bluuebunny/crossref_metadata_2025_split huggingface dataset:
Synthesis, Characterization and in Vitro Study of the Cytostatic and Antiviral Activity of New Polymeric Silver(I) Complexes with Ribbon Structures Derived from the Conjugated Heterocyclic Thioamide 2‐Mercapto‐3,4,5,6‐tetra‐ hydropyrimidine
Authors: Zachariadis Panagiotis C. , Hadjikakou Sotiris K. , Hadjiliadis Nick , Skoulika Stavroula , Michaelides Adonis , Balzarini Jan , De Clercq Erik 
Published: March 2004

AbstractSilver(I) bromide reacts with 2‐mercapto‐3,4,5,6‐tetrahydropyrimidine (StpmH2, C4H8N2S) in DMSO with an excess of triethylamine to give a water‐insoluble complex of formula [Ag6(μ2‐Br)6(μ2‐StpmH2)4(μ3‐StpmH2)2]n (1), while the reaction of silver(I) nitrate with StpmH2 under the same conditions gives a water‐insoluble complex of formula [{Ag4(μ2‐StpmH2)6}(NO3)4]n (2). The products were characterized by elemental analyses, and FT‐IR far‐IR, UV/Vis, 1H and 13C NMR spectroscopy. Crystal structures of complexes 1 and 2 were determined by X‐ray diffraction. Complex 1, C24H48Ag6N12S6, crystallizes in the triclinic system space group P$\bar 1$
, a = 8.041(1) Å, b = 12.838(4) Å, c = 13.281(2) Å, α = 68.40(1)°, β = 72.97(1)°, γ = 87.80(2), Z = 2, forming a one‐dimensional infinite ribbon structure by strong interatomic interactions of two μ2‐Br bonds with Ag(1). Complex 2, C24H48Ag4N16O12S6, crystallizes in the orthorhombic system, space group Cmc21, and a = 32.148(3) Å, b = 9.461(2) Å, c = 7.234(1) Å, α = β = γ = 90°, Z = 8, forming infinite Ag−S−Ag chains which are bridged to each other by a sulfur atom of μ2‐StpmH2 ligands. Complexes 1 and 2 were studied for their cytostatic activity against murine leukemia (L1210) and human T‐lymphocyte (Molt4/C8 and CEM) cells and for their antiviral activity against a wide variety of viruses. They are markedly cytostatic at 50% inhibitory concentration (IC50) values ranging from 3 to 17 μg/mL. None of the compounds showed appreciable antiviral activity at subtoxic concentrations. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
sp3‐C1‐Bridged 1,3‐Me2Cp/Amido Titanium and Zirconium Complexes and Their Reactivities towards Ethylene Polymerization
Authors: Kim Tae Ho , Won Young Chul , Lee Bun Yeoul , Shin Dong Mok , Chung Young Keun 
Published: March 2004

Abstractsp3‐C1‐Bridged Cp/amido titanium and zirconium complexes are synthesized by using novel fulvenes that have substituents in the 1‐, 4‐, and 6‐positions only. Thus, the nucleophilic attack of lithium tert‐butylamide on 1,4‐dimethyl‐6‐phenylfulvene, 6‐(2‐furyl)‐1,4‐dimethylfulvene and 6‐cyclohexyl‐1,4‐dimethylfulvene, and subsequent aqueous workup, yields tBuN(H)−CHR−C5H3Me2 (R = phenyl, 6; R = 2‐furyl, 7; R = cyclohexyl, 8) in 80, 88, and 60% yields, respectively. The reactions of the ligands with M(NMe2)4 (M = Ti or Zr) afford the desired bis(dimethylamido) complexes, which can be transformed by treatment with Me2SiCl2 to the dichloride complexes, (1,3‐Me2C5H2−CHR−NtBu−κN)MCl2 (M = Ti, R = phenyl, 10; M = Zr, R = phenyl, 11; M = Ti, R = 2‐furyl, 13; M = Zr, R = 2‐furyl, 14; M = Zr, R = cyclohexyl, 15). Complex 10 is very sensitive towards water, such that an oxo‐bridged dimer, (1,3‐Me2C5H2−CHPh−NHtBu)ZrCl2−μO−ZrCl2(1,3‐Me2C5H2−CHPh−NHtBu) (12), is easily formed by the abstraction of water from the solvents. The solid structures of (1,3‐Me2C5H2−CHPh−NtBu−κN)Zr(NMe2)2, 10, 11, and 12 have been determined by X‐ray crystallography. The complexes are active towards the polymerization of ethylene when activated with MAO. The activities are rather low and the molecular weight distributions are broad (Mw/Mn = 10−36). (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Synthesis and Characterization of the Diastereomers Λ‐ and Δ‐[Ru(bpy)2(m‐bpy‐Gly‐L‐His‐L‐Lys)]Cl2−1H NMR Studies on Their Interactions with the Deoxynucleotide Duplex d[(5′‐CGCGAATTCGCG‐3′)2]
Authors: Myari Alexandra , Hadjiliadis Nick , Garoufis Achilleas 
Published: March 2004

