Browsing by Author "Wong, Eric A."
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- Activity and mRNA abundance of enzymes for fatty acid synthesis and desaturation in mammary cell culturesJayan, Geetha C. Jr. (Virginia Tech, 1998-08-14)The effect of exogenous unsaturated fatty acids on cellular fatty acid biosynthesis in mammary cells was examined. Under normal situations, even though the diet of a dairy cow contains considerable amounts of unsaturated fatty acids, viz. oleic acid (18:1) and linoleic acid (18:2), the major 18-carbon fatty acid that enters the circulation post-ruminally for delivery to the mammary gland is saturated fatty acid, viz. stearic acid (18:0). This is due to extensive ruminal biohydrogenation of unsaturated fatty acids. Studies have indicated that saturated fatty acids such as 18:0 are enhancers and that certain unsaturated fatty acids are inhibitors of de novo fatty acid synthesis in tissues such as the liver and adipose tissue. The present study investigated the effect of cis and trans isomers of 18:1 and 18:2 on de novo fatty acid synthesis and desaturation in mouse and bovine mammary epithelial cell cultures, and compared it with the effect caused by 18:0. In the first experiment 12.5, 25, 50 or 100 micromoles stearic acid (SA), oleic acid (OA), elaidic acid (EA), trans-vaccenic acid (TVA), linoleic acid (LA) or conjugated linoleic acid (CLA) were supplemented in the media of mouse mammary epithelial (MME) cells that were grown to confluence in Dulbecco's modified Eagle's medium (DMEM). As indicated by cellular palmitic acid (16:0) content and fatty acid synthetase (FAS) activity, when compared with SA all unsaturated fatty acid treatments inhibited de novo fatty acid synthesis in MME cells. In addition, OA at all concentrations and LA and CLA at 50 and 100 micromoles inhibited cellular stearoyl-CoA desaturase (SCD) activity and mRNA abundance. However, EA and TVA, when compared with SA, enhanced SCD activity and mRNA abundance at 12.5 and 25 micromoles. In the second experiment 25, 50 or 100 micromoles SA, OA, TVA, LA or CLA were supplemented in the media of bovine mammary epithelial cells that were grown to confluence in DMEM. As indicated by cellular 16:0 content, acetyl-CoA carboxylase (ACC) activity and FAS activity, treatment with the unsaturated fatty acids inhibited de novo fatty acid synthesis at all concentrations, when compared with SA. Unsaturated fatty acid treatments also reduced the abundance of ACC and FAS mRNA in the cells. When compared with SA at all treatment-concentrations, OA and LA inhibited whereas TVA and CLA enhanced cellular SCD activity and mRNA abundance in the bovine cells. In both cell types, CLA and TVA appeared to be the most potent inhibitors of saturated fatty acid biosynthesis.
- Assessing Hepatic Gene Expression in Response to Xenobiotic Exposure in MiceBoorgula, Smitha (Virginia Tech, 2007-04-12)Xenobiotics are plant derived compounds metabolized by phase I and II liver enzymes. Phase I enzymes increase, and phase II enzymes decrease, xenobiotic toxicity. Xenobiotics considered were ergotamine, associated with fescue toxicosis, and sulforaphane, a phase II inducer. Hypothesized responses in liver gene expression and enzyme activity due to exposure to these xenobiotics were tested. Polymorphic mice were gavaged with sulforaphane, ergotamine or control over four daily dosing periods (2, 5, 8 and 11 d), with at least 5 mice per treatment. Mice were killed and livers collected 24 h after last dosing. With ergotamine, expression of phase II genes catecholâ Oâ amine methyltransferase 1 (P = 0.009) on d 8, and glutathioneâ Sâ transferase (Gst) mu1 (Gstm1; P = 0.049) on d 11 was increased, and sulfotransferase 5a1 on d 11 decreased (P = 0.02). Sulforaphane increased expression of cytochrome P450 1a2 on d 5 (P = 0.02) and flavin containing monooxygenases 1 on d 11 (P = 0.002), both phase I genes. It also increased expression of a phase II gene transcription factor (P = 0.03) and quinone reductase 02 (P = 0.007) on d 5, and Gstm1 on d 8 (P = 0.04) and d 11 (P = 0.01). Moreover, sulforaphane treated mice had higher (P < 0.05) Gstm1 expression across days. Among enzymes, only sufloraphane treated mice had higher (P < 0.05) Gst activity. The increase in both Gstm1 expression and Gst activity indicate a consistent benefit of sufloraphane on phase II enzyme activity.
