Experiments on the Entrainment of Sediment by Turbidity Currents with the Use of Submarine Sediment

Source: ICHE Conference Archive - https://mdi-de.baw.de/icheArchiv

Establishment of a Novel Fluorescence-Based Method to Evaluate Chaperone-Mediated Autophagy in a Single Neuron

Background: Chaperone-mediated autophagy (CMA) is a selective autophagy-lysosome protein degradation pathway. The role of CMA in normal neuronal functions and in neural disease pathogenesis remains unclear, in part because there is no available method to monitor CMA activity at the single-cell level. Methodology/Principal Findings: We sought to establish a single-cell monitoring method by visualizing translocation of CMA substrates from the cytosol to lysosomes using the HaloTag (HT) system. GAPDH, a CMA substrate, was fused to HT (GAPDH-HT); this protein accumulated in the lysosomes of HeLa cells and cultured cerebellar Purkinje cells (PCs) after labeling with fluorescent dye-conjugated HT ligand. Lysosomal accumulation was enhanced by treatments that activate CMA and prevented by siRNA-mediated knockdown of LAMP2A, a lysosomal receptor for CMA, and by treatments that inactivate CMA. These results suggest that lysosomal accumulation of GAPDH-HT reflects CMA activity. Using this method, we revealed that mutant cPKC, which causes spinocerebellar ataxia type 14, decreased CMA activity in cultured PCs. Conclusion/Significance: In the present study, we established a novel fluorescent-based method to evaluate CMA activity in a single neuron. This novel method should be useful and valuable for evaluating the role of CMA in various neurona

Imidazodiazepinediones: A New Class of Adenosine Receptor Antagonists

A series of imidazo[4,5-e][l-4]diazepine-5,8-diones were synthesized from hypoxanthines. Certain of these cyclic homologues of caffeine, theophylline, theobromine, 3-isobutyl-l-methylxanthine, and enprofylline were inhibitors of binding of adenosine analogues to rat brain A1 and A2 adenosine receptors and were antagonists of A2 adenosine receptors stimulatory to adenylate cyclase in rat PC12 cell membranes. Activity at adenosine receptors was lower than the corresponding xanthines, perhaps because imidazodiazepinediones contain a boat-shaped seven-membered ring rather than the planar heteroaryl ring system of the xanthines. The imidazodiazepinediones had low affinity for brain benzodiazepine sites. © 1990, American Chemical Society. All rights reserved

The Subcellular Dynamics of the Gs-Linked Receptor GPR3 Contribute to the Local Activation of PKA in Cerebellar Granular Neurons.

G-protein-coupled receptor (GPR) 3 is a member of the GPR family that constitutively activates adenylate cyclase. We have reported that the expression of GPR3 in cerebellar granular neurons (CGNs) contributes to neurite outgrowth and modulates neuronal proliferation and survival. To further identify its role, we have analyzed the precise distribution and local functions of GPR3 in neurons. The fluorescently tagged GPR3 protein was distributed in the plasma membrane, the Golgi body, and the endosomes. In addition, we have revealed that the plasma membrane expression of GPR3 functionally up-regulated the levels of PKA, as measured by a PKA FRET indicator. Next, we asked if the PKA activity was modulated by the expression of GPR3 in CGNs. PKA activity was highly modulated at the neurite tips compared to the soma. In addition, the PKA activity at the neurite tips was up-regulated when GPR3 was transfected into the cells. However, local PKA activity was decreased when endogenous GPR3 was suppressed by a GPR3 siRNA. Finally, we determined the local dynamics of GPR3 in CGNs using time-lapse analysis. Surprisingly, the fluorescent GPR3 puncta were transported along the neurite in both directions over time. In addition, the anterograde movements of the GPR3 puncta in the neurite were significantly inhibited by actin or microtubule polymerization inhibitors and were also disturbed by the Myosin II inhibitor blebbistatin. Moreover, the PKA activity at the tips of the neurites was decreased when blebbistatin was administered. These results suggested that GPR3 was transported along the neurite and contributed to the local activation of PKA in CGN development. The local dynamics of GPR3 in CGNs may affect local neuronal functions, including neuronal differentiation and maturation

Evaluation of the Effectiveness of Post-Stroke Metformin Treatment Using Permanent Middle Cerebral Artery Occlusion in Rats

