Individual Enzyme Activities Research Articles

Lower platelet mitochondrial function in severe septic patients than in controls

The oxidative phosphorylation system (OXPHOS) in septic patients has been scarcely analyzed in studies of small sample size and the results are apparently inconsistent. Previously, including 96 severe septic patients, we found that nonsurviving severe septic patients showed lower platelet respiratory complex IV (CIV) activity than surviving patients at the moment of severe sepsis diagnosis and during the first week of sepsis diagnosis. However, we did not examine this enzyme activity in normal individuals. Thus, the objective of this study was to compare the CIV activity between severe septic patients and healthy control individuals in a larger series of patients (including 198 severe septic patients).

Role of glutathione, glutathione transferase, and glutaredoxin in regulation of redox-dependent processes.

Over the last decade fundamentally new features have been revealed for the participation of glutathione and glutathione-dependent enzymes (glutathione transferase and glutaredoxin) in cell proliferation, apoptosis, protein folding, and cell signaling. Reduced glutathione (GSH) plays an important role in maintaining cellular redox status by participating in thiol-disulfide exchange, which regulates a number of cell functions including gene expression and the activity of individual enzymes and enzyme systems. Maintaining optimum GSH/GSSG ratio is essential to cell viability. Decrease in the ratio can serve as an indicator of damage to the cell redox status and of changes in redox-dependent gene regulation. Disturbance of intracellular GSH balance is observed in a number of pathologies including cancer. Consequences of inappropriate GSH/GSSG ratio include significant changes in the mechanism of cellular redox-dependent signaling controlled both nonenzymatically and enzymatically with the participation of isoforms of glutathione transferase and glutaredoxin. This review summarizes recent data on the role of glutathione, glutathione transferase, and glutaredoxin in the regulation of cellular redox-dependent processes.

Polysaccharidases in the Crystalline Styles of Selectively Bred Greenshell Mussel (Perna canaliculusGmelin) Families

Assays of chitinase, cellulase, and amylase activities in the crystalline styles of selected fast- and slow-growing families of the mussel Perna canaliculus were carried out on 2 occasions over 6 mo, during which time the shellfish grew in a normal mussel cultivation situation. The data did not support the hypothesis that differences in growth rate by these families could be related to differences in the expression of digestive enzymes in the styles, leading to improved nutritional efficiency by faster growing families. On the contrary, the styles of faster growing families in general exhibited lower rates of enzyme activity. Differences in enzyme activities between families appeared to be related to the size of the individuals rather than their genetic heritage. The crystalline styles of larger, more mature shellfish contained less moisture and less soluble protein, and the lower rates of enzyme activity in larger individuals may be indicative of a reallocation of style protein synthesis toward higher levels of insoluble structural proteins.

Molecular kinetics. Ras activation by SOS: allosteric regulation by altered fluctuation dynamics.

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras-guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average.

Outliers dominate signaling at cell membrane

Molecular Kinetics SOS enzymes act at cell membranes to activate Ras, a regulatory protein often overactive in cancer cells. Iversen et al. devised a system where they could observe the activity of individual enzymes at work. The single SOS molecules occupied stable states that varied greatly in their catalytic activity. Regulation appeared to occur by altering the time spent in active states. The overall activity of SOS was determined by just a few molecules that achieved the highest catalytic activity. The methods described should allow further detailed kinetic analysis of this and other signaling events that occur at the cell membrane — properties that it is not possible to discern from bulk biochemical measurements. Science , this issue p. [50][1] [1]: /lookup/doi/10.1126/science.1250373

Interspecific diversity in root antioxidative enzyme activities reflect root turnover strategies and preferred habitats in wetland graminoids

