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The ribonuclease A superfamily is a vertebrate-specific family of proteins that encompasses eight functional members in humans. The proteins are secreted by diverse innate immune cells, from blood cells to epithelial cells and their levels in our body fluids correlate with infection and inflammation processes. Recent studies ascribe a prominent role to secretory RNases in the extracellular space. Extracellular RNases endowed with immuno-modulatory and antimicrobial properties can participate in a wide variety of host defense tasks, from performing cellular housekeeping to maintaining body fluid sterility. Their expression and secretion are induced in response to a variety of injury stimuli. The secreted proteins can target damaged cells and facilitate their removal from the focus of infection or inflammation. Following tissue damage, RNases can participate in clearing RNA from cellular debris or work as signaling molecules to regulate the host response and contribute to tissue remodeling and repair. We provide here an overall perspective on the current knowledge of human RNases' biological properties and their role in health and disease. The review also includes a brief description of other vertebrate family members and unrelated extracellular RNases that share common mechanisms of action. A better knowledge of RNase mechanism of actions and an understanding of their physiological roles should facilitate the development of novel therapeutics.

Acute infection by Gram-negative pathogens can induce an exacerbated immune response that leads to lethal septic shock syndrome. Bacterial lipopolysaccharide (LPS) is a major pathogen-associated molecular pattern molecule that can initiate massive and lethal immune system stimulation. Therefore, the development of new and effective LPS-neutralizing agents is a top priority. The eosinophil cationic protein (ECP) is an antimicrobial protein secreted in response to infection, with a remarkable affinity for LPS. In the present study, we demonstrate that ECP is able to neutralize bacterial LPS and inhibit tumor necrosis factor-α production in human macrophages. We also characterized ECP neutralizing activity using progressively truncated LPS mutants, and conclude that the polysaccharide moiety and lipid A portions are required for LPS-mediated neutralization. In addition, we mapped the structural determinants required for the ECP-LPS interaction by nuclear magnetic resonance. Our results show that ECP is able to neutralize LPS and therefore opens a new route for developing novel therapeutic agents based on the ECP structural scaffolding., (© 2016 Federation of European Biochemical Societies.)

Human antimicrobial RNases, which belong to the vertebrate RNase A superfamily and are secreted upon infection, display a wide spectrum of antipathogen activities. In this work, we examined the antifungal activity of the eosinophil RNase 3 and the skin-derived RNase 7, two proteins expressed by innate cell types that are directly involved in the host defense against fungal infection. Candida albicans has been selected as a suitable working model for testing RNase activities toward a eukaryotic pathogen. We explored the distinct levels of action of both RNases on yeast by combining cell viability and membrane model assays together with protein labeling and confocal microscopy. Site-directed mutagenesis was applied to ablate either the protein active site or the key anchoring region for cell binding. This is the first integrated study that highlights the RNases' dual mechanism of action. Along with an overall membrane-destabilization process, the RNases could internalize and target cellular RNA. The data support the contribution of the enzymatic activity for the antipathogen action of both antimicrobial proteins, which can be envisaged as suitable templates for the development of novel antifungal drugs. We suggest that both human RNases work as multitasking antimicrobial proteins that provide a first line immune barrier., (© 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Eradication of established biofilm communities of pathogenic Gram-negative species is one of the pending challenges for the development of new antimicrobial agents. In particular, Pseudomonas aeruginosa is one of the main dreaded nosocomial species, with a tendency to form organized microbial communities that offer an enhanced resistance to conventional antibiotics. We describe here an engineered antimicrobial peptide (AMP) which combines bactericidal activity with a high bacterial cell agglutination and lipopolysaccharide (LPS) affinity. The RN3(5-17P22-36) peptide is a 30-mer derived from the eosinophil cationic protein (ECP), a host defense RNase secreted by eosinophils upon infection, with a wide spectrum of antipathogen activity. The protein displays high biofilm eradication activity that is not dependent on its RNase catalytic activity, as evaluated by using an active site-defective mutant. On the other hand, the peptide encompasses both the LPS-binding and aggregation-prone regions from the parental protein, which provide the appropriate structural features for the peptide's attachment to the bacterial exopolysaccharide layer and further improved removal of established biofilms. Moreover, the peptide's high cationicity and amphipathicity promote the cell membrane destabilization action. The results are also compared side by side with other reported AMPs effective against either planktonic and/or biofilm forms of Pseudomonas aeruginosa strain PAO1. The ECP and its derived peptide are unique in combining high bactericidal potency and cell agglutination activity, achieving effective biofilm eradication at a low micromolar range. We conclude that the designed RN3(5-17P22-36) peptide is a promising lead candidate against Gram-negative biofilms., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Pregnancy induced erythroblastopenia is an exceptional pathologic entity. Only isolated cases have been described in English literature. The aim of this study is to analyze the characteristics of this extremely rare disease through the description of a new case of pregnancy induced erythroblastopenia and through the study of the 17 cases reported before.

