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Biomedical subjects

T Kitagawa

Publications and source records attributed to T Kitagawa.

At least 217 records · Page 12Linked to original sources
PubMed1995

Determination of urinary acetylpolyamines by a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA).

A monoclonal antibody (mAb), ASPM-2, produced against N-(gamma-maleimidobutyryloxy)-succinimide (GMBS)-conjugated polyamine spermine [Spm; Fujiwara et al. (1994) Histochemistry 102, 397-404] was used for the development of an enzyme-linked immunosorbent assay (ELISA) for acetylpolyamines (Ac-PAs) in human urine. The ELISA is based on the principle of competition between an analyte and Spm-glutaraldehyde-bovine serum albumin conjugate-coated polystyrene microtiter wells for the mAb, followed by immunoreaction with biotinylated anti-mouse immunoglobulin and horseradish peroxidase-streptavidin. The ASPM-2 mAb showed strong immunoreaction with N1,N12-diacetylspermine (2Ac-Spm), N-monoacetylspermine (Ac-Spm), and N1-acetylspermidine (N1-Ac-Spd), the EC50 values being 29, 50, and 51 microM, respectively, but no cross-reaction with other PA-related compounds or amino acids. The method was used to measure urinary Ac-PA levels in healthy subjects and cancer patients, without pretreatment of the specimens, mean concentrations of 3.25 and 2.80 mumol per 24-h urine, respectively (as N1-Ac-Spd), being found. The ASPM-2 ELISA for N1-Ac-Spd, which is the PA most relevant to the analysis of human urine among the three Ac-PAs mentioned above, is specific and accurate, and can easily be used to analyze large numbers of specimens in parallel. It should thus have potential for studying the relationship between urinary N1-Ac-Spd levels and cancer.

Animals↗
PubMed1995

A tumor-associated glycosylation change in the glucose transporter GLUT1 controlled by tumor suppressor function in human cell hybrids.

Studies of human cell hybrids have provided evidence that the tumorigenicity of a cervical carcinoma (HeLa) is under the control of a putative tumor suppressor on chromosome 11. Using these human cell hybrids, we found a tumor-associated glycosylation change in the glucose transporter GLUT1, which is an N-linked glycoprotein at the plasma membrane. The non-tumorigenic HeLa x fibroblast cell hybrid CGL1 and the normal diploid fibroblast WI38 expressed the 50-55 kDa GLUT1, whereas in a tumorigenic segregant hybrid, CGL4, as well as in parental HeLa cells, GLUT1 glycosylation was altered and its molecular mass was about 70 kDa. However, the altered GLUT1 glycosylation was not observed in SV40-transformed WI38 cells, suggesting a correlation between this glycosylation change and a putative tumor suppressor function. Further investigations using glycosidases, glycosylation inhibitors and lectin-affinity chromatography demonstrated that the tumor-associated glycosylation change in GLUT1 was mainly due to the increase in N-acetyl-lactosamine repeats in the N-linked oligosaccharides. In accordance with the altered glycosylation, affinity for 2-deoxyglucose in the tumorigenic CGL4 cells increased 2-fold, but there was little change in the Vmax. These results suggest there may be a functional role for the modulation by glycosylation of GLUT1 in the tumorigenic behavior of CGL4 and HeLa cells.

Biological Transport↗
PubMed1995

N-terminal quarter part of tetracycline transporter from pACYC184 complements K+ uptake activity in K+ uptake-deficient mutants of Escherichia coli and Vibrio alginolyticus.

In an attempt to clone a gene encoding the K+ uptake system from Vibrio alginolyticus, two plasmids, pKT2 and pKT4, were derived from pACYC184. These plasmids allowed the growth of K+ uptake-deficient mutant strains of Escherichia coli TK420 and V. alginolyticus FS181 in a low K+ medium. The pKT2 and pKT4 had an insertion about 7 and 6 kb, respectively, from the genome of V. alginolyticus. We prepared deletion plasmids from both plasmids and found that the site of genes inserted in the two was not identical and that the active locus corresponded to the structural gene encoding the N-terminal quarter part of tetA(C) gene. The N-terminal region of tetA(C) gene was ligated in another vector plasmid pHG165 to produce pHGK23. pHGK23 complemented the growth of TK420 in the low K+ medium. It contained only 62 bp from the genome of V. alginolyticus, and the open reading frame was composed of 98 amino acid residues from the N-terminal quarter part of tetA(C) and 5 amino acid residues attached by gene fusion. Using the Na+ -loaded cells of TK420, pHGK23 was found to increase the activity of K+ uptake. These results show that the N-terminal side tetA(C) gene product functions as a K+ uptake system.

