bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of prolonged incubation with copper on endothelium‐dependent relaxation in rat isolated aorta
Authors: Chiarugi Alberto , Pitari Giovanni Mario , Costa Rosa , Ferrante Margherita , Villari Loredana , Amico‐Roxas Matilde , Godfraind Théophile , Bianchi Alfredo , Salomone Salvatore 
Published: September 2002

We investigated the effects of prolonged exposure to copper (Cu2+) on vascular functioning of isolated rat aorta.
Aortic rings were exposed to CuSO4 (3–24 h) in Dulbecco's modified Eagle medium with or without 10% foetal bovine serum (FBS) and then challenged with vasoconstrictors or vasodilators in the absence of Cu2+.
Exposure to 2 μM Cu2+ in the absence of FBS did not modify the response to phenylephrine (PE) or acetylcholine (ACh) in aortic rings incubated for 24 h. Identical exposure in the presence of FBS increased the contractile response to 1 μM PE by 30% (P<0.05) and impaired the relaxant response to 3 μM ACh or 1 μM A23187 (ACh, from 65.7±7.1 to 6.2±1.1%, n=8; A23187, from 74.6±8.2 to 12.0±0.8%, n=6; P<0.01 for both). Cu2+ exposure did not affect the relaxant response to NO‐donors.
Impairment of vasorelaxation appeared 3 h after incubation with 2 μM Cu2+ and required 12 h to attain a steady state. Vasorelaxation to ACh was partially restored by 1 mM tiron (intracellular scavenger of superoxide ions; maximum relaxation 34.2±6.4%, n=10, P<0.01 vs Cu2+ alone), whereas catalase, superoxide dismutase or cycloheximide were ineffective.
Twenty‐four hour‐exposure to 2 μM Cu2+ did not affect endothelium integrity or eNOS expression, and increased the Cu content in arterial rings from 6.8±1.1 to 18.9±2.9 ng mg−1 wet weight, n=8; P<0.01.
Our results show that, in the presence of FBS, prolonged exposure to submicromolar concentrations of Cu2+ impaired endothelium‐dependent vasorelaxation in aortic rings, probably through an intracellular generation of superoxide ions.

British Journal of Pharmacology (2002) 136, 1185–1193. doi:10.1038/sj.bjp.0704816
Calcium channel subtypes contributing to acetylcholine release from normal, 4‐aminopyridine‐treated and myasthenic syndrome auto‐antibodies‐affected neuromuscular junctions
Authors: Giovannini F , Sher E , Webster R , Boot J , Lang B 
Published: September 2002

Acetylcholine release at the neuromuscular junction relies on rapid, local and transient calcium increase at presynaptic active zones, triggered by the ion influx through voltage‐dependent calcium channels (VDCCs) clustered on the presynaptic membrane. Pharmacological investigation of the role of different VDCC subtypes (L‐, N‐, P/Q‐ and R‐type) in spontaneous and evoked acetylcholine (ACh) release was carried out in adult mouse neuromuscular junctions (NMJs) under normal and pathological conditions.
ω‐Agatoxin IVA (500 nM), a specific P/Q‐type VDCC blocker, abolished end plate potentials (EPPs) in normal NMJs. However, when neurotransmitter release was potentiated by the presence of the K+ channel blocker 4‐aminopyridine (4‐AP), an ω‐agatoxin IVA‐ and ω‐conotoxin MVIIC‐resistant component was detected. This resistant component was only partially sensitive to 1 μM ω‐conotoxin GVIA (N‐type VDCC blocker), but insensitive to any other known VDCC blockers. Spontaneous release was dependent only on P/Q‐type VDCC in normal NMJs. However, in the presence of 4‐AP, it relied on L‐type VDCCs too.
ACh release from normal NMJs was compared with that of NMJs of mice passively injected with IgGs obtained from patients with Lambert‐Eaton myasthenic syndrome (LEMS), a disorder characterized by a compromised neurotransmitter release. Differently from normal NMJs, in LEMS IgGs‐treated NMJs an ω‐agatoxin IVA‐resistant EPP component was detected, which was only partially blocked by calciseptine (1 μM), a specific L‐type VDCC blocker.
Altogether, these data demonstrate that multiple VDCC subtypes are present at the mouse NMJ and that a resistant component can be identified under ‘pharmacological’ and/or ‘pathological’ conditions.

