Preventive effect of grapefruit juice (Citrus Paradisi Macf.) on morphine withdrawal symptome in male rats

Background & Objective: Addiction to opiates such as morphine is one of major public health problems. It has been shown that in addicted animals, administration of antioxidant agents such as vitamin C can reduce the withdrawal symptoms (WDS). The aim of this study was to evaluate the preventional effect of grapefruit juice (Citrus Paradisi Macf.) on withdrawal symptoms in rats. Materials & Methods: In this experimental study, Sixteen male Wistar rats (250-300g) randomly divided into two groups (n=8). All animals were addicted by intraperitoneal (i.p) injection of morphine (the 1-3 days: 10 mg/kg, the 4-6 days: 20 mg/kg and the 7-9 days 40 mg/kg daily) for 9 days. The first group received 2 ml Citrus Paradisi Macf. orally 1 hour before morphine administration. The sham group received 2 ml of normal saline. Naloxone (10mg/kg, s.c) was administrated 45 minutes after of an additional dose of morphine (40 mg/kg) in the tenth day for withdrawal symptoms inducing. Then withdrawal symptoms such as frequency of wet-dog shaking, teeth chattering, defecation and penis licking were evaluated for 30 minutes. Results: All withdrawal symptoms including frequency of wet-dog shaking, teeth chattering, defecation and penis licking were reduced in the Citrus Paradisi Macf. group in comparison with the sham group significantly (p<0.05). Conclusion: Our results showed that presumably antioxidant activity of Citrus Paradisi Macf. can reduce withdrawal symptoms. Although the exact mechanisms of its effect in brain need to be elucidate

Cumulus Cell Role on Mouse Germinal Vesicle Oocyte Maturation, Fertilization, and Subsequent Embryo Development to Blastocyst Stage In Vitro

Objective: The purpose of this study is to investigate the effect of cumulus cells on maturation,fertilization and subsequent development of mouse germinal vesicle oocytes.Materials and Methods: A total of 470 germinal vesicle (GV) oocytes were obtained from26 ovaries of 3- 4 week old ICR female mice 48 hours after injection of 5 IU pregnant mareserum gonadotropin (PMSG). Collected oocytes were divided into two groups; group I: GVoocytes without cumulus cells (denuded oocyte), group II: GV oocytes with cumulus cells(cumulus-oocyte complex). The oocytes in both groups were cultured in TCM-199 mediumsupplemented with 10% fetal bovine serum (FBS) for 22- 24 hours in a humidified atmosphereof 5% CO2 in air at 37°C. Oocyte maturation was scored under inverted microscope.To do in vitro fertilization, matured oocytes from each group were placed in T6 mediumand capacitated spermatozoa were added. Then the fertilized oocytes were cultured andassessed for cleavage to the 2-cell stage 24 hours and production of blastocyst 120 hoursafter fertilization. Data was analyzed by chi-square test and differences in the values wereconsiderable significant when p<0.05.Results: Maturation, fertilization, cleavage and blastocyst rates in denuded oocytes were:76.32%, 57.49%, 51.15% and 19.14% respectively. In the cumulus-oocyte complex rateswere: 89.41%, 80.76%, 75.58% and 45.62% respectively; all in the cumulus-oocyte complexwere significantly higher than those of denuded oocytes (p<0.05).Conclusion: The present study indicates that cumulus cells have important role duringmaturation, fertilization and subsequent embryo development to the blastocyst stage

Amyloid beta1–42 (Aβ42) up-regulates the expression of sortilin via the p75NTR/RhoA signaling pathway

Sortilin is the co-receptor of p75NTR which signals the cell death induced by Aβ and proneurotrophins. We found that sortilin is increased in the AD brain and up-regulated by Aβ and pro-brain-derived neurotrophic factor (proBDNF). Aβ-induced upregulation of sortilin is mediated by p75NTR and the down-streaming RhoA-ROCK signaling pathway. The Aβ/Sortilinp/75NTR signaling may play a role in the pathogenesis of AD.

Effect of sortilin binding domain on APP lysosomal targeting.

