High Fos expression during the active phase in orexin neurons of a diurnal rodent, Tamias sibiricus barberi

To investigate whether a diurnal animal possesses the orexinergic system implicating vigilance and behavior, we examined Fos immunoreactivity (IR) in orexinergic neurons of Korean chipmunks raised under 12h light-dark cycles. Brain tissue, collected at four different zeitgeber times (ZT), was double-labeled with Fos and orexin-A antibodies. There was no difference in the number of orexin-IR neurons in the hypothalamus across all ZTs. However, more orexin-IR neurons expressing Fos-IR were found at ZTs 3 and 9 than ZTs 15 and 21. The results demonstrate circadian variations in the activation of orexin neurons corresponding with locomotor cycles, similarly seen in nocturnal rodents.     

Identification of AQP5 in lipid rafts and its translocation to apical membranes by activation of M3 mAChRs in interlobular ducts of rat parotid gland

Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. M₃ muscarinic acetylcholine receptor (mAChR)-induced changes in AQP5 localization in rat parotid glands were investigated with immunofluorescence or immunoelectron microscopy, detergent solubility, and gradient density floatation assays. Confocal microscopy revealed AQP5 localization in intracellular vesicles of interlobular duct cells in rat parotid glands and AQP5 trafficking to the APM 10 min after injection of the mAChR agonist cevimeline. Conversely, 60 min after injection, there was a diffuse pattern of AQP5 staining in the cell cytoplasm. The calcium ionophore A-23187 mimicked the effects of cevimeline. Immunoelectron microscopic studies confirmed that cevimeline induced AQP5 trafficking from intracellular structures to APMs in the interlobular duct cells of rat parotid glands. Lipid raft markers flotillin-2 and GM1 colocalized with AQP5 and moved with AQP5 in response to cevimeline. Under control conditions, the majority of AQP5 localized in the Triton X-100-insoluble fraction and floated to the light-density fraction on discontinuous density gradients. After 10-min incubation of parotid tissue slices with cevimeline or A-23187, AQP5 levels decreased in the Triton X-100-insoluble fraction and increased in the Triton X-100-soluble fraction. Thus AQP5 localizes in the intracellular lipid rafts, and M₃ mAChR activation induces AQP5 trafficking to the APM with lipid rafts via intracellular Ca²⁺ signaling and induces AQP5 dissociation from lipid rafts to nonrafts on the APM in the interlobular duct cells of rat parotid glands. translocation; aquaporin-5     

Systematic screening and identification of antigens recognized by monoclonal antibodies raised against the developing lateral olfactory tract

During development, olfactory bulb axons navigate a complex microenvironment composed of myriad molecules to construct a bundle called the lateral olfactory tract. The axons themselves also express thousands of different molecules. In the present study, we produced and characterized six monoclonal antibodies that label the lateral olfactory tract and its surroundings in a unique pattern. The labeling profiles suggested that the antigen molecules recognized by each antibody are heterogeneously distributed around the developing lateral olfactory tract. We developed an efficient screening method to identify the antigen molecules by combining expression of a cDNA library in COS-7 cells and the subsequent immunohistochemical staining of the cells. The systematic screening successfully identified specific cDNA clones for all of the monoclonal antibodies, which highly probably coded for the antigen molecules, and therefore unveiled the molecular nature of local components that embrace the developing lateral olfactory tract in mice.     

Frequent retention of heterozygosity for point mutations in p53 and Ikaros in N-ethyl-N-nitrosourea-induced mouse thymic lymphomas

In agreement with Knudson's two-hit theory, recent findings indicate that the inactivation of tumor suppressor genes is not only mediated by the loss of function but also by the dominant-negative or gain-of-function activity. The former generally accompanies loss of a wild-type allele whereas in the latter a wild-type allele is retained. N-Ethyl-N-nitrosourea (ENU), which efficiently induces point mutations, reportedly leads to the development of tumors by activating ras oncogenes. Little is known about how ENU affects tumor suppressor genes and, therefore, we examined ENU-induced mutations of p53 and Ikaros in thymic lymphomas and compared these with mutations of Kras. In addition, loss of heterozygosity was examined for chromosome 11 to which both p53 and Ikaros were mapped. The frequency of point mutations in p53 and Ikaros was 30% (8/27) and 19% (5/27), respectively, comparable to that observed in Kras (33%: 9/27). In total, 14 of the 27 thymic lymphomas examined (52%) harbored mutations in at least one of these genes. One Ikaros mutation was located at the splice donor site, generating a novel splice isoform lacking zinc finger 3, Ik (F3del). Interestingly, 90% (10/11) of the tumors with point mutations retained wild-type alleles of p53 and Ikaros. Sequence analysis revealed that the most common nucleic acid substitutions were T>A (4/8) in p53, T>C (4/5) in Ikaros and G>A/T (8/9) in Kras, suggesting that the spectrum of mutations was gene dependent. These results suggest that point mutations in tumor suppressor genes without loss of the wild-type allele play an important role in ENU-induced lymphomagenesis.     

