Biomedical subjects

K Itagaki

Publications and source records attributed to K Itagaki.

At least 19 recordsLinked to original sources

PubMed2000

Double ventricular response by a single ventricular extrastimulus to the inner loop of reentry in a patient without apparent heart disease.

In a patient without apparent heart disease, a ventricular extrastimulus delivered from the left ventricular apex where the electrogram was recorded 30 ms after the onset of the QRS complex during VT advanced the second QRS complex, but not the first QRS complex. The morphology of the second QRS complex was the same as that of VT. The postpacing interval was the same as the cycle length of the VT. These findings indicated that the site of stimulation was at the inner loop of the reentry circuit of the VT. A ventricular extrastimulus with a shorter coupling interval advanced the first and second QRS complexes, indicating that the ventricle was activated by antidromic and orthodromic activation from the extrastimulus. Radiofrequency ablation at that site of stimulation terminated the VT and no further VT could be induced.

Aged↗

PubMed1999

Methionine S-oxidation in human and rabbit liver microsomes: evidence for a high-affinity methionine S-oxidase activity that is distinct from flavin-containing monooxygenase 3.

Methionine has previously been shown to be S-oxidized by flavin-containing monooxygenase (FMO) forms 1, 2, and 3. The most efficient catalyst was FMO3, which has a Km value for methionine S-oxidation of approximately 4 mM, and exhibits high selectivity for formation of the d-diastereoisomer of methionine sulfoxide. The current studies provide evidence for an additional methionine S-oxidase activity in liver microsomes. Human and rabbit liver microsomes exhibited a biphasic response to methionine at concentrations ranging from 0.05 to 10 mM, as indicated by both Eadie-Hofstee plots and nonlinear regression. The low-affinity component of the biphasic response had Km values of approximately 3 and 5 mM for humans and rabbits, respectively, as well as high diastereoselectivity for methionine sulfoxide formation. The low-affinity activity in rabbit liver microsomes was inhibited by methimazole, S-allyl-l-cysteine, and by mild heat treatment, suggesting the activity is FMO3. The high-affinity component of the biphasic response had Km values of approximately 0.07 and 0.04 mM for humans and rabbits, respectively, as well as lower diastereoselectivity for methionine sulfoxide formation. Further characterization of the high-affinity activity in rabbit liver microsomes indicated lack of involvement of cytochrome P450s or reactive oxygen species. The high-affinity activity was inhibited 25% by potassium cyanide and greater than 50% by methimazole and S-allyl-l-cysteine. Mild heat treatment produced 85% inhibition of the low-affinity activity, but only 30% inhibition of the high-affinity activity. Both high- and low-affinity activities were decreased by 85% in flavin-depleted microsomes. Because these results suggested the additional S-oxidase activity has characteristics of an FMO, recombinant human FMO4 was evaluated as a potential catalyst of this activity. Recombinant FMO4 catalyzed S-oxidation of both methionine and S-allyl-l-cysteine, with similar diastereoselectivity to the high-affinity microsomal S-oxidase; however, the Km values for both reactions appeared to be greater than 10 mM. In summary, these studies provide evidence for two microsomal methionine S-oxidase activities. FMO3 is the predominant catalyst at millimolar concentrations of methionine. However, at micromolar methionine concentrations, there is an additional S-oxidase activity that is distinct from FMO3.

Adult↗

PubMed1999

The human T cell leukemia virus type I-tax gene is responsible for the development of both inflammatory polyarthropathy resembling rheumatoid arthritis and noninflammatory ankylotic arthropathy in transgenic mice.

