Biomedical subjects

M Fukuda

Publications and source records attributed to M Fukuda.

At least 451 records · Page 25Linked to original sources

PubMed1997

The first C2 domain of synaptotagmin is required for exocytosis of insulin from pancreatic beta-cells: action of synaptotagmin at low micromolar calcium.

The Ca2+- and phospholipid-binding protein synaptotagmin is involved in neuroexocytosis. Its precise role and Ca2+-affinity in vivo are unclear. We investigated its putative function in insulin secretion which is maximally stimulated by 10 microM cytosolic free Ca2+. The well-characterized synaptotagmin isoforms I and II are present in pancreatic beta-cell lines RINm5F, INS-1 and HIT-T15 as shown by Northern and Western blots. Subcellular fractionation and confocal microscopy revealed their presence mainly on insulin-containing secretory granules whereas only minor amounts were found on synaptic vesicle-like microvesicles. Antibodies or Fab-fragments directed against the Ca2+-dependent phospholipid binding site of the first C2 domain of synaptotagmin I or II inhibited Ca2+-stimulated, but not GTPgammaS-induced exocytosis from streptolysin-O-permeabilized INS-1 and HIT-T15 cells. Transient expression of wild-type synaptotagmin II did not alter exocytosis in HIT-T15 cells. However, mutations in the Ca2+-dependent phospholipid binding site of the first C2 domain (Delta180-183, D231S) again inhibited only Ca2+-, but not GTPgammaS-evoked exocytosis. In contrast, mutations in the IP4-binding sites of the second C2 domain (Delta325-341; K327,328, 332Q) did not alter exocytosis. Synaptotagmin II mutated in both C2 domains (Delta180-183/K327,328,332Q) induced greater inhibition than mutant Delta180-183, suggesting a discrete requirement for the second C2 domain. Thus, synaptotagmin isoforms regulate exocytotic events occurring at low micromolar Ca2+.

Animals↗

PubMed1997

Altered Golgi localization of core 2 beta-1,6-N-acetylglucosaminyltransferase leads to decreased synthesis of branched O-glycans.

Mucin type O-glycans with core 2 branches are distinct from nonbranched O-glycans, and the amount of core 2 branched O-glycans changes dramatically during T cell differentiation. This oligosaccharide is synthesized only when core 2 beta-1, 6-N-acetylglucosaminyltransferase (C2GnT) is present, and the expression of this glycosyltransferase is highly regulated. To understand how O-glycan synthesis is regulated by the orderly appearance of glycosyltransferases that form core 2 branched O-glycans, the subcellular localization of C2GnT was determined by using antibodies generated that are specific to C2GnT. The studies using confocal light microscopy demonstrated that C2GnT was localized mainly in cis to medial-cisternae of the Golgi. We then converted C2GnT to a trans-Golgi enzyme by replacing its Golgi retention signal with that of alpha-2,6-sialyltransferase, which resides in trans-Golgi. Chinese hamster ovary cells expressing wild type C2GnT and the chimeric C2GnT were then subjected to oligosaccharide analysis. The results obtained clearly indicate that the conversion of C2GnT into a trans-Golgi enzyme resulted in a substantial decrease of core 2 branched oligosaccharides. These results, taken together, strongly suggest that the predominance of core 2 branched oligosaccharides in those cells expressing C2GnT is due to the fact that C2GnT is located earlier in the Golgi than alpha-2,3-sialyltransferase that competes with C2GnT for the common substrate. Furthermore, alteration of Golgi localization renders the chimeric C2GnT much less efficient in synthesizing core 2 branched oligosaccharides, indicating the critical role of orderly subcellular localization of glycosyltransferases.

