Disruption of cagA, the apoprotein gene of chromoprotein antibiotic C-1027, eliminates holo-antibiotic production, but not the cytotoxic chromophore.

C-1027 is a chromoprotein of the nine-membered enediyne antitumour antibiotic family, comprising apoprotein to stabilize and transport the enediyne chromophore. The disruption of apoprotein gene cagA within the C-1027 biosynthetic gene cluster abolished C-1027 holo-antibiotic production detected by an antibacterial assay, as well as the expression of the apoprotein and C-1027 chromophore extracted following protein precipitation of the culture supernatant. Complementation of the cagA-disrupted mutant AKO with the intact cagA gene restored C-1027 production, suggesting that cagA is indispensable for holo-antibiotic production. Overexpression of cagA in the wild-type strain resulted in a significant increase in C-1027 production as expected. Surprisingly, electrospray ionization (ESI)-MS and ESI-MS/MS analyses suggested that the AKO mutant still produced the C-1027 enediyne chromophore [m/z=844 (M+H)(+)] and its aromatized product [m/z=846 (M+H)(+)]. Consistent with this, the results from gene expression analysis using real-time reverse transcriptase-PCR showed that transcripts of the positive regulator sgcR3 and the structural genes sgcA1, sgcC4, sgcD6 and sgcE were readily detected in the AKO mutant as well as in the wild-type and the complementation strain. These results provided, for the first time, evidence suggesting that the apoprotein of C-1027 is not essential in the self-resistance mechanism for the enediyne chromophore.

Global diastolic strain rate for the assessment of left ventricular relaxation and filling pressures.

BACKGROUND: Diastolic strain rate (SR) measurements that comprise all left ventricular (LV) segments are advantageous over myocardial velocity for assessment of diastolic function. Mitral early diastolic velocity (E)/SR ratio during the isovolumetric relaxation (IVR) period can be used to estimate LV filling pressures. METHODS AND RESULTS: Simultaneous echocardiographic imaging and LV pressure measurements (7F catheters) were performed in 7 adult dogs. Loading conditions were altered by saline infusion and caval occlusion, and lusitropic state was changed by dobutamine and esmolol infusion. A curve depicting global SR was derived from each of the 3 apical views, and SR was measured during IVR (SR(IVR)) and early LV filling (SR(E)). SR(IVR) had a strong correlation with time constant of LV pressure decay during the IVR period (tau) (r=-0.83, P<0.001), whereas SR(E) was significantly related to LV end-diastolic pressure (r=0.52, P=0.005) in the experimental stages where tau was <40 ms. In 50 patients with simultaneous right heart catheterization and echocardiographic imaging, mitral E/SR(IVR) ratio had the best correlation with mean wedge pressure (r=0.79, P<0.001), as well as in 24 prospective patients (r=0.84, P=0.001). E/SR(IVR) was most useful in patients with ratio of E to mitral annulus early diastolic velocity (E/Ea ratio) 8 to 15 and was more accurate than E/Ea in patients with normal ejection fraction and regional dysfunction (both P<0.01). CONCLUSIONS: Global SR(IVR) by 2-dimensional speckle tracking is strongly dependent on LV relaxation. E/SR(IVR) can predict LV filling pressures with reasonable accuracy, particularly in patients with normal ejection fraction and in those with regional dysfunction.

Conformation studies on and assessment by spectral analysis of the protein-chromophore interaction of the macromolecular antitumor antibiotic C-1027.

Characterization of the secondary structure of the antitumor antibiotic C-1027 has been made from a comparison of C-1027 and its apoprotein by various analytical means. The results indicated the antibiotic to be abundant in beta-structure by measurements of Fourier-transform infrared (FT-IR) spectroscopy and the circular dichroism (CD) spectrum, and by a prediction of the secondary structure based on the amino acid sequence of the peptide. In comparison of the IR spectra of their proteins in D2O, the apoprotein exhibited a faster H-D exchange than C-1027, indicating an increase in the "non-motile parts" of the beta-sheets formed through the protein-chromophore interaction in holo-C-1027. The prediction of hydropathic index indicated the hydrophobic residues of the apoprotein to be predominantly located in the beta-sheet structures, suggesting hydrophobic interaction in the binding between chromophore and apoprotein. Further, the interaction between chromophore and apoprotein was detected by a fluorescence method. The result showed the dissociation constant (Kd) to be 6.88 x 10(-5) M, indicating that the chromophore is tightly bound to the protein moiety.