Biomedical subjects
P Robert
Publications and source records attributed to P Robert.
Altered filterability of fresh sickle cell trait donor blood.
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Mapping of HCG-receptor complexes.
Molecular forms of the porcine LH/CG receptor (pLHR) and complexes between hCG and either the full-length pLHR or its extracellular domain (ectodomain) have been produced in various recombinant systems. In COS cells and in the baculovirus insect cells system, the co-expression of the ecto- and endo-domains reconstituted a functional receptor where the association of the two domains seems to depend upon the presence of disulfide bridges. According to previous observations [39], synthetic peptides mimicking three regions of the ectodomain (21-38, 100-115, 250-272) were found to inhibit hormone binding and stimulation of cAMP production. Antisera raised against these peptides contained anti-peptide antibodies (Ab) able to interfere with hormone signalling. Moreover, the results of peptide mapping indicated that some peptides stretches may be more involved in signalling rather than in binding. Immunochemical mapping based on monoclonal antibodies (mAbs) was used to probe the hCG-ectodomain complex. It appeared that mAbs directed to epitopes present on the 'beta-tip' of hCG (assembled from the beta subunit loops 3 and 1, and previously designated site IIIb) and on the 'alpha-tip' (alpha subunit loops 1 and 3, site IIIa) bound to hCG-receptor complexes, whereas a conformational epitope (defined by the alpha-beta interface between beta seat belt C-terminus and alpha loop 2, site II) was masked. Interestingly, we and others previously reported that, in the hCG-full length receptor complex, site IIIa was shielded to mAb binding. A peptide mimicking the second extracellular loop (EL2) of the receptor endodomain was found to prevent the binding of a mAb directed to site IIIa, suggesting that this region of the endodomain may be interacting with the 'alpha-tip'. In the full-length, membrane anchored pLHR, the EL2 peptide inhibited hCG-induced cAMP production, but not binding. The possibility of inhibiting stimulation without inhibition of binding gives support to the 'negative specificity' hypothesis [6]. Thus, the ectodomain of the glycoprotein hormone receptors might be considered as a screening device preventing access of any glycoprotein hormone to the signalling peptide keys of the endodomain, which otherwise would be sensitive to any alpha subunit stimulation. Finally, antibody binding to site IIIa on the hCG-ectodomain complex was also hindered by an anti-peptide mAb directed against a peptide encoded by the eighth exon (pE x 8) of the LHR. This suggests that pEx8 is vicinal to the alpha-tip of hCG and to EL2 in the hCG-full length receptor complex. Altogether, these observations help to build up a topological model of the hCG-receptor complex.
Changes in liver core temperature during preservation and rewarming in human and porcine liver allografts.
Liver core temperature during organ procurement, storage, and rewarming has not been reported in human orthotopic liver transplantations (OLT). We have shown in the rat that optimal temperature for liver storage is not 4 degrees C but 0 degree C to 1 degree C. Therefore, a study was undertaken in humans and in pigs to determine the pattern of temperature change during OLT. The porcine studies were performed, because it was not possible to follow human grafts during the period that they were sterilely packaged. Temperature depression in humans was rapid during organ perfusion, remained stable during organ dissection, and decreased again slightly, when after excision, the organ was perfused again. Temperature depression during the period of perfusion with University of Wisconsin (UW) solution was curvilinear with the initial rapid temperature depression followed by a period of slower temperature depression. Volume perfused versus time was linear during these periods and the relationship between temperature depression and volume infused was curvilinear. At the time of packaging, 65 +/- 12 minutes after start of cold perfusion, the liver core temperature was 5.7 degrees C +/- 1.3 degrees C. Studies in the pig showed that it took 75 to 100 minutes for liver core temperature to decrease below 5 degrees C, and core temperature reached a plateau at 1 degree C at 195 +/- 75 minutes after packaging. During the rewarming period in humans, while vascular anastomoses were being constructed, there was a rapid linear increase in temperature from 0.8 degree C, when the graft was removed from the cold, to 17.2 degrees C +/- 3.1 degrees C at 45.5 +/- 4.4 minutes later, just before portal reperfusion commenced. These studies show that it takes only a short time to cool livers down to 10 degrees C, but after flushing is stopped, temperature depression is markedly reduced, and ideal temperatures are not reached before packaging. Rewarming of livers during performance of vascular anastomoses is rapid and reaches temperatures at which substantial hepatic metabolism is occurring.
[Serotonin syndrome in acute poisoning with antidepressive agents].
