Comparison of the molecular effects of the mycotoxins beta-zearalenol and deoxynivalenol in porcine endometrial cells--a review

The mycotoxins beta-zearalenol (beta-ZOL) and deoxynivalenol (DON) produce toxic effects that result in diseases in humans and animals. The molecular mechanisms that control the mycotoxin-mediated effects are far from being completely understood. Various results show that these mycotoxins could inhibit cell proliferation. In the present short communication, the influence of beta-ZOL and DON on the abundance and phosphorylation state of kinases that are included in regulation of the initiation of mRNA translation (which is correlated with cell proliferation) was compared in porcine endometrial cells (PEC). Our results indicate that these mycotoxins modulate the expression and phosphorylation of these factors in a different manner. Whereas beta-ZOL mainly had an impact on the biological activity of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), protein kinase B (Akt), eukaryotic initiation factor 4E (eIF4E) and its repressor 4E binding protein 1 (4E-BP1), DON reduced the abundance of p38 MAPk, Akt and specific 4E-BP1 bands. In summary, these results indicate that beta-ZOL influences molecular events that are included in the initiation of mRNA translation in the porcine endometrium but DON does not alter such processes clearly.     

The Embryonic Pregnancy Signal Oestradiol Influences Gene Expression at the Level of Translational Initiation in Porcine Endometrial Cells

In the pig, conceptus-derived oestrogens (days 11 and 12 of pregnancy) seem to be a critical component of the signalling mechanism for maternal recognition of pregnancy. Embryonic oestrogens can mediate effects on endometrial function by interactions with epithelial and stromal oestrogen receptors (ER). Recent data demonstrate that cell membrane ER interacts with the phosphatidylinositol 3-kinase/Akt pathway in several types of cells. The protein kinase Akt is involved in the control of cell growth, survival and proliferation. One distinct function of the Akt signalling cascade is its ability to phosphorylate the eukaryotic initiation factor-4E (eIF-4E)-binding protein 1 (4E-BP1). This phosphorylation suppresses the inhibitory effect of 4E-BP1 on the translation initiation factor eIF4E and in such a way potentially stimulates gene expression at the level of translational initiation. The aim of the present study was to examine if embryonic oestradiol (E(2)) transmits its effect by such a mechanism. Endometrial cells of cyclic gilts (day 13 of the oestrous cycle, n = 4) were cultured and supplemented with vehicle (control), E(2) (50 and 100 pm/l) or with the selective ER modulator raloxifen (10 and 1000 nm/l), and incubated for 24 h. The cell viability was detected by MTT assay, the abundance and phosphorylation of Akt, 4E-BP1 and ERalpha was analysed by Western blotting. Incubation with E(2) or raloxifen did not alter endometrial cell viability. The phosphorylation of Akt at Ser(473) seems to be increased by E(2) (p < 0.05) and decreased by raloxifen (p > 0.05). Raloxifen (1000 nm/l) induced a band shift in 4E-BP1 to the highest electrophoretic mobility which reflects a decrease in phosphorylation (p < 0.05), whereas an influence of E(2) on 4E-BP1 phosphorylation could not be detected. The decrease (p < 0.05) of the abundance of the 80 kDa ERalpha form both by E(2) and raloxifen indicates that the E(2)-stimulated Akt phosphorylation and the inhibition of 4E-BP1 phosphorylation by raloxifen is an E(2) ER-transmitted process. Therefore, embryonic oestrogens can potentially transmit their effect by influencing signalling cascades which modulate gene expression at the level of translational initiation.     

