Publications by authors named "Nancy B Schwartz"

29 Publications

  • Page 1 of 1

Grant application outcomes for biomedical researchers who participated in the National Research Mentoring Network's Grant Writing Coaching Programs.

PLoS One 2020 9;15(11):e0241851. Epub 2020 Nov 9.

Department of Family and Preventive Medicine, University of Utah School of Medicine, Salt Lake City, Utah, United States of America.

Background: A diverse research workforce is essential for catalyzing biomedical advancements, but this workforce goal is hindered by persistent sex and racial/ethnic disparities among investigators receiving research grants from the National Institutes of Health (NIH). In response, the NIH-funded National Research Mentoring Network implemented a Grant Writing Coaching Program (GCP) to provide diverse cohorts of early-career investigators across the United States with intensive coaching throughout the proposal development process. We evaluated the GCP's national reach and short-term impact on participants' proposal submissions and funding outcomes.

Methods: The GCP was delivered as six similar but distinct models. All models began with an in-person group session, followed by a series of coaching sessions over 4 to 12 months. Participants were surveyed at 6-, 12- and 18-months after program completion to assess proposal outcomes (submissions, awards). Self-reported data were verified and supplemented by searches of public repositories of awarded grants when available. Submission and award rates were derived from counts of participants who submitted or were awarded at least one grant proposal in a category (NIH, other federal, non-federal).

Results: From June 2015 through March 2019, 545 investigators (67% female, 61% under-represented racial/ethnic minority, URM) from 187 different institutions participated in the GCP. Among them, 324 (59% of participants) submitted at least one grant application and 134 (41% of submitters) received funding. A total of 164 grants were awarded, the majority being from the NIH (93, 56%). Of the 74 R01 (or similar) NIH research proposals submitted by GCP participants, 16 have been funded thus far (56% to URM, 75% to women). This 22% award rate exceeded the 2016-2018 NIH success rates for new R01s.

Conclusion: Inter- and intra-institutional grant writing coaching groups are a feasible and effective approach to supporting the grant acquisition efforts of early-career biomedical investigators, including women and those from URM groups.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0241851PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652313PMC
January 2021

Vascular dimorphism ensured by regulated proteoglycan dynamics favors rapid umbilical artery closure at birth.

Elife 2020 09 10;9. Epub 2020 Sep 10.

Department of Biomedical Engineering, Cleveland Clinic Lerner Research Institute, Cleveland, United States.

The umbilical artery lumen closes rapidly at birth, preventing neonatal blood loss, whereas the umbilical vein remains patent longer. Here, analysis of umbilical cords from humans and other mammals identified differential arterial-venous proteoglycan dynamics as a determinant of these contrasting vascular responses. The umbilical artery, but not the vein, has an inner layer enriched in the hydrated proteoglycan aggrecan, external to which lie contraction-primed smooth muscle cells (SMC). At birth, SMC contraction drives inner layer buckling and centripetal displacement to occlude the arterial lumen, a mechanism revealed by biomechanical observations and confirmed by computational analyses. This vascular dimorphism arises from spatially regulated proteoglycan expression and breakdown. Mice lacking aggrecan or the metalloprotease ADAMTS1, which degrades proteoglycans, demonstrate their opposing roles in umbilical vascular dimorphism, including effects on SMC differentiation. Umbilical vessel dimorphism is conserved in mammals, suggesting that differential proteoglycan dynamics and inner layer buckling were positively selected during evolution.
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http://dx.doi.org/10.7554/eLife.60683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529456PMC
September 2020

Leveraging a collaborative consortium model of mentee/mentor training to foster career progression of underrepresented postdoctoral researchers and promote institutional diversity and inclusion.

PLoS One 2020 1;15(9):e0238518. Epub 2020 Sep 1.

Department of Pediatrics and Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, United States of America.

Changing institutional culture to be more diverse and inclusive within the biomedical academic community is difficult for many reasons. Herein we present evidence that a collaborative model involving multiple institutions of higher education can initiate and execute individual institutional change directed at enhancing diversity and inclusion at the postdoctoral researcher (postdoc) and junior faculty level by implementing evidence-based mentoring practices. A higher education consortium, the Big Ten Academic Alliance, invited individual member institutions to send participants to one of two types of annual mentor training: 1) "Mentoring-Up" training for postdocs, a majority of whom were from underrepresented groups; 2) Mentor Facilitator training-a train-the-trainer model-for faculty and senior leadership. From 2016 to 2019, 102 postdocs and 160 senior faculty and administrative leaders participated. Postdocs reported improvements in their mentoring proficiency (87%) and improved relationships with their PIs (71%). 29% of postdoc respondents transitioned to faculty positions, and 85% of these were underrepresented and 75% were female. 59 out of the 120 faculty and administrators (49%) trained in the first three years provided mentor training on their campuses to over 3000 undergraduate and graduate students, postdocs and faculty within the project period. We conclude that early stage biomedical professionals as well as individual institutions of higher education benefited significantly from this collaborative mentee/mentor training model.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0238518PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7462290PMC
October 2020

The Role of in Prenatal and Postnatal Murine Chondrocytes and Trabecular Bone.

JBMR Plus 2020 Feb 17;4(2):e10254. Epub 2019 Dec 17.

Department of Pediatrics University of Chicago Chicago IL USA.

Osteoarthritis and osteoporosis are widely prevalent and have far-reaching public health implications. There is increasing evidence that epigenetics, in particular, histone 3 lysine 79 methyltransferase , plays an important role in the cartilage and bone biology. In this study, we evaluated the role of in the articular cartilage, growth plate, and trabecular bone utilizing conditional KO mouse models. We generated chondrocyte-specific constitutive and inducible conditional KO mouse lines using - and systems. Prenatal deletion of in mouse chondrocytes led to perinatal mortality, accelerated ossification, and dysregulation of expression. Postnatal deletion of in mouse chondrocytes resulted in trabecular bone loss decreased extracellular matrix production, and disruption of the growth plate. In addition, pharmacological inhibition of DOT1L in a progeria mouse model partially rescued the abnormal osseous phenotype. In conclusion, is important in maintaining the growth plate, extracellular matrix production, and trabecular bone. © 2019 The Authors. published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbm4.10254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017886PMC
February 2020

Comparisons and Approaches of PREP Programs at Different Stages of Maturity: Challenges, Best Practices and Benefits.

