Three-dimensional assembly and disassembly of Fe3O4-decorated porous carbon nanocomposite with enhanced transversal relaxation for magnetic resonance sensing of bisphenol A.

The design and construction of a novel magnetic resonance sensor (MRS) is presented for bisphenol A (BPA) detection. The MRS has been built based on the core component of magnetic Fe3O4 nanoparticles (~ 40 nm), which were uniformly distributed in nanoporous carbon (abbreviated as Fe3O4@NPC). The synthesis was derived from the calcination of the metal organic framework (MOF) precursor of Fe-MIL-101 at high temperature. Fe3O4@NPC was confirmed with enhanced transversal relaxation with r2 value of 118.2 mM-1 s-1, which was around 1.7 times higher than that of the naked Fe3O4 nanoparticle. This enhancement is attributed to the excellent proton transverse relaxation rate of Fe3O4@NPC caused by the reduced self-diffusion coefficient of water molecules in the vicinity of Fe3O4 nanoparticles in the nanoporous carbon. BPA antibody (Ab) and antigen (Ag)-ovalbumin (OVA) were immobilized onto the Fe3O4@NPC to form Ab-Fe3O4@NPC and Ag-Fe3O4@NPC, respectively. These two composites can cause the three-dimensional assembly of Fe3O4@NPC via immunological recognition. The presence of BPA can compete with antigen-OVA to combine with Ab-Fe3O4@NPC, thereby breaking the assembly process (disassembly). The difference in the change of the T2 value before and after adding BPA can thus be used to monitor BPA. The proposed MRS not only revealed a wide linear range of BPA concentration from 0.05 to 50 ng mL-1 with an extremely low detection limit of 0.012 ng mL-1 (S/N = 3), but also displayed high selectivity towards matrix interferences. The recoveries of BPA ranged from 95.6 to 108.4% for spiked tea π, and 93.4 to 104.7% for spiked canned oranges samples, respectively, and the RSD (n = 3) was less than 4.4% for 3 successive assays. The versatility of Fe3O4@NPC with customized relaxation responses provides the possibility for the adaptation of magnetic resonance platforms for food safety development. The magnetic Fe3O4 nanoparticles are uniformly dispersed in the nanoporous carbon (Fe3O4@NPC), which derived from the calcinating of the metal organic framework (MOF) precursor of Fe-MIL-101. And the magnetic Fe3O4@NPCs are adopted for the construction of magnetic resonance sensor (MRS) for bisphenol A (BPA) detection.

Polycaprolactone/silk fibroin electrospun nanofibers-based lateral flow test strip for quick and facile determination of bisphenol A in breast milk.

This study aimed to develop a sensitive lateral flow test strip for the detection of bisphenol A (BPA) in breast milk. Conventional nitrocellulose test membrane was coated with the coaxial nanofiber, consisting of the inner polycaprolactone (PCL) and the outer PCL/silk fibroin (SF) mixture, to decrease the flow rate of the breast milk in the lateral flow assay (LFA). The nanofiber was prepared by using coaxial electrospinning, and BPA antibody was immobilized physically to the nanofiber. This nanofiber was used as a test membrane in the LFA. Color changes on the test membrane were evaluated as the signal intensity of the BPA. Breast milk creates a background on surfaces due to its structural properties. This background was detected by comparing the signal intensity with the signal intensity of water. The higher signal intensity was found in water samples when compared to breast milk samples. Although the detection limit is 2 ng/ml in both coaxial PCL/SF nanofiber and nitrocellulose (NC) test membranes, the color intensity increased with the increasing BPA concentration in the coaxial PCL/SF nanofiber. As a new dimension, the coaxial PCL/SF nanofiber provided higher color intensity than the NC membrane. In conclusion, a sensitive onsite method was developed for the detection of BPA in breast milk by using new coaxial PCL/SF nanofiber as a test membrane in LFA.

A novel method for rapid and quantitative detection of bisphenol A in urine.

