Search

Organisms have seasonal physiological changes in response to day length. Long-day stimulation induces thyroid-stimulating hormone beta subunit (TSHβ) in the pars tuberalis (PT), which mediates photoperiodic reactions like day-length measurement and physiological adaptation. However, the mechanism of TSHβ induction for day-length measurement is largely unknown. To screen candidate upstream molecules of TSHβ, which convey light information to the PT, we generated Luciferase knock-in mice, which quantitatively report the dynamics of TSHβ expression. We cultured brain slices containing the PT region from adult and neonatal mice and measured the bioluminescence activities from each slice over several days. A decrease in the bioluminescence activities was observed after melatonin treatment in adult and neonatal slices. These observations indicate that the experimental system possesses responsiveness of the TSHβ expression to melatonin. Thus, we concluded that our experimental system monitors TSHβ expression dynamics in response to external stimuli.

Most kidney nephron components, including glomeruli and renal tubules, derive from the metanephric mesenchyme. The overall differentiation into each component finishes at birth, but the molecular events linking the perinatal and adult kidneys remain elusive. Dullard was cloned from Xenopus kidneys, and encodes a phosphatase that negatively regulates BMP signalling. Here we report that Dullard deletion in the murine metanephric mesenchyme leads to failure of nephron maintenance after birth, resulting in lethality before adulthood. The nephron components are lost by massive apoptosis within 3 weeks after birth, leading to formation of a large hollow with a thin-layered cortex and medulla. Phosphorylated Smad1/5/8 is upregulated in the mutant nephrons, probably through cell-autonomous inhibitory effects of Dullard on BMP signalling. Importantly, administration of the BMP receptor kinase inhibitor LDN-193189 partially rescued the defects caused by Dullard deletion. Thus, Dullard keeps BMP signalling at an appropriate level, which is required for nephron maintenance in the postnatal period.

Live imaging is a powerful approach to understanding the dynamic processes that occur during development. Periimplantation mouse embryos are transparent, making them suitable for live imaging. In this chapter we describe the culturing and live imaging of mouse embryos, and also introduce the reporter mouse lines for organelle labeling we recently established.

Dullard/Ctdnep1 is a member of the serine/threonine phosphatase family of the C-terminal domain of eukaryotic RNA polymerase II. Embryos lacking Dullard activity fail to form primordial germ cells (PGCs). In the mouse, the formation of PGCs is influenced by BMP4 and WNT3 activity. Although Dullard is reputed to negatively regulate BMP receptor function, in this study we found mutations in Dullard had no detectable effect on BMP4 and p-Smad activity. Furthermore Dullard mutations did not influence the dosage-dependent inductive effect of Bmp4 in PGC formation. However, Dullard may function as a positive regulator of WNT signalling. Combined loss of one copy each of Dullard and Wnt3 had a synergistic effect on the reduction of PGC numbers in the compound heterozygous embryo. In addition, loss of Dullard function was accompanied by down-regulation of WNT/β-catenin signalling activity and a reduction in the level of Dishevelled 2 (Dvl2). Therefore, Dullard may play a role in the fine-tuning of WNT signalling activity by modulating the expression of ligands/antagonists and the availability of Dvl2 protein during specification of the germ cell lineage.

今日ヘルペスウイルス感染症の治療薬として, acyclovirが一般的に使用されている. しかし腎機能低下症例において, ときに重篤で多彩な神経症状を呈するacyclovir脳症を生じるとの報告を散見する.今回透析患者において, acyclovirを経口投与開始後, 多彩な枢神経症状を呈し, acyclovir脳症を疑われ, 血液浄化法の施行により症状の改善を認めた2症例を経験した. 症例1は, 43歳, 男性. 35歳時よりIgA腎症による慢性腎不全にてCAPD療法施行中, 帯状疱疹の診断にてacyclovir 4,000mg/日経口開始. 翌日より多彩な中枢神経症状を認め, 直接血液吸着の施行により症状の改善を認めた. 症例2は, 48歳, 男性. 38歳時より慢性腎不全にて血液透析施行中, 帯状疱疹の診断にてacyclovir 4,000mg/日経口開始. 2日後より多彩な中枢神経症状を認め, 血液濾過透析の施行により症状の改善を認めた. 後日両症例ともacyclovirの血中濃度が46.8μmol/l, 41.1μmol/lと高値を示し, 確定診断がついた. なお症例2においてヘルペス感染症に対し, vidarabineを使用し, 重篤な副作用もなく, 軽快した.透析患者にacyclovirを投与する際は, acyclovir脳症の発現に十分な注意を要し, 不幸にもacyclovir脳症を生じたときは, 早急に適切な血液浄化法を行う必要があり, 血液浄化法は有効な治療法であると思われた. また血液透析患者におけるvidarabineの投与法として, 血液濾過透析を併用し, 通常使用量の半量, 約6mg/kgを血液濾過透析後に, 4時間をかけ持続点滴する方法で, 血中濃度の蓄積も認めず, 明らかな副作用も認めず, 使用することができ, 安全な方法と思われた.