Ethn Dis 2020 16;30(1):55-64. Epub 2020 Jan 16.

Albert Einstein College of Medicine, Bronx, NY.

The Postbaccalaureate Research Education Programs (PREP) are designed to provide research training and educational opportunities for recent baccalaureate graduates from targeted groups defined by NIH who would benefit by academic enhancements between the completion of undergraduate studies and admission to a PhD program. These programs offer exposure to the biomedical science community in a way that helps post-undergraduate individuals visualize future careers as well-trained, enthusiastic leaders in biomedical research who represent and will promote diversity in science. Specifically, PREPs provide the preparation and skills required for entrance into, and successful completion of, a PhD program via in-depth exposure to a research setting, which helps to refine the post-undergraduate's research interests, assists in providing a realistic understanding of the end results one can expect from research, and offers a forum for discussion with lab peers and mentors about possible career paths. Beyond the lab, PREPs offer programmatic activities to develop analytical, writing, and oral presentation skills necessary for a competitive graduate school application and success in graduate school thereafter. Individual mentoring increases the post-undergraduate's confidence and familiarity with members of the research community, so that pursuit of a PhD becomes a realistic and less-intimidating path. Interventions and developmental activities are matched to the background preparation, research experience, and learning style of each post-undergraduate. As with all training programs, there is no perfect model and each program must fit in and adapt to their respective institutional environments and cultures. Thus, in this article, we provide perspectives and approaches developed by a long-standing program in existence almost since the beginning of the PREP program along with one PREP at an early stage of maturity, having just been through one renewal.
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http://dx.doi.org/10.18865/ed.30.1.55DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6970524PMC
February 2021

Global Brain Transcriptome Analysis of a Tpp1 Neuronal Ceroid Lipofuscinoses Mouse Model.

ASN Neuro 2019 Jan-Dec;11:1759091419843393

1 Department of Pediatrics, Biological Sciences Division, The University of Chicago, IL, USA.

In humans, homozygous mutations in the TPP1 gene results in loss of tripeptidyl peptidase 1 (TPP1) enzymatic activity, leading to late infantile neuronal ceroid lipofuscinoses disease. Using a mouse model that targets the Tpp1 gene and recapitulates the pathology and clinical features of the human disease, we analyzed end-stage (4 months) transcriptional changes associated with lack of TPP1 activity. Using RNA sequencing technology, Tpp1 expression changes in the forebrain/midbrain and cerebellum of 4-month-old homozygotes were compared with strain-related controls. Transcriptional changes were found in 510 and 1,550 gene transcripts in forebrain/midbrain and cerebellum, respectively, from Tpp1-deficient brain tissues when compared with age-matched controls. Analysis of the differentially expressed genes using the Ingenuity™ pathway software, revealed increased neuroinflammation activity in microglia and astrocytes that could lead to neuronal dysfunction, particularly in the cerebellum. We also observed upregulation in the production of nitric oxide and reactive oxygen species; activation of leukocyte extravasation signals and complement pathways; and downregulation of major transcription factors involved in control of circadian rhythm. Several of these expression changes were confirmed by independent quantitative polymerase chain reaction and histological analysis by mRNA in situ hybridization, which allowed for an in-depth anatomical analysis of the pathology and provided independent confirmation of at least two of the major networks affected in this model. The identification of differentially expressed genes has revealed new lines of investigation for this complex disorder that may lead to novel therapeutic targets.
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http://dx.doi.org/10.1177/1759091419843393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475859PMC
May 2020

United States National Postdoc Survey results and the interaction of gender, career choice and mentor impact.

Elife 2018 12 18;7. Epub 2018 Dec 18.

Department of Pediatrics and Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, United States.

The postdoctoral community is an essential component of the academic and scientific workforce, but a lack of data about this community has made it difficult to develop policies to address concerns about salaries, working conditions, diversity and career development, and to evaluate the impact of existing policies. Here we present comprehensive survey results from 7,603 postdocs based at 351 US academic and non-academic (e.g. hospital, industry and government lab) institutions in 2016. In addition to demographic and salary information, we present multivariate analyses on factors influencing postdoc career plans and satisfaction with mentorship. We further analyze gender dynamics and expose wage disparities. Academic research positions remain the predominant career choice, although women and US citizens are less likely than their male and non-US citizen counterparts to choose academic research positions. Receiving mentorship training has a significant positive effect on postdoc satisfaction with mentorship. Quality of and satisfaction with postdoc mentorship also appear to heavily influence career choice.
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http://dx.doi.org/10.7554/eLife.40189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298783PMC
December 2018

Proteoglycans in brain development and pathogenesis.

FEBS Lett 2018 12 23;592(23):3791-3805. Epub 2018 Mar 23.

Department of Pediatrics, Biological Sciences Division, The University of Chicago, IL, USA.

