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International audience; Background: In ruminants, physiological and nutritional changes occur peripartum. We investigated if gastro-intestinal microbiota, rumen metabolism and antioxidant status were affected around parturition and what could be the impact of a daily supplementation of a live yeast additive in late gestating ewes. Methods: Rumen, feces and blood samples were collected from 2 groups of 14 ewes one month and a few days before parturition, and 2 weeks postpartum. Results: In the control ewes close to parturition, slight changes in the ruminal microbiota were observed, with a decrease in the concentration F. succinogenes and in the relative abundance of the Fibrobacteres phylum. Moreover, a decrease in the alpha-diversity of the bacterial community and a reduced relative abundance of the Fibrobacteres phylum were observed in their feces. Control ewes were prone to oxidative stress, as shown by an increase in malondialdehyde (MDA) concentration, a lower total antioxidant status, and higher glutathione peroxidase (GPx) activity in the blood. In the yeast supplemented ewes, most of the microbial changes observed in the control group were alleviated. An increase in GPx activity, and a significant decrease in MDA concentration were measured. Conclusions: The live yeast used in this study could stabilize gastro-intestinal microbiota and reduce oxidative stress close to parturition

International audience; This study aimed to determine how ageing and cooking, each one applied to the beef meat most suitable (panfried or grilled ribeye steak, braised chuck and fried or roasted rump steak), induce changes in lipid content, fatty acid (FA) composition and lipid oxidation of muscles from 16 cattle representative of animals raised for France meat production. The fattiest muscle (ribeye) was the richest in saturated and monounsaturated FA leading to poor nutritional indexes. In contrast, the leanest muscle (rump) had the highest proportion of polyunsaturated FA and the highest levels of peroxidation without exceeding critical limits. The impact of cooking methods seemed mainly linked to the moisture loss increasing meat fat content and the culinary fat addition whose FA composition marked the meat. Cooking methods induced oxidation phenomena that could exceed the limit thresholds. In conclusion, short cooking time of rump steak was the best combination to meet nutritional expectations.

International audience; Many studies on beef nutritional qualities require the quantification of intramuscular fat. To reduce the sample amount, solvent use and time of analysis, two alternative methods to the Folch et al. (1957) reference method were studied: a miniaturised Folch's method and a near-infrared spectroscopic method. Performances and acceptability limits were evaluated with accuracy profiles for each of the methods. Equations to correct bias between the alternative and reference methods were calculated. Uncertainties associated with measurements were determined, and the validity domains were defined. From a previous set of studies, the ability of each method to discriminate samples from bovines of different breeds or receiving diverse treatments was tested. The validity domain of the miniaturised Folch's method ranged from 1.9 to 13.8 g of total lipids/100 g of tissue, and that of the near-infrared spectroscopic method ranged from 4.8 to 13.8 g of total lipids/100 g of tissue, with less than 20% difference from the reference method's results. Thus, the two alternative methods could be used depending on the research objectives: the miniaturised Folch's method could be used for detailed quantification of intramuscular fat and the near-infrared spectroscopic method for a quick classification of a large number of muscles. The precise knowledge of uncertainties associated with each measurement was determined, and perfect continuity with the results obtained so far with the reference Folch's method was confirmed.

Skin aspartic acid protease (SASPase) is believed to be a key enzyme involved in filaggrin processing during epidermal terminal differentiation. Since little is known about the regulation of SASPase function, the aim of this study was to identify involved protein partners in the process. Yeast two hybrid analyses using SASPase as bait against a human reconstructed skin library identified that the N-terminal domain of filaggrin 2 binds to the N-terminal fragment of SASPase. This interaction was confirmed in reciprocal yeast two hybrid screens and by Surface Plasmon Resonance analyses. Immunohistochemical studies in human skin, using specific antibodies to SASPase and the N-terminal domain of filaggrin 2, showed that the two proteins partially co-localized to the stratum granulosum. In vitro enzymatic assays showed that the N-terminal domain of filaggrin 2 enhanced the autoactivation of SASPase to its 14 kDa active form. Taken together, the data suggest that the N-terminal domain of filaggrin 2 regulates the activation of SASPase that may be a key event upstream of filaggrin processing to natural moisturizing factors in the human epidermis.

