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The orderly differentiation of cell lineages within gastric glands is regulated by a complicated interplay of local mucosal growth factors and hormones. Histamine secreted from enterochromaffin-like cells plays an important role in not only stimulated gastric acid secretion but also coordination of intramucosal growth and lineage differentiation. We have examined histidine-decarboxylase (HDC)-deficient mice, which lack endogenous histamine synthesis, to evaluate the influence of histamine on differentiation of fundic mucosal lineages and the development of metaplasia following induction of acute oxyntic atrophy. Stomachs from HDC-deficient mice and wild-type mice were evaluated at 8 wk and 12 mo of age. DMP-777 was administrated orally to 6-wk-old mice for 1 to 14 days. Sections of gastric mucosa were stained with antibodies against Mist1, intrinsic factor, H/K-ATPase, trefoil factor 2 (TFF2), chromogranin A, and Ext1 and for the cell cycle marker phospho-histone H3. HDC-deficient mice at 8 wk of age demonstrated a prominent increase in chief cells expressing Mist1 and intrinsic factor. Importantly Mist1-positive mature chief cells were present in the midgland region as well as at the bases of fundic glands, indicating a premature differentiation of chief cells. Mice dually deficient for both HDC and gastrin showed a normal distribution of chief cells in fundic glands. Treatment of HDC-deficient mice with DMP-777 led to loss of parietal cells and an accelerated and exaggerated emergence of mucous cell metaplasia with the presence of dual intrinsic factor and TFF2-expressing cells throughout the gland length, indicative of the emergence of spasmolytic polypeptide-expressing metaplasia (SPEM) from chief cells. These findings indicate that histamine, in concert with gastrin, regulates the appropriate differentiation of chief cells from mucous neck cells as they migrate toward the bases of fundic glands. Nevertheless, histamine is not required for emergence of SPEM following acute oxyntic atrophy. Show less

Histologic grade is currently the best predictor of clinical course in chondrosarcoma patients. Grading suffers, however, from extensive interobserver variability and new objective markers are needed. Hence, we have investigated DNA copy numbers in chondrosarcomas with the purpose of identifying markers useful for prognosis and subclassification. The overall pattern of genomic imbalances was assessed in a series of 67 chondrosarcomas using array comparative genomic hybridization. Statistical analyses were applied to evaluate the significance of alterations detected in subgroups based on clinical data, morphology, grade, tumor size, and karyotypic features. Also, the global gene expression profiles were obtained in a subset of the tumors. Genomic imbalances, in most tumors affecting large regions of the genome, were found in 90% of the cases. Several apparently distinctive aberrations affecting conventional central and peripheral tumors, respectively, were identified. Although rare, recurrent amplifications were found at 8q24.21-q24.22 and 11q22.1-q22.3, and homozygous deletions of loci previously implicated in chondrosarcoma development affected the CDKN2A, EXT1, and EXT2 genes. The chromosomal imbalances in two distinct groups of predominantly near-haploid and near-triploid tumors, respectively, support the notion that polyploidization of an initially hyperhaploid/hypodiploid cell population is a common mechanism of chondrosarcoma progression. Increasing patient age as well as tumor grade were associated with adverse outcome, but no copy number imbalance affected metastasis development or tumor-associated death. Despite similarities in the overall genomic patterns, the present findings suggest that some regions are specifically altered in conventional central and peripheral tumors, respectively. Show less

Hereditary multiple exostosis (HME) is an autosomal dominant bone disorder, characterized by short stature and the presence of multiple benign tumors mainly at the ends of long bones. HME is genetically heterogeneous with two known genes on 8q24 (EXT1) and 11p11 (EXT2), and a third minor locus mapped to 19p (EXT3). The majority of EXT1 and EXT2 mutations result in premature protein truncation and loss of function. We analyzed two autosomal dominant HME families of Indian origin. Linkage analysis using fluorescently labeled microsatellite markers at the candidate gene regions was performed. Mutation analysis was carried out by bidirectional sequencing of purified PCR products. We found linkage in one family to EXT1 and in the other family to EXT2. Mutation screening in the EXT1 gene revealed a novel frameshift mutation, a single base deletion in exon 1 (c.142delC). This mutation segregated in all affected members and was absent in the unaffected family members and 60 unrelated controls. In the second family, a previously unreported stop mutation, the substitution c.817C>T, was observed in the EXT2 gene in all affected members and in none of the unaffected family members and 90 unrelated controls. Our findings expand the mutation spectrum of EXT1 and EXT2 and highlight the genetic and phenotypic heterogeneity of HME. Show less

