The analysis of thiodiglycollic acid by selected ion monitoring

A selected ion monitoring assay for thiodiglycollic acid in urine is described. Urine samples are analysed by combined gas chromatography-mass spectrometry as their dibutyl esters using pimelic acid as an internal standard. Rapid analysis was achieved by the simplification of sample preparation. The assay has proved to be reliable, with a detection limit of less than 0.5 mumol/l. The excretion of large amounts of thiodiglycollic acid in premature babies urine has been confirmed, with the greatest excretion occurring from those neonates born with a gestational age of 30 wk or less.     

Cytotoxic lipid esters from Peucedanum ledebourielloides

Two cancer cell growth inhibitory esters, 1,2-dipalmitoyl-3-glucosyl glycerol (1) and 1,6-dihydroxy-hexane-bis-palmitoyl ester (2), together with arachidic acid-2-hydroxy-glycerol ester, daucosterol, and oleanolic acid, were isolated from the roots of Peucedanum ledebourielloides (Apiaceae family). The structures were determined by spectroscopic analyses. The esters 1 and 2 displayed significant activity against the SGC-7901, HT-29, and HL-60 cancer cell lines.     

Dolastatin 10, a powerful cytostatic peptide derived from a marine animal. Inhibition of tubulin polymerization mediated through the vinca alkaloid binding domain

Dolastatin 10, a cytostatic peptide containing several unique amino acid subunits, was isolated from the marine shell-less mollusk Dolabella auricularia (Pettit GR, Kamano Y, Herald CL, Tuinman AA, Boettner FE, Kizu H, Schmidt JM, Baczynskyj L, Tomer KB and Bontems RJ, J Am Chem Soc 109: 6883-6885, 1987). Since our preliminary studies demonstrated that dolastatin 10 inhibited tubulin polymerization and the binding of radiolabeled vinblastine to tubulin, an initial characterization of the properties of dolastatin 10 included a comparison to other antimitotic drugs interfering with vinca alkaloid binding to tubulin (vinblastine, maytansine, rhizoxin, and phomopsin A). Dolastatin 10 inhibited the growth of L1210 murine leukemia cells in culture, with a concordant rise in the mitotic index, and its IC50 value for cell growth was 0.5 nM. Comparable values for the other drugs were 0.5 nM for maytansine, 1 nM for rhizoxin, 20 nM for vinblastine, and 7 microM for phomopsin A. IC50 values were also obtained for the polymerization of purified tubulin in glutamate: 1.2 microM for dolastatin 10, 1.4 microM for phomopsin A, 1.5 microM for vinblastine, 3.5 microM for maytansine, and 6.8 microM for rhizoxin. Dolastatin 10 and vinblastine were comparable in their effects on microtubule assembly dependent on microtubule-associated proteins. Preliminary studies indicated that dolastatin 10, like vinblastine, causes formation of a cold-stable tubulin aggregate at higher drug concentrations. We confirmed that rhizoxin, phomopsin A, and maytansine also inhibit the binding of radiolabeled vinblastine and vincristine to tubulin. Dolastatin 10 and phomopsin A were the strongest inhibitors of these reactions, and rhizoxin the weakest. Dolastatin 10, phomopsin A, maytansine, vinblastine, and rhizoxin all inhibited tubulin-dependent GTP hydrolysis. The greatest inhibition of hydrolysis was observed with dolastatin 10 and phomopsin A, and the least inhibition with rhizoxin.     

Influence of the substrate binding characteristics of fibronectin on corneal epithelial cell outgrowth. Student Research Award in the Doctoral Degree Candidate Category, Fourth World Biomaterials Congress (18th annual meeting of the Society for Biomaterials), Berlin, Germany, April 24-28, 1992.

