Agriculture in the Face of Changing Markets, Institutions and Policies: Challenges and Strategies

Since the late 1980s, agriculture in Central and Eastern European Countries (CEECs) has been under considerable adjustment pressure due to changing political, economic and institutional environments. These changes have been linked to the transition process, as well as the ongoing integration into the European Union and the world market. Reduced subsidies, increased environmental and food quality demands, as well as structural changes in the supply, processing and food retailing sector call for major structural adjustments and the improvement of farmersâ managerial abilities. Though such changes always carry significant threats to farms, they also offer new opportunities for the farms' entrepreneurial engagement. Upcoming changes in the agricultural environment and their possible consequences for farm structures across Europe are thus still timely subjects. The objective of the IAMO Forum 2006 is to contribute to the success of agriculture in the CEECs, as well as their neighboring countries, in todayâs increasingly competitive environment. Concrete questions the conference focuses on are: What are the most suitable farm organizations, cooperative arrangements and contractual forms? How to improve efficiency and productivity? Where do market niches lie and what are the new product demands? This book contains 33 invited and selected contributions. These papers will be presented at the IAMO Forum 2006 in order to offer a platform for scientists, practitioners and policy-makers to discuss challenges and potential strategies at the farm, value chain, rural society and policy levels in order to cope with the upcoming challenges. IAMO Forum 2006, as well as this book, would not have been possible without the engagement of many people and institutions. We thank the authors of the submitted abstracts and papers, as well as the referees, for their evaluation of the abstracts from which the papers were selected. In particular, we would like to express our thanks to OLIVER JUNGKLAUS, GABRIELE MEWES, KLAUS REINSBERG and ANGELA SCHOLZ, who significantly contributed to the organization of the Forum. Furthermore, our thanks goes to SILKE SCHARF for her work on the layout and editing support of this book, and to JIM CURTISS, JAMIE BULLOCH, and DÃNALL Ã MEARÃIN for their English proof-reading. As experience from previous years documents, the course of the IAMO Forum continues to profit from the support and engagement of the IAMO administration, which we gratefully acknowledge. Last but not least, we are very grateful to the Robert Bosch Foundation, the Federal Ministry of Nutrition, Agriculture and Consumer Protection (BMELV), the German Research Foundation (DFG), the Haniel Foundation and the Leibniz Institute of Agricultural Development in Central and Eastern Europe (IAMO) for their respective financial support.Agribusiness, Community/Rural/Urban Development, Farm Management, Industrial Organization, International Development, Labor and Human Capital, Land Economics/Use, Productivity Analysis,

p53 Regulates Cell Cycle and MicroRNAs to Promote Differentiation of Human Embryonic Stem Cells

Multiple studies show that tumor suppressor p53 is a barrier to dedifferentiation; whether this is strictly due to repression of proliferation remains a subject of debate. Here, we show that p53 plays an active role in promoting differentiation of human embryonic stem cells (hESCs) and opposing self-renewal by regulation of specific target genes and microRNAs. In contrast to mouse embryonic stem cells, p53 in hESCs is maintained at low levels in the nucleus, albeit in a deacetylated, inactive state. In response to retinoic acid, CBP/p300 acetylates p53 at lysine 373, which leads to dissociation from E3-ubiquitin ligases HDM2 and TRIM24. Stabilized p53 binds CDKN1A to establish a G1 phase of cell cycle without activation of cell death pathways. In parallel, p53 activates expression of miR-34a and miR-145, which in turn repress stem cell factors OCT4, KLF4, LIN28A, and SOX2 and prevent backsliding to pluripotency. Induction of p53 levels is a key step: RNA-interference-mediated knockdown of p53 delays differentiation, whereas depletion of negative regulators of p53 or ectopic expression of p53 yields spontaneous differentiation of hESCs, independently of retinoic acid. Ectopic expression of p53R175H, a mutated form of p53 that does not bind DNA or regulate transcription, failed to induce differentiation. These studies underscore the importance of a p53-regulated network in determining the human stem cell state

p53 regulates cell cycle and microRNAs to promote differentiation of human embryonic stem cells.