- Autocrine mechanisms of action of insulin-like growth factor-I (IGF-I) and hormonal regulation of expression of IGF-finding proteins in mammary epithelial cellsRomagnolo, Donato (Virginia Tech, 1993)Limited information is available concerning the molecular and cellular mechanisms that regulate expression of insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBPs) genes in mammary epithelial cells. To test the hypothesis that IGF-I affects growth of bovine mammary epithelial cells through an autocrine and/or paracrine pathway, several cell lines were developed expressing an ovine exon-2 containing IGF-I cDNA under the control of the mouse mammary tumor virus-long terminal repeat (pMMTV-IGF-I), early simian virus (pSV40-IGF-I), and herpes simplex thymidine kinase (pTK-IGF-I) promoters. Stably transfected clones were generated by cotransfection of clonal MAC-T cells with the IGF-I expression vectors and a plasmid conferring resistance to hygromycin-B (HYG-B), using a calcium phosphate precipitation procedure. Induction of the MMTV-LTR with the glucocorticoid dexamethasone (DEX) was required for enhanced expression of IGF-I in MD-IGF-I (MD=Mammary Derived) cells, whereas SV40-IGF-I cells constitutively expressed the highest levels of IGF-I, followed by TK-IGF-I cells. Activity of the MMTV promoter in MD-IGF-I cells was coordinately regulated by lactogenic hormones and extracellular matrix. Acute secretion of DEX-induced recombinant IGF-I by MD-IGF-I cells stimulated cell proliferation through an autocrine/paracrine pathway and triggered the expression of IGFBP-3. Neither acute nor constitutive expression of IGF-I affected expression of type 1 IGF receptor mRNAs, but down-regulated cell surface receptor levels, in the order SV40-> TK- > MD-IGF-I. Secretion of IGF-I-induced IGFBP-3 potentiated the mitogenic actions of IGF-I as evidenced by enhancement of [³H]thymidine uptake into DNA of parental MAC-T cells. This study provides evidence that local production of IGF-I can stimulate cell proliferation of bovine mammary epithelial cells through an autocrine/paracrine mode of action. We suggest that secretion of IGF-I-induced IGFBP-3 by bovine mammary epithelial cells enhances cell responsiveness to IGF-I, but does not prevent down-regulation of the IGF-I receptor in cells constitutively expressing IGF-I.
- Cellular Events During Coccidial Infection in ChickensSu, Shengchen (Virginia Tech, 2016-09-21)Avian coccidiosis is caused by the intestinal protozoa Eimeria. The parasite's site of infection in the intestine is site specific. Eimeria acervulina mainly affects the duodenum, E. maxima the jejunum, and E. tenella the ceca. Lesions in the intestinal mucosa cause reduced feed efficiency and body weight gain in Eimeria-challenged chickens. My previous studies showed that the growth reduction may be due to changes in expression of digestive enzymes and nutrient transporters in the intestine. This can also lead to diminished intracellular pools of nutrients and inhibit pathogen replication. In this dissertation, further analysis of cellular events was performed. Expression of host defense peptides (HDPs), apoptosis and autophagy related genes were examined in Eimeria challenged broilers. The results showed that upon Eimeria infection, LEAP2 was consistently downregulated in the target tissues, while the avian beta-defensins (AvBDs) showed many variations in expression patterns. Downregulation of LEAP2 may be a mechanism for Eimeria to combat the host defense system, and to promote its survival inside the host cell. The in situ hybridization results showed that LEAP2 was expressed only along the villus in the small intestine and not in the crypt. This is the first time LEAP2 has been localized to epithelial cells of the chicken intestine. Eimeria infection can also induce an anti-apoptotic and anti-autophagy state in the host cells. This condition can be both favorable and unfavorable to parasite survival and replication inside the host cell. A comparison of gene expression between Ross and Eimeria resistant Fayoumi (line M5.1 and M15.2) chickens challenged with Eimeria maxima was conducted. The comparison among different lines of chickens showed differential gene expression patterns in lines with different resistance to Eimeria. The similar body weight reduction indicated that there may not be a significant Eimeria resistant line among the Ross, Fayoumi M5.1 and M15.2 birds. The interaction between Eimeria and the host cell is very complex. Studying the mechanisms behind the changes of gene expression during Eimeria infection may give rise to potential therapeutic targets of coccidiosis.
- Centennial Review: The chicken yolk sac is a multifunctional organWong, Eric A.; Uni, Z. (Elsevier, 2020-12-08)The yolk sac (YS) consists of the yolk, which supplies nutrients, and the YS tissue, which surrounds the yolk and provides essential metabolic functions for the developing embryo. The YS tissue is derived from the midgut of the embryo and consists of a layer of endodermal epithelial cells (EEC) in contact with the yolk contents, a mesodermal layer that contains the vascular system and an outer ectodermal layer. The YS tissue is a multifunctional organ that provides essential functions such as host immunity, nutrient uptake, carbohydrate and lipid metabolism, and erythropoiesis. The YS tissue plays a role in immunity by the transport of maternal antibodies in the yolk to the embryonic circulation that feeds the developing embryo. In addition, the YS tissue expresses high mRNA levels of the host defense peptide, avian b-defensin 10 during mid embryogenesis. Owing to its origin, the YS EEC share some functional properties with intestinal epithelial cells such as expression of transporters for amino acids, peptides, monosaccharides, fatty acids, and minerals. The YS tissue stores glycogen and expresses enzymes for glycogen synthesis and breakdown and glucogenesis. This carbohydrate metabolism may play an important role in the hatching process. The mesodermal layer of the YS tissue is the site for erythropoiesis and provides erythrocytes before the maturation of the bone marrow. Other functions of the YS tissue involve synthesis of plasma proteins, lipid transport and cholesterol metabolism, and synthesis of thyroxine. Thus, the YS is an essential organ for the growth, development, and health of the developing embryo. This review will provide an overview of the studies that have investigated the functionalities of the YS tissue at the cellular and molecular levels with a focus on chickens.
