bluuebunny/crossref_metadata_2025_split huggingface dataset:
The human hyaluronan synthase genes: putative mediators of renal fibrosis
Authors: Bowen Timothy , Monslow Jamie , Davies Malcolm , Williams John D. 
Published: September 2009

Introduction  The glycosaminoglycan hyaluronan (HA) is a key component of the vertebrate extracellular matrix and is synthesized by the HA synthase (HAS) enzymes HAS1, HAS2 and HAS3 at the plasma membrane. Accumulating evidence emphasizes the relevance of HA metabolism in clinical nephrological processes such as renal fibrosis (Strutz 2001) and peritoneal mesothelial wound healing (Yung et al. 2000). In the present study, the genomic sequences and organization of the genes encoding the human HAS isoforms were deduced, in silico, from reference cDNA and genomic sequence data and subsequently supported by in vitro data. The region immediately upstream of each HAS gene was then screened for promoter activity.Materials and methods  The in silico methods used in the present study have been described in detail elsewhere (Williams et al. 2002). The programmes BLAST and BLAST2 were used to identify genomic DNA sequences containing the reference cDNA sequences for HAS1, HAS2 and HAS3. Intron/exon boundary sequences were confirmed by sequencing of PCR products from genomic DNA, and each PCR‐amplified promoter region was cloned and analysed by luciferase assay (Hoogendoorn et al. 2003).Results  The HAS1 gene comprised five exons, with the translation start site situated 9 bp from the 3′‐end of of exon 1. In contrast, the genomic structures for HAS2 and both HAS3 variants spanned four exons, exon 1 forming a discrete 5′‐untranslated region and the translation start site located at nucleotide 1 of exon 2. Luciferase analysis of approximately 500 bp of the flanking genomic sequence of each HAS gene showed constitutive promoter activity.Discussion  Excessive HA synthesis in the renal cortex is characteristic of pathological processes such as the inflammation and early matrix expansion that precede renal fibrosis (Strutz 2001). The regulation of expression of the human HAS genes is therefore of great interest. We have used in vitro and in silico methods to deduce the genomic structures of the human HAS genes. Furthermore, we have demonstrated that the sequences immediately upstream of these genomic loci are transcriptionally active. We are continuing with more detailed analysis of these promoters, the data from which will help to evaluate the role of the HAS genes in renal fibrosis.
Bone morphogenetic protein‐1 cleaves prodecorin in vitro and in cellulo
Authors: Gouizi Hanane , Guarrigue‐Antar Laure , Richardson Susan , Kadler Karl 
Published: September 2009

Introduction  Decorin is a ubiquitously distributed small leucine‐rich protein (SLRP) with pivotal roles in collagen fibrillogenesis, regulation of cell matrix deposition as well as cell growth, adhesion and migration. It is expressed in the proform but found mainly in the processed form in various tissues. However, the role of propeptide of prodecorin is still unclear. A possible function is in the trafficking of prodecorin to specific compartment(s) in the cell where it may bind/interact with other ECM molecules prior to secretion. BMP‐1 is a metalloproteinase that is responsible for the processing of various ECM molecules including procollagens and prolysyloxidase. Probiglycan, a SLRP with 57% homology to prodecorin, has also been shown to be a substrate of BMP‐1, which led to the hypothesis that prodecorin could also be processed by the same enzyme. This would emphasize the important role of BMP‐1 in the deposition of the ECM.Materials and methods  To aid detection, Flag‐tag (DYKDDDDK) was introduced into the prodecorin sequence immediately 5′ of the propeptide sequence. This was achieved via a two‐step PCR reaction. The triple‐alanine mutant was generated using the same method replacing the putative BMP‐1 cleavage site EDE (30–32) with AAA residues. Transient and stable transfectants were generated in HT1080 human fibroblast cells. These were chosen because of their ability to express and secrete the mutant prodecorin. Purification of decorin was achieved using anion exchange chromatography. BMP‐1‐Flag was expressed in, and purified from, transfected 293‐EBNA cells.Results  BMP‐1 was assayed for cleavage of prodecorin in vitro. Addition of BMP‐1 to the assay resulted in removal of the prodomain. Addition of EDTA restores detection of prodecorin by anti‐Flag antibody. HT1080 cells doubly transfected with Flag‐tagged prodecorin and BMP‐1‐Flag secreted decorin in the mature form. Control cells secreted uncleaved prodecorin. HT1080 cells doubly transfected with Flag‐tagged triple‐alanine prodecorin mutant and BMP‐1‐Flag did not show cleavage of prodecorin. It was observed that the GAG‐chain addition to this mutant was affected by the mutation.Discussion  BMP‐1 cleaves prodecorin in vitro and in cellulo. The cleaved bond is possibly with the E‐DE (30–32) sequence, but this will be confirmed by cleavage of prodecorin and sequencing of the mature protein. These residues are also important in the initiation and stability of the glycosaminoglycan chain of decorin, because mutation of these results in production of mainly unglycanated prodecorin. This suggests a possible role of the prodomain in the initiation of the GAG chain.
Proteomics of tendon ECM assembly
Authors: Richardson Susan H. , Thornton Dave J. , Kadler Karl E. 
Published: September 2009