We previously reported that inflammatory arthropathy resembling rheumatoid arthritis (RA) develops among transgenic mice carrying the long terminal repeat (LTR)-env-pX-LTR region of human T cell leukemia virus type I (LTR-pX-Tg mice). Because four genes are encoded in this region, we produced transgenic mice that only express the tax gene to examine its role in the development of arthritis. Transgenic mice were produced by constructing DNAs that express the tax gene alone under the control of either its own LTR or CD4 enhancer/promoter and by microinjecting them into C3H/HeN-fertilized ova. We produced seven transgenic mice carrying the LTR-tax gene and nine mice carrying the CD4-tax and found that one of the LTR-tax-Tg mice and five of CD4-tax-Tg mice developed RA-like inflammatory arthropathy similar to LTR-pX-Tg mice, indicating that the tax gene is arthritogenic. On the other hand, the other two LTR-tax-Tg mice had ankylotic changes caused by new bone formation without inflammation. In these ankylotic mice, tax mRNA, inflammatory cytokine mRNA, and autoantibody levels except for TGF-beta1 level were lower than those in LTR-pX- or CD4-tax-Tg mice. These results show that Tax is responsible for the development of inflammatory arthropathy resembling RA and that this protein also causes ankylotic arthropathy.

Animals↗

PubMed1999

Species and sex differences in expression of flavin-containing monooxygenase form 3 in liver and kidney microsomes.

Flavin-containing monooxygenase (FMO) 3 is the predominant FMO isoform in adult human liver; however, little is known about its expression in common laboratory species. Studies have shown FMO3 levels to be sex-dependent in mouse liver, but not in human liver. The current study was undertaken to determine the expression of FMO3 in liver and kidney microsomes from multiple species, and to determine whether the sex dependence seen in mouse liver extends to other species and/or tissues. FMO3 had previously been shown to be the major FMO involved in methionine S-oxidation in rat and rabbit liver microsomes. In this study, species differences in FMO3 levels were assessed in liver microsomes from humans, rats, dogs, mice, and rabbits, and in kidney microsomes from rats, dogs, mice, and rabbits, by comparing methionine S-oxidase activities. Species differences were noted in male liver microsomes, with rabbits having 3-fold higher methionine S-oxidase activity than mice and dogs and 1.5-fold higher activity than humans and rats. Species differences were also noted in male and female kidney microsomes, with rats exhibiting 2- to 6-fold higher methionine S-oxidase activity than the other species. Sex differences in FMO3 levels were assessed using methionine S-oxidase activity and immunoblotting, and were noted only in liver microsomes from mice and dogs, with females having higher levels than males. Results also show that FMO3 orthologs from multiple species are catalytically similar with regard to methionine, S-allyl-L-cysteine, and S-(1,2-dichlorovinyl)-L-cysteine S-oxidations.

Animals↗

PubMed1998

The development of autoimmune inflammatory arthropathy in mice transgenic for the human T cell leukemia virus type-1 env-pX region is not dependent on H-2 haplotypes and modified by the expression levels of Fas antigen.

Previously, we reported that human T cell leukemia virus type-1 env-pX region-introduced transgenic (pX-Tg) mice develop an inflammatory polyarthropathy. Although autoimmune pathogenesis was suggested, the detailed mechanisms remain to be elucidated. In this report, we examined effects of the MHC and fas genes on the development of the disease. When pX-Tg mice were backcrossed with different inbred strains, the incidence of arthritis differed among strains; 64% and 72% in BALB/cAn (H-2d), 25% and 46% in C3H/HeN (H-2k), and 0% and 2% in C57BL/6J (H-2b) background at 3 and 6 months of age, respectively. Rheumatoid factor levels in the serum correlated with the susceptibility to the disease, whereas IL-1beta and MHC gene expression were similarly elevated in all of these strains, suggesting involvement of immune regulatory genes in this strain difference. However, introduction of the H-2d locus into C57BL/6J pX-Tg mice did not increase the incidence of arthritis, and substitution of the BALB/cAn H-2 locus with the H-2b did not decrease it. The results indicate that the H-2 locus is not the major determinant of the disease. Then, since previous study indicated a defect in Fas-mediated apoptosis of transgenic T cells, the effects of fas gene modification on the disease were examined. The incidence increased when these pX-Tg mice were crossed with lpr/lpr mice, while it decreased when crossed with fas-transgenic mice. These observations suggest that aberration of Fas-mediated apoptosis of peripheral lymphocytes, rather than negative selection in the thymus, is involved in the development of autoimmune arthropathy in pX-Tg mice.

