Marc Torrent

Beatriz G De

La Torre

Victoria Nogués

David Andreu

Ester Boix
email: ester.boix@uab.es

; De La Torre

; Nogués

M Victòria

; Andreu

Bactericidal and membrane disruption activities of the eosinophil cationic protein are largely retained in an N-terminal fragment

Keywords: Antimicrobial peptides, host defense, electron microscopy, lipopolysaccharides, liposomes transmission electron microscopy; SEM, scanning electron microscopy

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Host defense proteins and peptides of the innate immune system are potential candidates for chemotherapeutic development. Among those, antimicrobial peptides (AMPs) are characterized by a variety of primary and secondary structures [START_REF] Powers | The relationship between peptide structure and antibacterial activity[END_REF] and by a broad spectra of activity. Their fast, non-specific mechanism of action [START_REF] Lohner | Molecular mechanisms of membrane perturbation by antimicrobial peptides and the use of biophysical studies in the design of novel peptide antibiotics[END_REF], mostly at the plasma membrane and cell surface level, tends to make the development of resistant strains rather unlikely [START_REF] Rivas | Amphibian antimicrobial peptides and Protozoa: Lessons from parasites[END_REF]. On the basis of these features, several native AMPs or their synthetic analogues have been proposed as alternatives to conventional antibiotics; and some of them are currently in clinical trials, mostly for topical applications [4]. Some AMPs are derived from host defense proteins by limited proteolysis that releases in vivo active fragments, often corresponding to the N or C terminus [START_REF] Gudmundsson | Neutrophil antibacterial peptides, multifunctional effector molecules in the mammalian immune system[END_REF]. Proteolytic processing is very frequent in immunological cascade events, where local cleavage can release the active peptides in the area of infection or inflammation. There are also multiple examples of synthetic peptides corresponding to a protein N-or C-terminus that display antimicrobial activity [START_REF] During | The non-enzymatic microbicidal activity of lysozymes[END_REF]. Thus lactoferricin, a naturally occurring bactericidal peptide derived from the N-terminus of lactoferrin, is very effective against some antibiotic resistant strains [START_REF] Nibbering | Human lactoferrin and peptides derived from its N terminus are highly effective against infections with antibiotic-resistant bacteria[END_REF]. Likewise, bactericidal permeability-increasing protein (BPI) is expressed in neutrophils and displays cytotoxicity against Gramnegative bacteria. A recombinant fragment corresponding to the BPI N-terminus is currently under phase III clinical trials [START_REF] Canny | Bactericidal/permeability-increasing protein (BPI) and BPI homologs at mucosal sites[END_REF]. Other examples of AMPs resulting from proteolysis include cathelicidins from neutrophils [START_REF] Zanetti | Cathelicidins: a novel protein family with a common proregion and a variable C-terminal antimicrobial domain[END_REF], and cryptdins, α-defensins secreted by intestinal Paneth cells, whose bactericidal activity requires activation of a precursor by a metalloprotease [10]. We are currently working on eosinophil cationic protein (ECP) as a model of the potential involvement of mammalian RNases in the host defense system. ECP is a secretory ribonuclease (RNase 3) found in the eosinophilic leukocyte and potentially involved in innate immunity. ECP can also be expressed at lower level by activated neutrophils. Although the role of eosinophils during infection is controversial, eosinophils are activated and selectively release their content at the inflammation areas [START_REF] Hogan | Eosinophils: biological properties and role in health and disease[END_REF]. Some authors have reported antimicrobial activities for eosinophils, and proposed a complementary action together with neutrophils during bacterial infections. Besides, the protein local secretion, which can harm the own host tissues, may also participate in immunoregulation and tissue remodeling processes [12,[START_REF] Gomes | Eosinophil-fibroblast interactions induce fibroblast IL-6 secretion and extracellular matrix gene expression: implications in fibrogenesis[END_REF]. Although a wealth of structural and functional data have been reported, the physiological role of ECP remains elusive [14]. Its cytotoxic activity is effective against a wide range of pathogens, suggesting a relatively non-specific mechanism of action. Although there is no evidence of direct in vivo involvement of ECP in the host response to bacterial infection, ECP kills both Gram-negative and Gram-positive strains at a low micromolar range and its activity depends on its action both at the bacterial cell wall and cytoplasmic membrane levels [START_REF] Lehrer | Antibacterial properties of eosinophil major basic protein and eosinophil cationic protein[END_REF][16][17][START_REF] Torrent | Comparison of the membrane interaction mechanism of two antimicrobial RNases: RNase 3/ECP and RNase 7[END_REF]. ECP belongs to the vertebrate RNase A superfamily, several members of which are endowed with antimicrobial properties (Fig. 1S) [19][START_REF] Pizzo | Ribonucleases with angiogenic and bactericidal activities from the Atlantic salmon[END_REF][START_REF] Hooper | Angiogenins: a new class of microbicidal proteins involved in innate immunity[END_REF], a family that may have started off playing a physiological role in the host immune system [START_REF] Cho | Zebrafish ribonucleases are bactericidal: implications for the origin of the vertebrate RNase A superfamily[END_REF][START_REF] Rosenberg | RNase A ribonucleases and host defense: an evolving story[END_REF][START_REF] Pizzo | The success of the RNase scaffold in the advance of biosciences and in evolution[END_REF]. Interestingly, ECP antibacterial activity is not shared by eosinophil derived neurotoxin (EDN), a closely related eosinophil ribonuclease [25]. Antimicrobial RNases, as innate immune proteins with anti-infective and immunomodulatory properties, present substantial therapeutic potential in the drug development industry, both in the search of alternative antibiotics and for the treatment of inflammatory disorders. In order to identify the structural determinants of ECP cytotoxic mechanism of action, we have searched the ECP sequence for potential active domains using a theoretical A c c e p t e d M a n u s c r i p t approach to spot key antimicrobial regions (Torrent et al., unpublished results), and have identified a potentially active region at the N-terminus. To validate our hypothesis, we have synthesized three peptides representative of this region (Fig. 1) and assessed their properties both on a synthetic membrane model and on bacterial cell cultures.