Bisphenol A (BPA) is classified as an endocrine disruptor (ED) and it can interact with variety of hormone receptors leading to hormonal disruption and increased risk of various adverse health effects. Reducing human exposure to BPA is one of the main challenges of public health, as it is constantly present in daily life. A low-cost and commonly applied method to enable determination of BPA in the patient's body has yet to be developed. Currently available techniques are expensive, time-consuming, and require access to highly equipped analytical chemistry laboratories. Here we describe a fast and cheap engineered lateral flow assay of our design, to detect of BPA in urine samples. The technology not only provides an opportunity to perform rapid medical diagnostics without the need for an access to the central laboratory but also a means for self-diagnosis by the patient. The addition of ß-glucuronidase improves the sensitivity of detection as it releases the free BPA from glucuronide complexes in urine. This invention may become a demonstrated analytical means for lowering human exposure to BPA and probably also to other EDs and consequently, may be useful in decrease of the risk for several lifestyle diseases.

Plant Phenolics as Pathogen-Carrier Immunogenicity Modulator Haptens.

BACKGROUND: Pathogens use multiple mechanisms to disrupt cell functioning in their host and allow pathogenesis. These mechanisms involve communication between the pathogen and the host cell through protein-protein interactions. METHODS: Protein-protein interactions chains referred to as signal transduction pathways are the processes by which a chemical or physical signal transmits through a cell as series of molecular events so the pathogen needs to intercept these molecular pathways at few positions to induce pathogenesis such as pathogen viability, infection or hypersensitivity. RESULTS: The pathogen nodes of interception are not necessarily the most immunogenic; so that novel immunogenicity-improvement strategies need to be developed thought a chemical conjugation of the pathogen-carrier nodes to develop an efficient immune response in order to block pathogenesis. On the other hand, if pathogen-carriers are immunogens; toleration ought to be induced by this conjugation avoiding hypersensitivity. Thus, this paper addresses the biological plausibility of plant-phenolics as pathogen-carrier immunogenicity modulator haptens. CONCLUSION: The plant-phenolic compounds have in their structure functional groups such as hydroxyl, carbonyl, carboxyl, ester, or ether, capable of reacting with the amino or carbonyl groups of the amino acids of a pathogen-carrier to form conjugates. Besides, the varied carbon structures these phenolic compounds have; it is possible to alter the pathogen-carrier related factors that determine the immunogenicity: 1) Structural complexity, 2) Molecular size, 3) Structural heterogeneity, 4) Accessibility to antigenic determinants or epitopes, 5) Optical configuration, 6) Physical state, or 7) Molecular rigidity.

Environmental Pollution as a Risk Factor in Testicular Tumour Development: Focus on the Interaction between Bisphenol A and the Associated Immune Response.

Bisphenol A (BPA) is an endocrine disruptor to which animals and humans are highly exposed. Many reports have established a relationship between BPA exposure and breast cancer incidence, especially during critical periods of development. However, its effects on the immune response in testicular tumour growth have not yet been described. Thus, we wanted to analyse the effect of perinatal BPA exposure in pregnant female mice and the immune response modulation and tumour growth in an intratesticular cancer model in offspring male mice. Pregnant female mice were exposed to a dose of 250 mg/kg/day/body weight of BPA in their drinking water. In adulthood, male offspring underwent intrascrotal inoculation with 4T1 cancer cells. On day 21 after inoculation, mice were euthanised, and serum was obtained to measure BPA levels using HPLC coupled to mass spectrometry. The percentages of immune cell populations in peripheral lymph nodes (PLN), the spleen and tumours were evaluated by flow cytometry. In addition, the tumour expression of IL-10, TNF-α and TGF-ß was analysed by RT-PCR. Of note, we found detectable circulating levels of BPA in the offspring of mothers exposed to it while pregnant. Remarkably, BPA treatment promoted tumour growth by about 75% compared to mice coming from female mice that did not receive the compound. Perinatal exposure to BPA modulated the percentages of different immune cells in the spleen and PLN. In addition, the expression of inflammatory-related cytokines (IL-10 and TNF-α) in the tumours was significantly enhanced compared to control and vehicle groups. In conclusion, the perinatal BPA administration in pregnant female mice modulated different cellular and molecular immune components that resulted in outstanding testicular tumour size in male offspring.

