Dynamics of the T4 bacteriophage DNA packasome motor: endonuclease VII resolvase release of arrested Y-DNA substrates

Conserved bacteriophage ATP-based DNA translocation motors consist of a multimeric packaging terminase docked onto a unique procapsid vertex containing a portal ring. DNA is translocated into the empty procapsid through the portal ring channel to high density. In vivo the T4 phage packaging motor deals with Y- or X-structures in the replicative concatemer substrate by employing a portal-bound Holliday junction resolvase that trims and releases these DNA roadblocks to packaging. Here using dye-labeled packaging anchored 3.7-kb Y-DNAs or linear DNAs, we demonstrate FRET between the dye-labeled substrates and GFP portal-containing procapsids and between GFP portal and single dye-labeled terminases. We show using FRET-fluorescence correlation spectroscopy that purified T4 gp49 endonuclease VII resolvase can release DNA compression in vitro in prohead portal packaging motor anchored and arrested Y-DNA substrates. In addition, using active terminases labeled at the N- and C-terminal ends with a single dye molecule, we show by FRET distance of the N-terminal GFP-labeled portal protein containing prohead at 6.9 nm from the N terminus and at 5.7 nm from the C terminus of the terminase. Packaging with a C-terminal fluorescent terminase on a GFP portal prohead, FRET shows a reduction in distance to the GFP portal of 0.6 nm in the arrested Y-DNA as compared with linear DNA; the reduction is reversed by resolvase treatment. Conformational changes in both the motor proteins and the DNA substrate itself that are associated with the power stroke of the motor are consistent with a proposed linear motor employing a terminal-to-portal DNA grip-and-release mechanism.     

Apoptosis control by death and decoy receptors

The death receptors Fas and tumor necrosis factor receptor 1 (TNFR1) trigger apoptosis upon engagement by their cognate death ligands. Recently, researchers have discovered several novel homologues of Fas and TNFR1: DR 3, 4, 5, and 6 function as death receptors that signal apoptosis, whereas DcR 1, 2, and 3 act as decoys that compete with specific death receptors for ligand binding. Further, mouse gene knockout studies have enabled researchers to delineate some of the signaling pathways that connect death receptors to the cell's apoptotic machinery.     

mE10, a novel caspase recruitment domain-containing proapoptotic molecule

Apoptotic signaling is mediated by homophilic interactions between conserved domains present in components of the death pathway. The death domain, death effector domain, and caspase recruitment domain (CARD) are examples of such interaction motifs. We have identified a novel mammalian CARD-containing adaptor molecule termed mE10 (mammalian E10). The N-terminal CARD of mE10 exhibits significant homology (47% identity and 64% similarity) to the CARD of a gene from Equine Herpesvirus type 2. The C-terminal region is unique. Overexpression of mE10 in MCF-7 human breast carcinoma cells induces apoptosis. Mutational analysis indicates that CARD-mediated mE10 oligomerization is essential for killing activity. The C terminus of mE10 bound to the zymogen form of caspase-9 and promoted its processing to the active dimeric species. Taken together, these data suggest a model where autoproteolytic activation of pro-caspase-9 is mediated by mE10-induced oligomerization.     

Myodegeneration in EDA-A2 transgenic mice is prevented by XEDAR deficiency

EDA-A1 and EDA-A2 are members of the tumor necrosis factor family of ligands. The products of alternative splicing of the ectodysplasin (EDA) gene, EDA-A1 and EDA-A2 differ by an insertion of two amino acids and bind to distinct receptors. The longer isoform, EDA-A1, binds to EDAR and plays an important role in sweat gland, hair, and tooth development; mutations in EDA, EDAR, or the downstream adaptor EDARADD cause hypohidrotic ectodermal dysplasia. EDA-A2 engages the receptor XEDAR, but its role in the whole organism is less clear. We have generated XEDAR-deficient mice by gene targeting and transgenic mice expressing secreted forms of EDA-A1 or EDA-A2 downstream of the skeletal muscle-specific myosin light-chain 2 or skin-specific keratin 5 promoter. Mice lacking XEDAR were indistinguishable from their wild-type littermates, but EDA-A2 transgenic mice exhibited multifocal myodegeneration. This phenotype was not observed in the absence of XEDAR. Skeletal muscle in EDA-A1 transgenic mice was unaffected, but their sebaceous glands were hypertrophied and hyperplastic, consistent with a role for EDA-A1 in the development of these structures. These data indicate that XEDAR-transduced signals are dispensable for development of ectoderm-derived organs but might play a role in skeletal muscle homeostasis.     

