A nontoxicPseudomonas exotoxin a induces active immunity and passive protective antibody againstPseudomonas exotoxin a intoxication

Pseudomonas exotoxin A (PE) is one of the most potent cytotoxic agents produced byPseudomonas aeruginosa. In this study, we examined the possibility of using PE with a deletion of 38 carboxyl-terminal amino acid residues, designated PE(576–613), for active immunization against PE-mediated disease. We first examined the toxic effects of PE and PE(576–613) on 5- and 9-week-old ICR mice. The results show that the subcutaneous administration of PE(576–613) at a dose of 250 µg was still nontoxic to 5- and 9-week-old ICR mice, while native PE was lethal at a dose of 0.5 and 1 µg, respectively. PE(576–613) was then used to immunize ICR mice. The minimum dose of PE(576–613) that could effectively induce anti-PE antibodies in 5- and 9-week-old ICR mice was found to be 250 ng. However, immunization with 250 ng PE(576–613) failed to protect the immunized mice from a lethal dose of PE. The effective immunization dose of PE(576–613) that could protect mice against a 2 µg PE challenge was found to be 15 µg. In addition, sera obtained from PE(576–613)-immunized ICR mice were able to neutralize PE intoxication and effectively protect mice from PE. Thus, PE(576–613) may be used as an alternative route to new PE vaccine development.     

铜绿假单胞菌外毒素A的生产,分离纯化和鉴定

通过对培养基组成、种子活化、接种量和培养条件进行优化,使铜绿假单胞菌外毒素Ａ（ＰＥ）产量达到每毫升５－１０μｇ和１９２小鼠ＬＤ５０,不低于国外报道水平。经二步纯化,ＰＥ蛋白回收率为３３．３３％（ＰＥ）和１６．６７％（ＬＤ５０）,提纯系数为４３８．５（ＰＥ）或２１８．５（ＬＤ５０）,ＳＤＳ－ＰＡＧＥ呈现一条带,相对分子质量为６６０００,琼脂糖扩散鉴定与兔抗ＰＥ产生一条沉淀线,小鼠半数致死量为０．１５     

A hyman hybrid hybridoma producing a bispecific monoclonal antibody that can target tumor cells for attack by Pseudomonas aeruginosa exotoxin A

By fusing a human hybridoma producing an IgG2 antibody against human A431 epidermoid carcinoma cells with an Epstein-Barr virus-transformed human B lymphocyte producing an IgG2 antibody against Pseudomonas aeruginosa exotoxin A, we established a hybrid hybridoma producing a bispecific monoclonal antibody reacting with both A431 cells and the exotoxin. Human IgG was purified from the culture supernatant of the hybrid hybridoma, and the bispecific monoclonal antibody in the IgG preparation was further separated from the two parental antibodies by hydroxyapatite high-performance liquid chromatography. The human bispecific monoclonal antibody thus obtained efficiently targeted the antibody-reative cells, A431, for attack by the exotoxin in vitro.Abbreviations bs mAb Bispecific Monoclonal Antibody - HRP Horseradish Peroxidase - MHA Mixed Hemadsorption Assay - MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide - PEA Pseudomonas aeruginosa Exotoxin A - PEG Polyethylene Glycol     

Interspecific hybrids between Brassica napus L. and B. oleracea L. developed by embryo culture

Summary Interspecific hybrids between Brassica napus and B. oleracea are difficult to produce, and previous attempts to transfer economic characters from one species to the other have largely been unsuccessful. In these studies, oilseed rape cv. Tower (2n38) (B. napus) was crossed with broccoli and kale (2n18) (B. oleracea), and hybrid plants were developed from embryos in culture by either organogenesis or somatic embryogenesis. In rape × broccoli, F₁ plants were regenerated from hybrid embryos and the plants produced viable selfed seeds. F₅ plants (2n38) homozygous for white flower colour were selected for high oil content (47%) and Line 15; a selection from these plants produced fertile hybrids with rape, broccoli and kale without embryo culture. In reciprocal crosses between oilseed rape cv. Tower and an aphid resistant diploid kale, 28 and 56 chromosome F₁ hybrid plants were regenerated from somatic embryos. The 56 chromosome plants were self-fertile and it was concluded from F₂ segregation ratios that a single dominant gene controls resistance to cabbage aphid in kale. The 28 chromosome F₁'s were self-sterile, but these and the 56 chromosome F₁'s could be backcrossed to rape and kale. A cross between the F₁ (2n56) and a forage rape resulted in the selection of a cabbage aphid (Brevicoryne brassicae L.) resistant line (Line 3). Both Line 15 and Line 3 can serve as bridges for gene interchange between B. campestris, B. napus and B. oleracea, which has not been possible hitherto. Hybridisations between rape and tetraploid kale produced F₁ plants with 37 chromosomes. One F₂ plant possessed coronal scales and the inheritance was shown to be controlled by a single recessive gene unlinked to petal colour.This paper is dedicated to Mr. T. P. Palmer, a colleague and close friend who retired from the DSIR as Assistant Director of the Crop Research Division in September 1984     

