Nutritional management of hyperlipaemia in a jenny: a brief report.

An adult jenny (5-years-old, non-pregnant) was presented to the Veterinary Teaching Hospital (VTH) of the University of Sassari, with a recent history of appetite loss, extreme underweight condition and reluctance to move. On physical inspection, emaciation [body condition score, BCS: 3/9], muscular waste [muscular condition score, MCS: 1/5], loose/running faeces [faecal score, FS: 2/8], and a general state of mild dehydration were found. Blood analyses outlined a general undernourishment condition [circulating albumins, ALB: 17.6 g/L (21.6-31.6 g/L)] with underlying systemic inflammatory profile and moderate increase in circulating enzymes to explore liver function [aspartate amino-transferase, AST: 657 u/L (279-430 u/L); alanine amino-transferase ALT: 60 u/L (5-14 u/L); gamma-glutamyl-transferase, γ-GT: 87 IU/L (14-69 IU/L); total bilirubin close to the upper limit, TB: 0.20 mg/dL(0.07-0.21 mg/dL)]and hyperlipaemia [TG: 8.70 mmol/L (0.60-2.87 mmol/L)], following fat depots mobilisation, with total cholesterol closed to the lower limit of the physiological range. Hyper-phosphataemia was linked to haemolytic anaemia [P:1.81 mmol/L (0.77-1.39 mmol/L) and red blood cells, RBC: 4.14 1012/L (4.40-7.10 1012)] aligned with the TB to the upper limit. On ultrasound abdominal imaging, enlarged and hyper-echogenic liver was observed. Based on the clinical evaluation, a condition of hepatic lipidosis was diagnosed, requiring dedicated nutritional treatment to solve the extreme emaciation along with the metabolic disorder in support of medical therapy. A two-step feeding protocol was planned to support treatments aiming at immediate re-hydration (Ringer lactate solution 2 ml/kg/8 h). The nutritional objectives were meant at first to restart the voluntary feed intake. Gradual increasing energy provision through a palatable hay-based diet was planned to cover one fourth of daily metabolizable energy requirement calculated on the expected metabolic weight, adjusted according to the daily intake of feed and clinical condition. At the conclusion of this first 7-day phase, circulating blood parameters were closer to the reference values and the BCS moved from 3 to 4 out of 9. Bowel motility was restored, and faecal score improved (4/8). In the second phase, allowance to pasture and a combination diet with compound mixed feed were designed. Within four weeks of starting the nutritional plan, blood parameters were re-established to reference values. The gradual feed provision calculated in this two-phase approach proved successful in support of the overall clinical improvement observed after four weeks of treatment, in a severely undernourished jenny with compromised liver functions.

Characterization of OAR1 and OAR18 QTL associated with muscle depth in British commercial terminal sire sheep.

This study aimed at verifying previously identified QTL affecting growth and carcass traits on ovine chromosome 18 (OAR18) in Texel sheep (n = 1844), and on OAR1 in Charollais (n = 851) and Suffolk (n = 998) sheep. The QTL were investigated using regression and variance component mapping (VCA) of body weight, muscle and fat depth measurements. In addition, the mode of inheritance of the Texel OAR18 QTL was explored, using data from 4376 Texel sheep, fitting VCA models testing for additive and imprinting effects. We also simulated a 480-sheep population with different QTL imprinting models and various available levels of marker information to understand the behaviour of the VCA results under different assumed genetic models. In summary, the previously identified QTL were successfully verified using both interval mapping and VCA in the three breeds. We propose a polar overdominance mode of inheritance for the OAR18 QTL in Texel sheep, and we present methods to dissect the QTL mode of inheritance, using the Texel OAR18 QTL as an example.

Risk assessment for recrudescence of avian influenza in caged layer houses following depopulation: the effect of cleansing, disinfection and dismantling of equipment.

