Signaling pathways involved in liver injury and regeneration in rabbit hemorrhagic disease, an animal model of virally-induced fulminant hepatic failure

Management of fulminant hepatic failure (FHF) continues to be one challenging problem, and experimental animal models resembling its clinical conditions are still needed. Rabbit hemorrhagic disease (RHD) fullfils many requirements of an animal model of FHF. This work investigated changes in MAPK, NF-κB, AP-1 and STAT pathways during RHD-induced liver injury. Rabbits were infected with 2 × 10⁴ hemagglutination units of an RHD virus isolate. Apoptosis was documented by the presence of caspase-3 activity and substantial PARP proteolysis at 36 and 48 h postinfection (pi). Infection induced a marked and maintained expression of TNF-α from 12 h pi, while there was only a transitory increase in IL-6 expression. Expression of phosphorylated (p)-JNK, p-p38 and p-ERK1/2 was significantly elevated at 12 h pi. At 48 h pi p-JNK expression was maintained at a maximum level, while that of p-p38 returned to normality and there was no p-ERK1/2 expression. Activation of NF-κB and AP-1 and increased expression of VCAM-1 and COX-2 were observed. No significant changes were detected in activation of STAT1 and STAT3, while SOCS3 expression increased significantly. The current findings suggest that activation of JNK is an essential component in liver injury mediated by the RHD virus and that lack of activation of STAT3, probably mediated by SOCS3 over-expression, would contribute to the inhibition of the regenerative response. Data show the presence of molecular mechanisms contributing to liver damage and the lack of regeneration and they support the usefulness of this model to investigate novel therapeutical modalities in FHF.     

Activated Vitamin D3 and Pro-activated Vitamin D3 Attenuate Induction of Permanent Changes Caused by Neonatal Estrogen Exposure in the Mouse Vagina

Exposure of mice to a high dose of estrogens including diethylstilbestrol (DES) during the neonatal period modifies the developmental plan of the genital tract, which leads to various permanent changes in physiology, morphology and gene expression. These changes include development of an abnormal vaginal epithelium lined with hyperplastic mucinous cells accompanied by Tff1 gene expression in mice. Here, the influence of vitamin D on the direct effect of estrogen on the developing mouse vagina was examined. The mid-vagina of neonatal mice was cultured in a serum-free medium containing estradiol-17β (E₂) and various concentrations of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D) ex vivo and then was transplanted under the renal capsule of ovariectomized host mice for 35 days. Exposure to E₂ alone caused the vaginal tissue to develop estrogen-independent epithelial hyperplasia and to express TFF1 mRNA, while addition of a low nanomolar amount of 1,25(OH)₂D added at the same time as E₂ to the culture medium attenuated the effects of estrogen. Expression of vitamin D receptor was also evident in the neonatal mouse vagina. Interestingly, addition of 25-hydroxyvitamin D₃, a pro-activated form of vitamin D, at the micromolar level was found to be potent in disrupting the developmental effects of E₂, while cholecalciferol was not at least at the dose examined. Correspondingly, expression of Cyp27B1, a kidney-specific 25-hydroxyvitamin D hydroxylase, was evident in the neonatal mouse vagina when examined by RT-PCR. In addition, simultaneous administration of 1,25(OH)₂D successfully attenuated DES-induced ovary-independent hyperplasia in the vagina in neonatal mice in vivo. Thus, manipulation of vitamin D influenced the harmful effects of estrogens on mouse vaginal development.     