Animals↗

PubMed1998

Isoform specificity of trimethylamine N-oxygenation by human flavin-containing monooxygenase (FMO) and P450 enzymes: selective catalysis by FMO3.

In the present study, we expressed human flavin-containing monooxygenase 1 (FMO1), FMO3, FMO4t (truncated), and FMO5 in the baculovirus expression vector system at levels of 0.6 to 2.4 nmol FMO/mg of membrane protein. These four isoforms, as well as purified rabbit FMO2, and eleven heterologously expressed human P450 isoforms were examined for their capacity to metabolize trimethylamine (TMA) to its N-oxide (TMAO), using a new, specific HPLC method with radiochemical detection. Human FMO3 was by far the most active isoform, exhibiting a turnover number of 30 nmol TMAO/nmol FMO3/min at pH 7.4 and 0.5 mM TMA. None of the other monooxygenases formed TMAO at rates greater than 1 nmol/nmol FMO/min under these conditions. Human fetal liver, adult liver, kidney and intestine microsomes were screened for TMA oxidation, and only human adult liver microsomes provided substantial TMAO-formation (range 2.9 to 9.1 nmol TMAO/mg protein/min, N = 5). Kinetic studies of TMAO formation by recombinant human FMO3, employing three different analytical methods, resulted in a Km of 28 +/- 1 microM and a Vmax of 36.3 +/- 5.7 nmol TMAO/nmol FMO3/min. The Km determined in human liver microsomes ranged from 13.0 to 54.8 microM. Therefore, at physiological pH, human FMO3 is a very specific and efficient TMA N-oxygenase, and is likely responsible for the metabolic clearance of TMA in vivo in humans. In addition, this specificity provides a good in vitro probe for the determination of FMO3-mediated activity in human tissues, by analyzing TMAO formation at pH 7.4 with TMA concentrations not higher than 0.5 mM.

Chromatography, High Pressure Liquid↗

PubMed1998

Photochemical identification of transmembrane segment IVS6 as the binding region of semotiadil, a new modulator for the L-type voltage-dependent Ca2+ channel.

To identify the binding domain of a new Ca2+ antagonist semotiadil on L-type Ca2+ channels from skeletal muscle, photolabeling was carried out by using an azidophenyl derivative of [3H]semotiadil. Photoincorporation was observed in several polypeptides of membrane triad preparations; the only specific photoincorporation was in the alpha1 subunit of the Ca2+ channel. After solubilization and purification, the photolabeled alpha1 subunit was subjected to proteolytic and CNBr cleavage followed by antibody mapping. Specific labeling was associated solely with the region of transmembrane segment S6 in repeat IV. Quantitative immunoprecipitation was found in the tryptic and the Lys-C/Glu-C fragments of 6.6 and 6.1 kDa, respectively. Further CNBr cleavage of the Lys-C digests produced two smaller fragments of 3.4 and 1.8 kDa that were included in the tryptic and Lys-C/Glu-C fragments. The smallest labeled fragments were: Tyr1350-Met1366 and Leu1367-Met1381 containing IVS6, a possible pore-forming region. The data suggest that semotiadil binds to a region that is overlapped with but not identical to those for phenylalkylamines, dihydropyridines and benzothiazepines. The present study also provides evidence that region IV represents an important component of a binding pocket for Ca2+ antagonists.

Animals↗

PubMed1996

Expression and characterization of a modified flavin-containing monooxygenase 4 from humans.

