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Biomedical subjects

T Kanazawa

Publications and source records attributed to T Kanazawa.

At least 19 recordsLinked to original sources
PubMed1999

Mutations of Arg198 in sarcoplasmic reticulum Ca2+-ATPase cause inhibition of hydrolysis of the phosphoenzyme intermediate formed from inorganic phosphate.

Arg198 of sarcoplasmic reticulum Ca2+-ATPase was substituted with lysine, glutamine, glutamic acid, alanine, and isoleucine by site-directed mutagenesis. Kinetic analysis was performed with microsomal membranes isolated from COS-1 cells which were transfected with the mutated cDNAs. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme was determined by first phosphorylating the Ca2+-ATPase with 32Pi and then diluting the sample with non-radioactive Pi. This rate was reduced substantially in the mutant R198Q, more strongly in the mutants R198A and R1981, and most strongly in the mutant R198E, but to a much lesser extent in R198K. The reduction in the rate of dephosphorylation was consistent with the observed decrease in the turnover rate of the Ca2+-ATPase accompanied by the steady-state accumulation of the ADP-insensitive phosphoenzyme formed from ATP. These results indicate that the positive charge and high hydrophilicity of Arg198 are critical for rapid hydrolysis of the ADP-insensitive phosphoenzyme.

Adenosine Diphosphate↗
PubMed1999

Formation of the ADP-insensitive phosphoenzyme intermediate in the sarcoplasmic reticulum Ca2+-ATPase of which both Cys344 and Cys364 are modified by N-ethylmaleimide.

Sarcoplasmic reticulum vesicles were pretreated with N-ethylmaleimide under the conditions in which both Cys344 and Cys364 (SHD) of the Ca2+-ATPase are selectively modified. Effects of the modification on the transition of the phosphoenzyme intermediate (EP) from ADP-sensitive form to ADP-insensitive form and on the formation of ADP-insensitive EP from Pi were examined without added K+. At pH 7.0-8.0 in totally aqueous media, the EP transition and the EP formation from Pi were almost completely inhibited by the SHD modification. The formation of ADP-insensitive EP from ATP and from Pi in the SHD-modified enzyme occurred to some extent at pH 6.0-6.5 and were greatly increased by addition of dimethyl sulfoxide at pH 6. 0-8.0. The inhibition by the SHD modification of the EP formation from Pi in the absence of dimethyl sulfoxide was attributed to a decrease in the equilibrium constant for the EP formation from the enzyme-Pi-Mg complex. When 40% (v/v) dimethyl sulfoxide was present, almost all the phosphorylation sites in the SHD-modified enzyme were phosphorylated with ATP at pH 6.0 or with Pi at pH 6.0-7.0, and all the EP formed was ADP-insensitive. These results lead to the possibility that the previously reported exclusion of water from the catalytic site upon the EP transition and upon the EP formation from the enzyme-Pi-Mg complex is inhibited by the SHD modification. The present study has revealed the conditions in which the enzyme is released from the inhibition by this modification. The modification of SHD, which brackets the phosphorylation site (Asp351), may provide a useful tool for the analysis of conformational changes at the phosphorylation site occurring in the catalytic cycle.

Adenosine Diphosphate↗
PubMed1999

Role of diffusion-weighted echo-planar MRI in distinguishing between brain brain abscess and tumour: a preliminary report.

Our purpose was to evaluate diffusion-weighted (DW) echo-planar MRI in differentiating between brain abscess and tumour. We examined two patients with surgically confirmed pyogenic brain abscess and 18 with metastatic brain tumours or high-grade glioma, using a 1.5 T system. The apparent diffusion coefficient (ADC) of each necrotic or solid contrast-enhancing lesion was measured with two different b values (20 and 1200 s/mm2). All capsule-stage brain abscesses (4 lesions) and zones of cerebritis (2 lesions) were identified on high-b-value DWI as markedly high-signal areas of decreased ADC (range, 0.58-0.70 [(10-3 mm2/s; mean, 0.63)]). All cystic or necrotic portions of brain tumours (14 lesions) were identified on high-b-value DWI as low-signal areas of increased ADC (range, 2.20-3.20 [(10-3 mm2/s; mean, 2.70)]). Solid, contrast-enhancing portions of brain tumours (19 lesions) were identified on high-b-value DWI as high-signal areas of sightly decreased or increased ADC (range, 0.77-1.29 [(10-3 mm2/s; mean, 0.94)]). Our preliminary results indicate that DW echo-planar MRI be used for distinguishing between brain abscess and tumour.