IGF-1 stimulates protein synthesis by enhanced signaling through mTORC1 in bovine mammary epithelial cells

Using the MAC-T cell line as a model, the effects of insulin-like growth factor (IGF)-1 on the regulation of protein synthesis through the mammalian target of rapamycin complex 1 (mTORC1) signaling in bovine mammary epithelial cells were evaluated. Global rates of protein synthesis increased by 47% within 30 min of IGF-1 treatment. The effect of IGF-1 on protein synthesis was associated with enhanced association of the eukaryotic initiation factor (eIF) 4E with eIF4G and a concomitant reduction of eIF4E association with eIF4E-binding protein-1 (4E-BP1). There was a progressive increase in the phosphorylation state of ribosomal protein S6 kinase-1, a downstream target of mTORC1 in response to IGF-1. In addition, IGF-1 stimulated mTORC1 kinase activity toward 4E-BP1 in vitro. Phosphorylation on Ser473 of Akt was induced by IGF-1 within 5 min and remained elevated throughout a 30-min time course. The effect of IGF-1 on Akt phosphorylation was also concentration dependent. Activation of Akt by IGF-1 led to increased phosphorylation of tuberous sclerosis complex 2 on Thr1426, without any change in its association with tuberous sclerosis complex 1. Phosphorylation of proline-rich Akt substrate of 40-kDa (PRAS40) at Thr246 was stimulated by IGF-1. The amount of PRAS40 associated with mTORC1 decreased in response to IGF-1, and PRAS40 binding to mTORC1 was inversely related to its phosphorylation level. Overall, these results suggest that activation of the PI3K-Akt pathway by IGF-1 stimulated global protein synthesis in bovine mammary epithelial cells through changes in the phosphorylation and association state of components of the mTORC1 signaling pathway.     

槲皮素对猪肠上皮细胞利用蛋白质的作用机制

本试验旨在研究槲皮素促进猪肠上皮细胞利用蛋白质的作用及机制。猪肠上皮细胞孵育48 h后试验组分别用含0.1、0.2、0.4、0.8和1.6 mg/L槲皮素的二甲基亚砜(DMSO)溶液处理72 h,对照组采用0.2%DMSO处理。采用二喹啉甲酸(BCA)测定受试细胞中蛋白质的含量;采用实时荧光定量PCR(RT-qPCR)法测定氨基酸和小肽转运载体以及哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关基因的mRNA相对表达量;采用Western blot法测定mTOR信号通路相关基因的蛋白表达。结果表明:与对照组相比,1)0.4和0.8 mg/L槲皮素均极显著增加猪肠上皮细胞中蛋白质的含量(P<0.01)。2)1.6 mg/L槲皮素极显著提高猪肠上皮细胞中兴奋性氨基酸转运载体1(EAAC1)、谷氨酰胺载体2(ASCT2)、氨基酸转运载体A2(ATA2)、L型氨基酸转运载体2(LAT2)、阳离子氨基酸转运载体1(CAT1)、b 0,+系统氨基酸转运载体(rBAT)、y+L系统氨基酸转运载体1(y+LAT1)、y+L系统氨基酸转运载体2(y+LAT2)和寡肽转运载体1(PepT1)mRNA相对表达量(P<0.01)。3)0.4 mg/L槲皮素极显著降低猪肠上皮细胞中结节性硬化复合物1(TSC1)mRNA相对表达量(P<0.01);0.8 mg/L槲皮素极显著增加mTOR和核糖体蛋白S6(RPS6)mRNA相对表达量并极显著降低TSC1 mRNA相对表达量(P<0.01);1.6 mg/L槲皮素极显著增加mTOR、真核起始因子4E结合蛋白1(4E-BP1)、真核细胞翻译起始因子4E(eIF4E)、真核细胞翻译起始因子4B(eIF4B)、真核细胞翻译起始因子4A(eIF4A)和RPS6 mRNA相对表达量(P<0.01)。4)0.1和1.6 mg/L槲皮素极显著提高猪肠上皮细胞中mTOR、eIF4E和eIF4A蛋白表达量并极显著降低4E-BP1蛋白表达量(P<0.01)。由此可见,槲皮素可通过调控氨基酸转运载体、小肽转运载体及mTOR信号通路相关基因的表达来促进猪肠上皮细胞对蛋白质的利用。     

The effect of dietary protein levels on the expression of genes coding for four selected protein translation initiation factors in muscle tissue of Wujin pig

