bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of rapid volume changes of substrate‐adherent cells by conventional microscopy 3D imaging
Authors: BOUDREAULT F. , GRYGORCZYK R.
Published: August 2004

Precise measurement of rapid volume changes of substrate‐adherent cells is essential to understand many aspects of cell physiology, yet techniques to evaluate volume changes with sufficient precision and high temporal resolution are limited. Here, we describe a novel imaging method that surveys the rapid morphology modifications of living, substrate‐adherent cells based on phase‐contrast, digital video microscopy. Cells grown on a glass substrate are mounted in a custom‐designed, side‐viewing chamber and subjected to hypotonic swelling. Side‐view images of the rapidly swelling cell, and at the end of the assay, an image of the same cell viewed from a perpendicular direction through the substrate, are acquired. Based on these images, off‐line reconstruction of 3D cell morphology is performed, which precisely measures cell volume, height and surface at different points during cell volume changes. Volume evaluations are comparable to those obtained by confocal laser scanning microscopy (ΔVolume ≤ 14%), but our method has superior temporal resolution limited only by the time of single‐image acquisition, typically ∼100 ms. The advantages of using standard phase‐contrast microscopy without the need for cell staining or intense illumination to monitor cell volume make this system a promising new tool to investigate the fundamentals of cell volume physiology.
Secular Trends in Substance Use: The Conflict and Young People in Northern Ireland
Authors: Higgins Kathryn , Percy Andrew , Mc Crystal Patrick
Published: August 2004

The resolution of political conflict has led some to suggest that Northern Ireland will now face a range of social problems that have been ignored or suppressed by the Troubles. One such area is adolescent drug use. In this article, a review of a range of data sources shows that drug use, with few exceptions, has increased since the emergence of the ongoing peace process. Social and political changes and enhanced paramilitary involvement in the drugs trade appear to have somehow created an environment where drug use has flourished. In reviewing current drug policy and practice, the article highlights the lack of prevention, treatment, and harm reduction services established in Northern Ireland as a cause for concern.
The Development of Intergroup Forgiveness in Northern Ireland
Authors: McLernon Frances , Cairns Ed , Hewstone Miles , Smith Ron
Published: August 2004

As societies like Northern Ireland, Israel, and South Africa strive to resolve social conflict, there is growing theoretical and empirical interest in the role of intergroup forgiveness. This study examined intergroup forgiveness among 340 young adults in Northern Ireland. A short form of the Enright Forgiveness Inventory explored possible influences on propensity to forgive. All participants were Catholic and female (mean age 17.36 years), and had experienced verbal or physical injury or bereavement due to the Northern Irish political violence. Overall forgiveness levels were low in comparison with previous studies of interpersonal forgiveness but similar to previous studies of intergroup forgiveness in Northern Ireland. The strongest (negative) predictor of forgiveness was the perceived degree of hurt caused by the injury.
Processing Constraints, Categorial Analysis, and the Second Language Acquisition of the Chinese Adjective Suffix ‐de(ADJ)
Authors: Zhang Yanyin
Published: August 2004

The study investigates the second language (L2) acquisition of the adjective marker ‐de(ADJ) in Chinese. It explores the interaction between processing constraints as represented in processability theory (Pienemann, 1998) and the learner's categorial analysis of Chinese adjectives and stative verbs (which cross‐categorize) in the acquisition process. An examination of 3 longitudinal L2 data sets revealed more stative verbs (Vstatives) than adjectives in the learners' L2 Chinese. Furthermore, there appeared to be a correlation between the number of adjectives in the L2 samples and the developmental schedule of ‐de(ADJ). The reasons for the findings were explored through the processing procedures required for the structures in which the adjective and the Vstative occur and the impact they had on learners' categorial analysis of adjectives and Vstatives in L2 Chinese.
Semantic Transfer and Its Implications for Vocabulary Teaching in a Second Language
Authors: Jiang Nan
Published: August 2004

This study investigated semantic transfer in second language (L2) learning and provided a replication of the author's study (Jiang, 2002) in a different English as a Second Language (ESL) population. Korean ESL speakers were asked to perform a semantic judgment task in which they decided whether or not 2 English words were related in meaning. Two types of related word pairs served as critical stimuli: word pairs whose 2 members shared or did not share the same Korean translation. The Korean ESL speakers responded to the same‐translation pairs significantly faster than to the different‐translation pairs whereas no such same‐translation effect was found among native speakers of English. The same‐translation effect found in L2 speakers was taken as evidence for the presence of first language semantic structures in L2 lexical representations and their continued involvement in L2 processing. Pedagogical implications for L2 vocabulary teaching are discussed.
MLJ Reviews
Authors: Liskin‐Gasparro Judith E.
Published: August 2004

Books reviewed in this article:

Bowker, Lynne, & Jennifer Pearson. Working with Specialized Language.

Brown, James D., & Theodore S. Rodgers. Doing Second Language Research.

De Kock, Josse, & Carmen Gómez Molina. Lingüística aplicada. La lengua: meta, materia y referencia en investigación, enseñanza y estilística.

Granger, Sylviane, Joseph Hung, & Stephanie Petch‐Tyson. (Eds.). Computer Learner Corpora, Second Language Acquisition, and Foreign Language Teaching.

Gubbins, Paul, & Mike Holt. (Eds.). Beyond Boundaries: Language and Identity in Contemporary Europe.

Haverkate, Henk. The Syntax, Semantics and Pragmatics of Spanish Mood.

