A new tandem repeat in bovine fibrinogen Aalpha gene

In this study, we describe intraspecies variation in the alphaC connector region of the bovine fibrinogen Aalpha gene. Sequencing and genotyping of six bovine breeds revealed 7 to 10 tandem repeats in the alphaC connector region. In addition, we observed length differences between B. indicus and B. taurus, with the B. indicus having longer fibrinogen alphaC connectors (10-repeat alleles) than B. taurus (7- and 9-repeats). The difference in tandem repeats may be related to the function of blood coagulation system.     

Synergistic effects of MAP2 and MAP1B knockout in neuronal migration, dendritic outgrowth, and microtubule organization

MAP1B and MAP2 are major members of neuronal microtubule-associated proteins (MAPs). To gain insights into the function of MAP2 in vivo, we generated MAP2-deficient (map2(-/-)) mice. They developed without any apparent abnormalities, which indicates that MAP2 is dispensable in mouse survival. Because previous reports suggest a functional redundancy among MAPs, we next generated mice lacking both MAP2 and MAP1B to test their possible synergistic functions in vivo. Map2(-/-)map1b(-/-) mice died in their perinatal period. They showed not only fiber tract malformations but also disrupted cortical patterning caused by retarded neuronal migration. In spite of this, their cortical layer maintained an "inside-out" pattern. Detailed observation of primary cultures of hippocampal neurons from map2(-/-)map1b(-/-) mice revealed inhibited microtubule bundling and neurite elongation. In these neurons, synergistic effects caused by the loss of MAP2 and MAP1B were more apparent in dendrites than in axons. The spacing of microtubules was reduced significantly in map2(-/-)map1b(-/-) mice in vitro and in vivo. These results suggest that MAP2 and MAP1B have overlapping functions in neuronal migration and neurite outgrowth by organizing microtubules in developing neurons both for axonal and dendritic morphogenesis but more dominantly for dendritic morphogenesis.     

Localization of cytochrome P-450 in the colonic mucosa of 3-methylcholanthrene-pretreated and untreated rats An immunohistochemical study

Summary Immunohistochemical localization of cytochrome P-450 in the colonic mucosa of 3-methylcholanthrene-pretreated and untreated rats was studied by indirect fluorescent antibody staining technique. A polyclonal antibody for cytochrome P-450_MC purified from hepatic microsomes of 3-methylcholanthrene-pretreated rats was used for this experiment. A strong immunofluorescence was found to be localized in the cytoplasm of the surface epithelium of the mucosa in the colon of 3-methylcholanthrene-pretreated rats. A faint immunofluorescence was also observed in the epithelium of untreated rats. 7-Ethoxycoumarin O-deethylase activity of colonic microsomes was significantly enhanced by 3-methylcholanthrene-pretreatment in parallel with an increase in the intensity of immunostaining for cytochrome P-450_MC in Western blotting analysis. This is the first report on the localization of cytochrome P-450 in the colonic mucosa.     

Indicators of hippocampal neurogenesis are altered by 56Fe-particle irradiation in a dose-dependent manner

The health risks to astronauts exposed to high-LET radiation include possible cognitive deficits. The pathogenesis of radiation-induced cognitive injury is unknown but may involve loss of neural precursor cells from the subgranular zone (SGZ) of the hippocampal dentate gyrus. To address this hypothesis, adult female C57BL/6 mice received whole-body irradiation with a 1 GeV/nucleon iron-particle beam in a single fraction of 0, 1, 2 and 3 Gy. Two months later mice were given BrdU injections to label proliferating cells. Subsequently, hippocampal tissue was assessed using immunohistochemistry for detection of proliferating cells and immature neurons. Routine histopathological methods were used to qualitatively assess tissue/cell morphology in the hippocampal formation and adjacent areas. When compared to controls, irradiated mice showed progressively fewer BrdU-positive cells as a function of dose. This observation was confirmed by Ki-67 immunostaining in the SGZ showing reductions in a dose-dependent fashion. The progeny of the proliferating SGZ cells, i.e. immature neurons, were visualized by doublecortin staining and were significantly reduced by irradiation, with the decreases ranging from 34% after 1 Gy to 71% after 3 Gy. Histopathology showed that in addition to cell changes in the SGZ, (56)Fe particles induced a chronic and diffuse astrocytosis and changes in pyramidal neurons in and around the hippocampal formation. The present data provide the first evidence that high-LET radiation has deleterious effects on cells associated with hippocampal neurogenesis.     

