Equine sperm-neutrophil binding.

When mares are inseminated repeatedly, protein molecules from the seminal plasma (SP) prevent sperm-neutrophil binding and reduced fertility. The molecule(s) responsible for sperm-neutrophil binding is not known and the identification of beneficial SP proteins is complicated by their large numbers and abundant variation. We examined several important aspects of sperm-neutrophil binding to ultimately facilitate the identification and isolation of the molecule(s) responsible. First, we raised anti-equine P-selectin antibodies to determine the involvement of this adhesion molecule in sperm-neutrophil binding. While these antibodies identified equine P-selectin, they did not inhibit sperm-neutrophil binding. However, acrosome-reacted equine sperm expressed a molecule similar to the ligand recognition unit of P-selectin. Second, we attempted to characterize SP protein binding to equine sperm and gauge their affinity. Biotinylated SP proteins were incubated with fresh sperm, washed over a viscous medium, electrophoresed, and probed with avidin. Several SP proteins bound to sperm with a strong affinity to withstand these treatments. This finding may prove valuable for future attempts to identify and characterize specific SP molecules. Lastly, we compared the secretions from male sex organs/glands on sperm motility, sperm-neutrophil binding, and their protein profile. We expected fewer proteins from individual organs/glands, which would facilitate isolation and identification of target molecules. While each secretion had a varying effect on motility and sperm-neutrophil binding, the protein profile was as complex as that seen in whole SP, indicating that collection of proteins from individual sources will not facilitate this work. Together, these experiments answer several important questions related to sperm-neutrophil binding, sperm-SP proteins interaction, and the complexity of the SP proteome.

A noncytolytic α toxin recombinant protein protects turkeys against Clostridium septicum challenge.

Clostridium septicum and its associated cytolytic α toxin, along with several other clostridial species, has been implicated as the causative agent of gangrenous dermatitis. A recombinant noncytolytic C. septicum α toxin (NCAT) peptide was developed for use as a vaccine and demonstrated to be safe at concentrations as high as 1 mg/ml. NCAT, used as a purified antigen, partially purified antigen, or in combination with native antigens, was compared to salt-fractionated α toxin combined with denatured C septicum bacteria (native) in a vaccination trial. Three-day-old poults were placed into one of five groups and received two, 0.2-ml vaccinations 5 wk apart. Subcutaneous challenge with 3.2 x 10(7) log phase C. septicum resulted in 78% to 95% of the vaccinated birds surviving challenge compared to 48% of sham-injected controls. By ELISA analysis on NCAT-coated plates, birds receiving vaccines containing the recombinant NCAT peptide showed significantly higher blood serum antibody concentrations than did birds receiving vaccines containing native antigens or alum controls. Additionally, high levels of maternally transferred antibodies reactive to NCAT-purified antigens found in the pre-immune sera from naive 3-day-old poults suggest that the tertiary structure of the NCAT peptide has a high homology to the native protein structure. In conclusion, our study showed that the use of a vaccine comprised of a noncytolytic recombinant α toxin peptide antigen provided clinical protection equal to the use of vaccines formulated with inactivated native proteins at a reduced overall cost.

Laser microdissection coupled with RNA-seq analysis of porcine enterocytes infected with an obligate intracellular pathogen (Lawsonia intracellularis).

BACKGROUND: Lawsonia intracellularis is an obligate intracellular bacterium and the etiologic agent of proliferative enteropathy. The disease is endemic in pigs, emerging in horses and has been described in various other species including nonhuman primates. Cell proliferation is associated with bacterial replication in enterocyte cytoplasm, but the molecular basis of the host-pathogen interaction is unknown. We used laser capture microdissection coupled with RNA-seq technology to characterize the transcriptional responses of infected enterocytes and the host-pathogen interaction. RESULTS: Proliferative enterocytes was associated with activation of transcription, protein biosynthesis and genes acting on the G1 phase of the host cell cycle (Rho family). The lack of differentiation in infected enterocytes was demonstrated by the repression of membrane transporters related to nutrient acquisition. The activation of the copper uptake transporter by infected enterocytes was associated with high expression of the Zn/Cu superoxide dismutase by L. intracellularis. This suggests that the intracellular bacteria incorporate intracytoplasmic copper and express a sophisticated mechanism to cope with oxidative stress. CONCLUSIONS: The feasibility of coupling microdissection and RNA-seq was demonstrated by characterizing the host-bacterial interactions from a specific cell type in a heterogeneous tissue. High expression of L. intracellularis genes encoding hypothetical proteins and activation of host Rho genes infers the role of unrecognized bacterial cyclomodulins in the pathogenesis of proliferative enteropathy.

