Characterization of cultured insect cells selected by <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> crystal toxin

Summary Selection for resistance against Bacillus thuringiensis (Bt) Cry1Ac10 in the Trichoplusia ni (Hübner) cell line BTI-TN-5B1-4 (TnH5) was tested, and the development of resistance in the selected cells was like a S-form curve. Monitoring at the Cry1Ac10 50th challenge, the resistance ratio was 1, 294-fold as many as that of initial cells. But the resistance to Cry1Ac10 declined gradually when the selection was relaxed. The resistance declined rapidly at the low level of resistance and slowly at the high level of resistance. This resistant cell had high resistance to all the tested solubilized trypsin-treated mixture of crystal multitoxins of B. thuringiensis subsp. aizawai GC-91, an engineering bacterium of Bt, B. thuringiensis subsp. aizawai HD-133 and B. thuringiensis subsp. kurstaki HD-1, and low cross-resistance (19.7-fold) to activated Cry1C. Both N-acetyl-d-galactosamine (GalNAc) and tunicamycin did not inhibit the toxicity of Cry1Ac10 to the susceptible TnH5 cells. Comparison of the total proteins of the selected resistant cells with that of the nonselected susceptible cells by two-dimensional electrophoresis analysis showed that were obvious differences among the 11 protein expression. These results strongly suggest that there exists an unknown mechanism of resistance in the cell line that was different from the reported mechanisms in insects.     

A DNA vaccine co-expressing antigen and an anti-apoptotic molecule further enhances the antigen-specific CD8+ T-cell immune response

We have shown that DNA encoding the anti-apoptotic protein Bcl-xL enhances E7-specific CD8+ T-cell responses and DNA encoding pro-apoptotic protein caspase-3 suppresses E7-specific CD8+ T-cell responses when co-administered intradermally via gene gun with DNA encoding human papillomavirus type 16 (HPV-16) E7 linked to the sorting signal of the lysosome-associated membrane protein type 1 (LAMP-1). E7 and LAMP-1 are linked to form the chimeric Sig/E7/LAMP-1 (SEL). Because co-administration does not ensure delivery of both constructs to a single cell, we used pVITRO, a mammalian expression vector with double promoters, to ensure expression of both molecules in the same cell. We vaccinated C57BL/6 mice with pVITRO-SEL-Bcl-xL, pVITRO-SEL-mtBcl-xL, pVITRO-SEL, or pVITRO-SEL-caspase-3 intradermally via gene gun and intramuscularly via injection. We demonstrated that vaccination with pVITRO achieved similar results to a co-administration strategy: that Bcl-xL enhanced the E7-specific CTL response and caspase-3 suppressed the E7-specific CTL response. In addition, we found intradermal vaccination elicited significantly higher numbers of E7-specific CD8+ T cells compared to intramuscular vaccination. Thus, intradermal vaccination with a pVITRO vector combining an anti-apoptotic strategy (Bcl-xL) and an intracellular targeting strategy (SEL) further enhances the E7-specific CD8+ T-cell response and guarantees co-expression of both encoded molecules in transfected cells.T.W.K. and C.-F.H. contributed equally to this work.     

Local and systemic inhibition of lung tumor growth after nanoparticle-mediated mda-7/IL-24 gene delivery

The human melanoma differentiation associated gene-7 (mda-7), also known as interleukin-24 (IL-24), is a novel gene with tumor suppressor, antiangiogenic, and cytokine properties. In vitro adenovirus-mediated gene transfer of the human mda-7/IL-24 gene (Ad-mda-7) results in ubiquitous growth suppression of human cancer cells with minimal toxicity to normal cells. Intratumoral administration of Ad-mda-7 to lung tumor xenografts results in growth suppression via induction of apoptosis and antiangiogenic mechanisms. Although these results are encouraging, one limitation of this approach is that its locoregional clinical application-systemic delivery of adenoviruses for treatment of disseminated cancer is not feasible at the present time. An alternative approach that is suitable for systemic application is non-viral gene delivery. We recently demonstrated that DOTAP:cholesterol (DOTAP:Chol) nanoparticles effectively deliver tumor suppressor genes to primary and disseminated lung tumors. In the present study, therefore, we evaluated nanoparticle-mediated delivery of the human mda-7/IL-24 gene to primary and disseminated lung tumors in vivo. We demonstrate that DOTAP:Chol efficiently delivers the mda-7/IL-24 gene to human lung tumor xenografts, resulting in suppression of tumor growth. Growth-inhibitory effects were observed in both primary (P=0.001) and metastatic lung tumors (P=0.02). Furthermore, tumor vascularization was reduced in mda-7/IL-24-treated tumors. Finally, growth was also inhibited in murine syngenic tumors treated with DOTAP:Chol-mda-7 nanoparticles (P=0.01). This is the first report demonstrating (1) systemic therapeutic effects of mda-7/IL-24 in lung cancer, and (2) antitumor effects of human mda-7 in syngeneic cancer models. Our findings are important for the development of mda-7/IL-24 treatments for primary and disseminated cancers.     

