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Biomedical subjects

A Takashima

Publications and source records attributed to A Takashima.

At least 73 records · Page 4Linked to original sources
PubMed1998

Purification and characterization of two muconate cycloisomerase isozymes from aniline-assimilating Frateuria species ANA-18.

Two muconate cycloisomerases (MC I and MC II, EC 5.5.1.1) were purified to homogeneity from an aniline-grown Frateuria sp. ANA-18. MC I and MC II were similar in molecular mass, optimal pH, and pH stability but different in thermostability, and some other enzymatic properties. NH2-terminal amino acid sequences were different between the two isozymes, indicated that these are encoded by different genes. Different inducible production of MC I and MC II suggested that two catechol branches involved in the beta-ketoadipate pathway function in Frateuria sp. ANA-18.

Amino Acid Sequence↗
PubMed1998

T cell-mediated terminal maturation of dendritic cells, a critical transition into fully potent antigen presenting cells.

Dendritic cells (DC) are a special subset of leukocytes characterized by their potent ability to activate immunologically naive T cells. We established a series of long-term DC lines, termed "XS lines", from the epidermis of newborn mouse skin. These lines retain many important features of resident DC in epidermis, i.e., Langerhans cells (LC), including surface phenotype, antigen (Ag) presenting capacity, and cytokine and cytokine receptor profiles. Working with XS lines, we have observed recently that DC undergo rapid and profound changes during Ag presentation. Specifically, upon Ag-specific interaction with T cell clones, XS52 cells: a) begin to secrete several cytokines (IL-1 beta, IL-6, and TNF alpha), b) lose the expression of CSF-1 receptor and proliferative responsiveness to CSF-1, c) elevate the expression of B7-2 (CD86), and d) lose the phagocytotic and adhesive capacities. We propose that these changes represent "T cell-mediated terminal maturation" of DC, a critical transition into a fully professional Ag presenting cell.

Animals↗
PubMed1997

Cloning of cDNA and expression of the gene encoding rat presenilin-2.

We have cloned the rat homologue of the presenilin-2 (PS-2) cDNA. PS-2 is responsible for chromosome 1-linked familial Alzheimer's disease. Sequence analysis predicted that the rat PS-2 encodes a 448 amino acid (aa) protein, and there was a very high degree of amino acid identity between rat and human PS-2 (95%). All the mutated codons in PS-2 and PS-1 in chromosome 1- or 14-linked familial Alzheimer's disease patients were conserved in rat PS-2. The expression of PS-2 was weaker than that of PS-1. The alternatively spliced short form of PS-2 mRNA, which was detected in human tissues was not detected in various rat tissues. During brain development, the expression level of both PS-2 and PS-1 increased but decreased in the adult. No remarkable change was observed in neural differentiation of PC12 cells.

Alternative Splicing↗
PubMed1997

Different effects of Alzheimer-associated mutations of presenilin 1 on its processing.

Presenilin 1 (PS 1) shows missense mutations in most early-onset familial Alzheimer's disease (FAD). Transfection of cDNA for wild type PS 1 into rat pheochromocytoma PC12 cells generated a 47 kDa full-size PS 1 protein, which was processed into a 28 kDa N-terminal fragment and a 19 kDa C-terminal fragment. We prepared selected Alzheimer-associated mutations (Gly384Ala, Leu392Val, and Cys410Tyr) of PS 1, which localized after a possible cleavage site. By transient expression in PC12 cells and rat glioma cell line, C6, we examined their influence on the processing of PS 1. Cys410Tyr inhibited proteolytic processing of PS 1, while Gly384Ala and Leu392Val did not. Thus, the Alzheimer related mutations can be divided into two groups in terms of their effect on the proteolytic cleavage of PS 1.

Alzheimer Disease↗
PubMed1997

Ultraviolet light suppresses IFN-gamma-induced IL-7 gene expression in murine keratinocytes by interfering with IFN regulatory factors.

