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Biomedical subjects

M Yamada

Publications and source records attributed to M Yamada.

At least 793 records · Page 44Linked to original sources
PubMed1997

Bcl-2 relieves the trans-repressive function of the glucocorticoid receptor and inhibits the activation of CPP32-like cysteine proteases.

Glucocorticoid induces apoptosis in immature lymphocytes which is inhibitable by Bcl-2. Although glucocorticoid-mediated signal transduction is well understood, the mechanism of the induction of apoptosis by the activated glucocorticoid receptor as well as the inhibition of apoptosis by Bcl-2 remains enigmatic. Here we report that overexpressed Bcl-2 relieves the glucocorticoid receptor-mediated repressive function on the AP-1 activity and completely inhibits the activation of CPP32-like cysteine proteases. In contrast, glucocorticoid receptor-mediated transactivation was not affected by Bcl-2. This suggests that glucocorticoid may induce apoptosis by repressing transactivation by AP-1 which is relieved by Bcl-2. Furthermore, we report evidence that, in contrast with CPP32-like proteases, ICE-like proteases are not involved in this apoptotic pathway.

Apoptosis↗
PubMed1997

Targeted disruption of the gene encoding the classical nitroreductase enzyme in Salmonella typhimurium Ames test strains TA1535 and TA1538.

The gene encoding the 'classical nitroreductase' (CNR) of Salmonella typhimurium was disrupted. In this manner, cnr null mutant derivatives of strains TA1535 and TA1538 were constructed, and named YG7131 and YG7127, respectively. In both strain backgrounds, cnr gene disruption reduced nitrofurazone-reductase activity. This reduction almost completely eliminated the nitroreductase activity of strain TA1538. In contrast, the nitroreductase activity of strain TA1535 was much higher than that in TA1538. In this background, cnr gene disruption resulted in a reduction in nitroreductase activity by a similar absolute amount as in TA1538, but representing only about one-quarter of the original activity of TA1535. The results suggest that S. typhimurium has originally at least two distinct nitroreductases, one of which is already deficient in strain TA1538; the CNR is present in both TA1535 and TA1538. Also, these two strains (including their derivatives, TA98 and TA100) are not isogenic with regard to nitroreductase activity. After the introduction of plasmid pKM101, the sensitivities of the strains YG7132 and YG7128, the cnr-null mutants of TA98 and TA100, respectively, against several nitro compounds were compared with those of the conventional cnr-deficient strains TA98NR and TA100NR and the wild-type strains TA98 and TA100. The mutagenicities of 2-nitrofluorene and 1-nitropyrene in YG7132 or TA98NR were ten-fold lower than those of the compounds in TA98. Similarly, the mutagenicity of 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide in strain YG7128 or TA100NR was substantially lower than that of the compound in TA100. However, the mutagenicity of 2-nitronaphthalene in YG7128 was between those observed with TA100 and TA100NR, suggesting that a nitroreductase in S. typhimurium other than CNR is involved in the metabolic activation of this compound. The cnr gene of S. typhimurium positively hybridized with DNA at 13 min on the E. coli chromosome where the nfsB and nfnB genes of E. coli are mapped. These results suggest that the cnr gene of S. typhimurium is a counterpart of the nfsB and nfnB genes of E. coli, and that the newly constructed cnr-deletion strains are useful to assess the role of nitroreductases in the metabolic activation of mutagenic nitro compounds.

Blotting, Southern↗
PubMed1997

Gene organization and transcriptional regulation of the gntRKU operon involved in gluconate uptake and catabolism of Escherichia coli.

We cloned and characterized the gntRKU operon encoding part of the GntI system involved in gluconate uptake and catabolism by Escherichia coli. The operon was shown to encode its repressor, a thermoresistant gluconate kinase, and a low affinity gluconate permease. CAT fusion analysis revealed that the operon has a promoter for gntR and another for gntKU, and that the gntR gene is constitutively expressed, while that of gntKU is regulated positively by the cAMP-CRP complex and negatively by GntR. Read-through transcription from the gntR promoter into gntK was decreased in the presence of GntR, although GntR did not repress its own promoter. In addition, transcriptional attenuation was observed after the gntK gene, so gntU expression is reduced presumably to modulate the production of the low affinity gluconate permease according to the available concentration of gluconate.

