R-phycoerythrin Research Articles

Extraction and Purification of R-phycoerythrin from Marine Red Algae.

This chapter focuses on the recovery of an R-Phycoerythrin (R-PE)-enriched fraction from marine algae. Since R-PE is a proteinaceous pigment, we have developed a simple and rapid two-step method devoted to the extraction and purification of R-PE from marine red algae. Here we describe a phosphate buffer extraction followed by anion exchange chromatography carried on a DEAE Sepharose Fast Flow column. To ensure the quality and quantity of R-PE recovery, we also indicate different methods to monitor each fraction obtained, such as spectrophotometric indicators, gel filtration, and SDS-PAGE analysis.

Abstract 5098: GFP compatibility with EdU cell proliferation assay

Abstract Examining cell proliferation in GFP expressing cells is of general interest in many aspects of biology including regenerative medicine, stem cells, developmental biology and some fields of cancer research. However, visualization of GFP expression is not readily compatible with commonly used proliferation assays which incorporate a thymidine analog to directly measure S-phase proliferation of DNA. With the BrdU (bromo-deoxyuridine) assay, an antibody based detection method, alcohol for fixation and hydrochloric acid for DNA denaturation is commonly used. Neither chemical is compatible with GFP fluorescence. For imaging applications there are some methods to avoid the use of HCl as a denaturant with the BrdU assay, however; they are typically “home-brew” methods used to partially digest the DNA, adding extra steps, and not easily performed. The much faster and reliable EdU (ethynyl-deoxyuridine) cell proliferation assay which uses click chemistry for detection of S-phase proliferation of DNA, uses formaldehyde based fixation and avoids the use of HCl for DNA denaturation. However, the use of copper to catalyse the click reaction also negatively affects GFP fluorescence. Anti-GFP antibodies can be used in the click reaction work flow, to “retrieve” the lost GFP fluorescence but are not convenient and add extra steps. We present recent improvements to the click chemistry based EdU cell proliferation assay which minimizes the loss of GFP and other fluorescent proteins signals and avoids the need for “work around” methods. The resulting click reaction is both more rapid and brighter than the “classic” click EdU assay. The modifications preserve most of the GFP fluorescence and permit multiplex detection with EdU with no change in the work flow of the classic click EdU assay. We optimized components in the improved click reaction conditions and tested compatibility with various fluorescent proteins. Examples of click chemistry and GFP/RFP/mCherry compatibility are presented using the EdU cell proliferation assay in both cell culture and in GFP expressing tissue. Additionally, improvements are demonstrated in with other applications of click chemistry assays used for imaging as well as with on flow cytometry platforms where GFP, R-phycoerythrin (R-PE), or other fluorescent proteins are commonly combined with cell proliferation assays. The use of the modified click reaction is an enabling improvement over originally described copper based click reactions and will further enhance the utility of EdU based cell proliferation. Citation Format: Scott T. Clarke, Zhichao Song, Kelvin Y. Kwan, Carolyn DeMarco, Aleksey Rukavishnikov, Upinder Singh, Kyle Gee. GFP compatibility with EdU cell proliferation assay. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5098. doi:10.1158/1538-7445.AM2014-5098

An integrated approach for enhanced protein conjugation and capture with viral nanotemplates and hydrogel microparticle platforms via rapid bioorthogonal reactions.

We demonstrate significantly enhanced protein conjugation and target protein capture capacity by exploiting tobacco mosaic virus (TMV) templates assembled with hydrogel microparticles. Protein conjugation results with a red fluorescent protein R-Phycoerythrin (R-PE) show significantly enhanced protein conjugation capacity of TMV-assembled particles (TMV-particles) compared to planar substrates or hydrogel microparticles. In-depth examination of protein conjugation kinetics via tetrazine (Tz)-trans-cyclooctene (TCO) cycloaddition and strain-promoted alkyne-azide cycloaddition (SPAAC) reaction demonstrates that TMV-particles provide a less hindered environment for protein conjugation. Target protein capture results using an anti-R-PE antibody (R-Ab)-R-PE pair also show substantially improved capture capacity of R-Ab conjugated TMV-particles over R-Ab conjugated hydrogel microparticles. We further demonstrate readily controlled protein and antibody conjugation capacity by simply varying TMV concentrations, which show negligible negative impact of densely assembled TMVs on protein conjugation and capture capacity. Combined, these results illustrate a facile postfabrication protein conjugation approach with TMV templates assembled onto hydrogel microparticles for improved and controlled protein conjugation and sensing platforms. We anticipate that our approach can be readily applied to various protein sensing applications.

