Publications by authors named "Johannes Boonstra"

33 Publications

Collaborative Learning in Higher Education: Evoking Positive Interdependence.

CBE Life Sci Educ Winter 2016;15(4)

Department of Biology, Utrecht University, 3584 CH Utrecht, The Netherlands.

Collaborative learning is a widely used instructional method, but the learning potential of this instructional method is often underused in practice. Therefore, the importance of various factors underlying effective collaborative learning should be determined. In the current study, five different life sciences undergraduate courses with successful collaborative-learning results were selected. This study focuses on factors that increased the effectiveness of collaboration in these courses, according to the students. Nine focus group interviews were conducted and analyzed. Results show that factors evoking effective collaboration were student autonomy and self-regulatory behavior, combined with a challenging, open, and complex group task that required the students to create something new and original. The design factors of these courses fostered a sense of responsibility and of shared ownership of both the collaborative process and the end product of the group assignment. In addition, students reported the absence of any free riders in these group assignments. Interestingly, it was observed that students seemed to value their sense of achievement, their learning processes, and the products they were working on more than their grades. It is concluded that collaborative learning in higher education should be designed using challenging and relevant tasks that build shared ownership with students.
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http://dx.doi.org/10.1187/cbe.16-07-0219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5132366PMC
February 2017

Transient Intervals of Hyper-Gravity Enhance Endothelial Barrier Integrity: Impact of Mechanical and Gravitational Forces Measured Electrically.

PLoS One 2015 4;10(12):e0144269. Epub 2015 Dec 4.

Department of Physiology, Institute for Cardiovascular Research (ICaR-VU), VU University Medical Center, Amsterdam, The Netherlands.

Background: Endothelial cells (EC) guard vascular functions by forming a dynamic barrier throughout the vascular system that sensitively adapts to 'classical' biomechanical forces, such as fluid shear stress and hydrostatic pressure. Alterations in gravitational forces might similarly affect EC integrity, but remain insufficiently studied.

Methods: In an unique approach, we utilized Electric Cell-substrate Impedance Sensing (ECIS) in the gravity-simulators at the European Space Agency (ESA) to study dynamic responses of human EC to simulated micro- and hyper-gravity as well as to classical forces.

Results: Short intervals of micro- or hyper-gravity evoked distinct endothelial responses. Stimulated micro-gravity led to decreased endothelial barrier integrity, whereas hyper-gravity caused sustained barrier enhancement by rapid improvement of cell-cell integrity, evidenced by a significant junctional accumulation of VE-cadherin (p = 0.011), significant enforcement of peripheral F-actin (p = 0.008) and accompanied by a slower enhancement of cell-matrix interactions. The hyper-gravity triggered EC responses were force dependent and nitric-oxide (NO) mediated showing a maximal resistance increase of 29.2±4.8 ohms at 2g and 60.9±6.2 ohms at 4g vs. baseline values that was significantly suppressed by NO blockage (p = 0.011).

Conclusion: In conclusion, short-term application of hyper-gravity caused a sustained improvement of endothelial barrier integrity, whereas simulated micro-gravity weakened the endothelium. In clear contrast, classical forces of shear stress and hydrostatic pressure induced either short-lived or no changes to the EC barrier. Here, ECIS has proven a powerful tool to characterize subtle and distinct EC gravity-responses due to its high temporal resolution, wherefore ECIS has a great potential for the study of gravity-responses such as in real space flights providing quantitative assessment of a variety of cell biological characteristics of any adherent growing cell type in an automated and continuous fashion.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0144269PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4670102PMC
June 2016

Causes and Consequences of Age-Related Changes in DNA Methylation: A Role for ROS?

Biology (Basel) 2014 Jun 18;3(2):403-25. Epub 2014 Jun 18.

Science Department, University College Utrecht, Campusplein 1, 3584 ED Utrecht, The Netherlands.

Recent genome-wide analysis of C-phosphate-G (CpG) sites has shown that the DNA methylome changes with increasing age, giving rise to genome-wide hypomethylation with site‑specific incidences of hypermethylation. This notion has received a lot of attention, as it potentially explains why aged organisms generally have a higher risk of age-related diseases. However, very little is known about the mechanisms that could cause the occurrence of these changes. Moreover, there does not appear to be a clear link between popular theories of aging and alterations in the methylome. Some of the most fruitful of these theories attribute an important role to reactive oxygen species, which seem to be responsible for an increase in oxidative damage to macromolecules, such as DNA, during the lifetime of an organism. In this review, the connection between changes in DNA methylation and these reactive oxygen species is discussed, as well as the effect of these changes on health. Deeper insights into the nature, causes and consequences of the aging methylome might provide a deeper understanding of the molecular mechanisms of aging and eventually contribute to the development of new diagnostic and therapeutic tools.
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http://dx.doi.org/10.3390/biology3020403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4085615PMC
June 2014

Ground-based facilities for simulation of microgravity: organism-specific recommendations for their use, and recommended terminology.

Astrobiology 2013 Jan 19;13(1):1-17. Epub 2012 Dec 19.

