The Shumiya cataract rat (SCR) is a hereditary cataractous strain. Previous crossing experiments revealed that one genetically recessive locus and one copy of a recessive gene associated with embryonic lethality were a prerequisite for cataract onset in the SCR. In this study, we revealed unique molecular genetic features of cataract onset in the SCR by identifying a gene responsible for cataract and embryonic lethality in the SCR. Genetic linkage analysis and positional cloning revealed mutations of the gene for lanosterol synthase (Lss) on chromosome 20 in SCR, which functions in the cholesterol biosynthesis pathway. Accordingly, the cataract in SCR could be classified under cholesterol deficiency-associated cataracts. Cataract onset in SCR was uniquely regulated by a specific combination of different mutant (Lss^l) and polymorphic alleles (Lss^s) on the Lss locus. Lss^s was a hypomorphic allele with a G to A nucleotide substitution in exon 4 that reduced LSS activity to about 40% of normal by substituting an amino acid (D139N) . The G to A nucleotide substitution also caused aberrant splicing, which led to transcripts with a deletion of a sequence corresponding to the last 47 nucleotides of the exon 4. Thus, the Lss^s allele was unique in that one nucleotide substitution acted synergistically to reduce enzyme activity. Lss^l was a null allele with a deletion of thirteen consecutive nucleotides (1405-1407del) plus a single nucleotide insertion (1419-1420insT), leading to a deletion of four consecutive amino acids (H469-C472del) . Thus, Lss^s and Lss^l could be assumed to be cataract and embryonic lethal alleles, respectively. These observations indicated that the cataract onset in the SCR complied with a threshold model. The prerequisite for cataract onset was the Lss^s/Lss^l genotype, which reduced LSS activity below the threshold, that is about 12-24% of normal. Furthermore, the linkage analysis revealed that a locus on chromosome 15 of normal ACT rat strain (Cats2^A) suppressed cataract onset with incomplete penetrance, indicating that cataract in SCR is an oligogenic trait.The through elucidation of the mechanisms for cataract in SCR might afford an excellent insight into the studies of threshold diseases as well as of other oligogenic traits.

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The basic helix-loop-helix leucine zipper transcription factor MITF is important for the development of mast cells. The mutant tg/tg mice, which do not express MITF, lack mast cells in tissues. For the development of mast cells, not only mast cell precursors but also environmental factors are necessary. The mutant W/W^v mice that also lack mast cells like tg/tg mice possess normal environment but abnormal mast cell precursors. Since MITF is expressed in both mast cells and tissues where mast cells develop, the tg/tg mice may show abnormalities in both mast cell precursor and tissue environment. We investigated the abnormalities of mast cell precursors and environment in tg/tg mice by skin and bone marrow transplantations. When bone marrow cells of tg/tg mice were transplanted to W/W^v mice, mast cells did not develop in tissues. The number of developing mast cells in the skin from W/W^v mice was much lower when grafted to tg/tg mice than when grafted to normal (+ / +) mice. These indicated that mast cell precursors of tg/tg mice did not develop in tissues. When bone marrow cells of + / + mice were transplanted, the number of developing mast cells was significantly lower in tg/tg mice than in W/W^v mice. When the skin was grafted to + / + mice, the magnitude of increase of mast cell number was lower in the graft from tg/tg mice than in the graft from W/W^v mice. These showed that the tissue environment for mast cell development was defective in tg/tg mice. MITF was essential for both mast cell precursors and tissue environment.

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Transforming growth factor (TGF) -β constitutes a superf amily of multifunctional cytokines that regulate a variety of cellular responses including cell proliferation, apoptosis and extracellular matrix deposition. TGF-β1 is reportedly upregulated in tubulointerstitial fibrosis as characterized by epithelilal-mesenchymal transition (EMT) in the renal tubules and excessive accumulation of extracellular matrix with monocyte influx. Here we show that mice lacking Smad3, a key mediator of TGF-β signaling, are protected against tubulointerstitial fibrosis induced by unilateral ureteral obstruction (UUO) . Primary culture of renal tubular epithelial cells from Smad3-null or wild-type mice demonstrated that TGF-β1-induced EMT of the renal tubules and autoinduction of TGF-β1 require Smad3 signaling. Cyclic stretching of cultured renal tubular epithelial cells that mimics distention of the renal tubules by UUO induces EMT and upregulation of TGF-βl in a Smad3-dependent manner. Exogenous bone marrow monocytes accelerate EMT of the cultured renal tubular epithelial cells and renal tubules in the obstructed kidney by UUO dependent on TGF-β1/Smad3 signaling. These data indicate that the Smad3 pathway is essential for the pathogenesis of renal tubulointerstitial fibrosis and suggests that small-molecule inhibitors of this pathway may have clinical application in the treatment of fibroinflammatory nephropathy.

