Multicentre, prospective, double-blind, randomised controlled clinical trial comparing different non-opioid analgesic combinations with morphine for postoperative analgesia: the OCTOPUS study.

BACKGROUND: Head-to-head comparisons of combinations of more than one non-opioid analgesic (NOA) with morphine alone, for postoperative analgesia, are lacking. The objective of this multicentre, randomised, double-blind controlled trial was to compare the morphine-sparing effects of different combinations of three NOAs-paracetamol (P), nefopam (N), and ketoprofen (K)-for postoperative analgesia. METHODS: Patients from 10 hospitals were randomised to one of eight groups: control (C) received saline as placebo, P, N, K, PN, PK, NK, and PNK. Treatments were given intravenously four times a day during the first 48 h after surgery, and morphine patient-controlled analgesia was used as rescue analgesia. The outcome measures were morphine consumption, pain scores, and morphine-related side-effects evaluated 24 and 48 h after surgery. RESULTS: Two hundred and thirty-seven patients undergoing a major surgical procedure were included between July 2013 and November 2016. Despite a failure to reach a calculated sample size, 24 h morphine consumption [median (inter-quartile range)] was significantly reduced in the PNK group [5 (1-11) mg] compared with either the C group [27 (11-42) mg; P<0.05] or the N group [21 (12-29) mg; P<0.05]. Results were similar 48 h after surgery. Patients experienced less pain in the PNK group compared with the C, N, and P groups. No difference was observed in the incidence of morphine-related side-effects. CONCLUSIONS: Combining three NOAs with morphine allows a significant morphine sparing for 48 h after surgery associated with superior analgesia the first 24 h when compared with morphine alone. CLINICAL TRIAL REGISTRATION: EudraCT: 2012-004219-30; NCT01882530.

On the effect of alkaline pH and cofactor availability in the conformational and oligomeric state of Escherichia coli glutamate decarboxylase.

Escherichia coli glutamate decarboxylase (EcGad) is a homohexameric pyridoxal 5'-phosphate (PLP)-dependent enzyme. It is the structural component of the major acid resistance system that protects E. coli from strong acid stress (pH < 3), typically encountered in the mammalian gastrointestinal tract. In fact EcGad consumes one proton/catalytic cycle while yielding γ-aminobutyrate and carbon dioxide from the decarboxylation of l-glutamate. Two isoforms of Gad occur in E. coli (GadA and GadB) that are 99% identical in sequence. GadB is the most intensively investigated. Prompted by the observation that some transcriptomic and proteomic studies show EcGad to be expressed in conditions far from acidic, we investigated the structural organization of EcGadB in solution in the pH range 7.5-8.6. Small angle X-ray scattering, combined with size exclusion chromatography, and analytical ultracentrifugation analysis show that the compact and entangled EcGadB hexameric structure undergoes dissociation into dimers as pH alkalinizes. When PLP is not present, the dimeric species is the most abundant in solution, though evidence for the occurrence of a likely tetrameric species was also obtained. Trp fluorescence emission spectra as well as limited proteolysis studies suggest that PLP plays a key role in the acquisition of a folding necessary for the canonical catalytic activity.

Aromatic amino-acids and subunit assembly in the hemoglobins from Scapharca inaequivalvis: a fluorescence and CD study of the apoproteins.

The dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis have a unique assembly that places the heme-carrying E and F helices in the inside of the molecule. These helices form the intersubunit contact in the dimer, which represents the structural unit since the tetramer is a dimer of dimers. The E and F helices are highly conserved and contain about 70% of the phenylalanine and tyrosine residues, while the tryptophan residues are near the tetramer contact. The spectroscopic properties (circular dichroism and intrinsic fluorescence) of the aromatic amino-acid residues in the two globins indicate that heme removal brings about a larger conformational change in the tetrameric than in the dimeric protein and that the tryptophan residues acquire a more rigid conformation in the tetramer.

Subunit interactions in the dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis.

