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Patients with celiac disease continue to be exposed to gluten despite efforts to maintain a gluten-free diet (GFD). Gluten exposure in those with celiac disease leads to pathological changes in the small intestine that may or may not be associated with gastrointestinal distress. While several studies have investigated a GFD, little is known about sources of gluten contamination that prevent proper maintenance of such a diet by celiac patients. In this study, we investigate common food practices that could lead to gluten cross-contact. Three different practices were examined for gluten cross-contact: gluten-free foods fried in a fryer also used for gluten containing foods, gluten-free bread toasted in a toaster also used for gluten-containing bread, and popular sandwich spreads applied with a knife used on gluten-containing bread (mayonnaise, jam, and peanut butter). We used the ALLER-TEK™ Gluten ELISA test kit and the sandwich ELISA RIDASCREEN Gliadin test kit, which is endorsed for determination of gluten content and used for the evaluation of food cross-contact. Using both kits gave the advantage of using the 401.2 antibody as well as the better established R5 antibody, providing increased confidence in our results. We found these practices resulted in small amounts of gluten cross-contact, although the majority of the results (93.6%) showed no significant cross-contact. Mayonnaise and peanut butter samples were contaminated with gluten above the limit designated by the FDA as gluten-free20 kg/mg (ppm).

In this research, we explored various immobilized enzyme support materials, including the novel nylon-6 fiber membrane (NFM), and evaluated the increase in surface area and its effect on enzyme binding potential. We also manipulated incubation and reaction conditions and assessed the subsequent effects on activity and stability of β-galactosidase, with comparisons between various solid support materials and free (dissolved) enzyme. Nylon-6 fiber membranes were created by electrospinning and were compared with other materials as solid supports for enzyme binding. The other materials included polyvinylidene fluoride 5-kDa nanofiltration dairy membranes, nylon-6 pellets, and silica glass beads. Scanning electron microscopy revealed the large surface area of NFM, which correlated with greater enzyme activity compared with the relatively flatter surfaces of the other solid support materials. Enzyme activity was measured spectrophotometrically with the color-changing substrate o-nitrophenyl-β-d-galactopyranoside. Compared with the other solid supports, NFM had greater maximum enzyme binding potential. Across pH conditions ranging from 3.5 to 6.0 (including the optimal pH of 4.0-5.0), enzyme activity was maintained on the membrane-immobilized samples, whereas free enzyme did not maintain activity. Altering the storage temperature (4, 22, and 50°C) affected enzyme stability (i.e., the ability of the enzyme to maintain activity over time) of free and polyvinylidene fluoride membrane samples. However, NFM samples maintained stability across the varying storage temperatures. Increasing the immobilization solution enzyme concentration above the maximum enzyme binding capacity had no significant effect on enzyme stability for membrane-immobilized samples; however, both had lower mean stability than free enzyme by approximately 74%. With further development, β-galactosidase immobilized on NFM or other membranes could be used in continuous processing in the dairy industry for a combination of filtration and lactose hydrolysis-creating products that are reduced in lactose and increased in sweetness, with no requirement for "added sugars" on the nutrition label and no enzyme listed as final product ingredient.

Invertase catalyzes the hydrolysis of sucrose to glucose and fructose. Invertase from Saccharomyces cerevisiae has been used in studies of the enzyme catalytic mechanism for more than a century. Is...

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: &gamma, tocotrienol (&gamma, T3), &alpha, tocopherol ether acetate (&alpha, 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 &micro, M &gamma, T3, 30 &micro, M &alpha, TEA, and 25 nm DOC was most effective in the treatment of PC-3 cells. We also found that the combination of &gamma, T3 and &alpha, 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.

