• Acetylation of the Pro-Apoptotic Factor, p53 in the Hippocampus following Cerebral Ischemia and Modulation by Estrogen

      Raz, Limor; Zhang, Quan-Guang; Han, Dong; Dong, Yan; De Sevilla, Liesl; Brann, Darrell W; Institute of Molecular Medicine and Genetics (2011-10-26)
      Background: Recent studies demonstrate that acetylation of the transcription factor, p53 on lysine373 leads to its enhanced stabilization/activity and increased susceptibility of cells to stress. However, it is not known whether acetylation of p53 is altered in the hippocampus following global cerebral ischemia (GCI) or is regulated by the hormone, 17b-estradiol (17b2E2), and thus, this study examined these issues.
    • alpha-Actinin interacts with rapsyn in agrin-stimulated AChR clustering.

      Dobbins, G Clement; Luo, Shiwen; Yang, Zhihua; Xiong, Wen C; Mei, Lin; Institute of Molecular Medicine and Genetics; Department of Neuroscience and Regenerative Medicine (2009-01-13)
      : AChR is concentrated at the postjunctional membrane at the neuromuscular junction. However, the underlying mechanism is unclear. We show that alpha-actinin, a protein known to cross-link F-actin, interacts with rapsyn, a scaffold protein essential for neuromuscular junction formation. alpha-Actinin, rapsyn, and surface AChR form a ternary complex. Moreover, the rapsyn-alpha-actinin interaction is increased by agrin, a factor known to stimulate AChR clustering. Downregulation of alpha-actinin expression inhibits agrin-mediated AChR clustering. Furthermore, the rapsyn-alpha-actinin interaction can be disrupted by inhibiting Abl and by cholinergic stimulation. Together these results indicate a role for alpha-actinin in AChR clustering.
    • BDNF-induced recruitment of TrkB receptor into neuronal lipid rafts

      Suzuki, Shingo; Numakawa, Tadahiro; Shimazu, Kazuhiro; Koshimizu, Hisatsugu; Hara, Tomoko; Hatanaka, Hiroshi; Mei, Lin; Lu, Bai; Kojima, Masami; Institute of Molecular Medicine and Genetics (2004-12-20)
      Brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity but the underlying signaling mechanisms remain unknown. Here, we show that BDNF rapidly recruits full-length TrkB (TrkB-FL) receptor into cholesterol-rich lipid rafts from nonraft regions of neuronal plasma membranes. Translocation of TrkB-FL was blocked by Trk inhibitors, suggesting a role of TrkB tyrosine kinase in the translocation. Disruption of lipid rafts by depleting cholesterol from cell surface blocked the ligand-induced translocation. Moreover, disruption of lipid rafts prevented potentiating effects of BDNF on transmitter release in cultured neurons and synaptic response to tetanus in hippocampal slices. In contrast, lipid rafts are not required for BDNF regulation of neuronal survival. Thus, ligand-induced TrkB translocation into lipid rafts may represent a signaling mechanism selective for synaptic modulation by BDNF in the central nervous system.
    • Ceramide in Stem Cell Differentiation and Embryo Development: Novel Functions of a Topological Cell-Signaling Lipid and the Concept of Ceramide Compartments

      Bieberich, Erhard; Institute of Molecular Medicine and Genetics (2010-12-29)
      In the last two decades, the view on the function of ceramide as a sole metabolic precursor for other sphingolipids has completely changed. A plethora of studies has shown that ceramide is an important lipid cell-signaling factor regulating apoptosis in a variety of cell types. With the advent of new stem cell technologies and knockout mice for specific steps in ceramide biosynthesis, this view is about to change again. Recent studies suggest that ceramide is a critical cell-signaling factor for stem cell differentiation and cell polarity, two processes at the core of embryo development. This paper discusses studies on ceramide using in vitro differentiated stem cells, embryo cultures, and knockout mice with the goal of linking specific developmental stages to exciting and novel functions of this lipid. Particular attention is devoted to the concept of ceramide as a topological cell-signaling lipid: a lipid that forms distinct structures (membrane domains and vesicles termed â sphingosomeâ ), which confines ceramide-induced cell signaling pathways to localized and even polarized compartments.
    • Ceramide-mediated Regulation of Cell Polarity in Primitive Ectoderm Cells: A novel role for sphingolipids in morphogenesis

