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Phospholipase Cγ2 (PLCγ2) plays a critical role in B cells differentiation by acting downstream BCR-mediated cell signaling. After binding of antigen to the B cell receptor (BCR), PLCγ2 is translocated to the cell membrane where it forms a signaling protein complex with the immunoglobulin α (Igα) and Igβ subunits of BCR, the protein tyrosine kinases Lyn, Syk and Btk and the adaptor protein B cell linker (BLNK). In the complex, PLCγ2 docks on BLNK and is activated by phosphorylation of Y759 by Btk kinase. PLCγ2 tyrosine-phosphorylation enables the phospholipase active site to gain access to and to hydrolyze its membrane-located substrate, phosphatidylinsositol 4,5-bisphosphate to inositol 1,4,5- triphosphate (IP3) and diacylglycerol (DAG) to trigger calcium release from intra-cellular stores. Although the activation of PLCg2 by tyrosine-phosphorylation is well understood, the converse process of PLCγ2 dephosphorylation and the protein tyrosine phosphatase(s) (PTPs) responsible for that are not yet clarified. In this work, we used a straightforward method, recently developed by us, for identification of PTPs that specifically dephosphorylates a given protein phosphotyrosine site. Therefore, we used a synthetic phosphopeptide containing the phosphotyrosine Y759 of PLCγ2 replaced by difluoro-phosphonomethyl phenyl alanine (F2pmF) to pulldown PTPs from cell lysates where PLCγ2 is endogenously expressed (e.g. Raji and HEK293T cells). Finally, the captured PTPs were identified by nanoLC-MS/MS. Analysis of the mass spectrometry results revealed two PTPs identified with high confidence: PTPN2 (TC-PTP) and PTPN1(PTP1B). Next, we found by in vitro tests that the identified PTPs directly dephosphorylated PLCγ2 co-expressed with Btk kinase in cell lysates. In addition, each of D/A and/or C/S trapping mutants of both PTPs were able to pulldown PLCγ2 when they were co-expressed in HEK293T cells. In summary, our results suggest that TC-PTP and PTP1B are specific PTPs for PLCγ2 dephosphorylation. [ABSTRACT FROM AUTHOR]

Member of the miR-15 precursor family, mmu-miR-195 is a conserved small non-coding RNA known for its role in cell cycle and cell death/apoptosis regulation. miR-195 is differentially expressed in various organs and tissues of the postnatal mouse. Recent analyses have associated miR-195 with different diseases such as cancer, chronic obstructive pulmonary disease, heart failure, Parkinson disease. We evaluated the expression of the mature and precursor mmu-miR-195 in the heart of male and female, wild-type mice at different stages of pre-natal and post-natal life. Quantitative Real-Time PCR (qRT-PCR) analysis revealed coordinated, concordant changes of the mature form, which are not paralleled by the pri-miRNA changes. Furthermore, we used a combined machine learning algorithm for miR targets prediction, and a clustering and topological community detection (CTCD) approach followed by gene ontology analysis to describe the impact of miR-195 changes upon heart transcriptome. We show that miR-195 regulates multiple novel signaling pathways associated with cardiac maturation and provide data validating - both at mRNA and protein level – our predictions. [ABSTRACT FROM AUTHOR]

In the carrot case, which is an allogamous species, due to its protandry suitable for insect pollination, in order to assure the necessary pollen for obtaining of the comercial hybrid seed, it is compulswy a synchronism during the flower phenophase in male sterile and male fertile lines used as partners in the hybridization process. Our investigations aimed to establish the receptivity period of the stigma for pollination according to the morphological flower type (hermaphrodyte one, petaloid one or brown anthers one) and temperature conditions. Experiments were carried out during the 1997- 2006 period, the biological material consiting in 9 inbreed lines, namely 3 for each flower type. On each plant, summing up 15 for each line were manually pollinated the flowers located on 3 equivalent inflorescences having a simultaneous blossom. The receptivity period was recorded as the time interval when the seeds were born and developed. The study of these multiple comparison revealed that the receptivity stage for pollen of stigma is influenced by the flower morphological type as well of the temperature during the blossom phenophase. For the same conditions, the lines of petaloidal type had got the longest receptivity period (18 days). This adaptation of the plants of petaloid type, wich are less visited by the pollinating insects (Litvinova,1988), is determined by the structural changes of the flowers (Erickson and Struckmeyer, 1986). For all these flower types the receptivity period was shorter under the temperature conditions higher than 30°C during the blossom phenophase during the 2004-2006 period. As a conclusion, one could state that male-sterile lines of petaloid type used as female parent assure in the most efficient way the pollen plenty from the main and equivalent inflorescences of the hermaphrodyte flowers evenif they are earlier. [ABSTRACT FROM AUTHOR]

Superiority of the carrot hybrids by comparison with the varieties is revealed by the uniformity and quality of the roots, their productive potential as well a high rate of biochemical compounds accumulation (Sycheva, 1986; Barbara Michalik end al., 1988 Gauchene, 1989). During the 2004-2006 period, 10 F1 carrot hybrid combinations were studied in order to establish of the intensity of behaviour of heterosis phenomenon (e.g. suppositional one, real one and competitive one) in some biochemical traits (total carotenes content and soluble dry matter) in order to identify the most valuable perspective hybrids. So, it was performed by comparison of the hybrid value with the average of parental lines and with the best parent as well as with the best grown variety Nantes for each heterosis type, namely suppositional one (Hi), real one (Hr), and competitive one (Hc) which were computed in per cent (%). For the total carotenoid content, the highest values were recorded for the suppositional heterosis (HMV 9- 118%), while for the real heterosis by comparison with the best parental line. The values recorded were smaller of 100, so practically the carotenoid contents of the hybrids was revealed to by intermediary between the parental forms. Regarding the intensity of behaviour of heterosis for the trait soluble dray matter content, it was found that the values ranged between 125 (HMV2) and 109,1% (HMV10) for the suppositional heterosis, but for real one the values ranged between 105,8 (HMV2) and 97% for HMV10. The results above mentioned led to the conclusion that for these biochemical traits the values of the hybrids are intermediary between the parental forms and that is why is necessary to use as parents some inbred lines having high values for the total caroten content and soluble dry matter. [ABSTRACT FROM AUTHOR]

Protein tyrosine phosphatases (PTP) are a large group of enzymes which work together with protein tyrosine kinases to control the tyrosine phosphorylation of proteins, thus playing a major role in cellular signaling. Here, we provide detailed protocols for expression and purification of the catalytic domain of RPTPμ and full length Eya3 as well as the extracellular region of PTPBR7. Methods are described for evaluation of the purity of the recombinant proteins thus obtained. For the purified Eya3 phosphatase we provide protocols for enzyme activity assay using either chromogenic, fluorescent, or peptide substrates. Determination of kinetic parameters by different graphical and computer-based procedures is also described.