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Even if the Origin of Life cannot be discovered, prebiotic models can limit the number of possible scenarios. In this context, formamide chemistry furnishes a general and robust probe to test a top-down approach to LUCA, the Last Common Universal Ancestor.

This research evaluated the formation of free radicals in the oxidative degradation of biodiesel in contact with metallic copper and metal alloys via EPR using the spin trapping PBN, identifying and quantifying the formation of free radicals in commercial biodiesel samples. The biodiesel samples were analyzed in contact with carbon steel, silver steel, metallic copper and blackberry extract. The lowest values of oxidative stability were obtained for biodiesel in contact with metallic copper, followed by carbon steel and silver steel. The highest values of radicals formed were obtained for the sample containing blackberry extract. It was possible to identify radical species such as OH and CHOHCH3 during the decomposition of biodiesel.

The very first origin of RNA is fraught with uncertainty. RNA world might have been preceded by an "unknown-polymer" world, or it might have started its polymerization destiny from highly activated precursors as phosphoramidated nucleotides. It might even have taken origin outside this planet. Each one of these scenarios has its appeals and drawbacks. Alternatively, RNA might have originated by self-polymerization of simple pre- biotically plausible compounds, i.e., 3',5' cyclic nucleotides. In this work, we review our observations about spontaneous polymerization of 3', 5' cyclic ribonucleotides and in particular of 3',5' cyclic cGMP. The reaction requires neither template, nor enzymatic activities, is thermodynamically favored, and selectively yields 3',5'-bonded oligoribonucleotides containing as many as 25 nucleotides.

A natural sample of the mineral goethite was characterized by X-band Electron Spin Resonance (ESR), vibrating sample Magnetometry and Mossbauer spectroscopy techniques, with the main objective of studying the magnetic transition from the antiferromagnetic to the paramagnetic state that this mineral undergoes upon reaching a certain critical temperature (Neel temperature). Although an ESR signal was not expected in goethite samples at room temperature, due to its antiferromagnetic arrangement, a resonance line was observed. This behavior was attributed to the existence of vacancies in the mineral structure. Increasing the temperature from RT to 352 K no significant change occurred in the ESR spectra. From 352 K, the goethite spectrum showed an additional ESR line, the intensity of this line grew until it stabilized at around 444 K. The appearance of this new resonance line, and its evolution with temperature, was attributed to two subsequent effects: firstly the spin flop effect from 352 to 372 K; and secondly, the transition to the paramagnetic state at T N = 372 K. Magnetometry and Mossbauer measurements corroborated this assumption and the transition temperature was identified by magnetometry as T N = 370 K. Simulations of the goethite ESR spectra in both antiferromagnetic and paramagnetic states were performed by deconvolution of the resonance lines into two contributions, one from species with vacancies in their vicinity, and another with complete vicinity.

Melanins are biopigments ubiquitous in flora and fauna, exhibiting a range of interesting functional properties such as UV-Vis photoprotection, thermoregulation, hydration-dependent electrical conduction and metal chelation. In the human body, melanins can be found in the skin, hair, middle ear, retina and heart. The metal chelation properties of neuromelanin, a subclass of melanins found in the dopaminergic neurons of the brain, may be involved in Parkinson's disease. Considering synthetic and natural (from the ink sac of the cuttlefish) thin films of eumelanin, the subclass of melanins most investigated by materials scientists, we report on two types of resistive switching (standard and hybrid), with different ON/OFF ratios, observed in planar gold/eumelanin/gold structures. The resistive switching process, based on the formation of conductive filaments, was studied by means of transient electrical measurements, scanning electron microscopy, atomic force microscopy and time of flight-secondary ion mass spectrometry. We observed an extended correlation among the factors affecting the two different types of switch: primarily the eumelanin's hydration level as well as the presence of chelating groups in the eumelanin molecular structure and the electrical bias. This work contributes to the development of environmentally benign organic resistive switching devices, namely electrochemical metallization memory cells making use of the biocompatible, biodegradable and abundant eumelanin biopigment as the ion conductive layer.

Goethite is an antiferromagnetic material that frequently presents a superimposed weak ferromagnetic component. In this research, two goethite samples were prepared using distinct synthesis routes, being later subjected to heat treatment. Magnetization dynamics were analyzed as a function of applied magnetic field and temperature. Moreover, the magnetic measurements were correlated with the resulting microstructures, obtained through scanning and transmission electron microscopy. The results indicate an enhanced ferromagnetic behavior in those goethite samples, with greater presence of defects and imperfections. Besides that, it was observed a reduction of magnetic fluctuations after the healing of the samples via heat treatment. Despite what is vastly proposed in the literature to explain the origin of the magnetic component in goethite, our analysis showed that, for the studied samples, this effect is caused by a magnetic mismatch in the antiferromagnetic lattice. This mismatch is possibly caused by defects as grain boundaries, voids and grain rotations, which may lead to dislocations and consequently to magnetic mismatches in the lattice.