Microarray analysis of early and late passage chicken embryo fibroblast cells.

Primary cultured cells derived from normal tissue have a limited lifespan due to replicative senescence and show distinct phenotypes such as irreversible cell cycle arrest and enlarged morphology. Studying senescence-associated genetic alterations in chicken cells will provide valuable knowledge of cellular growth characteristics, when compared with normal and rapidly growing cell lines. Microarray analysis of early- and late-passage (passage 4 and 18, respectively) primary chicken embryo fibroblast (CEF) cells was performed with a 4X44K chicken oligo microarray. A total of 1,888 differentially expressed genes were identified with a 2-fold level cutoff that included 272 upregulated and 1,616 downregulated genes in late-passage senescent CEF cells. Bioinformatic analyses were performed using Ingenuity Pathway Analysis (IPA, http://www.ingenuity.com). Of the 1,888 differentially expressed genes in senescent CEF cells, 458 were identified as functionally known genes and only 61 genes showed upregulation. Because senescent cells generally showed the deactivated states of most cellular mechanisms for proliferation and energy metabolism, intensified analysis on upregulated genes revealed that the molecular mechanisms in senescent CEF cells are characterized by the suppression of cell cycle and proliferation, progression of cell death including apoptosis, and increased expression of various secreting factors. These regulatory pathways may be opposite to those found in the immortal CEF cell line, such as the DF-1 immortal line. Further comparison of differentially expressed genes between senescent and immortal DF-1 CEF cells showed that 35 genes overlapped and were oppositely regulated. The global gene expression profiles may provide insight into the cellular mechanisms that regulate cellular senescence and immortalization of CEF cells.

Establishment of an immortal chicken embryo liver-derived cell line.

A continuously growing immortal cell substrate can be used for virus propagation, diagnostic purposes, and vaccine production. The aim of this study was to develop an immortal chicken cell line for efficient propagation of avian infectious viruses. From the various chicken embryo cells that were tested for life span extension, an immortalized chicken embryo liver (CEL) cell line, named CEL-im, was derived spontaneously without either oncogenic viruses or carcinogenic chemical treatment. Currently, CEL-im cells are growing 0.8 to 1.1 population doublings per day and have reached 120 passages. The CEL-im cell line is permissive for poultry infectious viruses, including avian metapneumovirus (AMPV), Marek's disease virus serotype 1 (MDV-1), and infectious laryngotracheitis virus. The CEL-im cells produced high AMPV titer (>10(5) pfu/mL), whereas very low titers (~10 pfu/mL) for MDV-1 and infectious laryngotracheitis virus were produced. To identify genetic alterations in the immortal CEL-im cell line, telomerase activity and mRNA expression for major cell cycle regulatory genes were determined during the immortalizing process. The CEL-im cell line has negative telomerase activity, and when compared with the primary passage 2 CEL cell counterpart, mRNA expression of tumor suppressor protein p53, mouse double minute 2 (Mdm2), cyclin dependent kinase (CDK) inhibitor p21 (p21(WAF)), and CDK inhibitor p16 (p16(INK4)) were downregulated in the CEL-im cell line, whereas retinoblastoma (Rb), transcription factor E2F, member 1 (E2F-1), and alternative reading frame of p16(INK4) (ARF) were upregulated. These results are similar to genetic alterations found previously in immortal chicken embryo fibroblast (CEF) cell lines that showed efficient propagation of MDV-1. Therefore, this newly established CEL-im cell line can serve as an alternative cell substrate for the propagation of poultry viruses, such as AMPV.

Genome-wide differential gene expression in immortalized DF-1 chicken embryo fibroblast cell line.

