Biomedical subjects

Toshihiko Iwanaga

Publications and source records attributed to Toshihiko Iwanaga.

At least 19 recordsLinked to original sources

PubMed2003

Production and characterization of monoclonal antibodies against midgut of ixodid tick, Haemaphysalis longicornis.

There are concerted efforts toward development of tick vaccines to replace current chemical control strategies that have serious limitations [Parasitologia 32 (1990) 145; Infectious Disease Clinics of North America (1999) 209-226]. In this study, monoclonal antibodies (mAbs) specific to Haemaphysalis longicornis midgut proteins were produced and characterized. Eight antibody-secreting hybridomas were cloned and the mAbs typed as IgG1, IgG2a and IgG2b. On immunoblots, all mAbs reacted with a midgut protein band of about 76 kDa. All mAbs uniformly immunogold-stained the surface or epithelial layers of H. longicornis midgut and endosomes. Adult ticks (50%) that fed on an ascitic mouse producing the IgGs developed a red coloration and did not oviposit. As such, the 76 kDa protein that reacted with the mAbs could, therefore, be a potential candidate for tick vaccine development.

Animals↗

PubMed2003

Purification and identification of a binding protein for pancreatic secretory trypsin inhibitor: a novel role of the inhibitor as an anti-granzyme A.

Pancreatic secretory trypsin inhibitor (PSTI) is a potent trypsin inhibitor that is mainly found in pancreatic juice. PSTI has been shown to bind specifically to a protein, distinct from trypsin, on the surface of dispersed cells obtained from tissues such as small intestine. In the present study, we affinity-purified the binding protein from the 2% (w/v) Triton X-100-soluble fraction of dispersed rat small-intestinal cells using recombinant rat PSTI. Partial N-terminal sequencing of the purified protein gave a sequence that was identical with the sequence of mouse granzyme A (GzmA), a tryptase produced in cytotoxic lymphocytes. We confirmed the formation of an affinity-cross-linked complex between (125)I-labelled PSTI and recombinant rat GzmA (rGzmA). In situ hybridization and immunostaining revealed the existence of GzmA-expressing intraepithelial lymphocytes in the rat small intestine. We concluded that the PSTI-binding protein isolated from the dispersed cells is GzmA that is produced in the lymphocytes of the tissue. The rGzmA hydrolysed the N -alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT), and the BLT hydrolysis was inhibited by PSTI. Sulphated glycosaminoglycans, such as fucoidan or heparin, showed almost no effect on the inhibition of rGzmA by PSTI, whereas they decreased the inhibition by antithrombin III. In the present paper, we propose a novel role of PSTI as a GzmA inhibitor.

Animals↗

PubMed2003

Expression of estrogen receptor alpha (ER alpha) in the rat temporomandibular joint.

Numerous epidemiological studies have pointed out a higher frequency of temporomandibular disorder (TMD) in women than in men, which indicates the involvement of a sex hormone, such as estrogen, in the pathogenesis of TMD. Although estrogen is known to play pivotal roles in osteoarthrosis or rheumatoid arthritis in systemic joints, there have been few reports about the role of estrogen in the temporomandibular joint (TMJ). The effect of estrogen is generally mediated by the estrogen receptors (ERs) ER alpha (the predominant type) and ER beta. In this study we examined the expression of ER alpha protein and mRNA in the TMJ of adult male rats by immunocytochemistry and in situ hybridization histochemistry. Intense ER alpha immunoreactivity was localized in the synovial lining cells, stromal cells in the articular disc, and chondrocytes in the TMJ. These ER alpha-immunopositive synovial lining cells are characteristic of cytoplasmic processes identified with confocal and immunoelectron microscopy, which indicates that they are synovial type B cells. In situ hybridization histochemistry confirmed intense signals for ER alpha in the synovial lining cells and the sublining fibroblasts at mRNA levels. The nuclei of chondrocytes showed an intense immunoreaction for ER alpha in the maturative and hypertrophic layers of the articular cartilage. In addition to the nuclear localization of ER alpha, a weak immunoreaction appeared in the cytoplasm of some ER alpha-positive cells. These findings support the hypothesis that TMJ tissue-at least in the male rat-has the potential to be an estrogen target tissue.