Percutaneous laser ablation (PLA) may be useful in treating patients with metachronous metastatic lymph nodes in the neck.Our objective was to assess PLA as a treatment of difficult-to-treat metachronous cervical lymph node metastases from papillary thyroid carcinoma.We conducted a retrospective analysis of prospectively collected data at a public hospital.Fifteen patients with previous resection of papillary thyroid carcinoma with elevated serum levels of thyroglobulin (Tg) or anti-Tg antibodies (TgAbs) and 24 metachronous nodal metastases treated between September 2010 and April 2012 were followed with [¹⁸F]fluorodeoxyglucose (¹⁸FDG) positron emission tomography (PET)/computed tomography (CT) and contrast-enhanced ultrasound (CEUS).Intervention was PLA.Technique feasibility and technical success were evaluated. Tg/TgAb serum levels and ¹⁸FDG-PET/CT, and CEUS appearance were assessed at 6 and 12 months and compared with baseline. Complications were recorded.PLA was always feasible, and technical success was achieved in all patients. At 6 months, local control was achieved in 11 of 15 patients (73%), with 6 (40%) having serum Tg/TgAb normalized (P = .017 vs baseline). Whereas 20 of 24 (83%) nodes were negative at ¹⁸FDG-PET/CT and CEUS (P.001 vs baseline), 4 were ¹⁸FDG-PET/CT-positive (3 also CEUS-positive). At the 12-month follow-up, local control was achieved in 10 of 14 patients (71.4%). Sixteen of 20 nodes (80%) were negative at ¹⁸FDG-PET/CT and CEUS (P.001 vs baseline), 4 were ¹⁸FDG-PET/CT-positive (2 also CEUS-positive). Four of 10 (40%) patients had normalization of serum Tg/TgAb (P = .098 vs baseline). No major complications occurred.PLA is potentially feasible, safe, and effective for the treatment of metachronous cervical nodal metastases from papillary thyroid carcinoma. This procedure may reduce or delay a large number of highly invasive repeat neck dissections.

In this work, poly (3-methylthiophene) (P3MT) or poly (3-octylthiophene) (P3OT) films were synthesized electrochemically with polydiphenylamine (PDFA), in layers and blended copolymers, in non-aqueous media through the oxidation of the monomers, using a standard three-electrode cell in acetonitrile with 0.100 mol L −1 LiClO 4 . The polymeric thin films were deposited on platinum plates for optimal quality control of the process. The electrochemical behaviors of as-prepared and partial dedoped films obtained by these methods was verified by cyclic voltammetry (CV) and have been characterized by UV–vis reflectance, resonance Raman, electron paramagnetic resonance (EPR) and photoluminescence (PL) spectroscopy. Based on data obtained by UV–vis reflectance spectroscopy, it was possible to characterize the chemical species in the polymer matrix using the resonant Raman method. Both Raman and PL spectra results led to the characterization of three structures (aromatic, semi-quinone and quinone forms of thiophene rings) which formed the homopolymers and copolymers chain. The EPR measurements were taken to quantify the semi-quinone species stabilization at 298 and 77 K temperatures. The ionization potential ( I P ), electron affinity ( E.A. ) and gap energy ( E g ) parameters were determined based on the technical analyses carried out.

In hemodialysis patients, vertebral fractures were associated with elevated sclerostin levels, suggesting that sclerostin could reflect bone fragility in these patients. Fragility fractures are common in hemodialysis patients. The aims of our study were to determine the prevalence of vertebral fracture and analyze associations between sclerostin serum levels and vertebral fractures in hemodialysis patients. Ninety-two hemodialysis patients and 100 controls matched for age and sex were studied. Bone mineral density was measured by ultrasonography at non-dominant heel. The markers of bone turnover included serum osteocalcin, C-terminal telopeptide, and sclerostin. All participants underwent radiography of the thoracic and lumbar spine to ascertain the presence of vertebral fractures. Bone ultrasound parameters at calcaneus were significantly lower in hemodialysis patients compared with controls; bone turnover markers and parathyroid hormone level were significantly higher, while serum of 25-OH-D3 was significantly lower in hemodialysis group. One or more moderate or severe vertebral fractures were found in 38 hemodialysis patients, whereas in control group, 10 patients had a vertebral fracture. In hemodialysis group, the comparison between patients with and without vertebral fractures showed that the patients with vertebral fractures had the serum sclerostin levels statistically higher than patients without vertebral, while serum levels of 25-OH-D3 was significantly lower in patients with vertebral fractures compared to the patients without vertebral fractures. Multivariate analysis disclosed that sclerostin levels were associated with an increased risk of vertebral fractures in hemodialysis patients after adjusting for multiple variables. Our data shows high prevalence of vertebral fractures in hemodialysis patients and that it is associated with elevated sclerostin levels, reflecting bone fragility in these patients.

