Crystal structures of reduced and oxidized DsbA: investigation of domain motion and thiolate stabilization

Background: The redox proteins that incorporate a thioredoxin fold have diverse properties and functions. The bacterial protein-folding factor DsbA is the most oxidizing of the thioredoxin family. DsbA catalyzes disulfide-bond formation during the folding of secreted proteins, The extremely oxidizing nature of DsbA has been proposed to result from either domain motion or stabilizing active-site interactions in the reduced form. In the domain motion model, hinge bending between the two domains of DsbA occurs as a result of redox-related conformational changes. Results: We have determined the crystal structures of reduced and oxidized DsbA in the same crystal form and at the same pH (5.6). The crystal structure of a lower pH form of oxidized DsbA has also been determined (pH 5.0). These new crystal structures of DsbA, and the previously determined structure of oxidized DsbA at pH 6.5, provide the foundation for analysis of structural changes that occur upon reduction of the active-site disulfide bond. Conclusions: The structures of reduced and oxidized DsbA reveal that hinge bending motions do occur between the two domains. These motions are independent of redox state, however, and therefore do not contribute to the energetic differences between the two redox states, instead, the observed domain motion is proposed to be a consequence of substrate binding. Furthermore, DsbA's highly oxidizing nature is a result of hydrogen bond, electrostatic and helix-dipole interactions that favour the thiolate over the disulfide at the active site.

Iron(III) and iron(II) complexes of 1-thia-4,7-diazacyclononane ([9]aneN(2)S) and 1,4-dithia-7-azacyclononane ([9]aneNS(2)). X-Ray structural analyses, magnetic susceptibility, Mossbauer, EPR and electronic spectroscopy

The complexes [Fe([9]aneN(2)S)(2)][ClO4](2), [Fe([9]aneN(2)S)(2)][ClO4](3) and [Fe([9]aneNS(2))(2)][ClO4](2) ([9]aneN(2)S = 1-thia-4. 7-diazacyclononane and [9]aneNS(2) = 1,4-dithia-7-azacyclononane) have been prepared and the latter two characterised by X-ray crystallography. The Mossbauer spectra (isomer shift/mm s(-1), quadrupole splitting/mm s(-1), 4.2 K) for [Fe([9]aneN(2)S)(2)][ClO4](2) (0.52, 0.57), [Fe([9]aneN(2)S)(2)][ClO4](3) (0.25, 2.72) and [Fe([9]aneNS(2))(2)][ClO4](2) (0.43, 0.28) are typical for iron(II) and iron(III) complexes. Variable-temperature susceptibility measurements for [Fe([9]aneN(2)S)(2)][ClO4](2) (2-300 K) revealed temperature-dependent behaviour in both the solid state [2.95 mu(B) (300 K)-0.5 mu(B) (4.2 K)] and solution (Delta H degrees 20-22 kJ mol(-1), Delta S degrees 53-60 J mol(-1) K-1). For [Fe([9]aneN(2)S)(2)][ClO4](3) in the solid state [2.3 mu(B) (300 K)-1.9 mu(B) (4.2 K)] the magnetic data were fit to a simple model (H = -lambda L . S + mu L-z) to give the spin-orbit coupling constant (lambda) of -260 +/- 10 cm(-1). The solid-state X-band EPR spectrum of [Fe([9]aneN(2)S)(2)][ClO4](3) revealed axial symmetry (g(perpendicular to) = 2.607, g(parallel to) = 1.599). Resolution of g(perpendicular to) into two components at Q-band frequencies indicated a rhombic distortion. The low-temperature single-crystal absorption spectra of [Fe([9]aneN(2)S)(2)][ClO4](2) and [Fe([9]aneNS(2))(2)][ClO4](2) exhibited additional bands which resembled pseudotetragonal low-symmetry splitting of the parent octahedral (1)A(1g) --> T-1(2g) and (1)A(1g) ---> T-1(1g) transitions. However, the magnitude of these splittings was too large, requiring 10Dq for the thioether donors to be significantly larger than for the amine donors. Instead, these bands were tentatively assigned to weak, low-energy S --> Fe-II charge-transfer transitions. Above 200 K, thermal occupation of the high-spin T-5(2g) ground state resulted in observation of the T-5(2g) --> E-5(g) transition in the crystal spectrum of [Fe([9]aneN(2)S)(2)][ClO4](2). From a temperature-dependence study, the separation of the low-spin (1)A(1g) and high-spin T-5(2g) ground states was approximately 1700 cm(-1). The spectrum of the iron(III) complex [Fe([9]aneN(2)S)(2)][ClO4](3) is consistent with a low-spin d(5) configuration.