      Krishnamurthy, Kannan; Institute of Molecular Medicine and Genetics (2009-01)
      Ceramide is considered a key sphingolipid, regulating a variety of critical cellular processes. To facilitate the study of ceramide localization and its interaction with cellular proteins, we have developed a novel antibody against ceramide, raised in rabbit (rabbit IgG). The novel antibody specifically recognizes ceramide in lipid overlay assays and detects ceramide containing different fatty acid chain lengths (i.e. C2-, C16-, C18-, C20- and C24 ceramide). The new antibody was compared with the commercially available anti-ceramide mouse IgM antibody in immunocytochemistry experiments to study the localization of ceramide. Although both antibodies stain similar regions on the cell membrane, the rabbit IgG reveals the distribution of ceramide in intracellular compartments that are not well identified with the commercially available antibody. Pharmacological depletion or increase of ceramide levels results in a corresponding change in staining intensity, confirming the specificity of the antibody. These results indicate that the rabbit IgG is a suitable antibody to determine both the localization of ceramide, and its interaction with proteins by immunocytochemistry. To investigate the role of ceramide in early embryonic development, we used embryoid bodies (EBs) differentiated from mouse embryonic stem cells as a model. The primitive ectoderm cell layer of EBs represents the primitive ectoderm of the early embryo. In mammals, the primitive ectoderm is an epithelium of polarized cells that undergoes gastrulation and differentiates into all embryonic tissues. We find that in primitive ectoderm cells, ceramide was elevated and asymmetrically distributed to the apico-lateral cell membrane, where it was co-distributed with Cdc42 and F-actin. Pharmacological or siRNAmediated inhibition of ceramide biosynthesis impaired primitive ectoderm formation and concomitantly increased apoptosis in EBs. Primitive ectoderm formation was restored by incubation with ceramide or a ceramide analog, indicating that the observed defect was due to loss of ceramide. Ceramide depletion also prevented membrane translocation of atypical PKC (aPKC), interaction of aPKC with Cdc42, and phosphorylation of GSK-3|3. Recombinant aPKC, when bound to ceramide-containing lipid vesicles, formed a complex with the polarity protein Par6 and Cdc42. Taken together, our data suggest a novel mechanism by which a ceramide-induced, apico-lateral polarity complex with aPKC regulates primitive ectoderm cell polarity and morphogenesis.
    • Characterization of Cervical and Head and Neck Squamous Cell Carcinomas by Proteomic Analysis

      Merkley, Mark A.; Institute of Molecular Medicine and Genetics (2009-06)
      (First Paragraph) The oral cavity, oropharynx, larynx, esophagus, and ano-genital orifices are lined with stratified squamous nonkeratinized epithelium, which forms the barrier between the underlying tissue and the external environment. The proliferative nature of this epithelium, together with its potential exposure to environmental insults such as tobacco carcinogens, alcohol, or oncogenic viruses, makes it susceptible to carcinogenesis. Indeed, carcinomas of stratified squamous nonkeratinized epithelium are among the most common and deadly cancers worldwide. In particular head and neck squamous cell carcinoma and cervical squamous cell carcinoma together account for about 1 million new cases annually, worldwide. These cancers arise from similar tissues and share common risk factors, although they differ in that effective population-based screening exists only for cervical cancer.
    • Cleft palate is caused by CNS dysfunction in Gad1 and Viaat knockout mice.