Johnson, Karen E., & Paula R. Golombek. Teachers' Narrative Inquiry as Professional Development.

Kikuchi, Masato. A Systematic Approach to Business Japanese.

Lazaraton, Anne. A Qualitative Approach to the Validation of Oral Language Tests.

Leaver, Betty Lou, & Boris Shekhtman. (Eds.). Developing Professional‐Level Language Proficiency.

Medgyes, Peter. Laughing Matters: Humour in the Language Classroom.

Miquel, Lourdes, & Neus Sans. ¿Eres tú, María?

Miquel, Lourdes, & Neus Sans. La llamada de La Habana.

Miquel, Lourdes, & Neus Sans. Lejos de casa.

Miquel, Lourdes, & Neus Sans. Poderoso caballero.

Miquel, Lourdes, & Neus Sans. Por amor al arte.

Miquel, Lourdes, & Neus Sans. Una nota falsa.

Miquel, Lourdes, & Neus Sans. Vacaciones al sol.

Nakajima, Kazuko. (Ed.). Learning Japanese in the Network Society.

Salaberry, M. Rafael, & Yasuhiro Shirai. (Eds.). The L2 Acquisition of Tense‐Aspect Morphology.

Sampaio, João, & Barbara Mcintyre. Colloquial Portuguese: The Complete Course for Beginners.

Schleppegrell, Mary J., & M. Cecilia Colombi. (Eds.). Developing Advanced Literacy in First and Second Languages: Meaning with Power.

Sullivan, Joann Hammadou. (Ed.). Research in Second Language Learning, Vol. 1: Literacy and the Second Language Learner.
We See in the Dark
Authors: Sorensen Roy
Published: August 2004

Do we need light to see? I argue that the black experience of a man in a perfectly dark cave is a representation of an absence of light, not an absence of representation. There is certainly a difference between his perceptual knowledge and that of his blind companion. Only the sighted man can tell whether the cave is dark just by looking. But perhaps he is merely inferring darkness from his failure to see. To get an unambiguous answer, I switch the focus from perceptual knowledge to non‐epistemic seeing. My conclusion is that we see even in the limiting case of absolute darkness – regardless of whether we believe we are seeing. We see little of pratical interest. But in terms of basic information, we see about as much as we do when the lights are on. Depending on what has gone before and after, we may even see ordinary objects.
Odious Comparisons: Incommensurability, the Case Study, and “Small N's” in Sociology
Authors: Steinmetz George
Published: August 2004

Case studies and “small-N comparisons” have been attacked from two directions, positivist and incommensurabilist. At the same time, some authors have defended small-N comparisons as allowing qualitative researchers to attain a degree of scientificity, yet they also have rejected the case study as merely “idiographic.” Practitioners of the case study sometimes agree with these critics, disavowing all claims to scientificity. A related set of disagreements concerns the role and nature of social theory in sociology, which sometimes is described as useless and parasitic and other times as evolving in splendid isolation from empirical research. These three forms of sociological activity—comparative analysis, studies of individual cases, and social theory—are defended here from the standpoint of critical realism. In this article I first reconstruct, in very broad strokes, the dominant epistemological and ontological framework of postwar U.S. sociology. The next two sections discuss several positivist and incommensurabilist criticisms of comparison and case studies. The last two sections propose an understanding of comparison as operating along two dimensions, events and structures, and offer an illustration of the difference and relationship between the two.
Reasons Why
Authors: Tilly Charles
Published: August 2004

Reasons—organized answers to the question “Why does (did, should) X do Y?”—vary between formulas and cause-effect accounts in one dimension and between popular and specialized statements on the other. Conventions, explanatory stories, codified justifications, and technical accounts all qualify as reasons. Choices among types of reasons and contents within each type vary as a function of social relations between givers and receivers. As professional analysts of reasons for social processes as well as of reasons that social actors provide for their actions, sociologists face serious challenges to their credibility. They can reply to those challenges by (1) building records of effective intervention in social affairs; (2) educating audiences in the logic of social science; (3) incorporating their own explanations into widely available explanatory stories; or (4) confining their conversation to each other. Sociologists who want to influence public understanding must adopt some combination of Options 1 to 3.
Presence of immunoglobulin M antibodies around the glomerular capillaries and in the mesangium of normal and passive Heymann nephritis rats
Authors: Barabas Arpad Z. , Cole Chad D. , Barabas Arpad D. , Cowan Jord M. , Yoon Chang Soon , Waisman David M. , Lafreniere Rene
Published: August 2004