Amino Acid Sequence↗
PubMed1997

Sulphonylurea receptor 2B and Kir6.1 form a sulphonylurea-sensitive but ATP-insensitive K+ channel.

1. We analysed the K+ channel composed of the sulphonylurea receptor 2B (SUR2B) and an inwardly rectifying K+ channel subunit Kir6.1 coexpressed in a mammalian cell line, HEK293T, with the patch clamp technique. 2. In the cell-attached configuration, K+ channel openers (pinacidil and nicorandil) activated approximately 33 pS K+ channels (approximately 145 mM external K+), which were inhibited by the sulphonylurea glibenclamide. 3. Although SUR2B forms an ATP-sensitive K+ channel with Kir6.2, whose amino acid sequence is approximately 70% homologous with that of Kir6.1, the K+ channel composed of SUR2B and Kir6.1 surprisingly did not spontaneously open on patch excision in the absence of intracellular ATP. 4. In inside-out patches, uridine diphosphate and guanosine diphosphate induced channel activity, which was inhibited by glibenclamide but not ATP. Intracellular ATP on its own activated the channels. K+ channel openers and intracellular nucleotides synergistically activated the channel. 5. Therefore, the K+ channel composed of SUR2B and Kir6.1 is not a classical ATP-sensitive K+ channel but closely resembles the nucleotide diphosphate-dependent K+ channel in vascular smooth muscle cells.

ATP-Binding Cassette Transporters↗
PubMed1997

Generation of free radicals during lipid hydroperoxide-triggered apoptosis in PC12h cells.

The compound 13-L-hydroperoxylinoleic acid (LOOH) triggered the death of clonal rat pheochromocytoma PC12h cells (LD50 = about 8 microM). LOOH induced nuclear condensation and DNA fragmentation, which was prevented by cycloheximide (a protein synthesis inhibitor) and NGF, indicating that LOOH triggered apoptosis in PC12h cells. LOOH produced reactive oxygen species (ROS) in PC12h cells in a time- and dose-dependent manner, as measured by flow cytometry using the ROS-specific fluorescent indicator, 6-carboxy-2,7-dichorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA). Antioxidants such as N,N'-diphenyl-p-phenylenediamine (DPPD), vitamin E and N-acetylcysteine, and a ferric iron chelator, deferoxamine, inhibited the LOOH-triggered apoptosis and simultaneously decreased the generation of ROS, whereas an inhibitor of glutathione synthesis, buthionine sulfoximine (BSO), enhanced the apoptosis and increased the generation of ROS. These results indicate that LOOH triggers the apoptosis of PC12h cells by increasing the production of ROS. A confocal analysis with the Ca(2+)-specific fluorescent indicator, fluo-3, demonstrated that LOOH at concentrations up to 200 microM, did not increase the intracellular Ca2+ concentration. These data indicate that LOOH induces apoptosis of PC12h cells through the enhanced production of ROS, not through increasing the permeability of Ca2+.

Animals↗
PubMed1997

X-linked spinal and bulbar muscular atrophy with myasthenic symptoms.

We describe a patient with X-linked spinal and bulbar muscular atrophy (X-SBMA) and myasthenic symptoms. The diagnosis of X-SBMA was established by demonstration of the increased number of CAG repeats in the androgen receptor gene on the X chromosome. This patient was characterized by the clinical symptoms of fatigability, decremental motor responses to repetitive nerve stimulation, and improvement of the myasthenic symptoms with oral administration of pyridostigmine. No serum antibody to acetylcholine receptor was detected. It is suggested that, in the process of chronic denervation and reinnervation of X-SBMA, reinnervated motor endplates may be associated with the defect of neuromuscular transmission.

Genetic Linkage↗
PubMed1997

Targeted killing of migrating glioma cells by injection of HTK-modified glioma cells.