- Changes with age in density of goblet cells in the small intestine of broiler chicksReynolds, K. L.; Cloft, Sara E.; Wong, Eric A. (Elsevier, 2020-05-01)Goblet cells secrete mucin 2 (Muc2), which is a major component of the mucus that lines the intestinal tract and creates a protective barrier between pathogens and the intestinal epithelial cells and thus are important for chick health. The objectives of this study were to determine the age-specific and intestinal segment–specific expression of Muc2 mRNA and changes in the number of goblet cells from late embryogenesis to early after hatch. Small intestinal samples from the duodenum, jejunum, and ileum were collected from Cobb 500 broilers at embryonic day 19 (e19), day of hatch (doh), and day 2 and 4 after hatch. Cells expressing Muc2 mRNA and mucin glycoprotein were detected by in situ hybridization or alcian blue and periodic acid–Schiff staining, respectively. Along the villi, there were many more cells expressing Muc2 mRNA than those stained for mucin glycoprotein. In the crypt, cells expressing Muc2 mRNA did not stain for mucin glycoprotein. There was an increase in the density of goblet cells in the villi and Muc2 mRNA expressing cells in the crypts of the jejunum and ileum from e19 to doh and day 2 to day 4, with no change between doh and day 2. In contrast, in the duodenum, the density of goblet cells in the villi and Muc2 mRNA expressing cells in the crypts remained constant from e19 to day 4. At day 4, the villi in the ileum had a greater density of goblet cells than the duodenum. In the crypt, the ileum had a greater density of Muc2 mRNA expressing cells than the duodenum at doh, and the ileum and jejunum both had greater densities of Muc2 mRNA expressing cells than the duodenum at day 4. These results indicate that the population of goblet cells has reached a steady state by doh in the duodenum, whereas in the jejunum and ileum, a steady-state population was not reached until after hatch.
- Characterization of Gene Expression During Adenosine 3':5'-Cyclic Monophosphate Induced Neuroendocrine Differentiation in Human Prostatic AdenocarcinomaGoodin, Jeremy Lee (Virginia Tech, 2002-04-03)The LNCaP cell line is a versatile and useful model that is suitable for the study of human prostate cancer in vitro. The elevation of LNCaP intracellular cAMP levels through the addition of membrane permeable cAMP analogues, phosphodiesterase inhibitors, adenylate cyclase activators, or components of the cAMP signal transduction pathway can induce reversible neuroendocrine differentiation. Elucidation of those genes that are differentially expressed between undifferentiated prostate cancer cells and prostate cancer cells that have been induced to differentiate may present new insights for the molecular mechanisms governing neuroendocrine differentiation, early detection of prostate cancer, and/or potential targets for gene therapy. In this study, differential display PCR was used to identify 226 differentially expressed PCR products. Twelve of the differential display PCR products were confirmed by Northern blot analysis and cloned. DNA sequencing and database comparisons were performed. Among the differentially expressed genes, the human ribosomal S3a gene was identified as down regulated in response to LNCaP differentiation. In order to better ascertain the mechanism by which HRS3a gene expression is decreased during differentiation, the promoter region for this gene was analyzed. Electrophoretic mobility shift assay, antibody supershift assays, site-directed mutagenesis, and luciferase reporter gene analysis were employed to authenticate the roles of several transcription factors in the regulation of the HRS3a gene. Two cyclic AMP response elements, a Sp1 element and a GA-binding protein element, were involved in the regulation of HRS3a gene expression. In order to ascertain the effect of HRS3a down regulation in LNCaP cells, antisense phosphorothioate oligonucleotides were designed to inhibit HRS3a gene expression. Treatment of LNCaP cells with antisense HRS3a oligonucleotides did not influence cAMP induced neuroendocrine differentiation but antisense treatment did result in a decrease in LNCaP cell growth. In addition, it was determined that morphological changes associated with cAMP induced differentiation of LNCaP cells from the epithelial to the neuroendocrine state may not require alterations in gene expression nor the expression of novel proteins.
- Characterization of Pituitary Protein Expression Patterns During Stages in the Reproductive Cycle of Turkey HensSpellerberg, Amy Marie (Virginia Tech, 2003-04-11)Improvements in turkey reproductive efficiency is a very desirable goal for the turkey industry. The ability to maintain turkey hens in the egg-laying (LAY) stage and produce one additional egg per hen a year is estimated to save the turkey industry approximately $1.5 million dollars per year. Overall protein expression generated by tissues of the hypothalamic-hypophyseal complex, namely the anterior pituitary, of the mature turkey hen have a profound impact on reproductive cycling (Scanes, 2000). One of the key physiological factors produced by the anterior pituitary and shown to play a significant role in the regulation of egg laying is the protein prolactin (Prl). The objectives for this study are to examine the overall protein expression patterns from turkey hen pituitary tissue during the nonphotostimulated (NPS), photostimulated (PS), and egg laying (LAY) stages. Attempts to isolate transcription factors that regulate the expression of Prl using an affinity chromatography technique or southwestern screening of a bacteriophage expression library were not successful. A global analysis of protein expression, using two-dimensional polyacrylamide gels (2D gels), was conducted using whole cell, cytoplasmic and nuclear protein extracts from pituitary tissue collected during the NPS, PS and LAY reproductive stages. Approximately 1,046 proteins ranging in pI from 4.6-8.2 and molecular weights between 100 kDa-6kDa were resolved. Protein expression patterns were replicated and verified using pituitaries harvested from NPS, PS and LAY stage turkey hens from another laboratory. Proteins showing considerable changes (563 proteins increased in expression and 98 proteins decreased in expression from the NPS to the LAY stage) in their expression between the reproductive stages were grouped in analysis sets for future identification. These proteins may prove to be important to the reproductive cycling of the turkey hen and warrant future investigation. The results of this study contribute to the overall understanding of the role that the pituitary, as a critical part of the hypothalamic-hypophyseal complex, plays in turkey hen reproductive cycling.