RESULTS

Antimicrobial activity

Sequence analysis of ECP suggests that the N-terminus might contain an antimicrobial domain. Applying a predictive strategy based on a previous peptide library highthroughput screening results [26], a bactericidal propensity value was assigned to each amino acid (Torrent et al., submitted). Scanning on ECP spotted a main potential active region at the N-terminus (residues 30 to 45). To test this hypothesis, the full N-terminal domain, ECP , as well as two smaller representative peptides, ECP(1-19) and ECP , have been chosen for this study (Fig. 1). ECP includes the first ECP α-helix elements (α1, α2 and α3) and the first β1 region. The ECP(24-45) peptide spans the main predicted sequence and includes the Trp35-Arg36 residues previously identified to be determinant for cytotoxicity and membrane lysis activity [16,27,28]. The ECP(1-19) peptide, which corresponds to the first α1 domain, represents the ECP homolog of the S-peptide in RNase A [29]. The bactericidal properties of the three peptides against two representative Gramnegative and -positive strains (E. coli and S. aureus, respectively) have been compared with those of the full ECP sequence. Analysis of the half inhibitory concentration (IC 50 ) and the minimal inhibitory concentration (MIC) values (Table I) shows the 1-45 segment to be practically equivalent to the entire native sequence in terms of antimicrobial activity against both strains. Within the 1-45 segment, activity is shown to be mostly confined to residues 24-45, while the 1-19 peptide requires about 10-20 times higher concentrations to display significant bactericidal activity. As shown in Table 1, ECP and the three tested peptides displayed similar active ranges against both E. coli and S. aureus. The bactericidal effects of the peptides on either strain have been further analyzed to better characterize their distinct properties. ECP can depolarize the cytoplasmic membrane of both E. coli and S. aureus cells [17]. The evaluation of the protein capacity to depolarize the bacteria cytoplasmic cell is an indirect method to assess its ability to interact at the bacteria surface and alter the inner membrane. Previous results on ECP depolarization capacity indicated that its activity is higher in the Gram-negative tested strain, probably as a consequence of the easier access of the protein to the cytoplasmic membrane. In fact, ECP can alter by itself the Gram-negative strain outer membrane structure [17]. We have now compared the protein depolarization capacity with the corresponding N-terminal peptides (Fig. 2). ECP is the most active segment while ECP retains an intermediate activity and ECP [START_REF] Powers | The relationship between peptide structure and antibacterial activity[END_REF][START_REF] Lohner | Molecular mechanisms of membrane perturbation by antimicrobial peptides and the use of biophysical studies in the design of novel peptide antibiotics[END_REF][START_REF] Rivas | Amphibian antimicrobial peptides and Protozoa: Lessons from parasites[END_REF](4)[START_REF] Gudmundsson | Neutrophil antibacterial peptides, multifunctional effector molecules in the mammalian immune system[END_REF][START_REF] During | The non-enzymatic microbicidal activity of lysozymes[END_REF][START_REF] Nibbering | Human lactoferrin and peptides derived from its N terminus are highly effective against infections with antibiotic-resistant bacteria[END_REF][START_REF] Canny | Bactericidal/permeability-increasing protein (BPI) and BPI homologs at mucosal sites[END_REF][START_REF] Zanetti | Cathelicidins: a novel protein family with a common proregion and a variable C-terminal antimicrobial domain[END_REF](10)[START_REF] Hogan | Eosinophils: biological properties and role in health and disease[END_REF](12)[START_REF] Gomes | Eosinophil-fibroblast interactions induce fibroblast IL-6 secretion and extracellular matrix gene expression: implications in fibrogenesis[END_REF](14)[START_REF] Lehrer | Antibacterial properties of eosinophil major basic protein and eosinophil cationic protein[END_REF](16)(17)[START_REF] Torrent | Comparison of the membrane interaction mechanism of two antimicrobial RNases: RNase 3/ECP and RNase 7[END_REF](19) shows the worst performance, as previously observed for the corresponding bactericidal values (Table I). However, significant differences between the two strains are also found. While in E. coli ECP(1-45) and ECP A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. microscopy (TEM). In E. coli, ECP promotes outer membrane detachment, alteration of the overall cell shape and partial loss of cell content [17]. Comparison of the peptides with ECP confirms that both ECP(1-45) and ECP are very active against E. coli (Fig. 3). TEM micrographs include many hypodense damaged cells with vacuolization, local outer membrane detachment and loss of characteristic baton-shaped morphology. In contrast, practically no damaged cells can be seen upon treatment with ECP(1-19). For S. aureus (Fig. 2S), TEM micrographs also show some hypodense cells for ECP and ECP , although the overall damage is comparatively much more reduced for this strain. ECP(1-45) relative damage is similar to ECP, while ECP(24-45) is slightly less active. As above, no significant effect is visible for ECP [START_REF] Powers | The relationship between peptide structure and antibacterial activity[END_REF][START_REF] Lohner | Molecular mechanisms of membrane perturbation by antimicrobial peptides and the use of biophysical studies in the design of novel peptide antibiotics[END_REF][START_REF] Rivas | Amphibian antimicrobial peptides and Protozoa: Lessons from parasites[END_REF](4)[START_REF] Gudmundsson | Neutrophil antibacterial peptides, multifunctional effector molecules in the mammalian immune system[END_REF][START_REF] During | The non-enzymatic microbicidal activity of lysozymes[END_REF][START_REF] Nibbering | Human lactoferrin and peptides derived from its N terminus are highly effective against infections with antibiotic-resistant bacteria[END_REF][START_REF] Canny | Bactericidal/permeability-increasing protein (BPI) and BPI homologs at mucosal sites[END_REF][START_REF] Zanetti | Cathelicidins: a novel protein family with a common proregion and a variable C-terminal antimicrobial domain[END_REF](10)[START_REF] Hogan | Eosinophils: biological properties and role in health and disease[END_REF](12)[START_REF] Gomes | Eosinophil-fibroblast interactions induce fibroblast IL-6 secretion and extracellular matrix gene expression: implications in fibrogenesis[END_REF](14)[START_REF] Lehrer | Antibacterial properties of eosinophil major basic protein and eosinophil cationic protein[END_REF](16)(17)[START_REF] Torrent | Comparison of the membrane interaction mechanism of two antimicrobial RNases: RNase 3/ECP and RNase 7[END_REF](19). Complementary analysis by SEM further illustrated the effect of the peptides on the bacterial surface. For ECP, previous data indicated a high ability to aggregate E.coli cells and to damage their wall envelope [17]. This cell agglutination ability is retained by ECP(1-45) (Fig. 3), while no aggregates are observed upon incubation with the shorter peptides ECP and ECP [START_REF] Powers | The relationship between peptide structure and antibacterial activity[END_REF][START_REF] Lohner | Molecular mechanisms of membrane perturbation by antimicrobial peptides and the use of biophysical studies in the design of novel peptide antibiotics[END_REF][START_REF] Rivas | Amphibian antimicrobial peptides and Protozoa: Lessons from parasites[END_REF](4)[START_REF] Gudmundsson | Neutrophil antibacterial peptides, multifunctional effector molecules in the mammalian immune system[END_REF][START_REF] During | The non-enzymatic microbicidal activity of lysozymes[END_REF][START_REF] Nibbering | Human lactoferrin and peptides derived from its N terminus are highly effective against infections with antibiotic-resistant bacteria[END_REF][START_REF] Canny | Bactericidal/permeability-increasing protein (BPI) and BPI homologs at mucosal sites[END_REF][START_REF] Zanetti | Cathelicidins: a novel protein family with a common proregion and a variable C-terminal antimicrobial domain[END_REF](10)[START_REF] Hogan | Eosinophils: biological properties and role in health and disease[END_REF](12)[START_REF] Gomes | Eosinophil-fibroblast interactions induce fibroblast IL-6 secretion and extracellular matrix gene expression: implications in fibrogenesis[END_REF](14)[START_REF] Lehrer | Antibacterial properties of eosinophil major basic protein and eosinophil cationic protein[END_REF](16)(17)[START_REF] Torrent | Comparison of the membrane interaction mechanism of two antimicrobial RNases: RNase 3/ECP and RNase 7[END_REF](19). On the other hand, significant surface wall damage is patent for cells treated with both ECP(1-45) and ECP , while little effect is registered upon incubation with ECP [START_REF] Powers | The relationship between peptide structure and antibacterial activity[END_REF][START_REF] Lohner | Molecular mechanisms of membrane perturbation by antimicrobial peptides and the use of biophysical studies in the design of novel peptide antibiotics[END_REF][START_REF] Rivas | Amphibian antimicrobial peptides and Protozoa: Lessons from parasites[END_REF](4)[START_REF] Gudmundsson | Neutrophil antibacterial peptides, multifunctional effector molecules in the mammalian immune system[END_REF][START_REF] During | The non-enzymatic microbicidal activity of lysozymes[END_REF][START_REF] Nibbering | Human lactoferrin and peptides derived from its N terminus are highly effective against infections with antibiotic-resistant bacteria[END_REF][START_REF] Canny | Bactericidal/permeability-increasing protein (BPI) and BPI homologs at mucosal sites[END_REF][START_REF] Zanetti | Cathelicidins: a novel protein family with a common proregion and a variable C-terminal antimicrobial domain[END_REF](10)[START_REF] Hogan | Eosinophils: biological properties and role in health and disease[END_REF](12)[START_REF] Gomes | Eosinophil-fibroblast interactions induce fibroblast IL-6 secretion and extracellular matrix gene expression: implications in fibrogenesis[END_REF](14)[START_REF] Lehrer | Antibacterial properties of eosinophil major basic protein and eosinophil cationic protein[END_REF](16)(17)[START_REF] Torrent | Comparison of the membrane interaction mechanism of two antimicrobial RNases: RNase 3/ECP and RNase 7[END_REF](19). SEM micrographs of ECP-treated S. aureus cells did not reveal any aggregate, as already reported [17]. Surface wall damage for S. aureus cells is only mild compared to the treated E. coli cells and some local blebs appear. Cells incubated with ECP(1-45) show nearly the same pattern, while with ECP(24-45) wall damage is quite mild, and undetectable with ECP(1-19) (Fig. 2S). Outer membrane detachment, visualized by electron microscopy, is considered to be related to ECP uncommonly high ability to bind lipopolysaccharides (LPS), the main component of Gram-negative bacterial surfaces, and its antigenic portion, the lipid A [17]. We have now compared the LPS binding capacity of ECP and its N-terminal fragments. A decreasing order of LPS binding abilities, from ECP(1-45) to ECP(1-19) is registered (see Table II and Fig. 3S). The displacement ability of ECP(1-45) is considerably lower than that of ECP, but still high if compared with the positive control polymyxin B (Fig. 3S). On the contrary, ECP(1-19) does not have any significant affinity to LPS. The LPS binding process can also be followed by the analysis of the intrinsic fluorescence spectra (see Table II). The recorded blue shifts suggest that Trp residues are involved in the interaction and that Trp35 might be the one which mostly alters its environment upon binding. On the other hand, while the native protein does not modify its overall structure upon LPS binding, as assessed by CD spectra, we observe significant changes for the peptides, with an increase in their secondary structure content (Fig. 6). The near UV spectra indicate that changes at the aromatic residues local environments take place in the presence of LPS. A careful inspection reveals that while ECP(1-19) does not modify significantly its overall profile, both ECP(1-45) and ECP register an altered profile. In particular, ECP(1-45) shifts drastically the bands in the characteristic ranges for Tyr and Trp, suggesting the involvement of Tyr33 and Trp35 exposed residues in the LPS binding. Interestingly, both ECP(1-45) and ECP(24-45) peptide far UV spectra in the presence of LPS indicate a much more structured conformation upon binding (Fig. 6). Additionally, in order to identify potential interactions between the protein and the Nterminal peptides at the Gram-positive bacteria surface, we have also analyzed the changes at the intrinsic fluorescence spectra in the presence of lipoteichoic acids (LTA). Electrostatic interactions between the polypeptide cationic residues and the negative charged groups of LTA are expected. Results confirmed the binding of LTA and the contribution of Trp residues (Table II). Pronounced shifts are mainly recorded for ECP and ECP (24-45) peptides, with comparable values, while the blue shift

