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PDF file - 236K, 4NQO-water consumptions

PDF file - 48K, Hematological indices of wild-type and Nrf2-KO mice treated with 4NQO

PDF file - 31K, Sequences of primers and probes for RT-PCR

PDF file - 725K, The effects of 4NQO treatment on the body weights of wild-type, Keap1-KD and Nrf2-KO mice

PDF file - 78K

PDF file - 720K, A model for the role of the Keap1-Nrf2 system in upper aerodigestive tract carcinogenesis induced by 4NQO

PDF file - 1357K, Representative hematoxylin-eosin-stained sections of esophageal tumors after experiments with 4NQO

The background of this study is to develop a method to improve the fluidity of high viscosity oil left in sunken ships. We focused on emulsification to control the rheological properties of the oil. As a first step towards the goal, experiments and modeling were conducted to understand the viscosity properties of oil-water emulsions. In the experiments, oil-water emulsions were prepared using several surfactants to measure viscosity properties. The results showed a continuous change in viscosity with increasing water content as well as a discontinuous change in viscosity due to phase inversion that occurs at a certain water content. In the theoretical modelling, a new layer-stacking model was developed and applied to the experimental results. This model, though extremely simplifying the structure of the emulsion, was found to be able to well explain the continuous and discontinuous viscosity characteristics of the emulsion.

While the vascular healing process after drug-eluting stent implantation is not fully elucidated, it is generally accepted that macrophages play an important role in inflammation. It is also known that macrophages involved in the pathogenesis of atherosclerosis may stem from several origins, that is, monocyte-derived macrophages versus resident macrophages. However, little is known about the role of human macrophages on reperfusion of culprit coronary arteries in patients with atherosclerotic disease who have sustained acute coronary syndrome. In this present case report, we describe the presence of cluster of differentiation (CD) 163-positive macrophages in close proximity to the stent struts at the luminal site 72 hours after drug-eluting stent implantation in the culprit coronary lesion for ST elevation myocardial infarction by postmortem examination.

In the robust control of systems with dynamic uncertainty, there are mainly two methodologies: one based on the gain bound of uncertainty and the other based on the phase bound. The well-known small-gain approach belongs to the former and is able to conduct nonconservative robust performance design by using $\mu$ -synthesis. Meanwhile, the study on the latter case is still under way, and only a positive-real method was proposed by the authors recently to handle the robust performance synthesis. This paper aims at improving the positive-real method. The key idea is to model a passive uncertainty not only by its phase bound but also utilizing its largest gain. This is achieved by applying the notion of output strict passivity well known in nonlinear control. Furthermore, the negative-imaginary uncertainty can be treated in this framework more naturally. A case study on a vibration system validates the advantage of the present method.

The gyroscopic g factor, g ′ , of FeCo nanogranules embedded in a matrix of MgF 2 (FeCo-MgF 2 ) was determined by measuring the magnetic-field generation from a rotating sample due to the Barnett effect. The g ′ value of the FeCo-MgF 2 is estimated to be 1.76 ± 0.11. The orbital contribution to the magnetic moment in the FeCo nanogranules was found to be quite large compared with that in bulk FeCo, being consistent with a density-functional-theory calculation that shows that the orbital magnetic moment may increase at the FeCo/MgF 2 interfaces. The result suggests that the orbital magnetic moment is enhanced by symmetry breaking at the surface of the FeCo nanogranules.

We report direct observation of gyromagnetic reversal, which is the sign change of gyromagnetic ratio in a ferrimagnet Ho$_3$Fe$_5$O$_{12}$, by using the Barnett effect measurement technique at low temperatures. The Barnett effect is a phenomenon in which magnetization is induced by mechanical rotation through the coupling between rotation and total angular momentum $J$ of electrons. The magnetization of Ho$_3$Fe$_5$O$_{12}$ induced by mechanical rotation disappears at 135~K and 240~K. The temperatures correspond to the magnetization compensation temperature $T_{\rm M}$ and the angular momentum compensation temperature $T_{\rm A}$, respectively. Between $T_{\rm M}$ and $T_{\rm A}$, the magnetization flips over to be parallel against the angular momentum due to the sign change of gyromagnetic ratio. This study provides an unprecedented technique to explore the gyromagnetic properties., 4pages,4figures

