David Hamilton Young

Physiological changes in suspension-cultured soybean cells elicited by treatment with chitosan

Abstract Addition of soluble chitosan to suspension-cultured Glycine max cells inhibited the rate of increase in cell fresh weight, increased their glyceollin content and altered their cell wall composition. The amounts of wall-bound phenolic compounds and callose increased and the walls became more resistant to degradation by fungal enzymes. Poly-L-lysine caused the same effects as chitosan, whereas the monomers L-lysine and D-glucosamine were not active. All the reactions observed could be partly suppressed by Na-polygalacturonate.

Novel Fungitoxicity Assays for Inhibition of Germination-Associated Adhesion of Botrytis cinerea and Puccinia recondita Spores

ABSTRACT Botrytis cinerea and Puccinia recondita spores adhere strongly to polystyrene microtiter plates coincident with germination. We developed assays for inhibition of spore adhesion in 96-well microtiter plates by using sulforhodamine B staining to quantify the adherent spores. In both organisms, fungicides that inhibited germination strongly inhibited spore adhesion, with 50% effective concentrations (EC50s) comparable to those for inhibition of germination. In contrast, fungicides that acted after germination in B. cinerea inhibited spore adhesion to microtiter plates only at concentrations much higher than their EC50s for inhibition of mycelial growth. Similarly, in P. recondita the ergosterol biosynthesis inhibitors myclobutanil and fenbuconazole acted after germination and did not inhibit spore adhesion. The assays provide a rapid, high-throughput alternative to traditional spore germination assays and may be applicable to other fungi.

Identification of ceramide-phosphorylethanolamine in Oomycete plant pathogens: Pythium ultimum, phytophthora infestans, and Phytophthora capsici

Cellular lipids were extracted from three species of Oomycete plant pathogens (Pythium ultimum, Phytophthora infestans, and Ph. capsici) and analyzed via normal-phase high-performance liquid chromatography with flame-ionization detection. The most abundant polar lipids in each of the three species were the polar membrane lipids, phosphatidylethanolamine (PE), phosphatidylcholine, and a phosphosphingolipid that eluted soon after PE. Structural analysis via mass spectrometry and nuclear magnetic resonance spectrometry revealed that the phosphosphingolipid was ceramide phosphorylethanolamine (Cer-PE). The most abundant molecular species of Cer-PE in P. ultimum had a molecular weight of 670.5, contained an unusual 19-carbon branched triunsaturated sphingoid (C19-Δ4, 8, 10, 9-methyl long-chain base) and palmitic acid as the amidelinked fatty acid. The most abundant molecular species of Cer-PE in Ph. infestans has a molecular weight of 714.5, contained a common 16-carbon 1,3 di-OH sphingoid, and erucic (cis 13-docosenoic, C22-Δ13) acid as the amide-linked fatty acid. The Cer-PE in Ph. capsici comprised a mixture of each of the two molecular species found in P. ultimum and Ph. infestans.

Chlorophyll-derived porphyrins co-chromatograph with phospholipids in high performance liquid chromatographic separations of plant lipid classes

Total lipid extracts were prepared from the leaves of potatoes, tomatoes, grapes and spinach, and each was analyzed by normal-phase high performance liquid chromatography (HPLC). When these total lipid extracts were subjected to mild alkaline hydrolysis and the hydrolysates analyzed by normal-phase HPLC, distinct peaks were detected in the region where common phospholipids typically elute (i.e. at 48, 50, and 68 min). The ultra violet-visible spectra of these peak fractions revealed that each exhibited absorption maxima at 400 and 660 nm, suggesting that the peaks were porphyrins, most likely derived from chlorophylls. Mild alkaline hydrolysis apparently cleaved the ester bond of the chlorophylls and released the porphyrin and phytol components. This explanation was verified when commercially prepared chlorophylls a and b were subjected to the same alkaline hydrolysis conditions and identical peaks at 48 and 68 min were observed. Experiments with buffered (pH 6.0) aqueous homogenates of potato and tomato revealed that similar chlorophyll-derived porphyrins were generated by endogenous enzymes. With the increasing popularity of HPLC as a tool for plant lipid analysis, users of this methodology should be cautioned as to the occurence of these non-phospholipid peaks in the retention time region where phospholipids commonly elute.