Proteoglycans are diverse, complex extracellular/cell surface macromolecules composed of a central core protein with covalently linked glycosaminoglycan (GAG) chains; both of these components contribute to the growing list of important bio-active functions attributed to proteoglycans. Increasingly, attention has been paid to the roles of proteoglycans in nervous tissue development due to their highly regulated spatio/temporal expression patterns, whereby they promote/inhibit neurite outgrowth, participate in specification and maturation of various precursor cell types, and regulate cell behaviors like migration, axonal pathfinding, synaptogenesis and plasticity. These functions emanate from both the environments proteoglycans create around cells by retaining ions and water or serving as scaffolds for cell shaping or motility, and from dynamic interactions that modulate signaling fields for cytokines, growth factors and morphogens, which may bind to either the protein or GAG portions. Also, genetic abnormalities impacting proteoglycan synthesis during critical steps of brain development and response to environmental insults and injuries, as well as changes in microenvironment interactions leading to tumors in the central nervous system, all suggest roles for proteoglycans in behavioral and intellectual disorders and malignancies.
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http://dx.doi.org/10.1002/1873-3468.13026DOI Listing
December 2018

Glial cell responses in a murine multifactorial perinatal brain injury model.

Brain Res 2018 02 21;1681:52-63. Epub 2017 Dec 21.

Department of Pediatrics, Biological Sciences Division, The University of Chicago, Chicago, IL, USA; Department of Biochemistry and Molecular Biology, Biological Sciences Division, The University of Chicago, Chicago, IL, USA.

The impact of traumatic brain injury during the perinatal period, which coincides with glial cell (astrocyte and oligodendrocyte) maturation was assessed to determine whether a second insult, e.g., increased inflammation due to remote bacterial exposure, exacerbates the initial injury's effects, possibly eliciting longer-term brain damage. Thus, a murine multifactorial injury model incorporating both mechanisms consisting of perinatal penetrating traumatic brain injury, with or without intraperitoneal injection of lipopolysaccharide (LPS), an analog of remote pathogen exposure has been developed. Four days after injury, gene expression changes for different cell markers were assessed using mRNA in situ hybridization (ISH) and qPCR. Astrocytic marker mRNA levels increased in the stab-alone and stab-plus-LPS treated animals indicating reactive gliosis. Activated microglial/macrophage marker levels, increased in the ipsilateral sides of stab and stab-plus LPS animals by P10, but the differences resolved by P15. Ectopic expression of glial precursor and neural stem cell markers within the cortical injury site was observed by ISH, suggesting that existing precursors and neural stem cells migrate into the injured areas to replace the cells lost in the injury process. Furthermore, single exposure to LPS concomitant with acute stab injury affected the oligodendrocyte population in both the injured and contralateral uninjured side, indicating that after compromise of the blood-brain barrier integrity, oligodendrocytes become even more susceptible to inflammatory injury. This multifactorial approach should lead to a better understanding of the pathogenic sequelae observed as a consequence of perinatal brain insult/injury, caused by combinations of trauma, intrauterine infection, hypoxia and/or ischemia in humans.
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http://dx.doi.org/10.1016/j.brainres.2017.12.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5780221PMC
February 2018

Delivery and tracking of quantum dot peptide bioconjugates in an intact developing avian brain.

ACS Chem Neurosci 2015 Mar 5;6(3):494-504. Epub 2015 Mar 5.

#Departments of Pediatrics, Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, United States.

Luminescent semiconductor ∼9.5 nm nanoparticles (quantum dots: QDs) have intrinsic physiochemical and optical properties which enable us to begin to understand the mechanisms of nanoparticle mediated chemical/drug delivery. Here, we demonstrate the ability of CdSe/ZnS core/shell QDs surface functionalized with a zwitterionic compact ligand to deliver a cell-penetrating lipopeptide to the developing chick embryo brain without any apparent toxicity. Functionalized QDs were conjugated to the palmitoylated peptide WGDap(Palmitoyl)VKIKKP9GGH6, previously shown to uniquely facilitate endosomal escape, and microinjected into the embryonic chick spinal cord canal at embryo day 4 (E4). We were subsequently able to follow the labeling of spinal cord extension into the ventricles, migratory neuroblasts, maturing brain cells, and complex structures such as the choroid plexus. QD intensity extended throughout the brain, and peaked between E8 and E11 when fluorescence was concentrated in the choroid plexus before declining to hatching (E21/P0). We observed no abnormalities in embryonic patterning or embryo survival, and mRNA in situ hybridization confirmed that, at key developmental stages, the expression pattern of genes associated with different brain cell types (brain lipid binding protein, Sox-2, proteolipid protein and Class III-β-Tubulin) all showed a normal labeling pattern and intensity. Our findings suggest that we can use chemically modified QDs to identify and track neural stem cells as they migrate, that the choroid plexus clears these injected QDs/nanoparticles from the brain after E15, and that they can deliver drugs and peptides to the developing brain.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5056627PMC
http://dx.doi.org/10.1021/acschemneuro.5b00022DOI Listing
March 2015

Aggrecan is required for growth plate cytoarchitecture and differentiation.

Dev Biol 2014 Dec 18;396(2):224-36. Epub 2014 Oct 18.

Department of Pediatrics, The University of Chicago, Chicago IL 60637, USA; Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA. Electronic address:

The proteoglycan aggrecan is a prominent component of the extracellular matrix in growth plate cartilage. A naturally occurring, recessive, perinatally lethal mutation in the aggrecan core protein gene, cmd(bc) (Acan(cmd-Bc)), that deletes the entire protein-coding sequence provided a model in which to characterize the phenotypic and morphologic effects of aggrecan deletion on skeletal development. We also generated a novel transgenic mouse, Tg(COL2A1-ACAN), that has the chick ACAN coding sequence driven by the mouse COL2A1 promoter to enable the production of cmd(bc)/cmd(bc); Tg(COL2A1-ACAN) rescue embryos. These were used to assess the impact of aggrecan on growth plate organization, chondrocyte survival and proliferation, and the expression of mRNAs encoding chondrocyte differentiation markers and growth factors. Homozygous mutant (cmd(bc)/cmd(bc)) embryos exhibited severe defects in all skeletal elements with deformed and shortened (50%) limb elements. Expression of aggrecan in rescue embryos reversed the skeletal defects to varying degrees with a 20% increase in limb element length and near-full reversal (80%) of size and diameter of the ribcage and vertebrae. Aggrecan-null growth plates were devoid of matrix and lacked chondrocyte organization and differentiation, while those of the rescue embryos exhibited matrix production concomitant with partial zonation of chondrocytes having proliferative and hypertrophic morphologies. Deformation of the trachea, likely the cause of the mutation's lethality, was reduced in the rescue embryos. Aggrecan-null embryos also had abnormal patterns of COL10A1, SOX9, IHH, PTCH1, and FGFR3 mRNA expression in the growth plate. Expression of chick aggrecan in the rescue embryos notably increased COLX expression, accompanied by the reappearance of a hypertrophic zone and IHH expression. Significantly, in transgenic rescue embryos, the cell death and decreased proliferation phenotypes exhibited by the mutants were reversed; both were restored to wild-type levels. These findings suggest that aggrecan has a major role in regulating the expression of key growth factors and signaling molecules during development of cartilaginous tissue and is essential for proper chondrocyte organization, morphology, and survival during embryonic limb development.
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http://dx.doi.org/10.1016/j.ydbio.2014.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4261049PMC
December 2014

