• Effect of pre-germinated brown rice intake on diabetic neuropathy in streptozotocin-induced diabetic rats.

      Usuki, Seigo; Ito, Yukihiko; Morikawa, Keiko; Kise, Mitsuo; Ariga, Toshio; Rivner, Michael; Yu, Robert K.; Institute of Molecular Medicine and Genetics; Department of Neurology (2008-02-19)
      ABSTRACT: BACKGROUND: To study the effects of a pre-germinated brown rice diet (PR) on diabetic neuropathy in streptozotocin (STZ)-induced diabetic rats. METHODS: The effects of a PR diet on diabetic neuropathy in STZ-induced diabetic rats were evaluated and compared with those fed brown rice (BR) or white rice (WR) diets with respect to the following parameters: blood-glucose level, motor-nerve conduction velocity (NCV), sciatic-nerve Na+/K+-ATPase activity, and serum homocysteine-thiolactonase (HTase) activity. RESULTS: Compared with diabetic rats fed BR or WR diets, those fed a PR diet demonstrated significantly lower blood-glucose levels (p < 0.001), improved NCV (1.2- and 1.3-fold higher, respectively), and increased Na+/K+-ATPase activity (1.6- and 1.7-fold higher, respectively). The PR diet was also able to normalize decreased serum homocysteine levels normally seen in diabetic rats. The increased Na+/K+-ATPase activity observed in rats fed PR diets was associated with elevations in HTase activity (r = 0.913, p < 0.001). The in vitro effect of the total lipid extract from PR bran (TLp) on the Na+/K+-ATPase and HTase activity was also examined. Incubation of homocysteine thiolactone (HT) with low-density lipoprotein (LDL) in vitro resulted in generation of HT-modified LDL, which possessed high potency to inhibit Na+/K+-ATPase activity in the sciatic nerve membrane. The inhibitory effect of HT-modified LDL on Na+/K+-ATPase activity disappeared when TLp was added to the incubation mixture. Furthermore, TLp directly activated the HTase associated with high-density lipoprotein (HDL). CONCLUSION: PR treatment shows efficacy for protecting diabetic deterioration and for improving physiological parameters of diabetic neuropathy in rats, as compared with a BR or WR diet. This effect may be induced by a mechanism whereby PR intake mitigates diabetic neuropathy by one or more factors in the total lipid fraction. The active lipid fraction is able to protect the Na+/K+-ATPase of the sciatic-nerve membrane from the toxicity of HT-modified LDL and to directly activate the HTase of HDL.
    • Lectin-based food poisoning: a new mechanism of protein toxicity.

      Miyake, Katsuya; Tanaka, Toru; McNeil, Paul L.; Institute of Molecular Medicine and Genetics; Department of Cellular Biology and Anatomy (2007-08-01)
      BACKGROUND: Ingestion of the lectins present in certain improperly cooked vegetables can result in acute GI tract distress, but the mechanism of toxicity is unknown. In vivo, gut epithelial cells are constantly exposed to mechanical and other stresses and consequently individual cells frequently experience plasma membrane disruptions. Repair of these cell surface disruptions allows the wounded cell to survive: failure results in necrotic cell death. Plasma membrane repair is mediated, in part, by an exocytotic event that adds a patch of internal membrane to the defect site. Lectins are known to inhibit exocytosis. We therefore tested the novel hypothesis that lectin toxicity is due to an inhibitory effect on plasma membrane repair. METHODS AND FINDINGS: Repair of plasma membrane disruptions and exocytosis of mucus was assessed after treatment of cultured cell models and excised segments of the GI tract with lectins. Plasma membrane disruptions were produced by focal irradiation of individual cells, using a microscope-based laser, or by mechanical abrasion of multiple cells, using a syringe needle. Repair was then assessed by monitoring the cytosolic penetration of dyes incapable of crossing the intact plasma membrane. We found that cell surface-bound lectins potently inhibited plasma membrane repair, and the exocytosis of mucus that normally accompanies the repair response. CONCLUSIONS: Lectins potently inhibit plasma membrane repair, and hence are toxic to wounded cells. This represents a novel form of protein-based toxicity, one that, we propose, is the basis of plant lectin food poisoning.
    • 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.
    • Influence of DNA Ends on Structure and Function of the DNA-dependent Protein Kinase