Multiple studies show that tumor suppressor p53 is a barrier to dedifferentiation; whether this is strictly due to repression of proliferation remains a subject of debate. Here, we show that p53 plays an active role in promoting differentiation of human embryonic stem cells (hESCs) and opposing self-renewal by regulation of specific target genes and microRNAs. In contrast to mouse embryonic stem cells, p53 in hESCs is maintained at low levels in the nucleus, albeit in a deacetylated, inactive state. In response to retinoic acid, CBP/p300 acetylates p53 at lysine 373, which leads to dissociation from E3-ubiquitin ligases HDM2 and TRIM24. Stabilized p53 binds CDKN1A to establish a G(1) phase of cell cycle without activation of cell death pathways. In parallel, p53 activates expression of miR-34a and miR-145, which in turn repress stem cell factors OCT4, KLF4, LIN28A, and SOX2 and prevent backsliding to pluripotency. Induction of p53 levels is a key step: RNA-interference-mediated knockdown of p53 delays differentiation, whereas depletion of negative regulators of p53 or ectopic expression of p53 yields spontaneous differentiation of hESCs, independently of retinoic acid. Ectopic expression of p53R175H, a mutated form of p53 that does not bind DNA or regulate transcription, failed to induce differentiation. These studies underscore the importance of a p53-regulated network in determining the human stem cell state

p53 drives differentiation of hESCs.

<p>(A) AP staining. hESCs transfected with non-target (siControl) or siRNA specific to p53 (<i>siTP53</i>) or p21 (<i>siCDKN1A</i>) were treated with RA and stained for AP (blue colonies). (B and C) hESCs transfected and treated as in (A) were used in Western blotting (B) and qRT-PCR (C) analyses. The blots in (B) were quantitated, and average density of three different blots is plotted (bottom panel) (*, <i>p</i><0.05) (mean ± SEM). (D) OCT4 + SSEA4 staining. hESCs transfected with siRNA followed by RA treatment were stained for SSEA4 and OCT4 and subjected to dual flow cytometry analysis. Triplicate samples were analyzed in each experiment, and data analyzed with FACSDiva software. Decreases in fraction of OCT4-positive cells, as compared to siControl untreated, are indicated in red. (E) AP staining. hESCs transfected with <i>HDM2</i> or <i>TRIM24</i> siRNA were treated with RA and stained for AP. (F) Quantified AP-stained colonies. Date shown are for 50 colonies per treatment in three separate experiments (in [A] and [E]), scored as undifferentiated, partially differentiated, or fully differentiated colonies, mean ± SEM. (G) OCT4 + SSEA4 staining and flow cytometry analysis as in (D) after transfection with siRNA targeting <i>HDM2</i> or <i>TRIM24</i>. (H) Cell cycle analysis. hESCs transfected with <i>HDM2</i> or <i>TRIM24</i> siRNA were stained with PI and subjected to cell cycle analysis. (Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s003" target="_blank">Figures S3</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s005" target="_blank">S5</a>.).</p

The consequence of p53 accumulation in hESCs.

<p>(A) Cell cycle analysis. hESCs transfected with non-target (siControl) or siRNA specific to p53 (<i>siTP53</i>) or p21 (<i>siCDKN1A</i>) and treated with RA were stained with PI and subjected to flow cytometry analysis to determine DNA content. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#s2" target="_blank">Results</a> quantitated as fold change in cell cycle are shown. (B) qRT-PCR. RNA from hESCs treated with RA for 4 d or Adr for 6 h were subjected to qRT-PCR assay using primers specific for human <i>CDKN1A</i>. (C) ChIP. p53-bound chromatin was immunoprecipitated from hESCs, and p53 enrichment on <i>CDKN1A</i> was analyzed by qRT-PCR using primers encompassing p53REs (*, <i>p</i><0.05). Scheme representing location of p53RE and primers used for ChIP-qRT-PCR are shown on the top (asterisk indicates the 3′ end of the gene). (D) hESCs treated with RA or Adr were lysed, and cell lysates were blotted for γ-H2AX. (E) Apoptosis assay. hESCs treated as in (D) were stained with Annexin V and PI, and percent apoptotic cells was determined by flow cytometry (mean ± SEM). (Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s003" target="_blank">Figures S3</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s004" target="_blank">S4</a>.).</p

