Salmonella vector induces protective immunity against Lawsonia and Salmonella in murine model using prokaryotic expression system.

BACKGROUND: Lawsonia intracellularis is the causative agent of proliferative enteropathy and is associated with several outbreaks, causing substantial economic loss to the porcine industry. OBJECTIVES: In this study, we focused on demonstrating the protective effect in the mouse model through the immunological bases of two vaccine strains against porcine proliferative enteritis. METHODS: We used live-attenuated Salmonella Typhimurium (ST) secreting two selected immunogenic LI antigens (Lawsonia autotransporter A epitopes and flagellin [FliC]-peptidoglycan-associated lipoprotein-FliC) as the vaccine carrier. The constructs were cloned into a Salmonella expression vector (pJHL65) and transformed into the ST strain (JOL912). The expression of immunogenic proteins within Salmonella was evaluated via immunoblotting. RESULTS: Immunizing BALB/c mice orally and subcutaneously induced high levels of LI-specific systemic immunoglobulin G and mucosal secretory immunoglobulin A. In immunized mice, there was significant upregulation of interferon-γ and interleukin-4 cytokine mRNA and an increase in the subpopulations of cluster of differentiation (CD) 4+ and CD 8+ T lymphocytes upon splenocytes re-stimulation with LI antigens. We observed significant protection in C57BL/6 mice against challenge with 106.9 times the median tissue culture infectious dose of LI or 2 × 109 colony-forming units of the virulent ST strain. Immunizing mice with either individual vaccine strains or co-mixture inhibited bacterial proliferation, with a marked reduction in the percentage of mice shedding Lawsonia in their feces. CONCLUSIONS: Salmonella-mediated LI gene delivery induces robust humoral and cellular immune reactions, leading to significant protection against LI and salmonellosis.

Knockdown of Y-box binding protein 1 induces autophagy in early porcine embryos.

Y-box binding protein 1 (YBX1) plays important roles in RNA stabilization, translation, transcriptional regulation, and mitophagy. However, its effects on porcine preimplantation embryos remain unclear. In this study, we knocked down YBX1 in the one-cell (1C) stage embryo via small interfering RNA microinjection to determine its function in porcine embryo development. The mRNA level of YBX1 was found to be highly expressed at the four-cell (4C) stage in porcine embryos compared with one-cell (1C) and two-cell (2C) stages. The number of blastocysts was reduced following YBX1 knockdown. Notably, YBX1 knockdown decreased the phosphatase and tensin homolog-induced kinase 1 (PINK1) and parkin RBR E3 ubiquitin protein ligase (PRKN) mRNA levels. YBX1 knockdown also decreased PINK1, active mitochondria, and sirtuin 1 levels, indicating reduced mitophagy and mitochondrial biogenesis. Furthermore, YBX1 knockdown increased the levels of glucose-regulated protein 78 (GRP78) and calnexin, leading to endoplasmic reticulum (ER) stress. Additionally, YBX1 knockdown increased autophagy and apoptosis. In conclusion, knockdown of YBX1 decreases mitochondrial function, while increasing ER stress and autophagy during embryonic development.

Profiling of differentially expressed proteins between fresh and frozen-thawed Duroc boar semen using ProteinChip CM10.

Many studies have been conducted to improve technology for semen cryopreservation in pigs. However, computer-assisted analysis of sperm motility and morphology is insufficient to predict the molecular function of frozen-thawed semen. More accurate expression patterns of boar sperm proteins may be derived using the isobaric tags for relative and absolute quantification (iTRAQ) technique. In this study, the iTRAQ-labeling system was coupled with liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis to identify differentially expressed CM10-fractionated proteins between fresh and frozen-thawed boar semen. A total of 76 protein types were identified to be differentially expressed, among which 9 and 67 proteins showed higher and lower expression in frozen-thawed than in fresh sperm samples, respectively. The classified functions of these proteins included oxidative phosphorylation, mitochondrial inner membrane and matrix, and pyruvate metabolic processes, which are involved in adenosine triphosphate (ATP) synthesis; and sperm flagellum and motile cilium, which are involved in sperm tail structure. These results suggest a possible network of biomarkers associated with survival after the cryopreservation of Duroc boar semen.