      Oh, Won-Jong; Westmoreland, Joby J; Summers, Ryan; Condie, Brian G.; Institute of Molecular Medicine and Genetics (2010-03-24)
      BACKGROUND: Previous studies have shown that disruption of GABA signaling in mice via mutations in the Gad1, Gabrb3 or Viaat genes leads to the development of non-neural developmental defects such as cleft palate. Studies of the Gabrb3 and Gad1 mutant mice have suggested that GABA function could be required either in the central nervous system or in the palate itself for normal palatogenesis. METHODOLOGY/PRINCIPAL FINDINGS: To further examine the role of GABA signaling in palatogenesis we used three independent experimental approaches to test whether Gad1 or Viaat function is required in the fetal CNS for normal palate development. We used oral explant cultures to demonstrate that the Gad1 and Viaat mutant palates were able to undergo palatogenesis in culture, suggesting that there is no defect in the palate tissue itself in these mice. In a second series of experiments we found that the GABA(A) receptor agonist muscimol could rescue the cleft palate phenotype in Gad1 and Viaat mutant embryos. This suggested that normal multimeric GABA(A) receptors in the CNS were necessary for normal palatogenesis. In addition, we showed that CNS-specific inactivation of Gad1 was sufficient to disrupt palate development. CONCLUSIONS/SIGNIFICANCE: Our results are consistent with a role for Gad1 and Viaat in the central nervous system for normal development of the palate. We suggest that the alterations in GABA signaling lead to non-neural defects such as cleft palate as a secondary effect due to alterations in or elimination of fetal movements.
    • The Clinical Phenotype of An Extended Pedigree with Late-onset Alzheimer's Disease

      Fennell, Eleanor M.; Institute of Molecular Medicine and Genetics (2009-03)
      Alzheimer's disease (AD) is a devastating neurodegenerative disease with a multifaceted etiology. This retrospective exploratory study used the family history method to develop a phenotype based on the personal history of cognitive decline, changes in behavioral characteristics, and the medical, social and environmental history of the five AD affected family members in one large extended family. Group family interviews provided a description of the phenotype and identified medical and environmental risk modifiers. Average age-of-onset for AD in 5 of 12 siblings, (all homozygous APOE4/4) was 69.2 years (range 66-72 years). Fisher's exact test identified neuropsychiatric behaviors as common phenotypic manifestations; delusions (p = 0.0455), hallucinations (p = 0.0101), irritability (p = 0.0455), personality change (p = 0.0013), pacing (p = 0.0013), aggressiveness (p = 0.0455), and poor judgment (p = 0.0013). Environmental variables that emerged as significant were a less than an eighth grade education (p — 0.0152), presence of stroke/TIA (p = 0.0455), presence of osteoarthritis (p = 0.0455), and vitamin B12 deficiency (p = 0.0013). Risk of stroke/TIA may be related to the increased risk from APOE4 while vitamin B12 deficiency may be associated with advanced age. There was no significant protective benefit from the management of hypertension (p = 0.5758), use of statins to control cholesterol (p = 0.9545), use of Vitamin C (p = 1.000), and/or Vitamin E (p = 0.9899) among family members. Potential modifiable health practices among the AD unaffected siblings demonstrated that 85% continued to engage in a sedentary lifestyle (p = 0.6818), 57% were overweight (p = 0.6894), 57% (p = 0.6894) consumed an unhealthy diet, and 56% smoked (p = 0.1591). Single nucleotide polymorphism (SNP) microarray Clinical Phenotype of Alzheimer's Disease analysis indicated that several SNPs in the gene, transient receptor potential cation channel, subfamily C, member 4 associated protein, (TRPC4AP), were significant. All five of the affected siblings and three unaffected siblings exhibited one haplotype; the unaffected siblings with the same haplotype as affected siblings were younger in age and did not have any cognitive problems at the time of the study (Poduslo, Huang, Huang, Smith, 2008).
    • Comparison and Avoidance of Toxicity of Penetrating Cryoprotectants

