2-Amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP), one of the heterocyclic amines produced most abundantly during cooking of meat and fish, has been widely used for colon and mammary gland carcinogenesis experiments in rats. On the other hand, PhIP shows weak carcinogenicity in the colon of mice, but, instead, induces lymphoma and leukemia at a high incidence after long-term exposure. Recently it has been reported that colonic adenocarcinomas could be rapidly induced in mice when dextran sodium sulfate (DSS), a potent inducer for colitis is applied to mice after PhIP exposure. In the present study, we investigated the effects of inflammation on colon carcinogenesis using that protocol slightly modified.
C57BL/6J mice were given a single i.g. administration (200 mg/kg BW) of PhIP, followed by treatment with 1, 1.5, or 2% DSS in drinking water for 4-6 days. Sequential observation revealed that at all the sacrifice time-points (5, 10, 15, and 20 weeks after PhIP administration), adenoma and well-differentiated adenocarcinoma developed in the caecum and colon, and their incidence increased depending upon the intensity of colitis. The neoplastic cells showed β-catenin accumulation in their cytoplasm and nucleus. Mutations in the β-catenin gene were also detected in several tumors, but not in all tumors, suggesting that other processes for Wnt-signal activation could be involved in the development of colon tumors induced by the combined treatment with PhIP and DSS.
To further analyze the consequences of inflammatory responses to DSS exposure, C57BL/6J mice were given 2% DSS for 4 days, and histopathological evaluation was performed at weeks 0, 1, 2, and 4 after DSS removal. Prominent mucosal ulceration with regenerative hyperplasia was observed, especially at 2 weeks after DSS removal, and acute inflammation progressed to chronicity with time. Therefore, it was suggested that colitis induced by DSS influenced epithelial proliferation over a long period of time. This mouse model will serve as a useful tool for the analysis of colon carcinogenesis, especially inflammatory bowel disease-associated neoplasia in humans.

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Ubiquitin carboxyl-terminal hydrolase L1 is exclusively expressed in neurons, testis and ovary, which mediating various biological activities. However, the role of UCH-L1 in oocytes in ovary remains still unknown. Here, we report the expression pattern of UCH-L1 proteins in the mouse ovaries and embryos. UCH-L1 proteins were continuously expressed in plasma membrane of oocytes in ovary and fertilized eggs. To further investigate the role of UCHL1 in embryos, we analyzed the fertilization rate of UCHL1-deficient embryos of gad female mice. gad mice showed significantly increased polyspermy rate in in vitro fertilization experiment, though the rate of fertilization are not significantly different from wild-type mice, In addition, the litter size of gad female mice was significantly reduced compared with wild-type mice. Thus, it is suggested UCH-L1 functions in the block to polyspermy in mice oocytes.

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Serum/glucocorticoid regulated kinase 3 (SGK3), also known as serum/glucocorticoid regulated kinase-like (SGKL) or cytokine-independent survival kinase (CISK), is a member of the AGC kinase family, and is considered to be involved in various intracellular processes, including membrane trafficking, cell survival and apoptosis. Mutation in the Sgk3 gene causes both defective hair follicle development and hair cycle alteration in mice. Sgk3-mutant YPC mice (YPC-Sgk3^ypc/Sgk3^ypc) was strongly suggested to express SGK3 protein whose function was altered, and in the hair follicles of YPC, the aberrant differentiation and poor proliferation of hair matrix keratinocytes during the period of postnatal hair follicle development resulted in a complete lack of hair medulla and weak hair, and, surprisingly, the length of postnatal hair follicle development and anagen term was shown to be dramatically shortened. Also, phosphorylation of GSK3β at Ser9 and the nuclear accumulation of β-catenin were reduced in the developing YPC hair follicle, and this suggests that the phosphorylation of GSK3β and WNT-β-catenin pathway takes part in the SGK3-dependent regulation of hair follicle development. Moreover, the above-mentioned features, especially the hair cycling pattern, are different from those in other Sgk3-null mutant strains, and this suggests that the various patterns of dysfunction in the SGK3 protein may result in phenotypic variation. Our results indicate that SGK3 is a very important and characteristic molecule that plays critical roles in both hair follicle morphogenesis and hair cycling.