Human RNase 6 is a cationic secreted protein that belongs to the RNase A superfamily. Its expression is induced in neutrophils and monocytes upon bacterial infection, suggesting a role in host defence. We present here the crystal structure of RNase 6 obtained at 1.72 Å (1 Å=0.1 nm) resolution, which is the first report for the protein 3D structure and thereby setting the basis for functional studies. The structure shows an overall kidney-shaped globular fold shared with the other known family members. Three sulfate anions bound to RNase 6 were found, interacting with residues at the main active site (His(15), His(122) and Gln(14)) and cationic surface-exposed residues (His(36), His(39), Arg(66) and His(67)). Kinetic characterization, together with prediction of protein-nucleotide complexes by molecular dynamics, was applied to analyse the RNase 6 substrate nitrogenous base and phosphate selectivity. Our results reveal that, although RNase 6 is a moderate catalyst in comparison with the pancreatic RNase type, its structure includes lineage-specific features that facilitate its activity towards polymeric nucleotide substrates. In particular, enzyme interactions at the substrate 5' end can provide an endonuclease-type cleavage pattern. Interestingly, the RNase 6 crystal structure revealed a novel secondary active site conformed by the His(36)-His(39) dyad that facilitates the polynucleotide substrate catalysis., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

Human Ribonuclease 6 is a secreted protein belonging to the ribonuclease A (RNaseA) superfamily, a vertebrate specific family suggested to arise with an ancestral host defense role. Tissue distribution analysis revealed its expression in innate cell types, showing abundance in monocytes and neutrophils. Recent evidence of induction of the protein expression by bacterial infection suggested an antipathogen function in vivo. In our laboratory, the antimicrobial properties of the protein have been evaluated against Gram-negative and Gram-positive species and its mechanism of action was characterized using a membrane model. Interestingly, our results indicate that RNase6, as previously reported for RNase3, is able to specifically agglutinate Gram-negative bacteria as a main trait of its antimicrobial activity. Moreover, a side by side comparative analysis with the RN6(1-45) derived peptide highlights that the antimicrobial activity is mostly retained at the protein N-terminus. Further work by site directed mutagenesis and structural analysis has identified two residues involved in the protein antimicrobial action (Trp1 and Ile13) that are essential for the cell agglutination properties. This is the first structure-functional characterization of RNase6 antimicrobial properties, supporting its contribution to the infection focus clearance.

Knowledge on the contribution of protein glycosylation in host defense antimicrobial peptides is still scarce. We have studied here how the post-translational modification pattern modulates the antimicrobial activity of one of the best characterized leukocyte granule proteins. The human eosinophil cationic protein (ECP), an eosinophil specific granule protein secreted during inflammation and infection, can target a wide variety of pathogens. Previous work in human eosinophil extracts identified several ECP native forms and glycosylation heterogeneity was found to contribute to the protein biological properties. In this study we analyze for the first time the antimicrobial activity of the distinct native proteins purified from healthy donor blood. Low and heavy molecular weight forms were tested on Escherichia coli cell cultures and compared with the recombinant non-glycosylated protein. Further analysis on model membranes provided an insight towards an understanding of the protein behavior at the cytoplasmic membrane level. The results highlight the significant reduction in protein toxicity and bacteria agglutination activity for heavy glycosylated fractions. Notwithstanding, the lower glycosylated fraction mostly retains the lipopolysaccharide binding affinity together with the cytoplasmic membrane depolarization and membrane leakage activities. From structural analysis we propose that heavy glycosylation interferes with the protein self-aggregation, hindering the cell agglutination and membrane disruption processes. The results suggest the contribution of post-translational modifications to the antimicrobial role of ECP in host defense., (© 2014 FEBS.)

