bluuebunny/crossref_metadata_2025 huggingface dataset:
AM3, a natural glycoconjugate, induces the functional maturation of human dendritic cells
Authors: Martín‐Vilchez S , Molina‐Jiménez F , Alonso‐Lebrero J L , Sanz‐Cameno P , Rodríguez‐Muñoz Y , Benedicto I , Roda‐Navarro P , Trapero M , Aragoneses‐Fenoll L , González S , Pivel J P , Corbí A L , López‐Cabrera M , Moreno‐Otero R , Majano P L
Published: April 2008

Background and purpose: Dendritic cells (DCs) are dedicated antigen‐presenting cells able to initiate specific immune responses and their maturation is critical for the induction of antigen‐specific T‐lymphocyte responses. Here, we have investigated the effects of Inmunoferon‐active principle (AM3), the active agent of a commercial immunomodulatory drug, on human monocyte‐derived DCs (MDDCs).

Experimental approach: MDDCs derived from healthy and hepatitis C virus (HCV)‐infected patients were stimulated with AM3. We analysed the expression of cell surface proteins by flow cytometry, that of cytokine production by ELISA, and the expression of chemokines and chemokine receptors by RNase protection assays. T‐lymphocyte proliferation was assessed in mixed lymphocyte reactions, protein expression by western blot and luciferase‐based reporter methods, and Toll‐like receptor (TLR)‐blocking antibodies were employed to analyse TLR activity.

Key results: In MDDCs, AM3 induced or enhanced expression of CD54, CD83, CD86, HLA‐DR, chemokines and chemokine receptors, interleukin (IL)‐12p70 and IL‐10. Furthermore, AM3 stimulated MDDCs to increase proliferation of allogenic T cells. AM3 triggered nuclear translocation of NF‐κB and phosphorylation of p38 mitogen‐activated protein kinase. AM3 promoted NF‐κB activation in a TLR‐4‐dependent manner, and blocking TLR‐4 activity attenuated the enhanced expression of CD80, CD83 and CD86 induced by AM3. AM3 enhanced the expression of maturation‐associated markers in MDDCs from HCV‐infected patients and increased the proliferation of T lymphocytes induced by these MDDCs.

Conclusions and implications: These results underline the effects of AM3 in promoting maturation of MDDCs and suggest that AM3 might be useful in regulating immune responses in pathophysiological situations requiring DC maturation.

British Journal of Pharmacology (2008) 154, 698–708; doi:10.1038/bjp.2008.87; published online 14 April 2008
Effects of imatinib mesylate on spontaneous electrical and mechanical activity in smooth muscle of the guinea‐pig stomach
Authors: Hashitani H , Hayase M , Suzuki H
Published: April 2008

Background and purpose:

Effects of imatinib mesylate, a Kit receptor tyrosine kinase inhibitor, on spontaneous activity of interstitial cells of Cajal (ICC) and smooth muscles in the stomach were investigated.

Experimental approach:

Effects of imatinib on spontaneous electrical and mechanical activity were investigated by measuring changes in the membrane potential and tension recorded from smooth muscles of the guinea‐pig stomach. Its effects on spontaneous changes in intracellular concentration of Ca2+ ([Ca2+]i) (Ca2+ transients) were also examined in fura‐2‐loaded preparations.

Key results:

Imatinib (1–10 μM) suppressed spontaneous contractions and Ca2+ transients. Simultaneous recordings of electrical and mechanical activity demonstrated that imatinib (1 μM) reduced the amplitude of spontaneous contractions without suppressing corresponding slow waves. In the presence of nifedipine (1 μM), imatinib (10 μM) reduced the duration of slow waves and follower potentials in the antrum and accelerated their generation, but had little affect on their amplitude. In contrast, imatinib reduced the amplitude of antral slow potentials and slow waves in the corpus.

Conclusions and implications:

Imatinib may suppress spontaneous contractions of gastric smooth muscles by inhibiting pathways that increase [Ca2+]i in smooth muscles rather than by specifically inhibiting the activity of ICC. A high concentration of imatinib (10 μM) reduced the duration of slow waves or follower potentials in the antrum, which reflect activity of ICC distributed in the myenteric layers (ICC‐MY), and suppressed antral slow potentials or corporal slow waves, which reflect activity of ICC within the muscle bundles (ICC‐IM), presumably by inhibiting intracellular Ca2+ handling.

British Journal of Pharmacology (2008) 154, 451–459; doi:10.1038/bjp.2008.91; published online 14 April 2008
Inhibition of the rapid component of the delayed rectifier potassium current in ventricular myocytes by angiotensin II via the AT1 receptor
Authors: Wang Y H , Shi C X , Dong F , Sheng J W , Xu Y F
Published: April 2008

Background and purpose: There is increasing evidence that angiotensin II (Ang II) is associated with the occurrence of ventricular arrhythmias. However, little is known about the electrophysiological effects of Ang II on ventricular repolarization. The rapid component of the delayed rectifier K+ current (IKr) plays a critical role in cardiac repolarization. Hence, the aim of this study was to assess the effect of Ang II on IKr in guinea‐pig ventricular myocytes.

Experimental approach: The whole‐cell patch‐clamp technique was used to record IKr in native cardiocytes and in human embryonic kidney (HEK) 293 cells, co‐transfected with human ether‐a‐go‐go‐related gene (hERG) encoding the α‐subunit of IKr and the human Ang II type 1 (AT1) receptor gene.

Key results: Ang II decreased the amplitude of IKr in a concentration‐dependent manner with an IC50 of 8.9 nM. Action potential durations at 50% (APD50) and 90% (APD90) repolarization were prolonged 20% and 16%, respectively by Ang II (100 nM). Ang II‐induced inhibition of the IKr was abolished by the AT1 receptor blocker, losartan (1 μM). Ang II decreased hERG current in HEK293 cells and significantly delayed channel activation, deactivation and recovery from inactivation. Moreover, PKC inhibitors, stausporine and Bis‐1, significantly attenuated Ang II‐induced inhibition of IKr.

