Delta opioid receptor selectivity induced by conformational constraints in linear enkephalin-related peptides: 1H 400-MHz NMR study and theoretical calculations

Introduction into the structure of the linear hexapeptide DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr) or DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr) of tert-butyl groups as constraints different from cyclization leads to a large increase in the selectivity for delta opioid binding site in the case of DSTBULET [Tyr-D-Ser-(OtBu)-Gly-Phe-Leu-Thr] (Ki delta = 6.14 nM; Ki mu = 374 nM) and BUBU [Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(OtBu)] (Ki delta = 4.68 nM; Ki mu = 475 nM) or a loss of affinity for DTTBULET [Tyr-D-Thr(OtBu)-Gly-Phe-Leu-Thr] (Ki delta = 866 nM; Ki mu = 4500 nM). This puzzling behavior is studied here by 400-MHz 1H NMR spectroscopy in DMSO-d6 solution and by theoretical calculations. When DSLET and DTLET are compared, the reduction in energetically accessible phi and psi angles induced by the tert-butyl group in the D-Ser2 residue decreases the degree of freedom in the N-terminal part of the peptides. For DSTBULET and BUBU, the rigidification of the backbone evidenced by the appearance of the large NOE's of Phe4 NH-Gly3 alpha and Gly3 NH-alpha and by the loss of the C7 folding around the D-Ser2 residue found in DSLET could explain the drastic loss of affinity for mu opioid receptors. In DTTBULET, a large change in the spatial orientation around the D-Thr2 (OtBu) residue forces the aromatic rings far from each other.(ABSTRACT TRUNCATED AT 250 WORDS)     

Slow heat rate increases yeast thermotolerance by maintaining plasma membrane integrity.

Thermal resistance of Saccharomyces cerevisiae was found to be drastically dependent on the kinetics of heat perturbation. Yeasts were found to be more resistant to a plateau of 1 h at 50 degrees C after a slope of temperature increase (slow and linear temperature increments) than after a shock (sudden temperature change). Thermotolerance was mainly acquired between 40-50 degrees C during a heat slope, i.e., above the maximal temperature of growth. The death of the yeasts subjected to a heat shock might be related to the loss of membrane integrity: intracellular contents extrusion, i.e., membrane permeabilization, was found to precede cell death. However, the permeabilization did not precede cell death during a heat slope and, therefore, membrane permeabilization was a consequence rather than a cause of cell death. During a slow temperature increase, yeasts which remain viable may have time to adapt their plasma membrane and thus maintain membrane integrity.     

Action potentials of the rabbit, guinea pig, dog and albino rat working ventricular myocardium under interpolated extrasystole conditions during postnatal ontogenesis

Experiments were carried out on the working myocardium of the right heart ventricle of newborn and adult rabbits, guinea-pigs, dogs and albino rats. In the dog, the guinea-pig and the rabbit, after ten action potentials (AP) elicited with 1 Hz frequency we always interpolated an extrasystole at an interval (TE) of 100-900 ms. In albino rats we used a basic frequency of 2 Hz and a TE of 30-370 ms from the last regular AP. Using glass microelectrodes, we recorded the extrasystolic AP (EAP) and the next subsequent AP (2AP). The results were evaluated by constructing graphs of the correlations of the duration of the plateau phase (D0) to TE and of the duration of repolarization to -60 mV level (D60) to the TE. In the myocardium of newborn rabbits, guinea-pigs and dogs, with short TE both D0 and D60 of the EAP are shorter than in the steady state (SS), while for the 2AP the same parameters are influenced only a little. As the TE lengthens, the EAP gradually acquire a length corresponding more to the SS. With TE longer than half the duration of the cycle in the steady state the EAP return to normal, while the 2AP become shorter. The effect of extrasystole on the rat EAP and 2AP diminished with advancing age. In the myocardium of adult rabbits and adult guinea-pigs, and slightly in the myocardium of adult dogs and newborn rats, we observed that the duration of the EAP, with certain TE, was greater than in the steady state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Homologies between paraflagellar rod proteins from trypanosomes and euglenoids revealed by a monoclonal antibody

A Nonidet P 40 insoluble fraction was isolated from Trypanosoma brucei and was used to raise a monoclonal antibody (5E9). The antigen was localized by indirect immunofluorescence in the flagellum of T. brucei and of two species of euglenoids, Euglena gracilis and Distigma proteus. In immunoblot analysis, 5E9 appeared to bind to paraflagellar rod proteins PFR1 and PFR2 of T. brucei (72000 and 75000 mol. wt.) and of E. gracilis (67000 and 76000 mol. wt.). The presence of a common epitope in paraflagellar rod proteins from species of trypanosomes and euglenoids shows that despite distinct structures of the rods some identical domain exists in the proteins that could be involved in their supramolecular assembly into a similar organelle. The antigenic determinant defined by 5E9 was also shown to be present in a 87000 molecular weight polypeptide located in the proximal part of the flagellum of Crithidia oncopelti in which a paraflagellar rod is not detectable at the ultrastructural level.     

The relationship of hormone-sensitive and hormone-insensitive phosphatidylinositol to phosphatidylinositol 4,5-bisphosphate in the WRK-1 cell

We have previously characterized two distinct pools of phosphatidylinositol (PI) in the WRK-1 rat mammary tumor cell, one whose metabolism is enhanced in response to vasopressin and another which is insensitive to hormonal manipulation. The purpose of the present study was to examine the relationship between cellular phosphatidylinositol 4,5-bisphosphate (PIP2) and each of the two PI pools. We have found that in WRK-1 cells, vasopressin induces the rapid loss of PIP2 and the accumulation of inositol phosphates. By making use of kinetic differences in 32Pi uptake into the two pools of PI and assessing radioactivity levels in the 1-phosphate of PIP2, we have determined that hormone-sensitive PI is the precursor of approximately 60% of the cellular PIP2; the remainder is synthesized from the hormone-insensitive pool. Additional data indicate that PIP2 derived from hormone-sensitive PI is likewise hormone-sensitive, while that synthesized from hormone-insensitive PI remains stable over a long period of time and is not affected by the presence of vasopressin.