Flavohemoglobins (FlavoHb) play a key role in bacterial resistance to nitrosative stress and NO signaling modulation. In this study, we cloned, expressed, and characterized the flavoHb from the opportunistic pathogen, Staphylococcus aureus. The higher amino-acid sequence homology is shared with that from Saccharomyces cerevisiae which was therefore used to build a model structure by homology modeling. Interestingly, the high sequence homology with S. cerevisiae did not correlate with the enzymatic and kinetic properties which are much similar to those of Escherichia coli. In vitro and aerobically, we showed that S. aureus and Ralstonia eutropha flavoHbs accept cytochrome c and oxygen as substrates. Based on this feature, we investigated the preferences for both substrates depending on miconazole or econazole addition and found that the inhibitor chemical composition is determinant.

Objectives: We wanted to measure the impact of going from a two-step screening for gestational diabetes mellitus (50g oral glucose tolerance test then 100g OGTT) to a one-step screening 75g OGTT (WHO's recommendations)., Patients and Methods: A prospective study was carried out among patients who consulted between July 1st, 2008 and October 31st, 2009. The screening was performed in the first trimester if risk factors were identified and between 24 and 28 weeks of gestation (WG)., Results: During our period of study of 15 months, 706 pregnant women were included. The prescription of a screening test was performed in 403 women, i.e. 57% of cases. Out of the 403-screened women, 33 had gestational diabetes mellitus (GDM) i.e. a 8.2% prevalence rate. In univariate analysis, the following are considered to be risk factors: age, diabetes family history and macrosomia history in a previous pregnancy. Between 24 and 28 WG, 34.34% of the screening tests were achieved. The 75g OGTT is prescribed in 96.2% of cases as a screening test of GDM but fasting blood glucose is still prescribed in 3.8% of cases. Also, before 12 WG, 75g OGTT represent 64.7% of the prescribed tests., Discussion: Despite the simplification of the GDM screening procedure, our work shows no significant difference of the screening rate and prevalence of GDM between our present study and the first work done in the same service (57% versus 61%, P=0.7 and 8.2% versus 7.7%, P=0.9). The GDM risk factors found are also identical between the two studies: age, type 2 diabetes family history and macrosomia history. Moreover, there is a statistically significant improvement in the screening age (23±6.7 versus 20.9 WG±6.8 in the first half of 2008, P<0.001) and the number of tests ordered during the period between 24 and 28 WG (34.34% versus 23.9% in the first half of 2008, P<0.001). For the GDM screening tests between 24 and 28 WG, 75g OGTT replaced the 50g OGTT (test used in the first study), but fasting blood glucose is prescribed in 3.8% of cases knowing that normal fasting blood glucose alone done between 24 and 28 WG has a poor sensitivity and do not exclude the diagnosis of GDM (Cosson, 2006) [11]. Also, before 12 WG, 75g OGTT represents 64.7% of the prescribed tests. However, there is to date no data to validate load testing before 24 WG., Conclusion: Our comparative study revealed maintenance of low implementation of universal screening of GDM despite the simplification of the protocol. Between 24 and 28 WG, 75g OGTT replaced the 50g OGTT, but fasting blood glucose was still prescribed. A sensitization meeting involving all intervening persons is to be organized with proposals for corrective actions. The final objective is the systematic screening of gestational diabetes mellitus with the use of fasting blood glucose in the first trimester and the 75g OGTT between 24 and 28 weeks of gestation., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

The ribonuclease (RNase) A superfamily lineage includes distant members with antimicrobial properties, suggesting a common ancestral host-defense role. In an effort to identify the minimal requirements for the eosinophil cationic protein (ECP or RNase 3) antimicrobial properties we applied site-directed mutagenesis on its closest family homolog, the eosinophil-derived neurotoxin (EDN or RNase 2). Both eosinophil secretion proteins are involved in human immune defense, and are reported as being among the most rapidly evolving coding sequences in primates. Previous studies in our laboratory defined two regions at the N-terminus involved in the protein antimicrobial action, encompassing residues 8-16 and 34-36. Here, we demonstrate that switching two single residues is enough to provide EDN with ECP antipathogen properties. That is, the EDN double-mutant Q34R/R35W displays enhanced bactericidal activity, particularly towards Gram-negative bacteria, and a significant increase in its affinity towards the bacterial outer membrane lipopolysaccharides. Moreover, we confirmed the direct contribution of residue W35 in lipopolysaccharide binding, membrane interaction and permeabilization processes. Furthermore, additional T13 to I substitution provides EDN with an exposed hydrophobic patch required for protein self-aggregation and triggers bacterial agglutination, thereby increasing the final antimicrobial activity by up to 20-fold. Our results highlight how single selected mutations can reshape the entire protein function. This study provides an example of how structure-guided protein engineering can successfully reproduce an evolution selection process towards the emergence of new physiological roles., (© 2013 FEBS.)