Conclusions and implications: Ang II produces an inhibitory effect on IKr/hERG currents via AT1 receptors linked to the PKC pathway in ventricular myocytes. This is a potential mechanism by which elevated levels of Ang II are involved in the occurrence of arrhythmias in cardiac hypertrophy and failure.

British Journal of Pharmacology (2008) 154, 429–439; doi:10.1038/bjp.2008.95; published online 14 April 2008
Novel consequences of voltage‐dependence to G‐protein‐coupled P2Y1 receptors
Authors: Gurung I S , Martinez‐Pinna J , Mahaut‐Smith M P
Published: April 2008

Background and purpose:

Emerging evidence suggests that activation of G‐protein‐coupled receptors (GPCRs) can be directly regulated by membrane voltage. However, the physiological and pharmacological relevance of this effect remains unclear. We have further examined this phenomenon for P2Y1 receptors in the non‐excitable megakaryocyte using a range of agonists and antagonists.

Experimental approach:

Simultaneous whole‐cell patch clamp and fura‐2 fluorescence recordings of rat megakaryocytes, which lack voltage‐gated Ca2+ influx, were used to examine the voltage‐dependence of P2Y1 receptor‐evoked IP3‐dependent Ca2+ mobilization.

Results:

Depolarization transiently and repeatedly enhanced P2Y1 receptor‐evoked Ca2+ mobilization across a wide concentration range of both weak, partial and full, potent agonists. Moreover, the amplitude of the depolarization‐evoked [Ca2+]i increase displayed an inverse relationship with agonist concentration, such that the greatest potentiating effect of voltage was observed at near‐threshold levels of agonist. Unexpectedly, depolarization also stimulated an [Ca2+]i increase in the absence of agonist during exposure to the competitive antagonists A3P5PS and MRS2179, or the allosteric enhancer 2,2′‐pyridylisatogen tosylate. A further effect of some antagonists, particularly suramin, was to enhance the depolarization‐evoked Ca2+ responses during co‐application of an agonist. Of several P2Y1 receptor inhibitors, only SCH202676, which has a proposed allosteric mechanism of action, could block ADP‐induced voltage‐dependent Ca2+ release.

Conclusions and implications:

The ability of depolarization to potentiate GPCRs at near‐threshold agonist concentrations represents a novel mechanism for coincidence detection. Furthermore, the induction and enhancement of voltage‐dependent GPCR responses by antagonists has implications for the design of therapeutic compounds.

British Journal of Pharmacology (2008) 154, 882–889; doi:10.1038/bjp.2008.97; published online 14 April 2008
Genetic disruption of calpain correlates with loss of membrane blebbing and differential expression of RhoGDI-1, cofilin and tropomyosin
Authors: Larsen Anna K. , Lametsch René , Elce John S. , Larsen Jørgen K. , Thomsen Bo , Larsen Martin R. , Lawson Moira A. , Greer Peter A. , Ertbjerg Per
Published: April 2008

Dynamic regulation of the actin cytoskeleton is important for cell motility, spreading and the formation of membrane surface extensions such as lamellipodia, ruffles and blebs. The ubiquitous calpains contribute to integrin-mediated cytoskeletal remodelling during cell migration and spreading, by cleavage of focal adhesion components and signalling molecules. In the present study, the live-cell morphology of calpain-knockout and wild-type cells was examined by time-lapse fluorescence microscopy, and a role of calpain in mediating the formation of sporadic membrane blebs was established. Membrane blebbing was significantly reduced in calpain-knockout cells, and genetic rescue fully restored the wild-type phenotype in knockout cells. Proteomic comparison of wild-type and knockout cells identified decreased levels of RhoGDI-1 (Rho GDP-dissociation inhibitor) and cofilin 1, and increased levels of tropomyosin in calpain-knockout cells, suggesting a role of calpain in regulating membrane extensions involving these proteins. RhoGDI, cofilin and tropomyosin are known regulators of actin filament dynamics and membrane extensions. The reduced levels of RhoGDI-1 in calpain-knockout cells observed by proteome analysis were confirmed by immunoblotting. Genetic rescue of the calpain-knockout cells enhanced RhoGDI-1-expression 2-fold above that normally present in wild-type cells. These results suggest a regulatory connection between calpain and RhoGDI-1 in promoting formation of membrane blebs.
Akt substrate TBC1D1 regulates GLUT1 expression through the mTOR pathway in 3T3-L1 adipocytes
Authors: Zhou Qiong L. , Jiang Zhen Y. , Holik John , Chawla Anil , Hagan G. Nana , Leszyk John , Czech Michael P.
Published: April 2008