Samples of Araucaria area soil from Parana state, Brazil, were separated by particle size fractionation and investigated by electron paramagnetic resonance (EPR) in X-Band of 9.5 GHz at room temperature and 77 K, infra-red spectroscopy and X-ray diffractometry. The paramagnetic species in the soil samples were identified by comparison with EPR spectra of some minerals studied recently by our group, several soil types and/or soil components investigated in the literature. The value of g = 2.1 (ΔH = 85 mT) indicated the presence of ferrihydrite. Hematite was identified by g = 2.1 (ΔH = 100 mT) and g = 4.3 for Fe 3+ lines of the concentrated dominium and diluted dominium. Kaolinite was identified by IR and EPR with the resonance at g = 4.3 attributed to Fe 3+ ions in isolated sites of tetrahedral and octahedral symmetry with rhombic distortion. The resonances at g = 3.7 and g = 4.9 were attributed to Fe 3+ in more highly symmetrical environment than rhombic symmetry, but not in axial symmetry. Three signals around g = 2 were attributed to radiation defects, plus additional resonances at g = 2.8 and 9.0. Signals less intense than those at g = 2.1, 3.7, and 6.5, observed for clear grains of soil, were attributed to presence of Fe 3+ in quartz which was identified by IR and XDR.

In this work, poly(3-octylthiophene) (P3OT) films were synthesized electrochemically in non-aqueous media through the oxidation of the monomer, (3-octylthiophene), using a standard three-electrode cell in acetonitrile with 0.05 mol L S1 LiClO4 or 0.05 mol L S1 Et4NBF4. The polymeric films were deposited on fluorine tin oxide (FTO). The partial dedoping was obtained in NH4OH solution, providing a good chemical stability of the formed material. The films obtained through this method have been characterized by Fourier-transform infrared spectroscopy (FT-IR), electron paramagnetic resonance (EPR), UV–Vis absorption, and photoluminescence (PL) spectroscopy. The FT-IR and EPR spectra together gave the results that led to characterization of two structures (pristine and non-pristine forms of thiophene rings) while forming the P3OT polymer chain. These results were associated with the stabilization of pristine chains and mixed chains (non-pristine structures) in the polymeric film. Their bands in the PL spectra are wide and asymmetric and their adjustments by Gaussian functions was necessary; this was the main indication that there are two distinct contributions to the emission spectra. These two contributions are attributed to the emission by mixed chains (Gaussian centered at higher energy) and by pristine chains (Gaussian of lower energy) present in the formed polymeric material. Copyright 2010 John Wiley & Sons, Ltd.

Petroleum of Arabian and Colombian origin was studied by electron paramagnetic resonance (EPR) spectroscopy at X- (9 GHz), Q- (34 GHz) and W-bands (94 GHz). The experiments were performed at room temperature (about 300 K) and at 77 K (W-band only). The asymmetry in the lines corresponding to free radicals was observed more intensely in the W-band spectra. The values of the line width ΔH in the spectra increased linearly with the microwave frequency utilized in the EPR experiments. A mathematical simulation of the free radical signal for the EPR spectra in three bands with a set of parameters corresponding to a single species was attempted, but this was not exactly coincident with the experimental signals, suggesting that the hyperfine interaction of the unpaired electron with its neighborhood corresponds to more than one species of radical in the molecular structure of the petroleum asphaltene.

We used the cDNA microarray technique to monitor simultaneously possible changes induced by hypergravity in the expression level of thousands of hippocampal genes. We tested the mRNA level of about 5000 genes in the hippocampus of mice subjected to 1.09 g (1g) or to 1.85 g (2g) for five repeated 1-h daily rotations in a centrifuge (g = 9.81 m/s2). Data were compared with those obtained for mice kept stationary (C). The ratios 1g/C and 2g/C identified genes affected by rotation and rotation + hypergravity, respectively, whereas 2g/1g ratio identified those affected by hypergravity. We found that about 200 genes were affected by rotation and/or rotation + hypergravity. Almost all the genes affected by rotation + hypergravity were up-regulated, only five being down-regulated. The modulated genes code for proteins involved in a wide range of cellular functions (DNA/RNA metabolism, protein processing, intermediate metabolism, cytoskeleton and motility, cell cycle and apoptosis, signal transduction, neuronal structure/function), suggesting that rotation + hypergravity may affect several aspects of the hippocampal function in order to compensate for environmental changes. Six genes directly or indirectly involved in synaptic transmission and plasticity (proSAAS, neuroblastoma ras oncogene, ESTs moderately similar to thymosin beta-10, syndet, inhibin beta E and Ngfi-A binding protein 2) were found to be significantly modulated by hypergravity and unaffected or only slightly affected by rotation. The modulation by hypergravity of these genes suggests that this stimulus might induce plastic remodelling of the hippocampal circuits, possibly both at structural and functional level.

