bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bone marrow cells carrying the env‐pX transgene play a role in the severity but not prolongation of arthritis in human T‐cell leukaemia virus type‐I transgenic rats: a possible role of articular tissues carrying the transgene in the prolongation of arthritis
Authors: Abe Asami , Ishizu Akihiro , Ikeda Hitoshi , Hayase Hiroko , Tsuji Takahiro , Miyatake Yukiko , Tsuji Muneharu , Fugo Kazunori , Sugaya Toshiaki , Higuchi Masato , Matsuno Takeo , Yoshiki Takashi 
Published: August 2004

SummaryTransgenic rats carrying the env‐pX gene of human T‐cell leukaemia virus type‐I (env‐pX rats) were immunized with type II collagen (CII), and chronological alterations of arthritis were compared with findings of collagen‐induced arthritis (CIA) in wildtype Wistar–King–Aptekman–Hokudai (WKAH) rats. Arthritis induced by CII in env‐pX rats was more severe and persisted longer than CIA in WKAH rats. To determine whether the phenomenon is caused mainly by the transgene‐carrying lymphocytes or articular tissues, we immunized lethally irradiated env‐pX and WKAH rats with reciprocal bone marrow cell (BMC) transplantation. A severe but transient arthritis was induced by CII in WKAH rats reconstituted by env‐pX BMC (w/tB/CII rats). On the other hand, in env‐pX rats reconstituted by WKAH BMC, arthritis persisted longer than in w/tB/CII rats, although the degree was less at an early phase after CII immunization. These findings suggest that articular tissues rather than the BMCs carrying the env‐pX transgene play a role in the prolongation of arthritis in env‐pX rats, although BMCs carrying the transgene are associated with the severity of arthritis. When inflammatory cytokines in synovial cells isolated from env‐pX rats before they developed arthritis were examined, interleukin‐6 (IL‐6) was detected at a higher level than in synovial cells from WKAH rats, thus suggesting the critical role of IL‐6 in env‐pX arthritis.
Immunohistochemical analysis of heme oxygenase‐1 in preneoplastic and neoplastic lesions during chemical hepatocarcinogenesis§
Authors: Caballero Fabiana , Meiss Roberto , Gimenez Alejandra , Batlle Alcira , Vazquez Elba 
Published: August 2004

SummaryHeme oxygenase (HO) breaks down the pro‐oxidant heme into carbon monoxide, iron and the antioxidant biliverdin. The isoform HO‐1 plays an effective role to counteract oxidative damage and to control inflammation. Prolonged cellular damage due to chronic inflammation is one of the reasons leading to the development of tumours. The aim of this work was to investigate HO‐1 expression and localization along the different stages of chemically induced hepatocarcinogenesis (HCC) and the occurring morphological changes. To provoke sustained oxidative stress and chronic inflammation, CF1 mice received dietary p‐dimethylaminoazobenzene (DAB, 0.5%, w/w) during a whole period of 14 months. HO‐1 expression increased along the experimental trial in morphologically normal hepatocytes in DAB‐treated animals. HO‐1 expression diminished in altered hepatic foci (AHF) and oval cells and early preneoplastic lesions. Otherwise, marked HO‐1 overexpression was detected in Kupffer cells and macrophages surrounding necrotic and nodular areas. Adenomas showed decreased HO‐1 immunostaining. In hepatocellular carcinomas, an inverse relationship was found between the immunohistochemical expression of HO‐1 and the degree of tumour differentiation, being negative in poorly differentiated tumours. In our experimental model, down modulation of HO‐1 expression correlated with malignancy progression. Thus, our data point to activation of HO‐1 as a potential therapeutic tool.
MS strategies for high throughput glycomics and glyco‐proteomics
Authors: Dell Anne , Sutton‐Smith Mark , Panico Maria , Chalabi Sara , Wong Nyet‐Kui , Hitchen Paul , Jang‐Lee Jihye , North Simon , Ledger Victoria , Parry Simon , Haslam Stuart , Morris Howard 
Published: August 2004

