Ketamine in chronic pain: A Delphi survey.

BACKGROUND: There is no recommendation in Europe for the use of ketamine in patients with chronic pain. The heterogeneity of practice highlights the need to seek the advice of experts in order to establish a national consensus. This Delphi survey aimed to reach a national consensus on the use of ketamine in chronic pain in Pain clinics. METHODS: A collaborative four-round internet-based questionnaire was used. It was created after literature search on ketamine administration in chronic pain and included about 96 items. It discussed utility and advantages, adverse events and deleterious aspects, methods of administration, concomitant treatments and assessment of results. RESULTS: Twenty-eight experts completed all rounds of the survey with a total of 81.3% items reaching a consensual answer. Neuropathic pain represents the first indication to use ketamine, followed, with a good to moderate utility, by other situations (fibromyalgia, complex regional pain syndrome, central neuropathic pain, peripheral neuropathic pain, nociceptive pain, sensitization, opioid withdrawal, palliative care, depression). Experts agreed on the rare occurrence of adverse events. Concerning routes of administration, intravenous infusion with doses of 0.5-0.9 mg/kg/d for 4 days of treatment is preferred. Place of care is hospital, as in- or out-patient, with a quarterly administration of ketamine. Finally, ketamine effectiveness is assessed 1 month after infusion, and experts encourage combination with non-pharmacological treatment. CONCLUSIONS: This Delphi survey established a consensus of pain specialists on the use of ketamine in refractory chronic pain, thus providing a basis for future comparative trials. SIGNIFICANCE: This Delphi survey in chronic pain reached agreement on four main aspects: (1) Priority to treat neuropathic pain with evaluation of effectiveness at 1 month; (2) No deleterious effects in the majority of listed diseases/situations with the absence or <3% of suggested adverse events; (3) 0.5-0.9 mg/kg/d IV infusion; (4) Combination with non-pharmacological treatment.

Effect of (5'S)-5',8-cyclo-2'-deoxyadenosine on the conformation of di and trinucleotides. A NMR and DFT study.

5',8-Purine cyclonucleosides constitute an important class of oxidatively generated tandem lesions whose formation involves initial hydroxyl radical-mediated hydrogen atom abstraction from the 5-hydroxymethyl group of 2-deoxyribose followed by intramolecular cyclization. The present study deals with the synthesis of the 5'S diastereomer of 5',8-cyclo-2'-deoxyadenosine containing di- and tri-oligodeoxynucleotides as an attempt to delineate the conformational changes induced in the DNA fragments by the presence of a rigid modified nucleoside. For this purpose, extensive 1D and 2D NMR measurements that were completed by DFT theoretical calculations were performed. As a striking result, it was found that the covalent bond between C(5') and C(8) in the investigated purine cyclonucleoside induces an unusual West ((0)T(1)) conformation of the furanose ring. Thus it can be postulated that the rigid structure of the tandem lesion would strongly perturb the global geometry of oligonucleotides at the site of the modification and therefore affect the enzymatic activity of DNA polymerases and repair enzymes.

The possible role of an [FeFe]-hydrogenase-like protein in the plant responses to changing atmospheric oxygen levels.

The knockdown of a [FeFe]-hydrogenase-like gene in the model plants Medicago truncatula and Arabidopsis thaliana resulted in a mutant with a dwarf phenotype. Surprisingly, the phenotype is undistinguishable from wild type under hypoxic conditions. The heterologous expression of the plant gene in Escherichia coli indicates that the resulting protein probably coordinates two [Fe-S] clusters with different magnetic properties. Sequence alignment analysis indicates that these two clusters would be topologically equivalent to the mesial and proximal [Fe-S] centers of [FeFe]-hydrogenases. A possible role of the gene product in oxygen signaling pathways is discussed.

Human iron regulatory protein 2 is easily cleaved in its specific domain: consequences for the haem binding properties of the protein.

Mammalian IRPs (iron regulatory proteins), IRP1 and IRP2, are cytosolic RNA-binding proteins that post-transcriptionally control the mRNA of proteins involved in storage, transport, and utilization of iron. In iron-replete cells, IRP2 undergoes degradation by the ubiquitin/proteasome pathway. Binding of haem to a 73aa-Domain (73-amino-acid domain) that is unique in IRP2 has been previously proposed as the initial iron-sensing mechanism. It is shown here that recombinant IRP2 and the 73aa-Domain are sensitive to proteolysis at the same site. NMR results suggest that the isolated 73aa-Domain is not structured. Iron-independent cleavage of IRP2 within the 73aa-Domain also occurs in lung cancer (H1299) cells. Haem interacts with a cysteine residue only in truncated forms of the 73aa-Domain, as shown by a series of complementary physicochemical approaches, including NMR, EPR and UV-visible absorption spectroscopy. In contrast, the cofactor is not ligated by the same residue in the full-length peptide or intact IRP2, although non-specific interaction occurs between these molecular forms and haem. Therefore it is unlikely that the iron-dependent degradation of IRP2 is mediated by haem binding to the intact 73aa-Domain, since the sequence resembling an HRM (haem-regulatory motif) in the 73aa-Domain does not provide an axial ligand of the cofactor unless this domain is cleaved.