Mammalian PIG-X and yeast Pbn1p are the essential components of glycosylphosphatidylinositol-mannosyltransferase I

Within the endoplasmic reticulum (ER), mannoses and glucoses, donated from dolichol-phosphate-mannose and -glucose, are transferred to N-glycan and GPI-anchor precursors, and serine/threonine residues in many proteins. Glycosyltransferases that mediate these reactions are ER-resident multitransmembrane proteins with common characteristics, forming a superfamily of >10 enzymes. Here, we report an essential component of glycosylphosphatidylinositol-mannosyltransferase I (GPI-MT-I), which transfers the first of the four mannoses in the GPI-anchor precursors. We isolated a Chinese hamster ovary (CHO) cell mutant defective in GPI-MT-I but not its catalytic component PIG-M. The mutant gene, termed phosphatidylinositolglycan-class X (PIG-X), encoded a 252-amino acid ER-resident type I transmembrane protein with a large lumenal domain. PIG-X and PIG-M formed a complex, and PIG-M expression was <10% in the absence of PIG-X, indicating that PIG-X stabilizes PIG-M. We found that Saccharomyces cerevisiae Pbn1p/YCL052Cp, which was previously reported to be involved in autoprocessing of proproteinase B, is the functional homologue of PIG-X; Pbn1p is critical for Gpi14p/YJR013Wp function, the yeast homologue of PIG-M. This is the first report of an essential subcomponent of glycosyltransferases using dolichol-phosphate-monosaccharide.     

Comparisons of germination traits of alpine plants between fellfield and snowbed habitats

We examined the seed-germination responses of 27 alpine species with reference to habitat type (fellfield and snowbed), temperature (five regimes), and light requirement. About 70% of species showed >40% germination at warm temperatures without cold stratification. However, a moist-chilling treatment markedly improved the germination percentages in most species, especially under cool conditions. Thus, cold stratification effectively reduced the temperature requirement for germination. Patterns of germination response within species were not consistent between the fellfield and snowbed habitats for species inhabiting both habitats. For interspecific comparisons, there were no significant differences in germination responses to the temperature regimes and the cold stratification between the fellfield and snowbed species. Also, germination speed and the length of germinating period did not differ between fellfield and snowbed species. Most species (86%) showed a requirement for light for germination without cold stratification. Although the extent of the light requirement was reduced after cold stratification in some species, the light requirement of most small-seeded species remained. The combination of cold stratification and the light requirement is a major factor determining the seedling emergence and formation of seed banks in alpine plants. However, habitat-specific patterns of germination traits were less clear, suggesting similar germination traits in fellfield and snowbed plants, at least under controlled conditions in the laboratory.     

Serotonin transporter polymorphisms affect human blood glucose control

We measured the effect of nutritional intervention on clinical data, including fasting blood glucose (FBG), and their association with polymorphisms of the serotonin transporter-linked polymorphic region (5-HTTLPR) which might affect adherence. Enrolled in the intervention program were 264 Japanese women not on medication for diabetes, hypercholesterolemia or hypertension. The 5-HTTLPR allele (S and L) frequencies among the subjects differed markedly from those of Caucasians: SS (n = 183), LS (n = 69), and LL (n = 12). The decrease in FBG (DeltaFBG) from the beginning to the end of the program (11 weeks; short-term study), and DeltaFBG from the beginning to a follow-up check performed between 2002 and 2004 (average of 23 years later; long-term study) was calculated. The SS homozygotes of 5-HTTLPR showed larger DeltaFBG (P = 0.01 and P < 0.0001 in the short- and long-term studies, respectively) than DeltaFBG with other genotypes.