Altrenogest affects expression of galectin-3 and fibroblast growth factor 9 in the reproductive tract of sows.

A synthetic progestin altrenogest (ALT) is used to synchronize the estrus cycle of swine for fixed-time artificial insemination (AI) and has been shown to improve follicular development and reproductive performances in post-weaning sows. However, the effects of ALT treatment on reproductive tracts, including the ovaries, oviducts and uterus have not been yet clarified. In this study, we examined the expression of genes involved in endometrial responses in ALT-treated sows. ALT did not significantly alter luteinizing hormone (LH), follicle-stimulating hormone (FSH) and estradiol profiles in blood compared to untreated control. Quantitative RT-polymerase chain reaction (qRT-PCR) analysis showed that the expression of genes encoding galectin-3 (LGALS3) and fibroblast growth factor 9 (FGF9) was upregulated in the reproductive tracts of ALT-treated sows, including the ovaries, oviducts and uteri. Moreover, ALT treatment induced the expression of FGF9 and galectin-3 proteins, and promoted their localization to the luminal epithelium of the oviducts and uterus. Our findings suggest that the enhancement of reproductive performance shown by ALT-treated sows is associated with the upregulation of galectin-3 and FGF9, which are essential for endometrial receptivity, successful implantation, and pregnancy.

Genetic relationship between purebred and synthetic pigs for growth performance using single step method.

OBJECTIVE: The objective of this study was to estimate the genetic correlation (rpc) of growth performance between purebred (Duroc and Korean native) and synthetic (WooriHeukDon) pigs using a single-step method. METHODS: Phenotypes of 15,902 pigs with genotyped data from 1,792 pigs from a nucleus farm were used for this study. We estimated the rpc of several performance traits between WooriHeukDon and purebred pigs: day of target weight (DAY), backfat thickness (BF), feed conversion rate (FCR), and residual feed intake (RFI). The variances and covariances of the studied traits were estimated by an animal multi-trait model that applied the Bayesian inference. RESULTS: rpc within traits was lower than 0.1 for DAY and BF, but high for FCR and RFI; in particular, rpc for RFI between Duroc and WooriHeukDon pigs was nearly 1. Comparison between different traits revealed that RFI in Duroc pigs was associated with different traits in WooriHeukDon pigs. However, the most of rpc between different traits were estimated with low or with high standard deviation. CONCLUSION: The results indicated that there were substantial differences in rpc of traits in the synthetic WooriHeukDon pigs, which could be caused by these pigs having a more complex origin than other crossbred pigs. RFI was strongly correlated between Duroc and WooriHeukDon pigs, and these breeds might have similar single nucleotide polymorphism effects that control RFI. RFI is more essential for metabolism than other growth traits and these metabolic characteristics in purebred pigs, such as nutrient utilization, could significantly affect those in synthetic pigs. The findings of this study can be used to elucidate the genetic architecture of crossbred pigs and help develop new breeds with target traits.

The expression and localization of V-ATPase and cytokeratin 5 during postnatal development of the pig epididymis.

