Nonspecific, Reversible Inhibition of Voltage-Gated Calcium Channels by CaMKII Inhibitor CK59

Investigation of kinase-related processes often uses pharmacological inhibition to reveal pathways in which kinases are involved. However, one concern about using such kinase inhibitors is their potential lack of specificity. Here, we report that the calcium–calmodulin-dependent kinase II (CaMKII) inhibitor CK59 inhibited multiple voltage-gated calcium channels, including the L-type channel during depolarization in a dose-dependent manner. The use of another CaMKII inhibitor, cell-permeable autocamtide-2 related inhibitory peptide II (Ant-AIP-II), failed to similarly decrease calcium current or entry in hippocampal cultures, as shown by ratiometric calcium imaging and whole-cell patch clamp electrophysiology. Notably, inhibition due to CK59 was reversible; washout of the drug brought calcium levels back to control values upon depolarization. Furthermore, the IC₅₀ for CK59 was approximately 50 μM, which is only fivefold larger than the reported IC₅₀ values for CaMKII inhibition. Similar nonspecific actions of other CaMKII inhibitors KN93 and KN62 have previously been reported. In the case of all three kinase inhibitors, the IC₅₀ for calcium current inhibition falls near that of CaMKII inhibition. Our findings demonstrate that CK59 attenuates activity of voltage-gated calcium channels, and thus provide more evidence for caution when relying on pharmacological inhibition to examine kinase-dependent phenomena.     

Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein

Hepatitis C virus (HCV) co-opts hepatic lipid pathways to facilitate its pathogenesis. The virus alters cellular lipid biosynthesis and trafficking, and causes an accumulation of lipid droplets (LDs) that gives rise to hepatic steatosis. Little is known about how these changes are controlled at the molecular level, and how they are related to the underlying metabolic states of the infected cell. The HCV core protein has previously been shown to independently induce alterations in hepatic lipid homeostasis. Herein, we demonstrate, using coherent anti-Stokes Raman scattering (CARS) microscopy, that expression of domain 2 of the HCV core protein (D2) fused to GFP is sufficient to induce an accumulation of larger lipid droplets (LDs) in the perinuclear region. Additionally, we performed fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotides [NAD(P)H], a key coenzyme in cellular metabolic processes, to monitor changes in the cofactor’s abundance and conformational state in D2-GFP transfected cells. When expressed in Huh-7 human hepatoma cells, we observed that the D2-GFP induced accumulation of LDs correlated with an increase in total NAD(P)H fluorescence and an increase in the ratio of free to bound NAD(P)H. This is consistent with an approximate 10 fold increase in cellular NAD(P)H levels. Furthermore, the lifetimes of bound and free NAD(P)H were both significantly reduced – indicating viral protein-induced alterations in the cofactors’ binding and microenvironment. Interestingly, the D2-expressing cells showed a more diffuse localization of NAD(P)H fluorescence signal, consistent with an accumulation of the co-factor outside the mitochondria. These observations suggest that HCV causes a shift of metabolic control away from the use of the coenzyme in mitochondrial electron transport and towards glycolysis, lipid biosynthesis, and building of new biomass. Overall, our findings demonstrate that HCV induced alterations in hepatic metabolism is tightly linked to alterations in NAD(P)H functional states.     

Design of the Intravenous Magnesium Efficacy in Acute Stroke (IMAGES) trial [ISRCTN19943732]

The Intravenous Magnesium Efficacy in Acute Stroke (IMAGES) trial is a multicentre,randomised, placebo-controlled trial of magnesium sulphate (MgSO₄) funded by the UK Medical Research Council. When complete, it will be the largest single neuroprotective study undertaken to date. Conscious patients presenting within 12 h of acute stroke with limb weakness are eligible. The primary outcome measure is combined death and disability as measured using the Barthel Index at 90-day follow up. By randomizing 2700 patients, the study will have 84% power to detect a 5.5% absolute reduction in the primary end-point. By April 2000, 86 centres were participating, with representation in Canada, USA, Europe, South America, Singapore and Australia. So far, 1206 patients have been randomised, of whom 37% were treated within 6 h. Overall 3-month mortality was 20% and the primary outcome event rate was 43%. The study is ongoing and centres worldwide are encouraged to participate.     

