Browsing by Author "Mukwevho, Emmanuel"
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- ItemOpen AccessThe isolation and characterization of heat shock protein Hsp12 in Lipomyces starkeyi(2002) Mukwevho, Emmanuel; Brandt, Wolf F; Lindsey, George GThe stress response protein Hsp 12 is induced in S. cerevisiae cells upon exposure to salt stress, heat shock, ethanol, and upon entry to stationary phase (Mtwisha et aI., 1998). In this study, the occurrence of proteins related to Hsp12 was investigated in a number of yeasts (namely, Saccharomyces cerevisiae S288C, Schizosaccharomyces pombe, Debaromyces hansenii, Lipomyces starkeyi Y-2024, Saccharomyces cerevisiae IFO 23X7 (Kaokai), Zygosaccharomyces rouxii and Pichia sorbitophila. This was performed by selective protein extraction followed by SDS-P AGE and western blotting using a S. cerevisiae anti-Hsp 12 antibody. The results showed that almost all the yeasts investigated possessed a protein that had an identical migration to that of Hsp 12 with the exception of S. pombe, which contained a 9 kDa protein. Western blotting using the antiHsp 12 antibody cross-reacted only with the two S. cerevisiae species in addition to the 12 kDa protein from Lipomyces starkeyi of all the species investigated. MALDI-TOF peptide mass analysis after tryptic digestion of the L. starkeyi 12 kDa protein showed that a close sequence similarity existed to that of S. cerevisiae Hsp 12 and none to rest of the 12 kDa proteins isolated from all the other species investigated. In order to determine the sequence of the Hsp 12 protein, the L. starkeyi Hsp 12 gene was amplified using S. cerevisiae Hsp 12 primers. Gene sequencing of both S. cerevisiae and L. starkeyi Hsp 12 genes revealed three nucleotide differences existed between them. L. starkeyi Hsp 12 was found to be present in relatively small amounts during early growth stages but increased during log phase with a slight further increase during stationary phase. Increasing the salt concentration in the growth medium was found to induce Hsp 12. Increased levels of Hsp 12 appeared to confer a degree of protection during desiccation and subsequent rehydration of both L. starkeyi and S. cerevisiae.
- ItemOpen AccessRegulation of Glut-4 Expression in Skeletal Muscle cells: The Roles of Nuclear Respiratory Factor-1 and calcium/calmodulin dependent protein Kinase(2010) Mukwevho, Emmanuel; Ojuka, EdwardGLUT4 protein is the major glucose transporter in skeletal muscle and is vital in the maintenance of euglycemia (17; 108). Underexpression of GLUT4 or impairement of its translocation from intracellular compartments to the cell surface, are linked to diminished glucose transport, hyperglycemia and type II diabetes (59; 61; 153). Type II diabetes can be alleviated by increasing GLUT4 expression (223). Previous reports have shown that overexpression of NRF-1 and activation of CaMKII increases GLUT4 expression but the mechanisms involved have not be characterized (10; 173). Therefore, the objective of this thesis was to investigate the molecular mechanisms by which NRF-1 and CaMK II regulate GLUT4 expression in C2C12 myocytes. We engineered C2C12 cells that overexpressed NRF-1 in response to doxycycline (Dox) using a Tet-On gene expression system and assessed the effects of NRF-1 overexpression on: a) MEF2A, GLUT4 and δALAS proteins by western blot, and b) the binding of NRF-1 to mef2a and δalas genes and MEF2A to the glut4 gene, by chromatin immunoprecipitation assay (ChIP). The importance of MEF2A in NRF-1-induced increase in GLUT4 expression was investigated by silencing MEF2A expression using small interference RNA (siRNA). CaMK II was activated in wild-type C2C12 myocytes using 10 mM caffeine and was inhibited by 25 μ M KN93. Acetylation of histones in the vicinity of NRF-1 and MEF2A binding sites on the mef2a and glut4 genes, respectively, were assessed by ChIP assay. HDAC5 nuclear export was assessed by immunocytochemistry and mRNA levels by qRT-PCR. Overexpression of NRF-1 resulted in ~3-fold increases in mef2a-bound NRF-1 and glut4 -bound MEF2A at 6 h and 8 h post Dox treatment, respectively. MEF2A and GLUT4 proteins were both increased ~1.6-fold at 6 h and 18 h post Dox treatment. Silencing of MEF2A caused a marked downregulation of GLUT4 expression in NRF-1-overexpressing cells.