John Howl

Critical evaluation of ECV304 as a human endothelial cell model defined by genetic analysis and functional responses: A comparison with the human bladder cancer derived epithelial cell line t24/83

Early reports indicated that ECV304 was a spontaneously-transformed line derived from a Japanese human umbilical vein endothelial cells (HUVEC) culture. Many morphological, immunochemical, and genetic studies provided further evidence that ECV304 was a valuable biomedical research tool and could be used to study processes that include angiogenesis in vitro and signal transduction by a variety of G protein-coupled receptors. However, several distinct differences between ECV304 and HUVEC are now apparent and recent reports have indicated genetic similarity between ECV304 and T24/83, a human bladder cancer cell line. To further assess the utility of ECV304 as a human endothelial cell model, we compared the functional responses of ECV304 and T24/83 to a range of G protein-coupled receptor agonists. We also used DNA fingerprinting to karyotype both ECV304 and T24/83. Both ATP and uridine triphosphate (UTP) stimulated inositol phosphate metabolism in ECV304 without alteration of cAMP levels. Comparative data using selective P2Y receptor agonists indicated that this response, leading to calcium mobilization from intracellular stores, was predominantly mediated by the activation of P2Y2 receptors. Similar responses were recorded from both ECV304 and T24/83 cells. ECV304 expressed a relatively high basal activity of NOS that was reduced by L-NAME and stimulated by P2Y2 receptor agonists. In contrast, P2Y2 receptor activation did not induce prostaglandin synthesis in ECV304. Both ECV304 and T24/83 express receptors for adenosine, adrenaline, and calcitonin, which stimulate adenylate cyclase. Proliferation of ECV304 and T24/83 cells, measured by the incorporation of [3H]thymidine into DNA, was largely serum-independent. This was in contrast to parallel experiments with porcine and bovine aortic endothelial cells that indicated a marked serum-dependent increase in DNA synthesis. Genetic analysis confirmed that ECV304 and T24/83 are identical. ECV304 displays some endothelial characteristics and is useful for the study of receptor pharmacology. However, ECV304 is not of HUVEC origin and is therefore an inappropriate cell line to study endothelial cell biology.

Protein Kinase A-Dependent Step(s) in Hepatitis C Virus Entry and Infectivity

ABSTRACT Viruses exploit signaling pathways to their advantage during multiple stages of their life cycle. We demonstrate a role for protein kinase A (PKA) in the hepatitis C virus (HCV) life cycle. The inhibition of PKA with H89, cyclic AMP (cAMP) antagonists, or the protein kinase inhibitor peptide reduced HCV entry into Huh-7.5 hepatoma cells. Bioluminescence resonance energy transfer methodology allowed us to investigate the PKA isoform specificity of the cAMP antagonists in Huh-7.5 cells, suggesting a role for PKA type II in HCV internalization. Since viral entry is dependent on the host cell expression of CD81, scavenger receptor BI, and claudin-1 (CLDN1), we studied the role of PKA in regulating viral receptor localization by confocal imaging and fluorescence resonance energy transfer (FRET) analysis. Inhibiting PKA activity in Huh-7.5 cells induced a reorganization of CLDN1 from the plasma membrane to an intracellular vesicular location(s) and disrupted FRET between CLDN1 and CD81, demonstrating the importance of CLDN1 expression at the plasma membrane for viral receptor activity. Inhibiting PKA activity in Huh-7.5 cells reduced the infectivity of extracellular virus without modulating the level of cell-free HCV RNA, suggesting that particle secretion was not affected but that specific infectivity was reduced. Viral particles released from H89-treated cells displayed the same range of buoyant densities as did those from control cells, suggesting that viral protein association with lipoproteins is not regulated by PKA. HCV infection of Huh-7.5 cells increased cAMP levels and phosphorylated PKA substrates, supporting a model where infection activates PKA in a cAMP-dependent manner to promote virus release and transmission.

Peptide Synthesis and Applications

Part I: Common Strategies Fundamentals of Modern Peptide Synthesis Muriel Amblard, Jean-Alain Fehrentz, Jean Martinez, and Gilles Subra Chimerism: A Strategy to Expand the Utility and Applications of Peptides John Howl Part II: Synthetic Methodologies and Applications Modification of Peptides and Other Drugs Using Lipoamino Acids and Sugars Joanne T. Blanchfield and Istvan Toth Synthesis of Linear, Branched, and Cyclic Peptide Chimera Gabor Mezo and Ferenc Hudecz Synthesis of Cell-Penetrating Peptides for Cargo Delivery Margus Pooga and Ulo Langel Incorporation of the Phosphotyrosyl Mimetic 4(Phosphonodifluoromethyl)phenylalanine (F2Pmp) Into Signal Transduction-Directed Peptides Zhu-Jun Yao, Kyeong Lee, and Terrence R. Burke, Jr. Expressed Protein Ligation for Protein Semisynthesis and Engineering Zuzana Machova and Annette G. Beck-Sickinger Cellular Delivery of Peptide Nucleic Acid by Cell-Penetrating Peptides Kalle Kilk and Ulo Langel Quenched Fluorescent Substrate-Based Peptidase Assays Rebecca A. Lew, Nathalie Tochon-Danguy, Catherine A. Hamilton, Karen M. Stewart, Marie-Isabel Aguilar, and A. Ian Smith A Convenient Method for the Synthesis of Cyclic Peptide Libraries Gregory T. Bourne, Jonathon L. Nielson, Justin F. Coughlan, Paul Darwen, Marc R. Campitelli, Douglas A. Horton, Andreas Rhumann, Stephen G. Love, Tran T. Tran, and Mark L. Smythe High-Throughput Peptide Synthesis Michal Lebl and John Hachmann Backbone Amide Linker Strategies for the Solid-Phase Synthesis of C-Terminal Modified Peptides Jordi Alsina, Steven A. Kates, George Barany, and Fernando Albericio Synthesis of Peptide Bioconjugates Ferenc Hudecz Part III: Practical Guides Protein Identification by Mass Spectrometric Analyses of Peptides AshleyMartin Manual Solid-Phase Synthesis of Glutathione Analogs: A Laboratory-Based Short Course Ursel Soomets, Mihkel Zilmer, and Ulo Langel Index

