Anchored FRET sensors detect local caspase activation prior to neuronal degeneration

<p>Abstract</p> <p>Background</p> <p>Recent studies indicate local caspase activation in dendrites or axons during development and in neurodegenerative disorders such as Alzheimer's disease (AD). Emerging evidences point to soluble oligomeric amyloid-β peptide as a causative agent in AD.</p> <p>Results</p> <p>Here we describe the design of fluorescence resonance energy transfer (FRET)-based caspase sensors, fused to the microtubule associated protein tau. Specific caspase sensors preferentially cleaved by caspase-3, -6 or -9 were expressed in differentiated human neuroblastoma SH-SY5Y cells. The anchoring of the sensors resulted in high FRET signals both in extended neurites and soma and made analysis of spatiotemporal signal propagation possible. Caspase activation was detected as loss of FRET after exposure to different stimuli. Interestingly, after staurosporine treatment caspase-6 activation was significantly delayed in neurites compared to cell bodies. In addition, we show that exposure to oligomer-enriched amyloid-β peptide resulted in loss of FRET in cells expressing sensors for caspase-3 and -6, but not -9, in both soma and neurites before neurite degeneration was observed.</p> <p>Conclusions</p> <p>Taken together, the results show that by using anchored FRET sensors it is possible to detect stimuli-dependent differential activation of caspases and to distinguish local from global caspase activation in live neuronal cells. Furthermore, in these cells oligomer-enriched amyloid-β peptide induces a global, rather than local activation of caspase-3 and -6, which subsequently leads to neuronal cell death.</p

Particulate mercury in the atmosphere: Its significance, transport, transformation and sources

The importance of particulate mercury (Hg(p)) in the transport, chemistry and deposition of this toxic metal has long been underestimated and largely ignored. While it was once believed to constitute a small percentage of total atmospheric mercury, Hg(p) may contribute a significant portion of the deposition of this metal to adjacent natural waters. Recent measurements of Hg(p) in several urban/industrial areas have documented that Hg can be associated with large particles (>2.5 μm) and in concentrations similar to those of the vapor phase Hg (ng/m 3 ). As part of ongoing effort to diagnose the sources, transport and deposition of Hg to the Great Lakes and other Great Waters, the University of Michigan Air Quality Laboratory (UMAQL) has investigated the physical and chemical properties of particulate-phase Hg in both urban and rural locations. It appears that particulate Hg may be the one of the most difficult of the Hg measurements to perform, and perhaps the one of the most important for deposition and source apportionment studies. Particulate Hg concentrations measured in rural areas of the Great Lakes Region and Vermont ranged from 1 to 86 pg/m 3 whereas Hg(p) levels in urban/industrialized areas were in the range 15 pg/m 3 to 1.2 ng/m 3 .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43909/1/11270_2005_Article_BF01189664.pd

Long-term Atmospheric Mercury Wet Deposition at Underhill, Vermont

Section 112(m) of the 1990 Clean Air Act Amendments, referred to as the Great Waters Program, mandated an assessment of atmospheric deposition of hazardous air pollutants (HAPs) to Lake Champlain. Mercury (Hg) was listed as a priority HAP and has continued to be a high priority for a number of national and international programs. An assessment of the magnitude and seasonal variation of atmospheric Hg levels and deposition in the Lake Champlain basin was initiated in December 1992 which included event precipitation collection, as well as collection of vapor and particle phase Hg in ambient air. Sampling was performed at the Proctor Maple Research Center in Underhill Center, VT. The range in the annual volume-weighted mean concentration for Hg in precipitation was 7.8–10.5 ng/l for the 11-year sampling period and the average amount of Hg deposited with each precipitation event was 0.10 μg/m 2 . The average amount of Hg deposited through precipitation each year from 1993 to 2003 was 9.7 μg/m 2 /yr. A seasonal pattern for Hg in precipitation is clearly evident, with increased Hg concentrations and deposition observed during spring and summer months. While a clear trend in the 11-year event deposition record at Underhill was not observed, a significant decrease in the event max-to-monthly ratio was observed suggesting that a major source influence was controlled over time. Discrete precipitation events were responsible for significant fractions of the monthly and annual loading of Hg to the forested ecosystem in Vermont. Monthly-averaged temperatures were found to be moderately correlated with monthly volume-weighted mean Hg concentrations ( r 2 =0.61) and Hg deposition ( r 2 =0.67) recorded at the Vermont site. Meteorological analysis indicated the highest levels of Hg in precipitation were associated with regional transport from the west, southwest, and south during the warmer months.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44444/1/10646_2004_Article_6260.pd

Fe65 electrophoretic mobility shift due to phosphorylation is not detected in the nuclear fraction.

<p>A. Western blot analysis of Fe65 in cytoplasmic (C1) and nuclear (N1) fractions after incubation in the absence or presence of alkaline phosphatase. Two different exposure times are shown for optimal visualization of Fe65 in the different fractions. B. Western blot analysis of Fe65 in cytoplasmic (C1), nuclear (N1) and cytoplasmic and nuclear mixed (C1 + N1) fractions after incubation in the absence or presence of alkaline phosphatase. C. Western blot analysis showing the effect of alkaline phosphatase treatment of cytoplasmic fractions on endogenous Fe65 and p97Fe65-TAP (C2), p60Fe65-TAP (C3), or the deletion mutants ΔWWFe65-TAP (C4) and ΔPTB2Fe65-TAP. D. Representative western blot analysis of lysates from SK-N-AS cells overexpressing the different Fe65 constructs lacking the TAP tag. The results are from the same blot. Different exposure times are shown for optimal visualization.</p