Publications by authors named "Bhanu Prakash V L Telugu"

18 Publications

  • Page 1 of 1

SMARCAD1 Contributes to the Regulation of Naive Pluripotency by Interacting with Histone Citrullination.

Cell Rep 2017 03;18(13):3117-3128

Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

Histone citrullination regulates diverse cellular processes. Here, we report that SMARCAD1 preferentially associates with H3 arginine 26 citrullination (H3R26Cit) peptides present on arrays composed of 384 histone peptides harboring distinct post-transcriptional modifications. Among ten histone modifications assayed by ChIP-seq, H3R26Cit exhibited the most extensive genomewide co-localization with SMARCAD1 binding. Increased Smarcad1 expression correlated with naive pluripotency in pre-implantation embryos. In the presence of LIF, Smarcad1 knockdown (KD) embryonic stem cells lost naive state phenotypes but remained pluripotent, as suggested by morphology, gene expression, histone modifications, alkaline phosphatase activity, energy metabolism, embryoid bodies, teratoma, and chimeras. The majority of H3R26Cit ChIP-seq peaks occupied by SMARCAD1 were associated with increased levels of H3K9me3 in Smarcad1 KD cells. Inhibition of H3Cit induced H3K9me3 at the overlapping regions of H3R26Cit peaks and SMARCAD1 peaks. These data suggest a model in which SMARCAD1 regulates naive pluripotency by interacting with H3R26Cit and suppressing heterochromatin formation.
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http://dx.doi.org/10.1016/j.celrep.2017.02.070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5466819PMC
March 2017

Enhanced Development of Skeletal Myotubes from Porcine Induced Pluripotent Stem Cells.

Sci Rep 2017 02 6;7:41833. Epub 2017 Feb 6.

C.S. Bond Life Sciences Center, University of Missouri, Columbia, MO 6521, USA.

The pig is recognized as a valuable model in biomedical research in addition to its agricultural importance. Here we describe a means for generating skeletal muscle efficiently from porcine induced pluripotent stem cells (piPSC) in vitro thereby providing a versatile platform for applications ranging from regenerative biology to the ex vivo cultivation of meat. The GSK3B inhibitor, CHIR99021 was employed to suppress apoptosis, elicit WNT signaling events and drive naïve-type piPSC along the mesoderm lineage, and, in combination with the DNA methylation inhibitor 5-aza-cytidine, to activate an early skeletal muscle transcription program. Terminal differentiation was then induced by activation of an ectopically expressed MYOD1. Myotubes, characterized by myofibril development and both spontaneous and stimuli-elicited excitation-contraction coupling cycles appeared within 11 days. Efficient lineage-specific differentiation was confirmed by uniform NCAM1 and myosin heavy chain expression. These results provide an approach for generating skeletal muscle that is potentially applicable to other pluripotent cell lines and to generating other forms of muscle.
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http://dx.doi.org/10.1038/srep41833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5292944PMC
February 2017

MicroRNA-Mediated Reprogramming of Somatic Cells into Induced Pluripotent Stem Cells.

Methods Mol Biol 2015 ;1330:29-36

Department of Animal and Avian Sciences, University of Maryland, 2121 ANSC Building, College Park, MD, 20742, USA.

MicroRNAs or miRNAs belong to a class of small noncoding RNAs that play a crucial role in posttranscriptional regulation of gene expression. Nascent miRNAs are expressed as a longer transcript, which are then processed into a smaller 18-23-nucleotide mature miRNAs that bind to the target transcripts and induce cleavage or inhibit translation. MiRNAs therefore represent another key regulator of gene expression in establishing and maintaining unique cellular fate. Several classes of miRNAs have been identified to be uniquely expressed in embryonic stem cells (ESC) and regulated by the core transcription factors Oct4, Sox2, and Klf4. One such class of miRNAs is the mir-302/367 cluster that is enriched in pluripotent cells in vivo and in vitro. Using the mir-302/367 either by themselves or in combination with the Yamanaka reprogramming factors (Oct4, Sox2, c-Myc, and Klf4) has resulted in the establishment of induced pluripotent stem cells (iPSC) with high efficiencies. In this chapter, we outline the methodologies for establishing and utilizing the miRNA-based tools for reprogramming somatic cells into iPSC.
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http://dx.doi.org/10.1007/978-1-4939-2848-4_3DOI Listing
September 2016

Differentiation of trophoblast cells from human embryonic stem cells: to be or not to be?