Nitrogen Oxyanion-dependent Dissociation of a Two-component Complex That Regulates Bacterial Nitrate Assimilation

Nitrogen is an essential nutrient for growth and is readily available to microbes in many environments in the form of ammonium and nitrate. Both ions are of environmental significance due to sustained use of inorganic fertilizers on agricultural soils. Diverse species of bacteria that have an assimilatory nitrate/nitrite reductase system (NAS) can use nitrate or nitrite as the sole nitrogen source for growth when ammonium is limited. In Paracoccus denitrificans, the pathway-specific two-component regulator for NAS expression is encoded by the nasT and nasS genes. Here, we show that the putative RNA-binding protein NasT is a positive regulator essential for expression of the nas gene cluster (i.e. nasABGHC). By contrast, a nitrogen oxyanion-binding sensor (NasS) is required for nitrate/nitrite-responsive control of nas gene expression. The NasS and NasT proteins co-purify as a stable heterotetrameric regulatory complex, NasS-NasT. This protein-protein interaction is sensitive to nitrate and nitrite, which cause dissociation of the NasS-NasT complex into monomeric NasS and an oligomeric form of NasT. NasT has been shown to bind the leader RNA for nasA. Thus, upon liberation from the complex, the positive regulator NasT is free to up-regulate nas gene expression.     

Continuous bioprocessing: The real thing this time?: 10th Annual bioProcessUK Conference,December 3–4, 2013, London,UK

The Annual bioProcessUK Conference has acted as the key networking event for bioprocess scientists and engineers in the UK for the past 10 years. The following article is a report from the sessions that focused on continuous bioprocessing during the 10^th Annual bioProcessUK Conference (London, December 2013). These sessions were organized by the ‘EPSRC Centre for Innovative Manufacturing in Emergent Macromolecular Therapies’ hosted at University College London. A plenary lecture and workshop provided a forum for participants to debate topical issues in roundtable discussions with industry and academic experts from institutions such as Genzyme, Janssen, Novo Nordisk, Pfizer, Merck, GE Healthcare and University College London. The aim of these particular sessions was to understand better the challenges and opportunities for continuous bioprocessing in the bioprocessing sector.     

It’s a knockout: CCN3 suppresses neointimal thickening

The role of CCN proteins in vivo is only just becoming understood. A prototypical member of the CCN family, CCN3 suppresses proliferation. In a study in press, Shimoyama and colleagues show that mice lacking CCN3 have a hyperproliferative response to vascular injury. These data, along with other recent observations, suggest that CCN3 may represent a novel therapy for hyperproliferative diseases.     

The impact of ticks on pheasant territoriality

Pheasants are competent reservoir hosts for the Lyme disease spirochaete, Borrelia burgdorferi s.l., and carry large, but highly over-dispersed, infestations of the vector ticks, Ixodes ricinus . The effects of experimental reduction of tick infestation levels on the survival and territorial behaviour of male pheasants were studied. Over three years in two woodlands in southern England, birds were marked individually and half were fitted with a slow-release acaricide, which substantially reduced their tick burdens from March to August. Acaricide treatment affected reproductive success but had no discernible impact on the survival rates of male pheasants. The degree of wattle inflation by males, an indicator of territorial status and a correlate of harem acquisition, was significantly greater among treated males. In each year, a significantly higher proportion of treated (overall 44%) than control (22%) males acquired harems. Males that acquired females ranged over small areas on field edges. By contrast, those with no females ranged more widely in woods and the adjoining fields, increasing their exposure to questing ticks. The relative contribution of such roving males to tick-borne pathogen transmission may thus increase.