The purpose of this study was to determine mitochondrial biogenesis-related mRNA expression, binding of transcription factors to the peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) promoter, and subcellular location of PGC-1α protein in human skeletal muscle following exercise in a hot environment compared with a room temperature environment. Recreationally trained males (n = 11) completed two trials in a temperature- and humidity-controlled environmental chamber. Each trial consisted of cycling in either a hot (H) or room temperature (C) environment (33 and 20°C, respectively) for 1 h at 60% of maximum wattage (Wmax) followed by 3 h of supine recovery at room temperature. Muscle biopsies were taken from the vastus lateralis pre-, post-, and 3 h postexercise. PGC-1α mRNA increased post (P = 0.039)- and 3 h postexercise in C (P = 0.002). PGC-1α, estrogen-related receptor-α (ERRα), and nuclear respiratory factor 1 (NRF-1) mRNA was all lower in H than C post (P = 0.038, P < 0.001, and P = 0.030, respectively)- and 3 h postexercise (P = 0.035, P = 0.007, and P < 0.001, respectively). Binding of cAMP response element-binding protein (CREB) (P = 0.005), myocyte enhancer factor 2 (MEF2) (P = 0.047), and FoxO forkhead box class-O1 (FoxO1) (P = 0.010) to the promoter region of the PGC-1α gene was lower in H than C. Nuclear PGC-1α protein increased postexercise in both H and C (P = 0.029) but was not different between trials (P = 0.602). These data indicate that acute exercise in a hot environment blunts expression of mitochondrial biogenesis-related mRNA, due to decreased binding of CREB, MEF2, and FoxO1 to the PGC-1α promoter.

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) forms oligomeric structures important for optimal function in binding and internalization of Man-6-P-bearing extracellular ligands as well as lysosomal biogenesis and growth regulation. However, neither the mechanism of inter-receptor interaction nor the dimerization domain has yet been identified. We hypothesized that areas near the ligand binding domains of the receptor would contribute preferentially to oligomerization. Two panels of minireceptors were constructed that involved truncations of either the N- or C-terminal regions of the M6P/IGF2R encompassing deletions of various ligand binding domains. alpha-FLAG or alpha-Myc-based immunoprecipitation assays showed that all of the minireceptors tested were able to associate with a full-length, Myc-tagged M6P/IGF2R (WT-M). In the alpha-FLAG but not alpha-Myc immunoprecipitation assays, the degree of association of a series of C-terminally truncated minireceptors with WT-M showed a positive trend with length of the minireceptor. In contrast, length did not seem to affect the association of the N-terminally truncated minireceptors with WT-M, except that the 12th extracytoplasmic repeat appeared exceptionally important in dimerization in the alpha-FLAG assays. The presence of mutations in the ligand-binding sites of the minireceptors had no effect on their ability to associate with WT-M. Thus, association within the heterodimers was not dependent on the presence of functional ligand binding domains. Heterodimers formed between WT-M and the minireceptors demonstrated high affinity IGF-II and Man-6-P-ligand binding, suggesting a functional association. We conclude that there is no finite M6P/IGF2R dimerization domain, but rather that interactions between dimer partners occur all along the extracytoplasmic region of the receptor.

Peroxisome proliferator-activated receptor-α coactivator-1α (PGC-1α) mRNA is increased with both exercise and exposure to cold temperature. However, transcriptional control has yet to be examined during exercise in the cold. Additionally, the need for environmental cold exposure after exercise may not be a practical recovery modality. The purpose of this study was to determine mitochondrial-related gene expression and transcriptional control of PGC-1α following exercise in a cold compared with room temperature environment. Eleven recreationally trained males completed two 1-h cycling bouts in a cold (7°C) or room temperature (20°C) environment, followed by 3 h of supine recovery in standard room conditions. Muscle biopsies were taken from the vastus lateralis preexercise, postexercise, and after a 3-h recovery. Gene expression and transcription factor binding to the PGC-1α promoter were analyzed. PGC-1α mRNA increased from preexercise to 3 h of recovery, but there was no difference between trials. Estrogen-related receptor-α (ERRα), myocyte enhancer factor-2 (MEF2A), and nuclear respiratory factor-1 (NRF-1) mRNA were lower in cold than at room temperature. Forkhead box class-O (FOXO1) and cAMP response element-binding protein (CREB) binding to the PGC-1α promoter were increased postexercise and at 3 h of recovery. MEF2A binding increased postexercise, and activating transcription factor 2 (ATF2) binding increased at 3 h of recovery. These data indicate no difference in PGC-1α mRNA or transcriptional control after exercise in cold versus room temperature and 3 h of recovery. However, the observed reductions in the mRNA of select transcription factors downstream of PGC-1α indicate a potential influence of exercise in the cold on the transcriptional response related to mitochondrial biogenesis.