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The National Bio Resource Project for the Rat in Japan (NBRP-Rat) collects, preserves and distributes rat strains. More than 250 inbred strains have been deposited thus far into the NBRP-Rat, and are maintained as specific pathogen-free (SPF) rats or cryopreserved embryos. We are now comprehensively characterizing deposited strains as part of the Rat Phenome Project in order to reevaluate their value as models of human diseases. Phenotypic data is being collected for 7 categories, 109 parameters; Functional Observational Battery (FOB, neurobe-havior), behavior studies, blood pressure, biochemical blood tests, hematology, urology, and anatomy. Furthermore, genotypes are being determined for 370 SSLP markers distributed through the whole rat genome.
Here we report these large-scale, high-throughput screening data that have already been collected for 54 rat strains. This comprehensive, original phenotypic data can be systematically viewed by‘Strain Ranking’for each parameter. This allows investigators to explore the relationship between several rat strains, to identify new rat models, and to select the most suitable strains for specific experiments. The discovery of several potential models for human diseases, such as hypertension, hypotension, renal diseases, hyperlipemia, hematological disorders and neurological disorders illustrates the potential of many existing rat strains. All deposited strains and obtained data are freely available for any interested researcher worldwide at www. anim. med. kyoto-u. ac. jp/nbr.

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Atopic dermatitis (AD) is one of the most common and severe skin diseases, and is primarily a disease of infancy and childhood. Although a number of mouse models for human AD have been developed, they all have significant disadvantage, such as a different timing of onset and remission from human AD. We generated transgenic (Tg) rats overexpressing telomerase in ubiquitous tissues. The Tg rats developed skin lesions that are in accordance with those of human AD, and this Tg rats was named ‘Inaba’. Clinical signs and symptoms seen in Tg rats began at 2 weeks of age with superficial erosion and deep excoriation followed by itching, erythema, hemorrhage, edema, scaling and dryness of the skin. Their skin lesion was begun to improve by 8 weeks of age. The Inaba rats showed elevated serum IgE. The histopathological examination of dorsal skin of Inaba rat revealed significant histologic changes, including hyperkerathosis, acanthosis, parakerathosis, telangiectasia, spongiosis, and infiltration of inflammatory cells were observed. Thus, we consider that Inaba rat is a useful model to investigate the pathogenesis of AD especially appeared in infancy to childhood, and to develop a new generation of therapeutic strategies for the intervention of allergic diseases.

