Due to their unique three-dimensional structure, DNA or RNA oligonucleotide aptamers bind to various molecules with high affinity and specificity. Aptamers, alone or in combination with antibodies, can be used to sensitively quantify target molecules by quantitative real-time polymerase chain reaction (qPCR). However, the assays are often complicated and unreliable. In this study, we explored the feasibility of performing the entire assay on wells of routinely used polypropylene PCR plates. We found that polypropylene wells efficiently bind proteins. This allows the entire assay to be run in a single well. To minimize nonspecific binding of the assay components to the polypropylene wells, we tested various blocking agents and identified methylcellulose as an effective alternative to the commonly used BSA. Methylcellulose not only demonstrates comparable or superior blocking capabilities but also offers the advantage of a well-defined composition and non-animal origin. Our findings support the utilization of aptamers, either alone or in combination with antibodies, for sensitive quantification of selected molecules immobilized in polypropylene PCR wells in a streamlined one-well qPCR assay under well-defined conditions.

• Keywords
• DNA aptamers, blocking agents, immunoglobulins, methylcellulose, polymerase chain reaction, polypropylene wells,
• MeSH
• Aptamers, Nucleotide * MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Methylcellulose MeSH
• Polypropylenes MeSH
• Antibodies MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Aptamers, Nucleotide * MeSH
• Methylcellulose MeSH
• Polypropylenes MeSH
• Antibodies MeSH

C-terminal Src kinase (CSK) is a major negative regulator of Src family tyrosine kinases (SFKs) that play critical roles in immunoreceptor signaling. CSK is brought in contiguity to the plasma membrane-bound SFKs via binding to transmembrane adaptor PAG, also known as CSK-binding protein. The recent finding that PAG can function as a positive regulator of the high-affinity IgE receptor (FcεRI)-mediated mast cell signaling suggested that PAG and CSK have some non-overlapping regulatory functions in mast cell activation. To determine the regulatory roles of CSK in FcεRI signaling, we derived bone marrow-derived mast cells (BMMCs) with reduced or enhanced expression of CSK from wild-type (WT) or PAG knockout (KO) mice and analyzed their FcεRI-mediated activation events. We found that in contrast to PAG-KO cells, antigen-activated BMMCs with CSK knockdown (KD) exhibited significantly higher degranulation, calcium response, and tyrosine phosphorylation of FcεRI, SYK, and phospholipase C. Interestingly, FcεRI-mediated events in BMMCs with PAG-KO were restored upon CSK silencing. BMMCs with CSK-KD/PAG-KO resembled BMMCs with CSK-KD alone. Unexpectedly, cells with CSK-KD showed reduced kinase activity of LYN and decreased phosphorylation of transcription factor STAT5. This was accompanied by impaired production of proinflammatory cytokines and chemokines in antigen-activated cells. In line with this, BMMCs with CSK-KD exhibited enhanced phosphorylation of protein phosphatase SHP-1, which provides a negative feedback loop for regulating phosphorylation of STAT5 and LYN kinase activity. Furthermore, we found that in WT BMMCs SHP-1 forms complexes containing LYN, CSK, and STAT5. Altogether, our data demonstrate that in FcεRI-activated mast cells CSK is a negative regulator of degranulation and chemotaxis, but a positive regulator of adhesion to fibronectin and production of proinflammatory cytokines. Some of these pathways are not dependent on the presence of PAG.

• Keywords
• C-terminal Src kinase, LYN, SHP-1, STAT5, cytokines, degranulation, mast cell, phosphoprotein associated with glycosphingolipid-enriched microdomains,
• MeSH
• Analysis of Variance MeSH
• Bone Marrow Cells physiology   MeSH
• CSK Tyrosine-Protein Kinase MeSH
• Cytokines metabolism   MeSH
• Cell Degranulation MeSH
• Fibronectins metabolism   MeSH
• Phosphoproteins metabolism   MeSH
• Phosphorylation MeSH
• Genetic Vectors MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Mast Cells physiology   MeSH
• Membrane Proteins metabolism   MeSH
• Intercellular Signaling Peptides and Proteins MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Receptors, IgE metabolism   MeSH
• Signal Transduction immunology   MeSH
• src-Family Kinases metabolism   physiology   MeSH
• STAT5 Transcription Factor metabolism   MeSH
• Tyrosine metabolism   MeSH
• Protein Tyrosine Phosphatase, Non-Receptor Type 6 metabolism   MeSH
• Calcium metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CSK Tyrosine-Protein Kinase MeSH
• CSK protein, human MeSH Browser
• Cytokines MeSH
• Fibronectins MeSH
• Phosphoproteins MeSH
• lyn protein-tyrosine kinase MeSH Browser
• Membrane Proteins MeSH
• Intercellular Signaling Peptides and Proteins MeSH
• Pag protein, mouse MeSH Browser
• Pag1 protein, mouse MeSH Browser
• Ptpn6 protein, mouse MeSH Browser
• Receptors, IgE MeSH
• src-Family Kinases MeSH
• STAT5 Transcription Factor MeSH
• Tyrosine MeSH
• Protein Tyrosine Phosphatase, Non-Receptor Type 6 MeSH
• Calcium MeSH

