Didemnin B Conformation and dynamics of an antitumour and antiviral depsipeptide studied in solution by 1H and 13C. n.m.r. spectroscopy

The solution conformation of didemnin B, the most potent member of a family of depsipeptides that shows antitumour, antiviral, and immunosuppressive activity, has been studied in chloroform solution using n.m.r. spectroscopy. ¹H and ¹³C spectra have been assigned from analysis of a number of two-dimensional homonuclear and heteronuclear chemical shift correlation experiments which confirm the recently corrected primary structure of the molecule. The conformation of the peptide has been deduced from measurements of the temperature dependence of the NH chemical shifts, analysis of coupling constant data and primarily through NOE effects observed in the rotating frame. Interproton distance bounds determined from a quantitative analysis of the ROE data provide 41 constraints from which a family of closely related structures were calculated using distance geometry algorithms. A type II β-turn involving residues Thr⁶, Leu⁷, and Pro⁸ is well represented in the computed conformers as is a hydrogen bonding interaction between the NH of Leu³ and the carbonyl oxygen of Thr⁶. This latter interaction causes the linear portion of the structure to fold back over the depsipeptide ring, imparting to it a degree of structural stability as well as giving the molecule a somewhat globular character. Only one transannular hydrogen bond, between 1st¹ NH and Leu³ carbonyl, stabilizes the conformation of the depsipeptide, which has an irregular non-planar configuration. The small temperature coefficients (< 2.0 × 10^?3ppm/°C) for the NHs of 1st¹ and Leu³ are consistent with their involvement in these hydrogen bonding interactions. We find that many of the structural features observed in the crystalline form of didemnin B are conserved in solution. Analysis of the ¹³C spin-lattice relaxation rates of the protonated carbons reveals small variations in effective correlation times at specific sites in the molecule. The data suggests that the peptide segment encompassing residues Leu³through to Thr⁶ is in a more motionally restricted part of the structure.     

Problems associated with use of the benzyloxymethyl protecting group for histidines Formaldehyde adducts formed during cleavage by hydrogen fluoride 1

The use of N^α-tert.-butyloxycarbonyl-N^π benzyloxymethylhistidine in peptide synthesis resulted in significant levels of several different side products attributable to the generation of formaldehyde during the hydrogen fluoride cleavage reaction. Methylated impurities in a decapeptide were isolated and identified. These methylated impurities were attributed to the use of the benzyloxymethyl protecting group for the histidines, since the impurities did not form when the dinitrophenyl protecting group was used. Also, peptides containing benzyloxymethyl-protected histidines in addition to N-terminal cysteines quantitatively yielded their respective N-terminal thiazolidine derivatives upon isolation from standard hydrogen fluoride cleavage mixtures. Thiazolidine ring formation was circumvented by including in the cleavage reaction a formaldehyde scavenger such as cysteine hydrochloride or resorcinol.     

True Incidence of Pacemaker Syndrome

HELDMAN, D., ET AL.: True Incidence of Pacemaker Syndrome. Although the purported incidence of pacemaker syndrome according to the literature is only 5%–15%, this is based on a series of patients with VVI pacing. Increasing numbers of studies are being reported in which patients prefer the dual chamber mode despite little benefit being demonstrated on objective testing, suggesting that pacemaker syndrome may be more common than is generally reported. This study was designed to evaluate the reported symptoms in a series of patients programmed to both the VVI and one or more dual chamber modes. Forty unselected patients with dual chamber pacemakers were entered into a blind, randomized trial comparing the symptoms associated with VVI pacing to those associated with dual chamber pacing. Patients were randomized to either WI or dual chamber pacing. At the end of 1 week, questionnaires rating 16 different symptoms were completed. Blood pressure, LV function, presence of ventriculoatrial conduction, and ability to override the pacemaker were evaluated. The pacemaker was then programmed to the other mode. Overall, 12 of 16 symptoms were significantly worse in the VVI as compared to dual chamber mode. The most highly significant (p < 0.005) were shortness of breath, dizziness, fatigue, pulsations in the neck or abdomen, cough, and apprehension. Pacemaker syndrome was clinically recognized in 83% of patients paced in the WI mode with 65% of patients experiencing moderate to severe symptoms. There were no readily identified clinical, hemodynamic, or electrophysiological parameters that predicted which patients would develop pacemaker syndrome. Thus, when patients have an opportunity to experience both pacing modes in close proximity to one another, there is a high incidence of pacemaker syndrome in the VVI mode.     