Syndecan-1 is a proteoglycan that concentrates heparin-binding factors on the surface of multiple myeloma cells, and probably plays a major role in multiple myeloma biology. As heparan sulphate and chondroitin sulphate are the bioactive components of syndecan-1, we analysed the signature of genes encoding 100 proteins involved in synthesis of these chains, i.e. from precursor uptake to post-translational modifications, using Affymetrix microarrays. The expression of enzymes required for heparan sulphate and chondroitin sulphate biosynthesis was shown to increase in parallel with syndecan-1 expression, throughout the differentiation of memory B cells into plasmablasts and normal bone marrow plasma cells. Sixteen genes were significantly different between normal and malignant plasma cells, nine of these genes -EXT2, CHSY3, CSGALNACT1, HS3ST2, HS2ST1, CHST11, CSGALNACT2, HPSE, SULF2 - encode proteins involved in glycosaminoglycan chain synthesis or modifications. Kaplan-Meier analysis was performed in two independent series of patients: B4GALT7, CSGALNACT1, HS2ST1 were associated with a good prognosis whereas EXT1 was linked to a bad prognosis. This study provides an overall picture of the major genes encoding for proteins involved in heparan sulphate and chondroitin sulphate synthesis and modifications that can be implicated in normal and malignant plasma cells. Show less

The tumor suppressor genes EXT1 and EXT2 are involved in the formation of multiple osteochondromas, which can progress to become secondary peripheral chondrosarcomas. The most common chondrosarcoma subtype is primary central chondrosarcoma, which occurs in the medullar cavity of bone. The EXT1/EXT2 protein complex is involved in heparan sulfate proteoglycan (HSPG) biosynthesis, which is important for signal transduction of Indian hedgehog (IHH), WNT, and transforming growth factor (TGF)-beta. The role of EXT and its downstream targets in central chondrosarcomas is currently unknown. EXT1 and EXT2 were therefore evaluated in central chondrosarcomas at both the DNA and mRNA levels. Immunohistochemistry was used to assess HSPG (CD44v3 and SDC2), WNT (beta-catenin), and TGF-beta (PAI-1 and phosphorylated Smad2) signaling, whereas IHH signaling was studied both by quantitative polymerase chain reaction and in vitro. mRNA levels of both EXT1 and EXT2 were normal in central chondrosarcomas; genomic alterations were absent in these regions and in 30 other HSPG-related genes. Although HSPGs were aberrantly located (CD44v3 in the Golgi and SDC2 in cytoplasm and nucleus), this was not caused by mutation. WNT signaling negatively correlated with increasing histological grade, whereas TGF-beta positively correlated with increasing histological grade. IHH signaling was active, and inhibition decreased cell viability in one of six cell lines. Our data suggest that, despite normal EXT in central chondrosarcomas, HSPGs and HSPG-dependent signaling are affected in both central and peripheral chondrosarcomas. Show less

HS (heparan sulfate) proteoglycans are key regulators of vital processes in the body. HS chains with distinct sequences bind to various protein ligands, such as growth factors and morphogens, and thereby function as important regulators of protein gradient formation and signal transduction. HS is synthesized through the concerted action of many different ER (endoplasmic reticulum) and Golgi-resident enzymes. In higher organisms, many of these enzymes occur in multiple isoforms that differ in substrate specificity and spatial and temporal expression. In order to investigate how the structural complexity of HS has evolved, in the present study we focused on the starlet sea anemone (Nematostella vectensis), which belongs to the Anthozoa, which are considered to have retained many ancestral features. Members of all of the enzyme families involved in the generation and modification of HS were identified in Nematostella. Our results show that the enzymes are highly conserved throughout evolution, but the number of isoforms varies. Furthermore, the HS polymerases [Ext (exostosin) enzymes Ext1, Ext2 and Ext-like3] represent distinct subgroups, indicating that these three genes have already been present in the last common ancestor of Cnidaria and Bilateria. In situ hybridization showed up-regulation of certain enzymes in specific areas of the embryo at different developmental stages. The specific mRNA expression pattern of particular HS enzymes implies that they may play a specific role in HS modifications during larval development. Finally, biochemical analysis of Nematostella HS demonstrates that the sea anemone synthesizes a polysaccharide with a unique structure. Show less