Characterization of agarophytic seaweeds from the biorefinery context

The major seaweed components such as natural colorants (R-phycoerythrin (R-PE), R-phycocyanin (R-PC)), minerals, proteins, lipids, cellulose and agar which are of considerable commercial value were estimated in 15 different agarophytic seaweeds. R-PE and R-PC contents ranged from 138.33 ± 17.67 to 1039.43 ± 27.65 μg/g and 50.26 ± 6.63 to 818.2 ± 48 μg/g on fresh weight (fw) basis, respectively. Appreciable amounts of both macro-minerals (K, Na, Ca, Mg) and micro-minerals (Fe, Zn, Se, Mn) were estimated. The total lipid and protein contents were 0.65 ± 0.06% to 1.53 ± 0.07% and 4.75 ± 0.5% to 19.31 ± 3.5% on dry weight (dw) basis respectively while cellulose and agar contents varied from 3.7 ± 0.13% to 12.20 ± 0.45% and 9.17 ± 0.62% to 25.23 ± 0.50% dw, respectively. The overall finding of this study enable the selection and value addition of agarophytic feedstock for biorefinery.

Dielectrophoretic immobilisation of antibodies on microelectrode arrays

A silicon based chip device with a regular array of more than 100,000 cylindrical sub-microelectrodes has been developed for the dielectrophoretic (DEP) manipulation of nanoparticles and molecules in solution. It was fabricated by a standard CMOS (complementary metal oxide semiconductor) compatible process. The distribution of the electrical field gradient was calculated to predict the applicability of the setup. Heating due to field application was determined microscopically using a temperature sensitive fluorescent dye. Depending on voltage and frequency, temperature increase was found to be compatible with protein function. Successful field controlled immobilisation of biomolecules from solution was demonstrated with the autofluorescent protein R-phycoerythrin (RPE) and with fluorescently labelled IgG antibodies. Biological activity after DEP application was proven by immobilisation of an anti-RPE antibody and subsequent binding of RPE. These results demonstrate that the developed chip system allows the directed immobilisation of proteins onto microelectrodes by dielectrophoresis without the need for any chemical modification and that protein function is preserved. Being based on standard lithographical methods, further miniaturisation and on-chip integration of electronics towards a multiparameter single cell analysis system appear near at hand.

A low-cost, high-performance system for fluorescence lateral flow assays.

We demonstrate a fluorescence lateral flow system that has excellent sensitivity and wide dynamic range. The illumination system utilizes an LED, plastic lenses and plastic and colored glass filters for the excitation and emission light. Images are collected on an iPhone 4. Several fluorescent dyes with long Stokes shifts were evaluated for their signal and nonspecific binding in lateral flow. A wide range of values for the ratio of signal to nonspecific binding was found, from 50 for R-phycoerythrin (R-PE) to 0.15 for Brilliant Violet 605. The long Stokes shift of R-PE allowed the use of inexpensive plastic filters rather than costly interference filters to block the LED light. Fluorescence detection with R-PE and absorbance detection with colloidal gold were directly compared in lateral flow using biotinylated bovine serum albumen (BSA) as the analyte. Fluorescence provided linear data over a range of 0.4–4,000 ng/mL with a 1,000-fold signal change while colloidal gold provided non-linear data over a range of 16–4,000 ng/mL with a 10-fold signal change. A comparison using human chorionic gonadotropin (hCG) as the analyte showed a similar advantage in the fluorescent system. We believe our inexpensive yet high-performance platform will be useful for providing quantitative and sensitive detection in a point-of-care setting.

