Expression, purification, crystallization, and NMR studies of the helicase interaction domain of Escherichia coli DnaG primase

in Escherichia coli, the DnaG primase is the RNA polymerase that synthesizes RNA primers at replication forks. It is composed of three domains, a small N-terminal zinc-binding domain, a larger central domain responsible for RNA synthesis, and a C-terminal domain comprising residues 434-581 [DnaG(434-581)] that interact with the hexameric DnaB helicase. Presumably because of this interaction, it had not been possible previously to express the C-terminal domain in a stably transformed E coli strain. This problem was overcome by expression of DnaG(434-581) under control of tandem bacteriophage gimel-promoters, and the protein was purified in yields of 4-6 mg/L of culture and studied by NMR. A TOCSY spectrum of a 2 mM solution of the protein at pH 7.0, indicated that its structured core comprises residues 444-579. This was consistent with sequence conservation among most-closely related primases. Linewidths in a NOESY spectrum of a 0.5 mM sample in 10 mM phosphate, pH 6.05, 0.1 M NaCl, recorded at 36 degreesC, indicated the protein to be monomeric. Crystals of selenomethionine-substituted DnaG(434-581) obtained by the hanging-drop vapor-diffusion method were body-centered tetragonal, space group I4(1)22, with unit cell parameters a = b 142.2 Angstrom, c = 192.1 Angstrom, and diffracted beyond 2.7 Angstrom resolution with synchrotron radiation. (C) 2003 Elsevier Inc. All rights reserved.

The land assembly problem revisited

As in the standard land assembly problem, a developer wants to buy two adjacent blocks of land belonging to two different owners. The value of the two blocks of land to the developer is greater than the sum of the individual values of the blocks for each owner. Unlike the land assembly literature, however, our focus is on the incentive that each lot owner has to delay the start of negotiations, rather than on the public goods nature of the problem. An incentive for delay exists, for example, when owners perceive that being last to sell will allow them to capture a larger share of the joint surplus from the development. We show that competition at point of sale can cause equilibrium delay, and that cooperation at point of sale will eliminate delay. This suggests that strategic delay is another source for the inefficient allocation of land, in addition to the public-good type externality pointed out by Grossman and Hart [Bell Journal of Economics 11 (1980) 42] and O'Flaherty [Regional Science and Urban Economics 24 (1994) 287]. (C) 2004 Elsevier B.V. All rights reserved.

Sequence analysis, chromosomal distribution and long-range organization show that rapid turnover of new and old pBuM satellite DNA repeats leads to different patterns of variation in seven species of the Drosophila buzzatii cluster

We aimed to study patterns of variation and factors influencing the evolutionary dynamics of a satellite DNA, pBuM, in all seven Drosophila species from the buzzatii cluster (repleta group). We analyzed 117 alpha pBuM-1 (monomer length 190 bp) and 119 composite alpha/beta (370 bp) pBuM-2 repeats and determined the chromosome location and long-range organization on DNA fibers of major sequence variants. Such combined methodologies in the study of satDNAs have been used in very few organisms. In most species, concerted evolution is linked to high copy number of pBuM repeats. Species presenting low-abundance and scattered distributed pBuM repeats did not undergo concerted evolution and maintained part of the ancestral inter-repeat variability. The alpha and alpha/beta repeats colocalized in heterochromatic regions and were distributed on multiple chromosomes, with notable differences between species. High-resolution FISH revealed array sizes of a few kilobases to over 0.7 Mb and mutual arrangements of alpha and alpha/beta repeats along the same DNA fibers, but with considerable changes in the amount of each variant across species. From sequence, chromosomal and phylogenetic data, we could infer that homogenization and amplification events involved both new and ancestral pBuM variants. Altogether, the data on the structure and organization of the pBuM satDNA give insights into genome evolution including mechanisms that contribute to concerted evolution and diversification.

Colonization sequence of non-pollinating fig wasps associated with Ficus citrifolia in Brazil

Mutualisms, such as the fig-agaonid wasp association, are susceptible to colonization by parasitic species, which exploit the resources involved therein. In most cases, they oviposit into the figs from outside without providing any pollination service. In this study, we used several different methods (adhesive traps and direct standardized field observations) to assess the colonization sequence of a diverse fig wasp fauna associated with Ficus citrifolia, section Americana, in Brazil. They consistently showed a temporal partitioning in colonization among non-pollinating fig wasp species. Idarnes species belonging to the flavicollis and incerta groups colonized figs just before or during the fig receptive phase. In contrast, Idarnes females belonging to the carme group oviposited one to three weeks later, mainly in the middle of the inter-floral phase. Eurytoma, Heterandrium, Physothorax and Torymus were later colonizers, and laid eggs either in the middle or during the late inter-floral phase. The results suggest that these Neotropical fig wasps have different strategies of resource exploitation, even among species belonging to the same genus.

