Biomedical subjects

K Inagaki

Publications and source records attributed to K Inagaki.

At least 55 records · Page 3Linked to original sources

PubMed2000

Role of tyrosine 114 of L-methionine gamma-lyase from Pseudomonas putida.

L-Methionine gamma-lyase from Pseudomonas putida has a conserved tyrosine residue (Tyr114) in the active site as in all known sequences of y-family pyridoxal 5'-phosphate dependent enzymes. A mutant form of L-methionine y-lyase in which Tyr114 was replaced by phenylalanine (Y114F) resulted in 910-fold decrease in kcat for alpha,gamma-elimination of L-methionine, while the Km remained the same as the wild type enzyme. The Y114F mutant had the reduced kcat by only 28- and 16-fold for substrates with an electron-withdrawing group at the gamma-position, namely O-acetyl-L-homoserine and L-methionine sulfone, respectively, and also the similar reduction of kcat for alpha,beta-elimination and deamination substrates. The hydrogen exchange reactions of substrate and the spectral changes of the substrate-enzyme complex catalyzed by the mutant enzyme suggested that gamma-elimination process for L-methionine is the rate-limiting determination step in alpha,gamma-elimination overall reaction of the Y114F mutant. These results indicate that Tyr114 of L-methionine gamma-lyase is important in y-elimination of the substrate.

Carbon-Sulfur Lyases↗

PubMed2000

Sensitive detection of mediastinal lymph node metastasis of lung cancer with 11C-choline PET.

UNLABELLED: 11C-choline and FDG are PET tracers used to visualize various malignancies. In this study, we compared their capabilities in detecting mediastinal lymph node metastasis originating from non-small cell lung cancer (NSCLC). METHODS: Twenty-nine patients with biopsy-proven NSCLC were studied with PET. 11C-choline PET and FDG PET were performed from 5 and 40 min, respectively, after injection of 370 MBq tracer. PET data were analyzed in terms of the standardized uptake value (SUV). After the PET study, the patients underwent lung resection and mediastinal lymph node dissection. The resected specimens were examined pathologically, and the PET data were analyzed in reference to the pathologic data. RESULTS: With 11C-choline, the SUV in metastasis was similar to the SUV in the primary tumor, where the similarity of the SUV was 100% allowing for a 40% difference. With FDG, small metastases were invisible on the PET image. The SUV of FDG in metastasis was much smaller than that in the primary tumor, and the similarity of the SUV was only 19% allowing for a 40% difference. When pathologic findings were used as standards, the sensitivities of 11C-choline PET and FDG PET in detecting mediastinal lymph node metastasis were 100% and 75%, respectively. CONCLUSION: 11C-choline PET was very effective in detecting lymph node metastases in the mediastinum originating from NSCLC, with a sensitivity of 100%. 11C-choline PET promises to be useful not only before surgery but also after surgery.

Adult↗

PubMed2000

[Diagnostic efficacy of 18F-fluorodeoxy glucose-positron emission tomography in multiple solitary pulmonary nodules].

A 73-year-old man was admitted to our hospital for evaluation of abnormal lung shadows in the left lung. Chest computed tomography revealed a cavitary lesion with irregular edges in the right S10 and a nodular lesion with well-defined margins in the left S6. Histological examination of a specimen obtained by transbronchial lung biopsy revealed squamous cell carcinoma in the right S10 but no significant findings in the left S6. Thirdly, 18F-fluorodeoxyglucose-positron emission tomography (18F-FDG-PET) demonstrated that the nodular shadow in the left S6 was a low-uptake structure and that the cavitary lesion in the right S10 was a high-uptake lesion. We therefore considered that the nodular shadow in the left S6 was not one of neoplastic disease. Partial lung resection of the left S6 was carried out by videoassisted thoracoscopic surgery. The pathological diagnosis of the left S6 was epithelial granuloma with caseation. A culture of the same resected specimen was positive for Mycobacterium avium. The eventual clinical staging for the squamous cell carcinoma in the right S10 was cT2N0M0 (IB). Radical surgical treatment and right lower lobectomy were performed for the S10 lesion. We considered that 18F-FDG-PET was an effective noninvasive procedure for diagnosis of solitary or multiple solitary nodular shadows in the lung.