Combining chemotherapeutics to treat malignant tumors has been shown to be effective in preventing drug resistance, tumor recurrence, and reducing tumor size. We modeled combination drug therapy in PC-3 human prostate cancer cells using mixture design response surface methodology (MDRSM), a statistical technique designed to optimize compositions that we applied in a novel manner to design combinations of chemotherapeutics. Conventional chemotherapeutics (mitoxantrone, cabazitaxel, and docetaxel) and natural bioactive compounds (resveratrol, piperlongumine, and flavopiridol) were used in 12 different combinations containing three drugs at varying concentrations. Cell viability and cell cycle data were collected and used to plot response surfaces in MDRSM that identified the most effective concentrations of each drug in combination. MDRSM allows for extrapolation of data from three or more compounds in variable ratio combinations, unlike the Chou-Talalay method. MDRSM combinations were compared with combination index data from the Chou-Talalay method and were found to coincide. We propose MDRSM as an effective tool in devising combination treatments that can improve treatment effectiveness and increase treatment personalization, because MDRSM measures effectiveness rather than synergism, potentiation, or antagonism.

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. 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.

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., Highlights • Resveratrol (RES) derivatives increase [Ca2+]i more than RES in PC-3 cancer cells. • RES derivatives induce ER Ca2+ release to elevate [Ca2+]i. • RES derivatives inhibit plasma membrane Ca2+-ATPase to elevate [Ca2+]i. • RES derivatives decrease cell viability more than RES in PC-3 cancer cells.

This work demonstrates a new method for measuring the stability of enzyme activity by isothermal titration calorimetry (ITC). The peak heat rate observed after a single injection of the substrate solution into an enzyme solution is correlated with enzyme activity. Multiple injections of the substrate into the same enzyme solution over time show the loss of enzyme activity. The assay is autonomous, requiring very little personnel time, and is applicable to most media and enzymes.

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 treatmen...

Plasma membrane Ca2+-ATPase (PMCA) plays a vital role in maintaining cytosolic calcium concentration ([Ca2+]i). Given that many diseases have modified PMCA expression and activity, PMCA is an important potential target for therapeutic treatment. This study demonstrates that the non-toxic, naturally-occurring polyphenol resveratrol (RES) induces increases in [Ca2+]i via PMCA inhibition in primary dermal fibroblasts and MDA-MB-231 breast cancer cells. Our results also illustrate that RES and the fluorescent intracellular calcium indicator Fura-2, are compatible for simultaneous use, in contrast to previous studies, which indicated that RES modulates the Fura-2 fluorescence independent of calcium concentration. Because RES has been identified as a PMCA inhibitor, further studies may be conducted to develop more specific PMCA inhibitors from RES derivatives for potential therapeutic use.

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 cytoprotective effects of pigment epithelium-derived factor (PEDF) require interactions between an as of a yet undefined region with a distinct ectodomain on the PEDF receptor (PEDF-R). Here we characterized the area in PEDF that interacts with PEDF-R to promote photoreceptor survival. Molecular docking studies suggested that the ligand binding site of PEDF-R interacts with the neurotrophic region of PEDF (44-mer, positions 78-121). Binding assays demonstrated that PEDF-R bound the 44-mer peptide. Moreover, peptide P1 from the PEDF-R ectodomain had affinity for the 44-mer and a shorter fragment within it, 17-mer (positions 98-114). Single residue substitutions to alanine along the 17-mer sequence were designed and tested for binding and biological activity. Altered 17-mer[R99A] did not bind to the P1 peptide, whereas 17-mer[H105A] had higher affinity than the unmodified 17-mer. Peptides 17-mer, 17-mer[H105A], and 44-mer exhibited cytoprotective effects in cultured retina R28 cells. Intravitreal injections of these peptides and PEDF in the rd1 mouse model of retinal degeneration decreased the numbers of dying photoreceptors, 17-mer[H105A] being most effective. The blocking peptide P1 hindered their protective effects both in retina cells and in vivo. Thus, in addition to demonstrating that the region composed of positions 98-114 of PEDF contains critical residues for PEDF-R interaction that mediates survival effects, the findings reveal distinct small PEDF fragments with neurotrophic effects on photoreceptors.