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Allergy is a major health problem, and this is especially true for type I allergy, such as bronchial asthma and allergic rhinoconjunctivitis, with more than 20% of the world population being affected by these conditions. Affected individuals exhibit a high titer of immunoglobulin E (IgE) antibodies specific for allergens such as those associated with house dust mites and plant pollen. Exposure of such individuals to these allergens induces the activation of mast cells through antigen-antibody-mediated cross-linking of IgE receptors on the cell surface. Activated mast cells release allergic mediators, such as histamine, cysteinyl leukotrienes, and various cytokines. These substances not only mediate acute allergic responses but also induce the late phase of inflammation by stimulating local synthesis of various chemokines such as CCL11 (eotaxin) and CCL17 (TARC) and recruiting T helper 2 (Th2) lymphocytes and eosinophils. Repeated exposure to allergens enhances inflammation characterized by infiltration of these cells and can cause tissue remodeling. However, how this course of responses is regulated and why it becomes exaggerated in some population to develop diseases remains unknown. Prostaglandins (PGs) including PGD₂ and PGE₂ are produced during allergic reactions. We have shown PGD₂, which was released from activated mast cell, plays a role as a causative substance in allergic inflammation of airway. The concentrations of Th2 cytokines and the extent of lymphocyte accumulation in the lung of ovalbumin challenged PGD₂ receptor (DP) deficient mice were greatly reduced compared with those in wild-type animals. Moreover, DP deficient mice showed only marginal infiltration of eosinophils and failed to develop airway hyperreactivity. Whereas PGD₂ functions as an allergic mediator, aspirin-like drugs are generally ineffective in allergic disorders, suggesting that another PG-mediated pathway prevents the development of allergic reactions. Recently, we have shown that PGE₂ acting at the EP3 receptor plays such a role. Mice lacking EP3 developed allergic inflammation markedly more pronounced than that in wild-type mice or mice deficient in other EP subtypes. Conversely, an EP3-selective agonist suppressed the inflammation. This suppression was effective when the compound was administered 3 h after antigen challenge and was associated with inhibition of antigen-induced changes in gene expression. Thus, PGD₂ functions as a mast cell-derived mediator to trigger asthmatic responses. In contrast, the PGE₂-EP3 pathway negatively modulates these reactions.

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Aromatic hydrocarbon carcinogens, 7, 12-dimethylbenz [a] anthacene (DMBA) and 7, 8, 12-trimethylbenz [a] anthracene (TMBA) induce leukemia in the rat. Almost 30% of DMBA- and 16% of TMBA-induced leukemia reveal total or partial trisomy of #2 chromosome (+2 and 2q+, respectively) and many of the rest have normal diploid (2n) karyotype. Most of these leukemias are erythroblastic leukemia and they reveal a specific mutation of N-ras gene (A → T transversion at the second base of colon 61) . N-ras gene was assigned to chromosome band 2q34 in a commonly duplicated segment of +2 and 2q+. Although the 2n leukemias with N-ras mutation reveal loss of wild type N-ras allele (LOH), the +2 leukemias retain the wild type allele. This means that +2, 2q+, and LOH contribute to the dominancy of mutant N-ras gene.
This article reviews the general aspects of DMBA- and TMBA-induced rat leukemias focusing on the origin of the specific N-ras mutation and other specific genetic changes in leukemia cells.
DMBA and TMBA induce chromosome aberrations (CA) non-randomly distributed along chromosomes. Three major susceptible regions were found along the #2 chromosome including the N-ras locus. Some marker chromosomes in leukemias, 3 types of 2q+ and one of translocation type +2, had chromatid structures obviously formed by chromosome rearrangements at these DMBA-susceptible sites. The role of rDNA loci on #3, #11, and #12 chromosomes and some oncogenes (abl and H-ras) in translocations was also shown in leukemia cells. The susceptibility of the above chromosome regions to DMBA was markedly enhanced by erythropoietin (Ep) given a short time before DMBA treatment. The incidence of DMBA-leukemia was also enhanced by the preliminary anemia treatment. From these results, the authors propose that the change in susceptibility of target cells to chemical carcinogens is caused by transcription induced by Ep allowing the preferential binding of active carcinogen-metabolites to the specific genomic sites. Gene induction is therefore considered an essential step in carcinogenesis.

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Transgenic animals carrying human c-Ha-ras proto-oncogene, v-Ha-ras transgenic mice, pim-1 transgenic mice have been shown to exhibit increased carcinogen susceptibility. Based on this characteristics, studies aimed for application to medium-term screening with from 20 to 30 weeks duration for various environmental carcinogens are under way in many institutes. On the other hand, rat models are advantageous by larger organ size, abundant information regarding preneoplasias and virus-free constitution, we have generated transgenic rats carrying copies of the human c-Ha-ras proto-oncogene (Hras128) and shown to be extremely sensitive to mammary, and to a lesser extent, lesions in the urinary bladder, oesophagus and skin carcinogenesis. In most of mammary carcinomas, mutations of the transgene but not the endogenous H-ras gene are present, appearing to occur early in the process of carcinogenesis, which involves proliferation of cells in terminal end buds (TEBs) . Further findings suggest that this is independent of endogenous ovarian hormones, although inhibited by soy isoflavones. Although further studies of mechanistic aspect are clearly necessary, the model appears to have great potential for short-to long-term screening purposes, not only for modifiers active in the breast, but also other organs where tumors develop characterized by ras gene mutations.