Mapping proteoglycan functions with glycosidases.

Methods Mol Biol 2015 ;1229:443-55

Department of Pediatrics, University of Chicago, 5841S. Maryland Avenue, Chicago, IL, 60637, USA.

The intrinsic and extrinsic factors that contribute to stem and neuronal precursor cell maintenance and/or differentiation remain poorly understood. Proteoglycans, major residents of the stem cell microenvironment, modulate key signaling cues and are of particular importance. We have taken a loss-of-function approach, by developing a library of bacterial lyases and sulfatases to specifically remodel the ECM and test the functional role of glycosaminoglycans (GAGs) in cell self-renewal, maintenance, and differentiation.
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http://dx.doi.org/10.1007/978-1-4939-1714-3_34DOI Listing
June 2015

Chemistry and function of glycosaminoglycans in the nervous system.

Adv Neurobiol 2014;9:89-115

Department of Pediatrics, University of Chicago, Chicago, IL, 60637, USA,

The glycosaminoglycan (GAG) family is characterized by covalently linked repeating disaccharides forming long unbranched polysaccharide chains. Thus far in higher eukaryotes, the family consists of chondroitin sulfate (CS), heparin/heparan sulfate (HS), dermatan sulfate (DS), and hyaluronan (HA). All GAG chains (except HA) are characteristically modified by varying amounts of esterified sulfate. One or more GAG chains are usually found in nature bound to polypeptide backbones in the form of proteoglycans; HA is the exception and is not synthesized covalently bound to a protein. Proteoglycans, and especially their GAG components, participate in numerous biologically significant interactions with growth factors, chemokines, morphogens, guidance molecules, survival factors, and other extracellular and cell-surface components. These interactions are often critical to the basic developmental processes of cellular proliferation and differentiation, as well as to both the onset of disease sequelae and the prevention of disease progression. In the nervous system, GAG/proteoglycan-mediated interactions participate in proliferation and synaptogenesis, neural plasticity, and regeneration. This review focuses on the structure, chemistry, and function of GAGs in nervous system development, disease, and injury response.
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http://dx.doi.org/10.1007/978-1-4939-1154-7_5DOI Listing
January 2014

Forward genetics defines Xylt1 as a key, conserved regulator of early chondrocyte maturation and skeletal length.

Dev Biol 2014 Jan 23;385(1):67-82. Epub 2013 Oct 23.

Department of Genetics, Yale University, New Haven, CT 06520, United States.

The long bones of the vertebrate body are built by the initial formation of a cartilage template that is later replaced by mineralized bone. The proliferation and maturation of the skeletal precursor cells (chondrocytes) within the cartilage template and their replacement by bone is a highly coordinated process which, if misregulated, can lead to a number of defects including dwarfism and other skeletal deformities. This is exemplified by the fact that abnormal bone development is one of the most common types of human birth defects. Yet, many of the factors that initiate and regulate chondrocyte maturation are not known. We identified a recessive dwarf mouse mutant (pug) from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. pug mutant skeletal elements are patterned normally during development, but display a ~20% length reduction compared to wild-type embryos. We show that the pug mutation does not lead to changes in chondrocyte proliferation but instead promotes premature maturation and early ossification, which ultimately leads to disproportionate dwarfism. Using sequence capture and high-throughput sequencing, we identified a missense mutation in the Xylosyltransferase 1 (Xylt1) gene in pug mutants. Xylosyltransferases catalyze the initial step in glycosaminoglycan (GAG) chain addition to proteoglycan core proteins, and these modifications are essential for normal proteoglycan function. We show that the pug mutation disrupts Xylt1 activity and subcellular localization, leading to a reduction in GAG chains in pug mutants. The pug mutant serves as a novel model for mammalian dwarfism and identifies a key role for proteoglycan modification in the initiation of chondrocyte maturation.
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http://dx.doi.org/10.1016/j.ydbio.2013.10.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3895954PMC
January 2014

Neutral sphingomyelinase 2 deficiency increases hyaluronan synthesis by up-regulation of Hyaluronan synthase 2 through decreased ceramide production and activation of Akt.

J Biol Chem 2012 Apr 1;287(17):13620-32. Epub 2012 Mar 1.

Department of Pediatrics, University of Chicago, Chicago, Illinois 60637, USA.