Aged↗
PubMed1999

Thallium-201 scintigraphy in malignant mesothelioma.

A patient with malignant pleural mesothelioma was investigated using 201Tl scintigraphy. There was diffuse pleural tumour accumulation on planar scintigraphy. Single photon emission computed tomography (SPECT) demonstrated exact tumour location. It is concluded that planar scintigraphy using 201Tl may be useful in detecting mesothelioma. Clearer tumour localization is possible with SPECT.

Female↗
PubMed1998

Diffusion-weighted echo-planar MRI of lacunar infarcts.

We studied 35 patients with lacunar infarcts, using diffusion-weighted echo-planar imaging (DW-EPI) at 1.5 T. The relative apparent diffusion coefficient ratio (ADCR) of each lesion was calculated and lesion conspicuity on DW-EPI was compared to that on images acquired with fast fluid-attenuated inversion recovery and T2-weighted fast spin-echo sequences. Acute small infarcts (within 3 days) were identified with DW-EPI as an area of decreased ADCR (range 0.33-0.87; mean 0.67) and high signal, subacute small infarcts (4-30 days) as a high-signal or isointense areas of decreased or nearly normal ADCR (0.54-0.98; 0.73), and chronic small infarcts (> 30 days) as low- or high-signal areas of nearly normal or increased ADCR (0.97-1.92; 1.32). In three patients, small infarcts of the brain stem in the hyperacute phase (within 6 h) were seen only with DW-EPI. In five patients, fresh small infarcts adjacent to multiple old infarcts could be distinguished only with DW-EPI.

Aged↗
PubMed1998

Soil factors influencing the distribution of Oncomelania quadrasi, the intermediate host of Schistosoma japonicum, on Bohol Island, Philippines.

Soil conditions essential to the survival of Oncomelania quadrasi on Bohol Island in the Philippines were examined to clarify the factors limiting distribution of the snail and to develop a method for breeding large numbers of the snail in the laboratory. Soil samples in and around snail habitats were analysed and used for breeding experiments in the laboratory. Experiments using paddy soil derived from different parent materials revealed that the numbers of juvenile snails hatched varied widely between several soil samples. The best soils for reproduction generally had a pH of 5.6-7.9 and > 200 mg of available CaO/100 g. These soil factors, in addition to shade and moisture, determine the optimum conditions for the breeding of O. quadrasi in the field as well as in the laboratory. The determination of the optimum conditions for laboratory breeding of O. quadrasi and other intermediate snail hosts should facilitate detailed study of the hosts and the development of better methods to control or eradicate schistosomiasis and other snail-transmitted diseases.

Animals↗
PubMed1998

A plasmid isolated from phytopathogenic onion yellows phytoplasma and its heterogeneity in the pathogenic phytoplasma mutant.

A 3.6-kbp DNA fragment was cloned from the extrachromosomal DNA of a pathogenic plant mollicute, onion yellows phytoplasma (OY-W). Sequence analysis of the fragment revealed an open reading frame (ORF) encoding the replication (Rep) protein of rolling-circle replication (RCR)-type plasmids. This result suggests the existence of a plasmid (pOYW1) in OY-W that uses the RCR mechanism. This assumption was confirmed by detecting the single-stranded DNA (ssDNA) of a replication intermediate that is specifically produced by the RCR mechanism. This is the first report on the identification of the replication system of this plasmid and the genes encoded in it. With a DNA fragment including the Rep gene region of pOYW1 used as a probe, Southern and Northern (RNA) blot hybridizations were employed to examine the heterogeneity between the plasmids found in OY-W and a pathogenic mutant (OY-M) isolated from OY-W. Multiple bands were detected in the DNA and RNA extracted from both OY-W and OY-M infected plants, although the banding patterns were different. Moreover, the copy number of plasmids from OY-W was about 4.2 times greater than that from OY-M. These results indicate constructive heterogeneity between OY-W and OY-M plasmids, and the possibility of a relationship between the plasmid-encoded genes and the pathogenicity of the phytoplasma was suggested.