Structures of free and complexed forms of Escherichia coli xanthine-guanine phosphoribosyltransferase

Structures of free, substrate-bound and product-bound forms of Escherichia coli xanthine-guanine phosphoribosyltransferase (XGPRT) have been determined by X-ray crystallography. These are compared with the previously determined structure of magnesium and sulphate-bound XPRT. The structure of free XGPRT at 2.25 Angstrom resolution confirms the flexibility of residues in and around a mobile loop identified in other PRTases and shows that the cis-peptide conformation of Arg37 at the active site is maintained in the absence of bound ligands. The structures of XGPRT complexed with the purine base substrates guanine or xanthine in combination with cPRib-PP, an analog of the second substrate PRib-PP, have been solved to 2.0 Angstrom resolution. In these two structures the disordered phosphate-binding loop of uncomplexed XGPRT becomes ordered through interactions with the 5'-phosphate group of cPRib-PP. The cyclopentane ring of cPRib-PP has the C3 exo pucker conformation, stabilised by the cPRib-PP-bound Mg2+. The purine base specificity of XGPRT appears to be due to water-mediated interactions between the 2-exocyclic groups of guanine or xanthine and side-chains of Glu136 and Asp140, as well as the main-chain oxygen atom of Ile135. Asp92, together with Lys115, could help stabilise the N7-protonated tautomer of the incoming base and could act as a general base to remove the proton from N7 .when the nucleotide product is formed. The 2.6 Angstrom resolution structure of XGPRT complexed with product GMP is similar to the substrate-bound complexes. However, the ribose ring of GMP is rotated by similar to 24 degrees compared with the equivalent ring in cPRib-PP. This rotation results in the loss of all interactions between the ribosyl group and the enzyme in the product complex. (C) 1998 Academic Press.

Solution structure of methionine-oxidized amyloid beta-peptide (1-40). Does oxidation affect conformational switching?

The solution structure of A beta(1-40)Met(O), the methionine-oxidized form of amyloid beta-peptide A beta(1-40), has been investigated by CD and NMR spectroscopy. Oxidation of Met35 may have implications in the aetiology of Alzheimer's disease. Circular dichroism experiments showed that whereas A beta(1-40) and A beta(1-40)Met(O) both adopt essentially random coil structures in water (pH 4) at micromolar concentrations, the former aggregates within several days while the latter is stable for at least 7 days under these conditions. This remarkable difference led us to determine the solution structure of A beta(1-40)Met(O) using H-1 NMR spectroscopy. In a water-SDS micelle medium needed to solubilize both peptides at the millimolar concentrations required to measure NMR spectra, chemical shift and NOE data for A beta(1-40)Met(O) strongly suggest the presence of a helical region between residues 16 and 24. This is supported by slow H-D exchange of amide protons in this region and by structure calculations using simulated annealing with the program XPLOR. The remainder of the structure is relatively disordered. Our previously reported NMR data for A beta(1-40) in the same solvent shows that helices are present over residues 15-24 (helix 1) and 28-36 (helix 2), Oxidation of Met35 thus causes a local and selective disruption of helix 2. In addition to this helix-coil rearrangement in aqueous micelles, the CD data show that oxidation inhibits a coil-to-beta-sheet transition in water. These significant structural rearrangements in the C-terminal region of A beta may be important clues to the chemistry and biology of A beta(1-40) and A beta(1-42).

Epidermal iontophoresis: II. Application of the ionic mobility-pore model to the transport of local anesthetics

Purpose, An in vitro study was carried out to determine the iontophoretic permeability of local anesthetics through human epidermis. The relationship between physicochemical structure and the permeability of these solutes was then examined using an ionic mobility-pore model developed to define quantitative relationships. Methods. The iontophoretic permeability of both ester-type anesthetics (procaine, butacaine, tetracaine) and amide-type anesthetics (prilocaine, mepivacaine, lidocaine, bupivacaine, etidocaine, cinchocaine) were determined through excised human epidermis over 2 hrs using a constant d.c. current and Ag/AgCl electrodes. Individual ion mobilities were determined from conductivity measurements in aqueous solutions. Multiple stepwise regression was applied to interrelate the iontophoretic permeability of the solutes with their physical properties to examine the appropriateness of the ionic mobility-pore model and to determine the best predictor of iontophoretic permeability of the local anesthetics. Results. The logarithm of the iontophoretic permeability coefficient (log PCj,iont) for local anesthetics was directly related to the log ionic mobility and MW for the free volume form of the model when other conditions are held constant. Multiple linear regressions confirmed that log PCj,iont was best defined by ionic mobility (and its determinants: conductivity, pK(a) and MW) and MW. Conclusions. Our results suggest that of the properties studied, the best predictors of iontophoretic transport of local anesthetics are ionic mobility (or pK(a)) and molecular size. These predictions are consistent with the ionic mobility pore model determined by the mobility of ions in the aqueous solution, the total current, epidermal permselectivity and other factors as defined by the model.