Introduction  The ability of tendon to transduce tensile force to the skeleton relies on 10s of 1000s of long (>100 microns) extracellular collagen fibrils that are in parallel register with the long axis of the tendon. Electron microscopy studies of developing mouse‐tail tendon show that at E13.5, the tissue is predominantly composed of cells with little or no matrix. By E15.5, intracellular fibrils can be seen. The long collagen fibrils are deposited one‐by‐one by ‘finger‐like’ projections of the plasma membrane into hexagonally packed bundles of the fibrils. Moreover, the fibrils have a precise interfibrillar spacing of approximately 30 nm. The aim of this work was to identify proteins whose patterns of expression change during tail development in order to understand better the molecular basis of intracellular collagen fibril assembly, tubule trafficking and supramolecular organization of the fibrils in the embryonic mouse tendon.Materials and methods  Fresh embryonic tail tissue was harvested and proteins extracted into buffer containing urea, thiourea, detergent and DTT (DL‐dithiothreitol). Proteins (approximately 100 µg) were first separated by isoelectrofocusing on 24 cm, linear immobilized pH gradient strips (pH 4–7). Strips were reduced with 1% DTT and alkylated with 5% iodoacetamide before second dimension separation by SDS‐PAGE on an 11% gel. Gels were stained with either 0.1% colloidal Coomassie Blue or silver nitrate.Coomassie‐stained gels were used for MS analysis. Protein spots of interest were excised from the 2D gel, destained, reduced, alkylated and digested overnight with trypsin. The resulting peptides were then extracted and analysed by LC‐MS/MS. The data were used to search against SWISSPROT and Tremblnew public protein databases.Results  We have established reproducible extraction conditions and separation methods to obtain 2D gels containing >500 protein ‘spots’ that are visible by Coomassie Blue staining.Discussion  Our approach is to extract proteins from whole embryonic tail from both E13.5 and E15.5 tissues, separate the proteins by 2D‐gel electrophoresis, highlight key changes in the spot profile and identify these proteins by trypsin digestion, q‐TOF mass spectrometry and database matching. A surprising observation was the abundance of intracellular proteins involved in protein folding and trafficking. A poster will be presented that lists the first proteins that we have identified.
Identification, expression and tissue distribution of the three rat lysyl hydroxylase isoforms
Authors: Mercer D.K. , Nicol P.F. , Kimbembe C. , Robins S.P. 
Published: September 2009

Introduction  Lysyl hydroxylases (LHs) (procollagen‐lysine 2‐oxoglutarate 5‐dioxygenase; PLOD) catalyse the hydroxylation of lysine residues during the post‐translational modification of collagenous proteins.Results  In this poster, we describe the first identification and cloning of LH isoforms 2 and 3 from the rat, including both LH2‐splice variants (LH2a and LH2b). The rat LHs are expressed in almost all tissue and cell types examined, indicating a probable lack of tissue specificity for LH function. All LH isoforms were stably transfected into CHO‐K1 cells, and this represents the first example of recombinant LH production in a eukaryotic cell line. Expression and production of all LH isoforms led to an increase in total collagen synthesis. LH1 and LH2a expression and production led to an increase in total pyridinium cross‐link production.Discussion  Evidence that LH2a possesses telopeptide lysyl hydroxylase activity, previously thought to be a novel enzyme, is presented.
The role of glutamate signalling in rheumatoid arthritis
Authors: Flood S.L. , Duance V.C. , Mason D.J. 
Published: September 2009