Bisphenol A modified DNA: A possible immunogenic stimulus for anti-DNA autoantibodies in systemic lupus erythematosus.

Anti-DNA antibodies are now considered as a universal diagnostic feature for the patients with systemic lupus erythematosus (SLE) but the mechanism(s) involved in the generation of these autoantibodies remains to be investigated. Bisphenol A (BPA) is a synthetic phenol extensively used in the manufacturing of polycarbonated plastics. Upon mixing in the diet, it causes several health hazards. This study was undertaken to investigate the contribution of BPA induced DNA damage in SLE patients. Human DNA was modified by BPA in-vitro and the binding characteristics of SLE circulating immunoglobulin Gs (SLE-IgGs) with BPA damaged DNA (BPA-DNA) were screened and compared with the IgGs from normal healthy humans (NH-IgGs). Immunogenicity of BPA-DNA was determined by immunisation in rabbits. DNA from SLE patients (SLE-DNA) or healthy humans (NH-DNA) were isolated and their binding specificity with rabbit anti-BPA-DNA-IgGs was studied. Treatment of human DNA with BPA caused extensive damaged. Circulating SLE-IgGs showed strong recognition of BPA-DNA. BPA-DNA induced high titre antibodies in rabbits. Rabbit anti-BPA-DNA-IgGs showed strong cross reaction with isolated DNA from SLE patients. In short, we concluded that the structural alterations in DNA by BPA, generate neo-epitopes that may be a factor responsible for the induction of anti-DNA autoantibodies in SLE.

Sex-associated protective effect of early bisphenol-A exposure during enteric infection with Trichinella spiralis in mice.

Bisphenol A (BPA) is an endocrine disruptor compound with estrogenic activity, possessing affinity for both nuclear (ERα and ERß) and membrane estrogen receptors. The main source of BPA exposure comes from the contamination of food and water by plastic storage containers or disposable bottles, among others, in which case BPA is easily ingested. Exposure to BPA during early pregnancy leads to lifelong effects; however, its effect on the immune system has not been fully studied. Since endocrine and immune systems interact in a bidirectional manner, the disruption of the former may cause permanent alterations of the latter, thus affecting a future anti-parasitic response. In this study, neonate BALB/c mice were exposed to a single dose of BPA (250 µg/kg); once sexual maturity was reached, they were orally infected with Trichinella spiralis (T. spiralis). The analyses performed after 5 days of infection revealed a decreased parasitic load in the duodenum of mice in the BPA-treated group. Flow cytometry analyses also revealed changes in the immune cell subpopulations of the infected animals when compared to the BPA-treated group. RT-PCR analyses of duodenum samples showed an increased expression of TNF-α, IFN-γ, IL-4, IL-5, and IL-9 in the BPA-treated group. These findings show a new aspect whereby early-life exposure to BPA contributes to the protection against T. spiralis by modulating the anti-parasitic immune response.

A multiepitopic theoretical fusion construct based on in-silico epitope screening of known vaccine candidates for protection against wide range of enterobacterial pathogens.