The dissociation behaviour of oxygenated Scapharea inaequivalvis HbII, the tetrameric hemoglobin component contained in the erythrocytes of this bivalve mollusc, has been compared with that of oxygenated human hemoglobin, HbA. At neutral pH the molluscan protein dissociates reversibly into dimers as does HbA, although dissociation is less marked; moreover the dimer-tetramer association constant is not sensitive to the presence of inorganic phosphates and high salt concentrations. HbII dimers hybridize with HbA dimers in solution, pointing to an overall similarity of the dimer interfaces in these hemoglobins from distantly related species. The gel filtration behaviour of the dimeric hemoglobin component of the erythrocytes. HbI, indicates that at neutral pH the protein has very little tendency to dissociate into monomers even at micromolar concentrations. Hb I was found to contain small amounts (2-4%) of bound carbohydrates.

Studies on Scapharca hemoglobins. Properties of the dimeric protein reconstituted with Fe- or Co-porphyrin.

A native globin from the dimeric hemoglobin, hemoglobin I, of the mollusc Scapharca inaequivalvis has been obtained with the acid-acetone method. The globin has a lower sedimentation coefficient than the native protein at neutral pH; its reconstitution product with natural heme has the same physicochemical and functional properties as the native protein. proto- and meso-cobalt hemoglobin I have been prepared and characterized. proto-Cobalt hemoglobin I binds oxygen reversibly with a lower affinity and a lower cooperativity than native hemoglobin I; thus, the changes in the functional properties brought about by substitution of iron with cobalt are similar to those observed in human hemoglobin A. The EPR spectra of deoxy-proto-cobalt hemoglobin I and of the photolysis product of oxy-meso-cobalt hemoglobin I indicate that two histidine residues are the apical heme ligands. The broad signal at g = 2.38 in deoxy-proto-cobalt hemoglobin I points to a constrained structure of the heme site in this derivative which results from a distorted coordination of the hindered proximal histidine. A similar structure has been proposed previously for the alpha chains in deoxy-cobalt hemoglobin A.

Assembly of erythrocruorin from the earthworm Octolasium complanatum.

The spontaneous assembly of the earthworm erythrocruorin molecule (60 S) from its 1/12 subunits (10 S) obtained by alkaline dissociation is a long debated problem, since the 60 S to 10 S dissociation step has been regarded as essentially irreversible or as only partially reversible when freshly dissociated solutions are used. Erythrocruorin from the earthworm Octolasium complanatum has been reassembled from its 10 S subunits. "Age" of the subunits, pH, and divalent cation concentration are the factors that influence the assembly reaction. Of primary importance is the age of the subunits, i.e. their exposure time to the alkaline dissociating pH. Parallel sedimentation velocity and sodium dodecyl sulfate/polyacrylamide gel electrophoresis experiments on the dissociated and reassembled solutions indicate that two processes take place at alkaline pH values: disulfide exchange and limited proteolysis. These processes, whose relative importance differs in the various preparations, might be responsible for the loss of reassociating capacity of the 10 S subunits. With freshly dissociated subunits, reassembly up to 80% may be achieved at pH 6.2 to 6.5 in the absence of divalent cations; the presence of 25 to 50 mM-Ca2+ renders the reaction essentially pH-independent in the range 6.2 to 8. The effect of Ca2+ is discussed in the light of the presence of structure-stabilizing binding sites for divalent cations at the 10 S intersubunit's contact regions.

Thermodynamic properties of oxygen equilibria of dimeric and tetrameric hemoglobins from Scapharca inaequivalvis.

