Switching between the alternative structures and functions of a 2-Cys peroxiredoxin, by site-directed mutagenesis.

Members of the typical 2-Cys peroxiredoxin (Prx) subfamily represent an intriguing example of protein moonlighting behavior since this enzyme shifts function: indeed, upon chemical stimuli, such as oxidative stress, Prx undergoes a switch from peroxidase to molecular chaperone, associated to a change in quaternary structure from dimers/decamers to higher-molecular-weight (HMW) species. In order to detail the structural mechanism of this switch at molecular level, we have designed and expressed mutants of peroxiredoxin I from Schistosoma mansoni (SmPrxI) with constitutive HMW assembly and molecular chaperone activity. By a combination of X-ray crystallography, transmission electron microscopy and functional experiments, we defined the structural events responsible for the moonlighting behavior of 2-Cys Prx and we demonstrated that acidification is coupled to local structural variations localized at the active site and a change in oligomerization to HMW forms, similar to those induced by oxidative stress. Moreover, we suggest that the binding site of the unfolded polypeptide is at least in part contributed by the hydrophobic surface exposed by the unfolding of the active site. We also find an inverse correlation between the extent of ring stacking and molecular chaperone activity that is explained assuming that the binding occurs at the extremities of the nanotube, and the longer the nanotube is, the lesser the ratio binding sites/molecular mass is.

Random mutants of a Pleurotus ostreatus laccase as new biocatalysts for industrial effluents bioremediation.

AIMS: To select better performing laccase variants among the 2300 randomly mutated variants of Pleurotus ostreatus POXA1b laccase to develop improved laccase-based biocatalysts. METHODS AND RESULTS: Screening of collections of 2300 randomly mutated variants of POXA1b was performed by assaying activity towards the phenolic substrate 2,6-dimethoxyphenol. Two new variants endowed with higher enzyme activity than the wild-type laccase were characterized, and their ability to decolourize industrial dyes with complex trisazo-, polyazo- and stilbene-type structures, in the absence of mediators, was demonstrated. One of the mutants (2L4A) was also proved to be highly stable at both acidic and alkaline pH values (displaying a half-life of around 1 month at the pH levels of both 5 and 10). CONCLUSIONS: In comparison with the wild-type laccase, the new selected 2L4A mutant shows a significant increase in stability at acidic pH, whilst storing its high stability at alkaline pH. This variant also represents a more versatile enzyme with respect to both the variety of xenobiotics degraded and the operative conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This work represents the first example of improvement of a basidiomycete laccase for industrial effluents bioremediation by directed evolution.

Bio-remediation of colored industrial wastewaters by the white-rot fungi Phanerochaete chrysosporium and Pleurotus ostreatus and their enzymes.

The effect of Phanerochaete chrysosporium and Pleurotus ostreatus whole cells and their ligninolytic enzymes on models of colored industrial wastewaters was evaluated. Models of acid, direct and reactive dye wastewaters from textile industry have been defined on the basis of discharged amounts, economic relevance and representativeness of chemical structures of the contained dyes. Phanerochaete chrysosporium provided an effective decolourization of direct dye wastewater model, reaching about 45% decolourization in only 1 day of treatment, and about 90% decolourization within 7 days, whilst P. ostreatus was able to decolorize and detoxify acid dye wastewater model providing 40% decolourization in only 1 day, and 60% in 7 days. P. ostreatus growth conditions that induce laccase production (up to 130,000 U/l) were identified, and extra-cellular enzyme mixtures, with known laccase isoenzyme composition, were produced and used in wastewater models decolourization. The mixtures decolorized and detoxified the acid dye wastewater model, suggesting laccases as the main agents of wastewater decolourization by P. ostreatus. A laccase mixture was immobilized by entrapment in Cu-alginate beads, and the immobilized enzymes were shown to be effective in batch decolourization, even after 15 stepwise additions of dye for a total exposure of about 1 month.

Limits of DPUI application associated with the number of particles within actinide aerosols.

Dose per unit intake (DPUI) of radionuclides is obtained using International Commission on Radiological Protection (ICRP) models. After inhalation exposure, the first model calculates the fraction of activity deposited within the different regions of the respiratory tract, assuming that the aerosol contains an infinite number of particles. Using default parameters for workers, an exposure to one annual limit of intake (ALI) corresponds to an aerosol of 239PuO2 containing approximately 1 x 10(6) particles. To reach such an exposure, very low particle number might be involved especially for compounds having a high specific activity. This study provides examples of exposures to actinide aerosols for which the number of particles is too low for a standard application of the ICRP model. These examples, which involve physical studies of aerosols collected at the workplace and interpretation of bioassay data, show that the number of particles of the aerosol can be the main limit for the application of DPUI after inhalation exposure.