Molecular characterization of a 13-amino acid deletion in VP1 (1D) protein and novel amino acid substitutions in 3D polymerase protein of foot and mouth disease virus subtype A/Iran87

The nucleotide sequence of the VP1 (1D) and partial 3D polymerase (3D^pol) coding regions of the foot and mouth disease virus (FMDV) vaccine strain A/Iran87, a highly passaged isolate (~150 passages), was determined and aligned with previously published FMDV serotype A sequences. Overall analysis of the amino acid substitutions revealed that the partial 3D^pol coding region contained four amino acid alterations. Amino acid sequence comparison of the VP1 coding region of the field isolates revealed deletions in the highly passaged Iranian isolate (A/Iran87). The prominent G-H loop of the FMDV VP1 protein contains the conserved arginine-glycine-aspartic acid (RGD) tripeptide, which is a well-known ligand for a specific cell surface integrin. Despite losing the RGD sequence of the VP1 protein and an Asp₂₆→Glu substitution in a beta sheet located within a small groove of the 3D^pol protein, the virus grew in BHK 21 suspension cell cultures. Since this strain has been used as a vaccine strain, it may be inferred that the RGD deletion has no critical role in virus attachment to the cell during the initiation of infection. It is probable that this FMDV subtype can utilize other pathways for cell attachment.     

乳牛口服大量β-胡萝卜素对维生素D3及钙磷代谢的影响

６头健康成年荷斯坦乳牛被随机均分为２组。试验组牛第１周每头口服β－胡萝卜素２３．０ｇ,以后每周每头口服１１．５ｇ,对照组不做任何处理。试验期共８周。结果表明,试验组乳牛血清钙及无机磷水平呈下降趋势;血清维生素Ｄ３活性代谢产物２５－ＯＨ－Ｄ３和１,２５－（ＯＨ）２－Ｄ３均呈现明显下降;至第５周末,血液β－胡萝卜素水平最高,２５－ＯＨ－Ｄ３水平下降非常明显。据此认为,乳牛对β－胡萝卜素的吸收存在一阈值,大剂量的β－胡萝卜素进入机体,可能直接干扰维生素Ｄ３的羟化,使其活性代谢产物呈下降趋势,但由于吸收阈值的存在,其对维生素Ｄ３和骨骼钙磷代谢的干扰作用可得到一定程度的减缓。     

The localization and function of p38α mitogen‐activated protein kinase in rat oocytes

P38α mitogen‐activated protein kinase (MAPK), which is a member of the canonical MAPK family, is activated in response to various extracellular stresses and plays a role in multiple cellular processes. In this study, we investigated the expression, subcellular localization and functional roles of p38α MAPK during the meiotic maturation of rat oocytes. We found that p38α MAPK phosphorylation (p‐p38α MAPK, indicative of p38α MAPK activation) was low at the germinal vesicle (GV) stage, increased 3 hr after germinal vesicle breakdown (GVBD) and maintained its maximum at metaphase I (MI) or metaphase II (MII). The p‐p38α MAPK mainly accumulated in the GV and had no obvious expression in the nucleus. From GVBD to MII, p‐p38α MAPK was distributed in the cytoplasm around either the chromosomes or the spindle. We used SB203580, an inhibitor of p38α MAPK, to investigate the possible functional role of p38α MAPK during rat oocyte meiotic maturation. Treatment of GV stage oocytes with 20 μM SB203580 blocked p‐p38α MAPK activity, and the spindles appeared abnormal. Additionally, the rate of GVBD after 3 hr of culture with 20 μM SB203580 (58.8%) was significantly inhibited compared with the control (82.5%, p < .05), and the polar body extrusion rate after 12 hr of culture with SB203580 was also significantly decreased compared with the control (40.1% vs 73.3%, p < .05). Taken together, these data indicate that p38α MAPK may play a vital role in rat oocyte meiotic maturation.     

Involvement of the ERK1/2 MAPK pathway in insulin-induced S6K1 activation in avian cells

In mammals, insulin regulates S6K1, a key enzyme involved in the control of protein synthesis, via the well-documented phosphoinositide-3'kinase (PI3K) pathway. Conversely, S6K1 is activated by insulin in avian muscle despite the relative insulin insensitivity of the PI3K pathway in this tissue. Mitogen-activated protein kinase (MAPK) cascade is another insulin sensitive pathway. The aim of this study was to explore the potential involvement of the ERK1/2 MAPK pathway in the control of p70 S6 kinase (S6K1) in avian species. Firstly, we characterized ERK1/2 MAPK in various chicken tissues. ERK2 was the only isoform detected in avian species whatever the tissue studied. We also showed that ERK2 is activated in vivo by insulin in chicken muscle. The regulation and the role of ERK2 in insulin signaling were next investigated in chicken hepatoma cells (LMH) and primary myoblasts. Insulin stimulation led to ERK2 and S6K1 phosphorylation, and concomitantly increased kinase activity. U0126, an inhibitor of the ERK MAPK pathway, completely abolished insulin-induced S6K1 phosphorylation and activity in chicken myoblasts, whereas its effect was only partial in LMH cells. In conclusion, these results show that ERK1/2 MAPK is involved in the control of S6K1 by insulin in chicken cells, particularly myoblasts.     

