Maria-Nefeli Tsaloglou

Electroporation and lysis of marine microalga Karenia brevis for RNA extraction and amplification

We describe here a simple device for dielectrophoretic concentration of marine microalga Karenia brevis non-motile cells, followed by electric field-mediated lysis for RNA extraction. The lysate was purified using magnetic beads and pure RNA extracted. RNA quality was assessed off-chip by nucleic acid sequence-based amplification and the optimum conditions for lysis were determined. This procedure will form part of an integrated microfluidic system that is being developed with sub-systems for performing cell concentration and lysis, RNA extraction/purification and real-time quantitative RNA detection. The integrated system and its components could be used for a large range of applications including in situ harmful algal bloom detection, transcriptomics and point-of-care diagnostics.

On-chip real-time nucleic acid sequence-based amplification for RNA detection and amplification

Development of integrated systems for nucleic acid analyses is mainly driven by the requirement for fast and simple clinical and environmental diagnostics. The need for affordable and effective point-of-care diagnosis has inspired an entire field of biotechnology in micro and nano-fluidics. We are developing a microfluidic system that has individual sub-systems for performing cell concentration and lysis, RNA extraction/purification and real-time RNA detection. The system is being developed to analyse the rbcL gene of phytoplankton Karenia brevis, a species responsible for harmful algal blooms. This integrated system will perform sub-cellular analysis of RNA using nucleic acid sequence-based amplification and would be used in large scale biochemical analysis and experimentation. The device could potentially be used for the detection of any species with a known target nucleic acid sequence for in situ environmental monitoring, forensics or clinical diagnostics.

The effect of lipids on the enzymatic activity of 6-phosphofructo-1-kinase from B. stearothermophilus

6-Phosphofructo-1-kinase (PFK-1), a major regulatory enzyme in the glycolysis pathway, is a cytoplasmic enzyme with complicated allosteric kinetics. Here we investigate the effects of lipids on the activity of PFK from Bacillus stearothermophilus (BsPFK), to determine whether BsPFK shares any of the membrane binding or lipid binding properties reported for some mammalian PFKs. Our results show that large unilamellar vesicles (LUVs) composed of either the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or of 1:1 (mole ratio) DOPC and the fatty acid, oleic acid (OA), cause a three-fold increase in V(max), depending on the lipid concentration and vesicle composition, but no change in K(m). Further studies show lipids do not reverse the allosteric inhibitory effects of phosphoenolpyruvate (PEP) on BsPFK. SDS/PAGE studies do not show significant binding of the BsPFK tetramer to the surface of the phospholipid vesicles, suggesting that modulation of catalytic activity is due to binding of lipid monomers. By simulating the kinetics of BsPFK interaction with vesicles and lipid monomers we conclude that the change in BsPFK catalytic activity with respect to lipid concentration is consistent with monomer abstraction from vesicles rather than direct uptake of lipid monomers from solution.

A multi-parametric assessment of decontamination protocols for the subglacial Lake Ellsworth probe

Direct measurement and sampling of pristine environments, such as subglacial lakes, without introducing contaminating microorganisms and biomolecules from the surface, represents a significant engineering and microbiological challenge. In this study, we compare methods for decontamination of titanium grade 5 surfaces, the material extensively used to construct a custom-made probe for reaching, measuring and sampling subglacial Lake Ellsworth in West Antarctica. Coupons of titanium were artificially contaminated with Pseudomonas fluorescens bacteria and then exposed to a number of decontamination procedures. The most effective sterilants were (i) hydrogen peroxide vapour, and (ii) Biocleanse™, a commercially available, detergent-based biocidal solution. After each decontamination procedure the bacteria were incapable of proliferation, and showed no evidence of metabolic activity based on the generation of adenosine triphosphate (ATP). The use of ultraviolet irradiation or ethyl alcohol solution was comparatively ineffective for sterilisation. Hydrogen peroxide vapour and ultraviolet irradiation, which directly damage nucleic acids, were the most effective methods for removing detectable DNA, which was measured using 16S rRNA gene copy number and fluorescence-based total DNA quantification. Our results have not only been used to tailor the Ellsworth probe decontamination process, but also hold value for subsequent engineering projects, where high standards of decontamination are required.

Detection and quantification of the toxic microalgae Karenia brevis using lab on a chip mRNA sequence-based amplification

Now and again, the rapid proliferation of certain species of phytoplankton can give rise to Harmful Algal Blooms, which pose a serious threat to marine life and human health. Current methods of monitoring phytoplankton are limited by poor specificity or by the requirement to return samples to a highly resourced, centralised lab. The Lab Card is a small, microfluidic cassette which, when used in tandem with a portable Lab Card Reader can be used to sensitively and specifically quantify harmful algae in the field, from nucleic acid extracts using RNA amplification; a sensitive and specific method for the enumeration of potentially any species based on their unique genetic signatures. This study reports the culmination of work to develop a Lab Card-based genetic assay to quantify the harmful algae Karenia brevis using mRNA amplification by the Nucleic Acid Sequence Based Amplification (NASBA) method. K. brevis cells were quantified by amplification of the rbcL gene transcript in nucleic acid extracts of K. brevis cell samples. A novel enzyme dehydration and preservation method was combined with a pre-existing reagent Gelification method to prepare fully preserved Lab Cards with a shelf-life of at least six weeks prior to use. Using an internal control (IC), the Lab Card-based rbcL NASBA was demonstrated for the quantification of K. brevis from cell extracts containing between 50 and 5000 cells. This is the first demonstration of quantitation of K. brevis using IC-NASBA on a Lab Card.