      Szurek, Edyta A.; Eroglu, Ali; Institute of Molecular Medicine and Genetics; Department of Medicine; Department of Obstetrics and Gynecology; GHSU Cancer Center (2011-11-16)
      The objective of this study was to elucidate the toxicity of widely used penetrating cryoprotective agents (CPAs) to mammalian oocytes. To this end, mouse metaphase II (M II) oocytes were exposed to 1.5 M solutions of dimethylsulfoxide (DMSO), ethylene glycol (EG), or propanediol (PROH) prepared in phosphate buffered saline (PBS) containing 10% fetal bovine serum. To address the time- and temperature-dependence of the CPA toxicity, M II oocytes were exposed to the aforementioned CPAs at room temperature (RT, ,23uC) and 37uC for 15 or 30 minutes. Subsequently, the toxicity of each CPA was evaluated by examining post-exposure survival, fertilization, embryonic development, chromosomal abnormalities, and parthenogenetic activation of treated oocytes. Untreated oocytes served as controls. Exposure of MII oocytes to 1.5 M DMSO or 1.5 M EG at RT for 15 min did not adversely affect any of the evaluated criteria. In contrast, 1.5 M PROH induced a significant increase in oocyte degeneration (54.2%) and parthenogenetic activation (16%) under same conditions. When the CPA exposure was performed at 37uC, the toxic effect of PROH further increased, resulting in lower survival (15%) and no fertilization while the toxicity of DMSO and EG was still insignificant. Nevertheless, it was possible to completely avoid the toxicity of PROH by decreasing its concentration to 0.75 M and combining it with 0.75 M DMSO to bring the total CPA concentration to a cryoprotective level. Moreover, combining lower concentrations (i.e., 0.75 M) of PROH and DMSO significantly improved the cryosurvival of MII oocytes compared to the equivalent concentration of DMSO alone. Taken together, our results suggest that from the perspective of CPA toxicity, DMSO and EG are safer to use in slow cooling protocols while a lower concentration of PROH can be combined with another CPA to avoid its toxicity and to improve the cryosurvival as well.
    • Conditional Vascular Cell Adhesion Molecule 1 Deletion in Mice

      Koni, Pandelakis A.; Joshi, Sunil K.; Temann, Ulla-Angela; Olson, Dian; Burkly, Linda; Flavell, Richard A.; Institute of Molecular Medicine and Genetics; Department of Medicine (2001-03-19)
      We generated vascular cell adhesion molecule (VCAM)-1 “knock-in” mice and Cre recombinase transgenic mice to delete the VCAM-1 gene ( vcam-1 ) in whole mice, thereby overcoming the embryonic lethality seen with conventional vcam-1 –deficient mice. vcam-1 knock-in mice expressed normal levels of VCAM-1 but showed loss of VCAM-1 on endothelial and hematopoietic cells when interbred with a “TIE2Cre” transgene. Analysis of peripheral blood from conditional vcam-1– deficient mice revealed mild leukocytosis, including elevated immature B cell numbers. Conversely, the bone marrow (BM) had reduced immature B cell numbers, but normal numbers of pro-B cells. vcam-1 –deficient mice also had reduced mature IgD 1 B and T cells in BM and a greatly reduced capacity to support short-term migration of transferred B cells, CD4 1 T cells, CD8 1 T cells, and preactivated CD4 1 T cells to the BM. Thus, we report an until now unappreciated dominant role for VCAM-1 in lymphocyte homing to BM.
    • Contribution of â Virus-specific CD8+ Cytotoxic T Cells to Virus Clearance or Pathologic Manifestations of Influenza Virus Infection in a T Cell Receptor Transgenic Mouse Model