OBJECTIVE: We examined the localization and expression of H+ pumping vacuolar ATPase (V-ATPase) and cytokeratin 5 (KRT5) in the epididymis of pigs, expressed in clear and basal cells, respectively, during postnatal development. METHODS: Epididymides were obtained from pigs at 1, 7, 21, 60, 120, and 180 days of age; we observed the localization and expression patterns of V-ATPase and KRT5 in the different regions of these organs, namely, the caput, corpus, and cauda. The differentiation of epididymal epithelial cells was determined by immunofluorescence labeling using cell-type-specific markers and observed using confocal microscopy. RESULTS: At postnatal day 5 (PND5), the localization of clear cells commenced migration from the cauda toward the caput. Although at PND120, goblet-shaped clear cells were detected along the entire length of the epididymis, those labeled for V-ATPase had disappeared from the corpus to cauda and were maintained only in the caput epididymis in adult pigs. In contrast, whereas basal cells labeled for KRT5 were only present in the vas deferens at birth, they were detected in all regions of the epididymis at PND60. These cells were localized at the base of the epithelium; however, no basal cells characterized by luminally extending cell projections were observed in any of the adult epididymides examined. CONCLUSION: The differentiation of clear and basal cells progressively initiates in a retrograde manner from the cauda to the caput epididymis. The cell-type-specific distribution and localization of the epithelial cells play important roles in establishing a unique luminal environment for sperm maturation and storage in the pig epididymis.

Validation of mouse phosphoprotein enriched in astrocyte 15 (mPEA15) expressing transgenic pig as a potential model in diabetes translational research.

The present study aimed to investigate the characteristics of mPEA15 expressing transgenic pig (TG pig) as a potential model for diabetes. Expression analysis confirmed the ubiquitous expression of mPEA15 in TG pigs at F4. Oral glucose tolerance test results showed that restoration of normal glucose levels was significantly delayed in the TG pigs when compared with that in the wild-type pigs (WT pigs). Primary skeletal muscle cells isolated from TG pigs demonstrated reduced glucose uptake and reduced GLUT4 translocation to the plasma membrane in response to insulin treatment. Combined, these results suggest that mPEA15 expressing pigs has a glucose intolerance and insulin resistance which are known to mediate the pathophysiology of type 2 diabetes mellitus. Thus, mPEA15 transgenic pigs would serve as a promising model for diabetes translational research.

Expression patterns of male germ cell markers in cryptorchid pig testes.

Male germ cell apoptosis has been described in heat-damaged testes by cryptorchidism. In the present study, wild type pig testes were compared with cryptorchid testes via histological and immunohistological analyses. Spermatozoa were not detected in two cryptorchid testes and the diameters of seminiferous tubules were significantly reduced in cryptorchid pig testes compared with wild type pig testes. Cells expressing marker genes for undifferentiated spermatogonia, such as protein gene product 9.5 was significantly decreased in cryptochid pig testes. In addition, the numbers of cells expressing DEAD-box polypeptide 4 (VASA), synaptonemal complex protein 3, protamine, and acrosin (a biomarker of spermatocyte, spermatid, and spermatozoa) were significantly reduced in cryptochid pig testes. However, the number of vimentin-expressing Sertoli cells was not changed or was significantly increased in cryptorchid pig testes. This result indicates that male germ cells are specifically damaged by heat in cryptorchid pig testes and not Sertoli cells. These findings will facilitate the further study of spermatogenesis and the specific mechanisms by which cryptorchidism causes male infertility.

Potential use of transgenic domestic pigs expressing recombinant human erythropoietin in diabetes translation research.

Recently, diabetes mellitus (DM) has shown rapid global increases with about five million deaths annually. Animal models are imperative to understand disease mechanisms and develop diagnostic, preventive, and therapeutic interventions in translational research. Rodent and mini-pig models have been established and widely used for DM research. However, domestic pig models are limited in spite of advantages such as pharmacokinetic and physiopathological availability. This study examines the potential use of domestic pigs expressing recombinant human erythropoietin (rhEPO) as disease and therapeutic response models for DM. We previously generated transgenic pigs (n = 16, EPO Tg) in which rhEPO was expressed and circulated in all organs. Thirty-two pigs, including 16 controls, were fed high fat (HF) diets for 42 weeks. Subsequently, blood samples for chemical and metabolic analysis were collected after fasting for 24 h and glucose loading for oral glucose tolerance tests (OGTTs). We found increased activation of the PI3 K/Akt signaling pathway under hypoxic conditions after rhEPO treatment, and HF diet-inducible-obesity in the EPO Tg and control pigs. OGTTs showed lower fasting glucose levels in the EPO Tg pigs than in controls before and after the HF diet, suggesting that rhEPO may affect glucose concentrations. Insulin and C-peptide concentrations responded slowly to glucose administration and returned to initial levels after 2 h. The blood test results suggest that EPO might affect metabolic and chemical components such as glucose, high-density lipoprotein, glucagon, triglyceride, and free fatty acid. Our findings support the use of rhEPO transgenic domestic pigs as model animals for translational DM research.

