Role of Amphipathic Helix of a Herpesviral Protein in Membrane Deformation and T Cell Receptor Downregulation

Lipid rafts are membrane microdomains that function as platforms for signal transduction and membrane trafficking. Tyrosine kinase interacting protein (Tip) of T lymphotropic Herpesvirus saimiri (HVS) is targeted to lipid rafts in T cells and downregulates TCR and CD4 surface expression. Here, we report that the membrane-proximal amphipathic helix preceding Tip''s transmembrane (TM) domain mediates lipid raft localization and membrane deformation. In turn, this motif directs Tip''s lysosomal trafficking and selective TCR downregulation. The amphipathic helix binds to the negatively charged lipids and induces liposome tubulation, the TM domain mediates oligomerization, and cooperation of the membrane-proximal helix with the TM domain is sufficient for localization to lipid rafts and lysosomal compartments, especially the mutivesicular bodies. These findings suggest that the membrane-proximal amphipathic helix and TM domain provide HVS Tip with the unique ability to deform the cellular membranes in lipid rafts and to downregulate TCRs potentially through MVB formation.     

Effect of cola intake on insulin resistance in moderate fat-fed weaning male rats

In recent years, the prevalence of type 2 diabetes mellitus has dramatically increased in Korea as the diet has rapidly become westernized. We determined the effect of a long-term cola intake for insulin resistance in weaning male Sprague Dawley rats consuming a moderate fat diet. Thirty male pubs born from 6 female rats were randomized into cola or water drinking groups. The rats of the cola group were freely provided with 33 energy percent fat diets and cola for 28 weeks, while the rats of the control group had the same diet with water instead of cola. The daily caloric intake did not differ between groups, while the rats in the cola group consumed more carbohydrates. However, the mean body weight of the cola group was lower than that of the control group from the second week of the study. Whole body glucose disposal rates measured by euglycemic hyperinsulinemic clamp were higher in the cola group. Compared to the control group, glycogen contents and fraction velocity of glycogen synthase of the quadriceps muscle in the cola group were higher by 39.4% and 40.3%, respectively. Uncoupling protein (UCP)-2 and GLUT 4 contents of soleus and quadriceps muscles were higher in the cola group than the control group. In conclusion, insulin action improved with increased peripheral glucose utilization in weaning male rats drinking cola, which was partly due to lower body weight. This latter was possibly as a result of increased thermogenesis in muscles.     

Human iPSC‐derived neural precursor cells differentiate into multiple cell types to delay disease progression following transplantation into YAC128 Huntington's disease mouse model

ObjectivesTo investigate whether human HLA‐homozygous induced pluripotent stem cell (iPSC)‐derived neural precursor cells (iPSC‐NPCs) can provide functional benefits in Huntington’s disease (HD), we transplanted them into the YAC128 transgenic HD mouse model.Materials and MethodsCHAi001‐A, an HLA‐homozygous iPSC line (A*33:03‐B*44:03‐DRB1*13:02), was differentiated into neural precursor cells, and then, they were transplanted into 6 months‐old YAC128 mice. Various behavioural and histological analyses were performed for five months after transplantation.ResultsMotor and cognitive functions were significantly improved in transplanted animals. Cells transplanted in the striatum showed multipotential differentiation. Five months after transplantation, the donor cells had differentiated into neurons, oligodendrocytes and astrocytes. Transplantation restored DARPP‐32 expression, synaptophysin density, myelin basic protein expression in the corpus callosum and astrocyte function.ConclusionAltogether, these results strongly suggest that iPSC‐NPCs transplantation induces neuroprotection and functional recovery in a mouse model of HD and should be taken forward for clinical trials in HD patients.     

AP2α is essential for MUC8 gene expression in human airway epithelial cells

Mucins are high molecular weight proteins that make up the major components of mucus. Hypersecretion of mucus is a feature of several chronic inflammatory airway diseases. MUC8 is an important component of airway mucus, and its gene expression is upregulated in nasal polyp epithelium. Little is known about the molecular mechanisms of MUC8 gene expression. We first observed overexpression of activator protein‐2alpha (AP2α) in human nasal polyp epithelium. We hypothesized that AP2α overexpression in nasal polyp epithelium correlates closely with MUC8 gene expression. We demonstrated that phorbol 12‐myristate 13‐acetate (PMA) treatment of the airway epithelial cell line NCI‐H292 increases MUC8 gene and AP2α expression. In this study, we sought to determine which signal pathway is involved in PMA‐induced MUC8 gene expression. The results show that the protein kinase C and mitogen‐activating protein/ERK kinase (MAPK) pathways modulate MUC8 gene expression. PD98059 or ERK1/2 siRNA and RO‐31‐8220 or PKC siRNA significantly suppress AP2α as well as MUC8 gene expression in PMA‐treated cells. To verify the role of AP2α, we specifically knocked down AP2α expression with siRNA. A significant AP2α knock‐down inhibited PMA‐induced MUC8 gene expression. While dominant negative AP2α decreased PMA‐induced MUC8 gene expression, overexpressing wildtype AP2α increased MUC8 gene expression. Furthermore, using lentiviral vectors for RNA interference in human nasal polyp epithelial cells, we confirmed an essential role for AP2α in MUC8 gene expression. From these results, we concluded that PMA induces MUC8 gene expression through a mechanism involving PKC, ERK1/2, and AP2α activation in human airway epithelial cells. J. Cell. Biochem. 110: 1386–1398, 2010. © 2010 Wiley‐Liss, Inc.     