Amino Acid Sequence↗
PubMed1998

[A case of refractory pulmonary fistula, managed with bronchoscopic therapy using occluding spiral embolus].

We report a successful case of bronchoscopic therapy using occluding spiral embolus and fibrin glue for refractory pulmonary fistula. A 22-year-old female underwent left lower lobectomy for giant bulla of the lung. Air leakage began 6 days after lobectomy. Closing alveolar fistula was performed 12 days after first operation. Relapsing air leakage began 4 days after second operation. Bronchography revealed a fistula from left B1+2c. The insertion of embolus through bronchofiberscope, following administration of fibrin glue, was performed to close the fistula. This method is effective for refractory pulmonary fistula.

Adult↗
PubMed1998

[14C-oxalate autoradiographic studies on distribution of oxalate and calcium oxalate crystal fixation in rat kidney].

To elucidate the intrarenal distribution of oxalate and to reveal the first site of calcium oxalate crystal fixation along the nephron, we carried out 14C-oxalate renal microautoradiography by the thaw mount method 30, 60 and 90 min after intravenous administration of 14C-oxalate, in normal rats and in hyperoxaluric rats fed a vitamin B6-deficient diet for 4 and 9 weeks. In normal rats, radioactivity was observed in the entire kidney area at 30 min after the injection and at 90 min there was radioactivity only in the papilla, where radioactivity appeared in the parenchyma, mamely the epithelial cells of collecting ducts and the surrounding interstitial tissue. In hyperoxaluric rats, microautoradiograms showed the spotted deposits of 14C-oxalate in both renal papilla and medulla. In 600 x magnification of the autoradiograms, most of the deposits were detected in the epithelial cells of collecting ducts and the surrounding interstitial tissue. These deposits were identified by a polarizing microscope and X-ray microanalysis as crystal deposits of calcium oxalate. These results indicated that oxalate remained for an extended period of time in the epithelial cells of collecting ducts and in the surrounding interstitial tissue in renal papilla, and the calcium oxalate crystals fixed to the same tissue was the first step in the development of calcium oxalate stones.

Animals↗
PubMed1998

[Combined chemotherapy with low-dose cisplatin, tegafur and uracil in a case with neck recurrence of laryngeal cancer].

The patient was a 60-year-old man with progressive neck recurrence of laryngeal squamous cell carcinoma. As a previous treatment, he had undergone irradiation (primary: 60 Gy; neck: 45 Gy) after two cycles of neoadjuvant chemotherapy (cisplatin 100 mg/m2 daily; day 1:5-fluorouracil 1,000 mg/m2 daily; day 1-5) and planned neck dissection. For a neck recurrence, he had received five cycles of low-dose cisplatin (5 mg/body daily: day 1-5) with tegafur and uracil (600 mg/body daily: day 1-7) every week. As an outpatient, he then received ten cycles of low-dose cisplatin (10 mg/body daily: day 3, 6) with tegafur and uracil (600 mg/body daily: day 1-7) every week. The size of the tumor did not decrease with the above chemotherapy, but no remarkable growth of the cancer was seen, and no toxic effect of the chemotherapy was observed. He continued his job for 30 weeks just as before he had fallen sick. Chemotherapy of low-dose cisplatin with tegafur and uracil was suggested to be useful for the patient with recurrent laryngeal squamous cell carcinoma.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1997

Modification of histidine 5 in sarcoplasmic reticulum Ca2+-ATPase by diethyl pyrocarbonate causes strong inhibition of formation of the phosphoenzyme intermediate from inorganic phosphate.

