Polyomavirus-derived virus-like particles (VLPs) have been described as potential carriers for encapsidation of nucleic acids in gene therapy. Although VLPs can be generated in E. coli or insect cells, the yeast expression system should be advantageous as it is well established for the biotechnological generation of products for human use, especially because they are free of toxins hazardous for humans. We selected the yeast Saccharomyces cerevisiae for expression of the major capsid protein VP1 of a non-human polyomavirus, the hamster polyomavirus (HaPV). Two entire HaPV VP1-coding sequences, starting with the authentic and a second upstream ATG, respectively, were subcloned and expressed to high levels in Saccharomyces cerevisiae. The expressed VP1 assembled spontaneously into VLPs with a structure resembling that of the native HaPV capsid. Determination of the subcellular localization revealed a nuclear localization of some particles formed by the N-terminally extended VP1, whereas particles formed by the authentic VP1 were found mainly in the cytoplasmic compartment.

Skeletal myoblast cells have a high potential for cell therapy based repair of the injured myocardium. Stem cell differentiation markers are important for finding an appropriate cellular state optimal for engraftment. We followed differentiation-induced changes in cellular size, the distribution of mitochondria, adenine and pyridine nucleotide content, NAD(P)H fluorescence in skeletal myogenic stem cell populations derived from rabbit skeletal muscle. The fraction of larger size cells increased during differentiation. Mitochondrial distribution changed from perinuclear in undifferentiated cells to even in the cytoplasm of differentiated cells. The most remarkable feature of undifferentiated myoblasts was a very low NAD(P)H fluorescence which augmented after initiating cell differentiation, due to metabolic and physiological changes, but that did not correlate with an increase in cellular size. A significant increase of NAD(P)H concentration was chromatographically detected after 8 days of differentiation, although the NAD(P)H fluorescence increased by 42% as soon as after three days and 2.7-fold after five days of differentiation as compared with the control. We show that the early increase in fluorescence is determined by mitochondrial NADH, but upon a longer differentiation NADPH amount also increases. The total amount of adenine nucleotides (ATP, ADP, AMP) increased, but the ATP / ADP ratio decreased during differentiation. We suggest that an increase in NAD(P)H fluorescence may be useful for a non-invasive detection of the onset and course of myoblast differentiation. INtRODuctIONThe high therapeutical potential of skeletal myoblasts makes important a thorough understanding of cell differentiation process. The changes stem cells undergo during differentiation include transformations of cell shape, ultrastructure, biochemical composition and metabolism. The goal to employ cells for regenerative medicine implies the necessity of knowing how to track and control the differentiation process. Noninvasive and rapid methods to monitor the metabolic and viability state in differentiating cells are under extreme request. The pyridine dinucleotides NAD + / NADH and NADP + / NADPH are important indicators of cell viability and energy metabolism state. Taking into account that their reduced forms are natural cellular fluorophores, it is reasonable to verify how NAD(P)H fluorescence correlates with numerous changes in cell state during differentiation. This should help to estimate to what extent NAD(P)H fluorescence measurements may be useful for a noninvasive analysis.When a cell enters the differentiation process, it switches the metabolism from anaerobic to aerobic [1]. The expression of mitochondrial enzymes and the respiration rate increase and the mitochondrial distribution changes from perinuclear to even in the cell volume [2]. Although several authors have focused on changes in adenine nucleotides [1,2], the content of pyridine dinucleotides in stem cells and their changes during differentiation have not yet been...