[Evaluation of an "Emergency Thrombosis" care system in a university-hospital department of general emergencies]. / Évaluation de la mise en place d'une filière de soins « Urgence Thrombose ¼ dans un service hospitalo-universitaire d'Urgences générales.

GOAL: Describe the use of diagnostic, prognostic and therapeutic algorithms for venous thromboembolism (VTE), derived from the 2014 European guidelines, in a teaching hospital's emergencies department and compare two groups: the 2015 group "without a care path" and the 2017 group "with a care path". METHOD: Comparative and retrospective study of the characteristics of emergencies department patients admitted for VTE from January to June 2015 for the 2015 group and from January to June 2017 for the 2017 group. RESULTS: Seventy-nine patients were included in the 2015 group and 62 patients in the 2017 group. In 24% of cases a clinical probability rule was calculated in the 2017 group (vs. no score in 2015, P<0.05). In the 2015 group, 10% of patients did not have a D-Dimer measurement in case of low clinical probability (vs. 0% in 2017, P<0.05). For both groups, the severity score sPESI was not noted in the medical record. All patients with pulmonary embolism were hospitalized in both groups. A total of 36% of patients with deep vein thrombosis (DVT) were hospitalized in the 2015 group (vs. none in 2017, P<0.05). A total of 52.5% of patients were treated with direct oral anticoagulants (DOAS) in the 2017 group vs. 32.5% in the 2015 group (P<0.05). In 18% of cases DOAS were prescribed by emergency physicians in the 2017 group vs. 2.5% in the 2015 group (P<0.05). Mean hospital stay was 7.4 days in the 2017 group and 9.4 days in the 2015 group (P<0.05). CONCLUSION: We observed a change in clinical practices and prescriptions after the establishment of an "Emergency Thrombosis" care system. Indeed, improvement in the calculation of the clinical probability score, increase in the outpatient management of DVT, increase in prescribing DOAS and reducing the length of hospital stay were the main revisions. The implementation of standardized digitally calculated clinical and prognostic probability scores would optimize this care path, as well as allow a better distribution of the post-emergency consultations created for outpatients.

Purification of elongation factors EF-Tu and EF-G from Escherichia coli by covalent chromatography on thiol-sepharose.

The elongation factors EF-Tu and EF-G of Escherichia coli are involved in the transport of aminoacyl-tRNA to ribosomes and the translocation of ribosomes on mRNA, respectively. Both possess cysteine residues that are important for activity. We took advantage of this property to design a purification protocol based on thiol-Sepharose chromatography, a method involving thiol-disulfide interchange between protein thiol groups and the glutathione-2-pyridyl-disulfide conjugate of the affinity resin. Bacterial cells were lysed by a lysozyme-EDTA method, and the lysate supernatant was purified by chromatography on, first, DEAE-Sephacel and, then thiol-Sepharose. Both elongation factors were purified in a single procedure, since DEAE-Sephacel fractions containing both factors were loaded on the thiol-Sepharose column. Thiol-Sepharose chromatography efficiently separates each elongation factor from all contaminating proteins. The purified elongation factors were characterized by SDS-PAGE, protein sequencing, and biological activity. The specific reactivities of the elongation factors with thiol-Sepharose allow their efficient purification and suggest that they possess hitherto undiscovered properties connected with their reactive thiols.

Molecular identification of the role of voltage-gated K+ channels, Kv1.5 and Kv2.1, in hypoxic pulmonary vasoconstriction and control of resting membrane potential in rat pulmonary artery myocytes.

Hypoxia initiates pulmonary vasoconstriction (HPV) by inhibiting one or more voltage-gated potassium channels (Kv) in the pulmonary artery smooth muscle cells (PASMCs) of resistance arteries. The resulting membrane depolarization increases opening of voltage-gated calcium channels, raising cytosolic Ca2+ and initiating HPV. There are presently nine families of Kv channels known and pharmacological inhibitors lack the specificity to distinguish those involved in control of resting membrane potential (Em) or HPV. However, the Kv channels involved in Em and HPV have characteristic electrophysiological and pharmacological properties which suggest their molecular identity. They are slowly inactivating, delayed rectifier currents, inhibited by 4-aminopyridine (4-AP) but insensitive to charybdotoxin. Candidate Kv channels with these traits (Kv1.5 and Kv2.1) were studied. Antibodies were used to immunolocalize and functionally characterize the contribution of Kv1. 5 and Kv2.1 to PASMC electrophysiology and vascular tone. Immunoblotting confirmed the presence of Kv1.1, 1.2, 1.3, 1.5, 1.6, and 2.1, but not Kv1.4, in PASMCs. Intracellular administration of anti-Kv2.1 inhibited whole cell K+ current (IK) and depolarized Em. Anti-Kv2.1 also elevated resting tension and diminished 4-AP-induced vasoconstriction in membrane-permeabilized pulmonary artery rings. Anti-Kv1.5 inhibited IK and selectively reduced the rise in [Ca2+]i and constriction caused by hypoxia and 4-AP. However, anti-Kv1.5 neither caused depolarization nor elevated basal pulmonary artery tone. This study demonstrates that antibodies can be used to dissect the whole cell K+ currents in mammalian cells. We conclude that Kv2. 1 is an important determinant of resting Em in PASMCs from resistance arteries. Both Kv2.1 and Kv1.5 contribute to the initiation of HPV.