Aged↗

PubMed1999

Tyrosine phosphorylation of p62(Dok) induced by cell adhesion and insulin: possible role in cell migration.

Dok, a 62-kDa Ras GTPase-activating protein (rasGAP)-associated phosphotyrosyl protein, is thought to act as a multiple docking protein downstream of receptor or non-receptor tyrosine kinases. Cell adhesion to extracellular matrix proteins induced marked tyrosine phosphorylation of Dok. This adhesion-dependent phosphorylation of Dok was mediated, at least in part, by Src family tyrosine kinases. The maximal insulin-induced tyrosine phosphorylation of Dok required a Src family kinase. A mutant Dok (DokDeltaPH) that lacked its pleckstrin homology domain failed to undergo tyrosine phosphorylation in response to cell adhesion or insulin. Furthermore, unlike the wild-type protein, DokDeltaPH did not localize to subcellular membrane components. Insulin promoted the association of tyrosine-phosphorylated Dok with the adapter protein NCK and rasGAP. In contrast, a mutant Dok (DokY361F), in which Tyr361 was replaced by phenylalanine, failed to bind NCK but partially retained the ability to bind rasGAP in response to insulin. Overexpression of wild-type Dok, but not that of DokDeltaPH or DokY361F, enhanced the cell migratory response to insulin without affecting insulin activation of mitogen-activated protein kinase. These results identify Dok as a signal transducer that potentially links, through its interaction with NCK or rasGAP, cell adhesion and insulin receptors to the machinery that controls cell motility.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Molecular cloning and functional characterization of rat delta-6 fatty acid desaturase.

Mammalian cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) sequence informations. These fragments were subsequently used to screen a rat liver cDNA library, yielding a 1573-bp clone. Expression of DNA fragment containing either of two possible open reading frames (nucleotide numbers 97-1431 and 148-1431) of the isolated clone in yeast led to the accumulation of gamma-linolenic acid in the presence of exogenous linoleic acid. In this system, the addition of alpha-linolenic acid also resulted in the accumulation of its Delta-6 desaturated product whereas dihomo-gamma-linolenic acid failed to be a substrate. These results indicate that the protein encoded by the rat cDNA is Delta-6 fatty acid desaturase, and the first 17 amino acids corresponding to the coding region 97-147 of the clone are not required to function in yeast.

Amino Acid Sequence↗

PubMed1999

PI 3-kinase gamma and protein kinase C-zeta mediate RAS-independent activation of MAP kinase by a Gi protein-coupled receptor.

Receptors coupled to the inhibitory G protein Gi, such as that for lysophosphatidic acid (LPA), have been shown to activate MAP kinase through a RAS-dependent pathway. However, LPA (but not insulin) has now been shown to activate MAP kinase in a RAS-independent manner in CHO cells that overexpress a dominant-negative mutant of the guanine nucleotide exchange protein SOS (CHO-DeltaSOS cells). LPA also induced the activation of MAP kinase kinase (MEK), but not that of RAF1, in CHO-DeltaSOS cells. The RAS-independent activation of MAP kinase by LPA was blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K) or by overexpression of a dominant-negative mutant of the gamma isoform of PI3K. Furthermore, LPA induced the activation of the atypical zeta isoform of protein kinase C (PKC-zeta) in CHO-DeltaSOS cells in a manner that was sensitive to wortmannin or to the dominant-negative mutant of PI3Kgamma, and overexpression of a dominant-negative mutant of PKC-zeta inhibited LPA-induced activation of MAP kinase. These observations indicate that Gi protein-coupled receptors induce activation of MEK and MAP kinase through a RAS-independent pathway that involves PI3Kgamma-dependent activation of atypical PKC-zeta.

Animals↗

PubMed1999

Purification and characterization of NAD:Penicillamine ADP transferase from Bacillus sphaericus. A novel NAD-dependent enzyme catalyzing phosphoramide bond formation.