Production of human tissue-type plasminogen activator (htPA) using in vitro cultured transgenic pig mammary gland cells.

Tissue plasminogen activator (tPA) is a protein involved in the breakdown of blood clots. We have previously produced a human tPA (htPA)-overexpressing transgenic pig using a mammary gland-specific promoter. In this study, we have established a transgenic pig mammary gland cell line that produces recombinant htPA. The mammary gland cells grew well and retained their character over long periods of culture. There was no difference in the extent of apoptosis in transgenic cells compared to wild-type mammary gland cells. In addition, the transgenic mammary gland cells expressed and secreted htPA into the conditioned media at a concentration similar to that in milk. This transgenic cell line represents a simple and ethical method for recombinant htPA production.

Effects of Sex and Breed on Meat Quality and Sensory Properties in Three-way Crossbred Pigs Sired by Duroc or by a Synthetic Breed Based on a Korean Native Breed.

This study was conducted to determine the effects of breed and sex on meat quality and sensory properties of the loin in three-way crossbred pigs: Landrace× Yorkshire×Duroc (LYD) and Landrace×Yorkshire×Woori (LYW) black pig synthesized by Korean native breed. Carcass traits did not differ by breed. Carcass weight and backfat thickness were higher in castrates than in gilts (p<0.01). LYW showed significant high values in fat content, cooking loss, and water-holding capacity (WHC) than LYD (p<0.05). Redness and yellowness of the meat were higher in LYW than in LYD (p<0.01). Further, LYW had lower pH and shear force than LYD (p<0.001). Significant high scores in color and flavor were obtained in LYW or gilts compared to LYD or castrates by sensory panel, respectively (p<0.05). However, other sensory traits did not differ by breed or sex. Capric acid (C10:0) was higher in LYD than LYW (p<0.001). However, stearic acid (C18:0) and saturated fatty acid (SFA) contents were higher in LYW than LYD (p<0.05). Eicosenoic acid (C20:2) and the n6/n3 ratio were higher in gilts than in castrates, whereas SFA content was higher in castrates than in gilts. These results suggest that certain physicochemical qualities of meat and sensory properties are improved in LYW compared to LYD. This study could provide basic data on meat quality of crossbred pigs with Woori black pig as a terminal sire.

Stage-specific expression of DDX4 and c-kit at different developmental stages of the porcine testis.

Spermatogenesis begins with spermatogonial stem cells (SSCs), which are located in the basement membrane of the adult testes. Previous studies have described specific biomarkers for undifferentiated porcine spermatogonia or SSCs; however, these markers are not sufficient to understand spermatogenesis at different developmental stages. The objective of this study was characterize the expression of DEAD-Box polypeptide 4 (DDX4, also known as VASA) and tyrosine-protein kinase kit (c-kit), as potential markers of male germ cells in the porcine testis. In porcine testis tissue at prepubertal stages (5, 30, and 60 days), DDX4 and c-kit protein expression was detected in the most undifferentiated spermatogonia, which also express protein gene product 9.5 (PGP9.5). However, in porcine testis tissues from pubertal and postpubertal stages (90, 120, and 150 days), DDX4 and c-kit were not detected in PGP9.5-positive undifferentiated spermatogonia. The DDX4 expression pattern was similar to that of c-kit in the porcine testis. In adult porcine testes, DDX4-expressing cells were located on the lumenal side, compared to synaptonemal complex protein 3-positive primary spermatocytes, but DDX-4 was not co-expressed with acrosin, a known acrosome marker. In addition, DDX4 was detected in PGP9.5-expressing porcine SSCs in culture. Based on our results, we suggest that DDX4 and c-kit are putative markers of undifferentiated spermatogonia in the prepubertal porcine testis. While in the postpubertal porcine testis, they are markers of differentiated spermatocytes. These findings may facilitate future studies of porcine spermatogenesis.