Chaperone properties of bacterial elongation factor EF-Tu.

Elongation factor Tu (EF-Tu) is involved in the binding and transport of the appropriate codon-specified aminoacyl-tRNA to the aminoacyl site of the ribosome. We report herewith that the Escherichia coli EF-Tu interacts with unfolded and denatured proteins as do molecular chaperones that are involved in protein folding and protein renaturation after stress. EF-Tu promotes the functional folding of citrate synthase and alpha-glucosidase after urea denaturation. It prevents the aggregation of citrate synthase under heat shock conditions, and it forms stable complexes with several unfolded proteins such as reduced carboxymethyl alpha-lactalbumin and unfolded bovine pancreatic trypsin inhibitor. The EF-Tu.GDP complex is much more active than EF-Tu.GTP in stimulating protein renaturation. These chaperone-like functions of EF-Tu occur at concentrations that are at least 20-fold lower than the cellular concentration of this factor. These results suggest that EF-Tu, in addition to its function in translation elongation, might be implicated in protein folding and protection from stress.

Induction of stress proteins by tobacco smoke in human monocytes: modulation by antioxidants.

Tobacco smoke (TS) induced in human monocytes the synthesis of both the classical heat shock proteins (HSP) (Hsp70, Hsp90, Hsp110) and the oxidation-specific stress protein (SP) heme oxygenase (HO). To determine the role of reactive oxygen species in SP induction by TS, we incubated the monocytes with various antioxidants before exposure to TS. Quercetin and N-acetylcysteine (NAC) both prevented the induction of HO by TS but not, or less so, than that of the classical HSP, while the nitric oxide synthase inhibitor L-nitroarginine had no effect. Thus, at least two mechanisms appear involved in SP induction by TS; (i) the induction of HO (oxidation-dependent), which was prevented by quercetin and NAC; and (ii) the induction of Hsp70, which was, at least in part, oxidation-independent. SP induction might represent an adequate biosensor for TS and other radical-mediated environmental exposures.

Defect in export and synthesis of the periplasmic galactose receptor MglB in dnaK mutants of Escherichia coli, and decreased stability of the mglB mRNA.

The high-affinity galactose permease, which comprises the periplasmic galactose receptor MglB, the membrane translocator MglC and the membrane-associated ATPase MglA, displayed a reduced activity in a dnaK temperature-sensitive mutant of Escherichia coli. This reduced transport activity correlated with a reduction in the quantity of MglB. At 42 degrees C, an accumulation of pre-MglB in the dnaK temperature-sensitive mutant reflected a defect in MglB export. In addition, an accumulation of pre-MglB in secB, secA and secY mutants suggested that SecB and the Sec translocase are also involved in export of the periplasmic galactose receptor. At 30 degrees C, there was no accumulation of pre-MglB in the dnaK mutant, but there was still a decreased amount of MglB in the periplasm. The reduction in MglB expression was not the result of a decrease in its stability, nor was it the result of a general defect in translation or transcription, since the MglA protein (which is expressed from the same operon as MglB) was synthesized in normal amounts. Two mRNAs are implicated in the expression of the mgl genes, a polycistronic mglBAC mRNA, and a more stable and more abundant mglB mRNA, produced by 3'-5' degradation of the mglBAC mRNA (R. W. Hogg, C. Voelker & I. von Carlowitz, 1991, Mol Gen Genet 229, 453-459). The mglB mRNA is protected against exonucleases by a REP (Repetitive Extragenic Palindrome) sequence located at its 3' extremity, which is responsible for the higher expression of MglB compared to MglA and MglC. The decreased MglB expression in the dnaK mutant at 30 degrees C in the present work correlated with a reduced stability of the mglB mRNA, which may have resulted from a defective stabilization by the REP sequence, or from a defect in translation of the mglB gene.

Reversal by GroES of the GroEL preference from hydrophobic amino acids toward hydrophilic amino acids.