Stroke is the second leading cause of death worldwide. Treatment options for ischemic stroke are limited, and the development of new therapeutic agents or combined therapies is imperative. Growing evidence suggests that metformin treatment, due to its anti-inflammatory action, exerts a neuroprotective effect against ischemia/reperfusion-induced brain damage. Experimental assessment has typically been performed in models of cerebral transient ischemia followed by long-term reperfusion. The aim of this study was to evaluate the neuroprotective effect of metformin treatment after permanent middle cerebral artery occlusion (pMCAO) without reperfusion in rats. Neurological deficits were assessed using the Longa scale, which offers a graded scale on body movement following pMCAO. Both infarct size and brain oedema area were measured by staining with 2,3,5-triphenyltetrazolium chloride. The number of neurons and total and activated microglia, as well as interleukin 10 (IL-10) production, in brain sections were evaluated by immunohistochemical staining. Our results show that metformin treatment improves the neurological state and reduces infarct size after 120 h of pMCAO. Metformin also prevents neuronal loss in the ischemic cortex but not in the striatum after 48 h of pMCAO. Moreover, post-stroke treatment with metformin significantly decreases the number of total and activated microglia at 48 h. The anti-inflammatory effect of metformin is associated with increased IL-10 production at 48 h after pMCAO. The results of the present study suggest that post-stroke treatment with metformin exerts anti-inflammatory and neuroprotective effects in a pMCAO model

Propofol induced diverse and subtype-specific translocation of PKC families

Propofol is the most commonly used anesthetic. Immunohistochemical studies have reported that propofol translocated protein kinase Cs (PKCs) in cardiomyocyte in a subtype-specific manner; however detailed features of the propofol-induced translocation of PKCs remain unknown. In this study, we performed real-time observation of propofol-induced PKC translocation in SH-SY5Y cells expressing PKCs fused with a fluorescent protein. Propofol unidirectionally translocated γPKC-GFP, a conventional PKC, and ζPKC-GFP, an atypical PKC, to the plasma membrane and nucleus, respectively, whereas the propofol-induced translocation of novel PKCs was diverse and subtype-specific among δPKC, εPKC and ηPKC. The propofol-induced translocation of εPKC-GFP was especially complicated and diverse, that is, 200 μM propofol first translocated εPKC-GFP to the perinuclear region. Thereafter, εPKC was translocated to the nucleus, followed by translocation to the plasma membrane. Analysis using a mutant εPKC in which the C1 domain was deleted demonstrated that the C1b domain of εPKC was indispensable for its translocation to the perinuclear region and plasma membrane, but not for its nuclear translocation. An in vitro kinase assay revealed that propofol increased the activities of the PKCs activities at the concentration that triggered the translocation. These results suggest that propofol could translocate PKCs to their appropriate target sites in a subtype-specific manner and concomitantly activated PKCs at these sites, contributing to its beneficial or adverse effects. Keywords: Propofol, Protein kinase C, PKC translocatio

The subcellular distribution of fluorescently tagged GPR3 in CGNs.

<p>(A) We employed a GPR3-HT expression vector to analyze the localization of GPR3. After transfecting the GPR3-HT vector into the CGNs, the cells were labeled with the TMR HaloTag ligand at forty-eight hours after transfection. A representative image was shown. (B) To evaluate the localization of GPR3 at the plasma membrane, the CGNs were co-transfected with the GPR3-HT and YFP-mem expression vectors, followed by TMR HaloTag ligand labeling. YFP-mem fluorescence was shown in the green pseudo-color to easily visualize the co-localization. (C-F) To evaluate the localization of GPR3 in the endoplasmic reticulum, Golgi body, and endosomes, the GPR3-mAGFL expression vector was transfected into the CGNs. Forty-eight hours after transfection, the cells were immunostained with various organelle markers: anti-KDEL antibody (endoplasmic reticulum), WGA633 (Golgi body), anti-Rab5 antibody (early endosome), and anti-Rab7 antibody (late endosome). Co-localization of GPR3 with the organelle markers was evaluated by confocal microscopy using built-in Z-stack analysis software. Scale bar = 10 μm.</p

The local PKA activity was modulated by the expression of GPR3 in the CGNs.

