• 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.
    • 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.
    • 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.
    • 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.
    • Ganglioside metabolism in a transgenic mouse model of Alzheimer's disease: expression of Chol-1a antigens in the brain

      Ariga, Toshio; Yanagisawa, Makoto; Wakade, Chandramohan; Ando, Susumu; Buccafusco, Jerry J; McDonald, Michael P; Yu, Robert K.; Institute of Molecular Medicine and Genetics; Department of Pharmacology and Toxicology (2010-10-4)
      The accumulation of Ab (amyloid b-protein) is one of the major pathological hallmarks in AD (Alzheimer’s disease). Gangliosides, sialic acid-containing glycosphingolipids enriched in the nervous system and frequently used as biomarkers associated with the biochemical pathology of neurological disorders, have been suggested to be involved in the initial aggregation of Ab. In the present study, we have examined ganglioside metabolism in the brain of a double- Tg (transgenic) mouse model of AD that co-expresses mouse/ human chimaeric APP (amyloid precursor protein) with the Swedish mutation and human presenilin-1 with a deletion of exon 9. Although accumulation of Ab was confirmed in the double-Tg mouse brains and sera, no statistically significant change was detected in the concentration and composition of major ganglio-N-tetraosyl-series gangliosides in the double-Tg brain. Most interestingly, Chol-1a antigens (cholinergic neuron-specific gangliosides), such as GT1aa and GQ1ba, which are minor species in the brain, were found to be increased in the double-Tg mouse brain. We interpret that the occurrence of these gangliosides may represent evidence for generation of cholinergic neurons in the AD brain, as a result of compensatory neurogenesis activated by the presence of Ab.
    • Regulation of prostaglandin synthesis and cell adhesion by a tryptophan catabolizing enzyme.

      Marshall, Brendan; Keskin, Derin B.; Mellor, Andrew L.; Institute of Molecular Medicine and Genetics (2002-11-19)
      BACKGROUND: The tryptophan catabolizing enzyme, indoleamine 2,3, dioxygenase (IDO) is one of two mammalian enzymes, which can catabolize the rarest essential amino acid, tryptophan. IDO is inducible by cytokines such as interferon-gamma and plays a role in inflammation and maternal tolerance of fetal allografts, although its exact mode of action is unclear. Therefore, we investigated the circumstances under which IDO is expressed in vitro together with the effects of overexpression of IDO on the growth and morphology of cells. RESULTS: Overexpression of IDO in the murine macrophage cell line RAW 264.7 and the murine fibrosarcoma cell line MC57, resulted in the growth of macroscopic cell foci, with altered cell adhesion properties. The expression of IDO was also detected during adhesion of wild type, nontransfected cells in tissue culture to standard cell growth substrates. Inhibition of this expression, likewise resulted in alterations in cell adhesion. Overexpression of IDO or inhibition of endogenous IDO expression was accompanied by changes in metalloproteinase expression and also in the expression and activity of the cyclooxygenase enzymes. In the case of RAW cells, IDO effects on cell growth could be reversed by adding back prostaglandins. CONCLUSIONS: These results suggest that catabolism of the rarest essential amino acid may regulate processes such as cell adhesion and prostaglandin synthesis.
    • Specific expression of lacZ and cre recombinase in fetal thymic epithelial cells by multiplex gene targeting at the Foxn1 locus.

      Gordon, Julie; Xiao, Shiyun; Hughes, Bernard; Su, Dong-ming; Navarre, Samuel P; Condie, Brian G.; Manley, Nancy R; Institute of Molecular Medicine and Genetics (2007-07-04)
      BACKGROUND: Thymic epithelial cells (TECs) promote thymocyte maturation and are required for the early stages of thymocyte development and for positive selection. However, investigation of the mechanisms by which TECs perform these functions has been inhibited by the lack of genetic tools. Since the Foxn1 gene is expressed in all presumptive TECs from the early stages of thymus organogenesis and broadly in the adult thymus, it is an ideal locus for driving gene expression in differentiating and mature TECs. RESULTS: We generated two knock-in alleles of Foxn1 by inserting IRES-Cre or IRES-lacZ cassettes into the 3' UTR of the Foxn1 locus. We simultaneously electroporated the two targeting vectors to generate the two independent alleles in the same experiment, demonstrating the feasibility of multiplex gene targeting at this locus. Our analysis shows that the knockin alleles drive expression of Cre or lacZ in all TECs in the fetal thymus. Furthermore, the knockin alleles express Cre or lacZ in a Foxn1-like pattern without disrupting Foxn1 function as determined by phenotype analysis of Foxn1 knockin/Foxn1 null compound heterozygotes. CONCLUSION: These data show that multiplex gene targeting into the 3' UTR of the Foxn1 locus is an efficient method to express any gene of interest in TECs from the earliest stage of thymus organogenesis. The resulting alleles will make possible new molecular and genetic studies of TEC differentiation and function. We also discuss evidence indicating that gene targeting into the 3' UTR is a technique that may be broadly applicable for the generation of genetically neutral driver strains.
    • VPS35 haploinsufficiency increases Alzheimerâ s disease neuropathology

      Wen, Lei; Tang, Fu-Lei; Hong, Yan; Luo, Shi-Wen; Wang, Chun-Lei; He, Wanxia; Shen, Chengyong; Jung, Ji-Ung; Xiong, Fei; Lee, Dae-hoon; et al. (2011-11-28)
      VPS35, a major component of the retromer complex, is important for endosome-to-Golgi retrieval of membrane proteins. Although implicated in Alzheimerâ s disease (AD), how VPS35 regulates AD-associated pathology is unknown. In this paper, we show that hemizygous deletion of Vps35 in the Tg2576 mouse model of AD led to earlier-onset AD-like phenotypes, including cognitive memory deficits, defective long-term potentiation, and impaired postsynaptic glutamatergic neurotransmission in young adult age. These deficits correlated well with an increase of β-amyloid peptide (Aβ) level in the mutant hippocampus. We further demonstrate that VPS35 is predominantly expressed in pyramidal neurons of young adult hippocampus and interacts with BACE1, a protease responsible for Aβ production. Loss of VPS35 function in the mouse hippocampus increased BACE1 activity. Suppression of VPS35 expression in culture decreased BACE1 trans-Golgi localization but enriched it in endosomes. These results demonstrate an essential role for VPS35 in suppression of AD neuropathology and in inhibition of BACE1 activation and Aβ production by promoting BACE1 endosome-to-Golgi retrieval.