Model depicting role of p53 in inducing differentiation of hESCs.

<p>In pluripotent hESCs, p53 is negatively regulated by HDM2 and TRIM24. Differentiation induces acetylation at Lys373 of p53 via CBP/p300, p53K373ac then activates transcription by binding to p53REs on <i>CDKN1A</i> (p21), <i>miR-34a</i>, and <i>miR-145</i>. Induction of p21 leads to p53-dependent elongation of G₁ phase, whereas induction of <i>miR-34a</i> supports G1 elongation, blocks deactivation of p53 by targeting the deacetylase SIRT1, and counteracts pluripotency by targeting <i>LIN28A</i>. On the other hand, <i>miR-145</i> targets OCT4, KLF4, and SOX2 and antagonize pluripotency. Thus, p53 exerts a cumulative pro-differentiation effect by elongating hESC G₁ phase via p21 and synergistically up-regulating <i>miR-34a</i> and <i>miR-145</i> to counteract pluripotency. Ub, ubiquitin.</p

p53 protein is induced during differentiation of hESCs.

<p>(A) qRT-PCR. RNA from hESCs treated with RA in medium without FGF for 5 d (R0–R5), were subjected to qRT-PCR assay (data are presented as mean ± SEM). (B and C) Western blot. Lysates (total cell lysate [TCL]) prepared from hESCs cultured as in (A) were analyzed by Western blotting, the blots in (B) were quantitated (C): the average density of three different blots is plotted (*, <i>p</i><0.05). (D) <i>TP53</i> qRT-PCR. RNA samples prepared as in (A) were subjected to qRT-PCR assay (mean ± SEM). (E) Immunofluorescence. hESCs in complete CM or treated with RA for 3 d were stained with antibodies against p53 and OCT4, and nuclei were stained with DAPI. Scale bar is 50 µm. (F) p53 nuclear localization. Nuclear extracts prepared from hESCs cultured as in (A) were analyzed by Western blotting. (Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s001" target="_blank">Figure S1</a>.).</p

Acetylation of Lys373 leads to stabilization of p53.

<p>(A) p53 acetylation. Equal amounts of p53 were immunoprecipitated by adjusting the amounts of total cell lysates prepared from hESCs and probed with p53K373ac antibody. (B) Immunofluorescence. hESCs treated with RA for 3 d were stained with antibodies against p53K373ac and OCT4; nuclei were stained with DAPI. (C) Co-immunoprecipitation. Cell lysates from RA-treated hESCs were immunoprecipitated with p53 followed by Western blotting. (D) p53 acetylation. p53 immunoprecipitated from hESCs cultured under differentiating conditions and treated with either circumin on day 2 or nicotinamide on day 4 and probed with p53K373ac antibody. (E) Co-immunoprecipitation. Cell lysates from differentiating hESCs were immunoprecipitated with HDM2 or TRIM24 antibodies and analyzed by Western blotting. (F) Endogenous p53 ubiquitination. hESCs cultured under differentiating conditions were treated with MG132 + RA or MG132 + Adr; endogenous p53 was immunoprecipitated and probed for ubiquitin (top panel). Same blot was reprobed with p53 antibody to show the equal p53 pull down (bottom panel). (Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s002" target="_blank">Figure S2</a>.) IP, mmunoprecipitation; Ub, ubiquitin; WB, Western blot.</p

DNA binding activity of p53 is required to induce differentiation of hESCs.