Amino Acid Sequence↗
PubMed1995

Non-insulin-dependent diabetes mellitus complicated with idiopathic hypoparathyroidism.

We report a case of non-insulin-dependent diabetes mellitus (NIDDM) complicated with idiopathic hypoparathyroidism. A 74-year-old male was hospitalized because of diplopia. He was revealed to have NIDDM. The levels of serum Ca and intact-PTH were 6.3 mg/dl and < 5 pg/ml, respectively. Brain computed tomography revealed abnormal calcification in the cerebral basal ganglia and the cerebellum. After recovery from hypocalcemia, the endogenous insulin secretion was normalized. It is suggested that the pathogenesis of NIDDM in this patient may have been related to an insulin secretory defect as a result of hypocalcemia in addition to the hereditary risk.

Aged↗
PubMed1995

Serial changes in the prevalence of islet cell antibodies and islet cell antibody titer in children with IDDM of abrupt or slow onset.

OBJECTIVE: To elucidate the significance of clinical and immunogenic heterogeneity in Japanese children with insulin-dependent diabetes mellitus (IDDM). RESEARCH DESIGN AND METHODS: Serial changes in the prevalence of islet cell antibodies (ICAs) and in ICA titer were monitored for 10 years after diagnosis in 34 IDDM children, 17 with abrupt onset and 17 with slow onset, whose durations of disease were > 5 years. RESULTS: In slow-onset IDDM children, enough beta-cell function was maintained in the early phase of the disease within 2 years after diagnosis. There was a high prevalence of ICAs in children with both forms of IDDM at the time of diagnosis (abrupt onset 94%; slow onset 82%). However, the decline in the frequency of ICAs in slow-onset IDDM seemed less marked than in abrupt-onset IDDM after a duration of > or = 1 year (47 vs. 82%, 1-3 years; 24 vs. 47%, 3-5 years; 24 vs. 47%, 5-7 years; 18 vs. 53%, 7-10 years, P < 0.05). In terms of changes in ICA titer, abrupt-onset IDDM children initially had high ICA levels of 160-320 Juvenile Diabetes Foundation units (JDF U), but these titers decreased rapidly after the 1st year. On the other hand, slow-onset IDDM children tended to continue to be ICA+ for a relatively long period with low titers of 20-40 JDF U. Among 12 children who remained ICA+ for > 5 years, slow onset was noted in 67% while abrupt onset was seen in only 33%. CONCLUSIONS: From these results, we speculated that changes in ICA titer reflect the slow autoimmune destruction of pancreatic beta-cells. It may be probable that immunogenic factors as well as environmental factors could affect the clinical features in the early phase of IDDM in children.

Age of Onset↗
PubMed1995

Papillary carcinoma of the thyroid gland with fibromatosis-like stroma: a case report and review of the literature.

A new case of a rare variant of papillary carcinoma of the thyroid gland with fibromatosis-like stroma is reported. The patient was a 43-yr-old woman who had a well demarcated tumor that showed an expansive growth from the left thyroid lobe into perithyroidal soft tissues. Histologically, the tumor was composed predominantly of a fibromatosis-like stroma in which were diffusely dispersed small follicles of papillary carcinoma. At the advancing front of extrathyroidal extension of the tumor, fibromyxomatous changes of soft tissues were preceded by infiltration of the papillary carcinoma component. Immunohistochemistry and electron microscopy showed that the stromal cells had a myofibroblastic nature. One metastatically involved lymph node did not show fibromatosis-like stroma. The patient has remained well with no evidence of recurrence for 1 yr.

Adult↗
PubMed1995

[Pharmacokinetic comparison of intraarterial and intraportal infusion of adriamycin in regional chemotherapy of the liver].

We investigated the adriamycin (ADR) pharmacokinetics following intraarterial and intraportal infusion under hepatic venous isolation and charcoal hemoperfusion (HVI.CHP). HVI.CHP was used to measure the first-pass amount of adriamycin through the liver and to reduce hepatic re-entry of the drug. Beagles underwent a 10-min ADR infusion (1 mg/kg) either through the hepatic artery (group I, n = 5) or the portal vein (group II, n = 5) under a 20-min HVI.CHP. During HVI.CHP, the hepatic venous flow rate and plasma ADR levels in prefilter (hepatic venous level), postfilter and peripheral blood were serially measured. Based on these measurements, the hepatic extraction ratio (HER) of ADR was calculated. Areas under the time-concentration curves of prefilter levels in groups I and II were 6.1 +/- 1.6 and 16.9 +/- 5.0 micrograms.min/ml, respectively, showing a significant difference between two groups (p < 0.01). On the contrary, HER of group I (81.2%) was significantly higher than that of group II (47.2%, p < 0.01). These results indicate that intraarterial infusion of ADR is superior to intraportal infusion in terms of local drug delivery to the liver and systemic drug toxicities.

