Studies using amphibians have contributed to the progress of life science including developmental biology and cell biology for more than one hundred years. Since the 1950s Xenopus laevis in particular has been used by scientists in many fields for experiments, resulting in the development of various techniques such as microsurgery on early embryos, biosynthesis of gene-encoded protein in oocytes by mRNA injection, misexpression experiments by mRNA injection into embryos, gene knockdown studies by injection of morpholino anti-sense oligonucleotide into fertilized eggs, transgenesis by the I-SceI meganuclease method, and so on. In this paper we will introduce Xenopus tropicalis as an alternative experimental animal. It has a shorter generation time and smaller diploid genome, together with whole-genome sequence data. The procedures available for Xenopus laevis can work well with Xenopus tropicalis, and embryos of both species develop at similar rates according to the developmental staging system of Nieuwkoop and Faber. Experimental systems of Xenopus tropicalis will pave the way for a new era of vertebrate genomics and genetics.

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In order to examine the influence of obesity on metabolic disorder and liver pathogenesis of the Fatty Liver Shionogi (FLS) mouse, which develops hereditary fatty liver and spontaneous liver tumors, we established a new congenic strain named FLS-Lep^ob. The Lep^ob gene of the C57BL/6JWakShi (B6)-Lep^ob/Lep^ob mouse was transferred into the genome of the FLS mouse, by backcross mating. FLS-Lep^ob/Lep^ob mice were maintained by intercrossing between Lep^ob-heterozygous littermates. The FLS-Lep^ob/Lep^ob mice of both sexes developed remarkable hyperphagia, obesity and type 2 diabetes mellitus. At 12 weeks of age, glucosuria was detected in all male and female FLS-Lep^ob/Lep^ob mice. Biochemical examination demonstrated that the FLS-Lep^ob/Lep^ob mice have severe hyperlipidemia and hyperinsulinemia. The livers of FLS-Lep^ob/Lep^ob mice showed microvesicular steatosis and deposition of large lipid droplets in hepatocytes throughout the lobules. The steatohepatitis-like lesions including the multifocal mononuclear cell infiltration and clusters of foamy cells were observed earlier in FLS-Lep^ob/ Lep^ob mice than in FLS mice. B6-Lep^ob/Lep^ob mice did not show hepatic inflammatory change. Furthermore, FLS-Lep^ob/Lep^ob mice developed multiple hepatic tumors including hepatocellular adenomas and carcinomas following steatohepatitis. In conclusion, the FLS-Lep^ob/Lep^ob mice developed steatohepatitis and hepatic tumors following hepatic steatosis. The FLS-Lep^ob/Lep^ob mouse with obesity and type 2 diabetes mellitus might be a useful animal model for human non-alcoholic steatohepatitis (NASH).

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Although chronic pancreatitis is a risk factor for pancreatic ductal adenocarcinoma (PDA), the relationship between chronic pancreatitis and PDA remains obscure. A critical obstacle to understanding the role of chronic pancreatitis is the lack of animal models. To develop one such model, mice were fed long-term with a choline deficient ethionine-supplemented (CDE) diet. Histological evaluation revealed that chronic pancreatitis, characterized by acinar atrophy, fibrosis and well-developed tubular complexes (TCs), was observed after 24 weeks of CDE diet treatment. Furthermore, expression of epidermal growth factor receptor (EGFR) and its ligands; serine protease inhibitor Kazal type 3 (Spink3) and transforming growth factor α (TGF α) and activation of K-Ras (GTP-Ras formation), which are frequently observed in human PDA, were indeed observed in parallel with TCs formation. Neoplastic lesions were not found after 54 weeks of treatment, suggesting that a continuation of CDE diet or another insult is required for the development of PDA.

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A reproducible and reliable myocardial infarction (MI) model with less inter-individual variation in ischemic size and ventricular function is essential in cardiovascular research. Little is known about whether the different ligation coordinates [whole length of left anterior descending artery (LAD) or diagonal branches] affect the inter-individual variation of ventricular function in the MI model. The present study compared the characteristics of the experimental swine MI model induced by surgical occlusion of LAD in two groups: group A (n=24), where ligation was performed below the second ventricular branch (D₂ branch), and group B (n=23), where ligation was performed at a distance one-third distal to the apex. Variation of ischemic size and left ventricular ejection fraction (LVEF) at 4 weeks after MI was compared between the two groups using the homoscedasticity F test and coefficient of variance (CV). Difficulty in identifying ventricular branches and the great variation of branching patterns encumbered the precise ligation of LAD in group A. The ischemic size and LVEF in group B were less variable than those of group A. There were significant correlations between the percentile of LAD ligation and infarct size or ventricular function. In conclusion, ligating LAD using its whole length rather than ventricular branches as coordinates may be more practical and advisable for establishing reproducible MI models, and this procedure may prove to help standardize the location of occlusion and infarct size.