Reproduction 2014 May 10;147(5):D1-12. Epub 2014 Apr 10.

Division of Animal Sciences.

It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.
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http://dx.doi.org/10.1530/REP-14-0080DOI Listing
May 2014

Role of stem cells in large animal genetic engineering in the TALENs-CRISPR era.

Reprod Fertil Dev 2013 ;26(1):65-73

Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742, USA.

The establishment of embryonic stem cells (ESCs) and gene targeting technologies in mice has revolutionised the field of genetics. The relative ease with which genes can be knocked out, and exogenous sequences introduced, has allowed the mouse to become the prime model for deciphering the genetic code. Not surprisingly, the lack of authentic ESCs has hampered the livestock genetics field and has forced animal scientists into adapting alternative technologies for genetic engineering. The recent discovery of the creation of induced pluripotent stem cells (iPSCs) by upregulation of a handful of reprogramming genes has offered renewed enthusiasm to animal geneticists. However, much like ESCs, establishing authentic iPSCs from the domestic animals is still beset with problems, including (but not limited to) the persistent expression of reprogramming genes and the lack of proven potential for differentiation into target cell types both in vitro and in vivo. Site-specific nucleases comprised of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regulated interspaced short palindromic repeats (CRISPRs) emerged as powerful genetic tools for precisely editing the genome, usurping the need for ESC-based genetic modifications even in the mouse. In this article, in the aftermath of these powerful genome editing technologies, the role of pluripotent stem cells in livestock genetics is discussed.
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http://dx.doi.org/10.1071/RD13258DOI Listing
August 2014

Model systems for studying trophoblast differentiation from human pluripotent stem cells.

Cell Tissue Res 2012 Sep 17;349(3):809-24. Epub 2012 Mar 17.

Division of Animal Sciences & Bond Life Sciences Center, University of Missouri-Columbia, 65211, USA.

This review focuses on a now well-established model for generating cells of the trophoblast (TB) lineage by treating human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) with the growth factor BMP4. We first discuss the opposing roles of FGF2 and BMP4 in directing TB formation and the need to exclude the former from the growth medium to minimize the co-induction of mesoderm and endoderm. Under these conditions, there is up-regulation of several transcription factors implicated in TB lineage emergence within 3 h of BMP4 exposure and, over a period of days and especially under a high O(2) gas atmosphere, gradual appearance of cell types carrying markers for more differentiated TB cell types, including extravillous TB and syncytioTB. We describe the potential value of including low molecular weight pharmaceutical agents that block activin A (INHBA) and FGF2 signaling to support BMP4-directed differentiation. We contend that the weight of available evidence supports the contention that BMP4 converts human ESC and iPSC of the so-called epiblast type unidirectionally to TB. We also consider the argument that BMP4 treatment of human ESC in the absence of exogenous FGF2 leads only to the emergence of mesoderm derivatives to be seriously flawed. Instead, we propose that, when signaling networks supporting pluripotency ESC or iPSC become unsustainable and when specification towards extra-embryonic mesoderm and endoderm are rendered inoperative, TB emerges as a major default state to pluripotency.
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http://dx.doi.org/10.1007/s00441-012-1371-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3429771PMC
September 2012

Generation of colonies of induced trophoblast cells during standard reprogramming of porcine fibroblasts to induced pluripotent stem cells.

Biol Reprod 2011 Oct 6;85(4):779-87. Epub 2011 Jul 6.