Diffuse distribution of small, faintly staining, beaded deposits of rat immunoglobulin M (IgM) around the glomerular capillary blood vessels, and a more intensely staining larger deposition in the mesangium, were observed on the kidney sections of normal rats. As glomerular‐fixed nephritogenic antigens are known to be present on the epithelial aspect of the glomerular basement membrane (GBM), especially at the soles of foot processes and at the slit pores, it was assumed that the IgM antibodies were directed against these antigens. Investigation by immunofluorescent antibody double‐staining techniques of rat kidney sections obtained from normal and rabbit anti‐FX1A‐injected rats stained for the nephritogenic antigen showed that a number of antigenic sites in the glomeruli and in the mesangium shared antibody hits by heterologous rabbit IgG and autologous rat IgM antibodies. Most sites in the glomeruli stained specifically for rat IgM or rabbit IgG, but preferentially for the latter. The intensely fluorescent mesangial deposits stained mainly for rat IgM, indicating that at these sites the antigenic material was virtually saturated, while areas at the entry to the mesangial space also stained for rabbit IgG, indicating that at these locations free nephritogenic epitopes were still available for reaction with the anti‐FX1A antibody. Western blot analysis have shown that the rabbit anti‐rat FX1A IgG and the rat anti‐rat KF3 IgM antibodies are directed against the same renal tubular‐derived antigen with a molecular weight of 70,000. These experimental findings collectively demonstrate that the heterologous IgG and autologous IgM antibodies are directed against the same nephritogenic antigen, which is found in the glomeruli, the mesangium and the proximal convoluted tubules. Thus, the IgM autoantibody has a possible physiological role but, in addition, there is evidence of active immunophagocytic events, manifested in a rapid and continuous entrapment and expulsion of macromolecules after their processing by the mesangial cells of normal and passive Heymann nephritis rats.
Conformations of cell surface oligosaccharides and recognition by lectins from pathogens
Authors: Imberty Anne
Published: August 2004

Pseudomonas aeruginosa is essentially a saprophytic bacterium that can turn into an aggressive pathogen when in contact with damaged and inflamed tissues such as burns and lungs of cystic fibrosis (CF) patients. In all of these cases, the host cell surfaces present altered glycosylations. The most studied example is that of CF where point mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, results in altered ion movements and also influences the N‐glycosylation of the CFTR protein and other cell surface glycoproteins. Increased fucosylation and decreased sialylation with higher levels of Lewis x and Lewis a epitopes correlate to P. aeruginosa‐specific binding. In addition, both the mucus covering the airway epithelia of CF patients and their salivary mucins display higher levels of sialylated and sulfated Lewis oligosaccharides that attract P. aeruginosa.

The galactose‐ and fucose‐binding (PA‐IL and PA‐IIL) lectins of P. aeruginosa contribute to the virulence of this pathogenic bacterium, which is a major cause of morbidity and mortality in CF patients. Database searching in newly sequenced genomes indicates that the presence of these lectins is characteristics of high environmental adaptability of some opportunistic bacteria. The crystal structures of PA‐IL and PA‐IIL lectins have been solved in complex with the corresponding monosaccharides. Molecular modelling studies were used in order to predict the binding mode of PA‐IIL with complex oligosaccharides. Combination of theoretical studies with binding assays give clear indication that the histo‐blood group epitope Lewis a could be the biological ligand present in the lung of CF patients.
Some new insights on the molecular structure and function of heparan sulfate
Authors: Gallagher John
Published: August 2004

It is well established that the heparan sulfate (HS) polysaccharide contains sulfated domains (S‐domains) composed of sequences of N‐ and O‐sulfated disaccharides separated by regions of low sulfation enriched in N‐acetylated disaccharide units. Recent work using the enzyme K5 lyase has shed new light on the molecular organization of HS. K5 lyase is produced by bacteriophage K5A and it facilitates phage infectivity by degrading the polysaccharide coat of Escherichia coli K5; the K5 polysaccharide has an identical structure to the polymeric precursor of HS, named heparan, composed of GlcNAc 1‐4 GlcA repeat units. We found that K5 lyase reduced the average size of HS derived from mouse 3T3 cells from 45 kDa to 7 kDa. Studies of its substrate specificity indicated a strong preference for sequences of four or more N‐acetylated disaccharide units. N‐sulfation, the initial step in the conversion of heparan to HS, inhibited the action of the enzyme. One important consequence of this was that the regions of alternating N‐acetylated and N‐sulfated disaccharides, the NA/NS domains (GlcA 1‐4 GlcNAc 1‐4 GlcA 1‐4 GlcNS‐), that are common in HS are not degraded by K5 lyase. On the basis of these and other findings, we can refine our original model of HS to define these domains as the predicted transition zones between the highly sulfated S‐domains and the N‐acetylated sections of the polymer chain. The close apposition of NA/NS‐ and S‐domains, with their distinctive sulfation patterns, creates composite regions of hypervariable structure with an enhanced potential for imparting specificity to HS–protein interactions. The spacing of the K5 lyase cleavage sites is very similar to that of the heparinase I sites (i.e. the S‐domains) indicating near maximal separation of the unmodified and highly modified regions. The model is suggestive of a regulated mechanism of HS biosynthesis and has implications for the proposed role of HS as a template for the assembly of multiprotein signalling complexes on the cell surface.
Phage display‐derived antibodies: new tools to study heparan sulfate diversity
Authors: Van Kuppevelt Toin
Published: August 2004

Heparan sulfates comprise a heterogeneous group of polysaccharides involved in many basic cellular phenomena including proliferation, migration and differentiation. Their biosynthesis allows for the formation of a large number of different structures. Specific HS structures are likely involved in the binding of HS to proteins, such as growth factors. Anti‐HS antibodies, obvious tools to study structural heterogeneity, are difficult to obtain due to the poor immunogenicity of HS. To circumvent this, we have adopted antibody phage display technology, because this system allows for the generation of antibodies against self‐antigens.

A human semisynthetic antibody phage display library (Nissim et al. 1994) and a semisynthetic library in which the CDR 3 region of the antibodies contained an XBBXBX amino acid sequence (a consensus heparin‐binding site) were used to select antibodies to HS using biopanning strategies.