British Journal of Pharmacology (2002) 136, 1135–1145. doi:10.1038/sj.bjp.0704818
Characterization of the anandamide induced depolarization of guinea‐pig isolated vagus nerve
Authors: Kagaya Manabu , Lamb Jasmine , Robbins Jon , Page Clive P , Spina Domenico 
Published: September 2002

There is considerable interest in elucidating potential endogenously derived agonists of the vanilloid receptor and the role of anandamide in this regard has received considerable attention. In the present study, we have used an electrophysiological technique to investigate the mechanism of activation of vanilloid receptors in an isolated vagal preparation.
Both capsaicin and anandamide depolarized de‐sheathed whole vagal nerve preparations that was antagonized by the VR1 antagonist, capsazepine (P<0.05) whilst this response was unaltered by the cannabinoid (CB1) selective antagonist SR141716A or the CB2 selective antagonist, SR144528, thereby ruling out a role for cannabinoid receptors in this response.
The PKC activator, phorbol‐12‐myristate‐13‐acetate (PMA) augmented depolarization to both anandamide and capsaicin and this response was significantly inhibited with the PKC inhibitor, bisindolylmaleimide (BIM) (P<0.05).
The role of lipoxygenase products in the depolarization to anandamide was investigated in the presence of the lipoxygenase inhibitor, 5,8,11‐Eicosatriynoic acid (ETI). Depolarization to anandamide and arachidonic acid was significantly inhibited in the presence of ET1 (P<0.05). However, in the absence of calcium depolarization to anandamide was not inhibited by ETI.
Using confocal microscopy we have demonstrated the presence of vanilloid receptors on both neuropeptide containing nerves and nerves that did not stain for sensory neuropeptides.
These results demonstrate that anandamide evokes depolarization of guinea‐pig vagus nerve, following activation of vanilloid receptors, a component of which involves the generation of lipoxygenase products. Furthermore, these receptors are distributed in both neuropeptide and non‐neuropeptide containing nerves.

British Journal of Pharmacology (2002) 137, 39–48. doi:10.1038/sj.bjp.0704840
Direct and indirect inhibition by nociceptin/orphanin FQ on noradrenaline release from rodent cerebral cortex in vitro
Authors: Siniscalchi Anna , Rodi Donata , Morari Michele , Marti Matteo , Cavallini Sabrina , Marino Silvia , Beani Lorenzo , Bianchi Clementina 
Published: September 2002

The modulation exerted by nociceptin/orphanin FQ (NC) on noradrenaline (NE) release in rodent cerebral cortex slices and synaptosomes was studied.
Rat, mouse and guinea‐pig cortical slices and synaptosomes were preincubated with 0.1 μM [3H]‐NE and superfused. NE release was evoked by 2 min of electrical (3 Hz) stimulation in slices and by 1 min pulse of 10 mM KCl in synaptosomes.
In rat cortical slices, 0.01–3 μM NC reduced the evoked [3H]‐NE efflux (Emax−54%), with a bell‐shaped concentration‐response curve, which regained its monotonic nature in the presence of either 0.1 μM naloxone (NX) or 30 μM bicuculline. In synaptosomes, the NC effect curve was sygmoidal in shape and reached a plateau at 1 μM concentration.
In the rat, both 1 μM [Phe1ψ(CH2‐NH)Gly2]NC(1‐13)NH2 and 10 μM [Nphe1]NC(1‐13)NH2 (NPhe) antagonised NC‐induced inhibition, without per se modifying [3H]‐NE efflux. The effects of 0.3–1 μM NC concentrations were partially prevented by 1 μM NX; 1 μM D‐Phe‐Cys‐Thr‐D‐Trp‐Orn‐Thr‐Pen‐Thr‐NH2 (CTOP) was also an effective antagonist, but 0.1 μM norbinaltorphimine was not.
In the mouse cerebral cortex, NC‐induced inhibition of NE release (pEC50 6.87, Emax−61%, in the slices) was prevented by Nphe but was NX‐insensitive. In guinea‐pig cortical slices, NC effect (pEC50 6.22, Emax−38%) was prevented by Nphe, but was NX‐insensitive.
These findings demonstrate that NC inhibits NE release from rodent cerebral cortex via presynaptically located ORL1 receptors. In the rat, μ opioid and GABAA receptors are involved as well.

