Use of focussed beam reflectance measurement (FBRM) for monitoring changes in biomass concentration

The potential of focussed beam reflectance measurement (FBRM) as a tool to monitor changes in biomass concentration was investigated in a number of biological systems. The measurement technique was applied to two morphologically dissimilar plant cell suspension cultures, Morinda citrifolia and Centaurea calcitrapa, to a filamentous bacteria, Streptomyces natalensis, to high density cultures of Escherichia coli and to a murine Sp2/0 hybridoma suspension cell line, 3-2.19. In all cases, the biomass concentration proved to be correlated with total FBRM counts. The nature of the correlation varied between systems and was influenced by the concentration, nature, size and morphology of the particle under investigation.     

Going forward: Increasing the accessibility of imaging mass spectrometry

The driving force behind the high and increasing popularity of imaging mass spectrometry is its demonstrated potential for the determination of new diagnostic/prognostic biomarkers and its ability to simultaneously trace the distributions of pharmaceuticals and their metabolites in tissues without the need to develop expensive radioactively-labeled analogues. Both of these applications would benefit from standardized methods, for the development of novel MS-based molecular histology tests and governmental-approved MS-based assays for pharmaceutical development. In addition, the broader scientific community would benefit from the increased accessibility of the technique. Currently imaging MS studies are individual endeavors, utilizing the individual expertise and infrastructure of a single laboratory and their immediate collaborators. A wide array of tissue preparation, data acquisition and data analysis techniques has been developed but lacks an international collaborative structure and data sharing capabilities. Such a collaborative framework would enable methodological exchange and detailed comparisons of analytical capabilities, to explore synergies between the different methods and result in the development of robust standardized methods. Here we describe the activities of a new European imaging MS network that will explicitly compare and contrast existing methods to provide best practice guidelines for the entire healthcare research community.     

GLP-1 and related peptides cause concentration-dependent relaxation of rat aorta through a pathway involving KATP and cAMP

Increasing evidence from both clinical and experimental studies indicates that the insulin-releasing hormone, glucagon-like peptide-1 (GLP-1) may exert additional protective/reparative effects on the cardiovascular system. The aim of this study was to examine vasorelaxant effects of GLP-1(7-36)amide, three structurally-related peptides and a non-peptide GLP-1 agonist in rat aorta. Interestingly, all GLP-1 compounds, including the established GLP-1 receptor antagonist, exendin (9-39) caused concentration-dependent relaxation. Mechanistic studies employing hyperpolarising concentrations of potassium or glybenclamide revealed that these relaxant effects are mediated via specific activation of ATP-sensitive potassium channels. Further experiments using a specific membrane-permeable cyclic AMP (cAMP) antagonist, and demonstration of increased cAMP production in response to GLP-1 illustrated the critical importance of this pathway. These data significantly extend previous observations suggesting that GLP-1 may modulate vascular function, and indicate that this effect may be mediated by the GLP-1 receptor. However, further studies are required in order to establish whether GLP-1 related agents may confer additional cardiovascular benefits to diabetic patients.     

Phosphorylation of murine caspase-9 by the protein kinase casein kinase 2 regulates its cleavage by caspase-8

Previous studies from our laboratory had indicated that cytochrome c-independent processing and activation of caspase-9 by caspase-8 contributed to early amplification of the caspase cascade in tumor necrosis factor (TNF)-alpha-treated murine cells. Here we show that murine caspase-9 is phosphorylated by casein kinase 2 (CK2) on a serine near the site of caspase-8 cleavage. CK2 has been shown to regulate cleavage of the pro-apoptotic Bid protein by phosphorylating serine residues near its caspase-8 cleavage site. Similarly, CK2 modification of Ser(348) on caspase-9 appears to render the protease refractory to cleavage by active caspase-8. This phosphorylation did not affect the ability of caspase-9 to autoprocess. Substitution of Ser(348) abolished phosphorylation but not cleavage, and a phospho-site mutant promoted apoptosis in TNF-alpha-treated caspase-9 knock-out mouse embryo fibroblasts. Furthermore, inhibition of CK2 activity and RNA interference-mediated knockdown of the kinase accelerated caspase-9 activation, whereas phosphatase inhibition delayed both caspase-9 activation and death in response to TNF receptor occupation. Taken together, these studies show that TNF receptor cross-linking promotes dephosphorylation of caspase-9, rendering it susceptible to processing by activated caspase-8 protein. Thus, our data suggest that modification of procaspase-9 to protect it from inappropriate cleavage and activation is yet another mechanism by which the oncogenic kinase CK2 promotes survival.     

