Christopher Larson

CCAAT enhancer-binding protein (C/EBP) and AML1 (CBF alpha2) synergistically activate the macrophage colony-stimulating factor receptor promoter.

Transcription factors play a key role in the development and differentiation of specific lineages from multipotential progenitors. Identification of these regulators and determining the mechanism of how they activate their target genes are important for understanding normal development of monocytes and macrophages and the pathogenesis of a common form of adult acute leukemia, in which the differentiation of monocytic cells is blocked. Our previous work has shown that the monocyte-specific expression of the macrophage colony-stimulating factor (M-CSF) receptor is regulated by three transcription factors interacting with critical regions of the M-CSF receptor promoter, including PU.1 and AML1.PU.1 is essential for myeloid cell development, while the AML1 gene is involved in several common leukemia-related chromosome translocations, although its role in hematopoiesis has not been fully identified. Along with AML1, a third factor, Mono A, interacts with a small region of the promoter which can function as a monocyte-specific enhancer when multimerized and linked to a heterologous basal promoter. Here, we demonstrate by electrophoretic mobility shift assays with monocytic nuclear extracts, COS-7 cell-transfected factors, and specific antibodies that the monocyte-enriched factor Mono A is CCAAT enhancer-binding protein (C/EBP). C/EBP has been shown previously to be an important transcription factor involved in hepatocyte and adipocyte differentiation; in hematopoietic cells, C/EBP is specifically expressed in myeloid cells. In vitro binding analysis reveals a physical interaction between C/EBP and AML1. Further transfection studies show that C/EBP and AML1 in concert with the AML1 heterodimer partner CBF beta synergistically activate M-CSF receptor by more then 60 fold. These results demonstrate that C/EBP and AML1 are important factors for regulating a critical hematopoietic growth factor receptor, the M-CSF receptor, suggesting a mechanism of how the AML1 fusion protein could contribute to acute myeloid leukemia. Furthermore, they demonstrate physical and functional interactions between AML1 and C/EBP transcription factor family members.

Structure of the human NF‐κB p52 homodimer‐DNA complex at 2.1 Å resolution

The crystal structure of human NF‐κB p52 in its specific complex with the natural κB DNA binding site MHC H‐2 has been solved at 2.1 Å resolution. Whereas the overall structure resembles that of the NF‐κB p50‐DNA complex, pronounced differences are observed within the ‘insert region’. This sequence segment differs in length between different Rel proteins. Compared with NF‐κB p50, the compact α‐helical insert region element is rotated away from the core of the N‐terminal domain, opening up a mainly polar cleft. The insert region presents potential interaction surfaces to other proteins. The high resolution of the structure reveals many water molecules which mediate interactions in the protein‐DNA interface. Additional complexity in Rel protein‐DNA interaction comes from an extended interfacial water cavity that connects residues at the edge of the dimer interface to the central DNA bases. The observed water network might acount for differences in binding specificity between NF‐κB p52 and NF‐κB p50 homodimers.

A multifunctional plasmid for protein expression by ECPCR: overproduction of the p50 subunit of NF-κB

We report the construction of a new T7-based transcription vector, pLM1, designed for use in the ECPCR method of protein overproduction. The utility of pLMq is illustrated by overproduction of the 50 kDa DNA-binding subunit of the human transcription factor NF-κB in E. coli. pLM1-p50 directs high-level expression of soluble, active NF-κB p50.

Limited proteolysis and site-directed mutagenesis of the NF-κB p50 DNA-binding subunit

Tryptic digestion was used to obtain an active 41 kDa fragment of NF-κB. The major proteolysis products were comprised of residues 2-364 (p502–364), 2–362 (p502–362) and 2–366 (p502–366). By site-directed truncation mutagenesis, overproducers for p502–364 and p502–366 were constructed. These vectors direct high level expression of homogeneous, active protein.

The design and synthesis of novel α-ketoamide-based p38 MAP kinase inhibitors

We have identified a novel series of potent p38 MAP kinase inhibitors through structure-based design which due to their extended molecular architecture bind, in addition to the ATP site, to an allosteric pocket. In vitro ADME and in vivo PK studies show these compounds to have drug-like characteristics which could result in the development of an oral treatment for inflammatory conditions.

Studies of Polonium Removal from Molten Lead-Bismuth for Lead-Alloy-Cooled Reactor Applications

Abstract The isotope 210Po is the main product of neutron activation in fast reactors cooled by molten lead-bismuth eutectic (LBE). The isotope 210Po is a pure alpha emitter with a half-life of 138.38 days. For typical values of the neutron flux the 210Po concentration in the coolant can reach 1–10 Ci/kg. While exposure of plant personnel to Po is prevented under normal operating conditions because the primary system is sealed, Po does pose a radiological hazard during maintenance activities for which access to submerged structures is required as well as during accidents resulting in breach of the primary-system barrier. Obviously, continuous removal of Po from the LBE reduces this hazard. Therefore, it is important to understand the mechanisms by which Po is formed in and released from the LBE. We summarize research performed at the Idaho National Engineering and Environmental Laboratory and the Massachusetts Institute of Technology to investigate the basic chemistry of four mechanisms of Po release, which could serve as the basis for a coolant cleanup system in LBE-cooled reactors. The mechanisms explored are lead polonide evaporation, formation of polonium hydride, rare-earth filtering, and alkaline extraction. For the key chemical species involved expressions are given for useful quantities such as formation energy, release, and deposition rates. It is concluded that the most promising removal mechanism is alkaline extraction, although a more systematic investigation of this mechanism is needed.

A DNA-bending protein interacts with an essential upstream regulatory element of the human embryonic beta-like globin gene.

The mammalian beta-like globin gene family has served as an important model system for analysis of tissue- and developmental state-specific gene regulation. Although the activities of a number of regulatory proteins have been implicated in the erythroid cell-specific transcription of globin genes, the mechanisms that restrict their expression to discrete stages of development are less well understood. We have previously identified a novel regulatory element (PRE II) upstream from the human embryonic beta-like globin gene (epsilon) that synergizes with other sequences to confer tissue- and stage-specific expression on a minimal epsilon-globin gene promoter in cultured embryonic erythroid cells. Binding of an erythroid nuclear protein (PRE II-binding factor [PRE-IIBF]) to the PRE II control element is required for promoter activation. Here we report on some of the biochemical properties of PREIIBF, including the characterization of its specificity and affinity for DNA. The embryonic and adult forms of PREIIBF recognize their cognate sequences with identical specificities, supporting our earlier conclusion that they are very similar proteins. PREIIBF binds DNA as a single polypeptide with an Mr of approximately 80,000 to 85,000 and introduces a bend into the target DNA molecule. These results suggest a mechanism by which PREIIBF may contribute to the regulation of the embryonic beta-like globin gene within the context of a complex locus.

Optimization of α-ketoamide based p38 inhibitors through modifications to the region that binds to the allosteric site

We have optimized a novel series of potent p38 MAP kinase inhibitors based on an alpha-ketoamide scaffold through structure based design that due to their extended molecular architecture bind, in addition to the ATP site, to an allosteric pocket. In vitro ADME, in vivo PK and efficacy studies show these compounds to have drug-like characteristics and have resulted in the nomination of a development candidate which is currently in phase II clinical trials for the oral treatment of inflammatory conditions.

'Reverse' α-ketoamide-based p38 MAP kinase inhibitors

We have identified a second series of potent p38 inhibitors. As with our first generation series, these compounds are based on an alpha-ketoamide scaffold. The reversal of the ketoamide order, however, introduces more chemical flexibility and in addition results in improve potencies against p38.