We report a case of severe serotonin syndrome after self-poisoning with two antidepressant drugs, paroxetine (a selective inhibitor of serotonin reuptake) and moclobemide (a reversible inhibitor of MAO-A). The serotonin syndrome is characterized by neuromuscular, behavioural, and autonomic changes. It occurs with the use of drugs able to increase serotonergic transmission in brain by acting on biosynthesis, reuptake, catabolism or release of serotonin. Treatment is symptomatic. The incidence of severe cases seems to have increased, probably due to the use of new antidepressant "specific" inhibitors of the serotonin reuptake.
Assessment of qualitative functional parameters of stored red blood cells from donors with sickle cell trait (AS) or with heterozygote (AC) status.
We have compared the storage quality of three groups of 10 red blood cell concentrates of the AA, AS and AC haemoglobin phenotype. The 30 samples were drawn on CPD on day 0, and were centrifuged 6 hours later. Leukocytes were removed on D1 and the samples were kept at +4 degrees C for 42 days in SAG-mannitol. Viability tests were performed using a series of in vitro tests, including: ATP, 2,3-DPG, K+, glucose, lactic acid, pH, osmotic fragility tests and erythrocytic morphology determination. Results demonstrated a good functional quality for AS and AC haemoglobin RBCs. Higher 2,3-DPG levels were found in Hb AS and Hb AC. A lower osmotic fragility in Hb AC and AS RBCs versus Hb AA was observed; no significant difference was found in terms of ATP levels and other parameters. In addition no variation in S and C haemoglobin levels and no sickling were observed. In conclusion, these results indicate an overall good quality for haemoglobin AS and AC RBCs. Further in vivo studies must now be performed in order to confirm the transfusional quality of haemoglobin AS and AC RBCs.
Characterization of a monoclonal antibody reacting with the free human luteinizing hormone beta-subunit.
Measurement of serum luteinizing hormone (hLH) is important for the detection and follow-up of patients with pathological processes of the reproductive axis. Detection of the uncombined form of the beta-subunit of hLH, or free hLH beta, has proved to be of clinical interest in the recognition of gonadotroph adenomas. As no monoclonal antibody specific for the free hLH beta is at present available, we elicited monoclonal antibodies using free hLH beta as an immunogen. An antibody, named BLH01, was selected for its specific binding to the free beta-subunit, and its antibody-binding site was characterized at the molecular level, emphasizing the importance of amino acid residues located between the disulfide-bonded Cys93 and Cys100. This region has been demonstrated as being particularly critical for the specific binding of lutropic hormones to their receptors. Topographic assignment of the epitope recognized by BLH01 was then achieved by cross-matching studies based on a library of antibodies directed to hLH beta, and the location of some epitopes on the three-dimensional model of the beta-subunit is proposed. A two-site immunoassay based on BLH01 as capture antibody was then developed. This assay, using BLH01, may constitute a simple, sensitive and highly specific procedure for assessing the clinical usefulness of measuring free hLH beta, particularly for the diagnosis and follow-up of patients presenting with pituitary adenomas.
[Fatty acid composition and cholesterol content in naturally canned jurel, sardine, salmon, and tuna].
To obtain more information about fatty acid profile and cholesterol content of fat extracted from canned fish in brine habitually consumed in Chile, four different species Jurel (Trachurus murphyi), Sardine (Sardinops sagax), Salmon (Oncorhynchus kisutch) and Tuna (Thunnus alalunga) were analyzed. The GLC of fatty acid methyl esters showed that the main group of fatty acids belongs to polyunsaturated, being omega-3 family the more important. The principal representants were eicosapentaenoic (EPA) and docosahexaenoic acids (DHA), with percentages between 5%-11% and 12%-22% respectively. Omega-6 family was represented mainly by arachidonic acid (AA) with percentages between 2%-4%. Cholesterol content was similar to the values found in other animal origen meats. The figures were between 41-86 mg of cholesterol per 100 g of edible product, Tuna in brine, was the product with the lowest content of cholesterol. The calculated amount of EPA, DHA and total omega-3 fatty acids indicated values between 95-604, 390-1163 and 609-2775 mg respectively per 100 g of edible product. Due these results is important to emphasize the consumption of this type of canned fish in brine, that they really represent a good dietary source of mainly polyunsaturated omega-3 fatty acids. The international recommendations indicate to increase the consumption of fish, due the beneficial effects described in relation with cardiovascular disease, which is the mean cause of death in Chile, country with a wide variety of marine origen foods, but with a contradictory answer about its consumption which is not incorporated in the current diet.
