Panax quinquefolius (North American Ginseng) Polysaccharides as Immunomodulators: Current Research Status and Future Directions.

Panax quinquefolius (North American ginseng, NAG) is a popular medicinal plant used widely in traditional medicine. NAG products are currently available in various forms such as roots, extracts, nutraceuticals, dietary supplements, energy drinks, etc. NAG polysaccharides are recognized as one of the major bioactive ingredients. However, most NAG reviews are focused on ginsenosides with little information on polysaccharides. NAG polysaccharides have demonstrated a therapeutic activity in numerous studies, in which many of the bioactivities involve regulation of the immune response. The purpose of this review is to summarize the structural features and the immunomodulatory properties of crude, partially purified, and pure polysaccharides isolated from NAG. Receptors of the innate immune system that potentially bind to NAG polysaccharides and the respective signal transduction pathways initiated by these compounds are discussed. Major challenges, recent innovations, and future directions in NAG polysaccharide research are also summarized.

Effect of addition of adjuvants on physical and chemical characteristics of Bt bioinsecticide mixture.

Bacillus thuringiensis (Bt) is the main bacterium used in the formulation of bioinsecticides because it produces toxins and spores that are toxic to several orders of insects. The efficacy of Bt bioinsecticide is influenced by the quality of its application. The association with other crop protection products, such as adjuvants, can affect the physical and chemical parameters of the mixture. This study evaluated the physical and chemical parameters, volume median diameter (VMD), uniformity coefficient of droplets (SPAN), percentage of volume in drift droplets (%V <100 µm), contact angle, surface tension, potential of hydrogen (pH) and electrical conductivity (E.C.) of Bt bioinsecticides in concentrated suspension (SC), and wettable powder (WP) formulations associated with adjuvants. The largest droplet diameter and smallest values of drift droplets were found in the WP formulation with lower drift potential. The addition of mineral oil and surfactant to the mixtures of bioinsecticide reduced contact angle values and surface tension of the droplets, resulting in greater spreading of droplets in leaves. The addition of lecithin and propionic-acid-based adjuvants lowered the pH in both formulations. The adjuvants used in this study affected the physical and chemical characteristics of the mixtures, improving or impairing the quality of Bt bioinsecticide applications.

Montmorillonite improved the intestinal mucosal barrier functions of laying hens in late production.

This study was conducted to investigate the effects of dietary supplementation with montmorillonite (MMT) on performance, intestinal endotoxin concentration, gut mucosal oxidation status, intestinal morphology and permeability, and immunological barrier function of laying hens during late production. Four hundred and eighty 75-week-old laying hens (Lohmann Brown) were randomly assigned to five treatments with eight replicates per treatment and 12 hens in each replicate. The hens were fed the basal diet supplemented with 0 (control), 0.3, 0.6, 0.9, or 1.2 g MMT/kg for 70 days. Compared with the control, supplemented with 0.9 g MMT/kg increased egg mass significantly (p < 0.05) during weeks 1-5 of the experiment. Supplemented with 0.6 and 0.9 g MMT/kg also increased the endotoxin concentration in the ileal digesta (p < 0.05), but decreased the MDA concentration in the ileum significantly (p < 0.05). The T-AOC in the jejunum of the group fed 0.3 g MMT/kg was significantly increased (p < 0.05). Compared with the control, the villus height:crypt depth of ileum from the groups fed 0.6, 0.9, and 1.2 g MMT/kg increased significantly (p < 0.05). The sIgA concentration of jejunum in the groups fed 0.6 and 0.9 g MMT/kg was higher (p < 0.05) than the control. The MMT supplementation linearly increased (p < 0.05) the mRNA expression of claudin-1 and claudin-5 in the jejunum. Dietary MMT supplementation down-regulated the mRNA expression of NF-κB P65 and TNF-α in the jejunum in a linear and quadratic manner (p < 0.05). The IL-1ß mRNA expression of jejunum in the group fed 0.6 g MMT/kg was lower (p < 0.05) than the control. In conclusion, dietary supplementation with MMT may improve the gut barrier functions and suggests that 0.9 g/kg of MMT in diets may be the optimal supplemental level for laying hens in late production.