Fucci technology makes possible the distinction between live cells in the G1 and S/G2/M phases by dual-color imaging. This technology relies upon ubiquitylation-mediated proteolysis, and transgenic mice expressing Fucci provide a powerful model system with which to study the coordination of the cell cycle and development. The mice were initially generated using the CAG promoter; lines expressing the G1 and S/G2/M phase probes that emitted orange (mKO2) and green (mAG) fluorescence, respectively, were separately constructed. Owing to cell type-biased strength of the CAG promoter as well as the positional effects of random transgenesis, however, we noticed some variability in Fucci expression levels. To control more reliably the expression of cell cycle probes, we used different genetic approaches to create two types of reporter mouse lines with Fucci2 and Rosa26 transcriptional machinery. Fucci2 is a recently developed Fucci derivative, which emits red (mCherry) and green (mVenus) fluorescence and provides better color contrast than Fucci. A new transgenic line, R26p-Fucci2, utilizes the Rosa26 promoter and harbors the G1 and S/G2/M phase probes in a single transgene to preserve their co-inheritance. In the other R26R-Fucci2 approach, the two probes are incorporated into Rosa26 locus conditionally. The Cre-mediated loxP recombination technique thus allows researchers to design cell-type-specific Fucci2 expression. By performing time-lapse imaging experiments using R26p-Fucci2 and R26-Fucci2 in which R26R-Fucci2 had undergone germline loxP recombination, we demonstrated the great promise of these mouse reporters for studying cell cycle behavior in vivo.

Summary In the process of neuronal wiring, axons derived from the same functional group typically extend together, resulting in fascicle formation. How these axons communicate with one another remains largely unknown. Here, we show that protocadherin-17 (Pcdh17) supports this group extension by recruiting actin polymerization regulators to interaxonal contact sites. Pcdh17 is expressed by a subset of amygdala neurons, and it accumulates at axon-axon boundaries because of homophilic binding. Pcdh17 knockout in mice suppressed the extension of these axons. Ectopically expressed Pcdh17 altered the pattern of axon extension. In in vitro cultures, wild-type growth cones normally migrate along other axons, whereas Pcdh17 null growth cones do not. Pcdh17 recruits the WAVE complex, Lamellipodin, and Ena/VASP to cell-cell contacts, converting these sites into motile structures. We propose that, through these mechanisms, Pcdh17 maintains the migration of growth cones that are in contact with other axons, thereby supporting their collective extension.

Global gene deletion studies in mice and humans have established the pivotal role of runt related transcription factor-2 (Runx2) in both intramembranous and endochondral ossification processes during skeletogenesis. In this study, we for the first time generated mice carrying a conditional Runx2 allele with exon 4, which encodes the Runt domain, flanked by loxP sites. These mice were crossed with α1(I)-collagen-Cre or α1(II)-collagen-Cre transgenic mice to obtain osteoblast-specific or chondrocyte-specific Runx2 deficient mice, respectively. As seen in Runx2(-/-) mice, perinatal lethality was observed in α1(II)-Cre;Runx2(flox/flox) mice, but this was not the case in animals in which α1(I)-collagen-Cre was used to delete Runx2. When using double-staining with Alizarin red for mineralized matrix and Alcian blue for cartilaginous matrix, we observed previously that mineralization was totally absent at embryonic day 15.5 (E15.5) throughout the body in Runx2(-/-) mice, but was found in areas undergoing intramembranous ossification such as skull and clavicles in α1(II)-Cre;Runx2(flox/flox) mice. In newborn α1(II)-Cre;Runx2(flox/flox) mice, mineralization impairment was restricted to skeletal areas undergoing endochondral ossification including long bones and vertebrae. In contrast, no apparent skeletal abnormalities were seen in mutant embryo, newborn, and 3-week-old to 6-week old-mice in which Runx2 had been deleted with the α1(I)-collagen-Cre driver. These results suggest that Runx2 is absolutely required for endochondral ossification during embryonic and postnatal skeletogenesis, but that disrupting its expression in already committed osteoblasts as achieved here with the α1(I)-collagen-Cre driver does not affect overtly intramembranous and endochondral ossification. The Runx2 floxed allele established here is undoubtedly useful for investigating the role of Runx2 in particular cells.

Meiotic pachytene checkpoints monitor the failure of homologous recombination and synapsis to ensure faithful chromosome segregation during gamete formation. To date, the molecular basis of the mammalian pachytene checkpoints has remained largely unknown. We here report that mouse HORMAD1 is required for a meiotic prophase checkpoint that eliminates asynaptic oocytes. Hormad1-deficient mice are infertile and show an extensive failure of homologous pairing and synapsis, consistent with the evolutionarily conserved function of meiotic HORMA domain proteins. Unexpectedly, Hormad1-deficient ovaries contain a normal number of oocytes despite asynapsis and consequently produce aneuploid oocytes, indicating a checkpoint failure. By the analysis of Hormad1/Spo11 double mutants, the Hormad1 deficiency was found to abrogate the massive oocyte loss in the Spo11-deficient ovary. The Hormad1 deficiency also causes the eventual loss of pseudo sex body in the Spo11-deficient ovary and testis. These results suggest the involvement of HORMAD1 in the repressive chromatin domain formation that is proposed to be important in the meiotic prophase checkpoints. We also show the extensive phosphorylation of HORMAD1 in the Spo11-deficient testis and ovary, suggesting an involvement of novel DNA damage-independent phosphorylation signaling in the surveillance mechanism. Our present results provide clues to HORMAD1-dependent checkpoint in response to asynapsis in mammalian meiosis.

The bioactive lysophospholipid mediator sphingosine-1-phosphate (S1P) promotes the egress of newly formed T cells from the thymus and the release of immature B cells from the bone marrow. It has remained unclear, however, where and how S1P is released. Here, we show that in mice, the S1P transporter spinster homolog 2 (Spns2) is responsible for the egress of mature T cells and immature B cells from the thymus and bone marrow, respectively. Global Spns2-KO mice exhibited marked accumulation of mature T cells in thymi and decreased numbers of peripheral T cells in blood and secondary lymphoid organs. Mature recirculating B cells were reduced in frequency in the bone marrow as well as in blood and secondary lymphoid organs. Bone marrow reconstitution studies revealed that Spns2 was not involved in S1P release from blood cells and suggested a role for Spns2 in other cells. Consistent with these data, endothelia-specific deletion of Spns2 resulted in defects of lymphocyte egress similar to those observed in the global Spns2-KO mice. These data suggest that Spns2 functions in ECs to establish the S1P gradient required for T and B cells to egress from their respective primary lymphoid organs. Furthermore, Spns2 could be a therapeutic target for a broad array of inflammatory and autoimmune diseases.