Animals↗

PubMed2003

Immunohistochemical demonstration of acidic mammalian chitinase in the mouse salivary gland and gastric mucosa.

Acidic mammalian chitinase (AMCase) is the sole chitinolytic enzyme that has been identified thus far in the gastrointestinal tract of mammals. AMCase mRNA expression has been demonstrated in the salivary gland and stomach of mice and in the stomach of humans, while a bovine homologue of AMCase is produced in the liver and secreted into the blood. The present study using antibody raised against bovine AMCase demonstrates the cellular distribution of AMCase in salivary and gastric secretions at the protein level. Immunostaining using mouse tissues detected intense immunoreactivity for AMCase in serous-type secretory cells of the parotid gland and von Ebner's gland. Gastric chief cells, localized at the bottom of gastric glands, were also immunoreactive for AMCase. Electron-microscopically, the immunoreactivity was localized in granules in the apical cytoplasm of these secretory cells, and not in other structures. Western blot analysis confirmed the existence of AMCase in the parotid gland and stomach, and in their secretions in mice. However, no immunoreactive band was clearly detectable in immunoblots of the human parotid saliva and gastric juice. At least in the mouse, AMCase is secreted into the saliva and gastric juice, and may function as a digestive enzyme or play a defensive role against chitinous pathogens.

Animals↗

PubMed2003

Milk sphingomyelin accelerates enzymatic and morphological maturation of the intestine in artificially reared rats.

OBJECTIVES: Sphingomyelin (SPM) is the dominant phospholipid, comprising 38% of total human milk phospholipids. Although little is known about the nutritional importance of SPM during the neonatal period, SPM may affect the growth and development of tissues in the newborn infant through mechanisms regulating cell proliferation and differentiation. We evaluated the effect of sphingomyelin (SPM) in artificially reared rats as a suitable model of gut maturation in the suckling infant. METHODS: Seven-day-old Sprague-Dawley rat pups were cannulated intragastrically and reared artificially on milk containing 0.5% SPM or 0.5% phosphatidylcholine (PC) for 1 week. RESULTS: Intestinal lactase activity in the SPM group was significantly lower than that in the control or PC group. Upon histologic examination, intestinal villi were found to be occupied with vacuolated cells in the control and the PC group, whereas the vacuolated cells were restricted to the tip of villi in the SPM group. The Auerbach nerve plexus area of the ileum in the SPM group was significantly greater, possibly due to accelerated development, than that in the control group or PC group. CONCLUSIONS: The present results suggest that SPM, the dominant phospholipid in milk, plays an important role in neonatal gut maturation during the suckling period.

Animals↗

PubMed2003

Bacterial lipopolysaccharide-induced expression of the IkappaB protein MAIL in B-lymphocytes and macrophages.

Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL), a recently cloned nuclear IkappaB protein induced by lipopolysaccharide (LPS) stimulation in lymphoid organs, is involved in the regulation of inflammatory responses. The present in situ hybridization and immunohistochemical analyses revealed the distinct expression of the MAIL mRNA and protein in B-lymphocytes of the white pulp of the spleen and cortical lymphoid follicles of lymph nodes in LPS-injected mice. MAIL signals were also localized in F4/80-positive macrophages in these organs. LPS clearly induced MAIL expression in cultured B-lymphocytes and monocytes/macrophages, but only faintly so in T-lymphocytes, fibroblasts, and endothelial cells. MAIL was also induced by inflammatory cytokines such as interleukin-1 and -6, and tumor necrosis factor in cultured cells. Northern blot, Western blot, and in situ hybridization analyses showed that the major expression product of the Mail gene was a long splicing variant (MAIL-L) rather than a short one, both in lymphoid organs and cultured cells. These results collectively indicate that LPS induces MAIL-L predominantly in B-lymphocytes and macrophages.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Passive immunization with monoclonal antibodies: effects on Haemaphysalis longicornis tick infestation of BALB/c mice.

