Showing papers by "Kyoto University published in 2000"

Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation.

Abstract: PD-1 is an immunoinhibitory receptor expressed by activated T cells, B cells, and myeloid cells. Mice deficient in PD-1 exhibit a breakdown of peripheral tolerance and demonstrate multiple autoimmune features. We report here that the ligand of PD-1 (PD-L1) is a member of the B7 gene family. Engagement of PD-1 by PD-L1 leads to the inhibition of T cell receptor-mediated lymphocyte proliferation and cytokine secretion. In addition, PD-1 signaling can inhibit at least suboptimal levels of CD28-mediated costimulation. PD-L1 is expressed by antigen-presenting cells, including human peripheral blood monocytes stimulated with interferon gamma, and activated human and murine dendritic cells. In addition, PD-L1 is expressed in nonlymphoid tissues such as heart and lung. The relative levels of inhibitory PD-L1 and costimulatory B7-1/B7-2 signals on antigen-presenting cells may determine the extent of T cell activation and consequently the threshold between tolerance and autoimmunity. PD-L1 expression on nonlymphoid tissues and its potential interaction with PD-1 may subsequently determine the extent of immune responses at sites of inflammation.

Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis

Abstract: During carcinogenesis of pancreatic islets in transgenic mice, an angiogenic switch activates the quiescent vasculature Paradoxically, vascular endothelial growth factor (VEGF) and its receptors are expressed constitutively Nevertheless, a synthetic inhibitor (SU5416) of VEGF signalling impairs angiogenic switching and tumour growth Two metalloproteinases, MMP-2/gelatinase-A and MMP-9/gelatinase-B, are upregulated in angiogenic lesions MMP-9 can render normal islets angiogenic, releasing VEGF MMP inhibitors reduce angiogenic switching, and tumour number and growth, as does genetic ablation of MMP-9 Absence of MMP-2 does not impair induction of angiogenesis, but retards tumour growth, whereas lack of urokinase has no effect Our results show that MMP-9 is a component of the angiogenic switch

Immunologic Self-Tolerance Maintained by Cd25+Cd4+Regulatory T Cells Constitutively Expressing Cytotoxic T Lymphocyte–Associated Antigen 4

Abstract: This report shows that cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) plays a key role in T cell–mediated dominant immunologic self-tolerance. In vivo blockade of CTLA-4 for a limited period in normal mice leads to spontaneous development of chronic organ-specific autoimmune diseases, which are immunopathologically similar to human counterparts. In normal naive mice, CTLA-4 is constitutively expressed on CD25+CD4+ T cells, which constitute 5–10% of peripheral CD4+ T cells. When the CD25+CD4+ T cells are stimulated via the T cell receptor in vitro, they potently suppress antigen-specific and polyclonal activation and proliferation of other T cells, including CTLA-4–deficient T cells, and blockade of CTLA-4 abrogates the suppression. CD28-deficient CD25+CD4+ T cells can also suppress normal T cells, indicating that CD28 is dispensable for activation of the regulatory T cells. Thus, the CD25+CD4+ regulatory T cell population engaged in dominant self-tolerance may require CTLA-4 but not CD28 as a costimulatory molecule for its functional activation. Furthermore, interference with this role of CTLA-4 suffices to elicit autoimmune disease in otherwise normal animals, presumably through affecting CD25+CD4+ T cell–mediated control of self-reactive T cells. This unique function of CTLA-4 could be exploited to potentiate T cell–mediated immunoregulation, and thereby to induce immunologic tolerance or to control autoimmunity.

Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase

Abstract: Autosomal recessive juvenile parkinsonism (AR-JP), one of the most common familial forms of Parkinson disease, is characterized by selective dopaminergic neural cell death and the absence of the Lewy body, a cytoplasmic inclusion body consisting of aggregates of abnormally accumulated proteins. We previously cloned PARK2, mutations of which cause AR-JP (ref. 2), but the function of the gene product, parkin, remains unknown. We report here that parkin is involved in protein degradation as a ubiquitin-protein ligase collaborating with the ubiquitin-conjugating enzyme UbcH7, and that mutant parkins from AR-JP patients show loss of the ubiquitin-protein ligase activity. Our findings indicate that accumulation of proteins that have yet to be identified causes a selective neural cell death without formation of Lewy bodies. Our findings should enhance the exploration of the molecular mechanisms of neurodegeneration in Parkinson disease as well as in other neurodegenerative diseases that are characterized by involvement of abnormal protein ubiquitination, including Alzheimer disease, other tauopathies, CAG triplet repeat disorders and amyotrophic lateral sclerosis.

The Vienna classification of gastrointestinal epithelial neoplasia

Abstract: Background—Use of the conventional Western and Japanese classification systems of gastrointestinal epithelial neoplasia results in large diVerences among pathologists in the diagnosis of oesophageal, gastric, and colorectal neoplastic lesions. Aim—To develop common worldwide terminology for gastrointestinal epithelial neoplasia. Methods—Thirty one pathologists from 12 countries reviewed 35 gastric, 20 colorectal, and 21 oesophageal biopsy and resection specimens. The extent of diagnostic agreement between those with Western and Japanese viewpoints was assessed by kappa statistics. The pathologists met in Vienna to discuss the results and to develop a new consensus terminology. Results—The large diVerences between the conventional Western and Japanese diagnoses were confirmed (percentage of specimens for which there was agreement and kappa values: 37% and 0.16 for gastric; 45% and 0.27 for colorectal; and 14% and 0.01 for oesophageal lesions). There was much better agreement among pathologists (71% and 0.55 for gastric; 65% and 0.47 for colorectal; and 62% and 0.31 for oesophageal lesions) when the original assessments of the specimens were regrouped into the categories of the proposed Vienna classification of gastrointestinal epithelial neoplasia: (1) negative for neoplasia/dysplasia, (2) indefinite for neoplasia/dysplasia, (3) non-invasive low grade neoplasia (low grade adenoma/ dysplasia), (4) non-invasive high grade neoplasia (high grade adenoma/ dysplasia, non-invasive carcinoma and suspicion of invasive carcinoma), and (5) invasive neoplasia (intramucosal carcinoma, submucosal carcinoma or beyond). Conclusion—The diVerences between Western and Japanese pathologists in the diagnostic classification of gastrointestinal epithelial neoplastic lesions can be resolved largely by adopting the proposed terminology, which is based on cytological and architectural severity and invasion status. (Gut 2000;47:251‐255)

Structural determinants of water permeation through aquaporin-1.

Abstract: Human red cell AQP1 is the first functionally defined member of the aquaporin family of membrane water channels. Here we describe an atomic model of AQP1 at 3.8A resolution from electron crystallographic data. Multiple highly conserved amino-acid residues stabilize the novel fold of AQP1. The aqueous pathway is lined with conserved hydrophobic residues that permit rapid water transport, whereas the water selectivity is due to a constriction of the pore diameter to about 3 A over a span of one residue. The atomic model provides a possible molecular explanation to a longstanding puzzle in physiology-how membranes can be freely permeable to water but impermeable to protons.

Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors

Abstract: Interaction between endothelial cells and mural cells (pericytes and vascular smooth muscle) is essential for vascular development and maintenance Endothelial cells arise from Flk1-expressing (Flk1+) mesoderm cells, whereas mural cells are believed to derive from mesoderm, neural crest or epicardial cells and migrate to form the vessel wall Difficulty in preparing pure populations of these lineages has hampered dissection of the mechanisms underlying vascular formation Here we show that Flk1+ cells derived from embryonic stem cells can differentiate into both endothelial and mural cells and can reproduce the vascular organization process Vascular endothelial growth factor promotes endothelial cell differentiation, whereas mural cells are induced by platelet-derived growth factor-BB Vascular cells derived from Flk1+ cells can organize into vessel-like structures consisting of endothelial tubes supported by mural cells in three-dimensional culture Injection of Flk1+ cells into chick embryos showed that they can incorporate as endothelial and mural cells and contribute to the developing vasculature in vivo Our findings indicate that Flk1+ cells can act as 'vascular progenitor cells' to form mature vessels and thus offer potential for tissue engineering of the vascular system