Multiple studies have suggested that the protein kinase Akt/PKB (protein kinase B) is required for insulin-stimulated glucose transport in skeletal muscle and adipose cells. In an attempt to understand links between Akt activation and glucose transport regulation, we applied mass spectrometry-based proteomics and bioinformatics approaches to identify potential Akt substrates containing the phospho-Akt substrate motif RXRXXpS/T. The present study describes the identification of the Rab GAP (GTPase-activating protein)-domain containing protein TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1], which is closely related to TBC1D4 [TBC domain family, member 4, also denoted AS160 (Akt substrate of 160 kDa)], as an Akt substrate that is phosphorylated at Thr590. RNAi (RNA interference)-mediated silencing of TBC1D1 elevated basal deoxyglucose uptake by approx. 61% in 3T3-L1 mouse embryo adipocytes, while the suppression of TBC1D4 and RapGAP220 under the same conditions had little effect on basal and insulin-stimulated deoxyglucose uptake. Silencing of TBC1D1 strongly increased expression of the GLUT1 glucose transporter but not GLUT4 in cultured adipocytes, whereas the decrease in TBC1D4 had no effect. Remarkably, loss of TBC1D1 in 3T3-L1 adipocytes activated the mTOR (mammalian target of rapamycin)-p70 S6 protein kinase pathway, and the increase in GLUT1 expression in the cells treated with TBC1D1 siRNA (small interfering RNA) was blocked by the mTOR inhibitor rapamycin. Furthermore, overexpression of the mutant TBC1D1-T590A, lacking the putative Akt/PKB phosphorylation site, inhibited insulin stimulation of p70 S6 kinase phosphorylation at Thr389, a phosphorylation induced by mTOR. Taken together, our data suggest that TBC1D1 may be involved in controlling GLUT1 glucose transporter expression through the mTOR-p70 S6 kinase pathway.
Cyto-insectotoxins, a novel class of cytolytic and insecticidal peptides from spider venom
Authors: Vassilevski Alexander A. , Kozlov Sergey A. , Samsonova Olga V. , Egorova Natalya S. , Karpunin Dmitry V. , Pluzhnikov Kirill A. , Feofanov Alexei V. , Grishin Eugene V.
Published: April 2008

Eight linear cationic peptides with cytolytic and insecticidal activity, designated cyto-insectotoxins (CITs), were identified in Lachesana tarabaevi spider venom. The peptides showed antibiotic activity towards Gram-positive and Gram-negative bacteria at micromolar concentrations as well as toxicity to insects. The primary structures of the toxins were established by direct Edman sequencing in combination with enzymatic and chemical polypeptide degradation and MS. CITs represent a novel class of cytolytic molecules and spider venom toxins. They are the first example of molecules showing equally potent antimicrobial and insecticidal effects. Analysis of L. tarabaevi venom gland expressed sequence tag database revealed the primary structures of the protein precursors; eight peptides homologous with the purified toxins were additionally predicted. CIT precursors share a conventional prepropeptide structure with an acidic prosequence and a processing motif common to most spider toxin precursors. The most abundant peptide, CIT 1a, was chemically synthesized, and its lytic activity on different bacterial strains, human erythrocytes and lymphocytes, insect cells, planar lipid bilayers and lipid vesicles was characterized. The spider L. tarabaevi is suggested to have evolved to rely on a unique set of linear cytolytic toxins, as opposed to the more common disulfide-containing spider neurotoxins.
Ubiquitin-mediated proteasomal degradation of non-synonymous SNP variants of human ABC transporter ABCG2
Authors: Nakagawa Hiroshi , Tamura Ai , Wakabayashi Kanako , Hoshijima Kazuyuki , Komada Masayuki , Yoshida Takashi , Kometani Satoshi , Matsubara Takayoshi , Mikuriya Kenta , Ishikawa Toshihisa
Published: April 2008

Clinical relevance is implicated between the genetic polymorphisms of the ABC (ATP-binding cassette) transporter ABCG2 (ABC subfamily G, member 2) and the individual differences in drug response. We expressed a total of seven non-synonymous SNP (single nucleotide polymorphism) variants in Flp-In-293 cells by using the Flp (flippase) recombinase system. Of these, ABCG2 F208S and S441N variants were found to be expressed at markedly low levels, whereas their mRNA levels were equal to those of the other SNP variants and ABCG2 WT (wild-type). Interestingly, protein expression levels of the ABCG2 F208S and S441N variants increased 6- to 12-fold when Flp-In-293 cells were treated with MG132, a proteasome inhibitor. Immunoprecipitation followed by immunoblot analysis showed that the ABCG2 F208S and S441N variant proteins were endogenously ubiquitinated in Flp-In-293 cells, and treatment with MG132 significantly enhanced the level of these ubiquitinated variants. Immunofluorescence microscopy demonstrated that MG132 greatly affected the ABCG2 F208S and S441N variants in terms of both protein levels and intracellular distribution. Immunoblot analysis revealed that those variants were N-glycosylated; however, their oligosaccharides were immature compared with those present on ABCG2 WT. The ABCG2 F208S and S441N variant proteins do not appear to be processed in the Golgi apparatus, but undergo ubiquitin-mediated protein degradation in proteasomes, whereas ABCG2 WT is sorted to the plasma membrane and then degraded via the lysosomal pathway. The present study provides the first evidence that certain genetic polymorphisms can affect the protein stability of ABCG2. Control of proteasomal degradation of ABCG2 would provide a novel approach in cancer chemotherapy to circumvent multidrug resistance of human cancers.
Gq-dependent signalling by the lysophosphatidic acid receptor LPA3 in gastric smooth muscle: reciprocal regulation of MYPT1 phosphorylation by Rho kinase and cAMP-independent PKA
Authors: Sriwai Wimolpak , Zhou Huiping , Murthy Karnam S.
Published: April 2008