Development of the mammalian telencephalon is precisely organized by a combination of extracellular signaling events derived from signaling centers and transcription factor networks. Using gene expression profiling of the developing mouse dorsal telencephalon, we found that the DM domain transcription factor Dmrta2 (doublesex and mab-3-related transcription factor a2) is involved in the development of the dorsal telencephalon. Consistent with its medial-high/lateral-low expression pattern in the dorsal telencephalon, Dmrta2 null mutants demonstrated a dramatic reduction in medial cortical structures such as the cortical hem and the choroid plexus, and a complete loss of the hippocampus. In this mutant, the dorsal telencephalon also showed a remarkable size reduction, in addition to abnormal cell cycle kinetics and defective patterning. In contrast, a conditional Dmrta2 deletion in the telencephalon, which was accomplished after entry into the neurogenic phase, resulted in only a slight reduction in telencephalon size and normal patterning. We also found that Dmrta2 expression was decreased by a dominant-negative Tcf and was increased by a stabilized β-catenin form. These data suggest that Dmrta2 plays pivotal roles in the early development of the telencephalon via the formation of the cortical hem, a source of Wnts, and also in the maintenance of neural progenitors as a downstream of the Wnt pathway.

症例は61歳男性. 主訴は意識障害. 分裂病にて近医で投薬されていた. 1992年11月4日自宅で倒れているところを発見され当院へ搬送される. 来院時意識レベル300, 体温28.0℃, 血圧90/60mmHg, 脈拍70/minであった. 白血球および血小板数の減少, 高窒素血症, GOT, GPTおよびアミラーゼの上昇を認めた. 偶発性低体温症と診断し, 保存的治療を開始したが, 入院5日目頃より尿量の低下を認め, 血中尿素窒素104mg/dl, 血清クレアチニン7.7mg/dlとなった. この時点で血液透析療法を開始したが, 同時期に緊張性気胸, 肝機能障害も合併し多臓器不全に陥った. しかし,保存的治療と血液透析療法にて増悪を阻止することができた.

Although GPRC5B and GPRC5C are categorized into the G protein-coupled receptor family C, including glutamate receptors, GABA receptors, and taste receptors, their physiological functions remain unknown. Since both receptors are expressed in the brain and evolutionarily conserved from fly to human, it is conceivable that they have significant biological roles particularly in the central nervous system (CNS). We generated GPRC5B- and GPRC5C-deficient mice to examine their roles in the CNS. Both homozygous mice were viable, fertile, and showed no apparent histological abnormalities, though GPRC5B-deficient mice resulted in partial perinatal lethality. We demonstrated that the expressions of GPRC5B and GPRC5C are developmentally regulated and differentially distributed in the brain. GPRC5B-deficient mice exhibited altered spontaneous activity pattern and decreased response to a new environment, while GPRC5C-deficient mice have no apparent behavioral deficits. Thus, GPRC5B has important roles for animal behavior controlled by the CNS. In contrast, GPRC5C does not affect behavior, though it has a high sequence similarity to GPRC5B. These findings suggest that family C, group 5 (GPRC5) receptors in mammals are functionally segregated from their common ancestor.

Collapsin response mediator proteins (CRMPs) are a family of cytosolic phosphoproteins that consist of 5 members (CRMP 1-5). CRMP2 and CRMP4 regulate neurite outgrowth by binding to tubulin heterodimers, resulting in the assembly of microtubules. CRMP2 also mediates the growth cone collapse response to the repulsive guidance molecule semaphorin-3A (Sema3A). However, the role of CRMP4 in Sema3A signaling and its function in the developing mouse brain remain unclear. We generated CRMP4-/- mice in order to study the in vivo function of CRMP4 and identified a phenotype of proximal bifurcation of apical dendrites in the CA1 pyramidal neurons of CRMP4-/- mice. We also observed increased dendritic branching in cultured CRMP4-/- hippocampal neurons as well as in cultured cortical neurons treated with CRMP4 shRNA. Sema3A induces extension and branching of the dendrites of hippocampal neurons; however, these inductions were compromised in the CRMP4-/- hippocampal neurons. These results suggest that CRMP4 suppresses apical dendrite bifurcation of CA1 pyramidal neurons in the mouse hippocampus and that this is partly dependent on Sema3A signaling.

Although various function of chemerin have been suggested, its physiological role remains to be elucidated. Here we show that chemerin-deficient mice are glucose intolerant irrespective of exhibiting reduced macrophage accumulation in adipose tissue. The glucose intolerance was mainly due to increased hepatic glucose production and impaired insulin secretion. Chemerin and its receptor ChemR23 were expressed in β-cell. Studies using isolated islets and perfused pancreas revealed impaired glucose-dependent insulin secretion (GSIS) in chemerin-deficient mice. Conversely, chemerin transgenic mice revealed enhanced GSIS and improved glucose tolerance. Expression of MafA, a pivotal transcriptional factor for β-cell function, was downregulated in chemerin-deficient islets and a chemerin-ablated β-cell line and rescue of MafA expression restored GSIS, indicating that chemerin regulates β-cell function via maintaining MafA expression. These results indicate that chemerin regulates β-cell function and plays an important role in glucose homeostasis in a tissue-dependent manner.

