Autophagy-dependent rhodopsin degradation prevents retinal degeneration in Drosophila.

Recent studies have demonstrated protective roles for autophagy in various neurodegenerative disorders, including the polyglutamine diseases; however, the role of autophagy in retinal degeneration has remained unclear. Accumulation of activated rhodopsin in some Drosophila mutants leads to retinal degeneration, and although it is known that activated rhodopsin is degraded in endosomal pathways in normal photoreceptor cells, the contribution of autophagy to rhodopsin regulation has remained elusive. This study reveals that activated rhodopsin is degraded by autophagy in collaboration with endosomal pathways to prevent retinal degeneration. Light-dependent retinal degeneration in the Drosophila visual system is caused by the knockdown or mutation of autophagy-essential components, such as autophagy-related protein 7 and 8 (atg-7/atg-8), or genes essential for PE (phosphatidylethanolamine) biogenesis and autophagosome formation, including Phosphatidylserine decarboxylase (Psd) and CDP-ethanolamine:diacylglycerol ethanolaminephosphotransferase (Ept). The knockdown of atg-7/8 or Psd/Ept produced an increase in the amount of rhodopsin localized to Rab7-positive late endosomes. This rhodopsin accumulation, followed by retinal degeneration, was suppressed by overexpression of Rab7, which accelerated the endosomal degradation pathway. These results indicate a degree of cross talk between the autophagic and endosomal/lysosomal pathways. Importantly, a reduction in rhodopsin levels rescued Psd knockdown-induced retinal degeneration. Additionally, the Psd knockdown-induced retinal degeneration phenotype was enhanced by Ppt1 inactivation, which causes infantile neuronal ceroid lipofuscinosis, implying that autophagy plays a significant role in its pathogenesis. Collectively, the current data reveal that autophagy suppresses light-dependent retinal degeneration in collaboration with the endosomal degradation pathway and that rhodopsin is a key substrate for autophagic degradation in this context.

[Effectiveness of network meeting system following practical pharmacy training].

We established a network meeting system program and used it to review a prior pharmaceutical practice training session. Pharmacists at Tokai University Hachioji Hospital gave lectures about dispensing and other tasks performed by clinical pharmacists to third-year undergraduate students at Tokyo University of Pharmacy and Life Sciences. After the lectures, discussions were held using the network meeting system, after which a questionnaire was completed by the students. The questionnaire was answered by 530 students, of whom approximately 90% expressed interest in the program, 80% noted approval of the media used in the system, and 94% thought that the program was useful. Thus, we concluded that the students were motivated by the program to remember what they had learned in the lectures. We also found that the quality of data communication had an effect on the interests and motivation of the students. Based on their evaluation of the media, it was considered that improvements in communication regarding the system were necessary, though the evaluation of the utility of the program was not influenced by the quality of data communication. As a result, we concluded that our network meeting system program was useful to review prior learning of pharmaceutical practice.

Versatile roles of R-Ras GAP in neurite formation of PC12 cells and embryonic vascular development.

Ras GTPase-activating proteins (GAP) are negative regulators of Ras that convert active Ras-GTP to inactive Ras-GDP. R-Ras GAP is a membrane-associated molecule with stronger GAP activity for R-Ras, an activator of integrin, than H-Ras. We found that R-Ras GAP is down-regulated during neurite formation in rat pheochromocytoma PC12 cells by nerve growth factor (NGF), which is blocked by the transient expression of R-Ras gap or dominant negative R-ras cDNA. By establishing a PC12 subclone that stably expresses exogenous R-Ras GAP, it was found that NGF reduced endogenous R-Ras GAP but not exogenous R-Ras GAP, suggesting that down-regulation of R-Ras GAP occurs at the transcription level. To clarify the physiological role of R-Ras GAP, we generated mice that express mutant Ras GAP with knocked down activity. While heterozygotes are normal, homozygous mice die at E12.5-13.5 of massive subcutaneous and intraparenchymal bleeding, probably due to underdeveloped adherens junctions between capillary endothelial cells. These results show essential roles of R-Ras GAP in development and differentiation: its expression is needed for embryonic development of blood vessel barriers, whereas its down-regulation facilitates NGF-induced neurite formation of PC12 cells via maintaining activated R-Ras.

Prefabricated engineered skin flap using an arteriovenous vascular bundle as a vascular carrier in rabbits.

