The colloidal gold nanoparticle-based lateral flow immunoassay for fast and simple detection of plant-derived doping agent, higenamine.

Higenamine (HM), an alkaloid found in various plant species, is obtained when norcoclaurine synthase selectively condenses dopamine and 4-hydroxyphenylacetaldehyde to give (S)-higenamine ((S)-HM). The World Anti-doping Agency has listed HM as a prohibited agent in athletics. As a result, many commercial, academic, and regulatory bodies across the globe are invested in finding a rapid method for (S)-HM detection. In the current study, a lateral flow immunoassay (LFA) was developed in which the relevant biosensor was generated as a conjugate of the monoclonal antibody against (S)-HM (namely, MAb E8) and colloidal gold nanoparticles. The HM-γ-globulin conjugates and rabbit anti-mouse IgG antibodies were placed in the test and control zones, respectively. The free (S)-HM molecules in the samples and the immobilized HM-γ-globulin conjugates competitively reacted with the developed biosensor in the LFA. An inverse relationship existed between the biosensors' visible response, which was noted by the variation in the intensity of a pinkish spot in the test zone, and the content of the free (S)-HM. The limit of detection of the developed LFA was 156 ng/mL. Various validation methods confirmed that the LFA exhibited sufficient sensitivity, selectivity, repeatability, and reliability, making it ideal for (S)-HM detection in plant samples and plant-containing products. The developed system required only a small sample volume (20 µL) and a concise sample preparation time compared with conventional LFAs. Thus, the LFA reported in this study could serve as a rapid response kit for the detection of (S)-HM in plant samples.

Early Pep-13-induced immune responses are SERK3A/B-dependent in potato.

Potato plants treated with the pathogen-associated molecular pattern Pep-13 mount salicylic acid- and jasmonic acid-dependent defense responses, leading to enhanced resistance against Phytophthora infestans, the causal agent of late blight disease. Recognition of Pep-13 is assumed to occur by binding to a yet unknown plasma membrane-localized receptor kinase. The potato genes annotated to encode the co-receptor BAK1, StSERK3A and StSERK3B, are activated in response to Pep-13 treatment. Transgenic RNAi-potato plants with reduced expression of both SERK3A and SERK3B were generated. In response to Pep-13 treatment, the formation of reactive oxygen species and MAP kinase activation, observed in wild type plants, is highly reduced in StSERK3A/B-RNAi plants, suggesting that StSERK3A/B are required for perception of Pep-13 in potato. In contrast, defense gene expression is induced by Pep-13 in both control and StSERK3A/B-depleted plants. Altered morphology of StSERK3A/B-RNAi plants correlates with major shifts in metabolism, as determined by untargeted metabolite profiling. Enhanced levels of hydroxycinnamic acid amides, typical phytoalexins of potato, in StSERK3A/B-RNAi plants are accompanied by significantly decreased levels of flavonoids and steroidal glycoalkaloids. Thus, altered metabolism in StSERK3A/B-RNAi plants correlates with the ability of StSERK3A/B-depleted plants to mount defense, despite highly decreased early immune responses.

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.

Antiophidic solanidane steroidal alkaloids from Solanum campaniforme.

Three new solanidane alkaloids bearing a 22,23-epoxy ring (1-3) and four known compounds were isolated from leaves of Solanum campaniforme. The structures were determined using spectroscopic techniques, including 1D and 2D NMR, and HRESIMS experiments. The antiophidic activity of the alkaloids was tested against Bothrops pauloensis venom. Compounds 1-3 completely inhibited myotoxicity without inhibiting phospholipase A2 activity of the venom, while hemorrhage and skin necrosis were significantly reduced in the presence of alkaloids 1 and 2.

Baccatin III, a synthetic precursor of taxol, enhances MHC-restricted antigen presentation in dendritic cells.