<p>A: APP colocalization with lysosome. HEK293 cells were co-transfected with APP770-YFP and different sortilin constructs. The expressed APP and sortilin constructs excluding Sort-FL were visualized by either YFP or CFP fluorescence. Sort-FL-myc/His was immunostained with anti-sortilin, followed by staining with Cy3 conjugated secondary antibodies. Lysosomes were immunostained with lysosomal antibody (Lamp1), followed by staining with Cy5 conjugated secondary antibodies. B: Sortilin constructs sorting ASM to lysosomes. HEK293 cells were transfected with different sortilin constructs. The endogenous ASM was immunostained with mouse anti-ASM (Abcam), followed by staining with Cy3 conjugated secondary antibodies. Lysosome were immunostained with lysosomal antibody (Lamp1), followed by staining with Alexa 488 conjugated secondary antibodies. Sortilin constructs excluding Sort-FL were visualized by CFP fluorescence. Sort-FL-myc/His was immunostained with anti-sortilin, followed by staining with Cy5 conjugated secondary antibodies. Plotted colocalization is indicated at bottom. The percentage of colocalization is represented as mean± SEM (n = 20). The colocalization is compared with Sort-FL. The star (*) indicates <i>p</i><0.01. Scale bar 7.5 µm.</p

Effect of sortilin on APP lysosomal degradation.

<p>APP lysosomal targeting. HEK293 cells were co-transfected with APP-YFP and sortilin-pcDNA3.1 constructs or pcDNA3.1 (mock DNA) plasmid in 1∶1 molar ratio for 24 hours and then treated with DMSO or Bafilomycin AI, a lysosomal inhibitor, (BafA1, 4 µM, Sigma) for 6 h <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063049#pone.0063049-Kwon1" target="_blank">[30]</a>. Cell lysates were harvested and APP level was examined by WB with mouse anti-APP-N’ (22c11). Transfected APP-YFP served to monitor transfection efficiency. The APP level was plotted after correction as to the corresponding β-actin and transfection efficiency.</p

Lack of sortilin reduces APP distribution in lysosome and increases APP distribution in lipid rafts.

<p>Colocalization of APP with cell organelles in cortical neurons. Wild type (WT) and sortilin knockout (KO) mouse cortical neurons were immunostained for APP with mouse anti-APP-N’ (22c11) and followed by staining with Cy3 conjugated secondary antibodies (red) and cell organelles: Golgi, Early endosome, late endosome and lysosome immunostained with Giantin, EEA1, anti-mannose 6 phosphate receptor (for late endosome) and Lamp1, followed by staining with Alexa 488 conjugated secondary antibodies (green). Cell nuclei are stained by DAPI (blue). The colocalization is indicated in merged panels (yellow). Snapshots of colocalization are shown on the right of each panel. The percentage of colocalization (Col.) is plotted and is represented as mean± SEM (n = 20). The colocalization of APP with cell organelles is compared between WT and KO neurons. The star (*) indicates <i>p</i><0.01. Scale bar 10 µm.</p

Mapping APP binding to sortilin and the binding sequences.

