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Biomedical subjects

M Fukuda

Publications and source records attributed to M Fukuda.

At least 523 records · Page 29Linked to original sources
PubMed1996

Behavior of the cell cycle-associated proteins in an unusual G0-arrestable cancer cell line.

An adenocarcinoma cell line which has the ability to arrest in G0 phase under exhausted culture conditions was established. Using the cell line, we investigated the expression of cell cycle-associated proteins including cdc2, cdk2, cyclin A, cyclin Dl, and Rb during entry into or withdrawal from the cell cycle. MAP kinase expression was also investigated as one of the most downstream proteins of the signal transduction of growth factors. The cells in the quiescent state did not express cdc2. In contrast, cdk2 was expressed weakly, and cyclin A and cyclin D1 were strongly expressed in the quiescent cells. The expression of cdk2 and cyclin D1 in the quiescent cells was reduced after stimulation by renewal of the medium and then increased, accompanied by Rb phosphorylation and cdc2 expression around the G1/S transition. Cdc2 and the hyperphosphorylated form of Rb disappeared as the cells became quiescent. MAP kinase expression was unchanged throughout all the phases analyzed. The results indicate that down-regulation of neither cdk2, cyclin A, cyclin D1, nor MAP kinase is necessary to arrest cells in G0, but that only Rb dephosphorylation and down-regulation of cdc2 are accompanied by an arrest of cell proliferation in G0 in the cell line.

Adenocarcinoma↗
PubMed1996

Possible roles of tumor-associated carbohydrate antigens.

Recent progress in the studies on the roles of carbohydrates has brought about critical discoveries, which allow us to have working hypotheses for understanding the roles of tumor-associated carbohydrate antigens. In this report, I focus my description on three different aspects of this progress. I discuss: (a) the immunological response to oligosaccharides aberrantly expressed under pathological conditions; (b) possible roles of carbohydrate-dependent cell adhesion during tumor metastasis; and (c) the roles of carbohydrates in modulating the functions of proteins that attach those carbohydrates. These three areas in the roles of carbohydrates will likely be the target for continuous research efforts to reveal the roles of tumor-associated carbohydrates.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Phospholipid composition dependence of Ca2+-dependent phospholipid binding to the C2A domain of synaptotagmin IV.

Synaptotagmins I and II are Ca2+- and phospholipid-binding proteins of synaptic vesicles that may function as Ca2+ receptors for neurotransmitter release via their first C2 domains. Herein, we describe the phospholipid binding properties of C2A domains of multiple synaptotagmins (II-VI). We demonstrate that all synaptotagmins can bind negatively charged phospholipids (phosphatidylserine (PS) and phosphatidylinositol (PI)) in a Ca2+-dependent manner, although it was previously reported that synaptotagmins IV and VI do not bind phospholipids. The Ca2+-dependent interaction of the C2A domain of synaptotagmin IV with PS was found to have two components with EC50 values of approximately 5 and 120 microM free Ca2+ and exhibited positive cooperativity (Hill coefficient of approximately 2 for both components). This value is lower than that of the C2A domain of synaptotagmin II (Hill coefficient of approximately 3). All other isoforms bound PS with high affinity (EC50 of 0.3-1 microM free Ca2+; Hill coefficient of 3-3.5). In addition, the C2A domain of synaptotagmin IV cannot bind liposomes consisting of PS (or PI) and phosphatidylcholine, PC (or phosphatidylethanolamine, PE) (1:1, w/w), indicating that the binding to negatively charged phospholipids is inhibited by the presence of PC or PE. In contrast, other isoforms bound all of the liposomes, which include either PS or PI, in a Ca2+-dependent manner. Mutational analysis indicated that this phospholipid composition-dependent Ca2+ binding of synaptotagmin IV results in the substitution of Asp for Ser at position 244. The cytoplasmic domain of synaptotagmin IV also shows this unique phospholipid binding. However, it binds PS with a positive cooperativity and an affinity similar to those of the C2A domains of other isoforms. Our results suggest that synaptotagmin IV is also a potential Ca2+ sensor for neurotransmitter release.

Amino Acid Sequence↗
PubMed1996

The influence of blood gas changes on hyperthermia-induced seizures in developing rats.