"Seed money" and publication output in Mexican research: a case study of ifs grantees

Este estudio determina, a través de un análisis de publicación, el efecto de los recursos otorgados por la International Foundation of Science (IFS) a investigadores mexicanos para su desarrollo académico a comienzos de sus carreras. Desde 1974 el IFS ha apoyado 4000 investigadores jóvenes en países en desarrollo, que lleven a cabo investigaciones relevantes en el uso sostenible de los recursos naturales biológicos. En marzo 2000 se recibieron 105 listas de publicaciones de los 138 actuales y anteriores mexicanos subvencionados, se codificaron según tipo y formato de las publicaciones, idioma de publicación, publicación en revistas de cobertura internacional, coautorías y colocación del autor. Estas variables fueron analizadas en relación a cuándo se otorgó el financiamiento, número de donaciones recibidas, área de investigación del proyecto financiado, y membresía en el Sistema Nacional de Investigadores (SNI). La tendencia de publicación muestra que el apoyo del IFS contribuyó a aumentar el volumen de publicaciones, con más frecuencia en inglés, y más a menudo en revistas científicas de corriente principal, además de incrementar la visibilidad internacional y su contribución a la internacionalización de la ciencia mexicana. Se sugiere que los investigadores becados del IFS fueron capaces de establecerse como científicos bona fide en México, incluso aquellos en las universidades e instituciones de investigación de menor ranking. Se redujo la probabilidad de fuga de cerebros y se contribuyó a fortalecer la investigación nacional. Sin embargo, se concluye que más que un factor crucial para el desarrollo de sus investigaciones, la beca IFS es un facilitador importante para el avance de la carrera de los becarios

Characterization of a naphthalene dioxygenase endowed with an exceptionally broad substrate specificity toward polycyclic aromatic hydrocarbons.

In Sphingomonas CHY-1, a single ring-hydroxylating dioxygenase is responsible for the initial attack of a range of polycyclic aromatic hydrocarbons (PAHs) composed of up to five rings. The components of this enzyme were separately purified and characterized. The oxygenase component (ht-PhnI) was shown to contain one Rieske-type [2Fe-2S] cluster and one mononuclear Fe center per alpha subunit, based on EPR measurements and iron assay. Steady-state kinetic measurements revealed that the enzyme had a relatively low apparent Michaelis constant for naphthalene (K(m) = 0.92 +/- 0.15 microM) and an apparent specificity constant of 2.0 +/- 0.3 mM(-)(1) s(-)(1). Naphthalene was converted to the corresponding 1,2-dihydrodiol with stoichiometric oxidation of NADH. On the other hand, the oxidation of eight other PAHs occurred at slower rates and with coupling efficiencies that decreased with the enzyme reaction rate. Uncoupling was associated with hydrogen peroxide formation, which is potentially deleterious to cells and might inhibit PAH degradation. In single turnover reactions, ht-PhnI alone catalyzed PAH hydroxylation at a faster rate in the presence of organic solvent, suggesting that the transfer of substrate to the active site is a limiting factor. The four-ring PAHs chrysene and benz[a]anthracene were subjected to a double ring-dihydroxylation, giving rise to the formation of a significant proportion of bis-cis-dihydrodiols. In addition, the dihydroxylation of benz[a]anthracene yielded three dihydrodiols, the enzyme showing a preference for carbons in positions 1,2 and 10,11. This is the first characterization of a dioxygenase able to dihydroxylate PAHs made up of four and five rings.

Drosophila nuclear receptor E75 is a thiolate hemoprotein.