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Many neuropharmacological agents modulate the activity and conformation of heptahelical G protein-coupled receptors and activate ligand-specific signaling pathways. The hallucinogenic chemical 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI), a serotonin receptor 2A (5-HT2AR) agonist, evokes extracellular signal-regulated kinase 1/2 (ERK1/2) signaling and head-twitch behavior. We previously reported that the senescence accelerated-prone mouse 6 (SAMP6) exhibited altered emotional behavior and increased levels of a serotonin-biosynthesizing enzyme compared to the senescence accelerated-resistant mouse 1 (SAMR1); however, the mechanism underlying the relationship between specific receptor signaling and behavioral phenotypes was unclear. In this study, we performed head-twitch tests and examined the total and phosphorylated levels of ERK1/2 and cAMP-responsive element-binding protein (CREB) in the bilateral somatosensory cortex to assess the differences between SAMP6 and SAMR1 using DOI. Although DOI dose-dependently increased the head-twitch response in both strains, the responses of SAMP6 given 0.3 and 1.0?mg/kg DOI were significantly greater than those of SAMR1 given DOI at the same doses. Although no dose-dependent increase in total ERK1/2 and total CREB expression was detected in response to DOI, the levels of phospho-ERK1/2 and -CREB increased in both strains. The phospho-ERK1/2 and -CREB levels in SAMP6 given 0.3 and 1.0?mg/kg DOI were significantly higher than those in SAMR1 given DOI at the same doses. These results indicate that SAMP6 increases DOI-dependent ERK1/2-CREB signaling leading to more head-twitch responses than SAMR1, and that SAMP6 could provide a useful model for examining the relationship between 5-HT2AR regulatory signaling and behavioral phenotypes.

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The full-length cDNA sequence of canine excitatory amino acid transporter (EAAT) 5 was determined in samples taken from the canine retina. The sequence was 2,467 bp long and was predicted to encode the 560 amino acid polypeptides. The deduced amino acid sequence of canine EAAT5 showed similarities of 91.8 and 92.7% to those of humans and rats, respectively. In canine, EAAT5 has a 49.4% identity with EAAT1, 43.7% with EAAT2, 46.4% with EAAT3, and 45.7% with EAAT4. RT-PCR analysis revealed EAAT5 expression in primary lens epithelial cell culture and the cerebellum, and Western blot analysis detected a single band of 60 kDa which confirmed EAAT5 protein expression in these cells. In addition, all subtypes of EAAT were detected in canine lens epithelial cells, indicating the pivotal role of EAATs in supplying glutamate, the precursor of antioxidant glutathione in the lens.

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Apolipoprotein B-48 (apoB-48) is produced by the small intestine, is associated with chylomicrons, and appears to be a suitable marker for clinical studies of postprandial dynamics of lipoproteins. It is also associated with cardiovascular risk factors. We have developed an assay system to quantify immuno-reactive apoB-48 in rabbit serum or plasma. A microtiter-plate was coated with monoclonal antibody raised against human apoB-48 C-terminal specific decapeptide that has high homology to the rabbit C-terminal sequence. Appropriate ELISA standard curves were obtained using apoB-48 extracted from rabbit serum by immuno-affinity chromatography. No cross-reactivity was found with apoB-100 in western blot analyses. Intra- and inter-assay CVs were less than 3%. Recovery of rabbit apoB-48 spiked in serum was within 93.4–105%. ApoB-48 levels in healthy controls rabbits fed a normal diet were within the range of 0.903–1.09 μg/ml (mean ± SD: 1.03 ± 0.084 μg/ml). In healthy animals, the blood apoB-48 level was markedly increased by a high fat diet and in the postprandial state in parallel with serum triglyceride. Ezetimibe, cholesterol absorption inhibitor, given orally to rabbits fed on a high fat diet blocked further increase of blood levels of apoB-48 and triglyceride. This method for measuring apoB-48 using the monoclonal antibody is simple, reliable, and suitable for routine analyses.