      Moskophidis, Demetrius; Kioussis, Dimitris; Institute of Molecular Medicine and Genetics (1998-07-20)
      The ability of influenza virus to evade immune surveillance by neutralizing antibodies (Abs) directed against its variable surface antigens provides a challenge to the development of effective vaccines. CD8+ cytotoxic T lymphocytes (CTLs) restricted by class I major histocompatibility complex molecules are important in establishing immunity to influenza virus because they recognize internal viral proteins which are conserved between multiple viral strains. In contrast, protective Abs are strain-specific. However, the precise role of effector CD8+ CTLs in protection from influenza virus infection, critical for understanding disease pathogenesis, has not been well defined. In transgenic mice with a very high frequency of antiinfluenza CTL precursors, but without protective Abs, CD8+ CTLs conferred protection against low dose viral challenge, but exacerbated viral pathology and caused mortality at high viral dose. The data suggest a dual role for CD8+ CTLs against influenza, which may present a challenge to the development of effective CTL vaccines. Effector mechanisms used by CD8+ CTLs in orchestrating clearance of virus and recovery from experimental influenza infection, or potentiation of lethal pathology, are discussed.
    • A critical role for the programmed death ligand 1 in fetomaternal tolerance

      Guleria, Indira; Khosroshahi, Arezou; Ansari, Mohammed Javeed; Habicht, Antje; Azuma, Miyuki; Yagita, Hideo; Noelle, Randolph J.; Coyle, Anthony; Mellor, Andrew L.; Khoury, Samia J.; et al. (2005-07-18)
      Fetal survival during gestation implies that tolerance mechanisms suppress the maternal immune response to paternally inherited alloantigens. Here we show that the inhibitory T cell costimulatory molecule, programmed death ligand 1 (PDL1), has an important role in conferring fetomaternal tolerance in an allogeneic pregnancy model. Blockade of PDL1 signaling during murine pregnancy resulted in increased rejection rates of allogeneic concepti but not syngeneic concepti. Fetal rejection was T cell– but not B cell–dependent because PDL1-specific antibody treatment caused fetal rejection in B cell–deficient but not in RAG-1 –deficient females. Blockade of PDL1 also resulted in a significant increase in the frequency of IFN- –producing lymphocytes in response to alloantigen in an ELISPOT assay and higher IFN- levels in placental homogenates by ELISA. Finally, PDL1-deficient females exhibited decreased allogeneic fetal survival rates as compared with littermate and heterozygote controls and showed evidence of expansion of T helper type 1 immune responses in vivo. These results provide the first evidence that PDL1 is involved in fetomaternal tolerance.
    • Critical Roles of Pten in B Cell Homeostasis and Immunoglobulin Class Switch Recombination

      Suzuki, Akira; Kaisho, Tsuneyasu; Ohishi, Minako; Tsukio-Yamaguchi, Manae; Tsubata, Takeshi; Koni, Pandelakis A.; Sasaki, Takehiko; Mak, Tak Wah; Nakano, Toru; Institute of Molecular Medicine and Genetics (2003-03-3)
      Pten is a tumor suppressor gene mutated in human cancers. We used the Cre-loxP system to generate a B cellâ specific mutation of Pten in mice (bPtenflox/floxmice). bPtenflox/flox mice showed elevated numbers of B1a cells and increased serum autoantibodies. Among B2 cells in bPtenflox/flox spleens, numbers of marginal zone B (MZB) cells were significantly increased while those of follicular B (FOB) cells were correspondingly decreased. Pten-deficient B cells hyperproliferated, were resistant to apoptotic stimuli, and showed enhanced migration. The survival kinase PKB/Akt was highly activated in Pten-deficient splenic B cells. In addition, immunoglobulin class switch recombination was defective and induction of activation-induced cytidine deaminase (AID) was impaired. Thus, Pten plays a role in developmental fate determination of B cells and is an indispensable regulator of B cell homeostasis.
    • The Current State of Proteomics in GI Oncology

      Lin, Ying; Dynan, William S.; Lee, Jeffrey R.; Zhu, Zhao-Hua; Schade, Robert R.; Institute of Molecular Medicine and Genetics; Department of Pathology (2009-03-23)
      Keywords: Clinical proteomics
    • Cytotoxic effects of G(M1) ganglioside and amyloid beta-peptide on mouse embryonic neural stem cells.

