Effect of flumazenil on diaphragm electrical activation during weaning from mechanical ventilation after acute respiratory distress syndrome.

BACKGROUND: Diaphragm electrical activation (EAdi) and the ratio of tidal volume to EAdi (VT/EAdi) may provide clinical information on neuroventilatory efficiency (NVE) in patients being weaned from mechanical ventilation. We tested the hypothesis that residual sedation could interfere with respiratory recovery, by assessing the effects of flumazenil on EAdi and VT/EAdi ratio. METHODS: This observational study included 13 patients breathing with pressure-support ventilation (PSV) after a long period of controlled mechanical ventilation (i.e. >4 days) plus midazolam-based sedation for acute respiratory distress syndrome. EAdi and respiratory patterns were compared before and after a bolus of flumazenil, which was given because neurological status needed to be evaluated. RESULTS: Flumazenil induced a significant increase in EAdi [+71 (41-123)%, P=0.0002] and VT [+17 (8-32)%, P=0.0005], resulting in significantly decreased NVE [-34 (15-43)%]. The increased VT was significantly correlated with the increased EAdi (ρ=0.70, P=0.009). CONCLUSIONS: During weaning from mechanical ventilation, the diaphragmatic contribution to the breathing process may be reduced by residual midazolam-induced ventilatory depression. The increased EAdi with reversal of residual sedation was associated with a proportional increase in VT. These findings should be considered by the attending physician when interpreting daily EAdi and VT changes during weaning from mechanical ventilation.

Neuro-ventilatory efficiency during weaning from mechanical ventilation using neurally adjusted ventilatory assist.

BACKGROUND: Neuro-ventilatory efficiency (NVE), defined as the tidal volume to electrical diaphragm-activity ratio (VT/EAdi) at the beginning and end of the weaning process after acute hypoxaemic respiratory failure, may provide valuable information about patient recovery. METHODS: This observational study included 12 patients breathing with neurally adjusted ventilatory assist (NAVA). When a spontaneous breathing trial (SBT) with pressure support of 7 cm H2O and PEEP was unsuccessful, NAVA was used and the level was adjusted to obtain an EAdi of ∼60% of maximal EAdi during SBT. VT and EAdi were recorded continuously. We compared changes in NVE between NAVA and SBT at the first failed and first successful SBT. RESULTS: When patients were switched from NAVA to SBT, NVE was significantly reduced during both unsuccessful and successful SBT (-56 and -38%, respectively); however, this reduction was significantly lower when SBT was successful (P=0.01). Between the first and last day of weaning, we observed that NVE decreased with NAVA [40.6 (27.7-89.5) vs 28.8 (18.6-46.7); P=0.002] with a significant decrease in NAVA level, whereas it remained unchanged during SBT [15.4 (10.7-39.1) vs 19.5 (11.6-29.6); P=0.50] with significant increases in both EAdi and VT and no difference in respiratory rhythm. CONCLUSIONS: These results suggest that in patients after respiratory failure and prolonged mechanical ventilation, changes in VT and NVE, between SBTs are indicative of patient recovery. Larger clinical trials are needed to clarify whether changes in NVE reliably predict weaning in patients ventilated with NAVA.

[Changes in kaliemia following rapid sequence induction with succinylcholine in critically ill patients]. / Variation de kaliémie après une induction en séquence rapide avec la succinylcholine chez les patients de réanimation.

OBJECTIVE: Evaluate the changes in potassium following rapid sequence induction with succinylcholine in critically ill-patients and determine whether hospital length of stay could influence the succinylcholine-induced hyperkaliemia. STUDY DESIGN: Prospective and observational study. PATIENTS AND METHODS: After approval by our local ethical committee, we prospectively included 36 patients admitted from more than 24hours in ICU and who required succinylcholine for rapid tracheal intubation (1mg/kg). Serum potassium was measured before, 5 and 30min after succinylcholine. The incidence of life-threatening hyperkaliemia (≥6.5mmol/L) was noted. RESULTS: We could observe significant and transient increase in serum potassium (median increase of 0.45 [0.20-0.80] mmol/L at five minutes). A significant relationship was observed between the ICU length of stay and arterial potassium increase (r=0.37, P<0.05). From the ROC curve, a threshold of 12 days had an 86% sensitivity and 69% specificity in discriminating patients in whom the potassium increase was more than 1.5mmol/L. CONCLUSION: Induction with succinylcholine is followed by significant but transient hyperkaliema. The ICU length of stay before giving succinylcholine could influence significantly the amplitude of potassium increase.