[Combination of propofol-ketamine-vecuronium for total intravenous anesthesia under hazardous conditions].
A total intravenous technique using propofol, ketamine and vecuronium was successfully used on 29 patients treated for elective surgery at the UNPROFOR French Medical Group (Sarajevo, Bosnia Herzegovina). Operative conditions were satisfactory for the surgeons. Recovery was fast and emergence reactions very limited. No hypoxaemia was observed during the immediate postoperative period. The use of a propofol/ketamine/vecuronium combination is possible in field anaesthesia especially when opiates and inhalational agents are not available.
Impairment of recognition memory with, but not without, conscious recollection in schizophrenia.
OBJECTIVE: To test the hypothesis that there is a link between the memory deficit associated with schizophrenia and an impairment of consciousness, an experiential approach was used to assess recognition memory and awareness in schizophrenic patients and normal subjects. METHOD: On a recognition memory task with low- and high-frequency words, the schizophrenic (N = 30) and normal (N = 30) subjects gave "remember" responses to recognized items that were accompanied by conscious recollection and "know" responses to items that were recognized on the basis of familiarity without any recollective experience. RESULTS: Schizophrenia selectively impaired recognition based on recollective experience, as measured by "remember" responses, but had no effects on "know" responses. In the comparison group, low-frequency words, relative to high-frequency words, enhanced conscious recollection but not familiarity. The schizophrenic patients did not display the same word-frequency effect. CONCLUSIONS: These results indicate that schizophrenia affects differentially two means of access to the personal past: it impairs recognition memory with, but not without, conscious recollection. They suggest that the impairment of conscious recollection observed in schizophrenic patients could be due to a failure of elaborative processing of information.
[Efficacy of antidepressants and thymoregulators in the long-term evolution of depression].
Official recommendations pointed out the long term maintenance treatment of recurrent unipolar depression on the basis of a significant effect of antidepressants and mood-stabilizers versus placebo. The results of controlled studies, mainly using imipramine or lithium salts, have not been encouraging in term of long term prognosis, due to the limited success rate for maintenance phase ranging from 30 to 48%. The "Pittsburgh study" maximized the recurrence potential by defining patients selection on at least 3 previous episodes of unipolar depression, with the immediate previous episode being no more than 2.5 years earlier, beginning the experimental maintenance therapy phase after patients remain relatively symptom free for a total of 20 weeks, and scheduled the imipramine withdrawal in the randomly assigned placebo group by a progressive reduction of 33% per week. Survival analysis in the 5 years maintenance outcome of previously imipramine responders patients demonstrated an increased risk of depressive recurrence at the beginning of discontinuation but did not prove a true prophylactic effect. Furthermore these findings do not prove that treatment are not more effective than placebo, suggesting at least a revisitation of the clinical concept of response. The putative efficacy of biological treatment should be an operational criteria to elicit vulnerability markers, mainly in the field of sleep research.
Citalopram in doses of 20-60 mg is effective in depression relapse prevention: a placebo-controlled 6 month study.
The aim of the present study was to determine whether, in patients with depression who had responded favourably to the selective serotonin reuptake inhibitor citalopram, there was a therapeutic benefit in continuation treatment. Three hundred and ninety-one depressive patients were included in an open short-term citalopram treatment period. Only patients who responded to treatment at 8 weeks (total score of 12 or less on the MADRS scale) were randomized to the 24 week double-blind phase. Seventy-four patients were treated with placebo and 152 with citalopram at the same constant dose to which the patient had responded in the first phase. Relapse was defined as a total score of 25 or more on the MADRS scale. Twenty-one patients (13.8%) continuing to receive citalopram relapsed compared with 18 patients (24.3%) receiving placebo. The log rank test for survival data used to test the quality of relapse hazards between the placebo group and the citalopram group showed that patients treated with citalopram had a significantly lower relapse rate (p = 0.04). The results of this study are in general agreement with those of other studies on antidepressants, and support the hypothesis that full dose continuation is more effective than placebo in preventing relapse of depression.
Incline, speed, and distance assessment during unconstrained walking.
Body accelerations during human walking were recorded by a portable measuring device. A new method for parameterizing body accelerations and finding the pattern of walking is outlined. Two neural networks were designed to recognize each pattern and estimate the speed and incline of walking. Six subjects performed treadmill walking followed by self-paced walking on an outdoor test circuit involving roads of various inclines. The neural networks were first "trained" by known patterns of treadmill walking. Then the inclines, the speeds, and the distance covered during overground walking (outdoor circuit) were estimated. The results show a good agreement between actual and predicted variables. The standard deviation of estimated incline was less than 2.6% and the maximum of the coefficient of variation of speed estimation is 6%. To the best of our knowledge, these results constitute the first assessment of speed, incline and distance covered during level and slope walking and offer investigators a new tool for assessing levels of outdoor physical activity.