Animals↗

PubMed1997

A biotinylated perfringolysin O derivative: a new probe for detection of cell surface cholesterol.

theta-Toxin is a cholesterol-binding, pore-forming cytolysin of Clostridium perfringens. To detect cell surface cholesterol, we prepared a theta-toxin derivative, BC theta by biotinylation of a protease-nicked theta-toxin, which has the same binding affinity for cholesterol as theta-toxin without cytolytic activity. Human erythrocytes, V79 cells and human umbilical vein endothelial cells (HUVEC), were stained with BC theta coupled with FITC-avidin, and then the cells were analyzed by either flow cytometry or laser confocal microscopy. The fluorescence intensity increased in both intact and briefly fixed cells when treated with BC theta. BC theta-treated V79 cells were stained by neither trypan blue nor propidium iodide, indicating that BC stained just the outer surface of the plasma membrane of vital cells. Treatment of the cells with digitonin, a cholesterol-sequestering reagent, decreased the fluorescence intensity to the background level, indicating that BC theta staining is specific for cholesterol. The fluorescence intensity of erythrocytes pre-permeabilized with a small amount of theta-toxin increased more than ten-fold, suggesting higher cholesterol contents in the inner layer of the plasma membrane. When cells were cultured with cholesterol-depleted medium, the fluorescence intensity stained by BC theta decreased remarkably in V79 cells, but did not change in HUVEC. This indicates that cell surface cholesterol may be provided in different ways with these two cell lines. These results suggest that BC theta can be a useful probe for visualizing cell surface cholesterol and for evaluating the effects of cellular events on the topology and distribution of cholesterol.

Animals↗

PubMed1997

Characterization of the pleckstrin homology domain of Btk as an inositol polyphosphate and phosphoinositide binding domain.

We previously reported that the pleckstrin homology (PH) domain of Bruton's tyrosine kinase (Btk) binds Ins(1,3,4,5)P4 and that missense mutations in this domain which cause either human X-linked agammaglobulinemia (XLA) or murine X-linked immunodeficiency (Xid) also dramatically reduce the Ins(1,3,4,5)P4 binding activity. In this paper, we describe the inositol phosphate binding specificity of the Btk PH domain and different inositol polyphosphate binding properties among the PH domains of Tec family kinases. Our results suggest that certain inositol phosphates and/or phosphoinositides are physiological ligands of some Tec family kinases and that Tec family members are differently regulated by inositol molecules.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1997

Expression of stable human O-glycan core 2 beta-1,6-N-acetylglucosaminyltransferase in Sf9 insect cells.

UDP-GlcNAc:Galbeta1-3GalNAc-R (GlcNAc to GalNAc) beta-1, 6-N-acetylglucosaminyltransferase (C2GnT) catalyses the formation of O-glycan core 2. Purification and characterization of C2GnT from natural sources has been hampered by the instability of this enzyme. We have been able to prepare a stable partly purified recombinant human C2GnT by expression of a truncated form of the enzyme in the baculovirus/Spodoptera frugiperda 9 (Sf9) insect cell system. C2GnT activity was secreted into the Sf9 culture medium (15 pmol/min per microl; approx. 0.2 mg/l) and was stable at 4 degrees C either in solution or after lyophilization. Endoglycosidase H and N-glycanase F treatment of the radiolabelled C2GnT indicated the presence of N-glycans at both potential N-glycosylation sites. The elimination of one or both of the two potential N-glycosylation sites or treatment of the virus-infected insect cells with tunicamycin resulted in loss of enzyme activity due in part to protein degradation.

Animals↗

PubMed1997

Mutation rate of GB virus C/hepatitis G virus over the entire genome and in subgenomic regions.

A patient on maintenance hemodialysis was infected with a recently discovered putative non-A to -E hepatitis virus designated GB virus C (GBV-C) or hepatitis G virus (HGV) by transfusion. The viral isolate was recovered from the patient soon after she turned positive for GBV-C/HGV RNA in serum (GS185) and 8.4 years thereafter (GS193), and the entire nucleotide sequences were determined. They both had a genomic length of 9391 nucleotides with a defective C gene made of only 42 nucleotides. Between GS185 and GS193, 31 (0.33%) nucleotides were different, which changed 5 (0.18%) of the encoded 2842 amino acids. Thus, GBV-C/HGV was estimated to have a mutation rate of 3.9 x 10(-4) base substitutions per site per year. Nucleotide conversions were distributed over subgenomic regions, except in the 5' untranslated region of 552 nucleotides and a defective short C gene, which were conserved in sequence. The change in the putative envelope genes (E1 and E2) was no different from that in the entire genome with only 6 (0.35%) nucleotide substitutions among the 1730, just 1 of which induced an amino acid conversion. Taken along with the comparison of the two isolates with the reported five GBV-C or HGV isolates, these results indicate that GBV-C/HGV would not have hypervariable regions and would use a strategy for viral persistence that is different from immune escape.

