Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease

A nitric oxide synthase (NOS)-like activity has been demonstrated in human red blood cells (RBCs), but doubts about its functional significance, isoform identity and disease relevance remain. Using flow cytometry in combination with the NO-imaging probe DAF-FM we find that all blood cells form NO intracellularly, with a rank order of monocytes > neutrophils > lymphocytes > RBCs > platelets. The observation of a NO-related fluorescence within RBCs was unexpected given the abundance of the NO-scavenger oxyhemoglobin. Constitutive normoxic NO formation was abolished by NOS inhibition and intracellular NO scavenging, confirmed by laser-scanning microscopy and unequivocally validated by detection of the DAF-FM reaction product with NO using HPLC and LC-MS/MS. Employing immunoprecipitation, ESI-MS/MS-based peptide sequencing and enzymatic assay we further demonstrate that human RBCs contain an endothelial NOS (eNOS) that converts L-3H-Arginine to L-3H-Citrulline in a Ca2+/Calmodulin-dependent fashion. Moreover, in patients with coronary artery disease, red cell eNOS expression and activity are both lower than in age-matched healthy individuals and correlate with the degree of endothelial dysfunction. Thus, human RBCs constitutively produce NO under normoxic conditions via an active eNOS isoform the activity of which is compromised in patients with coronary artery disease.

Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity

Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or incomplete inactivation. Non-infectious empty capsids are structural mimics of authentic particles with no associated risk and constitute an alternate vaccine candidate. Capsids self-assemble from the processed virus structural proteins, VP0, VP3 and VP1, which are released from the structural protein precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown to be efficient but linkage of the cognate 3C protease to the C-terminus reduces expression significantly. Inactivation of the 3C enzyme in a P1-2A-3C cassette allows expression and intermediate levels of 3C activity resulted in efficient processing of the P1-2A precursor into the structural proteins which assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine.

The effect of copper(II), iron(II) sulphate and vitamin C combinations on the weak antimicrobial activity of (+)-catechin against Staphylococcus aureus and other microbes

Few attempts have been made to improve the activity of plant compounds with low antimicrobial efficacy. (+)-Catechin, a weak antimicrobial tea flavanol, was combined with putative adjuncts and tested against different species of bacteria. Copper(II) sulphate enhanced (+)-catechin activity against Pseudomonas aeruginosa but not Staphylococcus aureus, Proteus mirabilis or Escherichia coli. Attempts to raise the activity of (+)-catechin against two unresponsive species, S. aureus and E. coli, with iron(II) sulphate, iron(III) chloride, and vitamin C, showed that iron(II) enhanced (+)-catechin against S. aureus, but not E. coli; neither iron(III) nor combined iron(II) and copper(II), enhanced (+)-catechin activity against either species. Vitamin C enhanced copper(II) containing combinations against both species in the absence of iron(II). Catalase or EDTA added to active samples removed viability effects suggesting that active mixtures had produced H2O2via the action of added metal(II) ions. H2O2 generation by (+)-catechin plus copper(II) mixtures and copper(II) alone could account for the principal effect of bacterial growth inhibition following 30 minute exposures as well as the antimicrobial effect of (+)-catechin–iron(II) against S. aureus. These novel findings about a weak antimicrobial flavanol contrast with previous knowledge of more active flavanols with transition metal combinations. Weak antimicrobial compounds like (+)-catechin within enhancement mixtures may therefore be used as efficacious agents. (+)-Catechin may provide a means of lowering copper(II) or iron(II) contents in certain crop protection and other products.

Persistence of anticancer activity in berry extracts after simulated gastrointestinal digestion and colonic fermentation.

Fruit and vegetable consumption is associated at the population level with a protective effect against colorectal cancer. Phenolic compounds, especially abundant in berries, are of interest due to their putative anticancer activity. After consumption, however, phenolic compounds are subject to digestive conditions within the gastrointestinal tract that alter their structures and potentially their function. However, the majority of phenolic compounds are not efficiently absorbed in the small intestine and a substantial portion pass into the colon. We characterized berry extracts (raspberries, strawberries, blackcurrants) produced by in vitro-simulated upper intestinal tract digestion and subsequent fecal fermentation. These extracts and selected individual colonic metabolites were then evaluated for their putative anticancer activities using in vitro models of colorectal cancer, representing the key stages of initiation, promotion and invasion. Over a physiologically-relevant dose range (0-50 µg/ml gallic acid equivalents), the digested and fermented extracts demonstrated significant anti-genotoxic, anti-mutagenic and anti-invasive activity on colonocytes. This work indicates that phenolic compounds from berries undergo considerable structural modifications during their passage through the gastrointestinal tract but their breakdown products and metabolites retain biological activity and can modulate cellular processes associated with colon cancer.

