Biomedical subjects

P Legrand

Publications and source records attributed to P Legrand.

At least 55 records · Page 3Linked to original sources

PubMed1998

The secondary structure and architecture of human elastin.

The presented work constitutes the first structural characterization of both insoluble human elastin and its solubilized form, kappa-elastin. Structural data were reached following the use of Fourier transform infrared, near infrared Fourier transform Raman and circular dichroism optical spectroscopic methods and their quantitative analysis permitted us to estimate approximately 10% alpha-helices, approximately 35% beta-strands and approximately 55% undefined conformations in the global secondary structure of insoluble human elastin in the solid state. Following the use of the LINK method, the probable local distribution of the secondary-structure elements along the sequence was determined and compared to that obtained for bovine elastin, the historical standard of elastin. This comparison led us to propose a globular architecture for the human elastomer and permitted us to delineate some elements of its structure-elasticity relationship.

Amino Acid Sequence↗

PubMed1998

FT-IR and X-ray spectroscopic investigations of Na-diclofenac-cyclodextrins interactions.

The association of DCF-Na (the salt of the 2-[(2,6-dichlorophenyl)amino]-phenyl-acetic acid) with beta-CD (cyclodextrin) in some therapeutic formulas can contribute to the optimisation of the physico-chemical and pharmaceutical properties of the parent drug. The understanding of the interaction between DCF with beta-CD represents the objective of this study. FT-IR spectroscopy is one of the methods which clarify the nature of these interactions in complexes of such type. Therefore the changes in FT-IR spectra of binary dispersed systems DCF/beta-CD in physical mixture and coprecipitate from methanol (molar ratios: 1/1, 1/2, 2/3, 3/4, 7/4) were analysed. The analysis of the broadening of the X-ray powder diffraction line has been applied to investigate the average effective crystallite size, the mean square of the microstrain caused by distortions within beta-CD crystallite and the fault probability in the binary dispersed DCF/beta-CD coprecipitate system.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1998

Selective screening of carriers for control of methicillin-resistant Staphylococcus aureus (MRSA) in high-risk hospital areas with a high level of endemic MRSA.

Screening for methicillin-resistant Staphylococcus aureus (MRSA) carriage in patients at risk was evaluated as part of a control program in a 26-bed medical intensive care unit (ICU) of a university hospital with a high level of endemic MRSA. Control measures included isolation and barrier precautions, skin decolonization with chlorhexidine of patients from whom MRSA was recovered, and mupirocin treatment of nasal carriers of MRSA. Of 3,686 patients admitted during a 4-year period, 44% were screened, which occurred during admission for 38%; MRSA was recovered from 293 patients (8%). There were 150 imported cases and 143 ICU-acquired cases, of which 51% and 45%, respectively, were first identified through screening. Nasal swab cultures identified 84% of MRSA carriers. The incidence of all ICU-acquired cases and of acquired colonization or infection decreased from 5.8% and 5.6% to 2.6% and 1.4% (P = .002 and P < .001), respectively, whereas that of imported cases remained unchanged (range, 3.8% to 4.3%; P = .8). Selective screening for nasal carriage during admission to high-risk areas may contribute to identification of a substantial proportion of cases of MRSA and to early implementation of effective control measures.

Carrier State↗

PubMed1997

Oncogenic activation of RET by two distinct FMTC mutations affecting the tyrosine kinase domain.

Multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC) are two dominantly inherited disorders caused by germline mutations of the RET proto-oncogene. The RET gene codes for a receptor tyrosine kinase. The majority of MEN2A and FMTC mutations are clustered in the extra-cellular cysteine-rich domain and result in constitutive activation of the tyrosine kinase through the formation of disulfide-bonded RET homodimers. Recently, two novel point mutations have been identified in the germline of five distinct FMTC families. Both mutations occur within the catalytic domain of the RET kinase and lead to the substitution of either glutamic acid 768 or valine 804 by an aspartic acid and a leucine respectively. We have introduced each FMTC mutation in two RET isoforms: RET51 the long isoform (1114 aa) and RET9 the short isoform (1072 aa) which differ in the C-terminal region of the protein. The RET51 isoform carrying either E768D or V804L mutation was autophosphorylated, displayed a transforming activity upon expression in Rat1 fibroblasts and induced neuronal differentiation of PC12 cells. However, the transforming capacity of these RET51-FMTC mutants was found to be severalfold less potent compared to the same isoform carrying either the MEN2A mutation (C634R) or the MEN2B mutation (M918T). In contrast, RET9 containing mutations E768D or V804L was not autophosphorylated, exhibited a poor oncogenic potential in fibroblasts and did not promote neuritic outgrowth upon expression in PC12 cells. Overall, these findings demonstrate that mutations E768D and V804L are gain-of-function mutations that confer to the long RET isoform the capacity to exert a biological effect, although these mutations are more weakly activating than the MEN2A and MEN2B mutations. These results may provide a biochemical basis as to why the phenotypic consequences of these mutations are restricted to thyroid C-cells.

3T3 Cells↗

PubMed1997

Role of aspartate 70 and tryptophan 82 in binding of succinyldithiocholine to human butyrylcholinesterase.

The atypical variant of human butyrylcholinesterase has Gly in place of Asp 70. Patients with this D70G mutation respond abnormally to the muscle relaxant succinyldicholine, experiencing hours of apnea rather than the intended 3 min. Asp 70 is at the rim of the active site gorge 12 A from the active site Ser 198. An unanswered question in the literature is why the atypical variant has a 10-fold increase in Km for compounds with a single positive charge but a 100-fold increase in Km for compounds with two positive charges. We mutated residues Asp 70, Trp 82, Trp 231, Glu 197, and Tyr 332 and expressed mutant enzymes in mammalian cells. Steady-state kinetic parameters for hydrolysis of butyrylthiocholine, benzoylcholine, succinyldithiocholine, and o-nitrophenyl butyrate were determined. The wild type and the D70G mutant had identical k(cat) values for all substrates. Molecular modeling and molecular dynamics suggested that succinyldicholine could bind in two consecutive orientations in the active site gorge; formation of one complex caused a conformational change in the omega loop involving Asp 70 and Trp 82. We propose the formation of three enzyme-substrate intermediates preceding the acyl-enzyme intermediate; kinetic data support this contention. Substrates with a single positive charge interact with Asp 70 just once, whereas substrates with two positive charges, for example succinyldithiocholine, interact with Asp 70 in two complexes, thus explaining the 10- and 100-fold increases in Km in the D70G mutant.

Aspartic Acid↗

PubMed1997

Acute bacterial pneumonia in rats increases alveolar epithelial fluid clearance by a tumor necrosis factor-alpha-dependent mechanism.

To study the rate and regulation of alveolar fluid clearance in acute pneumonia, we created a model of Pseudomonas aeruginosa pneumonia in rats. To measure alveolar liquid and protein clearance, we instilled into the airspaces a 5% bovine albumin solution with 1.5 microCi of 125I-human albumin, 24 h after intratracheal instillation of bacteria. The concentration of unlabeled and labeled protein in the distal airspaces over 1 h was used as an index of net alveolar fluid clearance. Since there was histologic evidence of alveolar epithelial injury, several methods were used to measure alveolar fluid clearance, including the use of experiments in rats with blood flow and the use of experiments in rats without blood flow, so that movement across the epithelial barrier would be minimized in the latter group. The results with each method were identical. We found that P. aeruginosa pneumonia increased alveolar liquid clearance over 1 h by 48% in studies with blood flow, and by 43% in rats without blood flow, compared with respective controls (P < 0.05). In both studies, this increase was inhibited with amiloride. However, propranolol had no inhibitory effect, thus ruling out a catecholamine-dependent mechanism to explain the increase in alveolar fluid clearance. An antitumor necrosis factor-alpha neutralizing antibody, instilled into the lung 5 min before bacteria, prevented the increase in alveolar liquid clearance in rats with pneumonia (P < 0.05). Also, TNFalpha (5 microg) instilled in normal rats increased alveolar liquid clearance by 43% over 1 h compared with control rats (P < 0.05). In normal rats instilled with TNFalpha, propranolol had no inhibitory effect. In conclusion, gram-negative pneumonia markedly upregulates net alveolar epithelial fluid clearance, in part by a TNFalpha-dependent mechanism. This finding provides a novel mechanism for the upregulation of alveolar epithelial sodium and fluid transport from the distal airspaces of the lung.