      Yanagisawa, Makoto; Ariga, Toshio; Yu, Robert K.; Institute of Molecular Medicine and Genetics; Institute of Neuroscience (2010-03-22)
      AD (Alzheimer's disease) is a neurodegenerative disease and the most common form of dementia. One of the pathological hallmarks of AD is the aggregation of extracellular Abetas (amyloid beta-peptides) in senile plaques in the brain. The process could be initiated by seeding provided by an interaction between G(M1) ganglioside and Abetas. Several reports have documented the bifunctional roles of Abetas in NSCs (neural stem cells), but the precise effects of G(M1) and Abeta on NSCs have not yet been clarified. We evaluated the effect of G(M1) and Abeta-(1-40) on mouse NECs (neuroepithelial cells), which are known to be rich in NSCs. No change of cell number was detected in NECs cultured in the presence of either G(M1) or Abeta-(1-40). On the contrary, a decreased number of NECs were cultured in the presence of a combination of G(M1) and Abeta-(1-40). The exogenously added G(M1) and Abeta-(1-40) were confirmed to incorporate into NECs. The Ras-MAPK (mitogen-activated protein kinase) pathway, important for cell proliferation, was intact in NECs simultaneously treated with G(M1) and Abeta-(1-40), but caspase 3 was activated. NECs treated with G(M1) and Abeta-(1-40) were positive in the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay, an indicator of cell death. It was found that G(M1) and Abeta-(1-40) interacted in the presence of cholesterol and sphingomyelin, components of cell surface microdomains. The cytotoxic effect was found also in NSCs prepared via neurospheres. These results indicate that Abeta-(1-40) and G(M1) co-operatively exert a cytotoxic effect on NSCs, likely via incorporation into NEC membranes, where they form a complex for the activation of cell death signalling.
    • Deoxycholate promotes survival of breast cancer cells by reducing the level of pro-apoptotic ceramide.

      Krishnamurthy, Kannan; Wang, Guanghu; Rokhfeld, Dmitriy; Bieberich, Erhard; Institute of Molecular Medicine and Genetics; Student Research and Training (STAR) Program, School of Graduate Studies (2009-02-23)
      INTRODUCTION: At physiologic concentration in serum, the bile acid sodium deoxycholate (DC) induces survival and migration of breast cancer cells. Here we provide evidence of a novel mechanism by which DC reduces apoptosis that is induced by the sphingolipid ceramide in breast cancer cells. METHODS: Murine mammacarcinoma 4T1 cells were used in vitro to determine apoptosis and alteration of sphingolipid metabolism by DC, and in vivo to quantify the effect of DC on metastasis. RESULTS: We found that DC increased the number of intestinal metastases generated from 4T1 cell tumors grafted into the fat pad. The metastatic nodes contained slowly dividing cancer cells in immediate vicinity of newly formed blood vessels. These cells were positive for CD44, a marker that has been suggested to be expressed on breast cancer stem cells. In culture, a subpopulation (3 +/- 1%) of slowly dividing, CD44+ cells gave rise to rapidly dividing, CD44- cells. DC promoted survival of CD44+ cells, which was concurrent with reduced levels of activated caspase 3 and ceramide, a sphingolipid inducing apoptosis in 4T1 cells. Z-guggulsterone, an antagonist of the farnesoid-X-receptor, obliterated this anti-apoptotic effect, indicating that DC increased cell survival via farnesoid-X-receptor. DC also increased the gene expression of the vascular endothelial growth factor receptor 2 (Flk-1), suggesting that DC enhanced the initial growth of secondary tumors adjacent to blood vessels. The Flk-1 antagonist SU5416 obliterated the reduction of ceramide and apoptosis by DC, indicating that enhanced cell survival is due to Flk-1-induced reduction in ceramide. CONCLUSIONS: Our findings show, for the first time, that DC is a natural tumor promoter by elevating Flk-1 and decreasing ceramide-mediated apoptosis of breast cancer progenitor cells. Reducing the level or effect of serum DC and elevating ceramide in breast cancer progenitor cells by treatment with Z-guggulsterone and/or vascular endothelial growth factor receptor 2/Flk-1 antagonists may thus be a promising strategy to reduce breast cancer metastasis.
    • Design, Synthesis, and Initial Evaluation of D-Glyceraldehyde Crosslinked Gelatin-Hydroxyapatite as a Potential Bone Graft Substitute Material