Magnetohydrodynamic generators use magnetic fields to convert the kinetic energy of conducting fluids into electricity. Fluid motion is now shown to generate spin currents, which can induce electric voltages without applying magnetic fields. Magnetohydrodynamic generation1,2,3,4 is the conversion of fluid kinetic energy into electricity. Such conversion, which has been applied to various types of electric power generation, is driven by the Lorentz force acting on charged particles and thus a magnetic field is necessary3,4. On the other hand, recent studies of spintronics5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23 have revealed the similarity between the function of a magnetic field and that of spin–orbit interactions in condensed matter. This suggests the existence of an undiscovered route to realize the conversion of fluid dynamics into electricity without using magnetic fields. Here we show electric voltage generation from fluid dynamics free from magnetic fields; we excited liquid-metal flows in a narrow channel and observed longitudinal voltage generation in the liquid. This voltage has nothing to do with electrification or thermoelectric effects, but turned out to follow a universal scaling rule based on a spin-mediated scenario. The result shows that the observed voltage is caused by spin-current6 generation from a fluid motion: spin hydrodynamic generation. The observed phenomenon allows us to make mechanical spin-current and electric generators, opening a door to fluid spintronics.

Objective Fcγ receptor type IIb (FcγRIIb) is a major negative regulator of B cells, and the lack of FcγRIIb expression has been reported to induce systemic lupus erythematosus (SLE) in mice of the C57BL/6 (B6) genetic background. The 129 strain–derived Sle16 locus on the telomeric region of chromosome 1 including polymorphic Fcgr2b confers the predisposition to systemic autoimmunity when present on the B6 background. We undertook this study to examine the effect of the Sle16 locus on autoimmune disease in FcγRIIb-deficient B6 mice. Methods We established 2 lines of FcγRIIb-deficient B6 congenic mouse strains (KO1 and KO2) by selective backcrossing of the originally constructed FcγRIIb-deficient mice on a hybrid (129 × B6) background into a B6 background. Although both lack FcγRIIb expression, the KO1 and KO2 strains carry different lengths of the 129 strain–derived telomeric chromosome 1 segment flanked to the null-mutated Fcgr2b gene; the KO1 strain carries a 129 strain–derived ∼6.3-Mb interval distal from the null-mutated Fcgr2b gene within the Sle16 locus, while this interval in the KO2 strain is of B6 origin. Results Unexpectedly, both strains failed to develop SLE; instead, the KO1 strain, but not the KO2 strain, spontaneously developed severe rheumatoid arthritis (RA) with an incidence reaching >90% at age 12 months. Conclusion The current study shows evidence that the epistatic interaction between the Fcgr2b-null mutation and a polymorphic gene(s) in the 129 strain–derived interval located in the distal Sle16 locus contributes to RA susceptibility in a new mouse model with the B6 genetic background, although the participation of other genetic polymorphisms cannot be totally excluded.

The chicken Ig-β locus is organized by three cell-type-specific genes and two ubiquitously expressed genes. B-cell-specific DNase I hypersensitive sites (DHS) in that locus, including three present inside the flanking gene, were grouped into six regions and deleted. The deletions decreased Ig-β mRNA content to

Recent studies indicate that several transcription units assemble to form a 'transcription factory' where active transcription occurs in the nuclei. Previously, we generated chicken B-lymphocyte-derived DT40 cells lacking six transcriptional regulatory regions scattered in and around the immunoglobulin (Ig)-β gene. The deletions caused a complete shut down of transcription and epigenetic regulation of the Ig-β gene, demonstrating that the scattered regulatory regions cooperated in the transcriptional and epigenetic regulation of the gene. However, the in vivo 3-dimensional spatial relationships between the Ig-β promoter and these six regulatory regions were not investigated. In this study, we used chromosome conformation capture (3C) technology and demonstrated that the Ig-β promoter physically interacted with the scattered regulatory regions. We found that the Ig-β promoter also interacted with two downstream promoters of ubiquitously expressed genes, rad motif 1 (RDM1) and Plekhm1, to form a transcription factory, but not with three ubiquitously expressed genes, BAF60b, p45/SUG, and RRMJ3, located upstream of the Ig-β gene. In this factory, the chromatin from the three promoters and the scattered regulatory regions of the Ig-β gene formed a complex structure with many chromatin loops.

Problem statement: Mast cells develop from bone marrow-derived progenitor cells and are distributed in the skin or mucosa where they play proinflammatory roles in the first line of defense. Since some tumors in humans and experimental animals exhibited infiltration of increased mast cells, we investigated the contribution of mast cells to the override of tumor rejection. Approach: MRL/N-1 cells are malignant fibrous histiocytoma-like cells established from the spleen of a Fas ligand (FasL)-deficient MRL/Mp-FasLgld/gld (MRL/gld) mouse and are implantable in Fas-deficient MRL/Mp-Faslpr/lpr (MRL/lpr) mice. MRL/N-1 cells were implanted in MRL/gld, MRL/lpr and MRL/+mice after antibody treatments or with mast cells or macrophages and the tumor growth was observed. Results: MRL/N-1 cells were rejected by Fas-intact syngeneic MRL/+ mice in CD8+ T cell-mediated manner. This rejection was inhibited by the co-implanted mast cells. MRL/N-1 cells transfected with FasL were rejected by MRL/+ and MRL/gld mice. Conclusion: Mast cells abrogate the rejection of MRL/N-1 tumor cells and that this tumor rejection is mediated by CD8+ T cells and dependent on host Fas-FasL axis.