Following an outbreak of highly pathogenic avian influenza virus (HPAIV) in a poultry house, control measures are put in place to prevent further spread. An essential part of the control measures based on the European Commission Avian Influenza Directive 2005/94/EC is the cleansing and disinfection (C&D) of infected premises. Cleansing and disinfection includes both preliminary and secondary C&D, and the dismantling of complex equipment during secondary C&D is also required, which is costly to the owner and also delays the secondary cleansing process, hence increasing the risk for onward spread. In this study, a quantitative risk assessment is presented to assess the risk of re-infection (recrudescence) occurring in an enriched colony-caged layer poultry house on restocking with chickens after different C&D scenarios. The risk is expressed as the number of restocked poultry houses expected before recrudescence occurs. Three C&D scenarios were considered, namely (i) preliminary C&D alone, (ii) preliminary C&D plus secondary C&D without dismantling and (iii) preliminary C&D plus secondary C&D with dismantling. The source-pathway-receptor framework was used to construct the model, and parameterisation was based on the three C&D scenarios. Two key operational variables in the model are (i) the time between depopulation of infected birds and restocking with new birds (TbDR) and (ii) the proportion of infected material that bypasses C&D, enabling virus to survive the process. Probability distributions were used to describe these two parameters for which there was recognised variability between premises in TbDR or uncertainty due to lack of information in the fraction of bypass. The risk assessment estimates that the median (95% credible intervals) number of repopulated poultry houses before recrudescence are 1.2 × 104 (50 to 2.8 × 106), 1.9 × 105 (780 to 5.7 × 107) and 1.1 × 106 (4.2 × 103 to 2.9 × 108) under C&D scenarios (i), (ii) and (iii), respectively. Thus for HPAIV in caged layers, undertaking secondary C&D without dismantling reduces the risk by 16-fold compared to preliminary C&D alone. Dismantling has an additional, although smaller, impact, reducing the risk by a further 6-fold and thus around 90-fold compared to preliminary C&D alone. On the basis of the 95% credible intervals, the model demonstrates the importance of secondary C&D (with or without dismantling) over preliminary C&D alone. However, the extra protection afforded by dismantling may not be cost beneficial in the context of reduced risk of onward spread.

p.Asn176Lys and p.Met137Thr dimorphisms of the PRNP gene significantly decrease the susceptibility to classical scrapie in ARQ/ARQ sheep.

In this study, we investigated the susceptibility to scrapie of Sarda breed sheep carrying the genotype ARQ/ARQ with additional polymorphisms at the PRNP gene. To do this, we examined 256 scrapie-affected sheep and 320 flock-mate negative controls from 24 flocks. Logistic regression analysis demonstrated that sheep carrying the ARQ/ARQ genotype with additional dimorphisms had lower risk of becoming scrapie affected when compared with those with ARQ/ARQ(wildtype) genotype. ARQ/ARQ genotypes that were detected with heterozygous or homozygous p.Asn176Lys and p.Met137Thr dimorphisms were associated with the lowest susceptibility to the disease. A significant lower risk was also associated with the p.Arg154His dimorphism, while p.Leu141Phe had a protective effect that was not statistically significant.

Visualization of cleavage furrow proteins in fixed dividing spermatocytes.

Cytokinesis separates the cytoplasmic organelles and the duplicated genome into two daughter cells at the end of cell division. In animal cell cytokinesis, assembly and constriction of the contractile apparatus must be finely coordinated with plasma membrane remodeling and vesicle trafficking at the cleavage furrow. Accurate control of these events during cell cleavage is a fundamental task in all organisms and is also essential for maintaining ploidy and preventing neoplastic transformation. Drosophila male meiosis provides a well-suited cell system for exploring the molecular mechanisms underlying cytokinesis, combining the powerful tools of Drosophila genetics with unique cytological characteristics. Remarkably the large size of male meiotic cells highly facilitates cytological analysis of cytokinesis. Here we describe the main procedures that we use for fixing and visualizing cleavage furrow proteins in male meiotic cells. Moreover, we detail our protocol to detect protein interactions in fixed dividing spermatocytes by applying in situ proximity ligation assay.

Effects in dogs with behavioural disorders of a commercial nutraceutical diet on stress and neuroendocrine parameters.