The objective of this study was to investigate the regulatory mechanism underlying the increased muscle protein accumulation in pigs while were fed a high protein diet. The eukaryotic initiation factors (eIFs) have been reported to involve in muscle protein synthesis. We investigated the mRNA and protein expression levels of eIF2B1, 4A1, 4B and 4E in Wujin pigs fed either a high protein (HP: 18%) or a low protein (LP: 14%) diet at 30, 60 or 100 kg body weight, based on real‐time PCR and western blotting analyses. Our results indicated that the expression levels of eIF2B1 mRNA and protein were increased by HP diet at all body weight. The HP diet showed higher mRNA and protein levels of eIF4B gene at 60 and 100 kg. The protein expression of eIF4E phosphorylation was increased by HP diet only at 30 kg. These data suggested that the HP diet promoted porcine muscle protein accumulation mainly by up‐regulating eIF2B1, 4B and 4E rather than 4A1 expression along the growth stages.     

Inhibition of host protein synthesis in B95a cells infected with the HL strain of measles virus

The shut-off of host protein synthesis in virus-infected cells is one of the important mechanisms for viral replication. In this report, we showed that the HL strain of measles virus (MeV-HL) as well as other field isolates, which were isolated from human blood lymphocytes using B95a cells, induce the shut-off in B95a cells. Since the Edmonston strain of MeV failed to induce the shut-off in B95a cells, the ability to induce the shut-off was considered to be dependent on virus strains. Although, the modification of eukaryotic translation initiation factors (eIF) including eIF4G, eIF4E, and 4E-BP1 was reported for shut-off by various viruses, the involvement of these eIFs was not observed in MeV-HL-infected B95a cells. Instead, the accumulation of phosphorylated eIF2alpha was found to coincide to the decrease of host protein synthesis, suggesting the involvement of phosphorylation of eIF2alpha in inhibition of translation as one of the mechanisms of the shut-off.     

Profiles of maternal origin factors during transition from embryonic diapause to implantation in roe deer

The aim was to evaluate in female roe deer: (a) PAG mRNA relative abundance in endometrial uterine tissue for determination of the duration of embryonic diapause, (b) mRNA relative abundance of progesterone, estradiol, and prolactin (P4, E2, and PRL) receptors (PGR, ESR, and PRLR) during diapause and after implantation in the endometrium; (c) concentration of P4, E2, and PRL in the blood, and (d) a noninvasive method of hormone detection by measurement of P4 and E2 concentrations in feces. A total of fifteen individuals were obtained post mortem during hunting seasons and divided into three experimental groups (November, December, January). The results did not reveal mRNA relative abundance for PAGs in the endometrium or detectable PAG concentrations in the serum of all examined females. Concentration of PRL and mRNA relative abundance for PRLR long isoform in the endometrium was the highest in January (p < .01). mRNA relative abundance for PGR, P4 concentration in the endometrium, serum, and feces was the highest in January (p < .01). Endometrial origin PRL and P4 may be responsible for the termination of this process and pregnancy development after implantation.     

Differential regulation of protein synthesis in skeletal muscle and liver of neonatal pigs by leucine through an mTORC1-dependent pathway

Neonatal growth is characterized by a high protein synthesis rate that is largely due to an enhanced sensitivity to the postprandial rise in insulin and amino acids, especially leucine. The mechanism of leucine’s action in vivo is not well understood. In this study, we investigated the effect of leucine infusion on protein synthesis in skeletal muscle and liver of neonatal pigs. To evaluate the mode of action of leucine, we used rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) complex-1 (mTORC1). Overnight-fasted 7-day-old piglets were treated with rapamycin for 1 hour and then infused with leucine (400 μmol·kg -1 ·h -1 ) for 1 hour. Leucine infusion increased the rate of protein synthesis, and ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) phosphorylation in gastrocnemius and masseter muscles (P < 0.05), but not in the liver. The leucine-induced stimulation of protein synthesis and S6K1 and 4E-BP1 phosphorylation were completely blocked by rapamycin, suggesting that leucine action is by an mTORC1-dependent mechanism. Neither leucine nor rapamycin had any effect on the activation of the upstream mTORC1 regulators, AMP-activated protein kinase and protein kinase B, in skeletal muscle or liver. The activation of eIF2a and elongation factor 2 was not affected by leucine or rapamycin, indicating that these two pathways are not limiting steps of leucine-induced protein synthesis. These results suggest that leucine stimulates muscle protein synthesis in neonatal pigs by inducing the activation of mTORC1 and its downstream pathway leading to mRNA translation.     

