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PubMed2003

Generation and sequence characterization of a normalized cDNA library from swine ovarian follicles.

Ovulation rate is a major factor determining litter size in swine and is, therefore, a trait of economic importance to the pork industry. The dynamics of follicle development, which in turn are dictated by a balance between follicle recruitment, maturation, selection, and atresia, are a major determining factor of ovulation rate. The role of several genes expressed in the ovaries during these processes has been described, but studies utilizing large-scale genomic approaches have yet to be conducted to examine gene expression in this tissue more globally. We have developed a normalized cDNA library from swine ovarian follicles in various stages of development, ranging from 2.0 to 10.0 mm in diameter, collected from gilts from divergent genetic lines selected for high and low ovulation rates, during the 7 initial days of the follicular phase of the estrous cycle. EST sequences were obtained from 5231 distinct clones derived from this library. In total, 3479 unique sequence clusters were obtained, of which 2661 singletons (76.5%) were observed. BLASTN searches with the primary sequences from the clusters obtained resulted in 1037 sequences not matching (E <1.0(-06) any of the sequences in the nt database (29.8% novelty rate). This resource will facilitate the use of cDNA microarrays in functional genomics studies aiming at unraveling the genetic and physiological mechanisms underlying follicle maturation and ovulation rate in swine.

Animals↗
PubMed1997

Construction of a swine YAC library allowing an efficient recovery of unique and centromeric repeated sequences.

A swine DNA genomic library was constructed in yeast artificial chromosome (YAC) using the pYAC4 vector and the AB1380 strain. The DNA prepared from two Large White males was partially digested with EcoRI and size selected after both digestion and ligation. The YAC library contained 33792 arrayed clones with an average size of 280 kb as estimated by analysis of 2% of the clones, thus representing a threefold coverage of the swine haploid genome. The library was organized in pools to facilitate the PCR screening. The complexity of the library was tested both for unique and centromeric repeated sequences. In all, 20 out of 22 primer sets allowed the characterization of one to six clones containing specific unique sequences. These sequences are known to be on Chromosomes (Chrs) 1, 2, 5, 6, 7, 8, 13, 14, 15, 17, and X. Eight additional clones carrying centromeric repeat units were also isolated with a single primer set. The sequencing of 37 distinct repeat units of about 340 bp subcloned from these eight YACs revealed high sequence diversity indicating the existence of numerous centromeric repeat unit subfamilies in swine. Furthermore, the analysis of the restriction patterns with selected enzymes suggested a higher order organization of the repeat units. According to preliminary FISH experiments on a small number of randomly chosen YACs and YACs carrying specific sequences, the chimerism appeared to be low. In addition, primed in situ labeling experiments favored the idea that the YACs with centromeric repeat sequences were derived from a subset of metacentric and submetacentric chromosomes.

Animals↗
PubMed1997

Genomic mapping of chemokine and transforming growth factor genes in swine.

Five chemokine genes, transforming growth factors alpha, beta 2 and 3 (TGFBA, TGFB-2, and TGFB-3), interleukin 8 (IL-8), and monocyte chemoattractant protein 2 (MCP-2), were mapped to porcine linkage groups on Chromosomes 3q, 10p, 7q, 8, and 12q, respectively. Restriction fragment length polymorphisms (RFLPs) for these genes were developed by Southern blot hybridization after digestion of porcine genomic DNA with BamHI and MspI (TGFBA), BamHI and PvuII (TGFB-2), HindIII (TGFB-3), BglII (IL-8), and PstI (MCP-2) and used to genotype the USDA-MARC Swine Reference Population pigs. Sufficient informative meioses, 61 (TGFBA), 58 (TGFB-2), 28 (TGFB-3), 38 (IL-8), and 156 (MCP-2), were available to pursue two-point pairwise linkage analysis with over 1,000 existing loci in the USDA-MARC genome database to establish initial linkage (LOD > 3). Multi-point analysis with CRIMAP determined the most likely order for each new marker. The assignment of the five chemokine genes in swine concurs with previous porcine/human chromosomal homologies based on results from ZOO-FISH and chromosomal painting experiments. These findings add five new informative Type I markers within a single gene family to the swine genome and may help us understand the genetic basis for disease resistance in livestock.