The present study characterized the signalling pathways initiated by the bioactive lipid, LPA (lysophosphatidic acid) in smooth muscle. Expression of LPA3 receptors, but not LPA1 and LPA2, receptors was demonstrated by Western blot analysis. LPA stimulated phosphoinositide hydrolysis, PKC (protein kinase C) and Rho kinase (Rho-associated kinase) activities: stimulation of all three enzymes was inhibited by expression of the Gαq, but not the Gαi, minigene. Initial contraction and MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation induced by LPA were abolished by inhibitors of PLC (phospholipase C)-β (U73122) or MLCK (myosin light-chain kinase; ML-9), but were not affected by inhibitors of PKC (bisindolylmaleimide) or Rho kinase (Y27632). In contrast, sustained contraction, and phosphorylation of MLC20 and CPI-17 (PKC-potentiated inhibitor 17 kDa protein) induced by LPA were abolished selectively by bisindolylmaleimide. LPA-induced activation of IKK2 {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase 2} and PKA (protein kinase A; cAMP-dependent protein kinase), and degradation of IκBα were blocked by the RhoA inhibitor (C3 exoenzyme) and in cells expressing dominant-negative mutants of IKK2(K44A) or RhoA(N19RhoA). Phosphorylation by Rho kinase of MYPT1 (myosin phosphatase targeting subunit 1) at Thr696 was masked by phosphorylation of MYPT1 at Ser695 by PKA derived from IκB degradation via RhoA, but unmasked in the presence of PKI (PKA inhibitor) or C3 exoenzyme and in cells expressing IKK2(K44A). We conclude that LPA induces initial contraction which involves activation of PLC-β and MLCK and phosphorylation of MLC20, and sustained contraction which involves activation of PKC and phosphorylation of CPI-17 and MLC20. Although Rho kinase was activated, phosphorylation of MYPT1 at Thr696 by Rho kinase was masked by phosphorylation of MYPT1 at Ser695 via cAMP-independent PKA derived from the NF-κB pathway.
A CD study of uncoupling protein-1 and its transmembrane and matrix-loop domains
Authors: Jelokhani-Niaraki Masoud , Ivanova Marina V. , McIntyre Bonnie L. , Newman Cheryl L. , McSorley Fern R. , Young Elizabeth K. , Smith Matthew D.
Published: April 2008

Conformations of the prototypic UCP-1 (uncoupling protein-1) and its TM (transmembrane) and ML (matrix-loop) domains were studied by CD spectroscopy. Recombinant, untagged mouse UCP-1 and a hexahistidine-tagged version of the protein were obtained in high purity following their overexpression in Escherichia coli. The TM and ML domains of hamster UCP-1 were chemically synthesized. Conformations of both recombinant UCP-1 proteins were dominantly helical (40–50%) in digitonin micelles. Binding of the purine nucleotides GDP and GTP to UCP-1, detected in the near-UV CD region, supported the existence of the functional form of the protein in digitonin micelles. All individual TM and ML peptides, except the third ML domain, adopted helical structures in aqueous trifluoroethanol, which implies that, in addition to six TM segments, at least two of the ML domains of the UCP-1 can form helical structures in membrane interface regions. TM and ML domains interacted with vesicles composed of the main phospholipids of the inner membrane of mitochondria, phosphatidylcholine, phosphatidylethanolamine and cardiolipin, to adopt dominantly β- and/or unordered conformations. Mixtures of UCP-1 peptide domains spontaneously associated in aqueous, phospholipid vesicles and digitonin micelle environments to form ordered conformations, which exhibited common features with the conformations of the full-length proteins. Thermal denaturations of UCP-1 and its nine-peptide-domain assembly in digitonin were co-operative but not reversible. Assembly of six TM domains in lipid bilayers formed ion-conducting units with possible helical bundle conformations. Consequently, covalent connection between peptide domains, tight domain interactions and TM potential are essential for the formation of the functional conformation of UCP-1.
Identification of androgen-selective androgen-response elements in the human aquaporin-5 and Rad9 genes
Authors: Moehren Udo , Denayer Sarah , Podvinec Michael , Verrijdt Guy , Claessens Frank
Published: April 2008

The AR (androgen receptor) is known to influence the expression of its target genes by binding to different sets of AREs (androgen-response elements) in the DNA. One set consists of the classical steroid-response elements which are partial palindromic repeats of the 5′-TGTTCT-3′ steroid-receptor monomer-binding element. The second set contains motifs that are AR-specific and that are proposed to be partial direct repeats of the same motif. On the basis of this assumption, we used an in silico approach to identify new androgen-selective AREs in the regulatory regions of known androgen-responsive genes. We have used an extension of the NUBIScan algorithm to screen a collection of 85 known human androgen-responsive genes compiled from literature and database searches. We report the evaluation of the most promising hits resulting from this computational search by in vitro DNA-binding assays using full-size ARs and GRs (glucocorticoid receptors) as well as their isolated DBDs (DNA-binding domains). We also describe the ability of some of these motifs to confer androgen-, but not glucocorticoid-, responsiveness to reporter-gene expression. The elements found in the aquaporin-5 and the Rad9 (radiation-sensitive 9) genes showed selective AR versus GR binding in band-shift assays and a strong activity and selectivity in functional assays, both as isolated elements and in their original contexts. Our data indicate the validity of the hypothesis that selective AREs are recognizable as direct 5′-TGTTCT-3′ repeats, and extend the list of currently known selective elements.
The Ser186 phospho-acceptor site within ERK4 is essential for its ability to interact with and activate PRAK/MK5
Authors: Perander Maria , Åberg Espen , Johansen Bjarne , Dreyer Bo , Guldvik Ingrid J. , Outzen Heidi , Keyse Stephen M. , Seternes Ole-Morten
Published: April 2008

ERK (extracellular-signal-regulated kinase) 4 [MAPK (mitogen-activated protein kinase) 4] and ERK3 (MAPK6) are atypical MAPKs. One major difference between these proteins and the classical MAPKs is substitution of the conserved T-X-Y motif within the activation loop by a single phospho-acceptor site within an S-E-G motif. In the present study we report that Ser186 of the S-E-G motif in ERK4 is phosphorylated in vivo. Kinase-dead ERK4 is also phosphorylated on Ser186, indicating that an ERK4 kinase, rather than autophosphorylation, is responsible. Co-expression of MK5 [MAPK-activated protein kinase 5; also known as PRAK (p38-regulated/activated kinase)], a physiological target of ERK4, increases phosphorylation of Ser186. This is not dependent on MK5 activity, but does require interaction between ERK4 and MK5 suggesting that MK5 binding either prevents ERK4 dephosphorylation or facilitates ERK4 kinase activity. ERK4 mutants in which Ser186 is replaced with either an alanine residue or a phospho-mimetic residue (glutamate) are unable to activate MK5 and Ser186 is also required for cytoplasmic anchoring of MK5. Both defects seem to reflect an impaired ability of the ERK4 mutants to interact with MK5. We find that there are at least two endogenous pools of wild-type ERK4. One form exhibits reduced mobility when analysed using SDS/PAGE. This is due to MK5-dependent phosphorylation and only this retarded ERK4 species is both phosphorylated on Ser186 and co-immunoprecipitates with wild-type MK5. We conclude that binding between ERK4 and MK5 facilitates phosphorylation of Ser186 and stabilization of the ERK4–MK5 complex. This results in phosphorylation and activation of MK5, which in turn phosphorylates ERK4 on sites other than Ser186 resulting in the observed mobility shift.
Interaction of acetylcholinesterase with the G4 domain of the laminin α1-chain
Authors: Johnson Glynis , Swart Chrisna , Moore Samuel W.
Published: April 2008

