EGF-induced inhibition of glucose transport is mediated by PKC and MAPK signal pathways in primary cultured chicken hepatocytes

EGF is a regulator of a wide variety of processes in various cell systems. Hepatocytes are important sites in the body's metabolism and function. Glucose transporter 2 (GLUT2) is a major transporter that is expressed strongly in hepatocytes. Therefore, this study examined the effect of EGF on GLUT2 and its related signal cascades in primary cultured chicken hepatocytes. EGF decreased [(3)H]deoxyglucose uptake in a dose- and time-dependent manner (>10 ng/ml, 2 h). AG-1478 (an EGF receptor antagonist) and genistein and herbimycin A (tyrosine kinase inhibitors) blocked the EGF-induced decrease in [(3)H]deoxyglucose uptake, which correlated with the GLUT2 expression level. In addition, the EGF-induced decrease in GLUT2 protein expression was inhibited by staurosporine, H-7, or bisindolylmaleimide I (PKC inhibitors), PD-98059 (a MEK inhibitor), SB-203580 (a p38 MAPK inhibitor), and SP-600125 (a JNK inhibitor), suggesting a role of both PKC and MAPKs (p44/42 MAPK, p38 MAPK, and JNK). In particular, EGF increased the translocation of PKC isoforms (PKC-alpha, -beta(1), -gamma, -delta, and -zeta) from the cytosol to the membrane fraction and increased the activation of p44/42 MAPK, p38 MAPK, and JNK. Moreover, PKC inhibitors blocked the EGF-induced phosphorylation of three MAPKs. In conclusion, EGF decreases the GLUT2 expression level via the PKC-MAPK signal cascade in chicken hepatocytes.     

Redox regulation of RhoA

We have previously shown that redox agents including superoxide anion radical and nitrogen dioxide can react with GXXXXGK(S/T)C motif-containing GTPases (i.e., Rac1, Cdc42, and RhoA) to stimulate guanine nucleotide release. We now show that the reaction of RhoA with redox agents leads to different functional consequences from that of Rac1 and Cdc42 due to the presence of an additional cysteine (GXXXCGK(S/T)C) in the RhoA redox-active motif. While reaction of redox agents with RhoA stimulates guanine nucleotide dissociation, RhoA is subsequently inactivated through formation of an intramolecular disulfide that prevents guanine nucleotide binding thereby causing RhoA inactivation. Thus, redox agents may function to downregulate RhoA activity under conditions that stimulate Rac1 and Cdc42 activity. The opposing functions of these GTPases may be due in part to their differential redox regulation. In addition, the results presented herein suggest that the platinated-chemotherapeutic agent, cisplatin, which is known for targeting nucleic acids, reacts with RhoA to produce a RhoA thiol-cisplatin-thiol adduct, leading to inactivation of RhoA. Similarly, certain arsenic complexes (i.e., arsenate and arsenic trioxide) may inactivate RhoA by bridging the cysteine residues in the GXXXCGK(S/T)C motif. Thus, in addition to redox agents, platinated-chemotherapeutic agents and arsenic complexes may modulate the activity of GTPases containing the GXXXCGK(S/T)C motif (i.e., RhoA and RhoB).     

Isolation and Characterization of a Lytic Myoviridae Bacteriophage PAS-1 with Broad Infectivity in Aeromonas salmonicida

To search for candidate control agents against Aeromonas salmonicida subsp. salmonicida infections in aquaculture, one bacteriophage (phage), designated as PAS-1, was isolated from the sediment samples of the rainbow trout (Oncorhynchus mykiss) culture farm in Korea. The PAS-1 was morphologically classified as Myoviridae and possessed approximately 48 kb of double-strand genomic DNA. The phage showed broad host ranges to other subspecies of A. salmonicida as well as A. salmonicida subsp. salmonicida including antibiotic-resistant strains. Its latent period and burst size were estimated to be approximately 40 min and 116.7 PFU/cell, respectively. Furthermore, genomic and structural proteomic analysis of PAS-1 revealed that the phage was closely related to other Myoviridae phages infecting enterobacteria or Aeromonas species. The bacteriolytic activity of phage PAS-1 was evaluated using three subspecies of A. salmonicida strain at different doses of multiplicity of infection, and the results proved to be efficient for the reduction of bacterial growth. Based on these results, PAS-1 could be considered as a novel Aeromonas phage and might have potentiality to reduce the impacts of A. salmonicida infections in aquaculture.     