Animals↗
PubMed2005

A novel mucosal vaccine against foot-and-mouth disease virus induces protection in mice and swine.

Epitopes of a foot-and-mouth disease virus (FMDV) capsid protein VP1 complex and a chimera of 6xHis-tagged cholera toxin B subunit (hCTB) were expressed in Hansenula polymorpha and used together as a mucosal vaccine. Antibody and cytokine responses to VP1-hCTB vaccine and protection against FMDV were evaluated by ELISA and a virus challenge test in mice, respectively. VP1-hCTB directly enhanced the expression of interleukin-5 (IL-5) both in serum and supernatants of cultured spleen cells. After challenging suckling mice with 10(5) FMDV (=50% lethal dosage per mouse) a greater protection was seen after intraperitoneal and intranasal vaccinations than after oral vaccination. In swine immunized with VP1-hCTB, immune responses were achieved after three administrations, and the vaccine protected swine (80%) when challenged with 10(6.5) FMDV (=50% infectious dosage per swine). These results demonstrated the possibility of using CTB as a mucosal adjuvant to elicit protective immune responses against FMDV.

Adjuvants, Immunologic↗
PubMed1981

Purification and enzymatic properties of an L-leucine aminopeptidase from swine liver.

An L-leucine aminopeptidase (alpha-aminoacyl-peptide hydrolase (cytosol), EC 3.4.11.1), having a specificity toward the substrate L-leucine amide, but not toward L-leucyl beta-naphthylamide or L-leucyl p-nitroanilide, has been purified 332-fold from swine liver, with a yield of 8.6%. This is the first purification of this enzyme from hepatic tissue. The purified enzyme submitted to analytical electrophoresis on cellulose acetate strips or in polyacrylamide gel showed a single band after straining with Ponceau S Red dye or Amido black, respectively. Purified swine liver L-leucine aminopeptidase, a cytosol enzyme, exhibited a molecular weight of 268 000 +/- 50 000 by gel filtration. It hydrolyzed L-leucine amide substrate and L-leucyl peptides. It was activated by Mg2+ and Mn2+ and inhibited by Co2+ and Zn2+. The optimum pH was 10. It was rather sensitive to heat elevation. Swine liver L-leucine aminopeptidase was inhibited by EDTA, citric acid, isocaproic acid, dodecylamine, aliphatic alcohols and p-chloromercuribenzoate but unaffected by monoiodoacetic acid and diisopropyl fluorophosphate.

Animals↗
PubMed1989

Prostaglandin-E2 9-ketoreductase from swine kidney. Production of antisera and application to development of a radioimmunoassay.

Prostaglandin-E2 9-ketoreductase (PGE2-9-KR, EC 1.1.1.189), the enzyme which catalyzes the reaction from prostaglandin E2 (PGE2) to prostaglandin F2 alpha (PGF2 alpha), was purified 580-fold from swine kidney. The molecular mass of the enzyme determined by SDS-gel electrophoresis was 33 kDa. Antiserum against the purified enzyme was raised in three rabbits. The antiserum was able to precipitate PGE2-9-KR from swine kidney and to crossreact with pGE2-9-KR from several reproductive organ tissues, such as rabbit ovary, rabbit corpus luteum, rabbit endometrium and human decidua vera. When swine kidney PGE2-9-KR was labelled with 125I and incubated with affinity-purified antiserum in the presence of increasing amounts of unlabelled enzyme, competitive binding of the unlabelled enzyme to the antibody was observed. A radioimmunoassay for the quantitation of the enzyme was developed. The standard curve was linear from 5 to 500 ng enzyme. The intra- and interassay coefficients of variation were 6.4 and 13.2%, respectively. The assay may be useful for the quantitation of PGE2-9-KR in several tissues under various physiological conditions.

Animals↗
PubMed1983

Anti-proliferative effect of pyridinolcarbamate and of aspirin in the early stages of atherogenesis in swine.