Fever induces seizures in infants with febrile convulsions or epilepsy. Hyperpnea induced by fever may contribute to the induction of these seizures. In order to examine this possibility, we evaluated the effect of changes in arterial blood gas tension on hyperthermia-induced seizures in developing rats. Electrical seizure discharges were induced by application of infra-red rays on the skull of rats under mechanical ventilation with different respiratory conditions. There was positive correlation between pCO(2) and the seizure threshold (ST) defined as a latency from the start of hyperthermia to the occurrence of seizures: ST (seconds, s) = 2.36 pCO(2) + 0.05 (R(2) = 0.80, P < 0.001). Seizure duration (SD) was longer at lower pCO(2) level: 18 (6-33) (median, range) s at pCO(2) ranging from 23 to 26 mmHg vs. 0 (0-7) s at pCO(2) ranging from 35 to 57 mmHg (P < 0.01). Hypoxia significantly increased ST: 84 (61-100) s at P0(2) ranging from 53 to 76 mmHg vs. 60 (51-72) s at P0(2) ranging from 87 to 131 mmHg (P < 0.01). Hyperoxia prolonged SD: 27 (10-30) s at P02 ranging from 100 to 170 mmHg vs. 9 (0-23) at P0(2) ranging from 53 to 93 mmHg (P < 0.02). Hypocarbia caused by fever-induced hyperpnea probably contributes to the generation of fever-induced seizures.

Animals↗
PubMed1996

Sequential detection of tumor cells in the peripheral blood and bone marrow of patients with stage IV neuroblastoma by the reverse transcription-polymerase chain reaction for tyrosine hydroxylase mRNA.

BACKGROUND: The aim of this study was to evaluate the changes of tumor cell contamination in bone marrow (BM) and peripheral blood (PB) during the clinical course of patients with advanced neuroblastoma by detecting tyrosine hydroxylase (TH) mRNA to clarify the appropriate source and time for harvesting hematopoietic stem cells for transplantation. METHODS: A total of 15 patients with Stage IV neuroblastoma were studied. All 15 patients had peripheral blood stem cell (PBSC) samples and BM samples examined for TH mRNA by using the reverse transcription-polymerase chain reaction (RT-PCR) at the time of harvest. Nine of the 15 patients, also had BM and PB samples examined sequentially. RESULTS: Comparing the 45 paired samples concurrently drawn, 16 of 28 BM samples (57.1%) and 4 of 28 PB samples (14.2%) obtained during complete remission (CR) were positive for TH mRNA (P < 0.01), whereas 17 of 17 BM samples (100%) and 14 of 17 PB samples (82.3%) obtained before CR was achieved were positive (not significant). The incidence of TH mRNA positivity was significantly lower in the samples obtained during CR than those obtained before CR was achieved (P < 0.0001 for PB samples, P < 0.01 for BM samples). At the time of PBSC harvesting, the incidence of TH mRNA positivity was lower in PBSC samples (3 of 15, 20%) than in BM samples obtained concurrently (10 of 15, 66.7%; P < 0.03). CONCLUSIONS: These findings show that there is a substantial risk of tumor cell contamination in harvested PBSCs, although its incidence was lower than that in BM samples. We recommend that PBSCs would be better harvested during remission and should be examined for tumor contamination before use as a stem cell source.

Base Sequence↗
PubMed1996

Expression cloning of a human GT3 synthase. GD3 AND GT3 are synthesized by a single enzyme.

Gangliosides of the C series such as GT3 are polysialylated glycosphingolipids whose synthesis is developmentally regulated. Here we report the expression cDNA cloning and characterization of GT3 synthase that adds the second alpha-2,8-sialic acid to GD3, NeuNAcalpha2-->8NeuNAcalpha2-->3Galbeta1-->4Glc-->Cer, thus forming GT3, NeuNAcalpha2-->8NeuNAcalpha2-->8NeuNAc alpha2-->3Galbeta1--> 4Glc-->Cer. Unexpectedly, the cloned cDNA was found to be identical to the cDNA that encodes GD3 synthase. The newly identified enzyme was therefore named GD3/GT3 synthase (GD3/GT3ST). GD3/GT3ST synthesized GT3 most efficiently when GM3, NeuNAcalpha2-->3Galbeta1-->4Glc-->Cer, was incubated as an acceptor, indicating that GD3/GT3ST is a polysialyltransferase that can transfer more than one sialic acid residue via alpha-2,8 linkage to gangliosides. Moreover, a longer period of incubation of GD3 with GD3/GT3ST produced a significant amount of GT3 and higher polysialogangliosides. Among various cell lines expressing GD3/GT3ST, higher polysialogangliosides including GT3 were detected only in cell lines where the amount of GD3/GT3 mRNA is sufficiently high. The expression of GD3/GT3ST mRNA among human tissues is highly restricted to fetal and adult brains. The GD3/GT3ST gene was found to be located at chromosome 12, region p12. Taken together, these results indicate that C series polysialogangliosides are synthesized by a ganglioside-specific polysialyltransferase, GD3/GT3ST, that is specifically expressed in neural tissues.