The PYRIN domain: a member of the death domain-fold superfamily

PYRIN domains were identified recently as putative protein-protein interaction domains at the N-termini of several proteins thought to function in apoptotic and inflammatory signaling pathways. The approximately 95 residue PYRIN domains have no statistically significant sequence homology to proteins with known three-dimensional structure. Using secondary structure prediction and potential-based fold recognition methods, however, the PYRIN domain is predicted to be a member of the six-helix bundle death domain-fold superfamily that includes death domains (DDs), death effector domains (DEDs), and caspase recruitment domains (CARDs). Members of the death domain-fold superfamily are well established mediators of protein-protein interactions found in many proteins involved in apoptosis and inflammation, indicating further that the PYRIN domains serve a similar function. An homology model of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1, a member of the Apaf-1/Ced-4 family of proteins, was constructed using the three-dimensional structures of the FADD and p75 neurotrophin receptor DDs, and of the Apaf-1 and caspase-9 CARDs, as templates. Validation of the model using a variety of computational techniques indicates that the fold prediction is consistent with the sequence. Comparison of a circular dichroism spectrum of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1 with spectra of several proteins known to adopt the death domain-fold provides experimental support for the structure prediction.     

Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy

The green fluorescent protein (GFP) and other intrinsically fluorescent proteins (IFPs) are popular reporters because they allow visualization of cellular constituents in living specimens. IFP technology makes it possible to view dynamic processes in living cells, but extended observation, using fluorescence microscopy (both wide-field and confocal), can result in significant light energy exposure. Therefore, it is possible that cells experience light-induced damage that alters cell physiology and confounds observations. To understand the impact that extended viewing has on cells, we obtained quantitative information about the effect of light energy dose and observation conditions on tobacco BY-2 cell physiology. Our results show a non-linear relationship between the excitation light intensity and mitotic arrest, and the frequency of mitotic arrest is dependent on the presence of an IFP that absorbs the excitation light. Moreover, fluorescence microscopy induces the production of reactive oxygen species (ROS), as assayed using BY-2 cells loaded with oxidation-sensitive dyes, and the level of ROS production increases if the cells express an IFP that absorbs the excitation light energy. The dye oxidation follows sigmoidal kinetics and is reversible if the cells are exposed to low irradiation levels. In addition, the dye oxidation rate shows a non-linear relationship to the excitation light intensity, and a good correlation exists between photobleaching, mitotic arrest, and dye oxidation. The data highlight the importance of ROS scavenging for normal mitotic progression, and provide a reference for judiciously choosing conditions that avoid photobleaching that can lead to ROS accumulation and physiological damage.     

Reversal of slow flow phenomenon during primary stenting by bail-out administration of abciximab

BACKGROUND: Slow flow or no reflow phenomenon is increasingly being recognized as a serious problem during coronary angioplasty and stenting. This phenomenon is seen more often during angioplasty in highly thrombogenic milieux, especially in a setting of acute myocardial infarction. The treatment of this complication is often not satisfactory. In this study the authors assessed the efficacy of abciximab, a potent antiplatelet drug, in treating slow flow or no reflow phenomenon during primary percutaneous transluminal coronary angioplasty (PTCA) for acute myocardial infarction (AMI). METHODS: Twenty-one instances of persistent slow flow phenomenon were encountered in 131 consecutive patients subjected to primary PTCA for AMI (16%). It was more common in patients presenting with AMI complicated by cardiogenic shock (nine of 21, 43%). Of these 21 cases of slow flow, 10 patients were given injection abciximab during the procedure of primary PTCA as a bail-out measure after encountering the complication of slow flow or no reflow. A pre-discharge coronary angiography was carried out in all patients who survived. RESULTS: In seven of 10 patients in the abciximab group flow had improved to TIMI-3. In contrast, in the non-abciximab group TIMI flow improved in only four of 11 patients. Patients with persistent slow flow had significantly higher mortality at the first 30-day follow-up than patients with TIMI-3 flow (33% versus 1.8%, p<0.001). CONCLUSION: In this small nonrandomized study significant improvement in coronary flow was achieved by using intravenous abciximab after observing slow flow or no reflow phenomenon during primary PTCA. More frequent use of this drug in this milieu might help in preventing the development of this complication. Larger studies are warranted to confirm this life-saving beneficial effect of bail-out administration of abciximab during primary angioplasty.