The Unusually Stable Quaternary Structure of Human Cu,Zn-Superoxide Dismutase 1 Is Controlled by Both Metal Occupancy and Disulfide Status

The eukaryotic copper,zinc superoxide dismutases are remarkably stable dimeric proteins that maintain an intrasubunit disulfide bond in the reducing environment of the cytosol and are active under a variety of stringent denaturing conditions. The structural interplay of conserved disulfide bond and metal-site occupancy in human copper,zinc superoxide dismutase (hSOD1) is of increasing interest as these post-translational modifications are known to dramatically alter the catalytic chemistry, the subcellular localization, and the susceptibility of the protein to aggregation. Using biophysical methods, we find no significant change in the gross secondary or tertiary structure of the demetallated form upon reduction of the disulfide. Interestingly, reduction does lead to a dramatic change in the quaternary structure, decreasing the monomer-to-dimer equilibrium constant by at least four orders of magnitude. This reduced form of hSOD1 is monomeric, even at concentrations well above the physiological range. Either the addition of Zn(II) or the formation of the disulfide leads to a shift in equilibrium that favors the dimeric species, even at low protein concentrations (i.e. micromolar range). We conclude that only the most immature form of hSOD1, i.e. one without any post-translational modifications, favors the monomeric state under physiological conditions. This finding provides a basis for understanding the selectivity of mitochondrial SOD1 import and may be relevant to the toxic properties of mutant forms of hSOD1 that can cause the familial form of amyotrophic lateral sclerosis

Solution Structure of the Yeast Copper Transporter Domain Ccc2a in the Apo and Cu(I)-loaded States

Ccc2 is an intracellular copper transporter in Saccharomyces cerevisiae and is a physiological target of the copper chaperone Atx1. Here we describe the solution structure of the first N-terminal MTCXXC metal-binding domain, Ccc2a, both in the presence and absence of Cu(I). For Cu(I)-Ccc2a, 1944 meaningful nuclear Overhauser effects were used to obtain a family of 35 structures with root mean square deviation to the average structure of 0.36 +/- 0.06 A for the backbone and 0.79 +/- 0.05 A for the heavy atoms. For apo-Ccc2a, 1970 meaningful nuclear Overhauser effects have been used with 35 (3)J(HNHalpha) to obtain a family of 35 structures with root mean square deviation to the average structure of 0.38 +/- 0.06 A for the backbone and 0.82 +/- 0.07 A for the heavy atoms. The protein exhibits a betaalphabetabetaalphabeta, ferrodoxin-like fold similar to that of its target Atx1 and that of a human counterpart, the fourth metal-binding domain of the Menkes protein. The overall fold remains unchanged upon copper loading, but the copper-binding site itself becomes less disordered. The helical context of the copper-binding site, and the copper-induced conformational changes in Ccc2a differ from those in Atx1. Ccc2a presents a conserved acidic surface which complements the basic surface of Atx1 and a hydrophobic surface. These results open new mechanistic aspects of copper transporter domains with physiological copper donor and acceptor proteins

Characterization of the Binding Interface between the Copper Chaperone Atx1 and the First Cytosolic Domain of Ccc2 ATPase

The interaction of the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase, Ccc2a, was investigated by NMR in solution. In particular, a solution of Cu(I)-15NAtx1 was titrated with apo-Ccc2a, and, vice versa, a solution of Cu(I)-15NCcc2a was titrated with apo-Atx1. By following the 15N and 1H chemical shifts, a new species is detected in both experiments. This species is the same in both titrations and is in fast exchange with the parent species on the NMR time scale. Nuclear relaxation data are consistent with the formation of an adduct. Judging from the nuclear Overhauser effect spectroscopy patterns, the structure of Cu(I)-15NCcc2a in the presence of apo-Atx1 is not significantly altered, whereas Cu(I)-15NAtx1 in the presence of apo-Ccc2a experiences some changes with respect to both the apoproteins and the Cu(I)-loaded proteins. The structure of the Cu(I)-15NAtx1 moiety in the adduct was obtained from 1137 nuclear Overhauser effects to a final root mean square deviation to the mean structure of 0.76 +/- 0.13 A for the backbone and 1.11 +/- 0.11 A for the heavy atoms. 15N and 1H chemical shifts suggest the regions of interaction that, together with independent information, allow a structural model of the adduct to be proposed. The apo form of Atx1 displays significant mobility in loops 1 and 5, the N-terminal part of helix alpha1, and the C-terminal part of helix alpha2 on the ms-micros time scale. These regions correspond to the metal binding site. Such mobility is largely reduced in the free Cu(I)-Atx1 and in the adduct with apo-Ccc2a. The analogous mobility of Ccc2a in both Cu(I) and apo forms is reduced with respect to Atx1. Such an adduct is relevant as a structural and kinetic model for copper transfer from Atx1 to Ccc2a in physiological conditions