The outgrowth of corneal epithelial cells onto a polymeric substrate is expected to be the primary event in the epithelialization of a synthetic corneal graft. Circular corneal buttons (5 mm) were punched from excised rabbit corneas and placed onto bare substrates or substrates preadsorbed with fibronectin (fn), albumin, or binary mixtures of both fn and albumin. Cell outgrowth areas were measured after culturing the buttons for 4 days in serum-free medium. Fibronectin adsorption to the materials was measured from pure and binary solutions with 125I-radiolabeled fibronectin. A parameter thought to be related to the binding strength of fn to polymeric substrates was measured in parallel experiments by partial elution of the adsorbed fn by 3% sodium dodecyl sulfate (SDS). Following pure solution fibronectin adsorption a range of outgrowth areas was measured (from 0.86 +/- 0.03 cm2 for glass to 1.49 +/- 0.03 cm2 for TCPS). On all of the materials tested cell outgrowth areas increased following fn preadsorption and decreased following albumin preadsorption relative to bare surfaces (p less than 0.05). Following preadsorption with binary protein mixtures cell outgrowth areas increased with fibronectin adsorption, however, the outgrowth areas were not determined solely by the concentration of fn adsorbed onto the surfaces. This result suggested that the biological efficiency of the adsorbed fibronectin was substrate-dependent. When the cell outgrowth data were cross-plotted against fn retention following SDS elution, the outgrowth areas were found to increase along with increases in fn retention. Based on these data we suggest that epithelial cell outgrowth may be partially governed by the tightness of binding between the fn molecules and the underlying substrate.     

Structure-activity studies with chiral isomers and with segments of the antimitotic marine peptide dolastatin 10

Eighteen configurational isomers of the antimitotic peptide dolastatin 10 (Bai et al., Biochem Pharmacol 39: 1941-1949, 1990) derived from Dolabella auricularia, together with segments obtained as precursors in its synthesis (Pettit et al., J Am Chem Soc 111: 5463-5465, 1989), were examined as inhibitors of tubulin polymerization and as inhibitors of growth of L1210 murine leukemia cells in culture. Dolastatin 10 consists of four amino acids (in order from the amino terminus: dolavaline, valine, dolaisoleucine, and dolaproine), three unique to D. auricularia, linked to an unusual primary amine (dolaphenine, probably derived from phenylalanine) at what would otherwise be its carboxyl terminus. Dolastatin 10 has nine asymmetric carbon atoms, and available isomers included alternate configurations at five positions (positions 9 and 10 in the dolaproine moiety and positions 18, 19 and 19a in the dolaisoleucine moiety). For tubulin polymerization, only alterations at positions 18 and 19 resulted in loss of inhibitory activity of the isomer. In addition, a tripeptide containing dolavaline, valine and dolaisoleucine with all asymmetric carbons identical configurationally to those in dolastatin 10 was found to be about 30% as effective as dolastatin 10 in inhibiting tubulin polymerization. Cytotoxic effects were much more sensitive to alterations in the dolastatin 10 structure. The only modification which did not lead to reduced cytotoxicity was reversal of configuration at position 19a in the dolaisoleucine moiety. Both this isomer and dolastatin 10 had IC50 values of less than 1 nM. Several other isomers had IC50 values with the L1210 cells in the range of 30-90 nM, but these did not correlate well with their inhibitory effects on tubulin polymerization. The tripeptide effective as an inhibitor of tubulin polymerization had no activity against the L1210 cells.     

Dynamics of ablation plume particles generated during excimer laser corneal ablation

Background and Objective: Although the empirical characteristics of ArF excimer laser corneal ablation have been well documented, the exact ablation mechanisms are not well understood. The present paper reports a quantitative analysis of corneal ablation plumes using in situ time resolved laser light scattering and Raman spectroscopy. Study Design/Materials and Methods: Bovine corneas were used as the ArF excimer laser ablation targets. Light scattering data were recorded from the ablation plume as a function of height above the tissue surface and as function of delay time with respect to the ablative ArF laser pulse. Results: Raman spectra of the ablation plume allow identification of the particles as water. Mean plume particle diameters are found to decrease with height, while the particle volume fractions are relatively constant. The total volume of plume particles correlates well with the total volume of water in the ablated corneal tissue. Conclusion: The finding of a non-evolving plume composed of water spherules, combined with the excellent agreement between total volume of water in the plume and the content of water in the ablated corneal tissue, support the concept of photodecomposition or “cold ablation” for corneal tissue during ArF excimer laser ablation. © 1995 Wiley-Liss, Inc.