Biomedical subjects
S Shimizu
Publications and source records attributed to S Shimizu.
Beta-ureidopropionase with N-carbamoyl-alpha-L-amino acid amidohydrolase activity from an aerobic bacterium, Pseudomonas putida IFO 12996.
beta-Ureidopropionase of aerobic bacterial origin was purified to homogeneity from Pseudomonas putida IFO 12996. The enzyme shows a broad substrate specificity. In addition to beta-ureidopropionate (Km 3.74 mM, Vmax 4.12 U/mg), gamma-ureido-n-butyrate (Km 11.6 mM, Vmax 19.4 U/mg), and several N-carbamoyl-alpha-amino acids, such as N-carbamoylglycine (Km 0.68 mM, Vmax 9.14 x 10(-2) U/mg), N-carbamoyl-L-alanine (Km 1.56 mM, Vmax 1.00 U/mg), N-carbamoyl-L-serine (Km 75.1 mM, Vmax 3.78 U/mg), and N-carbamoyl-DL-alpha-amino-n-butyrate (Km 2.81 mM, Vmax 1.08 U/mg), are also hydrolyzed. The hydrolysis of N-carbamoyl-alpha-amino acids is strictly L enantiomer specific. N-Formyl-L-alanine and N-acetyl-L-alanine are also hydrolyzed by the enzyme, but the rate of hydrolysis is lower than the rate for N-carbamoyl-L-alanine. The enzyme requires a divalent metal ion, such as Co2+, Ni2+ or Mn2+, for activity, and is significantly affected by sulfhydryl reagents. The enzyme consists of two polypeptide chains with identical relative molecular mass M(r) 45000. The broad substrate specificity and metal ion dependence of the enzyme show that the beta-ureidopropionase of this aerobic bacterium is quite different from the beta-ureidopropionases of mammals and anaerobic bacteria.
Leukemic meningitis in a patient with splenic lymphoma with villous lymphocytes (SLVL). Meningitis as a possible initial manifestation of SLVL.
BACKGROUND: Splenic lymphoma with villous lymphocytes (SLVL) is a low grade, non-Hodgkin's lymphoma with a stable or slowly progressive clinical course. To the authors' knowledge, central nervous system involvement has not been described previously in patients with SLVL. METHODS: Morphologic, immunocytochemical, and immunohistochemical analyses were conducted to determine the nature of villous lymphocytes in the peripheral blood, spleen, and cerebrospinal fluid (CSF) of a patient with massive splenomegaly. RESULTS: A diagnosis of SLVL was made, based on tartrate-resistant acid phosphatase-negative peripheral villous lymphocytosis with CD19+, CD20+, HLA-DR+ phenotypes, and the involvement of spleen white pulp with these cells. Mononuclear cells in the CSF showed the same morphologic and immunocytochemical features seen in the villous lymphocytes in the peripheral blood and spleen. Splenectomy and intrathecal chemotherapy were successful in clearing leukemic cells from the CSF. CONCLUSION: In this patient with SLVL in whom leukemic meningitis developed, meningitis was found to be a possible initial manifestation of SLVL.
Autocrine factor enhancing the secretion of M(r) 95,000 gelatinase (matrix metalloproteinase 9) in serum-free medium conditioned with murine metastatic colon carcinoma cells.
We previously reported that murine tumor cells with a high spontaneous metastatic potential to the lung secrete higher amounts of M(r) 95,000 gelatinase (matrix metalloproteinase 9, MMP9) than do poorly metastatic cells. The present study, conducted to clarify the mechanisms underlying the increase in MMP9, revealed an autocrine factor that enhances the secretion of M(r) 95,000 gelatinase (MMP9). The secretion of MMP9 by highly metastatic colon carcinoma LuM1 cells, detected by zymography, was augmented 10-fold when cultured in medium supplemented with serum-free medium conditioned with LuM1 cells. Because the secretion of M(r) 60,000 gelatinase (MMP2), as well as total protein, by the same cells was not affected under these conditions, the augmentation appears specific for MMP9. The steady-state level of MMP9 mRNA was elevated in LuM1 cells cultured in the presence of the supernatant. The amount of the factor in the culture medium increased with time in culture, indicating that it was produced by the LuM1 cells. It was found to be heat stable but sensitive to trypsin digestion. Conditioned medium from poorly metastatic NM11 cells did not stimulate the secretion of gelatinases by NM11 cells, suggesting that autocrine stimulation of MMP9 secretion is a characteristic of metastatic cells. This factor could account for the augmented secretion of MMP9 by murine tumor cells with spontaneous metastatic potential to the lung.