Hereditary multiple exostoses (HME) is an autosomal dominant orthopaedic disorder most frequently caused by mutations in the EXT1 gene. The aim of the present study is to determine the underlying molecular defect of HME in two multigenerational Tunisian families with 21 affected members and to examine the degree of intrafamilial variability. Linkage analysis was performed using three microsatellite markers encompassing the EXT1 locus and mutation screening was carried out by direct sequencing. In family 1, evidence for linkage to EXT1 was obtained on the basis of a maximum LOD score of 4.26 at theta = 0.00 with D8S1694 marker. Sequencing of the EXT1 revealed a heterozygous G > T transversion (c.1019G>T) in exon 2, leading to a missense mutation at the codon 340 (p.Arg340Leu). In family 2 we identified a novel heterozygous 1 bp deletion in the exon 1 (c.529₅₃₁delA) leading to a premature codon stop and truncated EXT1 protein expression (p.Lys177LysfsX15). This mutation was associated with the evidence of an intrafamilial clinical variability and considered to be a novel disease-causing mutation in the EXT1 gene. These findings provide additional support for the involvement of EXT1 gene in the HME disease. Show less

Heparan sulfate (HS) proteoglycans influence embryonic development and adult physiology through interactions with protein ligands. The interactions depend on HS structure, which is determined largely during biosynthesis by Golgi enzymes. How biosynthesis is regulated is more or less unknown. During polymerization of the HS chain, carried out by a complex of the exostosin proteins EXT1 and EXT2, the first modification enzyme, glucosaminyl N-deacetylase/N-sulfotransferase (NDST), introduces N-sulfate groups into the growing polymer. Unexpectedly, we found that the level of expression of EXT1 and EXT2 affected the amount of NDST1 present in the cell, which, in turn, greatly influenced HS structure. Whereas overexpression of EXT2 in HEK 293 cells enhanced NDST1 expression, increased NDST1 N-glycosylation, and resulted in elevated HS sulfation, overexpression of EXT1 had opposite effects. Accordingly, heart tissue from transgenic mice overexpressing EXT2 showed increased NDST activity. Immunoprecipitaion experiments suggested an interaction between EXT2 and NDST1. We speculate that NDST1 competes with EXT1 for binding to EXT2. Increased NDST activity in fibroblasts with a gene trap mutation in EXT1 supports this notion. These results support a model in which the enzymes of HS biosynthesis form a complex, or a GAGosome. Show less

Hereditary multiple exostoses (HME) is an autosomal-dominant disorder characterized by the development of benign tumours, multiple osteochondromas (exostoses), growing outward from the metaphyses of long bones. Birth prevalence is estimated to be one in 50,000, and the severity of the disease is variable. Osteochondromas may cause complications including pain, deformities and shortening of the long bones, restricted motion of joints, nerve or blood vessel compression, and malignant transformation (5% of cases) in adulthood. HME is a genetically heterogeneous disorder and is associated with mutations in EXT1 or EXT2 genes, which are both tumour suppressor genes. EXT genes encode glycosyltransferases, termed 'exostosins', which are involved in the biosynthesis of heparan sulphate. Enchondromatosis (or Ollier disease) is characterized by the presence of intra-osseous benign cartilaginous tumours. The estimated prevalence of the disease is one in 100,000. An asymmetrical distribution of cartilage lesions is observed in the disease. The number, size and location of the enchondromas can be extremely variable between patients. Clinical problems caused by enchondromas include skeletal deformities, limb length discrepancy, pain and the potential risk for malignant change to chondrosarcoma (20-50% of cases). The condition in which multiple enchondromas is associated with haemangiomas is known as 'Maffucci syndrome'. Ollier disease and Maffucci syndrome are not usually inherited disorders. Show less

Multiple osteochondromas (MO) is an autosomal-dominant skeletal disorder characterized by the formation of multiple cartilage-capped protuberances. MO is genetically heterogeneous and is associated with mutations in the EXT1 and EXT2 genes. In this study we describe extensive mutation screening in a set of 63 patients with clinical and radiographical diagnosis of MO. Denaturing high-performance liquid chromatography analysis revealed mutations in 43 patients. Additional deletion analysis by fluorescence in situ hybridization and a newly developed multiplex ligation-dependent probe amplification probe set identified one patient with an intragenic EXT1 translocation, three patients with a partial EXT1 deletion, and one patient with a partial EXT2 deletion. Thirty-six patients harbored an EXT1 mutation (57%), and 12 had an EXT2 mutation (19%). We show that our optimized denaturing high-performance liquid chromatography/sequencing/multiplex ligation-dependent probe amplification protocol represents a reliable and highly sensitive diagnostic strategy for mutation screening in MO patients. Clinical analysis showed no clear genotype-phenotype correlation in our cohort of MO patients. Show less

