R Mishra

Cell-Penetrating Peptides and Peptide Nucleic Acid-Coupled MRI Contrast Agents: Evaluation of Cellular Delivery and Target Binding

Molecular imaging of cells and cellular processes can be achieved by tagging intracellular targets such as receptors, enzymes, or mRNA. Seeking to visualize the presence of specific mRNAs by magnetic resonance (MR) imaging, we coupled peptide nucleic acids (PNA) with gadolinium-based MR contrast agents using cell-penetrating peptides for intracellular delivery. Antisense to mRNA of DsRed2 protein was used as proof of principle. The conjugates were produced by continuous solid-phase synthesis followed by chelation with gadolinium. Their cellular uptake was confirmed by fluorescence microscopy and spectroscopy as well as by MR imaging of labeled cells. The cell-penetrating peptide D-Tat(57-49) was selected over two other derivatives of HIV-1 Tat peptide, based on its superior intracellular delivery of the gadolinium-based contrast agents. Further improved delivery of conjugates was achieved upon coupling peptide nucleic acids (antisense to mRNA of DsRed2 protein and nonsense with no natural counterpart). Significant enhancement in MR contrast was obtained in cells labeled with concentrations as low as 2.5 μM of these agents. Specific binding of the targeting PNA containing conjugate to its complementary oligonucleotide sequence was proven by in vitro cell-free assay. In contrast, a lack of specific enrichment was observed in transgenic cells containing the target due to nonspecific vesicular entrapment of contrast agents. Preliminary biodistribution studies showed conjugate-related fluorescence in several organs, especially the liver and bladder, indicating high mobility of the agent in spite of its high molecular weight. No conjugate related toxicity was observed. These results are encouraging, as they warrant further molecular optimization and consecutive specificity studies in vivo of this new generation of contrast agents.

Synthesis and Characterization of a Cell-Permeable Bimodal Contrast Agent Targeting β-Galactosidase

Noninvasive monitoring of intracellular targets such as enzymes, receptors, or mRNA by means of magnetic resonance imaging (MRI) is increasingly gaining relevance in various research areas. A vital prerequisite for their visualization is the development of cell-permeable imaging probes, which can specifically interact with the target that characterizes the cellular or molecular process of interest. Here, we describe a dual-labeled probe, Gd-DOTA-k(FR)-Gal-CPP, designed to report the presence of intracellular β-galactosidase (β-gal) enzyme by MRI. This conjugate consists of a galactose based core serving as cleavable spacer, incorporated between the cell-penetrating peptide D-Tat(49-57) and reporter moieties (Gd-DOTA, fluorescein (FR)). We employed a facile building block approach to obtain our bimodal probe, Gd-DOTA-k(FR)-Gal-CPP. This strategy involved the preparation of the building blocks and their subsequent assembly using Fmoc-mediated solid phase synthesis, followed by the complexation of ligand 14 with GdCl(3). Gd-DOTA-k(FR)-Gal-CPP showed a considerably higher relaxivity enhancement (16.8±0.6 mM(-1)s(-1), 123 MHz, ∼21°C) relative to the commercial Gd-DOTA (4.0±0.12 mM(-1)s(-1), 123MHz, ∼21 °C). The activation of Gd-DOTA-k(FR)-Gal-CPP was based on a cellular retention strategy that required enzymatic cleavage of the delivery vector from galactose moiety following the cell internalization to achieve a prolonged accumulation of the reporter components (Gd-DOTA/FR) in the β-gal expressing cells. Cellular uptake of Gd-DOTA-k(FR)-Gal-CPP in β-gal expressing C6/LacZ and enzyme deficient parental C6 rat glioma cells was confirmed by fluorescence spectroscopy, MR imaging and ICP-AES measurements. All methods showed higher accumulation of measured reporters in C6/LacZ cells compared to enzyme deficient parental C6 cells. Fluorescence microscopy of cells labeled with Gd-DOTA-k(FR)-Gal-CPP indicated a predominantly vesicular localization of the green fluorescent conjugate around cell nuclei. This cellular distribution was most likely responsible for the observed non-specific background signal in the enzyme deficient C6 cells. Even though the specific accumulation of our bimodal probe has to be further improved, it could be already used for cell imaging by MRI and optical modalities.

