<Emphasis Type="Italic">Prays oleae</Emphasis> Midgut Putative Receptor of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Vegetative Insecticidal Protein Vip3LB Differs from that of Cry1Ac Toxin

Vegetative insecticidal protein (Vip) is a class of insecticidal proteins produced by many Bacillus thuringiensis strains during their vegetative growth stage. The vip3LB gene of B. thuringiensis strain BUPM95, which encodes a protein active against the Lepidoptera olive tree pathogenic insect Prays oleae, was cloned into pET-14b vector and overexpressed in Escherichia coli. The expressed Vip3LB protein, found in the E. coli cytoplasmic fraction, was purified and used to produce anti-Vip3LB antibodies. Using the midgut extract of P. oleae, the purified Vip3LB bound to a 65-kDa protein, whereas Cry1Ac toxin bound to a 210-kDa midgut putative receptor. This result justifies the importance of the biological pest control agent Vip3LB that could be used as another alternative particularly in case of resistance to Cry toxins.     

Effects on capacitance by overexpression of membrane proteins

Functional Channelrhodopsin-2 (ChR2) overexpression of about 10⁴ channels/μm² in the plasma membrane of HEK293 cells was studied by patch-clamp and freeze-fracture electron microscopy. Simultaneous electrorotation measurements revealed that ChR2 expression was accompanied by a marked increase of the area-specific membrane capacitance (C_m). The C_m increase apparently resulted partly from an enlargement of the size and/or number of microvilli. This is suggested by a relatively large C_m of 1.15 ± 0.08 μF/cm² in ChR2-expressing cells measured under isotonic conditions. This value was much higher than that of the control HEK293 cells (0.79 ± 0.02 μF/cm²). However, even after complete loss of microvilli under strong hypoosmolar conditions (100 mOsm), the ChR2-expressing cells still exhibited a significantly larger C_m (0.85 ± 0.07 μF/cm²) as compared to non-expressing control cells (0.70 ± 0.03 μF/cm²). Therefore, a second mechanism of capacitance increase may involve changes in the membrane permittivity and/or thickness due to the embedded ChR2 proteins.     

Overexpression of alginate lyase of <Emphasis Type="Italic">Pseudoalteromonas elyakovii</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>, purification,and characterization of the recombinant alginate lyase

The alyPEEC gene encoding alginate lyase from marine bacterium Pseudoalteromonas elyakovii IAM 14594 was subcloned into pBAD24 with arabinose promoter and sequenced, and overexpressed in TOP10 strain of E. coli after arabinose induction. Expression levels of alyPEEC gene in E. coli cells were over 39.6-fold higher than those in P. elyakovii IAM 14594 cells. The molecular mass of purified alginate lyase from the engineered E. coli cells was estimated to be 32.0 kDa. Optimum pH and temperature of the alginate lyase activity were 7.0 and 30 °C, respectively. The enzyme was unstable on heating and in acidic and alkaline solution. The enzyme activity was stimulated by the MgCl₂, NaCl, KCl, CaCl₂, BaCl₂ and MnCl₂, but was inhibited by the addition of 1.0 mM of EGTA, EDTA, SDS, ZnSO₄, AgNO₃, and CoCl₂. All the alginate, polyM and polyG could be converted into oligosaccharides with more than tetrasaccharides by the purified recombinant alginate lyase, suggesting that the recombinant alginate lyase produced by the engineered E. coli has highly potential application in seaweed genetics, food and pharmaceutical industries.     

Down‐regulation of BnDA1, whose gene locus is associated with the seeds weight,improves the seeds weight and organ size in Brassica napus

Brassica napus L. is an important oil crop worldwide and is the main raw material for biofuel. Seed weight and seed size are the main contributors to seed yield. DA1 (DA means big in Chinese) is an ubiquitin receptor and negatively regulates seed size. Down‐regulation of AtDA1 in Arabidopsis leads to larger seeds and organs by increasing cell proliferation in integuments. In this study, BnDA1 was down‐regulated in B. napus by over expressed of AtDA1^R358K, which is a functional deficiency of DA1 with an arginine‐to‐lysine mutation at the 358th amino acid. The results showed that the biomass and size of the seeds, cotyledons, leaves, flowers and siliques of transgenic plants all increased significantly. In particular, the 1000 seed weight increased 21.23% and the seed yield per plant increased 13.22% in field condition. The transgenic plants had no negative traits related to yield. The candidate gene association analysis demonstrated that the BnDA1 locus was contributed to the seeds weight. Therefore, our study showed that regulation of DA1 in B. napus can increase the seed yield and biomass, and DA1 is a promising target for crop improvement.     

