Steroidal bivalent ligands for the estrogen receptor: Design,synthesis, characterization and binding affinities

Steroidal bivalent ligands for the estrogen receptor (ER) were designed using crystal structures of ERα dimers as a template. The syntheses of several 17α-ethynylestradiol-based bivalent ligands with varying linker compositions and lengths are described. The binding affinities of these bivalent ligands for ERα and ERβ were determined. In the two series of bivalent ligands that we synthesized, there is a clear correlation between linker length and binding affinity, both of which reach a maximum at the same tether length. Further studies are underway to explore aspects of bivalent ligand and control compound binding to the ERs and their effects on ER dimer formation; these results will be reported in a subsequent publication.     

Synthesis and evaluation of aryl-substituted diarylpropionitriles,selective ligands for estrogen receptor β, as positron-emission tomographic imaging agents

We have investigated halogen-substituted non-steroidal estrogens with selective binding affinity for the estrogen receptor β (ERβ that might be used for imaging the levels of this ER-subtype in breast tumors by positron emission tomography (PET). Based on diarylpropionitrile (DPN, 1a), a compound previously reported that has a 72-fold binding selectivity for ERβ, we developed a series of DPN analogs having methyl-, hydroxyl-, and halogen substituents, including fluoroethyl and fluoropropyl groups. In competitive radiometric binding assays with [³H]estradiol, all of these DPN analogs showed high ERβ/ERα selectivity; while the selectivity varied, in some cases it reached nearly 300-fold (RBA: ERα, 0.023%; ERβ, 6.25%). The absolute ERβ binding affinities, however, were not sufficient to merit further consideration for developing these ligands as PET imaging agents.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Hexazonium pararosaniline as a fixative for animal tissues

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a “fixative” action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.     

Inactive proteinases in silkmoth moulting gel

The moulting gel of silkmoths lacks proteolytic activity but contains an inactive form of the proteinases which are later found in the moulting fluid. These inactive enzymes are activatable in vitro by dilution, activation proceeding most rapidly at low ionic strength. Activation proceeds as a first-order process and is not autocatalytic. Approximately the full amount of proteinases ultimately found in moulting fluid are already present in the gel. Moulting gel does not inhibit the active proteinases of moulting fluid; moreover, the proteolytic activity elicited by dilution of the moulting gel does not disappear upon reconcentration. These observations suggest that the proteinases in moulting gel are not inhibited by a stable, dissociable inhibitor; they may be present either as compartmentalized active enzymes or as proenzymes. Several possible mechanisms for the in vivo activation at the time of gel to fluid transformation are discussed.     

In vitro evaluation of native entomopathogenic fungi and neem (Azadiractha indica) extracts on Spodoptera frugiperda

The control of Spodoptera frugiperda is based on synthetic insecticides, so some alternatives are the use of entomopathogenic fungi (EF) and neem extract. The objective of the study was to evaluate in vitro effectiveness of native EF and neem extracts on S. frugiperda larvae. Six EF were identified by DNA sequencing of ITS regions from three EF (Fusarium solani, Metarrhizium robertsii, Nigrospora spherica and Penicillium citrinum). They were evaluated in concentrations of 1 × 10⁸ spores/ mL. In addition, a second bioassay was carried out evaluating only F. solani, M. robertsii and N. sphaerica and the addition of vegetable oil. On the other hand, extraction of secondary metabolites from neem seed (Azadirachta indica) was carried out by performing, mass (g) and solvent volume (mL ethanol and water) combinations, which were subjected to microwaves and ultrasound. Subsequently, these extracts were evaluated in concentrations of 3%, 4% and 5%. A survival analysis was performed for each of the bioassays. With respect to the results of the first bioassay, F. solani obtained a probability of survival of 0.476 on the seventh day, while in the second bioassay, M. robertsii obtained 0.488 survival probability. This suggests that the expected percentage of larvae that stay alive on the sixth day is 48.8%. However, in the evaluation of the neem extract the combination 1:12/70% to 4% caused 84% mortality of larvae. The use of native HE and neem extracts has potential for the control of S. frugiperda.     

Immunolocalization of MMP-19 in the human intervertebral disc: implications for disc aging and degeneration

Matrix metalloproteinases (MMPs) degrade components of the extracellular matrix of the disc, but the presence of MMP-19 has not been explored. In other tissues, MMP-19 is known to act in proteolysis of the insulin-like growth factor (IGF) binding protein-3, thereby exposing this protein to make it available to influence cell behavior. MMP-19 also has been shown to inhibit capillary-like formation and thus play a role in the avascular nature of the disc. Using immunohistochemistry, normal discs from six subjects aged newborn through 10 years and 20 disc specimens from control donors or surgical patients aged 15-76 (mean age 40.2 years) were examined for immunolocalization of MMP-19; six Thompson grade I discs, five Thompson grade II, eight Thompson grade III, five Thompson grade IV, and one Thompson grade V discs were analyzed. The results indicate that in discs from young subjects, MMP-19 was uniformly localized in the outer annulus. In discs from adult donors and surgical patients, outer and inner annulus cells only occasionally showed MMP-19 localization. The greatest expression of MMP-19 was observed in young discs, and little expression was seen in older or degenerating discs. Because MMP-19 has been shown to regulate IGF-mediated proliferation in other tissues, its decline in the aging/degenerating disc may contribute to the age-related decrease in disc cell numbers.