The cysteinyl leukotrienes (LTs), LTC(4), LTD(4), and LTE(4), are potent inflammatory mediators and are involved in allergic reactions, such as bronchoconstriction, eosinophilic inflammation, and allergic cell proliferation. The present study aimed to elucidate the role of constitutively produced cysteinyl LTs in mast cell activation. We used a newly developed quantification method based on mass spectrometry to detect cysteinyl LTs in the cultured medium of mouse bone marrow-derived mast cells (BMMCs), which were obtained by interleukin (IL)-3-conditioned culture of mouse bone marrow. BMMCs were stimulated with immunoglobulin (Ig) E and antigen (IgE/Ag) or lipopolysaccharide for 1 or 24 h. This new quantification method revealed that unstimulated BMMCs produced and secreted LTB4 and LTE4 after 24 h of incubation. The treatment of unstimulated BMMCs for 2 h with montelukast, an antagonist of a cysteinyl LT receptor, CysLT1, resulted in the suppression of a downstream signaling event of this receptor, i.e., the decrease in phosphorylation of extracellular responsive kinases. Thus, cysteinyl LTs constitutively simulate BMMCs through the CysLT1 receptor in an autocrine manner. Treatment of BMMCs for 3 weeks with montelukast, which caused long-term inhibition of the autocrine cyteinyl LT-derived signal, significantly attenuated the IgE/Ag-dependent degranulation, as judged by the decrease in the release of beta-hexosaminidase, an enzyme contained in the granules, whereas the production of cytokines, such as IL-6 and tumor necrosis factor-alpha, were largely unaffected. In conclusion, an autocrine signal derived from constitutively produced cysteinyl LTs predisposes mast cells to the degranulation upon allergic stimulation.

Pathogens are sensed by Toll-like receptors (TLRs) expressed in leukocytes in the innate immune system. However, excess stimulation of TLR pathways is supposed to be connected with provocation of autoimmunity. We show that paired immunoglobulin (Ig)-like receptor B (PIR-B), an immunoreceptor tyrosine-based inhibitory motif–harboring receptor for major histocompatibility class I molecules, on relatively primitive B cells, B-1 cells, suppresses TLR9 signaling via Bruton's tyrosine kinase (Btk) dephosphorylation, which leads to attenuated activation of nuclear factor κB p65RelA but not p38 or Erk, and blocks the production of natural IgM antibodies, including anti-IgG Fc autoantibodies, particularly rheumatoid factor. The autoantibody production in PIR-B–deficient (Pirb−/−) mice was further augmented in combination with the Faslpr mutation, which might be linked to the development of autoimmune glomerulonephritis. These results show the critical link between TLR9-mediated sensing and a simultaneously evoked, PIR-B–mediated inhibitory circuit with a Btk intersection in B-1 cells, and suggest a novel way toward preventing pathogenic natural autoantibody production.

The endosomal sorting complex required for transport (ESCRT) proteins form multimolecular complexes that control multivesicular body formation, endosomal sorting, and transport ubiquitinated membrane proteins (including cell-surface receptors) to the endosomes for degradation. There is accumulating evidence that endosomal dysfunction is linked to neural cell degeneration in vitro, but little is known about the relationship between neural disorders and ESCRT proteins in vivo. Here we specifically deleted the hrs gene, ESCRT-0, in the neurons of mice by crossing loxP-flanked hrs mice with transgenic mice expressing the synapsin-I Cre protein (SynI-cre). Histological analyses revealed that both apoptosis and a loss of hippocampal CA3 pyramidal neurons occurred in the hrs(flox/flox);SynI-cre mice. Notably, the hrs(flox/flox);SynI-cre mice accumulated ubiquitinated proteins, such as glutamate receptors and an autophagy-regulating protein, p62. These molecules are particularly prominent in the hippocampal CA3 neurons and cerebral cortex with advancing age. Accordingly, we found that both locomotor activity and learning ability were severely reduced in the hrs(flox/flox);SynI-cre mice. These data suggest that Hrs plays an important role in neural cell survival in vivo and provide an animal model for neurodegenerative diseases that are known to be commonly affected by the generation of proteinaceous aggregates.