British Journal of Pharmacology (2002) 136, 1178–1184. doi:10.1038/sj.bjp.0704841
Drug resistance in HIV‐1 protease: Flexibility‐assisted mechanism of compensatory mutations
Authors: Piana Stefano , Carloni Paolo , Rothlisberger Ursula 
Published: September 2002

AbstractThe emergence of drug‐resistant variants is a serious side effect associated with acquired immune deficiency syndrome therapies based on inhibition of human immunodeficiency virus type 1 protease (HIV‐1 PR). In these variants, compensatory mutations, usually located far from the active site, are able to affect the enzymatic activity via molecular mechanisms that have been related to differences in the conformational flexibility, although the detailed mechanistic aspects have not been clarified so far. Here, we perform multinanosecond molecular dynamics simulations on L63P HIV‐1 PR, corresponding to the wild type, and one of its most frequently occurring compensatory mutations, M46I, complexed with the substrate and an enzymatic intermediate. The quality of the calculations is established by comparison with the available nuclear magnetic resonance data. Our calculations indicate that the dynamical fluctuations of the mutated enzyme differ from those in the wild type. These differences in the dynamic properties of the adducts with the substrate and with the gem‐diol intermediate might be directly related to variations in the enzymatic activity and therefore offer an explanation of the observed changes in catalytic rate between wild type and mutated enzyme. We anticipate that this “flexibility‐assisted” mechanism might be effective in the vast majority of compensatory mutations, which do not change the electrostatic properties of the enzyme.
The SPOT technique as a tool for studying protein tyrosine phosphatase substrate specificities
Authors: Espanel Xavier , Huguenin‐Reggiani Martine , van Huijsduijnen Rob Hooft 
Published: September 2002

AbstractThe activity of protein tyrosine phosphatases (PTPs) is restricted by their substrate specificities. The analysis of PTP specificity was greatly helped by the discovery that “substrate‐trapping” PTP mutants, such as PTP‐1B D181A, stably and specifically bind their substrates. We have set up a PTP substrate specificity assay based on the SPOT technique, which involves the microsynthesis of (phospho)peptides on membranes. To validate this approach, substrate trapping PTP‐1B was tested on its cognate ligand, the autophosphorylated insulin receptor (IR). On SPOT membranes, IR peptides with phosphotyrosine 1163 were efficiently bound by PTP1B D181A, and dephosphorylated by PTP‐1B. Phosphotyrosine 1163 was preferred over the neighboring 1158 and 1162 phosphotyrosines. PTP‐1B also recognized IR‐like motifs in Trk autophosphorylation domains, and STAT 5 phosphopeptides. Using a gridded 20‐by‐20 SPOT library, we show that peptides with the YZM motif (Z: phosphotyrosine) are the strongest ligands for PTP‐1B D181A, but not the optimal substrates for dephosphorylation by wild‐type PTP1B. In addition we show that PTP‐1B and PTP‐β dephosphorylation efficiency is strongly modulated by the introduction of phospho‐serine or phospho‐threonine in their cognate phospho‐tyrosine substrates. Altogether our data illustrate that the SPOT technique is a highly efficient tool for the study of PTP substrate specificity.
A kinetic study of β‐lactoglobulin amyloid fibril formation promoted by urea
Authors: Hamada Daizo , Dobson Christopher M. 
Published: September 2002

