Proteomic profile evolution during steatosis development in ducks.

We investigated a protein profile evolution during steatosis in ducks using 2-dimensional electrophoresis gels to better understand the mechanisms underlying liver steatosis at the level of hepatic proteins in waterfowl. Two-dimensional electrophoresis gels were performed in the liver at different stages of steatosis in the duck. Mule ducks were slaughtered after 0, 14, or 23 meals of overfeeding, according to commercial conditions. Thirty-one proteic spots were differentially expressed between 3 or 2 durations of overfeeding: 3 spots were differentially expressed between the 3 times and 28 spots were differentially expressed between 2 times. The identified proteins (14) could be regrouped into 5 categories: enzymes, translation factors, proteins involved in cell structure, proteins with antioxidant properties, and proteins that can link calcium. This study opens new research areas in the understanding of steatosis in waterfowl, such as cell structure and oxidative stress.

Early post-mortem sarcoplasmic proteome of porcine muscle related to protein oxidation.

Oxidative deterioration or modifications of proteins which appear during meat storage and processes can result in the impairment of technological, sensorial and nutritional qualities. Improving the quality involves a better understanding of the biochemical mechanisms responsible for protein oxidation in meat. For that purpose, an analysis was conducted to investigate the relationships between the early post-mortem sarcoplasmic proteome, which contains the majority of enzymes involved in the oxidative process, and protein oxidation generated during meat storage and cooking. This study was performed in Longissimus lumborum pig muscle. In order to have sufficient variability in the proteome and in the meat oxidation level, five groups of 10 animals issued from two different breeds and raised in three different rearing systems were analysed. Protein oxidation was estimated by the measurement of carbonyl groups after 1 and 4days of refrigerated storage, and after 100°C experimental cooking of the 4days aged meat. Significant correlations (p<0.05) were observed between the level of carbonyl groups and the intensities of 104 spots of the 2D electrophoresis, out of which 52 were clearly identified. The possible involvement of some proteins in the muscle oxidative stress leading to protein oxidation is discussed.

Deciphering the protein digestion of meat products for the elderly by in vitro food oral processing and gastric dynamic digestion, peptidome analysis and modeling.

The elderly population will increase sharply in the future, along with an emerging range of specific nutritional needs that include adapted food. We aimed to develop a workflow to study the fate of a food, objectify the bioavailability of nutrients in the case of the digestive physiology of the elderly, and model the fate of proteins in the stomach. Pork frankfurters were subjected to in vitro normal and deficient mastication and gastric digestion, mimicking adult and elderly food oral and digestive processing. Swallowable food boluses were characterized for granulometric and rheological properties. Biochemical analyses were conducted on the bolus and on the digesta. Macronutrients, label-free peptide quantification and identification were performed, and modeling was applied to protein digestion kinetics. After deficient mastication, the food bolus was harder with more large particles, lower free iron release and more protein oxidation. The amount of peptides released in the stomach progressively increased, but to a lower extent for the elderly digestive condition and irrespective of masticatory efficiency. 592 peptides were identified from 67 proteins. Different trajectories were observed for adult and elderly digestive conditions, and two groups of meat proteins were identified based on the rate of hydrolysis. Designing suitable foods requires in vitro tools to evaluate the possible benefit for the elderly. Besides the well-known notion of Food Oral Processing (FOP), our work broadens the concept by extending oral activity to digestion when working in a nutritional context. This new concept is named Food Oral and Digestive Processing, FODP.

Lactobacillus role during conditioning of refrigerated and vacuum-packaged Argentinean meat.

The role of Lactobacillus strains with bioprotective and technological potential on raw beef during 15days of storage under vacuum at 7°C was investigated. The assayed strains were able to grow on the meat, Lactobacillus curvatus CRL705 and Lactobacillus sakei 23K showing the highest competitiveness. A net increase of amino acids was determined in inoculated samples when compared to the control, this being maximal for Lactobacillus plantarum CRL681. Although an important endogenous activity of meat sarcoplasmic proteins was observed, the disappearance of protein bands and the generation of a new one were detected as a consequence of Lactobacillus growth. A synergistic effect of Lactobacillus in combination with the muscle proteolytic enzyme complex can be suggested. From the studied strains, the bacteriocin producer L. curvatus CRL705 may be considered as a good candidate to contribute to meat ageing by means of small peptides and free amino acids generation while improving shelf life.

Pig Longissimus lumborum proteome: Part I. Effects of genetic background, rearing environment and gender.