Tick vaccine development plays an important role in current tick control strategies. Previously, we have produced three different isotypes of monoclonal antibodies (mAbs) which recognized a midgut protein of adult Haemaphysalis longicornis. These mAbs, typed as IgG1, 2a, and 2b, reacted with a 76 kDa surface protein of midgut cells. We speculated that the 76 kDa protein may be an unknown antigen for a tick vaccine and the three mAbs may work as probes to clone the protein. In this study, to test whether these three isotypes have anti-tick effects and if so which works more effectively, we conducted passive immunization in BALB/c mice with each of the mAbs, and infested the mice with adult ticks. All isotypes significantly reduced the number of hatched larvae, compared to controls, however, no differences in the magnitude of the reduction were observed among the three.

Animals↗

PubMed2002

Piccolo, a Ca2+ sensor in pancreatic beta-cells. Involvement of cAMP-GEFII.Rim2. Piccolo complex in cAMP-dependent exocytosis.

We have previously shown that cAMP-binding protein cAMP-guanidine nucleotide exchange factor II (GEFII) (or Epac2) interacting with Rim2 is involved in cAMP-dependent, protein kinase A-independent exocytosis in pancreatic beta-cells. The action of the cAMP-GEFII.Rim2 complex requires both intracellular cAMP and Ca(2+). Although Rim2 has C(2) domains, its role as a Ca(2+) sensor has remained unclear. In the present investigation, we have discovered that Piccolo, a CAZ (cytoskeletal matrix associated with the active zone) protein in neurons that is structurally related to Rim2, also binds to cAMP-GEFII and that it forms both homodimer and heterodimer with Rim2 in a Ca(2+)-dependent manner, whereas Rim2 alone does not form the homodimer. The association of Piccolo.Rim2 heterodimerization is stronger than Piccolo. Piccolo homodimerization. Treatment of pancreatic islets with antisense oligodeoxynucleotides against Piccolo inhibits insulin secretion induced by cAMP analog 8-bromo-cyclic AMP plus high glucose stimulation. These results suggest that Piccolo serves as a Ca(2+) sensor in exocytosis in pancreatic beta-cells and that the formation of a cAMP-GEFII.Rim2. Piccolo complex is important in cAMP-induced insulin secretion. In addition, this study suggests that CAZ proteins similar to those in neurons are also function in pancreatic beta-cells.

Animals↗

PubMed2002

Glucocorticoid inhibits expression of V-1, a catecholamine biosynthesis regulatory protein, in cultured adrenal medullary cells.

V-1 acts as a positive and coordinate regulator of gene expression of catecholamine biosynthetic enzymes in PC12D cells. The present study was conducted to investigate the mechanism controlling expression of V-1 in the adrenal gland. Immunohistochemical analysis demonstrated that noradrenergic chromaffin cells more highly expressed V-1 than adrenergic chromaffin cells preferentially expressing the glucocorticoid receptor in rat adrenal glands. Western blotting showed that in cultured bovine adrenal medullary cells, dexamethasone, a synthetic glucocorticoid, inhibited expression of V-1, and that this inhibition was prevented by RU-486, a glucocorticoid receptor antagonist. These results suggest that adrenal expression of V-1 is differentially controlled by glucocorticoids through the specific receptor, and that thereby V-1 regulates catecholamine biosynthesis in a catecholaminergic phenotype-dependent manner.

Adrenal Medulla↗

PubMed2002

Epididymis is a novel site of erythropoietin production in mouse reproductive organs.

The epididymis consists of the interstitial tissue and the ductus epididymidis, an extremely tortuous duct, in which spermatozoa exported from the testis gain motility and fertilizing capacity. We found that the cultured mouse epididymis produces erythropoietin (Epo). The content of Epo mRNA in the epididymis from the adult mouse (8-week-old) amounts to 40% of that in the kidney. The epididymal Epo mRNA dramatically increased upon growth; its level increased 120-fold from the age of 3 weeks to 7 weeks when they complete sexual maturation, while the increase in the total RNA was 3-fold. Hypoxia induced a 5-fold increase in the epididymal Epo mRNA transiently, which is much lower than the induction in the kidney (28-fold). In situ hybridization technique elucidated that the site of Epo production was located in the interstitial space between ductus epididymidis. The epididymal Epo may have an unidentified function in the male reproductive organ.

