bluuebunny/crossref_metadata_2025 huggingface dataset:
New methodology applied to deriving total ozone and other atmospheric variables from global irradiance spectra
Authors: Kerr James B. , Davis John M.
Published: November 2007

A new sampling and analysis method for acquiring low‐noise spectra using the Brewer spectrophotometer is applied to ground‐based spectral measurements of global ultraviolet radiation. The new technique substantially reduces noise caused by changing atmospheric conditions that can occur during the sampling period. Routine measurements made at Toronto between 1996 and 2006 were used to develop a statistical model for determining total ozone and other variables from the global spectral data. Long‐term comparison of global measurements with direct measurements demonstrates that global scan data can be used to measure total ozone with an accuracy better than ±1% for a wide variety of total ozone amounts and vertical profiles under clear skies. Other information such as ozone temperature and instrument wavelength stability can also be extracted. The measurements compared with model results show good agreement for clear‐sky conditions. The results indicate that atmospheric aerosols and clouds can enhance absorption of UV radiation by ozone. For aerosol optical depth of one, the absorption enhancement is about 2.0% at air mass value of 1 and drops to 0.5% enhancement for air mass values between 2 and 3. For clouds the enhancement is generally small in winter months but can be substantial in summer months. The statistical relationship used for measuring total ozone using spectral global irradiance data is given. Comparisons of total ozone and ozone temperature derived from global scans with those from direct Sun scans are shown, and the effects of aerosols and clouds on derived total ozone from global data are discussed.
An investigation of water uptake on clays minerals using ATR‐FTIR spectroscopy coupled with quartz crystal microbalance measurements
Authors: Schuttlefield Jennifer D. , Cox David , Grassian Vicki H.
Published: November 2007

It is becoming increasingly clear that the heterogeneous chemistry of mineral dust aerosol is a function of relative humidity (f(RH)) as water on the surface of the mineral dust particles can enhance or inhibit its reactivity depending on the reaction. Since clay minerals make up a significant component of the mineral dust aerosol, it is important to understand water uptake on this large fraction of dust present in the Earth's atmosphere. In this study, bulk and surface properties of several types and sources of clay minerals are characterized using a variety of techniques, including surface area measurements, attenuated total reflection Fourier transform infrared (ATR‐FTIR) spectroscopy, scanning electron microscopy coupled with energy dispersive X‐ray (SEM‐EDX) analysis, X‐Ray diffraction (XRD), Mössbauer spectroscopy, and X‐ray photoelectron spectroscopy (XPS). For these well‐characterized clays, ATR‐FTIR spectroscopy is combined with quartz crystal microbalance (QCM) mass measurements to investigate water uptake as a f(RH). Similar measurements were also done for α‐Al2O3 and synthetic NaY zeolite for comparison. Water uptake on the clay minerals, although variable and dependent on both the type and source of the clay, is greater than that found for α‐Al2O3, a metal oxide, and in many cases similar or greater than NaY zeolite, a crystalline porous material. The presence of cations with large hydration energies significantly increases the water uptake capacity for the clay minerals. The atmospheric implications of these results are discussed.
Transition and pattern diversity in arid and semiarid grassland: A modeling study
Authors: Zeng Xiaodong , Zeng Xubin
Published: November 2007

Abrupt transitions between large‐scale grassland and desert in arid and semiarid regions have been observed in nature and reproduced by modeling studies. Observations also show the existence of nonuniform fine‐scale vegetation patterns along the transition zone. This paper attempts to better understand these observations from two very different spatial scales. By explicitly introducing horizontal interaction terms into our previous dynamical grassland model, vegetation patterns with high diversities are found in the transition zone, and the system possesses an infinite number of equilibrium states in response to a given climatic forcing. The transition can be elucidated in two ways. In terms of the vegetation formations, the ecosystem undergoes the transition from uniform grassland to regular and irregular vegetation patterns, and then to pure desert as the moisture index (i.e., the ratio of precipitation over potential evaporation) decreases. In terms of biomass, the transition from grassland to desert goes through a narrow range of moisture index under which grassland is most fragile, as indicated by erratic vegetation patterns and large variation of average biomass. The existence of this range, however, has not been reported in previous modeling studies, and still needs to be validated using observational data.
Thorsellia anophelis is the dominant bacterium in a Kenyan population of adult Anopheles gambiae mosquitoes
Authors: Briones Aurelio M , Shililu Josephat , Githure John , Novak Robert , Raskin Lutgarde
Published: November 2007

Anopheles gambiae mosquitoes are not known to harbor endosymbiotic bacteria. Here we show, using nucleic acid-based methods, that 16S rRNA gene sequences specific to a recently described mosquito midgut bacterium, Thorsellia anophelis, is predominant in the midgut of adult An. gambiae s.l. mosquitoes captured in residences in central Kenya, and also occurs in the aquatic rice paddy environment nearby. PCR consistently detected T. anophelis in the surface microlayer of rice paddies, which is also consistent with the surface-feeding behavior of A. gambiae s.l. larvae. Phylogenetic analysis of cloned environmental 16S rRNA genes identified four major Thorsellia lineages, which are closely affiliated to an insect endosymbiont of the genus Arsenophonus. Physiological characterizations support the hypothesis that T. anophelis is well adapted to the female anopheline midgut by utilizing blood and tolerating the alkaline conditions in this environment. The results suggest that aquatically derived bacteria such as T. anophelis can persist through mosquito metamorphosis and become well-established in the adult mosquito midgut.
Interspecific competition and siderophore-mediated cooperation in Pseudomonas aeruginosa
Authors: Harrison Freya , Paul Jon , Massey Ruth C , Buckling Angus
Published: November 2007

