Reaction of KHP with excess NaOH or TRIS as standard reactions for calibration of titration calorimeters from 0 to 60 °C.

Calibration of titration calorimeters is an ongoing problem, particularly with calorimeters with reaction vessel volumes < 10 mL in which an electrical calibration heater is positioned outside the calorimetric vessel. Consequently, a chemical reaction with a known enthalpy change must be used to accurately calibrate these calorimeters. This work proposes the use of standard solutions of potassium acid phthalate (KHP) titrated into solutions of excess sodium hydroxide (NaOH) or excess tris(hydroxymethyl)aminomethane (TRIS) as standard reactions to determine the collective accuracy of the relevant variables in a determination of the molar enthalpy change for a reaction. KHP is readily available in high purity, weighable for easy preparation of solutions with accurately known concentrations, stable in solution, not compromised by side reactions with common contaminants such as atmospheric CO2, and non-corrosive to materials used in calorimeter construction. Molar enthalpy changes for these reactions were calculated from 0 to 60 °C from reliable literature data for the pKa of KHP, the molar enthalpy change for protonation of TRIS, and the molar enthalpy change for ionization of water. The feasibility of using these reactions as enthalpic standards was tested in several calorimeters; a 50 mL CSC 4300, a 185 µL NanoITC, a 1.4 mL VP-ITC, and a TAM III with 1 mL reaction vessels. The results from the 50 mL CSC 4300, which was accurately calibrated with an electric heater, verified the accuracy of the calculated standard values for the molar enthalpy changes of the proposed reactions.

Thermal characterization and separation of whey proteins by differential scanning calorimetry.

Most commercially available whey products contain a mixture of 6-7 whey proteins; however, there is an increased focus on using the individual whey proteins for their unique biological activities. Before extracting individual whey proteins for use, it is important to quantify how much of a particular protein is present in whey mixtures as well as if the protein is still structurally folded. We first characterized the denaturation temperature and enthalpy values for the six purified whey proteins at six pHs (3-9) and under ion chelation using a nano-differential scanning calorimeter (DSC). From the individual protein scans, we determined the optimal condition for detecting all 6 proteins on a single DSC scan was whey in an EDTA MOPs pH 6.7 buffer.

Metabolomics of acid whey derived from Greek yogurt.

Acid whey, a byproduct of Greek yogurt production, has little commercial value due to its low protein content and is also environmentally harmful when disposed of as waste. However, as a product of microbial fermentation, acid whey could be a rich source of beneficial metabolites associated with fermented foods. This study increases understanding of acid whey composition by providing a complete metabolomic profile of acid whey. Commercial and laboratory-made Greek yogurts, prepared with 3 different bacterial culture combinations, were evaluated. Samples of uncultured milk and cultured whey from each batch were analyzed. Ultra-high-performance liquid chromatography-tandem mass spectrometry metabolomics were used to separate and identify 477 metabolites. Compared with uncultured controls, acid whey from fermented yogurt showed decreases in some metabolites and increases in others, presumably due to the effects of microbial metabolism. Additional metabolites appeared in yogurt whey but not in the uncultured control. Therefore, the effect of microbial fermentation is complex, leading to increases or decreases in potentially bioactive bovine metabolites while generating new microbial compounds that may be beneficial. Metabolite production was significantly affected by combinations of culturing organisms and production location. Differences between laboratory-made and commercial samples could be caused by different starting ingredients, environmental factors, or both.

γ-Tocotrienol and α-Tocopherol Ether Acetate Enhance Docetaxel Activity in Drug-Resistant Prostate Cancer Cells.

