IMAGEtags: Quantifying mRNA Transcription in Real Time with Multiaptamer Reporters.

Cell communications are essential to the organization, development, and maintenance of multicellular organisms. Much of this communication involves changes in RNA transcription and is dynamic. Most methods for studying transcription require interrupting the continuity of cellular function by sacrificing the communicating cells and capturing gene expression information by periodic sampling of individual cells or the population. The IMAGEtag technology to quantify RNA levels in living cells, demonstrated here in yeast, allows individual cells to be tracked over time as they respond to different environmental cues. IMAGEtags are short RNAs consisting of strings of a variable number of tandem aptamers that bind small-molecule ligands. The aptamer strings can vary in length and in configuration of aptamer constituents, such as to contain multiples of the same aptamer or two or more different aptamers that alternate in their occurrence. A minimum effective length is about five aptamers. The maximum length is undefined. The small-molecule ligands are enabled for imaging as fluorophore conjugates. For each IMAGEtag, two fluorophore conjugates are provided, which are FRET pairs. When a cell expresses an RNA containing an IMAGEtag sequence, the aptamers bind their ligands and bring the fluorophores into sufficiently close proximity to allow FRET. The background fluorescence of both fluorophores is minimal in the FRET channel. These features endow IMAGEtags with the sensitivity to report on mRNA expression levels in living cells.

Evaluation of corn furan fatty acid putative endocrine disruptors on reproductive performance in adult female chickens.

Based on evidence from rodent models, it was hypothesized that furan fatty acids found in corn would inhibit reproduction in the laying hen. An isomeric mixture of furan fatty acids [9, (12)-oxy-10,13-dihydroxystearic acid and 10, (13)-oxy-9,12-dihydroxystearic acid] was administered for a period of 3 wk via the diet (1 and 3 ppm) at levels greater than those in corn to 20-wk-old pullets. There were no overt indications of acute or chronic toxicity (no effects on mortality, feed intake, or average daily gain). Similarly, there was no dose-dependent effect on reproductive parameters [egg production, egg weight, shell thickness, ovarian weight, number or weight of large yolky preovulatory follicles, and number of small yellow follicles (4-8 mm in diameter)]. The present data do not suggest that furan fatty acids are a cause of concern to the poultry industry.

Synthesis of hydroxy and methoxy perylene quinones, their spectroscopic and computational characterization, and their antiviral activity.

Hydroxy and methoxy perylene quinones are synthesized in an attempt to isolate the essential spectroscopic and biological features of light-induced antiviral agents such as hypericin and hypocrellin. Unlike their naturally occurring counterparts, these synthetic quinones bear the carbonyl, hydroxyl, and methoxy groups in the "bay region." The hydroxy and methoxy compounds have rich absorption spectra with broad features in the visible (approximately 450-800 nm) and relatively more intense and narrow features at wavelengths < or = 350 nm. High-level ab initio quantum mechanical calculations assign the features in the absorption spectra to electronic transitions from S0 to S2 and to higher-lying electronic states. The calculations indicate that in the ground state the trans dihydroxy isomer is 12.5 kcal/mol lower in energy than the cis dihydroxy isomer and is thus the only species present. The lowest-energy trans methoxy ground state isomer and the lowest-energy cis methoxy ground state isomer are found to be degenerate. An additional cis methoxy isomer 6.3 kcal/mol higher in energy than the global minimum is assumed to contribute to the spectrum and is also considered. Finally, the synthetic compounds exhibit similar light-induced antiviral activity to each other, but significantly less than that of hypericin.

Toward the molecular flashlight: preparation, properties, and photophysics of a hypericin-luciferin tethered molecule.

The synthesis of a molecule containing hypericin and luciferin moieties joined by a tether is reported. The light-induced (in vitro) antiviral activity as well as the photophysical properties of this new compound are measured and compared with those of the parent compounds, hypericin and pseudohypericin. This tethered molecule exhibits excited-state behavior that is very similar to that of its parent compounds and antiviral activity that is identical, within experimental error, to that of its more closely related parent compound, pseudohypericin. The implications for a photodynamic therapy that is independent of external light sources are discussed.

Is Change in Electrical Potential or pH a Hatching Signal for Heterodera glycines?