Triennial Growth Symposium: leucine acts as a nutrient signal to stimulate protein synthesis in neonatal pigs

The postprandial increases in AA and insulin independently stimulate protein synthesis in skeletal muscle of piglets. Leucine is an important mediator of the response to AA. We have shown that the postprandial increase in leucine, but not isoleucine or valine, acutely stimulates muscle protein synthesis in piglets. Leucine increases muscle protein synthesis by modulating the activation of mammalian target of rapamycin (mTOR) complex 1 and signaling components of translation initiation. Leucine increases the phosphorylation of mTOR, 70-kDa ribosomal protein S6 kinase-1, eukaryotic initiation factor (eIF) 4E-binding protein-1, and eIF4G; decreases eIF2α phosphorylation; and increases the association of eIF4E with eIF4G. However, leucine does not affect the upstream activators of mTOR, that is, protein kinase B, adenosine monophosphate-activated protein kinase, and tuberous sclerosis complex 1/2, or the activation of translation elongation regulator, eukaryotic elongation factor 2. The action of leucine can be replicated by α-ketoisocaproate but not by norleucine. Interference by rapamycin with the raptor-mTOR interaction blocks leucine-induced muscle protein synthesis. The acute leucine-induced stimulation of muscle protein synthesis is not maintained for prolonged periods, despite continued activation of mTOR signaling, because circulating AA fall as they are utilized for protein synthesis. However, when circulating AA concentrations are maintained, the leucine-induced stimulation of muscle protein synthesis is maintained for prolonged periods. Thus, leucine acts as a nutrient signal to stimulate translation initiation, but whether this translates into a prolonged increase in protein synthesis depends on the sustained availability of all AA.     

Contribution of MX dynamin, oligoadenylate synthetase, and protein kinase R to anti-paramyxovirus activity of type 1 interferons in vitro

OBJECTIVE: To determine the contribution of MX dynamin, oligoadenylate synthetase (OAS), and double-stranded RNA-dependent protein kinase R (PKR) to the antiviral effects of type 1 interferons (IFNs) against bovine parainfluenza-3 virus (PI-3V) infection of Vero cells. SAMPLE POPULATION: Vero cell cultures. PROCEDURES: PI-3V yield was first compared between control and transfected type 1 IFNs-incompetent Vero cells expressing recombinant OAS or MX proteins. Afterwards, phosphorylation of eukaryotic initiation factor 2 alpha (eIF2alpha) was used to scale the degree of PKR activation upon infection of Vero cells by PI-3V. RESULTS: Overexpression of OAS did not result in significantly decreased viral replication. Phosphorylated eIF2alpha forms, the hallmark of PKR activation, were not increased in IFNalpha-primed infected Vero cells. Although human MXA contributed to partial blockade of replication of bovine PI-3V, the antiviral effect was not as strong as that of IFNalpha. CONCLUSIONS AND CLINICAL RELEVANCE: The powerful anti-Paramyxovirus activity of type 1 IFNs is mediated by noncanonic pathways.     