A large number of antibodies were selected, sequenced and characterized. A number of chemical groups in HS, important for antibody recognition, were identified. Antibodies revealed a large and regulated domain diversity of HS in a number of tissues (Dennissen et al. 2002). The up/down‐regulation of specific HS structures in various pathologies including cancer and nephropathies were identified (Jenniskens et al. 2003; Smetsers et al. 2003). Transfection of cells with cDNAs encoding the antibodies could be used to block HS in vitro and to study its physiological relevance.

It is concluded that phage display‐derived antibodies to HS may provide new tools to study the role of HS in health and disease.
The role of proteoglycans in regeneration and plasticity
Authors: Fawcett James
Published: August 2004

Chondroitin sulfate proteoglycans (CSPGs) are up‐regulated in glial scar tissue and inhibit axon regeneration. Not only are the protein cores up‐regulated, but also there is more glycosaminoglycan (GAG) attached to them. The final stage of GAG synthesis is sulfation, which can occur in three positions. Both 6‐sulfated GAG and the sulfotransferase that sulfates N‐acetylgalactosamine in the 6 position is specifically up‐regulated in glial scar tissue, in inhibitory glial cells and in astrocytes treated with tumour growth factor α (TGF‐α) and TGF‐β. Removal of GAG chains by digestion with chondroitinase or inhibition of GAG synthesis with chlorate or β‐D‐xylosides, removes much of the inhibition from CSPGs in vitro. We therefore tested to see whether GAG digestion by chondroitinase would promote axon regeneration in vivo. We first treated mechanical lesions of the nigrostriatal tract and saw regeneration of about 4% of axons back to their target. Next, dorsal column lesions of the spinal cord at C4 were treated. Both sensory and corticospinal axons regenerated in treated cords, and there was rapid return of function in beam and grid walking tests. The return of function was so rapid that we hypothesized that some of it might be due to enhanced plasticity. Many neuronal cell bodies and dendrites are coated in thick perineuronal nets of inhibitory CSPGs and tenascin‐R, which would certainly be expected to prevent the formation of new synapses. We therefore tested the effects of chondroitinase treatment in a plasticity model: ocular dominance shift in the visual cortex following monocular deprivation. Monocular deprivation in adult animals normally produces no ocular dominance shift. However, in adult animals in which the cortex was treated with chondroitinase, there was a large shift in response to monocular deprivation.
Modulation of heparan sulfate/chemokine interactions using RNA interference
Authors: Arzoumanian Hrant , Bennet Terry , Watson Zara , Carter Noel
Published: August 2004

Introduction  The glycosaminoglycan heparan sulfate (HS) on the luminal surface of the vascular endothelium plays a vital role in promoting leucocyte migration during inflammation via specific interaction chemokines. HS is modified by sulfation and this appears to regulate chemokine binding. We previously observed that under pro‐inflammatory conditions [tumour necrosis factor α (TNF‐α) and interferon‐γ (IFN‐γ)], the endothelial cell line HMEC‐1 showed an increased sulfation HS. This promoted a significant increase binding of the chemokine RANTES and transendothelial migration of leucocytes. TNF‐α and IFN‐γ clearly modulated HS biosynthesis, in part by up‐regulating the NDST 1 and 2 genes responsible for N‐sulfation of the glycan, resulting in structural changes that favour HS/chemokine interactions (Carter et al. 2003). We have attempted to down‐regulate the expression of the gene NDST1 by RNA interference in order to investigate the structural and functional effects of reduced HS N‐sulfation.

Materials and methods  A double‐stranded interference RNA was designed and synthesized in vitro to target the sulfotransferase NDST‐1 and used to tranfect the HMEC‐1 cell line. A number of assays measured its effect on HS structure and function. Cell surface N‐sulfation was measured by flow cytometry using the antibody F58 10E4 (Seikagaku, Japan). Cell surface binding of the chemokine MCP‐1 was visualized by immunohistochemistry. The functional effect of MCP‐1 binding was measured using a trans‐endothelial migration assay.

Results  Surprisingly, the transfected cells appeared to show a small but marked increase in cell surface N‐sulfation. This was consistent with previous results transfecting a full‐length NDST‐1 antisense construct into the cell line, A549 (unpublished). The increase in N‐sulfation observed was supported functional date. Namely, transfected cells show an increased binding of the chemokine MCP‐1 and this promoted a statistically significant increase in transendothelial migration of leucocytes.

Discussion  The results observed were contrary to expectations. It would appear that the interference RNA molecule is in some way interfering in the regulation of the NDST‐1 expression leading to an increase not decrease in gene expression. This has yet to be verified but we are beginning real time expression studies to validate this. We have hypothesized two possible reasons for this observation; endothelial cells may be able to sense a reduction in NDST activity and up‐regulate gene expression. Or the presence of the interference RNA itself promotes NDST‐1 expression. The latter is supported by growing evidence that short double‐stranded RNAs are thought to up‐regulate the pro‐inflammatory STAT transcription factor family (Shuey et al. 2002).
Identification of major heparin‐binding proteins in plasma using electrophoresis and mass spectrometry
Authors: Killeen Ruth , Wait Robin , Begum Shajna , Gray Elaine , Mulloy Barbara
Published: August 2004

Introduction  The anticoagulant glycosaminoglycan heparin binds to the plasma protein antithrombin, potentiating its activity and so inhibiting the coagulation cascade. Among other therapeutic applications, it is used to prevent and treat deep vein thrombosis. As heparin binding is an attribute shared by many proteins other than antithrombin, sequestration by plasma proteins could potentially reduce the available concentration of therapeutically administered heparin, and thus modulates its biological activity. Although the heparin‐binding ability of some abundant plasma proteins is well characterized, the binding potential of the vast majority of plasma constituents is unknown. We have therefore initiated a systematic study of the heparin‐binding plasma proteome, using affinity capture on immobilized heparin, followed by salt elution and protein identification by mass spectrometry.