[Influence of the fascia iliaca compartment block on postoperative cognitive status in the elderly]. / Bloc iliofascial et troubles cognitifs postopératoires chez la personne âgée.

OBJECTIVE: The aim of this study was to assess the influence of a regional analgesia technique on the incidence of postoperative cognitive dysfunction (POCD) after hip surgery, in elderly patients. PATIENTS AND METHODS: Patients, aged over 65 years, were assigned in two groups according to the anaesthesia technique: group NKT (general anaesthesia with target concentration infusion of propofol and remifentanil, with a 0.1 mg/kg-bolus of morphine at the end of surgery), group KT (preoperative iliaca compartment block with catheter and then general anaesthesia without bolus of morphine). Postoperative analgesia was similar in both groups: paracetamol, tramadol, and subcutaneous morphine if verbal pain scale equal or greater than 2 (0.1 mg/kg). POCD was defined as a decrease in Mini Mental Status (MMSE) equal or greater than 2 points and was monitored during 2 days. Consumption of opioids, pain scores and side effects were recorded. RESULTS: Sixty-five patients were included: 34 in NKT group and 31 in KT group. MMSE scores were higher in the KT group at day 1 and day 2 (p=0.01 and 0.0004, respectively). POCD was less frequent in group KT at day 2 (6 % vs 41 % ; p=0.001) and pain scores were lower during the first 48 hours (p=0.03). Remifentanil consumption was lower in KT group (0.43+/-0.18 mg vs 0.61+/-0.25 mg, p=0.002). Total amount of morphine, including the bolus in NKT group, was significantly lower in KT group (7 [5-17] mg vs 0 [0-5] mg, p<10(-6)). CONCLUSION: Postoperative analgesia by iliaca compartment block with catheter seems to provide a decrease in the incidence of POCD after hip surgery in elderly patients. STUDY DESIGN: Prospective, observational study.

[Stomach rupture associated with noninvasive ventilation]. / Rupture gastrique au décours d'une séance de ventilation non invasive.

Noninvasive positive-pressure ventilation (NIPPV) is a safe method to treat acute respiratory failure and is known to decrease the need for intubation and the length of ICU-stay. Few severe complications have been reported even when the indications are respected. Some rare cases of gastric distension were recently described. We report the case of a gastric perforation associated with NIPPV. The treatment was closure with a primary interrupted two-layer suture. Recovery was complete and the patient was transferred to the ward on Day 11.

[Cranial subdural haematoma after spinal anaesthesia treated by blood patch]. / Hématome sous-dural aigu intracrânien après rachianesthésie traité par blood patch.

The authors report a case of subdural haematoma after spinal anaesthesia. A 36-year-old woman underwent phlebectomy under spinal anaesthesia. Two days later, she complains of severe headache without neurological signs, not responding to bed rest and analgesics. Magnetic resonance imaging showed a small acute subdural haematoma in the right parieto-occipital region. On the forth day, she was given a blood-patch, which improved rapidly the patient. Recovery was complete.

[Use of activated recombinant FVII in the control of haemorrhage following trauma]. / Contrôle d'une hémorragie traumatique persistante par le facteur VII activé recombinant.

Uncontrolled haemorrhage is a major cause of death in trauma patients: sometimes inaccessible to surgery and often associated with coagulopathy. We report a case of severe blunt pelvic trauma associated with suicide. The conventional treatments were unsuccessful and embolization was impossible. The patient required massive blood product transfusion. A 100 microg/kg recombinant activated factor VII dose was infused, twice. After administration of the first dose, the blood requirement decreased dramatically. Further work and trials are required to assess the safety profile and dose regimen for this drug.