Adrenergic beta-Antagonists↗

PubMed1997

Modulation of nitric oxide production in RAW 264.7 cells by transforming growth factor-beta and interleukin-10: differential effects on free and encapsulated immunomodulator.

The mouse macrophage cell line RAW 264.7 can be stimulated to produce nitric oxide (NO) by muramyltripeptide cholesterol included within biodegradable poly(D,L-lactide) nanocapsules (NC MTPChol). The aim of this work was to determine whether one or both of the cytokines transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) could be responsible for feedback control seen at high concentrations. Activated RAW 264.7 cells produced TGFbeta1. When exogenous TGF-beta1 was added during stimulation, a dose-dependent inhibition of NO production was observed when NC MTP-Chol was used, whereas activation by the soluble muramyl dipeptide (MDP) was not affected. Furthermore, addition of a blocking antibody to TGF-beta arrested the fall in NO production seen at high concentrations of NC MTP-Chol. Addition of IL-10 during RAW 264.7 cell activation also reduced NO production; however, in this case, both NC MTP-Chol and MDP were equally affected. The presence of anti-IL-10 antibody during activation significantly increased NO production.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1997

Octanoate metabolism in primary culture of chicken hepatocytes: evidence for high-capacity octanoate esterification.

[14C]Octanoate metabolism was measured in chicken primary hepatocytes, as a function of time in culture. [14C]Octanoate was essentially converted to oxidative products (CO2 and ketone bodies) in freshly isolated cells while [14C]palmitate was exclusively esterified in triacylglycerols (TG). In contrast, in cultured cells, [14C] octanoate was exclusively recovered in TG as radiolabeled octanoate (33%), but also as newly synthetised fatty acids mainly palmitate (37%), stearate (18%), oleate (4.4%), decanoate (2.4%), and myristate (2.2%). Therefore, it may be suggested that the enhanced lipogenic conditions induced by culture enhance octanoate flux toward TG synthesis in this model. It is further suggested that chicken hepatocytes in primary culture could be a useful model to study regulatory mechanisms of several lipogenic enzymes.

Animals↗

PubMed1997

Reemergence of gentamicin-susceptible strains of methicillin-resistant Staphylococcus aureus: roles of an infection control program and changes in aminoglycoside use.

The spread of methicillin-resistant Staphylococcus aureus (MRSA) in our hospital in the 1980s correlated with increasing acquisition of resistance to antibiotics including gentamicin, rifampin, and fluoroquinolones. During the period 1993-1995, there was a major change in clinical MRSA isolates: the percentage of aminoglycoside-resistant MRSA isolates decreased from 75% to 52%, while the proportion of heterogeneous MRSA strains susceptible to gentamicin, rifampin, and tetracycline increased gradually from 4.9% to 27.5%. We used five epidemiological markers (i.e., antibiotyping, phage typing, pulsed-field gel electrophoresis, and restriction analysis of PCR amplified coagulase and protein A genes) to characterize recent isolates. With use of these techniques, we confirmed the persistence of the aminoglycoside-resistant MRSA clone and identified a clone of erythromycin-susceptible strains among the gentamicin-susceptible isolates and found that the remaining strains were diverse. These changes were due to the introduction of various MRSA strains from outside the hospital, while implementation of infection control measures in 1991 could have led to reduced transmission of the aminoglycoside-resistant MRSA strain. Changes in antibiotic prescribing patterns that resulted in reduced selective pressure from gentamicin may have contributed to the spread of gentamicin-susceptible MRSA strains.