Flowerlike Mg(OH)2 Cross-Nanosheets for Controlling Cry1Ac Protein Loss: Evaluation of Insecticidal Activity and Biosecurity.

Bacillus thuringiensis (Bt) can produce Cry proteins during the sporulation phase, and Cry protein is effective against lepidopteran, coleopteran, and dipteran insects and nematodes. However, Cry protein tends to be discharged into soil and nontarget plants through rainwater runoff, leading to reduced effective period toward target insects. In the present study, nano-Mg(OH)2 (magnesium hydroxide nanoparticles, MHNPs) were synthesized to control the loss of Cry1Ac protein and deliver protein to Helicoverpa armigera (Lepidoptera: Noctuidae). The results showed that Cry1Ac protein could be loaded onto MHNPs through electrostatic adsorption, and both MHNPs and Cry protein were stable during the adsorption process. Meanwhile, the Cry1Ac-loaded MHNPs could remain on the surface of cotton leaves, resulting in enhanced adhesion of Cry1Ac protein by 59.50% and increased pest mortality by 75.00%. Additionally, MHNPs could be slowly decomposed by acid medium and MHNPs showed no obvious influence on cotton, Bt, Escherichia coli, and H. armigera. Therefore, MHNPs could serve as an efficient nanocarrier for delivery of Cry1Ac protein and be used as a potential adjuvant for biopesticide in agricultural applications.

Dietary Tryptophan Enhanced the Expression of Tight Junction Protein ZO-1 in Intestine.

Dietary amino acids provide various beneficial effects for our health. The aim of the present study was to assess the effects of tryptophan (Trp) supplementation on barrier function. Ninety-six healthy finishing pigs (initial body weight 51.49 ± 1.12 kg) were randomly allocated into 2 treatment groups, control group, and 0.2% Trp group. The control group was fed the basal diet, and 0.2% Trp group was fed basal diet plus 0.2% Trp. The trial period is 60 d. Compared with control group, the mRNA abundance of claudin-3 and zonula occluden-1 (ZO-1) in the jejunum in 0.2% Trp group (P < 0.05) was increased. According to immunohistochemistry and immunoblotting test, the expression of ZO-1 in jejunum in 0.2% Trp group was also significantly increased compared with the control group (P < 0.05). These results revealed that Trp enhanced the expression of tight junction protein ZO-1 in the intestine of pig model. Trp may be potential and beneficial dietary functional factor for regulating the intestinal development and inhibiting intestinal aging.

Development of selectable marker-free transgenic potato plants expressing cry3A against the Colorado potato beetle (Leptinotarsa decemlineata Say).

BACKGROUND: Elimination of selectable marker genes (SMGs) is important for the safe assessment and commercial use of transgenic plants. The destructive and invasive Colorado potato beetle (CPB) poses a serious threat to potato production. In response to this need, selectable marker-free transgenic potato lines expressing cry3A were developed to control the damage and spread of CPB. RESULTS: We simultaneously introduced cry3A and npt II genes harboured in different plasmids into the potato genome using the Agrobacterium-mediated cotransformation method. Four selectable marker-free transgenic potato (CT) lines expressing cry3A were developed by self-crossing segregation and molecular analyses, including Southern blot, western blot and enzyme-linked immunosorbent assay (ELISA) assays. CT lines were used in a resistance bioassay against CPB in the laboratory and field. In the laboratory, CT lines exhibited high resistance to CPB, and 100% mortality of first-instar larvae occurred 6 days after infestation. In the field, untransformed plant leaves were almost entirely consumed, with an average of 155 larvae present per plant 25 days after inoculation. However, CT lines showed no damage symptoms, with approximately 2.5 larvae surviving per plant. CONCLUSION: We successfully eliminated SMGs from the transgenic potato lines expressing cry3A in order to decrease CPB damage, control the spread of this pest eastwards and alleviate the concern regarding the safe assessment of regulatory requirements. © 2015 Society of Chemical Industry.