- Characterization of plasmids among the three species of GluconobacterBrookman, Lori L. (Virginia Tech, 1995-07-10)The genus Gluconobacter consists of acetic acid bacteria which have the ability to generate acidic products from their substrates, particularly acetic acid from ethanol. For this reason, the gluconobacters live in acidic, sugary environments such as flowers, honey bees, fruits, cider, vinegar, wine and beer. The gluconobacters carry out a strictly respiratory type of metabolism using only oxygen as a terminal electron acceptor. They do not completely oxidize a substrate to carbon dioxide. Instead, they partially oxidize the substrate using membrane-bound dehydrogenases and excrete the product into the surrounding growth medium. It is these limited oxidations that make the gtuconobacters industrially useful. Although much is known about the physiology of the limited oxidations in the gluconobacters, little is known of their genetics, particularly, their plasmids. The overall purpose of this dissertation was to determine if Gluconobacter plasmids correlate with oxidative capability and/or antibiotic resistance. To achieve this goal, I first needed a way to screen strains of Gluconobacter for their ability to oxidize many different substrates. 'developed an assay that used an unusual artificial electron acceptor, tetranitroblue tetrazolium (TNBT) and then tested the ability of six strains to oxidize 13 chemical compounds. Although most strains were able to oxidize the 13 compounds tested, they accomplished this with varying extents of oxidation. These differences were noted even with strains representing the same species.
- Characterizing the cargo binding and regulatory function of the tail domain in Ncd motor proteinLonergan, Natalie Elaine (Virginia Tech, 2009-10-09)Non-claret disjunctional (Ncd) is a kinesin-14 microtubule motor protein involved in the assembly and stability of meiotic and mitotic spindles in Drosophila oocytes and early embryos, respectively. Ncd functions by cross-linking microtubules through the tail and motor domains. It was originally believed that the role of the Ncd tail domain was to only statically bind microtubules. However, the Ncd tail domain has recently been shown to have properties that stabilize and bundle microtubules, and contribute to the overall motility of the Ncd protein. Continued characterization of the Ncd tail domain is essential to understanding the complete role of Ncd in cell division. This work explored the regulatory function and microtubule binding properties of the Ncd tail domain. Ncd activity is regulated during interphase by nuclear sequestration. GFP-Ncd fusion proteins, containing full length Ncd, individual Ncd domains, or combinations of Ncd domains, were used to identify the presence of a nuclear localization signal (NLS) in the Ncd polypeptide. The nuclear localization of only the GFP fusion proteins containing the Ncd tail sequence indicates that the NLS is contained within the tail domain. Subsequent, experiments performed with GFP fusion proteins containing segments of the tail domain indicate that essential NLS amino acid segments may span the length of the tail domain. Attempts to characterize the microtubule binding properties of the Ncd tail domain, using bacterially expressed MBP-Ncd tail-stalk, were unsuccessful. MBP-Ncd tail-stalk proteins aggregated under binding assay conditions, preventing an accurate determination of the stoichiometric binding relationship between Ncd and the tubulin dimer.
- Cloning and characterization of glycogen synthase from Dictyostelium discoideumWilliamson, Brian (Virginia Tech, 1995)In Dictyostelium, glycogen metabolism plays a major role in development. Undifferentiated cells contain stores of glycogen that are broken down and converted to structural components in differentiated cells. The enzyme that synthesizes this developmentally important pool of glycogen is glycogen synthase. I have cloned the entire coding and 1.3 kb of upstream noncoding region, of glycogen synthase, using PCR amplification and genomic library screening. In order to clone the 3’ portion of the gene it was necessary to develop a new technique, enrichment-PCR, that relies on the base composition of the Dictyostelium genome. Due to the high A+T content of the Dictyostelium genome, a polyT primer and a gene specific primer were used to amplify an unknown DNA fragment, flanking a known sequence. Analysis of the complete coding region showed that glycogen synthase possesses three introns that contain the consensus splice sites for Dictyostelium. The luciferase reporter gene was used to study the transcriptional regulation of glycogen synthase. I defined the cis-acting elements that are required for proper transcriptional regulation of glycogen synthase by using promoter/luciferase fusions of varying sizes. Using the luciferase reporter system a putative promoter element was identified. Additional luciferase constructs were made to identify the specific nucleotide involved in transcription of the glycogen synthase gene. Small defined deletions are often necessary for reporter gene analysis. We have developed a deletion cassette that can expand the functionality of any commonly-used vector. The deletion cassette confers the ability to make small specific sequential deletions of the DNA flanking the cassette. We have shown that this altered vector (pDNBL) now has the ability to create 2, 4, 5 or 9 bp deletions. A number of experimental approaches were taken to study the regulation of glycogen synthase. Homologous recombination was used to try to generate a glycogen synthase (-) cell line. In addition, I have constructed a green fluorescent protein (GFP) vector (pNV) based on the pVTL2 vector. This reporter gene is useful for monitoring the expression of a particular gene in vivo.