Abstract 470: Development of Control Cells in a Cell-Based Assay for Salt Sensitivity

Salt sensitivity of blood pressure (BP) affects 25% of the world’s population and has a cardiovascular risk that is similar to hypertension, but can be present in normotensive individuals. Salt sensitivity can be detected with a two week controlled diet, which is difficult to administer in the clinical setting. Our previous method of determining an individual’s salt sensitivity status through the use of confocal microscopy is challenging as a clinical assay due to its complexity. We therefore developed a rapid method of diagnosis based on exfoliated renal proximal tubule cells (RPTC) in urine. Our streamlined approach made use of an Accuri™ C6 (BD Biosciences) flow cytometer to measure monensin (MON)-stimulated dopamine-1 receptor (D1R) and angiotensin-2 receptor (AT2R) recruitment to the plasma membrane. In order to control for method-induced variability and biological variation, we developed external controls. Two different immortalized cell lines, i22 (D1R coupled) and i19 (D1R uncoupled) were chosen for their consistent level of response to stimulation by MON, a sodium ionophore. These cell lines were each labeled with D1R or AT2R antibodies directly conjugated to R-Phycoerythrin (RPE) (Ex: 535nm, Em: 575nm) to obtain a basal level of surface receptor. Afterwards, the cells were stimulated with MON (1μM) to elicit a Na+ -induced receptor recruitment response. Finally, the cells were stained with directly labeled D1R or AT2R antibodies conjugated to Allophycocyanin (APC) (Ex: 650 nm, Em: 660 nm), after which they were read on the Accuri. Normally coupled i22 cells showed a significantly higher (p<0.005) recruitment ratio (Stimulated RFU (APC) / Basal RFU (RPE)) for D1R of 0.42 (SEM ± 0.012, n=7) than did uncoupled i19 cells, which showed a recruitment ratio of 0.36 (SEM = ± 0.006, n=7). Normally coupled i22 cells showed a significantly higher (p<0.05) recruitment ratio for AT2R of 0.45 (SEM ± 0.018, n=6) than did uncoupled i19 cells, which showed a recruitment ratio of 0.39 (SEM = ± 0.013, n=6). This controlled cell-based assay will facilitate testing in large cohorts to validate its use as a means to determine each individual’s salt sensitivity index.

Further studies and biological activities of macromolecular protein R-Phycoerythrin from Portieria hornemannii

In the present study, the purified R-Phycoerythrin (R-PE) from a red alga Portieria hornemannii was subjected to the analysis of stability under the influence of different agents. Among the various inhibitors tested on R-PE EDTA at lower concentrations (<1mM) supported the activity of R-PE. When R-PE was exposed to different organic solvents, ethanol supported the activity at the maximum followed by acetone, ethyl acetate, chloroform and methanol. Citric acid, as a preservative maintained the stability of R-PE both under 0±5°C and 30±5°C with 59.34% and 56.23% respectively, on 30th day. Thermal decomposition of the R-PE began near 60°C. Maximum weight loss was occurred between 150°C and 500°C. Complete weight loss was recorded around 875°C. Thermal denaturation was observed between 19°C and 40°C. Moderate to low antioxidant activities were observed in R-PE in relation to total antioxidant activities. After characterization, R-PE was taken for in vitro anticancer studies against selected cancer cell lines. Further studies involving AO/EB fluorescence staining and phase contrast microscope revealed characteristic apoptotic features like cell shrinkage, membrane blebbing, and nuclear DNA fragmentation, etc. Likewise, FACS analysis revealed the cell cycle distribution pattern of A549 and HepG2 cells.

Nanochannel pH Gradient Electrofocusing of Proteins

We demonstrate matrix-free pH gradient electrofocusing of proteins within an 85 nm deep nanochannel. In contrast to conventional isoelectric focusing where the fluid does not move, this pH gradient method traps protein molecules flowing through a channel by balancing electric forces due to pH-dependent protein charge and viscous drag forces caused by electro-osmosis. The nanoscale depth of the device and the low voltage used limit convection relative to diffusion, thus producing a stable focused band of protein. R-Phycoerythrin (RPE) and Dylight labeled streptavidin (Dyl-Strep) were focused within a nanochannel using applied voltages between 0.4 and 1.6 V. Concentration enhancement factors of over 380 have been achieved within 5 min. Varying the buffer pH (between 2.7 and 7.2) at the boundaries of the nanochannel affected the shape of the focused bands. For RPE, a pH span of 4.5 (pH 2.7 to 7.2) yielded the narrowest peak while a span of 2.4 (pH 2.7 to 5.1) produced a significantly wider peak. Such matrix-free nanofluidic devices with pH gradient electrofocusing may enable on-chip integration of orthogonal separation techniques with mass spectrometry offering labor savings and enhanced performance.

