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H Sugiyama

Publications and source records attributed to H Sugiyama.

At least 19 recordsLinked to original sources
PubMed1997

Interactions of deglycosylated cobalt(III)-pepleomycin (green form) with DNA based on NMR structural studies,.

Pepleomycin (PEP)1 is a metalloglycopeptide antitumor antibiotic that has improved pharmacological properties than does bleomycin (BLM). Both PEP and BLM bind to and degrade DNA in a sequence-selective manner. The binding interactions of HO2--Co(III)-CodPEP (CodPEP) with CGTACG have been studied by 2D NMR and molecular modeling. Inspection of the 2D-NMR data revealed 60 notable intermolecular NOEs between CodPEP and CGTACG which place the drug's metal binding domain and peptide linker in the minor groove of the DNA close to G8 and T9. On the basis of the NOEs, the drug's DNA binding domain is located close to the T9.A4 and A10.T3 base pairs. Intercalation of the bithiazole tail between these base pairs is indicated by the loss of DNA symmetry upon complexation with CodPEP, by a break in the sequential connectivity at the TpA steps, and by the upfield shift of the bithiazole H-H5 and H-H5' proton resonances. Intercalation of the bithiazole moiety unfolds the CodPEP molecule and exposes its hydroperoxide group to the DNA. The hydroperoxide group in the refined model of CodPEP-CGTACG is close to the C4' proton of T9, consistent with cleavage at this position. The NOE pattern between the pyrimidine ring of CodPEP and G8 of DNA suggests a specific pairing recognition via hydrogen bonds between these groups, thus establishing a 5'-GT-3' sequence preference. The structural elucidation of the free CodPEP and CoPEP [Caceres-Cortes et al. (1997) Eur. J. Biochem. 244, 818-828], and of the complex of CodPEP-CGTACG afford a plausible mechanism for the recognition and its subsequent cleaving of DNA by the drug. The process involves the unfolding of the compact CodPEP, recognition of a guanine base using the metal binding domain, threading of the bithiazole tail between base pairs, and finally positioning of the HO2- group close to the T or C found 3' to the specific G site.

Antibiotics, Antineoplastic↗
PubMed1997

Phosphorylation and calmodulin binding of the metabotropic glutamate receptor subtype 5 (mGluR5) are antagonistic in vitro.

Metabotropic glutamate receptors, which are members of a G protein-coupled receptor family, mediate the glutamate responses by coupling to the intracellular signal transduction pathway. We herein report that calmodulin (CaM) interacts with the metabotropic glutamate receptor subtype 5 (mGluR5) in a Ca2+-dependent manner in vitro. CaM is capable of binding on two distinct sites in the COOH-terminal intracellular region of the receptor with different affinities. The CaM binding domains are separated by an alternatively spliced exon cassette present in one of the splicing isoforms of mGluR5. By using fusion proteins and synthetic peptides we showed that protein kinase C phosphorylates both CaM binding regions. This phosphorylation is inhibited by the binding of CaM to the receptor, and conversely the binding is inhibited by the phosphorylation. These antagonisms of the CaM binding and phosphorylation thus suggest the possibility that they regulate the receptor responses in vivo.

Amino Acid Sequence↗
PubMed1997

Overexpression of wild type p70 S6 kinase interferes with cytokinesis.

p70 S6 kinase (p70s6k) is a serine/threonine kinase which is activated through an unidentified pathway by mitogenic stimuli. Here we demonstrate that stable- and transient-overexpression of wild type (WT)-p70s6k results in perturbation of cytokinesis in NIH3T3 cells. Cells overexpressing WT-p70s6k demonstrated a slow growth rate and lower viability, whereas cells overexpressing a mutant T229A-p70s6k had a similar growth rate and viability as those of parental NIH3T3 cells. Moreover, WT-p70s6k cells demonstrated several abnormalities in cellular morphology; both the cytoplasm and the nuclei were large and a proportion of the cells was multinucleated. Flow cytometric analysis of propidium iodide-stained cells demonstrated that about 50% of the cells had 4N or greater content of DNA. When cell division in WT-p70s6k cells was monitored by time-lapse video microscopy, cytokines was often incomplete after mitosis, producing binucleated cells. Addition of rapamycin to WT-p70s6k cells, which inactivates endogenous and transfected p70s6k, failed to reverse any of the morphological abnormalities. These indicate that overexpression of p70s6k but not increases in activity of p70s6k, is responsible for the phenotype. A molecule which interacts with p70s6k may be involed in this regulation of cytokinesis.