Antioxidant enzymes protect cells against oxidative stress and are associated with stress tolerance and longevity. In animals, variation in their activities has been shown to relate to species ecology, but in plants, comparative studies with wild species are rare. We investigated activities of five antioxidant enzymes – ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), peroxidase (POX), and superoxide dismutase (SOD) – in roots of four perennial graminoid wetland species over a growing season to find out whether differences in root turnover or habitat preferences would be associated with variation in seasonal patterns of antioxidant enzyme activities. The investigated species differ in their root turnover strategies (fine roots senesce in the fall or fine roots survive the winter) and habitat preferences (nutrient-poor vs. productive wetlands). Roots were collected both in the field and from garden-grown plants. Antioxidant enzyme activities were higher and lipid peroxidation rates lower in species with annual root systems, and for species of the nutrient-poor wetland, compared with perennial roots and species of productive wetlands, respectively. There was variation in the activities of individual antioxidant enzymes, but discriminant analyses with all enzymes revealed a clear picture, indicating consistent associations of antioxidant enzyme activities with the type of root turnover strategy and with the preferred habitat. We conclude that antioxidant enzyme activities in plant roots are associated with the species' ecological strategies and can be used as traits for the characterization of the species' position along plant economics spectrum.

Effects of Cadmium and Mercury on the Upper Part of Skeletal Muscle Glycolysis in Mice

The effects of pre-incubation with mercury (Hg2+) and cadmium (Cd2+) on the activities of individual glycolytic enzymes, on the flux and on internal metabolite concentrations of the upper part of glycolysis were investigated in mouse muscle extracts. In the range of metal concentrations analysed we found that only hexokinase and phosphofructokinase, the enzymes that shared the control of the flux, were inhibited by Hg2+ and Cd2+. The concentrations of the internal metabolites glucose-6-phosphate and fructose-6-phosphate did not change significantly when Hg2+ and Cd2+ were added. A mathematical model was constructed to explore the mechanisms of inhibition of Hg2+ and Cd2+ on hexokinase and phosphofructokinase. Equations derived from detailed mechanistic models for each inhibition were fitted to the experimental data. In a concentration-dependent manner these equations describe the observed inhibition of enzyme activity. Under the conditions analysed, the integral model showed that the simultaneous inhibition of hexokinase and phosphofructokinase explains the observation that the concentrations of glucose-6-phosphate and fructose-6-phosphate did not change as the heavy metals decreased the glycolytic flux.

Consumption of green coffee reduces blood pressure and body composition by influencing 11β-HSD1 enzyme activity in healthy individuals: a pilot crossover study using green and black coffee.

Dietary polyphenols may have a protective role against the development of CVD. Thus, we aimed to investigate the effects of green coffee (GC), rich in chlorogenic acid, and black coffee (BC) on cardiovascular markers. A randomised pilot crossover study was performed on healthy subjects who consumed both coffees for 2 weeks. We measured anthropometry, blood pressure, and arterial elasticity after each intervention and collected urine samples to monitor antioxidant capacity. Free cortisol and cortisone levels were obtained from urine and analysed by specific ELISA methods. Systolic blood pressure (P = 0.018) and arterial elasticity (P = 0.001) were significantly reduced after GC. BMI (P = 0.04 for BC; P = 0.01 for GC) and abdominal fat (P = 0.01 for BC; P = 0.009 for GC) were also significantly reduced with no changes in energy intake. Urinary free cortisol was significantly reduced from 125.6 ± 85.9 nmol/day to 76.0 ± 54.9 nmol/day following GC and increased to 132.1 ± 89.1 nmol/day after BC. Urinary free cortisone increased by 18% following BC and 9% following GC (nonsignificant). Cortisol/cortisone ratio (indicating 11β-HSD1 activity) was reduced after GC (from 3.5 ± 1.9 to 1.7 ± 1.04, P = 0.002). This suggests that GC can play a role in reducing cardiovascular risk factors. Further research including hypertensive and overweight individuals will now be justified to clarify whether GC could have a therapeutic role in CVD.