Thermal diffusion in molecular dynamics simulations of thin film diamond deposition

Molecular dynamics simulations of carbon atom depositions are used to investigate energy diffusion from the impact zone. A modified Stillinger-Weber potential models the carbon interactions for both sp2 and sp3 bonding. Simulations were performed on 50 eV carbon atom depositions onto the (111) surface of a 3.8 x 3.4 x 1.0 nm diamond slab containing 2816 atoms in 11 layers of 256 atoms each. The bottom layer was thermostated to 300 K. At every 100th simulation time step (27 fs), the average local kinetic energy, and hence local temperature, is calculated. To do this the substrate is divided into a set of 15 concentric hemispherical zones, each of thickness one atomic diameter (0.14 nm) and centered on the impact point. A 50-eV incident atom heats the local impact zone above 10 000 K. After the initial large transient (200 fs) the impact zone has cooled below 3000 K, then near 1000 K by 1 ps. Thereafter the temperature profile decays approximately as described by diffusion theory, perturbed by atomic scale fluctuations. A continuum model of classical energy transfer is provided by the traditional thermal diffusion equation. The results show that continuum diffusion theory describes well energy diffusion in low energy atomic deposition processes, at distance and time scales larger than 1.5 nm and 1-2 ps, beyond which the energy decays essentially exponentially. (C) 1998 Published by Elsevier Science S.A. All rights reserved.

Predictors of local recurrence after treatment of ductal carcinoma in situ - A meta-analysis

lBACKGROUND. Management of patients with ductal carcinoma in situ (DCIS) is a dilemma, as mastectomy provides nearly a 100% cure rate but at the expense of physical and psychologic morbidity. It would be helpful if we could predict which patients with DCIS are at sufficiently high risk of local recurrence after conservative surgery (CS) alone to warrant postoperative radiotherapy (RT) and which patients are at sufficient risk of local recurrence after CS + RT to warrant mastectomy. The authors reviewed the published studies and identified the factors that may be predictive of local recurrence after management by mastectomy, CS alone, or CS + RT. METHODS. The authors examined patient, tumor, and treatment factors as potential predictors for local recurrence and estimated the risks of recurrence based on a review of published studies. They examined the effects of patient factors (age at diagnosis and family history), tumor factors (sub-type of DCIS, grade, tumor size, necrosis, and margins), and treatment (mastectomy, CS alone, and CS + RT). The 95% confidence intervals (CI) of the recurrence rates for each of the studies were calculated for subtype, grade, and necrosis, using the exact binomial; the summary recurrence rate and 95% CI for each treatment category were calculated by quantitative meta-analysis using the fixed and random effects models applied to proportions. RESULTS, Meta-analysis yielded a summary recurrence rate of 22.5% (95% CI = 16.9-28.2) for studies employing CS alone, 8.9% (95% CI = 6.8-11.0) for CS + RT, and 1.4% (95% CI = 0.7-2.1) for studies involving mastectomy alone. These summary figures indicate a clear and statistically significant separation, and therefore outcome, between the recurrence rates of each treatment category, despite the likelihood that the patients who underwent CS alone were likely to have had smaller, possibly low grade lesions with clear margins. The patients with risk factors of presence of necrosis, high grade cytologic features, or comedo subtype were found to derive the greatest improvement in local control with the addition of RT to CS. Local recurrence among patients treated by CS alone is approximately 20%, and one-half of the recurrences are invasive cancers. For most patients, RT reduces the risk of recurrence after CS alone by at least 50%. The differences in local recurrence between CS alone and CS + RT are most apparent for those patients with high grade tumors or DCIS with necrosis, or of the comedo subtype, or DCIS with close or positive surgical margins. CONCLUSIONS, The authors recommend that radiation be added to CS if patients with DCIS who also have the risk factors for local recurrence choose breast conservation over mastectomy. The patients who may be suitable for CS alone outside of a clinical trial may be those who have low grade lesions with little or no necrosis, and with clear surgical margins. Use of the summary statistics when discussing outcomes with patients may help the patient make treatment decisions. Cancer 1999;85:616-28. (C) 1999 American Cancer Society.

