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Biomedical subjects

S Inoue

Publications and source records attributed to S Inoue.

At least 775 records · Page 43Linked to original sources
PubMed1994

Identification and structural determination of the KDN-containing N-linked glycan chains consisting of bi- and triantennary complex-type units of KDN-glycoprotein previously isolated from rainbow trout vitelline envelopes.

KDN-gp, which is the unique glycoprotein of the rainbow trout egg envelope, was shown to have a small amount of N-linked oligosaccharide units in addition to a large number of O-linked glycan units. Structural analysis based on chemical analysis in combination with 400 MHz 1H NMR spectroscopy revealed the presence of fully KDNosylated bi- and triantennary complex-type oligosaccharide chains, mostly fucosylated at the innermost GlcNAc residue and bisected by the GlcNAc residue linked beta 1-->4 to the beta-Man residue. The structures thus determined represent the first demonstration of N-linked glycan unit containing the KDN residues in the KDN-containing glycoproteins (see Chart 1). The KDN-gp of the rainbow trout egg envelope is a molecule that is present in the second layer of the vitelline envelope but is exposed to the outer surface around the micropyle through which sperm can get in at fertilization. Like human hematopoietic cell surface glycoproteins such as glycophorin A and leukosialin, KDN-gp, which is now characterized to contain N-linked complex-type glycan chains as minor components, is heavily O-glycosylated with alpha 2-->8-linked oligo/polyKDN-containing glycan units attached O-glycosidically to Ser/Thr residues. Although little is known about the functional roles of these glycan chains, KDN-gp appears to form a model for further study on the function of cell surface receptor for sperm in fertilization.

Animals↗
PubMed1994

Basic structure of basement membranes is a fine network of "cords," irregular anastomosing strands.

A three-dimensional network of irregular anastomosing strands, referred to as "cords," was found to be the main component of the lamina densa of a) common, "thin" basement membranes in tissues from diverse origins including foot pad epidermis, trachea, jejunum, seminiferous tubule and vas deferens of the rat, monkey seminiferous tubule, and mouse ciliary process, b) a "double" basement membrane, the rat glomerular basement membrane, and c) "thick" basement membranes including rat Reichert's membrane, mouse lens capsule and the Engelbreth-Holm-Swarm (EHS) tumor matrix. The average thickness of the cords was 3.2-4.8 nm, 4 nm, and 4.7-5 nm, respectively, in these three types of basement membranes. The mean diameter of the intercordal spaces, or openings of the network, averaged 14 nm with a range from 8 nm in the glomerular basement membrane to 21.9 nm in the lens capsule. After cryofixation followed by freeze substitution or freeze drying, similar cord networks were observed in all basement membranes examined which included two thin basement membranes, that of the rat epididymis and seminiferous tubules, and three thick basement membranes, that is, the lens capsule and the EHS tumor matrix of the mouse, and rat Reichert's membrane. In addition, following the co-incubation of laminin, type IV collagen and heparan sulfate proteoglycan at 35 degrees C, a precipitate was formed which was found to contain lamina densa-like sheets and large semisolid masses. Both types of structures were found to be made up of a network of 3 nm wide cords, which resembled that of natural basement membranes. With the immunoperoxidase technique, these cords were stained for major basement membrane components including laminin, type IV collagen, heparan sulfate proteoglycan, entactin, and fibronectin. Ribbon-like "double tracks" 4.5 nm in width and being distributed along cords have been identified as the form taken by heparan sulfate proteoglycan in basement membranes. Following mild plasmin treatment, most of the cord components were digested away leaving behind a network of fine filaments found to contain type IV collagen. Each cord, therefore, is organized by a type IV collagen core filament which is surrounded by a plasmin-sensitive sheath containing other basement membrane components. Two types of minor structural components, that is, 7-10 nm wide straight "basotubules" and 3.5 nm wide particulate structures referred to as "pentosomes" were associated with cord network in some basement membranes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Lamina lucida of basement membrane: an artefact.