Amino Acid Sequence↗

PubMed1997

Behavioral and P3 amplitude enhancement in schizophrenia following feedback training.

In order to clarify the remediability of behavioral and electrophysiological abnormalities in schizophrenia, hit rate, reaction time, and P3 amplitude from auditory event-related potentials were evaluated before and after feedback training of a task in 14 schizophrenics and 12 age-matched normal controls. Although mean changes in the three indices due to the training were nonsignificant in both schizophrenic and normal control groups as a whole, the changes in hit rate and reaction time correlated significantly with the P3 amplitude change in the schizophrenic (r = 0.60 and -0.58, respectively) but not in the normal control group. The P3 amplitude change also correlated with the P3 amplitude before the training only in the schizophrenic group (r = -0.68), suggesting that the training was more effective for the schizophrenic patients with marked P3 amplitude reduction. The observed P3 amplitude increase due to training may represent an electrophysiological correlate of a remediable aspect of behavioral deficits in schizophrenics, which may underlie the effectiveness of nonpharmacological treatments.

Adult↗

PubMed1997

Subtyping of D20S85 STR alleles by single-strand conformation polymorphism (SSCP) analysis.

During a population study of STR locus D20S85, we discovered two types of sequence variations by direct sequencing of the alleles: two transitions each of G to A and A to G occur in the 5' flanking region in the individuals possessing allele 6 and some of those possessing allele 7 [1]. Using single-strand conformation polymorphism (SSCP) analysis, we were able to distinguish two subtypes of allele 7 from each other. This analysis method enables rapid screening for STR alleles of the same length with different sequences, and should find application to other complex STR loci because of the practical advantage of simplicity in comparison to sequencing.

Forensic Medicine↗

PubMed1997

Evaluation of synergism by a novel three-dimensional model for the combined action of cisplatin and etoposide on the growth of a human small-cell lung-cancer cell line, SBC-3.

Although the combination of cisplatin and etoposide has been used as standard therapy for small-cell lung cancer, it is difficult to demonstrate combination effects between cisplatin and etoposide in vitro. We therefore adopted a 3-dimensional (3-D) model to analyze the combination effects of anticancer drugs, and compared the results of analysis by the new 3-D model with those obtained from traditional 2-D models for the cisplatin-etoposide combination. In this study, using a human small-cell lung-cancer cell line (SBC-3), 3-D model analysis clearly identified a relationship depending on the concentrations of both drugs, and demonstrated that peak synergy occurred at the higher concentrations of cisplatin and etoposide. Antagonistic interactions were noted with a nadir at low concentrations of etoposide and cisplatin. In contrast, 2-D models such as combination index and isobologam analysis fail to characterize the complex interactions between cisplatin and etoposide, since their joint effects are concentration-dependent. Combination index (CI) plots show that synergy is evident only for molar ratios of cisplatin: etoposide of 2:1 to 1:5. On isobologram analysis, synergy could be detected when great inhibitory effects on cell growth were present (high endpoint), but not with small inhibitory effects (lower endpoints). Thus, either synergy or antagonism may occur, but depend on the selection of variables, such as the molar ratios or the endpoints chosen for the experiments. This could explain the inconsistency in the in vitro combination effects reported to date. The 3-D model, which compensates for the above deficiencies of 2-D models, can facilitate rigorous analysis of drug interactions over the entire clinical dose range, using microcomputers and sophisticated graphics programs. This direct and pragmatic method offers investigators a practical new tool with which to analyze drug combinations for cancer chemotherapy.

Antineoplastic Agents↗

PubMed1997

Regulation by bivalent cations of phospholipid binding to the C2A domain of synaptotagmin III.