      Jovanovic, Marko; Institute of Molecular Medicine and Genetics (2006-12)
      Non-homologous end joining is a major DNA double-strand break repair pathway in mammalian cells. The DNA-dependent protein kinase (DNA-PK), consisting of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Ku heterodimer, is hypothesized to be a key regulator of the pathway. Available data suggest DNA-PKcs may exert this regulatory function by controlling access to the DNA termini and by phosphorylation of itself and other proteins. I further characterized DNA-PK-DNA interaction by studying binding of DNA-PKcs and Ku to oligonucleotides with chemically defined end structures under conditions that preclude synapsis between opposing DNA ends. Binding of DNA-PKcs to DNA varied with the end structure in a manner that suggests that partial melting of DNA ends is necessary for the formation of a stable, enzymatically active complex. Unexpectedly, these studies also revealed that ATP, as well as its nonhydrolyzable analog AMP-PNP, have an allosteric effect on the interaction of DNA-PKcs with DNA.
    • 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.
    • Regulation of osteoclast function and bone mass by RAGE

      Zhou, Zheng; Immel, David; Xi, Cai-Xia; Bierhaus, Angelika; Feng, Xu; Mei, Lin; Nawroth, Peter; Stern, David M.; Xiong, Wen-Cheng; Institute of Molecular Medicine and Genetics; et al. (2006-04-17)
      The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily that has multiple ligands and is implicated in the pathogenesis of various diseases, including diabetic complications, neurodegenerative disorders, and inflammatory responses. However, the role of RAGE in normal physiology is largely undefined. Here, we present evidence for a role of RAGE in osteoclast maturation and function, which has consequences for bone remodeling. Mice lacking RAGE had increased bone mass and bone mineral density and decreased bone resorptive activity in vivo. In vitroâ differentiated RAGE-deficient osteoclasts exhibited disrupted actin ring and sealing zone structures, impaired maturation, and reduced bone resorptive activity. Impaired signaling downstream of αvβ3 integrin was observed in RAGEâ /â bone marrow macrophages and precursors of OCs. These results demonstrate a role for RAGE in osteoclast actin cytoskeletal reorganization, adhesion, and function, and suggest that the osteosclerotic-like phenotype observed in RAGE knockout mice is due to a defect in osteoclast function.
    • Genomic and Functional Analysis of Vesicular Inhibitory Amino Acid Transporter During Mouse Embryogenesis

      Oh, Won-Jong; Institute of Molecular Medicine and Genetics (2006-01)
      The specification of particular neuronal phenotypes during embryonic development requires the appropriate activation and regulation of genes encoding the proteins required for neurotransmitter synthesis, vesicular packaging and re-uptake from the synaptic cleft. Each neurotransmitter is packaged into synaptic vesicles by its own distinct vesicular transporter. In addition, neurotransmitter packaging is well controlled by other co-factors (reviewed in Ahnert-Hilger et al., 2003). Components of GABAergic neurons GABAergic neurons are the principal inhibitory neurons in the mammalian central nervous system (CNS), where GABA is synthesized from glutamate by two glutamate decarboxylases (GAD), namely GAD65 (Gad2) and GAD67 (Gad1) (Erlander et al., 1991). GABA is then loaded into synaptic vesicles by the vesicular inhibitory amino acid transporter (VIAAT, also known as VGAT). Four GABA transporters (GAT 1-4) are responsible for the re-uptake of GABA from the synaptic cleft through the plasma membrane. Inhibitory GABAergic transmission is mediated by binding of GABA to its ionotropic receptors, GABAA and GABAC, which are ligand-gated chloride channels, and its metabotropic receptor, GABAB (Fig. 1).
    • 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.
    • 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.
    • Signaling in the Late Phase of T Cell Activation