The "bystander effect" describes the killing of nearby unmodified cells and herpes simplex thymidine kinase (HTK)-transduced cells by ganciclovir (GCV) treatment. This effect is thought to be produced by contact between these cells. In this study, we showed that injected glioma cells migrated rapidly to a place distant from the injection point whereas injected virus-producing fibroblast cells did not migrate in a murine brain model. Moreover, the initially injected glioma cells and glioma cells injected at a later time mix very well, even at a place distant from the injection point. This suggested that glioma cells migrating after injection could be targeted by HTK-modified glioma cells introduced in a second injection and be killed together by GCV treatment. Therefore, we injected HTK-modified glioma cells 3 days after injection of wild glioma cells to investigate whether wild-type glioma cells that migrated to a place distant from the injection point could also be killed by GCV treatment. Tumor growth was suppressed after the GCV treatment. Suppression of tumor growth of wild glioma cells is not solely mediated by the immune response, which may be triggered by the killing of HTK-modified glioma cells with GCV, because inoculation of HTK-modified glioma to the contralateral side followed by GCV treatment did not cure the initial wild glioma. Moreover, the migration of the second inoculum of glioma cells is necessary for effective killing, because early administration of GCV resulted in insufficient killing.

Animals↗
PubMed1997

Protein kinase C and c-myc gene activation pathways in thrombopoietin signal transduction.

Thrombopoietin (TPO) is the major regulator of the proliferation and differentiation of megakaryocyte precursors through interaction with its receptor encoded by the c-mpl protooncogene. We established the human TPO-dependent leukemia cell line, UT-7/TPO (Blood 87, 4552, 1996). In these cells, TPO activated protein kinase C (PKC) in a time dependent manner. Subsequently, the c-myc gene was transiently induced to a maximal level 60-90 minutes after TPO exposure. In addition, we found that stimulating UT-7/TPO cells with TPO rapidly induces the significant accumulation of inositol 1, 4, 5-trisphosphate (Ins-P3), leading to the mobilization of calcium from intracellular stores. Taken together, the activation of PKC and subsequent c-myc gene induction are involved in the TPO-induced cellular response(s), presumably through the activation of PLC.

Calcium↗
PubMed1997

Differential human nucleotide excision repair of paired and mispaired cisplatin-DNA adducts.

In order to understand the action of the chemotherapeutic drug cisplatin, it is necessary to determine why some types of cisplatin-DNA intrastrand crosslinks are repaired better than others. Using cell extracts and circular duplex DNA, we compared nucleotide excision repair of uniquely placed 1,2-GG, 1,2-AG, and 1,3-GTG cisplatin-crosslinks, and a 2-acetylaminofluorene lesion. The 1,3 crosslink and the acetylaminofluorene lesion were repaired by normal cell extracts approximately 15-20 fold better than the 1,2 crosslinks. No evidence was found for selective shielding of 1,2 cisplatin crosslinks from repair by cellular proteins. Fractionation of cell extracts to remove putative shielding proteins did not improve repair of the 1,2-GG crosslink, and cell extracts did not selectively inhibit access of UvrABC incision nuclease to 1,2-GG crosslinks. The poorer repair of 1,2 crosslinks in comparison to the 1,3 crosslink is more likely a consequence of different structural alterations of the DNA helix. In support of this, a 1,2-GG-cisplatin crosslink was much better repaired when it was opposite one or two non-complementary thymines. Extracts from cells defective in the hMutSalpha mismatch binding activity also showed preferential repair of the 1,3 crosslink over the 1,2 crosslink, and increased repair of the 1,2 adduct when opposite thymines, showing that hMutSalphais not involved in the differential NER of these substrates in vitro. Mismatched cisplatin adducts could arise by translesion DNA synthesis, and improved repair of such adducts could promote cisplatin-induced mutagenesis in some cases.

Animals↗
PubMed1997

Selective recognition of a cisplatin-DNA adduct by human mismatch repair proteins.

The antitumor agent cis-diamminedichloroplatinum(II) (cisplatin) introduces cytotoxic DNA damage predominantly in the form of intrastrand crosslinks between adjacent purines. Binding assays using a series of duplex oligonucleotides containing a single 1,2 diguanyl intrastrand crosslink indicate that human cell extracts contain factors that preferentially recognise this type of damage when the complementary strand contains T opposite the 3', and C opposite the 5'guanine in the crosslink. Under the conditions of the band-shift assay used, little binding is observed if the positions of the T and C are reversed in the complementary strand. Similarly, duplexes containing CC or TT opposite the crosslink are recognised relatively poorly. The binding activity is absent from extracts of the colorectal carcinoma cell lines LoVo and DLD-1 in which the hMutSalpha mismatch recognition complex is inactivated by mutation. Extensively purified human hMutSalpha exhibits the same substrate preference and binds to the mismatched platinated DNA at least as well as to an identical unplatinated duplex containing a single G.T mismatch. It is likely, therefore, that human mismatch repair may be triggered by 1,2 diguanyl intrastrand crosslinks that have undergone replicative bypass.