Functional evidence for purinergic inhibitory neuromuscular transmission in the mouse internal anal sphincter

The neurotransmitter(s) underlying nitric oxide synthase (NOS)-independent neural inhibition in the internal anal sphincter (IAS) is still uncertain. The present study investigated the role of purinergic transmission. Contractile and electrical responses to electrical field stimulation of nerves (0.1-5 Hz for 10-60 s) were recorded in strips of mouse IAS. A single stimulus generated a 28-mV fast inhibitory junction potential (IJP) and relaxation. The NOS inhibitor N(omega)-nitro-l-arginine (l-NNA) reduced the fast IJP duration by 20%. Repetitive stimulation at 2.5-5 Hz caused a more sustained IJP and sustained relaxation. l-NNA reduced relaxation at 1 Hz and the sustained IJP at 2.5-5 Hz. All other experiments were carried out in the presence of NOS blockade. IJPs and relaxation were significantly reduced by the P2 receptor antagonists 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid (PPADS) (100 microM), by desensitization of P2Y receptors with adenosine 5'-[beta-thio]diphosphate (ADP-betaS) (10 microM), and by the selective P2Y1 receptor blocker 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate (MRS2179) (10 microM). Relaxation and IJPs were also significantly reduced by the K(+) channel blocker apamin (1 microM). Removal of extracellular potassium (K(o)) increased IJP amplitude to 205% of control, whereas return of K(o) 30 min later hyperpolarized cells by 19 mV and reduced IJP amplitude to 50% of control. Exogenous ATP (3 mM) relaxed muscles in the presence of TTX (1 microM) and hyperpolarized cells by 15 mV. In conclusion, these data suggest that purinergic transmission significantly contributes to NOS-independent neural inhibition in the mouse IAS. P2Y1 receptors, as well as at least one other P2 receptor subtype, contribute to this pathway. Purinergic receptors activate apamin-sensitive K(+) channels as well as other apamin-insensitive conductances leading to hyperpolarization and relaxation.     

Direct analysis of a GPCR-agonist interaction by surface plasmon resonance

Despite their clinical importance, detailed analysis of ligand binding at G-protein coupled receptors (GPCRs) has proved difficult. Here we successfully measure the binding of a GPCR, neurotensin receptor-1 (NTS-1), to its ligand, neurotensin (NT), using surface plasmon resonance (SPR). Specific responses were observed between NT and purified, detergent-solublised, recombinant NTS-1, using a novel configuration where the biotinylated NT ligand was immobilised on the biosensor surface. This SPR approach shows promise as a generic approach for the study of ligand interactions with other suitable GPCRs.     

Structure-activity relationships of a peptide inhibitor of the human FcRn:human IgG interaction

A family of five peptides was previously discovered by phage display techniques that binds to the human neonatal Fc receptor (FcRn) and inhibits the human IgG:human FcRn protein-protein interaction [Proc. Nat. Acad. Sci. U.S.A.2008, 105, 2337-2342]. The consensus peptide motif consists of the sequence GHFGGXY where X is preferably a hydrophobic amino acid, and also includes a disulfide bridge enclosing 11-amino acids in varying positions about the consensus sequence. We describe herein the structure-activity relationships of one of the five peptides in binding to FcRn using surface plasmon resonance and IgG:FcRn competition ELISA assays. Modifications of the peptide length, cyclization, and the incorporation of amino acid substitutions and dipeptide mimetics were studied. The most potent analogs exhibited a 50- to 100-fold improvement of in vitro activity over that of the phage-identified peptide sequence.     

Staphylococcus aureus‐mediated blood–brain barrier injury: an in vitro human brain microvascular endothelial cell model

Blood–brain barrier (BBB) disruption constitutes a hallmark event during pathogen‐mediated neurological disorders such as bacterial meningitis. As a prevalent opportunistic pathogen, Staphylococcus aureus (SA) is of particular interest in this context, although our fundamental understanding of how SA disrupts the BBB is very limited. This paper employs in vitro infection models to address this. Human brain microvascular endothelial cells (HBMvECs) were infected with formaldehyde‐fixed (multiplicity of infection [MOI] 0–250, 0–48 hr) and live (MOI 0–100, 0–3 hr) SA cultures. Both Fixed‐SA and Live‐SA could adhere to HBMvECs with equal efficacy and cause elevated paracellular permeability. In further studies employing Fixed‐SA, infection of HBMvECs caused dose‐dependent release of cytokines/chemokines (TNF‐α, IL‐6, MCP‐1, IP‐10, and thrombomodulin), reduced expression of interendothelial junction proteins (VE‐Cadherin, claudin‐5, and ZO‐1), and activation of both canonical and non‐canonical NF‐κB pathways. Using N‐acetylcysteine, we determined that these events were coupled to the SA‐mediated induction of reactive oxygen species (ROS) within HBMvECs. Finally, treatment of HBMvECs with Fixed‐ΔSpA (MOI 0–250, 48 hr), a gene deletion mutant of Staphylococcal protein A associated with bacterial infectivity, had relatively similar effects to Newman WT Fixed‐SA. In conclusion, these findings provide insight into how SA infection may activate proinflammatory mechanisms within the brain microvascular endothelium to elicit BBB failure.