      Chang, Jing-Wen; Institute of Molecular Medicine and Genetics (2004-12)
      Engagement of the T cell antigen receptor (TCR) induces multiple signaling pathways, including the activation of extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase (MAPK). We previously reported the importance of sustained ERK activation for interleukin-2 (IL-2) production. Inhibition o f ERK activation from 2 to 6 hours after TCR stimulation significantly impaired IL-2 production and activation of the nuclear factor-kappaB (NF-kB) family transcription factor, c-Rel, whereas inhibition during the first 4 hours had no effect. Loss o f the adaptor protein, She, results in impaired ERK activation during the late phase of TCR stimulation, and leads to severely reduced IL-2 production and c-Rel activation. These data suggest a novel activation process following TCR stimulation that involves She and late ERK activation-dependent regulation of c-Rel activation and IL-2 production. To further understand the mechanisms underlying this pathway, we employed a two-dimensional differential in-gel electrophoresis/ mass spectrometry (2D-DIGE/MS)-based proteomics approach. This approach to identify members of a Shc-containing signaling complex revealed alpha tubulin and beta actin as She associated proteins. Furthermore, we identified proteins whose expression and modification are triggered by TCR stimulation and are under control of the ERK signaling pathway, by comparing TCR-stimulated samples with or without MEK (MAPK kinase) inhibitor treatment. Here we report heterogeneous nuclear ribonucleoprotein K (hnRNP-K) as a novel downstream target of ERK in TCR signaling. Functional studies using small RNA interference showed that hnRNPK regulated IL-2 production at the transcriptional level. We also showed that knockdown o f hnRNP-K expression specifically impaired NF-kB activity, but caused a relatively minor effect on activating protein-1 (AP-1) activity and expression of CD69 or CD25. Biochemical analysis showed that knockdown of hnRNP-K caused enhanced proteolysis of the protooncogene Vav. MEK inhibitor treatment during the late phase of stimulation also enhanced proteolysis of Vav. Moreover, knockdown of hnRNP-K impaired Vav-mediated transcriptional activation of IL-2 gene. Taken together; these results indicate that ERK signaling modulates IL-2 production by regulating Vav activity through the function of hnRNP-K. We also examined changes in phosphoprotein profiles upon TCR stimulation and MEK inhibitor treatment. Results obtained from these three different approaches provide a further understanding of the mechanisms that regulate late phase T cell activation as well as the components required for full activation o f T cells.
    • Selective apoptosis of pluripotent mouse and human stem cells by novel ceramide analogues prevents teratoma formation and enriches for neural precursors in ES cellâ derived neural transplants

      Bieberich, Erhard; Silva, Jeane; Wang, Guanghu; Krishnamurthy, Kannan; Condie, Brian G.; Institute of Molecular Medicine and Genetics (2004-11-22)
      The formation of stem cellâ derived tumors (teratomas) is observed when engrafting undifferentiated embryonic stem (ES) cells, embryoid bodyâ derived cells (EBCs), or mammalian embryos and is a significant obstacle to stem cell therapy. We show that in tumors formed after engraftment of EBCs into mouse brain, expression of the pluripotency marker Oct-4 colocalized with that of prostate apoptosis response-4 (PAR-4), a protein mediating ceramide-induced apoptosis during neural differentiation of ES cells. We tested the ability of the novel ceramide analogue N-oleoyl serinol (S18) to eliminate mouse and human Oct-4(+)/PAR-4(+) cells and to increase the proportion of nestin(+) neuroprogenitors in EBC-derived cell cultures and grafts. S18-treated EBCs persisted in the hippocampal area and showed neuronal lineage differentiation as indicated by the expression of β-tubulin III. However, untreated cells formed numerous teratomas that contained derivatives of endoderm, mesoderm, and ectoderm. Our results show for the first time that ceramide-induced apoptosis eliminates residual, pluripotent EBCs, prevents teratoma formation, and enriches the EBCs for cells that undergo neural differentiation after transplantation.
    • 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.
    • Regulation of cell death in mitotic neural progenitor cells by asymmetric distribution of prostate apoptosis response 4 (PAR-4) and simultaneous elevation of endogenous ceramide

      Bieberich, Erhard; MacKinnon, Sarah; Silva, Jeane; Noggle, Scott A; Condie, Brian G.; Institute of Molecular Medicine and Genetics; Department of Medicine (2003-08-4)
      Cell death and survival of neural progenitor (NP) cells are determined by signals that are largely unknown. We have analyzed pro-apoptotic signaling in individual NP cells that have been derived from mouse embryonic stem cells. NP formation was concomitant with elevated apoptosis and increased expression of ceramide and prostate apoptosis response 4 (PAR-4). Morpholino oligonucleotide-mediated antisense knockdown of PAR-4 or inhibition of ceramide biosynthesis reduced stem cell apoptosis, whereas PAR-4 overexpression and treatment with ceramide analogs elevated apoptosis. Apoptotic cells also stained for proliferating cell nuclear antigen (a nuclear mitosis marker protein), but not for nestin (a marker for NP cells). In mitotic cells, asymmetric distribution of PAR-4 and nestin resulted in one nestin(â )/PAR-4(+) daughter cell, in which ceramide elevation induced apoptosis. The other cell was nestin(+), but PAR-4(â ), and was not apoptotic. Asymmetric distribution of PAR-4 and simultaneous elevation of endogenous ceramide provides a possible mechanism underlying asymmetric differentiation and apoptosis of neuronal stem cells in the developing brain.
    • Impaired membrane resealing and autoimmune myositis in synaptotagmin VIIâ deficient mice

