Novel role and mechanism of glutathione peroxidase-4 in nutritional pancreatic atrophy of chicks induced by dietary selenium deficiency.

Nutritional pancreatic atrophy (NPA) is a classical Se/vitamin E deficiency disease of chicks. To reveal molecular mechanisms of its pathogenesis, we fed day-old chicks a practical, low-Se diet (14 µg Se/kg), and replicated the typical symptoms of NPA including vesiculated mitochondria, cytoplasmic vacuoles, and hyaline bodies in acinar cells of chicks as early as day 18. Target pathway analyses illustrated a > 90% depletion (P < 0.05) of glutathione peroxidase 4 (GPX4) protein and up-regulated apoptotic signaling (cytochrome C/caspase 9/caspase 3) in the pancreas and(or) acinar cells of Se deficient chicks compared with Se-adequate chicks. Subsequently, we overexpressed and suppressed GPX4 expression in the pancreatic acinar cells and observed an inverse (P < 0.05) relationship between the GPX4 production and apoptotic signaling and cell death. Applying pull down and mass spectrometry, we unveiled that GPX4 bound prothymosin alpha (ProTalpha) to inhibit formation of apoptosome in the pancreatic acinar cells. Destroying this novel protein-protein interaction by silencing either gene expression accelerated H2O2-induced apoptosis in the cells. In the end, we applied GPX4 shRNA to silence GPX4 expression in chick embryo and confirmed the physiological relevance of the GPX4 role and mechanism shown ex vivo and in the acinar cells. Altogether, our results indicated that GPX4 depletion in Se-deficient chicks acted as a major contributor to their development of NPA due to the lost binding of GPX4 to ProTalpha and its subsequent inhibition on the cytochrome c/caspase 9/caspase 3 cascade in the acinar cells. Our findings not only provide a novel molecular mechanism for explaining pathogenesis of NPA but also reveal a completely new cellular pathway in regulating apoptosis by selenoproteins.

The protective impact of Trans-Cinnamaldehyde (TCA) against the IL-1b induced inflammation in in vitro osteoarthritis model by regulating PI3K/AKT pathways.

INTRODUCTION: Osteoarthritis (OA) is a severe joint degeneration disease in elderly people described by the advanced degradation of articular cartilage, which ultimately leads to chronic pain. Trans-cinnamaldehyde (TCA) exerted its anti-inflammatory function in numerous disease syndromes; however, its role in the pathogenesis of OA remains unknown. The current research aimed to explore the potential protective impact of TCA in the progression of osteoarthritis in vitro. MATERIAL AND METHODS: Human knee articular chondrocytes were treated with 10 ng/ml IL-1b alone for 24 h or in a combination in a pretreatment with TCA at different concentrations (2, 5, 10 µg/mL, 24 h). The viability and cell apoptosis were determined by CCK-8 assay and flow cytometry methods. The protein levels of IL-8, PGE2, and TNF-a and the levels of phosphorylated AKT and PI3K were evaluated using ELISA assay. Moreover, RT-qPCR was used to measure the relative mRNA expression of MMP-13, iNOS, COX-2, and ADAMTS-5 in IL-1b-induced chondrocytes. RESULTS: Our results revealed that the treatment with TCA had no effect on chondrocytes' proliferation and apoptosis. Moreover, the protein levels of IL-8, TNF-a, and PGE2 were considerably reduced in IL-1b-induced chondrocytes treated with different concentrations of TCA. Furthermore, the mRNA expression of MMP-13, iNOS, COX-2, and ADAMTS-5 and the phosphorylation of AKT and PI3K were markedly reduced in IL-1b-induced chondrocytes with the increase in the concentration of TCA. CONCLUSIONS: Trans-cinnamaldehyde inhibited the inflammation induced by IL-1b in chondrocytes through the PI3K/AKT pathway, which suggests that TCA might serve as a potential therapeutic agent for osteoarthritis treatment.

Characterization and Antioxidant Activity of the Exopolysaccharide Produced by Bacillus amyloliquefaciens GSBa-1.

