Interactions between molecular chaperone P20 and Cyt2Ba7 toxin inBacillus thuringiensis

P20 or 20-kilodalton protein is a molecular chaperone protein inBacillus thuringiensis(Bt) which can increase yields and facilitates crystal formation of various insecticidal crystal proteins (ICPs). In previous studies, aB. thuringiensisinsecticidal protein gene,cyt2Ba7, was cloned, expressed but its expression level is very low inB. thuringiensis. In this study, various expression vectors were constructed by incorporatingp20 in forward or reverse direction in the upstream ofcyt2Ba7 and transformed into aB. thuringiensisacrystalliferous strain 4Q7. The result showed that in the presence of P20, the expression of Cyt2Ba7 was significantly increased. Especially whenp20 gene was reversely inserted in the upstream ofcyt2Ba7 gene, the expression of Cyt2Ba7 was increased ∼3.2 times meanwhile more and bigger crystals were observed under electron microscopy. By using purified Cyt2Ba7, P20 protein and P20-specific antiserum, immunoblotting and ligand blot analysis demonstrated a strong binding affinity between P20 and Cyt2Ba7. These results reveal that P20 can promote the crystal formation and enhance the expression of Cyt2Ba7 as a molecular chaperone, which can be a powerful tool to boost the ICPs production inB. thuringiensisand help develop more effective insect control strategies.

Insecticidal Specificity of Cry1Ah to Helicoverpa armigera Is Determined by Binding of APN1 via Domain II Loops 2 and 3

ABSTRACT Bacillus thuringiensis Cry1Ah protein is highly toxic against Helicoverpa armigera but shows no toxicity against Bombyx mori larvae. In contrast, the closely related Cry1Ai toxin showed the opposite phenotype: high activity against B. mori but no toxicity against H. armigera. Analysis of binding of Cry1Ah to brush border membrane vesicle (BBMV) proteins from H. armigera and B. mori by surface plasmon resonance revealed association of toxin binding with insect specificity. Pulldown experiments identified aminopeptidase N1 (APN1) as a Cry1Ah binding protein that was not observed in the assays using B. mori BBMV proteins. The APN1 Cry1Ah binding region was narrowed to the region from A548 to S798 (fragment H3) by expressing four different APN1 fragments in Escherichia coli and analyzing Cry1Ah binding by ligand blot. Binding competition experiments of Cry1Ah to APN1 fragment H3 using synthetic peptides corresponding to four predicted domain II loop regions showed that loop 2 and loop 3 have additive effects on binding to APN1 fragment H3. Moreover, switching of loop 2 and loop 3 regions from Cry1Ah to Cry1Ai toxins showed that loop 2 and loop 3 are both involved in specificity and toxicity against H. armigera. IMPORTANCE Domain II loop regions have been shown to be involved in binding to larval gut proteins mediating insect specificity. The modification of loop regions is a direct and effective method to construct new Cry toxin variants to increase toxicity or modify specificity. Our results show that the exchange of loop regions from one toxin into another is a successful scheme for modification of B. thuringiensis Cry toxin specificity.

Development of a Ligand Blot Assay Using Biotinylated Live Cells

Adhesive interactions between cells are critical to a variety of processes, including host-pathogen relationships. The authors have developed a new technique for the observation of binding interactions in which molecules obtained from excised tissues are resolved by gel electrophoresis and transferred to a membrane. Biotinylated live cells are then kept in contact with that membrane, and their interactions with proteins of interest are detected by peroxidase-labeled streptavidin, followed by a biotin-streptavidin detection system. The adhesion proteins can eventually be identified by cutting the relevant band(s) and performing mass spectrometry or other amino acid—sequencing methods. The technique described here allows for the identification of both known and novel adhesion molecules capable of binding to live cells, among a complex mixture and without previous isolation or purification. This is especially important for the analysis of host-parasite interactions and may be extended to other types of cell-cell interactions. ( Journal of Biomolecular Screening 2007:1006-1010)

Identification of a 71 kDa protein as a putative non-genomic membrane progesterone receptor in boar spermatozoa

A putative non-genomic progesterone receptor was identified by Western blot analysis from the membrane fraction but not the cytosolic fraction of boar spermatozoa using monoclonal antibody (mAb) C-262. When the membrane and the cytosolic fractions of boar liver, kidney, uterus and spermatozoa were analyzed with mAb C-262, protein bands with molecular masses of 86 and 120 kDa were detected from the cytosolic fraction of the uterus, whereas a 71 kDa protein was detected from the membrane fraction of spermatozoa. Apparently, while the 86 and 120 kDa proteins from the uterus correspond to the genomic progesterone receptor isoforms A and B in boar, the 71 kDa protein of the sperm membrane fraction seems to be a novel membrane-associated progesterone receptor. Ligand blot assay of the membrane and the cytosolic fractions of boar spermatozoa performed with peroxidase-conjugated progesterone revealed that only the 71 kDa membrane protein binds specifically to progesterone, reinforcing the results obtained from the Western blot analysis. Also ligand blot assays performed in the presence of mAb C-262 demonstrated that mAb C-262 inhibited progesterone binding to the 71 kDa protein in a dose-dependent manner. Ligand blot assays performed in the presence of free progesterone, RU486 or estrogen revealed that binding of peroxidase-conjugated progesterone to the 71 kDa protein was inhibited by free progesterone and RU486 in a dose-dependent manner but not by estrogen, which further confirms that progesterone binds to the 71 kDa protein specifically. Furthermore, the progesterone-induced acrosome reaction was inhibited by mAb C-262 in a dose-dependent manner. These results strongly imply that spermatozoa possess a progesterone receptor in a membrane-bound form and can be influenced by progesterone via non-genomic progesterone receptor.

IGF-I, IGF-II, 'free' IGF-I and IGF-binding proteins-2 to -6 during high-dose oestrogen treatment in constitutionally tall girls

OBJECTIVE: To investigate the effect of high-dose oestrogen treatment on IGF-I, IGF-II, free-dissociable IGF-I and the IGF-binding proteins (IGFBP)-2 to -6 in girls with constitutional tall stature. METHODS: In patient cohort 1, blood samples were drawn before and after 3 months of daily oral treatment with 0.1 mg ethinyloestradiol. In cohort 2, samples were collected at the end of the treatment period and 3 to 6 months afterwards. IGFs and IGFBPs were analysed by specific immunoassays and by Western ligand blot. RESULTS: Total IGF-I decreased significantly on oestrogen treatment and increased again after oestrogen withdrawal. Ligand blot analysis showed a clear reduction in a 34 kDa band, corresponding to IGFBP-2, and a strong induction of a 24 kDa band, corresponding to the non-glycosylated form of IGFBP-4. These changes were confirmed by specific immunological methods. The serum levels of IGFBP-3, IGFBP-5 and IGFBP-6 remained unchanged during the first 3 months of treatment. In cohort 2, IGFBP-3 and IGFBP-6 increased after oestrogen withdrawal. Free-dissociable IGF-I fell to 35+/-4% during oestrogen therapy and rose again when the treatment was stopped. CONCLUSIONS: Oestrogens modulate the serum concentrations of several components of the IGF system. The fall in total IGF-I is not explained by a decrease in IGFBPs but probably results from a decreased synthesis.