BACKGROUND: When compared to primary chicken embryo fibroblast (CEF) cells, the immortal DF-1 CEF line exhibits enhanced growth rates and susceptibility to oxidative stress. Although genes responsible for cell cycle regulation and antioxidant functions have been identified, the genome-wide transcription profile of immortal DF-1 CEF cells has not been previously reported. Global gene expression in primary CEF and DF-1 cells was performed using a 4X44K chicken oligo microarray. RESULTS: A total of 3876 differentially expressed genes were identified with a 2 fold level cutoff that included 1706 up-regulated and 2170 down-regulated genes in DF-1 cells. Network and functional analyses using Ingenuity Pathways Analysis (IPA, Ingenuity® Systems, http://www.ingenuity.com) revealed that 902 of 3876 differentially expressed genes were classified into a number of functional groups including cellular growth and proliferation, cell cycle, cellular movement, cancer, genetic disorders, and cell death. Also, the top 5 gene networks with intermolecular connections were identified. Bioinformatic analyses suggested that DF-1 cells were characterized by enhanced molecular mechanisms for cell cycle progression and proliferation, suppressing cell death pathways, altered cellular morphogenesis, and accelerated capacity for molecule transport. Key molecules for these functions include E2F1, BRCA1, SRC, CASP3, and the peroxidases. CONCLUSIONS: The global gene expression profiles provide insight into the cellular mechanisms that regulate the unique characteristics observed in immortal DF-1 CEF cells.

A method for the rapid isolation of virus from cultured cells.

A simple and efficient collection method using hypotonic burst to isolate virions from infected cultured cells is described. Distilled water treatment of avian metapneumovirus (AMPV)-infected cells with thorough mixing and repeated pipeting was considerably faster for virion collection in avian cells compared to the widely used freeze-thaw (F-T) method (30 min vs. 3-4 h). This method was also more effective for virion collection. The total number of virions recovered from AMPV-infected immortal turkey turbinate cells by the novel water lysis method was 3-fold higher than by the F-T method. This simple water lysis method can be applied to virion collection for other RNA viruses such as the paramyxoviruses that are used to infect cultured cells.

Species-specific deletion of the viral attachment glycoprotein of avian metapneumovirus.

The avian metapneumovirus (AMPV) genome encodes the fusion (F), small hydrophobic (SH), and attachment glycoprotein (G) as envelope glycoproteins. The F and G proteins mainly function to allow viral entry into host cells during the early steps of the virus life cycle. The highly variable AMPV G protein is a major determinant for distinguishing virus subtypes. Sequence analysis was used to determine if any differences between avian or mammalian cell propagated subtype C AMPV could be detected for the 1.8kb G gene. As a result, the complete 1.8kb G gene was found to be present when AMPV was propagated in our immortal turkey turbinate (TT-1) cell line regardless of passage number. Surprisingly, AMPV propagated for 15 or more passages in mammalian Vero cells revealed an essentially deleted G gene in the viral genome, resulting in no G gene mRNA expression. Although the Vero cell propagated AMPV genome contained a small 122 nucleotide fragment of the G gene, no other mRNA variants were detected from either mammalian or avian propagated AMPV. The G gene truncation might be caused by cellular molecular mechanisms that are species-specific. The lack of viral gene deletions suggests that avian cell propagated AMPV will provide a better alternative host for live recombinant vaccine development based on a reverse genetics system.

Regulation of the cd38 promoter in human airway smooth muscle cells by TNF-alpha and dexamethasone.