Ultra‐high sensitivity mass spectrometric strategies for defining the primary structures of highly complex mixtures of glycopolymers are revolutionizing structural glycobiology in the postgenomic era. MS strategies incorporating MALDI‐MS and ES‐MS/MS enable very complex mixtures from biological extracts and/or glycopolymer digests to be screened, thereby revealing the types of glycans present and, importantly, providing clues to structures that are likely to be functionally important.This lecture will overview strategies employed in our laboratory which enable the glycome of cells, tissues and organs to be examined and the glycoforms of individual glycoproteins to be identified. The types of structural problems that can be solved with current technology will be exemplified by some of our recent research in the following areas:

Development and application of glycomics screening strategies for examining glycosylation changes in cells and tissues of knockout mice.
Defining changes in glycosylation of murine T and B cells upon cytokine activation.
Characterization of the ovarian cancer antigen, CA125.
Sequencing and site analysis of N‐ and O‐glycans in murine ZP3, the putative sperm receptor.
Characterization of mucin glycosylation in cystic fibrosis‐related research.
Glyco‐proteomic investigations of changes in O‐glycosylation of CD8 during T‐cell development.
Glyco‐proteomic analyses of novel glycosylation of bacterial proteins.
Biosynthesis of heparan sulfate – how regulated does it need to be?
Authors: Lindahl Ulf 
Published: August 2004

Heparan sulfate proteoglycans (HSPGs) are ubiquitous in animals. The functional significance of the structural variability exhibited by the constituent HS chains remains a fundamental question in cell and developmental biology. The polysaccharides influence cell behaviour, by modulating the activities of signalling proteins or through interactions in the extracellular matrix. HS is synthesized as a linear polymer of alternating glucuronate (GlcA) and N‐acetylglucosamine (GlcNAc) units. The resultant [GlcA‐GlcNAc]n polymer is partially modified through a series of N‐deacetylation/N‐sulfation, GlcA C5‐epimerization and O‐sulfation reactions (Esko & Lindahl 2001) and may be further modified by selective removal of O‐sulfate groups (Ai et al. 2003). Owing to the variable extent of these reactions in different cells and tissues, and at different developmental stages, the mature HS products display different structures, typically expressed through sulfated domains of variable length and composition (Esko & Lindahl 2001; Casu & Lindahl 2001). These domains provide binding sites for protein ligands and thus constitute the structural basis for the diverse functional roles of HS. The regulated diversity of HS structure is demonstrated through compositional analysis of HS from various organs as well as by immunohistochemical recognition of HS epitopes (van Kuppevelt, T. H et al. 1998). So far, a precise structure–function relation has been defined only for the antithrombin‐binding pentasaccharide sequence (Casu & Lindahl 2001).HS is essential throughout development, as mouse embryos devoid of HS‐polymerizing enzymes fail in gastrulation (Gallagher 2001). By contrast, targeted disruption of genes encoding polymer‐modifying enzymes is associated with surprisingly mild phenotypes. Embryos lacking N‐deacetylase/N‐sulfotransferase‐1, 2‐O‐sulfotransferase (Gallagher 2001) or GlcA C5‐epimerase (Li et al. 2003), thus show major defects incompatible with postnatal life, but also apparently normal organ systems known to rely on HS‐binding morphogens or growth factors for their development. Moreover, transgenic mice overexpressing heparanase appear essentially normal (Zcharia et al. 2004), in spite of an overall drastic reduction in HS chain size. How stringent are structure/function relations in HS–protein interactions?
An ultrastructural investigation of dermatopontin‐knockout mouse corneas
Authors: Cooper L.J. , Bentley A.J. , Nieduszynski I.A. , Fullwood N.J. , Ellis T.S. , Thomson A. , Utani1 A. , Sinkai H. , Brown G.M. 
Published: August 2004