The inability to obtain flavin-containing monooxygenase 4 (FMO4) in heterologous systems has hampered efforts to characterize this isoform of the FMO gene family. Neither the human nor the rabbit ortholog of FMO4, each of which has been cloned and sequenced, has been expressed. Attempts to achieve expression of FMO4 have been made with Escherichia coli, baculovirus, yeast, and COS systems. The cDNAs encoding FMO4 have extended coding regions compared with those encoding other FMO isoforms. The derived amino acid sequences of FMO1, -2, -3, and -5 from all species examined contain about the same number of residues (531-535 residues), whereas the derived sequences of human and rabbit FMO4 contain 558 and 555 residues, respectively. We have investigated whether the elongation of the FMO4 coding region is related to the inability to achieve expression. The cDNA encoding human FMO4 has been modified by a single base change that introduces a stop codon at the consensus position. This modification allows for expression in E. coli. Lack of expression of intact FMO4 is caused by a problem that occurs following transcription, a problem that is overcome completely by relocation of the stop codon 81 bases to 5' of its normal position. Truncated FMO4 is expressed as an active enzyme with characteristics typical of an FMO isoform. Possible functional changes resulting from altering the 3'-end of an FMO were investigated with human FMO3. Elongation of the coding region of the FMO3 cDNA to the next available stop codon (FMO3*) resulted in the expression of an enzyme with properties very similar to those of unmodified FMO3. Elongation of FMO3 lowered the level of expression in E. coli but did not eliminate it. As with FMO4, the difference in expression levels between FMO3 and elongated FMO3 (FMO3*) appears to be related to translation rather than transcription. The functional characteristics of FMO3 and FMO3* are not significantly different.

Amino Acid Sequence↗

PubMed1995

Autoimmunity induction by human T cell leukemia virus type 1 in transgenic mice that develop chronic inflammatory arthropathy resembling rheumatoid arthritis in humans.

We recently reported on an inflammatory arthropathy resembling rheumatoid arthritis that develops in high incidence among transgenic mice that carry the env-pX region of the human T cell leukemia virus type 1 genome. In an effort to elucidate the pathogenesis of this disease, we found that genes for inflammatory cytokines, including IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, transforming growth factor-beta 1, IFN-gamma, and IL-2, as well as MHC genes were activated in transgenic joints. Serum levels of IL-1 beta and IL-6 were also elevated. Interestingly, these mice produced Ab against IgG, type II collagen (IIC), and heat shock proteins accompanied by IgG hypergammaglobulinemia. The cellular immune response to IIC as well as that to heat shock proteins were activated. Moreover, these mice became immunologically responsive to exogenously administered IIC and developed arthritis, in contrast to their nontransgenic littermates, which showed little response to IIC. Taken together, the results suggest that human T cell leukemia virus type 1 can cause immune system hyperreactivity and induce autoimmunity. The possibility that elevated cytokine and/or MHC gene expression are involved in the development of autoimmunity and arthropathy are discussed.

Animals↗

PubMed1995

Augmentation of c-fos and c-jun expression in transgenic mice carrying the human T-cell leukemia virus type-I tax gene.

To analyze the effect of human T-cell leukemia virus type I (HTLV-I) on cellular gene expression and its relation to tumorigenesis, two lines of transgenic mice carrying the long terminal repeat (LTR)-env-pX-LTR regions of the HTLV-I genome were produced. The transgene was expressed in many organs, including the brain, salivary gland, spleen, thymus, skin, muscle, and mammary gland. We found that the expression of the c-fos and c-jun genes, but not of the lyn and c-myc genes, was augmented 2- to 20-fold in histologically normal skin and muscle of these mice. The augmentation was tissue specific, suggesting the involvement of a cellular factor in the transgene action. In these mice, a three to seven times higher incidence of tumors was seen as compared with the control mice. These tumors included mesenchymal tumors, such as fibrosarcoma, neurofibroma, and lipoma, and adenocarcinomas of the mammary gland, salivary gland, and lung. The c-fos and c-jun genes were also activated in these tumors. The possible roles of elevated c-fos and c-jun gene expression in tumorigensis are discussed.

Animals↗

PubMed1994

[Evaluation of the effective use of the "health notebook"].