Baccatin III, a precursor for the semisynthesis of taxol, is widely considered to be an inactive derivative of taxol. Here we show that baccatin III efficiently enhances MHC-restricted antigen presentation in dendritic cells. Baccatin III increased both class I- and class II-restricted presentation of exogenous OVA in bone marrow-derived dendritic cells (BM-DCs). Baccatin III also increased class I-restricted presentation of virus-encoded endogenous OVA in BM-DCs. Baccatin III did not affect the phagocytic activity of BM-DCs. The antigen presentation-enhancing activity of baccatin III was examined further with nanoparticles containing OVA and baccatin III. Inclusion of baccatin III to nanoparticles containing OVA greatly enhanced their capacity to induce class I-restricted OVA peptide presentation in DCs both in vitro and in vivo. Accordingly, nanoparticles containing both OVA and baccatin III were much more efficient in inducing an OVA-specific CTL response in mice compared to those containing OVA only. These results demonstrate that baccatin III exerts immunomodulatory activities in vivo as well as in vitro on the MHC-restricted antigen presentation.

Astragalus polysaccharide and oxymatrine can synergistically improve the immune efficacy of Newcastle disease vaccine in chicken.

Three hundred and sixty 14-day-old chickens were divided into seven groups. The chickens, except for blank control group, were vaccinated with Newcastle disease vaccine, repeated at 28 days old. At the same time of the first vaccination, the chickens in three astragalus polysaccharide-oxymatrine (AP-OM) groups were orally administrated respectively with the mixture of AP-OM at high, medium and low concentrations, in astragalus polysaccharide (AP) group and oxymatrine (OM) group, with corresponding medicine, in non-medicine (NM) control group, with equal volume of physiological saline, once a day for 3 successive days. On 14, 21, 28, 35 and 42 days after the first vaccination, the changes of peripheral lymphocyte proliferation and serum antibody titers of the chickens were determined by MTT method and hemagglutination inhibition test. On 14, 28 and 42 days after the first vaccination, the serum IL-2 concentration was determined by Enzyme-linked Immunosorbent Assay (ELISA). The results showed that at most time points, the lymphocyte proliferation, antibody titers and IL-2 concentrations of 5 medicine-administrating groups were significantly higher than that of corresponding NM group. At some time points, the lymphocyte proliferation, antibody titers and IL-2 concentrations in high and medium doses of AP-OM groups were significantly or numberly higher than those in AP group and OM group. It indicated that AP-OM could significantly improve the immune efficacy of Newcastle disease vaccine, astragalus polysaccharide and oxymatrine possessed synergistical immunoenhancement.

Characterization of cereal toxicity for celiac disease patients based on protein homology in grains.

BACKGROUND AND AIMS: Celiac disease is caused by T-cell responses to wheat gluten-derived peptides. The presence of such peptides in other widely consumed grains, however, has hardly been studied. METHODS: We have performed homology searches to identify regions with sequence similarity to T-cell stimulatory gluten peptides in the available gluten sequences: the hordeins of barley, secalins of rye, and avenins of oats. The identified peptides were tested for T-cell stimulatory properties. RESULTS: With 1 exception, no identical matches with T-cell stimulatory gluten peptides were found in the other grains. However, less stringent searches identified 11 homologous sequences in hordeins, secalins, and avenins located in regions similar to those in the original gluten proteins. Seven of these 11 peptides were recognized by gluten-specific T-cell lines and/or clones from patients with celiac disease. Comparison of T-cell stimulatory sequences with homologous but non-T-cell stimulatory sequences indicated key amino acids that on substitution either completely or partially abrogated the T-cell stimulatory activity of the gluten peptides. Finally, we show that single nucleotide substitutions in gluten genes will suffice to induce these effects. CONCLUSIONS: These results show that the disease-inducing properties of barley and rye can in part be explained by T-cell cross-reactivity against gluten-, secalin-, and hordein-derived peptides. Moreover, the results provide a first step toward a rational strategy for gluten detoxification via targeted mutagenesis at the genetic level.

Neurotropic, immunological and gastric effects of low doses of Atropa belladonna L., Gelsemium sempervirens L. and Poumon histamine in stressed mice.

Previous studies realized in the laboratory have indicated that application of experimental stress (such as unavoidable footshock) induced significant behavioral, gastric and immunological alterations in mice. The aim of this study was to evaluate effects of low doses of Atropa belladonna L., Gelsemium sempervirens L. and Poumon histamine on stress-induced behavioral, immunological and gastric alterations. Locomotor, postural and exploratory activities have been evaluated by two behavioral tests: light/dark box and staircase tests. Immunological studies were investigated to count white blood cells subpopulations (lymphocytes, neutrophils, monocytes and basophils) by coulter counter. The severity of gastric erosions was evaluated by microscopic technique in mice after experimental stress. The results have demonstrated that low doses of G. sempervirens L. and A. belladonna L. had a significant neurotropic and protective effects on behavioral and gastric alterations induced by experimental stress. The immunological protective effects observed were probably induced via their neurotropic effects. The P. histamine showed a significant immunoprotective and gastroprotective effect in mice exposed to experimental stress.