Oxygen equilibrium curves of the dimeric and tetrameric hemoglobin components of Scapharca inaequivalvis were determined at several temperatures. The oxygen equilibrium curves were analyzed by the two-step and four-step oxygen equilibrium schemes of Adair for the dimeric and tetrameric hemoglobins, respectively. The enthalpy and entropy changes for each oxygenation step were determined by the temperature dependences of the Adair constants using van't Hoff equations. Neither dimeric nor tetrameric hemoglobins release protons or anions upon oxygenation under the experimental conditions of the present study. The enthalpy and entropy changes are non-uniform with respect to the oxygenation step and do not need to be corrected for the oxygenation-linked release of protons and anions. For the tetrameric hemoglobin, enthalpy-entropy compensation was observed between the first, second and third steps of oxygenation. The present results suggest that the origin of the co-operative oxygenation is primarily entropic for both hemoglobin components of S. inaequivalvis. Comparison of these data with those obtained on other hemoglobins shows that no simple generalization can be made as to the thermodynamic nature of co-operativity in oxygen binding.

The homodimeric hemoglobin from Scapharca can be locked into new cooperative structures upon reaction of Cys92, located at the subunit interface, with organomercurials.

In the cooperative, homodimeric hemoglobin from Scapharca inaequivalvis, HbI, the subunit interface is formed by the heme-carrying E and F helices and contains the only cysteine residue of the globin chain (Cys92, F2) in an area which changes from hydrophilic to hydrophobic upon oxygenation. Binding of organomercurials to HbI is cooperative and entails major quaternary rearrangements. The reaction of Cys92 with p-chloromercuri-benzoate (PMB) and p-nitro-o-chloromercuriphenol (PN), a sensitive reporter of the cysteine microenvironment at neutral pH values, has been followed in stopped flow experiments. Kinetic evidence for the cooperativity of mercurial binding has been obtained and the rate of the corresponding conformational transition has been estimated. As expected PN, but not PMB, is able to monitor the oxygen-linked change of the cysteine microenvironment. The modification of Cys92 with PN has unique functional effects. In PN-reacted HbI cooperativity is maintained, albeit to a different extent, depending on the ligation state of the protein during mercaptide formation. It may be envisaged that PN locks the protein into new, cooperative, quaternary structures stabilized by hydrogen bonding interactions between the ionized nitrophenol moiety and the contralateral subunit.

Calcium- and pH-linked oligomerization of sorcin causing translocation from cytosol to membranes.

Sorcin, a cytosolic calcium-binding protein containing a pair of EF-hand motifs, undergoes a Ca2(+)-dependent translocation to the cell membrane. The underlying conformational change is similar at pH 6.0 and 7.5 and consists in an increase in overall hydrophobicity that involves the aromatic residues and in particular the two tryptophan residues which become less exposed to solvent. The concomitant association from dimers to tetramers indicates that the tryptophan residues, which are located between the EF-hand sites, become buried at the dimer-dimer interface. Ca2(+)-bound sorcin displays a striking difference in solubility as a function of pH that has been ascribed to the formation of calcium-stabilized aggregates.

The sorcin-annexin VII calcium-dependent interaction requires the sorcin N-terminal domain.

Surface plasmon resonance experiments show that at neutral pH the stability of the complex between sorcin and annexin VII (synexin) increases dramatically between 3 and 6 microM calcium; at the latter cation concentration the K(D) value is 0.63 microM. In turn, the lack of complex formation between the sorcin Ca(2+) binding domain (33-198) and synexin maps the annexin binding site to the N-terminal region of the sorcin polypeptide chain. Annexin VII likewise employs the N-terminal domain, more specifically the first 31 amino acids, to interact with sorcin [Brownawell, A.M. and Creutz, C.E. (1997) J. Biol. Chem. 272, 22182-22190]. The interaction may involve similar structural motifs in the two proteins, namely GGYY and GYGG in sorcin and GYPP in synexin.

Calcium-dependent translocation of sorcin to membranes: functional relevance in contractile tissue.