3T3 Cells↗
PubMed1997

The Wilms tumor suppressor gene WT1 induces G1 arrest and apoptosis in myeloblastic leukemia M1 cells.

WT1 was isolated as a tumor suppressor gene of Wilms tumor. However, high expression of WT1 correlates with poor prognosis in acute leukemia. In addition suppression of WT1 expression by WT1 anti-sense oligonucleotide inhibits proliferation of leukemia cells, suggesting that WT1 is important for their proliferation. To further elucidate the biological significance of WT1 in leukemic cell growth, we overexpressed exogenous WT1 in murine M1 myeloblastic leukemia cells using the isopropyl-beta-D-thiogalactoside (IPTG)-controlled expression system. We found that induction of one splicing variant of WT1 [WT1-17AA(+)-KTS(-)] in M1 cells induces cell cycle arrest and apoptotic cell death. These results suggest that the role of WT1 is different depending on the type of leukemia cell in which it is expressed.

Animals↗
PubMed1997

Photoaffinity labeling of peroxisome proliferator binding proteins in rat hepatocytes; dehydroepiandrosterone sulfate- and bezafibrate-binding proteins.

To detect the cellular sites which directly interact with peroxisome proliferators (PPs) and mediate their inducing effect on peroxisomal enzymes in rat hepatocytes, two kinds of radiolabeled ligands, AD12 (7alpha-N-(4-azido-2-hydroxy-5-iodo[125I]benzyl)-aminomethyl-5-and rostene-3beta-ol-17-one-O-3-sulfate) and BZ5 (2-[p-[2-(4'-azido-3',5'-diiodo[125I]benzamido-2'-hydroxy)ethyl]phenoxy] -2-methylpropionic acid), were developed for photoaffinity labeling. These compounds were derivatives of dehydroepiandrosterone sulfate (DHEAS) and bezafibrate, respectively, with an azido group as the photoreactive functional group. Upon UV-irradiation following incubation with rat liver cytosol and nuclei, both the ligands effectively radiolabeled several proteins analyzed by SDS-polyacrylamide gel electrophoresis/radioluminography. When [125I]AD12 was used at a concentration of 0.2 microM, two cytosolic proteins with molecular masses of 55 and 28 kDa and a nuclear protein of 40 kDa were specifically labeled, as coincubation with a 1000-fold excess of DHEAS inhibited labeling. Photoaffinity labeling of the cytosolic 28-kDa protein was also affected by Wy-14,643, but not by unsulfated dehydroepiandrosterone or androsterone sulfate, consistent with our previous findings obtained in competitive binding studies of [3H]DHEAS-binding detected in rat liver cytosol (Yamada et al. (1994) Biochim. Biophys. Acta 1224, 139-146). On the other hand, [125I]BZ5 specifically labeled a cytosolic protein of 31 kDa, which was inhibited by coincubation with bezafibrate, clofibric acid and Wy-14,643, but not with DHEAS. Thus, [125I]AD12 and [125I]BZ5 labeled several proteins which recognized DHEAS and bezafibrate, respectively, in rat liver cytosol and nuclei, providing a useful means to investigate PP-binding proteins.

Affinity Labels↗
PubMed1997

Synapse-selective impairment of NMDA receptor functions in mice lacking NMDA receptor epsilon 1 or epsilon 2 subunit.