- Cloning, Expression, and Developmental and Dietary Regulations of a Chicken Intestinal Peptide Transporter and Characterization and Regulation of an Ovine Gastrointestinal Peptide Transporter Expressed in a Mammalian Cell LineChen, Hong (Virginia Tech, 2001-09-28)To study peptide absorption in chickens, an intestinal peptide transporter cDNA (cPepT1) was isolated from a chicken cDNA library. The cDNA was 2,914-bp and encoded a protein of 714 amino acid residues. Twenty-three di-, tri-, and tetra-peptides were used for functional analysis of cPepT1 in Xenopus oocytes and Chinese hamster ovary (CHO) cells. For most di- and tripeptides tested, the Kt was in the micromolar range, except Lys-Lys and Lys-Trp-Lys. Northern analysis demonstrated that cPepT1 is expressed strongly in the small intestine, and at lower levels in kidney and cecum. These results demonstrated the presence and functions of a peptide transporter in chickens. cPepT1 mRNA abundance was evaluated in response to developmental and dietary regulations. In Experiment 1, eggs at incubation day 18 (E18) and Cobb chicks after hatch (d 0) were sampled before treatments. Three groups of chicks were fed diets containing 12, 18, or 24% crude protein (CP). Feed intake of chicks fed the 18 or 24% CP diets was restricted to that of chicks fed the 12% CP diet. In Experiment 2, a fourth group with free access to the 24% CP diet was added. cPepT1 mRNA abundance was quantified from northern blots. By d 0, there was a 50-fold increase in cPepT1 mRNA abundance compared with E 18. In chicks fed the 12% CP diet, cPepT1 mRNA abundance decreased throughout the 35 d. Chicks fed 18 or 24% CP diets showed an increase in cPepT1 mRNA abundance with time. In chicks with free access to the 24% CP diet, cPepT1 mRNA decreased until d 14 but returned to an intermediate level at d 35. Our results indicate that cPepT1 mRNA is regulated by both dietary protein and developmental stage. To investigate the kinetics of an ovine peptide transporter (oPepT1), CHO cells were transfected with oPepT1 cDNA. Uptake of Gly-Sar by transfected cells was pH-dependent, concentration-dependent, and saturable. Competition studies showed that all di-, tri-, and tetra-peptides inhibited uptake of Gly-Sar. Pretreatment of the cells with staurosporine resulted in an increase in peptide transport. This increase was blocked by pretreatment with PMA. The results indicate that protein kinase plays a role in oPepT1 function.
- Cloning, Sequencing and Expression of a Porcine Intestinal Peptide Transporter in a Mammalian Cell LineKlang, Judith Elisa (Virginia Tech, 2002-12-10)Absorption of dietary proteins can be met through the uptake of free amino acids or as small peptides. A peptide transport protein, PepT1, is responsible for the absorption of intact peptides arising from digestion of dietary proteins. PepT1 is driven by a H+-coupled transport system that allows for the absorption of small peptides through the intestinal brush border membrane. Screening of a porcine intestinal cDNA library with a sheep PepT1 cDNA probe resulted in the identification of three porcine PepT1 (pPepT1) cDNAs of varying sizes and sequences. Each variant cDNA isolated was cloned into a mammalian expression vector, sequenced, and expressed in Chinese hamster ovary (CHO) cells. Peptide transport was assessed by uptake studies using the radiolabeled dipeptide [3H]-Gly-Sar. Only one of the three cDNAs encoding for a protein of 708 amino acids induced H+-dependent peptide transport activity. Through computer analysis, a putative protein structure for pPepT1 was developed. The transporter has an unusual 13 transmembrane structure with the N-terminus located extracellularly and the C-terminus located intracellularly. Seven glycosylation sites and three protein kinase C phosphorylation sites are located throughout the protein. Expression of pPepT1 activity in CHO cells had a optimal peptide uptake at 18-24 hours. The transporter showed optimal uptake at a pH of 5.5-6.0. Eighteen different unlabeled dipeptides and tripeptides were found to inhibit the uptake of [3H] -Gly-Sar in competition studies. The IC50 of 13 of the dipeptides and two tripeptides ranged between 0.015 to 0.4 mmol/L. The exceptions were Lys-Lys, Arg-Lys, and Lys-Trp-Lys, which showed IC50 values greater than 1.37 mmol/L and appear to be poor substrates for pPepT1. All three of the tetrapeptides examined showed very high IC50 values and inhibition of the uptake of Gly-Sar was too small to measure even at a 10mM concentration. Dipeptides and tripeptides appear to be substrates for the porcine intestinal peptide transporter while tetrapeptides do not appear to be transported.