Mimicking dominant negative inhibition of prion replication through structure-based drug design.

Recent progress determining the structure of the host-encoded prion protein (PrP(C)) and the role of auxiliary molecules in prion replication permits a more rational approach in the development of therapeutic interventions. Our objective is to identify a new class of lead compounds that mimic the dominant negative PrP(C) mutants, which inhibit an abnormal isoform (PrP(Sc)) formation. A computational search was conducted on the Available Chemicals Directory for molecules that mimic both the spatial orientation and basic polymorphism of PrP residues 168, 172, 215, and 219, which confer dominant negative inhibition. The search revealed 1,000 potential candidates that were visually analyzed with respect to the structure of this four-residue epitope on PrP(C). Sixty-three compounds were tested for inhibition of PrP(Sc) formation in scrapie-infected mouse neuroblastoma cells (ScN2a). Two compounds, Cp-60 (2-amino-6-[(2-aminophenyl)thio]-4-(2-furyl)pyridine-3, 5-dicarbonitrile) and Cp-62 (N'1-(¿5-[(4, 5-dichloro-1H-imidazol-1-yl)methyl]-2-furyl¿carbonyl)-4 methoxybenzene-1-sulfonohydrazide), inhibited PrP(Sc) formation in a dose-dependent manner and demonstrated low levels of toxicity. A substructure search of the Available Chemicals Directory based on Cp-60 identified five related molecules, three of which exhibited activities comparable to Cp-60. Mimicking dominant negative inhibition in the design of drugs that inhibit prion replication may provide a more general approach to developing therapeutics for deleterious protein-protein interactions.

Metal chelating properties of adenylate kinase from Paracoccus denitrificans.

Zinc, a common element of adenylate kinases from Gram-positive bacteria, binds to a structural motif consisting of three or four cysteine residues, Cys-X2-Cys-X16-Cys-X2-Cys/Asp. The enzyme from Paracoccus denitrificans, a Gram-negative bacterium, has structural features much similar to those of adenylate kinases from Gram-positive organisms [Spurgin, P., Tomasselli, A.G., and Schiltz, E. (1989) Eur. J. Biochem., 179, 621-628]. However, adenylate kinase isolated from this bacterium was not reported to bind metal. These findings prompted us to clone the corresponding gene of P. denitrificans, and to characterize the enzyme overproduced in Escherichia coli. The deduced primary structure of adenylate kinase from P. denitrificans revealed two differences from that previously published: Cys was found at position 130 instead of His, and His was found at position 138 instead of Gly. The recombinant enzyme is a dimer which binds either zinc or iron, in a metal/monomer ratio of one. The dissociating sulfhydryl reagent, p-(hydroxy-mercuri)phenylsulfonate, released the metal from the protein, confirming that thiols are involved in zinc- or iron-binding. The iron-chelated form of recombinant P. denitrificans adenylate kinase, which is essentially under reduced form, transfers electrons to the oxidized cytochrome c. In conclusion, the absence of metal in the enzyme isolated from P. denitrificans is not related to the protein structure but most probably due to the physiological properties of the host organism.

Genetically engineered zinc-chelating adenylate kinase from Escherichia coli with enhanced thermal stability.

In contrast with adenylate kinase from Gram-negative bacteria, the enzyme from Gram-positive organisms harbors a structural Zn2+ bound to 3 or 4 Cys residues in the structural motif Cys-X2-Cys-X16-Cys-X2-Cys/Asp. Site-directed mutagenesis of His126, Ser129, Asp146, and Thr149 (corresponding to Cys130, Cys133, Cys150, and Cys153 in adenylate kinase from Bacillus stearothermophilus) in Escherichia coli adenylate kinase was undertaken for determining whether the presence of Cys residues is the only prerequisite to bind zinc or (possible) other cations. A number of variants of adenylate kinase from E. coli, containing 1-4 Cys residues were obtained, purified, and analyzed for metal content, structural integrity, activity, and thermodynamic stability. All mutants bearing 3 or 4 cysteine residues acquired zinc binding properties. Moreover, the quadruple mutant exhibited a remarkably high thermal stability as compared with the wild-type form with preservation of the kinetic parameters of the parent enzyme.