MEDOR, a didactic tool to support interpretation of bioassay data after internal contamination by actinides.

A didactic software, MEthodes DOsimètriques de REférence (MEDOR), is being developed to provide help in the interpretation of biological data. Its main purpose is to evaluate the pertinence of the application of different models. This paper describes its first version that is focused on inhalation exposure to actinide aerosols. With this tool, sensitivity analysis on different parameters of the ICRP models can be easily done for aerosol deposition, in terms of activity and particle number, actinide biokinetics and doses. The user can analyse different inhalation cases showing either that dose per unit intake cannot be applied if the aerosol contains a low number of particles or that an inhibition of the late pulmonary clearance by particle transport can occur which contributes to a 3-4 fold increase in effective dose as compared with application of default parameters. This underlines the need to estimate systematically the number of deposited particles, as well as to do chest monitoring as long as possible.

Modelling of bioassay data from a Pu wound treated by repeated DTPA perfusions: biokinetics and dosimetric approaches.

The aim of this study is to model plutonium (Pu) excretion from the analysis of a well-documented Pu wound case involving repeated diethylene-triamine-penta-acetic acid (DTPA) perfusions up to 390 d and monitoring up to 3109 d. Three modelling approaches were simultaneously applied involving: (1) release of soluble Pu from the wound, estimated with the ICRP66 dissolution model, (2) systemic behaviour of Pu by using ICRP67 model, but also two new models recently reported and (3) additional 'Pu-DTPA' compartments which transfer Pu directly to urinary compartment from blood, interstitial fluids and liver. The best fit of simulations to biological data was obtained by using the new Leggett's systemic model and assuming the presence of three DTPA compartments. Calculations have shown that DTPA treatments have contributed to a 3-fold reduction of the effective dose. Thus, reduction of doses associated with the DTPA treatments can be estimated by modelling which is useful to improve the efficacy of a DTPA treatment schedule based on a diminution of risk.

Hair dosimetry following neutron irradiation.

Use of hair as a biological dosimeter of neutron exposure was proposed a few years ago. To date, the (32)S(n,p)(32)P reaction in hair with a threshold of 2.5 MeV is the best choice to determine the fast neutron dose using body activation. This information is essential with regards to the heterogeneity of the neutron transfer to the organism. This is a very important parameter for individual dose reconstruction from the surface to the deeper tissues. This evaluation is essential to the adapted management of irradiated victims by specialized medical staff. Comparison exercises between clinical biochemistry laboratories from French sites (the CEA and COGEMA) and from the IRSN were carried out to validate the measurement of (32)P activity in hair and to improve the techniques used to perform this examination. Hair was placed on a phantom and was irradiated at different doses in the SILENE reactor (Valduc, France). Different parameters were tested: variation of hair type, minimum weight of hair sample, hair wash before measurement, delivery period of results, and different irradiation configurations. The results obtained in these comparison exercises by the different laboratories showed an excellent correlation. This allowed the assessment of a dose-activity relationship and confirmed the feasibility and the interest of (32)P measurement in hair following fast neutron irradiation.

Stapes surgery in relation to age.

This prospective study aims to evaluate the efficacy of stapedotomy in relation to age. Eighty-four ears of 82 consecutive patients who underwent stapedotomy were studied. Patients were divided into five groups according to their age. In each patient, we evaluated the pre- and postoperative auditory thresholds, according to the Committee on Hearing and Equilibrium of the American Academy of Otolaryngology - Head and Neck Surgery guidelines. Statistically significant (P < 0.05) differences between the pre- and postoperative air conduction thresholds were observed in all groups. Statistically significant reductions of air-bone gap were observed at lower-medium frequencies (250, 500 and 1000 Hz) in the elderly as well as in the younger patients. We did not find a higher susceptibility of the inner ear to surgical trauma in the elderly in comparison to the younger patients. Our data show that stapedotomy results in older adults are comparable to those obtained in the younger, without an increased incidence of complications.

Unilateral endolymphatic hydrops: what about the contralateral ear?