Interaction with lipid bilayers

The correlation between ECP cytotoxic activity and its membrane destabilizing capacity has been previously reported [27,28]. Detailed analysis of ECP action using large and giant unilamellar vesicles as membrane models revealed aggregation and leakage activities at a nanomolar range [16,[START_REF] Torrent | Comparison of the membrane interaction mechanism of two antimicrobial RNases: RNase 3/ECP and RNase 7[END_REF] When the peptide spectra are recorded in the presence of the DOPC/DOPG LUVs, a significant blue shift is observed in all cases, confirming the interaction with the lipid bilayer. Shifts for ECP and ECP are more pronounced than for ECP [START_REF] Powers | The relationship between peptide structure and antibacterial activity[END_REF][START_REF] Lohner | Molecular mechanisms of membrane perturbation by antimicrobial peptides and the use of biophysical studies in the design of novel peptide antibiotics[END_REF][START_REF] Rivas | Amphibian antimicrobial peptides and Protozoa: Lessons from parasites[END_REF](4)[START_REF] Gudmundsson | Neutrophil antibacterial peptides, multifunctional effector molecules in the mammalian immune system[END_REF][START_REF] During | The non-enzymatic microbicidal activity of lysozymes[END_REF][START_REF] Nibbering | Human lactoferrin and peptides derived from its N terminus are highly effective against infections with antibiotic-resistant bacteria[END_REF][START_REF] Canny | Bactericidal/permeability-increasing protein (BPI) and BPI homologs at mucosal sites[END_REF][START_REF] Zanetti | Cathelicidins: a novel protein family with a common proregion and a variable C-terminal antimicrobial domain[END_REF](10)[START_REF] Hogan | Eosinophils: biological properties and role in health and disease[END_REF](12)[START_REF] Gomes | Eosinophil-fibroblast interactions induce fibroblast IL-6 secretion and extracellular matrix gene expression: implications in fibrogenesis[END_REF](14)[START_REF] Lehrer | Antibacterial properties of eosinophil major basic protein and eosinophil cationic protein[END_REF](16)(17)[START_REF] Torrent | Comparison of the membrane interaction mechanism of two antimicrobial RNases: RNase 3/ECP and RNase 7[END_REF](19), suggesting that Trp35 does undergo a more pronounced change on its microenvironment when the peptides associate to the lipid bilayers. In fact, the similarity between spectra of ECP(24-45) and ECP suggests that Trp10 contribution to the final signal is minor. The interaction with lipid bilayers has been further investigated by fluorescence quenching experiments using brominated phospholipids. For that purpose, LUVs made up of DOPC:DOPG with PC dibromo derivates at several positions remote from the head group (6,7; 9,10 and 11,12, respectively) have been prepared. This methodology allows an estimation of the degree of peptide insertion into the lipid bilayers. The quenching efficiency of the differently brominated lipids has been recorded by comparing the respective fluorescence quenching slopes. For all peptides, the presence of either Trp10 or Trp35 has allowed to monitor changes upon membrane interaction. The Stern-Volmer quenching slopes show a steep decrease from the 6,7-to the 11,12dibrominated LUVs, indicating a diminishing interaction between the Trp residues and the bromine-labeled phospholipid (Fig. 5S). From these data, an average insertion of ca. 10 Å (from the bilayer center) can be estimated for the Trp residues of both ECP and ECP(1-45), similar to ECP [16], while a slightly deeper insertion (ca. 8Å) is found for the ECP(1-19) peptide (Fig. 4). Another way to evaluate lipid bilayer destabilization is determining the ability to aggregate lipid vesicles and to trigger the leakage and release of aqueous content. Thus, ECP promotes the release of encapsuled ANTS/DPX at 1/300 protein/lipid ratio and above [16]. In tune with above trends, the leakage ability of ECP(1-45) is similar to ECP, that of ECP(24-45) is slightly lower, while ECP(1-19) requires much higher peptide/lipid ratios to induce significant leakage (Fig. 5.A). Liposome aggregation can also be monitored by the increase of scattering intensity at 470 nm. By this method, ECP has detectable LUV aggregation above 1/1500 protein/lipid molar ratio [16], which is mostly retained by ECP(1-45) but somehow surprisingly lost in the ECP(24-45), while partial aggregating behavior is noted for ECP(1-19) (Fig. 5.B). Finally, we have used circular dichroism (CD) to investigate the conformational changes brought about by peptide-membrane interaction (Fig. 6). The CD spectrum of ECP in aqueous solution has been reported [START_REF] Nikolovski | Thermal unfolding of eosinophil cationic protein/ribonuclease 3: a nonreversible process[END_REF], with the inferred secondary structure values showing some deviation from those derived from X-ray crystallography [START_REF] Boix | Crystal structure of eosinophil cationic protein at 2.4 A resolution[END_REF]. Those differences were mainly attributed to the contribution of aromatic amino acid side chains to the spectral profile. Comparison of the near UV spectra of ECP and the peptides (Fig. 6) reveals significant changes at the aromatic residues local environment for ECP and ECP . We have also analyzed the far UV spectra for ECP and the derived peptides in the absence and presence of SDS (Fig. 6). ECP data indicates that no overall conformation changes have taken place. In contrast, for the three peptides some significant differences were observed. SDS induced mainly the transition to more structured conformations for the three peptides, where a reduction of the β-sheet content and a significant increase in the α-helical content is registered. Thus we can conclude that when exposed to a lipid environment the peptides undergo conformational reorganization, increasing considerably their structured content. Comparison of the α-helix content for each peptide indicates an increase of 25, 35 and 70% for ECP(1-19), ECP(24-45) and ECP(1-45) respectively. This is in tune with the respective abilities to disrupt lipid bilayers (Fig. 5.A). On the other hand, prediction of potential transmembrane segments using the MPEx software identifies a segment from residues 9 to 27 with a high tendency to form α-helix in a lipid environment. This might explain why the peptide with the highest activity on lipid bilayers is ECP(1-45), which includes this segment in its entirety.

DISCUSSION

ECP is an eosinophil-secreted protein involved in inflammation processes with diverse antipathogen properties [14,[START_REF] Venge | Eosinophil cationic protein (ECP): molecular and biological properties and the use of ECP as a marker of eosinophil activation in disease[END_REF]. In previous works we have characterized its bactericidal activity, and identified some structural determinants for its cytotoxic capacity [16][17][START_REF] Torrent | Comparison of the membrane interaction mechanism of two antimicrobial RNases: RNase 3/ECP and RNase 7[END_REF]28]. ECP diverged from the other eosinophil RNase, EDN, under an unusual evolutive pressure and acquired a higher cationicity and cytotoxicity [START_REF] Zhang | Positive Darwinian selection after gene duplication in primate ribonuclease genes[END_REF]. We have now identified an N-terminal region that retains both the bactericidal and membrane-disrupting activities of ECP. The selected 1-45 segment fulfills the common structural-chemical criteria for AMPs [START_REF] Powers | The relationship between peptide structure and antibacterial activity[END_REF][START_REF] Brown | Cationic host defense (antimicrobial) peptides[END_REF]36]. It includes a high number of Arg residues which confer the three synthetic peptides a net positive charge [+2, +5 and +8 for ECP [START_REF] Powers | The relationship between peptide structure and antibacterial activity[END_REF][START_REF] Lohner | Molecular mechanisms of membrane perturbation by antimicrobial peptides and the use of biophysical studies in the design of novel peptide antibiotics[END_REF][START_REF] Rivas | Amphibian antimicrobial peptides and Protozoa: Lessons from parasites[END_REF](4)[START_REF] Gudmundsson | Neutrophil antibacterial peptides, multifunctional effector molecules in the mammalian immune system[END_REF][START_REF] During | The non-enzymatic microbicidal activity of lysozymes[END_REF][START_REF] Nibbering | Human lactoferrin and peptides derived from its N terminus are highly effective against infections with antibiotic-resistant bacteria[END_REF][START_REF] Canny | Bactericidal/permeability-increasing protein (BPI) and BPI homologs at mucosal sites[END_REF][START_REF] Zanetti | Cathelicidins: a novel protein family with a common proregion and a variable C-terminal antimicrobial domain[END_REF](10)[START_REF] Hogan | Eosinophils: biological properties and role in health and disease[END_REF](12)[START_REF] Gomes | Eosinophil-fibroblast interactions induce fibroblast IL-6 secretion and extracellular matrix gene expression: implications in fibrogenesis[END_REF](14)[START_REF] Lehrer | Antibacterial properties of eosinophil major basic protein and eosinophil cationic protein[END_REF](16)(17)[START_REF] Torrent | Comparison of the membrane interaction mechanism of two antimicrobial RNases: RNase 3/ECP and RNase 7[END_REF](19), ECP and ECP , respectively] as well as a high pI (predicted as 12, 11.5 and 11.9). On the other hand, the studied N-terminus includes two Cys residues, which were substituted in the synthetic peptides by Ser to avoid potential formation of intra or inter disulfide bridges. Although the peptides cannot reproduce the full scenario in the context of the disulfide-bonded protein structure, the use of synthetic peptides, with Cys substitutions or deletions, is an useful approach, as reported for other antimicrobial proteins [START_REF] Bulet | Anti-microbial peptides: from invertebrates to vertebrates[END_REF]. In fact, some antimicrobial proteins do retain activity at their denatured state [START_REF] During | The non-enzymatic microbicidal activity of lysozymes[END_REF]. We can also find the report of an antimicrobial RNase A homolog, the salmon RNase, which preserves activity when fully denatured [START_REF] Pizzo | Ribonucleases with angiogenic and bactericidal activities from the Atlantic salmon[END_REF]. Moreover, the characterization of protein derived peptides was also successfully applied to the avian leukocyte RNase A homologues (Fig. 1S), where one of the peptides outside the protein scaffold retained the bactericidal activity [START_REF] Nitto | Evolution and function of leukocyte RNase A ribonucleases of the avian species, Gallus gallus[END_REF]. A cationic cluster at the N-terminus was also identified for the skin derived RNase 7, another human RNase A homolog (Fig. 1S) that contributes to the innate immunity at the skin chemical barrier [START_REF] Huang | The flexible and clustered lysine residues of human ribonuclease 7 are critical for membrane permeability and antimicrobial activity[END_REF][START_REF] Schroder | Antimicrobial skin peptides and proteins[END_REF]. Comparison of the bactericidal and membrane-destabilizing activities of ECP and the three synthetic peptides indicates that the ECP N-terminus retains part of the sequence determinants involved at the different levels of ECP's cytotoxic capacity. The longest 1-45 peptide displays nearly all the bactericidal activity of the whole protein against E. coli and S. aureus strains, and partially retains the LPS binding and depolarization abilities. Additionally, ECP has almost the same membrane interaction capacity than ECP, with nearly equivalent leakage and liposome aggregation activities. For its part, the inner ECP(24-45) peptide can partially but not fully reproduce the bactericidal and membrane destabilization capacities of the intact protein and the N-terminal ECP(1-19) short peptide has almost completely lost its toxicity for bacterial cells. Altogether, the results obtained reveal once again that membrane disruption cannot by itself explain the toxicity of ECP towards bacterial cells. Besides, the observed differences on the activity against Gram-negative and Gram-positive strains also suggest the involvement of strain-specific determinants in the killing events. Electron microscopy illustrates diverse abilities of the peptides at the bacterial wall surface. Similar bactericidal activities do not readily translate to bacteria wall damage, suggesting that cell viability is lost before any cell surface destruction is visualized. Although ECP and its derived peptides are bactericidal on both E. coli and S. aureus, bacterial wall damage is much more pronounced on the Gram-negative strain. ECP shows a specific capacity to destroy the E. coli outer membrane and to agglutinate the cells [17], which is only retained by the full 1-45 fragment. The propensity of ECP and its derived peptides to bind lipid bilayers may also be facilitated by their hydrophobicity and aggregation tendency. We used the Aggrescan software [41] to screen the ECP sequence for the presence of hydrophobic patches that can favour aggregation. One such sequence is found at the ECP N-terminus (residues 8 to 16), and is not present in the other eosinophil RNase (EDN). An N-terminal aggregation domain is also identified in the skin-derived RNase 7, though not in other screened human RNases (results not shown). An exposed hydrophobic patch may promote both protein-protein and also protein-lipid bilayer interactions. The fact that ECP(1-19) partially retains a lipid aggregation ability, while the internal segment ECP does not, also supports the presence of a potential "aggregation-promoting" domain at the ECP N-terminus end. For its part, the other short peptide ECP(24-45) seems to include a key region for bactericidal action, as well as for specific binding to the bacteria wall. Both ECP(1-45) and ECP(24-45) retain part of ECP's affinity for LPS, where Tyr33 and Trp35 residues might participate in the interaction. Recent results on ECP heparin binding affinity indicate that charged and aromatic residues in the 34-38 region are involved in this selective ability [42], providing a key domain for the protein anchoring to the cell surface. Recent work by Döring and coworkers [START_REF] Ulrich | Posttranslational tyrosine nitration of eosinophil granule toxins mediated by eosinophil peroxidase[END_REF] on the Tyr nitration process activated during eosinophil maturation identified in ECP a unique Tyr nitration at residue 33, and suggested that Tyr nitration may modulate ECP aggregation and/or its interaction with other eosinophil proteins.

