P2X7 in Cancer: From Molecular Mechanisms to Therapeutics.

P2X7 is a transmembrane receptor expressed in multiple cell types including neurons, dendritic cells, macrophages, monocytes, B and T cells where it can drive a wide range of physiological responses from pain transduction to immune response. Upon activation by its main ligand, extracellular ATP, P2X7 can form a nonselective channel for cations to enter the cell. Prolonged activation of P2X7, high levels of extracellular ATP over an extended time period can lead to the formation of a macropore, leading to depolarization of the plasma membrane and ultimately to cell death.

Heat shock protein complex vaccination induces protection against Helicobacter pylori without exogenous adjuvant.

Background: The development of a vaccine against the human gastric pathogen Helicobacter pylori, the main causative agent of gastric adenocarcinoma, has been hampered by a number of issues, including the lack of a mucosal adjuvant for use in humans. Heat shock proteins (Hsp), highly conserved molecules expressed by both bacteria and mammalian species, possess a range of functions, including acting as chaperones for cellular proteins and the ability to activate innate immune receptors. Hsp complex (HspC) vaccines, containing Hsp derived from pathogenic bacteria, are immunostimulatory without addition of an exogenous adjuvant and can induce immunity against their chaperoned proteins.

Heat-shock proteins as dendritic cell-targeting vaccines--getting warmer.

Heat-shock proteins (hsp) provide a natural link between innate and adaptive immune responses by combining the ideal properties of antigen carriage (chaperoning), targeting and activation of antigen-presenting cells (APC), including dendritic cells (DC). Targeting is achieved through binding of hsp to distinct cell surface receptors and is followed by antigen internalization, processing and presentation. An improved understanding of the interaction of hsp with DC has driven the development of numerous hsp-containing vaccines, designed to deliver antigens directly to DC.

An ultra scale-down characterization of low shear stress primary recovery stages to enhance selectivity of fusion protein recovery from its molecular variants.

Fusion proteins offer the prospect of new therapeutic products with multiple functions. The primary recovery is investigated of a fusion protein consisting of modified E2 protein from hepatitis C virus fused to human IgG1 Fc and expressed in a Chinese hamster ovary (CHO) cell line. Fusion protein products inevitably pose increased challenge in preparation and purification.

Measuring Ca²⁺ changes in multiwell format using the Fluorometric Imaging Plate Reader (FLIPR(®)).

The Fluorometric Imaging Plate Reader (FLIPR) has made a significant contribution to drug discovery programs. The key advantage of FLIPR over conventional plate readers is the ability to measure fluorescence emission from multiple wells (96 wells or 384 wells) simultaneously and with high temporal resolution. Consequently, FLIPR has been used extensively to record dynamic intracellular processes such as changes in intracellular Ca(2+) ion concentration, membrane potential, and pH.

Ratiometric Ca²⁺ measurements using the FlexStation(®)Scanning Fluorometer.

The FlexStation( ® ) Scanning Fluorometer is a fluorescence plate reader that can measure intracellular Ca(2+) concentration using both single-wavelength and dual-wavelength fluorescent probes. The FlexStation uses a Xenon flashlamp and monochromators for both excitation and emission light to allow the use of a wide range of fluorescent indicators. The system incorporates a fluid transfer system for addition of test compounds from a source plate to the cell plate during data acquisition.

TRPM2 is elevated in the tMCAO stroke model, transcriptionally regulated, and functionally expressed in C13 microglia.

We report the detailed expression profile of TRPM2 mRNA within the human central nervous system (CNS) and demonstrate increased TRPM2 mRNA expression at 1 and 4 weeks following ischemic injury in the rat transient middle cerebral artery occlusion (tMCAO) stroke model. Microglial cells play a key role in pathology produced following ischemic injury in the CNS and possess TRPM2, which may contribute to stroke-related pathological responses. We show that TRPM2 mRNA is present in the human C13 microglial cell line and is reduced by antisense treatment.

Tissue distribution profiles of the human TRPM cation channel family.

