Influence of Mn2+ ion coordination on tRNAVal1 macrostructure and determination of some coordination sites of Mn2+ ions in tRNAVal1

Electron paramagnetic resonance spectroscopy has been used to study the coupling of Mn²⁺ ions with the tRNA^Val₁ modified with a spin label at four pseudouridylic residues and with the valyl-tRNA^Val₁ modified with a spin label at the α-amine group of the valyl residue. A sharp increase of spin-label mobility has been found in these samples, due to the conformational transition induced by the first and second Mn²⁺ ions. Analysis of dipole–dipole couplings of spin labels with the coordinated ions revealed a definite order in the occupation of ion coordination sites in the tRNA. For some valyl-tRNA^Val₁ molecules, the second Mn²⁺ ions were shown to coordinate on the α-amine group of the valyl residue at a distance of 15–25 Å from a spin label. As a result of the conformational transition, a coordination site appeared in the tRNA at one of the pseudouridylic residues, its distance from the spin label being less than 10 Å. It has been suggested that the conformational transition induced by ions excluded some bases from the system of hydrogen bonds at the level of the tRNA tertiary structure. As a result, these bases acquired sufficient sterical freedom to participate in the Mn²⁺ ion coordination.     

Human VPS34 is required for internal vesicle formation within multivesicular endosomes.

After internalization from the plasma membrane, activated EGF receptors (EGFRs) are delivered to multivesicular bodies (MVBs). Within MVBs, EGFRs are removed from the perimeter membrane to internal vesicles, thereby being sorted from transferrin receptors, which recycle back to the plasma membrane. The phosphatidylinositol (PI) 3'-kinase inhibitor, wortmannin, inhibits internal vesicle formation within MVBs and causes EGFRs to remain in clusters on the perimeter membrane. Microinjection of isotype-specific inhibitory antibodies demonstrates that the PI 3'-kinase required for internal vesicle formation is hVPS34. In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation. We showed previously that tyrosine kinase-negative EGFRs fail to accumulate on internal vesicles of MVBs but are recycled rather than delivered to lysosomes. Therefore, we conclude that selection of EGFRs for inclusion on internal vesicles requires tyrosine kinase but not PI 3'-kinase activity, whereas vesicle formation requires PI 3'-kinase activity. Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs. Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.     

Purification and partial sequence analysis of pp185, the major cellular substrate of the insulin receptor tyrosine kinase

Insulin stimulates the tyrosine phosphorylation of a 185-kDa putative cytosolic substrate protein (pp185) in diverse cell types. After intravenous insulin infusion into the live intact rat, pp185 and the 95-kDa insulin receptor beta-subunit were the major proteins that tyrosine phosphorylated in liver, skeletal muscle, and adipose tissue. Both proteins were maximally phosphorylated within 30 s, and both increased in phosphotyrosine content in parallel with increasing insulin dose. However, pp185 tyrosine phosphorylation was transient, with almost complete dephosphorylation within 2-3 min despite continued insulin stimulation. To identify pp185 directly, we purified pp185 from insulin-stimulated rat liver, using a denaturation-based extraction procedure that blocks endogenous protein phosphatases and thus allows a high yield, single step isolation of phosphotyrosyl proteins by anti-phosphotyrosine antibody immunoaffinity absorption. From 50 rat livers, 50-100 pmol of pp185 was isolated. Edman degradation of seven internal tryptic peptide fragments of pp185 yielded novel amino acid sequences, indicating that pp185 is a new protein. Antipeptide antibodies were raised which specifically recognize a single, 185-kDa insulin-stimulated phosphotyrosyl protein in liver, skeletal muscle, adipose tissue, and several cultured cell lines. These results indicate that pp185 is expressed in a variety of insulin-responsive tissues, is the major protein rapidly tyrosine phosphorylated under physiological conditions in the intact animal, and also provide a route for cloning the pp185 gene and elucidating the function of pp185 in insulin signal transduction.     

Measurement of total respiratory impedance in infants by the forced oscillation technique

The forced oscillation technique according to Làndsér et al. (J. Appl. Physiol. 41:101-106, 1976) was modified for use in infants. Adaptations, including a flexible tube to connect the infant to the measuring system and a bias flow to avoid rebreathing, did not influence impedance values. The linearity of the respiratory system was assessed and confirmed by 1) applying pseudo-random noise oscillations at three different amplitudes to 7 infants and 2) comparing in 12 infants impedance values obtained with pseudo-random noise and with sinusoidal oscillations at 12 and 32 Hz. Intersubject variability, averaged for all frequencies, was 6%. In 17 infants the relative error (+/- SD) between two series of five measurements within a time interval of 15 min was 0.5 +/- 5.7%. No statistically significant difference was found between impedance values before and after repositioning of the infant's head, whereas rotation resulted in a decrease in resistance and no effect on reactance. Our results indicate that the infant-adapted forced pseudo-random noise oscillation technique has the potential to give valuable information about ventilatory lung function in infants.