An organic solvent-tolerant bacterium and its organic solvent-stable protease.
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Endopeptidases↗
Biomedical subjects
F Watanabe
Publications and source records attributed to F Watanabe.
CPP32/Yama/apopain cleaves the catalytic component of DNA-dependent protein kinase in the holoenzyme.
DNA-dependent protein kinase (DNA-PK) is composed of a 460-kDa catalytic component (p460) and a DNA-binding component Ku protein. Immunoblot analysis after treatment of Jurkat cells with anti-Fas antibody demonstrated the cleavage of p460 concomitantly with an increase in CPP32/Yama/apopain activity. Recombinant CPP32/Yama/apopain specifically cleaved p460 in the DNA-PK preparation that had been purified from Raji cells into 230- and 160-kDa polypeptides, the latter of which was detected in anti-Fas-treated Jurkat cells. The regulatory component Ku protein was not significantly affected by CPP32/Yama/apopain. DNA-PK activity was decreased with the disappearance of p460 in the incubation of DNA-PK with CPP32/Yama/apopain. These results suggest that the catalytic component of DNA-PK is one of the target proteins for CPP32/Yama/apopain in Fas-mediated apoptosis.
Amino Acid Sequence↗
A Case of Breast Cancer Metastatic to the Pituitary Gland.
A case of breast cancer that developed pituitary metastasis 22 years after mastectomy is reported. The pituitary metastasis was associated with hypopituitarism, impairment of the visual field and later diabetes insipidus. The serum levels of CA15-3 and NCC-ST-439, tumor markers of breast cancer, were increased, and CA15-3 (DF3) and NCC-ST-439 were demonstrated in the resected pituitary metastatic lesion immunohistochemically.
Journal Article↗
Autoantibodies to DNA-dependent protein kinase. Probes for the catalytic subunit.
DNA-dependent protein kinase (DNA-PK) is an important nuclear enzyme which consists of a catalytic subunit known as DNA-PKcs and a regulatory component identified as the Ku autoantigen. In the present study, we surveyed 312 patients in a search for this specificity. 10 sera immunoprecipitated a large polypeptide which exactly comigrated with DNA-PKcs in SDS-PAGE. Immunoblot analysis demonstrated that this polypeptide was recognizable by a rabbit antiserum specific for DNA-PKcs. Although the patient sera did not bind to biochemically purified DNA-PKcs in immunoblots or ELISA, they were able to deplete DNA-PK catalytic activity from extracts of HeLa cells in a dose-dependent manner. We conclude that these antibodies should be useful probes for studies which aim to define the role of DNA-PK in cells. Since six sera simultaneously contained antibodies to the Ku protein, these studies suggest that relatively intact forms of DNA-PK complex act as autoantigenic particles in selected patients.
Antigens, Nuclear↗
Enzymatic and fluorescence studies of four single-tryptophan mutants of rat testis fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase.
In order to determine environments around four tryptophan residues, located in the N-terminus, in the kinase and in the phosphatase domains of rat testis Fru 6-P,2-kinase:Fru 2,6-bisphosphatase, mutant enzymes containing a single tryptophan were constructed by site-directed mutagenesis. The kinetic constants of these mutant enzymes were similar to those of the wild-type enzyme. The sum of the fluorescence intensities of the enzymes was 1.5 x that of the wild-type enzyme, and Trp 299, Trp 64, Trp 15, and Trp 320 contributed 38%, 28%, 17%, and 17%, respectively. The fluorescence polarization of the wild-type enzyme was significantly lower than any of the mutant enzymes, suggesting proximity of two tryptophan residues in the wild-type enzyme. The polarization in the presence of Fru 6-P affected only Trp 15, which suggested that it is located near the Fru 6-P binding site, but Trp 64 is not. Inactivation of both enzyme activities and unfolding of these enzymes in guanidine were monitored by activity assays and fluorescence intensities and maxima. Both Fru 6-P,2-kinase and Fru 2,6-bisphosphatase activities of all these enzymes were inactivated between 0.7 and 1 M guanidine. Enzymes containing Trp 64 or Trp 15 showed biphasic fractional unfolding curves, but those of Trp 299 or Trp 320 showed gradual steady changes. Fluorescence quenching by iodide indicated that Trp 64 was not accessible and that other Trp residues were only slightly accessible to solvent. These results suggest that all the Trp residues are in heterogeneous environments and that none are exposed on the protein surface.
Animals↗
Accumulation of methylmalonic acid caused by vitamin B12-deficiency disrupts normal cellular metabolism in rat liver.
