Alpha-thiol deoxynucleotide triphosphates (S-dNTPs) as radioprotective agents: A novel approach.

In this study, the ability of a mixture of four different alpha-thiol deoxynucleotide triphosphates (S-dNTPs) each at a concentration of 10μM when incorporated into the genomic DNA of proliferating human HL-60 and Mono-Mac-6 (MM-6) cells in vitro to provide protection from 2, 5, and 10 Gy of gamma radiation was investigated. Incorporation of the four different S-dNTPs into nuclear DNA at 10 μM concentration for five days was validated by agarose gel electrophoretic band shift analysis. S-dNTP-treated genomic DNA reacted with BODIPY-iodoacetamide demonstrated a band shift to higher molecular weight to confirm the presence of sulfur moieties in the resultant phosphorothioate DNA backbones.

Functions of the Hsp90-Binding FKBP Immunophilins.

The Hsp90 chaperone is known to interact with a diverse array of client proteins. However, in every case examined, Hsp90 is also accompanied by a single or several co-chaperone proteins. One class of co-chaperone contains a tetratricopeptide repeat (TPR) domain that targets the co-chaperone to the C-terminal region of Hsp90.

Management of Hsp90-Dependent Protein Folding by Small Molecules Targeting the Aha1 Co-Chaperone.

Hsp90 plays an important role in health and is a therapeutic target for managing misfolding disease. Compounds that disrupt co-chaperone delivery of clients to Hsp90 target a subset of Hsp90 activities, thereby minimizing the toxicity of pan-Hsp90 inhibitors. Here, we have identified SEW04784 as a first-in-class inhibitor of the Aha1-stimulated Hsp90 ATPase activity without inhibiting basal Hsp90 ATPase.

Nuclear Receptors: A Historical Perspective.

In this chapter, we summarize the birth of the field of nuclear receptors. These receptors exhibit a multitude of roles in cell biology and hence have attracted a great deal of interest in the drug discovery field. It is not certain whether these receptors evolved independently or an ancestral protein acquired various functions upon binding to preexisting small molecules, ligands.

The Nuclear Receptor Field: A Historical Overview and Future Challenges.

In this article we summarize the birth of the field of nuclear receptors, the discovery of untransformed and transformed isoforms of ligand-binding macromolecules, the discovery of the three-domain structure of the receptors, and the properties of the Hsp90-based heterocomplex responsible for the overall structure of the oligomeric receptor and many aspects of the biological effects. The discovery and properties of the subfamily of receptors called orphan receptors is also outlined. Novel molecular aspects of the mechanism of action of nuclear receptors and challenges to resolve in the near future are discussed.

Aptamer-Magnetic Bead Quantum Dot Sandwich Assays for Foodborne Pathogen Detection: Pros, Cons, and Lessons Learned.

DNA and RNA aptamers have been extensively investigated as potential competitors for antibodies for a variety of applications including food safety testing. Ultrasensitive fluorescence detection of foodborne pathogenic bacteria as low as 1-10 cells/mL has been achieved using aptamers coupled to quantum dots in clear pristine buffers for environmental sample detection. Quantum dots offer other advantages, including single UV or blue light source multiplex (multicolored) detection.

Further characterization and independent validation of a DNA aptamer-quantum dot-based magnetic sandwich assay for Campylobacter.

Previously reported DNA aptamers developed against surface proteins extracted from Campylobacter jejuni were further characterized by aptamer-based Western blotting and shown to bind epitopes on proteins weighing ~16 and 60 kD from reduced C. jejuni and Campylobacter coli lysates. Proteins of these approximate weights have also been identified in traditional antibody-based Western blots of Campylobacter spp.

Inactivation of ID4 promotes a CRPC phenotype with constitutive AR activation through FKBP52.

Castration-resistant prostate cancer (CRPC) is the emergence of prostate cancer cells that have adapted to the androgen-depleted environment of the prostate. In recent years, targeting multiple chaperones and co-chaperones (e.g.

Studies of DNA Aptamer OliGreen and PicoGreen Fluorescence Interactions in Buffer and Serum.

Spectrofluorometric and emission peak titration and timed studies of OliGreen (OG) and PicoGreen (PG) were conducted in Tris EDTA (TE) buffer, pooled rat and fetal bovine serum with two different aptamers of 72 and 192 bases in length to determine if OG or PG were suitable for aptamer pharmacokinetic (PK) studies in sera. Results indicated that OG and PG detected the single-stranded (ss) and double-stranded (ds) stem-loop structures of the two aptamers quite well in TE with reliable standard curves having exponential character (or several linear detection regions) up to 1 μg/ml of aptamer DNA with detection limits of ~1 ng/ml. The intensity of OG and PG staining appeared to correlate with the number and percentage of ss and ds bases in each aptamer.

The FKBP52 Cochaperone Acts in Synergy with β-Catenin to Potentiate Androgen Receptor Signaling.

FKBP52 and β-catenin have emerged in recent years as attractive targets for prostate cancer treatment. β-catenin interacts directly with the androgen receptor (AR) and has been characterized as a co-activator of AR-mediated transcription. FKBP52 is a positive regulator of AR in cellular and whole animal models and is required for the development of androgen-dependent tissues.