Magnetic vortex core observation in circular dots of permalloy

Abstract: Spin structures of nanoscale magnetic dots are the subject of increasing scientific effort, as the confinement of spins imposed by the geometrical restrictions makes these structures comparable to some internal characteristic length scales of the magnet. For a vortex (a ferromagnetic dot with a curling magnetic structure), a spot of perpendicular magnetization has been theoretically predicted to exist at the center of the vortex. Experimental evidence for this magnetization spot is provided by magnetic force microscopy imaging of circular dots of permalloy (Ni 80 Fe 20 ) 0.3 to 1 micrometer in diameter and 50 nanometers thick.

The induction of tolerance by dendritic cells that have captured apoptotic cells.

Abstract: What makes a protein immunogenic, particularly for strong T cell–mediated immunity? To a first approximation, this determination seems to be made by dendritic cells (DCs). Immature DCs, as in skin ([1][1])([2][2])([3][3])([4][4]), lung ([5][5]), blood ([6][6])([7][7]), and spleen ([7][7])([8][8

Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor

Abstract: The metabotropic glutamate receptors (mGluRs) are key receptors in the modulation of excitatory synaptic transmission in the central nervous system. Here we have determined three different crystal structures of the extracellular ligand-binding region of mGluR1--in a complex with glutamate and in two unliganded forms. They all showed disulphide-linked homodimers, whose 'active' and 'resting' conformations are modulated through the dimeric interface by a packed alpha-helical structure. The bi-lobed protomer architectures flexibly change their domain arrangements to form an 'open' or 'closed' conformation. The structures imply that glutamate binding stabilizes both the 'active' dimer and the 'closed' protomer in dynamic equilibrium. Movements of the four domains in the dimer are likely to affect the separation of the transmembrane and intracellular regions, and thereby activate the receptor. This scheme in the initial receptor activation could be applied generally to G-protein-coupled neurotransmitter receptors that possess extracellular ligand-binding sites.

Control of endodermal endocrine development by Hes-1.

Abstract: Development of endocrine cells in the endoderm involves Atonal and Achaete/Scute-related basic helix-loop-helix (bHLH) proteins. These proteins also serve as neuronal determination and differentiation factors, and are antagonized by the Notch pathway partly acting through Hairy and Enhancer-of-split (HES)-type proteins. Here we show that mice deficient in Hes1 (encoding Hes-1) display severe pancreatic hypoplasia caused by depletion of pancreatic epithelial precursors due to accelerated differentiation of post-mitotic endocrine cells expressing glucagon. Moreover, upregulation of several bHLH components is associated with precocious and excessive differentiation of multiple endocrine cell types in the developing stomach and gut, showing that Hes-1 operates as a general negative regulator of endodermal endocrine differentiation.

Complex Phenotype of Mice Lacking Occludin, a Component of Tight Junction Strands

Abstract: Occludin is an integral membrane protein with four transmembrane domains that is exclusively localized at tight junction (TJ) strands. Here, we describe the generation and analysis of mice carrying a null mutation in the occludin gene. Occludin -/- mice were born with no gross phenotype in the expected Mendelian ratios, but they showed significant postnatal growth retardation. Occludin -/- males produced no litters with wild-type females, whereas occludin -/- females produced litters normally when mated with wild-type males but did not suckle them. In occludin -/- mice, TJs themselves did not appear to be affected morphologically, and the barrier function of intestinal epithelium was normal as far as examined electrophysiologically. However, histological abnormalities were found in several tissues, i.e., chronic inflammation and hyperplasia of the gastric epithelium, calcification in the brain, testicular atrophy, loss of cytoplasmic granules in striated duct cells of the salivary gland, and thinning of the compact bone. These phenotypes suggested that the functions of TJs as well as occludin are more complex than previously supposed.

