Presence of a putative vesicular inositol 1,4,5-trisphosphate-sensitive nucleoplasmic Ca2+ store

The inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are widely localized in both the heterochromatin and euchromatin regions. We found recently the presence of nucleoplasmic complexes that are composed of phospholipids, IP(3)R/Ca(2+) channels, and Ca(2+) storage protein chromogranin B (CGB). Close examination and 3D image reconstruction of these complexes revealed numerous vesicular structures with an average diameter of approximately 50 nm that are primarily interspersed between the heterochromatins. IP(3) rapidly released Ca(2+) from these structures, but other inositol phosphates, inositol 1,4-bisphosphate, inositol 1,3,4-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate, failed to release Ca(2+). Addition of heparin or IP(3)R antibody blocked the IP(3)-induced Ca(2+) releases, indicating the release of Ca(2+) through the IP(3)R/Ca(2+) channels. Given the presence of the IP(3)R/Ca(2+) channels and Ca(2+) storage protein CGB in these vesicular structures, we postulate that these vesicles are the IP(3)-sensitive nucleoplasmic Ca(2+) stores. Abundance of the vesicular Ca(2+) stores between the heterochromatins appeared to imply critical roles these vesicular Ca(2+) stores play in controlling the Ca(2+) concentrations of the chromosomes.     

Minibrain/Dyrk1a Regulates Food Intake through the Sir2-FOXO-sNPF/NPY Pathway in Drosophila and Mammals

Feeding behavior is one of the most essential activities in animals, which is tightly regulated by neuroendocrine factors. Drosophila melanogaster short neuropeptide F (sNPF) and the mammalian functional homolog neuropeptide Y (NPY) regulate food intake. Understanding the molecular mechanism of sNPF and NPY signaling is critical to elucidate feeding regulation. Here, we found that minibrain (mnb) and the mammalian ortholog Dyrk1a target genes of sNPF and NPY signaling and regulate food intake in Drosophila melanogaster and mice. In Drosophila melanogaster neuronal cells and mouse hypothalamic cells, sNPF and NPY modulated the mnb and Dyrk1a expression through the PKA-CREB pathway. Increased Dyrk1a activated Sirt1 to regulate the deacetylation of FOXO, which potentiated FOXO-induced sNPF/NPY expression and in turn promoted food intake. Conversely, AKT-mediated insulin signaling suppressed FOXO-mediated sNPF/NPY expression, which resulted in decreasing food intake. Furthermore, human Dyrk1a transgenic mice exhibited decreased FOXO acetylation and increased NPY expression in the hypothalamus, as well as increased food intake. Our findings demonstrate that Mnb/Dyrk1a regulates food intake through the evolutionary conserved Sir2-FOXO-sNPF/NPY pathway in Drosophila melanogaster and mammals.     

Three-dimensional analysis of abnormal ultrastructural alteration in mitochondria of hippocampus of APP/PSEN1 transgenic mouse

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder. The deterioration of subcellular organelles, including the mitochondria, is another major ultrastructural characteristic of AD pathogenesis, in addition to amyloid plaque deposition. However, the three-dimensional (3-D) study of mitochondrial structural alteration in AD remains poorly understood. Therefore, ultrastructural analysis, 3-D electron tomography, and immunogold electron microscopy were performed in the present study to clarify the abnormal structural alterations in mitochondria caused by the progression of AD in APP/PSEN1 transgenic mice, expressing human amyloid precursor protein, as a model for AD. Amyloid β (Aβ) plaques accumulated and dystrophic neurites (DN) developed in the hippocampus of transgenic AD mouse brains. We also identified the loss of peroxiredoxin 3, an endogenous cytoprotective antioxidant enzyme and the accumulation of Aβ in the hippocampal mitochondria of transgenic mice, which differs from those in age-matched wild-type mice. The mitochondria in Aβ plaque-detected regions were severely disrupted, and the patterns of ultrastructural abnormalities were classified into three groups: disappearance of cristae, swelling of cristae, and bulging of the outer membrane. These results demonstrated that morpho-functional alterations of mitochondria and AD progression are closely associated and may be beneficial in investigating the function of mitochondria in AD pathogenesis.     

Proteomic analysis of the extraembryonic tissue from cloned porcine embryos

Cloned animals developed from somatic cell nuclear transfer (SCNT) embryos are useful resources for agricultural and medical applications. However, the birth rate in the cloned animals is very low, and the cloned animals that have survived show various developmental defects. In this report, we present the morphology and differentially regulated proteins in the extraembryonic tissue from SCNT embryos to understand the molecular nature of the tissue. We examined 26-day-old SCNT porcine embryos at which the sonogram can first detect pregnancy. The extraembryonic tissue from SCNT embryos was abnormally small compared with the control. In the proteomic analysis with the SCNT extraembryonic tissue, 39 proteins were identified as differentially regulated proteins. Among up-regulated proteins, Annexins and Hsp27 were found. They are closely related to the processes of apoptosis. Among down-regulated proteins, Peroxiredoxins and anaerobic glycolytic enzymes were identified. In the Western blot analysis, antioxidant enzymes and the antiapoptotic Bcl-2 protein were down-regulated, and caspases were up-regulated. In the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay with the placenta from SCNT embryos, apoptotic trophoblasts were observed. These results demonstrate that a major reason for the low birth rate of cloned animals is due to abnormal apoptosis in the extraembryonic tissue during early pregnancy.     