Adenosine Triphosphatases↗
PubMed1997

Identification of binding domains for basic fibroblast growth factor in proteoglycan macrophage colony-stimulating factor.

We recently demonstrated that proteoglycan macrophage colony-stimulating factor (PG-M-CSF) binds basic fibroblast growth factor (bFGF) and neutralizes the biological activity of bFGF. In this study, we identified the binding sites of PG-M-CSF for bFGF. We examined the binding of bFGF to overlapping 12-mer peptides with the sequence of the putative binding region. High affinity binding was detected at two peaks; one consisted of the three adjacent peptides, 212-223, 213-224 and 214-225 and the other, of the three adjacent peptides, 246-257, 247-258 and 248-259. The synthetic peptide (212VDPGSAKQRPPRST225) did not inhibit bFGF binding to another peptide (246PQPRPSVGAFNPGM259), and vice versa. However, both peptides inhibited the bFGF-induced but not platelet-derived growth factor-induced stimulation of DNA synthesis in murine Balb/c 3T3 cells.

3T3 Cells↗
PubMed1997

Enhancement of macrophage colony-stimulating factor-induced growth and differentiation of human monocytes by interleukin-10.

Interleukin-10 (IL-10) has been reported to be a negative cytokine for monocytes/macrophages. In the present study, we showed that IL-10 is rather a positive cytokine and augments the growth and differentiation of human monocytes stimulated with macrophage colony-stimulating factor (M-CSF). Highly purified adherent human monocytes were cultured for 7 days with M-CSF in the presence or absence of IL-10. The number of recovered cells increased in the culture of monocytes with M-CSF + IL-10 compared to the culture with M-CSF alone. IL-10 alone was not enough to maintain the survival and differentiation of monocytes into macrophages. Morphological change cultured in M-CSF was also accelerated by addition of IL-10, and macrophages cultured in M-CSF + IL-10 were more elongated compared to macrophages cultured with M-CSF alone. Binding of 125I-M-CSF to monocytes incubated with M-CSF + IL-10 was about 1.7-fold higher than that to monocytes incubated with M-CSF alone. In accordance with the binding study, Northern blot analysis showed that the levels of the expression of c-fms, M-CSF receptor, mRNA in macrophages cultured in M-CSF + IL-10 were higher than that in macrophages cultured in M-CSF alone. Macrophages cultured in M-CSF + IL-10 expressed higher level of Fc gamma RI, II, III, and showed augmented Fc gamma receptor mediated phagocytosis. The former also produced higher level of H2O2 and O2-, when stimulated with zymosan, and of IL-6 when stimulated with lipopolysaccharide compared to the latter. These results taken together suggest that IL-10 augments the growth and differentiation of human monocytes cultured in M-CSF.

Antigens, Surface↗
PubMed1997

Suppressive effects of human herpesvirus 6 on in vitro colony formation of hematopoietic progenitor cells.

Human herpesvirus 6 (HHV-6) has been reported to be involved in bone marrow failure after bone marrow transplantation (BMT). To elucidate the role of HHV-6 in the marrow failure, we examined the comparative effect of two variants of HHV-6 (HHV-6A and HHV-6B) and human herpesvirus 7 (HHV-7) on in vitro colony formation of hematopoietic progenitor cells in methylcellulose semi-solid media. Progenitor cells prepared from cord blood mononuclear cells (CBMNCs) were infected with one of these viruses at various multiplicity of infection (MOI), and were subjected to methylcellulose colony assay. Formation of both granulocyte/macrophage (CFU-GM) and erythroid (BFU-E) colonies was MOI-dependently suppressed after infection with the Z29 strain of HHV-6B. Although HHV-6A suppressed the formation of BFU-E colonies as efficiently as HHV-6B, the former did not exhibit significant suppressive effect on the formation of CFU-GM colonies at an MOI 1. HHV-7 had no effect on hematopoietic colony formation at all. Based on frequent positivity of viral DNA in single colonies obtained from HHV-6-infected progenitor cells by polymerase chain reaction and in situ hybridization, direct effects of HHV-6 on the hematopoietic progenitor cells are suggested as the cause of the suppression rather than indirect effects via accessory cells of the bone marrow.