Murine gammaherpesvirus 68 (MHV68) is used as a model to study gammaherpesvirus pathogenesis both in tissue culture systems and in vivo. We used a gene-trapping approach to get insight into cellular factors involved in MHV68 infection. By generating a library of gene-trapped CHO cells, we were able to isolate several clones that exhibited various degrees of resistance to MHV68-induced cytopathic effect. Clones that showed the highest degree of resistance were affected at the early stage of the viral cycle, with the vast majority of these clones being deficient for heparan sulfate (HS) expression at the cell surface. Heparan sulfate expression could be restored in all the HS-deficient clones by expression of EXT1, an enzyme that is essential for the biosynthesis of HS. Consistent with the role of HS in viral entry, HS-deficient CHO cells did not support viral internalization. Cell surface heparan sulfate proteoglycans (HSPG) are mostly composed of HS chains attached to two families of core proteins, the transmembrane syndecans and the GPI-anchored glypicans. Treatment of CHO cells with phosphatidylinositol-specific phospholipase C (PI-PLC) did not significantly affect the level of HS expression, indicating that the glypicans are not a major source of HSPG in CHO cells. By contrast, treatment of CHO cells with PMA, a drug known to accelerate syndecan shedding, resulted in a decrease in both HS expression and susceptibility to MHV68; these effects were abolished by TIMP-3, a specific inhibitor of syndecan shedding. All together, our results confirm the essential role of HS in MHV68 infection and identify the syndecans as a major source of HSPG used by the virus as coreceptors to infect CHO cells. Show less

The tricho-rhino-phalangeal syndrome type II (TRPS II) is characterized by sparse scalp hair, a long nose with a bulbous tip, a long flat philtrum, cone-shaped epiphyses of the phalanges, retarded bone age in infancy and multiple cartilaginous exostoses. All patients have a hemizygous deletion on chromosome 8q23.3-24.11 which spans at least the 2.8 Mb-region from TRPS1 through EXT1. Only patients with deletions that extend beyond this interval tend to have mental retardation. Here we describe a 14.5-year-old girl with mental retardation and TRPS II. Her facial features are only mild, but she has the typical skeletal features including cone-shaped epiphyses at the phalanges, retarded bone age, multiple exostoses and short stature. She is the first patient with TRPS II and a molecularly proven mosaic interstitial deletion in 8q22.3-q24.13. The deletion is one of the largest ever found in TRPS II, and spans 19.79 Mb and 50 genes or loci including TRPS1 and EXT1. The degree of mosaicism is 7% in lymphocytes from peripheral blood and 97% in skin fibroblasts. Show less

Multiple osteochondromas (MO; also referred to as hereditary multiple exostoses [HME] in the literature) is an autosomal dominant disorder characterized by benign, cartilage-capped bone tumors that grow from the metaphyses of long bones. Two genes are associated with this disease: EXT1 on 8q24.11-q24.13 and EXT2 on 11p12-p11. Mutations in EXT1 and EXT2 are found in 54-96% of patients with MO and are generally more frequent in EXT1 than in EXT2. We previously studied 43 Japanese families with MO using single-strand conformation polymorphism analysis for EXT1 and EXT2, and reported 23 families (54%) with mutations and 20 families (46%) with no mutations in these genes. Among the families with mutations, 17 families (40%) had mutations in EXT1, and 6 families (14%) had mutations in EXT2. Here we examined the same 43 Japanese families using denaturing high-performance liquid chromatography as an alternative technique. We detected five mutations, three of which are novel, in seven families in addition to the previously described mutations. In summary, we detected mutations in EXT1 or EXT2 in 30 (70%) out of 43 families. Our result suggests the presence of other gene(s) responsible for MO, at least in Japanese patients. Show less

Osteochondroma is the most common benign bone tumor and usually occurs in the metaphyseal region of the long bones. This tumor takes the form of a cartilage-capped bony outgrowth on the surface of the bone. The vast majority (85%) of osteochondromas present as solitary, nonhereditary lesions. Approximately 15% of osteochondromas occur as multiple lesions in the context of hereditary multiple osteochondromas (HMOs), a disorder that is inherited in an autosomal dominant manner. Most lesions appear in children and adolescents as painless, slow-growing masses. However, depending on the location of the osteochondroma, significant symptoms may occur as a result of complications such as fracture, bony deformity, mechanical joint problems and vascular or neurologic compromise. Malignant transformation of osteochondromas can occur later in adulthood but rarely metastasize. The treatment of choice for osteochondroma is surgical unless the skeleton is still immature. Pathogenetic analysis showed that HMOs are caused by mutations in either of two genes: exostosis (multiple)-1 (EXT1), which is located on chromosome 8q24.11-q24.13 or exostosis (multiple)-2 (EXT2), which is located on chromosome 11p11-12. Recently, biallelic inactivation of the EXT1 locus was described in nonhereditary osteochondromas. The EXT1 and EXT2 proteins function in the biosynthesis of heparin sulfate proteoglycans (HSPGs) which are multifunctional proteins involved in several growth signaling pathways in the normal epiphyseal growth plate. Reduced EXT1 or EXT2 expression in osteochondromas is associated with disordered cellular distribution of HSPGs, resulting in defective endochondral ossification which is likely to be involved in the formation of osteochondromas. Here the clinical, radiological, pathological and pathogenetic features and the treatment modalities of osteochondroma are reviewed. Show less