Introduction  The corneal stroma is composed of a network of heterotypic collagen fibrils, proteoglycans and matrix proteins. Transparency of the tissue principally requires the uniformity of fibril diameters and interfibrillar distances and the presence of a quasi‐hexagonal lattice arrangement of parallel fibrils. Keratan sulfate proteoglycans (KSPGs) have a crucial role and the KS chains are clearly required for the maintenance of transparency. Undersulfation of corneal KS results in tissue opacity and the lumican (a KSPG) knockout mouse shows corneal opacity and the disruption of collagen fibril diameters and interfibrillar distances (Chakravarti et al. 1998). Biochemical analysis has shown that dermatopontin is an abundant component of the extracellular matrix and that it interacts with KSPGs via the KS chains. This study aims to determine whether dermatopontin has a direct role in corneal matrix organization by investigating the corneal ultrastructure of dermatopontin‐null (dpt–/–) mouse corneas.Materials and methods  Conventional light microscopy was used to compare the corneal thickness of dpt–/– mice (Takeda et al. 2002) with that of the wild‐type. Collagen fibril distribution was studied using transmission electron microscopy and the datasets analysed using SIS‐pro image analysis software to determine fibrillar volume, shape factor, fibril diameter and spacing.Results  Light microscopy demonstrated that dpt–/– corneas show a 24% reduction in average stromal thickness compared to wild‐type (P < 0.001). The epithelium and Descemet's membrane appear normal. Examination of dpt–/– stroma by transmission electron microscopy indicates a significant disruption to lamellar organization in the posterior region while the central and anterior regions appear largely unaffected compared to wild‐type. The collagen fibrils in dpt–/– stroma show a pronounced increase in interfibrillar spacing as well as exhibiting a lower fibril volume fraction. There is no apparent difference in fibril diameter between dpt–/– and wild‐type mice.Discussion  Collectively, these data suggest that dermatopontin plays a key role in collagen fibril organization and deposition. Like the cornea from lumican‐knockout mice (Chakravarti et al. 1998), the defects in collagen organization in dpt–/– cornea appear to be most severe in the posterior stroma. It is likely that dermatopontin interacts with the KS chains on lumican and this interaction is involved in the maintenance of stromal architecture.
PVA combined with 0.8% alginate: a potential 3‐dimensional vehicle to deliver and retain cells during autologous repair of connective tissues?
Authors: Gargiulo B. , Menage J. , Evans H. , Urban J.P.G. , Caterson B. , Curtis C. , Eisenstein S.M. , Roberts S. 
Published: August 2004

Introduction  Autologous chondrocyte implantation (ACI) is routinely used for the repair of articular cartilage defects. A similar method may be employed to treat degenerate intervertebral discs or other connective tissues. A system in which cells could not only be delivered, but also retained would offer advantages compared to ACI. Such a vehicle would also allow a homogeneous distribution of cells throughout the defect and enhance nutrient penetration to the seeded cells.Materials and methods  Bovine nucleus cells were isolated via enzyme digestion and expanded in number to passage 3. The cells were resuspended in 0.8% alginate and loaded into polyvinyl alcohol (PVA) cubes. These constructs were placed into a solution of calcium chloride to ‘gel’ the alginate. Constructs were cultured in DMEM + 10% FBS within 15‐ml conical tubes rotated at 37 °C for up to 28 days. Cell distribution/morphology and proliferation were assessed on H&E and Ki‐67 stained sections, respectively. The re‐expression of a disc cell phenotype was assessed using toluidine blue staining and immunohistochemistry (with antibodies to collagen types I, II, IIA, VI and X and to the glycosaminoglycans, chondroitin‐4‐ and ‐6‐sulfate and keratan sulfate. RT‐PCR was performed using oligonucleotide primers to collagen types I, II and X, aggrecan, link protein and small leucine‐rich PGs.Results  H&E staining of 10‐µm thick cryosections revealed an even distribution of loaded cells throughout the scaffold at day 1 being maintained through to day 28. Toluidine blue staining showed the presence of GAGs, increasing with time. Ki‐67 staining indicated that approximately 5% of cells were proliferating at all time points. Immunohistochemistry demonstrated the production of collagen types I, II, IIA, VI and X, and the glycosaminoglycans, chondroitin‐4‐, ‐6‐sulfates and keratan sulfate. RT‐PCR results showed mRNA expression of fibromodulin throughout the experiment, lumican at days 14, 21 and 28. Types II and X collagen were present at days 21 and 28.Discussion  Combining 0.8% alginate with PVA retained 100% of the seeded cells and allowed an even distribution of cells throughout the scaffold. The immunohistochemistry and RT‐PCR demonstrated that the system allowed the bovine nucleus cells to express phenotypic markers expressed by disc cells in vivo. These preliminary results indicate that the PVA/alginate system could act as a suitable delivery device for cells during autologous repair of the intervertebral disc or other connective tissues such as meniscus.
An interaction between opticin and heparan sulfate may provide the molecular basis for vitreoretinal adhesion
Authors: John Hindson V. , Gallagher John T. , Bishop Paul N. 
Published: August 2004