Beneficial effects of cyclosporine on reoxygenation injury in hypoxic rat liver.
The effect of CsA on hypoxia-reoxygenation injury was studied in perfused rat livers. CsA did not attenuate hypoxic injury, as assessed by the release of lactate dehydrogenase and mitochondrial aspartate aminotransferase. During reoxygenation, the release of lactate dehydrogenase was also not affected by CsA. However, the release of mitochondrial aspartate aminotransferase into the cytosol, which indicates mitochondrial injury, was significantly reduced by CsA. The effect of CsA on mitochondrial function during hypoxia-reoxygenation was also investigated. CsA administration increased both the respiratory control ratio and the adenine nucleotide content after reoxygenation in both isolated mitochondria and perfused livers. In addition, glucose production by perfused livers after reoxygenation was increased by CsA. We conclude that the beneficial effect of CsA on hypoxia-reoxygenation injury may be partly due to protection of the mitochondria against reoxygenation injury.
[Endocrine pathological analysis of primary pigmented nodular adrenocortical disease].
We have studied eleven cases of the adrenal with primary pigmented nodular adrenocortical disease (PPNAD) by immunohistochemistry of all steroidogenic enzymes involved in cortisol biosynthesis, by in situ hybridization of P450C17 in seven cases in order to localize the sites of steroidogenesis and by chromosome suppression in situ hybridization of genomic DNA in five cases in order to determine possible genetic abnormalities of the disorder. Immunoreactivity of all the enzymes examined was intense in almost all the cells in adrenocortical nodules while internodular adrenal cortex, including the cases without cortical atrophy, was negative for the enzymes with the exception of 3 beta HSD. In situ hybridization studies of P450C17 yielded results consistent with those of immunohistochemistry. These results may be consistent with autonomous cortisol production by the nodular cells and indicate that almost all of the cells in the cortical nodules produce cortisol, which can explain the presence of hypercortisolism despite small sizes of adrenals in PPNAD. Chromosome suppression in situ hybridization analysis demonstrated possible genetic defects in chromosome 16 in this disorder.
Hairpin loop and second kringle domain are essential sites for heparin binding and biological activity of hepatocyte growth factor.
Hepatocyte growth factor (HGF) has a strong affinity for heparin. About one fourth of HGF secreted from MRC-5 human embryonic lung fibroblast cells was found to be associated with heparin and heparan sulfate proteoglycan on the cell surface and extracellular matrix. To identify heparin-binding sites within the HGF molecule, we constructed variously deleted mutant HGFs and examined their binding ability to an immobilized heparin column. Native HGF and mutant HGFs, including d-K1 (deletion of the first kringle domain), d-K3 (deletion of the third kringle domain), d-K4 (deletion of the fourth kringle domain), d-beta (deletion of beta-chain), and HK1K2 (consisting of the N-terminal hairpin loop and the first two kringle domains), tightly bound to a heparin column, but d-H (deletion of the N-terminal hairpin loop) and d-K2 (deletion of the second kringle domain) markedly decreased binding ability to the column. These observations suggest that the N-terminal hairpin loop and the second kringle domain are essential for the heparin-binding of HGF. The finding that HK1K2 competed the binding of 125I-HGF to immobilized heparin provided additional evidence that the N-terminal half of HGF alpha-chain is the principal heparin-binding site. The hairpin loop in HGF possesses a cluster of basic amino acid residues and a highly positive net charge, when compared with hairpin loop structures in the other proteins, plasminogen and HGF-like protein. The second kringle domain in HGF has the basic amino acid cluster in the central region. Thus, it is likely that the basic clusters in these domains cooperatively contribute to the binding of HGF to the anionic heparin or heparan sulfate molecule.
Gelatinases and metalloproteinase inhibitor secreted by murine colonic carcinoma cells with differing metastatic potential.
Gelatinase activity and inhibitory activity against collagenase were measured in serum-free medium conditioned by murine colonic carcinoma cells with different spontaneous metastatic potentials to the lung. The medium conditioned with poorly metastatic NM11 cells gave higher inhibitory activity than that conditioned with highly metastatic LuM1 cells, while the level of secreted gelatinases in the same medium was lower in NM11 medium than in LuM1 case. Northern analysis showed the higher gene expression of both tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 in NM11 cells than in LuM1 cells, suggesting that both TIMPs are responsible for the increase of inhibitory activity in NM11 conditioned medium. Examination of the balance of gelatinases and inhibitor revealed that the amount of inhibitor exceeded that of gelatinases in the medium conditioned with NM11 cells. In contrast, the medium conditioned with LuM1 cells contained excess amounts of gelatinases. The results indicated a close correlation between the balance of gelatinases and inhibitors and the metastatic behavior of murine tumor cells.