Determination of complete sequence information of the human ABO blood group orthologous gene in pigs and breed difference in blood type frequencies.

The sequence information of the genomic form of the human ABO blood group orthologous gene (erythrocyte antigen A, EAA) is not complete in pigs. Therefore, we cloned and characterized the nucleotide sequence of EAA intron 7, which is critical to understand genetic difference between A and 0 blood groups in pigs, covering complete genomic sequence information of EAA excluding a ~560bp unsequencible gap. We also analyzed genetic polymorphisms within EAA intron 7 and exon 8. We found difference in A0 blood group frequencies among pig breeds. In addition, we designed a new genomic DNA-based A0 blood group typing method and improved the accuracy and simplicity of the typing.

ß2-microglobulin gene duplication in cetartiodactyla remains intact only in pigs and possibly confers selective advantage to the species.

Several ß2-microglobulin (B2M) -bound protein complexes undertake key roles in various immune system pathways, including the neonatal Fc receptor (FcRn), cluster of differentiation 1 (CD1) protein, non-classical major histocompatibility complex (MHC), and well-known MHC class I molecules. Therefore, the duplication of B2M may lead to an increase in the biological competence of organisms to the environment. Based on the pig genome assembly SSC10.2, a segmental duplication of ~45.5 kb, encoding the entire B2M protein, was identified in pig chromosome 1. Through experimental validation, we confirmed the functional duplication of the B2M gene with a completely identical coding sequence between two copies in pigs. Considering the importance of B2M in the immune system, we performed the phylogenetic analysis of B2M duplication in ten mammalian species, confirming the presence of B2M duplication in cetartioldactyls, like cattle, sheep, goats, pigs and whales, but non-cetartiodactyl species, like mice, cats, dogs, horses, and humans. The density of long interspersed nuclear element (LINE) at the edges of duplicated blocks (39 to 66%) was found to be 2 to 3-fold higher than the average (20.12%) of the pig genome, suggesting its role in the duplication event. The B2M mRNA expression level in pigs was 12.71 and 7.57 times (2-ΔΔCt values) higher than humans and mice, respectively. However, we were unable to experimentally demonstrate the difference in the level of B2M protein because species specific anti-B2M antibodies are not available. We reported, for the first time, the functional duplication of the B2M gene in animals. The identification of partially remaining duplicated B2M sequences in the genomes of only cetartiodactyls indicates that the event was lineage specific. B2M duplication could be beneficial to the immune system of pigs by increasing the availability of MHC class I light chain protein, B2M, to complex with the proteins encoded by the relatively large number of MHC class I heavy chain genes in pigs. Further studies are necessary to address the biological meaning of increased expression of B2M.

NANOG gene expression is regulated by the ETS transcription factor ETV4 in human embryonic carcinoma NCCIT cells.

We demonstrated that ETV4 is a transcriptional activator of the NANOG gene in human embryonic carcinoma NCCIT cells. The endogenous expression of NANOG and ETV4 in naïve cells was significantly down-regulated upon differentiation and by shRNA-mediated knockdown of ETV4. NANOG transcription was significantly upregulated by ETV4 overexpression. A putative ETS binding site (EBS) is present in the region (-285 to -138) of the proximal promoter. Site-directed mutagenesis of the putative EBS (-196AGGATT-191) abolished NANOG promoter activity and ETV4 interacted with this putative EBS both in vivo and in vitro. Our data provide the molecular details of ETV4-mediated NANOG gene expression.

