Biomedical subjects
Publications and source records attributed to S Toyoshima.
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Routine analysis of the carbohydrate moieties of a glycoprotein is critical for ensuring the consistent quality of biopharmaceutical products. Fluorophore-assisted carbohydrate electrophoresis (FACE) is a recently introduced method for the separation, by polyacrylamide gel electrophoresis, of oligosaccharides labeled with 8-amino-naphthalene-1,3,6-trisulphonic acid (ANTS). In this study, we have evaluated the applicability of the FACE method to analysis of the carbohydrate moieties of three different recombinant human erythropoietins (rHuEPOs), two Chinese hamster ovary (CHO) cell-derived rHuEPOs, and one baby hamster kidney (BHK) cell-derived rHuEPO. The N-linked oligosaccharides released from the rHuEPOs were labeled with ANTS. Enzymic sequence analysis of the N-linked oligosaccharides was performed by the FACE method. The results showed that the FACE method was useful for the analysis of sialo-, asialo-, and agalacto-oligosaccharides in the same gel. Its usefulness for rapidly and reliably revealing the oligosaccharide profiles of given glycoproteins, and its reproducibility were also confirmed in this study. In conclusion, the method can be used for evaluating the quality consistency of recombinant glycoprotein products in terms of the carbohydrate moiety.
Pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase is an NADPH-dependent enzyme that catalyses the reduction of ketones on steroids and aldehydes and ketones on various xenobiotics, like its homologue carbonyl reductase. 3alpha/beta,20beta-Hydroxysteroid dehydrogenase and carbonyl reductase are members of the short-chain dehydrogenases/reductase family, in which a tyrosine residue and a lysine residue have been identified as catalytically important. In pig 20beta-hydroxysteroid dehydrogenase these residues are tyrosine-194 and lysine-198. Here we report the effect on the reduction of two ketone and two aldehyde substrates by pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase in which tyrosine-194 has been mutated to phenylalanine and cysteine, and lysine-198 has been mutated to isoleucine and arginine. Mutants with phenylalanine-194 or isoleucine-198 are inactive. Depending on the substrate, the mutant with cysteine-194 has a catalytic efficiency of 0.4-1% and the mutant with arginine-198 has a catalytic efficiency of 4-23% of the wild-type enzyme. We also mutated tyrosine-81 and tyrosine-253 to phenylalanine. Although both tyrosines are conserved in 3alpha/beta,20beta-hydroxysteroid dehydrogenase and carbonyl reductase, depending on the substrate, the mutant enzymes are as active as, or more active than, wild-type enzyme.
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We report herein an unusual case of a composite glandular-neuroendocrine carcinoma of the hilar bile duct. A 71-year-old Japanese woman was admitted to our hospital suffering from general fatigue, progressive jaundice, and a high fever. Computed tomography and angiography findings revealed a solid hypervascular mass in the hepatic hilus. Thus, a subsegmentectomy of the liver (S4, S5) and bile duct resection with lymph node dissection were performed. A tumor measuring 6.0 x 3.0 cm was found to be located in the bile duct of the hepatic hilus. Histologically, the tumor was composed of well-differentiated adenocarcinoma and small cell neuroendocrine carcinoma cells, with a histological transition between the two components. Grimelius' method revealed the presence of diffuse positive tumor cells in neuroendocrine carcinoma. The neuroendocrine tumor cells were also diffusely immunoreactive to chromogranin A. To the best of our knowledge, only 22 previous cases of composite glandular-neuroendocrine carcinoma in the biliary tract have been reported; however, this is the first case report of a clearly composite tumor of the hilar bile duct.
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.
We herein report a case of secretory carcinoma of the breast in a 73-year-old woman. Secretory carcinoma is an extremely rare tumor and demonstrates distinctive pathologic characteristics. This tumor frequently occurs in either children or adolescents, and thus has been called juvenile carcinoma. We encountered this rare tumor in an elderly woman. Aspiration biopsy cytology was performed twice, but the cytological diagnosis was not carcinoma. Such pathologic characteristics as mild atypia, the absence of hyperchromasia, and a minimal degree of pleomorphism in the tumor cells can thus lead to a cytological misdiagnosis. An excisional biopsy was performed and secretory carcinoma was finally diagnosed. Consequently, a modified radical mastectomy (Kodama's method) was performed 7 days later. We describe this very rare tumor's clinicopathologic characteristics.
Two patients with malignant melanoma were evaluated using Tl-201 scintigraphy. Planar scintigraphy showed tumor accumulation, and SPECT Tl-201 imaging revealed exact tumor localization. The findings indicate the utility of Tl-201 to detect the primary lesion and to identify postoperative recurrence in malignant melanoma.
We have isolated an NADPH-dependent reductase from neonatal pig testes that metabolizes androgens and a variety of xenobiotics. This enzyme is distinct from 3alpha/beta,20beta-hydroxysteroid dehydrogenase or its homologue, carbonyl reductase, as judged by its immunological and molecular properties and its much narrower specificity for steroids. This reductase and 3alpha/beta,20beta-hydroxysteroid dehydrogenase may be part of a mechanism for regulating androgen levels the neonatal pig testes. Interestingly, we could not find multiple isoforms of 3alpha/beta,20beta-hydroxysteroid dehydrogenase/carbonyl reductase in pig testes unlike human and rat testes and other organs in which multiple isoforms are expressed.
