WorldWideScience

Sample records for cell somatic electromotility

  1. Outer Hair Cell Somatic Electromotility In Vivo and Power Transfer to the Organ of Corti

    OpenAIRE

    Ramamoorthy, Sripriya; Nuttall, Alfred L.

    2012-01-01

    The active amplification of sound-induced vibrations in the cochlea, known to be crucial for auditory sensitivity and frequency selectivity, is not well understood. The outer hair cell (OHC) somatic electromotility is a potential mechanism for such amplification. Its effectiveness in vivo is putatively limited by the electrical low-pass filtering of the cell's transmembrane potential. However, the transmembrane potential is an incomplete metric. We propose and estimate two metrics to evaluate...

  2. A membrane bending model of outer hair cell electromotility.

    OpenAIRE

    Raphael, R. M.; Popel, A S; Brownell, W. E.

    2000-01-01

    We propose a new mechanism for outer hair cell electromotility based on electrically induced localized changes in the curvature of the plasma membrane (flexoelectricity). Electromechanical coupling in the cell's lateral wall is modeled in terms of linear constitutive equations for a flexoelectric membrane and then extended to nonlinear coupling based on the Langevin function. The Langevin function, which describes the fraction of dipoles aligned with an applied electric field, is shown to be ...

  3. Modeling high-frequency electromotility of cochlear outer hair cell in microchamber experiment

    Science.gov (United States)

    Liao, Zhijie; Popel, Aleksander S.; Brownell, William E.; Spector, Alexander A.

    2005-04-01

    Cochlear outer hair cells (OHC) are critically important for the amplification and sharp frequency selectivity of the mammalian ear. The microchamber experiment has been an effective tool to analyze the OHC high-frequency performance. In this study, the OHC electrical stimulation in the microchamber is simulated. The model takes into account the inertial and viscous properties of fluids inside and outside the cell as well as the viscoelastic and piezoelectric properties of the cell composite membrane (wall). The closed ends of the cylindrical cell were considered as oscillatory rigid plates. The final solution was obtained in terms of Fourier series, and it was checked against the available results of the microchamber experiment. The conditions of the interaction between the cell and pipette was analyzed, and it was found that the amount of slip along the contact surface has a significant effect on the cell electromotile response. The cell's length changes were computed as a function of frequency, and their dependence on the viscosities of both fluids and the cell wall was analyzed. The distribution of the viscous losses inside the fluids was also estimated. The proposed approach can help in a better understanding of the high-frequency OHC electromotility under experimental and physiological conditions. .

  4. Monitoring Milk Somatic Cell Counts

    Directory of Open Access Journals (Sweden)

    Gheorghe Şteţca

    2014-11-01

    Full Text Available The presence of somatic cells in milk is a widely disputed issue in milk production sector. The somatic cell counts in raw milk are a marker for the specific cow diseases such as mastitis or swollen udder. The high level of somatic cells causes physical and chemical changes to milk composition and nutritional value, and as well to milk products. Also, the mastitic milk is not proper for human consumption due to its contribution to spreading of certain diseases and food poisoning. According to these effects, EU Regulations established the maximum threshold of admitted somatic cells in raw milk to 400000 cells / mL starting with 2014. The purpose of this study was carried out in order to examine the raw milk samples provided from small farms, industrial type farms and milk processing units. There are several ways to count somatic cells in milk but the reference accepted method is the microscopic method described by the SR EN ISO 13366-1/2008. Generally samples registered values in accordance with the admissible limit. By periodical monitoring of the somatic cell count, certain technological process issues are being avoided and consumer’s health ensured.

  5. Reprogrammed Pluripotent Stem Cells from Somatic Cells

    OpenAIRE

    Kim, Jong Soo; Choi, Hyun Woo; Choi, Sol; Do, Jeong Tae

    2011-01-01

    Pluripotent stem cells, such as embryonic stem (ES) cells, can differentiate into all cell types. So, these cells can be a biological resource for regenerative medicine. However, ES cells known as standard pluripotent cells have problem to be used for cell therapy because of ethical issue of the origin and immune response on the graft. Hence, recently reprogrammed pluripotent cells have been suggested as an alternative source for regenerative medicine. Somatic cells can acquire the ES cell-li...

  6. Reprogrammed pluripotent stem cells from somatic cells.

    Science.gov (United States)

    Kim, Jong Soo; Choi, Hyun Woo; Choi, Sol; Do, Jeong Tae

    2011-06-01

    Pluripotent stem cells, such as embryonic stem (ES) cells, can differentiate into all cell types. So, these cells can be a biological resource for regenerative medicine. However, ES cells known as standard pluripotent cells have problem to be used for cell therapy because of ethical issue of the origin and immune response on the graft. Hence, recently reprogrammed pluripotent cells have been suggested as an alternative source for regenerative medicine. Somatic cells can acquire the ES cell-like pluripotency by transferring somatic cell nuclei into oocytes, by cell fusion with pluripotent cells. Retroviral-mediated introduction of four factors, Oct4, Sox2, Klf4 and c-Myc can successfully reprogram somatic cells into ES cell-like pluripotent stem cells, known as induced pluripotent stem (iPS) cells. These cells closely resemble ES cells in gene expression pattern, cell biologic and phenotypic characteristics. However, to reach the eventual goal of clinical application, it is necessary to overcome the major drawbacks such as low reprogramming efficiency and genomic alterations due to viral integration. In this review, we discuss the current reprogramming techniques and mechanisms of nuclear reprogramming induced by transcription factor transduction. PMID:24298328

  7. China Succeeded in Somatic Cell Cloning

    Institute of Scientific and Technical Information of China (English)

    Song Jianlan

    2002-01-01

    @@ Chinese scientists have succeeded in cloning a colony of cattle from fully differentiated somatic cells. The news was announced jointly by the Chinese Academy of Sciences (CAS), National Natural Science Foundation of China (NSFC) and the government of Shandong Province at a press conference held on March 7, 2002.

  8. Somatic Cell Nuclear Transfer in the Mouse

    Science.gov (United States)

    Kishigami, Satoshi; Wakayama, Teruhiko

    Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals since “Dolly,” the first animal cloned from an adult cell was reported in 1997. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, this technique has been proven to work in more than 10 mammalian species. Among them, the mouse provides the best model for both basic and applied research of somatic cloning because of its abounding genetic resources, rapid sexual maturity and propagation, minimal requirements for housing, etc. This chapter describes a basic protocol for mouse cloning using cumulus cells, the most popular cell type for NT, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. In particular, we focus on a new, more efficient mouse cloning protocol using trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which increases both in vitro and in vivo developmental rates from twofold to fivefold. This new method including TSA will be helpful to establish mouse cloning in many laboratories.

  9. Somatic cell genetic approaches to Down's syndrome.

    Science.gov (United States)

    Patterson, D; Jones, C; Scoggin, C; Miller, Y E; Graw, S

    1982-01-01

    Somatic cell genetic analysis of mutants of Chinese hamster ovary cells with deficient purine synthesis and of hybrids between these mutants and human cells is described. Data are presented substantiating that two genes for enzymes of purine synthesis, AdeC and AdeG, can be coordinately regulated in mammalian cells. Analysis of a human-hamster hybrid cell, Ade C/21, which contains a normal complement of hamster chromosomes and human chromosome 21 as its only human genetic component recognizable by electrophoretic and immunogenetic techniques demonstrates that genes associated with the presence of human chromosome 21 and required for the synthesis of specific polypeptides and specific human lethal cell surface antigens can be detected in these hybrids. PMID:6217778

  10. Direct reprogramming of somatic cells: an update

    Directory of Open Access Journals (Sweden)

    Phuc Van Pham

    2015-03-01

    Full Text Available Direct epigenetic reprogramming is a technique that converts a differentiated adult cell into another differentiated cell and mdash;such fibroblasts to cardiomyocytes and mdash;without passage through an undifferentiated pluripotent stage. This novel technology is opening doors in biological research and regenerative medicine. Some preliminary studies about direct reprogramming started in the 1980s when differentiated adult cells could be converted into other differentiated cells by overexpressing transcription-factor genes. These studies also showed that differentiated cells have plasticity. Direct reprogramming can be a powerful tool in biological research and regenerative medicine, especially the new frontier of personalized medicine. This review aims to summarize all direct reprogramming studies of somatic cells by master control genes as well as potential applications of these techniques in research and treatment of selected human diseases. [Biomed Res Ther 2015; 2(3.000: 231-240

  11. Somatic Cell Count, Importance and Effect Factors in Dairy Cattle

    OpenAIRE

    İbrahim Aytekin; Saim Boztepe

    2014-01-01

    The somatic cell count (SCC) is commonly used as a measure of udder health and milk quality. Thus, to determine the milk quality standards in many countries, it legally determined as an indicator of somatic cell count raw milk and determines the level of payments to milk producers. The present study investigated that the somatic cell count is an indicator of udder health status, diagnosis of subclinical mastitis, health and quality of milk and milk products, its importance and effect factors ...

  12. Aneuploidy in mammalian somatic cells in vivo.

    Science.gov (United States)

    Cimino, M C; Tice, R R; Liang, J C

    1986-01-01

    Aneuploidy is an important potential source of human disease and of reproductive failure. Nevertheless, the ability of chemical agents to induce aneuploidy has been investigated only sporadically in intact (whole-animal) mammalian systems. A search of the available literature from the EMCT Aneuploidy File (for years 1970-1983) provided 112 papers that dealt with aneuploidy in mammalian somatic cells in vivo. 59 of these papers did not meet minimal criteria for analysis and were rejected from subsequent review. Of the remaining 53 papers that dealt with aneuploidy induction by chemical agents in mammalian somatic cells in vivo, only 3 (6%) contained data that were considered to be supported conclusively by adequate study designs, execution, and reporting. These 3 papers dealt with 2 chemicals, one of which, mercury, was negative for aneuploidy induction in humans, and the other, pyrimethamine, was positive in an experimental rodent study. The majority of papers (94%) were considered inconclusive for a variety of reasons. The most common reasons for calling a study inconclusive were (a) combining data on hyperploidy with those on hypoploidy and/or polyploidy, (b) an inadequate or unspecified number of animals and/or cells per animal scored per treatment group, and (c) poor data presentation such that animal-to-animal variability could not be assessed. Suggestions for protocol development are made, and the future directions of research into aneuploidy induction are discussed. PMID:3941670

  13. The histone chaperone CAF-1 safeguards somatic cell identity

    OpenAIRE

    Cheloufi, Sihem; Elling, Ulrich; Hopfgartner, Barbara; Youngsook L. Jung; Murn, Jernej; Ninova, Maria; Hubmann, Maria; Badeaux, Aimee I; Ang, Cheen Euong; Tenen, Danielle; Wesche, Daniel J; Abazova, Nadezhda; Hogue, Max; Tasdemir, Nilgun; Brumbaugh, Justin

    2016-01-01

    Cellular differentiation involves profound remodeling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNAi screens targeting chromatin factors during transcription factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPSCs). Remarkably, subunits of the chromatin assembly ...

  14. The histone chaperone CAF-1 safeguards somatic cell identity

    OpenAIRE

    Cheloufi, Sihem; Elling, Ulrich; Hopfgartner, Barbara; Youngsook L. Jung; Murn, Jernej; Ninova, Maria; Hubmann, Maria; Badeaux, Aimee I; Ang, Cheen Euong; Tenen, Danielle; Wesche, Daniel J; Abazova, Nadezhda; Hogue, Max; Tasdemir, Nilgun; Brumbaugh, Justin

    2015-01-01

    Cellular differentiation involves profound remodeling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNAi screens targeting chromatin factors during transcription factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPSCs). Remarkably, subunits of the chromatin assembly ...

  15. Cytogenetic analysis of human somatic cell haploidization.

    Science.gov (United States)

    Galat, V; Ozen, S; Rechitsky, S; Kuliev, A; Verlinsky, Y

    2005-02-01

    Despite recent interest in the derivation of female and male gametes through somatic cell nuclear transfer, there is still insufficient data on chromosomal analysis of these gametes resulting from haploidization, especially involving a human nuclear donor and recipient oocytes. The objective of this study was to investigate the fidelity of chromosomal separation during haploidization of human cumulus cells by in-vitro matured human enucleated MII oocytes. A total of 129 oocytes were tested 4-7, 8-14, or 15-21 h after nuclear transfer (NT) followed by electro-stimulation, resulting in 71.3% activation efficiency on average. Haploidization was documented by the formation of two separate groups of chromosomes, originating from either polar body/pronucleus (PB/PN), or only 2PN, which were tested by 5-colour FISH, or DNA analysis for copy number of chromosomes 13, 16, 18, 21, 22 and X. Two PN were formed more frequently than PB/PN, irrespective of incubation time. In agreement with recent reports on mouse oocytes, as many as 90.2% of the resulting haploid sets tested showed abnormal chromosome segregation, suggesting unsuitability of the resulting artificial gametes for practical application at the present time. PMID:15823223

  16. Alternative lengthening of telomeres in normal mammalian somatic cells

    OpenAIRE

    Neumann, Axel A.; Watson, Catherine M.; Noble, Jane R.; Hilda A Pickett; Tam, Patrick P.L.; Reddel, Roger R

    2013-01-01

    Alternative lengthening of telomeres (ALT), a mechanism involving the replication of new telomeric DNA from a DNA template, is used by some cancer cells to lengthen their telomeres. Reddel and colleagues now show that ALT activity exists in normal somatic tissues as well. A telomere with a DNA tag is found to be intertelomerically copied in normal somatic cells but not germline cells, providing important implications for understanding telomere maintenance and its evolution.

  17. The histone chaperone CAF-1 safeguards somatic cell identity

    OpenAIRE

    Cheloufi, Sihem; Ninova, Maria; Aravin, Alexei

    2015-01-01

    Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromat...

  18. Relationship between lactoferrin, minerals, and somatic cells in bovine milk

    OpenAIRE

    Soyeurt, Hélène; Arnould, Valérie; Bruwier, Damien; Dardenne, Pierre; Romnee, Jean-Michel; Gengler, Nicolas

    2008-01-01

    Selection for increased mastitis resistance is hampered by lack of available data. Currently, somatic cell count or score are proven indicators. However, it should be a priority to increase the number of available indicator traits for mastitis resistance. The aim of this research was to study the relationships among potential indicator traits as lactoferrin content, concentrations of major minerals in milk (calcium, Ca; sodium, Na; phosphore, P), and somatic cell count. Firs...

  19. Mouse cloning and somatic cell reprogramming using electrofused blastomeres

    Institute of Scientific and Technical Information of China (English)

    Amjad Riaz; Xiaoyang Zhao; Xiangpeng Dai; Wei Li; Lei Liu; Haifeng Wan; Yang Yu; Liu Wang; Qi Zhou

    2011-01-01

    Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem(ES)cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved.Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.

  20. File list: Oth.Gon.10.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.10.AllAg.Testicular_somatic_cells mm9 TFs and others Gonad Testicular somat...ic cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Gon.10.AllAg.Testicular_somatic_cells.bed ...

  1. File list: Oth.Gon.20.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.20.AllAg.Testicular_somatic_cells mm9 TFs and others Gonad Testicular somat...ic cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Gon.20.AllAg.Testicular_somatic_cells.bed ...

  2. File list: Pol.Gon.05.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Gon.05.AllAg.Testicular_somatic_cells mm9 RNA polymerase Gonad Testicular somat...ic cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Gon.05.AllAg.Testicular_somatic_cells.bed ...

  3. Somatic Cell Dedifferentiation/Reprogramming for Regenerative Medicine

    OpenAIRE

    Ramesh, Thiyagarajan; Lee, Sun-Hee; Lee, Choon-Soo; Kwon, Yoo-Wook; Cho, Hyun-Jai

    2009-01-01

    The concept of dedifferentiation or reprogramming of a somatic cell into a pluripotent embryonic stem cell-like cell (ES-like cell), which give rise to three germ layers and differentiate various cell types, opens a new era in stem cell biology and provides potential therapeutic modality in regenerative medicine. Here, we outline current dedifferentiation/reprogramming methods and their technical hurdles, and the safety and therapeutic applications of reprogrammed pluripotent stem cells in re...

  4. Generation of bovine transgenics using somatic cell nuclear transfer

    Directory of Open Access Journals (Sweden)

    Stice Steven L

    2003-11-01

    Full Text Available Abstract The ability to produce transgenic animals through the introduction of exogenous DNA has existed for many years. However, past methods available to generate transgenic animals, such as pronuclear microinjection or the use of embryonic stem cells, have either been inefficient or not available in all animals, bovine included. More recently somatic cell nuclear transfer has provided a method to create transgenic animals that overcomes many deficiencies present in other methods. This review summarizes the benefits of using somatic cell nuclear transfer to create bovine transgenics as well as the possible opportunities this method creates for the future.

  5. The histone chaperone CAF-1 safeguards somatic cell identity.

    Science.gov (United States)

    Cheloufi, Sihem; Elling, Ulrich; Hopfgartner, Barbara; Jung, Youngsook L; Murn, Jernej; Ninova, Maria; Hubmann, Maria; Badeaux, Aimee I; Euong Ang, Cheen; Tenen, Danielle; Wesche, Daniel J; Abazova, Nadezhda; Hogue, Max; Tasdemir, Nilgun; Brumbaugh, Justin; Rathert, Philipp; Jude, Julian; Ferrari, Francesco; Blanco, Andres; Fellner, Michaela; Wenzel, Daniel; Zinner, Marietta; Vidal, Simon E; Bell, Oliver; Stadtfeld, Matthias; Chang, Howard Y; Almouzni, Genevieve; Lowe, Scott W; Rinn, John; Wernig, Marius; Aravin, Alexei; Shi, Yang; Park, Peter J; Penninger, Josef M; Zuber, Johannes; Hochedlinger, Konrad

    2015-12-10

    Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting. PMID:26659182

  6. Buffalo milk: proteins electrophoretic profile and somatic cell count

    Directory of Open Access Journals (Sweden)

    S. Mattii

    2011-03-01

    Full Text Available Water buffalo milk differs from the cow’s milk for greater fat and protein content, very important features in cheese making. Proteins, casein and whey-proteins in particular, are the most important factors determining cheese yield. Several previous research discussed the rule of SCC in cow milk production (Varisco, 1999 and the close relationship existing between cow’s milk cheese yield and somatic cell count (Barbano, 2000. In particular the inverse correlation between cheese yields and somatic cells’content have been demonstrated. In Italy the regulation in force DPR 54/97 acknowledges what expressed in EEC 46/92 Directive (Tripodi, 1999 without fixing the limit threshold of somatic cells for buffalo’s milk....

  7. Oocyte-somatic cells interactions, lessons from evolution

    Directory of Open Access Journals (Sweden)

    Charlier Cathy

    2012-10-01

    Full Text Available Abstract Background Despite the known importance of somatic cells for oocyte developmental competence acquisition, the overall mechanisms underlying the acquisition of full developmental competence are far from being understood, especially in non-mammalian species. The present work aimed at identifying key molecular signals from somatic origin that would be shared by vertebrates. Results Using a parallel transcriptomic analysis in 4 vertebrate species - a teleost fish, an amphibian, and two mammals - at similar key steps of developmental competence acquisition, we identified a large number of species-specific differentially expressed genes and a surprisingly high number of orthologous genes exhibiting similar expression profiles in the 3 tetrapods and in the 4 vertebrates. Among the evolutionary conserved players participating in developmental competence acquisition are genes involved in key processes such as cellular energy metabolism, cell-to-cell communications, and meiosis control. In addition, we report many novel molecular actors from somatic origin that have never been studied in the vertebrate ovary. Interestingly, a significant number of these new players actively participate in Drosophila oogenesis. Conclusions Our study provides a comprehensive overview of evolutionary-conserved mechanisms from somatic origin participating in oocyte developmental competence acquisition in 4 vertebrates. Together our results indicate that despite major differences in ovarian follicular structure, some of the key players from somatic origin involved in oocyte developmental competence acquisition would be shared, not only by vertebrates, but also by metazoans. The conservation of these mechanisms during vertebrate evolution further emphasizes the important contribution of the somatic compartment to oocyte quality and paves the way for future investigations aiming at better understanding what makes a good egg.

  8. File list: His.Gon.20.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Gon.20.AllAg.Testicular_somatic_cells mm9 Histone Gonad Testicular somatic cell...s SRX591729,SRX591717 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Gon.20.AllAg.Testicular_somatic_cells.bed ...

  9. Somatic cell count distributions during lactation predict clinical mastitis

    NARCIS (Netherlands)

    Green, M.J.; Green, L.E.; Schukken, Y.H.; Bradley, A.J.; Peeler, E.J.; Barkema, H.W.; Haas, de Y.; Collis, V.J.; Medley, G.F.

    2004-01-01

    This research investigated somatic cell count (SCC) records during lactation, with the purpose of identifying distribution characteristics (mean and measures of variation) that were most closely associated with clinical mastitis. Three separate data sets were used, one containing quarter SCC (n = 14

  10. Mouse cloning and somatic cell reprogramming using electrofused blastomeres

    OpenAIRE

    Riaz, Amjad; Zhao, Xiaoyang; Dai, Xiangpeng; Li, Wei; Liu, Lei; Wan, Haifeng; Yu, Yang; Wang, Liu; Zhou, Qi

    2010-01-01

    Mouse cloning from fertilized eggs can assist development of approaches for the production of “genetically tailored” human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to s...

  11. File list: Unc.Gon.20.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Gon.20.AllAg.Testicular_somatic_cells mm9 Unclassified Gonad Testicular somatic... cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Gon.20.AllAg.Testicular_somatic_cells.bed ...

  12. Generation of bovine transgenics using somatic cell nuclear transfer

    OpenAIRE

    Stice Steven L; Hodges Craig A

    2003-01-01

    Abstract The ability to produce transgenic animals through the introduction of exogenous DNA has existed for many years. However, past methods available to generate transgenic animals, such as pronuclear microinjection or the use of embryonic stem cells, have either been inefficient or not available in all animals, bovine included. More recently somatic cell nuclear transfer has provided a method to create transgenic animals that overcomes many deficiencies present in other methods. This revi...

  13. Reprogramming to pluripotency can conceal somatic cell chromosomal instability.

    Directory of Open Access Journals (Sweden)

    Masakazu Hamada

    Full Text Available The discovery that somatic cells are reprogrammable to pluripotency by ectopic expression of a small subset of transcription factors has created great potential for the development of broadly applicable stem-cell-based therapies. One of the concerns regarding the safe use of induced pluripotent stem cells (iPSCs in therapeutic applications is loss of genomic integrity, a hallmark of various human conditions and diseases, including cancer. Structural chromosome defects such as short telomeres and double-strand breaks are known to limit reprogramming of somatic cells into iPSCs, but whether defects that cause whole-chromosome instability (W-CIN preclude reprogramming is unknown. Here we demonstrate, using aneuploidy-prone mouse embryonic fibroblasts (MEFs in which chromosome missegregation is driven by BubR1 or RanBP2 insufficiency, that W-CIN is not a barrier to reprogramming. Unexpectedly, the two W-CIN defects had contrasting effects on iPSC genomic integrity, with BubR1 hypomorphic MEFs almost exclusively yielding aneuploid iPSC clones and RanBP2 hypomorphic MEFs karyotypically normal iPSC clones. Moreover, BubR1-insufficient iPSC clones were karyotypically unstable, whereas RanBP2-insufficient iPSC clones were rather stable. These findings suggest that aneuploid cells can be selected for or against during reprogramming depending on the W-CIN gene defect and present the novel concept that somatic cell W-CIN can be concealed in the pluripotent state. Thus, karyotypic analysis of somatic cells of origin in addition to iPSC lines is necessary for safe application of reprogramming technology.

  14. Mitochondria, cellular stress resistance, somatic cell depletion and lifespan.

    Science.gov (United States)

    Robb, Ellen L; Page, Melissa M; Stuart, Jeffrey A

    2009-03-01

    The causes of aging and determinants of maximum lifespan in animal species are multifaceted and complex. However, a wealth of experimental data suggests that mitochondria are involved both in the aging process and in regulating lifespan. Here we outline a somatic cell depletion (SCD) model to account for correlations between: (1) mitochondrial reactive oxygen species and lifespan; (2) mitochondrial antioxidant enzymes and lifespan; (3) mitochondrial DNA mutation and lifespan and (4) cellular stress resistance and lifespan. We examine the available data from within the framework of the SCD model, in which mitochondrial dysfunction leading to cell death and gradual loss of essential somatic cells eventually contributes to the decline in physiological performance that limits lifespan. This model is useful in explaining many of the mitochondrial manipulations that alter maximum lifespan in a variety of animal species; however, there are a number of caveats and critical experiments outstanding, and these are outlined in this review. PMID:20021396

  15. Somatic cell bovine cloning: Effect of donor cell and recipients

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Adult somatic cell nuclear transfer was conducted by using cultured ear fibroblast cells obtained from a Holstein female cow (GN) and a Galoway herd bull (GLV). The percentages of reconstructed eggs developed into blastocysts were similar in GN (23.98%, 123 of 513) and in GLV groups (29.55%, 138 of 467). However, the rate of reconstructed female (GN) embryos developed into term was higher than that of male (GLV) (8.02% and 1.82%, respectively). Three kinds of cows, Luxi Yellow cows, Holstein heifers and Holstein cows with normal reproductive records were used as recipients. When the reconstructed embryos from GN were transferred, there was no difference in the pregnancy rate among three kinds of recipients, but the abortion rate of Luxi Yellow cows was significantly higher (85.71%) than in the other two groups (14.29% and 0%, respectively; P < 0.05). And the percentages of newborn calves in transferred embryos were significantly different between Luxi Yellow cows and Holstein breed (1.54%, 10.39% and 20.0%, respectively, P < 0.05). However, when reconstructed embryos from GLV were transferred, there was no difference among three kinds of recipients in the pregnancy rate, the abortion rate and the delivery rate.

  16. Buffalo milk: proteins electrophoretic profile and somatic cell count

    OpenAIRE

    S. Mattii; B. Tommei; Pasquini, M.

    2011-01-01

    Water buffalo milk differs from the cow’s milk for greater fat and protein content, very important features in cheese making. Proteins, casein and whey-proteins in particular, are the most important factors determining cheese yield. Several previous research discussed the rule of SCC in cow milk production (Varisco, 1999) and the close relationship existing between cow’s milk cheese yield and somatic cell count (Barbano, 2000). In particular the inverse correlation between cheese ...

  17. Cytogenetic effects of irradiation on somatic and germ cells

    OpenAIRE

    Egozcue, Josep; Álvarez Arpal, Ricardo; Barquinero, J. F.; BARRIOS, L; Caballín, M. R.; Genescà i Garrigosa, Anna; Miró, Rosa; Ponsa Arjona, Immaculada; Tusell Padrós, Laura

    1999-01-01

    This paper summarizes the results obtained in two of the research projects carried out in our laboratory within the radiation protection programs of the Consejo de Seguridad Nuclear and the European Union. These two research lines are fundamentally interconnected, since the analysis of the cytogenetic effects of radiation on somatic cells studies the consequences of occupational or accidental exposure to radiation for the individual, especially from the point of view of developing some type o...

  18. Normal somatic cell count and subclinical mastitis in Murrah buffaloes.

    Science.gov (United States)

    Dhakal, I P

    2006-03-01

    This study was conducted to investigate the normal somatic cell count (SCC) and to define subclinical mastitis in Murrah buffaloes. Data were collected from 60 clinically normal buffaloes stationed at five farms of Chitwan Nepal and Buffalo Research Center, Hissar, India. Somatic cell count was measured using the Newman-Lampert staining technique. The upper limit of SCC was determined >or=200 000/ml of milk based on the mean +/- 2SD of a total SCC. Abnormal data of the SCC was repeatedly removed, which lie beyond the values of more than mean + 2SD until all the data come to lie within (mean + 2SD). Averages of SCC of right front and right hind quarters were significantly higher than left front and left hind quarters. Nearly 94% of California mastitis test (CMT) negative quarters were having somatic cells >or=200 000/ml. The mean SCC of CMT positive quarter was significantly higher (P CMT negative quarters. Subclinical mastitis was diagnosed on the basis of samples with SCCs >or=200 000/ml with positive bacterial cultures. Subclinical mastitis was found in 21.7% buffaloes and 8% of the quarter foremilk samples. Neutrophil counts were significantly higher in subclinical mastitis milk. PMID:16626405

  19. Multipotent somatic stem cells contribute to the stem cell niche in the Drosophila testis.

    Science.gov (United States)

    Voog, Justin; D'Alterio, Cecilia; Jones, D Leanne

    2008-08-28

    Adult stem cells reside in specialized microenvironments, or niches, that have an important role in regulating stem cell behaviour. Therefore, tight control of niche number, size and function is necessary to ensure the proper balance between stem cells and progenitor cells available for tissue homeostasis and wound repair. The stem cell niche in the Drosophila male gonad is located at the tip of the testis where germline and somatic stem cells surround the apical hub, a cluster of approximately 10-15 somatic cells that is required for stem cell self-renewal and maintenance. Here we show that somatic stem cells in the Drosophila testis contribute to both the apical hub and the somatic cyst cell lineage. The Drosophila orthologue of epithelial cadherin (DE-cadherin) is required for somatic stem cell maintenance and, consequently, the apical hub. Furthermore, our data indicate that the transcriptional repressor escargot regulates the ability of somatic cells to assume and/or maintain hub cell identity. These data highlight the dynamic relationship between stem cells and the niche and provide insight into genetic programmes that regulate niche size and function to support normal tissue homeostasis and organ regeneration throughout life. PMID:18641633

  20. Mutation of mitochondria genome: trigger of somatic cell transforming to cancer cell.

    Science.gov (United States)

    Jianping, Du

    2010-01-01

    Nearly 80 years ago, scientist Otto Warburg originated a hypothesis that the cause of cancer is primarily a defect in energy metabolism. Following studies showed that mitochondria impact carcinogenesis to remodel somatic cells to cancer cells through modifying the genome, through maintenance the tumorigenic phenotype, and through apoptosis. And the Endosymbiotic Theory explains the origin of mitochondria and eukaryotes, on the other hands, the mitochondria also can fall back. Compared to chromosome genomes, the mitochondria genomes were not restricted by introns so they were mutated(fall back) easy. The result is that mitochondria lose function and internal environment of somatic cell become acid and evoked chromosome genomes to mutate, in the end somatic cells become cancer cells. It is the trigger of somatic cell transforming to cancer cell that mitochondria genome happen mutation and lose function. PMID:20181100

  1. Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming.

    Science.gov (United States)

    Krijger, Peter Hugo Lodewijk; Di Stefano, Bruno; de Wit, Elzo; Limone, Francesco; van Oevelen, Chris; de Laat, Wouter; Graf, Thomas

    2016-05-01

    Forced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here we studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. We find large-scale topologically associated domain (TAD) repositioning and alterations of tissue-restricted genomic neighborhoods and chromatin loops, effectively erasing the somatic-cell-specific genome structures while establishing an embryonic stem-cell-like 3D genome. Yet, early passage iPSCs carry topological hallmarks that enable recognition of their cell of origin. These hallmarks are not remnants of somatic chromosome topologies. Instead, the distinguishing topological features are acquired during reprogramming, as we also find for cell-of-origin-dependent gene expression patterns. PMID:26971819

  2. A stochastic model of epigenetic dynamics in somatic cell reprogramming

    Directory of Open Access Journals (Sweden)

    Max eFloettmann

    2012-06-01

    Full Text Available Somatic cell reprogramming has dramatically changed stem cell research inrecent years. The high pace of new findings in the field and an ever increasingamount of data from new high throughput techniques make it challengingto isolate core principles of the process. In order to analyze suchmechanisms, we developed an abstract mechanistic model of a subset of theknown regulatory processes during cell differentiation and production of inducedpluripotent stem cells. This probabilistic Boolean network describesthe interplay between gene expression, chromatin modifications and DNAmethylation. The model incorporates recent findings in epigenetics and reproducesexperimentally observed reprogramming efficiencies and changes inmethylation and chromatin remodeling. It enables us to investigate in detail,how the temporal progression of the process is regulated. It also explicitlyincludes the transduction of factors using viral vectors and their silencing inreprogrammed cells, since this is still a standard procedure in somatic cellreprogramming. Based on the model we calculate an epigenetic landscape.Simulation results show good reproduction of experimental observations duringreprogramming, despite the simple stucture of the model. An extensiveanalysis and introduced variations hint towards possible optimizations of theprocess, that could push the technique closer to clinical applications. Fasterchanges in DNA methylation increase the speed of reprogramming at theexpense of efficiency, while accelerated chromatin modifications moderatelyimprove efficiency.

  3. Somatic cell genotoxicity at the glycophorin A locus in humans

    International Nuclear Information System (INIS)

    We have developed an assay for detecting variant erythrocytes that occur as a result of in vivo allele loss at the glycophorin A (GPA) locus on chromosome 4 in humans. This gene codes for an erythroid- specific cell surface glycoprotein, and with our assay we are able to detect rare variant erythrocytes that have lost expression of one of the two GPA alleles. Two distinctly different variant cell types are detected with this assay. One variant cell type (called N OE) is hemizygous. Our assay also detects homozygous variant erythrocytes that have lost expression of the GPA(M) allele and express the GPA(N) allele at twice the heterozygous level. The results of this assay are an enumeration of the frequency of N OE and NN variant cell types for each individual analyzed. These variant cell frequencies provide a measure of the amount of somatic cell genotoxicity that has occurred at the GPA locus. Such genotoxicity could be the result of (1) reactions of toxic chemicals to which the individual has been exposed, or (2) high energy radiation effects on erythroid precursor cells, or (3) errors in DNA replication or repair in these cells of the bone marrow. Thus, the GPA-based variant cell frequency can serve as a biodosimeter that indicates the amount of genotoxic exposure each individual has received. Because two very different kinds of variant cells are enumerated, different kinds of genotoxicity should be distinguishable. Results of the GPA somatic genotoxicity assay may also provide valuable information for cancer-risk estimation on each individual. 16 refs

  4. Generation of cloned calves from different types of somatic cells

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    Six types of bovine somatic cell lines,including a granulosa cell line of Chinese red-breed yellow cattle(YGR),a granulosa cell line of Holstein cow(HGR),two skin fibroblast cell lines of two adult Holstein cows respectively(AFB1 and AFB2),a skin fibroblast cell line(FFB)and an oviduct epithelial cell line(FOV)of a Holstein fetus,were established.Somatic cell nuclear transfer(SCNT)was carried out using these cells as nuclei donor,and a total of 12 healthy calves were cloned.The effects of different types of donor cells on developmental potential of bovine SCNT embryos were investigated.(i)There was no significant difference in development rates to the blastocyst stage for SCNT embryos from YGR and HGR(33.2% and 35.1%,respectively).Pregnancy rates of them were 33.3% and 30.2%,respectively; and birth rates were 16.7%and 11.6%,respectively.(ii)Development rates to the blastocyst stage for SCNT embryos from diffetent individuals(AFB1 and AFB2)differed significantly(27.9% and 39.4%,respectively,P <0.05).Pregnancy rates of them were 36.2% and 36.4%,respectively; and birth rates were 14.9% and 27.3%,respectively.(iii)There was significant difference in development rates to the blastocyst stage for SCNT embryos from FFB and FOV of the same fetus(37.9% and 41.5%,respectively,P < 0.05).Pregnancy rates of them were 45.7% and 24.1%,respectively; and birth rates were 22.9 % and 10.3%,respectively.Finally,developmental potential of bovine SCNT embryos from all four types of somatic cells from Holstein cows(HGR,AFB,FFB and FOV)were compared.For in vitro development stage,development rates to the blastocyst stage for SCNT embryos from HGR,AFB,FFB and FOV were 35.1%A,29.4%B,37.9%A and 41.5%C,respectively(pABC<0.05); for in vivo development stage,pregnancy rates of them were 30.2%,36.2%,45.7%and 24.1%,respectively; and birth rates of them were 11.6%,17.2%,22.9% and 10.3% respectively.

  5. Somatic cell count control strategies in dairy ewes

    OpenAIRE

    Spanu, Carlo

    2010-01-01

    The consumption of milk products, especially made from raw milk, have been reported to be associated with food borne diseases. Since most sheep’s milk products are made from raw milk, it is clear how udder health is an important prerequisite to produce hygienic milk. Ewes with mastitis, particularly in their subclinical form, serve as reservoir of pathogens that can be shed into the milk and constitute a potential risk for human health. Milk somatic cell count (SCC) is not a public health con...

  6. Reconstruction of human embryos derived from somatic cells

    Institute of Scientific and Technical Information of China (English)

    LU Changfu; LIN Ge; XIE Changqing; GONG Fei; ZHOU Hong; TAN Yueqiu; LU Guangxiu

    2003-01-01

    Reconstruction of human nuclear transfer embryos is a necessary step of therapeutic cloning. In this study we injected somatic cell nuclei into MⅡ oocytes and activated reconstructed oocytes with calcium ionophore A23187 (CaA) and 6-dimethylaminopurine (6-DMAP). After oocyte activation and 2PN formation, we removed the female PN. By using this method, we avoided the application of DNA fluorescent stain and ultraviolet light for oocyte enucleation, and over elimination of ooplasm was also mitigated. Some reconstructed embryos developed into theblastocyst stage in vitro.

  7. Human somatic cell mutagenesis creates genetically tractable sarcomas.

    Science.gov (United States)

    Molyneux, Sam D; Waterhouse, Paul D; Shelton, Dawne; Shao, Yang W; Watling, Christopher M; Tang, Qing-Lian; Harris, Isaac S; Dickson, Brendan C; Tharmapalan, Pirashaanthy; Sandve, Geir K; Zhang, Xiaoyang; Bailey, Swneke D; Berman, Hal; Wunder, Jay S; Izsvák, Zsuzsanna; Iszvak, Zsuzsanna; Lupien, Mathieu; Mak, Tak W; Khokha, Rama

    2014-09-01

    Creating spontaneous yet genetically tractable human tumors from normal cells presents a fundamental challenge. Here we combined retroviral and transposon insertional mutagenesis to enable cancer gene discovery starting with human primary cells. We used lentiviruses to seed gain- and loss-of-function gene disruption elements, which were further deployed by Sleeping Beauty transposons throughout the genome of human bone explant mesenchymal cells. De novo tumors generated rapidly in this context were high-grade myxofibrosarcomas. Tumor insertion sites were enriched in recurrent somatic copy-number aberration regions from multiple cancer types and could be used to pinpoint new driver genes that sustain somatic alterations in patients. We identified HDLBP, which encodes the RNA-binding protein vigilin, as a candidate tumor suppressor deleted at 2q37.3 in greater than one out of ten tumors across multiple tissues of origin. Hybrid viral-transposon systems may accelerate the functional annotation of cancer genomes by enabling insertional mutagenesis screens in higher eukaryotes that are not amenable to germline transgenesis. PMID:25129143

  8. Evaluation of milk yield in tsigaiewes by somatic cell count

    Directory of Open Access Journals (Sweden)

    Martina Vršková

    2015-08-01

    Full Text Available The objective of our research was to study daily milk production which was affected by somatic cell count (SCC. The study was performed on a selected flock of purebred Tsigai ewes (326 animals. Regular milk yield recording was performed during the evening milking in around the middle of April, May and June. Milk samples were analyzed for basic milk composition (fat, protein and lactose and somatic cells count. SCC were evaluated using decadic logarithm (logSCC.According to animals, the dairy ewes were divided into the four groups on the basis of individual SCC (G1 = SCC <100 × 103 cells.mL-1, G2 = SCC between 100 – 300 × 103 cells.mL-1, G3 = SCC between 300 – 600 × 103 cells.mL-1, G4 = SCC >600 × 103 cells.mL-1 to study the frequency of distribution of animals in selected group of ewes throughout experimental period. The average daily milk production in selected flock of Tsigai was 421.02 mL. We reached the highest daily milk production in April 476.40 ml and the highest content of fat and protein in June, while milk production was the lowest. From this flock of purebred Tsigai 76% of eweswere below SCC 300 × 103 cells.mL-1. This SCC indicated a good health status of experimental ewes, at which 61% sheep were at the first lactation. We found a tendency to lower milk production by a higher SCC. With the increasing SCC decreased lactose content from 4.78% (G1 to 4.32% (G4. Reduced lactose content refers to the occurrence of mastitis and there is a need for performing bacteriological examination in milk.

  9. Somatic cell genetics approach to dissecting mammalian DNA repair

    International Nuclear Information System (INIS)

    This review article examines the application of the methods and concepts of somatic cell genetics to the study of DNA repair. The first steps of this approach involve classical procedures of mutant isolation, complementation analysis, and mapping of genes using hybrid cells. Subsequent steps utilize the techniques of DNA-mediated gene transfer and methodologies of the recombinant DNA field. Several human repair genes have been cloned, but they have not been used to overproduce proteins thus far. This article highlights the more important developments and attempts to review in detail all of the isolated mutant cell lines that may be altered in the repair processes. Faster methods of gene cloning are greatly needed because the procedures for making secondary transformants from total genomic DNA are tedious

  10. Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells

    OpenAIRE

    Chung, H.J.; Hassan, M. M.; Park, J O; Kim, H. J.; S.T. Hong

    2015-01-01

    Recent advances have raised hope that transplantation of adherent somatic cells could provide dramatic new therapies for various diseases. However, current methods for transplanting adherent somatic cells are not efficient enough for therapeutic applications. Here, we report the development of a novel method to generate quasi-natural cell blocks for high-efficiency transplantation of adherent somatic cells. The blocks were created by providing a unique environment in which cultured cells gene...

  11. Gnotobiotic Miniature Pig Interbreed Somatic Cell Nuclear Transfer for Xenotransplantation.

    Science.gov (United States)

    Hwang, Jeong Ho; Kim, Sang Eun; Gupta, Mukesh Kumar; Lee, HoonTaek

    2016-08-01

    Transgenic animal producing technology has improved consistently over the last couple of decades. Among the available methods, somatic cell nuclear transfer (SCNT) technology was officially the most popular. However, SCNT has low efficiency and requires a highly skilled individual. Additionally, the allo-SCNT nuclear reprogramming mechanism is poorly understood in the gnotobiotic miniature pig, which is a candidate for xenotransplantation, making sampling in oocytes very difficult compared to commercial hybrid pigs. Therefore, interbreed SCNT (ibSCNT), which is a combination of miniature pig and commercial pig (Landrace based), was analyzed and was found to be similar to SCNT in terms of the rate of blastocyst formation (12.6% ± 2.9% vs. 15.5% ± 2.2%; p > 0.05). However, a significantly lower fusion rate was observed in the ibSCNT compared to normal SCNT with Landrace pig somatic cells (29.6% ± 0.8% vs. 65.0% ± 4.9%). Thus, the optimization of fusion parameters was necessary for efficient SCNT. Our results further revealed that ibSCNT by the whole-cell intracytoplasmic injection (WCICI) method had a significantly higher blastocyst forming efficiency than the electrofusion method (31.1 ± 8.5 vs. 15.5% ± 2.2%). The nuclear remodeling and the pattern of changes in acetylation at H3K9 residue were similar in both SCNT and ibSCNT embryos. PMID:27459580

  12. Agronomic traits and RAPD analysis of two mutants derived from rice somatic cell culturing

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Genetic variation, including agronomic trait variation, often occurs in somatic cell culturing. In this study, we compared the main agronomic traits of two rice mutants, M3 and M14, which were derived from Shenxiangjing 5 somatic cell culturing. Significant differences were found between the two mutants and the wild rice Shenxiangjing 5 (Table 1). Results were as follows:

  13. Production of transgenic calves by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    GONG Guochun; WAN Rong; HUANG Yinghua; LI Ning; DAI Yunping; FAN Baoliang; ZHU Huabing; WANG Lili; WANG Haiping; TANG Bo; LIU Ying; LI Rong

    2004-01-01

    Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector (pCE-EGFP-IRES-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation, and a transgenic cell line was obtained. Somatic cell nuclear transfer (SCNT) was carried out using the transgenic cells as nuclei donor. A total of 424 SCNT embryos were reconstructed and 208 (49.1%) of them developed to blastocyst stage. 17 blastocysts on D 7 after reconstruction were transferred to 17 surrogate calves, and 5 (29.4%) recipients were found to be pregnant. Three of them maintained to term and delivered three cloned calves. PCR and Southern blot analysis confirmed the integration of transgene in all of the three cloned calves. In addition, expression of EGFP was detected in biopsy isolated from the transgenic cloned calves and fibroblasts derived from the biopsy. Our results suggest that transgenic calves could be efficiently produced by SCNT using transgenic cells as nuclei donor. Furthermore, all cloned animals could be ensured to be transgenic by efficiently pre-screening transgenic cells and SCNT embryos using the constructed double marker selective vector.

  14. Somatic cell and factors which affect their count in milk

    Directory of Open Access Journals (Sweden)

    Zrinka Čačić

    2003-01-01

    Full Text Available Milk quality is determined by chemical composition, physical characteristics and hygienic parameters. The main indicators of hygienic quality of milk are total number of microorganisms and somatic cell count (SCC. Environmental factors have the greatest influence on increasing SCC. The most important environmental parameters are status of udder infection, age of cow, stage of lactation, number of lactation, breed, housing, geographicalarea and seasons, herd size, stress, heavy physical activity and, milking. A farmer (milk producer himself can control a great number of environmental factors using good management practise and permanent education. Since SCC participate in creating the price of milk, it is necessary to inform milk producers how to organise their production so that they would produce maximum quantity of good hygienic quality milk.

  15. New Rapid Method of DNA Isolation from Milk Somatic Cells.

    Science.gov (United States)

    Pokorska, Joanna; Kułaj, Dominika; Dusza, Magdalena; Żychlińska-Buczek, Justyna; Makulska, Joanna

    2016-04-01

    Isolation of genomic DNA is one of the basic steps in many different molecular analyses. There are a few reports on methods of DNA isolation from milk, but many of them are time consuming and expensive, and require relatively large volumes of raw milk. In this study a rapid, sensitive, and efficient method of DNA extraction from milk somatic cells of various mammals (cattle, sheep, goats, horses) is presented. It was found that milk is a good source of genomic DNA, and to obtain a sufficient amount and quality of DNA, suitable for molecular analysis such as PCR, 10 mL of raw milk is sufficient. Thanks to this method, stress in animals can be reduced during collection of researched material. Therefore, this method could be widely used in molecular analyses. PMID:26913552

  16. CYTOLOGICAL QUALITY OF GOAT MILK ON THE BASIS OF THE SOMATIC CELL COUNT

    Directory of Open Access Journals (Sweden)

    Henryka BERNACKA

    2007-07-01

    Full Text Available The aim of the present paper was to evaluate the cytological quality of goat milk based on the somatic cell count in respective months of lactation. Besides there was defined the effect of somatic cell on the milk production and chemical composition of milk. The research covered goats of color improved breed in the 2nd and 3rd lactation. Daily milk yield, chemical composition of milk and its somatic cell count were defined based on monthly morning and evening control milkings from both teats, following the A4 method applied in District Animal Evaluation Stations. The research indicated that the greater the somatic cell count in milk, the lower the daily milk yield, however the greater the somatic cell count, the greater the percentage content of fat and dry matter and the lower the content of lactose.

  17. Transient Acquisition of Pluripotency During Somatic Cell Transdifferentiation with iPSC Reprogramming Factors

    OpenAIRE

    Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D.; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R.; Greenleaf, William J.; Massarwa, Rada

    2015-01-01

    Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors 1,2 . Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote linea...

  18. Factors Affecting the Development of Somatic Cell Nuclear Transfer Embryos in Cattle

    OpenAIRE

    Akagi, Satoshi; Matsukawa, Kazutsugu; TAKAHASHI, Seiya

    2014-01-01

    Nuclear transfer is a complex multistep procedure that includes oocyte maturation, cell cycle synchronization of donor cells, enucleation, cell fusion, oocyte activation and embryo culture. Therefore, many factors are believed to contribute to the success of embryo development following nuclear transfer. Numerous attempts to improve cloning efficiency have been conducted since the birth of the first sheep by somatic cell nuclear transfer. However, the efficiency of somatic cell cloning has re...

  19. File list: InP.Gon.50.AllAg.Testicular_somatic_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Gon.50.AllAg.Testicular_somatic_cells mm9 Input control Gonad Testicular somati...c cells SRX591728,SRX591716 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Gon.50.AllAg.Testicular_somatic_cells.bed ...

  20. Propagation of elite rescue dogs by somatic cell nuclear transfer.

    Science.gov (United States)

    Oh, Hyun Ju; Choi, Jin; Kim, Min Jung; Kim, Geon A; Jo, Young Kwang; Choi, Yoo Bin; Lee, Byeong Chun

    2016-01-01

    The objective of the present study was to compare the efficiency of two oocyte activation culture media to produce cloned dogs from an elite rescue dog and to analyze their behavioral tendencies. In somatic cell nuclear transfer procedure, fused couplets were activated by calcium ionophore treatment for 4 min, cultured in two media: modified synthetic oviduct fluid (mSOF) with 1.9 mmol/L 6-dimethylaminopyridine (DMAP) (SOF-DMAP) or porcine zygote medium (PZM-5) with 1.9 mmol/L DMAP (PZM-DMAP) for 4 h, and then were transferred into recipients. After embryo transfer, pregnancy was detected in one out of three surrogate mothers that received cloned embryos from the PZM-DMAP group (33.3%), and one pregnancy (25%) was detected in four surrogate mothers receiving cloned embryos from the SOF-DMAP group. Each pregnant dog gave birth to one healthy cloned puppy by cesarean section. We conducted the puppy aptitude test with two cloned puppies; the two cloned puppies were classified as the same type, accepting humans and leaders easily. The present study indicated that the type of medium used in 6-DMAP culture did not increase in cloning efficiency and dogs cloned using donor cells derived from one elite dog have similar behavioral tendencies. PMID:26387964

  1. Utilization of zinc methionine supplementation in Friesian cows: somatic cell count in milk and mastitis

    International Nuclear Information System (INIS)

    Full text: Two hundreds and forty lactating Friesian cows on the 1st to 8th of lactation and different stages of lactation were used to study some factors affecting on somatic cell count and its effects on milk yield and composition. Also, 12 normal cows, 15 subclinical and 15 clinical mastitis cows were used to study the effect of zinc methionine supplementation on somatic cell count and mastitis. Cows were divided into three similar groups, the first groups was unsupplemented, while the second and third groups were supplemented with 5 and 10 gm zinc methionine / head / day, respectively. Subclinical and clinical mastitis cows were intramammary injected by antibiotic Gentamast (Gentamicin 100 mg) till complete recovery. The obtained results showed that winter season showed significantly (P < 0.05) the highest somatic cell count followed by summer season, while the lowest value was in autumn season. Somatic cell count tended to decrease with the progress of lactation up to the peak period and increased significantly (P < 0.05) thereafter and also with the progress number of lactation. The percentages of normal, subclinical and clinical mastitis cows were 77.71, 15.82 and 6.46%, respectively. Milk yield and composition and its output decreased significantly (P < 0.05) with increasing somatic cell count. Zinc methionine supplementation resulted in significant (P < 0.05) decrease in somatic cell count in milk. Zinc methionine supplementation for subclinical and clinical mastitis cows led to significant decrease (P < 0.05) on somatic cell count, electrical conductivity, recovery time and the cost of therapy compared with unsupplemented group. It could be concluded that increasing somatic cell count decreased milk yield and composition. Zinc methionine supplementation at the level of 5 g per head daily to lactating Friesian cows reduced somatic cell count in milk, recovery time and therapy cost of mastitis. (author)

  2. Embryonic Development following Somatic Cell Nuclear Transfer Impeded by Persisting Histone Methylation

    OpenAIRE

    Matoba, Shogo; Liu, Yuting; Lu, Falong; Iwabuchi, Kumiko A.; Inoue, Azusa; Zhang, Yi

    2014-01-01

    Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major epigenetic barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed n...

  3. Privileged Communication Embryonic Development Following Somatic Cell Nuclear Transfer Impeded by Persisting Histone Methylation

    OpenAIRE

    Matoba, Shogo; Liu, Yuting; Lu, Falong; Iwabuchi, Kumiko A.; Shen, Li; Inoue, Azusa; Zhang, Yi

    2014-01-01

    Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major epigenetic barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed n...

  4. An unregulated regulator: Vasa expression in the development of somatic cells and in tumorigenesis.

    Science.gov (United States)

    Poon, Jessica; Wessel, Gary M; Yajima, Mamiko

    2016-07-01

    Growing evidence in diverse organisms shows that genes originally thought to function uniquely in the germ line may also function in somatic cells, and in some cases even contribute to tumorigenesis. Here we review the somatic functions of Vasa, one of the most conserved "germ line" factors among metazoans. Vasa expression in somatic cells is tightly regulated and often transient during normal development, and appears to play essential roles in regulation of embryonic cells and regenerative tissues. Its dysregulation, however, is believed to be an important element of tumorigenic cell regulation. In this perspectives paper, we propose how some conserved functions of Vasa may be selected for somatic cell regulation, including its potential impact on efficient and localized translational activities and in some cases on cellular malfunctioning and tumorigenesis. PMID:27179696

  5. Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm

    International Nuclear Information System (INIS)

    The mechanisms governing nuclear reprogramming have not been fully elucidated yet; however, recent studies show a universally conserved ability of both oocyte and egg components to reprogram gene expression in somatic cells. The activation of genes associated with pluripotency by oocyte/egg components may require the remodeling of nuclear structures, such that they can acquire the features of early embryos and pluripotent cells. Here, we report on the remodeling of the nuclear lamina of mammalian cells by Xenopus oocyte and egg extracts. Lamin A/C is removed from somatic cells incubated in oocyte and egg extracts in an active process that requires permeable nuclear pores. Removal of lamin A/C is specific, since B-type lamins are not changed, and it is not dependent on the incorporation Xenopus egg specific lamin III. Moreover, transcriptional activity is differentially regulated in somatic cells incubated in the extracts. Pol I and II transcriptions are maintained in cells in oocyte extracts; however, both activities are abolished in egg extracts. Our study shows that components of oocyte and egg extracts can modify the nuclear lamina of somatic cells and that this nuclear remodeling induces a structural change in the nucleus which may have implications for transcriptional activity. These experiments suggest that modifications in the nuclear lamina structure by the removal of somatic proteins and the incorporation of oocyte/egg components may contribute to the reprogramming of somatic cell nuclei and may define a characteristic configuration of pluripotent cells

  6. The uranyl influence on a mutation process in germ and somatic cells of mice

    International Nuclear Information System (INIS)

    The mutagenic effect of uranyl was revealed by the chromosome rearrangement test in germ and somatic cells of mice. The effect value depended on duration of substance administration into organism. (authors)

  7. Chinmo is sufficient to induce male fate in somatic cells of the adult Drosophila ovary.

    Science.gov (United States)

    Ma, Qing; de Cuevas, Margaret; Matunis, Erika L

    2016-03-01

    Sexual identity is continuously maintained in specific differentiated cell types long after sex determination occurs during development. In the adult Drosophila testis, the putative transcription factor Chronologically inappropriate morphogenesis (Chinmo) acts with the canonical male sex determinant DoublesexM (Dsx(M)) to maintain the male identity of somatic cyst stem cells and their progeny. Here we find that ectopic expression of chinmo is sufficient to induce a male identity in adult ovarian somatic cells, but it acts through a Dsx(M)-independent mechanism. Conversely, the feminization of the testis somatic stem cell lineage caused by loss of chinmo is enhanced by expression of the canonical female sex determinant Dsx(F), indicating that chinmo acts in parallel with the canonical sex determination pathway to maintain the male identity of testis somatic cells. Consistent with this finding, ectopic expression of female sex determinants in the adult testis disrupts tissue morphology. The miRNA let-7 downregulates chinmo in many contexts, and ectopic expression of let-7 in the adult testis is sufficient to recapitulate the chinmo loss-of-function phenotype, but we find no apparent phenotypes upon removal of let-7 in the adult ovary or testis. Our finding that chinmo is necessary and sufficient to promote a male identity in adult gonadal somatic cells suggests that the sexual identity of somatic cells can be reprogrammed in the adult Drosophila ovary as well as in the testis. PMID:26811385

  8. Auxin gradients trigger de novo formation of stem cells during somatic embryogenesis

    OpenAIRE

    Su, Ying Hua; Zhang, Xian Sheng

    2009-01-01

    Single or a group of somatic cells could give rise to the whole plant, which require hormones, or plant growth regulators. Although many studies have been done during past years, how hormones specify cell fate during in vitro organogenesis is still unknown. To uncover this mechanism, Arabidopsis somatic embryogenesis has been recognized as a model for studying in vitro plant organogenesis. In this paper, we showed that establishment of auxin gradients within embryonic callus is essential for ...

  9. Reproductional indicator influence on the somatic cell count of cow's milk

    OpenAIRE

    Jonikaitė, Inga

    2007-01-01

    Research data show that the somatic cell count increases during the transition period when dairy cows are transferred from barns to pastures (month of May) and during the transition period when dairy cows are transferred from pasture to barn (month of October). During these period’s feedstuff composition changes, as does the temperature, microclimate parameters, which also have an influence on cows with Sub-clinical mastitis. Somatic cell counts are lowest in 1st lactation cows. 1st lactat...

  10. The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Athanasia D Panopoulos; Margaret Lutz; W Travis Berggren; Kun Zhang; Ronald M Evans; Gary Siuzdak; Juan Carlos Izpisua Belmonte; Oscar Yanes; SergioRuiz; Yasuyuki S Kida; Dinh Diep; Ralf Tautenhahn; Aida Herrerias; Erika M Batchelder; Nongluk Plongthongkum

    2012-01-01

    Metabolism is vital to every aspect of cell function,yet the metabolome of induced pluripotent stem cells (iPSCs)remains largely unexplored.Here we report,using an untargeted metabolomics approach,that human iPSCs share a pluripotent metabolomic signature with embryonic stem cells (ESCs) that is distinct from their parental cells,and that is characterized by changes in metabolites involved in cellular respiration.Examination of cellular bioenergetics corroborated with our metabolomic analysis,and demonstrated that somatic cells convert from an oxidative state to a glycolytic state in pluripotency.Interestingly,the bioenergetics of various somatic cells correlated with their reprogramming efficiencies.We further identified metabolites that differ between iPSCs and ESCs,which revealed novel metabolic pathways that play a critical role in regulating somatic cell reprogramming.Our findings are the first to globally analyze the metabolome of iPSCs,and provide mechanistic insight into a new layer of regulation involved in inducing pluripotency,and in evaluating iPSC and ESC equivalence.

  11. Plant Hormones Increase Efficiency of Reprogramming Mouse Somatic Cells to Induced Pluripotent Stem Cells and Reduce Tumorigenicity

    OpenAIRE

    Alvarez Palomo, Ana Belén; McLenachan, Samuel; Requena Osete, Jordi; Menchón, Cristina; Barrot, Carme; Chen, Fred; Munné-Bosch, Sergi; Michael J. Edel

    2013-01-01

    Reprogramming of somatic cells into induced pluripotent stem (iPS) cells by defined pluripotency and self-renewal factors has taken stem cell technology to the forefront of regenerative medicine. However, a number of challenges remain in the field including efficient protocols and the threat of cancer. Reprogramming of plant somatic cells to plant embryonic stem cells using a combination of two plant hormones was discovered in 1957 and has been a routine university laboratory practical for ov...

  12. Limiting replication stress during somatic cell reprogramming reduces genomic instability in induced pluripotent stem cells

    OpenAIRE

    Ruiz, Sergio; Lopez Contreras, Andres J.; Gabut, Mathieu; Marion, Rosa M.; Guti??rrez Mart??nez, Paula; Bua, Sabela; Ram??rez, Oscar; Olalde, I??igo; Rodrigo Perez, Sara; Li, Han; Marqu??s i Bonet, Tom??s, 1975-; Serrano, Manuel; Blasco, Maria A; Batada, Nizar N; Fern??ndez Capetillo, Oscar

    2015-01-01

    The generation of induced pluripotent stem cells (iPSC) from adult somatic cells is one of the most remarkable discoveries in recent decades. However, several works have reported evidence of genomic instability in iPSC, raising concerns on their biomedical use. The reasons behind the genomic instability observed in iPSC remain mostly unknown. Here we show that, similar to the phenomenon of oncogene-induced replication stress, the expression of reprogramming factors induces replication stress....

  13. Mutation of mitochondria genome: trigger of somatic cell transforming to cancer cell

    OpenAIRE

    Jianping Du

    2010-01-01

    Abstract Nearly 80 years ago, scientist Otto Warburg originated a hypothesis that the cause of cancer is primarily a defect in energy metabolism. Following studies showed that mitochondria impact carcinogenesis to remodel somatic cells to cancer cells through modifying the genome, through maintenance the tumorigenic phenotype, and through apoptosis. And the Endosymbiotic Theory explains the origin of mitochondria and eukaryotes, on the other hands, the mitochondria also can fall back. Compare...

  14. Factors affecting somatic cell count in dairy goats: a review

    Directory of Open Access Journals (Sweden)

    Rocío Jiménez-Granado

    2014-02-01

    Full Text Available Somatic cell count (SCC in monitoring udder health has been described in numerous studies as a useful method for the diagnosis of intramammary infection (IMI, and it is considered in standards of quality and hygiene of cow’s milk in many countries. However, several authors have questioned the validity of SCC as a reliable IMI diagnosis tool in dairy goats. This review attempts to reflect the importance of different infectious and non-infectious factors that can modify SCC values in goat milk, and must, therefore, be taken into account when using the SCC as a tool in the improvement of udder health and the quality of milk in this species. In dairy goats, some investigations have shown that mammary bacterial infections are a major cause of increased SCC and loss of production. In goats however, the relationship between bacterial infections and SCC values is not as simple as in dairy cattle, since non-infectious factors also have a big impact on SCC. Intrinsic factors are those that depend directly on the animal: time and number of lactation (higher SCC late in lactation and in aged goats, prolificity (higher SCC in multiple births, milking time (higher SCC in evening compared to morning milking and number of milkings per day, among others. Extrinsic factors include: milking routine (lower SCC in machine than in manual milking, seasonality and food. In addition, milk secretion in goats is mostly apocrine and therefore characterized by the presence of epithelial debris or cytoplasmic particles, which makes the use of DNA specific counters mandatory. All this information is of interest in order to correctly interpret the SCC in goat milk and to establish differential SCC standards.

  15. Factors affecting somatic cell count in dairy goats: a review

    Energy Technology Data Exchange (ETDEWEB)

    Jimenez-Granda, R.; Sanchez-Rodriguez, M.; Arce, C.; Rodriguez-Estevez, V.

    2014-06-01

    Somatic cell count (SCC) in monitoring udder health has been described in numerous studies as a useful method for the diagnosis of intramammary infection (IMI), and it is considered in standards of quality and hygiene of cows milk in many countries. However, several authors have questioned the validity of SCC as a reliable IMI diagnosis tool in dairy goats. This review attempts to reflect the importance of different infectious and non-infectious factors that can modify SCC values in goat milk, and must, therefore, be taken into account when using the SCC as a tool in the improvement of udder health and the quality of milk in this species. In dairy goats, some investigations have shown that mammary bacterial infections are a major cause of increased SCC and loss of production. In goats however, the relationship between bacterial infections and SCC values is not as simple as in dairy cattle, since non-infectious factors also have a big impact on SCC. Intrinsic factors are those that depend directly on the animal: time and number of lactation (higher SCC late in lactation and in aged goats), prolificity (higher SCC in multiple births), milking time (higher SCC in evening compared to morning milking) and number of milkings per day, among others. Extrinsic factors include: milking routine (lower SCC in machine than in manual milking), seasonality and food. In addition, milk secretion in goats is mostly apocrine and therefore characterized by the presence of epithelial debris or cytoplasmic particles, which makes the use of DNA specific counters mandatory. All this information is of interest in order to correctly interpret the SCC in goat milk and to establish differential SCC standards. (Author)

  16. Regenerative therapy for neuronal diseases with transplantation of somatic stem cells

    OpenAIRE

    Kanno, Hiroshi

    2013-01-01

    Pluripotent stem cells, which are capable of differentiating in various species of cells, are hoped to be donor cells in transplantation in regenerative medicine. Embryonic stem (ES) cells and induced pluripotent stem cells have the potential to differentiate in approximately all species of cells. However, the proliferating ability of these cells is high and the cancer formation ability is also recognized. In addition, ethical problems exist in using ES cells. Somatic stem cells with the abil...

  17. Embryonic stem cells and somatic cells differ in mutation frequency and type

    Science.gov (United States)

    Cervantes, Rachel B.; Stringer, James R.; Shao, Changshun; Tischfield, Jay A.; Stambrook, Peter J.

    2002-01-01

    Pluripotent embryonic stem (ES) cells have been used to produce genetically modified mice as experimental models of human genetic diseases. Increasingly, human ES cells are being considered for their potential in the treatment of injury and disease. Here we have shown that mutation in murine ES cells, heterozygous at the selectable Aprt locus, differs from that in embryonic somatic cells. The mutation frequency in ES cells is significantly lower than that in mouse embryonic fibroblasts, which is similar to that in adult cells in vivo. The distribution of spontaneous mutagenic events is remarkably different between the two cell types. Although loss of the functional allele is the predominant mutation type in both cases, representing about 80% of all events, mitotic recombination accounted for all loss of heterozygosity events detected in somatic cells. In contrast, mitotic recombination in ES cells appeared to be suppressed and chromosome loss/reduplication, leading to uniparental disomy (UPD), represented more than half of the loss of heterozygosity events. Extended culture of ES cells led to accumulation of cells with adenine phosphoribosyltransferase deficiency and UPD. Because UPD leads to reduction to homozygosity at multiple recessive disease loci, including tumor suppressor loci, in the affected chromosome, the increased risk of tumor formation after stem cell therapy should be viewed with concern. PMID:11891338

  18. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

    OpenAIRE

    Page Grier P; Kasinathan Poothappillai; Wang Zhongde; Rodriguez-Osorio Nelida; Robl James M; Memili Erdogan

    2009-01-01

    Abstract Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT) is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clo...

  19. Transcriptional Reprogramming of Gene Expression in Bovine Somatic Cell Chromatin Transfer Embryos

    OpenAIRE

    Rodriguez-Osorio, N.; Wang, Zhongde; Page, G. P.; Robl, J M; Memili, E.

    2009-01-01

    Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT) is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from ...

  20. [Antiviral activity of interferon and its inducers in human lymphoblastoid and somatic cells].

    Science.gov (United States)

    Novokhatskiĭ, A S; Labzo, S S; Tsareva, A A

    1979-04-01

    The antiviral effect of interferon inductors, such as poly-I--poly-C, phage f2 RNA replicative form and low molecular inductor GSN and their influence on cellular DNA synthesis were studied in the cultures of lymphoblastoid (inplanting lines Raji Namalva) and somatic human cells. The Semliki forest virus used as the test organism multiplicated well in cells Raji accumulating up to 9 lg BOU/ml. The two-strand RNA was less active in the lymphoid cells than in the somatic ones. GSN was 10 times more active and less toxic in cells Raji as compared to the fibroblasts. The lymphoblastoid interferon had higher antiviral activity as compared to the fibroblast interferon in the system of Raji--Semliki forest virus than in the system of the human embryon fibroblast--Venezuela Horse Encephalytic Virus. Romantadin actively inhibited (100 times) production of the alfavirus in both the somatic and lymphoblastoid cells. PMID:220908

  1. The Epigenetic Reprogramming Roadmap in Generation of iPSCs from Somatic Cells

    DEFF Research Database (Denmark)

    Brix, Jacob; Zhou, Yan; Luo, Yonglun

    2015-01-01

    Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is a comprehensive epigenetic process involving genome-wide modifications of histones and DNA methylation. This process is often incomplete, which subsequently affects iPSC reprograming, pluripotency, and differentiation...... capacity. Here we review the epigenetic changes with a focus on histone modification (methylation and acetylation) and DNA modification (methylation) during iPSC induction. We look at changes in specific epigenetic signatures, aberrations and epigenetic memory during reprogramming and small molecules...... influencing the epigenetic reprogramming of somatic cells. Finally, we discuss how to improve iPSC generation and pluripotency through epigenetic manipulations....

  2. Factors Affecting on Somatic Cells Count in Slovak Simmental Dairy Cows

    Directory of Open Access Journals (Sweden)

    Jozef Bujko

    2014-11-01

    Full Text Available The aim this work was to analyse factors affecting on the somatic cells count in Slovak Simmental dairy cows. Data were analysed using the SAS version 9.1.3. and linear model with fixed effects of herd, years and months controls, sire and breeding types. The analyses by the effect on somatic cells count was the highest effect of herd-years-months of control R2 = 0.151316 and effect of sire R2 = 0.054182. These effects were high statistical significant P<0.01. Correlation coefficients between milk in kg, fat, protein, lactose in % with somatic cells count were r= -0.25096, r= 0.02593, r= 0.22321and r=-0.39567.

  3. Hexavalent chromium induces apoptosis in male somatic and spermatogonial stem cells via redox imbalance

    OpenAIRE

    Joydeep Das; Min-Hee Kang; Eunsu Kim; Deug-Nam Kwon; Yun-Jung Choi; Jin-Hoi Kim

    2015-01-01

    Hexavalent chromium [Cr(VI)], an environmental toxicant, causes severe male reproductive abnormalities. However, the actual mechanisms of toxicity are not clearly understood and have not been studied in detail. The present in vitro study aimed to investigate the mechanism of reproductive toxicity of Cr(VI) in male somatic cells (mouse TM3 Leydig cells and TM4 Sertoli cells) and spermatogonial stem cells (SSCs) because damage to or dysfunction of these cells can directly affect spermatogenesis...

  4. Number and importance of somatic cells in goat’s milk

    OpenAIRE

    Lidija Kozačinski; Majić, T.; Željka Cvrtila; Mirza Hadžiosmanović

    2001-01-01

    Goat’s milk samples were examined on mastitis using stable procedure (California-mastitis test). 427 of the examined milk samples (46.82%) had positive reaction from 1 to 3 while other 485 samples (53.18%) had negative reaction on the mastitis test, indicating that no illness of mammary gland occurred. Number of somatic cells, counted using “Fossomatic” counter, was 1.3x106/ml average. By comparing the results of mastitis-test evaluation (CMT) with the number of somatic cells and findings of ...

  5. The Drosophila BCL6 homolog ken and barbie promotes somatic stem cell self-renewal in the testis niche

    OpenAIRE

    Issigonis, Melanie; Matunis, Erika

    2012-01-01

    Stem cells sustain tissue regeneration by their remarkable ability to replenish the stem cell pool and to generate differentiating progeny. Signals from local microenvironments, or niches, control stem cell behavior. In the Drosophila testis, a group of somatic support cells called the hub creates a stem cell niche by locally activating the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway in two adjacent types of stem cells: germline stem cells (GSCs) and somat...