CGP 41251, a novel protein kinase inhibitor with in vitro selectivity for protein kinase C, strongly inhibits immunological activation of human skin mast cells and human basophils.

The process of high-affinity IgE receptor (Fc epsilon RI)-mediated signal transduction in human basophils and mast cells is accompanied by activation of protein kinase C (PKC). The present study investigated the effects of a novel protein kinase inhibitor with in vitro selectivity for PKC (CGP 41251) in comparison with the potent but non-selective PKC inhibitor staurosporine on the activation of human peripheral basophilic leukocytes and enzymatically isolated human skin mast cells. CGP 41251 exerted strong concentration-dependent inhibitory effects on Fc epsilon RI-mediated histamine release from both cell populations. In addition, the IgE-mediated generation of arachidonic acid metabolites (leukotriene C4/D4 and prostaglandin E2) from human basophils was also significantly inhibited by this compound. Its action was not significantly different from the action of staurosporine. Direct activation of cellular PKC by the phorbol ester 12-o-tetradecanoyl-phorbol-13-acetate and subsequent histamine release from basophils was also inhibited by both compounds. CGP 41251 did not suppress N-formyl-met-leu-phe- or A23187-induced activation of basophils, whereas A23187-induced mediator release from human skin mast cells was inhibited in a concentration-dependent fashion. We conclude that an increase of in vitro selectivity for PKC does not significantly enhance inhibitory effects on immunological activation of histamine-containing cells. Moreover, nonimmunological pathways of signal transduction in basophils and mast cells appear to be mediated by distinct biochemical events.

Stimulation of neutrophil respiratory burst and degranulation by Japanese encephalitis virus-induced macrophage derived factor.

The effects of Japanese encephalitis virus (JEV) and JEV-induced macrophage derived neutrophil chemotactic factor (MDF) on respiratory burst and granule exocytosis in neutrophils were studied and were compared with N-formyl-methionyl-leucyl-phenylalanine (FMLP). JEV-stimulated neutrophils released reactive oxygen metabolites with maximum activity between days 7 and 9. The response in mice was sensitive to anti-JEV-antisera treatment. Stimulation by MDF resulted in quick release of superoxide and granule enzymes from neutrophils upon both in-vivo and in-vitro stimulation in a dose dependent manner. The effect was abrogated by the MDF-specific antisera treatment. These responses were similar in kinetics and magnitude to those produced in response to FMLP. The respiratory burst elicited by MDF was abrogated by staurosporine, indicating that neutrophil activation and signal transduction by MDF and FMLP are dependent on protein kinase C.

[Iga and IgG antireticuline in celiac disease. Their application as diagnostic markers of the disease]. / Estudio de la reactividad IgA e IgG antirreticulina en la enfermedad celíaca. Su aplicación como marcador diagnóstico de la enfermedad.

We report the results of indirect immunofluorescent (IFI) detection of IgA and IgG antireticulin antibodies (IgA-ARA and IgG-ARA, respectively) in 283 serum samples from pediatric patients with coeliac disease (with and without gluten containing diets), patients with non-coeliac gastrointestinal disease, patients without gastrointestinal disease (control group) and patients with an increased risk for coeliac disease (diabetes mellitus, dermatitis herpetiformis or first grade relatives of coeliac patients). Our results indicate that IgA-ARA is a reproducible marker, with high positive (99-100%) and negative (100%) prediction values, when it is applied to children who have been on gluten containing diets for a long time (more than six months). The IgA-ARA measurement is not applicable in cases of selective IgA deficiency. Although IgG-ARA has a high predictive positive value, its low predictive negative value makes it a poor diagnostic tool. In the risk groups, our results suggest that these antibodies are useful in patient selection for intestinal biopsy.

Cytoskeletal elements regulate the distribution of nerve growth factor receptors in PC12 cells.

Nerve growth factor receptor (NGFR)-like immunoreactivity (IR) was studied in PC12 cells treated for 96 hr with NGF (40 ng/ml), using immunogold labeling and electron microscopic morphometric analysis. The cells were exposed to the anti-NGFR antibody 192-IgG, followed by immunoglobulin (IgG) conjugated with colloidal gold. PC12 cells exhibited occasional gold label (positive NGFR-IR) on all surfaces. Cells treated with colcemid (0.05 micrograms/ml) or cytochalasin D (2 micrograms/ml), which limit microtubule (MT) and microfilament (MF) formation, respectively, displayed an increased NGFR-IR in terms of gold labeling. NGFR-IR was also seen on taxol (0.85 micrograms/ml)-exposed cells, an agent that promotes MT assembly. Cells treated simultaneously with cytochalasin D and taxol had a dramatically augmented NGFR-IR on their surfaces, which exceeded levels obtained with either agent alone. Prominent NGFR-IR was localized frequently in coated endocytotic vesicles, in smooth endoplasmic reticulum, and in secondary multivesicular lysosomes, in both treated and untreated cells. The results suggest that a large number of NGFRs (positive NGFR-IR) in PC12 cells are cryptic and not available for ligand binding. Changes in cytoskeletal organization that may affect mobility of integral membrane proteins can modulate the distribution of NGFR-IR on neuronal surfaces.

Protein kinase C activation modulates tumour necrosis factor-alpha priming of human neutrophils for zymosan-induced leukotriene B4 release.

Neutrophil (PMN) activation by the yeast component zymosan involves the complement receptor type 3 (CD11b/CD18). Recombinant human tumour necrosis factor-alpha (rhTNF-alpha) augmented the zymosan-stimulated leukotriene B4 (LTB4) release from PMN, reaching a fourfold increase at 10(-9) M. Co-incubation of PMN with 10(-9) M rhTNF-alpha and staurosporine resulted in a further dose-dependent increase, which became significantly greater than a purely additive effect at a staurosporine concentration of 10 nM. This synergy was maintained at all doses of staurosporine tested. In addition, doses of phorbol 12-myristate 13-acetate (PMA) that do not activate protein kinase C (PKC) (below 10(-9) M) also augmented the zymosan-stimulated release of LTB4. However, doses of PMA above 10(-9) M progressively inhibited the response to levels below that of zymosan alone. Staurosporine at 50 nM completely prevented, and 10(-9) M rhTNF-alpha partially but significantly (P less than 0.02 at 10(-8) M PMA, P less than 0.01 at 10(-7) M PMA) reversed, this high-dose PMA inhibition. PKC activation thus opposes the priming effect of rhTNF-alpha on neutrophils, while PKC inhibition may enhance the ability of rhTNF-alpha to prime PMN for zymosan activation. The combined effect of rhTNF-alpha and staurosporine suggests an intracellular synergy rather than simply a direct action due to increased zymosan receptor expression. Thus there appear to be mechanisms whereby the responses of neutrophils may be augmented without activating PKC. Indeed, kinase activation may even exert a degree of feedback control that is antagonized by rhTNF-alpha treatment.

The immunomodulating preparation Ukrain does not induce anaphylactic sensitization in mice and guinea pigs.

The ability of Chelidonium majus L. alkaloids derivative Ukrain to induce an anaphylactic sensitization was tested on mice and guinea pigs. The levels of IgE antibody in the mouse sera, and IgG1a, IgG1b as well as IgE antibody levels in guinea pig sera, were evaluated by passive cutaneous anaphylaxis (PCA) tests. Ukrain alone or adsorbed on aluminium hydroxide gel (alum) introduced into BALB/c mice in several subcutaneous injections was unable to stimulate measurable anti-Ukrain IgE antibody response. Moreover, Ukrain introduced together with ovalbumin (OA) into mice in the course of immunization with OA induced lower anti-OA antibody response as compared to the response induced by OA alone. Ukrain adsorbed on alum and injected subcutaneously into guinea pigs did not induce measurable IgG1a, IgG1b and IgE antibody response. The present results suggest that the immunomodulating preparation Ukrain could be therapeutically safe at least as far its inability to induce anaphylaxis is concerned.

Comparative immunopharmacology and toxicology of the bisbenzylisoquinoline alkaloids tetrandrine and berbamine.