Both intra- and interspecific interactions between microbes are likely to play an important role in determining the severity of microbial infections. Here, we study the impact of interactions between coinfecting opportunistic pathogens Staphylococcus aureus and Pseudomonas aeruginosa on both phenotypic and genetic changes in a P. aeruginosa social trait, the production of iron-scavenging siderophores. Siderophores are facultatively upregulated in response to iron limitation and play a key role in determining the virulence of microbial infections. Siderophore production is metabolically expensive to individual producers but benefits the group as a whole because siderophores can be used by all cells in the vicinity with siderophore receptors. Hence, populations of siderophore producers can be invaded by nonproducing cheats. Previous work has shown that P. aeruginosa can lyse S. aureus, supplying a source of free iron. We therefore hypothesized that the presence of S. aureus might result in facultative downregulation of siderophore production, and in turn, reduced selection for siderophore cheats. We tested this hypothesis by evolving P. aeruginosa in the presence and absence of free iron and S. aureus, in a fully factorial design. Iron had the expected effect: siderophore production was downregulated and cheats evolved less readily, but the presence of S. aureus instead increased facultative siderophore production and selection for cheats. This is probably because the S. aureus had the net effect of competing for iron, rather than acting as an iron source. This study demonstrates that interspecific competition can have a marked effect on intraspecific social interactions.
Alternative assembly pathways of the amyloidogenic yeast prion determinant Sup35–NM
Authors: Hess Simone , Lindquist Susan L , Scheibel Thomas
Published: November 2007

The self‐perpetuating conformational change of the translation termination factor Sup35 is associated with a prion phenomenon of Saccharomyces cerevisiae. In vitro, the prion‐determining region (NM) of Sup35 assembles into amyloid‐like fibres through a mechanism of nucleated conformational conversion. Here, we describe an alternative assembly pathway of NM that produces filaments that are composed of β‐strands and random coiled regions with several‐fold smaller diameters than the amyloid fibres. NM filaments are not detectable with either thioflavin T or Congo Red and do not show SDS or protease resistance. As filaments do not self‐convert into fibres and do not act as seed, they are not intermediates of amyloid fibre formation. Instead, they represent a stable off‐pathway form. Similar to mammalian prion proteins, Sup35 contains oligopeptide repeats located in the NM region. We found that the number of repeats determines the partitioning of the protein between filaments and amyloid‐like fibres. Low numbers of repeats favour the formation of the filamentous structure, whereas high numbers of repeats favour the formation of amyloid‐like fibres.
The twin-arginine transport system: moving folded proteins across membranes
Authors: Sargent F.
Published: November 2007

The Tat (twin-arginine transport) pathway is a protein-targeting system dedicated to the transmembrane translocation of fully folded proteins. This system is highly prevalent in the cytoplasmic membranes of bacteria and archaea, and is also found in the thylakoid membranes of plant chloroplasts and possibly also in the inner membrane of plant mitochondria. Proteins are targeted to a membrane-embedded Tat translocase by specialized N-terminal twin-arginine signal peptides bearing an SRRXFLK amino acid motif. The genes encoding components of the Tat translocase were discovered approx. 10 years ago, and, since then, research in this area has expanded on a global scale. In this review, the key discoveries in this field are summarized, and recent studies of bacterial twin-arginine signal-peptide-binding proteins are discussed.
Biochemistry and functional significance of collagen cross-linking
Authors: Robins S.P.
Published: November 2007

The biophysical characteristics of vascular tissues are dependent largely on the properties of fibrillar collagens. Considering the predominant structural component, collagen type I, the present review describes the mechanisms of formation and maturation of lysyl oxidase-mediated cross-linking, leading to an understanding of how intracellular collagen-modifying enzymes affect the patterns of cross-links produced. An important distinction is made between the enzyme-mediated cross-linking, essential for optimum tissue function, and the non-enzymatic aging processes that generally lead to structural changes deleterious to function. Finally, the extracellular matrix of vascular tissue is a multicomponent system and the role of other major constituents, such as elastin and glycosaminoglycans, in modifying tissue properties should be considered. Some details of newer methods being developed to quantify these constituents will be presented.
Cross-link breakers as a new therapeutic approach to cardiovascular disease
Authors: Susic D.
Published: November 2007

Fibrillar proteins, such as collagens type I and III, and elastin are components of the extracellular matrix. They form an intricate widespread network that provides a basis for maintaining the physical structure of the heart and vessels and also play an important role in determining cardiovascular function. Physiologically, collagen and elastin fibres are enzymatically cross-linked to form matrix. In addition to these enzymatically formed cross-links, collagen fibres may be linked non-enzymatically, most notably by formation of AGEs (advanced glycation end-products). AGEs are formed by a reaction between reducing sugars and body proteins; they are formed increasingly in diabetes mellitus and hypertension and they accumulate with aging. There are several mechanisms whereby AGEs may affect cardiovascular structure and function. These include increased myocardial and vascular stiffness and (upon reaction with their receptors) inflammatory reactions, release of growth factors and cytokines, and increased oxidative stress. Therefore breaking AGEs appears as a promising tool in the therapy of cardiovascular injury related to diabetes, hypertension and aging. Breakers of AGE cross-links have been developed and one of them, alagebrium, has been extensively studied. This brief review discusses the formation of AGEs, their role in mediating cardiovascular injury, as well as the results of experimental and clinical studies involving alagebrium.
Mast cells and degradation of pericellular and extracellular matrices: potential contributions to erosion, rupture and intraplaque haemorrhage of atherosclerotic plaques
Authors: Kovanen P.T.
Published: November 2007

