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Oocyte apoptosis can be used as an indicator of oocyte quality and development competency. Phospholipase C (PLC) is a critical enzyme that participates in phosphoinositide metabolic regulation and performs many functions, including the regulation of reproduction. In this study, we aimed to explore whether PLC participates in the regulation of apoptosis in porcine oocytes and investigated its possible mechanism. In porcine oocytes, 0.5 μM U73122 (the PLC inhibitor) was considered to be the best concentration to facilitate maturation, and 0.5 μM m-3M3FBS (the PLC activator) was regarded as the most appropriate concentration to inhibit maturation. The percentage of cleavage and blastocysts treated with 0.5 μM U73122 was lower than that of the control group. Furthermore, the percentage of cleavage and blastocysts treated with 0.5 μM m-3M3FBS was higher than that of the control group. The relative PLC messenger RNA (mRNA) expression tested by a quantitative real-time polymerase chain reaction was found to be inhibited by 0.5 μM U73122 or activated by 0.5 μM m-3M3FBS. The relative mRNA abundance of BAK, BAX, CASP3, CASP8, and TP53 and protein abundance of Bak, cleaved caspase-3, caspase-8, and P53 was activated by U73122 or inhibited by m-3M3FBS, while the relative mRNA and protein level of BCL6 showed the opposite trend. The intracellular Ca2+ concentration increased and the expression of PLCB1 protein also increased in porcine oocytes when they were cultured with 0.5 μM m-3M3FBS for 44 hours. The abundance of proteins PKCβ and CAMKIIα and the expression of several downstream genes (CDC42, NFATc1, NFATc2, NFκB, and NLK) were activated by m-3M3FBS or inhibited by U73122. Our findings indicate that PLC inhibits apoptosis and maturation in porcine oocytes. The intracellular Ca2+ concentration, two Ca2+ -sensitive proteins, and several downstream genes were positively regulated by PLC.

Glycogen synthase kinase beta (GSK3β) plays an important role in skeletal muscle growth, regeneration, and repair. However, the mechanism of GSK3β regulating MyHC2a expression is currently not clear. In this study, GSK3β inhibition promoted skeletal muscle satellite cells (SMSCs) differentiation and increased expression of MyoD, MyoG, MyHC1, and MyHC2a genes. Then we cloned approximately 1.1 kb of goat MyHC2a gene promoter. The deletion fragment (-514/+55) of MyHC2a promoter exhibited the highest level of promoter activity, and a NFATc2 element in this region was responsible for MyHC2a promoter activity. Treatment of SB216713 significantly decreased the transcriptional activity of the fragment (-514/+55). Furthermore, GSK3β inhibition had no effect on the luciferase activity of MyHC2a promoter after mutating the NFATc2-binding site. These results demonstrated that GSK3β inhibition promoted SMSCs differentiation and regulated the MyHC2a gene expression through NFATc2 in goat-differentiated SMSCs.

The family of nuclear factor of activated T cells (NFAT) transcription factors plays a critical role in mediating immune responses. Our previous clinical pharmacogenetic studies suggested that NFATC2 is associated with the risk of hypersensitivity reactions to the chemotherapeutic agent L-asparaginase (ASNase) that worsen outcomes during the treatment of pediatric acute lymphoblastic leukemia. We therefore hypothesized that the genetic inhibition of NFATC2 would protect against the development of anti-ASNase antibodies and ASNase hypersensitivity. Our study demonstrates that ASNase-immunized NFATC2-deficient mice are protected against ASNase hypersensitivity and develop lower antigen-specific and total immunoglobulin E (IgE) levels compared with wild-type (WT) controls. Furthermore, ASNase-immunized NFATC2-deficient mice develop more CD4+ regulatory T cells, fewer CD4+ interleukin-4-positive (IL-4+) cells, higher IL-10/TGF-β1 levels, and lower IL-4/IL-13 levels relative to WT mice. Basophils and peritoneal mast cells from ASNase-immunized, but not naive, NFATC2-deficient mice had lower FceRI expression and decreased IgE-mediated mast cell activation than WT mice. Furthermore, ASNase-immunized, but not naive, NFATC2-deficient mice developed less severe shock than WT mice after induction of passive anaphylaxis or direct histamine administration. Thus, inhibition of NFATC2 protects against ASNase hypersensitivity by impairing T helper 2 responses, which may provide a novel strategy for attenuating hypersensitivity and the development of antidrug antibodies, including to ASNase.

CD4+ 25+ regulatory T cells (Tregs) play a role in controlling the development and progression of autoimmunity. The transcription factors Foxp3 and NFATC2 (NFAT1) play key roles in regulating the development and function of Tregs. The present study examined the involvement of Tregs in the pathophysiology of autoimmune neutropenia in children. Tregs were analysed by flow cytometry, based on the expressions of CD4, CD25, and intracellular Foxp3. The expressions of FOXP3 and NFATC2 mRNA in the CD4+ 25+ cells were determined by quantitative real-time polymerase chain reaction. The percentage of CD4+ 25(high) Tregs in patients with autoimmune neutropenia was significantly lower than that in age-matched healthy subjects. The intracellular expression of Foxp3 of CD4+ 25+ cells in patients similarly decreased in comparison to that in healthy subjects. The expression of FOXP3 and NFATC2 mRNA of CD4+ 25+ cells in patients also significantly decreased in comparison to that in healthy subjects. These results suggest that the deficiency of Tregs might thus play an important role in the immunopathophysiology of autoimmune neutropenia in children.