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Accumulating evidence indicates that host reaction including inflammatory responses is a critical component of tumor development. To investigate the role of microenvironment for gastrointestinal tumor development, we have constructed series of genetically engineered mouse models, such as Apc^Δ716 mice, COX-2 gene knockout mice, and COX-2/m PGES-1 transgenic mice. Although homozygous Apc^Δ716 mice were embryonically lethal, heterozygotes developed benign polyps in entire intestinal tract caused by loss of the wild-type Apc gene. Accordingly, activation of Wnt signaling triggered by disruption of Apc gene is a direct cause for intestinal polyp development. We next constructed compound mutants of the Apc^Δ716 with COX-2 knockout mice. COX-2 is a key enzyme for prostaglandin biosynthesis and induced in the stromal cells of variety of tumor tissues including Apc^Δ716 polyps. Homozygous disruption of COX-2 genes in the Apc^Δ716 mice reduced the number of polyps dramatically by 80% compared with COX-2 wild-type mice. Moreover, treatment of Apc^Δ716 mice with a COX-2 selective inhibitor, rofecoxib, also suppressed intestinal polyposis. These results, taken together, indicate that induction of COX-2 in the stromal cells is responsible for tumor cell proliferation (promotion), whereas Wnt activation in the epithelial cells is required for transformation (initiation) . To further investigate the role of COX-2 pathway in the microenvironment, we constructed transgenic mice (K19-C2mE mice) expressing both COX-2 and mPGES-1 simultaneously. The K19-C2mE mice developed hyperplastic tumors in the glandular stomach with submucosal inflammatory responses. Antibiotic treatment suppressed inflammatory cell infiltration and mucosal hyperplasia, suggesting that host responses caused by bacterial infection is responsible for epithelial hyperplasia. Importantly, mucosal macrophages were still found after treatment with antibiotics, indicating that macrophage recruitment was the primary effect of increased level of PGE₂. Furthermore, mucosal macrophages in the K19-C2mE mice were activated by bacterial infection, producing large amount of proinflammatory cytokines such as TNF-α. It is possible that these cytokines as downstream of COX-2 pathway contribute to enhanced proliferation of the epithelial cells in the tumor promotion stage.

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The cataract were, macroscopically, found in the right eye of F344 male rat at 13 weeks-old, but no abnormality in the left eye. At 60 weeks-old, the degeneration and thinning of lens epithelial cells in the anterior lentil. The denucleation of epithelial cells and swelling of lens fibers in the equator lentis were found.

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The DRH is an inbred rat strain established by selective mating of the 3'-Me-DAB resistant progeny of closed colony Donryu rats. Genetic analysis shows that two semi-dominant QTLs, Drh1 and Drh2, are responsible for strong resistance to chemical-induced hepatocarcinogenesis in DRH rats. To evaluate the effect of the single Drh1 locus on various stages of liver carcinogenesis, we constructed a speed congenic strain DRH.F344-Drh1 by transferring a susceptible Drh1 allele of F344 to DRH rats by marker-assisted backcrossing. After oral administration of 3'-DAB for 8 weeks, the quantitative parameters of fibrosis, enzyme altered foci, GST-P expression and proliferation of liver cells in DRH.F344-Drh1 rats were intermediate between F344 and DRH. In the liver of carcinogen-fed DRH rats, there was intensive apoptosis as detected by TUNEL stain, but not in the liver of F344 and DRH.F344-Drh1 rats. Injection of lead nitrate (100 μmol/kg. B. W.) induced a wave of liver cell proliferation, as seen by BrdU uptake within a few days in F344 and DRH.F344-Drh1 rats, but not in DRH rats. Instead, there were numerous TUNEL-positive nuclei in the DRH liver after lead nitrate injection. The major role of Drh1 is effective removal of the hepatocytes newly recruited to proliferate after chemical injury.

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[in Japanese]

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