Animals↗
PubMed1995

[Pharmacokinetic evaluation of complete infrarenal inferior vena caval isolation and charcoal hemoperfusion for intra-arterial chemotherapy of pelvic tumors].

Regional chemotherapy for malignant pelvic tumors is disappointing due to the poor tumor response and dose-limiting systemic toxicity. This study was designed to determine whether a novel venous isolation-charcoal hemoperfusion system could limit systemic exposure to chemotherapeutic agents after regional arterial infusion. Adriamycin (2 mg/kg) was continuously administered in the internal iliac arteries of beagles under complete isolation of the infrarenal inferior vena cava and extracorporeal charcoal hemoperfusion (IVCI.CHP). Control beagles received adriamycin at the same dosage without IVCI.CHP. Plasma adriamycin concentrations were measured in the left carotid artery (systemic level) at intervals of up to 40 minutes after the start of drug infusion. In animals with IVCI.CHP, the drug levels were also determined at the inlet and outlet of a CHP filter. The mean drug extraction ratios by the CHP filter averaged 88%. As a result, animals with IVCI.CHP showed a significant reduction in systemic drug levels at all measuring time points compared to controls. The peak systemic levels of adriamycin were 0.8 +/- 0.1 and 2.3 +/- 0.8 micrograms/ml, respectively, in IVCI.CHP and control animals (p < 0.01). IVCI.CHP accomplished a significant reduction in systemic exposure to adriamycin after iliac arterial infusion. This novel system will allow regional delivery of high-dose cytotoxic agents with little systemic toxicity for pelvic tumors.

Animals↗
PubMed1995

[A case of malignant fibrous histiocytoma treated with intraarterial chemotherapy under complete venous isolation and charcoal hemoperfusion].

Herein reported is a case with malignant fibrous histiocytoma (MFH) at the left elbow treated successfully with intraarterial chemotherapy under complete brachial venous isolation and charcoal hemoperfusion (BVI-CHP). A 56-year-old man was admitted to our institution because of local recurrence at the left elbow 6 months after extended local resection combined with systemic chemotherapy. We treated the patient with a 15-min intraarterial infusion of adriamycin (100 mg/body) and cisplatin (30 mg/body) under a concomitant 30-min BVI-CHP. Two weeks after the first treatment, he received a repeated intraarterial infusion of adriamycin (80 mg/body) and cisplatin (50 mg/body) under BVI-CHP. The tumor became necrotic one week after the first treatment, resulting in 60% reduction in tumor diameter. In addition, angiography demonstrated a remarkable shrinkage of the tumor stain. Despite repeated intraarterial high-dose infusions of chemotherapeutic agents, systemic toxicities, such as leukopenia, nausea/vomiting and alopecia, were not observed. These results indicate that this approach offers a novel therapeutic option for malignant tumors in the extremities.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1995

[A case of rheumatoid arthritis associated with pernicious anemia and bronchiolitis obliterans organizing pneumonia].

A case of rheumateid arthritis (RA) with pernicious anemia (PA) and wandering multiple patchy densities in bilateral lung fields is reported. A 72-year-old woman was hospitalized in February 1994, because of cough. She had already advanced RA (Class IV, Stage IV). She showed macrocytic and hyperchromic anemia as follows ; red-cell count (RBC), 176 x 10(4)/microliters; hemoglobin (Hb),7.2 g/dl; hematocrit (Ht), 21.0% ; MCV, 119.3 fl; and MCH, 40.9 pg. Chest roentgenogram revealed multiple patchy densities in bilateral lung fields and there was no response to the administration of antibiotic agents. From these clinical pictures bronchiolitis obliterans organizing pneumonia (BOOP) was highly suspected. After steroid injection into the joint space, the abnormal lung shadows disappeared. Anemia had been recovering spontaneously, but recurred in July. The results of blood examination were as follows ; RBC, 162 x 10(4)/microliters; Hb, 6.7ng/dl; Ht, 19.1%; MCV, 117.9 fl; and MCH, 41.4 pg. Anti-intrinsic factor antibody was positive. The level of serum vitamin B12 was low, 76 pg/ml. Sternal bone marrow aspiration showed magaloblastic changes with hypersegmentation of granulocytes. PA was diagnosed and improvement was noted after the intramuscular administration of vitamin B12. Subjective symptoms based on RA did not change during the clinical course. It is suggested that the pathogenesis about the combination of RA, BOOP and PA is related to common immunological abnormalities in our patient. A case of RA with PA and BOOP has not been reported previously, thus this case is considered clinically valuable.