      Florschutz, Anthony V; Institute of Molecular Medicine and Genetics (2012-07)
      Utilization of bone grafts for the treatment of skeletal pathology is a common practice in orthopaedic, craniomaxillofacial, dental, and plastic surgery. Autogenous bone graft is the established archetype but has disadvantages including donor site morbidity, limited supply, and prolonging operative time. In order to avoid these and other issues, bone graft substitute materials are becoming increasingly prevalent among surgeons for reconstructing skeletal defects and arthrodesis applications. Bone graft substitutes are biomaterials, biologies, and guided tissue/bone regenerative devices that can be used alone or in combinations as supplements or alternatives to autogenous bone graft. There is a growing interest and trend to specialize graft substitutes for specific indications and although there is good rationale for this indication-specific approach, the development and utility of a more universal bone graft substitute may provide a better answer for patients and surgeons. The aim of the present research focuses on the design, synthesis, and initial evaluation of D-glyceraldehyde crosslinked gelatin-hydroxyapatite composites for potential use as a bone graft substitutes. After initial establishment of rational material design, gelatinhydroxyapatite scaffolds were fabricated with different gelatin:hydroxyapatite ratios and crosslinking concentrations. The synthesized scaffolds were subsequently evaluated on the basis of their swelling behavior, porosity, density, percent composition, mechanical properties, and morphology and further assessed with respect to cell-biomaterial interaction and biomineralization in vitro. Although none of the materials achieved mechanical properties suitable for structural graft applications, a reproducible material design and synthesis was achieved with properties recognized to facilitate bone formation. Select scaffold formulations as well as a subset of scaffolds loaded with recombinant human bone morphogenetic protein-2 were implanted ectopically in a rodent animal model and histologically evaluated for biocompatibility, degradation, and bone formation in vivo. The gelatin-hydroxyapatite scaffolds retained dimensional structure over 28 days and did not elicit any undesirable systemic or local effects. Distinct areas of mineralization and osteoid/bone were noted in all the implanted scaffolds and quantitative differences were primarily dependent on the presence of hydroxyapatite.
    • Directed neuronal differentiation of human embryonic stem cells.

      Schulz, Thomas C; Palmarini, Gail M; Noggle, Scott A; Weiler, Deborah A; Mitalipova, Maisam M; Condie, Brian G.; Institute of Molecular Medicine and Genetics (2004-05-10)
      BACKGROUND: We have developed a culture system for the efficient and directed differentiation of human embryonic stem cells (HESCs) to neural precursors and neurons.HESC were maintained by manual passaging and were differentiated to a morphologically distinct OCT-4+/SSEA-4- monolayer cell type prior to the derivation of embryoid bodies. Embryoid bodies were grown in suspension in serum free conditions, in the presence of 50% conditioned medium from the human hepatocarcinoma cell line HepG2 (MedII). RESULTS: A neural precursor population was observed within HESC derived serum free embryoid bodies cultured in MedII conditioned medium, around 7-10 days after derivation. The neural precursors were organized into rosettes comprised of a central cavity surrounded by ring of cells, 4 to 8 cells in width. The central cells within rosettes were proliferating, as indicated by the presence of condensed mitotic chromosomes and by phosphoHistone H3 immunostaining. When plated and maintained in adherent culture, the rosettes of neural precursors were surrounded by large interwoven networks of neurites. Immunostaining demonstrated the expression of nestin in rosettes and associated non-neuronal cell types, and a radial expression of Map-2 in rosettes. Differentiated neurons expressed the markers Map-2 and Neurofilament H, and a subpopulation of the neurons expressed tyrosine hydroxylase, a marker for dopaminergic neurons. CONCLUSION: This novel directed differentiation approach led to the efficient derivation of neuronal cultures from HESCs, including the differentiation of tyrosine hydroxylase expressing neurons. HESC were morphologically differentiated to a monolayer OCT-4+ cell type, which was used to derive embryoid bodies directly into serum free conditions. Exposure to the MedII conditioned medium enhanced the derivation of neural precursors, the first example of the effect of this conditioned medium on HESC.
    • Disruption-induced mucus secretion: repair and protection.