1. We have explored the effects of targeted disruption of the N-methyl-D-aspartate (NMDA) receptor epsilon 1 or epsilon 2 subunit gene on NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) and long-term potentiations (LTPs) at the two types of synapse in mouse hippocampal CA3 pyramidal neurons: those formed by the commissural/associational (C/A) and fimbrial (Fim) inputs. 2. Electrophysiological experiments were performed in hippocampal slices prepared from both wild-type and epsilon 1- or epsilon 2-disrupted mice using extracellular and whole-cell patch recording techniques. To assess the epsilon 1, epsilon 2 and zeta 1 subunit expression at cellular levels, we performed non-isotopic in situ hybridization with digoxigenin-labelled cRNA probes. 3. We could record EPSCs in response to the stimulations to either of the C/A and Fim afferents from a single CA3 pyramidal neuron. The epsilon 1, epsilon 2 and zeta 1 subunits were expressed together in individual CA3 neurons. 4. The epsilon 1 subunit disruption selectively reduced NMDA EPSCs and LTP in the C/A-CA3 synapse without significantly affecting those in the Fim-CA3 synapse, whereas the epsilon 2 subunit mutation diminished NMDA EPSCs and LTP in the Fim-CA3 synapse with no appreciable functional modifications in the C/A-CA3 synapse. 5. These results suggest that NMDA receptors with different subunit compositions function within a single CA3 pyramidal cell in a synapse-selective manner.

Animals↗
PubMed1997

Structures of cobalt(III)-pepleomycin and cobalt(III)-deglycopepleomycin (green forms) determined by NMR studies.

Pepleomycin (PEP) is a metalloglycopeptide that has stronger anticancer activity and less pulmonary toxicity than bleomycin (BLM). PEP, like BLM, exerts its action by binding to and degrading DNA in the presence of oxygen and certain metals. Obtaining detailed structural information of PEP and PEP-DNA complexes is crucial to understanding its anticancer activity. The structures of two green forms of cobalt-PEP species, HO2-Co(III)-PEP (denoted CoPEP) and deglycosylated HO2-Co(III)-PEP (denoted CodPEP) have been obtained by NOE restrained refinements. Earlier studies of the related HO2-Co(III)-BLM A2 proposed that two chiral conformers (form A or B) could exist with either the beta-aminoalanine primary amine (A,NH2) or the mannose carbamoyl nitrogen (M,NH2) as the axial ligand. Analysis of our NOESY data shows convincingly that form A is the most probable conformer with the mannose carbamoyl M,NH2 and the beta-aminoalanine primary amine A,NH2 as the axial ligands in CoPEP and CodPEP, respectively. The NOE cross-peaks resulting from the interactions between the N-terminus (i.e., the metal-binding domain) and the C-terminus of CoPEP and CodPEP have similar patterns, suggesting that they both adopt compact structures with the bithiazole group folded back over the N-terminus.

Antibiotics, Antineoplastic↗
PubMed1997

Cloning of cDNA and estrogen-induced hepatic gene expression for choriogenin H, a precursor protein of the fish egg envelope (chorion).

A cDNA for choriogenin H (Chg H; formerly high-molecular weight spawning female-specific substances, or H-SF), a precursor protein of the inner layer subunits of egg envelope (chorion) of the teleost fish, Oryzias latipes, was cloned and analyzed. The clone consisted of 1913 bp and contained an open reading frame encoding a signal peptide of 22 aa and Chg H protein of 569 aa. The Chg protein possessed three potential N-glycosylation sites and Pro-X-Y repeat sequences in the first two-fifths of the N terminus. There were amino acid sequence similarities between Chg H and a gene product expressed in the liver of female winter flounder during vitellogenesis. Moreover, the amino acid sequence of Chg H is similar to that of ZP2 rather than ZP3 of zona pellucida of some mammals. Northern blot analysis indicated that gene expression for Chg H occurred only in the livers of spawning female fish and 17beta-estradiol-treated male fish, but not in the ovary of the spawning female fish. Gene expression for Chg H and Chg L (formerly low-molecular weight spawning female-specific substance, or L-SF) was induced and increased in parallel in the male fish liver after 17beta-estradiol treatment.

Amino Acid Sequence↗
PubMed1997

Aberrant overexpression of the Wilms tumor gene (WT1) in human leukemia.