Centro de Investigaciones Biológicas (CSIC), Madrid, Spain.

Research in microgravity is indispensable to disclose the impact of gravity on biological processes and organisms. However, research in the near-Earth orbit is severely constrained by the limited number of flight opportunities. Ground-based simulators of microgravity are valuable tools for preparing spaceflight experiments, but they also facilitate stand-alone studies and thus provide additional and cost-efficient platforms for gravitational research. The various microgravity simulators that are frequently used by gravitational biologists are based on different physical principles. This comparative study gives an overview of the most frequently used microgravity simulators and demonstrates their individual capacities and limitations. The range of applicability of the various ground-based microgravity simulators for biological specimens was carefully evaluated by using organisms that have been studied extensively under the conditions of real microgravity in space. In addition, current heterogeneous terminology is discussed critically, and recommendations are given for appropriate selection of adequate simulators and consistent use of nomenclature.
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http://dx.doi.org/10.1089/ast.2012.0876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3549630PMC
January 2013

The influence of reactive oxygen species on cell cycle progression in mammalian cells.

Gene 2012 Dec 31;511(1):1-6. Epub 2012 Aug 31.

University College Utrecht, P O Box 80145, 3508 TC Utrecht, The Netherlands.

Cell cycle regulation is performed by cyclins and cyclin dependent kinases (CDKs). Recently, it has become clear that reactive oxygen species (ROS) influence the presence and activity of these enzymes and thereby control cell cycle progression. In this review, we first describe the discovery of enzymes specialized in ROS production: the NADPH oxidase (NOX) complexes. This discovery led to the recognition of ROS as essential players in many cellular processes, including cell cycle progression. ROS influence cell cycle progression in a context-dependent manner via phosphorylation and ubiquitination of CDKs and cell cycle regulatory molecules. We show that ROS often regulate ubiquitination via intermediate phosphorylation and that phosphorylation is thus the major regulatory mechanism influenced by ROS. In addition, ROS have recently been shown to be able to activate growth factor receptors. We will illustrate the diverse roles of ROS as mediators in cell cycle regulation by incorporating phosphorylation, ubiquitination and receptor activation in a model of cell cycle regulation involving EGF-receptor activation. We conclude that ROS can no longer be ignored when studying cell cycle progression.
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http://dx.doi.org/10.1016/j.gene.2012.08.038DOI Listing
December 2012

MAPK uncouples cell cycle progression from cell spreading and cytoskeletal organization in cycling cells.

Cell Mol Life Sci 2013 Jan 25;70(2):293-307. Epub 2012 Aug 25.

Department of Cell Biology, Faculty of Sciences, University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands.

Integrin-mediated cytoskeletal tension supports growth-factor-induced proliferation, and disruption of the actin cytoskeleton in growth factor-stimulated cells prevents the re-expression of cyclin D and cell cycle re-entry from quiescence. In contrast to cells that enter the cell cycle from G0, cycling cells continuously express cyclin D, and are subject to major cell shape changes during the cell cycle. Here, we investigated the cell cycle requirements for cytoskeletal tension and cell spreading in cycling mammalian cells that enter G1-phase from mitosis. Disruption of the actin cytoskeleton at progressive time-points in G1-phase induced cell rounding, FA disassembly, and attenuated both integrin signaling and growth factor-induced p44/p42 mitogen-activated protein kinase activation. Although cyclin D expression was reduced, the expression of cyclin A and entry into S-phase were not affected. Moreover, expression of cyclin B1, progression through G2- and M-phase, and commitment to a new cell cycle occurred normally. In contrast, cell cycle progression was strongly prevented by inhibition of MAPK activity in G1-phase, whereas cell spreading, cytoskeletal organization, and integrin signaling were not impaired. MAPK inhibition also prevented cytoskeleton-independent cell cycle progression. Thus, these results uncouple the requirements for cell spreading and cytoskeletal organization from MAPK signaling, and show that cycling mammalian cells can proliferate independently of actin stress fibers, focal adhesions, or cell spreading, as long as a threshold level of MAPK activity is sustained.
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http://dx.doi.org/10.1007/s00018-012-1130-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3535415PMC
January 2013

Kinetics of PKCε activating and inhibiting llama single chain antibodies and their effect on PKCε translocation in HeLa cells.

PLoS One 2012 20;7(4):e35630. Epub 2012 Apr 20.

Cell Biology, Department of Biology, University of Utrecht, Utrecht, The Netherlands.

