Effect of Plasma Activated Liquid (PAL) on bacteria viability.

<p>A: 1: control bacteria incubated for 2 hours in PBS and % of surviving cells was evaluated by the CFU method. 2: <i>E</i>. <i>coli</i> exposed to He plasma for 10 min with 2 hr storage at 4°C. 3: Bacteria exposed to PAL (PBS treated for 10 min He plasma) for 2 hr at 4°C. 4: Bacteria exposed to He plasma for 10 min and incubated for 2 hours with non-plasma treated PBS at 4°C. 5: Bacteria exposed to plasma for 10 min and then incubated for 2 hours at 4°C with PAL (PBS treated for 10 min He plasma). 6: Bacteria exposed to PAL (PBS treated for 10 min He plasma and left at room temperature for 2 hours) for 2 hours at 4°C. The values are means ± SEM of 3 separate experiments (*p<0.01 and ** p<0.05 vs control). B: Same experimental procedures using He-N₂ plasma. C: Same experimental procedures using He-O₂ plasma.</p

Respiration was similar in brain mitochondrial preparations from 24-month-old <i>PARK2^−/−</i> and wild type mice examined with standard oxygraphy.

<p>(<b>A</b>) Representative experiment with a crude mitochondrial pellet from striatum. Blue curve = oxygen (O₂) concentration expressed as nmol/mL; red curve = rate of oxygen consumption expressed as nmoles O₂/mL and min; ADP = addition of 400 µM ADP; OM = addition of 1 µg/mL oligomycin, a complex V inhibitor; KCN = addition of 1 mM KCN (non-respiratory oxygen consumption); (<b>B, C, D</b>) Bars showing state 3 respiration (respiration in the presence of substrates +ADP – non-respiratory oxygen consumption) and state 4 respiration (respiration in the presence of oligomycin – non-respiratory oxygen consumption) on complex I substrates (glutamate+malate); black bars = <i>PARK2^−/−</i>, white bars = wild type; data are expressed as nmoles O₂/mL and mg proteins and are shown as means ± SD; two animals, one per genotype, were analyzed the same day; the numbers between brackets indicate the number of individual animals of each genotype; (B) results from crude mitochondrial pellets from striatum (n = 6), (C) crude mitochondrial pellets from cortex (n = 7) and (D) purified mitochondria from whole brain (n = 2).</p

High resolution respirometry reveals a respiration defect in <i>PARK2^−/−</i> mice.

<p>(<b>A</b>) Representative experiment with striatal post-nuclear supernatant. Blue curve = oxygen concentration (O₂) expressed as nmol/mL; red curve = rate of oxygen consumption expressed as nmoles O₂/mL and min; successive additions: ADP = 1 mM ADP in the medium with 10 mM glutamate+5 mM malate; OM = 1 µg/mL oligomycin, cccp = successive additions of 1.25 µM (up to the maximal respiration rate independent from ATP synthase capacity); KCN = 1 mM KCN (non-respiratory oxygen consumption); (<b>B, C, D</b>) Bars showing state 3 respiration, state 4 respiration and respiratory reserve on complex I substrates (glutamate+malate) in post-nuclear supernatants from (B) striatum, (C) midbrain and (D) liver of 24- and 9-month-old <i>PARK2^−/−</i> (black bars) and wild type mice (white bars); all data are expressed as nmoles O₂/mL and mg proteins and shown as means ± SD; two animals, one per genotype, were analyzed the same day; the data were obtained from 6 to 8 individual animals of each genotype. * <i>p</i><0.05 using Mann and Whitney test. In liver, state 3 respiration rate and respiratory reserve significantly decreased with age in both wild type mice (p = 0.011 and <0.001 when comparing state 3 respiration and respiratory reserve respectively between 9 and 24 months of age using Mann and Whitney test) and in <i>PARK2^−/−</i> mice (p = 0.019 and <0.001 when comparing state 3 respiration and respiratory reserve respectively between 9 and 24 months of age using Mann and Whitney test).</p

Maximal respiration is reduced in striatal neurons from <i>PARK2^−/−</i> mice.

<p>Representative experiments with neurons or astrocytes from striatum or cortex: the traces represent the evolution of the respiration rate (in pmol O₂/minute) in Seahorse plates seeded with cells from <i>PARK2^−/−</i> (black circles) and wild type (white circles) mice. Sequential additions are: oligomycin (OM) (0.25 µg/ml for neurons and 0.5 µg/ml for astrocytes), fccp (FP) (3 µM for cortical neurons and 1 µM for other cells), and rotenone+antimycine (RA) (50 nM rotenone+150 µg/ml antimycin A). Bars below the traces show the means and SD of basal respiration, maximal respiration and spare respiratory capacity (SRC = maximal – basal respiration) of 3 independent tests, each performed in 6 to 12 independent wells. * <i>p</i><0.05 using Mann and Whitney test.</p

The inner mitochondrial membrane potential is normal in midbrain and striatum of <i>PARK2</i>^−/− mice.

<p>Dark circles = <i>PARK2^−/−</i>, white circles = wild type; ΔΨm = inner mitochondrial membrane potential expressed as mV after transformation of the fluorescent rhodamine 123 signal using the Nernst equation, as explained in the methods section; A = ΔΨm in the presence of glutamate+malate; B = A+1 mM ADP; C = B+1 µM oligomycin; D = C+1 µM cccp; E = C+2 µM cccp; F = C+4 µM cccp;G = C+8 µM cccp. The data were obtained in parallel to the analysis of respiration; two animals, one per genotype, were analyzed the same day; the data obtained from seven 9-month-old mice and six 24-month-old ones of each genotype, they are expressed as mean and SD.</p

Increased mitochondrial Mn superoxide dismutase (SOD2) in the striatum of <i>PARK2</i>^−/− mice.

<p>Western blot analysis of proteins from crude mitochondrial pellets from striatum (A) and midbrain (B) of <i>PARK2</i>^−/− and wild-type mice, at 12 and 24 months of age; loading control was the outer membrane protein Voltage-Dependent Anion Channel (VDAC); (C) densitometric analysis of the SOD2 signal shown in A and B was normalized to VDAC signal and quantification was expressed as means and SD and as % of the mean of wild-type samples on the membrane shown in A and B; * = p<0.05 using Mann and Whitney test; † and ‡: samples from 12-month-old <i>PARK2^−/−</i> and wild type mice respectively showing that the SOD2 steady-state level does not significantly change with age.</p