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The Matsumoto Eosinophilia Shinshu (MES) rat strain develops hereditary blood eosinophilia and eosinophil-related inflammatory lesions in organs due to the mutant Cyba^mes gene. We hypothesized that a new eosinophilia model with a different phenotype could be established by changing the genetic background of rats. We bred and characterized a congenic strain, in which the mutant Cyba^mes gene was introduced into the background of a BN strain (BN.MES-Cyba^mes). The congenic rats showed robust proliferation of eosinophils in the bone marrow. Nonetheless, blood eosinophil levels of the rats remained within the normal range. In addition, the rats manifested focal necrosis with eosinophilic infiltration in the liver, a phenotype rarely observed in the original MES rat strain. These results imply the presence of genetic polymorphisms between MES and BN strains which modulate the mobilization of eosinophils to the peripheral circulation and organs. The newly established BN.MES-Cyba^mes congenic rat strain, together with the original MES strain, will provide useful models for elucidating the molecular genetic mechanisms involved in the development and trafficking of eosinophils.

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The renin-angiotensin system plays a central role in the pathological mechanisms of diabetic nephropathy and is regulated by renal expression of cyclooxygenase-2 (COX-2). In the present study, the kidneys of diabetic KK-A^y mice, a model of human type 2 diabetes, were investigated histologically and immunohistochemically at 8, 12, 16, and 20 weeks of age, and changes in renal lesions and expression of COX-2 and renin were evaluated quantitatively. Glomerular damage, characterized by expansion of mesangial matrices and nodular lesions, was observed in the kidneys of these mice. The glomerular sclerosis score gradually increased with age and was significantly higher than those of age-matched control C57BL/6 mice at 12, 16, and 20 weeks of age. Although mild tubulointerstitial damage was observed, there was no significant change in the interstitial fibrosis score. These findings were considered early diabetic nephropathy changes. COX-2-positive signals were consistently detected in the macula densa cells of the thick ascending limbs in all KK-A^y mice, with a slightly higher score observed at 8 weeks of age. No COX-2-positive signals were detected in C57BL/6 mice. Renin-positive signals were commonly detected in the juxtaglomerular arterioles, and the scores in KK-A^y mice increased at 16 weeks and decreased at 20 weeks of age. The present study demonstrated activation of renal COX-2 and renin expression in diabetic KK-A^y mice at different stages. This finding suggests that these two enzymes contribute to the development and progression of diabetic nephropathy via different mechanisms.

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The inhibitory effects of yogurt consisting of milk fermented by Lactobacillus delbrueckii subsp. bulgaricus strain 2038 and Streptococcus salivarius subsp. thermophilus strain 1131 on formation of colonic aberrant crypt foci (ACF) in rats and also on development of colorectal tumors in transgenic mice harboring human prototype c-Ha-ras genes (rasH2 mice) were examined. F344 rats and rasH2 mice were fed commercial diet containing freeze-dried yogurt or starter medium (non-fermented milk). Rats were inoculated orally with heterocyclic amine 2-amino-methyl-6-phenylimidazo[4,5-b]pyridine hydrochloride (PhIP) for two weeks. The rats were necropsied 14 days after the PhIP treatment, and ACF in the colon and rectum were counted. RasH2 mice were injected with 1,2-dimethylhydrazine dihydrochloride (DMH) for 20 weeks. Three weeks after the last injection of DMH, rasH2 mice were necropsied to determine the number and the size of colorectal tumors. Yogurt supplementation in diet significantly reduced the number of ACF and aberrant crypts (ACs) in rats fed control diet (P<0.01), but not in rats fed non-fermented milk diet. On the other hand, rasH2 mice receiving the yogurt-supplemented diet had significantly reduced numbers of tumors induced by DMH compared with those fed the non-fermented milk-supplemented diet (P<0.05). These results demonstrate that the yogurt used in this study appears to have tumor-suppressing properties, and rasH2 mice are a useful model for the evaluation of antitumor activities of foods.