Synaptotagmins are Ca2+-and phospholipid-binding proteins of synaptic vesicles that might function as Ca2+ receptors for neurotransmitter release via their first C2 (C2A) domain. Here we describe the effect of Mg2+ on phospholipid binding to the C2A domains of multiple synaptotagmins (II-VI), and demonstrate that only synaptotagmin III can bind negatively charged phospholipids [phosphatidylserine (PS) and phosphatidylinositol] in a Mg2+-dependent manner. The Mg2+-dependent interaction with PS was found to have an EC50 of approx. 30 microM Mg2+, which is comparable to that of Sr2+ and Ba2+ (EC50 values of approx. 10 microM). This binding property of the C2A domain is specific to synaptotagmin III, because none of the C2A domains of other proteins, such as rabphilin 3A, Doc2alpha, Doc2beta or Gap1(m), showed phospholipid binding activity in the presence of 1 mM Mg2+. Our results suggest that synaptotagmin III is involved in presynaptic functions different from those of synaptotagmins I and II.

Amino Acid Sequence↗

PubMed1997

Interaction of MAP kinase with MAP kinase kinase: its possible role in the control of nucleocytoplasmic transport of MAP kinase.

The mitogen-activated protein kinase (MAPK) cascade consisting of MAPK and its direct activator, MAPK kinase (MAPKK), is essential for signaling of various extracellular stimuli to the nucleus. Upon stimulation, MAPK is translocated to the nucleus, whereas MAPKK stays in the cytoplasm. It has been shown recently that the cytoplasmic localization of MAPKK is determined by its nuclear export signal (NES) in the near N-terminal region (residues 33-44). However, the mechanism determining the subcellular distribution of MAPK has been poorly understood. Here, we show that introduction of v-Ras, active STE11 or constitutively active MAPKK can induce nuclear translocation of MAPK in mammalian cultured cells. Furthermore, we show evidence suggesting that MAPK is localized to the cytoplasm through its specific association with MAPKK and that nuclear accumulation of MAPK is accompanied by dissociation of a complex between MAPK and MAPKK following activation of the MAPK pathway. We have identified the MAPK-binding site of MAPKK as its N-terminal residues 1-32. Moreover, a peptide encompassing the MAPK-binding site and the NES sequence of MAPKK has been shown to be sufficient to retain MAPK to the cytoplasm. These findings reveal the molecular basis regulating subcellular distribution of MAPK, and identify a novel function of MAPKK as a cytoplasmic anchoring protein for MAPK.

Animals↗

PubMed1997

Expression cloning and characterization of a cDNA encoding a novel membrane protein required for the formation of O-acetylated ganglioside: a putative acetyl-CoA transporter.

By expression cloning using COS-1 cells stably transfected with GD3-synthase (COS-1/GD3+) as a recipient cell line, we have isolated a cDNA, termed AT-1, encoding a novel protein required for the formation of O-acetylated (Ac) gangliosides. The cDNA encodes a protein with multitransmembrane spanning domains with a leucine zipper motif. It consists of 549 amino acids and has a molecular mass of 60.9 kDa. Although both O-Ac-GD3 and O-Ac-GT3 were barely detectable in recipient cells or cells transfected with the vector alone, their amount increased significantly in transfectants containing AT-1. When semi-intact cells prepared by treatment with streptolysin O were incubated with [Ac-14C]-Ac-CoA, increased incorporation of radioactivity was found in those cells transfected with AT-1 when compared with the mock transfectants. Northern blot analysis showed two major transcripts of 3.3 and 4.3 kb in all tissues examined. Immunohistochemical study with an antibody specific to the AT-1 protein suggested that it is most probably expressed in the endoplasmic reticulum membrane. Based on these results, the protein encoded by AT-1 is suggested to be an Ac-CoA transporter that is involved in the process of O-acetylation.

Acetylation↗

PubMed1997

Analysis of Cdc2 and Cyclin D1 Expression in Breast Cancer by Immunoblotting.

