A missense mutation in melanocortin 1 receptor is associated with the red coat colour in donkeys.

The seven donkey breeds recognised by the French studbook are characterised by few coat colours: black, bay and grey. Normand bay donkeys seldom give birth to red foals, a colour more commonly seen and recognised in American miniature donkeys. Red resembles the equine chestnut colour, previously attributed to a mutation in the melanocortin 1 receptor gene (MC1R). We used a panel of 124 donkeys to identify a recessive missense c.629T>C variant in MC1R that showed a perfect association with the red coat colour. This variant leads to a methionine to threonine substitution at position 210 in the protein. We showed that methionine 210 is highly conserved among vertebrate melanocortin receptors. Previous in silico and in vitro analyses predicted this residue to lie within a functional site. Our in vivo results emphasised the pivotal role played by this residue, the alteration of which yielded a phenotype fully compatible with a loss of function of MC1R. We thus propose to name the c.629T>C allele in donkeys the e allele, which further enlarges the panel of recessive MC1R loss-of-function alleles described in animals and humans.

In vitro and in vivo evaluation of the haematopoietic potential of skeletal muscle in a non-human primate model.

This study was aimed at evaluating the in vitro and in vivo haematopoietic potential in macaque skeletal muscle cells. Biopsy samples showed the presence of CD34(+) (7.6%), CD90(+) (8.4%), CD117(+), CD31(+), side population (SP) cells (7-10%) and a low number of CD45(+) cells. In clonogenic and long-term culture-initiating cell assays, no haematopoietic potential could be detected in either total mononuclear cells or SP cells. Regarding in vivo studies, two animals were transplanted with unfractionated fresh muscle cells after lethal irradiation. Both animals died early after transplant without any evidence of haematopoietic reconstitution. In two other monkeys, harvested muscle cells were frozen and secondarily marked using a green fluorescent protein (GFP)-lentiviral vector. After sublethal irradiation, both animals were transplanted with GFP-expressing muscle cells followed by a bone marrow rescue. Both animals had haematopoietic reconstitution at days 22 and 25, but no GFP-expressing haematopoietic cells could be detected by flow cytometry, either in the blood or in clonogenic cells from marrow aspirates. Using PCR assays, GFP(+) cells were detected in a single marrow sample of one animal at 41 days after transplantation. These results strongly suggest that as opposed to murine muscle, the non-human primate skeletal muscle does not harbour cells with a straightforward haematopoietic potential.

Delayed gastric emptying and altered antrum protein metabolism during activity-based anorexia.

BACKGROUND: Anorexia nervosa, a restrictive eating disorder, is often associated with gastrointestinal disorders, particularly a delayed gastric emptying. However, the mechanisms remained poorly documented. Thus, we aimed to evaluate gastric emptying and antrum protein metabolism in the Activity-Based Anorexia model (ABA). METHODS: Females C57Bl/6 mice were randomized into 3 groups: Control, ABA, and Limited Food Access (LFA). Food access has been progressively limited from 6 h/day at day 6 to 3 h/day at day 9 and until day 17. ABA mice had free access to an activity wheel. Gastric emptying was assessed. On gastric extracts, a proteomic analysis was performed, as well as an evaluation of protein synthesis and protein oxidation. KEY RESULTS: Both LFA and ABA mice exhibited a delayed gastric emptying compared with Controls (P < .05). Proteomic approach revealed 15 proteins that were differentially expressed. Among these proteins, we identified 2 clusters of interest contributing to (i) the organization of muscle fiber with ACTA2, VCL, KRT19, KRT8, and DES proteins and (ii) "heat shock proteins" with STIP1, HSPD1, and HSPA8 proteins. ABA mice specifically exhibited an increased rate of gastric oxidized proteins. CONCLUSIONS AND INFERENCES: Delayed gastric emptying observed in anorectic conditions appears to be secondary to malnutrition. However, an oxidative stress is specifically present in the stomach of ABA mice. Its role remains to be further studied.

[Preliminary study of cardiorespiratory decondittioning in athletes after anterior cruciate ligament reconstruction]. / Etude préliminaire de la désadaptation cardiorespiratoire après une ligamentoplastie de genou chez le sportif.

