The role of β18-β19 loop structure in insecticidal activity of Cry1Ac toxin from Bacillus thuringiensis

The β18-β19 loop in domain Ⅲ of Cry1Ac toxin is unique among Bacillus thuringlensis Cry proteins. In this study, the role of the loop structure in insecticidal activity of Cry1Ac toxin was investigated. Alanine scanning mutations within the loop were initially generated and most mutants were over-expressed and reduced toxicity at different degrees, except mutant N546A that showed almost 2 times enhanced toxicity against Helicoverpa armigera larvae. Further mutagenic analysis of N546 revealed that a charged amino acid in this position would cause very unfavorable influence on insecticidal activity. In addition, the deletion of N546 led to protein instability because of destruction of the loop integrity. Besides, mutant W544F was much more toxic than W544Y, indicating that hydrophobic nature of the position was important for maintaining the stability and activity of Cry1Ac protein. These findings are the first biological evidence for a structural function of β18-β19 loop in insecticidal activity of the Cry1Ac toxin.     

Transgenic tobacco plants expressing synthetic Cry1Ac and Cry1Ie genes are more toxic to cotton bollworm than those containing one gene

Transgenic tobacco plants carrying Cry1Ac, Cry1Ie or both genes were obtained. In the leaves of transgenic plants carrying both genes, the contents of Cry1Ac and Cry1Ie proteins were 0.173% and 0.131% of the total proteins, respectively. Cry1Ac protein content was 0.182% and Cry1Ie protein con- tent was 0.124% of the total proteins in the leaves of transgenic plants containing only one Bt gene. Fresh leaves of transgenic tobacco and wild-type plants were used for the insect bioassay against wild-type and Cry1Ac-resistant cotton bollworm (Helicoverpa armigera). The bioassay results showed that transgenic plants carrying both genes were significantly more toxic to wild-type and Cry1Ac-resistant cotton bollworm than those carrying Cry1Ac or Cry1Ie alone. This study indicates that the higher toxicity of transgenic tobacco plants carrying both genes is caused by the cooperative function of both Bt proteins, thus providing a potential way to delay the development of insect resis- tance to transgenic crops.     

Involvement of DEG5 and DEG8 proteases in the turnover of the photosystem II reaction center D1 protein under heat stress in Arabidopsis thaliana

Deg5,deg8 and the double mutant,deg5deg8 of Arabidopsis thaliana were used to study the physiological role of the DEG proteases in the repair cycle of photosystem II (PSII) under heat stress. PSII activity in deg mutants showed increased sensitivity to heat stress, and the extent of this effect was greater in the double mutant, deg5deg8, than in the single mutants, deg5 and deg8. Degradation of the D1 protein was slower in the mutants than in the WT plants. Furthermore, the levels of other PSII reaction center proteins tested remained relatively stable in the mutant and WT plants following high-temperature treatment. Thus, our results indicate that DEG5 and DEG8 may have synergistic function in degradation of D1 protein under heat stress.     

Analysis of proteins that interact with nucleocapsid protein of SARS-CoV using 1 5-mer phage-displayed library

Analysis of proteins that interact with N protein of SARS-CoV using 15-mer phage-displayed library will help to explore the virus pathogenesis and to develop new drugs and vaccines against SARS. In this study, we cloned, expressed and purified N protein of SARS-CoV. This 46-kD N protein was verified by SDS-PAGE and Western-blot. Then, the peptides binding-specific to N protein were identified using 15-mer phage-displayed library. Surprisingly, all of the 89 clones from monoclonal ELISA were positive （S/N〉2.1） and the result was further confirmed experimentally once again. Six N protein-binding peptides, designated separately as SNA1, SNA2, SNA4, SNA5, SNA9 and SNG11, were selected for sequencing. Sequence analysis suggested that SNA5 shared approximatively 100% sequence identity to SNA4, SNA2, SNA9 and SNA1. In addition, the binding specificity of the 15-mer peptides with the SARS-CoV N protein was further demonstrated by blocking ELISA using the synthetical 15-mer peptide according to the deduced amino acid sequence of SNA5. Also, the deduced amino sequence of SNA5 was compared with proteins in translated database using the tblastx program, and the results showed that the proteins with the highest homology were Ubiquinol-cytochrome c reductase iron-sulfur subunits （UCRI or UQCR）, otherwise known as the Rieske iron-sulfur proteins （RISP）. Notablely, in the [2Fe-2S] redox centre of UCRI, there were 6 residues [GGW（Y）F（Y）CP] compatible to the residues （position 2→7, GGWFCP7） of the NH2-terminal of the 15-mer peptide, which indicated higher binding specificity between the N protein of SARS-CoV and the redox centre of UCRI to some extent. Here, the possible molecular mechanisms of SARS-CoV N protein in the pathogenesis of SARS are discussed.     