In view of all above, we submit that the 1-45 sequence of ECP embraces a main protein functional domain. We have proven that the ECP(1-45) peptide reproduces most of ECP's antimicrobial properties. The inner ECP(24-45) sequence retains the key bactericidal region, but loses some of ECP's membrane destabilizing capacities. One can plausibly propose that the first 19 residues are also necessary, either for preserving native-like conformation, or for promoting aggregation or both. While devoid of significant bactericidal action, this 1-19 region retains membrane-destabilizing properties, in tune with data suggesting that it includes an aggregation patch. In any event, ECP-derived peptides can provide a model, as a novel source of AMPs as alternative antibiotics against resistant strains. The ECP E. coli cells agglutinating activity is indeed a selected property in the search of alternative antibiotics, as a mechanism to induce bacteria clearance by the host phagocytic cells [44]. Given the pharmaceutical interest on the development of new LPS binders for treatment of immune disorders, the partial LPS-binding ability at the 24-45 stretch also offers a starting template for the design of new peptide-derived immunomodulator drugs.

MATERIALS AND METHODS

Materials

1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2 dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG); 1-palmitoyl-2-stearoyl-(6,7, 9,10, and 11,12)dibromo-sn glycero-3-phosphocholine (6,7-diBrPC, 9,10-diBrPC, and 11,12-diBrPC) were from Avanti Polar Lipids (Birmingham, AL). 8-aminonaphthalene-1,3,6trisulfonic acid disodium salt (ANTS) and p-xylene -bis -pyridinium bromide (DPX) were from Molecular Probes, Invitrogen (Carlsbad, CA).

Bovine pancreatic ribonuclease A, type XII-A; Polymyxin B sulfate, lipoteichoic acids from S. aureus, and LPS from E. coli serotype 0111:B4 were purchased from Sigma-Aldrich (St. Louis, MO). BODIPY TR cadaverine, BC (5-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4adiaza-s-indacene 3yl) phenoxy)acetyl)amino) pentylamine, hydrochloride;) and 3,3 dipropylthiacarbocyanine (diSC 3 [START_REF] Gudmundsson | Neutrophil antibacterial peptides, multifunctional effector molecules in the mammalian immune system[END_REF]) were purchased from Molecular Probes (Eugene, OR). PD-10 desalting columns with Sephadex G-25 were from Amersham Pharmacia Biotech, Uppsala, Sweden. Escherichia coli BL21DE3, Novagen (Madison, WI) and Staphylococcus aureus 502 A, ATCC (Rockville, MD) strains were used.

Peptides

Peptide ECP(1-19) was obtained from NeoMPS (Strasbourg, France). ECP and ECP(1-45), with Cys residues at positions 23 and 37 replaced by Ser, were prepared by Fmoc solid phase peptide synthesis methods [START_REF] Fields | Solid phase peptide synthesis utilizing 9fluorenylmethoxycarbonyl amino acids[END_REF]. All peptides were purified by HPLC to ca. 95% homogeneity and satisfactorily characterized by MALDI-TOF mass spectrometry. Further experimental details can be found in the Supplementary material file (Figs. S6-S8).

Expression and Purification of ECP

Wild-type ECP was obtained from a human ECP synthetic gene [46]. Protein expression in the E. coli BL21(DE3) strain, folding of the protein from inclusion bodies and the purification steps were carried out as previously described [46].

IC 50 determination

Antimicrobial activity was expressed as the half inhibitory concentration (IC 50 ). IC 50 of each peptide was determined from 2 independent experiments performed in triplicate for each peptide concentration. Peptides were dissolved in Na 2 HPO 4 phosphate buffer, 10 mM, pH 7.5 and serially diluted from 0.2 μM to 10 μM. Bacteria were incubated at 37 ºC overnight in LB and diluted to give approximately 5x10 5 CFU/ml. In each assay peptide solutions were added to each bacteria dilution, incubated for 4 hours and samples were plated on Petri dishes and incubated at 37ºC overnight. The colony forming units (CFU) in each Petri dish was counted, the average and the standard error calculated and represented in a semi -logarithmic plot.

MIC determination

Antimicrobial activity was expressed as the minimal inhibitory concentration (MIC), which is defined as the lowest concentration of peptides that completely inhibits microbial growth. MIC of each peptide was determined from 2 independent experiments performed in triplicate for each peptide concentration. Peptides were dissolved in Na 2 HPO 4 phosphate buffer, 10 mM, pH 7.5 and serially diluted from 0.2 μM to 100 μM. Bacteria were incubated at 37 ºC overnight in LB and diluted to give approximately 5x10 5 CFU/ml. In each assay peptide solutions were added to each bacteria dilution, incubated for 4 hours and samples were plated on Petri dishes and incubated at 37ºC overnight.

Liposome preparation

Large unilamellar vesicles (LUV) of a defined size (about 200 nm) were prepared from a vacuum-drying lipids chloroform solution by extrusion through 800, 400 and 200 nm polycarbonate membranes as previously described [16]. The lipid suspension was frozen and thawed several times prior extrusion. Liposomes containing DOPC/DOPG (3:2 molar ratio) were obtained. A 1 mM stock solution of liposome suspension in 10 mM Tris-HCl, 0.1 M NaCl, pH 7.4 was prepared.

Fluorescence measurements

Tryptophan (Trp) fluorescence emission spectra were recorded using a 280nm excitation wavelength. Slits were set at 2 nm for excitation and 5-10 nm for emission. Emission spectra were recorded from 300 to 400 nm at a scan rate of 60 nm/min, in a 10 x 10 mm cuvette with stirring immediately after sample mixing. Protein and peptide spectra at 0.5 μM in 10 mM Hepes, pH 7.4 buffer were obtained at 25 ºC in the absence and presence of 200 μM liposome suspension, 200 μM LPS (assuming a 90000 g/mol molecular weight) or 200 μM LTA (calculated from a 2200 MW reference value). Fluorescence measurements were performed on a Cary Eclipse Spectrofluorimeter. Spectra in the presence of liposomes were corrected for light scattering, by subtracting the corresponding LUV background. For each condition 3 spectra were averaged. The fluorescence spectra were also calculated as a function of the frequency scale (wave number) and adjusted using a log-normal function as detailed previously [16].

Depth-dependent fluorescence quenching experiments using labelled brominated lipids

Quenching of protein Trp residues by brominated lipids was introduced to analyze the relative location of both Trp10 and Trp35 residues upon membrane interaction. LUV of DOPC/DOPG (3:2 molar ratio) were prepared as previously described [16], containing either (6,7)-, (9,10)-, or (11,12)-Br 2 -PC. Brominated lipids were mixed at increasing percentage from 20% up to 80% with unlabelled DOPC/DOPG vesicles. The emission fluorescence spectra were recorded at 25ºC using the following conditions: 10 mM Tris-HCl, pH 7.4, buffer, 0.5 μM protein concentration and 200 μM liposome concentration, excitation wavelength of 280 nm, excitation and emission slits set to 2 and 10 nm respectively. For each condition 3 spectra were averaged. The quenching efficiency of brominated lipids on protein Trp fluorescence was determined by calculating the area under the fluorescence spectra in range of 320 to 380 nm. Relative fluorescence intensities (F 0 /F) were compared, where F 0 is the fluorescence intensity in the absence

ANTS/DPX liposome leakage assay

The ANTS/DPX liposome leakage fluorescence assay was performed as described previously [16,28]. Briefly, a unique population of large unilamelar vesicles (LUV) of DOPC/DOPG (3:2 molar ratio) lipids was obtained containing 12.5 mM ANTS, 45 mM DPX, 20 mM NaCl, 10 mM Tris-HCl, pH 7.5. The ANTS/DPX liposome suspension was diluted to 30 μM final concentration and was incubated at room temperature in the presence of the ECP or the synthetic peptides. The leakage activity was assayed at different polypeptide concentrations, up to 8 μM, by following the release of the liposome content. Fluorescence was measured using a 386 nm excitation wavelength and 535 nm emission wavelength. Slits were set at 5 nm and 10 nm for excitation and emission respectively. The percentage of leakage (%L) produced by the proteins after 1 h of incubation with the liposomes was calculated with the following equation: (%L)= 100(Fp -F 0 )/(F 100 -F 0 ); where Fp is the final fluorescence intensity after addition of the protein (1 h), F 0 and F 100 are the fluorescence intensities before addition of the protein and after addition of 0.5% Triton X100. For each protein concentration three calculated leakage values were averaged. Leakage could not be quantified for those samples where the assay conditions trigger the LUV precipitation, as LUV samples incubated with high ECP protein concentrations.

LUV liposome aggregation

Aggregation of LUV lipid vesicles was monitored by measuring the scattering intensity with the excitation and emission wavelengths set at 470 nm using a Cary Eclypse spectrofluorimeter and cuvettes with an optical path of 1 cm. Prior to the addition of ECP and peptides, the vesicles were allowed to equilibrate for 15 min at room temperature. The buffer used was 10 mM HEPES, pH 7.4. Final assay conditions were: 200 μM lipid concentration and from 0.04 to 4 μM polypeptide range concentration, in 10 mM HEPES, pH 7.4 buffer. After 30 min of incubation at room temperature, the signal was read at 90º from the excitation beam with slits at 5 and 10 nm.

Circular dichroism spectroscopy

The far-UV circular dichroism (CD) spectra were collected with a JASCO J-715 spectropolarimeter. Spectra were recorded from 250 up to 190 nm, at 1 nm intervals, 1 nm bandwidth, with a scan speed of 10 nm/min, at 25ºC. 0.2 cm pathlength cuvettes were used. Mean-residue ellipticity [θ] (deg cm 2 dmol -1 ) was calculated using the formula:

[ ] ( ) cl MRW 10 θ θ =
where MRW is the mean residue molecular weight of the protein, c is the protein concentration and l is the cell path length. Data of 4 consecutive scans was averaged and a linear smoothing from each 5 consecutive measurements was applied. ECP and peptides spectra in the absence and presence of SDS and LPS were recorded. 4-8 μM ECP or peptide concentrations diluted in phosphate buffer 5 mM, pH 7,5, and 1 mM SDS and 1 mM LPS (assuming a 90000 g/mol molecular weight) concentration were used. Samples were centrifuged for 5 min at 10.000 g before use. For near -UV CD, spectra were recorded from 340 up to 255 nm, at 1 nm intervals, 1 nm bandwidth, with a scan speed of 10 nm/min, at 25ºC and 1 cm pathlength cuvettes were used. Data of 4 consecutive scans was averaged. ECP and peptides spectra in the absence and presence of LPS were recorded. 80-100 μM ECP or peptide concentrations diluted in phosphate buffer 5 mM, pH 7,5, and 1 mM LPS (assuming a 90000 g/mol molecular weight) concentration were used. Percentage of each secondary structure was estimated using the JASCO software, based on the calculation method described by Yang and coworkers [START_REF] Yang | Calculation of protein conformation from circular dichroism[END_REF].