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To establish the cutoff values for screening ENU-induced behavioral mutations, normal variations in mouse behavioral data were examined in home-cage activity (HA), open-field (OF), and passive-avoidance (PA) tests. We defined the normal range as one that included more than 95% of the normal control values. The cutoffs were defined to identify outliers yielding values that deviated from the normal by less than 5% for C57BL/6J, DBA/2J, DBF₁, and N₂ (DXDB) progenies. Cutoff values for G1-phenodeviant (DBF₁) identification were defined based on values over ± 3.0 SD from the mean of DBF₁ for all parameters assessed in the HA and OF tests. For the PA test, the cutoff values were defined based on whether the mice met the learning criterion during the 2nd (at a shock intensity of 0.3 mA) or the 3rd (at a shock intensity of 0.15 mA) retention test. For several parameters, the lower outliers were undetectable as the calculated cutoffs were negative values. Based on the cutoff criteria, we identified 275 behavioral phenodeviants among 2,646 G1 progeny. Of these, 64 were crossed with wild-type DBA/2J individuals, and the phenotype transmission was examined in the G2 progeny using the cutoffs defined for N₂ mice. In the G2 mice, we identified 15 novel dominant mutants exhibiting behavioral abnormalities, including hyperactivity in the HA or OF tests, hypoactivity in the OF test, and PA deficits. Genetic and detailed behavioral analysis of these ENU-induced mutants will provide novel insights into the molecular mechanisms underlying behavior.

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The objective of the present study was to conduct the genetic characterization of nine experimental chicken lines based on multilocus microsatellite analysis. Commercial chicken lines were also analyzed in order to compare their levels of genetic uniformity with those of the experimental lines. In six experimental lines, more than 80% of genotyped loci showed fixed allele for all individuals in each line, whereas only 17.5% of genotyped loci were fixed in commercial lines, at the maximum. One of experimental lines (GSN/1) was categorized as a highly inbred line on the basis of all individuals having the same, single allele at every microsatellite locus. Genetic information obtained from the present study should be helpful for the utilization and management of experimental chicken resources.

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We investigated the effects of a bioartificial endocrine pancreas (Bio-AEP) produced by mouse β cells on sexual dysfunction of streptozotocin (STZ)-induced diabetic female rats. Female rats were administered STZ (60 mg/kg BW, i.v.) at the age of 10 weeks and transplanted with a Bio-AEP including mouse β cells at the age of 14 weeks (STZ+Bio-AEP group). Lordosis and proceptive sexual behavior of female rats were observed. The results showed that after the Bio-AEP transplant blood glucose recovered from 380–450 mg/dl induced by streptozotocin to 140–230 mg/dl and suppressed lordosis and proceptive behavior also recovered. These results suggest that it is possible to reverse sexual dysfunction by continuous administration of mouse insulin.

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The effects of progesterone (P4) used in physiological studies and in delayed parturition in reproductive engineering were examined. A dose of 0.25, 0.5, 1, or 2 mg of P4 was repeatedly administered to Jcl:MCH(ICR) mice on days 17 and 18 of gestation, and plasma concentrations of P4 were investigated. The P4 concentrations in mothers and fetuses after administration of exogenous P4 were no differences between doses of 1 and 2 mg. Jcl:MCH(ICR) mothers administered a P4 dose of 1 mg did not give birth. Therefore, we consider 2 mg of P4 is an overdose and that it is evident that a dose of 1 mg P4 is sufficient to induce delayed parturition.

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The Spontaneously Diabetic Torii-Lepr^fa (SDT-fa/fa) rat, a new model of obese type 2 diabetes, shows obesity, hyperglycemia, and hyperlipidemia from 6 weeks of age. Diabetic complications such as nephropathy and cataract are observed with aging; however, blood pressure change with age has not previously been examined. In this study, blood pressure was periodically measured and the change was investigated. Blood pressure in male SDT-fa/fa rats was elevated at 8, 16, and 24 weeks of age, whereas the heart rate was not changed. In addition to hyperglycemia, hyperlipidemia, and proteinuria, hyperleptinemia and increased urine angiotensinogen were observed in SDT-fa/fa rats. Blood pressure and heart rate in the male original SDT (SDT-+/+) rat did not significantly change. In conclusion, the SDT-fa/fa rat is a promising model, showing significant hypertension with diabetes mellitus.

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A survey on the number of live laboratory animals reared in research institutions, including universities, biomedical institutes, testing laboratories, pharmaceutical companies, and animal breeders, was conducted on June 1, 2009. One thousand seventy-four replies were recovered from 1,593 institutions, and 471 of them had used live laboratory animals from June 1, 2008 to May 31, 2009. A total of 11,337,334 live laboratory animals were being maintained on June 1, 2009 in Japan.

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