Protein kinase D isoforms are expressed in rat and mouse primary sensory neurons and are activated by agonists of protease-activated receptor 2

Serine proteases generated during injury and inflammation cleave protease-activated receptor 2 (PAR(2)) on primary sensory neurons to induce neurogenic inflammation and hyperalgesia. Hyperalgesia requires sensitization of transient receptor potential vanilloid (TRPV) ion channels by mechanisms involving phospholipase C and protein kinase C (PKC). The protein kinase D (PKD) serine/threonine kinases are activated by diacylglycerol and PKCs and can phosphorylate TRPV1. Thus, PKDs may participate in novel signal transduction pathways triggered by serine proteases during inflammation and pain. However, it is not known whether PAR(2) activates PKD, and the expression of PKD isoforms by nociceptive neurons is poorly characterized. By using HEK293 cells transfected with PKDs, we found that PAR(2) stimulation promoted plasma membrane translocation and phosphorylation of PKD1, PKD2, and PKD3, indicating activation. This effect was partially dependent on PKCepsilon. By immunofluorescence and confocal microscopy, with antibodies against PKD1/PKD2 and PKD3 and neuronal markers, we found that PKDs were expressed in rat and mouse dorsal root ganglia (DRG) neurons, including nociceptive neurons that expressed TRPV1, PAR(2), and neuropeptides. PAR(2) agonist induced phosphorylation of PKD in cultured DRG neurons, indicating PKD activation. Intraplantar injection of PAR(2) agonist also caused phosphorylation of PKD in neurons of lumbar DRG, confirming activation in vivo. Thus, PKD1, PKD2, and PKD3 are expressed in primary sensory neurons that mediate neurogenic inflammation and pain transmission, and PAR(2) agonists activate PKDs in HEK293 cells and DRG neurons in culture and in intact animals. PKD may be a novel component of a signal transduction pathway for protease-induced activation of nociceptive neurons and an important new target for antiinflammatory and analgesic therapies.

Post-endocytic sorting of calcitonin receptor-like receptor and receptor activity-modifying protein 1

Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene-related peptide (CGRP). Although CGRP induces endocytosis of CLR/RAMP1, little is known about post-endocytic sorting of these proteins. We observed that the duration of stimulation with CGRP markedly affected post-endocytic sorting of CLR/RAMP1. In HEK and SK-N-MC cells, transient stimulation (10(-7) M CGRP, 1 h), induced CLR/RAMP1 recycling with similar kinetics (2-6 h), demonstrated by labeling receptors in living cells with antibodies to extracellular epitopes. Recycling of CLR/RAMP1 correlated with resensitization of CGRP-induced increases in [Ca(2+)](i). Cycloheximide did not affect resensitization, but bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPases, abolished resensitization. Recycling CLR and RAMP1 were detected in endosomes containing Rab4a and Rab11a, and expression of GTPase-defective Rab4aS22N and Rab11aS25N inhibited resensitization. After sustained stimulation (10(-7) M CGRP, >2 h), CLR/RAMP1 trafficked to lysosomes. RAMP1 was degraded approximately 4-fold more rapidly than CLR (RAMP1, 45% degradation, 5 h; CLR, 54% degradation, 16 h), determined by Western blotting. Inhibitors of lysosomal, but not proteasomal, proteases prevented degradation. Sustained stimulation did not induce detectable mono- or polyubiquitination of CLR or RAMP1, determined by immunoprecipitation and Western blotting. Moreover, a RAMP1 mutant lacking the only intracellular lysine (RAMP1K142R) internalized and was degraded normally. Thus, after transient stimulation with CGRP, CLR and RAMP1 traffic from endosomes to the plasma membrane, which mediates resensitization. After sustained stimulation, CLR and RAMP1 traffic from endosomes to lysosomes by ubiquitin-independent mechanisms, where they are degraded at different rates.

Localization of calcitonin receptor-like receptor and receptor activity modifying protein 1 in enteric neurons, dorsal root ganglia, and the spinal cord of the rat

Calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene related peptide (CGRP) and intermedin. Although CGRP is widely expressed in the nervous system, less is known about the localization of CLR and RAMP1. To localize these proteins, we raised antibodies to CLR and RAMP1. Antibodies specifically interacted with CLR and RAMP1 in HEK cells coexpressing rat CLR and RAMP1, determined by Western blotting and immunofluorescence. Fluorescent CGRP specifically bound to the surface of these cells and CGRP, CLR, and RAMP1 internalized into the same endosomes. CLR was prominently localized in nerve fibers of the myenteric and submucosal plexuses, muscularis externa and lamina propria of the gastrointestinal tract, and in the dorsal horn of the spinal cord of rats. CLR was detected at low levels in the soma of enteric, dorsal root ganglia (DRG), and spinal neurons. RAMP1 was also localized to enteric and DRG neurons and the dorsal horn. CLR and RAMP1 were detected in perivascular nerves and arterial smooth muscle. Nerve fibers containing CGRP and intermedin were closely associated with CLR fibers in the gastrointestinal tract and dorsal horn, and CGRP and CLR colocalized in DRG neurons. Thus, CLR and RAMP1 may mediate the effects of CGRP and intermedin in the nervous system. However, mRNA encoding RAMP2 and RAMP3 was also detected in the gastrointestinal tract, DRG, and dorsal horn, suggesting that CLR may associate with other RAMPs in these tissues to form a receptor for additional peptides such as adrenomedullin.

Prehistoric activity in Peterstone Great Wharf Palaeochannels: field survey 2005-6

This is an interim report on research carried out at the intertidal site of Peterstone Great Wharf, located on the Wentlooge Levels, c. 7 km east of Cardiff. The project is the first detailed survey and excavation of a site originally recorded in 1996-7 as part of a larger survey of the intertidal zone from Cardiff to the Second Severn Crossing. The 1997 survey produced important evidence for prehistoric human activity preserved within four palaeochannels. Significant erosion has taken place since then. A new survey of the foreshore has identified additional palaeochannels not seen in 1997 which form part of a more complex system of inter-cutting channels, many containing wood structures including short lines of timbers on the channel edge. Artefacts include a wooden axe handle, antler artefacts, an animal bone assemblage and some pottery of Beaker and Bronze Age date. The finds are thought to derive from a nearby, possibly eroded, settlement. The channels have trapped artefacts and preserved evidence of a range of activities, including what are interpreted as possible boat landings and fishing structures.

Gut microbial activity, implications for health and disease: the potential role of metabolite analysis

Microbial metabolism of proteins and amino acids by human gut bacteria generates a variety of compounds including phenol, indole, and sulfur compounds and branched chain fatty acids, many of which have been shown to elicit a toxic effect on the lumen. Bacterial fermentation of amino acids and proteins occurs mainly in the distal colon, a site that is often fraught with symptoms from disorders including ulcerative colitis (UC) and colorectal cancer (CRC). In contrast to carbohydrate metabolism by the gut microbiota, proteolysis is less extensively researched. Many metabolites are low molecular weight, volatile compounds. This review will summarize the use of analytical methods to detect and identify compounds in order to elucidate the relationship between specific dietary proteinaceous substrates, their corresponding metabolites, and implications for gastrointestinal health.

Fermentable carbohydrate alters hypothalamic neuronal activity and protects against the obesogenic environment

Obesity has become a major global health problem. Recently, attention has focused on the benefits of fermentable carbohydrates on modulating metabolism. Here, we take a system approach to investigate the physiological effects of supplementation with oligofructose-enriched inulin (In). We hypothesize that supplementation with this fermentable carbohydrate will not only lead to changes in body weight and composition, but also to modulation in neuronal activation in the hypothalamus. Male C57BL/6 mice were maintained on a normal chow diet (control) or a high fat (HF) diet supplemented with either oligofructose-enriched In or corn starch (Cs) for 9 weeks. Compared to HF+Cs diet, In supplementation led to significant reduction in average daily weight gain (mean ± s.e.m.: 0.19 ± 0.01 g vs. 0.26 ± 0.02 g, P < 0.01), total body adiposity (24.9 ± 1.2% vs. 30.7 ± 1.4%, P < 0.01), and lowered liver fat content (11.7 ± 1.7% vs. 23.8 ± 3.4%, P < 0.01). Significant changes were also observed in fecal bacterial distribution, with increases in both Bifidobacteria and Lactobacillius and a significant increase in short chain fatty acids (SCFA). Using manganese-enhanced MRI (MEMRI), we observed a significant increase in neuronal activation within the arcuate nucleus (ARC) of animals that received In supplementation compared to those fed HF+Cs diet. In conclusion, we have demonstrated for the first time, in the same animal, a wide range of beneficial metabolic effects following supplementation of a HF diet with oligofructose-enriched In, as well as significant changes in hypothalamic neuronal activity.