Mast cells are present in advanced human atherosclerotic plaques, where they are thought to exert multiple effects on their neighbouring cells and on the extracellular matrix of the plaque. Extensive efforts at delineating their role(s) in atherosclerotic plaques have unravelled mechanisms by which plaque mast cells may render advanced atherosclerotic plaques susceptible to erosion, rupture or intraplaque haemorrhage and so modulate their stability. In these mechanisms, the key effector molecules are mast-cell-derived neutral proteases and pro-inflammatory cytokines. These effector molecules are synthesized and stored in the cytoplasmic secretory granules of mast cells and, once the mast cells are activated to degranulate, are released into the microenvironment surrounding the activated mast cells. In the plaques, the key target cells are endothelial cells and smooth muscle cells and their pericellular matrices. In addition, the various components of the extracellular matrix of the plaques, notably collagen, are degraded when the released mast cell proteases activate matrix metalloproteinases in the plaques. By rendering the plaque susceptible to erosion, to rupture or to intraplaque haemorrhage, the mast cells may contribute to the onset of acute atherothrombotic complications of coronary atherosclerosis, such as myocardial infarction.
Dr Brian Gibberd (1931–2006): a pioneering clinician in Refsum's disease
Authors: Wierzbicki A.S. , Lloyd M.D.
Published: November 2007

Branched-chain fatty acids are common components of the human diet (phytanic acid) or are produced endogenously (bile acids), and are also used as medicines (ibuprofen). Owing to their branched-chain structure, they are metabolized in peroxisomes. In the case of phytanic acid, the presence of a 3-methyl group prevents β-oxidation, and instead it undergoes one round of α-oxidation to allow further metabolism. Defects in this process give rise to neurological diseases and cancer. Dr Brian F. Gibberd was one of the first U.K. physicians to recognize the importance of these peroxisomal metabolic pathways in clinical medicine, and pioneered their study. This obituary recognizes his many achievements in neurology and especially in the treatment of peroxisomal disorders. The following four papers from this mini-symposium entitled ‘Advances in peroxisomal α-, β- and ω-oxidation’ describe work done in this area as part of a collaborative study in which Dr Gibberd played a key role. This work was presented as part of the Cardiovascular Bioscience focused topic at the Life Sciences 2007 conference, and this mini-symposium was dedicated to Dr Gibberd and his important contributions to this field.
Peroxisomes, Refsum's disease and the α- and ω-oxidation of phytanic acid
Authors: Wanders R.J.A. , Komen J.C.
Published: November 2007

In the present paper, we describe the current state of knowledge regarding the enzymology of the phytanic acid α-oxidation pathway. The product of phytanic acid α-oxidation, i.e. pristanic acid, undergoes three cycles of β-oxidation in peroxisomes after which the products, including 4,8-dimethylnonanoyl-CoA, propionyl-CoA and acetyl-CoA, are exported from the peroxisome via one of two routes, including (i) the carnitine-dependent route, mediated by CRAT (carnitine acetyltransferase) and CROT (carnitine O-octanoyltransferase), and (ii) the free acid route, mediated by one or more of the peroxisomal ACOTs (acyl-CoA thioesterases). We also describe our recent data on the ω-oxidation of phytanic acid, especially since pharmacological up-regulation of this pathway may form the basis of a new treatment strategy for ARD (adult Refsum's disease). In patients suffering from ARD, phytanic acid accumulates in tissues and body fluids due to a defect in the α-oxidation system.
Peroxisomal disorders affecting phytanic acid α-oxidation: a review
Authors: Wierzbicki A.S.
Published: November 2007

Peroxisomes are involved in the synthesis and degradation of complex fatty acids. They contain enzymes involved in the α-, β- and ω-oxidation pathways for fatty acids. Investigation of these pathways and the diseases associated with mutations in enzymes involved in the degradation of phytanic acid have led to the clarification of the pathophysiology of Refsum's disease, rhizomelic chondrodysplasia and AMACR (α-methylacyl-CoA racemase) deficiency. This has highlighted the role of an Fe(II)- and 2-oxoglutarate-dependent oxygenases [PhyH (phytanoyl-CoA 2-hydroxylase), also known as PAHX], thiamin-dependent lyases (phytanoyl-CoA lyase) and CYP (cytochrome P450) family 4A in fatty acid metabolism. The differential regulation and biology of these pathways is suggesting novel ways to treat the neuro-ophthalmological sequelae of Refsum's disease. More recently, the discovery that AMACR and other peroxisomal β-oxidation pathway enzymes are highly expressed in prostate and renal cell cancers has prompted active investigation into the role of these oxidation pathways and the peroxisome in the progression of obesity- and insulin resistance-related cancers.
Ion channel switching and activation in smooth-muscle cells of occlusive vascular diseases
Authors: Beech D.J.
Published: November 2007