XAS characterization of the RuO(2)-Ta(2)O(5) system local (crystal) structure

The influence of the preparation method on the structural properties of the RuO(2)-Ta(2)O(5) system was investigated. Both thin films on Ti substrates and powder samples of nominal composition Ti/RuO(2)-Ta(2)O(5) (Ru:Ta = 100:0, 90:10, 80:20, 30:70, and 0:100 at.%) were prepared through thermal decomposition of polymeric precursors (DPP). The thin films and powder samples were investigated using X-ray absorption spectroscopy (XAS). XANES analyses showed that Ru and Ta are present in the Ru(IV) and Ta(V) oxidation states. EXAFS signals of all the samples were analyzed, to obtain the average bond length (r), coordination number, and the Debye-Waller factor (sigma(2)) for each Ru-O, Ru-Ru, Ta-O nearest-neighbor. The first shell Ru-O distance was found at 1.91-1.92 angstrom with coordination number of 1.8-2.1, and at 2.01-2.02 angstrom with coordination number of 3.9-4.1. The Ta-O distance obtained for all the samples and in both modes (transmission and fluorescence) had significantly different values from the theoretical ones. The results revealed that the local structure around both the Ru and Ta sites are similar, and that they consist of distorted M-O(6) octahedra (where M = Ru or Ta). (C) 2010 Elsevier B.V. All rights reserved.

Local delivery of EGF-liposome mediated bone modeling in orthodontic tooth movement by increasing RANKL expression

Aims: It has long been demonstrated that epidermal growth factor (EGF) has catabolic effects oil bone. Thus. we examined the role of EGF in regulating mechanically induced bone modeling in a rat model of orthodontic tooth movement. Main methods: The maxillary first molars of rats were moved mesially using an orthodontic appliance attached to the maxillary incisor teeth. Rats were randomly divided into 4 groups: (G1) administration of PBS (Phosphate buffer saline Solution (n = 24); (G2) administration of empty liposomes (it = 24): (Q) administration 20 rig of EGF Solution (n = 24): and (G4) 20 ng of EGF-liposomes Solution (it = 24). Each Solution was injected in the mucosa of the left first molar adjacent to the appliance. At days 5, 10, 14 and 2 1 after drug administration. 6 animals of each group were sacrificed. Histomorphometric analysis was used to quantify osteoclasts (Tartrate-resistant acid phosphatase (TRAP) + cells) and tooth movement. Using immunohistochemistry assay we evaluated the RANKL (receptor activator of nuclear factor kappa B ligand) and epidermal growth factor receptor (EGFR) expression. Key findings: The EGF-liposome administration showed an increased tooth movement and osteoclast numbers compared to controls (p<0.05). This was correlated with intense RANKL expression. Both osteoblasts and osteoclasts expressed EGFR. Significance: Local delivery of EGF-liposome stimulates, osteoclastogenesis and tooth movement. (C) 2009 Elsevier Inc. All rights reserved.

QUADRANT-SEARCHING: A NEW TECHNIQUE TO REDUCE THE SEARCH SPACE OF THE INVERSE PROBLEM OF EIT USING BEM TO THE DIRECT PROBLEM

The image reconstruction using the EIT (Electrical Impedance Tomography) technique is a nonlinear and ill-posed inverse problem which demands a powerful direct or iterative method. A typical approach for solving the problem is to minimize an error functional using an iterative method. In this case, an initial solution close enough to the global minimum is mandatory to ensure the convergence to the correct minimum in an appropriate time interval. The aim of this paper is to present a new, simple and low cost technique (quadrant-searching) to reduce the search space and consequently to obtain an initial solution of the inverse problem of EIT. This technique calculates the error functional for four different contrast distributions placing a large prospective inclusion in the four quadrants of the domain. Comparing the four values of the error functional it is possible to get conclusions about the internal electric contrast. For this purpose, initially we performed tests to assess the accuracy of the BEM (Boundary Element Method) when applied to the direct problem of the EIT and to verify the behavior of error functional surface in the search space. Finally, numerical tests have been performed to verify the new technique.

Existence Results for Second Order Differential Equations with Nonlocal Conditions in Banach Spaces

This work is concerned with implicit second order abstract differential equations with nonlocal conditions. Assuming that the involved operators satisfy sonic compactness properties, we establish the existence of local mild solutions, the existence of global mild solutions and the existence of asymptotically almost periodic solutions.

Local inflammation is crucial for T cell mediated rejection of skin graft expressing foreign antigen

Most of the skin grafts from (K14hGH.FVB C57BL/6) F1 mice, which express foreign antigen (human growth hormone, hGH) in skin keratinocytes driven by keratin 14 promoter, were spontaneously rejected by syngeneic wild type F1 recipients and hGH-specific immune responses such as antibody and hGHspecific T cells were generated in these recipients. Interestingly, a 2nd F1 hGH-expressing skin graft was rejected by graft primed recipients, but was not rejected from such recipients if CD4+ or CD8+ T cells were depleted prior to the placement of the 2nd graft. Surprisingly, this 2nd graft retained healthy even after CD4+ or CD8+ T cells were allowed to recover so that the animal could reject a freshly placed 3rd F1 hGH-expressing graft. Furthermore, inflammatory response induced by topical treatment with imiquimod could lead to the rejection of some well-healed 2nd grafts. This result indicates that both CD4+ and CD8+ T cells are required for the rejection and the ability of effector T cells to reject a graft is determined by local factors in the graft which are presumably determined by inflammation induced by surgery or imiquimod treatment. Taken together, our results suggest that in addition to CD4+ and CD8+ T cells, local environmental factors induced by inflammation are also crucial for effector T cell functions leading to graft destruction. The understanding of these local factors will lead to more effective immunotherapy for established, epithelial cancer in the future.