Development of a fluorescent enzyme-linked DNA aptamer-magnetic bead sandwich assay and portable fluorometer for sensitive and rapid listeria detection.

A fluorescent DNA aptamer-magnetic bead sandwich assay was developed to detect listeriolysin O (LLO) protein from pathogenic Listeria bacteria using a peroxidase-linked system, Amplex Ultra Red (AUR; derivatized resazurin) substrate, and a custom-designed handheld fluorometer. The assay is highly sensitive with demonstrated limits of detection (LODs) in the range of 4 to 61 L. monocytogenes cells or the equivalent LLO produced by 4 to 61 cells on average in separate titration trials.

Functions of the Hsp90-binding FKBP immunophilins.

Hsp90 functionally interacts with a broad array of client proteins, but in every case examined Hsp90 is accompanied by one or more co-chaperones. One class of co-chaperone contains a tetratricopeptide repeat domain that targets the co-chaperone to the C-terminal region of Hsp90. Within this class are Hsp90-binding peptidylprolyl isomerases, most of which belong to the FK506-binding protein (FKBP) family.

Solution formulation development and efficacy of MJC13 in a preclinical model of castration-resistant prostate cancer.

MJC13, a novel FKBP52 targeting agent, has potential use for the treatment of castration-resistant prostate cancer. The purpose of this work was to develop a solution formulation of MJC13, and obtain its efficacy profile in a human prostate cancer xenograft mouse model. Preformulation studies were conducted to evaluate the physicochemical properties.

Bioavailability and fate of sediment-associated trenbolone and estradiol in aquatic systems.

Endocrine disrupting effects in aquatic organisms have been observed in systems influenced by steroid hormones. Associating endocrine disruption with aqueous concentrations of steroids alone may overlook the influence of source-sink dynamics in sediments on steroid hormone bioavailability. The objective of this study was to determine the fate of 17β-estradiol and 17β-trenbolone in two field sediments and to evaluate the corresponding bioavailability of the compounds to the fathead minnow (Pimephales promelas).

The cochaperone SGTA (small glutamine-rich tetratricopeptide repeat-containing protein alpha) demonstrates regulatory specificity for the androgen, glucocorticoid, and progesterone receptors.

Steroid hormone receptors are ligand-dependent transcription factors that require the ordered assembly of multichaperone complexes for transcriptional activity. Although heat shock protein (Hsp) 90 and Hsp70 are key players in this process, multiple Hsp70- and Hsp90-associated cochaperones associate with receptor-chaperone complexes to regulate receptor folding and activation. Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) was recently characterized as an Hsp70 and Hsp90-associated cochaperone that specifically regulates androgen receptor activity.

Development of a fluorescent enzyme-linked DNA aptamer-magnetic bead sandwich assay and portable fluorometer for sensitive and rapid leishmania detection in sandflies.

A fluorescent peroxidase-linked DNA aptamer-magnetic bead sandwich assay is described which detects as little as 100 ng of soluble protein extracted from Leishmania major promastigotes with a high molarity chaotropic salt. Lessons learned during development of the assay are described and elucidate the pros and cons of using fluorescent dyes or nanoparticles and quantum dots versus a more consistent peroxidase-linked Amplex Ultra Red (AUR; similar to resazurin) fluorescence version of the assay. While all versions of the assays were highly sensitive, the AUR-based version exhibited lower variability between tests.

LOSS of Mrp1 alters detoxification enzyme expression in a tissue- and hormonal-status-specific manner.

The multidrug resistance-associated protein1 (MRP1/ABCC1) is a member of the ABCC transporter subfamily that mediates the efflux of pharmaceuticals, xenobiotics and steroid hormones, typically as glutathione, glucuronide or sulfate conjugates. Since loss of one transporter can be compensated by increasing the expression of other transporters and conjugation enzymes, we sought to examine compensatory changes in phase I, II and III enzyme expression in extrahepatic tissues, including the kidney, lungs and small intestine of intact or castrated Mrp1(-/-) male mice. In the kidney, the expression of several P450s, sulfotransferase 1a1 (Sult), glucuronosyltransferases (Ugt) and Mrps2-4, were significantly changed owing to castration alone.

Regulation of steroid hormone receptor function by the 52-kDa FK506-binding protein (FKBP52).

The large FK506-binding protein FKBP52 has been characterized as an important positive regulator of androgen, glucocorticoid and progesterone receptor signaling pathways. FKBP52 associates with receptor-Hsp90 complexes and is proposed to have roles in both receptor hormone binding and receptor subcellular localization. Data from biochemical and cellular studies have been corroborated in whole animal models as fkbp52-deficient male and female mice display characteristics of androgen, glucocorticoid and/or progesterone insensitivity.

Mice lacking Mrp1 have reduced testicular steroid hormone levels and alterations in steroid biosynthetic enzymes.

The multidrug resistance-associated protein 1 (MRP1/ABCC1) is a member of the ABC active transporter family that can transport several steroid hormone conjugates, including 17beta-estradiol glucuronide, dehydroepiandrosterone sulfate (DHEAS), and estrone 3-sulfate. The present study investigated the role that MRP1 plays in maintaining proper hormone levels in the serum and testes. Serum and testicular steroid hormone levels were examined in both wild-type mice and Mrp1 null mice.