<p>(A) hESCs stably expressing p53WT and mutant p53 (p53R175H and p53R175P) under control of tet-inducible promoter cultured in CM + FGF were treated with 100 ng/ml Dox for 2 d. p53 and OCT4 protein levels were analyzed by blotting. (B) AP staining. hESCs in (A) treated with Dox for 2 d (2D) or 4 d (4D) and AP stained. (Arrows indicate differentiated cells.) (C) qRT-PCR. hESCs treated with Dox for 1 d (1D) or 2 d (2D). RNA analyzed by qRT-PCR assay for expression of exogenous <i>TP53, CDKN1A, OCT4</i>, and <i>AFP</i> (*, <i>p</i><0.05) (mean ± SEM). (D) Cell cycle analysis. hESCs treated with RA for 1 d or Dox for 1 d or 2 d, stained with PI, and subjected to cell cycle analysis (mean ± SEM). (E) hESCs stably expressing p53WT were transfected with siRNA and treated with Dox for 2 d. p53, p21, and OCT4 protein levels were analyzed. (F and G) hESCs expressing p53WT were treated with Dox for 2 d or 4 d, and lysed to analyze protein (F) or RNA (G) for various differentiation markers; AFP and GATA4 (endoderm), Brachyury (mesoderm), and PAX6 (ectoderm). (H) p53 acetylation. Lysates from hESCs treated with RA and Dox-inducible p53WT treated with Dox were blotted for p53K373ac and p53. (Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001268#pbio.1001268.s006" target="_blank">Figure S6</a>.).</p

Agriculture in the Face of Changing Markets, Institutions and Policies: Challenges and Strategies

Since the late 1980s, agriculture in Central and Eastern European Countries (CEECs) has been under considerable adjustment pressure due to changing political, economic and institutional environments. These changes have been linked to the transition process, as well as the ongoing integration into the European Union and the world market. Reduced subsidies, increased environmental and food quality demands, as well as structural changes in the supply, processing and food retailing sector call for major structural adjustments and the improvement of farmers’ managerial abilities. Though such changes always carry significant threats to farms, they also offer new opportunities for the farms' entrepreneurial engagement. Upcoming changes in the agricultural environment and their possible consequences for farm structures across Europe are thus still timely subjects. The objective of the IAMO Forum 2006 is to contribute to the success of agriculture in the CEECs, as well as their neighboring countries, in today’s increasingly competitive environment. Concrete questions the conference focuses on are: What are the most suitable farm organizations, cooperative arrangements and contractual forms? How to improve efficiency and productivity? Where do market niches lie and what are the new product demands? This book contains 33 invited and selected contributions. These papers will be presented at the IAMO Forum 2006 in order to offer a platform for scientists, practitioners and policy-makers to discuss challenges and potential strategies at the farm, value chain, rural society and policy levels in order to cope with the upcoming challenges. IAMO Forum 2006, as well as this book, would not have been possible without the engagement of many people and institutions. We thank the authors of the submitted abstracts and papers, as well as the referees, for their evaluation of the abstracts from which the papers were selected. In particular, we would like to express our thanks to OLIVER JUNGKLAUS, GABRIELE MEWES, KLAUS REINSBERG and ANGELA SCHOLZ, who significantly contributed to the organization of the Forum. Furthermore, our thanks goes to SILKE SCHARF for her work on the layout and editing support of this book, and to JIM CURTISS, JAMIE BULLOCH, and DÓNALL Ó MEARÁIN for their English proof-reading. As experience from previous years documents, the course of the IAMO Forum continues to profit from the support and engagement of the IAMO administration, which we gratefully acknowledge. Last but not least, we are very grateful to the Robert Bosch Foundation, the Federal Ministry of Nutrition, Agriculture and Consumer Protection (BMELV), the German Research Foundation (DFG), the Haniel Foundation and the Leibniz Institute of Agricultural Development in Central and Eastern Europe (IAMO) for their respective financial support