The C57BL/6, 129, and B6,129 mouse strains or stocks have been commonly used to generate targeted mutant mice. The pathology of these mice is not well characterized. In studies of these aging mice, we found high incidences of hyalinosis (eosinophilic cytoplasmic change) in the glandular stomach, respiratory tract, bile duct, and gall bladder of B6,129 CYP1A2-null and wild-type mice as well as in both sexes of the background 129S4/SvJae strain. The gastric lesions of the glandular stomach were found in 95.7% of female CYP1A2-null mice as well as in 45.7% of female 129S4/SvJae animals. The eosinophilic protein isolated from characteristic hyaline gastric lesions was identified as Ym2, a member of the chitinase family. Immunohistochemistry, using rabbit polyclonal antibodies to oligopeptides derived from the Ym1 sequence, detected focal to diffuse reactivity within both normal and abnormal nasal olfactory and respiratory epithelium, pulmonary alveolar macrophages, bone marrow myeloid cells, and the squamous epithelium of the forestomach and epithelium of the glandular stomach. Alveolar macrophages in acidophilic pneumonia, a major cause of death of aging 129 mice, and in mice with the me mutation also were highly immunoreactive. CYP1A2 carries out oxidation and N-hydroxylation of arylamine carcinogens and heterocyclic amine food mutagens which, when followed by O-esterification by acetate or sulfate transferases, results in unstable electrophilic derivatives that can cause cell toxicity, cell death, or cell transformation. 1-3 CYP1A2 catalyzes caffeine 3-demethylation and metabolically activates aflatoxin B 1 to its ultimate carcinogenic intermediate 4 and paradoxically catalyzing the hydroxylation of aflatoxin B 1 to aflatoxin M 1 in a detoxification pathway. 5 In addition, CYP1A2 was found to be involved in both hamster and human catechol estrogen metabolism. 6 Although CYP1A2 is constitutively expressed in the liver of mice, rats, and humans and is inducible by ligands of the aryl hydrocarbon receptor (Ahr) in all mammalian species analyzed, no expression has been demonstrated in the stomach, even after treating animals with known inducers of this enzyme. 7 However, the mechanisms involved in CYP1A2 gene regulation are less well established than CYP1A1 because it is difficult to induce expression in vitro and the sensitivities of current methodologies may simply be too low under normal in vivo conditions. The mammalian conservation of the CYP1A subfamily argues for an essential function in metabolism not limited to activation and detoxification of exogenous chemicals and toxins. As with most of the cytochromes P-450, determination of function has primarily been a consequence of exposures to man-made compounds, but it must be remembered that their evolution predates industrialization. The genes of the two recognized members of the CYP1A subfamily,

Brain-specific angiogenesis inhibitor 2 (BAI2) is a member of adhesion-G protein-coupled receptors (GPCRs). BAI2 is dominantly expressed in the brain and its physiological ligands and functions are still unclear. Adhesion-GPCRs, including BAI2, commonly have a long N-terminal extracellular region (ECR) containing the GPCR proteolysis site (GPS) and the cleavage of the ECR at the GPS domain is suspected to be important for their function. In this study, we analyzed the proteolytic processing of BAI2 and its activation mechanism. Several cleaved C-terminal fragments of BAI2 were identified in mouse hippocampus. We confirmed that mutation in the GPS domain caused inhibition of the proteolysis of BAI2, which indicated the possibility that BAI2 was cleaved at the GPS domain. The association of the ECR putatively cleaved at the GPS domain and the C-terminal seven-transmembrane (7TM) fragment was detected by co-immunoprecipitation. We also found that furin prohormone convertase cleaved BAI2 at another site in the ECR. Additionally, the C-terminal fragment cleaved at the GPS domain specifically activated the nuclear factor of activated T-cells (NFAT) pathway. These results suggest that BAI2 is a functional GPCR regulated by proteolytic processing and activates the NFAT pathway.

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Microsomal epoxide hydrolase (mEH) is a conserved enzyme that is known to hydrolyze many drugs and carcinogens, and a few endogenous steroids and bile acids. mEH-null mice were produced and found to be fertile and have no phenotypic abnormalities thus indicating that mEH is not critical for reproduction and physiological homeostasis. mEH has also been implicated in participating in the metabolic activation of polycyclic aromatic hydrocarbon carcinogens. Embryonic fibroblast derived from the mEH-null mice were unable to produce the proximate carcinogenic metabolite of 7,12-dimethylbenz[a]anthracene (DMBA), a widely studied experimental prototype for the polycylic aromatic hydrocarbon class of chemical carcinogens. They were also resistant to DMBA-mediated toxicity. Using the two-stage initiation-promotion skin cancer bioassay, the mEH-null mice were found to be highly resistant to DMBA-induced carcinogenesis. In a complete carcinogenesis bioassay, the mEH mice were totally resistant to tumorigenesis. These data establish in an intact animal model that mEH is a key genetic determinant in DMBA carcinogenesis through its role in production of the ultimate carcinogenic metabolite of DMBA, the 3,4-diol-1,2-epoxide.Microsomal epoxide hydrolase (mEH) 1 is a critical phase I biotransformation enzyme that catalyzes hydrolysis of a large number of epoxide intermediates (1, 2). mEH is highly conserved in different mammalian species, is expressed in the embryo (3-5) and multiple organs, and is active toward some endogenous epoxy-steroids (6) and bile acids (7) thus suggesting that it plays a critical physiological role. mEH is usually thought to play a pivotal role in protection against the toxicity of reactive epoxide intermediates, because metabolism of epoxides by this enzyme results in the production of less reactive and less toxic dihydrodiol intermediates of drugs such as phenytoin and carbamazepine (8, 9) and epoxides of environmental toxins (10, 11). In contrast to this protective effect, mEH is thought to be required for the metabolic activation of the potent carcinogen 7,12-dimethylbenz[a]anthracene (DMBA), a widely studied experimental prototype for the polycylic aromatic hydrocarbon class of chemical carcinogens (12). P450s and mEH metabolize DMBA to both inert metabolites and metabolites that are electrophilic and capable of producing DNA adducts (Fig. 1). Cytochrome P450 CYP1B1 oxidizes DMBA to the 3,4-epoxide (13). This is followed by hydrolysis of the epoxide by mEH to the proximate carcinogenic metabolite, DMBA-3,4-diol. This metabolite can be further oxidized by either CYP1A1 or CYP1B1 to the principal ultimate carcinogenic metabolite, DMBA-3,4-diol-1,2-epoxide, that is capable of producing DNA adducts (14 -17). Other ring hydroxylations and methyl hydroxylations of DMBA result in inactive metabolites that do not bind DNA. Based on this scheme, mEH should be a critical enzyme in the pathway leading to the carcinogenic activity of DMBA. However a role for mEH in DMBA carcinogenesis has not been established i...

