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Biomedical subjects

Y Ueda

Publications and source records attributed to Y Ueda.

At least 595 records · Page 33Linked to original sources
PubMed1993

The role of acidic amino-acid residues in catalytic and adsorptive sites of Bacillus cereus sphingomyelinase.

By the modification of acidic amino-acid residues with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate), the activity of sphingomyelinase of Bacillus cereus was decreased by 80-90%. Also, the reduction of Cys residues in the sphingomyelinase molecule by dithiothreitol caused a drastic decrease in enzymatic activity, whereas the sphingomyelinase activity was not affected by treatment with p-chloromercuribenzenesulfonic acid. Actually, no inactivation of sphingomyelinase activity was observed after selective modification of basic amino-acid residues such as Lys, His and Arg, and of the uncharged amino-acid residues Ser and Thr. The treatment of the sphingomyelinase molecule with Woodward's reagent K or dithiothreitol also brought about the inhibition of the specific adsorption of sphingomyelinase toward intact erythrocyte membranes. However, the extent of inhibition in the enzyme adsorption, 20-50%, was less than that observed in the sphingomyelinase activity. These results suggest that acidic amino-acid residues, such as Asp and Glu, in the sphingomyelinase molecule are involved in the catalytic sites and the adsorptive sites. Apparently, the disruption of disulfide linkage in the sphingomyelinase molecule by dithiothreitol destabilized its structure, resulting in a drastic decrease in sphingomyelin-hydrolyzing activity and specific adsorption of sphingomyelinase towards erythrocyte membranes.

Amino Acid Sequence↗
PubMed1993

Synthesis and expression of a DNA encoding the Fv domain of an anti-lysozyme monoclonal antibody, HyHEL10, in Streptomyces lividans.

A secretory production system for the Fv domain of a monoclonal antibody (mAb) against hen egg-white lysozyme (HEL) was established in Streptomyces lividans using a chemically synthesized gene. The synthetic DNAs encoding the Fv fragments (VH and VL) of the anti-HEL mAb, HyHEL10, were fused to DNA encoding the signal peptide of Streptomyces subtilisin inhibitor (SPssi) in an SPssi::VH-SPssi::VL dicistronic arrangement. The genes were expressed under the control of the ssi promoter using S. lividans as host. Each Fv fragment was accurately processed and secreted into the growth medium. No inclusion bodies were produced. The Fv fragments were isolated from culture supernatant by a two-step purification (affinity chromatography and gel filtration) with a high yield (approx. 1 microgram/ml). Purified Fv fragments bound to HEL specifically, and completely inhibited the catalytic activity of HEL at a molar ratio of 1.25 for Fv vs. HEL.

Amino Acid Sequence↗
PubMed1993

Regulation of apolipoprotein B production and secretion in response to the change of intracellular cholesteryl ester contents in rabbit hepatocytes.

In the present study, we investigated the mechanisms that regulate apolipoprotein B-100 (apoB) secretion in response to the change of intracellular cholesteryl ester contents by adding low density lipoprotein (LDL) and an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase (pravastatin) in rabbit hepatocyte culture system. LDL caused a significant dose-dependent increase in apoB secretion. On the other hand, the addition of pravastatin decreased apoB secretion significantly. LDL caused a dose-dependent increase in cellular cholesteryl ester, while cellular cholesteryl ester was decreased by pravastatin significantly. Therefore, these results indicate that the change of apoB secretion was in parallel with the change of cellular cholesteryl ester contents. Cellular contents of free cholesterol, triglyceride, and phospholipid did not change. To investigate intracellular degradation of apoB prior to secretion, pulse-chase experiments were performed. It was shown that the addition of pravastatin accelerated intracellular degradation of apoB, while LDL slowed the apoB intracellular degradation rate. We also investigated whether the change of cellular cholesteryl ester could affect the apoB mRNA level. Northern blot analysis and solution hybridization RNase protection assay demonstrated that neither LDL nor pravastatin caused a significant change in cellular apoB mRNA level. We conclude that intracellular cholesteryl ester contents play a critical role in apoB secretion, and the intracellular apoB degradation rate could be the main mechanism that regulates apoB secretion in response to the change of intracellular cholesteryl ester level.

