Single nucleotide polymorphism C994g of the cytochrome P450 gene possess pleiotropic eff ects in Bos taurus, L.

The chronic consumption of mycotoxin-contaminated forage tends to the weight loss of young cattle, decreasing the reproductive success of adult animals, milk yield, and tolerance to high temperatures. The aim of this paper was to study the eff ects of the SNP C994G in the CYP3A28 gene on the productive and reproductive characteristics of dairy and beef breeds. Cows were measured by body weight dynamics, exterior, reproduction, milk production traits, bone mineral density, bulls were analyzed estimating their progeny traits. Dairy cows received dietary betaine supplement with the measurement of homocysteine levels. Molecular genetic was performed by PCR-RFLP method. The effects of SNP C994G were shown in relation to the udder size, the constitution and birth weight of Abredin-Angus cows, as well as the fat and protein content of Ukrainian Red-and-White Dairy cows milk. The higher sensitivity of the GG genotype to less homocysteine level after the addition of betaine was shown. Given the identifi ed correspondence, pleiotropic eff ects of CYP3A28 gene can be concluded. A microscopic analysis of forage supported the findings in relation to greater efficiency of the CC genotype under conditions of low mycotoxins contamination, to which CYP3A28 is selective

Alternative splicing affecting the sh3a domain controls the binding properties of intersectin 1 in neurons

a b s t r a c t Intersectin 1 (ITSN1) is a conserved adaptor protein implicated in endocytosis, regulation of actin cytoskeleton rearrangements and mitogenic signaling. Its expression is characterized by multiple alternative splicing. Here we show neuron-specific expression of ITSN1 isoforms containing exon 20, which encodes five amino acid residues in the first SH3 domain (SH3A). In vitro binding experiments demonstrated that inclusion of exon 20 changes the binding properties of the SH3A domain. Endocytic proteins dynamin 1 and synaptojanin 1 as well as GTPase-activating protein CdGAP bound the neuron-specific variant of the SH3A domain with higher affinity than ubiquitously expressed SH3A. In contrast, SOS1, a guanine nucleotide exchange factor for Ras, and the ubiquitin ligase Cbl mainly interact with the ubiquitously expressed isoform. These results demonstrate that alternative splicing leads to the formation of two pools of ITSN1 with potentially different properties in neurons, affecting ITSN1 function as adaptor protein. Ó 2008 Elsevier Inc. All rights reserved. Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein involved in clathrin-mediated endocytosis, apoptosis, signal transduction, and regulation of cytoskeletal rearrangements ITSN1 has been shown to interact with several essential endocytic proteins, including dynamin, epsin, Eps15, and synaptojanin Previously, we found seven additional splice variants of the human and mouse ITSN1 genes In view of the potential link between alternative exon 20 usage and the binding properties of ITSN1, we have investigated the interaction of two SH3A domain splice variants with the main ITSN1 protein partners. The results presented here highlight differences in the binding properties of alternatively spliced SH3A domains of ITSN1 and showed how alternative splicing can regulate the functions of ITSN1 as adaptor protein. Materials and methods Expression constructs. The cDNA sequences corresponding to the proline-rich region (PRD) of dynamin 1 (residues 742-851, GenBank Accession No. NP_0010053360), SOS1-IsfI (residues 0006-291X/$ -see front matter

Adaptor proteins intersectin 1 and 2 bind similar proline-rich ligands but are differentially recognized by SH2 domain-containing proteins.

BACKGROUND: Scaffolding proteins of the intersectin (ITSN) family, ITSN1 and ITSN2, are crucial for the initiation stage of clathrin-mediated endocytosis. These proteins are closely related but have implications in distinct pathologies. To determine how these proteins could be separated in certain cell pathways we performed a comparative study of ITSNs. METHODOLOGY/PRINCIPAL FINDINGS: We have shown that endogenous ITSN1 and ITSN2 colocalize and form a complex in cells. A structural comparison of five SH3 domains, which mediated most ITSNs protein-protein interactions, demonstrated a similarity of their ligand-binding sites. We showed that the SH3 domains of ITSN2 bound well-established interactors of ITSN1 as well as newly identified ITSNs protein partners. A search for a novel interacting interface revealed multiple tyrosines that could be phosphorylated in ITSN2. Phosphorylation of ITSN2 isoforms but not ITSN1 short isoform was observed in various cell lines. EGF stimulation of HeLa cells enhanced tyrosine phosphorylation of ITSN2 isoforms and enabled their recognition by the SH2 domains of the Fyn, Fgr and Abl1 kinases, the regulatory subunit of PI3K, the adaptor proteins Grb2 and Crk, and phospholipase C gamma. The SH2 domains mentioned were unable to bind ITSN1 short isoform. CONCLUSIONS/SIGNIFICANCE: Our results indicate that during evolution of vertebrates ITSN2 acquired a novel protein-interaction interface that allows its specific recognition by the SH2 domains of signaling proteins. We propose that these data could be important to understand the functional diversity of paralogous ITSN proteins