Regulation of protein metabolism by insulin: value of different approaches and animal models

Insulin induces protein accretion by stimulating protein synthesis and inhibiting proteolysis. However, the mechanisms of regulation of protein metabolism by insulin are complex and still not completely understood. The use of approaches combining hyperinsulinemic clamp and isotopic methods, or measurement of the activation of intracellular kinases involved in insulin signaling, in addition to the use of different animal models in a comparative physiology process, provide better understanding of the potential regulation of protein metabolism by insulin. Studies using the clamp technique in lactating goats have shown a clear inhibitory effect of insulin on proteolysis, with an interaction between the effects of insulin and amino acids. Such studies revealed that the insulin-inhibited proteolysis is improved in lactating goats, this adaptative process limiting the mobilization of body protein under the conditions of amino acid deficit which occurs during early lactation. Insulin signaling studies in growing chickens have also provided some interesting features of insulin regulation compared to mammals. Refeeding or insulin injection leads to the activation of the early steps of insulin receptor signaling in the liver but not in the muscle. Muscle p70 S6 kinase, a kinase involved in the insulin activation of protein synthesis, was found to be markedly activated in response to insulin and to refeeding, suggesting that other signaling pathways than those classically described in mammalian muscles may be involved in signal transduction. Finally, although the role of insulin has been doubtful and has long been considered to be minor in ruminants and in avian species, this hormone clearly regulates protein metabolism in both species.     

Role of uncoupling protein 1 in the anti-obesity effect of beta3-adrenergic agonist in the dog

We have reported that chronic treatment with beta3-adrenoceptor agonists reduces body fat content and induces the expression of mitochondrial thermogenic uncoupling protein 1 (UCP1) in adipose tissue in the dog. To evaluate the role of UCP1 in the anti-obesity effect of the agonists, we isolated adipocytes from subcutaneous fat pad of beagles before and after a 2-week treatment with AJ-9677, a specific beta3-adrenoceptor agonist, and examined their thermogenic activity in vitro. Histological and protein analysis revealed that adipose tissues before the treatment were composed of unilocular cells filled with a single large droplet, while the tissues after the treatment contained many smaller and some multilocular adipocytes expressing UCP1 and abundant mitochondrial proteins. Before the treatment, oxygen consumption rate was very low and did not change even when the cells were stimulated by AJ-9677. Two-week AJ-9677 treatment increased basal oxygen consumption rate by 7-fold, and produced a clear responsiveness to AJ-9677 stimulation. Thus, chronic treatment with AJ-9677 induced UCP1 in adipocytes, where oxygen consumption increased in response to AJ-9677 stimulation. It was suggested that UCP1-dependent energy expenditure in adipose tissue contributes to the anti-obesity effect of beta3-adrenoceptor agonist in dogs.     

Refeeding and insulin activate the AKT/p70S6 kinase pathway without affecting IRS1 tyrosine phosphorylation in chicken muscle