<p>(<b>A</b>) Determining APP 1-287 and APP 713-770 binding to sortilin. HEK293 cells were co-transfected with sort-FL-myc and APP 1-287YFP or APP 1-542YFP or APP 541-671YFP or APP713-770YFP (lanes 1–4). Sort-T-myc was used for the co-transfection with the same APP-YFP constructs (lanes 5–8). Lysates from each co-transfection were used as input (lane 9–12). Cell lysates immunoprecipitated with α-Myc (lanes 1–8) and inputs were blotted with goat (gt)-α-GFP. The co-IP blot was re-probed with α-Myc for sortilin constructs (lower). APP 1-542YFP (87 kDa), APP 1-287YFP (58 kDa), APP 541-671YFP (42 kDa), APP 713-770YFP (33 kDa), Sort-myc (115 kDa) and Sort-T-myc (110 kDa) are indicated by arrows. (<b>B</b>) Determining N terminal binding sites between APP and sortilin. HEK293 cells were co-transfected with APP 1-287YFP and Sort 78-385CFP. Cell lysates were immunoprecipitated with α-APP-N’ for APP1-287YFP (lane 1) and mouse IgG (mIgG) as a control for non-specific binding (lane 2), and blotted with gt-α-GFP. Lysate was used as input (lane 3). APP1-287YFP (58 kDa) and Sort78-385CFP (61 kda) are indicated by arrows. (<b>C</b>) Determining APP 1-141 binding to sortilin. HEK293 cells were co-transfected with APP 1-141YFP and Sort-FL-myc. Cell lysates were immunoprecipitated with α-Myc for Sort-FL-myc (lane 1) and mIgG (lane 2), and blotted with gt-α-GFP for APP1-141YFP. The direct Immunoprecipitated APP1-141YFP by α-GFP was used as input (lane 3). Also, cell lysates were immunoprecipitated with rabbit-α-GFP for APP1-141YFP (lane 4) and rabbite IgG (rIgG) as control (lane 5), and blotted with α-Myc for sortilin. The direct immunoprecipitated Sort-FL-myc by α-Myc was used as input (lane 6). APP1-141YFP (41 kDa) and Sort-FL-myc (115 kDa) are indicated by arrows. (<b>D</b>) Determining C terminal binding sites between APP and sortilin. HEK293 cells were co-transfected with APP 713-770YFP and Sort del.MS2-CFP. Cell lysates were immunoprecipitated with α-APP-C’ for APP 713-770YFP (lane 1) and rIgG (lane 3), and blotted with α-Sort C’. Lysate was used as input (lane 2). Sort del.MS2-CFP (34 kDa) is indicated by arrow. (<b>E</b>) Determining Sort-MS1 (del.MS2) interaction with APP NPTYKFFE motif. Sort del.MS2-CFP was used with APP 713-770-YFP or APP 713-770 mut-YFP for the co-transfection of HEK293 and then subjected to FRET. APP 713-770 mut-YFP construct contains a mutated NPT<u>Y</u>KF<u>F</u>E motif where Y and F (underlined) are substituted with A. FRET efficiency representing the protein-protein interaction was determined from a photobleached region of interest (ROI). Three independent experiments were performed. Bars represent mean± SEM (n = 6 ROI×3). The star (*) indicates <i>p</i><0.01. Abbreviation: negative control: NC; positive control: PC.</p

Sortilin and APP are allied <i>in vitro and in vivo</i>.

<p>(<b>A</b>) Colocalization of sortilin and APP. Mouse cortical neuron (upper) and brain cortex (lower) were immunostained for APP (red) and sortilin (green). Colocalization of sortilin and APP was indicated in merged panels (yellow). DAPI stained cell nuclei (blue). Plotted colocalization: 92% mean± SEM, n = 20, in cortical neurons and 95% mean± SEM, n = 3, in brain cortexes. Scale bar 25 µm for cortical neurons, 5 µm for enlarged image and 100 µm for brain cortex. (<b>B</b>) Co-IP of sortilin with APP in co-transfected HEK293 cells. HEK293 cells growing in 10 cm culture dishes were co-transfected with APP770-YFP/Sort-FL-myc/His (lane 1, 4) or Sort-T-myc/His (lane 2, 5). Cell lysates were immunoprecipitated with rabbit anti-GFP (α-GFP) for APP and blotted with mouse anti-Myc (α-Myc) for sortilin. Mixed lysates were used for IgG (lane 3). Sort-FL-myc.His (Sort-myc), Sort-T-myc.His (sort-T-myc) and APP770-YFP (APP-YFP) are indicated by arrows. (<b>C</b>) Co-IP of sortilin with APP in APPSwe/PS1dE9 transgenic mouse brain lysate. Mouse brain lysates were subjected to immunoprecipitation with rabbit anti-APP C’ (α-APP C’) and blotted with rabbit anti-sortilin (α-Sort) (left panel) or immunoprecipitation with α-Sort and blotted with α-APP C’ (right panel). Sortilin and APP are indicated by arrows. (<b>D</b>) Control for Co-IP using pEYFP. HEK293 cells were co-transfected with pEYFP/Sort-myc. Co-IP was performed using α-GFP and blotted with α-sort and α-GFP. Rabbit IgG (IgG) was used as a control for non-specific binding.</p

Effect of sortilin on APP distribution in lipid rafts.

<p>A: HEK293 cells co-transfected with APP695 and sortilin constructs were lysed in Triton-X-100 buffer and subjected to discontinuous sucrose density gradient ultracentrifugation fractionation. Equal volumes from each fraction were examined by WB for APP and flotillin-1. B: Lack of sortilin increases APP distribution within lipid rafts in sortilin KO mice. Mouse brains were homogenized in Triton-X-100 buffer and subjected to discontinuous sucrose density gradient ultracentrifugation fractionation. Equal volumes from each fraction were examined by WB for APP and flotillin-1. Bars represent mean± SEM (n = 3) from three independent experiments. The star (*) indicates <i>p</i><0.01.</p