ABSTRACT This study explored the possibilities that changes in the egg shell/lipid layer electrical potential or pH communicate external hatching conditions to the Heterodera glycines second-stage juvenile (J2) within the mature egg and that electrophysiology could measure effects of chemicals on emergence. Potentials were measured following application of the emergence inducers (ZnSO(4) and ZnCl(2)), ions that do not affect emergence, or synthetic emergence inhibitors. Results were compared with pH measurements and emergence bioassays. Healthy appearing eggs had negative resting potentials. Application of ZnSO(4) caused a smooth depolarization. However, eggs containing J2 and immature eggs depolarized to a similar degree when ZnSO(4) was added. In addition, ZnSO(4), synthetic emergence inhibitors, and CaCl(2) caused similar depolarization, and some depolarization was measured in dye-permeable eggs and empty shells. Results suggest that change in cation surface charge contributed to depolarization and that Cl penetrated the egg shell/lipid layer without causing potential changes. In bioassays, zinc consistently stimulated emergence to a greater degree than H(2)O, other cations, or buffers, and counteracted emergence inhibitors. Zinc-caused emergence stimulation was independent of pH. In summary, it is concluded that depolarization and pH are not emergence signals and electrophysiology is unlikely to measure effectiveness of emergence stimulators or inhibitors.

Identification of a vibrational frequency corresponding to H-atom translocation in hypericin.

Using time-resolved infrared spectroscopy, ab initio quantum mechanical calculations and synthetic organic chemistry a region in the infrared spectrum of triplet hypericin has been found between 1400 and 1500 cm-1 corresponding to the translocation of the hydrogen atom between the enol and the keto oxygens, O...H...O. This result is discussed in the context of the photophysics of hypericin and of eventual measurements to observe directly the excited-state H-atom transfer.

Tumor cell toxicity of hypericin and related analogs.

A series of hypericin analogs were found to differ in their cytotoxic activity induced by ambient light levels. These analogs vary in their ability to partition into cells, to generate singlet oxygen as well as in other photophysical properties. The data suggest that the biological activity of hypericin is due to a combination of factors whose roles may vary under different circumstances.

Deprotonation of benzylic ethers using a hindered phosphazene base. A synthesis of benzofurans from ortho-substituted benzaldehydes.

The hindered nonionic phosphazene base P(4)-t-Bu efficiently deprotonates o-arylmethoxy benzaldehydes, leading to a direct synthesis of benzofurans. Strong ionic bases such as LDA, LiTMP, and KH failed.

Synthesis and endothelin receptor binding activity of synthetic analogues of RES-1149-2.

A direct synthesis of analogues 6 and 7 is described. The key transformations are the addition of dibromomethyl lithium to a ketone and the subsequent mild hydrolysis to a hemiacetal.

Phenanthrenequinone antiretroviral agents.

Compounds 3 and 5 are the first phenanthrenequinones to exhibit significant virucidal activity against the retrovirus equine infectious anemia virus. They differ from hypericin in that their virucidal activity is not light dependent.

Construction of the tetracyclic skeleton of leucothol A. 1,2,4a,5,6,7,8,9,10,10a,11,11a-dodecahydro-11-hydroxy-11-methyl-7- phenylthiomethyl-5aH-5a,8-methano-cyclohepta[b] naphthalen-10-one.

The title compound, C24H30O2S, is a tetracyclic intermediate for the synthesis of leucothol A and the first example of an ethanoanthracene skeleton to be synthetized. The single-crystal X-ray structure of this compound is presented.

Chemiluminescent activation of the antiviral activity of hypericin: a molecular flashlight.

Hypericin is a naturally occurring photosensitizer that displays potent antiviral activity in the presence of light. The absence of light in many regions of the body may preclude the use of hypericin and other photosensitizers as therapeutic compounds for the treatment of viral infections in vivo. The chemiluminescent oxidation of luciferin by the luciferase from the North American firefly Photinus pyralis was found to generate sufficiently intense and long-lived emission to induce antiviral activity of hypericin. Light-induced virucidal activity of hypericin was demonstrated against equine infectious anemia virus, a lentivirus structurally, genetically, and antigenically related to the human immunodeficiency virus. The implications for exploiting chemiluminescence as a "molecular flashlight" for effecting photodynamic therapy against virus-infected cells and tumor cells are discussed.

Photosensitization is required for inactivation of equine infectious anemia virus by hypericin.

Hypericin, a photoreactive polycyclic quinone, was found to dramatically reduce infectivity of cell-free stocks of equine infectious anemia virus. However, the antiviral activity of hypericin was completely dependent on the presence of light. Short periods of photosensitization resulted in a partial loss of reverse transcriptase activity and complete inhibition of viral infectivity. These results suggest that the photodynamic effect of hypericin interferes with more than one stage in the virus replication cycle.

Antiretroviral activity of synthetic hypericin and related analogs.

Hypericin and pseudohypericin are naturally occurring polycyclic quinones which have recently been shown to inhibit the infectivity of several retroviruses, including human immunodeficiency virus. To better understand the antiviral mechanisms of these compounds, hypericin and a series of analogous quinones were synthesized and tested for anti-retroviral activity against equine infectious anemia virus (EIAV). Treatment of EIAV-infected cells with hypericin reduced the production of infectious virus by 99.99%. None of the analogs were found to inhibit virus replication. These results suggest that the complete ring structure of hypericin is required, but not sufficient, for antiviral activity.