Dysregulation of PKCε is involved in several serious diseases such as cancer, type II diabetes and Alzheimer's disease. Therefore, specific activators and inhibitors of PKCε hold promise as future therapeutics, in addition to being useful in research into PKCε regulated pathways. We have previously described llama single chain antibodies (VHHs) that specifically activate (A10, C1 and D1) or inhibit (E6 and G8) human recombinant PKCε. Here we report a thorough kinetic analysis of these VHHs. The inhibiting VHHs act as non-competitive inhibitors of PKCε activity, whereas the activating VHHs have several different modes of action, either increasing V(max) and/or decreasing K(m) values. We also show that the binding of the VHHs to PKCε is conformation-dependent, rendering the determination of affinities difficult. Apparent affinities are in the micromolar range based on surface plasmon resonance studies. Furthermore, the VHHs have no effect on the activity of rat PKCε nor can they bind the rat form of the protein in immunoprecipitation studies despite the 98% identity between the human and rat PKCε proteins. Finally, we show for the first time that the VHHs can influence PKCε function also in cells, since an activating VHH increases the rate of PKCε translocation in response to PMA in HeLa cells, whereas an inhibiting VHH slows down the translocation. These results give insight into the mechanisms of PKCε activity modulation and highlight the importance of protein conformation on VHH binding.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035630PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334965PMC
August 2012

Inhibition of protein kinase B activity induces cell cycle arrest and apoptosis during early G₁ phase in CHO cells.

Cell Biol Int 2012 Apr;36(4):357-65

Cellular Dynamics, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase.
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http://dx.doi.org/10.1042/CBI20110092DOI Listing
April 2012

Attachment of HeLa cells during early G1 phase.

Histochem Cell Biol 2011 Oct 11;136(4):399-411. Epub 2011 Aug 11.

Department of Cell Biology, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands.

Both growth factor directed and integrin dependent signal transduction were shown to take place directly after completion of mitosis. The local activation of these signal transduction cascades was investigated in early G1 cells. Interestingly, various key signal transduction proteins were found in blebs at the cell membrane within 30 min after mitosis. These membrane blebs appeared in round, mitotic-like cells and disappeared rapidly during spreading of the cells in G1 phase. In addition to tyrosine-phosphorylated proteins, the blebs contained also phosphorylated FAK and phosphorylated MAP kinase. The formation of membrane blebs in round, mitotic cells before cell spreading is not specific for mitotic cells, because similar features were observed in trypsinized cells. Just before cell spreading also these cells exhibited membrane blebs containing active signal transduction proteins. Inhibition of signal transduction did not affect membrane bleb formation, suggesting that the membrane blebs were formed independent of signal transduction.
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http://dx.doi.org/10.1007/s00418-011-0852-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3174367PMC
October 2011

An undergraduate course to bridge the gap between textbooks and scientific research.

CBE Life Sci Educ 2011 ;10(1):83-94

Science Department, University College Utrecht, Utrecht, The Netherlands.

This article reports on a one-semester Advanced Cell Biology course that endeavors to bridge the gap between gaining basic textbook knowledge about cell biology and learning to think and work as a researcher. The key elements of this course are 1) learning to work with primary articles in order to get acquainted with the field of choice, to learn scientific reasoning, and to identify gaps in our current knowledge that represent opportunities for further research; 2) formulating a research project with fellow students; 3) gaining thorough knowledge of relevant methodology and technologies used within the field of cell biology; 4) developing cooperation and leadership skills; and 5) presenting and defending research projects before a jury of experts. The course activities were student centered and focused on designing a genuine research program. Our 5-yr experience with this course demonstrates that 1) undergraduate students are capable of delivering high-quality research designs that meet professional standards, and 2) the authenticity of the learning environment in this course strongly engages students to become self-directed and critical thinkers. We hope to provide colleagues with an example of a course that encourages and stimulates students to develop essential research thinking skills.
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http://dx.doi.org/10.1187/cbe.10-08-0100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3046892PMC
June 2011

The development of activating and inhibiting camelid VHH domains against human protein kinase C epsilon.

Eur J Pharm Sci 2011 Mar 8;42(4):332-9. Epub 2011 Jan 8.

Cellular Dynamics, Department of Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands.

The 10 isozymes of the protein kinase C (PKC) family can have different roles on the same biological process, making isozyme specific analysis of function crucial. Currently, only few pharmacological compounds with moderate isozyme specific effects exist thus hampering research into individual PKC isozymes. The antigen binding regions of camelid single chain antibodies (VHHs) could provide a solution for obtaining PKC isozyme specific modulators. In the present study, we have successfully selected and characterized PKCɛ specific VHH antibodies from two immune VHH libraries using phage display. The VHHs were shown to exclusively bind to PKCɛ in ELISA and immunoprecipitation studies. Strikingly, five of the VHHs had an effect on PKCɛ kinase activity in vitro. VHHs A10, C1 and D1 increased PKCɛ kinase activity in a concentration-dependent manner (EC(50) values: 212-310nM), whereas E6 and G8 inhibited PKCɛ activity (IC(50) values: 103-233nM). None of these VHHs had an effect on the activity of the other novel PKC isozymes PKCδ and PKCθ. To our knowledge, these antibodies are the first described VHH activators and inhibitors for a protein kinase. Furthermore, the development of PKCɛ specific modulators is an important contribution to PKC research.
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http://dx.doi.org/10.1016/j.ejps.2010.12.012DOI Listing
March 2011

Novel role of cPLA(2)alpha in membrane and actin dynamics.

Cell Mol Life Sci 2010 May 29;67(9):1547-57. Epub 2010 Jan 29.