  6. Breaching the kinetic barrier to in vitro somatic stem cell propagation

    OpenAIRE

    Merok, Joshua R.; Sherley, James L.

    2001-01-01

    Abstract Here we have reviewed the conventional definitions and fundamental characteristics of the two basic types of stem cells, embryonic stem cells (ESCs) and somatic stem cells (SSCs). By taking into account the often-overlooked asymmetric cell kinetics of SSCs, we consider the evidence that should SSCs retain these growth kinetics in vitro, a natural kinetic barrier to SSC propagation exists. Recent discoveries showing that the tumor suppressor gene p53 can act as a regulator of asymmetr...

  7. Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

    Institute of Scientific and Technical Information of China (English)

    YING CHEN; QING ZHANG YANG; DA YUAN CHEN; MIN KANG WANG; JIN SONG LI; SHAO LIANG HUANG; XIANG YIN KONG; YAO ZHOU SHI; ZHI QIANG WANG; JIA HUI XIA; ZHI GAO LONG; ZHI XU HE; ZHI GANG XUE; WEN XIANG DING; HUI ZHEN SHENG; AILIAN LIU; KAI WANG; WEN WEI MAO; JIAN XIN CHU; YONG LU; ZHENG FU FANG; YING TANG SHI

    2003-01-01

    To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PGR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.

  8. Genetic aspects of somatic cell count and udder health in the Italian Valle del Belice dairy sheep

    NARCIS (Netherlands)

    Riggio, V.

    2012-01-01

    Mastitis is an inflammation of the udder, which leads to economic loss, mainly consisting of discarded milk, reduced milk production and quality, and increased health costs. Somatic cell count (SCC), and therefore somatic cell score (SCS), is widely used as indicator of mastitis. In this thesis, I f

  9. A Comparative View on Human Somatic Cell Sources for iPSC Generation

    Directory of Open Access Journals (Sweden)

    Stefanie Raab

    2014-01-01

    Full Text Available The breakthrough of reprogramming human somatic cells was achieved in 2006 by the work of Yamanaka and Takahashi. From this point, fibroblasts are the most commonly used primary somatic cell type for the generation of induced pluripotent stem cells (iPSCs. Various characteristics of fibroblasts supported their utilization for the groundbreaking experiments of iPSC generation. One major advantage is the high availability of fibroblasts which can be easily isolated from skin biopsies. Furthermore, their cultivation, propagation, and cryoconservation properties are uncomplicated with respect to nutritional requirements and viability in culture. However, the required skin biopsy remains an invasive approach, representing a major drawback for using fibroblasts as the starting material. More and more studies appeared over the last years, describing the reprogramming of other human somatic cell types. Cells isolated from blood samples or urine, as well as more unexpected cell types, like pancreatic islet beta cells, synovial cells, or mesenchymal stromal cells from wisdom teeth, show promising characteristics for a reprogramming strategy. Here, we want to highlight the advantages of keratinocytes from human plucked hair as a widely usable, noninvasive harvesting method for primary material in comparison with other commonly used cell types.

  10. Longitudinal Analysis of Somatic Cell Count for Joint Genetic Evaluation of Mastitis and Recovery Liability

    DEFF Research Database (Denmark)

    Welderufael, Berihu Gebremedhin; de Koning, D J; Janss, Luc;

    Abstract Text: Better models of genetic evaluation for mastitis can be developed through longitudinal analysis of somatic cell count (SCC) which usually is used as a proxy for mastitis. Mastitis and recovery data with weekly observations of SCC were simulated for daughter groups of 60 and 240 per...

  11. STUDY REGARDING THE CORELATION BETWEEN SOMATIC CELLS COUNT AND MAJOR CHEMICAL COMPOUNDS IN RAW MILK

    Directory of Open Access Journals (Sweden)

    S. ACATINCĂI

    2013-12-01

    Full Text Available This study approaches the dynamic of somatic cells number and chemical composition of milk during 13 months of control. The study also investigates the correlations between the number of somatic cells and some chemical parameters in milk. Studies were carried out on Romanian Black and White cows between March 2005 and March 2006 at the Didactical farm of the Banat University of Agricultural Sciences Timisoara. As quality indicator, the number of somatic cells has different values among the controls. Average values for the 13 months of control, with the exception of three controls, were below maximum limit admitted from 1th of January 2007 (600000 SCC/ml milk. There weren’t any significant differences for SCC between the two seasons. Chemical parameters in milk varied in close limits and the differences were not significant, with one exception for fat percent. Fat percent is higher (p<0.05 in the cold season 3.87% compared with 3.55% during the warm season. Somatic cells number is weak correlated with lactose and strong correlated with proteins.

  12. Genetic correlations between pathogen-specific mastitis and somatic cell count in Danish Holsteins

    DEFF Research Database (Denmark)

    Sørensen, Lars Peter; Mark, Thomas; Madsen, P.;

    2009-01-01

    The aim of this study was to estimate genetic correlations (r(a)) between 2 lactation average somatic cell count (LASCC) traits and 6 different mastitis traits in 226,482 first-parity Danish Holstein cows that calved between 1998 and 2008. The LASCC traits were defined from 5 to either 170 d (LASCC...

  13. Relationship between somatic cell count status and subsequent clinical mastitis in Dutch dairy cows

    NARCIS (Netherlands)

    Borne, van den B.H.P.; Vernooij, J.C.M.; Lupindu, A.M.; Schaik, van G.; Frankena, K.; Lam, T.J.G.M.; Nielen, M.

    2011-01-01

    High composite somatic cell counts (CSCC) in dairy cows may develop into clinical mastitis (CM), suggesting that prevention or intervention of high CSCC may prevent CM later in lactation. The objective of this study was to quantify the relationship between high CSCC in dairy cows and the first subse

  14. Impact of selection for decreased somatic cell score on productive life and culling for mastitis

    Science.gov (United States)

    Impact of continued selection for decreased somatic cell score (SCS) was examined to determine if such selection resulted in greater mastitis susceptibility and shorter productive life (PL). Holstein artificial-insemination bulls with a predicted transmitting ability (PTA) for SCS based on >=35 daug...

  15. Number and importance of somatic cells in goat’s milk

    Directory of Open Access Journals (Sweden)

    Lidija Kozačinski

    2001-04-01

    Full Text Available Goat’s milk samples were examined on mastitis using stable procedure (California-mastitis test. 427 of the examined milk samples (46.82% had positive reaction from 1 to 3 while other 485 samples (53.18% had negative reaction on the mastitis test, indicating that no illness of mammary gland occurred. Number of somatic cells, counted using “Fossomatic” counter, was 1.3x106/ml average. By comparing the results of mastitis-test evaluation (CMT with the number of somatic cells and findings of mastitis agents in milk showed that higher number of somatic cells is not the only indication of goat’s mammary gland illness. Mastitis-test is method that can exclude inflammation of goat’s mammary gland, but every positive reaction should be confirmed or eliminate with bacteriological examination. Based on the results of this research, it has been shown that the limit for somatic cells number in goat's milk can be over 1 000 000/ml.

  16. The ups and downs of somatic cell nucleus transfer (SCNT) in humans

    OpenAIRE

    Fulka, Josef; Langerova, Alena; Loi, Pasqualino; Ptak, Grazyna; Albertini, David; Fulka, Helena

    2013-01-01

    Achieving successful somatic cell nuclear transfer (SCNT) in the human and subhuman primate relative to other mammals has been questioned for a variety of technical and logistical issues. Here we summarize the gradual evolution of SCNT technology from the perspective of oocyte quality and cell cycle status that has recently led to the demonstration of feasibility in the human for deriving chromosomally normal stem cells lines. With these advances in hand, prospects for therapeutic cloning mus...

  17. A matter of identity — Phenotype and differentiation potential of human somatic stem cells

    Directory of Open Access Journals (Sweden)

    S.E.P. New

    2015-07-01

    Full Text Available Human somatic stem cells with neural differentiation potential can be valuable for developing cell-based therapies, including treatment of birth-related defects, while avoiding issues associated with cell reprogramming. Precisely defining the “identity” and differentiation potential of somatic stem cells from different sources, has proven difficult, given differences in sets of specific markers, protocols used and lack of side-by-side characterization of these cells in different studies. Therefore, we set to compare expression of mesenchymal and neural markers in human umbilical cord-derived mesenchymal stem cells (UC-MSCs, pediatric adipose-derived stem cells (p-ADSCs in parallel with human neural stem cells (NSCs. We show that UC-MSCs at a basal level express mesenchymal and so-called “neural” markers, similar to that we previously reported for the p-ADSCs. All somatic stem cell populations studied, independently from tissue and patient of origin, displayed a remarkably similar expression of surface markers, with the main difference being the restricted expression of CD133 and CD34 to NSCs. Expression of certain surface and neural markers was affected by the expansion medium used. As predicted, UC-MSCs and p-ADSCs demonstrated tri-mesenchymal lineage differentiation potential, though p-ADSCs display superior chondrogenic differentiation capability. UC-MSCs and p-ADSCs responded also to neurogenic induction by up-regulating neuronal markers, but crucially they appeared morphologically immature when compared with differentiated NSCs. This highlights the need for further investigation into the use of these cells for neural therapies. Crucially, this study demonstrates the lack of simple means to distinguish between different cell types and the effect of culture conditions on their phenotype, and indicates that a more extensive set of markers should be used for somatic stem cell characterization, especially when developing therapeutic

  18. Testicular Somatic Cells, not Gonocytes, Are the Major Source of Functional Activin A during Testis Morphogenesis

    OpenAIRE

    Archambeault, Denise R.; Tomaszewski, Jessica; Childs, Andrew J.; Anderson, Richard A.; YAO, HUMPHREY HUNG-CHANG

    2011-01-01

    Proper development of the seminiferous tubules (or testis cords in embryos) is critical for male fertility. Sertoli cells, somatic components of the seminiferous tubules, serve as nurse cells to the male germline, and thus their numbers decide the quantity of sperm output in adulthood. We previously identified activin A, the protein product of the activin βA (Inhba) gene, as a key regulator of murine Sertoli cell proliferation and testis cord expansion during embryogenesis. Although our genet...

  19. Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos

    International Nuclear Information System (INIS)

    Embryonic stem cells were isolated from rabbit blastocysts derived from fertilization (conventional rbES cells), parthenogenesis (pES cells) and nuclear transfer (ntES cells), and propagated in a serum-free culture system. Rabbit ES (rbES) cells proliferated for a prolonged time in an undifferentiated state and maintained a normal karyotype. These cells grew in a monolayer with a high nuclear/cytoplasm ratio and contained a high level of alkaline phosphate activity. In addition, rbES cells expressed the pluripotent marker Oct-4, as well as EBAF2, FGF4, TDGF1, but not antigens recognized by antibodies against SSEA-1, SSEA-3, SSEA-4, TRA-1-10 and TRA-1-81. All 3 types of ES cells formed embryoid bodies and generated teratoma that contained tissue types of all three germ layers. rbES cells exhibited a high cloning efficiency, were genetically modified readily and were used as nuclear donors to generate a viable rabbit through somatic cell nuclear transfer. In combination with genetic engineering, the ES cell technology should facilitate the creation of new rabbit lines

  20. Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Masahito [Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Umeyama, Kazuhiro [Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); International Cluster for Bio-Resource Research, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Matsunari, Hitomi [Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Takayanagi, Shuko [Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Haruyama, Erika; Nakano, Kazuaki; Fujiwara, Tsukasa; Ikezawa, Yuka [Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Nakauchi, Hiromitsu [Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, Tokyo University, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); and others

    2010-11-05

    Research highlights: {yields} EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. {yields} ZFNs induced targeted mutations in porcine primary cultured cells. {yields} Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

  1. Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

    International Nuclear Information System (INIS)

    Research highlights: → EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. → ZFNs induced targeted mutations in porcine primary cultured cells. → Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

  2. Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

    Institute of Scientific and Technical Information of China (English)

    Lan LI; Wei SHEN; Lingjiang MIN; Qingyu PAN; Yujiang SUN; Jixian DENG; Qingjie PAN

    2008-01-01

    Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropri-ate post-translational modifications.The nuclear transfer of transgenic somatic cells is a powerful method to pro-duce mammary gland bioreactors.We established an effi-cient gene transfer and nuclear transfer approach in goat somatic cells.Gene targeting vector pGBC2LF was con-structed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene.Goat fetal fibroblasts were transfected with lin-earized pGBC2LF and 14 cell lines were positive accord-ing to PCR and Southern blot.The transgenic cells were used as donor cells of nuclear transfer and some of recon-structed embryos could develop into blastocyst in vitro.

  3. Differential nanoreprotoxicity of silver nanoparticles in male somatic cells and spermatogonial stem cells

    Directory of Open Access Journals (Sweden)

    Zhang XF

    2015-02-01

    Full Text Available Xi-Feng Zhang,* Yun-Jung Choi,* Jae Woong Han, Eunsu Kim, Jung Hyun Park, Sangiliyandi Gurunathan, Jin-Hoi Kim Department of Animal Biotechnology, Konkuk University, Seoul, South Korea *These authors contributed equally to this work Background: Silver nanoparticles (AgNPs possess unique physical, chemical, and biological properties. AgNPs have been increasingly used as anticancer, antiangiogenic, and antibacterial agents for the treatment of bacterial infections in open wounds as well as in ointments, bandages, and wound dressings. The present study aimed to investigate the effects of two different sizes of AgNPs (10 nm and 20 nm in male somatic Leydig (TM3 and Sertoli (TM4 cells and spermatogonial stem cells (SSCs. Methods: Here, we demonstrate a green and simple method for the synthesis of AgNPs using Bacillus cereus culture supernatants. The synthesized AgNPs were characterized using ultraviolet and visible absorption spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and transmission electron microscopy (TEM. The toxicity of the synthesized AgNPs was evaluated by the effects on cell viability, metabolic activity, oxidative stress, apoptosis, and expression of genes encoding steroidogenic and tight junction proteins. Results: AgNPs inhibited the viability and proliferation of TM3 and TM4 cells in a dose- and size-dependent manner by damaging cell membranes and inducing the generation of reactive oxygen species, which in turn affected SSC growth on TM3 and TM4 as feeder cells. Small AgNPs (10 nm were more cytotoxic than medium-sized nanoparticles (20 nm. TEM revealed the presence of AgNPs in the cell cytoplasm and nucleus, and detected mitochondrial damage and enhanced formation of autosomes and autolysosomes in the AgNP-treated cells. Flow cytometry analysis using Annexin V/propidium iodide staining showed massive cell death by apoptosis or necrosis. Real-time polymerase chain reaction and western blot analyses

  4. Role of ooplasm in nuclear and nucleolar remodeling of intergeneric somatic cell nuclear transfer embryos during the first cell cycle

    DEFF Research Database (Denmark)

    Østrup, Olga; Strejcek, Frantisek; Petrovicova, Ida;

    2011-01-01

    intergeneric SCNT embryos were compared to their parthenogenetic counterparts to assess the effects of the introduced somatic cell. Despite the absence of morphological remodeling (premature chromatin condensation, nuclear envelope breakdown), reconstructed embryos showed nuclear and nucleolar precursor body......Initially, development of the zygote is under control of the oocyte ooplasm. However, it is presently unknown if and to what extent is the ooplasm able to interact with a transferred somatic cell from another species in the context of interspecies somatic cell nuclear transfer (SCNT). Here, one......-cell stage embryos were processed at different points in time post activation (2 hpa, 4 hpa, 8 hpa, and 12 hpa) for detailed nuclear and nucleolar analysis by TEM, and immunofluorescence for visualization of nucleolar proteins related to transcription (UBF) and processing (fibrillarin). Bovine and porcine...

  5. Production of transgenic7blastocyst of sheep by somatic cell cloning

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Five samples from primary cultures of five sheep ovarian granulosa cells were transfeeted by pEGFP N1 DNA. Five transgenic positive cell lines, each from one of the five samples above, were used as donor nuclei for somatic nucleus transfer. A total of 352 in vitro matured and enucle ated sheep oocytes were fused electrically with transgenic granulosa cells and 329 reconstructed embryos were ob tained after activation by Ionomycin/6-DMAP, and these embryos were cultured in SOFaaBSA medium for 7 d. The result shows that 312 embryos (94.8%) had gone through cleavage and among them 63 (19.1%) had developed to the blastocyst stage. Expression of GFP gene was detected in various stages of early embryonic development by sampling randomly. Blastocyst rates given by the four cells treated with 0.5% FCS starvation was 19.6% (55/280) and it had not shown difference significantly (P>0.05) with the result ob tained with another cell line that had not gone through se rum starvation (16.3%, 8/49). This experiment indicates that sheep transgenic embryos up to the blastocyst stage can be produced effectively by the combination of gene transfection in somatic cells in culture and somatic cell cloning.

  6. The ultrastructure of the kinetochore and kinetochore fiber in Drosophila somatic cells

    OpenAIRE

    Maiato, Helder; Hergert, Polla J.; Moutinho-Pereira, Sara; Dong, Yimin; VandenBeldt, Kristin J.; Rieder, Conly L.; McEwen, Bruce F.

    2006-01-01

    Drosophila melanogaster is a widely used model organism for the molecular dissection of mitosis in animals. However, despite the popularity of this system, no studies have been published on the ultrastructure of Drosophila kinetochores and kinetochore fibers (K-fibers) in somatic cells. To amend this situation, we used correlative light (LM) and electron microscopy (EM) to study kinetochores in cultured Drosophila S2 cells during metaphase, and after colchicine treatment to depolymerize all m...

  7. Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model*

    OpenAIRE

    Tong, Guo-qing; Heng, Boon-chin; Ng, Soon-chye

    2007-01-01

    This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 µmol/L strontium chloride for 5 h and sub...

  8. Differential staining of interspecific chromosomes in somatic cell hybrids by alkaline Giemsa stain.

    Science.gov (United States)

    Friend, K K; Chen, S; Ruddle, F H

    1976-03-01

    Staining of chromosome preparations of Chinese hamster-human hybrid cells and mouse-chimpanzee hybrids with alkaline Giemsa has yielded color differentiation of the interspecific chromosomes. Bicolor chromosomes, indicating apparent translocations also are observed for each of these hybrids. The specific color differences observed provide a rapid means of recognizing and aiding in the identification of the interspecific chromosomes and apparent translocations in these somatic cell hybrids. PMID:1028166

  9. Direct and indirect measurement of somatic cell count as indicator of intramammary infection in dairy goats

    OpenAIRE

    Olofsson Ida; Persson Ylva

    2011-01-01

    Abstract Background Mastitis is the most important and costly disease in dairy goat production. Subclinical mastitis is common in goats and is mainly caused by contagious bacteria. Several methods to diagnose subclinical mastitis are available. In this study indirect measurement of somatic cell count (SCC) by California Mastitis Test (CMT) and direct measurement of SCC using a portable deLaval cell counter (DCC) are evaluated. Swedish goat farmers would primarily benefit from diagnostic metho...

  10. Uncoupled embryonic and extra-embryonic tissues compromise blastocyst development after somatic cell nuclear transfer

    OpenAIRE

    Degrelle, Severine; Jaffrézic, Florence; Campion, Evelyne; Le Cao, Kim-Anh; Le Bourhis, Daniel; Richard, Christophe; Rodde, Nathalie; Fleurot, Renaud; Everts, Robin E.; Lecardonnel, Jérôme; Heyman, Yvan; Vignon, Xavier; Yang, Xiangzhong; Tian, Xiuchun C.; Lewin, Harris A

    2012-01-01

    Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulatin...

  11. Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

    International Nuclear Information System (INIS)

    In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells were tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.

  12. A molecular roadmap of reprogramming somatic cells into iPS cells.

    Science.gov (United States)

    Polo, Jose M; Anderssen, Endre; Walsh, Ryan M; Schwarz, Benjamin A; Nefzger, Christian M; Lim, Sue Mei; Borkent, Marti; Apostolou, Effie; Alaei, Sara; Cloutier, Jennifer; Bar-Nur, Ori; Cheloufi, Sihem; Stadtfeld, Matthias; Figueroa, Maria Eugenia; Robinton, Daisy; Natesan, Sridaran; Melnick, Ari; Zhu, Jinfang; Ramaswamy, Sridhar; Hochedlinger, Konrad

    2012-12-21

    Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we examined defined intermediate cell populations poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcriptional wave and can be rescued by elevated expression of all four factors. The establishment of bivalent domains occurs gradually after the first wave, whereas changes in DNA methylation take place after the second wave when cells acquire stable pluripotency. This integrative analysis allowed us to identify genes that act as roadblocks during reprogramming and surface markers that further enrich for cells prone to forming iPSCs. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming. PMID:23260147

  13. Roles of small molecules in somatic cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Jian-bin SU; Duan-qing PEI; Bao-ming QIN

    2013-01-01

    The Nobel Prize in Physiology and Medicine 2012 was awarded to Sir John B GURDON and Shinya YAMANAKA for their discovery that mature cells can be reprogrammed to become pluripotent.This event reaffirms the importance of research on cell fate plasticity and the technology progress in the stem cell field and regenerative medicine.Indeed,reprogramming technology has developed at a dazzling speed within the past 6 years,yet we are still at the early stages of understanding the mechanisms of cell fate identity.This is particularly true in the case of human induced pluripotent stem ceils (iPSCs),which lack reliable standards in the evaluation of their fidelity and safety prior to their application.Along with the genetic approaches,small molecules nowadays become convenient tools for modulating endogenous protein functions and regulating key cellular processes,including the mesenchymal-to-epithelial transition,metabolism,signal transduction and epigenetics.Moreover,small molecules may affect not only the efficiency of clone formation but also the quality of the resulting cells.With increasing availability of such chemicals,we can better understand the biology of stems cells and further improve the technology of generation of stem cells.

  14. Somatic mutation and cell differentiation in neoplastic transformation

    Energy Technology Data Exchange (ETDEWEB)

    Huberman, E.; Collart, F.R.

    1987-01-01

    In brief, the authors suggest that tumor formation may result from continuous expression of growth facilitating genes that, as a result of irreversible changes during the initiation step, are placed under the control of genes expressed during normal differentiation. Thus, to understand carcinogenesis, we must decipher the processes that lead to the acquisition of a mature phenotype in both normal and tumor cells and characterize the growth dependency of tumor cells to inducers of cell differentiation. Furthermore, the growth of a variety of tumors may be controlled through the use of inducers of maturation that activate genes located beyond the gene that is altered during tumor initiation. 22 refs., 3 figs.

  15. Somatic mutation and cell differentiation in neoplastic transformation

    International Nuclear Information System (INIS)

    In brief, the authors suggest that tumor formation may result from continuous expression of growth facilitating genes that, as a result of irreversible changes during the initiation step, are placed under the control of genes expressed during normal differentiation. Thus, to understand carcinogenesis, we must decipher the processes that lead to the acquisition of a mature phenotype in both normal and tumor cells and characterize the growth dependency of tumor cells to inducers of cell differentiation. Furthermore, the growth of a variety of tumors may be controlled through the use of inducers of maturation that activate genes located beyond the gene that is altered during tumor initiation. 22 refs., 3 figs

  16. Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells.

    Directory of Open Access Journals (Sweden)

    Jakub Cieslak

    Full Text Available Apart from the well-known role of somatic cell count as a parameter reflecting the inflammatory status of the mammary gland, the composition of cells isolated from milk is considered as a valuable material for gene expression studies in mammals. Due to its unique composition, in recent years an increasing interest in mare's milk consumption has been observed. Thus, investigating the genetic background of horse's milk variability presents and interesting study model. Relying on 39 milk samples collected from mares representing three breeds (Polish Primitive Horse, Polish Cold-blooded Horse, Polish Warmblood Horse we aimed to investigate the utility of equine milk somatic cells as a source of mRNA and to screen the best reference genes for RT-qPCR using geNorm and NormFinder algorithms. The results showed that despite relatively low somatic cell counts in mare's milk, the amount and the quality of the extracted RNA are sufficient for gene expression studies. The analysis of the utility of 7 potential reference genes for RT-qPCR experiments for the normalization of equine milk somatic cells revealed some differences between the outcomes of the applied algorithms, although in both cases the KRT8 and TOP2B genes were pointed as the most stable. Analysis by geNorm showed that the combination of 4 reference genes (ACTB, GAPDH, TOP2B and KRT8 is required for apropriate RT-qPCR experiments normalization, whereas NormFinder algorithm pointed the combination of KRT8 and RPS9 genes as the most suitable. The trial study of the relative transcript abundance of the beta-casein gene with the use of various types and numbers of internal control genes confirmed once again that the selection of proper reference gene combinations is crucial for the final results of each real-time PCR experiment.

  17. Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells

    Directory of Open Access Journals (Sweden)

    H.J. Chung

    2015-05-01

    Full Text Available Recent advances have raised hope that transplantation of adherent somatic cells could provide dramatic new therapies for various diseases. However, current methods for transplanting adherent somatic cells are not efficient enough for therapeutic applications. Here, we report the development of a novel method to generate quasi-natural cell blocks for high-efficiency transplantation of adherent somatic cells. The blocks were created by providing a unique environment in which cultured cells generated their own extracellular matrix. Initially, stromal cells isolated from mice were expanded in vitro in liquid cell culture medium followed by transferring the cells into a hydrogel shell. After incubation for 1 day with mechanical agitation, the encapsulated cell mass was perforated with a thin needle and then incubated for an additional 6 days to form a quasi-natural cell block. Allograft transplantation of the cell block into C57BL/6 mice resulted in perfect adaptation of the allograft and complete integration into the tissue of the recipient. This method could be widely applied for repairing damaged cells or tissues, stem cell transplantation, ex vivo gene therapy, or plastic surgery.

  18. Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells.

    Science.gov (United States)

    Chung, H J; Hassan, M M; Park, J O; Kim, H J; Hong, S T

    2015-05-01

    Recent advances have raised hope that transplantation of adherent somatic cells could provide dramatic new therapies for various diseases. However, current methods for transplanting adherent somatic cells are not efficient enough for therapeutic applications. Here, we report the development of a novel method to generate quasi-natural cell blocks for high-efficiency transplantation of adherent somatic cells. The blocks were created by providing a unique environment in which cultured cells generated their own extracellular matrix. Initially, stromal cells isolated from mice were expanded in vitro in liquid cell culture medium followed by transferring the cells into a hydrogel shell. After incubation for 1 day with mechanical agitation, the encapsulated cell mass was perforated with a thin needle and then incubated for an additional 6 days to form a quasi-natural cell block. Allograft transplantation of the cell block into C57BL/6 mice resulted in perfect adaptation of the allograft and complete integration into the tissue of the recipient. This method could be widely applied for repairing damaged cells or tissues, stem cell transplantation, ex vivo gene therapy, or plastic surgery. PMID:25742639

  19. Evidence for estrogen receptor expression in germ cell and somatic cell subpopulations in the ovary of the newly hatched chicken.

    Science.gov (United States)

    Méndez, M C; Chávez, B; Echeverría, O; Vilchis, F; Vázquez Nin, G H; Pedernera, E

    1999-10-01

    Estrogens are involved in the gonadal morphogenesis of vertebrates, and almost all hormonal effects of 17beta-estradiol are mediated through specific receptors. At the time of sexual differentiation in the chicken, or even before, there is evidence of the presence of estrogen receptors and the secretion of 17beta-estradiol. However, no information is available regarding the cellular types that express the estrogen receptor in the immature chick ovary. The present study analyzes estrogen receptor expression in germ and somatic cells of the ovary in the newly hatched chicken. Highly purified cell subpopulations of germ and somatic cells were evaluated for specific 17beta-estradiol nuclear binding. In addition, the estrogen receptor was localized at the ultrastructural level by the immunogold technique. Finally, reverse transcription and polymerase chain reaction procedures detected a steady-state level of mRNA for the estrogen receptor. Somatic cells including typical steroidogenic cells showed specific 17beta-estradiol nuclear binding, displayed the estrogen receptor, and possessed estrogen receptor transcripts. The same result was observed in primary oocytes, together with the ultrastructural localization of estrogen receptor in extended chromatin filaments. Our experimental data support the hypothesis that estrogens are involved in the function of somatic and germ cells subpopulations in the immature chicken ovary. PMID:10555548

  20. Telomere Elongation and Naive Pluripotent Stem Cells Achieved from Telomerase Haplo-Insufficient Cells by Somatic Cell Nuclear Transfer

    Directory of Open Access Journals (Sweden)

    Li-Ying Sung

    2014-12-01

    Full Text Available Haplo-insufficiency of telomerase genes in humans leads to telomere syndromes such as dyskeratosis congenital and idiopathic pulmonary fibrosis. Generation of pluripotent stem cells from telomerase haplo-insufficient donor cells would provide unique opportunities toward the realization of patient-specific stem cell therapies. Recently, pluripotent human embryonic stem cells (ntESCs have been efficiently achieved by somatic cell nuclear transfer (SCNT. We tested the hypothesis that SCNT could effectively elongate shortening telomeres of telomerase haplo-insufficient cells in the ntESCs with relevant mouse models. Indeed, telomeres of telomerase haplo-insufficient (Terc+/− mouse cells are elongated in ntESCs. Moreover, ntESCs derived from Terc+/− cells exhibit naive pluripotency as evidenced by generation of Terc+/− ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency. These data suggest that SCNT could offer a powerful tool to reprogram telomeres and to discover the factors for robust restoration of telomeres and pluripotency of telomerase haplo-insufficient somatic cells.

  1. Aberrant histone H4 acetylation in dead somatic cell-cloned calves

    Institute of Scientific and Technical Information of China (English)

    Lei Zhang; Shaohua Wang; Qiang Li; Xiangdong Ding; Yunping Dai; Ning Li

    2008-01-01

    In somatic cell-cloned animals, inefficient epigenetic reprogramming can result in an inappropriate gene expression and histone H4 acetylation is one of the key epigenetic modifications regulating gene expression. In this study, we investigated the levels of histone H4 acetylation of 11 development-related genes and expression levels of 19 genes in lungs of three normal control calves and nine aber-rant somatic cell-cloned calves. The results showed that nine studied genes had decreased acetylation levels in aberrant clones (p 0.05). Whereas 13 genes had significantly decreased expression (p 0.05), and only one gene had higher expression level in clones (p < 0.05). Furthermore, FGFR, GHR, HGFR and IGF1 genes showed lowered levels of both histone H4 acetylation and expression in aberrant clones than in controls, and the level of histone H4 acetylation was even more lowered in aberrant clones than those in controls. It was suggested that the lower levels of histone H4 acetylation in aberrant clones caused by the previous memory of cell differentiation might not support enough chromatin reprogramming, thus affecting appropriate gene expressions, and growth and development of the cloned calves. To our knowledge, this is the first study on how histone H4 acetylation affects gene expression in organs of somatic cell-cloned calves.

  2. Flow-cytometric measurements of somatic cell mutations in Thorotrast patients

    International Nuclear Information System (INIS)

    Exposure to ionizing radiation is a well-recognized risk factor for cancer development. Because ionizing radiation can induce mutations, an accurate way of measuring somatic mutation frequencies could be a useful tool for evaluating cancer risk. In the present study, we have examined in vivo somatic mutation frequencies at the erythrocyte glycophorin A and T-cell receptor loci in 18 Thorotrast patients. These persons have been continuously irradiated with alpha particles emitted from the internal deposition of thorium dioxide and thus have increased risks of certain malignant tumors. When compared with controls, the Thorotrast patients showed a significantly higher frequency of mutants at the lymphocyte T-cell receptor loci but not at the erythrocyte glycophorin A loci. (author)

  3. Increased somatic cell mutant frequency in atomic bomb survivors

    International Nuclear Information System (INIS)

    Frequencies of mutant T-cells in peripheral blood, which are deficient in the activity of hypoxanthine guanine phosphoribosyltransferase (HPRT) were determined for atomic bomb survivors by direct clonal assay using a previously reported method. Results from 30 exposed survivors (exposed to more than 1 rad) and 17 age- and sex-matched controls (exposed to less than 1 rad) were analyzed. The mean mutant frequency (Mf) in the exposed (5.2 x 10-6; range 0.8 - 14.4 x 10-6) was significantly higher than in controls (3.4 x 10-6; range 1.3 - 9.3 x 10-6), a fact not attributable to lower nonmutant cell cloning efficiencies in the exposed group since cell cloning efficiencies were virtually identical in both groups. An initial analysis of the data did not reveal a significant correlation between individual Mfs and individual radiation dose estimates when the latter were defined by the original, tentative estimates (T65D), even though there was a significant positive correlation of Mfs with individual frequency of lymphocytes bearing chromosome aberration. However, reanalysis using the newer revised individual dose estimates (DS86) for 27 exposed survivors and 17 controls did reveal a significant but shallow positive correlation between T-cell Mf values and individual exposure doses. These results indicate that HPRT mutation in vivo in human T-cells could be detected in these survivors 40 years after the presumed mutational event. (author)

  4. Rescue of marker phenotypes mediated by somatic cell hybridization

    International Nuclear Information System (INIS)

    The effect of irradiation prior to virus-induced cell fusion on the frequency of hybrid production has been measured as a function of radiation dose. The Chinese hamster line wg3h (HGPRT-) was crossed with the TK- mutants; Chinese hamster A23 or mouse 3T34E, and hybrids were selected in HAT medium. Irradiation of one (marker rescue) or both (mutual rescue) partners before fusion yielded qualitatively different results. After X-irradiation, marker rescue curves were of single-hit type, with D0 values about five-fold greater than the irradiated parent cell. Mutual rescue curves were of the multi-hit type, with zero-dose extrapolation value (n) greater than that of the more resistant partner, but no significant alteration in D0. Qualitatively similar results were obtained after U.V.- irradiation, but the probability of rescue per surviving parent cell was

  5. Progress toward generating a ferret model of cystic fibrosis by somatic cell nuclear transfer

    OpenAIRE

    Engelhardt John F; Li Ziyi

    2003-01-01

    Abstract Mammalian cloning by nuclear transfer from somatic cells has created new opportunities to generate animal models of genetic diseases in species other than mice. Although genetic mouse models play a critical role in basic and applied research for numerous diseases, often mouse models do not adequately reproduce the human disease phenotype. Cystic fibrosis (CF) is one such disease. Targeted ablation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in mice does not...

  6. Production of Cloned Korean Native Pig by Somatic Cell Nuclear Transfer

    OpenAIRE

    Hwang, In-Sul; Kwon, Dae-Jin; Oh, Keun Bong; Ock, Sun-A; Chung, Hak-Jae; Cho, In-Cheol; Lee, Jeong-Woong; Im, Gi-Sun; Hwang, Seongsoo

    2015-01-01

    The Korean native pig (KNP) have been considered as animal models for animal biotechnology research because of their relatively small body size and their presumably highly inbred status due to the closed breeding program. However, little is reported about the use of KNP for animal biotechnology researches. This study was performed to establish the somatic cell nuclear transfer (SCNT) protocol for the production of swine leukocyte antigens (SLA) homotype-defined SCNT KNP. The ear fibroblast ce...

  7. Machine Learning as an aid to management decisions on high somatic cell counts in dairy farms

    OpenAIRE

    Goyache, Félix; Díez, Jorge; López, Secundino; Pajares-Bernaldo, Gerardo; Santos, Begoña; Fernández, Iván; Prieto, Miguel

    2005-01-01

    High somatic cell counts (SCC) is associated with mastitis infection, in dairy herds, worldwide. This work describes Machine Learning (ML) techniques designed to improve the information offered to farmers on animals producing high SCCs according to particular herd profiles. The analysed population included 71 dairy farms in Asturias (Northern Spain) and a total of 2,407 lactating cows. Four sources of information were available: a) a questionnaire survey describing facilities, milking routine...

  8. Mastitis diagnosis in dairy goats through somatic cell counts and Californian Mastitis Test. Preliminary results

    OpenAIRE

    Mendonça, Álvaro; Valentim, Ramiro; Nunes, Manuel; Correia, Teresa Montenegro; Trigo, Margarida; Maurício, Raimundo; Costa, Cristina; Coelho, Alípio

    2004-01-01

    The aim of this work was to evaluate somatic cell count (SCC) and Californian mastitis test (CMT) reliability as methods to survey mastitis in Serrana goats. Microbiological diagnosis, SCC and CTM were performed on 2028 samples, collected from individual glands during a lactation period. According to results CMT (predictive negative value = 69.5%) may be used as a cheap and practical method for sub clinical mastitis survey in Serrana goats. Decision on SCC use will depend on additional resear...

  9. Histocompatibility genes and Somatic Cell Count (SCC) in Italian Holstein Friesian

    OpenAIRE

    Longeri, M.; A. B. Samoré; I. Taboni; Strillacci, M G; Zanotti, M.

    2011-01-01

    Mastitis is a dairy cattle disease leading to great economic losses in milk production, management costs and veterinary treatments. This character generally shows an unfavourable genetic correlation with milk production. However, genetic factors influencing mastitis susceptibility independent from those influencing milk production could exist. Therefore, it should be possible to select at the same time against mastitis and for high milk production. The “somatic cell count” (SCC) c...

  10. Genetic analysis for mastitis resistance and milk somatic cell score in French Lacaune dairy sheep

    OpenAIRE

    Barillet, Francis; Rupp, Rachel; Mignon-Grasteau, S.; Astruc, J.M.; Jacquin, M.

    2001-01-01

    Genetic analysis for mastitis resistance was studied from two data sets. Firstly, risk factors for different mastitis traits, i.e. culling due to clinical or chronic mastitis and subclinical mastitis predicted from somatic cell count (SCC), were explored using data from 957 first lactation Lacaune ewes of an experimental INRA flock composed of two divergent lines for milk yield. Secondly, genetic parameters for SCC were estimated from 5 272 first lactation Lacaune ewes recorded among 38 flock...

  11. Genetic analysis for mastitis resistance and milk somatic cell score in French Lacaune dairy sheep

    OpenAIRE

    Astruc Jean-Michel; Mignon-Grasteau Sandrine; Rupp Rachel; Barillet Francis; Jacquin Michèle

    2001-01-01

    Abstract Genetic analysis for mastitis resistance was studied from two data sets. Firstly, risk factors for different mastitis traits, i.e. culling due to clinical or chronic mastitis and subclinical mastitis predicted from somatic cell count (SCC), were explored using data from 957 first lactation Lacaune ewes of an experimental INRA flock composed of two divergent lines for milk yield. Secondly, genetic parameters for SCC were estimated from 5 272 first lactation Lacaune ewes recorded among...

  12. Use of California mastitis test, somatic cells count and bacteriological findings in diagnostics of subclinical mastitis

    OpenAIRE

    Varatanović N.; Podžo M.; Mutevelić T.; Podžo K.; Čengić B.; Hodžić A.; Hodžić E.

    2010-01-01

    We have performed diagnostics of sub clinical mastitis in three different cow breeds with comparison of California mastitis test results, somatic cells count at quarter level and with bacteriological findings confirmation in order to justify their appliance in mastitis diagnostics. In total, 90 cows or 360 quarters of mammary gland have been examined. In 63.3 % of the examined cows, with different racial origin, positive reaction to California mastitis test have been established. Usually, pos...

  13. Effect of somatic cell count and lactation stage on sheep milk quality

    OpenAIRE

    Emilia Duranti; Arianna Bolla; Anna Caroli; Elena Budelli; Mariano Pauselli; Carmen Casoli; Leonardo Bianchi

    2010-01-01

    In order to evaluate the effects of mammary health status and lactation phase on the qualitative parameters of ovinemilk, 213 individual milk samples were repeatedly collected from 40 primiparous Sarda ewes on a monthly basis. Yield,physico-chemical characteristics, casein fractions quantitative distribution, somatic cell count (SCC), cheese making propertiesand plasmin-plasminogen activity were determined on each sample. Repeated individual milk SCC were used as amarker of udder health statu...

  14. Phenotypes of Aging Postovulatory Oocytes After Somatic Cell Nuclear Transfer in Mice.

    Science.gov (United States)

    Lee, Ah Reum; Shimoike, Takashi; Wakayama, Teruhiko; Kishigami, Satoshi

    2016-06-01

    Oocytes rapidly lose their developmental potential after ovulation, termed postovulatory oocyte aging, and often exhibit characteristic phenotypes, such as cytofragmentation, abnormal spindle shapes, and chromosome misalignments. Here, we reconstructed mouse oocytes using somatic cell nuclear transfer (SCNT) to reveal the effect of somatic cell-derived nuclei on oocyte physiology during aging. Normal oocytes started undergoing cytofragmentation 24 hours after oocyte collection; however, this occurred earlier in SCNT oocytes and was more severe at 48 hours, suggesting that the transferred somatic cell nuclei affected oocyte physiology. We found no difference in the status of acetylated α-tubulin (Ac-Tub) and α-tubulin (Tub) between normal and SCNT aging oocytes, but unlike normal oocytes, aging SCNT oocytes did not have astral microtubules. Interestingly, aging SCNT oocytes displayed more severely scattered chromosomes or irregularly shaped spindles. Observations of the microfilaments showed that, in normal oocytes, there was a clear actin ring beneath the plasma membrane and condensed microfilaments around the spindle (the actin cap) at 0 hours, and the actin filaments started degenerating at 1 hour, becoming completely disrupted and distributed to the cytoplasm at 24 hours. By contrast, in SCNT oocytes, an actin cap formed around the transplanted nuclei within 1 hour of SCNT, which was still present at 24 hours. Thus, SCNT oocytes age in a similar but distinct way, suggesting that they not only contain nuclei with abnormal epigenetics but are also physiologically different. PMID:27253626

  15. Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins

    Directory of Open Access Journals (Sweden)

    Ki-Eun Park

    2016-05-01

    Full Text Available The pig is an ideal large animal model for genetic engineering applications. A relatively short gestation interval and large litter size makes the pig a conducive model for generating and propagating genetic modifications. The domestic pig also shares close similarity in anatomy, physiology, size, and life expectancy, making it an ideal animal for modeling human diseases. Often, however, the technical difficulties in generating desired genetic modifications such as targeted knockin of short stretches of sequences or transgenes have impeded progress in this field. In this study, we have investigated and compared the relative efficiency of CRISPR/Cas ribonucleoproteins in engineering targeted knockin of pseudo attP sites downstream of a ubiquitously expressed COL1A gene in porcine somatic cells and generated live fetuses by somatic cell nuclear transfer (SCNT. By leveraging these knockin pseudo attP sites, we have demonstrated subsequent phiC31 integrase mediated integration of green fluorescent protein (GFP transgene into the site. This work for the first time created an optimized protocol for CRISPR/Cas mediated knockin in porcine somatic cells, while simultaneously creating a stable platform for future transgene integration and generating transgenic animals.

  16. Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins.

    Science.gov (United States)

    Park, Ki-Eun; Park, Chi-Hun; Powell, Anne; Martin, Jessica; Donovan, David M; Telugu, Bhanu P

    2016-01-01

    The pig is an ideal large animal model for genetic engineering applications. A relatively short gestation interval and large litter size makes the pig a conducive model for generating and propagating genetic modifications. The domestic pig also shares close similarity in anatomy, physiology, size, and life expectancy, making it an ideal animal for modeling human diseases. Often, however, the technical difficulties in generating desired genetic modifications such as targeted knockin of short stretches of sequences or transgenes have impeded progress in this field. In this study, we have investigated and compared the relative efficiency of CRISPR/Cas ribonucleoproteins in engineering targeted knockin of pseudo attP sites downstream of a ubiquitously expressed COL1A gene in porcine somatic cells and generated live fetuses by somatic cell nuclear transfer (SCNT). By leveraging these knockin pseudo attP sites, we have demonstrated subsequent phiC31 integrase mediated integration of green fluorescent protein (GFP) transgene into the site. This work for the first time created an optimized protocol for CRISPR/Cas mediated knockin in porcine somatic cells, while simultaneously creating a stable platform for future transgene integration and generating transgenic animals. PMID:27240344

  17. Genetic aspects of somatic cell count and udder health in the Italian Valle del Belice dairy sheep

    OpenAIRE

    Riggio, V

    2012-01-01

    Mastitis is an inflammation of the udder, which leads to economic loss, mainly consisting of discarded milk, reduced milk production and quality, and increased health costs. Somatic cell count (SCC), and therefore somatic cell score (SCS), is widely used as indicator of mastitis. In this thesis, I focus on the genetic parameters of SCS as indicator of mastitis, and on the possibilities of using this trait for selection for mastitis resistance in the Valle del Belice dairy sheep. In Chapter 1,...

  18. Genetic relationship of lactation persistency with milk yield, somatic cell score, reproductive traits, and longevity in Slovak Holstein cattle

    OpenAIRE

    Strapáková, Eva; Candrák, Juraj; Strapák, Peter

    2016-01-01

    The objective of this study was to estimate the breeding values (BVs) of lactation persistency, the test day of milk yield, the somatic cell score, reproductive traits (calving interval, days open), longevity in Slovak Holstein dairy cattle. BVs were used for the detection of relationships among the persistency of lactation and other selected traits. Data for the estimation of BVs of milk production and somatic cell score were collected from 855 240 cows. BVs for reproductive t...

  19. Potential of primary kidney cells for somatic cell nuclear transfer mediated transgenesis in pig

    Directory of Open Access Journals (Sweden)

    Richter Anne

    2012-11-01

    Full Text Available Abstract Background Somatic cell nuclear transfer (SCNT is currently the most efficient and precise method to generate genetically tailored pig models for biomedical research. However, the efficiency of this approach is crucially dependent on the source of nuclear donor cells. In this study, we evaluate the potential of primary porcine kidney cells (PKCs as cell source for SCNT, including their proliferation capacity, transfection efficiency, and capacity to support full term development of SCNT embryos after additive gene transfer or homologous recombination. Results PKCs could be maintained in culture with stable karyotype for up to 71 passages, whereas porcine fetal fibroblasts (PFFs and porcine ear fibroblasts (PEFs could be hardly passaged more than 20 times. Compared with PFFs and PEFs, PKCs exhibited a higher proliferation rate and resulted in a 2-fold higher blastocyst rate after SCNT and in vitro cultivation. Among the four transfection methods tested with a GFP expression plasmid, best results were obtained with the NucleofectorTM technology, resulting in transfection efficiencies of 70% to 89% with high fluorescence intensity, low cytotoxicity, good cell proliferation, and almost no morphological signs of cell stress. Usage of genetically modified PKCs in SCNT resulted in approximately 150 piglets carrying at least one of 18 different transgenes. Several of those pigs originated from PKCs that underwent homologous recombination and antibiotic selection before SCNT. Conclusion The high proliferation capacity of PKCs facilitates the introduction of precise and complex genetic modifications in vitro. PKCs are thus a valuable cell source for the generation of porcine biomedical models by SCNT.

  20. The glycophorin A assay for somatic cell mutations in humans

    International Nuclear Information System (INIS)

    In this report we briefly review our past experience and some new developments with the GPA assay. Particular emphasis will be placed on two areas that affect the utility of the GPA assay for human population monitoring. The first is our efforts to simplify the GPA assay to make it more generally available for large population studies. The second is to begin to understand some of the characteristics of human hemopoiesis which affect the accumulation and expression of mutant phenotype cells. 11 refs., 4 figs

  1. Cell fusion of bone marrow cells and somatic cell reprogramming by embryonic stem cells

    OpenAIRE

    Bonde, Sabrina; Pedram, Mehrdad; Stultz, Ryan; Zavazava, Nicholas

    2010-01-01

    Bone marrow transplantation is a curative treatment for many diseases, including leukemia, autoimmune diseases, and a number of immunodeficiencies. Recently, it was claimed that bone marrow cells transdifferentiate, a much desired property as bone marrow cells are abundant and therefore could be used in regenerative medicine to treat incurable chronic diseases. Using a Cre/loxP system, we studied cell fusion after bone marrow transplantation. Fused cells were chiefly Gr-1+, a myeloid cell mar...

  2. Cats cloned from fetal and adult somatic cells by nuclear transfer.

    Science.gov (United States)

    Yin, X J; Lee, H S; Lee, Y H; Seo, Y I; Jeon, S J; Choi, E G; Cho, S J; Cho, S G; Min, W; Kang, S K; Hwang, W S; Kong, I K

    2005-02-01

    This work was undertaken in order to study the developmental competence of nuclear transfer (NT) into cat embryos using fetal fibroblast and adult skin fibroblast cells as donor nuclei. Oocytes were recovered by mincing the ovaries in Hepes-buffered TCM199 and selecting the cumulus oocyte complexes (COCs) with compact cumulus cell mass and dark color. Homogenous ooplasm was cultured for maturation in TCM199+10% fetal bovine serum (FBS) for 12 h and used as a source of recipient cytoplast for exogenous somatic nuclei. In experiment 1, we evaluated the effect of donor cell type on the reconstruction and development of cloned embryos. Fusion, first cleavage and blastocyst developmental rate were not different between fetal fibroblasts and adult skin cells (71.2 vs 66.8; 71.0 vs 57.6; 4.0 vs 6.1% respectively; P < 0.05). In experiment 2, cloned embryos were surgically transferred into the oviducts of recipient queens. One of the seven recipient queens was delivered naturally of 2 healthy cloned cats and 1 stillborn from fetal fibroblast cells of male origin 65 days after embryo transfer. One of three recipient queens was delivered naturally of 1 healthy cloned cat from adult skin cells of female origin 65 days after embryo transfer. The cloned cats showed genotypes identical to the donor cell lines, indicating that adult somatic cells can be used for feline cloning. PMID:15695619

  3. Bacteria-human somatic cell lateral gene transfer is enriched in cancer samples.

    Directory of Open Access Journals (Sweden)

    David R Riley

    Full Text Available There are 10× more bacterial cells in our bodies from the microbiome than human cells. Viral DNA is known to integrate in the human genome, but the integration of bacterial DNA has not been described. Using publicly available sequence data from the human genome project, the 1000 Genomes Project, and The Cancer Genome Atlas (TCGA, we examined bacterial DNA integration into the human somatic genome. Here we present evidence that bacterial DNA integrates into the human somatic genome through an RNA intermediate, and that such integrations are detected more frequently in (a tumors than normal samples, (b RNA than DNA samples, and (c the mitochondrial genome than the nuclear genome. Hundreds of thousands of paired reads support random integration of Acinetobacter-like DNA in the human mitochondrial genome in acute myeloid leukemia samples. Numerous read pairs across multiple stomach adenocarcinoma samples support specific integration of Pseudomonas-like DNA in the 5'-UTR and 3'-UTR of four proto-oncogenes that are up-regulated in their transcription, consistent with conversion to an oncogene. These data support our hypothesis that bacterial integrations occur in the human somatic genome and may play a role in carcinogenesis. We anticipate that the application of our approach to additional cancer genome projects will lead to the more frequent detection of bacterial DNA integrations in tumors that are in close proximity to the human microbiome.

  4. Similar GABAA receptor subunit composition in somatic and axon initial segment synapses of hippocampal pyramidal cells.

    Science.gov (United States)

    Kerti-Szigeti, Katalin; Nusser, Zoltan

    2016-01-01

    Hippocampal pyramidal cells (PCs) express many GABAAR subunit types and receive GABAergic inputs from distinct interneurons. Previous experiments revealed input-specific differences in α1 and α2 subunit densities in perisomatic synapses, suggesting distinct IPSC decay kinetics. However, IPSC decays evoked by axo-axonic, parvalbumin- or cholecystokinin-expressing basket cells were found to be similar. Using replica immunogold labeling, here we show that all CA1 PC somatic and AIS synapses contain the α1, α2, β1, β2, β3 and γ2 subunits. In CA3 PCs, 90% of the perisomatic synapses are immunopositive for the α1 subunit and all synapses are positive for the remaining five subunits. Somatic synapses form unimodal distributions based on their immunoreactivity for these subunits. The α2 subunit densities in somatic synapses facing Cav2.1 (i.e. parvalbumin) or Cav2.2 (cholecystokinin) positive presynaptic active zones are comparable. We conclude that perisomatic synapses made by three distinct interneuron types have similar GABAA receptor subunit content. PMID:27537197

  5. Transient Acquisition of Pluripotency During Somatic Cell Transdifferentiation with iPSC Reprogramming Factors

    Science.gov (United States)

    Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D.; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R.; Greenleaf, William J.; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H.

    2015-01-01

    Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors1,2. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation3–6. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced transdifferentiation pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by different methods. PMID:26098448

  6. The Transcriptional Consequences of Somatic Amplifications, Deletions, and Rearrangements in a Human Lung Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Lucy F Stead

    2012-11-01

    Full Text Available Lung cancer causes more deaths, worldwide, than any other cancer. Several histologic subtypes exist. Currently, there is a dearth of targeted therapies for treating one of the main subtypes: squamous cell carcinoma (SCC. As for many cancers, lung SCC karyotypes are often highly anomalous owing to large somatic structural variants, some of which are seen repeatedly in lung SCC, indicating a potential causal association for genes therein. We chose to characterize a lung SCC genome to unprecedented detail and integrate our findings with the concurrently characterized transcriptome. We aimed to ascertain how somatic structural changes affected gene expression within the cell in ways that could confer a pathogenic phenotype. We sequenced the genomes of a lung SCC cell line (LUDLU-1 and its matched lymphocyte cell line (AGLCL to more than 50x coverage. We also sequenced the transcriptomes of LUDLU-1 and a normal bronchial epithelium cell line (LIMM-NBE1, resulting in more than 600 million aligned reads per sample, including both coding and non-coding RNA (ncRNA, in a strand-directional manner. We also captured small RNA (<30 bp. We discovered significant, but weak, correlations between copy number and expression for protein-coding genes, antisense transcripts, long intergenic ncRNA, and microRNA (miRNA. We found that miRNA undergo the largest change in overall expression pattern between the normal bronchial epithelium and the tumor cell line. We found evidence of transcription across the novel genomic sequence created from six somatic structural variants. For each part of our integrated analysis, we highlight candidate genes that have undergone the largest expression changes.

  7. Extracellular matrix of dental pulp stem cells: Applications in pulp tissue engineering using somatic MSCs

    Directory of Open Access Journals (Sweden)

    Sriram eRavindran

    2014-01-01

    Full Text Available Dental Caries affects approximately 90% of the world’s population. At present, the clinical treatment for dental caries is root canal therapy. This treatment results in loss of tooth sensitivity and vitality. Tissue engineering can potentially solve this problem by enabling regeneration of a functional pulp tissue. Dental pulp stem cells (DPSCs have been shown to be an excellent source for pulp regeneration. However, limited availability of these cells hinders its potential for clinical translation. We have investigated the possibility of using somatic mesenchymal stem cells from other sources for dental pulp tissue regeneration using a biomimetic dental pulp extracellular matrix (ECM incorporated scaffold. Human periodontal ligament stem cells (PDLSCs and human bone marrow stromal cells (HMSCs were investigated for their ability to differentiate towards an odontogenic lineage. In vitro real-time PCR results coupled with histological and immunohistochemical examination of the explanted tissues confirmed the ability of PDLSCs and HMSCs to form a vascularized pulp-like tissue. These findings indicate that the dental pulp stem derived ECM scaffold stimulated odontogenic differentiation of PDLSCs and HMSCs without the need for exogenous addition of growth and differentiation factors. This study represents a translational perspective toward possible therapeutic application of using a combination of somatic stem cells and extracellular matrix for pulp regeneration.

  8. Somatically Acquired LINE-1 Insertions in Normal Esophagus Undergo Clonal Expansion in Esophageal Squamous Cell Carcinoma.

    Science.gov (United States)

    Doucet-O'Hare, Tara T; Sharma, Reema; Rodić, Nemanja; Anders, Robert A; Burns, Kathleen H; Kazazian, Haig H

    2016-09-01

    Squamous cell carcinoma of the esophagus (SCC) is the most common form of esophageal cancer in the world and is typically diagnosed at an advanced stage when successful treatment is challenging. Understanding the mutational profile of this cancer may identify new treatment strategies. Because somatic retrotransposition has been shown in tumors of the gastrointestinal system, we focused on LINE-1 (L1) mobilization as a source of genetic instability in this cancer. We hypothesized that retrotransposition is ongoing in SCC patients. The expression of L1 encoded proteins is necessary for retrotransposition to occur; therefore, we evaluated the expression of L1 open reading frame 1 protein (ORF1p). Using immunohistochemistry, we detected ORF1p expression in all four SCC cases evaluated. Using L1-seq, we identified and validated 74 somatic insertions in eight tumors of the nine evaluated. Of these, 12 insertions appeared to be somatic, not genetically inherited, and sub-clonal (i.e., present in less than one copy per genome equivalent) in the adjacent normal esophagus (NE), while clonal in the tumor. Our results indicate that L1 retrotransposition is active in SCC of the esophagus and that insertion events are present in histologically NE that expands clonally in the subsequent tumor. PMID:27319353

  9. Developmental potential of human oocytes reconstructed by transferring somatic cell nuclei into polyspermic zygote cytoplasm

    International Nuclear Information System (INIS)

    The generation of patient-specific nuclear transfer embryonic stem cells holds huge promise in modern regenerative medicine and cell-based drug discovery. Since human in vivo matured oocytes are not readily available, human therapeutic cloning is developing slowly. Here, we investigated for the first time whether human polyspermic zygotes could support preimplantation development of cloned embryos. Our results showed that polyspermic zygotes could be used as recipients for human somatic cell nuclear transfer (SCNT). The preimplantation developmental potential of SCNT embryos from polyspermic zygotes was limited to the 8-cell stage. Since ES cell lines can be derived from single blastomeres, these results may have important significance for human ES cells derived by SCNT. In addition, confocal images demonstrated that all of the SCNT embryos that failed to cleave showed abnormal microtubule organization. The results of the present study suggest that polyspermic human zygotes could be used as a potential source of recipient cytoplasm for SCNT.

  10. Developmental potential of human oocytes reconstructed by transferring somatic cell nuclei into polyspermic zygote cytoplasm

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Yong; Chen, Xinjie; Luo, Yumei; Chen, Xiaolin; Li, Shaoying; Huang, Yulin [Institute of Gynecology and Obstetrics, The Third Affiliated Hospital of Guangzhou Medical College, Duobao Road 63, Guangzhou, Guangdong (China); Sun, Xiaofang, E-mail: xiaofangsun@hotmail.com [Institute of Gynecology and Obstetrics, The Third Affiliated Hospital of Guangzhou Medical College, Duobao Road 63, Guangzhou, Guangdong (China)

    2009-04-24

    The generation of patient-specific nuclear transfer embryonic stem cells holds huge promise in modern regenerative medicine and cell-based drug discovery. Since human in vivo matured oocytes are not readily available, human therapeutic cloning is developing slowly. Here, we investigated for the first time whether human polyspermic zygotes could support preimplantation development of cloned embryos. Our results showed that polyspermic zygotes could be used as recipients for human somatic cell nuclear transfer (SCNT). The preimplantation developmental potential of SCNT embryos from polyspermic zygotes was limited to the 8-cell stage. Since ES cell lines can be derived from single blastomeres, these results may have important significance for human ES cells derived by SCNT. In addition, confocal images demonstrated that all of the SCNT embryos that failed to cleave showed abnormal microtubule organization. The results of the present study suggest that polyspermic human zygotes could be used as a potential source of recipient cytoplasm for SCNT.

  11. Somatic cell nuclear transfer: Infinite reproduction of a unique diploid genome

    International Nuclear Information System (INIS)

    In mammals, a diploid genome of an individual following fertilization of an egg and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual ending. Even as cultured cells from the individual, they cannot normally proliferate in perpetuity because of the 'Hayflick limit'. However, Dolly, the sheep cloned from an adult mammary gland cell, changes this scenario. Somatic cell nuclear transfer (SCNT) enables us to produce offspring without germ cells, that is, to 'passage' a unique diploid genome. Animal cloning has also proven to be a powerful research tool for reprogramming in many mammals, notably mouse and cow. The mechanism underlying reprogramming, however, remains largely unknown and, animal cloning has been inefficient as a result. More momentously, in addition to abortion and fetal mortality, some cloned animals display possible premature aging phenotypes including early death and short telomere lengths. Under these inauspicious conditions, is it really possible for SCNT to preserve a diploid genome? Delightfully, in mouse and recently in primate, using SCNT we can produce nuclear transfer ES cells (ntES) more efficiently, which can preserve the eternal lifespan for the 'passage' of a unique diploid genome. Further, new somatic cloning technique using histone-deacetylase inhibitors has been developed which can significantly increase the previous cloning rates two to six times. Here, we introduce SCNT and its value as a preservation tool for a diploid genome while reviewing aging of cloned animals on cellular and individual levels

  12. Are we Genomic Mosaics? Variations of the Genome of Somatic Cells can Contribute to Diversify our Phenotypes

    OpenAIRE

    Astolfi, P.A.; Salamini, F.; Sgaramella, V

    2010-01-01

    Theoretical and experimental evidences support the hypothesis that the genomes and the epigenomes may be different in the somatic cells of complex organisms. In the genome, the differences range from single base substitutions to chromosome number; in the epigenome, they entail multiple postsynthetic modifications of the chromatin. Somatic genome variations (SGV) may accumulate during development in response both to genetic programs, which may differ from tissue to tissue, and to environmental...

  13. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

    Directory of Open Access Journals (Sweden)

    Page Grier P

    2009-04-01

    Full Text Available Abstract Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT. Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively. However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research.

  14. Genomic stability of lyophilized sheep somatic cells before and after nuclear transfer.

    Directory of Open Access Journals (Sweden)

    Domenico Iuso

    Full Text Available The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT. Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.

  15. Polycomb Group Proteins: Multi-Faceted Regulators of Somatic Stem Cells and Cancer

    Science.gov (United States)

    Sauvageau, Martin; Sauvageau, Guy

    2016-01-01

    Polycomb Group (PcG) proteins are transcriptional repressors that epigenetically modify chromatin and participate in the establishment and maintenance of cell fates. These proteins play important roles in both stem cell self-renewal and in cancer development. Our understanding of their mechanism of action has greatly advanced over the past 10 years, but many unanswered questions remain. In this review, we present the currently available experimental data that connect PcG protein function with some of the key processes which govern somatic stem cell activity. We also highlight recent studies suggesting that a delicate balance in PcG gene dosage is crucial for proper stem cell homeostasis and prevention of cancer stem cell development. PMID:20804967

  16. The effects of dry-off therapy on milk somatic cell count in Saanen goats

    OpenAIRE

    BAŞTAN, Ayhan; SALAR, SEÇKİN; ACAR, Duygu BAKİ; DEMİREL, MÜRŞİDE AYŞE; CENGİZ, Mehmet; DARBAZ, İSFENDİYAR; BULUT, GAYE

    2015-01-01

    The aim of this study was to determine the effectiveness of dry-off antibiotic therapy and teat sealant on somatic cell count (SCC) in Saanen goats. The goats were randomly divided into 3 groups. In Groups I (n = 50) and II (n = 50), the goats were treated with intramammary antibiotics and a combination of intramammary antibiotics and internal teat sealant at dry-off, respectively. The animals in Group III (n = 50) were designated as the control group. For the SCC analysis and bacteriological...

  17. Effect of weather conditions on somatic cell score in Sicilian Valle del Belice ewes

    Directory of Open Access Journals (Sweden)

    B. Portolano

    2010-04-01

    Full Text Available Mastitis susceptibility of Valle del Belice ewes from the south of Sicily to temperature, humidity, precipitation, solar radiation, sun hours, air pressure, wind-speed and wind-direction measured by a public weather station was investigated using 65,848 test-day somatic cell score (SCS records of 5,237 ewes. All weather parameters had an effect on SCS in a regression approach. Extreme values of maximum and minimum temperaturehumidity indices resulted in increased SCS. Higher precipitation, solar radiation and sun hours resulted in increased SCS, whereas higher air pressure and wind speed resulted in reduced SCS.

  18. Seasonal and Milking-to-Milking Variations in Cow Milk Fat, Protein and Somatic Cell Counts

    OpenAIRE

    Elena Raluca PAVEL; Constantin GAVAN

    2011-01-01

    The first objective of this study was to examine milking-to-milking variations in milk fat, protein and SCC (somatic cell count). The second objective of this study was to examine variations of milk components (fat, protein and SCC) over a period of six months (April-September 2010) at Agricultural Research Development Station Simnic. A total of 128 milk samples (64 morning milking and 64 evening milking ones) from milk bulk tank commingled from 90�4 Holstein cows, were collected and analyzed...

  19. Characterizing somatic hypermutation and gene conversion in the chicken DT40 cell system.

    Science.gov (United States)

    Kothapalli, Nagarama; Fugmann, Sebastian D

    2011-01-01

    The secondary immunoglobulin gene diversification processes, somatic hypermutation (SHM), immunoglobulin gene conversion (GCV), and class switch recombination, are important for efficient humoral immune responses. They require the action of activation-induced cytidine deaminase, an enzyme that deaminates cytosine in the context of single-stranded DNA. The chicken DT40 B-cell line is an important model system for exploring the mechanisms of SHM and GCV, as both processes occur constitutively without the need for stimulation. In addition, standard gene targeting strategies can be used for defined manipulations of the DT40 genome. Thus, these cells represent an excellent model of choice for genetic studies of SHM and GCV. Problems arising from defects in early B-cell development that are of concern when using genetically engineered mice are avoided in this system. Here, we describe how to perform gene targeting in DT40 cells and how to determine the effects of such modifications on SHM and GCV. PMID:21701980

  20. Germline stem cell arrest inhibits the collapse of somatic proteostasis early in Caenorhabditis elegans adulthood.

    Science.gov (United States)

    Shemesh, Netta; Shai, Nadav; Ben-Zvi, Anat

    2013-10-01

    All cells rely on highly conserved protein folding and clearance pathways to detect and resolve protein damage and to maintain protein homeostasis (proteostasis). Because age is associated with an imbalance in proteostasis, there is a need to understand how protein folding is regulated in a multicellular organism that undergoes aging. We have observed that the ability of Caenorhabditis elegans to maintain proteostasis declines sharply following the onset of oocyte biomass production, suggesting that a restricted protein folding capacity may be linked to the onset of reproduction. To test this hypothesis, we monitored the effects of different sterile mutations on the maintenance of proteostasis in the soma of C. elegans. We found that germline stem cell (GSC) arrest rescued protein quality control, resulting in maintenance of robust proteostasis in different somatic tissues of adult animals. We further demonstrated that GSC-dependent modulation of proteostasis requires several different signaling pathways, including hsf-1 and daf-16/kri-1/tcer-1, daf-12, daf-9, daf-36, nhr-80, and pha-4 that differentially modulate somatic quality control functions, such that each signaling pathway affects different aspects of proteostasis and cannot functionally complement the other pathways. We propose that the effect of GSCs on the collapse of proteostasis at the transition to adulthood is due to a switch mechanism that links GSC status with maintenance of somatic proteostasis via regulation of the expression and function of different quality control machineries and cellular stress responses that progressively lead to a decline in the maintenance of proteostasis in adulthood, thereby linking reproduction to the maintenance of the soma. PMID:23734734

  1. Driving folliculogenesis by the oocyte-somatic cell dialog: Lessons from genetic models.

    Science.gov (United States)

    Monniaux, Danielle

    2016-07-01

    This review focuses on the role of the dialog between the oocyte and its companion somatic cells in driving folliculogenesis from the primordial to the preovulatory follicle stage. Mouse and sheep genetic models have brought complementary evidence of these cell interactions and their consequences for ovarian function. In mouse, the deletion of genes encoding connexins has shown that functional gap junction channels between oocytes and granulosa cells and between granulosa cells themselves maintain the follicle in a functionally integrated state. Targeted deletions in oocytes or granulosa cells have revealed the cell- and stage-specific role of ubiquist factors belonging to the phosphatidylinositol 3 kinase signaling pathway in primordial follicle activation, oocyte growth and follicle survival. Various models of transgenic mice and sheep carrying natural loss-of-function mutations associated with sterility have established that the oocyte-derived factors, bone morphogenetic protein (BMP) 15 and growth differentiation factor 9 orchestrate follicle development, support cumulus metabolism and maturation and participate in oocyte meiosis arrest. Unexpectedly in sheep, mutations resulting in the attenuation of BMP signaling lead to enhanced ovulation rate, likely resulting from a lowered follicular atresia rate and the enhancement of FSH-regulated follicular maturation. Both the activation level of BMP signaling and an adequate equilibrium between BMP15 and growth differentiation factor 9 determine follicle survival, maturation, and development toward ovulation. The physiological approaches which were implemented on genetic animal models during the last 20 years have opened up new perspectives for female fertility by identifying the main signaling pathways of the oocyte-somatic cell dialog. PMID:27155734

  2. Transcription factor Oct1 is a somatic and cancer stem cell determinant.

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    Jessica Maddox

    Full Text Available Defining master transcription factors governing somatic and cancer stem cell identity is an important goal. Here we show that the Oct4 paralog Oct1, a transcription factor implicated in stress responses, metabolic control, and poised transcription states, regulates normal and pathologic stem cell function. Oct1(HI cells in the colon and small intestine co-express known stem cell markers. In primary malignant tissue, high Oct1 protein but not mRNA levels strongly correlate with the frequency of CD24(LOCD44(HI cancer-initiating cells. Reducing Oct1 expression via RNAi reduces the proportion of ALDH(HI and dye efflux(HI cells, and increasing Oct1 increases the proportion of ALDH(HI cells. Normal ALDH(HI cells harbor elevated Oct1 protein but not mRNA levels. Functionally, we show that Oct1 promotes tumor engraftment frequency and promotes hematopoietic stem cell engraftment potential in competitive and serial transplants. In addition to previously described Oct1 transcriptional targets, we identify four Oct1 targets associated with the stem cell phenotype. Cumulatively, the data indicate that Oct1 regulates normal and cancer stem cell function.

  3. Cigarette smoking during early pregnancy reduces the number of embryonic germ and somatic cells

    DEFF Research Database (Denmark)

    Mamsen, Linn; Lutterodt, M C; Andersen, Elisabeth Anne Wreford;

    2010-01-01

    stereological methods were used to estimate gonadal cell numbers in histological sections. Results were also evaluated in the context of previously published data on ovaries from our laboratory. RESULTS: A significant reduction in the number of germ cells by 55% [95% confidence interval (CI) 74-21% reduction, P......BACKGROUND: Cigarette smoking during pregnancy is associated with negative reproductive consequences for male fetuses in adult life such as reduced testicular volume and sperm concentration. The present study evaluates the number of germ and somatic cells present in human embryonic first......-trimester gonads in relation to maternal smoking. METHODS: The study includes 24 human first-trimester testes, aged 37-68 days post-conception, obtained from women undergoing legal termination of pregnancy. A questionnaire was used to obtain information about smoking and drinking habits during pregnancy. Validated...