The exopolysaccharide (EPS) produced by Bacillus amyloliquefaciens GSBa-1 was isolated and purified by ethanol precipitation, and DEAE-cellulose and Sepharose CL-6B chromatographies. The molecular mass of the purified EPS was determined to be 54 kDa. Monosaccharide analysis showed that the EPS was composed of predominantly glucose, and it was further confirmed by NMR spectroscopy to be α-glucan that consisted of a trisaccharide repeating unit with possible presence of two α-(1→3) and one α-(1→6) glucosidic linkages. Microstructural analysis showed that the EPS appeared as ellipsoid or globose with a smooth surface. The EPS had a degradation temperature at 240°C. Furthermore, the EPS had strong DPPH and hydroxyl radical scavenging activities, and moderate superoxidant anion scavenging and metal ion-chelating activities. This is the first characterization of a glucan produced by B. amyloliquefaciens with strong antioxidant activity. The results of this study suggest the potential of the EPS from B. amyloliquefaciens GSBa-1 to serve as a natural antioxidant for application in functional products.

Characterization of Selenoprotein M and Its Response to Selenium Deficiency in Chicken Brain.

Selenoprotein M (SelM) may function as thiol disulfide oxidoreductase that participates in the formation of disulfide bonds and can be implicated in calcium responses. SelM may have a functional role in catalyzing free radicals and has been associated with Alzheimer's disease (AD). However, studies of SelM in chicken remain very limited. In this study, two groups of day-old broiler chicks (n = 40/group) were fed a corn-soy basal diet (BD, 13 µg Se/kg) and BD supplemented with Se (as sodium selenite) at 0.3 mg/kg. The brain was collected at 14, 21, 28, and 42 days of age. We performed a sequence analysis and predicted the structure and function of SelM. We also investigated the effects of Se deficiency on the expression of Selt, Selw, and Selm and the Se status in the chicken brain. The results show that Se deficiency induced the lower (P < 0.05) Se content, glutathione peroxidase (GPx), and catalase (CAT) activities; increased (P < 0.05) malondialdehyde (MDA) content; and reduced (P < 0.05) the expression of Selm messenger RNA (mRNA) and protein abundance of SelM in the brain. However, there were no significant brain Selt and Selw mRNA levels by dietary Se deficiency in chicks. The different regulations of these three redox (Rdx) protein expressions by Se deficiency represent a novel finding of the present study. Our results demonstrated that SelM may have an important role in protecting against oxidative damage in the brain of chicken, which might shed light on the role of SelM in human neurodegenerative disease. More studies are needed to confirm our conclusion.

Characterization and Expression of Chicken Selenoprotein U.

Selenoprotein U (SelU) may regulate a myriad of biological processes through its redox function. In chicks, neither the nucleotide sequence nor the amino acid sequence is known. The main objectives of this study were to clone and characterize the chicken Selu gene and investigate Selu messenger RNA (mRNA) and protein expression in chicken tissues. The coding sequence (CDS) of Selu contained 387 bases with a typical mammalian selenocysteine insertion sequence (SECIS) located in the 3'-untranslated region. The deduced amino acid sequence of chicken SelU contains 224 amino acids with UAA as the stop codon. Like all SelU genes identified in different species, chicken SelU contains one well-conserved selenocysteine (Sec) at the 85th position encoded by the UGA codon. The SECIS element was with the conserved denosine (--AAA--) rather than the motif cytidine (--CC--) motif. Moreover, the expression pattern of Selu mRNA in muscle, liver, kidney, heart, spleen, lung, testis, and brain was analyzed with real-time quantitative PCR in young male chickens fed a Se-deficient corn-soybean meal basal diet supplemented with 0.0 and 0.3 mg Se/kg in the form of sodium selenite. We found that the abundance of Selu mRNA in muscle, liver, kidney, heart, spleen, and lung was downregulated (P < 0.05) by Se deficiency. However, it was not affected by dietary Se concentrations in testis and brain. Furthermore, protein abundance of SelU in these seven tissues was consistent with the mRNA abundance. Hence, we suggest that Selu might play an important role in the biochemical function of Se in birds.