In tissues prepared with chemical fixation followed by conventional dehydration, basement membranes have been observed to be laminated structures composed of a lamina lucida and lamina densa as well as a poorly limited transitional zone referred to as the pars fibroreticularis. Scattered attempts in the application of new techniques of tissue preparation such as cryofixation or freeze substitution for the study of the basement membrane structure have been made in recent years. From these studies, the possibility has arisen in which basement membranes are composed of only the lamina densa without a lamina lucida. In recent studies in this laboratory, the attempt was made to determine whether or not this lamina lucida is an artefact, and if so, which step in the conventional method of tissue preparation is responsible for its formation. Basement membranes from diverse sources in the mouse and rat including the testis, ductus epididymis, eye, thyroid, kidney, and skin, were observed after either cryofixation by slam freezing followed by freeze substitution, or aldehyde fixation followed by freeze substitution. The basement membranes after preservation with either of these two methods were composed of only the lamina densa with no lamina lucida. It indicates that an artefactual formation of the lamina lucida occurs during dehydration in conventional tissue preparation rather than during chemical fixation. In view of the well known superiority of freeze substitution over conventional dehydration, the lamina lucida of the basement membrane is likely to be an artefact. Therefore, it is concluded that the lamina lucida is an artefact formed during conventional tissue preparation, and in its original condition in the living state, the basement membrane is composed of a single layer made up of lamina densa material.

Animals↗
PubMed1994

Isolation and characterization of a phospholipase A2 inhibitor from the blood plasma of the Thailand cobra Naja naja kaouthia.

A phospholipase A2 (PLA2) inhibitory protein was purified from the blood plasma of the Thailand cobra Naja naja kaouthia by sequential chromatography on Sephadex G-200, DEAE Affi-Gel Blue, and Protein-Pak G-Butyl columns. The purified inhibitor was a glycoprotein with an apparent molecular mass of about 90 kDa and contained 25- and 31-kDa subunits with a molar ratio of 1:2. The 31-kDa subunit contained a glycosidic chain and the molecular mass was reduced to 28 kDa by N-glycosidase F treatment. The amino acid compositions of the two subunits were characterized by their high content of cysteine residues. The inhibitor inhibited group II as well as group I PLA2's. Since the fundamental properties were different from those of the two Crotalidae inhibitors already reported, the cobra inhibitor might be a new type of PLA2 inhibitor.

Agkistrodon↗
PubMed1994

Effects of 1 alpha,25-dihydroxyvitamin D3 on macrophage colony-stimulating factor production and proliferation of human monocytic cells.

1 alpha-25-Dihydroxyvitamin D3 [1 alpha,25(OH)2D3] stimulates the proliferation of human monocytes in vitro. In the present study, we investigated a possible role of macrophage colony-stimulating factor (M-CSF) in 1 alpha,25(OH)2D3-induced proliferation of human circulating monocytes and the effects of 1 alpha,25(OH)2D3 on M-CSF production by human monocytic cells. Both 1 alpha,25(OH)2D3 and recombinant human M-CSF increased 2.5-fold the nucleus number of human circulating monocytes on day 6 of the culture. These effects were inhibited by antihuman M-CSF antibody as well as by anti-c-fms antibody, although these antibodies themselves did not affect the nucleus number when added to control culture. These results indicated that M-CSF is required for 1 alpha,25(OH)2D3-stimulated monocyte proliferation. In addition, 1 alpha,25(OH)2D3 stimulated M-CSF secretion from human circulating monocytes. Secretion and mRNA expression of M-CSF by 12-0-tetradecanoylphorbol-13-acetate (TPA)-treated THP-1 cells (human monocytic leukemia cell line) and TPA-treated HL-60 cells (human promyelocytic leukemia cell line) were also increased by 1 alpha,25(OH)2D3. M-CSF secretion from TPA-treated THP-1 cells was increased by 1 alpha,25(OH)2D3 in a dose-dependent and metabolite-specific manner. The present study demonstrates that 1 alpha,25(OH)2D3 is a potent stimulator for M-CSF production by human monocytic cells and that the proliferative effect of 1 alpha,25(OH)2D3 on human monocytes may be attributed, at least in part, to the stimulated secretion of M-CSF.