International audience; The effects of extruded linseed and rapeseed on lipids and FA composition of total, polar and neutral lipids of longissimus thoracis (LT) and semitendinosus (ST) muscles were investigated in 21 Normand cull cows. Animals were assigned in a 100d finishing period to straw (30%) and concentrate (70%) based (C) or the same diet supplemented with linseed (L) or with rapeseed (66%) plus linseed (33%) (RL). Beef polar and neutral lipids were purified by liquid chromatography and their FA analysed by GLC. Trans and cis 18:1, purified by HPLC from total FA methyl esters, were analysed by GLC-MS. L and LR diets did not increase beef lipid deposition, but had modified FA composition of both LT and ST muscles in favouring deposition of 18:3n-3 and 9cis,11tr 18:2 (CIA), mainly to the detriment of 18:1 Delta 9 cis (neutral lipids) and 18:2n-6 (polar lipids). However, they did not favour deposition of LC n-3 PUFA in the two muscles, but had increased deposition of trans 18:1 significantly, especially of Delta 13tr to Delta 16tr isoforms to the detriment of Delta 10tr 18:1 (L diet) and of Delta 11tr 18:1 (RL diet). (C) 2014 Elsevier Ltd. All rights reserved.

International audience; Hydroxyalkenals are lipid oxidation end-products resulting from the oxidation of polyunsaturated fatty acids (PUFA). This study aimed at quantifying the production of 4-hydroxy-2-nonenal-protein adducts (HNE-P) via Michael addition from n-6 PUFA oxidation in the gastric digesta of mini-pigs after the consumption of meat-based meals with different plant antioxidant contents. Using the accuracy profile procedure, we validated an extraction protocol for the quantification of HNE-P by GC-MS/MS in gastric contents. The formation of HNE-P in the gastric compartment was observed for the first time, with concentrations ranging from less than 0.52 to 1.33 nmol HNE-P per 500 mg digesta. Nevertheless, most gastric HNE-P levels were below the limit of quantification of 0.52 nmol HNE-P per 500 mg digesta. In this animal study, the protective effect of plant antioxidant sources on HNE-P formation was not evidenced contrasting with the results using TBARS as markers.

International audience; The aim of this study was to assess the feasibility of near-infrared reflectance spectroscopy (NIRS) for predicting lamb meat fatty acid composition. We compared ground vs. intact non-ground meat samples to determine whether grinding and homogenisation of meat samples improved the performance of the predictions. We used 76 male lambs, of which 32 were pasture-fed and 44 stall-fed with concentrate and hay. The reflectance spectrum of Longissimus lumborum muscle was measured at wavelengths between 400 and 2500 nm. Predictions were better with ground than with intact muscle samples. NIRS accurately predicts several individual fatty acids (FA) (16:0, 18:0, 16:1 Delta 9 cis, 17:1 Delta 9 cis, 18:1 Delta 9 cis, 18:1 Delta 11 cis and 16:1 Delta 9 trans) and several FA groups (total linear saturated FA, total branched saturated FA, total saturated FA, total as monounsaturated FA (MUFA), total trans MUFA, total MUFA and total polyunsaturated PUFA). These results show the potential of NIRS as a rapid, and convenient tool to predict the major FA in lamb meat.