To clarify the relationship between intracellular concentrations of methylmalonic acid and metabolic and growth inhibition in vitamin B12-deficient rats, hepatic methylmalonic acid levels were assayed and inhibition of glucose and glutamic acid metabolism by methylmalonic acid was studied in isolated hepatocytes. Vitamin B12-deficient rats (14 weeks old) excreted more urinary methylmalonic acid and had lower body weights than the control rats. Hepatic methylmalonic acid levels (3.6(SD 1.30)-5.3 (SD 0.51) mumol/g tissue; 7.9 (SD 2.90)-11.8 (SD 1.14) mM) were increased and correlated with the extent of the growth retardation during vitamin B12-deficiency. Isolated hepatocytes and mitochondria from normally fed rats were labelled with [14C(U)]glucose and [14C(U)]glutamic acid respectively, in the presence or absence of 5 mM-methylmalonic acid. Although methylmalonic acid did not affect the incorporation of 14C into protein and organic acid fractions in the hepatocytes, it inhibited 14CO2 formation (an index of glucose oxidation by the Krebs cycle) by 25% and incorporation of 14C into the amino acid fraction by 30%. In the mitochondria, methylmalonic acid inhibited 14CO2 formation (indicating glutamic acid oxidation by the Krebs cycle) by 70%, but not the incorporation of 14C into the protein fraction. The incorporation of 14C into the organic acid fraction was significantly stimulated by the addition of methylmalonic acid. These results indicate that the unusual accumulation of methylmalonic acid caused by vitamin B12-deficiency disrupts normal glucose and glutamic acid metabolism in rat liver, probably by inhibiting the Krebs cycle.
Animals↗
Mitochondrial NADH- or NADPH-linked aquacobalamin reductase activity is low in human skin fibroblasts with defects in synthesis of cobalamin coenzymes.
Mammalian livers have been reported to contain NADH- and NADPH-linked aquacobalamin reductases, which are distributed in both mitochondria and microsomes. The four aquacobalamin reductase isozymes have been purified and characterized from rat liver. It is unclear which aquacobalamin reductase among the four reductase isozymes participates in the synthesis of cobalamin coenzymes. To clarify the physiological roles of the aquacobalamin reductase isozymes, human mutant fibroblasts (cblC and cblA cells) with defects in cobalamin reductases involved in the coenzyme synthesis were used. In the cblC cells, the activity of the mitochondrial NADH-linked aquacobalamin reductase was reduced significantly, compared with normal human fibroblasts but the mitochondrial NADPH-linked enzyme was not. The reduced specific activity of the NADH-linked enzyme was not due to reduction in levels of the enzyme, but in its affinity for NADH. Although there was not a significant difference in the mitochondrial NADH-linked enzyme activity between normal and cblA cells, the activity of the mitochondrial NADPH-linked enzyme was not detectable in the mutant cells. These results indicate that the defects in the mitochondrial NADH- and NADPH-linked aquacobalamin reductases underlie cblC and cblA disorders, respectively.
Cells, Cultured↗
Adenosylcobalamin-dependent methylmalonyl-CoA mutase isozymes in the photosynthetic protozoon Euglena gracilis Z.
The photosynthetic protozoon Euglena gracilis Z contains adenosylcobalamin-dependent methylmalonyl-CoA mutase (MCM) involved in propionate metabolism. The specific activity of the Euglena mutase was about 6.5-fold greater in propionate-adapted Euglena cells than in photoautotrophic cells (control). Although the control cells contained only one mutase (apparent M(r) 72,000), the propionate-adapted cells contained two mutases with M(r) values of 72,000 and 17,000; both enzymes were located in the mitochondria. These results provide evidence that propionate-adapted Euglena contains two MCM isozymes. The induced mutase (M(r) 17,000) permits photoassimilation of propionate.
Animals↗
Effect of penicillin G on the electroporation of Rhodococcus rhodochrous CF222.
Suitable conditions for the introduction of bacteriophage DNA into cells of Rhodococcus rhodochrous CF222 by electroporation were established, and penicillin G was found to enhance the transfection frequency. When conditions optimal for the parental strain were applied to its colony-morphological mutants, different transfection frequencies were observed. Penicillin G enhanced the transfection frequency of smooth and mucoidal mutants but not of rough mutants.
Bacteriophages↗
Ovarian epithelial borderline tumor and carcinoma in young women.