  4. The influence of somatic cell count on sheep milk composition and cheese-making properties

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    M. Todaro

    2010-01-01

    Full Text Available Somatic cell count (SCC is an important tool for monitoring intramammary infections in dairy cows. However, systematic generalization of this decision rule is not easy in small ruminants. Determination of SCC in sheep milk is important for the processors of milk (indicator of quality, for breeders (mastitis indicator and could be useful for selection as well. SCC value can be affected by some non-infective factors such as breed, stage of lactation, parity, type of lambing, type of milking, etc. (Bergonier et al., 1994, as well the health status of the udder (Fruganti et al., 1985; Ranucci et al., 1988. In addition, EC Directive 92/46, which regulates the production and commercialisation of milk and dairy products, imposes strict limits on SCC from dairy cattle but it does not dispel the uncertainty over recommended SCC levels in small ruminants.With the aim of knowing more about somatic cells count and their effects on milk quality and cheese-making properties an experimental trial was carried out.

  5. Estimation of (covariance components of nematode parasites resistance and somatic cell count in dairy sheep

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    Sara Casu

    2010-01-01

    Full Text Available Nematode parasites and mastitis are the major animal health constraints in sheep. The aim of this study was estimating the genetic (covariances of nematode parasites resistance and somatic cell count in dairy sheep. From 2000 to 2008, Somatic Cell Score (SCS and Faecal Egg Count (FEC records were available on an experimental population consisting of 949 backcross ewes and 806 their daughters. Data were processed independently for each subpopulation in order to adjust for specific environmental effects and to obtain lactation records for both traits to be used in the genetic analysis. Variance components estimation was performed by using the REML method applied to a bi-trait repeatability animal model. Heritabilities of lactation SCS (LSCS and FEC were 0.19 and 0.16. Genetic correlation was 0.21, whereas phenotypic correlation was 0.01. The estimated heritabilities confirm that both traits could be selected by the classical quantitative approach. The genetic correlation estimate between LSCS and FEC suggests that selection for one of the two traits would not have any detrimental effect on the other one.

  6. Genetic parameters for test day somatic cell score in Brazilian Holstein cattle.

    Science.gov (United States)

    Costa, C N; Santos, G G; Cobuci, J A; Thompson, G; Carvalheira, J G V

    2015-01-01

    Selection for lower somatic cell count has been included in the breeding objectives of several countries in order to increase resistance to mastitis. Genetic parameters of somatic cell scores (SCS) were estimated from the first lactation test day records of Brazilian Holstein cows using random-regression models with Legendre polynomials (LP) of the order 3-5. Data consisted of 87,711 TD produced by 10,084 cows, sired by 619 bulls calved from 1993 to 2007. Heritability estimates varied from 0.06 to 0.14 and decreased from the beginning of the lactation up to 60 days in milk (DIM) and increased thereafter to the end of lactation. Genetic correlations between adjacent DIM were very high (>0.83) but decreased to negative values, obtained with LP of order four, between DIM in the extremes of lactation. Despite the favorable trend, genetic changes in SCS were not significant and did not differ among LP. There was little benefit of fitting an LP of an order >3 to model animal genetic and permanent environment effects for SCS. Estimates of variance components found in this study may be used for breeding value estimation for SCS and selection for mastitis resistance in Holstein cattle in Brazil. PMID:26782564

  7. Progress toward generating a ferret model of cystic fibrosis by somatic cell nuclear transfer

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    Engelhardt John F

    2003-11-01

    Full Text Available Abstract Mammalian cloning by nuclear transfer from somatic cells has created new opportunities to generate animal models of genetic diseases in species other than mice. Although genetic mouse models play a critical role in basic and applied research for numerous diseases, often mouse models do not adequately reproduce the human disease phenotype. Cystic fibrosis (CF is one such disease. Targeted ablation of the cystic fibrosis transmembrane conductance regulator (CFTR gene in mice does not adequately replicate spontaneous bacterial infections observed in the human CF lung. Hence, several laboratories are pursuing alternative animal models of CF in larger species such as the pig, sheep, rabbits, and ferrets. Our laboratory has focused on developing the ferret as a CF animal model. Over the past few years, we have investigated several experimental parameters required for gene targeting and nuclear transfer (NT cloning in the ferret using somatic cells. In this review, we will discuss our progress and the hurdles to NT cloning and gene-targeting that accompany efforts to generate animal models of genetic diseases in species such as the ferret.

  8. Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young June; Ahn, Kwang Sung; Kim, Minjeong; Kim, Min Ju; Park, Sang-Min; Ryu, Junghyun; Ahn, Jin Seop; Heo, Soon Young; Kang, Jee Hyun; Choi, You Jung [Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan (Korea, Republic of); Choi, Seong-Jun [Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of); Shim, Hosup, E-mail: shim@dku.edu [Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan (Korea, Republic of); Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of); Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of)

    2014-10-03

    Highlights: • ATM gene-targeted pigs were produced by somatic cell nuclear transfer. • A novel large animal model for ataxia telangiectasia was developed. • The new model may provide an alternative to the mouse model. - Abstract: Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies.

  9. Behavioral observations of adolescent Holstein heifers cloned from adult somatic cells.

    Science.gov (United States)

    Savage, Amy F; Maull, John; Tian, X Cindy; Taneja, Maneesh; Katz, Larry; Darre, Michael; Yang, Xiangzhong

    2003-10-01

    Cloning using somatic cells offers many potential applications in biomedicine and basic research. The objective of this study was to test whether clones from the same genotype can be used as models to study the genetic influences of behavior. Specifically, several aspects of the behavior of four prepubertal heifers cloned from somatic cells of a 13-year-old Holstein cow along with age-matched control heifers were compared to determine whether juvenile clones from an aged adult behave similarly to their age-matched controls, and whether clones with identical genetic makeup exhibit any behavioral trends. Behavioral observations or behavior challenge tests were conducted to compare the following traits: vocalization, play behavior, movement frequencies, grooming, curiosity, and companion preference, as well as dominance and aggressiveness. From play behavior, movements and vocalization, we observed that these four juvenile clones of an aged genetic donor did not show behavioral indications of aging and were similar to their counterparts of comparable chronological age except that they tended to play less than controls. Behavioral trends were also observed in the clones that indicated that they exhibited higher levels of curiosity, more grooming activities and were more aggressive and dominant than controls. Furthermore, these four clones preferred each other or the donor as companions, which may indicate genetic kin recognition. PMID:12935849

  10. Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

    International Nuclear Information System (INIS)

    Highlights: • ATM gene-targeted pigs were produced by somatic cell nuclear transfer. • A novel large animal model for ataxia telangiectasia was developed. • The new model may provide an alternative to the mouse model. - Abstract: Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies

  11. Blood count and number of somatic cells in milk of cows infected with Coxiella burnetii

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    Radinović Miodrag

    2011-01-01

    Full Text Available The objective of the work was to examine the intensity of the local immune response of the mammary gland and the changes in the differential blood count of chronically infected cows. An experiment was performed on a group of cows with Q fever serologically proven using the ELISA test (IDEXX. Based on the ELISA test results, an experimental group of ten infected cows was formed. Blood was sampled from the experimental cows, and cumulative milk samples were taken. The number of erythrocytes was determined spectrophotometrically, and the number of leucocytes using the method according to Bürker - Türk. The blood analysis established an increased number of erythrocytes, while the number of leucocytes was within the limits of physiological values. The milk samples were used for the determination of the number of somatic cells using flow cytometric measurements. The processing of the milk samples established an average number of somatic cells of 853.000 /mL milk.

  12. EFFECT OF THE COW AGE GROUP AND LACTATION STAGE ON THE COUNT OF SOMATIC CELLS IN COW MILK

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    Beata SITKOWSKA

    2008-07-01

    Full Text Available The aim of the paper was to evaluate the effect of the cow age group and lactation stage on the count of somatic cells in cow milk. The analysis was made based on the breeding documentation of 11359 test yields from cows representing twelve herds in the Kujawy and Pomorze Province. All the animals researched calved for the first time in 2001. The numerical data were verified statistically with the analysis of variance following GLM procedure, considering the effect of the herd, father, sampling month, cow groups (primiparas, multiparas, lactation stage at which the sample was taken. The effect of these factors on basic milking yield and the content of somatic cells were mostly highly significant. It was observed that depending on the milk use length in cows during lactation, the content of somatic cells in milk increased, and the yield of milk, fat and protein decreased.

  13. Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 μmol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.

  14. PIWI proteins and PIWI-interacting RNAs function in Hydra somatic stem cells.

    Science.gov (United States)

    Juliano, Celina E; Reich, Adrian; Liu, Na; Götzfried, Jessica; Zhong, Mei; Uman, Selen; Reenan, Robert A; Wessel, Gary M; Steele, Robert E; Lin, Haifan

    2014-01-01

    PIWI proteins and their bound PIWI-interacting RNAs (piRNAs) are found in animal germlines and are essential for fertility, but their functions outside of the gonad are not well understood. The cnidarian Hydra is a simple metazoan with well-characterized stem/progenitor cells that provides a unique model for analysis of PIWI function. Here we report that Hydra has two PIWI proteins, Hydra PIWI (Hywi) and Hydra PIWI-like (Hyli), both of which are expressed in all Hydra stem/progenitor cells, but not in terminally differentiated cells. We identified ∼15 million piRNAs associated with Hywi and/or Hyli and found that they exhibit the ping-pong signature of piRNA biogenesis. Hydra PIWI proteins are strictly cytoplasmic and thus likely act as posttranscriptional regulators. To explore this function, we generated a Hydra transcriptome for piRNA mapping. piRNAs map to transposons with a 25- to 35-fold enrichment compared with the abundance of transposon transcripts. By sequencing the small RNAs specific to the interstitial, ectodermal, and endodermal lineages, we found that the targeting of transposons appears to be largely restricted to the interstitial lineage. We also identified putative nontransposon targets of the pathway unique to each lineage. Finally we demonstrate that hywi function is essential in the somatic epithelial lineages. This comprehensive analysis of the PIWI-piRNA pathway in the somatic stem/progenitor cells of a nonbilaterian animal suggests that this pathway originated with broader stem cell functionality. PMID:24367095

  15. Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer

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    Müller Mathias

    2007-12-01

    Full Text Available Abstract Background The mitochondrial DNA (mtDNA of the cloned sheep "Dolly" and nine other ovine clones produced by somatic cell nuclear transfer (SCNT was reported to consist only of recipient oocyte mtDNA without any detectable mtDNA contribution from the nucleus donor cell. In cattle, mouse and pig several or most of the clones showed transmission of nuclear donor mtDNA resulting in mitochondrial heteroplasmy. To clarify the discrepant transmission pattern of donor mtDNA in sheep clones we analysed the mtDNA composition of seven fetuses and five lambs cloned from fetal fibroblasts. Results The three fetal fibroblast donor cells used for SCNT harboured low mtDNA copy numbers per cell (A: 753 ± 54, B: 292 ± 33 and C: 561 ± 88. The ratio of donor to recipient oocyte mtDNAs was determined using a quantitative amplification refractory mutation system (ARMS PCR (i.e. ARMS-qPCR. For quantification of SNP variants with frequencies below 0.1% we developed a restriction endonuclease-mediated selective quantitative PCR (REMS-qPCR. We report the first cases (n = 4 fetuses, n = 3 lambs of recipient oocyte/nuclear donor mtDNA heteroplasmy in SCNT-derived ovine clones demonstrating that there is no species-effect hindering ovine nucleus-donor mtDNA from being transmitted to the somatic clonal offspring. Most of the heteroplasmic clones exhibited low-level heteroplasmy (0.1% to 0.9%, n = 6 indicating neutral transmission of parental mtDNAs. High-level heteroplasmy (6.8% to 46.5% was observed in one case. This clone possessed a divergent recipient oocyte-derived mtDNA genotype with three rare amino acid changes compared to the donor including one substitution at an evolutionary conserved site. Conclusion Our study using state-of-the-art techniques for mtDNA quantification, like ARMS-qPCR and the novel REMS-qPCR, documents for the first time the transmission of donor mtDNA into somatic sheep clones. MtDNA heteroplasmy was detected in seven of 12 clones

  16. Stochastic modeling indicates that aging and somatic evolution in the hematopoetic system are driven by non-cell-autonomous processes.

    Science.gov (United States)

    Rozhok, Andrii I; Salstrom, Jennifer L; DeGregori, James

    2014-12-01

    Age-dependent tissue decline and increased cancer incidence are widely accepted to be rate-limited by the accumulation of somatic mutations over time. Current models of carcinogenesis are dominated by the assumption that oncogenic mutations have defined advantageous fitness effects on recipient stem and progenitor cells, promoting and rate-limiting somatic evolution. However, this assumption is markedly discrepant with evolutionary theory, whereby fitness is a dynamic property of a phenotype imposed upon and widely modulated by environment. We computationally modeled dynamic microenvironment-dependent fitness alterations in hematopoietic stem cells (HSC) within the Sprengel-Liebig system known to govern evolution at the population level. Our model for the first time integrates real data on age-dependent dynamics of HSC division rates, pool size, and accumulation of genetic changes and demonstrates that somatic evolution is not rate-limited by the occurrence of mutations, but instead results from aged microenvironment-driven alterations in the selective/fitness value of previously accumulated genetic changes. Our results are also consistent with evolutionary models of aging and thus oppose both somatic mutation-centric paradigms of carcinogenesis and tissue functional decline. In total, we demonstrate that aging directly promotes HSC fitness decline and somatic evolution via non-cell-autonomous mechanisms. PMID:25564763

  17. Influence of somatic cell count on mineral content and salt equilibria of milk

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    Primo Mariani

    2010-01-01

    Full Text Available Aim of this research was to study the effect of somatic cell count on mineral content and salt equilibria at the level of quarter milk samples. Ten Italian Friesian cows, in which two homologous quarters (front quarters in 1 cow, rear quarters in 6 cows and both rear and front quarters in 3 cows were characterised by a milk SCC400,000 cells/mL (HC-milk, respectively, were selected. Cows were milked at quarter level during the morning milking and a single sample was collected from each selected quarter, thus, 26 quarter milk samples were collected. Compared to LC-milk, HC-milk was characterised by a lower content of phosphorus and potassium and by a higher content of both sodium and chloride. The equilibrium of calcium, phosphorus and magnesium between the colloidal and soluble phase of milk and the mineralisation degree of the casein micelles, were not different between HC and LC milk.

  18. Improving the development of early bovine somatic-cell nuclear transfer embryos by treating adult donor cells with vitamin C.

    Science.gov (United States)

    Chen, Huanhuan; Zhang, Lei; Guo, Zekun; Wang, Yongsheng; He, Rongjun; Qin, Yumin; Quan, Fusheng; Zhang, Yong

    2015-11-01

    Vitamin C (Vc) has been widely studied in cell and embryo culture, and has recently been demonstrated to promote cellular reprogramming. The objective of this study was to identify a suitable Vc concentration that, when used to treat adult bovine fibroblasts serving as donor cells for nuclear transfer, improved donor-cell physiology and the developmental potential of the cloned embryos that the donor nuclei were used to create. A Vc concentration of 0.15 mM promoted cell proliferation and increased donor-cell 5-hydroxy methyl cytosine levels 2.73-fold (P DNA methylation levels in donor cells, and improves the developmental competence of bovine somatic-cell nuclear transfer embryos. PMID:26212732

  19. Microprojectile delivery to DNA to leaf cells in Dactylis glomerata and its expression in somatic embryos

    International Nuclear Information System (INIS)

    Development of techniques for gene transfer by bombardment with DNA coated microprojectiles has greatly expanded the potential for obtaining transgenic plants in cereals and grasses and has been successfully used in several species. The leaf culture system in Dactylis glomerata L. (orchardgrass), in which embryos initiate and develop from single mesophyll cells, is especially attractive for gene transfer experiments. Tillers were selected from greenhouse grown plants of Embryogen-P orchardgrass, and leaf segments were plated on SH medium with 30 μM dicamba (SH30). Tungsten particles were coated with DNA plasmids containing the bar gene that encodes for phosphinotricin resistance (the active ingredient of the herbicides bialaphos and Basta), and the uidA gene that encodes the enzyme β-glucuronidase (GUS). Both genes were under control of either the CaMV35S or the maize ubiquitin Ubi1 promoter. Microprojectile bombardment was conducted with a particle inflow gun. Six of these plants showed no reaction when the leaves were treated with 0.01% Basta, indicating resistance to the herbicide. The leaf tissue from these plants produced somatic embryos when cultured on medium containing 1.5% bialaphos. Somatic embryos from the leaf tissue of the regenerated plans also stained blue when treated with X-gluc. Putative transformations for both genes were confirmed by polymerase chain reaction techniques. 5 refs, 2 figs

  20. Condensin II subunit dCAP-D3 restricts retrotransposon mobilization in Drosophila somatic cells.

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    Andrew T Schuster

    2013-10-01

    Full Text Available Retrotransposon sequences are positioned throughout the genome of almost every eukaryote that has been sequenced. As mobilization of these elements can have detrimental effects on the transcriptional regulation and stability of an organism's genome, most organisms have evolved mechanisms to repress their movement. Here, we identify a novel role for the Drosophila melanogaster Condensin II subunit, dCAP-D3 in preventing the mobilization of retrotransposons located in somatic cell euchromatin. dCAP-D3 regulates transcription of euchromatic gene clusters which contain or are proximal to retrotransposon sequence. ChIP experiments demonstrate that dCAP-D3 binds to these loci and is important for maintaining a repressed chromatin structure within the boundaries of the retrotransposon and for repressing retrotransposon transcription. We show that dCAP-D3 prevents accumulation of double stranded DNA breaks within retrotransposon sequence, and decreased dCAP-D3 levels leads to a precise loss of retrotransposon sequence at some dCAP-D3 regulated gene clusters and a gain of sequence elsewhere in the genome. Homologous chromosomes exhibit high levels of pairing in Drosophila somatic cells, and our FISH analyses demonstrate that retrotransposon-containing euchromatic loci are regions which are actually less paired than euchromatic regions devoid of retrotransposon sequences. Decreased dCAP-D3 expression increases pairing of homologous retrotransposon-containing loci in tissue culture cells. We propose that the combined effects of dCAP-D3 deficiency on double strand break levels, chromatin structure, transcription and pairing at retrotransposon-containing loci may lead to 1 higher levels of homologous recombination between repeats flanking retrotransposons in dCAP-D3 deficient cells and 2 increased retrotransposition. These findings identify a novel role for the anti-pairing activities of dCAP-D3/Condensin II and uncover a new way in which dCAP-D3/Condensin

  1. In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment

    International Nuclear Information System (INIS)

    Chimeric chloramphenicol acetyltransferase and β-galactosidase marker genes were coated onto fine gold particles and used to bombard a variety of mammalian tissues and cells. Transient expression of the genes was obtained in liver, skin, and muscle tissues of rat and mouse bombarded in vivo. Similar results were obtained with freshly isolated ductal segments of rat and human mammary glands and primary cultures derived from these explants. Gene transfer and transient expression were also observed in eight human cell culture lines, including cells of epithelial, endothelial, fibroblast, and lymphocyte origin. Using CHO and MCF-7 cell cultures as models, we obtained stable gene transfer at frequencies of 1.7 x 10-3 and 6 x 10-4, respectively. The particle bombardment technology thus provides a useful means to transfer foreign genes into a variety of mammalian somatic cell systems. The method is applicable to tissues in vivo as well as to isolated cells in culture and has proven effective with all cell or tissue types tested thus far. This technology may therefore prove to be applicable in various aspects of gene therapy

  2. Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

    Energy Technology Data Exchange (ETDEWEB)

    Ostrup, Olga, E-mail: osvarcova@gmail.com [Institute of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C (Denmark); Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo (Norway); Norwegian Center for Stem Cell Research, Oslo (Norway); Hyttel, Poul; Klaerke, Dan A. [Institute of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C (Denmark); Collas, Philippe, E-mail: philc@medisin.uio.no [Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo (Norway); Norwegian Center for Stem Cell Research, Oslo (Norway)

    2011-09-02

    Highlights: {yields} Xenopus egg extract remodels nuclei and alter cell growth characteristics. {yields} Ribosomal genes are reprogrammed within 6 h after extract exposure. {yields} rDNA reprogramming involves promoter targeting of SNF2H remodeling complex. {yields} Xenopus egg extract does not initiate stress-related response in somatic cells. {yields} Aza-cytidine elicits a stress-induced response in reprogrammed cells. -- Abstract: Extracts from Xenopus eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression. This occurs within hours of extract treatment and is distinct from a stress response. Egg extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus egg extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.

  3. Relationship between mastitis causative pathogens and somatic cell counts in milk of dairy cows

    Directory of Open Access Journals (Sweden)

    Sharaf Eldeen Idriss

    2013-12-01

    Full Text Available Milk somatic cell count is a key component of national and international regulation for milk quality and an indicator of udder health and of the prevalence of clinical and subclinical mastitis in dairy herds. The objective of this study was to evaluate the presence of mastitis pathogens in milk samples differed by somatic cell count (SCC in microbiologically positive samples. Also frequency of distribution of samples differed by SCC were studied in non infected samples as well. The milk samples were collected from individual quarters from the dairy farms located in Nitra region with problematic udder health of herd for SCC and bacteriological analysis. Totally, 390 milk samples were examined, and 288 (73.85% positive milk samples were detected. Four SCC groups of samples (400×103 /ml were used to identify presence of microorganisms in positive samples. The most frequently isolated pathogens in samples with high SCC >400×103 /ml according to year were Coagulase-negative Staphylococci (29.11 % in 2012, followed by Staphylococcus aureus (28.0% in 2010, yeasts (24.05% in 2012, Escherichia coli (22.78% in 2012, Bacillus sp. (20% in 2010 and Pseudomonas aerugenosa (11.88% in 2011. Coagulase-negative Staphylococci (66.67% were the predominantly identified in the samples with low SCC <100×103 cells/ml, followed by Bacillus spp (50%, Entrococcus spp. (33.33% and Staphylococcus aureus (16.67% and E. coli (16.67%. The results of this study indicated that the SCC of individual milk samples corresponded with the health status of the udder of dairy cows represented by presence of mastitis microorganisms in milk. However, the contamination of milk samples could be also connected with low SCC. On the ohter side the samples with high SCC were found out without presence of microorganism. The further study is needed to identify the reason of high SCC in milk from negative samples.

  4. BIX-01294 increases pig cloning efficiency by improving epigenetic reprogramming of somatic cell nuclei.

    Science.gov (United States)

    Huang, Jiaojiao; Zhang, Hongyong; Yao, Jing; Qin, Guosong; Wang, Feng; Wang, Xianlong; Luo, Ailing; Zheng, Qiantao; Cao, Chunwei; Zhao, Jianguo

    2016-01-01

    Accumulating evidence suggests that faulty epigenetic reprogramming leads to the abnormal development of cloned embryos and results in the low success rates observed in all mammals produced through somatic cell nuclear transfer (SCNT). The aberrant methylation status of H3K9me and H3K9me2 has been reported in cloned mouse embryos. To explore the role of H3K9me2 and H3K9me in the porcine somatic cell nuclear reprogramming, BIX-01294, known as a specific inhibitor of G9A (histone-lysine methyltransferase of H3K9), was used to treat the nuclear-transferred (NT) oocytes for 14-16 h after activation. The results showed that the developmental competence of porcine SCNT embryos was significantly enhanced both in vitro (blastocyst rate 16.4% vs 23.2%, P<0.05) and in vivo (cloning rate 1.59% vs 2.96%) after 50 nm BIX-01294 treatment. BIX-01294 treatment significantly decreased the levels of H3K9me2 and H3K9me at the 2- and 4-cell stages, which are associated with embryo genetic activation, and increased the transcriptional expression of the pluripotency genes SOX2, NANOG and OCT4 in cloned blastocysts. Furthermore, the histone acetylation levels of H3K9, H4K8 and H4K12 in cloned embryos were decreased after BIX-01294 treatment. However, co-treatment of activated NT oocytes with BIX-01294 and Scriptaid rescued donor nuclear chromatin from decreased histone acetylation of H4K8 that resulted from exposure to BIX-01294 only and consequently improved the preimplantation development of SCNT embryos (blastocyst formation rates of 23.7% vs 21.5%). These results indicated that treatment with BIX-01294 enhanced the developmental competence of porcine SCNT embryos through improvements in epigenetic reprogramming and gene expression. PMID:26604326

  5. Direct and indirect measurement of somatic cell count as indicator of intramammary infection in dairy goats

    Directory of Open Access Journals (Sweden)

    Olofsson Ida

    2011-03-01

    Full Text Available Abstract Background Mastitis is the most important and costly disease in dairy goat production. Subclinical mastitis is common in goats and is mainly caused by contagious bacteria. Several methods to diagnose subclinical mastitis are available. In this study indirect measurement of somatic cell count (SCC by California Mastitis Test (CMT and direct measurement of SCC using a portable deLaval cell counter (DCC are evaluated. Swedish goat farmers would primarily benefit from diagnostic methods that can be used at the farm. The purpose of the study was to evaluate SCC measured by CMT and DCC as possible markers for intramammary infection (IMI in goats without clinical symptoms of mastitis. Moreover to see how well indirect measurement of SCC (CMT corresponded to direct measurement of SCC (DCC. Method Udder half milk samples were collected once from dairy goats (n = 111, in five different farms in Northern and Central Sweden. Only clinically healthy animals were included in the study. All goats were in mid to late lactation at sampling. Milk samples were analyzed for SCC by CMT and DCC at the farm, and for bacterial growth at the laboratory. Results Intramammary infection, defined as growth of udder pathogens, was found in 39 (18% of the milk samples. No growth was found in 180 (81% samples while 3 (1% samples were contaminated. The most frequently isolated bacterial species was coagulase negative staphylococci (CNS (72% of all isolates, followed by Staphylococcus aureus (23% of all isolates. Somatic cell count measured by DCC was strongly (p = 0.000 associated with bacterial growth. There was also a very strong association between CMT and bacterial growth. CMT 1 was associated with freedom of IMI while CMT ≥2 was associated with IMI. Indirect measurement of SCC by CMT was well correlated with SCC measured by DCC. Conclusions According to the results, SCC measured with CMT or DCC can predict udder infection in goats, and CMT can be used as a

  6. Stem Cell Interaction with Somatic Niche May Hold the Key to Fertility Restoration in Cancer Patients

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    Deepa Bhartiya

    2012-01-01

    Full Text Available The spontaneous return of fertility after bone marrow transplantation or heterotopic grafting of cryopreserved ovarian cortical tissue has surprised many, and a possible link with stem cells has been proposed. We have reviewed the available literature on ovarian stem cells in adult mammalian ovaries and presented a model that proposes that the ovary harbors two distinct populations of stem cells, namely, pluripotent, quiescent, very small embryonic-like stem cells (VSELs, and slightly larger “progenitor” ovarian germ stem cells (OGSCs. Besides compromising the somatic niche, oncotherapy destroys OGSCs since, like tumor cells, they are actively dividing; however VSELs persist since they are relatively quiescent. BMT or transplanted ovarian cortical tissue may help rejuvenate the ovarian niche, which possibly supports differentiation of persisting VSELs resulting in neo-oogenesis and follicular development responsible for successful pregnancies. Postnatal oogenesis in mammalian ovary from VSELs may be exploited for fertility restoration in cancer survivors including those who were earlier deprived of gametes and/or gonadal tissue cryopreservation options.

  7. Relationship of Somatic Cell Count with Milk Yield and Composition in Chinese Holstein Population

    Institute of Scientific and Technical Information of China (English)

    GUO Jia-zhong; LIU Xiao-lin; XU A-juan; XIA Zhi

    2010-01-01

    The objective of this study was to analyze the relationship of somatic cell count(SCC)with milk yield,fat and protein percentage,fat and protein yield using analysis of variance and correlation analysis in Chinese Holstein population.The10 524 test-day records of 568 Chinese Holstein Cattle were obtained from 2 commercial herds in Xi'an region of China during February 2002 to March 2009.Milk yield,fat percentage,fat and protein yield initially increased and then dropped down with parity,whereas protein percentage decreased and SCC increased.Analysis of variance showed highly significant effects of different subclasses SCC on milk yield and composition(P0.05).The results of the present study first time provide the relevant base-line data for assessing milk production at Xi'an region of China.

  8. Communication between oocytes and somatic cells regulates volatile pheromone production in Caenorhabditis elegans.

    Science.gov (United States)

    Leighton, Daniel H W; Choe, Andrea; Wu, Shannon Y; Sternberg, Paul W

    2014-12-16

    Males of the androdioecious species Caenorhabditis elegans are more likely to attempt to mate with and successfully inseminate C. elegans hermaphrodites that do not concurrently harbor sperm. Although a small number of genes have been implicated in this effect, the mechanism by which it arises remains unknown. In the context of the battle of the sexes, it is also unknown whether this effect is to the benefit of the male, the hermaphrodite, or both. We report that successful contact between mature sperm and oocyte in the C. elegans gonad at the start of fertilization causes the oocyte to release a signal that is transmitted to somatic cells in its mother, with the ultimate effect of reducing her attractiveness to males. Changes in hermaphrodite attractiveness are tied to the production of a volatile pheromone, the first such pheromone described in C. elegans. PMID:25453110

  9. Herd level approach to high bulk milk somatic cell count problems in dairy cattle.

    Science.gov (United States)

    Barkema, Herman W; De Vliegher, Sarne; Piepers, Sofie; Zadoks, Ruth N

    2013-06-01

    Since the introduction of the standard mastitis prevention program in the late 1960s, enormous progress has been made in decreasing the average bulk milk somatic cell count (BMSCC). In many countries, reduction of BMSCC has been encouraged through premium payments or penalty systems. However, the success of the program depends heavily on consistent implementation of management practices. The approach to problem solving in a herd with high BMSCC must include the following elements: (1) problem definition using primary udder health parameters; (2) detection of cows causing the problem; (3) definition of short- and long-term goals; (4) formulation and implementation of a herd management plan; and (5) evaluation of the results. Findings and plans are recorded for use at follow-up visits. Every high BMSCC problem can be solved if farmers are sufficiently motivated, if farm advisors are sufficiently knowledgeable, and if farmer and advisors work together according to a jointly determined plan. PMID:23706026

  10. Genetic Analysis of Somatic Cell Score in Danish Holsteins Using a Liability-Normal Mixture Model

    DEFF Research Database (Denmark)

    Madsen, P; Shariati, M M; Ødegård, J

    2008-01-01

    Mixture models are appealing for identifying hidden structures affecting somatic cell score (SCS) data, such as unrecorded cases of subclinical mastitis. Thus, liability-normal mixture (LNM) models were used for genetic analysis of SCS data, with the aim of predicting breeding values for such cases...... of mastitis. Here, putative mastitis statuses and breeding values for liability to putative mastitis were inferred solely from SCS observations. In total, there were 395,906 test-day records for SCS from 50,607 Danish Holstein cows. Four different statistical models were fitted: A) a classical...... from IMI- udders relative to SCS from IMI+ udders. Further, the genetic correlation between SCS of IMI- and SCS of IMI+ was 0.61, and heritability for liability to putative mastitis was 0.07. Models B2 and C allocated approximately 30% of SCS records to IMI+, but for model B1 this fraction was only 10...

  11. Repression of germline RNAi pathways in somatic cells by retinoblastoma pathway chromatin complexes.

    Directory of Open Access Journals (Sweden)

    Xiaoyun Wu

    Full Text Available The retinoblastoma (Rb tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene.

  12. Repression of germline RNAi pathways in somatic cells by retinoblastoma pathway chromatin complexes.

    Science.gov (United States)

    Wu, Xiaoyun; Shi, Zhen; Cui, Mingxue; Han, Min; Ruvkun, Gary

    2012-01-01

    The retinoblastoma (Rb) tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene. PMID:22412383

  13. Reprogramming resistant genes: in-depth comparison of gene expressions among iPS, ES and somatic cells.

    Directory of Open Access Journals (Sweden)

    Natalia ePolouliakh

    2013-01-01

    Full Text Available Transcription factor based reprogramming reverts adult cells to an embryonic state, yielding potential for generating different tissue types. However, recent reports indicated the substantial differences in pattern of gene expression between induced pluripotent stem (iPS cells and embryonic stem (ES cells. In this study we compare gene expression signatures of different iPS and ES cell lines and relate expression profiles of differently expressed genes to their expression status in somatic cells. As a result, we discovered that genes resistant to reprogramming comprise two major clusters, which are reprogramming dependent ‘Induced Genes’ and somatic origin ‘Inherited Genes’, both exhibiting preferences in methylation marks. Closer look into the Induced Genes by means of the transcription regulation analysis predicted several groups of genes with various roles in reprogramming and transgene DNA binding model. We believe that our results are a helpful source for biologists for further improvement of iPS cell technology.

  14. Somatic cell count and biochemical components of milk related to udder health in buffaloes

    Directory of Open Access Journals (Sweden)

    S.T. Singh

    2010-02-01

    Full Text Available The 399 clinically healthy quarters from 101 Murrah buffaloes were analyzed for somatic cell count (SCC; DCC and microscope methods and biochemical composition of milk in relation to udder health. The udder health revealed specific subclinical mastitis (SSM in 7% and non-specific mastitis (NSM in 49% of quarters. Latent infections comprised 1%. Staphylococci (43%, streptococci (39% and corynebacteria (18% constituted chief etiological agents in SSM. Electrical conductivity increased significantly both in SSM and NSM compared to healthy quarters. Significant effects for SNF and density were seen in SSM only. DCC and microscope depicted similar cell counts with a correlation coefficient of 0.89. The correlations of DCC with CMT and EC were 0.85 and 0.51, respectively. Quarters with negative CMT reactions had DCC values of < 3 × 105 cells/ml. The DCC means for negative, trace, and +1 to 2 CMT scores were 122, 238, and 593 (× 103 cells/ml, respectively. Lactose with discrimination ability of 83.76% was found better indicator of udder inflammation in buffaloes. Buffaloes unlike cows have low numbers of quarter infections, respond similarly as cows to udder inflammation but at different levels, and DCC may be effectively employed for expressing milk cell count in this species.

  15. Bcl6 Is Required for Somatic Hypermutation and Gene Conversion in Chicken DT40 Cells

    Science.gov (United States)

    Williams, Alan M.; Maman, Yaakov; Alinikula, Jukka; Schatz, David G.

    2016-01-01

    The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID. PMID:26900682

  16. Bcl6 Is Required for Somatic Hypermutation and Gene Conversion in Chicken DT40 Cells.

    Science.gov (United States)

    Williams, Alan M; Maman, Yaakov; Alinikula, Jukka; Schatz, David G

    2016-01-01

    The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID. PMID:26900682

  17. GeneticParameters for Milk Somatic Cell Score and Relationships with Production Traits in Primparous Dairy Sheep

    NARCIS (Netherlands)

    Riggio, V.; Finocchiaro, R.; Kaam, van J.B.C.H.M.; Portolano, B.; Bovenhuis, H.

    2007-01-01

    A total of 13,066 first-lactation test-day records of 2,277 Valle del Belice ewes from 17 flocks were used to estimate genetic parameters for somatic cell scores (SCS) and milk production traits, using a repeatability test-day animal model. Heritability estimates were low and ranged from 0.09 to 0.1

  18. Effect of somatic cell count level on functional longevity in Valle del Belice dairy sheep assessed using survival analysis

    NARCIS (Netherlands)

    Riggio, V.; Maizon, D.O.; Portolano, B.; Bovenhuis, H.; Arendonk, van J.A.M.

    2009-01-01

    The objectives of this study were to evaluate the effect of somatic cell count (SCC) on functional longevity and to estimate the heritability of functional longevity using survival analysis in Valle del Belice dairy sheep. A total of 4,880 lactations of 2,190 ewes from 11 flocks were used. In this s

  19. Repeatability of differential goat bulk milk culture and associations with somatic cell count, total bacterial count, and standard plate count

    NARCIS (Netherlands)

    Koop, G.; Dik, N.; Nielen, M.; Lipman, L.J.A.

    2010-01-01

    The aims of this study were to assess how different bacterial groups in bulk milk are related to bulk milk somatic cell count (SCC), bulk milk total bacterial count (TBC), and bulk milk standard plate count (SPC) and to measure the repeatability of bulk milk culturing. On 53 Dutch dairy goat farms,

  20. Pathogen-specific effects of quantitative trait loci affecting clinical mastitis and somatic cell count in danish holstein cattle

    DEFF Research Database (Denmark)

    Sørensen, Lars Peter; Guldbrandtsen, Bernt; Thomasen, J.R.;

    2008-01-01

    The aim of this study was to investigate whether quantitative trait loci (QTL) affecting the risk of clinical mastitis (CM) and QTL affecting somatic cell score (SCS) exhibit pathogen-specific effects on the incidence of mastitis. Bacteriological data on mastitis pathogens were used to investigate...

  1. Influence of udder infection status on milk enzyme activities and somatic cell count throughout early lactation in goats

    DEFF Research Database (Denmark)

    Stuhr, T; Aulrich, K; Barth, K;

    2013-01-01

    At present the analysis of somatic cell count (SCC) used for the detection of intramammary infections (IMI) in bovine milk is also recommended for goat milk, but due to the various factors influencing SCC it allows only limited conclusions on the udder health of goats. The research on enzyme...

  2. Receiver-operating characteristic curves for somatic cell scores and California mastitis test in Valle del Be lice dairy sheep

    NARCIS (Netherlands)

    Riggio, V.; Pesce, L.L.; Morreale, S.; Portolano, B.

    2013-01-01

    Using receiver-operating characteristic (ROC) curve methodology this study was designed to assess the diagnostic effectiveness of somatic cell count (SCC) and the California mastitis test (CMT) in Valle del Belice sheep, and to propose and evaluate threshold values for those tests that would optimal

  3. Expression of members of immunoglobulin gene family in somatic cell hybrids between human B and T cells

    Energy Technology Data Exchange (ETDEWEB)

    Kozbor, D.; Burioni, R.; Ar-Rushdi, A.; Zmijewski, C.; Croce, C.M.

    1987-07-01

    Somatic cell hybrids were obtained between human T and B cells and tested for the expression of differentiated traits of both cell lineages. The T-cell parent SUP-T1 is CD3/sup -/, CD4/sup +/, CD1/sup +/, CD8/sup +/, is weakly positive for HLA class I determinants, and has an inversion of chromosome 14 due to a site-specific recombination event between an immunoglobulin heavy-chain variable gene and the joining segment of the T-cell receptor ..cap alpha.. chain. The B-cell parent, the 6-thioguanine- and ouabain-resistant mutant GM1500, is a lymphoblastoid cell line that secretes IgG2, K chains, and expresses B1, B532, and HLA class I and II antigens. All hybrids expressed characteristics of B cells (Ig/sup +/, B1/sup +/, B532/sup +/, EBNA/sup +/, HLA antigens), whereas only CD4 among the T-cell markers was expressed. The level of T-cell receptor ..beta..-chain transcript was greatly reduced and no RNA of the chimeric T-cell receptor ..cap alpha..-chain joining segment-immunoglobulin heavy-chain variable region was detected. Southern blot analysis indicated that absence of T-cell differentiation markers in the hybrids was not due to chromosomal loss. Rather, some B-cell-specific factor present in the hybrids may account for the suppression.

  4. Expression of members of immunoglobulin gene family in somatic cell hybrids between human B and T cells

    International Nuclear Information System (INIS)

    Somatic cell hybrids were obtained between human T and B cells and tested for the expression of differentiated traits of both cell lineages. The T-cell parent SUP-T1 is CD3-, CD4+, CD1+, CD8+, is weakly positive for HLA class I determinants, and has an inversion of chromosome 14 due to a site-specific recombination event between an immunoglobulin heavy-chain variable gene and the joining segment of the T-cell receptor α chain. The B-cell parent, the 6-thioguanine- and ouabain-resistant mutant GM1500, is a lymphoblastoid cell line that secretes IgG2, K chains, and expresses B1, B532, and HLA class I and II antigens. All hybrids expressed characteristics of B cells (Ig+, B1+, B532+, EBNA+, HLA antigens), whereas only CD4 among the T-cell markers was expressed. The level of T-cell receptor β-chain transcript was greatly reduced and no RNA of the chimeric T-cell receptor α-chain joining segment-immunoglobulin heavy-chain variable region was detected. Southern blot analysis indicated that absence of T-cell differentiation markers in the hybrids was not due to chromosomal loss. Rather, some B-cell-specific factor present in the hybrids may account for the suppression

  5. Assessment of imidacloprid-induced mutagenic effects in somatic cells of Swiss albino male mice.

    Science.gov (United States)

    Bagri, Preeti; Kumar, Vinod; Sikka, Anil K

    2016-10-01

    Pesticides are being used for plant protection to increase food protection and to reduce insect-borne diseases worldwide. Exposure to the pesticides may cause genotoxic effects on both the target and nontarget organisms, including man. Therefore, the mutagenicity evaluation of such pesticides has become a priority area of research. Imidacloprid (IMI), a neonicotinoid insecticide, is widely used in agriculture either alone or in combination with other insecticides. A combined approach employing micronucleus test (MNT) and chromosomal aberrations assay (CA) was utilized to assess the mutagenicity of imidacloprid in bone marrow of Swiss albino male mice. IMI suspension was prepared in 3% gum acacia and administered at doses of 5.5, 11 and 22 mg/kg body weight for 7, 14 and 28 days to mice. IMI treatment resulted in a dose and time-dependant increase in the frequencies of micronuclei per cell and chromosomal aberrations in bone marrow cells. A statistically significant increase in chromosomal aberrations and micronuclei/cell was found only after daily treatment of IMI at highest selected dose (22 mg/kg body weight) for longest selected time period (28 days) compared to the control group. Thus, daily exposure of imidacloprid at a dose level of 22 mg/kg body weight for 28 days caused mutagenic effects on the somatic cells of Swiss albino male mice. PMID:26823062

  6. Plasma α-tocopherol content and its relationship with milk somatic cells count in Italian commercial herds.

    Directory of Open Access Journals (Sweden)

    Adriano Pilotto

    2015-07-01

    Full Text Available This work was aimed to investigate relationship between plasma vitamin E concentration and milk somatic cell count in healthy cows in commercial herds. 49 multiparous cows from two commercial dairy herds were monitored from the day of dry off until 90 DIM. BCS was assessed and blood samples were collected at dry off, day 0, 30, 60 and 90 postpartum. Plasma was analyzed for α-tocopherol content. Quantification of NEFA, BOHB, Zn and Se was performed in serum samples. Milk production and composition was obtained from routinely test-day of Italian milk producers association. Somatic Cell Score (SCS was calculated and included in the dataset. Analysis of data was performed using MIXED repeated and CORR procedures of SAS.We did not observe a correlation between plasmatic vitamin E and somatic cell score, and this can be explained by the low level of somatic cell score (averages 1.64 and 1.26. The lowest value of vitamin E was observed at parturition (1.64 µg/ml and 1.95 µg/ml. A significant (P<0.01 negative (-20% correlation was observed between NEFA serum content and α-tocopherol plasma concentration. Serum selenium content was positively correlated (+42%, P<0.0001 to zinc concentration. Grouping cows on the basis of their plasma α-tocopherol content higher or lower than 3 μg/mL at dry off, SCS at 30 and 60 DIM tended to be higher in lactating animals with lower content of α-tocopherol (1.12 vs. 1.72, P=0.18 at 30d; 0.92 vs. 1.72, P=0.07 at 60d. However, plasma α-tocopherol content at dry off could be usefully correlated with somatic cell count in fresh cows.

  7. Improved development of somatic cell cloned mouse embryos by vitamin C and latrunculin A.

    Directory of Open Access Journals (Sweden)

    Anna Mallol

    Full Text Available Impaired development of embryos produced by somatic cell nuclear transfer (SCNT is mostly associated with faulty reprogramming of the somatic nucleus to a totipotent state and can be improved by treatment with epigenetic modifiers. Here we report that addition of 100 μM vitamin C (VitC to embryo culture medium for at least 16 h post-activation significantly increases mouse blastocyst formation and, when combined with the use of latrunculin A (LatA during micromanipulation and activation procedures, also development to term. In spite of this, no significant effects on pluripotency (OCT4 and NANOG or nuclear reprogramming markers (H3K14 acetylation, H3K9 methylation and DNA methylation and hydroxymethylation could be detected. The use of LatA alone significantly improved in vitro development, but not full-term development. On the other hand, the simultaneous treatment of cloned embryos with VitC and the histone deacetylase inhibitor psammaplin A (PsA, in combination with the use of LatA, resulted in cloning efficiencies equivalent to those of VitC or PsA treatments alone, and the effects on pluripotency and nuclear reprogramming markers were less evident than when only the PsA treatment was applied. These results suggest that although both epigenetic modifiers improve cloning efficiencies, possibly through different mechanisms, they do not show an additive effect when combined. Improvement of SCNT efficiency is essential for its applications in reproductive and therapeutic cloning, and identification of molecules which increase this efficiency should facilitate studies on the mechanism of nuclear reprogramming and acquisition of totipotency.

  8. Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cells in vitro model

    Science.gov (United States)

    Ma, Li-bing; He, Xiao-ning; Si, Wan-tong; Zheng, Yue-Mao

    2016-01-01

    Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos. PMID:26243608

  9. Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cells in vitro model.

    Science.gov (United States)

    He, Xiao-Ying; Ma, Li-Bing; He, Xiao-Ning; Si, Wan-Tong; Zheng, Yue-Mao

    2016-06-30

    Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos. PMID:26243608

  10. Green fluorescent protein (GFP) transgenic pig produced by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    LIU ZhongHua; SUN Shuang; LI YuTian; WANG HongBin; R S PRATHER; SONG Jun; WANG ZhenKun; TIAN JiangTian; KONG QingRan; ZHENG Zhong; YIN Zhi; GAO Li; MA HaiKun

    2008-01-01

    Transgenic somatic cell nuclear transfer is a very promising route for producing transgenic farm ani-mals. Research on GFP transgenic pigs can provide useful information for breeding transgenic pigs, human disease models and human organ xenotransplantation. In this study, a liposomal transfection system was screened and transgenic embryos were reconstructed by nuclear transfer of GFP positive cells into enucleated in vitro matured oocytes. The development of reconstructed embryos both in vitro and in vivo was observed, and GFP expression was determined. The results showed that porcine fe-tal-derived fibroblast cells cultured with 4.0 plJmL liposome and 1.6 pg/mL plasmid DNA for 6 h re-sulted in the highest transfection rate (3.6%). The percentage of GFP reconstructed embryos that de-veloped in vitro to the blastocyst stage was 10%. Of those the GFP positive percentage was 48%. Re-constructed transgenic embryos were transferred to 10 recipients. 5 of them were pregnant, and 3 de-livered 6 cloned piglets in which 4 piglets were transgenic for the GFP as verified by both GFP protein expression and GFP DNA sequence analysis. The percentage of reconstructed embryos that resulted in cloned piglets was 1.0%; while the percentage of piglets that were transgenic was 0.7%. This is the first group of transgenic cloned pigs born in China, marking a great progress in Chinese transgenic cloned pig research.

  11. Exome sequencing identifies somatic mutations of DDX3X in natural killer/T-cell lymphoma.

    Science.gov (United States)

    Jiang, Lu; Gu, Zhao-Hui; Yan, Zi-Xun; Zhao, Xia; Xie, Yin-Yin; Zhang, Zi-Guan; Pan, Chun-Ming; Hu, Yuan; Cai, Chang-Ping; Dong, Ying; Huang, Jin-Yan; Wang, Li; Shen, Yang; Meng, Guoyu; Zhou, Jian-Feng; Hu, Jian-Da; Wang, Jin-Fen; Liu, Yuan-Hua; Yang, Lin-Hua; Zhang, Feng; Wang, Jian-Min; Wang, Zhao; Peng, Zhi-Gang; Chen, Fang-Yuan; Sun, Zi-Min; Ding, Hao; Shi, Ju-Mei; Hou, Jian; Yan, Jin-Song; Shi, Jing-Yi; Xu, Lan; Li, Yang; Lu, Jing; Zheng, Zhong; Xue, Wen; Zhao, Wei-Li; Chen, Zhu; Chen, Sai-Juan

    2015-09-01

    Natural killer/T-cell lymphoma (NKTCL) is a malignant proliferation of CD56(+) and cytoCD3(+) lymphocytes with aggressive clinical course, which is prevalent in Asian and South American populations. The molecular pathogenesis of NKTCL has largely remained elusive. We identified somatic gene mutations in 25 people with NKTCL by whole-exome sequencing and confirmed them in an extended validation group of 80 people by targeted sequencing. Recurrent mutations were most frequently located in the RNA helicase gene DDX3X (21/105 subjects, 20.0%), tumor suppressors (TP53 and MGA), JAK-STAT-pathway molecules (STAT3 and STAT5B) and epigenetic modifiers (MLL2, ARID1A, EP300 and ASXL3). As compared to wild-type protein, DDX3X mutants exhibited decreased RNA-unwinding activity, loss of suppressive effects on cell-cycle progression in NK cells and transcriptional activation of NF-κB and MAPK pathways. Clinically, patients with DDX3X mutations presented a poor prognosis. Our work thus contributes to the understanding of the disease mechanism of NKTCL. PMID:26192917

  12. Use of domestic detergents in the California mastitis test for high somatic cell counts in milk.

    Science.gov (United States)

    Leach, K A; Green, M J; Breen, J E; Huxley, J N; Macaulay, R; Newton, H T; Bradley, A J

    2008-11-01

    The California mastitis test (CMT) is used on farms to identify subclinical mastitis by an indirect estimation of the somatic cell count (SCC) in milk. Four commercially available detergents were compared with a bespoke cmt fluid for their ability to detect milk samples with a scc above 200,000 cells/ml; differences between the interpretation of the results of the tests by eight operators were also investigated. The sensitivity and specificity of the test were affected by the type of detergent, and by the operators' interpretations. When used by the most sensitive operator, suitably diluted Fairy Liquid performed almost identically to cmt fluid in identifying milk samples with more than 200,000 cells/ml. The average sensitivities achieved by the eight operators for detecting this threshold were 82 per cent for Fairy Liquid and 84 per cent for cmt fluid, and the specificities were 93 and 91 per cent respectively. The other detergents contained less anionic surfactants and were less sensitive but similarly specific. PMID:18997186

  13. Determination of methyl methanesulfonate pretreatment effect in Drosophila melanogaster larvaes upon repair mechanisms in somatic cells

    International Nuclear Information System (INIS)

    To make evident the existence of SOS repair mecanism in somatic cells, larvaes of drosophila melanogaster with MWH markers for females and FLR markers for males were used. This larvaes received a pretreatment with MMS at concentrations of 0.0007% and 0.0014% during 24 hours and latter a treatment with gamma rays at different dosis. SMART program was used to make stastistical evaluations. Small spots were observed which can have two origins. First could be damage in the last part of third stage in which cells are in last divisions and second could be the damage to larvaes in early stages in shich pretreatment with MMS cause lesions which prevent the reproduction of the cells. Also big spots were observed which presence is due to recombination. It was detected than the bigger the concentration of MMS and radiation dose, the bigger the induced damage. In some groups such observation was impossible may be to technical problems as relative humidity, out of phase in the growth of larvaes giving place that treatment were given in three stages. For this reasons it was impossible to discriminate if drosophila melanogaster is wheter or not capable to induce a repair mechanism (Author)

  14. Looking into the Black Box: Insights into the Mechanisms of Somatic Cell Reprogramming

    Directory of Open Access Journals (Sweden)

    Jeffrey L. Wrana

    2011-01-01

    Full Text Available The dramatic discovery that somatic cells could be reprogrammed to induced pluripotent stem cells (iPSCs, by the expression of just four factors, has opened new opportunities for regenerative medicine and novel ways of modeling human diseases. Extensive research over the short time since the first iPSCs were generated has yielded the ability to reprogram various cell types using a diverse range of methods. However the duration, efficiency, and safety of induced reprogramming have remained a persistent limitation to achieving a robust experimental and therapeutic system. The field has worked to resolve these issues through technological advances using non-integrative approaches, factor replacement or complementation with microRNA, shRNA and drugs. Despite these advances, the molecular mechanisms underlying the reprogramming process remain poorly understood. Recently, through the use of inducible secondary reprogramming systems, researchers have now accessed more rigorous mechanistic experiments to decipher this complex process. In this review we will discuss some of the major recent findings in reprogramming, pertaining to proliferation and cellular senescence, epigenetic and chromatin remodeling, and other complex cellular processes such as morphological changes and mesenchymal-to-epithelial transition. We will focus on the implications of this work in the construction of a mechanistic understanding of reprogramming and discuss unexplored areas in this rapidly expanding field.

  15. DNA methylation in porcine preimplantation embryos developed in vivo and produced by in vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Deshmukh, Rahul Shahaji; Østrup, Olga; Østrup, Esben;

    2011-01-01

    DNA demethylation and remethylation are crucial for reprogramming of the differentiated parental/somatic genome in the recipient ooplasm upon somatic cell nuclear transfer. Here, we analyzed the DNA methylation dynamics during porcine preimplantation development. Porcine in vivo developed (IV), i...

  16. Effects of Histone Deacetylase Inhibitor Oxamflatin on In Vitro Porcine Somatic Cell Nuclear Transfer Embryos

    Science.gov (United States)

    Hou, Liming; Ma, Fanhua; Yang, Jinzeng; Riaz, Hasan; Wang, Yongliang; Wu, Wangjun; Xia, Xiaoliang; Ma, Zhiyuan; Zhou, Ying; Zhang, Lin; Ying, Wenqin; Xu, Dequan; Zuo, Bo; Ren, Zhuqing

    2014-01-01

    Abstract Low cloning efficiency is considered to be caused by the incomplete or aberrant epigenetic reprogramming of differentiated donor cells in somatic cell nuclear transfer (SCNT) embryos. Oxamflatin, a novel class of histone deacetylase inhibitor (HDACi), has been found to improve the in vitro and full-term developmental potential of SCNT embryos. In the present study, we studied the effects of oxamflatin treatment on in vitro porcine SCNT embryos. Our results indicated that the rate of in vitro blastocyst formation of SCNT embryos treated with 1 μM oxamflatin for 15 h postactivation was significantly higher than all other treatments. Treatment of oxamflatin decreased the relative histone deacetylase (HDAC) activity in cloned embryos and resulted in hyperacetylation levels of histone H3 at lysine 9 (AcH3K9) and histone H4 at lysine 5 (AcH4K5) at pronuclear, two-cell, and four-cell stages partly through downregulating HDAC1. The suppression of HDAC6 through oxamflatin increased the nonhistone acetylation level of α-tubulin during the mitotic cell cycle of early SCNT embryos. In addition, we demonstrated that oxamflatin downregulated DNA methyltransferase 1 (DNMT1) expression and global DNA methylation level (5-methylcytosine) in two-cell-stage porcine SCNT embryos. The pluripotency-related gene POU5F1 was found to be upregulated in the oxamflatin-treated group with a decreased DNA methylation tendency in its promoter regions. Treatment of oxamflatin did not change the locus-specific DNA methylation levels of Sus scrofa heterochromatic satellite DNA sequences at the blastocyst stage. Meanwhile, our findings suggest that treatment with HDACi may contribute to maintaining the stable status of cytoskeleton-associated elements, such as acetylated α-tubulin, which may be the crucial determinants of donor nuclear reprogramming in early SCNT embryos. In summary, oxamflatin treatment improves the developmental potential of porcine SCNT embryos in vitro. PMID

  17. Trichostatin A specifically improves the aberrant expression of transcription factor genes in embryos produced by somatic cell nuclear transfer

    OpenAIRE

    Kimiko Inoue; Mami Oikawa; Satoshi Kamimura; Narumi Ogonuki; Toshinobu Nakamura; Toru Nakano; Kuniya Abe; Atsuo Ogura

    2015-01-01

    Although mammalian cloning by somatic cell nuclear transfer (SCNT) has been established in various species, the low developmental efficiency has hampered its practical applications. Treatment of SCNT-derived embryos with histone deacetylase (HDAC) inhibitors can improve their development, but the underlying mechanism is still unclear. To address this question, we analysed gene expression profiles of SCNT-derived 2-cell mouse embryos treated with trichostatin A (TSA), a potent HDAC inhibitor t...

  18. Loss of Wild-Type ATRX Expression in Somatic Cell Hybrids Segregates with Activation of Alternative Lengthening of Telomeres

    OpenAIRE

    Kylie Bower; Napier, Christine E.; Cole, Sara L.; Dagg, Rebecca A.; Lau, Loretta M. S.; Duncan, Emma L; Moy, Elsa L.; Reddel, Roger R

    2012-01-01

    Alternative Lengthening of Telomeres (ALT) is a non-telomerase mechanism of telomere lengthening that occurs in about 10% of cancers overall and is particularly common in astrocytic brain tumors and specific types of sarcomas. Somatic cell hybridization analyses have previously shown that normal telomerase-negative fibroblasts and telomerase-positive immortalized cell lines contain repressors of ALT activity, indicating that activation of ALT results from loss of one or more unidentified repr...

  19. Proliferation of germ cells and somatic cells in first trimester human embryonic gonads as indicated by S and S+G2+M phase fractions

    DEFF Research Database (Denmark)

    Sørensen, K P; Lutterodt, M C; Mamsen, L S;

    2011-01-01

    The number of germ cells and somatic cells in human embryonic and foetal gonads has previously been estimated by stereological methods, which are time- and labour-consuming with little information concerning cell proliferation. Here, we studied whether flow cytometry could be applied as an easier...... method, also enabling estimation of the fraction of cells in S or S+G(2)+M (SG(2) M) cell-cycle phases as indicators of cell proliferation....

  20. Transcriptomic analysis of milk somatic cells in mastitis resistant and susceptible sheep upon challenge with Staphylococcus epidermidis and Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Tasca Christian

    2011-04-01

    Full Text Available Abstract Background The existence of a genetic basis for host responses to bacterial intramammary infections has been widely documented, but the underlying mechanisms and the genes are still largely unknown. Previously, two divergent lines of sheep selected for high/low milk somatic cell scores have been shown to be respectively susceptible and resistant to intramammary infections by Staphylococcus spp. Transcriptional profiling with an 15K ovine-specific microarray of the milk somatic cells of susceptible and resistant sheep infected successively by S. epidermidis and S. aureus was performed in order to enhance our understanding of the molecular and cellular events associated with mastitis resistance. Results The bacteriological titre was lower in the resistant than in the susceptible animals in the 48 hours following inoculation, although milk somatic cell concentration was similar. Gene expression was analysed in milk somatic cells, mainly represented by neutrophils, collected 12 hours post-challenge. A high number of differentially expressed genes between the two challenges indicated that more T cells are recruited upon inoculation by S. aureus than S. epidermidis. A total of 52 genes were significantly differentially expressed between the resistant and susceptible animals. Further Gene Ontology analysis indicated that differentially expressed genes were associated with immune and inflammatory responses, leukocyte adhesion, cell migration, and signal transduction. Close biological relationships could be established between most genes using gene network analysis. Furthermore, gene expression suggests that the cell turn-over, as a consequence of apoptosis/granulopoiesis, may be enhanced in the resistant line when compared to the susceptible line. Conclusions Gene profiling in resistant and susceptible lines has provided good candidates for mapping the biological pathways and genes underlying genetically determined resistance and susceptibility

  1. Rex Rabbit Somatic Cell Nuclear Transfer with In Vitro-Matured Oocytes.

    Science.gov (United States)

    Liu, Yong; Wang, Huili; Lu, Jinhua; Miao, Yiliang; Cao, Xinyan; Zhang, Ling; Wu, Xiaoqing; Wu, Fengrui; Ding, Biao; Wang, Rong; Luo, Mingjiu; Li, Wenyong; Tan, Jinghe

    2016-06-01

    Somatic cell nuclear transfer (SCNT) requires large numbers of matured oocytes. In vitro-matured (IVM) oocytes have been used in SCNT in many animals. We investigated the use of IVM oocytes in Rex rabbit SCNT using Rex rabbit ovaries obtained from a local abattoir. The meiotic ability of oocytes isolated from follicles of different diameters was studied. Rex rabbit SCNT was optimized for denucleation, activation, and donor cell synchronization. Rex rabbit oocytes grew to the largest diameter (110 μm) when the follicle diameter was 1.0 mm. Oocytes isolated from 0.7-mm follicles acquired maturation ability. More than 90% of these oocytes matured after IVC for 18 h. The developmental potential of oocytes isolated from >1-mm follicles was greater than that of oocytes isolated from 0.7- to 1.0-mm follicles. The highest activation rates for IVM Rex rabbit oocytes were seen after treatment with 2.5 μM ionomycin for 5 min followed by 2 mM 6-dimethylaminopurine (6-DMAP) and 5 μg/mL cycloheximide (CHX) for 1 h. Ionomycin induced the chromatin of IVM oocytes to protrude from the oocyte surface, promoting denucleation. Fetal fibroblast cells (FFCs) and cumulus cells (CCs) were more suitable for Rex rabbit SCNT than skin fibroblast cells (SFCs) (blastocyst rate was 35.6 ± 2.2% and 38.0 ± 6.0% vs. 19.7 ± 3.1%). The best fusion condition was a 2DC interval for 1 sec, 1.6 kV/cm voltages, and 40 μsec duration in 0.28 M mannitol. In conclusion, the in vitro maturation of Rex rabbit oocytes and SCNT procedures were studied systematically and optimized in this study. PMID:27159389

  2. Cytogenetic effects of leachates from tannery solid waste on the somatic cells of Vicia faba.

    Science.gov (United States)

    Chandra, Saurabh; Chauhan, L K S; Pande, P N; Gupta, S K

    2004-04-01

    The contamination of surface- and groundwater by the leaching of solid wastes generated by industrial activities as a result of water runoff and rainfall is a matter of great concern. The leachates from tannery solid waste (TSW), a major environmental pollutant, were examined for their possible genotoxic effects on the somatic cells of Vicia faba. Leachates were prepared from solid wastes procured from leather-tanning industrial sites, and V. faba seedlings were exposed to three test concentrations, 2.5%, 5%, and 10%, through soil and aqueous media for 5 days. The root tips examined for cytogenetic damage revealed that leachate of TSW significantly inhibited the mitotic index and induced significantly frequent chromosomal and mitotic aberrations (CA/MA) in a dose-dependent manner. The chemical analysis of TSW samples revealed that the chief constituents were chromium and nickel, which may cause genetic abnormalities. The frequency of aberrations was found to be higher in the root meristematic cells of Vicia faba exposed through the aqueous medium than those exposed through the soil medium. The results of the present study indicated that contamination of potable water bodies by leachates of TSW may cause genotoxicity. For the biomonitoring of complex mixtures of toxicants with the V. faba bioassay, the use of the aqueous medium seems to be a more promising method than the use of the soil medium. PMID:15037999

  3. Dairy Herd Mastitis Program in Argentina: Farm Clusters and Effects on Bulk Milk Somatic Cell Counts

    Directory of Open Access Journals (Sweden)

    C Vissio1*, SA Dieser2, CG Raspanti2, JA Giraudo1, CI Bogni2, LM Odierno2 and AJ Larriestra1

    2013-01-01

    Full Text Available This research has been conducted to characterize dairy farm clusters according to mastitis control program practiced among small and medium dairy producer from Argentina, and also to evaluate the effect of such farm cluster patterns on bulk milk somatic cell count (BMSCC. Two samples of 51 (cross-sectional and 38 (longitudinal herds were selected to identify farm clusters and study the influence of management on monthly BMSCC, respectively. The cross-sectional sample involved the milking routine and facilities assessment of each herd visited. Hierarchical cluster analysis was used to find the most discriminating farm attributes in the cross sectional sample. Afterward, the herd cluster typologies were identified in the longitudinal sample. Herd monthly BMSCC average was evaluated during 12 months fitting a linear mixed model. Two clusters were identified, the farms in the Cluster I applied a comprehensive mastitis program in opposite to Cluster II. Post-dipping, dry cow therapy and milking machine test were routinely applied in Cluster I. In the longitudinal study, 14 out of 38 dairy herds were labeled as Cluster I and the rest were assigned to Cluster II. Significant difference in BMSCC was found between cluster I and II (60,000 cells/mL. The present study showed the relevance and potential impact of promoting mastitis control practices among small and medium sized dairy producers in Argentina.

  4. PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells.

    Science.gov (United States)

    Chandrakanthan, Vashe; Yeola, Avani; Kwan, Jair C; Oliver, Rema A; Qiao, Qiao; Kang, Young Chan; Zarzour, Peter; Beck, Dominik; Boelen, Lies; Unnikrishnan, Ashwin; Villanueva, Jeanette E; Nunez, Andrea C; Knezevic, Kathy; Palu, Cintia; Nasrallah, Rabab; Carnell, Michael; Macmillan, Alex; Whan, Renee; Yu, Yan; Hardy, Philip; Grey, Shane T; Gladbach, Amadeus; Delerue, Fabien; Ittner, Lars; Mobbs, Ralph; Walkley, Carl R; Purton, Louise E; Ward, Robyn L; Wong, Jason W H; Hesson, Luke B; Walsh, William; Pimanda, John E

    2016-04-19

    Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor-AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration. PMID:27044077

  5. An Epigenetic Modifier Results in Improved In Vitro Blastocyst production after Somatic Cell Nuclear Transfer

    DEFF Research Database (Denmark)

    Zhang, Yunhai; Li, Juan; Villemoes, Klaus; Pedersen, Anette Møjbæk; Purup, Stig; Vajta, Gabor

    2007-01-01

    significantly improve blastocyst yield compared to the control (46.4 ± 4.6% vs 17.7 ± 4.9% for treated and untreated embryos, respectively; p < 0.05), whereas similar cleavage rate and total cell number per blastocyst were observed. In order to assess if the improvement is cell line specific, three cell lines...... were tested, and for all cell lines an enhancement in blastocyst development compared to their corresponding control was observed. Our data demonstrate that TSA treatment after somatic cell nuclear transfer in the pig can significantly improve the in vitro blastocyst production...

  6. A novel somatic MAPK1 mutation in primary ovarian mixed germ cell tumors.

    Science.gov (United States)

    Zou, Yang; Deng, Wei; Wang, Feng; Yu, Xiao-Hong; Liu, Fa-Ying; Yang, Bi-Cheng; Huang, Mei-Zhen; Guo, Jiu-Bai; Xie, Qiu-Hua; He, Ming; Huang, Ou-Ping

    2016-02-01

    A recent exome-sequencing study revealed prevalent mitogen-activated protein kinase 1 (MAPK1) p.E322K mutation in cervical carcinoma. It remains largely unknown whether ovarian carcinomas also harbor MAPK1 mutations. As paralogous gene mutations co‑occur frequently in human malignancies, we analyzed here a total of 263 ovarian carcinomas for the presence of MAPK1 and paralogous MAPK3 mutations by DNA sequencing. A previously unreported MAPK1 p.D321N somatic mutation was identified in 2 out of 18 (11.1%) ovarian mixed germ cell tumors, while no other MAPK1 or MAPK3 mutation was detected in our samples. Of note, OCC‑115, the MAPK1‑mutated sample with bilateral cancerous ovaries affected, harbored MAPK1 mutation in the right ovary while retained the left ovary intact, implicating that the genetic alterations underlying ovarian mixed germ cell tumor may be different, even in patients with similar genetic backgrounds and tumor microenvironments. The results of evolutionary conservation and protein structure modeling analysis implicated that MAPK1 p.D321N mutation may be pathogenic. Additionally, mutations in protein phosphatase 2 regulatory subunit α (PPP2R1A), ring finger protein 43 (RNF43), DNA directed polymerase ε (POLE1), ribonuclease type III (DICER1), CCCTC‑binding factor (CTCF), ribosomal protein L22 (RPL22), DNA methyltransferase 3α (DNMT3A), transformation/transcription domain‑associated protein (TRRAP), isocitrate dehydrogenase (IDH)1 and IDH2 were not detected in ovarian mixed germ cell tumors, implicating these genetic alterations may be not associated with MAPK1 mutation in the development of this malignancy. The present study identified a previously unreported MAPK1 mutation in ovarian mixed germ cell tumors for the first time, and this mutation may be actively involved in the tumorigenesis of this disease. PMID:26548627

  7. Cell-Specific mRNA Profiling of the Caenorhabditis elegans Somatic Gonadal Precursor Cells Identifies Suites of Sex-Biased and Gonad-Enriched Transcripts.

    Science.gov (United States)

    Kroetz, Mary B; Zarkower, David

    2015-12-01

    The Caenorhabditis elegans somatic gonad differs greatly between the two sexes in its pattern of cell divisions, migration, and differentiation. Despite decades of study, the genetic pathways directing early gonadal development and establishing sexual dimorphism in the gonad remain largely unknown. To help define the genetic networks that regulate gonadal development, we employed cell-specific RNA-seq. We identified transcripts present in the somatic gonadal precursor cells and their daughter cells of each sex at the onset of sexual differentiation. We identified several hundred gonad-enriched transcripts, including the majority of known regulators of early gonadal development, and transgenic reporter analysis confirmed the effectiveness of this approach. Before the division of the somatic gonad precursors, few sex-biased gonadal transcripts were detectable; less than 6 hr later, after their division, we identified more than 250 sex-biased transcripts, of which about a third were enriched in the somatic gonad compared to the whole animal. This indicates that a robust sex-biased developmental program, some of it gonad-specific, initiates in the somatic gonadal precursor cells around the time of their first division. About 10% of male-biased transcripts had orthologs with male-biased expression in the early mouse gonad, suggesting possible conservation of gonad sex differentiation. Cell-specific analysis also identified approximately 70 previously unannotated mRNA isoforms that are enriched in the somatic gonad. Our data illustrate the power of cell-specific transcriptome analysis and suggest that early sex differentiation in the gonad is controlled by a relatively small suite of differentially expressed genes, even after dimorphism has become apparent. PMID:26497144

  8. The Drosophila BCL6 homolog Ken and Barbie promotes somatic stem cell self-renewal in the testis niche.

    Science.gov (United States)

    Issigonis, Melanie; Matunis, Erika

    2012-08-15

    Stem cells sustain tissue regeneration by their remarkable ability to replenish the stem cell pool and to generate differentiating progeny. Signals from local microenvironments, or niches, control stem cell behavior. In the Drosophila testis, a group of somatic support cells called the hub creates a stem cell niche by locally activating the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway in two adjacent types of stem cells: germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Here, we find that ken and barbie (ken) is autonomously required for the self-renewal of CySCs but not GSCs. Furthermore, Ken misexpression in the CySC lineage induces the cell-autonomous self-renewal of somatic cells as well as the nonautonomous self-renewal of germ cells outside the niche. Thus, Ken, like Stat92E and its targets ZFH1 (Leatherman and Dinardo, 2008) and Chinmo (Flaherty et al., 2010), is necessary and sufficient for CySC renewal. However, ken is not a JAK-STAT target in the testis, but instead acts in parallel to Stat92E to ensure CySC self-renewal. Ken represses a subset of Stat92E targets in the embryo (Arbouzova et al., 2006) suggesting that Ken maintains CySCs by repressing differentiation factors. In support of this hypothesis, we find that the global JAK-STAT inhibitor Protein tyrosine phosphatase 61F (Ptp61F) is a JAK-STAT target in the testis that is repressed by Ken. Together, our work demonstrates that Ken has an important role in the inhibition of CySC differentiation. Studies of ken may inform our understanding of its vertebrate orthologue B-Cell Lymphoma 6 (BCL6) and how misregulation of this oncogene leads to human lymphomas. PMID:22580161

  9. Meiotic Recombination in Somatic Cell Nuclear Transfer Bulls and Their Offspring

    Science.gov (United States)

    In mammals, homologous chromosome pairing and recombination are essential events for meiosis. The generation of reciprocal exchanges of genetic material ensure both genetic diversity and the proper segregation of homologous chromosomes. With the advent of reproductive biotechnologies such as somat...

