bluuebunny/crossref_metadata_2025 huggingface dataset:
Dissolution of limestone fractures by cooling waters: Early development of hypogene karst systems
Authors: Andre Benjamin J. , Rajaram Harihar
Published: January 2005

Fracture dissolution in the early stages of karstification under hypogene conditions is investigated using a coupled numerical model of fluid flow, heat transfer, and reactive transport. Dissolution of calcite in the H2O‐CO2‐CaCO3 system along a cooling flow path is investigated using both equilibrium and kinetic models. During the very early stages of fracture growth, there is a positive feedback between flow, heat transfer, and dissolution. In this stage the dissolution rate is largely controlled by the retrograde solubility of calcite, and aperture growth is relatively uniform along the fracture length. There is a period of slow continuous increase in the mass flow rate through the fracture, which is followed by an abrupt rapid increase. We refer to the time when this rapid increase occurs as the maturation time. As the flow rate continues to increase after maturation, forced convective effects lead to higher fluid temperatures in the fracture, resulting in a negative feedback that slows the rate of fracture growth. The behavior of aperture growth before the maturation time can be described by a simple ordinary differential equation. The solution of this differential equation provides an estimate of the maturation time, in terms of the initial aperture, hydraulic and thermal gradients, and the change in solubility with temperature. The behavior before maturation in two‐dimensional variable aperture fractures is investigated using a simplified model. The maturation time is shown to decrease with the degree of aperture variability due to highly selective growth along preferential flow paths.
Sources of salinity and boron in the Gaza strip: Natural contaminant flow in the southern Mediterranean coastal aquifer
Authors: Vengosh Avner , Kloppmann Wolfram , Marei Amer , Livshitz Yakov , Gutierrez Alexis , Banna Mazen , Guerrot Catherine , Pankratov Irena , Raanan Hadas
Published: January 2005

Salinization in coastal aquifers is a global phenomenon resulting from the overexploitation of scarce water resources. The Gaza Strip is one of the most severe cases of salinization, as accelerated degradation of the water quality endangers the present and future water supply for over 1 million people. We investigate the chemical and isotopic (87Sr/86Sr, δ11B, δ18O, δ2H, and δ34SSO4) compositions of groundwater from the southern Mediterranean coastal aquifer (Israel) and the Gaza Strip in order to elucidate the origin of salinity and boron contamination. The original salinity in the eastern part of the aquifer is derived from discharge of saline groundwater from the adjacent Avedat aquitard (Na/Cl < 1, 87Sr/86Sr ∼ 0.7079, and δ11B ∼ 40‰). As the groundwater flows to the central part of the aquifer, a dramatic change in its composition occurs (Na/Cl > 1, high B/Cl, SO4/Cl, and HCO3, 87Sr/86Sr ∼ 0.7083; δ11B ∼ 48‰), although the δ18O‐δ2H slope is identical to that of the Avedat aquitard. The geochemical data suggest that dissolution of pedogenic carbonate and gypsum minerals in the overlying loessial sequence generated the Ca‐rich solution that triggered base exchange reactions and produced Na‐ and B‐rich groundwater. The geochemical data show that most of the salinization process in the Gaza Strip is derived from the lateral flow of the Na‐rich saline groundwater, superimposed with seawater intrusion and anthropogenic nitrate pollution. The methodology of identification of multiple salinity sources can be used to establish a long‐term management plan for the Gaza Strip and can also be implemented to understand complex salinization processes in other similarly stressed coastal aquifers.
The Drosophila protein kinase LK6 is regulated by ERK and phosphorylates the eukaryotic initiation factor eIF4E in vivo
Authors: PARRA-PALAU Josep L. , SCHEPER Gert C. , HARPER Daniel E. , PROUD Christopher G.
Published: January 2005

In Drosophila cells, phosphorylation of eIF4E (eukaryotic initiation factor 4E) is required for growth and development. In Drosophila melanogaster, LK6 is the closest homologue of mammalian Mnk1 and Mnk2 [MAPK (mitogen-activated protein kinase) signal-integrating kinases 1 and 2 respectively] that phosphorylate mammalian eIF4E. Mnk1 is activated by both mitogen- and stress-activated signalling pathways [ERK (extracellular-signal-regulated kinase) and p38 MAPK], whereas Mnk2 contains a MAPK-binding motif that is selective for ERKs. LK6 possesses a binding motif similar to that in Mnk2. In the present study, we show that LK6 can phosphorylate eIF4E at the physiological site. LK6 activity is increased by the ERK signalling pathway and not by the stress-activated p38 MAPK signalling pathway. Consistent with this, LK6 binds ERK in mammalian cells, and this requires an intact binding motif. LK6 can bind to eIF4G in mammalian cells, and expression of LK6 increases the phosphorylation of the endogenous eIF4E. In Drosophila S2 Schneider cells, LK6 binds the ERK homologue Rolled, but not the p38 MAPK homologue. LK6 phosphorylates Drosophila eIF4E in vitro. The phosphorylation of endogenous eIF4E in Drosophila cells is increased by activation of the ERK pathway but not by arsenite, an activator of p38 MAPK. RNA interference directed against LK6 significantly decreases eIF4E phosphorylation in Drosophila cells. These results show that LK6 binds to ERK and is activated by ERK signalling and it is responsible for phosphorylating eIF4E in Drosophila.
Expression of gp91phox/Nox2 in COS-7 cells: cellular localization of the protein and the detection of outward proton currents
Authors: MURILLO Isabel , HENDERSON Lydia M.
Published: January 2005