International audience; Seven- to 10-month-old calves are the typical production of Galician Blond (GB), the most important bovine local beef breed in Spain. As meat lipid repercussions on human health depend on their fatty acid (FA) profile, this study aimed at analysing the individual FA at weaning and at the end of the feeding finishing period, especially trans and cis 18:1 isomers in total lipids of the Longissimus thoracis muscle in GB male calves. Distribution of main FA in veal lipids was characterized by gas-liquid chromatography (GLC) analysis on a high polar glass capillary column. Total trans and cis 18:1 isomers were purified, from total FA (TFA) methyl esters, by preparative reversed-phase high-performance liquid chromatography, to allow subsequent specific analysis of their different isomers by GLC. Calves stayed with their mothers at pasture for 2 or 5 months in intensive or semi-extensive systems, followed by an indoor feeding period. The weaned (W) group was finished on concentrate and hay, whereas the non-weaned (NW) group was finished on concentrate and hay and it continued suckling until slaughter. The studied effects did not present interactions. The duration of the indoor finishing period hardly had a significant influence on the parameters analysed. Compared to W calves, NW calves had higher proportions (% TFA) of 18:3n-3 (+38%, P < 0.0001), 20:5n-3 (+22%, P = 0.005), 22:5n-3 (+13%, P = 0.042), cis monounsaturated FA (MUFA; 18%, P = 0.032), total MUFA (18%, P = 0.013), n-3 polyunsaturated FA (PUFA; +25%, P = 0.0001) and conjugated linoleic acid (CLA; +48%, P < 0.0001) to the detriment of 18:0 (-10%, P = 0.002), 18:2n-6 (-25%, P = 0.004) and n-6 PUFA (-20%, P = 0.011). With regard to cis and trans 18:1 isomers, NW calves had higher proportions (% total cis or trans 18:1) of Delta 11trans and Delta 16trans (+25% and +22%, respectively, P < 0.01) and Delta 13 + 14trans (+13%, P < 0.05) and lower proportions of Delta 6 to 8 and Delta 10trans (-28% and 58%, respectively, P < 0.001). NW calves' meat might be more suitable for human health than W calves' meat due to the increase of anti-atherogenous FA such as n-3 PUFA, CLA and 18:1 Delta 11trans.

International audience; The 'Organic' product label guarantees a production process that avoids the use of synthetic fertilisers, pesticides and hormones and minimises recourse to pharmaceuticals or veterinary drugs; however, the product's quality remains an issue that needs to be addressed in response to consumer demand. Consequently, this study was conducted to compare the sensory and nutritional qualities of meat and carcasses from pasture-fed lambs reared organically (O) or conventionally (C). Mean lamb growth profile was kept similar between the two treatments to avoid confounding effects with lamb age or weight at slaughter. The experiment was conducted over 3 years (2005 to 2007) with 12 O and 12 C lambs each year. The O and C treatments differed in the level of on-pasture mineral N fertilisation inducing a higher proportion of white clover in the organic pasture than the conventional pasture. Lambs were slaughtered when they attained a fat class of 2 to 3, and carcass and meat quality were evaluated. Lambs were slaughtered at an average weight and age of 35.3 kg and 156 days in the O treatment, respectively, and 35.2 kg and 155 days in the C treatment, respectively. Sensory evaluation indicated that loin chops from the O treatment had a higher level of abnormal fat odour compared with the C treatment. Carcasses from the O treatment had a softer subcutaneous fat one among 3 years (2007) compared to the C treatment. These results are probably due to a higher proportion of white clover in the diet. Organically reared lambs did offer the slight advantage of muscle fatty acid containing a higher level of stearic acid, which may have positive effects in the prevention of cardiovascular disease in humans. This may be the result of a higher rumen bio-hydrogenation of C18: 3n-3 due to differences in the botanical composition between the O and the C pasture. Production system had no effect on the colour characteristics of the meat and subcutaneous fat, except lightness of subcutaneous dorsal fat, which was slightly higher in the O lambs. There were no differences between O and C lambs in terms of colour stability and lipid oxidation of the meat during the 6-day refrigerated storage under gas-permeable film

Thomas-maleville Agnès. Regard d'un écrivain sur l'agglomération rouennaise : Hector Malot. In: Études Normandes, 56e année, n°4, 2007. Ecrivains normands : Hector Malo... et les autres. pp. 17-40.

Les acides gras polyinsaturés (AGPI) de type n-6 et n-3 sont connus pour leurs propriétés bénéfiques pour l'Homme. Mais ces composés sont également très sensibles à la peroxydation. Les produits terminaux de la lipoperoxydation sont le 4-hydroxy-2-nonénal (HNE) et le 4-hydroxy-2-hexénal (HHE) respectivement. Ces composés sont très réactifs ce qui leur permet de moduler l’expression génique en se fixant sur des protéines ou directement sur l’ADN. Par contre, ils peuvent aussi s’avérer cytotoxiques lorsqu’ils sont produits en excès. Ils existent sous deux formes, une forme libre et une forme liée notamment à des protéines. Nous avons mis au point une méthode d’extraction conjointe du HNE et du HHE sous leurs deux formes pour leur quantification par CPG-SM/SM. Nous avons également proposé différentes adaptations de la méthode en fonction des matrices testées. Enfin, certaines performances des méthodes proposées ont été évaluées par l'approche du profil d’exactitude.

