NMR Studies on Structure and Dynamics of the Monomeric Derivative of BS-RNase: New Insights for 3D Domain Swapping

Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state, these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able to dislocate both N- and C-termini, but usually this process requires denaturing conditions. In contrast, bovine seminal ribonuclease (BS-RNase), which is a homo-dimeric protein sharing 80% of sequence identity with RNase A, occurs natively as a mixture of swapped and unswapped isoforms. The presence of two disulfides bridging the subunits, indeed, ensures a dimeric structure also to the unswapped molecule. In vitro, the two BS-RNase isoforms interconvert under physiological conditions. Since the tendency to swap is often related to the instability of the monomeric proteins, in these paper we have analysed in detail the stability in solution of the monomeric derivative of BS-RNase (mBS) by a combination of NMR studies and Molecular Dynamics Simulations. The refinement of NMR structure and relaxation data indicate a close similarity with RNase A, without any evidence of aggregation or partial opening. The high compactness of mBS structure is confirmed also by H/D exchange, urea denaturation, and TEMPOL mapping of the protein surface. The present extensive structural and dynamic investigation of (monomeric) mBS did not show any experimental evidence that could explain the known differences in swapping between BS-RNase and RNase A. Hence, we conclude that the swapping in BS-RNase must be influenced by the distinct features of the dimers, suggesting a prominent role for the interchain disulfide bridges

Ciprofloxacin-loaded calcium alginate wafers prepared by freeze-drying technique for potential healing of chronic diabetic foot ulcers

Calcium alginate (CA) wafer dressings were prepared by lyophilization of hydrogels to deliver ciprofloxacin (CIP) directly to the wound site of infected diabetic foot ulcers (DFUs). The dressings were physically characterized by scanning electron microscopy (SEM), texture analysis (for mechanical and in vitro adhesion properties), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). Further, functional properties essential for wound healing, i.e., porosity, in vitro swelling index, water absorption (Aw), equilibrium water content (EWC), water vapor transmission rate (WVTR), evaporative water loss (EWL), moisture content, in vitro drug release and kinetics, antimicrobial activity, and cell viability (MTT assay) were investigated. The wafers were soft, of uniform texture and thickness, and pliable in nature. Wafers showed ideal wound dressing characteristics in terms of fluid handling properties due to high porosity (SEM). XRD confirmed crystalline nature of the dressings and FTIR showed hydrogen bond formation between CA and CIP. The dressings showed initial fast release followed by sustained drug release which can inhibit and prevent re-infection caused by both Gram-positive and Gram-negative bacteria. The dressings also showed biocompatibility (> 85% cell viability over 72 h) with human adult keratinocytes. Therefore, it will be a potential medicated dressing for patients with DFUs infected with drug-resistant bacteria

Les entreprises de pêche artisanale : essais de typologie

[fre] Cet article présente les résultats des tentatives d'élaboration d'une typologie des entreprises de pêche artisanale française. L'originalité de ces essais tient au rôle majeur qui est accordé aux variables économiques et ce en vue d'analyser ces flottilles au regard de données comptables sur un échantillon national puis régional. L'application théorique des ratios économiques et financiers au secteur des pêches artisanales permet d'en dresser un diagnostic plutôt sombre. La sélection d'une zone géographique et l'approche menée en terme d'analyse factorielle des correspondances multiples permettent de croiser les caractéristiques d'exploitation des bateaux avec les critères de rentabilité précédemment évoqués et ce afin d'étudier leurs liens. [eng] This paper presents some attempts for a French artisanal fishing enterprises typology. The originality of these attempts arises from the lead given to economic variables in order to analyze the fishing fleet using accountable datas both at national then regional level. The théorie use of economic and financial ratios (added value, solvency and financial autonomy) leads to the presentation of the artisanal fleet economic situation. The choice of a geographic area and the use of multiple-correspondance-analysis permits a cross analysis of fisheries features and return criteria.