Acute toxicity and oxidative damage induced by silica nanorattle in vivo

Hollow mesoporous nanomaterial is a kind of promising new drug delivery system due to their unique hollow structures.In order to evaluate the toxicity of silica nanorattle (SN) particles in vivo,40 female mice were used in this study to investigate the acute toxicity and oxidative damage.Mice were intravenously injected with SN suspension in sterile 5% glucose at 40,80 and 240 mg/kg,respectively.The control group was administrated with equal-volume 5% glucose.Weight,feed intake,hematology analysis,blood biochemical assay and histopathology diagnosis were examined.The activities of SOD,GSH and CAT were measured as well.The results demonstrated that the levels of ALT and AST in the mice treated with 240 mg/kg SN increased significantly as compared with the control group (P<0.05),whereas the contents of BUN and CREA changed unremarkably.The activity of SOD induced by SN in the liver decreased significantly (P<0.05).In summary,this study revealed that liver was the target organ of the SN.It also can be concluded that activity of SOD played an important role in liver injury caused by SN.     

Agrobacterium tumefaciens-mediated Transformation of CryⅠA(b) gene to Trichoderma harzianum

In this study, Cry ⅠA(b) gene was successfully transferred into the biocontrol fungus Trichoderma harzianum with an efficiency of 60-180 transformants per 10^6 spores by using Agrobacterium tumefaciens-mediated transformation. Putative transformants were analyzed to test the presence of Cry ⅠA(b) gene by Southern blot. Most transformants contained a single T-DNA copy. RT-PCR analysis showed that the Cry ⅠA(b) gene was transcribed. Antifungal activities and insecticidal activities of the transformants were examined. There was no obvious difference in antifungal activities between the transformants and their wild strains. The modified mortalities of the transformants T1 and T2 were 69.57% and 91.30%, respectively. The tranformation system mediated by A. tumefaciens proved to be a powerful tool for the filamentous fungi transformation and functional genomic study with its high transformation frequency, simplicity of T-DNA integration, and genetic stability of transformants.     

Structure modeling and spatial epitope analysis for HA protein of the novel H1N1 influenza virus

In recent months, a novel influenza virus H1N1 broke out around the world. With bioinformatics technology, the 3D structure of HA protein was obtained, and the epitope residues were predicted with the method developed in our group for this novel flu virus. 58 amino acids were identified as potential epitope residues, the majority of which clustered at the surface of the globular head of HA protein. Although it is located at the similar position, the epitope of HA protein for the novel H1N1 flu virus has obvious differences in the electrostatic potential compared to that of HA proteins from previous flu viruses.     

Ubi1 intron-mediated enhancement of the expression of Bt cry1Ah gene in transgenic maize (Zea mays L.)

The cry1Ah gene was one of novel insecticidal genes cloned from Bacillus thuringiensis isolate BT8. Two plant expression vectors containing cry1Ah gene were constructed. The first intron of maize ubiqutin1 gene was inserted between the maize Ubiquitin promoter and cry1Ah gene in one of the plant expressing vectors (pUUOAH). The two vectors were introduced into maize immature embryonic calli by microprojectile bombardment, and the reproductively plants were acquired. PCR and Southern blot analysis showed that foreign genes had been integrated into maize genome and inherited to the next generation stably. The ELISA assay to T1 and T2 generation plants showed that the expression of Cry1Ah protein in the construct containing the ubi1 intron (pUUOAH) was 20% higher than that of the intronless construct (pUOAH). Bioassay results showed that the transgenic maize harboring cry1Ah gene had high resistance to the Asian corn borers and the insecticidal activity of the transgenic maize containing the ubi1 intron was higher than that of the intronless construct. These results indicated that the maize ubi1 intron can enhance the expression of the Bt cry1Ah gene in transgenic maize efficiently     

Ubi1 intron-mediated enhancement of the expression of Bt cry1Ah gene in transgenic maize （Zea mays L.）

