Sir3-dependent assembly of supramolecular chromatin structures in vitro

Baculovirus-expressed recombinant Sir3p (rSir3p) has been purified to near homogeneity, and its binding to naked DNA, mononucleosomes, and nucleosomal arrays has been characterized in vitro. At stoichiometric levels rSir3p interacts with intact nucleosomal arrays, mononucleosomes, and naked DNA, as evidenced by formation of supershifted species on native agarose gels. Proteolytic removal of the core histone tail domains inhibits but does not completely abolish rSir3p binding to nucleosomal arrays. The linker DNA in the supershifted complexes remains freely accessible to restriction endonuclease digestion, suggesting that both the tail domains and nucleosomal DNA contribute to rSir3p–chromatin interactions. Together these data indicate that rSir3p cross-links individual nucleosomal arrays into supramolecular assemblies whose physical properties transcend those of typical 10-nm and 30-nm fibers. Based on these data we hypothesize that Sir3p functions, at least in part, by mediating reorganization of the canonical chromatin fiber into functionally specialized higher order chromosomal domains.

Association of RNase mitochondrial RNA processing enzyme with ribonuclease P in higher ordered structures in the nucleolus: a possible coordinate role in ribosome biogenesis.

RNase mitochondrial RNA processing enzyme (MRP) is a nucleolar ribonucleoprotein particle that participates in 5.8S ribosomal RNA maturation in eukaryotes. This enzyme shares a polypeptide and an RNA structural motif with ribonuclease P (RNase P), a nuclear endoribonuclease originally described in the nucleus that processes RNA transcripts to generate their mature 5' termini. Both enzymes are also located in mitochondria. This report further characterizes the relationship between RNase MRP and RNase P. Antisense affinity selection with biotinylated 2'-O-methyl oligoribonucleotides and glycerol gradient fractionation experiments demonstrated that small subpopulations of RNase MRP and RNase P associate with each other in vivo in macromolecular complex, possibly 60-80S preribosomes. This latter notion was supported by fluorescence in situ hybridization experiments with antisense oligonucleotides that localized that RNA components of RNase MRP and RNase P to the nucleolus and to discrete cytoplasmic structures. These findings suggest that small subpopulations of RNase MRP and RNase P are physically associated, and that both may function in ribosomal RNA maturation or ribosome assembly.

Evidence for nonrandom hydrophobicity structures in protein chains.

The question of whether proteins originate from random sequences of amino acids is addressed. A statistical analysis is performed in terms of blocked and random walk values formed by binary hydrophobic assignments of the amino acids along the protein chains. Theoretical expectations of these variables from random distributions of hydrophobicities are compared with those obtained from functional proteins. The results, which are based upon proteins in the SWISS-PROT data base, convincingly show that the amino acid sequences in proteins differ from what is expected from random sequences in a statistically significant way. By performing Fourier transforms on the random walks, one obtains additional evidence for nonrandomness of the distributions. We have also analyzed results from a synthetic model containing only two amino acid types, hydrophobic and hydrophilic. With reasonable criteria on good folding properties in terms of thermodynamical and kinetic behavior, sequences that fold well are isolated. Performing the same statistical analysis on the sequences that fold well indicates similar deviations from randomness as for the functional proteins. The deviations from randomness can be interpreted as originating from anticorrelations in terms of an Ising spin model for the hydrophobicities. Our results, which differ from some previous investigations using other methods, might have impact on how permissive with respect to sequence specificity protein folding process is-only sequences with nonrandom hydrophobicity distributions fold well. Other distributions give rise to energy landscapes with poor folding properties and hence did not survive the evolution.

Visualization of intermediate and transition-state structures in protein-tyrosine phosphatase catalysis.

Engineering site-specific amino acid substitutions into the protein-tyrosine phosphatase (PTPase) PTP1 and the dual-specific vaccinia H1-related phosphatase (VHR), has kinetically isolated the two chemical steps of the reaction and provided a rare opportunity for examining transition states and directly observing the phosphoenzyme intermediate. Changing serine to alanine in the active-site sequence motif HCXXGXXRS shifted the rate-limiting step from intermediate formation to intermediate hydrolysis. Using phosphorus 31P NMR, the covalent thiol-phosphate intermediate was directly observed during catalytic turnover. The importance of the conserved aspartic acid (D92 in VHR and D181 in PTP1) in both chemical steps was established. Kinetic analysis of D92N and D181N mutants indicated that aspartic acid acts as a general acid by protonating the leaving-group phenolic oxygen. Structure-reactivity experiments with native and aspartate mutant enzymes established that proton transfer is concomitant with P-O cleavage, such that no charge develops on the phenolic oxygen. Steady- and presteady-state kinetics, as well as NMR analysis of the double mutant D92N/S131A (VHR), suggested that the conserved aspartic acid functions as a general base during intermediate hydrolysis. As a general base, aspartate would activate a water molecule to facilitate nucleophilic attack. The amino acids involved in transition-state stabilization for cysteinylphosphate hydrolysis were confirmed by the x-ray structure of the Yersinia PTPase complexed with vanadate, a transition-state mimic that binds covalently to the active-site cysteine. Consistent with the NMR, x-ray, biochemical, and kinetic data, a unifying mechanism for catalysis is proposed.