The well-being of dogs can be affected by changes in human lifestyle, eating habits and increased stressors that lead to behavioural disorders including fear, hyperactivity and anxiety, followed by negative affective moods and poor welfare. This randomised, controlled clinical evaluation involved 69 dogs, 38 males and 31 females, of different breeds, with behavioural disorders related to anxiety and chronic stress. They were fed a control diet or a nutraceutical diet (ND group) for 45 days. Neuroendocrine (serotonin, dopamine, ß-endorphins, noradrenaline and cortisol) and stress (derivatives of reactive oxygen metabolites (dROMs) and biological antioxidant potential (BAP)) parameters related to behavioural disorders were evaluated at the beginning and end of the study period. Results showed a significant increase in serotonin, dopamine and ß-endorphins plasma concentrations (*P<0.05, *P<0.05 and **P<0.01, respectively) and a significant decrease in noradrenaline and cortisol plasma concentrations in the ND group (*P<0.05). dROMs significantly decreased in the ND group (*P<0.05) while BAP was not affected. This study demonstrated for the first time that a specific diet significantly and positively affected neuroendocrine parameters and dROMs. These results open significant perspectives concerning the use of diet and nutraceuticals in the treatment of behavioural disorders.

The analysis of the human high affinity IgE receptor Fc epsilon Ri alpha from multiple crystal forms.

We have solved the structure of the human high affinity IgE receptor, Fc epsilon RI alpha, in six different crystal forms, showing the structure in 15 different chemical environments. This database of structures shows no change in the overall shape of the molecule, as the angle between domains 1 and 2 (D1 and D2) varies little across the ensemble. However, the receptor has local conformational variability in the C' strand of D2 and in the BC loop of D1. In every crystal form, a residue inserts between tryptophan residues 87 and 110, mimicking the position of a proline from the IgE ligand. The different crystal forms reveal a distribution of carbohydrates lining the front and back surfaces of the structure. An analysis of crystal contacts in the different forms indicates regions where the molecule interacts with other proteins, and reveals a potential new binding site distal to the IgE binding site. The results of this study point to new directions for the design of molecules to inhibit the interaction of Fc epsilon RI alpha with its natural ligand and thus to prevent a primary step in the allergic response.

Responses of hematological parameters, beta-endorphin, cortisol, reactive oxygen metabolites, and biological antioxidant potential in horses participating in a traditional tournament.

Several concerns have been raised over the health of animals used in equestrian games that have their origins in historical or religious events and are currently held in many countries. This study investigated physiological stress response and health status of horses participating in the Sartiglia, a historical horse tournament held in the city of Oristano, Italy, which is principally based on the attempts of masked horsemen at a gallop to run a sword through a hole in a suspended silver star. Blood samples were collected from 21 horses the day before the tournament (D0), during the tournament (D1), and the day after the tournament (D2). Samples were analyzed for complete blood count and biochemical, hormonal, and oxidative stress assays. Data were analyzed using the mixed effect model with sampling session as one of the fixed effects. On the whole, blood parameters evidenced an optimal health status of horses at D0. Significant dehydration and increase of circulating glucose, enzymes, cortisol, and ß-endorphin were registered at D1 (P < 0.001) with a complete recovery of physiological values just at D2. The reactive oxygen metabolites (d-ROM), from which the prooxidant activity can be evaluated, showed an increase from D0 to D1 and D2. Concentration of biological antioxidant potential, which measured the antioxidant capacity, was characterized by the maximum level registered during the tournament and counteracted the simultaneous increase of d-ROM. It can be hypothesized that the tournament played an important role in causing high levels of oxidant markers not only because of the physical exercise represented by the gallop but also because the emotional stressors. In conclusion, the tournament caused significant changes of most parameters, which rapidly recovered to baseline values within the day after. These data will certainly be useful for a future implementation of tests in equine medicine and for the improvements of knowledge of changes of blood parameters and health of horses in similar tournaments.

Plasma proteins as early biomarkers of exposure to carcinogenic aromatic amines.