Aged↗
PubMed1994

Spectroscopic study of Ser92 mutants of human myoglobin: hydrogen bonding effect of Ser92 to proximal His93 on structure and property of myoglobin.

Neutron diffraction studies have demonstrated that the hydroxyl group oxygen of Ser92(F7) is hydrogen bonded to the proximal His93(48) N epsilon H proton in myoglobin (Mb) [Cheng, X., & Shoenborn, B. P. (1991) J. Mol. Biol. 220, 381-399]. In order to examine the importance of this hydrogen bond, Ser92 was replaced with Ala and Asp in human Mb. By comparing the optical, 1H-NMR, resonance Raman, and IR spectra of Mb(S92A) in several spin and oxidation states with those of wild-type Mb, it was found that the mutation causes a structural change on the heme proximal side but not on the distal side. Comparison of the NMR spectra of the cyanomet form of Mb(S92A) and Mb(WT) suggests that the imidazole plane of His93 rotates somewhat around the Fe-N delta (His93) bond upon loss of the hydrogen bond between His93 and Ser92. The 2D 1H-NMR measurements of the CO complexes show that mutation of Ser92 to Ala changes the relative position of the His97 imidazole group to the heme plane, but the change is not so drastic as reported in the crystal data of Ser92 mutant of pig Mb [Smerdon et al. (1993) Biochemistry 32, 5132-5138]. On the other hand, ligand (CO, O2) binding is only slightly affected by this mutation. From these results, we conclude that the Ser92-His93 hydrogen bond maintains the protein structure of the proximal heme pocket, but it does not strongly affect the electronic structure of the heme as well as of the His93 imidazole ring.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Monoxide↗
PubMed1994

Molecular structure of redox metal centers of the cytochrome bo complex from Escherichia coli. Spectroscopic characterizations of the subunit I histidine mutant oxidases.

A site-directed mutagenesis study on the conserved subunit I histidines of the cytochrome bo complex in Escherichia coli identified ligands of the low spin heme B and CuB centers; however, the assignment of the proximal ligand of the high spin heme O was ambiguous (Minagawa, J., Mogi, T., Gennis, R. B., and Anraku, Y. (1992) J. Biol. Chem. 267, 2096-2104). We have extended this work and characterized the metal centers in the purified histidine mutant oxidases by optical, EPR, and resonance Raman spectroscopies and by biochemical analysis. We found that the H284A and H333A oxidases contain two heme B molecules, which exhibit the gz = 2.99 low spin and cyanide-sensitive g perpendicular = 6 high-spin EPR signals, whereas the H419A oxidase contains only low spin heme B, which shows the gz component in a considerably higher magnetic field at g = 2.92. The CuB center was partially retained in the H284A oxidase but was almost completely lost in the H333A and H419A oxidases. Thus, we concluded that His419 is the proximal ligand of the high spin heme O and that His284 is located at the distal side of the high spin heme O. His284 plays an indispensable role in maintaining the structure of the Feo-CuB binuclear site suitable for the exogenous ligand bindings in the reduced state, since its substitution eliminated the CO binding activity. In addition, we found that His106 and His421 are in fact the axial ligands of the low spin heme B, although the H421A mutation perturbed the binuclear metal center seriously. Based upon experimental results with isotopic substitutions on iron in the oxidases, we assigned the Raman band at 208 cm-1 to the iron-histidine stretching mode (vFc(2+)-N(His)) of the wild-type ferrous cytochrome o, which was upshifted slightly by a loss of the CuB center in the H333A oxidase. A molecular structure of the metal centers and a possible mechanism of the electron transfer-coupled proton pumping in the cytochrome bo complex are proposed on the basis of our present findings.

Copper↗
PubMed1994

Selective resonance Raman observation of the "607 nm" form generated in the reaction of oxidized cytochrome c oxidase with hydrogen peroxide.