Cyclins and cyclin-dependent kinases may reflect the status of cell proliferation in cancer tissues. The authors sought to determine whether cdc2 and cyclin D1 are expressed in breast cancer and are useful as prognostic factors. Accumulation of cdc2 and cyclin D1 proteins was examined in 88 cases of breast cancer using immunoblotting techniques and correlations with clinicopathological factors and prognoses were investigated. Cdc2 and cyclin D1 proteins were observed in 27.3 % and 75.0 % of breast cancers studied, respectively. The incidence of lymph node metastasis was significantly high in cdc2/cyclin D1-double positive group and low in double negative group. On the other hand, the incidence of estrogen receptor (ER) negative cases was significantly higher in the cdc2-positive/cyclin D1-negative group. Relapse-free survival times of cdc2-positive cases were significantly shorter than those of Cdc2-negative cases. The relapse-free survival times of cyclin D1-positive cases also tended to be poorer than those of cyclin D1-negative cases. Multivariate analyses revealed cdc2 as the second most significant of the prognostic variables, following lymph node status. The three-year relapse-free survival rate of cdc2/cyclin D1-double positive cases was 58.9%, whereas that of cdc2/cyclin D1-doublue negative cases was 100%. Cdc2 and cyclin D1 represent the status of cell proliferation in breast cancer, and may be useful in breast cancer assessment.

Journal Article↗

PubMed1997

Human STX polysialyltransferase forms the embryonic form of the neural cell adhesion molecule. Tissue-specific expression, neurite outgrowth, and chromosomal localization in comparison with another polysialyltransferase, PST.

PST and STX are polysialyltransferases that form polysialic acid in the neural cell adhesion molecule (N-CAM), although it is not known why these two polysialyltransferases exist. In the present study, we have first isolated cDNA encoding human STX, which includes 5'-untranslated sequence. Northern blot analysis, using this cDNA and PST cDNA previously isolated by us, demonstrated that PST and STX are expressed in different fetal and adult tissues. STX is primarily expressed in embryonic tissues, but only modestly in adult heart, brain, and thymus. PST, on the other hand, is continuously expressed in adult heart, brain, thymus, spleen, small and large intestines, and peripheral blood leukocytes. In various parts of adult brain, the relative amount of PST and STX appears to be substantially different depending on the regions. The analysis by in situ hybridization of mouse adult brain, however, suggests that polysialic acid in the hippocampal formation is synthesized by both STX and PST. HeLa cells doubly transfected with the isolated STX cDNA and N-CAM cDNA supported neurite outgrowth much better than HeLa cells expressing N-CAM alone. However, polysialic acid synthesized by PST appears to be a better substratum than that synthesized by STX. Moreover, the genes for PST and STX were found to reside at chromosome 5, band p21 and chromosome 15, band q26, respectively. These results, taken together, strongly suggest that PST and STX are expressed distinctly in tissue-specific and cell-specific manners and that they apparently have distinct roles in development and organogenesis.

Adult↗

PubMed1997

The bphDEF meta-cleavage pathway genes involved in biphenyl/polychlorinated biphenyl degradation are located on a linear plasmid and separated from the initial bphACB genes in Rhodococcus sp. strain RHA1.

The bphACB genes responsible for the initial oxidation of the aromatic ring of biphenyl/polychlorinated biphenyls (PCB) to meta-cleavage product in Rhodococcus sp. RHA1 have been characterized. We cloned the 6.1 kb EcoRI fragment containing another extradiol dioxygenase gene (etbC) which was induced during the growth on ethylbenzene. The bphD, bphE and bphF encoding 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase, 2-hydroxypenta-2,4-dienoate hydratase and 4-hydroxy-2-oxovalerate aldolase, respectively, were found downstream of etbC. The deduced amino acid (aa) sequence of RHA1 bphD and bphE had 27-33% and 32-38% identity, respectively, with those of the corresponding genes in Pseudomonas. BphE and BphF are closely related to the corresponding homoprotocatechuate meta-cleavage pathway enzymes of Escherichia coli C. The bphD and bphF were expressed in E. coli and the BphD activity was detected. The etbCphDEF genes were transcribed in biphenyl and ethylbenzene growing cells. Pulsed field gel electrophoresis (PFGE) analysis indicated that RHA1 contains three large linear plasmids. Southern blot analysis indicated that the meta-cleavage pathway for biphenyl/PCB catabolism in RHA1 is directed by the 390 kb plasmid borne bphDEF genes located separately from bphACB gene cluster on the 1100 kb plasmid.