      Chakrabarti, Sabyasachi; Kobayashi, Koichi S.; Flavell, Richard A.; Marks, Carolyn B.; Miyake, Katsuya; Liston, David R.; Fowler, Kimberly T.; Gorelick, Fred S.; Andrews, Norma W.; Institute of Molecular Medicine and Genetics (2003-08-18)
      Members of the synaptotagmin family have been proposed to function as Ca2+ sensors in membrane fusion. Syt VII is a ubiquitously expressed synaptotagmin previously implicated in plasma membrane repair and Trypanosoma cruzi invasion, events which are mediated by the Ca2+-regulated exocytosis of lysosomes. Here, we show that embryonic fibroblasts from Syt VII-deficient mice are less susceptible to trypanosome invasion, and defective in lysosomal exocytosis and resealing after wounding. Examination of mutant mouse tissues revealed extensive fibrosis in the skin and skeletal muscle. Inflammatory myopathy, with muscle fiber invasion by leukocytes and endomysial collagen deposition, was associated with elevated creatine kinase release and progressive muscle weakness. Interestingly, similar to what is observed in human polymyositis/dermatomyositis, the mice developed a strong antinuclear antibody response, characteristic of autoimmune disorders. Thus, defective plasma membrane repair in tissues under mechanical stress may favor the development of inflammatory autoimmune disease.
    • Estrogen-astrocyte interactions: implications for neuroprotection.

      Dhandapani, Krishnan M.; Brann, Darrell W; Institute of Molecular Medicine and Genetics; Department of Neurology (2003-03-31)
      BACKGROUND: Recent work has suggested that the ovarian steroid 17beta-estradiol, at physiological concentrations, may exert protective effects in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and acute ischemic stroke. While physiological concentrations of estrogen have consistently been shown to be protective in vivo, direct protection upon purified neurons is controversial, with many investigators unable to show a direct protection in highly purified primary neuronal cultures. These findings suggest that while direct protection may occur in some instances, an alternative or parallel pathway for protection may exist which could involve another cell type in the brain. PRESENTATION OF THE HYPOTHESIS: A hypothetical indirect protective mechanism is proposed whereby physiological levels of estrogen stimulate the release of astrocyte-derived neuroprotective factors, which aid in the protection of neurons from cell death. This hypothesis is attractive as it provides a potential mechanism for protection of estrogen receptor (ER)-negative neurons through an astrocyte intermediate. It is envisioned that the indirect pathway could act in concert with the direct pathway to achieve a more widespread global protection of both ER+ and ER- neurons. TESTING THE HYPOTHESIS: We hypothesize that targeted deletion of estrogen receptors in astrocytes will significantly attenuate the neuroprotective effects of estrogen. IMPLICATIONS OF THE HYPOTHESIS: If true, the hypothesis would significantly advance our understanding of endocrine-glia-neuron interactions. It may also help explain, at least in part, the reported beneficial effects of estrogen in neurodegenerative disorders. Finally, it also sets the stage for potential extension of the hypothetical mechanism to other important estrogen actions in the brain such as neurotropism, neurosecretion, and synaptic plasticity.
    • 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.
    • Mechanisms of Neuroprotection by Estrogen and Selective Estrogen Receptor Modulators

      Dhandapani, Krishnan M.; Institute of Molecular Medicine and Genetics (2003-03)
      Specific Aim #1: To determine whether 17b-E2 and SERMs directly influence neuronal survival. Specific Aim #2: To determine whether astrocyte-derived TGF-b protects neurons from cell death. Specific Aim #3: To determine whether 17b-E2 or SERMs regulate the release of TGF-b from astrocytes. Specific Aim #4:To elucidate the mechanism of TGF-b mediated neuroprotection in GT1-7 Neurons. Specific Aim #5: To identify genes potentially mediating the neuroprotective effects of 17b-E2 and/or tamoxifen through the use of high density gene chip arrays.
    • 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.