To clarify the influence of age on clinical and histologic features, young women with ovarian epithelial borderline tumors and carcinomas were analyzed. A retrospective review of 93 patients with ovarian common epithelial borderline tumors and carcinomas was performed. Twenty-one of 93 patients (22.6%) were under age 40. Predominant symptoms in young patients were abdominal pain or abdominal distention, but 4 (19%) patients had no symptom. Eleven (11/21; 52.4%) patients were nulliparous and six patients received conservative surgery to preserve fertility. The rate of histologic type and stage did not significantly differ between the young and older groups. Although overall survival between young and older patients did not significantly differ, the survival of young patients with advanced disease (stage III and IV) was significantly worse than in older patients with advanced disease. (p < 0.05).
Adolescent↗
[K-ras gene mutations in adenomas from familial adenomatous polyposis].
A 18-year-old woman underwent total colectomy for familial adenomatous polyposis. In order to clarify the significance of K-ras mutations in early colorectal carcinogenesis, K-ras mutations were analyzed in multiple adenomas by PCR-SSCP method. A total of 256 adenomas were found throughout the entire colon and rectum, and the distribution was a sparse type. The correlation between K-ras gene and clinicopathological factors was examined in 90 adenomas. There was no correlation among K-ras mutations and anatomical distribution, or morphological classification, but K-ras mutation was more frequent in severe compared with slight atypia. We investigated the correlation between the size of adenoma in the horizontal and vertical directions and K-ras mutation. K-ras mutation was more frequent in the horizontal size greater than 6 mm in diameter, and also more frequent in vertical size greater than 20 mm in height. It was concluded that the adenomas detecting K-ras mutations might have proliferating potential, and would be applied to determine polypectomy.
Adenoma↗
[Determination of optimal technical factors for the single breath-hold pulmonary MR angiography and its clinical applications].
To optimize pulmonary MR angiography for the noninvasive evaluation of pulmonary vasculature, six healthy volunteers were examined using the fast radiofrequency spoiled gradient echo sequence (Fast SPGR) and standard body coil of a commercial 1.5T MR imaging system. The examinations by 2D Fast SPGR were performed under various TR, flip angles and slice thicknesses within the time of a single breath hold, and those by 3D Fast SPGR were performed under various flip angles and slab thicknesses. The most satisfactory results were obtained by 2D Fast SPGR with the parameters of TR 60 msec, TE 2.1 msec, flip angle 20 degrees slice thickness 10 mm, FOV 30-40 cm, matrix 256 x 192 and NEX1. For 3D Fast SPGR, TR 10 msec, TE 1.9 msec, flip angle 10 degrees, slab thickness 60mm (12 partitions), FOV 30-40 cm, matrix 256 x 128 and NEX1 were best. Concomitant injection of Gd-DTPA provided higher resolution of peripheral vessels. The MIP images showed pulmonary vasculature with resolution of vessels beyond 4 th order branches in 2D Fast SPGR and 5 th order branches in 3D Fast SPGR. 3D Fast SPGR with contrast enhancement was applied to patients with primary lung cancer in the hilum, malignant mediastinal tumor, pulmonary embolism, pulmonary arteriovenous fistula, pulmonary varix, and round atelectasis. Pulmonary MR angiography is considered to be a noninvasive and effective method not only for the evaluation of the tumor invasion to the central pulmonary vessels but also for the demonstration of other pulmonary vascular pathology.
Adult↗
Hepatic support strategies.
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Adolescent↗
[Dobutamine stress causes left ventricular outflow tract obstruction].