The extracellular pigment epithelium-derived factor (PEDF) displays retina survival activity by interacting with receptor proteins on cell surfaces. We have previously reported that PEDF binds and stimulates PEDF receptor (PEDF-R), a transmembrane phospholipase. However, the PEDF binding site of PEDF-R and its involvement in survival activity have not been identified. The purpose of this work is to identify a biologically relevant ligand-binding site on PEDF-R. PEDF bound the PEDF-R ectodomain L4 (Leu159–Met325) with affinity similar to the full-length PEDF-R (Met1–Leu504). Binding assays using synthetic peptides spanning L4 showed that PEDF selectively bound E5b (Ile193–Leu232) and P1 (Thr210–Leu249) peptides. Recombinant C-terminal truncated PEDF-R4 (Met1–Leu232) and internally truncated PEDF-R and PEDF-R4 (ΔHis203–Leu232) retained phospholipase activity of the full-length PEDF-R. However, PEDF-R polypeptides without the His203–Leu232 region lost the PEDF affinity that stimulated their enzymatic activity. Cell surface labeling showed that PEDF-R is present in the plasma membranes of retina cells. Using siRNA to selectively knock down PEDF-R in retina cells, we demonstrated that PEDF-R is essential for PEDF-mediated cell survival and antiapoptotic activities. Furthermore, preincubation of PEDF with P1 and E5b peptides blocked the PEDF·PEDF-R-mediated retina cell survival activity, implying that peptide binding to PEDF excluded ligand-receptor interactions on the cell surface. Our findings establish that PEDF-R is required for the survival and antiapoptotic effects of PEDF on retina cells and has determinants for PEDF binding within its L4 ectodomain that are critical for enzymatic stimulation. Background: PEDF has neurotrophic activity and interacts with PEDF-R, a membrane-linked lipase. Results: A PEDF-binding region of PEDF-R is required for PEDF-R enzymatic stimulation, and peptides derived from this region block PEDF·PEDF-R-mediated retinal survival activities. Conclusion: A ligand binding domain is identified in PEDF-R, a critical receptor for the survival activity of PEDF. Significance: The findings provide mechanistic insight into the survival activity of PEDF.

Low cancer survival rates and the serious side effects often associated with current chemotherapeutics highlight the need for new and effective nontoxic anticancer agents. Since 1997 when Jang and colleagues first described resveratrol's ability to inhibit carcinogenesis, it has consistently proven effective at tumor inhibition in diverse human cancer models. This finding has raised the hope that resveratrol would pioneer a novel class of nontoxic chemotherapeutics. As a consequence of initial basic and preclinical studies, resveratrol is now being extensively promoted in the unregulated nutraceutical sector. However, some fundamental aspects of resveratrol's action need to be understood before it can be developed into a clinically viable anticancer drug. These areas pertain to the key mechanism(s) by which resveratrol potentiates its antitumor effects. Current research suggests that these mechanisms might be through novel pathways, requiring an understanding of cellular uptake, sentinel targets, and in vivo biological networks. The metabolism of resveratrol and its bioavailablity also warrant further consideration in light of recent in vitro and in vivo studies. Finally, we need to appreciate the sorts of information about resveratrol that may translate between different disease entities. We present a critical discussion of these issues and suggest important experiments that could pave the way to the successful translation of resveratrol to the clinic.Clin Cancer Res; 16(24); 5942–8. ©2010 AACR.

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.

The molybdenum nitrogenase enzyme system, comprised of the MoFe protein and the Fe protein, catalyzes the reduction of atmospheric N(2) to NH(3). Interactions between these two proteins and between Fe protein and nucleotides (MgADP and MgATP) are crucial to catalysis. It is well established that salts are inhibitors of nitrogenase catalysis that target these interactions. However, the implications of salt effects are often overlooked. We have reexamined salt effects in light of a comprehensive framework for nitrogenase interactions to offer an in-depth analysis of the sources of salt inhibition and underlying apparent cooperativity. More importantly, we have identified patterns of salt activation of nitrogenase that correspond to at least two mechanisms. One of these mechanisms is that charge screening of MoFe protein-Fe protein interactions in the nitrogenase complex accelerates the rate of nitrogenase complex dissociation, which is the rate-limiting step of catalysis. This kind of salt activation operates under conditions of high catalytic activity and low salt concentrations that may resemble those found in vivo. While simple kinetic arguments are strong evidence for this kind of salt activation, further confirmation was sought by demonstrating that tight complexes that have previously displayed little or no activity due to the inability of Fe protein to dissociate from the complex are activated by the presence of salt. This occurs for the combination Azotobacter vinelandii MoFe protein with: (a) the L127Delta Fe protein; and (b) Clostridium pasteurianum Fe protein. The curvature of activation vs. salt implies a synergistic salt-protein interaction.