Enterobacterial pathogens that have acquired antibiotic resistance genes are a leading cause of community and hospital acquired infections. In such a situation vaccination is considered as a better option to prevent such infections. In the current study reverse vaccinology approach has been used to select peptides from already known immunogenic proteins to design a chimeric construct. We selected Yersiniabactin receptor of Escherichia coli UMN026 and Flagellin of Stenotrophomonas maltophila. B-cell linear epitopes were predicted using Bepipred prediction tool. Peptide binding with reference sets of 27 alleles of MHC class I and class II was also analyzed. The predicted peptides-MHC complexes were further validated using simulation dynamics. The in-silico construction of chimera was done by restriction mapping and codon optimization. Chimera was evaluated using the immunoinformatic approach as done for the selected proteins. From the 673 amino acids of FyuA protein, a region from 1 to 492 was selected for containing more linear epitopes and the processing scores obtained were significant for MHC class I and class II binding. Similarly, from Flagellin, a region between 60 and 328 amino acids was selected and the peptides present in the selected region showed lower percentile ranks for binding with MHC molecules. The simulation studies validated the predictions of peptide-MHC complexes. The selected gene fragments accommodating maximum part of these peptides were used to design a chimaeric construct of 2454 bp. From the immunoinformatic analysis, the chimera was found to be more immunogenic in terms of increased number of B-cell and T-cell epitopes along with increased coverage of global populations with allelic variability.

Development of dual-color total internal reflection fluorescence biosensor for simultaneous quantitation of two small molecules and their affinity constants with antibodies.

A novel dual-color total internal reflection fluorescence biosensor (DTB) was successfully developed for the simultaneous detection of two small molecules based on a simple optical structure and the time resolved effect of fiber optic switch. The DTB employed a single-multi mode fiber optic coupler instead of a sophisticated confocal optical system for the transmission of two excitation lights and dual-color fluorescence, and a photodiode detector instead of photomultiplier for the simultaneous detection of dual-color fluorescence. The compact optical design of DTB improved its optical transmission efficiency and detection sensitivity because of no requirement of numerous optical separation elements and rigorous optical alignment. The DTB was applied for the simultaneous detection of 2,4-Bisphenol-A (BPA) and 2,4-Dichlorophenoxyacetic acid (2,4-D) using one bifunctional fiber optic bio-probe modified by two hapten-protein conjugates. When the mixture of Cy5.5 labeled anti-2,4-D antibody and Pacific Blue dye labeled anti-BPA antibody was introduced over the surface of the bio-probe, they bound with their respective hapten-protein conjugate immobilized onto the bio-probe. Based on the time-resolved effect of fiber optic switch, two fluorescence dyes were alternatively excited by 635 nm and 405 nm laser lights and simultaneously detected by one photodiode detector. Taking indirect competitive immunoassay principle, BPA and 2,4-D were simultaneously detected using the DTB with high sensitivity, accuracy, and rapidity. The quantitation of affinity constants between small molecules and their antibodies was also achieved based on the proposed theory. The DTB provides a flexible and powerful platform for simultaneously sensitive quantitation of multiple targets and the affinity constants of biomolecular interactions.

A novel electrochemical immunosensor based on catalase functionalized AuNPs-loaded self-assembled polymer nanospheres for ultrasensitive detection of tetrabromobisphenol A bis(2-hydroxyethyl) ether.

A competitive immunosensor was established using an electrochemical amperometric strategy for sensitive detection of tetrabromobisphenol A bis(2-hydroxyethyl) ether (TBBPA-DHEE), an important derivative of tetrabromobisphenol A (TBBPA). In this system, the amplified electrochemical signal towards the reduction of hydrogen peroxide (H2O2) was recorded by amperometric method. Meanwhile, the synthetized catalase functionalized AuNPs-loaded self-assembled polymer nanospheres showed an excellent electrocatalytic ability to catalyse H2O2, which was beneficial for strengthening the electrochemical signals. Under the optimized conditions, this method displayed: (i) low detection limits (0.12 ng/mL, 7 times lower than the traditional ELISA with the same antibody); (ii) satisfactory accuracy (recoveries, 78-124%; RSD, 2.1-8.3%) and good agreement with the corresponding ELISA; (iii) low sample consumption (6 µL) and low cost. The proposed approach was applied for investigation of TBBPA-DHEE from environmental waters, and our results indicated that this immunosensor has great potential to detect the trace pollutants in aquatic environments.

Bisphenol A: A notorious player in the mosaic of autoimmunity.