Department of Cellular Dynamics, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Actin-directed processes such as membrane ruffling and cell migration are regulated by specific signal transduction pathways that become activated by growth factor receptors. The same signaling pathways that lead to modifications in actin dynamics also activate cPLA(2)alpha. Moreover, arachidonic acid, the product of cPLA(2)alpha activity, is involved in regulation of actin dynamics. Therefore, it was investigated whether cPLA(2)alpha plays a role in actin dynamics, more specifically during growth factor-induced membrane ruffling and cell migration. Upon stimulation of ruffling and cell migration by growth factors, endogenous cPLA(2)alpha and its active phosphorylated form were shown to relocate at protrusions of the cell membrane involved in actin and membrane dynamics. Inhibition of cPLA(2)alpha activity with specific inhibitors blocked growth factor-induced membrane and actin dynamics, suggesting an important role for cPLA(2)alpha in these processes.
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http://dx.doi.org/10.1007/s00018-010-0267-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2856858PMC
May 2010

The ROS-NOX connection in cancer and angiogenesis.

Crit Rev Eukaryot Gene Expr 2008 ;18(1):35-45

Department of Cell Architecture and Dynamics, Institute of Biomembranes, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Initially viewed as dangerous byproducts of aerobic life, reactive oxygen species (ROS) nowadays appear to be essential secondary messengers of many signaling cascades and cellular functions. The establishment of ROS as important signaling molecules has been confirmed by the existence of specialized ROS producing complexes expressed in nonphagocytic cells, the NADPH oxidase complex (NOX). Because of the diversity of their proteic targets (besides lipids and DNA), ROS have multiple and sometimes contradictory functions. In the present review, we focus on several different signaling pathways influenced by ROS and NOX in tumorigenesis, focusing on proliferation and angiogenesis. We review the ROS targets regulating proliferation, including cellular signaling (phosphatases, AP1, and nuclear factor-kappa B [NF-kappaB]) and cell cycle targets (CDC25, cyclin D, and forkhead proteins), and the role of NOX during proliferation. Finally, we review the direct and indirect involvement of ROS and NOX in (tumor) angiogenesis through the regulation of different biologic systems such as vascular endothelial growth factor, angiotensin II, hypoxia-inducible factor, AP1, and inflammation.
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http://dx.doi.org/10.1615/critreveukargeneexpr.v18.i1.30DOI Listing
April 2008

Identification of a restriction point at the M/G1 transition during the ongoing cell cycle.

Adv Enzyme Regul 2007 2;47:208-21. Epub 2007 Mar 2.

Cellular Architecture and Dynamics, Faculty of Sciences, Padualaan 8, University Utrecht, 3584 CH Utrecht, The Netherlands.

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http://dx.doi.org/10.1016/j.advenzreg.2006.11.001DOI Listing
November 2007

Focal adhesion signaling and actin stress fibers are dispensable for progression through the ongoing cell cycle.

J Cell Sci 2007 Jan 5;120(Pt 1):66-76. Epub 2006 Dec 5.

Cellular Architecture and Dynamics, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Prevention of cell spreading or disruption of actin filaments inhibits growth factor stimulated cell cycle re-entry from quiescence, mainly because of a failure to induce cyclin D expression. Ectopic cyclin D expression overrules anchorage-dependency, suggesting that cell spreading per se is not required as long as cyclin D is otherwise induced. We investigated whether cyclin D expression in cells exiting mitosis is sufficient to drive morphology-independent cell cycle progression in continuously cycling (i.e. not quiescent) cells. Disruption of post-mitotic actin reorganization did not affect substratum reattachment but abolished the formation of filopodia, lamellipodia and ruffles, as well as stress fiber organization, focal adhesion assembly and cell spreading. Furthermore, integrin-mediated focal adhesion kinase (FAK) autophosphorylation and growth factor stimulated p42/p44 mitogen activated protein kinase (MAPK) activation were inhibited. Despite a progressive loss of cyclin D expression in late G1, cyclin E and cyclin A were normally induced. In addition, cells committed to DNA synthesis and completed their entire cycle. Our results demonstrate that post-mitotic disruption of the actin cytoskeleton allows cell cycle progression independent of focal adhesion signaling, cytoskeletal organization and cell shape, presumably because pre-existing cyclin D levels are sufficient to drive cell cycle progression at the M-G1 border.
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http://dx.doi.org/10.1242/jcs.03301DOI Listing
January 2007

Degradation of the hexose transporter Hxt5p in Saccharomyces cerevisiae.

Biol Cell 2007 Jan;99(1):13-23

Cellular Architecture and Dynamics, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Background Information: Hxt5p is a member of a multigene family of hexose transporter proteins which translocate glucose across the plasma membrane of the yeast Saccharomyces cerevisiae. In contrast with other major hexose transporters of this family, Hxt5p expression is regulated by the growth rate of the cells and not by the external glucose concentration. Furthermore, Hxt5p is the only glucose transporter expressed during stationary phase. These observations suggest a different role for Hxt5p in S. cerevisiae. Therefore we studied the metabolism and localization of Hxt5p in more detail.