Although the primary function of AChE (acetylcholinesterase) is the synaptic hydrolysis of acetylcholine, it appears that the protein is also able to promote various non-cholinergic activities, including cell adhesion, neurite outgrowth and amyloidosis. We have observed previously that AChE is able to bind to mouse laminin-111 in vitro by an electrostatic mechanism. We have also observed that certain mAbs (monoclonal antibodies) recognizing AChE's PAS (peripheral anionic site) inhibit both laminin binding and cell adhesion in neuroblastoma cells. Here, we investigated the interaction sites of the two molecules, using docking, synthetic peptides, ELISAs and conformational interaction site mapping. Mouse AChE was observed on docking to bind to a discontinuous, largely basic, structure, Val2718-Arg-Lys-Arg-Leu2722, Tyr2738-Tyr2739, Tyr2789-Ile-Lys-Arg-Lys2793 and Val2817-Glu-Arg-Lys2820, on the mouse laminin α1 G4 domain. ELISAs using synthetic peptides confirmed the involvement of the AG-73 site (2719–2729). This site overlaps extensively with laminin's heparin-binding site, and AChE was observed to compete with heparan sulfate for laminin binding. Docking showed the major component of the interaction site on AChE to be the acidic sequence Arg90-Glu-Leu-Ser-Glu-Asp95 on the omega loop, and also the involvement of Pro40-Pro-Val42, Arg46 (linked to Glu94 by a salt bridge) and the hexapeptide Asp61-Ala-Thr-Thr-Phe-Gln66. Epitope analysis, using CLiPS™ technology, of seven adhesion-inhibiting mAbs (three anti-human AChE, one anti-Torpedo AChE and three anti-human anti-anti-idiotypic antibodies) showed their major recognition site to be the sequence Pro40-Pro-Met-Gly-Pro-Arg-Arg-Phe48 (AChE human sequence). The antibodies, however, also reacted with the proline-containing sequences Pro78-Gly-Phe-Glu-Gly-Thr-Glu84 and Pro88-Asn-Arg-Glu-Leu-Ser-Glu-Asp95. Antibodies that recognized other features of the PAS area but not the Arg90-Gly-Leu-Ser-Glu-Asp95 motif interfered neither with laminin binding nor with cell adhesion. These results define sites for the interaction of AChE and laminin and suggest that the interaction plays a role in cell adhesion. They also suggest the strong probability of functional redundancy between AChE and other molecules in early development, particularly heparan sulfate proteoglycans, which may explain the survival of the AChE-knockout mouse.
Endosomal NADPH oxidase regulates c-Src activation following hypoxia/reoxygenation injury
Authors: Li Qiang , Zhang Yulong , Marden Jennifer J. , Banfi Botond , Engelhardt John F.
Published: April 2008

c-Src has been shown to activate NF-κB (nuclear factor κB) following H/R (hypoxia/reoxygenation) by acting as a redox-dependent IκBα (inhibitory κB) tyrosine kinase. In the present study, we have investigated the redox-dependent mechanism of c-Src activation following H/R injury and found that ROS (reactive oxygen species) generated by endosomal Noxs (NADPH oxidases) are critical for this process. Endocytosis following H/R was required for the activation of endosomal Noxs, c-Src activation, and the ability of c-Src to tyrosine-phosphorylate IκBα. Quenching intra-endosomal ROS during reoxygenation inhibited c-Src activation without affecting c-Src recruitment from the plasma membrane to endosomes. However, siRNA (small interfering RNA)-mediated knockdown of Rac1 prevented c-Src recruitment into the endosomal compartment following H/R. Given that Rac1 is a known activator of Nox1 and Nox2, we investigated whether these two proteins were required for c-Src activation in Nox-deficient primary fibroblasts. Findings from these studies suggest that both Nox1 and Nox2 participate in the initial redox activation of c-Src following H/R. In summary, our results suggest that Rac1-dependent Noxs play a critical role in activating c-Src following H/R injury. This signalling pathway may be a useful therapeutic target for ischaemia/reperfusion-related diseases.
The guanine-nucleotide-exchange factor BopE from Burkholderia pseudomallei adopts a compact version of the Salmonella SopE/SopE2 fold and undergoes a closed-to-open conformational change upon interaction with Cdc42
Authors: Upadhyay Abhishek , Wu Huan-Lin , Williams Christopher , Field Terry , Galyov Edouard E. , van den Elsen Jean M. H. , Bagby Stefan
Published: April 2008