Two-dimensional gel electrophoresis (2DG) has been used to study the changes induced in dog plasma polypeptides by the known urinary bladder carcinogens, 4-aminobiphenyl (4-ABP) and 2-naphthylamine (2-NA). Treatment with 3-aminobiphenyl (3-ABP) and 1-naphthylamine (1-NA), both considered to be non-carcinogenic, were used as controls. The purpose of this study was: (1) to determine whether or not changes that occurred in the plasma protein patterns were specific to 4-ABP and/or other related carcinogenic arylamines; (2) to measure the time course in the changes of the major polypeptides during dosing and their resynthesis during a recovery period; and (3) to determine, by microsequencing, the biochemical identity of the affected proteins. The results indicate that only the most potent carcinogen, 4-ABP, had the effect of suppressing the expression of some proteins, while the other aromatic amines caused no discernible change in the 2DG patterns during a 12-week dosing period. The 4-ABP caused dramatic suppression of two sets of proteins. One set of three spots had an apparent molecular weight of 32.5 kDa, and a pI of 5.8-6.0. The major component in this group was identified as the beta-chain of haptoglobin. Expression of this protein decreased markedly during the first 2 weeks of treatment and recovered slowly after dosing stopped. Since haptoglobin functions to bind with free hemoglobin and facilitates its elimination from the blood stream, these results can be rationalized as a consequence of 4-ABP binding to hemoglobin in the erythrocyte, resulting in cell death and hemolysis. The 4-ABP modified hemoglobin then binds to haptoglobin and this tertiary complex is purged from the blood stream, resulting in the disappearance of free haptoglobin. A second set of spots (mol. wt., 65 kDa; pI, 6.5-6.6) disappeared much faster than the haptoglobin, and recovered more quickly. The major protein is about one-fifth the intensity of haptoglobin and appeared to be N-terminally blocked. Internal microsequencing of four fragments obtained from tryptic cleavage of the major spot of this group showed significant similarity to the serum albumin sequence of several species. This spot group is not the major serum albumin spot, however, since the latter is readily identified as the most abundant spot on the plasma map. During the course of this study, several other polypeptides in the 2DG map of dog plasma were identified and are presented here.

A method to define the carboxyl terminal of proteins.

Accurate definition of the carboxyl terminal of proteins is necessary for elucidating posttranslational processing at the C-terminal and more generally for characterizing protein primary structures. Here, we describe a strategy for isolating and characterizing the C-terminal peptide of a protein after proteolysis with endoprotease Lys-C. Isolation is achieved using anhydrotrypsin, a catalytically inert derivative of trypsin that binds peptides containing lysine or arginine residues at their C-termini without cleaving them. Rapid, accurate characterization of the isolated C-terminal peptide is achieved by mass spectrometry. Initial identification of the C-terminal peptide is obtained by comparing matrix-assisted laser desorption/ionization time-of-flight mass spectra of the digest prior to and after incubation with anhydrotrypsin. Characterization of the C-terminal sequence is achieved by capillary-HPLC electrospray ionization tandem mass spectrometry of the isolated peptide using a quadrupole ion trap mass spectrometer in the selective reaction monitoring mode. This strategy was successfully applied to the characterization of the C-terminal of proteins with molecular masses ranging up to 56 kDa.

Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification.

Although mass spectrometric peptide mapping has become an established technique for the rapid identification of proteins isolated by polyacrylamide gel electrophoresis (PAGE), the results of the identification procedure can sometimes be ambiguous. Such ambiguities become increasingly prevalent for proteins isolated as mixtures or when only very small amounts of the proteins are isolated. The quality of the identification procedure can be improved by increasing the number of peptides that are extracted from the gel. Here we show that cysteine alkylation is required to ensure maximal coverage in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mapping of proteins isolated by PAGE. In the described procedure, alkylation was performed prior to electrophoresis to avoid the adventitious formation of acrylamide adducts during electrophoresis. In this way, homogeneous alkylation was obtained with three different alkylating reagents (4-vinylpyridine, iodoacetamide, acrylamide). Cysteine alkylation was also used as a tool for the identification of cysteine-containing peptides. Using a 1:1 mixture of unlabeled acrylamide and deuterium-labeled acrylamide ([2,3,3'-D3]acrylamide), the proteins of interest were alkylated prior to electrophoretic separation. Peptide mixtures produced by trypsin digestion of the resulting protein bands were analyzed by MALDI-TOF MS, and the cysteine content of the peptides was inferred from the isotopic distributions. The cysteine content information was readily obtained and used to improve the protein identification process.

A post-translational modification of the photosystem II subunit CP29 protects maize from cold stress.