We have reported previously that gp91phox, expressed in CHO (Chinese hamster ovary) cells, functions as a voltage-dependent proton channel. However, others have reported that COS-7 cells expressing gp91phox failed to exhibit outward proton currents, and concluded that gp91phox does not function as a proton channel. To investigate this clear difference in findings, we have examined the expression and cellular localization of the fusion protein EGFP-C–91, in which gp91phox is fused to the C-terminus of enhanced green fluorescent protein. EGFP-C–91 was observed in the plasma membrane and intracellular membranes of 30% of the transfected COS-7 cells. In the remaining COS-7 cells, EGFP-C–91 was detected in the intracellular membranes only. In CHO cells EGFP-C–91 was present in both the plasma membrane and the intracellular membranes of all transfected cells. Under the whole-cell configuration, outward currents were recorded from COS-7 cells expressing gp91phox. These increased in magnitude and lost their ‘droop’ over time as the pipette solution equilibrated with the cell cytoplasm (50 min). The threshold activation voltage for the currents was shifted by ∼60 mV for a 1 unit difference in bath pH. Zn2+ inhibited the outward currents observed in COS-7 cells expressing gp91phox. The tail current reversal potential was −64 mV at a pHo (external pH) of 8.0, −40 mV at pHo 7.4 and −8 mV at pHo 7.0, indicating that the current arises from the movement of protons. Outward currents were exhibited by 37.5% of the COS-7 cells expressing gp91phox. Proton currents were recorded following the excision of inside-out patches from cells transfected with gp91phox. The presence of outward proton currents in COS-7 cells expressing gp91phox provides further support for our proposed role for gp91phox as the NADPH oxidase-associated proton channel.
C-terminal splicing of NTPDase2 provides distinctive catalytic properties, cellular distribution and enzyme regulation
Authors: WANG Carol J. H. , VLAJKOVIC Srdjan M. , HOUSLEY Gary D. , BRAUN Norbert , ZIMMERMANN Herbert , ROBSON Simon C. , SÉVIGNY Jean , SOELLER Christian , THORNE Peter R.
Published: January 2005

The present study provides functional characterization of alternative splicing of the NTPDase2 (ecto-nucleoside triphosphate diphosphohydrolase-2) involved in the regulation of extracellular nucleotide concentrations in a range of organ systems. A novel NTPDase2β isoform produced by alternative splicing of the rat NTPDase2 gene provides an extended intracellular C-terminus and distinguishes itself from NTPDase2α isoform in gaining several intracellular protein kinase CK2 (casein kinase 2) phosphorylation sites and losing the intracellular protein kinase C motif. The plasmids containing NTPDase2α or NTPDase2β cDNA were used to stably transfect Chinese-hamster ovary-S cells. Imaging studies showed that NTPDase2α was predominantly membrane-bound, whereas NTPDase2β had combined cell surface and intracellular localization. α and β isoforms showed variations in divalent cation dependence and substrate specificity for nucleoside-5′-triphosphates and nucleoside-5′-diphosphates. NTPDase2β exhibited reduced ATPase activity and no apparent ADPase activity. NTPDase2 isoforms demonstrated similar sensitivity to inhibitors such as suramin and pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid, and differential regulation by protein kinases. NTPDase2β was up-regulated by intracellular protein kinase CK2 phosphorylation, whereas NTPDase2α activity was down-regulated by protein kinase C phosphorylation. The results demonstrate that alternative coding of the intracellular C-terminal domain contributes distinctive phenotypic variation with respect to extracellular nucleotide specificity, hydrolysis kinetics, protein kinase-dependent intracellular regulation and protein trafficking. These findings advance the molecular physiology of this enzyme system by characterizing the contribution of the C-terminal domain to many of the enzyme's signature properties.
Topological assignment of the N-terminal extension of plasma gelsolin to the gelsolin surface
Authors: FOCK Ulrike , JOCKUSCH Brigitte M. , SCHUBERT Wolf-Dieter , HINSSEN Horst
Published: January 2005

The actin-binding protein gelsolin is highly conserved in vertebrates and exists in two isoforms, a cytoplasmic and an extracellular variant, generated by alternative splicing. In mammals, these isoforms differ only by an N-terminal extension in plasma gelsolin, a short sequence of up to 25 amino acids. Cells and tissues may contain both variants, as plasma gelsolin is secreted by many cell types. The tertiary structure of equine plasma gelsolin has been elucidated, but without any information on the N-terminal extension. In this paper, we present topographical data on the N-terminal extension, derived using a biochemical and immunological approach. For this purpose, a monoclonal antibody was generated that exclusively recognizes cytoplasmic gelsolin but not the extracellular variant and thus allows isoform-specific immunodetection and quantification of cytoplasmic gelsolin in the presence of plasma gelsolin. Using limited proteolysis and pepscan analysis, we mapped the binding epitope and localized it within two regions in segment 1 of the cytoplasmic gelsolin sequence: Tyr34–Ile45 and Leu64–Ile78. In the tertiary structure of the cytoplasmic variant, these sequences are mutually adjacent and located in the proximity of the N-terminus. We therefore conclude that the binding site of the antibody is covered by the N-terminal extension in plasma gelsolin and thus sterically hinders antibody binding. Our results allow for a topological model of the N-terminal extension on the surface of the gelsolin molecule, which was unknown previously.
Phosphorylation and desensitization of the lysophosphatidic acid receptor LPA1
Authors: AVENDAÑO-VÁZQUEZ S. Eréndira , GARCÍA-CABALLERO Agustín , GARCÍA-SÁINZ J. Adolfo
Published: January 2005