Expression of ricin A chain and ricin A chain-KDEL in Escherichia coli

Ricin and its A chains can be used to conjugate with monoclonal antibodies to prepare immunotoxins. Ricin A chain (RTA) and its modification RTA-KDEL (ER-retrieval signal) were expressed with the pKK223.3 system in Escherichia coli under control of a tac promoter. The recombinant proteins can be purified by one-step affinity chromatography on a column of Blue-Sepharose 6B. The toxicities of RTA and its mutant RTA-KDEL were evaluated by the MTT assay in HeLa, MCF, and ECV-304 cells following fluid-phase endocytosis. RTA-KDEL was somewhat more cytotoxic than RTA itself in the different cell lines. The results suggest that rRTA-KDEL may be useful for the synthesis of more potent immunotoxins.     

An improved strategy for automated electron microscopic tomography

A prediction-based scheme is proposed and implemented for automated electron microscopic tomography. By assuming that the sample follows a simple geometric rotation and that the optical system can be characterized in terms of an offset between the optical and mechanical axes, it is found that the image movement in the x, y, and z directions due to stage tilt can be dynamically predicted with desired accuracy (15 nm in x-y position and 100 nm in focus). Thus, the microscope optical system (beam/image shift and focus) can be automatically adjusted to compensate for the predicted image movement prior to taking the projected image at each tilt angle. As a consequence, it is not necessary to either record additional images for tracking and focusing during the course of data collections or to spend valuable setup time in a lengthy pre-calibration of stage motions. Furthermore, this scheme is also found to tolerate a significant degree of non-eucentricity and to be quite robust in the collection of regular and cryo low-dose images on thin or thick samples even at magnifications greater than 62000x and angular step as large as 10 degrees. For interested users the software can be freely downloaded for non-profit use at http://www.msg.ucsf.edu/tomography.     

Toxicity of <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> LP1-G Against Susceptible and Resistant <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis> and the Cloning of the Mosquitocidal Toxin Gene

Bacillus sphaericus LP1-G, belonging to flagellar serotype H3, has been found to have moderate toxicity against two resistant Culex quinquefasciatus colonies (RLCq1 and RLCq2) and the susceptible contrast (SLCq). With an aim of screening mosquitocidal acting factor, a partial genome library was prepared from a partial HindIII digest of the total DNA from Bacillus sphaericus LP1-G. Two thousand twenty Escherichia coli clones were screened for toxicity against susceptible SLCq, and a toxic clone, designated E-UL68, was chosen for further study. The recombinant E-UL68 performed toxicity against both susceptible and two resistant colonies, having the same level of toxicity as that of wide-type strain LP1-G. Sequence analysis revealed that the inserted fragment was composed of 3876 nucleotides and contained a complete gene, whose sequence was identical to that of the mtx gene from B. sphaericus SSII-1. Because the binary toxin produced during sporulation of strain LP1-G has no activity against the target mosquitoes, this indicates that the Mtx toxin or other active factors might perhaps be responsible for the toxicity of LP1-G against different colonies of mosquito larvae.Received: 7 October 2002 / Accepted: 4 November 2002     

Lead discovery of alpha,beta-unsaturated sulfones from a combinatorial library as inhibitors of inducible VCAM-1 expression

alpha,beta-Unsaturated sulfones have been discovered from a combinatorial library as leads for a new series of inhibitors of inducible VCAM-1 expression. Although not essential, further conjugation of the sulfonyl group to another vinyl group or a phenyl group increases the potency dramatically.     

Copper complex of hydroxyl-substituted triazamacrocyclic ligand and its antitumor activity

The protonation constants and the stability constants for the formation of copper (II) complex of the ligand [1,4,7] Triazecan-9-ol (L) were presented. Antitumor activity of CuL complex was reported. Preliminary pharmacological tests showed that it had antitumor activity against HXO-RB44 and BEL-7402 cell lines in vitro. Nuclei of [CuL]-stimulated BEL-7402 cells clearly exhibited condensation and break down into chromatin clumps typical of apoptosis. Also it exhibited perturbation effects to BEL-7402 cell lines cycle and further studies showed that it could cleave supercoiled DNA (pBR 322) to nicked and linear DNA.     

两种过滤特征基因选择算法的有效性研究

对基因表达谱进行特征基因选择不仅能改善疾病分类方法的效能,而且为寻找与疾病相关的特征基因提供新的途径．通过比较用调整p值的t检验、非参数评分两种特征基因选择算法后和未进行选择时支持向量机(SVM)分类器的分类性能、支持向量(SV)的吻合度、错分样本ID的吻合度和对样本均匀翻倍后的稳定性．结果发现：特征选择后线性、核函数为二阶多项式和径向基的SVM分类性能明显提高;特征选择前后的SV及错分样本ID的吻合度均较高;SVM的稳定性较好．由此得出结论：这两种特征选择算法具有一定的有效性．