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Biomedical subjects

M Fukuda

Publications and source records attributed to M Fukuda.

At least 649 records · Page 36Linked to original sources
PubMed1995

[A case of acute myelogenous leukemia accompanied with myelofibrosis and megakaryocyte-like giant bizarre blasts].

A 45-year-old man was admitted with high fever and leukocytosis in August 1993. The diagnosis of acute myelogenous leukemia (AML; M2) was made on the basis of morphological, cytochemical and immunological characteristics of the blasts in the bone marrow. The induction therapy with BHAC, daunorubicin, 6-MP was unsuccessful in achieving remission; the bone marrow biopsy specimen revealed the proliferation of the remaining leukemic cells and massive fibrosis accompanied with unusual megakaryocyte-like giant bizarre cells. These megakaryocyte-like giant cells were positive for myeloperoxidase and CD34, but not GPIIIa and factor VIII, indicating that those were derived from myelogenous stem cells. Following the low-dose Ara-C therapy, improvement of fibrosis and disappearance of these giant cells were observed in the bone marrow. After the reinduction therapy with high-dose Ara-C and MIT against markedly increased blasts, the patient died of systemic fungal infection. The presence of myelofibrosis and giant atypical blasts might allow resistance to therapy and poor prognosis.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1995

PCR amplification of D2S44 (YNH24) alleles.

We have successfully amplified D2S44 (YNH24) alleles by a method for long-distance PCR using a special polymerase enhancer, Taq Extender PCR Additive. The alleles amplified from DNA samples of 58 Japanese subjects ranged from 0.42 to 3.5 kb and were 1.5 kb shorter than those detected by Southern blotting of Hinf I-digested genomic DNAs. Although alleles longer than 3 kb were barely visible by ethidium bromide staining, we were able to visualize them clearly with SYBR GREEN I NUCLEIC ACIC GEL STAIN. PCR amplification of D2S44 alleles is much simpler than their restriction fragment length polymorphism (RFLP) analysis; therefore, our procedure is well-suited for use in medicolegal practice. With minor modifications, the method described here should be applicable to other loci of variable number of tandem repeats (VNTRs) that have been analyzed by Southern blotting.

Alleles↗
PubMed1995

Iron and copper deposition in chronic active hepatitis and liver cirrhosis; pathogenetic role in progressive liver cell damage.

Iron and copper deposition were examined in patients with chronic active viral hepatitis (CAH) and posthepatitic liver cirrhosis (LC) by Berlin blue, rhodanine, or Victoria blue staining and X-ray microanalysis. Considerable iron or copper deposition was demonstrated in the peripheral zones of hepatic lobules in both CAH (53% of specimens) and LC (63% of specimens). Frozen sections taken from the 2 CAH surgical sections with iron depositions were examined by photoncounting image analysis, and superoxide liberation from the metal granules were demonstrated. In areas of metal deposition, vacuolation of liver cell nuclei, accumulation of lipofuscin, and induction of metallothionein (69% of rhodanine- or Victoria blue-positive specimens) were often demonstrated, whereas induction of ferritin was found only in 14% of Berlin blue-positive specimens. The PCNA index was significantly lower in areas of metal deposition than in the adjacent areas without metal deposition, indicating lowered proliferative capability in the former. These results indicate that cell-mediated immune mechanisms causing the disturbance of bile secretion and heavy metal deposition in the peripheral zones of hepatic lobules may be involved in the progression of viral hepatitis from its acute phase to CAH and finally to LC phase, resulting in piecemeal necrosis. However, cholangitis could not be demonstrated in the present study.

Adult↗
PubMed1995

[Glial tumor cell proliferation and immune response in the brain].