Acetates↗
PubMed1993

Effect of a rare leucine codon, TTA, on expression of a foreign gene in Streptomyces lividans.

Streptomyces are bacteria with a very high chromosomal G+C composition (> 70 mol%) and extremely biased codon usage. In order to investigate the relationship between codon usage and gene expression in Streptomyces, we used ssi (Streptomyces subtilisin inhibitor) as a reporter gene and monitored its secretory expression in S. lividans. In consequence of alteration of the native codons of Leu, Lys and Ser of ssi to minor ones by site-directed mutagenesis, i.e., Leu79-Leu80: CTG-CTC to TTA-TTA, Lys89: AAG to AAA, Ser108-Ser109: TCG-AGC to TCT-TCT, respectively, the production of SSI was reduced remarkably in the case of TTA codons, while it was slightly increased in the case of AAA and almost the same in TCT codons. This conspicuous decrease found for Leu codon replacement was probably due to the low availability of intracellular tRNA(Leu) (UUA), a product of bldA which has been reported to be expressed only during the late stage of growth.

Amino Acid Sequence↗
PubMed1993

Differentiating and antitumor activities of 1 alpha,25-dihydroxyvitamin D3 in vitro and 1 alpha-hydroxyvitamin D3 in vivo on human osteosarcoma.

The differentiating and antitumor activities of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) in vitro and 1 alpha-hydroxyvitamin D3 (1 alpha(OH)D3) in vivo were studied with a human osteosarcoma cell line (OST strain). Anti-tumor activity was estimated with the use of 3-(4,5-dimethylthiazol)-2, 5-diphenyltetrazolium bromide (MTT) assay, colony-forming assay, and athymic mouse assay. The intracellular alkaline phosphatase (ALP) activity of tumor cells and production of bone Gla protein (BGP) in culture media were measured to mark osteoblastic differentiation. In addition, the combination of 1 alpha,25(OH)2D3 and cis-dichlorodiammineplatinum(II) (CDDP) was tested by the colony-forming assay and the measurement of ALP activity and BGP production for differentiating and antitumor effects. The assays revealed that 1 alpha,25(OH)2D3 exerted a dose-related, growth-inhibitory influence. In the colony-forming assay, the 1 alpha,25(OH)2D3-treated colonies were smaller than the untreated colonies. The ALP activity and the BGP production also increased in relation to dose. In the assay in athymic mice, the relative weight of tumors treated with 1 alpha(OH)D3 at 2.5 nmol/kg was significantly smaller than that of the controls, and no side effects were observed in the 1 alpha(OH)D3-treated mice. Marked tumor chondrogenesis was observed in human osteosarcoma treated with 1 alpha(OH)D3 in athymic mice. The combination of 1 alpha,25(OH)2D3 at 10(-8) M and CDDP at 2 micrograms/ml significantly enhanced both the differentiation and the growth inhibition in vitro. Our study apparently is the first demonstration that vitamin D3 metabolites have an antitumor and differentiating effect on human osteosarcoma cells in vitro and in athymic mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Effect of T cells on the complement production by monocytes.

We studied the role of T cells in the regulation of C3 or C4 production by monocytes. While cocultured T cells reduced C3 production, they enhanced C4 production by monocytes in a number-dependent manner. Interleukin-2 enhanced these effects of T cells on C3 and C4 production, indicating that activated T cells had greater effects on complement production by monocytes. CD4+ T cells had greater effects than those of CD8+ T cells on the C3 or C4 production by monocytes, indicating that CD4+ T cells were mainly involved in the regulation of C3 and C4 production by monocytes. T-cell-conditioned medium (T-CM) also decreased C3 and enhanced C4 production, but the effect of T-CM on C3 and C4 production was less remarkable than that of T cells. T cells separated by transwell inserts from monocytes had similar effects as those of T-CM on complement production. Anti-IFN-gamma antibody completely blocked the suppressing effect on C3 production and partially blocked the stimulating effect of T cells on C4 production. These results indicate that T cells regulate C3 or C4 production by monocytes via IFN-gamma as a humoral factor. Moreover, the stimulating effect of T cells on C4 production was partially blocked when monocytes and T cells were incubated with anti-CD2, anti-LFA-1, and anti-HLA-DR antibodies, and anti-CD2 partially blocked the suppressing effect of T cells on C3 production by monocytes. These results indicate that T cells modulate the C3 and C4 production by monocytes not only via a humoral factor but also via a direct interaction with monocytes.