Anti-Bacterial Agents↗

PubMed1997

Inhibiting delta9-desaturase activity impairs triacylglycerol secretion in cultured chicken hepatocytes.

The relationship between endogenous oleic acid produced by hepatic delta9-desaturase and the secretion of VLDL-triglycerides was investigated in a primary culture of chicken hepatocytes. When the fatty acid compositions of the secreted and intracellular triglycerides (TG) (or triacylglycerols) were compared, an imbalance between monoenes and saturated fatty acids was observed, with the secreted TG being significantly more unsaturated than the intracellular TG. The addition of a mixture of cyclopropenic fatty acids (specific inhibitors of fatty acid desaturation) to the culture medium of cells 24 h before measurement of their delta9-desaturase activity and TG secretion rate caused a significant impairment of both desaturase activity and TG secretion, without affecting total TG synthesis. However, the addition of oleic acid to the culture medium of cells treated with cyclopropenic fatty acids restored the TG secretion rate. Palmitic acid did not restore the TG secretion rate and linoleic acid partly restored the TG secretion rate. Finally, even in the presence of oleic acid in the culture medium of secreting cells, those which had been treated with cyclopropenic fatty acids had a significantly lower TG secretion rate than nontreated cells. Taken together, these results show that TG secretion is highly dependent on the delta9-desaturase activity and suggest that oversecretion of VLDL-TG in chickens and subsequent fattening could originate in a high hepatic delta9-desaturation of saturated fatty acids.

Animals↗

PubMed1997

Mycobacterium tuberculosis Des protein: an immunodominant target for the humoral response of tuberculous patients.

The phoA gene fusion methodology permitted the identification of a new Mycobacterium tuberculosis exported protein, Des. This protein has significant sequence similarities to plant acyl-acyl carrier protein desaturases, which are enzymes involved in general fatty acid biosynthesis as well as in mycolic acid biosynthesis in mycobacteria. As shown by Western blot and enzyme-linked immunosorbent assay experiments, the Des protein is a major B-cell antigen that was recognized by all the tuberculous M. tuberculosis- and M. bovis-infected human patients tested.

Amino Acid Sequence↗

PubMed1997

Release of amphotericin B from delivery systems and its action against fungal and mammalian cells.

Spectroscopic studies of four amphotericin B (AmB) lipid preparations--small negatively charged unilamellar vesicles "AmBisome", positively charged oligolamellar liposomes "Ampholiposomes", AmB Lipid Complex "L-AmpB33"--and AmB association with gamma cyclodextrin demonstrated that the composition of drug delivery system directly influences AmB organization. The aggregation state and release in external medium of AmB was monitored by circular dichroism and UV-visible absorption. AmB short-term activity against Candida albicans (K+ leakage) was found to be correlated with the amount of free AmB released from lipid preparations. These data seem to indicate that lipid composition influences anti-Candida albicans activity, by modulation of AmB binding to lipids.

Amphotericin B↗

PubMed1997

[Survival after 60 years of age among elderly retired miners].

This study analyses long term effects of working conditions at an advanced age and post retirement. A longitudinal study was done among 63,000 retired miners between 60 to 65 years old. The standardised mortality ratio (SMR) in the cohort was above that expected on the basis of concurrent mortality in the reference population (SMR = 111; 95% confidence interval (CI): 108-114). The excess mortality was observed in occupational groups with exposure to underground tasks (10 years of underground tasks: RR = 2; 95% CI: .1.7-2.5), but there was sign of a healthy worker effect after 25 underground years. The effects of modern technologies and of medical progress have changed working conditions and care has reduced mortality for retired manual workers: exposure to underground tasks before 1950: RR = 1.7; CI 95%: 1.4-2.0; after 1950: RR = 1.2; CI 95%: 1.1-1.2.

Aged↗

PubMed1996

Relationship between hepatic fatty acid desaturation and lipid secretion in the estrogenized chicken.

