Reliability of deep branch of ulnar nerve identification in interosseous-to-ulnar motor nerve transfer: A cadaver study of 20 wrists.

Reinnervation of the intrinsic hand muscles after proximal ulnar nerve repair is often unsatisfactory. Promising results have nevertheless been reported recently for supercharged end-to-side anterior interosseous to deep branch of the ulnar nerve (DBUN) transfer. The aim of this study was to determine whether the DBUN can be reliably identified without retrograde intraneural dissection from Guyon's canal. Twenty cadaveric wrists were dissected. In a first stage, nerve transfer was performed through a limited 4 cm incision without releasing Guyon's canal. In a second stage, correct identification of the DBUN was assessed by retrograde intraneural dissection from its point of exit from Guyon's canal. The DBUN was correctly identified in 18 of the 20 wrists (90%). Although anatomical landmarks provide valuable clues, identifying the DBUN by neurolysis is technically challenging. All the elements required for nerve transfer can be exposed through a 4 cm incision, but the DBUN was nevertheless incorrectly identified in 10% of cases. Guyon canal release seems advisable to guarantee correct DBUN identification.

Flow cytometric analysis of intraplatelet VASP phosphorylation for the detection of clopidogrel resistance in patients with ischemic cardiovascular diseases.

Interindividual variability of the inhibitory effect of clopidogrel on platelet functions leading to clopidogrel resistance has been described in some patients with ischemic cardiovascular disease. A reliable laboratory test is therefore needed to identify patients insufficiently protected by this antiplatelet treatment. The phosphorylation of vasodilator-stimulated phosphoprotein (VASP), an intraplatelet actin regulatory protein, is dependent on the level of activation of the platelet P2Y12 receptor, which is targeted by clopidogrel. The aim of this study was to use a flow cytometric VASP phosphorylation assay to evaluate the efficacy of clopidogrel therapy. The platelet reactivity index (PRI), expressed as a percentage, is the difference in VASP fluorescence intensity between resting (+PGE1) and activated (+ADP) platelets. In vitro, the PRI was strongly correlated with the inhibition of platelet aggregation induced by specific blockade of the P2Y12 receptor by the competitive antagonist AR-C69931MX (R = 0.72, P < 0.0001). Ex vivo, the PRI was 78.3 +/- 4.6% in 47 healthy donors, 79.0 +/- 4.1% in 34 patients not receiving clopidogrel and 61.1 +/- 17.0% in 33 patients treated with clopidogrel (P < 0.0001). In the clopidogrel group, the PRI values were widely dispersed (from 6.6 to 85.8%) and more than 30% of these patients had a PRI equivalent of values in patients not receiving clopidogrel. The flow cytometric analysis of VASP phosphorylation seems to be a suitable test to evaluate the efficacy of clopidogrel treatment. This assay demonstrated a wide interindividual variability of the inhibitory response of platelets to clopidogrel and showed that one-third of the patients treated appeared to be 'unprotected' by this therapy.

Solution structure of the squash trypsin inhibitor MCoTI-II. A new family for cyclic knottins.

The "knottin" fold is a stable cysteine-rich scaffold, in which one disulfide crosses the macrocycle made by two other disulfides and the connecting backbone segments. This scaffold is found in several protein families with no evolutionary relationships. In the past few years, several homologous peptides from the Rubiaceae and Violaceae families were shown to define a new structural family based on macrocyclic knottin fold. We recently isolated from Momordica cochinchinensis seeds the first known macrocyclic squash trypsin inhibitors. These compounds are the first members of a new family of cyclic knottins. In this paper, we present NMR structural studies of one of them, MCoTI-II, and of a beta-Asp rearranged form, MCoTI-IIb. Both compounds display similar and well-defined conformations. These cyclic squash inhibitors share a similar conformation with noncyclic squash inhibitors such as CPTI-II, and it is postulated that the main effect of the cyclization is a reduced sensitivity to exo-proteases. On the contrary, clear differences were detected with the three-dimensional structures of other known cyclic knottins, i.e., kalata B1 or circulin A. The two-disulfide cystine-stabilized beta-sheet motif [Heitz et al. (1999) Biochemistry 38, 10615-10625] is conserved in the two families, whereas in the C-to-N linker, one disulfide bridge and one loop are differently located. The molecular surface of MCoTI-II is almost entirely charged in contrast to circulin A that displays a well-marked amphiphilic character. These differences might explain why the isolated macrocyclic squash inhibitors from M. cochinchinensis display no significant antibacterial activity, whereas circulins and kalata B1 do.