Objective: The aim of this study was to evaluate the antifungal activity of the major fraction of fatty acids methyl esters (FAMEs) isolated from Linum usitatissimum L. seeds oil collected from Bechar department (Algeria)., Methods: The assessment of antifungal activity was carried out in terms of percentage of radial growth on solid medium (potatoes dextrose agar PDA) and biomass growth inhibition on liquid medium (potatoes dextrose broth PDB) against two fungi., Results: The FAMEs was found to be effective in inhibiting the radial mycelial growth of Aspergillus flavus more than Aspergillus ochraceus on all tested concentrations. The highest antifungal index was found to be (54.19%) compared to Aspergillus ochraceus (40.48%). The results of the antifungal activity of the FAMEs inhibition of biomass on liquid medium gave no discounted results, but this does not exclude the antifungal activity., Conclusions: We can assume that the observed antifungal potency may be due to the abundance of linoleic and α-linolenic acids in linseed oil which appears to be promising to treat fungal infections, storage fungi and food spoilage in food industry field.

Vertebrate secreted RNases are small cationic protein endowed with an endoribonuclease activity that belong to the RNase A superfamily and display diverse cytotoxic activities. In an effort to unravel their mechanism of action, we have analysed their nucleotide binding recognition patterns. General shared features with other nucleotide binding proteins were deduced from overall statistics on the available structure complexes at the Protein Data Bank and compared with the particularities of selected representative endoribonuclease families. Results were compared with other endoribonuclease representative families and with the overall protein-nucleotide interaction features. Preferred amino acids and atom types involved in pair bonding interactions were identified, defining the spatial motives for phosphate, base and ribose building blocks. Together with the conserved catalytic triad at the active site, variability was observed for secondary binding subsites that may contribute to the proper substrate alignment and could explain the distinct substrate preference patterns. Highly conserved binding patterns were identified for the pyrimidine and purine subsites at the main and secondary base subsites. Particular substitution could be ascribed to specific adenine or guanine specificities. Distribution of evolutionary conserved residues were compared to search for the structure determinants that underlie their diverse catalytic efficiency and those that may account for putative physiological substrate targets or other non-catalytic biological activities that contribute to the antipathogen role of the RNases involved in the host defence system. A side by side comparison with another endoribonuclease superfamily of secreted cytotoxic proteins, the microbial RNases, was carried on to analyse the common features and peculiarities that rule their substrate recognition. The data provides the structural basis for the development of applied therapies targeting cellular nucleotide polymers., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Protein-glycosaminoglycan interactions are essential in many biological processes and human diseases, yet how their recognition occurs is poorly understood. Eosinophil cationic protein (ECP) is a cytotoxic ribonuclease that interacts with glycosaminoglycans at the cell surface; this promotes the destabilization of the cellular membrane and triggers ECP's toxic activity. To understand this membrane destabilization event and the differences in the toxicity of ECP and its homologues, the high resolution solution structure of the complex between full length folded ECP and a heparin-derived trisaccharide (O-iPr-α-D-GlcNS6S-α(1-4)-L-IdoA2S-α(1-4)-D-GlcNS6S) has been solved by NMR methods and molecular dynamics simulations. The bound protein retains the tertiary structure of the free protein. The (2)S(0) conformation of the IdoA ring is preferably recognized by the protein. We have identified the precise location of the heparin binding site, dissected the specific interactions responsible for molecular recognition, and defined the structural requirements for this interaction. The structure reveals the contribution of Arg7, Gln14, and His15 in helix α1, Gln40 in strand β1, His64 in loop 4, and His128 in strand β6 in the recognition event and corroborates the previously reported participation of residues Arg34-Asn39. The participation of the catalytic triad (His15, Lys38, His128) in recognizing the heparin mimetic reveals, at atomic resolution, the mechanism of heparin's inhibition of ECP's ribonucleolytic activity. We have integrated all the available data to propose a molecular model for the membrane interaction process. The solved NMR complex provides the structural model necessary to design inhibitors to block ECP's toxicity implicated in eosinophil pathologies.

