9 research outputs found
Energy utilization in FDB fibers.
<p>The graph shows the estimates for the relative baseline change ± SEM after different treatment conditions (pretreatment/injection). Changes, displayed in %, are statistically valuated by the corresponding p-value. The increased SERCA activity observed after FK506 injection can be incompletely normalized by pretreatment with JTV-519.</p
The graph shows the estimates for the AUC (mean±SEM) for the four groups.
<p>Changes, displayed in %, are statistically valuated by the corresponding p-value. n: number of fibers measured per group. Control (n=42), FK506 (n=42), JTV-519 (n=53) and JTV-519 + FK506 (n= 44).</p
Temporal profile curves for calcium transients during single twitch activation (A) and tetanic stimulation (B).
<p>Each line corresponds to the average of all single fiber measurements that have been included in the statistical analysis. For improving the visibility of group differences, the time interval was adjusted accordingly. The red and black dashed lines indicate the drift of the baseline calcium during the tetanic stimulation. Treatment of fibers with 25 μM FK506 (red line) leads to an elevated inter-peak-baseline level compared to untreated control fibers (black line). </p
Detection of mhtt species in human samples using SEC-FRET technology.
<p>6.1. SEC-FRET profiles with the UV absorbance in the upper right corner in human fibroblasts (A) Healthy (Q7), (B) HD (Q68) (C) Detection of mhtt full length protein (FL) in HD fibroblast by Western blot analysis using MW1 or 2B7antibodies 6.2. SEC-FRET profiles with the UV absorbance in the upper right corner in human <i>post mortem</i> sensory and motor cortex (A) Healthy, (B) HD grade1. (C) Detection of mhtt full length (peak1) and mhtt fragments (peak2) protein in HD human post mortem brain by Western blot analysis using 4C9 (human poly-proline) or 2B7antibodies.</p
Analysis of <i>Hdh</i>Q150/− striatum.
<p>(A) Overlapped SEC-FRET profiles at 3, 4, 6 and 8 months of <i>Hdh</i>Q150/− striatum samples. (B) Detection of htt full-length protein (FL) by Western blot analysis in fractions 35–37 (peaks 1) and of mhtt fragments in fractions 44–49 (peak 2) using either 2B7 or MW1 antibodies. (C). Detection of mhtt fragments by Western Blot analysis of fractions 44, 45, 46 of <i>Hdh</i>Q150/− striatum samples at 3 and 8 months (D) Quantification of the insoluble mhtt by TR-FRET MW8-Tb and MW8-D2 detection. (E) Quantification of the soluble mhtt by TR-FRET 2B7-Tb and MW8-D2 detection. (F) Detection of mhtt fragments by Western Blot analysis of soluble (supernatant, 100000 g) and insoluble (pellet) brain extracts of <i>Hdh</i>Q150/− mice at 3, 8 and 12 months.</p
A model of how mutant htt aggregates in cells.
<p>After proteolytic cleavage of the full-length mutant htt protein, free monomeric N-terminal fragments are extremely short-lived, likely because the expanded polyQ region confers great conformational flexibility but misfolded states at the same time (k1). These facilitate unspecific protein-protein interactions that compromise cell function and viability. As a first line of defense, cells may therefore capture mutant htt monomeric fragments rapidly into soluble oligomers (k2). The pool size capacity of soluble oligomers may be limited by the availability of stabilization-factors (SF) of the protein quality control (chaperone, autophagy, proteosome, etc.). Chronic saturation of the soluble oligomer pool would likely increase the flux of mutant htt fragments into and out of the soluble oligomer pool into insoluble aggregates, perhaps the last resort and coping strategy cells can count on to prevent mutant htt fragments from impairing cell function (k3 and k4).</p
Inverse correlation of soluble and aggregated mhtt fragments analyzed by <i>Hdh</i>Q150/− forebrain tissue.