The multifactorial etiology of autoimmune diseases has been studied at large. Genetic risk factors and environmental agents play an integral role in the pathogenesis of autoimmune processes. In recent decades, Bisphenol A (BPA), an exogenous compound found in polycarbonate plastic, has gained attention for its harmful multifocal effects on a diverse subset of systemic pathways, potentially contributing to disease onset and exacerbation. BPA is a xenoestrogen used globally in the manufacture of daily use products including plastic storage containers, water and infant bottles, and food and drink packaging. BPA exhibits immune stimulatory activity bringing into question the association between its greater global presence and the increased prevalence of autoimmune diseases. The purpose of this multi-study analysis is to assess recent research investigating the underlying role of BPA in autoreactive mechanisms. Although research at present does not directly link BPA exposure to the development of autoimmune diseases, a large body of evidence supports the pro-inflammatory effects of BPA on the immune system. Further studies are required to elucidate the role of BPA in autoimmune pathogenesis, however caution should be taken in the use of BPA containing products by those affected or genetically susceptible to developing autoimmune diseases.

Alterations in human neutrophil function caused by bisphenol A.

Bisphenol A (BPA) is a synthetic, organic compound frequently present in consumer plastics, including plastic-lined cans, water bottles, toys, and teeth sutures. Previous studies have shown that BPA can produce adverse health effects that include defects in reproductive function and altered prenatal/childhood development. However, little is known regarding the effects of BPA on immune function. In this study, we assessed the effect of BPA on human neutrophils, a critical component of the innate immune system's defense against pathogens. We found that BPA induces a concentration-dependent increase in reactive oxygen species (ROS) generation by neutrophils, which is inhibited by the estrogen receptor-ß antagonist PHTPP. Furthermore, incubation with the membrane-permeable calcium chelator BAPTA-AM and/or removal of extracellular calcium inhibited BPA-induced ROS production, indicating that the process is calcium dependent. Transwell chemotaxis assays revealed that BPA exposure reduces the chemotactic capacity of neutrophils in a gradient of the bacterial cell wall component f-Met-Leu-Phe, a potent chemoattractant. Exposure to BPA also inhibits the ability of neutrophils to kill methicillin-resistant Staphylococcus aureus, a leading human pathogen. Our findings reveal that BPA alters the in vitro function of neutrophils, including ROS production, chemotaxis, and bacterial killing, and raises the possibility of altered innate immunity in vivo, especially in those with compromised immune function and who can be exposed to BPA in a wide variety of products.

Universal and reusable hapten/antibody-mediated portable optofluidic immunosensing platform for rapid on-site detection of pathogens.

A universal and reusable hapten-antibody-mediated portable optofluidic immunosensing platform (OIP) was developed for rapid on-site detection of pathogens. By using Escherichia coli O157:H7 (E. coli O157:H7) and bisphenol A-Bovine serum albumin (BPA-BSA)/anti-BPA antibody as a model pathogen and a mediated hapten-antibody, respectively, a novel immunoassay mechanism was proposed to detect pathogens. The BPA-BSA-modified immunosensor and E. coli O157:H7 were initially saturated with anti-BPA antibodies (mouse IgG) and anti-E. coli O157:H7 antibodies (mouse IgG), respectively. Then, the fluorescence-labeled secondary antibodies (goat anti-mouse IgG antibody) were incubated with E. coli O157:H7 with their antibodies. Next, the mixture was introduced into the immunosensor surface bound to the anti-BPA antibodies. A high concentration of E. coli O157:H7 in the sample reduced the number of fluorescence-labeled secondary antibodies bound to the immunosensor surface, thus resulting in the detection of low fluorescence signals. Under optimized conditions, the hapten-antibody-mediated OIP system exhibited a detection limit of 8 cfu/mL E. coli O157:H7 after concentrating 100 times by using centrifugation, and a test cycle, including prereaction, detection, and regeneration, was less than 1 h. The robustness of the hapten-carrier protein-modified immunosensor surface allowed multiple pathogen immunoassays. The proposed strategy demonstrated good recovery, precision, and accuracy through the evaluation of the spiked water samples. We expect that the new platform can be readily used for the detection of other pathogens in a variety of application fields ranging from environmental monitoring and food safety to medical diagnosis.