Antimicrobial RNases are small cationic proteins belonging to the vertebrate RNase A superfamily and endowed with a wide range of antipathogen activities. Vertebrate RNases, while sharing the active site architecture, are found to display a variety of noncatalytical biological properties, providing an excellent example of multitask proteins. The antibacterial activity of distant related RNases suggested that the family evolved from an ancestral host-defence function. The review provides a structural insight into antimicrobial RNases, taking as a reference the human RNase 3, also named eosinophil cationic protein (ECP). A particular high binding affinity against bacterial wall structures mediates the protein action. In particular, the interaction with the lipopolysaccharides at the Gram-negative outer membrane correlates with the protein antimicrobial and specific cell agglutinating activity. Although a direct mechanical action at the bacteria wall seems to be sufficient to trigger bacterial death, a potential intracellular target cannot be discarded. Indeed, the cationic clusters at the protein surface may serve both to interact with nucleic acids and cell surface heterosaccharides. Sequence determinants for ECP activity were screened by prediction tools, proteolysis and peptide synthesis. Docking results are complementing the structural analysis to delineate the protein anchoring sites for anionic targets of biological significance.

The human eosinophil cationic protein (ECP), also known as RNase 3, is an eosinophil secretion protein that is involved in innate immunity and displays antipathogen and proinflammatory activities. ECP has a high binding affinity for heterosaccharides, such as bacterial lipopolysaccharides and heparan sulfate found in the glycocalix of eukaryotic cells. We have crystallized ECP in complex with sulfate anions in a new monoclinic crystal form. In this form, the active site groove is exposed, providing an alternative model for ligand binding studies. By exploring the protein-sulfate complex, we have defined the sulfate binding site architecture. Three main sites (S1-S3) are located in the protein active site; S1 and S2 overlap with the phosphate binding sites involved in RNase nucleotide recognition. A new site (S3) that is unique to ECP is one of the key anchoring points for sulfated ligands. Arg 1 and Arg 7 in S3, together with Arg 34 and Arg 36 in S1, form the main basic clusters that assist in the recognition of ligand anionic groups. The location of additional sulfate bound molecules, some of which contribute to the crystal packing, may mimic the binding to extended anionic polymers. In conclusion, the structural data define a binding pattern for the recognition of sulfated molecules that can modulate the role of ECP in innate immunity. The results reveal the structural basis for the high affinity of ECP for glycosaminoglycans and can assist in structure-based drug design of inhibitors of the protein cytotoxicity to host tissues during inflammation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Antimicrobial proteins and peptides (AMPs) are essential effectors of innate immunity, acting as a first line of defense against bacterial infections. Many AMPs exhibit high affinity for cell wall structures such as lipopolysaccharide (LPS), a potent endotoxin able to induce sepsis. Hence, understanding how AMPs can interact with and neutralize LPS endotoxin is of special relevance for human health. Eosinophil cationic protein (ECP) is an eosinophil secreted protein with high activity against both Gram-negative and Gram-positive bacteria. ECP has a remarkable affinity for LPS and a distinctive agglutinating activity. By using a battery of LPS-truncated E. coli mutant strains, we demonstrate that the polysaccharide moiety of LPS is essential for ECP-mediated bacterial agglutination, thereby modulating its antimicrobial action. The mechanism of action of ECP at the bacterial surface is drastically affected by the LPS structure and in particular by its polysaccharide moiety. We have also analyzed an N-terminal fragment that retains the whole protein activity and displays similar cell agglutination behavior. Conversely, a fragment with further minimization of the antimicrobial domain, though retaining the antimicrobial capacity, significantly loses its agglutinating activity, exhibiting a different mechanism of action which is not dependent on the LPS composition. The results highlight the correlation between the protein's antimicrobial activity and its ability to interact with the LPS outer layer and promote bacterial agglutination.

The eosinophil cationic protein (ECP) is a human antimicrobial protein involved in the host immune defense that belongs to the pancreatic RNase A family. ECP displays a wide range of antipathogen activities. The protein is highly cationic and its bactericidal activity is dependant on both cationic and hydrophobic surface exposed residues. Previous studies on ECP by site-directed mutagenesis indicated that the RNase activity is not essential for its bactericidal activity. To further understand the ECP bactericidal mechanism, we have applied enzymatic and chemical limited cleavage to search for active sequence determinants. Following a search for potential peptidases we selected the Lys-endoproteinase, which cleaves the ECP polypeptide at the carboxyl side of its unique Lys residue, releasing the N-terminal fragment (0-38). Chemical digestion using cyanogen bromide released several complementary peptides at the protein N-terminus. Interestingly, ECP treatment with cyanogen bromide represents a new example of selective chemical cleavage at the carboxyl side of not only Met but also Trp residues. Recombinant ECP was denatured and carboxyamidomethylated prior to enzymatic and chemical cleavage. Irreversible denaturation abolishes the protein bactericidal activity. The characterization of the digestion products by both enzymatic and chemical approaches identifies a region at the protein N-terminus, from residues 11 to 35, that retains the bactericidal activity. The most active fragment, ECP(0-38), is further compared to ECP derived synthetic peptides. The region includes previously identified stretches related to lipopolysaccharide binding and bacteria agglutination. The results contribute to define the shortest ECP minimized version that would retain its antimicrobial properties. The data suggest that the antimicrobial RNase can provide a scaffold for the selective release of cytotoxic peptides., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