The extracellular pigment epithelium-derived factor (PEDF) displays retina survival activity by interacting with receptor proteins on cell surfaces. We have previously reported that PEDF binds and stimulates PEDF receptor (PEDF-R), a transmembrane phospholipase. However, the PEDF binding site of PEDF-R and its involvement in survival activity have not been identified. The purpose of this work is to identify a biologically relevant ligand-binding site on PEDF-R. PEDF bound the PEDF-R ectodomain L4 (Leu(159)-Met(325)) with affinity similar to the full-length PEDF-R (Met(1)-Leu(504)). Binding assays using synthetic peptides spanning L4 showed that PEDF selectively bound E5b (Ile(193)-Leu(232)) and P1 (Thr(210)-Leu(249)) peptides. Recombinant C-terminal truncated PEDF-R4 (Met(1)-Leu(232)) and internally truncated PEDF-R and PEDF-R4 (ΔHis(203)-Leu(232)) retained phospholipase activity of the full-length PEDF-R. However, PEDF-R polypeptides without the His(203)-Leu(232) region lost the PEDF affinity that stimulated their enzymatic activity. Cell surface labeling showed that PEDF-R is present in the plasma membranes of retina cells. Using siRNA to selectively knock down PEDF-R in retina cells, we demonstrated that PEDF-R is essential for PEDF-mediated cell survival and antiapoptotic activities. Furthermore, preincubation of PEDF with P1 and E5b peptides blocked the PEDF·PEDF-R-mediated retina cell survival activity, implying that peptide binding to PEDF excluded ligand-receptor interactions on the cell surface. Our findings establish that PEDF-R is required for the survival and antiapoptotic effects of PEDF on retina cells and has determinants for PEDF binding within its L4 ectodomain that are critical for enzymatic stimulation.

Resveratrol, a non-toxic 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. In this study we demonstrate that resveratrol alone, and not the glucuronidated or sulfated metabolites, is taken up into tumor cells, induces a rise in [Ca2+]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.

The rate of Fe(3+) release from horse spleen ferritin (HoSF) was measured using the Fe(3+)-specific chelator desferoxamine (DES). The reaction consists of two kinetic phases. The first is a rapid non-linear reaction followed by a slower linear reaction. The overall two-phase reaction was resolved into three kinetic events: 1) a rapid first-order reaction in HoSF (k(1)); 2) a second slower first-order reaction in HoSF (k(2)); and 3) a zero-order slow reaction in HoSF (k(3)). The zero-order reaction was independent of DES concentration. The two first-order reactions had a near zero-order dependence on DES concentration and were independent of pH from 6.8 to 8.2. The two first-order reactions accounted for 6-9 rapidly reacting Fe(3+) ions. Activation energies of 10.5±0.8, 13.5±2.0 and 62.4±2.1kJ/mol were calculated for the kinetic events associated with k(1), k(2), and k(3), respectively. Iron release occurs by: 1) a slow zero-order rate-limiting reaction governed by k(3) and corresponding to the dissociation of Fe(3+) ions from the FeOOH core that bind to an Fe(3+) binding site designated as site 1 (proposed to be within the 3-fold channel); 2) transfer of Fe(3+) from site 1 to site 2 (a second binding site in the 3-fold channel) (k(2)); and 3) rapid iron loss from site 2 to DES (k(1)).