Urokinase-type plasminogen activator receptor (uPAR) binding by the mannose 6-phosphate/insulinlike growth factor II receptor (Man-6-P/IGF2R) is considered important to Man-6-P/IGF2R tumor suppressor function via regulation of cell surface proteolytic activity. Our goal was to map the uPAR binding site of the Man-6-P/IGF2R by analyzing the uPAR binding characteristics of a panel of minireceptors containing different regions of the Man-6-P/IGF2R extracytoplasmic domain. Coimmunoprecipitation assays revealed that soluble recombinant uPAR (suPAR) bound the Man-6-P/IGF2R at two distinct sites, one localized to the amino-terminal end of the Man-6-P/IGF2R extracytoplasmic domain (repeats 1-3) and the other to the more carboxyl-terminal end (repeats 7-9). These sites correspond with the positions of the two Man-6-P binding domains of Man-6-P/ IGF2R. Indeed, the suPAR-Man-6-P/IGF2R interaction was inhibited by Man-6-P, and binding-competent su-PAR species represented a minor percentage (8 -30%) of the suPAR present. In contrast, Man-6-P/IGF2R binding of endogenous, full-length uPAR solubilized from plasma membranes of the prostate cancer cell line, PC-3, was not inhibited by Man-6-P. Further studies showed that very little (<5%) endogenous uPAR was Man-6-P/ IGF2R binding-competent. We conclude that, contrary to previous reports, the interaction between uPAR and Man-6-P/IGF2R is a low percentage binding event and that suPAR and full-length uPAR bind the Man-6-P/ IGF2R by different mechanisms.

A new lecture/laboratory course to offer advanced biochemical training for undergraduate and early graduate students has been developed in the Department of Chemistry at the University of Nebraska at Omaha. This unique course offers students an opportunity to work hands-on with modern instrumentation not normally found in a predominately undergraduate institution, and to complete an entire research project in a realistic timeframe via a time-intensive curriculum as a special summer session. The course content gives a strong background in protein structure/chemistry, purification principles, protocol development, optimization strategies, use and programming of an automated chromatography instrument, and characterization strategies with an emphasis on X-ray crystallography. The laboratory portion offers students the chance to purify a protein (human inosine triphosphate pyrophosphatase) from start to finish, program and use an Ä KTA fast protein liquid chromatography instrument, and to grow and analyze their own protein crystals using their purified protein. This innovative laboratory experience gives the participating students the opportunity to complete a miniresearch project in real time and enhances their overall understanding of important biochemical research techniques and the instrumentation involved, fostering a better understanding of the research process all within a classroom setting. Evaluations and feedback concerning this course indicated a positive learning environment, a retention of knowledge and skills, a belief that the skill set learned continues to be useful in current endeavors, and a sense of accomplishment in the completion of an actual research project within the confines of a class setting.Keywords: Active learning, curriculum design development and implementation, laboratory exercise, new course development, protein structure function and folding.One major downfall in any undergraduate laboratory course is the disconnection between a series of 3-hour miniaturized experiments and the realistic nature of true bench work. In a typical undergraduate course, the experiments are written to introduce students to working at a bench, calculations, and use of equipment, but are all designed with maximal successful outcome ratios for the students. In an actual research laboratory setting, students must deal with experiments that take extended timeframes to complete, multiple trials for optimization, and work in collaboration to optimize equipment and reagent use. Students quickly learn that research results are not as easily obtained as their former laboratory manuals made it seem. Many of our science majors at the undergraduate level are hoping to join graduate programs or work forces which require applicants with research experience, who are detailed notebook writers, and who have developed solid critical thinking skills. Graduate students may find that their research project involves the need for protein isolation and/or purification, a skill set that may be outside of their laboratory's area of...