The Cap-binding protein eIF4E promotes folding of a functional domain of yeast translation initiation factor eIF4G1.

The association of eucaryotic translation initiation factor eIF4G with the cap-binding protein eIF4E establishes a critical link between the mRNA and the ribosome during translation initiation. This association requires a conserved seven amino acid peptide within eIF4G that binds to eIF4E. Here we report that a 98-amino acid fragment of S. cerevisiae eIF4G1 that contains this eIF4E binding peptide undergoes an unfolded to folded transition upon binding to eIF4E. The folding of the eIF4G1 domain was evidenced by the eIF4E-dependent changes in its protease sensitivity and (1)H-(15)N HSQC NMR spectrum. Analysis of a series of charge-to-alanine mutations throughout the essential 55.4-kDa core of yeast eIF4G1 also revealed substitutions within this 98-amino acid region that led to reduced eIF4E binding in vivo and in vitro. These data suggest that the association of yeast eIF4E with eIF4G1 leads to the formation of a structured domain within eIF4G1 that could serve as a specific site for interactions with other components of the translational apparatus. They also suggest that the stability of the native eIF4E-eIF4G complex is determined by amino acid residues outside of the conserved seven-residue consensus sequence.

Regulation of inositol 1,4,5-trisphosphate receptors in rat basophilic leukemia cells. I. Multiple conformational states of the receptor in a microsomal preparation.

A detailed characterization of the inositol 1,4,5-trisphosphate (IP3) receptor in rat basophilic leukemia (RBL) cells, a neoplastic mast cell line, has been possible through the growth of solid RBL cell tumors which provide a rich source of IP3 receptor. Equilibrium binding studies show a 1.6 +/- 0.1 pmol/mg of protein maximal binding capacity for [3H]IP3 at optimal Ca2+ (10 microM). The specificity of the RBL cell IP3 receptor towards phosphoinositides, ATP and heparin parallels those previously described with excitable and nonexcitable tissues. [3H]IP3 binding is slightly enhanced from < 1 nM to 10 microM Ca2+ and inhibited by > 10 microM Ca2+. Kinetic and equilibrium studies provide evidence for at least two classes or conformational states of binding sites with pico- and nanomolar affinities. At nM concentrations of IP3, neither binding to the IP3 receptor nor IP3-induced Ca2+ efflux from permeabilized cells demonstrates cooperativity. In contrast, at pM concentrations, IP3 binding kinetics deviate from simple mass action suggesting a complex interaction among binding sites for IP3 on the receptor-channel oligomer. The mechanisms that regulate [3H]IP3 binding in RBL cells are unique when compared to what has been reported in other cells.

Regulation of inositol 1,4,5-trisphosphate receptors in rat basophilic leukemia cells. II. Modulation of the receptor in permeabilized cells by the cytosolic compartment.

Engagement of the IP3 receptor by its ligand releases Ca2+ from intracellular stores of the rat basophilic leukemia (RBL) cell. The IP3 receptor in washed permeabilized cells has high affinity (Kd = 1.2 +/- 0.3 nM) for [3H]IP3 and is not sensitive to physiological concentrations of Ca2+. Moreover, washed permeabilized cells only release small amounts of Ca2+ when stimulated with IP3. When [3H]IP3 binding to permeabilized cells is performed in the presence of cytosolic constituents (unwashed cells), the IP3 receptor has a lower affinity for [3H]IP3 (Kd from 20 to 100 nM) and has enhanced Ca2+ release. Cytosolic supernatant, prepared by centrifugation of permeabilized cells and added back to washed permeabilized cells, decreases [3H]IP3 binding in a dose-dependent manner and increases the amount of Ca2+ released by IP3. Depletion of either MgATP or IP3 in the cytosolic supernatant does not affect the supernatant's ability to decrease [3H]IP3 binding. Though MgATP competitively inhibits [3H]IP3 binding, it cannot fully account for the shift in Kd or the modulation of IP3-stimulated Ca2+ release in the presence of cytosol. These findings suggest that components present in the cytosolic supernatant modulate the function of the IP3 receptor by maintaining it in a low affinity state capable of promoting Ca2+ release.

A definition for paternalism.

Several definitions of paternalism from the contemporary literature are examined. These are all found to be more or less defective when tested against various counterexamples. An alternative definition is subsequently developed using two necessary conditions which taken together are considered sufficient to define paternalistic actions. Those conditions are (1) the paternalistic action is primarily intended to benefit the recipient, and (2) the recipient's consent or dissent is not a relevant consideration for the initiator.

KIE: Definitions of paternalism found in the works of Gerald Dworkin, Allen Buchanan, Bernard Gert and Charles Culver, and James Childress are analyzed and found defective when tested against various counterexamples. Hershey then develops an alternative definition using two necessary conditions which, when taken together, are sufficient to encompass any and all cases of paternalism. An action, initiated by a human individual or group with regard to another human individual or group, is paternalistic if, and only if, (1) the action is primarily intended by the initiator to benefit the recipient, and (2) the recipient's consent or dissent is not a relevant consideration for the initiator.

Relationship of cell-mediated cytotoxicity against melanoma cells to prognosis in melanoma patients.

The cell-mediated cytotoxicity (CMC) of blood mononuclear cells against cultured human melanoma cells was measured in patients after surgical removal of localized melanoma, at a time when they were considered on clinical grounds to be free of melanoma. It was found that the distribution of CMC values against melanoma cells in melanoma patients was different from that in control subjects, and several sub-populations of melanoma patients were evident on the basis of these measurements. No difference in distribution of CMC values was found against non-melanoma cells, which suggested the changes were specific for melanoma. The proportion of patients with recurrence of melanoma was compared between the patient groups with low, normal or high CMC values against cultured melanoma cells after surgery. Analysis for periods extending to 2 years showed that patients with low CMC values after surgery had a significantly higher incidence of recurrence from melanoma than patients with normal or high CMC values. These results suggest there may be a sub-group of melanoma patients who have intrinsically low CMC against melanoma cells, and that this may be an important predisposing factor in the development of recurrent melanoma.