A two-step immunomagnetic separation of somatic cell subpopulations for a gene expression profile study in bovine milk.

The objective of this study was to demonstrate the usefulness of an immunomagnetic method to purify subpopulations of milk somatic cells. The experiment was conducted on milk samples collected from healthy cows (n = 17) and from cows with clinical mastitis (n = 24) due to a Staphylococcus aureus natural infection. A two-step immunomagnetic purification was applied to simultaneously separate three somatic cell subpopulations from the same milk sample. Total RNA was extracted and qPCR was performed to determinate mRNA levels of innate immunity target genes in purified somatic cell subpopulations. Good quality and quantity of RNA allowed the reference gene analysis in each cell subpopulation. An up-regulation of the main genes involved in innate immune defence was detected in separated polymorphonuclear neutrophilic leucocytes-monocytes and lymphocytes of mastitic milk. These results and flow cytometric analysis suggest that the immunomagnetic purification is an efficient method for the isolation of the three populations from milk, allowing the cells to be studied separately.

A Health Status Update of Myocastor coypus in Northern Italy.

Myocastor coypus is a pest animal present in Africa, Europe, North America and Asia that causes agricultural and ecological damages. Moreover, it has to be considered as a potential risk for public health. Forty-four coypus from the "Parco Naturale La Mandria" (Piedmont region, Northwest Italy) have been analysed. A complete necropsy and a whole histological evaluation of the liver, kidney and lung have been carried out on all the animals. Moreover, the positivity to Hepatitis E Virus (HEV), Encephalomyocarditis virus (EMCV), Francisella spp., Toxoplasma gondii and Neospora caninum have been investigated. None of the animal were positive for HEV, EMCV, Francisella spp. or Neospora caninum. Two animals tested positive for Toxoplasma gondii. A high presence of histological lesions has been identified in different organs, suggesting that lesions could be induced by different pathogens. As previously reported, coypu can act as a host for several pathogens, including important agents for human and animal health, and surveillance is necessary to fully understand the biological role and the importance of coypu as a disease reservoir in our country.

Coping with Tissue Sampling in Suboptimal Conditions: Comparison of Different Tissue Preservation Methods for Histological and Molecular Analysis.

A high quality of samples is crucial for the success of the analysis and diagnostic purposes, and therefore the right method of conservation is vitally important for an optimal preservation of tissues. Indeed, the time to deliver the sample to the laboratory could be remarkably long, especially under suboptimal conditions, and the use of specific fixatives or cold storage may not be possible. Moreover, the portability and cost of storage equipment, their toxicity, and their ease of use play a central role when choosing the correct preservation method. The aim of this study was the identification of a reliable and economic method for tissue preservation, to be used in "in-field" sampling, suitable for both histological and molecular analysis. Punch biopsies were collected from six cattle livers. Comparisons among methods of preservation using RNAlater, silica beads, and under-vacuum was carried out. These methods were tested through considering different times and temperatures, assuming three days as a maximum time interval from sampling to laboratory and choosing 4 °C and 24 °C as references for refrigeration temperature and room temperature, respectively. Histologically, the integrity of nucleus, cytoplasm, preservation of liver structure, and easiness of recognition of inflammatory infiltrate were evaluated. The integrity of the extracted DNA and RNA was evaluated through PCR and by means of an automated electrophoresis station, respectively. RNAlater and silica beads poorly preserved the histological parameters evaluated, independently from the temperature. Conversely, the vacuum-sealed samples showed a good grade of preservation until 48 h. DNA quality was acceptable for each sample. RNA integrity showed promising results only for samples preserved with silica beads.

FKBP5 gene expression in skeletal muscle as a potential biomarker for illegal glucocorticoid treatment in veal calves.

For the current European legislation, the chemical analysis of drug residues is the exclusive accepted method to identify animals illicitly treated with growth promoters. Glucocorticoids and their metabolites are no detectable by LC/MS-MS methods in biological fluids when the growth promoter administration is discontinued several days prior to the slaughtering. The aim of this study was to elucidate the effect on the expression of genes belonging to the glucocorticoid pathway in three types of skeletal muscle of calves treated with prednisolone or dexamethasone in combination with estradiol. A gene expression change of glucocorticoid receptors (NR3C1 and NR3C2), their chaperones molecules (FKBP prolyl isomerase 4 and 5, FKBP4 and 5) and pre-receptor system (hydroxysteroid 11-beta dehydrogenases 1 and 2, HSD11B1 and 2) may indicate potential biomarkers of glucocorticoid treatment. In the biceps brachii muscle, the administration of dexamethasone with estradiol increased HSD11B2 (P < 0.01) and NR3C2 (P < 0.01) gene expression, whereas prednisolone administration increased HSD11B1 transcript levels (P < 0.05). In the longissimus lumborum muscle, NR3C2 gene expression decreased following prednisolone administration (P < 0.05). FKBP5 gene expression decreased in all considered muscles of calves administered with dexamethasone and estradiol (P < 0.01), whereas increased in the longissimus lumborum (P < 0.01) and vastus lateralis (P < 0.05) muscle of prednisolone-treated group (P < 0.05). The opposite effect of dexamethasone and prednisolone appears very promising to develop a low-cost screening test, because the expression analysis of a unique gene in a given tissue may distinguish the dispensed molecules.