Implication of MAPK1/MAPK3 signalling pathway in t(8;9)(p22;24)/PCM1‐JAK2 myelodysplastic/myeloproliferative neoplasms

Implication of <scp>MAPK</scp>1/<scp>MAPK</scp>3 signalling pathway in t(8;9)(p22;24)/<i><scp>PCM</scp>1‐<scp>JAK</scp>2</i> myelodysplastic/myeloproliferative neoplasms

Multiplex detection of plant pathogens using a microsphere immunoassay technology.

Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), chilli vein-banding mottle virus (CVbMV, potyvirus), watermelon silver mottle virus (WSMoV, tospovirus serogroup IV) and melon yellow spot virus (MYSV, tospovirus). An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE)-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA) when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour) was much shorter than that of ELISA (4 hours). This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection.

Simultaneous Detection of Clavibacter michiganensis subsp. nebraskensis and Pantoea stewartii subsp. stewartii Based on Microsphere Immunoreaction

Clavibacter michiganensis subsp. nebraskensis (Cmn) and Pantoea stewartii subsp. stewartii (Pss) are two plant pathogens that can cause tremendous agricultural economic losses. This novel method based on microsphere immunoreaction was developed for the simultaneous detection of Cmn and Pss in maize. This multiplex method was constructed based on microsphere immunodetection with fluorescent labels such as quantum dots (QDs) and R-phycoerythrin (R-PE) for the detection of Cmn and Pss. Captured QDs and R-PE serve as signal reporters for fluorescent readout. The principle of this method is based on a sandwich immunoreaction. Cmn and Pss captured by the microspheres were detected using flow cytometry. The limit of detection of this method was 10 times lower than the enzyme-linked immunosorbent assay (ELISA), and its analysis time (1 h) was much shorter compared with ELISA (6–8 h). The method, which has been proven to be an effective approach to multiplex detection of plant bacteria (Cmn and Pss as models), not only increased the varieties but also improved the sensitivity. The microsphere immunoreaction provides a universal method for the multiplex determination of microbes because of its high sensitivity, specificity, and speed. In the future, the method will be more fully validated in vivo to detect diversiform bacteria.

Trehalose Preserves the Integrity of Lyophilized Phycoerythrin–AntiHuman CD8 Antibody Conjugates and Enhances their Thermal Stability in Flow Cytometric Assays

An increasing number of publications report on the efficacy of trehalose in preserving organisms, cells, and macromolecules from adverse environmental conditions such as extreme temperatures and dryness. Although the mechanism by which this disaccharide exerts its protection is still debated, the implementation of trehalose as stabilizer is becoming a praxis in several preparative protocols from the pharmaceutical industry. We tested the ability of trehalose in protecting R-Phycoerythrin (R-PE), a pigment-protein complex widely used as fluorescent marker, from thermal denaturation. Once embedded into a dried trehalose matrix, R-PE retains its optical absorption-emission characteristics even when exposed to 70°C for h or when subjected to freeze-drying. We subsequently examined the protection exerted by trehalose on freeze-dried antihuman CD8-RPE (CD8-RPE) conjugated antibodies. Flow cytometric analysis showed that colyophilized trehalose-CD8-RPE preparations can be exposed for 4 weeks at 45°C without significant loss of functionality. Remarkably, even following 4 weeks incubation at 70°C, the preparations are still able to specifically recognize CD8(+) lymphocyte populations. These results show that colyophilization with trehalose makes possible the preparation of antibody-based diagnostic kits which can withstand breaks in the "cold chain" distribution, particularly suited for use in less-developed countries of the tropical areas.

Isolation and characterization of macromolecular protein R-Phycoerythrin from Portieria hornemannii