BACKGROUND: CD38 is expressed in human airway smooth muscle (HASM) cells, regulates intracellular calcium, and its expression is augmented by tumor necrosis factor alpha (TNF-alpha). CD38 has a role in airway hyperresponsiveness, a hallmark of asthma, since deficient mice develop attenuated airway hyperresponsiveness compared to wild-type mice following intranasal challenges with cytokines such as IL-13 and TNF-alpha. Regulation of CD38 expression in HASM cells involves the transcription factor NF-kappaB, and glucocorticoids inhibit this expression through NF-kappaB-dependent and -independent mechanisms. In this study, we determined whether the transcriptional regulation of CD38 expression in HASM cells involves response elements within the promoter region of this gene. METHODS: We cloned a putative 3 kb promoter fragment of the human cd38 gene into pGL3 basic vector in front of a luciferase reporter gene. Sequence analysis of the putative cd38 promoter region revealed one NF-kappaB and several AP-1 and glucocorticoid response element (GRE) motifs. HASM cells were transfected with the 3 kb promoter, a 1.8 kb truncated promoter that lacks the NF-kappaB and some of the AP-1 sites, or the promoter with mutations of the NF-kappaB and/or AP-1 sites. Using the electrophoretic mobility shift assays, we determined the binding of nuclear proteins to oligonucleotides encoding the putative cd38 NF-kappaB, AP-1, and GRE sites, and the specificity of this binding was confirmed by gel supershift analysis with appropriate antibodies. RESULTS: TNF-alpha induced a two-fold activation of the 3 kb promoter following its transfection into HASM cells. In cells transfected with the 1.8 kb promoter or promoter constructs lacking NF-kappaB and/or AP-1 sites or in the presence of dexamethasone, there was no induction in the presence of TNF-alpha. The binding of nuclear proteins to oligonucleotides encoding the putative cd38 NF-kappaB site and some of the six AP-1 sites was increased by TNF-alpha, and to some of the putative cd38 GREs by dexamethasone. CONCLUSION: The EMSA results and the cd38 promoter-reporter assays confirm the functional role of NF-kappaB, AP-1 and GREs in the cd38 promoter in the transcriptional regulation of CD38.

Enzymatic engineering of the porcine genome with transposons and recombinases.

BACKGROUND: Swine is an important agricultural commodity and biomedical model. Manipulation of the pig genome provides opportunity to improve production efficiency, enhance disease resistance, and add value to swine products. Genetic engineering can also expand the utility of pigs for modeling human disease, developing clinical treatment methodologies, or donating tissues for xenotransplantation. Realizing the full potential of pig genetic engineering requires translation of the complete repertoire of genetic tools currently employed in smaller model organisms to practical use in pigs. RESULTS: Application of transposon and recombinase technologies for manipulation of the swine genome requires characterization of their activity in pig cells. We tested four transposon systems- Sleeping Beauty, Tol2, piggyBac, and Passport in cultured porcine cells. Transposons increased the efficiency of DNA integration up to 28-fold above background and provided for precise delivery of 1 to 15 transgenes per cell. Both Cre and Flp recombinase were functional in pig cells as measured by their ability to remove a positive-negative selection cassette from 16 independent clones and over 20 independent genomic locations. We also demonstrated a Cre-dependent genetic switch capable of eliminating an intervening positive-negative selection cassette and activating GFP expression from episomal and genome-resident transposons. CONCLUSION: We have demonstrated for the first time that transposons and recombinases are capable of mobilizing DNA into and out of the porcine genome in a precise and efficient manner. This study provides the basis for developing transposon and recombinase based tools for genetic engineering of the swine genome.

Establishment of an immortal turkey turbinate cell line suitable for avian metapneumovirus propagation.

Until recently, there has not been a homologous avian cellular substrate which could continuously produce high titer avian metapneumovirus (AMPV); development of such a cell line should provide an excellent model system for studying AMPV infection. We have established a non-tumorigenic immortal turkey turbinate cell line (TT-1) to propagate sufficiently high AMPV titers. Currently, immortal TT-1 cells are growing continuously at 1.2-1.4 population doublings per day and are at passage 160. Kinetic analysis suggests that AMPV can infect and replicate more rapidly in TT-1 compared to Vero cells, although both cell types undergo apoptosis upon infection. The non-tumorigenic, reverse transcriptase negative TT-1 cell line can serve as an excellent homologous cellular substrate for virus propagation.

Contributions of differential p53 expression in the spontaneous immortalization of a chicken embryo fibroblast cell line.

BACKGROUND: The present study was carried out to determine whether the p53 pathway played a role in the spontaneous immortalization of the SC-2 chicken embryo fibroblast (CEF) cell line that has been in continuous culture for over three years. RESULTS: The SC-2 cell line emerged from an extended crisis period with a considerably slower growth rate than primary CEF cells. The phenotype of the SC-2 cells changed dramatically at about passage 80, appearing smaller than at earlier passages (e.g., passage 43) and possessing a small, compact morphology. This morphological change coincided with an increase in growth rate. Passage 43 SC-2 cells expressed undetectable levels of p53 mRNA, but by passage 95, the levels were elevated compared to primary passage 6 CEF cells and similar to levels in senescent CEF cells. However, the high level of p53 mRNA detected in passage 95 SC-2 cells did not correlate to functional protein activity. The expression levels of the p53-regulated p21WAF1 gene were significantly decreased in all SC-2 passages that were analyzed. Examination of the Rb pathway revealed that E2F-1 and p15INK4b expression fluctuated with increasing passages, with levels higher in passage 95 SC-2 cells compared to primary passage 6 CEF cells. CONCLUSION: The present study suggests that altered expression of genes involved in the p53 and Rb pathways, specifically, p53 and p21WAF1, may have contributed to the immortalization of the SC-2 CEF cell line.