Study of the effect of abiotic stress on the antioxidant enzyme activity of cereals under regulated environmental conditions

The impacts of climate modification were examined in terms of changes in the stress tolerance of winter wheat varieties. The enzyme reactions of two winter wheat varieties to drought stress, simulated by water withholding in three different phenophases, were analysed in a phytotron experiment in the Centre for Agricultural Research, Hungarian Academy of Sciences. Plants were raised either at ambient CO2 level or at twice this concentration. The quantities of glutathione reductase (GR), glutathione-S-transferase (GST), catalase (CAT), guaiacol peroxidase (POD) and ascorbate peroxidase (APX) were determined from leaf samples collected at the end of the drought treatment.The results showed that antioxidant enzymes may help to counterbalance the reactive oxygen species induced by stress during various stages of the vegetation period. Although there were substantial differences in the changes induced in the activity of individual enzymes by modifications in environmental factors, this activity and its response to stress depended not only on these factors, but also on the developmental stage of the plant. Modifications in enzyme activity could indicate that enhanced CO2 concentration delayed the development of drought stress up to first node appearance, and stimulated antioxidant enzyme activity when drought occurred during ripening.

Mining for hemicellulases in the fungus-growing termite Pseudacanthotermes militaris using functional metagenomics

BackgroundThe metagenomic analysis of gut microbiomes has emerged as a powerful strategy for the identification of biomass-degrading enzymes, which will be no doubt useful for the development of advanced biorefining processes. In the present study, we have performed a functional metagenomic analysis on comb and gut microbiomes associated with the fungus-growing termite, Pseudacanthotermes militaris.ResultsUsing whole termite abdomens and fungal-comb material respectively, two fosmid-based metagenomic libraries were created and screened for the presence of xylan-degrading enzymes. This revealed 101 positive clones, corresponding to an extremely high global hit rate of 0.49%. Many clones displayed either β-d-xylosidase (EC 3.2.1.37) or α-l-arabinofuranosidase (EC 3.2.1.55) activity, while others displayed the ability to degrade AZCL-xylan or AZCL-β-(1,3)-β-(1,4)-glucan. Using secondary screening it was possible to pinpoint clones of interest that were used to prepare fosmid DNA. Sequencing of fosmid DNA generated 1.46 Mbp of sequence data, and bioinformatics analysis revealed 63 sequences encoding putative carbohydrate-active enzymes, with many of these forming parts of sequence clusters, probably having carbohydrate degradation and metabolic functions. Taxonomic assignment of the different sequences revealed that Firmicutes and Bacteroidetes were predominant phyla in the gut sample, while microbial diversity in the comb sample resembled that of typical soil samples. Cloning and expression in E. coli of six enzyme candidates identified in the libraries provided access to individual enzyme activities, which all proved to be coherent with the primary and secondary functional screens.ConclusionsThis study shows that the gut microbiome of P. militaris possesses the potential to degrade biomass components, such as arabinoxylans and arabinans. Moreover, the data presented suggests that prokaryotic microorganisms present in the comb could also play a part in the degradation of biomass within the termite mound, although further investigation will be needed to clarify the complex synergies that might exist between the different microbiomes that constitute the termitosphere of fungus-growing termites. This study exemplifies the power of functional metagenomics for the discovery of biomass-active enzymes and has provided a collection of potentially interesting biocatalysts for further study.

Inter and intra plant variability of enzyme profiles including various phosphoesterases and sulfatase of six wastewater treatment plants

Biodegradation of organic wastewater constituents by activated sludge microorganisms is based on enzymatic processes. It is supposed that wastewater treatment plants (WWTP) differ in their enzymatic fingerprints. To determine such fingerprints, activated sludges from nine aerated tanks of six WWTPs were repeatedly sampled and analyzed for the activities of l-alanine aminopeptidase, esterase, α- and β-glucosidase, alkaline phosphatase, phosphodiesterase, phosphotriesterase, and sulfatase. In one WWTP the enzymatic activities and their variations within 1 week were assayed in various process stages. Mostly the enzymatic profiles were dominated by l-alanine aminopeptidase, followed by alkaline phosphatase. They differed in variable contributions of esterase, phosphodiesterase, α- and β-glucosidase. The sulfatase activity was generally low. For the first time phosphotriesterase activity was detected in various samples, but with limited analytical validity. Particle mass-related activities of individual enzymes varied between plants by factors 2-4 and up to 11, when related to suspension volumes.