Blood vessels are essential for animal life, allowing flow of oxygen and nutrients to tissues and removal of waste products. Consequently, inappropriate remodelling of blood vessels, resulting in occlusion, can lead to disabling or catastrophic events: heart attacks, strokes and claudication. An important cell type of remodelling is the VSMC (vascular smooth-muscle cell), a fascinating cell that contributes significantly to occlusive vascular diseases by virtue of its ability to ‘modulate’ to a cell that no longer contracts and arranges radially in the medial layer of the vessel wall but migrates, invades, proliferates and adopts phenotypes of other cells. An intriguing aspect of modulation is switching to different ion transport systems. Initial events include loss of the CaV1.2 (L-type voltage-gated calcium) channel and gain of the KCa3.1 (IKCa) potassium channel, which putatively occur to enable membrane hyperpolarization that increases rather than decreases a type of calcium entry coupled with cell cycle activity, cell proliferation and cell migration. This type of calcium entry is related to store- and receptor-operated calcium entry phenomena, which, in VSMCs, are contributed to by TRPC [TRP (transient receptor potential) canonical] channel subunits. Instead of being voltage-gated, these channels are chemically gated – importantly, by key phospholipid factors of vascular development and disease. This brief review focuses on the hypothesis that the transition to a modulated cell may require a switch from predominantly voltage- to predominantly lipid-sensing ion channels.
Current challenges in coronary stenting: from bench to bedside
Authors: Morton A.C. , Walker R.D. , Gunn J.
Published: November 2007

PCI (percutaneous coronary intervention) now outnumbers CABG (coronary artery bypass grafting) by more than 3:1 for the treatment of coronary heart disease. In this article, we discuss the current challenges faced by interventional cardiologists including restenosis and its treatment options and potential therapies for the future. The impact of stent geometry on restenosis and strategies to deal with challenging lesions such as bifurcations and lesions in the left main stem are also discussed.
Hypoxic inhibition of human cardiac fibroblast invasion and MMP-2 activation may impair adaptive myocardial remodelling
Authors: Morley M.E. , Riches K. , Peers C. , Porter K.E.
Published: November 2007

Cardiac fibroblasts account for up to two-thirds of the total number of cells in the normal heart and are responsible for extracellular matrix homoeostasis. In vitro, type I collagen, the predominant myocardial collagen, stimulates proteolytic activation of constitutively secreted proMMP-2 (pro-matrix metalloproteinase-2). This occurs at the cell membrane and requires formation of a ternary complex with MT1-MMP (membrane-type-1 MMP) and TIMP-2 (tissue inhibitor of metalloproteinases-2). Following MI (myocardial infarction), normally quiescent fibroblasts initiate a wound healing response by transforming into a proliferative and invasive myofibroblast phenotype. Deprivation of oxygen to the myocardium is an inevitable consequence of MI; therefore this reparative event occurs under chronically hypoxic conditions. However, species and preparation variations can strongly influence fibroblast behaviour, which is an important consideration when selecting experimental models for provision of clinically useful information.
The regulatory function of plasma-membrane Ca2+-ATPase (PMCA) in the heart
Authors: Oceandy D. , Stanley P.J. , Cartwright E.J. , Neyses L.
Published: November 2007

The PMCA (plasma-membrane Ca2+-ATPase) is a ubiquitously expressed calcium-extruding enzymatic pump important in the control of intracellular calcium concentration. Unlike in non-excitable cells, where PMCA is the only system for calcium extrusion, in excitable cells, such as cardiomyocytes, PMCA has been shown to play only a minor role in calcium homoeostasis compared with the NCX (sodium/calcium exchanger), another system of calcium extrusion. However, increasing evidence points to an important role for PMCA in signal transduction; of particular interest in cardiac physiology is the modulation of nNOS (neuronal nitric oxide synthase) by isoform 4b of PMCA. In the present paper, we will discuss recent advances that support a key role for PMCA4 in modulating the nitric oxide signalling pathway in the heart.
cAMP-specific phosphodiesterase-4D5 (PDE4D5) provides a paradigm for understanding the unique non-redundant roles that PDE4 isoforms play in shaping compartmentalized cAMP cell signalling
Authors: Lynch M.J. , Baillie G.S. , Houslay M.D.
Published: November 2007

The PDE4 (phosphodiesterase-4) enzyme family consists of a distinct array of N-terminal splice variant isoforms arising from four subfamily genes (4A, 4B, 4C and 4D). These all hydrolyse specifically the intracellular second messenger cAMP. Although identical in catalytic function, each isoform appears to serve a non-superfluous regulatory role. For example, a β-arrestin-sequestered subpopulation of the PDE4D5 isoform specifically regulates the phosphorylation of the β2-AR (β2-adrenergic receptor) by PKA (protein kinase A; also called cAMP-dependent protein kinase). This was elucidated by the use of novel technologies, including dominant-negative approaches, siRNA (small interfering RNA) knockdown and spot-immobilized peptide array analyses. Functional phenotypes uncovered using these methodologies have shown that β-arrestin-sequestered PDE4D5 shapes the spatial cAMP gradient around the membrane-bound β2-AR, regulating its phosphorylation by PKA and its ability to activate ERK (extracellular-signal-regulated kinase) through Gi in cardiomyocytes and HEK-293 (human embryonic kidney)-B2 cells. This approach has provided the very first identification of a non-redundant and specific role for a PDE isoform. The fact that phenotypes can be uncovered by displacing PDE4 isoforms from specific anchor sites using dominant-negative constructs and cell-permeable peptides points to novel means for developing therapeutics aimed at disrupting specifically sequestered PDE isoforms and even specifically sequestered subpopulations of individual isoforms.
Ryanodine receptor mutations in arrhythmias: advances in understanding the mechanisms of channel dysfunction
Authors: Thomas N.L. , George C.H. , Williams A.J. , Lai F.A.
Published: November 2007