AIM: The aim of this study was to analyze changes in cardiorespiratory fitness of athletes who had surgery following a lesion of the anterior cruciate ligament of the knee. METHODS: Two groups of 12 athletes at the regional level underwent surgical repair to rebuild the external anterior crossed ligament of the knee (central third bone patellar tendon bone autograft and doubled semitendinosus/doubled gracilis autograft techniques). All subjects were evaluated before and after surgery within 7 days: the first group underwent maximal incremental tests with the upper limbs, and the second group measurement of resting cardiac volumes. RESULTS: Surgery followed by a few days of confinement generated a quick and significant reduction in the maximal oxygen consumption (-7%, P<0.05) and peak aerobic power (-8%, P<0.05). End diastolic volume and stroke volume were reduced, by 23% and 27% respectively (P<0.05). A significant reduction of ejection fraction was also observed (P<0.05). The mean left ventricular ejection fraction was 65% before the surgery 60% after 7 days' of hospitalization. CONCLUSION: In sportsmen, 7 days of hospitalization due to surgery of the knee led to resting cardiac unsuitability characterized by a significant reduction in the stroke volume. These elements could involve decreased aerobic fitness and should encourage the hospital practitioner to propose a program of aerobic training in addition to conventional rehabilitation.

Chronic delivery of α-melanocyte-stimulating hormone in rat hypothalamus using albumin-alginate microparticles: effects on food intake and body weight.

Chronic delivery of neuropeptides in the brain is a useful experimental approach to study their long-term effects on various biological parameters. In this work, we tested albumin-alginate microparticles, as a potential delivery system, to study if continuous release in the hypothalamus of α-melanocyte-stimulating hormone (α-MSH), an anorexigenic neuropeptide, may result in a long-term decrease in food intake and body weight. The 2-week release of α-MSH from peptide-loaded particles was confirmed by an in vitro assay. Then, daily food intake and body weight were studied for 18 days in rats injected bilaterally into the paraventricular hypothalamic nucleus with particles loaded or not with α-MSH. A decrease in body weight gain, persisting throughout the study, was found in rats injected with α-MSH-charged particles as compared with rats receiving non-charged particles and with rats injected with the same dose of α-MSH in solution. Food intake was significantly decreased for 3 days in rats receiving α-MSH-loaded particles and it was not followed by the feeding rebound effect which appears after food restriction. The presence of α-MSH-loaded particles in the hypothalamus was confirmed by immunohistochemistry. In conclusion, our study validates albumin-alginate microparticles as a new carrier system for long-term delivery of neuropeptides in the brain and demonstrates that chronic delivery of α-MSH in the hypothalamus results in a prolonged suppression of food intake and a decrease of body weight gain in rats.

Site-directed mutagenesis and chemical modification of the two cysteine residues of the UDP-N-acetylmuramoyl:L-alanine ligase of Escherichia coli.

Site-directed mutagenesis and chemical modification of the two cysteine residues of the MurC L-alanine-adding enzyme from Escherichia coli were undertaken to study their possible role in activity and stability. Their replacement by alanine was not critical for activity. However, C230 played a role in enzyme stability and substrate binding. N-Ethylmaleimide alkylation led to monoalkylated and dialkylated proteins. The monoalkylated protein had mostly unmodified C230 residues. The extent of alkylation of C230 paralleled the loss of activity, whereas that of C426 did not. Protection against inactivation by beta,gamma-imidoadenosine 5'-triphosphate implied the involvement of C230 in the ATP binding site.

Adenylate cyclase activity in bovine and human corneal endothelium.

Adenylate cyclase activity was measured in particulate fractions prepared from bovine and human corneal endothelium. Bovine enzyme activity was stimulated by fluoride, 5'-guanylylimidodiphosphate (GppNp) and beta-adrenergic agonists. Drug stimulation patterns of the human enzyme appeared qualitatively similar, but activities were generally higher than those of the bovine enzyme. This study demonstrates the existence of a receptor-mediated adenylate cyclase system in bovine and human corneal endothelium and suggests a possible role for cycli AMP in corneal endothelial physiology.

Characterization of adenylate cyclase activity in bovine and human corneal epithelium.