Apoptosis in the porcine uterine endometrium during the estrous cycle, early pregnancy and post partum

The mammalian uterus changes dramatically during the estrous cycle, pregnancy, and involution post partum. Dynamic changes in the uterine endometrium are a type of homeostasis and proceed with proliferation and exclusion of cells. Homeostasis of the uterus is closely related to apoptosis involving various hormones and cytokines. The objective of the present study was to determine the morphological features and occurrence of apoptosis in the porcine endometrium during the estrous cycle, early pregnancy, and post partum. Cyclic changes in the morphology of the surface epithelium were observed during the estrous cycle. The heights of surface epithelia were significantly high on day 4 of the estrous cycle and the early pregnancy. The heights of the surface epithelium remained low from days 1 to 31 post partum. We then used terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling (TUNEL) of the 3'-terminal of fragmented DNA, which is effective for detection of apoptosis in various tissues. We found that apoptosis in the porcine endometrium contributed to homeostasis of the endometrium during the estrous cycle through control of cell proliferation and exclusion. Conversely, apoptosis on days 4 and 8 of gestation before the implantation window depended on the plasma estrogen and progesterone levels; however, suppressive homeostasis of apoptosis occurred at the time of implantation on days 15, 18 and 21 of gestation. Our study is the first to demonstrate apoptotic cell death in the porcine endometrium directly by TUNEL method. The results strongly suggest that uterine homeostasis is mainly controlled by apoptosis during the estrous cycle and early pregnancy.     

Regulation of protein degradation pathways by amino acids and insulin in skeletal muscle of neonatal pigs

Background

The rapid gain in lean mass in neonates requires greater rates of protein synthesis than degradation. We previously delineated the molecular mechanisms by which insulin and amino acids, especially leucine, modulate skeletal muscle protein synthesis and how this changes with development. In the current study, we identified mechanisms involved in protein degradation regulation. In experiment 1, 6- and 26-d-old pigs were studied during 1) euinsulinemic-euglycemic-euaminoacidemic, 2) euinsulinemic-euglycemic-hyperaminoacidemic, and 3) hyperinsulinemic-euglycemic-euaminoacidemic clamps for 2 h. In experiment 2, 5-d-old pigs were studied during 1) euinsulinemic-euglycemic-euaminoacidemic-euleucinemic, 2) euinsulinemic-euglycemic-hypoaminoacidemic-hyperleucinemic, and 3) euinsulinemic-euglycemic-euaminoacidemic-hyperleucinemic clamps for 24 h. We determined in muscle indices of ubiquitin-proteasome, i.e., atrogin-1 (MAFbx) and muscle RING-finger protein-1 (MuRF1) and autophagy-lysosome systems, i.e., unc51-like kinase 1 (UKL1), microtubule-associated protein light chain 3 (LC3), and lysosomal-associated membrane protein 2 (Lamp-2). For comparison, we measured ribosomal protein S6 (rpS6) and eukaryotic initiation factor 4E (eIF4E) activation, components of translation initiation.

Results

Abundance of atrogin-1, but not MuRF1, was greater in 26- than 6-d-old pigs and was not affected by insulin, amino acids, or leucine. Abundance of ULK1 and LC3 was higher in younger pigs and not affected by treatment. The LC3-II/LC3-I ratio was reduced and ULK1 phosphorylation increased by insulin, amino acids, and leucine. These responses were more profound in younger pigs. Abundance of Lamp-2 was not affected by treatment or development. Abundance of eIF4E, but not rpS6, was higher in 6- than 26-d-old-pigs but unaffected by treatment. Phosphorylation of eIF4E was not affected by treatment, however, insulin, amino acids, and leucine stimulated rpS6 phosphorylation, and the responses decreased with development.

Conclusions

The rapid growth of neonatal muscle is in part due to the positive balance between the activation of protein synthesis and degradation signaling. Insulin, amino acids, and, particularly, leucine, act as signals to modulate muscle protein synthesis and degradation in neonates.     