Eosinophil cationic protein (ECP) is a highly stable, cytotoxic ribonuclease with the ability to enter and disrupt membranes that participates in innate immune defense against parasites but also kills human cells. We have used NMR spectroscopy to characterize the binding of ECP to membrane and heparin mimetics at a residue level. We believe we have identified three Arg-rich surface loops and Trp(35) as crucial for membrane binding. Importantly, we have provided evidence that the interaction surface of ECP with heparin mimetics is extended with respect to that previously described (fragment 34-38). We believe we have identified new sites involved in the interaction for the first time, and shown that the N-terminal alpha-helix, the third loop, and the first and last beta-strands are key for heparin binding. We have also shown that a biologically active ECP N-terminal fragment comprising the first 45 residues (ECP1-45) retains the capacity to bind membrane and heparin mimetics, thus neither the ECP tertiary structure nor its high conformational stability are required for cytotoxicity., (Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The eosinophil cationic protein/RNase 3 and the skin-derived RNase 7 are two human antimicrobial RNases involved in host innate immunity. Both belong to the RNase A superfamily and share a high cationicity and a common structural architecture. However, they present significant divergence at their primary structures, displaying either a high number of Arg or Lys residues, respectively. Previous comparative studies with a membrane model revealed two distinct mechanisms of action for lipid bilayer disruption. We have now compared their bactericidal activity, identifying some features that confer specificity at the bacterial cell wall level. RNase 3 displays a specific Escherichia coli cell agglutination activity, which is not shared by RNase 7. The RNase 3 agglutination process precedes the bacterial death and lysis event. In turn, RNase 7 can trigger the release of bacterial cell content without inducing any cell aggregation process. We hypothesize that the RNase 3 agglutination activity may depend on its high affinity for lipopolysaccharides and the presence of an N-terminal hydrophobic patch, and thus could facilitate host clearance activity at the infection focus by phagocytic cells. The present study suggests that the membrane disruption abilities do not solely explain the protein bacterial target preferences and highlights the key role of antimicrobial action at the bacterial cell wall level. An understanding of the interaction between antimicrobial proteins and their target at the bacterial envelope should aid in the design of alternative peptide-derived antibiotics.

Eosinophil cationic protein (ECP)/human RNase 3, a member of the RNase A family, is a remarkably cytotoxic protein implicated in asthma and allergies. These activities are probably due to ECP's ability to interact with and disrupt membranes and depend on two Trp, 19 Arg, and possibly an extremely high conformational stability. Here, we have used NMR spectroscopy to assign essentially all (1)H, (15)N, and backbone (13)C resonances, to solve the 3D structure in aqueous solution and to quantify its residue-level stability. The NMR solution structure was determined on the basis of 2316 distance constraints and is well-defined (backbone RMSD = 0.81 A). The N-terminus and the loop composed of residues 114-123 are relatively well-ordered; in contrast, conformational diversity is observed for the loop segments 17-22, 65-68, and 92-95 and most exposed sidechains. The side chain NH groups of the two Trp and 19 Arg showed no significant protection against hydrogen/deuterium exchange. The most protected NH groups belong to the first and last two beta-strands, and curiously, the first alpha-helix. Analysis of their exchange rates reveals a strikingly high global stability of 11.8 kcal/mol. This value and other stability measurements are used to better quantify ECP's unfolding thermodynamics.

The eosinophil cationic protein (ECP) is an eosinophil-secreted RNase involved in the immune host defense, with a cytotoxic activity against a wide range of pathogens. The protein displays antimicrobial activity against both Gram-negative and Gram-positive strains. The protein can destabilize lipid bilayers, although the action at the membrane level can only partially account for its bactericidal activity. We have now shown that ECP can bind with high affinity to the bacteria-wall components. We have analyzed its specific association to lipopolysaccharides (LPSs), its lipid A component, and peptidoglycans (PGNs). ECP high-affinity binding capacity to LPSs and lipid A has been analyzed by a fluorescent displacement assay, and the corresponding dissociation constants were calculated using the protein labeled with a fluorophor. The protein also binds in vivo to bacteria cells. Ultrastructural analysis of cell bacteria wall and morphology have been visualized by scanning and transmission electron microscopy in both Escherichia coli and Staphylococcus aureus strains. The protein damages the bacteria surface and induces the cell population aggregation on E. coli cultures. Although both bacteria strain cells retain their shape and no cell lysis is patent, the protein can induce in E. coli the outer membrane detachment. ECP also activates the cytoplasmic membrane depolarization in both strains. Moreover, the depolarization activity on E. coli does not require any pretreatment to overcome the outer membrane barrier. The protein binding to the bacteria-wall surface would represent a first encounter step key in its antimicrobial mechanism of action.

