Gloria L. Silva

Synthesis of new fluorogenic cyanine dyes and incorporation into RNA fluoromodules.

A new fluorogenic cyanine dye was synthesized and found to have low fluorescence quantum yield in fluid solution and in the presence of double-stranded DNA but 80-fold enhanced fluorescence in viscous glycerol solution. An RNA aptamer selected for binding to the new dye exhibits K(d) = 87 nM and 60-fold fluorescence enhancement. The dye-aptamer pair is a fluoromodule that can be incorporated into fluorescent sensors and labels.

A rainbow of fluoromodules: a promiscuous scFv protein binds to and activates a diverse set of fluorogenic cyanine dyes.

Combined magnetic and fluorescence cell sorting were used to select Fluorogen Activating Proteins (FAPs) from a yeast surface-displayed library for binding to the fluorogenic cyanine dye Dimethyl Indole Red (DIR). Several FAPs were selected that bind to the dye with low nanomolar Kd values and enhance fluorescence more than 100-fold. One of these FAPs also exhibits considerable promiscuity, binding with high affinity to several other fluorogenic cyanine dyes with emission wavelengths covering most of the visible and near-IR regions of the spectrum. This significantly expands the number and wavelength range of scFv-based fluoromodules.

Modulation of the Multidrug-Resistance Phenotype by New Tropane Alkaloid Aromatic Esters from Erythroxylum pervillei

Nine tropane alkaloid aromatic esters (1-9) were isolated from the roots of Erythroxylum pervillei by following their potential to reverse multidrug-resistance with vinblastine-resistant oral epidermoid carcinoma (KB-V1) cells. All isolates, including seven new structures (3-9), were evaluated against a panel of human cancer cell lines, and it was found that alkaloids 3 and 5-9 showed the greatest activity with KB-V1 cells assessed in the presence of vinblastine, suggesting that these new compounds are potent modulators of P-glycoprotein. Confirmatory results were obtained with human ovarian adenocarcinoma (SKVLB) cells evaluated in the presence of adriamycin and synergistic studies performed with several cell lines from the NCI tumor panel. The structures of the new compounds were determined using spectroscopic techniques. Single-crystal X-ray analysis was performed on the monoester, tropane-3 alpha,6 beta,7 beta-triol 3-phenylacetate (1).

Alkaloids in Bufonid Toads (Melanophryniscus): Temporal and Geographic Determinants for Two Argentinian Species

Bufonid toads of the genus Melanophryniscus represent one of several lineages of anurans with the ability to sequester alkaloids from dietary arthropods for chemical defense. The alkaloid profile for Melanophryniscus stelzneri from a location in the province of Córdoba, Argentina, changed significantly over a 10-year period, probably indicating changes in availability of alkaloid-containing arthropods. A total of 29 alkaloids were identified in two collections of this population. Eight alkaloids were identified in M. stelzneri from another location in the province of Córdoba. The alkaloid profiles of Melanophryniscus rubriventris collected from four locations in the provinces of Salta and Jujuy, Argentina, contained 44 compounds and differed considerably between locations. Furthermore, alkaloid profiles of M. stelzneri and M. rubriventris strongly differed, probably reflecting differences in the ecosystem and hence in availability of alkaloid-containing arthropods.

ent-Pimarane type diterpenes from Gnaphalium gaudichaudianum

Fractionation of the methanol extract of Gnaphalium gaudichaudianum DC afforded one new and six known ent-pimarane diterpenes together with velutin, squalene and stigmasterol. The structure of the new compound was established on the basis of extensive 1D and 2D NMR spectroscopic data interpretation. Two of the isolated compounds exhibited moderate toxicity in the Artemia salina toxicity test.