The cardiac ryanodine receptor (RyR2) mediates rapid Ca2+ efflux from intracellular stores to effect myocyte contraction during the process of EC (excitation–contraction) coupling. It is now known that mutations in this channel perturb Ca2+ release function, leading to triggered arrhythmias that may cause SCD (sudden cardiac death). Resolving the precise molecular mechanisms by which SCD-linked RyR2 dysfunction occurs currently constitutes a burgeoning area of cardiac research. So far, defective channel phosphorylation, accessory protein binding, luminal/cytosolic Ca2+ sensing, and the disruption of interdomain interactions represent the main candidate mechanisms for explaining aberrant SR (sarcoplasmic reticulum) Ca2+ release via mutants of RyR2. It appears increasingly unlikely that a single exclusive common mechanism underlies every case of mutant channel dysfunction, and that each of these potential mechanisms may contribute to the resultant phenotype. The present review will consider very recent mechanistic developments in this field, including new observations from mutant RyR2 transgenic mouse models, peptide-probe studies, and the implications of functional and phenotypic heterogeneity of RyR2 mutations and polymorphisms.
Sarcoplasmic reticulum calcium leak and cardiac arrhythmias
Authors: Chelu M.G. , Wehrens X.H.T.
Published: November 2007

Ventricular arrhythmias deteriorating into sudden cardiac death are a major cause of mortality worldwide. The recent linkage of a genetic form of cardiac arrhythmia to mutations in the gene encoding RyR2 (ryanodine receptor 2) has uncovered an important role of this SR (sarcoplasmic reticulum) calcium release channel in triggering arrhythmias. Mutant RyR2 channels give rise to spontaneous release of calcium (Ca2+) from the SR during diastole, which enhances the probability of ventricular arrhythmias. Several molecular mechanisms have been proposed to explain the gain-of-function phenotype observed in mutant RyR2 channels. Despite considerable differences between the models discussed in the present review, each predicts spontaneous diastolic Ca2+ leak from the SR due to incomplete closure of the RyR2 channel. Enhanced SR Ca2+ leak is also observed in common structural diseases of the heart, such as heart failure. In heart failure, defective channel regulation in the absence of inherited mutations may also increase SR Ca2+ leak and initiate cardiac arrhythmias. Therefore inhibition of diastolic Ca2+ leak through SR Ca2+ release channels has emerged as a new and promising therapeutic target for cardiac arrhythmias.
Calcium in the heart: when it's good, it's very very good, but when it's bad, it's horrid
Authors: Roderick H.L. , Higazi D.R. , Smyrnias I. , Fearnley C. , Harzheim D. , Bootman M.D.
Published: November 2007

Ca2+ increases in the heart control both contraction and transcription. To accommodate a short-term increased cardiovascular demand, neurohormonal modulators acting on the cardiac pacemaker and individual myocytes induce an increase in frequency and magnitude of myocyte contraction respectively. Prolonged, enhanced function results in hypertrophic growth of the heart, which is initially also associated with greater Ca2+ signals and cardiac contraction. As a result of disease, however, hypertrophy progresses to a decompensated state and Ca2+ signalling capacity and cardiac output are reduced. Here, the role that Ca2+ plays in the induction of hypertrophy as well as the impact that cardiac hypertrophy and failure has on Ca2+ fluxes will be discussed.
Visualization of protein interactions inside the secretory pathway
Authors: Nyfeler B. , Hauri H.-P.
Published: November 2007

The ER (endoplasmic reticulum) is a major protein folding and modification organelle. In its lumen, the ER processes a third of all newly synthesized proteins. To accomplish this task, numerous resident proteins capture the nascent and newly synthesized proteins. The underlying luminal protein–protein interactions, however, are inherently difficult to analyse, mainly due to their transient nature and the rather specialized environment of the ER. To overcome these limitations, we developed a PCA (protein fragment complementation assay) based on the citrine variant of YFP (yellow fluorescent protein). YFP PCA was successfully applied to visualize the protein interactions of the cargo transport receptor ERGIC-53 (endoplasmic reticulum–Golgi intermediate compartment protein of 53 kDa) with its luminal interaction partner MCFD2 (multiple coagulation factor deficiency protein 2) and its cargo proteins cathepsin Z and cathepsin C in a specific manner. With the prospect of screening cDNA libraries for novel protein–protein interactions, YFP PCA is a promising emerging technique for mapping protein interactions inside the secretory pathway in a genome-wide setting.
Protein–protein interactions in the assembly and subcellular trafficking of the BACE (β-site amyloid precursor protein-cleaving enzyme) complex of Alzheimer's disease
Authors: Parsons R.B. , Austen B.M.
Published: November 2007

The correct assembly of the BACE (β-site amyloid precursor protein-cleaving enzyme or β-secretase) complex and its subsequent trafficking to cellular compartments where it associates with the APP (amyloid precursor protein) is essential for the production of Aβ (amyloid β-peptide), the protein whose aggregation into senile plaques is thought to be responsible for the pathogenesis of AD (Alzheimer's disease). These processes rely upon both transient and permanent BACE–protein interactions. This review will discuss what is currently known about these BACE–protein interactions and how they may reveal novel therapeutic targets for the treatment of AD.
Solid-state NMR spectroscopy as a tool for drug design: from membrane-embedded targets to amyloid fibrils
Authors: Middleton D.A.
Published: November 2007