Materials and methods  Five platelet‐depleted human plasma pools were fractionated by affinity chromatography on HiTrap heparin columns. Bound material was eluted stepwise with 0.5 m, 0.7 m, 1.2 m and 2.2 m NaCl. The approximate protein concentration of each plasma pool and fraction was determined by optical density reading at 280 nm. The fractions were separated by SDS‐PAGE and protein bands were visualized by staining with Coomassie blue or silver nitrate. Stained bands were excised, digested in gel with trypsin, and the liberated peptides were analysed by tandem electrospray mass spectrometry using a Micromass Q‐Tof. Proteins were identified by correlation of the uninterpreted tandem mass spectra to entries in the UniProt database.

Results  Over 50 proteins were identified in whole plasma. No systematic differences in protein composition were observed between the different plasma pools. Heparin‐binding proteins recovered in salt washes of 0.7 m or above included fibronectin, histidine‐rich glycoprotein, apolipoproteins, complement factors C3 and C4b, and antithrombin III. Antithrombin was the major protein eluting at 1.2 m NaCl or above.

Discussion  The rationale of this study was to investigate plasma for the presence of proteins potentially able to compete with antithrombin for therapeutically administered heparin. Of over 50 different plasma proteins identified in this preliminary study, only antithrombin remained bound to heparin above 1.2 m NaCl. We anticipate that use of larger volumes of albumin‐ and immunoglobulin‐depleted plasma will reveal low concentrations of additional high‐affinity binding proteins, but these are unlikely to bind significant amounts of heparin. However, in the present study, we detected several abundant proteins, which bound to immobilized heparin at 0.7 m NaCl, and these may reduce the bioavailability of heparin by nonspecific binding.
Heparan sulfate is essential for neuron migration and axon outgrowth in Caenorhabditis elegans
Authors: Kinnunen Tarja , Townsend Joanne , Turnbull Jeremy
Published: August 2004

Introduction  The importance of heparan sulfate proteoglycans (HSPGs) has been highlighted by a number of human genetic disorders associated with mutations in genes encoding for HSPG protein cores or biosynthetic enzymes required for heparan sulfate assembly.

Materials and methods  We have studied the role of HS and HSPGs in the nervous system development using the nematode Caenorhabditis elegans as a model organism. Our focus has been on the single C. elegans homologues of the vertebrate syndecans, sdn‐1 and heparan 2‐O‐sulfotransferase, hst‐2.

Results and discussion  C. elegans syndecan, sdn‐1 and heparan 2‐O‐sulfotransferase, hst‐2, expressions coincide with the start of morphogenesis in mid‐embryonic stages after most cell divisions have occurred. Sdn‐1 is expressed primarily in neuronal cells, whereas hst‐2 is more widely expressed in neurons, hypodermis (epidermis), pharynx and the gonad leader cells (DTC). A deletion mutant of sdn‐1 which lacks the putative heparan sulfate (HS) attachment sites has specific neuron migration and axon pathfinding defects. Hst‐2 mutants have partially overlapping yet diverse neuronal migration defects. In both sdn‐1 and hst‐2 mutants, the serotonergic hermaphrodite‐specific neurons (HSNs) fail to migrate to their correct position in the vicinity of the vulva and hence fail to innervate the vulval muscles causing sdn‐1 and hst‐2 mutants to become egg‐laying defective (egl) and accumulate embryos within the mother. Canal‐associated neurons (CANs) that, like the HSNs, have a stereotyped long‐distance postembryonic migration pattern also fail to migrate to their correct position in sdn‐1 mutants, whereas in hst‐2 mutants the CANs migrate normally. These results suggest that specific 2‐O‐sulfation of sdn‐1 HS is essential for HSN migration, whereas it is dispensable for CAN migration.

Hst‐2 mutants have additional cell migration defects that are not detected in sdn‐1 mutants suggesting that other HSPGs such as perlecan/unc‐52 and glypican/gpn‐1 may be responsible for these defects.
Studying the role of heparin in the formation of FGF1‐FGFR2 complexes using gel chromatography
Authors: Robinson Christopher J. , Harmer Nicholas J. , Blundell Tom L. , Gallagher John T.
Published: August 2004

Introduction  The fibroblast growth factors (FGFs) are a family of at least 24 proteins which have diverse roles in regulating cell proliferation, migration and differentiation during embryogenesis, angiogenesis and tissue repair. Four FGF receptor tyrosine kinases (FGFRs) have been identified, each of which binds a specific subset of the FGF family. FGF binding induces dimerization of the FGFRs leading to their activation through transphosphorylation. Heparin or heparan sulfate (HS) is essential for the activity of the FGFs, because they form an integral part of the signalling complex on the cell surface. Our group has been using gel filtration FPLC to study the role that heparin plays in the formation of FGF1‐FGFR2 complexes.

Methods  A Superdex 200 HR 10/30 FPLC column was calibrated using protein standards and used to estimate the size of protein complexes which form in the presence of heparin. The column was run in 150 mm NaCl, 50 mm HEPES, pH 7.2 buffer and complexes could be visualized by protein UV absorbance at 280 nm.

