Fluorine-NMR studies of chimpanzee hemoglobin

It has been demonstrated that 4-fluorophenylalanine, a known inhibitor of protein synthesis, becomes incorporated into hemoglobin when present in the diet of a chimpanzee. 19F-NMR spectra of various forms of this protein show well-resolved lines, each line presumably corresponding to a unique phenylalanine/fluorophenylalanine position of the primary sequence. Fluorine chemical shifts and, by implication, tertiary structures vary with the oxidation state and ligand.     

NMR studies of carbonic anhydrase-4-fluorobenzenesulfonamide complexes

Binding of 4-fluorobenzenesulfonamide to human carbonic anhydrases I and II has been studied by proton, fluorine, and nitrogen-15 nuclear magnetic resonance spectroscopy. All three types of experiments provide evidence that the stoichiometry of the interaction of this inhibitor with both enzymes is 2 mol of inhibitor bound per mole of enzyme. Observations which suggest that the bound forms are involved in an exchange process that is rapid at room temperature but slower at 2 degrees C are described. Nitrogen-15 shift data show that the bound inhibitors are present at the active site as anions. The proton experiments indicate appreciable reorganization of the tertiary structure of the protein upon binding. Saturation-transfer experiments to determine the rate of dissociation of the inhibitor-enzyme complex lead to the conclusion that the dissociation process is more complicated than a simple free-bound equilibrium.     

Regulation of rat ovarian cell growth and steroid secretion

A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin.     

A comparison of univariate and multivariate gene selection techniques for classification of cancer datasets

Background  

Gene selection is an important step when building predictors of disease state based on gene expression data. Gene selection generally improves performance and identifies a relevant subset of genes. Many univariate and multivariate gene selection approaches have been proposed. Frequently the claim is made that genes are co-regulated (due to pathway dependencies) and that multivariate approaches are therefore per definition more desirable than univariate selection approaches. Based on the published performances of all these approaches a fair comparison of the available results can not be made. This mainly stems from two factors. First, the results are often biased, since the validation set is in one way or another involved in training the predictor, resulting in optimistically biased performance estimates. Second, the published results are often based on a small number of relatively simple datasets. Consequently no generally applicable conclusions can be drawn.     

Effects of fluorine substitution on the structure and dynamics of complexes of dihydrofolate reductase (Escherichia coli).

Fluorine NMR experiments with a protein containing fluorinated amino acid analogs can often be used to probe structure and dynamics of the protein as well as conformational changes produced by binding of small molecules. The relevance of NMR experiments with fluorine-containing materials to characteristics of the corresponding native (nonfluorinated) proteins depends upon the extent to which these characteristics are altered by the presence of fluorine. The present work uses molecular dynamics simulations to explore the effects of replacement of tryptophan by 6-fluorotryptophan in folate and methotrexate complexes of the enzyme dihydrofolate reductase (DHFR) (Escherichia coli). Simulations of the folate-native enzyme complex produce local correlation times and order parameters that are generally in good agreement with experimental values. Simulations of the corresponding fluorotryptophan-containing system indicate that the structure and dynamics of this complex are scarcely changed by the presence of fluorinated amino acids. Calculations with the pharmacologically important methotrexate-enzyme complex predict dynamical behavior of the protein similar to that of the folate complex for both the fluorinated and native enzyme. It thus appears that, on the time scale sampled by these computer simulations, substitution of 6-fluorotryptophan for tryptophan has little effect on either the structures or dynamics of DHFR in these complexes.     

Segmental identity and cerebellar granule cell induction in rhombomere 1

Background  

Cerebellar granule cell precursors are specifically generated within the hindbrain segment, rhombomere 1, which is bounded rostrally by the midbrain/hindbrain isthmus and caudally by the boundary of the Hoxa2 expression domain. While graded signals from the isthmus have a demonstrable patterning role within this region, the significance of segmental identity for neuronal specification within rhombomere 1 is unexplored. We examined the response of granule cell precursors to the overexpression of Hoxa2, which normally determines patterns of development specific to the hindbrain. How much does the development of the cerebellum, a midbrain/hindbrain structure, reflect its neuromeric origin as a hindbrain segment?     

TMAO promotes fibrillization and microtubule assembly activity in the C-terminal repeat region of tau

Alzheimer's disease most closely correlates with the appearance of the neurofibrillary tangles (NFTs), intracellular fibrous aggregates of the microtubule-associated protein, tau. Under native conditions, tau is an unstructured protein, and its physical characterization has revealed no clues about the three-dimensional structural determinants essential for aggregation or microtubule binding. We have found that the natural osmolyte trimethylamine N-oxide (TMAO) induces secondary structure in a C-terminal fragment of tau (tau(187)) and greatly promotes both self-aggregation and microtubule (MT) assembly activity. These processes could be distinguished, however, by a single-amino acid substitution (Tyr(310) --> Ala), which severely inhibited aggregation but had no effect on MT assembly activity. The inability of this mutant to aggregate could be completely reversed by TMAO. We propose a model in which TMAO induces partial order in tau(187), resulting in conformers that may correspond to on-pathway intermediates of either aggregation or tau-dependent MT assembly or both. These studies set the stage for future high-resolution structural characterization of these intermediates and the basis by which Tyr(310) may direct pathologic versus normal tau function.