Esculetin exerts anti-proliferative effects against non-small-cell lung carcinoma by suppressing specificity protein 1 in vitro.

Esculetin, a coumarin derivative, is a phenolic compound isolated from Artemisia capillaris, Citrus limonia, and Euphorbia lathyris. Although it has been reported to have anti-inflammatory, anti-oxidant, and anti-proliferative activities in several human cancers, its anti-proliferative activity against non-small-cell lung carcinoma (NSCLC) and the molecular mechanisms involved have not been adequately elucidated. In this study, we used two NSCLC cell lines (NCI-H358 and NCI-H1299) to investigate the anti-proliferative activity and apoptotic effect of esculetin. Our data showed that esculetin-treated cells exhibited reduced proliferation and apoptotic cell morphologies. Intriguingly, the transcription factor specificity protein 1 (Sp1) was significantly suppressed by esculetin in a dose- and time-dependent manner. Furthermore, the levels of p27 and p21, two key regulators of the cell cycle, were up-regulated by the esculetin-mediated down-regulation of Sp1; the level of a third cell-cycle regulator, survivin, was decreased, resulting in caspase-dependent apoptosis. Therefore, we conclude that esculetin could be a potent anti-proliferative agent in patients with NSCLC.

Growth and Differentiation Factor 3 Is Transcriptionally Regulated by OCT4 in Human Embryonic Carcinoma Cells.

Growth and differentiation factor 3 (GDF3), a mammalian-specific transforming growth factor ß ligand, and OCT4, one of key stem cell transcription factors, are expressed in testicular germ cell tumors (TGCTs) as well as pluripotent stem cells. To understand the molecular mechanism by which OCT4 and GDF3 function in tumorigenesis as well as stemness, we investigated the transcriptional regulation of GDF3 mediated by OCT4 in human embryonic carcinoma (EC) NCCIT cells, which are pluripotent stem cells of TGCTs. GDF3 and OCT4 was highly expressed in undifferentiated NCCIT cells and then significantly decreased upon retinoic acid-induced differentiation in a time-dependent manner. Moreover, GDF3 expression was reduced by short hairpin RNA-mediated knockdown of OCT4 and increased by OCT4 overexpression, suggesting that GDF3 and OCT4 have a functional relationship in pluripotent stem cells. A promoter-reporter assay revealed that the GDF3 promoter (-1721-Luc) activity was significantly activated by OCT4 in a dose-dependent manner. Moreover, the minimal promoter (-183-Luc) was sufficient for OCT4-mediated transcriptional activation and provided a potential binding site for the direct interaction with OCT4. Collectively, this study provides the evidence about the regulatory mechanism of GDF3 mediated by OCT4 in pluripotent EC cells.

Kahweol induces apoptosis by suppressing BTF3 expression through the ERK signaling pathway in non-small cell lung cancer cells.

Kahweol, a diterpene molecule, has antiproliferative effects on several types of human cancer cells, but whether it has apoptotic effect in non-small cell lung cancer (NSCLC) is not known. To explore this possibility, we incubated cells from two NSCLC cell lines, NCI-H358 and NCI­H1299, with different concentrations of kahweol and used the MTS assay, DAPI staining, propidium iodide staining, Annexin V staining, immunocytochemical test, and western blot analysis to characterize this molecule and the signaling pathway underlying its effects. The kahweol-treated cells showed significantly decreased cell viability, increased nuclear condensation, and an increased number of Annexin V-positive NSCLC cells. Suppression of basic transcription factor 3 (BTF3) was followed by apoptosis induced by kahweol via the ERK-mediated signaling pathway in a dose- and time-dependent manner. In addition, kahweol modulated the protein expression of BTF3 genes involved in cell-cycle regulation and apoptosis-related proteins, resulting in apoptotic cell death. Our results collectively indicated that kahweol inhibited the proliferation of NSCLC cells through ERK-mediated signaling pathways and the downregulation of BTF3.