Structure-based design has gained credibility as a valuable component of the modern drug discovery process. The technique of SSNMR (solid-state NMR) promises to be a useful counterpart to the conventional experimental techniques of X-ray crystallography and solution-state NMR for providing structural features of drug targets that can guide medicinal chemistry towards drug candidates. This article highlights some recent SSNMR approaches from our group for identifying active compounds, such as enzyme inhibitors, receptor antagonists and peptide agents, that prevent the aggregation of amyloid proteins involved in neurodegenerative diseases. It is anticipated that the use of SSNMR in drug discovery will become more widespread in the wake of advances in hardware and methodological developments.
Magic-angle-spinning NMR spectroscopy applied to small molecules and peptides in lipid bilayers
Authors: Ader C. , Schneider R. , Seidel K. , Etzkorn M. , Baldus M.
Published: November 2007

ssNMR (solid-state NMR) spectroscopy provides increasing possibilities to study the structural and dynamic aspects of biological membranes. Here, we review recent ssNMR experiments that are based on MAS (magic angle spinning) and that provide insight into the structure and dynamics of membrane systems at the atomic level. Such methods can be used to study membrane architecture, domain formation or molecular complexation in a way that is highly complementary to other biophysical methods such as imaging or calorimetry.
Targeting the oncogenic protein kinase Cι signalling pathway for the treatment of cancer
Authors: Fields A.P. , Frederick L.A. , Regala R.P.
Published: November 2007

PKC (protein kinase C) isoenzymes are key signalling components involved in the regulation of normal cell proliferation, differentiation, polarity and survival. The aberrant regulation of PKC isoenzymes has been implicated in the development of many human diseases including cancer [Fields and Gustafson (2003) Methods Mol. Biol. 233, 519–537]. To date, however, only one PKC isoenzyme, the aPKC [atypical PKCι (protein kinase Cι)], has been identified as a human oncogene [Regala, Weems, Jamieson, Khoor, Edell, Lohse and Fields (2005) Cancer Res. 65, 8905–8911]. PKCι has also proven to be a useful prognostic marker and legitimate target for the development of novel pharmacological agents for the treatment of cancer. The PKCι gene resides at chromosome 3q26 and is a frequent target of tumour-specific gene amplification in multiple forms of human cancer. PKCι gene amplification in turn drives PKCι overexpression in these cancers. Genetic disruption of PKCι expression blocks multiple aspects of the transformed phenotype of human cancer cells including transformed growth in soft agar, invasion through Matrigel and growth of subcutaneous tumours in nude mice. Genetic dissection of oncogenic PKCι signalling mechanisms demonstrates that PKCι drives transformed growth by activating a PKCι → Rac1 → PAK (p21-activated kinase) → MEK [MAPK (mitogen-activated protein kinase) 1,2/ERK (extracellular-signal-regulated kinase) kinase] 1,2 signalling pathway [Regala, Weems, Jamieson, Copland, Thompson and Fields (2005) J. Biol. Chem. 280, 31109–31115]. The transforming activity of PKCι requires the N-terminal PB1 (Phox-Bem1) domain of PKCι, which serves to couple PKCι with downstream effector molecules. Hence, there exists a strong rationale for developing novel cancer therapeutics that target the PB1 domain of PKCι and thereby disrupt its interactions with effector molecules. Using a novel high-throughput drug screen, we identified compounds that can disrupt PB1–PB1 domain interactions between PKCι and the adaptor molecule Par6 [Stallings-Mann, Jamieson, Regala, Weems, Murray and Fields (2006) Cancer Res. 66, 1767–1774]. Our screen identified the gold compounds ATG (aurothioglucose) and ATM (aurothiomalate) as specific inhibitors of the PB1–PB1 domain interaction between PKCι and Par6 that exhibit anti-tumour activity against NSCLC (non-small-cell lung cancer) both in vitro and in vivo. Structural analysis, site-directed mutagenesis and modelling indicate that ATM specifically targets the PB1 domain of PKCι to mediate its anti-tumour activity [Erdogan, Lamark, Stallings-Mann, Lee, Pellechia, Thompson, Johansen and Fields (2006) J. Biol. Chem. 281, 28450–28459]. Taken together, our recent work demonstrates that PKCι signalling is required for transformed growth of human tumours and is an attractive target for development of mechanism-based cancer therapies. ATM is currently in Phase I clinical trials for the treatment of NSCLC.
Murine models for chronic lymphocytic leukaemia
Authors: Michie A.M. , Nakagawa R. , McCaig A.M.
Published: November 2007

CLL (chronic lymphocytic leukaemia) is characterized by the clonal outgrowth of B-lymphocytes with the distinctive phenotype: CD19hiCD5+CD23+IgMlo. These malignant B-cells accumulate in the PB (peripheral blood) and lymphoid organs, and are generally arrested at the G0/G1-phase of cell cycle and display a resistance to apoptosis. To date, most of the CLL research has been carried out using PB samples obtained from patients with established CLL, which have proved instrumental in characterizing the disease. However, while CLL cells appear to have a defect in apoptosis in vivo, they rapidly undergo apoptosis ex vivo, suggesting that CLL cells are dependent on microenvironmental signals to enhance cell survival. One approach used to define the cellular and molecular events that govern CLL has been the development of murine models that replicate the human disease. As well as providing a deeper understanding of the potential triggers for CLL, these models provide preclinical in vivo systems to test novel therapies. The focus of the present review will be to highlight the recent advances in the development of mouse models for CLL.
Competitive inhibitors and allosteric activators of protein kinase C isoenzymes: a personal account and progress report on transferring academic discoveries to the clinic
Authors: Budas G.R. , Koyanagi T. , Churchill E.N. , Mochly-Rosen D.
Published: November 2007