Metabolomics Analysis To Evaluate the Anti-Inflammatory Effects of Polyphenols: Glabridin Reversed Metabolism Change Caused by LPS in RAW 264.7 Cells.

Inflammation has been shown to play a critical role in the development of many diseases. In this study, we used metabolomics to evaluate the inflammatory effect of lipopolysaccharide (LPS) and the anti-inflammatory effect of glabridin (GB, a polyphenol from Glycurrhiza glabra L. roots) in RAW 264.7 cells. Multivariate statistical analysis showed that in comparison with the LPS group, the metabolic profile of the GB group was more similar to that of the control group. LPS impacted the amino acid, energy, and lipid metabolisms in RAW 264.7 cells, and metabolic pathway analysis showed that GB reversed some of those LPS impacts. Metabolomics analysis provided us with a new perspective to better understand the inflammatory response and the anti-inflammatory effects of GB. Metabolic pathway analysis can be an effective tool to elucidate the mechanism of inflammation and to potentially find new anti-inflammatory agents.

Correlation between antibodies to bisphenol A, its target enzyme protein disulfide isomerase and antibodies to neuron-specific antigens.

Evidence continues to increase linking autoimmunity and other complex diseases to the chemicals commonly found in our environment. Bisphenol A (BPA) is a synthetic monomer used widely in many forms, from food containers to toys, medical products and many others. The potential for BPA to participate as a triggering agent for autoimmune diseases is likely due to its known immunological influences. The goal of this research was to determine if immune reactivity to BPA has any correlation with neurological antibodies. BPA binds to a target enzyme called protein disulfide isomerase (PDI). Myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) are neuronal antigens that are target sites for neuroinflammation and neuroautoimmunity. We determined the co-occurrence of anti-MBP and anti-MOG antibodies with antibodies made against BPA bound to human serum albumin in 100 healthy human subjects. Correlation between BPA to PDI, BPA to MOG, BPA to MBP, PDI to MBP and PDI to MOG were all highly statistically significant (P < 0.0001). The outcome of our study suggests that immune reactivity to BPA-human serum albumin and PDI has a high degree of statistical significance with substantial correlation with both MBP and MOG antibody levels. This suggests that BPA may be a trigger for the production of antibodies against PDI, MBP and MOG. Immune reactivity to BPA bound to human tissue proteins may be a contributing factor to neurological autoimmune disorders. Further research is needed to determine the exact relationship of these antibodies with neuroautoimmunities. Copyright © 2016 The Authors Journal of Applied Toxicology Published by John Wiley & Sons Ltd.

The Association of Serum Bisphenol A with Thyroid Autoimmunity.

Introduction: Data on the association of bisphenol A (BPA) exposure and autoimmunity in humans is unclear. Objective: To elucidate the influence of BPA on thyroid autoimmunity, in the present study we assessed the association between serum BPA and thyroid autoantibodies. Methods: Serum samples from 2361 subjects, aged ≥15 years, from the Thai 4th National Health Examination Survey were measured for BPA, antithyroglobulin (TgAb), antithyroperoxidase (TPOAb) and antithyrotrophin receptor (TRAb) antibodies. Results: The proportion of subjects positive for TgAb, TPOAb and TRAb were 11.1%, 14.9% and 1.9%, respectively. With regard to BPA, 51.9% had serum BPA levels exceeding the detection limit of the assay (0.3). There was a significant increasing trend for subjects with TgAb (p < 0.05) and TPOAb (p < 0.001) positivity as BPA quartiles increased, particularly in the highest quartile. In contrast, no relationship between BPA quartiles and TRAb was found. Logistic regression analysis showed that age, gender and BPA quartiles were determinants of TPOAb or TgAb positivity, independent of BMI. However, only the association between BPA and TPOAb positivity was consistent in both men and women. Conclusions: BPA was independently associated with TPOAb positivity. However, its mechanism related to TPOAb positivity, subsequently leading to autoimmune thyroid disease, needs further investigation.