The mechanism of glial tumor cell proliferation and the immune response to glioma cells in the brain were examined both in vitro and in vivo experimental systems, using mouse malignant glioma cell line, 203-glioma. A fura-2 fluorescence image showed marked rise in the intracellular calcium ion concentration in mechanically stimulated single cells. The increased calcium spread to adjacent cells, probably due to some stimulating factor released from cells. Dye microinjection revealed no gap junction between cells. Antagonists of voltage-dependent calcium channels did not act on the calcium response. These suggest that calcium signaling in the glioma cells may be mediated via a membrane receptor but not through a gap junction. Depletion of extracellular calcium ion and addition of intracellular calcium blocker demonstrated that calcium signaling in stimulated cells may be related to both an influx of extracellular calcium and a redistribution of intracellular calcium from internal stores, whereas calcium transmission to adjacent cells may involve calcium influx alone. The splenic cytotoxic T lymphocyte (CTL) activity in intracerebral tumor-bearing hosts increased with a peak 2 weeks after tumor cell inoculation, but rapidly decreased concurrently with increased intracranial pressure. The major histocompatibility complex, MHC, class I antigen expression on tumor cells grafted intracerebrally was found to enhance markedly, resulting in an increase in susceptibility to CTL. It was suggested that there may be a positive correlation between the cell surface MHC class I antigen expression and sensitivity to CTL in glioma cells.

Animals↗
PubMed1994

Analysis of the Ras p21/mitogen-activated protein kinase signaling in vitro and in Xenopus oocytes.

Ras p21 in the GTP-bound form was shown to act as an upstream activator for mitogen-activated protein (MAP) kinase kinase (MAPKK) and MAP kinase, and Raf-1 was reported to act as a MAPKK kinase. Further, physical association between Ras and Raf-1 was demonstrated. Here we have shown that incubation of Xenopus immature oocyte extracts with Ras enhances the ability of endogenous Raf-1 to activate MAPKK. Moreover, a dominant negative form of Raf-1 blocked the Ras-induced activation of MAPKK and MAP kinase in the extracts, but not the cyclin A-dependent activation of MAP kinase. When the extracts were depleted of 45-kDa MAPKK with polyclonal anti-MAPKK antibody, no activation of MAP kinase occurred even after incubation with Ras. These results suggest that Ras can activate the MAPKK kinase activity of Raf-1 in the extracts and that MAPKK is indispensable for the Ras-induced MAP kinase activation. It is well known that Ras can induce oocyte maturation when injected into immature Xenopus oocytes. Co-injection of Ras with an anti-MAPKK antibody that inhibits the MAPKK activity prevented the Ras-induced germinal vesicle breakdown, suggesting that MAPKK mediates, at least, one of cellular functions of Ras.

Animals↗
PubMed1994

Expression of distinct fucosylated oligosaccharides and carbohydrate-mediated adhesion efficiency directed by two different alpha-1,3-fucosyltransferases. Comparison of E- and L-selectin-mediated adhesion.

Among five different human alpha 1 --> 3 fucosyltransferases cloned, fucosyltransferases III (Fuc-TIII) and IV (Fuc-TIV) differ significantly from each other. Fuc-TIII transfers a fucose to both sialylated and nonsialylated N-acetyllactosamine, but Fuc-TIV apparently transfers a fucose only to neutral N-acetyllactosamine. In this study, Chinese hamster ovary (CHO) cells were stably transfected with Fuc-TIII or Fuc-TIV, and the resultant cell lines, CHO-FTIII and CHO-FTIV, were compared for the carbohydrate structures and for their binding to E-selectin or L-selectin. CHO-FTIII and CHO-FTIV cells were labeled metabolically with [3H]galactose, and glycopeptides obtained from these cells were fractionated by serial lectin affinity chromatography. The fractionated glycopeptides were then subjected to various combinations of exoglycosidase treatment or endo-beta-galactosidase digestion. The results obtained can be summarized as follows. CHO-FTIII cells express sialyl Lewisx, Lewisx, and VIM-2 structures, whereas CHO-FTIV cells express only an Lex structure with a small amount of VIM-2 structure. When CHO-FTIII and CHO-FTIV cells were tested for adhesion to E-selectin expressed by tumor necrosis factor-activated endothelial cells and to an E-selectin chimeric protein, only CHO-FTIII cells were found to adhere well to E-selectin. Moreover, both CHO-FTIII and CHO-FTIV cells failed to adhere to an L-selectin chimeric protein. These results clearly indicate that FT-III and FT-IV direct distinctly different fucosylated oligosaccharides. This difference in oligosaccharide structures results in an entirely different efficiency in adhesion to E-selectin. The results also demonstrate that expression of sialyl Lex itself is not sufficient for L-selectin binding.