Desaturation of fatty acids is thought to facilitate their incorporation into glyceride and their subsequent secretion as lipoproteins. However, in the laying hen, the dramatic increase in hepatic lipogenesis is often paralleled by a liver steatosis that may affect egg production and even result in death. The balance between lipid secretion and storage, in relation to the fatty acid desaturation process, was therefore investigated in young male estrogenized chicken. Estrogen stimulation resulted in a dramatic increase in VLDL concentration (40.4 mg/ml versus 0.158 mg/ml in control) and hepatic lipid content (8.61 g/liver versus 1.47 g/liver in control). In estrogenized chickens, VLDL, total liver, and microsomes contained relatively more monounsaturated and less saturated and polyunsaturated fatty acids, whereas hepatic delta 9 desaturase activity was twofold higher. Moreover, in these birds, the proportion of monoenoic fatty acids was greater in VLDL (55%) than in the liver (50%), which was indicative of their preferential secretion. Therefore, under the influence of estrogen, fatty acid synthesis and desaturation are associated with and increased VLDL secretion, which limits the degree of hepatic accumulation of triglyceride and the risk of subsequent steatosis.

Animals↗

PubMed1996

Effect of insulin on triacylglycerol synthesis and secretion by chicken hepatocytes in primary culture.

A primary culture system of chicken hepatocytes was developed to study the effects of genetic, hormonal and nutritional factors on hepatic triglyceride (TG) secretion in the chicken, and the effect of insulin on TG synthesis and secretion examined. TG synthesis and secretion was measured using [3H]-glycerol incorporation into cellular and secreted TG. An additional step consisting of brief incubation of the monolayer with trypsin solution to improve harvesting the medium immediately adjacent to the cell monolayer, is also proposed. In our culture system, TG secretion occurred at least up to 75 hr of culture, showing a maximum between 40 and 60 hr of culture. No significant effect of insulin could be observed after 24 hr of culture. A clear stimulatory effect was observed with 10(-9) M insulin concentration after 48 hr. The mean ratio of the secretion rates in the presence or absence of insulin was 2.20 +/- 0.30 (SEM, n = 4). In contrast, the 10(-6) M concentration of insulin had no effect on TG secretion. The use of an additional trypsinization step enhanced the findings obtained by simple removal of the culture medium: a clear stimulatory effect of insulin on TG synthesis was observed after both 24 and 48 hr of culture. Analysis of TG fatty acid composition showed an imbalance of monoene versus saturated fatty acids between cellular and secreted TG, secreted TG being more monounsaturated than cellular TG. These results were obtained in the absence of exogenous fatty acid. An oleic acid supplement in the culture medium did not significantly influence TG secretion. In summary, a primary culture system for chicken hepatocyte was developed. A high level of TG secretion was seen in these cells with or without exogenous fatty acid and this secretion was stimulated by insulin. It was concluded that chicken hepatocytes in primary culture provide a useful model for studying regulation of TG secretion.

Animals↗

PubMed1996

Comparative internalization and recycling of different amphotericin B formulations by a macrophage-like cell line.

The amount of amphotericin B (AmB) associated with cultured murine macrophage-like J774 cells, after incubation with various AmB lipid formulations, was determined by absorption spectroscopy. Large, negatively charged, AmB-containing, multilamellar vesicles and small cholesteryl sulphate-AmB complexes both enhanced the amount of AmB associated with J774 cells at 37 degrees C (up to 500-fold the extracellular concentration). In contrast, AmB-containing, small, negatively charged vesicles (AmBisome), positively charged, oligolamellar vesicles and mixed micelles showed a lower association of the antibiotic with cells, compared with AmB added from a solution in dimethylsulphoxide or Fungizone. Experiments performed at 40 degrees C showed a large reduction of AmB uptake for AmB preparations and AmB added from a solution in dimethysulphoxide or Fungizone, suggesting a high percentage of internalization of the antibiotic. Experiments in the presence of cytochalasin B resulted in a decrease of AmB uptake mainly for the preparations of large diameter, suggesting that these formulations were taken up by phagocytosis. A comparative study with Chinese hamster ovary cells, a model of non-phagocytic cells, showed a reduction in the take up of AmB. This reduction was always more marked when AmB was incorporated in lipid formulations. On the other hand, accumulation of the antibiotic in J774 cells was shown to be followed by its release from the cells in an unbound form, the extent of release depending on the type of vector used. The results suggest that in some cases macrophages can be considered as reservoirs of antibiotic, releasing free AmB in the medium.