Results  In agreement with published data (Pellegrini et al. 2000), we found that 2 : 2 : 1 complexes formed spontaneously in solution between FGF1, FGFR2IIIc and heparin decasaccharides (dp10). Although heparin dp6–8 fragments could bind FGF1 and FGFR2 simultaneously, only dp10 and larger fragments efficiently formed 2 : 2 : 1 complexes. These complexes appear to be relatively stable, as they remain associated following prolonged incubation with heparinase. FGF1 was able to bind heparin saccharides strongly even in the absence of receptor. In contrast, FGFR2 had very little affinity for either FGF1 or heparin dp12 separately, but bound efficiently to the FGF1‐heparin complex. Dimerization of FGF1 appears to be essential for subsequent dimerization of FGFR2. Selectively desulfated heparin saccharides could only bind FGF1 singly and formed 1 : 1 : 1 complexes with FGFR2, which did not then associate with one another.

Discussion  Our data supports the idea that heparin plays a dual role in the formation of FGF1 signalling complexes, firstly by mediating interactions between FGF1 and FGFR2 and secondly by acting as a template for the dimerization of FGF1‐FGFR2 subunits. This technique could be easily adapted to study other heparin/HS‐mediated ligand–receptor interactions and could provide new insights into the mechanisms of formation of multiprotein signalling systems on the cell surface.
The analysis of the fibroblast growth factor ligand‐receptor complex using a quartz crystal microbalance‐dissipation
Authors: Rudd Tim , Duchesne Laurence , Fernig David G. , Nichols Richard J.
Published: August 2004

Introduction  The quartz crystal microbalance‐dissipation (QCM‐D) is a device that measures both the mass of adsorbates upon a surface and the energy dissipated by the surface. The latter quantity, known as dissipation (D), gives information about the viscoelastic properties of species adsorbed to the surface leading to insights into their shape and conformation.

Methods  The fibroblast growth factor (FGF) receptor‐ligand signalling complex is simulated within the QCM‐D by the immobilization of heparin‐derived oligosaccharides to a gold‐sputtered QCM‐D crystal surface, and then the surface is interrogated with various growth factors and a recombinant receptor. Fibroblast growth factor receptor 1 (FGFR1) extracellular domain (immunoglobulin loops II and III and the acid box of the FGFR1IIIc), FGF‐1, FGF‐2, FGF‐7 and hepatocyte growth factor/scatter factor (HGF/SF) all bind to immobilized heparin‐derived oligosaccharides with different changes in D relative to mass for the same surface.

Results  For example, complexes of FGF‐1 and octasaccharide are, on a mass basis, far more dissipative than complexes of HGF/SF. One interpretation of these results is that octasaccharides are relatively flexible, or at least contain hinge regions and that different FGFs have a preference for binding above the hinge (so more dissipative) or on the hinge (so more rigid). Binding of FGFR1 produced complexes that were less dissipative than the FGF‐1 complexes. Intriguingly, deglycosylation of the FGFR1 increased the amount of binding to heparin tetrasaccharide, and this complex on a mass basis is more dissipative.

Conclusion  By using the QCM‐D, we are able to gain insights into novel aspects of these protein‐glycosaminoglycan complexes.
Purification and characterization of endocan (endothelial cell‐specific molecule‐1), a circulating proteoglycan involved in tumour progression and inflammatory diseases
Authors: Sarrazin Stéphane , Bechard David , Lassalle Philippe , Lyon Malcolm , Scherpereel Arnaud , Lortat‐Jacob Hugues , Ouatas Taoufik , Delehedde Maryse
Published: August 2004

Introduction  By virtue of the multiplicity of their protein‐binding partners (e.g. growth factors, cytokines/chemokines), proteoglycans have been shown to be involved in the regulation of a large number of pathophysiological processes including cancer and inflammatory diseases. We have studied and characterized endocan, also called endothelial cell‐specific molecule‐1 (ESM‐1), which represents a new group of circulating proteoglycans. Endocan is mainly expressed by endothelial cells but also by epithelial cells from lung, gut and kidney. Structurally, endocan is constituted of a mature polypeptide of 165 amino acids with a single glycosaminoglycan chain covalently linked to the serine at position 137 (Béchard et al. 2001).

Methods and results  We showed that human umbilical vein endothelial cells expressed endocan specifically with a single chain of dermatan sulfate (DS) as glycosaminoglycan moiety. As shown by surface plasmon resonance, the DS chain directly interacts with cytokines and growth factors including hepatocyte growth factor/scatter factor and could be responsible for endocan's biological activities. Human embryonic kidney 293 cells, which have been genetically engineered to overexpress endocan, induce tumour growth when injected subcutaneously in SCID mice. Moreover, inflammatory cytokines such as TNF‐a and IL‐1 have been shown to increase the synthesis and the secretion of endocan from human umbilical vein endothelial cells.

Conclusion  These results suggest that circulating levels of endocan may represent a novel marker for cancer and inflammatory diseases. Further studies on its GAG structure could help us to better understand the biological activities of endocan and to design future glycomic‐based therapies.
Anti‐inflammatory actions of green tea catechins and ligands of peroxisome proliferator‐activated receptors
Authors: Trekli M. , Buttle D. , Guesdon F.
Published: August 2004

Introduction  Green tea catechins and peroxisome proliferator‐activated receptor (PPAR) agonists have been shown to reduce inflammation associated with collagen‐induced arthritis in mice (Haqqi et al. 1999; Cuzzocrea et al. 2003) and to inhibit other markers of inflammation (Vankemmelbeke et al. 2003; Sabatini et al. 2002; Singh et al. 2003; Jiang et al. 1998). We are investigating the mechanisms by which catechins and PPAR agonists interfere with the signalling pathways of the pro‐inflammatory cytokines, IL‐1 and TNF.