Results And Conclusions: Inhibition of HXT5 expression in stationary-phase cells by the addition of glucose, which increases the growth rate, led to a decrease in the amount of Hxt5 protein within a few hours. Addition of glucose to stationary-phase cells resulted in a transient phosphorylation of Hxt5p on serine residues, but no ubiquitination was detected. The decrease in Hxt5p levels is caused by internalization of the protein, as observed by immunofluorescence microscopy. In stationary-phase cells, Hxt5p was localized predominantly at the cell periphery and upon addition of glucose to the cells the protein translocated to the cell interior. Electron microscopy demonstrated that the internalized Hxt5p-HA (haemagglutinin) protein was localized to small vesicles, multivesicular bodies and the vacuole. These results suggest that internalization and degradation of Hxt5p in the vacuole occur in an ubiquitination-independent manner via the endocytic pathway.
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http://dx.doi.org/10.1042/BC20060040DOI Listing
January 2007

Signal transduction and actin in the regulation of G1-phase progression.

Crit Rev Eukaryot Gene Expr 2005 ;15(3):255-76

Department of Cell Architecture and Dynamics, Institute of Biomembranes, University of Utrecht, The Netherlands.

Regulation of cell proliferation is dependent on the integration of signal transduction systems that are activated by external signal molecules, such as growth factors and extracellular matrix components. Dependent on these signal transduction networks, the cells decide in the G1 phase to continue proliferation or, alternatively, to stop cell-cycle progression and undergo apoptosis, differentiation, or quiescence. The MAP kinase and PI-3 kinase pathways have been demonstrated to play an essential role in these G1-phase decisions. Interestingly, actin has been demonstrated to mutually interfere with signal transduction. In addition, it has been indicated that the FOXO transcription factors are involved in these decisions, as well. Actin has been demonstrated to play an important role in the regulation of G1-phase progression. Because of its properties as a structural protein, actin is essential in cytokinesis and in cell spreading and, thus, is involved in G1-phase progression. As an intermediate factor in signal transduction, actin is likely to be involved in cell-cycle regulation induced by external signal molecules. And, finally, actin has been demonstrated to play a direct role in transcription. These observations indicate a prominent role of actin in the regulation of G1-phase progression.
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http://dx.doi.org/10.1615/critreveukargeneexpr.v15.i3.70DOI Listing
March 2006

Role of signal transduction and actin in G1 phase progression.

Adv Enzyme Regul 2005 27;45:186-200. Epub 2005 Sep 27.

Department of Molecular Cell Biology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584CH Utrecht, The Netherlands.

Progression through the cell cycle of mammalian cells is dependent upon external factors such as growth- and ECM factors. These factors exert their effect predominantly during the G1 phase of the cell cycle. When cells are cultured in suspension or when growth factors are withdrawn from the medium, cells will stop cell cycle progression and enter a quiescent state. Cells will remain in this quiescent state until extracellular conditions change and cells are stimulated to re-enter the cell cycle. This stimulation is mediated by various signal transduction cascades such as the mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol 3-kinase (PI3-kinase) pathway. In Chinese hamster ovary cells at least two serum-dependent points exist during G1 phase that lead to diffent cellular responses. The first point is located immediately after mitosis and is suggested to link with apoptosis. The second point is located in late G1 phase and probably corresponds with cellular differentiation. Signal transduction is mutually related to the cytoskeleton, especially the actin microfilament system. The actin microfilament system influences signal transduction and several signal transduction pathways influence the actin structure. Here we describe the role of the MAPK and PI3-kinase activities and of actin microfilaments in progression through the cell cycle and their role in the two G1 checkpoints.
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http://dx.doi.org/10.1016/j.advenzreg.2005.02.015DOI Listing
February 2006

Rapid identification of target genes for 3-methyl-1-butanol production in Saccharomyces cerevisiae.

Appl Microbiol Biotechnol 2006 Mar 23;70(2):237-46. Epub 2005 Jul 23.

Department of Molecular Cell Biology, Utrecht University, The Netherlands.

Extracellular conditions determine the taste of fermented foods by affecting metabolite formation by the micro-organisms involved. To identify targets for improvement of metabolite formation in food fermentation processes, automated high-throughput screening and cDNA microarray approaches were applied. Saccharomyces cerevisiae was cultivated in 96-well microtiter plates, and the effects of salt concentration and pH on the growth and synthesis of the fusel alcohol-flavoured substance, 3-methyl-1-butanol, was evaluated. Optimal fermentation conditions for 3-methyl-1-butanol concentration were found at pH 3.0 and 0% NaCl. To identify genes encoding enzymes with major influence on product formation, a genome-wide gene expression analysis was carried out with S. cerevisiae cells grown at pH 3.0 (optimal for 3-methyl-1-butanol formation) and pH 5.0 (yeast cultivated under standard conditions). A subset of 747 genes was significantly induced or repressed when the pH was changed from pH 5.0 to 3.0. Expression of seven genes related to the 3-methyl-1-butanol pathway, i.e. LAT1, PDX1, THI3, ALD4, ILV3, ILV5 and LEU4, strongly changed in response to this switch in pH of the growth medium. In addition, genes involved in NAD metabolism, i.e. BNA2, BNA3, BNA4 and BNA6, or those involved in the TCA cycle and glutamate metabolism, i.e. MEU1, CIT1, CIT2, KDG1 and KDG2, displayed significant changes in expression. The results indicate that this is a rapid and valuable approach for identification of interesting target genes for improvement of yeast strains used in industrial processes.
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http://dx.doi.org/10.1007/s00253-005-0070-2DOI Listing
March 2006

Specific production rate of VHH antibody fragments by Saccharomyces cerevisiae is correlated with growth rate, independent of nutrient limitation.

