With muscle wasting, caspase-3 activation and the ubiquitin-proteasome system act synergistically to increase the degradation of muscle proteins. Whether proteasome activity is also elevated in response to catabolic conditions is unknown. We find that caspase-3 increases proteasome activity in myotubes but not in myoblasts. This difference is related to the cleavage of specific 19 S proteasome subunits. In mouse muscle or myotubes, caspase-3 cleaves Rpt2 and Rpt6 increasing proteasome activity. In myoblasts, caspase-3 cleaves Rpt5 to decrease proteasome activity. To confirm the caspase-3 dependence, caspase-3 cleavage sites in Rpt2, Rpt6, or Rpt5 were mutated. This prevented the cleavage of these subunits by caspase-3 as well as the changes in proteasome activity. During differentiation of myoblasts to myotubes, there is an obligatory, transient increase in caspase-3 activity, accompanied by a corresponding increase in proteasome activity and cleavage of Rpt2 and Rpt6. Therefore, differentiation changes the proteasome type from sensitivity of Rpt5 to caspase-3 in myoblasts to sensitivity of Rpt2 and Rpt6 in myotubes. This novel mechanism identifies a feed-forward amplification that augments muscle proteolysis in catabolic conditions. Indeed, we found that in mice with a muscle wasting condition, chronic kidney disease, there was cleavage of subunits Rpt2 and Rpt6 and stimulation of proteasome activity.The ubiquitin-proteasome system (UPS) 4 is responsible for the degradation of most proteins in the cytoplasm and nuclei of cells (1, 2). It is also involved in regulating many cell functions including control of the cell cycle, antigen presentation, ion transport, and muscle mass (3-6). One event that regulates proteolysis in the UPS is the rate of ubiquitin conjugation to protein substrates. For example, ␤TCF, the activated E3 enzyme, triggers degradation of IB and ␤-catenin, whereas activation of Atrogin-1/MAFbx or MuRF1 in muscle is closely linked to increased protein degradation (7,8).Rates of protein degradation in the UPS could also be regulated through changes in the proteolytic activity of the proteasome. For example, treatment of lymphoid cells with ␥-interferon increases the expression of proteasome subunits, LMP2 and 7, and these subunits are incorporated into proteasomes stimulating the breakdown of proteins into peptides that are more suitable for class 1 antigen presentation (9 -11). Other evidence suggests that the activity of the UPS can be determined by variations in proteasome activity. In several conditions that are associated with the loss of muscle mass, proteasome subunits are expressed at a higher level but it is not proven that changes in subunit expression accelerate the rate of muscle proteolysis (12-18). Alternatively, proteasome activity can be suppressed in association with changes in subunits. Sun et al. (19) reported that caspase-3 activation in Jurkat T cells or cancer cells causes cleavage of specific subunits of the 19 S regulatory complex of the proteasome: Rpt5, Rpn10, and Rpn2...

Using a model amphotropic recombinant retrovirus encoding the Escherichia coli lacZ gene and quantitative assays to measure virus infection, we have determined the effects of time and target cell number on infectivity. Infection of various numbers of NIH 3T3 fibroblasts showed that the extent of lacZ virus infection was dependent on virus concentration and independent of target cell number. These results demonstrate that multiplicity of infection is not an accurate predictor of the efficiency of retroviral infection. Varying the time of viral infection revealed that maximal infection occurred after greater than 24 h of exposure of the cells to the lacZ virus. Half-maximal infection occurred after 5 h of exposure. After 2 h of adsorption at 37؇C, the majority of infectious virus was not adsorbed to cells but was unbound and able to infect other cells. These results are discussed in terms of both their relevance to the fundamental biology of retrovirus infection and the use of recombinant retroviruses for retrovirus-mediated gene transfer with purposes of gene therapy.

No abstract

The Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech is currently making an intentional effort to vertically integrate entrepreneurial minded learning within the undergraduate curriculum. With funding from the Kern Family Foundation, the goals of this work are not only to better equip students to meet the demands of the modern marketplace but also to empower students to tell the story of their growth into entrepreneurially minded engineers. In order to tell this story, students engage in a portfolio process grounded in evidence and reflection. The structure of this story-centric curricular framework consists of a first-year launcher course where foundational topics such as design thinking, reflection, folio thinking, and entrepreneurial mindset are introduced. At the other end of the framework is a unique course called The Art of Telling Your Story . In this upper level course, students learn to tell their unique stories of their experiences and entrepreneurial mindset growth culminating in an ePortfolio. In between these two curricular bookends, students are involved in their core biomedical engineering classes. Five of these core courses are designated as gateway courses . In gateway courses, students complete signature assignments specifically created to foster entrepreneurial mindset and critical reflection. These signature assignments are also designed to produce meaningful artifacts and experiences that may later be used as part of students' stories and portfolios. In this paper, we describe the details of the structure and implementation of this vertically integrated curricular framework and present evidence of entrepreneurial mindset growth within freshmen and upper level students' ePortfolios.