In C9 cells, LPA (lysophosphatidic acid) induced inositol phosphate production, increased intracellular calcium concentration and inhibited adenylate cyclase activity. These responses were abolished in cells challenged with active phorbol esters. Action of phorbol esters was blocked by inhibitors of PKC (protein kinase C) and by its down-regulation. LPA1 receptor phosphorylation was observed in response to phorbol esters. The effect was rapid (t1/2∼1 min), intense (2-fold) and sustained (at least 60 min). PKC inhibitors markedly decreased the LPA1 receptor phosphorylation induced by phorbol esters. LPA1 receptor tagged with the green fluorescent protein internalized in response to PKC activation. In addition, LPA and angiotensin II were also capable of inducing LPA1 receptor phosphorylation, showing that LPA1 receptor can be subjected to homologous and heterologous desensitization.
Activation of serine/threonine protein phosphatase-1 is required for ceramide-induced survival of sympathetic neurons
Authors: PLUMMER Greg , PERREAULT Kathleen R. , HOLMES Charles F. B. , POSSE de CHAVES Elena I.
Published: January 2005

In sympathetic neurons, C6-ceramide, as well as endogenous ceramides, blocks apoptosis elicited by NGF (nerve growth factor) deprivation. The mechanism(s) involved in ceramide-induced neuronal survival are poorly understood. Few direct targets for the diverse cellular effects of ceramide have been identified. Amongst those proposed is PP-1c, the catalytic subunit of serine/threonine PP-1 (protein phosphatase-1). Here, we present the first evidence of PP-1c activation by ceramide in live cells, namely NGF-deprived sympathetic neurons. We first determined PP activity in cellular lysates from sympathetic neurons treated with exogenous ceramide and demonstrated a 2–3-fold increase in PP activity. PP activation was completely blocked by the addition of the specific type-1 PP inhibitor protein I-2 as well as by tautomycin, but unaffected by 2 nM okadaic acid, strongly indicating that the ceramide-activated phosphatase activity was PP-1c. Inhibition of PP activity by phosphatidic acid (which has been reported to be a selective inhibitor of PP-1c) and tautomycin (a PP-1 and PP-2A inhibitor), but not by 10 nM okadaic acid, abolished the anti-apoptotic effect of ceramide in NGF-deprived neurons, suggesting that activation of PP-1c is required for ceramide-induced neuronal survival. Ceramide was able to prevent pRb (retinoblastoma gene product) hyperphosphorylation by a mechanism dependent on PP-1c activation, suggesting that two consequences of NGF deprivation in sympathetic neurons are inhibition of PP-1c and subsequent hyperphosphorylation of pRb protein. These findings suggest a novel mechanism for ceramide-induced survival, and implicate the involvement of PPs in apoptosis induced by NGF deprivation.
Hepatic farnesyl diphosphate synthase expression is suppressed by polyunsaturated fatty acids
Authors: LE JOSSIC-CORCOS Catherine , GONTHIER Céline , ZAGHINI Isabelle , LOGETTE Emmanuelle , SHECHTER Ishaiahu , BOURNOT Paulette
Published: January 2005

Dietary vegetable oils and fish oils rich in PUFA (polyunsaturated fatty acids) exert hypocholesterolaemic and hypotriglyceridaemic effects in rodents. The plasma cholesterol-lowering properties of PUFA are due partly to a diminution of cholesterol synthesis and of the activity of the rate-limiting enzyme HMG-CoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase). To better understand the mechanisms involved, we examined how tuna fish oil and individual n−3 and n−6 PUFA affect the expression of hepatic FPP synthase (farnesyl diphosphate synthase), a SREBP (sterol regulatory element-binding protein) target enzyme that is subject to negative-feedback regulation by sterols, in co-ordination with HMG-CoA reductase. Feeding mice on a tuna fish oil diet for 2 weeks decreased serum cholesterol and triacylglycerol levels, by 50% and 60% respectively. Hepatic levels of FPP synthase and HMG-CoA reductase mRNAs were also decreased, by 70% and 40% respectively. Individual n−3 and n−6 PUFA lowered FPP synthase and HMG-CoA reductase mRNA levels in H4IIEC3 rat hepatoma cells to a greater extent than did stearate and oleate, with the largest inhibitory effects occurring with arachidonate, EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid). We observed a similar inhibitory effect on protein levels of FPP synthase. The suppressive effect of PUFA on the FPP synthase mRNA level was not due to a decrease in mRNA stability, but to transcription inhibition. Moreover, a lower nuclear availability of both SREBP-1 and SREBP-2 mature forms was observed in HepG2 human hepatoblastoma cells treated with arachidonate, EPA or DHA. Taken together, these data suggest that PUFA can down-regulate hepatic cholesterol synthesis through inhibition of HMG-CoA reductase and FPP synthase, at least in part through impairment of the SREBP pathway.
Electrochemical redox transformations of T1 and T2 copper sites in nativeTrametes hirsutalaccase at gold electrode
Authors: SHLEEV Sergey , CHRISTENSON Andreas , SEREZHENKOV Vladimir , BURBAEV Dosymzhan , YAROPOLOV Alexander , GORTON Lo , RUZGAS Tautgirdas
Published: January 2005

Mediatorless, electrochemically driven, redox transformations of T1 (type 1) and T2 copper sites in Trametes hirsuta laccase were studied by cyclic voltammetry and spectroelectrochemical redox titrations using bare gold electrode. DET (direct electron transfer) between the electrode and the enzyme was observed under anaerobic conditions. From analysis of experimental data it is concluded that the T2 copper site is in DET contact with gold. It was found that electron transfer between the gold surface and the T1 copper site progresses through the T2 copper site. From EPR measurements and electrochemical data it is proposed that the redox potential of the T2 site for high-potential ‘blue’ laccase is equal to about 400 mV versus NHE (normal hydrogen electrode) at pH 6.5. The hypothesis that the redox potentials of the T2 copper sites in low- and high-potential laccases/oxidases from totally different sources might be very similar, i.e. approx. 400 mV, is discussed.
Extracellular RNA is a natural cofactor for the (auto-)activation of Factor VII-activating protease (FSAP)
Authors: NAKAZAWA Fumie , KANNEMEIER Christian , SHIBAMIYA Aya , SONG Yutong , TZIMA Eleni , SCHUBERT Uwe , KOYAMA Takatoshi , NIEPMANN Michael , TRUSHEIM Heidi , ENGELMANN Bernd , PREISSNER Klaus T.
Published: January 2005