UV light is a potent stimulus for keratinocytes to release several cytokines. Recently, UV light was shown to inhibit keratinocyte release of IL-7, a growth factor for dendritic epidermal T cells. Since to date IL-7 is the only keratinocyte-derived cytokine down-regulated by UV light, we addressed the molecular mechanisms involved. IFN-gamma treatment of the murine keratinocyte cell line Pam 212 resulted in an up-regulation of IL-7 mRNA, while IL-7 transcripts were suppressed in cells exposed to UV before IFN-gamma. Because IFN-gamma induces IL-7 via activation of an IFN-stimulated response element (ISRE) located in the 5' upstream region of the IL-7 gene, bandshift assays were performed using the ISRE sequence from the IL-7 gene. Nuclear extracts from untreated cells revealed two bands, a slower migrating band identified by supershift analysis as IFN regulatory factor-2 (IRF-2), a transcriptional repressor, and a more rapidly migrating band identified as IRF-1, a transcriptional activator. IFN-gamma significantly induced IRF-1 binding, whereas UV treatment plus IFN-gamma decreased IRF-1 binding, suggesting that UV light suppresses IFN-gamma-induced expression of IL-7 by interfering with IRF-1. Chloramphenicol transferase assay confirmed functional relevance, showing that the minimal promoter sequence for the ISRE explicitly responded to IFN-gamma, which was suppressed by UV irradiation. Northern blot analysis using an IRF-1 cDNA probe revealed that UV light reduced IFN-gamma-induced IRF-1 mRNA. This study demonstrates that UV light can inhibit cytokine activities by interference with transcriptional activators. This newly described ability of UV light may contribute to its immunosuppressive properties.

Animals↗
PubMed1997

Activation of LA-N-2 cell phospholipases by single alanine substitution analogs of amyloid beta peptide (25-35).

A series of single alanine substituted analogs of amyloid beta peptide (25-35) were tested for their ability to activate the phospholipases of cultured LA-N-2 cells. Substitution of alanine for the amino acids 29-34 prevented the activation of phospholipases A2 and D. In addition substitution of alanine at 28 prevented phospholipase D but not phospholipase A2 activation. All the alanine substitutions, except for positions 33 and 35, blunted phospholipase C activations. There were no activations by scrambled amyloid beta peptide.

Alanine↗
PubMed1997

Nontoxic amyloid beta peptide 1-42 suppresses acetylcholine synthesis. Possible role in cholinergic dysfunction in Alzheimer's disease.

We show here that amyloid beta peptide1-42 (Abeta1-42) may play a key role in the pathogenesis of the cholinergic dysfunction seen in Alzheimer's disease (AD), in addition to its putative role in amyloid plaque formation. Abeta1-42 freshly solubilized in water (non-aged Abeta1-42), which was not neurotoxic without preaggregation, suppressed acetylcholine (ACh) synthesis in cholinergic neurons at very low concentrations (10-100 nM), although non-aged Abeta1-40 was ineffective. Non-aged Abeta1-42 impaired pyruvate dehydrogenase (PDH) activity by activating mitochondrial tau protein kinase I/glycogen synthase kinase-3beta, as we have already shown in hippocampal neurons (Hoshi, M., Takashima, A., Noguchi, K., Murayama, M., Sato, M., Kondo, S., Saitoh, Y., Ishiguro, K., Hoshino, T., and Imahori, K. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2719-2723). Neither choline acetyltransferase activity nor choline metabolism was affected. Therefore, the major cause of reduced ACh synthesis was considered to be an inadequate supply of acetyl-CoA owing to PDH impairment. Soluble Abeta1-42 increases specifically in AD brain (Kuo, Y.-M., Emmerling, M. R., Vigo-Pelfrey, C., Kasunic, T. C., Kirkpatrick, J. B., Murdoch, G. H., Ball, M. J., and Roher, A. E. (1996) J. Biol. Chem. 271, 4077-4081). This increase in soluble Abeta1-42 may disturb cholinergic function, leading to the deterioration of memory and cognitive function that is characteristic of AD.

Acetylcholine↗
PubMed1997

Mechanisms regulating telomerase activity in murine T cells.