The resistance of maize plants to cold stress has been associated with the appearance of a new chlorophyll a/b binding protein in the thylakoid membrane following chilling treatment in the light. The cold-induced protein has been isolated, characterized by amino acid sequencing, and pulse labeled with radioactive precursors, showing that it is the product of post-translational modification by phosphorylation of the minor chlorophyll a/b protein CP29 rather than the product of a cold-regulated gene or an unprocessed CP29 precursor. We show here that the CP29 kinase activity displays unique characteristics differing from previously described thylakoid kinases and is regulated by the redox state of a quinonic site. Finally, we show that maize plants unable to perform phosphorylation have enhanced sensitivity to cold-induced photoinhibition.

Glycosylation of human truncated Fc epsilon RI alpha chain is necessary for efficient folding in the endoplasmic reticulum.

The high affinity immunoglobulin E (IgE) receptor is an alpha beta gamma 2 tetrameric complex. The truncated extracellular segment (alpha t) of the heavily glycosylated alpha chain is sufficient for high affinity binding of IgE. Here we have expressed various alpha t mutants in eukaryotic and prokaryotic cells to analyze the role of glycosylation in the folding, stability, and secretion of alpha t. All seven N-linked glycosylation sites in alpha t are glycosylated and their mutations have an additive effect on the folding and secretion of alpha t. Mutation of the seven N-glycosylation sites (delta 1-7 alpha t) induces misfolding and retention of alpha t in the endoplasmic reticulum. Similarly, tunicamycin treatment reduces substantially the folding efficiency of wild-type alpha t. In contrast, no difference in folding efficiency is detected between wild-type alpha t and delta 1-7 alpha t expressed in Escherichia coli. In addition, maturation of N-linked oligosaccharides and addition of O-linked carbohydrates are not required for either the transport or the IgE-binding function of alpha t. Furthermore, complete enzymatic deglycosylation does not affect the stability and the IgE-binding capacity of alpha t. Therefore, glycosylation is not intrinsically necessary for proper folding of alpha t but is required for folding in the endoplasmic reticulum. Our data are compatible with the concept that specific interactions between N-linked oligosaccharides and the folding machinery of the endoplasmic reticulum are necessary for efficient folding of alpha t in eukaryotic cells.

Noninterfering synthetic peptides as internal controls for amino acid sequencing of sample unknowns.

Noninterfering synthetic peptides have been designed that may be used as internal sequencing standards (ISS-1 and ISS-2) by placing them in an amino acid sequencer with the sample. The peptides are composed of four unnatural amino acids which all yield phenylthiohydantoin derivatives having unique retention times compared with those obtained from the commonly observed natural residues. These internal standards indicate how the entire sequencing system was functioning during the actual analysis of an unknown by providing an initial yield and numerous repetitive yields. Verifying proper operation is extremely important when cycles appear blank due to the presence of modified amino acids or a blocked N-terminus. In addition, the ISS peptides can detect and identify different types of sequencing errors. This sometimes eliminates the need to rerun a sample due to blank cycles caused by mechanical malfunctions which result in failure to cleave the N-terminal residues. ISS-1 or ISS-2 may also be utilized during method development to compare different sample supports and to normalize sequencing data from proteins or peptides that have been treated differently.

Structural characterization of cytosolic NADP(+)-dependent isocitrate dehydrogenase from rat ovary.

Cytosolic NADP(+)-dependent isocitrate dehydrogenase was purified to homogeneity from superovulated rat ovaries. Amino acid sequence information was obtained by analyzing peptides generated by digestion with either cyanogen bromide or trypsin. Eleven peptides were sequenced and a total of 146 amino acids were identified. Nine of these peptides were found to be 60-100% identical with sequences from mitochondrial NADP(+)-dependent isocitrate dehydrogenase. Conservation of amino acids was observed for residues that were previously identified as potentially binding isocitrate-Mg2+. Circular dichroism measurements showed that the structure is composed of approximately 35% alpha-helix and 21% beta-sheet segments. Temperature denaturation studies indicated that the enzyme is more stable in the presence of isocitrate.

Keratin 14 protein in cultured nonparenchymal rat hepatic epithelial cells: characterization of keratin 14 and keratin 19 as antigens for the commonly used mouse monoclonal antibody OV-6.

