Increasing antioxidant levels in tomatoes through modification of the flavonoid biosynthetic pathway.

Flavonoids are a diverse group of phenolic secondary metabolites that occur naturally in plants and therefore form an integral component of the human diet. Many of the compounds belonging to this group are potent antioxidants in vitro and epidemiological studies suggest a direct correlation between high flavonoid intake and decreased risk of cardiovascular disease, cancer and other age-related diseases. Enhancing flavonoid biosynthesis in chosen crops may provide new raw materials that have the potential to be used in foods designed for specific benefits to human health. Using genetic modification, it was possible to generate several tomato lines with significantly altered flavonoid content and to probe the role and importance of several key enzymatic steps in the tomato flavonoid biosynthetic pathway. Most notably an up to 78-fold increase in total fruit flavonols was achieved through ectopic expression of a single biosynthetic enzyme, chalcone isomerase. In addition, chalcone synthase and flavonol synthase transgenes were found to act synergistically to up-regulate flavonol biosynthesis significantly in tomato flesh tissues.

Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols.

Tomatoes are an excellent source of the carotenoid lycopene, a compound that is thought to be protective against prostate cancer. They also contain small amounts of flavonoids in their peel ( approximately 5-10 mg/kg fresh weight), mainly naringenin chalcone and the flavonol rutin, a quercetin glycoside. Flavonols are very potent antioxidants, and an increasing body of epidemiological data suggests that high flavonoid intake is correlated with a decreased risk for cardiovascular disease. We have upregulated flavonol biosynthesis in the tomato in order to generate fruit with increased antioxidant capacity and a wider range of potential health benefit properties. This involved transformation of tomato with the Petunia chi-a gene encoding chalcone isomerase. Resulting transgenic tomato lines produced an increase of up to 78 fold in fruit peel flavonols, mainly due to an accumulation of rutin. No gross phenotypical differences were observed between high-flavonol transgenic and control lines. The phenotype segregated with the transgene and demonstrated a stable inheritance pattern over four subsequent generations tested thus far. Whole-fruit flavonol levels in the best of these lines are similar to those found in onions, a crop with naturally high levels of flavonol compounds. Processing of high-flavonol tomatoes demonstrated that 65% of flavonols present in the fresh fruit were retained in the processed paste, supporting their potential as raw materials for tomato-based functional food products.

Antibody fragment Fv4155 bound to two closely related steroid hormones: the structural basis of fine specificity.

BACKGROUND: The concentration of steroid glucuronides in serial samples of early morning urine (EMU) can be used to predict the fertile period in the female menstrual cycle. The monoclonal antibody 4155 has been used as a convenient means of measuring the concentration of steroid glucuronides in EMU, as it specifically recognises the steroid hormone estrone beta-D-glucuronide (E3G), with very high affinity, and the closely related hormone estriol 3-(beta-d-glucuronide) (EI3G), with reduced affinity. Although 4115 binds these hormones with different affinities, EI3G differs from E3G only in the addition of a hydroxyl group and reduction of an adjacent carbonyl. To investigate the structural basis of this fine binding specificity, we have determined the crystal structures of the variable fragment (Fv) of 4155 in complex with each of these hormones. RESULTS: Two crystal forms of the Fv4155-EI3G complex, at resolutions of 2.1 A and 2.5 A, and one form of the Fv4155-E3G complex, at 2.1 A resolution were solved and refined. The crystal structures show the E3G or EI3G antigen lying in an extended cleft, running form the centre of the antibody combining site down one side of the variable domain interface, and formed almost entirely from residues in the heavy chain. The binding cleft lies primarily between the heavy chain complementarity determining regions (CDRs), rather than in the interface between the heavy and light chains. In both complexes the binding of the glucuronic sugar, and rings A and B of the steroid, is specified by the shape of the narrow cleft. Analysis of the Fv structure reveals that five of the six CDR regions can be assigned to one of the predefined canonical structural classes. CONCLUSIONS: The difference in the binding affinity of Fv4155 for the two steroid hormones is accounted for by a subtle combination of a less favoured hydrogen-bond geometry, and a minor rearrangement of the water molecule network around the binding site. The rearrangement of water molecules results from the burial of the additional hydroxyl group of the EI3G in a hydrophobic environment.

Construction of a reshaped HMFG1 antibody and comparison of its fine specificity with that of the parent mouse antibody.

A human antibody with milk mucin specificity was obtained by transferring the complementarity determining regions (CDR) of the mouse antibody HMFG1 onto carefully selected human framework regions. The resulting reshaped human antibody, HuHMFG1, showed no difference in relative affinity for its antigen compared with the parent mouse HMFG1. The minimum epitope recognized by both the mouse and reshaped antibodies was demonstrated by epitope mapping to be identical, and consists of the tetramer PDTR. In a replacement net analysis, in which each of the amino acids was replaced in turn with the 19 other residues, it was determined that mouse HMFG1 and HuHMFG1 reacted with this series of synthetic peptides in an equivalent manner, indicating retention of identical fine specificity in the HuHMFG1 antibody. In contrast to other published reports, this was achieved without involvement of any framework residues in the binding site transfer. These data demonstrate that if well-matching human framework regions are employed grafting the CDR only can be sufficient to confer desired specificities to human antibodies and can, indeed, provide human analogues of mouse antibodies with virtually indistinguishable affinities and fine specificities relative to the mouse parent antibodies.

Reshaping human monoclonal antibodies for imaging and therapy.

Current problems in obtaining suitable monoclonal antibodies (MoAbs) for in vivo imaging and therapy can now be bypassed using protein-engineering rather than conventional hybridoma technology. Rodent antibody binding sites can be grafted onto human acceptor antibodies thus reshaping the human MoAbs into reagents with more desired specificities. We successfully exploited this technology to produce two reshaped human antibodies with the same specificities and very similar binding abilities as demonstrated for the mouse antibodies H17E2 and HMFG1.

EcoK selection vectors for shotgun cloning into M13 and deletion mutagenesis.

For shotgun cloning into M13 vectors, a double-stranded cassette of synthetic oligonucleotides containing a SmaI site within the two halves of an EcoK site, has been introduced into the vector M13mp8. Cloning of blunt end DNA into the SmaI site destroys the EcoK site, and recombinants are therefore preferentially selected on transfection into a K strain of E.coli. For deletion mutagenesis using synthetic oligonucleotides, an M13 vector with four copies of the EcoK cassette has been made to facilitate the joining of lacZ or a Factor Xa cleavage site to any protein reading frame.