Sorcin, a 22 kDa calcium binding protein present in abundance in cardiac tissue and in multi-drug resistant cells and previously described as a soluble protein, is now shown to undergo a calcium-dependent translocation process from the cytosol to cellular membranes in both systems. The translocation process takes place also in E. coli BL21 cells that express recombinant sorcin, r-sorcin, and can be exploited in the purification of the protein. Calcium binding to purified r-sorcin occurs at micromolar concentrations of the metal and is accompanied by a conformational change that renders the protein soluble in the non-ionic detergent Triton X-114. This finding suggests that lipids are the target of sorcin on cellular membranes. The possible significance of the calcium-dependent translocation of sorcin in the specialized functions of sorcin-expressing cells is discussed.

Amino acid sequence of the cooperative homodimeric hemoglobin from the mollusc Scapharca inaequivalvis and topology of the intersubunit contacts.

The dimeric hemoglobin (HbI) from Scapharca inaequivalvis is highly homologous to the other known dimeric Acid hemoglobins. The sequence has a distinctive hydrophobicity profile in the region corresponding to the E and F helices with respect to both the hemoglobin and myoglobin chains from vertebrates due to the presence of several additional hydrophobic residues. The characteristic topology of the E and F helices is conserved in all the known sequences of Arcid hemoglobins including that of the so-called alpha chain of the tetrameric component from Anadara trapezia. The rationale for this conservation lies in the unusual assembly of Arcid hemoglobins where the E and F helices are involved in the interdimeric contact. It is suggested that the extra hydrophobic residues play a major role in the assembly of the basic dimeric unit in these hemoglobins.

A cooperative hemoglobin with directly communicating hemes. The Scapharca inaequivalvis homodimer.

The unique functional properties of the homodimeric hemoglobin (HbI) extracted from the Arcid blood clam Scapharca inaequivalvis are discussed in the light of the unusual assembly of this protein. At variance with vertebrate hemoglobins, in S. inaequivalvis HbI, the heme-carrying E and F helices form the subunit interface and bring the heme groups almost into direct contact. This creates a new pathway for transferring information about the ligation state of the heme from one subunit to the other which allows cooperativity in the binding of heme ligands to be displayed by a homodimer. The tight coupling between the two subunits and the two heme groups also manifests itself in other reactions that are cooperative in S. inaequivalvis HbI, but not in human hemoglobin, namely, the cleavage of the proximal histidine-heme iron bond and the modification of specific residues located at the subunit interface.

Determination of methionine sulfoxide in proteins: comparison of a gas-chromatographic and electrophoretic method.

Two methods for the determination of methionine in proteins have been used to estimate the extent of methionine sulfoxide obtained upon exposure of proteins to oxidizing agents. Both methods are based on prior treatment with cyanogen bromide, which attacks methionines (but not the sulfoxide derivative) with the resultant formation of methyl thiocyanate and peptides. The amount of methyl thiocyanate is determined quantitatively by gas chromatography, while the number of peptides is ascertained by SDS-polyacrylamide gel electrophoresis. The gas chromatographic estimate of CH3SCN offers an accurate and precise method (down to nanogram values) for the quantitative determination of methionine sulfoxide in proteins. Due to its simplicity and the use of low-cost equipment, the electrophoretic method appears to be a valuable complement to the gas chromatographic method, and the two methods in conjunction provide novel results.

Identification of the site of ferrocyanide binding involved in the intramolecular electron transfer process to oxidized heme in Scapharca dimeric hemoglobin.

The cooperative homodimeric hemoglobin (HbI)2 from the mollusc Scapharca inaequivalvis is characterized by unusual properties of the ferric derivative. The dimeric aquomet form undergoes a pH-dependent reversible dissociation into a monomeric low-spin hemichrome. Moreover, in HbI oxidized with ferricyanide the ferrocyanide anion produced in the reaction remains bound to the oxidized protein with high affinity and forms an intramolecular redox couple with the heme iron. Thus, the reduced HbI-CO adduct is obtained readily in the presence of carbon monoxide. The ferrocyanide binding site of HbI has been identified by modifying the only cysteine residue of the polypeptide chain, Cys 92 (F2), which is located at the subunit interface near the proximal histidine (His 101, F11). In HbI modified with organomercurials the rate of oxidation by ferricyanide depends on the presence and position of a negatively charged group on the aromatic ring, indicating that the binding site of the ferrocyanide anion is located near Cys 92. The tendency to dissociate into the monomeric hemichrome of the various Cys 92-reacted proteins and the study of the intramolecular electron transfer reaction between bound ferrocyanide and the heme iron confirmed this location. The proposed binding site of the ferrocyanide anion comprises a cluster of positive charges at the subunit interface formed by Lys 96, Arg 53', Lys 65', and Arg 67' where apices indicate residues of the contralateral subunit.