J Biotechnol 2005 Aug;118(3):270-7

Department of Molecular Cell Biology and the Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Saccharomyces cerevisiae carrying a multicopy integrated expression vector containing the gene encoding a Llama antibody fragment, has been cultivated in continuous cultures both under carbon and nitrogen limiting conditions with galactose as the sole carbon source. VHH-R2 expression was under control of the inducible GAL7 promoter. Induction however, was independent of the galactose consumption rate and maximal at all growth rates. VHH-R2 was secreted with 70% efficiency at all growth rates and under both limitations. The specific production rate increased linear with increasing growth rate in a growth-associated manner. However, when grown under nitrogen limitation at growth rates above 0.09 h(-1), the extracellular VHH-R2 was less active or part of the VHH-R2 was in an inactive form. From our results we conclude that to obtain a maximal amount of VHH per kilogram biomass per hour, VHH production should be done in carbon limited continuous cultures at high specific growth rates.
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http://dx.doi.org/10.1016/j.jbiotec.2005.05.010DOI Listing
August 2005

Bat2p is essential in Saccharomyces cerevisiae for fusel alcohol production on the non-fermentable carbon source ethanol.

FEMS Yeast Res 2005 May;5(8):757-66

Department of Molecular Cell Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Branched-chain amino acids (BCAAs) are key substrates in the formation of fusel alcohols, important flavour components in fermented foods. The first step in the catabolic BCAA degradation is a transaminase step, catalyzed by a branched-chain amino acid transaminase (BCAAT). Saccharomyces cerevisiae possesses a mitochondrial and a cytosolic BCAAT, Bat1p and Bat2p, respectively. In order to study the impact of the BCAATs on fusel alcohol production derived from the BCAA metabolism, S. cerevisiae BCAAT-deletion mutants were constructed. The BCAA l-leucine was exogenously supplied during cultivations with mutants of S. cerevisiae. BAT1 deletion is not essential for fusel alcohol production, neither under glucose nor under ethanol growth conditions. The 3-methyl-1-butanol production rate of bat1Delta-cells on ethanol was decreased in comparison with that of wild-type cells, but the cells were still able to produce 3-methyl-1-butanol. However, drastic effects in fusel alcohol production were obtained in cells lacking BAT2. Although the constructed bat2Delta-single deletion strain and the bat1Deltabat2Delta-double deletion strain were still able to produce 3-methyl-1-butanol when grown on glucose, they were incapable of producing any 3-methyl-1-butanol when ethanol was the sole carbon source available. In the circumstances used, gene expression analysis revealed a strong upregulation of BAT2 gene activity in the wild type, when cells grew on ethanol as carbon source. Apparently, the carbon metabolism is able to influence the expression of BCAATs and interferes with the nitrogen metabolism. Furthermore, analysis of gene expression profiles shows that the expression of genes coding for other transaminases present in S. cerevisiae was influenced by the deletion of one or both BCAATs. Several transaminases were upregulated when a BCAAT was deleted. Strikingly, none of the known transaminases was significantly upregulated when BAT2 was deleted. Therefore we conclude that the expression of BAT2 is essential for 3-methyl-1-butanol formation on the non-fermentable carbon source, ethanol.
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http://dx.doi.org/10.1016/j.femsyr.2005.02.005DOI Listing
May 2005

Regulation of enzyme activity in vivo is determined by its cellular localization.

Adv Enzyme Regul 2004 ;44:61-73

Department of Molecular Cell Biology, Institute of Biomembranes, Utrecht University, Padualaan 8, CH 3584 Utrecht, The Netherlands.

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http://dx.doi.org/10.1016/j.advenzreg.2003.11.008DOI Listing
March 2005

HXT5 expression is under control of STRE and HAP elements in the HXT5 promoter.

Yeast 2004 Jul;21(9):747-57

Department of Molecular Cell Biology and the Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Hexose transporter (Hxt) proteins transport glucose across the plasma membrane in the yeast Saccharomyces cerevisiae. Recently, we have shown that expression of HXT5 is regulated by the growth rate of the cells. Because gene expression is regulated by binding of specific transcription factors to regulatory elements in the promoters of genes, the presence of putative regulatory elements in the promoter of HXT5 was determined by computer-assisted analysis. This revealed the presence of two putative stress-responsive elements (STREs), one putative post-diauxic shift (PDS) element and two putative Hap2/3/4/5p (HAP) complex binding elements. The involvement of these elements was studied by using mutations in a HXT5 promoter-LacZ fusion construct. Growth during various conditions that result in low growth rates of yeast cells revealed that the STRE most proximal to the translation initiation site seemed to be involved in particular in regulation of HXT5 expression during growth at decreased growth rates. In addition, the HAP elements seemed to be required during growth on non-fermentable carbon sources. The PDS element and, to a lesser extent, the other STRE showed particular involvement in regulation of HXT5 expression during growth on ethanol. Finally, it was shown that the PKA pathway, which is known to be involved in expression of STRE-regulated genes, was also involved in regulation of HXT5 expression. A possible mechanism by which expression of HXT5 could be regulated by the transcriptional regulatory elements in the promoter is discussed.
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http://dx.doi.org/10.1002/yea.1106DOI Listing
July 2004

Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells.