Associations of miR-181a with Health-Related Quality of Life, Cognitive Functioning, and Clinical Data of Patients with Different Grade Glioma Tumors

Gliomas are central nervous system tumors with a lethal prognosis. Small micro-RNA molecules participate in various biological processes, are tissue-specific, and, therefore, could be promising targets for cancer treatment. Thus, this study aims to examine miR-181a as a potent biomarker for the diagnosis and prognosis of glioma patients and, for the first time, to find associations between the expression level of miR-181a and patient quality of life (QoL) and cognitive functioning. The expression level of miR-181a was analyzed in 78 post-operative II-IV grade gliomas by quantitative real-time polymerase chain reaction. The expression profile was compared with patient clinical data (age, survival time after the operation, tumor grade and location, mutation status of isocitrate dehydrogenase 1 (IDH1), and promoter methylation of O-6-methylguanine methyltransferase). Furthermore, the health-related QoL was assessed using the Karnofsky performance scale and the quality of life questionnaires; while cognitive assessment was assessed by the Hopkins verbal learning test-revised, trail-making test, and phonemic fluency tasks. The expression of miR-181a was significantly lower in tumors of grade III and IV and was associated with IDH1 wild-type gliomas and a worse prognosis of patient overall survival. Additionally, a positive correlation was observed between miR-181a levels and functional status and QoL of glioma patients. Therefore, miR-181a is a unique molecule that plays an important role in gliomagenesis, and is also associated with changes in patients’ quality of life

Identification and characterization of a novel mammalian isoform of the endocytic adaptor ITSN1

a b s t r a c t a r t i c l e i n f o Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein engaged in clathrin-mediated endocytosis, cell signaling and actin cytoskeleton rearrangements. Two major ITSN1 isoforms were initially described, the ubiquitous short isoform (ITSN1-s) and the long isoform (ITSN1-l) expressed predominantly in neurons. Numerous alternative splicing events for ITSN1 pre-mRNA were later identified. Here we describe a novel isoform ITSN1-22a with an alternative C-terminus encoded by exon 22a. This exon is only found in placental mammals. The transcript of ITSN1-22a is detected in a wide range of human and mouse tissues. We show here that two alternative splicing events affect the coding sequence of the ITSN1-22a isoform. Moreover, alternative polyadenylation of these transcripts was demonstrated in human tissues. The protein encoded by the ITSN1-22a transcript possesses two EH domains, a coiled-coil region, an SH3A domain and a specific Cterminal domain (CTD) but lacks four SH3 domains in comparison with ITSN1-s. The level of ITSN1-22a protein varies in different mouse tissues and human cell lines. The highest amounts of this isoform occur in mouse brain, spleen, lung and the human B cell line DG75. ITSN1-22a binds via its CTD to the SH3 domain of the endocytic protein amphiphysin 1 and the SH3A domain of ITSN1. Furthermore association in vivo and codistribution of ITSN1-22a and ITSN1-s were demonstrated suggesting that these isoforms could function in concert. We have revealed differential binding of ITSN1-s and ITSN1-22a to the ubiquitin ligase Cbl. Both isoforms possess the SH3A domain capable of binding to Cbl; however ITSN1-22a in contrast to ITSN1-s did not interact with Cbl in vivo. In vitro binding experiments demonstrated that the CTD of ITSN1-22a negatively regulated its binding to Cbl; at the same time interaction with another partner, dynamin 1 was not affected by the presence of the CTD. These data suggest that intramolecular interaction within ITSN1-22a could specifically regulate its binding to protein partners. Thus, this novel mammalian ITSN1 isoform possesses a significantly altered domain structure and performs specific protein-protein interactions

Concentration and Methylation of Cell-Free DNA from Blood Plasma as Diagnostic Markers of Renal Cancer

The critical point for successful treatment of cancer is diagnosis at early stages of tumor development. Cancer cell-specific methylated DNA has been found in the blood of cancer patients, indicating that cell-free DNA (cfDNA) circulating in the blood is a convenient tumor-associated DNA marker. Therefore methylated cfDNA can be used as a minimally invasive diagnostic marker. We analysed the concentration of plasma cfDNA and methylation of six tumor suppressor genes in samples of 27 patients with renal cancer and 15 healthy donors as controls. The cfDNA concentrations in samples from cancer patients and healthy donors was measured using two different methods, the SYBR Green I fluorescence test and quantitative real-time PCR. Both methods revealed a statistically significant increase of cfDNA concentrations in cancer patients. Hypermethylation on cfDNA was detected for the LRRC3B (74.1%), APC (51.9%), FHIT (55.6%), and RASSF1 (62.9%) genes in patients with renal cancer. Promoter methylation of VHL and ITGA9 genes was not found on cfDNA. Our results confirmed that the cfDNA level and methylation of CpG islands of RASSF1A, FHIT, and APC genes in blood plasma can be used as noninvasive diagnostic markers of cancer

ITSN1 and ITSN2 are associated in cells.