A lipid mass spectrometry investigation on the effect of non-bilayer forming amphiphiles to the phospholipid metabolism of HeLa cells

Withdrawn PP2B-18 Co-ordinate induction of PPARa and SREBP2 in multifunctional protein 2 deficient mice K. Martens, E. Ver Loren Van Themaat, P. Van Veldhoven, A. Van Kampen and M. Baes Laboratory for Cell Metabolism and LIPIT, K. U. Leuven, BELGIUM, Bioinformatics Laboratory, AMC, Amsterdam, NETHERLANDS Introduction: Mice with inactivation of the D-specific multifunctional protein 2 (MFP2), a crucial enzyme of peroxisomal b-oxidation, develop multiple pathologies in diverse tissues already starting in the post-natal period and causing death before the age of 6 months. Methods and results: Gene expression profiling performed on liver of 2-day-old pups revealed up-regulation of PPARa responsive genes in knockout mice. Surprisingly, also genes involved in cholesterol biosynthesis were markedly induced. Real-time PCR confirmed the induction of PPARa target genes and of HMGCR and SREBP2, both involved in cholesterol synthesis, as well in 2-day-old pups as in adults. In accordance, the rate of cholesterol biosynthesis was markedly increased in liver of knockout mice but the hepatic cholesterol concentration was unaltered. In PPARa/MFP2 double knockout mice, upregulations of SREBP2 and HMGCR were less pronounced suggesting a link between PPARa induction and cholesterol synthesis. Conclusion: These data indicate that impaired peroxisomal b-oxidation causes an accumulation of PPARa ligands in the intact animal and a concomitant up-regulation of genes of cholesterol biosynthesis through increased expression of SREBP2. Since the hepatic cholesterol concentration was not different between the genotypes, it appears that the up-regulation was not triggered by a reduced level of cholesterol, neither resulted in increased cholesterol levels. PP2B-19 Dominant negative mutant of CREB inhibits TrkCinduced activation of the nur77 promoter J. Matuszyk and D. Klopotowska L.Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, POLAND nur77 (also termed NGFI-B, TR3, NR4A1) and its family members Nurr1 and Nor-1 are orphan nuclear receptors that play important roles in neuronal differentiation, memory consolidation, stress response, and apoptosis. The promoter region of the nur77 gene contains four near AP-1 (NAP) sites 5’-TGCGTCA. Results of Yoon & Lau [1] supported the hypothesis that JunD, but not CREB, binding to two proximal NAP elements is responsible for induction of the transcription of nur77 in response to nerve growth factor. However, the results of the present study indicate that A-CREB (the dominant negative mutant of CREB) inhibits the activation of the nur77 promoter in response to the activation of TrkC. In conclusion it is suggested that activation of the nur77 promoter in response to neurotrophins requires the co-operation of both CREB and JunD. Reference: 1. Yoon & Lau, MCB 1994; 14: 7731–7743. PP2B-20 Genome-wide location of glucocorticoid receptor binding sites D. Mitsiou, M. McCalman, M. Alexis and H. Stunnenberg Molecular Endocrinology Programme, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, GREECE, NCMLS, Department of Molecular Biology, Radboud University of Nijmegen, Nijmegen, NETHERLANDS Introduction: Glucocorticoids (GCs) are essential steroid hormones that regulate a variety of physiological processes including growth, differentiation, programmed cell death, glucose homeostasis and protein, lipid and carbohydrate metabolism. GCs exert their functions through the glucocorticoid receptor (GR), a ligand inducible transcription factor that binds to a variety of promoter elements, including glucocorticoid response elements (GREs), and regulates gene transcription in a cell-specific manner. The mechanisms by which GR selectively regulates cell-specific transcription and the global networks regulated by GR are not well established. In the present study we mapped the human GR binding sites on a genome-wide scale. Methods: To identify the global profile of GR binding sites we used chromatin immunoprecipitation (ChIP), with a monoclonal antibody against GR, combined with either hybridization on genomic microarrays covering the entire genome (ChIP-on-chip) or massively parallel sequencing (ChIP-seq). Results: We mapped the genome-wide GR binding sites in immortalized human hepatocytes (IHH) and HeLa cells and identified known and novel cis regulatory elements and target genes in cell-specific contexts. This study revealed the presence of GR binding sites in previously un-explored regions of the genome as well as networks of transcription factors underlying glucocorticoid signalling. Our data demonstrated distinct mechanisms of glucocorticoid-mediated gene regulation and shed light on the global networks modulated by GR. Conclusions: Analysis of GR chromatin occupancy in human genome substantially contributes to our understanding of the mechanisms and molecular networks underlying the diverse biological effects of glucocorticoids. 