Amyloid-beta peptide degradation in cell cultures by mycoplasma contaminants-0

L negative control template (Control -), a positive control template of genomic DNA (Control +), or with medium from mycoplasma-positive HEK293 cell cultures (Mycoplasma+ Medium). Assay was carried out according to manufacturer's instructions (MycoSensor PCR Assay Kit, Stratagene). The changes in intensity of the internal control amplification are due to competition with the mycoplasma-specific PCR. (B) Medium from mycoplasma-positive cells was centrifuged at 1000 × g for 10 min to pellet the cell debris. Conditioned medium from APP-transfected HEK293 cells was diluted 1:1 with either fresh medium (Control DMEM), or with unfiltered (Mycoplasma+ Medium) or 0.2 μm filtered [Mycoplasma+ Medium (Filtered)] medium from mycoplasma-positive cells. The resulting mixtures were incubated at 37°C for the indicated periods of time. sAPPα and Aβ were analyzed by WB, as described in Figure 1B.<p><b>Copyright information:</b></p><p>Taken from "Amyloid-beta peptide degradation in cell cultures by mycoplasma contaminants"</p><p>http://www.biomedcentral.com/1756-0500/1/38</p><p>BMC Research Notes 2008;1():38-38.</p><p>Published online 30 Jun 2008</p><p>PMCID:PMC2527505.</p><p></p

Effect of the R154H and G330D variants on CALHM1 gating and Ca²⁺ permeability.

<p><b>A–C</b>. Currents observed in oocytes expressing (<b>A</b>) WT-CALHM1, (<b>B</b>) G330D-CALHM1, and (<b>C</b>) R154H-CALHM1 in standard bath solution containing 2 mM Ca²⁺_o in response to voltage pulses from −80 mV to +60 mV; holding potential −40 mV. <b>D</b>. Following a series of voltage pulses, currents at −80 mV were measured to determine G-V relations in the presence and absence of Ca²⁺_o. For each oocyte, G_max was determined by fitting 0 mM Ca²⁺_o data with a Boltzmann function; all currents were then normalized to G_max. Normalized data were fit with Boltzmann functions with the assumption that Ca²⁺ does not affect G_max<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112484#pone.0112484-Ma1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112484#pone.0112484-Tanis1" target="_blank">[15]</a>. WT-CALHM1 0 mM Ca²⁺_o (red circles) V_0.5 =  −75.9 mV; R154H-CALHM1 0 mM Ca²⁺_o (blue circles) V_0.5 =  −82.8 mV; G330D-CALHM1 0 mM Ca²⁺_o (black circles) V_0.5 =  −78.8 mV; WT-CALHM1 2 mM Ca²⁺_o (red triangles) V_0.5 = 60.8 mV; R154H-CALHM1 2 mM Ca²⁺_o (blue triangles) V_0.5 = 62.4 mV; G330D-CALHM1 2 mM Ca²⁺_o (black triangles) V_0.5 = 62.8 mV. <b>E</b>. Changes in E_rev resulting from changing from 0 to 2 mM Ca²⁺_o solution in oocytes expressing WT-CALHM1 (red), G330D-CALHM1 (black), and R154H-CALHM1 (blue). n = 4–6 oocytes for each condition; Error bars, SE.</p

Effect of CALHM1 activation on Aβ levels.

<p><b>A–D</b>. APP-N2a (<b>A</b> and <b>B</b>) and APP-HEK293 (<b>C</b> and <b>D</b>) cells transfected with empty vector or WT-CALHM1 were subjected to Ca²⁺ add-back conditions in the absence (CaAB, <b>A</b> and <b>C</b>) or presence of 1% FBS (CaAB 1% FBS, <b>B</b> and <b>D</b>). Extracellular Aβ and sAPPα were analyzed by WB after the indicated periods of secretion. Cell lysates were probed using anti-Myc and anti-actin antibodies to detect CALHM1 and actin, respectively.</p

Effect of the CALHM1 G330D and R154H variants on Aβ levels.

