Beef extract supplementation promotes myoblast proliferation and myotube growth in C2C12 cells.

PURPOSE: We previously determined that the intake of beef extract for 4 weeks increases skeletal muscle mass in rats. Thus, this study aimed to clarify whether beef extract has a hypertrophic effect on muscle cells and to determine the signaling pathway underlying beef extract-induced myotube hypertrophy. METHODS: We assessed the effects of beef extract supplement on mouse C2C12 skeletal muscle cell proliferation and differentiation and myotube growth. In addition, the phosphorylation of Akt, ERK1/2, and mTOR following beef extract supplementation was examined by western blotting. Furthermore, the bioactive constituents of beef extract were examined using amino acid analysis and dialysis. RESULTS: In the proliferative stage, beef extract significantly increased myoblast proliferation. In the differentiation stage, beef extract supplementation did not promote myoblast differentiation. In mature myotubes, beef extract supplementation increased myotube diameter and promoted protein synthesis. Although Akt and ERK1/2 levels were not affected, beef extract supplementation increased mTOR phosphorylation, which indicated that the mTOR pathway mediates beef extract-induced myotube hypertrophy. The hypertrophic activity was observed in fractions of > 7000 Da. CONCLUSIONS: Beef extract promoted C2C12 myoblast proliferation and C2C12 myotube hypertrophy. Myotube hypertrophy was potentially induced by mTOR activation and active components in beef extract were estimated to be > 7000 Da.

Induction of avian ß-defensins by CpG oligodeoxynucleotides and proinflammatory cytokines in hen vaginal cells in vitro.

Immune function in the vagina of hen oviduct is essential to prevent infection by microorganisms colonizing in the cloaca. The aim of this study was to determine whether CpG oligodeoxynucleotides (CpG-ODN) stimulate the expression of avian ß-defensins (AvBDs) in hen vaginal cells. Specific questions were whether CpG-ODN affects the expression of AvBDs and proinflammatory cytokines and whether the cytokines affect AvBDs expression in vaginal cells. The dispersed vaginal cells of White Leghorn laying hens were cultured and stimulated by different doses of lipopolysaccharide (LPS), CpG-ODN, interleukin 1ß (IL1B), or IL6. The cultured cell population contained epithelial cells, fibroblast-like cells, and CD45-positive leukocytes. The immunoreactive AvBD3, -10, and -12 were localized in the mucosal epithelium in the section of the vagina. The expression of AvBDs, IL1B, and IL6 was analyzed by quantitative RT-PCR. RT-PCR analysis showed the expression of AvBD1, -3, -4, -5, -10, and -12 in the cultured vaginal cells without stimulation. Toll-like receptors (TLRs) 4 and 21, which recognize LPS and CpG-ODN respectively and IL1 and IL6 receptors (IL1R1 and IL6R) were also expressed in them. The expression of IL1B, IL6, and AvBD10 and -12 was upregulated by LPS, whereas only IL1B and IL6 were upregulated by CpG-ODN. IL1B stimulation upregulated AvBD1 and -3 expression, whereas IL6 stimulation did not cause changes in AvBDs expression. These results suggest that CpG-ODN derived from microbes upregulates the expression of IL1B and IL6 by interaction with TLR21 and then IL1B induces AvBD1 and -3 to prevent infection in the vagina.

Regulation of matrix metalloproteinase-1 and alpha-smooth muscle actin expression by interleukin-1 alpha and tumour necrosis factor alpha in hepatic stellate cells.

Hepatic stellate cells (HSCs) are key players in liver fibrosis and regeneration via collagen degradation and synthesis. These phenomena involve inflammatory cytokines released from non-parenchymal liver cells such as Kupffer cells. Although the effects of individual cytokines on many cell types have been investigated in various conditions, such as inflammation and tissue fibrosis, investigating the effect of combined cytokines would further our understanding of the regulatory mechanisms in tissue fibrosis. Here, we report the effect of multiple cytokine combinations on primary HSCs. We first examined the effect of individual cytokines and then the simultaneous exposure of different cytokines, including interleukin-6 (IL-6), IL-1 alpha (IL-1α), platelet-derived growth factor (PDGF), tumour necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-ß), on matrix metalloproteinase-1 (MMP1) gene expression in primary HSCs. We observed that the combination of all five cytokines induced higher levels of MMP1 gene expression. Of these cytokines, TNF-α and IL-1α were found to be the key cytokines for not only inducing MMP1 expression, but also increasing α-smooth muscle actin gene expression. In conclusion, the combined treatment of TNF-α and IL-1α on HSCs had an enhanced effect on the expression of the fibrotic genes, MMP1 and α-smooth muscle actin, so appears to be an important regulator for tissue regeneration. This finding suggests that stimulation with combined anti-fibrotic cytokines is a potential approach in the development of a novel therapy for the recovery of liver fibrosis.

An exocellular thermolysin-like metalloprotease produced by Vibrio fluvialis: purification, characterization, and gene cloning.

An exocellular metalloprotease produced by Vibrio fluvialis, an enteropathogenic vibrio, was purified and characterized. The metalloprotease (V. fluvialis protease [VFP]) was found to have very similar characteristics to V. vulnificus protease, including a molecular mass of 45kDa, sensitivity to chelating agents or competitive inhibitors for thermolysin-like metalloproteases, and the substrate specificity. The structural gene for VFP was also cloned, and its nucleotide sequence was determined. The deduced amino acid sequence confirmed that VFP was a member of the thermolysin family. VFP, like V. vulnificus protease, showed the haemagglutinating, permeability-enhancing and haemorrhagic activities in addition to the proteolytic activity toward oligopeptide, casein or elastin.