Since 1983, with the institution of the "Health Service Law for the Aged", the "health notebook" has been issued to people aged 40 years and over in order to aid in management of their health. Few people actually fill their health data in notebook by themselves. In order to develop effective use of the health notebook by residents and health professionals, the uses of the health notebooks by residents aged 40 years and over, public health nurses, and physicians were investigated. Three hundred and fifty four residents aged 40 and over, 41 public health nurses, and 18 physicians were studied in 1990, in Yamagata city. A majority of residents took their health notebooks with them to health consultations, and public health nurses used the notebooks to provide advice to them. Public health nurses effectively issued the health notebooks to residents using occasions where residents gathered. Some physicians reported that health notebooks were useful for motivating the people to maintain their health, while others preferred using a health card media. When comparing the health notebooks to the maternity passbooks, health notebooks need to be more easily utilized by users for recording information, and their value should be effectively explained to them. Furthermore, in order to promote self-care behaviors, greater use of health notebooks by all health professionals in indicated.

Adult↗

PubMed1993

Azidobutyryl clentiazem, a new photoactivatable diltiazem analog, labels benzothiazepine binding sites in the alpha 1 subunit of the skeletal muscle calcium channel.

[3H]Azidobutyryl clentiazem, a new photoactivable diltiazem derivative, has a higher binding affinity than azidobutyryl diltiazem. It can be covalently incorporated into the alpha 1 subunit of the skeletal muscle calcium channel by UV irradiation, which allows the benzothiazepine binding site to be determined. The photolabeled alpha 1 subunit and its proteolytic fragments were analyzed with a panel of sequence-directed antibodies. The results suggest that the linker region between segment S5 and S6 of domain IV is involved in benzothiazepine binding. This site is different from the dihydropyridine and verapamil binding sites.

Affinity Labels↗

PubMed1993

Identification of 1,4-dihydropyridine binding domains within the primary structure of the alpha 1 subunit of the skeletal muscle L-type calcium channel.

Calcium channel blockers are drugs that bind to the alpha 1 subunit of L-type calcium channels and selectively inhibit ion movements through these channels. Determination of the mechanism of channel blockade requires localization of drug-binding sites within the primary structure of the receptor. In this study the 1,4-dihydropyridine-binding site of the membrane bound receptor has been identified. The covalently labeled receptor was purified and digested with trypsin. The labeled peptide fragments were immunoprecipitated with sequence-directed antibodies. The data indicate the existence of at least three distinct dihydropyridine-binding domains within the primary structure of the alpha 1 subunit.

Affinity Labels↗

PubMed1993

Inflammatory polyarthritis in mice transgenic for human T cell leukemia virus type I.

OBJECTIVE: We have recently reported that arthropathy develops in high incidence among transgenic mice carrying the pX region of human T cell leukemia virus type I (HTLV-I). In the present study, the histopathologic features of the joints in these mice were examined in order to compare the animal disease with rheumatoid arthritis (RA) in humans. METHODS: Paraffin sections of limbs (right and left fingers, wrists, elbows, shoulders, toes, knees, and ankles) were stained with hematoxylin and eosin, periodic acid-Schiff, azan-Mallory, or phosphotungstic acid hematoxylin, and examined by light microscopy. RESULTS: Abnormalities of the limbs began to occur as early as 3 weeks of age, and the incidence gradually increased until the mice were 12 months old. The incidence of arthropathy was 22% (48 of 217) at 3 months of age and 28% (18 of 64) at 6 months. The severity of the histopathologic changes in the joints of the transgenic mice ranged from grade I to grade IV. CONCLUSION: The major histopathologic features in the joints of HTLV-I transgenic mice are similar to those in humans with RA. Thus, these mice may represent a useful model for the study of the disease in humans.

Animals↗

PubMed1992

Evidence for the existence of RNA of Ca(2+)-channel alpha 2/delta subunit in Xenopus oocytes.