[Change of Bt protein in soil after growing Bt corns and returning corn straws to soil and its effects on soil nutrients].

The spatiotemporal dynamics of Bt protein in soil and the change of soil nutrients in rhizosphere soil, root surface soil and soils at 0-20, 20-40 and 40-60 cm were measured in greenhouse experiments. Two Bt corns, 5422Bt1 and 5422CBCL, and their near isogenic non-Bt variety 5422 were grown for 90 days and the crop residues were retained to soil. Results showed that 1.59 and 2.78 ng x g(-1) Bt protein were detected in the rhizosphere soil with Bt corns 5422Bt1 and 5422CBCL immediately after harvest. However, there were only trace amounts of Bt protein (< 0.5 ng x g(-1)) were detected in root surface soil after 90 days and in bulk soil in the two Bt corn treatments after 30, 60 and 90 days. When corn residues returned to soil, Bt protein declined rapidly within 3 days and only trace amounts of Bt protein were measured after 7 days. There were no sig- nificant differences in organic matter, available nutrient (alkaline hydrolytic N, available P, available K) or total nutrient (total N, total P, total K) in root surface soils and soils at 0-20 cm, 20-40 cm and 40-60 cm among the Bt and non-Bt corns after 90 days. Sixty days after returning crop residues of 5422Btl to soil, the contents of organic matter and total N increased and the content of available K reduced significantly in the 0-20 cm soil depth. There were no significant differences in any other parameter at the 0-20 cm depth, neither for any parameter in the 20-40 cm and 40-60 cm soil depths compared to those in the non-Bt corn 5422 treatment. There were no significant differences in soil nutrient contents in Bt corn 5422CBCL treatment compared to those in non-Bt corn 5422 treatment except that available phosphorus content was reduced in root surface soils, and total P content increased at the 0-20 cm soil depth after 90 days. When crop residues of Bt corn 5422 CBCL were returned to soil, only available P content in the 0-20 cm soil layer was evidently higher compared to the soil receiving crop residues of non-Bt corn 5422. Results suggested that Bt protein released from root and crop residues of Bt corns would not accumulate in soil, and growing Bt corns and returning crop residues to soil would have no significant effect on soil nutrients in general.

Voluntary activation of the sympathetic nervous system and attenuation of the innate immune response in humans.

Excessive or persistent proinflammatory cytokine production plays a central role in autoimmune diseases. Acute activation of the sympathetic nervous system attenuates the innate immune response. However, both the autonomic nervous system and innate immune system are regarded as systems that cannot be voluntarily influenced. Herein, we evaluated the effects of a training program on the autonomic nervous system and innate immune response. Healthy volunteers were randomized to either the intervention (n = 12) or control group (n = 12). Subjects in the intervention group were trained for 10 d in meditation (third eye meditation), breathing techniques (i.a., cyclic hyperventilation followed by breath retention), and exposure to cold (i.a., immersions in ice cold water). The control group was not trained. Subsequently, all subjects underwent experimental endotoxemia (i.v. administration of 2 ng/kg Escherichia coli endotoxin). In the intervention group, practicing the learned techniques resulted in intermittent respiratory alkalosis and hypoxia resulting in profoundly increased plasma epinephrine levels. In the intervention group, plasma levels of the anti-inflammatory cytokine IL-10 increased more rapidly after endotoxin administration, correlated strongly with preceding epinephrine levels, and were higher. Levels of proinflammatory mediators TNF-α, IL-6, and IL-8 were lower in the intervention group and correlated negatively with IL-10 levels. Finally, flu-like symptoms were lower in the intervention group. In conclusion, we demonstrate that voluntary activation of the sympathetic nervous system results in epinephrine release and subsequent suppression of the innate immune response in humans in vivo. These results could have important implications for the treatment of conditions associated with excessive or persistent inflammation, such as autoimmune diseases.

Effects of central injection of anti-LPS antibody and blockade of TLR4 on GnRH/LH secretion during immunological stress in anestrous ewes.