BopE is a type III secreted protein from Burkholderia pseudomallei, the aetiological agent of melioidosis, a severe emerging infection. BopE is a GEF (guanine-nucleotide-exchange factor) for the Rho GTPases Cdc42 (cell division cycle 42) and Rac1. We have determined the structure of BopE catalytic domain (amino acids 78–261) by NMR spectroscopy and it shows that BopE78–261 comprises two three-helix bundles (α1α4α5 and α2α3α6). This fold is similar to that adopted by the BopE homologues SopE and SopE2, which are GEFs from Salmonella. Whereas the two three-helix bundles of SopE78–240 and SopE269–240 form the arms of a ‘Λ’ shape, BopE78–261 adopts a more closed conformation with substantial interactions between the two three-helix bundles. We propose that arginine and proline residues are important in the conformational differences between BopE and SopE/E2. Analysis of the molecular interface in the SopE78–240–Cdc42 complex crystal structure indicates that, in a BopE–Cdc42 interaction, the closed conformation of BopE78–261 would engender steric clashes with the Cdc42 switch regions. This implies that BopE78–261 must undergo a closed-to-open conformational change in order to catalyse guanine nucleotide exchange. In an NMR titration to investigate the BopE78–261–Cdc42 interaction, the appearance of additional peaks per NH for residues in hinge regions of BopE78–261 indicates that BopE78–261 does undergo a closed-to-open conformational change in the presence of Cdc42. The conformational change hypothesis is further supported by substantial improvement of BopE78–261 catalytic efficiency through mutations that favour an open conformation. Requirement for closed-to-open conformational change explains the 10–40-fold lower kcat of BopE compared with SopE and SopE2.
Specific transmembrane segments are selectively delayed at the ER translocon during opsin biogenesis
Authors: Ismail Nurzian , Crawshaw Samuel G. , Cross Benedict C. S. , Haagsma Anna C. , High Stephen
Published: April 2008

A site-specific cross-linking approach was used to study the integration of TM (transmembrane) segments 4–7 of the polytopic membrane protein, opsin, at the ER (endoplasmic reticulum). We found that although TM4 exits the ER translocon rapidly, TM segments 5, 6 and 7 are all retained at the translocon until opsin biosynthesis is terminated. Furthermore, although artificial extension of the nascent chain is not sufficient to release the C-terminal region of opsin from the translocon, substitution of the native TM segment 7 with a more hydrophobic TM segment results in its rapid lateral exit into the lipid bilayer. We conclude that the intrinsic properties of a TM segment determine the timing of its membrane integration rather than its relative location within the polypeptide chain. A pronounced and prolonged association of opsin TM5 with the translocon-associated component PAT-10 was also observed, suggesting that PAT-10 may facilitate the assembly of distinct opsin subdomains during membrane integration. The results of the present study strongly support a model in which the ER translocon co-ordinates the integration of selected TM segments in response to the specific requirements of the precursor being synthesized.
Rapid and extensive uptake and activation of hydrophobic triphenylphosphonium cations within cells
Authors: Ross Meredith F. , Prime Tracy A. , Abakumova Irina , James Andrew M. , Porteous Carolyn M. , Smith Robin A. J. , Murphy Michael P.
Published: April 2008

Mitochondria-targeted molecules comprising the lipophilic TPP (triphenylphosphonium) cation covalently linked to a hydrophobic bioactive moiety are used to modify and probe mitochondria in cells and in vivo. However, it is unclear how hydrophobicity affects the rate and extent of their uptake into mitochondria within cells, making it difficult to interpret experiments because their intracellular concentration in different compartments is uncertain. To address this issue, we compared the uptake into both isolated mitochondria and mitochondria within cells of two hydrophobic TPP derivatives, [3H]MitoQ (mitoquinone) and [3H]DecylTPP, with the more hydrophilic TPP cation [3H]TPMP (methyltriphenylphosphonium). Uptake of MitoQ by mitochondria and cells was described by the Nernst equation and was ∼5-fold greater than that for TPMP, as a result of its greater binding within the mitochondrial matrix. DecylTPP was also taken up extensively by cells, indicating that increased hydrophobicity enhanced uptake. Both MitoQ and DecylTPP were taken up very rapidly into cells, reaching a steady state within 15 min, compared with ∼8 h for TPMP. This far faster uptake was the result of the increased rate of passage of hydrophobic TPP molecules through the plasma membrane. Within cells MitoQ was predominantly located within mitochondria, where it was rapidly reduced to the ubiquinol form, consistent with its protective effects in cells and in vivo being due to the ubiquinol antioxidant. The strong influence of hydrophobicity on TPP cation uptake into mitochondria within cells facilitates the rational design of mitochondria-targeted compounds to report on and modify mitochondrial function in vivo.
Catalytically inactive phospholipase A2 homologue binds to vascular endothelial growth factor receptor-2 via a C-terminal loop region
Authors: Fujisawa Daisuke , Yamazaki Yasuo , Lomonte Bruno , Morita Takashi
Published: April 2008

VEGF (vascular endothelial growth factor) regulates neovascularization through binding to its receptor KDR (kinase insert domain-containing receptor; VEGF receptor-2). We recently identified a catalytically inactive PLA2 (phospholipase A2) homologue (KDR-bp) in the venom of eastern cottonmouth (Agkistrodon piscivorus piscivorus) as a third KDR-binding protein, in addition to VEGF165 and tissue inhibitor of metalloproteinase-3. KDR-bp binds to the extracellular domain of KDR with a Kd of 10−8 M, resulting in specific blockade of endothelial cell growth induced by VEGF165. Inactive PLA2 homologues are widely distributed in the venoms of Viperidae snakes and are known to act as myotoxins. In the present study, we demonstrated that KDR-binding ability is a common characteristic for inactive PLA2 homologues in snake venom, but not for active PLA2s such as neurotoxic and platelet aggregation-modulating PLA2s. To understand better the KDR and KDR-bp interaction, we resolved the binding region of KDR-bp using eight synthetic peptides designed based on the structure of KDR-bp. A synthetic peptide based on the structure of the C-terminal loop region of KDR-bp showed high affinity for KDR, but other peptides did not, suggesting that the C-terminal loop region of KDR-bp is involved in the interaction with KDR. The results of the present study provide insight into the binding of inactive PLA2 homologues to KDR, and may also assist in the design of novel anti-KDR molecules for anti-angiogenic therapy.
Apolipoprotein C-III: understanding an emerging cardiovascular risk factor
Authors: Ooi Esther M. M. , Barrett P. Hugh R. , Chan Dick C. , Watts Gerald F.
Published: April 2008

