Disruption of Drosophila melanogaster Lipid Metabolism Genes Causes Tissue Overgrowth Associated with Altered Developmental Signaling.

Developmental patterning requires the precise interplay of numerous intercellular signaling pathways to ensure that cells are properly specified during tissue formation and organogenesis. The spatiotemporal function of many developmental pathways is strongly influenced by the biosynthesis and intracellular trafficking of signaling components. Receptors and ligands must be trafficked to the cell surface where they interact, and their subsequent endocytic internalization and endosomal trafficking is critical for both signal propagation and its down-modulation. In a forward genetic screen for mutations that alter intracellular Notch receptor trafficking in Drosophila melanogaster, we recovered mutants that disrupt genes encoding serine palmitoyltransferase and acetyl-CoA carboxylase. Both mutants cause Notch, Wingless, the Epidermal Growth Factor Receptor (EFGR), and Patched to accumulate abnormally in endosomal compartments. In mosaic animals, mutant tissues exhibit an unusual non-cell-autonomous effect whereby mutant cells are functionally rescued by secreted activities emanating from adjacent wildtype tissue. Strikingly, both mutants display prominent tissue overgrowth phenotypes that are partially attributable to altered Notch and Wnt signaling. Our analysis of the mutants demonstrates genetic links between abnormal lipid metabolism, perturbations in developmental signaling, and aberrant cell proliferation

Co-transfection of EYFP-GH and ECFP-rab3B in an experimental pituitary GH3 cell: a role of rab3B in secretion of GH through porosome.

Recently, in order to elucidate the role of rab3B in porosome, we have observed the incorporation of rab3B in the secretion of GH through porosome under confocal laser scanning microscopy (CLSM). Transfected cells with GH-EYFP fusion protein and rab3B-ECFP fusion protein were observed under CLSM, which showed the colocalization of EYFP-GH and ECFP-rab3B in the budding configuration of secretory process. These structural and functional images of rab3B imply the incorporation of rab3B in the secretion of GH through porosome