The present study was designed to examine the effect of intracerebroventricular (icv) administration of antilipopolysaccharide (LPS) antibody and blockade of Toll-like receptor 4 (TLR4) during immune stress induced by intravenous (iv) LPS injection on the gonadotropin-releasing hormone/luteinizing hormone (GnRH/LH) secretion in anestrous ewes. Injection of anti-LPS antibody and TLR4 blockade significantly (P < 0.01) reduced the LPS dependent lowering amount of GnRH mRNA in the median eminence (ME). Moreover, blockade of TLR4 caused restoration of LH- ß transcription in the anterior pituitary decreased by the immune stress. However, there was no effect of this treatment on reduced LH release. The results of our study showed that the blockade of TLR4 receptor in the hypothalamus is not sufficient to unblock the release of LH suppressed by the immune/inflammatory challenges. This suggests that during inflammation the LH secretion could be inhibited directly at the pituitary level by peripheral factors such as proinflammatory cytokines and circulating endotoxin as well.

Application of capillary electrophoretic chips in protein profiling of plant extracts for identification of genetic modifications of maize.

In this study, the chip gel electrophoresis with LIF detection was applied in protein profiling of fractionated and total extracts of maize standards. The sensitivity of such determinations can be enhanced by lyophilization of extracts or employing filtering and preconcentration with cutoff filters. Combinatorial peptide ligand library applied for sample processing prior to the electrophoretic analysis was, especially, an effective pretreatment step in the determination of low-abundance proteins. Several repeatable differences were observed for protein profiles between maize standards not containing the genetically modified organisms (GMOs) and those containing GMO, which can be potentially employed for identification of GMO in maize samples and foods of maize origin.

Potato flour mediated solid-state fermentation for the enhanced production of Bacillus thuringiensis-toxin.

In this study, we explored the efficacy of raw potato flour (PF) as supplement to the conventional LB medium (LB control, designated as M1) for enhancing the concomitant production of endospores and δ-endotoxin from Bacillus thuringiensis subsp. kurstaki by solid-state fermentation (SSF). Of different concentrations and combinations of media tested, 10% (w/v) PF supplemented LB medium (M2) was found as the best source for the maximum yield of toxin. After 12 h submerged fermentation (SmF) at 37°C and 125 rpm, M2 was made into a wet-solid matter for SSF by removing the supernatant (1000 ×g, 10 min); the resultant pellet subsequently incubated statically (37°C) for the production of B. thuringiensis subsp. kurstaki toxin (Btk-toxin). In comparison to M1, yield of δ-endotoxin purified by sucrose density gradient centrifugation method from M2 was about 6-fold higher (53% recovery). This maximum yield from M2 was obtained at 48 h (as against 72 h from M1), thus the gestation period of M2 was reduced by 24 h with higher yield. In addition to the quantitative data, qualitative photomicrographs taken by image analyzer, scanning electron and fluorescent microscopes and digital camera showed physical evidences for the upper hand of SSF over conventional SmF for the enhanced production of Btk-toxin. SDS-PAGE image of the purified δ-endotoxin showed three major fractions with apparent MWs 66, 45 and 30 kDa. Briefly, if low-cost agricultural products like PF is used as supplement to LB, by SSF strategy, production of Btk-toxin could be enhanced to 6-fold in short gestation time without losing its entomotoxicity efficiency.

Effect of Zingiber officinale and propolis on microorganisms and endotoxins in root canals

The purpose of this study was to evaluate the effectiveness of glycolic propolis (PRO) and ginger (GIN) extracts, calcium hydroxide (CH), chlorhexidine (CLX) gel and their combinations as ICMs (ICMs) against Candida albicans,Enterococcus faecalis, Escherichia coli and endotoxins in root canals. Material and Methods: After 28 days of contamination with microorganisms, the canals were instrumented and then divided according to the ICM: CH+saline; CLX, CH+CLX, PRO, PRO+CH; GIN; GIN+CH; saline. The antimicrobial activity and quantification of endotoxins by the chromogenic test of Limulus amebocyte lysate were evaluated after contamination and instrumentation at 14 days of ICM application and 7 days after ICM removal. Results and Conclusion: After analysis of results and application of the Kruskal-Wallis and Dunn statistical tests at 5% significance level, it was concluded that all ICMs were able to eliminate the microorganisms in the root canals and reduce their amount of endotoxins; however, CH was more effective in neutralizing endotoxins and less effective against C. albicans and E. faecalis, requiring the use of medication combinations to obtain higher success.