Telomeres shorten with successive cell divisions in normal somatic cells, while telomerase plays an important role in maintaining their lengths. Although telomerase activity was originally described as being expressed exclusively by immortal cells and germline cells, a recently developed PCR-based technique (telomeric repeat amplification protocol (TRAP)) has revealed that normal peripheral blood leukocytes also exhibit this activity following mitogenic stimulation. In this study, we examined mechanisms by which mitogenic stimuli up-regulated telomerase activity in T cells. Splenic T cells freshly isolated from BALB/c mice exhibited only negligible telomerase activity. When stimulated with Con A or immobilized anti-CD3 mAb at 10 microg/ml, they acquired an increased telomerase activity and maximal proliferation. By contrast, T cells treated with a much lower concentration (0.03 microg/ml) of anti-CD3 mAb required exogenous IL-2 for telomerase activation and proliferation. Likewise, adult thymocytes treated with anti-CD3 mAb exhibited telomerase activation and proliferation only in the presence of exogenous IL-2, IL-4, IL-7, or IL-15. Furthermore, IL-2 alone was sufficient for telomerase activation in Con A blasts. These results illustrate a pathway through which cytokine receptors transduce telomerase activation signals in T cells. Although telomerase activation was concomitant with cell growth in normal T cells, we have identified T cell lines that showed discrepancies; the CTLL-2 line showed constitutive telomerase activity regardless of cell proliferative state, whereas the 7-17 line proliferated vigorously in response to IL-2, IL-7, or IL-15, without detectable telomerase activity. Thus, telomerase activity, which is ordinarily associated with proliferation in normal T cells, is not necessarily required or sufficient for cell growth.

Adult↗
PubMed1997

Regulation of cytokine expression in macrophages and the Langerhans cell-like line XS52 by calcitonin gene-related peptide.

Calcitonin gene-related peptide (CGRP) inhibits antigen presentation by Langerhans cells (LC) and macrophages, and LC are anatomically associated with CGRP-containing epidermal nerves. To determine whether CGRP may produce some of its functional effects through regulation of cytokine expression, we utilized enzyme-linked immunosorbent assay (ELISA) of conditioned supernatants to examine production of interleukin (IL)-10 and IL-1 beta protein in the LC-like cell line XS52 as well as the reverse transcriptase-polymerase chain reaction (RT-PCR) to examine levels of mRNA for IL-10, IL-1 beta, and the 40-kDa subunit (p40) of IL-12. CGRP augmented the lipopolysaccharide (LPS) and granulocyte-macrophage colony-stimulating factor (GM-CSF) -induced release of IL-10 protein and the induced expression of IL-10 mRNA in these cells. However, it suppressed the induction of release of IL-1 beta protein and the induction of mRNA for IL-12 p40 and IL-1 beta by LPS and GM-CSF. Regulation of cytokine expression in peritoneal macrophages was also examined. By ELISA, the LPS-induced expression of IL-10 was augmented by CGRP, whereas the induction of IL-1 beta was suppressed. Northern analysis demonstrated augmentation of LPS-induced IL-10 mRNA levels and inhibition of LPS-induced IL-1 beta mRNA by CGRP. CGRP inhibited the LPS-induced induction of IL-12 mRNA as assessed by RT-PCR. Up-regulation of B7-2 expression by LPS and GM-CSF was suppressed by CGRP in both XS52 cells and macrophages, as previously reported. This suppression, however, could be abrogated by co-culture with neutralizing antibodies to IL-10. Furthermore, the presence of neutralizing antibodies to IL-10 during exposure of epidermal cells (EC) to CGRP prevented the CGRP-mediated suppression of EC presentation of tumor-associated antigens (from the S1509a spindle cell carcinoma) for elicitation of delayed-type hypersensitivity in S1509a-immune mice. These data suggest that suppression of antigen-presenting function by CGRP is mediated, at least in part, by changes in cytokine expression that favor less robust antigen presentation for cell-mediated immunity.

Animals↗
PubMed1997

Presenilin 1 immunostaining using well-characterized antibodies in human tissues.

Using well-characterized monoclonal antibodies which recognize the N-terminus of presenilin 1 (PS1), we examined by immunohistochemistry brain tissues from patients with nonneurological conditions, Alzheimer's disease (AD) and cerebral infarction, as well as normal human liver tissues. The antibodies to PS1 did not reveal any positive staining in nonneurological conditions. In AD, the antibody showed positive staining of intraneuronal neurofibrillary tangles and neuropil threads. In cerebral infarcts, some macrophages were positively stained. In contrast to the C-terminal fragment of PS1, which has been claimed to be present in senile plaques, the N-terminal fragment binds to intracellular and intradendritic pathological structures and may play a role in tau phosphorylation in AD.

