Solution structure of marinostatin, a natural ester-linked protein protease inhibitor.

Marinostatin is a unique protein protease inhibitor containing two ester linkages. We have purified a 12-residue marinostatin [MST(1-12), (1)FATMRYPSDSDE(12)] and determined the residues involved in the formation of the ester linkages and the solution structure by (1)H NMR spectroscopy and restrained molecular dynamics calculation. The two ester linkages of MST(1-12) are formed between hydroxyl and carboxyl groups, Thr(3)-Asp(9) and Ser(8)-Asp(11), indicating that MST(1-12) has two cyclic regions which are fused at the residues of Ser(8) and Asp(9). A strong NOE cross-peak between Tyr(6) H(alpha) and Pro(7) H(alpha) was observed, indicating that the Pro(7) residue takes a cis-conformation. Well-converged structures and hydrogen-deuterium experiments of MST(1-12) showed that the backbone NH proton of the P1'residue, Arg(5), is hydrogen-bonded to the carbonyl oxygen of the ester linkage between Thr(3) and Asp(9). To reveal the significance of the ester linkages, a marinostatin analogue, MST-2SS ((1)FACMRYPCCSCE(12)) with two disulfide bridges of Cys(3)-Cys(9) and Cys(8)-Cys(11), was also synthesized. The inhibitory activity of MST-2SS was as strong as that of MST(1-12), and the Pro(7) residue of MST-2SS also takes a cis-conformation. However, the exchange rate of the Arg(5) NH proton of MST-2SS was about 100 times faster than that of MST(1-12), and the structure calculation of MST-2SS was not converged on account of the small number of NOEs, indicating that MST-2SS takes a more flexible structure. The hydrogen acceptability of the ester linkage formed by the P2 position residue, Thr(3), is crucial for suppressing the fluctuation of the reactive site and sustaining the inhibitory activity, which enables marinostatin to be one of the smallest protease inhibitors in nature.

Fractal and periodic heart rate dynamics in fetal sheep: comparison of conventional and new measures based on fractal analysis.

The physiological significance of spectral and fractal components of spontaneous heart rate (HR) variability in the fetus remains unclear. To examine the relationship between circadian rhythms in different measures of HR variability, R-R interval time series obtained by fetal ECGs were recorded continuously over 24 h in five pregnant sheep at 116-125 days gestation. Conventional measures of short-term (STV) and long-term variability (LTV), low-frequency (LF; 0.025-0.15 cycles/beat) and high-frequency (HF; 0.2-0.5 cycles/beat) spectral powers, the LF-to-HF ratio, and fractal dimension values were calculated from 24-h ECG recordings and quantified every 60 min. STV, LTV, and LF and HF spectral powers were minimal during the day but increased significantly to their highest values at night. We found a significant positive correlation between these measures, whereas the cosinor method showed significant similarity between their circadian rhythm patterns. Fetal R-R intervals also exhibited fractal structures. Fetal HR variability had a fractal structure, which was similar between day and night. These results suggested that the circadian rhythms exhibited by STV and LTV during the day were mainly due to changes in frequency components rather than to fractal components of fetal HR fluctuation.

V-shaped deceleration differs in the pattern of carotid blood flow from variable deceleration provoked by cord compression.

OBJECTIVE: To investigate whether V-shaped decelerations in fetal heart rate tracing are a physiologic response to fetal movements or secondary to cord compression. STUDY DESIGN: Six pregnant sheep and their fetuses (115-125 days of gestation) were surgically instrumented and studied. Fetal electrocardiogram, carotid blood flow, arterial blood pressure and fetal movement were continuously monitored for 24 hours. Following the undisturbed 24 hour recording, these parameters were monitored during umbilical cord compression (n = 6). Differences in these parameters between V-shaped decelerations and decelerations provoked by cord compressions were examined. RESULTS: Elevation of blood pressure and decreased carotid blood flow were observed coincidentally with the initiation of V-shaped decelerations. In cord compression, elevation of both blood pressure and carotid blood flow were followed by a decreased heart rate. V-shaped decelerations exhibited a different alteration of carotid blood flow compared to decelerations caused by umbilical cord compression. CONCLUSION: V-shaped deceleration is a physiologic response secondary mainly to fetal movements and is not caused by cord compression.