Glomerular deposition of fibrillar collagen is a characteristic finding of genetically distinct conditions, including nail-patella syndrome and collagen type III glomerulopathy. A case of familial nephropathy in which steroid-sensitive nephrotic syndrome and glomerular deposits of fibrillar collagen are associated with multiple exostoses due to mutation of the EXT1 gene is described. This gene encodes a glycosyltransferase required for synthesis of heparan sulfate glycosaminoglycans. There is deficiency of heparan sulfate and perlecan, together with accumulation of collagens, in the matrix of EXT1-associated osteochondromas. Similar glomerular basement membrane abnormalities could offer an explanation for both the renal ultrastructural changes and steroid-sensitive nephrotic syndrome. Show less

Identification of casual mutations in Hereditary Multiple Exostoses (HME) is important because of similar conditions in which multiple exostoses occur. Therefore mutation analysis can help to confirm the clinical diagnosis and to improve the management of therapy. HME is an inherited disorder of bone growth. HME can be referred to by various names such as Heredity Multiple Exostoses, Hereditary Multiple Osteochondromata, Multiple Carthaginous Exostoses, etc. People who have HME grow exostoses, or bony bumps, on their bones which can vary in size, location and number depending on the individual. HME is inherited in an autosomal dominant manner with an estimated prevalence of 1/50,000 in western countries. At least three loci (EXT1, EXT2 and EXT3) thought to be involved in this skeletal disease. Approximately 90% of affected families possess mutations in the coding regions of EXT1 and EXT2 genes and the majority of these mutations cause loss of function. EXT1 and EXT2 genes encode related members of a putative tumor suppressor family. In this first report from Iran we identified a frame shift mutation (1100-1101 insA) in exon 3 of EXT1 gene in a family being suspicious of HME. This mutation leads to a premature stop codon and previously not described. Additionally, we have found an unreported silent mutation in the exon six of EXT1 gene with uncertain significance. Show less

To compare the molecular basis difference between recurrent respiratory papillomatosis (RRP) and vocal cord polyp, to analyze the expression of glycan structural genes, and to discuss the pathopoiesis mechanism of RRP. The gene expressing profile between the 3 groups papilloma and the vocal cord polyp regarded as normal larynx epithelium were compared using mRNA parallel amplify and the human genome gene expressing microarray. Through cluster analysis, Gene Ontology function gene annotation and path way analysis, the relative gene of RRP and HPV infection were acquired. According to three microarrays results, total 567 expression changed genes related to HPV induce RRP were acquired. A serial change of glycan structure biosynthesis and degradation pathways was significant. The expression of dolichyl-phosphate mannosyltransferase polypeptide 1 (DPM1), asparagine-linked glycosylation 1 homolog (ALG1), fucosyltransferase 8 (FUT8) and alpha-mannosidase 1A (MAN1A) were regulated and beta-hexosaminidase (HEXB), beta1-galactosidase (GLB1), exostoses 1 (EXT1), fucosyltransferase (FUT) reduced expression and heparan sulfate 3-O-sulfotransferase 1 (HS3ST3A1) increased expression. The two related enzymes of the glycosphingolipids which is the main composed of the cell membrane, beta-3-N-acetylglucosaminyltransferase 4 (B3GNT4) and UDP-glucose ceramide glucosyltransferase (UGCG) increase expression, HEXB and GLB1 reduced expression. The alteration of the coding genes of glycan structure biosynthesis and degradation pathways were significantly and characteristically in pathopoiesis mechanism of RRP. This abnormality may be the beginning of tumor form HPV infection. Show less