To clarify whether the expression of the WT1 gene in leukemic cells is aberrant or merely reflects that in normal counterparts, the expression levels of the WT1 gene were quantitated for normal hematopoietic progenitor cells. Bone marrow (BM) and umbilical cord blood (CB) cells were fluorescence-activated cell sorting (FACS)-sorted into CD34+ and CD34- cell populations, and the CD34+ cells into nine subsets (CD34+ CD33-, CD34+ CD33+, CD34+ CD38-, CD34+ CD38+, CD34+ HLA-DR-, CD34+ HLA-DR+, CD34+ c-kit(high), CD34+ c-kit(low), and CD34+ c-kit-) according to the expression levels of CD34, CD33, CD38, HLA-DR, and c-kit. Moreover, acute myeloid leukemic cells were also FACS-sorted into four populations (CD34+ CD33-, CD34+ CD33+, CD34- CD33+, and CD34- CD33-). FACS-sorted normal hematopoietic progenitor and leukemic cells and FACS-unsorted leukemic cells were examined for the WT1 expression by quantitative reverse transcriptase-polymerase chain reaction. The WT1 expression in the CD34+ and CD34- cell populations and in the nine CD34+ subsets of BM and CB was at either very low (1.0 to 2.4 x 10(-2)) or undetectable (< 10(-2)) levels (the WT1 expression level of K562 cells was defined as 1.0), whereas the average levels of WT1 expression in FACS-sorted and -unsorted leukemic cells were 2.4 to 9.3 x 10(-1). Thus, the WT1 expression levels in normal hematopoietic progenitor cells were at least 10 times less than those in leukemic cells. Therefore, we could not find any normal counterparts of BM or CB that expressed the WT1 at levels comparable with those in leukemic cells. These results indicate an aberrant overexpression of the WT1 gene in leukemic cells and imply the involvement of this gene in human leukemogenesis.

Acute Disease↗
PubMed1997

Perturbation of the expression of the catalytic subunit C alpha of cyclic AMP-dependent protein kinase inhibits TCR-triggered secretion of IL-2 by T helper hybridoma cells.

The role of cAMP-dependent protein kinase (PKA) in the regulation of TCR-triggered IL-2 secretion was studied by transfecting T hybridoma cells with cDNA encoding the inhibitory regulatory subunit (RI alpha) of PKA with mutations in cAMP-binding sites (RI alpha(m)) or by pretreating T cells with catalytic subunit-alpha (C alpha) antisense mRNA oligonucleotides. Transfected RI alpha(m) was expected to compete with endogenous regulatory subunits and to irreversibly inactivate the catalytic subunit in RI alpha(m)-C alpha complexes. It was shown that C alpha and RI alpha are the major PKA subunits in T cells, thereby justifying the choice of RI alpha(m) and C alpha antisense oligos to modulate PKA activity in T lymphocytes. Perturbation of the expression of PKA subunits by RI alpha(m) resulted in transfectants with 1) no changes in basal PKA activity but inhibited cAMP-inducible PKA activity or 2) inhibited basal PKA activity but unaffected cAMP-inducible PKA activity. Transfectants with inhibited basal PKA activity had changed (inhibited) levels of TCR-triggered IL-2 production. The anti-C alpha antisense mRNA oligomers also inhibited basal PKA activity and TCR-triggered production of IL-2. The experiments described here and recently reported studies of the effects of C alpha inactivation on CTL effector functions and IFN-gamma secretion suggest that basal PKA activity could be required for the propagation of TCR-triggered signals needed for lymphokine secretion by T cells.

Animals↗
PubMed1997

Pigmented villonodular synovitis of the temporomandibular joint.

Pigmented villonodular synovitis is a proliferative disorder affecting the synovium and may be an aggressive local process with bone destruction and invasion of contiguous structures. Involvement of the temporomandibular joint is very rare. We report a rare case of pigmented villonodular synovitis of the temporomandibular joint that invaded the infratemporal fossa with bone destruction of the mandibular condyle. Magnetic resonance imaging was effective in delineating the extent of the lesion in the infratemporal fossa. Fine-needle aspiration cytology showed multinucleated giant cells and round or spindle-shaped cells with hemosiderin deposits. The tumor was resected en bloc through a combined temporocervical and infratemporal fossa approach. The postoperative histological diagnosis was a variant of pigmented villonodular synovitis. Fine-needle aspiration cytology and magnetic resonance imaging proved to be useful in the preoperative diagnosis of pigmented villonodular synovitis of the temporomandibular joint and in the preoperative planning that is required to obtain adequate surgical margins.