Sarcoplasmic reticulum vesicles were modified with diethyl pyrocarbonate (DEPC), a histidine-modifying reagent. Phosphoenzyme formation from Pi in the Ca2+-ATPase (reversal of hydrolysis of the phosphoenzyme intermediate) was almost completely inhibited by this modification. Tight binding of F- and Mg2+ and high affinity binding of vanadate in the presence of Mg2+, both of which produce transition state analogs for phosphoenzyme formation from the magnesium-enzyme-phosphate complex, were also inhibited. Formation of the phosphoenzyme from acetyl phosphate in the forward reaction was only weakly inhibited, but hydrolysis of the phosphoenzyme was strongly inhibited. The enzyme was protected by tight binding of F- and Mg2+ or by high affinity binding of vanadate in the presence of Mg2+ against the DEPC-induced inhibition of phosphoenzyme formation from Pi. The enzyme was also protected by tight binding of F- and Mg2+ against the DEPC-induced inhibition of phosphoenzyme hydrolysis. Peptide mapping of the tryptic digests, detection of peptides containing DEPC-modified histidine by UV absorption at 240 nm, amino acid analysis, sequencing, and mass spectrometry showed that His-5 was a single major residue protected by the above transition state analogs against the modification with DEPC. These results indicate that modification of His-5 with DEPC is responsible for the DEPC-induced inhibition of phosphoenzyme formation from Pi and of phosphoenzyme hydrolysis and suggest that His-5 is located in or very close to the catalytic site in the transition state for phosphoenzyme formation from the magnesium-enzyme-phosphate complex and is likely involved in the catalytic process of this reaction step.

Animals↗
PubMed1997

Inhibitory effects of spinorphin, a novel endogenous regulator, on chemotaxis, O2- generation, and exocytosis by N-formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated neutrophils.

To characterize the inflammatory effect of spinorphin, an endogenous peptide purified from bovine spinal cord, its effects on chemotaxis, O2- generation, and exocytosis by N-formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated human neutrophils (PMNs) in vitro were examined. At 10 microM, spinorphin significantly inhibited chemotaxis by FMLP-stimulated PMNs. Spinorphin at 100 microM also inhibited both O2- generation and exocytosis of beta-glucuronidase and collagenase by FMLP-stimulated PMNs. The mechanisms by which spinorphin inhibits these PMN functions were examined further. Spinorphin markedly suppressed the binding of FML[3H]P to its receptor on PMNs, as observed in a binding assay. However, other neuropeptides that were examined (angiotensin II and substance P) had no effect on FML[3H]P binding, suggesting the possibility that spinorphin plays a specific role in the inhibition of the binding between FMLP and its receptor. The suppression of FMLP binding also caused a decrease of the FMLP-induced intracellular calcium concentration [Ca2+]i, which acts as a second messenger leading to PMN functions. These results suggest that spinorphin may be a new endogenous inflammation-regulatory peptide that modulates the interaction of FMLP with its receptor.

CD13 Antigens↗
PubMed1997

Modification of arginine-198 in sarcoplasmic reticulum Ca2+-ATPase by 1,2-cyclohexanedione causes inhibition of formation of the phosphoenzyme intermediate from inorganic phosphate.

Sarcoplasmic reticulum vesicles were modified with 1,2-cyclohexanedione (CHD), a specific arginine-modifying reagent, in sodium borate (pH 8.0 or 8.8). Phosphoenzyme formation from Pi in the Ca2+-ATPase (reversal of hydrolysis of the phosphoenzyme intermediate) was almost completely inhibited by the modification with CHD. Tight binding of F- and Mg2+ and high affinity binding of vanadate in the presence of Mg2+, either of which produces a transition state analog for phosphoenzyme formation from the magnesium-enzyme-phosphate complex, were also markedly inhibited. In contrast, phosphoenzyme formation from acetyl phosphate in the forward reaction was unaffected. The enzyme was appreciably protected by tight binding of F- and Mg2+ or by high affinity binding of vanadate in the presence of Mg2+, but not by the presence of 20 mM MgCl2 alone or 150 mM Pi alone, against the CHD-induced inhibition of phosphoenzyme formation from Pi. Peptide mapping of the tryptic digests, detection of peptides containing CHD-modified arginyl residues with Girard's reagent T, sequencing, and mass spectrometry showed that Arg-198 was a single major residue protected by tight binding of F- and Mg2+ against the modification with CHD. These results indicate that modification of Arg-198 with CHD is responsible for at least a part (the portion reduced by the transition state analogs) of the CHD-induced inhibition of phosphoenzyme formation from Pi and suggest that Arg-198 is located in or close to the catalytic site in the transition state for phosphoenzyme formation from the magnesium-enzyme-phosphate complex.