Bioinformatic analysis tools for peptide characterization

Physicochemical properties of peptides (pI and net charge) were predicted using the Swiss-Prot expasy server (http://www.expasy.ch/sprot/). Transmembrane-prediction domain analysis was performed using the Membrane Protein Explorer server (MPEx; http://blanco.biomol.uci.edu/mpex). Sequences with aggregation propensity were predicted using the Aggrescan server [41].

Bacterial cytoplasmic membrane depolarization assay

Membrane depolarization was monitored as previously described [17], using the DiSC3(5) lipophilic dye that changes its fluorescence intensity in response to changes in transmembrane potential. E. coli and S. aureus cells were grown to mid-exponential phase and resuspended in 5 mM Hepes-KOH, 20 mM glucose, and 100 mM KCl at pH 7.2 to an OD 600 of 0.05. DiSC3(5) was added. Changes in the fluorescence due to the alteration of the cytoplasmic membrane potential were continuously monitored at 20 °C by fluorescence emission at 670 nm using an excitation wavelength of 620 nm. When the dye uptake was maximal, as indicated by a stable reduction in the fluorescence because of quenching of the accumulated dye at the membrane, polypeptide samples were added at a final concentration of 4 μM. All conditions were assayed in duplicate. The time necessary to reach a stabilized maximum fluorescence reading was recorded for each condition.

Fluorescent probe displacement assay for lipolysaccharide binding Lipolysaccharide (LPS) binding was assessed using the fluorescent probe BODIPY TR cadaverine (BC) as previously described [17]. BC binds strongly to native LPS, specifically recognizing the Lipid A portion. LPS binding assays were carried out in 5 mM Hepes buffer, pH 7.5. The displacement assay was performed by the addition of 1-2 μl aliquots of a stock solution of ECP and peptides to 1 ml of a continuously stirred mixture of LPS at 10 μg/ml and BC (10 μM). The BC excitation wavelength was 580 nm and emission wavelength was 620 nm. Excitation and emission slits were set at 2.5 nm and 20 nm, respectively. Final values correspond to an average of 4 replicates. Quantitative effective displacement values (ED 50 ) were calculated. The ED 50 was computed at the midpoint of the fluorescent signal versus the protein concentration of the displacement curve by a curve-fitting of the data to the equation

x ma F F F F OF - - = 0 0
where OF is the occupancy factor, F 0 the fluorescence intensity of BC alone, F max is the intensity in the presence of LPS at a saturation concentration and F the intensities of the LPS:BC mixtures at each displacer concentrations. Polymyxin B (PMB) and RNase A were used as positive and negative controls, respectively.

Scanning electron microscopy (SEM)

One-ml cultures of E. coli and S. aureus were grown at 37 °C to mid-exponential phase (OD 600 of 0.4) and incubated with 10 μM of ECP and the derived peptides in phosphate-buffered saline (PBS) at room temperature. Sample aliquots were taken up to 4 h of incubation and prepared for SEM analysis as previously described [17]. The micrographs were viewed at 15 kV accelerating voltage in a Hitachi S-570 scanning electron microscope, and a secondary electron image of cells for topography contrast was collected at several magnifications.

Transmission Electron Microscopy (TEM).

Mid-logarithmic phase E. coli and S. aureus cells (OD 600 ∼ 0.4) were incubated with 10 μM ECP and peptides for 4 h. After treatment, bacterial pellets were prefixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer at pH 7.4 for 2 h at 4 °C and post-fixed in 1% osmium tetroxide buffered in 0.1 M cacodylate at pH 7.4 for 2 h at 4 °C. The samples were dehydrated in acetone (50, 70, 90, 95, and 100%). The cells were immersed in EPON resin, and ultrathin sections were examined in a JEOL JEM 2011 microscope (Jeol Ltd., Tokio, Japan). A c c e p t e d M a n u s c r i p t

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Table II. Maximum position of intrinsec fluorescence spectra of polypeptides in the absence and presence of liposomes, LPS and LTA.

Fluorescent emission spectra recorded from 300 to 400 nm using a 280 nm excitation wavelength. The data were fitted with a log-normal

Biochemical

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Biochemical

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  corresponding to the ECP(1-19) is only minor, suggesting the involvement of Trp35 in the interaction.

  . In the present work, we have characterized the interaction of ECP N-terminal peptides with LUV vesicles made up of DOPC/DOPG phospholipids. The intrinsic fluorescence signals of the peptides were first analyzed. ECP includes two Trp residues, Trp10 and Trp35, both present in the studied ECP peptides, that have been classified into different spectral classes [30]. Comparison of ECP with single Trp mutants indicates that most of the blue shift in the fluorescence spectrum is due to the association of solvent-exposed Trp35 with the lipid bilayer [16]. The intrinsic fluorescence spectra of the peptides (Fig. 4S) indicate that, as expected, both Trp residues are now much more solvent-exposed than in their respective locations within the protein. Of the two, Trp10 is the residue experiencing a more pronounced change, from a buried location in ECP [16] to a more exposed position in ECP(1-19).

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Biochemical

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Biochemical

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Biochemical

  Journal Immediate Publication. Published on 18 May 2009 as manuscript BJ20082330 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082330 A c c e p t e d M a n u s c r i p t of quencher. To calculate the depth of insertion of the protein in the lipid bilayer the distribution analysis (DA) was employed, as previously detailed [16].

Biochemical

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Biochemical

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Figure 1 .

 1 Figure 1. Graphical representation of the 3D structure of ECP drawn with Pymol (DeLano Scientific LLC) and ECP primary sequence and its secondary elements drawn with ESPript. The main chains of regions 1-19, 24-45 and 1-45 are labeled in grey tones and the corresponding primary sequence is boxed.

Figure 2 .

 2 Figure 2. Depolarization activity on E. coli (A) and S. aureus (B) cells determined by the DiSC 3[START_REF] Gudmundsson | Neutrophil antibacterial peptides, multifunctional effector molecules in the mammalian immune system[END_REF] dye assay. Bacterial cells were grown at 37 ºC to mid-exponential phase (OD 600 = 0.4), and resuspended in 5 mM Hepes-KOH, 20 mM glucose, 100 mM KCl, pH 7.2, to an OD 600 of 0.05. DiSC 3[START_REF] Gudmundsson | Neutrophil antibacterial peptides, multifunctional effector molecules in the mammalian immune system[END_REF] was added and when the dye uptake was maximal, polypeptides in 5 mM Hepes-KOH buffer pH 7.2 were added at a final concentration of 4 μM. The increase in fluorescence intensity (FI) registers the dye release upon reduction of the transmembrane potential. ECP (circles), ECP(1-45) (triangles), ECP(24-45) (squares) and ECP(1-19) (rhombus). No activity is registered for S. aureus cells incubated with ECP(1-19).

Figure 3 .

 3 Figure 3. Transmission electron microscopy of E. coli (left panel) and scanning electron microscopy of E. coli (right panel) incubated with 10 μM of ECP and the peptides for 3 h. From up to bottom: control cells, ECP(1-19), ECP(24-45), ECP(1-45) and ECP. Magnification scale is indicated at the bottom of each micrograph.

Figure 4 .

 4 Figure 4. Depth-dependent fluorescence quenching profiles calculated by distribution analysis to visualize changes in the Trp chromophore (F) fluorescence, normalized to its value in the absence of quenching (F 0 ), when the quencher is present at certain depths in the bilayer. h m is the average location depth of the Trp residues, calculated from the bilayer center. Liposomes containing DOPC/DOPG (3:2) were mixed with increasing molar ratios of (6,7)-Br 2 -PC, (9,10)-Br 2 -PC, or (11,12)-Br 2 -PC. Assay conditions were 0.5 μM final protein concentration and 200 μM liposome concentration. ECP(1-19) (closed squares); ECP(24-45) (closed triangles) and ECP(1-45) (closed circles).

Figure 5 .

 5 Figure5. Peptide action on lipid bilayers. A) Leakage activity was monitored by release of ANTS/DPX liposome content. Percentage of leakage (% L) produced by the polypeptides after 1 h incubation with liposomes at room temperature was determined. B) LUV aggregation as a function of polypeptide concentration. LUVs were incubated with of each peptide and aggregation was monitored as an increase of scattering intensity at 470 nm at 90º from the excitation beam after 30 min incubation at room temperature. ECP (empty circles), ECP(1-45) (filled squares), ECP(24-45) (filled triangles) and ECP(1-19) (filled circles).

Figure 6 .

 6 Figure 6. Circular dichroism far UV spectra of ECP (A), ECP(1-19) (B), ECP(24-45) (C), ECP(1-45) (D) in the absence (solid line) and presence of SDS (dotted line) and LPS (dashed line). Four consecutive scans were averaged. Mean-residue ellipticity [θ] (deg cm 2 dmol -1 ) was calculated. Top right insets include the CD near UV spectra in the absence (solid line) and presence (dashed line) of LPS.

Biochemical

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FIGURES Figure 1

 1 FIGURES

Figure 2 Biochemical

 2 Figure 2

Figure 3 Biochemical

 3 Figure 3

Figure 4 .

 4 Figure 4.

Figure 5 Biochemical

 5 Figure 5

  have both intermediate activity and ECP(1-19) is quite inactive, in S. aureus ECP(1-45) is nearly equipotent with ECP, and ECP(1-19) is totally inactive.For a better understanding of the cytotoxic activities of the peptides on bacteria we have analyzed their effects by electron microscopy. Potential damage on both the bacteria wall surface and the cytoplasmic membrane can be visualized by transmission electron

	Biochemical Journal Immediate Publication. Published on 18 May 2009 as manuscript BJ20082330
	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082330

Table I .