Involvement of the MAPK and PI3K pathways in chitinase 3-like 1-regulated hyperoxia-induced airway epithelial cell death

BackgroundExposure to 100% oxygen causes hyperoxic acute lung injury characterized by cell death and injury of alveolar epithelial cells. Recently, the role of chitinase 3-like 1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, in oxidative stress was demonstrated in murine models. High levels of serum CHI3L1 have been associated with various diseases of the lung, such as asthma, chronic obstructive pulmonary disease, and cancer. However, the role of CHI3L1 in human airway epithelial cells undergoing oxidative stress remains unknown. In addition, the signaling pathways associated with CHI3L1 in this process are poorly understood.PurposeIn this study, we demonstrate the role of CHI3L1, along with the MAPK and PI3K signaling pathways, in hyperoxia-exposed airway epithelial cells.MethodThe human airway epithelial cell line, BEAS-2B, was exposed to >95% oxygen (hyperoxia) for up to 72 h. Hyperoxia-induced cell death was determined by assessing cell viability, Annexin-V FITC staining, caspase-3 and -7 expression, and electron microscopy. CHI3L1 knockdown and overexpression studies were conducted in BEAS-2B cells to examine the role of CHI3L1 in hyperoxia-induced apoptosis. Activation of the MAPK and PI3K pathways was also investigated to determine the role of these signaling cascades in this process.ResultsHyperoxia exposure increased CHI3L1 expression and apoptosis in a time-dependent manner. CHI3L1 knockdown protected cells from hyperoxia-induced apoptosis. In contrast, CHI3L1 overexpression promoted cell death after hyperoxia exposure. Finally, phosphorylation of ERK1/2, p38, and Akt were affected by CHI3L1 knockdown.ConclusionThis study indicates that CHI3L1 is involved in hyperoxia-induced cell death, suggesting that CHI3L1 may be one of several cell death regulators influencing the MAPK and PI3K pathways during oxidative stress in human airway epithelial cells.     

Hepatic expression of cytochrome P450 in type 2 diabetic Goto-Kakizaki rats

Although hepatic expression of cytochrome P450 (CYP) changes markedly in diabetes, the role of ketone bodies in the regulation of CYP in diabetes is controversial. The present study was performed to determine the expression and activity of CYP in non-obese type II diabetic Goto-Kakizaki (GK) rats with normal levels of ketone bodies. In the present study, basal serum glucose levels increased 1.95-fold in GK rats, but acetoacetate and β-hydroxybutyrate levels were not significantly different. Hepatic expression of CYP reductase and CYP3A2 was up-regulated in the GK rats, and consequently, activities of CYP reductase and midazolam 4-hydroxylase, mainly catalyzed by CYP3A2, increased. In contrast, hepatic expression of CYP1A2 and CYP3A1 was down-regulated and the activities of 7-ethoxyresorufin-O-deethylase and 7-methoxyresorufin-O-demethylase, mainly catalyzed by CYP1A, also decreased in GK rats. Hepatic levels of microsomal protein and total CYP and hepatic expression of cytochrome b(5), CYP1B1, CYP2B1 and CYP2C11 were not significantly different between the GK rats and normal Wistar rats. Moreover, the expression and activity of CYP2E1, reported to be up-regulated in diabetes with hyperketonemia, were not significantly different between GK rats and control rats, suggesting that elevation of ketone bodies plays a critical role in the up-regulation of hepatic CYP2E1 in diabetic rats. Our results showed that the expression of hepatic CYP is regulated in an isoform-specific manner. The present results also show that the GK rat is a useful animal model for the pathophysiological study of non-obese type II diabetes with normal ketone body levels.     

