Lanthanide-binding-tags (LBTs) are valuable tools for investigation of protein structure, function, and dynamics by NMR spectroscopy, X-ray crystallography and luminescence studies. We have inserted LBTs into three different loop positions (denoted L, R, and S) of the model protein interleukin-1β and varied the length of the spacer between the LBT and the protein (denoted 1-3). Luminescence studies demonstrate that all nine constructs bind Tb 3+ tightly in the low nanomolar range. No significant change in the fusion protein occurs from insertion of the LBT, as shown by two X-ray crystallographic structures of the IL1β-S1 and IL1β-L3 constructs and for the remaining constructs by comparing 1 H-15 N-HSQC NMR spectra with wild-type IL1β. Additionally, binding of LBT-loop IL1β proteins to their native binding partner in vitro remains unaltered. X-ray crystallographic phasing was successful using only the signal from the bound lanthanide. Large residual dipolar couplings (RDCs) could be determined by NMR spectroscopy for all LBT-loopconstructs and revealed that the LBT-2 series were rigidly incorporated into the interleukin-1β structure. The paramagnetic NMR spectra of loop-LBT mutant IL1β-R2 were assigned and the Δχ tensor components were calculated based on RDCs and pseudocontact shifts (PCSs). A structural model of the IL1β-R2 construct was calculated using the paramagnetic restraints. The current data provide support that encodable LBTs serve as versatile biophysical tags when inserted into loop regions of proteins of known structure or predicted via homology modelling.

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BRCA1, a breast and ovarian cancer-suppressor gene, exerts tumor-suppressing functions that appear to be associated, at least in part, with its DNA repair, checkpoint, and mitotic regulatory activities. Earlier work from our laboratory also suggested an ability of BRCA1 to communicate with the inactive X chromosome (Xi) in female somatic cells (Ganesan et al., 2002). Xiao et al. (2007) (this issue of Cell) have challenged this conclusion. Here we discuss recently published data from our laboratory and others and present new results that, together, provide further support for a role of BRCA1 in the regulation of XIST concentration on Xi in somatic cells.

Cancer is a leading cause of death in children with tumors of the central nervous system, the most commonly encountered solid malignancies in this population. Radiotherapy (RT) is an integral part of managing brain tumors, with excellent long-term survival overall. The tumor histology will dictate the volume of tissue requiring treatment and the dose. However, radiation in developing children can yield functional deficits and/or cosmetic defects and carries a risk of second tumors. In particular, children receiving RT are at risk for neurocognitive effects, neuroendocrine dysfunction, hearing loss, vascular anomalies and events, and psychosocial dysfunction. The risk of these late effects is directly correlated with the volume of tissue irradiated and dose delivered and is inversely correlated with age. To limit the risk of developing these late effects, improved conformity of radiation to the target volume has come from adopting a volumetric planning process. Radiation beam characteristics have also evolved to achieve this end, as exemplified through development of intensity modulated photons and the use of protons. Understanding dose limits of critical at-risk structures for different RT modalities is evolving. In this review, we discuss the physical basis of the most common RT modalities used to treat pediatric brain tumors (intensity modulated radiation therapy and proton therapy), the RT planning process, survival outcomes for several common pediatric malignant brain tumor histologies, RT-associated toxicities, and steps taken to mitigate the risk of acute and late effects from treatment.