Rearrangement of the rheumatoid factor-related germline gene Vg and bcl-2 expression in lymphoproliferative disorders in patients with Sjögren's syndrome.
Of 250 patients with Sjögren's syndrome (SS) (190 with 1 degree SS and 60 with 2 degrees SS), 60 patients demonstrated lymphoproliferative disorders (LPD): 41 patients had monoclonal gammopathy (MG), 4 had pseudolymphoma, 3 had in situ monoclonal lymphoproliferation, and 12 had malignant lymphoma. Rearrangement of the rheumatoid factor (RF)-related germline gene Vg (associated with the monoclonal RF SF18/2) was demonstrated in 24 of 50 genomic DNAs from the peripheral blood leukocytes of SS patients. It was found in none of 20 DNAs from normal subjects. This suggests an almost 50% incidence of a germline Ig gene rearrangement in SS patients without clinical MG. Lymphocytes composing the lymphoepithelial lesion (LEL) in the major salivary glands of SS patients expressed the oncogene bcl-2 protein in 4 out of 6 patients. The progression of SS from benign to malignant lymphoproliferation may be related to suppression of apoptotic death by bcl-2. These findings suggest that (i) RF clones are activated in SS with little or no somatic mutation resulting in monoclonal proliferation and (ii) the LEL in the salivary gland is one site for monoclonal B cell proliferation and emerging malignant lymphoma.
Tissue distribution of ERp61 and association of its increased expression with IgG production in hybridoma cells.
A protein of molecular weight 60 kDa was purified from the culture medium of a murine colon carcinoma cell line, colon26, and its partial amino-acid sequence determined. Extremely high homology was found with the deduced sequence from cDNA of rat ERp61, earlier found to be an endoplasmic reticulum (ER)-resident protein with redox activity and a similar structure to protein disulfide isomerase (PDI). Western blotting analysis showed that colon26 cells secrete a significant amount of ERp61 into culture medium, although most remains intracellular. The thiol:protein disulfide oxidoreductase activity of the purified mouse ERp61 was demonstrated by insulin-reduction assay. The ER location of the protein in fibroblasts was immunocytochemically confirmed by double staining for ERp61 and another ER-resident protein, PDI or Hsp47. Immunohistochemical studies of murine tissues showed a ubiquitous distribution of ERp61 in a wide variety of cell types. However, it was particularly abundant in plasma cells, mucus-secreting cells in various tissues, neuroendocrine cells including neurons, and follicular epithelia of thyroid gland that actively synthesize and secrete proteins containing cysteine residues. Furthermore, a high correlation was observed between intracellular amounts of ERp61 and immunoglobulin production by hybridoma cells. These results indicate that ERp61 may be involved in disulfide bond formation for such proteins.
Changes in biliary glutathione level during ischemia-reperfusion of rat liver.
Changes in hepatic and biliary glutathione levels were studied in rat liver treated with tert-butyl hydroperoxide (t-BuOOH) and subjected to ischemia-reperfusion. Immediately after t-BuOOH administration, the oxidized glutathione (GSSG) values and reduced glutathione (GSSG/GSH) ratio in the bile increased dose-dependently and then returned to control level within 10 min, whereas the hepatic ATP level and bile flow rate were not affected by t-BuOOH at doses of up to 1.0 mmol/kg. These data suggested that the liver remains viable on treatment with up to 1.0 mmole/kg t-BuOOH, and that hepatocytes can rapidly dismute t-BuOOH at up to this dose. The hepatic GSH and GSSG levels did not vary appreciably during ischemia for 10 or 30 min or during subsequent reperfusion, but the GSSG/GSH ratio increased after ischemia for 30 min. The rate of bile flow and the biliary level of GSH decreased after ischemia for 30 min in proportion to the decrease in the hepatic ATP level. However, the biliary GSSG concentration did not vary on reperfusion, although GSSG secretion into the bile is also related to the hepatic ATP level. As a result, the GSSG/GSH ratio in the bile increased during reperfusion after ischemia for 30 min. This increased ratio is thought to reflect oxidation of hepatic GSH by hydroperoxide produced during reperfusion. The GSSG/GSH ratio in the bile after 30 min ischemia corresponded to that observed after a small dose (0.07 mmole/kg body wt) of t-BuOOH, which hepatocytes could dismute rapidly without loss of their viability.(ABSTRACT TRUNCATED AT 250 WORDS)
Krabbe's disease with giant lamellar bodies in Purkinje cells.