Characterization of bioactive cell penetrating peptides from human cytochrome c: protein mimicry and the development of a novel apoptogenic agent.

Cell penetrating peptides (CPPs) with intrinsic biological activities offer a novel strategy for the modulation of intracellular events. QSAR analysis identified CPPs within human cytochrome c. Two such sequences, Cyt c(77-101) and Cyt c(86-101), induced tumor cell apoptosis, thus mimicking the role of Cyt c as a key regulator of programmed cell death. Quantitative analyses confirmed that Cyt c(77-101) is an extremely efficient CPP. Thus, Cyt c(77-101) was selected for modification to incorporate target-specific peptidyl motifs. Chimeric N-terminal extension with a target mimetic of FG nucleoporins significantly enhanced the apoptogenic potency of Cyt c(77-101) to a concentration readily achievable in vivo. Moreover, this construct, Nup153-Cyt c, facilitates the dramatic redistribution of nuclear pore complex proteins and thus propounds the nuclear pore complex as a novel target for the therapeutic induction of apoptosis.

Mitoparan and target-selective chimeric analogues : Membrane translocation and intracellular redistribution induces mitochondrial apoptosis

Mastoparan, and structurally-related amphipathic peptides, may induce cell death by augmentation of necrotic and/or apoptotic pathways. To more precisely delineate cytotoxic mechanisms, we determined that [Lys(5,8)Aib(10)]mastoparan (mitoparan) specifically induces apoptosis of U373MG and ECV304 cells, as demonstrated by endonuclease and caspase-3 activation and phosphatidylserine translocation. Live cell imaging confirmed that, following translocation of the plasma membrane, mitoparan specifically co-localizes with mitochondria. Complementary studies indicated that mitoparan induces swelling and permeabilization of isolated mitochondria, through cooperation with a protein of the permeability transition pore complex VDAC, leading to the release of the apoptogenic factor, cytochrome c. N-terminal acylation of mitoparan facilitated the synthesis of chimeric peptides that incorporated target-specific address motifs including an integrin-specific RGD sequence and a Fas ligand mimetic. Significantly, these sychnologically-organised peptides demonstrated further enhanced cytotoxic potencies. We conclude that the cell penetrant, mitochondriotoxic and apoptogenic properties of mitoparan, and its chimeric analogues, offer new insights to the study and therapeutic induction of apoptosis.

Osteoblasts derived from load-bearing bones of the rat express both L- and T-like voltage-operated calcium channels and mRNA for α1C, α1D and α1G subunits

Abstract. Voltage operated calcium channels (VOCCs) are implicated in osteoblastic mechano- and hormonal transduction. Very little, however, is known about the expression of VOCCs in osteoblasts of load-bearing bones. Here we describe two types of whole-cell calcium current in rat femoral explant-derived osteoblasts. The first is high-voltage activated and sensitive to nifedipine, Bay K8644 and FPL 64176. The second is low-voltage activated and is sensitive to micromolar concentrations of Ni2+. The properties of these two currents are consistent with those of L-type and T-type calcium currents respectively. T-type currents were detected in most cells on the day of passage, the level of expression being significantly lower on subsequent days. L-type currents were also most common on the day of passage but were detected consistently throughout the 4-day period of study. The reverse transcription polymerase chain reaction with non-specific primers directed against all L-type VOCC α1 subunits and then with specific primers directed against sequences from rat brain α1C (L-type), α1D (L-type) and α1G (T-type) VOCC subunits detected transcripts of appropriate size in all four cases. Products from the three sets of specific primer pairs (α1C, α1D, α1G) were sequenced and were identical to their respective rat brain templates.

The many futures for cell-penetrating peptides: how soon is now?