Based on the hypothesis that the complex pathological and immunological manifestations of rheumatoid arthritis (RA) and the related diseases are under the control of multiple gene loci with allelic polymorphism, a recombinant congenic mouse strain was prepared between an MRL/Mp-lpr/lpr (MRL/lpr) strain, which develops arthritis resembling RA, and a non-arthritic strain C3H/HeJ-lpr/lpr (C3H/lpr). In MRL/lpr x (MRL/lpr x C3H/lpr) F1 mice, the mice developing severe arthritis were selected based on joint swelling to further continue intercrosses, and then an McH-lpr/lpr-RA1 (McH/lpr-RA1) strain was established and its histopathological phenotypes of joints and autoimmune traits were analyzed. Arthritis in McH/lpr-RA1 mice developed at a higher incidence by 20 weeks of age, compared with that in the MRL/lpr mice, who had severe synovitis (ankle, 60.3%; knee, 65.1%), and also fibrous and fibrocartilaginous lesions of articular ligamenta resembling enthesopathy (ankle, 79.4%; knee, 38.1%), resulting in ankylosis. The lymphoproliferative disorder was less, and serum levels of IgG and IgG autoantibodies including anti-dsDNA and rheumatoid factor were lower than those of both MRL/lpr and C3H/lpr strains. McH/lpr-RA1 mice may provide a new insight into the study of RA regarding the common genomic spectrum of seronegative RA and enthesopathy.

Aging readily affects immune system under the influence of environmental and/or intrinsic factors while accelerating the development of various immune disorders including autoimmune diseases. Little is known about molecular and cellular mechanisms connecting between immune senescence and development of autoimmune diseases. Here, we first show strain-specific and aging-sensitive onset of B-cell abnormality in a lupus-prone MRL/Mp.Fas(lpr) (MRL/lpr) strain of mice. This abnormality was characterized by the regression of B lymphopoiesis in the bone marrow of this strain. We next examined the association between the B-cell regression and onset of autoimmune diseases in aged (MRL/lpr x C3H/He.Fas(lpr)) F2 mice, in which pathologic phenotypes, such as glomerulonephritis, vasculitis, sialoadenitis and arthritis, variously developed. We also searched whole genome to identify genetic loci linked to the B-cell regression by using the same F2 mice. The B-cell regression manifested in the spleen of F2 mice was retrospectively evaluated by reverse transcriptase-based PCR quantification. The results demonstrated that the onset of autoimmune diseases in the F2 mice was not associated with the aging-sensitive B-cell regression. The genetic study identified a significant locus responsible for the B-cell regression in the vicinity of D5Mit233 (29 cM). This is first evidence for the presence of a genetic locus that affects B lymphopoiesis in an aging-sensitive manner.

Sex-related differences (SrD) are a general characteristic of human autoimmune diseases. There is an increasing body of evidence that suggests a link between sex-related hormones and autoimmune onsets. Here, through a genetic approach using a lupus mouse model, we attempted to show the involvement of genetic factors in the development of SrD in autoimmune diseases. Using MRL/lpr x (MRL/lpr x C57BL/6.Fas(lpr))F1 (MBN2) mice, the whole genome was searched to identify linkage loci to autoimmune phenotypes inherited from a lupus MRL/Mp.Fas(lpr) (MRL/lpr) strain of mice, which exhibits glomerulonephritis, splenomegaly and antinuclear autoantibody. The genome-wide association study confirmed four linkage loci on chromosomes 4, 7, 13, and 17. Furthermore, differential analyses performed using male and female groups of MBN2 mice revealed that two loci located on chromosomes 4 (41-72 cM, MRL/lpr allele) and 7 (4-21 cM, B6/lpr allele) were male specific and suppressed autoimmune phenotypes. Notably, the sum effect of the two loci adequately explained a range of SrD developed in the MBN2 mice. Our present findings suggest the presence of a male-predominant mechanism underlying the development of SrD in autoimmunity, depending on the effects of autosomal loci under an undefined male-specific condition.

Background: The incidence and characteristics of spontaneous ankylosis in the ankle of specific F 1 mice descended from two Fas -deficient strains were reported. Here the coincidence of synovial proliferation and ankylosis in the descendent F 2 mice is reported. Aim: To clarify whether the two distinct manifestations are genetically different. Methods: An arthropathic group of mice (MCF 2 ) were bred by intercrossing MRL/Mp. Fas lpr - sap – /sap – and C3H/He. Fas lpr mice. All mice were killed by bleeding under anaesthesia when they were 6 months old. Pathological grades for synovial proliferation were determined by microscopical examination. To obtain a linkage locus, the whole genome of male MCF 2 mice was scanned by using 73 microsatellite markers. Results: Synovial proliferation was equally observed in male and female MCF 2 mice. No correlation was observed between the grades of synovial proliferation and the ankylosis occurring in the MCF 2 mice. A suggestive susceptibility locus was shown in the middle of chromosome 11. This locus was an MRL allele with a recessive inheritance mode. Conclusion: The pathogenic mechanisms of synovial proliferation and ankylosis are genetically different. The present locus is overlapped with some loci associated with rheumatoid arthritis and with others associated with experimental arthritides.