IN VITRO ANTIOXIDANT ACTIVITIES OF THE FERMENTED MARINE MICROALGA PAVLOVA LUTHERI (HAPTOPHYTA) WITH THE YEAST HANSENULA POLYMORPHA1

Microalgae are major primary producers of organic matter in aquatic environments through their photosynthetic activities. Fermented microalga (Pavlova lutheri Butcher) preparation (FMP) is the product of yeast fermentation by Hansenula polymorpha. It was tested for the antioxidant activities including lipid peroxidation inhibitory activity, free‐radical‐scavenging activity, inhibition of reactive oxygen species (ROS) on mouse macrophages (RAW264.7 cell), and inhibited myeloperoxidase (MPO) activity in human myeloid cells (HL60). FMP exhibited the highest antioxidant activity on free‐radical scavenging, inhibitory intracellular ROS, and inhibited MPO activity. MTT [3‐(4,5‐dimethyl‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay showed no cytotoxicity in mouse macrophages (RAW264.7 cell), human myeloid cells (HL60), and human fetal lung fibroblast cell line (MRC‐5). Furthermore, the antioxidative mechanism of FMP was evaluated by protein expression levels of antioxidant enzyme (superoxide dismutase [SOD] and glutathione [GSH]) using Western blot. The results obtained in the present study indicated that FMP is a potential source of natural antioxidant.     

ATM mediates interdependent activation of p53 and ERK through formation of a ternary complex with p-p53 and p-ERK in response to DNA damage

DNA damage in eukaryotic cells induces signaling pathways mediated by the ATM, p53 and ERK proteins, but the interactions between these pathways are not completely known. To address this issue, we performed a time course analysis in human embryonic fibroblast cells treated with DNA-damaging agents. DNA damage induced the phosphorylation of p53 at Ser 15 (p-p53) and the phosphorylation of ERK (p-ERK). Inhibition of p53 by a dominant negative mutant or in p53(-/-) fibroblast cells abolished ERK phosphorylation. ERK inhibitor prevented p53 phosphorylation, indicating that phosphorylations of p53 and p-ERK are interdependent each other. A time course analysis showed that ATM interacted with p-p53 and p-ERK in early time (0.5 h) and interaction between ATM-bound p-p53 and p-ERK or ATM-bound p-ERK and p-p53 occurred in late time (3 h) of DNA damage. These results indicate that ATM mediates interdependent activation of p53 and ERK through formation of a ternary complex between p-p53 and p-ERK in response to DNA damage to cause growth arrest.     

Genetic variants of the <Emphasis Type="Italic">FABP4</Emphasis> gene are associated with marbling scores and meat quality grades in Hanwoo (Korean cattle)

This study was aimed to search new genetic variants in the bovine FABP4 gene as molecular markers for meat quality and carcass traits. PCR–RFLP analysis revealed that three SNPs located at nucleotide positions g.2834C>G, g.3533T>A, and g.3691G>A were identified based on a GenBank accession number (NC_007312.4). Sequence analysis revealed that SNPs were located in intron 1 (g.2834C>G) and 2 (g.3533T>A), and an exon 3 (g.3691G>A), showing allele frequencies as 0.592, 0.579, and 0.789, respectively. Genetic variabilities of heterozygosity (He) and polymorphic information contents (PIC) were estimated for g.2834C>G (0.608 and 0.531), g.3533T>A (0.615 and 0.539), and g.3691G>A (0.498 and 0.401) loci, respectively. A SNP located in the exon 3 of FABP4 was characterized and associated with desirable increases of MS (marbling scores) and MG (meat quality grades) in Hanwoo. The statistical analysis revealed that additive effects by GG genotypes in g.3691G>A SNP were significantly greater than AA genotypes in MS and MG traits. These findings suggest that the FABP4g.3691G>A SNP will be a useful candidate locus to maximize economic benefits for cattle populations.