Hypotension during dobutamine stress echocardiography is caused by ischemia as well as non-ischemic causes. Whether sigmoid interventricular septum seen in the aged can cause hypotension during dobutamine stress echocardiography was investigated in eight men and four women with sigmoid interventricular septum (aged 53 to 76 years, mean 67 +/- 7 years). At peak dobutamine dose, seven patients (group H) showed a hypotensive response (defined as 5 mmHg or greater decrease in systolic blood pressure from the peak systolic blood pressure; mean = - 17 +/- 13 mmHg), while five subjects (group N) did not. No subject showed regional wall motion abnormalities. Before dobutamine infusion, group H had smaller left ventricular end-systolic dimension (26 +/- 3 vs 30 +/- 3 mm) than group N, but no difference was found in left ventricular end-diastolic dimension (44 +/- 3 vs 47 +/- 4 mm), percentage fractional shortening (40 +/- 6% vs 34 +/- 7%), diastolic aorto-septal angle (82 +/- 10 vs 95 +/- 11 degrees), systolic aorto-septal angle (94 +/- 10 vs 101 +/- 11 degrees), peak left ventricular outflow velocity (1.3 +/- 0.2 vs 1.2 +/- 0.3 m/sec) or peak left ventricular outflow pressure gradient (7 +/- 2 vs 6 +/- 3 mmHg). Group H had a lower peak dobutamine dose than group N (33 +/- 8 vs 40 +/- 7 micrograms/kg/min), but under the peak dose of dobutamine infusion group H showed smaller left ventricular end-systolic dimension (20 +/- 3 vs 26 +/- 4 mm), systolic mitral annulus diameter (19 +/- 3 vs 23 +/- 2 mm), diastolic aorto-septal angle (72 +/- 16 vs 92 +/- 6 degrees), and systolic aorto-septal angle (84 +/- 12 vs 100 +/- 6 degrees), and higher heart rate (114 +/- 10 vs 79 +/- 16 bpm), percentage fractional shortening (53 +/- 8% vs 43 +/- 7%), peak left ventricular outflow velocity (3.2 +/- 0.8 vs 1.7 +/- 0.3 m/sec), and peak left ventricular outflow pressure gradient (43 +/- 23 vs 12 +/- 5 mmHg). In addition, systolic anterior motion of the mitral valve with septal contact developed in 86% of group H and 0% of group N. Thus, about half of the patients with sigmoid interventricular septum show hyperresponse to dobutamine and develop dynamic left ventricular outflow tract obstruction as well as systemic arterial hypotension even without regional left ventricular wall motion abnormalities.
Aged↗
[Liver autoreaction in hepatitis virus infection].
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Chronic Disease↗
Increased nitric oxide in the exhaled air of patients with decompensated liver cirrhosis.
OBJECTIVE: To determine if nitric oxide output in exhaled air is increased in patients with liver cirrhosis. DESIGN: Cross-sectional study. SETTING: A university hospital. PATIENTS: 50 patients with liver cirrhosis, 6 patients with chronic hepatitis, and 15 healthy controls. MEASUREMENTS: Nitric oxide in exhaled air was measured using a chemiluminescence analyzer. Cardiac index was determined using echocardiography. RESULTS: Patients with decompensated liver cirrhosis had higher levels of exhaled nitric oxide output (Child C patients, 190 +/- 11 nL/min per m2 body surface area) than controls (97 +/- 8 nL/min per m2 body surface area; P < 0.001), whereas patients with compensated liver cirrhosis or chronic hepatitis had levels of nitric oxide output similar to those found in controls. Cardiac index was greater in patients with liver cirrhosis (Child C patients, 4.3 +/- 0.3 L/min per m2 body surface area) than in controls (2.9 +/- 0.2 L/min per m2 body surface area; P < 0.001). Cardiac index correlated with nitric oxide levels (r = 0.621; P < 0.001). CONCLUSIONS: Increased nitric oxide output in exhaled air is associated with systemic circulatory disturbances in patients with liver cirrhosis.
Aged↗
Hexose phosphate binding sites of fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase.
A previous chemical modification study [Kitamura et al. (1989) J. Biol. Chem. 264, 6344-6438] has shown that N-bromoacetylethanolamine phosphate labeled specifically Cys107 of rat liver Fru 6-P,2-kinase:Fru 2.6-Pase and the corresponding Cys of the bovine heart enzyme, leading to inactivation of kinase activity. Since Fru 6-P provided protection against the inactivation, this region of the enzyme was thought to be a Fru 6-P binding site of the kinase enzyme. To examine this possibility, oligonucleotide-directed mutagenesis has been used to alter several residues in expressed rat testis Fru 6-P,2-kinase:Fru 2,6-Pase. The change of Lys100, Lys103, and Asp112 caused at most a 2-fold increase in KmF6P and a 2-3-fold increase in KmATP, suggesting that these residues are not involved in the direct binding of Fru 6-P. However, change of Arg102 to Leu and to Lys resulted in a 325x and 22x, respectively, increase in KmF6P, and change of Arg102 to Glu resulted in nearly complete loss of the kinase activity. Change of Cys105 to Ala or Ser increased KmF6P about 10x. The Vmax of all these mutated enzymes except the one that changed Arg102 to Glu (R102E) was increased 10% to 85%. The kinetic parameters of Fru 2,6-Pase were not altered by these changes. R102E formed several polymeric forms of the enzyme, including a tetramer. Both R102E and an additional derivative that substituted Lys for Arg102 (R102K) were slightly more susceptible to guanidine inactivation than the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