Abstract A16: Genetic polymorphisms in the carcinogen metabolism pathway and lung cancer risk

Abstract Alterations affecting genes involved in the carcinogen metabolism are associated with cancer susceptibility since these can affect the detoxification capacity of the gene product. Genes regulating the carcinogen metabolism are also highly effective in the pathways involved in lung cancer development. The microsomal epoxide hydroxylase gene EPHX1 participates in the detoxification and activation of the carcinogenic compounds in the cigarette smoke. Two polymorphic changes in the EPHX1 gene result in slow and fast alleles with low and high enzyme activity. This differential effect suggests that these polymorphisms may affect individual lung cancer risk. Myeloperoxidase (MPO) is an oxidative enzyme released in response to cigarette smoke from the lung tissue. A G>A polymorphism located in the 5' untranslated region of the MPO gene that may be responsible for reduced transcriptional activity due to the decreased binding affinity for the SP1 transcription factor. In individuals carrying the A allele metabolic procarcinogen compounds are not effectively activated. Therefore, this variant is thought to play a protective role in lung cancer. The NAD(P)H dehydrogenase (NQO1) gene is an important mediator of lung cancer and codes for a cytoplasmic electron reductase which prevents the production of reactive radical species by detoxification of the quinones and their derivatives. Mutations in the NQO1 gene have been associated with susceptibility to various forms of cancer. A C to T substitution at codon 187 of the gene results in almost undetectable enzyme activity in the homozygote individuals carrying the variant allele. In this study we investigated the association of the EPHX1, MPO and NQO1 gene variants with lung cancer in 126 patients and 64 healthy individuals. Gene expression analysis was performed using the LightCycler 480 Real Time PCR system and hybridization probes. To analyze the genotypes the EPHX1, MPO and NQO1 genes were amplified by polymerase chain reaction and the presence of the gene variants was investigated by restriction fragment length polymorphism after digestion with specific restriction enzymes. No significant differences in the distribution of the genotypes and allele frequencies were observed between the patients and the control group for the MPO gene and the two different polymorphic variants of the EPHX1 gene. The NQO1 expression levels were higher in the patients when compared to the controls but the difference was not significant. On the other hand, a significant association was observed between the CC genotype and NQO1 expression. Citation Format: Nejat Dalay, Nur Buyru, Zubeyde Yalniz. Genetic polymorphisms in the carcinogen metabolism pathway and lung cancer risk. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A16.

Untargeted plasma metabolite profiling reveals the broad systemic consequences of xanthine oxidoreductase inactivation in mice.

A major challenge in systems biology is integration of molecular findings for individual enzyme activities into a cohesive high-level understanding of cellular metabolism and physiology/pathophysiology. However, meaningful prediction for how a perturbed enzyme activity will globally impact metabolism in a cell, tissue or intact organisms is precluded by multiple unknowns, including in vivo enzymatic rates, subcellular distribution and pathway interactions. To address this challenge, metabolomics offers the potential to simultaneously survey changes in thousands of structurally diverse metabolites within complex biological matrices. The present study assessed the capability of untargeted plasma metabolite profiling to discover systemic changes arising from inactivation of xanthine oxidoreductase (XOR), an enzyme that catalyzes the final steps in purine degradation. Using LC-MS coupled with a multivariate statistical data analysis platform, we confidently surveyed >3,700 plasma metabolites (50–1,000 Da) for differential expression in XOR wildtype vs. mice with inactivated XOR, arising from gene deletion or pharmacological inhibition. Results confirmed the predicted derangements in purine metabolism, but also revealed unanticipated perturbations in metabolism of pyrimidines, nicotinamides, tryptophan, phospholipids, Krebs and urea cycles, and revealed kidney dysfunction biomarkers. Histochemical studies confirmed and characterized kidney failure in xor-nullizygous mice. These findings provide new insight into XOR functions and demonstrate the power of untargeted metabolite profiling for systemic discovery of direct and indirect consequences of gene mutations and drug treatments.