Endogenous cholinergic tone modulates spontaneous network level neuronal activity in primary cortical cultures grown on multi-electrode arrays

Background Cortical cultures grown long-term on multi-electrode arrays (MEAs) are frequently and extensively used as models of cortical networks in studies of neuronal firing activity, neuropharmacology, toxicology and mechanisms underlying synaptic plasticity. However, in contrast to the predominantly asynchronous neuronal firing activity exhibited by intact cortex, electrophysiological activity of mature cortical cultures is dominated by spontaneous epileptiform-like global burst events which hinders their effective use in network-level studies, particularly for neurally-controlled animat (‘artificial animal’) applications. Thus, the identification of culture features that can be exploited to produce neuronal activity more representative of that seen in vivo could increase the utility and relevance of studies that employ these preparations. Acetylcholine has a recognised neuromodulatory role affecting excitability, rhythmicity, plasticity and information flow in vivo although its endogenous production by cortical cultures and subsequent functional influence upon neuronal excitability remains unknown. Results Consequently, using MEA electrophysiological recording supported by immunohistochemical and RT-qPCR methods, we demonstrate for the first time, the presence of intrinsic cholinergic neurons and significant, endogenous cholinergic tone in cortical cultures with a characterisation of the muscarinic and nicotinic components that underlie modulation of spontaneous neuronal activity. We found that tonic muscarinic ACh receptor (mAChR) activation affects global excitability and burst event regularity in a culture age-dependent manner whilst, in contrast, tonic nicotinic ACh receptor (nAChR) activation can modulate burst duration and the proportion of spikes occurring within bursts in a spatio-temporal fashion. Conclusions We suggest that the presence of significant endogenous cholinergic tone in cortical cultures and the comparability of its modulatory effects to those seen in intact brain tissues support emerging, exploitable commonalities between in vivo and in vitro preparations. We conclude that experimental manipulation of endogenous cholinergic tone could offer a novel opportunity to improve the use of cortical cultures for studies of network-level mechanisms in a manner that remains largely consistent with its functional role.

A mesoscopic modelling approach to anaesthetic action on brain electrical activity

Relating the measurable, large scale, effects of anaesthetic agents to their molecular and cellular targets of action is necessary to better understand the principles by which they affect behavior, as well as enabling the design and evaluation of more effective agents and the better clinical monitoring of existing and future drugs. Volatile and intravenous general anaesthetic agents (GAs) are now known to exert their effects on a variety of protein targets, the most important of which seem to be the neuronal ion channels. It is hence unlikely that anaesthetic effect is the result of a unitary mechanism at the single cell level. However, by altering the behavior of ion channels GAs are believed to change the overall dynamics of distributed networks of neurons. This disruption of regular network activity can be hypothesized to cause the hypnotic and analgesic effects of GAs and may well present more stereotypical characteristics than its underlying microscopic causes. Nevertheless, there have been surprisingly few theories that have attempted to integrate, in a quantitative manner, the empirically well documented alterations in neuronal ion channel behavior with the corresponding macroscopic effects. Here we outline one such approach, and show that a range of well documented effects of anaesthetics on the electroencephalogram (EEG) may be putatively accounted for. In particular we parameterize, on the basis of detailed empirical data, the effects of halogenated volatile ethers (a clinically widely used class of general anaesthetic agent). The resulting model is able to provisionally account for a range of anaesthetically induced EEG phenomena that include EEG slowing, biphasic changes in EEG power, and the dose dependent appearance of anomalous ictal activity, as well as providing a basis for novel approaches to monitoring brain function in both health and disease.

Modeling electrocortical activity through improved local approximations of integral neural field equations

Neural field models of firing rate activity typically take the form of integral equations with space-dependent axonal delays. Under natural assumptions on the synaptic connectivity we show how one can derive an equivalent partial differential equation (PDE) model that properly treats the axonal delay terms of the integral formulation. Our analysis avoids the so-called long-wavelength approximation that has previously been used to formulate PDE models for neural activity in two spatial dimensions. Direct numerical simulations of this PDE model show instabilities of the homogeneous steady state that are in full agreement with a Turing instability analysis of the original integral model. We discuss the benefits of such a local model and its usefulness in modeling electrocortical activity. In particular, we are able to treat “patchy” connections, whereby a homogeneous and isotropic system is modulated in a spatially periodic fashion. In this case the emergence of a “lattice-directed” traveling wave predicted by a linear instability analysis is confirmed by the numerical simulation of an appropriate set of coupled PDEs.

Understanding the transition to seizure by modeling the epileptiform activity of general anesthetic agents

A central difficulty in modeling epileptogenesis using biologically plausible computational and mathematical models is not the production of activity characteristic of a seizure, but rather producing it in response to specific and quantifiable physiologic change or pathologic abnormality. This is particularly problematic when it is considered that the pathophysiological genesis of most epilepsies is largely unknown. However, several volatile general anesthetic agents, whose principle targets of action are quantifiably well characterized, are also known to be proconvulsant. The authors describe recent approaches to theoretically describing the electroencephalographic effects of volatile general anesthetic agents that may be able to provide important insights into the physiologic mechanisms that underpin seizure initiation.