A 2×2×2 factorial experiment on Longissimus lumborum of 24 pigs found that rearing environment (indoors or outdoors), breed of sire (Duroc or Large White), and gender (female or castrated male) influenced 22, 10, and 88 proteins of the soluble fraction, respectively, containing 220 matched spots in total. Some proteins were influenced by more than one main effect. Outdoor rearing resulted in lower levels of enzymes of the glycolytic pathway suggesting a more oxidative metabolism. Breed of sire slightly altered the balance of enzymes of the glycolytic pathway. Gender had profound effects. In particular, different enzyme levels suggest a more lipid oriented energy metabolism, and a higher extractability of myofibrillar proteins suggest altered control of the contractile apparatus, in castrated males. Differences in extractability did not explain the profound gender effects. Glycogen content, ultimate pH, drip and thawing losses showed main or interactive effects of the three treatment factors.

Pig Longissimus lumborum proteome: Part II: Relationships between protein content and meat quality.

Gender, rearing environment and breed of sire influenced 50.5% of the matched protein spots of the soluble fraction and some meat quality traits [Kwasiborski, A., Sayd, T., Chambon, C., Santé-Lhoutellier, V., Rocha, D., & Terlouw, C. (2008). Muscle proteome in pigs: Part I: Effects of genetic background, rearing environment and gender. Meat Science]. Multiple regression analyses determined that 1 or 2 proteins explained between 24% and 85% of variability in Longissimus meat quality. Regression models differed between treatment groups, but relationships between proteins and meat quality traits seemed to be related to common underlying mechanisms. Thus, proteins retained in models for ultimate pH, lightness, drip, thawing and cooking loss were related to the glycolytic pathway, phosphate transfer, or fibre type composition. Another model for thawing loss retained proteins related to denaturation of myofibrils or lipid content. The models for redness involved proteins related to post-mortem oxidative activity. Thus, proteins correlated with meat quality traits were related to biochemical mechanisms known to be involved in meat quality. Relative contributions of these mechanisms may vary according to gender, sire breed or rearing environment.

Combined in vivo and in silico approaches for predicting the release of bioactive peptides from meat digestion.

We studied the kinetics of peptide release during the gastric digestion of meat proteins in vivo, in view to predicting the release of bioactive peptides further on in the digestive tract. Six mini pigs fitted with gastric cannulas received a meal with cooked beef as protein source. Digesta was collected at regular time intervals up to 5½â€¯h. The peptides generated by the gastric digestion of meat were identified and quantified using label-free LC MS, thereafter subjected to in silico digestion mimicking the action of intestinal enzymes. Three clusters of proteins presenting similar evolutions according to their dynamic hydrolysis were obtained. This study clearly improves the in silico prediction of the intestinal release of bioactive peptides by mapping meat protein degradation in the stomach in an in vivo model. Knowledge of the conformation of the peptides released in the stomach further improves this prediction.

Visual and visuospatial short-term memory in mild cognitive impairment and Alzheimer disease: role of attention.

It has been proposed that visual recognition memory and certain attentional mechanisms are impaired early in Alzheimer disease (AD). Little is known about visuospatial recognition memory in AD. The crucial role of the hippocampus on spatial memory and its damage in AD suggest that visuospatial recognition memory may also be impaired early. The aim of the present study was to evaluate which modality, i.e. visual or visuospatial, is more implicated in the early memory impairment in AD. First, to determine onset of memory impairment, we compared the performances of patients with AD to those with amnestic mild cognitive impairment (MCI). Second, to determine the relative contribution of attentional impairment on the performance of MCI and AD patients, we tested the influence of a distractor in the interval between the memory image and recognition tests. Results showed that visuospatial short-term deficits appear earlier than visual short-term ones. In addition to mnemonic deficits, results showed attentional deficiency in both MCI and AD patients. Deficits of performances in visual modality seemed of attentional origin whereas those of visuospatial modality seemed of memory origin. The combination of attentional and mnemonic evaluation is likely to be a promising approach to finding predictive markers that distinguish MCI patients that convert to AD.

Behavioral and immunohistological effects of cholinergic damage in immunolesioned rats: alteration of c-Fos and polysialylated neural cell adhesion molecule expression.

