bluuebunny/crossref_metadata_2025 huggingface dataset:
Clustering of death receptors in lipid rafts initiates neutrophil spontaneous apoptosis
Authors: Scheel-Toellner D. , Wang K. , Assi L.K. , Webb P.R. , Craddock R.M. , Salmon M. , Lord J.M.
Published: October 2004

Neutrophils die by apoptosis spontaneously within 12–24 h of their release from the bone marrow. The mechanism regulating entry of neutrophils into apoptosis at the end of their life-span is currently under debate. Our data suggest that neutrophil apoptosis involves a novel mechanism of caspase 8 activation that is indirectly regulated by accumulation of reactive oxygen species. We detected early activation of caspase 8 upstream of caspase 3 activation, suggesting death receptor signalling. The CD95 DISC (death-inducing signalling complex) was detected in neutrophils, but blocking antibodies to death receptors did not inhibit apoptosis, suggesting a novel mechanism for caspase 8 activation. Death receptor clustering in ceramide-rich lipid rafts is thought to be an early event in their signalling, so we investigated the role of ceramide generated by ASM (acid sphingomyelinase) in neutrophil apoptosis. Ceramide was generated early in neutrophil apoptosis, and ASM activity was required for neutrophil apoptosis. Moreover, neutrophil apoptosis was significantly delayed in ASM−/− mice compared with their wild-type littermates. CD95 DISC components were present in lipid rafts in neutrophils, and were progressively clustered in cultured neutrophils. Generation of ceramide was blocked by desferrioxamine, suggesting that hydroxyl radicals are important for the activation of ASM. This observation was in line with our earlier observation of a precipitous drop in reduced glutathione in the aging neutrophil.
Post-transcriptional processing of cellular RNAs in herpes simplex virus-infected cells
Authors: Taddeo B. , Esclatine A. , Roizman B.
Published: October 2004

In HSV-1 (herpes simplex virus 1)-infected cells, the UL41 gene product carried with the virion has been shown to mediate the degradation of mRNA, leading to the shut-off of cellular protein synthesis. Analysis of the RNAs accumulating in cells infected with HSV-1 revealed the accumulation of RNAs encoding numerous cellular proteins both associated with and independent of activation of the NF-κB (nuclear factor κB) pathway. Studies on the activation of NF-κB and the expression and fate of selected cellular transcripts revealed the following. (i) In HSV-1-infected cells, NF-κB is activated by activated protein kinase R. Furthermore, the blockade of NF-κB translocation by suppression of protein kinase R activation does not render the cell more susceptible to apoptosis induced by viral gene expression. (ii) A number of mRNA up-regulated in infected cells [e.g. IκBα (inhibitory κBα), the immediate-early response protein IEX-1 and c-fos] are partially degraded and not translated. The degradation is UL41-dependent and results in deadenylation, endonucleolytic cleavage and 3′–5′ degradation. The 5′-portion resulting from the endonucleolytic cleavage tends to linger in the infected cells. To date, the RNAs processed in this manner contained ARE (AU-rich elements) in their 3′-untranslated domains. RNAs lacking ARE were expressed and not degraded in this manner. (iii) Tristetraprolin and T-cell internal antigen-1, cellular proteins involved in the degradation of ARE-containing RNAs, are induced and activated in infected cells and tristetraprolin interacts physically with the UL41 protein.
Pleckstrin homology domains: not just for phosphoinositides
Authors: Lemmon M.A.
Published: October 2004

PH domains (pleckstrin homology domains) are the 11th most common domain in the human genome and are best known for their ability to target cellular membranes by binding specifically to phosphoinositides. Recent studies in yeast have shown that, in fact, this is a property of only a small fraction of the known PH domains. Most PH domains are not capable of independent membrane targeting, and those capable of doing so (approx. 33%) appear, most often, to require both phosphoinositide and non-phosphoinositide determinants for their subcellular localization. Several recent studies have suggested that small GTPases such as ARF family proteins play a role in defining PH domain localization. Some others have described a signalling role for PH domains in regulating small GTPases, although phosphoinositides may also play a role. These findings herald a change in our perspective of PH domain function, which will be significantly more diverse than previously supposed.
Lipid rafts clustering and signalling by listeriolysin O
Authors: Gekara N.O. , Weiss S.
Published: October 2004

Listeriolysin O, the major virulent determinant of Listeria monocytogenes, is known for forming pores on cholesterol-rich membranes. In the present study, we reveal its other facet, rafts clustering. By immunofluorescence microscopy, we show that the glycosylphosphatidylinositol-anchored proteins CD14 and CD24, which normally exhibit uniform distribution on J774 cells, undergo clustering upon treatment with LLO. The non-raft marker transferrin receptor is unaffected by such treatment. Rafts clustering might explain the induction of tyrosine phosphorylation observed on LLO-treated cells.
N-glycosylated catalytic unit meets O-glycosylated propeptide: complex protein architecture in a fungal hexosaminidase
Authors: Plíhal O. , Sklenář J. , Kmoníčková J. , Man P. , Pompach P. , Havlíček V. , Křen V. , Bezouška K.
Published: October 2004

