The replication licensing factor (RLF) is an essential initiation factor that is involved in preventing re-replication of chromosomal DNA in a single cell cycle. In Xenopus egg extracts, it can be separated into two components: RLF-M, a complex of MCM/P1 polypeptides, and RLF-B, which is currently unpurified. In this paper we investigate variations in RLF activity throughout the cell cycle. Total RLF activity is low in metaphase, due to a lack of RLF-B activity and the presence of an RLF inhibitor. RLF-B is rapidly activated on exit from metaphase, and then declines during interphase. The RLF inhibitor present in metaphase extracts is dependent on the activity of cyclin-dependent kinases (Cdks). Affinity depletion of Cdks from metaphase extracts removed the RLF inhibitor, while Cdc2/cyclin B directly inhibited RLF activity. In metaphase extracts treated with the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP), both cyclin B and the RLF inhibitor were stabilized although the extracts morphologically entered interphase. These results are consistent with studies in other organisms that invoke a key role for Cdks in preventing re-replication of DNA in a single cell cycle.

Abstract. Using immunodepletion of cyclin E and the inhibitor protein p21 wAF/cIP1, we demonstrate that the cyclin E protein, in association with Cdk2, is required for chromosomal replication in Xenopus extracts. The requirement for cyclin E occurs early in the replication process and cyclin E/Cdk2 is not required for the elongation phase of replication on single-stranded substrates. Although cyclin E/Cdk2 is likely to be the major target by which p21 inhibits the initiation of sperm DNA replication, p21 can inhibit single-stranded replication through a mechanism dependent on PCNA. While the cyclin E/Cdk2 complex appears to have a role in the initiation of DNA replication, another Cdk kinase, possibly cyclin A/Cdk, may be involved in a later step controlling the switch from initiation to elongation. The provision of a large maternal pool of cyclin E protein shows that regulators of replication are constitutively present, which explains the lack of a protein synthesis requirement for replication in the early embryonic cell cycle.

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Synchrotron X-ray radiography, due to its high temporal and spatial resolutions, provides a valuable means for understanding the in operando water transport behaviour in polymer electrolyte membrane fuel cells. The purpose of this study is to address the specific artefact of imaging sample movement, which poses a significant challenge to synchrotron-based imaging for fuel cell diagnostics. Specifically, the impact of the micrometer-scale movement of the sample was determined, and a correction methodology was developed. At a photon energy level of 20 keV, a maximum movement of 7.5 µm resulted in a false water thickness of 0.93 cm (9% higher than the maximum amount of water that the experimental apparatus could physically contain). This artefact was corrected by image translations based on the relationship between the false water thickness value and the distance moved by the sample. The implementation of this correction method led to a significant reduction in false water thickness (to ∼0.04 cm). Furthermore, to account for inaccuracies in pixel intensities due to the scattering effect and higher harmonics, a calibration technique was introduced for the liquid water X-ray attenuation coefficient, which was found to be 0.657 ± 0.023 cm(-1) at 20 keV. The work presented in this paper provides valuable tools for artefact compensation and accuracy improvements for dynamic synchrotron X-ray imaging of fuel cells.

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