A Radioligand Binding Assay for Antitubulin Activity in Tumor Cells

The benzamide RH-5854 is shown to be highly potent toward tumor cells and to arrest nuclear division by a highly specific covalent binding to the β-subunit of tubulin in the colchicine binding region. Binding of 3H-RH-5854 to β-tubulin in HCT-116 colon cancer cells is saturable and has been exploited in the development of a cell-based competitive binding assay, which allows antitubulin effects to be detected inwhole cells. 3H-RH-5854 binding is strongly inhibited by preincubating the cells with compounds that bind to the colchicine site andwith paclitaxel. Binding of 3H-RH-5854 is enhanced by preincubating the cells with vinblastine but not by other agents that bind at or near the vinblastine site (ansamitocin P-3 and phomopsin A). Various cytotoxic agents that do not act on tubulin do not affect binding of 3H-RH-5854 in HCT-116 cells, demonstrating specificity of the assay for detection of antitubulin activity. As an alternative to traditional assays that employ isolated brain tubulin, the 3HRH-5854 binding assay enables screening for antitubulin effects directly in tumor cells, providing an assay that accounts for cell-specific criteria that influence sensitivity such as different tubulin isotypes, tubulin mutations, drug metabolism, and efflux mechanisms.

1,4-Bis-(2,3-epoxypropylamino)-9,10-anthracenedione and related compounds as potent antifungal and antimicrobial agents

1,4-Bis-(2,3-epoxypropylamino)-9,10-anthracenedione (1) and related derivatives were assayed for antifungal, antimicrobial, and plant growth regulatory activity. Compound 1 was extremely potent against several strains of fungi, bacteria, and algae (MIC <2 ppm) and controlled two types of downy mildew by 90–100% at a dose of 12 ppm.

Synthesis of (±)-2R,4R,5S-2-methoxy-4-nitro-5-(2,3,4-trimethoxyphenyl)cyclohexanone and (±)-2R,4S,5R-2-methoxy-5-nitro-4-(2,3,4-trimethoxyphenyl)cyclohexanone as colchicine mimetics

Abstract Colchicine mimetic (±)-4S,5R-4-nitro-5-(2,3,4-trimethoxyphenyl)cyclohexene ( 1 ) was epoxidized to afford a mixture of epoxides. The epoxides were separately converted in two steps, with high stereoselectivity, to two regioisomeric α-methoxyketones. One regioisomer, (±)-2R,4S,5R-2-methoxy-5-nitro-4-(2,3,4-trimethoxyphenyl)cyclohexanone ( 17 ), proved to be about 12-fold more potent than synthetic precursor 1 against HCT-116 tumor cells while the other regioisomer, (±)-2R,4R,5S-2-methoxy-4-nitro-5-(2,3,4-trimethoxyphenyl)cyclohexanone ( 16 ), and the synthetic intermediates tested showed no improvement in potency.

Synthesis and Antifungal Activity of an Acivicine-based Dipeptide Library

Javier Garcia, Blanca Martı́nez-Teipel, Ernesto Nicolás, David H. Young, Enrique L. Michelotti* & Fernando Albericio* 1 Department of Organic Chemistry, University of Barcelona, Marti i Franqués 1-11, 08028-Barcelona, Spain; 2 Rohm and Haas Company, 727 Norristown Road, Spring House, PA 19477-0904, USA; 3 Barcelona Science Park, University of Barcelona, Josep Samitier 1, 08028-Barcelona, Spain; 4 Current address: Adolor Corporation, 700 Pennsylvania Drive Exton, PA 19341, USA; 5 Current address: Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA; 6 Current address: Locus Pharmaceuticals, 512 Townshipline Rd Blue Bell, PA 19422, USA (*Authors for correspondence, e-mail: emichelotti@locuspharma.com, Fax: 1215358 2020; e-mail: albericio@pcb.ub.es, Fax: 3493403 7088)