The aim of this study was to determine the brain structures as well as the plasticity events associated with the behavioral effects of cholinergic damage. Rats were submitted to injection of 192 IgG-saporin in the medial septum/diagonal band of Broca complex and the nucleus basalis magnocellularis. The immunohistochemical expression of c-Fos protein and PSA-NCAM (polysialylated neural cell adhesion molecule) and the behavioral performances in the nonmatching-to-position task were assessed at various post-lesion times. Thus, 3 days after injection of the immunotoxin, increased c-Fos labeling was observed in the areas of infusion, indicating these cells were undergoing some plastic changes and/or apoptotic processes. A drastic increase was observed in the number of PSA-NCAM positive cells and in their dendritic arborization in the dentate gyrus. At 7 days post-lesion, no behavioral deficit was observed in immunolesioned rats despite the drastic loss of cholinergic neurons. These neurons showed decreased c-Fos protein expression in the piriform and entorhinal cortex and in the dentate gyrus. In the latter, PSA-NCAM induction was high, suggesting that remodeling occurred, which in turn might contribute to sustaining some mnemonic function in immunolesioned rats. At 1 month, cholinergic neurons totally disappeared and behavioral deficits were drastic. c-Fos expression showed no change. In contrast, the increased PSA-NCAM-labeling observed at short post-lesion times was maintained but the plastic changes due to this molecule could not compensate the behavioral deficit caused by the immunotoxin. Thus, as the post-lesion time increases, a gradual degeneration process should occur that may contribute to mnemonic impairments. This neuronal loss leads to molecular and cellular alterations, which in turn may aggravate cognitive deficits.

Small peptides (<5kDa) found in ready-to-eat beef meat.

Dietary proteins can have biological properties, many attributed to bioactive peptides (2-50 amino acids). Since little is known about peptides in meat, we investigated the postmortem occurrence of low molecular weight peptides (<5kDa) in bovine Pectoralis profundus muscle, after 14 days storage at 4°C and vacuum cooking for 90min at 75°C. The study combined quantitative (amino acid analysis) and qualitative approaches (mass spectrometry). Eighty-nine percent of peptidic amino acids in fresh muscle corresponded to carnosine, anserine and glutathione. Levels of these compounds were lower in cooked meat compared to fresh muscle. Concomitantly, numerous larger compounds, most probably peptides, were generated in a very reproducible manner during ageing and even more during cooking of meat. Seven peptides (fragments of troponin T, nebulin, procollagen and cypher proteins) were identified in cooked meat extracts.

Quantification of peptides released during in vitro digestion of cooked meat.

We aimed to identify and quantify the peptides generated during in vitro digestion of cooked meat by liquid chromatography coupled with high resolution mass spectrometer. A total of 940 non-redundant peptides in the gastric compartment and 989 non-redundant peptides in the intestinal compartment were quantified and identified. Among the 71 different proteins identified, 43 meat proteins were found in the two digestive compartments, 20 proteins were specific to the gastric compartment and 8 proteins to the intestinal compartment. In terms of estimation, the proteins involved in muscle contraction and structure were preferentially enzymatically hydrolyzed in the small intestine. The effect of cooking provided different but less clear patterns of digestion. To the best of our knowledge, this constitutes the highest number of peptides identified in beef meat digests and provides a comprehensive database for meat protein digestion associated with cooking conditions. Such quantitative and qualitative differences may have important nutritional consequences.

Proteome changes in rat serum after a chronic ingestion of enriched uranium: Toward a biological signature of internal contamination and radiological effect.

The civilian and military use of uranium results in an increased risk of human exposure. The toxicity of uranium results from both its chemical and radiological properties that vary with isotopic composition. Validated biomarkers of health effects associated with exposure to uranium are neither sensitive nor specific to uranium radiotoxicity and/or radiological effect. This study aimed at investigating if serum proteins could be useful as biomarkers of both uranium exposure and radiological effect. Male Sprague-Dawley rats were chronically exposed through drinking water to low levels (40mg/L, corresponding to 1mg of uranium per animal per day) of either 4% (235)U-enriched uranium (EU) or 12% EU during 6 weeks. A proteomics approach based on two-dimensional electrophoresis (2D-DIGE) and mass spectrometry (MS) was used to establish protein expression profiles that could be relevant for discriminating between groups, and to identify some differentially expressed proteins following uranium ingestion. It demonstrated that the expressions of 174 protein spots over 1045 quantified spots were altered after uranium exposure (p<0.05). Using both inferential and non-supervised multivariate statistics, we show sets of spots features that lead to a clear discrimination between controls and EU exposed groups on the one hand (21 spots), and between 4% EU and 12% EU on the other hand (7 spots), showing that investigation of the serum proteome may possibly be of relevance to address both uranium contamination and radiological effect. Finally, using bioinformatics tools, pathway analyses of differentially expressed MS-identified proteins find that acute phase, inflammatory and immune responses as well as oxidative stress are likely involved in the response to contamination, suggesting a physiological perturbation, but that does not necessarily lead to a toxic effect.