Blotting, Northern↗
PubMed1994

Developmental expression of trout egg polysialoglycoproteins and the prerequisite alpha 2,6-, and alpha 2,8-sialyl and alpha 2,8-polysialyltransferase activities required for their synthesis during oogenesis.

The developmental expression of the alpha 2,6- and alpha 2,8-linked sialic acid (Sia) residues in trout egg polysialoglycoproteins (PSGPs) was studied by correlating the temporal expression of these sugar residues, and the prerequisite sialyltransferases responsible for their synthesis, during oogenesis. The following new findings are reported. 1) Disialylated glycoproteins were identified in ovaries 4-6 months prior to ovulation. Three months prior to ovulation, a second more highly sialylated glycoprotein appeared. Structural studies confirmed that the two glycoproteins were discrete molecular species, designated PSGP(low Sia) and PSGP(high Sia), which differed only in their Sia content. PSGP(low Sia) contained mostly disialyl (Sia alpha 2,8-Sia alpha 2,6-) side chains, whereas PSGP(high Sia) contained alpha 2,8-linked oligo/polySia side chains ranging in length from 2 to over 20 Sia residues. The average degree of polymerization ([DP]av) was 6. 2) Biosynthetic studies using CMP-[14C]Neu5Ac indicated that three sialyltransferase activities were responsible for synthesis of the polysialyl residues of PSGPs: (i) alpha-N-acetylgalactosaminide alpha 2,6-sialyltransferase (alpha 2,6-ST), which catalyzed formation of the Sia residues alpha 2,6-linked to the proximal GalNAc residues in asialo-PSGP; (ii) alpha 2,6-sialoside alpha 2,8-sialyltransferase (alpha 2,8-ST or "initiase"), which catalyzed transfer of the first alpha 2,8-Sia residue to the alpha 2,6-linked Sia residue; and (iii) an alpha 2,8-polysialyltransferase (alpha 2,8-polyST or "polymerase"), responsible for synthesis of the alpha 2,8-linked poly/oligo Sia chains in PSGP(high Sia). Expression of these enzyme activities increased in accordance with the developmental appearance of each PSGP. 3) Structural characterization of the [14C]Sia-labeled side chains of each PSGP at different stages of development confirmed that synthesis of the disialyl unit containing a single alpha 2,8-Sia residue occurred before alpha 2,8-polysialylation. 4) In ovaries, 96% of the sialyltransferase activities were found in the Golgi-derived immature cortical vesicles or as soluble enzymes released from the fragile vesicles. Less than 4% of the activities were localized in the membrane (Golgi) fraction. In mature eggs, the sialyltransferases were also detected as soluble enzymes, and within the cortical vesicles.

Amino Acid Sequence↗
PubMed1994

Structural studies of a novel type of pentaantennary large glycan unit in the fertilization-associated carbohydrate-rich glycopeptide isolated from the fertilized eggs of Oryzias latipes.

In a previous report (Kitajima, K., Inoue, S., and Inoue, Y. (1989) Dev. Biol. 132, 544-553), we found the presence of a heavily glycosylated polyprotein, "H-hyosophorin," isolated from the unfertilized eggs of Oryzias latipes. We now report our detailed analysis of the structure of the N-glycan chain in L-hyosophorin, the smallest repeating unit of H-hyosophorin, which was isolated from the fertilized eggs of O. latipes and formed from H-hyosophorin upon fertilization. The N-glycan structures were defined by a combination of compositional analysis, methylation analysis, selective chemical degradation (i.e. mild methanolysis, periodate-Smith degradation, and hydrazinolysis-nitrous acid deamination), enzymatic (endo-beta-galactosidase, peptide:N-glycanase, and Newcastle disease virus sialidase) digestion, and instrumental analyses (one- and two-dimensional proton nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry) which revealed novel and unique features: (a) the presence of highly branched poly-N-acetylactosamino pentaantennary structures; (b) the presence of a beta-galactosylated Lewis X antigenic epitope, Gal beta 1-->4 Gal beta 1-->4 (Fuc alpha 1-->3) GlcNAc beta 1-->; (c) the presence of a beta-galactosylated sialyl Lewis X structure, Gal beta 1-->4 (Neu5Ac alpha 2-->3) Gal beta 1-->4(Fuc alpha 1-->3) GlcNAc beta 1-->; (d) the presence of Gal beta 1-->4 Gal beta 1--> and Gal beta 1--> 4Gal beta 1-->4Gal beta 1--> as the major and minor groupings, respectively; and (e) the presence of the branched Gal residues, -->4GlcNAc beta 1-->3(Gal beta 1-->4) Gal beta 1-->. This study represents the first detailed investigation regarding the nature of highly branched complex asparagine-linked pentaantennary glycans in glycoproteins. The unique expression of such bulky multiantennary glycan units on proteins could be essential during early embryogenesis.