Curcuminoid-phospholipid complex induces apoptosis in mammary epithelial cells by STAT-3 signaling.

Curcumin (from the rhizome of Curcuma longa) is well documented for its medicinal properties in Indian and Chinese systems of medicine where it is widely used for the treatment of several diseases. Epidemiological observations are suggestive that curcumin consumption may reduce the risk of some form of cancers and provide other protective biological effects in humans. These biological properties have been attributed to curcuminoids that have been widely studied for their anti-inflammatory, anti-angiogenic, antioxidant, wound healing and anti-cancer effects. In this study we have investigated on the effect of a curcumin phospholipid complex on mammary epithelial cell viability. HC11 and BME-UV cell lines, validated models to study biology of normal, not tumoral, mammary epithelial cells, were used to analyse these effects. We report that curcumin acts on STAT-3 signal pathway to reduce cell viability and increase apoptosis evaluated by the the amount of activated caspase 3. Further it reduces MAPK and AKT activations. JSI-124, a STAT-3 inhibitor (100 nM) was able to block the negative effect of curcumin on cell viability and caspase 3 activation. Finally the negative effect of cucumin on cell viability has been impaired in STAT-3i HC11, where STAT-3 protein was greatly reduced by shRNA-interference. These results indicate that curcumin presents a potential adverse effect to normal mammary epithelial cells and that it has a specific effect on signal trasduction in mammary epithelium.

17ß-estradiol upregulates oxytocin and the oxytocin receptor in C2C12 myotubes.

BACKGROUND: The endocrinology of skeletal muscle is highly complex and many issues about hormone action in skeletal muscle are still unresolved. Aim of the work is to improve our knowledge on the relationship between skeletal muscle and 17ß-estradiol. METHODS: The skeletal muscle cell line C2C12 was treated with 17ß-estradiol, the oxytocin peptide and a combination of the two hormones. The mRNA levels of myogenic regulatory factors, myosin heavy chain, oxytocin, oxytocin receptor and adipogenic factors were analysed in C2C12 myotubes. RESULTS: It was demonstrated that C2C12 myoblasts and myotubes express oxytocin and its receptor, in particular the receptor levels physiologically increase in differentiated myotubes. Myotubes treated with 17ß-estradiol overexpressed oxytocin and oxytocin receptor genes by approximately 3- and 29-fold, respectively. A decrease in the expression of fatty acid binding protein 4 (0.62-fold), a fat metabolism-associated gene, was observed in oxytocin-treated myotubes. On the contrary, fatty acid binding protein 4 was upregulated (2.66-fold) after the administration of the combination of 17ß-estradiol and oxytocin. 17ß-estradiol regulates oxytocin and its receptor in skeletal muscle cells and they act in a synergic way on fatty acid metabolism. DISCUSSION: Oxytocin and its receptor are physiologically regulated along differentiation. 17ß-estradiol regulates oxytocin and its receptor in skeletal muscle cells. 17ß-estradiol and oxytocin act in a synergic way on fatty acid metabolism. A better understanding of the regulation of skeletal muscle homeostasis by estrogens and oxytocin peptide could contribute to increase our knowledge of muscle and its metabolism.

Development and Application of a Screening Method of Absolute Quantitative PCR To Detect the Abuse of Sex Steroid Hormone Administration in Male Bovines.

A methodology for the absolute quantification of regucalcin gene through quantitative PCR was set up to confirm that the decrease of regucalcin gene expression in the testis is an effective biomarker for tracing sex steroid hormone treatment in bovine husbandry. On the basis of TaqMan technology, an external standard curve was generated. Using in vivo experiments, a ROC curve was developed to calculate the criterion value, specificity, and sensitivity for this potential biomarker. Then, regucalcin gene expression was assessed in veal calves and beef intended for human consumption. In 11 of 54 calves and in 5 of 70 beef cattle the regucalcin gene was expressed under their respective cutoff. Additionally, a mild decrease of regucalcin protein expression was revealed by immunohistochemistry in subjects tested positive via qPCR. These preliminary results suggest that this transcriptomics test may be employed as a novel diagnostic screening tool, improving significantly the overall efficacy of food control.

Regucalcin Expression as a Diagnostic Tool for the Illicit Use of Steroids in Veal Calves.

It has been previously demonstrated that sex steroid hormone treatment down-regulates regucalcin gene expression in the accessory sex glands and testis of prepubertal and adult male bovines. The aim of this study was to investigate whether low doses of sex steroid hormones combined with other drugs significantly affect regucalcin gene expression in the accessory sex glands and testis of veal calves. The regucalcin expression was down-regulated in the bulbo-urethral glands of estrogen-treated calves, whereas it was up-regulated in the prostate of estrogen-treated calves. Only the testis of androgen-treated calves showed a down-regulation of the regucalcin expression. Thus, the administration of sex steroid hormones, even in low doses and combined with other molecules, could affect regucalcin expression in target organs. Particularly, the specific response in the testis suggests regucalcin expression in this organ as a first molecular biomarker of illicit androgen administration in bovine husbandry.