      Miyake, Katsuya; Tanaka, Tomoaki; McNeil, Paul L.; Institute of Molecular Medicine and Genetics; Department of Cellular Biology and Anatomy (2006-08-28)
      When a cell suffers a plasma membrane disruption, extracellular Ca(2+) rapidly diffuses into its cytosol, triggering there local homotypic and exocytotic membrane fusion events. One role of this emergency exocytotic response is to promote cell survival: the internal membrane thus added to the plasma membrane acts as a reparative "patch." Another, unexplored consequence of disruption-induced exocytosis is secretion. Many of the cells lining the gastrointestinal tract secrete mucus via a compound exocytotic mechanism, and these and other epithelial cell types lining the digestive tract are normally subject to plasma membrane disruption injury in vivo. Here we show that plasma membrane disruption triggers a potent mucus secretory response from stomach mucous cells wounded in vitro by shear stress or by laser irradiation. This disruption-induced secretory response is Ca(2+) dependent, and coupled to cell resealing: disruption in the absence of Ca(2+) does not trigger mucus release, but results instead in cell death due to failure to reseal. Ca(2+)-dependent, disruption-induced mucus secretion and resealing were also demonstrable in segments of intact rat large intestine. We propose that, in addition to promoting cell survival of membrane disruptions, disruption-induced exocytosis serves also the important protective function of liberating lubricating mucus at sites of mechanical wear and tear. This mode of mechanotransduction can, we propose, explain how lubrication in the gastrointestinal tract is rapidly and precisely adjusted to widely fluctuating, diet-dependent levels of mechanical stress.
    • Dynamic expression of a glutamate decarboxylase gene in multiple non-neural tissues during mouse development.

      Maddox, Dennis M; Condie, Brian G.; Institute of Molecular Medicine and Genetics; Department of Cellular Biology and Anatomy (2003-10-29)
      BACKGROUND: Glutamate decarboxylase (GAD) is the biosynthetic enzyme for the neurotransmitter gamma-aminobutyric acid (GABA). Mouse embryos lacking the 67-kDa isoform of GAD (encoded by the Gad1 gene) develop a complete cleft of the secondary palate. This phenotype suggests that this gene may be involved in the normal development of tissues outside of the CNS. Although Gad1 expression in adult non-CNS tissues has been noted previously, no systematic analysis of its embryonic expression outside of the nervous system has been performed. The objective of this study was to define additional structures outside of the central nervous system that express Gad1, indicating those structures that may require its function for normal development. RESULTS: Our analysis detected the localized expression of Gad1 transcripts in several developing tissues in the mouse embryo from E9.0-E14.5. Tissues expressing Gad1 included the tail bud mesenchyme, the pharyngeal pouches and arches, the ectodermal placodes of the developing vibrissae, and the apical ectodermal ridge (AER), mesenchyme and ectoderm of the limb buds. CONCLUSIONS: Some of the sites of Gad1 expression are tissues that emit signals required for patterning and differentiation (AER, vibrissal placodes). Other sites correspond to proliferating stem cell populations that give rise to multiple differentiated tissues (tail bud mesenchyme, pharyngeal endoderm and mesenchyme). The dynamic expression of Gad1 in such tissues suggests a wider role for GABA signaling in development than was previously appreciated.