The formation of malodorous dimethyl oligosulphides in treated groundwater: the role of biofilms and potential precursors.

Water distributed from the Wanneroo Groundwater Treatment Plant intermittently contains dimethyl trisulphide (DMTS). The compound is responsible for a "swampy odour" in the water. DMTS production from potential precursors was insignificant in the absence of biofilms when compared with DMTS production from precursors in the presence of biofilms in a biofilm reactor. Greatest dimethyl disulphide (DMDS) and DMTS production (> 3000 ng L-1 DMTS) occurred in the reactors when supplied with methane thio-containing compounds, such as methionine, S-methyl cysteine and methyl-3-(methylmercapto)-propionate. Abiotic DMTS production from oligosulphides also occurred through the addition of the methylating agents, methyl iodide or methyl-p-toluene sulphonate. Significant DMTS production also occurred with Wanneroo water that contained added omega-thio-containing compounds such as cysteine (1400 ng L-1 DMTS), and 3-mercaptopropionate (210 ng L-1). Biomethylation, a ubiquitous response by microorganisms for the detoxification of toxic compounds, generated DMDS/TS from biofilm oligosulphides. Biofilms exposed to the toxic compounds selenate or 2,4,6-trichlorophenol methylated oligosulphides in addition to the toxins. Sodium sulphide also stimulated DMTS production. Easily Biodegradable Dissolved Organic Carbon (BDOC) probably contributed indirectly to DMTS production by the biofilms, although whether this was a result of its stimulation of greater microbial activity or consumption of oxygen, or both, remains unresolved. Stagnation of water in the biofilm reactors also increased DMTS production, which was concomitant with depletion of oxygen concentrations in the bulk water. Many processes, such as degradation of methane thio-containing compounds, methylation of sulphides and oligosulphides, and changes in contributions of different metabolic pathways upon depletion of oxygen concentrations upon water stagnation, probably contribute simultaneously to "swampy odour" production in the distribution system.

Conformation and ion channel properties of a five-helix Bundle protein.

The primary amphipathic peptide Ac-Met-Gly-Leu-Gly-Leu-Trp-Leu-Leu-Val-Leu10-Ala-Ala-Ala-Leu-Gln-Gly-Ala-Lys-Lys-Lys20-Arg-Lys-Val-NH-CH2-CH2-SH called SPM was able to induce formation of ion channels into planar lipid bilayers with main conductance values of 75 and 950 pS in 1 M KCl. The 75 pS value can be attributed to an aggregate composed of five monomers since the corresponding five-unit bundle (5-SPM) also presented a 70 pS channels under the same conditions. The upper 950 pS level would be generated by a hexameric aggregate. Ion channels induced by both SPM and its pentameric bundle are slightly cation selective but not voltage-dependent. The structural studies showed that the SPM and 5-SPM possess mainly an alpha-helical structure (approximately 40%) and are strongly embedded in the bilayer. This behaviour and the strong hydrophobic interactions occurring between helices in the bundle induce a strong stabilization of 5-SPM in the bilayer and would be responsible for the stepwise current fluctuations observed during the incorporation of 5-SPM into the membrane.

Squash trypsin inhibitors from Momordica cochinchinensis exhibit an atypical macrocyclic structure.