 I IC50 and minimal inhibitory concentration (MIC) of ECP and peptides. Bacteria were incubated at 37 ºC overnight in LB and diluted to give approximately 5x10 5 CFU/ml. Final values were determined from 2 independent experiments performed in triplicate for each peptide concentration. Biochemical Journal Immediate Publication. Published on 18 May 2009 as manuscript BJ20082330 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082330

	Polypeptide	Bactericidal activity	Bactericidal activity
		(E. coli)	(S. aureus)
		IC 50 (μM)	MIC (μM)	IC 50 (μM)	MIC (μM)
	ECP	0.045±0.007	0.30-0.50	0.045±0.004	0.30-0.50
	R1R45	0.058±0.009	0.5-0.75	0.060±0.004	0.50-0.75
	T24R45	0.282±0.010	5-10	0.173±0.002	1.00-2.00
	R1R19	2.866±0.053	50-100	3.579±2.452	50-100

  function as previously detailed [16]. Quantitative effective displacement values (ED 50 ) for LPS binding, calculated from Fig. 3S curves, are also included. N.D., no displacement is detected.

	Polypeptide	Control	DOPC:DOPG 3:2		LPS	LTA		LPS binding
		λ max (nm)	λ max (nm)	λ shift (nm)	λ max (nm)	λ shift (nm)	λ max (nm) λ shift (nm)	ED 50 (μM)
	ECP	344	339	5	340	4	340	4	0.080±0.001
	R1R45	352	342	10	344	8	342	10	0.780±0.001
	T24R45	355	341	14	346	9	344	11	1.525±0.004
	R1R19	339	336	3	337	2	337	2	N.D.

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© 2009 The Authors Journal compilation © 2009 Portland Press Limited

THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082330 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

ACKNOWLEDGMENTS

Scanning electron microscopy was performed at the Servei de Microscopia of the Universitat Autònoma de Barcelona (UAB). We thank Francisca Cardoso and Francesc Bohils for their assistance in electron micrography preparation. Fluorescence assays were done at the Laboratori d'Anàlisi i Fotodocumentació, Fac. Biociències, UAB. CD spectra were recorded at the Servei d'Anàlisi Química, Fac. Ciències, UAB. This project was funded by a grant from Ministerio de Educación y Cultura (Grants BMC2003-08485-C02-01 and BFU2006-15543-C02-01) and by the Fundació La Marató de TV3 (TV3-031110). M.T. was the recipient of a predoctoral fellowship from the Generalitat de Catalunya. Work at Universitat Pompeu Fabra was supported by the Spanish Ministries of Health (Fondo de Investigaciones Sanitarias grant PI040885), Education and Science (BIO2005-07592-CO2-02 and PET2006-00139-00), and by Generalitat de Catalunya (SGR00494).


Christophe Beuste

Sylvain Miraux

Véronique J Deschodt-Arsac

Eric Thiaudiere

Jean-Michel Franconi

Philippe H Diolez

Laurent M Arsac

Modular regulation analysis

Keywords: Metabolic control analysis, 31 P-MR Spectroscopy, ATP turnover, hypoxic conditioning, oxidative phosphorylation Biochemical

Introduction

As an effective oxygen sink, mitochondria house the ultimate step of the fate of oxygen pathway from air through the cardiovascular and pulmonary systems. Oxygen is used for substrate oxidation by mitochondria and is therefore involved in the main mechanism responsible for energy supply in moderately exercising skeletal muscle. An intriguing question for physiologists over the last three decades has been the influence of acute hypoxia over muscle contraction in an integrated context. Accordingly, the effect of breathing gas mixtures with varied fractions of inspired oxygen on muscle respiration has been extensively studied. Acute and severe reduction in oxygen delivery typically results in a wide range of metabolic disturbance, e.g. decrease in the steady level of phosphocreatine (PCr) [START_REF] Haseler | Phosphocreatine hydrolysis during submaximal exercise: the effect of FIO2[END_REF] and alteration in other energetic intermediates (ATP, Pi, and ADP) [START_REF] Hogan | Role of O2 in regulating tissue respiration in dog muscle working in situ[END_REF], ultimately culminating in a total loss of contractile function [START_REF] Bylund-Fellenius | Energy metabolism in relation to oxygen partial pressure in human skeletal muscle during exercise[END_REF][START_REF] Idstrom | Energy metabolism in relation to oxygen supply in contracting rat skeletal muscle[END_REF]. A less marked perturbation by acute hypoxia of the bioenergetic system of contraction in skeletal muscle is generally reported in humans as well as in rodents after exposure to hypoxia for several weeks as well as in humans indigenous to high altitudes [START_REF] Allen | Simultaneous 31P MRS of the soleus and gastrocnemius in Sherpas during graded calf muscle exercise[END_REF][START_REF] Matheson | Skeletal muscle metabolism and work capacity: a 31P-NMR study of Andean natives and lowlanders[END_REF]. To date yet, the hypoxia-induced responses of integrative regulatory features of muscle energetics have not been systematically studied.

Recent successful analyses suggest that an improved understanding of the integrative regulation by O 2 could be gained from a modular approach of the energy metabolism of contracting muscle. In such integrative approaches, the energy metabolism of intact contracting muscle (either heart or skeletal muscle) can be conceptually divided into two modules: ATP/PCr-producer and ATP/PCr-consumer [START_REF] Arsac | In vivo modular control analysis of energy metabolism in contracting skeletal muscle[END_REF][START_REF] Calmettes | Modular control analysis of effects of chronic hypoxia on mouse heart[END_REF][START_REF] Diolez | Modular regulation analysis of heart contraction: application to in situ demonstration of a direct mitochondrial activation by calcium in beating heart[END_REF][10]. Modules communicate only via a group of energetic intermediates (ADP, ATP, Pi, Cr and PCr) whose concentrations are connected to each other and to the phosphorylation potential. Since muscle PCr dynamics can be detected non-invasively by 31P-MR spectroscopy with high signal/noise ratio, PCr concentration ([PCr]) is systematically the studied variable in the above experimental works.

The reactivity of each module to PCr is quantified by the elasticity coefficient (or elasticity).

Since the whole set of elasticities determine internal regulation in a metabolic pathway [11][START_REF] Brand | Regulation analysis of energy metabolism[END_REF][START_REF] Brand | Top-down elasticity analysis and its application to energy metabolism in isolated mitochondria and intact cells[END_REF][14][15] as well as in modular systems [7-10, 14, 16, 17], these parameters may be used to objective the regulation of ATP turnover or so-called 'energy demand-supply coupling' [2, A c c e p t e d M a n u s c r i p t 5, 6, 18] in contracting muscle of hypoxic animals. Additionally, once modular elasticities are known, the regulatory role of any effector on the energetic system can be quantified [START_REF] Diolez | Modular regulation analysis of heart contraction: application to in situ demonstration of a direct mitochondrial activation by calcium in beating heart[END_REF][START_REF] Brand | Regulation analysis of energy metabolism[END_REF]. So, the overall regulation analysis would likely make it possible to find out the origin of the less marked perturbation by O 2 of the energetic system of contraction after hypoxic conditioning.

In the present study, 31 P-MR spectroscopy was used to quantify in vivo the elasticity to PCr of ATP/PCr-producer and ATP/PCr-consumer modules in normoxic rats (N) vs. hypoxic conditioned rats (HC) exposed for 6 weeks to 10.5% O 2 . The determination of elasticity values then served for the determination of control exerted by each module on ATP turnover and contraction, as quantified by flux-control coefficients. At last, thanks to the determination of control distribution, the direct effect of O 2 on the producer module was quantified in N and HC rats to uncover regulatory features of acute hypoxia on the energetic system.

Experimental procedures

Animal preparation. Female Wistar rats weighting 280 to 340g were used for these experiments according to the guidelines of the National Research council's Guide for the Care and Use of Laboratory Animals. All animals were housed four to cage in an environmentally controlled facility (12h/12h light-dark cycle, 22°C) and received food and water ad libitum. They were separated in two groups: normoxic (N) vs. chronically hypoxic (CH), each group housed in specific compartments.

In the hypoxic compartment, long-term reduction in the fraction of inspired oxygen (FiO 2 ) to 10.5%, as achieved by mixing air with nitrogen, was used to simulate chronic hypoxia during six weeks. Circulating the gas mixture through a water volume into a mixing chamber preserved humidity. F i O 2 was continuously monitored in the hypoxic compartment with an alarm oxygen analyser (GasAlert Extreme, BW technologies, Calgary, Canada).

For experiments, rats were carefully taken out of their cage then anesthetized in a plexiglass box flushed by air containing 3% isoflurane. Throughout the experiment, anesthesia was Experimental set up. The experimental set up was described elsewhere [START_REF] Arsac | In vivo modular control analysis of energy metabolism in contracting skeletal muscle[END_REF]. Briefly, the anesthetized rat was placed in a supine position into a supraconducting magnet (4.7 Tesla, 47/50 Biospec Avance MR system Bruker, Ettlingen, Germany) so that the right segment of the lower limb was centred inside a 30 mm diameter 1 H Helmotz imaging coil tuned to the proton frequency (200.3MHz). A circular homebuilt transmit-receive 31 P probe (18mm) was placed horizontally under the gastrocnemius and tuned to the phosphorus frequency (81.1MHz). Proton shimming was achieved locally in a 20x20x20 mm 3 voxel by using the water proton resonance, with typical widths at half heights of 30-35Hz.

Before measurements in contracting muscles, a proton MR image was acquired axially by using the 1 H coil to verify muscle group position and to improve field homogeneity.

The plantar flexor muscles group was stimulated electrically (Compex 2 stimulator, Ecublens, Switzerland) directly with transcutaneous electrodes located at the knee and heel levels. For the study of moderate intensity of exercise, the current intensity was set at 6 mA in N rats as well as in CH rats.

A total of thirty 31 P spectra (100 s rectangular pulse, 60° flip angle in the middle of the coil; 64 accumulations 2.8 s recovery time; 3.3kHz bandwidth; 1024 data points) were acquired in 180s blocks throughout the experimental protocol.

Two 31 P-MRS spectra were acquired to assess the level of muscle phosphocreatine at rest. Then, during a warm-up period muscle stimulation was increased progressively and hypoxia was gradually initiated by F i O 2 reduction of inhaled gas (21% to 9% O 2 ). No measurements were taken into account for the subsequent analysis until contraction and spectra reached a steady state. Typically this transitory period lasted about 40 min (13 spectra).

Once the hypoxic (9% O 2 ) steady state was reached and maintained for 15 min, a first switch to air for additional 15 min (5 spectra) followed by a second switch to a lower electrical stimulation (5 additional spectra) provided modulations around the studied steady state of PCr The reaction was started by addition of reduced cytochrome c (50 M). Cox activity was expressed in mol. min -1 .g -1 of protein.