Core binding factor beta-smooth muscle myosin heavy chain chimeric protein involved in acute myeloid leukemia forms unusual nuclear rod-like structures in transformed NIH 3T3 cells.

Patients with the M4Eo subtype of acute myeloid leukemia almost invariably are found to have an inversion of chromosome 16 in their leukemic cells, which results in a gene fusion between the transcription factor called core binding factor beta (CBFbeta) on 16q and a smooth muscle myosin heavy chain (SMMHC) gene on 16p. Subcellular localizations of the wild-type CBFbeta and the CBFbeta-SMMHC fusion protein were determined by immunofluorescence of NIH 3T3 cells that overexpress wild-type or fusion protein. Normal CBFbeta showed an unexpected perinuclear pattern consistent with primary localization in the Golgi complex. The CBFbeta-SMMHC fusion protein had a very different pattern. Nuclear staining included rod-like crystalline structures as long as 11 microm. The heterodimeric partner of CBFbeta, CBFalpha, formed part of this complex. Cytoplasmic staining included stress fibers that colocalized with actin, probably as a consequence of the myosin heavy chain component of the fusion protein. Deletion of different regions of the CBFbeta portion of the fusion protein showed that binding to CBFalpha was not required for nuclear translocation. However, deletion of parts of the SMMHC domain of the fusion protein involved in myosin-mediated filament formation resulted in proteins that did not form rod-like structures. These observations confirm previous indirect evidence that the CBFbeta-SMMHC fusion protein is capable of forming macromolecular nuclear aggregates and suggests possible models for the mechanism of leukemic transformation.

Paired structures in knowledge representation.

In this position paper we propose a consistent and unifying view to all those basic knowledge representation models that are based on the existence of two somehow opposite fuzzy concepts. A number of these basic models can be found in fuzzy logic and multi-valued logic literature. Here it is claimed that it is the semantic relationship between two paired concepts what determines the emergence of different types of neutrality, namely indeterminacy, ambivalence and conflict, widely used under different frameworks (possibly under different names). It will be shown the potential relevance of paired structures, generated from two paired concepts together with their associated neutrality, all of them to be modeled as fuzzy sets. In this way, paired structures can be viewed as a standard basic model from which different models arise. This unifying view should therefore allow a deeper analysis of the relationships between several existing knowledge representation formalisms, providing a basis from which more expressive models can be later developed.

Using the damage from 2010 Haiti earthquake for calibrating vulnerability models of typical structures in Port-au-Prince (Haiti)

After the 2010 Haiti earthquake, that hits the city of Port-au-Prince, capital city of Haiti, a multidisciplinary working group of specialists (seismologist, geologists, engineers and architects) from different Spanish Universities and also from Haiti, joined effort under the SISMO-HAITI project (financed by the Universidad Politecnica de Madrid), with an objective: Evaluation of seismic hazard and risk in Haiti and its application to the seismic design, urban planning, emergency and resource management. In this paper, as a first step for a structural damage estimation of future earthquakes in the country, a calibration of damage functions has been carried out by means of a two-stage procedure. After compiling a database with observed damage in the city after the earthquake, the exposure model (building stock) has been classified and through an iteratively two-step calibration process, a specific set of damage functions for the country has been proposed. Additionally, Next Generation Attenuation Models (NGA) and Vs30 models have been analysed to choose the most appropriate for the seismic risk estimation in the city. Finally in a next paper, these functions will be used to estimate a seismic risk scenario for a future earthquake.

Probing large-scale wind structures in Vela X–1 using off-states with INTEGRAL

Vela X–1 is the prototype of the class of wind-fed accreting pulsars in high-mass X-ray binaries hosting a supergiant donor. We have analysed in a systematic way 10 years of INTEGRAL data of Vela X–1 (22–50 keV) and we found that when outside the X-ray eclipse, the source undergoes several luminosity drops where the hard X-rays luminosity goes below ∼3 × 1035 erg s−1, becoming undetected by INTEGRAL. These drops in the X-ray flux are usually referred to as ‘off-states’ in the literature. We have investigated the distribution of these off-states along the Vela X–1 ∼ 8.9 d orbit, finding that their orbital occurrence displays an asymmetric distribution, with a higher probability to observe an off-state near the pre-eclipse than during the post-eclipse. This asymmetry can be explained by scattering of hard X-rays in a region of ionized wind, able to reduce the source hard X-ray brightness preferentially near eclipse ingress. We associate this ionized large-scale wind structure with the photoionization wake produced by the interaction of the supergiant wind with the X-ray emission from the neutron star. We emphasize that this observational result could be obtained thanks to the accumulation of a decade of INTEGRAL data, with observations covering the whole orbit several times, allowing us to detect an asymmetric pattern in the orbital distribution of off-states in Vela X–1.