Three trypsin inhibitors (TIs), from the seeds of the squash Momordica cochinchinensis (MCo), have been isolated and purified using gel filtration, ion exchange chromatography, and reverse-phase HPLC. Their sequences could be determined only after proteolytic cleavages. In the case of MCoTI-I and -II, it was shown that their polypeptide backbones are cyclic, a structure that has never been described in squash TIs. They contain 34 amino acid residues with 3 disulfide bridges and measured molecular masses of 3453.0 and 3480.7, respectively. They are the largest known macrocyclic peptides containing disulfide bridges. Their sequences show strong homology to other squash TIs, suggesting a similar three-dimensional structure and an analogous mechanism of action. A model of MCoTI-II was constructed by analogy to the crystal structure of the complex between bovine trypsin and CMTI-I, indicating that the linker connecting the two termini is flexible and does not impose significant geometrical constraints. This flexibility allows an Asp-Gly peptide bond rearrangement to occur in this region, giving rise to two isoforms of MCoTI-II. Although the importance of cyclization is not clear, it might confer increased stability and resistance to proteolysis. A minor species, MCoTI-III, was also characterized as containing 30 amino acid residues with a molecular mass of 3379.6. This component possesses a linear backbone with a blocked N-terminus. MCoTIs represent interesting candidates for drug design, either by changing their specificity of inhibition or by using their structure as natural scaffolds bearing new binding activities.

Min-21 and min-23, the smallest peptides that fold like a cystine-stabilized beta-sheet motif: design, solution structure, and thermal stability.

Small disulfide-rich proteins provide examples of simple and stable scaffolds for design purposes. The cystine-stabilized beta-sheet (CSB) motif is one such elementary structural motif and is found in many protein families with no evolutionary relationships. In this paper, we present NMR structural studies and stability measurements of two short peptides of 21 and 23 residues that correspond to the isolated CSB motif taken from a 28-residue squash trypsin inhibitor. The two peptides contain two disulfide bridges instead of three for the parent protein, but were shown to fold in a native-like fashion, indicating that the CSB motif can be considered an autonomous folding unit. The 23-residue peptide was truncated at the N-terminus. It has a well-defined conformation close to that of the parent squash inhibitor, and although less stable than the native protein, it still exhibits a high T(m) of about 100 degrees C. We suggest that this peptide is a very good starting building block for engineering new bioactive molecules by grafting different active or recognition sites onto it. The 21-residue peptide was further shortened by removing two residues in the loop connecting the second and third cysteines. This peptide exhibited a less well-defined conformation and is less stable by about 1 kcal mol(-)(1), but it might be useful if a higher flexibility is desired. The lower stability of the 21-residue peptide is supposed to result from inadequate lengths of segments connecting the first three cysteines, thus providing new insights into the structural determinants of the CSB motif.

Design and synthesis of a peptide derived from positions 195-244 of human cdc25C phosphatase.

We have designed, synthesized and purified a 51 amino acid peptide derived from an essential domain of human cdc25C phosphatase. In vivo, differential phosphorylation of this domain regulates either the induction of mitotic processes, or the checkpoint arrest of eukaryotic cells in response to DNA damage. Peptide synthesis was achieved using the stepwise Fmoc strategy and resulted in an important yield of highly pure peptide. The final peptide was identified by amino acid analysis, electrospray mass spectrometry and nuclear magnetic resonance, which revealed that one of the two methionines within the peptide was oxidized into its sulphoxide derivative We investigated whether this 51 amino acid peptide folded into secondary structures in solution by circular dichroism and observed the formation of alpha helices in TFE. Finally, we verified that this peptide could bind to its biologically relevant 14-3-3 partner in vitro by fluorescence spectroscopy.

Interactions of primary amphipathic vector peptides with membranes. Conformational consequences and influence on cellular localization.

The conformations of two peptides produced by the combinations of a nuclear localization sequence and a sequence issued from the fusion protein gp41 of HIV 1 have been analyzed both in solution and in membranes or in membrane mimicking environments. Both are shown to be nonordered in water, alpha-helical when incorporated into SDS micelles where the helical domain concerns the hydrophobic part of the peptides. Interactions with lipids induce the formation of beta-sheet and the lipid-peptide interactions are governed by the nature of the lipid polar headgroups. A monolayer study shows that replacement of the sequence separating the two sequences with an arginine favors the lipid-peptide interactions which may contribute to the understanding of the different, nuclear and membrane associated, cellular localizations of the peptides.

Folding of the squash trypsin inhibitor EETI II. Evidence of native and non-native local structural preferences in a linear analogue.