Resonance Raman spectra were measured selectively for the "607 nm" form, which had been assigned to a peroxy intermediate formed in the reaction of oxidized cytochrome c oxidase with hydrogen peroxide at ambient temperature. A single oxygen isotope-sensitive band was found at 803 cm-1 for the reaction with H2(16)O2 (at 769 cm-1 with H2(18)O2) upon excitation at 607 nm, the wavelength of the difference absorption maximum characteristic of the "peroxy" intermediate. Upon excitation at shorter wavelengths (down to 580 nm), the Raman spectrum simply became weaker without yielding any new features. When H2(16)O18O was used, two bands were observed at 803 and 769 cm-1 (within an accuracy of 0.5 cm-1), but with only half the intensity of those observed with H2(16)O2 or H2(18)O2, which ruled out the possibility that the 803 cm-1 band arose from the O-O or Fe-O2 stretching of the FeIII(O-O-) heme. Conversely, the 34-cm-1 downshift with 18O is in good agreement with the calculated 16O/18O shift (35 cm-1) expected for the diatomic Fe = 16O oscillator at 803 cm-1. This band exhibited an upshift by 1.3 cm-1 in 2H2O, similar to the case of compound II of horseradish peroxidase at neutral pH, and indicative of the presence of a hydrogen bond to the FeIV = O oxygen. The 803/769 cm-1 pair of resonance Raman bands were also observed upon blue excitation, as is the case for the bands found in the dioxygen cycle of this enzyme (Ogura, T., Takahashi, S., Hirota, S., Shinzawa-Itoh, K., Yoshikawa, S., Appelman, E. H., and Kitagawa, T. (1993) J. Am. Chem. Soc. 115, 8527-8536). This observation provides the first direct characterization of the 607 nm form of this enzyme in its reaction with H2O2.

Animals↗
PubMed1994

Identification of a novel quinone-binding site in the cytochrome bo complex from Escherichia coli.

The cytochrome bo complex is a heme BO-type heme-copper quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as an electron transfer-linked proton pump. To study the protein-mediated electron transfer from substrates to metal centers, we carried out quantitative and qualitative analyses of a bound quinone in the purified oxidase and found that it has a novel high affinity ubiquinone-binding site distinct from the quinol oxidation site. Enzymatic and spectroscopic studies suggest that the quinone-binding site is located close to both the quinol oxidation site in subunit II and low-spin heme B in subunit I. The quinone-binding site of a bound ubiquinone-free oxidase was reconstituted with the potent quinol oxidation site inhibitor 2,6-dichloro-4-nitrophenol, which decreased the Vmax value of the ubiquinol-1 oxidase activity to one-fourth of the control activity. These results indicate that the quinone-binding site is essential for the catalytic functions of the cytochrome bo complex and mediates electron transfer from the quinol oxidation site to the low-spin heme.

Air↗
PubMed1994

New hapten-protein conjugation method using N-(m-aminobenzoyloxy) succinimide as a two-level heterobifunctional agent: thyrotropin-releasing hormone as a model peptide without free amino or carboxyl groups.

The use of a two-level heterobifunctional agent N-(m-aminobenzoyloxy)succinimide (m-ABS) allowed us to develop a new method for preparing hapten-protein conjugates. This was demonstrated by a conjugation between thyrotropin-releasing hormone (TRH) and bovine or human serum albumin (BSA or HSA). The conjugation is based on the principle that the succinimidyl ester group of m-ABS immediately acts on an epsilon-amino group of lysine residues of carrier protein BSA (or HSA) and a m-aminobenzoyl group incorporated into the protein is then activated by diazotization to a functional m-diazobenzoyl group (m-DB) acting on a histidyl group of TRH. The TRH-BSA containing about 3.5 mol of TRH per BSA molecule, elicited the production of TRH antibody in rabbits. A new type of enzyme-linked immunosorbent assay (ELISA) for TRH was developed using the antiserum, the solid-phase antigen TRH-HSA and the commercially available horseradish peroxidase-labeled goat anti-rabbit IgG/Fab' as a marker, revealing that the ELISA was monospecific to the hormone and measured as low as 50 pg of the hormone reproducibly. Also, using the antiserum by the indirect immunoperoxidase method the distribution of immunoreactive TRH in the rat brain was demonstrated in neurons of the paraventricular nucleus and neuronal processes of the median eminence. These results strongly suggested that the use of m-ABS provided a simple and efficient new method for preparing immunogens not only for the previously reported haptens with a primary amino group(s) (J. Immunol. Methods 134 (1990) 227), but also for haptens with an imidazole, phenolic, or indole group(s) in the molecule.

Amino Acid Sequence↗