In recent years the use of nanocrystals (NCs) as inorganic semiconductors in conjugated polymer-based hybrid solar cells (HSCs) results as a challenging target for researchers because of their high electron mobility and good mechanical robustness compared to fullerene derivatives, as well as their capability of harvesting photons in the visible to near-infrared region of the solar spectrum. Unfortunately HSCs still suffer from a major drawback, that is a poor control over the organic donor and inorganic acceptor domain size distributions. The domain size of the donor and acceptor materials should be comparable to the exciton diffusion length in order to increase the probability of exciton dissociation, and to improve charge carrier transport and collection at the device electrodes. The readiness of inorganic NCs to macrophase separate from non-polar conjugated polymers at higher loading concentrations, prevents the building of a controllable interpenetrating percolation network. A suitable nanostructuring compatibilizer can induce selective intermolecular interactions, leading to morphological order associated to an improvement in the interface processes, which consequently drive a strong rising in the performances of the device by decreasing recombination losses. For this purpose, block copolymers are particularly significant because they can self-assemble through phase separation by rationally tailoring the blocks using functional moieties with different physical-chemical behaviours. We report on the synthesis of a rod-coil diblock copolymer based on poly[2,6-(4,4-bis-(2-ethylhexyl)-4H-cyclopenta[2,1-b:3,4-b']dithiophene)-alt-4,7-(2,1,3-benzothiadiazole)] (PCPDTBT), a low band gap copolymer, and a coil block of poly(4-vinylpyridine) able to interact with semiconducting CdSe NCs with the aim of using it as coordinating material in the preparation of PCPDTBT/CdSe NCs-based hybrid solar cells.

Conjugated polymers are characterized by the delocalization of ?-electrons responsible of their electrical properties and structural rigidity. The manipulation of the chemical structures through the introduction of flexible chains allows to influence the morphology controlling the nanosegregation of the material. The use of nanocrystals (NCs) of inorganic semiconductors in "hybrid solar cells" offers high electron mobility enhancing the performance of the devices. The control of the active layer morphology at the nanoscale level ensures the further improve of the efficiency of the cells. The goal of this work is the evaluation of two approaches for the synthesis of a rod-coil copolymer based on poly[2,6-(4,4-bis-(2-ethylhexyl)-4H-cyclopenta[2,1-b:3,4-b']dithiophene)-alt-4,7-(2,1,3-benzothiadiazole)] rod-like block (electrondonor) and a coil block of poly(4-vinylpyridine) able to interact with semiconducting CdSe NCs (electronacceptor) with the aim of using it as both active and coordinating material for nanocomposites preparation with over 90% of NCs.

Although the crystal structure of nucleosome core particle is essentially symmetrical in the vicinity of the dyad, the linker histone binds asymmetrically in this region to select a single high-affinity site from potentially two equivalent sites. To try to resolve this apparent paradox we mapped to base-pair resolution the dyads and rotational settings of nucleosome core particles reassembled on synthetic tandemly repeating 20 bp DNA sequences. In agreement with previous observations, we observed (1) that the helical repeat on each side of the dyad cluster is 10 bp maintaining register with the sequence repeat and (2) that this register changes by 2 bp in the vicinity of the dyad. The additional 2 bp required to effect the change in the rotational settings is accommodated by an adjustment immediately adjacent to the dyad. At the dyad the hydroxyl radical cleavage is asymmetric and we suggest that the inferred structural asymmetry could direct the binding of the linker histone to a single preferred site.

Several cell adhesion molecules that mediate the binding of lymphocytes to high endothelial venules (HEV) from flowing blood have been identified but the regulation of lymphocyte migration across the HEV wall into the lymph node (LN) is far from understood. In this study we have used an in vitro model of lymphocyte migration across HEV, and analysed the roles of two integrins in the binding and transendothelial migration of T lymphocytes and T lymphoblasts. The adhesion of T lymphocytes to high endothelial cells (HEC) cultured from rat LN HEV differed from that of T lymphoblasts since the percentage of T lymphoblasts that adhered and transmigrated was higher and was not increased by IFN-gamma pretreatment of HEC. Antibodies to alpha(4) integrins, VCAM-1 or LFA-1 maximally inhibited T lymphocyte adhesion by 40-50%, whereas antibodies to ICAM-1 were less effective (20% inhibition). The effects of alpha(4) integrin and LFA-1 antibodies were additive, giving90% inhibition. T lymphocytes which adhered in the presence of LFA-1 antibody showed reduced levels of transmigration and, in the presence of alpha(4) integrin antibody, slightly increased transmigration. Antibodies to alpha(4) integrins, VCAM-1, LFA-1 or ICAM-1 had little effect on T lymphoblast adhesion (maxima of 10-30% inhibition) and T lymphoblasts transmigrated normally in the presence of either alpha(4) integrin or LFA-1 antibodies. However, the effects of alpha(4) integrin and LFA-1 antibodies on T lymphoblast adhesion were synergistic, giving90% inhibition of adhesion. These results suggest that the majority of T lymphoblasts use either alpha(4) integrins or LFA-1 to bind and transmigrate HEV, and the roles of these integrins on activated T cells are overlapping and redundant. In contrast, either integrin supports half-maximal binding of unactivated T lymphocytes to the surface of HEV and LFA-1 makes a larger contribution than alpha(4) integrins to transendothelial migration.