A peptide, corresponding to the entire sequence of the squash trypsin inhibitor EETI II (Ecballium elaterium trypsin inhibitor) in which the six cysteines, engaged in three disulphide bridges in native EETI II, have been replaced by six serines, has been synthesised. CD, Fourier-transform infrared spectroscopy (FTIR) and 1H-NMR studies of this peptide revealed that some secondary structures present in native EETI II are still populated in the absence of disulphide bonds. Native-like secondary structures were observed for segments 10-15 (helix), 16-19 and 22-25 (reverse turns) but no native tertiary interaction was detected. However, a non-native local interaction between the aromatic ring of Phe26 and the amide group of Gly28 was observed. It is hypothesised that the 10-15, 16-19 and 22-25 native-like local conformations could play a major role in the early folding of EETI II.

Body protein and lipid deficit in tumour-bearing rats in relation to age.

Cancer cachexia is among the most dramatic situations of depletion in body energy reserves. To ascertain whether the pattern of body composition alteration during tumour development is influenced by aging as in uncomplicated starvation, we compared the difference of body composition between Yoshida sarcoma bearing rats and young (200 g, 7 weeks) and adult (400 g, 13 weeks) control rats. After the same duration of tumour bearing, mass and composition of tumours were similar in adult and young rats, indicating that they are independent of host age. Food intake decreased to a remarkably similar value in both young and adults. Body water content was elevated in hosts of both ages. The relative deficit of body lipid vs controls was similar for both, the absolute lipid deficit being therefore larger in adult than in young tumour-bearing rats (14.3 +/- 4.4 g vs 6.8 +/- 0.9 g; P < 0.01). In contrast, there was a relatively larger deficit of body protein in young rats. Paradoxically, these rats still maintained a positive nitrogen balance whereas this balance was negative in adult tumour-bearing rats. In conclusion, as previously shown in uncomplicated undernutrition, the anorexia induced by Yoshida sarcoma development is still associated with some protein accretion in young rats whereas cachexia develops in adults.

Characterization and 2D NMR study of the stable [9-21, 15-27] 2 disulfide intermediate in the folding of the 3 disulfide trypsin inhibitor EETI II.

The three disulfide Ecballium elaterium trypsin inhibitor II (EETI II) reduction with dithiothreitol (DTT) and reoxidation of the fully reduced derivative have been examined. A common stable intermediate has been observed for both processes. Isolation and sequencing of carboxymethylated material showed that the intermediate lacks the [2-19] bridge. The NMR study showed a very strong structural conservation as compared to the native EETI II, suggesting that the bridges are the [9-21] and [15-27] native ones. The differences occurred in sections 2-7 (containing the free cysteine 2 and the Arg 4-Ile 5 active site) and 19-21 (containing the second free cysteine). Distance geometry calculations and restrained molecular dynamics refinements were also in favor of a [9-21, 15-27] arrangement and resulted in a well-conserved (7-28) segment.

[Evaluation of the cost-efficacy ratio of Pall BB 22 15 filters for the bacterial protection of anesthesia circuits]. / Evaluation du rapport coût-efficacité des filtres Pall BB 22 15 dans la protection bactérienne des circuits d'anesthésie.

The aim of this study is to assess wether the use of the bacteriological filter Pall BB 22 15 placed on the Y piece of the anesthesia equipment decreases contamination and furthermore to evaluate the cost of this practice versus changing anesthetic circuits after every patient. Randomized trials are conducted with three "Engström" machines in three cardiac surgery operating rooms. The Y pieces were examined with qualitative and quantitative bacteriological analysis. Use filters is less expensive than changing circuit for each patient for a comparable efficacy.

1H n.m.r. conformational studies on the C-terminal octapeptide of oxyntomodulin, a beta-turn locked by a salt bridge.