Single-nucleotide polymorphism studies have linked the chromosome 17q12-q21 region, where the human orosomucoid-like (ORMDL)3 gene is localized, to the risk of asthma and several other inflammatory diseases. Although mast cells are involved in the development of these diseases, the contribution of ORMDL3 to the mast cell physiology is unknown. In this study, we examined the role of ORMDL3 in antigen-induced activation of murine mast cells with reduced or enhanced ORMDL3 expression. Our data show that in antigen-activated mast cells, reduced expression of the ORMDL3 protein had no effect on degranulation and calcium response, but significantly enhanced phosphorylation of AKT kinase at Ser 473 followed by enhanced phosphorylation and degradation of IκBα and translocation of the NF-κB p65 subunit into the nucleus. These events were associated with an increased expression of proinflammatory cytokines (TNF-α, IL-6, and IL-13), chemokines (CCL3 and CCL4), and cyclooxygenase-2 dependent synthesis of prostaglandin D2. Antigen-mediated chemotaxis was also enhanced in ORMDL3-deficient cells, whereas spreading on fibronectin was decreased. On the other hand, increased expression of ORMDL3 had no significant effect on the studied signaling events, except for reduced antigen-mediated chemotaxis. These data were corroborated by increased IgE-antigen-dependent passive cutaneous anaphylaxis in mice with locally silenced ORMDL3 using short interfering RNAs. Our data also show that antigen triggers suppression of ORMDL3 expression in the mast cells. In summary, we provide evidence that downregulation of ORMDL3 expression in mast cells enhances AKT and NF-κB-directed signaling pathways and chemotaxis and contributes to the development of mast cell-mediated local inflammation in vivo.

• Keywords
• Chemotaxis, Degranulation, Mast cell, ORMDL3 knockdown, Proinflammatory cytokines, Prostaglandin D2, RNA interference,
• MeSH
• Chemotaxis * MeSH
• Cytokines genetics   immunology   MeSH
• Cell Degranulation * MeSH
• Down-Regulation MeSH
• Cells, Cultured MeSH
• Mast Cells cytology   immunology   metabolism   MeSH
• Membrane Proteins genetics   immunology   MeSH
• RNA, Messenger genetics   MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Receptors, IgE immunology   MeSH
• Up-Regulation MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokines MeSH
• Membrane Proteins MeSH
• RNA, Messenger MeSH
• ORMDL3 protein, mouse MeSH Browser
• Receptors, IgE MeSH

The transmembrane adaptor protein PAG/CBP (here, PAG) is expressed in multiple cell types. Tyrosine-phosphorylated PAG serves as an anchor for C-terminal SRC kinase, an inhibitor of SRC-family kinases. The role of PAG as a negative regulator of immunoreceptor signaling has been examined in several model systems, but no functions in vivo have been determined. Here, we examined the activation of bone marrow-derived mast cells (BMMCs) with PAG knockout and PAG knockdown and the corresponding controls. Our data show that PAG-deficient BMMCs exhibit impaired antigen-induced degranulation, extracellular calcium uptake, tyrosine phosphorylation of several key signaling proteins (including the high-affinity IgE receptor subunits, spleen tyrosine kinase, and phospholipase C), production of several cytokines and chemokines, and chemotaxis. The enzymatic activities of the LYN and FYN kinases were increased in nonactivated cells, suggesting the involvement of a LYN- and/or a FYN-dependent negative regulatory loop. When BMMCs from PAG-knockout mice were activated via the KIT receptor, enhanced degranulation and tyrosine phosphorylation of the receptor were observed. In vivo experiments showed that PAG is a positive regulator of passive systemic anaphylaxis. The combined data indicate that PAG can function as both a positive and a negative regulator of mast cell signaling, depending upon the signaling pathway involved.

• MeSH
• Anaphylaxis genetics   MeSH
• Bone Marrow Cells metabolism   physiology   MeSH
• CSK Tyrosine-Protein Kinase MeSH
• Cell Degranulation MeSH
• Type C Phospholipases metabolism   MeSH
• Phosphoproteins genetics   MeSH
• Phosphorylation MeSH
• Intracellular Signaling Peptides and Proteins metabolism   MeSH
• Syk Kinase MeSH
• RNA, Small Interfering MeSH
• Mast Cells metabolism   physiology   MeSH
• Membrane Proteins genetics   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Proto-Oncogene Proteins c-fyn biosynthesis   MeSH
• Proto-Oncogene Proteins c-kit metabolism   MeSH
• Receptors, IgE metabolism   MeSH
• RNA Interference MeSH
• Signal Transduction MeSH
• src-Family Kinases biosynthesis   metabolism   MeSH
• Protein-Tyrosine Kinases metabolism   MeSH
• Calcium metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CSK Tyrosine-Protein Kinase MeSH
• Type C Phospholipases MeSH
• Phosphoproteins MeSH
• Fyn protein, mouse MeSH Browser
• Intracellular Signaling Peptides and Proteins MeSH
• Syk Kinase MeSH
• lyn protein-tyrosine kinase MeSH Browser
• RNA, Small Interfering MeSH
• Membrane Proteins MeSH
• Pag1 protein, mouse MeSH Browser
• Proto-Oncogene Proteins c-fyn MeSH
• Proto-Oncogene Proteins c-kit MeSH
• Receptors, IgE MeSH
• src-Family Kinases MeSH
• Syk protein, mouse MeSH Browser
• Protein-Tyrosine Kinases MeSH
• Calcium MeSH