High cellular organization of pyoverdine biosynthesis in Pseudomonas aeruginosa: clustering of PvdA at the old cell pole

Pyoverdine I (PVDI) is the major siderophore produced by Pseudomonas aeruginosa PAO1 to import iron. Its biosynthesis requires the coordinated action of cytoplasmic, periplasmic and membrane proteins. The individual enzymatic activities of these proteins are well known. However, their subcellular distribution in particular areas of the cytoplasm, periplasm, or within the membrane has never been investigated. We used chromosomal replacement to generate P.aeruginosa strains producing fluorescent fusions with PvdA, one of the initial enzymes in the biosynthetic pathway of PVDI in the cytoplasm, and PvdQ, involved in the maturation of PVDI in the periplasm. Cellular fractionation indicated that a substantial amount of PvdA-YFP was located in the membrane fraction. Epifluorescence microscopy imaging showed that PvdA-YFP was mainly clustered at the old cell pole of bacteria, indicating a polar segregation of the protein. Epifluorescence and TIRF imaging on cells expressing labelled PvdQ showed that this enzyme was uniformly distributed in the periplasm, in contrast with PvdA-YFP. The description of the intracellular distribution of these enzymes contributes to the understanding of the PVDI biosynthetic pathway.

Enzymes To Die For: Exploiting Nucleotide Metabolizing Enzymes for Cancer Gene Therapy

Suicide gene therapy is an attractive strategy to selectively destroy cancer cells while minimizing unnecessary toxicity to normal cells. Since this idea was first introduced more than two decades ago, numerous studies have been conducted and significant developments have been made to further its application for mainstream cancer therapy. Major limitations of the suicide gene therapy strategy that have hindered its clinical application include inefficient directed delivery to cancer cells and the poor prodrug activation capacity of suicide enzymes. This review is focused on efforts that have been and are currently being pursued to improve the activity of individual suicide enzymes towards their respective prodrugs with particular attention to the application of nucleotide metabolizing enzymes in suicide cancer gene therapy. A number of protein engineering strategies have been employed and our discussion here will center on the use of mutagenesis approaches to create and evaluate nucleotide metabolizing enzymes with enhanced prodrug activation capacity and increased thermostability. Several of these studies have yielded clinically important enzyme variants that are relevant for cancer gene therapy applications because their utilization can serve to maximize cancer cell killing while minimizing the prodrug dose, thereby limiting undesirable side effects.

Single-Molecule Analysis of the Functional Structure of Telomerase RNP

Telomerase is a cellular ribonucleoprotein (RNP) complex comprised of telomerase RNA, telomerase reverse transcriptase (TERT) and other protein cofactors. The activity of the telomerase enzyme is essential for the maintenance of genome stability and normal cell development. Despite the biomedical importance of telomerase activity, detailed structural models for the enzyme remain to be established. Here we report the development and application of a single-molecule assay for direct structural analysis of catalytically active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity, allowing precise discrimination between inactive, active and processive enzyme binding events. FRET signals from individual enzyme molecules during the active and processive binding events were then used to determine the global organization of telomerase RNA within catalytically active holoenzymes. Using this assay, we have identified an active conformation of telomerase among a heterogeneous population of enzymes with distinct structures. In particular, we have established that the phylogenetically conserved pseudoknot motif within telomerase RNA is properly folded in catalytically active enzymes. Interestingly, the pseudoknot motif is misfolded in the absence of proteins, and the protein subunits of the telomerase holoenzyme counteract RNA misfolding and allow a significant fraction of the RNPs to form the pseudoknot structure. Only those RNP complexes containing a properly folded pseudoknot are catalytically active. These results not only demonstrate the functional importance of various telomerase RNA regions such as the pseudoknot but also establish a direct method for elucidating how the structure of telomerase enables its function.