  10. TCPs, WUSs, and WINDs: Families of transcription factors that regulate shoot meristem formation, stem cell maintenance, and somatic cell differentiation

    Directory of Open Access Journals (Sweden)

    Miho eIkeda

    2014-09-01

    Full Text Available In contrast to somatic mammalian cells, which cannot alter their fate, plant cells can dedifferentiate to form totipotent callus cells and regenerate a whole plant, following treatment with specific phytohormones. However, the regulatory mechanisms and key factors that control differentiation-dedifferentiation and cell totipotency have not been completely clarified in plants. Recently, several plant transcription factors that regulate meristem formation and dedifferentiation have been identified and include members of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP, WUSCHEL (WUS, and WOUND INDUCED DEDIFFERENTIATION (WIND1 families. WUS and WIND positively control plant cell totipotency, while TCP negatively controls it. Interestingly, TCP is a transcriptional activator that acts as a negative regulator of shoot meristem formation, and WUS is a transcriptional repressor that positively maintains totipotency of the stem cells of the shoot meristem. We describe here the functions of TCP, WUS and WIND transcription factors in the regulation of differentiation-dedifferentiation by positive and negative transcriptional regulators.

  11. A somatic cell hybrid panel for pig regional gene mapping characterized by molecular cytogenetics.

    Science.gov (United States)

    Yerle, M; Echard, G; Robic, A; Mairal, A; Dubut-Fontana, C; Riquet, J; Pinton, P; Milan, D; Lahbib-Mansais, Y; Gellin, J

    1996-01-01

    A panel of 27 pig x rodent somatic cell hybrids was produced and characterized cytogenetically. The first step of this study consisted of hybridizing a SINE probe to GTG-banded metaphases of each hybrid clone in order to count and identify the normal pig chromosomes and to detect rearranged ones. The second step consisted of using the DNA of each clone as a probe after pIRS-PCR (porcine interspersed repetitive sequence-polymerase chain reaction) amplification to highly enrich it in pig sequences. These probes, hybridized to normal pig metaphase chromosomes, enabled the identification of the complete porcine complement in the hybrid lines. Whole chromosomes and fragments were characterized quickly and precisely, and results were compared. In addition to this cytogenetic characterization, molecular verification was also carried out by using primers specific to six microsatellites and to one gene previously mapped to pig chromosomes. The results obtained allow us to conclude that we have produced a panel that is informative for all porcine chromosomes. This panel constitutes a highly efficient tool to establish not only assignments of genes and markers but also regional localizations on pig chromosomes. PMID:8697807

  12. Comparing milk yield, chemical properties and somatic cell count from organic and conventional mountain farming systems

    Directory of Open Access Journals (Sweden)

    Marcello Bianchi

    2010-01-01

    Full Text Available A study was undertaken to investigate the effects of farming systems (organic vs. conventional, diet (hay/concentrate vs. pasture and their interaction on milk yield, gross composition and fatty acid (FA profile of dairy cows bred in mountainous areas. For this purpose four dairy farms (two organic and two conventional were chosen in the alpine territory of Aosta Valley (NW Italy; individual milk yield was recorded daily and bulk milk samples were collected monthly from February to September 2007 to cover dietary variations. Higher levels of milk production (P<0.05 and lower milk protein amounts (P<0.01 were observed in the organic farms with respect to the conventional ones, while no significant differences were noticed in milk fat and lactose contents and in somatic cell count. Concerning fatty acids, only small differences were detected between organic and conventional milk and such differences seemed to be related mainly to the stabled period. Diet affected almost all variables studied: pasture feeding provided a significant improvement in the fatty acid composition in both organic and conventional systems leading to lower hypercholesterolemic saturated fatty acids, higher mono- and polyunsaturated fatty acids and conjugated linoleic acid amounts (P<0.001.

  13. Effect of somatic cell count and lactation stage on sheep milk quality

    Directory of Open Access Journals (Sweden)

    Emilia Duranti

    2010-01-01

    Full Text Available In order to evaluate the effects of mammary health status and lactation phase on the qualitative parameters of ovinemilk, 213 individual milk samples were repeatedly collected from 40 primiparous Sarda ewes on a monthly basis. Yield,physico-chemical characteristics, casein fractions quantitative distribution, somatic cell count (SCC, cheese making propertiesand plasmin-plasminogen activity were determined on each sample. Repeated individual milk SCC were used as amarker of udder health status, allowing the definition of three classes: “Healthy” (H, “Infected” (I or “Doubtful” (D.Samples were grouped into 4 classes of days in milk (DIM. To evaluate the influence of mammary health status andphase of lactation, a mixed model was performed using the ewe as random effect. Milk physico-chemical parameters wereinfluenced both by the udder health status and by lactation phase. In particular, the udder health status adversely affectedαs1 and β1-casein fractions (Pand 64.60% in “H”, “D” and “I,” respectively. Lactation phase influenced the overall milk composition and technologicalcharacteristics. Plasmin activity was higher in the “I” group than in the others (16.1 vs 11.8 and 11.2 U/ml; Pit significantly (Pexert a detrimental effect on milk quality since they enhance its endogenous proteolytic activity.

  14. Inheritance of mitochondrial DNA in serially recloned pigs by somatic cell nuclear transfer (SCNT)

    International Nuclear Information System (INIS)

    Highlights: ► We success serial SCNT through the third generation using pig fibroblasts. ► Donor-specific mtDNA in the recloned pigs was detected. ► SCNT affect mtDNA mounts. -- Abstract: Somatic cell nuclear transfer (SCNT) has been established for the transmission of specific nuclear DNA. However, the fate of donor mitochondrial DNA (mtDNA) remains unclear. Here, we examined the fate of donor mtDNA in recloned pigs through third generations. Fibroblasts of recloned pigs were obtained from offspring of each generation produced by fusion of cultured fibroblasts from a Minnesota miniature pig (MMP) into enucleated oocytes of a Landrace pig. The D-loop regions from the mtDNA of donor and recipient differ at nucleotide sequence positions 16050 (A→T), 16062 (T→C), and 16135 (G→A). In order to determine the fate of donor mtDNA in recloned pigs, we analyzed the D-loop region of the donor’s mtDNA by allele-specific PCR (AS-PCR) and real-time PCR. Donor mtDNA was successfully detected in all recloned offspring (F1, F2, and F3). These results indicate that heteroplasmy that originate from donor and recipient mtDNA is maintained in recloned pigs, resulting from SCNT, unlike natural reproduction.

  15. Cloned pigs derived from somatic cell nuclear transfer embryos cultured in vitro at low oxygen tension

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Pig cloning has great potential to human xenotransplantation. The present study was designed to establish a more efficient system for producing cloned pigs by somatic cell nuclear transfer (SCNT). Our approach was as follows: SCNT embryos were reconstructed by using fetal fibroblasts of Chinese miniature pig as donors and in vitro matured oocytes of prepubertal gilts as recipients. Reconstructed embryos were induced by electrical fusion/activation and cultured in BSA-containing North Carolina State University 23 medium (NCSU-23) or Porcine Zygote Medium (PZM-3) at the gas condition of 5% CO2, 7% O2, 88% N2. A total of 230 cloned embryos were transferred to three surrogate sows, producing three piglets. One of them is apparently healthy. The clonal provenance of the piglet was indicated by its coat color and confirmed by DNA microsatellite analysis. These results indicate that the use of in vitro matured oocytes from prepubertal gilts as recipient, combined with cloned embryos cultured at low oxygen tension is an effective way to produce cloned pigs.

  16. Inheritance of mitochondrial DNA in serially recloned pigs by somatic cell nuclear transfer (SCNT)

    Energy Technology Data Exchange (ETDEWEB)

    Do, Minhwa; Jang, Won-Gu; Hwang, Jeong Hee; Jang, Hoon; Kim, Eun-Jung; Jeong, Eun-Jeong [Regenerative Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305 806 (Korea, Republic of); Shim, Hosup [Department of Physiology, Dankook University School of Medicine, Cheonan 330 714 (Korea, Republic of); Hwang, Sung Soo; Oh, Keon Bong; Byun, Sung June [Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Suwon (Korea, Republic of); Kim, Jin-Hoi [Department of Animal Biotechnology, Konkuk University, Seoul 143 701 (Korea, Republic of); Lee, Jeong Woong, E-mail: jwlee@kribb.re.kr [Regenerative Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305 806 (Korea, Republic of)

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer We success serial SCNT through the third generation using pig fibroblasts. Black-Right-Pointing-Pointer Donor-specific mtDNA in the recloned pigs was detected. Black-Right-Pointing-Pointer SCNT affect mtDNA mounts. -- Abstract: Somatic cell nuclear transfer (SCNT) has been established for the transmission of specific nuclear DNA. However, the fate of donor mitochondrial DNA (mtDNA) remains unclear. Here, we examined the fate of donor mtDNA in recloned pigs through third generations. Fibroblasts of recloned pigs were obtained from offspring of each generation produced by fusion of cultured fibroblasts from a Minnesota miniature pig (MMP) into enucleated oocytes of a Landrace pig. The D-loop regions from the mtDNA of donor and recipient differ at nucleotide sequence positions 16050 (A{yields}T), 16062 (T{yields}C), and 16135 (G{yields}A). In order to determine the fate of donor mtDNA in recloned pigs, we analyzed the D-loop region of the donor's mtDNA by allele-specific PCR (AS-PCR) and real-time PCR. Donor mtDNA was successfully detected in all recloned offspring (F1, F2, and F3). These results indicate that heteroplasmy that originate from donor and recipient mtDNA is maintained in recloned pigs, resulting from SCNT, unlike natural reproduction.

  17. Post-milking teat dip use in dairy herds with high or low somatic cell counts.

    Science.gov (United States)

    Erskine, R J; Eberhart, R J

    1991-12-15

    Milk samples for bacteriologic culture were submitted from 71 dairy herds, 24 with low somatic cell count (SCC) and 47 with high SCC and high prevalence of subclinical mastitis. At the time of sample submission to the Mastitis Diagnostic Laboratory of Pennsylvania State University, information regarding the herd mastitis control practices was collected. A combined program of post-milking teat dipping (PMTD) and antibiotic treatment of all cows at the start of the nonlactating period was practiced more frequently for herds with low SCC, (P less than 0.001) than for herds with high SCC. Among all herds for which PMTD was practiced, a higher proportion (P less than 0.001) of those for which chlorhexidine-based products were used had low SCC than high SCC. Conversely, a higher proportion of herds for which a dip with an acrylic latex barrier was used had high SCC rather than low SCC (P = 0.002). For herds with high prevalence of subclinical mastitis, and despite a program of PMTD and treatment of all cows at the start of the nonlactating period, a change to a different germicidal teat dip product may be indicated to help reduce prevalence of infection. PMID:1813466

  18. Association between BoLA-DRB3 and somatic cell count in Holstein cattle from Argentina.

    Science.gov (United States)

    Baltian, L R; Ripoli, M V; Sanfilippo, S; Takeshima, S N; Aida, Y; Giovambattista, G

    2012-07-01

    Different studies have proved that the resistance/susceptibility to mastitis is genetically determined. The major histocompatibility complex in cows is known as bovine lymphocyte antigen (BoLA). Genes from the BoLA have been associated with the occurrence of infectious diseases such as mastitis and leukosis, especially the BoLA-DRB gene. The object of the present study was to detect associations between BoLA-DRB3 alleles and somatic cell count (SCC), as an indicator of resistance/susceptibility to mastitis in Holstein cattle (N = 123) from La Pampa, Argentina. Fisher's exact test and Woolf-Haldane odds ratio were applied to study the association between SCC and BoLA-DRB3 allele frequencies. Significant association was noted between BoLA-DRB3.2*23 and *27 alleles (p < 0.05) and protective or susceptibility effects, respectively. In addition, alleles BoLA-DRB3.2*20 and *25 exhibit suggestive association with high SCC (p < 0.1). These results were partially in agreement with data reported from Japanese Holstein cattle, but differed from those published by other authors. A possible explanation for the contrasting results could be that the mastitis is a multifactor disease caused by different pathogens. Moreover, most of the studies were carried out using PCR-RFLP method, which has less resolution than PCR-SBT because PCR-RFLP defined alleles included more than one sequenced alleles. PMID:22531932

  19. Affinity maturation of anti-TNF-alpha scFv with somatic hypermutation in non-B cells.

    Science.gov (United States)

    Chen, Shaopeng; Qiu, Junkang; Chen, Chuan; Liu, Chunchun; Liu, Yuheng; An, Lili; Jia, Junying; Tang, Jie; Wu, Lijun; Hang, Haiying

    2012-06-01

    Activation-induced cytidine deaminase (AID) is required for the generation of antibody diversity through initiating both somatic hypermutation (SHM) and class switch recombination. A few research groups have successfully used the feature of AID for generating mutant libraries in directed evolution of target proteins in B cells in vitro. B cells, cultured in suspension, are not convenient for transfection and cloning. In this study, we established an AID-based mutant accumulation and sorting system in adherent human cells. Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells, and a stable cell clone (H1299-AID) was selected. Afterwards, anti-hTNF-α scFv (ATscFv) was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells. By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha, two ATscFv mutant gene clones were isolated. Compared with the wild type ATscFv, the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha. The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells, which makes directed evolution in mammalian cells easier and more efficient. PMID:22467272

  20. Effective Oocyte Vitrification and Survival Techniques for Bovine Somatic Cell Nuclear Transfer.

    Science.gov (United States)

    Park, Min Jee; Lee, Seung Eun; Kim, Eun Young; Lee, Jun Beom; Jeong, Chang Jin; Park, Se Pill

    2015-06-01

    Bovine somatic cell nuclear transfer (SCNT) using vitrified-thawed (VT) oocytes has been studied; however, the cloning efficiency of these oocytes is not comparable with that of nonvitrified (non-V) fresh oocytes. This study sought to optimize the survival and cryopreservation of VT oocytes for SCNT. Co-culture with feeder cells that had been preincubated for 15 h significantly improved the survival of VT oocytes and their in vitro developmental potential following SCNT in comparison to co-culture with feeder cells that had been preincubated for 2, 5, or 24 h (pEVT) group, 13.7%; VT group, 15.0%; p<0.05] and was comparable with that of the non-V group (25.9%). The reactive oxygen species level was significantly lower in the EAVT group than in the other vitrification groups (p<0.05). mRNA levels of maternal genes (ZAR1, BMP15, and NLRP5) and a stress gene (HSF1) were lower in the vitrification groups than in the non-V group (p<0.05), whereas the level of phospho-p44/42 mitogen-activated protein kinase did not differ among the groups. Among the vitrification groups, blastocysts in the EAVT group had the best developmental potential, as judged by their high mRNA expression of developmental potential-related genes (POU5f1, Interferon-tau, and SLC2A5) and their low expression of proapoptotic (CASP3) and stress (Hsp70) genes. This study demonstrates that SCNT using bovine frozen-thawed oocytes can be successfully achieved using optimized vitrification and co-culture techniques. PMID:25984830

  1. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Pang, Daxin, E-mail: pdx@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Ouyang, Hongsheng, E-mail: ouyh@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China)

    2011-07-29

    Highlights: {yields} Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. {yields} The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. {yields} A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 {mu}g/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

  2. Effects of Insulin-like Growth Factor-1 on Development of Somatic Cell Cloned Bovine Embryos.

    Science.gov (United States)

    Qu, Pengxiang; Li, Yanyan; Deng, Tengfei; Jia, Dan; Qing, Suzhu; Su, Jianmin; Zhang, Yong; Wang, Yongsheng

    2016-06-01

    The aim of this study was to assess the effect of insulin-like growth factor-1 (IGF-1) on the developmental competence of somatic cell nuclear transfer (SCNT) bovine embryos. First, the expression levels of IGF-1 receptor (IGF-1R) and IGF-1 in the oocytes and embryos of different developmental stages were examined. Then the effects of exogenous IGF-1 on the development of SCNT embryos were evaluated both in vitro and in vivo. The results showed that IGF-1 was not expressed in both IVF and SCNT embryos, whereas IGF-1R could be detected throughout the preimplantation stages in both protein and mRNA levels. Also, exogenous IGF-1 had no obvious impact on the developmental competence of IVF embryos. However, it could improve the developmental competence of SCNT embryos in terms of blastocyst developmental rate (31.3% vs. 43.2%, p < 0.05), total cell number (93.0 ± 9.9 vs. 101.0 ± 9.8, p < 0.05), ratio of inner cell mass (ICM) to trophectoderm (TE) (0.29 ± 0.006 vs. 0.39 ± 0.005, p < 0.05), and apoptosis index in day 7 blastocysts (2.5 ± 0.22 vs. 8.7 ± 0.41, p < 0.05) compared to the control group. Although no statistical difference in pregnancy rate and birth rate was observed after embryo transfer, there was an upward tendency in both examined terms in the IGF-1-supplemented group when compared with the control group. In conclusion, the present study showed that supplementing exogenous IGF-1 to the culture medium has an obvious positive effect on the development competence of SCNT embryos. PMID:27135251

  3. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

    International Nuclear Information System (INIS)

    Highlights: → Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. → The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. → A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 μg/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

  4. Somatic cell banking - An alternative technology for conservation of endangered sheep breeds

    International Nuclear Information System (INIS)

    cells but these were removed mechanically as well as enzymatically to get pure fibroblasts. Sub-culturing or 'splitting cells' was done periodically removing growth media, washing the plate, dissociating the cells and diluting cell suspension in fresh media. Standard growth curve: Whenever, a new batch of culture media supplement was introduced, it was checked for its efficacy for growth of cells in culture and compared with standard growth curve. Goat skin fibroblasts remained in lag phase for initial two days when they settled on the solid surface of culture vessel and then came to log phase when maximum growth took place spanning from the third to the seventh day. As the confluencey level increased and media supplement was depleted, cells stopped dividing and a plateau was attained from the eighth day onwards and then showed decline due to contact inhibition. Cell proliferation index: Under standard culture conditions, skin fibroblast cells divide once in 24 hours but it is rarely achieved in normal culturing. The population doubling time and cell proliferation rate per day were checked at regular interval for quality assessment. For this, ELISA based MTT assay, incorporation of 5-bromo-de-oxyuridine method, and flow cytometer methods were used. Evaluation of cells for ploidy level: During long-term culturing the cells are likely to develop one or other type of chromosomal abnormalities. It must be ensured that the cells in different passages be checked for normal ploidy so that viable clones can be developed from them. Cultures showing increased frequency of aneuploidy or polyploidy must be terminated from further passaging. DNA from cultured somatic cells can be isolated using available DNA isolation kits and checked for its quality on 2% agarose. Cryo-freezing of cells: Cells are best frozen as cell suspension. Healthy culture were always employed to provide the stock to freeze cells. The cells were frozen at controlled freezing rate. The cells were kept at -80

  5. Rapamycin treatment during in vitro maturation of oocytes improves embryonic development after parthenogenesis and somatic cell nuclear transfer in pigs

    OpenAIRE

    Lee, Joohyeong; Park, Jong-Im; Yun, Jung Im; LEE, YONGJIN; Yong, Hwanyul; Lee, Seung Tae; Park, Choon-Keun; Hyun, Sang-Hwan; Lee, Geun-Shik; Lee, Eunsong

    2015-01-01

    This study was conducted to investigate the effects of rapamycin treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Morphologically good (MGCOCs) and poor oocytes (MPCOCs) were untreated or treated with 1 nM rapamycin during 0-22 h, 22-42 h, or 0-42 h of IVM. Rapamycin had no significant effects on nuclear maturation and blastocyst formation after PA of MGCOCs. Blasto...

  6. Efficiency of somatic cell count and california mastitis test in the diagnosis of subclinical mastitis in terrincha ewes

    OpenAIRE

    Mendonça, Álvaro; Machado, M.; Tavares, A.; Quintas, Hélder; Valentim, Ramiro; Maurício, Raimundo; Cardoso, Manuel

    2012-01-01

    This study aimed to compare the efficiency of microbiological test with Californian Mastitis Test and somatic cell count in the diagnosis of Subclinical Mastitis (SM) in Terrincha sheep. Twenty-seven of a flock of about 200 Terrincha ewes (local breed) were studied for a period of 9 weeks (n > 497 samples). Milk samples were aseptically collected from each half udder once a week. At the same time, another sampled was collected from the bulk tank. After being transported to Lab under refrigera...

  7. STUDY REGARDING THE INFLUENCE OF SEASON AND LACTATION ORDER ON MILK YIELD, MAJOR COMPONENTS AND SOMATIC CELL COUNT OF MILK

    OpenAIRE

    S. ACATINCĂI; ADELA MARCU; L.T. CZISZTER; SIMONA BAUL; ANDREEA FERENCZ; D. GĂVOJDEAN

    2013-01-01

    The effects of lactation order and season on the milk production, chemical compositionand somatic cell number during a normal lactation (305 days) were studied. Researcheswere carried out on Romanian Black and White cows from the Didactical StationTimişoara. Cows calved in autumn and finished their lactation by the end of the nextyear. Milk production increased progressively in the second and third lactation, thusfat, protein and lactose yields increased, too. During the warm season (April-Se...

  8. A conditional Orco requirement in the somatic cyst cells for maintaining spermatids in a tight bundle in Drosophila testis

    Indian Academy of Sciences (India)

    Pankaj Dubey; Prakash Joti; Krishanu Ray

    2016-06-01

    Odorant receptors (OR) heterodimerizes with the OR co-receptor (Orco), forming specific odorant-gated cation channels, which are key to odor reception at the olfactory sensory neurons (OSN). Mammalian ORs are expressed in many other tissues, including testis. However, their biological implications are yet to be fully ascertained. In the mosquito, Orco is localized along the sperm tail and is indicated to maintain fidelity. Here, we show that orco expresses in Drosophila testis. The levels are higher in the somatic cyst cells. The orco-null mutants are perfectly fertile at 25°C. At 28°C, the coiled spermatid bundles are severely disrupted. The loss of Orco also disrupts the actin cap, which forms inside the head cyst cell at the rostral ends of the spermatid nuclei after coiling, and plays a key role in preventing the abnormal release of spermatids from the cyst enclosure. Both the defects are rescued by the somatic cyst cell-specific expression of the UAS-orco transgene. These results highlight a novel role of Orco in the somatic tissue during sperm release.

  9. Are we Genomic Mosaics? Variations of the Genome of Somatic Cells can Contribute to Diversify our Phenotypes.

    Science.gov (United States)

    Astolfi, P A; Salamini, F; Sgaramella, V

    2010-09-01

    Theoretical and experimental evidences support the hypothesis that the genomes and the epigenomes may be different in the somatic cells of complex organisms. In the genome, the differences range from single base substitutions to chromosome number; in the epigenome, they entail multiple postsynthetic modifications of the chromatin. Somatic genome variations (SGV) may accumulate during development in response both to genetic programs, which may differ from tissue to tissue, and to environmental stimuli, which are often undetected and generally irreproducible. SGV may jeopardize physiological cellular functions, but also create novel coding and regulatory sequences, to be exposed to intraorganismal Darwinian selection. Genomes acknowledged as comparatively poor in genes, such as humans', could thus increase their pristine informational endowment. A better understanding of SGV will contribute to basic issues such as the "nature vs nurture" dualism and the inheritance of acquired characters. On the applied side, they may explain the low yield of cloning via somatic cell nuclear transfer, provide clues to some of the problems associated with transdifferentiation, and interfere with individual DNA analysis. SGV may be unique in the different cells types and in the different developmental stages, and thus explain the several hundred gaps persisting in the human genomes "completed" so far. They may compound the variations associated to our epigenomes and make of each of us an "(epi)genomic" mosaic. An ensuing paradigm is the possibility that a single genome (the ephemeral one assembled at fertilization) has the capacity to generate several different brains in response to different environments. PMID:21358981

  10. A role for XLF in DNA repair and recombination in human somatic cells.

    Science.gov (United States)

    Fattah, Farjana Jahan; Kweon, Junghun; Wang, Yongbao; Lee, Eu Han; Kan, Yinan; Lichter, Natalie; Weisensel, Natalie; Hendrickson, Eric A

    2014-03-01

    Classic non-homologous end-joining (C-NHEJ) is required for the repair of radiation-induced DNA double-strand breaks (DSBs) in mammalian cells and plays a critical role in lymphoid V(D)J recombination. A core C-NHEJ component is the DNA ligase IV co-factor, Cernunnos/XLF (hereafter XLF). In patients, mutations in XLF cause predicted increases in radiosensitivity and deficits in immune function, but also cause other less well-understood pathologies including neural disorders. To characterize XLF function(s) in a defined genetic system, we used a recombinant adeno-associated virus-mediated gene targeting strategy to inactivate both copies of the XLF locus in the human HCT116 cell line. Analyses of XLF-null cells (which were viable) showed that they were highly sensitive to ionizing radiation and a radiomimetic DNA damaging agent, etoposide. XLF-null cells had profound DNA DSB repair defects as measured by in vivo plasmid end-joining assays and were also dramatically impaired in their ability to form either V(D)J coding or signal joints on extrachromosomal substrates. Thus, our somatic XLF-null cell line recapitulates many of the phenotypes expected from XLF patient cell lines. Subsequent structure:function experiments utilizing the expression of wild-type and mutant XLF cDNAs demonstrated that all of the phenotypes of an XLF deficiency could be rescued by the overexpression of a wild-type XLF cDNA. Unexpectedly, mutant forms of XLF bearing point mutations at amino acid positions L115 and L179, also completely complemented the null phenotype suggesting, in contrast to predictions to the contrary, that these mutations do not abrogate XLF function. Finally, we demonstrate that the absence of XLF causes a small, but significant, increase in homologous recombination, implicating XLF in DSB pathway choice regulation. We conclude that human XLF is a non-essential, but critical, C-NHEJ-repair factor. PMID:24461734

  11. Persistent chromosome aberrations in the somatic cells of A-bomb survivors, Hiroshima and Nagasaki

    International Nuclear Information System (INIS)

    Current status of knowledge on the radiation-induced chromosome aberrations persisting since their induction in 1945 to date in the somatic cells of A-bomb survivors in Hiroshima and Nagasaki is reviewed. Dose-response relationship for chromosome aberration frequencies observed with the use of the old A-bomb dosimetry system (T65D) is also demonstrable based on the new dosimetry system (DS86). Despite the fact that the remarkable decrease in the amount of neutron component relative to the total dose in Hiroshima, there still exist inter-city differences in aberration frequency per unit dose both for kerma and bone marrow dose; the dose-square term is smaller in Hiroshima than in Nagasaki. The differential contribution of neutron radiation may be responsible in some part for the observed difference between Hiroshima and Nagasaki, although proof still remains to be obtained. There is a wide variability of the frequency of cells with chromosome aberrations between survivors within a given dose range. Random errors in the dose estimates assigned to individual survivors seem responsible, to a large extent, for the observed overdispersions in aberration frequencies in both cities. New molecular biology-oriented techniques to differentially stain specific chromosomes using fluorescence in situ hybridization with chromosome-specific composite DNA probes seem extremely promising for future rapid, accurate and extensive screening of reciprocal translocations observed predominantly in A-bomb survivors. Such data may be utilized to establish a better biological dosimetry system, especially for those persons who are irradiated in vivo many years before cytogenetic examinations. (author)

  12. A proteomic perspective on the changes in milk proteins due to high somatic cell count.

    Science.gov (United States)

    Zhang, L; Boeren, S; van Hooijdonk, A C M; Vervoort, J M; Hettinga, K A

    2015-08-01

    Although cows with subclinical mastitis have no difference in the appearance of their milk, milk composition and milk quality are altered because of the inflammation. To know the changes in milk quality with different somatic cell count (SCC) levels, 5 pooled bovine milk samples with SCC from 10(5) to 10(6) cells/mL were analyzed qualitatively and quantitatively using both one-dimension sodium dodecyl sulfate PAGE and filter-aided sample preparation coupled with dimethyl labeling, both followed by liquid chromatography tandem mass spectrometry. Minor differences were found on the qualitative level in the proteome from milk with different SCC levels, whereas the concentration of milk proteins showed remarkable changes. Not only immune-related proteins (cathelicidins, IGK protein, CD59 molecule, complement regulatory protein, lactadherin), but also proteins with other biological functions (e.g., lipid metabolism: platelet glycoprotein 4, butyrophilin subfamily 1 member A1, perilipin-2) were significantly different in milk from cows with high SCC level compared with low SCC level. The increased concentration of protease inhibitors in the milk with higher SCC levels may suggest a protective role in the mammary gland against protease activity. Prostaglandin-H2 D-isomerase showed a linear relation with SCC, which was confirmed with an ELISA. However, the correlation coefficient was lower in individual cows compared with bulk milk. These results indicate that prostaglandin-H2 D-isomerase may be used as an indicator to evaluate bulk milk quality and thereby reduce the economic loss in the dairy industry. The results from this study reflect the biological phenomena occurring during subclinical mastitis and in addition provide a potential indicator for the detection of bulk milk with high SCC. PMID:26094216

  13. Economic consequences of mastitis and withdrawal of milk with high somatic cell count in Swedish dairy herds

    DEFF Research Database (Denmark)

    Nielsen, C; Østergaard, Søren; Emanuelson, U;

    2010-01-01

    The main aim was to assess the impact of mastitis on technical and economic results of a dairy herd under current Swedish farming conditions. The second aim was to investigate the effects obtained by withdrawing milk with high somatic cell count (SCC). A dynamic and stochastic simulation model, Sim...... the predicted bulk tank SCC exceeded 220 000, 200 000 or 180 000 cells/ml, and on cow-level information in three scenarios: withdrawal was initiated when the predicted SCC in an individual cow's milk exceeded 1 000 000, 750 000 or 500 000 cells/ml. The accuracy with which SCC was measured and...

  14. Diethylnitrosamine-induced expression of germline-specific genes and pluripotency factors, including vasa and oct4, in medaka somatic cells.

    Science.gov (United States)

    Shen, Jialing; Yokota, Shinpei; Yokoi, Hayato; Suzuki, Tohru

    2016-09-16

    Various methods have been developed to reprogram mammalian somatic cells into pluripotent cells as well as to directly reprogram somatic cells into other cell lineages. We are interested in applying these methods to fish, and here, we examined whether mRNA expression of germline-specific genes (vasa, nanos2, -3) and pluripotency factors (oct4, sox2, c-myc, nanog) is inducible in somatic cells of Japanese medaka (Oryzias latipes). We found that the expression of vasa is induced in the gut and regenerating fin by exposure to a carcinogen, diethylnitrosamine (DEN). Induction of vasa in the gut started on the 5th day of treatment with >50 ppm DEN. In addition, nanos2, -3, oct4, sox2, klf4, c-myc, and nanog were also expressed simultaneously in some vasa-positive gut and regenerating fin samples. Vasa-positive cells were detected by immunohistochemistry (IHC) in the muscle surrounding the gut and in the wound epidermis, blastema, and fibroblast-like cells in regenerating fin. In vasa:GFP transgenic medaka, green fluorescent protein (GFP) fluorescence appeared in the wound epidermis and fibroblast-like cells in the regenerating fin following DEN exposure, in agreement with the IHC data. Our data show that mRNA expression of genes relevant to germ cell specification and pluripotency can be induced in fish somatic cells by exposure to DEN, suggesting the possibility of efficient and rapid cell reprogramming of fish somatic cells. PMID:27514449

  15. Testicular germ cell tumours in dogs are predominantly of spermatocytic seminoma type and are frequently associated with somatic cell tumours

    DEFF Research Database (Denmark)

    Bush, J M; Gardiner, D W; Palmer, J S; Rajpert-De Meyts, E; Veeramachaneni, D N R

    Unlike seminomas in humans, seminomas in animals are not typically sub-classified as classical or spermatocytic types. To compare testicular germ cell tumours (TGCT) in dogs with those of men, archived tissues from 347 cases of canine testicular tumours were morphologically evaluated and characte......Unlike seminomas in humans, seminomas in animals are not typically sub-classified as classical or spermatocytic types. To compare testicular germ cell tumours (TGCT) in dogs with those of men, archived tissues from 347 cases of canine testicular tumours were morphologically evaluated and...... characterized using human classification criteria. Histopathological and immunohistological analysis of PLAP, KIT, DAZ and DMRT1 expression revealed that canine seminomas closely resemble human spermatocytic seminomas. In addition, a relatively frequent concomitant presence of somatic cell tumours was noted in...... canine TGCT. None of the canine TGCT evaluated demonstrated the presence of carcinoma in situ cells, a standard feature of human classical seminomas, suggesting that classical seminomas either do not occur in dogs or are rare in occurrence. Canine spermatocytic seminomas may provide a useful model for...

  16. Cells with Stem Cell Characteristics in Somatic Compartments of the Ovary

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    Katarzyna Kossowska-Tomaszczuk

    2013-01-01

    Full Text Available Antral follicular growth in the ovary is characterized by rapid expansion of granulosa cells accompanied by a rising complexity of their functionality. Within two weeks the number of human granulosa cells increases from less than 500,000 to more than 50 millions cells per follicle and differentiates into groups of cells with a variety of specialized functions involved in steroidogenesis, nursing the oocyte, and forming a functional syncitium. Both the rapid proliferation and different specialized functions of the granulosa cells can only be explained through the involvement of stem cells. However, luteinizing granulosa cells were believed to be terminally differentiated cells. Only recently, stem and progenitor cells with FSH-receptor activity were identified in populations of luteinizing granulosa cells obtained during oocyte collected for assisted reproduction. In the presence of the leukaemia-inhibiting factor (LIF, it was possible to culture a subpopulation of the luteinizing granulosa cells over prolonged time periods. Furthermore, when embedded in a matrix consisting of collagen type I, these cells continued to express the FSH receptor over prolonged time periods, developed globular formations that surrogated as follicle-like structures, providing a promising tool for reproductive biology.

  17. A Study of the Somatic Cell Count of Kosovo Bulk Milk Farm Management and Perspective

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    HYSEN BYTYQI

    2014-06-01

    Full Text Available The aim of this study was to determine the effects of the somatic cell count (SCC in bulk milk farm management and its commercial perspective according to the milk quality standards in Kosovo. A 2069 raw bulk milk samples were taken from a milk collection points in four regions of Kosovo, with two months visits throughout a year. All samples were analyzed by using “FossomaticMinor” equipment, while for the results obtained and identification of different variables effect of SCC on raw bulk milk a general linear model was used. The effect of all variables was considered as a fixed. The overall results show that herd, region, and month of the year (P smaller than 0.0001, respectively, had a significant effect on the presence of SCC. Based on the country existing milk standards for raw milk, the results gained show about 29.6 % belong to extra class milk (SCC/mL less than 300.000, followed by milk quality class IIId, Ist and IId, 24.3%, 8.5%, 8.2%. Of concern is the fact that about 29.5% of total bulk milk analyzed tend to be out of milk quality standards, poor quality ((SCC/mL more than 600.000. The overall mean of SCC on milk was high 772.475 per mL milk, indicating negative farm profit correlation, poor animal health and food safety. The result obtained can be used for assessing raw milk quality and controlling herd management programs.

  18. Genetic analysis for mastitis resistance and milk somatic cell score in French Lacaune dairy sheep

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    Astruc Jean-Michel

    2001-07-01

    Full Text Available Abstract Genetic analysis for mastitis resistance was studied from two data sets. Firstly, risk factors for different mastitis traits, i.e. culling due to clinical or chronic mastitis and subclinical mastitis predicted from somatic cell count (SCC, were explored using data from 957 first lactation Lacaune ewes of an experimental INRA flock composed of two divergent lines for milk yield. Secondly, genetic parameters for SCC were estimated from 5 272 first lactation Lacaune ewes recorded among 38 flocks, using an animal model. In the experimental flock, the frequency of culling due to clinical mastitis (5% was lower than that of subclinical mastitis (10% predicted from SCC. Predicted subclinical mastitis was unfavourably associated with the milk yield level. Such an antagonism was not detected for clinical mastitis, which could result, to some extent, from its low frequency or from the limited amount of data. In practice, however, selection for mastitis resistance could be limited in a first approach to selection against subclinical mastitis using SCC. The heritability estimate of SCC was 0.15 for the lactation mean trait and varied from 0.04 to 0.12 from the first to the fifth test-day. The genetic correlation between lactation SCC and milk yield was slightly positive (0.15 but showed a strong evolution during lactation, i.e. from favourable (-0.48 to antagonistic (0.27. On a lactation basis, our results suggest that selection for mastitis resistance based on SCC is feasible. Patterns for genetic parameters within first lactation, however, require further confirmation and investigation.

  19. Heritability estimates associated with alternative definitions of mastitis and correlations with somatic cell score and yield.

    Science.gov (United States)

    Vallimont, J E; Dechow, C D; Sattler, C G; Clay, J S

    2009-07-01

    The objectives of this study were to compare alternative mastitis definitions and to estimate genetic correlations of producer-recorded mastitis with somatic cell score (SCS) and yield. Cow health events and lactation records from June 2002 through October 2007 were provided by Dairy Records Management Systems (Raleigh, NC). First- through fifth-lactation records from cows calving between 20 and 120 mo of age and that calved in a herd-year with at least 1% of cows with a clinical mastitis event were retained. The edited data contained 118,516 lactation records and 1,072,741 test-day records of 64,893 cows. Mastitis occurrence (1 = at least one mastitis event during lactation or test-day interval, 0 = no mastitis events), number of mastitis events during lactation, SCS, and yield were analyzed with animal models (single trait) or sire-maternal grandsire models (multiple trait) in ASREML. Comparisons were made among models assuming a normal distribution, a binary distribution, or Poisson distribution (for total episodes). The overall incidence of clinical mastitis was 15.4%; and heritability estimates ranged from 0.73% (test-day interval mastitis with a linear model) to 11.07% (number of mastitis episodes with a Poisson model). Increased mastitis incidence was genetically correlated with higher SCS (range 0.66 to 0.88) and was generally correlated with higher yield (range -0.03 to 0.40), particularly during first lactation (0.04 to 0.40). Significant genetic variation exists for clinical mastitis; and health events recorded by producers could be used to generate genetic evaluations for cow health. Sires ranked similarly for daughter mastitis susceptibility regardless of how mastitis was defined; however, test-day interval mastitis and a total count of mastitis episodes per lactation allow a higher proportion of mastitis treatments to be included in the genetic analysis. PMID:19528618

  20. Longitudinal study of reproductive performance of female cattle produced by somatic cell nuclear transfer.

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    Irina A Polejaeva

    Full Text Available The objective of this study was to determine whether or not reproductive performance in cattle produced by somatic cell nuclear transfer (SCNT is significantly different from that of their genetic donors. To address this question, we directed two longitudinal studies using different embryo production procedures: (1 superovulation followed by artificial insemination (AI and embryo collection and (2 ultrasound-guided ovum pick-up followed by in vitro fertilization (OPU-IVF. Collectively, these two studies represent the largest data set available for any species on the reproductive performance of female clones and their genetic donors as measured by their embryo production outcomes in commercial embryo production program. The large-scale study described herein was conducted over a six-year period of time and provides a unique comparison of 96 clones to the 40 corresponding genetic donors. To our knowledge, this is the first longitudinal study on the reproductive performance of cattle clones using OPU-IVF. With nearly 2,000 reproductive procedures performed and more than 9,200 transferable embryos produced, our observations show that the reproductive performance of cattle produced by SCNT is not different compared to their genetic donors for the production of transferable embryos after either AI followed by embryo collection (P = 0.77 or OPU-IVF (P = 0.97. These data are in agreement with previous reports showing that the reproductive capabilities of cloned cattle are equal to that of conventionally produced cattle. In conclusion, results of this longitudinal study once again demonstrate that cloning technology, in combination with superovulation, AI and embryo collection or OPU-IVF, provides a valuable tool for faster dissemination of superior maternal genetics.

  1. Somatic-cell mutation induced by short exposures to cigarette smoke in urate-null, oxidative stress-sensitive Drosophila.

    Science.gov (United States)

    Uchiyama, Tomoyo; Koike, Ryota; Yuma, Yoko; Okamoto, Keinosuke; Arimoto-Kobayashi, Sakae; Suzuki, Toshinori; Negishi, Tomoe

    2016-01-01

    We previously reported that a urate-null strain of Drosophila is hypersensitive to cigarette smoke (CS), and we suggested that CS induces oxidative stress in Drosophila because uric acid is a potent antioxidant. Although the carcinogenic risk of CS exposure is widely recognized; documentation of in vivo genotoxic activity of environmental CS, especially gaseous-phase CS, remains inconclusive. To date, somatic-cell mutations in Drosophila resulting from exposure to CS have not been detected via the somatic mutation and recombination test (wing spot test) with wild-type flies, a widely used Drosophila assay for the detection of somatic-cell mutation; moreover, genotoxicity has not been documented via a DNA repair test that involves DNA repair-deficient Drosophila. In this study, we used a new Drosophila strain (y v ma-l; mwh) to examine the mutagenicity induced by gaseous-phase CS; these flies are urate-null due to a mutation in ma-l, and they are heterozygous for multiple wing hair (mwh), a mutation that functions as a marker for somatic-cell mutation. In an assay with this newly developed strain, a superoxide anion-producing weed-killer, paraquat, exhibited significant mutagenicity; in contrast, paraquat was hardly mutagenic with a wild-type strain. Drosophila larvae were exposed to CS for 2, 4 or 6h, and then kept at 25°C on instant medium until adulthood. After eclosion, mutant spots, which consisted of mutant hairs on wings, were scored. The number of mutant spots increased significantly in an exposure time-dependent manner in the urate-null females (ma-l (-/-)), but not in the urate-positive females (ma-l (+/-)). In this study, we showed that short-term exposure to CS was mutagenic in this in vivo system. In addition, we obtained suggestive data regarding reactive oxygen species production in larva after CS exposure using the fluorescence probe H2DCFDA. These results suggest that oxidative damage, which might be countered by uric acid, was partly responsible

  2. Nuclear transfer of synchronized African wild cat somatic cells into enucleated domestic cat oocytes

    Science.gov (United States)

    Gomez, M.C.; Jenkins, J.A.; Giraldo, A.; Harris, R.F.; King, A.; Dresser, B.L.; Pope, C.E.

    2003-01-01

    The African wild cat is one of the smallest wild cats and its future is threatened by hybridization with domestic cats. Nuclear transfer, a valuable tool for retaining genetic variability, offers the possibility of species continuation rather than extinction. The aim of this study was to investigate the ability of somatic cell nuclei of the African wild cat (AWC) to dedifferentiate within domestic cat (DSH) cytoplasts and to support early development after nuclear transplantation. In experiment 1, distributions of AWC and DSH fibroblasts in each cell-cycle phase were assessed by flow cytometry using cells cultured to confluency and disaggregated with pronase, trypsin, or mechanical separation. Trypsin (89.0%) and pronase (93.0%) yielded higher proportions of AWC nuclei in the G0/G1 phase than mechanical separation (82.0%). In contrast, mechanical separation yielded higher percentages of DSH nuclei in the G0/G1 phase (86.6%) than pronase (79.7%) or trypsin (74.2%) treatments. In both species, pronase induced less DNA damage than trypsin. In experiment 2, the effects of serum starvation, culture to confluency, and exposure to roscovitine on the distribution of AWC and DSH fibroblasts in various phases of the cell cycle were determined. Flow cytometry analyses revealed that the dynamics of the cell cycle varied as culture conditions were modified. Specifically, a higher percentage of AWC and DSH nuclei were in the G0/G1 phase after cells were serum starved (83% vs. 96%) than were present in cycling cells (50% vs. 64%), after contact inhibition (61% vs. 88%), or after roscovitine (56% vs. 84%) treatment, respectively. In experiment 3, we evaluated the effects of cell synchronization and oocyte maturation (in vivo vs. in vitro) on the reconstruction and development of AWC-DSH- and DSH-DSH-cloned embryos. The method of cell synchronization did not affect the fusion and cleavage rate because only a slightly higher percentage of fused couplets cleaved when donor nuclei

  3. Ultrastructural comparison of porcine putative embryonic stem cells derived by in vitro fertilization and somatic cell nuclear transfer

    Science.gov (United States)

    YOO, Hyunju; KIM, Eunhye; HWANG, Seon-Ung; YOON, Junchul David; JEON, Yubyeol; PARK, Kyu-Mi; KIM, Kyu-Jun; JIN, Minghui; LEE, Chang-Kyu; LEE, Eunsong; KIM, Hyunggee; KIM, Gonhyung; HYUN, Sang-Hwan

    2016-01-01

    The ultrastructure of porcine putative embryonic stem cells and porcine fetal fibroblasts (PFFs) was analyzed by transmission electron microscopy. The aim of this study was to compare the features of organelles in in vitro fertilization (IVF) derived porcine embryonic stem cells (IVF-pESCs) and somatic cell nuclear transfer (SCNT) derived pESCs (SCNT-pESCs). Also, the features of organelles in high-passage IVF-pESCs were compared with those in low-passage cells. The ultrastructure of PFFs showed rare microvilli on the cell surfaces, polygonal or irregular nuclei with one to two reticular-shaped nucleoli and euchromatin, low cytoplasm-to-nucleus ratios, rare ribosomes, rare rough endoplasmic reticulum, elongated mitochondria, rich lysosomes and rich phagocytic vacuoles. IVF-pESCs showed rare microvilli on the cell surfaces, round or irregular nuclei with one to two reticular-shaped nucleoli and euchromatin, low cytoplasm-to-nucleus ratios, rich ribosomes, long stacks of rough endoplasmic reticulum, elongated mitochondria, rare lysosomes and rare autophagic vacuoles. By contrast, SCNT-pESCs showed rich microvilli with various lengths and frequencies on the cell surfaces, polygonal nuclei with one reticular shaped nucleoli and heterochromatin, high cytoplasm-to-nucleus ratios, rare ribosomes, rare rough endoplasmic reticulum, round mitochondria, rich lysosomes and rich phagocytic vacuoles with clear intercellular junctions. Furthermore, high-passage IVF-pESCs showed irregularly shaped colonies, pyknosis and numerous lysosomes associated with autophagic vacuoles showing signs of apoptosis. In conclusion, this study confirms that the ultrastructural characteristics of pESCs differ depending on their origin. These ultrastructural characteristics might be useful in biomedical research using pESCs, leading to new insights regarding regenerative medicine and tissue repair. PMID:26821870

  4. Rapid and Efficient Direct Conversion of Human Adult Somatic Cells into Neural Stem Cells by HMGA2/let-7b

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    Kyung-Rok Yu

    2015-01-01

    Full Text Available A recent study has suggested that fibroblasts can be converted into mouse-induced neural stem cells (miNSCs through the expression of defined factors. However, successful generation of human iNSCs (hiNSCs has proven challenging to achieve. Here, using microRNA (miRNA expression profile analyses, we showed that let-7 microRNA has critical roles for the formation of PAX6/NESTIN-positive colonies from human adult fibroblasts and the proliferation and self-renewal of hiNSCs. HMGA2, a let-7-targeting gene, enables induction of hiNSCs that displayed morphological/molecular features and in vitro/in vivo differentiation potential similar to H9-derived NSCs. Interestingly, HMGA2 facilitated the efficient conversion of senescent somatic cells or blood CD34+ cells into hiNSCs through an interaction with SOX2, whereas other combinations or SOX2 alone showed a limited conversion ability. Taken together, these findings suggest that HMGA2/let-7 facilitates direct reprogramming toward hiNSCs in minimal conditions and maintains hiNSC self-renewal, providing a strategy for the clinical treatment of neurological diseases.

  5. Live imaging of Drosophila gonad formation reveals roles for Six4 in regulating germline and somatic cell migration

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    Jarman Andrew P

    2007-05-01

    Full Text Available Abstract Background Movement of cells, either as amoeboid individuals or in organised groups, is a key feature of organ formation. Both modes of migration occur during Drosophila embryonic gonad development, which therefore provides a paradigm for understanding the contribution of these processes to organ morphogenesis. Gonads of Drosophila are formed from three distinct cell types: primordial germ cells (PGCs, somatic gonadal precursors (SGPs, and in males, male-specific somatic gonadal precursors (msSGPs. These originate in distinct locations and migrate to associate in two intermingled clusters which then compact to form the spherical primitive gonads. PGC movements are well studied, but much less is known of the migratory events and other interactions undergone by their somatic partners. These appear to move in organised groups like, for example, lateral line cells in zebra fish or Drosophila ovarian border cells. Results We have used time-lapse fluorescence imaging to characterise gonadal cell behaviour in wild type and mutant embryos. We show that the homeodomain transcription factor Six4 is required for the migration of the PGCs and the msSGPs towards the SGPs. We have identified a likely cause of this in the case of PGCs as we have found that Six4 is required for expression of Hmgcr which codes for HMGCoA reductase and is necessary for attraction of PGCs by SGPs. Six4 affects msSGP migration by a different pathway as these move normally in Hmgcr mutant embryos. Additionally, embryos lacking fully functional Six4 show a novel phenotype in which the SGPs, which originate in distinct clusters, fail to coalesce to form unified gonads. Conclusion Our work establishes the Drosophila gonad as a model system for the analysis of coordinated cell migrations and morphogenesis using live imaging and demonstrates that Six4 is a key regulator of somatic cell function during gonadogenesis. Our data suggest that the initial association of SGP clusters

  6. Development capacity of pre- and postpubertal pig oocytes evaluated by somatic cell nuclear transfer and parthenogenetic activation

    DEFF Research Database (Denmark)

    Skovsgaard, Hanne; Li, Rong; Liu, Ying;

    2013-01-01

    Most of the porcine oocytes used for in vitro studies are collected from gilts. Our aims were to study development capacity of gilt v. sow oocytes (pre- and postpubertal respectively) using 2 techniques illustrating development competence [parthenogenetic activation (PA) and somatic cell nuclear...... transfer (SCNT)], and to describe a simple method to select the most competent oocytes. Inside-ZP diameter of in vitro-matured gilt oocytes was measured (µm; small ≤110; medium >110; large ≥120). Gilt and sow oocytes were morphologically grouped as good (even cytoplasm, smooth cell membrane, visible...

  7. Somatically Hypermutated Plasmodium-Specific IgM(+) Memory B Cells Are Rapid, Plastic, Early Responders upon Malaria Rechallenge.

    Science.gov (United States)

    Krishnamurty, Akshay T; Thouvenel, Christopher D; Portugal, Silvia; Keitany, Gladys J; Kim, Karen S; Holder, Anthony; Crompton, Peter D; Rawlings, David J; Pepper, Marion

    2016-08-16

    Humoral immunity consists of pre-existing antibodies expressed by long-lived plasma cells and rapidly reactive memory B cells (MBC). Recent studies of MBC development and function after protein immunization have uncovered significant MBC heterogeneity. To clarify functional roles for distinct MBC subsets during malaria infection, we generated tetramers that identify Plasmodium-specific MBCs in both humans and mice. Long-lived murine Plasmodium-specific MBCs consisted of three populations: somatically hypermutated immunoglobulin M(+) (IgM(+)) and IgG(+) MBC subsets and an unmutated IgD(+) MBC population. Rechallenge experiments revealed that high affinity, somatically hypermutated Plasmodium-specific IgM(+) MBCs proliferated and gave rise to antibody-secreting cells that dominated the early secondary response to parasite rechallenge. IgM(+) MBCs also gave rise to T cell-dependent IgM(+) and IgG(+)B220(+)CD138(+) plasmablasts or T cell-independent B220(-)CD138(+) IgM(+) plasma cells. Thus, even in competition with IgG(+) MBCs, IgM(+) MBCs are rapid, plastic, early responders to a secondary Plasmodium rechallenge and should be targeted by vaccine strategies. PMID:27473412

  8. Whole-exome sequencing identifies a somatic missense mutation of NBN in clear cell sarcoma of the salivary gland.

    Science.gov (United States)

    Zhang, Lei; Jia, Zhen; Mao, Fengbiao; Shi, Yueyi; Bu, Rong Fa; Zhang, Baorong

    2016-06-01

    Clear cell sarcoma (CCS) is a rare, low-grade carcinoma commonly located in the distal extremities of young adults involving tendons and aponeuroses. CCS is characterized by its poor prognosis due to late diagnosis, multiple local recurrence, propensity to late metastases, and a high rate of tumor-related mortality. The genetic cause for CCS is thought to be EWSR1 gene translocation. However, CCS lacking a translocation may have other, as yet uncharacterized, genetic mutations that can cause the same pathological effect. A combination of whole‑exome sequencing and Sanger sequencing of cancer tissue and venous blood from a patient diagnosed with CCS of the salivary gland revealed a somatic missense mutation, c.1061C>T (p.P354L), in exon 9 of the Nibrin gene (NBN). This somatic missense mutation led to the conversion of proline to leucine (p.P354L), resulting in deleterious effects for the NBN protein. Multiple-sequence alignments showed that codon 354, where the mutation (c.1061C>T) occurs, is located within a phylogenetically conserved region. In conclusion, we here report a somatic missense mutation c.1061C>T (p.P354L) in the NBN gene in a patient with CCS lacking an EWSR1-ATF1 fusion. Our findings broaden the genotypic spectrum of CCS and provide new molecular insight that should prove useful in the future clinical genetic diagnosis of CCS. PMID:27109316

  9. Activation of ribosomal RNA genes in porcine embryos produced in vitro or by somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Bjerregaard, Bolette; Pedersen, Hanne Gervi; Jakobsen, Anne Sørig;

    2007-01-01

    The onset of ribosomal RNA (rRNA) synthesis occurs during the second half of the third cell cycle, that is, at the four-cell stage, in porcine embryos developed in vivo. In the present study the onset of rRNA synthesis was investigated in porcine embryos produced in vitro (IVP) or by somatic cell...... were equal proportions of transcriptionally active and inactive embryos and essentially all embryos that developed to the 16-cell stage (n = 21) and further to the blastocyst stage (n = 19) contained only transcriptionally active cells. In conclusion, porcine embryos produced in vitro had an......-cell stage (n = 45), 38% of the embryos contained 1-3 nuclei with signs of rRNA transcription, indicating an asynchronous transcription initiation. This pattern continued in the following stages, as 78% (n = 47), 47% (n = 42) and 83% (n = 37) of the embryos revealed a mixture of transcriptionally inactive...

  10. Effects of injectable trace mineral supplementation in lactating dairy cows with elevated somatic cell counts.

    Science.gov (United States)

    Ganda, E K; Bisinotto, R S; Vasquez, A K; Teixeira, A G V; Machado, V S; Foditsch, C; Bicalho, M; Lima, F S; Stephens, L; Gomes, M S; Dias, J M; Bicalho, R C

    2016-09-01

    Objectives of this clinical trial were to evaluate the effects of injectable trace mineral supplementation (ITMS) on somatic cell count (SCC), linear score (LS), milk yield, milk fat and protein contents, subclinical mastitis cure, and incidence of clinical mastitis in cows with elevated SCC. Holstein cows from a commercial dairy farm in New York were evaluated for subclinical mastitis, defined as SCC ≥200×10(3) cells/mL on the test day preceding enrollment. Cows with a history of treatment for clinical mastitis in the current lactation and those pregnant for more than 150d were not eligible for enrollment. Cows fitting inclusion criteria were randomly allocated to 1 of 2 treatment groups. Cows assigned to ITMS (n=306) received 1 subcutaneous injection containing zinc (300mg), manganese (50mg), selenium (25mg), and copper (75mg) at enrollment (d 0). Control cows (CTRL; n=314) received 1 subcutaneous injection of sterile saline solution. Following treatment, visual assessment of milk was performed daily, and cows with abnormal milk (i.e., presence of flakes, clots, or serous milk) were diagnosed with clinical mastitis (CM). Chronic clinical mastitis was defined as cows with 3 or more cases of CM. Milk yield, milk fat and protein contents, SCC, and LS were evaluated once monthly. Additionally, randomly selected animals were sampled to test serum concentrations of selected minerals on d0 and 30 (n=30 cows/treatment). Treatment did not affect serum concentrations of calcium, magnesium, phosphorus, potassium, copper, iron, manganese, selenium, and zinc on d30. Injectable supplementation with trace minerals did not improve overall cure of subclinical mastitis (CTRL=42.8 vs. ITMS=46.5%), although a tendency was observed in cows with 3 or more lactations (CTRL=27.1 vs. ITMS=40.0%). Supplementation did not reduce treatment incidence of CM (CTRL=48.2 vs. ITMS=41.7%); however, it tended to reduce the proportion of cows diagnosed with chronic CM (CTRL=16.9 vs. ITMS=12

  11. Comparison of the efficiency of Banna miniature inbred pig somatic cell nuclear transfer among different donor cells.

    Directory of Open Access Journals (Sweden)

    Hongjiang Wei

    Full Text Available Somatic cell nuclear transfer (SCNT is an important method of breeding quality varieties, expanding groups, and preserving endangered species. However, the viability of SCNT embryos is poor, and the cloned rate of animal production is low in pig. This study aims to investigate the gene function and establish a disease model of Banna miniature inbred pig. SCNT with donor cells derived from fetal, newborn, and adult fibroblasts was performed, and the cloning efficiencies among the donor cells were compared. The results showed that the cleavage and blastocyst formation rates did not significantly differ between the reconstructed embryos derived from the fetal (74.3% and 27.4% and newborn (76.4% and 21.8% fibroblasts of the Banna miniature inbred pig (P>0.05. However, both fetal and newborn fibroblast groups showed significantly higher rates than the adult fibroblast group (61.9% and 13.0%; P<0.05. The pregnancy rates of the recipients in the fetal and newborn fibroblast groups (60% and 80%, respectively were higher than those in the adult fibroblast group. Eight, three, and one cloned piglet were obtained from reconstructed embryos of the fetal, newborn, and adult fibroblasts, respectively. Microsatellite analyses results indicated that the genotypes of all cloning piglets were identical to their donor cells and that the genetic homozygosity of the Banna miniature inbred pig was higher than those of the recipients. Therefore, the offspring was successfully cloned using the fetal, newborn, and adult fibroblasts of Banna miniature inbred pig as donor cells.

  12. A protocol for embryonic stem cell derivation by somatic cell nuclear transfer into human oocytes

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Dieter Egli & Gloryn Chia ### Abstract Here we describe detailed methods that allowed us to derive embryonic stem cell lines by nuclear transfer of fibroblasts from a newborn and from a type 1 diabetic adult. The protocol is based on the insight that 1) agents for cell fusion can act as potent mediators of oocyte activation by compromising maintaining plasma membrane integrity; minimizing the concentration at which they are used, and at least transiently remove calcium f...

  13. Effect of Somatic Cell Types and Culture Medium on in vitro Maturation, Fertilization and Early Development Capability of Buffalo Oocytes

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    H. Jamil*, H. A. Samad, N. Rehman, Z. I. Qureshi and L. A. Lodhi

    2011-04-01

    Full Text Available This study was designed to evaluate the efficacy of different somatic cell types and media in supporting in vitro maturation (IVM, in vitro fertilization (IVF and early embryonic development competence of buffalo follicular oocytes. Cumulus oocyte complexes were collected for maturation from follicles (>6mm of buffalo ovaries collected at the local abattoir. Oocytes were co-cultured in tissue culture medium (TCM-199 with either granulosa cells, cumulus cells, or buffalo oviductal epithelial cells (BOEC @ 3x106 cells/ml or in TCM-199 without helper cells (control at 39°C and 5%CO2 in humidified air. Fresh semen was prepared in modified Ca++ free Tyrode medium. Fertilization was carried out in four types of media: i Tyrode lactate albumin pyruvate (TALP, ii TALP+BOEC, iii modified Ca++ free Tyrode and iv modified Ca++ free Tyrode+BOEC. Fertilized oocytes were cultured for early embryonic development in TCM-199 with and without BOEC. Higher maturation rates were observed in the granulosa (84.24% and cumulus cells (83.44% than BOEC co culture system (73.37%. Highest fertilization rate was obtained in modified Ca++ free Tyrode with BOEC co culture (70.42%, followed by modified Ca++ free Tyrode alone (63.77%, TALP with BOEC (36.92% and TALP alone (10.94%. Development of early embryos (8-cell stage improved in TCM-199 with BOEC co culture than TCM-199 alone. From the results of this study, it can be concluded that addition of somatic cells (granulosa cells, cumulus cells results in higher maturation rates of buffalo follicular oocytes than BOEC co culture system, while fertilization rate improved in modified Ca++ free Tyrode with and without BOEC. Addition of BOEC to TCM-199 improved the developmental capacity of early embryo.

  14. Effects of Scriptaid on Cell Cycle and Histone Acetylation of Ovine Nuclear Donor Cumulus Cells and their Ability to Support the Development of Somatic Cell Nuclear Transfer Embryos

    Directory of Open Access Journals (Sweden)

    Hui Cao

    2015-10-01

    Full Text Available Compelling evidence suggests that histone deacetylase inhibitor (HDACi influences the development of somatic cell nuclear transfer (SCNT embryos. The current study was conducted to determine the effect of pretreatment of donor cumulus cells with Scriptaid (a novel HDACi on cell cycle, histone acetylation and cloning embryos development in ovine. First, we optimized the efficiency of Scriptaid in a dose (0, 0.1, 0.2, 0.4 and 0.8 μmol/L and time-dependent (0, 12, 24, 36, and 48 h manner on the developmental capacity of these embryos. Then, we quantitatively assessed the alterations of acetylation levels in histone H3 lysine 9 (acH3K9 and histone H4 lysine 12 (acH4K12 of cumulus cells and SCNT embryos by immunofluorescence staining. Furthermore, we detected the proportion of G0/G1 phase cells in cumulus cells. We found a significantly improved blastocyst development rates of cloning embryos derived from donor cumulus cells pretreated with a mild dose (0.2 μmol/L of Scriptaid for 24 hours (21/86 [24.39%] vs. 11/85 [12.91%]; P<0.05. Meanwhile, the levels of acH3K9 and acH4K12 were also improved significantly in cumulus cells and SCNT embryos (P<0.05. Moreover, more cumulus cells pretreated with Scriptaid were in G0/G1 phase compared with control group (84.22% vs. 75.96%, P<0.05. In conclusion, donor cumulus cells treated with Scriptaid is beneficial to early development of SCNT embryos, ascending acH3K9/ acH4K12 and G0/G1 phase cells proportion of cumulus cell. Scriptaid can be used to improve the efficiency of somatic cell nuclear transfer in ovine.

  15. An integrated inspection of the somatic mutations in a lung squamous cell carcinoma using next-generation sequencing.

    Directory of Open Access Journals (Sweden)

    Lucy F Stead

    Full Text Available Squamous cell carcinoma (SCC of the lung kills over 350,000 people annually worldwide, and is the main lung cancer histotype with no targeted treatments. High-coverage whole-genome sequencing of the other main subtypes, small-cell and adenocarcinoma, gave insights into carcinogenic mechanisms and disease etiology. The genomic complexity within the lung SCC subtype, as revealed by The Cancer Genome Atlas, means this subtype is likely to benefit from a more integrated approach in which the transcriptional consequences of somatic mutations are simultaneously inspected. Here we present such an approach: the integrated analysis of deep sequencing data from both the whole genome and whole transcriptome (coding and non-coding of LUDLU-1, a SCC lung cell line. Our results show that LUDLU-1 lacks the mutational signature that has been previously associated with tobacco exposure in other lung cancer subtypes, and suggests that DNA-repair efficiency is adversely affected; LUDLU-1 contains somatic mutations in TP53 and BRCA2, allelic imbalance in the expression of two cancer-associated BRCA1 germline polymorphisms and reduced transcription of a potentially endogenous PARP2 inhibitor. Functional assays were performed and compared with a control lung cancer cell line. LUDLU-1 did not exhibit radiosensitisation or an increase in sensitivity to PARP inhibitors. However, LUDLU-1 did exhibit small but significant differences with respect to cisplatin sensitivity. Our research shows how integrated analyses of high-throughput data can generate hypotheses to be tested in the lab.

  16. Retroperitoneal teratoma with somatic malignant transformation: A papillary renal cell carcinoma in a testicular germ cell tumour metastasis following platinum-based chemotherapy

    OpenAIRE

    Zeh Nina; Wild Peter J; Bode Peter K; Kristiansen Glen; Moch Holger; Sulser Tullio; Hermanns Thomas

    2013-01-01

    Abstract Background Malignant transformation describes the phenomenon in which a somatic component of a germ cell teratoma undergoes malignant differentiation. A variety of different types of sarcoma and carcinoma, all non-germ cell, have been described as a result of malignant transformation. Case presentation A 33-year-old man presented with a left testicular mass and elevated tumour markers. Staging investigations revealed retroperitoneal lymphadenopathy with obstruction of the left ureter...

  17. Evaluation of the recombination in somatic cells induced by radiation in different stages of Drosophila larval development

    International Nuclear Information System (INIS)

    The mitotic recombination can happen spontaneously and its frequency is very low, however the recombination rate of a cell can be increased by the exposure to agents which cause damage to DNA. This type of agents are knew commonly as recombinogens. The ionizing radiation and a numerous chemical agents can be mentioned (Vogel, 1992). The objective of this work is to determine if the mutation/recombination rate induced by gamma rays varies with the development stage. In order to realize this investigation it was used the mutation and somatic recombination test of Drosophila wing (Graf and col. 1984). The mwh/ mwh and flr3/TM3, Ser stocks were used. (Author)

  18. Body-weight and chromosome aberrations induced by X-rays in somatic cells of Drosophila melanogaster

    International Nuclear Information System (INIS)

    Body-weight has been shown to influence the final expression of genetic damage by X-rays in Drosophila melanogaster. If larvae of Drosophila were raised up to the third instar in media containing different amounts of the same nutrient and in different conditions of crowding a positive correlation was observed between body-weight and frequency of chromosome aberrations induced by a given dose of X-rays in the somatic cells of their nerve ganglia. This effect, present in both sexes, is most plausibly attributed to a different capacity of big and small larvae for repairing radiation damage. (orig.)

  19. The chromosome content and genotype of two wheat cell lines and of their somatic fusion product with oat

    OpenAIRE

    Xiang, Fengning; Wang, Junfeng; Xu, Chunhui; Xia, Guangmin

    2010-01-01

    Somatic hybridization seeks to genetically combine phylogenetically distant parents. An effective system has been established in bread wheat (Triticum aestivum L.) involving protoplasts from a non-totipotent cell line adapted to in vitro culture (T1) in combination with totipotent protoplasts harvested from embryogenic calli (T2). Here, we report the karyotype and genotype of T1 and T2. Line T1 carries nine A (A-genome of wheat), seven B (B-genome of wheat) and eight D (D-genome of wheat) gen...

  20. Somatic Cells Count and Its Genetic Association with Milk Yield in Dairy Cattle Raised under Thai Tropical Environmental Conditions

    OpenAIRE

    Jattawa, D.; Koonawootrittriron, S.; Elzo, M. A.; Suwanasopee, T.

    2012-01-01

    Somatic cells count (SCC), milk yield (MY) and pedigree information of 2,791 first lactation cows that calved between 1990 and 2010 on 259 Thai farms were used to estimate genetic parameters and trends for SCC and its genetic association with MY. The SCC were log-transformed (lnSCC) to make them normally distributed. An average information-restricted maximum likelihood procedure was used to estimate variance components. A bivariate animal model that considered herd-yr-season, calving age, and...

  1. Electrospun poly (3-hydroxybutyrate-co-3-hydroxyvalerate)/hydroxyapatite scaffold with unrestricted somatic stem cells for bone regeneration.

    Science.gov (United States)

    Biazar, Esmaeil; Heidari Keshel, Saeed

    2015-01-01

    The combination of scaffolds and cells can be useful in tissue reconstruction. In this study, nanofibrous poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/nanohydroxyapatite (nano-HAp) scaffolds, filled with unrestricted somatic stem cells (USSCs), were used for healing calvarial bone in rat model. The healing effects of these scaffolds, with and without stem cells, in bone regeneration were investigated by computed tomography (CT) analysis and pathology assays after 28 days of grafting. The results of CT analysis showed that bone regeneration on the scaffolds, and the amounts of regenerated new bone for polymer/nano-HAp scaffold with USSC, was significantly greater than the scaffold without cell and untreated control samples. Therefore, the combination of scaffold especially with USSC could be considered as a useful method for bone regeneration. PMID:25710767

  2. Neuronal generation from somatic stem cells: current knowledge and perspectives on the treatment of acquired and degenerative central nervous system disorders.

    Science.gov (United States)

    Corti, S; Locatelli, F; Strazzer, S; Guglieri, M; Comi, G P

    2003-06-01

    Stem cell transplantation through cell replacement or as vector for gene delivery is a potential strategy for the treatment of neurodegenerative diseases. Several studies have reported the transdifferentiation of different somatic stem cells into neurons in vitro or after transplantation into animal models. This observation has pointed out the perspective of using an ethical and accessible cell source to "replace" damaged neurons or provide support to brain tissue. However, recent findings such as the cell fusion phenomenon have raised some doubts about the real existence of somatic stem cell plasticity. In this review, we will discuss current evidence and controversial issues about the neuroneogenesis from various sources of somatic cells focusing on the techniques of isolation, expansion in vitro as well as the inductive factors that lead to transdifferentiation in order to identify the factors peculiar to this process. The morphological, immunochemical, and physiological criteria to correctly judge whether the neuronal transdifferentation occurred are critically presented. We will also discuss the transplantation experiments that were done in view of a possible clinical therapeutic application. Animal models of stroke, spinal cord and brain trauma have improved with Mesenchymal Stem Cells or Bone Marrow transplantation. This improvement does not seem to depend on the replacement of the lost neurons but may be due to increased expression levels of neurotrophic factors, thus suggesting a beneficial effect of somatic cells regardless of transdifferentiation. Critical understanding of available data on the mechanisms governing the cell fate reprogramming is a necessary achievement toward an effective cell therapy. PMID:12762483

  3. DNA Methylation and Somatic Mutations Converge on the Cell Cycle and Define Similar Evolutionary Histories in Brain Tumors.