Comparison of avian cell substrates for propagating subtype C avian metapneumovirus.

Avian metapneumovirus (AMPV) is a respiratory viral pathogen that causes turkey rhinotracheitis (TRT) or swollen head syndrome (SHS) in chickens. AMPV was first isolated in South Africa during the early 1970s and has subsequently spread worldwide during the 1980s to include Europe, Asia, and South America. In 1996, a genetically distinct AMPV subgroup C was isolated in the US following an outbreak of TRT. Vero cells are currently the best available substrate for AMPV propagation but are of non-avian origin. A number of different avian cell substrates have been compared to determine which is the most suitable for the propagation of AMPV to sufficiently high titers. Of the cell substrates tested, primary turkey turbinate and kidney and chicken kidney cells produced titers equal to or greater than Vero cells. Turkey turbinate and kidney epithelial cells that were life-span extended by the ectopic expression of human telomerase catalytic subunit (HTERT) initially displayed AMPV titers comparable to Vero cell controls, but declined in virus production with increased passage in culture. Interestingly, plaques emanating from Vero propagated virus were relatively small and dispersed, when analyzed by immunofluorescent assays (IFA), while both turkey turbinate and kidney cell propagated AMPV produced larger plaques. Even with these differences, there were no changes in the predicted amino acid sequences of the nucleocapsid (N) and phosphoprotein (P) genes of AMPV propagated in either turkey turbinate or Vero host cells. However, the fusion (F) gene showed 11 amino acid differences (98.7% identity) between the two host cell types. These results suggest that AMPV propagated in homologous avian cellular substrates may produce more infectious virus with possibly more effective fusion activity, compared to Vero cell propagation.

Activation of the intrinsic mitochondrial apoptotic pathway in swine influenza virus-mediated cell death.

The mitochondrial pathway of swine influenza virus (SIV)-induced apoptosis was investigated using porcine kidney (PK-15) cells, swine testicle (ST) cells, and HeLa cervical carcinoma cells which are known not to support viral replication. As judged by cell morphology, annexin V staining, and DNA fragmentation, PK-15 and ST cells infected with three different subtypes of SIV (H1N1, H3N2, and H1N2) were obviously killed by apoptosis, not necrosis. SIV infection in PK-15 and HeLa cells was shown to decrease the cellular levels of Bcl-2 protein compared to that of mock-infected control cells at 24 h post-infection, whereas expression levels of Bax protein increased in the PK-15 cells, but did not increase in HeLa cells by SIV infection. Cytochrome c upregulation was also observed in cytosolic fractions of the PK-15 and HeLa cells infected with SIV. Apoptosome (a multi-protein complex consisting of cytochrome c, Apaf-1, caspase-9, and ATP) formation was confirmed by immunoprecipitation using cytochrome c antibody. Furthermore, SIV infection increased the cellular levels of TAJ, an activator of the JNK- stressing pathway, and the c-Jun protein in the PK-15 and HeLa cells. Taken together, these results suggest that the mitochondrial pathway should be implicated in the apoptosis of PK-15 cells induced by SIV infection.

Expression analysis and mitochondrial targeting properties of the chicken manganese-containing superoxide dismutase.