The cry1Ah gene was one of novel insecticidal genes cloned from Bacillus thuringiensis isolate BT8. Two plant expression vectors containing cry1Ah gene were constructed. The first intron of maize ubiqutinl gene was inserted between the maize Ubiquitin promoter and cry1Ah gene in one of the plant expressing vectors （pUUOAH）. The two vectors were introduced into maize immature embryonic calli by microprojectile bombardment, and the reproductively plants were acquired. PCR and Southern blot analysis showed that foreign genes had been integrated into maize genome and inherited to the next generation stably. The ELISA assay to T1 and T2 generation plants showed that the expression of CrylAh protein in the construct containing the ubil intron （pUUOAH） was 20% higher than that of the intronless construct （pUOAH）. Bioassay results showed that the transgenic maize harboring cry1Ah gene had high resistance to the Asian corn borers and the insecticidal activity of the transgenic maize containing the ubil intron was higher than that of the intronless construct. These results indicated that the maize ubil intron can enhance the expression of the Bt cry1Ah gene in transgenic maize efficiently     

Construction of a new plant expression vector containing two insect resistant genes and its expression in transgenic tobacco plants

A new plant expression vector (pBS29K-BA) containing two insect resistant genes, a synthetic chimeric gene BtS29K encoding the activated insecticidal protein Cry1Ac and a gene API-BA encoding the arrowhead (Sagittaria sagittifolia L.) proteinase inhibitor (API) A and B, is constructed. Transgenic tobacco plants expressing these two genes are obtained through Agrobacterium-mediated transformation of tobacco leaf discs. The average expression levels of Cry1Ac and API-BA proteins in transgenic plants are of 3.2 μg and 4.9 μg per gram fresh leaf respectively. The results of insecticidal assay of transgenic plants indicate that the pBS29K-BA transformed plants are more resistant to insect damage than the plants expressing the Cry1Ac gene or API-BA gene alone.     

Production of β-1,3-glucanase and chitinase of two biocon trol agents and their possible modes of action

Pichia membranefaciens Hansen and Candida guilliermondii(Cast) Langeronet Guerra are two antagonists of R. stolonifer on harvested nectarine and peach fruits. In this study, β-1,3-glucanase and chitinase activities of the antagonists were induced in vitro and in vivo. The highest β-1,3-glucanase activity was detected in Lilly-Barnett minimal salt medium supplemented with glucose in combination with CWP of R. stolonifer as a carbon source. The β-1,3-glucanase activity of P. membranefaciens reached the maximum level,being 114.0 SU (specific activity unit), and that of C. guilliermondii reached 103.2 SU. The lowest β-1,3-glucanase activity was observed in the medium containing glucose as sole carbon source. P. membranefaciens was able to produce significantly higher levels of chitinase (exochitinase and endochitinase) in vitro than C. guilliermondii grown in Czapeck minimal medium. An increase in β-1,3-glucanase and chitinase activity was also triggered by wounding, adding of carbon sources and yeast cells. The results showed that both β-1,3-glucanase and chitinase from P. membranefaciens and C. guilliermondii exhibited some effects on controlling R.stolonifer, and might have a synergistic activity against R.stolonifer.     

Triphenyltin inhibition of the proteasome activity and its influence on substrate protein levels in nerve cells

Triphenyltin (TPT) widely exists as environmental pollutant, but its toxicity towards nerve cells and the related mechanism remain unclear. In this research, SHSY-5Y cells were exposed to TPT, and the cellular proteasome activity, Iκb proteins, Bax and α-synuclein levels were investigated. The results show that TPT can inhibit the cellular proteasome activity, and result in the accumulation of ubiquitinized proteins. TPT exposure can change the protein levels of IκB, Bax, and α-synuclein, affect NFκB pathwa...     

Important amino acid in the hemagglutinin glycoprotein of measles virus (MV) that governs hemadsorption

Two strains of measles virus IMA and SMD that were isolated using B95a cell line show hemadsorption negative. After adaptation to Vero cells, these strains gain the ability to agglutinate AGM-RBC and show hemadsorption positive. Sequence analysis of these two kinds of virus reveals that the two strains all have a Ser (HAD negative) to Gly (HAD positive) mutation at position 546 in the H protein. Site-directed mutagenesis and expression in COS cells were used to confirm that the mutation Ser→Gly is responsible for hemadsorption alteration. Then the monoclonal antibody against CD46 was used to identify that this mutation also governs the binding function of MV H protein to CD46 receptor. The data provide a new important amino acid of MV H protein that governs the hemadsorption and binding to CD46 receptor.     

