The Pineapple EST sequencing and microarray project

We have initiated an EST sequencing project to survey a range of expressed sequences from green fruit, yellow fruit, roots, and root-knot nematode infected root/gall tissues. In total, 5681 edited EST sequences were retrieved. Clone redundancy was high in the fruit libraries, with the combined fruit 1548 clone sequences clustering into just 634 contigs comprising 191 consensus sequences and 443 singletons. Half of all fruit EST clone sequences clustered within approximately 14 and 9% of contigs from green unripe and yellow ripe libraries respectively, indicating that a small subset of genes dominates the majority of the transcriptome. The root and root/gall libraries had lower levels of redundancy than the fruit libraries. Half of the root/gall ESTs clustered within approximately 40% of all contigs, indicating the roots possess a more complex transcriptome. Contig assembly and cluster analysis revealed major differences in the abundant gene sequences expressed between the unripe green and the ripe yellow fruit tissues, or gene sequences expressed between the weeks 1-4 and weeks 5-10 nematode infected gall vascular cylinder libraries.

Latest developments in the microarray analysis of human brain mRNA

Microarrays are used to monitor the expression of thousands of gene transcripts. This technique requires high-quality RNA, which can be extracted from a variety sources, including autopsy brain tissue. Most nucleic acids and proteins are reasonably stable post mortem. However, their abundance and integrity can exhibit marked intraand inter-subject variability, so care must be taken when comparisons between case-groups are made. We will review issues associated with the sampling of RNA from autopsy brain tissue in relation to various ante- and post-mortem factors.

Oxygen free radicals denature human IgG and increase its reactivity with rheumatoid factor antibody

Rheumatoid inflammation is characterised by the production of rheumatoid factor antibodies directed against denatured IgG. Oxygen free radicals have the potential to denature all manner of proteins and can be generated by activated phagocytic cells in the inflamed joint. By modifying routine ELISA and nephelometric procedures for measuring rheumatoid factor, (i.e. substituting free radical altered IgG for rabbit and heat aggregated IgG as antigens) we have observed that oxygen radicals, generated by (1) UV light and (2) PMA-activated neutrophils, give rise to monomeric and polymeric forms of IgG which have increased reactivity towards IgM and IgA polyclonal rheumatoid factor antibodies. We conclude that free radical alteration of IgG may be a stimulus to the formation of immune complexes with rheumatoid factor antibody, thereby promoting and amplifying tissue damage during rheumatoid inflammation.

Novel monoclonal antibody recognition of oxidative DNA damage adduct, deoxycytidine-glyoxal

Glyoxal, a reactive aldehyde, is a decomposition product of lipid hydroperoxides, oxidative deoxyribose breakdown, or autoxidation of sugars, such as glucose. It readily forms DNA adducts, generating potential carcinogens such as glyoxalated deoxycytidine (gdC). A major drawback in assessing gdC formation in cellular DNA has been methodologic sensitivity. We have developed an mAb that specifically recognizes gdC. Balb/c mice were immunized with DNA, oxidatively modified by UVC/hydrogen peroxide in the presence of endogenous metal ions. Although UVC is not normally considered an oxidizing agent, a UVC/hydrogen peroxide combination may lead to glyoxalated bases arising from hydroxyl radical damage to deoxyribose. This damaging system was used to induce numerous oxidative lesions including glyoxal DNA modifications, from which resulted a number of clones. Clone F3/9/H2/G5 showed increased reactivity toward glyoxal-modified DNA greater than that of the immunizing antigen. ELISA unequivocally showed Ab recognition toward gdC, which was confirmed by gas chromatography-mass spectrometry of the derivatized adduct after formic acid hydrolysis to the modified base. Binding of Ab F3/9 with glyoxalated and untreated oligomers containing deoxycytidine, deoxyguanosine, thymidine, and deoxyadenosine assessed by ELISA produced significant recognition (p 0.0001) of glyoxal-modified deoxycytidine greater than that of untreated oligomer. Additionally, inhibition ELISA studies using the glyoxalated and native deoxycytidine oligomer showed increased recognition for gdC with more than a 5-fold difference in IC50 values. DNA modified with increasing levels of iron (II)/EDTA produced a dose-dependent increase in Ab F3/9 binding. This was reduced in the presence of catalase or aminoguanidine. We have validated the potential of gdC as a marker of oxidative DNA damage and showed negligible cross-reactivity with 8-oxo-2'-deoxyguanosine or malondialdehyde-modified DNA as well as its utility in immunocytochemistry. Formation of the gdC adduct may involve intermediate structures; however, our results strongly suggest Ab F3/9 has major specificity for the predominant product, 5-hydroxyacetyl-dC.