Selenoproteins protect against avian nutritional muscular dystrophy by metabolizing peroxides and regulating redox/apoptotic signaling.

Nutritional muscular dystrophy (NMD) of chicks is induced by dietary selenium (Se)/vitamin E (Vit. E) deficiencies and may be associated with oxidative cell damage. To reveal the underlying mechanisms related to the presumed oxidative cell damage, we fed four groups of 1-day-old broiler chicks (n = 40/group) with a basal diet (BD; 10 µg Se/kg; no Vit. E added, -Se -Vit. E) or the BD plus all-rac-α-tocopheryl acetate at 50mg/kg (-Se +Vit. E), Se (as sodium selenite) at 0.3mg/kg (+Se -Vit. E), or both of these nutrients (+Se +Vit. E) for 6 weeks. High incidences of NMD (93%) and mortality (36%) of the chicks were induced by the BD, starting at week 3. Dietary Se deficiency alone also induced muscle fiber rupture and coagulation necrosis in the pectoral muscle of chicks at week 3 and thereafter, with increased (P < 0.05) malondialdehyde, decreased (P < 0.05) total antioxidant capacity, and diminished (P < 0.05) glutathione peroxidase activities in the muscle. To link these oxidative damages of the muscle cells to the Se-deficiency-induced NMD, we first determined gene expression of the potential 26 selenoproteins in the muscle of the chicks at week 2 before the onset of symptoms. Compared with the +Se chicks, the -Se chicks had lower (P < 0.05) muscle mRNA levels of Gpx1, Gpx3, Gpx4, Sepp1, Selo, Selk, Selu, Selh, Selm, Sepw1, and Sep15. The -Se chicks also had decreased (P < 0.05) production of 6 selenoproteins (long-form selenoprotein P (SelP-L), GPx1, GPx4, Sep15, SelW, and SelN), but increased levels (P < 0.05) of the short-form selenoprotein P in muscle at weeks 2 and 4. Dietary Se deficiency elevated (P < 0.05) muscle p53, cleaved caspase 3, cleaved caspase 9, cyclooxygenase 2 (COX2), focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K), phospho-Akt, nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (p38 MAPK), phospho-p38 MAPK, phospho-JNK, and phospho-ERK and decreased (P < 0.05) muscle procaspase 3, procaspase 9, and NF-κB inhibitor α. In conclusion, the downregulation of SelP-L, GPx1, GPx4, Sep15, SelW, and SelN by dietary Se deficiency might account for induced oxidative stress and the subsequent peroxidative damage of chick muscle cells via the activation of the p53/caspase 9/caspase 3, COX2/FAK/PI3K/Akt/NF-κB, and p38 MAPK/JNK/ERK signaling pathways. Metabolism of peroxides and redox regulation are likely to be the mechanisms whereby these selenoproteins prevented the onset of NMD in chicks.

Identification of novel nuclear localization signal within the ErbB-2 protein.

ErbB2, a member of the receptor tyrosine kinase family, is frequently over-expressed in breast cancer. Proteolysis of the extracellular domain of ErbB2 results in constitutive activation of ErbB2 kinase. Recent study reported that ErbB2 is found in the nucleus. Here, we showed that ErbB2 is imported into the nucleus through a nuclear localization signal (NLS)-mediated mechanism. The NLS sequence KRRQQKIRKYTMRR (aa655-668) contains three clusters of basic amino acids and it is sufficient to target GFP into the nucleus. However, mutation in any basic amino acid cluster of this NLS sequence significantly affects its nuclear localization. Furthermore, it was found that this NLS is essential for the nuclear localization of ErbB2 since the intracellular domain of Erb2 lacking NLS completely abrogates its nuclear translocation. Taken together, our study identified a novel nuclear localization signal and reveals a novel mechanism underlying ErbB2 nuclear trafficking and localization.