Mechanism of electrical conductivity of transparent InGaZnO 4

Abstract: The electronic structure of ${\mathrm{InGaZnO}}_{4},$ which has a layered structure with alternating laminated layers of ${\mathrm{InO}}_{2}$ and ${\mathrm{GaZnO}}_{2},$ was calculated in order to investigate the mechanism of electrical conductivity. In the crystal structure obtained through relaxation calculations using classical two-center potentials, the Ga ion in the ${\mathrm{GaZnO}}_{2}$ layer has pentagonal coordination forming a bipyramid with five oxygen ions, while the Zn ion in the same layer has tetrahedral coordination, losing a bond with the oxygen at the top of one pyramid. The molecular orbitals of model clusters for the relaxed structure, which were calculated by the discrete variational $X\ensuremath{\alpha}$ method using a model cluster, show strong two-dimensional structures. The electronic states at the edge of the conduction band are the result of overlapping between In $5s$ orbitals, and delocalize in the ${\mathrm{InO}}_{2}$ layer. The energy in the Ga $4s$ and Zn $4s$ states in the ${\mathrm{GaZnO}}_{2}$ layer was too large to be doped with electrons. The In $5s$ states are considered to be conduction paths for carrier electrons. A very high conductivity can be expected in the case where dopant ions are introduced into the ${\mathrm{GaZnO}}_{2}$ layers.

Trapping and emission of photons by a single defect in a photonic bandgap structure

Abstract: By introducing artificial defects and/or light-emitters into photonic bandgap structures1,2, it should be possible to manipulate photons. For example, it has been predicted2 that strong localization (or trapping) of photons should occur in structures with single defects, and that the propagation3,4 of photons should be controllable using arrays of defects. But there has been little experimental progress in this regard, with the exception of a laser5 based on a single-defect photonic crystal. Here we demonstrate photon trapping by a single defect that has been created artificially inside a two-dimensional photonic bandgap structure. Photons propagating through a linear waveguide are trapped by the defect, which then emits them to free space. We envisage that this phenomenon may be used in ultra-small optical devices whose function is to selectively drop (or add) photons with various energies from (or to) optical communication traffic. More generally, our work should facilitate the development of all-optical circuits incorporating photonic bandgap waveguides and resonators.

Full three-dimensional photonic bandgap crystals at near-infrared wavelengths

Abstract: An artificial crystal structure has been fabricated exhibiting a full three-dimensional photonic bandgap effect at optical communication wavelengths. The photonic crystal was constructed by stacking 0.7-micrometer period semiconductor stripes with the accuracy of 30 nanometers by advanced wafer-fusion technique. A bandgap effect of more than 40 decibels (which corresponds to 99.99% reflection) was successfully achieved. The result encourages us to create an ultra-small optical integrated circuit including a three-dimensional photonic crystal waveguide with a sharp bend.

Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases.

Abstract: Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide++ + dihydrochloride] is widely used as a specific inhibitor of the Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) family of protein kinases. This study examined the inhibition mechanism and profile of actions of Y-27632 and a related compound, Y-30141 [(+)-(R)-trans- 4-(1-aminoethyl)-N-(1H-pyrrolo[2, 3-b]pyridin-4-yl)cyclohexan-ecarboxamide dihydrochloride]. Y-27632 and Y-30141 inhibited the kinase activity of both ROCK-I and ROCK-II in vitro, and this inhibition was reversed by ATP in a competitive manner. This suggests that these compounds inhibit the kinases by binding to the catalytic site. Their affinities for ROCK kinases as determined by K(i) values were at least 20 to 30 times higher than those for two other Rho effector kinases, citron kinase and protein kinase PKN. [(3)H]Y-30141 was taken up by cells in a temperature- and time-dependent and saturable manner, and this uptake was competed with unlabeled Y-27632. No concentrated accumulation was found, suggesting that the uptake is a carrier-mediated facilitated diffusion. Y-27632 abolished stress fibers in Swiss 3T3 cells at 10 microM, but the G(1)-S phase transition of the cell cycle and cytokinesis were little affected at this concentration. Y-30141 was 10 times more potent than Y-27632 in inhibiting the kinase activity and stress fiber formation, and it caused significant delay in the G(1)-S transition and inhibition of cytokinesis at 10 microM.

AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1

Abstract: The Wnt signalling pathway is essential for development and organogenesis1,2,3. Wnt signalling stabilizes β-catenin, which accumulates in the cytoplasm, binds to T-cell factor (TCF; also known as lymphocyte enhancer-binding factor, LEF) and then upregulates downstream genes4,5,6. Mutations in CTNNB1 (encoding β-catenin) or APC (adenomatous polyposis coli) have been reported in human neoplasms including colon cancers and hepatocellular carcinomas7,8,9,10,11,12,13 (HCCs). Because HCCs tend to show accumulation of β-catenin more often than mutations in CTNNB1 , we looked for mutations in AXIN1, encoding a key factor for Wnt signalling, in 6 HCC cell lines and 100 primary HCCs. Among the 4 cell lines and 87 HCCs in which we did not detect CTNNB1 mutations, we identified AXIN1 mutations in 3 cell lines and 6 mutations in 5 of the primary HCCs. In cell lines containing mutations in either gene, we observed increased DNA binding of TCF associated with β-catenin in nuclei. Adenovirus mediated gene transfer of wild-type AXIN1 induced apoptosis in hepatocellular and colorectal cancer cells that had accumulated β-catenin as a consequence of either APC, CTNNB1 or AXIN1 mutation, suggesting that axin may be an effective therapeutic molecule for suppressing growth of hepatocellular and colorectal cancers.

ATF6 Activated by Proteolysis Binds in the Presence of NF-Y (CBF) Directly to the cis-Acting Element Responsible for the Mammalian Unfolded Protein Response

Abstract: Transcription of genes encoding molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) is induced by accumulation of unfolded proteins in the ER. This intracellular signaling, known as the unfolded protein response (UPR), is mediated by the cis-acting ER stress response element (ERSE) in mammals. In addition to ER chaperones, the mammalian transcription factor CHOP (also called GADD153) is induced by ER stress. We report here that the transcription factor XBP-1 (also called TREB5) is also induced by ER stress and that induction of CHOP and XBP-1 is mediated by ERSE. The ERSE consensus sequence is CCAAT-N9-CCACG. As the general transcription factor NF-Y (also known as CBF) binds to CCAAT, CCACG is considered to provide specificity in the mammalian UPR. We recently found that the basic leucine zipper protein ATF6 isolated as a CCACG-binding protein is synthesized as a transmembrane protein in the ER, and ER stress-induced proteolysis produces a soluble form of ATF6 that translocates into the nucleus. We report here that overexpression of soluble ATF6 activates transcription of the CHOP and XBP-1 genes as well as of ER chaperone genes constitutively, whereas overexpression of a dominant negative mutant of ATF6 blocks the induction by ER stress. Furthermore, we demonstrated that soluble ATF6 binds directly to CCACG only when CCAAT exactly 9 bp upstream of CCACG is bound to NF-Y. Based on these and other findings, we concluded that specific and direct interactions between ATF6 and ERSE are critical for transcriptional induction not only of ER chaperones but also of CHOP and XBP-1.