β-N-Acetylhexosaminidase from a filamentous fungus Aspergillus oryzae is a secreted enzyme known to be an important component of the binary chitinolytic system. Cloning of the hexA gene and sequencing of the enzyme revealed its unique preproprotein structure. While the enzyme's zincin-like and catalytic domain had significant similarities with members of the glycohydrolase 20 family, the propeptide was unique for the fungal enzyme. Detailed pulse–chase and inhibition studies revealed that propeptide was processed during the biosynthesis of the enzyme. Moreover, the presence of propeptide was necessary for enzyme activation, dimerization and secretion. The catalytic unit was N-glycosylated, and the propeptide was O-glycosylated, both in their C-terminal parts. Deglycosylation experiments revealed that the N-glycosylation increased the stability and solubility of the enzyme. In contrast, O-glycosylated propeptide was necessary to attain the full enzymic activity.
Molecular mechanisms in clathrin-mediated membrane budding revealed through subcellular proteomics
Authors: Ritter B. , Blondeau F. , Denisov A.Yu. , Gehring K. , McPherson P.S.
Published: October 2004

Subcellular proteomics is a powerful new approach that combines subcellular fractionation and MS (mass spectrometry) to identify the protein complement of cellular compartments. The approach has been applied to isolated organelles and major suborganellar structures and each study has identified known proteins not previously understood to associate with the compartment and novel proteins that had been described only as predicted open-reading frames from genome sequencing data. We have utilized subcellular proteomics to analyse the protein components of CCVs (clathrin-coated vesicles) isolated from adult brain. Accounting for identified fragmented peptides allows for a quantitative assessment of protein complexes associated with CCVs, and the identification of many of the known components of post-fusion synaptic vesicles demonstrates that a main function for brain CCVs is to recycle synaptic vesicles. In addition, we have identified a number of novel proteins that participate in CCV formation and function at the trans-Golgi network and the plasma membrane. Characterization of two of these proteins, NECAP1 and NECAP2, has led to the identification of a new consensus motif that mediates protein interactions with the clathrin adaptor protein 2. These studies highlight the ability of proteomics to reveal new insights into the mechanisms and functional roles of subcellular compartments.
Moving proteins from the cytosol into mitochondria
Authors: Chacinska A. , Rehling P.
Published: October 2004

Mitochondria of the yeast Saccharomyces cerevisiae contain at least 750 different proteins, which perform diverse roles. Most of these proteins (approx. 99%) are translated on cytosolic ribosomes, and their import into mitochondria is essential for mitochondrial function. Proteinaceous machineries of great complexity, the so-called translocases, in the mitochondrial membranes mediate the import of these proteins.
The role of SHIP1 in macrophage programming and activation
Authors: Rauh M.J. , Sly L.M. , Kalesnikoff J. , Hughes M.R. , Cao L.-P. , Lam V. , Krystal G.
Published: October 2004

The SHIP1 (SH2-containing inositol-5′-phosphatase 1) acts as a negative regulator of proliferation, survival and end cell activation in haemopoietic cells. It does so, at least in part, by translocating to membranes after extracellular stimulation and hydrolysing the phosphoinositide 3-kinase-generated second messenger, PtdIns(3,4,5)P3 to PtdIns(3,4)P2. SHIP1−/− mice have, as a result, an increased number of neutrophils and monocyte/macrophages because their progenitors display enhanced survival and proliferation. These mice also suffer from osteoporosis because of an increased number of hyperactive osteoclasts and a significant neutrophil infiltration of the lungs. Interestingly, SHIP1−/− mice do not display endotoxin tolerance and we have found that lipopolysaccharide-induced endotoxin tolerance is contingent on up-regulating SHIP1, through the production of autocrine-acting transforming growth factor-β, in bone-marrow-derived macrophages and mast cells. Intriguingly, unlike bone-marrow-derived macrophages, SHIP1−/− peritoneal and alveolar macrophages produce 10-fold less NO than wild-type macrophages because these in vivo-generated macrophages have very high arginase I levels and this enzyme competes with inducible nitric oxide synthase for the substrate L-arginine. It is probable that, in the face of chronically increased PtdIns(3,4,5)P3 levels in their myeloid progenitors, SHIP1−/− mice display a skewed development away from M1 (killer) macrophages (which have high inducible nitric oxide synthase levels and produce NO to kill microorganisms and tumour cells), towards M2 (healing) macrophages (which have high arginase levels and produce ornithine to promote host-cell growth and collagen formation). This skewing probably occurs to avoid septic shock and suggests that the phosphoinositide 3-kinase pathway plays a critical role in programming macrophages.
Glycogen synthase kinase 3β and the regulation of axon growth
Authors: Goold R.G. , Gordon-Weeks P.R.
Published: October 2004

One of the earliest hallmarks that distinguish growing axons from dendrites is their growth rate; axons grow faster than dendrites. In vertebrates, where axons are required to grow for considerable distances, particularly in the peripheral nervous system, a fast axon growth rate is a requisite property. In neurons that respond to the neurotrophin growth factor/nerve growth factor with increased axon growth rates, two distinct intracellular signalling pathways are recruited: the MAPK (mitogen-activated protein kinase) pathway and the phosphatidylinositol-3 kinase pathway. The activation of either pathway leads to changes in microtubule dynamics within growing axons and growth cones and these underlie fast axon growth rates. Microtubule dynamics is regulated by microtubule-associated proteins and in the MAPK pathway this function is subserved by microtubule-associated protein 1B, whereas in the phosphatidylinositol-3 kinase pathway, adenomatous polyposis coli is the regulating microtubule-associated protein.
Mechanisms of integration of cells and extracellular matrices by integrins
Authors: Humphries M.J. , Travis M.A. , Clark K. , Mould A.P.
Published: October 2004