The paramagnetic centres of Cu 2+ in ammonium bromide crystals were studied at 300 and 77 K with X-band and Q-band EPR. We determined the spin Hamiltonian parameters, contact interaction and covalent bonding parameters of the paramagnetic centres of the NH 4 Br:Cu 2+ . The superhyperfine interaction to the Cu paramagnetic centres in NH 4 Br is much stronger than in NH 4 Cl. The results obtained for the contact interaction (with a new assignment that we gave to the EPR spectra) are in agreement with those found for other copper complexes. The comparison of the results of the covalent bonding parameters with those of other compounds showed a strong covalency of NH 4 Br:Cu 2+ , suggesting that orbital admixtures in this complex are large and apparently much bigger than estimates based on overlap effects alone with no covalent bonding. The Cu 2+ wave function of the NH 4 Br written as an admixture of |3 z 2 − r 2 〉 and | x 2 − y 2 〉 orbitals is not suitable to describe the system. A real model must take into account the admixtures with the ligand orbitals. The d–s mixing is high in the CuBr 4 (NH 3 ) 2 2− centre, indicating strong bonding between the Cu 2+ ion and the two NH 3 molecules. In the case of the CuBr 4 (NH 3 )(H 2 O) 2− and CuBr 4 (H 2 O) 2 2− centres the binding between the Cu 2+ ion and the H 2 O molecules is much weaker.

In vivo nucleotide-level mapping of nucleosomes in the promoter of the methanol oxidase (MOX) gene in the yeast Hansenula polymorpha is reported. The 4 nucleosomes analyzed are organized in families; they localize in alternative positions along a unique rotational phase, and the linker regions can be occupied by alternative nucleosomes. This organization underscores a substantial freedom of choice by histone octamers when nucleating on a promoter region.

Ammonium bromide crystals doped with Cu2+ and grown in basic solution (pH approximately=8) were studied at 300 K and 77 K with X-band EPR to elucidate divergent spin Hamiltonian parameters. Careful measurements of the perpendicular part of the spectra indicated resolved 16 lines (300 K) and 19 lines (77 K). They were interpreted combining 4 hyperfine and 13 ligand (Br) hyperfine lines. We obtained Aperpendicular to =(33+or-2)*10-4 cm-1 (300 K) and Aperpendicular to =(67+or-2)*10-4 cm-1 (77 K). These values are not so different from the known values assigned to the analogous centre in NH4Cl and ND4Cl. The discrepancy reported is due to the difficulty in ascribing the Br hyperfine parameter now determined as Aperpendicular to '=(47+or-3)*10-4 cm-1 (300 and 77 K). Compared with that of Cl compounds it is about five times greater, which is of the same order as the hyperfine interaction ratio for free Br and Cl atoms.

Inactivation of the nonessential TOP1 gene, which codes for Saccharomyces cerevisiae DNA topoisomerase I, affects the rate of transcription starting at the ADH2 promoter. For both the chromosomal gene and the plasmid-borne promoter, mRNA accumulation is kinetically favored in the mutant relative to a wild-type isogenic strain. The addition of ethanol causes in wild-type yeast strains a substantial increase in linking number both on the ADH2-containing plasmid and on the resident 2 microns DNA. Evidence has been obtained that such an in vivo increase in linking number depends on (i) the activity of DNA topoisomerase I and of no other enzyme and (ii) ethanol addition, not on the release from glucose repression. A direct cause-effect relationship between the change in supercoiling and alteration of transcription cannot be defined. However, the hypothesis that a metabolism-induced modification of DNA topology in a eukaryotic cell plays a role in regulating gene expression is discussed.

Cleavage sites of eukaryotic DNA topoisomerase I on curved linear DNAs are clustered, map on the same side of the curve (the external one) and their distribution has the same period as the helical repeat, as observed on curved DNA tracts of Crithidia fasciculata, of Saccharomyces cerevisiae ARS1, of pT7CAT and on synthetic DNAs. The effects of the tridimensional context on both the cleavage and the topoisomerization reactions of DNA topoisomerase I were determined using serial DNA constructs made with inserts in which synthetic curves lie in a plane and in which the orientation of the planes of curvature is shifted by 72 degrees, 144 degrees, 216 degrees, 288 degrees and 360 degrees. The insertion of a curve markedly changes the reaction properties of the surrounding sequences.

Here, we show for the first time that gua-nine, adenine, and hypoxanthine can be produced from forma-mide in a single model prebiotic reaction at lower tempera-tures than previously reported, if formamide is subjected to UVirradiation during heating; this observation relaxes the require-ments for prebiotic purine nucleobase formation. The yieldand diversity of purines produced in heated/UV-irradiated for-mamide are further enhanced by the presence of inorganiccatalysts, as solids or as dissolved ions. We also analyzed theproducts of formamide solutions to which specific hydrogencyanide (HCN) condensation products

A new interstitial Cu2+ centre was found in NH4Br crystals grown in acidic solution (pH

The synthesis of prebiotic molecules is a major problem in chemical evolution as well as in any origin-of-life theory. We report here a plausible new prebiotic synthesis of naturally occurring purine and pyrimidine derivatives from formamide under catalytic conditions. In the presence of CaCO 3 and different inorganic oxides, namely silica, alumine, kaolin, and zeolite (Y type), neat formamide undergoes the formation of purine, adenine, cytosine, and 4(3H)-pyrimidinone, from acceptable to good yields. The role of catalysts showed to be not limited to the improvement of the yield but it is also relevant in providing a high selectivity in the products distribution.