Interaction of lactoferrin with Escherichia coli cells and correlation with antibacterial activity.

It has been established that the antimicrobial activity of lactoferrin towards Escherichia coli is enhanced by a direct contact between the protein and the microbial cell and that, in the case of E. coli K-12 strains, an antibacterial activity of lactoferrin unrelated to iron withdrawal is present. Evidence is now reported that lactoferrin binds to surface structures expressed in E. coli K-12 strains grown in either an "excess" or "stress" of iron. Under the experimental conditions used, lactoferrin binding both in the apo and in the iron-saturated form yields a maximum of 1.6 X 10(5) bound molecules/E. coli K-12 cell; the amount of lactoferrin bound does not depend on the expression of the iron-regulated outer membrane proteins. In contrast, lactoferrin does not bind to E. coli clinical isolates. Apo-lactoferrin (at 500 micrograms/ml in a chemically defined medium) inhibits the growth of E. coli K-12 strains but not of clinical isolates. These findings suggest that the antibacterial activity of the protein could be associated to its binding to the cell surface.

Two different crystal forms of sorcin, a penta-EF-hand Ca2+-binding protein.

Sorcin is a 198 amino-acid Ca(2+)-binding protein that belongs to the penta-EF-hand family. Its Ca(2+)-binding domain (residues 33-198) has been crystallized in the absence of Ca(2+) in two different crystal forms. Two complete data sets have been collected on a synchrotron source under cryocooling conditions from crystals grown using ammonium sulfate as precipitant: monoclinic crystals in space group C2, with unit-cell parameters a = 130.93, b = 103.85, c = 78.55 A, beta = 118.0 degrees, diffracting to 2.1 A, and tetragonal crystals in space group P42(1)2, with unit-cell parameters a = b = 103.33, c = 79.15, diffracting to 2.7 A. Crystals were also grown using PEG 6000 as precipitating agent. They also belong to space group C2, diffract to 2.8 A and their unit-cell parameters are very similar to the first form. Structure determination by molecular replacement has been initiated. Structural information should be useful for elucidating the interaction of sorcin with membrane targets.

Metal regulation of siderophore synthesis in Pseudomonas aeruginosa and functional effects of siderophore-metal complexes.

Pseudomonas aeruginosa synthesizes two siderophores, pyochelin and pyoverdin, characterized by widely different structures, physicochemical properties, and affinities for Fe(III). Titration experiments showed that pyochelin, which is endowed with a relatively low affinity for Fe(III), binds other transition metals, such as Cu(II), Co(II), Mo(VI), and Ni(II), with appreciable affinity. In line with these observations, Fe(III) and Co(II) at 10 microM or Mo(VI), Ni(II), and Cu(II) at 100 microM repressed pyochelin synthesis and reduced expression of iron-regulated outer membrane proteins of 75, 68, and 14 kDa. In contrast, pyoverdin synthesis and expression of the 80-kDa receptor protein were affected only by Fe(III). All of the metals tested, except Mo(VI), significantly promoted P. aeruginosa growth in metal-poor medium; Mo(VI), Ni(II), and Co(II) were more efficient as pyochelin complexes than the free metal ions and the siderophore. The observed correlation between the affinity of pyochelin for Fe(III), Co(II), and Mo(VI) and the functional effects of these metals indicates that pyochelin may play a role in their delivery to P. aeruginosa.