Cell Communication↗
PubMed1993

Characterization of vaccinia surface antigen expressed by recombinant baculovirus.

The gene encoding the vaccinia surface antigen (S antigen) was inserted into a baculovirus transfer vector and a recombinant virus was isolated. The S antigen was expressed on the surface of Spodoptera frugiperda cells (Sf cells) infected with the recombinant baculovirus. Recombinant proteins were detected in immunoblotting with anti-vaccinia serum and have the apparent molecular weight of 40 and 50-kDa. The 50-kDa polypeptide was tunicamycin sensitive and was thus glycosylated. The glycosylated 50-kDa polypeptide was also secreted into culture supernatants. Antiserum directed against the expressed S antigen specifically reacted to the authentic S antigen that is located on the mammalian cells infected with vaccinia virus, but did not affect the production of infectious progeny virus. Antisera against both the terminal regions of the S antigen were prepared by immunizing rabbits with the recombinant fusion proteins produced in the bacterial expression vector. The S antigen on the cell surface reacted by immunofluorescence with anti-C-terminus serum but not or very weakly with anti N-terminus serum, indicating that the hydrophobic N-terminus functions both as a signal and a membrane anchor sequence. Interleukin (IL)-1 alpha failed to bind to the S antigen expressed on insect and mammalian cells, although the homology between IL-1 receptor and the S antigen has been reported by computer analysis of amino acid sequence.

3T3 Cells↗
PubMed1993

Chemotherapeutic effect on osteosarcoma on basis of collagen analysis: a proposal of the induction of osteosarcoma differentiation.

The authors studied effect of chemotherapy on osteosarcoma by collagen analysis. As a result of this case study we propose the induction of osteosarcoma differentiation by chemotherapy. Treatment of a conventional osteosarcoma with two intra-arterial infusions of cisplatin and the T-12 protocol of Rosen resulted in sclerotic changes and good margination accompanied by the disappearance of the soft-tissue component from the X-rays. More than 90% tumour destruction was histologically demonstrated; tumour bone and osteoid increased after the chemotherapy, and the viable area of the tumour resembled an osteoblastoma. Before the chemotherapy, immunolocalization determined collagen types I and V to be diffusely present in the bone and osteoid. After the chemotherapy, the antibody to type I collagen was diffusely present, but the antibody to type V collagen occurred only on the surface of the increased bone and osteoid as in normal bone. When osteosarcoma cells were treated in vitro with methotrexate or cisplatin, collagen production increased significantly. It is thus believed that tumour cells were directly stimulated with these chemotherapeutic agents to produce collagen. The findings suggested that some anticancer agents might not only be cytotoxic to but also differentiate osteosarcoma cells.

Adolescent↗
PubMed1993

Immunohistochemical localization of collagenous proteins in cartilaginous tumors: characteristic distribution of type IX collagen.

The distinctive tissue localization of collagen types, particularly of type IX collagen in human cartilaginous tumors (10 cases of enchondroma and 15 cases of chondrosarcoma including 3 cases of secondary chondrosarcoma) was examined immunohistochemically using affinity-purified antibodies against types I, II, III, V, VI, and IX collagen, in comparison with that in human fetal cartilage. In fetal cartilage matrix, types II and IX collagen were diffusely distributed, while types I, III, and V collagens were not present. In the matrices of enchondromas and primary chondrosarcomas, types II and IX collagens were also diffusely distributed, but with some areas of irregular type IX collagen deposits. The secondary chondrosarcoma simulated normal fetal cartilage in the distribution pattern of types II and IX collagen, unlike the pattern in primary chondrosarcoma, where types II and IX collagen were decreased and poorly immunostained, whereas non-cartilaginous interstitial collagens (I, III, and V) appeared diffusely in the matrix, increasing with the grade of malignancy. These findings suggest that neoplastic cartilage is characterized initially by an uneven distribution of type IX collagen, prior to any alteration of other types of collagen; the diverse expressions of intercellular components in cartilaginous tumors may be one indicator for malignancy.