A strain of Bacillus sphaericus isolated from a local soil sample has been found to use beta,beta-dimethyl-DL-cysteine (DL-penicillamine) as the sole nitrogen source. Crude cell extract of the bacterium showed potent penicillamine-consuming activity only in the presence of NAD, which, however, was not used as an electron acceptor. Characterization of reaction products revealed that penicillamine was derivatized to a phosphoramide adduct with the ADP moiety of NAD, whereas the nicotinamide-ribose group was released and hydrolyzed spontaneously to ribose and nicotinamide. The phosphoramide product, ADP-penicillamine, caused potent product inhibition on the purified enzyme, and adenylate deaminase was found to be effective in converting the inhibitory product into inosine-diphosphate-penicillamine and thereby maintained the catalysis for several hours. The novel enzyme, termed as NAD:penicillamine ADP transferase, showed a single band on SDS-polyacrylamide gel electrophoresis with a mass of approximately 42 kDa. The native enzyme was monomeric. The enzyme showed high substrate specificity to NAD (Km = 13.0 mM) and L-penicillamine (Km = 6.5 mM); other nucleotides such as NADP, NAD(P)H, AMP, ADP, and ADP-ribose did not substitute for NAD, and L-valine, L-cysteine, L-homocysteine, L-cystine, L-leucine, and L-isoleucine did not serve as the substrate. Kinetic studies suggested an Ordered Bi Bi mechanism, with NAD as the first substrate to bind and ADP-L-penicillamine as the last product released. The novel NAD-dependent enzyme may catalyze the first step in penicillamine degradation in the strain of B. sphaericus.

Bacillus↗

PubMed1999

Epithelioid hemangioendothelioma with marked liver deformity and secondary Budd-Chiari syndrome: pathological and radiological correlation.

A case of malignant epithelioid hemangioendothelioma of the liver in a 48-year-old woman with severe portal hypertension and marked deformity of the liver is presented. This woman had a history of mild liver dysfunction since the age of 30 years, and abdominal distention, esophageal varices, splenomegaly and ascites since October 1996. Imaging examinations revealed liver deformity with severe atrophy of the left lobe and the anterior segment of the right lobe. Celiac arteriography showed narrowing and upward deviation of the proper hepatic artery, and occlusion of the left and right anterior hepatic arteries. Since March 1997, hepatic venography showed stenosis in the right hepatic vein truncus. Budd-Chiari syndrome was clinically diagnosed. She died in June 1997. The autopsy disclosed massive tumor embolism in the left and right anterior portal branches, few in the hepatic artery, and occlusion of the left and right anterior hepatic arteries. The extensive tumor embolism resulted in portal hypertension, and atrophy of the left lobe. The anterior segment of the right lobe was probably caused by the occlusion of both the hepatic arteries and the portal veins. The posterior segment of the right lobe, without massive tumor embolism in its portal branch, appeared hypertrophic.

Budd-Chiari Syndrome↗

PubMed1999

[Clinical investigation on endobronchial tuberculosis].

In order to assess the clinical features and clinical courses of endobronchial tuberculosis, which included trachea to segmental bronchus, we studied 34 cases of patients who were admitted to TB ward of International Medical Center of Japan from 1994 to 1997. We noticed a higher incidence in females and in the main bronchus. Cough was the most common complaint seen in 97% of cases. The duration of symptoms before the initiation of antituberculous chemotherapy was long (on the average 6 months), and they were often treated as bronchial asthma or bronchitis. Bronchoscopic examination is necessary for diagnosis. The scars sometimes gave rise to severe stenosis, especially when the lesion developed to an advanced stage or circumscribed the lumen before treatment. We tried INH inhalation with systemic chemotherapy. Although rapid improvement was suggested by this method, yet no significant difference was seen in the results for the efficacious prevention of stenosis. Five cases required surgical intervension (bronchoplasty and lobectomy) in order to avoid atelectasis or secondary infection. Early diagnosis and appropriate treatment are most important, and bronchoscopic examination is essential in early diagnosis.