Ghrelin Strongly Stimulates Growth Hormone Release in Humans

Abstract: Ghrelin is a recently identified endogenous ligand for the GH secretagogue receptor and is involved in a novel system for regulating GH release. However, little is known about its GH-releasing activity and other endocrine effects in humans. To address this issue, we studied the GH, ACTH, cortisol, PRL, LH, FSH, and TSH responses to synthetic human ghrelin. In four normal male adults (28-37 yr), iv ghrelin administration released GH in a dose-dependent manner and 0.2, 1.0, and 5.0 microg/kg ghrelin produced 43.3 +/- 6.0, 81.5 +/- 12.7, and 107.0 +/- 10.7 ng/mL of the GH peak values at 30 min, respectively. ACTH, cortisol, and PRL levels were also elevated after ghrelin injection, while the lowest dose (0.2 microg/kg) resulted in only minimum peak values of these hormones (22.8 +/- 3.0 pg/mL, 9.4 +/- 1.9 microg/dL, and 4.6 +/- 0.6 ng/mL, respectively). There were no significant changes in LH, FSH, or TSH levels. This is the first study showing evidence that ghrelin strongly stimulates GH release in humans.

Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants.

Abstract: A rice gene encoding a calcium-dependent protein kinase (CDPK), OsCDPK7, was induced by cold and salt stresses. To elucidate the physiological function of OsCDPK7, we generated transgenic rice plants with altered levels of the protein. The extent of tolerance to cold and salt/drought stresses of these plants correlated well with the level of OsCDPK7 expression. Therefore, OsCDPK7 was shown to be a positive regulator commonly involved in the tolerance to both stresses in rice. Over-expression of OsCDPK7 enhanced induction of some stress-responsive genes in response to salinity/drought, but not to cold. Thus, it was suggested that the downstream pathways leading to the cold and salt/drought tolerance are different from each other. It seems likely that at least two distinct pathways commonly use a single CDPK, maintaining the signalling specificity through unknown post-translational regulation mechanisms. These results demonstrate that simple manipulation of CDPK activity has great potential with regard to plant improvement.

A chromatin remodelling complex involved in transcription and DNA processing

Abstract: The packaging of the eukaryotic genome in chromatin presents barriers that restrict the access of enzymes that process DNA. To overcome these barriers, cells possess a number of multi-protein, ATP-dependent chromatin remodelling complexes, each containing an ATPase subunit from the SNF2/SWI2 superfamily. Chromatin remodelling complexes function by increasing nucleosome mobility and are clearly implicated in transcription. Here we have analysed SNF2/SWI2- and ISWI-related proteins to identify remodelling complexes that potentially assist other DNA transactions. We purified a complex from Saccharomyces cerevisiae that contains the Ino80 ATPase. The INO80 complex contains about 12 polypeptides including two proteins related to the bacterial RuvB DNA helicase, which catalyses branch migration of Holliday junctions. The purified complex remodels chromatin, facilitates transcription in vitro and displays 3' to 5' DNA helicase activity. Mutants of ino80 show hypersensitivity to agents that cause DNA damage, in addition to defects in transcription. These results indicate that chromatin remodelling driven by the Ino80 ATPase may be connected to transcription as well as DNA damage repair.

A conserved docking motif in MAP kinases common to substrates, activators and regulators

Abstract: Mitogen-activated protein kinases (MAPKs) are specifically phosphorylated and activated by the MAPK kinases, phosphorylate various targets such as MAPK-activated protein kinases and transcription factors, and are inactivated by specific phosphatases. Recently, docking interactions via the non-catalytic regions of MAPKs have been suggested to be important in regulating these reactions. Here we identify docking sites in MAPKs and in MAPK-interacting enzymes. A docking domain in extracellular-signal-regulated kinase (ERK), a MAPK, serves as a common site for binding to the MAPK kinase MEK1, the MAPK-activated protein kinase MNK1 and the MAPK phosphatase MKP3. Two aspartic acids in this domain are essential for docking, one of which is mutated in the sevenmaker mutant of Drosophila ERK/Rolled. A corresponding domain in the MAPKs p38 and JNK/SAPK also serves as a common docking site for their MEKs, MAPK-activated protein kinases and MKPs. These docking interactions increase the efficiency of the enzymatic reactions. These findings reveal a hitherto unidentified docking motif in MAPKs that is used in common for recognition of their activators, substrates and regulators.