FSAP (Factor VII-activating protease) is a new plasma-derived serine protease with putative dual functions in haemostasis, including activation of coagulation Factor VII and generation of urinary-type plasminogen activator (urokinase). The (auto-)activation of FSAP is facilitated by polyanionic glycosaminoglycans, such as heparin or dextran sulphate, whereas calcium ions stabilize the active form of FSAP. In the present study, extracellular RNA was identified and characterized as a novel FSAP cofactor. The conditioned medium derived from various cell types such as smooth muscle cells, endothelial cells, osteosarcoma cells or CHO (Chinese-hamster ovary) cells contained an acidic factor that initiated (auto-)activation of FSAP. RNase A, but not other hydrolytic enzymes (proteases, glycanases and DNase), abolished the FSAP cofactor activity, which was subsequently isolated by anion-exchange chromatography and unequivocally identified as RNA. In purified systems, as well as in plasma, different forms of natural RNA (rRNA, tRNA, viral RNA and artificial RNA) were able to (auto-)activate FSAP into the two-chain enzyme form. The specific binding of FSAP to RNA (but not to DNA) was shown by mobility-shift assays and UV crosslinking, thereby identifying FSAP as a new extracellular RNA-binding protein, the KD estimated to be 170–350 nM. Activation of FSAP occurred through an RNA-dependent template mechanism involving a nucleic acid size of at least 100 nt. In a purified system, natural RNA augmented the FSAP-dependent Factor VII activation several-fold (as shown by subsequent Factor Xa generation), as well as the FSAP-mediated generation of urokinase. Our results provide evidence for the first time that extracellular RNA, present at sites of cell damage or vascular injury, can serve an important as yet unrecognized cofactor function in haemostasis by inducing (auto-)activation of FSAP through a novel surface-dependent mechanism.
Crystal structure of human carbonic anhydrase II at 1.95 Å resolution in complex with 667-coumate, a novel anti-cancer agent
Authors: LLOYD Matthew D. , PEDERICK Richard L. , NATESH Ramanathan , WOO L. W. Lawrence , PUROHIT Atul , REED Michael J. , ACHARYA K. Ravi , POTTER Barry V. L.
Published: January 2005

CA (carbonic anhydrase) catalyses the reversible hydration of carbon dioxide into bicarbonate, and at least 14 isoforms have been identified in vertebrates. The role of CA type II in maintaining the fluid and pH balance has made it an attractive drug target for the treatment of glaucoma and cancer. 667-Coumate is a potent inhibitor of the novel oncology target steroid sulphatase and is currently in Phase 1 clinical trials for hormone-dependent breast cancer. It also inhibits CA II in vitro. In the present study, CA II was crystallized with 667-coumate and the structure was determined by X-ray crystallography at 1.95 Å (1 Å=0.1 nm) resolution. The structure reported here is the first for an inhibitor based on a coumarin ring and shows ligation of the sulphamate group to the active-site zinc at 2.15 Å through a nitrogen anion. The first two rings of the coumarin moiety are bound within the hydrophobic binding site of CA II. Important residues contributing to binding include Val-121, Phe-131, Val-135, Leu-141, Leu-198 and Pro-202. The third seven-membered ring is more mobile and is located in the channel leading to the surface of the enzyme. Pharmacokinetic studies show enhanced stability of 667-coumate in vivo and this has been ascribed to binding of CA II in erythrocytes. This result provides a structural basis for the stabilization and long half-life of 667-coumate in blood compared with its rapid disappearance in plasma, and suggests that reversible binding of inhibitors to CA may be a general method of delivering this type of labile drug.
The endo-β-agarases AgaA and AgaB from the marine bacterium Zobellia galactanivorans: two paralogue enzymes with different molecular organizations and catalytic behaviours
Authors: JAM Murielle , FLAMENT Didier , ALLOUCH Julie , POTIN Philippe , THION Laurent , KLOAREG Bernard , CZJZEK Mirjam , HELBERT William , MICHEL Gurvan , BARBEYRON Tristan
Published: January 2005

Two β-agarase genes, agaA and agaB, were functionally cloned from the marine bacterium Zobellia galactanivorans. The agaA and agaB genes encode proteins of 539 and 353 amino acids respectively, with theoretical masses of 60 and 40 kDa. These two β-agarases feature homologous catalytic domains belonging to family GH-16. However, AgaA displays a modular architecture, consisting of the catalytic domain (AgaAc) and two C-terminal domains of unknown function which are processed during secretion of the enzyme. In contrast, AgaB is composed of the catalytic module and a signal peptide similar to the N-terminal signature of prokaryotic lipoproteins, suggesting that this protein is anchored in the cytoplasmic membrane. Gel filtration and electrospray MS experiments demonstrate that AgaB is a dimer in solution, while AgaAc is a monomeric protein. AgaAc and AgaB were overexpressed in Escherichia coli and purified to homogeneity. Both enzymes cleave the β-(1→4) linkages of agarose in a random manner and with retention of the anomeric configuration. Although they behave similarly towards liquid agarose, AgaAc is more efficient than AgaB in the degradation of agarose gels. Given these organizational and catalytic differences, we propose that, reminiscent of the agarolytic system of Pseudoalteromonas atlantica, AgaA is specialized in the initial attack on solid-phase agarose, while AgaB is involved with the degradation of agarose fragments.
Inhibition of Rac activation as a mechanism for negative regulation of actin cytoskeletal reorganization and cell motility by cAMP
Authors: NAGASAWA Shin-ya , TAKUWA Noriko , SUGIMOTO Naotoshi , MABUCHI Hiroshi , TAKUWA Yoh
Published: January 2005