Wnt-3a is a ligand that activates the beta-catenin-dependent pathway in Wnt signaling, which is implicated in numerous physiological events such as morphogenesis. So far, heparan sulfate (HS) proteoglycans have been highlighted as a low affinity receptor for morphogens containing Wnts. Here we show the importance of chondroitin sulfate (CS) proteoglycans in the efficient signaling of Wnt-3a and the structural features of CS required for the regulation of Wnt-3a signaling. Wnt-3a signaling was depressed in a mouse L cell mutant, called sog9, which is defective in the EXT1 gene encoding the HS-synthesizing enzyme and the chondroitin 4-O-sulfotransferase (C4ST-1) gene compared with parental L cells. The transfection of sog9 cells with C4ST-1 resulted in the recovery of Wnt-3a signaling, whereas the expression of EXT1 in sog9 cells could not restore Wnt-3a signaling. In addition, the expression level of introduced C4ST-1 correlated with the recovery of Wnt-3a signaling accompanied by the increased expression of the E disaccharide unit of CS. Interestingly, molecular interaction analyses using Biacore revealed that squid CS-E (rich in the E disaccharide unit) bound strongly to Wnt-3a (K(d)=13.2 nm) to the same extent as heparin from bovine lung (K(d)=8.43 nm). In contrast, other CS isoforms as well as HS isolated from bovine kidney showed little binding activity to Wnt-3a. Moreover, exogenously added CS-E potently inhibited the accumulation of beta-catenin induced by Wnt-3a. These results suggest that CS-E-like structures synthesized by C4ST-1 participate in Wnt-3a signaling and modulate the physiological events caused by Wnt-3a signals. Show less

The concept that the glomerular filtration barrier exhibits charge selectivity is a basic tenet of renal physiology. Heparan sulfate is a major contributor of glomerular anionic charge. In a new study, prevention of podocytes from synthesizing heparan sulfate, via mutation of Ext1, causes only mild, statistically insignificant albuminuria, despite dramatic alterations in glomerular anionic charge. Show less

Podocytes synthesize the majority of the glomerular basement membrane components with some contribution from the glomerular capillary endothelial cells. The anionic charge of heparan sulfate proteoglycans is conferred by covalently attached heparan sulfate glycosaminoglycans and these are thought to provide critical charge selectivity to the glomerular basement membrane for ultrafiltration. One key component in herparan sulfate glycosaminoglycan assembly is the Ext1 gene product encoding a subunit of heparan sulfate co-polymerase. Here we knocked out Ext1 gene expression in podocytes halting polymerization of heparin sulfate glycosaminoglycans on the proteoglycan core proteins secreted by podocytes. Glomerular development occurred normally in these knockout animals but changes in podocyte morphology, such as foot process effacement, were seen as early as 1 month after birth. Immunohistochemical analysis showed a significant decrease in heparan sulfate glycosaminoglycans confirmed by ultrastructural studies using polyethyleneimine staining. Despite podocyte abnormalities and loss of heparan sulfate glycosaminoglycans, severe albuminuria did not develop in the knockout mice. We show that the presence of podocyte-secreted heparan sulfate glycosaminoglycans is not absolutely necessary to limit albuminuria suggesting the existence of other mechanisms that limit albuminuria. Heparan sulfate glycosaminoglycans appear to have functions that control podocyte behavior rather than be primarily an ultrafiltration barrier. Show less

We have examined the role of heparan sulphate in lymphocyte development and activation in mice by conditionally deleting the genes encoding the heparan sulphate biosynthetic enzymes N-deacetylase/N-sulphotransferase-1 and -2 (Ndst1 and Ndst2) and glucuronic acid/N-acetylglucosamine co-polymerase-1 (Ext1) in T cells and B cells, respectively. Ndst1 and Ndst2 are the only Ndst isoforms in T cells. In T-cell Ndst-deficient mice there were normal ratios of CD4(+)/CD8(+) cells in the blood, spleen and thymus, indicating no dramatic effect on development. However, Ndst-deficient T cells were hyperresponsive to low-level activation, suggesting that cell surface heparan sulphate plays a role in T-cell proliferation. The hyperresponsive state correlated with a decrease in cell surface heparan sulphate that occurs in response to activation in wild-type cells. There was a slight change in the number of developing B cells in B-cell Ext1-deficient mice, but the alteration did not cause a change in antibody production. These findings demonstrate that cell surface heparan sulphate may not play a crucial role in lymphocyte development, but can modulate the sensitivity of T cells to activation. Show less

Langer-Giedion syndrome results from a microdeletion at 8q24.1 encompassing the EXT1 and the adjacent TRPS1 gene. We report on a boy with an oligo array-cgh characterized small microdeletion involving EXT1 alone but with some features of Langer-Giedion syndrome suggesting a functional disturbance of TRPS1. This boy, in addition to a mild Langer-Giedion like phenotype, also had some unusual features including prominent toe pads and fat pads on the soles of his feet similar to those described in Pierpont syndrome. Show less