Proteases play a pivotal role in epidermal differentiation and desquamation. Separation of a total protein extract from human reconstructed epidermis by two-dimensional gel electrophoresis and subsequent peptide analysis of a specific protein spot identified a new protein exhibiting similarities with the retroviral aspartic protease family. Cloning of the corresponding full-length cDNA revealed an open reading frame encoding for a new protease of 343 amino acids, containing a putative aspartic protease catalytic domain. We named this protein Skin ASpartic Protease (SASPase). RT-PCR and northern blot analysis of various human tissues revealed that SASPase was specifically expressed within the epidermis. Immunohistochemical analysis showed a particularly intense expression restricted to the granular layers, whereas in diseased skin, its expression was changed. Western blot analysis, using a monoclonal antibody, revealed the expression of two forms of the enzyme: a 28 kDa putative proform and the active 14 kDa form. Recombinant truncated SASPase (SASP28) was generated from a prokaryotic expression system in Escherichia coli as a fusion protein with GST. SASP28 degraded insulin and to a lesser extent casein with a pH optimum of 5. As seen for retroviral proteases, an auto-activation processing was evidenced, generating a 14 kDa protein (SASP14). Site-directed mutagenesis inhibited auto-activation of the enzyme. Indinavir, a potent HIV protease inhibitor used in AIDS therapy, had a significant inhibitory effect on rSASPase auto-activation, which could explain its side effects on skin.

Introduction et but de l’etude L’utilisation de la spectroscopie proche infrarouge (SPIR) pour determiner la qualite nutritionnelle d’une viande est en developpement. Parmi les parametres mesures, la quantification rapide et a moindre cout des acides gras (AG) est d’un interet particulier pour les filieres de differenciation des viandes portant sur leur qualite lipidique, comme la filiere Bleu-Blanc-Coeur. Des travaux chez le bovin ont montre que certains acides gras pouvaient etre mesures par SPIR sur des echantillons broyes finement a l’azote liquide (Mourot et al. , 2013). Cependant ce mode de preparation des echantillons est contraignant et onereux. L’objectif de ce travail est donc de tester les performances de prediction des acides gras de la viande bovine a l’aide d’un equipement de laboratoire et d’etudier l’effet du mode de preparation de l’echantillon. Materiel et methodes 76 bavettes de flanchet (Rectus abdominis) issues de gros bovins de types genetiques differents (lait ou viande), de categories animales differentes (taurillons, vaches, bœufs) et consommant des rations diversifiees a base d’herbe ou d’ensilage de mais avec ou sans supplementation lipidique a base de graines de lin extrudees ont ete prelevees. Les mesures de spectres infrarouges ont ete acquises en suivant un gradient croissant d’homogeneite de l’echantillon : (i) decoupe en cubes (B0), (ii) broye avec un hachoir a viande (B1), (iii) broye avec un broyeur a couteaux (B2) et (iv) broye finement dans l’azote liquide (B3). Les profils en acides gras (g/100 g de viande) ont ete determines par Chromatographie en Phase Gazeuse, sur les echantillons broyes a l’azote liquide. Les modeles de calibrations ont ete developpes par PLS et valides par validation croisee, sur le logiciel R et sur l’ensemble du spectre. Les performances des modeles pour les differents acides gras sont evaluees sur la base des criteres suivants : erreur de validation croisee (SECV) ; coefficient de determination en validation croisee (R 2 CV) et rapport de performances (RPD : rapport entre l’ecart-type des valeurs d’acides gras obtenues par chromatographie et le SECV). Un RPD superieur a 10 est considere comme etant similaire a la methode de reference. Pour la prediction des teneurs en acides gras des viandes, un RPD de 2,5 est considere comme satisfaisant pour des analyses de routine (Guy et al. , 2011). Resultats et Analyse statistique Les resultats montrent des performances de prediction des acides gras croissantes avec le gradient de broyage des echantillons, et ce quels que soient les acides gras. Les predictions sur les echantillons B0 ou B1 sont sensiblement les memes et ne sont pas satisfaisantes (RPD 2 > 0,85 et RPD > 2,5) et le sont d’autant mieux pour le broyage B3. Pour les autres acides et notamment les acides gras polyinsatures (AGPI) n-6 ou n-3, les performances ne sont pas satisfaisantes a des fins quantitatives (RPD Conclusion La SPIR peut etre utilisee comme methode de mesure rapide, fiable et simple a mettre en œuvre pour la determination de certains acides gras de la viande. L’homogeneite de l’echantillon est une condition importante quant a la qualite des predictions obtenues.