Digestion of cooked meat proteins is slightly affected by age as assessed using the dynamic gastrointestinal TIM model and mass spectrometry.

In humans, meat ensures the supply of proteins with high nutritional value and indispensable amino acids. The main goal of the present study was to compare the degradation of meat proteins in adult and elderly digestive conditions. Cooked meat was subjected to in vitro digestion in the dynamic multi-compartmental TIM (TNO gastroIntestinal Model) system. Digestibility and bioaccessibility were determined using nitrogen balance and digestion products were identified using mass spectrometry. The TIM model was adapted according to in vivo data to mimic the specific digestive conditions of elderly people. Meat protein digestibility and bioaccessibility were around 96 and 60% respectively and were not influenced by age (P > 0.05). As much as 800 peptides were identified in the duodenal and jejunal compartments issued from 50 meat proteins with a percentage of coverage varying from 13 to 69%. Six proteins, mainly from the cytosol, were differentially hydrolyzed under the adult and elderly digestive conditions. Pyruvate kinase was the only protein clearly showing a delay in its degradation under elderly digestive conditions. This study provides significant insights into the understanding of meat protein dynamic digestion. Such data will be helpful to design in vivo studies aiming to evaluate dietary strategies that can attenuate muscle mass loss and more generally maintain a better quality of life in the elderly population.

Time course of behavioral changes following basal forebrain cholinergic damage in rats: Environmental enrichment as a therapeutic intervention.

The present experiment was designed to study changes in behavior following immunolesioning of the basal forebrain cholinergic system. Rats were lesioned at 3 months of age by injection of the 192 IgG-saporin immunotoxin into the medial septum area and the nucleus basalis magnocellularis, and then tested at different times after surgery (from days 7-500) on a range of behavioral tests, administered in the following order: a nonmatching-to-position task in a T-maze, an object-recognition task, an object-location task, and an open-field activity test. The results revealed a two-way interaction between post-lesion behavioral testing time and memory demands. In the nonmatching-to-position task, memory deficits appeared quite rapidly after surgery, i.e. at a post-lesion time as short as 1 month. In the object-recognition test, memory impairments appeared only when rats were tested at late post-lesion times (starting at 15 months), whereas in the object-location task deficits were apparent at early post-lesion times (starting from 2 months). Taking the post-operative time into account, one can hypothesize that at the shortest post-lesion times, behavioral deficits are due to pure cholinergic depletion, while as the post-lesion time increases, one can speculate the occurrence of a non-cholinergic system decompensation process and/or a gradual degeneration process affecting other neuronal systems that may contribute to mnemonic impairments. Interestingly, when middle-aged rats were housed in an enriched environment, 192 IgG-saporin-lesioned rats performed better than standard-lesioned rats on both the nonmatching-to-position and the object-recognition tests. Environment enrichment had significant beneficial effects in 192 IgG-saporin-lesioned rats, suggesting that lesioned rats at late post-lesion times (over 1 year) still have appreciable cognitive plasticity.

Down-regulation of mitochondrial mRNAs in the mdx mouse model for Duchenne muscular dystrophy.

In our search for genes up- or down-regulated genes in the mdx mouse model for Duchenne muscular dystrophy, we isolated a down-regulated mitochondrial DNA clone. In addition to this clone, all protein-coding mitochondrial genes tested had tissue-specific and age independent down-regulated expression. This implied mechanisms at the RNA level since no change in the mitochondrial DNA contents were detected. Cytochrome c oxidase activity showed the same range of down-regulated expression. These data provide a molecular basis for energetic metabolism modifications in mdx mice.

Therapeutic effectiveness of iodine-131 MIBG metastases of a nonsecreting paraganglioma.

This case report describes the treatment of the bone metastases of a nonfunctioning sympathetic paraganglioma, with [131I]MIBG. After primary tumor excision and unsuccessful external radiotherapy, the patient received three therapeutic doses of [131I]MIBG, resulting in a reduction of the number and volume of metastases, and an improvement of the general condition. At 3 yr following [131I]MIBG therapy, the patient remained in remission. [131I]MIBG appears to be an efficient and safe agent for treating malignant sympathetic paraganglioma.

Whole body quantitative autoradiographic study of the biodistribution of iobitridol in rats.

