Effect of essential fatty acids on circulating T cell subsets in patients with colorectal cancer

The effect of essential fatty acids (EFA), given orally as dietary supplements, on the responsiveness in vitro of peripheral blood lymphocytes (PBL), to the mitogen concanavalin A have been studied in 10 patients with localized and 14 patients with advanced colorectal cancer. The degree of lymphocyte activation was assessed by measuring the amount of tritiated [³H]thymidine incorporated into newly synthesised lymphocyte DNA. The results were expressed as stimulation indices. T cell responses to concanavalin A stimulation showed a significant reduction of stimulation indices following EFA supplementation, in both the localized (P=0.026) and advanced (P=0.016) tumour groups, when compared with pretreatment activity in vitro. Mixing experiments, using EFA-supplemented and non-EFA-supplemented lymphocytes with concanavalin A, suggest no enhancement of T suppressor cell activity. Cell surface marker analysis (fluorescence-activated cell sorting for CD phenotyping) revealed a reduction of absolute numbers of CD4⁺ and CD8⁺ lymphocytes following EFA supplementation. The stimulation indices returned to presupplementation values 3 months following cessation of EFA intake. There was no significant change of these indices in the control (no EFA supplementation) advanced tumour group tested. This study suggests that EFA supplementation in patients with colorectal cancer selectively reduces circulating PBL. and T cell subset (including suppressor cells) numbers and/or activity. Such effects may have an important outcome in patients with malignant disease.This work was supported by grants from the Grampian Health Broad, the Royal College of Surgeons of Edinburgh, and Scottish Hospital Endownment Research Trust     

A radiometric assay for methyl red azoreductase DT-diaphorase [EC 1.6.99.2]

A rapid radiometric assay for measuring oxygen insensitive methyl red azoreductase has been developed. [¹⁴C]Methyl red was synthesized from [U-¹⁴C]aniline; the final product had a radiochemical purity of ≥98%. [¹⁴C]Methyl red was then used as the substrate to assay azoreductase activity using a modification of a previously published procedure. Results obtained by the radiometric assay were comparable to those obtained using the fluorescent procedure. The radiometric assay is quick, simple, and reliable. Methyl red azoreductase has been shown to be identical with DT-diaphorase [EC 1.6.99.2]. The assay described can therefore be used to assay DT-diaphorase activity and does not suffer from the limitations, such as lack of specificity and low sensitivity, usually associated with DT-diaphorase assays.     

Revisiting the use of Iprodione and Trichoderma in the integrated management of onion white rot

The biocontrol properties of Trichoderma species are well documented, but their effectiveness in antagonism of the problematic Sclerotium cepivorum, the causal agent of white rot in Allium species, appears limited with reports of significant control only relating to deliberately-mutated strains of Trichoderma. Our previous studies have indicated the possibility of using selected naturally-occurring strains of the antagonist in the suppression of other diseases; now in vitro and controlled environment in vivo studies have indicated that a degree of control of Onion White Rot is possible, and that the selected antagonist strains can be used in integrated treatments with Iprodione to good effect. The possible value of such treatments is considered in light of other approaches to the suppression of this continuing problem.     

Sinorhizobium meliloti cells require biotin and either cobalt or methionine for growth

Sinorhizobium meliloti is usually cultured in rich media containing yeast extract. It has been suggested that some components of yeast extract are also required for growth in minimal medium. We tested 27 strains of this bacterium and found that none were able to grow in minimal medium when methods to limit carryover of yeast extract were used during inoculation. By fractionation of yeast extract, two required growth factors were identified. Biotin was found to be absolutely required for growth, whereas previously the need for this vitamin was considered to be strain specific. All strains also required supplementation with cobalt or methionine, consistent with the requirement for a vitamin B(12)-dependent homocysteine methyltransferase for methionine biosynthesis.     

Cell signal mechanisms,conjugated linoleic acids (CLAs) and anti-tumorigenesis

Fats have been adversely implicated in the aetiology of many forms of cancer yet evidence is accumulating that certain types of fatty acids have anticancer properties. This is well documented for fish-oil fatty acids of the n-3 family. Recently, fatty acids found to occur naturally in ruminant-derived food products were found to have anticancer properties. These fatty acids were identified as conjugated linoleic acids (CLAs) derived from the parent linoleic acid by its partial hydrogenation by rumen bacteria. Studies with tumour-bearing animals have shown that consumption of CLAs particularly with regard to breast and prostate cancer is beneficial. Studies with cancer cells have also shown that these fatty acids can inhibit cell proliferation and induce cell death. However, little is known regarding the mechanisms of action of these CLAs. In particular, which cellular signal mechanisms are regulated by CLAs which can explain their anticancer properties. We have shown that CLAs specifically up-regulate cell signal systems at the level of gene expression (mRNA, protein) in human breast and prostate cancer cells which are responsible for the induction of apoptosis or programmed cell death. These findings support the anticancer effects of CLA found in animal models and indicate similar effects could occur in man.     

A spatiotemporal characterization of the effect of p53 phosphorylation on its interaction with MDM2

The interaction of p53 and MDM2 is modulated by the phosphorylation of p53. This mechanism is key to activating p53, yet its molecular determinants are not fully understood. To study the spatiotemporal characteristics of this molecular process we carried out Brownian dynamics simulations of the interactions of the MDM2 protein with a p53 peptide in its wild type state and when phosphorylated at Thr18 (pThr18) and Ser20 (pSer20). We found that p53 phosphorylation results in concerted changes in the topology of the interaction landscape in the diffusively bound encounter complex domain. These changes hinder phosphorylated p53 peptides from binding to MDM2 well before reaching the binding site. The underlying mechanism appears to involve shift of the peptide away from the vicinity of the MDM2 protein, peptide reorientation, and reduction in peptide residence time relative to wild-type p53 peptide. pThr18 and pSr20 p53 peptides experience reduction in residence times by factors of 13.6 and 37.5 respectively relative to the wild-type p53 peptide, indicating a greater role for Ser20 phosphorylation in abrogating p53 MDM2 interactions. These detailed insights into the effect of phosphorylation on molecular interactions are not available from conventional experimental and theoretical approaches and open up new avenues that incorporate molecular interaction dynamics, for stabilizing p53 against MDM2, which is a major focus of anticancer drug lead development.     

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO₂ anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO₂ in Drosophila melanogaster. The impact of CO₂ anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.     

Ground wētā in vines of the Awatere Valley,Marlborough: biology,density and distribution

The endemic New Zealand ground wētā (Hemiandrus sp. ‘promontorius’) has a Naturally Uncommon conservation status. This is because of the paucity of information on its density and distribution. Here, the biology, density and distribution of a population of this wētā found in and around vineyards in the Awatere Valley, Marlborough was studied. Wētā density was assessed in vineyards, paddocks and shrublands in this valley. Soil moisture, penetration resistance, pH and organic matter were recorded at locations with and without wētā. Wētā density in vineyards was significantly higher than in either paddocks or shrub habitats. In vineyards, the density of this insect was significantly higher under-vines than in the inter-rows. Higher numbers of this wētā were found in moist soils that required lower force to burrow. Females laid an average of 55 eggs between March and April, which hatched in September. These findings highlight the intersection between agriculture and conservation.