AbstractThe formation of fibrillar aggregates by β‐lactoglobulin in the presence of urea has been monitored by using thioflavin T fluorescence and transmission electron microscopy (TEM). Large quantities of aggregated protein were formed by incubating β‐lactoglobulin in 3–5 M urea at 37°C and pH 7.0 for 10–30 days. The TEM images of the aggregates in 3–5 M urea show the presence of fibrils with diameters of 8–10 nm, and increases in thioflavin T fluorescence are indicative of the formation of amyloid structures. The kinetics of spontaneous fibrillogenesis detected by thioflavin T fluorescence show sigmoidal behavior involving a clear lag phase. Moreover, addition of preformed fibrils into protein solutions containing urea shows that fibril formation can be accelerated by seeding processes that remove the lag phase. Both of these findings are indicative of nucleation‐dependent fibril formation. The urea concentration where fibril formation is most rapid, both for seeded and unseeded solutions, is ∼5.0 M, close to the concentration of urea corresponding to the midpoint of unfolding (5.3 M). This result indicates that efficient fibril formation involves a balance between the requirement of a significant population of unfolded or partially unfolded molecules and the need to avoid conditions that strongly destabilize intermolecular interactions.
Cannabinoid receptor‐G protein interactions: Gαi1‐bound structures of IC3 and a mutant with altered G protein specificity
Authors: Ulfers Amy L. , McMurry Jonathan L. , Miller Alexander , Wang Ligong , Kendall Debra A. , Mierke Dale F. 
Published: September 2002

AbstractThe structure of the C‐terminal region of the third cytoplasmic loop (IC3) of the cannabinoid receptor one (CB1) bound to Gαi1 has been determined using transferred nuclear Overhauser effects (NOEs). The wild‐type IC3 sequence is helical when associated with Gαi1. In contrast, a peptide containing the amino‐acid inversion, Ala341‐Leu342 adopts a single turn. These findings correlate with the attenuated Gi association of CB1 with the Ala341‐Leu342 mutation previously observed in vivo and the diminished stimulation of Gαi1 GTPase activity by the corresponding peptide demonstrated in vitro here. These results, the first to report the structure of a GPCR domain while associated with G protein, imply the C‐terminus of CB1 IC3, a region with high‐sequence conservation among G‐protein coupled receptors, must be helical for efficient coupling and activation of the Gi protein.
Gamma Interferon-Dependent Protection of the Mouse Upper Respiratory Tract following Parenteral Immunization with a Respiratory Syncytial Virus G Protein Fragment
Authors: Plotnicky-Gilquin Helene , Cyblat-Chanal Dominique , Aubry Jean-Pierre , Champion Thierry , Beck Alain , Nguyen Thien , Bonnefoy Jean-Yves , Corvaïa Nathalie 
Published: September 2002

ABSTRACTThe protective mechanisms induced in the mouse upper respiratory tract (URT) after intraperitoneal immunization with G2Na, a recombinant respiratory syncytial virus (RSV) G protein fragment (amino acid residues 130 to 230), were investigated. This protection was recently shown to be mediated by CD4+T cells and to be critically dependent on the cysteines and amino acids 193 and 194 (H. Plotnicky-Gilquin, A. Robert, L. Chevalet, J.-F. Haeuw, A. Beck, J.-Y. Bonnefoy, C. Brandt, C.-A. Siegrist, T. N. Nguyen, and U. F. Power, J. Virol. 74:3455-3463, 2000). On G2Na, we identified a domain (amino acid residues 182 to 198) responsible for the T-helper-cell activity. This region coincided with a peptide designed AICK (residues 184 to 198) which includes the previously identified murine and human T-helper-cell epitope on the native G protein (P. W. Tebbey, M. Hagen, and G. E. Hancock, J. Exp. Med. 188:1967-1972, 1998). Immunization with AICK, in alum or complete Freund's adjuvant, significantly reduced nasal RSV titers in normal BALB/c mice. However, although lung protection was induced, in contrast to the case with live RSV, neither AICK nor G2Na was able to prevent nasal infection in gamma interferon (IFN-γ)-knockout mice. Anti-IFN-γ neutralizing antibodies partially inhibited URT protection after administration to G2Na-immunized BALB/c mice. Furthermore, while purified CD4+T cells from BALB/c mice immunized with G2Na or AICK significantly reduced lung and nasal infection of naive recipient mice after adoptive transfer, the cells from IFN-γ-knockout mice had no effect. Together, these results demonstrated for the first time that the T-helper-cell epitope of RSV G protein induces URT protection in mice after parenteral immunization through a Th1-type, IFN-γ-dependent mechanism.
Exchange of the C-Terminal Part of VP3 from Very Virulent Infectious Bursal Disease Virus Results in an Attenuated Virus with a Unique Antigenic Structure
Authors: Boot Hein J. , ter Huurne A. Agnes H. M. , Hoekman Arjan J. W. , Pol Jan M. , Gielkens Arno L. J. , Peeters Ben P. H. 
Published: September 2002