We have recently reported that cell lines of nonparenchymal origin isolated from rat liver and pancreas, which have been suggested to be the progeny of a facultative stem cell compartment in vivo, express an unusual combination of keratins (K). These cell lines express K8 and K14 but not K18 and K5, their normal partners in filament formation (Bisgaard HC, Thorgeirsson SS, J Cell Physiol 147:333-343, 1991). However, upon spontaneous transformation and differentiation toward a hepatoblastlike progeny, K14 expression is abrogated and replaced by expression of K18 (Wirth et al., Electrophoresis 13:305-332, 1992). In the study presented here, we confirmed by protein sequence analysis that K14 was a major component of the intermediate filaments in a nonparenchymal cell line of hepatic origin. Immunocytochemical analysis of the cells in monolayer demonstrated that K8 as well as K14 were incorporated in the cellular cytoskeleton. Further analysis by immunoprecipitation showed that filament complexes were formed between K8 and K14 as atypical partners. Thus, we concluded that in some nonparenchymal cell lines isolated from rat liver, K8 and K14 form a major intermediate filament network. Finally, we showed that an antibody widely used in studies of the cell lineages of hepatic and pancreatic tissues and their neoplasms, the mouse monoclonal antibody OV-6, recognizes a common epitope in K14 and K19.

A conformational rearrangement upon binding of IgE to its high affinity receptor.

One of the critical steps in the allergic reaction is the binding of immunoglobulin E (IgE) to its high affinity receptor (FcepsilonRI). FcepsilonRI is a tetrameric complex composed of an alpha-chain, a beta-chain, and a dimeric gamma-chain. The extracellular portion of the alpha-chain (alpha-t) is sufficient for the binding of IgE. The Fc portion of IgE contains two copies of the FcepsilonRI binding sites. In contrast, the binding stoichiometry is 1:1. Previously, it was hypothesized that the binding of FcepsilonRI to IgE results in a conformational change in IgE that precludes the binding of a second molecule (Presta, L., Shields, R., O'Connel, L., Lahr, S., Porter, J. , Gorman, C., and Jardieu, P.(1994) J. Biol. Chem. 269, 26368-26373). Here we characterize the secondary structure of IgE and alpha-t and analyze their interaction by circular dichroism spectroscopy. Binding experiments show that when IgE interacts with alpha-t there is a 15-26% decrease of the negative ellipticity at 217 nm. Together, the absence of an alpha-helix element in alpha-t and the small contribution of alpha-t to the spectra of the complex indicate that upon binding, a major conformational rearrangement must occur on IgE. In addition, we analyze the thermal unfolding of alpha-t, IgE, and their complex. Despite the several domains that constitute IgE and alpha-t, these molecules unfold cooperatively with two-state kinetics.

Cytosolic NADP(+)-dependent isocitrate dehydrogenase. Isolation of rat cDNA and study of tissue-specific and developmental expression of mRNA.

Immunoscreening and DNA hybridization were used to isolate a 1.72-kilobase pair cDNA encoding cytosolic NADP(+)-dependent isocitrate dehydrogenase from a rat liver, lambda gt11 cDNA library. The identity of the cDNA was confirmed by comparison of the deduced amino acid sequence with sequences of peptides obtained from purified ovarian cytosolic isocitrate dehydrogenase. The 1.72-kilobase pair cDNA sequence translated into a protein of 414 amino acid residues with a molecular mass of 46,681 Da. The amino acid sequence contains a tripeptide (AKL) at the COOH terminus which represents a possible peroxisomal targeting sequence. The deduced amino acid sequence of the rat liver cytosolic isocitrate dehydrogenase showed 70 and 59% identity with sequences reported for NADP(+)-dependent isocitrate dehydrogenases from porcine mitochondria and yeast cytosol respectively. Northern blot analysis demonstrated a 13-fold increase in expression of cytosolic NADP(+)-dependent isocitrate dehydrogenase mRNA during the gonadotropin-induced development of the immature rat ovary. In comparative studies, the cytosolic and mitochondrial isocitrate dehydrogenase mRNAs were found to differ in size (2.2 and 1.8 kilobases, respectively) and to be differentially expressed in various tissues of the rat. Distinct digestion patterns were also obtained in Southern blot analysis of rat genomic DNA.