Animals↗
PubMed1994

The inositol high-polyphosphate series blocks synaptic transmission by preventing vesicular fusion: a squid giant synapse study.

Presynaptic injection of inositol 1,3,4,5-tetraphosphate, inositol 1,3,4,5,6-pentakisphosphate, or inositol 1,2,3,4,5,6-hexakisphosphate--which we denote here the inositol high-polyphosphate series (IHPS)--is shown to block synaptic transmission when injected into the preterminal of the squid giant synapse. This effect is not produced by injection of inositol 1,4,5-trisphosphate. The synaptic block is characterized by a time course in the order of 15-45 min, depending on the injection site in the preterminal fiber; the fastest block occurs when the injection is made at the terminal release site. Presynaptic voltage clamp during transmitter release demonstrates that IHPS block did not modify the presynaptic inward, calcium current. Analysis of synaptic noise at the postsynaptic axon shows that both the evoked and spontaneous transmitter release are blocked by the IHPS. Tetanic stimulation of the presynaptic fiber at frequencies of 100 Hz indicates that block is accompanied by gradual reduction of the postsynaptic response, demonstrating that the block interferes with vesicular fusion rather than with vesicular docking. These results, in combination with the recently demonstrated observation that the IHPS bind the C2B domain in synaptotagmin [Fukada, M., Aruga, J., Niinobe, M., Aimoto, S. & Mikoshiba, K. (1994) J. Biol. Chem. 269, 29206-29211], suggest that IHPS elements are involved in vesicle fusion and exocytosis. In addition, a scheme is proposed in which synaptotagmin triggers transmitter release directly by promoting the fusion of synaptic vesicles with the presynaptic plasmalemma, in agreement with the very rapid nature of transmitter release in chemical synapses.

Animals↗
PubMed1994

Synaptotagmin is an inositol polyphosphate binding protein: isolation and characterization as an Ins 1,3,4,5-P4 binding protein.

We isolated a binding protein for inositol 1,3,4,5-tetrakisphosphate (InsP4) from detergent-solubilized mouse cerebellar membrane fractions by sequential column chromatographies. Partial amino acid sequencing of the purified sample revealed that the protein is essentially identical to rat synaptotagmin II, an integral membrane protein of synaptic vesicles. Immunoprecipitation experiment of [3H]InsP4 binding activity of the purified protein using polyclonal antibody against the C2A domain of rat synaptotagmin II also revealed that mouse synaptotagmin II is the InsP4 binding protein (IP4BP). Scatchard analysis of InsP4 binding to the IP4BP/synaptotagmin indicates a single binding site with a Kd of 30 nM. The present finding that InsP4 binds strongly to synaptotagmin II suggests an important role for inositol polyphosphates in the regulation of neurotransmitter release.

Amino Acid Sequence↗
PubMed1994

Inositol-1,3,4,5-tetrakisphosphate binding to C2B domain of IP4BP/synaptotagmin II.

IP4BP/Synaptotagmin II is an inositol-1,3,4,5-tetrakisphosphate (IP4) or inositol polyphosphate-binding protein, which is accumulated at nerve terminals. Here we report a novel function of the C2B domain, which was originally thought to be responsible for Ca(2+)-dependent binding to phospholipid membranes. A study of deletion mutants showed that about 30 amino acids of the central region of the C2B domain of mouse IP4BP/synaptotagmin II (315 IHLMQNGKRLKKKKTTVKKKTLNPYFNESFSF 346) are essential for inositol polyphosphate binding. This binding domain includes a sequence corresponding to the squid Pep20 peptide, which is also known to be essential for neurotransmitter release (Bommert, K., Charlton, M. P., DeBello, W. M., Chin, G. J., Betz, H., and Augustine, G. J. (1993) Nature 363, 163-165), suggesting that inositol polyphosphate has some effect on neurotransmitter release. Rabphilin 3A, another neuronal protein containing C2 domains, cannot bind IP4, indicating that the IP4 binding property is specific to the C2B domain of synaptotagmin. Phospholipid and IP4 binding experiments clearly indicated that the C2A and C2B domains have different functions. The C2A domain binds phospholipid in a Ca(2+)-dependent manner, but the C2B domain binds inositol polyphosphate and phospholipid irrespective of the presence of Ca2+. Our data suggest that the C2B domain of synaptotogamin is the inositol polyphosphate sensor at the synaptic vesicle and may be involved in synaptic function.