AbstractThe synthesis and characterization of the diastereomeric complexes Λ‐ and Δ‐[Ru(bpy)2(m‐bpy‐GHK)]Cl2, (GHK = glycine‐L‐histidine‐L‐lysine, m‐bpy = 4′‐methyl‐2,2′‐bipyridine) as well as their binding properties to the deoxynucleotide duplex d(5′‐CGCGAATTCGCG‐3′)2studied by means of NMR, ESI‐MS and CD spectroscopy, are reported. The ROESY spectrum of Λ‐[Ru(bpy)2(m‐bpy‐GHK)]Cl2shows intramolecular cross peaks between the bpy H3 or H3′ protons and the aromatic H2 and H5 of the histidine imidazole ring, indicating that the peptide adopts an orientation with the imidazole ring close to the bpy ligand, possibly interacting by π‐stacking. The absence of intramolecular cross peaks between the peptide and the bipyridine ligands in the ROESY spectrum of Δ‐[Ru(bpy)2(m‐bpy‐GHK)]Cl2on the other hand, shows that in this case the peptide is far from the two bpy ligands, having a different orientation from the Λ isomer. The isomers interact with the oligonucleotide duplex differently. Δ‐[Ru(bpy)2(m‐bpy‐GHK)]Cl2binds in the oligonucleotide major groove close to the central part of the sequence. Λ‐[Ru(bpy)2(m‐bpy‐GHK)]Cl2binds, probably non selectively, approaching the helix from the minor groove. It can be concluded that the peptide (GHK) binding leads the rest of the isomer to interact with the oligonucleotide in the case of the Δ isomer. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Reactivity of 2‐Aminothiazole and 2‐ or 6‐Aminobenzothiazole Derivatives Towards the Triphenylbismuth Diacetate/Catalytic Copper Diacetate Phenylation System
Authors: Miloudi Abdellah , El‐Abed Douniazad , Boyer Gérard , Finet Jean Pierre , Galy Jean Pierre , Siri Didier 
Published: March 2004

AbstractThe copper diacetate catalysed reaction of triphenylbismuth diacetate with 6‐aminobenzothiazole compounds afforded selectively the 6‐phenylamino derivatives in good to high yields. A similar reaction with 2‐aminothiazole or 2‐aminobenzothiazole compounds gave mixtures of the monophenylated and diphenylated products 2‐phenylaminothiazole and 2‐(N‐phenylamino)‐3‐N′‐phenylthiazole derivatives, respectively, with the diphenyl product being predominant. Semi‐empirical calculations with the SAM1/D and CHAIN methods performed on the evolution of a 2‐aminobenzothiazole − copper(III) intermediate were in good qualitative agreement with the experimental results. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Synthesis of Polyfluorinated Nitrogen‐Containing Heterocycles from Hemifluorinated Enones or Organofluorosilicon Building Blocks as Synthetic Equivalents
Authors: Chanteau Frédéric , Didier Benoît , Dondy Boniface , Doussot Pascale , Plantier‐Royon Richard , Portella Charles 
Published: March 2004