Fibroblasts from the fro/fro mouse, with a deletion in the Smpd3 gene coding for the active site of neutral sphingomyelinase 2 (NSMase2), secreted increased amounts of hyaluronan (HA). This was reversed by transfection with the Smpd3 gene, suggesting a connection between sphingolipid and glycosaminoglycan metabolism. The deficiency of NSMase2 resulted in storage of sphingomyelin (SM) and cholesterol with a 50% reduction in ceramides (Cer). RT-PCR and Western blot analysis showed that increased HA secretion resulted from increased hyaluronan synthase 2 (HAS2) activity localized to sphingolipid-enriched lipid rafts. Although cholesterol levels were also elevated in lipid rafts from mouse fibroblasts deficient in lysosomal acid SMase activity (deletion of the Smpd1(-/-) gene), there was no increase in HA secretion. We then showed that in fro/fro fibroblasts, the reduced ceramide was associated with decreased phosphorylation of protein phosphatase 2A (PP2A) and increased phosphorylation of its substrate Akt-p, together with PI3K, PDK1, mTOR (mammalian target of rapamycin), and p70S6K, although PTEN was unaffected. Exogenous ceramide, as well as inhibitors of Akt (Akt inhibitor VIII), PI 3-kinase (LY294002 and wortmannin), and mTOR (rapamycin) reduced secretion of HA, whereas the NSMase2 inhibitor GW4869 increased HA synthesis and secretion. We propose that NSMase2/Cer are the key mediators of the regulation of HA synthesis, via microdomains and the Akt/mTOR pathway.
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http://dx.doi.org/10.1074/jbc.M111.304857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3340193PMC
April 2012

Proteoglycans: gene cloning.

Methods Mol Biol 2012 ;836:3-21

Departments of Pediatric, The University of Chicago, Chicago, IL, USA.

Aggrecan is a large proteoglycan that plays roles in numerous tissues during vertebrate development and adult life. The 6,327-nt chick aggrecan coding sequence had been determined from overlapping clones, but a full-length cDNA, needed for use in transgenic expression studies, had not been constructed. The strategy employed to do so was to generate two overlapping cDNA subfragments that shared a unique restriction site in the overlap and then join them at that site. These subfragments were obtained and cloned into the TOPO-TA vector pCR2.1. Digestion of the two constructs with the shared-site enzyme, XbaI, produced vector/5'-cDNA and 3'-cDNA fragments with XbaI-ends; these were ligated to produce the final full-length cDNA.
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http://dx.doi.org/10.1007/978-1-61779-498-8_1DOI Listing
May 2012

Astrocyte precursor response to embryonic brain injury.

Brain Res 2011 May 9;1389:35-49. Epub 2011 Mar 9.

Department of Pediatrics, The University of Chicago Medical Center, 5841 S. Maryland Avenue, MC 5058, Chicago, IL 60637, USA.

Penetrating traumatic insult during pregnancy is a leading cause of human fetal demise; in particular, trauma to the brain may lead to devastating long-term cognitive sequelae. Perinatal brain injury involves glial precursors, but the neural mechanisms controlling astrocyte ontogeny after injury remain incompletely understood, partly due to a lack of appropriate markers and animal models. We analyzed astrocyte precursor response to injury at the beginning (E11) and peak (E15) of gliogenesis in an avian tectal model of penetrating embryonic brain trauma, without confounding maternal and sibling effects. At both ages, lateral ventricular dilatation, necrotic foci, periventricular cysts and intraventricular hemorrhages were observed distal to stab wounds two days after a unilateral stab injury to optic tecta. Neuronal (TUBB3) and oligodendrocyte precursor (PLP) markers were down-regulated, even far-removed from the wound site. In contrast, the mature astrocyte marker, GFAP, was up-regulated at the wound site, around necrotic areas and cysts, plus in usual areas of GFAP expression. Increased inflammatory response and apoptotic cell death were also confirmed in the injured tecta. Increased expression of NFIA, SOX9 and GLAST at the wound site and in the ventricular zone (VZ) of the injured tecta indicated an astroglial precursor response. However, cell division increased in the VZ only in early (E11) injury, but not later (E15), indicating that in late injury the astrogliogenesis occurring after acute injury is predominantly due to precursor differentiation rather than precursor proliferation. The inability to replenish the glial precursor pool during the critical period of vulnerability to injury may be an important cause of subsequent developmental abnormalities.
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http://dx.doi.org/10.1016/j.brainres.2011.03.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3097075PMC
May 2011

Sulfation of chondroitin sulfate proteoglycans is necessary for proper Indian hedgehog signaling in the developing growth plate.

Development 2009 May 15;136(10):1697-706. Epub 2009 Apr 15.

Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.

In contrast to the functional role of heparan sulfate proteoglycans (HSPGs), the importance of chondroitin sulfate proteoglycans (CSPGs) in modulating signaling pathways involving hedgehog proteins, wingless-related proteins and fibroblast growth factors remains unclear. To elucidate the importance of sulfated CSPGs in signaling paradigms required for endochondral bone formation, the brachymorphic (bm) mouse was used as a model for undersulfated CSPGs. The bm mouse exhibits a postnatal chondrodysplasia caused by a mutation in the phosphoadenosine phosphosulfate (PAPS) synthetase (Papss2) gene, leading to reduced levels of PAPS and undersulfated proteoglycans. Biochemical analysis of the glycosaminoglycan (GAG) content in bm cartilage via sulfate labeling and fluorophore-assisted carbohydrate electrophoresis revealed preferential undersulfation of chondroitin chains (CS) and normal sulfation of heparan sulfate chains. In situ hybridization and immunohistochemical analysis of bm limb growth plates showed diminished Indian hedgehog (Ihh) signaling and abnormal Ihh protein distribution in the extracellular matrix. Consistent with the decrease in hedgehog signaling, BrdU incorporation exhibited a significant reduction in chondrocyte proliferation. Direct measurements of Ihh binding to defined GAG chains demonstrated that Ihh interacts with CS, particularly chondroitin-4-sulfate. Furthermore, co-immunoprecipitation experiments showed that Ihh binds to the major cartilage CSPG aggrecan via its CS chains. Overall, this study demonstrates an important function for CSPGs in modulating Ihh signaling in the developing growth plate, and highlights the importance of carbohydrate sulfation in regulating growth factor signaling.
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http://dx.doi.org/10.1242/dev.030742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2673757PMC
May 2009

Aggrecan modulation of growth plate morphogenesis.