Characterization of inflammatory responses to eccentric exercise in humans

Eccentric exercise commonly results in muscle damage. The primary sequence of events leading to exercise-induced muscle damage is believed to involve initial mechanical disruption of sarcomeres, followed by impaired excitation-contraction coupling and calcium signaling, and finally, activation of calcium-sensitive degradation pathways. Muscle damage is characterized by ultrastructural changes to muscle architecture, increased muscle proteins and enzymes in the bloodstream, loss of muscular strength and range of motion and muscle soreness. The inflammatory response to exercise-induced muscle damage is characterized by leukocyte infiltration and production of pro-inflammatory cytokines within damaged muscle tissue, systemic release of leukocytes and cytokines, in addition to alterations in leukocyte receptor expression and functional activity. Current evidence suggests that inflammatory responses to muscle damage are dependent on the type of eccentric exercise, previous eccentric loading (repeated bouts), age and gender. Circulating neutrophil counts and systemic cytokine responses are greater after eccentric exercise using a large muscle mass (e.g. downhill running, eccentric cycling) than after other types of eccentric exercise involving a smaller muscle mass. After an initial bout of eccentric exercise, circulating leukocyte counts and cell surface receptor expression are attenuated. Leukocyte and cytokine responses to eccentric exercise are impaired in elderly individuals, while cellular infiltration into skeletal muscle is greater in human females than males after eccentric exercise. Whether alterations in intracellular calcium homeostasis influence inflammatory responses to muscle damage is uncertain. Furthermore, the effects of antioxidant supplements are variable, and the limited data available indicates that anti-inflammatory drugs largely have no influence on inflammatory responses to eccentric exercise. In this review, we compare local versus systemic inflammatory responses, and discuss some of the possible mechanisms regulating the inflammatory responses to exercise-induced muscle damage in humans.

Local immunological factors associated with recurrence of mucosal leishmaniasis

Recurrence of mucosal leishmaniasis (ML) is frequent, but the causative mechanisms are unknown. Our aim was to compare cellular and cytokine patterns of lesions from ML that evolved to recurrence or cure in order to determine the risk factor associated with recurrence. Lesions were evaluated by immunohistochemistry before and after therapy, and patients were followed-up for five years. Higher levels of CD4(+) T and IFN-gamma-producing cells were detected in active lesions and decreased after therapy. Macrophages and IL-10 were markedly increased in cured patients. Conversely, CD8(+) T and NK cells were higher in relapsed than in cured cases. Notably, a decrease in these cells in addition to decreased IL-10 and IFN-gamma was also observed after therapy. These data suggest that exacerbated CD8(+) activity, in addition to a poor regulatory response, could underlie an unfavorable fate with regard to ML. These markers may be useful for predicting the prognosis of ML in lesion studies. (C) 2008 Elsevier Inc. All rights reserved.

Three-dimensional electrical impedance, tomography: A topology optimization approach

Electrical impedance tomography is a technique to estimate the impedance distribution within a domain, based on measurements on its boundary. In other words, given the mathematical model of the domain, its geometry and boundary conditions, a nonlinear inverse problem of estimating the electric impedance distribution can be solved. Several impedance estimation algorithms have been proposed to solve this problem. In this paper, we present a three-dimensional algorithm, based on the topology optimization method, as an alternative. A sequence of linear programming problems, allowing for constraints, is solved utilizing this method. In each iteration, the finite element method provides the electric potential field within the model of the domain. An electrode model is also proposed (thus, increasing the accuracy of the finite element results). The algorithm is tested using numerically simulated data and also experimental data, and absolute resistivity values are obtained. These results, corresponding to phantoms with two different conductive materials, exhibit relatively well-defined boundaries between them, and show that this is a practical and potentially useful technique to be applied to monitor lung aeration, including the possibility of imaging a pneumothorax.

Surgical Correction of Aesthetically Deformed Eyebrows Using Local Transposition Flaps

Eyebrow positions differ in many ways. They vary in shape, thickness, length, and distance between the eyebrows, making the face more or less harmonious. When a large distance exists between the eyebrows and the medial brow is slanting downward, the glabellar area is larger, giving the face an awkward appearance. To correct this deformity, the authors propose using two Z-plasties to allow transposition of flaps in the region of the medial brow. The Z-shaped flap is outlined at the medial third of the brow, in the glabellar region, with the eyebrow centered in the lower portion of the ""Z"" and the hairless skin in the upper portion. The flaps then are transposed and sutured. Transposition of the flaps, lifting the brow flap to the glabellar region, results in horizontal positioning of the medial and central third of the eyebrow. The proposed transpositioning of ""Z"" flaps in this region corrects this type of deformity of the medial and central portions of the eyebrows, with an aesthetically satisfactory result.