AbstractA series of polyfluorinated heterocycles has been prepared by heterocyclisation of hemifluorinated enones or organofluorosilicon synthetic equivalents with different bis(nucleophiles). These polyfluorinated building blocks were obtained by treatment of acylsilanes with perfluoro organometallic reagents. The method is general and has been applied to aliphatic, aromatic and carbohydrate derivatives, to give oxazolidines, imidazolidines, benzodia‐ and ‐thiazepines, quinolines and pyrimidines bearing both a fluorine atom and a perfluoroalkyl group in vicinal positions. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Umpolung of the 5‐Alkyl‐2‐dimethylamino‐1,3‐dithiolium‐4‐thiolate Mesoion and Its Application in the Synthesis of Some New Tetrathiafulvalenes
Authors: Hellberg Jonas , Dahlstedt Emma , Woldegiorgis Andreas 
Published: March 2004

AbstractThe 5‐alkyl‐2‐dimethylamino‐1,3‐dithiolium‐4‐thiolate mesoion could be umpoled with sulfuryl chloride to yield a dicationic electrophile 3 that reacted with various electron‐rich aromatic substrates to yield arylthio‐substituted 1,3‐dithiolium salts 13−25. Two of these compounds have been transformed to the corresponding symmetrical tetrathiafulvalenes 43 and 44, and their cyclovoltammetric behaviour recorded. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Improved Protocol for the Diastereoselective Cyclopropanation of Alkenes using Geminal Dizinc Carbenoids: A Study on the Effect of Zinc Iodide
Authors: Fournier Jean‐François , Charette André B. 
Published: March 2004

AbstractA mixture of ZnI2, EtZnI·2OEt2 and CHI3 produces a gem‐dizinc carbenoid that is an efficient cyclopropanating reagent. The presence of ZnI2 allows for shorter reaction times and cleaner reactions, particularly with less reactive substrates. This modification improves the scope of the reaction and it raises important mechanistic issues about this reaction. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Cre recombinase‐mediated gene targeting of mesenchymal cells
Authors: Florin Lore , Alter Heike , Gröne Hermann‐Josef , Szabowski Axel , Schütz Günther , Angel Peter 
Published: March 2004

AbstractLoss‐of‐function approaches by the Cre/loxP technology have provided powerful tools for functional analyses of genes of interest expressed preferentially in a particular tissue. Here we describe the generation of transgenic mouse lines expressing Cre recombinase under the control of the promoter/enhancer unit of the gene for the α2 chain of collagen type I (Col1α2). As an expression vector, we used a P1‐derived artificial chromosome (PAC), which harbors ∼100 kb carrying the col1α2 gene. The improved coding sequence of the Cre recombinase was introduced to replace the first exon of col1α2. Cre expression was determined by immunohistochemistry and Cre‐mediated onset of β‐galactosidase expression in ROSA26R‐Cre reporter mice. In four analyzed transgenic lines, Cre recombinase was efficiently expressed during embryogenesis and in adult animals in cells of mesenchymal origin, such as dermal fibroblasts, mesenchymal cells of blood vessel walls, and cells in fibrous connective tissues surrounding internal organs. genesis 38:139–144, 2004. © 2004 Wiley‐Liss, Inc.
Cell type‐specific conditional regulation of the c‐myc proto‐oncogene by combining Cre/loxP recombination and tamoxifen‐mediated activation
Authors: Jäger Richard , Maurer Jochen , Jacob Andrea , Schorle Hubert 
Published: March 2004