International audience; Fat entering food during frying needs to be monitored to control the nutritional properties of the products: fat penetration and fatty acid (FA) composition. The large difference between the apparent diffusion coefficients of lipids and meat fibers allows the use of diffusion-weighted magnetic resonance imaging (DWI) to measure oil uptake profiles. This method, in association with analysis of FAs by gas-liquid chromatography. predicts nutritional changes. Beef samples from finishing cows given control feed or high FA supplemented feed were fried in olive oil at 130 degrees C and 180 degrees C. Frying oil penetration was quantified by computing oil signal profiles from 3D DWI. Oil penetration was deeper at 180 degrees C (5 mm) than at 130 degrees C (2.5 mm), consistent with oil penetration processes. Oil penetration evaluated with DWI was correlated (R-2=0.82) with biochemical analysis of FA composition. These results highlight the predominance of oil uptake over animal feed effects in the first millimeters of in-plane fried meat.

International audience

In atopic dermatitis (AD), the skin barrier is disturbed, and the expression of calcium-dependent S100 proteins and the calcium gradient is also altered in the epidermis. The calmodulin-like skin protein (CLSP), which is expressed in the differentiated epidermis, is believed to modulate the function of calcium-dependent proteins involved in barrier formation and is significantly increased in the epidermis of psoriatic patients. We, therefore, investigated the CLSP level in skin biopsies taken from patients with acute exacerbated and non-exacerbated AD as well as from healthy control subjects. Immunohistochemical, Western blot and ELISA analyses showed significant increases (P < 0.03) in CLSP level in the epidermis from patients with acute exacerbated AD as compared to that from patients with non-exacerbated AD and from control subjects. Such increased expression of CLSP may help re-establish a functional epidermal barrier in acute AD.

International audience; Physicochemical characteristics and oxidative stability during storage were determined in Gastrocnemius pars interna (GN) and Iliofiburalis (IF) muscles of Rhea americana. Glycolytic potential (GP) and pH decline of muscles were measured within the first 24 h post mortem. Colour, lipid and protein stability were determined during storage of meat, i.e. 5 days under air-packaging at 4 °C, or 28 days under vacuumpackaging at 4 °C. In parallel, anti-oxidant status of muscles was estimated by measuring α-tocopherol content and anti-oxidant enzyme activities (superoxide dismutase and catalase), while pro-oxidant status was evaluated by determining haeminic iron and long chain fatty acids (especially polyunsaturated fatty acids). The ultimate pH was similar in both muscles, but the GP value was significantly higher in IF than in GN muscle. Haeminic iron and alpha-tocopherol content differed between muscles, with 30% more haeminic iron (pb0.05) and 134% more alpha-tocopherol (pb0.001) in IF than GN muscle. The IF muscle presented higher lipid content and lower PUFA/SFA ratio (polyunsaturated fatty acids/saturated fatty acids) than GN muscle. With storage under air-packaging, lipid and protein oxidation of rhea muscles increased up to 275% and 30%, respectively. This increase was more rapidly and marked in IF muscle. The IF also showed high level of metmyoglobin accumulation after 3 days of storage (47%) and was rejected by 1 consumer out of 2 in sensorial analysis. Under vacuum-packaging, both muscles showed a high stability of colour and no oxidation of lipids and proteins.

Effectiveness of GF-120 (Dow Chemical) Fruit Fly Bait containing the insecticide spinosad in controlling mango-infesting fruit flies (Diptera: Tephritidae) was assessed by comparing treated orchards with untreated orchards. Twelve mango, Mangifera indica L., plantations located in six villages (two similar orchards per village: one orchard treated and orchard untreated) scattered in the Borgou department (northern Benin) were monitored weekly with fly traps, and the fruit was sampled twice for larval infestation at the beginning and in the middle of May in both 2006 and 2007. The two main mango fruit fly pests are Ceratitis cosyra (Walker) and Bactrocera invadens Drew, Tsuruta & White, an invasive species that recently spread throughout West Africa. In both the 2006 and 2007 seasons, C. cosyra had the earliest peak of abundance, and the difference between treated and untreated orchards, in terms of mean number of flies trapped per week and per trap, was significant only in 2007. B. invadens populations quickly increased with the onset of the rains, from mid-May onward, with no significant difference between treated and untreated orchards. In 2006 and 2007, the larval infestation by B. invadens was significantly lower in plots treated with GF-120 than in untreated control plots. GF-120 provided an 81% reduction in the number of pupae per kilogram of fruit after weekly applications for 7 wk in 2006 and an 89% reduction after 10 wk of weekly applications in 2007. The possibility of integrating GF120 bait sprays in an integrated pest management package is discussed in relation to market requirements.