CyLoP-1: A Novel Cysteine-Rich Cell-Penetrating Peptide for Cytosolic Delivery of Cargoes

Cell-penetrating peptides (CPPs) may have impli-cations in biomedical sciences by improving the delivery of a wide variety of drugs through the membrane barrier. CPPs are generally taken up by endocytotic pathways, and vesicular encapsulation is a limiting factor in the area of intracellular targeting. A novel, cationic cysteine-rich CPP, CyLoP-1, has been developed exhibiting distinguished diffused cytosolic distribution along with endosomal uptake at low micromolar concentrations. Comparative uptake analysis with known CPPs showed CyLoP-1 as a promising delivery vector to access the cytosol in a variety of cell types. In addition to the positively charged residues, the presence of cysteines and tryptophans proved to be essential to maintain its functionality. Also, the oxidation status of the cysteines played an important role for the uptake efficiency of CyLoP-1, with the disulfide-containing form being more effective. The distinct feature of CyLoP-1 to enter the cytosol was further explored by the covalent attachment of cargoes of different nature and sizes. In particular, induction of caspase-3 activity (indicating apoptosis) by a CyLoP-1-SmacN7 conjugate proved successful delivery of the pro-apoptotic cargo to its site of action in the cytosol. Efficient intracellular delivery into the entire cytosol already at low micromolar concentrations makes CyLoP-1 a promising candidate for cytosolic delivery of cargoes of small sizes. Thus, this peptide might prove to be useful for efficient transmembrane delivery of agents directed to cytosolic targets.

Microscopic Visualization of Metabotropic Glutamate Receptors on the Surface of Living Cells Using Bifunctional Magnetic Resonance Imaging Probes

A series of bimodal metabotropic glutamate-receptor targeted MRI contrast agents has been developed and evaluated, based on established competitive metabotropic Glu receptor subtype 5 (mGluR5) antagonists. In order to directly visualize mGluR5 binding of these agents on the surface of live astrocytes, variations in the core structure were made. A set of gadolinium conjugates containing either a cyanine dye or a fluorescein moiety was accordingly prepared, to allow visualization by optical microscopy in cellulo. In each case, surface receptor binding was compromised and cell internalization observed. Another approach, examining the location of a terbium analogue via sensitized emission, also exhibited nonspecific cell uptake in neuronal cell line models. Finally, biotin derivatives of two lead compounds were prepared, and the specificity of binding to the mGluR5 cell surface receptors was demonstrated with the aid of their fluorescently labeled avidin conjugates, using both total internal reflection fluorescence (TIRF) and confocal microscopy.

MR contrast agent composed of cholesterol and peptide nucleic acids: Design, synthesis and cellular uptake

A new mRNA targeting contrast agent consisting of three main functional domains, (i) gadolinium based magnetic resonance reporter part, (ii) antisense peptide nucleic acids targeted to mRNA, and (iii) cholesterol as the delivery vector, was developed and synthesized. The new contrast agent showed efficient cellular uptake and significant contrast enhancement at very low labeling concentrations (0.5 microM). However, after uptake into cells the agent was located predominantly in endosomes like a similar cell penetrating peptide conjugated probe. Our results indicate that this newly developed contrast agent could be used for the labeling of cells for optical as well as magnetic resonance imaging.

CPP or Cholesterol Conjugated Antisense PNA for Cellular Delivery

Gonadotropin Releasing Hormone (pGlu-His-Trp-Ser-Tyr-Gly-Leu- Arg-Pro-Gly-NH2, GnRH) plays a signifi cant role in the controlling of gonadotropins and steroids hormones. A large number of linear GnRH analogues has been synthesized and tested for several medical uses. Leuprolide acetate (pGlu-His-Trp-Ser-Tyr-(D)Leu-Leu-Arg-Pro-NHEt, LPA) is a potent GnRH agonist and is used to treat a wide range of sex hormone related disorders, including prostatic cancer, endometriosis and precocious puberty. Despite its widespread use, only limited information based on spectroscopic evidence regarding the solution conformation of Leuprolide are known. Moreover, non crystallographic data is available for the receptor of GnRH (G protein-coupled receptor). The aim of this study was to characterize the conformation of Leuprolide and its modifi ed linear analogue (pGlu-His-Trp-Ser-Tyr(OMe)-(D)Leu-Leu- Arg-Aze-NHEt) in DMSO solution (which simulates better the receptor environment) using Nuclear Magnetic Resonance (NMR) and Molecular Modeling techniques. By using both NMR and Molecular Modeling we have characterized the secondary structural preferences of these GnRH analogues.