Materials and methods  Epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC), epicatechin (EC) and fenofibrate were from Sigma Aldrich, Poole, UK. Rosiglitazone was from Cayman Chemical, Ann Arbor, MI, USA. Cytokines and IL‐8 ELISA were from NIBSC. IL‐6 ELISA from eBiosciences (San Diego, CA, USA). Cells used were primary fibroblasts from inflamed periodontal tissue or osteoarthritic chondrocytes from discarded tissue following surgery.

Results  We show the significant suppression of cytokine‐induced IL‐6 and IL‐8 release by EGCG, which is dose‐ and cell‐type dependent. The other three catechins have a much lesser effect. Preliminary results show that PPAR agonists also interfere with TNF‐induced IL‐8 production.

Discussion  Green tea has a long history of human consumption, and epidemiological studies have shown that it can reduce inflammatory diseases (Greenwald et al. 2002). Our results confirm that EGCG has the strongest anti‐inflammatory effect, compared with the other three catechins. They also indicate that the mechanism of action of EGCG is cell‐type dependent. This could be because the compound targets components of pro‐inflammatory signalling pathways whose expression is restricted to certain differentiated cell types. Alternatively, the different sensitivities of cells to EGCG could reflect differences in their ability to metabolize the compound. Further experiments are needed to discriminate between these hypotheses.
Analysis of CD44 hyaluronan‐binding domain mutants by NMR
Authors: Wright A.J. , Banerji S. , Noble M. , Jackson D.G. , Campbell I.D. , Day A.J.
Published: August 2004

Introduction  Hyaluronan (HA) is a ubiquitous high molecular mass glycosaminoglycan composed of a repeating disaccharide. CD44, the major cell surface receptor for HA, has a HA‐binding domain (CD44_HABD) at the N‐terminus of the protein, the 3D structure of which has been determined by both NMR and X‐ray crystallography (Teriete et al. 2004); NMR spectra collected on the protein in complex with HA oligosaccharides has allowed us to predict how they may thread across the interaction surface. Amino acids previously implicated in HA binding include R41, Y42, R78 and Y79 (Peach et al. 1993; Bajorath et al. 1998), which form a cluster on the surface of the Link module‐like region (Teriete et al. 2004), as well as residues in the C‐terminal extension (R150, R154, K158 and R162) (Peach et al. 1993). The position of the putative‐binding residues in the C‐terminal segment, and NMR data, led to the hypothesis of two modes of HA binding (Teriete et al. 2004). Here, this hypothesis is tested by NMR studies of single‐site mutants in the context of the CD44_HABD construct.

Materials and methods  Four mutants of CD44_HABD were made, each with a single residue substitution (R150A, R154A, K158A and R162A). These constructs were expressed as 15N‐labelled proteins in Escherichia coli, refolded and purified to homogeneity. 1H–15N HSQC spectra were acquired on the mutants in the presence of varying concentrations of HA hexasaccharide (HA6) and compared to the wild‐type construct to determine changes of protein fold and ligand binding.

Results  The mutants R150A, R154A and K158A have similar HSQC spectra to wild‐type CD44_HABD except for local chemical shift perturbations around the altered residue. Conversely, the R162A mutant has widespread chemical shift differences compared to wild‐type indicating that this mutation disrupts the fold. On binding HA6, the R150A, R154A and K158A mutants all experience shift perturbations similar to that seen with the wild‐type protein.

Discussion  The interaction of HA6 with wild‐type CD44_HABD has been found to cause a significant conformational change in the protein (Teriete et al. 2004). The results here indicate that this ligand‐induced rearrangement can also occur in the R150A, R154A and R158A mutants. Therefore, these mutations do not seem to affect the binding of CD44 to HA6. It has been shown previously that the R162A mutation has reduced affinity for HA compared to wild‐type protein (Peach et al. 1993). This loss of function may be due to the perturbation of the protein fold, and it is possible therefore that R162 does not participate directly in binding. Work is in progress to test the functional activity of these CD44_HABD mutants using ELISA‐like assays and to investigate their chemical shift perturbation in the presence of longer oligosaccharides.
Equity Dilution: An Alternative Perspective on Mortgage Default
Authors: LaCour‐Little Michael
Published: August 2004

Empirical research on mortgage default in the single‐family market has focused on the value of the borrower's put option using house price indices to estimate contemporaneous loan‐to‐value ratio or the probability of negative equity. But since the borrower possesses the option to increase leverage by taking on additional debt secured by junior liens subsequent to loan origination (a phenomenon termed here equity dilution), even a perfect house price adjustment cannot be expected to accurately measure changes in borrower equity over time. Since junior liens are generally unobservable to senior debt holders, proxies are required in empirical applications. This paper employs an independent estimate of junior lien probability developed from the 1998 Survey of Consumer Finances combined with loan level mortgage performance data to examine the role junior liens play in increasing default risk.
Do Riskier Borrowers Borrow More?
Authors: Harrison David M. , Noordewier Thomas G. , Yavas Abdullah
Published: August 2004