<p>(A-B) The local PKA activity was compared between the soma and neurite tips in GPR3-transfected CGNs. The CGNs were transfected with Mock+control siRNA, pGPR3-HT+control siRNA, Mock+GPR3 siRNA, and pGPR3-HT+GPR3 siRNA, respectively. Forty-eight hours after transfection, the FRET/CFP images were captured using a fluorescent microscope. After capturing the images, the FRET/CFP intensity ratio was calculated and compared between the soma and neurite tips. (A) Representative FRET/CFP images from CGNs in each condition were shown (left raw). Magnifications of neurite tip were also shown (right raw) (B) The ratios of the PKA activity (tip to soma) in each condition were analyzed identically. The data represent the means ± SEM for each condition (n = 7). The asterisk (*) represents p < 0.005. (C-D) The PKA activity at the neurite tips was analyzed in CGNs transfected with Mock+control siRNA, pGPR3-HT+control siRNA, Mock+GPR3 siRNA, and pGPR3-HT+GPR3 siRNA, respectively. Forty-eight hours after transfection, the FRET/CFP images were captured using a fluorescent microscope. After capturing the images, the cells were treated with 1 mM dbcAMP for 15 min, and FRET/CFP images were captured again in the same cell to evaluated fully activated PKA. (C) Representative FRET/CFP images from the neurite tips of CGNs in each condition were shown. (D) The FRET/CFP ratio in each group were analyzed identically. The FRET/CFP ratios are expressed as the percentage of the maximum level stimulated by 1 mM dbcAMP in each cell. The data represent the means ± SEM for each condition (n = 9). The asterisk (*) represents p < 0.005.</p

The GPR3-mediated modulation of PKA activity was analyzed with a PKA FRET indicator.

<p>(A-C) HEK293 cells were co-transfected with the AKAR3-EV and GPR3 expression plasmids. AKAR3-EV and the mock vector were co-transfected as a control. Forty-eight hours after transfection, the CFP and FRET images were captured using a fluorescent microscope. Some of the AKAR3-EV/mock vector co-transfected cells were treated with 1 mM dbcAMP 15 min before the images were captured. The FRET/CFP ratios in the images were analyzed by the MetaMorph software. (A) Representative images of cells transfected with either the mock vector (top) or the GPR3 vector (bottom) are shown. (B) The YFP/CFP ratio in each group were analyzed identically in each dish (n = 8). The values are expressed as the percentage of the FRET/CFP ratio in the GPR3-transfected or dbcAMP-treated cells compared to the mock-transfected cells, i.e., the percentage of the control vector-transfected cells. The means ± SEM were calculated for each condition. The asterisk (*) represents p < 0.001 compared to the cells transfected with the mock vector. (C) The FRET/CFP ratio in the cytoplasm and plasma membrane were evaluated in a single GPR3-transfected cell. The asterisk (*) represents p < 0.001 compared to the values for the cytoplasm. (D-E) The PKA activity in GPR3-expressing CGNs was evaluated by AKAR3-EV. CGNs were co-transfected with mock/AKAR3-EV or pGPR3-HT/AKAR3-EV expression plasmids. Forty-eight hours after transfection, the CGNs were stained with organelle-specific markers for the plasma membrane, ER, Golgi body, lysosome, and mitochondria. (D) Representative images of CGNs stained with CellMask (plasma membrane), ER tracker (ER), WGA633 (Golgi body), LysoTracker (lysosome), and MitoTracker (mitochondria) were shown (middle raw). The CFP/YFP ratios in the images were visualized and analyzed by the MetaMorph software (right raw). (E) The FRET/CFP ratios were compared in each organelle of the mock/AKAR3-EV- or pGPR3-HT/AKAR3-EV-transfected CGNs. The FRET/CFP ratios were significantly increased in the plasma membrane of the GPR3-HT-expressing CGNs compared to the mock-transfected CGNs. The data represent the means ± SEM for each condition (n = 6). The asterisk (*) represents p < 0.05. (F) The PKA activity in the plasma membrane around soma was analyzed in CGNs transfected with control siRNA+Mock, GPR3 siRNA+Mock, and GPR3 siRNA+ pGPR3-HT, respectively. Forty-eight hours after transfection, the FRET/CFP images were taken. The FRET/CFP ratio in each group were analyzed identically. The data represent the means ± SEM for each condition (n = 10). The asterisk (*) represents p < 0.05 and the double asterisk (**) represents p < 0.005.</p