bluuebunny/crossref_metadata_2025_split huggingface dataset:
Amino acid residues important for substrate specificity of the amino acid permeases Can1p and Gnp1p in Saccharomyces cerevisiae
Authors: Regenberg Birgitte , Kielland‐Brandt Morten C. 
Published: January 2005

AbstractDeletion of the general amino acid permease gene GAP1 abolishes uptake of L‐citrulline in Saccharomyces cerevisiae, resulting in the inability to grow on L‐citrulline as sole nitrogen source. Selection for suppressor mutants that restored growth on L‐citrulline led to isolation of 21 mutations in the arginine permease gene CAN1. One similar mutation was found in the glutamine‐asparagine permease gene GNP1. L‐[14C]citrulline uptake measurements confirmed that suppressor mutations in CAN1 conferred uptake of this amino acid, while none of the mutant permeases had lost the ability to transport L‐[14C]arginine. Substrate specificity seemed to remain narrow in most cases, and broad substrate specificity was only observed in the cases where mutations affect two proline residues (P148 and P313) that are both conserved in the amino acid–polyamine–choline (APC) transporter superfamily. We found mutations affecting six predicted domains (helices III and X, and loops 1, 2, 6 and 7) of the permeases. Helix III and loop 7 are candidates for domains in direct contact with thetransported amino acid. Helix III was affected in both CAN1 (Y173H, Y173D) and GNP1 (W239C) mutants and has previously been found to be important for substrate preference in other members of the family. Furthermore, the mutations affecting loop 7 (residue T354, S355, Y356) are close to a glutamate side chain (E367) potentially interacting with the positively charged substrate, a notion supported by conservation of the side chain in permeases for cationic substrates. Copyright © 2001 John Wiley & Sons, Ltd.
Coherent structures in oscillatory boundary layers
Authors: COSTAMAGNA PAOLA , VITTORI GIOVANNA , BLONDEAUX PAOLO 
Published: January 2005

The dynamics of the vortex structures appearing in an oscillatory boundary layer
(Stokes boundary layer), when the flow departs from the laminar regime, is investigated by means of flow visualizations and a quantitative analysis of the velocity and
vorticity fields. The data are obtained by means of direct numerical simulations of the
Navier–Stokes and continuity equations. The wall is flat but characterized by small
imperfections. The analysis is aimed at identifying points in common and differences
between wall turbulence in unsteady flows and the well-investigated turbulence structure in the steady case. As in Jimenez & Moin (1991), the goal is to isolate the basic
flow unit and to study its morphology and dynamics. Therefore, the computational
domain is kept as small as possible.The elementary process which maintains turbulence in oscillatory boundary layers
is found to be similar to that of steady flows. Indeed, when turbulence is generated,
a sequence of events similar to those observed in steady boundary layers is observed.
However, these events do not occur randomly in time but with a repetition time scale
which is about half the period of fluid oscillations. At the end of the accelerating
phases of the cycle, low-speed streaks appear close to the wall. During the early
part of the decelerating phases the strength of the low-speed streaks grows. Then the
streaks twist, oscillate and eventually break, originating small-scale vortices. Far from
the wall, the analysis of the vorticity field has revealed the existence of a sequence of
streamwise vortices of alternating circulation pumping low-speed fluid far from the
wall as suggested by Sendstad & Moin (1992) for steady flows. The vortex structures
observed far from the wall disappear when too small a computational domain is
used, even though turbulence is self-sustaining. The present results suggest that the
streak instability mechanism is the dominant mechanism generating and maintaining
turbulence; no evidence of the well-known parent vortex structures spawning offspring
vortices is found. Although wall imperfections are necessary to trigger transition to
turbulence, the characteristics of the coherent vortex structures, for example the
spacing of the low-speed streaks, are found to be independent of wall imperfections.
The World Health Organization adult ADHD self-report scale (ASRS): a short screening scale for use in the general population
Authors: KESSLER RONALD C. , ADLER LENARD , AMES MINNIE , DEMLER OLGA , FARAONE STEVE , HIRIPI EVA , HOWES MARY J. , JIN ROBERT , SECNIK KRISTINA , SPENCER THOMAS , USTUN T. BEDIRHAN , WALTERS ELLEN E. 
Published: January 2005

Background. A self-report screening scale of adult attention-deficit/hyperactivity disorder (ADHD), the World Health Organization (WHO) Adult ADHD Self-Report Scale (ASRS) was developed in conjunction with revision of the WHO Composite International Diagnostic Interview (CIDI). The current report presents data on concordance of the ASRS and of a short-form ASRS screener with blind clinical diagnoses in a community sample.Method. The ASRS includes 18 questions about frequency of recent DSM-IV Criterion A symptoms of adult ADHD. The ASRS screener consists of six out of these 18 questions that were selected based on stepwise logistic regression to optimize concordance with the clinical classification. ASRS responses were compared to blind clinical ratings of DSM-IV adult ADHD in a sample of 154 respondents who previously participated in the US National Comorbidity Survey Replication (NCS-R), oversampling those who reported childhood ADHD and adult persistence.Results. Each ASRS symptom measure was significantly related to the comparable clinical symptom rating, but varied substantially in concordance (Cohen's κ in the range 0·16–0·81). Optimal scoring to predict clinical syndrome classifications was to sum unweighted dichotomous responses across all 18 ASRS questions. However, because of the wide variation in symptom-level concordance, the unweighted six-question ASRS screener outperformed the unweighted 18-question ASRS in sensitivity (68·7% v. 56·3%), specificity (99·5% v. 98·3%), total classification accuracy (97·9% v. 96·2%), and κ (0·76 v. 0·58).Conclusions. Clinical calibration in larger samples might show that a weighted version of the 18-question ASRS outperforms the six-question ASRS screener. Until that time, however, the unweighted screener should be preferred to the full ASRS, both in community surveys and in clinical outreach and case-finding initiatives.
Karl Barth: from fighter against the ‘Roman heresy’ to leading thinker for the ecumenical movement
Authors: Welker Michael 
Published: January 2005

Karl Barth saw himself as a ‘Randfigur’, a boundary figure, in ecumenical theology, while important members of the ecumenical movement regarded him as a ‘Wegbereiter de Okumene des 20. Jahrhunderts’, a pioneer of the ecumene in the twentieth century. Which characterisation is correct?The article sheds light on Karl Barth as an ‘ecumenical theologian’ in eight different phases of his life: his wrestling with Roman Catholicism in Göttingen and Munster, particularly with the help of the Munich Jesuit Erich Przywara; his encounter and interaction with ecumenical leaders such as Visser't Hooft and Pierre Maury at the beginning of the Nazi dictatorship and his disappointment about the failing resistance of the ecumenical institutions against Hitler; his search for a clear ecumenical course during the Second World War and the Cold War thereafter; his contribution to the meeting of the World Council of Churches in Amsterdam in 1948 and in the preparation of this meeting; his complex and complicated dealing with the ‘fundamental ecumenical question’ of church and Israel; the reception of his theology in Roman Catholicism in the 1950s and 1960s through von Balthasar, Kung and other young theologians and Barth's interaction with them; Barth's engagement with Vatican II and his trip to Rome; finally, his personal ‘ecumenical existence’ in the last years of his life.The contribution explores continuities and discontinuities in his stance towards ‘ecumenical theology’ – ecumenical theology in its various meanings. It depicts Barth in his journey from a fighter against the ‘Roman heresy’ to a critical pioneer of ecumenical theology in general and the institutionalised ecumene in particular.
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.
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.