While it is self-evident that all extracellular molecules are an integral part of a multicellular organism, it is paradoxical that they are often considered to be dissociated from cells. The reality is that a continuum of dynamic, bi-directional interactions links the intracellular environment through cell-surface receptors to multimolecular extracellular assemblies. These interactions not only control the behaviour of individual cells, but also determine tissue architecture. Adhesion receptor function is partly determined by an ability to tether the contractile cytoskeleton to the plasma membrane, but there is also evidence that integrin receptors modulate signalling events that are essential for cellular differentiation. A major challenge is now to integrate work at the atomic, molecular and cellular levels, and obtain holistic insights into the mechanisms controlling cell adhesion. In the present study, we review current knowledge of the molecular mechanisms employed by cells to integrate with the extracellular matrix. Two main topics are covered: the adaptation of integrin structure for bi-directional signalling and the integration of integrin signalling with other receptors.
Regulation of cellular processes by PPARγ ligands in neuroblastoma cells is modulated by the level of retinoblastoma protein expression
Authors: Emmans V.C. , Rodway H.A. , Hunt A.N. , Lillycrop K.A.
Published: October 2004

Neuroblastoma is a childhood cancer, which spontaneously regresses. This has led to a search for agents that mimic this process. We show that both natural and synthetic ligands of PPARγ (peroxisome-proliferator-activated receptor γ) inhibit the growth of neuroblastoma cells in vitro. The degree of PPAR activation was attenuated however in the presence of the retinoblastoma protein. Addition of trichostatin A, a histone deacetylase inhibitor, abolished retinoblastoma protein repression of PPAR activity. Moreover, enhanced growth inhibition was observed when neuroblastoma cells were treated with a PPARγ ligand and a histone deacetylase inhibitor, suggesting a combination therapy to treat neuroblastoma might prove more effective than using either agent alone.
Heterodimers and family-B GPCRs: RAMPs, CGRP and adrenomedullin
Authors: Conner A.C. , Simms J. , Hay D.L. , Mahmoud K. , Howitt S.G. , Wheatley M. , Poyner D.R.
Published: October 2004

RAMPs (receptor activity-modifying proteins) are single-pass transmembrane proteins that associate with certain family-B GPCRs (G-protein-coupled receptors). Specifically for the CT (calcitonin) receptor-like receptor and the CT receptor, this results in profound changes in ligand binding and receptor pharmacology, allowing the generation of six distinct receptors with preferences for CGRP (CT gene-related peptide), adrenomedullin, amylin and CT. There are three RAMPs: RAMP1–RAMP3. The N-terminus appears to be the main determinant of receptor pharmacology, whereas the transmembrane domain contributes to association of the RAMP with the GPCR. The N-terminus of all members of the RAMP family probably contains two disulphide bonds; a potential third disulphide is found in RAMP1 and RAMP3. The N-terminus appears to be in close proximity to the ligand and plays a key role in its binding, either directly or indirectly. BIBN4096BS, a CGRP antagonist, targets RAMP1 and this gives the compound very high selectivity for the human CGRP1 receptor.
Dimerization of α1-adrenoceptors
Authors: Milligan G. , Pediani J. , Fidock M. , López-Giménez J.F.
Published: October 2004

Three distinct genes encode α1-adrenoceptors. Although homodimers of each subtype have been reported, certain but not all combinations of heterodimers of the α1-adrenoceptors appear to form. Key studies in this field are reviewed and the approaches that have been applied to monitoring the selectivity and the basis of α1-adrenoceptor dimerization are discussed.
GPCR-interacting proteins (GIPs): nature and functions
Authors: Bockaert J. , Roussignol G. , Bécamel C. , Gavarini S. , Joubert L. , Dumuis A. , Fagni L. , Marin P.
Published: October 2004

The simplistic idea that seven transmembrane receptors are single monomeric proteins that interact with heterotrimeric G-proteins after agonist binding is definitively out of date. Indeed, GPCRs (G-protein-coupled receptors) are part of multiprotein networks organized around scaffolding proteins. These GIPs (GPCR-interacting proteins) are either transmembrane or cytosolic proteins. Proteomic approaches can be used to get global pictures of these ‘receptosomes’. This approach allowed us to identify direct but also indirect binding partners of serotonin receptors. GIPs are involved in a wide range of functions including control of the targeting, trafficking and signalling of GPCRs. One of them, Shank, which is a secondary and tertiary partner of metabotropic and ionotropic glutamate receptors, respectively, can induce the formation of a whole functional glutamate ‘receptosome’ and the structure to which it is associated, the dendritic spine.
Molecular mechanisms of GABAB receptor activation: new insights from the mechanism of action of CGP7930, a positive allosteric modulator
Authors: Binet V. , Goudet C. , Brajon C. , Corre L. Le , Acher F. , Pin J.-P. , Prézeau L.
Published: October 2004