It has recently become clear that signals mediated by members of the TNFR superfamily, including lymphotoxin-β receptor (LTβR), receptor activator for NF-κB (RANK), and CD40, play essential roles in organizing the integrity of medullary thymic epithelial cells (mTECs) required for the establishment of self-tolerance. However, details of the mechanism responsible for the unique and cooperative action of individual and multiple TNFR superfamily members during mTEC differentiation still remain enigmatic. In this study, we show that the LTβR signal upregulates expression of RANK in the thymic stroma, thereby promoting accessibility to the RANK ligand necessary for mTEC differentiation. Cooperation between the LTβR and RANK signals for optimal mTEC differentiation was underscored by the exaggerated defect of thymic organogenesis observed in mice doubly deficient for these signals. In contrast, we observed little cooperation between the LTβR and CD40 signals. Thus, the LTβR signal exhibits a novel and unique function in promoting RANK activity for mTEC organization, indicating a link between thymic organogenesis mediated by multiple cytokine signals and the control of autoimmunity.

microRNA-9-2andmicroRNA-9-3double-mutant mice demonstrate thatmicroRNA-9(miR-9) controls neural progenitor proliferation and differentiation in the developing telencephalon by regulating the expression of multiple transcription factors. As suggested by our previous study, the Foxg1 expression was elevated, and the production of Cajal-Retzius cells and early-born neurons was suppressed in themiR-9-2/3double-mutant pallium. At embryonic day 16.5 (E16.5), however, the Foxg1 expression was no longer elevated. The expression of an AU-rich RNA-binding protein Elavl2 increased at E16.5, Elav2 associated withFoxg13′ untranslated region (UTR), and it countered theFoxg1suppression by miR-9. Later, progenitor proliferation was reduced in themiR-9-2/3double-mutant pallium with the decrease in Nr2e1 and Pax6 expression and the increase in Meis2 expression. The analyses suggest thatmicroRNA-9indirectly inhibitsPax6expression by suppressing Meis2 expression. In contrast, together with Elavl1 and Msi1,microRNA-9targetsNr2e1mRNA 3′ UTR to enhance the expression. Concomitantly, cortical layers were reduced, each cortical projection was malformed, and the tangential migration of interneurons into the pallium was impaired in themiR-9-2/3double mutants. miR-9 also targetsGsh23′ UTR, and Gsh2, as well as Foxg1, expression was elevated in themiR-9-2/3double-mutant subpallium. The subpallium progenitor proliferation was enhanced, and the development of basal ganglia including striatum and globus pallidus was suppressed. Pallial/subpallial boundary shifted dorsally, and the ventral pallium was lost. Corridor was malformed, and thalamocortical and corticofugal axons were misrouted in themiR-9-2/3double mutants.

The biocompatibilities in blood purification therapy are defined as “a concept to stipulate safety of blood purification therapy by an index based on interaction in the body arising from blood purification therapy itself.” The biocompatibilities are associated with not only materials to be used but also many factors such as sterilization method and eluted substance. It is often evaluated based on impacts on cellular pathways and on humoral pathways. Since the biocompatibilities of blood purification therapy in particular hemodialysis are not just a prognostic factor for dialysis patients but a contributory factor for long-term complications, it should be considered with adequate attention. It is important that blood purification therapy should be performed by consistently evaluating not only risks associated with these biocompatibilities but also the other advantages obtained from treatments. In this paper, the biocompatibilities of membrane and adsorption material based on Japanese original which are used for blood purification therapy are described.

Neurons have highly polarized structures that determine what parts of the soma elaborate the axon and dendrites. However, little is known about the mechanisms that establish neuronal polarityin vivo. Cerebellar Purkinje cells extend a single primary dendrite from the soma that ramifies into a highly branched dendritic arbor. We used the zebrafish cerebellum to investigate the mechanisms by which Purkinje cells acquire these characteristics. To examine dendritic morphogenesis in individual Purkinje cells, we marked the cell membrane using a Purkinje cell-specific promoter to drive membrane-targeted fluorescent proteins. We found that zebrafish Purkinje cells initially extend multiple neurites from the soma and subsequently retract all but one, which becomes the primary dendrite. In addition, the Golgi apparatus specifically locates to the root of the primary dendrite, and its localization is already established in immature Purkinje cells that have multiple neurites. Inhibiting secretory trafficking through the Golgi apparatus reduces dendritic growth, suggesting that the Golgi apparatus is involved in the dendritic morphogenesis. We also demonstrated that in a mutant of an atypical protein kinase C (aPKC), Prkci, Purkinje cells retain multiple primary dendrites and show disrupted localization of the Golgi apparatus. Furthermore, a mosaic inhibition of Prkci in Purkinje cells recapitulates the aPKC mutant phenotype. These results suggest that the aPKC cell autonomously controls the Golgi localization and thereby regulates the specification of the primary dendrite of Purkinje cells.

Mdga1, encoding a GPI-anchored immunoglobulin superfamily molecule containing an MAM domain, is expressed by a specific subset of neurons, including layer II/III projection neurons, in the mouse neocortex. To investigate the function of Mdga1 in corticogenesis, we generated Mdga1-deficient mice and backcrossed them to obtain a congenic background. Gross anatomy of the Mdga1-deficient brain at postnatal day (P) 14 showed no obvious phenotype. However, the migration of Mdga1-mutant neurons to the superficial cortical plate was clearly delayed. Most Mdga1-mutant neurons reached the lower portion of the upper cortical layer by embryonic day 18.5 and stayed there through P0. By P7, the location of the mutant cells was the same as wild-type. The location of Cux2-expressing upper-layer neurons in the cortical plate was largely unaffected. These observations indicated that Mdga1 is involved in the migration and positioning of a subset of cortical neurons and suggested that the radial migration of upper-layer neurons might be differentially regulated.