Comparison of the bisbenzylisoquinolines tetrandrine and berbamine shows that both drugs are equipotent in terms of enhancement of antibody responses and suppression of delayed-type hypersensitivity (DTH) responses to sheep red blood cell antigens. Both compounds are also equally active when given to mice during the induction and expression phases of DTH. Using a model of experimental brucellosis in mice, it was found that both compounds did not affect antibody responses, while they caused equipotent suppression of DTH. By contrast, berbamine but not tetrandrine caused significant suppression of spleen weight. Also, berbamine caused a significantly greater enhancement of spleen colony counts of Brucella abortus than tetrandrine. Short-term toxicology studies showed no toxic effects at bioactive doses.

An investigation of the irritant and allergenic properties of daffodils (Narcissus pseudonarcissus L., Amaryllidaceae). A review of daffodil dermatitis.

Irritancy of daffodil flowers and bulbs was assessed using various fresh plant preparations, solvent extracts and some of the known Amaryllidaceae alkaloids on guinea pigs. Sensitization was also carried out on guinea pigs using these plant preparations, solvent extracts and 7 fractions obtained after preparative chromatography of the bulb ether extract. Only 1 fraction, containing 2 alkaloids, was capable of inducing delayed hypersensitivity in the animals; the sensitivity achieved, however, was weak. The substances were identified as masonin and homolycorin, which acted as elicitors, but masonin may also be a sensitizer. While homolycorin is a known daffodil constituent, masonin has not been found previously in Narcissus pseudonarcissus. 3 other alkaloids as well as chelidonic acid and isorhamnetin were non-elicitors in the sensitized guinea pigs.

Antibody recognition of substituted ammonium ions. Modulation by the counterion.

Antibodies directed against elliptinium acetate, a quaternary ammonium compound with antineoplastic activity in man, were obtained in rabbits, after conjugation of the drug with haemocyanin. These antibodies are specific for the quaternary ammonium structure. However, the recognition of the drug can be markedly decreased (ten-fold) by changing the associated counterion. These observations were extended to other ellipticine derivatives that exist in two forms: acetate and chloride. In each case, the recognition of the acetate form was 7-11-fold higher than that of the chloride form. These results could be explained by high-energy strengths existing between the cation and the anion, resulting in a paired-ion antigen. This represents the first identification of antibodies directed to a paired-ion structure, with specificity for both the cation and anion used for immunization. Such results are relevant in the construction of immunoassays for quaternary ammonium compounds.

Polyclonal antibodies to elliptinium acetate: structure-activity relationships and comparison with human anti-elliptinium IgM.

In order to elucidate the immune-mediated hemolytic disease induced in man by elliptinium acetate, a quaternary ammonium compound with antineoplastic activity, polyclonal antibodies directed against this hapten were raised in rabbits. The coupling step between drug and carrier was performed according to a putative human in vivo hapten conjugation mechanism. Structure-activity relationships of the resulting IgG were compared with the epitope site recognized by human anti-elliptinium IgM by using a panel of twelve elliptinium acetate analogues. Although both antibodies were directed principally against the quaternary ammonium ion, a poor correlation between the cross-reactivity indices was obtained. In fact, it appeared that both antibodies recognized specifically the ammonium group plus different regions of the molecule: the indole ring for human antibodies, the N-alkyl group and its vicinity for rabbit ones. The specificity of the obtained rabbit polyclonal antisera is discussed, with regard to the conjugation mechanism of the drug occurring in man.

Human antibodies to the antineoplastic drug elliptinium: characterization and structure-activity relationships.

Immune-mediated hemolytic disease is a phenomenon rarely encountered with cancer chemotherapeutic agents. Elliptinium, a tetracyclic ammonium compound used in breast and kidney cancer, can induce antibodies that may result in clinical hemolysis. This study reports the characterization of the elliptinium haptenic determinant by use of two different methodologies: a hemagglutination test and a radioimmunoassay. Binding of 12 analogues or derivatives of elliptinium was also studied. Good correlation between the two methods was obtained, indicating that the determinant is most likely located on the quaternary ammonium-containing ring. Furthermore, the hydrophilicity of the drug appears to be an important factor in the antibody reaction. The mechanism of the binding of elliptinium to its antibodies is discussed.