The GABAB (γ-aminobutyric acid-B) receptor is composed of two subunits, GABAB1 and GABAB2. Both subunits share structural homology with other class-III G-protein-coupled receptors. They contain two main domains, a heptahelical domain typical of all G-protein-coupled receptors and a large ECD (extracellular domain). It has not been demonstrated whether the association of these two subunits is always required for function. However, GABAB2 plays a major role in coupling with G-proteins, and GABAB1 has been shown to bind GABA. To date, only ligands interacting with GABAB1-ECD have been identified. In the present study, we explored the mechanism of action of CGP7930, a compound described as a positive allosteric regulator of the GABAB receptor. We have shown that it can weakly activate the wild-type GABAB receptor, but also the GABAB2 expressed alone, thus being the first described agonist of GABAB2. CGP7930 retains its weak agonist activity on a GABAB2 subunit deleted of its ECD. Thus the heptahelical domain of GABAB2 behaves similar to a rhodopsin-like receptor. These results open new strategies for studying the mechanism of activation of GABAB receptor and examine any possible role of GABAB2.
Structure and chemistry of the Sir2 family of NAD+-dependent histone/protein deactylases
Authors: Marmorstein R.
Published: October 2004

The yeast Sir2 (silent information regulator-2) protein functions as an NAD+-dependent histone deacetylase to silence gene expression from the mating-type locus, tolomeres and rDNA and also promotes longevity and genome stability in response to calorie restriction. Homologues of yeast Sir2 have been identified in the three domains of bacteria, archaea and eukaryotes; in mammalian cells, Sir2 proteins also deacetylate non-histone proteins such as the p53 tumour suppressor protein, α-tubulin and forkhead transcription factors to mediate diverse biological processes including metabolism, cell motility and cancer. We have determined the X-ray crystal structure of a Sir2 homologue from yeast Hst2 (yHst2), in various liganded forms, including the yHst2/acetyl-Lys-16 histone H4/NAD+ ternary complex; we have also performed related biochemical studies to address the conserved mode of catalysis by these enzymes as well as the distinguishing features that allow different members of the family to target their respective cognate substrates. These studies have implications for the structure-based design of Sir2-specific small molecule compounds, which might modulate Sir2 function for therapeutic application.
RNA interference and double-stranded-RNA-activated pathways
Authors: Sledz C.A. , Williams B.R.G.
Published: October 2004

RNAi (RNA interference) has become a powerful tool to determine gene function. Different methods of expressing the short ds (double-stranded) RNA intermediates required for interference in mammalian systems have been developed, including the introduction of si (short interfering) RNAs by direct transfection or driven from transfected plasmids or lentiviral vectors encoding sh (short hairpin) RNAs. Although RNAi relies upon a high degree of specificity, recent findings suggest that off-target non-specific effects can be encountered. We found that transfection of siRNAs can results in an interferon-mediated activation of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathway and global up-regulation of interferon-stimulated genes. This effect is mediated in part by the dsRNA-dependent protein kinase PKR, as this kinase is activated by the 21 bp siRNA, and is required in response to the siRNAs. However, the transcription factor IRF3 (interferon-regulatory factor 3) is also activated by siRNA as a primary response, resulting in the stimulation of genes independent of an interferon response. In cells deficient in IRF3, this response is blunted, but can be restored by re-introduction of IRF3. Thus siRNAs induce complex signalling responses in target cells, leading to effects beyond the selective silencing of specific genes.
ArabidopsisDNA double-strand break repair pathways
Authors: West C.E. , Waterworth W.M. , Sunderland P.A. , Bray C.M.
Published: October 2004

DSBs (double-strand breaks) are one of the most serious forms of DNA damage that can occur in a cell's genome. DNA replication in cells containing DSBs, or following incorrect repair, may result in the loss of large amounts of genetic material, aneuploid daughter cells and cell death. There are two major pathways for DSB repair: HR (homologous recombination) uses an intact copy of the damaged region as a template for repair, whereas NHEJ (non-homologous end-joining) rejoins DNA ends independently of DNA sequence. In most plants, NHEJ is the predominant DSB repair pathway. Previously, the Arabidopsis NHEJ mutant atku80 was isolated and found to display hypersensitivity to bleomycin, a drug that causes DSBs in DNA. In the present study, the transcript profiles of wild-type and atku80 mutant plants grown in the presence and absence of bleomycin are determined by microarray analysis. Several genes displayed very strong transcriptional induction specifically in response to DNA damage, including the characterized DSB repair genes AtRAD51 and AtBRCA1. These results identify novel candidate genes that encode components of the DSB repair pathways active in NHEJ mutant plants.
Cell cycle-regulated transcription in fission yeast
Authors: McInerny C.J.
Published: October 2004

A fundamental process in biology is the mechanism by which cells duplicate and divide to produce two identical daughter cells. The fission yeast, Schizosaccharomyces pombe, has proved to be an excellent model organism to study the role that gene expression plays in this process. The basic paradigm emerging is that a number of groups of genes are expressed in successive waves at different cell cycle times. Transcription of a particular group is controlled by a common DNA motif present in each gene's promoter, bound by a transcription factor complex. Each motif and transcription factor complex is specific to the time in the cell cycle when the group of genes is expressed. Examples of this are the MBF (MCB-binding factor)/MCB (MluI cell cycle box) system controlling gene expression at the start of S-phase, and PBF (PCB-binding factor)/PCB (Pombe cell cycle box) regulation of transcription at the end of mitosis. In some cases, these transcription control systems also operate during the alternative form of cell division, meiosis.
Vitamin A regulates proliferation and apoptosis of human T- and B-cells
Authors: Blomhoff H.K.
Published: October 2004

Vitamin A is known to protect against infections, but it is not established how vitamin A metabolites stimulate the immune system. We have investigated the effects of physiological levels of retinoic acid on the function of normal human T- and B-cells. Surprisingly, we found that the proliferation of B-cells was inhibited by retinoids, and that this was due to rapid inhibition of the cell cycle machinery regulating G1-to-S transition. In contrast, the proliferation of T-cells was enhanced by physiological levels of retinoic acid, and the effect was due to induction of IL-2 (interleukin 2). The ‘non-death-receptor’-mediated apoptosis of normal T-cells induced by prolonged (but single) stimulation of the cells was also prevented by retinoid acid, and also this effect was mediated via enhanced production of IL-2. The induction of IL-2 was at the transcriptional level, and all the effects of vitamin A on both B-and T-cells were mediated via the nuclear retinoic acid receptors (RARs), and not retinoid X receptors (RXRs).
Dietary lipids and gene expression
Authors: Roche H.M.
Published: October 2004

Nutrition is a key environmental factor that is particularly involved in the pathogenesis and progression of several polygenic, diet-related diseases. Nutrigenomics refers to the interaction between nutrition and the human genome. Dietary fatty acids interact with multiple nutrient-sensitive transcription factors. This explains the molecular basis of some of the health effects associated with altered dietary fatty acid composition. The metabolic syndrome is a very common condition, characterized by insulin resistance, abdominal obesity, dyslipidaemia and hypertension. It often precedes Type 2 diabetes mellitus, and is associated with a greater risk of cardiovascular disease. Several lines of evidence suggest that the interaction between nutrient-derived metabolic stressors and pro-inflammatory signals play an important role in the aetiology of insulin resistance and the development of the metabolic syndrome. This paper will address the interaction between several nutrient-sensitive transcription factors, including SREBP (sterol-regulatory-element-binding protein) and NFκB (nuclear factor κB), demonstrating how this interaction may be altered with dietary fatty acid interventions.
Folate and DNA methylation during in utero development and aging
Authors: McKay J.A. , Williams E.A. , Mathers J.C.
Published: October 2004

DNA methylation is one of several epigenetic mechanisms that play a regulatory role in genome programming and imprinting during embryogenesis. Aberrant DNA methylation has been implicated in the pathogenesis of a number of diseases associated with aging, including cancer and cardiovascular and neurological diseases. Evidence is accumulating that dietary factors in utero modulate disease risk in later life. Although folic acid is a key component of DNA methylation, the impact of folic acid availability in utero on DNA methylation patterns and disease risk in adulthood is at present poorly characterized. This review describes the relationship between folic acid and DNA methylation, and the association between DNA methylation during in utero development and aging.
Oligomeric assembly of dopamine D1 and glutamate NMDA receptors: molecular mechanisms and functional implications
Authors: Fiorentini C. , Missale C.
Published: October 2004

In the striatum, dopamine D1R (D1 receptor) activation potentiates NMDA (N-methyl-D-aspartate) transmission and is required for NMDA-mediated long-term potentiation at corticostriatal synapses. By using a combination of co-immunoprecipitation, pull-out with glutathione S-transferase-fusion proteins and bioluminescence resonance energy transfer, we have reported that the D1R forms a heteromeric complex with the NMDAR (NMDA receptor) and that this mechanism is crucial to recruit the D1R to the postsynaptic density. By using confocal and radioligand-binding assay, we also demonstrated that the interaction with NMDAR abolishes agonist-mediated D1R sequestration, indicating that oligomerization with NMDAR could represent a novel regulatory mechanism modulating D1R cellular trafficking and desensitization.
The third extracellular loop of G-protein-coupled receptors: more than just a linker between two important transmembrane helices
Authors: Lawson Z. , Wheatley M.
Published: October 2004

GPCRs (G-protein-coupled receptors) are a large family of structurally related proteins, which mediate their effects by coupling with G-proteins. Despite responding to a range of very diverse stimuli, these receptors exhibit a conserved tertiary structure comprising a bundle of seven TM (transmembrane) helices linked by alternating ECLs (extracellular loops) and ICLs (intracellular loops). The hydrophobic environment formed by the cluster of TM helices is functionally important. For example, the 11-cis retinal chromophore of rhodopsin forms a protonated Schiff base linkage to a lysine in TM7, deep within the helical bundle, and small ligands, such as amine neurotransmitters and non-peptide analogues of peptide hormones, also bind within the corresponding region of their cognate receptors. In addition, activation of GPCRs involves relative movement of TM helices to present G-protein interaction sites across the intracellular face of the receptor. Consequently, it might be assumed that the ECLs of the GPCR are inert peptide linkers that merely connect important TM helices. Focusing on ECL3 (third ECL), it is becoming increasingly apparent that this extracellular domain can fulfil a range of important roles with respect to GPCR signalling, including agonist binding, ligand selectivity and receptor activation.
Lipids in health and disease
Authors: Shepherd J.
Published: October 2004

The evidence linking cholesterol levels in the blood to vascular risk is now incontrovertible and the introduction of HMG CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitor (or statin) therapy into clinical practice has now revolutionized the management of lipid disorders and silenced at a stroke the critics of cholesterol control as a means to vascular disease prevention. Statins were the first lipid-lowering agents, which, within a framework of a clinical trial, actually extended life by mechanisms that probably go beyond cholesterol alone. Their benefits are so impressive that some enthusiasts have been emboldened to write that they ‘are to atherosclerosis what penicillin was to infectious disease’. But is Nature as easily tamed as we might imagine? Some individuals show a modest or even poor response to statin therapy. The recent discovery of ezetimibe, a highly efficient and precise cholesterol absorption inhibitor, has proven to be a very effective cholesterol lowering alternative for them and combining statins with ezetimibe, thereby inhibiting cholesterol absorption and endogenous synthesis, takes us to realms of cholesterol lowering capability that could not have been dreamt of a decade ago.
Use of mass spectrometry-based lipidomics to probe PITPα (phosphatidylinositol transfer protein α) function inside the nuclei of PITPα+/+ and PITPα−/− cells
Authors: Hunt A.N. , Alb J.G. , Koster G. , Postle A.D. , Bankaitis V.A.
Published: October 2004

The mammalian phospholipid exchange protein PITPα (phosphatidylinositol transfer protein alpha), found in both extranuclear and endonuclear compartments, is thought in part to facilitate nuclear import of the PtdIns (phosphatidylinositol) consumed in the generation of proliferation-associated endonuclear diacylglycerol accumulations. Unlike phosphatidylcholine, endonuclear PtdIns is not synthesized in situ. However, despite progressive postnatal lethality of PITPα ablation in mice, PITPα−/− MEF (mouse embryonic fibroblasts) lack an obviously impaired proliferative capacity. We used ESI-MS (tandem electrospray ionization-MS) to monitor incorporation of the deuterated phospholipid precursors, choline-d9 and inositol-d6, into molecular species of whole cell and endonuclear phosphatidylcholine and PtdIns over 24 h to assess the contribution of PITPα to the nuclear import of PtdIns into MEF cells. In cells labelled for 1, 3, 6, 12 and 24 h fractional inositol-d6 incorporation into whole-cell PtdIns species was consistently higher in PITPα−/− MEF implying greater flux through its biosynthetic pathway. Moreover, endonuclear accumulation of PtdIns-d6 was apparent in the PITPα−/− cells and mirrored that in PITPα+/+ cells. Together, these results suggest that the essential endonuclear PtdIns import via PITPα can be accommodated by other mechanisms.
Immunodetection of UCP1 in rat thymocytes
Authors: Carroll A.M. , Haines L.R. , Pearson T.W. , Brennan C. , Breen E.P. , Porter R.K.
Published: October 2004

Thymi were dissected from rats and connective tissue was removed. Mitochondria were purified from isolated thymocytes and immunoblot analysis was performed using an antibody specific for uncoupling protein 1, which detected a 32.5 kDa protein associated with mitochondria from the thymocytes. This implies that rat thymocytes contain uncoupling protein 1.
Nitric oxide, hypoxia and brain inflammation
Authors: Mander P. , Brown G.C.
Published: October 2004

NO (nitric oxide) acutely and potently inhibits mitochondrial cytochrome oxidase in competition with oxygen, thereby raising the apparent KM for oxygen of mitochondria and neurons into the physiological or pathological range. We find that NO from an NO donor or glial inducible NOS (nitric oxide synthase) highly sensitizes neurons to hypoxia-induced death, probably via the NO–oxygen competition at cytochrome oxidase. Thus the NO from neuronal NOS during excitotoxicity or the NO from inducible NOS during inflammation may sensitize the brain to hypoxic/ischaemic damage.
Cell-penetrating peptides are excluded from the mitochondrial matrix
Authors: Ross M.F. , Murphy M.P.
Published: October 2004

CPPs (cell-penetrating peptides) facilitate cellular uptake of covalently attached macromolecules, through an as yet controversial mechanism that either involves direct membrane passage or a type of endocytosis. We investigated the potential of the CPPs penetratin and Tat to act as mitochondria-targeting vectors by testing whether they were internalized by isolated mitochondria, and by mitochondria within cells in culture. We also tested peptides conjugated to the mitochondria-targeting moiety triphenylphosphonium. We found no evidence for mitochondrial uptake by penetratin, Tat or their triphenylphosphonium conjugates. This result suggests that CPPs are unsuitable as mitochondria-targeting vectors, and implies an endocytic mode of cellular uptake for CPPs.
Epigenetic approaches to cancer therapy
Authors: Plumb J.A. , Steele N. , Finn P.W. , Brown R.
Published: October 2004

Histone deacetylation and DNA methylation have a central role in the control of gene expression, including transcriptional repression of tumour suppressor genes. Loss of DNA mismatch repair due to methylation of the hMLH1 gene promoter results in resistance to cisplatin in vitro and in vivo. The cisplatin-resistant cell line A2780/cp70 is 8-fold more resistant to cisplatin than the non-resistant cell line, and has the hMLH1 gene methylated. Treatment with an inhibitor of DNA methyltransferase, DAC (2-deoxy-5′-azacytidine), results in a partial reversal of DNA methylation, re-expression of MLH1 (mutL homologue 1) and sensitization to cisplatin both in vitro and in vivo. PXD101 is a novel hydroxamate type histone deacetylase inhibitor that shows antitumour activity in vivo and is currently in phase I clinical evaluation. Treatment of A2780/cp70 tumour-bearing mice with DAC followed by PXD101 results in a marked increase in the number of cells that re-express MLH1. Since the clinical use of DAC may be limited by toxicity and eventual re-methylation of genes, we suggest that the combination of DAC and PXD101 could have a role in increasing the efficacy of chemotherapy in patients with tumours that lack MLH1 expression due to hMLH1 gene promoter methylation.
The role of TFIIB conformation in transcriptional regulation
Authors: Elsby L.M. , Roberts S.G.E.
Published: October 2004

Transcription by RNA polymerase II requires the assembly of the general transcription factors at the promoter to form a preinitiaiton complex. TFIIB (transcription factor IIB) plays a central role in this process, mediating the recruitment of RNA polymerase II and positioning it over the transcription start site. The assembly of TFIIB at the promoter can be a limiting event and several activator proteins have been shown to target TFIIB recruitment in the process of transcriptional stimulation. TFIIB is composed of two domains that engage in an intramolecular interaction. Indeed, the conformation of TFIIB has been found to underpin the function of this general transcription factor. Here we discuss our current understanding of TFIIB conformation and its role in transcription control.
What is in a filopodium? Starfish versus hedgehogs
Authors: Passey S. , Pellegrin S. , Mellor H.
Published: October 2004

Many cell types can generate thin actin-based protrusive structures, which are often classified under the general term of ‘filopodia’. However, a range of filopodia-like structures exists that differ both morphologically and functionally. In this brief review, we discuss the different types of filopodial structures, together with the actin-binding proteins and signalling pathways involved in their formation. Specifically, we highlight the differences between the filopodial extensions induced by the Rho GTPases Cdc42 and Rif.
Annexin A2 expression during cellular differentiation in myeloid cell lines
Authors: Gilmore W.S. , Olwill S. , McGlynn H. , Alexander H.D.
Published: October 2004

Annexin A2 is a calcium-dependent, phospholipid-binding protein found on many cell types. It consists of a short hydrophobic tail (Ser2-Asn32), which dictates its function, and a core domain (Phe33-Asp339), which is involved in phospholipid binding. Annexin A2 has been implicated in a number of biochemical processes, including cell proliferation, foetal immune tolerance, ion-channel activation, cell–cell interactions and the bridging of membranes. Annexin A2 is reported to be a powerful activator of plasminogen and, therefore, is implicated in many normal and pathological processes such as haemostasis and metastasis. Myeloid cell lines are used, extensively, to study many aspects of cellular proliferation, differentiation and function. In the present study, we have used flow cytometry and real-time PCR to investigate the role of annexin A2 expression in the proliferation and differentiation of a number of myeloid cell lines. The results demonstrated that annexin A2 expression was affected when the cells were induced to differentiate by stimulation with all-trans-retinoic acid. Annexin A2 may, therefore, be an important player in cellular differentiation and its disorders.
Immunoglobulin A1 proteases: a structure–function update
Authors: Parsons H.K. , Vitovski S. , Sayers J.R.
Published: October 2004

IgA1 (immunoglobulin A1) antibodies are the first line of defence against microbial pathogens such as Neisseria meningitidis and Haemophilus influenzae. However, these bacteria secrete a site-specific protease that is capable of cleaving human IgA1 and interacting with other host components. The IgA proteases are released by the type V secretion pathway, which involves translocation through two membranes and an autolytic, post-translational processing step. Results reported recently throw light on the type V secretion pathway and on the roles of the multifunctional IgA protease. The IgA1 protease-recognition sequence is present within the IgA1 hinge region as well as in the variable sequence connecting the IgA1 protease to its translocator domain. Recent results suggest that neisserial IgA1 proteases are capable of cleaving substrates lacking the classical recognition sequence. This review will cover recent advances in the IgA protease field.
Impact of the end of range damage from low energy Ge preamorphizing implants on the thermal stability of shallow boron profiles
Authors: Camillo-Castillo R. A. , Law M. E. , Jones K. S.
Published: October 2004

A fundamental understanding of the effect of scaling amorphous layers on the thermal stability of active concentrations is required for the formation of ultrashallow junctions. A study on the influence of boron on the evolution of the end of range defects for samples containing shallow amorphous layers formed by low energy germanium implants is conducted. Czochralski grown (100) silicon wafers are preamorphized with 1×1015cm−2, 10keV Ge+ and subsequently implanted with 1×1015cm−2, 1keV B+ such that high boron levels are attained in the end of range region. A sequence of anneals are performed at 750°C, under nitrogen ambient for times ranging from 1s to 6h and the end of range defect evolution is imaged via plan-view transmission electron microscopy (TEM). Defect analyses are conducted utilizing quantitative TEM which indicates substantial differences in the defect evolution for samples with boron in the end of range. The extended defects observed are very unstable and undergo a fast dissolution. In contrast, stable defects are observed in the experimental control in which the evolution follows an Ostwald ripening behavior. Secondary ion mass spectroscopy analyses confirm the ephemeral nature of the defects observed and also demonstrates drastic reductions in interstitial supersaturation. In addition, uphill-type diffusion is observed to occur for a short time frame, which emphasizes a transient interstitial supersaturation. Correlation of this data with sheet resistance and active dose measurements conducted on a Hall measurement system strongly indicates the formation of boron interstitial clusters. The high boron concentrations and supersaturation levels attained at the anneal temperature enables the cluster formation. An estimate of the boron concentrations trapped in the clusters is determined from the active dose obtained from the Hall measurements and indicates concentrations much higher than those available in the end of range. This suggests an interstitial migration from the end of range to regions of higher boron levels. Since the end of range is in the vicinity of the highly doped layer it is not isolated from the strain effects induced by the high initial activation levels. Hence it is proposed that the tensile strain stimulates the interstitial migration from the end of range to the boron-doped layer. Consequently, the end of range defects dissolve as the interstitial supersaturation falls below levels required to sustain their evolution.
Highly resolved quantized double-layer charging of relatively larger dodecanethiol-passivated gold quantum dots
Authors: Chaki Nirmalya K. , Kakade Bhalchandra , Sharma Jadab , Mahima Subhramannia , Vijayamohanan Kunjukrishna P. , Haram Santosh K.
Published: October 2004

Monolayer-protected quantum dots (Q-dots) show multivalent redox property, popularly known as the quantized double-layer (QDL) charging phenomena. In this report, we demonstrate the QDL behavior of the larger-sized Au Q-dots (ca.3.72nm) protected with dodecanethiol using differential pulse voltammetry (DPV) and cyclic voltammetry (CV). The voltammetric results show that the QDL property is evident even for these larger-sized Q-dots as reflected by a large population of well-resolved charging events in a narrow potential range with an almost equidistant voltage (ΔV) spacing. The theoretical calculation of the variation of charging energy with size using the well-known concentric sphere capacitance model facilitates the understanding of electrochemical behavior of these sidelined larger-sized Au Q-dots. The calculated capacitance value is in well agreement with the experimentally obtained value of 1.6aF.
Photorefractive effect and photochromism in the Fe-doped relaxor ferroelectric crystal Pb(Zn1∕3Nb2∕3)O3-PbTiO3
Authors: Sato Y. , Abe S. , Fujimura R. , Ono H. , Oda K. , Shimura T. , Kuroda K.
Published: October 2004

The photorefractive properties of the iron-doped Pb(Zn1∕3Nb2∕3)O3-PbTiO3 (PZN-PT) relaxor ferroelectric single crystals were investigated by two-beam coupling experiments. The Fe:0.01wt%- and the Fe:0.1wt%-doped 0.91PZN-0.09PT single crystals were grown with the flux solution method. The Extinction spectrum, the electro-opric coefficient, the light-induced absorption change, and the photoconductivity were measured in these samples. The maximum gain coefficient reached to 21cm−1, which is about twice as large as that of undoped PZN-PT. The grating formation rate decreases with the Fe concentration.
Calculations using the helical filamentary structure for current distributions of a six around one superconducting strand cable and a multifilamentary composite
Authors: Tominaka Toshiharu
Published: October 2004

The current and magnetic-field distributions within a twisted six around one (6-1) superconducting strand cable and a twisted simple superconducting multifilamentary composite have been studied with the assumption that the current distribution within each strand or each filament is uniform. For this purpose, the vector potential and the magnetic field due to a helical conductor with a circular cross section is calculated from the numerical integration of the analytical expression for a helical thin conductor. The current distributions among each strand or each filament are obtained by minimizing the magnetic energy for the case of the insulated strands or filaments, and by requiring zero magnetic flux enclosed between any pair of strands or filaments for the case of the noninsulated strands or filaments. For the case of the untwisted insulated strands or filaments, it is confirmed that the calculated results coincide with those due to the inductance. The screening circulation currents among strands within a multistrand cable or filaments within a composite are obtained as the difference between the current distributions for the case with the noninsulated strand or filament and for the case with the imaginarily insulated strand or filament. As a result, it is revealed that the negative current in the inner strands or filaments is a universal feature of the current distributions for superconducting multistrand cables and multifilamentary composites during the current sweep. Finally, the magnetic field distributions of a twisted 6-1 superconducting strand cable and a twisted simple superconducting multifilamentary composite have been obtained.
Improved three-dimensional optical model for thin-film silicon solar cells
Authors: Springer J. , Poruba A. , Vanecek M.
Published: October 2004

We present an optical model for thin-film silicon solar cells (both single and multijunction) with nanorough surfaces/interfaces. For these cells, the optical absorptance within each layer and the total reflectance are computed taking into account roughness, angular distribution of scattered light, thicknesses, and optical constants of all layers. In the model, we combine coherent approach, scattering theory, and Monte Carlo tracing method. Results of the model are shown to be in good agreement with the experimentally measured spectral response and the total reflectance of solar cells. Some predictions of the ultimate solar cell performance based on the model are presented as well.