Blood analysis. The hematocrit (Ht) was determined using blood collected from rat heart and centrifuged in micro capillary tube.

Results

Hypoxic conditioned (HC) rats exposed to 10.5% O 2 during 6-weeks had higher hematocrit than control normoxic (N) rats (52±3% vs. 42±3%, P<0.05), thus indicating improved oxygen transport capacity. HC rats had also lower Cox activity (250±12 vs 343±27 mol•min -1 •g -1 , P<0.05) possibly indicating a lower maximal oxidative capacity in the soleus muscle. Taken together, these results indicate that our rats exposed for 6 weeks to 10.5% O 2 were effectively conditioned.

The present elasticity analysis was based on simultaneous recordings of steady levels of contraction and PCr during a steady state exercise modulated by acute hypoxia (9% O 2 ) and slight decrease in electrical stimulation as illustrated in figure 2. Figure 3 provides a comparative overview of averaged changes in PCr and contraction due to modulations by 9%O 2 and lowering electrical stimulation in N rats and HC rats. Solid lines represent the effect of each modulation around the studied steady state called 'reference'. As quantified below by the elasticity analysis, figure 3 shows similar slopes (solid lines) in HC and N rats in response to each modulation. It also worth noting that acute hypoxia (9%O 2 ) induced a lower decrease in both PCr and contraction in HC rats when compared to N rats.

Averaged elasticities of the ATP/PCr-producer and ATP/PCr-consumer module in each population (HC vs. N) are shown in table 1. One main finding of the present study was the absence of difference between elasticities in HC rats and in N rats.

Flux-control coefficients calculated from these experimental elasticities are reported in table 1. A very similar control distribution was quantified in N and HC rats. In the present study where moderate work intensity was stimulated, the control over ATP turnover and contraction was dominantly on the consumer (C c =65%). Yet, since the producer had substantial control over contraction (C p =35%), any external effector that can affect directly the producer activity is expected to alter contraction in some extent. Acute hypoxia (9% O 2 ) was just the effector whose direct effect on producer was quantified here. Since contraction was less inhibited by acute hypoxia in HC rats while the producer, which is the target of acute hypoxia, had similar control over contraction, a lower direct effect of acute hypoxia on A c c e p t e d M a n u s c r i p t producer must be concluded. Accordingly, the quantified direct effect of acute hypoxia on the ATP/PCr-producer module in HC rats was only 70% of the direct effect in N rats (table 1).

Discussion

The present study demonstrates that, in moderately contracting skeletal muscle, internal regulatory features of ATP turnover as determined here by elasticity coefficients in vivo are likely not affected by long-term exposure to 10.5% O 2 . In agreement with previous works e.g.

[6] we additionally observed a lower impact of acute hypoxia (9%O 2 ) on contraction in hypoxic conditioned animals, which can be viewed as a positive adaptation to chronic hypoxia. The application of regulation analysis indicates that in absence of change in internal regulation of ATP turnover, this adaptation in HC rats is fully explained by a lower direct effect of ambient hypoxia on the producer.

There is a growing appreciation of the concept that an improved understanding of complex regulatory processes could be gained from integrative determination of modular elasticity coefficients in intact organs. By conceptually defining heart energetics as a two-module system, ATP/PCr-producer and ATP/PCr-consumer, Diolez and colleagues [START_REF] Diolez | Modular regulation analysis of heart contraction: application to in situ demonstration of a direct mitochondrial activation by calcium in beating heart[END_REF] highlighted the routes taken by regulatory effects of calcium in beating heart. Korzeniewski and colleagues [10] incorporated elasticity coefficients in the Proportional Activation Approach and demonstrated so, that both ATP/PCr-producer and ATP/PCr-consumer modules are directly activated to a similar extent during physiological activation of the heart by adrenaline.

As regards skeletal muscle, Jeneson and colleagues [START_REF] Jeneson | A metabolic control analysis of kinetic controls in ATP free energy metabolism in contracting skeletal muscle[END_REF] and recently Arsac and colleagues [START_REF] Arsac | In vivo modular control analysis of energy metabolism in contracting skeletal muscle[END_REF] proposed theoretical and experimental approaches respectively for the assessment in vivo, of elasticity coefficients of ATP/PCr-producer and ATP/PCr-consumer modules. Although such methods are usually crude, they have been demonstrated to significantly strengthen the field of physiological regulations in skeletal muscle [START_REF] Jeneson | Regulation analysis of contractile ATPase flux in skeletal muscle[END_REF]. Since in the present study, the producer elasticity reached similar values in normoxic rats and hypoxic conditioned rats, we conclude that hypoxic conditioning has no effect on the reactivity of oxidative phosphorylation to energetic intermediates (ADP, ATP, Pi, PCr, Cr and the phosphorylation of tissue respiration is likely unaltered by chronic hypoxia. Although our approach actually did not evaluate variations in all these key regulators but focused on PCr as representative, the degree to which PCr is broken down in our conditions fits well with changes of the energy charge of the cell [START_REF] Hogan | Role of O2 in regulating tissue respiration in dog muscle working in situ[END_REF]18,21,22]. Force output, as a surrogate of ATP turnover, was the other critical variable measured in the present approach. For reliable MCA, one should assume that contractile efficiency is not affected by hypoxia, incidentally fatigue due to prolonged work, or stimulation intensity. In humans cycling at submaximal intensities, no change in efficiency was observed at sea level, acute, and chronic altitude of 4300m [START_REF] Wolfel | Oxygen transport during steady-state submaximal exercise in chronic hypoxia[END_REF]. As well, in electrically stimulated canine gastrocnemius under control, ischemic and hypoxemic conditions, there was clearly no effect of a lower O 2 delivery on efficicency, as indicated by proportional decrease in muscle force output and O 2 uptake [START_REF] Hogan | Effect of gradual reduction in O2 delivery on intracellular homeostasis in contracting skeletal muscle[END_REF]. In steady states prolonged for hours (>2h, personal results) we noted no decline in force output or in PCr concentration, even at the highest (sub-maximal) intensities used in these experiments. We concluded that electrical stimulation intensities generate non-fatiguing conditions and that efficiency therefore is unlikely to change with respect to time in our conditions. At last, an obvious candidate for a change in efficiency due to different stimulation intensities is a shift in the recruitment of oxidative and glycolytic fibers. Our previous T 2 -weighted images (based on 1 H MR imaging) indicated that fibers of the mixed gastrocnemius are recruited at whatever electrical intensity [START_REF] Arsac | In vivo modular control analysis of energy metabolism in contracting skeletal muscle[END_REF]. Furtherrmore, muscle pH during the periods of interest reached 6.97±0.04 in N rats and 6.94±0.07 in HC rats with no value below 6.87, thus indicating that ATP synthesis is fully, or at least to a large extent, oxidative in every experiment.

Although the producer elasticity is a critical parameter that likely reveals kinetic or thermodynamic 'driving functions' for mitochondrial metabolism, the comprehensive understanding of ATP turnover regulation requires paying attention to the reactivity of ATP/PCr consumption as well. In the present study, we obtained similar consumer elasticities in N and HC rats. Therefore, similar p as well as similar c in both populations suggest that The absence of changes in elasticity to the intermediates of ATP production and ATP consumption in hypoxic animals is not documented elsewhere in the literature. Our results in normoxic animals can yet be compared to experiments based on electrically stimulated skeletal muscle of animals in situ. Hogan and colleagues [START_REF] Hogan | Role of O2 in regulating tissue respiration in dog muscle working in situ[END_REF] obtained concomitant recordings of contraction and PCr levels in electrically stimulated canine gastrocnemius in situ at two levels of stimulation and under normal and low O 2 conditions. So, the elasticity of conceptual producer and consumer modules in their normoxic animals can be calculated from concomitant changes in oxygen uptake and [PCr] in response to hypoxemia and electrical stimulation. The calculated values of -1.1 for the producer elasticity and 1.2 for the consumer elasticity are consistent with the present quantification in N rats (table 1). These similarities in elasticity coefficients obtained in intact muscle in previous studies [START_REF] Hogan | Role of O2 in regulating tissue respiration in dog muscle working in situ[END_REF][START_REF] Arsac | In vivo modular control analysis of energy metabolism in contracting skeletal muscle[END_REF] as well as in the present study indicate that, although the proposed experimental methods might be crude, elasticity coefficients can be determined in intact muscle with fine consistency. Therefore, we exclude that HC rats might have altered elasticities undetected by our present approach and point definitively to the absence of alteration in internal regulatory features of ATP turnover and contraction in our hypoxic conditioned rats.

The theory of metabolic control analysis has shown that control over the flux through a given pathway can be distributed over many steps in the pathway and that the degree of control of any given step can be quantified by its flux control coefficient [15]. In the present study, the control over ATP turnover and contraction derived from elasticity values was effectively distributed between the two modules in each population (table 1). The quantification of fluxcontrol coefficients highlights similar C p , amounting to 35% in N and HC rats. The critical consequence for our understanding of regulation by O 2 of muscle energetics is that any direct effect of O 2 on the producer activity will be transmitted to contraction with similar 'strength' (35%) in N and HC rats. Therefore similar control strength over the flux by the producer module in N and HC rats has to be analyzed in concert with the overall effect of 9% O 2 on contraction (so-called global effect in the framework of regulation analysis). This global A c c e p t e d M a n u s c r i p t effect was only -9±6% in HC rats but amounted to -17±14% in N rats (figure 3). Since this difference in global effect cannot be explained by different control by the producer over contraction, it is fully explained by a lower direct effect of 9% O 2 on the producer module, amounting to -31% in HC rats but -44% in N rats (P<0.05). It is therefore concluded that the target of hypoxic conditioning in the integrated energetic system of contraction in vivo is not the sensitivity of ATP/PCr producing processes to energetic intermediates -as reflected by elasticity coefficients -but the sensitivity of these processes to ambient O 2 . This is a critical advantage of regulation analysis to quantify direct effects of external effectors on a metabolic pathway. The 30% lower direct effect of O 2 on mitochondrial ATP/PCr production quantified here in HC rats was obtained after 6 weeks at 10.5% O 2 in initially healthy rats. Different conditioning patterns may generate different strength of adaptation. Similarly, any initial pathology in the cardio-respiratory system of the animal may result in more or less severe change in the direct effect of acute hypoxia. Modular regulation analysis likely has the potential to quantify these particular integrated responses.