INTRODUCTION: The early recognition of incipient Meniere's disease (MD) in the asymptomatic ear of a patient with known unilateral disease has profound implications for patient management and follow-up, but the criteria for a right and precocious diagnosis is still controversial. MATERIALS AND METHODS: We evaluated forty-nine patients with MD, selected according to Committee on Hearing and Equilibrium guidelines. All patients underwent laminar tonal audiometry, stapedial reflex study, Glycerol dehydration test, Auditory Brainstem Response (ABR) vestibular examination. MRI was performed in 14 patients. RESULTS: A raised hearing threshold in the contralateral ear was found in 27 patients, but only 7 (14.3%) fulfilled the requirements to be considered affected by bilateral MD. The average delay of occurrence in the contralateral ear was 7 years (from 5 to 12 years). The glycerol test was positive only in 4 patients with unilateral MD and moderate hearing loss. It was not positive in any case of bilateral MD. The membranous endolymphatic duct and sac is not well visualised with MRI on the affected side in the majority of patients. CONCLUSION: A MRI study must be included in the diagnostic protocol for MD and with improvements in this imaging modality will possibly allow detection of variations in the size of inner ear structures. Glycerol dehydration test was useful only in selected cases. A full assessment of incipient disease in the asymptomatic ear in unilateral Meniere's disease should be undertaken. A conservative approach in surgical treatment of unilateral MD is recommended because of the possibility of evolution of a bilateral form, which can occur even 10 years after the onset of the disease.

Control of heme reactivity by diffusion: structural basis and functional characterization in hemoglobin mutants.

The effect of mutagenesis on O(2), CO, and NO binding to mutants of human hemoglobin, designed to modify some features of the reactivity that hinder use of hemoglobin solutions as blood substitute, has been extensively investigated. The kinetics may be interpreted in the framework of the Monod-Wyman-Changeux two-state allosteric model, based on the high-resolution crystallographic structures of the mutants and taking into account the control of heme reactivity by the distal side mutations. The mutations involve residues at topological position B10 and E7, i.e., Leu (B10) to Tyr and His (E7) to Gln, on either the alpha chains alone (yielding the hybrid tetramer Hbalpha(YQ)), the beta chains alone (hybrid tetramer Hbbeta(YQ)), or both types of chains (Hb(YQ)). Our data indicate that the two mutations affect ligand diffusion into the pocket, leading to proteins with low affinity for O(2) and CO, and especially with reduced reactivity toward NO, a difficult goal to achieve. The observed kinetic heterogeneity between the alpha(YQ) and beta(YQ) chains in Hb(YQ) has been rationalized on the basis of the three-dimensional structure of the active site. Furthermore, we report for the first time an experiment of partial CO binding, selective for the beta chains, to high salt crystals of the mutant Hb(YQ) in the T-state; these crystallographic data may be interpreted as "snapshots" of the initial events possibly occurring on ligand binding to the T-allosteric state of this peculiar mutant Hb.

Crystallization and preliminary X-ray diffraction studies of a monooxygenase from Streptomyces coelicolor A3(2) involved in the biosynthesis of the polyketide actinorhodin.

The aromatic monooxygenase ActVA-Orf6 from Streptomyces coelicolor A3(2) that catalyses an unusual oxidation on the actinorhodin biosynthetic pathway has been crystallized. The crystals diffract to 1.73 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 46.95, b = 59.29, c = 71.67 A. Solvent-content (44%) and self-rotation function calculations predict the presence of two molecules in the asymmetric unit. Structure determination should provide further insight into the enzyme mechanism and aid in the design of biosynthetic pathways to produce new polyketide natural products with novel functionality.

Modulation of ligand binding in engineered human hemoglobin distal pocket.

Functional and structural studies on hemoglobin and myoglobin from different animals and engineered variants have enlightened the great importance of the physico-chemical properties of the side-chains at topological position B10 and E7. These residues proved to be crucial to the discrimination and stabilisation of gaseous ligands. In view of the data obtained on the high oxygen affinity hemoglobin from Ascaris worms and a new mutant of sperm whale myoglobin, we selected the two mutations Leu B10-->Tyr and His E7-->Gln as potentially relevant to control ligand binding parameters in the alpha and beta-chains of human hemoglobin. Here, we present an investigation of three new mutants: HbalphaYQ (alpha2YQbeta2A), HbbetaYQ (alpha2Abeta2YQ) and HbalphabetaYQ (alpha2YQbeta2YQ). They are characterised by a very low reactivity for NO, O2 and CO, and a reduced cooperativity. Their functional properties are not inconsistent with the behaviour expected for a two-state allosteric model. Proteins with these substitutions may be considered as candidates for the synthesis of a possible "blood substitute", which should yield an O2 adduct stable to autoxidation and slowly reacting with NO. The mutant HbalphabetaYQ is particularly interesting because the rate of reaction of NO with the oxy and deoxy derivatives is reduced. A structural interpretation of our data is presented based on the 3D structure of deoxy HbalphabetaYQ determined by crystallography at 1.8 A resolution.