The properties of adenylate cyclase from bovine and human corneal epithelium were investigated. Adrenergic drugs were the most effective stimulatory agents tested in bovine tissue, causing greater activation than did fluoride. Isoproterenol was the most potent agonist, followed by epinephrine and norepinephrine. Phenylephrine and dopamine also stimulated adenylate cyclase through beta-adrenergic receptors at relatively high concentrations. Enzyme stimulation by all the adrenergic drugs tested was completely inhibited by 1 microM propranolol or 0.1 microM timolol. The GTP analogue, GppNp, produced considerable activation and caused an augmented response when combined with isoproterenol, but not with fluoride. Prostaglandins E1, E2, or F2 alpha produced a small but significant stimulation over control which was not sensitive to propranolol inhibition. Adenylate cyclase from human corneal epithelium exhibited qualitatively similar characteristics to those of the bovine enzyme. Fluoride was the most effective stimulatory agent, followed by isoproterenol, phenylephrine, and dopamine. Prostaglandins failed to stimulate adenylate cyclase activity in human corneal epithelial preparations.

Identification of a neutralizing domain in the external envelope glycoprotein of simian immunodeficiency virus.

Two murine monoclonal antibodies (MAbs), designated MATG2014 and MATG2033, were generated. They are reactive with the external envelope glycoprotein gp130 of the simian immunodeficiency virus of macaque monkey (SIVmac251), and display a cell-free virus neutralizing activity in vitro. In addition, MATG2014 cross-reacts with HIV-2Rod gp140. Epitope mapping of these MAbs was performed by screening and SIVmac peptide library expressed in yeast and confirmed using synthetic peptides. MATG2014 and MATG2033 recognize two overlapping epitopes localized in an 18 residue domain between amino acid 171 and 188 of the SIVmac251 gp130. Sera from experimentally SIV-infected macaques are immunoreactive with this neutralizing domain. Sequence comparison with related SIV and HIV-2 viral strains indicates a low variability of this region, consistent with the cross-reactivity of MATG2014 with HIV-2Rod gp140. This domain should then be considered in designing experimental vaccines.

In vivo behaviour of hydroxyapatite coatings on titanium implants: a quantitative study in the rabbit.

The aim of this study was to evaluate quantitatively the behaviour of in vivo hydroxyapatite coated implants (HA) in the rabbit over time, and to compare the results with observations made on titanium plasma spray implants (TPS). Results were analysed according to the percentage of bone contact. Eighteen HA cylindrical implants (3.25 x 8 mm) and 6 TPS cylindrical implants from Steri-Oss were placed in the epiphysis of the femur in 24 white rabbits. Each rabbit received one implant. Three rabbits with one HA implant (n = 3) and 1 rabbit with one TPS implant (n = 1) were sacrificed after implantation periods of 2, 4, 6, 8, 10 and 12 months. Implants were cut along the long axis and prepared for histological and histomorphometrical evaluations. Measurements of coating thickness and percentage of bone contact were performed with scanning electron microscopy analysis on the sides of the implant, in 3 different types of bone, namely cortical, trabecular and marrow. In cortical bone, dense bone was apposed to the HA implants: from 92.3 +/- 5.5% at 2 months to 89.6 +/- 6.5% at 1 year, with no significant regression of HA thickness (P = 0.37). TPS coating showed less bone contact, but thickness was stable (P = 0.46). In trabecular zone, where bone contact was less pronounced, a significant regression of HA coatings thickness (P < 0.05) was observed. Nevertheless TPS coatings were stable (P = 0.81). Histomorphometrical results demonstrated that a highly significant regression (P < 0.0001) of HA thickness was observed in the marrow area, where the bone-to-implant contact never exceeded 7.6% from 2 to 12 months. TPS coating did not reveal any sign of resorption (P = 0.88), despite a rare bone contact. Histological analysis revealed inflammatory and giant cells, principally in the marrow area in contact with HA coating, but always in restrictive numbers. We conclude that bone contact protected the HA coating from resorption.

Isolation and characterization of gamma delta T lymphocyte cell lines from Sinclair swine peripheral blood.