Dev Biol 2009 May 3;329(2):242-57. Epub 2009 Mar 3.

Departments of Pediatrics and Biochemistry and Molecular Biology, Committee on Developmental Biology, The University of Chicago, Chicago, IL 60637, USA.

Chick and mouse embryos with heritable deficiencies of aggrecan exhibit severe dwarfism and premature death, demonstrating the essential involvement of aggrecan in development. The aggrecan-deficient nanomelic (nm) chick mutant E12 fully formed growth plate (GP) is devoid of matrix and exhibits markedly altered cytoarchitecture, proliferative capacity, and degree of cell death. While differentiation of chondroblasts to pre-hypertrophic chondrocytes (IHH expression) is normal up to E6, the extended periosteum expression pattern of PTCH (a downstream effector of IHH) indicates altered propagation of IHH signaling, as well as accelerated down-regulation of FGFR3 expression, decreased BrdU incorporation and higher levels of ERK phosphorylation, all indicating early effects on FGF signaling. By E7 reduced IHH expression and premature expression of COL10A1 foreshadow the acceleration of hypertrophy observed at E12. By E8, exacerbated co-expression of IHH and COL10A1 lead to delayed separation and establishment of the two GPs in each element. By E9, increased numbers of cells express P-SMAD1/5/8, indicating altered BMP signaling. These results indicate that the IHH, FGF and BMP signaling pathways are altered from the very beginning of GP formation in the absence of aggrecan, thereby inducing premature hypertrophic chondrocyte maturation, leading to the nanomelic long bone growth disorder.
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http://dx.doi.org/10.1016/j.ydbio.2009.02.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2810547PMC
May 2009

Sulfate activation enzymes: phylogeny and association with pyrophosphatase.

J Mol Evol 2009 Jan 6;68(1):1-13. Epub 2008 Dec 6.

Department of Pediatrics, University of Chicago, 5841 South Maryland Avenue, WCH C519A, Chicago, IL, 60637, USA,

The enzymes catalyzing the first two reactions in the sulfate activation pathway, ATP-sulfurylase (S) and APS-kinase (K), are fused as 'KS' in animals but are fused as 'SK' in select bacteria and fungi. We have discovered a novel triple fusion protein of K, S, and pyrophosphatase (P) in several protozoan genomes within the Stramenopile lineage. These triple domain fusion proteins led us to hypothesize that pyrophosphatase enzymes and sulfate activation enzymes physically interact to impact the thermodynamics of the sulfate activation pathway. In support of this hypothesis, we demonstrate through biochemical assays that separately encoded KS and P proteins physically interact and that KS/P complexes activate more sulfate than KS alone. We also conclude on the basis of phylogenetic analyses that all known KS fusions originate from a single fusion event early in the eukaryotic lineage. Strikingly, our analyses support the same conclusion for all known SK fusions. These observations indicate that the patchwork of fused and nonfused S and K genes observed in modern-day eukaryotes and prokaryotes are the result of the two ancestral fusion genes evolving by an assortment of gene fissions, duplications, deletions, and horizontal transfers in different lineages. Our integrative use of genomics, phylogenetics, and biochemistry to characterize pyrophosphatase as a new member of the sulfate activation pathway should be effective at identifying new protein members and connections in other molecular pathways.
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http://dx.doi.org/10.1007/s00239-008-9181-6DOI Listing
January 2009

Glial migratory streams in the developing hindbrain: a slice culture approach.

J Neurosci Methods 2009 Feb 2;177(1):30-43. Epub 2008 Oct 2.

Department of Pediatrics, 5841 S. Maryland Avenue, MC 5058, The University of Chicago, Chicago, IL 60637, USA.

Compared to our knowledge of neurogenesis, relatively little is known about glial cell specification and migration during central nervous system development. We have established a novel chick hindbrain slice preparation which permits examination of gliogenesis in its native environment, providing a means to study the signaling pathways involved in glial cell specification and migration during development. Cells in the hindbrain slice preparations mature in a manner which is similar to in vivo developmental timing and patterning paradigms. To demonstrate the utility of this approach, we examined the effect of the retinoic acid signaling pathway on cells in these slices, showing that addition of exogenous trans-retinoic acid to slice cultures promotes expression of a marker of mature astrocytes, glial fibrillary acidic protein (GFAP), while the inhibition of endogenous retinoic acid synthesis reduces GFAP expression; the results suggest a role for retinoic acid in modulating glial differentiation. Using these hindbrain slice cultures, we have used two different approaches to label glial progenitors specifically at the ventricular zone and have observed for the first time the ventrally-directed migration of these cells from the ventricular zone of the hindbrain. This slice culture system is thus an innovative and robust tool for examining glial cell migration and the extracellular molecular and signaling pathways which regulate glial differentiation.
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http://dx.doi.org/10.1016/j.jneumeth.2008.09.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2677068PMC
February 2009

Aggrecan is expressed by embryonic brain glia and regulates astrocyte development.

Dev Biol 2008 Mar 22;315(1):114-24. Epub 2008 Jan 22.

Department of Pediatrics, 5841 S. Maryland Ave., MC 5058, The University of Chicago, Chicago, IL 60637, USA.