シナプスマーカーVGLUT1, VGAT を用いた嗅球神経回路の新たな形態学的解析

嗅覚の一次中枢である嗅球の投射ニューロン（僧帽細胞，房飾細胞）は，その樹状突起上で樹状突起間シナプスを形成したのちに，高次の脳中枢に情報を伝達する．従って，僧帽細胞の突起上のシナプスの分布を明らかにすることは，匂い情報調節を解析する上で必須である．従来，シナプス結合を形態学的に同定するものは高解像度な電子顕微鏡による解析であった．しかし，電子顕微鏡では，観察できる領域は限られ，広範囲の解析を行うのは困難であり，僧帽細胞を厳密に同定することもできない．Vesicular glutamate transporter（VGLUT1）やvesicular GABA transporter（VGAT）などのシナプスマーカーの有用性が最近多くの脳領域で報告されており，これらのマーカーにより電子顕微鏡による解析以上に，嗅球でも信頼性の高いシナプスの定量ができるかもしれない．本研究では，新たなマーカーによる，樹状突起間シナプスの同定の有用性をマウスの嗅球で検討した．まず，嗅球を抗VGLUT1抗体，抗VGAT 抗体で単染色し，電子顕微鏡で観察すると，非対称性シナプス，対称性シナプスのシナプス終末がそれぞれ標識されることが確認できた．次に単一の僧帽細胞をウイルスベクター注入により標識し，抗VGLUT1抗体，抗VGAT 抗体で多重染色して, 僧帽細胞とVGLUT1，VGAT との共存部位を共焦点レーザー顕微鏡で観察した．次いで同部位を電子顕微鏡で同定し，微細構造を解析した．その結果，VGLUT1陽性部位のうち82％に非対称性シナプス，VGAT 陽性部位のうち79% に対称性シナプスが電子顕微鏡で同定でき，VGLUT1，VGAT が，嗅球のシナプスの質的な解析や定量のためのマーカーとして信頼できると結論づけた．また，抗VGLUT1抗体，抗VGAT 抗体で二重染色したものを，高解像度でモンタージュ撮影すると，VGLUT1が外網状層を中心，VGAT が糸球体層に多く分布し，外網状層に中等度分布するなど，新たな知見が得られた．これらの結果は，VGLUT1とVGAT が，シナプスの同定や広範囲の脳領域のシナプスの分布を解析するために有用なマーカーであるということを示している．The olfactory bulb (OB) is the primary center of the olfactory system in the brain. The projection neurons of the OB, mitral cells and tufted cells, form dendro-dendritic synapses with interneurons on their dendrites and relay olfactory information to higher brain regions. Thus, it is necessary to identify the distribution of synapses on mitral cell dendrites to study the processing of olfactory information. High-resolution analysis by electron microscopy (EM) enables morphological identification of synapses; however, examination of large areas is difficult with EM because of its narrow observation range. Further, mitral cells cannot be clearly identified with EM. Vesicular glutamate transporter 1 (VGLUT1) and vesicular GABA transporter (VGAT) are synapse markers and the usefulness of them for estimating synaptic distributions has been reported in many brain regions. Using these markers for quantitative analysis of synapses on dendrites of OB projection neurons may provide higher reliability than EM analysis. In this study, we examined the usefulness of these markers for identification of dendro-dendritic synapses in the mouse OB. First, the OB was single-immunolabeled for VGLUT1 or VGAT and processed for EM. Using EM analysis, we observed VGLUT1 labeling in asymmetrical synapses and VGAT labeling in symmetrical synapses. Second, individual mitral cells were selectively labeled by viral injection and single or double-labeled for VGLUT1, VGAT or both. Labeled sites on mitral cells were analyzed with confocal laser scanning microscopy and subsequently examined with EM. We identified that 82% of VGLUT1-positive and 79% of VGAT-positive sites corresponded to asymmetrical synapses and symmetrical synapses identified by EM, respectively. Thus, we conclude that these synaptic markers are reliable for qualitative observation and quantification of synapses in the OB. Finally, the OB was doublelabeled for VGLUT1 and VGAT, and examined by confocal microscopy with high-resolution montage imaging to analyze distributions of the synaptic markers. VGLUT1 was mainly distributed in the external plexiform layer. VGAT was mainly distributed in the glomerular layer and moderately in the external plexiform layer. These findings indicate that VGLUT1 and VGAT are reliable presynaptic markers for individual synapses and for distribution throughout a brain region

PHOSPHATEMIC INDEX EVALUATES PHOSPHORUS LOAD

Objective: Dietary phosphorus (P) restriction is crucial to treat hyperphosphatemia and reduce cardiovascular disease risk and mortality in patients with chronic kidney disease (CKD) and the wider population. Various methods for dietary P restriction exist, but the bioavailability of P in food should also be considered when making appropriate food choices to maintain patients’ quality of life. Here, we propose the ‘‘Phosphatemic Index’’ (PI) as a novel tool for evaluating dietary P load based on P bioavailability; we also evaluated the effect of continuous intake of different PI foods in mixed meals on serum intact fibroblast growth factor 23 concentration. Design and Methods: A 2-stage crossover study was conducted: Study 1: 20 healthy participants consumed 10 different foods containing 200 mg of P, and the PI was calculated from the area under the curve of a time versus serum P concentration curve; Study 2: 10 healthy participants consumed 4 different test meals (low, medium, or high PI meals or a control) over a 5-day period. Results: Study 1 showed milk and dairy products had high PI values, pork and ham had medium PI values, and soy and tofu had low PI values. In Study 2, ingestion of high PI test meals showed higher fasting serum intact fibroblast growth factor 23 levels and lower serum 1,25-dihydroxyvitamin D levels compared with ingestion of low PI test meals. Conclusion: These findings suggest that the PI can usefully evaluate the dietary P load of various foods and may help to make appropriate food choices for dietary P restriction in CKD patients