Evidence for a role of RUNX1 as recombinase cofactor for TCRß rearrangements and pathological deletions in ETV6-RUNX1 ALL.

T-cell receptor gene beta (TCRß) gene rearrangement represents a complex, tightly regulated molecular mechanism involving excision, deletion and recombination of DNA during T-cell development. RUNX1, a well-known transcription factor for T-cell differentiation, has recently been described to act in addition as a recombinase cofactor for TCRδ gene rearrangements. In this work we employed a RUNX1 knock-out mouse model and demonstrate by deep TCRß sequencing, immunostaining and chromatin immunoprecipitation that RUNX1 binds to the initiation site of TCRß rearrangement and its homozygous inactivation induces severe structural changes of the rearranged TCRß gene, whereas heterozygous inactivation has almost no impact. To compare the mouse model results to the situation in Acute Lymphoblastic Leukemia (ALL) we analyzed TCRß gene rearrangements in T-ALL samples harboring heterozygous Runx1 mutations. Comparable to the Runx1+/- mouse model, heterozygous Runx1 mutations in T-ALL patients displayed no detectable impact on TCRß rearrangements. Furthermore, we reanalyzed published sequence data from recurrent deletion borders of ALL patients carrying an ETV6-RUNX1 translocation. RUNX1 motifs were significantly overrepresented at the deletion ends arguing for a role of RUNX1 in the deletion mechanism. Collectively, our data imply a role of RUNX1 as recombinase cofactor for both physiological and aberrant deletions.

Developing research priorities for palliative care of people with intellectual disabilities in Europe: a consultation process using nominal group technique.

BACKGROUND: Empirical knowledge around palliative care provision and needs of people with intellectual disabilities is extremely limited, as is the availability of research resources, including expertise and funding. This paper describes a consultation process that sought to develop an agenda for research priorities for palliative care of people with intellectual disabilities in Europe. METHODS: A two-day workshop was convened, attended by 16 academics and clinicians in the field of palliative care and intellectual disability from six European countries. The first day consisted of round-table presentations and discussions about the current state of the art, research challenges and knowledge gaps. The second day was focused on developing consensus research priorities with 12 of the workshop participants using nominal group technique, a structured method which involved generating a list of research priorities and ranking them in order of importance. RESULTS: A total of 40 research priorities were proposed and collapsed into eleven research themes. The four most important research themes were: investigating issues around end of life decision making; mapping the scale and scope of the issue; investigating the quality of palliative care for people with intellectual disabilities, including the challenges in achieving best practice; and developing outcome measures and instruments for palliative care of people with intellectual disabilities. CONCLUSIONS: The proposal of four major priority areas and a range of minor themes for future research in intellectual disability, death, dying and palliative care will help researchers to focus limited resources and research expertise on areas where it is most needed and support the building of collaborations. The next steps are to cross-validate these research priorities with people with intellectual disabilities, carers, clinicians, researchers and other stakeholders across Europe; to validate them with local and national policy makers to determine how they could best be incorporated in policy and programmes; and to translate them into actual research studies by setting up European collaborations for specific studies that require such collaboration, develop research proposals and attract research funding.

Identification of the chromosomal replication origin from Thermus thermophilus and its interaction with the replication initiator DnaA.

The chromosomal replication origin oriC and the gene encoding the replication initiator protein DnaA from Thermus thermophilus have been identified and cloned into an Escherichia coli vector system. The replication origin is composed of 13 characteristically arranged DnaA boxes, binding sites for the DnaA protein, and an AT-rich stretch, followed by the dnaN gene. The dnaA gene is located upstream of the origin and expresses a typical DnaA protein that follows the division into four domains, as with other members of the DnaA protein family. Here, we report the purification of Thermus-DnaA (Tth-DnaA) and characterize the interaction of the purified protein with the replication origin, with regard to the binding kinetics and stoichiometry of this interaction. Using gel retardation assays, surface plasmon resonance (SPR) and electron microscopy, we show that, unlike the E. coli DnaA, Tth-DnaA does not recognize a single DnaA box, instead a cluster of three tandemly repeated DnaA boxes is the minimal requirement for specific binding. The highest binding affinities are observed with full-length oriC or six clustered, tandemly repeated DnaA boxes. Furthermore, high-affinity DNA-binding of Tth-DnaA is dependent on the presence of ATP. The Thermus DnaA/oriC interaction will be compared with oriC complex formation generated by other DnaA proteins.

Isolation and some properties of a cathepsin E type proteinase from rat spleen.

Cathepsin E (EC 3.4.23.--) has been isolated from rat spleen. The procedure included autolysis at pH 4.2 which was probably the reason why we isolated a polypeptide of Mr 42 kDa instead of 90 kDa. The latter is reported in the literature to be the Mr of native cathepsin E. The enzyme dissociates under reducing conditions in two identical monomers. In our preparation a mechanism different from reduction must be active producing the 42 kDa polypeptide. This enzyme was hard to distinguish from cathepsin D (EC 3.4.23.5.) which shows similar properties such as size, substrate specificity, stability in 6 M urea, and dependence of the activity on pH. The clear distinction between the two enzymes was proven on the basis of immunochemical reactions. Antibodies to both cathepsins, D and E, did not show any crossreaction with the nonrelated antigen.

Proteolysis in Euglena gracilis : II. Soluble and particle-bound acidic proteinase activities of the cysteine and aspartic types during growth and chloroplast development.

Endoproteolytic activities (EC 3.4.22. and 23.) of cell-free extracts of Euglena gracilis, measured by autolysis and azocaseinolysis, vary considerably during the culture growth cycle. They are high in the lag phase, drop sharply up to the mid-logarithmic phase, and then rise again reaching the initial high levels in the stationary phase. This pattern has been observed for both the soluble and the particulate proteolytic activities of four cell types differing with regard to the developmental state of the chloroplast: dark-grown, light-induced, and light-grown wild-type cells, as well as light-grown apoplastic W3BUL mutant cells, all on a glucose-based medium. Therefore, the activity of the main intracellular proteinases is neither directly nor indirectly light-regulated, but seems to be controlled by the availability of nutrients. Endogenous inhibitors of proteinases could not be detected. Cysteine proteinase activity has been found in the soluble and the particulate fractions, but aspartic proteinase activity in the latter ones only. Different cysteine proteinases may be present in the two fractions, during the different growth phases, and in the four cell types studied.