Development of inactivated trivalent vaccine for the teratogenic Aino,Akabane and Chuzan viruses

Aino, Akabane and Chuzan viruses are arthropod-borne (arbo) viruses transmitted by blood-sucking insects like mosquitoes and Culicoides biting midges. These arbovirus infections are mainly associated with abortion, stillbirth and congenital defects in pregnant cattle, sheep and goats, which induces a considerable economic loss in livestock industry. The viruses seem to be widely distributed in Southeast Asia and Australia. As a control strategy, an inactivated trivalent vaccine against Aino, Akabane and Chuzan virus was developed by using binary ethylenimine or formalin as an inactivating agent. The newly developed trivalent vaccine is evaluated for its safety and immunogenicity in animals such as mice, guinea pigs and cattle. The immune responses were significantly detected within 2-weeks after second vaccination without any side effects. Since the field application of experimental vaccine also revealed increased antibodies in inoculated cattle, we demonstrated that these trivalent vaccines could be used as a vaccine to control the arboviral infections in ruminants.     

Mucosa-associated epithelial chemokine/CCL28 expression in the uterus attracts CCR10+ IgA plasma cells following mucosal vaccination via estrogen control

Previous studies demonstrated cross talk between mucosal and reproductive organs during secretory IgA Ab induction. In this study, we aimed to clarify the underlying mechanisms of this cross talk. We found significantly higher titers of Ag-specific secretory IgA Ab in the vaginal wash after mucosal vaccination by both the intranasal (i.n.) and the intravaginal routes but not by the s.c. route. Interestingly, Ag-specific IgA Ab-secreting cells (ASCs) were found mainly in the uterus but not in the cervix and vaginal canal after i.n. vaccination. The fact that most Ag-specific IgA ASCs isolated from the uteri of vaccinated mice migrated toward mucosa-associated epithelial chemokine (MEC)/CCL28 suggests dominant expression of CCR10 on the IgA ASCs. Further, IgA ASCs in the uteri of vaccinated mice were reduced drastically in mice treated with neutralizing anti-MEC/CCL28 Ab. Most intriguingly, the female sex hormone estrogen directly regulated MEC/CCL28 expression and was augmented by i.n. vaccination with cholera toxin or stimulators for innate immunity. Further, blockage of estrogen function in the uterus by oral administration of the estrogen antagonist raloxifene significantly inhibited migration of Ag-specific IgA ASCs after i.n. vaccination with OVA plus cholera toxin. Taken together, these data strongly suggest that CCR10(+) IgA ASCs induced by mucosal vaccination via the i.n. route migrate into the uterus in a MEC/CCL28-dependent manner and that estrogen might have a crucial role in the protection against genital infection by regulating MEC/CCL28 expression in the uterus.     

Preparation of new tetraaza macrocyclic nickel(II) and copper(II) complexes bearing N-propionic methyl ester groups

A 14-membered tetraaza macrocycle, 2,13-bis(2-carbomethoxyethyl)-5,16-dimethyl-2,6,13,17-tetraazatricyclo[16.4.0.^1.180^7.12]docosane (L²) bearing two N-CH₂CH₂COOMe groups, and its nickel(II) and copper(II) complexes have been prepared and characterized. The nickel(II) and copper(II) complexes of 2-(2-carbomethoxyethyl)-5,16-dimethyl-2,6,13,17-tetraazatricyclo[16.4.0.^1.180^7.12]docosane (L³) containing one N-CH₂CH₂COOMe group have also been prepared. The crystal structure of [NiL²](ClO₄)₂ shows that the complex has a slightly distorted trans-octahedral coordination geometry with two relatively short axial Ni-O (N-CH₂CH₂COOMe group) bonds (2.136(3) Å). In various solvents, however, a considerable proportion of [NiL²]²⁺ exists as a square-planar form, in which the functional pendant arms are not involved in coordination. The proportion of the square-planar isomer varies with solvents in the order of nitromethane ? acetonitrile < H₂O < DMF ? DMSO. In the case of [CuL²](ClO₄)₂, only one N-CH₂CH₂COOMe group is involved in coordination. The N-CH₂CH₂COOMe group of [NiL³](ClO₄)₂ is not directly involved in coordination even in the solid state, though the functional group of [CuL³](ClO₄)₂ is coordinated to the metal ion.