RATIONALE AND OBJECTIVES: Iobitridol is a new nonionic low-osmolality contrast medium. During preclinical development of this agent, it was of interest to verify that it behaves like other urographic and angiographic contrast agents (i.e., as a tracer of extracellular fluid). METHODS: Male and female rats were imaged using a quantitative autoradiographic method after intravenous administration of iodine-125-labeled product at a dose of 300 mg iodine/kg. RESULTS: The radioactivity was rapidly distributed with substantial uptake in the thyroid, kidneys, and skin after 10 minutes. The central nervous system showed no uptake. The radioactivity was rapidly eliminated (i.e., after 24 and 48 hours, only traces were found) except in the thyroid (because of free radiolabeled iodides present in small quantities in the administered solution). The considerable renal uptake after administration can be attributed to urinary excretion of the radioactivity (86% of the administered dose after 24 hours). Total elimination was achieved after 48 hours. No sex-related effects were observed. CONCLUSION: The absence of a target organ, the abundant and rapid urinary elimination, and the absence of transfer across the blood-brain barrier suggest that iobitridol is a tracer of extracellular fluid.

Superparamagnetic iron oxide-enhanced magnetic resonance imaging of experimental liver tumors after mitomycin C administration.

The influence of mitomycin C chemotherapy on superparamagnetic iron oxide (SPIO) uptake by the liver was studied in rats (n = 70). This commonly used antineoplastic drug induces a decrease in the phagocytic function of the macrophage-phagocytic system (MPS). The plasma clearance of SPIO measured by relaxometry followed a biexponential model. The fast component half-life increased from 2.9 minutes in controls to 4.5 minutes in mitomycin C-treated animals. The slow component half-life increased from 11.3 to 36.7 minutes. Nevertheless, the magnetic resonance imaging (MRI) diagnostic efficacy 2 hours after infusion of the superparamagnetic agent AMI 25 (n = 10) was as satisfactory in the treated group as in the untreated one. These MRI results were consistent with the relaxometric T2* liver measurements, which were identical in both groups.

Evaluation of intrarenal distribution of ultrasmall superparamagnetic iron oxide particles by magnetic resonance imaging and modification by furosemide and water restriction.

RATIONALE AND OBJECTIVES: The steady-state intrarenal distribution of ultrasmall superparamagnetic iron oxide (USPIO) particles in the cortex, the outer medulla (OM), and the inner medulla (IM) was investigated using magnetic resonance imaging in the normal rabbit kidney and in situations that modify the corticopapillary osmotic gradient. METHODS: Experiments were performed on rabbit kidneys with T2-weighed spin-echo sequence and T2-weighted gradient-recalled-echo sequence. The intravenous dose was 27 mumole/kg of iron in all rabbits. Three groups were studied: normally hydrated rabbits (n = 5), after water restriction (n = 5) to increase the osmotic gradient, and after furosemide injection (n = 5) to decrease the osmotic gradient. The signal intensity (SI) was quantified by region of interest placed on the cortex, the OM and the IM. RESULTS: In normally hydrated rabbits, a maximal decrease of the SI after USPIO was noted in the medulla, without significant difference between the OM and the IM on spin-echo sequences. The decrease of the SI was maximal in the IM on gradient-recalled echo sequences. In dehydrated animals, the maximum SI loss was in the OM. The furosemide-induced transient enhancement of the IM lasted a few minutes. CONCLUSIONS: The observed SI changes due to the distribution of the USPIO between OM and IM were not based on modifications of the osmotic gradient. The authors suggest that these SI changes were mostly dependent on the vascular density.

Gd-DOTA. Pharmacokinetics and tolerability after intravenous injection into healthy volunteers.

The pharmacokinetics of Gd-DOTA meglumine in humans were evaluated in six healthy male volunteers. The agent was injected intravenously at 0.1 mmol/kg over approximately 2 minutes. Its behavior was found to be similar to that of urographic and angiographic iodinated contrast media with a plasma elimination half-life of 91 +/- 14 minutes (mean +/- standard deviation [SD]), a small distribution volume of 171.0 +/- 19.7 mL/kg and rapid urinary excretion. The results suggest rapid passive extravascular diffusion of gadolinium (Gd)-DOTA in the interstitial space without intracellular penetration, followed by a rapid urinary excretion via glomerular filtration. Furthermore, the results are consistent with animal data that showed that the compound does not cross the normal blood brain barrier. Its plasma pharmacokinetics appeared to be similar to those reported for Gd-DTPA. No relevant biological effects were seen with Gd-DOTA, especially in regard to serum iron and bilirubin levels.