Adult↗

PubMed1999

[Evaluation of parathyroid imaging methods with 99mTc-MIBI--the comparison of planar images obtained using a pinhole collimator and a parallel-hole collimator].

Parathyroid scintigraphy with 99mTc-MIBI was performed using two kinds of collimators, namely, a pinhole one and a parallel-hole one, to evaluate which one was more suitable for the detection of hyperfunctioning parathyroid lesions. In the studies using 99mTc source, the pinhole collimator showed better efficiency and spatial resolution in the distance where the parathyroid scan are actually performed. In the phantom study, the nodular activities modeling parathyroid lesions were visualized better on the images obtained using the pinhole collimator. In clinical studies for 30 patients suspicious of hyperparathyroidism, hyperfunctioning parathyroid nodules were better detected when the pinhole collimator was used. In conclusion, the pinhole collimator was thought to be more suitable for parathyroid scintigraphy with 99mTc-MIBI than the parallel-hole collimator.

Aged↗

PubMed1999

[Body surface ultrasonography-guided bronchofiberscopy].

Transbronchial lung biopsies and cytologic studies under ultrasonographic guidance from the body surface were conducted in 39 patients whose lesions were adjacent to the thoracic wall. In 26 patients, biopsy, curettage, or brushing forceps were visualized in the mass or infiltrative lesion by thoracic echogram. Positive findings were obtained in 23 patients, for a conclusive diagnostic rate of 88.5%. Of the 13 patients in whom forceps could not be visualized by echogram, 10 had positive findings, for a diagnostic rate of 76.9%. For visualization by thoracic echogram, abnormal lung lesions must be in direct contact with the thoracic wall. Occasionally, diagnostic procedures may be impeded by anatomical structures such as shoulder joints or scapula. Despite these disadvantages, the ultrasonography-guided bronchofiberscope is quite useful because it facilitates real-time confirmation of the positioning of the forceps relative to the lesions. It is also useful in cases when the peripheral lesions are too small or vague to be demonstrated by fluoroscopy alone, because the echo probe can be the target of the forceps instead of the missing shadows. The diagnostic rate should be higher when the forceps are visualized in the lesions ultrasonographically.

Adult↗

PubMed1998

Cardiac endothelin-1 plays a critical role in the functional deterioration of left ventricles during the transition from compensatory hypertrophy to congestive heart failure in salt-sensitive hypertensive rats.

BACKGROUND: To investigate whether endogenous ET-1 participates in an adaptive process of left ventricular hypertrophy (LVH) or a maladaptive process from LVH to congestive heart failure (CHF), we used a Dahl salt-sensitive (DS) rat model, in which systemic hypertension caused compensated concentric LVH at the age of 11 weeks followed by marked LV dilatation and global hypokinesis at the age of 17 weeks. METHODS AND RESULTS: By specific sandwich enzyme immunoassay, serum and myocardial ET-1 levels at the LVH stage were not elevated compared with age-matched Dahl salt-resistant (DR) rats, despite the marked increase of LV/body weight ratio (LV/BW). However, at the CHF stage, serum and LV ET-1 levels increased by 3. 8-fold and 5.4-fold, respectively. LV ET-1 contents had close relationships with the fractional shortening (r=0.763) and the systolic wall stress (r=0.858) measured by in vivo transthoracic echocardiography. Immunohistochemistry demonstrated that the remarkably increased ET-1 in LV is located mainly in cardiomyocytes. By competitive reverse transcriptase-polymerase chain reaction, LV prepro-ET-1 mRNA levels increased by 4.1-fold in CHF rats. We randomized 11-week-old LVH rats to chronic treatment with the endothelin receptor antagonist bosentan (Bos, 100 mg. kg-1. d-1, n=14), the alpha1-receptor antagonist doxazosin (Dox, 1 mg. kg-1. d-1, n=12), or vehicle (Cont, n=14). Bos treatment did not alter the LV geometry and function at 15 weeks; however, it attenuated the decrease of LV fractional shortening by 51% (P<0.01) without reducing the LV/BW at 17 weeks. Conversely, Dox, which decreased the blood pressure to the same extent as Bos, did not affect the progression of LV dysfunction. Bos (93%; P<0.0001 versus Cont) but not Dox (42%; P=0.8465 versus Cont) ameliorated the survival rate at 17 weeks (Cont; 36%). CONCLUSIONS: The accelerated myocardial synthesis of ET-1 contributes directly to LV contractile dysfunction during the transition from LVH to CHF. Unelevated levels of LV ET-1 at the established LVH stage and lack of effects on LV mass by chronic bosentan treatment suggest that myocardial growth is mediated through alternative pathways. These studies indicate that chronic ET antagonism may provide an additional strategy for heart failure therapy in humans.