Introduction et but de l’etude Le projet SpecMeat vise a mettre au point un outil analytique utilisable en abattoir, base sur la spectroscopie proche infrarouge (SPIR), pour apprecier la composition en acides gras (AG) de la viande bovine. Il permettrait ainsi d’orienter precocement les carcasses vers les circuits commerciaux les plus adaptes (filiere Bleu-Blanc Cœur, par exemple) sur la base de leur composition en AG. La methode de reference pour la mesure de la composition en AG, la chromatographie en phase gazeuse (CPG), est destructrice, longue et couteuse. Des travaux precurseurs ont montre que la SPIR est une methode de mesure alternative (Mairesse et al. , 2012 ; Riovanto et al. , 2012), qui, apres une phase de calibration specifique de chaque parametre a mesurer, presente l’interet d’etre rapide, non destructive, peu couteuse et souvent utilisable en ligne. L’etude rapportee ici a pour objectif de determiner le tissu de la carcasse d’un gros bovin a mesurer pour une calibration optimale de la SPIR sur la composition en AG. Materiel et methodes Sept tissus dont 4 muscles i) Longissimus thoracis, ii) Infraspinatus, iii) Diaphragma, iv) Rectus abdominis, et 3 tissus adipeux : i) le gras d’epaule, ii) le gras sous-cutane intercostal, iii) le gras intermusculaire de la 5 e cote ont ete preleves sur 78 carcasses de gros bovins de differents types genetiques (lait, viande), de differentes categories (jeunes bovins, vaches, bœufs), alimentes avec des rations a base d’herbe ou d’ensilage de mais avec ou sans supplementation en graines de lin extrudees. Des mesures spectrales (C 0 ) ont ete realisees sur la carcasse avant prelevements puis sur les tissus broyes finement dans l’azote liquide (B 3 ). Les spectres ont ete acquis a l’aide d’un spectrometre portable ASD LabSpec 4 (350–2 500 nm). Les profils en acides gras ont ete determines par CPG apres extraction des lipides totaux et transmethylation (Guy et al. , 2011). Les modeles de calibration ont ete developpes par PLS et valides par validation croisee sur le logiciel R, pour l’ensemble du spectre. Les performances du modele de predictions pour les differents acides gras sont evaluees sur la base des criteres suivants : erreur de validation croisee (SE CV ) et coefficient de determination en validation croisee (R 2 CV ). Resultats et Analyse statistique Pour les tissus adipeux, quels que soient les acides gras consideres, ils ne sont pas correctement predits a partir des mesures SPIR C 0 (R 2 CV 3 (R 2 CV 3 : R 2 CV > 0,78) par rapport aux muscles bruts presents sur la carcasse (C 0 : 0, 66 > R 2 CV > 0,29). Les AG polyinsatures (AGPI) semblent mal predits par la SPIR portable. Le Diaphragma , muscle plus gras que les 3 autres (10,2 % de lipides vs . 5,9 % en moyenne pour les autres), enregistre les performances de calibration les plus faibles des muscles, celles des 3 autres muscles etant tres proches. Conclusion La SPIR portable presente un potentiel interessant pour une utilisation sur le terrain en vue de classer et orienter les carcasses sur la base de leur composition en AG. De facon a optimiser la prediction de la composition en AG, il semble preferable de realiser les mesures sur des muscles maigres comme le Rectus abdominis. Remerciements : Cette etude a ete realisee avec le soutien financier du 14 e FUI, des regions Bretagne et Pays de la Loire, du Conseil General des Cotes-d’Armor, de Lannion-Tregor-Communaute et du FEDER (Projet SpecMeat).