Adult↗
PubMed1996

Synergism between cisplatin and topoisomerase I inhibitors, NB-506 and SN-38, in human small cell lung cancer cells.

Topoisomerase I-targeting anticancer agents such as 7-ethyl-10-[4-(1-piperidyl)-1-piperidyl]carbonyloxy-camptothecin (CPT-11) and 6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13-(beta-D- glucopyranosyl)-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-di one (NB-506) have been developed and show strong antitumor activity against various cancers. We examined the interaction of these drugs and cisplatin (CDDP), and biochemical mechanisms of synergism between them. Interaction of drugs in human small cell lung cancer cells, SBC-3, was analyzed using the isobologram method. Combinations of CDDP with NB-506, CPT-11, and an active metabolite of CPT-11, 7-ethyl-10-hydroxy-CPT (SN-38), showed synergistic effects. Formation of DNA interstrand cross-links (ICLs) on the cells was analyzed using an alkaline elution assay and increased ICLs were observed by simultaneous exposure to CDDP (1.5 microM) and NB-506 (10 nM) compared with that in response to CDDP alone. DNA repair after ICL formation induced by 3-h exposure to CDDP (1.5 microM) was reduced by NB-506 (10 nM) exposure. On the other hand, a higher concentration of CDDP (150 microM) enhanced the topoisomerase I inhibitory activity of NB-506 and SN-38 determined by relaxation of supercoiled Escherichia coli DNA. These biological interactions might result in synergistic interactions between CDDP and NB-506 or SN-38. Topoisomerase I inhibitors and CDDP may be a key regimen for cancer chemotherapy and merit further examination.

Antineoplastic Agents↗
PubMed1996

Three-dimensional structures of free form and two substrate complexes of an extradiol ring-cleavage type dioxygenase, the BphC enzyme from Pseudomonas sp. strain KKS102.

The crystal structure of an enzyme having polychlorinated-biphenyl degrading activity, the BphC enzyme from Pseudomonas sp. strain KKS102, has been solved as a free form at 1.8 A resolution. This is the first three-dimensional structure among the extradiol-type dioxygenases. Based on 34,387 reflections (10.0 to 1.8 A, completeness 87.8%), a current R-factor of 20.4% (with a free R-factor of 24.3%) was obtained with a model obeying standard geometry within 0.011 A in bond lengths and 1.91 degrees in bond angles. The BphC enzyme is a homo-octamer and each subunit is composed of two domains: Domain 1 (N-terminal part) and Domain 2 (C-terminal part). Each domain contains two repetitions of a novel folding motif (the "beta alpha beta beta beta" motif) each consisting of ca 55 amino acid residues. A single Fe ion in the active site coordinates the side-chains of three amino acid residues (His145, His209 and Glu260) and two solvent molecules. The coordination geometry is that of a square pyramid. In addition to the free form of the BphC enzyme, we have solved two three-dimensional structures of the BphC enzyme complexed with its substrates, 2,3-dihydroxybiphenyl (2,3-DHBP) or 3-methylcatechol (3-MCT). These substrates were found intact in the active site probably because of the oxidation of the Fe ion into ferric form (as judged by EPR spectra) in the present crystals. In both of the two substrate complexes, the two hydroxyl groups of the substrate, together with the three enzymatic side-chain ligands, were found to form a penta-coordinated system around the Fe ion roughly arranged in a trigonal bipyramidal configuration. The active site structures appear to be essentially consistent with the reaction mechanism proposed so far.

Amino Acid Sequence↗
PubMed1996

Decreased expression of nucleoside diphosphate kinase alpha isoform, an nm23-H2 gene homolog, is associated with metastatic potential of rat mammary-adenocarcinoma cells.