Adaptation, Physiological↗

PubMed1998

Structure of 3-isopropylmalate dehydrogenase in complex with 3-isopropylmalate at 2.0 A resolution: the role of Glu88 in the unique substrate-recognition mechanism.

BACKGROUND: 3-Isopropylmalate dehydrogenase (IPMDH) and isocitrate dehydrogenase (ICDH) belong to a unique family of bifunctional decarboxylating dehydrogenases. Although the ICDH dimer catalyzes its reaction under a closed conformation, known structures of the IPMDH dimer (without substrate) adopt a fully open or a partially closed form. Considering the similarity in the catalytic mechanism, the IPMDH dimer must be in a fully closed conformation during the reaction. A large conformational change should therefore occur upon substrate binding. RESULTS: We have determined the crystal structure of IPMDH from Thiobacillus ferrooxidans (Tf) complexed with 3-isopropylmalate (IPM) at 2.0 A resolution by the molecular replacement method. The structure shows a fully closed conformation and the substrate-binding site is quite similar to that of ICDH except for a region around the gamma-isopropyl group. The gamma group is recognized by a unique hydrophobic pocket, which includes Glu88, Leu91 and Leu92 from subunit 1 and Val193' from subunit 2. CONCLUSIONS: A large movement of domain 1 is induced by substrate binding, which results in the formation of the hydrophobic pocket for the gamma-isopropyl moiety of IPM. A glutamic acid in domain 1, Glu88, participates in the formation of the hydrophobic pocket. The C beta and C gamma atoms of Glu88 interact with the gamma-isopropyl moiety of IPM and are central to the recognition of substrate. The acidic tip of Glu88 is likely to interact with the nicotinamide mononucleotide (NMN) ribose of NAD+ in the ternary complex. This structure clearly explains the substrate specificity of IPMDH.

3-Isopropylmalate Dehydrogenase↗

PubMed1998

Direct injection determination of theophylline and caffeine in blood serum by high-performance liquid chromatography using an ODS column coated with a zwitterionic bile acid derivative.

An ODS column dynamically coated with zwitterionic bile acid derivative, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), was evaluated for direct injection determination of drugs in blood serum by HPLC. Polar functional groups such as sulfonate, ammonium and the three hydroxyl groups in CHAPS protruding towards an aqueous mobile phase formed a hydrophilic layer over the ODS reversed-phase surface, which resulted in high molecular mass compounds such as proteins being prevented from penetrating into the internal hydrophobic region. The bulk of the proteins were eluted as an unretained or nearly unretained band by using 0.2 mM sodium hydrogenphosphate solution (pH 7.4) as the mobile phase. In contrast, small molecules such as some inorganic anions and aromatic compounds were retained and thereby separated from one another. It was confirmed that the ODS column modified with CHAPS acts as a restricted access-type column with a hydrophobic interior and a hydrophilic exterior. Hence biological fluids could be directly injected into the CHAPS-coated ODS column. The present HPLC system using the CHAPS-coated ODS column was applied to the determination of theophylline and caffeine in human blood serum. The detection limits for the two drugs with UV absorption at 273 nm were 0.2 and 0.5 mg l-1 (injection volume 20 microliters) and the relative standard deviations of peak area measurements were < 1.4% and 2.2%, respectively, for 10 replicate measurements of serum spiked with 5 mg l-1 of each of the drugs.