When young swine are fed a suitable hyperlipidemic (HL) diet the number of cells in intimal cell masses (ICM) of large and medium-sized arteries begins to increase in number; by 90 days on the diet the cell increase is several-fold compared to no significant increase in cell numbers in ICM of mash-fed controls. The objective of the current study was to see if the cell increase associated with the HL diet could be reduced by pyridinolcarbamate or aspirin. Arteries studied were the abdominal aorta and the coronaries; time on HL diet was 90 days. Both drugs resulted in significant reductions in the increases in cells in ICM associated with the HL diet in both of the arterial beds that were studied. The reduction was most prominent in the abdominal aorta where the increase in ICM cells in the untreated HL diet-fed swine was from an average of 242 +/- 50 cells per cross-section in the mash-fed controls to 1925 +/- 556; in the pyridinolcarbamate-treated swine the increase was only to 641 +/- 214 and in the aspirin-treated group to 770 +/- 146.

Animals↗
PubMed1986

Atherosclerotic lesions in coronary arteries of hyperlipidemic swine. Part 2. Endothelial cell kinetics and leukocyte adherence associated with early lesions.

The role of endothelial cells (EC) in the development and progression of early swine intimal cell mass (ICM)-derived coronary artery lesions in 55 swine fed either a mash or hyperlipidemic diet for 14, 49 or 90 days was investigated. Characteristics studied were endothelial cell turnover (using tritiated thymidine autoradiography), adhesion of leukocytes (presumably chiefly monocytes) to endothelium, and the presence or absence of endothelial cell denudation. The major findings were: An increased adherence of leucocytes to endothelial cells over ICM-lesions in the HL-90 day group compared to the corresponding mash value at 90 days as well as to that of each of the other HL and mash groups. Significant positive correlation between the labeling indices (LI) of endothelial cells lying over coronary artery lesion cells and the labeling indices of the underlying ICM-lesion cells; also, a significant positive correlation between the labeling indices of endothelial cells over lesions of the abdominal aorta and those of the coronary arteries. At 90 days the endothelial cell LI over lesions in the HL group was significantly higher than the corresponding values in the mash group. Since the EC increase rates by growth in the two groups are also significantly different, the differences in LIs reflect at least in part EC growth differences and no strong conclusion can be made regarding possible cell turnover differences. No frank endothelial denudation was found. The findings suggest that in swine coronary arteries participation by monocytes from circulating blood is a factor in the early progression of the lesion as well as smooth muscle cell proliferation.

Animals↗
PubMed1986

Intimal cell mass-derived atherosclerotic lesions in the abdominal aorta of hyperlipidemic swine. Part 2. Investigation of endothelial cell changes and leukocyte adherence associated with early smooth muscle cell proliferative activity.

We have investigated several aspects of endothelial cell (EC) behavior during the initiation and early development of intimal cell mass (ICM)-derived atherosclerotic lesions in the distal abdominal aortas of young swine fed hyperlipidemic (HL) diets for 0, 14, 49, or 90 days. By scanning electron microscopy no breaks in endothelial integrity or other abnormalities were observed even at 90 days on diet after lesions were well established. Also, counts of leukocytes adherent to the endothelium by both scanning and light microscopy revealed no greater numbers in HL than in mash control swine. Estimates of individual EC losses over ICM-lesions in the HL swine (based on calculations from tritiated thymidine labeling indices and EC growth rates determined by counts) suggested a loss of approximately one per 100 EC/day. This loss over ICM lesion was not significantly greater than that over ICM in mash controls, but was significantly greater than that of EC not over ICM lesions in the same HL animal. In any event, the estimated loss seems to be too trivial for the endothelial barrier to be compromised even transiently in a biologically significant fashion. A significant correlation was observed between tritiated thymidine labeling indices of cells within the ICM lesions and of those of the overlying EC that was not observed with the ICM in the controls. Possibly the positive correlation may be a result of the abluminal surface of the overlying EC being exposed to the abnormal milieu of the ICM lesion. It is emphasized that results reported here apply only to ICM-derived lesions at an early stage of development and that they do not contradict results obtained by others with other lesion types such as those derived from monocytes. Furthermore, in later stages of development of ICM-derived lesions in the same model we know that extensive endothelial cell damage can be demonstrated. Also, functional changes in endothelial permeability may have been present in early stages that would not have been detected with the methods used in this study.