AbstractDevelopment of inducible genetic switches for in vivo use with transgenic mice has revolutionized many areas in modern molecular biology. Combining two techniques, Cre/loxP‐based genetic recombination and ligand‐dependent activation of a chimeric protein, we generated transgenic mice which allow for the spatiotemporal control of expression and of activity of the proto‐oncogene c‐myc. To these ends, the gene encoding the tamoxifen‐inducible c‐mycERT fusion protein (mycERT) was inserted in the ubiquitously active ROSA 26 gene locus by gene targeting. In the resulting ROSAMER allele, generalized transcription of the mycERT gene is prevented by a preceding transcriptional stop sequence which is flanked by loxP sites. Crosses of ROSAMER transgenic mice with Mox2 cre transgenic mice revealed tight control of mycERT transcription in various tissues unless the transcriptional stop sequence was removed by cre‐mediated excision. Furthermore, we were able to demonstrate tamoxifen‐dependent activation of the MycERT protein in embryonic fibroblasts derived from such mice. As a proof of principle, we demonstrate that primary neural crest cultures established from ROSAMER mice maintain their proliferative capacity in a 4‐OHT‐dependent manner. Furthermore, we demonstrate that such neural crest cells retain their differentiation potential as shown by expression of NF 160, a marker of neuronal differentiation upon 4‐OHT withdrawal. The transgenic mice produced may thus be valuable tools for studying the cell type‐specific effects of c‐myc activity in development and disease. genesis 38:145–150, 2004. © 2004 Wiley‐Liss, Inc.
Myelin‐, reactive glia‐, and scar‐derived CNS axon growth inhibitors: Expression, receptor signaling, and correlation with axon regeneration
Authors: Sandvig Axel , Berry Martin , Barrett Lee B. , Butt Arthur , Logan Ann 
Published: March 2004

AbstractAxon regeneration is arrested in the injured central nervous system (CNS) by axon growth‐inhibitory ligands expressed in oligodendrocytes/myelin, NG2‐glia, and reactive astrocytes in the lesion and degenerating tracts, and by fibroblasts in scar tissue. Growth cone receptors (Rc) bind inhibitory ligands, activating a Rho‐family GTPase intracellular signaling pathway that disrupts the actin cytoskeleton inducing growth cone collapse/repulsion. The known inhibitory ligands include the chondroitin sulfate proteoglycans (CSPG) Neurocan, Brevican, Phosphacan, Tenascin, and NG2, as either membrane‐bound or secreted molecules; Ephrins expressed on astrocyte/fibroblast membranes; the myelin/oligodendrocyte‐derived growth inhibitors Nogo, MAG, and OMgp; and membrane‐bound semaphorins (Sema) produced by meningeal fibroblasts invading the scar. No definitive CSPG Rc have been identified, although intracellular signaling through the Rho family of G‐proteins is probably common to all the inhibitory ligands. Ephrins bind to signalling Ephs. The ligand‐binding Rc for all the myelin inhibitors is NgR and requires p75NTR for transmembrane signaling. The neuropilin (NP)/plexin (Plex) Rc complex binds Sema. Strategies for promoting axon growth after CNS injury are thwarted by the plethora of inhibitory ligands and the ligand promiscuity of some of their Rc. There is also paradoxical reciprocal expression of many of the inhibitory ligands/Rc in normal and damaged neurons, and NgR expression is restricted to a limited number of neuronal populations. All these factors, together with an incomplete understanding of the normal functions of many of these molecules in the intact CNS, presently confound interpretive acumen in regenerative studies. © 2004 Wiley‐Liss, Inc.
Astrocyte cultures from conditional connexin43‐deficient mice
Authors: Theis Martin , Speidel Dina , Willecke Klaus 
Published: March 2004

AbstractConnexin43 (Cx43) mainly provides the molecular basis for astrocytic gap junctions. Interastrocytic coupling is thought to mediate extracellular ion homeostasis, long‐range signaling, and neuroprotection in the brain. Cx43 has been implicated in astrocytic growth control and is also expressed in other cell types in the brain, such as leptomeningeal and vascular cells. Cx43 function has been studied in astrocyte cultures of Cx43‐deficient mice, which lack Cx43 in all cell types. We have generated conditionally deficient mice with an astrocyte‐directed inactivation of Cx43, which leaves expression in other cell types unaffected. Other connexins have been detected in astrocytes. For the study of astrocytes lacking Cx45 and Cx26 in vitro, which deficiencies are embryonic lethal, conditionally deficient astrocyte cultures are essential. In the present study, we describe the developmental kinetics of Cx43 inactivation and loss of intercellular communication in astrocyte cultures derived from conditional Cx43‐deficient mice. Conditional ablation of Cx43 is efficient, reaches a plateau at 4 weeks in culture, but retains Cx43 expression in contaminating nonastrocytic cells. Our findings indicate that conditional knockout astrocytes are a promising tool for the study of embryonic lethal genes in astrocyte cultures. © 2004 Wiley‐Liss, Inc.
Microglia enhance dorsal root ganglion outgrowth in Schwann cell cultures
Authors: Hynds Dianna L. , Rangappa Nagarathnamma , Beest Julia Ter , Snow Diane M. , Rabchevsky Alexander G. 
Published: March 2004