The nm23 gene [encoding nucleoside diphosphate kinase (NDPK)] may act as a metastasis suppressor in certain tumor cells. We investigated the role of NDPK isoforms (alpha and beta) in the metastatic processes, using rat mammary-adenocarcinoma cell lines of poor (MTC) and high (MTLn3) spontaneous metastatic potential respectively. In these cell lines, as in most rat tissues, the alpha isoform (nm23-H2 homolog) was more highly expressed than the beta isoform (nm23-H1 homolog) at the mRNA and protein levels. When examined by Northern- and Western-blot analyses, expression of the 2 isoforms was reduced in highly metastatic MTLn3 cells compared with poorly metastatic MTC cells. The reduced expression was also associated with diminished NDPK-enzyme activity in the cell extracts. Southern-blot and RT-PCR-SSCP analyses suggested that the 2 genes were not grossly altered or mutated in their translation regions. MTLn3 cell clones transfected with NDPKalpha or NDPKbeta cDNA were all tumorigenic when implanted into the mammary fat pad of syngeneic rats. Among those, only clones transfected with the NDPKalpha gene exhibited reduced lung metastasis in a spontaneous metastasis assay.

Adenocarcinoma↗
PubMed1996

A human polysialyltransferase directs in vitro synthesis of polysialic acid.

Polysialic acid (PSA) is a linear homopolymer of alpha-2,8-linked sialic acid residues whose expression is developmentally regulated and modulates the adhesive property of the neural adhesion molecule, N-CAM. Recently, hamster and human cDNAs encoding polysialyltransferase (PST-1 for the hamster enzyme and PST for the human enzyme) were cloned, and by using the human cDNA it was demonstrated that the expression of PSA in N-CAM facilitates neurite outgrowth (Nakayama, J., Fukuda, M.N., Fredette, B., Ranscht, B., and Fukuda, M. (1995) Proc. Natl. Acad. Sci. U.S.A., 92, 7031-7035; Eckhardt, M.A., Mühlenhoff, M., Bethe, A., Koopman, J., Frosch, M., and Gerardy-Schahn, R. (1995) Nature 373, 715-718.) Although these studies demonstrated that PST-1 and PST synthesize PSA in cultured cells, it was not shown that they could catalyze the polycondensation of alpha-2,8-linked sialic acid on a glycoconjugate template containing alpha-2,3-linked sialic acid. Here we demonstrate that PSA formation by PST is independent from the presence of N-CAM in vivo. We then develop an in vitro assay of PSA synthesis using glycoproteins other than N-CAM as acceptors and a soluble PST as an enzyme source. The soluble PST, produced as a chimeric protein fused with protein A, was incubated with rat alpha 1-acid glycoprotein, fetuin or human alpha 1-acid glycoprotein as acceptors together with the donor substrate CMP-[14C]NeuNAc. Incubation of fetuin with the soluble PST, in particular, resulted in a high molecular weight product that was susceptible to PSA-specific endoneuraminidase. Polysialylated products were not formed when alpha-2,3-linked sialic acid was removed from the acceptor fetuin before incubation. These results establish that a single enzyme, PST, alone can catalyze both the addition of the first alpha-2,8-linked sialic acid to alpha-2,3-linked sialic acid and the polycondensation of all alpha-2,8-linked sialic acids, yielding PSA.

Animals↗
PubMed1996

Infection with GB virus C (GBV-C) in patients with chronic liver disease or on maintenance hemodialysis in Indonesia.

RNA of a non-A to E hepatitis virus identified recently and designated provisionally GB virus C(GBV-C), was sought in patients in Indonesia by reverse-transcription polymerase chain reaction with nested primers deduced from a helicase-like region. GBV-C RNA was detected in 32 (55%) of 58 patients on maintenance hemodialysis at a frequency significantly higher (P < 0.001) than that in seven (5%) of 149 patients with chronic liver disease. Co-infection with hepatitis C virus was observed in 26 (81%) of the 32 patients on hemodialysis and in five (71%) of the seven patients with liver disease who were infected with GBV-C. Complete identity was observed in a sequence of 100 base pairs in the helicase-like region for GBV-C cDNA clones from some patients on maintenance hemodialysis. These results indicate that the patients on hemodialysis would be at high risk for GBV-C infection, which would be transmitted by transfusion and patient-to-patient routes.

Antigens, Viral↗
PubMed1996

Adenocarcinoma in the anal canal associated with a fistula: report of a case.