Introduction  Rheumatoid arthritis (RA) is characterized by joint inflammation and destruction mediated by enhanced secretion of degradative enzymes and cytokines. Inflammation of the joint is accompanied by elevated levels of glutamate within the synovial fluid (McNearney et al. 2000). Glutamate has been shown to modulate bone cell phenotype (Chenu et al. 1998). We hypothesize that the elevated glutamate in synovial fluid that accompanies RA can induce phenotypic changes associated with synovial joint destruction.Methods  The mRNA expression of glutamate receptors and transporters was investigated in tissues of the rat knee joint by RT‐PCR to determine which cell types are potentially responsive to glutamate. To determine whether extracellular glutamate affects synoviocyte phenotype, we have investigated the effect of different glutamate concentrations on human, primary, RA synoviocytes' enzyme and cytokine expression. Matrix metalloproteinases (MMPs) and tissue inhibibitors of MMPs (TIMPs) protein expression was determined by zymography, and IL‐6 release by RA synoviocytes was measured using an IL‐6 ELISA (R & D systems).Results  RT‐PCR has revealed glutamate transporter (GLAST‐1) and ionotropic and metabotropic receptor mRNA expression in several tissues of the rat knee joint. Pro‐MMP2, TIMP1 and TIMP2 activity were all increased in the presence of high extracellular glutamate concentrations and in the presence of glutamate transporter inhibitors. The presence of glutamate transporter inhibitors also increased production of the cytokine IL‐6 but only at low glutamate concentrations.Discussion  Our data show that many cells of the synovial joint have potential for responding to glutamate signals. Primary synoviocytes from RA patients increase MMP and TIMP expression in response to high extracellular glutamate and in the presence of glutamate transporter inhibitors which also increase IL‐6 production. We postulate therefore that the high levels of glutamate present in RA synovial fluid are having a pro‐inflammatory effect on synoviocytes. This may be mediated through the cytokine IL‐6, the synovial fluid levels of which correlate with RA disease activity. We have previously shown that IL‐6 treatment of RA synoviocytes induces the glutamate transporter (GLAST‐1) expression therefore indicating a feedback mechanism between IL‐6 and GLAST‐1 expression. We are currently investigating the effect of ionotropic glutamate receptor antagonists on the pheontype of RA synoviocytes to elucidate the specific signalling mechanisms involved.
Regulation of the small GTPase RhoA by syndecan‐4 and integrins in focal adhesion formation
Authors: Yoneda Atsuko , Dovas Athanassios , Multhaupt Hinke A.B. , Couchman John R. 
Published: September 2009

Introduction  The transmembrane heparan sulfate proteoglycan, syndecan‐4, and integrins are important receptors for focal adhesion (FA) formation on fibronectin (FN) substrates. The small GTPase RhoA is also known to regulate FA and stress fiber formation. It has been suggested that syndecan‐4 and integrins co‐operatively regulate the assembly of FA in a Rho‐dependent manner, but the mechanism is unclear. Here, we examined the function of RhoA and the Rho effector kinases ROCKs in syndecan‐4 signalling on the process of FA formation and the possible mechanism by which syndecan‐4 may regulate RhoA activity.Methods  Primary rat embryonic fibroblasts (REFs) were seeded on FN or ‘RGD’‐containing integrin‐binding domain of FN and lysed at various time points. The amount of active form of RhoA in each lysate was analysed by pull‐down experiments.Results and discussion  The relative activities of RhoA showed one peak in the process of FA formation on FN, whereas no peak was obtained on the integrin‐binding domain. The one peak of RhoA activity on integrin‐binding domain was restored by addition of heparin‐binding domain into medium. These results suggested that a signal through syndecan‐4 link to the Rho pathway. Both ROCK‐I and ‐II isozymes were present in REF cell lysates and each could be specifically immunoprecipitated. The ROCK kinase activities in immunoprecipitates were analysed using GST‐myosin light chain as a substrate. The amount of ROCK‐I and ‐II activities changed through the adhesion process on FN and appeared to be independently regulated. Therefore, one or both ROCKs may be downstream of a syndecan‐4‐mediated signalling response through RhoA. The core protein of syndecan‐4 can directly bind to and activate PKC‐α. We found that PKC‐α could phosphorylate Rho‐Guanine Nucleotide Dissociation Inhibitor (GDI) in vitro. It has been suggested that PKC‐α‐mediated phosphorylation of Rho GDI stimulates GDI dissociation, thereby resulting in Rho activation. It is possible that syndecan‐4 regulates Rho/ROCK pathway through PKC‐α activation on the process of FA formation.
NADPH oxidases and MMP‐9/TIMP‐1 balance in human glomeruli: putative links to diabetic nephropathy
Authors: Millet C. , Trocmé C. , Vergnaud S. , Papacatzis S. , Descotes J.L. , Morel F. , Zaoui P. 
Published: September 2009