The basis for the choice of translational position of a histone octamer on DNA is poorly understood. To gain further insights into this question we have studied the translational and rotational settings of core particles assembled on a simple repeating 20 bp positioning sequence. We show that the translational positions of the core particles assembled on this sequence are invariant with respect to the DNA sequence and occur at 20 bp intervals. Certain modifications of the original sequence reduce the spacing of possible dyads to 10 bp. At least one of these alters both the translational and rotational settings. We conclude that the translational position of a core particle is specified by sequence determinants additional to those specifying rotational positioning. The rotational settings on either side of the dyads of core particles assembled on the wild-type and a mutant sequence differ by +2 bp, corresponding to an overall helical periodicity of approximately 10.15 bp. The average helical periodicity of the central two to four turns is 10.5-11 bp whilst that of the flanking DNA is closer to 10 bp. The DNA immediately flanking the dyad is also characterised by a more extensive susceptibility to cleavage by hydroxyl radical.

Although the crystal structure of nucleosome core particle is essentially symmetrical in the vicinity of the dyad, the linker histone binds asymmetrically in this region to select a single high-affinity site from potentially two equivalent sites. To try to resolve this apparent paradox we mapped to base-pair resolution the dyads and rotational settings of nucleosome core particles reassembled on synthetic tandemly repeating 20 bp DNA sequences. In agreement with previous observations, we observed (1) that the helical repeat on each side of the dyad cluster is 10 bp maintaining register with the sequence repeat and (2) that this register changes by 2 bp in the vicinity of the dyad. The additional 2 bp required to effect the change in the rotational settings is accommodated by an adjustment immediately adjacent to the dyad. At the dyad the hydroxyl radical cleavage is asymmetric and we suggest that the inferred structural asymmetry could direct the binding of the linker histone to a single preferred site.

In Saccharomyces cerevisiae, imbalance of the genes coding for the heterochromatin components Sir3p and histone H4 (namely, overdosage of SIR3 and lack of one of the two genes coding for H4) causes modifications in telomere length and telomere sequence organization, favoring the insertion of Y' elements into a stably shortened (C1-3A)n repeat tract. We report here that the newly inserted Y' elements are unstable and are lost with high frequency, generating clonal subpopulations with short telomeres, as revealed by the analysis of a specific telomere (LIII) and of the overall population of telomeres. Moreover, the growth rates of the subpopulations with and without Y' elements on LIII are different, the Y'-less individuals reproducing 20% more slowly than individuals bearing Y' elements. When grown together with Y'-bearing individuals, the subpopulations with the normal LIII telomere (which are viable and genetically stable if grown alone) are rapidly competed out. Hence, genetic imbalance for the structural components of heterochromatin results in a complex and rapidly changing mixture of subpopulations in such cultures. Thus, in situations where subpopulations are allowed to compete, heterochromatin-based differential growth rates result in neo-Darwinian clonal selection.

Telomeric heterochromatin plays an essential role in telomere function, including the regulation of telomere length. We observe that in Saccharomyces cerevisiae an imbalance in the dosage of genes for two protein components of heterochromatin (namely Sir3p and histone H4) causes modifications in telomere length and telomere sequence organization. The effects of Sir3p/H4 imbalance were analyzed in yeast strains in which the wild-type SIR3 gene (normally a single-copy gene) was either absent or present in 20-30 copies, and both histone H4 genes (HHF1 and HHF2) were present or HHF1 was deleted, thus covering a wide range of viable gene-dosage combinations. Modifications of telomeres and of subtelomeric regions were identified by analyzing both the overall telomere population and by focusing on two single telomeric regions: the left telomere of chromosome III (LIII) and the right telomere of chromosome XI (RXI). The modifications induced by alteration of the Sir3p/H4 ratio consist of a reduction in the length and an increase in the instability of the terminal block of (C(1-3)A)n repeats and in susceptibility to insertion of Y' elements into this repeat element. Restoration of the wild-type gene ratio (by removal of the extra copies of SIR3 or by complementation with the missing second copy of HHF) restored the original telomere organization, both with respect to the length of the (C(1-3)A)n repeat stretch and the absence of Y' elements. This behavior shows that the stability of the wild-type sequence organization requires maintenance of the normal structure of telomeric heterochromatin.