Gene 2004 Aug;337:1-13

Department of Cell Biology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Cell cycle progression is regulated by a wide variety of external factors, amongst them are growth factors and extracellular matrix factors. During the last decades evidence has been obtained that reactive oxygen species (ROS) may also play an important role in cell cycle progression. ROS may be generated by external and internal factors. In this overview we describe briefly the generation of ROS and their effects on processes that have been demonstrated to play an essential role in cell cycle progression, including such systems as signal transduction cascades, protein ubiquitination and degradation, and the cytoskeleton. These different effects of ROS influence cell cycle progression dependent upon the amount and duration of ROS exposure. Activation of growth factor stimulated signaling cascades by low levels of ROS result in increased cell cycle progression, or, in case of prolonged exposure, to a differentiation like growth arrest. From many studies it seems clear that the cyclin kinase inhibitor protein p21 plays a prominent role, leading to cell cycle arrest at higher but not directly lethal levels of ROS. Dependent upon the nature of p21 induction, the cell cycle arrest may be transient, coupled to repair processes, or permanent. At high concentrations of ROS all of the above processes are activated, in combination with enhanced damage to the building blocks of the cell, leading to apoptosis or even necrosis.
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http://dx.doi.org/10.1016/j.gene.2004.04.032DOI Listing
August 2004

Activation of cytosolic phospholipase A2 in Her14 fibroblasts by hydrogen peroxide: a p42/44(MAPK)-dependent and phosphorylation-independent mechanism.

Biochim Biophys Acta 2004 Mar;1636(2-3):183-95

Department of Molecular Cell Biology, Institute and Graduate School of Biomembranes, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of diseases as well as various normal cellular processes. It has been suggested that ROS function as mediators of signal transduction, given that they can mimic growth factor-induced signaling. The ROS H2O2 has been reported to activate phospholipase A2 (PLA2) and, therefore, we investigated if and through which pathway ROS activate cytosolic PLA2 (cPLA2) in Her14 fibroblasts. cPLA2 was activated concentration-dependently by H2O2 in a transient manner. In addition, the lipophilic cumene hydroperoxide was shown to induce cPLA2 activity in the same manner. H2O2-induced cPLA2 activity in Her14 cells was partially phosphorylation-dependent, which was mediated through the Raf-MEK-p42/44(MAPK) pathway and occurred partially through a phosphorylation-independent mechanism. ROS can lead to changes in the (micro) viscosity of membranes due to the presence oxidized lipids, thereby increasing the substrate availability for cPLA2. In support of this, treatment of Her14 cells with H2O2 induced lipid peroxidation time-dependently as determined from degradation of lipid arachidonate and linoleate and the formation of aldehydic degradation products. Furthermore, H2O2 induced translocation of cPLA2 to the membrane fraction in a calcium-independent fashion, with a concomitant increase in cPLA2 activity. Collectively, the results suggest that oxidative stress-induced cPLA2 activity is partially phosphorylation-dependent and is further increased due to increased substrate availability by the action of ROS on membranes.
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http://dx.doi.org/10.1016/j.bbalip.2003.12.008DOI Listing
March 2004

The role of cytosolic phospholipase A2 in cell cycle progression.

Prog Cell Cycle Res 2003 ;5:181-90

Department of Molecular Cell Biology, University Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands.

Cytosolic phospholipase A2 (cPLA2) cleaves fatty acids from the sn-2 position of phospholipids resulting in the release of arachidonic acid. cPLA2 is activated upon phosphorylation, followed by a Ca(2+)-dependent translocation to cellular membranes. The activity of cPLA2 has been demonstrated to fluctuate during the M, G1 and S-phases of the cell cycle, and it has been shown recently that inhibition of the relatively high activity during the initial phases of the cell cycle results in a drastic reduction in S-phase entry. These findings demonstrate that in addition to a role in the inflammatory response, cPLA2 plays also an important regulatory role in cell cycle progression.
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December 2003

Progression through the G1-phase of the on-going cell cycle.