2B. Nuclear Receptors and Control of Transcription Abstracts FEBS Journal 275 (Suppl. 1) 99–437 (2008) a 2008 The Authors Journal compilation a 2008 FEBS 139 PP2B-21 CD40 ligation induces immunoproteasome gene expression via the co-ordinated action of NF-jB and of NF-jB mediated de novo synthesis of IRF1 A. Moschonas, M. Kouraki, P. Knox, E. Thymiakou, D. Kardassis and A. G. Eliopoulos Molecular and Cellular Biology Laboratory, School of Medicine, University of Crete, Heraklion, GREECE, Laboratory of Biochemistry, School of Medicine, University of Crete, Heraklion, GREECE Interferon regulatory factors (IRFs) comprise a family of pleiotropic transcription factors which influence development, immune homeostasis and disease pathogenesis. In this study, we demonstrate that stimulation of CD40, a TNF receptor family member with a pivotal role in adaptive and innate immunity, rapidly induces the expression of IRF-1 in carcinoma cells. Using a combination of small molecule kinase inhibitors, RNA interference and CD40 mutagenesis approaches, we show that p65 (RelA) NF-jB but not MAPK signaling is required for the CD40 ligand-induced up-regulation of IRF-1. Chromatin immunoprecipitation and gel-shift assays demonstrated recruitment of p65 NF-jB to the IRF-1 promoter. When fused to a luciferase reporter gene, the IRF-1 promoter responds to CD40 stimulation whereas mutations which abolish NF-jB binding render it unresponsive. Evaluation of NF-jB pathway components suggests the involvement of TAK1, IKKb and IjBa in IRF-1 promoter regulation. NF-jB and de novo synthesized IRF-1 converge to regulate immunoproteasome gene expression, as evident by the recruitment of both transcription factors to the promoter regions of transporter for antigen processing (TAP)-1, TAP-2, tapasin and the low molecular mass polypeptide (LMP)-2 and LMP10. Moreover, the RNA interference-mediated knock-down of IRF-1 reduced, whereas inhibition of NF-jB abolished the effects of CD40 on TAP-1 up-regulation. Collectively, these data reveal a novel mechanism of IRF-1 induction by CD40 which ensures that IRF-1 functions concurrently with NF-jB to facilitate immunoproteasome gene transcription. PP2B-22 Inhibition of the ubiquitous transcription factor Yin Yang 1 by phytosteryl ferulates and their therapeutic potentials R. Nagasaka, K. Ohara and H. Ushio Tokyo University of Marine Science and Technology, Tokyo, JAPAN Rice bran oil accepted worldwide contains a lot of phytosteryl ferulates, one of hydroxycinnamic acid derivatives ubiquitously found in plants, compared with other plant oil. We have reported that phytosteryl ferulates inihibited DNA-binding of NF-jB. In this study, we evaluated the effects of phytosteryl ferulates on DNA binding activities of over 300 transcription factors in RAW 264.7 macrophages. It suggested that phytosteryl ferulates would inhibit one ubiquitous transcription factor Yin Yang 1 (YY1) activation. The transcription factor YY1 is known to have a fundamental role in normal biologic processes such as embryogenesis, differentiation, replication, and cellular proliferation. YY1 exerts its effects on genes involved in these processes via its ability to initiate, activate, or repress transcription depending upon the context in which it binds. It is also generally accepted that the transcription factor acts as an initiator of tumorigenesis. Thus, phytosteryl ferulates might prevent metabolic and immunological diseases by NFjB inhibition and cancer by YY1 repression. The potential clinical significance of the functions will be discussed in this paper, in special the regulation of and the resistance to metabolism and cancer, subsuming complicated cell-cell interactions. PP2B-23 The effects of phytosteryl ferulates on multimeric form of adiponectin secreted from 3T3-L1 adipocytes K. Ohara, R. Nagasaka and H. Ushio Tokyo University of Marine Science and Technology, Tokyo, JAPAN Adiponectin has been postulated to play an important role in the modulation of glucose and lipid metabolisms in insulin-sensitive tissues in mammals. Adiponectin secreted from adipocytes and circulating in blood forms a wide range of multimers from trimers and hexamers to high molecular weight (HMW) multimers, and the ratios among them are closely correlated with insulin sensitivity. Rice bran contains a lot of plant sterols and their ferulic acid esters as compared with other plant oil. It is reported that the phytosteryl ferulates have the LDL cholesterol lowering effect in some mammals. We have recently demonstrated that phytosteryl ferulates enhance adiponectin secretion from adipocytes. In this study, we have investigated the effect of phytosteryl ferulates on the adiponectin multimers secreted from 3T3-L1 adipocytes. Mouse 3T3-L1 cells differentiated to adipocytes were treated with phytosteryl ferulates. The culture media was subjected to SDSPAGE under non-reducing, non-heat-denaturing conditions, followed by western blotting with anti-adipon