Optimization of expression and purification of two biologically active chimeric fusion proteins that consist of human interleukin-13 and Pseudomonas exotoxin in Escherichia coli

We have previously reported that a variety of solid human tumor cell lines express a large number of receptors for interleukin-13 (IL-13). These receptors could be targeted with a chimeric fusion protein consisting of human IL-13 and a truncated form of Pseudomonas exotoxin (PE). We describe here optimization of critical steps involved in high yield expression of two recombinant chimeric fusion proteins for obtaining highly purified and biologically active cytotoxins in Escherichia coli. The chimeric constructs of human IL-13 and two 38 kDa truncated PEs: (i) PE38 and (ii) PE38QQR, (three lysine residues in PE38 at 590, 606, and 613 substituted with two glutamine and one arginine) were used for protein expression in pET prokaryotic expression vector system with kanamycin as a selection antibiotic. Our results suggest that fresh transformation of E. coli and induction by isopropyl-beta-D-thiogalactopyranoside (IPTG) for 6 h resulted in maximum protein expression. To further improve the yield, we used a genetically modified E. coli strain, BL21(DE3)pLysS, which carries a plasmid for lysozyme with a weak promoter that inhibits T7 RNA polymerase and minimizes protein production in the absence of IPTG. Use of this strain eliminated the need for lysozyme digestion of the induced bacteria to release inclusion bodies, which resulted in expression of purer protein as compared to the conventional BL21(DE3) strain. Additional protocol optimizations included 16 h solubilization of inclusion bodies, constitution of refolding buffer, and timing of dialysis. These proteins were finally purified by Q-Sepharose, mono-Q, and gel filtration chromatography. Between 14-22 and 21-28 mg highly purified and biologically active protein was obtained from 1L of BL21 (DE3) and BL21 (DE3) pLysS bacteria culture, respectively. As IL-13R targeting for brain tumor therapy offers an exciting treatment option, optimization of production of IL-13PE will enhance production of clinical grade material for Phase III clinical trials.     

Unusual sequence of an abscisic acid-inducible mRNA which accumulates late in Brassica napus seed development

We have analyzed the nucleotide sequence and accumulation of an mRNA which is prevalent in seeds of Brassica napus L. During normal development, the mRNA begins to accumulate during late embryogeny, is stored in dry seeds, and becomes undetectable in seedlings within 24 hours after imbibition. Moreover, abscisic acid treatment of embryos precociously induces or enhances accumulation of the mRNA. Nucleotide sequencing studies show that the deduced 30 kDa polypeptide has an unusual primary structure; the polypeptide possesses direct amino acid sequence repeats and is virtually entirely hydrophilic with the exception of a hydrophobic carboxyl-terminal region. Based upon the expression pattern and predicted polypeptide sequence, we conclude that the mRNA is encoded by a late embryogenesis-abundant (Lea) gene in B. napus.Abbreviations ABA abscisic acid - bp base pairs - DAF days after flowering - HAI hours after the start of imbibition - kb kilobase (pairs)     

Non-zygotic embryos of Brassica napus L. contain embryo-specific storage proteins

The storage-protein content of non-zygotic and zygotic embryos of B. napus was compared, using antibodies to guantitate 12S storage protein in extracts by rocket immunoelectrophoresis. Non-zygotic embryos were induced from microspores in anther culture and on the hypocotyls of zygotic embryos in culture. All embryo-like structures were found to contain 12S storage protein, whereas preculture anthers, anthers from which embryos had been removed, and regenerated shoots did not have detectable 12S storage protein. In zygotic embryos, 12S storage protein was first detected at the cotyledon stage, but microsporic embryos contained storage protein at the globular and heart stages. Storage protein levels in microsporic and hypocotyl embryos were low relative to those in zygotic embryos. The largest microsporic embryo had a storage protein concentration of 13 g mg^-1 fresh weight, almost 10 times lower than a mature zygotic embryo. Thus, although storage proteins are present in both zygotic and non-zygotic embryos, the timing and extent of accumulation differ.     