IL-33, a member of the IL-1-related cytokines, is considered to be a proallergic cytokine that is especially involved in Th2-type immune responses. Moreover, like IL-1α, IL-33 has been suggested to act as an “alarmin” that amplifies immune responses during tissue injury. In contrast to IL-1, however, the precise roles of IL-33 in those settings are poorly understood. Using IL-1- and IL-33-deficient mice, we found that IL-1, but not IL-33, played a substantial role in induction of T cell-mediated type IV hypersensitivity such as contact and delayed-type hypersensitivity and autoimmune diseases such as experimental autoimmune encephalomyelitis. Most notably, however, IL-33 was important for innate-type mucosal immunity in the lungs and gut. That is, IL-33 was essential for manifestation of T cell-independent protease allergen-induced airway inflammation as well as OVA-induced allergic topical airway inflammation, without affecting acquisition of antigen-specific memory T cells. IL-33 was significantly involved in the development of dextran-induced colitis accompanied by T cell-independent epithelial cell damage, but not in streptozocin-induced diabetes or Con A-induced hepatitis characterized by T cell-mediated apoptotic tissue destruction. In addition, IL-33-deficient mice showed a substantially diminished LPS-induced systemic inflammatory response. These observations indicate that IL-33 is a crucial amplifier of mucosal and systemic innate, rather than acquired, immune responses.

The neurobiological mechanisms of emotional modulation and the molecular pathophysiology of anxiety disorders are largely unknown. The fibroblast growth factor (FGF) family has been implicated in the regulation of many physiological and pathological processes, which include the control of emotional behaviors. The present study examined mice with a targeted deletion of the fgf-bp3 gene, which encodes a novel FGF-binding protein, in animal models relevant to anxiety. To define the behavioral consequences of FGF-BP3 deficiency, we evaluated fgf-bp3-deficient mice using anxiety-related behavioral paradigms that provide a conflict between the desire to explore an unknown area or objects and the aversion to a brightly lit open space. The fgf-bp3-deficient mice exhibited alterations in time spent in the central area of the open-field arena, were less active in the lit areas of a light/dark transition test, and had a prolonged latency to feed during a novelty-induced hypophagia test. These changes were associated with alterations in light-induced orbitofrontal cortex (OFC) activation in an extracellular signal-regulated kinase (ERK) pathway-dependent manner. These results demonstrate that FGF-BP3 is a potent mediator of anxiety-related behaviors in mice and suggest that distinct pathways regulate emotional behaviors. Therefore, FGF-BP3 plays a critical role in the regulation of emotional states and in the development of anxiety disorders and should be investigated as a therapeutic target for anxiety disease in humans.

Amyloidosis of the external auditory canal is extremely rare. Only 12 cases, including the present case, have been reported, and this is the second reported case attributed to dialysis. This case involved a 56-year-old man who had been on dialysis for 12 years. A tumor was visible in the right external auditory canal and caused pain. The tumor was resected at another hospital, but subsequently recurred with rapid increase in size due to infection. We therefore performed further resection at our hospital. We report our experiences herein, together with a discussion of the literature.

In early June 2004, a 50-year-old female was admitted to the hospital for slight fever, general fatigue, hemoptysis, dyspnea, and renal dysfunction (serum creatinine[Cr] : 6.05 mg/dL). She had been treated with prednisolone (PSL : 10-20 mg/day) for RA. She was diagnosed with Goodpasture syndrome based on a high titer of anti-glomerular basement membrane antibody (87 EU), and pulmonary hemorrhage. The renal and pulmonary impairments were markedly improved by the pulse therapy, plasma exchange and temporary hemodialysis. However, the Cr level remained at 2.0 mg/dL, indicating nephrotic syndrome. Light microscopy with Periodic acid-Shiff(PAS) staining demonstrated global sclerosis in three of ten glomeruli. Five glomeruli showed the formation of cellular, and fibrocellular crescents, and the formation of fibrous crescents. Tubular damage and interstitial fibrosis were severe. Immunofluorescence microscopy disclosed major depositions of IgG in a linear pattern along the glomerular basement membrane(GBM). Electron microscopy revealed foot process effacement (50%)and no electron-dense deposits. Therefore, we diagnosed Goodpasture syndrome associated with minimal change nephrotic syndrome (MCNS). Some reports have dealt with the association of RA and Goodpasture syndrome with D-penicillamine, and of RA and antineutrophil cytoplasmic antibodies (ANCA)-related vasculitis with pulmonary hemorrhage, but none has dealt with cases complicated with RA and Goodpasture syndrome associated with MCNS. Accordingly, whether or not there is a causal relationship between RA and Goodpasture syndrome remains obscure, but since the number of reported cases is small, experience with more cases is necessary to clarify this matter.

Mapping the Neuronal Map In vertebrates, sensory information is topographically represented as a neural map in the brain. How is the neural map formed in the brain? Nearly a half-century ago, Roger Sperry proposed the “chemoaffinity” model, in which the positional cues on the target determine the axonal projection site, thereby establishing the topographic neural map. However, molecular mechanisms of topographic map formation remain controversial. Imai et al. (p. 585 , published online 9 July; see the Perspective by Miyamichi and Luo ) now report that the topographic map is formed by axon-axon interactions before the axons reach the target. In the mouse olfactory system, the topography of the map is determined by the relative expression levels of a guidance receptor, Neuropilin-1, and its repulsive ligand, Semaphorin-3A, expressed in axons. Topographic organization occurs even in the absence of the target, the olfactory bulb. These findings require that Sperry's model, which suggests that only the targets determine the topography of neural maps, needs to be reconsidered.