AbstractTransplantation of cellular populations to facilitate regrowth of damaged axons is a common experimental therapy for spinal cord injury. Schwann cells (SC) or microglia grafted into injury sites can promote axonal regrowth of central projections of dorsal root ganglion (DRG) sensory neurons. We sought to determine whether the addition of microglia or microglia‐derived secretory products alters DRG axon regrowth upon cultures of SC. Rat DRG explants were grown on monolayers consisting of either SC, microglia, SC exposed to microglia‐conditioned medium (MCM), or co‐cultures with different relative concentrations of microglia. Image analysis revealed that, compared to SC alone, the extent of neurite outgrowth was significantly greater on SC‐microglia co‐cultures. Immunocytochemistry for extracellular matrix molecules showed that microglial cells stained positively for growth‐promoting thrombospondin, whereas laminin and the inhibitory chondroitin sulfate proteoglycans (CSPGs) were localized primarily to SC. Notably, immunoreactivity for CSPGs appeared reduced in areas associated with DRG outgrowth in co‐cultures and SC exposed to MCM. These results show that microglia or their secreted products can augment SC‐mediated DRG regrowth in vitro, indicating that co‐grafting SC with microglia provides a novel approach to augment sensory fiber regeneration after spinal cord injury. © 2004 Wiley‐Liss, Inc.
Translocation of protein kinase C‐βII in astrocytes requires organized actin cytoskeleton and is not accompanied by synchronous RACK1 relocation
Authors: Pascale Alessia , Alkon Daniel L. , Grimaldi Maurizio 
Published: March 2004

AbstractProtein kinase C (PKC)‐βII is the most abundant PKC isoform in astrocytes. Upon activation, this isoform of PKC translocates from the cytosol to the plasma membrane (PM). In this study, we investigated in astrocytes the modality of PKC‐βII translocation as far as the participation of the receptor for activated C kinase‐1 (RACK1) and the requirement for intact cytoskeleton in the process. In astrocytes, Western blots and immunocytochemistry coupled to confocal microscopic quantitative analysis showed that after 5 min of phorbol‐12‐myristate‐13‐acetate (PMA) exposure, native PKC‐βII, but not PKC‐βI, is relocated efficiently from the cytosol to the PM. Translocation of PKC‐βII was not associated with synchronous RACK1 relocation. Furthermore, the quantity of PM‐associated PKC‐βII that co‐immunoprecipitated with PM‐bound RACK1 increased following PMA exposure, indicating a post activation binding of the two proteins in the PM. Because RACK1 and PKC‐βII relocation seemed not to be synchronous, we hypothesized that an intermediate interaction with the cytoskeleton was taking place. In fact, we were able to show that pharmacological disruption of actin‐based cytoskeleton greatly deranged PKC‐βII translocation to the PM. The requirement for intact actin cytoskeleton was specific, because depolymerization of tubulin had no effect on the ability of the kinase to translocate to the PM. These results indicate that in astrocytes, RACK1 and PKC‐βII synchronous relocation is not essential for relocation of PKC‐βII to the PM. In addition, we show for the first time that the integrity of the actin cytoskeleton plays a specific role in PKC‐βII movements in these cells. We hypothesize that in glial cells, rapidly occurring changes of actin cytoskeleton arrangement may be involved in the fast reprogramming of PKC targeting to specific PM location to phosphorylate substrates in different cellular locations. Published 2003 Wiley‐Liss, Inc.
Apotransferrin promotes the differentiation of two oligodendroglial cell lines
Authors: Paez Pablo M. , García Corina I. , Davio Carlos , Campagnoni Anthony T. , Soto Eduardo F. , Pasquini Juana M. 
Published: March 2004