Study aim  Glomerular basement membrane thickening, the hallmark of diabetic nephropathy, is thought to be related to an enhanced oxidative stress and reduced matrix proteolysis. Our study concerned the mRNA and protein expression of NADPH oxidase (NOX) components, MMP‐2, MMP‐9 and TIMP‐1 in freshly isolated human glomeruli as well as enzymatic activities and their modulation by glucose, H2O2 and angiotensin‐2.Material and methods  NOX, cytosolic and membrane‐bound associated proteins and mRNA were analysed by RT‐PCR and Western blotting after glomerular extraction. Oxidase activity was identified by cytochrome c reduction and chemiluminescence. Gelatinases and inhibitors were semiquantitatively assessed by RT‐PCR, gelatin zymography and ELISA in a model of glomerular conditioned survival.Results  NOX‐2, NOX‐4 and membrane‐bound and cytosolic factors could be observed in freshly extracted glomeruli (RNA + protein). p40phox, p67phox and p47phox molecular weights were increased compared to their phagocytic counterparts advocating for specific glomerular analogues, and a slight specific oxidase activity was retrieved in isolated glomeruli. Also, mRNA coding for MMP‐2, ‐9 and TIMP‐1, ‐2 were detected. High glucose concentrations (25 mm) reduced TIMP‐1 release in glomerular survival media and MMP‐2 activity in glomerular extracts. On the opposite, angiotensin‐2 significantly induced MMP‐2 and ‐9 activities in the survival media as well as H2O2 in glomerular extracts, while addition of 25 mm glucose blunted these findings.Conclusion  Glomerular matrix remodelling, the backbone of renal fibrosis in diabetic patients, could be induced by H2O2 from specific glomerular NADPH oxidases under the influence of extra‐cellular glucose and angiotensin‐2 and could participate in the control of MMP activities.
Up‐regulated lactate, TGF‐β and collagen synthesis in the ischaemic skin of patients with peripheral vascular disease
Authors: Dalton S.J , Whiting C.V , Mitchell D.C. , Tarlton J.F. 
Published: September 2009

Introduction  We have previously shown that dermal hypoxia alters collagen turnover in chronically ischaemic skin; however, no mechanism for this has been determined. In cultured human dermal fibroblasts, hypoxia causes an up‐regulation of collagen synthesis, probably mediated by TGF‐β (Falanga et al. 2002) in the presence of increased lactate. Here, we examine whether these processes are present in vivo in chronically ischaemic skin.Materials and methods  Paired biopsies of uninjured skin were harvested at below knee amputation from 16 patients with a history of peripheral vascular disease (PVD), following the quantification of ischaemia, using the ankle brachial pressure index (ABPI) and by lactate measurement. Nonischaemic samples were taken proximally from the amputation resection margin and ischaemic samples from a predetermined distal site. Site‐matched biopsies were taken for control at total knee replacement and varicose vein operations. Lactate levels were measured using enzymatic determination (Sigma, UK), collagen type‐I synthesis was determined by immunoassay for released C‐terminal propeptide (PICP) (Prolagen C, Quidel) and TGF‐β by ELISA. TGF‐β RI and RII were localized using immunohistochemistry.Results  The ABPI in all patients with PVD was <0.4 indicating severe ischaemia. Levels of lactate were elevated in the ischaemic tissue of these patients when compared to nonischaemic samples (P < 0.001), and an up‐regulation of collagen type‐I synthesis was demonstrated in the ischaemic samples (P < 0.01). Levels of TGF‐β were also raised (P < 0.05). TGF‐β RI and RII were expressed on dermal fibroblasts, keratinocytes and endothelial cells.Discussion  Increased lactate levels resulting from hypoxic metabolism have been demonstrated in skin flaps of animal models (Hoopes & Im 1978); however, lactate levels in human tissue, as a direct assessment of chronic ischaemia, have not previously been reported. Hypoxia, lactate and TGF‐β have been shown to stimulate collagen synthesis in vitro (Falanga et al. 2002; Cerbon‐Ambriz et al. 1991) but not in vivo in PVD. These findings are consistent with the hypothesis that chronic hypoxia leads to changes in the ECM of uninjured but ischaemic skin and may predispose it to dermal failure.
The importance of elastin and fibulin‐5 on spine development: a study of elastin KO and fibulin‐5 KO on the development of vertebral body and intervertebral disc
Authors: Yu Jing , Mcleans Sean , Yanagisawa Hiromi , Winlove C. Peter , Roberts Sally , Mecham Robert P. , Urban Jill 
Published: September 2009