The chromatin organization of eukaryotic telomeres is essential for telomeric function and is currently receiving great attention. In yeast, the structural organization of telomeres involves a complex interplay of telomeric proteins that results in the formation of heterochromatin. This telomeric heterochromatin involves homotypic and heterotypic protein interactions that have been summarized in a general model. Recent analyses have focused on the study of the structural complexity at yeast telomeres to the level of specific nucleosomes and of the distribution of protein complexes in a natural telomeric region (LIII). In this report, we further analyze the structural complexity of LIII and the implication of this structure on telomeric silencing. It is shown that the establishment of repressive heterochromatin structures at LIII requires the recruitment of Sir3p through interaction with the N terminus of histone H4. The establishment of such structures does not require acetylation of any of four lysines located in the H4 N terminus (lysines 5, 8, 12, and 16).

We have determined the sites that are preferentially cleaved by Mn(T4MPyP) (where T4MPyP is the dianion of 5, 10, 15, 20, tetrakis (4-N-methylpyridine)porphyrin) on synthetic DNAs and on both intrinsically curved and average-shaped natural DNA sequences. On the basis of cleavage selectivity and of DNase I footprinting we show that the recognition specificity by this compound is based on steric properties: the preferred conformation is a DNA minor groove narrower than average and dimensionally defined. This conclusion is reached on the basis of: (i) the localization of the preferential cleavage sites at the 3' extremity of short A-tracts, known to undergo minor groove directional narrowing; (ii) the effects of temperature on cleavage specificity on curved sequences; (iii) the localization of cleavage sites in synthetic constructs whose crystal and solution structure was previously defined, and in programmed sequence variants thereoff; (iv) the effects of base substitutions on cleavage efficiency; (v) DNase I footprinting analysis. Several of these evidences argue against the possibility that Mn(T4MPyP)/DNA site selection occurs on the basis of electrostatic potential effects.Mn(T4MPyP) provides a tool for the analysis of DNA conformation whose selectivity is complementary to that of DNase I and hydroxyl radicals.

The effects of the rotational information of DNA in determining the in vivolocalization of nucleosomal core particles (ncps) have been studied in the Saccharomyces cerevisiae5 S rRNA repeat gene. The distribution of the phased series of flexibility signals present in this DNA has been altered by inserting in its centre a 25 bp tract. The effects of such alteration on the in vivodistribution of the helically phased, alternatively located ncps have been determined relative to a reference 21 bp insertion mutant. The results show that the answers provided in vitroand in vivoby the yeast 5 S rRNA gene sequence to specific modifications of the DNA rotational frame are similar, thus pointing to the relevance of DNA rotational information in vivo.

We have defined the in vivo heterochromatin structure of the left telomere of Saccharomyces cerevisiae chromosome III (LIII). Analysis of heterochromatin of a single telomere was so far lacking, due to the difficulties intrinsic to the highly repetitive nature of telomeric sequences. In LIII, the terminal (C1-3A)n repetitive sequences are followed by a complete X element and by the single copy Ty5-1 retrotransposon. Both the telosome and the X element exhibit overall resistance to micrococcal nuclease digestion reflecting their tight chromatin structure organization. The X element contains protein complexes and irregularly distributed but well localized nucleosomes. In contrast, a regular array of phased nucleosomes is associated with the promoter region of Ty5-1 and with the more centromere-proximal sequences. The lack of a structural component of yeast telomeres, the SIR3 protein, does not alter the overall tight organization of the X element but causes a nucleosome rearrangement within the promoter region of Ty5-1 and releases Ty5-1 silencing. Thus, Sir3p links the modification of the heterochromatin structure with loss of transcriptional silencing.

Induction of transcription in eukaryotic promoters is accompanied by removal or remodeling of nucleosomes. Given that this process causes release of torsional stress, the question is asked relative to its fate and to its effects on local DNA conformation. Is it dispersed by free rotation through surrounding nucleosomes or does it stay locally to be used in the modulation or activation of the transcription machinery? The results of the calculations relative to the onset of writhing suggest that the free energy made available by removal of nucleosomes is in the range of values that corresponds to the transition linking difference, thus pointing to a possible regulatory mechanism for the local use of free energy in promoters.

We report on a chemical method that allows DNA sequencing by a single reaction. It is based on treatment of 5′-end-labeled DNA with N-methylformamide in the presence of manganese. This method allows the manipulation of samples to be kept to a minimum and consists of a single chemical step that requires about 30 minutes to complete base degradation, phosphodiester bond cleavage and denaturation. Examples of one-treatment, one-lane DNA sequencing of both radioactively and fluorescently 5′-end-labeled DNAs are reported.