Targeting local tissues by transdermal application: Understanding drug physicochemical properties that best exploit protein binding and blood flow effects

The targeting of topically applied drug molecules into tissues below a site of application requires an understanding of the complex interrelationships between the drug, its formulation, the barrier properties of the skin, and the physiological processes occurring below the skin that are responsible for drug clearance from the site, tissue, and/or systemic distribution and eventual elimination. There is still a certain amount of controversy over the ability of topically applied drugs to penetrate into deeper tissues by diffusion or whether this occurs by redistribution in the systemic circulation. The major focus of our work in this area has been in determining how changes in drug structure and physicochemical properties, such as protein binding and lipophilicity, affect drug clearance into the local dermal microcirculation and lymphatics, as well as subsequent distribution into deeper tissues below an application site. The present study outlines our recent thinking on the drug molecule optimal physical attributes, in terms of plasma and tissue partitioning behaviour, that offer the greatest potential for deep tissue targeting. Drug Dev. Res. 46:309-315, 1999. (C) 1999 Wiley-Liss, Inc.

Structure-activity relationships of omega-conotoxins MVIIA, MVIIC and 14 loop splice hybrids at N and P/Q-type calcium channels

The omega-conotoxins are a set of structurally related, four-loop, six cysteine containing peptides, that have a range of selectivities for different subtypes of the voltage-sensitive calcium channel (VSCC). To investigate the basis of the selectivity displayed by these peptides, we have studied the binding affinities of two naturally occurring omega-conotoxins, MVIIA and MVIIC and a series of 14 MVIIA/MVIIC loop hybrids using radioligand binding assays for N and P/Q-type Ca2+ channels in rat brain tissue. A selectivity profile was developed from the ratio of relative potencies at N-type VSCCs (using [I-125]GVIA radioligand binding assays) and P/Q-type VSCCs (using [I-125]MVIIC radioligand binding assays). in these peptides, loops 2 and 4 make the greatest contribution to VSCC subtype selectivity, while the effects of loops 1 and 3 are negligible. Peptides with homogenous combinations of loop 2 and 4 display clear selectivity preferences, while those with heterogeneous combinations of loops 2 and 4 are less discriminatory. H-1 NMR spectroscopy revealed that the global folds of MVIIA, MVIIC and the 14 loop hybrid peptides were similar; however, several differences in local structure were identified. Based on the binding data and the 3D structures of MVIIA, GVIA and MVIIC, we have developed a preliminary pharmacophore based on the omega-conotoxin residues most Likely to interact with the N-type VSCC. (C) 1999 Academic Press.

Variable temperature and pressure study of the aquation reactions of cobalt(III) and chromium(III) penta- and tetra-amines

Preparation of a series of specific penta- and tetra-amine derivatives of Co-III and Cr-III with a neutral leaving ligand has been carried out in order to accomplish a fine tuning of the associativeness/dissociativeness of their substitution reactions. Spontaneous aquation reactions of the neutral ligands have been studied at variable temperature and pressure. Although rate constants and thermal activation parameters show an important degree of scatter, the values determined for the activation volumes of the substitution process illustrate the mechanistic fine tuning that may be achieved for these reactions. In all cases, in the absence of important steric constraints in the molecule, electronic inductive effects seem to be the most important factor accounting for the dissociative shifts observed both for pentaamine (i.e.Delta V double dagger=+4.0 or +14.0 cm(3) mol(-1) and +5.2 or +16.5 cm(3) mol(-1) for the aquation of cis- or trans-[Co(MeNH2)(NH3)(4)(DMF)](3+) and cis- or trans-[CoL15(DMF)](3+) respectively, where L-15 represents a pentaamine macrocyclic ligand), and tetraamine systems (i.e.Delta V double dagger=+4.1 or +8.4 cm(3) mol(-1) and -10.8 or -7.4 cm(3) mol(-1) for the aquation of cis-[Co(NH3)(4)Cl(DMAC)](2+) (DMAC=dimethylacetamide) or cis-[Co(en)(2)Cl(DMAC)](2+) and cis-[Cr(NH3)(4)Cl(DMF)](2+) or cis -[Cr(en)(2)Cl(DMF)](2+)). From the results, clear evidence is obtained which indicates that, only when the situation is borderline I-a/I-d, or the steric demands are increased dramatically, dissociative shifts are observed; in all other cases electronic inductive effects seem to be dominant for such a tuning of the substitution process.