Diverse cellular transformation capability of overexpressed genes in human hepatocellular carcinoma

For isolation of novel cellular transforming genes that potentially participated in hepatocarcinogenesis, we conducted anchorage-independent growth (AIG) assays on 10 human liver cancer cell lines and observed strong AIG capabilities in PLC5 and Huh7 but negligible in Tong cells. After cloning of genes by differential subtractive chain reactions (DSC) from strong AIG to AIG negative cells, we sequenced 2304 clones and identified 245 genes. After four stringent criteria for selection of transforming genes among DSC clones, our results of quantitative RT-PCR analysis indicated that six genes, DDX3, EIF3S2, CLIC1, HDGF, GPC3, and HSPCA were overexpressed in 64%, 62%, 60%, 58%, 49%, and 47%, respectively, of 45 hepatocellular carcinoma (HCC) tissues. The results of cellular transformation capability by AIG assays indicated that the transfectants of EIF3S2 showed the strongest (> 100-fold), DDX3 and CLIC1 were moderate, GPC3 and HSPCA were weak, and HDGF was none in forming colonies in soft agar. Together, our results suggested that Tong is a suitable human cell line for screening of overexpressed and/or cellular transforming genes. In addition, our results suggested that diverse functions of cellular transforming genes in various biological pathways could transform human Tong cells and potentially reveal new targets for drug development of HCC.     

Expression, regulation and function of the homeobox gene empty spiracles in brain and ventral nerve cord development of Drosophila

We analyse the role of the empty spiracles (ems) gene in embryonic brain and ventral nerve cord development. ems is differentially expressed in the neurectoderm of the anterior head versus the trunk region of early embryos. A distal enhancer region drives expression in the deutocerebral brain anlage and a proximal enhancer region drives expression in the VNC and tritocerebral brain anlage. Mutant analysis indicates that in the anterior brain ems is necessary for regionalized neurogenesis in the deutocerebral and tritocerebral anlagen. In the posterior brain and VNC ems is necessary for correct axonal pathfinding of specific interneurons. Rescue experiments indicate that the murine Emx2 gene can partially replace the fly ems gene in CNS development.     

Cloning, sequencing and expression in Escherichia coli of a thermophilic lipase from Bacillus thermoleovorans ID-1

A thermophilic lipase of Bacillus thermoleovorans ID-1 was cloned and sequenced. The lipase gene codes 416 amino acid residues and contains the conserved pentapeptide Ala-X-Ser-X-Gly as other Bacillus lipase genes. The optimum temperature of the lipase is 75 degrees C, which is higher than other known Bacillus lipases. For expression in Escherichia coli, the lipase gene was subcloned in pET-22b(+) vector with a strong T7 promoter. Lipase activity was approximately 1.4-fold greater than under the native promoter.     

Constitutive activity of human angiotensin II type-1 receptors by Gq overexpression

We have developed an inducible HEK293/Tet-On cell line that transiently expresses both FLAG-tagged human angiotensin II type-I receptors (FLAG-hAT(1)R) and G(q)alpha G protein subunits in response to doxycycline. High and tightly regulated levels of FLAG-hAT(1)R (740+/-57 fmol/mg protein) and G(q)alpha (36-fold increase compared with non-induced cells) overexpression were consistently achieved. We investigated the possibility of using an inducible system to increase the proportion of constitutively active wild-type FLAG-hAT(1)Rs by overexpressing G(q)alpha. Following doxycycline treatment, we observed no significant change in the apparent binding affinity or potency (coupling efficiency) of angiotensin II, though significant increases in the intrinsic activity of several partial agonists were observed, indicative of constitutive activity. DUP753 (10 microM), a suggested inverse agonist, did not inhibit the enhanced level of basal (agonist-independent) activity. The data suggest that the resting equilibrium of hAT(1) receptors between the inactive (R) and active (R*) forms is predominantly weighted towards the inactive conformation.