- Conversion of equine umbilical cord matrix mesenchymal stem cells to the trophectoderm lineage using the Yamanaka reprogramming factorsReinholt, Brad M. (Virginia Tech, 2015-07-21)Induced pluripotent stem (iPS) cells that possess embryonic stem (ES) cell-like properties are generated through the use of the Yamanaka transcription factors, OCT4, SOX2, KLF4, and MYC (OSKM). Advanced transgene delivery methods utilizing non-integrating viruses for transduction of target cells has provided new opportunities for regenerative medicine in humans and other species. We sought to use this technology to generate equine iPS cells to address challenges in equine regenerative medicine. Umbilical cord matrix mesenchymal stromal cells (MSC) were transduced with the non-integrating Sendai virus encoding for the OSKM transcription factors. The cells initially were cultured on mouse embryonic feeder cells supplemented with LIF (10 ng/mL) and FGF2 (4 ng/mL). Transduction generated 21 initial colonies. Of these, four survived beyond 20 passages. The transduced equine cells morphologically resembled ES cells and expressed cell surface antigens indicative of ES cells. Molecular evaluation revealed the cells maintained expression of endogenous OSKM while the exogenous OSK transgenes were extinguished, but MYC was maintained. The transduced equine cells did not express the ES marker NANOG, but did express the trophectoderm markers CDX2 and TFAP2A. Both OCT4 and CDX2 were colocalized to the nucleus. The transduced equine cells were termed equine induced trophoblast (iTr) cells. Culture of the iTr cell in suspension resulted in formation of blastocyst-like spheres rather than solid cell aggregates indicative of ES and iPS cells. The iTr cells were transitioned to a feeder free monolayer culture. Transformation of the iTr cells to the spherical arrangement stimulated expression of genes that mark differentiation of trophoblast cells and up-regulated 250 transcripts over the monolayer arrangement. The iTr monolayer arrangement up-regulated 50 transcripts compared to the spherical arrangement. The iTr spheres respond to BMP4, EGF, and FGF2 by phosphorylating signal transduction proteins. Addition of BMP4, EGF, or FGF2 in combined pairs was able to alter TFAP2A, NEU1, and SLC35A1 expression. The generation of iTr cells by transduction of the Yamanaka reprogramming factors is not unique to equine cells. However, this report marks the generation of the first equine trophoblast cell line capable of recapitulating early equine trophoblast development. The new iTr line could prove valuable in gaining greater understanding of equine trophectoderm development.
- Cytokine mRNA Expression in the Small Intestine of Weanling Pigs Fed Diets Supplemented with Specialized Protein or Peptide SourcesZhao, J.; Harper, Allen F.; Webb, Kenneth E. Jr.; Kuehn, Larry Alexander; Gilbert, Elizabeth R.; Xiao, X.; Wong, Eric A. (2008-12)Cytokines play a central role in the mucosal immune response and are involved in regulation of nutrient absorption, metabolism and animal growth This study investigated the effect of diet manipulation with specialized protein or peptide sources on expression of cytokine (IL-1, IL-6, IL-10, and TNF-alpha) mRNA abundance in different intestinal regions and at different ages post-weaning in piglets. A total of 48 (17 days of age, 6.16 +/- 0.34 kg BW) weanling pigs were fed either a corn-soy/whey protein basal diet, the basal diet supplemented with spray-dried plasma protein (SDPP), or the basal diet supplemented with Peptiva (R), a hydrolyzed marine plant protein. A fourth treatment group was fed the SDPP diet, but the feed intake level was limited (SDPP-LF). Pigs were killed at 3 and 10 d, and intestinal cytokine mRNA was measured by real-time PCR using the relative quantification method. The SDPP-LF group exhibited an increased TNF-alpha mRNA abundance compared with the ad libitum SDPP group (p<0.05). The TNF-alpha and IL-10 mRNA abundance increased from the proximal to distal part of the intestine, and the mRNA abundance was greater (p<0.01) in the distal intestine as compared with the proximal and middle intestine. The cytokines IL-1-beta, IL-10 and TNF-alpha mRNA abundance also increased from d3 to d10 postweaning (p<0.01). In summary, restricted feeding increased the TNF-alpha mRNA abundance in the small intestine, however neither SDPP nor peptide supplementation affected cytokine mRNA expression. Abundance of mRNA for most cytokines examined in this study increased with age post-weaning, suggesting that during 10 d after weaning the mucosal immune system is still under development.