Life tables of the processionary caterpillar Ochrogaster lunifer Herrich-Schaffer (Lepidoptera : Thaumetopoeidae) at local and regional scales

A major ongoing debate in population ecology has surrounded the causative factors underlying the abundance of phytophagous insects and whether or not these factors limit or regulate herbivore populations. However, it is often difficult to identify mortality agents in census data, and their distribution and relative importance across large spatial scales are rarely understood. Were, we present life tables for egg batches and larval cohorts of the processionary caterpillar Ochrogaster lunifer Herrich-Schaffer, using intensive local sampling combined with extensive regional monitoring to ascertain the relative importance of different mortality factors at different localities. Extinction of entire cohorts (representing the entire reproductive output of one female) at natural localities was high, with 82% of the initial 492 cohorts going extinct. Mortality was highest in the egg and early instar stages due to predation from dermestid beetles, and while different mortality factors (e.g. hatching failure, egg parasitism and failure to establish on the host) were present at many localities, dermestid predation, either directly observed or inferred from indirect evidence, was the dominant mortality factor at 89% of localities surveyed. Predation was significantly higher in plantations than in natural habitats. The second most important mortality factor was resource depletion, with 14 cohorts defoliating their hosts. Egg and larval parasitism were not major mortality agents. A combination of predation and resource depletion consistently accounted for the majority of mortality across localities, suggesting that both factors are important in limiting population abundance. This evidence shows that O. lunifer is not regulated by natural enemies alone, but that resource patches (Acacia trees) ultimately, and frequently, act together to limit population growth.

Contemporary strategies for rural community development in Australia: a governmentality perspective

Contemporary strategies for rural development in Australia are based upon notions of self-help and bottom-up, community-based initiatives which are said to 'empower' the individual from the imposing structures of government intervention. While such strategies are not entirely new to Australia, they have, it seems, been inadequately theorised to date and are generally regarded, in rather functionalist terms, as indicative of attempts to cut back on public expenditure. Harnessing itself to the 'governmentality' perspective, this paper explores government and 'expert' discourses of rural community development in Queensland and suggests, instead, that these strategies are indicative of an advanced liberal form of rule which seeks to 'govern through community'. With this in mind, three basic research questions are identified as worthy of further exploration; how are the notions of self-governing individuals and communities constructed in political discourse; what political rationalities are used to justify current levels of(non) intervention and finally; what are the discourses, forms and outcomes of empowerment at the local level? The paper concludes by arguing that while the empowering effects of self-help are frequently cited as its greatest virtue, it is not so much control as the added burden of responsibility that is being devolved to local people. Given the emphasis of the governmentality perspective on strategies for 'governing at a distance', however, these conclusions can hardly be unexpected. (C) 2000 Elsevier Science Ltd, All rights reserved.

Finite element modelling of dissipative structures for nonequilibrium chemical reactions in fluid-saturated porous media

We use the finite element method to model and predict the dissipative structures of chemical species for a nonequilibrium chemical reaction system in a fluid-saturated porous medium. In particular, we explore the conditions under which dissipative structures of the species may exist in the Brusselator type of nonequilibrium chemical reaction. Since this is the first time the finite element method and related strategies have been used to study the chemical instability problems in a fluid-saturated porous medium, it is essential to validate the method and strategies before they are put into application. For this purpose, we have rigorously derived the analytical solutions for dissipative structures of chemical species in a benchmark problem, which geometrically is a square. Comparison of the numerical solutions with the analytical ones demonstrates that the proposed numerical method and strategy are robust enough to solve chemical instability problems in a fluid-saturated porous medium. Finally, the related numerical results from two application examples indicate that both the regime and the magnitude of pore-fluid flow have significant effects on the nature of the dissipative structures that developed for a nonequilibrium chemical reaction system in a fluid-saturated porous medium. The motivation for this study is that self-organization under conditions of pore-fluid flow in a porous medium is a potential mechanism of the orebody formation and mineralization in the upper crust of the Earth. (C) 2000 Elsevier Science S.A. All rights reserved.

Computation of bifurcation boundaries for power systems: A new Delta-plane method

This paper is devoted to the problems of finding the load flow feasibility, saddle node, and Hopf bifurcation boundaries in the space of power system parameters. The first part contains a review of the existing relevant approaches including not-so-well-known contributions from Russia. The second part presents a new robust method for finding the power system load flow feasibility boundary on the plane defined by any three vectors of dependent variables (nodal voltages), called the Delta plane. The method exploits some quadratic and linear properties of the load now equations and state matrices written in rectangular coordinates. An advantage of the method is that it does not require an iterative solution of nonlinear equations (except the eigenvalue problem). In addition to benefits for visualization, the method is a useful tool for topological studies of power system multiple solution structures and stability domains. Although the power system application is developed, the method can be equally efficient for any quadratic algebraic problem.