Objective: To identify the genetic loci regulating the incidence and severity of renal autoimmune vasculitis developed in murine lupus. Methods: Vasculitis of renal arteries was histopathologically evaluated in MRL/Mp- Fas lpr (MRL/lpr), C57BL/6- Fas lpr (B6/lpr), (MRL/lpr×B6/lpr) F 1 , and MRL/lpr×(MRL/lpr×B6/lpr) F 1 backcross mice. Using genomic DNA samples of the backcross mice, genome-wide scans, association studies, and linkage analyses were carried out based on genotypes of polymorphic microsatellite markers. Correlations of vasculitis grade and levels of various autoantibodies were also evaluated. Results: Two recessive susceptibility loci of the MRL allele were identified on chromosomes 4 and 1, which had previously been defined as the autoimmune related loci termed Arvm1 and Sle-1/Nba2 , respectively. The former was epistatic to the latter in a female specific manner. The titre of antinuclear autoantibody (ANA) in IgG class, but not ANA in IgM class or anti-dsDNA in either IgG or IgM class, correlated significantly with vasculitis grade. Conclusions: The present loci have been reported in previous studies using a different set of murine strains, suggesting that they are of importance in the development of autoimmune vasculitis in murine models. The concomitance of autoimmune vasculitis and IgG ANA suggests a shared genetic factor regulating these traits.

Objective Crescent formation in the renal glomerulus is a typical manifestation of progressive glomerulopathy associated with fatal renal failure; therefore, its prevention is of clinical importance. Little is known about the pathogenic mechanism for crescent formation. This study was undertaken in an attempt to identify the events that are critical for crescent formation in immune complex crescentic glomerulonephritis (CGN) by analyzing a novel mutant strain of mice. Methods A spontaneous mutant strain of mice was isolated from the autoimmune-prone strain EOD, which stably develops fatal CGN. The mutant phenotypes were assessed histopathologically, hematologically, and immunologically. The mutation was searched for with positional cloning using microsatellite markers. Results Compared with wild-type EOD (WT-EOD) mice, mutant EOD (mut-EOD) mice showed marked improvement in CGN in conjunction with an improvement in spontaneous mortality. In WT-EOD mice, an inverse correlation between blood urea nitrogen concentration and blood platelet count and massive accumulation of platelets in the glomerulus were evident, suggesting that an accumulation of platelets in the glomerulus contributes to the progression of CGN. The mutant platelets showed an abnormal aggregation in response to collagen and thrombin, associated with a bleeding tendency in mut-EOD mice. Genetic analysis revealed a deleterious mutation in the cappuccino gene (cno), which encodes a protein that belongs to a complex called the biogenesis of lysosome-related organelle complex 1 and is profoundly involved in platelet function. Morphologic examination revealed a partial defect in dense body formation in the δ-granule of platelets. Conclusion The present findings suggest that platelet functions have a critical role in crescent formation in autoimmune GN.

Ultracentrifuge experiments were performed on a miscible InPb alloy to study sedimentation of constitutional atoms under a strong gravitational field (maximum acceleration: 106 g level, temperature: below melting point (solid), starting state: inter- mediate aphase (f.c.t., c0/a0 1) appeared at the highestand lowestgravitational field sides of the sample, respectively. These observation results showed that the graded structure was con- tinuous in atomic scale, and was formed by sedimentation of constitutional atoms (substitutional atoms). It was also found that the composition change reached steady state within 60 hours. Based on a simulation of sedimentation process, the diffusion coefficient for sedimentation was estimated to be about 8 times larger than that for usual diffusion.