A new role for Zinc limitation in bacterial pathogenicity: modulation of α-hemolysin from uropathogenic Escherichia coli.

Metal limitation is a common situation during infection and can have profound effects on the pathogen's success. In this report, we examine the role of zinc limitation in the expression of a virulence factor in uropathogenic Escherichia coli. The pyelonephritis isolate J96 carries two hlyCABD operons that encode the RTX toxin α-hemolysin. While the coding regions of both operons are largely conserved, the upstream sequences, including the promoters, are unrelated. We show here that the two hlyCABD operons are differently regulated. The hly II operon is efficiently silenced in the presence of zinc and highly expressed when zinc is limited. In contrast, the hly I operon does not respond to zinc limitation. Genetic studies reveal that zinc-responsive regulation of the hly II operon is controlled by the Zur metalloregulatory protein. A Zur binding site was identified in the promoter sequence of the hly II operon, and we observe direct binding of Zur to this promoter region. Moreover, we find that Zur regulation of the hly II operon modulates the ability of E. coli J96 to induce a cytotoxic response in host cell lines in culture. Our report constitutes the first description of the involvement of the zinc-sensing protein Zur in directly modulating the expression of a virulence factor in bacteria

Identification of a New Epitope in uPAR as a Target for the Cancer Therapeutic Monoclonal Antibody ATN-658, a Structural Homolog of the uPAR Binding Integrin CD11b (αM)

The urokinase plasminogen activator receptor (uPAR) plays a role in tumor progression and has been proposed as a target for the treatment of cancer. We recently described the development of a novel humanized monoclonal antibody that targets uPAR and has anti-tumor activity in multiple xenograft animal tumor models. This antibody, ATN-658, does not inhibit ligand binding (i.e. uPA and vitronectin) to uPAR and its mechanism of action remains unclear. As a first step in understanding the anti-tumor activity of ATN-658, we set out to identify the epitope on uPAR to which ATN-658 binds. Guided by comparisons between primate and human uPAR, epitope mapping studies were performed using several orthogonal techniques. Systematic site directed and alanine scanning mutagenesis identified the region of aa 268–275 of uPAR as the epitope for ATN-658. No known function has previously been attributed to this epitope Structural insights into epitope recognition were obtained from structural studies of the Fab fragment of ATN-658 bound to uPAR. The structure shows that the ATN-658 binds to the DIII domain of uPAR, close to the C-terminus of the receptor, corroborating the epitope mapping results. Intriguingly, when bound to uPAR, the complementarity determining region (CDR) regions of ATN-658 closely mimic the binding regions of the integrin CD11b (αM), a previously identified uPAR ligand thought to be involved in leukocyte rolling, migration and complement fixation with no known role in tumor progression of solid tumors. These studies reveal a new functional epitope on uPAR involved in tumor progression and demonstrate a previously unrecognized strategy for the therapeutic targeting of uPAR

Co- and multimorbidity patterns in primary care based on episodes of care: results from the German CONTENT project