    Science.gov (United States)

    Mazor, Tali; Pankov, Aleksandr; Johnson, Brett E; Hong, Chibo; Hamilton, Emily G; Bell, Robert J A; Smirnov, Ivan V; Reis, Gerald F; Phillips, Joanna J; Barnes, Michael J; Idbaih, Ahmed; Alentorn, Agusti; Kloezeman, Jenneke J; Lamfers, Martine L M; Bollen, Andrew W; Taylor, Barry S; Molinaro, Annette M; Olshen, Adam B; Chang, Susan M; Song, Jun S; Costello, Joseph F

    2015-09-14

    The evolutionary history of tumor cell populations can be reconstructed from patterns of genetic alterations. In contrast to stable genetic events, epigenetic states are reversible and sensitive to the microenvironment, prompting the question whether epigenetic information can similarly be used to discover tumor phylogeny. We examined the spatial and temporal dynamics of DNA methylation in a cohort of low-grade gliomas and their patient-matched recurrences. Genes transcriptionally upregulated through promoter hypomethylation during malignant progression to high-grade glioblastoma were enriched in cell cycle function, evolving in parallel with genetic alterations that deregulate the G1/S cell cycle checkpoint. Moreover, phyloepigenetic relationships robustly recapitulated phylogenetic patterns inferred from somatic mutations. These findings highlight widespread co-dependency of genetic and epigenetic events throughout brain tumor evolution. PMID:26373278

  4. Genotoxic effects of bisphenol A on somatic cells of female mice, alone and in combination with X-rays.

    Science.gov (United States)

    Gajowik, Aneta; Radzikowska, Joanna; Dobrzyńska, Małgorzata M

    2013-10-01

    Bisphenol A (BPA), a monomer used in the manufacture of epoxy, polycarbonate, and polystyrene resins, is a xenoestrogen present in many consumer products. We investigated the effects of 2-week exposure to BPA, either alone or in combination with X-rays, on the induction of DNA damage in somatic cells of female mice in vivo. The micronucleus and alkaline comet assays were used to evaluate genotoxicity. BPA induced DNA strand breaks in lung cells but not in bone marrow lymphocytes, liver, kidney, or spleen cells. Induction of micronuclei was observed only in polychromatic reticulocytes of peripheral blood. Levels of damage following combination exposure to ionizing radiation plus BPA depended on tissue, assay, and time. PMID:23954285

  5. Loss of l(3)mbt leads to acquisition of the ping-pong cycle in Drosophila ovarian somatic cells.

    Science.gov (United States)

    Sumiyoshi, Tetsutaro; Sato, Kaoru; Yamamoto, Hitomi; Iwasaki, Yuka W; Siomi, Haruhiko; Siomi, Mikiko C

    2016-07-15

    In Drosophila germ cells, PIWI-interacting RNAs (piRNAs) are amplified through a PIWI slicer-dependent feed-forward loop termed the ping-pong cycle, yielding secondary piRNAs. However, the detailed mechanism remains poorly understood, largely because an ex vivo model system amenable to biochemical analyses has not been available. Here, we show that CRISPR-mediated loss of function of lethal (3) malignant brain tumor [l(3)mbt] leads to ectopic activation of the germ-specific ping-pong cycle in ovarian somatic cells. Perinuclear foci resembling nuage, the ping-pong center, appeared following l(3)mbt mutation. This activation of the ping-pong machinery in cultured cells will greatly facilitate elucidation of the mechanism underlying secondary piRNA biogenesis in Drosophila. PMID:27474440

  6. Analyses of genetic relationships between linear type traits, fat-to-protein ratio, milk production traits, and somatic cell count in first-parity Czech Holstein cows

    DEFF Research Database (Denmark)

    Zink, V; Zavadilová, L; Lassen, Jan;

    2014-01-01

    Genetic and phenotypic correlations between production traits, selected linear type traits, and somatic cell score were estimated. The results could be useful for breeding programs involving Czech Holstein dairy cows or other populations. A series of bivariate analyses was applied whereby (co......)variance components were estimated using average information (AI-REML) implemented via the DMU statistical package. Chosen phenotypic data included average somatic cell score per a 305-day standard first lactation as well as the production traits milk yield, fat yield, protein yield, fat percentage, and protein...... and protein yield. In total, 27 098 somatic cell score records were available. The strongest positive genetic correlation between production traits and linear type traits was estimated between udder width and fat yield (0.51 ± 0.04), while the strongest negative correlation estimated was between body...

  7. Comparative expression profiling of E. coli and S. aureus inoculated primary mammary gland cells sampled from cows with different genetic predispositions for somatic cell score

    OpenAIRE

    Ponsuksili Siriluck; Kühn Christa; Wellnitz Olga; Griesbeck-Zilch Bettina; Repsilber Dirk; Hartmann Anja; Brand Bodo; Meyer Heinrich HD; Schwerin Manfred

    2011-01-01

    Abstract Background During the past ten years many quantitative trait loci (QTL) affecting mastitis incidence and mastitis related traits like somatic cell score (SCS) were identified in cattle. However, little is known about the molecular architecture of QTL affecting mastitis susceptibility and the underlying physiological mechanisms and genes causing mastitis susceptibility. Here, a genome-wide expression analysis was conducted to analyze molecular mechanisms of mastitis susceptibility tha...

  8. Transient acid treatment cannot induce neonatal somatic cells to become pluripotent stem cells [v1; ref status: indexed, http://f1000r.es/3dq

    Directory of Open Access Journals (Sweden)

    Mei Kuen Tang

    2014-05-01

    Full Text Available Currently, there are genetic- and chemical-based methods for producing pluripotent stem cells from somatic cells, but all of them are extremely inefficient.  However, a simple and efficient technique has recently been reported by Obokata et al (2014a, b that creates pluripotent stem cells through acid-based treatment of somatic cells.  These cells were named stimulus-triggered acquisition of pluripotency (STAP stem cells. This would be a major game changer in regenerative medicine if the results could be independently replicated. Hence, we isolated CD45+ splenocytes from five-day-old Oct4-GFP mice and treated the cells with acidified (pH 5.7 Hank’s Balanced Salt Solution (HBSS for 25 min, using the methods described by Obokata et al 2014c. However, we found that this method did not induce the splenocytes to express the stem cell marker Oct4-GFP when observed under a confocal microscope three to six days after acid treatment. qPCR analysis also confirmed that acid treatment did not induce the splenocytes to express the stemness markers Oct4, Sox2 and Nanog.  In addition, we obtained similar results from acid-treated Oct4-GFP lung fibroblasts. In summary, we have not been able to produce STAP stem cells from neonatal splenocytes or lung fibroblasts using the acid-based treatment reported by Obokata et al (2014a, b, c.

  9. A correlative study on the frequencies of radiation-induced chromosome aberrations in somatic and germ cells of mammals

    International Nuclear Information System (INIS)

    A series of investigations on the correlation between the frequencies of radiation-induced chromosome aberrations in somatic and germ cells of mouse and rhesus monkey is described. In the mouse the induction of reciprocal translocations in bone-marrow cells was compared with that in spermatogonia (as scored in the descending spermatocytes). In the rhesus monkey frequencies of radiation-induced chromosome aberrations in spermatogonia and peripheral blood lymphocytes were studied. Furthermore the effect of multigeneration irradiation (69 generations with 200 rads X-rays) on the sensitivity for translocation induction in spermatogonia of male mice was studied. Frequencies of dicentric chromosomes and chromosomal deletions in cultured peripheral blood lymphocytes of 5 different types of mice were determined following in vitro irradiation with doses of 100 and/or 200 rad X-rays. To obtain more insight into the processes underlying translocation induction in spermatogonia of the mouse, fractionation experiments were conducted

  10. Loss of wild-type ATRX expression in somatic cell hybrids segregates with activation of Alternative Lengthening of Telomeres.

    Directory of Open Access Journals (Sweden)

    Kylie Bower

    Full Text Available Alternative Lengthening of Telomeres (ALT is a non-telomerase mechanism of telomere lengthening that occurs in about 10% of cancers overall and is particularly common in astrocytic brain tumors and specific types of sarcomas. Somatic cell hybridization analyses have previously shown that normal telomerase-negative fibroblasts and telomerase-positive immortalized cell lines contain repressors of ALT activity, indicating that activation of ALT results from loss of one or more unidentified repressors. More recently, ATRX or DAXX was shown to be mutated both in tumors with telomere lengths suggestive of ALT activity and in ALT cell lines. Here, an ALT cell line was separately fused to each of four telomerase-positive cell lines, and four or five independent hybrid lines from each fusion were examined for expression of ATRX and DAXX and for telomere lengthening mechanism. The hybrid lines expressed either telomerase or ALT, with the other mechanism being repressed. DAXX was expressed normally in all parental cell lines and in all of the hybrids. ATRX was expressed normally in each of the four telomerase-positive parental cell lines and in every telomerase-positive hybrid line, and was abnormal in the ALT parental cells and in all but one of the ALT hybrids. This correlation between ALT activity and loss of ATRX expression is consistent with ATRX being a repressor of ALT.

  11. The Yb body, a major site for Piwi-associated RNA biogenesis and a gateway for Piwi expression and transport to the nucleus in somatic cells.

    Science.gov (United States)

    Qi, Hongying; Watanabe, Toshiaki; Ku, Hsueh-Yen; Liu, Na; Zhong, Mei; Lin, Haifan

    2011-02-01

    Despite exciting progress in understanding the Piwi-interacting RNA (piRNA) pathway in the germ line, less is known about this pathway in somatic cells. We showed previously that Piwi, a key component of the piRNA pathway in Drosophila, is regulated in somatic cells by Yb, a novel protein containing an RNA helicase-like motif and a Tudor-like domain. Yb is specifically expressed in gonadal somatic cells and regulates piwi in somatic niche cells to control germ line and somatic stem cell self-renewal. However, the molecular basis of the regulation remains elusive. Here, we report that Yb recruits Armitage (Armi), a putative RNA helicase involved in the piRNA pathway, to the Yb body, a cytoplasmic sphere to which Yb is exclusively localized. Moreover, co-immunoprecipitation experiments show that Yb forms a complex with Armi. In Yb mutants, Armi is dispersed throughout the cytoplasm, and Piwi fails to enter the nucleus and is rarely detectable in the cytoplasm. Furthermore, somatic piRNAs are drastically diminished, and soma-expressing transposons are desilenced. These observations indicate a crucial role of Yb and the Yb body in piRNA biogenesis, possibly by regulating the activity of Armi that controls the entry of Piwi into the nucleus for its function. Finally, we discovered putative endo-siRNAs in the flamenco locus and the Yb dependence of their expression. These observations further implicate a role for Yb in transposon silencing via both the piRNA and endo-siRNA pathways. PMID:21106531

  12. Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells

    Directory of Open Access Journals (Sweden)

    Sharma Shikhar

    2012-01-01

    Full Text Available Abstract Background DNA methylation, histone modifications and nucleosome occupancy act in concert for regulation of gene expression patterns in mammalian cells. Recently, G9a, a H3K9 methyltransferase, has been shown to play a role in establishment of DNA methylation at embryonic gene targets in ES cells through recruitment of de novo DNMT3A/3B enzymes. However, whether G9a plays a similar role in maintenance of DNA methylation in somatic cells is still unclear. Results Here we show that G9a is not essential for maintenance of DNA methylation in somatic cells. Knockdown of G9a has no measurable effect on DNA methylation levels at G9a-target loci. DNMT3A/3B remain stably anchored to nucleosomes containing methylated DNA even in the absence of G9a, ensuring faithful propagation of methylated states in cooperation with DNMT1 through somatic divisions. Moreover, G9a also associates with nucleosomes in a DNMT3A/3B and DNA methylation-independent manner. However, G9a knockdown synergizes with pharmacologic inhibition of DNMTs resulting in increased hypomethylation and inhibition of cell proliferation. Conclusions Taken together, these data suggest that G9a is not involved in maintenance of DNA methylation in somatic cells but might play a role in re-initiation of de novo methylation after treatment with hypomethylating drugs, thus serving as a potential target for combinatorial treatments strategies involving DNMTs inhibitors.

  13. Animal models of melanoma: a somatic cell gene delivery mouse model allows rapid evaluation of genesimplicated in human melanoma%Animal models of melanoma: a somatic cell gene delivery mouse model allows rapid evaluation of genes implicated in human melanoma

    Institute of Scientific and Technical Information of China (English)

    Andrea J. McKinney; Sheri L. Holmen

    2011-01-01

    The increasing incidence and mortality associated with advanced stages of melanoma are cause for concern. Few treatment options are available for advanced melanoma and the 5-year survival rate is less than 15%. Targeted therapies may revolutionize melanoma treatment by providing less toxic and more effective strategies. However, maximizing effectiveness requires further understanding of the molecular alterations that drive tumor formation, progression, and maintenance, as well as elucidating the mechanisms of resistance. Several different genetic alterations identified in human melanoma have been recapitulated in mice. This review outlines recent progress made in the development of mouse models of melanoma and summarizes what these findings reveal about the human disease. We begin with a discussion of traditional models and conclude with the recently developed RCAS/TVA somatic cell gene delivery mouse model of melanoma.

  14. Milk Yield Traits, Somatic Cell Score, Milking Time and Age at Calving of Pure Holstein Versus Crossbred Cow

    Directory of Open Access Journals (Sweden)

    Francesca Malchiodi

    2011-09-01

    Full Text Available Pure Holstein (HO, n=430, crosses between Swedish Red and HO (SRxHO, n=41, Montbeliarde and HO (MOxHO, n=18, and MO and SRxHO (MOxSH, n=53 were compared for milk, fat and protein yield, fat and protein percentage, somatic cell count (SCC, milking time (MT, and age at first and second calving. A total of 180,933 test-day information for milk yield and MT, and 5,249 for fat and protein percentage and SCC were recorded on first and second parity cows milked in one herd of Cremona province (northern Italy. Somatic cell count and MT were log-transformed to somatic cells score (SCS and LnMT, respectively, before statistical investigation. Production traits, LnMT and SCS were analyzed through a mixed model that included fixed effects of test-day, parity, days in milk (DIM, genotype and interaction between parity and genotype, and the random effects of cow nested within genotype and residual, whereas the model for age at calving included year and month of calving and genotype as fixed effects, and residual as random. MOxHO and pure HO cows differed only for age at second calving (70 d higher for purebreds; P<0.05. Holsteins produced more milk (+2.86 kg/d; P<0.01 and protein yield (+0.05 kg/d; P<0.05 than SRxHO crossbreds, but lower protein percentage (-0.09%; P<0.01, and age at second calving was 44 d (P<0.01 higher than SRxHO. Also, HO produced more milk and fat than MOxSH cows (+1.61 and +0.08 kg/d, respectively; P<0.05, but lower protein percentage (-0.11%; P<0.001, and calved later, both at first and second calving (+24 and +43 d, respectively; P<0.05. Results indicated that crossbred cows can compete with the cosmopolitan breed for several traits.

  15. Full-term development of gaur-bovine interspecies somatic cell nuclear transfer embryos: effect of trichostatin A treatment.

    Science.gov (United States)

    Srirattana, Kanokwan; Imsoonthornruksa, Sumeth; Laowtammathron, Chuti; Sangmalee, Anawat; Tunwattana, Wanchai; Thongprapai, Thamnoon; Chaimongkol, Chockchai; Ketudat-Cairns, Mariena; Parnpai, Rangsun

    2012-06-01

    Trichostatin A (TSA) has previously been used in somatic cell nuclear transfer (SCNT) to improve the cloning efficiency in several species, which led our team to investigate the effects of TSA on the full-term development of bovine SCNT and gaur-bovine interspecies SCNT (gaur iSCNT; gaur somatic cells as donors and bovine oocytes as recipients) embryos. Treatment with 50 nM TSA for 10 h after fusion had no positive effects on the rates of fusion, cleavage, or the development to eight-cell or morula stages in both bovine SCNT and gaur iSCNT embryos. However, TSA treatment significantly enhanced the blastocyst formation rate in bovine SCNT embryos (44 vs. 32-34% in the TSA-treated and TSA-untreated groups, respectively), but had no effects on gaur iSCNT embryos. The fresh blastocysts derived from bovine SCNT and gaur iSCNT embryos (fresh groups), as well as vitrified bovine SCNT blastocysts (vitrified group), were transferred to bovine recipients. We found that TSA treatment increased the pregnancy rates only in recipients receiving fresh bovine SCNT embryos. In recipients receiving TSA-treated bovine SCNT embryos, three cloned calves from the fresh group and twin cloned calves from the vitrified group were delivered; however, no calf was born from the TSA-untreated bovine SCNT embryos. In contrast, one gaur iSCNT calf was born from a recipient receiving blastocysts from the TSA-untreated group. In summary, TSA improved the preimplantation development and pregnancy rates of bovine SCNT embryos, but did not have any beneficial effect on gaur iSCNT embryos. However, one gaur iSCNT calf reached full-term development. PMID:22578161

  16. Identification of murine B cell lines that undergo somatic hypermutation focused to A:T and G:C residues

    Science.gov (United States)

    Bhattacharya, Palash; Grigera, Fernando; Rogozin, Igor B.; McCarty, Thomas; Morse, Herbert C.; Kenter, Amy L.

    2016-01-01

    Activation-induced deaminase (AID) is the master regulator of class switch recombination (CSR) and somatic hypermutation (SHM), but the mechanisms regulating AID function are obscure. The differential pattern of switch plasmid activity in three IgM+/AID+ and two IgG+/AID+ B cell lines prompted an analysis of global gene expression to discover the origin of these cells. Gene profiling suggested that the IgG+/AID+ B cell lines derived from germinal center B cells. Analysis of SHM potential demonstrates that the IgVκ domains are inducibly diversified at high rate during in vitro culture. The mutation spectra focused to A:T base pairs, revealing a component of the hypermutation program that occurs preferentially during phase 2 of SHM. The A:T error spectra were analyzed and were not characteristic of polymerase η activity. A differential pattern of three consensus motifs used for A:T base substitutions was observed in WT and Polη-, Msh2- and Msh6-deficient B cells. Strikingly, mutations in our B cell lines recapitulated the mutable motif profile for Polη and Msh2 deficiency, respectively, and suggest that an additional pathway for the generation of A:T mutations in SHM is conserved in mouse and human. PMID:18081040

  17. Whole Exome Sequencing Identifies Frequent Somatic Mutations in Cell-Cell Adhesion Genes in Chinese Patients with Lung Squamous Cell Carcinoma.

    Science.gov (United States)

    Li, Chenguang; Gao, Zhibo; Li, Fei; Li, Xiangchun; Sun, Yihua; Wang, Mengyun; Li, Dan; Wang, Rui; Li, Fuming; Fang, Rong; Pan, Yunjian; Luo, Xiaoyang; He, Jing; Zheng, Liangtao; Xia, Jufeng; Qiu, Lixin; He, Jun; Ye, Ting; Zhang, Ruoxin; He, Minghui; Zhu, Meiling; Hu, Haichuan; Shi, Tingyan; Zhou, Xiaoyan; Sun, Menghong; Tian, Shilin; Zhou, Yong; Wang, Qiaoxiu; Chen, Longyun; Yin, Guangliang; Lu, Jingya; Wu, Renhua; Guo, Guangwu; Li, Yingrui; Hu, Xueda; Li, Lin; Asan; Wang, Qin; Yin, Ye; Feng, Qiang; Wang, Bin; Wang, Hang; Wang, Mingbang; Yang, Xiaonan; Zhang, Xiuqing; Yang, Huanming; Jin, Li; Wang, Cun-Yu; Ji, Hongbin; Chen, Haiquan; Wang, Jun; Wei, Qingyi

    2015-01-01

    Lung squamous cell carcinoma (SQCC) accounts for about 30% of all lung cancer cases. Understanding of mutational landscape for this subtype of lung cancer in Chinese patients is currently limited. We performed whole exome sequencing in samples from 100 patients with lung SQCCs to search for somatic mutations and the subsequent target capture sequencing in another 98 samples for validation. We identified 20 significantly mutated genes, including TP53, CDH10, NFE2L2 and PTEN. Pathways with frequently mutated genes included those of cell-cell adhesion/Wnt/Hippo in 76%, oxidative stress response in 21%, and phosphatidylinositol-3-OH kinase in 36% of the tested tumor samples. Mutations of Chromatin regulatory factor genes were identified at a lower frequency. In functional assays, we observed that knockdown of CDH10 promoted cell proliferation, soft-agar colony formation, cell migration and cell invasion, and overexpression of CDH10 inhibited cell proliferation. This mutational landscape of lung SQCC in Chinese patients improves our current understanding of lung carcinogenesis, early diagnosis and personalized therapy. PMID:26503331

  18. Immune competence of the mammary gland as affected by somatic cell and pathogenic bacteria in ewes with subclinical mastitis.

    Science.gov (United States)

    Albenzio, M; Santillo, A; Caroprese, M; Ruggieri, D; Ciliberti, M; Sevi, A

    2012-07-01

    Immune competence of the ewe mammary gland was investigated by monitoring the leukocyte differential count, cytokine pattern, and endogenous proteolytic enzymes in milk samples with different somatic cell counts (SCC) and pathogenic bacteria. Furthermore, the leukocyte differential count and T-lymphocyte populations were evaluated in ewe blood. A total of 1,500 individual milk samples were randomly selected from the pool of the samples collected during sampling and grouped into 5 classes of 300 samples each, on the basis of SCC. Classes were 2,000,000 cells/mL. Microbiological analyses of ewe milk were conducted to detect mastitis-related pathogens. Sheep whose udders were without clinical abnormalities, and whose milk was apparently normal but with at least 10(3)cfu/mL of the same pathogen were considered to have subclinical mastitis and therefore defined as infected. Polymorphonuclear neutrophilic leukocytes (PMNL) and macrophages increased with SCC, whereas lymphocytes decreased. Milk samples with SCC >1,000,000 cells/mL showed differences in leukocyte populations between uninfected and infected ewes, with higher percentages of PMNL and macrophages and lower percentages of lymphocytes in infected animals. Nonviable PMNL levels were the highest in ewe milk samples with SCC 500,000 cells/mL, nonviable PMNL were higher in uninfected ewes than in infected ones. In infected animals giving milk with SCC >1,000,000 cells/mL, a higher CD4(+)/CD8(+) ratio was observed, suggesting that the presence of pathogens induced an activation of both CD4(+) and CD8(+). The levels of tumor necrosis factor-α and IL-12 were higher in infected than uninfected ewes, irrespective of SCC. Plasmin activity increased along with SCC and was always higher in infected than uninfected animals; cathepsin D increased starting from 1,001,000 cells/mL in milk samples from noninfected ewes and starting from 301,000 cells/mL in milk samples from infected animals. The associations between somatic

  19. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    International Nuclear Information System (INIS)

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further

  20. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

    2014-02-21

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

  1. Cytogenetic sequelae in sex and somatic cells at mice subjected to chronic irradiation simulating occupational conditions of radiation effect

    International Nuclear Information System (INIS)

    Cytogenetic effect of chronic irradiation in low doses to study chronic radiation effect upon man under professional conditions has been investigated. The experiments have been carried out on white mice, subjected to chronic effect of 60Co gamma irradiation (during 15 - 19 months in doses of 6, 17 and 50 mrad for 6 - 7 hrs a day). It is shown, that under effect of chronic irradiation, modelling the conditions of professional irradiation effect, in sex and somatic cells of the mice chromosomal aberrations appear, which depend on the age of animals and magnitude of every day dose. However direct dependence of these changes on the magnitude of the total dose has not been established

  2. Genetic Parameters For The Somatic Cells Count In The Milk Of Buffaloes Using Ordinary Test Day Models

    Directory of Open Access Journals (Sweden)

    H. Tonhati

    2010-02-01

    Full Text Available The buffaloes dairy milk production (BDMP has increased in the last 20 years, mainly for the manufacturing of mozzarella cheese, which is recognized by its high nutritional quality. However, this quality can be affected by several factors i. e. high somatic cells count (SCC provokes changes in the milk’s constituents. As in bovine dairy milk, the SCC is used as diagnostic tool for milk quality; because it enables the diagnosis of sub-clinic mastitis and also allows the selection of individuals genetically resistant to that disease. Based on it, we collected information about SCC and BDMP along the lactation in Murrah breed buffaloes, during the period between 1997 and 2005. Curves were designed to estimate genetic parameters. These parameters were estimated by ordinary test-day models. There were observed variations in the estimated heritability for both characteristics .The lowest score for somatic cells count (SSCC was seen at first month (0.01 and the highest at sixth months (0.29 The genetic correlation between these traits varied from -1 at the 1 and 9th months to 0.31 and 0.30 in the2 and 4th month of lactation. Phenotypic correlations were all negative (-0.07 in the second month and up to -0.35 in the eighth month of lactation. These results showed that environmental factors are more important than genetics in explain SCC, for this reason, selection for genetic resistance to mastitis in buffalos based in SCC should not be done. In the other hand, negative phenotypic correlations demonstrated that as the SCC increased, the milk production decreased.

  3. Somatic cell mutations at the glycophorin A locus in erythrocytes of atomic bomb survivors: Implications for radiation carcinogenesis

    International Nuclear Information System (INIS)

    To clarify the relationship between somatic cell mutations and radiation exposure, the frequency of hemizygous mutant erythrocytes at the glycophorin A (GPA) locus was measured by flow cytometry for 1,226 heterozygous atomic bomb (A-bomb) survivors in HIroshima and Nagasaki. For statistical analysis, both GPA mutant frequency and radiation dose were log-transformed to normalize skewed distributions of these variables. The GPA mutant frequency increased slightly but significantly with age at testing and with the number of cigarettes smoked. Also, mutant frequency was significantly higher in males than in females even with adjustment for smoking and was higher to Hiroshima than in Nagasaki. These characteristics of background GPA mutant frequency are qualitatively similar to those of background solid cancer incidence or mortality obtained from previous epidemiological studies of survivors. An analysis of the mutant frequency dose response using a descriptive model showed that the doubling dose is about 1.20 Sv [95% confidence interval (CI): 0.95-1.56], whereas the minimum dose for detecting a significant increase in mutant frequency is about 0.24 Sv (95% CI: 0.041-0.51). No significant effects of sex, city or age at the time of exposure on the dose response were detected. Interestingly, the doubling dose of the GPA mutant frequency was similar to that of solid cancer incidence in A-bomb survivors. This observation is in line with the hypothesis that radiation-induced somatic cell mutations are the major cause of excess cancer risk after radiation. 49 refs., 6 figs., 2 tabs

  4. Generation of porcine fetal fibroblasts expressing the tetracycline-inducible Cas9 gene by somatic cell nuclear transfer.

    Science.gov (United States)

    Liu, Guoqian; Liu, Kai; Wei, Hengxi; Li, Li; Zhang, Shouquan

    2016-09-01

    Cas9 endonuclease, from so-called clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems of Streptococcus pyogenes, type II functions as an RNA-guided endonuclease and edits the genomes of prokaryotic and eukaryotic organisms, including deletion and insertion by DNA double‑stranded break repair mechanisms. In previous studies, it was observed that Cas9, with a genome‑scale lentiviral single‑guide RNA library, could be applied to a loss‑of‑function genetic screen, although the loss‑of‑function genes have yet to be verified in vitro and this approach has not been used in porcine cells. Based on these observations, lentiviral Cas9 was used to infect porcine primary fibroblasts to achieve cell colonies carrying Cas9 endonuclease. Subsequently, porcine fetal fibroblasts expressing the tetracycline‑inducible Cas9 gene were generated by somatic cell nuclear transfer, and three 30 day transgenic porcine fetal fibroblasts (PFFs) were obtained. Polymerase chain reaction (PCR), reverse transcription‑PCR and western blot analysis indicated that the PFFs were Cas9‑positive. In addition, one of the three integrations was located near to known functional genes in the PFF1 cell line, whereas neither of the integrations was located in the PFF1 or PFF2 cell lines. It was hypothesized that these transgenic PFFs may be useful for conditional genomic editing in pigs, and for generating ideal modified porcine models. PMID:27430306

  5. Generation of porcine fetal fibroblasts expressing the tetracycline-inducible Cas9 gene by somatic cell nuclear transfer

    Science.gov (United States)

    Liu, Guoqian; Liu, Kai; Wei, Hengxi; Li, Li; Zhang, Shouquan

    2016-01-01

    Cas9 endonuclease, from so-called clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems of Streptococcus pyogenes, type II functions as an RNA-guided endonuclease and edits the genomes of prokaryotic and eukaryotic organisms, including deletion and insertion by DNA double-stranded break repair mechanisms. In previous studies, it was observed that Cas9, with a genome-scale lentiviral single-guide RNA library, could be applied to a loss-of-function genetic screen, although the loss-of-function genes have yet to be verified in vitro and this approach has not been used in porcine cells. Based on these observations, lentiviral Cas9 was used to infect porcine primary fibroblasts to achieve cell colonies carrying Cas9 endonuclease. Subsequently, porcine fetal fibroblasts expressing the tetracycline-inducible Cas9 gene were generated by somatic cell nuclear transfer, and three 30 day transgenic porcine fetal fibroblasts (PFFs) were obtained. Polymerase chain reaction (PCR), reverse transcription-PCR and western blot analysis indicated that the PFFs were Cas9-positive. In addition, one of the three integrations was located near to known functional genes in the PFF1 cell line, whereas neither of the integrations was located in the PFF1 or PFF2 cell lines. It was hypothesized that these transgenic PFFs may be useful for conditional genomic editing in pigs, and for generating ideal modified porcine models. PMID:27430306

  6. Genotoxic and biochemical effects of Yohimbe after short-term treatment in somatic and germ cells of Swiss Albino Mice

    International Nuclear Information System (INIS)

    Yohimbe was evaluated for its effects on cytological and biochemical toxicity in male Swiss albino mice. Adult male mice were mice were treated with different doses (750, 1500 and 3000 mg yohombe/kg., body weight/day) in form of an aqueous suspension for 7 consecutive days by gavage. The following parameters were evaluated: (i) cytological studies on micronucleus test, (ii) cytological analysis of spermatozoa abnormalities, (iii) Cytogentic analysis of meiotic chromosomes in the tests, (iv) quantification of proteins, ribose nucleic acid (RNA) and deoxyribose nucleic acid (DNA) in hepatic and testicular cells and (v) estimation of malondialdehyde (MDA) and nonprotein sulfhydryl (NP-SH) in hepatic and testicular cells. The treatment caused significant changes in the frequency of micronuclei in the femoral cells and induced spermatozoal abnormalities and testicular chromosomal aberrations. The study on biochemical parameters showed an increase of MDA and depletion of NP-SH, proteins, RNA and DNA in both hepatic and testicular cells. The data elucidated the role of free radical species in cytological and biochemical changes in both somatic and germ cells of Swiss albino mice. The exact mechanism of the genesis of lipid peroxides is not known, however, this might be related to the influence of yohimbine (the principal constituent of yohimbe) to enhance some catecholamines, including norepineprine which possess destructive stimuli on biological systems. It is suggested that, in view of the observed cytological and biochemical effects of yohimbe, it may be subjected to a thorough evaluation of toxicity before making it available for human use. (author)

  7. Genetic parameters for somatic cell score according to udder infection status in Valle del Belice dairy sheep and impact of imperfect diagnosis of infection.

    NARCIS (Netherlands)

    Riggio, V.; Portolano, B.; Bovenhuis, H.; Scatassa, S.; Bishop, S.C.

    2010-01-01

    Background Somatic cell score (SCS) has been promoted as a selection criterion to improve mastitis resistance. However, SCS from healthy and infected animals may be considered as separate traits. Moreover, imperfect sensitivity and specificity could influence animals' classification and impact on es

  8. Genetic parameters for somatic cell score according to udder infection status in Valle del Belice dairy sheep and impact of imperfect diagnosis of infection

    NARCIS (Netherlands)

    Riggio, V.; Portolano, B.; Bovenhuis, H.; Scatassa, S.; Caracappa, S.; Bishop, S.C.

    2011-01-01

    BACKGROUND: Somatic cell score (SCS) has been promoted as a selection criterion to improve mastitis resistance. However, SCS from healthy and infected animals may be considered as separate traits. Moreover, imperfect sensitivity and specificity could influence animals' classification and impact on e

  9. Resistance to penicillin of Staphylococcus aureus isolates from cows with high somatic cell counts in organic and conventional dairy herds in Denmark

    DEFF Research Database (Denmark)

    Bennedsgaard, Torben W.; Thamsborg, Stig M.; Aarestrup, Frank Møller;

    2006-01-01

    . Risk of infection was estimated based on somatic cell count, milk production, breed, age and lactation stage. Results: The high-risk cows represented about 49% of the cows in the herds. The overall prevalence of SA and SAr among these cows was 29% (95% confidence interval: 24%-34%) and 4% (95...

  10. A precise, low-cost milk sampler to enable the analysis of fat, protein, lactose and somatic cells in milk from individual cows

    NARCIS (Netherlands)

    Clarke, T.; Hannah, M.C.; Wientjes, H.A.

    2007-01-01

    Less than half of Australian dairy farmers undertake production recording that normally requires the collection of milk samples for the measurements of fat, protein and lactose percentages and somatic cell count. Usually the milk samples are collected from individual animals on a `one-day-per-month¿

  11. Stimulation of Activin A/Nodal signaling is insufficient to induce definitive endoderm formation of cord blood-derived unrestricted somatic stem cells

    OpenAIRE

    Filby, Caitlin E.; Williamson, Robert; van Kooy, Peter; Pébay, Alice; Dottori, Mirella; Elwood, Ngaire J.; Zaibak, Faten

    2011-01-01

    Introduction Unrestricted somatic stem cells (USSC) derived from umbilical cord blood are an attractive alternative to human embryonic stem cells (hESC) for cellular therapy. USSC are capable of forming cells representative of all three germ line layers. The aim of this study was to determine the potential of USSC to form definitive endoderm following induction with Activin A, a protein known to specify definitive endoderm formation of hESC. Methods USSC were cultured for (1) three days with ...

  12. Influence of somatic cell donor breed on reproductive performance and comparison of prenatal growth in cloned canines.

    Science.gov (United States)

    Jeong, Yeon Woo; Kim, Joung Joo; Hossein, Mohammad Shamim; Hwang, Kyu Chan; Hwang, In-sung; Hyun, Sang Hwan; Kim, Nam-Hyung; Han, Ho Jae; Hwang, Woo Suk

    2014-06-01

    Using in vivo-flushed oocytes from a homogenous dog population and subsequent embryo transfer after nuclear transfer, we studied the effects of donor cells collected from 10 different breeds on cloning efficiency and perinatal development of resulted cloned puppies. The breeds were categorized into four groups according to their body weight: small (≤9 kg), medium (>9-20 kg), large (>20-40 kg), and ultra large (>40 kg). A total of 1611 cloned embryos were transferred into 454 surrogate bitches for production of cloned puppies. No statistically significant differences were observed for initial pregnancy rates at Day 30 of embryo transfer for the donor cells originated from different breeds. However, full-term pregnancy rates were 16.5%, 11.0%, 10.0%, and 7.1% for the donor cells originated from ultra-large breed, large, medium, and small breeds, respectively, where pregnancy rate in the ultra-large group was significantly higher compared with the small breeds (P pups significantly increased proportional to breed size. The highest litter size was examined in ultra-large breeds. There was no correlation between the number of embryo transferred and litter size. Taken together, the efficiency of somatic cell cloning and fetal survival after embryo transfer may be affected significantly by selecting the appropriate genotype. PMID:24613602

  13. Correlation between somatic cell count and chemical composition of cooled raw milk in properties of Rio Grande do Norte, Brazil

    Directory of Open Access Journals (Sweden)

    Adriano Henrique do Nascimento Rangel

    2014-06-01

    Full Text Available Due to the damage caused by subclinical mastitis in loss of production and quality of milk, the present study aimed to verify the correlation between somatic cell count (SCC and the chemical composition of cooled raw milk collected in the Agreste region of Rio Grande do Norte, Brazil, in drought and rain seasons. Samples were collected in seven dairy farms during morning time, between January 2010 and March 2012, and sent to the Brazilian et of Milk Quality Laboratory (ESALQ/USP. The contents of protein, fat, lactose, casein, total solids, nonfat dry extract and urea nitrogen, besides of SCC and total bacterial count were performed. Data were submitted to analysis of variance, correlation analysis and comparison of means by Tuckey test , 5%. The average SCC was 604,000 cells/mL and had significant variation in the dry period (558 000 cells/mL and rainy (650 000 cells/mL. The SCC was positively correlated with fat and total solids but negatively with the lactose cow’s milk of bulk tank, regardless of the season in the Agreste of Rio Grande do Norte.

  14. Local Genome Topology Can Exhibit an Incompletely Rewired 3D-Folding State during Somatic Cell Reprogramming.

    Science.gov (United States)

    Beagan, Jonathan A; Gilgenast, Thomas G; Kim, Jesi; Plona, Zachary; Norton, Heidi K; Hu, Gui; Hsu, Sarah C; Shields, Emily J; Lyu, Xiaowen; Apostolou, Effie; Hochedlinger, Konrad; Corces, Victor G; Dekker, Job; Phillips-Cremins, Jennifer E

    2016-05-01

    Pluripotent genomes are folded in a topological hierarchy that reorganizes during differentiation. The extent to which chromatin architecture is reconfigured during somatic cell reprogramming is poorly understood. Here we integrate fine-resolution architecture maps with epigenetic marks and gene expression in embryonic stem cells (ESCs), neural progenitor cells (NPCs), and NPC-derived induced pluripotent stem cells (iPSCs). We find that most pluripotency genes reconnect to target enhancers during reprogramming. Unexpectedly, some NPC interactions around pluripotency genes persist in our iPSC clone. Pluripotency genes engaged in both "fully-reprogrammed" and "persistent-NPC" interactions exhibit over/undershooting of target expression levels in iPSCs. Additionally, we identify a subset of "poorly reprogrammed" interactions that do not reconnect in iPSCs and display only partially recovered, ESC-specific CTCF occupancy. 2i/LIF can abrogate persistent-NPC interactions, recover poorly reprogrammed interactions, reinstate CTCF occupancy, and restore expression levels. Our results demonstrate that iPSC genomes can exhibit imperfectly rewired 3D-folding linked to inaccurately reprogrammed gene expression. PMID:27152443

  15. Fanca deficiency reduces A/T transitions in somatic hypermutation and alters class switch recombination junctions in mouse B cells.

    Science.gov (United States)

    Nguyen, Thuy Vy; Riou, Lydia; Aoufouchi, Saïd; Rosselli, Filippo

    2014-06-01

    Fanconi anemia is a rare genetic disorder that can lead to bone marrow failure, congenital abnormalities, and increased risk for leukemia and cancer. Cells with loss-of-function mutations in the FANC pathway are characterized by chromosome fragility, altered mutability, and abnormal regulation of the nonhomologous end-joining (NHEJ) pathway. Somatic hypermutation (SHM) and immunoglobulin (Ig) class switch recombination (CSR) enable B cells to produce high-affinity antibodies of various isotypes. Both processes are initiated after the generation of dG:dU mismatches by activation-induced cytidine deaminase. Whereas SHM involves an error-prone repair process that introduces novel point mutations into the Ig gene, the mismatches generated during CSR are processed to create double-stranded breaks (DSBs) in DNA, which are then repaired by the NHEJ pathway. As several lines of evidence suggest a possible role for the FANC pathway in SHM and CSR, we analyzed both processes in B cells derived from Fanca(-/-) mice. Here we show that Fanca is required for the induction of transition mutations at A/T residues during SHM and that despite globally normal CSR function in splenic B cells, Fanca is required during CSR to stabilize duplexes between pairs of short microhomology regions, thereby impeding short-range recombination downstream of DSB formation. PMID:24799500

  16. Novel somatic mutations in large granular lymphocytic leukemia affecting the STAT-pathway and T-cell activation

    International Nuclear Information System (INIS)

    T-cell large granular lymphocytic (T-LGL) leukemia is a clonal disease characterized by the expansion of mature CD3+CD8+ cytotoxic T cells. It is often associated with autoimmune disorders and immune-mediated cytopenias. Our recent findings suggest that up to 40% of T-LGL patients harbor mutations in the STAT3 gene, whereas STAT5 mutations are present in 2% of patients. In order to identify putative disease-causing genetic alterations in the remaining T-LGL patients, we performed exome sequencing from three STAT mutation-negative patients and validated the findings in 113 large granular lymphocytic (LGL) leukemia patients. On average, 11 CD8+ LGL leukemia cell-specific high-confidence nonsynonymous somatic mutations were discovered in each patient. Interestingly, all patients had at least one mutation that affects either directly the STAT3-pathway (such as PTPRT) or T-cell activation (BCL11B, SLIT2 and NRP1). In all three patients, the STAT3 pathway was activated when studied by RNA expression or pSTAT3 analysis. Screening of the remaining 113 LGL leukemia patients did not reveal additional patients with same mutations. These novel mutations are potentially biologically relevant and represent rare genetic triggers for T-LGL leukemia, and are associated with similar disease phenotype as observed in patients with mutations in the STAT3 gene

  17. Genotoxic damage induced by isopropanol in germinal and somatic cells of Drosophila melanogaster.

    Science.gov (United States)

    Palermo, Ana María; Mudry, Marta Dolores

    2011-12-24

    Isopropanol (isopropyl alcohol, 2-propanol, IPA) is a volatile solvent widely used in domestic or industrial environments and reported as innocuous in various test systems. The aim of this work was to search for in vivo genotoxic effects of IPA in Drosophila melanogaster, studying its ability to induce nondisjunction (ND) in females, sex linked recessive lethals (SLRL) in males, and somatic mutation and/or recombination (SMART) in larvae. Treatments were acute (60min) and were administered via inhalation. IPA had low toxicity in adult flies (75% IPA mortality index, MI=12.7% (females) and 2.6% (males)) and larvae (MI=14.3%, 75% IPA). Female fertility was severely affected during the first 24h (brood I, BI) after treatment, but, afterwards, control values were recovered. IPA induced a 50-fold increase of ND (%) in 24h old females, and a six-fold rise in 4-5 d old BI offspring. Nondisjunction frequencies (%) in the offspring of broods II to V (24h in each case) were similar to control values. IPA doses of 25% and 50% (v/v), tested in 24h old females, showed a significant dose-dependent increase of ND(%)in BI only, with control values in subsequent broods. Flies gave normal offspring when kept in regular media for 24h before mating. The eye spot test (SMART) showed a significant increase at 50% IPA (pDNA directly, but perturbations of the nuclear membrane may be responsible for induction of ND. PMID:22001194

  18. Antimutagenic and antirecombinagenic activities of noni fruit juice in somatic cells of Drosophila melanogaster.

    Science.gov (United States)

    Franchi, Leonardo P; Guimarães, Nilza N; De Andrade, Laise R; De Andrade, Heloísa H R; Lehmann, Maurício; Dihl, Rafael R; Cunha, Kênya S

    2013-01-01

    Noni, a Hawaiian name for the fruit of Morinda citrifolia L., is a traditional medicinal plant from Polynesia widely used for the treatment of many diseases including arthritis, diabetes, asthma, hypertension and cancer. Here, a commercial noni juice (TNJ) was evaluated for its protective activities against the lesions induced by mitomycin C (MMC) and doxorrubicin (DXR) using the Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster. Three-day-old larvae, trans-heterozygous for two genetic markers (mwh and flr3 ), were co-treated with TNJ plus MMC or DXR. We have observed a reduction in genotoxic effects of MMC and DXR caused by the juice. TNJ provoked a marked decrease in all kinds of MMC- and DXR-induced mutant spots, mainly due to its antirecombinagenic activity. The TNJ protective effects were concentration-dependent, indicating a dose-response correlation, that can be attributed to a powerful antioxidant and/or free radical scavenger ability of TNJ. PMID:23828338

  19. Recombinagenic activity of integerrimine, a pyrrolizidine alkaloid from Senecio brasiliensis, in somatic cells of Drosophila melanogaster.

    Science.gov (United States)

    Campesato, V R; Graf, U; Reguly, M L; de Andrade, H H

    1997-01-01

    Integerrimine (ITR), a pyrrolizidine alkaloid from Senecio brasiliensis, was tested for genotoxicity using the wing somatic mutation and recombination test (SMART) in Drosophila melanogaster. The compound was administered by chronic feeding (48 hours) of 3-day-old larvae. Two different crosses involving the markers flare (flr) and multiple wing hairs (mwh) were used, that is, the standard (ST) cross and the high bioactivation (HB) cross, which has a high cytochrome P450-dependent bioactivation capacity. In both crosses, the wings of two types of progeny were analyzed, that is, inversion-free marker heterozygotes and balancer heterozygotes carrying multiple inversions. ITR was found to be equally potent in inducing spots in a dose-related manner in the marker heterozygotes of both crosses. This indicates that the bioactivation capacity present in larvae of the ST cross is sufficient to reveal the genotoxic activity of ITR. In the balancer heterozygotes of both crosses, where all recombinational events are eliminated due to the inversions, the frequencies of induced spots were considerably reduced which documents the recombinagenic activity of ITR. Linear regression analysis of the dose response relationships for both genotypes shows that 85% to 90% of the wing spots are due to mitotic recombination. PMID:9020312

  20. Characteristics of the somatic hypermutation in the Camelus dromedarius T cell receptor gamma (TRG) and delta (TRD) variable domains.

    Science.gov (United States)

    Ciccarese, Salvatrice; Vaccarelli, Giovanna; Lefranc, Marie-Paule; Tasco, Gianluca; Consiglio, Arianna; Casadio, Rita; Linguiti, Giovanna; Antonacci, Rachele

    2014-10-01

    In previous reports, we had shown in Camelus dromedarius that diversity in T cell receptor gamma (TRG) and delta (TRD) variable domains can be generated by somatic hypermutation (SHM). In the present paper, we further the previous finding by analyzing 85 unique spleen cDNA sequences encoding a total of 331 mutations from a single animal, and comparing the properties of the mutation profiles of dromedary TRG and TRD variable domains. The transition preference and the significant mutation frequency in the AID motifs (dgyw/wrch and wa/tw) demonstrate a strong dependence of the enzymes mediating SHM in TRG and TRD genes of dromedary similar to that of immunoglobulin genes in mammals. Overall, results reveal no asymmetry in the motifs targeting, i.e. mutations are equally distributed among g:c and a:t base pairs and replacement mutations are favored at the AID motifs, whereas neutral mutations appear to be more prone to accumulate in bases outside of the motifs. A detailed analysis of clonal lineages in TRG and TRD cDNA sequences also suggests that clonal expansion of mutated productive rearrangements may be crucial in shaping the somatic diversification in the dromedary. This is confirmed by the fact that our structural models, computed by adopting a comparative procedure, are consistent with the possibility that, irrespective of where (in the CDR-IMGT or in FR-IMGT) the diversity was generated by mutations, both clonal expansion and selection seem to be strictly related to an enhanced structural stability of the γδ subunits. PMID:24836674

  1. Somatic Embryogenesis in Peach Palm Using the Thin Cell Layer Technique: Induction, Morpho-histological Aspects and AFLP Analysis of Somaclonal Variation

    Science.gov (United States)

    Steinmacher, D. A.; Krohn, N. G.; Dantas, A. C. M.; Stefenon, V. M.; Clement, C. R.; Guerra, M. P.

    2007-01-01

    Background and Aims The thin cell layer (TCL) technique is based on the use of very small explants and has allowed enhanced in vitro morphogenesis in several plant species. The present study evaluated the TCL technique as a procedure for somatic embryo production and plantlet regeneration of peach palm. Methods TCL explants from different positions in the shoot apex and leaf sheath of peach palm were cultivated in MS culture medium supplemented with 0–600 µm Picloram in the presence of activated charcoal. The production of primary calli and embryogenic calli was evaluated in these different conditions. Histological and amplified fragment length polymorphism (AFLP) analyses were conducted to study in vitro morphogenetic responses and genetic stability, respectively, of the regenerated plantlets. Key Results Abundant primary callus induction was observed from TCLs of the shoot meristem in culture media supplemented with 150–600 µm Picloram (83–97 %, respectively). The production of embryogenic calli depends on Picloram concentration and explant position. The best response observed was 43 % embryogenic callus production from shoot meristem TCL on 300 µm Picloram. In maturation conditions, 34 ± 4 somatic embryos per embryogenic callus were obtained, and 45·0 ± 3·4 % of these fully developed somatic embryos were converted, resulting in plantlets ready for acclimatization, of which 80 % survived. Histological studies revealed that the first cellular division events occurred in cells adjacent to vascular tissue, resulting in primary calli, whose growth was ensured by a meristematic zone. A multicellular origin of the resulting somatic embryos arising from the meristematic zone is suggested. During maturation, histological analyses revealed bipolarization of the somatic embryos, as well as the development of new somatic embryos. AFLP analyses revealed that 92 % of the regenerated plantlets were true to type. The use of TCL explants considerably improves the

  2. Efficient production of omega-3 fatty acid desaturase (sFat-1)-transgenic pigs by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Omega-3(ω-3) fatty acid desaturase transgenic pigs may improve carcass fatty acid composition. The use of transgenic pigs is also an excellent large animal model for studying the role of ω-3 fatty acids in the prevention and treatment of coronary heart disease and cancer. Transgenic pigs carrying synthesized fatty acid desaturase-1 gene (sFat-1) from Caenorhabditis briggsae by somatic cell nuclear transfer (SCNT) were produced for the first time in China. Porcine fetal fibroblast cells were transfected with a sFat-1 expression cassette by the liposome-mediated method. Transgenic embryos were reconstructed by nuclear transfer of positive cells into enucleated in vitro matured oocytes. A total of 1889 reconstructed embryos were transferred into 10 naturally cycling gilts. Nine early pregnancies were established, 7 of which went to term. Twenty-one piglets were born. The cloning efficiency was 1.1% (born piglets/transferred embryos). The integration of the sFat-1 gene was confirmed in 15 live cloned piglets by PCR and Southern blot except for 2 piglets. Expression of the sFat-1 gene in 12 of 13 piglets was detected with RT-PCR. The data demonstrates that an efficient system for sFat-1 transgenic cloned pigs was developed, which led to the successful production of piglets expressing the sFat-1 gene.

  3. Epstein-Barr virus nuclear protein EBNA3C directly induces expression of AID and somatic mutations in B cells.

    Science.gov (United States)

    Kalchschmidt, Jens S; Bashford-Rogers, Rachael; Paschos, Kostas; Gillman, Adam C T; Styles, Christine T; Kellam, Paul; Allday, Martin J

    2016-05-30

    Activation-induced cytidine deaminase (AID), the enzyme responsible for induction of sequence variation in immunoglobulins (Igs) during the process of somatic hypermutation (SHM) and also Ig class switching, can have a potent mutator phenotype in the development of lymphoma. Using various Epstein-Barr virus (EBV) recombinants, we provide definitive evidence that the viral nuclear protein EBNA3C is essential in EBV-infected primary B cells for the induction of AID mRNA and protein. Using lymphoblastoid cell lines (LCLs) established with EBV recombinants conditional for EBNA3C function, this was confirmed, and it was shown that transactivation of the AID gene (AICDA) is associated with EBNA3C binding to highly conserved regulatory elements located proximal to and upstream of the AICDA transcription start site. EBNA3C binding initiated epigenetic changes to chromatin at specific sites across the AICDA locus. Deep sequencing of cDNA corresponding to the IgH V-D-J region from the conditional LCL was used to formally show that SHM is activated by functional EBNA3C and induction of AID. These data, showing the direct targeting and induction of functional AID by EBNA3C, suggest a novel role for EBV in the etiology of B cell cancers, including endemic Burkitt lymphoma. PMID:27217538

  4. Relationships of survival time, productivity and cause of death with telomere lengths of cows produced by somatic cell nuclear transfer.

    Science.gov (United States)

    Konishi, Kazuyuki; Yonai, Miharu; Kaneyama, Kanako; Ito, Satoshi; Matsuda, Hideo; Yoshioka, Hajime; Nagai, Takashi; Imai, Kei

    2011-10-01

    The reproductive ability, milk-producing capacity, survival time and relationships of these parameters with telomere length were investigated in 4 groups of cows produced by somatic cell nuclear transfer (SCNT). Each group was produced using the same donor cells (6 Holstein (1H), 3 Holstein (2H), 4 Jersey (1J) and 5 Japanese Black (1B) cows). As controls, 47 Holstein cows produced by artificial insemination were used. The SCNT cows were artificially inseminated, and multiple deliveries were performed after successive rounds of breeding and conception. No correlation was observed between the telomere length and survival time in the SCNT cows. Causes of death of SCNT cows included accidents, accident-associated infections, inappropriate management, acute mastitis and hypocalcemia. The lifetime productivity of SCNT cows was superior to those of the controls and cell donor cows. All SCNT beef cows with a relatively light burden of lactation remained alive and showed significantly prolonged survival time compared with the cows in the SCNT dairy breeds. These results suggest that the lifetime productivity of SCNT cows was favorable, and their survival time was more strongly influenced by environmental burdens, such as pregnancy, delivery, lactation and feeding management, than by the telomere length. PMID:21666348

  5. Effects of different nuclear transfer and activation methods on the development of mouse somatic cell cloned embryos

    Institute of Scientific and Technical Information of China (English)

    Wang ErYao; YU Yang; Li XueMei; JIAO LiHong; Wang Liu

    2007-01-01

    A group of adult somatic cell cloned mice were obtained by using cumulus cells as nuclei donor cells. To study the effect of different nuclear transfer (NT) and activation methods on the development of mouse cloned embryos, embryos were reconstructed using two traditional NT methods (electrofusion and direct injection) and four activation treatments (electric pulse, ethanol, SrCl2 and electric pulse combined with SrCl2). The data showed that the efficiency of reconstruction using the direct injection method is significantly higher (90.7%) than that of the electrofusion method (49.7%). Parthenogenetic embryos can develop to blastocyst stage with three activation conditions, including ethanol, electric pulse and SrCl2; however, the rates of development to blastocyst after ethanol and electric pulse activation (52.4%, 54.2%) are significantly lower than after SrCl2 activation (76.9%). Treatment of embryos for 6 h with 10 mmol/L SrCl2 was found to be the best condition for activation of parthenogenetic as well as reconstructed embryos. By contrast, reconstructed embryos failed to develop to blastocyst stage after being activated by ethanol. The use of either injection or electrofusion for embryo reconstruction affected the pre-implantation development. However, after transfer in pseudopregnant mice, cloned mice were obtained from both methods.

  6. DNA methylation status of H19 and Xist genos in lungs of somatic cell nuclear transfer bovines

    Institute of Scientific and Technical Information of China (English)

    CHEN Jie; LI DongJie; LIU YanQin; ZHANG Cui; DAI YunPing; LI ShiJie; LINing

    2008-01-01

    In somatic cell nuclear transfer (SCNT) technologies, the donor cell's nuclei need to be epigenetically reprogrammed for embryonic development. The incomplete reprogramming of donor cell nuclei has been implicated as a primary reason for the low efficiency of SCNT. DNA methylation is a major epige- netic modification of the genome that regulates crucial aspects of genome function, including estab-lishment of genomic imprinting. In order to make sure whether the DNA methylation reprogramming is efficient in SCNT animals, we analyzed the DNA methylation status of two imprinting genes, H19 and Xist, in lungs of deceased SCNT bovines that died within 48 h of birth using bisulfite sequencing analysis. Our findings demonstrated that cloned bovines showed significantly lower DNA methylation of H19 than controls (P<0.05), and three tested CpGs sites (1, 2, 3) exhibited unmethylation in one cloned bovine (9C3); however, Xist showed similar DNA methylation levels between clones and con- trols, and both showed hypermethylation (96.11% and 86.67%).

  7. The effects of storage temperature on goat milk somatic cell count using the DeLaval counter.

    Science.gov (United States)

    Sanchez-Macias, Davinia; Castro, Noemi; Moreno-Indias, Isabel; Morales-delaNuez, Antonio; Briggs, Heather; Capote, Juan; Argüello, Anastasio

    2010-10-01

    This study investigated the influence of storage temperature and storage time on goat milk somatic cell counts (SCCs) determined using the DeLaval cell counter (DCC). SCCs were measured in 40 Majorera goat milk samples using the DCC device. Samples were grouped from high score (>2,750 x 10(3) cells/mL) to low score (milk sample was divided into four aliquots and stored at four different temperatures (4 degrees C, 21 degrees C, 36 degrees C or 45 degrees C). The SCC was recorded every hour for 12 hours. Storage of goat milk with a high SCC for 5, 5, 2 or 1 hour at 4 degrees C, 21 degrees C, 36 degrees C or 45 degrees C, respectively, decreased the SCC value compared to fresh milk. The goat milk SCC was lower after 1 hour of storage than that determined for fresh milk at any tested temperature in low-SCC samples. The data presented herein suggest that regardless of storage temperature, goat milk samples should not be stored for more than 1 hour before measurement of SCC with a DCC device. PMID:20419471

  8. An investigation on somatic cell count in milk samples collected from dairy farms at Tabriz region of Iran

    Directory of Open Access Journals (Sweden)

    Rahim Beheshti,

    2011-08-01

    Full Text Available The aim of present study was somatic cell count in milk samples collected from dairy farms at Tabriz region, Northwest of Iran. Three flocks selected based on high productivity and similar characteristics (use of family labour, Holstein herds and average production between upper than 11 kg/cow/day. Milk samples obtained from three parity classes were collect individually from the cows in the second and fifth month of lactation in two seasons: autumn-winter and spring-summer. Results show higher SCC for dairy cattle with second or upper milking. Cows at fifth or upper lactation period had 1000- 5000 ×103 cells/ml commonly but at first lactation there was no any cow with 1000-2500 ×103 cells/ml. In conclusion, incidence of high SCC rate (1000-5000×103 is considerably high during fifth or upper parity but 250 to 750 ×103 SCC cows are considerably low in number compared with first parity cows. Cows at first lactation commonly had 250-500×103 SCC at Tabriz regional farms.

  9. Chromatin dynamics in pollen mother cells underpin a common scenario at the somatic-to-reproductive fate transition of both the male and female lineages in Arabidopsis

    OpenAIRE

    She, Wenjing; Baroux, Célia

    2015-01-01

    Unlike animals, where the germline is established early during embryogenesis, plants set aside their reproductive lineage late in development in dedicated floral organs. The specification of pollen mother cells (PMC) committed to meiosis takes place in the sporogenous tissue in anther locules and marks the somatic-to-reproductive cell fate transition toward the male reproductive lineage. Here we show that Arabidopsis PMC differentiation is accompanied by large-scale changes in chromatin organ...

  10. Mapping of Microsatellite SW943 to Porcine Chromosome 12p11-(2/3p13) Using Primed in situ Synthesis and Somatic Cell Hybrid Panel

    Institute of Scientific and Technical Information of China (English)

    LIU Bang; WANG Yong-qiang; ZHANG Qing-de; YU Mei; ZHAO Shu-hong; XIONG Tong-an; LI Kui

    2002-01-01

    The porcine microsatellite SW943 was regionally localized on 12p11-(2/3p13) by the two methods: the Primed in situ (PRINS) labelling on the pachytene bivalents of pigs using the Dig-11-dUTP as the report molecule and pig × rodent Somatic Cell Hybrid PaneI(SCHP) which contains 27 cell lines through PCR amplification. Advantages and disadvantages of the two methods for physical mapping of microsatellites were also discussed.

  11. Cellular heredity in haploid cultures of somatic cells, March 1968-April 1981. Final report

    International Nuclear Information System (INIS)

    An account is given of the development and application to cell-culture genetics of unique haploid cell lines from frog embryo developed in this laboratory. Since 1968, the main aim of this project has been to develop the haploid cell system for studies of mutagenesis in culture, particularly by ultraviolet radiation. In the course of this work we isolated chromosomally stable cell lines, derived and characterized a number of variants, and adapted cell hybridization and other methods to this material. Particular emphasis was placed on ultraviolet photobiology, including studies of cell survival, mutagenesis, and pathways of repair of uv-damaged DNA. Although at present less widely used for genetic experiments than mammalian cell lines, the frog cells offer the advantages of authentic haploidy and a favorable repertory of DNA repair pathways for study of uv mutagenesis

  12. Stem Cell Interaction with Somatic Niche May Hold the Key to Fertility Restoration in Cancer Patients

    OpenAIRE

    Deepa Bhartiya; Kalpana Sriraman; Seema Parte

    2012-01-01

    The spontaneous return of fertility after bone marrow transplantation or heterotopic grafting of cryopreserved ovarian cortical tissue has surprised many, and a possible link with stem cells has been proposed. We have reviewed the available literature on ovarian stem cells in adult mammalian ovaries and presented a model that proposes that the ovary harbors two distinct populations of stem cells, namely, pluripotent, quiescent, very small embryonic-like stem cells (VSELs), and slightly larger...

  13. Somatic cell banking - An alternative technology for the conservation of endangered sheep breeds

    International Nuclear Information System (INIS)

    Skin samples from ear pinna of 10 male and 10 female sheep were collected and cultured in DMEM+Ham's F12 nutrient medium. Cell viability was 95 to 100% in different cultures. Mean cell proliferation rates were 0.94-0.67 and 1.15-0.56 for males and females in different passages, respectively. Cell proliferation rates were highest in first passage and then showed an age-related decline. Average cell doubling time was 30 h in males and 29.6 h in females. Skin fibroblast cell growth curves were in lag phase for the first 2 days, entered log phase (3rd to 7th days) and plateaued on day 8. Diploid chromosomal counts in proliferating cells up to the 5th passage were normal (2N=54), with no gross chromosomal aberrations recorded. Cells frozen from cycling cells at 80-90% confluency showed superior post-thaw growth compared with cells from overconfluent cultures. DMSO at 10% (v/v) in freezing media was optimal. Controlled-rate freezing at -1 deg. C/min showed better post-thaw cell viability and growth potential. Direct plating of thawed cells without removing DMSO and other contents of the freezing medium gave better post-thaw survival and proliferation rates. (author)

  14. Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells

    Directory of Open Access Journals (Sweden)

    Han Yong-Mahn

    2009-07-01

    Full Text Available Abstract Background Interspecies somatic cell nuclear transfer (iSCNT has been proposed as a tool to address basic developmental questions and to improve the feasibility of cell therapy. However, the low efficiency of iSCNT embryonic development is a crucial problem when compared to in vitro fertilization (IVF and intraspecies SCNT. Thus, we examined the effect of donor cell species on the early development of SCNT embryos after reconstruction with bovine ooplasm. Results No apparent difference in cleavage rate was found among IVF, monkey-bovine (MB-iSCNT, and bovine-bovine (BB-SCNT embryos. However, MB-iSCNT embryos failed to develop beyond the 8- or 16-cell stages and lacked expression of the genes involved in embryonic genome activation (EGA at the 8-cell stage. From ultrastructural observations made during the peri-EGA period using transmission electron microscopy (TEM, we found that the nucleoli of MB-iSCNT embryos were morphologically abnormal or arrested at the primary stage of nucleologenesis. Consistent with the TEM analysis, nucleolar component proteins, such as upstream binding transcription factor, fibrillarin, nucleolin, and nucleophosmin, showed decreased expression and were structurally disorganized in MB-iSCNT embryos compared to IVF and BB-SCNT embryos, as revealed by real-time PCR and immunofluorescence confocal laser scanning microscopy, respectively. Conclusion The down-regulation of housekeeping and imprinting genes, abnormal nucleolar morphology, and aberrant patterns of nucleolar proteins during EGA resulted in developmental failure in MB-iSCNT embryos. These results provide insight into the unresolved problems of early embryonic development in iSCNT embryos.

  15. Evaluation of genotoxic potential of Hypericum triquetrifolium extract in somatic and germ cells of male albino mice

    Directory of Open Access Journals (Sweden)

    Bushra M. A. Mohammed,

    2011-04-01

    Full Text Available Hypericum triquetrifolium aqueous extract were studied for the first time for its toxic and the possible clastogenic effects in vivo on the bone marrow and spermatozoa cells of Swiss albino mice. The lethal dose of the aqueous extract was considered to be 10.33 g/kg of the body weight, injected subcutaneously. The doses which were chosen for treatments were 2, 1, and 0.25 g/kg. H. triquetrifolium extract induce statistically significant increases in the average numbers of micronucleus(MN at the dose 2 g/kg and chromosome aberrations at the doses 2 and 1 g/kg ,the majority of aberrations observed were chromatid breaks, centromeric breaks, acentric fragments. The extract was found to inhibit mitotic index (MI in a dose-dependent manner. Moreover the plant extract showed a significant induction of sperm abnormalities in all concentrations used comparing with the untreated animals. The most frequent types of sperm abnormalities of the treated groups were; amorphous, pseudo-droplet defect, bent mid piece defect and corkscrew mid piece defect. However, the lowest dose 0.25 g/kg body weight was the most effective one which markedly increased the corkscrew midpiece defect. The results indicated that the mixture of the compounds found in the aqueous extract caused cytotoxicity and induced different cytogenetic effects in both somatic and germ cells of male albino mice.

  16. Human unrestricted somatic stem cells loaded in nanofibrous PCL scaffold and their healing effect on skin defects.

    Science.gov (United States)

    Bahrami, Hoda; Keshel, Saeed Heidari; Chari, Aliakbar Jafari; Biazar, Esmaeil

    2016-09-01

    Unrestricted somatic stem cells (USSCs) loaded in nanofibrous polycaprolactone (PCL) scaffolds can be used for skin regeneration when grafted onto full-thickness skin defects of rats. Nanofibrous PCL scaffolds were designed by the electrospinning method and crosslinked with laminin protein. Afterwards, the scaffolds were evaluated by scanning electron microscopy, and physical and mechanical assays. In this study, nanofibrous PCL scaffolds loaded with USSCs were grafted onto the skin defects. The wounds were subsequently investigated 21 days after grafting. Results of mechanical and physical analyses showed good resilience and compliance to movement as a skin graft. In animal models; study samples exhibited the most pronounced effect on wound closure, with statistically significant improvement in wound healing being seen at 21 days post-operatively. Histological examinations of healed wounds from all samples showed a thin epidermis plus recovered skin appendages in the dermal layer for samples with cell. Thus, the graft of nanofibrous PCL scaffolds loaded with USSC showed better results during the healing process of skin defects in rat models. PMID:26140614

  17. Modulatory effects of the antioxidant ascorbic acid on the direct genotoxicity of doxorubicin in somatic cells of Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Edson José Fragiorge

    2007-03-01

    Full Text Available In this study two different crosses involving the wing cell markers mwh and flr³ (standard (ST cross and high bioactivation (HB cross, the latter being characterized by a high constitutive level of cytochrome P450 which leads to an increased sensitivity to a number of promutagens and procarcinogens were used to investigate the modulatory effects of ascorbic acid (AA combined with the antitumor agent doxorubicin (DXR in Drosophila melanogaster. We observed that the two different concentrations of AA (50 or 100 mM had no effect on spots frequencies, while DXR treatments (0.2 or 0.4 mM gave positive results for all types of spots, when compared to negative control. For marker-heterozygous (MH flies, a protective effect was observed with the lower concentration of AA (50 mM that was able to statistically decrease the frequency of spots induced by DXR (0.2 mM, while an enhanced frequency of spots induced by DXR was observed with the higher concentration of AA (100 mM, when compared to DXR treatment (p < 0.05. These results suggest that AA may interfere with free radicals generated by DXR and with other possible reactive metabolites. The efficiency of AA in protecting the somatic cells of D. melanogaster against mutation and recombination induced by DXR is dependent on the dose used and the protection is directly related to the activity of cytochrome P450 enzymes.

  18. Biodosimetry of Chernobyl cleanup workers from Estonia and Latvia using the glycophorin A in vivo somatic cell mutation assay

    International Nuclear Information System (INIS)

    The reactor accident at Chernobyl in 1986 necessitated a massive environmental cleanup that involved over 600,000 workers from all 15 Republics of the former Soviet Union. To determine whether the whole-body radiation received by workers in the course of these decontamination activities resulted in a detectable biological response, over 1,500 blood samples were obtained from cleanup workers sent from two Baltic countries, Estonia and Latvia. Here we report the results of studies of biodosimetry using the glycophorin A (GPA) locus in vivo somatic cell mutation assay applied to 734 blood samples from these workers, to 51 control samples from unexposed Baltic populations and to 94 samples from historical U.S. controls. The data reveal inconsistent evidence that the protracted radiation exposures received by these workers resulted in a significant dose-associated increase in GPA locus mutations compared with the controls. Taken together, these data suggest that the average radiation exposure to these workers does not greatly exceed 10 cGy, the minimum levels at which radiation effects might be detectable by the assay. Although the protracted nature of the exposure may have reduced the efficiency of induction of GPA locus mutations, it is likely that the estimated physical doses for these cleanup worker populations (median reported dose 9.5 cGy) were too low to result in radiation damage to erythroid stem cells that can be detected reliably by this method. 25 refs., 2 figs., 3 tabs

  19. Cellular heredity in haploid cultures of somatic cells. Progress report, August 1978-September 1979

    International Nuclear Information System (INIS)

    During the past year we have continued our studies of the relation of ultraviolet mutagenesis to DNA repair in cultures of the haploid frog cell line ICR 2A. Our method of irradiation of cells in suspension was improved by construction of an improved detector with major sensitivity to the 254 nm Hg resonance line, to give better estimates of actual exposure of the cells. Using this method, dose-response and dose-fractionation studies on irradiation of ouabain resistance were carried out. The uv induction of this phenotype in the ICR 2A cell line was found to be less than that necessary for adequate analysis of dose-response curves. Cell fusion experiments using frog and mouse cells revealed an enhancement of mutagenesis in the mouse parent that will be explored in further work

  20. Cellular heredity in haploid cultures of somatic cells. Progress report, August 1978-September 1979

    International Nuclear Information System (INIS)

    During the past year, studies were continued on the relation of ultraviolet mutagenesis to DNA repair in cultures of the haploid frog cell line ICR 2A. The method of irradiation of cells in suspension was improved by construction of an improved detector with major sensitivity to the 254 nm Hg resonance line, to give better estimates of actual exposure of the cells. Using this method, dose-response and dose-fractionation studies on irradiation of ouabain resistance were carried out. The uv induction of this phenotype in the ICR 2A cell line was found to be less than that necessary for adequate analysis of dose-response curves. Cell fusion experiments using frog and mouse cells revealed an enhancement of mutagenesis in the mouse parent that will be explored in further work

  1. The Determination of Somatic Cell Count and Some Components of Raw Milk Evaluated By a Private Company in Trakya

    Directory of Open Access Journals (Sweden)

    A. R. Onal

    2007-05-01

    Full Text Available The aim of this study was to determine the amount of bulk tank somatic cell counts. Chemical and microbiological compositions of raw milk produced in Trakya were also reached in order to evaluate the structure of milk production. For this purpose 36 raw milk samples were collected from bulk milk tank within three different location of Trakya (18 samples from Edirne, 10 from Tekirdağ and 8 from Kırklareli. The arithmetic means and standard errors of fat percentages, non-fat dry matter, protein percentages, BTSCC (Bulk Tank Milk Somatic Cell Count and TB (Total Bacteria for Edirne, Tekirdağ and Kırklareli provinces were; 3.70 0.052, 3.60 0.098, 3.76 0.064; 8.34 0.025, 8.50 0.035, 8.39 0.038; 3.05 0.012, 3.09 0.019, 3.05 0.016; 308.555 26.510 SCC/ml (log 5.459 0.04 SCC/ml, 350.200 53.627 SCC/ml (Log 5.500 0.06 SCC/ml, 254.500 37.645 SCC/ml (Log 5.370 0.06 SCC/ml; 479.481 51.777 cfu/ml (Log 5.630 0.05 cfu/ml, 435.716 91.194 cfu/ml (Log 5.5230.12 cfu/ml, 446.958 81.515 cfu/ml (Log 5.602 0.075 cfu/ml respectively. Consequentially, the correlation coefficient for LogBTSCC and fat percentage, non-fat dry matter, protein percentage and LogTB were found to be 0.036, 0.251, 0.421 and 0.219 respectively. A significant (p<0.05 correlation coefficient was obtained between LogBTSCC and protein percentage.

