Search

Adenylosuccinate synthetase (ADSS) catalyzes the key step of AMP synthesis. Vertebrates have two isozymes of ADSS, which are named ADSS1 and ADSS2, respectively. In this study, we cloned porcine ADSS1 and ADSS2 genes and comparatively analyzed their sequence, chromosome mapping, mRNA distribution and subcellular localization. According to our results, the ADSS1 gene was predominantly expressed in the striated muscle tissues, while ADSS2 gene distributed widely in all the tissues detected. Additionally, ADSS1 gene was up-regulated significantly along with porcine muscle growth, and ADSS2 gene expression was more constant during the muscle development. Porcine ADSS1 gene was assigned to SSC7q and the linked marker was SSC12B09, ADSS2 gene was mapped on SSC10p and the linked marker was SW497, and porcine ADSS2 protein was subcellular localized in mitochondria. Moreover, we found that one single nucleotide polymorphism (SNP, T/C(70)) in the ninth intron of ADSS2 gene was significantly associated with average daily gain trait (ADG, P<0.05) and loin muscle area trait (P<0.05).

Calsarcins comprise a novel family of muscle-specific calcineurin-interacting proteins and play an important role in modulating both the function and substrate specificity of calcineurin in muscle cells. In this study, we cloned and characterized calsarcins from pig muscle. The deduced amino acid sequences of porcine calsarcin-1 (CS-1), calsarcin-2 (CS-2), and calsarcin-3 (CS-3) share the same putative calcineurin and alpha-actinin binding regions. Radiation hybrid mapping data indicate that CS-1 and CS-2 map to q2.1-2.5 of pig chromosome 8 (SSC8) and q2.4 of pig chromosome 14 (SSC14), respectively. The mRNA expressions of both CS-1 and CS-2 are regulated in skeletal muscle similarly during postnatal development but not during prenatal development, indicating differences in function, additionally demonstrated by minute differences in cellular localization within Pig Kidney Epithelial cells (PK15). We provide the first evidence that CS-1 is abundantly expressed in porcine heart and has an expression pattern similar to the human gene. This result suggests that the pig may be a suitable animal model to study the function of calsarcins in human heart disease.

Background: Pig (Sus scrofa) is a major source of dietary proteins for human consumption and is becoming a valuable model in agricultural and biomedical research. The recently developed isobaric tag for relative and absolute quantitation (iTRAQ) method allows sensitive and accurate protein quantification. Here, we performed the first iTRAQ-based quantitative proteomic analyses of Landrace (LR) and Wuzhishan (WZS) pig longissimus dorsi muscle tissues during early embryonic development. Results: The iTRAQ-based early embryonic longissimus dorsi muscle study of LR and WZS ranging from 21 to 42 days post coitus (dpc) identified a total of 4431 proteins from17,214 peptides, which were matched with 36,4025 spectra at a false discovery rate of 5 %. In both WZS and LR, the largest amount of differentially expressed proteins (DEPs) were found between 28 and 35 dpc. 252 breed-DEPs were selected by GO analysis, including 8 myofibrillar proteins. Only MYHCI/IIA mRNA were detected due to early embryonic stages, and significantly higher expression of them were found in WZS during these 4 stages. MYHCI was first found in WZS at 28 dpc and expressed in both breeds at later stages, while MYHCII protein was not detected until 35 dpc in both breeds. Thus, 33 myogenic breed-DEPs selected from last two stages were analyzed by STRING, which showed that some myofibrillar proteins (MYH1, TPM4, MYH10, etc.) and functional proteins (CSRP2, CASQ2, OTC, etc.), together with candidate myogenic proteins (H3F3A, HDGFRP2, etc.), probably participate in the regulatory network of myofiber formation. Conclusion: Our iTRAQ-based early embryonic longissimus dorsi muscle study of LR and WZS provides new data on the in vivo muscle development distinctions during early embryonic development, which contributes to the improved understanding in the regulation mechanism of early myogenesis in agricultural animals. [ABSTRACT FROM AUTHOR]

Highlights: [•] Overexpression of miR-344 inhibited 3T3-L1 preadipocyte differentiation. [•] GSK3β was confirmed to be the target of miR-344. [•] Knockdown of endogenous GSK3β repressed 3T3-L1 preadipocyte differentiation. [•] Overexpression of GSK3β could overcome the effect of miR-344. [•] miR-344 activated Wnt/β-catenin signaling and repressed preadipocyte differentiation. [ABSTRACT FROM AUTHOR]