cAMP has been found to play a role in mediating the negative regulation of cell motility, although its underlying molecular mechanism remains poorly understood. By using CHO (Chinese-hamster ovary) cells that express the EP2 subtype of PGE2 (prostaglandin E2) receptors, we provide evidence that an increase in cellular cAMP content leads to inhibition of cellular Rac activity, which serves as a mechanism for this negative regulation. In CHO cells expressing EP2, but not in vector control cells, PGE2 dose-dependently inhibited chemotaxis towards IGF-I (insulin-like growth factor-I), which is a Rac-dependent process, with the maximal 75% inhibition observed at 10−8 M PGE2. EP2 stimulation failed to inhibit tyrosine phosphorylation either of IGF-I receptor or IRS-1 (insulin receptor substrate-1), or activation of phosphoinositide 3-kinase or Akt in response to IGF-I, but potently and dose-dependently inhibited IGF-I-induced activation of cellular Rac activity and membrane ruffling. However, PGE2 failed to inhibit Val12-Rac-induced membrane ruffling. Similar to the case of CHO cells, PGE2 inhibited PDGF (platelet-derived growth factor)-induced Rac activation and chemotaxis in vascular smooth muscle cells endogenously expressing EP2. The inhibitory effects of PGE2 on IGF-I-induced chemotaxis, membrane ruffling and Rac activation were faithfully reproduced by a low concentration of forskolin, which induced a comparable extent of cAMP elevation as with 10−8 M PGE2, and were potentiated by isobutylmethylxanthine. The protein kinase A inhibitor Rp isomer of adenosine 3′,5′-cyclic monophosphorothioate reduced PGE2 inhibition of Rac activation and chemotaxis. These results indicate that EP2 mediates Rac inhibition through a mechanism involving cAMP and protein kinase A, thereby inhibiting membrane ruffling and chemotaxis.
Syncollin is required for efficient zymogen granule exocytosis
Authors: WÄSLE Barbara , TURVEY Matthew , LARINA Olga , THORN Peter , SKEPPER Jeremy , MORTON A. Jennifer , EDWARDSON J. Michael
Published: January 2005

Syncollin is a 13 kDa protein that is present in the exocrine pancreas, where the majority of the protein is tightly attached to the luminal surface of the zymogen granule membrane. We have addressed the physiological role of syncollin by studying the phenotype of syncollin KO (knockout) mice. These mice show pancreatic hypertrophy and elevated pancreatic amylase levels. Further, secretagogue-stimulated amylase release from pancreatic lobules of syncollin KO mice was found to be reduced by about 45% compared with wild-type lobules, and the delivery of newly synthesized protein to zymogen granules was delayed, indicating that the mice have a pancreatic secretory defect. As determined by two-photon imaging, the number of secretagogue-stimulated exocytotic events in acini from syncollin KO mice was reduced by 50%. This reduction was accounted for predominantly by a loss of later, ‘secondary’ fusion events between zymogen granules and other granules that had already fused with the plasma membrane. We conclude that syncollin is required for efficient exocytosis in the pancreatic acinar cell, and that it plays a particularly important role in compound exocytosis.
The recombinant dihydropyridine receptor II–III loop and partly structured ‘C’ region peptides modify cardiac ryanodine receptor activity
Authors: DULHUNTY Angela F. , KARUNASEKARA Yamuna , CURTIS Suzanne M. , HARVEY Peta J. , BOARD Philip G. , CASAROTTO Marco G.
Published: January 2005

A physical association between the II–III loop of the DHPR (dihydropryidine receptor) and the RyR (ryanodine receptor) is essential for excitation–contraction coupling in skeletal, but not cardiac, muscle. However, peptides corresponding to a part of the II–III loop interact with the cardiac RyR2 suggesting the possibility of a physical coupling between the proteins. Whether the full II–III loop and its functionally important ‘C’ region (cardiac DHPR residues 855–891 or skeletal 724–760) interact with cardiac RyR2 is not known and is examined in the present study. Both the cardiac DHPR II–III loop (CDCL) and cardiac peptide (Cc) activated RyR2 channels at concentrations >10 nM. The skeletal DHPR II–III loop (SDCL) activated channels at ≤100 nM and weakly inhibited at ≥1 μM. In contrast, skeletal peptide (Cs) inhibited channels at all concentrations when added alone, or was ineffective if added in the presence of Cc. Ca2+-induced Ca2+ release from cardiac sarcoplasmic reticulum was enhanced by CDCL, SDCL and the C peptides. The results indicate that the interaction between the II–III loop and RyR2 depends critically on the ‘A’ region (skeletal DHPR residues 671–690 or cardiac 793–812) and also involves the C region. Structure analysis indicated that (i) both Cs and Cc are random coil at room temperature, but, at 5 °C, have partial helical regions in their N-terminal and central parts, and (ii) secondary-structure profiles for CDCL and SDCL are similar. The data provide novel evidence that the DHPR II–III loop and its C region interact with cardiac RyR2, and that the ability to interact is not isoform-specific.
Depletion of iNOS-derived nitric oxide by prostaglandin H synthase-2 in inflammation-activated J774.2 macrophages through lipohydroperoxidase turnover
Authors: CLARK Stephen R. , ANNING Peter B. , COFFEY Marcus J. , ROBERTS Andrew G. , MARNETT Lawrence J. , O'DONNELL Valerie B.
Published: January 2005