Contains fulltext : 69171.pdf (publisher's version ) (Open Access)BACKGROUND: Due to technological progress and improvements in medical care and health policy the average age of patients in primary care is continuously growing. In equal measure, an increasing proportion of mostly elderly primary care patients presents with multiple coexisting medical conditions. To properly assess the current situation of co- and multimorbidity, valid scientific data based on an appropriate data structure are indispensable. CONTENT (CONTinuous morbidity registration Epidemiologic NeTwork) is an ambitious project in Germany to establish a system for adequate record keeping and analysis in primary care based on episodes of care. An episode is defined as health problem from its first presentation by a patient to a doctor until the completion of the last encounter for it. The study aims to describe co- and multimorbidity as well as health care utilization based on episodes of care for the study population of the first participating general practices. METHODS: The analyses were based on a total of 39,699 patients in a yearly contact group (YCG) out of 17 general practices in Germany for which data entry based on episodes of care using the International Classification of Primary Care (ICPC) was performed between 1.1.2006 and 31.12.2006. In order to model the relationship between the explanatory variables (age, gender, number of chronic conditions) and the response variables of interest (number of different prescriptions, number of referrals, number of encounters) that were applied to measure health care utilization, we used multiple linear regression. RESULTS: In comparison to gender, patients' age had a manifestly stronger impact on the number of different prescriptions, the number of referrals and number of encounters. In comparison to age (beta = 0.043, p < 0.0001), multimorbidity measured by the number of patients' chronic conditions (beta = 0.51, p < 0.0001) had a manifestly stronger impact the number of encounters for the observation period. Moreover, we could observe that the number of patients' chronic conditions had a significant impact on the number of different prescriptions (beta = 0.226, p < 0.0001) as well as on the number of referrals (beta = 0.3, p < 0.0001). CONCLUSION: Documentation in primary care on the basis of episodes of care facilitates an insight to concurrently existing health problems and related medical procedures. Therefore, the resulting data provide a basis to obtain co- and multimorbidity patterns and corresponding health care utilization issues in order to understand the particular complex needs caused by multimorbidity

НАПРАВЛЕНИЯ СОВЕРШЕНСТВОВАНИЯ ОБРУДОВАНИЯ ДЛЯ ФЛОТАЦИОННОГО ОБОГАЩЕНИЯ ТОНКОДИСПЕРСНыХ МАТЕРИАЛОВ

Проблема и ее связь с научными и практическими задачами. В связи с тем, что в поступающем на обогатительные фабрики сырье содержится до 30% ма-териала крупностью менее 1 мм, роль процесса флотации существенно возрас-тает. Этому способствует и возможность создания достаточно простых замкну-тых водно-шламовых схем, включающих флотацию в качестве основного эле-мента очистки оборотных вод. Многими исследованиями, которые проводились ранее и продолжают выполняться и в настоящее время, установлены направле-ния совершенствования этого достаточно сложного физико-химического про-цесс

Chlorhexidine versus povidone–iodine skin antisepsis before upper limb surgery (CIPHUR) : an international multicentre prospective cohort study

Introduction Surgical site infection (SSI) is the most common and costly complication of surgery. International guidelines recommend topical alcoholic chlorhexidine (CHX) before surgery. However, upper limb surgeons continue to use other antiseptics, citing a lack of applicable evidence, and concerns related to open wounds and tourniquets. This study aimed to evaluate the safety and effectiveness of different topical antiseptics before upper limb surgery. Methods This international multicentre prospective cohort study recruited consecutive adults and children who underwent surgery distal to the shoulder joint. The intervention was use of CHX or povidone–iodine (PVI) antiseptics in either aqueous or alcoholic form. The primary outcome was SSI within 90 days. Mixed-effects time-to-event models were used to estimate the risk (hazard ratio (HR)) of SSI for patients undergoing elective and emergency upper limb surgery. Results A total of 2454 patients were included. The overall risk of SSI was 3.5 per cent. For elective upper limb surgery (1018 patients), alcoholic CHX appeared to be the most effective antiseptic, reducing the risk of SSI by 70 per cent (adjusted HR 0.30, 95 per cent c.i. 0.11 to 0.84), when compared with aqueous PVI. Concerning emergency upper limb surgery (1436 patients), aqueous PVI appeared to be the least effective antiseptic for preventing SSI; however, there was uncertainty in the estimates. No adverse events were reported. Conclusion The findings align with the global evidence base and international guidance, suggesting that alcoholic CHX should be used for skin antisepsis before clean (elective upper limb) surgery. For emergency (contaminated or dirty) upper limb surgery, the findings of this study were unclear and contradict the available evidence, concluding that further research is necessary