Reticulocytes and reticulated platelets: simultaneous measurement in whole blood by flow cytometry.

Reticulated platelets are a fraction of newly released circulating elements characterized by a residual amount of RNA. It has been suggested that the reticulated platelet count, providing an estimate of thrombopoiesis in the same way as erythrocyte reticulocyte count is a measure of erythropoiesis, may be useful in the study of thrombocytopenic disorders. Reticulated red cells and platelets can be analyzed by flow cytometry using specific stains for nucleic acids such as Thiazole Orange and Auramine-O. The aim of our work was to perform the simultaneous evaluation of reticulated elements in whole blood using a standard flow cytometer and to correlate the results obtained with a dedicated cytometer. A group of 14 patients with abnormal absolute reticulocyte counts (range 1.1-11%) and a group of 41 patients showing a platelet discrimination error when analyzed with a dedicated flow cytometer (Sysmex R1000) were enrolled. Linear amplification of both scatter and fluorescence was used to perform reticulocyte count. A gate was set on platelet dimensions, and logarithmic amplification of scatter and fluorescence was used to count reticulated platelets. A good correlation was obtained both for results of reticulocyte count (r2 = 0.9825) and for reticulated platelets (r2 = 0.8717) between our method and those using dedicated instruments. These data show that reticulated platelet count may be easily introduced in clinical laboratories that routinely perform reticulocyte count by flow cytometry.

Free energy of burying hydrophobic residues in the interface between protein subunits.

We have obtained an experimental estimate of the free energy change associated with variations at the interface between protein subunits, a subject that has raised considerable interest since the concept of accessible surface area was introduced by Lee and Richards [Lee, B. & Richards, F. M. (1971) J. Mol. Biol. 55, 379-400]. We determined by analytical ultracentrifugation the dimer-tetramer equilibrium constant of five single and three double mutants of human Hb. One mutation is at the stationary alpha1 beta1 interface, and all of the others are at the sliding alpha1 beta2 interface where cleavage of the tetramer into dimers and ligand-linked allosteric changes are known to occur. A surprisingly good linear correlation between the change in the free energy of association of the mutants and the change in buried hydrophobic surface area was obtained, after corrections for the energetic cost of losing steric complementarity at the alphabeta dimer interface. The slope yields an interface stabilization free energy of -15 +/- 1.2 cal/mol upon burial of 1 A2 of hydrophobic surface, in very good agreement with the theoretical estimate given by Eisenberg and McLachlan [Eisenberg, D. & McLachlan, A. D. (1986) Nature (London) 319, 199-203].

Genotypic characterization of a nosocomial outbreak of VanA Enterococcus faecalis.

Despite growing concern about vancomycin-resistant enterococci (VRE) as nosocomial pathogens, especially in the United States, in Italy VRE still represent an uncommon and occasional experience for most diagnostic laboratories. We report a genotypic characterization of the first reported nosocomial outbreak of VRE in Italy. Some experiments, including plasmid analysis and pulsed-field gel electrophoresis (PFGE) assays, aimed at investigating the genetic relatedness of the VRE isolates. Other experiments, based on hybridization and polymerase chain reaction (PCR) assays, aimed at characterizing the vancomycin resistance determinants. Over a 6-month period, 21 VRE, all identified as Enterococcus faecalis, were isolated from eight patients (all treated earlier with glycopeptide antibiotics) in a neurosurgical intensive care unit. All isolates had the same biochemical profile and antibiotic susceptibility pattern, including high-level resistance to aminoglycosides and vancomycin and teicoplanin MICs of 256 and 128 micrograms/ml, respectively. Three plasmids, one strongly hybridizing with a vanA probe, were detected in all but the last of the 21 VRE isolates. The last isolate of the cluster lacked the smallest of the three plasmids. Similar restriction profiles were obtained after plasmid DNA digestion with several endonucleases, with minor differences appreciated only in the first and last isolates. Analysis of genomic DNA restriction fragment patterns by PFGE confirmed that the reported cluster of VRE isolations was due to a single nosocomial strain of E. faecalis, despite some modifications in plasmid DNA at the beginning and at the end of the outbreak. Completely different PFGE patterns were yielded by vancomycin-susceptible E. faecalis strains isolated during the same period from inpatients in the same intensive care unit. Hybridization experiments with vanA and vanS-vanH probes and DNA amplification assays using 14 PCR primer pairs specific for vanA cluster genes (vanR, vanS, vanH, vanA, and vanY), orf1, orf2, vanB, and vanC showed identical organization of resistance determinants in all epidemic VRE isolates. This organization appeared to be the same as that described for Tn1546 in VanA prototype strain E. faecium BM4147.