Iron uptake regulation in Pseudomonas aeruginosa

The Pseudomonas genus belongs to the γ division of Proteobacteria and many species produce the characteristic yellow–green siderophore pyoverdine, and often a second siderophore, of lower affinity for iron. These bacteria are known for their ability to colonize different ecological niches and for their versatile metabolism. It is therefore not surprising that they are endowed with the capacity to take up exogenous xenosiderophores via different TonB-dependent receptors. Uptake of iron is controlled by the central regulator Fur, and via extracytoplasmic sigma factors or other types of regulators (two-component systems, AraC regulators). In this review the Fur regulon (experimentally proven and/or predicted) of P. aeruginosa will be presented. An interesting feature revealed by this analysis of Fur-regulated genes is the overlap between the iron and the sulfur regulons as well with the quorum sensing system.     

Transfer of resistance against Phoma lingam to Brassica napus by asymmetric somatic hybridization combined with toxin selection

Summary Irradiated mesophyll protoplasts from nine different accessions of B. juncea, B. nigra and B. carinata, all resistant to Phoma lingam, were used as gene donors in fusion experiments with hypocotyl protoplasts isolated from B. napus as the recipient. A toxin, sirodesmin PL, was used to select those fusion products in which the resistant gene(s) was present. In the fusion experiments different gene donors, various irradiation dosages and toxin treatments were combined. Symmetric and asymmetric hybrid plants were obtained from the cell cultures with and without toxin selection. Isozymes were used to verify hybrid characters in the symmetric hybrids, whereas two DNA probes were used to identify donor-DNA in the asymmetric hybrids. Resistance to P. lingam was expressed in all symmetric hybrids, and in 19 of 24 toxin-selected asymmetric hybrids, while all the unselected asymmetric hybrids were susceptible.     

Heterologous expression and regulation of the lysA genes of Pseudomonas aeruginosa and Escherichia coli

Summary Chlorsulfuron-resistant mutants of Arabidopsis thaliana were isolated by screening for growth of seedlings in the presence of the herbicide. Both whole plants and derived tissue cultures were resistant to concentrations of the herbicide approximately 300-fold higher than that required to prevent growth of the wild-type. The resistance is due to a single dominant nuclear mutation at a locus designated csr which has been genetically mapped to chromosome-3. Acetohydroxy acid synthase activity in extracts from chlorsulfuron-resistant plants was much less-susceptible to inhibition by chlorsulfuron and a structurally related inhibitor than the activity in wild-type extracts. This suggests that the csr locus is the structural gene for acetohydroxy acid synthase.     

Development and storage-protein synthesis in Brassica napus L. embryos in vivo and in vitro

Immature embryos of Brassica napus were cultured in vitro with and without various concentrations of germination inhibitors, and the progress of embryogeny was monitored by comparing accumulation of storage proteins in culture with the normal accumulation in seeds. The two major B. napus storage proteins (12S and 1.7S) were purified from seed extracts and analyzed by rocket immunoelectrophoresis (12S protein) or by sodium lauryl sulfate polyacrylamide gel electrophoresis (1.7S protein). During embryo development within seeds both the 12S and 1.7S proteins were first detected when the cotyledons were well developed (embryo dry weight, 0.4 mg), and each storage protein accumulated at an average rate of 26 g d^-1 during maximum deposition. Accumulation of the 1.7S protein stopped when the water content of the embryo began to decline (embryo DW, 2.7 mg), but accumulation of the 12S protein continued until seed maturity (embryo DW, 3.6 mg). At the end of embryo development the 12S and the 1.7S proteins comprised approx. 60 and 20% of the total salt-soluble protein, respectively. When embryos were removed from seeds at day 27, just as storage protein was starting to accumulate, and placed in culture on a basal medium, they precociously germinated within 3d, and incorporation of amino acids into the 12S storage protein dropped from 3% of total incorporation to less than 1%. If 10^-6 M abscisic acid (ABA) was included in the medium, amino-acid incorporation into the 12S protein increased from 3% of total incorporation when embryos were placed into culture to 18%, 5d later, and the accumulation rate (27.1±2.6 g embryo^-1 d^-1) matched the maximum rate observed in the seed. High osmotica, such as 0.29 M sucrose or mannitol, added to the basal medium, also inhibited precocious germination, but there was a lag period before 12S-protein synthesis rates equaled the rates on ABA media. These results indicate that some factor in the seed environment is necessary for storage-protein synthesis to proceed, and that ABA is a possible candidate.Abbreviations ABA abscisic acid - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - SDS sodium lauryl sulfate     