Bond Life Sciences Center and Division of Animal Sciences, University of Missouri, Columbia, Missouri, USA.

During reprogramming of porcine mesenchymal cells with a four-factor (POU5F1/SOX2/KLF4/MYC) mixture of vectors, a fraction of the colonies had an atypical phenotype and arose earlier than the recognizable porcine induced pluripotent stem (iPS) cell colonies. Within days after each passage, patches of cells with an epithelial phenotype formed raised domes, particularly under 20% O(2) conditions. Relative to gene expression of the iPS cells, there was up-regulation of genes for transcription factors associated with trophoblast (TR) lineage emergence, e.g., GATA2, PPARG, MSX2, DLX3, HAND1, GCM1, CDX2, ID2, ELF5, TCFAP2C, and TEAD4 and for genes required for synthesis of products more typical of differentiated TR, such as steroids (HSD17B1, CYP11A1, and STAR), pregnancy-associated glycoproteins (PAG6), and select cytokines (IFND, IFNG, and IL1B). Although POU5F1 was down-regulated relative to that in iPS cells, it was not silenced in the induced TR (iTR) cells over continued passage. Like iPS cells, iTR cells did not senesce on extended passage and displayed high telomerase activity. Upon xenografting into immunodeficient mice, iTR cells formed nonhemorrhagic teratomas composed largely of layers of epithelium expressing TR markers. When cultured under conditions that promoted embryoid body formation, iTR cells formed floating spheres consisting of a single epithelial sheet whose cells were tethered laterally by desmosome-like structures. In conclusion, reprogramming of porcine fibroblasts to iPS cells generates, as a by-product, colonies composed of self-renewing populations of TR cells, possibly containing TR stem cells.
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http://dx.doi.org/10.1095/biolreprod.111.092809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3184293PMC
October 2011

Leukemia inhibitory factor (LIF)-dependent, pluripotent stem cells established from inner cell mass of porcine embryos.

J Biol Chem 2011 Aug 24;286(33):28948-28953. Epub 2011 Jun 24.

Division of Animal Sciences, University of Missouri, Columbia, Missouri 65211-7310; Department of Biochemistry, University of Missouri, Columbia, Missouri 65211-7310. Electronic address:

The pig is important for agriculture and as an animal model in human and veterinary medicine, yet despite over 20 years of effort, there has been a failure to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of leukemia inhibitory factor-dependent, so-called naive type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. The alkaline phosphatase-positive colonies resulting from reprogramming resemble mouse embryonic stem cells in colony morphology, cell cycle interval, transcriptome profile, and expression of pluripotent markers, such as POU5F1, SOX2, and surface marker SSEA1. They are dependent on leukemia inhibitory factor signaling for maintenance of pluripotency, can be cultured over extended passage, and have the ability to form teratomas. These cells derived from the inner cell mass of pig blastocysts are clearly distinct from the FGF2-dependent "primed" induced pluripotent stem cells described recently from porcine mesenchymal cells. The data are consistent with the hypothesis that the up-regulation of KLF4, as well as POU5F1, is required to create and stabilize the naive pluripotent state and may explain why the derivation of embryonic stem cells from pigs and other ungulates has proved so difficult.
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http://dx.doi.org/10.1074/jbc.M111.229468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3190702PMC
August 2011

Differentiation of induced pluripotent stem cells of swine into rod photoreceptors and their integration into the retina.

Stem Cells 2011 Jun;29(6):972-80

Department of Ophthalmology, The second Xiangya Hospital, Central South University, Changsha, Hunan, People's Republic of China.

