Specific Uncoupling of GRB2 from the Met Receptor DIFFERENTIAL EFFECTS ON TRANSFORMATION AND MOTILITY

The biological effects of hepatocyte growth factor/scatter factor are mediated by autophosphorylation of its receptor, the Met tyrosine kinase, on two carboxyl-terminal tyrosines. These phosphotyrosines (Y1349VHVNATY1356VNV) are multifunctional docking sites for several effectors. Grb2, the adaptor for the Ras guanyl-nucleotide exchanger SOS, binds to Tyr1356 in the YVNV motif. By site-directed mutagenesis we either abrogated or duplicated the Grb2 consensus, without interfering with the other effectors. Loss of the link with Grb2 severely impaired transformation. The same mutation, however, had no effect on the "scattering" response, indicating that the level of signal which can be reached by Grb2-independent routes is permissive for motility. Duplication of the Grb2 binding site enhanced transformation and left motility unchanged. Thus, two Met-mediated biological responses, motility and growth, can be dissociated on the basis of their differential requirement for a direct link with Ras

Quantification of aflatoxin M1 carry-over rate from feed to soft cheese

From January to December 2016, samples of milk and feeds of dairy cattle were monthly collected. The concentration of mycotoxins in all matrices was determined using the enzymatic immunoassay technique. The average concentration of aflatoxin B1 (AFB1), deoxynivalenol (DON) and zearalenone (ZEA) in feed was 3.01, 218.5 and 467 ug/kg, respectively. The average AFB1 carry-over rate was 0.84% with a variation between 0.05 to 5.93%. Particle size of the feed (P = 0.030) and individual milk production (P = 0.001) affected this rate. Mini-soft cheeses were produced using milk naturally contaminated with aflatoxin M1 (AFM1) as raw material to study its distribution both in whey and in cheese. The average level of AFM1 in milk was 0.014 μg/l. None of milk samples exceeded the maximum level accepted for AFB1 by the Southern Common Market (MERCOSUR) legislation (0.5 μg/l) and only 5.5% of samples exceeded the European Union (UE) regulations (0.05 μg/l). After the cheese elaboration, the concentration of AFM1 was determined in whey and in cheese. The greatest proportion (60%) was detected in whey while 40% AFM1 remained in the cheese. However, the concentration of AFM1 was higher in the cheese compared to the original milk.Fil: Costamagna, D.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Gaggiotti, M.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Chiericatti, C. A.. Universidad Nacional del Litoral. Facultad de Ingeniería Química; ArgentinaFil: Costabel, L.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Audero, Gabriela María de Luján. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Taverna, Miguel Angel. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Signorini Porchietto, Marcelo Lisandro. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentin

Transgenic mouse lines for non-invasive ratiometric monitoring of intracellular chloride

Chloride is the most abundant physiological anion and participates in a variety of cellular processes including trans-epithelial transport, cell volume regulation, and regulation of electrical excitability. The development of tools to monitor intracellular chloride concentration ([Cli]) is therefore important for the evaluation of cellular function in normal and pathological conditions. Recently, several Cl-sensitive genetically encoded probes have been described which allow for non-invasive monitoring of [Cli]. Here we describe two mouse lines expressing a CFP-YFP-based Cl probe called Cl-Sensor. First, we generated transgenic mice expressing Cl-Sensor under the control of the mouse Thy1 mini promoter. Cl-Sensor exhibited good expression from postnatal day two (P2) in neurons of the hippocampus and cortex, and its level increased strongly during development. Using simultaneous whole-cell monitoring of ionic currents and Cl-dependent fluorescence, we determined that the apparent EC50 for Cli was 46 mM, indicating that this line is appropriate for measuring neuronal [Cli] in postnatal mice. We also describe a transgenic mouse reporter line for Cre-dependent conditional expression of Cl-Sensor, which was targeted to the Rosa26 locus and by incorporating a strong exogenous promoter induced robust expression upon Cre-mediated recombination. We demonstrate high levels of tissue-specific expression in two different Cre-driver lines targeting cells of the myeloid lineage and peripheral sensory neurons. Using these mice the apparent EC50 for Cli was estimated to be 61 and 54 mM in macrophages and DRG, respectively. Our data suggest that these mouse lines will be useful models for ratiometric monitoring of Cli in specific cell types in vivo. © 2013 Batti, Mukhtarov, Audero, Ivanov, Paolicelli, Zurborg, Gross, Bregestovski and Heppenstall

