Fluorescence and NMR studies of intramolecular stacking of mRNA cap-analogues.

Intramolecular stacking of a series of new synthesized dinucleotide mRNA cap analogues has been investigated in aqueous buffers by means of fluorescence and 1H-NMR at various pH and temperatures, and compared with that for 7-methylguanosine(5')ppp(5')guanosine (m7GpppG), as well as its hypermethylated derivative m(3)2,2,7GpppG. Thermodynamic parameters for intramolecular self-association stabilized by stacking were established by temperature-dependent fluorescence quenching, taking into account collisional deactivation of the excited states. Relative orientations of the stacked bases in the cap analogues were determined with the aid of a program GEOSHIFT (Stolarski et al., Biochim. Biophys. Acta (1996) 1293, 97), based on ring-current anisotropy. 1D-soft-TOCSY experiments were applied to extract the exact values of vicinal coupling constants, and hence to resolve solution conformation of the cap molecules. Stacking interaction has been discussed in detail in terms of the cap structural features, e.g., types of bases and length of the 5',5'-phosphate bridges, and regarding the interactions stabilizing intramolecular stacking.

Stability of the residual structure in unfolded BPTI in different conditions of temperature and solvent composition measured by disulphide kinetics and double mutant cycle analysis.

The folding funnel model proposes a clear description of the protein folding process. To test this model, additional data on the structures populated in different stages of folding and their influence on further folding are required. Here, we use the double mutant strategy and disulphide formation kinetics measurements to study the impact on folding of the residual structure in unfolded bovine pancreatic trypsin inhibitor (BPTI). We show how five amino acid residues stabilise a folding initiation site, possibly a beta-hairpin, and influence the shape of the upper region of the folding funnel in BPTI in different conditions of temperature and solvent composition. Our data provide experimental evidence for the mechanism by which a fast search for a proper chain topology is made possible early in the folding of proteins. The results apply to proteins in general, not necessarily just to disulphide bonded proteins, since cysteine residues are used here merely as reporter groups.

The effect of menadione on sarcoplasmic reticulum Ca2+ and contractions of single guinea-pig cardiomyocytes.

We investigated the effect of 2-methyl-1,4-naphtoquinone (Menadione) on sarcoplasmic reticulum (SR) Ca2+ content and electrically stimulated contractions (ESCs) of single isolated myocytes of guinea-pig ventricular myocardium. The contractures initiated by means of microinjections of caffeine into the close vicinity of the cell were used as an indirect index of the SR Ca2+ content. Superfusion of the cells for 45 min with Menadione resulted in gradual disappearance of contractile responses to caffeine, prolongation of time to peak amplitude of ESCs by 48 +/- 15% and complete inhibition of postrest and postextrasystolic potentiation. These results are consistent with those of Floreani and Carpenedo (7) who found that Menadione strongly inhibits the SR Ca2+ ATPase. Despite depletion of the SR Ca2+ the amplitude of ESCs did not change which suggests that contractions were initiated in the cells treated with Menadione by Ca2+ derived from the sources other than the SR.

[A map for recording of 15 precordial leads improves the sensitivity of the exercise test]. / Rejestracja mapy 15 odprowadzen przedsercowych zwieksza czulosc testu wysilkowego.

Authors analyzed ecg recordings of 112 exercise tests performed in males aged 29-62. 15 precordial leads map (inclusive of V1-V6 standard leads) was used to record ecg tracings. In 22 patients (19.5%) ischemic, electrocardiographic changes were only observed at non-standard leads. Obtained data indicate higher sensitivity of the exercise test with ecg recordings enriched by additional leads.

[Two types of electromechanical coupling in myocardial myocytes]. / Dwa typy sprzezenia elektromechanicznego w miocytach serca.

Sarcoplasmic reticulum (SR) of isolated myocytes of guineapigs deprived of Ca2+ by means of short exposure of the cell to caffeine does not re-uptake Ca in the quiescent cell. In rat Ca2+ SR content is promptly recovered under the same experimental conditions. The results of this study confirm the two types of excitation-contraction coupling in isolated myocyes of gineapig and rat hearts: without Ca uptake and with Ca uptake between excitations, respectively.

Net Ca2+ influx and sarcoplasmic reticulum Ca2+ uptake in resting single myocytes of the rat heart: comparison with guinea-pig.

We tested Shattock and Bers' (1989) hypothesis according to which in rat cardiac myocytes net Ca2+ influx during diastole via Na/Ca exchange provides the main route of entry of Ca2+ available for activation of contractions. We used injections of caffeine into the close vicinity of the single, isolated rat or guinea-pig ventricular myocytes in order to release Ca2+ from sarcoplasmic reticulum (SR). The cells responded to caffeine with a transient contracture, the amplitude of which was regarded as a relative index of SR Ca2+ content. Application of caffeine deprived the SR of Ca2+. This was manifested by a very small (rat) or absent (guinea-pig) contractile response to the second application of caffeine and by a decrease of the amplitude of the first post caffeine contraction to 8 +/- 3% (rat) or to 16 +/- 6% (guinea-pig) of control. In the rat myocytes SR deprived of Ca2+ was able to recover its Ca2+ store even in the resting cell. This was indicated by the time dependent recovery of contractile response to the second application of caffeine and of the amplitude of the post-caffeine electrically evoked contractions. The recovery of post-caffeine contractile responses was completely inhibited by Ca2+ free solution, by 5.0 mM Ni+ and by low K+ (1.0 mM) hyperpolarising solution superfused from the first application of caffeine or during rest. The recovery was enhanced by superfusion of the cells with low Na+ (50%) solution. These results show that there is a considerable net Ca2+ influx by means of Na/Ca exchange and then the SR Ca2+ uptake in the resting rat myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Inotropic and electrophysiological effects of AB--2, a new antiarrhythmic agent, on isolated cardiac preparations.