Structural and energetic factors of the increased thermal stability in a genetically engineered Escherichia coli adenylate kinase.

Several variants of Escherichia coli adenylate kinase, designed to bind a Zn2+ ion, were produced by site-directed mutagenesis. The metal binding and enzymatic properties of the engineered variants have been described (Perrier, V., Burlacu-Miron, S., Bourgeois, S., Surewicz, W. K., and Gilles, A.-M. (1998) J. Biol. Chem. 273, 19097-19101). Here we report the structural properties and stability changes in a 4-Cys variant which binds a Zn2+ ion and has an increased thermal stability. CD studies indicate a very similar secondary structure content in the wild type and the engineered variant. NMR analysis revealed that the topology of the parallel beta-sheet, belonging to the protein core, and of the peripheral antiparallel beta-sheet are also conserved. The small local changes observed in the neighborhood of the substitution sites reflect a more compact state of the metal-binding domain. The Zn2+-bound quadruple mutant shows an increased thermal stability, reflected in a 9 degreesC increase of the mid-temperature of the first cooperative unfolding step. Binding of a bisubstrate analog P1, P5-di(adenosine-5')-pentaphosphate increases, by about 7 degreesC, the midpoint of this transition in both wild type and modified variant. The NMR data suggest that the peripheral domains involved in substrate binding unfold during the first denaturation step. Urea denaturation experiments indicate an increased resistance against chemical unfolding of the Zn2+-binding variant. In contrast, the Gibbs free energy of unfolding (at physiologically relevant conditions) of the quadruple mutant is lower than that of the wild type.

A new non-heme iron environment in Paracoccus denitrificans adenylate kinase studied by electron paramagnetic resonance and electron spin echo envelope modulation spectroscopy.

Adenylate kinase from the Gram-negative bacterium Paracoccus denitrificans (AKden) has structural features highly similar to those of the enzyme from Gram-positive organisms. Atomic absorption spectroscopy of the recombinant protein, which is a dimer, revealed the presence of two metals, zinc and iron, each binding most probably to one monomer. Under oxidizing conditions, the electron paramagnetic resonance (EPR) spectrum of AKden at 4.2 K consists of features at g = 9.23, 4.34, 4.21, and 3.68. These features are absent in the ascorbate-reduced protein and are characteristic of a S = 5/2 spin system in a rhombic environment with E/D = 0.24 and are assigned to a non-heme Fe3+ (S = 5/2) center. The zero-field splitting parameter D (D = 1.4 +/- 0.2 cm-1) was estimated from the temperature dependence of the EPR spectra. These EPR characteristic as well as the difference absorption spectrum (oxidized minus reduced) of AKden are similar to those reported for the non-heme iron protein rubredoxin. Nevertheless, the redox potential of the Fe2+/Fe3+ couple in AKden was measured at +230 +/- 30 mV, which is more positive than the redox potential of the non-heme iron in rubredoxin. Binding of cyanide converts the iron from the high-spin (S = 5/2) to the low-spin (S = 1/2) spin state. The EPR spectrum of the non-heme Fe3+(S = 1/2) in the presence of cyanide has g values of 2.45, 2.18, and 1.92 and spin-Hamiltonian parameters R/lambda = 7. 4 and R/mu = 0.56. The conversion of the non-heme iron to the low-spin (S = 1/2) state allowed the study of its local environment by electron spin echo envelope modulation spectroscopy (ESEEM). The ESEEM data revealed the existence of 14N or 15N nuclei coupled to the low-spin iron after addition of KC14N or KC15N respectively. This demonstrated that iron in AKden has at least one labile coordination position that can be easily occupied by cyanide. Other possible magnetic interactions with nitrogen(s) from the protein are discussed.