Probing the alpha 1 beta 2 interface of human hemoglobin by mutagenesis. Role of the FG-C contact regions.

The allosteric transition of hemoglobin involves an extensive reorganization of the alpha 1 beta 2 interface, in which two contact regions have been identified. This paper concerns at the effect of two mutations located in the "switch" (alpha C3 Thr --> Trp) and the "flexible joint" (beta C3 Trp --> Thr). We have expressed and characterized one double and two single mutants: Hb alpha T38W/beta W37T, Hb beta W37T, and Hb alpha T38W, whose structure has been determined by crystallography. We present data on: (i) the interface structure in the contact regions, (ii) oxygen and CO binding kinetics and cooperativity, (iii) dissociation rates of deoxy tetramers and association rates of deoxy dimers, and (iv) the effect of NaI on deoxy tetramer dissociation rate constant. All the mutants are tetrameric and T-state in the deoxygenated derivative. Reassociation of deoxygenated dimers is not modified by interface mutations. DeoxyHb alpha T38W/beta W37T dissociate much faster. We propose a binding site for I- at the switch region. The single mutants binds O2 cooperatively; the double one is almost non-cooperative, a feature confirmed by CO binding. The functional data, analyzed with the two-state model, indicate that these mutations reduce the value of the allosteric constant LO.

In vitro conjugative transfer of VanA vancomycin resistance between Enterococci and Listeriae of different species.

In a study designed to gain data on the in vitro transferability of vancomycin resistance from enterococci of the VanA phenotype to listeriae of different species, three clinical Enterococcus isolates-Enterococcus faecium LS10, Enterococcus faecalis LS4, and Enterococcus faecalis A3208, all harboring a plasmid that strongly hybridized with a vanA probe-were used as donors in transfer experiments. Strains of five Listeria species were used as recipients. From Enterococcus faecium LS10, glycopeptide resistance was transferred to Listeria monocytogenes, Listeria ivanovii, and Listeria welshimeri recipients, whereas no transfer occurred to Listeria seeligeri or Listeria innocua strains. From the two Enterococcus faecalis isolates, no transfer occurred to any Listeria recipient. MICs of both vancomycin and teicoplanin were > or = 256 mg/l for all transconjugants tested. Furthermore, all transconjugants harbored a plasmid that strongly hybridized with the vanA probe, with vanA consistently located in an EcoRI fragment of about 4 kb. Exposure of Listeria transconjugants to vancomycin resulted in synthesis of a membrane protein similar in size (39 kDa) to a vancomycin-induced membrane protein of Enterococcus faecium LS10. In retransfer experiments with Listeria transconjugants used as donors, glycopeptide resistance was transferred to all Listeria recipients tested, including strains of Listeria innocua and Listeria seeligeri, which were unable to receive the resistance from Enterococcus faecium LS10. The frequency of vanA transfer to listerial recipients was greater in retransfer experiments than in the primary matings. These findings suggest that the vanA resistance determinant might spread to the established pathogen Listeria monocytogenes, both directly from a resistant enterococcus and through strains of nonpathogenic Listeria species acting as intermediate resistance vehicles.

Use of primers selective for vancomycin resistance genes to determine van genotype in enterococci and to study gene organization in VanA isolates.

Vancomycin resistance in enterococci is an emerging therapeutic problem. Resistance is not always detected by standard microbiological methods. Oligonucleotide primers for PCR were designed to target amplification of defined regions of genes of the vanA cluster, as well as vanB and vanC1. These primers correctly identified 30 vancomycin-resistant isolates tested (17 VanA, 7 VanB, and 6 Enterococcus gallinarum). No amplification was observed with Enterococcus casseliflavus or vancomycin-susceptible strains. Using PCR and Southern blotting, we found that all 17 VanA isolates had orf-1, orf-2, vanR, vanS, vanH, vanA, and vanY genes in the same sequence and that the intergenic distances in the vanR-vanA segments were the same. The described methods should be applicable to the rapid detection of the different vancomycin resistance genotypes in enterococci.