Menin stimulates homology-directed DNA repair.

Menin, the nuclear protein encoded by the Multiple Endocrine Neoplasia type 1 (MEN1) gene, acts as a tumor suppressor. It interacts with a large number of proteins involved in chromatin modification, transcription, cell cycle checkpoint and DNA repair, though its exact function is not clear. We report that in human cells menin stimulates homology-directed (HD) DNA repair induced by the rare endonuclease I-SceI and it accumulates with Chk1 at the site of the double strand break. In addition, menin and Chk1 interact in vivo. Deletion of the first 228 amino acids of menin impairs the interaction with Chk1 and the ability to stimulate HD repair, suggesting that the complex menin-Chk1 on the damaged chromatin facilitates homologous recombination.

Loop dynamics of the extracellular domain of human tissue factor and activation of factor VIIa.

In the crystal structure of the complex between the soluble extracellular domain of tissue factor (sTF) and active-site-inhibited VIIa, residues 91 and 92 in the Pro(79)-Pro(92) loop of sTF interact with the catalytic domain of VIIa. It is not known, however, whether this loop has a role in allosteric activation of VIIa. Time-resolved fluorescence anisotropy measurements of probes covalently bound to sTF mutants E84C and T121C show that binding uninhibited Factor VIIa affects segmental motions in sTF. Glu(84) resides in the Pro(79)-Pro(92) loop, and Thr(121) resides in the turn between the first and second antiparallel beta-strands of the sTF subdomain that interacts with the Gla and EGF1 domains of VIIa; neither Glu(84) nor Thr(121) makes direct contact with VIIa. Probes bound to T121C report limited segmental flexibility in free sTF, which is lost after VIIa binding. Probes bound to E84C report substantial segmental flexibility in the Pro(79)-Pro(92) loop in free sTF, which is greatly reduced after VIIa binding. Thus, VIIa binding reduces dynamic motions in sTF. In particular, the decrease in the Pro(79)-Pro(92) loop motions indicates that loop entropy has a role in the thermodynamics of the protein-protein interactions involved in allosteric control of VIIa activation.

p85 regulatory subunit of PI3K mediates cAMP-PKA and estrogens biological effects on growth and survival.

Cyclic adenosine 3'5' monophosphate (cAMP) and protein kinase A (PKA) cooperate with phosphatidylinositol 3' kinase (PI3K) signals in the control of growth and survival. To determine the molecular mechanism(s) involved, we identified and mutagenized a specific serine (residue 83) in p85alpha(PI3K), which is phosphorylated in vivo and in vitro by PKA. Expression of p85alpha(PI3K) mutants (alanine or aspartic substitutions) significantly altered the biological responses of the cells to cAMP. cAMP protection from anoikis was reduced in cells expressing the alanine version p85alpha(PI3K). These cells did not arrest in G1 in the presence of cAMP, whereas cells expressing the aspartic mutant p85D accumulated in G1 even in the absence of cAMP. S phase was still efficiently inhibited by cAMP in cells expressing both mutants. The binding of PI3K to Ras p21 was greatly reduced in cells expressing p85A in the presence or absence of cAMP. Conversely, expression of the aspartic mutant stimulated robustly the binding of PI3K to p21 Ras in the presence of cAMP. Mutation in the Ser 83 inhibited cAMP, but not PDGF stimulation of PI3K. Conversely, the p85D aspartic mutant amplified cAMP stimulation of PI3K activity. Phosphorylation of Ser 83 by cAMP-PKA in p85alpha(PI3K) was also necessary for estrogen signaling as expression of p85A or p85D mutants inhibited or amplified, respectively, the binding of estrogen receptor to p85alpha and AKT phosphorylation induced by estrogens. The data presented indicate that: (1) phosphorylation of Ser 83 in p85alpha(PI3K) is critical for cAMP-PKA induced G1 arrest and survival in mouse 3T3 fibroblasts; (2) this site is necessary for amplification of estrogen signals by cAMP-PKA and related receptors. Finally, these data suggest a general mechanism of PI3K regulation by cAMP, operating in various cell types and under different conditions.