Background: The spontaneous onset of macroscopic arthropathy in the ankle of the particular F 1 mice descended from two Fas -deficient strains of mice; a mutant substrain of MRL/Mp. Fas lpr (MRL/rpl) and C3H/He. Fas lpr (C3H/lpr) was recently observed. Aim: To histopathologically characterise and genetically interpret the unique inheritance mode of disease in this arthropathy model. Methods: MRL/rpl, C3H/lpr, (MRL/rpl × C3H/lpr; MC) F 1 , (C3H/lpr × MRL/rpl; CM) F 1 and MCF 2 mice were bred under specific pathogen-free conditions. Histopathological grade of arthropathy was determined at 6 months by examination under a light microscope. To search for a linkage locus to the arthropathy, the whole genome of selected 48 male MCF 2 mice with 71 polymorphic microsatellite markers was scanned, followed by quantitative trait locus analysis. Results: The incidence of microscopically defined arthropathy in the male and female MCF 1 groups was 100% and 19.4%, respectively. No incidence was observed in the parental strains, MRL/rpl and C3H/lpr, and in CMF 1 mice. In the MCF 1 mice, the arthropathy mainly affected the ankle joints and was histopathologically characterised by marked entheseal proliferation with chondrocytic differentiation and ossification in the ankle joints, the manifestations similar to ankylosing enthesitis reported previously. An MRL/rpl-derived autosomal dominant susceptibility locus was mapped in the distal of D7Mit68 (60 cM) to the ankylosis onset. Conclusion: The MCF 1 mice stably develop spontaneous ankylosing disorders in the ankle, with a male predominance. The unique inheritance mode of ankylosis is possibly interpreted by the genetic interaction between the autosomal dominant locus and a Y-linked locus.

Ultracentrifuge experiments were performed on a miscible alloy of In-Pb system to study the sedimentation of atoms and phase equilibrium under a strong gravitational field (maximum acceleration: 10 6 g level, temperature: below melting point (solid), starting state: intermediate � -phase (fct, c0=a0 1) appeared at the strong- and weak-gravitational field sides of the sample, respectively, from starting state of � -phase, and the continuous changes in lattice parameters were observed in each phases. These observation results showed that the graded structure was continuous in atomic scale, and was formed by the sedimentation of substitutional solute atoms. Composition profiles of the centrifuged samples for 31.3 h, 60 h and 150 h showed that the composition change reached the steady state within 60 hours. As a result of the simulation of sedimentation process, the diffusion coefficient for sedimentation was estimated to be about 8 times larger than that for usual diffusion. The present result indicated that the diffusion mechanism of the sedimentation in this system was different from the conventional ones.

The rdw rat was initially reported as having hereditary dwarfism caused by pituitary dysfunction. Subsequent studies on the rdw rat, however, have demonstrated that the primary cause of rdw dwarfism is present in the thyroid gland but not in the pituitary gland. The primary cause of rdw rat disorders is a missense mutation of the thyroglobulin (Tg) gene by a one-point mutation. In the present study, we attempted to rescue the dwarfism of the rdw rats using a diet supplemented with thyroid powder (T-powder) and a thyroid graft (T-graft). The infants of the rdw rat were successfully raised to a mature stage body weight, accompanied by elevation of serum growth hormone (GH) and prolactin (PRL), by the T-powder. Furthermore, the T-graft successfully increased the body weight with fertility. The serum GH and PRL levels in the T-graft rdw rat significantly increased. The serum thyroid-stimulating hormone (TSH) levels in the T-graft rdw rat were significantly decreased but were significantly higher than those in the control rat. The GH and PRL mRNA expression in the rdw rat with the T-graft was virtually the same as that of the control, but the TSH beta mRNA differed from that of the control rats. Thus, the dwarfism in the rdw rat is rescued by thyroid function compensation, such as that afforded by T-powder and T-graft.

Using Ig-beta and growth hormone producing cells with liver-derived cells for controls, sensitivity of chromatin to DNase I was measured by real-time PCR at eleven targets in rat Ig-beta/growth hormone locus where four cell type-specific genes and two ubiquitously expressed genes are present in a compact 88-kb region. Chromatin situated at the promoter of actively-transcribed gene and placed at cell type-specific DNase I hypersensitive sites with enhancer activity was sensitive to DNase I. In the case of inactive gene, chromatin located in these regions was resistant to DNase I. Unexpectedly, however, chromatin placed in the transcribed intron was resistant to DNase I in two genes. DNase I sensitive chromatin was shown not to distribute locus-widely but rather to localize at the promoter and the enhancer of actively-transcribed genes in this locus.

An atomic-scale graded structure had been formed in an all-proportion miscible alloy in bismuth (Bi)-antimony (Sb) (70:30 in mol%) system by the sedimentation of substitutional solute atoms under a strong gravitational field of 1 million G level in maximum acceleration at 220–240□ (Mashimo et al. 1997, Mashimo et al. 2001). The large and long crystals whose grain sizes were several mm long and several hundreds of μm wide were oriented along the direction of gravity in the high gravity region larger than about 0.7×106 G, while those of the small crystals in the low gravity region smaller than it were several 10 Pin. It was found that the large strain existed in the large crystals by back-reflection Laue method. It was found that the c axis of hexagonal structure of a large crystal was roughly parallel to the direction of gravity by Transmission Electron Microscopy (TEM) observation.