Animals↗
PubMed1994

The effect of KN-62, Ca2+/calmodulin dependent protein kinase II inhibitor on cell cycle.

The isoquinolinesulfonamide derivative, KN-62, is a potent and specific inhibitor of Ca2+/calmodulin dependent protein kinase II (CaM kinase II) (Tokumitsu, H., Chijiwa, T., Hagiwara, M., Mizutani, A., Terasawa, M., and Hidaka, H.(1990) J. Biol. Chem. 265, 4315-4320). KN-62 inhibits growth of K562 cells, in a dose-dependent manner. Flow cytometric analysis demonstrates that the treatment of K562 cells with 10 microM KN-62 causes an accumulation of cells in S phase. Immunoblotting studies showed that specific antibodies against CaM kinase II recognized the 65 kDa of protein in K562 cells. This protein showed protein kinase activity as examined by the activity gel method. The inhibition of this enzyme activity by KN-62 was dose-dependent. The immunoprecipitates with the antibodies from K562 cells phosphorylates the synthetic peptide substrates, syntide-2. These results suggest that CaM kinase II plays an important role in the mechanisms for the cell growth in K562 cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Calcium ion binding of three different types of oligo/polysialic acids as studied by equilibrium dialysis and circular dichroic methods.

Ca2+ binding properties of three different types of oligo/polysialic acid chains, i.e., oligo/poly(Neu5Ac), oligo/poly(Neu5Gc), and oligo/poly(KDN), were studied by equilibrium dialysis and circular dichroism. Colominic acid, high molecular weight polysialoglycoprotein (H-PSGP), low molecular weight polysialoglycoprotein (L-PSGP), and 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN) glycoprotein were found to bind calcium ions with about 8-100 times the affinity of sialic acid monomer. Analysis by equilibrium dialysis of the binding of Ca2+ to colominic acid was biphasic, and the high-affinity interaction was shown to change with the degree of polymerization. Specific binding of Ca2+ to polysialic acid (polySia) caused characteristic effects in the circular dichroism spectrum. A pronounced decrease in the circular dichroism of polySia at 205 nm was observed upon addition of calcium. H-PSGP was found to bind calcium ions with 3-fold higher affinity than L-PSGP.

Animals↗
PubMed1994

Monoclonal antibody specific for alpha 2-->8-linked oligo deaminated neuraminic acid (KDN) sequences in glycoproteins. Preparation and characterization of a monoclonal antibody and its application in immunohistochemistry.

Two particular types of sialoglycoproteins have been detected in fish: polysialoglycoproteins containing alpha 2-->8-linked polysialic acid (-->8Neu5Gc alpha 2-->)n present in unfertilized Salmonidae fish eggs, and glycoproteins bearing oligo/polymers of deaminated neuraminic acids (KDN) found in the vitelline envelope of the eggs and ovarian fluid. We report the preparation and characterization of a monoclonal antibody specifically recognizing oligo/polymers of KDN sequences in glycoproteins and its application in immunohistochemistry. Fusion of spleen cells from a BALB/c mouse immunized with a KDN-rich glycoprotein (KDN-gp) containing (-->8KDN alpha 2-->)n-->6(KDN alpha 2-->3Gal beta 1-->3G alpha lNA-c alpha 1-->3) GalNAc alpha 1-->residues, with mouse myeloma cells yielded a hybrid cell line producing a monoclonal antibody that bound to KDN-gp, but not to KDN-gp depleted of KDN residues. The specificity of the monoclonal antibody, designated mAb.kdn8kdn, was determined by an enzyme-linked immunosorbent assay using KDN-gp samples that varied in KDN content. These antigens were prepared by the selective removal of KDN residues from the native KDN-gp. The mAb.kdn8kdn reacted most strongly with the intact KDN-gp and less strongly with KDN-gp samples containing decreased numbers of KDN residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Immunocytochemical identification of androgen receptor in mouse osteoclast-like multinucleated cells.