PGHS-2 (prostaglandin H synthase-2) is induced in mammalian cells by pro-inflammatory cytokines in tandem with iNOS [high-output (‘inducible’) nitric oxide synthase], and is co-localized with iNOS and nitrotyrosine in human atheroma macrophages. Herein, murine J774.2 macrophages incubated with lipopolysaccharide and interferon γ showed induction of PGHS-2 and generated NO using iNOS that could be completely depleted by 12(S)-HPETE [12(S)-hydroperoxyeicosatetraenoic acid; 2.4 μM] or hydrogen peroxide (500 μM) (0.42±0.084 and 0.38±0.02 nmol·min−1·106 cells−1 for HPETE and H2O2 respectively). COS-7 cells transiently transfected with human PGHS-2 also showed HPETE- or H2O2-dependent NO decay (0.44±0.016 and 0.20±0.04 nmol·min−1·106 cells−1 for 2.4 μM HPETE and 500 μM H2O2 respectively). Finally, purified PGHS-2 consumed NO in the presence of HPETE or H2O2 (168 and 140 μM·min−1·μM enzyme−1 for HPETE and H2O2 respectively), in a haem-dependent manner, with 20 nM enzyme consuming up to 4 μM NO. Km (app) values for NO and 15(S)-HPETE were 1.7±0.2 and 0.45±0.16 μM respectively. These data indicate that PGHS-2 catalytically consumes NO during peroxidase turnover and that pro-inflammatory cytokines simultaneously upregulate NO synthesis and degradation pathways in murine macrophages. Catalytic NO consumption by PGHS-2 represents a novel interaction between NO and PGHS-2 that may impact on the biological effects of NO in vascular signalling and inflammation.
Contribution of aldehyde dehydrogenase to mitochondrial bioactivation of nitroglycerin: evidence for the activation of purified soluble guanylate cyclase through direct formation of nitric oxide
Authors: KOLLAU Alexander , HOFER Alexandra , RUSSWURM Michael , KOESLING Doris , KEUNG Wing Ming , SCHMIDT Kurt , BRUNNER Friedrich , MAYER Bernd
Published: January 2005

Vascular relaxation to GTN (nitroglycerin) and other antianginal nitrovasodilators requires bioactivation of the drugs to NO or a related activator of sGC (soluble guanylate cyclase). Conversion of GTN into 1,2-GDN (1,2-glycerol dinitrate) and nitrite by mitochondrial ALDH2 (aldehyde dehydrogenase 2) may be an essential pathway of GTN bioactivation in blood vessels. In the present study, we characterized the profile of GTN biotransformation by purified human liver ALDH2 and rat liver mitochondria, and we used purified sGC as a sensitive detector of GTN bioactivity to examine whether ALDH2-catalysed nitrite formation is linked to sGC activation. In the presence of mitochondria, GTN activated sGC with an EC50 (half-maximally effective concentration) of 3.77±0.83 μM. The selective ALDH2 inhibitor, daidzin (0.1 mM), increased the EC50 of GTN to 7.47±0.93 μM. Lack of effect of the mitochondrial poisons, rotenone and myxothiazol, suggested that nitrite reduction by components of the respiratory chain is not essential to sGC activation. However, since co-incubation of sGC with purified ALDH2 led to significant stimulation of cGMP formation by GTN that was completely inhibited by 0.1 mM daidzin and NO scavengers, ALDH2 may convert GTN directly into NO or a related species. Studies with rat aortic rings suggested that ALDH2 contributes to GTN bioactivation and showed that maximal relaxation to GTN occurred at cGMP levels that were only 3.4% of the maximal levels obtained with NO. Comparison of sGC activation in the presence of mitochondria with cGMP accumulation in rat aorta revealed a slightly higher potency of GTN to activate sGC in vitro compared with blood vessels. Our results suggest that ALDH2 catalyses the mitochondrial bioactivation of GTN by the formation of a reactive NO-related intermediate that activates sGC. In addition, the previous conflicting notion of the existence of a high-affinity GTN-metabolizing pathway operating in intact blood vessels but not in tissue homogenates is explained.
Monitoring the formation of dynamic G-protein-coupled receptor–protein complexes in living cells
Authors: PFLEGER Kevin D. G. , EIDNE Karin A.
Published: January 2005

GPCRs (G-protein-coupled receptors) play an extremely important role in transducing extracellular signals across the cell membrane with high specificity and sensitivity. They are central to many of the body's endocrine and neurotransmitter pathways, and are consequently a major drug target. It is now clear that GPCRs interact with a range of proteins, including other GPCRs. Identifying and elucidating the function of such interactions will significantly enhance our understanding of cellular function, with the promise of new and improved pharmaceuticals. Biophysical techniques involving resonance energy transfer, namely FRET (fluorescence resonance energy transfer) and BRET (bioluminescence resonance energy transfer), now enable us to monitor the formation of dynamic GPCR–protein complexes in living cells, in real time. Their use has firmly established the concept of GPCR oligomerization, as well as demonstrating GPCR interactions with GPCR kinases, β-arrestins, adenylate cyclase and a subunit of an inwardly rectifying K+ channel. The present review examines recent technological advances and experimental applications of FRET and BRET, discussing particularly how they have been adapted to extract an ever-increasing amount of information about the nature, specificity, stoichiometry, kinetics and agonist-dependency of GPCR–protein interactions.
Direct control of caveolin-1 expression by FOXO transcription factors
Authors: van den HEUVEL A. Pieter J. , SCHULZE Almut , BURGERING Boudewijn M. T.
Published: January 2005