AbstractWe have previously shown that addition of apotransferrin (aTf) accelerates maturation of oligodendroglial cells (OLGcs) in primary cultures. In this work, we examined the effect of aTf on two conditionally immortalized cell lines: N19 and N20.1. These cells proliferate at 34°C and differentiate into mature OLGcs at 39°C. In vitro addition of aTf to both cell lines at the differentiation temperature for 7 days showed increased expression of galactocerebroside, O4, and myelin basic protein (MBP) and a drop in the percentage of BrdU+ cells. The effect on MBP expression was particularly interesting in the less mature N19 cells. These cells do not express either MBP mRNAs or proteins, so aTf induced, rather than modulated, MBP expression in this cell line. In addition, even though MBP mRNAs for all four isoforms were induced, only the 17 and 21.5 kDa appeared to be translated. OLGc differentiation has been shown to be stimulated by the cAMP‐CREB pathway. In N19 cells, following a pulse of aTf, there was a 10‐fold increase in cAMP levels accompanied by elevated levels of pCREB. In the more mature N20.1 cells, there were no changes in cAMP levels. We conclude that addition of aTf to immature OLGc lines can enhance their expression of differentiated markers, such as MBP. The action of aTf on MBP gene expression in the least mature line is likely to be mediated by the cAMP pathway. In the N20.1 cells, it appears that different signals and/or mechanisms are involved in modulating myelin lipid and MBP expression. The results suggest that aTf can influence OLGc gene expression and differentiation through multiple mechanisms depending on the maturation of the cell. © 2004 Wiley‐Liss, Inc.
GABAA receptors in Müller glial cells of the human retina
Authors: Biedermann Bernd , Bringmann Andreas , Franze Kristian , Faude Frank , Wiedemann Peter , Reichenbach Andreas 
Published: March 2004

AbstractThe present study was aimed at characterizing the GABAA receptor‐mediated currents in acutely isolated glial (Müller) cells of the human retina and investigating their subcellular localization across the Müller cell membrane. Extracellular application of GABA evoked two current responses in human Müller cells: a fast transient GABAA receptor‐mediated current that inactivated within 10 s and that was independent of extracellular Na+, and a sustained current that was dependent on extracellular Na+ and that was mediated by high‐affinity GABA transporters. The receptor current was half‐maximally activated at a GABA concentration of 32 μM, while the transporter current showed an affinity constant of 7.9 μM GABA. The receptor currents were blocked by bicuculline and picrotoxin and were also activated by muscimol or by other amino acids. The receptor currents are Cl− currents, as indicated by the close relationship between the reversal potential of these currents and the Cl− equilibrium potential. Using perforated‐patch recordings, a mean intracellular Cl− concentration of 37 ± 12 mM was determined in human Müller cells. Using electrophysiological and fluorescence imaging methods, it was revealed that GABAA receptors are unevenly distributed across the Müller cell membrane, with higher densities at the endfoot, at the soma, and at the distal sclerad end of the cells. It is concluded that GABAA receptor expression may allow a sensing of retinal GABAergic neuronal signal transmission by Müller cells. © 2004 Wiley‐Liss, Inc.
Astrocyte modulation of in vitro β‐amyloid neurotoxicity
Authors: Paradisi Silvia , Sacchetti Benedetto , Balduzzi Maria , Gaudi Simona , Malchiodi‐Albedi Fiorella 
Published: March 2004

AbstractIn Alzheimer's disease brain, β‐amyloid (Aβ) deposition is accompanied by astrocyte activation, whose role in the pathogenesis of the disease is still unclear. To explore the subject, we compared Aβ neurotoxicity in pure hippocampal cultures and neuronal‐astrocytic cocultures, where astrocytes conditioned neurons but were not in contact with them or Aβ. In the presence of astrocytes, neurons were protected from Aβ neurotoxicity. Neuritic dystrophy was reduced, synapses were partially preserved, and apoptosis was contrasted. The protection disappeared when astrocytes were also treated with Aβ, suggesting that Aβ‐astrocyte interaction is deleterious for neurons. This was supported by comparing Aβ neurotoxicity in pure neurons and neurons grown on astrocytes. In this case, where astrocytes were also in contact with Aβ, neuritic damage was enhanced and expression of synaptic vesicle proteins decreased. Our results suggest that astrocytes can protect neurons from Aβ neurotoxicity, but when they interact with Aβ, the protection is undermined and neurotoxicity enhanced. © 2004 Wiley‐Liss, Inc.