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The Ataxia and Male Sterility (AMS) mouse is a mutant that exhibits ataxia due to a near-complete loss of the cerebellar Purkinje cells and male sterility because of severe oligospermia due to the degeneration and death of differentiating germ cells in the seminiferous tubules. The disease-responsible ams gene was shown to be allelic to Nna1, of which the classical mutation, pcd, has been shown by others to be a severe allele for the Purkinje Cell Degeneration mouse, by mating heterozygous pairs from each mutant strain. A RT-PCR analysis to sequentially cover the whole transcript of the Nna1 gene revealed that no obvious differences in either the size or quantity of the mRNA. By sequencing the cDNA base pairs a point mutation of G to C was detected at 2423rd by in exon 17. This missense mutation caused 808th arginie proline and it appeared to affect the protein structure and stability, because the transfection of the cDNA construct of the wild-type, but not of the mutant, led to the translation and stable expression of the protein.
To investigate how ams mutation affects the cellular processes, we devoted our efforts to the morphological analysis of the mode of cell death. We herein describe the different types of cell death in the AMS mouse. The causes of the acute pathological atrophy of the AMS cerebellum were not only the primary near-complete loss of the Purkinje cells but also the secondary damage to other cells. Dying Purkinje cells lacked the characteristics of classical apoptosis except for their light microscopic morphology, but their death was considered to be autonomous death triggered by the direct effect of the ams mutation, because of their acute and near-complete disappearance and the particular changes observed in the cytoplasm. In contrast, in the granular layer, typical apoptotic bodies were recognized by electron microscopy, and a substantial number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) -positive cells and activated caspase-3-positive cells were observed. Granule cell death was considered to be target-related apoptosis which was induced after post-synaptic Purkinje cell death, because the age-dependent changes in the TUNEL-positive cell counts followed that of Purkinje cell loss and the peak value was still noted 1 week after the total loss of Purkinje cells. Our results indicate that both the total and partial losses of Purkinje cells and granule cells, respectively, contributed to the atrophy of the AMS cerebellum. Another study on the photic injury of the photoreceptor cell of the AMS retina revealed that the cells had lost the ability to resist such injurious factors while, in addition, the process of apoptotic cell death was also different from that of classical apoptosis. Since the ams mutation affects the dying process of many cell types in different manners, Nnal is likely to act as an important regulator of the life of cells.

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8-Oxoguanine (8-oxoG) has been paid attention due to its abundance and premutagenic property among a variety of oxidative DNA damages. The premutagenic 8-oxoG is known to cause its specific type of mutation after DNA replication, a GC to TA transversion, one of the mechanisms by which oxidative stress is involved in the aging process and the induction and progression of various diseases including carcinogenesis. As repair system against such premutagenicity of 8-oxoG, homolog of MutM and MutT in bacteria has been identified as Mmh/OGG1 and MH1 in mammals, respectively. The present study was investigated the repair system for 8-oxoG formation in hepatocarcinogenecity of pentachlorophenol (PCP), a wood preservative, using Mmh/OGG1 deficient mice. The deficient homo (KO), hetero and wild mice derived from C57BL strain were fed PCP at 600 ppm in diet for 26 weeks after initiation of single intraperitoneal injection of diethylnitrosoamine (DEN) at 100 mg/kg, and compared hepatic cancer development and hepatotoxicity to relevant controls. Although PCP treatment induced severe hepatotoxicity with inflammation, the treatment did not increase liver neoplastic lesion development with or without DEN initiation unexpectedly. Interestingly, the hepatocellualr proliferating activity in the homo mice given both of DEN and PCP treatment was significantly lower than that in the hetero and wild animals. In addition, increases of mRNA expression in major drug induced metabolic enzymes, cytochrome P450 1A2 and glutathione S-transferase were depressed in homo mice treated with PCP and DEN treatment. These results suggest that the hepatocarcinogenecity of PCP did not appear in Mmh/OGG1 gene deficient mice. Mechanisms of the negative results remain fully undetermined, but a genetic background in which C57BL mouse strain has resistant strain to hepatocarcinogenesis and any compensation pathway by other gene repair gene might be related. Lower potentials to drug-metabolism and cell proliferation activity in homo mice might accelerate the resistance.

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We are trying to develop a new molecular therapy (chemical chaperone therapy) for lysosomal diseases, particularly with severe neurological manifestations. Fabry disease (generalized vasculopathy) was the first target for therapeutic experiment using galactose and 1-deoxygalactonojirimycin, followed by β-galactosidase deficiency disorders (G_M1-gangliosi-dosis and Morquio B disease) using a newly developed chemical compound N-octyl-4-epi-β-valienamine (NOEV) for brain pathology, which serves as a chemical chaperone in somatic cells. NOEV is a potent inhibitor of lysosomal β-galactosidase in vitro. However, addition in the culture medium at low concentrations restored mutant enzyme activity and reduced substrate storage remarkably in human or murine fibroblasts. Oral administration of NOEV to the G_M1-gangliosidosis model mouse corrected pathological changes in the brain morphologically and biochemically. Preliminary clinical observations suggested prevention of phenotypic expression during short-term experiments. For monitoring the clinical effect of this new therapy, we developed a neurological assessment system for experimental mice, and hope to confirm the effect of this compound for disease model mice in the near future. Chemical chaperone therapy will be useful for certain patients with G_M1-gangliosidosis and potentially other lysosomal storage diseases with central nervous system involvement.