Enzymatic and nonenzymatic formation of reactive oxygen species from 6-anilino-5,8-quinolinequinone

The nonenzymatic and enzymatic formation of reactive oxygen species (ROS) from LY83583 (6-anilino-5,8-quinolinequinone) was investigated by electron paramagnetic resonance (EPR) spectroscopy. In the presence of thiol compounds such as glutathione and L-cysteine, LY83583 underwent a one-electron reduction due to low redox potential (-0.3+/-0.01 V vs. SCE), followed by formation of LY83583 semiquinone anion radical. This species was characterized by EPR spectroscopy under an argon atmosphere at neutral pH. Under an aerobic condition, this species interacts with molecular oxygen to form a superoxide anion radical. GSH-conjugated LY83583 was also identified by NMR and FAB-MS. When LY83583 was applied to PC12 cells, ROS formation was completely inhibited by both the flavoenzyme inhibitor DPI and the DT-diaphorase inhibitor dicumarol. On the other hand, ROS generation occurred independent of intracellular GSH level. These results indicate that LY83583 can generate ROS both enzymatically and nonenzymatically, although the enzymatic formation is dominant over the nonenzymatic system in PC12 cells.     

Molecular characterization of MHC class I and beta-2 microglobulin in a clonal strain of ginbuna crucian carp, Carassius auratus langsdorfii

Clonal ginbuna crucian carp is, a naturally gynogenetic fish, and is a useful model animal for studying T-cell-mediated immunity. To gain molecular information on MHC class I molecules from this species, we have identified four types of MHC class I (caauUA-S3n, caauUF-S3n, caauZE-S3n, and caauZB-S3n) and five beta 2-microglobulin (β(2)m) (caauβ2m-1a, caauβ2m-1b, caauβ2m-2, caauβ2m-3a and caauβ2m-3b) by an expressed sequence tag (EST) analysis and using homology cloning with degenerated primers. Like UA class I genes in other cyprinid fish, the caauUA-S3n shows features of classical MHC class I, such as conservation of all key amino acids interacting with antigenic peptides, and ubiquitous tissue expression. A phylogenetic analysis shows that the β(2)m-1 and β(2)m-2 isoforms are clustered with those of other cyprinid fishes, while β(2)m-3 isoforms make a cluster that is separated from a common ancestor of salmonid and cyprinid fishes. This finding suggests that the β(2)m isoforms of ginbuna cruician carp comprise two lineages and may possess different functions. The MHC class I and β(2)m sequences from one clonal strain will facilitate our understanding of the interaction of MHC class I with β(2)m in teleosts.     

In vitro induction of luteinizing hormone synthesis by estrogen in organ-cultured pituitary glands of the Japanese eel,Anguilla japonica

An organ culture method for pituitary glands isolated from immature Japanese eels (Anguilla japonica) was developed. This method could conserve the histological features of the pituitary glands for at least 21 days. The ability to synthesize gonadotropic hormone (GTH) in cultured eel pituitary glands was examined by detecting luteinizing hormone (LH) beta protein immunohistochemically. In a basal medium (Leibovitz L-15), LH beta-immunoreactive cells were very scarce, but after addition of estradiol-17beta (E2) a large number of immunoreactive cells appeared, particularly in the proximal pars distalis. The stimulatory effects of E2 on LH beta synthesis were dose (1-100 ng/ml)- and time (1.5-7 days)-dependent. Thus, in contrast with previous reports of the lack of a direct effect of E2 on GTH synthesis in primary cultured eel pituitary cells, the present results clearly indicate that E2 can stimulate GTH synthesis in immature eel pituitary glands. This organ culture method is useful to examine the actions of steroids and also other endocrine factors on the eel pituitary gland.