BACKGROUND: The authors previously described induction of spontaneous tissue generation by implanting a collagen matrix and a ligated pedicle (arteriovenous bundle) into a hollow porous chamber in vivo in the rabbit. They hypothesized that increased tissue volume could be obtained by the application of basic fibroblast growth factor (bFGF) and/or by increasing the chamber size and porosity. METHODS: In rabbits, a saphenous arteriovenous pedicle and a collagen sponge were inserted into a porous chamber in the groin. Small-volume pore chambers (experiment 1, n = 7) and larger-volume, wider pore chambers (experiment 2, n = 13) were compared, and each was compared with and without bFGF. An additional three flaps of experiment 2 with bFGF were skin grafted, microsurgically transplanted to the ear, and evaluated at 6 months for stability. RESULTS: All patent chambers grew tissue; chambers with bFGF were almost filled, and those without were only half-filled. Histomorphometric analysis confirmed a significant difference. The larger-volume, larger-pore chambers produced more than twice the volume of tissue as the smaller chambers did, and this was significant. Tissue volume in both the control and bFGF groups of experiment 2 was significantly greater than that in the respective groups of experiment 1. Histology, angiography, and scanning electron microscopy confirmed greater vascularity in the bFGF groups and demonstrated vascular connections penetrating the chamber pores linking with angiogenic sprouts, probably from the vasa vasorum of the pedicle, to contribute to new growth. Transplanted flaps survived and appeared normal 6 months later. CONCLUSIONS: Patent pedicles, bFGF, large pore size, and larger-volume chambers all seemed to contribute to increased tissue growth in this model. The tissue is stable long term.

Mechanism of neurotrophic action of nobiletin in PC12D cells.

Nobiletin is a nonpeptide compound with a low molecular weight from a citrus fruit and has the activity to rescue bulbectomy-induced memory impairment. Here we describe that nobiletin itself induces neurite outgrowth in PC12D cells, a rat pheochromocytoma cell line, like NGF, and the molecular mechanism of its neurotrophic action. As cultured in the presence of nobiletin or NGF for 48 h and then assayed using a scanning electron microscope, PC12D cells treated with nobiletin showed morphology with flatter and larger cell bodies than the cells cultured with NGF. Nobiletin-induced neurite outgrowth was inhibited by PD98059 and U0126 but not K252a. Consistently, nobiletin caused a concentration-dependent enhancement of Erk/MAP kinase phosphorylation and a sustained increment of phosphorylation of MEK and Erk/MAP kinase, resulting in a stimulation of CREB phosphorylation and CRE-mediated transcription. This compound also increased intracellular cAMP and CRE-mediated transcription in the presence of forskolin and enhanced PKA activity to stimulate phosphorylation of multiple PKA substrates in PC12D cells. Furthermore, nobiletin preferentially inhibited Ca2+/CaM-dependent phosphodiesterase in vitro. This compound failed to stimulate phosphorylation of Erk5, which is known to be induced by NGF/TrkA signaling. These results suggest that nobiletin induces neurite outgrowth by activating a cAMP/PKA/MEK/Erk/MAP kinase-dependent but not TrkA-dependent signaling pathway coupling with CRE-mediated gene transcription and may thus become a novel type of biochemical probe for elucidation of the molecular mechanism of neuronal differentiation.

PVR plays a critical role via JNK activation in thorax closure during Drosophila metamorphosis.

PVR, the Drosophila homolog of the PDGF/VEGF receptor, has been implicated in border cell migration during oogenesis and hemocyte migration during embryogenesis. It was earlier shown that Mbc, a CDM family protein, and its effector, Rac, transduced the guidance signal from PVR during border cell migration. Here we demonstrate that PVR is also required for the morphogenetic process, thorax closure, during metamorphosis. The results of genetic and biochemical experiments indicate that PVR activates the JNK pathway. We present evidence showing Crk (an adaptor molecule), Mbc, ELMO (a homolog of Caenorhabditis elegans CED-12 and mammalian ELMO), and Rac to be mediators of JNK activation by PVR. In addition, we suppose that not only Rac but also Cdc42 is activated and involved in JNK activation downstream of PVR.

Enhanced phosphorylation and enzymatic activity of phosphoglucomutase by the Btk29A tyrosine kinase in Drosophila.

The Drosophila Btk29A tyrosine kinase is suggested to be involved in diverse processes, although its target proteins are unknown. In the present study, we investigated substrates of Btk29A tyrosine kinase by expressing a catalytically activated form of Btk29A-P1 (Btk-EG) in Drosophila compound eyes. Expression in eye disks led to the development of the rough-eye phenotype and increased tyrosine phosphorylation of a 65-kDa protein. Partial amino acid sequence analysis of this protein showed that it was phosphoglucomutase. Phosphoglucomutase activity in heads from Btk-EG-expressing flies was higher than that in controls, suggesting that the levels of tyrosine phosphorylation and activity of the enzyme are associated with Btk29A tyrosine kinase activity.