Cell-free nuclear import

Transmission electron microscopy (TEM) is important to monitor cell-free sperm decondensation and nuclear formation. To prepare specimens for TEM, incubation mixture aliquots were fixed for 45 min in 2.5% (v/v) paraformaldehyde, 3.1% (v/v) glutaraldehyde, 0.02% (w/v) picric acid in 30 mAf NaHP04, pH 7.5. After fixation, samples were embedded in 2% (w/v) low-gelling-temperature agarose and postfixed for 15 min in 1% (w/v) OSO4. Samples may be dehydrated either in ethanol and propylene oxide or acetone and embedded for sectioning in Spurr s low-viscosity epoxy resin (Spurr, 1%9). Before examination, sections of about 70-nm thickness should be stained, e.g., with uranyl acetate and Reynolds lead citrate (Re)molds, 1%3). 

As described above, if dATP is mixed with extracts from proliferating cells, the extracts have the ability to induce nuclear apoptosis mediated by caspase-3 activation in a cell-free reaction. Using this system, Wang s group have purified the factors responsible for processing procaspase-3 and identified three gene products, namely Apaf-1, cytochrome c and procaspase-9. Thus, biochemical approaches using cell-free systems for apoptosis has demonstrated several important aspects in the field of apoptosis. 

Belloncik S, Akoury WE, Cheroutre M (1997), Importance of cholesterol for nuclear polyhedrosis virus (NPV) replication in cell cultures adapted to serum-free medium, In Maramorosh K, Mitsubashi J (Eds), Invertebrate Cell Culture Novel Directions and Biotechnology Applications, Science Publishers, Enfield, NH, pp. 141-147. 

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