A Rapid Non Invasive L-DOPA-13C Breath Test for Optimally Suppressing Extracerebral AADC Enzyme Activity - Toward Individualizing Carbidopa Therapy in Parkinson's Disease

Peripheral carbidopa (CD) levels directly impact on central dopamine (DA) production in Parkinson disease (PD) through extracerebral inhibition of dopa decarboxylase (AADC) resulting in an increase in levodopa (LD) bioavailability. Recent data suggests that higher CD doses than those presently used in PD treatment may result in improved clinical response. Optimizing CD doses in individual patients may, therefore, result in ideal individualized treatment. A single center, randomized, double-blind study was carried out recruiting 5 Parkinson’s disease (PD) patients already on LD/CD and 1 treatment näve PD patient using stable isotope labeled LD-1-¹³C as a substrate for a noninvasive breath test to evaluate individual AADC enzyme activity. Each patient was studied five times, receiving 200 mg LD-¹³C at each visit along with one of five randomized CD doses (0, 25, 50, 100 and 200 mg). The metabolite ¹³CO₂ in breath was measured for evaluating AADC enzyme activity and plasma metabolite levels for LD-¹³C and homovanillic acid (HVA) were measured for 4 hours. HVA in plasma and ¹³CO₂ in breath are metabolic products of LD. We found a significant positive correlation of ¹³CO₂ DOB AUC0-240 with serum HVA AUC0-240 following the oral dose of LD-1-¹³C for all 5 doses of CD (r² = 0.9378). With increasing inhibition of AADC enzyme activity with CD, we observed an increase in the plasma concentration of LD.We found an inverse correlation of the 13CO2 DOB AUC with serum LD-¹³C AUC. Our studies indicate the optimal dose of CD for maximal suppression of AADC enzyme activity can be determined for each individual from ¹³CO₂ generation in breath. The LD-breath test can be a useful noninvasive diagnostic tool for evaluation of AADC enzyme activity using the biomarker ¹³CO₂ in breath, a first step in personalizing CD doses for PD patients.

Chymase-Dependent Generation of Angiotensin II from Angiotensin-(1-12) in Human Atrial Tissue

Since angiotensin-(1-12) [Ang-(1-12)] is a non-renin dependent alternate precursor for the generation of cardiac Ang peptides in rat tissue, we investigated the metabolism of Ang-(1-12) by plasma membranes (PM) isolated from human atrial appendage tissue from nine patients undergoing cardiac surgery for primary control of atrial fibrillation (MAZE surgical procedure). PM was incubated with highly purified 125I-Ang-(1-12) at 37°C for 1 h with or without renin-angiotensin system (RAS) inhibitors [lisinopril for angiotensin converting enzyme (ACE), SCH39370 for neprilysin (NEP), MLN-4760 for ACE2 and chymostatin for chymase; 50 µM each]. 125I-Ang peptide fractions were identified by HPLC coupled to an inline γ-detector. In the absence of all RAS inhibitor, 125I-Ang-(1-12) was converted into Ang I (2±2%), Ang II (69±21%), Ang-(1-7) (5±2%), and Ang-(1-4) (2±1%). In the absence of all RAS inhibitor, only 22±10% of 125I-Ang-(1-12) was unmetabolized, whereas, in the presence of the all RAS inhibitors, 98±7% of 125I-Ang-(1-12) remained intact. The relative contribution of selective inhibition of ACE and chymase enzyme showed that 125I-Ang-(1-12) was primarily converted into Ang II (65±18%) by chymase while its hydrolysis into Ang II by ACE was significantly lower or undetectable. The activity of individual enzyme was calculated based on the amount of Ang II formation. These results showed very high chymase-mediated Ang II formation (28±3.1 fmol×min−1×mg−1, n = 9) from 125I-Ang-(1-12) and very low or undetectable Ang II formation by ACE (1.1±0.2 fmol×min−1×mg−1). Paralleling these findings, these tissues showed significant content of chymase protein that by immunocytochemistry were primarily localized in atrial cardiac myocytes. In conclusion, we demonstrate for the first time in human cardiac tissue a dominant role of cardiac chymase in the formation of Ang II from Ang-(1-12).