The chaperones GroEL/hsp60 are present in all prokaryotes and in mitochondria and chloroplasts of eukaryotic cells. They are involved in protein folding, protein targeting to membranes, protein renaturation, and control of protein-protein interactions. They interact with many polypeptides in an ATP-dependent manner and possess a peptide-dependent ATPase activity. GroEL/hsp60 cooperates with GroES/hsp10, and the productive folding of proteins by GroEL generally requires GroES, which appears to regulate the binding and release of substrate proteins by GroEL. In a recent study, we have shown that GroEL interacts preferentially with the side chains of hydrophobic amino acids (Ile, Phe, Val, Leu, and Trp) and more weakly with several polar or charged amino acids, including the strongest alpha-helix and beta-sheet formers (Glu, Gln, His, Thr, and Tyr). In this study, we show that GroES reduces the specificity of GroEL for hydrophobic amino acids and increases its specificity for hydrophilic ones. This shift by GroES of the GroEL specificity from hydrophobic amino acids toward hydrophilic ones might be of importance for its function in protein folding.

Localization of DnaK (chaperone 70) from Escherichia coli in an osmotic-shock-sensitive compartment of the cytoplasm.

The chaperone DnaK can be released (up to 40%) by osmotic shock, a procedure which is known to release the periplasmic proteins and a select group of cytoplasmic proteins (including thioredoxin and elongation factor Tu) possibly associated with the inner face of the inner membrane. As distinct from periplasmic proteins, DnaK is retained within spheroplasts prepared with lysozyme and EDTA. The ability to isolate DnaK with a membrane fraction prepared under gentle lysis conditions supports a peripheral association between DnaK and the cytoplasmic membrane. Furthermore, heat shock transiently increases the localization of DnaK in the osmotic-shock-sensitive compartment of the cytoplasm. We conclude that DnaK belongs to the select group of cytoplasmic proteins released by osmotic shock, which are possibly located at Bayer adhesion sites, where the inner and outer membranes are contiguous.

Specific interaction of the Escherichia coli chaperone GroEL (60-KDA heat shock protein) with the liganded form of the galactose binding protein.

The Escherichia coli chaperone GroEL interacts more strongly with the liganded form of the galactose binding protein (the galactose binding protein-galactose complex), than with its unliganded form. This specific interaction is reflected by the stimulation of the ATPase activity of GroEL by the liganded galactose binding protein. Interactions between native proteins and chaperones could be more frequent than generally suspected, and may help to detect protein conformational changes.

The MglA component of the binding protein-dependent galactose transport system of Salmonella typhimurium is a galactose-stimulated ATPase.

Binding protein-dependent transport systems mediate the accumulation of several ions, sugars, amino acids, and peptides in Gram-negative bacteria by using the energy of ATP hydrolysis and belong to a superfamily of membrane proteins which extends to eukaryotic cells and includes the multidrug resistance P-glycoprotein and the cystic fibrosis transmembrane conductance regulator. The binding protein-dependent galactose transport system of Salmonella typhimurium comprises four proteins which have been characterized previously by molecular cloning experiments (51,000-dalton MglA protein, with a stable proteolytic product of 38,000 daltons, 33,000-dalton MglB protein, 29,000-dalton MglC protein, 21,000-dalton MglE protein). By using a MglA hyperproducing strain, we have purified a galactose-stimulated ATPase which shows a single band in polyacrylamide gels under nondenaturing conditions and shows three bands at 51,000, 38,000, and 15,000 daltons on sodium dodecyl sulfate-polyacrylamide gels (our results suggest that the bands at 38,000 and 15,000 daltons represent proteolytic products of the 51,000-dalton protein). The ATPase activity coincides with the purified protein during the two last chromatographic steps of the purification procedure, and it cannot be isolated from a strain which does not contain the mglA gene. The MglA ATPase is stimulated 3-fold by galactose and hydrolyzes ATP to ADP and Pi (Km ATP = 60 microM, Ka galactose = 0.3 mM, Vmax = 140 nmol/min/mg of protein). The gamma-phosphate of ATP is transferred neither to galactose nor to the protein itself. Vanadate, N-ethylmaleimide and 5-methoxyindole-2-carboxylic acid, a specific inhibitor of binding protein-dependent transport systems, inhibit the MglA ATPase.

Reconstitution of the binding protein-dependent galactose transport of Salmonella typhimurium in proteoliposomes.

Binding protein-dependent transport systems mediate the accumulation of diverse substrates in bacteria. The binding protein-dependent galactose transport of Salmonella typhimurium has been reconstituted in proteoliposomes. The proteoliposomes were made with proteins solubilized and renatured from inclusion bodies produced by a bacterial strain containing a plasmid with the mgl (methylgalactose permease) operon of Salmonella typhimurium. Galactose transport is dependent both on the addition of the purified galactose binding protein to the transport assay, and on ATP. The interaction between the liganded galactose binding protein and proteoliposomes displays Michaelis type kinetics with a Km of around 15 microM. Galactose transport is coupled to ATP hydrolysis with a stoichiometry (ATP/galactose) of 2.5:1. Galactose transport in proteoliposomes is not significantly inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone, but is inhibited by 0.5 mM vanadate. The present reconstitution of galactose transport in proteoliposomes suggests that the MglA, MglC and MglE proteins have been solubilized and renatured in an active form from the inclusion bodies of the mgl hyperproducing strain.