Absence of a regenerative pathway for damaged retina following injury or disease has led to experiments using stem cell transplantation for retinal repair, and encouraging results have been obtained in rodents. The swine eye is a closer anatomical and physiological match to the human eye, but embryonic stem cells have not been isolated from pig, and photoreceptor differentiation has not been demonstrated with induced pluripotent stem cells (iPSCs) of swine. Here, we subjected iPSCs of swine to a rod photoreceptor differentiation protocol consisting of floating culture as embryoid bodies followed by differentiation in adherent culture. Real-time PCR and immunostaining of differentiated cells demonstrated loss of expression of the pluripotent genes POU5F1, NANOG, and SOX2 and induction of rod photoreceptor genes RCVRN, NRL, RHO, and ROM1. While these differentiated cells displayed neuronal morphology, culturing on a Matrigel substratum triggered a further morphological change resulting in concentration of rhodopsin (RHO) and rod outer segment-specific membrane protein 1 in outer segment-like projections resembling those on primary cultures of rod photoreceptors. The differentiated cells were transplanted into the subretinal space of pigs treated with iodoacetic acid to eliminate rod photoreceptors. Three weeks after transplantation, engrafted RHO+ cells were evident in the outer nuclear layer where photoreceptors normally reside. A portion of these transplanted cells had generated projections resembling outer segments. These results demonstrate that iPSCs of swine can differentiate into photoreceptors in culture, and these cells can integrate into the damaged swine neural retina, thus, laying a foundation for future studies using the pig as a model for retinal stem cell transplantation.
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http://dx.doi.org/10.1002/stem.637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263955PMC
June 2011

Porcine induced pluripotent stem cells analogous to naïve and primed embryonic stem cells of the mouse.

Int J Dev Biol 2010 ;54(11-12):1703-11

Division of Animal Sciences, University of Missouri, Columbia, MO, USA.

Authentic or naïve embryonic stem cells (ESC) have probably never been derived from the inner cell mass (ICM) of pig blastocysts, despite over 25 years of effort. Recently, several groups, including ours, have reported induced pluripotent stem cells (iPSC) from swine by reprogramming somatic cells with a combination of four factors, OCT4 (POU5F1)/SOX2/KLF4/c-MYC delivered by retroviral transduction. The porcine (p) iPSC resembled human (h) ESC and the mouse "Epiblast stem cells" (EpiSC) in their colony morphology and expression of pluripotent genes, and are likely dependent on FGF2/ACTIVIN/NODAL signaling, therefore representing a primed ESC state. These cells are likely to advance swine as a model in biomedical research, since grafts could potentially be matched to the animal that donated the cells for re-programming. The objective of the present work has been to develop naïve piPSC. Employing a combination of seven reprogramming factors assembled on episomal vectors, we successfully reprogrammed porcine embryonic fibroblasts on a modified LIF-medium supplemented with two kinase inhibitors; CHIR99021, which inhibits GSK-3beta, and PD0325901, a MEK inhibitor. The derived piPSC bear a striking resemblance to naïve mESC in colony morphology, are dependent on LIF to maintain an undifferentiated phenotype, and express markers consistent with pluripotency. They exhibit high telomerase activity, a short cell cycle interval, and a normal karyotype, and are able to generate teratomas. Currently, the competence of these lines for contributing to germ-line chimeras is being tested.
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http://dx.doi.org/10.1387/ijdb.103200btDOI Listing
September 2011

An examination of the proteolytic activity for bovine pregnancy-associated glycoproteins 2 and 12.

Biol Chem 2010 Feb-Mar;391(2-3):259-70

Division of Animal Sciences, University of Missouri, 163 ASRC, Columbia, MO 65211, USA.