Introduction  Opticin is a member of the extracellular matrix small leucine‐rich repeat (SLRP) proteoglycan/protein family, which was originally identified in the eye associated with the collagen fibrils of the vitreous humour. A putative heparin/heparan sulfate (HS) binding motif (RKERKRR) was identified at the N‐terminus of human opticin, but this is absent in the bovine form. Furthermore, the strength of attachment between the vitreous and the retina was observed to be species‐dependent and related to the presence or absence of this motif. We hypothesized that opticin cross‐links the collagen fibrils of the vitreous to HS proteoglycans in the inner limiting lamina (a basement membrane on the inner surface of the retina), contributing towards vitreoretinal adhesion.Materials and methods  Recombinant human and bovine opticin were expressed in 293‐EBNA cells and purified to apparent homogeneity. Solid phase assays and surface plasmon resonance studies were used to characterize interactions between immobilized heparin/HS and opticin.Results  Solid phase and BIAcore data revealed that human opticin binds heparin/HS and binds to heparin with a dissociation constant of approximately 20 nm. By contrast bovine opticin, which lacks the basic cluster, bound severalfold less tightly. Competition studies with heparin oligosaccharides indicated that the heparin/HS binding site is greater than 6 monosaccharides in length. Heparin, HS, chondroitin sulfate A (CS‐A), dermatan sulfate and hyaluronan all competed with heparin for binding to human opticin but CS‐C did not.Discussion  Work to date suggests that the N‐terminal sequence RKERKRR contributes significantly to the binding of opticin to heparin/HS. Vitreoretinal adhesion plays a key role in a number of eye diseases and inhibitors of the opticin–HS interaction could be of therapeutic value.
ADAMTS‐4 activity on a proteoglycan vs. a polypeptide is controlled by the ancillary domains
Authors: Jones Gavin C. , Vankemmelbeke Mireille N. , Verheijen Jan H. , DeDood Claudia , Day Anthony J. , Buttle David J. 
Published: August 2004

Introduction  In combination with the catalytic domain, the ancillary domains of the ADAMTS' are proposed to regulate activity via interactions with sulfated GAGs, the extracellular matrix (ECM) and cell surface. Interactions with both GAGs and the ECM have been attributed to the thrombospondin (TSP) type 1 motifs and spacer region (Kuno and Matsushima 1998; Flannery et al. 2002). ADAMTS‐1, ‐4 and ‐5, all undergo cleavage within their respective spacer regions (Flannery et al. 2002; Rodriguez‐Manzaneque et al. 2000; Georgiadis et al. 2002), an event which has been reported to increase activity towards the interglobular domain of aggrecan and decrease the heparin affinity of ADAMTS‐4 (Flannery et al. 2002; Gao et al. 2002).Materials and methods  V5‐ and His‐tagged recombinant human ADAMTS‐4 constructs terminating after the catalytic (▵DTS), disintegrin‐like (▵TS), TSP (▵S) or spacer region (Full) were expressed in High‐Five cells. Proteoglycanase activities of the resultant proteins were assayed with solution digests of aggrecan and a polyacrylamide‐entrapped aggrecan particle assay. Proteolytic activity was measured using a novel, nonglycosylated, reporter substrate assay.Results  All forms of ADAMTS‐4 were active to varying degrees in the reporter substrate assay. Digestion of aggrecan in solution digests was apparent in all proteins with the exception of the catalytic domain in isolation (▵DTS). Activity towards aggrecan decreased with increasing truncation of the protein.Discussion  Removal of the cysteine‐rich‐spacer domain and further C‐terminal truncations decrease the activity of ADAMTS‐4 towards aggrecan, whilst the proteolytic activity remains intact. Cleavages releasing the ancillary domains of ADAMTS' may therefore alter the catalytic activity of these enzymes against proteoglycans and also nonglycosylated polypeptides. More information is required about potential substrates for the processed forms of ADAMTS‐4.
Conformation of glycosaminoglycans by ion mobility mass spectrometry
Authors: Jin Lan , Barran Perdita E. , Lyon Malcolm , Uhrín Dušan 
Published: August 2004

