Dominant-negative mutant hepatocyte nuclear factor 1α induces diabetes in transgenic-cloned pigs

Pigs have been recognized as an excellent biomedical model for investigating a variety of human health issues. We developed genetically modified pigs that exhibit the apparent symptoms of diabetes. Transgenic cloned pigs carrying a mutant human hepatocyte nuclear factor 1α gene, which is known to cause the type 3 form of maturity-onset diabetes of the young, were produced using a combined technology of intracytoplasmic sperm injection-mediated gene transfer and somatic cell nuclear transfer. Although most of the 22 cloned offspring obtained died before weaning, four pigs that lived for 20–196 days were diagnosed as diabetes mellitus with nonfasting blood glucose levels greater than 200 mg/dl. Oral glucose tolerance test on a cloned pig also revealed a significant increase of blood glucose level after glucose loading. Histochemical analysis of pancreas tissue from the cloned pigs showed small and irregularly formed Langerhans Islets, in which poor insulin secretion was detected.     

Pearl bodies of Cayratia japonica (Thunb.) Gagnep. (Vitaceae) as alternative food for a predatory mite Euseius sojaensis (Ehara) (Acari: Phytoseiidae)

On the young leaves, shoots, and buds of Cayratia japonica (Thunb.) Gagnep. (Vitaceae), we observed nutritious bodies called pearl bodies and hypothesized that they are utilized by generalist predators as alternative foods. Some ambulate organisms consume pearl bodies in the wild and the predatory mite Euseius sojaensis (Ehara) (Acari: Phytoseiidae) was considered as a primary candidate. Pearl bodies promoted E. sojaensis settlement on C. japonica leaves and E. sojaensis could prey on the phytophagous mite Tetranychus kanzawai Kishida (Acari: Tetranychidae) when the predators settle on a leaf before the prey. In addition, the presence of pearl bodies did not reduce predation of E. sojaensis on T. kanzawai. This was seemingly because food quality of T. kanzawai was higher than pearl bodies. These results implied that pearl bodies on C. japonica leaves are utilized by E. sojaensis as alternative foods.     

Mouse hepatoblasts at distinct developmental stages are characterized by expression of EpCAM and DLK1: Drastic change of EpCAM expression during liver development

Hepatoblasts are hepatic progenitor cells that expand and give rise to either hepatocyte or cholangiocytes during liver development. We previously reported that delta-like 1 homolog (DLK1) is expressed in the mouse liver primordium at embryonic day (E) 10.5 and that DLK1⁺ cells in E14.5 liver contain high proliferative and bipotential hepatoblasts. While the expression of epithelial cell adhesion molecule (EpCAM) in hepatic stem/progenitor cells has been reported, its expression profile at an early stage of liver development remains unknown. In this study, we show that EpCAM is expressed in mouse liver bud at E9.5 and that EpCAM⁺DLK1⁺ hepatoblasts form hepatic cords at the early stage of hepatogenesis. DLK1⁺ cells of E11.5 liver were fractionated into EpCAM⁺ and EpCAM⁻ cells; one forth of EpCAM⁺DLK1⁺ cells formed a colony in vitro whereas EpCAM⁻DLK1⁺ cells rarely did it. Moreover, EpCAM⁺DLK1⁺ cells contained cells capable of forming a large colony, indicating that EpCAM⁺DLK1⁺ cells in E11.5 liver contain early hepatoblasts with high proliferation potential. Interestingly, EpCAM expression in hepatoblasts was dramatically reduced along with liver development and the colony-forming capacities of both EpCAM⁺DLK1⁺ and EpCAM⁻DLK1⁺ cells were comparable in E14.5 liver. It strongly suggested that most of mouse hepatoblasts are losing EpCAM expression at this stage. Moreover, we provide evidence that EpCAM⁺DLK1⁺ cells in E11.5 liver contain extrahepatic bile duct cells as well as hepatoblasts, while EpCAM⁻DLK1⁺ cells contain mesothelial cell precursors. Thus, the expression of EpCAM and DLK1 suggests the developmental pathways of mouse liver progenitors.     

Effect of sphingomyelin headgroup size on molecular properties and interactions with cholesterol

Sphingomyelins (SMs) and sterols are important constituents of the plasma membrane and have also been identified as major lipid components in membrane rafts. Using SM analogs with decreasing headgroup methylation, we systemically analyzed the effect of headgroup size on membrane properties and interactions with cholesterol. An increase in headgroup size resulted in a decrease in the main phase transition. Atom-scale molecular-dynamics simulations were in agreement with the fluorescence anisotropy experiments, showing that molecular areas increased and acyl chain order decreased with increasing headgroup size. Furthermore, the transition temperatures were constantly higher for SM headgroup analogs compared to corresponding phosphatidylcholine headgroup analogs. The sterol affinity for phospholipid bilayers was assessed using a sterol-partitioning assay and an increased headgroup size increased sterol affinity for the bilayer, with a higher sterol affinity for SM analogs as compared to phosphatidylcholine analogs. Moreover, the size of the headgroup affected the formation and composition of cholesterol-containing ordered domains. Palmitoyl-SM (the largest headgroup) seemed to attract more cholesterol into ordered domains than the other SM analogs with smaller headgroups. The ordering and condensing effect of cholesterol on membrane lipids was also largest for palmitoyl-SM as compared to the smaller SM analogs. The results show that the size of the SM headgroup is crucially important for SM-SM and SM-sterol interactions. Our results further emphasize that interfacial electrostatic interactions are important for stabilizing cholesterol interactions with SMs.     

