Effect of different peptides on the membrane potential of rat liver mitochondria measured by safranine O.

<p>Panel <b>A</b>. Shown are traces of fluorescence in the medium described in “Materials and Methods.” In all traces, 5 mM of succinate and 1 µM of rotenone were supplemented about 150 s before the addition of a peptide at t = 0 s. Control, no other additions. gA, [Glu1]gA, [Glu3]gA, [Lys1]gA, and [Lys3]gA show traces after the addition of 5 nM (i.e. about 10 nanogram/ml) of a corresponding peptide at t = 0 s. Trace “excess [Glu1]gA” was measured with 1 µg/ml peptide. Panel <b>B.</b> Dose dependence of the effect of [Glu1]gA on mitochondrial membrane potential.</p

Micrographs of rat kidney cell culture loaded with Lysotracker Green in the absence (A), and in the presence of 0.1 mg/ml (B) of [Glu1]gA.

<p>Micrographs of rat kidney cell culture loaded with Lysotracker Green in the absence (A), and in the presence of 0.1 mg/ml (B) of [Glu1]gA.</p

Single-channel recordings (A, D) and the corresponding current histograms (B, E) of gramicidin A (picogram/ml, A, B, and C) and [Glu1]gA (2 nanogram/ml, D, E, and F) at a voltage of 100 mV applied to the DPhPC/decane membrane.

<p>The solution was 100 mM HCl. (<b>C</b>, <b>F</b>) Open state duration histograms fitted by a single exponential with a time constant of 116 ms (<b>C</b>) and 16 ms (<b>F</b>). The records were filtered at 100 Hz (<b>A</b>, <b>B</b>, and <b>C</b>) or 1000 Hz (<b>D</b>, <b>E</b>, and <b>F</b>).</p

Effect of [Glu1]gA on mitochondrial membrane potential in renal cells.

<p>Confocal images of cultured renal cells loaded with TMRE in control (A) and after treatment with 0.01 mg/ml [Glu1]gA (B). Diagram (C) presents the mean intensity of TMRE fluorescence through 10 confocal images for each [Glu1]gA (solid bars) or gA (hatched bars) concentration.</p

Effect of liposomes (1 µg/ml DPhPC) on the induction of electrical current on planar lipid bilayer by gA (2 nanogram/ml, panel A) and [Glu1]gA (200 nanogram/ml, panel B).

<p>The peptides and liposomes were added at one side of the membrane. The solution was 100 mM KCl, 10 mM Tris, 10 mM MES, pH = 7.0, BLM voltage was 50 mV.</p

Dissipation of the pH gradient on membranes of pyranine-loaded liposomes by gA and [Glu1]gA (both 2 µg/ml).

<p>Inner liposomal pH was estimated from pyranine fluorescence intensity measured at 505 nm upon excitation at 455 nm. Nigericin (1 µM) was added at 400–420 s to equilibrate the pH. Control, a record without peptides.</p

Do Extracellular Vesicles Derived from Mesenchymal Stem Cells Contain Functional Mitochondria?

Extracellular vesicles (EV) derived from stem cells have become an effective complement to the use in cell therapy of stem cells themselves, which has led to an explosion of research into the mechanisms of vesicle formation and their action. There is evidence demonstrating the presence of mitochondrial components in EV, but a definitive conclusion about whether EV contains fully functional mitochondria has not yet been made. In this study, two EV fractions derived from mesenchymal stromal stem cells (MSC) and separated by their size were examined. Flow cytometry revealed the presence of mitochondrial lipid components capable of interacting with mitochondrial dyes MitoTracker Green and 10-nonylacridine orange; however, the EV response to the probe for mitochondrial membrane potential was negative. Detailed analysis revealed components from all mitochondria compartments, including house-keeping mitochondria proteins and DNA as well as energy-related proteins such as membrane-localized proteins of complexes I, IV, and V, and soluble proteins from the Krebs cycle. When assessing the functional activity of mitochondria, high variability in oxygen consumption was noted, which was only partially attributed to mitochondrial respiratory activity. Our findings demonstrate that the EV contain all parts of mitochondria; however, their independent functionality inside EV has not been confirmed, which may be due either to the absence of necessary cofactors and/or the EV formation process and, probably the methodology of obtaining EV