Eight members of the TRP-melastatin (TRPM) subfamily have been identified, whose physiological functions and distribution are poorly characterized. Although tissue expression and distribution patterns have been reported for individual TRPM channels, comparisons between individual studies are not possible because of variations in analysis techniques and tissue selection. We report here a comparative analysis of the expression patterns of all of the human TRPM channels in selected peripheral tissues and the central nervous system (CNS) using two distinct but complimentary approaches: TaqMan and SYBR Green real-time quantitative reverse transcription polymerase chain reaction (RT-PCR).

Characterisation of recombinant rat TRPM2 and a TRPM2-like conductance in cultured rat striatal neurones.

TRPM2, a member of the TRP ion channel family, is expressed both in the brain and immune cells of the monocyte lineage. Functionally, it is unique in its activation by intracellular ADP-ribose and both oxidative and nitrosative stress. To date studies of this channel have concentrated on human recombinant channels and rodent native preparations.

Measuring Ca²+ changes in multiwell format using the Fluorometric Imaging Plate Reader.

The Fluorometric Imaging Plate Reader (FLIPR®; Molecular Devices, Sunnyvale, CA) has made a significant contribution to drug discovery programs in the pharmaceutical industry since the first commercial instruments were introduced 9 yr ago. The key advantage of FLIPR over conventional plate readers is its ability to measure fluorescence emission from multiple wells (96- or 384-well) simultaneously and with high temporal resolution. Consequently, FLIPR has been used extensively to record dynamic intracellular processes such as changes in intracellular Ca(2+) ion concentration, membrane potential, and pH.

Ratiometric Ca²+ measurements using the FlexStation® Scanning Fluorometer.

Many commercial organizations currently use the Fluorometric Imaging Plate Reader (FLIPR®: Molecular Devices, Sunnyvale, CA) to conduct high-throughput measurements of intracellular Ca(2+) concentration (see Chapter 7 ), taking advantage of its rapid kinetics, reliability, and compatibility for automation. For the majority of industrial applications, the primary limitation of FLIPR (i.e.

TRPM2 channel opening in response to oxidative stress is dependent on activation of poly(ADP-ribose) polymerase.

1. TRPM2 (melastatin-like transient receptor potential 2 channel) is a nonselective cation channel that is activated under conditions of oxidative stress leading to an increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) and cell death. We investigated the role of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) on hydrogen peroxide (H(2)O(2))-mediated TRPM2 activation using a tetracycline-inducible TRPM2-expressing cell line.

Activation of vanilloid receptor 1 by resiniferatoxin mobilizes calcium from inositol 1,4,5-trisphosphate-sensitive stores.

1 Capsaicin and resiniferatoxin (RTX) stimulate Ca2+ influx by activating vanilloid receptor 1 (VR1), a ligand-gated Ca2+ channel on sensory neurones. We investigated whether VR1 activation could also trigger Ca2+ mobilization from intracellular Ca2+ stores. 2 Human VR1-transfected HEK293 cells (hVR1-HEK293) were loaded with Fluo-3 or a mixture of Fluo-4 and Fura Red and imaged on a fluorometric imaging plate reader (FLIPR) and confocal microscope respectively.

Identification and characterisation of functional bombesin receptors in human astrocytes.

Reverse transcription polymerase chain reaction (RT-PCR) demonstrated the presence of bombesin BB2 receptor mRNA but not bombesin BB1 receptor or bombesin BB3 receptor mRNA in cultured human astrocytes. Neuromedin C hyperpolarised human astrocytes in whole-cell current and voltage clamp recordings and increased the intracellular free Ca(2+) ion concentration ([Ca(2+)](i)) in single astrocytes. Treatment with neuromedin C caused larger and more frequent increases in [Ca(2+)](i) than those triggered by neuromedin B, with 96% and 78% of cells responding, respectively.

Activation of astrocyte intracellular signaling pathways by interleukin-1 in rat primary striatal cultures.

The striatum has been implicated as the site of action mediating neurotoxic effects of interleukin-1 (IL-1) during ischemia. However, the molecular mechanisms underlying these events have yet to be fully addressed. In the present study, primary cultures of rat striatal cells were used as a model for the study of IL-1 signaling pathways in the striatum.