Determination of the molecules that regulate astrocyte development has been hindered by the paucity of markers that identify astrocytic precursors in vivo. Here we report that the chondroitin sulfate proteoglycan aggrecan both regulates astrocyte development and is expressed by embryonic glial precursors. During chick brain development, the onset of aggrecan expression precedes that of the astrocytic marker GFAP and is concomitant with detection of the early glial markers GLAST and glutamine synthetase. In co-expression studies, we established that aggrecan-rich cells contain the radial glial markers nestin, BLBP and GLAST and later in embryogenesis, the astroglial marker GFAP. Parallel in vitro studies showed that ventricular zone cultures, enriched in aggrecan-expressing cells, could be directed to a GFAP-positive fate in G5-supplemented differentiation media. Analysis of the chick aggrecan mutant nanomelia revealed marked increases in the expression of the astrocyte differentiation genes GFAP, GLAST and GS in the absence of extracellular aggrecan. These increases in astrocytic marker gene expression could not be accounted for by changes in precursor proliferation or cell death, suggesting that aggrecan regulates the rate of astrocyte differentiation. Taken together, these results indicate a major role for aggrecan in the control of glial cell maturation during brain development.
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http://dx.doi.org/10.1016/j.ydbio.2007.12.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2408532PMC
March 2008

Got sulfate? Luring axons this way and that.

Chem Biol 2007 Feb;14(2):119-20

Department of Pediatrics, The University of Chicago, Chicago, IL 60637, USA.

Glycosaminoglycans are sulfated in complex and changing patterns that affect neural development. These sugars mediate interactions between macromolecules, and their biological contributions are of high interest. In this issue of Chemistry & Biology, Shipp and Hsieh-Wilson describe microarrays to probe these complex modifications.
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http://dx.doi.org/10.1016/j.chembiol.2007.02.001DOI Listing
February 2007

APBP-1, a DNA/RNA-binding protein, interacts with the chick aggrecan regulatory region.

J Biol Chem 2005 Oct 12;280(42):35606-16. Epub 2005 Aug 12.

Department of Pediatrics, University of Chicago, Chicago, Illinois 60637, USA.

Expression of the extracellular proteoglycan aggrecan is both cell-specific and developmentally regulated. Previous studies identified six functionally defined cis elements in the aggrecan promoter region which were shown to repress aggrecan gene expression (1). Using competition electrophoretic mobility shift assays (EMSAs) we have now identified in nuclear extracts a functional repressor cis element, (T/C)TCCCCT(A/C)RRC, which occurs at multiple locations within the chick aggrecan regulatory region. We purified the factor that binds to this cis element and established that it, APBP-1 (aggrecan promoter-binding protein-1), is a 19-kDa protein that has significant homology to CIRP (cold inducible RNA-binding protein). Recombinantly expressed APBP-1 mimics the native cis element-trans factor interaction in EMSAs. In situ hybridization demonstrates that aggrecan and APBP-1 RNA expression are restricted to complementary tissues in the developing limb, and Northern blot analysis of chick limb bud mRNA shows that APBP-1 mRNA expression is inversely correlated with aggrecan mRNA expression. Functional analyses by transient transfections and Northern blot analyses suggest APBP-1 has the capacity to repress aggrecan expression, indicating that this factor may be important regulator of aggrecan gene expression.
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http://dx.doi.org/10.1074/jbc.M505380200DOI Listing
October 2005

Proteoglycans in brain development.

Glycoconj J 2004 ;21(6):329-41

Department of Pediatrics, Committee on Developmental Biology, The University of Chicago, Chicago, IL 60637, USA.

Proteoglycans, as part of the extracellular or cell-surface milieu of most tissues and organ systems, play important roles in morphogenesis by modulating cell-matrix or cell-cell interactions, cell adhesiveness, or by binding and presenting growth and differentiation factors. Chondroitin sulfate proteoglycans which constitute the major population of proteoglycans in the central nervous system may influence formation of neuronal nuclei, establishment of boundaries for axonal growth and act as modulators of neuronal outgrowth during brain development, as well as during regeneration after injury. There is a paucity of information on the role of chondroitin sulfate proteoglycans in central nervous system organogenesis. In the chick embryo, aggrecan has a regionally specific and developmentally regulated expression profile during brain development. By Northern and Western blot analysis, aggrecan expression is first detected in chick brain on embryonic day 7 (E7), increases from E7 to E13, declines markedly after E16, and is not evident in hatchling brains. The time course and pattern of aggrecan expression observed in ventricular zone cells suggested that it might play a role in gliogenesis. We have analyzed the role of aggrecan during brain development using a aggrecan-deficient model, nanomelia. In nanomelic chicks, expression and levels of neurocan and brevican is not affected, indicating a non-redundant role for these members of the aggrecan gene family. Our analysis of the aggrecan-deficient model found a severely altered phenotype which affects cell behavior in a neuronal culture paradigm and expression of astrocytic markers in vivo . Taken together our results suggest a function for aggrecan in the specification of a sub-set of glia precursors that might give rise to astrocytes in vivo.
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http://dx.doi.org/10.1023/B:GLYC.0000046278.34016.36DOI Listing
May 2005

Aggrecan regulates telencephalic neuronal aggregation in culture.

Brain Res Dev Brain Res 2003 Jul;143(2):207-16

Departments of Pediatrics and Biochemistry & Molecular Biology, Committee on Developmental Biology, The University of Chicago, 5841 S. Maryland Avenue, MC 5058, Chicago, IL 60637, USA.

Proteoglycans have been suggested to play roles in pattern formation in the developing central nervous system. In the chick embryo, aggrecan, a chondroitin sulfate proteoglycan, has a regionally-specific and developmentally-regulated expression profile. Telencephalic neuronal cultures, when aggregated, exhibit aggrecan expression patterns comparable to those observed in vivo. The chicken mutation nanomelia produces a truncated aggrecan species that cannot be processed further and is not secreted. Neurons from normal and nanomelic chick embryo telencephalon were scored for aggregate formation and analyzed for distribution of aggrecan protein and expression of aggrecan mRNA. Distinctly different pattern formation, with respect to aggregate size (smaller) and number (fewer) were observed in poly-L-lysine plated neuronal cultures derived from nanomelic embryos when compared to those derived from normal embryos. Significantly, the nanomelic phenotype was subsequently rescued upon addition of the brain-specific form of aggrecan. Modulation of neuronal aggregate formation was mimicked by treatment with chondroitinase ABC but not other glycanases, and was rescued by addition of chondroitin 6-sulfate to the culture media. Lastly, although broad and diffuse distribution of aggrecan among the cell aggregates in the culture paradigm was observed by immunocytochemistry, mRNA in situ hybridization revealed that only a small population of cells in the center of the aggregates was responsible for the production of the secreted aggrecan found associated with neuronal aggregates. These studies suggest a function for aggrecan as a diffusible signal in CNS histomorphogenesis.
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http://dx.doi.org/10.1016/s0165-3806(03)00133-0DOI Listing
July 2003

Developmental expression of the HNK-1 carbohydrate epitope on aggrecan during chondrogenesis.