Introduction  Idiopathic scoliosis is the most common scoliosis and generally develops during juvenile or adolescent growth spurt (Stehbens 2003). It was reported that elastic fibre system might play a role in some idiopathic scoliosis patients (Hadley‐Miller et al. 1994). Indeed, transgenic mice with defects in elastic fibre system (including elastin null and fibulin‐5 null) appear to develop severe kyphoscoliosis. The aim of this study was to understand how such defects exert their effect on the spinal column.Materials and methods  Newborn elastin KO and 16‐week‐old fibulin‐5 KO mice spines were fixed in 10% formalin. Paraffin‐embedded sections (20 µm) were stained with haematoxylin & eosin (H&E) and alcian blue after the sections were dewaxed and rehydrated. The sections were examined by light microscopy.Results  H&E staining revealed that those newborn elastin KO mice seem to have a delayed ossification in vertebral bodies compared to that of wild‐type. Also, cell morphology in IVD appears very much different. Cells in outer annular (OA) and endplate regions are much round‐like comparing fibroblast‐like cells in wild‐type. In addition, GAG expressions showed by alcian blue staining appear much sparse and irregular in the matrix of IVD in newborn elastin KO mice. Fibulin‐5 KO mice (16 weeks) seem to have many cell clusters or clones in the growth plate, which is an indication of abnormal growth. Our results reveal the importance of elastin and fibulin‐5 on the development of spine.Discussion  Kypho‐scoliosis is a spinal deformity. Several different spinal tissues, e.g. muscle, vertebrae, IVD and ligament, are involved in the stability and load carriage of the spinal column.Some gene defects in these load‐bearing structures can lead to the scoliotic deformity, e.g. elastin null, fibulin‐5 null, collagen‐II null (Aszodi et al. 1998), perlecan null (Costell et al. 1999) and LTBP3‐null (Dabovic et al. 2002) as well as a mutant in a muscle‐specific protein (Blanco et al. 2001), but other defects [such as collagen‐IX KO's (Kimura et al. 1996)] do not. The relationship between defects in these structures and development of scoliotic is still unclear.
Factors affecting oxygen concentration gradients across articular cartilage
Authors: Zhou Shengda , Cui Zhanfeng , Urban Jill P.G. 
Published: September 2009

Introduction  Although it is commonly stated that oxygen concentrations fall to below 1% oxygen in the deep zone of articular cartilage, the evidence for this is sketchy and there is little information on the oxygen concentrations found in cartilage in vivo. The oxygen levels could be important as a number of studies have showed that low oxygen concentrations affect cartilage metabolism adversely (Grimshaw & Mason 2000; Lee & Urban 1997). As an initial step in determining oxygen concentration profiles across cartilage, we have measured the rate of oxygen consumption by chondrocytes. We then used this information to calculate the effect of various factors, such as presence of serum, cell density and perfusion from the subchondral bone on oxygen gradients across the joint.Methods  Chondrocytes were isolated from the metacarpal–phalangeal joint of adult steers and cultured for 7 days in alginate beads. The rate of oxygen consumption was measured in a respiration chamber using an oxygen electrode. The effect of oxygen tension, time in culture and serum addition on oxygen consumption was investigated. This data was used to predict the oxygen tension profiles across articular cartilage. The variation in oxygen tension with distance across the joint was predicted by solving a one‐dimensional reaction–diffusion equation using a finite‐difference method. The effect of influx of oxygen from the subchondral bone on oxygen profiles was estimated.Results  Oxygen consumption rates were significantly (2.5 fold) greater on the first day after isolation; on the second and subsequent days of culture, rates were similar to those reported by others (c. 10 nmol. million cells−1 h−1) (Bywaters 1937). The presence of serum increased the rate by around 50%. The consumption rate was relatively independent of oxygen tension between 5 and 21% oxygen, but below 5% oxygen, consumption rates fell in a concentration‐dependent manner. Calculations of the oxygen profile across cartilage showed that the concentration was sensitive to consumption rate and was also critically dependent on the extent of supply from the subchondral bone. Calculations also showed that growth factor addition led to a fall in oxygen tension throughout the tissue.Discussion  Here, we show that oxygen concentration profiles across cartilage are affected by cellular metabolic rates; addition of serum or growth factors for instance, by increasing consumption rates, can lower oxygen concentrations within the tissue significantly. Transport from the subchondral bone can also strongly affect the profile of oxygen across cartilage. Although its influence is usually ignored, there is anatomical and MRI evidence (Bashir et al. 1997) that this transport route is significant. Changes known to occur in the subchondral bone in osteoarthritis could alter transport into cartilage via this route and hence affect concentrations of oxygen and other important nutrients across the joint.