Inflammatory and degranulation effect of yellow sand on RBL-2H3 cells in relation to chemical and biological constituents.

Recent studie pointed out that allergic diseases have increased during the Asian dust storm event (ADSE) in Japan. Daily observations and the atmospheric concentrations of yellow sand (YS) aerosol have been increasing. In this study, YS samples collected from three sites of Japan during ADSE in 2009-2010 were used. The particles were analyzed by X-ray photoelectron spectroscopy (XPS) and X-ray fluorescence-energy dispersive spectrometer (XRF-EDS). We investigate ability of YS extract on enhancing the chemical mediator release and cytokine production from rat basophilic leukemia (RBL-2H3) cells. The dust particles at Fukuoka and Tsukuba were abundant in aluminum (Al), iron (Fe), potassium (K) and titan (Ti) than those at Naha. Concentration of the trace endotoxin and Cryptomeria japonica pollen allergen (Cry j 1) were measured in YS extract. After exposure of RBL-2H3 cells to YS extract, the ß-hexosaminidase (ß-hex) release, tumor necrosis factor-alpha (TNF-α) production were enhanced in RBL-2H3 cells. This process depends on endotoxin, Cry j 1 and other allergen present in the YS extract. YS water extract also show a strong cytotoxic effect on the cells. This data suggest that low levels of endotoxin and Cry j 1 in YS may cause allergy during the ADSE.

Evaluation of the resistance of transgenic potato plants expressing various levels of Cry3A against the Colorado potato beetle (Leptinotarsa decemlineata Say) in the laboratory and field.

BACKGROUND: The Colorado potato beetle (CPB), Leptinotarsa decemlineata Say, is a destructive pest. The CPB is a quarantine pest in China, but has now invaded the Xinjiang Uygur Autonomous Region and is continuing to spread eastwards. To control the damage and overspreading, transgenic potato plants expressing Cry3A toxin were developed, and their resistance to CPB was evaluated by bioassays in the laboratory and field in 2009, 2010 and 2011. RESULTS: The insect resistance of the high-dose (HD) transgenic lines was significantly greater than the middle-dose (MD) and low-dose (LD) transgenic lines regarding leaf consumption, biomass accumulation and mortality. The HD and MD transgenic lines showed 100% mortality when inoculated with first- and second-instar larvae; however, the LD transgenic lines showed about 50% mortality. The HD transgenic lines exhibited a significantly higher yield than the MD and LD transgenic lines owing to their high CPB resistance. CONCLUSION: Commercially available transgenic potato plants with above 0.1% Cry3A of total soluble protein and NT control refugia could control damage, delay adaptation and halt dispersion eastwards. The two HD transgenic lines developed in this study, PAH1 and PAH2, are ideal for use as cultivars or germplasm to breed new cultivars.

Molecular modeling of Bt Cry1Ac (DI-DII)-ASAL (Allium sativum lectin)-fusion protein and its interaction with aminopeptidase N (APN) receptor of Manduca sexta.