Animals↗

PubMed2002

Immunochemical characterization and measurement of neuronal type nitric oxide synthase in human neuroblastoma NB-OK-1 cell using novel anti-synthetic peptide antibody and specific immunoassay system.

We developed a sensitive and specific immunoassay system for human neuronal nitric oxide synthase (hnNOS) using synthetic hnNOS(998-1024) peptide and anti-hnNOS(998-1024) antibody. The novel antibody and radioimmunoassay system revealed a typical nNOS protein in human neuroblastoma NB-OK-1 cell (160 kDa, 180 fmol/10(6) cells). The kinetic parameters of the enzyme were K(m)=4.88 microM and V(max)=4.34 pmol/min/mg protein for L-arginine. On incubation of NB-OK-1 cell for 24 h, betamethasone phosphate decreased both nNOS-immunoreactivity (nNOS-IR) and enzymatic activity in the cell dose-dependently. On the other hand, pituitary adenylate cyclase activating polypeptide(1-38) (PACAP38) increased both nNOS-IR and enzymatic activity at concentrations of 10(-10) and 10(-9) M, but inversely decreased both at 10(-7) M. These suggest the positive and negative implications of endogenous NO in proliferation and differentiation of the cell, which support mitogenic activity of NO generated by nNOS in the cell. The present findings also provided evidence that the quantitative change of nNOS protein controls the integrated activity of the enzyme in the cell and, in turn, substantiate the validity and reliability of the present immunoassay system for hnNOS and its practical usefulness.

Amino Acid Sequence↗

PubMed2002

Identification, tissue expression, and functional characterization of Otx3, a novel member of the Otx family.

Transcription factors containing a homeodomain play an important role in the organogenesis of vertebrates. We have isolated a novel homeodomain transcription factor, Otx3, which is structurally and functionally related to Otx1 and Otx2, transcription factors that are critical in brain morphogenesis. Mouse Otx3 is a protein composed of 376 amino acids. Otx3 mRNA was expressed in mouse embryos from 10.5 to 13.5 days postcoitum (dpc) and in adult cerebellum as assessed by Northern blotting. Whole-mount in situ hybridization of mouse embryos from 9.5 to 11.5 dpc revealed strong expression of Otx3 mRNA in the diencephalon, mesencephalon, metencephalon, myelencephalon, and developing eye, indicating an expression pattern largely overlapping but distinct from those of Otx1 and Otx2. In addition, Otx3 was shown by electrophoretic mobility shift assay to bind to the TAATCC motif, the consensus binding sequence for Otx1, Otx2, and Crx. Results of a transcription reporter assay suggest that Otx3 functions as a transcription repressor by binding to this motif. These results suggest that Otx3 is a novel member of the Otx family and may be involved in the development of the central nervous system.

Amino Acid Sequence↗

PubMed2002

Cloning and characterization of a novel mammalian zinc transporter, zinc transporter 5, abundantly expressed in pancreatic beta cells.

Intracellular homeostasis for zinc is achieved through the coordinate regulation of specific transporters engaged in zinc influx, efflux, and intracellular compartmentalization. We have identified a novel mammalian zinc transporter, zinc transporter 5 (ZnT-5), by virtue of its similarity to ZRC1, a zinc transporter of Saccharomyces cerevisiae, a member of the cation diffusion facilitator family. Human ZnT-5 (hZnT-5) cDNA encodes a 765-amino acid protein with 15 predicted membrane-spanning domains. hZnT-5 was ubiquitously expressed in all tested human tissues and abundantly expressed in the pancreas. In the human pancreas, hZnT-5 was expressed abundantly in insulin-containing beta cells that contain zinc at the highest level in the body. The hZnT-5 immunoreactivity was found to be associated with secretory granules by electron microscopy. The hZnT-5-derived zinc transport activity was detected using the Golgi-enriched vesicles prepared from hZnT-5-induced HeLa/hZnT-5 cells in which exogenous hZnT-5 expression is inducible by the Tet-on gene regulation system. This activity was dependent on time, temperature, and concentration and was saturable. Moreover, zinc at a high concentration (10 mm) inhibited the growth of yeast expressing hZnT-5. These results suggest that ZnT-5 plays an important role for transporting zinc into secretory granules in pancreatic beta cells.