<p>(A) Characterization of the anti-ITSN2 antibodies produced. Nontransfected HEK293 cells or cells expressing GFP-ITSN1-S or GFP-ITSN2-S were lysed 24 h post-transfection. Total cell lysates were resolved by SDS-PAGE with subsequent immunoblot analysis using the anti-ITSN2 antibodies obtained or the commercially available anti-Intersectin/ESE-1. (B) HEK293 cells were plated on coverslips and fixed. Endogenous ITSNs were stained with anti-ITSN1/m and anti-ITSN2 antibodies, and visualized with Alexa 488-conjugated or Texas Red-conjugated secondary antibodies, respectively. Higher magnification of the area enclosed by a rectangle is shown below each image. Scale bar: 10 µm. (C) Lysates of HEK293 cells were subjected to immunoprecipitation using anti-ITSN2 antibodies (left panels). Conversely immunoprecipitation was performed using rabbit polyclonal antibodies against ITSN1 (right panels). In both cases immunoprecipitated material was probed with antibodies specific to ITSN1 and ITSN2. Representative data from three independent experiments are shown. IP, immunoprecipitation; NRS, normal rabbit serum; WB, Western blotting.</p

ITSN2 is phosphorylated on tyrosine residues.

<p>(A) Line graphs show the total number of tyrosine residues in ITSN1 and ITSN2 in a range of vertebrate taxonomic groups. Amino acid sequences of ITSN1 and ITSN2 orthologues in fish (<i>Danio rerio, Oreochromis niloticus</i> and <i>Tetraodon nigroviridis</i>), amphibians (<i>Xenopus tropicalis</i> and <i>Xenopus laevis</i>), reptiles (<i>Anolis carolinensis</i>), birds (<i>Gallus gallus, Meleagris gallopavo</i> and <i>Taeniopygia guttata</i>), rodents (<i>Mus musculus, Rattus norvegicus</i> and <i>Cricetulus griseus</i>) and primates (<i>Homo sapiens, Pan troglodita</i> and <i>Pongo abelii</i>) were obtained from the NCBI protein database (http: //ncbi.nlm.nih.gov). The average number of tyrosine residues per taxon is plotted, and error bars represent standard deviations. (B) Schematic representation of ITSNs domain organization and distribution of conserved tyrosine residues in ITSN1 and ITSN2 of various vertebrates. Tyrosine residues located within domain and interdomain regions are shown as dark blue and light blue boxes, respectively. Tyrosine residues that could be phosphorylated according to phosphoproteomic data (<a href="http://www.phosphosite.org" target="_blank">www.phosphosite.org</a>) are indicated by green circles. The number above each circle indicates the position of the residue in the amino acid sequence of human ITSN. Abbreviations are defined as follows: Homo, <i>Homo sapiens</i>; Mus, <i>Mus musculus</i>; Gallus, <i>Gallus gallus</i>; Anolis, <i>Anolis carolinensis</i>; Xenopus, <i>Xenopus laevis</i>. (C) Lysates of growing HEK293, HeLa, MCF-7 and MBA-MB-231 cells were subjected to immunoprecipitation with rabbit polyclonal anti-ITSN2 (left panel) or anti-ITSN1 (right panel) antibodies. Normal rabbit serum (NRS) was used as control. Precipitated proteins were analyzed by Western blotting using anti-phosphotyrosine antibodies and antibodies against ITSN1 or ITSN2. The experiments were repeated at least twice with reproducible results. IP, immunoprecipitation; WB, Western blotting.</p

Differences within ligand-binding sites of the SH3 domains of human ITSN1 and ITSN2.

§<p>LBS (ligand-binding site) of the SH3 domain consists of 15 amino acid residues <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070546#pone.0070546-Larson1" target="_blank">[38]</a>;</p>¥<p>ITSN1 → ITSN2;</p>¶<p>for intracellular proteins according to Betts and Russell <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070546#pone.0070546-Betts1" target="_blank">[42]</a>;</p>*<p>mismatch is not conserved in orthologues.</p