Inducible Gene Manipulations in Brain Serotonergic Neurons of Transgenic Rats

The serotonergic (5-HT) system has been implicated in various physiological processes and neuropsychiatric disorders, but in many aspects its role in normal and pathologic brain function is still unclear. One reason for this might be the lack of appropriate animal models which can address the complexity of physiological and pathophysiological 5-HT functioning. In this respect, rats offer many advantages over mice as they have been the animal of choice for sophisticated neurophysiological and behavioral studies. However, only recently technologies for the targeted and tissue specific modification of rat genes - a prerequisite for a detailed study of the 5-HT system - have been successfully developed. Here, we describe a rat transgenic system for inducible gene manipulations in 5-HT neurons. We generated a Cre driver line consisting of a tamoxifen-inducible CreERT2 recombinase under the control of mouse Tph2 regulatory sequences. Tissue-specific serotonergic Cre recombinase expression was detected in four transgenic TPH2-CreERT2 rat founder lines. For functional analysis of Cre-mediated recombination, we used a rat Cre reporter line (CAG-loxP.EGFP), in which EGFP is expressed after Cre-mediated removal of a loxP-flanked lacZ STOP cassette. We show an in-depth characterisation of this rat Cre reporter line and demonstrate its applicability for monitoring Cre-mediated recombination in all major neuronal subpopulations of the rat brain. Upon tamoxifen induction, double transgenic TPH2-CreERT2/CAG-loxP.EGFP rats show selective and efficient EGFP expression in 5-HT neurons. Without tamoxifen administration, EGFP is only expressed in few 5-HT neurons which confirms minimal background recombination. This 5-HT neuron specific CreERT2 line allows Cre-mediated, inducible gene deletion or gene overexpression in transgenic rats which provides new opportunities to decipher the complex functions of the mammalian serotonergic system

Identification of diagnostic serum protein profiles of glioblastoma patients

Diagnosis of a glioblastoma (GBM) is triggered by the onset of symptoms and is based on cerebral imaging and histological examination. Serum-based biomarkers may support detection of GBM. Here, we explored serum protein concentrations of GBM patients and used data mining to explore profiles of biomarkers and determine whether these are associated with the clinical status of the patients. Gene and protein expression data for astrocytoma and GBM were used to identify secreted proteins differently expressed in tumors and in normal brain tissues. Tumor expression and serum concentrations of 14 candidate proteins were analyzed for 23 GBM patients and nine healthy subjects. Data-mining methods involving all 14 proteins were used as an initial evaluation step to find clinically informative profiles. Data mining identified a serum protein profile formed by BMP2, HSP70, and CXCL10 that enabled correct assignment to the GBM group with specificity and sensitivity of 89 and 96%, respectively (p < 0.0001, Fischer’s exact test). Survival for more than 15 months after tumor resection was associated with a profile formed by TSP1, HSP70, and IGFBP3, enabling correct assignment in all cases (p < 0.0001, Fischer’s exact test). No correlation was found with tumor size or age of the patient. This study shows that robust serum profiles for GBM may be identified by data mining on the basis of a relatively small study cohort. Profiles of more than one biomarker enable more specific assignment to the GBM and survival group than those based on single proteins, confirming earlier attempts to correlate single markers with cancer. These conceptual findings will be a basis for validation in a larger sample size

Angiopoietin-1 promotes functional neovascularization that relieves ischemia by improving regional reperfusion in a swine chronic myocardial ischemia model

10.1007/s11373-006-9082-xJournal of Biomedical Science134579-59