Effects of AB--2, a compound structurally related to quinidine, on the force of contraction and intracellular potentials were studied in isolated cardiac preparations obtained from guinea pigs, rabbits, cats and dogs. AB--2 in concentrations of 3 and 6 microM increased the force of contraction of both ventricular and atrial muscle. This effect was absent in reserpinized preparations. In concentrations of 24-96 microM, AB--2 induced a concentration-dependent depression of contraction (ED50 approximately equal to 55 microM). Electrophysiological effects consisted of: 1) concentration-dependent reduction of maximum rate of depolarization (Km for guinea pig ventricular and atrial muscles was 72 and 111 microM, resp.) with no change in the resting membrane potential; 2) shortening of action potential duration in ventricular and Purkinje fibers while prolongation in atrial muscle; 3) reduction of pacemaker activity in Purkinje fibers. It is concluded that electrophysiological effects of AB--2 are similar to those of Class I antiarrhythmic agents.

The effects of blocking the Na-Ca exchange at intervals throughout the physiological contraction-relaxation cycle of single cardiac myocyte.

The method of rapid superfusion of the single isolated ventricular myocytes of guinea-pig heart was used in order to inhibit the Na-Ca exchange throughout the physiological contraction-relaxation cycle. Superfusion of the cell at selected intervals during the contraction with the Na,Ca-free solution resulted in increase in its amplitude, increase in time to peak shortening and in delay of relaxation, albeit the cells relaxed before reperfusion of normal Tyrode solution. The largest increase in amplitude of contraction (to 134 +/- 16%) was observed when the effective exchange of the cell's environment was attained approximately 50 ms after the pulse stimulating contraction. The effects declined promptly when the delay was increased beyond 100 ms. In the cells treated with 10 mM caffeine superfusion with the Na,Ca-free solution after the delay of 50-100 ms resulted in decrease in extent of shortening. Increase in delay resulted in slight increase in extent of shortening with respect to control and strong inhibition of relaxation. The strongest effects were observed when the delay was approximately 200 ms. Superfusion of the normal cells and of the cells treated with caffeine between contractions resulted in slight potentiation of the next beat. It is concluded that Na-Ca exchange provides an important mechanism of relaxation and outward Ca2+ transport in the physiological contraction of the ventricular cardiomyocyte.

A new approach to the direct measurement of tension within the outer layers of the left ventricular wall of the dog heart.

Tension was measured within the outer layers of the left ventricular wall of the dog heart with the strain-gauge force transducer coupled to the wall at diastole without the distortion of its geometry. The transducer still attached to the wall or its segments was calibrated with the passive forces and with the known active force on the isolated, beating heart. The differences between the readings of the transducer and the calibrating force did not exceed 20% and in most experiments they were much smaller. The transducer was practically insensitive to the force perpendicular to the measured one. The proposed method seems to be more reliable than the formerly used by these and other authors.

Excitation- and rest-dependent shifts of Ca in guinea-pig ventricular myocardium.

The rest- and excitation-dependent shifts of Ca and 45Ca in the isolated, perfused ventricles of guinea-pig hearts were investigated. As much as 50% of the total Ca content (2.2 mmol/kg ww) found in the ventricular muscle stimulated at a steady rate of 60/min, was released into perfusate during 4 min of rest. In the preparations perfused with 45Ca containing solution during the 4 min of rest or during the last 20 s of rest only, a single beat resulted in extra uptake of 0.359 and 0.287 mmol of labelled calcium (45Ca) per kg ww, respectively. Single post-rest excitation evoked in the ventricles which were previously perfused with radioactive solution for 64 min, resulted in increase in tissue 45Ca content by 0.229 mmol/kg ww. In these preparations, the gain in 45Ca is equivalent to the net Ca uptake. Continued post-rest stimulation at the rate of 60/min resulted in recovery of pre-rest content of 45Ca and of total Ca. Gain of 45Ca was paralleled by recovery of contractile force. Uptake of 45Ca in the preparations stimulated at the steady rate of 60/min was 0.137 mmol/kg ww and its value did not depend on the number of beats during exposure to the isotope. Thus 45Ca uptake over a number of steady-state beats may be regarded as equal to the uptake in a single beat. This uptake is by orders of magnitude larger than reported previously by other authors. It is proposed that contraction is triggered by Ca influx into the excited cells (Ca1), and that the response of contractile proteins to this trigger is controlled by a large intracellular Ca2 fraction whose volume is rate-dependent.