Identification of enzyme activity quantitative trait loci in a Solanum lycopersicum x Solanum pennellii introgression line population.

Activities of 28 enzymes from central carbon metabolism were measured in pericarp tissue of ripe tomato fruits from field trials with an introgression line (IL) population generated by introgressing segments of the genome of the wild relative Solanum pennellii (LA0716) into the modern tomato cultivar Solanum lycopersicum M82. Enzyme activities were determined using a robotized platform in optimized conditions, where the activities largely reflect the level of the corresponding proteins. Two experiments were analyzed from years with markedly different climate conditions. A total of 27 quantitative trait loci were shared in both experiments. Most resulted in increased enzyme activity when a portion of the S. lycopersicum genome was substituted with the corresponding portion of the genome of S. pennellii. This reflects the change in activity between the two parental genotypes. The mode of inheritance was studied in a heterozygote IL population. A similar proportion of quantitative trait loci (approximately 30%) showed additive, recessive, and dominant modes of inheritance, with only 5% showing overdominance. Comparison with the location of putative genes for the corresponding proteins indicates a large role of trans-regulatory mechanisms. These results point to the genetic control of individual enzyme activities being under the control of a complex program that is dominated by a network of trans-acting genes.

Combinations of Polymorphisms in Genes Involved in the 5-Fluorouracil Metabolism Pathway Are Associated with Gastrointestinal Toxicity in Chemotherapy-Treated Colorectal Cancer Patients

The purpose of this study was to investigate whether specific combinations of polymorphisms in genes encoding proteins involved in 5-fluorouracil (5-FU) pharmacokinetics and pharmacodynamics are associated with increased risk of treatment-induced toxicity. We analyzed two cohorts of 161 and 340 patients, the exploration and validation cohort, respectively. All patients were treated similarly with 5-FU-based adjuvant chemotherapy. We analyzed 13 functional polymorphisms and applied a four-fold analysis strategy using individual polymorphisms, haplotypes, and phenotypic enzyme activity or expression classifications based on combinations of functional polymorphisms in specific genes. Furthermore, multifactor dimensionality reduction analysis was used to identify a genetic interaction profile indicating an increased risk of toxicity. Alleles associated with low activity of methylene tetrahydrofolate reductase (MTHFR) were associated with decreased risk of toxicity [OR(Exploration) 0.39 (95% CI: 0.21-0.71, P = 0.003), OR(Validation) 0.63 (95% CI: 0.41-0.95, P = 0.03)]. A specific combination of the MTHFR 1298A>C and thymidylate synthase (TYMS) 3'-UTR (untranslated region) ins/del polymorphisms was significantly associated with increased toxicity in both cohorts [OR(Exploration) 2.40 (95% CI: 1.33-4.29, P = 0.003), OR(Validation) 1.81 (95% CI: 1.18-2.79, P = 0.007)]. The specific combination was also associated with increased cumulative incidence and earlier occurrence of severe toxicity during treatment. Our results indicate that MTHFR activity and a specific combination of the MTHFR 1298A>C and TYMS 3'-UTR ins/del polymorphisms are possible predictors of 5-FU treatment-related toxicity.