  2. Chromatin dynamics in Pollen Mother Cells underpin a common scenario at the somatic-to-reproductive fate transition of both the male and female lineages in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Wenjing eShe

    2015-04-01

    Full Text Available Unlike animals, where the germline is established early during embryogenesis, plants set aside their reproductive lineage late in development in dedicated floral organs. The specification of pollen mother cells (PMCs committed to meiosis takes place in the sporogenous tissue in anther locules and marks the somatic-to-reproductive cell fate transition towards the male reproductive lineage. Here we show that Arabidopsis PMCs differentiation is accompanied by large-scale changes in chromatin organization. This is characterized by significant increase in nuclear volume, chromatin decondensation, reduction in heterochromatin, eviction of linker histones and the H2AZ histone variant. These structural alterations are accompanied by dramatic, quantitative changes in histone modifications levels compared to that of surrounding somatic cells that do not share a sporogenic fate. All these changes are highly reminiscent of those we have formerly described in female megaspore mother cells (MMCs. This indicates that chromatin reprogramming is a common underlying scenario in the somatic-to-reproductive cell fate transition in both male and female lineages.

  3. Somatic-cell selection is a major determinant of the blood-cell phenotype in heterozygotes for glucose-6-phosphate dehydrogenase mutations causing severe enzyme deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Filosa, S.; Giacometti, N.; Wangwei, C.; Martini, G. [Istituto Internazionale di Genetica e Biofisica, Naples (Italy)] [and others

    1996-10-01

    X-chromosome inactivation in mammals is regarded as an essentially random process, but the resulting somatic-cell mosaicism creates the opportunity for cell selection. In most people with red-blood-cell glucose-6-phosphate dehydrogenase (G6PD) deficiency, the enzyme-deficient phenotype is only moderately expressed in nucleated cells. However, in a small subset of hemizygous males who suffer from chronic nonspherocytic hemolytic anemia, the underlying mutations (designated class I) cause more-severe G6PD deficiency, and this might provide an opportunity for selection in heterozygous females during development. In order to test this possibility we have analyzed four heterozygotes for class I G6PD mutations: two with G6PD Portici (1178G{r_arrow}A) and two with G6PD Bari (1187C{r_arrow}T). We found that in fractionated blood cell types (including erythroid, myeloid, and lymphoid cell lineages) there was a significant excess of G6PD-normal cells. The significant concordance that we have observed in the degree of imbalance in the different blood-cell lineages indicates that a selective mechanism is likely to operate at the level of pluripotent blood stem cells. Thus, it appears that severe G6PD deficiency affects adversely the proliferation or the survival of nucleated blood cells and that this phenotypic characteristic is critical during hematopoiesis. 65 refs., 6 figs., 3 tabs.

  4. In Vitro and In Vivo Hepatic Differentiation of Adult Somatic Stem Cells and Extraembryonic Stem Cells for Treating End Stage Liver Diseases

    Directory of Open Access Journals (Sweden)

    Chenxia Hu

    2015-01-01

    Full Text Available The shortage of liver donors is a major handicap that prevents most patients from receiving liver transplantation and places them on a waiting list for donated liver tissue. Then, primary hepatocyte transplantation and bioartificial livers have emerged as two alternative treatments for these often fatal diseases. However, another problem has emerged. Functional hepatocytes for liver regeneration are in short supply, and they will dedifferentiate immediately in vitro after they are isolated from liver tissue. Alternative stem-cell-based therapeutic strategies, including hepatic stem cells (HSCs, embryonic stem cells (ESCs, induced pluripotent stem cells (iPSCs, and mesenchymal stem cells (MSCs, are more promising, and more attention has been devoted to these approaches because of the high potency and proliferation ability of the cells. This review will focus on the general characteristics and the progress in hepatic differentiation of adult somatic stem cells and extraembryonic stem cells in vitro and in vivo for the treatment of end stage liver diseases. The hepatic differentiation of stem cells would offer an ideal and promising source for cell therapy and tissue engineering for treating liver diseases.

  5. [Prevalence of subclinical udder infections and individual somatic cell counts in three dairy goat herds during a full lactation].

    Science.gov (United States)

    Schaeren, W; Maurer, J

    2006-12-01

    For dairy goats, both the determination of the somatic cell counts (SCC) and the interpretation of these values may be a problem. Several investigations have shown that SCC for goat's milk, even from not infected mammary halves, are often higher than for cows milk. In the three herds examined about 40% of mammary halves and 30% of the goats were infected. However large differences between the three herds could be observed. In most cases, infections were caused by coagulase negative staphylococci (CNS) or corynebacteria. The SCC of individual milk samples from goats without any udder infection hardly differed from those of goats with at least one udder half infected with CNS. In 20% and 30% of the cases the SCC was higher than 750'000 cells/ml, respectively. The relation between California Mastitis Test (CMT) reactions and udder infections was not very close. Over 20% of mammary halves infected with CNS showed negative CMT reactions. On the other hand, 25% of samples from mammary halves without a proven infection reacted positively. The large differences in individual cell counts on herd and animal level indicate that production and breeding systems might be important reasons for the higher SCC. As a consequence, the most common methods for or the control of udder health and udder infections (SCC, California Mastitis Test) are of limited value for goats. Since there was only a weak relation between milk quality properties and SCC, any arguments for the introduction of legal limits below 1 million cells per ml can hardly be found. PMID:17263081

  6. Production of transgenic cashmere goat embryos expressing red fluorescent protein and containing IGF1 hair-follicle-cell specific expression cassette by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    BOU; ShorGan

    2009-01-01

    In the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1 (IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasts cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h. Parthenogenetic ooctyes were used as a model to investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO- Faa and CR1aa; 86.3% vs 83.9%, P>0.05 and 23.1% vs 17.2%,P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P<0.05). After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly ex- pressing red fluorescence. Two of the red fluorescent blastocysts were randomly selected to identify transgene by polymerase chain reaction. Both were positive. These results showed that: (i) RFP and Neor genes were correctly expressed indicating that transgenic somatic cell lines and positive trans- genic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and

  7. Production of transgenic cashmere goat embryos expressing red fluorescent protein and containing IGF1 hair-follicle-cell specific expression cassette by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    GUO XuDong; YANG DongShan; Ao XuDong; WU Xia; LI GuangPeng; WANG LingLing; BAO MingTao; XUE Lian; BOU ShorGan

    2009-01-01

    In the present study, cashmere goat fetal flbroblasta were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1(IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasta cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h.Parthenogenetic ooctyes were used as a model to Investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO-Faa and CR1aa; 86.3% va 83.9%, P>0.05 and 23.1% vs 17.2%, P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P<0.05).After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly ex-pressing red fluorescence. Two of the red fluorescent blastocysta were randomly selected to identify transgene by polymeraee chain reaction. Both were positive. These results showed that: (i) RFP and Neo genes were correctly expressed indicating that transgenlc somatic cell lines and positive trans-genic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and

  8. Radiation-induced changes to mammalian cells as a precipitating factor in somatic radiation injuries

    International Nuclear Information System (INIS)

    Radiation-induced inhibitions of proliferation were assessed in cell cultures examined for their colony-forming abilities as well as from changes of growth curves. The results of those measurements, along with simulating calculations, underlined the fact that the colony-forming capacity of a cell can by no means be equated with cell survival, unless due attention is given to the size of the colony formed. It is the size of the colony that provides a measure of the damage done to the irradiated cell. Cells counts are the most reliable method to ascertain the course of proliferation following radiation exposure. The difference between the two methods mentioned became particularly evident in studies with radiation protection substances. Dithiothreitol (DTT) and mercaptopropionyl glycine (MPG) were on the basis of colony formation clearly shown to offer protection against radiation. The growth curves, however, revealed that the proliferation of cells irradiated in the presence of radiation protection substances was even more strongly inhibited than that of cells influenced by irradiation alone. The neutral elution method failed to provide irrefutable evidence that the rate of double strand breaks was reduced by those two substances. Cysteamine and DTT were, however, able to inhibit radiation-induced changes to the proteins of human erythrocyte membranes. (orig./MG)

  9. Processed pseudogenes acquired somatically during cancer development

    OpenAIRE

    Cooke, Susanna L.; Shlien, Adam; Marshall, John; Pipinikas, Christodoulos P; Martincorena, Inigo; Tubio, Jose M. C.; Li, Yilong; Menzies, Andrew; Mudie, Laura; Ramakrishna, Manasa; Yates, Lucy; Davies, Helen; Bolli, Niccolo; Bignell, Graham R; Tarpey, Patrick S.

    2014-01-01

    Cancer evolves by mutation, with somatic reactivation of retrotransposons being one such mutational process. Germline retrotransposition can cause processed pseudogenes, but whether this occurs somatically has not been evaluated. Here we screen sequencing data from 660 cancer samples for somatically acquired pseudogenes. We find 42 events in 17 samples, especially non-small cell lung cancer (5/27) and colorectal cancer (2/11). Genomic features mirror those of germline LINE element retrotransp...

  10. Effect of Oxytocin Administration before Milking on Milk Production, Somatic Cells Count and fat Contents in Milk of Nili-Ravi Buffaloes

    Directory of Open Access Journals (Sweden)

    Muhammad Saleem Akhtar*, Laeeq Akbar Lodhi1, Abdul Asim Farooq, M. Mazhar Ayaz, Maqbool Hussain, Mushtaq Hussain Lashari and Zafar Iqbal Chaudhary

    2012-06-01

    Full Text Available This study was escorted to know the effect of oxytocin administration before milking on milk production, somatic cells count and fat contents in milk of buffaloes. Twenty lactating Nili-Ravi buffaloes were randomly divided into two groups. Group A (n = 10 buffaloes were treated intramuscularly with 30 IU of oxytocin daily before the start of milking for the period of 7 days, whereas group B (n = 10 buffaloes were given no treatment and served as control. Milk samples were collected from all buffaloes 7 days before (Phase I, during (Phase II and after (Phase III the treatment. There were significantly higher (P<0.05 milk production (liters during phase-II in group A (8.57±0.07 liters buffaloes as compare to group B (8.40±0.04 liters whereas non-significant differences were recorded in the mean milk production between group A and B during phase-I (8.46 vs 8.43 liters and III (8.54 liters. Somatic cells count varied from 72.96 to 97.01 × 103 and 71.86 to 77.14 × 103 cells per ml in group A and B, respectively. Mean somatic cells count were significantly higher (P<0.05 in group A as compared to group B during phases II of study. During phase I, II and III, there were non-significant differences in fat percentage between two groups of buffaloes. It was concluded that milk production and somatic cells count in milk of Nili-Ravi buffalo were affected by oxytocin injection before milking whereas there was no effect of oxytocin on milk fat percentage.

  11. Origin of the somatic cells in the rat gonad: An autoradiographic approach

    International Nuclear Information System (INIS)

    Gonads from rats of 11 and 12 days of gestation that had been given a pulse of 3H-thymidine were studied at different times using autoradiographic techniques. When the animals were labeled before the formation of the gonadal blastema (11 days), the percentage of labeled cells in this structure was greater than in the coelomic epithelium and mesenchyme. However, when 3H-thymidine was injected after the gonadal blastema was already established (12 days), very few of these cells took up the compound; they remained in this conditions until the sexual differentiation of the gonad. From this characteristic, which is an indication of the early differentiation of the blastemal cells, one may conclude that the seminiferous cords arise from this initial cellular line. On the other hand, there appears to be a contribution of coelomic epithelial cells during the formation of the sex cords in the ovary

  12. Comparative pluripotency analysis of mouse embryonic stem cells derived from wild-type and infertile hermaphrodite somatic cell nuclear transfer blastocysts

    Institute of Scientific and Technical Information of China (English)

    FAN Yong; YAO RuQiang; YU Yang; LI ZanDong; WANG Liu; ALICE Jouneau; ZHOU Qi; TONG Man; ZHAO ChunLi; DING ChenHui; HAO Jie; LV Zhuo; DAI XiangPeng; HAI Tang; LI XueMei

    2008-01-01

    Therapeutic cloning, whereby embryonic stem cells (ESCs) are derived from patient-specific cloned blastocysts via somatic cell nuclear transfer (SCNT), holds great promise for treating many human diseases using regenerative medicine. Teratoma formation and germline transmission have been used to confirm the pluripotency of mouse stem cells, but human embryonic stem cells (hESCs) have not been proven to be fully pluripotent owing to the ethical impossibility of testing for germ line transmission, which would be the strongest evidence for full pluripotency. Therefore, formation of differentiated cells from the three somatic germ layers within a teratoma is taken as the best indicator of pluripotency in hESC lines. The possibility that these lines lack full multi- or pluripotency has not yet been evaluated.In this study, we established 16 mouse ESC lines, including 3 genetically defective nuclear transfer-ESC (ntESC) lines derived from SCNT blastocysts of infertile hermaphrodite F1 mice and 13 ntESC lines derived from SCNT blastocysts of normal F1 mice. We found that the defective ntESCs expressed all in vitro markers of pluripotency and could form teratomas that included derivatives from all three germ layers, but could not be transmitted via the germ line, in contrast with normal ntESCs. Our results indicate that teratoma formation assays with hESCs might be an insufficient standard to assess full pluripotency, although they do define multipotency to some degree. More rigorous standards are required to assess the safety of hESCs for therapeutic cloning.

  13. Effect of estrone on somatic and female gametophyte cell division and differentiation in Arabidospis thaliana cultured in vitro

    Directory of Open Access Journals (Sweden)

    Piotr Żabicki

    2014-04-01

    Full Text Available The aim of the study was to determine the effect of the mammalian female sex hormone estrone on differentiation of somatic tissues and on induction of autonomous endosperm in culture of female gametophyte cells of Arabidopsis thaliana ecotype Columbia (Col-0. In culture, estrone-stimulated development of autonomous endosperm (AE occurred in 14.7% of unpollinated pistils. The AE represented development stages similar to those of young endosperm after fertilization and AE of fis mutants in vivo. In the majority of ovules the AE was in a few-nucleate young stage. Some ovules showed more advanced stages of AE development, with nuclei and cytoplasm forming characteristic nuclear cytoplasmic domains (NCDs. Sporadically, AE was divided into regions characteristic for Arabidopsis endosperm formed after fertilization. Direct organogenesis (caulogenesis, rhizogenesis, callus proliferation and formation of trichome-like structures were observed during in vitro culture of hypocotyls and cotyledons of 3-day-old seedlings cultured on medium supplemented with estrone for 28 days. Histological analysis showed adventitious root formation and changes in explant anatomy caused by estrone.

  14. Single nucleotide polymorphisms in candidate genes and their relation with somatic cell scores in Argentinean dairy cattle.

    Science.gov (United States)

    Nani, Juan P; Raschia, Maria A; Carignano, Hugo; Poli, Mario A; Calvinho, Luis F; Amadio, Ariel F

    2015-11-01

    The prevention and control of bovine mastitis by enhancing natural defenses in animals is important to improve the quality of dairy products. Mastitis resistance is a complex trait which depends on genetic components, as well as environmental and physiological factors. The limitations of classical control measures have led to the search for alternative approaches to minimize the use of antibiotics by selecting naturally resistant animals. Polymorphisms in genes associated with the innate immune system are strong candidates to be evaluated as genetic markers. In this work, we evaluated a set of single nucleotide polymorphisms (SNPs) in candidate genes for health and production traits, and determined their association with the somatic cell score (SCS) as an indicator of mastitis in Argentinean dairy cattle. We evaluated 941 cows: Holstein (n = 677) and Holstein × Jersey (n = 264) crossbred, daughters from 22 bulls from 14 dairy farms located in the central dairy area of Argentina. Two of the 21 successfully genotyped markers were found to be significantly associated (p < 0.05) with the SCS: GHR_140 and OPN_8514C-T. The heterozygote genotype for GHR_140 showed a favorable effect in reducing the SCS. On the other hand, heterozygote genotypes for OPN8514C-T caused an increase in the SCS; moreover, combined genotypes for OPN SNPs showed an even larger effect. These findings can contribute to the design of effective marker-assisted selection programs. PMID:25783851

  15. Stochastic anomaly of methylome but persistent SRY hypermethylation in disorder of sex development in canine somatic cell nuclear transfer.

    Science.gov (United States)

    Jeong, Young-Hee; Lu, Hanlin; Park, Chi-Hun; Li, Meiyan; Luo, Huijuan; Kim, Joung Joo; Liu, Siyang; Ko, Kyeong Hee; Huang, Shujia; Hwang, In Sung; Kang, Mi Na; Gong, Desheng; Park, Kang Bae; Choi, Eun Ji; Park, Jung Hyun; Jeong, Yeon Woo; Moon, Changjong; Hyun, Sang-Hwan; Kim, Nam Hyung; Jeung, Eui-Bae; Yang, Huanming; Hwang, Woo Suk; Gao, Fei

    2016-01-01

    Somatic cell nuclear transfer (SCNT) provides an excellent model for studying epigenomic reprogramming during mammalian development. We mapped the whole genome and whole methylome for potential anomalies of mutations or epimutations in SCNT-generated dogs with XY chromosomal sex but complete gonadal dysgenesis, which is classified as 78, XY disorder of sex development (DSD). Whole genome sequencing revealed no potential genomic variations that could explain the pathogenesis of DSD. However, extensive but stochastic anomalies of genome-wide DNA methylation were discovered in these SCNT DSD dogs. Persistent abnormal hypermethylation of the SRY gene was observed together with its down-regulated mRNA and protein expression. Failure of SRY expression due to hypermethylation was further correlated with silencing of a serial of testis determining genes, including SOX9, SF1, SOX8, AMH and DMRT1 in an early embryonic development stage at E34 in the XY(DSD) gonad, and high activation of the female specific genes, including FOXL2, RSPO1, CYP19A1, WNT4, ERα and ERβ, after one postnatal year in the ovotestis. Our results demonstrate that incomplete demethylation on the SRY gene is the driving cause of XY(DSD) in these XY DSD dogs, indicating a central role of epigenetic regulation in sex determination. PMID:27501986

  16. Receiver-operating characteristic curves for somatic cell scores and California mastitis test in Valle del Belice dairy sheep.

    Science.gov (United States)

    Riggio, Valentina; Pesce, Lorenzo L; Morreale, Salvatore; Portolano, Baldassare

    2013-06-01

    Using receiver-operating characteristic (ROC) curve methodology this study was designed to assess the diagnostic effectiveness of somatic cell count (SCC) and the California mastitis test (CMT) in Valle del Belice sheep, and to propose and evaluate threshold values for those tests that would optimally discriminate between healthy and infected udders. Milk samples (n=1357) were collected from 684 sheep in four flocks. The prevalence of infection, as determined by positive bacterial culture was 0.36, 87.7% of which were minor and 12.3% major pathogens. Of the culture negative samples, 83.7% had an SCCCMT results were evaluated, the estimated area under the ROC curve was greater for glands infected with major compared to minor pathogens (0.88 vs. 0.73), whereas the area under the curve considering all pathogens was similar to the one for minor pathogens (0.75). The estimated optimal thresholds were 3.00 (CMT), 2.81 (SCS for the whole sample), 2.81 (SCS for minor pathogens), and 3.33 (SCS for major pathogens). These correctly classified, respectively, 69.0%, 73.5%, 72.6% and 91.0% of infected udders in the samples. The CMT appeared only to discriminate udders infected with major pathogens. In this population, SCS appeared to be the best indirect test of the bacteriological status of the udder. PMID:23317658

  17. DNA-damaging potency and genotoxicity of aflatoxin M1 in somatic cells in vivo of Drosophila melanogaster.

    Science.gov (United States)

    Shibahara, T; Ogawa, H I; Ryo, H; Fujikawa, K

    1995-05-01

    Aflatoxin M1 (AFM1), a metabolic hydroxylation product of aflatoxin B1 (AFB1), and the parent compound were comparatively assayed for DNA-damaging potency and genotoxicity in vivo in Drosophila melanogaster using, respectively, the mei-9a mei-41D5 DNA repair test and the mwh/flr3 wing spot test. In the repair test, larval stock, consisting of meiotic recombination-deficient double mutant mei-9a mei-41D5 males and repair-proficient females, was exposed to the test agents, and the preferential killing of the mutant larvae was taken as evidence of the DNA-damaging effect. In this test, AFM1 was registered as a DNA-damaging agent with an activity approximately 3-fold lower than that of AFB1. In the wing spot test, where larval flies, trans-heterozygous for the somatic cell markers mwh and flr3, were treated and the wings were inspected at adulthood for spots manifesting the phenotypes of the markers, AFM1 exerted a genotoxic effect compatible to that of AFB1. Based on these results and other data, we predict that AFM1 may be genotoxic in mammalian in-vivo systems as well. PMID:7666765

  18. Stochastic anomaly of methylome but persistent SRY hypermethylation in disorder of sex development in canine somatic cell nuclear transfer

    Science.gov (United States)

    Jeong, Young-Hee; Lu, Hanlin; Park, Chi-Hun; Li, Meiyan; Luo, Huijuan; Kim, Joung Joo; Liu, Siyang; Ko, Kyeong Hee; Huang, Shujia; Hwang, In Sung; Kang, Mi Na; Gong, Desheng; Park, Kang Bae; Choi, Eun Ji; Park, Jung Hyun; Jeong, Yeon Woo; Moon, Changjong; Hyun, Sang-Hwan; Kim, Nam Hyung; Jeung, Eui-Bae; Yang, Huanming; Hwang, Woo Suk; Gao, Fei

    2016-01-01

    Somatic cell nuclear transfer (SCNT) provides an excellent model for studying epigenomic reprogramming during mammalian development. We mapped the whole genome and whole methylome for potential anomalies of mutations or epimutations in SCNT-generated dogs with XY chromosomal sex but complete gonadal dysgenesis, which is classified as 78, XY disorder of sex development (DSD). Whole genome sequencing revealed no potential genomic variations that could explain the pathogenesis of DSD. However, extensive but stochastic anomalies of genome-wide DNA methylation were discovered in these SCNT DSD dogs. Persistent abnormal hypermethylation of the SRY gene was observed together with its down-regulated mRNA and protein expression. Failure of SRY expression due to hypermethylation was further correlated with silencing of a serial of testis determining genes, including SOX9, SF1, SOX8, AMH and DMRT1 in an early embryonic development stage at E34 in the XYDSD gonad, and high activation of the female specific genes, including FOXL2, RSPO1, CYP19A1, WNT4, ERα and ERβ, after one postnatal year in the ovotestis. Our results demonstrate that incomplete demethylation on the SRY gene is the driving cause of XYDSD in these XY DSD dogs, indicating a central role of epigenetic regulation in sex determination. PMID:27501986

  19. Spatial patterns of recorded mastitis incidence and somatic cell counts in Swedish dairy cows: implications for surveillance

    Directory of Open Access Journals (Sweden)

    Cecilia Wolff

    2011-11-01

    Full Text Available Clinical mastitis (CM is the most common veterinary treated disease in Swedish dairy cattle. To investigate if the distribution of veterinary registered cases of CM in Sweden follows that of the spatial distribution of cows with high somatic cell counts (SCCs, the spatial distribution of CM odds was estimated from available records and compared with udder health measures based on measurements of SCC derived from official milk recording. The study revealed areas with significantly lower odds for CM but with a high proportion of cows with a poor udder health score, suggesting an under-reporting of CM. We also found areas of significantly higher odds for CM despite a low proportion of cows with a poor udder health score, suggestive of over-treatment of mastitis. The results should enable targeted studies of reasons for discrepancies, e.g. farmers’ and veterinarians’ attitudes to mastitis treatment and disease recording in areas with a deficit or excess of registered CM cases. High quality disease records for dairy cattle are of interest not only for the dairy management but also for disease surveillance, monitoring of use of antibiotics and food safety purposes.

  20. Changes in Some Biochemical Parameters and Somatic Cell Counts in the Milk of Buffalo and Cattle Suffering from Mastitis

    Directory of Open Access Journals (Sweden)

    Riaz Hussain§, Muhammad Tariq Javed and Ahrar Khan*

    2012-06-01

    Full Text Available The study was conducted on a total of 592 buffaloes and 453 cattle in their different stages of lactation to investigate the biochemical changes occurring in milk due to mastitis. California Mastitis Test (CMT was used to diagnose the mammary gland infection. The results revealed significant (P<0.0001 increase in pH, electrical conductivity, malondialdehyde and total dissolved solids, while decrease in fat, protein, lactose and solids not fat in milk samples of both mastitic buffaloes and cattle. The total somatic cell and neutrophil counts were significantly higher, while the macrophage and lymphocytes were lower in the milk of mastitic animals. The enzymes including lactate dehydrogenase, aspartate aminotransferase and alkaline phosphatase along with sodium were significantly higher in mastitic than healthy buffaloes. It was similar in cattle as well, with the exception of aspartate aminotransferase which was non-significant in cattle. The values of potassium, phosphorous, calcium, magnesium, zinc and iron were significantly higher in the milk of mastitic animals. The copper levels were significantly (P<0.0001 lower in mastitic than in healthy buffaloes, while it showed non-significant difference in cattle. The investigation of enzymes, lipid peroxidation product and milk electrical conductivity in present study appeared suitable diagnostic tools for identification of mastitis.

  1. Spindle formation and microtubule organization during first division in reconstructed rat embryos produced by somatic cell nuclear transfer.

    Science.gov (United States)

    Tomioka, Ikuo; Mizutani, Eiji; Yoshida, Tomoyuki; Sugawara, Atsushi; Inai, Kentaro; Sasada, Hiroshi; Sato, Eimei

    2007-08-01

    The present study was conducted to demonstrate the spindle formation and behavior of chromosomes and microtubules during first division in reconstructed rat embryos produced by somatic cell nuclear transfer (SCNT) with cumulus cell nuclei. To demonstrate the effect of oocyte aging after ovulation on the cleavage of SCNT embryos, micromanipulation was carried out 11, 15 and 18 h after injection of hCG. SCNT oocytes were activated by incubation in culture medium supplemented with 5 microM ionomycin for 5 min followed by treatment with 2 mM 6-dimethylaminopurine (6-DMAP) in mR1ECM for 2-3 h. For immunocytochemical observation, the SCNT embryos were incubated with monoclonal anti-alpha-tubulin antibody and then fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Cleavage rates were significantly higher for oocytes collected after 15 and 18 h rather than for those collected 11 h after injection of hCG (56 and 53%, respectively vs. 28%; P<0.05). Premature chromosome condensation occurred before activation of the SCNT oocytes, but adequate spindle formation was only rarely observed. The distribution of microtubules in SCNT embryos after activation was different from those of fertilized and parthenogenic oocytes, i.e., a dense microtubule organization shaped like a ring was observed. Eighteen to 20 h post-activation, most SCNT embryos were in the 2-cell stage, but no nucleoli were clearly visible, which was quite different from the fertilized oocytes. In addition, first division with and without small cellular bodies containing DNA was observed in the rat SCNT embryos in some cases. The present study suggests that reorganization of transferred nuclei in rat SCNT embryos may be inadequate in terms of formation of the mitotic assembly and nucleolar reorganization. PMID:17446658

  2. In Vitro Developmental Potential of Cloned Embryos Derived from Bovine Somatic Cells and Rabbits Oocyte

    Institute of Scientific and Technical Information of China (English)

    LIU Ya; LI Bin; ZHAO Huan; CHENG Li-zi; ZHANG Xiao-rong; CHEN Da-yuan; ZHANG Yun-hai; ZHANG Zhi-guo; JING Ren-tao; WANG Cun-li; ZHANG Mei-lin; LI Dong-wei

    2003-01-01

    180 reconstituted embryos were produced by nuclear transplantation using bovine ear fibroblasts at G0 or non-G0 stage as donor nuclei and oocytes collected from superovulated multiparous or young rabbits as recipients. After cultivation in two kinds of medium M199+ 10%FBS or RD+ 10%FBS, 112 of them developed to 2-cell stage (62.2%) and 26 to morula stage (14.4%) and 20 of them eventually developed to blastocyst stage (11. 1% ). There is no significant difference for the cleavage rates in two groups of reconstituted embryos derived from G0-stage and non-G0 stage donor cells respectively. However, G0-stage donor cells could result in higher rate of 8-cell - 16-cell stage embryos significantly (P<0.05), as well as higher rate of blastocysts (P<0.01). It seems that using two different culture systems had no significant effects on the cleavage rate, morula rate or blastocyst rate (P>0.05).

  3. Effect of weightlessness conditions on the somatic embryogenesis in the culture of carrot cells

    Science.gov (United States)

    Butenko, R. G.; Dmitriyeva, N. N.; Ongko, V.; Basyrova, L. V.

    1977-01-01

    A carrot cell culture seeded in Petri dishes in the United States and transported to the USSR was subjected to weightlessness for 20 days during the flight of Kosmos 782. The controls were cultures placed on a centrifuge (1 g) inside the satellite and cultures left on ground in the U.S.S.R. and the United States. A count of structures in the dishes after the flight showed that the number of developing embryonic structures and the extent of their differentiation in weightlessness did not reliably differ from the number and extent of differentiation in structures developed on the ground. Structures with long roots developed in weightlessness. Analysis of the root zones showed that these roots differed by the increased size of the zone of differentiated cells. The increased size of the zones of differentiated cells can indicate earlier development of embryonic structures.

  4. Cellular heredity in haploid cultures of somatic cells. Comprehensive report, April 1975--June 1977

    International Nuclear Information System (INIS)

    This report reviews genetic studies carried out since 1975 on a haploid cultured cell line from frog embryos (ICR 2A). Although a single chromosome set would be expected to facilitate recovery of recessive mutants, experiments suggested that cell culture variants might arise through processes more complex than the selection of simple mutational changes. Therefore, the objectives of the work reported here have been to throw light on just how cell culture variants arise in this system. First, we have continued to characterize the ICR 2A line, with emphasis on stability of karyotype and DNA content. Second, we have studied in detail the origin of two classes of drug-resistant variants. Bromodeoxyuridine resistance of the thymidine deficiency type has been shown to arise through sequential loss of two forms of thymidine-phosphorylating enzyme; loss of the second form of enzyme is complex, suggesting that changes more complex than simple recessive mutations may be involved. Another form of resistance, in which tolerance of high levels of bromodeoxyuridine is found in cells that continue to express thymidine kinase, remains under study. Variants resistant to microtubule inhibitors were isolated. It was found that these haploid strains have properties distinguishing them from analogous resistant strains isolated from diploid mammalian cell cultures in other laboratories. In order to understand better how mutagens are involved in the origin of cell culture variants, we have examined the effect of different forms of DNA repair on the frequency of drug-resistant colonies induced by ultraviolet radiation. Preliminary experiments suggest that the frequency of such colonies is greater when repair takes place through (presumably error-prone) dark repair than when (error-free) photoreversal is allowed to occur. Such experiments can determine whether new phenotypes arise from alterations in DNA, and thus whether, in a broad sense, they are likely to be mutational in nature

  5. Cellular heredity in haploid cultures of somatic cells. Comprehensive report, April 1975--June 1977. [UV radiation

    Energy Technology Data Exchange (ETDEWEB)

    Freed, J.J.

    1977-07-01

    This report reviews genetic studies carried out since 1975 on a haploid cultured cell line from frog embryos (ICR 2A). Although a single chromosome set would be expected to facilitate recovery of recessive mutants, experiments suggested that cell culture variants might arise through processes more complex than the selection of simple mutational changes. Therefore, the objectives of the work reported here have been to throw light on just how cell culture variants arise in this system. First, we have continued to characterize the ICR 2A line, with emphasis on stability of karyotype and DNA content. Second, we have studied in detail the origin of two classes of drug-resistant variants. Bromodeoxyuridine resistance of the thymidine deficiency type has been shown to arise through sequential loss of two forms of thymidine-phosphorylating enzyme; loss of the second form of enzyme is complex, suggesting that changes more complex than simple recessive mutations may be involved. Another form of resistance, in which tolerance of high levels of bromodeoxyuridine is found in cells that continue to express thymidine kinase, remains under study. Variants resistant to microtubule inhibitors were isolated. It was found that these haploid strains have properties distinguishing them from analogous resistant strains isolated from diploid mammalian cell cultures in other laboratories. In order to understand better how mutagens are involved in the origin of cell culture variants, we have examined the effect of different forms of DNA repair on the frequency of drug-resistant colonies induced by ultraviolet radiation. Preliminary experiments suggest that the frequency of such colonies is greater when repair takes place through (presumably error-prone) dark repair than when (error-free) photoreversal is allowed to occur. Such experiments can determine whether new phenotypes arise from alterations in DNA, and thus whether, in a broad sense, they are likely to be mutational in nature.

  6. Investigation of risk factors of bovine mastitis in Ethiopia; Isolation of mastitis causing agents and determination of the content of somatic cells in milk

    OpenAIRE

    Frese, Mathias Lutz

    2010-01-01

    In this thesis, the risk factors of bovine mastitis in different milk production systems in Ethiopia were investigated. Furthermore, mastitis causing agents were isolated after California Mastitis Test (CMT) was used as the field test. Somatic cells were counted and compared with the CMT. Low milk production and low quality of milk are apparently related to a lack of proper hygienic measures throughout the farm clusters.

  7. Relations between electrical conductivity, somatic cell count, California mastitis test and some quality parameters in the diagnosis of subclinical mastitis in dairy cows

    OpenAIRE

    KAŞIKÇI, Güven; ÇETİN, Ömer; BİNGÖL, Enver Barış; GÜNDÜZ, Mehmet Can

    2012-01-01

    This study was conducted to determine the effectiveness of the electrical conductivity (EC) method on the diagnosis of subclinical mastitis in dairy cows comparing with somatic cell count (SCC) and California mastitis test (CMT), and also to investigate the effect of these values on the amount of total viable bacteria, density, freezing point, and mineral substances. A total of 386 milk samples collected from quarters of 188 cows at 10 different farms were used as materials. Of the samples, 2...

  8. Somatic Cell Fusions Reveal Extensive Heterogeneity in Basal-like Breast Cancer

    DEFF Research Database (Denmark)

    Su, Ying; Subedee, Ashim; Bloushtain-Qimron, Noga;

    2015-01-01

    heterogeneity in basal-like breast cancers that correlates with clinical outcome. We also found that protein extracts of basal-like cells are sufficient to induce a luminal-to-basal phenotypic switch, implying a trigger of basal-like autoregulatory circuits. We determined that KDM6A might be required for...

  9. Radiogenotoxicological effect of signal nuclide 134Cs on somatic and germ cells

    Institute of Scientific and Technical Information of China (English)

    ZhuShou-Peng; XiaFen; 等

    1997-01-01

    Chromosome at aberation rates in bone marrow cells and micronucleus formation in bone marrow polychromatic erythrocytes both rise with increase in radioactivities of 134Cs,and can be fitted to power functions of radioactivities of 134Cs.In spermatogonia 134Cs mainly induced chromatid breakage,and abnormalities in sperm can also be experessed as power functions of radioactivities of 134 Cs.

  10. Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer

    NARCIS (Netherlands)

    Peifer, Martin; Fernandez-Cuesta, Lynnette; Sos, Martin L.; George, Julie; Seidel, Danila; Kasper, Lawryn H.; Plenker, Dennis; Leenders, Frauke; Sun, Ruping; Zander, Thomas; Menon, Roopika; Koker, Mirjam; Dahmen, Ilona; Mueller, Christian; Di Cerbo, Vincenzo; Schildhaus, Hans-Ulrich; Altmueller, Janine; Baessmann, Ingelore; Becker, Christian; de Wilde, Bram; Vandesompele, Jo; Boehm, Diana; Ansen, Sascha; Gabler, Franziska; Wilkening, Ines; Heynck, Stefanie; Heuckmann, Johannes M.; Lu, Xin; Carter, Scott L.; Cibulskis, Kristian; Banerji, Shantanu; Getz, Gad; Park, Kwon-Sik; Rauh, Daniel; Gruetter, Christian; Fischer, Matthias; Pasqualucci, Laura; Wright, Gavin; Wainer, Zoe; Russell, Prudence; Petersen, Iver; Chen, Yuan; Stoelben, Erich; Ludwig, Corinna; Schnabel, Philipp; Hoffmann, Hans; Muley, Thomas; Brockmann, Michael; Engel-Riedel, Walburga; Muscarella, Lucia A.; Fazio, Vito M.; Groen, Harry; Timens, Wim; Sietsma, Hannie; Thunnissen, Erik; Smit, Egbert; Heideman, Danielle A. M.; Snijders, Peter J. F.; Cappuzzo, Federico; Ligorio, Claudia; Damiani, Stefania; Field, John; Solberg, Steinar; Brustugun, Odd Terje; Lund-Iversen, Marius; Saenger, Joerg; Clement, Joachim H.; Soltermann, Alex; Moch, Holger; Weder, Walter; Solomon, Benjamin; Soria, Jean-Charles; Validire, Pierre; Besse, Benjamin; Brambilla, Elisabeth; Brambilla, Christian; Lantuejoul, Sylvie; Lorimier, Philippe; Schneider, Peter M.; Hallek, Michael; Pao, William; Meyerson, Matthew; Sage, Julien; Shendure, Jay; Schneider, Robert; Buettner, Reinhard; Wolf, Juergen; Nuernberg, Peter; Perner, Sven; Heukamp, Lukas C.; Brindle, Paul K.; Haas, Stefan; Thomas, Roman K.

    2012-01-01

    Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor prognosis(1-3). We sequenced 29 SCLC exomes, 2 genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4 +/- 1 protein-changing mutations per million base pairs. Therefore, we conducted integrated analys

  11. Differences during the first lactation between cows cloned by somatic cell nuclear transfer and noncloned cows.

    Science.gov (United States)

    Montazer-Torbati, F; Boutinaud, M; Brun, N; Richard, C; Neveu, A; Jaffrézic, F; Laloë, D; LeBourhis, D; Nguyen, M; Chadi, S; Jammes, H; Renard, J-P; Chat, S; Boukadiri, A; Devinoy, E

    2016-06-01

    Lactation performance is dependent on both the genetic characteristics and the environmental conditions surrounding lactating cows. However, individual variations can still be observed within a given breed under similar environmental conditions. The role of the environment between birth and lactation could be better appreciated in cloned cows, which are presumed to be genetically identical, but differences in lactation performance between cloned and noncloned cows first need to be clearly evaluated. Conflicting results have been described in the literature, so our aim was to clarify this situation. Nine cloned Prim' Holstein cows were produced by the transfer of nuclei from a single fibroblast cell line after cell fusion with enucleated oocytes. The cloned cows and 9 noncloned counterparts were raised under similar conditions. Milk production and composition were recorded monthly from calving until 200d in milk. At 67d in milk, biopsies were sampled from the rear quarter of the udder, their mammary epithelial cell content was evaluated, and mammary cell renewal, RNA, and DNA were then analyzed in relevant samples. The results showed that milk production did not differ significantly between cloned and noncloned cows, but milk protein and fat contents were less variable in cloned cows. Furthermore, milk fat yield and contents were lower in cloned cows during early lactation. At around 67 DIM, milk fat and protein yields, as well as milk fat, protein, and lactose contents, were also lower in cloned cows. These lower yields could be linked to the higher apoptotic rate observed in cloned cows. Apoptosis is triggered by insulin-like factor growth binding protein 5 (IGFBP5) and plasminogen activator inhibitor (PAI), which both interact with CSN1S2. During our experiments, CSN1S2 transcript levels were lower in the mammary gland of cloned cows. The mammary cell apoptotic rate observed in cloned cows may have been related to the higher levels of DNA (cytosine-5

  12. Prediction of the herd somatic cell count of the following month using a linear mixed effect model.

    Science.gov (United States)

    Lievaart, J J; Barkema, H W; van den Broek, J; Heesterbeek, J A P; Kremer, W D J

    2010-01-01

    An accurate prediction of the average somatic cell count (SCC) for the next month would be a valuable tool to support udder health management decisions. A linear mixed effect (LME) model was used to predict the average herd SCC (HSCC) for the following month. The LME model included data on SCC, herd characteristics, season, and management practices determined in a previous study that quantified the contribution of each factor for the HSCC. The LME model was tested on a new data set of 101 farms and included data from 3 consecutive years. The farms were split randomly in 2 groups of 50 and 51 farms. The first group of 50 farms was used to check for systematic errors in predicting monthly HSCC. An initial model was based on older data from a different part of the Netherlands and systematically overestimated HSCC in most months. Therefore, the model was adjusted for the difference in average HSCC between the 2 sets of farms (from the previous and current study) using the data from the first group of 50 farms. Subsequently, the data from the second group of 51 farms were used to independently assess this final model. A null model (no explanatory variables included) predicted 48 and 59% of the HSCC within the predetermined range of 20,000 and 30,000 cells/mL, respectively. The final LME model predicted 72 and 81% of the HSCC of the next month correctly within these 2 ranges. These outcomes indicate that the final LME model was a valid additional tool for farmers that could be useful in their short-term decisions regarding udder health management and could be included in dairy herd health programs. PMID:20059921

  13. Astaxanthin Normalizes Epigenetic Modifications of Bovine Somatic Cell Cloned Embryos and Decreases the Generation of Lipid Peroxidation.

    Science.gov (United States)

    Li, R; Wu, H; Zhuo, W W; Mao, Q F; Lan, H; Zhang, Y; Hua, S

    2015-10-01

    Astaxanthin is an extremely common antioxidant scavenging reactive oxygen species (ROS) and blocking lipid peroxidation. This study was conducted to investigate the effects of astaxanthin supplementation on oocyte maturation, and development of bovine somatic cell nuclear transfer (SCNT) embryos. Cumulus-oocyte complexes were cultured in maturation medium with astaxanthin (0, 0.5, 1.0, or 1.5 mg/l), respectively. We found that 0.5 mg/l astaxanthin supplementation significantly increased the proportion of oocyte maturation. Oocytes cultured in 0.5 mg/l astaxanthin supplementation were used to construct SCNT embryos and further cultured with 0, 0.5, 1.0 or 1.5 mg/l astaxanthin. The results showed that the supplementation of 0.5 mg/l astaxanthin significantly improved the proportions of cleavage and blastulation, as well as the total cell number in blastocysts compared with the control group, yet this influence was not concentration dependent. Chromosomal analyses revealed that more blastomeres showed a normal chromosomal complement in 0.5 mg/l astaxanthin treatment group, which was similar to that in IVF embryos. The methylation levels located on the exon 1 of the imprinted gene H19 and IGF2, pluripotent gene OCT4 were normalized, and global DNA methylation, H3K9 and H4K12 acetylation were also improved significantly, which was comparable to that in vitro fertilization (IVF) embryos. Moreover, we also found that astaxanthin supplementation significantly decreased the level of lipid peroxidation. Our findings showed that the supplementation of 0.5 mg/l astaxanthin to oocyte maturation medium and embryo culture medium improved oocyte maturation, SCNT embryo development, increased chromosomal stability and normalized the epigenetic modifications, as well as inhibited overproduction of lipid peroxidation. PMID:26280670

  14. Crystal Structures of Proto-oncogene Kinase Pim1: A Target of Aberrant Somatic Hypermutations in Diffuse Large Cell Lymphoma

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Abhinav; Mandiyan, Valsan; Suzuki, Yoshihisa; Zhang, Chao; Rice, Julie; Tsai, James; Artis, Dean R.; Ibrahim, Prabha; Bremer, Ryan (Plexxikon); (Plexxikon)

    2010-07-19

    Pim1, a serine/threonine kinase, is involved in several biological functions including cell survival, proliferation, and differentiation. While pim1 has been shown to be involved in several hematopoietic cancers, it was also recently identified as a target of aberrant somatic hypermutation in diffuse large cell lymphoma (DLCL), the most common form of non-Hodgkin's lymphoma. The crystal structures of Pim1 in apo form and bound with AMPPNP have been solved and several unique features of Pim1 were identified, including the presence of an extra {beta}-hairpin in the N-terminal lobe and an unusual conformation of the hinge connecting the two lobes of the enzyme. While the apo Pim1 structure is nearly identical with that reported recently, the structure of AMPPNP bound to Pim1 is significantly different. Pim1 is unique among protein kinases due to the presence of a proline residue at position 123 that precludes the formation of the canonical second hydrogen bond between the hinge backbone and the adenine moiety of ATP. One crystal structure reported here shows that changing P123 to methionine, a common residue that offers the backbone hydrogen bond to ATP, does not restore the ATP binding pocket of Pim1 to that of a typical kinase. These unique structural features in Pim1 result in novel binding modes of AMP and a known kinase inhibitor scaffold, as shown by co-crystallography. In addition, the kinase activities of five Pim1 mutants identified in DLCL patients have been determined. In each case, the observed effects on kinase activity are consistent with the predicted consequences of the mutation on the Pim1 structure. Finally, 70 co-crystal structures of low molecular mass, low-affinity compounds with Pim1 have been solved in order to identify novel chemical classes as potential Pim1 inhibitors. Based on the structural information, opportunities for optimization of one specific example are discussed.

  15. Somatic cell chromosome changes in a population exposed to low levels of ionizing radiation

    International Nuclear Information System (INIS)

    The analysis of chromosomes from the cells of 897 plutonium workers is reported. Within three years, the number of controls alone analyzed for this study approximated the largest plutonium cytogenetic studies today including workers plus controls (81 compared to 84 in a 1979 French study and 94 in a 1982 British report). The number of subjects analyzed in the first three years were: new employees - 245; new employees assigned to plutonium work areas - 7; workers with less than 3% of maximum permissible systemic burden (MPSB) - 35; workers with less than 50% MPSB - 274; workers with greater than 50% of MPSB - 65; follow-up familial congenital cytogenetics at worker request (through Medical) - 6; polymorphic/variant chromosome constitutions - 242; re-sampling of workers with elevated aberration yields - 26; cell sample study - 28; sister-chromatid-exchange (SCE) study - 23; beryllium workers at Rocky Flats - 10; Hanford worker analyses - 5). 20 refs., 3 figs., 5 tabs

  16. B cell-autonomous somatic mutation deficit following bone marrow transplant

    OpenAIRE

    Glas, A M

    2000-01-01

    The bone marrow is the major haematopoietic organ and is critically involved in the production of all formed blood elements in postnatal life. The bone marrow contains rapidly dividing cells and therefore is sensitive to DNA damaging agents. In certain types of cancers where a high dose of radiation and chemotherapeutic agents are needed, a bone marrow transplant is necessary to "rescue" the patient from the lethal side effects of radiation and chemotherapy. However, the immune system of tran...

  17. Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells

    OpenAIRE

    Sharma Shikhar; Gerke Daniel S; Han Han F; Jeong Shinwu; Stallcup Michael R; Jones Peter A; Liang Gangning

    2012-01-01

    Abstract Background DNA methylation, histone modifications and nucleosome occupancy act in concert for regulation of gene expression patterns in mammalian cells. Recently, G9a, a H3K9 methyltransferase, has been shown to play a role in establishment of DNA methylation at embryonic gene targets in ES cells through recruitment of de novo DNMT3A/3B enzymes. However, whether G9a plays a similar role in maintenance of DNA methylation in somatic cells is still unclear. Results Here we show that G9a...

  18. DNA methylation in porcine preimplantation embryos developed in vivo or produced by in vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Deshmukh, Rahul Shahaji; Østrup, Olga; Østrup, Esben;

    2011-01-01

    vitro fertilized (IVF), somatic cell nuclear transfer (SCNT) and parthenogenetically activated (PA) embryos were evaluated for DNA methylation quantification at different developmental stages. Fertilized (IV and IVF) one-cell stages lacked a substantial active demethylation of the paternal genome....... Embryos produced under in vitro conditions had higher levels of DNA methylation than IV. A lineage-specific DNA methylation (hypermethylation of the inner cell mass and hypomethylation of the trophectoderm) was observed in porcine IV late blastocysts, but was absent in PA- and SCNT-derived blastocysts...

  19. DNA methylation in porcine preimplantation embryos developed in-vivo or produced by in-vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Deshmukh, Rahul Shahaji; Østrup, Olga; Østrup, Esben;

    2011-01-01

    vitro fertilized (IVF), somatic cell nuclear transfer (SCNT) and parthenogenetically activated (PA) embryos were evaluated for DNA methylation quantification at different developmental stages. Fertilized (IV and IVF) one-cell stages lacked a substantial active demethylation of the paternal genome....... Embryos produced under in vitro conditions had higher levels of DNA methylation than IV. A lineage-specific DNA methylation (hypermethylation of the inner cell mass and hypomethylation of the trophectoderm) was observed in porcine IV late blastocysts, but was absent in PA- and SCNT-derived blastocysts...

  20. Radiation induced chromosome aberrations in somatic and germ cells of the male marmoset

    International Nuclear Information System (INIS)

    The induction of chromosome aberrations by low LET radiations was studied in peripheral lymphocytes and spermatogonial stem cells of the male marmoset. The data showed that there was no significant difference in the sensitivity of the lymphocytes whether they were irradiated in vitro or in vivo, but the frequency of heritable translocations recovered in the primary spermatocytes was considerably lower than that calculated to occur in the lymphocytes. The data are used to make estimates of human genetic risk from radiation based on limited interspecific comparisons

  1. Regularities of the formation of comutagenesis in irradiated human somatic cells

    International Nuclear Information System (INIS)

    The pattern (regularity) of co-mutagenic effects formation in healthy individuals' irradiated cells (T-lymphocytes) under verapamil and ascorbic acid treatment is studied. It is determined that the additional action of the investigated drugs in concentrations greater than the therapeutic one potentiate the effects of low doses of ionizing radiation by 1.5 times. Ascorbic acid and verapamil show co-mutagenic effects under high-dose irradiation (2.0 Gy), increasing the chromosomal aberration level by 1.4 times, regardless of the concentration of drugs.

  2. Sexual Fate Change of XX Germ Cells Caused by the Deletion of SMAD4 and STRA8 Independent of Somatic Sex Reprogramming.

    Science.gov (United States)

    Wu, Quan; Fukuda, Kurumi; Kato, Yuzuru; Zhou, Zhi; Deng, Chu-Xia; Saga, Yumiko

    2016-09-01

    The differential programming of sperm and eggs in gonads is a fundamental topic in reproductive biology. Although the sexual fate of germ cells is believed to be determined by signaling factors from sexually differentiated somatic cells in fetal gonads, the molecular mechanism that determines germ cell fate is poorly understood. Herein, we show that mothers against decapentaplegic homolog 4 (SMAD4) in germ cells is required for female-type differentiation. Germ cells in Smad4-deficient ovaries respond to retinoic acid signaling but fail to undergo meiotic prophase I, which coincides with the weaker expression of genes required for follicular formation, indicating that SMAD4 signaling is essential for oocyte differentiation and meiotic progression. Intriguingly, germline-specific deletion of Smad4 in Stra8-null female germ cells resulted in the up-regulation of genes required for male gonocyte differentiation, including Nanos2 and PLZF, suggesting the initiation of male-type differentiation in ovaries. Moreover, our transcriptome analyses of mutant ovaries revealed that the sex change phenotype is achieved without global gene expression changes in somatic cells. Our results demonstrate that SMAD4 and STRA8 are essential factors that regulate the female fate of germ cells. PMID:27606421

  3. The influence of selenium and zinc addition in food on concentration of these elements in blood and milk, on somatic cells number and histological characteristics of cows udders

    Directory of Open Access Journals (Sweden)

    Davidov Ivana

    2014-01-01

    Full Text Available The experiment included 30 cows of Holstein-Friesian breed, out of which 15 were receiving selenium and zinc in optimal doses before calving, while the others had never been supplemented with these micronutrients. There was analysed the concentration of selenium and zinc in blood and milk serum as well as the average number of somatic cells in corresponding lactation. After the cows exclusion from production, histological characteristics of cows udders were examined. The results of the investigation have shown that addition of selenium and zinc before calving has a positive effect on the values of these microelements in the blood and milk during the period of early lactation, that is, the concentration of these elements was significantly higher in the blood and milk of the cows that obtained selenium and zinc supplements. Also, in these cows there was significantly lower number of somatic cells during the following lacation period. In the parenchyma of the udder there was found less pronounced infiltration of leukocytes, notably thicker keratin layer of ductus papillaris and less expressed repairing processes that indicate a chronic inflammation of the udder in the samples after exclusion of the cows from production. There was a significant positive correlation between selenium in blood and milk, while there was not observed such a correlation for zinc. On the other hand, there was a significant negative correlation between the concentration of selenium in the blood and milk with the average number of somatic cells and the degree of infiltration of leukocytes, while its influence on the keratin layer of ductus papillarus was not shown. Zinc from blood and udder had a negative correlation with the number of somatic cells, had a positive correlation with the thickness of ductus papillaris keratin layer and had no influence on the level of leukocyte infiltration of udder parenchyma. Zinc demonstrates a positive influence on the formation of ductus

  4. Digital PCR for quantification of recurrent and potentially actionable somatic mutations in circulating free DNA from patients with diffuse large B-cell lymphoma.

    Science.gov (United States)

    Camus, Vincent; Sarafan-Vasseur, Nasrin; Bohers, Elodie; Dubois, Sydney; Mareschal, Sylvain; Bertrand, Philippe; Viailly, Pierre-Julien; Ruminy, Philippe; Maingonnat, Catherine; Lemasle, Emilie; Stamatoullas, Aspasia; Picquenot, Jean-Michel; Cornic, Marie; Beaussire, Ludivine; Bastard, Christian; Frebourg, Thierry; Tilly, Hervé; Jardin, Fabrice

    2016-09-01

    Diffuse large B-cell lymphoma (DLBCL) is an aggressive and heterogeneous malignancy harboring frequent targetable activating somatic mutations. Emerging evidence suggests that circulating cell-free DNA (cfDNA) can be used to detect somatic variants in DLBCL using Next-Generation Sequencing (NGS) experiments. In this proof-of-concept study, we chose to develop simple and valuable digital PCR (dPCR) assays for the detection of recurrent exportin-1 (XPO1) E571K, EZH2 Y641N, and MYD88 L265P mutations in DLBCL patients, thereby identifying patients most likely to potentially benefit from targeted therapies. We demonstrated that our dPCR assays were sufficiently sensitive to detect rare XPO1, EZH2, and MYD88 mutations in plasma cfDNA, with a sensitivity of 0.05%. cfDNA somatic mutation detection by dPCR seems to be a promising technique in the management of DLBCL, in addition to NGS experiments. PMID:26883583

  5. Candidate gene association analysis for milk yield, composition, urea nitrogen and somatic cell scores in Brown Swiss cows.

    Science.gov (United States)

    Cecchinato, A; Ribeca, C; Chessa, S; Cipolat-Gotet, C; Maretto, F; Casellas, J; Bittante, G

    2014-07-01

    The aim of this study was to investigate 96 single-nucleotide polymorphisms (SNPs) from 54 candidate genes, and test the associations of the polymorphic SNPs with milk yield, composition, milk urea nitrogen (MUN) content and somatic cell score (SCS) in individual milk samples from Italian Brown Swiss cows. Milk and blood samples were collected from 1271 cows sampled once from 85 herds. Milk production, quality traits (i.e. protein, casein, fat and lactose percentages), MUN and SCS were measured for each milk sample. Genotyping was performed using a custom Illumina VeraCode GoldenGate approach. A Bayesian linear animal model that considered the effects of herd, days in milk, parity, SNP genotype and additive polygenic effect was used for the association analysis. Our results showed that 14 of the 51 polymorphic SNPs had relevant additive effects on at least one of the aforementioned traits. Polymorphisms in the glucocorticoid receptor DNA-binding factor 1 (GRLF1), prolactin receptor (PRLR) and chemokine ligand 2 (CCL2) were associated with milk yield; an SNP in the stearoyl-CoA desaturase (SCD-1) was related to fat content; SNPs in the caspase recruitment domain 15 protein (CARD15) and lipin 1 (LPIN1) affected the protein and casein contents; SNPs in growth hormone 1 (GH1), lactotransferrin (LTF) and SCD-1 were relevant for casein number; variants in beta casein (CSN2), GH1, GRLF1 and LTF affected lactose content; SNPs in beta-2 adrenergic receptor (ADRB2), serpin peptidase inhibitor (PI) and SCD-1 were associated with MUN; and SNPs in acetyl-CoA carboxylase alpha (ACACA) and signal transducer and activator of transcription 5A (STAT5A) were relevant in explaining the variation of SCS. Although further research is needed to validate these SNPs in other populations and breeds, the association between these markers and milk yield, composition, MUN and SCS could be exploited in gene-assisted selection programs for genetic improvement purposes. PMID:24804775

  6. Effects of milk somatic cell counts on some physicochemical and functional characteristics of skim and whole milk powders.

    Science.gov (United States)

    Sert, Durmuş; Mercan, Emin; Aydemir, Serdar; Civelek, Mustafa

    2016-07-01

    The aim of this work was to study the influence of milk somatic cell count (SCC) levels on spray-dried milk powders. For this reason, 3 cow milks with different SCC (700,000 SCC/mL) were processed into skim (SMP) and whole milk powder (WMP). The effect of SCC on the physicochemical and functional characteristics of the milk powders and textural properties of set-type yogurts produced from reconstituted milk powders with different SCC was evaluated. A crucial difference was noted between milk powders depending on different SCC. Protein values and ash content of powder samples decreased correlatively with increasing SCC. The hydroxymethylfurfural content of SMP was higher than WMP. We noted an increase in hydroxymethylfurfural content of both SMP and WMP depending on elevated SCC. Solubility index of SMP and WMP was 1.280 to 1.632 and 0.940 to 1.208mL, respectively; with increasing SCC, solubility index was affected adversely. The highest foam stability was determined in SMP containing >700,000 SCC. Bulk density of SMP and WMP was between 0.682 and 0.708 and 0.660 to 0.685g/cm(3), respectively. An increase was observed in scorched particle of both SMP and WMP depending on increasing SCC. We found significant differences in particle size distribution of milk powders produced from milk with SCC at different levels. Although WMP had more uniform and big particle structure, SMP had more specific area. A negative correlation was noted between yogurt texture and SCC. Results indicate that milk SCC has negative influences on milk powder quality. PMID:27179852

  7. Different screening tests and milk somatic cell count for the prevalence of subclinical bovine mastitis in Bangladesh.

    Science.gov (United States)

    Hoque, Md Nazmul; Das, Ziban Chandra; Talukder, Anup Kumar; Alam, Mohammad Shah; Rahman, Abu Nasar Md Aminoor

    2015-01-01

    Identification of cows with subclinical mastitis (SCM) is an important tool for sustainable dairying and implementing effective mastitis control strategies. A total of 892 quarters milk samples from 228 lactating cows were screened by California mastitis test (CMT), White side test (WST), Surf field mastitis test (SFMT), and somatic cell count (SCC) to study the prevalence of bovine SCM in some selected areas of Bangladesh. Out of 228 cows, 148 (64.9%), 138 (60.5%), 132 (57.9%), and 164 (71.9%) were found positive for SCM by CMT, WST, SFMT, and SCC, respectively. The prevalence of bovine SCM was diagnosed 45.7, 40.2, 36.6, and 29.6% in Chittagong, Sirajgonj, Mymensingh, and Gazipur districts, respectively, based on a combination of all tests. The overall quarter-wise prevalence of SCM was 45.7, 43.5, 41.2, and 55.0% for CMT, WST, SFMT, and SCC. Single quarters and left front quarters were more prone to SCM (P CMT, WST, SFMT, and SCC was 65.8, 57.9, 51.0, and 82.5%; specificity 76.2, 72.4, 69.5, and 89.4%; percentage accuracy 70.0, 64.8, 59.9, and 85.2%; positive predictive value 75.2, 69.8, 64.9, and 92.7%, respectively. The categories of CMT reactions were strongly correlated with SCC (P tests (SCC>CMT>WST>SFMT). Thus, CMT was concluded to be the most accurate (r = 0.782) field diagnostic test after laboratory test like SCC (r = 0.924). However, the use of any single test may not be reliable in diagnosing SCM, while the result of CMT supported by SCC might be used effectively to pinpoint diagnosis of SCM in dairy animals than alone. PMID:25326717

  8. Short communication: Genetic correlation of bovine leukosis incidence with somatic cell score and milk yield in a US Holstein population.

    Science.gov (United States)

    Abdalla, E A; Weigel, K A; Byrem, T M; Rosa, G J M

    2016-03-01

    Bovine leukosis (BL) is a retroviral disease caused by the bovine leukosis virus (BLV), which affects only cattle. Dairy cows positive for BL produce less milk and have more days open than cows negative for BL. In addition, the virus also affects the immune system and causes weaker response to vaccines. Heritability estimates of BL incidence have been reported for Jersey and Holstein populations at about 0.08, indicating an important genetic component that can potentially be exploited to reduce the prevalence of the disease. However, before BL is used in selection programs, it is important to study its genetic associations with other economically important traits such that correlated responses to selection can be predicted. Hence, this study aimed to estimate the genetic correlations of BL with milk yield (MY) and with somatic cell score (SCS). Data of a commercial assay (ELISA) used to detect BLV antibodies in milk samples were obtained from Antel BioSystems (Lansing, MI). The data included continuous milk ELISA scores and binary milk ELISA results for 11,554 cows from 112 dairy herds across 16 US states. Continuous and binary milk ELISA were analyzed with linear and threshold models, respectively, together with MY and SCS using multitrait animal models. Genetic correlations (posterior means ± standard deviations) between BL incidence and MY were 0.17±0.077 and 0.14±0.076 using ELISA scores and results, respectively; with SCS, such estimates were 0.20±0.081 and 0.17±0.079, respectively. In summary, the results indicate that selection for higher MY may lead to increased BLV prevalence in dairy herds, but that the inclusion of BL (or SCS as an indicator trait) in selection indexes may help attenuate this problem. PMID:26778307

  9. EFFECT OF SEVERITY OF SUB-CLINICAL MASTITIS ON SOMATIC CELL COUNT AND LACTOSE CONTENTS OF BUFFALO MILK

    Directory of Open Access Journals (Sweden)

    A. SHARIF, T. AHMAD, M. Q. BILAL1, A. YOUSAF AND G. MUHAMMAD

    2007-07-01

    Full Text Available This study was conducted to determine the effect of severity of sub-clinical mastitis on somatic cell count (SCC and lactose contents of milk in 100 apparently healthy dairy buffaloes. Surf Field Mastitis Test (SFMT was used to determine the severity of sub-clinical mastitis which was graded as Negative (N, Traces (T, mild clumping (P1, moderate clumping (P2 and heavy clumping (P3. Mean milk SCC (x 105 at SFMT scores N, T, P1, P2 and P3 were 2.06 + 1.09, 3.73 + 0.96, 9.69 + 4.05, 31.97 + 10.26 and 121.01 + 23.71 per ml, respectively. Using the same scoring, mean values of milk lactose were 5.10 + 0.09, 4.81 + 0.10, 4.66 + 0.08, 3.92 + 0.05 and 2.66 + 0.37 percent, respectively. Percent increases of mean SCC in T, P1, P2 and P3 groups with respect to N (control were 81.47, 370.51, 1451.71 and 5773.41, respectively. Percent decreases of mean lactose in T, P1, P2 and P3 groups with respect to N (control were 5.54, 8.52, 22.98 and 47.81, respectively. Statistical analysis indicated non-significant difference of mean SCC in N and T groups, while there was highly significant (P<0.01 difference in mean SCC among P1, P2 and P3 groups and also with respect to N. Similarly, there was a significant (P<0.05 difference of mean lactose among T, P1, P2 and P3 groups and also with respect to control/ negative group.

  10. Spontaneous squamous cell carcinoma induced by the somatic inactivation of retinoblastoma and Trp53 tumor suppressors.

    Science.gov (United States)

    Martínez-Cruz, Ana Belén; Santos, Mirentxu; Lara, M Fernanda; Segrelles, Carmen; Ruiz, Sergio; Moral, Marta; Lorz, Corina; García-Escudero, Ramón; Paramio, Jesús M

    2008-02-01

    Squamous cell carcinomas (SCC) represent the most aggressive type of nonmelanoma skin cancer. Although little is known about the causal alterations of SCCs, in organ-transplanted patients the E7 and E6 oncogenes of human papillomavirus, targeting the p53- and pRb-dependent pathways, have been widely involved. Here, we report the functional consequences of the simultaneous elimination of Trp53 and retinoblastoma (Rb) genes in epidermis using Cre-loxP system. Loss of p53, but not pRb, produces spontaneous tumor development, indicating that p53 is the predominant tumor suppressor acting in mouse epidermis. Although the simultaneous inactivation of pRb and p53 does not aggravate the phenotype observed in Rb-deficient epidermis in terms of proliferation and/or differentiation, spontaneous SCC development is severely accelerated in doubly deficient mice. The tumors are aggressive and undifferentiated and display a hair follicle origin. Detailed analysis indicates that the acceleration is mediated by premature activation of the epidermal growth factor receptor/Akt pathway, resulting in increased proliferation in normal and dysplastic hair follicles and augmented tumor angiogenesis. The molecular characteristics of this model provide valuable tools to understand epidermal tumor formation and may ultimately contribute to the development of therapies for the treatment of aggressive squamous cancer. PMID:18245467

  11. Integrative genome analyses identify key somatic driver mutations of small cell lung cancer

    Science.gov (United States)

    Peifer, Martin; Fernández-Cuesta, Lynnette; Sos, Martin L; George, Julie; Seidel, Danila; Kasper, Lawryn H; Plenker, Dennis; Leenders, Frauke; Sun, Ruping; Zander, Thomas; Menon, Roopika; Koker, Mirjam; Dahmen, Ilona; Müller, Christian; Di Cerbo, Vincenzo; Schildhaus, Hans-Ulrich; Altmüller, Janine; Baessmann, Ingelore; Becker, Christian; de Wilde, Bram; Vandesompele, Jo; Böhm, Diana; Ansén, Sascha; Gabler, Franziska; Wilkening, Ines; Heynck, Stefanie; Heuckmann, Johannes M; Lu, Xin; Carter, Scott L; Cibulskis, Kristian; Banerji, Shantanu; Getz, Gad; Park, Kwon-Sik; Rauh, Daniel; Grütter, Christian; Fischer, Matthias; Pasqualucci, Laura; Wright, Gavin; Wainer, Zoe; Russell, Prudence; Petersen, Iver; Chen, Yuan; Stoelben, Erich; Ludwig, Corinna; Schnabel, Philipp; Hoffmann, Hans; Muley, Thomas; Brockmann, Michael; Engel-Riedel, Walburga; Muscarella, Lucia A; Fazio, Vito M; Groen, Harry; Timens, Wim; Sietsma, Hannie; Thunnissen, Erik; Smit, Egbert; Heideman, Daniëlle AM; Snijders, Peter JF; Cappuzzo, Federico; Ligorio, Claudia; Damiani, Stefania; Field, John; Solberg, Steinar; Brustugun, Odd Terje; Lund-Iversen, Marius; Sänger, Jörg; Clement, Joachim H; Soltermann, Alex; Moch, Holger; Weder, Walter; Solomon, Benjamin; Soria, Jean-Charles; Validire, Pierre; Besse, Benjamin; Brambilla, Elisabeth; Brambilla, Christian; Lantuejoul, Sylvie; Lorimier, Philippe; Schneider, Peter M; Hallek, Michael; Pao, William; Meyerson, Matthew; Sage, Julien; Shendure, Jay; Schneider, Robert; Büttner, Reinhard; Wolf, Jürgen; Nürnberg, Peter; Perner, Sven; Heukamp, Lukas C; Brindle, Paul K; Haas, Stefan; Thomas, Roman K

    2016-01-01

    Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor survival1–3. We sequenced 29 SCLC exomes, two genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4±1 protein-changing mutations per million basepairs. Therefore, we conducted integrated analyses of the various data sets to identify pathogenetically relevant mutated genes. In all cases we found evidence for inactivation of TP53 and RB1 and identified recurrent mutations in histone-modifying genes, CREBBP, EP300, and MLL. Furthermore, we observed mutations in PTEN, in SLIT2, and EPHA7, as well as focal amplifications of the FGFR1 tyrosine kinase gene. Finally, we detected many of the alterations found in humans in SCLC tumors from p53/Rb1-deficient mice4. Our study implicates histone modification as a major feature of SCLC, reveals potentially therapeutically tractable genome alterations, and provides a generalizable framework for identification of biologically relevant genes in the context of high mutational background. PMID:22941188

  12. Herd management and social variables associated with bulk tank somatic cell count in dairy herds in the eastern United States.