Sinclair miniature swine represent a breed of miniature swine which display a significant incidence of inheritable melanoma which undergo a developmentally regulated spontaneous regression. In an attempt to characterize the host cellular immune response to the melanoma, lymphocyte cell lines have been generated from peripheral blood and designated as peripheral blood lymphocyte cell lines (PBLCLs). The cell lines were expanded in vitro without the addition of exogenous mediators, cloned by limiting dilution, and characterized by flow microfluorimetry, Western, and Northern blot analysis. The cell lines were shown to be CD2-, CD4-, CD8-, and slg-, a phenotype consistent with a null cell population described in swine. The null cell population in swine has been reported to consist of a subpopulation of cells which express the gamma delta T cell receptor (TCR) heterodimer, swine gamma delta T lymphocytes. The PBLCLs were further analyzed by flow microfluorimetry and observed to express the IL-2R, swine MHC Class II antigens, and the endothelial lymphocyte adhesion marker (CD44), which can function as a homing receptor for the skin. In addition, the PBLCLs were observed to express the antigen which is recognized by mAb 86D, an antibody that has been reported to recognize an external epitope on a subset of gamma delta TCR bearing swine T lymphocytes. Western blot analysis of Triton X-114 phase fractions of a PBLCL revealed a protein recognized by the W6 antibody, an antibody which recognizes a conserved region of the C delta chain. Furthermore, Southern and Northern blot analysis indicated that the PBLCL have rearranged the TCR gamma chain gene and express mRNA from the TCR gamma and delta chain genes prior to and following treatment with ionomycin or Concanavalin A. Therefore, the data indicates that the PBLCLs represent swine gamma delta T lymphocyte cell lines which should enable us to enhance our understanding of the role of gamma delta T lymphocytes in the porcine immune system.

Evaluation of a diphenylphosphorylazide-crosslinked collagen membrane for guided bone regeneration in mandibular defects in rats.

In the present study, the potential of a diphenylphosphorylazide-crosslinked type I bovine collagen membrane was evaluated in the healing of mandibular bone defects applying the biological concept of guided bone regeneration. The experiment was carried out on 25 Wistar rats. After exposing the mandibular ramus bilaterally, 5 mm diameter full-thickness circular bone defects were surgically created. While the defect on one side was covered by the membrane (experimental), the defect on the other side was left uncovered (control) before closure of the overlying soft tissues. The rats were sacrificed in groups of 5 after 7, 15, 30, 90, and 180 days of healing. Although at early stages of healing similar amounts of bone formation were observed in the experimental and control defects, after 1 month of healing, most of the experimental defects were completely closed with new bone, while in the control defects, only limited amounts of new bone were observed at the rims and in the lingual aspect of the lesions. In the 90- and 180-day animals, all experimental defects were completely closed, while in the control defects, no statistically significant increase in bone regeneration was observed. The increase in percentage of bone regeneration in the experimental defects was statistically significant between the 15-day specimens as compared with the 7-day specimens (P < 0.01) and likewise between 30-day and 15-day specimens (P < 0.001). It can be concluded that a DPPA-crosslinked collagen membrane yields biocompatibility, ad hoc mechanical hindrance, and handling characteristics suitable for guided bone regeneration applications in this experimental model.

Culture of gingival fibroblasts on bioabsorbable regenerative materials in vitro.

BACKGROUND: The use of membranes in guided tissue regeneration (GTR) can limit the apical migration of gingival cells and favor the establishment of new attachment by periodontal ligament fibroblasts. However, gingival recession during healing following GTR has been described as a frequent complication. The purpose of this study was to determine if gingival fibroblasts are affected by the composition of the bioabsorbable membranes used in mucogingival surgery. METHODS: Two type of bioabsorbable regenerative materials were used as cell carriers. Wistar rat gingival fibroblasts (RGF) were obtained from attached gingiva, cut into small fragments, and placed in culture dishes. When confluent, cells were detached using trypsin and identified as "first transferred cells" (P1). At the third passage (P3), cell count, trypan blue exclusion test, acid phosphatase activity, DNA synthesis, phase contrast microscopy, and scanning electron microscopy were performed. The cells were then placed in wells containing the membranes and incubated for 72 hours. RESULTS: When examined under microscopy, the control wells (without membranes) showed one cell type with the elongated appearance characteristic of fibroblasts. The wells with membranes showed an altered cell morphology with a high proportion of cell fragments regardless of the type of membrane used. CONCLUSIONS: These results suggest that cell carrier membranes could affect RGF morphology and thus alter gingival tissue healing following GTR.

Oral status and nutrition in the institutionalized elderly.