The pregnancy-associated glycoproteins (PAGs) represent a complex group of putative aspartic peptidases expressed exclusively in the placentas of species in the Artiodactyla order. The ruminant PAGs segregate into two classes: the 'ancient' and 'modern' PAGs. Some of the modern PAGs possess alterations in the catalytic center that are predicted to preclude their ability to act as peptidases. The ancient ruminant PAGs in contrast are thought to be peptidases, although no proteolytic activity has been described for these members. The aim of the present study was to investigate (1) if the ancient bovine PAGs (PAG-2 and PAG-12) have proteolytic activity, and (2) if there are any differences in activity between these two closely related members. Recombinant bovine PAG-2 and PAG-12 were expressed in a baculovirus expression system and the purified proteins were analyzed for proteolytic activity against a synthetic fluorescent cathepsin D/E substrate. Both proteins exhibited proteolytic activity with acidic pH optima. The k(cat)/K(m) for bovine PAG-2 was 2.7x10(5) m(-1) s(-1) and for boPAG-12 it was 6.8x10(4) m(-1) s(-1). The enzymes were inhibited by pepstatin A with a K(i) of 0.56 and 7.5 nm for boPAG-2 and boPAG-12, respectively. This is the first report describing proteolytic activity in PAGs from ruminant ungulates.
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http://dx.doi.org/10.1515/BC.2010.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2969838PMC
June 2010

The promise of stem cell research in pigs and other ungulate species.

Stem Cell Rev Rep 2010 Mar;6(1):31-41

Division of Animal Sciences, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, 65211, USA.

Despite two decades of effort, establishment of pluripotent embryonic stem cells (ESC) from ungulates such as cattle and pigs has remained an elusive goal, with true ESC only successfully isolated from rodents and primates. The many reports describing ESC-like cultures from other "difficult" species has largely depended upon adopting strategies successful for mouse and human and have not yet produced a cell type that both proliferated continuously in culture without differentiation and demonstrated full pluripotent potential. These difficulties may have been exacerbated in ungulates by the lack of specific markers exclusive to inner cell mass (ICM) and its derivative the epiblast and by unique features of their preimplantation development. Especially important may have been the choice of culture condition, including growth factors, for establishing and sustaining the ESC. Recent modifications to culture medium, notably the inclusion of particular protein kinase inhibitors, have permitted ESC derivation from rat and previously "non-permissive" mouse strains. These conditions appear to stabilize the biochemical networks that sustain pluripotency and to render the cells dependent upon LIF signaling. In addition, the recent successful generation of induced pluripotent stem cells (iPSC) from pig by procedures that should be easily adapted to other species, is also likely to advance the area quickly. The pig is a particularly desirable species to create pluripotent cell lines because of its value as a biomedical model in transplantation at a time when there is mounting pressure to rush stem cells to the clinic before their safety has been adequately tested in animals.
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http://dx.doi.org/10.1007/s12015-009-9101-1DOI Listing
March 2010

Induced pluripotent stem cells from swine (Sus scrofa): why they may prove to be important.

Cell Cycle 2009 Oct 21;8(19):3078-81. Epub 2009 Oct 21.

Division of Animal Sciences, University of Missouri, Columbia, MO, USA.

Three recent papers, published almost simultaneously by different groups, have described the generation of induced pluripotent stem (iPS) cells from the pig, a species whose size, anatomy and physiology render them attractive as clinical models for the human. The approach used in each case was to infect somatic cells with integrating retroviral vectors designed to express four reprogramming genes (POU5F1, SOX2, cMYC and KLF4). The cell lines generated met the standard criteria for pluripotency, including the ability to differentiate along multiple tissue lineages. In most respects, the porcine iPS cells more resembled human embryonic stem cells and human iPS cells than their murine equivalents. Provided such porcine iPS cells can be "personalized" to specific pigs and then coaxed to differentiate along specific lineages, it should be possible to use such animals to test transplantation therapies with iPS cells for safety and efficacy before the procedures are applied to human patients.
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http://dx.doi.org/10.4161/cc.8.19.9589DOI Listing
October 2009

Derivation of induced pluripotent stem cells from pig somatic cells.

Proc Natl Acad Sci U S A 2009 Jul 18;106(27):10993-8. Epub 2009 Jun 18.

Division of Animal Sciences, University of Missouri, Columbia, MO 65211, USA.