<p><b>A</b>. APP-N2a cells were transiently transfected with empty vector or WT-, G330D-, and R154H-CALHM1, as well as W114A-CALHM1 (CALHM1 dead mutant control, see Ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112484#pone.0112484-DresesWerringloer2" target="_blank">[13]</a>). Cells were then subjected to Ca²⁺ add-back as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112484#pone-0112484-g003" target="_blank">Fig. 3A</a>. Extracellular Aβ and sAPPα were analyzed by WB after 1 hr of secretion. Cell lysates were probed using anti-Myc, anti-BACE1, and anti-actin antibodies to detect CALHM1, BACE1, and actin, respectively. <b>B</b>. Densitometric analyses and quantification of Aβ levels in 3 independent measurements as in (<b>A</b>). Error bars, SEM; *<i>P</i><0.01, **<i>P</i><0.001, relative to vector-transfected cells; ^#<i>P</i><0.05, ^##<i>P</i><0.001, relative to WT-CALHM1-transfected cells (ANOVA with Bonferroni correction).</p

Effect of the CALHM1 G330D and R154H variants on Ca²⁺ influx in mammalian cells.

<p><b>A</b>. Ca²⁺_i measurements with Fluo-4 loading and Ca²⁺ add-back in HT-22 cells transiently transfected with WT-, G330D-, R154H-, and W114A-CALHM1 or empty vector. Cells were incubated in Ca²⁺-free buffer (0 CaCl₂) for 30 min, and then challenged with physiological Ca²⁺_o concentration (1.4 mM CaCl₂) to monitor the restoration of Ca²⁺_i levels. RFU, relative fluorescence units. <b>B</b>. Peak of Ca²⁺_i concentration measurements after Ca²⁺ add-back expressed as ΔF/F₀. Error bars, SEM. *<i>P</i><0.01, **<i>P</i><0.001, relative to vector-transfected cells; ^#<i>P</i><0.01, ^##<i>P</i><0.001, relative to WT-CALHM1-transfected cells (ANOVA with Bonferroni correction, n = 3 independent experiments as in (<b>A</b>)).</p

Brain histopathological studies of the control RA and KI hypoxia groups.

<p>(A) high-power (40X) H&E staining of cortical brains with normal structure for both the control RA and KI hypoxia groups, while the WT hypoxia group showed injury damage (id). (B) H&E, high-power (60X) view of neurons from the dentate gyrus in the hippocampus with no ischemic damage in both the control and KI hypoxia groups, while the WT hypoxia group showed injury damage (id). (C) H&E, high-power (40X) view of Purkinje cells and granular cells in the cerebellum with no ischemic damage in both the control and KI hypoxia groups, while WT hypoxia group showed injury damage (id).</p

Brain PET scan with an FDG standardized uptake value, showing a cross and longitudinal sections of brain uptake.

<p>Brain PET scan with an FDG standardized uptake value, showing a cross and longitudinal sections of brain uptake.</p

Quantitative Assessment of Western blot for molecular markers including: GFAP, IBA1, MIF, pAMPK, and pACC in adult mouse brain groups (WT and KI) after exposure to hypoxia (10% for 10 days), in comparison with the RA control groups (WT and KI).

<p>Data are mean of 10± SEM animals per group. *P<0.05 versus the KI hypoxia and WT hypoxia groups.</p><p>* P<0.05 (H WT vs. H KI).</p><p>Quantitative Assessment of Western blot for molecular markers including: GFAP, IBA1, MIF, pAMPK, and pACC in adult mouse brain groups (WT and KI) after exposure to hypoxia (10% for 10 days), in comparison with the RA control groups (WT and KI).</p

PET scan data showing FDG in adult mice brain groups (WT and KI) after exposure to hypoxia (10% for 10 days), in comparison with the RA control groups (RA-WT and RA-KI).

<p>Data are mean ± SEM of 10 animals per group. *P<0.05 versus the KI hypoxia and WT hypoxia groups.</p><p>PET scan data showing FDG in adult mice brain groups (WT and KI) after exposure to hypoxia (10% for 10 days), in comparison with the RA control groups (RA-WT and RA-KI).</p

Activated caspase 3 ELISA in adult mouse brain groups (WT and KI), after exposure to hypoxia (10% for 10 days) in comparison with the RA control group (WT and KI).

<p>Data are mean of 10 animals per group ± SEM. *P<0.05 versus the KI hypoxia and WT hypoxia groups.</p