Animals↗
PubMed1987

Failure of foreign serum injections to induce immune vasculitis and to accelerate spontaneous or cholesterol-induced atherosclerosis in swine.

Experiments were conducted with domestic swine in an attempt to accelerate and/or modify spontaneous or cholesterol-induced atherosclerosis through immune complex-produced changes in the arterial wall. For the purpose of comparison, another experiment was carried out with rabbits. Swine differed completely from rabbits in that various experimental designs failed to produce immune complex arteritis and fibroproliferative arteriosclerosis. In addition, two foreign serum infusions (human plasma and horse serum on the 30th and 60th day, respectively, 350 mg/kg foreign protein/infusion/animal) did not produce a significant effect on spontaneous arteriosclerosis and cholesterol-induced atherosclerosis (4 months atherogenic diet) in the arterial system of swine. In the aortic trifurcation, however, it was found that antigen administration slightly enhanced cholesterol-dependent atherogenesis.

Anaphylaxis↗
PubMed1988

The "turning off" of excessive cell replicative activity in advanced atherosclerotic lesions of swine by a regression diet.

We studied progression of atherosclerotic lesions in the coronaries and abdominal aortas of swine fed hyperlipidemic (HL) diets producing serum cholesterol levels of about 700 mg/dl for up to 18 months with killings at 9, 13.5 and 18 months on diet. We studied changes in lesions in subsets given after 9 months on the HL diet a low fat, low cholesterol mash diet with killings at 4.5 and 9 months on the regression diet. Lesion cell numbers were evaluated using mean nuclear profiles per cross-section (Np/Cx) in an anatomically defined portion of artery as an index. Lesion sizes were evaluated using mean cross-sectional area (area/Cx) as an index. Tritiated thymidine labeling indices (LI) were used as an index of cell proliferative activity in the coronaries. We also determined the percentage of lesions occupied by lipid rich calcific necrotic debris. For further comparisons all of the same values were determined for normal intimal cell masses (ICM) in control groups fed a low fat, low cholesterol mash diet throughout. The atherosclerotic lesions in the HL swine appeared to arise mainly in the ICM. These became lesions and increased tremendously in size and cell numbers. The greatest increase was in the abdominal aorta even when lesion values were normalized by being expressed as percentages of the wall (intima + media). Lipid-rich calcific necrotic debris occupied about 25% of the lesion by 9 months and the percentage was similar at 18 months although size of lesions and their necrotic regions had increased 3-fold over the 9-month interval. Lesion [3H]thymidine LIs were 3-4-fold greater than in the control ICM. In the period 9-18 months on an HL diet lesions increased about 3-fold in size and cell numbers. In the swine put on a regression diet for the 9-18 month period growth of the lesions stopped completely. In fact, there was a numerical decrease in lesion sizes and cell numbers and this was statistically significant for the left anterior descending coronary. The lesion growth stoppage was partly accounted for by the "turning off" of excessive DNA synthesis activity of the lesion cells as indicated by the return of [3H] thymidine LIs to the normal values in the mash controls. Another factor was the lack of increase and in fact marked reduction in the size of the regions of lipid rich necrotic debris.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Modification of lipoprotein patterns and retardation of atherogenesis by a fish oil supplement to a hyperlipidemic diet for swine.