The different clinical entities of osteochondromas, hereditary multiple exostoses (HME) and non-familial solitary exostosis, are known to express localized exostoses in their joint metaphyseal cartilage. In the current study biopsies of osteochondromas patients were screened with respect to a number of cellular and molecular parameters. Specifically, cartilaginous biopsy samples of nine HME patients, 10 solitary exostosis patients and 10 articular cartilages of control subjects were collected and cell cultures were established. Results obtained showed that one of the two HME samples that underwent DNA sequencing analysis (HME-1) had a novel mutation for an early stop codon, which led to an aberrant protein, migrating at a lower molecular weight position. The EXT-1 mRNA and protein levels in chondrocyte cultures derived from all nine HME patients were elevated, compared with solitary exostosis patients or control subjects. Furthermore, cell cultures of HME patients had significantly decreased pericellular heparan sulphate (HS) in comparison with cultures of solitary exostosis patients or control subjects. Immunohistochemical staining of tissue sections and Western blotting of cell cultures derived from HME patients revealed higher levels of heparanase compared with solitary exostosis patients and of control subjects. Further investigations are needed to determine whether the low pericellular HS levels in HME patients stem from decreased biosynthesis of HS, increased degradation or a combination of both. In conclusion, it appears that due to a mutated glycosyltransferase, the low content of pericellular HS in HME patients leads to the anatomical deformations with exostoses formation. Hence, elevation of HS content in the pericellular regions should be a potential molecular target for correction. Show less

Multiple osteochondromas (MO) is characterised by development of two or more cartilage capped bony outgrowths (osteochondromas) of the long bones. The prevalence is estimated at 1:50,000, and it seems to be higher in males (male-to-female ratio 1.5:1). Osteochondromas develop and increase in size in the first decade of life, ceasing to grow when the growth plates close at puberty. They are pedunculated or sessile (broad base) and can vary widely in size. The number of osteochondromas may vary significantly within and between families, the mean number of locations is 15-18. The majority are asymptomatic and located in bones that develop from cartilage, especially the long bones of the extremities, predominantly around the knee. The facial bones are not affected. Osteochondromas may cause pain, functional problems and deformities, especially of the forearm, that may be reason for surgical removal. The most important complication is malignant transformation of osteochondroma towards secondary peripheral chondrosarcoma, which is estimated to occur in 0.5-5%. MO is an autosomal dominant disorder and is genetically heterogeneous. In almost 90% of MO patients germline mutations in the tumour suppressor genes EXT1 or EXT2 are found. The EXT genes encode glycosyltransferases, catalyzing heparan sulphate polymerization. The diagnosis is based on radiological and clinical documentation, supplemented with, if available, histological evaluation of osteochondromas. If the exact mutation is known antenatal diagnosis is technically possible. MO should be distinguished from metachondromatosis, dysplasia epiphysealis hemimelica and Ollier disease. Osteochondromas are benign lesions and do not affect life expectancy. Management includes removal of osteochondromas when they give complaints. Removed osteochondromas should be examined for malignant transformation towards secondary peripheral chondrosarcoma. Patients should be well instructed and regular follow-up for early detection of malignancy seems justified. For secondary peripheral chondrosarcoma, en-bloc resection of the lesion and its pseudocapsule with tumour-free margins, preferably in a bone tumour referral centre, should be performed. Show less

Multiple osteochondromas (MO) is an autosomal-dominant inherited disorder. The two genes responsible (EXT1 and EXT2) have been identified. We investigated 12 MO families for phenotype details and the genetic basis by cosegregation and mutation analysis (seven novel pathogenic mutations [five frameshift, one splice site, and one gross deletion] and one novel missense polymorphism). We found EXT1 to be responsible in seven families (19 affected members) and EXT2 in four families (17 affected members). One family remains undetermined. We found a tendency to a more severe phenotype in EXT1 families. As a novel finding, we could identify a single parameter (ulna/height ratio) that separates EXT1 family from EXT2 family in our series. Show less

Proteoglycans carrying heparan sulphate (HS) chains are ubiquitously expressed at cell surfaces and in extra-cellular matrices, and HS chains interact with numerous proteins, including growth factors, morphogens and extra-cellular-matrix proteins. These interactions form the basis of HS-related biological phenomena. Thus, the biosynthesis of HS regulates key events in embryonic development and homeostasis, and deranged HS biosynthesis could cause diseases. EXT1 and EXT2 genes encoding the polymerase responsible for HS biosynthesis are known as causative genes of hereditary multiple exostoses, a dominantly inherited genetic disorder characterized by the formation of multiple cartilaginous tumours. In this review, we will summarize HS biosynthesis in several model animals, the effects on cellular functions by alteration of HS biosynthesis, and HS-associated diseases. This review suggests that HS biosynthetic enzymes would be potential candidates for drug targets in various diseases. Show less