We have analyzed at both low and high resolution the distribution of nucleosomes over the Saccharomyces cerevisiae ADH2 promoter region in its chromosomal location, both under repressing (high-glucose) conditions and during derepression. Enzymatic treatments (micrococcal nuclease and restriction endonucleases) were used to probe the in vivo chromatin structure during ADH2 gene activation. Under glucose-repressed conditions, the ADH2 promoter was bound by a precise array of nucleosomes, the principal ones positioned at the RNA initiation sites (nucleosome +1), at the TATA box (nucleosome -1), and upstream of the ADR1-binding site (UAS1) (nucleosome - 2). The UAS1 sequence and the adjacent UAS2 sequence constituted a nucleosome-free region. Nucleosomes -1 and +1 were destabilized soon after depletion of glucose and had become so before the appearance of ADH2 mRNA. When the transcription rate was high, nucleosomes -2 and +2 also underwent rearrangement. When spheroplasts were prepared from cells grown in minimal medium, detection of this chromatin remodeling required the addition of a small amount of glucose. Cells lacking the ADR1 protein did not display any of these chromatin modifications upon glucose depletion. Since the UAS1 sequence to which Adr1p binds is located immediately upstream of nucleosome - 1, Adr1p is presumably required for destabilization of this nucleosome and for aiding the TATA-box accessibility to the transcription machinery.

Histone octamers (hos) and DNA topoisomerase I contribute, along with other proteins, to the higher order structure of chromatin. Here we report on the similar topological requirements of these two protein model systems for their interaction with DNA. Both histone octamers and topoisomerase I positively and consistently respond to DNA supercoiling and curvature, and to the spatial accessibility of the preferential interaction sites. These findings (1) point to the relevance of the topology-related DNA conformation in protein interactions and define the particular role of the helically phased rotational information; and (2) help to solve the apparent paradoxical behaviour of ubiquitous and abundant proteins that interact with defined DNA sites in spite of the lack of clear sequence consensuses. Considering firstly, that the interactions with DNA of both DNA topoisomerase I and histone octamers are topology-sensitive and that upon their interaction the DNA conformation is modified; and secondly, that similar behaviours have also been reported for DNA topoisomerase II and histone H1, a topology-based functional correlation among all these determinants of the higher order structure of chromatin is here suggested.

The effects of the rotational information of DNA in determining the in vitro localization of nucleosomal core particles (ncps) have been studied in the Saccharomyces cerevisiae 5S rRNA repeat gene. We have altered the distribution of the phased series of flexibility signals present on this DNA by inserting a 25-bp tract, and we have analyzed the effects of this mutation on the distribution and on the frequencies of ncps, as compared with the wild type and a reference 21-bp insertion mutant. The variation of the standard free energy of nucleosome reconstitution was determined. The results show that the DNA rotational information is a major determinant of ncps positioning, define how many rotationally phased signals are required for the formation of a stable particle, and teach how to modify their distribution through the alteration of the rotational signals.

Resting lymphocytes preferentially recirculate via the blood and lymphatics, migrating to the lymph nodes and spleen for efficient antigen encounter. Following activation, lymphocytes display altered migration pathways in that they preferentially return (“home”) to their site of activation. Activated lymphocytes also migrate to non-lymphoid organs and sites of inflammation, ensuring efficient antigen elimination. The receptor mechanisms that regulate migration are, as yet, unclear but appear to be altered upon lymphocyte activation and by the action of cytokines on endothelial cells. In order to study the role of activated T lymphocytes in lymphocyte recirculation and migration into inflammatory sites, a reproducible means of generating large numbers of lymphoblasts is required. We have investigated the use of a bispecific antibody (BsAb) constructed by Tutt et al.,1 consisting of chemically crosslinked mouse Fab’ fragments from R73 (anti-rat αs T cell receptor) and MRC-OX54 (anti-rat CD2) to stimulate the proliferation of rat T cells.

Several simple proteins and complex protein systems exist which do not recognize a defined sequence but--rather--a specific DNA conformation. We describe experiments and principles for two of these systems: nucleosomes and eukaryotic DNA topoisomerase I. Evidences are summarized that describe the effects of negative DNA supercoiling on nucleosome formation and the influence of DNA intrinsic curvature on their localization. The function of the DNA rotational information in nucleosome positioning and in the selection of multiple alternative positions on the same helical phase are described. This function suggests a novel genetic regulatory mechanism, based on nucleosome mobility and on the correlation between in vitro and in vivo positions. We observe that the same rules that determine the in vitro localization apply to the in vivo nucleosome positioning, as determined by a technique that relies on the use of nystatin and on the import of active enzymes in living yeast cells. The sensitivity of DNA topoisomerase I to the topological condition of the DNA substrate is reviewed and discussed taking into account recent experiments that describe the effect of the DNA tridimensional context on the reaction. These topics are discussed in the following order: (i) Proteins that look for a consensus DNA conformation; (ii) Nucleosomes; (iii) Negative supercoiling and nucleosomes; (iv) DNA curvature/bending and nucleosomes; (v) Multiple positioning; (vi) Multiple nucleosomes offer a contribution to the solution of the linking number paradox; (vii) Rotational versus translational information; (viii) A regulatory mechanism; (ix) DNA topoisomerase I; (x) DNA topoisomerase I and DNA supercoiling: a regulation by topological feedback; (xi) DNA topoisomerase I and DNA curvature; (xii) The in-and-out problem in the accessibility of DNA information; (xiii) The integrating function of the free energy of supercoiling.