Pharmacological characterization of three chicken melanocortin-3 receptor mutants.

The melanocortin-3 receptor (MC3R) is a G protein-coupled receptor and potentially important in production traits. Three naturally occurring mutations (M54L, G104S, and L151R) in chicken MC3R (cMC3R) were reported previously to be associated with production traits. Here, we inserted the full-length cMC3R coding sequence into pcDNA3.1(+) and generated the 3 mutations by site-directed mutagenesis. The total and cell surface expression of the receptors was measured by flow cytometry. We analyzed the pharmacological characteristics, including binding and cyclic adenosine monophosphate (cAMP) and mitogen-activated protein kinase (MAPK) signaling, using 6 ligands ([Nle4, D-Phe7]-α-melanocyte stimulating hormone (MSH), α-, ß-, γ-, and D-Trp8-γ-MSHs, and agouti-related peptide). All mutants had similar total and cell surface expression as the wild-type (WT) cMC3R. M54L had similar pharmacological properties as the WT cMC3R. G104S did not exhibit any specific binding but had minimal response to α-, ß-, γ-, and D-Trp8-γ-MSH, although it generated 24% WT response when stimulated by NDP-MSH. Although L151R had normal binding, the responses to agonists were reduced to approximately 25% of that of the WT. In MAPK signaling, all 3 mutants showed significantly increased agonist-stimulated phosphorylation of extracellular signal-regulated protein kinases 1/2, indicating the existence of biased signaling at G104S and L151R. In summary, our studies demonstrated that although all 3 mutations are significantly associated with production traits, only G104S and L151R had severe defects in receptor pharmacology. How M54L might cause production trait differences remains to be investigated.

Clinical significance of TBX2 in esophageal squamous cell carcinomas and its role in cell migration and invasion.

OBJECTIVE: To explore the role of T-box 2 (TBX2) in esophageal squamous cell carcinomas (ESCC). PATIENTS AND METHODS: Quantitative real-time polymerase chain reaction (PCR) and Western blot (WB) assays were used to detect the expression level of TBX2 in tissues and cells. Transwell assays were conducted for determination of cell invasion and migration. RESULTS: The results suggested that the TBX2 was upregulated in ESCC tissues. Further, high expression of TBX2 expression was associated with tumor size, differentiation, distant metastasis, and TNM stage. In our in-vitro study, we decreased the expression of TBX2 in ESCC cells by transfection using LipofectamineTM 3000. The results from the transwell assay suggested that the downregulation of TBX2 could significantly suppress cell migration and invasion. Besides, WB results indicated that epithelial-mesenchymal transition (EMT)-related protein expressions were also changed after transfection. CONCLUSIONS: TBX2, as an oncogene, could promote the progress of ESCC by affecting the transfer ability in tumor cells.

Glutamine protects myocardial ischemia-reperfusion injury in rats through the PI3K/Akt signaling pathway.

OBJECTIVE: To study the protective mechanism of glutamine (Gln) on myocardial ischemia-reperfusion (IR) injury in rats through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. MATERIALS AND METHODS: A total of 30 healthy SD rats weighing 200-300 g were used in this experiment. They were randomly divided into 3 groups: sham group (n=10), myocardial IR injury group (IR group, n=10), IR+Gln group (n=10). The protein expression levels of phosphorylated Akt (p-Akt), total Akt (t-Akt), phosphorylated mammalian target of rapamycin (p-mTOR), mTOR, proliferating cell nuclear antigen (PCNA), P21, and Tubulin were determined by Western blotting (WB). Quantitative Polymerase Chain Reaction (qPCR) was applied to detect the messenger ribonucleic acid (mRNA) levels of Akt and mTOR. 3-(4,5)-dimethylthiazol(-z-y1)-3,5-diphenyl tetrazolium bromide (MTT) test was utilized to examine the proliferation ability of cardiomyocytes in vitro. Besides, the contents of the inflammatory cytokines, tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) were measured via enzyme-linked immunosorbent assay (ELISA). Cell apoptosis in each group was examined through Hoechst staining. RESULTS: Compared with those in the sham group, ratios of p-AKT/AKT, p-mTOR/mTOR, and the level of PCNA extremely significantly decreased, but the level of P21 notably increased in IR group (p<0.01). In comparison with those in the IR group, ratios of p-AKT/AKT, p-mTOR/mTOR, and the level of PCNA were remarkably raised, while the level of P21 was remarkably reduced in IR+Gln group (p<0.05). QRT-PCR results manifested that there were no significant differences in the mRNA levels of Akt and mTOR among the three groups [no significant difference (NS)]. Moreover, the cell proliferation ability in IR group was remarkably lower than that in the sham group (p<0.01), while it was enhanced in the IR+Gln group compared with that in the IR group (p<0.05). Additionally, IR group had significantly elevated expression levels of TNF-α and IL-6 compared with the sham group (p<0.01), whereas the IR+Gln group had notably decreased expression levels of TNF-α and IL-6 compared with IR group (p<0.05). In comparison with that in the sham group, the apoptosis in IR group was significantly raised (p<0.01), and compared with that in the IR group, the apoptosis in the IR+Gln group prominently decreased (p<0.05). The contents of the inflammatory cytokines, TNF-α, and IL-6 presented the same trends among the three groups. CONCLUSIONS: Gln activates the PI3K/Akt signaling pathway by increasing the levels of p-AKT and p-mTOR. Gln can increase the PCNA level and reduce the P21 level, so as to enhance the proliferation ability of cardiomyocytes. Besides, Gln reduces the levels of inflammatory cytokines, TNF-α, and IL-6, and inhibits cell apoptosis. Finally, Gln can protect cells from myocardial IR injury by activating the PI3K/Akt signaling pathway.