An autopsy case of Krabbe's disease in a child aged 6 years 7 months 1s reported. Histologically, globoid cells, loss of myelin, oligodendroglia and axons, and marked gliosis were observed in the white matter. In addition, there was severe neuronal loss in the thalamus, pontine nucleus, dentate nucleus and olivary nuclei. The cerebellar cortex showed extensive loss of granular cells and moderate loss of Purkinje cells. There were numerous eosinophilic inclusion bodies, ranging from 2 to 15 microns in diameter, in the dendrites of Purkinje cells. The ultrastructural findings for the inclusion bodies were consistent with those of giant lamellar bodies. In addition, smaller lamellar bodies were frequently observed in the perikarya and dendrites. Although loss of granular cells was more prominent in the hemispheres than in the vermis, the inclusion bodies were observed in hemispheres but were infrequently observed in the vermis. They were found in degenerated Purkinje cells which had lost afferent fibers. It is considered that these giant lamellar body inclusions are an unusual type of degenerative structure specific to Purkinje cells.
Analysis of schizont antigen of Leucocytozoon caulleryi by SDS-PAGE and western blot.
To investigate the molecular weight of the schizont antigen of Leucocytozoon caulleryi, the schizont antigen was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. Thirty to 35 protein bands were detected by SDS-PAGE under reduced condition and ranged from 25 to 300 kDa. Most of them reacted with the sera of infected chickens by Western blot. The bands corresponding to polypeptides of 44, 58, 67 and 180 kDa reacted strongly, however, the bands of 30, 50 and 78 kDa did not react.
Long-term follow-up of coronary narrowing with spasm.
In order to clarify the fate of coronary narrowing with spasm, repeat angiograms of coronary narrowing with and without spasm were compared. The mean interval between the first and second angiograms was 3.6 years (range, 1.1-8.5 years). Improvement of narrowing was more frequent in the vasospastic group (23%) than in the group without spasm (3%, P < 0.005). The cause of this improvement in the vasospastic group may have been the resolution of the spasm in the first angiogram, but the presence of intravascular thrombus or bleeding or edema of the coronary arterial wall may have resolved in the second angiogram.
Enzymatic asymmetric synthesis of alpha-methyl arylalkylamines and alpha-methyl arylalkylalcohols by arylalkyl acylamidases.
With the novel microbial enzyme, 'arylalkyl acylamidase', optically active alpha-methyl arylalkylamines and alpha-methyl arylalkylalcohols have been obtained through enantioselective hydrolysis of their racemic amides and esters. (S)-Enantiomers of 1-methylbenzylamine, 1-methyl-3-phenylpropylamine and 1-methyl-3-phenylpropanol of high optical purity (> 94% e.e.) were synthesized with the cells of Nocardia erythropolis IAM 1440 or Cellulomonas fimi AKU 671. (R)-Enantiomer of 1-methyl-3-phenylpropylamine and (S)-enantiomer of 1-methyl-2-phenylpropanol of high optical purity (> 95% e.e.) were synthesized with the crude preparation of arylalkyl acylamidase of Pseudomonas putida Sc2 AKU 881.
Diagnostic ERCP.
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Endoscopic ultrasonography for early gastric cancer.
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Primary pulmonary-artery sarcoma. Report of a case with complete resection and graft replacement, and review of 47 surgically treated cases reported in the literature.
A case of pulmonary leiomyosarcoma originating in the left pulmonary artery stem in a 28-year-old Japanese man is reported. The patient complained of recurrent hemoptysis. The diagnosis was made at exploratory thoracotomy. Two days following thoracotomy, under cardio-pulmonary bypass, the upper one-third of the pulmonary trunk and part of the right pulmonary artery stem were resected and left pneumonectomy was performed. A Dacron prosthetic 23 mm composite graft was inserted to replace the pulmonary trunk and a portion of the right pulmonary artery. Postoperative course was uneventful. The patient died about 8 months after surgery because of right lung metastases. In our review of surgically treated forty-eight cases, complete resection was performed in 11, incomplete resection in 23, and embolectomy in 6, whereas 8 were unresectable. A localisation often not allowing adequate resection of the tumor or the advanced stage of the tumor at surgery are considered the main causes of the poor prognosis of the pulmonary sarcoma.