Cloning and sequencing of the equine testicular follitropin receptor.
To investigate the possibility that specific structural determinants within the equine follitropin receptor (eFSHR) are critical to the enhanced specificity of this receptor compared to other FSHRs, we used the RACE-PCR technique to clone the eFSHR from equine testis. Sequence analysis revealed that the eFSHR is highly homologous to other mammal FSHRs, but it presents 10 unique amino acid residue replacements in the extracellular domain. Furthermore, a potential N-glycosylation site was detected at a position not encountered in other receptors. Northern blot analysis identified three transcripts of 4.2 kb, 2.3 kb and 1.0 kb in horse testis.
Conformation changes of beta-lactoglobulin: an ATR infrared spectroscopic study of the effect of pH and ethanol.
Fourier transform infrared spectroscopy has been applied to investigate the secondary structural changes of beta-lactoglobulin in water/ethanol mixtures. The studies were carried out at two different pHs and at high protein concentrations. The spectra were recorded using an attenuated total reflection cell. The amide I band of beta-lactoglobulin in water reveals large amounts of intra extended beta-sheet structure. About 20% ethanol, beta-lactoglobulin unfolds and beta-strand formation is observed. alpha-Helices are built up by increasing the ethanol concentration up to 30%. In 50% ethanol, beta-lactoglobulin gels providing the apparent pH are neutral. The secondary structural changes of beta-lactoglobulin were observed on the similarity maps obtained by Principal Component Analysis.
Immunochemical mapping of human lutropin: I. Delineation of a conformational antigenic determinant.
Lutropin (LH), follitropin (FSH), thyrotropin (TSH) and choriogonadotropin (CG) are assembled of two non-covalently alpha (alpha) and beta (beta) subunits. We studied the discontinuous antigenic regions recognized by a monoclonal anti-hLH antibody designated as LH05 which binds to hLH, hCG and hTSH and does not cross-react with either the free subunits or hFSH. LH05 and an antibody designated HT13, recognizing an epitope partly comprizing the alpha64-76 region, did not bind simultaneously to hCG. Furthermore, LH05 was unable to combine with an anti-peptide antibody (LHP03) directed to residues 43-52 of hLHbeta. Thus, LH05 recognizes an epitope partly overlapping with those recognized by HT13 and LHP03. Using various hybrid molecules, we showed that the human alpha-subunit plays a critical role in the assembly of the epitope that, in contrast, contains amino acid residues conserved in the various beta-subunit of several species. Together, our results suggest that the amino acids Leu49-Pro50, Tyr59-Arg60 and Leu86-Ser87 in the hLHbeta and the alpha64-76 region are probably included in the epitope recognized by LH05 which appeared to be not accessible on the CG/LH receptor.
Immunochemical mapping of human lutropin: II. Characterization of two monoclonal antipeptide antibodies reacting with the native beta-subunit.
To investigate the epitopes present on the beta-subunit of the human lutropin (hLHbeta) and their topographical relationship at the surface of the molecule, we produced two monoclonal antipeptide antibodies, designated LHP03 and LHP04, capable of binding to the radiolabeled 125I-hLHbeta and directed to the 43-52 and 110-117 regions of the hLHbeta, respectively. Analysis of the accessibility of the epitopes on hLH and on the beta-subunit of human chorionic gonadotropin (hCGbeta), equine LH (eLHbeta) and ovine LH (oLHbeta) indicated that: (i) LHP03 binds to both the free hLHbeta subunit and dimeric hLH whereas LHP04 binds preferentially to the free hLHbeta, (ii) LHP03 recognizes weakly the hCGbeta and oLHbeta in comparison to hLHbeta and (iii) LHP04 binds oLHbeta as well as hLHbeta but does not bind to hCGbeta and eLHbeta. The topographical relationship of epitopes recognized by LHP03 and monoclonal antibodies recognizing dimer specific epitopes on hLH allowed us to localize discontinuous antigenic sites that overlaps or are located outside the hHLbeta(43-52) region. Together, our results demonstrated that the hHLbeta(43-52) portion is accessible on both the free hLHbeta subunit and hLH whereas the COOH-terminal portion, hHLbeta(110-117), is probably buried at the alpha/beta interface of the hormone.