Manganese-containing superoxide dismutase (MnSOD) is a major detoxifying enzyme that functions in cellular oxygen metabolism by converting O(2)(-) to H(2)O(2). A cDNA encoding the chicken MnSOD (cMnSOD) has been isolated from a chicken embryo fibroblast (CEF) cell cDNA library. The cloned cMnSOD is 1102 bp in length with an open reading frame (ORF) of 224 amino acids that includes a 26-amino-acid 5'-proximal mitochondrial targeting sequence (MTS). The mature 198-amino-acid region of the cMnSOD is highly conserved among various mammalian species. Two cMnSOD mRNA species (1.2 and 1.0 kb) were expressed in most of the tissues and organs analyzed, with the highest expression levels found in brain, kidney, and heart tissues. Compared to earlier stages of development, expression of cMnSOD was highest in day 13 embryonic heart tissue, and was maintained until post-hatch. Exogenously introduced cMnSOD-GFP fusion constructs (which included the MTS) clearly accumulated in the mitochondria of chicken cells, as expected. Surprisingly, the cMnSOD MTS signal, which displays little similarity to mammalian MTS sequences, enabled cMnSOD-GFP fusion proteins to target mitochondria not only from different cell types (fibroblastic and epithelial), but from a number of mammalian species (human, mouse, and pig). This suggests that specific amino acid motifs within the MTS domain may be more important than the overall sequence similarities for mitochondrial targeting.

Expression of the chicken homologue of the mouse double minute 2 gene.

While the p53 tumor suppressor plays a crucial role in regulating cell cycle checkpoints and apoptosis by acting as either a transcriptional activator or repressor in a variety of mammalian cells, its evolutionarily conserved functions remain to be elucidated in non-mammalian species. In the present study, we determined the functional role of p53 in avian cells by analyzing the expression pattern of the chicken homologue (CDM2) of mouse double minute 2, one of the transcriptional target genes of p53. CDM2 displayed considerable conservation in the p53 binding region as well as the nuclear localization and nuclear export signals and was found to be abundantly expressed in the reproductive organs (testis and ovarian follicles) and in the immune organs (bone marrow, bursa and thymus). CDM2 expression exhibited an early serum-response pattern consistent with its mammalian counterparts and was dramatically downregulated in most of the p53-downregulated immortal chicken embryo fibroblast (CEF) cells analyzed. Expression of CDM2 was shown to be transcriptionally upregulated in the primary CEF cells where p53 was activated by either mitomycin C treatment or by the exogenous transfection of the chicken p53 cDNA. Together, the current studies demonstrate that the expression of MDM2 homologues may be biologically conserved in mammalian and avian cells.

Modulation of p53 expression and its role in the conversion to a fully immortalized chicken embryo fibroblast line.

We have established a spontaneously immortalized chicken embryo fibroblast (CEF) cell line (SC-1) that has been in continuous culture for more than three years. This is only the second report of a spontaneously immortalized reverse transcriptase (RT)-negative chicken cell line. The SC-1 cells emerged from crisis (at about passage 29-31) with a slower growth rate than primary cells. Passage 50 SC-1 cells expressed similar levels of p53 mRNA, but slightly lower levels of p53 protein than passage 6 CEF cells. By passage 120, p53 mRNA levels were significantly decreased in the SC-1 cells, while protein levels were slightly increased compared to passage 6 CEF cells. However, functional analysis of p53 revealed reduced activity in later passage SC-1 cells. Other p53-related genes including p21WAF1, p27Kip1, MDM-2, and the p16INK4a alternate reading frame (ARF) sequence showed similar patterns of differential mRNA expression. Levels of p15INK4b mRNA and protein were dramatically decreased in SC-1 cells, suggesting that the Rb pathway also has been compromised. Telomerase expression was undetectable in SC-1 cells. Fluorescence-activated cell sorting analysis showed that SC-1 and primary cells contained a similar proportion of G0/G1 phase cells, unlike the only other spontaneously immortalized chicken cell line (DF-1). The present study suggests that alterations in the p53 and Rb pathways cause fluctuations in expression levels of important cell-cycle regulatory genes during crucial transition periods as the SC-1 spontaneously immortalized chicken fibroblast cells progress toward becoming a fully committed cell line.

Deregulation of catalase, not MnSOD, is associated with necrotic death of p53-defective DF-1 cells under antimycin A-induced oxidative stress.