Adenocarcinoma in the anal canal associated with an anal fistula is extremely rare, and in most cases its origin is difficult to ascertain because the primary sites have already been destroyed before any diagnosis of malignancy is able to be made. We report herein the case of a 62-year-old man found to have papillary adenocarcinoma with partial mucinous carcinoma associated with an anal fistula. The tumor was not exposed to the mucosal surface of the anal canal or rectum and an abdominoperineal resection was carried out. Macroscopic findings suggested that the tumor had developed from the anal fistula; however, the tumor showed a positive results when tested for O-acetylated sialic acids. This test also proved positive in the mucus of normal rectal mucosa, but not in the mucus of the anal glands. We speculated that the results of these tests may indicate that this tumor could have originated from the rectal mucosa, from where it migrated into the anal fistula.

Adenocarcinoma, Mucinous↗
PubMed1996

Association of Trp64Arg mutation of the beta3-adrenergic-receptor with NIDDM and body weight gain.

A possible pathogenic mutation in the beta 3-adrenergic-receptor gene (Trp64Arg) has been reported to be associated with an earlier age of onset of non-insulin-dependent diabetes mellitus (NIDDM) and clinical features of the insulin resistance syndrome in Pima Indian, Finnish and French subjects. Since marked heterogeneity has been reported in the association of mutations of candidate genes with NIDDM between Japanese and other ethnic groups, we investigated the association of Trp64Arg with NIDDM in Japanese subjects. The allele frequency of the mutation (Arg) was slightly, but not significantly, higher in NIDDM than in control subjects (70 out of 342 alleles [20.5%] vs 40 out of 248 [16.1%], respectively, p > 0.2). When our data were combined with those of Pima Indian and Finnish subjects, however, the Arg/Arg genotype was significantly associated with NIDDM as compared with the other two genotypes (p < 0.005, relative risk [RR] 2.13, 95% confidence interval [CI] 1.28-3.55). The Arg allele was also associated with NIDDM (p < 0.05, RR 1.27, 95% CI 1.06-1.52). Japanese subjects homozygous for the mutation had a significantly higher body mass index (mean +/- SD: 25.5 +/- 3.9 kg/m2) than heterozygotes (22.6 +/- 4.1, p < 0.05) and normal homozygotes (22.8 +/- 3.8, p < 0.05). NIDDM patients homozygous for the mutation tended to have an earlier age of onset of NIDDM than those with other genotypes. These data suggest that the Trp64Arg mutation not only contributes to weight gain and age-at-onset of NIDDM but is also associated with susceptibility to NIDDM.

Age of Onset↗
PubMed1996

Improvement by eicosanoids in cancer cachexia induced by LLC-IL6 transplantation.

Cachexia frequently occurs in the late stages of cancer, and is difficult to manage. We previously reported that interleukin-6 (IL-6) cDNA transfection into Lewis lung carcinoma (LLC-IL6) induced cachexia-like symptoms in C57BL/6 mice. This was thought to be a useful experimental model of cancer cachexia. We have examined the effects of two eicosanoids, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), in order to evaluate whether they could relieve cachexia. LLC-IL6-bearing animals were divided into three treatment groups receiving DHA, EPA or water as the control; 80-microliter samples of these compounds (purity > 95%) were administered orally by catheter daily starting 7 days after tumor transplantation. Tumor growth curves were similar in the three groups. There were no differences in water or food intake in the three groups. However, body weight, a marker of cachexia, was significantly higher in treated mice than in the control group. Sixteen days after tumor transplantation, the mean body weight was 17.45 g (P < 0.05), 17.2 g and 16.41 g in the groups receiving DHA, EPA and water respectively. The eicosanoids did not affect serum levels of IL-6. Ubiquitination of muscle protein, a marker of proteolysis coupled to cachexia, was compared in LLC-IL6- and LLC-transplanted mice. The eicosanoids prevented the ubiquitination of approximately 180 kDa protein. These results suggest that eicosanoids may prevent the cachexia mediated by IL-6.

Animals↗
PubMed1996

The Y-27H39 polymorphism in a Japanese population.

We studied 184 Japanese males for the tetranucleotide TAGA repeat polymorphism at the Y-27H39 locus on the Y chromosome, and discovered a new allele, tentatively named F. Direct sequencing of Y-27H39 alleles revealed that the new allele (206 nt) is larger than allele E (202 nt) by 1 repeat unit. The six alleles differ only in the number of repeats, the flanking sequences being constant. The allele frequencies are different in Japanese and Caucasians.

Alleles↗