Amino Acid Sequence↗

PubMed2002

Hemodynamics in vasculogenic mimicry and angiogenesis of inflammatory breast cancer xenograft.

In the present study, we examined hemodynamics in vasculogenic mimicry (VM) and angiogenesis of inflammatory breast cancer (IBC) xenografts (WIBC-9), having previously reported on the unique histological features and molecular basis of these processes (K. Shirakawa et al., Cancer Res., 61: 445-451, 2001). Histologically, the WIBC-9 xenografts exhibited invasive ductal carcinoma with a hypervascular structure (angiogenesis) in the tumor margin and VM without endothelial cells, central necrosis, or fibrosis in the tumor center. Results of molecular analysis indicated that WIBC-9 had a vasculogenic phenotype, including expression of Flt-1 and Tie-2. Comparison of WIBC-9 with an established non-IBC xenograft (MC-5), using time-coursed dynamic micromagnetic resonance angiography analysis (with our newly developed intravascular macromolecular magnetic resonance imaging contrast agent), electromicroscopy, and immunohistochemistry, demonstrated blood flow and a VM-angiogenesis junction in the central area of the WIBC-9 tumor. It has previously been considered impossible to prove a connection between VM and angiogenesis using angiography, because there are no intravascular macromolecular magnetic resonance imaging contrast agents that do not exhibit significant leakage through the vascular wall. In the present study, laser-captured microdissection was performed in regions of WIBC-9 tumors that exhibited VM without endothelial cells, central necrosis, or fibrosis, revealing expression of human-Flt-1 and human-Tie2 and the absence of human-CD31, human-endothelin B receptor, and human-thrombin receptor. These facts led us to hypothesize that the VM of WIBC-9 involves hemodynamics that serve to feed WIBC-9 cells, and this in turn suggests a connection between VM and angiogenesis.

Animals↗

PubMed2002

Identification of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine.

We have revealed that 100-200 clusters, filled with closely packed lymphocytes, can be found throughout the length of the antimesenteric wall of the mouse small intestine. They are composed of a large B cell area, including a germinal center, and epithelia overlying the clusters contain M cells. A large fraction of B cells displays B220+ CD19+ CD23+ IgM(low)IgD(high)CD5(-)Mac-1(-) phenotype, and the composition of IgA+ B cells is smaller but substantial. To our knowledge, these clusters are the first identification of isolated lymphoid follicles (ILF) in mouse small intestine. ILF can be first detected at 7 (BALB/c mice) and 25 (C57BL/6 mice) days after birth, and lymphoid clusters equivalent in terms of cellular mass to ILF are present in germfree, athymic nude, RAG-2(-/-), TCR-beta(-/-), and Ig mu-chain mutant (mu(-/-)) mice, although c-kit+ cells outnumber B220+ cells in germfree and athymic nude mice, and most lymphoid residents are c-kit+ B220(-) in RAG-2(-/-), TCR-beta(-/-), and mu(-/-) mice. ILF develop normally in the progeny of transplacentally manipulated Peyer's patch (PP)-deficient mice, and decreased numbers of conspicuously atrophied ILF are present in IL-7Ralpha(-/-) PP(null) mice. Neither ILF nor PP are detectable in lymphotoxin alpha(-/-) and aly/aly mice that retain well-developed cryptopatches (CP) and thymus-independent subsets of intraepithelial T cells, whereas ILF, PP, CP, and thymus-independent subsets of intraepithelial T cells disappear from common cytokine receptor gamma-chain mutant mice. These findings indicate that ILF, PP, and CP constitute three distinct organized gut-associated lymphoid tissues that reside in the lamina propria of the mouse small intestine.

Animals↗

PubMed2002

Expression of galanin receptor-1 (GALR1) in the rat trigeminal ganglia and molar teeth.