One of distinct genetic alterations in spontaneously immortalized DF-1 cells was found to be dysfunction of p53 and E2F-1 as well as altered antioxidant gene expression (upregulation of MnSOD and downregulation of catalase). We have characterized the cellular responses of primary and immortal DF-1 cells to oxidative stress and found that DF-1 cells were more sensitive to oxidative stress than their primary counterparts when treated with antimycin A. The increased DF-1 cell death by oxidative stress was accompanied by an increase in the levels of intracellular superoxide anions and hydrogen peroxide. The cell death in DF-1 cells by antimycin A showed none of the hallmarks of apoptosis, but displayed a significantly increased necrotic cell population. Anti-apoptotic Bcl-2 failed to inhibit oxidative-induced necrotic cell death in the DF-1 cells. However, this necrotic cell death was significantly decreased by treatment with hydrogen peroxide scavengers such as sodium pyruvate and N-acetyl-cysteine. Interestingly, overexpression of human catalase in DF-1 cells endowed cells resistant to the oxidative stress by antimycin A treatment, although the downregulation of MnSOD by an antisense strategy showed no evident change in the cytotoxic effect caused by antimycin A. Taken together, the present study might provide new therapeutic approach for tumor cells having the loss of p53 function and the altered antioxidant functions.

Establishment of life-span extended bovine fibroblast cells carrying the characterization of primary cells.

Although primary bovine embryonic fibroblast (BEF) cells have previously been used as nucleus-donors for nuclear transfer (NT), it has now been proposed to use BEF cells to generate cloned cows that were genetically modified by transgenic or a knock-out system. A major limitation to gene targeting somatic cells, however, is the overall life-span of the cell. In this study, we first examined in vitro life-span of primary BEF cells. Primary BEF cells were found to be replicative senescent at passage 10th-12th, similar to primary murine embryonic fibroblast cells. To overcome this short in vitro life-span, we have optimized culture conditions to extend the life-span and determined growth characteristics of BEF cell lines. Two life-span extended BEF cell lines (designated CGFR -BO-1 and CGFR-BO-2) were shown to grow much faster than their parental primary counterparts. Both cell lines did not display any potential for abnormal growth such as foci formations in either soft-agar or confluent culture condition. In cloning experiments using these cell lines as a nuclear donor, the reconstructed karyoblasts underwent apoptosis, reprogramming and development in the blastocyst stage, at a similar frequency to those observed with parental as well as adult primary fibroblasts. Furthermore, these cell lines targeted with green fluorescence protein (GFP) were successfully transduced, selected and reprogrammed by NT to develop into a blastocyst stage with GFP expression. Our results suggested methods to extend life-span of donor cells with tremendous implications for the genetic engineering of bovine fibroblast cells.

Comparative transcriptional analysis of homologous pathogenic and non-pathogenic Lawsonia intracellularis isolates in infected porcine cells.

Lawsonia intracellularis is the causative agent of proliferative enteropathy. This disease affects various animal species, including nonhuman primates, has been endemic in pigs, and is an emerging concern in horses. Non-pathogenic variants obtained through multiple passages in vitro do not induce disease, but bacterial isolates at low passage induce clinical and pathological changes. We hypothesize that genes differentially expressed between pathogenic (passage 10) and non-pathogenic (passage 60) L. intracellularis isolates encode potential bacterial virulence factors. The present study used high-throughput sequencing technology to characterize the transcriptional profiling of a pathogenic and a non-pathogenic homologous L. intracellularis variant during in vitro infection. A total of 401 genes were exclusively expressed by the pathogenic variant. Plasmid-encoded genes and those involved in membrane transporter (e.g. ATP-binding cassette), adaptation and stress response (e.g. transcriptional regulators) were the categories mostly responsible for this wider transcriptional landscape. The entire gene repertoire of plasmid A was repressed in the non-pathogenic variant suggesting its relevant role in the virulence phenotype of the pathogenic variant. Of the 319 genes which were commonly expressed in both pathogenic and non-pathogenic variants, no significant difference was observed by comparing their normalized transcription levels (fold change±2; p<0.05). Unexpectedly, these genes demonstrated a positive correlation (r(2) = 0.81; p<0.05), indicating the involvement of gene silencing (switching off) mechanisms to attenuate virulence properties of the pathogenic variant during multiple cell passages. Following the validation of these results by reverse transcriptase-quantitative PCR using ten selected genes, the present study represents the first report characterizing the transcriptional profile of L. intracellularis. The complexity of the virulence phenotype was demonstrated by the diversity of genes exclusively expressed in the pathogenic isolate. The results support our hypothesis and provide the basis for prospective mechanistic studies regarding specific roles of target genes involved in the pathogenesis, diagnosis and control of proliferative enteropathy.