A conserved arginine-containing motif crucial for the assembly and enzymatic activity of the mixed lineage leukemia protein-1 core complex

The mixed lineage leukemia protein-1 (MLL1) belongs to the SET1 family of histone H3 lysine 4 methyltransferases. Recent studies indicate that the catalytic subunits of SET1 family members are regulated by interaction with a conserved core group of proteins that include the WD repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5), and the absent small homeotic-2-like protein (Ash2L). It has been suggested that WDR5 functions to bridge the interactions between the catalytic and regulatory subunits of SET1 family complexes. However, the molecular details of these interactions are unknown. To gain insight into the interactions among these proteins, we have determined the biophysical basis for the interaction between the human WDR5 and MLL1. Our studies reveal that WDR5 preferentially recognizes a previously unidentified and conserved arginine-containing motif, called the "Win" or WDR5 interaction motif, which is located in the N-SET region of MLL1 and other SET1 family members. Surprisingly, our structural and functional studies show that WDR5 recognizes arginine 3765 of the MLL1 Win motif using the same arginine binding pocket on WDR5 that was previously shown to bind histone H3. We demonstrate that WDR5's recognition of arginine 3765 of MLL1 is essential for the assembly and enzymatic activity of the MLL1 core complex in vitro.     

Identification of potent RORβ modulators: Scaffold variation

We sought to develop RORβ-selective probe molecules in order to investigate the function of the receptor in vitro and in vivo and its role in the pathophysiology of disease. To accomplish this, we modified a potent dual RORβ/RORγ inverse agonist from the primary literature with the goal of improving selectivity for RORβ vs RORγ. Truncation of the Western portion of the molecule ablated activity at RORγ and led to a potent series of RORβ modulators. Continued exploration of this series investigated alternate replacement cores for the aminothiazole ring. Numerous suitable replacements were found during the course of our SAR investigations and are reported herein.     

Chemotactic behavior of myoblasts

Earlier studies have suggested that myogenic cells of somite origin migrate into the developing limb, but little is known about the factors affecting the pattern of migration. In order to understand the migratory behavior of myogenic cells, embryonic skeletal muscle cells were tested for their ability to migrate chemotactically using a modified Boyden chamber assay system. It is shown here, for the first time, that embryonic skeletal muscle cells have the capacity to migrate toward a gradient of platelet-derived growth factor (PDGF) and PDGF-like factors present in serum and chick embryo extract (CEE). On the other hand, nonmyogenic limb mesenchyme cells do not exhibit such a response. A hypothesis is proposed here that chemotactic factors from the already patterned vasculature might influence the distribution of skeletal muscle cells during early limb development.     

A simple procedure for NMR measurements of intra- and extracellular sodium in intact tissues

The total Na+ and both the intra- and extracellular Na+ content of excised rat and frog tissues was quantitated by 23Na NMR at 95.51 MHz. An external capillary containing 33 mM Na7[Dy(P3O10)2], resonating about 30 ppm upfield relative to the 0.00 ppm of the intracellular Na+, was inserted into the tissues. The capillary was calibrated against a concentration range of pure NaCl solution, for measurement of intracellular Na+, and against the same concentrations of NaCl solutions containing 4-6 mM K7[Dy(P3O10)2] in 50 mM histidine. Cl and 100 mM choline. Cl, for measurement of extracellular Na+. Spectra were recorded on tissues first in the absence of the shift reagent for determination of the total Na+. After addition of a K7[Dy(P3O10)2] solution to the sample, the 23Na spectra were recorded immediately so that data accumulation was completed within 15 min. Under these conditions, the extracellular Na+ resonated from 10 to 20 ppm upfield relative to the intracellular Na+, and no loss in the intensity of the extracellular Na+ resonance occurred due to the lability of dysprosium(III)tripolyphosphate (cf. Matwiyoff et al., Magn. Reson. Med. 3: 164, 1986). The intra- and extracellular Na+ content of the tissue was calculated from the integrated areas of the respective Na+ resonances and that of the calibrated capillary, from the known weight of the tissue, and from the known volumes of the solutions added.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two-dimensional proton magnetic resonance of human muscle extracts

In continuation of our previous work on one-dimensional (1D) proton nuclear magnetic resonance (1H-NMR) of normal and diseased human muscle extracts we recorded the two-dimensional (2D) J-correlated proton magnetic resonance spectra of these extracts. Significant differences between normal and diseased muscle extracts, not observed in the 1D 1H-NMR spectra, were seen from their 2D connectivity contour patterns. Taurine was not present in cerebral palsy muscle extract while both normal and scoliosis muscles contained this metabolite. Only the normal muscle had carnitine. Carnosine was present in all muscles. alpha-Ketoglutarate was found only in the diseased muscle extracts. While the amino acids lysine, cysteine and glutamine were common to normal and diseased muscles, threonine was seen only in the diseased muscles. Additional small differences were detected in the 2D patterns of human muscle extracts.