Aged↗
PubMed1997

5-Azacytidine induces toxicity in PC12 cells by apoptosis.

5-Azacytidine (5 Az)is a potent inhibitor of DNA methylation, and it may allow inactive genes to become expressed. In a previous study, we demonstrated that 5 Az administered to the dam induced apoptosis in the brains of fetal mice. In this study, the 5 Az-induced apoptosis was further characterized in differentiated PC 12 cells as a model for neuronal apoptosis. Cell death, determined by the activity of released lactate dehydrogenase (LDH) into the medium, occurred from 24 to 48 hrs after 5 Az treatment. Toxicity for differentiated PC 12 cells was observed on treatment with more than 10(-1) micrograms/ml of 5 Az, and it reached the maximal level at 10 micrograms/ml. Cycloheximide, an inhibitor of protein synthesis, prevented 5 Az toxicity, suggesting that this cell death required protein synthesis which could be related to the activation of a dormant gene(s). Electrophoresis of DNA from 5 Az-treated cells evoked ladder formation, indicating the cleavage of DNA into nucleosomes. Scanning electron microscopy demonstrated bleb formation, the so-called apoptotic bodies on the cell surface. The biochemical and morphological findings indicated that 5 Az-induced cell death occurred in the form of apoptosis. 5 Az-induced cell death was prevented by treatment with cAMP but not by treatment with high K+ or deoxycytidine. These results suggest that a cAMP-sensitive mechanism is involved in 5 Az-induced cell death. PC 12 cells should be of value in elucidating the molecular mechanism of 5 Az-induced neuronal apoptosis.

Animals↗
PubMed1997

S-adenosylmethionine decarboxylase activity is decreased in the rat cortex after traumatic brain injury.

S-Adenosyl-L-methionine decarboxylase (SAMdc) and L-ornithine decarboxylase (ODC) are major enzymes regulating polyamine synthesis. Following ischemia, putrescine content increases as a result of posttraumatic activation of ODC and inhibition of SAMdc. These alterations are thought to mediate edema and cell death. The purpose of this study was to quantify SAMdc activity and edema in the brain following controlled cortical impact injury. Anesthetized adult male rats underwent a right parietal craniectomy and were subjected to cortical impact injury. Tissues were obtained from three bilateral regions: parietal cortex, motor area (CPm); parietal cortex, somatosensory area (CPs); and the pyriform cortex (CPF). SAMdc activity was determined in the postmitochondrial fraction from homogenates of fresh, unfrozen tissues by measuring the decarboxylation of S-adenosyl-L-[carboxyl-14C]methionine. Basal SAMdc activity was determined in unoperated rats, and regional differences were noted: Activity was lower in the CPF than in the CPm and CPs. SAMdc activity decreased to the greatest extent in the ipsilateral CPm (impact site) from 1 to 72 h following traumatic brain injury. Significant edema was found in the ipsilateral CPm 1, 8, 16, 24, and 48 h after injury. Decreased SAMdc activity impairs the conversion of putrescine to polyamines and may contribute to delayed pathological changes in the brain after traumatic injury.

Adenosylmethionine Decarboxylase↗
PubMed1997

Expression of B7-1 by Pam 212 squamous cell carcinoma enhances tumor cell interactions with dendritic epidermal cells but does not affect in vivo tumor growth.