Amphotericin B↗

PubMed1995

Bovine elastin and kappa-elastin secondary structure determination by optical spectroscopies.

Elastin is the macromolecular polymer of tropoelastin molecules responsible for the elastic properties of tissues. The understanding of its specific elasticity is uncertain because its structure is still unknown. Here, we report the first experimental quantitative determination of bovine elastin secondary structures as well as those of its corresponding soluble kappa-elastin. Using circular dichroism and Fourier transform infrared and near infrared Fourier transform Raman spectroscopic data, we estimated the secondary structure contents of elastin to be approximately 10% alpha-helices, approximately 45% beta-sheets, and approximately 45% undefined conformations. These values were very close to those we had previously determined for the free monomeric tropoelastin molecule, suggesting thus that elastin would be constituted of a closely packed assembly of globular beta structural class tropoelastin molecules cross-linked to form the elastic network (liquid drop model of elastin architecture). The presence of a strong hydration shell is demonstrated for elastin, and its possible contribution to elasticity is discussed.

Amino Acids↗

PubMed1995

Frequency of bacteremia associated with transesophageal echocardiography in intensive care unit patients: a prospective study of 139 patients.

OBJECTIVE: To determine the occurrence rate of bacteremia associated with transesophageal echocardiography in intensive care unit (ICU) patients. DESIGN: A prospective study of 139 patients undergoing transesophageal echocardiography. SETTING: The medical ICU of a tertiary referral teaching hospital. PATIENTS: One hundred thirty-nine ICU patients (mean age 58 yrs) who underwent transesophageal echocardiography. INTERVENTIONS: Blood samples were systematically drawn for aerobic and anaerobic culture at the following times: before (blood culture 1), at the end of (blood culture 2), and 30 mins after (blood culture 3) transesophageal echocardiography examinations. MEASUREMENTS AND MAIN RESULTS: The mean duration of transesophageal echocardiography was 35 mins (range 7 to 120). One hundred thirty-four patients received mechanical ventilation; 125 patients had a nasogastric tube. Fifty-one patients had one or more underlying conditions that usually justify antimicrobial prophylaxis of bacterial endocarditis before high-risk procedures. Fifty-six patients did not receive any antibiotic treatment at the time of transesophageal echocardiography. In 114 patients, the three blood cultures were negative. In six patients, transesophageal echocardiography was performed during a preexisting bacteremia. A contamination (only one positive blood culture of the three sampling times) with coagulase-negative staphylococci occurred in four patients at blood culture 1, five patients at blood culture 2, and six patients at blood culture 3. Contamination with Corynebacterium species occurred in two patients at blood culture 2. In one patient receiving cefotaxime and netilmicin, blood culture 1 was sterile and blood cultures 2 and 3 yielded coagulase-negative staphylococci. In one patient receiving no antibiotic treatment, blood culture 1 was sterile and blood cultures 2 and 3 yielded Enterococcus faecalis. None of these two patients received a specific antibiotic treatment or developed any secondary septic focus. CONCLUSIONS: The overall frequency of bacteremia induced by transesophageal echocardiography in ICU patients was 1.4% (two of 139 patients) (95% confidence interval 0.2% to 5.1%). The frequency did not differ whether patients received antibiotics before transesophageal echocardiography (one [1.2%] of 83 patients) or not (one [1.8%] of 56 patients) (p = .96). Therefore, routine antimicrobial prophylaxis does not appear justified before transesophageal echocardiography in ICU patients.

Adult↗