Fluorescence in situ hybridization (FISH) was applied to 15 alveolar rhabdomyosarcomas (A-RMSs) and 14 embryonal RMSs (E-RMSs) to detect N-myc (also called MYCN) oncogene amplification. The results were compared with histologic characteristics and clinical factors. The number of surviving patients in each subtype was 5 of 15 with A-RMS and 5 of 14 with E-RMS. N-myc amplification was detected in 9 of the 15 A-RMSs but in none of the 14 E-RMSs. Tumor cells exhibiting N-myc amplification were identified only in the alveolar area in two A-RMSs, and they demonstrated a histologic mixture of alveolar and embryonal patterns. The remaining seven cases with an amplified N-myc showed a conventional alveolar pattern. Among the 15 A-RMSs, the survival rate of patients with tumors showing nonamplified N-myc and amplified N-myc oncogene was 4 (66%) of 6 and 1(11%) of 9, respectively (P < .05). No significant difference was observed between the other clinical findings (age, primary sites, clinical stages) of the N-myc-amplified and nonamplified tumors. Therefore, we concluded that the N-myc gene amplification, which is characteristic of a particular subtype of A-RMS, might be useful as a prognostic factor for an unfavorable outcome.
We herein report the successful surgical treatment of 2 cases of chronic expanding hematoma in the chest. The first patient, who had undergone thoracoplasty 42 years earlier due to tuberculosis, became aware of a slowly growing mass protruding in the lateral thoracic wall. The second patient, who had tuberculous pleurisy 36 years earlier, was referred to our department because of a slowly expanding intrathoracic mass revealed by a roentgenogram. The tumors, which were encapsulated chronic hematomas, were both surgically resected. These cases are rare because of the development of a very large mass after undergoing treatment for tuberculosis more than 30 years previously.
OBJECTIVE: To evaluate signals from breast lesions on both colour and spectral Doppler US, and to correlate findings with angiographic and histopathological features including microvessel density. MATERIALS AND METHODS: One hundred and sixteen breast lesions in 113 patients were evaluated with colour Doppler ultrasonography. Subjective and semi-quantitative assessment of colour signals as well as spectral Doppler analysis were performed and compared. Comparison of colour Doppler features with angiographic and histopathological findings were also carried out. RESULTS: Subjective evaluation revealed that colour signals were more commonly found in malignant (89%) than benign (56%) lesions. Malignant lesions demonstrated a more elaborate vascular network (51%) compared to 12.5% in benign lesions. Spectral Doppler analysis revealed slightly higher specificity values for the pulsatility and resistivity indices, respectively, but lower sensitivity and accuracy values compared to qualitative assessment. Colour Doppler patterns corresponded well with angiograhic images, however, the correlation between colour Doppler parameters and microvessel density was not significant. CONCLUSION: Despite the increased examination time required, spectral analysis does not appear to contribute significantly to the differentiation between malignant and benign breast tumours. Features such as the density, pattern and predominant site of colour signals may be more useful for the evaluation of breast lesions on colour Doppler imaging.
The carbonyl reductase activity exhibited by pig testicular 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) was examined using a recombinant enzyme. Kinetic parameters were obtained for 48 carbonyl group-containing substrates, including aromatic aldehydes, aromatic ketones, cycloketones, quinones, aliphatic aldehydes and aliphatic ketones. 20 beta-HSD showed a high affinity towards quinones, such as 9,10-phenanthrenequinone, alpha-naphthoquinone and menadione (Km values of 4, 2 and 5 microM, respectively), and the substrate utilization efficiency (Vmax/Km) of the enzyme against these quinones was very high. Cyclohexanone and 2-methylcyclohexanone were also reduced with a high Vmax/Km value, but not cyclopentanone or 2-methylcyclopentanone. Various aromatic aldehydes and ketones including benzaldehyde- and acetophenone-derivatives were reduced by 20 beta-HSD. Especially, 4-nitrobenzaldehyde and 4-nitroacetophenone were reduced with high Vmax/Km values in the related compounds. The enzyme also reduced the pyridine-derivatives, 2-, 3-, and 4-benzoylpyridine, with the Vmax/Km value for 2-benzoylpyridine being the highest. 20 beta-HSD reduced aliphatic aldehydes and aliphatic ketones, but was more effective on the former. The correlation between the structure of carbonyl compounds and their substrate Vmax/Km is discussed.
