Fatal steamer accident; blunt force injuries and drowning in post-mortem MSCT and MRI.

Steamer accidents, through contact with the bucket wheel, are very seldom today. No publication of such a kind of fatal accident could be found in literature. We present the case of a fatal steamer accident, in which the findings of a blunt traumatization of a person by the ship was completely documented by post-mortem combined multi-slice computed tomography (MSCT) and magnetic resonance imaging (MRI) examinations. A rupture of the aorta was detected using both radiological methods without use of radiopaque material. Radiological examination revealed a comminuted fracture of the thorax vertebrae at the same level as the aortic rupture. Injuries of the soft tissues of the back, caused by the bucket wheel of the steamer, were also diagnosed. In addition to the signs of blunt force trauma the findings of drowning such as an over inflation of the lungs, fluid in the stomach and duodenum were revealed. Furthermore, algological analysis detected diatoms in the lung tissue and blood from the left heart. Therefore, the cause of death was considered being a combination of fatal hemorrhage, caused by the aortic rupture, and drowning. We conclude that virtual autopsy using combined post-mortem MSCT and MRI is a useful tool for documentation, visualisation and analysis of the findings of blunt force trauma and drowning with a large potential in forensic medicine.

Chlorophyll breakdown: Pheophorbide a oxygenase is a Rieske-type iron-sulfur protein, encoded by the accelerated cell death 1 gene

Chlorophyll (chl) breakdown during senescence is an integral part of plant development and leads to the accumulation of colorless catabolites. The loss of green pigment is due to an oxygenolytic opening of the porphyrin macrocycle of pheophorbide (pheide) a followed by a reduction to yield a fluorescent chl catabolite. This step is comprised of the interaction of two enzymes, pheide a oxygenase (PaO) and red chl catabolite reductase. PaO activity is found only during senescence, hence PaO seems to be a key regulator of chl catabolism. Whereas red chl catabolite reductase has been cloned, the nature of PaO has remained elusive. Here we report on the identification of the PaO gene of Arabidopsis thaliana (AtPaO). AtPaO is a Rieske-type iron–sulfur cluster-containing enzyme that is identical to Arabidopsis accelerated cell death 1 and homologous to lethal leaf spot 1 (LLS1) of maize. Biochemical properties of recombinant AtPaO were identical to PaO isolated from a natural source. Production of fluorescent chl catabolite-1 required ferredoxin as an electron source and both substrates, pheide a and molecular oxygen. By using a maize lls1 mutant, the in vivo function of PaO, i.e., degradation of pheide a during senescence, could be confirmed. Thus, lls1 leaves stayed green during dark incubation and accumulated pheide a that caused a light-dependent lesion mimic phenotype. Whereas proteins were degraded similarly in wild type and lls1, a chl-binding protein was selectively retained in the mutant. PaO expression correlated positively with senescence, but the enzyme appeared to be post-translationally regulated as well.

Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation.

Death-associated protein kinase 2 (DAPK2) is a Ca(2+)/calmodulin-dependent Ser/Thr kinase that possesses tumor-suppressive functions and regulates programmed cell death, autophagy, oxidative stress, hematopoiesis, and motility. As only few binding partners of DAPK2 have been determined, the molecular mechanisms governing these biological functions are largely unknown. We report the identification of 180 potential DAPK2 interaction partners by affinity purification-coupled mass spectrometry, 12 of which are known DAPK binding proteins. A small subset of established and potential binding proteins detected in this screen was further investigated by bimolecular fluorescence complementation (BiFC) assays, a method to visualize protein interactions in living cells. These experiments revealed that α-actinin-1 and 14-3-3-β are novel DAPK2 binding partners. The interaction of DAPK2 with α-actinin-1 was localized at the plasma membrane, resulting in massive membrane blebbing and reduced cellular motility, whereas the interaction of DAPK2 with 14-3-3-β was localized to the cytoplasm, with no impact on blebbing, motility, or viability. Our results therefore suggest that DAPK2 effector functions are influenced by the protein's subcellular localization and highlight the utility of combining mass spectrometry screening with bimolecular fluorescence complementation to identify and characterize novel protein-protein interactions.

Defying death: Cellular survival strategies following plasmalemmal injury by bacterial toxins

The perforation of the plasmalemma by pore-forming toxins causes an influx of Ca(2+) and an efflux of cytoplasmic constituents. In order to ensure survival, the cell needs to identify, plug and remove lesions from its membrane. Quarantined by membrane folds and isolated by membrane fusion, the pores are removed from the plasmalemma and expelled into the extracellular space. Outward vesiculation and microparticle shedding seem to be the strategies of choice to eliminate toxin-perforated membrane regions from the plasmalemma of host cells. Depending on the cell type and the nature of injury, the membrane lesion can also be taken up by endocytosis and degraded internally. Host cells make excellent use of an initial, moderate rise in intracellular [Ca(2+)], which triggers containment of the toxin-inflicted damage and resealing of the damaged plasmalemma. Additional Ca(2+)-dependent defensive cellular actions range from the release of effector molecules in order to warn neighbouring cells, to the activation of caspases for the initiation of apoptosis in order to eliminate heavily damaged, dysregulated cells. Injury to the plasmalemma by bacterial toxins can be prevented by the early sequestration of bacterial toxins. Artificial liposomes can act as a decoy system preferentially binding and neutralizing bacterial toxins.

TRAIL signaling is mediated by DR4 in pancreatic tumor cells despite the expression of functional DR5

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and agonistic anti-DR4/TRAIL-R1 and anti-DR5/TRAIL-R2 antibodies are currently under clinical investigation for treatment of different malignancies. TRAIL activates DR4 and DR5 and thereby triggers apoptotic and non-apoptotic signaling pathways, but possible different roles of DR4 or DR5 in these responses has poorly been addressed so far. In the present work, we analyzed cell viability, DISC formation as well as IL-8 and NF-kappaB activation side by side in responses to TRAIL and agonistic antibodies against DR4 (mapatumumab) and against DR5 (lexatumumab) in pancreatic ductal adenocarcinoma cells. We found that all three reagents are able to activate cell death and pro-inflammatory signaling. Death-inducing signaling complex (DISC) analysis revealed that mapatumumab and lexatumumab induce formation of homocomplexes of either DR4 or DR5, whereas TRAIL additionally stimulated the formation of heterocomplexes of both receptors. Notably, blocking of receptors using DR4- and DR5-specific Fab fragments indicated that TRAIL exerted its function predominantly via DR4. Interestingly, inhibition of PKC by Goe6983 enabled DR5 to trigger apoptotic signaling in response to TRAIL and also strongly enhanced lexatumumab-mediated cell death. Our results suggest the existence of mechanisms that silence DR5 for TRAIL- but not for agonistic-antibody treatment.