Physical exercise ameliorates metabolic disorders such as type 2 diabetes mellitus and obesity, but the molecular basis of these effects remains elusive. In the present study, we found that exercise up-regulates heparin-binding epidermal growth factor-like growth factor (HB-EGF) in skeletal muscle. To address the metabolic consequences of such gain of HB-EGF function, we generated mice that overexpress this protein specifically in muscle. The transgenic animals exhibited a higher respiratory quotient than did wild-type mice during indirect calorimetry, indicative of their selective use of carbohydrate rather than fat as an energy substrate. They also showed substantial increases in glucose tolerance, insulin sensitivity, and glucose uptake by skeletal muscle. These changes were accompanied by increased kinase activity of Akt in skeletal muscle and consequent inhibition of Forkhead box O1-dependent expression of the pyruvate dehydrogenase kinase 4 gene. Furthermore, mice with a high level of transgene expression were largely protected from obesity, hepatic steatosis, and insulin resistance, even when maintained on a high-fat diet. Our results suggest that HB-EGF produced by contracting muscle acts as an insulin sensitizer that facilitates peripheral glucose disposal.

We report on a case of factor VIII inhibitor-positive acquired hemophilia in which combined therapy of plasma exchange (PE) and steroids was effective. The patient, a 68-year-old man, had undergone hemodialysis since April 1998, due to chronic renal failure caused by diabetic nephropathy. The hemostasis of blood access sites gradually became difficult after the initiation of dialysis and the prolongation of activated partial thromboplastin time (APTT) (74.5 s), and a decrease in factor VIII (0.02%) and an abnormally high concentration of factor VIII inhibitor (111 U/ml) were found. Under the diagnosis of factor VIII inhibitor-positive acquired hemophilia, 3 consecutive PE were performed, followed by a large dose administration of gamma globulin. However, the effect of this therapy disappeared within 20 days. Then the PE therapy was performed again accompanied by pulse methylprednisolone therapy. After that, factor VIII inhibitor was suppressed and the patient's hemostatic defect continued to improve even after the reduction of the steroid dose. These results suggest that PE is very effective in treating factor VIII inhibitor-positive acquired hemophilia.

Hepcidin has recently been recognized as a hormone essential to the negative regulation of iron. Synthesis of hepcidin is increased by iron overload or inflammation, and decreased by iron deficiency, anemia and erythropoietin. Dialysis patients frequently suffer the effects of both hepcidin increasing and decreasing factors. In this study, we investigated, for the first time, pro-hepcidin in dialysis patients while minimizing or manipulating these factors. We measured the serum pro-hepcidin in 23 hemodialysis patients without inflammation (the HD group) and 10 age-matched healthy volunteers. Those patients in the HD group were assigned to an iron-deficiency group (the ID group) or a non-iron deficiency subgroup (non-ID group). The HD group was followed up for two months. Iron therapy was performed in the ID group during the follow-up period. At the end of this time we evaluated the influence of iron therapy. Pro-hepcidin was similar in the HD groups and the healthy controls (295.1 [241.9, 413.7] vs. 301.7 [280.5, 383.5] ng/mL; not significant) despite the presence of hepcidin-decreasing factors. Pro-hepcidin in the ID group was significantly lower than in the non-ID group (262.6 [233.1, 295.1] vs. 359.2 [282.3, 446.5] ng/mL; P

We report a case of tako-tsubo cardiomyopathy associated with brain metastasis of seminoma. This disease is characterized by transient cardiac wall motion abnormalities, electrocardiographic changes and minimal myocardial enzymatic release. During the hospital days, acute congestive heart failure suddenly appeared. The electrocardiogram showed a ST segment elevation in V1-3 and a giant negative T wave in I, aVL, aVF and V3-6. The echocardiogram revealed left ventricular dysfunction with severe hypokinesis to akinesis of anterior and apical wall regions, and hyperkinesis of the basal wall despite the lack of cardiac enzymatic abnormalities. With proper treatment, the patient's overall condition, wall motion and electrocardiographic abnormalities greatly improved.

Gene targeting of mouse S ushi- i chi-related r etrotransposon h omologue 11 / Z inc finger CCHC domain-containing 16 (Sirh11/Zcchc16) causes abnormal behaviors related to cognition, including attention, impulsivity and working memory. Sirh11/Zcchc16 encodes a CCHC type of zinc-finger protein that exhibits high homology to an LTR retrotransposon Gag protein. Upon microdialysis analysis of the prefrontal cortex region, the recovery rate of noradrenaline (NA) was reduced compared with dopamine (DA) after perfusion of high potassium-containing artificial cerebrospinal fluid in knockout (KO) mice. These data indicate that Sirh11/Zcchc16 is involved in cognitive function in the brain, possibly via the noradrenergic system, in the contemporary mouse developmental systems. Interestingly, it is highly conserved in three out of the four major groups of the eutherians, euarchontoglires, laurasiatheria and afrotheria, but is heavily mutated in xenarthran species such as the sloth and armadillo, suggesting that it has contributed to brain evolution in the three major eutherian lineages, including humans and mice. Sirh11/Zcchc16 is the first SIRH gene to be involved in brain function, instead of just the placenta, as seen in the case of Peg10, Peg11/Rtl1 and Sirh7/Ldoc1., Author Summary Retrotransposon-derived DNA sequences occupy approximately 40% of the mammalian genome, compared with only 1.5% of protein coding genes. They have been commonly considered “junk DNA” and even potentially harmful for host organisms. However, a series of knockout (KO) mouse analyses demonstrated that at least some of the LTR retrotransposon- and retrovirus-derived sequences play essential roles in the current mammalian developmental system as endogenous genes, such as Peg10, Peg11/Rtl1, Sirh7/Ldoc1, SYNCYTINs and FEMATRIN-1, which are active in multiple aspects of placental function. Here we demonstrate that another LTR retrotransposon-derived gene, Sirh11/Zcchc16, plays an important role in cognitive function in the brain. Sirh11/Zcchc16 KO mice exhibit abnormal behaviors related to cognition, including attention, impulsivity and working memory, possibly due to the locus coeruleus-noradrenaline (LC-NA) system, suggesting that human SIRH11/ZCCHC16 may be involved in X-linked intellectual disability and/or attention-deficit/hyperactivity disorder. Comparative genome analysis demonstrates that SIRH11/ZCCHC16 was acquired in a common eutherian ancestor, suggesting that it contributed to eutherian brain evolution because it confers a critically important advantage in the competition that occurs in daily life. This study provides further insight into the impact of LTR retrotransposon-derived genes on mammalian evolution.