Protein kinase B can phoshorylate and thereby inactivate the FOXO (forkhead box O) family of transcription factors. When active, FOXO factors can bind to DNA in promoter sequences and subsequently regulate gene expression. We have used DNA microarray analysis to identify potential gene targets of FOXO. In the present study we demonstrate that caveolin-1 is directly controlled by FOXO. Firstly, caveolin-1 expression was increased upon induction or over-expression of FOXO factors at both mRNA and protein levels. Second, we show that endogenous regulation of FOXO activity regulates caveolin-1 levels and that this can be inhibited by dominant-negative FOXO. Third, FOXO activates transcription from the caveolin-1 promoter, and using chromatin immunoprecipitations we demonstrated that this activation occurs via direct interaction of FOXO with the promoter. Finally, we demonstrate FOXO-mediated attenuation of EGF (epidermal growth factor)-induced signalling, which in part is mediated by caveolin-1 expression, as suggested by previous studies [Park, Park, Cho, Kim, Ko, Seo and Park (2000) J. Biol. Chem. 275, 20847–20852]. These findings suggest a novel mechanism by which FOXO factors can exert their cellular effects via transcriptional activation of caveolin-1.
Structure–function analysis of water-soluble inhibitors of the catalytic domain of exotoxin A fromPseudomonas aeruginosa
Authors: YATES Susan P. , TAYLOR Patricia L. , JØRGENSEN René , FERRARIS Dana , ZHANG Jie , ANDERSEN Gregers R. , MERRILL A. Rod
Published: January 2005

The mono-ADPRT (mono-ADP-ribosyltransferase), Pseudomonas aeruginosa ETA (exotoxin A), catalyses the transfer of ADP-ribose from NAD+ to its protein substrate. A series of water-soluble compounds that structurally mimic the nicotinamide moiety of NAD+ was investigated for their inhibition of the catalytic domain of ETA. The importance of an amide locked into a hetero-ring structure and a core hetero-ring system that is planar was a trend evident by the IC50 values. Also, the weaker inhibitors have core ring structures that are less planar and thus more flexible. One of the most potent inhibitors, PJ34, was further characterized and shown to exhibit competitive inhibition with an inhibition constant Ki of 140 nM. We also report the crystal structure of the catalytic domain of ETA in complex with PJ34, the first example of a mono-ADPRT in complex with an inhibitor. The 2.1 Å (1 Å=0.1 nm) resolution structure revealed that PJ34 is bound within the nicotinamide-binding pocket and forms stabilizing hydrogen bonds with the main chain of Gly-441 and to the side-chain oxygen of Gln-485, a member of a proposed catalytic loop. Structural comparison of this inhibitor complex with diphtheria toxin (a mono-ADPRT) and with PARPs [poly(ADP-ribose) polymerases] shows similarity of the catalytic residues; however, a loop similar to that found in ETA is present in diphtheria toxin but not in PARP. The present study provides insight into the important features required for inhibitors that mimic NAD+ and their binding to the mono-ADPRT family of toxins.
A novel NADPH:(bound) NADP+ reductase and NADH:(bound) NADP+ transhydrogenase function in bovine liver catalase
Authors: GAETANI Gian F. , FERRARIS Anna M. , SANNA Paola , KIRKMAN Henry N.
Published: January 2005

Many catalases have the shared property of containing bound NADPH and being susceptible to inactivation by their own substrate, H2O2. The presence of additional (unbound) NADPH effectively prevents bovine liver and human erythrocytic catalase from becoming compound II, the reversibly inactivated state of catalase, and NADP+ is known to be generated in the process. The function of the bound NADPH, which is tightly bound in bovine liver catalase, has been unknown. The present study with bovine liver catalase and [14C]NADPH and [14C]NADH revealed that unbound NADPH or NADH are substrates for an internal reductase and transhydrogenase reaction respectively; the unbound NADPH or NADH cause tightly bound NADP+ to become NADPH without becoming tightly bound themselves. This and other results provide insight into the function of tightly bound NADPH.
Deficiency of PDK1 in liver results in glucose intolerance, impairment of insulin-regulated gene expression and liver failure
Authors: MORA Alfonso , LIPINA Christopher , TRONCHE François , SUTHERLAND Calum , ALESSI Dario R.
Published: January 2005

The liver plays an important role in insulin-regulated glucose homoeostasis. To study the function of the PDK1 (3-phosphoinositide-dependent protein kinase-1) signalling pathway in mediating insulin's actions in the liver, we employed CRE recombinase/loxP technology to generate L(liver)-PDK1−/− mice, which lack expression of PDK1 in hepatocytes and in which insulin failed to induce activation of PKB in liver. The L-PDK1−/− mice were not insulin-intolerant, possessed normal levels of blood glucose and insulin under normal feeding conditions, but were markedly glucose-intolerant when injected with glucose. The L-PDK1−/− mice also possessed 10-fold lower levels of hepatic glycogen compared with control littermates, and were unable to normalize their blood glucose levels within 2 h after injection of insulin. The glucose intolerance of the L-PDK1−/− mice may be due to an inability of glucose to suppress hepatic glucose output through the gluconeogenic pathway, since the mRNA encoding hepatic PEPCK (phosphoenolpyruvate carboxykinase), G6Pase (glucose-6-phosphatase) and SREBP1 (sterol-regulatory-element-binding protein 1), which regulate gluconeogenesis, are no longer controlled by feeding. Furthermore, three other insulin-controlled genes, namely IGFBP1 (insulin-like-growth-factor-binding protein-1), IRS2 (insulin receptor substrate 2) and glucokinase, were regulated abnormally by feeding in the liver of PDK1-deficient mice. Finally, the L-PDK1−/− mice died between 4–16 weeks of age due to liver failure. These results establish that the PDK1 signalling pathway plays an important role in regulating glucose homoeostasis and controlling expression of insulin-regulated genes. They suggest that a deficiency of the PDK1 pathway in the liver could contribute to development of diabetes, as well as to liver failure.
Effect of eprosartan on catecholamines and peripheral haemodynamics in subjects with insulin-induced hypoglycaemia
Authors: CHRISTENSEN Marina , IBSEN Hans , WORCK René
Published: January 2005