    Science.gov (United States)

    Schewe, R L; Kayitsinga, J; Contreras, G A; Odom, C; Coats, W A; Durst, P; Hovingh, E P; Martinez, R O; Mobley, R; Moore, S; Erskine, R J

    2015-11-01

    The ability to reduce somatic cell counts (SCC) and improve milk quality depends on the effective and consistent application of established mastitis control practices. The US dairy industry continues to rely more on nonfamily labor to perform critical tasks to maintain milk quality. Thus, it is important to understand dairy producer attitudes and beliefs relative to management practices, as well as employee performance, to advance milk quality within the changing structure of the dairy industry. To assess the adoption rate of mastitis control practices in United States dairy herds, as well as assess social variables, including attitudes toward employees relative to mastitis control, a survey was sent to 1,700 dairy farms in Michigan, Pennsylvania, and Florida in January and February of 2013. The survey included questions related to 7 major areas: sociodemographics and farm characteristics, milking proficiency, milking systems, cow environment, infected cow monitoring and treatment, farm labor, and attitudes toward mastitis and related antimicrobial use. The overall response rate was 41% (21% in Florida, 39% in Michigan, and 45% in Pennsylvania). Herd size ranged from 9 to 5,800 cows. Self-reported 3-mo geometric mean bulk tank SCC (BTSCC) for all states was 194,000 cells/mL. Multivariate analysis determined that proven mastitis control practices such as the use of internal teat sealants and blanket dry cow therapy, and not using water during udder preparation before milking, were associated with lower BTSCC. Additionally, farmer and manager beliefs and attitudes, including the perception of mastitis problems and the threshold of concern if BTSCC is above 300,000 cells/mL, were associated with BTSCC. Ensuring strict compliance with milking protocols, giving employees a financial or other penalty if BTSCC increased, and a perceived importance of reducing labor costs were negatively associated with BTSCC in farms with nonfamily employees. These findings highlight the

  13. Composition, functional properties and sensory characteristics of Mozzarella cheese manufactured from different somatic cell counts in milk

    Directory of Open Access Journals (Sweden)

    Evelise Andreatta

    2009-10-01

    Full Text Available In the present study, composition, functional properties and sensory characteristics of Mozzarella cheese produced from milk with somatic cell counts (SCC at low (800,000 cells/mL levels were investigated. Three batches of cheese were produced for each SCC category. The cheeses were vacuum packed in plastic bags and analysed after 2, 9, 16, 23 and 30 days of storage at 4ºC. SCC level did not affect the moisture, fat, total protein and ash content, mesophilic and psychrotrophic bacteria, and sensory parameters of Mozzarella cheese. However, meltability increased in cheese manufactured from high SCC milk. Results indicated that raw milk used to produce Mozzarella cheese should not contain high SCC (>800,000 cells/mL in order to avoid changes in the functional properties of the Mozzarella cheese.No presente estudo foram investigadas a composição, as propriedades funcionais e as características sensoriais do queijo Mussarela produzido a partir de leite com contagens de células somáticas (CCS em níveis baixos (800.000 CS/mL. Foram produzidos 3 lotes de queijo para cada CCS. Os queijos foram embalados a vácuo e analisados após 2, 9, 16, 23 e 30 dias de armazenamento a 4ºC. O nível de CS não afetou a umidade, os teores de gordura, proteína total e cinzas, os níveis de bactérias mesófilas e psicrotróficas, e os parâmetros sensoriais do queijo Mussarela. Entretanto, houve aumento da capacidade de derretimento no queijo fabricado com leite de alta CCS. Os resultados indicam que o leite cru utilizado para a produção de queijo Mussarela não deve conter níveis de CS acima de 800.000/mL, para evitar alterações nas propriedades funcionais do queijo Mussarela.

  14. Classical Mus musculus Igκ Enhancers Support Transcription but not High Level Somatic Hypermutation from a V-Lambda Promoter in Chicken DT40 Cells

    OpenAIRE

    Kothapalli, Naga Rama; Norton, Darrell D.; Fugmann, Sebastian D.

    2011-01-01

    Somatic hypermutation (SHM) of immunoglobulin genes is initiated by activation-induced cytidine deaminase (AID) in activated B cells. This process is strictly dependent on transcription. Hence, cis-acting transcriptional control elements have been proposed to target SHM to immunoglobulin loci. The Mus musculus Igκ locus is regulated by the intronic enhancer (iE/MAR) and the 3′ enhancer (3′E), and multiple studies using transgenic and knock-out approaches in mice and cell lines have reported s...

  15. Impact of Somatic Mutations in the D-Loop of Mitochondrial DNA on the Survival of Oral Squamous Cell Carcinoma Patients

    OpenAIRE

    Lin, Jin-Ching; Wang, Chen-Chi; Jiang, Rong-San; Wang, Wen-yi; Liu, Shih-An

    2015-01-01

    Objectives The aim of this study was to investigate somatic mutations in the D-loop of mitochondrial DNA (mtDNA) and their impact on survival in oral squamous cell carcinoma patients. Materials and Methods Surgical specimen confirmed by pathological examination and corresponding non-cancerous tissues were collected from 120 oral squamous cell carcinoma patients. The sequence in the D-loop of mtDNA from non-cancerous tissues was compared with that from paired cancer samples and any sequence di...

  16. Near Infrared Microspectroscopy, Fluorescence Microspectroscopy, Infrared Chemical Imaging and High Resolution Nuclear Magnetic Resonance Analysis of Soybean Seeds, Somatic Embryos and Single Cells

    CERN Document Server

    Baianu, I C; Hofmann, N E; Korban, S S; Lozano, P; You, T; AOCS 94th Meeting, Kansas

    2002-01-01

    Novel methodologies are currently being developed and established for the chemical analysis of soybean seeds, embryos and single cells by Fourier Transform Infrared (FT-IR), Fourier Transform Near Infrared (FT-NIR) Microspectroscopy, Fluorescence and High-Resolution NMR (HR-NMR). The first FT-NIR chemical images of biological systems approaching one micron resolution are presented here. Chemical images obtained by FT-NIR and FT-IR Microspectroscopy are presented for oil in soybean seeds and somatic embryos under physiological conditions. FT-NIR spectra of oil and proteins were obtained for volumes as small as two cubic microns. Related, HR-NMR analyses of oil contents in somatic embryos are also presented here with nanoliter precision. Such 400 MHz 1H NMR analyses allowed the selection of mutagenized embryos with higher oil content (e.g. ~20%) compared to non-mutagenized control embryos. Moreover, developmental changes in single soybean seeds and/or somatic embryos may be monitored by FT-NIR with a precision ...

  17. Embryo aggregation does not improve the development of interspecies somatic cell nuclear transfer embryos in the horse.

    Science.gov (United States)

    Gambini, Andrés; De Stéfano, Adrián; Jarazo, Javier; Buemo, Carla; Karlanian, Florencia; Salamone, Daniel Felipe

    2016-09-01

    The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as enucleated oocytes donors in our iSCNT study. We then assessed whether embryo aggregation could improve the in vitro development of ZF equine iSCNT embryos after reconstruction with porcine, bovine, and feline ooplasm. Bovine- and porcine-aggregated PA blastocysts had significantly larger diameters compared with nonaggregated embryos. On the other hand, feline- and bovine-aggregated PA embryos had higher blastocyst cell number. Embryo aggregation of equine-equine SCNT was found to be beneficial for embryo development as we have previously reported, but the aggregation of three ZF reconstructed embryos did not improve embryo developmental rates on iSCNT. In vitro embryo development of nonaggregated iSCNT was predominantly arrested around the stage when transcriptional activation of the embryonic genome is reported to start on the embryo of the donor species. Nevertheless, independent of embryo aggregation, equine blastocyst-like structures could be obtained in our study using domestic feline-enucleated oocytes. Taken together, these results reported that embryo aggregation enhance in vitro PA embryo development and embryo quality but effects vary depending on the species. Embryo aggregation also improves, as expected, the in vitro embryo development of equine-equine SCNT embryos; however, we did not observe positive effects on equine iSCNT embryo development. Among oocytes

  18. Development of porcine embryos reconstituted with somatic cells and enucleated metaphase I and II oocytes matured in a protein-free medium

    Directory of Open Access Journals (Sweden)

    Gibbons John R

    2001-07-01

    Full Text Available Abstract Background Many cloned animals have been created by transfer of differentiated cells at G0/G1 or M phase of the cell cycle into enucleated M II oocytes having high maturation/meiosis/mitosis-promoting factor activity. Because maturation/meiosis/mitosis-promoting factor activity during oocyte maturation is maximal at both M I and M II, M I oocytes may reprogram differentiated cell nuclei as well. The present study was conducted to examine the developmental ability in vitro of porcine embryos reconstructed by transferring somatic cells (ear fibroblasts into enucleated M I or M II oocytes. Results Analysis of the cell cycle stages revealed that 91.2 ± 0.2% of confluent cells were at the G0/G1 phase and 54.1 ± 4.4% of nocodazole-treated cells were at the G2/M phase, respectively. At 6 h after activation, nuclear swelling was observed in 50.0-88.9% and 34.4-39.5% of embryos reconstituted with confluent cells and nocodazole-treated cells regardless of the recipient oocytes, respectively. The incidence of both a swollen nucleus and polar body was low (6.3-10.5% for all nocodazole-treated donor cell regardless of the recipient oocyte. When embryos reconstituted with confluent cells and M I oocytes were cultured, 2 (1.5% blastocysts were obtained and this was significantly (P Conclusions Porcine M I oocytes have a potential to develop into blastocysts after nuclear transfer of somatic cells.

  19. Next generation sequencing of patients with mut methylmalonic aciduria: Validation of somatic cell studies and identification of 16 novel mutations.

    Science.gov (United States)

    Chu, Jordan; Pupavac, Mihaela; Watkins, David; Tian, Xia; Feng, Yanming; Chen, Stella; Fenter, Remington; Zhang, Victor W; Wang, Jing; Wong, Lee-Jun; Rosenblatt, David S

    2016-08-01

    Mutations in the MUT gene, which encodes the mitochondrial enzyme methylmalonyl-CoA mutase, are responsible for the mut form of methylmalonic aciduria (MMA). In this study, a next generation sequencing (NGS) based gene panel was used to analyze 53 patients that had been diagnosed with mut MMA by somatic cell complementation analysis. A total of 54 different mutations in MUT were identified in 48 patients; 16 novel mutations were identified, including 1 initiation site mutation (c.2T>C [p.M1?]), 1 missense mutation (c.566A>T [p.N189I]), 2 nonsense mutations (c.129G>A [p.W43*] and c.1975C>T [p.Q659*]), 2 mutations affecting splice sites (c.753+3A>G and c.754-2A>G), 8 small insertions, deletions, and duplications (c.29dupT [p.L10Ffs*39], c.55dupG [p.V19Gfs*30], c.631_633delGAG [p.E211del], c.795_796insT [p.M266Yfs*7], c.1061delCinsGGA [p.S354Wfs*20], c.1065_1068dupATGG [p.S357Mfs*5], c.1181dupT [p.L394Ffs*30], c.1240delG [p.E414Kfs*17]), a large insertion (c.146_147ins279), and a large deletion involving exon 13. Phenotypic rescue and cDNA analysis were used to confirm that the c.146_147ins279 and c.631_633delGAG mutations were associated with the decreased methylmalonyl-CoA mutase function observed in the patient fibroblasts. In five patients, the NGS panel did not confirm the diagnosis made by complementation analysis. One of these patients was found to carry 2 novel mutations (c.433G > A [p.E145K] and c.511A>C [p.N171H]) in the SUCLG1 gene. PMID:27233228

  20. Exploring the characteristics and dynamics of Ontario dairy herds experiencing increases in bulk milk somatic cell count during the summer.

    Science.gov (United States)

    Shock, D A; LeBlanc, S J; Leslie, K E; Hand, K; Godkin, M A; Coe, J B; Kelton, D F

    2015-06-01

    Regionally aggregated bulk milk somatic cell count (BMSCC) data from around the world shows a repeatable cyclicity, with the highest levels experienced during warm, humid seasons. No studies have evaluated this seasonal phenomenon at the herd level. The objectives of this study were to define summer seasonality in BMSCC on an individual herd basis, and subsequently to describe the characteristics and dynamics of herds with increased BMSCC in the summer. The data used for this analysis were from all dairy farms in Ontario, Canada, between January 2000 and December 2011 (n≈4,000 to 6,000 herds/yr). Bulk milk data were obtained from the milk marketing board and consisted of bulk milk production, components (fat, protein, lactose, other solids), and quality (BMSCC, bacterial count, inhibitor presence, freezing point), total milk quota of the farm, and milk quota and incentive fill percentage. A time-series linear mixed model, with random slopes and intercepts, was constructed using sine and cosine terms as predictors to describe seasonality, with herd as a random effect. For each herd, seasonality was described with reference to 1 cosine function of variable amplitude and phase shift. The predicted months of maximal and minimal BMSCC were then calculated. Herds were assigned as low, medium, and high summer increase (LSI, MSI, and HSI, respectively) based on percentiles of amplitude in BMSCC change for each of the 4 seasons. Using these seasonality classifications, 2 transitional repeated measures logistic regression models were built to assess the characteristics of MSI and HSI herds, using LSI herds as controls. Based on the analyses performed, a history of summer BMSCC increases increased the odds of experiencing a subsequent increase. As herd size decreased, the odds of experiencing HSI to MSI in BMSCC increased. Herds with more variability in daily BMSCC were at higher odds of experiencing MSI and HSI in BMSCC, as were herds with lower annual mean BMSCC. Finally

  1. The number of oogonia and somatic cells in the human female embryo and fetus in relation to whether or not exposed to maternal cigarette smoking

    DEFF Research Database (Denmark)

    Lutterodt, M C; Sørensen, K P; Larsen, K B;

    2009-01-01

    utero exposure to cigarette smoking. METHODS: Twenty-nine human first-trimester ovaries from legal abortions [aged 38-64 days post-conception (p.c.)] were collected. Mothers filled out a questionnaire about their smoking habits and delivered a urine sample for cotinine analysis. The ovarian cell numbers...... were estimated using stereological methods. RESULTS: A non-linear correlation between the numbers of oogonia and somatic cells in relation to age of the embryo/fetus was shown in 28 ovaries, including the first estimates performed in ovaries younger than 47 days p.c. Prenatal exposure to smoke showed a...... significant decrease in the number of somatic cells (P < or = 0.01). The number of oogonia was not significantly associated with prenatal exposure to maternal smoking (P < or = 0.09). The ratio between the two cell types decreased considerably from 1:45 to 1:23 from 38 to 46 days p.c. and was not affected by...

  2. Locus BoLA-DRB3 is just an ordinary site of the polygene when explaining genetic variance of somatic cell count and milk yield.

    Science.gov (United States)

    Oprzadek, Jolanta; Sender, Grazyna; Pawlik, Adrianna; Lukaszewicz, Marek

    2015-11-01

    The study aimed at clarifying the problem of the hitherto contradictory results regarding usefulness of BoLA-DRB3 locus as a marker in selection against mastitis and for milk yield. Treating the BoLA-DRB3 locus effect as random was proposed in place of considering it fixed. Somatic cell counts and milk yields recorded monthly on a test day (22,424) of 619 Polish Holstein cows genotyped for BoLA-DRB3 were analysed with an animal model including a random effect for genotype at this locus. The BoLA-DRB3 alleles were defined as restriction patterns obtained with three endonucleases. Two alternative BoLA-DRB3 additive genotype (co)variance structures were constructed for 161 genotypes recorded. One was based on the allelic similarity of the genotypes resulting in element values of 0 (no common allele), 0.5 (one allele in common), and 1 (diagonal). The other considered restriction site similarity (up to 3 in 1 allele) giving element values of 0 (no common restriction sites) and then increasingly in steps of 1/6 up to 6/6 (diagonal), where the numerator represents the number of common sites between genotypes. The DRB3 variance component for the natural logarithm of somatic cell count did not exceed 0.006 of the polygenic additive component or 0.003 for milk yield. Hence, unless we fail to detect the causative site or to properly define traits being the projection of a site, the effect of the genotype at the BoLA-DRB3 locus does not explain variation in somatic cell count and milk yield at a degree expected of a genetic marker. PMID:26333653

  3. Determination of the Effects of Some Environmental Factors on Raw Milk Somatic Cell Count of Brown Swiss Cows Raised in Farmer Condition

    OpenAIRE

    Aziz Şahin; Muzaffer Kaşıkcı

    2015-01-01

    In the current research, it was aimed to determine Somatic Cell Count (SCC) and the factors effecting on SCC in milk samples of Brown Swiss cattle raised at different farm conditions in Yıldızeli district of Sivas province in Turkey. Raw milk samples were collected at morning milking in months of May and November in year 2012. In total, 244 milk samples from 122 Brown Swiss cattle were analyzed. The effects of calving age, farm and test month on SCC were statistically significant. In the pres...

  4. A comparative study on the frequencies of radiation-induced chromosome aberrations in the somatic and germ cells in mouse and monkey

    International Nuclear Information System (INIS)

    Two systems were mainly used for studying the relationship between radiation induced chromosome aberration frequencies in somatic and germ cells. The first consists of reciprocal translocation induced in bone-marrow cells of mice compared to reciprocal translocation induced spermatogonia (scored in descending spermatocytes) of the same mice. Dose-response curves for induced aberrations in both cell types (0-100-200-300-400-500 and 600 R X-rays) and dose rate effects indicated that (130-1.92-0.0287 R/min) of a 400 R γ-ray exposure of the two cell types mitotically dividing germ cells respond to radiation similarly to mitotic dividing germ cells. Clonal proliferation or selective elimination of aberration-carrying cells, and other post-irradiation factors can, however, cause great differences in absolute aberration frequencies. A similar study was attempted, using the rhesus monkey as a second system. Its bone-marrow cells were proved unsuitable for induced reciprocal translocations. Stimulated peripheral blood lymphocytes were studied instead. Following 100, 200 and 300 R of X-rays, the frequencies of induced dicentric chromosomes were compared to those of induced reciprocal translocations in spermatogonia. Human peripheral blood was studied similarly. It was concluded that: (a) The absolute frequencies of chromosome aberrations in somatic and germ cells of the rhesus monkey are low compared to most other mammalian species. (b) The ratio between dicentric frequencies and reciprocal translocation frequencies at 100 R and 200 R differed significantly from 4:1 reported for mouse and Chinese hamster and 2:1 for marmoset and man. (c) Although the numbers of 'effective chromosome arms' in man and rhesus monkey are similar (81 vs 83), the rhesus monkey showed at all doses a lower rate of induction of dicentrics in blood lymphocytes than man, reaching statistical significance at the 300 R level

  5. Ophiobolin A from Bipolaris oryzae perturbs motility and membrane integrities of porcine sperm and induces cell death on mammalian somatic cell lines.

    Science.gov (United States)

    Bencsik, Ottó; Papp, Tamás; Berta, Máté; Zana, Annamária; Forgó, Péter; Dombi, György; Andersson, Maria A; Salkinoja-Salonen, Mirja; Vágvölgyi, Csaba; Szekeres, András

    2014-09-01

    Bipolaris oryzae is a phytopathogenic fungus causing a brown spot disease in rice, and produces substance that strongly perturbs motility and membrane integrities of boar spermatozoa. The substance was isolated from the liquid culture of the fungal strain using extraction and a multi-step semi-preparative HPLC procedures. Based on the results of mass spectrometric and 2D NMR techniques, the bioactive molecule was identified as ophiobolin A, a previously described sesterterpene-type compound. The purified ophiobolin A exhibited strong motility inhibition and viability reduction on boar spermatozoa. Furthermore, it damaged the sperm mitochondria significantly at sublethal concentration by the dissipation of transmembrane potential in the mitochondrial inner membrane, while the plasma membrane permeability barrier remained intact. The study demonstrated that the cytotoxicity of ophiobolin A toward somatic cell lines is higher by 1-2 orders of magnitude compared to other mitochondriotoxic mycotoxins, and towards sperm cells unique by replacing the progressive motility by shivering tail beating at low exposure concentration. PMID:25251540

  6. Ophiobolin A from Bipolaris oryzae Perturbs Motility and Membrane Integrities of Porcine Sperm and Induces Cell Death on Mammalian Somatic Cell Lines

    Directory of Open Access Journals (Sweden)

    Ottó Bencsik

    2014-09-01

    Full Text Available Bipolaris oryzae is a phytopathogenic fungus causing a brown spot disease in rice, and produces substance that strongly perturbs motility and membrane integrities of boar spermatozoa. The substance was isolated from the liquid culture of the fungal strain using extraction and a multi-step semi-preparative HPLC procedures. Based on the results of mass spectrometric and 2D NMR techniques, the bioactive molecule was identified as ophiobolin A, a previously described sesterterpene-type compound. The purified ophiobolin A exhibited strong motility inhibition and viability reduction on boar spermatozoa. Furthermore, it damaged the sperm mitochondria significantly at sublethal concentration by the dissipation of transmembrane potential in the mitochondrial inner membrane, while the plasma membrane permeability barrier remained intact. The study demonstrated that the cytotoxicity of ophiobolin A toward somatic cell lines is higher by 1–2 orders of magnitude compared to other mitochondriotoxic mycotoxins, and towards sperm cells unique by replacing the progressive motility by shivering tail beating at low exposure concentration.

  7. Papillary renal cell carcinoma with a somatic mutation in MET in a patient with autosomal dominant polycystic kidney disease.

    Science.gov (United States)

    Zhang, Wanying; Tan, Adrian Y; Blumenfeld, Jon; Liu, Genyan; Michaeel, Alber; Zhang, Tuo; Robinson, Brian D; Salvatore, Steven P; Kapur, Sandip; Donahue, Stephanie; Bobb, Warren O; Rennert, Hanna

    2016-01-01

    Autosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 and PKD2 and is characterized by proliferation of renal tubular epithelium and progressive chronic kidney disease. Derangements in similar cellular signaling pathways occur in ADPKD and renal malignancies, although an association of these disorders has not been established. Herein, we present a case of papillary RCC (pRCC) incidentally discovered in a patient with ADPKD following bilateral native nephrectomy during renal transplantation. Whole exome sequencing of the pRCC found a somatic missense mutation in MET proto-oncogene, p.Val1110Ile, not present in kidney cyst epithelium or non-cystic tissue. RNA sequencing demonstrated increased mRNA expression of MET and pathway-related genes, but no significant copy number variation of MET was detected. Genetic analysis of PKD genes from peripheral blood lymphocytes and renal cyst epithelium identified a constitutional PKD1 germline mutation, p.Trp1582Ser, predicted to be pathogenic. Unique somatic mutations in PKD1 were also detected in 80% of the renal cysts analyzed, but not in the pRCC. These results suggest that, in this patient, the pRCC utilized a signaling pathway involving MET that was distinct from the pathogenesis of ADPKD. This is the first report of PKD1 mutations and a somatic mutation of the MET oncogene in a pRCC in ADPKD. PMID:26718059

  8. Nanog1 in NTERA-2 and recombinant NanogP8 from somatic cancer cells adopt multiple protein conformations and migrate at multiple M.W species.

    Directory of Open Access Journals (Sweden)

    Bigang Liu

    Full Text Available Human Nanog1 is a 305-amino acid (aa homeodomain-containing transcription factor critical for the pluripotency of embryonic stem (ES and embryonal carcinoma (EC cells. Somatic cancer cells predominantly express a retrogene homolog of Nanog1 called NanogP8, which is ~99% similar to Nanog at the aa level. Although the predicted M.W of Nanog1/NanogP8 is ∼35 kD, both have been reported to migrate, on Western blotting (WB, at apparent molecular masses of 29-80 kD. Whether all these reported protein bands represent authentic Nanog proteins is unclear. Furthermore, detailed biochemical studies on Nanog1/NanogpP8 have been lacking. By combining WB using 8 anti-Nanog1 antibodies, immunoprecipitation, mass spectrometry, and studies using recombinant proteins, here we provide direct evidence that the Nanog1 protein in NTERA-2 EC cells exists as multiple M.W species from ~22 kD to 100 kD with a major 42 kD band detectable on WB. We then demonstrate that recombinant NanogP8 (rNanogP8 proteins made in bacteria using cDNAs from multiple cancer cells also migrate, on denaturing SDS-PAGE, at ~28 kD to 180 kD. Interestingly, different anti-Nanog1 antibodies exhibit differential reactivity towards rNanogP8 proteins, which can spontaneously form high M.W protein species. Finally, we show that most long-term cultured cancer cell lines seem to express very low levels of or different endogenous NanogP8 protein that cannot be readily detected by immunoprecipitation. Altogether, the current study reveals unique biochemical properties of Nanog1 in EC cells and NanogP8 in somatic cancer cells.

  9. Conditions Favorable for the Somatic Embryogenesis in Carrot Cell Culture Enhance Expression of the roIC Promoter-GUS Fusion Gene 1

    Science.gov (United States)

    Fujii, Nobuharu; Uchimiya, Hirofumi

    1991-01-01

    We obtained carrot (Daucus carota) cells possessing the 5′-noncoding sequence of the ORF12 gene (roIC) of TL-DNA of the Ri plasmid and a structural gene of bacterial β-glucuronidase by Agrobacterium-mediated transformation. When such cells were cultured in medium containing 2,4-dichlorophenoxyacetic acid, substantial reduction in β-glucuronidase activity was observed. Upon transferring the cells from a 2,4-D-containing medium to one devoid of 2,4-dichlorophenoxyacetic acid, enhanced expression of β-glucuronidase in somatic embryo development was recorded. Activation by gibberillic acid and suppression by abscisic acid of β-glucuronidase activities, in concord with embryogenesis, were also noted. Images Figure 2 PMID:16667958

  10. Conditions Favorable for the Somatic Embryogenesis in Carrot Cell Culture Enhance Expression of the roIC Promoter-GUS Fusion Gene.

    Science.gov (United States)

    Fujii, N; Uchimiya, H

    1991-01-01

    We obtained carrot (Daucus carota) cells possessing the 5'-noncoding sequence of the ORF12 gene (roIC) of TL-DNA of the Ri plasmid and a structural gene of bacterial beta-glucuronidase by Agrobacterium-mediated transformation. When such cells were cultured in medium containing 2,4-dichlorophenoxyacetic acid, substantial reduction in beta-glucuronidase activity was observed. Upon transferring the cells from a 2,4-D-containing medium to one devoid of 2,4-dichlorophenoxyacetic acid, enhanced expression of beta-glucuronidase in somatic embryo development was recorded. Activation by gibberillic acid and suppression by abscisic acid of beta-glucuronidase activities, in concord with embryogenesis, were also noted. PMID:16667958

  11. Refined positioning of a quantitative trait locus affecting somatic cell score on chromosome 18 in the German Holstein using linkage disequilibrium.

    Science.gov (United States)

    Baes, C; Brand, B; Mayer, M; Kühn, C; Liu, Z; Reinhardt, F; Reinsch, N

    2009-08-01

    Combined linkage and linkage disequilibrium analysis (LALD) was conducted to more accurately map a previously reported quantitative trait locus (QTL) affecting somatic cell score on bovine chromosome 18. A grand-daughter design consisting of 6 German Holstein grandsire families with 1,054 progeny-tested genotyped sons was used in this study. Twenty microsatellite markers, 5 single nucleotide polymorphisms, and an erythrocyte antigen marker with an average marker spacing of 1.95 cM were analyzed along a chromosomal segment of 50.80 cM. Variance components were estimated and restricted maximum likelihood test statistics were calculated at the midpoint of each marker interval. The test statistics calculated in single-QTL linkage analysis exceeded the genome-wide significance threshold at several putative QTL positions. Using LALD, we were successful in assigning a genome-wide significant QTL to a confidence interval of 10.8 cM between the markers ILSTS002 and BMS833. The QTL in this marker interval was estimated to be responsible for between 5.89 and 13.86% of the genetic variation in somatic cell score. In contrast to the single-QTL linkage analysis model, LALD analyses with a 2-QTL model confirmed the position of one QTL, but gave no conclusive evidence for the existence or position of a second QTL. Ultimately, the QTL position was narrowed down considerably compared with previous results with a refined confidence interval of less than 11 cM. PMID:19620688

  12. Subcutaneous injection of thymopentin in the area of the supramammary lymph node to reduce milk somatic cell count in subclinically mastitic cows.

    Science.gov (United States)

    Guan, R; Xu, W; Pan, T; Su, X; Hu, S

    2016-02-01

    The objective of this study was to evaluate the therapeutic effects of thymopentin (TP-5) injections on subclinical intramammary infection (IMI) in lactating cows. In Experiment I, 40 cows were randomly divided into four groups. The cows in groups 1, 2, and 3 received subcutaneous injections of TP-5 in the region of the supramammary lymph node at doses of 1, 2, and 4 mg, respectively, for 3 days. In Experiment II, 20 cows were randomly divided into two groups. The cows in group 1 were treated with injections of TP-5 (4 mg) for 3 days in the same area as in Experiment I. Group 4 in Experiment I and group 2 in Experiment II were not treated and served as control groups. Milk samples were collected before and after treatment for bacteriological examination and analysis of the somatic cell count and level of N-acetyl-β-d-glucosaminidase (NAGase). The results showed that treatment with TP-5 significantly reduced the somatic cell count (SCC) and NAGase activity of the milk and numerically reduced IMI. A dose of 4 mg was found to be optimal for the reduction of SCC and NAGase in milk. Therefore, further study of the use of TP-5 in the treatment of bovine mastitis is warranted. PMID:25976252

  13. Interval and composite interval mapping of somatic cell score, yield, and components of milk in dairy cattle.

    Science.gov (United States)

    Rodriguez-Zas, S L; Southey, B R; Heyen, D W; Lewin, H A

    2002-11-01

    Single-marker, interval-mapping (IM) and composite interval mapping (CIM) were used to detect quantitative trait loci (QTL) controlling milk, fat and protein yields, and somatic cell score (SCS). A granddaughter design was used to combine molecular genetic information with predicted transmitting abilities (PTA) and estimated daughter yield deviations (DYD) from eight Dairy Bull DNA Repository Holstein families. Models that included and excluded weights accounting for the uncertainty of the response variable were evaluated in each trait, family and phenotype (DYD and PTA) combination. The genotypic information consisted of 174 microsatellite markers along 29 Bos taurus autosomes. The average number of informative markers per autosome was three and the number of informative sons per family and marker varied between 21 and 173. Within-family results from the least squares single-marker analyses were used in expectation-maximization likelihood IM and CIM implemented with QTL Cartographer. Different CIM model specifications, offering complementary control on the background QTL outside the interval under study, were evaluated. Permutation techniques were used to calculate the genome-wide threshold test statistic values based on 1,000 samples. Results from the DYD and PTA analyses were highly consistent across traits and families. The minor differences in the estimates from the models that accounted for or ignored the uncertainty of the DYD (variance) and PTA (inverse of reliability) may be associated to the elevated and consistent precision of the DYD and PTA among sons. The CIM model best supported by the data had 10 markers controlling for background effects. On autosome (BTA) three, a QTL at 32 cM influencing protein yield was located in family five and a QTL at 74 cM for fat yield was located in family eight. Two map positions associated with SCS were detected on BTA 21, one at 33 cM in family one and the other at 84 cM in family three. A QTL for protein yield was

  14. Microbiological quality and somatic cell count in bulk milk of dromedary camels (Camelus dromedarius): descriptive statistics, correlations, and factors of variation.

    Science.gov (United States)

    Nagy, P; Faye, B; Marko, O; Thomas, S; Wernery, U; Juhasz, J

    2013-09-01

    The objectives of the present study were to monitor the microbiological quality and somatic cell count (SCC) of bulk tank milk at the world's first large-scale camel dairy farm for a 2-yr period, to compare the results of 2 methods for the enumeration of SCC, to evaluate correlation among milk quality indicators, and to determine the effect of specific factors (year, season, stage of lactation, and level of production) on milk quality indicators. The study was conducted from January 2008 to January 2010. Total viable count (TVC), coliform count (CC), California Mastitis Test (CMT) score, and SCC were determined from daily bulk milk samples. Somatic cell count was measured by using a direct microscopic method and with an automatic cell counter. In addition, production parameters [total daily milk production (TDM, kg), number of milking camels (NMC), average milk per camel (AMC, kg)] and stage of lactation (average postpartum days, PPD) were recorded for each test day. A strong correlation (r=0.33) was found between the 2 methods for SCC enumeration; however, values derived using the microscopic method were higher. The geometric means of SCC and TVC were 394×10(3) cells/mL and 5,157 cfu/mL during the observation period, respectively. Somatic cell count was >500×10(3) cells/mL on 14.6% (106/725) and TVC was >10×10(3) cfu/mL on 4.0% (30/742) of the test days. Both milk quality indicators had a distinct seasonal pattern. For log SCC, the mean was lowest in summer and highest in autumn. The seasonal pattern of log TVC was slightly different, with the lowest values being recorded during the spring. The monthly mean TVC pattern showed a clear difference between years. Coliform count was CMT score (r=0.26), and between log TVC and CC in yr 1 (r=0.30). All production parameters and stage of lactation showed strong seasonal variation. Log SCC was negatively correlated with TDM (r=-0.35), AMC (r=-0.37), and NMC (r=-0.15) and positively correlated with PPD (r=0.40). Log TVC

  15. 玉屏风散对奶牛产奶量及体细胞数的影响%The Influence on Milk Production of Cow and Number of Somatic Cells of Yupingfeng Powder

    Institute of Scientific and Technical Information of China (English)

    车淑芬; 廖智慧; 吕昌洪

    2014-01-01

    本试验选取玉屏风散,通过检测产奶量与体细胞数的变化,分析其对奶牛产奶量及体细胞数的影响,确定其在奶牛生产中发挥的作用。结果表明,玉屏风散能显著提高产奶量,降低体细胞数,达到了防病保健作用。%This experiment takes Yupingfeng powder, through the detection of milk production and the change of the number of somatic cells, analysis of the influence of cow milk production and somatic cell count, then deter-mine Yupingfeng powder,s role in dairy production. The results show that, Yupingfeng powder can significantly improve milk yield, reduce the somatic cell number ,to achieve the prevention health care function.

  16. Long-lived antigen-induced IgM plasma cells demonstrate somatic mutations and contribute to long-term protection.

    Science.gov (United States)

    Bohannon, Caitlin; Powers, Ryan; Satyabhama, Lakshmipriyadarshini; Cui, Ang; Tipton, Christopher; Michaeli, Miri; Skountzou, Ioanna; Mittler, Robert S; Kleinstein, Steven H; Mehr, Ramit; Lee, Francis Eun-Yun; Sanz, Ignacio; Jacob, Joshy

    2016-01-01

    Long-lived plasma cells are critical to humoral immunity as a lifelong source of protective antibodies. Antigen-activated B cells-with T-cell help-undergo affinity maturation within germinal centres and persist as long-lived IgG plasma cells in the bone marrow. Here we show that antigen-specific, induced IgM plasma cells also persist for a lifetime. Unlike long-lived IgG plasma cells, which develop in germinal centres and then home to the bone marrow, IgM plasma cells are primarily retained within the spleen and can develop even in the absence of germinal centres. Interestingly, their expressed IgV loci exhibit somatic mutations introduced by the activation-induced cytidine deaminase (AID). However, these IgM plasma cells are probably not antigen-selected, as replacement mutations are spread through the variable segment and not enriched within the CDRs. Finally, antibodies from long-lived IgM plasma cells provide protective host immunity against a lethal virus challenge. PMID:27270306

  17. Other somatic and fetal effects

    International Nuclear Information System (INIS)

    This chapter reviews the evidence for fetal and other radiation-induced somatic effects. The following studies are included: human epidemiological studies of cancer in childhood following exposure in utero; animal and human studies of the effects on growth and development; life-shortening studies in laboratory animals; cataracts of the human eye lens; radiosensitivity of germinal cells of the mammalian testis and ovary. 85 ref

  18. A comparative study of the frequencies of radiation-induced chromosome aberrations in somatic and germ cells of the Rhesus monkey (Macaca mulatta)

    International Nuclear Information System (INIS)

    Frequencies of radiation-induced chromosome aberrations in spermatogonia peripheral blood lymphocytes and bone-marrow cells of the rhesus monkey (Macaca mulatta) and in human blood lymphocytes, where determined at different exposures of X-rays. The dose-response curve for the induction of reciprocal translocations in spermatogonia suggested a 'hump' at about 200 rad. The absolute frequencies of chromosome aberrations in somatic and germ cells of the rhesus monkey were low in comparison with most other mammalian species and the ratio between aberrations in the two tissues was 25 to 1 at the 100 rad level. Although the numbers of 'effective chromosome arms' in man and rhesus monkey are similar (81 vs. 83), the rhesus monkey showed a lower rate of induction of dicentrics in blood lymphocytes than man at all doses reaching statistical significance at the 300 rad level

  19. Bright/Arid3A Acts as a Barrier to Somatic Cell Reprogramming through Direct Regulation of Oct4, Sox2, and Nanog

    Directory of Open Access Journals (Sweden)

    Melissa Popowski

    2014-01-01

    Full Text Available We show here that singular loss of the Bright/Arid3A transcription factor leads to reprograming of mouse embryonic fibroblasts (MEFs and enhancement of standard four-factor (4F reprogramming. Bright-deficient MEFs bypass senescence and, under standard embryonic stem cell (ESC culture conditions, spontaneously form clones that in vitro express pluripotency markers, differentiate to all germ lineages, and in vivo form teratomas and chimeric mice. We demonstrate that BRIGHT binds directly to the promoter/enhancer regions of Oct4, Sox2, and Nanog to contribute to their repression in both MEFs and ESCs. Thus, elimination of the BRIGHT barrier may provide an approach for somatic cell reprogramming.

  20. Mice deficient in carbonic anhydrase type 8 exhibit motor dysfunctions and abnormal calcium dynamics in the somatic region of cerebellar granule cells.

    Science.gov (United States)

    Lamont, Matthew G; Weber, John T

    2015-06-01

    The waddles (wdl) mouse is characterized by a namesake "side-to-side" waddling gait due to a homozygous mutation of the Car8 gene. This mutation results in non-functional copies of the protein carbonic anhydrase type 8. Rota-rod testing was conducted to characterize the wdl mutations' effect on motor output. Results indicated that younger homozygotes outperformed their older cohorts, an effect not seen in previous studies. Heterozygotes, which were thought to be free of motor impairment, displayed motor learning deficiencies when compared with wild type performance. Acute cerebellar slices were then utilized for fluorescent calcium imaging experiments, which revealed significant alterations in cerebellar granule cell somatic calcium signaling when exposed to glutamate. The contribution of GABAergic signaling to these alterations was also verified using bath application of bicuculline. Changes in somatic calcium signals were found to be applicable to an in vivo scenario by comparing group responses to electrical stimulation of afferent mossy fiber projections. Finally, intracellular calcium store function was also found to be altered by the wdl mutation when slices were treated with thapsigargin. These findings, taken together with previous work on the wdl mouse, indicate a widespread disruption in cerebellar circuitry hampering proper neuronal communication. PMID:25721739

  1. The Co-Injection of Somatic Cells with Embryonic Stem Cells Affects Teratoma Formation and the Properties of Teratoma-Derived Stem Cell-Like Cells

    OpenAIRE

    Gong, Seung Pyo; Kim, Boyun; Kwon, Hyo Sook; Yang, Woo Sub; Jeong, Jae-Wook; Ahn, Jiyeon; Lim, Jeong Mook

    2014-01-01

    The aim of this study was to assess the biological reactions triggered by stem cell transplantation related to phenotypic alteration, host-to-cell response, chromosomal stability, transcriptional alteration, and stem cell-like cell re-expansion. B6CBAF1 mouse embryonic stem cells (ESCs) were injected subcutaneously into homologous or heterologous (B6D2F1) recipients, and heterologous injections were performed with or without co-injection of B6D2F1 fetal fibroblasts. All homologous injections ...

  2. Management of somatic symptoms

    DEFF Research Database (Denmark)

    Schröder, Andreas; Dimsdale, Joel

    2014-01-01

    recognition and effective management of patients with excessive and disabling somatic symptoms. The clinical presentation of somatic symptoms is categorized into three groups of patients: those with multiple somatic symptoms, those with health anxiety, and those with conversion disorder. The chapter provides...... information to assist with making a diagnosis and differential diagnosis. Management includes ways to improve the physician–patient interaction that will benefit the patient, a step-care model based on illness severity and complexity, and psychological and pharmacologic treatment. The chapter is enhanced by...... figures and tables that summarize health anxiety, symptoms, differential diagnoses, and management strategies, as well as by case studies and examples....

  3. Endomembrane-associated RSD-3 is important for RNAi induced by extracellular silencing RNA in both somatic and germ cells of Caenorhabditis elegans

    Science.gov (United States)

    Imae, Rieko; Dejima, Katsufumi; Kage-Nakadai, Eriko; Arai, Hiroyuki; Mitani, Shohei

    2016-01-01

    RNA silencing signals in C. elegans spread among cells, leading to RNAi throughout the body. During systemic spread of RNAi, membrane trafficking is thought to play important roles. Here, we show that RNAi Spreading Defective-3 (rsd-3), which encodes a homolog of epsinR, a conserved ENTH (epsin N-terminal homology) domain protein, generally participates in cellular uptake of silencing RNA. RSD-3 is previously thought to be involved in systemic RNAi only in germ cells, but we isolated several deletion alleles of rsd-3, and found that these mutants are defective in the spread of silencing RNA not only into germ cells but also into somatic cells. RSD-3 is ubiquitously expressed, and intracellularly localized to the trans-Golgi network (TGN) and endosomes. Tissue-specific rescue experiments indicate that RSD-3 is required for importing silencing RNA into cells rather than exporting from cells. Structure/function analysis showed that the ENTH domain alone is sufficient, and membrane association of the ENTH domain is required, for RSD-3 function in systemic RNAi. Our results suggest that endomembrane trafficking through the TGN and endosomes generally plays an important role in cellular uptake of silencing RNA.

  4. Identification of Valid Housekeeping Genes for Real-Time Quantitative PCR Analysis of Collapsed Lung Tissues of Neonatal Somatic Cell Nuclear Transfer-Derived Cattle

    DEFF Research Database (Denmark)

    Liu, Yan; Zhang, Yunhai; Jiang, Qiuling;

    2015-01-01

    Cloned calves produced by somatic cell nuclear transfer frequently suffer alveolar collapse as newborns. To study the underlying pathophysiological mechanisms responsible for this phenomenon, the expression profiles of numerous genes involved in lung development need to be investigated......), peptidylprolyl isomerase A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA-box binding protein (TBP), and 5.8S ribosomal RNA (5.8S rRNA)-were selected and evaluated as candidates. Their gene expression levels in the collapsed lungs of deceased neonate cloned calves and normal lung derived from...... genes by NormFinder. Taking these results into account, we conclude that 5.8S rRNA and PPIA could be the most reliable reference genes for studying the genes involved in alveolar collapse. Moreover, 5.8S rRNA could be represented as a uniform reference gene in similar cases....

  5. The nonlinear effect of somatic cell count on milk composition, coagulation properties, curd firmness modeling, cheese yield, and curd nutrient recovery.

    Science.gov (United States)

    Bobbo, T; Cipolat-Gotet, C; Bittante, G; Cecchinato, A

    2016-07-01

    The aim of this study was to investigate the relationships between somatic cell count (SCC) in milk and several milk technological traits at the individual cow level. In particular, we determined the effects of very low to very high SCC on traits related to (1) milk yield and composition; (2) coagulation properties, including the traditional milk coagulation properties (MCP) and the new curd firming model parameters; and (3) cheese yield and recovery of milk nutrients in the curd (or loss in the whey). Milk samples from 1,271 Brown Swiss cows from 85 herds were used. Nine coagulation traits were measured: 3 traditional MCP [rennet coagulation time (RCT, min), curd firming rate (k20, min), and curd firmness after 30 min (a30, mm)] and 6 new curd firming and syneresis traits [potential asymptotic curd firmness at infinite time (CFP, mm), curd firming instant rate constant (kCF, % × min(-1)), syneresis instant rate constant (kSR, % × min(-1)), rennet coagulation time estimated using the equation (RCTeq, min), maximum curd firmness achieved within 45 min (CFmax, mm), and time at achievement of CFmax (tmax, min)]. The observed cheese-making traits included 3 cheese yield traits (%CYCURD, %CYSOLIDS, and %CYWATER, which represented the weights of curd, total solids, and water, respectively, as a percentage of the weight of the processed milk) and 4 nutrient recoveries in the curd (RECFAT, RECPROTEIN, RECSOLIDS, and RECENERGY, which each represented the percentage ratio between the nutrient in the curd and milk). Data were analyzed using a linear mixed model with the fixed effects of days in milk, parity, and somatic cell score (SCS), and the random effect of herd-date. Somatic cell score had strong influences on casein number and lactose, and also affected pH; these were traits characterized by a quadratic pattern of the data. The results also showed a negative linear relationship between SCS and milk yield. Somatic cell score influenced almost all of the tested

  6. Efficacy of Indigenous Herbal Preparation on Altered Milk pH, Somatic Cell Count and Electrolyte Profile in Subclinical Mastitis in Cows

    Directory of Open Access Journals (Sweden)

    A.Y. Kolte

    Full Text Available Comparative efficacy of three different locally prepared indigenous herbal paste were evaluated in subclinical 24 mastitic cows with reference to restoring altered milk pH, somatic cell count and milk electrolyte profile. The study revealed that all the treatment were found effective in restoring the altered milk constituents in subclinical mastitis with increased in the milk production. T3 (oots of Withania somnifera (Ashwagandha, Asparagus reacemosus (Shatavari, Curcuma-amada (Ambe Haldi and fresh leaves of Ocimum sanctum (Tulsi in equal quantities was found more effective than T5 ( T3 and T4 in combination in equal quantities and T4 (fresh roots of Glycerrhiza glabra (Jeshathamadh, Nardostachys jatamansi (Jatamansi, leaves of Riccinus communis (Yerand, bark of Ficus racemosa (Umber and rhizome of Curcuma longa (Haldi in equal quantities [Veterinary World 2008; 1(8.000: 239-240

  7. Somatic symptom disorder

    Science.gov (United States)

    American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders . 5th ed. Arlington, VA: American Psychiatric Publishing, 2013. Ferri F. Somatization disorder. In: Ferri FF, ed. Ferri's Clinical Advisor ...

  8. Yeast mother cell-specific ageing, genetic (in)stability, and the somatic mutation theory of ageing

    OpenAIRE

    Laun, Peter; Bruschi, Carlo V.; Dickinson, J. Richard; Rinnerthaler, Mark; Heeren, Gino; Schwimbersky, Richard; Rid, Raphaela; Breitenbach, Michael

    2007-01-01

    Yeast mother cell-specific ageing is characterized by a limited capacity to produce daughter cells. The replicative lifespan is determined by the number of cell cycles a mother cell has undergone, not by calendar time, and in a population of cells its distribution follows the Gompertz law. Daughter cells reset their clock to zero and enjoy the full lifespan characteristic for the strain. This kind of replicative ageing of a cell population based on asymmetric cell divisions is investigated as...

  9. DNA methylation patterns in tissues from mid-gestation bovine foetuses produced by somatic cell nuclear transfer show subtle abnormalities in nuclear reprogramming

    Directory of Open Access Journals (Sweden)

    Lee Rita SF

    2010-03-01

    Full Text Available Abstract Background Cloning of cattle by somatic cell nuclear transfer (SCNT is associated with a high incidence of pregnancy failure characterized by abnormal placental and foetal development. These abnormalities are thought to be due, in part, to incomplete re-setting of the epigenetic state of DNA in the donor somatic cell nucleus to a state that is capable of driving embryonic and foetal development to completion. Here, we tested the hypothesis that DNA methylation patterns were not appropriately established during nuclear reprogramming following SCNT. A panel of imprinted, non-imprinted genes and satellite repeat sequences was examined in tissues collected from viable and failing mid-gestation SCNT foetuses and compared with similar tissues from gestation-matched normal foetuses generated by artificial insemination (AI. Results Most of the genomic regions examined in tissues from viable and failing SCNT foetuses had DNA methylation patterns similar to those in comparable tissues from AI controls. However, statistically significant differences were found between SCNT and AI at specific CpG sites in some regions of the genome, particularly those associated with SNRPN and KCNQ1OT1, which tended to be hypomethylated in SCNT tissues. There was a high degree of variation between individuals in methylation levels at almost every CpG site in these two regions, even in AI controls. In other genomic regions, methylation levels at specific CpG sites were tightly controlled with little variation between individuals. Only one site (HAND1 showed a tissue-specific pattern of DNA methylation. Overall, DNA methylation patterns in tissues of failing foetuses were similar to apparently viable SCNT foetuses, although there were individuals showing extreme deviant patterns. Conclusion These results show that SCNT foetuses that had developed to mid-gestation had largely undergone nuclear reprogramming and that the epigenetic signature at this stage was not a

  10. Quality of raw cow milk in Republic of Macedonia determined through the testing of somatic cell count and total viable count

    Directory of Open Access Journals (Sweden)

    Angelovski Ljupco

    2008-11-01

    Full Text Available Somatic cells count and total viable count are criteria used to estimate the compliance of raw cow milk with the Book of rules for demands for safety and hygiene and procedures for official controls of milk and milk products, Official Gazette of RM 157/2007. According to the given demands, raw milk operators are obliged to conduct all procedures and to guarantee that milk is in compliance with the criteria laid down in Book of rules. At the same time, Republic of Macedonia have to fulfill EU criteria laid down in Directive 92/46 (Council directive 92/46/EEC laying down the health rules for the production and placing on the market of raw milk, heat-treated milk and milkbased products for quality of raw milk as part of implementation of community legislation and milk production. The independent laboratory for milk quality control at FVM-Skopje, in frame of its activities in the period February- August 2008 has conducted a study for obtaining preliminary results for the situation with raw milk quality produced in R. of Macedonia for somatic cells counts and total viable count. In the study we analyzed 2065 samples for TVC and 1625 samples for SCC of raw milk samples produced in different parts of the country. From the tested samples only 41,8% fulfill criteria for SCC and 41,45% criteria for TVC lay down in Book of rules for 2008. Assessment of the results in light of Council Directive it is obvious that only 42,7% of the samples for SCC and 10,7% for TVC fulfill the criteria of Council Directive having in mind different requirements vs. Book of rules.

  11. Somatic Embryogenesis in Chestnut

    OpenAIRE

    CORREDOIRA, E.; Ballester, A.; Viéitez Madriñán, Francisco Javier; Viéitez Martín, Ana María

    2005-01-01

    Somatic embryogenesis is an important biotechnological tool that demonstrates significant benefits when applied to forest tree species; clonal propagation, cryostorage of valuable germoplasm and genetic transformation are among the most promising of its applications. In this chapter, the state of the art of somatic embryogenesis in chestnut (an important economical tree species of the genus Castanea) is assessed and discussed. The factors affecting the induction (type of explant, growth condi...

  12. Stem Cell Information: Glossary

    Science.gov (United States)

    ... Neurons Oligodendrocyte Parthenogenesis Passage Pluripotent Polar body Preimplantation Proliferation Regenerative medicine Reproductive cloning Signals Somatic cell Somatic cell nuclear transfer (SCNT) Somatic (adult) stem cell Stem cells Stromal cells Subculturing Surface markers ...

  13. Human cord blood-derived unrestricted somatic stem cells promote wound healing and have therapeutic potential for patients with recessive dystrophic epidermolysis bullosa.

    Science.gov (United States)

    Liao, Yanling; Itoh, Munenari; Yang, Albert; Zhu, Hongwen; Roberts, Samantha; Highet, Alexandra M; Latshaw, Shaun; Mitchell, Kelly; van de Ven, Carmella; Christiano, Angela; Cairo, Mitchell S

    2014-03-01

    Human umbilical cord blood (CB)-derived unrestricted somatic stem cells (USSCs) have previously been demonstrated to have a broad differentiation potential and regenerative beneficial effects when administered in animal models of multiple degenerative diseases. Here we demonstrated that USSCs could be induced to express genes that hallmark keratinocyte differentiation. We also demonstrated that USSCs express type VII collagen (C7), a protein that is absent or defective in patients with an inherited skin disease, recessive dystrophic epidermolysis bullosa (RDEB). In mice with full-thickness excisional wounds, a single intradermal injection of USSCs at a 1-cm distance to the wound edge resulted in significantly accelerated wound healing. USSC-treated wounds displayed a higher density of CD31(+) cells, and the wounds healed with a significant increase in skin appendages. These beneficial effects were demonstrated without apparent differentiation of the injected USSCs into keratinocytes or endothelial cells. In vivo bioluminescent imaging (BLI) revealed specific migration of USSCs modified with a luciferase reporter gene, from a distant intradermal injection site to the wound, as well as following systemic injection of USSCs. These data suggest that CB-derived USSCs could significantly contribute to wound repair and be potentially used in cell therapy for patients with RDEB. PMID:23394106

  14. Protection by Panax ginseng C.A. Meyer against the genotoxicity of doxorubicin in somatic cells of Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Denise G. Pereira

    2008-12-01

    Full Text Available Panax ginseng is one of the most widely prescribed herbal medicines for the treatment of cancer, diabetes, chronic inflammation, and neurodegenerative and cardiovascular diseases. Since the use of alternative medicines in combination with conventional therapy may increase the risk of unwanted interactions, we investigated the possible genotoxicity of a water-soluble form of the dry root of P. ginseng (2.5, 5.0 or 10.0 mg/mL and its ability to protect against the genotoxicity of doxorubicin (DOX; 0.125 mg/mL by using the Drosophila melanogaster wing somatic mutation and recombination test (SMART with standard and high-bioactivation crosses of flies. Panax ginseng was not genotoxic at the concentrations tested, whereas DOX-induced genotoxicity in marker-heterozygous flies resulted mainly from mitotic recombination. At low concentrations, P. ginseng had antirecombinogenic activity that was independent of the concentration of extract used. Recombination events may promote cancer, but little is known about the ability of P. ginseng to inhibit such recombination or modulate DNA repair mechanisms.

  15. Absence of genotoxic activity from milk and water boiled in microwave oven in somatic cells from Drosophila melanogaster

    International Nuclear Information System (INIS)

    This paper reports an experiment for evaluation of the possible genotoxic effects of food prepared in a microwave oven, through the mutation test and somatic recombination, in wings of Drosophila melanogaster. Two crossing have been performed: a standard cross-ST and a high bioactivation cross - HB resulting in marked trans -heterozygote descendents (MH) and balanced heterozygotes (BH). The 72 hours larvas were fed with water and milk boiled both in the microwave oven and in the traditional way. The MH individual wings were analyzed, where the spots can be induced either by mutation or mitotic recombination. The experiment presented negative results related to the genotoxic effects of the water and milk boiled using the microwave oven, in MH descendents of both crossing. Therefore, under these experimental conditions, genotoxic activity were not presented by milk and water boiled in the microwave oven. However, an extensive study using different techniques is necessary to investigate the action of the food prepared in the microwave oven on the genetic material

  16. Genetic parameters for somatic cell score and production traits in the ifrst three lactations of Chinese Holstein cows

    Institute of Scientific and Technical Information of China (English)

    ZHAO Fu-ping; GUO Gang; WANG Ya-chun; GUO Xiang-yu; ZHANG Yuan; DU Li-xin

    2015-01-01

    The objectives of this study were to estimate genetic parameters of lactation average somatic cel scores (LSCS) and examine genetic associations between LSCS and production traits in the ifrst three lactations of Chinese Holstein cows using single-parity multi-trait animal model and multi-trait repeatability animal model. There were totaly 273605 lactation records of Chinese Holstein cows with ifrst calving from 2001 to 2012. Heritability estimates for LSCS ranged from 0.144 to 0.187. Genetic correlations between LSCS and 305 days milk, protein percentage and fat percentage were –0.079,–0.082 and –0.135, respectively. Phenotypic correlation between LSCS and 305 days milk yield was negative (–0.103 to –0.190). Genetic correlation between 305 days milk and fat percentage or protein percentage was highly negative. Genetic correlation between milk fat percentage and milk protein percentage was highly favorable. Heritabilities of production traits decreased with increase of parity, whereas heritability of LSCS increased with increase of parity.

  17. Determination of the Effects of Some Environmental Factors on Raw Milk Somatic Cell Count of Brown Swiss Cows Raised in Farmer Condition

    Directory of Open Access Journals (Sweden)

    Aziz Şahin

    2015-07-01

    Full Text Available In the current research, it was aimed to determine Somatic Cell Count (SCC and the factors effecting on SCC in milk samples of Brown Swiss cattle raised at different farm conditions in Yıldızeli district of Sivas province in Turkey. Raw milk samples were collected at morning milking in months of May and November in year 2012. In total, 244 milk samples from 122 Brown Swiss cattle were analyzed. The effects of calving age, farm and test month on SCC were statistically significant. In the present study, the average SCC was determined as 1209696±111361 cells/ml. About 56.6% of milk from November month and about 48.4 % of milk samples from Brown Swiss were containing over 500.000 cell/ ml. As a result, Brown Swiss cattle identified SCC in raw milk samples were significantly higher than the values specified in the legislation of the European Union Commission and Turkish Food Codex.

  18. Effect of leptin on oocyte maturation and subsequent pregnancy rate of cloned embryos reconstructed by somatic cell nuclear transfer in pigs

    Institute of Scientific and Technical Information of China (English)

    Hengxi Wei; Qiuyan Li; Jun Li; Yan Li; Yunping Dai; Yufang Ma; Kai Xue; Ning Li

    2008-01-01

    Cloning pigs by somatic cell nuclear transfer (SCNT) has wide applications in basic research,human medicine and agricultural production.To improve cloning efficiency,the effect of two basic maturation media,NCSU-23 and TCMI99,was compared,and TCM199 was selected for the following experiments with leptin.We systematically studied the effects of leptin supplementation on oocytes in vitro maturation (IVM),in vitro development of parthenogenetically activated (Phi) and SCNT embryos and/n vivo develop-ment of SCNT embryos after embryo transfer (ET).The results showed that supplementation of 100 or 200 ng/ml leptin into the mat-uration medium did not greatly affect nuclear maturation of oocytes,or cleavage rates of PA and SCNT (P<0.05).Blastocyst rates of PA and SCNT embryos were significantly improved when 100 or 200 ng/ml leptin was added to maturation medium,and the number of cells in PA blastocysts was also improved (P<0.05).The number of cells in blastocyst of SCNT was improved,when 100 ng/ml leptin was added (P<0.05).Furthermore,supplementation of 100 or 200 ng/ml leptin to the IVM medium may improve pregnancy rate and the delivery rate in pig cloning.

  19. Classical Mus musculus Igκ enhancers support transcription but not high level somatic hypermutation from a V-lambda promoter in chicken DT40 cells.

    Science.gov (United States)

    Kothapalli, Naga Rama; Norton, Darrell D; Fugmann, Sebastian D

    2011-01-01

    Somatic hypermutation (SHM) of immunoglobulin genes is initiated by activation-induced cytidine deaminase (AID) in activated B cells. This process is strictly dependent on transcription. Hence, cis-acting transcriptional control elements have been proposed to target SHM to immunoglobulin loci. The Mus musculus Igκ locus is regulated by the intronic enhancer (iE/MAR) and the 3' enhancer (3'E), and multiple studies using transgenic and knock-out approaches in mice and cell lines have reported somewhat contradictory results about the function of these enhancers in AID-mediated sequence diversification. Here we show that the M. musculus iE/MAR and 3'E elements are active solely as transcriptional enhancer when placed in the context of the IGL locus in Gallus gallus DT40 cells, but they are very inefficient in targeting AID-mediated mutation events to this locus. This suggests that either key components of the cis-regulatory targeting elements reside outside the murine Igκ transcriptional enhancer sequences, or that the targeting of AID activity to Ig loci occurs by largely species-specific mechanisms. PMID:21533098

  20. Classical Mus musculus Igκ enhancers support transcription but not high level somatic hypermutation from a V-lambda promoter in chicken DT40 cells.

    Directory of Open Access Journals (Sweden)

    Naga Rama Kothapalli

    Full Text Available Somatic hypermutation (SHM of immunoglobulin genes is initiated by activation-induced cytidine deaminase (AID in activated B cells. This process is strictly dependent on transcription. Hence, cis-acting transcriptional control elements have been proposed to target SHM to immunoglobulin loci. The Mus musculus Igκ locus is regulated by the intronic enhancer (iE/MAR and the 3' enhancer (3'E, and multiple studies using transgenic and knock-out approaches in mice and cell lines have reported somewhat contradictory results about the function of these enhancers in AID-mediated sequence diversification. Here we show that the M. musculus iE/MAR and 3'E elements are active solely as transcriptional enhancer when placed in the context of the IGL locus in Gallus gallus DT40 cells, but they are very inefficient in targeting AID-mediated mutation events to this locus. This suggests that either key components of the cis-regulatory targeting elements reside outside the murine Igκ transcriptional enhancer sequences, or that the targeting of AID activity to Ig loci occurs by largely species-specific mechanisms.

  1. Adaptation of the interspersed repetitive sequence polymerase chain reaction to the isolation of mouse DNA probes from somatic cell hybrids on a hamster background

    International Nuclear Information System (INIS)

    A strategy for the rapid isolation of DNA probes from radiation-fusion Chinese hamster cell hybrids containing overlapping portions of the murine X chromosome based on the interspersed repetitive sequence polymerase chain reaction (IRS-PCR) previously used with human somatic cell hybrids has been developed. This specific amplification of mouse DNA on a hamster background depends on the use of primers directed to the B2 short interspersed repeat element family and the R repeat, from the long interspersed repeat element family, L1. Two sets of amplification conditions, which gave specific amplification of mouse DNA from either a mouse X-monochromosomal hybrid or irradiation-fusion hybrids having reduced X content, were defined. The mouse X-only chromosome hybrid yielded approximately 20 discrete reproducible bands, while the irradiation-fusion hybrids yielded between 1 and 10 discrete products. Comparison of different irradiation-fusion hybrids has allowed the definition of both specific and shared products corresponding to different regions within the overlapping X-chromosome fragments present within these hybrids. Use of such hybrids and the IRS-PCR technique has allowed the isolation of probes corresponding to the central region of the mouse X chromosome that contains the X-inactivation center. The method should be widely applicable to the isolation of mouse DNA sequences from mouse hybrid cell lines on either human or Chinese hamster backgrounds

  2. Lowering storage temperature during ovary transport is beneficial to the developmental competence of bovine oocytes used for somatic cell nuclear transfer.

    Science.gov (United States)

    Wang, Y S; Zhao, X; Su, J M; An, Z X; Xiong, X R; Wang, L J; Liu, J; Quan, F S; Hua, S; Zhang, Y

    2011-03-01

    The objective of this study was to determine the effect of storage temperature during ovary transport on the developmental competence of bovine oocytes for use in somatic cell nuclear transfer (SCNT). Ovaries obtained from a slaughterhouse were stored in physiological saline for 3-4h at one of the three temperatures: 15 °C, 25 °C, or 35 °C. The developmental competence of oocytes used for SCNT was ascertained by cleavage and blastocyst formation rate, total cell number, apoptosis index, and the relative abundance of Bax and Hsp70.1 in day 7 blastocysts. Ovaries stored at 35 °C for 3-4h reduced the recovery rate of grade I and II oocytes compared with those stored at 25 °C or 15 °C (45.1±0.7% vs. 76.7±1.2% or 74.8±2.0%, Povaries stored at 15 °C, however, produced blastocysts with higher cell numbers (97.3±8.6 vs. 80.2±10.8 or 77.4±11.7; Povaries stored at 15 °C was lower than those stored at 25 °C or 35 °C (Pquality and developmental competence of oocytes used for SCNT due to the alleviation of stresses on the oocytes compared with those subjected to storage temperatures of 25 °C or 35 °C. PMID:21333472

  3. Characterization of Regenerative Phenotype of Unrestricted Somatic Stem Cells (USSC) from Human Umbilical Cord Blood (hUCB) by Functional Secretome Analysis.

    Science.gov (United States)

    Schira, Jessica; Falkenberg, Heiner; Hendricks, Marion; Waldera-Lupa, Daniel M; Kögler, Gesine; Meyer, Helmut E; Müller, Hans Werner; Stühler, Kai

    2015-10-01

    Stem cell transplantation is a promising therapeutic strategy to enhance axonal regeneration after spinal cord injury. Unrestricted somatic stem cells (USSC) isolated from human umbilical cord blood is an attractive stem cell population available at GMP grade without any ethical concerns. It has been shown that USSC transplantation into acute injured rat spinal cords leads to axonal regrowth and significant locomotor recovery, yet lacking cell replacement. Instead, USSC secrete trophic factors enhancing neurite growth of primary cortical neurons in vitro. Here, we applied a functional secretome approach characterizing proteins secreted by USSC for the first time and validated candidate neurite growth promoting factors using primary cortical neurons in vitro. By mass spectrometric analysis and exhaustive bioinformatic interrogation we identified 1156 proteins representing the secretome of USSC. Using Gene Ontology we revealed that USSC secretome contains proteins involved in a number of relevant biological processes of nerve regeneration such as cell adhesion, cell motion, blood vessel formation, cytoskeleton organization and extracellular matrix organization. We found for instance that 31 well-known neurite growth promoting factors like, e.g. neuronal growth regulator 1, NDNF, SPARC, and PEDF span the whole abundance range of USSC secretome. By the means of primary cortical neurons in vitro assays we verified SPARC and PEDF as significantly involved in USSC mediated neurite growth and therewith underline their role in improved locomotor recovery after transplantation. From our data we are convinced that USSC are a valuable tool in regenerative medicine as USSC's secretome contains a comprehensive network of trophic factors supporting nerve regeneration not only by a single process but also maintained its regenerative phenotype by a multitude of relevant biological processes. PMID:26183719

  4. Energy output from a single outer hair cell

    CERN Document Server

    Iwasa, Kuni H

    2016-01-01

    Electromotility of outer hair cells (OHCs) has been extensively studied with in vitro experiments because of its physiological significance in the cochlear amplifier, which provides the exquisite sensitivity and frequency selectivity of the mammalian ear. However, these studies have been performed largely under load-free conditions or with static load, while these cells function in vivo in a dynamic environment, receiving electrical energy to enhance mechanical oscillation in the inner ear. This gap leaves uncertainties in addressing a key issue, how much mechanical energy an OHC provides. The present report is an attempt of bridging the gap by introducing a simple one-dimensional model for electromotility of OHC in a dynamic environment. This model incorporates a feedback loop involving the receptor potential and the mechanical load on OHC, and leads to an analytical expression for the membrane capacitance, which explicitly describes the dependence on the elastic load, viscous drag, and the mass. The derived...

  5. SOMATIC EMBRYOGENESIS AND PLANT REGENERATION FROM IMMATURE EMBRYO INDUCED CALLUS OF MAIZE (ZEA MAYS L.)

    OpenAIRE

    Anna Preťová; Miroslava Jakubeková; Bohuš Obert

    2012-01-01

    Somatic embryogenesis is the process by which somatic cells, under induction conditions, generate embryogenic cells, which go through a series of morphological and biochemical changes that result in the formation of a somatic embryo. These characteristics have designated somatic embryogenesis into a model system for the study of morphological, physiological, molecular and biochemical events occurring during the onset and development of embryogenesis in higher plants. In our experiments somati...

  6. Casein fractions of ultra high temperature milk with different somatic cell counts Frações de caseína de leite longa-vida com diferentes contagens de células somáticas

    OpenAIRE

    Andrezza Maria Fernandes; Fernanda Bovo; Thais Santos Moretti; Roice Eliana Rosim; César Gonçalves de Lima; Carlos Augusto Fernandes de Oliveira

    2008-01-01

    The objective of this work was to evaluate the effect of somatic cell counts (SCC) in casein fractions of ultra high temperature (UHT) milk. Raw milks were categorized in SCC groups of low (200,000-320,000 cells mL-1), intermediate (380,000-560,000 cells mL-1) and high cells (600,000-800,000 cells mL-1). Five replicates of UHT milks within each SCC category were analyzed for casein fractions after 8, 30, 60, 90 and 120 days of storage through high performance liquid chromatography. SCC showed...

  7. Identification of a two-marker-haplotype on Bos taurus autosome 18 associated with somatic cell score in German Holstein cattle

    Directory of Open Access Journals (Sweden)

    Reinsch Norbert

    2009-09-01

    Full Text Available Abstract Background The somatic cell score (SCS is implemented in routine sire evaluations in many countries as an indicator trait for udder health. Somatic cell score is highly correlated with clinical mastitis, and in the German Holstein population quantitative trait loci (QTL for SCS have been repeatedly mapped on Bos taurus autosome 18 (BTA18. In the present study, we report a refined analysis of previously detected QTL regions on BTA18 with the aim of identifying marker and marker haplotypes in linkage disequilibrium with SCS. A combined linkage and linkage disequilibrium approach was implemented, and association analyses of marker genotypes and maternally inherited two-marker-haplotypes were conducted to identify marker and haplotypes in linkage disequilibrium with a locus affecting SCS in the German Holstein population. Results We detected a genome-wide significant QTL within marker interval 9 (HAMP_c.366+109G>A - BMS833 in the middle to telomeric region on BTA18 and a second putative QTL in marker interval 12-13 (BB710 - PVRL2_c.392G>A. Association analyses with genotypes of markers flanking the most likely QTL positions revealed the microsatellite marker BMS833 (interval 9 to be associated with a locus affecting SCS within the families investigated. A further analysis of maternally inherited two-marker haplotypes and effects of maternally inherited two-marker-interval gametes indicated haplotype 249-G in marker interval 12-13 (BB710 - PVRL2_c.392G>A to be associated with SCS in the German Holstein population. Conclusion Our results confirmed previous QTL mapping results for SCS and support the hypothesis that more than one locus presumably affects udder health in the middle to telomeric region of BTA18. However, a subsequent investigation of the reported QTL regions is necessary to verify the two-QTL hypothesis and confirm the association of two-marker-haplotype 249-G in marker interval 12-13 (BB710 - PVRL2_c.392G>A with SCS. For this

  8. Finding of coagulase negative staphylococci in the herd with an increased number of somatic cells in milk and their antimicrobial susceptibility

    Directory of Open Access Journals (Sweden)

    Katić Vera

    2013-01-01

    Full Text Available Coagulase-negative staphylococci (CNS are generally considered to be opportunistic pathogens. Controlling CNS mastitis is difficult because the epidemiology is not clear, and the CNS group consists of about 40 different Staphylococcus species. Therefore, the aim of this study was to determine the prevalence of coagulasenegative staphylococci in milk of the cows with subclinical mastitis, as well as to determine different CNS species isolated from quarter milk samples for their susceptibility to antimicrobials used commonly for mastitis therapy. On the farm where there was found an increase of somatic cells in bulk milk, 112 dairy cows were examined by mastitis test. From 52 udder quarters where mastitis test showed an increase of somatic cells, milk samples were taken for bacteriological examination. For isolating the causes of mastitis there was used blood agar. Identification of the causative agents of mastitis was carried out on the basis of colony appearance on blood agar and their physiological characteristics. Coagulasepositive staphylococci sensitivity which cause mastitis was tested by Kirby Bauer method. For susceptibility testing there were used commercially produced discs containing: 10 IU penicillin, amoxicillin/clavulanic acid (20 +10 μg, cloxacillin 25 μg, 30 μg amoxicillin, cephalexin 30 μg, ceftiofur 30 μg, 15 μg lincomycin, gentamicin and tetracycline 30 μg. The sensitivity of microorganisms was evaluated on the basis of inhibition zone diameter recommended by the manufacturer and was labeled as sensitive (S moderately sensitive (I or resistant (R. Coagulase-negative staphylococci were isolated from 61.53% of samples from cows with subclinical mastitis, making them the most common cause of subclinical mastitis. The highest resistance of coagulase-negative staphylococci was found to penicillin G (58.33% of isolates. Full sensitivity of coagulase-negative staphylococci was found to amoxicillin / clavulanic acid (100% of

  9. Chromosome aberrations in somatic and germ cells following exposure to 300 R X-ray to rabbits

    International Nuclear Information System (INIS)

    A comparison is made of the chromosome aberrations yield in peripheral blood lymphocytes and spermatogonia of rabbits following exposure to 300 R X-rays. Cytogenetic analysis of lymphocytes immediately after irradiation revealed 28,0 per cent aberrant cells, including 0,17 dicentrics per cell. 120 days later less aberrations in primary spermatocytes at diakinesis - metaphasis I - 0,025 translocations per cell - were found. 60 days after irradiation the aberration rate in lymphocytes drops to the spontaneous level - 0,16 per cent aberrant cells - but analysis of karyotyped metaphases show anomalies which are unidentifiable after simple microscopic scoring. (A.B.)

