558 was identical to that of methemoglobin, suggesting that the axial-ligand type in both hemoproteins may be the same, i.e. histidine is the fifth ligand. The ratio of the low-spin to high-spin heme in cytochrome b558 was evaluated by magnetic circular dichroism spectroscopy. The pH of cytochrome b558 was varied to form different ratios of the low-spin to high-spin states of the heme, and its O2-. generating activity was examined in cell-free systems. O2-. forming activity decreased concomitant with loss of the low-spin heme, which provides direct evidence that the low-spin state of cytochrome b558 is essential to generate O2-. and the heme retains the low-spin state through the redox cycle.

Animals↗