Two cDNAs coding for the porcine CD51 (αv) integrin subunit: Cloning, expression analysis, adhesion assays and chromosomal localization

Yubero, Noemí; Jiménez-Marín, Ángeles; Barbancho, M.; Garrido, Juan J.

doi:10.1016/j.gene.2011.04.006

Cited by 6 publications

(6 citation statements)

References 62 publications

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“…This is in concordance with the high conservation of the GVLGG sequence in the transmembrane region of the porcine (and other mammal) α ΙIb chains, since fibrinogen binding to α IIb β 3 is a prerequisite for platelets aggregation (Bennett, 2005). It is worthy to note that the porcine α v integrin, also present in platelets membranes, contains an AVLGG sequence in the transmembrane region, as well as in all their mammalian homologous with which it was compared (Yubero et al, 2011).…”

Section: Discussionsupporting

confidence: 69%

“…The phylogenetic tree of CD41 family of proteins showed that the closest to porcine CD41 were those of cows and horses, and that the clusters of domestic mammals showed the less divergence in evolution. However, compared with other αmammal integrins, like α v which show 90% of identity (Yubero et al, 2011), α IIb integrins show lower level of conservation, which could be associated with the number of β chains with which they can form receptors: only one (β 3 ) for α IIb , and at least five for α v. Porcine CD41 conserves all the main structural characteristics that define their functions in other species. The extracellular domain shows that porcine CD41 belongs to α integrins lacking I domain, a domain present in the NH 2 extreme of some integrins, like α 1, α 2 or β 2, which contains the functional sites to bind to ligands (Dickeson & Santoro, 1998;Humphries, 2000).…”

Section: Discussionmentioning

confidence: 95%

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Molecular Cloning, Characterization, Expression Analysis and Chromosomal Localization of the Gene Coding for the Porcine αIIb Subunit of the αIIbβ3 Integrin Platelet Receptor

Esteso¹,

Jiménez-Marín²,

Sanz³

et al. 2011

Molecular Cloning - Selected Applications in Medicine and Biology

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Molecular Cloning, Characterization, Expression Analysis and Chromosomal Localization of the Gene Coding for the Porcine αIIb Subunit of the αIIbβ3 Integrin Platelet Receptor 111 RNA isolation, RT-PCR and RACETotal RNA from platelets, cells or tissues was purified according to the M-MLV Reverse Transcriptase system (Invitrogene) using the random primers pd(N) 6 -5'-PO 3 NA + Salt (Pharmacia Biotech). RNA samples were kept at -80ºC after controlling the quality on a denaturing agarose gel. 5 µg RNA, resuspended in 9.5 µl water, were heated for 3 min at 65ºC in the presence of random hexamers (7.5 µM final concentration), and then cooled in ice. RNA was reverse transcribed using 1 µl Moloney murine leukemia virus reverse transcriptase (200 units/µl) (GibcoBRL) for 1 h at 42ºC in a final volume of 20 µl containing 4 µl of 5X reverse transcriptase buffer, 0,5 µl ribonuclease inhibitor (50 U/µl) (Roche), 1 µl 20 mM dNTP (Pharmacia) and 2 µl 0.1 M dithiothreitol. After 10 min at room temperature, 1 h at 42 º C, and 10 min at 95ºC, DEPC H 2 O were added until a final volume of 100 µl. 2 µl of this mixture were subjected to PCR using 1µl Tth DNA polymerase (1U/µl) (Biotools) and 2,5 µl each CD41-specific primer (20µM) (see Table 1) in a final volume of 50 µl containing 5 µl 10x buffer, 2 µl MgCl 2 (50 mM), and 8 µl dNTP MIX (1,25 mM each) (Biotools). The amplification consisted in 35 cycles of PCR and each cycle consisted of incubations at 94ºC for 1 min, TmºC for 1 min, and 72ºC for 1 min. The amplifications were electrophoresed on 1% agarose/1X TAE gel. RT-PCR on RNA18S cDNA was used as a control. For RACE (Rapid Amplification of cDNA Ends), 1 µg total RNA from platelet was used to reverse-transcribe using 1 µl Moloney murine leukemia virus reverse transcriptase (200 units/µl) (GibcoBRL) for 1 h at 42ºC in a final volume of 20 µl containing 4 µl of 10X reverse transcriptase buffer, 1,0 µl ribonuclease inhibitor (50 U/µl) (Roche), 4 µl 2,5 mM dNTP (Pharmacia) and 2 µl 3' RACE ADAPTER (20 µM) in a final volume of 20 µL. 3' CD41 cDNAs were obtained by PCR using a specific porcine CD41 primer and the anchor primer provided in the kit (Table 1). DNA sequencing and sequences analysisSequencing was performed using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) on a thermal DNA cycler GeneAmp PCR System 2400 (Applied Biosystems, Foster City, CA, USA), according to the instructions of the manufacturer, and analysed on an ABI PRISM 3100 Sequencer (Applied Biosystems, Foster City, CA, USA). Porcine CD41 sequence has been deposited at GenBank under number JF808665. Sequences were analyzed using the analysis software LaserGene (DNAstar, Londo, UK) and the analysis tools provided by the expasy web site . Primers design was performed with Oligo 6 (MBI, Cascade, CO, USA) and Amplify 3 (). Multiple alignment among CD41 peptide sequences from Sus scrofa (GenBank accession no. JF808665 ), Homo sapiens (GenBank...