The concurrence of visceral obesity, insulin resistance and dyslipidaemia comprises the concept of the metabolic syndrome. The metabolic syndrome is an escalating problem in developed and developing societies that tracks with the obesity epidemic. Dyslipidaemia in the metabolic syndrome is potently atherogenic and, hence, is a major risk factor for CVD (cardiovascular disease) in these subjects. It is globally characterized by hypertriglyceridaemia, near normal LDL (low-density lipoprotein)-cholesterol and low plasma HDL (high-density lipoprotein)-cholesterol. ApoC-III (apolipoprotein C-III), an important regulator of lipoprotein metabolism, is strongly associated with hypertriglyceridaemia and the progression of CVD. ApoC-III impairs the lipolysis of TRLs [triacylglycerol (triglyceride)-rich lipoproteins] by inhibiting lipoprotein lipase and the hepatic uptake of TRLs by remnant receptors. In the circulation, apoC-III is associated with TRLs and HDL, and freely exchanges among these lipoprotein particle systems. However, to fully understand the complex physiology and pathophysiology requires the application of tracer methodology and mathematical modelling. In addition, experimental evidence shows that apoC-III may also have a direct role in atherosclerosis. In the metabolic syndrome, increased apoC-III concentration, resulting from hepatic overproduction of VLDL (very-LDL) apoC-III, is strongly associated with delayed catabolism of triacylglycerols and TRLs. Several therapies pertinent to the metabolic syndrome, such as PPAR (peroxisome-proliferator-activated receptor) agonists and statins, can regulate apoC-III transport in the metabolic syndrome. Regulating apoC-III metabolism may be an important new therapeutic approach to managing dyslipidaemia and CVD risk in the metabolic syndrome.
Hypertension increases middle cerebral artery resting tone in spontaneously hypertensive rats: role of tonic vasoactive factor availability
Authors: González José M. , Somoza Beatriz , Conde M. Victoria , Fernández-Alfonso Maria S. , González M. Carmen , Arribas Silvia M.
Published: April 2008

The present study explores the contribution of alterations in resting tone to cerebral artery narrowing in SHRs (spontaneously hypertensive rats) and the role of hypertension development. Young pre-hypertensive and adult fully hypertensive SHRs and age-matched Wistar–Kyoto rat controls were used. The contribution of basal vasoactive factors to resting tone was studied in middle cerebral arteries with pressure myography. Basal NO and O2− (superoxide anion) availability were determined with fluorescent indicators using confocal microscopy and lucigenin-enhanced chemiluminescence. Basal O2− was also assessed in mesenteric resistance arteries. Middle cerebral arteries from adult rats, but not young pre-hypertensive rats, had augmented myogenic responses and resting tone and decreased relaxation to sodium nitroprusside compared with their normotensive counterparts. Cerebral arteries from adult SHRs also had an increase in tonic NO associated with a decrease in basal O2− availability. Basal O2− was instead increased in mesenteric arteries from SHRs. The present results indicate that large cerebral arteries from SHRs have an increase in their resting tone as a consequence of sustained hypertension and that this is related to a decrease in NO responsiveness. We suggest that this increase in resting tone and myogenic responses could act as a protective mechanism against the development of stroke in SHRs. The present study also demonstrates some unusual findings regarding the current understanding of the NO/O2− balance in hypertension with important differences between vascular beds and draws attention to the complexity of this balance in cardiovascular health and disease.
Urocortin 2 combined with angiotensin-converting enzyme inhibition in experimental heart failure
Authors: Rademaker Miriam T. , Charles Christopher J. , Nicholls M. Gary , Richards A. Mark
Published: April 2008

Ucn2 (urocortin 2) is a recently discovered peptide with therapeutic potential in heart failure. As any new treatment is likely to be used in conjunction with standard ACEI (angiotensin-converting enzyme inhibitor) therapy, it is important that the combined effects of these agents are assessed. In the present study, we investigated the effects of Ucn2 and an ACEI (captopril) administered for 3 h, both separately and together, in eight sheep with pacing-induced heart failure. Ucn2 and captopril alone both increased CO (cardiac output; Ucn2>captopril) and decreased arterial pressure (captopril>Ucn2), left atrial pressure (Ucn2>captopril) and peripheral resistance (Ucn2=captopril) relative to controls. Compared with either treatment alone, combined treatment further improved CO and reduced peripheral resistance and cardiac preload, without inducing further falls in blood pressure. In contrast with the marked increase in plasma renin activity observed with captopril alone, Ucn2 administration reduced renin activity, whereas the combined agents resulted in intermediate renin levels. All active treatments decreased circulating levels of aldosterone (Ucn2+captopril>Ucn2=captopril), endothelin-1 and the natriuretic peptides (Ucn2+captopril=Ucn2>captopril), whereas adrenaline (epinephrine) fell only with Ucn2 (Ucn2+captopril=Ucn2), and vasopressin increased during captopril alone. Ucn2, both separately and in conjunction with captopril, increased urine output, sodium and creatinine excretion and creatinine clearance. Conversely, captopril administered alone adversely affected these renal indices. In conclusion, co-treatment with Ucn2 and an ACEI in heart failure produced significantly greater improvements in haemodynamics, hormonal profile and renal function than achieved by captopril alone. These results indicate that dual treatment with these two agents is beneficial.
Polarized pump-probe measurements of electronic motion via a conical intersection
Authors: Farrow Darcie A. , Qian Wei , Smith Eric R. , Ferro Allison A. , Jonas David M.
Published: April 2008