Our study was not designed to specifically identify the very mechanisms responsible for the lower sensitivity to ambient O 2 of ATP/PCr production in hypoxic rats. In our conceptual definition of the two-module system, the producer module includes all the steps from O 2 and substrates uptake to phosphorylation. So, the concept of direct effect of O 2 quantified here embodies integrative properties of cellular steps as well as steps involved in the transfer of O 2 from lung to mitochondria. The increase in hematocrit in HC rats (52 vs. Elasticities determine the regulation of ATP turnover in contracting muscle, are uniquely determined by MCA, and may thus be used to uncover the origin of the observed dysfunctions under hypoxia conditions [START_REF] Calmettes | Modular control analysis of effects of chronic hypoxia on mouse heart[END_REF].

In conclusion, the present study provides a quantitative approach of the effects of acute and long-term hypoxia on muscle energetics. In vivo assessments of elasticity coefficients and direct effect of O 2 on ATP production illustrate how internal and external regulation of ATP turnover and contraction can be uncovered. We anticipate that the approach is useful to detect dysfunctions or improved functions at system levels [START_REF] Noble | The future: putting Humpty-Dumpty together again[END_REF] and brings new tools for integrative physiology [START_REF] Strange | The end of "naive reductionism": rise of systems biology or renaissance of physiology?[END_REF].

Elasticity Coefficients

ATP/PCr-producer -1.6 ± 1.4 (20) -1.7 ± 0.8 (17) ATP/PCr -consumer 0.8 ± 0.6 (28) 1.1 ± 0.7 (23) Flux Control Coefficients ATP/PCr -producer 34 ± 23 % (20) 35 ± 17 % (17) ATP/PCr -consumer 66 ± 23 % (20) 65 ± 17 % (17) Direct effect of acute hypoxia on ATP/PCr -Producer -44 ± 23 % (20) * -31 ± 13 % (17) (squares) and HC rats (circles). Therefore, slopes provide a rough illustration of elasticities.

Slopes look similar in N and HC rats, as confirmed by the actual calculation of elasticity coefficients (table 1) thanks to control analysis. Arrows show the more marked effect of acute hypoxia in N rats, studied here by tools of regulation analysis.

Biochemical

  Journal Immediate Publication. Published on 19 Feb 2009 as manuscript BJ20082385 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082385

Biochemical

  Journal Immediate Publication. Published on 19 Feb 2009 as manuscript BJ20082385 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082385 A c c e p t e d M a n u s c r i p t maintained by continuous inhalation of a gas mixture containing 1.5% isoflurane delivered in a facemask.

Biochemical

  Journal Immediate Publication. Published on 19 Feb 2009 as manuscript BJ20082385 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082385 A c c e p t e d M a n u s c r i p t and muscle work rate. The elasticity coefficient of the producer and consumer blocks, fluxcontrol coefficients, and direct effect of O 2 on producer (see below) were determined around this steady state. System variables. The conceptual system of muscle energetics used here is composed of two modules, ATP/PCr-producer and ATP/PCr-consumer (fig 1). The analysis of the system requires the measurements of two variables: the intermediate and the flux through each module; the later is equal to the overall flux through the system under steady state conditions. The representative 'intermediate' in the system was PCr concentration, as assessed by 31 P MR spectroscopy; muscle work rate represented the 'flux' as assessed by plantar flexion force exerted on a pedal [7]. Concentrations of metabolic intermediates were assessed from MR-spectroscopy spectra. MRS spectra were routinely deconvolved into Lorentzian lines (Igor Pro Wavemetrics, Lake Oswego, Oregon, USA). The phosphocreatine, Pi, and -ATP peak areas were calculated and were converted to concentrations assuming [ATP]=8.2 mM. Contraction resulting from muscle electrical stimulation was measured with a homebuilt ergometer consisting in a foot pedal connected to a hydraulic piston. A hydraulic circuit filled with water connected the piston to a force transducer (ref. N° MLT0699, AD Instruments powerLab Systems) that was placed outside the magnet. Changes in pressure induced by pedal stroke were recorded at 200Hz. The magnitude of strokes was computed as a function of time (Igor Pro, Wavemetrics, Oregon, USA) to provide the work rate (contraction) signal. Modular Control Analysis. The experimental setup made it possible to quantify concomitant changes in steady levels of PCr and contraction during modulations around the studied steady state called 'reference'. Following the principles of Metabolic Control Analysis, the modulation by acute hypoxia of steady levels of PCr and contraction made it possible to determine two parameters critical for our understanding of integrative regulation Biochemical Journal Immediate Publication. Published on 19 Feb 2009 as manuscript BJ20082385 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082385 A c c e p t e d M a n u s c r i p t of the modular system by acute hypoxia: (i) the consumer elasticity ( c ) and (ii) the role of oxygen as an external effector of the producer module. The consumer elasticity was determined by relative change in contraction divided by relative change in PCr, induced by acute hypoxia. The direct effect of acute hypoxia on the producer was determined by the relative change in contraction divided by the flux-control coefficient of the producer (see below). The modulation by electrical stimulation allowed the quantification of the producer elasticity ( p ). The producer elasticity was determined by the relative change in contraction divided by the relative change in PCr, induced by lowering electrical stimulation. Summation and connectivity theorems allowed the calculation of flux-control coefficients from the experimental elasticities. The flux-control coefficient of the producer (C p ) was calculated as c /( cp ); the flux control coefficient of the consumer (C c ) equals: p /( pc ). Enzymatic analysis. Once all the non-invasive measurements were performed, the animal was sacrificed by the injection of a lethal dose of pentobarbital. A portion of the rat soleus muscle (about 80 mg) was rapidly excised with scissors and immediately cooled in ice-cold buffer consisting of 25mM Sucrose, 5mM MOPS and 0.2mM EDTA. Whole tissue homogenates were prepared in the same buffer by hand homogenisation using a Polytron (PT 1200E; Kinematica AG) at 4°C. An aliquot of the homogenate (150 l) was used for measuring cytochrome c oxidase (Cox) activity, by following the decrease in absorbance at 550nm in spectrometer (Cary 50 UV-Vis spectrophotometer; Varian Inc.) with chemically reduced horse heart cytochrome c as electron donor. The reaction mixture contained (in mM): 100 KCl, 40 saccharose, 10 KH 2 PO 4 , 5 MgCl 2 /H 2 O, 1 EGTA and 0.1% (w/v) BSA (pH= 7.2).

  Biochemical Journal Immediate Publication. Published on 19 Feb 2009 as manuscript BJ20082385 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082385 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 19 Feb 2009 as manuscript BJ20082385 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082385

Biochemical

  Journal Immediate Publication. Published on 19 Feb 2009 as manuscript BJ20082385 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082385A c c e p t e d M a n u s c r i p t potential) during moderate exercise. In other words, the substrate feedback control[19, 20]

Biochemical

  Journal Immediate Publication. Published on 19 Feb 2009 as manuscript BJ20082385 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082385 A c c e p t e d M a n u s c r i p t internal kinetic functions involved in the regulation of muscle ATP turnover in vivo are essentially not altered in hypoxic conditioned rats, as compared to normoxic rats.

Biochemical

  Journal Immediate Publication. Published on 19 Feb 2009 as manuscript BJ20082385 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082385

  42%) indicates improved O 2 transport capacity; the lower Cox activity might indicate down-regulations in cell metabolism. Yet, these responses are not sufficient to assess in what extent the lower sensitivity of ATP production to O 2 is due to adaptations in systemic O 2 delivery [23] or due to cellular adaptations to chronic hypoxia [25]. A better understanding might come from in vivo assessments of muscle PO 2 as achieved for instance by proton MR spectroscopy of deoxymyoglobin [26]. Unfortunately, due the low sensitivity of magnetic resonance, we are not aware of deoxymyoglobin assessments in small muscles of animals. An interesting relationship to other 31 P MR approaches could be mentioned. As force is supposed to reflect the oxidative ATP rate (called J) in our conditions and assuming Biochemical Journal Immediate Publication. Published on 19 Feb 2009 as manuscript BJ20082385 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082385A c c e p t e d M a n u s c r i p t (reasonably, in this largely aerobic exercise) usual linearity of J to PCr, the location of the hypoxia point in N and HC rats in figure3correspond to a decreased J/ PCr, which would correspond to a reduced rate constant (increased time constant) of PCr kinetics during onset or offset of exercise (recovery from stimulation). Such a decease in PCr recovery rate constant has been noted in a variety of experimental and pathological muscle-hypoxic states, and amounts to about -50% in healthy humans breathing 10% O 2[27]. This value could be compared to -44%, the direct effect of hypoxia on producer assessed here by regulation analysis in N rats. We are not aware of assessment of PCr recovery rate constants in HC animals breathing hypoxic mixtures. At last, it is worth noting that PCr recovery rate (or time) constants say nothing about the elasticity of ATP/PCr production and ATP/PCr consumption.

Biochemical

  Journal Immediate Publication. Published on 19 Feb 2009 as manuscript BJ20082385 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082385 A c c e p t e d M a n u s c r i p t Normoxic rats (N) Hypoxic conditioned rats (HC)

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Typical recordings in a hypoxic conditioned (HC) rat. Recordings were obtained after the transitory period (not shown). Lowest peaks of PCr (front) indicate the period during which the HC rat inhaled 9%O 2 ; correspondingly contraction was low as indicated by pedal strokes due to plantar flexion. Highest peaks of PCr (back) were obtained during the lowelectrical stimulation period; correspondingly contraction was low. The 'reference' steady state is characterized by PCr-peaks of intermediate height and intermediate level of contraction. Elasticities were calculated from averaged pedal strokes and spectra. Pi, inorganic phosphate, NTP, nucleosides triphosphate.

Figure 3 .

 3 Figure 3. Effects of modulations on PCr and contraction. Lines indicate effects of acute hypoxia and lowering electrical stimulation on steady values of PCr and contraction in N rats

Biochemical 1 PCr 2

 12 figure[START_REF] Haseler | Phosphocreatine hydrolysis during submaximal exercise: the effect of FIO2[END_REF]

Table 1 .

 1 Coefficients determined by the control and regulation analyses Values are mean ± standard deviation. The number of experiments is indicated in parentheses. See text for the definition of the calculated coefficients. * P<0.05 Biochemical Journal Immediate Publication. Published on 19 Feb 2009 as manuscript BJ20082385 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082385 A c c e p t e d M a n u s c r i p t

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited