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By admin, on April 17th, 2012
PLoS One. 2012; 7(4): e35373
Watts SD, Suchland KL, Amara SG, Ingram SL
Regulation of chloride gradients is a major mechanism by which excitability is regulated in neurons. Disruption of these gradients is implicated in various diseases, including cystic fibrosis, neuropathic pain and epilepsy. Relatively few studies have addressed chloride regulation in neuronal processes because probes capable of detecting changes in small compartments over a physiological range are limited.In this study, a palmitoylation sequence was added to a variant of the yellow fluorescent protein previously described as a sensitive chloride indicator (YFPQS) to target the protein to the plasma membrane (mbYFPQS) of cultured midbrain neurons. The reporter partitions to the cytoplasmic face of the cellular membranes, including the plasma membrane throughout the neurons and fluorescence is stable over 30-40 min of repeated excitation showing less than 10% decrease in mbYFPQS fluorescence compared to baseline. The mbYFPQS has similar chloride sensitivity (k(50) = 41 mM) but has a shifted pKa compared to the unpalmitoylated YFPQS variant (cytYFPQS) that remains in the cytoplasm when expressed in midbrain neurons. Changes in mbYFPQS fluorescence were induced by the GABA(A) agonist muscimol and were similar in the soma and processes of the midbrain neurons. Amphetamine also increased mbYFPQS fluorescence in a subpopulation of cultured midbrain neurons that was reversed by the selective dopamine transporter (DAT) inhibitor, GBR12909, indicating that mbYFPQS is sensitive enough to detect endogenous DAT activity in midbrain dopamine (DA) neurons.The mbYFPQS biosensor is a sensitive tool to study modulation of intracellular chloride levels in neuronal processes and is particularly advantageous for simultaneous whole-cell patch clamp and live-cell imaging experiments.
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A sensitive membrane-targeted biosensor for monitoring changes in intracellular chloride in neuronal processes.
By admin, on April 15th, 2012
J Biol Chem. 2012 Apr 12;
Ma X, Zhao Y, Daaka Y, Nie Z
lower case Greek beta2-Adrenergic receptors (lower case Greek beta2AR) regulate cellular functions through G protein-transduced and lower case Greek betaArrestin-transduced signals. lower case Greek beta2ARs have been shown to regulate cancer cell migration, but the underlying mechanisms are not well understood. Here, we report that lower case Greek beta2AR regulates formation of focal adhesions, whose dynamic remodeling is critical for directed cell migration. lower case Greek beta2AR induces activation of RhoA, which is dependent on lower case Greek betaArrestin2 but not Gs. lower case Greek betaArrestin2 forms a complex with p115RhoGEF, a guanine nucleotide exchange factor for RhoA that is well known to be activated by G12/13- coupled receptors. Our results show that lower case Greek betaArrestin2 forms a complex with p115RhoGEF in the cytosol in resting cells. Upon lower case Greek beta2AR activation, both lower case Greek betaArrestin2 and p115RhoGEF translocate to the plasma membrane, with concomitant activation of RhoA and formation of focal adhesions and stress fibers. Activation of RhoA and focal adhesion remodeling may explain, at least in part, the role of lower case Greek beta2ARs in cell migration. These results suggest that lower case Greek betaArrestin2 may serve as a convergence point for non-G12/13 and non-Gq protein-coupled receptors to activate RhoA.
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Acute activation of beta2-adrenergic receptor regulates focal adhesions through betaArrestin2- and p115RhoGEF-mediated activation of RhoA.
By admin, on April 15th, 2012
J Biol Chem. 2012 Apr 12;
Ma X, Zhao Y, Daaka Y, Nie Z
lower case Greek beta2-Adrenergic receptors (lower case Greek beta2AR) regulate cellular functions through G protein-transduced and lower case Greek betaArrestin-transduced signals. lower case Greek beta2ARs have been shown to regulate cancer cell migration, but the underlying mechanisms are not well understood. Here, we report that lower case Greek beta2AR regulates formation of focal adhesions, whose dynamic remodeling is critical for directed cell migration. lower case Greek beta2AR induces activation of RhoA, which is dependent on lower case Greek betaArrestin2 but not Gs. lower case Greek betaArrestin2 forms a complex with p115RhoGEF, a guanine nucleotide exchange factor for RhoA that is well known to be activated by G12/13- coupled receptors. Our results show that lower case Greek betaArrestin2 forms a complex with p115RhoGEF in the cytosol in resting cells. Upon lower case Greek beta2AR activation, both lower case Greek betaArrestin2 and p115RhoGEF translocate to the plasma membrane, with concomitant activation of RhoA and formation of focal adhesions and stress fibers. Activation of RhoA and focal adhesion remodeling may explain, at least in part, the role of lower case Greek beta2ARs in cell migration. These results suggest that lower case Greek betaArrestin2 may serve as a convergence point for non-G12/13 and non-Gq protein-coupled receptors to activate RhoA.
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Acute activation of beta2-adrenergic receptor regulates focal adhesions through betaArrestin2- and p115RhoGEF-mediated activation of RhoA.
By admin, on April 9th, 2012
Cell Commun Signal. 2012 Apr 3; 10(1): 9
Engels N, Yigit G, Emmerich CH, Czesnik D, Schild D, Wienands J
ABSTRACT: BACKGROUND: The latent membrane protein (LMP) 2A of Epstein-Barr virus (EBV) is expressed during different latency stages of EBV-infected B cells in which it triggers activation of cytoplasmic protein tyrosine kinases. Early studies revealed that an immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic N-terminus of LMP2A can trigger a transient increase of the cytosolic Ca2+ concentration similar to that observed in antigen-activated B cells when expressed as a chimeric transmembrane receptor. Even so, LMP2A was subsequently ascribed an inhibitory rather than an activating function because its expression seemed to partially inhibit B cell antigen receptor (BCR) signaling in EBV-transformed B cell lines. However, the analysis of LMP2A signaling has been hampered by the lack of cellular model systems in which LMP2A can be studied without the influence of other EBV-encoded factors. RESULTS: We have reanalyzed LMP2A signaling using B cells in which LMP2A is expressed in an inducible manner in the absence of any other EBV signaling protein. This allowed us for the first time to monitor LMP2A signaling in statu nascendi as it occurs during the EBV life cycle in vivo. We show that mere expression of LMP2A not only stimulated protein tyrosine kinases but also induced phospholipase C-gamma2-mediated Ca2+ oscillations followed by activation of the extracellular signal-regulated kinase (Erk) mitogen-activated protein kinase pathway and induction of the lytic EBV gene bzlf1. Furthermore, expression of the constitutively phosphorylated LMP2A ITAM modulated rather than inhibited BCR-induced Ca2+ mobilization. CONCLUSION: Our data establish that LMP2A expression has a function beyond the putative inhibition of the BCR by generating a ligand-independent cellular activation signal that may provide a molecular switch for different EBV life cycle stages and most probably contributes to EBV-associated lymphoproliferative disorders.
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Epstein-Barr virus LMP2A signalling in statu nascendi mimics a B cell antigen receptor-like activation signal.
By admin, on April 9th, 2012
Cell Commun Signal. 2012 Apr 3; 10(1): 9
Engels N, Yigit G, Emmerich CH, Czesnik D, Schild D, Wienands J
ABSTRACT: BACKGROUND: The latent membrane protein (LMP) 2A of Epstein-Barr virus (EBV) is expressed during different latency stages of EBV-infected B cells in which it triggers activation of cytoplasmic protein tyrosine kinases. Early studies revealed that an immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic N-terminus of LMP2A can trigger a transient increase of the cytosolic Ca2+ concentration similar to that observed in antigen-activated B cells when expressed as a chimeric transmembrane receptor. Even so, LMP2A was subsequently ascribed an inhibitory rather than an activating function because its expression seemed to partially inhibit B cell antigen receptor (BCR) signaling in EBV-transformed B cell lines. However, the analysis of LMP2A signaling has been hampered by the lack of cellular model systems in which LMP2A can be studied without the influence of other EBV-encoded factors. RESULTS: We have reanalyzed LMP2A signaling using B cells in which LMP2A is expressed in an inducible manner in the absence of any other EBV signaling protein. This allowed us for the first time to monitor LMP2A signaling in statu nascendi as it occurs during the EBV life cycle in vivo. We show that mere expression of LMP2A not only stimulated protein tyrosine kinases but also induced phospholipase C-gamma2-mediated Ca2+ oscillations followed by activation of the extracellular signal-regulated kinase (Erk) mitogen-activated protein kinase pathway and induction of the lytic EBV gene bzlf1. Furthermore, expression of the constitutively phosphorylated LMP2A ITAM modulated rather than inhibited BCR-induced Ca2+ mobilization. CONCLUSION: Our data establish that LMP2A expression has a function beyond the putative inhibition of the BCR by generating a ligand-independent cellular activation signal that may provide a molecular switch for different EBV life cycle stages and most probably contributes to EBV-associated lymphoproliferative disorders.
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Epstein-Barr virus LMP2A signalling in statu nascendi mimics a B cell antigen receptor-like activation signal.
By admin, on April 7th, 2012
Cell Calcium . 2012 Apr 3; Giorgi C, Baldassari F, Bononi A, Bonora M, De Marchi E, Marchi S, Missiroli S, Patergnani S, Rimessi A, Suski JM, Wieckowski MR, Pinton P Mitochondria are key decoding stations of the apoptotic process.
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Mitochondrial Ca(2+) and apoptosis.
By admin, on April 7th, 2012
Cell Calcium . 2012 Apr 3; Giorgi C, Baldassari F, Bononi A, Bonora M, De Marchi E, Marchi S, Missiroli S, Patergnani S, Rimessi A, Suski JM, Wieckowski MR, Pinton P Mitochondria are key decoding stations of the apoptotic process. In support of this view, a large body of experimental evidence has unambiguously revealed that, in addition to the well-established function of producing most of the cellular ATP, mitochondria play a fundamental role in triggering apoptotic cell death.
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Mitochondrial Ca(2+) and apoptosis.
By admin, on April 6th, 2012
PLoS One. 2012; 7(3): e34037
Lama A, Pané-Farré J, Chon T, Wiersma AM, Sit CS, Vederas JC, Hecker M, Nakano MM
Amicoumacin A exhibits strong antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), hence we sought to uncover its mechanism of action. Genome-wide transcriptome analysis of S. aureus COL in response to amicoumacin A showed alteration in transcription of genes specifying several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. The most highly induced gene was lrgA, encoding an antiholin-like product, which is induced in cells undergoing a collapse of Δψ. Consistent with the notion that LrgA modulates murein hydrolase activity, COL grown in the presence of amicoumacin A showed reduced autolysis, which was primarily caused by lower hydrolase activity. To gain further insight into the mechanism of action of amicoumacin A, a whole genome comparison of wild-type COL and amicoumacin A-resistant mutants isolated by a serial passage method was carried out. Single point mutations generating codon substitutions were uncovered in ksgA (encoding RNA dimethyltransferase), fusA (elongation factor G), dnaG (primase), lacD (tagatose 1,6-bisphosphate aldolase), and SACOL0611 (a putative glycosyl transferase). The codon substitutions in EF-G that cause amicoumacin A resistance and fusidic acid resistance reside in separate domains and do not bring about cross resistance. Taken together, these results suggest that amicoumacin A might cause perturbation of the cell membrane and lead to energy dissipation. Decreased rates of cellular metabolism including protein synthesis and DNA replication in resistant strains might allow cells to compensate for membrane dysfunction and thus increase cell survivability.
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Response of Methicillin-Resistant Staphylococcus aureus to Amicoumacin A.
By admin, on April 4th, 2012
PLoS One. 2012; 7(3): e33253
Forster S, Thumser AE, Hood SR, Plant N
Fluorescent tracer dyes represent an important class of sub-cellular probes and allow the examination of cellular processes in real-time with minimal impact upon these processes. Such tracer dyes are becoming increasingly used for the examination of membrane transport processes, as they are easy-to-use, cost effective probe substrates for a number of membrane protein transporters. Rhodamine 123, a member of the rhodamine family of flurone dyes, has been used to examine membrane transport by the ABCB1 gene product, MDR1. MDR1 is viewed as the archetypal drug transport protein, and is able to efflux a large number of clinically relevant drugs. In addition, ectopic activity of MDR1 has been associated with the development of multiple drug resistance phenotype, which results in a poor patient response to therapeutic intervention. It is thus important to be able to examine the potential for novel compounds to be MDR1 substrates. Given the increasing use rhodamine 123 as a tracer dye for MDR1, a full characterisation of its spectral properties in a range of in vitro assay-relevant media is warranted. Herein, we determine λmax for excitation and emission or rhodamine 123 and its metabolite rhodamine 110 in commonly used solvents and extraction buffers, demonstrating that fluorescence is highly dependent on the chemical environment: Optimal parameters are 1% (v/v) methanol in HBSS, with λex = 505 nm, λem = 525 nm. We characterise the uptake of rhodamine 123 into cells, via both passive and active processes, and demonstrate that this occurs primarily through OATP1A2-mediated facilitated transport at concentrations below 2 µM, and via micelle-mediated passive diffusion above this. Finally, we quantify the intracellular sequestration and metabolism of rhodamine 123, demonstrating that these are both cell line-dependent factors that may influence the interpretation of transport assays.
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Characterization of Rhodamine-123 as a Tracer Dye for Use In In vitro Drug Transport Assays.
By admin, on March 28th, 2012
Mol Cell Biol. 2012 Mar 26;
Maerz S, Dettmann A, Seiler S
Nuclear Dbf2p-related (NDR) kinases and associated proteins are recognized as conserved network that regulates eukaryotic cell polarity. NDR kinases require association with MOB adaptor proteins and phosphorylation of two conserved residues in the activation segment and hydrophobic motif for activity and function. We demonstrate that the Neurospora crassa NDR kinase COT1 forms inactive dimers via a conserved N-terminal extension, which is also required for the interaction of the kinase with MOB2 to generate heterocomplexes with basal activity. Basal kinase activity also requires autophosphorylation of the COT1-MOB2 complex in the activation segment, while hydrophobic motif phosphorylation of COT1 by the germinal center kinase POD6 fully activates COT1 through induction of a conformational change. Hydrophobic motif phosphorylation is also required for plasma membrane association of the COT1-MOB2 complex. MOB2 further restricts the membrane-associated kinase complex to the hyphal apex to promote polar cell growth. These data support an integrated mechanism of NDR kinase regulation in vivo, in which kinase activation and cellular localization of COT1 are coordinated by dual phosphorylation and interaction with MOB2.
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Hydrophobic motif phosphorylation of the Neurospora crassa NDR kinase COT1 coordinates kinase activation with polar localization of COT1.
By admin, on March 24th, 2012
PLoS One. 2012; 7(3): e33377
He J, Yu T, Pan J, Li H
Store-operated Ca(2+) channels are a major Ca(2+) entry pathway in nonexcitable cells, which drive various essential cellular functions. Recently, STIM1 and Orai proteins have been identified as the major molecular components of the Ca(2+) release-activated Ca(2+) (CRAC) channel. As the key subunit of the CRAC channel, STIM1 is the ER Ca(2+) sensor and is essential for the recruitment and activation of Orai1. However, the mechanisms in transmission of information of STIM1 to Orai1 still need further investigation. Bimolecular fluorescence complementation (BiFC) is one of the most advanced and powerful tools for studying and visualising protein-protein interactions in living cells. We utilised BiFC and acceptor photobleaching fluorescence resonance energy transfer (FRET) experiments to visualise and determine the state of STIM1 in the living cells in resting state. Our results demonstrate that STIM1 exists in an oligomeric form in resting cells and that rather than the SAM motif, it is the C-terminus (residues 233-474) of STIM1 that is the key domain for the interaction between STIM1s. The STIM1 oligomers (BiFC-STIM1) and wild-type STIM1 colocalised and had a fibrillar distribution in resting conditions. Depletion of ER Ca(2+) stores induced BiFC-STIM1 distribution to become punctate, an effect that could be prevented or reversed by 2-APB. After depletion of the Ca(2+) stores, BiFC-STIM1 has the ability to form puncta that colocalise with wild-type STIM1 or Orai1 near the plasma membrane. Our data also indicate that the function of BiFC-STIM1 was not altered compared with that of wild-type STIM1.
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Visualisation and Identification of the Interaction between STIM1s in Resting Cells.
By admin, on March 22nd, 2012
Appl Biochem Biotechnol. 2012 Mar 21;
John L, John GJ, Kholia T
Leishmaniasis is a group of diseases with a spectrum of clinical manifestations ranging from cutaneous ulcers to visceral leishmaniasis, which results from the bite of an infected sandfly to human. Attempts to develop an effective vaccine have been shown to be feasible but no vaccine is in active clinical use. This study adopts a Reverse Vaccinology approach to identify common vaccine candidates from both highly pathogenic species Leishmania major and Leishmania infantum. Total proteome of both species were compared to identify common proteins, which are further taken for sub-cellular localization and transmembrane helices prediction. Plasma membrane proteins having only one transmembrane helix were first identified and analyzed which are non-homologous in human and mouse in order to avoid molecular mimicry with other proteins. Selected proteins were analyzed for their binding efficiency to both major histocompatibility complex (MHC) class I and class II alleles. As a result, 19 potential epitopes are screened in this study using different approaches, which can be further verified through in vivo experiments in MHC compatible animal models. This study demonstrates that Reverse Vaccinology approach has potential in discovering various immunogenic antigens from in silico analysis of pathogen's genome or proteome instead of culturing the whole organism by conventional methods.
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A Reverse Vaccinology Approach for the Identification of Potential Vaccine Candidates from Leishmania spp.
By admin, on March 22nd, 2012
By admin, on March 22nd, 2012
By admin, on March 22nd, 2012
Anaesth Intensive Care. 2012 Jan; 40(1): 46-70
Andrews DT, Royse C, Royse AG
This review summarises the most recent data in support of the role of the mitochondrial permeability transition pore (mPTP) in ischaemia-reperfusion injury, how anaesthetic agents interact with this molecular channel, and the relevance this holds for current anaesthetic practice. Ischaemia results in damage to the electron transport chain of enzymes and sets into play the assembly of a non-specific mega-channel (the mPTP) that transgresses the inner mitochondrial membrane. During reperfusion, uncontrolled opening of the mPTP causes widespread depolarisation of the inner mitochondrial membrane, hydrolysis of ATP, mitochondrial rupture and eventual necrotic cell death. Similarly, transient opening of the mPTP during less substantial ischaemia leads to differential swelling of the intermembrane space compared to the mitochondrial matrix, rupture of the outer mitochondrial membrane and release of pro-apoptotic factors into the cytosol. Recent data suggests that cellular protection from volatile anaesthetic agents follows specific downstream interactions with this molecular channel that are initiated early during anaesthesia. Intravenous anaesthetic agents also prevent the opening of the mPTP during reperfusion. Although by dissimilar mechanisms, both volatiles and propofol promote cell survival by preventing uncontrolled opening of the mPTP after ischaemia. It is now considered that anaesthetic-induced closure of the mPTP is the underlying effector mechanism that is responsible for the cytoprotection previously demonstrated in clinical studies investigating anaesthetic-mediated cardiac and neuroprotection. Manipulation of mPTP function offers a novel means of preventing ischaemic cell injury. Anaesthetic agents occupy a unique niche in the pharmacological armamentarium available for use in preventing cell death following ischaemia-reperfusion injury.
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The mitochondrial permeability transition pore and its role in anaesthesia-triggered cellular protection during ischaemia-reperfusion injury.
By admin, on March 22nd, 2012
Anaesth Intensive Care. 2012 Jan; 40(1): 46-70
Andrews DT, Royse C, Royse AG
This review summarises the most recent data in support of the role of the mitochondrial permeability transition pore (mPTP) in ischaemia-reperfusion injury, how anaesthetic agents interact with this molecular channel, and the relevance this holds for current anaesthetic practice. Ischaemia results in damage to the electron transport chain of enzymes and sets into play the assembly of a non-specific mega-channel (the mPTP) that transgresses the inner mitochondrial membrane. During reperfusion, uncontrolled opening of the mPTP causes widespread depolarisation of the inner mitochondrial membrane, hydrolysis of ATP, mitochondrial rupture and eventual necrotic cell death. Similarly, transient opening of the mPTP during less substantial ischaemia leads to differential swelling of the intermembrane space compared to the mitochondrial matrix, rupture of the outer mitochondrial membrane and release of pro-apoptotic factors into the cytosol. Recent data suggests that cellular protection from volatile anaesthetic agents follows specific downstream interactions with this molecular channel that are initiated early during anaesthesia. Intravenous anaesthetic agents also prevent the opening of the mPTP during reperfusion. Although by dissimilar mechanisms, both volatiles and propofol promote cell survival by preventing uncontrolled opening of the mPTP after ischaemia. It is now considered that anaesthetic-induced closure of the mPTP is the underlying effector mechanism that is responsible for the cytoprotection previously demonstrated in clinical studies investigating anaesthetic-mediated cardiac and neuroprotection. Manipulation of mPTP function offers a novel means of preventing ischaemic cell injury. Anaesthetic agents occupy a unique niche in the pharmacological armamentarium available for use in preventing cell death following ischaemia-reperfusion injury.
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The mitochondrial permeability transition pore and its role in anaesthesia-triggered cellular protection during ischaemia-reperfusion injury.
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