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Section: Discussionsupporting

confidence: 69%

Section: Discussionmentioning

confidence: 95%

Molecular Cloning, Characterization, Expression Analysis and Chromosomal Localization of the Gene Coding for the Porcine αIIb Subunit of the αIIbβ3 Integrin Platelet Receptor

Esteso¹,

Jiménez-Marín²,

Sanz³

et al. 2011

Molecular Cloning - Selected Applications in Medicine and Biology

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“…27,28 CD51 labels the integrin V alpha subunit, which can form heterodimers with at least five distinct beta subunits. 29,30 CD140α, platelet-derived growth factor receptor (PDGFR) α is a tyrosine kinase receptor and found to be expressed on mesenchymal-derived cells. 31 As such, the use of CD51/CD140α identified a large subset of perivascular Nestin + cells highly enriched for MSCs in both human and mouse bone marrow in vivo .…”

Section: Discussionmentioning

confidence: 99%

Single CD271 marker isolates mesenchymal stem cells from human dental pulp

et al. 2015

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Mesenchymal stem cells (MSCs) are a promising tool in regenerative medicine due to their capacity to differentiate into multiple lineages. In addition to MSCs isolated from bone marrow (BMSCs), adult MSCs are isolated from craniofacial tissues including dental pulp tissues (DPs) using various stem cell surface markers. However, there has been a lack of consensus on a set of surface makers that are reproducibly effective at isolating putative multipotent dental mesenchymal stem cells (DMSCs). In this study, we used different combinations of surface markers (CD51/CD140α, CD271, and STRO-1/CD146) to isolate homogeneous populations of DMSCs from heterogeneous dental pulp cells (DPCs) obtained from DP and compared their capacity to undergo multilineage differentiation. Fluorescence-activated cell sorting revealed that 27.3% of DPCs were CD51+/CD140α+, 10.6% were CD271+, and 0.3% were STRO-1+/CD146+. Under odontogenic conditions, all three subsets of isolated DMSCs exhibited differentiation capacity into odontogenic lineages. Among these isolated subsets of DMSCs, CD271+ DMSCs demonstrated the greatest odontogenic potential. While all three combinations of surface markers in this study successfully isolated DMSCs from DPCs, the single CD271 marker presents the most effective stem cell surface marker for identification of DMSCs with high odontogenic potential. Isolated CD271+ DMSCs could potentially be utilized for future clinical applications in dentistry and regenerative medicine.

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“…For immunohistochemistry, heat-mediated antigen retrieval in 0.01 M citric acid and labeling were performed as described elsewhere [12], employing the anti-vimentin monoclonal antibody (Chemicon/Millipore), a monoclonal antibody specific for porcine macrophages (clone 4E9/11) [13] and a rabbit antiserum against the somatic antigen of S. typhimurium.…”

Section: Histological Analysismentioning

confidence: 99%

Proteomic analysis of porcine mesenteric lymph-nodes after Salmonella typhimurium infection

Martins¹,

Collado-Romero²,

Martínez-Gomáriz³

et al. 2012

Journal of Proteomics

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Two cDNAs coding for the porcine CD51 (αv) integrin subunit: Cloning, expression analysis, adhesion assays and chromosomal localization

Cited by 6 publications

References 62 publications

Molecular Cloning, Characterization, Expression Analysis and Chromosomal Localization of the Gene Coding for the Porcine αIIb Subunit of the αIIbβ3 Integrin Platelet Receptor

Molecular Cloning, Characterization, Expression Analysis and Chromosomal Localization of the Gene Coding for the Porcine αIIb Subunit of the αIIbβ3 Integrin Platelet Receptor

Single CD271 marker isolates mesenchymal stem cells from human dental pulp

Proteomic analysis of porcine mesenteric lymph-nodes after Salmonella typhimurium infection

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