Expression of androgen receptor (AR) in mouse osteoclast-like multi-nucleated cells (OCs) was examined with immunocytochemical techniques. Murine OCs were obtained by co-culturing mouse osteoblastic cells and bone marrow cells. Three preparations of polyclonal anti-AR antibody which were raised in rabbit against different parts of the human AR were employed for the experiments. Specific staining for AR was demonstrated in the nuclei and the perinuclear area of mouse OCs. This is the first report demonstrating the presence of AR in osteoclast-like cells.

Animals↗
PubMed1994

Localization of follistatin, an activin-binding protein, in bone tissues.

We have previously reported that activin-A has a mitogenic effect on osteoblast-like MC3T3-E1 cells and that these cells produce an activin-binding protein, follistatin (Hashimoto, M., Shoda, A., Inoue, S., Yamada, R., Kondo, T., Sakurai, T., Ueno. N., and Muramatsu, M. (1992) J. Biol. Chem. 267, 4999-5004). Activin-A was also detected abundantly in bone matrix tissues and was found to act as an osteoclast differentiation factor. Here, we studied the expression of follistatin in bone tissues by in situ hybridization with a follistatin cRNA probe. Follistatin transcript was detected in the osteoblasts and a fraction of osteocyte population in the developing mouse mandible. Immunohistochemistry using anti-follistatin antibody supported this observation at the protein level. Follistatin mRNA was also detected in the callus of repairing bone after fracture. These findings suggest that activin and follistatin may play significant roles in the bone system in vivo.

Animals↗
PubMed1994

Characterization of the fibrillar layer at the epithelial-mesenchymal junction in tooth germs.

A characteristic layer containing numerous fibrils is associated with the basement membrane of the inner enamel epithelium during the early stages of odontogenesis. However, its nature is not well understood. In this study, the layer was examined with high-resolution electron microscopy and immuno-histochemical staining. Tooth germs of monkeys (Macaca fuscata) were studied and each fibril in the layer was found to be a tubular structure, 8-9 nm in width, resembling a "basotubule", the tubular structure previously observed in various basement membranes. The space between the fibrils was filled with a network formed by irregular anastomosing strands with an average thickness of 4 nm; these strands resembled the "cords" forming the network in the lamina densa of basement membranes. After immunoperoxidase staining, fine threads immunoreactive for laminin staining were seen winding along the strands of the network, and 1.5-nm wide filaments, immunoreactive for type IV collagen, took the form of a network arrangement. The 5-nm-wide ribbon-like structures associated with the strands were identified as heparan sulfate proteoglycan by immunostaining. These results are similar to those obtained for the cord network of the lamina densa. The "fibrillar layer" therefore represents a highly specialized lamina fibroreticularis of the basement membrane of the inner enamel epithelium, and rich in basotubules.

Animals↗
PubMed1994

Facilitation of liver regeneration after partial hepatectomy by ventromedial hypothalamic lesions in rats.

Whether or not the hypothalamus is involved in initiating hepatic DNA synthesis after partial hepatectomy is unclear. To determine the role of the ventromedial hypothalamic nuclei in liver regeneration after partial hepatectomy, we studied hepatic DNA synthesis during liver regeneration in rats with bilateral lesions of these nuclei. Lesions of the ventromedial hypothalamus accelerated the increase in hepatic DNA synthesis and raised the peak level of thymidine incorporation after partial hepatectomy. These effects of hypothalamic lesions were completely inhibited by hepatic vagotomy. Thus, lesions of the ventromedial hypothalamus appear to promote hepatic regeneration by increasing vagal stimulation of the liver.