The state of acetylation in H3 and H4 histones and dimethylation in the H3 histone Lys4 residue were examined by chromatin immunoprecipitation (ChIP) at 11 targets in the rat Ig-beta/growth hormone locus. Marked enhancement of the acetylation of histones H3 and H4 and the dimethylation of H3 Lys4 was observed in the chromatin situated close to the promoter of an actively transcribed gene. Chromatin positioned near a cell-type-specific DNase I-hypersensitive site with enhancer activity had the same histone modifications as the active promoter. In one transcribed intron, chromatin with fewer histone modifications was found, and in another transcribed intron, chromatin with markedly enhanced modifications was found. In most cases, no appreciable difference in the acetylation of histones H3 and H4 was found at prominently enhanced targets. However, different acetylation levels of H3 and H4 were found at one target. The targets with enhanced dimethylation of the H3 Lys4 residue coincided with those with prominently enhanced acetylation of histones H3 and H4.

Objective To clarify the mode of inheritance of the tissue distribution of vasculitis in MRL/Mp-lpr/lpr (MRL/lpr) lupus-prone mice and to identify the susceptibility loci. Methods Vasculitis in individual MRL/lpr, C3H/HeJ-lpr/lpr (C3H/lpr), (MRL/lpr × C3H/lpr)F1, and (MRL/lpr × C3H/lpr)F2 intercross mice was analyzed by histopathologic grading of main branches of the aorta and of medium-sized arteries in the lower limbs. Genomic DNA samples from F2 intercross mice were examined by simple sequence-length polymorphism analysis, and the polymorphic microsatellite markers highly associated with vasculitis in each tissue were determined as vasculitis susceptibility loci. Results A susceptibility locus with significant linkage to vasculitis of main branches of the aorta was mapped on chromosome 4 at D4Mit213 (map position 13.3cM) selectively in males, while vasculitis of medium-sized arteries in the lower limbs was mapped to different chromosomes: at D8Mit31 on chromosome 8 (map position 33.0) selectively in females and at D5Mit36 on chromosome 5 (map position 65.0). All of these were different from the previously defined loci governing susceptibility to vasculitis involving the kidneys. Conclusion Systemic vasculitis in MRL/lpr mice is genetically controlled with cumulative effects of multiple gene loci, each of which has tissue specificity.

A Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) plays an essential role in bone accrual and eye development. Here, we show that LRP5 is also required for normal cholesterol and glucose metabolism. The production of mice lacking LRP5 revealed that LRP5 deficiency led to increased plasma cholesterol levels in mice fed a high-fat diet, because of the decreased hepatic clearance of chylomicron remnants. In addition, when fed a normal diet, LRP5-deficient mice showed a markedly impaired glucose tolerance. The LRP5-deficient islets had a marked reduction in the levels of intracellular ATP and Ca 2+ in response to glucose, and thereby glucose-induced insulin secretion was decreased. The intracellular inositol 1,4,5-trisphosphate (IP3) production in response to glucose was also reduced in LRP5−/− islets. Real-time PCR analysis revealed a marked reduction of various transcripts for genes involved in glucose sensing in LRP5−/− islets. Furthermore, exposure of LRP5+/+ islets to Wnt-3a and Wnt-5a stimulates glucose-induced insulin secretion and this stimulation was blocked by the addition of a soluble form of Wnt receptor, secreted Frizzled-related protein-1. In contrast, LRP5-deficient islets lacked the Wnt-3a-stimulated insulin secretion. These data suggest that Wnt/LRP5 signaling contributes to the glucose-induced insulin secretion in the islets.

To search for novel regulatory regions, we examined the features of chromatin structure in the rat B29/Ig-β gene and its flanking regions by determining DNase I hypersensitive sites (DHS) in plasmacytoma-derived Y3 cells. Six Y3 cell-specific DHS were detected at −8.6, promoter, +0.7, +4.4, +6.0, and +8.7 kb. The DHS at +4.4, +6.0, and +8.7 kb were present in the intergenic region between B29/Ig-β and growth hormone (GH) genes and were mapped inside conserved sequences in rat and humans. In transient transfection into Y3 cells, 2.9-kb DNA containing the +4.4 and +6.0-kb DHS demonstrated six times more enhancing activity than B29/Ig-β promoter alone. Three intergenic DHS each possessed enhancing activity that was highest in the +4.4-kb region. In the electrophoretic mobility shift assay, a major band shift was demonstrated with Y3 nuclear extract and 0.3-kb DNA containing the +4.4-kb region with a conserved 0.22-kb sequence. By footprint analysis, 20 bases in the middle of the 0.3-kb DNA were protected by Y3 nuclear extract in which the consensus binding site for the OCT family was present. Deletion of the footprinted region reduced enhancing activity to that of the B29/Ig-β promoter alone. The sequence responsible for the major band shift and transcriptional enhancing activity in the conserved +4.4-kb region thus coincided with the 20-bp footprinted region.