A novel and label-free immunosensor for bisphenol A using rutin as the redox probe.

In this work, a new and label-free electrochemical immunosensor for sensitive detection of bisphenol A was reported. MWCNTs and gold nanoparticles (AuNPs) were modified on glassy carbon electrode surface to enhance current response. The Anti-BPA was immobilized on the modified electrode through AuNPs. Rutin was used for the first time as the redox probe to construct electrochemical immunosensor of bisphenol A. The peak current change due to the specific immuno-interaction between anti-BPA and BPA on the modified electrode surface was utilized to detect bisphenol A. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) were employed to trace the assembly process of the electrochemical immunosensor. Experimental factors affecting the sensitivity of the immunosensor were examined in terms of incubation time and pH of phosphate buffer solution (PBS). Under optimized conditions, the linear range of calibration curve based on the relationship between current response and BPA concentration was from 1.0×10(-8)-1.0×10(-6)M with detection limit of 8.7×10(-9)M (S/N=3). The proposed immunosensor showed good reproducibility, selectivity, stability and was successfully applied to the determination of BPA in real sample.

In vitro effect of 4-pentylphenol and 3-methyl-4-nitrophenol on murine splenic lymphocyte populations and cytokine/granzyme production.

Gasoline exhaust particles (GEP) and diesel exhaust particles (DEP) are considered to be some of the most important air pollutants. Among the many constituents in these pollutant particles, 4-pentylphenol (PP) and 3-methyl-4-nitrophenol (PNMC) are considered important phenolics in GEP and DEP, respectively. The aim of this study was to investigate the effect of in vitro exposure to commercially-supplied PP and PNMC on populations of, and production of interleukin (IL)-2, IL-4 and granzyme-B by, mouse splenic lymphocytes. After in vitro exposure to PP or PNMC for 48 h, splenocyte viability was measured, cell phenotypes, e.g. B-cell (CD19), T-cells (CD3), T-cell subsets (CD4 and CD8), were quantified by flow cytometry and production of IL-2, IL-4 and granzyme-B was assessed via ELISA. The oxidative toxicity of PP and PNMC toward the splenocytes was also evaluated using measures of hydroxyl radical and malondiadehyde production and changes in glutathione peroxidase and superoxide dismutase activities. Results showed that in vitro exposure to PP and PNMC inhibited splenic cell parameters in a dose-related manner. Exposure to PP and PNMC decreased splenic T-lymphocyte populations and splenocyte production of cytokines and granzyme B, as well as induced oxidative stress in the splenocytes. The results also showed that the percentages of CD3(+) T-cells overall and of CD4(+) and CD8(+) T-cells therein, among exposed splenocytes, were reduced; neither compound appeared to affect levels of CD19(+) B-cells. Overall, the suppressive effects of PP were stronger than PNMC. The data here provide support for the proposal that PP-/PNMC-induced toxicity in splenocytes may be due at least in part to oxidative damage and that PP and PNMC - as components of GEP and DEP - might significantly impact on splenic T-cell formation/release of cytokines/granzymes in situ.

Secondary metabolites in plants: transport and self-tolerance mechanisms.

Plants produce a host of secondary metabolites with a wide range of biological activities, including potential toxicity to eukaryotic cells. Plants generally manage these compounds by transport to the apoplast or specific organelles such as the vacuole, or other self-tolerance mechanisms. For efficient production of such bioactive compounds in plants or microbes, transport and self-tolerance mechanisms should function cooperatively with the corresponding biosynthetic enzymes. Intensive studies have identified and characterized the proteins responsible for transport and self-tolerance. In particular, many transporters have been isolated and their physiological functions have been proposed. This review describes recent progress in studies of transport and self-tolerance and provides an updated inventory of transporters according to their substrates. Application of such knowledge to synthetic biology might enable efficient production of valuable secondary metabolites in the future.