- Delayed access to feed affects broiler small intestinal morphology and goblet cell ontogenyLiu, Kuan-Ling; Jia, Meiting; Wong, Eric A. (2020-11)Broilers are often deprived of feed and water for up to 48 h after hatch. This delayed access to feed (DAF) can inhibit small intestine development. The objective of this study was to determine the effects of DAF on small intestinal morphology, mRNA abundance of the goblet cell marker Muc2 and absorptive cell marker PepT1, and the distribution of goblet cells in young broilers. Cobb 500 chicks, hatching within a 12-h window, were randomly allocated into 3 groups: control with no feed delay (ND), 24-h feed delay (DAF24), and 36-h feed delay (DAF36). Morphology, gene expression, and in situ hybridization analyses were conducted on the duodenum, jejunum, and ileum at 0, 24, 36, 72, 120, and 168 h after hatch. Statistical analysis was performed using a t test for ND and DAF24 at 24 h. A 2-way ANOVA and Tukey's HSD test (P < 0.05) were used for ND, DAF24, and DAF36 from 36 h. At 24 to 36 h, DAF decreased the ratio of villus height/crypt depth (VH/CD) in the duodenum but increased VH/CD in the ileum due to changes in CD, whereas at 72 h, DAF decreased VH/CD due to a decrease in VH. The mRNA abundance of PepT1 was upregulated, while Muc2 mRNA was downregulated in DAF chicks. Cells expressing Muc2 mRNA were present along the villi and in the crypts. The ratio of the number of goblet cells found in the upper half to the lower half of the villus was greater in DAF chicks than in ND chicks, suggesting that DAF affected the appearance of new goblet cells. The number of Muc2 mRNA-expressing cells in the crypt, however, was generally not affected by DAF. In conclusion, DAF transiently affected small intestinal morphology, upregulated PepT1 mRNA, downregulated Muc2 mRNA, and changed the distribution of goblet cells in the villi. By 168 h, however, these parameters were not different between ND, DAF24, and DAF36 chicks.
- Delayed access to feed affects broiler small intestinal morphology and intestinal cell ontogenyLiu, Kuan-Ling (Virginia Tech, 2019-08-01)In the broiler industry, chicks are often deprived of feed and water up to 48 h posthatch. This delayed access to feed (DAF) has been found to inhibit small intestinal development, compromising growth of the chick. To further understand the impact of DAF on small intestines at the molecular level, many developmental genes that regulate intestinal development were investigated. The objective of this study was to determine the effect of DAF on early posthatch broiler small intestinal morphology, which includes villus height (VH) and crypt depth (CD), and to quantify changes in regulatory genes, such as Olfactomedin 4 (Olfm4), Marker of Ki-67 (Ki-67), Peptide Transporter 1 (PepT1), and Mucin 2 (Muc2), in response to DAF. The Olfm4 mRNA can clearly identify stem cells in the intestinal crypt, which allows VH and CD to be measured, while Ki-67 marks the proliferating cells. The peptide transporter PepT1 is located on intestinal epithelial cells and plays a critical role in transporting di- and tripeptides. Muc2, which is secreted from goblet cells, forms mucus that lines the intestinal epithelial cells acting as a layer of protective coating. Cobb 500 chicks, hatching within a 12 h window, were randomly allocated into three experimental groups: control with no feed delay (ND), 24 h feed delay (D24), and 36 h feed delay (D36). Quantification of Olfm4, Ki-67, PepT1, and Muc2 mRNA abundance were investigated by quantitative PCR, in duodenum, jejunum, and ileum at 0 h, 24 h, 36 h, 72 h, 120 h, and 168 h posthatch. Additionally, localization of cells expressing each gene was visualized using in-situ hybridization at all listed times except 168 h posthatch. Statistical analysis was performed using JMP Pro 14, and significant differences between treatments within a collection day were determined by t-test and one-way ANOVA (P < 0.05). In the ND group, duodenal CD at 0 h was greatest compared to all other time points. With DAF, the duodenal VH of D36 chicks was lower at 36 h (P < 0.001) and 72 h (P = 0.002) compared to ND chicks. In the jejunum and ileum, the VH of D36 chicks was lower at 120 h (P = 0.005) and 72 h (P = 0.03), respectively, compared to ND chicks. In contrast, the VH of D24 chicks at 24 h was greater than ND (P = 0.004) in the jejunum. There was no difference between treatments by 168 h in all intestinal segments. The CD was also lower in DAF groups compared to ND but only in the jejunum and ileum. In contrast, duodenal CD was greater in D24 chicks at 24 h (P = 0.039) and in D36 chicks at 36 h (P < 0.0001) compared to ND chicks, but the difference was no longer significant by 72 h. The VH/CD ratio was lower in all three segments, except the ileum displayed a greater VH/CD ratio in D24 and D36 chicks at 24 h and 36 h, respectively, compared to ND chicks. The mRNA abundance of Olfm4 and Ki-67 was greater in DAF groups upon refeeding, but not until 120 h. The PepT1 mRNA abundance was greater in DAF groups while the abundance of Muc2 mRNA was lower. This difference in mRNA abundance level was more prominent in the duodenum and jejunum. From the analysis of number and distribution of goblet cells found in the upper half and lower half of the villi, expressed as a ratio (VU/VL), a greater ratio was observed in delayed groups compared to ND. In summary, while DAF resulted in altered small intestinal morphology with an effect more pronounced in D36 than D24 chicks, upon refeeding, some genes important to intestinal development were upregulated as a response to the treatment.