DSC study of the thermal stability of S-protein and S-peptide/S-protein

The thermal denaturation of S-protein is investigated at pH 7.0 by means of DSC measurements. The process is reversible and can be assimilated to a two-state transition. The low values of denaturation temperature and enthalpy, between 38.5 and 40.0 °C and 165 and 180 kJ mol-1, respectively, demonstrate that the loss of S-peptide strongly decreases the structural stability. The interaction between S-peptide and S-protein is thermodynamically characterized, at pH 7.0, by studying the thermal stability of S-peptide/ S-protein complexes at different molar ratios. A two-dimensional nonlinear regression analysis of the excess heat capacity surface as a function of temperature and S-peptide concentration enables us to determine the thermodynamic parameters of binding equilibrium. The values obtained are Kb(38.6 °C) ) (1.10 ( 0.15) × 10 6 M-1, ∆bH(38.6 °C) ) (-185 ( 10) kJ mol-1, and ∆bCp ) (-3.5 ( 0.5) kJ K-1 mol-1. These figures result in satisfactory agreement with literature values

Wound dressings as growth factor delivery platforms for chronic wound healing

Introduction: Years of tissue engineering research have clearly demonstrated the potential of integrating growth factors (GFs) into scaffolds for tissue regeneration, a concept that has recently been applied to wound dressings. The old concept of wound dressings that only take a passive role in wound healing has now been overtaken, and advanced dressings which can take an active part in wound healing, are of current research interest. Areas covered: In this review we will focus on the recent strategies for the delivery of GFs to wound sites with an emphasis on the different approaches used to achieve fine tuning of spatial and temporal concentrations to achieve therapeutic efficacy. Expert opinion: The use of GFs to accelerate wound healing and reduce scar formation is now considered a feasible therapeutic approach in patients with a high risk of infections and complications. The integration of micro–and nanotechnologies into wound dressings could be the key to overcome the inherent instability of GFs and offer adequate control over the release rate. Many investigations have led to encouraging outcomes in various in vitro and in vivo wound models, and it is expected that some of these technologies will satisfy clinical needs and will enter commercialization

Temperature-induced denaturation of ribonuclease S: a thermodynamic study

In this paper the thermal denaturation of ribonuclease S, the product of mild digestion of ribonuclease A by subtilisin, is deeply investigated by means of DSC and CD measurements. It results that at whatever pH in the range 4-7.5 the process is fully reversible but not well represented by the simple two-state N T D transition. Actually, a two-state model that considers both unfolding and dissociation, NL T D + L*, well accounts for the main features of the process: the tail present in the low-temperature side of DSC peaks and the marked dependence of denaturation temperature on protein concentration. This mechanism is strictly linked to the exact stoichiometry of RNase S. An excess of the protein component of RNase S, the so-called S-protein, shifts the system toward a more complex behavior, that deserves a separate treatment in the accompanying paper [Graziano, G., Catanzano, F., Giancola, C., & Barone, G. (1996) Biochemistry 35, 13386-13392]. The thermodynamic analysis leads to the conclusion that the difference in thermal stability between RNase S and RNase A is due to entropic effects, i.e., a greater conformational flexibility of both backbone and side chains in RNase S. The process becomes irreversible at pH 8.0-8.5, probably due to side-reactions occurring at high temperature. Finally, the influence of phosphate ion on the stability of RNase A and RNase S at pH 7.0 is studied and explained in terms of its binding on the active site of ribonucleases. The analysis enables us to obtain an estimate of the apparent association constant and binding enthalpy also

A Reassessment of the Molecular Origin of Cold Denaturation

The existence of cold denaturation is now firmly demonstrated by its direct observation for several globular proteins in aqueous solution. But the physico-chemical explanation of this intriguing phenomenon is still unsatisfactory. In this paper we deepen our understanding of cold denaturation by taking advantage of the theoretical model developed by Ikegami and using thermodynamic data on the transfer to water of liquid N-alkyl amides. The analysis leads to the conclusion that the presence of water is fundamental to determine the existence of cold denaturation due to its strong energetic interaction with the amino acid residues previously buried in the protein's interior