Characterization of the Molecular Mechanism Underlying Gibberellin Perception Complex Formation in Rice

The DELLA protein SLENDER RICE1 (SLR1) is a repressor of gibberellin (GA) signaling in rice (Oryza sativa), and most of the GA-associated responses are induced upon SLR1 degradation. It is assumed that interaction between GIBBERELLIN INSENSITIVE DWARF1 (GID1) and the N-terminal DELLA/TVHYNP motif of SLR1 triggers F-box protein GID2-mediated SLR1 degradation. We identified a semidominant dwarf mutant, Slr1-d4, which contains a mutation in the region encoding the C-terminal GRAS domain of SLR1 (SLR1^G576V). The GA-dependent degradation of SLR1^G576V was reduced in Slr1-d4, and compared with SLR1, SLR1^G576V showed reduced interaction with GID1 and almost none with GID2 when tested in yeast cells. Surface plasmon resonance of GID1-SLR1 and GID1-SLR1^G576V interactions revealed that the GRAS domain of SLR1 functions to stabilize the GID1-SLR1 interaction by reducing its dissociation rate and that the G576V substitution in SLR1 diminishes this stability. These results suggest that the stable interaction of GID1-SLR1 through the GRAS domain is essential for the recognition of SLR1 by GID2. We propose that when the DELLA/TVHYNP motif of SLR1 binds with GID1, it enables the GRAS domain of SLR1 to interact with GID1 and that the stable GID1-SLR1 complex is efficiently recognized by GID2.     

Enrichment of regulatory CD4(+)CD25(+) T cells by inhibition of phospholipase D signaling

Antigen stimulation of lymphocytes induces upregulation of phospholipase D (PLD) activity, but the biological significance of PLD-mediated signaling in T cells has not been well established. Here we demonstrate that PLD signaling is essential for proliferation of mouse CD8(+) T cells and CD4(+)CD25(-) T cells, but is not required for proliferation of CD4(+)CD25(+) regulatory T cells. We exploited this observation to develop an efficient method to enrich for regulatory T cells starting from preparations of total CD4(+) T lymphocytes. Inhibition of PLD signaling blocked effector T-cell proliferation after T cell-antigen receptor (TCR) engagement, but had no significant effect on the proliferation of CD4(+)CD25(+) T cells with regulatory functions. Consequently, cells expanded in vitro for one week by antigen receptor stimulation with PLD signal inhibition were markedly enriched for regulatory T cells.     

Low pKa lysine residues at the active site of sarcosine oxidase from Corynebacterium sp. U-96

Sarcosine oxidase from Corynebacterium sp. U-96 is inactivated by iodoacetamide with the modification of two specific residues. Comparing the amino acid sequence and mass spectra of the peptide fragments containing the modified residues with those from the native enzyme, the modified residues were identified to be lysine. The pKa of these residues were estimated to be 8.5 and 6.7 from the pH dependence of inactivation in the presence and absence of the competitive inhibitor, acetate. These estimated pKa values are much lower than that of the epsilon-amino group of lysine residue. There may be unique microenvironments around these residues that activate their -amino groups to be susceptible to iodoacetamide. A possible role of the lysine residue with pKa 6.7 is discussed.     

Non-genomic modulation of dopamine release by bisphenol-A in PC12 cells

An endocrine disruptor chemical, bisphenol-A (BPA), is reported to have several short-term actions in various tissues and/or cells; however, the mechanisms of these actions have not been fully elucidated. We investigated short-term actions evoked by BPA in pheochromocytoma PC12 cells. BPA elicited dopamine release in PC12 cells in a dose-dependent manner. A selective N-type calcium channel antagonist (omega-conotoxin GVIA) and a ryanodine receptor blocker (ryanodine) inhibited the BPA-induced dopamine release. The expression of ryanodine receptor mRNA was detected by RT-PCR in PC12 cells. Subsequently, in order to prove whether membrane receptors participate in BPA-evoked dopamine release, a guanine nucleotide-binding protein inhibitor [guanosine 5'-(beta-thio) diphosphate], cyclic AMP antagonist (Rp-cAMPS) or protein kinase A inhibitor (H7 or H89) was added to PC12 cells prior to BPA-treatment. All of these agents suppressed BPA-evoked dopamine release, indicating that multiple signaling pathways may be involved in BPA-evoked dopamine release in PC12 cells. In conclusion, we demonstrated that BPA induced dopamine release in a non-genomic manner through guanine nucleotide-binding protein and N-type calcium channels. These findings illustrate a novel function of BPA and suggest that exposure to BPA influences the function of dopaminergic neurons.