Amino Acid Sequence↗
PubMed1994

The pH-dependent swinging-out of the distal histidine residue in ferric hemoglobin of a midge larva (Tokunagayusurika akamusi).

Hemoglobin VII (TaVII) is a major component in the larval hemolymph of Tokunagayusurika akamusi, a common midge (Diptera) found in Japan. This protein contains 150 amino-acid residues including the usual distal histidine at position 64. When the aquomet-form was placed in acidic pH range, its Soret peak was considerably blue-shifted and accompanied by a marked decrease in intensity, indicative of the protein being converted into a structure quite similar to that of Aplysia myoglobin lacking the distal histidine residue. The pH-dependent magnetic circular dichroism spectra in the Soret region have also revealed that TaVII hemoglobin is in an equilibrium between a hexacoordinate and a pentacoordinate structure for its ferric heme iron. We attribute this to a transition from an iron-ligated water molecule that is hydrogen-bonded to the distal histidine, to a water-free iron with the histidine swung-out of the heme pocket. Furthermore, this process was described by the involvement of a single dissociable group with pKa = 6.3 in 0.1 M KCl at 25 degrees C.

Animals↗
PubMed1994

Effect of suramin on p34cdc2 kinase in vitro and in extracts from human H69 cells: evidence for a double mechanism of action.

We examined the effect of suramin, an anticancer agent and a functional analog of naturally occuring glycosaminoglycans, on p34cdc2 kinase. We find that suramin strongly inhibits the catalytic activity of purified p34cdc2 kinase (IC50 approximately 4 microM), whereas it only weakly inhibits the p13-agarose precipitated kinase activity from nuclear and cytoplasmic extracts of the asynchronous H69 human small cell lung cancer cells. We also find that the tyrosine phosphorylation of p34cdc2 kinase in the nuclear extract is increased about twice when the extracts are preincubated with 50 microM of suramin prior to the p13-agarose precipitation. We propose that this increase might result from the inhibitory effect of suramin towards p34cdc2-specific tyrosine phosphatases. These results suggest both a direct and an indirect effect of suramin on p34cdc2 kinase. We also find that heparin is a potent inhibitor of purified cdc2 kinase (IC50 approximately 3.5 micrograms/ml). Therefore, glycosaminoglycans might be physiological regulators of p34cdc2 kinase in vivo.

Antineoplastic Agents↗
PubMed1994

Contribution of gene conversion to the retention of the sequence for M blood group type determinant in glycophorin E gene.

M and N blood group antigens are presented by glycophorins A (GPA) and B (GPB) of the erythrocyte membrane. GPA expresses M or N blood group antigen depending on the allelic gene (GPAM gene or GPAN gene), while GPB expresses only the N antigen. M or N blood group antigen is specified by the first and fifth NH2-terminal amino acid residues in the mature proteins, encoded by the second exon of these genes. Glycophorin E (GPE) gene, another member of this gene family, has a genomic structure very similar to that of GPB. However, a predicted product of GPE gene carries M blood group antigen. To delineate evolutionary events leading to the formation of the sequences for M or N blood group type determinant in the members of human glycophorin gene family, the nucleotide sequences of a 1.45-kilobase pair region including the 3' part of intron 1, exon 2, and the entire intron 2 were compared for GPAM, GPAN, GPB, and GPE genes. Encompassing exon 2, there were identical sequences stretches of 139 and 138 base pairs between GPAM and GPE genes and between GPAN and GPB genes, respectively. The data of the entire sequences in this region revealed that the divergence of the sequence between GPE and GPA genes (2.14-2.49%) is less than that of the sequence between GPE and GPB genes (3.10%). The higher rate of divergence in this region was observed between GPA and GPB genes (3.79-3.86%). These results strongly suggest that GPE gene acquired the sequence for M blood group type determinant from GPAM gene through gene conversion after duplication of a progenitor gene common to GPB and GPE genes.