J Cell Biochem 2003 Oct;90(2):244-52

Department of Molecular Cell Biology, Institute of Biomembranes, University Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Cell cycle progression is dependent upon the action of cyclins and their partners the cyclin dependent kinases (CDKs). Each cell cycle phase has its own characteristic cyclin-CDK combination, cyclin D-CDK4,6 and cyclin E-CDK2 being responsible for progression through G(1)-phase into S-phase. Progression through G(1)-phase is regulated by signal transduction cascades activated by polypeptide growth factors and by extracellular matrix (ECM) components. Studies aiming to unravel the molecular mechanism by which these extracellular components activate the cyclin-CDK complexes in the G(1)-phase, are usually performed using serum-starved cells (G(0) cells). These cells are activated by addition of growth factors, or the cells are detached from the substratum by trypsinization and subsequently allowed to re-attach. An alternative approach, however, is to study the effects of growth factors and attachment in the ongoing cell cycle by synchronization of the cells by the mitotic shake-off method. These cells are not serum starved and not actively detached from the substratum. In this contribution it is shown that both methods yield significant different results. These observations demonstrate that data obtained with model systems should be interpreted with care, especially if the findings are used to explain cell cycle progression in cells in an intact organism.
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http://dx.doi.org/10.1002/jcb.10617DOI Listing
October 2003

Identification of salt-induced genes of Zygosaccharomyces rouxii by using Saccharomyces cerevisiae GeneFilters.

FEMS Yeast Res 2002 Dec;2(4):525-32

Department of Molecular Cell Biology and the Institute of Biomembranes, Utrecht University, Padualaan 8, The Netherlands.

Yeast GeneFilters containing all Saccharomyces cerevisiae open reading frame (ORF) sequences were used to elucidate gene activity in the osmotolerant yeast Zygosaccharomyces rouxii. Labelled cDNA derived from Z. rouxii was targeted to spotted S. cerevisiae ORFs. Approximately 90-100% homology of Z. rouxii genes with those of S. cerevisiae was required for definitive identification of the cDNAs hybridised to GeneFilter. Hybridised labelled cDNAs were visualised as small spots on the microarray, providing simultaneous information on homologous genes present in Z. rouxii and on their level of gene activity. Cross-hybridisation of the GeneFilters displayed 155 as yet unidentified genes of Z. rouxii hybridising to S. cerevisiae ORFs. From those 155 genes, the activity of 86 genes was influenced as a result of NaCl stress. In comparison with S. cerevisiae 24% of Z. rouxii genes revealed a different transcription behaviour following NaCl stress. All of these genes had no previously defined function in osmotic-stress response in Z. rouxii. Therefore, cross-hybridisation of GeneFilters proves to be an appropriate and straightforward method for screening transcripts in Z. rouxii, which provides an extension of the knowledge of genes present in a yeast genus other than S. cerevisiae.
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http://dx.doi.org/10.1111/j.1567-1364.2002.tb00119.xDOI Listing
December 2002

Trehalose and glycogen accumulation is related to the duration of the G1 phase of Saccharomyces cerevisiae.

FEMS Yeast Res 2003 May;3(3):261-8

Department of Molecular Cell Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Several factors may control trehalose and glycogen synthesis, like the glucose flux, the growth rate, the intracellular glucose-6-phosphate level and the glucose concentration in the medium. Here, the possible relation of these putative inducers to reserve carbohydrate accumulation was studied under well-defined growth conditions in nitrogen-limited continuous cultures. We showed that the amounts of accumulated trehalose and glycogen were regulated by the growth rate imposed on the culture, whereas other implicated inducers did not exhibit a correlation with reserve carbohydrate accumulation. Trehalose accumulation was induced at a dilution rate (D)
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http://dx.doi.org/10.1111/j.1567-1364.2003.tb00168.xDOI Listing
May 2003

HXT5 expression is determined by growth rates in Saccharomyces cerevisiae.

Yeast 2002 Sep;19(12):1029-38

Department of Molecular Cell Biology and the Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

In the yeast Saccharomyces cerevisiae, hexose transporter (Hxt) proteins transport glucose across the plasma membrane. The Hxt proteins are encoded by a multigene family with 20 members, of which Hxt1-4p and Hxt6-7p are the major hexose transporters. The remaining Hxt proteins have other or unknown functions. In this study, expression of HXT5 under different experimental set-ups is determined. In glucose-grown batch cultures, HXT5 is expressed prior to glucose depletion. Independent of the carbon source used in batch cultures, HXT5 is expressed after 24 h of growth and during growth on ethanol or glycerol, which indicates that growth on glucose is not necessary for expression of HXT5. Increasing the temperature or osmolarity of the growth medium also induces expression of HXT5. In fed-batch cultures, expression of HXT5 is only observed at low glucose consumption rates, independent of the extracellular glucose concentration. The only common parameter in these experiments is that an increase of HXT5 expression is accompanied by a decrease of the growth rate of cells. To determine whether HXT5 expression is determined by the growth rate, cells were grown in a nitrogen-limited continuous culture, which enables modulation of only the growth rate of cells. Indeed, HXT5 is expressed only at low dilution rates. Therefore, our results indicate that expression of HXT5 is regulated by growth rates of cells, rather than by extracellular glucose concentrations, as is the case for the major HXTs. A possible function for Hxt5p and factors responsible for increased expression of HXT5 upon low growth rates is discussed.
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http://dx.doi.org/10.1002/yea.895DOI Listing
September 2002
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