Conventional wisdom in the mortgage industry holds that loan‐to‐value (LTV) ratios are positively correlated with mortgage default rates. However, not all empirical studies of mortgage loan performance support this view. This paper offers a theoretical signaling model of why the correlation between LTV ratios and default risk is contingent upon the default costs of the borrower. Specifically, the model proposes that when default costs are high there exists a separating equilibrium in which risky borrowers will self‐select into lower LTV loans to reduce the probability of facing a costly default, while safe borrowers will self‐select into higher LTV loans as a signal of their enhanced creditworthiness. This adverse selection process gives rise to the possibility of higher default probabilities for lower LTV loans. Conversely, when default costs are low the conventional result, in which risky borrowers select higher LTV loans than safe borrowers, is obtained. Empirical results, based on a sample of 859 single‐family residential mortgage loans drawn from the portfolio of a national mortgage lender, are consistent with the separating equilibria predicted by the model.
What Factors Determine International Real Estate Security Returns?
Authors: Hamelink Foort , Hoesli Martin
Published: August 2004

We use constrained cross‐sectional regressions to disentangle the effects of various factors on international real estate security returns. Besides a common factor, pure country, property type, size and value/growth factors are considered. The value/growth measure that is used in this paper provides for each security the relative importance of the value and growth components, rather than a binary classification. The value/growth factor is found to be volatile and to have a substantial effect on returns over the period February 1990–April 2003. Country factors are the dominant factors, and size is shown to have a negative impact on returns. Statistical factors derived by means of cluster analysis explain about one third of specific returns on international real estate securities. The implication for portfolio managers is that failing to recognize the importance of the various factors leads to the portfolio being exposed to systematic risk.
Maintenance of anaesthesia with sevoflurane or isoflurane effects on adverse airway events in smokers
Authors: Wild M. R. , Gornall C. B. , Griffiths D. E. , Curran J.
Published: August 2004

Patients who smoke are at risk of coughing and other adverse airway events during induction of anaesthesia. We have studied the incidence of adverse airway events in smokers under isoflurane or sevoflurane anaesthesia after induction with propofol. Smokers inhaling isoflurane had a 45% incidence of adverse airway events compared to 10% in those inhaling sevoflurane (p = 0.013).
Potential immaturity of the T‐cell and antigen‐presenting cell interaction in cord blood with particular emphasis on the CD40‐CD40 ligand costimulatory pathway
Authors: Han P. , Mcdonald T. , Hodge G.
Published: August 2004

There are reports of immaturity of the neonatal immune system, which may explain the low incidence of graft‐versus‐host‐disease (GVHD) after cord blood transplantation. The CD40 ligand (CD40L)–CD40 interaction is important in regulating the cellular immune response. We hypothesized that the neonatal immune system may show immaturity in this interaction. We studied the function of the CD40L–CD40 interaction in the T‐cell interaction with B cells and monocytes in cord blood compared with adult blood in vitro. Consistent with previous reports, CD4+ T cells do not express CD40L after T‐cell activation. In whole blood, adult monocytes, but not neonatal monocytes, were activated following T‐cell activation. However, the activation of adult monocytes was not dependent on the CD40L–CD40 interaction. Using the CD40L trimer (Lt), we showed that cord B cells have comparable responses to CD40 ligation to those of the adult B cells. Both cord and adult monocytes do not respond as well as B cells and this is probably related to low density of expression of CD40. However, interferon‐γ up‐regulated CD40 on adult monocytes but not on cord monocytes. This potentiated the adult monocyte response to CD40 ligation by CD40Lt. Our findings suggest that the neonatal CD40L–CD40 pair is immature in the cellular immune response involving monocytes and that interferon‐γ fails to activate neonatal monocytes for a response to CD40L. These findings suggest that in the inflammatory microenvironment of cord blood transplantation neonatal monocytes may play a minor role in the effector arm of the immune response. This finding may be one of several mechanisms for the low incidence of GVHD that is observed following cord blood transplantation. Also the ligand‐receptor immaturity may contribute to the increased susceptibility of newborns to certain infections.
Monocytosis and accelerated activation of lymphocytes in C1q‐deficient autoimmune‐prone mice
Authors: Trendelenburg Marten , Manderson Anthony P. , Fossati‐Jimack Liliane , Walport Mark J. , Botto Marina
Published: August 2004

C1q deficiency has been shown to accelerate spontaneous autoimmunity in mice. We studied the time course of activation of monocytes and lymphocytes in autoimmune and non‐autoimmune mice in the presence or absence of C1q as a disease accelerator. Autoimmune MRL\Mp.C1qa−\− and non‐autoimmune C57BL\6.C1qa−\− mice were analysed at various time points between 6 and 33 weeks of age and compared to strain‐ and age‐matched C1q‐sufficient controls. Splenic and peritoneal leucocytes were analysed by flow cytometry and plasma levels of immunoglobulin M (IgM), total IgG, IgG subclasses and IgM autoantibodies were measured. Both C1q‐deficient strains had significantly more splenic monocytes than their controls at all time points analysed. In addition, MRL\Mp.C1qa−\− but not C57BL/6.C1qa−\− mice developed splenic hypercellularity starting at about 12–17 weeks old, had signs of accelerated CD4+ T‐cell activation and showed a marked increase in splenic plasma cells and total serum IgM levels from about 22 weeks of age. The accelerated CD4+ T‐cell activation was not due to a direct inhibitory effect of C1q on T cells. These data show that C1q deficiency causes splenic monocytosis together with accelerated T‐cell activation in an autoimmune‐prone mouse strain.