Regucalcin expression in bovine tissues and its regulation by sex steroid hormones in accessory sex glands.

Regucalcin (RGN) is a mammalian Ca2+-binding protein that plays an important role in intracellular Ca2+ homeostasis. Recently, RGN has been identified as a target gene for sex steroid hormones in the prostate glands and testis of rats and humans, but no studies have focused on RGN expression in bovine tissues. Thus, in the present study, we examined RGN mRNA and protein expression in the different tissues and organs of veal calves and beef cattle. Moreover, we investigated whether RGN expression is controlled through sex steroid hormones in bovine target tissues, namely the bulbo-urethral and prostate glands and the testis. Sex steroid hormones are still illegally used in bovine husbandry to increase muscle mass. The screening of the regulation and function of anabolic sex steroids via modified gene expression levels in various tissues represents a new approach for the detection of illicit drug treatments. Herein, we used quantitative PCR, western blot and immunohistochemistry analyses to demonstrate RGN mRNA and protein expression in bovine tissues. In addition, estrogen administration down-regulated RGN gene expression in the accessory sex glands of veal calves and beef cattle, while androgen treatment reduced RGN gene expression only in the testis. The confirmation of the regulation of RGN gene expression through sex steroid hormones might facilitate the potential detection of hormone abuse in bovine husbandry. Particularly, the specific response in the testis suggests that this tissue is ideal for the detection of illicit androgen administration in veal calves and beef cattle.

Epidermal growth factor and hepatocyte growth factor cooperate to enhance cell proliferation, scatter, and invasion in murine mammary epithelial cells.

The development of the mammary gland requires an integrated response to specific growth factors and steroid hormones. Hepatocyte growth factor (HGF) and its tyrosine kinase receptor, MET, are expressed and temporally regulated during mammary development and differentiation. Epidermal growth factor receptor (EGFR) and its ligands have also been implicated in mammary gland growth and morphogenesis. Since both cytokines seem to exert a morphogenic program in this tissue, we have investigated the possible concerted action of EGF and HGF on the HC11 cell line, a widely used model of nontumorigenic mammary cells. Western blot analysis indicated that HC11 expressed MET and EGFR, and showed ERK1/2 and AKT activation following HGF or EGF treatment. Analysis by real-time PCR and western blot showed that after an EGF but not HGF or insulin-like growth factor-I treatment, HC11 mammary cells exhibited an increase in MET expression at both the mRNA and protein levels, which was dependent on the AKT pathway. Simultaneous treatment with HGF and EGF increased proliferation, scatter, and invasion as assessed by cell count, cell cycle, scatter, and transwell assays. AKT inhibition did not influence the cooperation on proliferation or invasion after HGF+EGF treatment, while ERK1/2 inhibition abolished MET/EGFR cooperation on proliferation. HGF+EGF treatment increased the duration of ERK1/2 and AKT activation compared to HGF or EGF alone. All these data indicate that a crosstalk between the EGF and HGF pathways in mammary epithelial cells may modulate the development of the mammary gland.

Effects of feeding Spodoptera littoralis on lima bean leaves. III. Membrane depolarization and involvement of hydrogen peroxide.

In response to herbivore (Spodoptera littoralis) attack, lima bean (Phaseolus lunatus) leaves produced hydrogen peroxide (H(2)O(2)) in concentrations that were higher when compared to mechanically damaged (MD) leaves. Cellular and subcellular localization analyses revealed that H(2)O(2) was mainly localized in MD and herbivore-wounded (HW) zones and spread throughout the veins and tissues. Preferentially, H(2)O(2) was found in cell walls of spongy and mesophyll cells facing intercellular spaces, even though confocal laser scanning microscopy analyses also revealed the presence of H(2)O(2) in mitochondria/peroxisomes. Increased gene and enzyme activations of superoxide dismutase after HW were in agreement with confocal laser scanning microscopy data. After MD, additional application of H(2)O(2) prompted a transient transmembrane potential (V(m)) depolarization, with a V(m) depolarization rate that was higher when compared to HW leaves. In transgenic soybean (Glycine max) suspension cells expressing the Ca(2+)-sensing aequorin system, increasing amounts of added H(2)O(2) correlated with a higher cytosolic calcium ([Ca(2+)](cyt)) concentration. In MD and HW leaves, H(2)O(2) also triggered the increase of [Ca(2+)](cyt), but MD-elicited [Ca(2+)](cyt) increase was more pronounced when compared to HW leaves after addition of exogenous H(2)O(2). The results clearly indicate that V(m) depolarization caused by HW makes the membrane potential more positive and reduces the ability of lima bean leaves to react to signaling molecules.