International audience; The aim of this study was to validate the measurement of glucose appearance rate using [6,6-2H2]glucose i.v. infusion in lactating dairy cows. Sample enrichments were analysed by gas chromatography/mass spectrometry. Linearity (enriched solutions) and specificity (enriched plasma) were good: for enrichments ranging between 1.6 and 6.3 mol% excess, the slopes were about 1 and the ordinates at the origin were not different from zero. For a plasma enriched at 3.74 mol% excess, repeatability and long term intralaboratory reproducibility coefficients of variation were 1.31 and 1.90%, respectively. The appearance rates were calculated by two models. The values provided by the steady-state model were not different from those provided by the non-steady-state Steele model. Both models can be used because the treatment effects were similarly discriminated regardless of the model. In our experiments analysing the nutritional effects on Ra in mid-lactating cows, the precision of the method (1.90%) was not the limiting factor to detect a significant difference in Ra compared to the statistical precision obtained with the experimental scheme (4 × 4 and 5 × 5 Latin square design). We conclude that in lactating dairy cows, the measurement of glucose fluxes with this method is relevant and minimally invasive for the animals.

Desquamation is described as a protease-dependent phenomenon where serine proteases with a basic pH optimum play a key role. Recently proteases with an acidic pH optimum were identified in the stratumcorneum and associated with desquamation, e.g., cathepsin D and the stratum corneum thiol protease. The purpose of this study was to investigate if human stratum corneum contains proteases different from the above, exhibiting similar properties. After gel filtration, we identified four distinct proteolytic activities in a human stratum corneum extract, a cathepsin-E-like activity (80 kDa), a cathepsin-D activity (40 kDa), a yet unknown cathepsin-L-like form (28 kDa) exhibiting the highest caseinolytic activity, and a chymotrypsin-like protein (24 kDa) containing the acidic activity of the well described stratum corneum chymotryptic enzyme. We named the new 28 kDa protease stratum corneum cathepsin-L-like enzyme. Characterization of stratum corneum cathepsin-L-like enzyme provided clear evidence that this new protease, despite its membership to the cathepsin-L-like family, is distinct from cathepsin L and from the recently described stratum corneum thiol protease. Its ability to hydrolyze corneodesmosin, a marker of corneocyte cohesion, was in favor of a role of stratum corneum cathepsin-L-like enzyme in the desquamation process. A more detailed analysis did not allow us to identify stratum corneum cathepsin-L-like enzyme at the molecular level but revealed that stratum corneum thiol protease is identical with the recently described cathepsin L2 protease. Reverse transcription polymerase chain reaction studies and the use of a specific antibody revealed that, in contrast to earlier reports, expression of stratum corneum thiol protease in human epidermis is not related to keratinocyte differentiation. Our results indicate that the stratum corneum thiol protease is probably expressed as a pro-enzyme in the lower layers of the epidermis and in part activated by a yet unidentified mechanism in the upper layers during keratinocyte differentiation.

A protein exhibiting endoglycosidase activity was purified from plantar stratum corneum to apparent homogeneity in two sequential column chromatographic steps. Protein sequencing revealed its identity with the recently cloned human heparanase 1, an enzyme, the expression of which is reported to be related to the metastasic potential of tumor cells. By using a heparanase 1 specific antibody we were able to demonstrate that, in the plantar stratum corneum, heparanase 1 exists in two forms, the active 50 kDa protein and the inactive 63 kDa form, probably a proform of the enzyme. The antibody also decorated numerous degradation fragments. Reverse transcription polymerase chain reaction studies as well as immunohistochemical analysis using reconstructed and normal human epidermis demonstrated clearly a keratinocyte differentiation related expression of heparanase 1. Interestingly, the antibody also strongly decorated dendritic cells, which after double labeling could be identified to be a subpopulation of the epidermal Langerhans cells. Based on our findings and the known history of this enzyme, we advanced the hypothesis that heparanase 1 has multiple physiologic functions in the epidermis: (i) it plays an important role in epidermal differentiation, possibly by modulating the liberation of heparan sulfate bound (growth) factors; (ii) in the stratum corneum, the endoglycosidase activity of heparanase 1 might be indispensable and represent the first step in the desquamation process; and (iii) in Langerhans cells, its catalytic activity is required for the trans-tissue migration of these cells.