Bone Marrow Diseases↗
PubMed1997

Properties of primary murine stroma induced by macrophage colony-stimulating factor.

The ability of purified human macrophage colony-stimulating factor (M-CSF) to accelerate the formation of stromal cells from murine bone marrow cells was investigated. The liquid culture of the marrow cells with M-CSF resulted in the formation of monolayers of macrophages on day 7. When the M-CSF was removed on that day and the residual adherent cells were cultured in the absence of M-CSF for an additional 7 days, many colonies appeared with cells that were morphologically distinguishable from M-CSF-derived macrophages. The appearance of the colonies was dependent on the concentration of M-CSF used at the beginning of the culture. Each colony was isolated as a single clone and analyzed. All clones were negative for esterase staining. These cells did not express M-CSF receptor mRNA and did not show a mitogenic response to M-CSF. On the contrary, these cells could be stimulated to proliferate by fibroblast growth factor and platelet-derived growth factor. The polymerase chain reaction analysis of these cells demonstrated constitutive expression of mRNA for M-CSF, stem cell factor, and interleukin (IL)-1, but not IL-3. Some clones expressed mRNA for granulocyte/M-CSF and IL-6. We also examined the ability of the cells to maintain murine bone marrow high proliferative potential colony-forming cells (HPP-CFC) in a coculture system. Most of the clones showed a significant increase in total HPP-CFC numbers after 2 weeks of coculture, although the extent of stimulation differed among clones. These results suggested that the colonies established by M-CSF were composed of functional stromal cells that were phenotypically different from macrophages.

Animals↗
PubMed1997

Characterization of a deletion mutation involving exons 3-7 of the WASP gene detected in a patient with Wiskott-Aldrich syndrome.

A case of Wiskott-Aldrich syndrome (WAS) suspected to have a deletion mutation in the WAS protein (WASP) gene had previously been reported (Ariga et al., 1997). Genomic polymerase chain reaction (PCR) suggested that exons 3-7 of the WASP gene were included in the deletion. Present Southern blot studies confirm that the deletion is approximately 2.0 kb in length, involving exons 3-7 and seemed to have been created by the fusion of introns 2 and 7. To characterize the deletion mutation in detail, we analyzed the PCR-amplified fragments of introns 2 and 7 from normal individuals and the fragment suspected of including the deletion junction from the patient. Sequencing of the patient fragment revealed that the deletion mutation involving exons 3-7 of the WASP gene did, indeed, result from the fusion of introns 2 and 7.

Base Sequence↗
PubMed1997

Regional and cellular expression of the dentatorubral-pallidoluysian atrophy gene in brains of normal and affected individuals.

Dentatorubral-pallidoluysian atrophy is an autosomal dominant neurodegenerative disorder characterized by dementia and spinocerebellar degeneration. Recently, part of the gene responsible for this disorder was cloned containing a CAG repeat, and predominant neuronal expression of the gene was proved only through Northern blot analysis in rats. In this study, we investigated the regional and cellular expression of the dentatorubral-pallidoluysian atrophy gene in the central nervous system of normal and affected humans, as well as in rat brains. In normal control human subjects, the gene messenger RNA was present in all brain regions examined, with the highest levels seen in the cerebellum, hippocampus, substantia nigra, and pontine nuclei. Its expression in the striatum, globus pallidus, and cerebral cortex was intermediate. The gene was expressed predominantly in neurons; a low but significant level of expression was also seen in glial cells. Neurons susceptible to degeneration in dentatorubral-pallidoluysian atrophy did not selectively express high or low levels of its gene messenger RNA. In brains affected by the disorder, the distribution and levels of gene messenger RNA were comparable to those of the normal controls in all the areas. In the rat brains, gene messenger RNA expression was very similar to that in human brain. It was also expressed predominantly in neurons, while low-level expression was observed in glial cells. It is apparent from these results that the presence of expanded trinucleotide repeats in dentatorubral-pallidoluysian atrophy does not result in the absence of its gene messenger RNA expression or in altered patterns or levels of expression.

Adult↗