R-Phycoerythrin (R-PE) is one of the three phycobiliproteins which are extensively used as fluorescent probes, and it is prepared from red macro-algae. This macromolecular protein has gained importance in many biotechnological applications in food science, immunodiagnostic, therapy, cosmetics, protein and cell labeling, and analytical processes. In the present investigation, R-PE was isolated and purified from a red alga Portieria hornemannii. R-PE extracted and purified through ammonium sulfate precipitation (55%) followed by Q-Sepharose column chromatography had yielded a maximum purity of 5.2%. R-PE exhibited a typical “three-peak” with absorption maxima at 499, 545 and 565nm. CD spectrum of R-PE yielded the following secondary structure data: alpha helix (14.30%), beta helix (28.10%), turn helix (19.20%) and random coil helix (38.40%). The molecular mass of R-PE was 240kDa under Native-PAGE. Three different subunits such as α, β and γ of 16kDa, 21kDa and 39kDa were segregated under SDS-PAGE. On two dimensional gel electrophoresis, one basic and four acidic subunits were detected. Five different tryptic peptides were assigned under MALDI-TOF. The sequences of N-terminus of R-PE of 10 different amino acids are Met Lys Gln Met Trp Asp Arg Met Val Val. The preparative procedures of the R-PE extraction and purification established based on the experiments exhibit advantages and can offer a reference for R-PE preparation from other marine red macro-alga P. hornemannii.

Antitumor function and mechanism of phycoerythrin from Porphyra haitanensis

The anti-tumor effect of R-Phycoerythrin (R-PE) from Porphyra haitanensis was studied using cell line HeLa as an in vitro model and Sarcoma-180 (S180) tumor-bearing mice as an in vivo model. The results showed that the combination treatment of R-PE and photodynamic therapy PDT) significantly inhibited the growth of HeLa cells up to 81.5%, with a fair dose-effect relationship, but did not inhibit endothelial cells. The annexin v-fitc/PI fluorescence staining experiments demonstrated that at doses between 0~60µg/mL, apoptosis cells and later stage apoptosis cells or necrosis cells increased significantly as the R-PE dosage increased. DNA electrophoresis showed that after R-PE+PDT treatment of HeLa cells for 24 hours, a light "smear" band between 100~400bp appeared to indicate the degradation of genomic DNA. The QRT-PCR results showed that R-PE+PDT treatment increased caspase-3 and caspase-10 gene expression and decreased the Bcl-2 gene expression level significantly as the R-PE dose increased, implying that R-PE promoted HeLa cell apoptosis. Compared with untreated S180 tumor-bearing mice, R-PE injection significantly inhibited the growth of S180 in tumor-bearing mice up to 41.3% at a dose of 300mg-kg⁻¹. Simultaneously, the significant increase of superoxide dismutase (SOD) activity in serum (p < 0.01) and the decrease of the malondialdehyde (MDA) level in liver suggests that R-PE improved the anti-oxidant ability of the S180 tumor-bearing mice, which may related to its antitumor effect. In addition, the R-PE caused a significant increase (p < 0.05) in the spleen index and thymus index, and a significant increase (p < 0.01) in lymphocyte proliferation, NK cell kill activity and the TNF-α level in the serum of S180 tumor-bearing mice. These results strongly suggest that the antitumor effect of R-PE from Porphyra haitanensis functioned by increasing the immunity and antioxidant ability of S180 tumor-bearing mice, promoting apoptosis by increasing protease gene expression and TNF-α secretion.

Variation in the Biochemical Composition of the Edible Seaweed Grateloupia turuturu Yamada Harvested from Two Sampling Sites on the Brittany Coast (France): The Influence of Storage Method on the Extraction of the Seaweed Pigment R‐Phycoerythrin

Numerous studies have demonstrated that the biochemical content of seaweeds varies according to seasonality in a restricted area. In this study, the influence of sampling site on the biochemical composition of the edible red seaweed Grateloupia turuturu Yamada was investigated, but not its variation over time. Some differences in water‐soluble protein (7.19 ± 0.74 mg · g−1 dw and 19.15 ± 0.47 mg · g−1 dw), water‐soluble carbohydrate (16.05 ± 0.82 mg · g−1 dw and 41.57 ± 0.66 mg · g−1 dw), and lipid contents (28.07 ± 5.12 mg · g−1 dw and 54.35 ± 2.05 mg · g−1 dw) were recorded between the two sites chosen on the Brittany coast (France). The yield of R‐phycoerythrin (R‐PE) contained in the seaweed also varied according to the sampling site (1.16 ± 0.33 mg · g−1 dw versus 4.39 ± 0.15 mg · g−1 dw). In addition, the effect of storage conditions on the preservation of R‐PE was studied. The results demonstrated that freezing is the best preservation method in terms of R‐PE extraction yield and purity index. In conclusion, this study shows that the sampling site influences the biochemical content of the red seaweed Grateloupia turuturu. Moreover, the extraction yield of R‐phycoerythrin and its purity index depend on both the sampling site and the sample storage method.