For reasons that are unclear the production of embryonic stem cells from ungulates has proved elusive. Here, we describe induced pluripotent stem cells (iPSC) derived from porcine fetal fibroblasts by lentiviral transduction of 4 human (h) genes, hOCT4, hSOX2, hKLF4, and hc-MYC, the combination commonly used to create iPSC in mouse and human. Cells were cultured on irradiated mouse embryonic fibroblasts (MEF) and in medium supplemented with knockout serum replacement and FGF2. Compact colonies of alkaline phosphatase-positive cells emerged after approximately 22 days, providing an overall reprogramming efficiency of approximately 0.1%. The cells expressed porcine OCT4, NANOG, and SOX2 and had high telomerase activity, but also continued to express the 4 human transgenes. Unlike human ESC, the porcine iPSC (piPSC) were positive for SSEA-1, but negative for SSEA-3 and -4. Transcriptional profiling on Affymetrix (porcine) microarrays and real time RT-PCR supported the conclusion that reprogramming to pluripotency was complete. One cell line, ID6, had a normal karyotype, a cell doubling time of approximately 17 h, and has been maintained through >220 doublings. The ID6 line formed embryoid bodies, expressing genes representing all 3 germ layers when cultured under differentiating conditions, and teratomas containing tissues of ectoderm, mesoderm, and endoderm origin in nude mice. We conclude that porcine somatic cells can be reprogrammed to form piPSC. Such cell lines derived from individual animals could provide a means for testing the safety and efficacy of stem cell-derived tissue grafts when returned to the same pigs at a later age.
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http://dx.doi.org/10.1073/pnas.0905284106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2698893PMC
July 2009

Characterization of the bovine pregnancy-associated glycoprotein gene family--analysis of gene sequences, regulatory regions within the promoter and expression of selected genes.

BMC Genomics 2009 Apr 24;10:185. Epub 2009 Apr 24.

Division of Animal Sciences, University of Missouri, Columbia, MO 65211, USA.

Background: The Pregnancy-associated glycoproteins (PAGs) belong to a large family of aspartic peptidases expressed exclusively in the placenta of species in the Artiodactyla order. In cattle, the PAG gene family is comprised of at least 22 transcribed genes, as well as some variants. Phylogenetic analyses have shown that the PAG family segregates into 'ancient' and 'modern' groupings. Along with sequence differences between family members, there are clear distinctions in their spatio-temporal distribution and in their relative level of expression. In this report, 1) we performed an in silico analysis of the bovine genome to further characterize the PAG gene family, 2) we scrutinized proximal promoter sequences of the PAG genes to evaluate the evolution pressures operating on them and to identify putative regulatory regions, 3) we determined relative transcript abundance of selected PAGs during pregnancy and, 4) we performed preliminary characterization of the putative regulatory elements for one of the candidate PAGs, bovine (bo) PAG-2.

Results: From our analysis of the bovine genome, we identified 18 distinct PAG genes and 14 pseudogenes. We observed that the first 500 base pairs upstream of the translational start site contained multiple regions that are conserved among all boPAGs. However, a preponderance of conserved regions, that harbor recognition sites for putative transcriptional factors (TFs), were found to be unique to the modern boPAG grouping, but not the ancient boPAGs. We gathered evidence by means of Q-PCR and screening of EST databases to show that boPAG-2 is the most abundant of all boPAG transcripts. Finally, we provided preliminary evidence for the role of ETS- and DDVL-related TFs in the regulation of the boPAG-2 gene.

Conclusion: PAGs represent a relatively large gene family in the bovine genome. The proximal promoter regions of these genes display differences in putative TF binding sites, likely contributing to observed differences in spatial and temporal expression. We also discovered that boPAG-2 is the most abundant of all boPAG transcripts and provided evidence for the role of ETS and DDVL TFs in its regulation. These experiments mark the crucial first step in discerning the complex transcriptional regulation operating within the boPAG gene family.
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http://dx.doi.org/10.1186/1471-2164-10-185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2682831PMC
April 2009

The genome sequence of taurine cattle: a window to ruminant biology and evolution.