An estimation of the proton conductance of the inner membrane of turnip (Brassica napus L.) mitochondria

The permeability of the inner membrane of turnip mitochondria to H⁺ and OH^- ions has been investigated using an acid pulse technique. The rate of decay of a H⁺ pulse across the inner membrane is exponential having first-order kinetics and gives t 1/2 values of approx 54 s at neutral pH and at 25° C. Valinomycin or 1799 alone have little effect on t 1/2 values, whereas in combination, values of <15 s are observed. Nigericin produces a similar effect. The effective proton conductance of the inner membrane near pH 7 at 25° C is 0.27 nmol H⁺ min^-1 mg protein^-1 mV^-1. The results suggest that at neutral pH, the inner membrane of plant mitochondria is relatively impermeable to H⁺ and OH^- ions.     

The Brassica napus extA extensin gene is expressed in regions of the plant subject to tensile stresses

The expression of extA, an extensin gene from Brassica napus L. (oilseed rape) was examined in transgenic Nicotiana tabacum L. (tobacco) and untransformed Brassica juncea L. and B. napus tissues. Northern analysis showed that this gene maintained its normal pattern of expression when transferred to tobacco. In transgenic tobacco plants containing an extA promoter/-glucuronidase coding sequence fusion, expression of extA was detected in the external and internal phloem of the main stem. High expression levels were seen in cortical parenchyma cells at the point where the axillary flowering branch joined the main stem. Expression was greatest in regions where the maximum tensile stress would seem to be exerted on the main stem by the weight of the axillary branch. It was confirmed that this expression pattern was due to tensile stress by using weights to induce expression of the fusion gene in axillary flowering stalks. In B. juncea pods, in-situ hybridisation studies showed that the extensin gene was strongly expressed in cells of the carpel walls within which considerable tensile stresses develop.Abbreviations GUS -glucuronidase - X-GLUC 5-bromo-4-chloro-3-indolyl glucuronide - SSC saline sodium citrate We thank the Biotechnology and Biological Sciences Research Council for funding this research, and Mrs. W. Grail for her expertise in histochemical technique. Ms. J. Spence is the recipient of a Biotechnology and Biological Sciences Research Council post-graduate studentship award.     

Effects of an antisense napin gene on seed storage compounds in transgenic Brassica napus seeds

To manipulate the quantity and quality of storage components in Brassica napus seeds, we have constructed an antisense gene for the storage protein napin. The antisense gene was driven by the 5-flanking region of the B. napus napin gene to express antisense RNA in a seed-specific manner. Seeds of transgenic plants with antisense genes often contained reduced amounts of napin. In some transgenic plants, no accumulation of napin was observed. However, the total protein content of transgenic and wild-type seeds did not differ significantly. Seeds lacking napin accumulated 1.4 to 1.5 times more cruciferin than untransformed seeds, although the oleosin content was not affected. Fatty acid content and composition in the seeds of transgenic plants were also analyzed by gas chromatography. Though the total fatty acid content of the transformants was the same as that of non-transformants, there was a reduction in 18:1 contents and a concomitant increase of 18:2 in seeds with reduced napin levels. This observed change in fatty acid composition was inherited in the next generation.     

T-DNA tagging in Brassica napus as an efficient tool for the isolation of new promoters for selectable marker genes

A simple strategy to identify and isolate new promoters suitable for driving the expression of selectable marker genes is described. By employing a Brassica napus hypocotyl transformation protocol and a promoterless gus::nptII tagging construct, a series of 20 kanamycin-resistant tagged lines was produced. Most of the regenerated plants showed hardly any GUS activity in leaf, stem and root tissues. However, expression was readily restored in callus tissue induced on in vitro leaf segments. Genomic sequences upstream of the gus::nptII insertions were isolated via plasmid rescue. Three clones originating from single copy T-DNA lines were selected for further evaluation. The rescued plasmids were cloned as linear fragments in binary vectors and re-transformed to Brassica napus hypocotyl and Solanum tuberosum stem segments. The new sequences maintained their promoter activity, demonstrated by transient and stable GUS activity after transformation. Furthermore, the promoters provided sufficient expression of the nptII gene to yield transgenic plants when using kanamycin as selective agent. Database searching (BLASTN) revealed that the promoters have significant homology with three Arabidopsis BAC clones, one Arabidopsis cDNA and one Brassica napus cDNA. The results presented in this paper illustrate the strength of combined methods for identification, isolation and testing of new plant promoters.