Characterization of the separate kinase domain of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

The bifunctional enzvme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase consists of two dis tinct domains which catalyze the synthesis and hydrolysis of fructose-2, 6-bisphosphate, respectively. In this work the properties of the separate 6-phosphofructo-2-kinase domain were investigated. Purification of the expressed separate do main or isolation of this domain from purified glutathione S-transferase (GST) fusion protein with thrombin cleavage led to the loss of its kinase activity. Thus the domain in the GST-tagged form was characterized. The two forms of the do main with different lengths (amino acids 1 ～ 249 and 1 ～ 286) were very similar in kinetic property and could catalyze the formation of fructose-2,6-bisphosphate with a kcat 4-fold lower than that of the full-length enzyme. In addition, the domain was much more sensitive to guanidine inactivation and heat denaturation, and less stable at pH values below 7 than the full-length enzyme. The results suggest that the separate kinase domain of the bifunctional enzyme is far less perfect in structure in the absence of the bisphosphatase domain, though it still possesses the 6-phosphofructo-2-kinase activity.     

β-Adrenergic activation enhances NMDA-induced current in pyramidal cells of the basolateral nucleus of amygdala

NMDA receptor （NMDA-R） in the amygdala complex is critical for both long-term potentiation （LTP） and formation of conditioned fear memory. It is reported that activation of β-adrenoceptors （β-AR） in the amygdala facilitates LTP and enhances memory consolidation. The present study examined the regulatory effect of β-AR activation on NMDA-R mediated current in pyramidal cells of the basolateral nucleus of amygdala （BLA）, using whole-cell recording technique. Bath application of the β-AR agonist isoproterenol enhanced NMDA-induced current, and this facilitatory effect was blocked by co-administered propranolol, a β-AR antagonist. The facilitatory effect of isoproterenol on NMDA-induced current could not be induced when the protein kinase A （PKA） inhibitor Rp-cAMPs was added in electrode internal solution.The present results suggest that β-AR activation in the BLA could modulate NMDA-R activity directly and positively, probably via PKA.     

Isolation and mutation rate of the spontaneous PUR box and purR mutants

In our previous research in purine metabolism in Salmonella typhimurium, it was observed that the mutation frequency of the PUR box was one order of magnitude higher than that of purR under mutagenesis. In order to investigate further into this phenomenon, large amounts of independent PUR box and purR spontaneous mutants were isolated on lactose minimal medium by using a super-repressing mutant of purR (purRs). The mutational regions of 5 PUR box mutants and 4 purR mutants were cloned. Nucleotide sequence analysis showed that all the mutants had mutations at the expected sites. The comparison of the two types of mutations indicated that, although the purR gene was two orders of magnitude larger than the box). Thus, we concluded that high mutation frequency of the PUR box did not result from mutagenesis. Under spontaneous conditions, the mutation frequency of PUR box was also high. Some tentative explanations of this interesting phenomenon are given in this report.     

Development of the model of pancreatic β-cell membrane chromatography and its chromatographic characteristics

A new model of pancreatic β-cell membrane chromatography has been established by using a β-cell membrane stationary phase （β-CMSP） prepared by immobilizing the β-cell membrane onto the surface of silica carrier. The protein level and K^＋, Na^＋-ATP enzymatic activity of the β-CMSP were detected respectively. The surface characteristics of the β-CMSP were tested by using the scanning electron microscope and surface energy spectrometer. In this model, the column （10 mm × 2 mm, I.D.） packed with β-CMSP, 25 mmol/ammonium sulfate buffer solution （pH 7.4） as mobile phase with the flow rate of 0.2 mL/min at 37 ± 0.5℃ were used in the following studies. The retention characteristics of the sulfonylureas （gliquidone, glibenclamide, gliclazide and glipizide） were investigated under the chromatographic conditions above. The affinities of the sulfonylureas on β-cell membrane and receptor will be expressed by using the logk＇ values （the logarithm of capacity factor of a solute） in the model. The correlation of the affinity with the pharmacological effect of the sulfonylureas was analyzed also.