Pictorial Demonstration of the Simultaneous Binding of Multiple Unrelated Antigens to Individual Polyreactive Antibody‐Producing B Cells

To the editor: Polyreactive antibodies are a major component of the natural antibody repertoire and are capable of binding multiple structurally unrelated antigens (e.g. proteins, peptides, carbohydrates, DNA) [1–4]. About 15% of the B cells in human peripheral blood and up to 50% of the B cells in cord blood make polyreactive antibodies. Sequence analysis revealed that many of these antibodies are germline or near germline in configuration and have low antigen-binding affinities as compared to monoreactive antibodies [4]. The ability of polyreactive antibodies to bind multiple unrelated antigens is thought to be owing to the greater flexibility of their antigen-binding pockets as compared to the more rigid antigen-binding pockets of monoreactive antibodies. Although not as well studied, previously we showed by FACS analysis that receptors on the surface of the B cells that make polyreactive antibodies also can bind structurally unrelated antigens [4]. In the present experiments, using fluorochrome-labelled antigens and ImageStream, we demonstrate pictorially, for the first time, that a single polyreactive B cell can bind simultaneously multiple structurally unrelated antigens. Approximately 1.0 × 106, 8- to 12-week-old, C57BL/6 mouse peritoneal cells in a volume of 100 μl were incubated with one, two or three fluorochrome-labelled antigens along with FITC-conjugated antibody to B-220 (BD Biosciences, San Jose, CA, USA) and the death marker 7-AAD (BD Biosciences) for 30 min at 4°C and then analysed by FACS Calibur (BD, San Jose, CA, USA) or by ImageStream (Amins Corporation, Seattle, WA). Beta-galactosidase (beta-gal) and thyroglobulin (Tg) were conjugated with either R-phycoerythrin (R-PE) or allophycocyanin (APC), and insulin-biotin (Sigma-Aldrich, St Louis, MO, USA) was labelled with streptavidin-FITC, streptavidin-PE or streptavidin-APC. Only live B-220 cells that failed to take up 7-AAD were used to evaluate the binding of fluorochrome-labelled antigens. All experiments were carried out in compliance with institutional guideline and approved by the NIDCR ACUC (Bethesda, MD). FACS analysis revealed that 17.0% of the peritoneal B-220 cells bound both Tg and insulin, 11.0% insulin and beta-gal and 14.1% Tg and beta-gal. The binding of multiple fluorochrome-labelled antigens to individual polyreactive B cells then was pictorially evaluated by ImageStream technology. Approximately 50,000 images were saved using the same samples as in the flow cytometry experiments. Fig. 1A–D show individual B-220 positive cells to which (A) none of the antigens bound; (B) one antigen bounds; (C) two antigens bound; and (D) three antigens bound. Binding of multiple unrelated antigens to individual peritoneal B-220 cells as demonstrated by ImageStream. (A) Cells that failed to bind any of the three antigens (i.e. Tg, beta-gal and insulin); cells that bound (B) one antigen, (C) two antigens and (D) three antigens. Representative pictures from 50,000 images. It has long been thought, based on the clonal selection theory, that the receptors on the surface of antibody-producing B cells bind only their cognate antigen or a closely related antigen. However, with the discovery of polyreactive antibodies and with our previous FACS findings [4], it has become clear that the B cells that make these antibodies can bind to their surface multiple unrelated antigens. The experiments reported here confirm and extend these findings and show pictorially, for the first time, that at least three different and unrelated antigens can simultaneously bind to the same B cell. These findings have broad implications in terms of the role of polyreactive B cells in natural defence against bacterial [5] and viral infections [6]. In addition, these findings support the idea that polyreactive B cells may be involved in the presentation of antigens to T cells and could transport self-antigens to the thymus for initiation and/or maintenance of immunological tolerance [4]. Still unanswered is the question as to which of the many endogenous host antigens stimulate polyreactive B cells to secrete polyreactive antibodies and to keep the level of these antibodies relatively constant over time. Therefore, it is not unreasonable to speculate that polyreactive B cell stimulation and antibody secretion may depend on the number, density and affinity of endogenous host antigens that randomly bind to polyreactive B cell receptors. If this proves to be the case, it would provide new insight into the nature of the antigens that trigger and perpetuate the proliferation of polyreactive B cells in both natural immune defence and in diseases, such as chronic lymphocytic leukaemia in which many of the leukaemic cells produce polyreactive antibodies [7]. This work was supported by the Intramural Research Program of the National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland. The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. YX, ZZ and ALN designed the experiments. YX performed the experiments. ALN and YX wrote the paper. The authors report no conflict of interest.