Characterization of metal and nucleotide liganded forms of adenylate kinase by electrospray ionization mass spectrometry.

Complexes of adenylate kinase from Escherichia coli, Bacillus subtilis, and Bacillus stearothermophilus with the bisubstrate nucleotide analog P1,P5-di(adenosine 5')-pentaphosphate and with metal ions (Zn2+ and/or Mg2+) were analyzed by electrospray ionization mass spectrometry. P1,P5-di(adenosine 5')-pentaphosphate. adenylate kinase complex was detected in the positive mode at pH as low as 3.8. Binding of nucleotide to adenylate kinase stabilizes the overall structure of the protein and preserves the Zn2+ chelated form of the enzyme from the gram-positive organisms. In this way, it is possible in a single mass spectrometry experiment to screen metal-chelating adenylate kinases, without use of radioactively labeled compounds. Binding of Mg2+ to enzyme via P1,P5-di(adenosine 5')-pentaphosphate was also demonstrated by mass spectrometry. Although no amino acid side chain in adenylate kinase is supposed to interact with Mg2+, Asp93 in porcine muscle cytosolic enzyme, equivalent to Asp84 in the E. coli adenylate kinase, was proposed to stabilize the nucleotide.Mg2+ complex via water molecules.

Fatty acid and carotenoid composition of Rhodotorula strains.

Lipid content and composition of fatty acids with 6-25 carbon atoms were studied on strains of the 13 pink or red yeast species belonging to the genus Rhodotorula. The total amount of lipid represented an average of 13% of the dry weight. The neutral and polar lipid fractions were analyzed separately. For all the strains studied, the major fatty acids in both fractions were oleic, linoleic and palmitic acids, which formed 80% of the total number of fatty acids. A notable amount of arachidonic acid, a precursor of eicosanoid hormones, was found in R. acheniorum, R. aurantiaca and R. bacarum. Depending on the strain, 1-10 carotenoid pigments were detected; beta-carotene was always the major carotenoid present.

Zinc chelation and structural stability of adenylate kinase from Bacillus subtilis.

Adenylate kinase from Bacillus subtilis, like the enzyme from Bacillus stearothermophilus, contains a structural zinc atom. Cys153 in the enzyme from B. stearothermophilus, which is involved in the zinc coordination, is replaced in the adenylate kinase from B. subtilis by an aspartic acid residue. Therefore, we were interested in establishing whether this difference has an impact on the structure, the metal chelation, and the overall stability of these proteins. We also were interested in determining whether His138, which is conserved in many adenylate kinases, can act as a fourth partner in the metal chelation and, in general, whether His can successfully replace Cys or Asp in coordinating zinc in the adenylate kinase from B. subtilis. The adk gene from B. subtilis was cloned by polymerase chain reaction. The wild-type protein, together with several variants obtained by site-directed mutagenesis, were expressed in Escherichia coli and analyzed by biochemical and physicochemical methods. The H138N and D153C mutants of adenylate kinase from B. subtilis exhibited properties similar to those of the wild-type protein, indicating that His138 is not involved in metal coordination and that Asp153, just like Cys in the analogous position in the enzyme from B. stearothermophilus, can participate in zinc chelation. This is the first experimental evidence indicating that aspartic acid can be involved in the coordination of a structural zinc atom. On the other hand, the D153H and D153T variants showed significant changes in their zinc-binding properties. Dialysis of the latter proteins against buffer (in both the presence and the absence of 2 mM EDTA) resulted in removal of the metal ion and loss of enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Zinc, a structural component of adenylate kinases from gram-positive bacteria.

The recent finding that Bacillus stearothermophilus adenylate kinase contains a zinc atom coordinated to four cysteines prompted us to investigate the metal-binding properties of the enzyme from various bacteria. We conclude that zinc was present only in adenylate kinase from gram-positive species and that this property is correlated with the presence of three or four Cys residues in the sequence Cys-X2-Cys-X16-Cys-X2-Cys/Asp, in which X stands for different amino acid residues.