Bronchodilator Agents↗

PubMed1998

[Estimation of adverse drug reactions by the evaluation scores of subjective symptoms (complaints) and background of patients. I. Drug-induced liver disorders].

The purpose of this study was to develop, implement, and assess an estimation procedure for preventing drug-induced liver disorders. We have built a database for CARPIS (case reports of adverse drug reaction and poisoning information services) since 1987, and the case reports of adverse drug reaction accumulated in the CARPIS database to be total about 11,000. We studied the estimation procedure by evaluating the subjective symptoms, backgrounds and laboratory data of patients in 199 cases cumulated in the CARPIS database. The evaluation scores were created on the basis of the subjective symptoms and backgrounds of the patients. Then, we re-estimated 199 cases with the evaluation scores, among which were 165 cases (82.9%) in 199 cases drug-induced liver disorders. For the rest of 34 cases (17.1%) drug-induced liver disorders could not be estimated from our evaluation scores. These 34 cases were either those which the subjective symptoms were not described in their reports, because the patients were hospitalized for other diseases and drug-induced liver disorders were discovered by a clinical examination, or cases of infants for whom it was difficult to confirm the subjective symptoms. The validity of this evaluation scores were sensitivity = 82.9%, specificity = 91.0% and predictive value of positive test = 94.8%. To apply this evaluation scores onto the clinical practice, we prepared an evaluation form for the subjective symptoms and backgrounds of the patients with drug suspecting-induced liver disorders.

Adverse Drug Reaction Reporting Systems↗

PubMed1998

Gene cloning and characterization of an acidic xylanase from Acidobacterium capsulatum.

The gene xynA encoding an acid endo-beta-1,4-xylanase from an acidophilic bacterium, Acidobacterium capsulatum 161, was cloned and expressed in Eschrichia coli. The nucleotide sequence of the 1.6-kb DNA fragment containing xynA was analyzed, revealing an open reading frame of 1,215 bp encoding a peptide of 405 amino acid residues. The deduced amino acid sequence of XynA was very similar to other xylanases that are from the glycosyl hydrolase family 10. XynA was purified to homogeneity by SDS-polyacrylamide gel electrophoresis from E. coli transformants. The molecular mass and isoelectric point of XynA were 41 kDa and 7.3, respectively. The xylanase activity of the cloned XynA is an endo-acting enzyme that shows optimal activity at pH 5.0 and 65 degrees C, and is stable pH between 3.0 and 8.0. The K(m) and Vmax with oat spelt xylan as a substrate at pH 5.0 and 30 degrees C are 3.5 mg/ml and 403 mumol/min/mg.

Amino Acid Sequence↗

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

Intrapulmonary satellite nodule of lung cancer as a T factor.

OBJECTIVE: We conducted a validation of the treatment of satellite nodules in the UICC TNM classification of 1993 and 1997. PATIENTS AND METHODS: The clinical records of the 352 patients with T2-4 lung cancer who underwent curative resection were reviewed. The UICC classification of 1993 was used to classify the pathologic stage. No satellite nodule was found in 305 cases (PM0 group), 39 cases had a satellite nodule in the same lobe as that of the primary tumor (PM1 group), and 8 cases had a satellite nodule in another ipsilateral lobe (PM2 group). Outcome was compared based on the T classification. RESULTS: The differences of the survival rates between the PM0 and PM1 groups were not statistically significant for T2 and T3 cases. For T4 cases, the 5-year survival rate was 0% in the PM0 group, 33.3% in the PM1 group, and 0% in the PM2 group. CONCLUSION: It was appropriate for T2 and T3 cases that the rank of T classification was up-staged for a satellite nodule in the same lobe according to the 1993 classification. For T4 cases, however, the PM1 group had a significantly better outcome. If all PM1 cases with any T and N factor are included in T4 according to the 1997 classification, it may elevate the survival rate of T4 better than that of T4 by the former classification.

Adenocarcinoma↗