The heterodimeric hypoxia-inducible factor (HIF)-1 is a master transcriptional regulator of oxygen homeostasis and a possible target for gene therapy of ischemic disease. Although the role of oxygen concentration in HIF-1a protein stabilization is well established, it is less clear whether and how oxygen-regulated mechanisms contribute to HIF-1a protein modifications, nuclear translocation, heterodimerization with the b-subunit, recruitment of cofactors, and gene trans-activation. Because the HIF-1a protein is proteolytically degraded under normoxic conditions, we established two HeLa Tet-Off cell lines (HT42 and HT43), which inducibly overexpress high levels of HIF-1a under normoxic conditions, allowing to distinguish hypoxia-dependent from hypoxia-independent activation mechanisms. Using these cells, we found that normoxically induced HIF-1a is localized to the nucleus, binds DNA, and trans-activates reporter and endogenous target genes. The levels of p53 expression remained unaffected. The MAP kinase inhibitor PD98059 attenuated HIF-1a protein modifications and trans-activation ability but not protein stabilization and DNA-binding activity. Because overexpressed HIF-1a is fully localized to the nucleus but displays only partial DNA-binding and trans-activation activity, mitogen-activated protein kinase-dependent phosphorylation might be required for full HIF-1 activation. HIF-1a protein was also overexpressed in vivo, following the transplantation of HT42 cells into nude mice, demonstrating the feasibility of HIF-1a gene transfer.

Using differential cDNA library screening techniques based on metastatic and nonmetastatic rat mammary adenocarcinoma cell lines we previously cloned and sequenced the metastasis-associated gene mta1. Using homology to the rat MTA1 gene we cloned the human MTA1 gene and found it to be overexpressed in a variety of human cell lines. We found a close similarity between the human MTA1 and rat MTA1 genes, as shown by 88% and 96% identities of the nucleotide and predicted amino acid sequences, respectively. Both genes encode novel proteins that contain a proline-rich region (SH3 binding motif), a putative zinc finger motif, a leucine zipper motif, and five copies of the SPXX motif often found in gene regulatory proteins. Using Southern blot analysis, the MTA1 gene was found to be highly conserved among all species examined; and using Northern blot analysis, MTA1 transcripts were found in virtually all cell lines of human origin that were analyzed, including melanoma and breast, cervix and ovarian carcinoma cells and normal breast epithelial cells. However, the expression level of the MTA1 gene in a normal breast epithelial cell was approximately 50% of that found in rapidly growing breast adenocarcinoma cell lines and an atypical mammary cell line. Experimental inhibition of MTA1 protein expression using antisense phosphorothioate oligonucleotides resulted in growth inhibition of human MDA-MB-231 breast cancer cells with relatively high expression of the MTA1 gene. Furthermore, the MTA1 protein was localized in the nuclei of cells transfected using a mammalian expression vector containing the full-length MTA1 gene. The results suggest that the MTA1 protein may function in cellular signaling processes important in the progression and growth of cancer cells, possibly as a nuclear regulatory factor.

An Arabidopsis thaliana cDNA clone that encodes a putative receptor-like protein kinase gene (At-RLK3) was characterized. The deduced 667-amino acid protein consists of an amino-terminal signal sequence, an extracellular domain, a single transmembrane domain, and a cytoplasmic domain with characteristics of serine/threonine protein kinase. Because of the original features of its extracellular domain, the At-RLK3 protein is a member of a new class of receptor-like protein kinases. The At-RLK3 gene is present as a single copy within the Arabidopsis genome and its transcripts are detected in root, stem, leaf and flower. In cultured cells, the At-RLK3 gene is activated upon oxidative stress and salicylic acid treatment. In plants, the gene appears to be differentially regulated during various plant-pathogen interactions: upon inoculation with strains of Pseudomonas syringae pv. tomato harboring or not, different avr genes, At-RLK3 transcripts accumulate transiently at similar levels during both compatible and incompatible interactions. This gene is, however, preferentially expressed during the incompatible interaction induced by the soil-borne vascular bacteria, Ralstonia solanacearum. The involvement of At-RLK3 in signal transduction pathways during pathogen attack is discussed.

The rapid activation of gene expression by growth factors frequently involves the nucleocytoplasmic redistribution of proteins which regulate the transcription of discrete sets of genes. In particular, it has become apparent that protein kinases and transcription factors including protein kinase A, mitogen activated protein kinase, protein kinase C, NF-kappa B and Stat91, can rapidly migrate from the cytosol to the nucleus in response to a wide variety of extracellular stimuli. Furthermore, it is assumed that these proteins represent important components of the signal transduction pathways which operate between the plasma membrane and the nucleus. Here two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been used to detect proteins which undergo rapid growth factor stimulated nucleocytoplasmic redistribution. Epidermal growth factor and insulin influenced the association of 15 individual protein spots with the nucleus. Estimates of the abundance of these proteins revealed that they represented approximately 1/1000 to 1/5000 of total nuclear protein. To permit identification by microsequencing, five of the protein spots have been recovered from multiple 2-D PAGE gels and concentrated by one-dimensional elution-concentration gel electrophoresis. The data obtained demonstrate that 2-D PAGE can be used to: (i) analyse putative regulators of growth factor activated gene transcription, and (ii) purify sufficient amounts of these proteins to facilitate identification by microsequence analysis.