Direct antigen presentation of tumor-associated antigens by tumor cells to T lymphocytes may induce clonal anergy as a mechanism of escape from immune surveillance. B7-1 is a costimulatory molecule for the activation of both CD4+ and CD8+ T lymphocytes that prevents the induction of clonal anergy. Thus, the transfer of B7-1 genes into tumor cells can induce protective immunity and lead to tumor rejection of some tumors in model systems of in vivo tumor growth; however, there is no information on whether stable expression of B7-1 can affect the in vivo growth of squamous cell carcinoma, a common skin cancer. Here, we study how the stable cell surface expression of high levels of B7-1 by Pam 212, a murine squamous cell carcinoma, affects tumor cell-lymphocyte interactions (lymphocyte proliferation and cytotoxicity). Consistent with its costimulatory role, we demonstrate that B7-1 can efficiently induce dendritic epidermal T-cell proliferation in three different dendritic epidermal T-cell cell lines. In addition, B7-1 enhances dendritic epidermal T-cell cytolytic activity against Pam 212 cells in an in vitro 51Cr-release assay, which was blocked by CTLA-4/Ig fusion protein. In contrast to dendritic epidermal T cells, the expression of B7-1 does not alter Pam 212 interactions with either cytotoxic T-lymphocytes, natural killer, or lymphokine-activated killer cells. B7-1 expression by Pam 212 cells did not alter its ability to grow tumors in vivo, as their rate of tumor growth was the same as vector-transfected Pam 212 cells, which were B7-1 negative. Our studies indicate that B7-1 gene transfer into Pam 212 does not alter its tumorigenicity, because it does not alter tumor cell-lymphocyte interactions with cytotoxic T lymphocytes, natural killer cells, and lymphokine-activated killer cells. Further studies of B7-1 modified Pam 212 and dendritic epidermal T cells will clarify whether T-cell receptor-gamma/delta-bearing T lymphocytes can play a role in immunotherapy of Pam 212 squamous cell carcinoma.

Animals↗
PubMed1997

Strain differences in sensitivity to the promoting effect of sodium L-ascorbate in a two-stage rat urinary bladder carcinogenesis model.

Rat strain differences in sensitivity to the promoting effect of sodium L-ascorbate (SA) on the development of urinary bladder tumors were investigated. In experiment 1, WS/Shi (WS), ODS/Shiod/od (ODS), and LEW/Crj (LEW) rats were initiated with 0.05% N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in their drinking water and subsequently given basal Oriental MF diet (M) with or without a 5% SA supplement. In LEW rats the SA treatment increased the induction of neoplastic lesions in the urinary bladder, whereas WS and ODS animals proved unresponsive to its promoting effects. In experiment 2, WS and F344 rats were maintained on two kinds of commercial basal diets, M and CLEA CA-1 (C), during administration of SA, since dietary factors can influence promoting effects. Feeding M during the promotion period in F344 rats yielded significantly more neoplastic lesions than feeding C, but in WS rats no such dietary influence was apparent. In experiment 3, strain differences in biosynthesis of alpha-2u-globulin (alpha 1a-g) were assessed because both alpha 2a-g in the urine and administration of sodium salts of organic acids such as SA have been reported to be involved in tumor promotion. Immunohistochemical analysis of renal tubules and Western blotting analysis of urine revealed the presence of alpha 2a-g in all three strains examined. These data suggest that differences in susceptibility to promotion are due to genetic factors rather than dietary factors and the ability to synthesize alpha 2a-g.

Animals↗
PubMed1997

5-Azacytidine (5Az) induces apoptosis in PC12 cells: a model for 5Az-induced apoptosis in developing neuronal cells.

Our previous in vivo and in vitro studies showed that 5-azacytidine (5Az), a cytidine analog, induced apoptosis in developing neuronal cells in mice. To develop a system in which the precise molecular mechanism of 5Az-induced apoptosis in developing neuronal cells could be elucidated, we carried out the present study with PC12 cells. These cells are derived from a rat pheochromocytoma and extrude neurites in response to nerve growth factor (NGF). Light microscopy showed dose-dependent pyknotic and karyorrhectic changes in undifferentiated PC12 cells. Although they were less sensitive to 5Az, NGF-treated differentiated cells showed the same changes. Analysis by the TUNEL method (an in situ method for the detection of apoptosis) showed positive signals in the pyknotic and fragmented nuclei of these cells. Transmission electron microscopy revealed margination, segmentation, and condensation of nuclear chromatin, cell body shrinkage, and cytoplasmic vacuolization. Scanning electron microscopy demonstrated bleb formation on the cell surface. These pathomorphological changes are typical of apoptosis. 5Az seemed to affect cells that were in the proliferative stage; when the cells were terminally differentiated, their sensitivity to 5Az appeared to decline. PC12 cells could be used as a pathomorphological and biochemical model for studies of 5Az-induced neuronal cell apoptosis at the molecular and genetic level.

Animals↗