ANG II (angiotensin II) facilitates catecholamine release from the adrenal medulla and neuronal NE (noradrenaline) release. Since animal experiments point to specific sympatho-inhibitory properties of the AT1 (ANG II type 1)-receptor blocker EPRO (eprosartan), the primary aim of this study was to clarify if EPRO inhibits sympathetic reactivity in humans as determined by the effect of EPRO on insulin-induced catecholamine release. Sixteen healthy male volunteers were randomized in a double-blind cross-over study to receive a single dose of EPRO (600 mg) compared with placebo, followed by insulin-induced hypoglycaemia [0.15 IU (international unit)/kg of body weight; intravenous bolus] on two study days 1 week apart. From baseline to the end of hypoglycaemia (170 min), the sympatho-adrenal reactivity was mapped by invasive continuous blood pressure monitoring and repeated measurements of FBF (forearm blood flow), arterial and venous concentrations of glucose, catecholamines [EPI (adrenaline) and NE (noradrenaline)], renin, ANG II and aldosterone. EPRO induced an 8–10-fold increase in plasma renin and ANG II concentrations compared with placebo. Plasma glucose decreased equally during placebo and EPRO from baseline 5.9 mmol/l to 1.9 mmol/l and 2.1 mmol/l respectively, inducing a 17-fold increase in arterial EPI concentration at peak. The AUC (area under the curve) during hypoglycaemia for arterial EPI concentrations was 314±48 nmol·min·l−1 in placebo compared with 254±26 nmol·min·l−1 following EPRO treatment (P=0.14). EPRO attenuated the corresponding AUC for the EPI-induced pulse pressure response (4670±219 mmHg·min in EPRO compared with 5004±266 mmHg·min in placebo; P=0.02). Moreover, EPRO caused a less pronounced increase in FBF compared with placebo (402±30 compared with 479±46 ml·100 g−1 of body weight; P=0.04). Musculocutaneous NE release was not affected by EPRO and the AUC for NE release was 51.69±15.5 pmol·min−1·100 g−1 of body weight in placebo compared with 39.35±18.2 pmol·min−1·100 g−1 of body weight after EPRO treatment (P=0.57). In conclusion, EPRO did not significantly inhibit sympathetic reactivity compared with placebo; however, it blunted the haemodynamic responses elicited by the sympatho-adrenal stimulation which only tended to be attenuated by this drug.
Reproducibility of blood flow and post-occlusive reactive hyperaemia as measured by venous occlusion plethysmography
Authors: THIJSSEN Dick H. J. , BLEEKER Michiel W. P. , SMITS Paul , HOPMAN Maria T. E.
Published: January 2005

Venous occlusion plethysmography is commonly used as a tool to assess BF (blood flow) and VR (vascular resistance) at baseline and during PORH (post-occlusive reactive hyperaemia). However, little is known about the reproducibility of this method. The purpose of the present study was to investigate short- (hours) and medium (week)-term reproducibility of forearm, calf and thigh BF and VR at baseline and during PORH. Reproducibility was assessed by the CV (coefficient of variation). In eight subjects, baseline BF and VR of the forearm, calf and thigh were measured using venous occlusion plethysmography (50 mmHg). PORH and minimal VR were measured after 13 min of arterial occlusion (220 mmHg). Reproducibility of baseline forearm and calf BF was acceptable and in agreement with previous studies (CV, 12.9–21.2%). Short- and medium-term reproducibility of thigh BF was good (CV, 5.9% and 8.7% respectively). Baseline VR showed acceptable-to-good reproducibility for forearm, calf and thigh (8.3–22.5%). Forearm PORH showed a CV of 6.1% (short term) and 8.6% (medium term); this was 6.1% (short term) and 6.4% (medium term) for the calf and 6.4% (short term) and 8.0% (medium term) for the thigh. Minimal VR showed good-to-acceptable reproducibility (CV, 6.1–11.7%). In conclusion, forearm, calf and thigh BF and PORH measured by plethysmography have an acceptable-to-good short- and medium-term reproducibility. Short- and medium-term reproducibility of forearm and calf baseline BF are acceptable and thigh baseline BF has a good short- and medium-term reproducibility. Therefore plethysmography is a suitable low-cost tool to assess thigh baseline BF and PORH.
Compound heterozygosity for mutations Asp611→Tyr in KCNQ1 and Asp609→Gly in KCNH2 associated with severe long QT syndrome
Authors: YAMAGUCHI Masato , SHIMIZU Masami , INO Hidekazu , TERAI Hidenobu , HAYASHI Kenshi , KANEDA Tomoya , MABUCHI Hiroshi , SUMITA Ryo , OSHIMA Tohru , HOSHI Naoto , HIGASHIDA Haruhiro
Published: January 2005

LQTS (long QT syndrome) is an inherited cardiac disorder characterized by prolongation of QT interval, torsades de pointes and sudden death. We have identified two heterozygous missense mutations in the KCNQ1 and KCNH2 (also known as HERG) genes [Asp611→Tyr (D611Y) in KCNQ1 and Asp609→Gly (D609G) in KCNH2] in a 2-year-old boy with LQTS. The aim of the present study was to characterize the contributions of the mutations in the KCNQ1 and KCNH2 genes relative to the clinical manifestations and electrophysiological properties of LQTS. Six of 11 carriers of D611Y in KCNQ1 had long QT intervals. D609G in KCNH2 was detected only in the proband. Studies on the electrophysiological alterations due to the two missense mutations revealed that the D611Y mutation in KCNQ1 did not show a significant suppression of the currents compared with wild-type, but the time constants of current activation in the mutants were increased compared with that in the wild-type. In contrast, the D609G mutation in KCNH2 showed a dominant-negative suppression. Our results suggest that the mild phenotype produced by the D611Y mutation in KCNQ1 became more serious by addition of the D609G mutation in KCNH2 in the proband.