Prostate cancer is the second most commonly diagnosed cancer in men, and metastatic prostate cancer is currently incurable. Prostate cancer frequently becomes resistant to standard of care treatments, and the administration of chemotherapeutic drugs is often accompanied by toxic side effects. Combination therapy is one tool that can be used to combat therapeutic resistance and drug toxicity. Vitamin E (VE) compounds and analogs have been proposed as potential non-toxic chemotherapeutics. Here we modeled combination therapy using mixture design response surface methodology (MDRSM), a statistical technique designed to optimize mixture compositions, to determine whether combinations of three chemotherapeutic agents: γ-tocotrienol (γ-T3), α-tocopherol ether acetate (α-TEA), and docetaxel (DOC), would prove more effective than docetaxel alone in the treatment of human prostate cancer cells. Response surfaces were generated for cell viability, and the optimal treatment combination for reducing cell viability was calculated. We found that a combination of 20 µM γ-T3, 30 µM α-TEA, and 25 nm DOC was most effective in the treatment of PC-3 cells. We also found that the combination of γ-T3 and α-TEA with DOC decreased the amount of DOC required to reduce cell viability in PC-3 cells and ameliorated therapeutic resistance in DOC-resistant PC-3 cells.

Resveratrol derivatives increase cytosolic calcium by inhibiting plasma membrane ATPase and inducing calcium release from the endoplasmic reticulum in prostate cancer cells.

Resveratrol (RES) is a putative chemotherapeutic naturally found in grapes, peanuts, and Japanese knotweed. Previous studies demonstrate that RES modulates calcium signaling as part of its chemotherapeutic activity. In this study, we determined the chemotherapeutic activity of three RES esters that have been modified at the 4' hydroxyl by the addition of pivalate, butyrate, and isobutyrate. All of the RES derivatives disrupted the calcium signaling in prostate cancer cells more than the parent compound, RES. Further, we demonstrate that the RES derivatives may disrupt the calcium homeostasis by activating calcium release from the endoplasmic reticulum and inhibiting plasma membrane Ca2+-ATPase. The pivalated and butyrated RES derivatives decreased cell viability significantly more than RES. Because pivalated and butyrated RES are more effective than RES at targeting calcium signaling pathways, pivalated and butyrated RES may serve as more effective chemotherapeutics.

Calorimetric Methods for Measuring Stability and Reusability of Membrane Immobilized Enzymes.

The aim of this work is to develop calorimetric methods for characterizing the activity and stability of membrane immobilized enzymes. Invertase immobilized on a nylon-6 nanofiber membrane is used as a test case. The stability of both immobilized and free invertase activity was measured by spectrophotometry and isothermal titration calorimetry (ITC). Differential scanning calorimetry was used to measure the thermal stability of the structure and areal concentration of invertase on the membrane. This is the 1st demonstration that ITC can be used to determine activity and stability of an enzyme immobilized on a membrane. ITC and spectrophotometry show maximum activity of free and immobilized invertase at pH 4.5 and 45 to 55 °C. ITC determination of the activity as a function of temperature over an 8-h period shows a similar decline of activity of both free and immobilized invertase at 55 °C. PRACTICAL APPLICATION: Enzyme-catalyzed reactions occur in mild and environmentally friendly conditions, but are usually too costly to use in food manufacturing. When free enzymes are used, they are used once and replaced for each reaction, but enzymes immobilized on a solid support can be reused and have the additional advantage of being removed from the product. In this study, new calorimetric methods that are universally applicable to characterizing immobilized enzymes are used to determine the activity, stability, and reusability of invertase immobilized on a nanofiber support.

The Effects of 4'-Esterified Resveratrol Derivatives on Calcium Dynamics in Breast Cancer Cells.

Triple-negative breast cancer is a highly aggressive subtype of breast cancer. Frequently, breast cancer cells modulate their calcium signaling pathways to optimize growth. Unique calcium pathways in breast cancer cells could serve as a way to target tumorigenic cells without affecting normal tissue. Resveratrol has previously been shown to activate calcium signaling pathways. We use cell viability, single-cell calcium microscopy, and RT-PCR assays to determine the activity and mechanism of three different 4'-esterified resveratrol derivatives. We demonstrate that two of the derivatives reduce cell viability more effectively than resveratrol in MDA-MB-231 human breast cancer cells. The derivatives also activate similar pro-apoptotic calcium signaling pathways. In particular, the pivalated and butyrated resveratrol derivatives are intriguing putative chemotherapeutics because they are more effective at decreasing cell viability in vitro and inhibiting the plasma membrane Ca2+-ATPase, a protein that is often modulated in breast cancer.