Immunomodulatory Properties of the Protein Fraction from Phorphyra columbina

The phycobiliproteins from Rhodophyta , R-phycoerythrin (R-PE) and C-phycocyanin (C-PC), have been shown to exert immunomodulatory effects. This study evaluated the effects of a Phorphyra columbina protein fraction (PF) and R-PE and C-PC on rat primary splenocytes, macrophages, and T-lymphocytes in vitro. PF featured various protein species, including R-PE and C-PC. PF showed mitogenic effects on rat splenocytes and was nontoxic to cells except at 1 g L(-1) protein. IL-10 secretion was enhanced by PF in rat splenocytes, macrophages, and especially T-lymphocytes, whereas it was markedly diminished by R-PE and C-PC. The production of pro-inflammatory cytokines by macrophages was inhibited. The effect of PF on IL-10 was evoked by JNK/p38 MAPK and NF-κB-dependent pathways in macrophages and T-lymphocytes. It was concluded that PF has immunomodulatory effects on macrophages and lymphocytes that appear to be predominantly anti-inflammatory via up-regulated IL-10 production and cannot be accounted for by R-PE and C-PC.

Species-Specific Identification of Seven Vegetable Oils Based on Suspension Bead Array

Species adulteration of vegetable oils has become a main form of adulteration in vegetable oils, severely violating consumer rights and causing disorder in the market. A reliable method of species authentication of vegetable oils is desirable. This paper reports a novel method for identification of seven species of vegetable oils based on suspension bead array. One pair of universal primers and seven species-specific probes were designed targeting rbcl gene of the chloroplast. Each probe was coupled to a unique color-coded microsphere. Biotinylated PCR amplicons of seven oils were hybridized to the complementary probes on microsphere sets. Bound amplicons were detected fluorometrically using a reporter dye, streptavidin-R-phycoeryt hrin (SA-PE). A sample could be analyzed less than 1 h after PCR amplification. With the exception of olive probe, all probes showed no cross-reactivity with other species. Absolute detection limit of the seven probes ranged from 0.01 ng/μL to 0.0001 ng/μL. Detection limit in DNA mixture was from 10% to 5%. Detection of vegetable oils validated the effectiveness of the method. The suspension bead array as a rapid, sensitive, and high-throughput technology has potential to identify more species of vegetable oils with increased species of probes.

Synthesis of NaYF4:Yb/Er/Gd up-conversion luminescent nanoparticles and luminescence resonance energy transfer-based protein detection

High-quality NaYF4:Yb/Er/Gd up-conversion nanoparticles (UCNPs) were first synthesized by a solvothermal method using rare earth stearate, sodium fluoride, ethanol, water, and oleic acid as precursors. Doped Gd3+ ions can promote the transition of NaYF4 from cubic to hexagonal phase, shorten the reaction time, and reduce the reaction temperature without reducing the luminescence intensity of NaYF4:Yb/Er UCNPs. X-ray diffraction, infrared spectroscopy, transmission electron microscopy, and luminescence spectroscopy were applied to characterize the UCNPs. The nanoparticles exhibited small size and excellent green up-conversion photoluminescence, making them suitable for biological applications. After the surfaces of NaYF4:Yb/Er/Gd UCNPs were modified with amino groups through the Stöber method, they could be brought close enough to the analytically important protein called R-phycoerythrin (R-PE) bearing multiple carboxyl groups so that energy transfer could occur. A luminescence resonance energy transfer (LRET) system was developed using NaYF4:Yb/Er/Gd UCNPs as an energy donor and R-PE as an energy acceptor. As a result, a detection limit of R-PE of 0.5μg/ml was achieved by the LRET system with a relative standard deviation of 2.0%. Although this approach was first used successfully to detect R-PE, it can also be extended to the detection of other biological molecules.