ABSTRACT
Infectious bursal disease virus (IBDV) is the major viral pathogen in the poultry industry. Live attenuated serotype 1 vaccine strains are commonly used to protect susceptible chickens during their first 6 weeks of life. Wild-type serotype 1 IBDV strains are highly pathogenic only in chickens, whereas serotype 2 strains are apathogenic in chickens and other birds. Here we describe the replacement of the genomic double-stranded RNA (dsRNA) encoding the N- or C-terminal part of VP3 of serotype 1 very virulent IBDV (vvIBDV) (isolate D6948) with the corresponding part of serotype 2 (isolate TY89) genomic dsRNA. The modified virus containing the C-terminal part of serotype 2 VP3 significantly reduced the virulence in specific-pathogen-free chickens, without affecting the distinct bursa tropism of serotype 1 IBDV strains. Furthermore, by using serotype-specific antibodies we were able to distinguish bursas infected with wild-type vvIBDV from bursas infected with the modified vvIBDV. We are currently evaluating the potential of this recombinant strain as an attenuated live vaccine that induces a unique serological response (i.e., an IBDV marker vaccine).
Life Cycle Heterogeneity in Animal Models of Human Papillomavirus-Associated Disease
Authors: Peh Woei Ling , Middleton Kate , Christensen Neil , Nicholls Philip , Egawa Kiyofumi , Sotlar Karl , Brandsma Janet , Percival Alan , Lewis Jon , Liu Wen Jun , Doorbar John 
Published: September 2002

ABSTRACT
Animal papillomaviruses are widely used as models to study papillomavirus infection in humans despite differences in genome organization and tissue tropism. Here, we have investigated the extent to which animal models of papillomavirus infection resemble human disease by comparing the life cycles of 10 different papillomavirus types. Three phases in the life cycles of all viruses were apparent using antibodies that distinguish between early events, the onset of viral genome amplification, and the expression of capsid proteins. The initiation of these phases follows a highly ordered pattern that appears important for the production of virus particles. The viruses examined included canine oral papillomavirus, rabbit oral papillomavirus (ROPV), cottontail rabbit papillomavirus (CRPV), bovine papillomavirus type 1, and human papillomavirus types 1, 2, 11, and 16. Each papillomavirus type showed a distinctive gene expression pattern that could be explained in part by differences in tissue tropism, transmission route, and persistence. As the timing of life cycle events affects the accessibility of viral antigens to the immune system, the ideal model system should resemble human mucosal infection if vaccine design is to be effective. Of the model systems examined here, only ROPV had a tissue tropism and a life cycle organization that resembled those of the human mucosal types. ROPV appears most appropriate for studies of the life cycles of mucosal papillomavirus types and for the development of prophylactic vaccines. The persistence of abortive infections caused by CRPV offers advantages for the development of therapeutic vaccines.
Epstein-Barr Virus-Induced Changes in B-Lymphocyte Gene Expression
Authors: Carter Kara L. , Cahir-McFarland Ellen , Kieff Elliott 
Published: September 2002