Drosophila E75 is a member of the nuclear receptor superfamily. These eukaryotic transcription factors are involved in almost all physiological processes. They regulate transcription in response to binding of rigid hydrophobic hormone ligands. As it is the case for many nuclear receptors, the E75 hormone ligand was originally unknown. Recently, however, it was shown that the ligand binding domain (LBD) of E75 contains a tightly bound heme prosthetic group and is gas responsive. Here we have used site-directed mutagenesis along with UV-visible and electron paramagnetic resonance (EPR) spectroscopies to characterize and assign the heme iron axial ligands in E75. The F370Y mutation and addition of hemin to the growth medium during expression of the protein in Escherichia coli were necessary to produce good yields of heme-enriched E75 LBD. EPR studies revealed the presence of several species containing a strongly iron bound thiolate. The involvement of cysteines 396 and 468 in heme binding was subsequently shown by single and double mutations. Using a similar approach, we have also established that the sixth iron ligand of a well-defined coordination conformation, which accounts for approximately half of the total species, is histidine 574. The other iron coordination pairs are discussed. We conclude that E75 is a new example of a thiolate hemoprotein and that it may be involved in hormone synthesis regulation.

The [Fe-Fe]-hydrogenase maturation protein HydF from Thermotoga maritima is a GTPase with an iron-sulfur cluster.

The active site of [Fe-Fe]-hydrogenases is composed of a di-iron complex, where the two metal atoms are bridged together by a putative di(thiomethyl)amine molecule and are also ligated by di-nuclear ligands, namely carbon monoxide and cyanide. Biosynthesis of this metal site is thought to require specific protein machinery coded by the hydE, hydF, and hydG genes. The HydF protein has been cloned from the thermophilic organism Thermotoga maritima, purified, and characterized. The enzyme possesses specific amino acid signatures for GTP binding and is able to hydrolyze GTP. The anaerobically reconstituted TmHydF protein binds a [4Fe-4S] cluster with peculiar EPR characteristics: an S = 1/2 signal presenting a high field shifted g-value together with a S = 3/2 signal, similar to those observed for [4Fe-4S] clusters ligated by only three cysteines. HYSCORE spectroscopy experiments were carried out to determine the nature of the fourth ligand of the cluster, and its exchangeability was demonstrated with the formation of a [4Fe-4S]-imidazole complex.

Biochemical characterization of the HydE and HydG iron-only hydrogenase maturation enzymes from Thermatoga maritima.

Fe-only hydrogenases contain a di-iron active site complex, in which the two Fe atoms have carbon monoxide and cyanide ligands and are linked together by a putative di(thiomethyl)amine molecule. We have cloned, purified and characterized the HydE and HydG proteins, thought to be involved in the biosynthesis of this peculiar metal site, from the thermophilic organism Thermotoga maritima. The HydE protein anaerobically reconstituted with iron and sulfide binds two [4Fe-4S] clusters, as characterized by UV and EPR spectroscopy. The HydG protein binds one [4Fe-4S] cluster, and probably an additional one. Both enzymes are able to reductively cleave S-adenosylmethionine (SAM) when reduced by dithionite, confirming that they are Radical-SAM enzymes.

Crystallographic and spectroscopic evidence for high affinity binding of FeEDTA(H2O)- to the periplasmic nickel transporter NikA.

Because nickel is both essential and toxic to a great variety of organisms, its detection and transport is highly regulated. In Escherichia coli and other related Gram-negative bacteria, high affinity nickel transport depends on proteins expressed by the nik operon. A central actor of this process is the periplasmic NikA transport protein. A previous structural report has proposed that nickel binds to NikA as a pentahydrate species. However, both stereochemical considerations and X-ray absorption spectroscopic results are incompatible with that interpretation. Here, we report the 1.8 A resolution structure of NikA and show that it binds FeEDTA(H2O)- with very high affinity. In addition, we provide crystallographic evidence that a metal-EDTA complex was also bound to the previously reported NikA structure. Our observations strongly suggest that nickel transport in E. coli requires the binding of this metal ion to a metallophore that bears significant resemblance to EDTA. They also provide a basis for the potential use of NikA in the bioremediation of toxic transition metals and the design of artificial metalloenzymes.

An Isc-type extremely thermostable [2Fe-2S] ferredoxin from Aquifex aeolicus. Biochemical, spectroscopic, and unfolding studies.

Analysis of the genome of the hyperthermophilic bacterium Aquifex aeolicus has revealed the presence of a previously undetected gene potentially encoding a plant- and mammalian-type [2Fe-2S] ferredoxin. Expression of that gene in Escherichia coli has yielded a novel thermostable [2Fe-2S] ferredoxin (designated ferredoxin 5) whose sequence is most similar to those of ferredoxins involved in the assembly of iron-sulfur clusters (Isc-Fd). It nevertheless differs from the latter proteins by having deletions near its N- and C-termini, and no cysteine residues other than those involved in [2Fe-2S] cluster coordination. Resonance Raman, low-temperature MCD and EPR studies show close spectral similarities between ferredoxin 5 and the Isc-Fd from Azotobacter vinelandii. Mössbauer spectra of the reduced protein were analyzed with an S = 1/2 spin Hamiltonian and interpreted in the framework of the ligand field model proposed by Bertrand and Gayda. The redox potential of A. aeolicus ferredoxin 5 (-390 mV) is in keeping with its relatedness to Isc-Fd. Unfolding experiments showed that A. aeolicus ferredoxin 5 is highly thermostable (T(m) = 106 degrees C at pH 7), despite being devoid of features (e.g., high content of charged residues) usually associated with extreme thermal stability. Searches for genes potentially encoding plant-type [2Fe-2S] ferredoxins have been performed on the sequenced genomes of hyperthermophilic organisms. None other than the two proteins from A. aeolicus were retrieved, indicating that this otherwise widely distributed group of proteins is barely represented among hyperthermophiles.