Evaluation of four reagents for delipidation of serum.

Four reagents, Aerosil 380, Freon 113, Dextran sulfate 500-S, and a mixed organic solvent were tested for their abilities to produce optically clear, pooled human serum. Aerosil-380, a silicon dioxide, removed 95% of serum cholesterol and triglycerides, and 80% of the free fatty acids. A mixed organic solvent (n-butanol:diisopropyl ether) was equally effective, but also removed nearly all endogenous alkaline phosphatase and lactate dehydrogenase. Freon-113 and Dextran sulfate 500-S removed about half of the serum cholesterol and triglycerides. The serum content of several non-lipid components was unaffected by Aerosil-380, Freon-113, and Dextran sulfate treatments; however, the mixed organic solvent removed 69% of the endogenous calcium. Light scattering data revealed that treatment with all reagents except the mixed organic solvent resulted in optically-clear serum products.

Linearization of data for saturation-type competitive protein binding assay and radioimmunoassay.

Most of the commonly-performed competitive protein-binding radioassay methods utilized in the clinical laboratory are based on the principle of saturation analysis. Although many different methods for linearization of saturation-type assays have been proposed, the algebraic equivalency of all of these methods has not been adequately documented. In this manuscript we have shown the physical and mathematical basis for various methods for linearlization of saturation type assays and the algebraic equivalency of these linearization methods. We have also shown that key parameters such as slope and intercept may be dependent on different components of the assays system with differnt linearization methods. An understanding of these key parameters can help the analyst to evaluate changes in these key parameters and to integrate these parameters in a complete quality control system.

Optimization of T assay: a model study.

Factors involved in the optimization of competitive binding radioassays have been analyzed using a thyroxine (T4) radioassay as a model system. The effects of kinetic errors are minimized by the use of the lowest possible incubation temperature for the separation step and by the use of labeled T4 of high purity (which is not consistently available from commerical sources). The use of labeled T4 containing significant quantities of labeled triiodothyronine (T3) can lead to a marked increase in bias and a decrease in precision with relatively small errors in the separation step due to the relatively short half-life of the T3-binder complex. The determination of the dissociation rate constant (not to be confused with the affinity constant) allows one to make useful estimates of the tolerances that can be allowed in the separation step in order to achieve a desired level of accuracy and precison.

An evaluation of three commercially prepared anion-exchange resin columns for separation of tetraiodothryonine in serum.

Three different commercially prepared anion-exchange resin columns for thyroxine (T4) separation from serum for the "T4 by column" assay were evaluated using the protocol of the supplier. The claims by the suppliers for the distribution and recovery of T4 in the first and second thyroxine-containing eluates were experimentally evaluated by the addition of a tracer quantity of purified [125I]T4. The average experimentally determined elution ratios of recoverable T4 (sum of T4 in first and second eluates) with these commercial columns were: Bio-Rad, 92.2:7.8 (90:10 claimed); Oxford, 87.8:12.2 (94:6 claimed); and Curtis Nuclear, 88.7:11.3 (80:20 claimed). The percent recoveries of T4 in the first thyroxine-containing eluate were: Oxford, 82.3 + 9.70 (x +/- 1 S.D.); Bio-Rad, 91.75 +/- 2.09; and Curtis Nuclear, 74.20 +/- 6.14. Mean serum T4 values obtained by the column method with all commercial columns tested were lower than competitive protein binding radio-assay (CPBR) values if the former values were not corrected for recovery. When individual recovery correction factors were applied to column results, improved correlation and better correspondence of "T4 by column" mean values with the CPBR values were noted. It is concluded that the largest part of the total variability of the "T4 by column" assay is contributed by the chromatographic step when the colorimetric step is performed with an automated technique.