Science 2009 Apr;324(5926):522-8

To understand the biology and evolution of ruminants, the cattle genome was sequenced to about sevenfold coverage. The cattle genome contains a minimum of 22,000 genes, with a core set of 14,345 orthologs shared among seven mammalian species of which 1217 are absent or undetected in noneutherian (marsupial or monotreme) genomes. Cattle-specific evolutionary breakpoint regions in chromosomes have a higher density of segmental duplications, enrichment of repetitive elements, and species-specific variations in genes associated with lactation and immune responsiveness. Genes involved in metabolism are generally highly conserved, although five metabolic genes are deleted or extensively diverged from their human orthologs. The cattle genome sequence thus provides a resource for understanding mammalian evolution and accelerating livestock genetic improvement for milk and meat production.
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http://dx.doi.org/10.1126/science.1169588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943200PMC
April 2009

Acid peptidase activity released from in vitro produced porcine embryos: a candidate marker to predict developmental competence.

Mol Reprod Dev 2009 Apr;76(4):417-28

Division of Animal Sciences, University of Missouri, Columbia, Missouri 65211, USA.

The ability to efficiently create high quality embryos, competent to produce normal viable offspring in vitro, facilitates diverse technological advancements in animal agriculture and assisted reproduction. Current methods for evaluation of embryos are predominantly based on morphological characteristics which are prone to potential bias of the scorer. Metabolic and genetic markers have also been explored for quality assessment, but they are cost prohibitive or require longer periods of time for evaluation. We hypothesized that secreted enzymes could provide another means of embryo quality assessment. In this report, we provide evidence that medium conditioned by porcine embryos often has proteolytic activity that operates in acidic conditions (acid peptidase activity or APA). The APA could be inhibited by pepstatin A, suggesting that the activity is derived from one or more aspartic peptidases. We also provide evidence that single embryos, incubated for as few as 24 hr, released enough APA that it was possible to measure it accurately at day 5 of culture. We also observed that such activity on day 6 could be positively correlated with advanced developmental stage and embryo quality. In addition, those embryos that were graded identically by morphological evaluations often differed in the amount of APA--with some being significantly higher than the experimental threshold value. Therefore, the APA of embryos might serve as an additional marker for evaluation of embryos.
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http://dx.doi.org/10.1002/mrd.20965DOI Listing
April 2009

Characterization of the peptidase activity of recombinant porcine pregnancy-associated glycoprotein-2.

J Biochem 2008 Dec 3;144(6):725-32. Epub 2008 Oct 3.

Division of Animal Sciences, University of Missouri, Columbia, MO 65211, USA.

The pregnancy-associated glycoproteins (PAGs) belong to the aspartic peptidase family. They are expressed exclusively in trophoblasts of even-toed ungulates such as swine, cattle, sheep, etc. In pigs, two distinct PAG transcripts (and some variants) have been described. One of the transcripts, porcine PAG-1 (poPAG-1) may not be capable of acting as a peptidase. The second transcript, poPAG-2, possesses a conserved catalytic centre and has been predicted, but not shown, to have proteolytic activity. The thrust of this work was to test such a possibility. PoPAG-2 was expressed as a recombinant protein with an amino-terminal 'FLAG-tag' in a Baculoviral expression system. The expressed proteins were affinity purified by using an anti-FLAG antibody. The purified preparations were then analysed for proteolytic activity against a fluorescent substrate. Porcine PAG-2 had optimal proteolytic activity around pH 3.5. Against this substrate, it had a k(cat)/K(m) of 1.2 microM(-1) s(-1) and was inhibited by the aspartic peptidase inhibitor, pepstatin A, with a K(i) of 12.5 nM. Since the proteolytic activity of PAGs in the pig has now been established, the search for putative substrates to gain insight into the physiological role of PAGs will likely be the focus of future investigations.
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http://dx.doi.org/10.1093/jb/mvn127DOI Listing
December 2008