We have studied the effect of addition of 30 ml cod liver oil (FO) daily to a highly atherogenic butter (BT) diet for swine on lesion development in the coronary arteries and aorta, plasma lipoprotein (LP) patterns, plasma levels of thiobarbituric acid-reactive substances (TBARS) and on tritiated thymidine-labeling indices ([3H]TdR LI) of smooth muscle cells (SMC) and monocyte/macrophages (M/M phi) in the atherosclerotic lesions. Seventeen male Yorkshire swine (11.1 +/- 0.4 kg) were divided into 3 groups: BT (n = 6), BT + FO (n = 6) and mash (n = 5). They were fed the respective diets for 4 months. Terminally, fasting plasma was obtained and cholesterol contents were determined in various fractions of lipoproteins separated by density gradient ultracentrifugation, Pevikon block electrophoresis and immunoelectrophoresis. Apoprotein (B, A-I, E and C) contents of the plasma and lipoprotein fractions were determined by polyacrylamide gel electrophoresis and densitometry of gels stained with Coomassie blue. Swine were injected intramuscularly with 0.5 mCi/kg of [3H]TdR 2 h before death. The aorta and coronary arteries were perfusion fixed in situ under anesthesia. Samples were obtained for microscopic morphometry, autoradiography and immunohistochemistry from distal abdominal aorta, thoracic aorta, and proximal coronary arteries; left main (LM), left anterior descending (LAD), left circumflex (LCX), right main (RM), and right coronary artery (RCA). On the BT diet without FO there was extensive atherosclerotic (AS) lesion development, which was drastically reduced by the addition of FO to the BT diet in all sites by from 71 to 94%. The overall plasma cholesterol (CH) levels were reduced only modestly by the FO (816 +/- 64 to 629 +/- 14 mg/dl) but the distribution of CH in the various lipoprotein classes was remarkably altered. The CH in the large lipoprotein molecules containing both B and E apoproteins was reduced from 488 +/- 84 to 204 +/- 17 mg/dl by the FO with an almost corresponding increase in the conventional LDL molecules containing apo B only (158 +/- 29 to 344 +/- 15 mg/dl). We offer the hypothesis that the large apo B,E containing molecules are much more atherogenic than the smaller apo B containing molecules. This hypothesis is supported by a highly significant correlation between extent of lesion development in all arterial sites and plasma levels of CH in apo B,E containing lipoproteins. Plasma TBARS were elevated by the BT + FO diet but seemed to have no significant effect on the lesions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1980

Effects of superoxide dismutase on cholesterol 7 alpha-hydroxylation in swine.

Effects of carbon monoxide, nitrogen, ferricytochrome c and p-hydroxymercuribenzoate were studied on cholesterol 7 alpha-hydroxylase activity of swine hepatic microsomes. The results suggest that a microsomal electron transport system is involved in hepatic microsomal cholesterol 7 alpha-hydroxylation in swine. Cholesterol 7 alpha-hydroxylation is inhibited by superoxide dismutase in the standard assay system containing a NADPH generating system. Superoxide dismutase also inhibited cholesterol 7 alpha-hydroxylation in the system where superoxides were generated by enzymatic or nonenzymatic means in the absence of NADPH-generating system. The current study suggests that superoxide anion may be an important factor in the cholesterol 7 alpha-hydroxylation of swine.

Animals↗
PubMed1989

The entry of African swine fever virus into Vero cells.

The entry of African swine fever virus into Vero cells has been investigated by both biochemical and morphological techniques. A quantitative electron microscopy analysis of the early steps of the infection has shown that African swine fever virus enters Vero cells by a receptor-mediated endocytosis mechanism. The internalization of virus particles is a temperature- and energy-dependent process, since it did not take place at 4 degrees or in the presence of NaF and 2,4-dinitrophenol. To determine the involvement of acidic intracellular vacuoles in the virus entry pathway we have tested the effect of lysosomotropic agents in the infection. Chloroquine, dansylcadaverine, amantadine, methylamine, and ammonium chloride inhibited African swine fever virus production in Vero cells. Dansylcadaverine and chloroquine did not inhibit virus adsorption and internalization; however, in the presence of these drugs, virus particles were retained in cytoplasmic vacuoles and early viral RNA and protein synthesis were not detected, indicating that these compounds inhibit an early step in the infectious cycle, probably the uncoating of the virus particle.

African Swine Fever Virus↗
PubMed1990

Genetic variation and multigene families in African swine fever virus.

The genome of a virulent strain (LIS57) of African swine fever virus differs from that of the Vero-cell-adapted strain (BA71V) in several deletions located in the variable regions. The region which contains the most differences is located 8-20 kb from the left end. The DNA sequence of this region was obtained from LIS57 virus DNA and compared with the overlapping sequences of BA71V virus. This comparison revealed that the changes in the variable regions result in differences in the number of genes which belong to the multigene families 360 and 110. Virus isolate LIS57 contains at least 8 genes of the multigene family 360 and 12 genes of the multigene family 110, instead of the 6 and 5 genes, respectively, found in BA71V virus strain. The position of the deletions indicates that new combinations of multigene family members in African swine fever virus DNA may arise by in-frame recombination between homologous genes. These data indicate that the evolution of the multigene families 360 and 110 in African swine fever virus DNA has involved different processes, including gene duplication, divergence of duplicated genes, and gene deletion.