Exploration of the diaphorase activity of neutrophil NADPH oxidase.

In the O2- generating flavocytochrome b, the membrane-bound component of the neutrophil NADPH oxidase, electrons are transported from NADPH to O2 in the following sequence: NADPH --> FAD --> heme b -->O2. Although p-iodonitrotetrazolium (INT) has frequently been used as a probe of the diaphorase activity of the neutrophil flavocytochrome b, the propensity of its radical to interact reversibly with O2 led us to question its specificity. This study was undertaken to reexamine the interaction of INT with the redox components of the neutrophil flavocytochrome b. Two series of inhibitors were used, namely the flavin analog 5-deaza FAD and the heme inhibitors bipyridyl and benzylimidazole. The following results indicate that INT reacts preferentially with the hemes rather than with the FAD redox center of flavocytochrome b and is not therefore a specific probe of the diaphorase activity of flavocytochrome b. First, in anaerobiosis, reduced heme b in activated membranes was reoxidized by INT as efficiently as by O2 even in the presence of concentrations of 5-deaza FAD which fully inhibited the NADPH oxidase activity. Second, the titration curve of dithionite-reduced heme b in neutrophil membranes obtained by oxidation with increasing amounts of INT was strictly superimposable on that of dithionite-reduced hemin. Third, INT competitively inhibited the O2 uptake by the activated NADPH oxidase in a cell-free system. Finally, the heme inhibitor bipyridyl competitively inhibited the reduction of INT in anaerobiosis, and the oxygen uptake in aerobiosis.

Formation, Properties, and Characterization of a Fully Reduced Fe(II)Fe(II) Form of Spinach (and Parsley) [2Fe-2S] Ferredoxin with the Macrocyclic Complex [Cr(15-aneN(4))(H(2)O)(2)](2+) as Reductant.

Reduction of spinach and parsley ferredoxin FdI in the Fe(III)Fe(III) state with the 1,4,8,12-tetraazacyclopentadecane complex [Cr(15-aneN(4))(H(2)O)(2)](2+), here written as Cr(II)L, provides the first evidence for two 1-equiv steps yielding an Fe(II)Fe(II) product. Rate constants (25 degrees C) for spinach FdI are 2760 and 660 M(-)(1) s(-)(1), respectively, at pH 7.5, I = 0.100 M (NaCl). An important observation is that the Cr(III)L generated in the first step remains attached to the Fe(II)Fe(III) product and perturbs the protein active site sufficiently to make the second stage possible. The second Cr(II)L reduction is of the "outer-sphere" type, and the Cr(III)L generated is not attached to the protein. Anaerobic reoxidation of the fully reduced protein with [Co(NH(3))(6)](3+) is rapid and can be achieved with approximately 80% recovery of the Fe(III)Fe(III) oxidation state over 40 min. Air oxidation yields the Cr(III)L product Fe(III)Fe(III).Cr(III)L (Fe:Cr = 2:1). With Anabaena variabilis only a one-step reduction is observed and there is no Cr(III)L attachment. From a comparison of amino acid sequences with spinach (and parsley) FdI, a likely point of Cr(III)L attachment is indicated. Comparisons are made with dithionite as reductant. In addition, square-wave voltammetry on spinach Fe(III)Fe(III).Cr(III)L gives two reduction potentials -273 and -410 mV vs NHE. The different redox products have been characterized by EPR. Using (1)H NMR line-broadening techniques, evidence for Cr(III)L binding at a surface site close to Tyr-25/Tyr-82 is obtained. Also from investigations with redox-inactive [Cr(en)(3)](3+) as a competitive inhibitor for Cr(II)L reduction of spinach Fe(III)Fe(III), Tyr-25/Tyr-82 is proposed as the site for Cr(II)L reduction. From an extension of studies to include reduction of Fe(III)Fe(III).Cr(III)L with Cr(II)L, evidence is obtained for a second reaction site when that at Tyr-25/Tyr-82 is no longer available.