Natural products induce a G protein-mediated calcium pathway activating p53 in cancer cells.

Paclitaxel, etoposide, vincristine and doxorubicin are examples of natural products being used as chemotherapeutics but with adverse side effects that limit their therapeutic window. Natural products derived from plants and having low toxicity, such as quercetin, resveratrol, epigallocatechin gallate and piceatannol, have been shown to inhibit tumor cell growth both in vitro and in pre-clinical models of cancer, but their mechanisms of action have not been fully elucidated, thus restricting their use as prototypes for developing synthetic analogs with improved anti-cancer properties. We and others have demonstrated that one of the earliest and consistent events upon exposure of tumor cells to these less toxic natural products is a rise in cytoplasmic calcium, activating several pro-apoptotic pathways. We describe here a G protein/inositol 1,4,5-trisphosphate pathway (InsP3) in MDA-MB-231 human breast cancer cells that mediates between these less toxic natural products and the release of calcium from the endoplasmic reticulum. Further, we demonstrate that this elevation of intracellular calcium modulates p53 activity and the subsequent transcription of several pro-apoptotic genes encoding PIG8, CD95, PIDD, TP53INP, RRM2B, Noxa, p21 and PUMA. We conclude from our findings that less toxic natural products likely bind to a G protein coupled receptor that activates a G protein-mediated and calcium-dependent pathway resulting selectively in tumor cell death.

The sustained delivery of resveratrol or a defined grape powder inhibits new blood vessel formation in a mouse model of choroidal neovascularization.

The objective of this study was to determine whether resveratrol or a defined, reconstituted grape powder can attenuate the formation of new blood vessels in a mouse model of choroidal neovascularization (CNV). To accomplish this objective, C57BL/6J mice were randomized into control or treatment groups which received either resveratrol or grape powder by daily oral gavage, resveratrol or grape powder delivered ad libitum through the drinking water, or resveratrol by slow release via implanted osmotic pumps. A laser was used to rupture Bruch's membrane to induce CNV which was then detected in sclerochoroidal eyecups stained with antibodies against intercellular adhesion molecule-2. CNV area was measured using fluorescence microscopy and Image J software. Ad libitum delivery of both resveratrol and grape powder was shown to significantly reduce the extent of CNV by 68% and 57%, respectively. Parallel experiments conducted in vitro demonstrated that resveratrol activates p53 and inactivates Akt/protein kinase B in choroidal endothelial cells, contributing to its anti-proliferative and anti-migratory properties. In addition resveratrol was shown to inhibit the formation of endothelial cell networks, augmenting its overall anti-angiogenic effects. The non-toxic nature of resveratrol makes it an especially attractive candidate for the prevention and/or treatment of CNV.

Resveratrol metabolites do not elicit early pro-apoptotic mechanisms in neuroblastoma cells.

Resveratrol, a nontoxic polyphenol, has been shown to inhibit tumor growth in a xenograft mouse model of neuroblasoma. However, resveratrol is rapidly metabolized, mainly to its glucuronidated and sulfated derivatives. This study demonstrates that resveratrol alone, and not the glucuronidated or sulfated metabolites, is taken up into tumor cells, induces a rise in [Ca(2+)](i), and ultimately leads to a decrease in tumor cell viability. A new water-soluble resveratrol formulation was delivered directly at the site of the tumor in a neuroblastoma mouse model. The amount of unmodified resveratrol associated with the tumor increased more than 1000-fold. The increase of unmodified resveratrol associated with the tumor resulted in tumor regression. The number of residual tumor cells that remained viable also decreased as the ratio of the metabolites relative to unmodified resveratrol declined.