A general acid-base catalytic mechanism is responsible for the cleavage of the phosphodiester bonds of the RNA by ribonuclease A (RNase A). The main active site is formed by the amino acid residues His12, His119, and Lys41, and the process follows an endonucleolytic pattern that depends on the existence of a noncatalytic phosphate-binding subsite adjacent, on the 3'-side, to the active site; in this region the phosphate group of the substrate establishes electrostatic interactions through the side chains of Lys7 and Arg10. We have obtained, by means of site-directed mutagenesis, RNase A variants with His residues both at positions 7 and 10. These mutations have been introduced with the aim of transforming a noncatalytic binding subsite into a putative new catalytic active site. The RNase activity of these variants was determined by the zymogram technique and steady-state kinetic parameters were obtained by spectrophotometric methods. The variants showed a catalytic efficiency in the same order of magnitude as the wild-type enzyme. However, we have demonstrated in these variants important effects on the substrate's cleavage pattern. The quadruple mutant K7H/R10H/H12K/H119Q shows a clear increase of the exonucleolytic activity; in this case the original native active site has been suppressed, and, as consequence, its activity can only be associated to the new active site. In addition, the mutant K7H/R10H, with two putative active sites, also shows an increase in the exonucleolytic preference with respect to the wild type, a fact that may be correlated with the contribution of the new active site.

Eosinophil cationic protein (ECP)/ribonuclease 3 is a member of the RNase A superfamily involved in inflammatory processes mediated by eosinophils. ECP is bactericidal, helminthotoxic, and cytotoxic to tracheal epithelium cells and to several mammalian cell lines although its RNase activity is low. We studied the thermal stability of ECP by fourth-derivative UV absorbance spectra, circular dichroism, differential scanning calorimetry, and Fourier transform infrared spectroscopy. The T (1/2) values obtained with the different techniques were in very good agreement (T (1/2) approximately 72 degrees C), and the stability was maintained in the pH range between 5 and 7. The ECP calorimetric melting curve showed, in addition to the main transition, a pretransitional conformational change with a T (1/2) of 44 degrees C. Both calorimetric transitions disappeared after successive re-heatings, and the ratio DeltaH versus DeltaH (vH) of 2.2 indicated a significant deviation from the two-state model. It was observed that the thermal unfolding was irreversible. The unfolding process gives rise to changes in the environment of aromatic amino acids that are partially maintained in the refolded protein with the loss of secondary structure and the formation of oligomers. From the thermodynamic analysis of ECP variants, the contribution of specific amino acids, such as Trp10 and the region 115-122, to thermal stability was also determined. The high thermal stability of ECP may contribute to its resistance to degradation when the protein is secreted to the extracellular medium during the immune response.

The cleavage pattern of oligocytidylic acid substrates by bovine pancreatic ribonuclease A (RNase A) was studied by means of reversed-phase HPLC. Oligocytidylic acids, ranging from dinucleotides to heptanucleotides, were obtained by RNase A digestion of poly(C). They were identified by MALDI-TOF mass spectrometry; it was confirmed that all of them corresponded to the general structure (Cp)(n)C>p, in which C>p indicates a 2',3'-cyclic phosphate. This is a confirmation of the proposed mechanism for RNase A, wherein the so-called hydrolytic (or second) step is in fact a special case of the reverse of transphosphorylation (first step). The patterns of cleavage for the oligonucleotide substrates show that the native enzyme has no special preference for endonucleolytic or exonucleolytic cleavage, whereas a mutant of the enzyme (K7Q/R10Q-RNase A) lacking p(2) (a phosphate binding subsite adjacent, on the 3' side, to the main phosphate binding site p(1)) shows a clear exonucleolytic pattern; a mutant (K66Q-RNase A) lacking p(0) (a phosphate binding subsite adjacent, on the 5' side, to the main phosphate binding site p(1)) shows a more endonucleolytic pattern. This indicates the important role played by the subsites on the preference for the bond cleaved. Molecular modeling shows that, in the case of the p(2) mutant, the amide group of glutamine can form a hydrogen bond with the 2',3'-cyclic terminal phosphate, whereas the distance to a 3',5'-phosphodiester bond is too long to form such a hydrogen bond. This could explain the preference for exonucleolytic cleavage shown by the p(2) mutant.