Adolescent↗
PubMed1993

Borderline chondrosarcoma of long and flat bones.

We reviewed histological and clinical findings of six cases of borderline chondrosarcoma and examined the expression of collagen types I, II, III, V, and VI by immunohistochemical analysis of these tumors. Borderline chondrosarcoma is defined as a cartilaginous tumor of bone resembling enchondroma on the basis of histomorphology. Clinically the tumor causes intermittent vague pain unrelated to physical activities. On radiographs borderline chondrosarcoma is characterized by evidence of endosteal erosion. We observed local recurrences in two cases treated by intralesional excision and marginal excision, and one of those cases died of inoperable local tumor recurrence. In our histological analysis based on tissue patterns, there were enchondromatous patterns in five cases, and chondrosarcomatous patterns in four cases. In the second recurrent tumor in one case, a chondrosarcomatous pattern was newly observed, and the recurrent tumor was found to be a low-grade chondrosarcoma cytologically in the other case. In the tumor matrix immunoreactivity for collagen types II and VI was predominant, with collagen types I, III, and V showing heterogeneous expression in some cases. In all cases rimming of tumor lobules with collagen types I and V was absent. Immunoreactivity for collagen type II in the cytoplasm of tumor cells was found in four cases and all three recurrent tumors. Borderline chondrosarcoma, as defined by histology, clinical symptoms and radiological appearance, shows a collagen distribution pattern similar to that of low-grade chondrosarcoma. These findings are in accordance with the clinical outcome of borderline chondrosarcoma which parallels that of low-grade chondrosarcoma. Thus borderline chondrosarcoma may be best treated by wide en-bloc excision rather than curettage.

Adult↗
PubMed1993

Analysis of mutant P53 protein in osteosarcomas and other malignant and benign lesions of bone.

Alterations of tumour suppressor genes are considered crucial steps in the development of human cancers. Expressions of p53 protein, a product of the tumour suppressor gene altered most commonly in human cancers examined so far, were investigated immunohistochemically in 18 osteosarcomas and 40 other malignant and benign lesions of bone. A monoclonal antibody clone PAb240, which recognizes a common conformational epitope of mutant p53 proteins, stained nuclei of tumour cells in 12 of 18 osteosarcomas (67%). Six tumours (33%) particularly showed positive immunoreactions in more than half of the tumour cells. PAb240 also stained tumour cells in a small number of other malignant bone tumours, such as malignant fibrous histiocytoma, chondrosarcoma, and Ewing's sarcomas. Furthermore, a small number of cells of giant-cell tumours were positively stained. In contrast, PAb240 was completely negative in 21 benign bone tumours and reactive lesions examined. Another monoclonal antibody clone PAb1801, which reacts with both wild- and mutant-type p53 protein, reacted in nuclei of tumour cells of 7 osteosarcomas (39%). Most of those also reacted with PAb240. PAb1801 was expressed much more frequently in other malignant bone tumours and giant-cell tumours. In addition, PAb1801 showed intranuclear positive reactions in tumour cells of a benign chondroblastoma, and reactive cells such as actively proliferating preosteoblasts in a myositis ossificans and osteoclast-like giant cells in a giant-cell tumour. The immunoelectron-microscopic observation that p53 protein was localized in euchromatic areas of nuclei of osteosarcoma cells supported the specificity of immunoreaction for p53 protein, indicating an active role of p53 protein in the regulation of DNA synthesis and transcription. These findings suggest that point mutation of the p53 gene is frequently involved in the development of osteosarcomas. PAb240 may be a useful tool not only in screening point mutations of the p53 gene in osteosarcomas but also in the differential diagnosis between osteosarcomas and reactive bone-forming lesions. Expressions of mutant p53 protein were not correlated with any clinical or pathological factors examined, although the results should be confirmed in studies of a large number of osteosarcomas.

Adolescent↗