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Chorea-acanthocytosis (ChAc) is a human hereditary neurodegenerative disorder with autosomal recessive transmission, in which selective degeneration of striatum has been reported in brain pathology. Clinically, ChAc shows Huntington's disease-like neuropsychiatric symptoms and red blood cell acanthocytosis, and much variation in symptoms are observed even in brother cases. Recently, we identified the gene, CHAC (VPS13A) encoding a protein named chorein in which a deletion mutation was found in Japanese ChAc families. Then we identified the mouse CHAC cDNA sequence and the exon-intron structures of the gene, and produced a ChAc-model mouse introducing #60-61 exons-deletion corresponding to a human disease mutation by gene-targeting technique. The mice began to show acanthocytosis and motor disturbance after becoming old age. In behavioral observations, locomotor activity was significantly decreased, and the contact time at social interaction test was decreased significantly in the model mice. In the brain pathology, many apoptotic cells were observed in the striatum of the mutant mice. In neurochemical determination, dopamine-metabolite, HVA concentration decreased significantly in the portion including midbrain of the mutant mice. These findings are well consistent with the human results reported elsewhere and indicate that the ChAc-model mice showed mild phenotype with late adult onset. The ChAc-model mouse therefore provides a good model system to study the human disease. The mice are produced as hybrid of 129 and C57Bl/B6, and there are much variations in the degree of degeneration in the sriatum and other phenotypes, which might suggest the existence of modifier genes.

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Mouse models for human diseases have been shown to be very useful for analyzing patho-physiology of disease development and devising a new way of treatment including drug development. In this review, we will demonstrate this by two examples.
Among several spontaneous mutants showing tremor, the quaking viable (qk^v) mutation is an autosomal recessive mutation that exhibits pleiotropic effects on myelination and spermatogenesis (male sterility) . Homozygous mutant animals exhibit a characteristic tremor or ‘quaking’ of the hindlimbs by postnatal 10 day, and tonic type seizures at later stages. qkI, encoding an RNA binding protein, was identified as a candidate for this mutation. Through the binding to pre-mRNA, QKI may be involved in processing, localization, and protein expression of mRNA for myelin proteins. In addition, we found a novel function of qkI in smooth muscle cell (SMC) differentiation during embryonic blood vessel formation.
A mouse model of Rubinstein-Taybi syndrome (RTS) was generated by an insertional mutation into the cyclic AMP response element-binding protein (CREB) -binding protein (Crebbp) gene. Heterozygous Crebbp-deficient mice, which had truncated CBP protein containing the CREB-binding domain, showed clinical features of RTS, such as growth retardation (100%), retarded osseous maturation (100%), hypoplastic maxilla with narrow palate (100%), cardiac anomalies (15%) and skeletal abnormalities (7%) . Tests for memory storage such as the step-through-type passive avoidance test showed that mice were deficient in long-term memory (LTM) . From this observation, it is expected that drugs that modulate CREB function by enhancing cAMP signaling might yield an effective treatment for the memory defect (s) of Crebbp^+/- mice. Actually Bourtchouladze et al. designed a cell-based drug screen and discovered inhibitors of phosphodiesterase 4 (PDE4) to be particularly effective enhancers of CREB function. They demonstrated that the inhibitor abolished the long-term memory defect of Crebbp^+/- mice, suggesting that PDE4 inhibitors may be used to treat the cognitive dysfunction of RTS patients. Taken together, these results indicate that mouse models for human diseases are useful for functional genomic, medical genomics and pharmacogenomics.

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Among 200 species of NHPs, several monkeys and great apes are used for biomedical research. They are chimpanzees, and rhesus, cynomolgus and Japanese macaques which belong to the Old World monkeys, and the New World monkeys such as squirrel monkeys and common marmosets.
NHPs have advantages over other biomedical research mammals such as rodents in the ability to more reliably extrapolate their data directly to humans, because they have a closer genetic, morphological, and physiological relationship to humans than other experimental animals. Thus, infectious, senile or neurological disease models have been developed using NHPs. They are also used for translational studies of new medical technologies.
However, NHP also have some weak points, such as: they are expensive, hard to handle, the absence of genetic and biological controls, the high risk of zoonoses, need for compliance for CITES, legal quarantine before use etc. Despite these, and other disadvantages, NHP are still necessary for biomedical researches.
The key point of any NHP animal model is as follows. 1) The same animal model cannot be obtained in rodents. That is if the model is obtainable in rodents, then there is no need to develop a NHP animal model because it is easier to get results from rodents than from NHPs. 2) The NHP model covers data, which cannot be obtained from patients. That is if the data can be obtained from patients directly, it is easier to get results from patients than from NHPs. Only the NHP models which have the above tow characters may be a breakthrough of biomedical sciences.
I would like to introduce several NHP animal models including AID, EHEC, AZD, AMD, TCDD exposure and Gene therapy models in which we are conducting our projects in this symposium.

