ATR Prevents Ca\u3csup\u3e2+\u3c/sup\u3e Overload-Induced Necrotic Cell Death Through Phosphorylation-Mediated Inactivation of PARP1 Without DNA Damage Signaling

Hyperactivation of PARP1 is known to be a major cause of necrotic cell death by depleting NAD+/ATP pools during Ca2+ overload which is associated with many ischemic diseases. However, little is known about how PARP1 hyperactivity is regulated during calcium overload. In this study we show that ATR kinase, well known for its role in DNA damage responses, suppresses ionomycin, glutamate, or quinolinic acid-induced necrotic death of cells including SH-SY5Y neuronal cells. We found that the inhibition of necrosis requires the kinase activity of ATR. Specifically, ATR binds to and phosphorylates PARP1 at Ser179 after the ionophore treatments. This site-specific phosphorylation inactivates PARP1, inhibiting ionophore-induced necrosis. Strikingly, all of this occurs in the absence of detectable DNA damage and signaling up to 8 hours after ionophore treatment. Furthermore, little AIF was released from mitochondria/cytoplasm for nuclear import, supporting the necrotic type of cell death in the early period of the treatments. Our results reveal a novel ATR-mediated anti-necrotic mechanism in the cellular stress response to calcium influx without DNA damage signaling

Analysis of Global Sumoylation Changes Occurring during Keratinocyte Differentiation

Sumoylation is a highly dynamic process that plays a role in a multitude of processes ranging from cell cycle progression to mRNA processing and cancer. A previous study from our lab demonstrated that SUMO plays an important role in keratinocyte differentiation. Here we present a new method of tracking the sumoylation state of proteins by creating a stably transfected HaCaT keratinocyte cell line expressing an inducible SNAP-SUMO3 protein. The SNAP-tag allows covalent fluorescent labeling that is denaturation resistant. When combined with two-dimensional gel electrophoresis, the SNAP-tag technology provides direct visualization of sumoylated targets and can be used to follow temporal changes in the global cohort of sumoylated proteins during dynamic processes such as differentiation. HaCaT keratinocyte cells expressing SNAP-SUMO3 displayed normal morphological and biochemical features that are consistent with typical keratinocyte differentiation. SNAP-SUMO3 also localized normally in these cells with a predominantly nuclear signal and some minor cytoplasmic staining, consistent with previous reports for untagged SUMO2/3. During keratinocyte differentiation the total number of proteins modified by SNAP-SUMO3 was highest in basal cells, decreased abruptly after induction of differentiation, and slowly rebounded beginning between 48 and 72 hours as differentiation progressed. However, within this overall trend the pattern of change for individual sumoylated proteins was highly variable with both increases and decreases in amount over time. From these results we conclude that sumoylation of proteins during keratinocyte differentiation is a complex process which likely reflects and contributes to the biochemical changes that drive differentiation

Consequences of Chinese aid in Sub-Saharan Africa

China's position of non-interference in foreign governments' affairs, while currently good for Chinese business, may threaten to increase international terrorism, deepen regime corruption, and erode U.S. political relevance in sub-Saharan Africa. China has empowered private enterprises, which can monopolize African market sectors, marginalize African businesses, and exacerbate local social conditions. Using non-violent uprising and violent resistance events from Social Conflict Analysis Database (SCAD), World Governmental Indicators (WGI), and World Development Indicators (WDI) databases, this study seeks to determine to what extent China's long-term economic goals may challenge U.S. security objectives in Africa. Observations from African states will form the base for analysis to establish a fundamental correlation between Chinese direct investment and Beijing's foreign policy in Africa. This study illustrates that China's foreign policy is not reflected in the actions of its state-owned enterprises and non-government organizations, increasing the potential for friction and conflict, and that China's investment approach inherently requires the support of the host nation and may affect our African partners' alignment with U.S. policy objectives. This study also highlights a significant gap in data regarding the state of our partners in Africa.http://archive.org/details/consequencesofch1094551612Major, United States ArmyLieutenant Commander, United States NavyApproved for public release; distribution is unlimited

HaCaT SNAP-SUMO3 cells display normal biochemical and physical characteristics of keratinocyte differentiation.