Limitations of the Infant Mouse Test for Escherichia coli Heat Stable Enterotoxin

A study was undertaken to evaluate the response of different test systems to preparations of heat-stable enterotoxin (ST) derived from Eschericihia coli strains recovered from diarrheal disease of humans, pigs and calves. Sterile broth culture supernatants of enterotoxigenic strains of E. coli were heated at 65°C for 30 minutes and tested for the presence of heat-stable enterotoxin. Three test systems, namely, ligated intestine of weaned pigs, ligated intestine of rabbits and the infant mouse test were used in attempts to detect ST in the culture supernatants. Two patterns of reaction were observed in response to ST-containing preparations: either the preparation elicited a response in the three tests or the preparation elicited a reaction only in the ligated pig intestine. A response in all three tests were observed for 5/5 human ST-producing E. coli, 5/5 bovine enterotoxigenic E. coli, 5/5 “atypical” porcine enterotoxigenic E. coli, 3/3 St⁺LT^- porcine E. coli of serogroup O138:K81 and 4/24 LT⁺ST⁺ porcine E. coli. A response only in the ligated pig intestine was obtained with 5/5 ST⁺LT^- porcine E. coli belonging to serogroups other than O138:K81 and to 20/24 ST⁺LT⁺ E. coli from pigs. The results are consistent with the view that there are two kinds of ST, one of which (ST1) reacts in all three tests and the other (ST2) which reacts only in the ligated pig intestine. The findings underscore the limitations of the infant mouse test as a means of detecting ST in porcine isolates of E. coli, since the test fails to detect ST produced by a large number of these E. coli strains. There appeared to be a relationship between kind(s) of ST produced and the animal species from which the producing organism was recovered.     

Cloning and expression of bovine and porcine interleukin-2 in baculovirus and analysis of species cross-reactivity

The cDNAs encoding bovine and porcine interleukin-2 (IL-2) have been expressed using the baculovirus Autographa californica nuclear polyhedrosis virus as a vector in insect cells. Insect cells infected with recombinant viruses secreted bovine and porcine IL-2 into the culture medium, with biological activities for maintaining the proliferation of homologous cells. When the activities of these two IL-2 proteins and commercially available human IL-2 were tested on heterologous cells differences were found. Recombinant bovine (rb)IL-2 only supported the growth of bovine lymphocytes and was not active on human, mouse or porcine lymphocytes. Recombinant porcine (rp)IL-2 and recombinant human (rh)IL-2 supported the proliferation of human, bovine, porcine and murine cells. However, the proliferative response of human lymphocytes to rpIL-2 was only 50% of that seen with rhIL-2. Sequence differences at the predicted p55 and p75 contact binding sites may explain this.     

Conservation of deduced amino acid sequence of FimH among Escherichia coli of bovine, porcine and avian disease origin

The FimH subunit of type 1 pili mediates adhesion of Escherichia coli to epithelium in different animal hosts. In this study, we sequenced and analyzed the fimH genes of 24 E. coli strains from bovine and porcine clinical cases. The obtained sequences were compared among each other and also with 24 known fimH sequences from avian E. coli strains. This comparison revealed a substantial homology (>99%) among strains from the different animal species origins. Moreover, specific mutations were found, some of which were present more frequently in avian strains or in bovine and porcine strains.     

Bacterial adherence in mastitis caused by Escherichia coli.

The possible role of bacterial adherence in the pathogenesis of experimental mastitis in the mouse was examined with four strains of Escherichia coli. Two of these strains had a known adhesion antigen (K88) and two did not. The K88 antigen did not play a significant role in the virulence or infectivity of E. coli either in the murine or bovine mammary gland. Two E. coli strains, W1 (K88+) and J2 (K88-) were virulent in the mouse but did not adhere to epithelial cells. Both these strains produced clinical mastitis in the cow. A third strain, D282 (K88-), produced mild disease in the mouse but was avirulent in the cow. The fourth strain, 233/ID (K88+), was avirulent in both the mouse and the cow. Strains D282 and 233/1D were killed rapidly by bovine serum whilst J2 and W1 were more resistant. All strains were more sensitive than the control resistant strain E. coli P4, which is known to be highly virulent for the lactating udder.