Pervilleines B and C, new tropane alkaloid aromatic esters that reverse the multidrug-resistance in the hollow fiber assay

P-Glycoprotein (Pgp)-mediated drug efflux can yield a multidrug-resistance phenotype that is associated with poor response to cancer chemotherapy. Pervilleines B and C (PB and PC), two new tropane alkaloid aromatic esters obtained from a chloroform extract of the roots of Erythroxylum pervillei as the result of bioactivity-guided fractionation, were found to restore the vinblastine (VLB) sensitivity of cultured multidrug-resistant KB-V1 cells, with 50% inhibitory concentration values of 0.17 microM in each case. To explore the potential relevance of this response, KB-V1 cells were placed in hollow fibers and implanted into NCr nu/nu mice. Cell growth was not significantly inhibited when VLB or PB or PC were administered as single agents, but when used in combination with vinblastine inhibition of up to 77.7% was observed. Equimolar doses of verapamil were less effective. These data suggest that PB and PC are effective inhibitors of Pgp and should be further evaluated for clinical utility.

NOVEL CYTOTOXIC RING-A SECO-CYCLOARTANE TRITERPENES FROM GARDENIA CORONARIA AND G. SOOTEPENSIS

Abstract Coronalolide methyl ester (1), coronalolide (2), and coronalolic acid (3) were isolated from the leaves and/or stems of Gardenia coronaria. A further compound, methyl coronalolate acetate (4), was purified from the stems after methylation. The novel compounds 1–4 have the rare ring-A seco-cycloartane carbon skeleton and their structures were assigned on the basis of spectral data and molecular modeling, as well as X-ray crystallography performed on 1. Compounds 1 and 2 were also isolated from the leaves of Gardenia sootepensis and showed broad cytotoxic activity when evaluated against a panel of human cancer cell lines.

Fluorescent DNA Nanotags Featuring Covalently Attached Intercalating Dyes: Synthesis, Antibody Conjugation and Intracellular Imaging

We have synthesized fluorescent DNA duplexes featuring multiple thiazole orange (TO) intercalating dyes covalently attached to the DNA via a triazole linkage. The intercalating dyes stabilize the duplex against thermal denaturation and show bright fluorescence in the green region of the spectrum. The emission color can be changed to orange or red by addition of energy-accepting Cy3 or Cy5 dyes attached covalently to the DNA duplex. The dye-modified DNA duplexes were then attached to a secondary antibody for intracellular fluorescence imaging of centrosomes in Drosophila embryos. Bright fluorescent foci were observed at the centrosomes in both the donor (TO) and acceptor (Cy5) channels, because the energy transfer efficiency is moderate. Monitoring the Cy5 emission channel significantly minimized the background signal because of the large shift in emission wavelength allowed by energy transfer.

Antifungal activity of Heterothalamus alienus metabolites

The chemical study of Heterothalamus alienus gave rutin, spathulenol (1), (1R,7S)‐germacra‐4(15),5,10(14)‐trien‐1β‐ol (2), sakuranetin (3), padmatin 3‐acetate (4), (2R,3R)‐dihydroquercetin‐7,3′,4′‐trimethyl ether (5), (2R,3R)‐dihydroquercetin‐7,4′‐dimethyl ether (6), (2R,3R)‐3‐acetoxy‐5,7,4′‐trihydroxyflavanone (7), as the main components of an antifungal extract of the aerial parts of the plant. Compound 2 showed moderate activity, with Epidermophyton floccosum being the most susceptible species (MIC = 100 µg/mL); compound 3 showed the best antifungal behavior having a broad spectrum of action and the lowest MICs. This flavanone along with flavanolol 5 showed very good activity against standardized (MIC = 31.2 µg/mL) as well as clinical isolates of Trichophyton rubrum and T. mentagrophytes (MIC ranges 31.2–62.5 µg/mL and 31.2–125 µg/mL, respectively) and demonstrated not only fungistatic but also fungicide properties. Flavanolol 6 was active against all the dermatophytes tested with MICs of 62.5–250 µg/mL. Rutin, spathulenol (1) and the 3‐acetylated flavanones 4 and 7 were inactive or marginally active against the fungal panel. Copyright

Ambrosanolides and secoambrosanolides from Ambrosia tenuifolia

Abstract Populations of Ambrosia tenuifolia from four regions of North Central Argentina differed substantially in sesquiterpene lactone content. Lactones and lactone derivatives isolated were peruvin, ambrosic acid, cumanin, psilostachyin, psilostachyins B and C, dihydropsilostachyin C, altamisin and altamisic acid.