Amino Acid Sequence↗
PubMed1997

An enzyme-linked immunosorbent assay for detecting proteolytic activity of hepatitis C virus proteinase.

An ELISA method for the quantitation in vitro of HCV serine proteinase activity was developed. A peptide substrate, Ac-Gly-Glu-Ala-Gly-Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Ser-Tyr-Thr-Trp-Thr-L ys (biotin) -OH (Sub-1), was hydrolyzed by a recombinant NS3 proteinase fused with maltose binding protein (MBP-NS3) into a product with a free amino moiety at the N-terminus. The product was immobilized, and the amino moiety was analyzed by digoxigenin labeling followed by immunological reaction with anti-digoxigenin-alkaline phosphatase conjugate and then the colorimeteric reaction. This method is suited for the high throughput screening of inhibitors, and the screening can be accelerated by automatic operation.

Amino Acid Sequence↗
PubMed1997

Stoichiometry of phosphorylation to fluorescein 5-isothiocyanate binding in the Ca2+-ATPase of sarcoplasmic reticulum vesicles.

In an attempt to establish the stoichiometry of phosphorylation in the Ca2+-ATPase of sarcoplasmic reticulum (SR) vesicles, phosphorylation by ATP (or Pi) or labeling by fluorescein 5-isothiocyanate (FITC) was performed with the SR vesicles under the conditions in which almost all the phosphorylation sites or FITC binding sites are phosphorylated or labeled. The resulting vesicles were solubilized in lithium dodecyl sulfate and then the Ca2+-ATPase was purified by size exclusion high performance liquid chromatography. Peptide mapping and sequencing of the tryptic digest of the purified enzyme showed that Lys-515 of the Ca2+-ATPase was exclusively labeled with FITC, in agreement with the previously reported findings. The content of the phosphoenzyme from ATP (4.57 nmol/mg of Ca2+-ATPase protein) or from Pi (4.94 nmol/mg of Ca2+-ATPase protein) in the purified enzyme was approximately half the content of the FITC binding site (8.17-8.25 nmol/mg of Ca2+-ATPase protein) and also half the content of the Ca2+-ATPase molecule (9.06 nmol/mg of Ca2+-ATPase protein) calculated from its molecular mass (110,331 Da). These results show that there is one specific FITC binding site per molecule of the Ca2+-ATPase (in agreement with the previously reported findings) and that the stoichiometry of phosphorylation to FITC binding is approximately 0. 5:1.0. All the above findings lead to the conclusion that only half of the Ca2+-ATPase molecules present in the SR vesicles can be phosphorylated. FITC binding completely inhibited the ATP-induced phosphorylation before the binding reached its maximum level. This finding indicates that FITC preferentially binds to a part of the Ca2+-ATPase molecules and that this binding is primarily responsible for the inhibition of phosphorylation, suggesting an intermolecular ATPase-ATPase interaction.

Adenosine Triphosphate↗
PubMed1997

Extracardiac direct total cavopulmonary connection.

We report a successful extracardiac direct total cavopulmonary connection without prosthetic materials performed in a 3-year-old boy with tricuspid atresia, infundibular pulmonary stenosis, and normally positioned great arteries. The transected pulmonary trunk was bought down posterolaterally with respect to the right atrium and was anastomosed end-to-end with the inferior vena cava.

Child, Preschool↗