Alleles↗
PubMed1994

Identification of the bphA and bphB genes of Pseudomonas sp. strains KKS102 involved in degradation of biphenyl and polychlorinated biphenyls.

The nucleotide sequence of the upstream region of the bphC gene from Pseudomonas sp. strain KKS102 was determined. Four genes were found in this region. Deduced amino acid sequences of the first, second, third and fourth genes showed significant homology with a large subunit of iron-sulfur protein, a small subunit of iron-sulfur protein, ferredoxin and dihydrodiol dehydrogenase, respectively, from other bacteria which degrade biphenyl/polychlorinated biphenyls, toluene and benzene. E. coli, in which the four genes, bphC and the gene for ferredoxin reductase from benzene degrading bacterium were expressed, was able to produce meta-cleavage compounds from chlorinated biphenyls. These results show that these gene products are functional in both biphenyl and polychlorinated biphenyls degradation.

Alcohol Oxidoreductases↗
PubMed1994

Increased expression of nucleoside diphosphate kinases/nm23 in human diploid fibroblasts transformed by SV40 large T antigen or 60Co irradiation.

When the expression levels of nucleoside diphosphate (NDP) kinase/nm23 were examined in four human normal diploid fibroblast cell lines in comparison with their corresponding immortalized cells transformed by SV40 large T antigen or 60Co irradiation, mRNA levels of the two isoforms (NDP kinase A/nm23-H1, NDP kinase B/nm23-H2) were increased in the immortalized cell lines. The increase was found to be associated with increased translation products. Furthermore, the cell extracts prepared from these immortalized cell lines demonstrated slightly higher enzyme activity than those from their normal counterparts. Neither the growth state nor the in vitro aging largely affected their expression in a normal cell line (TIG-3) examined. The results suggest possible involvement of NDP kinases/nm23 in acquiring an infinite growth property of these cells.

Antigens, Polyomavirus Transforming↗
PubMed1994

Transcriptional activation of human leukosialin (CD43) gene by Sp1 through binding to a GGGTGG motif.

Human leukosialin (CD43) is expressed on the surface of hematopoietic cells in cell-type specific and differentiation-stage-specific manners. Previously we found that the sequence from -53 to -40 was critically involved in the promoter function [Kudo, S. & Fukuda, M. (1991) J. Biol. Chem. 266, 8483-8489]. A transient-expression assay using a chloramphenicol acetyltransferase reporter gene revealed that the promoter could confer a high basal transcriptional activity in both leukosialin-producing and non-producing cells. The transcription factor interacting with the promoter sequence was determined by DNase I footprinting and gel-mobility-shift assays. The nuclear extracts from both leukosialin-producing Jurkat cells and non-producing Hela cells showed a footprint on the 5' flanking region from -58 to -34. Gel-mobility-shift assays revealed that DNA-protein complexes were formed with both nuclear extracts, and these complex formations were inhibited by an oligonucleotide containing the Sp1-binding consensus sequence. Prior incubation of anti-Sp1 antibody with nuclear extracts in this assay resulted in the supershift of the band for the DNA-protein complex. In addition, the footprint produced by the purified Sp1 transcription factor was identical to those produced by nuclear extracts of Jurkat and Hela cells. The mutational analyses revealed that the binding affinities of Sp1 to mutated promoter sequences were parallel to the transcriptional activity of these promoter sequences. Transient expression analyses in Drosophila Schneider cells demonstrated that cotransfection with Sp1 expression plasmid increased the transcriptional activity. These results establish that Sp1 can bind to the promoter and positively regulates the expression of the leukosialin gene. Even the stable expression of CAT constructs in non-producing Hela cells showed high transcriptional activity. The leukosialin expression thus appears to be regulated by the unique mechanism, that is the repression of high basal transcriptional activity rather than the activation of the basal transcriptional level. Tissue-specific expression is probably achieved by suppression of the basal transcriptional activity in non-producing cells.

Animals↗