In an effort to identify Otx2 targets in mouse anterior neuroectoderm we identified a gene, mShisa, which is homologous to xShisa1 that we previously reported as a head inducer in Xenopus. mShisa encodes an antagonist against both Wnt and Fgf signalings; it inhibits these signalings cell-autonomously as xShisa1 does. The mShisa expression is lost or greatly reduced in Otx2 mutant visceral endoderm, anterior mesendoderm and anterior neuroectoderm. However, mShisa mutants exhibited no defects in head development. Shisa is composed of five subfamilies, but normal head development in mShisa mutants is unlikely to be explained in terms of the compensation of mShisa deficiency by its paralogues or by known Wnt antagonists in anterior visceral endoderm and/or anterior mesendoderm.

In chronic kidney disease (CKD), several abnormalities in bone and mineral metabolism develop in the majority of patients. The parathyroid plays a very important role in regulating bone and mineral metabolism; thus, control of parathyroid function is one of the main targets of the management of CKD-mineral and bone disorder (CKD-MBD). In the development of secondary hyperparathyroidism, it has recently been suggested that fibroblast growth factor 23 (FGF23) plays a crucial role, both as a phosphaturic factor and as a suppressor of active vitamin D (1,25D) production in the kidney. FGF23 is originally secreted to prevent hyperphosphatemia in CKD, but this occurs at the expense of low 1,25D and hyperparathyroidism (“trade-off” hypothesis revisited). Furthermore, recent data suggest that FGF23 could be another useful marker for the prognosis of hyperparathyroidism, because a high serum level may reflect the cumulative dose of vitamin D analogues previously administered. We have also demonstrated that severe hyperparathyroidism was associated with the production and secretion of a new form of parathyroid hormone (PTH) molecule, which can be detected by third-generation assays for PTH, but not by the second-generation assays. For the regression of already established nodular hyperplasia, the more advanced type of parathyroid hyperplasia, it is certainly necessary, in the near future, to develop new agents that specifically induce apoptosis in parathyroid cells. Until such agents are developed, prevention and early recognition of nodular hyperplasia is mandatory for the effective and safe management of hyperparathyroidism in CKD.

A 71-year-old woman was admitted to the Wakayama Medical University Hospital with dizziness and loss of body balance. She had started hemodialysis at the age of 70. During the 33 days before admission, she received oral tizanidine hydrochloride at 3 mg/day for leg cramps. An admission electrocardiogram (ECG) demonstrated sinus bradycardia of 47 bpm. A 24-h ECG showed a total number of heartbeats of 68,779 and an average heart rate of 48 bpm. The maximum RR interval was 3720 msec. The electrophysiology test demonstrated slight sinus node dysfunction. There was no major organic heart disease. We suspected that tizanidine was the cause of bradycardia and stopped administration of this drug. After discontinuation symptoms gradually disappeared. The serum concentration of the tizanidine showed a higher trough of 1.78 ng/mL. In conclusion, because there was a disappearance of symptoms and a lightening of bradycardia due to the discontinuation of this medication, tizanidine was strongly suspected as the cause of severe bradycardia.

Plasma exchange (PE) is often performed in combination with hemodialysis (HD) or hemodiafiltration. However, most methods were developed for the treatment of renal failure, so various problems may arise during treatment of liver failure (LF). In this study, we investigated the impact of PE alone and in combination with HD, and we assessed the complications of using PE + HD for the treatment of LF. After the exchange of 1 L of fresh frozen plasma (FFP), we measured serum electrolytes, HCO(3) (-), citrate, and acetate at 3 points in the circuit: A) the plasma separator inflow; B) after mixing of FFP/the dialyzer inflow; and C) the dialyzer outflow. Serum levels of human hepatocyte growth factor (HGF), acetate, and citrate were also measured before and after PE + HD. The levels of K(+), Ca(++), HCO(3) (-), and acetate were significantly decreased, and citrate was increased, between A and B. K(+) and citrate were decreased, while Ca(++), HCO(3) (-), and acetate showed an increase between B and C. Comparison of A with C revealed insufficient correction of the Ca(++) and citrate levels by HD. After PE + HD, serum levels of acetate and citrate were increased, while HGF was decreased. We concluded that i) when PE is performed, HD is also necessary for correction, but achieves insufficient correction of Ca(++) and citrate, ii) PE is non-selective and not only removes toxins but also beneficial substances such as HGF, iii) accumulation of acetate occurred, even with bicarbonate dialysate, since it also contains acetate for acidification.