The use of transthoracic color and pulsed Doppler echocardiography to detect intramyocardial coronary artery flow in humans was evaluated in 18 normal healthy subjects (mean age 54 years) and in 16 patients with hypertrophic cardiomyopathy (HCM; mean age 59 years) to measure the intramyocardial coronary artery flow velocity at the ventricular septum and the apex using a 10-5 or 7-4 MHz transducer. Linear inflow color Doppler signals which passed the interventricular septum were demonstrated in 15 of 18 normal subjects (83%) and 15 of 16 patients with HCM (94%). The phasic flow velocities measured by pulsed Doppler echocardiography consisted of two forward flow signals in mid-systole (S-wave) and holodiastole (D-wave), and were obtained in 11 of 18 in normal subjects (61%) and 14 of 16 patients with HCM (88%). The mean peak velocities of the S- and D-waves in patients with HCM (mean [+/-SD] 27 +/- 9 and 86 +/- 23 cm/sec, respectively) were significantly (p < 0.05) higher than those in normal subjects (18 +/- 4 and 54 +/- 11 cm/sec, respectively). At the apex, linear inflow color Doppler signals which passed the myocardium perpendicularly during the whole diastole were demonstrated in 14 of 18 normal subjects (78%) and all 16 patients with HCM (100%). The phasic flow velocities were measured by pulsed Doppler echocardiography in 10 of 18 normal subjects (56%) and 15 of 16 patients with HCM (94%). The mean peak velocities in patients with HCM (74 +/- 27 cm/sec) were significantly (p < 0.05) higher than those in normal subjects (33 +/- 13 cm/sec). Transthoracic color and pulsed Doppler echocardiography can detect intramyocardial coronary artery flow in humans at the interventricular septum and the apex noninvasively.
A 52-year-old Japanese woman presented with complaints of back pain, loss of appetite, and weight loss; her diagnosis was a mass in the pancreatic tail. A distal pancreatectomy combined with lymph node dissection was performed. The tumor measured 10.0 x 9.5 x 8.0 cm and consisted of a cystic mass and a solid area. Histologically, the cystic mass represented a typical mucinous cystadenocarcinoma, whereas the solid portion was composed of anaplastic tumor cells with round eosinophilic intracytoplasmic inclusions, as seen in malignant rhabdoid tumor of the kidney. The hyaline-like intracytoplasmic inclusions were weakly positive for periodic acid-Schiff and were resistant to diastase. There was a gradual transition between the mucinous cystadenocarcinoma and the anaplastic carcinoma showing rhabdoid features. Additionally, the tumor cells of the anaplastic carcinoma were immunoreactive to both epithelial membrane antigen and vimentin. These findings suggest that the anaplastic carcinoma with rhabdoid features developed from the mucinous cystadenocarcinoma of the pancreas.
It was previously reported that vaccination with synthetic peptides corresponding to the CDR2 or CDR3 region of T cell receptor (TCR) protected susceptible animals from the development of experimental autoimmune encephalomyelitis (EAE). However, recent studies by several research groups have revealed that TCR peptide therapy often confers little or no protection from autoimmune disease. In the present study, we attempted to find more appropriate peptides that is capable of conferring effective protection against the development of EAE. Four peptides corresponding to parts of the V beta region (13-23, 24-36, 39-59, and 64-74) were selected by epitope scanning and hydrophilicity searching, and their protective abilities were tested. All these peptides were, however, ineffective in protecting rats from the disease. We also generated three different synthetic peptides corresponding to the TCR J region of encephalitogenic T cells. Vaccination with the J-region peptides did not protect animals from the development of EAE. Rather, one of the peptides (V beta-DSS-J beta 2.6) enhanced the clinical severity of EAE and induced fatal disease in some rats. Taken together, TCR peptide therapy appears to be generally ineffective and elucidation of the mechanism by which EAE is enhanced after TCR peptide vaccination should provide insight into the pathogenesis of this disease.
A human macrophage calcium-dependent (C-type) lectin cDNA clone was obtained from a library derived from IL-2-treated peripheral blood monocytes. The cDNA cloning was based on the structural homology to hepatic asialoglycoprotein receptors. The nucleotide sequence of this cDNA clone was homologous to those of the galactose- and N-acetylgalactosamine-specific C-type macrophage lectins of rodents. In the putative carbohydrate recognition domain, deduced amino acid sequence revealed 60 and 63% homology to galactose- and N-acetylgalactosamine-specific C-type macrophage lectins of mice and rats, respectively. The cDNA clone was ligated into a mammalian expression vector and transfected into COS-1 cells. In the lysates of these cells, an MR 38,000 component, which bound to galactose-Sepharose, was identified after electrophoretic separation by its interaction with polyclonal antisera against synthetic polypeptides representing a portion of the carbohydrate recognition domain. The carbohydrate-binding specificity of the recombinant macrophage lectin was investigated by comparing elution profiles of various glycopeptides having defined carbohydrate structures from immobilized macrophage lectins. When N-terminal octapeptides from human glycophorin A that bore NeuAc alpha 2-3 Gal beta 1-3 (NeuAc alpha 2-6) GalNAc and its sequentially deglycosylated derivatives were compared, glycopeptides carrying three constitutive GalNAc-Ser/Thr (Tn Ag) strongly bound to the recombinant human macrophage lectin. This is the first study to demonstrate that human macrophage cell surface lectin recognizes Tn Ag, a well-known human carcinoma-associated epitope.