The expression of galanin receptor-1 (GALR1) was investigated in the rat trigeminal ganglion by using immunocytochemistry and in situ hybridization. In addition, the regional distribution of GALR1-immunoreactive pulpal nerves and their ultrastructure were examined in the molar teeth. In the trigeminal ganglion, the immunoreactivity for GALR1 was recognizable in about 30% of the total number of neurons. Most of the cell bodies were small to medium in size. Analysis of serially cut sections alternately stained with GALR1 and galanin antisera demonstrated that some GALR1-positive cells displayed immunoreactivity for galanin. In situ hybridization analysis, expression of GALR1 mRNA was detected in trigeminal ganglion cells. The cell size distribution was similar to that of GALR1-immunoreactive cells. In the dental pulp, a small number of nerve fibers displayed immunoreactivity for GALR1. The labeled fibers formed terminal arbors in the coronal pulp around and within the odontoblast cell layer, but never penetrated into the predentin and dentin. Ultrastructurally, GALR1 immunoreactivity in the dental pulp was confined to the axoplasm of unmyelinated nerve fibers. The present study provided new evidence that unmyelinated primary afferents innervating dental pulp possessed galanin receptor, and suggests the existence of nociceptive primary afferents functioning as autocrine cells.

Animals↗

PubMed2002

Mouse model of Prinzmetal angina by disruption of the inward rectifier Kir6.1.

The inwardly rectifying K(+) channel Kir6.1 forms K(+) channels by coupling with a sulfonylurea receptor in reconstituted systems, but the physiological roles of Kir6.1-containing K(+) channels have not been determined. We report here that mice lacking the gene encoding Kir6.1 (known as Kcnj8) have a high rate of sudden death associated with spontaneous ST elevation followed by atrioventricular block as seen on an electrocardiogram. The K(+) channel opener pinacidil did not induce K(+) currents in vascular smooth-muscle cells of Kir6.1-null mice, and there was no vasodilation response to pinacidil. The administration of methylergometrine, a vasoconstrictive agent, elicited ST elevation followed by cardiac death in Kir6.1-null mice but not in wild-type mice, indicating a phenotype characterized by hypercontractility of coronary arteries and resembling Prinzmetal (or variant) angina in humans. The Kir6.1-containing K(+) channel is critical in the regulation of vascular tonus, especially in the coronary arteries, and its disruption may cause Prinzmetal angina.

Angina Pectoris, Variant↗

PubMed2002

Downregulation of P2X3 receptor-dependent sensory functions in A/J inbred mouse strain.

There is large variability in the various pain responses including those to tissue injury among inbred mouse strains. However, the determinant factors for the strain-specific differences remain unknown. The P2X3 sensory-specific ATP-gated channel has been implicated as a damage-sensing molecule that evokes a pain sensation by receiving endogenous ATP from injured tissue. In this study, to clarify the contribution of the sensory P2X3 signalling to strain-specific differences in tissue injury pain, we examined whether the P2X3-mediated in vivo and in vitro responses in dorsal root ganglion (DRG) neurons are changed in the A/J inbred mouse strain, which is known to be resistant to tissue injury pain caused by formalin. Here we found that A/J mice exhibited a low magnitude of nocifensive behaviour induced by the P2X agonist alpha,beta-methylene ATP (alpha beta meATP) into the hindpaw compared with C57BL/6 J mice. This behaviour was blocked by P2X3 antisense oligodeoxynucleotides. The low magnitude of the in vivo pain sensation could be observed similarly in the in vitro response; the increase in the intracellular Ca(2+) increase by alpha beta meATP in capsaicin-sensitive DRG neurons from A/J mice was significantly lower than that from C57BL/6 J mice. In A/J DRG neurons the P2X3 protein level was significantly lower compared with C57BL/6 J DRG neurons. The change in P2X3 protein was selective because P2X2 protein was expressed equally in both strains. The present study suggests that the downregulation of sensory P2X3 could be one of the molecular predispositions to low sensitivity to tissue injury pain in the A/J inbred mouse strain.

Adenosine Triphosphate↗