Ba(2+)-currents (IBa) through voltage-dependent Ca(2+)-channels were studied in Xenopus oocytes injected with RNA from several excitable tissues, using the two-electrode voltage-clamp technique. Previous studies have shown that the expression of cardiac Ca(2+)-channels can be suppressed by an hybrid-arrest procedure that includes co-injection of the tissue-derived RNA with an 'antisense' oligonucleotide complementary to a part of RNA coding for the Ca(2+)-channel alpha 1 subunit. In this study, this method was used to investigate the role of the alpha 2/delta subunit. Co-injection of RNA extracted from either rabbit heart, rat brain or rat skeletal muscle (SkM) with 'antisense' oligonucleotides complementary to the alpha 2/delta subunit RNA did not substantially affect the expression of IBa in the oocytes. Using the Northern blot hybridization method, it was shown that native oocytes contain large amounts of alpha 2/delta subunit RNA of Ca(2+)-channel. It is proposed that te oligonucleotide treatment fails to eliminate the alpha 2/delta RNA because of the vast excess of endogenous alpha 2/delta RNA. These results impose a limit on the use of the hybrid-arrest method.

Animals↗

PubMed1992

Native-type DHP-sensitive calcium channel currents are produced by cloned rat aortic smooth muscle and cardiac alpha 1 subunits expressed in Xenopus laevis oocytes and are regulated by alpha 2- and beta-subunits.

Native tissue-like L-type voltage-dependent calcium channels (L-VDCC's) were expressed by in vitro transcribed cRNA injection of rat aorta or rabbit cardiac alpha 1 subunit into Xenopus laevis oocytes. Co-injection of VSM-alpha 1 with the cloned skeletal muscle beta-subunit (SK-beta) of the L-type VDCC significantly increased the expressed peak current amplitude without significant changes in kinetics. Similar results were obtained by co-injection of cardiac alpha 1 (DSHT-alpha 1) the cloned skeletal alpha 2-subunit (SK-alpha 2) or with SK-beta. The oocytes co-expressing cRNA's retained L-type VDCC pharmacology.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1992

Beta-subunit expression is required for cAMP-dependent increase of cloned cardiac and vascular calcium channel currents.

In contrast to vascular smooth muscle (VSM), cAMP-dependent phosphorylation increases L-type voltage dependent Ca(2+)-channel (L-VDCC) activity in heart. To investigate whether this difference depends on the tissue-specific alpha 1-subunit of the L-VDCC or its regulation by other subunits, we used a Xenopus laevis oocyte expression system. Injection of cAMP into oocytes expressing cardiac alpha 1 or VSM alpha 1 alone had no effect on L-VDCC activity. However, cAMP increased L-VDCC activity 2-fold in oocytes co-expressing cardiac alpha 1 or VSM alpha 1 with the skeletal muscle beta-subunit. These results suggest that the presence of the beta-subunit is required for cAMP-mediated increase of L-VDCC activity and that the characteristics of tissue-specific beta-subunits may explain differential regulation of L-VDCC activity.

Animals↗

PubMed1990

[Investigation on clinical efficacy and passage into ascites of cefminox in diffuse peritonitis associated with infantile acute appendicitis].

Cefminox (CMNX), one of newly developed cephamycin antibiotics, was administered to 7 cases of diffuse peritonitis associated with infantile acute appendicitis to determine its concentrations in the blood, the appendiceal tissue and the purulent ascites and simultaneously to investigate its clinical efficacy. CMNX was intravenously injected at a dose of 20 mg/kg (at a maximum amount of 1.0 g/body) before operation and intravenously by bolus injection or by drip infusion twice daily after operation for 3 to 11 days in a total dose of 2.36 to 14.06 g. Fourteen strains of bacteria were isolated from the purulent ascites: Escherichia coli was isolated from 6 cases but superinfections in 5 cases. MICs of CMNX against these isolated organisms were at or lower than 3.13 micrograms/ml for 12 out of 14 strains. CMNX penetrated into the appendiceal tissue and the purulent ascites very well. The concentrations in the pus reached higher than those in the tissue in about 1 hour after administration and were found to reach as high as 9.63 micrograms/ml in 5 hours after administration. Its clinical efficacies were excellent in 4 cases, good in 2 cases and poor in 1 case. No subjective or objective adverse reactions were observed nor any abnormalities were found in laboratory examinations.

Acute Disease↗