Biopsy, Needle↗
PubMed1997

Lineage- and differentiation stage-specific expression of LSM-1 (LPAP), a possible substrate for CD45, in human hematopoietic cells.

CD45, a transmembrane tyrosine phosphatase, is found on almost all nucleated hematopoietic cells and plays a crucial role in lymphocyte activation and differentiation. We recently achieved isolation of the human LSM-1 (hLSM-1) gene, whose product is a possible substrate for CD45, and we raised antibodies against the hLSM-1 protein. hLSM-1 expression in hematopoietic cells was examined with Northern and Western blot, fluorescence-activated cell sorter, and immunocytochemical analyses. It was found that in the lymphoid lineage, T and B lymphocytes as well as NK cells expressed LSM-1, whereas terminally differentiated plasma cells did not. As for the myeloid lineage, immature myeloid cells expressed LSM-1, whereas terminally differentiated granulocytes and monocytes did not. In the erythroid lineage, normal erythroblasts expressed very low levels of LSM-1, while erythroid cell lines (K562 and HEL) did not. Megakaryocytes did not express LSM-1. Both CD34+/CD33- and CD34+/CD33+ hematopoietic progenitor cells weakly expressed LSM-1. These results showed that LSM-1 is expressed in a lineage- and differentiation stage-specific fashion.

Blotting, Western↗
PubMed1997

A novel cDNA clone of Schistosoma japonicum encoding the 34,000 Dalton eggshell precursor protein.

A cDNA clone encoding the 34 kDa eggshell protein of Schistosoma japonicum was isolated from an adult female cDNA library with a rabbit antiserum raised against the 34 kDa female worm fraction. A 230 bp-insert of this clone (Sj23A) was introduced in frame into the expression plasmid vector, pMAL-c2, and the recombinant fusion protein of the Sj23A transiation product was induced in Escherichia coli. The antiserum raised against the recombinant protein reacted only with the native 34 kDa protein of mature female worms, which localized in the vitelline cells of the vitelline glands. By reverse transcription-polymerase chain reaction (RT-PCR) analysis, it was found that the gene corresponding to the Sj23A was expressed exclusively in mature female worms. The clone Sj23A showed a high degree of homology to the genes for the eggshell precursor proteins of Fasciola hepatica. At the deduced polypeptide level, the Sj23A also had similarities with the F. hepatica-protein sequence, the amino acid composition [high glycine (16%), lysine (12%) and tyrosine (11%)] and the presence of tyrosine residues flanked by glycine. The clone Sj23A also shared an extensive sequence homology with 3 S. mansoni expression sequence tags (ESTs). The present results suggest that the protein encoded by the female-specific Sj23A gene of S. japonicum is widely conserved in trematodes and plays a significant role as a precursor involved in eggshell formation.

Amino Acid Sequence↗
PubMed1997

Secondary acute monocytic leukemia occurring during the treatment of a testicular germ cell tumor. A case report and review of the literature.

Secondary leukemia following chemotherapy or radiotherapy for mediastinal germ cell tumors is a well-described entity. It also may occur in patients with testicular germ cell tumors. We report a case of acute monocytic leukemia occurring in a 44-year-old man who received etoposide-based chemotherapy and radiotherapy for a recurrent, metastatic testicular germ cell tumor. The patient received 14 cycles of systemic chemotherapy for pulmonary and para-aortic lymph node metastases following his initial orchiectomy. The total amount of etoposide this patient received was 6,400 mg/m2. Leukemia occurred 11 years after orchiectomy. A literature review revealed 25 other reported cases of secondary leukemias after treatment for testicular carcinoma. It is not clear whether chemotherapy, radiotherapy or both are responsible for the secondary leukemias seen in these patients.

Adult↗
PubMed1997

The G alpha protein GL2 alpha improves the ability to detect the subthreshold expressions of receptors linked to phospholipase C in Xenopus oocytes.