Polarized femtosecond pump-probe spectroscopy is used to observe electronic wavepacket motion for vibrational wavepackets centered on a conical intersection. After excitation of a doubly degenerate electronic state in a square symmetric silicon naphthalocyanine molecule, electronic motions cause a ∼100fs drop in the polarization anisotropy that can be quantitatively predicted from vibrational quantum beat modulations of the pump-probe signal. Vibrational symmetries are determined from the polarization anisotropy of the vibrational quantum beats. The polarization anisotropy of the totally symmetric vibrational quantum beats shows that the electronic wavepackets equilibrate via the conical intersection within ∼200fs. The relationship used to predict the initial electronic polarization anisotropy decay from the asymmetric vibrational quantum beat amplitudes indicates that the initial width of the vibrational wavepacket determines the initial speed of electronic wavepacket motion. For chemically reactive conical intersections, which can have 1000 times greater stabilization energies than the one observed here, the same theory predicts electronic equilibration within 2fs. Such electronic movements would be the fastest known chemical processes.
Magnetic second harmonic generation at the Co2MnSi∕AlOx interface
Authors: Shelford L. R. , Liu Y. , Hicken R. J. , Sakuraba Y. , Oogane M. , Ando Y.
Published: April 2008

We have studied magnetic second harmonic generation (MSHG) at the Co2MnSi∕AlOx interface. The variation of the MSHG intensity was consistent with the nonvanishing components of the nonlinear susceptibility tensor expected for the (001) cubic surface. The difference in the MSHG asymmetry, the MSHG anisotropy, is found to have maximum value at an annealing temperature of 450°C, for which similar samples have previously been found to show optimum L21 site ordering and maximum tunnel magnetoresistance.
Partially linearized, fully size-extensive, and reduced multireference coupled-cluster methods. I. Formalism and mutual relationship
Authors: Li Xiangzhu , Paldus Josef
Published: April 2008

We describe a fully size-extensive alternative of the reduced multireference (RMR) coupled-cluster (CC) method with singles (S) and doubles (D) that generates a subset of higher-than-pair cluster amplitudes, using linearized CC equations from the full CC chain, projected onto the corresponding higher-than-doubly excited configurations. This approach is referred to as partially linearized (pl) MR CCSD method and characterized by the acronym plMR CCSD. In contrast to a similar CCSDT-1 method [Y. S. Lee et al., J. Chem. Phys. 81, 5906 (1984)] this approach also considers higher than triples (currently up to hexuples), while focusing only on a small subset of such amplitudes, referred to as the primary ones. These amplitudes are selected using similar criteria as in RMR CCSD. An extension considering secondary triples via the standard (T)-type corrections, resulting in the plMR CCSD(T) method, is also considered. The relationship of RMR and plMR CCSD and CCSD(T) approaches is discussed, and their performance and characteristics are the subject of the subsequent Part II of this paper.
Partially linearized, fully size-extensive, and reduced multireference coupled-cluster methods. II. Applications and performance
Authors: Li Xiangzhu , Paldus Josef
Published: April 2008

The partially linearized (pl), fully size-extensive multireference (MR) coupled-cluster (CC) method, fully accounting for singles (S) and doubles (D) and approximately for a subset of primary higher than doubles, referred to as plMR CCSD, as well as its plMR CCSD(T) version corrected for secondary triples, as described in Part I of this paper [X. Li and J. Paldus, J. Chem. Phys. 128, 144118 (2008)], are applied to the problem of bond breaking in the HF, F2, H2O, and N2 molecules, as well as to the H4 model, using basis sets of a DZ or a cc-pVDZ quality that enable a comparison with the full configuration interaction (FCI) exact energies for a given ab initio model. A comparison of the performance of the plMR CCSD/CCSD(T) approaches with those of the reduced MR (RMR) CCSD/CCSD(T) methods, as well as with the standard single reference (SR) CCSD and CCSD(T) methods, is made in each case. For the H4 model and N2 we also compare our results with the completely renormalized (CR) CC(2,3) method [P. Piecuch and M. Włoch, J. Chem. Phys. 123, 224105 (2005)]. An important role of a proper choice of the model space for the MR-type methods is also addressed. The advantages and shortcomings of all these methods are pointed out and discussed, as well as their size-extensivity characteristics, in which case we distinguish supersystems involving noninteracting SR and MR subsystems from those involving only MR-type subsystems. Although the plMR-type approaches render fully size-extensive results, while the RMR CCSD may slightly violate this property, the latter method yields invariably superior results to the plMR CCSD ones and is more easy to apply in highly demanding cases, such as the triple-bond breaking in the nitrogen molecule.
The photodissociation of the water dimer in the Ã band: A twelve-dimensional quasiclassical study
Authors: Avila G. , Kroes G. J. , van Hemert M. C.
Published: April 2008

The quasiclassical absorption spectrum of the water dimer in the Ã band was calculated taking into account motion in all degrees of freedom of the system. The ab initio excited state potentials employed were interpolated by the modified Shepard interpolation method using QMRCI energies and state-averaged MCSCF gradients and Hessians. The ground state vibrational wavefunction was variationally calculated using an adiabatic separation between the high and low frequency normal modes of the system. The calculated spectrum of water dimer shows a clear blueshift with respect to the monomer, but also a small red tail, in agreement with the prediction by Harvey et al. [J. Chem. Phys. 109, 8747 (1998)]. Previous three-dimensional model studies of the photodissociation of the water dimer by Valenzano et al. [J. Chem. Phys. 123, 034303 (2005)] did not show this red tail. A thorough analysis of the dependence of the spectrum on the modes coupled explicitly in the calculation of the spectrum shows that the red tail is due to coupling between the intramolecular stretch vibrations on different monomers.