p70 S6 kinase (p70S6K) is a key enzyme involved in the control of protein synthesis. We have previously shown that this kinase is insulin sensitive in chicken muscle despite a relative insulin resistance in the early steps of insulin receptor signaling in this tissue, particularly with no change in tyrosine phosphorylation of the insulin receptor substrate 1 (IRS1). The aim of the present study is to further study the p70S6K pathway in chicken muscle. By analyzing in silico several kinases involved in the protein kinase B (PKB also called AKT)/target of rapamycin (TOR)/p70S6K pathway in the chicken, we showed that the amino acid sequence of the proteins exhibited a very high identity with their homologs in mammalian species and Drosophila. We investigated the regulation of these kinases in vivo or in vitro. Refeeding and insulin treatment significantly (P<0.05) increased the phosphorylation and/or activity of kinases upstream of p70S6K such as AKT and TOR. Similarly, refeeding and insulin increased the phosphorylation of p70S6K on key residues (i.e. T389, T229 and T421/S424) and the phosphorylation of a p70S6K downstream target, the ribosomal protein S6 (by 3-10-fold, P<0.05). Interestingly, we also showed an increase in the phosphorylation level of IRS1 on S632/S635, sites involved in insulin resistance. In conclusion, the AKT/TOR/p70S6K pathway is activated by refeeding and insulin injection, which might negatively regulate IRS1 tyrosine phosphorylation. These results indicate some particularities of the insulin signaling in chicken muscle and suggest the involvement of p70S6K in these features.     

Changes in the C-reactive protein and 13,14-dihydro-15-keto-prostaglandin F2α concentrations of uterine lavage samples after the administration of povidone-iodine in cows

Changes in the C-reactive protein (CRP) and 13,14-dihydro-15-keto-prostaglandin F_2α (PGFM) concentrations of uterine lavage fluid were examined in cows given an intrauterine povidone-iodine (PI) infusion. The mean polymorphonuclear leukocyte (PMN) ratios (the ratio of PMN to total cells) and CRP concentration of uterine lavage fluid on the day after the treatment were significantly (P<0.05) greater in the PI infusion group (PMN: 53.0 ± 32.7%, CRP: 50.2 ± 32.3 ng/mL) than in the non-treatment control group (PMN: 7.9 ± 21.9%, CRP: 17.2 ± 5.9 ng/mL), whereas there was no significant difference in the mean PGFM concentration between the two groups. The present findings suggest that the uterine CRP level is a useful biomarker of local uterine inflammation in cows.     

Variation in the expression of Hsp27, αB-crystallin mRNA and protein in heart and liver of pigs exposed to different transport times

Twenty pigs were randomly divided into four groups of five pigs each (not transported – control, 1, 2 and 4 h of transportation). A significant increase of ALT, AST and CK in the blood serum and acute parenchyma cell lesions were observed and those were characterized by acute degenerations in the heart and liver. Hsp27 expression levels increased significantly in the heart after 2 h and in the liver after 4 h of transportation, accompanying with the hsp27 mRNA increasing significantly in the heart and liver after 1 h of transportation. αB-crystallin expression levels were fluctuant (not significantly) in the heart and liver during transporting, however, αB-crystallin mRNA increase notably in the heart after 1 h and decrease significantly in the liver at 1 and 2 h of transportation, respectively. In conclusion, the cellular damage to the heart and liver is highest after 1 h of transportation, Hsp27 and αB-crystallin play dissimilar roles and show tissue-specific response in different tissues during transportation.     

The canine prostate cancer cell line CHP‐1 shows over‐expression of the co‐chaperone small glutamine‐rich tetratricopeptide repeat‐containing protein α

Although androgen therapy resistance and poor clinical outcomes are seen in most canine prostate cancer cases, there are only a few tools for analysing canine prostate cancer by using a cell biological approach. Therefore, to evaluate androgen‐independent neoplastic cell growth, a new canine prostate cancer cell line (CHP‐1) was established in this study. CHP‐1 over‐expressed the co‐chaperone small glutamine‐rich tetratricopeptide repeat‐containing protein α (SGTA), which is over‐expressed in human androgen‐independent prostate cancer. The CHP‐1 xenograft also showed SGTA over‐expression. Although CHP‐1 shows poor androgen receptor (AR) signalling upon dihydrotestosterone stimulation, forced expression of AR enabled evaluation of AR signalling. Taken together, these results suggest that CHP‐1 will be a useful model for investigating the pathogenesis of androgen‐dependent and androgen‐independent canine prostate cancer.