The innate immune response provides the initial defence mechanism against infection by other organisms. However, an excessive immune response will cause damage to host tissues. In an attempt to identify microRNAs (miRNAs) that regulate the innate immune response in inflammation and homeostasis, we examined the differential expression of miRNAs using microarray analysis in the spleens of mice injected intraperitoneally with lipopolysaccharide (LPS) and saline, respectively. Following challenge, we observed 19 miRNAs up-regulated (1·5-fold) in response to LPS. Among these miRNAs, miR-1224, whose expression level increased 5·7-fold 6 hr after LPS injection and 2·3-fold after 24 hr, was selected for further study. Tissue expression patterns showed that mouse miR-1224 is highly expressed in mouse spleen, kidney and lung. Transfection of miR-1224 mimics resulted in a decrease in basal tumour necrosis factor-α (TNF-α) promoter reporter gene activity and a down-regulation of LPS-induced TNF-α mRNA in RAW264.7 cells. With public databases of miRNA target prediction, miR-1224 was shown to bind to the 3′ untranslated region (UTR) of Sp1 mRNA, whose coding product controls TNF-α expression at the transcriptional level. Furthermore, we found that in HEK-293 cells, the activity of the luciferase reporter bearing Sp1 mRNA 3′ UTR was down-regulated significantly when transfected with miR-1224 mimics. After transfection of miR-1224 in RAW264.7 cells, nucleus Sp1 protein level decreased, and when endogenous miR-1224 was blocked, the decrease was abolished. Therefore, we initially speculated that miR-1224 was a negative regulator of TNF-α in an Sp1-dependent manner, which was confirmed in vivo by chromatin immunoprecipitation assay, and might be involved in regulating the LPS-mediated inflammatory responses. [ABSTRACT FROM AUTHOR]

MicroRNAs are a class of highly conserved ∼20 nucleotides non-coding RNAs that post-transcriptionally regulate gene expression. Many miRNAs were studied in the development of skeletal muscle, such as miR-1, miR-206, and miR-133. In our previous study, miR-127-3p was found highly expressed in porcine fetal skeletal muscle, whereas the detailed functions of miR-127-3p in muscle development is still unclear. In this study, we detected that miR-127-3p also highly expressed in skeletal muscle, cardiac muscle of adult mice and proliferative C2C12 cell lines. Overexpression of miR-127-3p almost has no effects on differentiation of C2C12 cell lines. However, miR-127-3p significantly inhibited the cell proliferation of C2C12 cells. Moreover, we identified KMT5a as a target gene that was down-regulated in both mRNA and protein level when miR-127-3p mimics were introduced. Furthermore, KMT5a overexpression in miR-127-3p treated cells rescued the influence of miR-127-3p on C2C12 proliferation. In brief, our data reveals that miR-127-3p regulates the proliferation of myocytes through KMT5a. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are endogenous small non-coding RNAs which modulate gene expression at the post-transcriptional level by either translational inhibition or mRNA degradation. MicroRNAs play important roles in both innate and adaptive immune response, including TLR-triggered immune response. In this study, we found that the expression of miR-709 was up-regulated in primary macrophage and RAW264.7 cells during the stimulation of LPS. Overexpression of miR-709 in RAW264.7 cells led to reduced production and gene expression of inflammatory cytokines (IL-6, TNF-α, IL-1β) during activation by LPS, whereas knockdown of miR-709 had completely opposite effects. We used bioinformatics and experimental techniques to demonstrate that GSK-3β is a direct target of miR-709. miR-709 mimics decreased GSK-3β protein but not mRNA level. We also found that miR-709 regulated the LPS-induced inflammatory response by targeting GSK-3β and elevating β-catenin. In conclusion, our data revealed a novel role for miR-709 in regulation of inflammatory response by targeting GSK-3β. [ABSTRACT FROM AUTHOR]

Abstract: Tmod4 (Tropomodulin 4) is a member of Tmod family that plays important role in thin filament length regulation and myofibril assembly. We found that the expression levels of Tmod4 were higher in skeletal muscle and adipose tissues. However, the function and regulation of the Tmod4 gene in the myogenesis and adipogenesis remains unclear. In this study, we found that the expression of Tmod4 was decreased in myogenesis while increased in adipogenesis. Then, the transcriptional regulation analysis of Tmod4 promoter showed that Tmod4 could be regulated directly by myogenic factors and adipogenic factors. Furthermore, the roles of Tmod4 in the myogenesis and adipogenesis were confirmed by its over-expression in C2C12 cells and 3T3 cells, which suggested that Tmod4 could promote adipogenesis by up-regulating the adipogenic factors but moderately delay the myogenesis. These results indicated that the Tmod4 gene may play as a switch between myogenesis and adipogenesis, which resulted in the balanced development between skeletal muscle and adipose tissue. Therefore, the model for switch role of the Tmod4 in the balanced regulation between myogenesis and adipogenesis was proposed. It is showed that the expression of Tmod4 was activated in adipogenesis by adipogenic factors while inhibited in myogenesis by myogenic factors. Moreover, Tmod4 could promote adipogenesis by up-regulating the expression of adipogenic factors while moderately delaying the myogenesis. Our study provides an important basis for further understanding the regulation and function of porcine Tmod4 in muscle and fat development. [Copyright &y& Elsevier]