Brain-specific angiogenesis inhibitor 2 (BAI2) is a transmembrane protein that is predominantly expressed in the brain. Although BAI2 is supposed to correlate with antiangiogenesis in the brain, its psychiatric function is still unclear. In this study, we examined the influence of BAI2 gene disruption on mood-related behavior using BAI2-deficient mice. BAI2-deficient mice showed significant antidepressant-like behavior in the social defeat test and in the tail suspension test compared with wild-type mice. On the other hand, BAI2-deficient mice had normal basal locomotor activity in the home cage and in the open field test, and normal learning ability and memory retention in the Morris water maze test. Additionally, we found that hippocampal cell proliferation in BAI2-deficient mice was higher than that in wild-type mice. These results indicate that BAI2 has an important role related to depression and influences the hippocampal neurogenesis. BAI2 may be a novel therapeutic target for mood-related disorders.

Patients with aneurysmal subarachnoid hemorrhage (SAH) frequently have deficits in learning and memory that may or may not be associated with detectable brain lesions. We examined mediators of long-term potentiation after SAH in rats to determine what processes might be involved. There was a reduction in synapses in the dendritic layer of the CA1 region on transmission electron microscopy as well as reduced colocalization of microtubule-associated protein 2 (MAP2) and synaptophysin. Immunohistochemistry showed reduced staining for GluR1 and calmodulin kinase 2 and increased staining for GluR2. Myelin basic protein staining was decreased as well. There was no detectable neuronal injury by Fluoro-Jade B, TUNEL, or activated caspase-3 staining. Vasospasm of the large arteries of the circle of Willis was mild to moderate in severity. Nitric oxide was increased and superoxide anion radical was decreased in hippocampal tissue. Cerebral blood flow, measured by magnetic resonance imaging, and cerebral glucose metabolism, measured by positron emission tomography, were no different in SAH compared with control groups. The results suggest that the etiology of loss of LTP after SAH is not cerebral ischemia but may be mediated by effects of subarachnoid blood such as oxidative stress and inflammation.

These results suggest that [(11)C]PK-11195 PET imaging would be a useful tool for evaluating microglial activation in a rat brain injury model.

Quantitative small-animal PET of mice requires successful delivery of radiotracers into the venous system. Intravenous injection of radiotracers via lateral tail veins is the most commonly used method of administration and can be technically challenging. Evaluation of the quality of an intravenous injection is necessary to determine whether small-animal PET is quantitatively accurate. The purpose of this study was to evaluate and compare the quality of 50 consecutive intravenous injections into mouse tail veins using both quantitative and qualitative methods. Methods: During 18 F-FDG intravenous injection, qualitative assessment of the injection was performed and classified according to specific criteria as good, intermediate, or poor. Small-animal PET scans of the body and tail were acquired, and tail injection sites were quantitatively assessed in terms of percentage injected dose per gram and classified as low, medium, or high uptake of 18 F-FDG. Qualitative and quantitative methods were compared. To assess baseline amounts of 18 F-FDG in the tail without a tail injection, 3 additional mice were injected by the intraperitoneal method, imaged, and quantitatively assessed in the same manner. The in vivo imaging data were validated on 7 additional mice by sacrificing them after scans, removing their tails, rescanning the tails, and then measuring the tail radioactivity ex vivo in a g-counter and correlating it with the in vivo amount. Results: Validation of in vivo imaging to ex vivo data yielded an excellent correlation, with an r 2 value of 0.95. Comparison of qualitative and quantitative methods yielded 45 matching results (42 good and low, 2 intermediate and medium, and 1 poor and high). There were 5 cases of mismatching results (1 false-negative and 4 false-positive) between qualitative and quantitative methods. Low-uptake tail injections were comparable to the intraperitoneal injection values. Using qualitative methods, accuracy was true 90% (45/50) of the time. The overall rate of successful intravenous injections was 92% (46/50) using quantitative methods. Conclusion: Qualitative assessment is all that is necessary if the intravenous injection is classified as good. In intermediate, poor, or uncertain classifications, a scan of the tail should be performed for quantitative assessment.