Continuous production of clinical dextran using two-stage reactor

Summary Clinical dextran with desired molecular weight was produced continuously in the two-stage reactor. Cells ofLeuconostoc meseteroides B512F cultivated in the first reactor were transferred to the second reactor where sucrose and primer were added for clinical dextran production. By using this two-stage reactor, the fraction of desired clinical dextran increased significantly when observed with gel permeation chromatography.     

SYNCRIP, a member of the heterogeneous nuclear ribonucleoprotein family, is involved in mouse hepatitis virus RNA synthesis

Several cellular proteins, including several heterogeneous nuclear ribonucleoproteins (hnRNPs), have been shown to function as regulatory factors for mouse hepatitis virus (MHV) RNA synthesis as a result of their binding to the 5' and 3' untranslated regions (UTRs) of the viral RNA. Here, we identified another cellular protein, p70, which has been shown by UV cross-linking to bind both the positive- and negative-strand UTRs of MHV RNA specifically. We purified p70 with a a one-step RNA affinity purification procedure with the biotin-labeled 5'-UTR. Matrix-assisted laser desorption ionization (MALDI)-mass spectrometry identified it as synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP). SYNCRIP is a member of the hnRNP family and localizes largely in the cytoplasm. The p70 was cross-linked to the MHV positive- or negative-strand UTR in vitro and in vivo. The bacterially expressed SYNCRIP was also able to bind to the 5'-UTR of both strands. The SYNCRIP-binding site was mapped to the leader sequence of the 5'-UTR, requiring the UCUAA repeat sequence. To investigate the functional significance of SYNCRIP in MHV replication, we expressed a full-length or a C-terminally truncated form of SYNCRIP in mammalian cells expressing the MHV receptor. The overexpression of either form of SYNCRIP inhibited syncytium formation induced by MHV infection. Furthermore, downregulation of the endogenous SYNCRIP with a specific short interfering RNA delayed MHV RNA synthesis; in contrast, overexpression or downregulation of SYNCRIP did not affect MHV translation. These results suggest that SYNCRIP may be directly involved in MHV RNA replication as a positive regulator. This study identified an additional cellular hnRNP as an MHV RNA-binding protein potentially involved in viral RNA synthesis.     

Inhibitory effects of glycoprotein-120 (G-120) from Ulmus davidiana Nakai on cell growth and activation of matrix metalloproteinases

Excessive breakdown of extracellular matrix by metalloproteinases (MMPs) occurs in many pathological conditions. Consequently, methods for inhibiting MMP activity have therapeutic potential. In this study, we investigated the effect of G-120, a 120 kDa glycoprotein purified from the Oriental herbal plant, Ulmus davidiana Nakai (UDN), on the activity and production of several MMPs by evaluating its growth inhibitory effect on NIH 3T3 cells. Tritium uptake assays showed that proliferation of NIH 3T3 cells was strongly suppressed, and G-120-mediated inhibition of DNA synthesis proved to involve a cytostatic, rather than a cytotoxic, effect, as shown by cytotoxicity and apoptosis assays. More importantly, G-120 strongly reduced the gelatinolytic and collagenase activities of MMP proteins, as well as expression of MMP-2 and MMP-9. Electrophoretic mobility shift assays revealed that it suppressed the DNA binding activity of NF-kappaB. Collectively, our observations show that G-120 strongly inhibits the activation of MMPs and NF-kappaB.     

Expression patterns in soybean resistant to Phakopsora pachyrhizi reveal the importance of peroxidases and lipoxygenases

Soybean rust caused by Phakopsora pachyrhizi Sydow is a devastating foliar disease that has spread to most soybean growing regions throughout the world, including the USA. Four independent rust resistance genes, Rpp1–Rpp4, have been identified in soybean that recognize specific isolates of P. pachyrhizi. A suppressive subtraction hybridization (SSH) complementary DNA (cDNA) library was constructed from the soybean accession PI200492, which contains Rpp1, after inoculation with two different isolates of P. pachyrhizi that result in susceptible or immune reactions. Both forward and reverse SSH were performed using cDNA from messenger RNA pooled from 1, 6, 12, 24, and 48 h post-inoculation. A total of 1,728 SSH clones were sequenced and compared to sequences in GenBank for similarity. Microarray analyses were conducted on a custom 7883 soybean-cDNA clone array encompassing all of the soybean-rust SSH clones and expressed sequence tags from four other soybean cDNA libraries. Results of the microarray revealed 558 cDNA clones differentially expressed in the immune reaction. The majority of the upregulated cDNA clones fell into the functional category of defense. In particular, cDNA clones with similarity to peroxidases and lipoxygenases were prevalent. Downregulated cDNA clones included those with similarity to cell-wall-associated protein, such as extensins, proline-rich proteins, and xyloglucan endotransglycosylases. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.