PKC (protein kinase C) isoenzymes are related protein kinases, involved in many signalling events in normal state and in disease. Basic research into identifying the molecular basis of PKC selectivity led to simple strategies to identify selective competitive inhibitor peptides and allosteric agonist peptides of individual PKC isoenzymes. The strategies and rationale used to identify these peptide regulators of protein–protein interaction may be applicable to other signalling events. Importantly, the PKC-regulating peptides proved to be useful pharmacological tools and may serve as drugs or drug leads for a variety of human diseases.
Structure-based optimization of PKCθ inhibitors
Authors: Mosyak L. , Xu Z. , Joseph-McCarthy D. , Brooijmans N. , Somers W. , Chaudhary D.
Published: November 2007

PKCθ (protein kinase Cθ) is a central signalling molecule in the T-cell receptor activation pathway and is a target for treatment of a number of diseases. Several PKC inhibitors are in the drug-discovery pharmaceutical programmes today for the treatment of cancer, diabetes and arthritis. CD4+ T-lymphocytes also play a critical role in the initiation and progression of allergic airway inflammation. Our goal is the development of PKCθ antagonists as a means to control asthma and autoimmune diseases, using the strategy based on developing small-molecule agents that would block the enzyme's catalytic activity. Here, we discuss our work on the discovery of lead chemical series and review our X-ray structural and modelling approaches, including a structure-surrogate strategy that helped guide us in the lead compound optimizations.
Sodium regulation of GAF domain function
Authors: Cann M.J.
Published: November 2007

Cyclic nucleotide PDEs (phosphodiesterases) regulate cellular levels of cAMP and cGMP by controlling the rate of degradation. Several mammalian PDE isoforms possess N-terminal GAF (found in cGMP PDEs, Anabaena adenylate cyclases and Escherichia coli FhlA; where FhlA is formate hydrogen lyase transcriptional activator) domains that bind cyclic nucleotides. Similarly, the CyaB1 and CyaB2 ACs (adenylate cyclases) of the cyanobacterium Anabaena PCC 7120 bind cAMP through one (CyaB1) or two (CyaB2) N-terminal GAF domains and mediate autoregulation of the AC domain. Sodium inhibits the activity of CyaB1, CyaB2 and mammalian PDE2A in vitro through modulation of GAF domain function. Furthermore, genetic ablation of cyaB1 and cyaB2 gives rise to Anabaena strains defective in homoeostasis at limiting sodium. Sodium regulation of GAF domain function has therefore been conserved since the eukaryotic/prokaryotic divergence. The GAF domain is the first identified protein domain to directly sense and signal changes in environmental sodium.
The roles of PKCδ and ϵ isoenzymes in the regulation of myocardial ischaemia/reperfusion injury
Authors: Churchill E.N. , Mochly-Rosen D.
Published: November 2007

Reperfusion of ischaemic cardiac tissue is associated with increased apoptosis and oncosis, resulting in diminished heart function. Short bouts of ischaemia before the prolonged ischaemic event (ischaemic preconditioning) protect the heart from injury mediated by reperfusion. The PKC (protein kinase C) family of serine/threonine kinases are involved in many different signalling processes. Two calcium-insensitive isoforms of the novel PKC subfamily, PKCδ and ϵ, play opposing roles in ischaemia/reperfusion injury. Activation of PKCδ during reperfusion induces cell death through the regulation of mitochondrial function and induction of apoptosis and oncosis. In contrast, activation of PKCϵ before ischaemia protects mitochondrial function and diminishes apoptosis and oncosis. How can two highly homologous PKC isoenzymes play such opposing roles through the regulation of mitochondrial function? This review will highlight what is known about PKCδ and ϵ function during ischaemia/reperfusion injury and will suggest a novel regulatory pathway which determines the fate of the cell following ischaemic stress.
The C2 domains of classical/conventional PKCs are specific PtdIns(4,5)P2-sensing domains
Authors: Corbalán-García S. , Guerrero-Valero M. , Marín-Vicente C. , Gómez-Fernández J.C.
Published: November 2007

The C2 domains of cPKCs [classical/conventional PKCs (protein kinase Cs)] bind to membranes in a Ca2+-dependent manner and thereby act as cellular Ca2+ effectors. Recent findings have demonstrated that the C2 domain of cPKCs interacts specifically with PtdIns(4,5)P2 through its polybasic cluster located in the β3–β4-strands, this interaction being critical for the membrane localization of these enzymes in living cells. In addition, these C2 domains exhibit higher affinity to bind PtdIns(4,5)P2 than any other polyphosphate phosphatidylinositols. It has also been shown that the presence of PtdIns(4,5)P2 in model membranes decreases the Ca2+ concentration required for classical C2 domains to bind them. Overall, the studies reviewed here suggest a new mechanism of membrane docking by the C2 domains of cPKCs in which the local densities of phosphatidylserine and PtdIns(4,5)P2 on the inner leaflet of the plasma membrane are sufficient to drive Ca2+-activated membrane docking during a physiological Ca2+ signal.
Trafficking of ATP-sensitive potassium channels in health and disease
Authors: Sivaprasadarao A. , Taneja T.K. , Mankouri J. , Smith A.J.
Published: November 2007

