Human chromosomes 9, 12, and 15 contain the nucleation sites of stress-induced nuclear bodies

We previously reported the identification of a novel nuclear compartment detectable in heat-shocked HeLa cells that we termed stress-induced Src-activated during mitosis nuclear body (SNB). This structure is the recruitment center for heat shock factor 1 and for a number of RNA processing factors, among a subset of Serine-Arginine splicing factors. In this article, we show that stress-induced SNBs are detectable in human but not in hamster cells. By means of hamster>human cell hybrids, we have identified three human chromosomes (9, 12, and 15) that are individually able to direct the formation of stress bodies in hamster cells. Similarly to stress-induced SNB, these bodies are sites of accumulation of hnRNP A1-interacting protein and heat shock factor 1, are usually associated to nucleoli, and consist of clusters of perichromatin granules. We show that the p13-q13 region of human chromosome 9 is sufficient to direct the formation of stress bodies in hamster>human cell hybrids. Fluorescence in situ hybridization experiments demonstrate that the pericentromeric heterochromatic q12 band of chromosome 9 and the centromeric regions of chromosomes 12 and 15 colocalize with stress-induced SNBs in human cells. Our data indicate that human chromosomes 9, 12, and 15 contain the nucleation sites of stress bodies in heat-shocked HeLa cells.     

Detection of Tissue Factor Antigen and Coagulation Activity in Coronary Artery Thrombi Isolated from Patients with ST-Segment Elevation Acute Myocardial Infarction

Introduction

Although ruptured atherosclerotic plaques have been extensively analyzed, the composition of thrombi causing arterial occlusion in patients with ST-segment elevation acute myocardial infarction has been less thoroughly investigated. We sought to investigate whether coagulant active tissue factor can be retrieved in thrombi of patients with STEMI undergoing primary percutaneous coronary intervention.

Methods

Nineteen patients with ST-segment elevation acute myocardial infarction referred for primary percutaneous coronary intervention were enrolled in this study. Coronary thrombi aspirated from coronary arteries were routinely processed for paraffin embedding and histological evaluation (4 patients) or immediately snap frozen for evaluation of tissue factor activity using a modified aPTT test (15 patients). Immunoprecipitation followed by immunoblotting was also performed in 12 patients.

Results

Thrombi aspirated from coronary arteries showed large and irregular areas of tissue factor staining within platelet aggregates, and in close contact with inflammatory cells. Some platelet aggregates stained positive for tissue factor, whereas others did not. Monocytes consistently stained strongly for tissue factor, neutrophils had a more variable and irregular tissue factor staining, and red blood cells did not demonstrate staining for tissue factor. Median clotting time of plasma samples containing homogenized thrombi incubated with a monoclonal antibody that specifically inhibits tissue factor-mediated coagulation activity (mAb 5G9) were significantly longer than their respective controls (88.9 seconds versus 76.5 seconds, respectively; p<0.001). Tissue factor was also identified by immunoprecipitation in 10 patients, with significant variability among band intensities.

Conclusions

Active tissue factor is present in coronary artery thrombi of patients with ST-segment elevation acute myocardial infarction, suggesting that it contributes to activate the coagulation cascade ensuing in coronary thrombosis.     

Synthesis and biological evaluation of new conformationally biased integrin ligands based on a tetrahydroazoninone scaffold

The synthesis of new conformationally biased cyclic pentapeptides, incorporating the RGD sequence, and built around a tetrahydroazoninone scaffold, is reported. They exhibit interesting activity towards integrin alphaVbeta3 and a remarkable selectivity in comparison with integrin alphaVbeta5.     

Cryo-negative staining reveals conformational flexibility within yeast RNA polymerase I

The structure of the yeast DNA-dependent RNA polymerase I (RNA Pol I), prepared by cryo-negative staining, was studied by electron microscopy. A structural model of the enzyme at a resolution of 1.8 nm was determined from the analysis of isolated molecules and showed an excellent fit with the atomic structure of the RNA Pol II Delta4/7. The high signal-to-noise ratio (SNR) of the stained molecular images revealed a conformational flexibility within the image data set that could be recovered in three-dimensions after implementation of a novel strategy to sort the "open" and "closed" conformations in our heterogeneous data set. This conformational change mapped in the "wall/flap" domain of the second largest subunit (beta-like) and allows a better accessibility of the DNA-binding groove. This displacement of the wall/flap domain could play an important role in the transition between initiation and elongation state of the enzyme. Moreover, a protrusion was apparent in the cryo-negatively stained model, which was absent in the atomic structure and was not detected in previous 3D models of RNA Pol I. This structure could, however, be detected in unstained views of the enzyme obtained from frozen hydrated 2D crystals, indicating that this novel feature is not induced by the staining process. Unexpectedly, negatively charged molybdenum compounds were found to accumulate within the DNA-binding groove, which is best explained by the highly positive electrostatic potential of this region of the molecule, thus, suggesting that the stain distribution reflects the overall surface charge of the molecule.     

Application of selectively acylated glycosides for the α-galactosidase-catalyzed synthesis of disaccharides

4-Nitrophenyl alpha-D-galactopyranosyl-(1-->3)-6-O-acetyl-alpha-D-galactopyranoside was prepared in a transglycosylation reaction catalyzed by alpha-D-galactosidase from Talaromyces flavus using 4-nitrophenyl alpha-D-galactopyranoside as a glycosyl donor and 4-nitrophenyl 6-O-acetyl-alpha-D-galactopyranoside as an acceptor. 4-Nitrophenyl 6-O-acetyl-alpha-D-galactopyranoside and 4-nitrophenyl 6-O-acetyl-beta-D-galactopyranoside were prepared in a regioselective enzymic transesterification in pyridine-acetone catalyzed by the lipase PS from Burkholderia cepacia. A series of water-miscible organic solvents (acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, 2-methoxyethanol, pyridine, 2-methylpropan-2-ol, tetrahydrofuran, propargyl alcohol) were used as co-solvents in this enzymic reaction. Their influence on the activity and stability of the alpha-galactosidase from T. flavus was established. 2-Methylpropan-2-ol and acetone (increasing the solubility of the modified substrate acceptors and displaying the minimum impairment of the activity and stability of the enzyme) were used as co-solvents in transglycosylation reactions.     

Localization of the yeast RNA polymerase I-specific subunits

The spatial distribution of four subunits specifically associated to the yeast DNA-dependent RNA polymerase I (RNA pol I) was studied by electron microscopy. A structural model of the native enzyme was determined by cryo-electron microscopy from isolated molecules and was compared with the atomic structure of RNA pol II Delta 4/7, which lacks the specific polypeptides. The two models were aligned and a difference map revealed four additional protein densities present in RNA pol I, which were characterized by immunolabelling. A protruding protein density named stalk was found to contain the RNA pol I-specific subunits A43 and A14. The docking with the atomic structure showed that the stalk protruded from the structure at the same site as the C-terminal domain (CTD) of the largest subunit of RNA pol II. Subunit A49 was placed on top of the clamp whereas subunit A34.5 bound at the entrance of the DNA binding cleft, where it could contact the downstream DNA. The location of the RNA pol I-specific subunits is correlated with their biological activity.