Animals↗
PubMed1994

Does an animal peptide: N-glycanase have the dual role as an enzyme and a carbohydrate-binding protein?

Recently, we have reported purification and characterization of a de-N-glycosylating enzyme, peptide: N-glycanase (PNGase) found in C3H mouse fibroblast L-929 cells, and designated L-929 PNGase [Suzuki T, Seko A, Kitajima K, Inoue Y, Inoue S (1994) J Biol Chem 269, 17611-18]. The unique properties of L-929 PNGase are that the enzyme had a high affinity to the substrate glycopeptide (e.g. Km = 114 microM for fetuin derived glycopentapeptide) and that the PNGase-catalysed reaction is strongly inhibited by the released free oligosaccharides but not by the free peptides formed, suggesting that L-929 PNGase is able to bind to a certain type of carbohydrate chain. In this study, we report the new findings of the mannan-binding property of L-929 PNGase: the de-N-glycosylating enzyme activity of L-929 PNGase was inhibited by yeast mannan and triomannose, Man alpha 1-->3(Man alpha 1-->6)Man, but not by mannose and alpha-methyl-D-mannoside. Furthermore, L-929 PNGase was revealed to bind to the glycan moiety of yeast mannan by using mannan-conjugated Sepharose 4B gel as a ligand, suggesting that L-929 PNGase could serve not only as an enzyme but also as a carbohydrate recognition protein in vivo. Such 'dual' properties found for animal-derived L-929 PNGase are unique and are not shared with other previously characterized plant- and bacterial-origin PNGases--PNGase A and PNGase F, respectively.

Amidohydrolases↗
PubMed1994

Identification, characterization, and developmental expression of a novel alpha 2-->8-KDN-transferase which terminates elongation of alpha 2-->8-linked oligo-polysialic acid chain synthesis in trout egg polysialoglycoproteins.

A novel glycosyltransferase which catalyses transfer of deaminated neuraminic acid, KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) from CMP-KDN to the non-reducing termini of oligo-polysialyl chains of polysialoglycoprotein (PSGP), was discovered in the ovary of rainbow trout (Oncorhynchus mykiss). The KDN-transferase activity was optimal at neutral pH, and stimulated 2 to 2.5-fold by 2-5 mM Mg2+ or Mn2+. Expression of KDN-transferase was developmentally regulated in parallel with expression of the alpha 2-->8-polysialyltransferase, which catalyses synthesis of the oligo-polysialyl chains in PSGP. Incorporation of the KDN residues into the oligo-polysialyl chains prevented their further elongation, resulting in 'capping' of the oligo-polysialyl chains. This is the first example of a glycosyltransferase that catalyses termination of alpha 2-->8-polysialylation in glycoproteins.

Animals↗
PubMed1994

Nerve growth factor (NGF) restores depletions of calcitonin gene-related peptide and substance P in sensory neurons from diabetic mice in vitro.

Dorsal root ganglion neurons from streptozotocin (STZ)-induced diabetic, genetic diabetic and normal mice were cultured in serum-containing media with or without nerve growth factor (NGF). The immunocytochemical analysis carried out after 1 week in culture revealed that the ratios of neurons immunoreactive to calcitonin gene-related peptide (CGRP) in NGF-free medium in the STZ-diabetic mice (average 23.2%) were significantly lower than those in the normal mice (45.1%). The ratios of neurons immunoreactive to CGRP and substance P (SP) in the NGF-free medium were also lower in the genetic diabetic mice (23.6% and 21.8%) than those in the normal ones (40.7% and 34.2%). However, treatment with NGF restored these reduced immunoreactivities in the diabetic groups in a dose-dependent manner. These results show that NGF can be effective for the diabetes-induced depletion of CGRP and SP in sensory neurons, and suggest its possible role in the prevention and improvement of diabetic sensory neuropathy.

Animals↗