KATP channels (ATP-sensitive potassium channels), comprising four subunits each of Kir6.2 (inwardly rectifying potassium channel 6.2) and the SUR1 (sulfonylurea receptor 1), play a central role in glucose-stimulated insulin secretion by the pancreatic β-cell. Changes in the number of channels at the cell surface are associated with genetic diseases of aberrant insulin secretion, including CHI (congenital hyperinsulinism) and NDM (neonatal diabetes mellitus). The present review summarizes advances in our understanding of the vesicular trafficking of normal KATP channels and how genetic mutations in Kir6.2 interfere with such trafficking. A mutation, E282K, causing CHI, was found to disrupt a DXE [di-acidic ER (endoplasmic reticulum)-exit signal], thereby preventing its assembly into COPII (coatamer protein II)-coated vesicles and subsequent ER exit. The resultant decrease in the cell-surface density of the channel could explain the disease phenotype. Two mutations, Y330C and F333I, reported in patients with NDM, disrupted an endocytic traffic signal, thereby impairing CCV (clathrin-coated vesicle) formation and endocytosis. The consequent increase in the density of KATP channels, together with an attenuated sensitivity to ATP reported previously, may account for the severe form of NDM.
hERG channel trafficking: novel targets in drug-induced long QT syndrome
Authors: Dennis A. , Wang L. , Wan X. , Ficker E.
Published: November 2007

The cardiac potassium channel hERG (human ether-a-go-go-related gene) encodes the α-subunit of the rapid delayed rectifier current IKr in the heart, which contributes to terminal repolarization in human cardiomyocytes. Direct block of hERG/IKr channels by a large number of therapeutic compounds produces acLQTS [acquired LQTS (long QT syndrome)] characterized by drug-induced QT prolongation and torsades de pointes arrhythmias. The cardiotoxicity associated with unintended hERG block has prompted pharmaceutical companies to screen developmental compounds for hERG blockade and made hERG a major target in drug safety programmes. More recently, a novel form of acLQTS has been discovered that may go undetected in most conventional safety assays. Several therapeutic compounds have been identified that reduce hERG/IKr currents not by direct block but by inhibition of hERG/IKr trafficking to the cell surface. Important examples are antineoplastic Hsp90 (heat-shock protein 90) inhibitors such as (i) geldanamycin, (ii) the leukaemia drug arsenic trioxide, (iii) the antiprotozoical pentamidine, (iv) probucol, a cholesterol-lowering drug, and (v) fluoxetine, a widely used antidepressant. Increased awareness of drug-induced hERG trafficking defects will help to further reduce the potentially lethal adverse cardiac events associated with acLQTS.
Dynamic regulation of the voltage-gated Kv2.1 potassium channel by multisite phosphorylation
Authors: Mohapatra D.P. , Park K.-S. , Trimmer J.S.
Published: November 2007

Voltage-gated K+ channels are key regulators of neuronal excitability. The Kv2.1 voltage-gated K+ channel is the major delayed rectifier K+ channel expressed in most central neurons, where it exists as a highly phosphorylated protein. Kv2.1 plays a critical role in homoeostatic regulation of intrinsic neuronal excitability through its activity- and calcineurin-dependent dephosphorylation. Here, we review studies leading to the identification and functional characterization of in vivo Kv2.1 phosphorylation sites, a subset of which contribute to graded modulation of voltage-dependent gating. These findings show that distinct developmental-, cell- and state-specific regulation of phosphorylation at specific sites confers a diversity of functions on Kv2.1 that is critical to its role as a regulator of intrinsic neuronal excitability.
Localization and trafficking of cardiac voltage-gated potassium channels
Authors: Steele D.F. , Zadeh A. Dehghani , Loewen M.E. , Fedida D.
Published: November 2007

The proper trafficking and localization of cardiac potassium channels is profoundly important to the regulation of the regionally distinct action potentials across the myocardium. These processes are only beginning to be unravelled and involve modulators of channel synthesis and assembly, post-translational processing, various molecular motors and an increasing number of modifying enzymes and molecular anchors. The roles of anchoring proteins, molecular motors and kinases are explored and recent findings on channel internalization and trafficking are presented.
Ion channels in epilepsy
Authors: Mizielinska S.M.
Published: November 2007

Neuronal excitability is determined by the flux of ions through ion channels. Many types of ion channels are expressed in the central nervous system, each responsible for its own aspect of neuronal excitability, from postsynaptic depolarization to action potential generation to neurotransmitter release. These mechanisms are tightly regulated to create a balance between excitation and inhibition. Disruption of this balance is thought to be key in many neurological disorders, including epilepsy syndromes. More and more ion channel mutations are being identified through genetic studies; however, their incidence is still small, suggesting the presence of undiscovered mutations or other causative mechanisms. Understanding wild-type channel function during epileptic activity may also provide vital insights into the remaining idiopathic epilepsies and provide targets for future antiepileptic drugs.
Voltage clamp fluorimetry studies of mammalian voltage-gated K+ channel gating
Authors: Claydon T.W. , Fedida D.
Published: November 2007

VCF (voltage clamp fluorimetry) provides a powerful technique to observe real-time conformational changes that are associated with ion channel gating. The present review highlights the insights such experiments have provided in understanding Kv (voltage-gated potassium) channel gating, with particular emphasis on the study of mammalian Kv1 channels. Further applications of VCF that would contribute to our understanding of the modulation of Kv channels in health and disease are also discussed.