OBJECTIVES: To evaluate, in an elderly population, whether poor oral status might be a contributing factor to the development of undernutrition and might be associated with less eating pleasure, more subjective eating difficulty and increased mashed food consumption. METHODS: An oral examination and an evaluation of masticatory capacity were performed on 120 institutionalized elderly subjects. The nutritional assessment included serum albumin concentration, the Mini Nutritional Assessment and a questionnaire on eating habits. RESULTS: Edentulous subjects without dentures or with only one complete denture had significantly lower MNA scores than edentulous subjects with two complete dentures (p < 0.05). Edentulous subjects with two complete dentures more frequently reported taking pleasure from eating (p = 0.05), and had less frequent difficulties with hard foods (p = 0.01) than edentulous subjects without dentures or with only one complete denture. Mashed food consumption (p < 0.01) was also reported more frequently in edentulous subjects without dentures or with only one complete denture. Subjects with two complete dentures had similar or better MNA scores as dentate subjects with relatively few remaining teeth (10.4 +/- 7.8 teeth). About half of the subjects (53%) could not perform the masticatory test. These subjects had lower MNA scores (p = 0.001) and a larger proportion ate mashed food (p < 0.001) compared to those who were able to perform the test. CONCLUSIONS: Poor oral status (edentulous without dentures or with only one complete denture) increased difficulty in eating hard foods, increased mashed food consumption and decreased eating pleasure. It seemed also to put institutionalized subjects at higher risk of undernutrition.

Sugar-mediated crosslinking of alpha-biotinylated-Lys to cysteamine-agarose support: a method to isolate Maillard Lys-Lys-like crosslinks.

Advanced glycation end products (AGEs) and, specifically, protein-protein AGE crosslinks have long been studied for their potential role in aging, diabetic complications and Alzheimer disease. With few exceptions, the chemical nature of these structures remains unknown. We report here a simple approach that allows the preparation and isolation of milligram quantities of sugar-mediated AGE Lys-Lys-like crosslinks from glycation mixtures. The method is based on a sugar-dependent incorporation of N(alpha)-biotinyl-L-Lys into cysteaminyldisulfide Sepharose 6B (AE-S-S-Sepharose 6B). Glycation mixtures with six different sugars showed a time- and sugar-dependent decrease in the concentration of the support-bound primary amino groups and accounted for almost 90% loss of cysteaminyl amino groups at the end of the various incubation periods. 4-Hydroxyazobenzene-2-carboxylic acid-avidin assays indicated the incorporation of N(alpha)-biotinyl-L-Lys equal to 8% of the total support amino groups with methylglyoxal after 7 d and 1% with fructose and glucose after 1 mo of incubation. Treatment of the washed, sugar-modified supports with 2-mercaptoethanol released the bulk of the bound AGE modifications and the crosslinks. Subsequent fractionation of these preparations over a monomeric avidin column afforded a complete separation of sugar-mediated AGE modifications and the crosslinks. Depending on the sugar employed, micromolar amounts of biotinylated Lys-Lys-like crosslinks were generated by this two-step procedure from 8 mL of the original AE-S-S-Sepharose 6B.

A novel process for anaerobic composting of municipal solid waste.

A novel process has been developed and evaluated in a pilot-scale program for conversion of the biodegradable fraction of municipal solid waste (MSW) to methane via anaerobic composting. The sequential batch anaerobic composting (SEBAC) process employs leachate management to provide organisms, moisture, and nutrients required for rapid conversion of MSW and removal of inhibitory fermentation products during start-up. The biodegradable organic materials are converted to methane and carbon dioxide in 21-42 d, rather than the years required in landfills.

[Osseointegrated endosseous implants, University of Liège concepts. Various clinical applications]. / Les implants endo-osseux ostéo-intégrables, conception ULg. Quelques réalisations cliniques.

Based on fundamental experimental studies performed by the research teams of Professor Bränemark (Göteborg, Sweden), the use of dental implants has become a scientifically accepted treatment concept in Dentistry to replace lost or missing teeth in fully and partially edentulous patients. The use of dental implants was initiated by the discovery that dental implants made of titanium can be anchored in the jawbone with direct bone contact (osseointegration).

[How I investigate...the age of adolescents using panoramic radiography]. / Comment j'explore ... l'âge de l'adolescent au moyen de radiographies panoramiques.

Stage of dental development gives an assessment of young adults chronological age. This is used in forensic medicine when birth data are lacking or doubted. The following method improves the accuracy of age determination in comparison with the commonly used method of Nolla.

[Burning mouth syndrome]. / Le syndrome des sensations de brûlure buccale.

The BMS which involves the oral mucosa and produces sensations of pain and prickling is still poorly known by the physician. It differs from the other pathologies of the oral mucosa by its multifactorial etiology and by its high prevalence among postmenopausal women. The current treatments are somewhat disappointing and postmenopausal women suffering from BMS are advised to take a hormonal replacement therapy even though no direct relation between BMS and estrogen blood levels has been proved.