Cardio-facio-cutaneous (CFC) syndrome is one of the 'RASopathies', a group of phenotypically overlapping syndromes caused by germline mutations that encode components of the RAS-MAPK pathway. Germline mutations in BRAF cause CFC syndrome, which is characterized by heart defects, distinctive facial features and ectodermal abnormalities. To define the pathogenesis and to develop a potential therapeutic approach in CFC syndrome, we here generated new knockin mice (here Braf(Q241R/+)) expressing the Braf Q241R mutation, which corresponds to the most frequent mutation in CFC syndrome, Q257R. Braf(Q241R/+) mice manifested embryonic/neonatal lethality, showing liver necrosis, edema and craniofacial abnormalities. Histological analysis revealed multiple heart defects, including cardiomegaly, enlarged cardiac valves, ventricular noncompaction and ventricular septal defects. Braf(Q241R/+) embryos also showed massively distended jugular lymphatic sacs and subcutaneous lymphatic vessels, demonstrating lymphatic defects in RASopathy knockin mice for the first time. Prenatal treatment with a MEK inhibitor, PD0325901, rescued the embryonic lethality with amelioration of craniofacial abnormalities and edema in Braf(Q241R/+) embryos. Unexpectedly, one surviving pup was obtained after treatment with a histone 3 demethylase inhibitor, GSK-J4, or NCDM-32b. Combination treatment with PD0325901 and GSK-J4 further increased the rescue from embryonic lethality, ameliorating enlarged cardiac valves. These results suggest that our new Braf knockin mice recapitulate major features of RASopathies and that epigenetic modulation as well as the inhibition of the ERK pathway will be a potential therapeutic strategy for the treatment of CFC syndrome.

The Src family kinase Lyn initiates intracellular signal transduction by associating with a variety of immune receptors such as antigen receptor on B cells and high-affinity Fc receptor (FcR) for immunoglobulin Ig(E) (FcepsilonRI) on mast cells. Involvement of Lyn in the IgE-mediated immediate-type hypersensitivity is well documented, but the physiological significance of Lyn in IgG-dependent, type III low-affinity FcR for IgG (FcgammaRIII)-mediated responses is largely unknown. In this study, we generated a double-mutant mouse strain deficient in both type II FcR for IgG (FcgammaRIIB) and Lyn to exclude any involvement of inhibitory signaling by FcgammaRIIB, which otherwise downregulates FcgammaRIII-mediated cellular responses. FcgammaRIIB-deficient but Lyn-sufficient mice served as controls. The Lyn deficiency attenuated IgG-mediated systemic anaphylaxis in vivo, and significantly reduced calcium mobilization and degranulation responses of bone marrow-derived mast cells (BMMCs) in vitro. However, we found that either interleukin 4 or tumor necrosis factor alpha release by BMMCs was comparable to that from Lyn-deficient and control mice, and the reverse-passive Arthus reaction was equally induced in both mutant mice, indicating that Lyn is not involved in the onset of the IgG-mediated, FcgammaRIII-dependent late phase responses of mast cells. These findings provide us with insight into distinct signaling mechanisms in mast cells underlying the development of diverse pathologies as well as a therapeutic potential for selective treatment of allergic disorders.

Isolation and characterization were performed for cDNA encoding mouse testicular cell adhesion molecule-1 (TCAM-1) using 2908 bases coding for a protein having 548 amino acids (60 kDa). Mouse TCAM-1 protein was found to consist of seven domains for signal sequence, five immunoglobulin (Ig) domains, and the transmembrane plus cytoplasmic domain. TCAM-1 gene and the region linking it to growth hormone (GH) gene located downstream from the TCAM-1 gene were then analyzed. The mouse TCAM-1 gene was 11.6 kb in length with 8 exons; the same as for the 12.0 kb rat gene. The distance from the TCAM-1 to GH gene was 12.5 kb in the mouse genome, and 7.6 kb in the rat. By Northern hybridization, 3.1-kb TCAM-1 mRNA was detected in 17-day testis and would appear present in pachytene spermatocytes and round spermatids.Key words: mouse cell adhesion molecule, gene structure, expression.