- Demonstration of peptide and free amino acid absorption by sheep forestomach epithelium using parabiotic chambers and identification of H⁺/peptide and free amino acid transport proteins in sheep omasal epithelium and bo,+ amino acid transport proteins in pig jejunal epithelium by expression of mRNA in Xenopus laevis oocytesMatthews, James Clyde (Virginia Tech, 1995-04-12)The absorption of methionine and methionylglycine (Met-Gly) across sheep (average BW = 38 kg) ruminal and omasal epithelia was studied using parabiotic chambers. Ruminal tissue demonstrated a greater ability to accumulate both substrates. Omasal tissue demonstrated a greater ability to translocate methionine and Met-Gly and a greater total absorption of both. Intact Met-Gly was transferred across both tissues. More was hydrolyzed by omasal epithelia. Within tissues, the total absorption of substrates did not differ. Evidence for carrier-mediated absorption was not observed. The ability to express exogenous mRNA in defolliculated Xenopus laevis oocytes was developed using sucrose gradient size-fractionated poly(A)⁺ RNA (RNA) isolated from the jejunal epithelial tissue of pig (average BW = 33.8 kg). Compared to water-injected oocytes, RNA injected oocytes displayed greater rates of Nat-independent lysine and leucine absorption. RNA-induced uptake of lysine (Kt = 52 uM) and leucine (Kt = 97 uM) was inhibited by 5 mM leucine and lysine, respectively, by .2 mM cysteine, but not by 5 mM glutamate. RNA-induced lysine and leucine absorption also was inhibited when oocytes were injected with RNA plus DNA oligomers that were complementary to the cloned human kidney b⁰‘⁺ transporter. Oocytes were injected with RNA isolated from omasal epithelial tissue of sheep (average BW = 67.5 kg) to identify potential peptide and amino acid transport proteins. Injection of specific RNA fractions induced greater rates of glycylsarcosine (Gly-Sar) uptake, as compared to water injection of oocytes. Media pH of less than 6.5 was required for induced Gly-Sar uptake. Induced Gly-Sar uptake required a pH of less than 6.5, was saturable (Kt = .40 mM), and was inhibited by 5 mM carnosine, Met-Gly, glycylleucine, but not by glycine. The RNA-induced Gly-Sar absorption was completely inhibited when oocytes were co-injected with RNA and DNA oligomers that were complementary to the cloned rabbit H⁺/peptide cotransporter. When oocytes were assayed for their ability to absorb lysine, RNA-induced lysine absorption was determined to be Na⁺-independent and to display b⁰‘⁺-like transport activity. Collectively, these results indicate that sheep omasal epithelia possess the potential to absorb free and peptide-bound amino acids by non-mediated processes and possess mRNA that encode for Ht-dependent dipeptide and b⁰‘⁺ transport protein activity. mRNA that encodes for b⁰‘⁺-like transport activity was identified in the jejunal epithelium of growing pigs.
- Detection and Identification of Acinobacillus pleuropneumoniae serotype 5 by multiplex Polymerase Chain ReactionLo, Terry (Virginia Tech, 1997-07-31)Traditional serologic assays of Actinobacillus pleuropeumoniae often have problems with cross-reactivity. To avoid the complications of antibody-antigen reactions, a PCR assay was developed to detect Actinobacillus pleuropneumoniae and identify serotype 5 strains. Primers specific to the conserved capsular export region of A. pleuropneumoniae amplified a 0.7 kb DNA band in all strains with the exception of serotype 4. A second set of primers specific to the unique capsular biosynthesis region of serotype 5 amplified a unique 1.1 kb band for serotype 5 only. The sensitivity of this assay was determined to be less than 100 colony forming units. This PCR assay enables us to detect A. pleuronpeumoniae and definitively distinguishes serotype 5 strains from other serotyes.
- Developmental and Growth Hormone Regulation of the Expression of Liver-Enriched Transcription Factors in Bovine LiverEleswarapu, Satyanarayana Venkata (Virginia Tech, 2004-05-11)Liver gene expression changes during development and is affected by growth hormone (GH). These changes in gene expression may be due to the differential expression of the liver-enriched transcription factors (LETFs). To study the potential involvement of LETFs in the regulation of gene expression in the bovine liver, we cloned the cDNA fragments of nine bovine LETFs, including hepatocyte nuclear factor (HNF)-1Æ Ã , 1Æ Ã , 3Æ Ã , 3Æ Ã , 3Æ Ã , 6, albumin D-element binding protein (DBP), and CCAAT/enhancer-binding proteins (C/EBP) -Æ Ã and Æ Ã , and compared the expression levels of them between adult and fetal bovine liver and between GH-treated and untreated adult bovine liver. The mRNA abundance of the LETFs was determined by ribonuclease protection assay (RPA). The cloned bovine LETF cDNA sequences showed high degrees of similarity (79 % to 99 %) to the LETF sequences of other species. The mRNA levels of HNF-1Æ Ã , HNF-3Æ Ã , and HNF-6 were significantly higher (P < 0.05) in the fetal liver (n=3) than in the adult liver (n=7). There were significant increases (P < 0.05) in the mRNA expression of HNF-3Æ Ã and HNF-6 in the liver of cows 24 h (n=6) and 1w (n=6) after GH administration. The results of this study suggest that HNF-1Æ Ã , HNF-3Æ Ã , and HNF-6 may play a role in differential regulation of gene expression between the fetal and adult bovine liver and that HNF-3Æ Ã and HNF-6 may be also involved in GH regulation of gene expression in the bovine liver.