Dev Dyn 2003 Jan;226(1):42-50

Department of Pediatrics, The University of Chicago, Chicago, Illinois, USA.

Previously, we showed that the HNK-1 carbohydrate epitope is expressed on aggrecan synthesized in the notochord but not in mature cartilage. In the present study, we demonstrate that in immature cartilage (embryonic day 6) the HNK-1 epitope is also expressed predominantly on aggrecan proteoglycan molecules. This finding was verified by using an aggrecan-deficient mutant, the nanomelic chick, which lacks HNK-1 immunostaining in the extracellular matrix of dividing and hypertrophic chondrocytes as late as embryonic day 12. By using both biochemical and immunologic approaches, the initially prominent expression of the HNK-1 epitope is down-regulated as development of limb and vertebral cartilage proceeds, so that by embryonic day 14 no HNK-1 is detectable. Localization changes with development and the HNK-1-aggrecan matrix becomes restricted to dividing and hypertrophic chondrocytes and is particularly concentrated in the intraterritorial matrix. Concomitant with the temporal and spatial decreases in HNK-1, there is a significant increase in keratan-sulfate content and the aggrecan-borne HNK-1 epitope is closely associated with proteolytic peptides that contain keratan sulfate chains, rather than chondroitin sulfate chains or carbohydrate-free domains. Lastly, the diminution in HNK-1 expression is consistent with a reduction in mRNA transcripts specific for at least one of the key enzymes in HNK-1 oligosaccharide biosynthesis, the HNK-1 sulfotransferase. These findings indicate that the HNK-1 carbohydrate may be a common modifier of several proteoglycans (such as aggrecan) that are usually expressed early in development, and that HNK-1 addition to these molecules may be regulated by tissue- and temporal-specific expression of requisite sulfotransferases and glycosyltransferases.
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http://dx.doi.org/10.1002/dvdy.10214DOI Listing
January 2003

Identification and functional characterization of the novel BM-motif in the murine phosphoadenosine phosphosulfate (PAPS) synthetase.

J Biol Chem 2003 Jan 31;278(1):71-5. Epub 2002 Oct 31.

Department of Chemistry, The University of Chicago, Chicago, Illinois 60637, USA.

PAPS synthetase (SK) catalyzes the two sequential reactions of phosphoadenosine phosphosulfate (PAPS) synthesis. A functional motif in the kinase domain of mouse SK, designated the BM-motif ((86) LDGDNhRxhh(N/S)(K/R)(97)), was defined in the course of identifying the brachymorphic (bm) defect. Sequence comparison and the secondary structure predicted for APS kinase suggest that the BM-motif consists of a DGD-turn sequence flanked by other conserved residues. Mutational analysis of the DGD-turn revealed that a flexible and neutral amino acid is preferred at residue 88, that negatively charged residues are strictly required at positions 87 and 89, and that the active site is rigid. The reduction in kinase activity for all DGD-turn mutants, except G88A, was much less severe than the reduction in overall activity, indicating that the BM-motif may also be playing a role in adenosine phosphosulfate (APS) channeling. Two switch mutations, LD86DL and DN89ND, designed to test the positional constraints of Asp(87) and Asp(89), exhibited complete loss of both kinase and overall activities, while LD86DL also exhibited a significant (60%) loss of reverse sulfurylase activity, suggesting that this peptide region is interacting with the sulfurylase domain as well as functioning in the kinase reaction. Other residues targeted for mutational analysis were the highly conserved flanking Asn(90), Arg(92), and Lys(97). N90A resulted in a partial (30%) loss in kinase and overall activities, R92A exhibited total loss of kinase and overall activities, and K97A had no effect on any of the three activities. The complexity of the bifunctional SK in catalyzing the kinase reaction and channeling APS is illustrated by the strict requirements of this novel structural motif in the kinase active site.
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http://dx.doi.org/10.1074/jbc.M206688200DOI Listing
January 2003

Chondrodysplasias due to proteoglycan defects.

Glycobiology 2002 Apr;12(4):57R-68R

Department of Pediatrics, University of Chicago, MC 5058, 5826 S. Maryland Ave., Chicago, IL 60637, USA.

The proteoglycans, especially the large chondroitin sulfate proteoglycan aggrecan, have long been viewed as important components of the extracellular matrix of cartilage. The drastic change in expression during differentiation from mesenchyme to cartilage, the loss of tissue integrity associated with proteoglycan degradation in several disease processes and, most important, the demonstration of abnormalities in proteoglycan production concomitant with the aberrant growth patterns exhibited by the brachymorphic mouse, the cartilage matrix deficient mouse, and the nanomelic chick provide the strongest evidence that the proteoglycan aggrecan is essential during differentiation and for maintenance of the skeletal elements. More recently, mutations associated with proteoglycans other than aggrecan, especially the heparan sulfate proteoglycans, glypican and perlecan, suggest an important role for these molecules in skeletal development as well. This review focuses on the molecular bases of the hereditary proteoglycan defects in animal models, as well as of some human chondrodysplasias, that collectively are providing a better understanding of the role of proteoglycans in the development and maintenance of the skeletal elements.
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http://dx.doi.org/10.1093/glycob/12.4.57rDOI Listing
April 2002