Previous studies reported that dialysis with albumin dialysate (AD) was effective in removing albumin-binding toxins (ABT), and the Molecular Adsorption Recycling System (MARS) and Continuous Albumin Purification System (CAPS) have been developed. These blood purification therapies were categorized into the concept of extracorporeal albumin dialysis (ECAD). ECAD is defined as extracorporeal therapies using AD for the removal of not only water-soluble but also ABT. It was reported that symmetric as well as asymmetric membrane dialyzers had the effect of the removal of bilirubin by AD. The larger pore size membrane can remove more bilirubin. In the greater albumin concentration in AD, the removal capacity for bilirubin by AD increased. Bilirubin in AD could be removed by a charcoal and a bilirubin adsorption column, and its concentration in AD remained approximately constant. In clinical performance of CAPS, cellulose triacetate membrane, 5% AD, bilirubin adsorber columns, and charcoal adsorber columns were used. This system was applied continuously for 24 h for treatment. CAPS could control not only renal but also liver function during the 24 h, without any adverse effect. MARS removes many toxic substances including ABT, and has beneficial effect on brain, liver, renal, and cardiovascular functions, and improvement of 30-day survival were reported. ECAD may become a possible therapeutic tool in patients with the disease state of ABT accumulation as an artificial kidney and liver. However, several attempts such as the application of recombinant human albumin and acetate free dialysate, should be required.

Transcription of the CYP24 (25-hydroxyvitamin D(3)-24-hydroxylase) gene is known to be induced by 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). We studied the induction kinetics in detail in human skin-derived fibroblasts. While the basal transcription of this gene was very low, addition of 1,25(OH)2D3 increased the mRNA level by 50-fold within 1h. The induction reached as high as 20000-fold after 12h. DNA microarray analysis also indicated that the induction ratio of the CYP24 gene is exceptionally high among 3800 human genes examined. The increase of mRNA was caused by stimulation of the transcription, but not by stabilization of mRNA. 24(R),25-dihydroxyvitamin D3 (24,25(OH)2D3), a compound metabolically related to 1,25(OH)2D3, also stimulated the CYP24 gene transcription, though at much higher concentrations. However, this stimulation was significantly augmented by synergistic actions of 24,25(OH)2D3 and 1,25(OH)2D3, suggesting that 24,25(OH)2D3 or its metabolites might be playing some roles in the regulation of CYP24 gene transcription.

A 55-year-old man was admitted to our hospital because of myelopathy. He had a history of chronic renal failure due to polycystic kidney disease at the age of 39, being treated by hemodialysis for 9 years with several blood transfusions for the treatment of renal anemia. After cadaver renal transplantation at the age of 48, he discontinued hemodialysis. At 50 years of age, he had pulmonary tuberculosis and tuberculous arthritis of the left elbow joint. He has experienced difficulty in walking since he was 48 years old, with mild dysuria. Gait disturbance gradually aggravated after that, and urinary retention was observed. When he was 55 years old, being human T-cell lymphotropic virus type-1 (HTLV-1)-positive in the serum and cerebrospinal fluid, he was diagnosed as having HTLV-1-associated myelopathy (HAM). As active steroid therapy was unapplicable because of the history of pulmonary tuberculosis and immunosuppression for transplanted kidney, a series of plasma exchanges (PE) was performed with fresh frozen plasma as a replacement fluid. After PE, dyskinesia of the left leg and dysuria subjectively and objectively improved. These results suggest that PE seems to be one of the therapeutic tools for the treatment of HAM.

Bullous pemphigoid (BP) is an autoimmune disease caused by an antidermal basal lamina antibody. In recent years double filtration plasmapheresis (DFPP) has been reported to be an effective therapy for BP. We experienced 3 cases of BP treated by DFPP. DFPP resulted in an improvement in clinical symptoms and remission allowing a decrease in the required dose of corticosteroid. DFPP was found to be an effective treatment for all 3 patients without noticeable adverse events resulting from DFPP. From these results it is concluded that DFPP is worth considering as an option as treatment for BP patients who were unresponsive to conventional steroid therapy, those in whom corticosteroids should be reduced or discontinued because of complications such as diabetes mellitus and/or osteoporosis.

We experienced a case of thrombotic thrombocytopenic purpura (TTP) finally relieved after 74 sessions of plasma exchange (PE). The patient was a 56-year-old male. In August 1999, he was examined in emergency because of brown urine and a lowered level of consciousness. As TTP was suspected according to the laboratory findings of abnormally high lactate dehydrogenase and total bilirubin, decreased platelet counts, and numerous fragmented erythrocytes, he was admitted to the ICU of our hospital. Immediately after admission, PE was started consecutively. Upon concomitant use of antiplatelet drugs and prostacyclin, the level of platelet counts recovered to 100,000/microl once, but decreased again. Thus, in addition to the PE, prednisolone and vincristine were administrated, which elevated the level of platelet counts to 200,000 to 300,000/microl. Since the erythrocyte fragmentation was noted frequently, PE was continued twice a week. From the 60th day of admission onward, however, his body temperature rose above 40 degrees C with a rapid increase of C-reactive protein. A blood culture detected methicillin-resistant Staphylococcus aureus (MRSA) which derived from a left lung abscess. During the course of anti-MRSA treatment, he presented acute renal failure and acute hepatic dysfunction, but survived because of the combined therapy. He was discharged on the 180th day of admission. These results suggest that a combined therapy of steroid and vincristine is effective to treat TTP refractory to PE, but careful attention should be paid to the complications caused by immunosuppression.