Genetic engineering of Bacillus thuringiensis (Bt) Cry proteins has resulted in the synthesis of various novel toxin proteins with enhanced insecticidal activity and specificity towards different insect pests. In this study, a fusion protein consisting of the DI-DII domains of Cry1Ac and garlic lectin (ASAL) has been designed in silico by replacing the DIII domain of Cry1Ac with ASAL. The binding interface between the DI-DII domains of Cry1Ac and lectin has been identified using protein-protein docking studies. Free energy of binding calculations and interaction profiles between the Cry1Ac and lectin domains confirmed the stability of fusion protein. A total of 18 hydrogen bonds was observed in the DI-DII-lectin fusion protein compared to 11 hydrogen bonds in the Cry1Ac (DI-DII-DIII) protein. Molecular mechanics/Poisson-Boltzmann (generalized-Born) surface area [MM/PB (GB) SA] methods were used for predicting free energy of interactions of the fusion proteins. Protein-protein docking studies based on the number of hydrogen bonds, hydrophobic interactions, aromatic-aromatic, aromatic-sulphur, cation-pi interactions and binding energy of Cry1Ac/fusion proteins with the aminopeptidase N (APN) of Manduca sexta rationalised the higher binding affinity of the fusion protein with the APN receptor compared to that of the Cry1Ac-APN complex, as predicted by ZDOCK, Rosetta and ClusPro analysis. The molecular binding interface between the fusion protein and the APN receptor is well packed, analogously to that of the Cry1Ac-APN complex. These findings offer scope for the design and development of customized fusion molecules for improved pest management in crop plants.

Assessing the removal potential of soil-aquifer treatment system (soil column) for endotoxin.

Soil-aquifer treatment (SAT) of wastewater is an increasingly valued practice for replenishing aquifers due to ease of operation and low maintenance needs and therefore low cost. In this study, we investigated the fate of endotoxins through laboratory-scale SAT soil columns over a four month period. The effluent of rapid sand filtration was run through the columns under gravity flow conditions. Four SAT columns were packed with four different filter materials (fine sand, medium sand, coarse sand and very coarse sand). The effluent of rapid sand filtration (average dissolved organic carbon (DOC) = 4 mg l(-1) and average endotoxin concentration = 4 EU ml(-1)) was collected from a domestic wastewater treatment plant in Sapporo, Japan. DOC removal ranged from 12.5% to greater than 22.5% during the study, with DOC levels averaging 3.1 and 3.5 mg l(-1) for the SAT columns packed with different soils. Endotoxin transformation exhibited different profiles, depending on the time and soil type. Reduction in endotoxin concentration averaged 64.3% and was as high as 86.7% across the soil columns 1, 2, 3 and 4, respectively. While DOC removal was gradual, the reductions in endotoxin levels were rather rapid and most of the removal was achieved in the top layers. Soil with a larger grain size had lower efficiency in removing endotoxin. Tests were performed to evaluate the transformation of organic matter showing endotoxicity and to determine the mechanisms responsible for changes in the structural and size properties of dissolved organic matter (OM) during SAT. Dissolved OM was fractionated using Sep-Pack C18 Cartridges into hydrophobic and hydrophilic fractions. Dialysis tubes with different molecular weight cut-offs were used to perform size fractions of OM showing endotoxicity. Evaluation of the transformation of organic matter showing endotoxicity during SAT indicated that both hydrophobic and large molecules were reduced. Moreover, experimental findings showed that adsorption test data fit to the Freundlich isotherm and were affected by the particle grain size with higher adsorption capacity for fine and medium sand.

The oral toxicity of the transgenic Bt+CpTI cotton pollen to honeybees (Apis mellifera).

Transgenic insect-resistant cotton has been planted in China in a large scale and may have adverse impacts on honeybees. Pollens from the transgenic Cry1Ac+CpTI cotton Zhong-41 and the parental cotton Zhong-23 were collected from the field and their impacts on adult worker bees were assessed. Experimental results showed that Zhong-41 pollen had no acute oral toxic effect on worker bees. No significant differences were observed in the superoxide dismutase activity or in the longevity of worker bees fed with diets containing the two cotton pollens. The main reasons for the outcome may be the low expression level of the transgenic proteins Cry1Ac and CpTI in the pollen of Zhong-41 as well as the substantial equivalence in the amounts of gross protein and soluble saccharides for the two cotton pollens. The implications of these results are discussed and further work to be carried out is put forward.

ATR-FTIR study of lipopolysaccharides at mineral surfaces.