Varices of the entire colon are very rare. This rare cause of massive lower gastrointestinal hemorrhage is almost invariably associated with cirrhosis of the liver and consequent hypertension or portal venous obstruction. We report about a patient with massive lower gastrointestinal bleeding from extensive colonic varices. Despite extensive investigation and a follow-up of 3 years, the etiology of the colonic varices could not be determined. Only a few cases of apparent idiopathic (familial or non-familial) colonic varices have been described. Recognition of this abnormality is important, however, because colonic varices may be the cause of recurrent, frequently massive lower gastrointestinal hemorrhage. A misleading endoscopic diagnosis can lead to inappropriate biopsies, resulting in major bleeding.

Pancreatic ribonuclease A is an enzyme that binds up ribonucleic acid (RNA) along the multiple binding subsites that essentially recognize the negatively charged phosphates of the substrate. It is endoribonuclease that catalyse depolimerization of single-stranded RNA. This work gives additional support to the existence of the phosphate-binding site p2 and confirms the central role of Lys-7 in establishing and electrostatic interraction with a phosphate group of the substrate. In this work catalytic properties of recombinant ribonuclease K7H have been studied. This enzyme is a mutant enzyme which contains histidine instead of lysine in a position 7, amino-acid that participates in the main catalytic center of RNase A, named p1. It was obtained by site-directed mutagenesis. Kinetic parameters of K7H have determined with C > p i poli (C) as substrates at pH 5.5 i 7.5. Kinetic parameters of K7H for C > p and as a substrate at pH 5.5 have not altered, but at pH 7.5 were significantly increased. Value Km was also increased, that indicates decreasing of affinity. Increasing of catalysis was double. Results of kinetic parameters of K7H with poli (C) as a substrate in pH 5.5 have shown slight difference according to kinetic parameters of commercial RNase A with poli (C). Significant decreasing of values of all kinetic parameters for K7H were reaction at pH 7.5.

Pancreatic ribonuclease A (RNase A) is a endonuclease that catalyzes depolymerization of ribonucleic acid (RNA) releasing oligonucleotides. In the process of binding enzyme with substrate are involved several non-catalytic phosphate binding subsites, one of them is p2, additional to main catalytic site p1. RNaza A prefers binding and cleavage of longer substrate molecules, and 3',5'-phosphodiester bond should be some six-seven residues apart from the end of molecules of the chain of RNA. In this work is analysed endonuclease activity of recombinant pancreatic RNase A (K7H), that in position seven instead of a lysine there is a histidine, amino acid residue that participates in main catalytic site p1. Mutant enzyme is obtained by site-directed mutagenesis by Kunkel. Results of this investigation have shown that substitution of lysine by histidine in position seven of RNase A has produced total deletion of p2 subsite, and K7H has lost endonuclease activity, and has become exonuclease. These results confirm central role of Lys-7 in establishing p2 subsite and endonuclease activity of pancreatic RNase A.

The reduced diffusion coefficient, D/D0, of fluorescein-labelled globular proteins in the agarose gels Sepharose Cl-2B, 4B and 6B were measured by the FRAP method. Comparison of the partition coefficients of the native and the labelled proteins in the gel showed that the fluorescein residues did not introduce new interactions between the solute and the gel matrix. D/D0 decreased as a function of the Stokes radius. The variation of D/D0 as a function of the partition coefficient of the proteins in the gel did not agree with a previously published prediction. This is in contrast with the diffusion of globular proteins in ACA34 gel, in which the sieving matrix is made of cross-linked polyacrylamide.

Ganglioneuroblastoma a transitional tumor of sympathetic origin has not yet been described as involving orbit. It is characterized by a mixture of cells ranging from primitive neuroblast to well differentiated ganglion cells within a neurofibromatous tissue. The prognosis is uncertain, as the tumor may either undergo maturation into a ganglioneuroma or may metastasize widely and rapidly as in neuroblastoma. We may postulate a relationship between ganglioneuroblastoma and Recklinghausen's neurofibromatosis in view of the development of the tumor in conjunction with the phacomatosis.