ABSTRACT
The structure of death receptor 4 (DR4) in complex with TNF-related apoptosis-inducing ligand (TRAIL) has been determined at 3â Å resolution and compared with those of previously determined DR5-TRAIL complexes. Consistent with the high sequence similarity between DR4 and DR5, the overall arrangement of the DR4-TRAIL complex does not differ substantially from that of the DR5-TRAIL complex. However, subtle differences are apparent. In addition, solution interaction studies were carried out that show differences in the thermodynamics of binding DR4 or DR5 with TRAIL.
Subject(s)
Receptors, TNF-Related Apoptosis-Inducing Ligand/chemistry , TNF-Related Apoptosis-Inducing Ligand/chemistry , Amino Acid Sequence , Calorimetry , Crystallization , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Sequence Data , Protein Binding , Receptors, TNF-Related Apoptosis-Inducing Ligand/isolation & purification , TNF-Related Apoptosis-Inducing Ligand/isolation & purification , ThermodynamicsABSTRACT
Prolonged therapeutic hypothermia (32-34 degrees C for 12-24 h) improves the functional outcome of comatose cardiac arrest survivors. It is generally believed that rapidly achieving target temperature optimizes neuroprotection. However, this hypothesis has not been tested systematically. In this study, we compared the neuroprotective effect of prolonged hypothermia initiated between 0 and 8 h after reoxygenation using an in vitro model of simulated global brain ischemia. Organotypic hippocampal slices were prepared from 5-day-old Wistar rat pups and cultured for 1 week prior to analysis. Ischemia was simulated by normothermic oxygen-glucose deprivation (OGD). Hypothermia (33 degrees C) was initiated 0-8 h after reoxygenation and maintained until 24 h post-injury. CA1 regional cell death was quantified by propidium iodide (PI) fluorescence. Release of 14-3-3 beta protein was evaluated as a potential surrogate maker for neuroprotection. Hypothermia initiated 0, 1, 2, or 4 h after 30 min OGD reduced 24 h CA1 regional PI fluorescence by 47 +/- 34%, 85 +/- 4%, 88 +/- 3%, and 88 +/- 5% (P < 0.05 for all versus normothermic reoxygenation). Direct comparison of hypothermia initiated 4 or 8 h after reoxygenation revealed equivalent neuroprotection following 15 and 30 min OGD, but neither was protective after 60 min OGD. Hypothermia initiated 4 or 8 h after 30 min OGD reduced 14-3-3 beta release by 73 +/- 11% and 92 +/- 4%, respectively (P < 0.01 for both versus normothermic reoxygenation). In this model, the neuroprotective effect of prolonged post-ischemic hypothermia is both optimal and equivalent when initiated between 1 and 8 h after reoxygenation. These results suggest the need for further in vivo studies to define the therapeutic window within which prolonged hypothermia is optimally neuroprotective after cardiac arrest.
Subject(s)
Hypothermia, Induced , Ischemic Attack, Transient/therapy , 14-3-3 Proteins/analysis , Animals , Hippocampus/injuries , Hippocampus/pathology , In Vitro Techniques , Ischemic Attack, Transient/metabolism , Rats , Rats, Wistar , Temperature , Time FactorsABSTRACT
PAX3 and PAX7 encode transcription factors implicated in the pathogenesis of rhabdomyosarcoma (RMS), including alveolar RMS in which chromosomal translocations generate PAX3-FKHR and PAX7-FKHR fusions. Previous studies of wild-type PAX3 and PAX7 identified alternative splicing events that modify the paired box and generate 2 isoforms of PAX3 (Q+ and Q-) and 4 isoforms of PAX7 (Q+GL+, Q+GL-, Q-GL+, Q-GL-). In our study, we investigated alternative splicing of the wild-type and fusion forms of PAX3 and PAX7 in alveolar and embryonal RMS and assessed the functional implications. For PAX3 and PAX3-FKHR, the Q+ and Q- isoforms were consistently co-expressed in RMS tumors with slightly higher levels of the Q+ isoform. For PAX7 and PAX7-FKHR, there was a consistent pattern of co-expression of the 4 isoforms in RMS tumors: Q+GL- > Q+GL+ >/= Q-GL- > Q-GL+. DNA binding analysis demonstrated that PAX3 and PAX3-FKHR Q- isoforms exhibit higher affinity than corresponding Q+ isoforms for class I sites and no difference for class II sites. For PAX7 and PAX7-FKHR, the relative affinity was Q-GL- > Q+GL- > Q-GL+ >/= Q+GL+ for class I sites and Q-GL-, Q+GL- > Q-GL+, Q+GL+ for class II sites. Finally, the transcriptional activities of the PAX3-FKHR and PAX7-FKHR isoforms on reporter plasmids varied over a 5-fold and 50-fold range, respectively, in accord with the differences in DNA binding activity. In conclusion, these studies reveal that PAX3, PAX7 and their fusions with FKHR are each expressed in RMS tumors as a consistent mixture of functionally distinct isoforms.