Xenopus laevis oocytes showed no electrophysiological responses to acetylcholine (ACh) and had no significant cholinergic receptor sites when prepared under our conditions. However, they were found to acquire robust electrophysiological responsiveness to ACh when bovine GL2 alpha, which is a member of the Gq alpha family and is highly homologous to mouse G11 alpha, was expressed by mRNA injection. Further analyses indicated that GL2 alpha amplified the activity of endogenous muscarinic ACh receptors that are expressed at an otherwise undetectable level, and thus made their detection possible. Thus, GL2 alpha may prove to be an effective method for detecting the activities of phospholipase C-linked receptors which are only marginally expressed. The usefulness of this method was confirmed in the analyses of a chimeric receptor constructed from metabotropic glutamate receptor subtype 1 alpha and muscarinic ACh receptor subtype M1. The chimeric receptor showed no electrophysiological responses to ACh when expressed alone in oocytes, but became responsive to ACh when co-expressed with GL2 alpha.

Acetylcholine↗
PubMed1997

[Clinical evaluation of cefpirome sulfate for severe infections in patients with hematological disorders. Hanshin Study Group of Hematopoietic Disorders and Infections].

We investigated the therapeutic efficacy and safety of cefpirome sulfate (CPR) in treatment of hematopoietic disorder-associated infections. A total of 219 patients were admitted to 12 hospitals of Hanshin Study Group of hematopoietic disorders and infections between April 1994 and March 1996 and were enrolled in this study. Most patients received intravenously infused CPR at a dose of 1 or 2 g twice a day for 3 days or more. Twenty nine patients dropped out or were excluded and remaining 190 patients were adopted for the evaluation. A overall response rate was 58.4% (111/190). Among neutropenic patients, the response rate was 50% (8/16) in patients whose peripheral neutrophil counts (PNC) remained less than 100/microliter throughout the observation period and was 53.7% (22/41) in patients with PNC remained less than 500/microliter. In contrast, in patient whose PNC was below 500 before the treatment but exceeded 501/microliter during of at the end of the treatment, the response rate was as high as 78.4% (29/37). When G-CSF was combined, the response rate became significantly (P < 0.05) higher, 68.5% (50/73), as compared with that, 52.1% (61/117), in patients without it. In cases in which the causative organisms could be identified, the organisms were eliminated in 81.8% (9/11) of the patients infected with Gram-positive bacteria, whereas in 100% (12/12) in those infected with Gram-negative bacteria. Skin eruption developed in 6 patients during the treatment with CPR, and vascular pain and parosmia in one each other. These symptoms subsided soon after discontinuation or even without discontinuation of CPR. Abnormal laboratory findings, mainly liver dysfunction, i.e. elevation of slight degree of serum transaminase levels, were observed. The values, however, turned to normal immediately after the cessation or completion of the treatment. In conclusion, CPR is considered to be an antibiotic of value with high efficacy and safety in treatment of hematopoietic disorder-associated infections.

Adolescent↗
PubMed1997

[A case of hypertrophic spinal pachymeningitis].

A 79-year-old woman was admitted to our hospital complaining of gait disturbance. At the time of admission, she had incomplete monoparesis of the left lower extremity, with a sensory level of T6. MR images showed a thickened dura at the C6-T4 vertebral levels. The thickened dura showed low intensities on T1- and T2-weighted images, and was homogeneously enhanced by Gd-DTPA. Hypertrophic spinal pachymeningitis (HSP) was suspected, and laminectomy was performed from T2 to T3. The thickened dura was found and partially resected. Microscopically, the diagnosis was HSP. Steroid therapy was initiated after surgery. Postoperatively, the patient's physical status and radiographic images improved. The etiology of HSP is not clear in most cases. It is difficult to diagnose radiographically, although MR imaging can help differentiate it from other disorders. T1- and T2-weighted images of HSP show low intensities, and the T1-weighted image is homogeneously enhanced by Gd-DTPA. Treatment is early decompressive surgery with possible excision of the involved dura. Steroid therapy was effective in our case.

Aged↗