Studies of CPPs (cell-penetrating peptides), sequences that are also commonly designated as protein transduction domains, now extend to a second decade of exciting and far-reaching discoveries. CPPs are proven vehicles for the intracellular delivery of macromolecules that include oligonucleotides, peptides and proteins, low-molecular-mass drugs, nanoparticles and liposomes. The biochemical properties of different classes of CPP, including various sequences derived from the HIV-1 Tat (transactivator of transcription) [e.g. Tat-(48-60), GRKKRRQRRRPPQ], and the homeodomain of the Drosophila homeoprotein Antennapaedia (residues 43-58, commonly named penetratin, RQIKIWFQNRRMKWKK), also provide novel insights into the fundamental mechanisms of translocation across biological membranes. Thus the efficacy of CPP-mediated cargo delivery continues to provide valuable tools for biomedical research and, as witnessed in 2007, candidate and emerging therapeutics. Thus it is anticipated that the further refinement of CPP technologies will provide drug-delivery vectors, cellular imaging tools, nanoparticulate devices and molecular therapeutics that will have a positive impact on the healthcare arena. The intention of this article is to provide both a succinct overview of current developments and applications of CPP technologies, and to illustrate key developments that the concerted efforts of the many researchers contributing to the Biochemical Societys Focused Meeting in Telford predict for the future. The accompanying papers in this issue of Biochemical Society Transactions provide additional details and appropriate references. Hopefully, the important and eagerly anticipated biomedical and clinical developments within the CPP field will occur sooner rather than later.

Bradykinin receptors as a therapeutic target

Biologically-active kinins, including bradykinin (BK) and Lys0-BK (kallidin), are short-lived peptide mediators predominantly generated by the enzymatic action of kallikreins on kininogen precursors. A diverse spectrum of physiological and pathological actions attributed to local kinin production is a consequence of the activation of G-protein-coupled receptors (GPCRs). Currently, two major subtypes of kinin receptor, designated B1 and B2, are recognised, although there is much evidence for pharmacological heterogeneity, particularly within the B2 receptors. Considering these facts and the widespread distribution of kinin receptors in many human tissues, it is no surprise that the therapeutic potential of kinins and kinin receptor antagonists remains the focus of numerous investigations. Studies in animals and animal tissues, instrumental in elucidating the biological roles of kinins, are well-documented in numerous excellent reviews. Unfortunately, and despite the enormous potential illustrated by animal studies, attempts to develop kinin analogues as therapeutic agents to combat human disease have largely proven disappointing. Consequently, this review selectively focuses upon studies that are directly relevant to the targeting of human BK receptors as a therapeutic intervention. In addition to providing a succinct review of well-documented pathological conditions to which kinin receptors contribute, the authors have also included more recent data that illustrate new avenues for the therapeutic application of kinin analogues.

Biological Applications of the Receptor Mimetic Peptide Mastoparan

The receptor mimetic and mast cell degranulating peptide mastoparan (MP) translocates cell membranes as an amphipathic alpha-helix, a feature that is undoubtedly a major determinant of bioactivity through the activation of heterotrimeric G proteins. Chimeric combinations of MP with G protein-coupled receptor (GPCR) ligands has produced peptides that exhibit biological activities distinct from their composite components. Thus, chimeric peptides such as galparan and M391 differentially modulate GTPase activity, display altered binding affinities for appropriate GPCRs and possess disparate secretory properties. MP and MP-containing chimerae also bind and modulate the activities of various other intracellular protein targets and are valuable tools to manipulate and study enzymatic activity, calcium homeostasis and apoptotic signalling pathways. In addition, charge delocalisation within the hydrophilic face of MP has produced analogues, including [Lys5, Lys8,Aib10]MP, that differentially regulate mast cell secretion and/or cytotoxicity. Finally, the identification of cell penetrant variants of MP chimerae has enabled the effective intracellular delivery of non-permeable biomolecules and presents an opportunity to target novel intracellular therapeutic loci.

Charge delocalisation and the design of novel mastoparan analogues: enhanced cytotoxicity and secretory efficacy of [Lys5, Lys8, Aib10]MP

The formation of an amphipathic helix is a major determinant of the biological activity of the tetradecapeptide mastoparan (MP). To address the functional significance of lysyl residues at positions 4, 11 and 12 of MP, we synthesised five novel analogues using sequence permutation and arginine-substitution to delocalise cationic charge. Comparative bioassays determined cytotoxicity, beta-hexoseaminidase secretory efficacy and peptide-activated extracellular receptor-stimulated kinase (ERK)1/2 phosphorylation. The monosubstitution of individual lysine residues with arginine produced differential changes to the indices of cytotoxicity and secretion indicating that these conservative substitutions are compatible with membrane translocation and the selective binding and activation of intracellular proteins. More profound changes to the predicted hydrophilic face of MP, resulting from the relocation or substitution of additional lysyl residues, enhanced both the cytotoxicity and secretory efficacy of novel peptides. Significantly, the more amphipathic peptide [Lys5, Lys8, Aib10]MP was identified to be both the most cytotoxic and the most potent secretagogue of all the peptides compared here. Charge delocalisation within the hydrophilic face of MP analogues was also compatible with peptide-induced activation of ERK1/2 phosphorylation. Our data indicate that charge delocalisation is a suitable strategy to engineer more potent analogues of MP that differentially target intracellular proteins.