<p>(<b>A</b>) SNAP-SUMO3 HaCaT cells were plated and induced to differentiate with 2.38 mM calcium. SNAP-SUMO3 induction was started 48 hours prior to harvest for each time point. Samples were collected at 24 hour intervals from 0–144 hours and were analyzed on 8% gels followed by Western blotting for K1, involucrin, loricrin, and tubulin. The upper figure shows a time based progression of K1 expression between differentiating (HI) and basal (Low) cultures. The lower panel shows the differences at 144 hours for K1, involucrin, and loricrin expression between high and low calcium SNAP-SUMO3 HaCaT cells. (<b>B through E</b>) Time course comparison of the physical morphology of basal (<b>B and C</b>) and differentiating (<b>D and E</b>) SNAP-SUMO3 HaCaT keratinocytes (B and D) versus parental HaCaT cells (C. and E) using phase microscopy. Magnification is 200× except for the 72 hour samples which were at 320×.</p

Relative protein distribution on 2D gels.

<p>Relative protein distribution on 2D gels.</p

Overall characterization of spot statistics obtained from the 2D analysis.

<p>(<b>A</b>) The bar graph depicts the number of total detectable spots, statistically significant spots, and unique spots for each time point analyzed. (<b>B</b>) A bar graph showing the percentage of spots that were present in from 2–7 time points. Spot data used to derive the graphs in (<b>A</b>) and (<b>B</b>) are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030165#pone.0030165.s007" target="_blank">Table S1</a>.</p

Spot values over the course of differentiation for representative spots.

<p>(<b>A</b>) A set of bar graphs depicting individual proteins that showed statistically significant changes in spot value over the 0–144 hour period. (<b>B</b>) A set of bar graphs depicting representative spots that showed no statistically significant differences during differentiation. Data for both (<b>A</b>) and (<b>B</b>) are derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030165#pone.0030165.s007" target="_blank">Table S1</a>.</p

SNAP-SUMO3 is functional and capable of being detected in gel.

<p>(<b>A</b>) SNAP-SUMO3 HaCaT cells were divided into induced (+lanes) and uninduced (−lanes) groups, and the induced groups were treated for 48 hours with 1.0 ug/ml of tetracycline. At 48 hours post Tet addition, cells were labeled with SNAP-cell DAF, lysed with 4× sample buffer, and electrophoresed on 6% (left and middle panels) or 15% (right panel) SDS polyacrylamide gels. The gels were either imaged using a Fuji FLA-5100 (Fluorescent Scan) or were transferred to a PVDF membrane and detected using S-protein conjugated to HRP (Far Western Blot panel) or anti-SUMO2/3 (Western Blot panel). (<b>B</b>) Induced extracts prepared as in (<b>A</b>) were subjected to 2-fold serial dilutions, electrophoresed on an 8% SDS polyacrylamide gel, and then free SNAP-SUMO3 was detected either by fluorescent scan or by immunoblotting with anti-SUMO3.</p

SNAP-SUMO3 expression does not affect cell cycle distribution, but does slow cell doubling.

<p>(<b>A</b>) HaCaT cells were plated and divided into uninduced and induced groups. Induction of SNAP-SUMO3 was for 48 hours with 1.0 ug/ml of tetracycline. After 48 hours the cells were analyzed by FACS to determine cell cycle distributions. The experiment was performed three times and the results shown are the mean and standard error of the mean. (<b>B</b>) SNAP-SUMO3 HaCaT cells and parental HaCaT FRT/TR#8 cells were grown and induced as in (<b>A</b>). Triplicate cultures were prepared and counted each day and averages were taken. Error bars represent mean ± standard deviation.</p