The octapeptide Lys-Arg-Asn-Lys-Asn-Asn-Ile-Ala (Arg4 in the human sequence) is the C-terminal part of porcine oxyntomodulin, an endogeneous peptide which is a potent inhibitor of stimulated acid secretion. This octapeptide exhibits the whole range of biological activities of the parent hormone. In the present work we report an 1H n.m.r. investigation of the conformational properties of the octapeptides of pig and human sequences in dimethylsulfoxide-d6 (DMSO) solution. The various resonances were assigned on the basis of two-dimensional COSY and NOESY experiments. Other experiments such as (i) temperature and concentration dependence of the amide proton chemical shifts, (ii) effects of ionic strength, (iii) comparison of the spectra with different analogues, were performed. We showed that in DMSO, the conformation of the octapeptide is directly related to the ionisation state of the C-terminus carboxyl group of alanine. In carboxylic state, the peptide adopts an extended conformation, while in the carboxylate state the four last residues (Asn-Asn-Ile-Ala) are involved in a type II beta-turn structure probably locked by a salt bridge between the carboxyl group of Ala8 and the epsilon ammonium group of Lys4 (or the guanidinium group of Arg4). These observations provide an insight into the possible conformational tendencies of this peptide in biological media.

Low dose heparin versus low molecular weight heparin (Kabi 2165, Fragmin) in the prophylaxis of thromboembolic complications of abdominal oncological surgery.

Eighty patients undergoing pelvic or abdominal surgery for cancer were randomized in two groups for prevention of postoperative thromboembolism: 40 patients received a 15,000 IU day-1 Calciparine prophylaxis and 40 patients a 5000 anti-Xa U/d Fragmin prophylaxis for 10 days. In the Calciparine group, two patients (5%) developed postoperative pulmonary embolism but none developed it in the Fragmin group. Two patients in the Fragmin group (5%) developed isotopic DVT, which was not confirmed by phlebography. There was no deep vein thrombosis of the lower limbs in the two groups. Important postoperative bleeding (one patient in the Calciparine group and two patients in the Fragmin group) was similar in both groups. Moderate and minor bleeding were significantly lower in the Fragmin group. Haemoglobin and haematocrit changes, total blood loss and transfusion requirements were not different in both groups. It is concluded that, over a 10-day period, one daily 5000 U Fragmin prophylaxis was as effective and safe as three daily 5000 IU Calciparine injections.

Peptides mimicking the flap of human renin: synthesis, conformation, and antibody recognition.

Four peptides related to human renin flap region have been synthesized. Two of them are ring closed through appropriately designed disulfide bridges. Structure analysis involving IR and NMR techniques and recognition by polyclonal human renin antibodies provides support for a beta-hairpin secondary structure of the cyclized peptides identical with that presented by the flap section in the speculative human renin model [Blundell, T., Sibanda, B. L., & Pearl, L. (1983) Nature (London) 304, 273-275; Sibanda, B. L., Blundell, T., Hobart, P. M., Fogliano, M., Bindra, J. S., Dominy, B. W., & Chirgwin, J. M. (1984) FEBS Lett. 174, 102-111].

[Diffuse leiomyomatosis of the stomach]. / Diffuse Leiomyomatose des Magens.

Sonography and computed tomography were the first imaging methods enabling the visualization of intramurally growing tumours of the stomach. The article reports on a rare case of a diffuse leiomyoma that was diagnosed by means of computed tomography.

[Ductus arteriosus in neonatal cyanotic cardiopathies. Value of the recording of the cutaneous partial pressure of oxygen]. / Le canal artériel dans les cardiopathies cyanogènes néonatales. Intérêt de l'enregistrement de la pression partielle cutanée en oxygène.

Adaptation to extrauterine life is always precarious in the newborn with cyanotic cardiac malformations. Limited survival is possible in cases with obstructive lesions of the right heart providing ductal closure is delayed. This was monitored in 3 cyanotic neonates with ductal dependant cardiac malformations (tricuspid atresia, transposition of the great arteries with atresia or severe stenosis of the pulmonary valve) by continuous measurement of the cutaneous pO2. The value of cutaneous pO2 monitoring has already been established. It is a reliable indicator of arterial pO2 in the neonatal period. Its major advantage, apart from the possibility of continuous monitoring, is that it is non-invasive and may be performed at the bedside. Analysis of curves recorded over several hours or days showed the cyclical nature of cutaneous pO2, probably related to slow variations in ductal diameter equilibrating effective pulmonary flow, arterial pO2 and ductal constriction. Prolonged hyperoxygenation of these desaturated children does not usually improve their condition; progressive deterioration due to the constrictive effect of oxygen on the ductus may be observed. When a clear decrease in the amplitude of oscillation is recorded ductal closure is imminent and palliative surgery should be undertaken whenever possible.