Differentiating Antiproliferative and Chemopreventive Modes of Activity for Electron-Deficient Aryl Isothiocyanates against Human MCF-7 Cells.

The consumption of Brassica vegetables provides beneficial effects through organic isothiocyanates (ITCs), products of the enzymatic hydrolysis of glucosinolate secondary metabolites. The ITC l-sulforaphane (l-SFN) is the principle agent in broccoli that demonstrates several modes of anticancer action. While the anticancer properties of ITCs like l-SFN have been extensively studied and l-SFN has been the subject of multiple human clinical trials, the scope of this work has largely been limited to those derivatives found in nature. Previous studies have demonstrated that structural changes in an ITC can lead to marked differences in a compound's potency to 1) inhibit the growth of cancer cells, and 2) alter cellular transcriptional profiles. This study describes the preparation of a library of non-natural aryl ITCs and the development of a bifurcated screening approach to evaluate the dose- and time-dependence on antiproliferative and chemopreventive properties against human MCF-7 breast cancer cells. Antiproliferative effects were evaluated using a commercial MTS cell viability assay. Chemopreventive properties were evaluated using an antioxidant response element (ARE)-promoted luciferase reporter assay. The results of this study have led to the identification of 1) several key structure-activity relationships and 2) lead ITCs for continued development.

Identification of Redox and Glucose-Dependent Txnip Protein Interactions.

Thioredoxin-interacting protein (Txnip) acts as a negative regulator of thioredoxin function and is a critical modulator of several diseases including, but not limited to, diabetes, ischemia-reperfusion cardiac injury, and carcinogenesis. Therefore, Txnip has become an attractive therapeutic target to alleviate disease pathologies. Although Txnip has been implicated with numerous cellular processes such as proliferation, fatty acid and glucose metabolism, inflammation, and apoptosis, the molecular mechanisms underlying these processes are largely unknown. The objective of these studies was to identify Txnip interacting proteins using the proximity-based labeling method, BioID, to understand differential regulation of pleiotropic Txnip cellular functions. The BioID transgene fused to Txnip expressed in HEK293 identified 31 interacting proteins. Many protein interactions were redox-dependent and were disrupted through mutation of a previously described reactive cysteine (C247S). Furthermore, we demonstrate that this model can be used to identify dynamic Txnip interactions due to known physiological regulators such as hyperglycemia. These data identify novel Txnip protein interactions and demonstrate dynamic interactions dependent on redox and glucose perturbations, providing clarification to the pleiotropic cellular functions of Txnip.

Evaluating the effectiveness of a laboratory-based professional development program for science educators.

The process of developing effective science educators has been a long-standing objective of the broader education community. Numerous studies have recommended not only depth in a teacher's subject area but also a breadth of professional development grounded in constructivist principles, allowing for successful student-centered and inquiry-based instruction. Few programs, however, have addressed the integration of the scientific research laboratory into the science classroom as a viable approach to professional development. Additionally, while occasional laboratory training programs have emerged in recent years, many lack a component for translating acquired skills into reformed classroom instruction. Given the rapid development and demand for knowledgeable employees and an informed population from the biotech and medical industries in recent years, it would appear to be particularly advantageous for the physiology and broader science education communities to consider this issue. The goal of this study was to examine the effectiveness of a laboratory-based professional development program focused on the integration of reformed teaching principles into the classrooms of secondary teachers. This was measured through the program's ability to instill in its participants elevated academic success while gaining fulfillment in the classroom. The findings demonstrated a significant improvement in the use of student-centered instruction and other reformed methods by program participants as well as improved self-efficacy, confidence, and job satisfaction. Also revealed was a reluctance to refashion established classroom protocols. The combination of these outcomes allowed for construction of an experiential framework for professional development in applied science education that supports an atmosphere of reformed teaching in the classroom.

High-performance liquid chromatography-based method to evaluate kinetics of glucosinolate hydrolysis by Sinapis alba myrosinase.