African Swine Fever Virus↗
PubMed1990

Sequence and evolutionary relationships of African swine fever virus thymidine kinase.

The thymidine kinase gene of African swine fever virus was mapped in a 1.4-kb EcoRI-PstI fragment located in the left half of the Eco RI K fragment of African swine fever virus DNA by using degenerate oligonucleotide probes derived from regions of the thymidine kinase sequence conserved in several poxviruses, man, mouse, and chicken. The nucleotide sequence of this region revealed an open reading frame of 196 codons, whose translated amino acid sequence showed significant similarity to the thymidine kinases of vaccinia virus, variola virus, monkeypox virus, shope fibroma virus, fowlpox virus, capripox virus, man, mouse, and chicken. The similarity scores obtained after comparison of known thymidine kinase sequences indicated that the African swine fever virus thymidine kinase is more distantly related than the poxvirus thymidine kinases to their cellular homologs. The evolutionary implications of these findings are discussed.

African Swine Fever Virus↗
PubMedDate not supplied

Immunological effect of a synthetic growth hormone peptide on the growth performance in swine.

A mouse monoclonal antibody (mAb), designated PS-7.6, was previously shown to enhance the activity of porcine growth hormone (pGH) in promoting the growth of hypophysectomized (hypox) rats. Epitope mapping studies indicated that the region recognized by PS-7.6 resided within an amino acid sequence 54-95 of pGH. A peptide corresponding to this sequence was synthesized and found to induce swine antibodies capable of augmenting pGH activity in hypox rats. On the basis of these previous observations, an attempt was made in this study to determine whether or not the peptide pGH(54-95) could be used as a vaccine to elicit antibodies functionally similar to PS-7.6 mAb, thus potentiating the efficacy of endogenous GH in swine. Young pigs (15-20 kg) were immunized with pGH(54-95) that had been conjugated with ovalbumin (OVA) and boosted twice at 4-week intervals. Control animals were similarly immunized with OVA. The weight gains and feed consumption of these animals were closely monitored throughout the trials. A number of carcass parameters were also examined when these animals reached 110-120 kg, at which time they were killed. Results indicated that immunization with peptide significantly accelerated the daily weight gain during the growing phase of growth. However, this effect disappeared during the finishing phase of growth. The failure to prolong the initial growth effect by the peptide immunization apparently correlated with the kinetics of antibody production, because antibodies immunoreactive to the peptide and pGH were detected in these animals after immunization but gradually diminished. This idea was supported by the fact that antibodies obtained from pigs 5 and 9 weeks after the initial immunization potentiated the activity of pGH in hypox rats, whereas antibodies harvested at week 16 did not. Furthermore, carcass evaluation was performed at time of killing and showed that the leaf fat and loin eye muscle were also significantly improved by peptide immunization. Taken together, the present findings suggest that pGH(54-95) peptide can be employed as a potential growth-promoting vaccine to improve the performance of swine.

Aging↗
PubMed1992

Sequences of the swine 21-hydroxylase gene (CYP21) and a portion of the opposite-strand overlapping gene of unknown function previously described in human.

We sequenced a 4.8 kb BamHI swine genomic fragment comprising the entire 21-hydroxylase gene (CYP21) and its 5' and 3' flanking segments. The CYP21 coding sequence spanned 3050 bp and as in other species, comprised 10 exons separated by the corresponding introns. The deduced protein corresponded to 492 amino acid residues, 8 of which differed from a previously sequenced swine CYP21 enzyme. The 5' flanking region displayed several putative cis-acting elements which may be involved in either constitutive or cyclic adenosine 3',5'-monophosphate (cAMP) dependent transcriptional expression. We also characterized within the 5' region a 139 bp repetitive element of the short interspersed nucleotide element (SINE) family located on the opposite strand. In addition, we characterized the last five exons of a human-like opposite strand gene (OSG/X) located in the swine at the 3' end of CYP21. The sequenced part of this OSG/X displayed a very strong homology with its human counterpart.

Amino Acid Sequence↗