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Kagoshima University has developed the Clawn miniature swine strains of defined SLA, which are maintained and produced in Japan Farm Clawn Institute (JFCI) as the facility to supply 400-500 inbred Clawn miniature swine per year. We are planning to expand the facility to supply the miniature swine not only to the researchers in Japan but also those outside the country. Body weight of Clawn miniature pig is about 30 kg by 1 year old, and it is up to 50 kg like a human even if it grows up, but it is dimension of around a one-fifth from a quarter of edible pig. We are going to develop this miniature swine for medical use, including endoscopic surgical training and TG or KO miniature pig to generate “disease model” or “donor animal for cyto-and organ-xenotransplantation”. The studies concerning the nature of porcine endogenous retrovirus (PERV) and the control of its infection to mankind are essential to swine-to-human xenotransplantation. On the basis of these studies we will establish the scientific standard to keep the human security. In the future, we hope that our center will greatly contribute to an international network of miniature swine user in biomedical research and life science education.

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Patients with acute coronary syndromes (ACS), which include unstable angina, acute myocardial infarction, and sudden cardiac death, are estimated more than 20 million in the world. Pathological examinations have demonstrated that vulnerable coronary atheromatous plaque is important in the onset of ACS. However, the detail mechanism is still uncertain. Several clinical intervention trials have showed that statins, inhibitors of cholesterol biosynthesis, suppress the onset of ACS. On the other hand, in vitro studies suggested that statins suppressed smooth muscle cell (SMC) proliferation and induced the apoptosis, while SMC is important in stabilization of atheromatous plaque. Therefore, results of in vitro studies are contradictory to observation of clinical studies. To clarify the mechanism of ACS and preventive effects of statins on ACS, suitable animal models have been required. We developed coronary atherosclerosis-prone WHHLCA rabbits and myocardial infarction-prone WHHLMI rabbits by a technique of selective breeding and examined statins' effects on coronary atherosclerosis. The original WHHL rabbits showed hypercholesterolemia due to deficiency of the LDL receptors and aortic atherosclerosis while the coronary plaques were mild and the frequency was very low. WHHLCA rabbits were developed from the original WHHL rabbit and have advanced coronary plaques but incidence of myocardial infarction is very low. WHHLMI rabbits were developed from WHHLCA rabbits and the cumulative incidence of myocardial infarction is more than 90% at 30 months old. WHHLMI and WHHLCA rabbits closely resemble humans in the lipid metabolism and atherosclerosis although mice and rats are largely different. In WHHLCA and WHHLMI rabbits, the coronary atherosclerosis start to develop from 2 months old and becomes transitional lesions at about 10 months old. At above 20 months old, the coronary plaques become complicated lesions. In intervention studies using WHHLCA and WHHLMI rabbits, therefore, we start treatments from 10 months old for one year, and examined coronary plaques at the end of the experiment. This study design is compatible to human intervention treatments. As a result, statins prevent destabilization of coronary plaques due to depression of macrophages in the plaque. The results support the hypothesis derived from human clinical studies that statins would stabilize coronary plaques and stabilization of coronary plaque would be important to prevent ACS. In addition, our results also suggested that statins effects on arterial smooth muscle cells are very weak in vivo compared to in vitro studies. In conclusion, since WHHLCA and WHHLMI rabbits resemble humans in the characteristics of lipid metabolism and atherosclerosis, these rabbit strains are useful for studies of hypercholesterolemia, atherosclerosis, and development of hypolipidemic and anti-atherosclerotic drugs.

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Purpose: We have recently reported that DNA fragmentation without cell loss were found in the hippocampus in EL mice, an epileptic mutant of epilepsy. And abundance of neurotrophic factors were expressed during the early developmental stage.
In the present study, we used the EL mice to examine how the altered cyclin and the corresponding Cyclin Dependent Kinases (CDK) family are related to the cell proliferation during development, and further the epileptogenesis.
Methods: Developmental changes of cyclin family and corresponding CDK were examined during cell cycle by Western blotting in the hippocampus of EL mice. Also, an attempt was made to identify the cell proliferation by using systemic Bromodeoxy Uridine (BrdU) to labell dividing cells.
Results: As compared with the control DDY, EL shows up-regulation of each cell cycle specific Cyclins/CDK's during early developmental stage which suggests that reentry of cell cycle is promoted before the beginning of seizures, possibly because of the abundance of neurotrophic factors.
Conclusions: It is concluded that the activation of Cyclins/CDK's during early developmental stage before exhibiting seizures, suggesting the reentry of cell cycle and cell proliferation in the hippocampus of EL.

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