<p>SEC-FRET profiles with the UV absorbance in the upper right corner for (A) 3 months and (B) 12 months heterozygote <i>Hdh Q150/−</i> forebrain tissue. (C) Quantification of soluble mhtt by 2B7-Tb and MW1-D2 TR-FRET detection in supernatant fraction and of insoluble mhtt by MW8-Tb and MW8-D2 detection in resuspended pellet fraction of <i>Hdh Q150</i>/− mice at 3, 8 and 12 months. Delta F values (see experimental procedure) are mean of 4 independent measurements ± SEM. Delta F values are adjusted to protein concentration and then normalized to wild-type. The t-test was used to assess significance (D) Quantification of SEC fractions of peak 1 and of peak 2 by 2B7-Tb and MW1-D2 TR-FRET taken from <i>Hdh Q150</i>/− mice at 3, 8 and 12 months. Delta F values (mean of 4 independent measurements ± SEM) are expressed as the maximal peak value; The t-test was used to assess significance.</p
Analysis of 8 months <i>Hdh</i>Q150 forebrain identified invariant, soluble oligomeric pool of mhtt fragments.
<p>(A), (B), (C), Individual TR-FRET based detection profiles after SEC with the UV absorbance in the upper right corner for A: Wild type, B: Heterozygote <i>Hdh</i>Q150, C: Homozygote <i>Hdh</i>Q150 forebrain samples at 8 months. Molecular weight of TR-FRET peaks are indicated in (C). (D) Western Blot analysis to detect htt full-length in the fractions 32, 33, 34 for peak1 and mhtt fragments at kDa (red arrow) in fractions 45, 46, 47, 48, 49, 50 for peak 2 using either 2B7 or MW1 antibodies. Note, black arrow in wt (peak2) for MW1 detection is non-specific background and below the specific mhtt fragment band in <i>Hdh Q150</i>/− and <i>Hdh Q150/Q150</i> (E) Quantification of soluble mhtt by 2B7-Tb/MW1-D2 TR-FRET detection in supernatant fraction and of insoluble mhtt by MW8-Tb and MW8-D2 detection in resuspended and sonicated pellet fraction taken from wild type, <i>Hdh Q150</i>/− and <i>Hdh Q150/Q150</i> forebrain tissue. Delta F values (see experimental procedure) are mean of 4 independent measurements ± SEM. Delta F values are adjusted to protein concentration and then normalized to wild-type. The t-test was used to assess significance (ns P>0.05; * P<0.05; ** P<0.01, *** P<0.001) (F) Quantification of soluble mhtt in peak1 and peak 2 by 2B7-Tb/MW1-D2 TR-FRET detection for wild type, <i>Hdh Q150</i>/− and <i>Hdh Q150/Q150</i>. Delta F values (mean of 4 independent measurements ± SEM) are expressed as the maximal peak value. The t-test was used to assess significance (G) Immunostaining of aggregated mhtt in the striatum of 4 months old heterozygote (left) and 4 months old homozygote <i>Hdh</i>Q150 mice (right). Frozen sections were stained with Alexa 488-labeled MW8 and counterstained with DAPI to visualize cell nuclei (blue fluorescence).</p
Immunostaining of aggregated mhtt in the striatum of <i>Hdh</i>Q150/− mice.
<p>Frozen sections were stained with unlabeled MW8 and Alexa 594-labeled secondary antibody. In wt control and at 2 months of age only non-specific background fluorescence is visible. At 4 months of age diffuse granular staining is detectable, predominantly in the nucleus. At 6 months of age the diffuse nuclear staining is less pronounced and small nuclear inclusions are visible. At later stages the nuclear inclusions are significantly larger and the granular staining is strongly diminished at 10 months of age. Note, the large elongated and irregular shaped fluorescent structures visible in images taken from <i>Hdh</i>Q150<b>/−</b> mice at 8 and 10 months of age are blood vessels (non-specific staining of mouse IgG by the secondary antibody).</p