Kaposi's sarcoma-associated herpesvirus (KSHV) and its murine homolog, murine gammaherpesvirus 68 (MHV68), are lymphotropic viruses that establish latent infection in their host. Surprisingly, while B cells are the main viral reservoir in vivo, B-cell lines are poorly permissive to infection by either MHV68 or KSHV. Here, we report that most B-cell lines express very little to no cell surface heparan sulfate (HS), a glycosaminoglycan that is essential for infection by these viruses. We found that Ext1, a key enzyme in the biosynthesis of HS, was expressed at a low level in these cells. Transfection of B-cell lines with Ext1 restored high HS expression at the cell surface. Overexpression of Ext1 in murine A20 and M12 B-cell lines increased MHV68 surface binding and enhanced the efficiency of infection. Finally, although it was not sufficient to allow efficient infection, the expression of HS on BJAB cells promoted KSHV binding at the cell surface. Thus, our results indicate that MHV68 and KSHV cycles are blocked in B-cell lines at the binding step due to a lack of surface HS. Show less

Amplification of 8q is frequently found in gastroesophageal junction (GEJ) cancer. It is usually detected in high-grade, high-stage GEJ adenocarcinomas. Moreover, it has been implicated in tumor progression in other cancer types. In this study, a detailed genomic analysis of 8q was performed on a series of GEJ adenocarcinomas, including 22 primary adenocarcinomas, 13 cell lines and two xenografts, by array comparative genomic hybridization (aCGH) with a whole chromosome 8q contig array. Of the 37 specimens, 21 originated from the esophagus and 16 were derived from the gastric cardia. Commonly overrepresented regions were identified at distal 8q, i.e. 124-125 Mb (8q24.13), at 127-128 Mb (8q24.21), and at 141-142 Mb (8q24.3). From these regions six genes were selected with putative relevance to cancer: ANXA13, MTSS1, FAM84B (alias NSE2), MYC, C8orf17 (alias MOST-1) and PTK2 (alias FAK). In addition, the gene EXT1 was selected since it was found in a specific amplification in cell line SK-GT-5. Quantitative RT-PCR analysis of these seven genes was subsequently performed on a panel of 24 gastroesophageal samples, including 13 cell lines, two xenografts and nine normal stomach controls. Significant overexpression was found for MYC and EXT1 in GEJ adenocarcinoma cell lines and xenografts compared to normal controls. Expression of the genes MTSS1, FAM84B and C8orf17 was found to be significantly decreased in this set of cell lines and xenografts. We conclude that, firstly, there are other genes than MYC involved in the 8q amplification in GEJ cancer. Secondly, the differential expression of these genes contributes to unravel the biology of GEJ adenocarcinomas. Show less

The purpose of this study was to compare the effects of the Traditional acupuncture point ST.36 and 'Omura's ST.36 Point' ("True ST.36") needling on the isokinetic knee extension & flexion strength of young soccer players. The Bi-Digital O-Ring Test (B.D.O.R.T.) of Yoshiaki Omura, M.D.,Sc.D. was used to determine the "True ST.36". Young soccer players (N = 24) between 16-18 years of age (Mean = 16.92 +/- 0.65) were involved in the study. The extension & flexion strengths of dominant legs were measured with Cybex 350 Extremity System isokinetically. The testing velocity was 60 degrees/sec. The peak torque value in Newton meters (Nm) was evaluated. Subjects were tested 3 times. Extension & Flexion 1 (EXT1, FLEX1) without acupuncture application, EXT2 & FLEX2 after application on the traditional acupuncture point, ST.36 and EXT3 & FLEX3 after application onto the 'Omura's New Foot-point' ("True ST.36"). Before each test, subjects warmed up for 10 minutes by cycling on an isokinetic ergometer at 50 RPM, 75 Watts load followed by stretching exercises of lower extremity. Mean EXT1, EXT2, EXT3 values were 196.92 +/- 28.70: 210.00 +/- 23.00; 224.42 +/- 21.70 respectively, where FLEX1, FLEX2, FLEX3 were 140.88 +/- 22.45; 151.13 +/- 21.27; 161.00 +/- 22.23. Comparisons of EXT1-EXT2, EXT1-EXT3, EXT2-EXT3, FLEX1-FLEX2, FLEX1-FLEX3, FLEX2-FLEX3 strength values showed all very high significance (P < 0.001) in favor of 1) Needling on relevant points and 2) Omura's ST.36 Point ("True ST.36"). We conclude that B.D.O.R.T. can help to determine new (True) Acupuncture points and, both points were effective for increasing the isokinetic knee extension & flexion strength of young soccer players very significantly where as Omura's ST.36 Point ("True ST.36") was more effective than Traditional Acupuncture point, ST.36. Show less