Lipopolysaccharides (LPS) are ubiquitous in natural aqueous systems because of bacterial cell turnover and lysis. LPS sorption and conformation at the mineral/water interface are strongly influenced by both solution and surface chemistry. In this study, the interaction of LPS with various surfaces (ZnSe, GeO(2), alpha-Fe(2)O(3), alpha-Al(2)O(3)) that vary in surface charge and hydrophobicity was investigated using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The presence of Ca(2+) (versus Na(+)) in LPS solutions resulted in aggregate reorientation and increased sorptive retention. ATR-FTIR spectra of Na-LPS systems are consistent with reduced surface affinity and are similar to those of solution phase LPS. Ca-LPS spectra reveal hydrophobic interactions of the lipid A region at the ZnSe internal reflection element (IRE). However, pH-dependent charge controls Ca-LPS sorption to hydrophilic surfaces (GeO(2), alpha-Fe(2)O(3), and alpha-Al(2)O(3)), where bonding occurs principally via O-antigen functional groups. As a result of accumulation at the solid-liquid interface, spectra of Ca-LPS represent primarily surface-bound LPS. Variable-angle ATR-FTIR spectra of Ca-LPS systems show depth-dependent trends that occur at the spatial scale of LPS aggregates, consistent with the formation of vesicular structures.

Adjuvant effect of lemnan, pectic polysaccharide of callus culture of Lemna minor L. at oral administration.

A pectic polysaccharide, lemnan LMC, was extracted from the callus of duckweed Lemna minor L. and was tested for adjuvant properties at oral administration with protein antigen. Mice were orally immunized thrice with weekly interval with free hen's egg lysozyme or lysozyme with LMC. Lemnan LMC was shown to increase delayed type hypersensitivity and serum antilysozyme IgG responses. LMC was established to increase levels of both serum IgG1 and IgG2a subclasses. The concentration of malondialdehyde and myeloperoxidase activity were found to be higher in the tissue samples obtained from small intestine of mice immunized with mixture of lysozyme/LMC than those immunized with lysozyme only. Thus, lemnan appeared to be useful as the adjuvant for oral immunization.

Single amino acid mutations in the cadherin receptor from Heliothis virescens affect its toxin binding ability to Cry1A toxins.

Bacillus thuringiensis Cry protein exerts its toxic effect through a receptor-mediated process. Both aminopeptidases and cadherin proteins were identified as putative Cry1A receptors from Heliothis virescens and Manduca sexta. The importance of cadherin was implied by its correlation with a Cry1Ac resistant H. virescens strain (Gahan, L. J., Gould, F., and Heckel, D. G. (2001) Science 293, 857-860). In this study, the Cry1Ac toxin-binding region in H. virescens cadherin was mapped to a 40-amino-acid fragment, from amino acids 1422 to 1440. This site overlaps with a Cry1Ab toxin-binding site, amino acids 1363-1464 recently reported in M. sexta (Hua, G., Jurat-Fuentes, J. L., and Adang, M. J. (2004) J. Biol. Chem. 279, 28051-28056). Further, feeding of the anti-H. virescens cadherin antiserum or the partial cadherins, which contain the toxin-binding region, in combination with Cry1Ab/Cry1Ac reduced insect mortality by 25.5-55.6% to first instar H. virescens and M. sexta larvae, suggesting a critical function for this cadherin domain in insect toxicity. Mutations in this region, to which the Cry1Ac binds through its loop 3, resulted in the loss of toxin binding. For the first time, we show that the cadherin amino acids Leu(1425) and Phe(1429) are critical for Cry1Ac toxin interaction, and if substituted with charged amino acids, result in the loss of toxin binding, with a K(D) of < 10(-5) m. Mutation of Gln(1430) to an alanine, however, increased the Cry1Ac affinity 10-fold primarily due to an increase on rate. The L1425R mutant can result from a single nucleotide mutation, CTG --> CGG, suggesting that these mutants, which have decreased toxin binding, may lead to Cry1A resistance in insects.