  10. Arabidopsis thaliana Somatic Embryogenesis Receptor Kinase 1 protein is present in sporophytic and gametophytic cells and undergoes endocytosis

    DEFF Research Database (Denmark)

    Kwaaitaal, Mark Adrianus Cornelis J; de Vries, S C; Russinova, E

    2005-01-01

    in diverse cell types including the epidermis and the vascular bundles. In some cells, fluorescent receptors were seen in small vesicle-like compartments. After application of the fungal toxin Brefeldin A, the fluorescent receptors were rapidly internalized in the root meristem and root vascular...

  11. Discrete levels of Twist activity are required to direct distinct cell functions during gastrulation and somatic myogenesis.

    Directory of Open Access Journals (Sweden)

    Ming-Ching Wong

    Full Text Available Twist (Twi, a conserved basic helix-loop-helix transcriptional regulator, directs the epithelial-to-mesenchymal transition (EMT, and regulates changes in cell fate, cell polarity, cell division and cell migration in organisms from flies to humans. Analogous to its role in EMT, Twist has been implicated in metastasis in numerous cancer types, including breast, pancreatic and prostate. In the Drosophila embryo, Twist is essential for discrete events in gastrulation and mesodermal patterning. In this study, we derive a twi allelic series by examining the various cellular events required for gastrulation in Drosophila. By genetically manipulating the levels of Twi activity during gastrulation, we find that coordination of cell division is the most sensitive cellular event, whereas changes in cell shape are the least sensitive. Strikingly, we show that by increasing levels of Snail expression in a severe twi hypomorphic allelic background, but not a twi null background, we can reconstitute gastrulation and produce viable adult flies. Our results demonstrate that the level of Twi activity determines whether the cellular events of ventral furrow formation, EMT, cell division and mesodermal migration occur.

  12. No Correlates for Somatic Motility in Freeze-Fractured Hair-Cell Membranes of Lizards and Birds

    Science.gov (United States)

    Köppl, C.; Forge, A.; Manley, G. A.

    2003-02-01

    It is not known whether active processes in mammals and non-mammals are due to the same underlying mechanism. To address this, we studied the size and density of particles in hair-cell membranes in mammals, in a lizard, the Tokay gecko, and in a bird, the barn owl. We surmised that if the prominent particles described in mammalian outer-hair-cell membranes are responsible for cochlear motility, a similar occurrence in non-mammalian hair cells would argue for similar mechanisms. Particle densities differed, however, substantially from those of mammals, suggesting that non-mammals have no membrane-based motility.

  13. Confidence-based somatic mutation evaluation and prioritization.

    Directory of Open Access Journals (Sweden)

    Martin Löwer

    Full Text Available Next generation sequencing (NGS has enabled high throughput discovery of somatic mutations. Detection depends on experimental design, lab platforms, parameters and analysis algorithms. However, NGS-based somatic mutation detection is prone to erroneous calls, with reported validation rates near 54% and congruence between algorithms less than 50%. Here, we developed an algorithm to assign a single statistic, a false discovery rate (FDR, to each somatic mutation identified by NGS. This FDR confidence value accurately discriminates true mutations from erroneous calls. Using sequencing data generated from triplicate exome profiling of C57BL/6 mice and B16-F10 melanoma cells, we used the existing algorithms GATK, SAMtools and SomaticSNiPer to identify somatic mutations. For each identified mutation, our algorithm assigned an FDR. We selected 139 mutations for validation, including 50 somatic mutations assigned a low FDR (high confidence and 44 mutations assigned a high FDR (low confidence. All of the high confidence somatic mutations validated (50 of 50, none of the 44 low confidence somatic mutations validated, and 15 of 45 mutations with an intermediate FDR validated. Furthermore, the assignment of a single FDR to individual mutations enables statistical comparisons of lab and computation methodologies, including ROC curves and AUC metrics. Using the HiSeq 2000, single end 50 nt reads from replicates generate the highest confidence somatic mutation call set.

  14. Epithelial cells derived from human embryonic stem cells display p16INK4A senescence, hypermotility, and differentiation properties shared by many P63+ somatic cell types

    DEFF Research Database (Denmark)

    Dabelsteen, Sally; Hercule, Paula; Barron, Patricia;

    2009-01-01

    Human embryonic stem (hES) cells can generate cells expressing p63, K14, and involucrin, which have been proposed to be keratinocytes. Although these hES-derived, keratinocyte-like (hESderK) cells form epithelioid colonies when cultured in a fibroblast feeder system optimal for normal tissue-derived...... response previously characterized in keratinocytes. In organotypic culture, hESderK cells stratified and expressed involucrin and K10, as do epidermal keratinocytes in vivo. However, their growth requirements were less stringent than keratinocytes. We then extended the comparison to endoderm-derived, p63...

  15. Checkpoint kinase 1 negatively regulates somatic hypermutation.

    Science.gov (United States)

    Frankenberger, Samantha; Davari, Kathrin; Fischer-Burkart, Sabine; Böttcher, Katrin; Tomi, Nils-Sebastian; Zimber-Strobl, Ursula; Jungnickel, Berit

    2014-04-01

    Immunoglobulin (Ig) diversification by somatic hypermutation in germinal center B cells is instrumental for maturation of the humoral immune response, but also bears the risk of excessive or aberrant genetic changes. Thus, introduction of DNA damage by activation-induced cytidine deaminase as well as DNA repair by multiple pathways need to be tightly regulated during the germinal center response to prevent lymphomagenesis. In the present study, we show that DNA damage checkpoint signaling via checkpoint kinase 1 (Chk1) negatively regulates somatic hypermutation. Chk1 inhibition in human B cell lymphoma lines as well as inactivation of Chk1 alleles by gene targeting in DT40 B cells leads to increased somatic hypermutation. This is apparently due to changes in DNA repair pathways regulated by Chk1, such as a decreased homologous recombination efficiency that also leads to decreased Ig gene conversion in DT40. Our data show that Chk1 signaling plays a crucial role in regulation of Ig diversification and sheds unexpected light on potential origins of aberrant somatic hypermutation in B cell lymphomagenesis. PMID:24423870

  16. Identification of neurofibromatosis 1 (NF1) homologous loci by direct sequencing, fluorescence in situ hybridization, and PCR amplification of somatic cell hybrids

    Energy Technology Data Exchange (ETDEWEB)

    Purandare, S.M.; Neil, S.M.; Brothman, A. [Univ. of Utah, Salt Lake City (United States)]|[Jikei Univ., Tokyo (Japan)] [and others

    1995-12-10

    Using fluorescence in situ hybridization (FISH), we have identified seven NF1-related loci, two separate loci on chromosome 2, at bands 2q21 and 2q33-q34, and one locus each on five other chromosomes at bands 14q11.2, 15q11.2, 18p11.2, 21q11.2-q21, and 22q11.2. Application of PCR using NF1 primer pairs and genomic DNA from somatic cell hybrids confirmed the above loci, identified additional loci on chromosomes 12 and 15, and showed that the various loci do not share homology beyond NF1 exon 27b. Sequenced PCR products representing segments corresponding to NF1 exons from these loci demonstrated greater than 95% sequence identity with the NF1 locus. We used sequence differences between bona fide NF1 and NF1-homologous loci to strategically design primer sets to specifically amplify 30 of 36 exons within the 5{prime} end of the NF1 gene. These developments have facilitated mutation analysis at the NF1 locus using genomic DNA as template. 41 refs., 3 figs., 3 tabs.

  17. Comparison of California Mastitis Test (CMT), Somatic Cell Counts (SCC) and bacteriological examinations for detection of camel (Camelus dromedarius) mastitis in Ethiopia.

    Science.gov (United States)

    Abdel Gadir Atif, E; Hildebrandt, Goetz; Kleer, Josef N; Molla, Bayleyegn; Kyule, Moses N; Baumann, Maximilian P O

    2006-01-01

    A total of 956 quarter milk samples from 253 traditionally managed lactating camels were collected aseptically from Negele (Borena Region), Dire Dawa, and Gewane (Afar Region), Ethiopia, according to multi-stage sampling. The quarter milk samples were subjected to California Mastitis Test (CMT), Somatic Cell Counts (SCC) and bacteriological examinations. Five hundred and seventy one (59.7%) quarter milk samples had microorganisms. Of these, 428 (75.0%) had isolates that were identified as major pathogens (MAP) and 143 (25.0%) as minor pathogens (MIP). A positive correlation was found between CMT scores and bacteriological classes (MAP, MIP) (p-value = 0.00). Strong correlation (p-value = 0.00) between CMT scores and SCC was recorded. The differences among the median log SCC of bacteriological classes (MAP, MIP) were not significant (p-value = 0.24). Similarly, the application of the cut-off level of 2.5 x 10(5) ml(-1) indicated less agreement (p-value = 0.32) for bacteriological classes MAP and MIP. PMID:16450708

  18. Resistance to penicillin of Staphylococcus aureus isolates from cows with high somatic cell counts in organic and conventional dairy herds in Denmark

    Directory of Open Access Journals (Sweden)

    Vaarst Mette

    2006-11-01

    Full Text Available Abstract Background Quarter milk samples from cows with high risk of intramammary infection were examined to determine the prevalence of Staphylococcus aureus (SA and penicillin resistant SA (SAr in conventional and organic dairy herds and herds converting to organic farming in a combined longitudinal and cross-sectional study. Methods 20 conventional herds, 18 organic herds that converted before 1995, and 19 herds converting to organic farming in 1999 or 2000 were included in the study. Herds converting to organic farming were sampled three times one year apart; the other herds were sampled once. Risk of infection was estimated based on somatic cell count, milk production, breed, age and lactation stage. Results The high-risk cows represented about 49 % of the cows in the herds. The overall prevalence of SA and SAr among these cows was 29% (95% confidence interval: 24%–34% and 4% (95% confidence interval: 2%–5% respectively. The prevalence of penicillin resistance among SA infected cows was 12% (95% confidence interval: 6%–19% when calculated from the first herd visits. No statistically significant differences were observed in the prevalence of SAr or the proportion of isolates resistant to penicillin between herd groups. Conclusion The proportion of isolates resistant to penicillin was low compared to studies in other countries except Norway and Sweden. Based on the low prevalence of penicillin resistance of SA, penicillin should still be the first choice of antimicrobial agent for treatment of bovine intramammary infection in Denmark.

  19. Fatty acid content, health and risk indices, physicochemical composition, and somatic cell counts of milk from organic and conventional farming systems in tropical south-eastern Mexico.

    Science.gov (United States)

    Delgadillo-Puga, Claudia; Sánchez-Muñoz, Bernardo; Nahed-Toral, José; Cuchillo-Hilario, Mario; Díaz-Martínez, Margarita; Solis-Zabaleta, Roman; Reyes-Hernández, Aurora; Castillo-Domíguez, Rosa Maria

    2014-06-01

    Organic agriculture and livestock farming is claimed to promote animal welfare and can offer animal products with better hygienic-sanitary quality, based on principles of health, ecology, fairness, and care. However, no clear advantages of organic milk (OM) versus conventional milk (CM) from tropical conditions are available. The aims of the study were to determine fatty acid profile, health-promoting (HPI) and thrombogenic (TI) indices, physicochemical composition, and somatic cell counts (SCC) of OM and CM in tropical south-eastern Mexico. Female cross-breed cows (400-600 kg) were employed. CM had larger values of saturated fatty acids (SFA) and polyunsaturated fatty acid (PUFA) (63.6 %; 4.57 %) than OM (61.48 %; 4.22 %), while OM resulted in a larger value of monounsaturated fatty acid (MUFA) (34.3 %) than CM (31.7 %). HPI and TI showed that OM was more favorable than CM. Milk production and physicochemical composition (PC) as well as density had no significant difference, while SCC was significantly lower in OM than in CM on a monthly basis. These results showed that OM promotes a healthful and balanced diet, and is already produced by sustainable ecologic technologies employing traditional agrosilvopastoral management, which is more environmentally friendly and promotes ecological resilience. PMID:24715204

  20. A pathogen-specific approach towards udder health management in dairy herds: Using culture and somatic cell counts from routine herd investigations.

    Science.gov (United States)

    Petzer, Inge-Marié; Karzis, Joanne; Donkin, Edward F; Webb, Edward C

    2016-01-01

    A dedicated udder health diagnostic programme was developed and used over a 15-year period in South Africa to analyse milk samples based on microbiological and cytological patterns within various groups and for individual cows and udder quarters in dairy herds. These pathogen-specific analyses are utilised for pro-active improvement and management of udder health in South African commercial dairy herds. The programme acts as a monitoring tool and identifies management areas at risk and individual cows with udder disease and uses both quarter and composite milk samples. Intra-mammary infection (IMI) is a dynamic situation and depending on the time a milk sample is taken, false-negative results may be obtained. A new IMI and an infection that is curing may both have low somatic cell counts (SCCs), masking the true bacterial status. SCC in individual infected udder quarters may differ greatly depending on the causative bacterial species, its pathogenicity, the host immune status and the environmental factors involved. A pathogen-specific udder health approach was followed with repeated herd tests to take account of these udder health dynamics. The results of the herd IMI investigation are applied in practice to assist veterinarians, udder health consultants and managers to make informed and specific detailed decisions at both a herd and on an individual cow basis regarding udder health. PMID:27608503

  1. Composition, somatic cell count and casein fractions of ethanol unstable milks - doi: 10.4025/actascitechnol.v35i1.11481

    Directory of Open Access Journals (Sweden)

    Carlos Augusto Fernandes de Oliveira

    2013-01-01

    Full Text Available This study determined the composition, somatic cell count (SCC and relative percentage of a-S1, a-S2, b and k caseins in ethanol-unstable (UNA and stable bulk milk samples. The sampling plan involved farms that supplied milk to one dairy plant located in the northeast region of São Paulo, Brazil, in May (fall, July (winter, and September (spring 2007. Three hundred thirty-four bulk milk samples within an acceptable range of pH and acidity were included in the study and divided into two groups: a stable milk, when protein precipitation only occurred with 78% ethanol (v/v; and b unstable (UNA milk, when the precipitation occurred with 72% ethanol (v/v. From the total samples analyzed, 77 (23% were unstable in the 72% ethanol (v/v, although they have shown normal pH and acidity, and 41 (12.3% were stable in 78% ethanol (v/v. No differences were found between relative percentages of a and b-casein in UNA or stable milks. However, UNA samples showed higher SCC, as well as lower casein and lactose contents. Results indicated that the high SCC may be involved as a causal factor for the high incidence of UNA milks in the herds studied.  

  2. Occurrence of genes coding for MSCRAMM and biofilm-associated protein Bap in Staphylococcus spp. isolated from bovine subclinical mastitis and relationship with somatic cell counts.

    Science.gov (United States)

    Zuniga, Eveline; Melville, Priscilla A; Saidenberg, André B S; Laes, Marco A; Gonsales, Fernanda F; Salaberry, Sandra R S; Gregori, Fabio; Brandão, Paulo E; dos Santos, Franklin G B; Lincopan, Nilton E; Benites, Nilson R

    2015-12-01

    This study aimed to elucidate aspects of the epidemiology of bovine subclinical mastitis through the assessment of genes encoding MSCRAMM (microbial surface components recognizing adhesive matrix molecules - a group of adhesins) and protein Bap (implicated in biofilm formation), in coagulase-positive (CPS) and coagulase-negative (CNS) Staphylococcus isolated from subclinical mastitis. Milk samples were collected for microbiological exams, somatic cell count (SCC) and a survey of the genes coding for MSCRAMM (cna, eno, ebpS, fnbA, fnbB and fib) and biofilm-associated protein Bap (bap) in 106 Staphylococcus spp. isolates using PCR. The frequencies of occurrence of eno (82.1%), fnbA (72.6%), fib (71.7%) and bap (56.6%) were higher (P < 0.0001) compared with the other assessed genes (cna, ebpS and fnbB). The higher frequency of occurrence (P < 0.005) of the bap gene in CNS compared with CPS suggests that in these species biofilm formation is an important mechanism for the persistence of the infection. The medians of the SCCs in the samples where eno, fnbA, fib and bap genes were detected were higher compared with Staphylococcus without the assessed genes (P < 0.05) and negative samples (P < 0.01), which indicated that the presence of these MSCRAMM may be related to a higher intensity of the inflammatory process. PMID:26318876

  3. Somatic cell count and milk neutrophil viability of dairy heifers with specific CXCR1 genotypes following experimental intramammary infection with Staphylococcus chromogenes originating from milk.

    Science.gov (United States)

    Verbeke, Joren; Piccart, Kristine; Piepers, Sofie; Van Poucke, Mario; Peelman, Luc; De Visscher, Anneleen; De Vliegher, Sarne

    2015-06-01

    Previous observational studies suggest an association between polymorphism c.980A>G in the CXCR1 gene, encoding the chemokine (C-X-C motif) receptor 1, and the innate immunity and infection status of the mammary gland. Mammary glands of eight Holstein heifers were experimentally infected with a Staphylococcus chromogenes isolate originating from a chronic intramammary infection (IMI) to study differences between CXCR1 genotypes c.980AG and c.980GG. Quarters from heifers with genotypes c.980AG and c.980GG developed subclinical mastitis but showed differences in the early response at 6-18 h post challenge. Bacterial count at 18 h post challenge tended to be higher in quarters from c.980AG heifers compared to c.980GG heifers. Somatic cell count (SCC) was higher at 6 h post challenge and tended to be higher at 9 h post challenge in c.980AG heifers compared to c.980GG heifers. Milk production decreased similarly. Milk neutrophils of c.980AG heifers showed more apoptosis at 9 h post challenge and tended to show more necrosis at 6, 9 and 12 h post challenge than c.980GG heifers. Differences were less pronounced in the later stage (>18 h) of infection. The results demonstrate that CXCR1 polymorphism can influence SCC and milk neutrophil viability following experimental IMI. PMID:25933826

  4. Bovine subclinical intramammary infection caused by coagulase-negative staphylococci increases somatic cell count but has no effect on milk yield or composition.

    Science.gov (United States)

    Tomazi, T; Gonçalves, J L; Barreiro, J R; Arcari, M A; dos Santos, M V

    2015-05-01

    The aim of this study was to evaluate the effect of subclinical intramammary infection (IMI) caused by coagulase-negative staphylococci (CNS) as a group and by specific CNS species on milk yield and composition and somatic cell count (SCC) of dairy cows. Selection of cows with IMI caused by CNS was performed by microbiological cultures of composite samples collected from 1,242 dairy cows distributed in 21 dairy herds. After selection of cows, milk yield was measured and milk samples were collected at the mammary quarter level (i.e., 1,140 mammary samples collected from 285 cows) for analysis of milk composition and SCC. In total, 108 isolates of CNS were identified at the species level by PCR-RFLP analysis. Forty-one pairs of contralateral mammary quarters, with and without IMI, were used to evaluate the effect of CNS on milk yield and composition. Mammary quarters infected with CNS had higher geometric mean SCC (306,106 cells/mL) than noninfected contralateral mammary quarters (62,807 cells/mL). Intramammary infection caused by CNS had no effect on milk yield or on contents of fat, crude protein, casein, lactose, total solids, and solids-not-fat. Staphylococcus chromogenes was the most prevalent CNS species in this study and the only species that allowed within-cow evaluation. The IMI caused by S. chromogenes increased SCC but had no effect on milk yield and composition at the quarter level. In conclusion, subclinical mastitis caused by CNS increased the SCC but had no effect on milk yield and composition of dairy cows. PMID:25726098

  5. Effects of bovine subclinical mastitis caused by Corynebacterium spp. on somatic cell count, milk yield and composition by comparing contralateral quarters.

    Science.gov (United States)

    Gonçalves, Juliano Leonel; Tomazi, Tiago; Barreiro, Juliana Regina; Beuron, Daniele Cristine; Arcari, Marcos André; Lee, Sarah Hwa In; Martins, Cristian Marlon de Magalhães Rodrigues; Araújo Junior, João Pessoa; dos Santos, Marcos Veiga

    2016-03-01

    Subclinical mastitis caused by Corynebacterium spp. (as a group and at the species level) was investigated by evaluating contralateral (healthy and infected) mammary quarters for somatic cell count (SCC), milk yield and composition. Selection of cows with subclinical mastitis caused by Corynebacterium spp. was performed by microbiological culture of composite samples collected from 1242 dairy cows from 21 dairy herds. For each of the selected cows, milk yield was measured and milk samples were collected at the mammary quarter level (i.e., 1140 mammary samples collected from 285 cows) for analysis of milk composition and SCC. The identification of Corynebacterium spp. isolates was performed by 16S rRNA gene sequencing. One hundred and eighty Corynebacterium spp. isolates were identified, of which 167 (92.77%) were C.bovis and eight (4.44%) non-C.bovis; for five of the Corynebacterium spp. isolates (2.77%), sequencing of 16S rRNA genes did not allow identification at the species level. Mammary quarters infected with Corynebacterium spp. as a group had a higher geometric mean SCC (197,900 cells/mL) than healthy contralateral mammary quarters (85,800 cells/mL). Species of Corynebacterium non-C.bovis were infrequently isolated and did not change SCC, milk yield or milk solid contents when evaluated at the contralateral quarter level. Although C.bovis infection showed no effect on milk yield, fat, protein, casein or total solids in milk, it increased SCC and decreased lactose and milk solids non-fat content. PMID:26831159

  6. Events of elevated somatic cell counts in high-producing dairy cows are associated with daily body weight loss in early lactation.

    Science.gov (United States)

    van Straten, M; Friger, M; Shpigel, N Y

    2009-09-01

    The objective of this study was to determine associations between body weight (BW) and body condition score (BCS) variables indicating a more severe negative energy balance in early lactation and events of somatic cell counts (SCC) >250,000 cells/mL and SCC >400,000 cells/mL in dairy cows. We studied lactations from 634 primiparous and 1,086 multiparous Israeli Holstein dairy cows originating from 7 commercial dairy farms. Generalized mixed models with a random herd effect were used to quantify the effects of BW and BCS variables in early lactation on events of elevated SCC. Data were analyzed using 2 different approaches. In the first approach, only first events in a lactation were taken into account, whereas in the second approach, all events in a lactation were analyzed and repeated events from the same cow were accounted for. Although no associations were found between the different BW and BCS variables and first events of elevated SCC, associations were present between these variables and events of elevated SCC when all events were analyzed. The cumulative incidence of a lactation with multiple events of SCC >250,000 cells/mL was 8.8 and 27.7% for primiparous and multiparous cows, respectively. The odds of an event of SCC >250,000 cells/mL were 25% greater for cows belonging to the upper quartile in relative BW loss from calving to nadir BW (loss >12.3, 15.0, and 15.7% for first-, second-, and third- parity and greater cows, respectively) compared with cows losing less relative BW. Odds of an event were 44% greater for cows with ketosis when compared with cows without. The cumulative incidence of a lactation with multiple events of SCC >400,000 cells/mL was 4.1 and 14.3% for primiparous and multiparous cows, respectively. The odds of an event of SCC >400,000 cells/mL were 43% greater for cows belonging to the upper quartile in relative BW loss from calving to nadir BW compared with cows losing less relative BW. Odds of an event were 33% greater for cows with

  7. Optical reprogramming of human somatic cells using ultrashort Bessel-shaped near-infrared femtosecond laser pulses

    Science.gov (United States)

    Uchugonova, Aisada; Breunig, Hans Georg; Batista, Ana; König, Karsten

    2015-11-01

    We report a virus-free optical approach to human cell reprogramming into induced pluripotent stem cells with low-power nanoporation using ultrashort Bessel-shaped laser pulses. Picojoule near-infrared sub-20 fs laser pulses at a high 85 MHz repetition frequency are employed to generate transient nanopores in the membrane of dermal fibroblasts for the introduction of four transcription factors to induce the reprogramming process. In contrast to conventional approaches which utilize retro- or lentiviruses to deliver genes or transcription factors into the host genome, the laser method is virus-free; hence, the risk of virus-induced cancer generation limiting clinical application is avoided.

  8. Optical reprogramming of human somatic cells using ultrashort Bessel-shaped near-infrared femtosecond laser pulses.

    Science.gov (United States)

    Uchugonova, Aisada; Breunig, Hans Georg; Batista, Ana; König, Karsten

    2015-11-01

    We report a virus-free optical approach to human cell reprogramming into induced pluripotent stem cells with low-power nanoporation using ultrashort Bessel-shaped laser pulses. Picojoule near-infrared sub-20 fs laser pulses at a high 85 MHz repetition frequency are employed to generate transient nanopores in the membrane of dermal fibroblasts for the introduction of four transcription factors to induce the reprogramming process. In contrast to conventional approaches which utilize retro- or lentiviruses to deliver genes or transcription factors into the host genome, the laser method is virus-free; hence, the risk of virus-induced cancer generation limiting clinical application is avoided. PMID:26618522

  9. The relationship between HLA class II polymorphisms and somatic deletions in testicular B cell lymphomas of Dutch patients

    NARCIS (Netherlands)

    Riemersma, SA; Jordanova, ES; Haasnoot, GW; Drabbels, J; Schuuring, E; Schreuder, GMT; Kluin, PM

    2006-01-01

    Several risk factors including immune deficiencies, infections, and autoimmune diseases have been established for non-Hodgkin's lymphoma (NHL). For diffuse large B cell lymphoma (DLBCL), the most common type of lymphoma, no risk factors have been described, which may be due to the intrinsic heteroge

  10. Are in-line measurements of somatic cell counts equally or more useful for genetic evaluations as those from DHI?

    DEFF Research Database (Denmark)

    Sørensen, Lars Peter; Løvendahl, Peter

    2012-01-01

    and 1294 first parity cows (5 to 305 d in milk). Cell counts were analyzed using a 2-trait linear animal model. There were a total of 298,988 records for OCC and 7779 for test-day SCC. The fixed part of the model included lactation curves fitted as a Wilmink function, and the random parts included animal...

  11. In vivo and in vitro analysis of age-associated changes and somatic cellular senescence in renal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Birgit Berkenkamp

    Full Text Available Acute kidney injury is a major clinical problem and advanced age is associated with ineffective renal regeneration and poor functional outcome. Data from kidney injury models suggest that a loss of tubular epithelial proliferation contributes to a decrease in renal repair capacity with aging, but aging can also lead to a higher severity of inflammation and damage which may influence repair. In this study we tested intrinsic age-dependent changes in tubular epithelial proliferation in young and old mice, by injecting low-dose lead acetate as a non-injurious mitogen. In parallel, we explored in vitro techniques of studying cellular senescence in primary tubular epithelial cells (PTEC. Lead acetate induced tubular epithelial proliferation at a significantly higher rate in young as compared to old mice. Old kidneys showed significantly more senescence as demonstrated by increased p16 (INK4a, senescence associated β-galactosidase, and γH2AX(+/Ki-67(- cells. This was paralleled in old kidneys by a higher number of Cyclin D1 positive tubular cells. This finding was corroborated by a positive correlation between Cyclin D1 positivity and age in human renal biopsies. When tubular cells were isolated from mouse kidneys they rapidly lost their age-associated differences under culture conditions. However, senescence was readily induced in PTEC by γ-irradiation representing a future model for study of cellular senescence in the renal epithelium. Together, our data indicate that the tubular epithelium of aged kidney has an intrinsically reduced proliferative capacity probably due to a higher load of senescent cells. Moreover, stress induced models of cellular senescence are preferable for study of the renal epithelium in vitro. Finally, the positive correlation of Cyclin D1 with age and cellular senescence in PTEC needs further evaluation as to a functional role of renal epithelial aging.

  12. Developmental Outcome and Related Abnormalities in Goats: Comparison Between Somatic Cell Nuclear Transfer- and In Vivo-Derived Concepti During Pregnancy Through Term.

    Science.gov (United States)

    Martins, Leonardo Tondello; Neto, Saul Gaudêncio; Tavares, Kaio César Simiano; Calderón, Carlos Enrique Méndez; Aguiar, Luis Henrique; Lazzarotto, Cícera Regina; Ongaratto, Felipe Ledur; Rodrigues, Victor Hugo Vieira; Carneiro, Igor de Sá; Rossetto, Rafael; Almeida, Anderson Pinto; Fernandes, César Carneiro Linhares; Rondina, Davide; Dias, Ana Christina Oliveira; Chies, Jocelei Maria; Polejaeva, Irina A; Rodrigues, José Luiz; Forell, Fabiana; Bertolini, Luciana Relly; Bertolini, Marcelo

    2016-08-01

    Cloning by somatic cell nuclear transfer (SCNT) is characterized by low efficiency and the occurrence of developmental abnormalities, which are rather poorly studied phenomena in goats. This study aimed at comparing overall SCNT efficiency in goats by using in vitro-matured (IVM) or in vivo-matured oocytes and fibroblast donor cells (mock transfected, transgenic, or wild type), also characterizing symptoms of the Abnormal Offspring Syndrome (AOS) in development, comparing results with pregnancies produced by artificial insemination (AI) and in vivo-derived (IVD) embryos. The SCNT group had lower pregnancy rate (18.3%, 11/60), total number of concepti (20.0%, 12/60), term births (3.3%, 2/60), and live births (1.7%, 1/60) than both the IVD (77.8%, 7/9; 155.5%, 14/9; 122.2%, 11/9; 88.8%, 8/9) and the AI (71.4%, 10/14; 121.4%, 17/14; 100%, 14/14; 78.5%, 11/14) groups, respectively (p < 0.05). No SCNT pregnancies reached term using IVM oocytes, but in vivo-matured oocytes resulted in two term transgenic cloned kids. The proportion fetal membrane (FM) weight/birth weight reflected an increase in FM size and cotyledonary enlargement in clones, for disproportionally bigger newborns in relation to cotyledonary numbers. Overall, goat cloning showed losses and abnormality patterns similar to the AOS in cloned cattle and sheep, which have not been previously well recognized in goats. PMID:27362734

  13. Socioeconomic and technical assistance factors related to total bacteria count and somatic cell count of milk from bulk tanks in southern Minas Gerais State, Brazil

    Directory of Open Access Journals (Sweden)

    Marcel Gomes Paixão

    2015-07-01

    Full Text Available The purpose of this survey was to evaluate the socioeconomic and technical assistance profiles of dairy farmers from six districts in the south of Minas Gerais state, Brazil, and to identify the possible risk factors associated with total milk bacteria count (TBC above 43,000 CFU mL-1 and bulk milk somatic cell count (BMSCC above 595,000 cells mL-1. Most of the producers were between 41 and 60 years of age (48.9%, 74.2% did not reach high school, and 72.3% of the respondents were satisfied with their profession, although 63% would not recommend dairy farming to their children. Only 34.7% used periodic technical assistance, but 59.1% consulted it in cases of doubt. The risk factors found in the final multivariable regression models were: TBC (Did not consult technical assistance in case of doubt, OR 3.97, P=0.030; Retirement, OR 9.32, P=0.041 and BMSCC (Producer does not reside on farm, OR 4.06, P=0.046; Presence of technical assistance OR 3.29, P=0.041. It can be concluded that the search for emergency technical assistance, as reported by farmers, was effective against the TBC problems; however, it was ineffective for controlling mastitis in the herd and reducing BMSCC levels. The 10 step mastitis control program from the National Mastitis Council needs to be included on the surveyed farms, especially the permanent advisory technical assistance from veterinarians, aiming towards the establishment of goals for udder health status, reviews and records.

  14. I131 therapy induces persistent radiation-dose dependent increases in glycophorin a locus somatic mutations in bone marrow stem cells

    International Nuclear Information System (INIS)

    Patients with thyroid diseases treated with I131 receive known sub-acute marrow exposures to ionizing radiation of ∼2 to >200 cGy. Time-series sampling of peripheral blood from these patients, assayed for the frequency of erythrocytes expressing glycophorin A (GPA) allele-loss variant phenotypes, demonstrates the induction, accumulation, and long-term persistence of radiation-induced in vivo somatic mutations at this locus in erythroid marrow progenitor cells. Initial dosimetry and assay data from 5 patients yielded a linear GPA dose response of ∼6.5 induced variants/106 cells/Gy which is 1/3 to 1/4 of that previously observed for Hiroshima A-bomb survivors and individuals exposed at the Chernobyl nuclear reactor and Goiania Cs137 source accidents who predominantly received external exposures to ionizing radiation. The lower slope of the dose response observed in the I131 treated patients may reflect a reduced biological effectiveness of this exposure due to differences in the energy spectra of the γ radiation, internal versus external exposure, and/or protracted versus acute dose rate effects. Ongoing studies of I131 treated patients are designed to define the shape of the low dose response and limit of sensitivity of the GPA assay; parameters that are required for the application of the assay as a quantitative cumulative radiation biodosimeter in medical, occupational, and accidental exposure settings. This biodosimetric analysis of patients receiving very similar marrow exposures will also permit an assessment of the inter-individual variability in biological response to ionizing radiation

  15. Effects of the reproductive status on morphological oocyte quality and developmental competence of oocytes after in vitro fertilization and somatic cell nuclear transfer in cat.

    Science.gov (United States)

    Naoi, H; Agung, B; Karja, N W K; Wongsrikeao, P; Shimizu, R; Taniguchi, M; Otoi, T

    2008-04-01

    This study was conducted to examine the effects of the reproductive cycle of donor cat on the quality of oocytes at recovery and developmental competence of oocytes after in vitro fertilization (IVF) and somatic cell nuclear transfer (NT). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular or luteal stages. After collection of oocytes, the oocytes were classified into four grades according to the morphological condition of oocyte cytoplasm and cumulus cells. A total of 16 558 oocytes were obtained from 198 ovarian pairs. The total mean numbers of oocytes and the mean numbers of oocytes with high quality (grade I) were significantly higher in ovarian pairs at the inactive stage (111.1 and 19.0 oocytes, respectively) than in ovarian pairs at the follicular stage (67.1 and 11.4 oocytes, respectively). A significant difference in the proportions of oocytes with grade I out of the total examined oocytes was observed between the follicular and luteal stages of ovaries (14.9% vs 20.2%, p quality of oocytes at recovery, irrespective of the reproductive status of ovaries. Moreover, there were no significant differences in the proportions of cleavage and development to the blastocyst stage of IVF and NT embryos among three oestrous stages of ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries has no apparent effects on the in vitro development of oocytes after IVF and NT, but the quality of oocytes at recovery influences the development of IVF embryos. PMID:18325005

  16. Effects of some natural extracts on tradescantia somatic cell pink mutation frequencies induced by gamma-ray

    International Nuclear Information System (INIS)

    This study deals with the effect of some natural extracts on the pink mutations induced by radiation in Tradescantia 4430 stamen hair cells. Inflorescence cuttings, with or without pretreatments of natural extracts for 3 hours, were exposed to 1 Gy of gamma ray. Comparisons were made on the basis of pooled data during the peak interval between the mean pink mutation frequencies of the two experimental groups. Pretreatments of FB or FB-I resulted in two-fold increases of the pink mutation frequencies, compared to those of control group. Synergism between certain fractions and radiation was a possible cause of increased DNA damage. On the other hand, the extract PG in proper concentrations significantly reduced the pink mutation frequencies (p<0.05). The result meant that PG had a protective effect on radiation-induced cell damage. Tradescantia proved to be an excellent biological model system for assessing the radiomodification effects of natural materials

  17. Evaluation of genotoxic potential of Hypericum triquetrifolium extract in somatic and germ cells of male albino mice

    OpenAIRE

    Bushra M. A. Mohammed,; Sarbast K. Q. Kheravii

    2011-01-01

    Hypericum triquetrifolium aqueous extract were studied for the first time for its toxic and the possible clastogenic effects in vivo on the bone marrow and spermatozoa cells of Swiss albino mice. The lethal dose of the aqueous extract was considered to be 10.33 g/kg of the body weight, injected subcutaneously. The doses which were chosen for treatments were 2, 1, and 0.25 g/kg. H. triquetrifolium extract induce statistically significant increases in the average...

  18. Novel somatic mutations identified by whole-exome sequencing in muscle-invasive transitional cell carcinoma of the bladder

    OpenAIRE

    PAN, HUIXING; Xu, Xiaojian; WU, DEYAO; QIU, QIAOCHENG; Zhou, Shoujun; He, Xuefeng; Zhou, Yunfeng; QU, PING; Hou, Jianquan; He, Jun; Zhou, Jian

    2016-01-01

    Transitional cell carcinoma (TCC) is the one of the most commonly observed types of cancer globally. The identification of novel disease-associated genes in TCC has had a significant effect on the diagnosis and treatment of bladder cancer; however, there may be a large number of novel genes that have not been identified. In the present study, the exomes of two individuals who were diagnosed with muscle-invasive TCC (MI-TCC) were sequenced to investigate potential variants. Subsequently, follo...

  19. Targeting exogenous GDNF gene to the bovine somatic cell beta-casein locus for the production of transgenic bovine animals.

    Science.gov (United States)

    Zhang, X M; Luo, F H; Ding, H M; Li, B; Zhang, J J; Wu, Y J

    2015-01-01

    Considerable attention is currently being directed toward methods for producing recombinant human proteins in the mammary glands of genetically modified transgenic livestock. However, the expression of inserted genes in transgenic animals is variable and often very low because of the randomness of the site of transgene integration. One possible strategy to avoid the expression problem associated with random integration is to use site-specific integration by targeting integration to a high expression locus and, thereby, to improve expression of the transferred gene. In the present study, we focused on glial cell line-derived neurotrophic factor (GDNF), a novel type of neurotrophic factor first cloned in 1993. Research has shown that GDNF may have potential applications in the treatment of Parkinson's disease and other diseases of the central nervous system since it acts as a protective factor for central dopaminergic neurons. Here, we constructed a gene targeting vector to knock-in the human GDNF gene at the bovine beta-casein gene locus as a first step to producing transgenic animals with a high level of expression of human GDNF protein in their mammary glands. Bovine fetal fibroblast cells were transfected with linearized pNRTCNbG by electroporation. Three cell clones were identified with successful targeting to the beta-casein locus; and were confirmed using both polymerase chain reaction analysis and sequencing. Gene-targeted cells were used as nuclear donors; a total of 161 embryos were reconstructed, 23 of which developed to the blastocyst stage. These blastocysts were transferred to 8 recipient cows, but no offspring were obtained. PMID:26634460

  20. Somatic symptoms in depression

    OpenAIRE

    Kapfhammer, Hans-Peter

    2006-01-01

    Both painful and nonpainful somatic symptoms essentially characterize clinical states of depressive mood. So far, this well-established psychopathological knowledge has been appreciated only insufficiently by the official diagnostic sys-terms of the Diagnostic and Statistical Manual of Mental Disorders, 4th edition, Text Revision (DSM-IVTR) and the ICD-10 Classification of Mental and Behavioral Disorders. Clinical Descriptions and Diagnostic Guidelines (ICD-10). From a perspective of primary ...

  1. Comparison of epidermal keratinocytes and dermal fibroblasts as potential target cells for somatic gene therapy of phenylketonuria

    DEFF Research Database (Denmark)

    Christensen, Rikke; Güttler, Flemming; Jensen, Thomas G

    2002-01-01

    gene therapy. We have previously shown that overexpression of PAH and GTP-CH in primary human keratinocytes leads to high levels of phenylalanine clearance without BH(4) supplementation [Gene Ther. 7 (2000) 1971]. Here, we investigate the capacity of fibroblasts, another cell type from the skin, to...... metabolize phenylalanine. After retroviral gene transfer of PAH and GTP-CH both normal and PKU patient fibroblasts were able to metabolize phenylalanine, however, in lower amounts compared to genetically modified keratinocytes. Further comparative analyses between keratinocytes and fibroblasts revealed a...

  2. Defining origins of malignant B cells: a new circulating normal human IgM(+)D(+) B-cell subset lacking CD27 expression and displaying somatically mutated IGHV genes as a relevant memory population.

    Science.gov (United States)

    Weston-Bell, N; Townsend, M; Di Genova, G; Forconi, F; Sahota, S S

    2009-11-01

    In probing the cell of origin in malignant B cells, an imprint of somatic hypermutation (SHM) in immunoglobulin (Ig) variable (V) region genes delineates antigen encounter, and identifying the precise pathway generating SHM in the normal B-cell counterpart becomes relevant. SHM remains the definitive memory imprint in normal human B cells, but CD27 expression also delineates memory. Recently, dye extrusion adenosine triphosphate-binding transporter assays identified circulating isotype-switched memory B cells that lacked CD27, yet exhibited low levels of SHM. To extend findings, we report a pre-switched CD27(-ve) circulating memory B-cell population in normal blood using comparable assays, and isolated CD19(+)IgM(+)D(+)CD27(-ve) cells (>99% purity) for the analysis of IGHV5/IGHV3-IGHM transcripts. Of these (n=334), approximately 78% were germ line and naive B cell derived. Strikingly, 21.9% of the transcripts were mutated. They showed 3-5 mutations (13.5% of sequences) and >5 mutations (8.4% of sequences) per transcript. Accrual of mutations in a subset of CD19(+)IgM(+)D(+)CD27(-ve) cells define a new circulating pre-switched memory B-cell pool, present in substantial numbers in the population harboring naive B cells. These CD19(+)IgM(+)D(+)CD27(-ve) memory B cells may have a distinct lineage and function, and seem relevant to understanding origins of malignant B cells, in particular those of hairy cell leukemia cells, which display mutated V genes yet lack CD27 expression. PMID:19776762

  3. Adaptive response of the mutagenicity of the somatic andgerm cells in mice induced by in vivo 147Pm radiation

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    Effects of pretreatment of mousewith low doses of internally deposited 147Pm on the mutagenicity ofboththe bone marrow lymphocytes and the spermatoblasts induced by subsequenthigh dose of internally deposited 147Pm were studied. Kunming male mice were injected from the tail vein with low doses of 147Pm(NO3)3 of 0.37, 3.7 and 37 Bq/gbody weight. Three days later the mice were injected with a high dose of147Pm(NO3)3 of 18.5 kBq/g body weight. The animals were sacrificedat 24 hafter the high dose injection. The results show that the bone marrow lymphocytesand the spermatoblasts pre-exposed in vivo to low doses of 147Pmcan produce anadaptive response. The radioadapted cells had a resistance to themutagenicity induced by the subsequent high dose of 147Pm, that is, theproportion of both the bone marrow lymphocytes containing micronuclei andthe sperm shape abnormalities was significantly lower than those forthe pure high dose group (P<0.01). In addition, the induction of theadaptive response of the cells possesses the different dose ranges forthe different biological parameters. The 147Pm dose ranges of the adaptiveresponse are 0.37~3.7 and 0.37~37 Bq/g body weight for thefrequency of micronucleated bone marrow lymphocytes and abnormalsperms, respectively.

  4. Influence of ecological factors of the zone of the Chernobyl disaster on the somatic cells of mice and their posterity

    International Nuclear Information System (INIS)

    The main purpose of the present research is to study the reaction of cells of the hematopoietic system and carcinogenesis in the lungs of linear mice which were in the zone of the Chernobyl disaster for 1 month and their posterity (F1). It is established that the increase in frequency of micronucleated polychromatic erythrocytes in bone marrow for posterity F1 from mouse-parents being in the zone of the Chernobyl disaster had no statistical significance in comparison with the control groups. It is shown that the raising sensitivity of the posterity of linear mice, which were in the zone of the Chernobyl disaster, to the carcinogenic effect of urethane had more significance in comparison with the sensitivity of their parents. The estimate of the tumor process has shown that a spontaneous frequency of adenomas in the lungs for posterity F1 statistically increased more than 5 times in comparison with the similar parameter for the posterity of intact mice. (authors)

  5. Glucose transporter 5 is undetectable in outer hair cells and does not contribute to cochlear amplification

    OpenAIRE

    Wu, Xudong; Wang, Xiang; Gao, Jiangang; Yu, Yiling; Jia, Shuping; Zheng, Jing; Dallos, Peter; David Z Z He; Cheatham, Mary Ann; Zuo, Jian

    2008-01-01

    Glucose transporter 5 (Glut5) is a high-affinity fructose transporter. It was proposed to be a motor protein or part of the motor complex required for cochlear amplification in outer hair cells (OHCs). Here we show that, in contrast to previous reports, Glut5 is undetectable, and possibly absent, in OHCs harvested from wildtype mice. Further, Glut5-deficient mice display normal OHC morphology and motor function (i.e., nonlinear capacitance and electromotility) and normal cochlear sensitivity....

  6. Identification of Valid Housekeeping Genes for Real-Time Quantitative PCR Analysis of Collapsed Lung Tissues of Neonatal Somatic Cell Nuclear Transfer-Derived Cattle.

    Science.gov (United States)

    Liu, Yan; Zhang, Yunhai; Jiang, Qiuling; Rao, Man; Sheng, Zheya; Zhang, Yu; Du, Weihua; Hao, Haisheng; Zhao, Xueming; Xu, Zhe; Liu, Jianning; Zhu, Huabin

    2015-10-01

    Cloned calves produced by somatic cell nuclear transfer frequently suffer alveolar collapse as newborns. To study the underlying pathophysiological mechanisms responsible for this phenomenon, the expression profiles of numerous genes involved in lung development need to be investigated. Quantitative real-time PCR is commonly adopted in gene expression analysis. However, selection of an appropriate reference gene for normalization is critical for obtaining reliable and accurate results. Seven housekeeping genes-β-glucuronidase (GUSB), phosphoglycerate kinase 1 (PGK1), β-2-microglobolin (B2M), peptidylprolyl isomerase A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA-box binding protein (TBP), and 5.8S ribosomal RNA (5.8S rRNA)-were selected and evaluated as candidates. Their gene expression levels in the collapsed lungs of deceased neonate cloned calves and normal lung derived from normal calves were assessed. The ranking of gene expression stability was estimated by the geNorm, NormFinder, and BestKeeper programs. 5.8S rRNA and PPIA were determined to be the most stable reference genes by geNorm and BestKeeper, whereas the combination of GAPDH and TBP was suggested as reference genes by NormFinder. Taking these results into account, we conclude that 5.8S rRNA and PPIA could be the most reliable reference genes for studying the genes involved in alveolar collapse. Moreover, 5.8S rRNA could be represented as a uniform reference gene in similar cases. PMID:26393896

  7. Expression Profile of Genes as Indicators of Developmental Competence and Quality of In Vitro Fertilization and Somatic Cell Nuclear Transfer Bovine Embryos

    Science.gov (United States)

    Monteleone, Melisa Carolina; Mucci, Nicolas; Kaiser, German Gustavo; Brocco, Marcela; Mutto, Adrián

    2014-01-01

    Reproductive biotechnologies such as in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) enable improved reproductive efficiency of animals. However, the birth rate of in vitro-derived embryos still lags behind that of their in vivo counterparts. Thus, it is critical to develop an accurate evaluation and prediction system of embryo competence, both for commercial purposes and for scientific research. Previous works have demonstrated that in vitro culture systems induce alterations in the relative abundance (RA) of diverse transcripts and thus compromise embryo quality. The aim of this work was to analyze the RA of a set of genes involved in cellular stress (heat shock protein 70-kDa, HSP70), endoplasmic reticulum (ER) stress (immunoglobulin heavy chain binding protein, Bip; proteasome subunit β5, PSMB5) and apoptosis (BCL-2 associated X protein, Bax; cysteine aspartate protease-3, Caspase-3) in bovine blastocysts produced by IVF or SCNT and compare it with that of their in vivo counterparts. Poly (A) + mRNA was isolated from three pools of 10 blastocysts per treatment and analyzed by real-time RT-PCR. The RA of three of the stress indicators analyzed (Bax, PSMB5 and Bip) was significantly increased in SCNT embryos as compared with that of in vivo-derived blastocysts. No significant differences were found in the RA of HSP70 and Caspase-3 gene transcripts. This study could potentially complement morphological analyses in the development of an effective and accurate technique for the diagnosis of embryo quality, ultimately aiding to improve the efficiency of assisted reproductive techniques (ART). PMID:25269019

  8. Expression profile of genes as indicators of developmental competence and quality of in vitro fertilization and somatic cell nuclear transfer bovine embryos.

    Directory of Open Access Journals (Sweden)

    Maria Jesús Cánepa

    Full Text Available Reproductive biotechnologies such as in vitro fertilization (IVF and somatic cell nuclear transfer (SCNT enable improved reproductive efficiency of animals. However, the birth rate of in vitro-derived embryos still lags behind that of their in vivo counterparts. Thus, it is critical to develop an accurate evaluation and prediction system of embryo competence, both for commercial purposes and for scientific research. Previous works have demonstrated that in vitro culture systems induce alterations in the relative abundance (RA of diverse transcripts and thus compromise embryo quality. The aim of this work was to analyze the RA of a set of genes involved in cellular stress (heat shock protein 70-kDa, HSP70, endoplasmic reticulum (ER stress (immunoglobulin heavy chain binding protein, Bip; proteasome subunit β5, PSMB5 and apoptosis (BCL-2 associated X protein, Bax; cysteine aspartate protease-3, Caspase-3 in bovine blastocysts produced by IVF or SCNT and compare it with that of their in vivo counterparts. Poly (A + mRNA was isolated from three pools of 10 blastocysts per treatment and analyzed by real-time RT-PCR. The RA of three of the stress indicators analyzed (Bax, PSMB5 and Bip was significantly increased in SCNT embryos as compared with that of in vivo-derived blastocysts. No significant differences were found in the RA of HSP70 and Caspase-3 gene transcripts. This study could potentially complement morphological analyses in the development of an effective and accurate technique for the diagnosis of embryo quality, ultimately aiding to improve the efficiency of assisted reproductive techniques (ART.

  9. Correlations among the somatic cell count of individual bulk milk, result of the California Mastitis Test and bacteriological status of the udder in dairy cows.

    Science.gov (United States)

    Jánosi, Sz; Baltay, Zs

    2004-01-01

    In a survey of about 3000 dairy cows producing low somatic cell count (SCC) milk and kept on a large-scale dairy farm, California Mastitis Test (CMT) positivity was found in 2714 udder quarters of 1491 cows. Pathogenic microorganisms were isolated from 57.6% of these 2714 udder quarters during bacteriological examination. The commonest pathogens were coagulase-negative staphylococci (CNS, 41%) and Staphylococcus aureus (32.5%); however, udder infections caused by environmental streptococci (12.8%) and coliform bacteria (6.8%) were also common. All pathogens resulted in a significant increase of the SCC in individual bulk milk (IBM) samples. In the case of CNS, this SCC elevation in IBM was significantly lower than in the case of infection by the other pathogens. In spite of this, because of the high number of udder infections caused by CNS, the adverse effect exerted by CNS on dairy herds is considered to be substantial. It was found that 54.6% of all CMT-positive cows produced IBM of an SCC below 400 thousand per ml. The milk produced by 41% of the 315 cows excreting S. aureus also had an SCC below 400 thousand per ml. This poses a serious risk of infection to the healthy herdmates. At the same time, 11% of the infected cows produced IBM with an SCC below 100 thousand per ml. On the basis of these findings, only the regular analysis of SCC of IBM can be a reliable indicator of chronic intramammary infection. As the SCC of milk produced by CMT-positive cows (and especially of those excreting pathogens) tended to increase with advancing lactation, the authors suggest that an efficient drying-off therapy should be used to restore udder health and, whenever justified, culling of cows cannot be avoided either. PMID:15168749

  10. Prospective blinded study of somatic mutation detection in cell-free DNA utilizing a targeted 54-gene next generation sequencing panel in metastatic solid tumor patients

    Science.gov (United States)

    Lanman, Richard B.; Mortimer, Stefanie; Zill, Oliver A.; Kim, Kyoung-Mee; Jang, Kee Taek; Kim, Seok-Hyung; Park, Se Hoon; Park, Joon Oh; Park, Young Suk; Lim, Ho Yeong; Eltoukhy, Helmy; Kang, Won Ki; Lee, Woo Yong; Kim, Hee-Cheol; Park, Keunchil; Lee, Jeeyun; Talasaz, AmirAli

    2015-01-01

    Sequencing of the mutant allele fraction of circulating cell-free DNA (cfDNA) derived from tumors is increasingly utilized to detect actionable genomic alterations in cancer. We conducted a prospective blinded study of a comprehensive cfDNA sequencing panel with 54 cancer genes. To evaluate the concordance between cfDNA and tumor DNA (tDNA), sequencing results were compared between cfDNA from plasma and genomic tumor DNA (tDNA). Utilizing next generation digital sequencing technology (DST), we profiled approximately 78,000 bases encoding 512 complete exons in the targeted genes in cfDNA from plasma. Seventy-five patients were prospectively enrolled between February 2013 and March 2014, including 61 metastatic cancer patients and 14 clinical stage II CRC patients with matched plasma and tissue samples. Using the 54-gene panel, we detected at least one somatic mutation in 44 of 61 tDNA (72.1%) and 29 of 44 (65.9%) cfDNA. The overall concordance rate of cfDNA to tDNA was 85.9%, when all detected mutations were considered. We collected serial cfDNAs during cetuximab-based treatment in 2 metastatic KRAS wild-type CRC patients, one with acquired resistance and one with primary resistance. We demonstrate newly emerged KRAS mutation in cfDNA 1.5 months before radiologic progression. Another patient had a newly emerged PIK3CA H1047R mutation on cfDNA analysis at progression during cetuximab/irinotecan chemotherapy with gradual increase in allele frequency from 0.8 to 2.1%. This blinded, prospective study of a cfDNA sequencing showed high concordance to tDNA suggesting that the DST approach may be used as a non-invasive biopsy-free alternative to conventional sequencing using tumor biopsy. PMID:26452027

  11. Bayesian evaluation of budgets for endemic disease control: An example using management changes to reduce milk somatic cell count early in the first lactation of Irish dairy cows.

    Science.gov (United States)

    Archer, S C; Mc Coy, F; Wapenaar, W; Green, M J

    2014-01-01

    The aim of this research was to determine budgets for specific management interventions to control heifer mastitis in Irish dairy herds as an example of evidence synthesis and 1-step Bayesian micro-simulation in a veterinary context. Budgets were determined for different decision makers based on their willingness to pay. Reducing the prevalence of heifers with a high milk somatic cell count (SCC) early in the first lactation could be achieved through herd level management interventions for pre- and peri-partum heifers, however the cost effectiveness of these interventions is unknown. A synthesis of multiple sources of evidence, accounting for variability and uncertainty in the available data is invaluable to inform decision makers around likely economic outcomes of investing in disease control measures. One analytical approach to this is Bayesian micro-simulation, where the trajectory of different individuals undergoing specific interventions is simulated. The classic micro-simulation framework was extended to encompass synthesis of evidence from 2 separate statistical models and previous research, with the outcome for an individual cow or herd assessed in terms of changes in lifetime milk yield, disposal risk, and likely financial returns conditional on the interventions being simultaneously applied. The 3 interventions tested were storage of bedding inside, decreasing transition yard stocking density, and spreading of bedding evenly in the calving area. Budgets for the interventions were determined based on the minimum expected return on investment, and the probability of the desired outcome. Budgets for interventions to control heifer mastitis were highly dependent on the decision maker's willingness to pay, and hence minimum expected return on investment. Understanding the requirements of decision makers and their rational spending limits would be useful for the development of specific interventions for particular farms to control heifer mastitis, and other

  12. Generation of minipigs with targeted transgene insertion by recombinase-mediated cassette exchange (RMCE) and somatic cell nuclear transfer (SCNT)

    DEFF Research Database (Denmark)

    Jakobsen, Jannik Ejnar; Johansen, Marianne G; Schmidt, Mette;

    2013-01-01

    Targeted transgenesis using site-specific recombinases is an attractive method to create genetically modified animals as it allows for integration of the transgene in a pre-selected transcriptionally active genomic site. Here we describe the application of recombinase-mediated cassette exchange (...... enhanced human UbiC promoter, had an expression profile in various tissues similar to that of the GFP marker gene. The results show that RMCE can be done in a pre-selected transcriptionally active acceptor locus for targeted transgenesis in pigs.......Targeted transgenesis using site-specific recombinases is an attractive method to create genetically modified animals as it allows for integration of the transgene in a pre-selected transcriptionally active genomic site. Here we describe the application of recombinase-mediated cassette exchange...... (RMCE) in cells from a Göttingen minipig with four RMCE acceptor loci, each containing a green fluorescence protein (GFP) marker gene driven by a human UbiC promoter. The four RMCE acceptor loci segregated independent of each other, and expression profiles could be determined in various tissues. Using...

  13. microRNA-29b is a novel mediator of Sox2 function in the regulation of somatic cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Xudong Guo; Qidong Liu; Guiying Wang; Songcheng Zhu; Longfei Gao; Wujun Hong; Yafang Chen

    2013-01-01

    Fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs) by the application of Yamanaka factors (OSKM),but the mechanisms underlying this reprogramming remain poorly understood.Here,we report that Sox2 directly regulates endogenous microRNA-29b (miR-29b) expression during iPSC generation and that miR-29b expression is required for OSKM-and OSK-mediated reprogramming.Mechanistic studies show that Dnmt3a and Dnmt3b are in vivo targets of miR-29b and that Dnmt3a and Dnmt3b expression is inversely correlated with miR-29b expression during reprogramming.Moreover,the effect of miR-29b on reprogramming can be blocked by Dnmt3a or Dnmt3b overexpression.Further experiments indicate that miR-29b-DNMT signaling is significantly involved in the regulation of DNA methylation-related reprogramming events,such as mesenchymal-to-epithelial transition (MET)and Dlk1-Dio3 region transcription.Thus,our studies not only reveal that miR-29b is a novel mediator of reprogramming factor Sox2 but also provide evidence for a muitistep mechanism in which Sox2 drives a miR-29b-DNMT signaling axis that regulates DNA methylation-related events during reprogramming.

  14. Association between isolation of Staphylococcus aureus one week after calving and milk yield, somatic cell count, clinical mastitis, and culling through the remaining lactation.

    Science.gov (United States)

    Whist, Anne Cathrine; Osterås, Olav; Sølverød, Liv

    2009-02-01

    Cows with isolation of Staphylococcus aureus approximately 1 week after calving and milk yield, somatic cell count (SCC), clinical mastitis (CM), and culling risk through the remaining lactation were assessed in 178 Norwegian dairy herds. Mixed models with repeated measures were used to compare milk yield and SCC, and survival analyses were used to estimate the hazard ratio for CM and culling. On average, cows with an isolate of Staph. aureus had a significantly higher SCC than culture-negative cows. If no post-milking teat disinfection (PMTD) was used, the mean values of SCC were 42,000, 61,000, 68,000 and 77,000 cells/ml for cows with no Staph. aureus isolate, with Staph. aureus isolated in 1 quarter, in 2 quarters and more than 2 quarters respectively. If iodine PMTD was used, SCC means were 36,000; 63,000; 70,000 and 122,000, respectively. Primiparous cows testing positive for Staph. aureus had the same milk yield curve as culture-negative cows, except for those with Staph. aureus isolated in more than 2 quarters. They produced 229 kg less during a 305-d lactation. Multiparous cows with isolation of Staph. aureus in at least 1 quarter produced 94-161 kg less milk in 2nd and >3rd parity, respectively, and those with isolation in more than 2 quarters produced 303-390 kg less than multiparous culture-negative animals during a 305-d lactation. Compared with culture-negative cows, the hazard ratio for CM and culling in cows with isolation of Staph. aureus in at least 1 quarter was 2.0 (1.6-2.4) and 1.7 (1.5-1.9), respectively. There was a decrease in the SCC and in the CM risk in culture-negative cows where iodine PMTD had been used, indicating that iodine PMTD has a preventive effect on already healthy cows. For cows testing positive for Staph. aureus in more than 2 quarters at calving, iodine PMTD had a negative effect on the CM risk and on the SCC through the remaining lactation. PMID:18922193

  15. Polymorphisms in the 5' upstream region of the CXCR1 chemokine receptor gene, and their association with somatic cell score in Holstein cattle in Canada.

    Science.gov (United States)

    Leyva-Baca, I; Schenkel, F; Martin, J; Karrow, N A

    2008-01-01

    Identification of regulatory elements in 5' regions of chemokine genes is fundamental for understanding chemokine gene expression in response to infection diseases. The CXCR1 receptor is expressed on the surface of neutrophils and interacts primarily with CXCL8 (IL-8), the most potent chemoattractant for neutrophils. The aim of this study was to characterize the 5' upstream region (2.1 kb) of the bovine CXCR1 chemokine receptor gene for polymorphism content and to identify in silico potential transcription-factor binding sites. The 5' flanking region was found by mining the NCBI GenBank (www.ncbi.nlm.nih.gov/). A DNA sequence from the whole genome shotgun sequence project with reference number AC150887.4 contained the CXCR1 5' flanking sequence. Computer analysis revealed potential binding sites for the transcription factors nuclear factor kappaB (NF-kappaB), binding factor GATA-1, barbiturate inducible element (Barbie), nuclear factor of activated T-cells, and activator protein 1. Polymorphism discovery in this region was conducted by constructing an inclusive DNA pool including 2 phenotypic extreme groups, 20 bulls with high estimated breeding values (EBV) for somatic cell score (SCS), and 20 bulls with low EBV for SCS. Independent amplicons along the 5' flanking region of bovine CXCR1 were generated for polymorphism discovery by sequencing. Three novel single nucleotide polymorphisms (SNP), CXCR1c.-344T>C, CXCR1c.-1768T>A, and CXCR1c.-1830A>G, and a previously identified SNP in the coding region, CXCR1c.777G>C, were identified. The 4 SNP were genotyped in Canadian Holstein bulls (n = 338) using tetra-primer amplification refractory mutation system (ARMS)-PCR. Average allele substitution effects were estimated to investigate associations between the 4 SNP and EBV for SCS in first, second, and third and later lactations. Multiple trait analysis revealed that the SNP CXCR1c.-1768T>A was associated with EBV for SCS in the first and second lactations and over all 3

  16. Genetic parameters for somatic cell score according to udder infection status in Valle del Belice dairy sheep and impact of imperfect diagnosis of infection

    Directory of Open Access Journals (Sweden)

    Bovenhuis Henk

    2010-07-01

    Full Text Available Abstract Background Somatic cell score (SCS has been promoted as a selection criterion to improve mastitis resistance. However, SCS from healthy and infected animals may be considered as separate traits. Moreover, imperfect sensitivity and specificity could influence animals' classification and impact on estimated variance components. This study was aimed at: (1 estimating the heritability of bacteria negative SCS, bacteria positive SCS, and infection status, (2 estimating phenotypic and genetic correlations between bacteria negative and bacteria positive SCS, and the genetic correlation between bacteria negative SCS and infection status, and (3 evaluating the impact of imperfect diagnosis of infection on variance component estimates. Methods Data on SCS and udder infection status for 1,120 ewes were collected from four Valle del Belice flocks. The pedigree file included 1,603 animals. The SCS dataset was split according to whether animals were infected or not at the time of sampling. A repeatability test-day animal model was used to estimate genetic parameters for SCS traits and the heritability of infection status. The genetic correlation between bacteria negative SCS and infection status was estimated using an MCMC threshold model, implemented by Gibbs Sampling. Results The heritability was 0.10 for bacteria negative SCS, 0.03 for bacteria positive SCS, and 0.09 for infection status, on the liability scale. The genetic correlation between bacteria negative and bacteria positive SCS was 0.62, suggesting that they may be genetically different traits. The genetic correlation between bacteria negative SCS and infection status was 0.51. We demonstrate that imperfect diagnosis of infection leads to underestimation of differences between bacteria negative and bacteria positive SCS, and we derive formulae to predict impacts on estimated genetic parameters. Conclusions The results suggest that bacteria negative and bacteria positive SCS are

  17. Genotype by environment interaction for production, somatic cell score, workability, and conformation traits in Dutch Holstein-Friesian cows between farms with or without grazing.

    Science.gov (United States)

    van der Laak, M; van Pelt, M L; de Jong, G; Mulder, H A

    2016-06-01

    In this study, genotype by environment interaction was investigated for production traits, somatic cell score (SCS), workability traits, and conformation traits for Holstein-Friesian cows producing on farms with or without grazing in the Netherlands. Additionally, heritabilities and repeatabilities were estimated in both farm systems. Data were available for 1,019 Dutch farms, and farm type was known for those farms, 142 farms without grazing and 877 farms with grazing. The data set consisted of 428,600 test-day records for production from 49,412 cows, and from this data set a subset for SCS was created, consisting of 374,734 test-day records from 45,955 cows. For workability and conformation traits, the data set consisted of 30,180 cows. Bivariate mixed models with multiple fixed effects and random sire and random permanent environment effects were applied. The majority of sires had daughters in both farm types. The heritabilities for milk yield (0.27), fat yield (0.19), and protein yield (0.20) were higher in farms with grazing than in farms without grazing with heritabilities of 0.24 for milk yield, 0.18 for fat yield, and 0.18 for protein yield. Repeatability was lower in the grazing farms for milk yield, fat yield, and protein yield, probably because of alternating quality of dry matter intake during grazing. Genetic correlations between grazing and no grazing were 0.99, 0.98, 0.97, and 1.00 for milk yield, fat yield, protein yield, and SCS, respectively. Genetic correlations for workability traits and conformation traits between grazing and no grazing varied between 0.93 and 1.00. For all traits, genetic correlations were close to unity, indicating no genotype by environment interaction between farms with or without grazing for production traits, SCS, workability traits, and conformation traits in Dutch Holstein-Friesians. Therefore, the same sires can be used for farms both with and without grazing. PMID:27040792

  18. Too much of a good thing: Long-term treatment with salicylate strengthens outer hair cell function but impairs auditory neural activity

    OpenAIRE

    Chen, Guang-Di; Kermany, Mohammad Habiby; D’Elia, Alessandra; Ralli, Massimo; Tanaka, Chiemi; Bielefeld, Eric C.; Ding, Dalian; Henderson, Donald; Salvi, Richard

    2010-01-01

    Aspirin has been extensively used in clinical settings. Its side effects on auditory function, including hearing loss and tinnitus, are considered as temporary. A recent promising finding is that chronic treatment with high-dose salicylate (the active ingredient of aspirin) for several weeks enhances expression of the outer hair cell (OHC) motor protein (prestin), resulting in strengthened OHC electromotility and enhanced distortion product otoacoustic emissions (DPOAE). To follow up on these...

  19. Risk factors for bulk milk somatic cell counts and total bacterial counts in smallholder dairy farms in the 10th region of Chile.

    Science.gov (United States)

    van Schaik, G; Green, L E; Guzmán, D; Esparza, H; Tadich, N

    2005-01-01

    We investigated the principal management factors that influenced bulk milk somatic cell count (BMSCC) and total bacterial count (TBC) of smallholder dairy farms in the 10th region of Chile. One hundred and fifty smallholder milk producers were selected randomly from 42 milk collection centres (MCCs). In April and May of 2002, all farms were visited and a detailed interview questionnaire on dairy-cow management related to milk quality was conducted. In addition, the BMSCC and TBC results from the previous 2 months' fortnightly tests were obtained from the MCCs. The mean BMSCC and TBC were used as the dependent variables in the analyses and were normalised by a natural-logarithm transformation (LN). All independent management variables were categorised into binary outcomes and present (=1) was compared with absent (=0). Biserial correlations were calculated between the LNBMSCC or LNTBC and the management factors of the smallholder farms. Management factors with correlations with P0.05) factors. A random MCC effect was included in the models to investigate the importance of clustering of herds within MCC. In the null model for mean LNTBC, the random effect of MCCs was highly significant. It was explained by: milk collected once a day or less compared with collection twice a day, not cleaning the bucket after milking mastitic cows versus cleaning the bucket and cooling milk in a vat of water versus not cooling milk or using ice or a bulk tank to cool milk. Other factors that increased the LNTBC were a waiting yard with a soil or gravel floor versus concrete, use of plastic buckets for milking instead of metal, not feeding California mastitis test (CMT)-positive milk to calves and cows of dual-purpose breed. The final model explained 35% of the variance. The model predicted that a herd that complied with all the management practices had a mean predicted TBC of 105 colony forming units (cfu)/ml, whereas a herd that did not comply with any of these management factors had

  20. Buffalo (Bubalus bubalis) SCNT embryos produced from somatic cells isolated from frozen-thawed semen: effect of trichostatin A on the in vitro and in vivo developmental potential, quality and epigenetic status.

    Science.gov (United States)

    Selokar, Naresh L; Saini, Monika; Agrawal, Himanshu; Palta, Prabhat; Chauhan, Manmohan S; Manik, Radheysham; Singla, Suresh K

    2016-08-01

    This study examined the effects of trichostatin A (TSA) treatment of reconstructed buffalo embryos, produced by hand-made cloning using somatic cells isolated from over a decade old frozen-thawed semen, on their in vitro and in vivo developmental competence, quality and epigenetic status. Following treatment of reconstructed embryos with TSA (0, 50 or 75 nM) for 10 h prior to culture, the cleavage (100.0 ± 0, 94.5 ± 2.3 and 96.1 ± 1.2%, respectively) and blastocyst rate (50.6 ± 2.3, 48.4 ± 2.7 and 48.1 ± 2.6%, respectively), total cell number (275 ± 17.4, 289 ± 30.1 and 317 ± 24.2, respectively) and apoptotic index (5.6 ± 0.7, 3.4 ± 0.9 and 4.5 ± 1.4, respectively) were not significantly different among the three groups. However, TSA treatment increased (P buffalo embryos produced using somatic cells isolated from frozen-thawed semen improved their epigenetic status but not the in vitro developmental potential and offspring rate. PMID:26503476