Introduction  Glycosaminogycans (GAGs) are polysaccharides found on most animal cell surfaces and extracellular matrices. They constitute an important class of biologically active macromolecules that are implicated in the biological activity of morphogens, growth factors, cytokines, chemokines and enzymes. Intervention in signalling pathways they are involved in, represents a valuable therapeutical opportunity for cancer treatment, wound healing and specific organ regeneration. GAGs are linear polysaccharides with alternating uronic acid and hexosamine residues. Their polysaccharide precursors are extensively modified (epimerization, N‐deacetylation N‐, O‐sulfation) creating a wide heterogeneity to their structures. The binding specificity of GAGs is encoded in their primary structures but ultimately depends on how this basis set of functional groups is presented to a protein in three‐dimensional space.Methods  As a part of our efforts to characterize the conformation of GAGs, we present here our preliminary results of conformational studies of heparin‐derived oligosaccharides by ion mobility mass spectrometry. This MS technique is designed to study conformation of mass‐selected gas‐phase ions. When ions drift through an inert gas under the influence of a weak electric field, their arrival time depends on their collisional cross sections. From this data, the experimental cross sectional areas are easily derived. These are then compared with those obtained from candidate geometries generated from molecular modelling, and hence conformation(s) of studied compounds can be determined.Results and discussion  We have measured the cross sectional areas of four heparin oligosaccharides: one disaccharide and three tetrasaccharides. Amongst these are two fully sulfated species and also two tetrasaccharides each with one sulfate group missing. Our samples were prepared by enzymatic digestion of heparin and contain a nonreducing terminal unsaturated glycosyluronic acid ring. We employ the GLYCAM 2000 force field (Woods) and the AMBER (Case et al. 2002) suite of programs to model the gas‐phase conformations of the oligosaccharides. We have generated AMBER parameters for sulfate groups using ab initio calculations and are in the process of generating structures for which we calculate the cross sectional areas using a projection approximation (Wyttenbach et al. 1997). We present a comparison of experimental cross sections with those of calculated models. We also compare them with cross sections calculated using oligosaccharide fragments extracted from X‐ray and NMR structures of heparin oligosaccharides – protein complexes.
New insights into the alternating sequences of heparan sulfate
Authors: Murphy Kevin J. , Lyon Malcolm , Merry Catherine L. R. , Thompson James E. , Roberts Ian S. , Gallagher John T. 
Published: August 2004

Introduction  The sulfated domains (S‐domains) within HS are thought to be the primary binding site for protein ligands (Gallagher and Lyon, 2000). Studies on the binding of multimeric chemokines to HS have suggested that N‐acetylated sequences (NAc‐domains) act to space the S‐domains correctly so binding can occur at more than one HS binding site within a protein (Lortat‐Jacob et al. 1995; Stringer et al. 2002). So far, little functional significance has been ascribed to either alternating N‐sulfate/NAc or lone N‐sulfate types of sequence, although alternating sequences are thought to occur at the borders between S‐domains and NAc‐domains, forming transition zones between the two. Existing scission techniques used to excise oligosacchrides from the intact chain for structural and functional analysis disrupt the transition zones (heparinase III), or the S‐domains (heparinase I) or both (low pH nitrous acid).Materials and methods  Radiolabelled HS was isolated from cultured 3T3 fibroblasts HS. Its patterns of degradation using bacteriophage K5 lyase as well as conventional heparinases I/III and nitrous acid were compared. K5 lyase‐resistant fragments were characterized by size exclusion chromatography and strong‐anion exchange HPLC.Results  The work presented demonstrates how the enzyme K5‐lyase specifically cleaves only within NAc‐domains requiring sequences of at least five consecutive N‐acetylated disaccharides within the HS chain. The enzyme leaves intact S‐domains together with their flanking transition zones in situ.Discussion  The characterization of oligosaccharides generated by K5 lyase cleavage of the intact HS chain provides new insights into the domain structure of HS.