Download Robert Jones

Bioimaging Conference ‘New Frontiers in Cellular Imaging’
Poster presentations:
Title: MNS: A novel Photorhabdus protein that Makes Nematodes Sticky.
Robert Jones, Nick Waterfield - Dept Biology and Biochemistry
Abstract:
Photorhabdus are insect-pathogenic bacteria that live in a symbiotic relationship with
Heterorhabditid nematodes. MNS is small protein highly secreted from the bacterial cell, and
appears to clump nematodes together. Knock-out mutants also implicate the protein in biofilm
formation and the attachment of cells to surfaces, as studied by Transmission electron
microscopy, surface plasmon resonance and other techniques.
-------------------------------------------------------------------------------Title: ATP induces rapid pro-thrombic morphological changes in raw 264.7 macrophages in the
absence of cell death.
S.F. Moore, A.B. Mackenzie – Dept of Pharmacy & Pharmacology
Abstract:
1.
Activation of the P2X7 receptor (P2X7R) can couple to the externalisation of
phosphatidylserine (PS) accompanied by membrane blebbing and shedding of PS-rich
microvesicles. Non-apoptotic externalisation of PS is able to form an enzyme complex
with factor Xa, factor Va and prothrombin of the coagulation cascade. This
prothrombinase complex catalyses the generation of thrombin.
2.
We characterised the murine P2X7R expressed in RAW 264.7 macrophages, studied
ATP induced changes in cellular morphology and determined the pro-thrombotic potential
of ATP induced PS externalisation and microvesicle shedding.
3.
Microvesicles shed from RAW 264.7 macrophages stimulated with ATP (1 mM, 10 min)
were collected and concentrated by ultracentrifugation (100,000g, 90 min). The prothrombotic potential of microvesicles was determined using the chromogenic thrombin
substrate S-2238.
4.
Treatment of RAW 264.7 macrophages with ATP caused a dose-dependent in ethidium
bromide uptake (pEC50 = 2.80 ± 0.06). 1 mM ATP in NaCl-based saline evoked rapid
(<10 min) externalisation of PS accompanied by microvesicle shedding.
5.
Microvesicles released from ATP (1 mM, 10 min) stimulated macrophages were found to
increase the rate of thrombin generation (2.20 ± 0.24 fold). Pre-incubation with P2X7R
antagonists KN-62 (10 M) and BBG (1µM) attenuates ATP induced pro-thrombotic
responses (P<0.001 and P<0.01 respectively, n=9)
These data demonstrate that the pro-inflammatory P2X7R can also support and propagate rapid
increase in thrombin generation in the absence of cell death.
----------------------------------------------------------------Title: Penetration of fluorescein into the nail after iontophoresis
Julie Dutet – Department of Pharmacy & Pharmacology
-------------------------------------------------------------------Title: Localisation of alpha7 Nicotinic Acetylcholine Receptors in Relation to Glutamatergic Axon
Terminals in the Rat Prefrontal Cortex
A.J. Willcox, J. Srinivasan, I.W. Jones, J.E. Cluderay*, E. Southam* and S. Wonnacott. - Dept.
Biology and Biochemistry, and *Psychiatry CEDD, GlaxoSmithKline
Abstract:
The PFC is the central executive of the brain, playing an essential role in cognitive processes.
Glutamatergic and dopaminergic signalling is critical in mediating these functions. The release of
these two neurotransmitters is modulated by nicotinic acetylcholine receptors (nAChR), in
particular, presynaptic alpha7 nAChR have been shown to modulate excitatory amino acid
release (Rousseau, et al. Neuropharm. 2005; 49:59). In this study we aimed to map the precise
distribution of alpha7 homomeric nAChR in the rat prefrontal cortex (PFC).
Autoradiography with 125I-alpha-bungarotoxin (Bgt) and 3H-methyllycaconitine were used to map
the general distribution of alpha7 nAChR within the rat PFC. AlexaFluor 488-conjugated Bgt in
conjunction with other neuronal markers provided further analysis of regions of interest at higher
magnification. Retrograde tracing was used to define the origin of axonal projections to different
regions of the PFC.
Autoradiography and confocal microscopy showed alpha7 nAChR to be sparsely expressed in
the PFC. Labelling was generally evenly distributed, except for a slight but consistent increase in
the ventral PFC compared to all other regions of the PFC. Glutamatergic afferents to the PFC
differentially express 3 vesicular glutamate transporters (VGluT) 1, 2 or 3, with VGluT 1 > VGluT
2 > VGluT 3. A small percentage of alpha7 nAChR co-localises with each isoform, VGluT 1
(4.7%), 2 (11.65%) or 3 (0.3%). In addition, co-labelling with markers for dopaminergic,
GABAergic, serotonergic and cholinergic neurons has clarified the location of alpha7 nAChRs in
the rat PFC with respect to transmitter cross talk.
Supported by BBSRC
----------------------------------------------------------Title: Pak1T212(PO4) is enriched in axon tracts in the developing CNS.
Julia Zhong – Dept Pharmacy & Pharmacology
Abstract:
Five micron coronal sections of paraffin embedded E16 mouse forebrain were immunostained for
PakT212(PO4) antibody (PK18, Sigma, Green) or TAG1 (Red). Merge imagine (yellow colour)
indicate PK18 positive staining enriched at lateral olfactory tracts (revealed by TAG3 that they are
axon tracts.
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Title: Preliminary Confocal Laser Scanning Microscopy (CLSM) Results from Porcine Skin
Treated with Fluorescently Labelled Nanoparticle/Mesoparticle (NP/MP)
Xiao Wu – Dept Pharmacy & Pharmacology
Abstract:
In order to investigate the transport mechanism of NP/MP-associated "active" across the skin,
CLSM was used to visualize the fluorescence in porcine skin treated with Nile Red (NR) labelled
NP/MP formulation. From the confocal images, skin structures such as corneocytes, hair and hair
follicles could be seen. It revealed that NR was mainly accumulated in the lipid area of stratum
corneum (SC) which is the outermost layer of the skin, and could penetrate into deeper regions of
the skin via hair follicles. Additional confocal microscopy experiments to visualize skin disposition
of commercially available, fluorescently-labelled polystyrene NP of well-defined size is
progressing.
----------------------------------------------------------------------Title: Nanodissection of Single Mammalian Cells Using AFM: the Route to Nanosurgery
A.V. Moskalenko1, D.J. Burbridge1, S.N. Gordeev1 and S.V. Smirnov2 - 1Department of Physics,
& 2Department of Pharmacy and Pharmacology
Abstract:
Atomic Force Microscopy (AFM) is widely used in biology for imaging. Here we demonstrate that
AFM can be utilized for making precise dissections and, in perspective, nanosurgery of biological
cells. Using the Electron-Beam-Induced Deposition technique we designed a number of
nanotools (nanoscalpels, nanohooks etc.) that can be used for such applications.
--------------------------------------------------------------Title: Staphylococcus aureus proteins and immune evasion
Liz Clark – Department of Biology & Biochemistry
Abstract:
My poster is concerned with characterizing the interactions between S. aureus proteins and
human serum proteins. The poster contains some information on S. aureus, the complement
system and the structure and function of two particular S. aureus proteins.