Neurocardiac Responses to Vagoafferent Electrostimulation in Humans

To determine if cardiac vagal tone is enhanced by vagal electrostimulation (VES), we examined the heart rate autospectrum (HRA) in eight patients with implanted stimulators for complex partial seizures. In four patients the VES was activated at 30 Hz and 500-msec pulse (HiStim group) compared to 2 Hz and 130-msec pulse for the LoStim group (n = 4). Continuous ECG and respiratory waveforms were recorded for 45 minutes every 8 hours (7–8 AM; 3–4 PM 11–12 PM) during resting supine wakeful epochs both before and 15 days after surgical implantation. From the HRA cardiac sympathovagal balance was expressed as the ratio of the low frequency (LF) power to the high frequency (HF) power. RESULTS: There were no presurgical differences between the groups in heart rate, its variance, or the energies contained in any autospectral band. The LoStim group showed no significant change in heart rate (HR), HF peak power, or LF:HF ratios during 2 weeks of VES. Conversely, in the HiStim group, the LF:HF peak power ratio fan expression of sympathetic dominance) decreased from 2.5 ± 1.5 preimplant to 1.5 ± 0.49 (P < 0.02) with VES. During VES there was a significantly higher HF power in the HiStim compared to LoStim group. No diurnal variations in HRA values were seen for either group. CONCLUSIONS: (1) A relationship exists between selective vagal nerve electrostimulation and the HRA; and (2) high stimulation frequency of the vagus nerve in man is associated with sustained augmentation of cardiac vagal tone throughout a 24-hour cycle.     

A Minimal Model of the Single Capacitor Biphasic Defibrillation Waveform

A quantita tive model of the single capacitor biphasic defibrillation wave form is proposed. The primary hypothesis of this model is that the first phase leaves a residual charge on the membranes of the unsynchronized cells, which can then reinitiate fibrillation. The second phase diminishes this charge, reducing the potential for refibrillation. To suppress this potential refibrillation, a monophasic shock must be strong enoagh to synchronize a critical mass of nearly 100% of the myocytes. Since the biphasic waveform performs this protection function by removing the residual charge (with its second phase), its first phase may be of a lower strength than a monophasic shock of equivalent performance. A quantitative model was developed to calculate the residual membrane voltage, V_m, assuming a capacitive membrane being alternately charged and discharged by the first and second phases, respectively. It was further assumed that the amplitude of the first phase would be predicted by a minimum value plus a term proportional to V_m². The model was evaluated on the pooled data of three relevant published studies comparing biphasic waveforms. The model explained 79% of the variance in the first phase amplitude and predicted optimal durations for various defibrillator capacitances and electrode resistances. Assuming a first phase of opti mal duration, the optimal second phase duration appears to be about 2.5 msec for all capacitances and resistances now seen clinically. Conclusion: The effectiveness of the single capacitor biphasic waveform may be explained by the second phase "burping" of the deleterious residual charge of the first phase that, in turn, reduces the synchronization requirement and the amplitude requirements of the first phase.     

Local Repolarization Abnormalities Induced by Transcatheter Radiofrequency Ablation in Pigs

Radiofrequency (RF) ablation alters action potential repolarization of myocardial cells and, theoretically, tbis should induce ST-T segment changes in the ECG. Since these ECG abnormalities have been rarely reported in patients submitted to RF ablation we assess the ability of the procedure to caase ST-T segment changes in local electrograms. Epicardial ECG mapping was performed in 17 anesthetized open chest pigs submitted to endocardial (n = 9) or to epicardial (n = 8) unipolar radiofrequency ablation (500 kHz, 20 W for 5-10 s). To characterize the cellular electrophysiological alterations induced by RE ablation transmembrane action potentials were recorded at various distances from the ablation lesion; these were compared with seven control pigs. Endocardial RE ablation induced a transient (< 5 min) change of 6.1 ± 2.4 m V in T wave amplitude (baseline: 12.8 ± 5.6 mV, P < O.OOl) in 141 out of 269 epicardial electrodes. T wave changes were associated with shortening in local activation time (20.1 ± 2.3 ms at baseline vs 18.5 ± 2.5 ms at 60 s after ablation, P = 0.03). RE current caused persistent ST segment elevation at the center of the ablation lesion with no transmural expansion. Intracellular potentials along a 2-6-mm wide myocardial band bordering the RE lesion showed lower amplitude (101 ± 7.0 mV vs 71 ± 23 mV, P < 0.01) and shorter duration (254 ± 44 ms vs 156 ± 29 ms, P < 0.01) than control hearts. The center of the ablation lesion was electrically anexcitable. We concluded that RF ablation alters cellular electrophysiology in small areas surrounding the ablation lesion and this causes short-lasting transmural changes in T wave amplitude and nontransmural ST segment elevation.