• 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.
    • Glucocorticoid-Induced Leucine Zipper (GILZ) Antagonizes TNF-a Inhibition of Mesenchymal Stem Cell Osteogenic Differentiation

      He, Linlin; Yang, Nianlan; Isales, Carlos M.; Shi, Xing-Ming; Institute of Molecular Medicine and Genetics; Department of Orthopaedic Surgery; Department of Pathology (2012-03-2)
      Tumor necrosis factor-alpha (TNF-a) is a potent proinflammatory cytokine that inhibits osteoblast differentiation while stimulating osteoclast differentiation and bone resorption. TNF-a activates MAP kinase pathway leading to inhibition of osterix (Osx) expression. TNF-a also induces the expression of E3 ubiquitin ligase protein Smurf1 and Smurf2 and promotes degradation of Runx2, another key transcription factor regulating osteoblast differentiation and bone formation. We showed previously that overexpression of glucocorticoid (GC)-induced leucine zipper (GILZ) enhances osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). We and others also showed that GILZ is a GC effecter and mediates GC anti-inflammatory activity. In this study, we asked the question whether GILZ retains its osteogenic activity while functioning as an anti-inflammatory mediator. To address this question, we infected mouse bone marrow MSCs with retroviruses expressing GILZ and induced them for osteogenic differentiation in the presence or absence of TNF-a. Our results show that overexpression of GILZ antagonized the inhibitory effects of TNF-a on MSC osteogenic differentiation and the mRNA and protein expression of Osx and Runx2, two pivotal osteogenic regulators. Further studies show that these antagonistic actions occur via mechanisms involving GILZ inhibition of TNF-a-induced ERK MAP kinase activation and protein degradation. These results suggest that GILZ may have therapeutic potential as a novel anti-inflammation therapy.
    • IGF-1 Induction by Acylated Steryl β-Glucosides Found in a Pre-Germinated Brown Rice Diet Reduces Oxidative Stress in Streptozotocin-Induced Diabetes

      Usuki, Seigo; Tsai, Ying-Ying; Morikawa, Keiko; Nonaka, Shota; Okuhara, Yasuhide; Kise, Mitsuo; Yu, Robert K.; Institute of Molecular Medicine and Genetics (2011-12-14)
      Background: The pathology of diabetic neuropathy involves oxidative stress on pancreatic b-cells, and is related to decreased levels of Insulin-like growth factor 1 (IGF-1). Acylated steryl b-glucoside (PR-ASG) found in pre-germiated brown rice is a bioactive substance exhibiting properties that enhance activity of homocysteine-thiolactonase (HTase), reducing oxidative stress in diabetic neuropathy. The biological importance of PR-ASG in pancreatic b-cells remains unknown.
    • An Improved Test for Detecting Multiplicative Homeostatic Synaptic Scaling

      Kim, Jimok; Tsien, Richard W.; Alger, Bradley E.; Institute of Molecular Medicine and Genetics; Graduate Program in Neuroscience; Department of Neurology (2012-05-17)
      Homeostatic scaling of synaptic strengths is essential for maintenance of network "gain", but also poses a risk of losing the distinctions among relative synaptic weights, which are possibly cellular correlates of memory storage. Multiplicative scaling of all synapses has been proposed as a mechanism that would preserve the relative weights among them, because they would all be proportionately adjusted. It is crucial for this hypothesis that all synapses be affected identically, but whether or not this actually occurs is difficult to determine directly. Mathematical tests for multiplicative synaptic scaling are presently carried out on distributions of miniature synaptic current amplitudes, but the accuracy of the test procedure has not been fully validated. We now show that the existence of an amplitude threshold for empirical detection of miniature synaptic currents limits the use of the most common method for detecting multiplicative changes. Our new method circumvents the problem by discarding the potentially distorting subthreshold values after computational scaling. This new method should be useful in assessing the underlying neurophysiological nature of a homeostatic synaptic scaling transformation, and therefore in evaluating its functional significance.
    • A New Antifibrotic Target of Ac-SDKP: Inhibition of Myofibroblast Differentiation in Rat Lung with Silicosis

      Xu, Hong; Yang, Fang; Sun, Ying; Yuan, Yuan; Cheng, Hua; Wei, Zhongqiu; Li, Shuyu; Cheng, Tan; Brann, Darrell W; Wang, Ruimin; et al. (2012-07-3)
      Background: Myofibroblast differentiation, characterized by a-smooth muscle actin (a-SMA) expression, is a key process in organ fibrosis, and is induced by TGF-b. Here we examined whether an anti-fibrotic agent, N-acetyl-seryl-aspartyllysylproline (Ac-SDKP), can regulate induction of TGF-b signaling and myofibroblast differentiation as a potential key component of its anti-fibrotic mechanism in vivo and in vitro.