Isothiocyanates (ITCs) are one of several hydrolysis products of glucosinolates, plant secondary metabolites that are substrates for the thioglucohydrolase myrosinase. Recent pursuits toward the development of synthetic non-natural ITCs have consequently led to an exploration of generating these compounds from non-natural glucosinolate precursors. Evaluation of the myrosinase-dependent conversion of select non-natural glucosinolates to non-natural ITCs cannot be accomplished using established ultraviolet-visible (UV-Vis) spectroscopic methods. To overcome this limitation, an alternative high-performance liquid chromatography (HPLC)-based analytical approach was developed where initial reaction velocities were generated from nonlinear reaction progress curves. Validation of this HPLC method was accomplished through parallel evaluation of three glucosinolates with UV-Vis methodology. The results of this study demonstrate that kinetic data are consistent between both analytical methods and that the tested glucosinolates respond similarly to both Michaelis-Menten and specific activity analyses. Consequently, this work resulted in the complete kinetic characterization of three glucosinolates with Sinapis alba myrosinase, with results that were consistent with previous reports.

Structure-activity relationships in toll-like receptor-2 agonistic diacylthioglycerol lipopeptides.

The N-termini of bacterial lipoproteins are acylated with a (S)-(2,3-bisacyloxypropyl)cysteinyl residue. Lipopeptides derived from lipoproteins activate innate immune responses by engaging Toll-like receptor 2 (TLR2) and are highly immunostimulatory and yet without apparent toxicity in animal models. The lipopeptides may therefore be useful as potential immunotherapeutic agents. Previous structure-activity relationships in such lipopeptides have largely been obtained using murine cells, and it is now clear that significant species-specific differences exist between human and murine TLR responses. We have examined in detail the role of the highly conserved Cys residue as well as the geometry and stereochemistry of the Cys-Ser dipeptide unit. (R)-Diacylthioglycerol analogues are maximally active in reporter gene assays using human TLR2. The Cys-Ser dipeptide unit represents the minimal part-structure, but its stereochemistry was found not to be a critical determinant of activity. The thioether bridge between the diacyl and dipeptide units is crucial, and replacement by an oxoether bridge results in a dramatic decrease in activity.

Synthesis and evaluation of electron-rich curcumin analogues.

The natural product curcumin has long been recognized for its medicinal properties and is utilized for the treatment of many diseases. However, it remains unknown whether this activity is based on its presumably promiscuous scaffold, or if it results from the Michael acceptor properties of the alpha,beta-unsaturated 1,3-diketone moiety central to its structure. To probe this issue, electron-rich pyrazole and isoxazole analogues were prepared and evaluated against two breast cancer cell lines, which resulted in the identification of several compounds that exhibit low micromolar to mid nanomolar anti-proliferative activity. A conjugate addition study was also performed to compare the relative electrophilicity of the diketone, pyrazole and isoxazole analogues.

Detection of nonsterol isoprenoids by HPLC-MS/MS.

Isoprenoids constitute an important class of biomolecules that participate in many different cellular processes. Most available detection methods allow the identification of only one or two specific nonsterol isoprenoid intermediates following radioactive or fluorescent labeling. We here report a rapid, nonradioactive, and sensitive procedure for the simultaneous detection and quantification of the eight main nonsterol intermediates of the isoprenoid biosynthesis pathway by means of tandem mass spectrometry. Intermediates were analyzed by HPLC-MS/MS in the multiple reaction monitoring mode using a silica-based C(18) HPLC column. For quantification, their stable isotope-labeled analogs were used as internal standards. HepG2 cells were used to validate the method. Mevalonate, phosphomevalonate, and the six subsequent isoprenoid pyrophosphates were readily determined with detection limits ranging from 0.03 to 1.0mumol/L. The intra- and interassay variations for HepG2 cell homogenates supplemented with isoprenoid intermediates were 3.6-10.9 and 4.4-11.9%, respectively. Under normal culturing conditions, isoprenoid intermediates in HepG2 cells were below detection limits. However, incubation of the cells with pamidronate, an inhibitor of farnesyl pyrophosphate synthase, resulted in increased levels of mevalonate, isopentenyl pyrophosphate/dimethylallyl pyrophosphate, and geranyl pyrophosphate. This method will be suitable for measuring profiles of isoprenoid intermediates in cells with compromised isoprenoid biosynthesis and for determining the specificity of potential inhibitors of the pathway.