Benzene Derivatives↗

PubMed1997

Selective photoaffinity labeling of the inositol polyphosphate binding C2B domains of synaptotagmins.

Synaptotagmin (Syt) II, a synaptic vesicle protein containing two copies of highly conserved protein kinase C homology regions known as the C2A and C2B domains, acts as a Ca2+ sensor and provides both phospholipid and inositol polyphosphate (IPn) recognition domains important in endo- and exocytosis. Four photoaffinity analogues of IP3, IP4, and IP6 containing a P-1- or P-2-linked 4-benzoyldihydrocinnamidyl (BZDC) photophore were used to label glutathione S-transferase (GST) fusion constructs of the Syt II-C2A and C2B domains. The P-2-linked [3H]BZDC-IP6 showed efficient, IP6-displaceable labeling of the GST-Syt II-C2B. The rank order of photocovalent modification paralleled the order of competitive displacement: IP6 (P-2-linked) > IP4 > IP3. The P-1-linked [3H]BZDC-IP6 failed to label the C2B domains. The GST-Syt III-C2B domain, which lacks IP6 binding affinity, also failed to undergo labeling by P-2-linked [3H]BZDC-IP6. When mixtures of the 32-amino acid basic peptide corresponding to the essential IPn binding region of the Syt II-C2B domain and GST-Syt II-C2B were labeled by a stoichiometric amount of P-2-linked [3H]BZDC-IP6, the two polypeptides showed equivalent affinity for the photolabel. Although the CD spectrum of this 32-mer at two pH values showed a random coil, the photoaffinity analogue of IP6 appeared to induce a binding-compatible structure in the short peptide.

Affinity Labels↗

PubMed1997

Distinct roles of C2A and C2B domains of synaptotagmin in the regulation of exocytosis in adrenal chromaffin cells.

Synaptotagmin that contains two repeats of C2 regulatory domains is considered to be involved in neurotransmitter release. To reveal the roles of synaptotagmin in the regulation of exocytosis, we examined the effects of antibodies against C2A and C2B domains on Ca2+-evoked catecholamine (CA) release from digitonin-permeabilized adrenal chromaffin cells, resolving the Ca2+-evoked release into ATP-dependent priming and ATP-independent Ca2+-triggered steps. Anti-C2A antibody clearly reduced the ATP-independent release, suggesting that the C2A domain directly facilitate or promote Ca2+-triggered step, vesicular fusion. In contrast, anti-C2B antibody did not affect Ca2+-evoked release by itself, but significantly increased the spontaneous Ca2+-independent release. In addition, inositol high-polyphosphate series (IHPS) that bind the C2B domain inhibited both the ATP-independent Ca2+-evoked release and the spontaneous release in a dose-dependent manner. The inhibition by IHPS was totally reversed by anti-C2B antibody and significantly reversed by high concentration of Ca2+. These results suggest that IHPS binding to C2B domain arrests membrane fusion by presumably preventing interaction of synaptotagmin with phospholipids or with proteins of plasma membrane. Thus, IHPS binding to the C2B domain might keep the docked or primed vesicles away from spontaneous fusion at resting level of intracellular Ca2+. Binding of the increased intracellular Ca2+ to the C2A domain may facilitate or trigger the vesicular fusion by releasing this suppression by IHPS.

Adenosine Triphosphate↗

PubMed1997

The function of inositol high polyphosphate binding proteins.

The inositol phosphate metabolism network has been found to be much more complex than previously thought, as more and more inositol phosphates and their metabolizing enzymes have been discovered. Some of the inositol phosphates have been shown to have biological activities, but little is known about their signal transduction mechanisms except for that of inositol 1,4,5-trisphosphate. The recent discovery, however, of a number of binding proteins for inositol high polyphosphate [inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, or inositol hexakisphosphate] enables us to speculate on the physiological function of these compounds. In this article we focus on two major issues: (1) the roles of inositol high polyphosphates in vesicular trafficking, especially exocytosis, and (2) pleckstrin homology domain-containing IP4 binding proteins involved in the Ras signaling pathway.

Amino Acid Sequence↗