Biocytin-based contrast agents for molecular imaging: an approach to developing new in vivo neuroanatomical tracers for MRI

One of the most striking characteristic of the brain is its profuse neuronal connectivity. Not surprisingly, the function of the nervous system critically depends on the spatiotemporal pattern of intercommunication between different regions of the brain. Both macroand microscopic aspects of the wiring diagrams of brain circuits are relevant and need to be understood in order to cope with the complexity of the brain function. In this way, for instance, the long-range connections that carry the functional specification of cortical territories need to be studied together with the detailed microcircuits inside a cortical column. Moreover, the temporal dimension of these wiring diagrams must be investigated since neuronal networks are dynamic structures exhibiting context-dependent changes in synaptic weights (Canals et al., 2009) and numbers (Chklovskii et al., 2004). Investigations over the last decades strongly suggest that stimulus or task related neural activity is distributed over large parts of the brain, covering different cortical and sub-cortical areas. For a detailed understanding of brain function, it is of prime importance to understand the organization of the neuronal connections. To chart the anatomical connections between the various components of brain networks, the neuronal tract tracing technique has been proved to be very useful. Thus, experimental tools that allow the exploration of brain circuits at

Novel Peptide Delivering Directly into the Cytosol: Prospective Tool For Intracellular Targeting

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

Novel Cysteine-rich Cell Penetrating Peptide: Efficient Uptake and Cytosolic Localization

Gonadotropin Releasing Hormone (pGlu-His-Trp-Ser-Tyr-Gly-Leu- Arg-Pro-Gly-NH2, GnRH) plays a signifi cant role in the controlling of gonadotropins and steroids hormones. A large number of linear GnRH analogues has been synthesized and tested for several medical uses. Leuprolide acetate (pGlu-His-Trp-Ser-Tyr-(D)Leu-Leu-Arg-Pro-NHEt, LPA) is a potent GnRH agonist and is used to treat a wide range of sex hormone related disorders, including prostatic cancer, endometriosis and precocious puberty. Despite its widespread use, only limited information based on spectroscopic evidence regarding the solution conformation of Leuprolide are known. Moreover, non crystallographic data is available for the receptor of GnRH (G protein-coupled receptor). The aim of this study was to characterize the conformation of Leuprolide and its modifi ed linear analogue (pGlu-His-Trp-Ser-Tyr(OMe)-(D)Leu-Leu- Arg-Aze-NHEt) in DMSO solution (which simulates better the receptor environment) using Nuclear Magnetic Resonance (NMR) and Molecular Modeling techniques. By using both NMR and Molecular Modeling we have characterized the secondary structural preferences of these GnRH analogues.

Cell Penetrating Peptides Delivering Intracellular Targeted Agents for Molecular Imaging

Gonadotropin Releasing Hormone (pGlu-His-Trp-Ser-Tyr-Gly-Leu- Arg-Pro-Gly-NH2, GnRH) plays a signifi cant role in the controlling of gonadotropins and steroids hormones. A large number of linear GnRH analogues has been synthesized and tested for several medical uses. Leuprolide acetate (pGlu-His-Trp-Ser-Tyr-(D)Leu-Leu-Arg-Pro-NHEt, LPA) is a potent GnRH agonist and is used to treat a wide range of sex hormone related disorders, including prostatic cancer, endometriosis and precocious puberty. Despite its widespread use, only limited information based on spectroscopic evidence regarding the solution conformation of Leuprolide are known. Moreover, non crystallographic data is available for the receptor of GnRH (G protein-coupled receptor). The aim of this study was to characterize the conformation of Leuprolide and its modifi ed linear analogue (pGlu-His-Trp-Ser-Tyr(OMe)-(D)Leu-Leu- Arg-Aze-NHEt) in DMSO solution (which simulates better the receptor environment) using Nuclear Magnetic Resonance (NMR) and Molecular Modeling techniques. By using both NMR and Molecular Modeling we have characterized the secondary structural preferences of these GnRH analogues.