ABSTRACTTo elucidate the mechanisms by which Epstein-Barr virus (EBV) latency III gene expression transforms primary B lymphocytes to lymphoblastoid cell lines (LCLs), the associated alterations in cell gene expression were assessed by using 4,146 cellular cDNAs arrayed on nitrocellulose filters and real-time reverse transcription-PCR (RT-PCR). A total of 1,405 of the 4,146 cDNAs were detected using cDNA probes from poly(A)+RNA of IB4 LCLs, a non-EBV-infected Burkitt's lymphoma (BL) cell line, BL41, or EBV latency III-converted BL41 cells (BL41EBV). Thirty-eight RNAs were consistently twofold more abundant in the IB4 LCL and BL41EBV than in BL41 by microarray analysis. Ten of these are known to be EBV induced. A total of 23 of 28 newly identified EBV-induced genes were confirmed by real-time RT-PCR. In addition, nine newly identified genes and CD10 were EBV repressed. These EBV-regulated genes encode proteins involved in signal transduction, transcription, protein biosynthesis and degradation, and cell motility, shape, or adhesion. Seven of seven newly identified EBV-induced RNAs were more abundant in newly established LCLs than in resting B lymphocytes. Surveys of eight promoters of newly identified genes implicate NF-κB or PU.1 as potentially important mediators of EBV-induced effects through LMP1 or EBNA2, respectively. Thus, examination of the transcriptional effects of EBV infection can elucidate the molecular mechanisms by which EBV latency III alters B lymphocytes.
Mitochondrial Targeting and Membrane Anchoring of a Viral Replicase in Plant and Yeast Cells
Authors: Weber-Lotfi Frédérique , Dietrich André , Russo Marcello , Rubino Luisa 
Published: September 2002

ABSTRACT

Replication of the
Carnation Italian ringspot virus
genomic RNA in plant cells occurs in multivesicular bodies which develop from the mitochondrial outer membrane during infection. ORF1 in the viral genome encodes a 36-kDa protein, while ORF2 codes for the 95-kDa replicase by readthrough of the ORF1 stop codon. We have shown previously that the N-terminal part of ORF1 contains the information leading to vesiculation of mitochondria and that the 36-kDa protein localizes to mitochondria. Using infection, in vivo expression of green fluorescent protein fusions in plant and yeast cells, and in vitro mitochondrial integration assays, we demonstrate here that both the 36-kDa protein and the complete replicase are targeted to mitochondria and anchor to the outer membrane with the N terminus and C terminus on the cytosolic side. Analysis of deletion mutants indicated that the anchor sequence is likely to correspond approximately to amino acids 84 to 196, containing two transmembrane domains. No evidence for a matrix-targeting presequence was found, and the data suggest that membrane insertion of the viral proteins is mediated by an import receptor-independent signal-anchor mechanism relying on the two transmembrane segments and multiple recognition signals present in the N-terminal part of ORF1.
Phosphorylation of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein
Authors: Wootton Sarah K. , Rowland Raymond R. R. , Yoo Dongwan 
Published: September 2002

ABSTRACT

Porcine reproductive and respiratory syndrome virus (PRRSV) is a cytoplasmic RNA virus with the unique or unusual feature of having a nucleocapsid (N) protein that is specifically transported to the nucleolus of virus-infected cells. In this communication, we show that the N protein is a phosphoprotein. Phosphoamino acid analysis of authentic and recombinant N proteins demonstrated that serine residues were exclusively phosphorylated. The pattern of phosphorylated N protein cellular distribution in comparison with that of [
35
S]methionine-labeled N protein suggested that phosphorylation does not influence subcellular localization of the protein. Time course studies showed that phosphorylation occurred during, or shortly after, synthesis of the N protein and that the protein remained stably phosphorylated throughout the life cycle of the virus to the extent that phosphorylated N protein was found in the mature virion. Two-dimensional electrophoresis and acid-urea gel electrophoresis showed that one species of the N protein is predominant in virus-infected cells, suggesting that multiple phosphorylated isoforms of N do not exist.