Molecular cloning and characterization of an almond 9-hydroperoxide lyase, a new CYP74 targeted to lipid bodies

Oxylipin metabolism represents one of many defence mechanisms employed by plants. It begins with the oxygenation of polyunsaturated fatty acids by lipoxygenases to form fatty acid hydroperoxides that are substrates for several enzymes, including specialized cytochrome P450s known as CYP74s. The targeting of a new CYP74, a 9-hydroperoxide lyase (HPL) from almonds, to the endomembrane system and lipid bodies, both as enzyme activity in almond seeds and as GFP fusions transiently expressed in tobacco protoplasts, is described. Such association of a CYP74 with lipid bodies has not been reported previously. Also described are the properties of a 9-HPL gene, the developmental regulation of its expression, the production and characterization of recombinant 9-HPL in Escherichia coli, and the developmental correlation between gene expression, enzyme activity, and the appearance of volatile C9 aldehydes from HPL action.     

Immunodominance of poxviral-specific CTL in a human trial of recombinant-modified vaccinia Ankara

Many recombinant poxviral vaccines are currently in clinical trials for cancer and infectious diseases. However, these agents have failed to generate T cell responses specific for recombinant gene products at levels comparable with T cell responses associated with natural viral infections. The recent identification of vaccinia-encoded CTL epitopes, including a new epitope described in this study, allows the simultaneous comparison of CTL responses specific for poxviral and recombinant epitopes. We performed detailed kinetic analyses of CTL responses in HLA-A*0201 patients receiving repeated injections of recombinant modified vaccinia Ankara encoding a string of melanoma tumor Ag epitopes. The vaccine-driven CTL hierarchy was dominated by modified vaccinia Ankara epitope-specific responses, even in patients who had not received previous smallpox vaccination. The only recombinant epitope that was able to impact on the CTL hierarchy was the melan-A26-35 analog epitope, whereas responses specific for the weaker affinity epitope NY-ESO-1(157-165) failed to be expanded above the level detected in prevaccination samples. Our results demonstrate that immunodominant vaccinia-specific CTL responses limit the effectiveness of poxviruses in recombinant vaccination strategies and that more powerful priming strategies are required to overcome immunodominance of poxvirus-specific T cell responses.     

Genetics of postzygotic isolation in Eucalyptus: whole-genome analysis of barriers to introgression in a wide interspecific cross of Eucalyptus grandis and E. globulus

The genetic architecture of hybrid fitness characters can provide valuable insights into the nature and evolution of postzygotic reproductive barriers in diverged species. We determined the genome-wide distribution of barriers to introgression in an F(1) hybrid of two Eucalyptus tree species, Eucalyptus grandis (W. Hill ex Maiden.) and E. globulus (Labill.). Two interspecific backcross families (N = 186) were used to construct comparative, single-tree, genetic linkage maps of an F(1) hybrid individual and two backcross parents. A total of 1354 testcross AFLP marker loci were evaluated in the three parental maps and a substantial proportion (27.7% average) exhibited transmission ratio distortion (alpha = 0.05). The distorted markers were located in distinct regions of the parental maps and marker alleles within each region were all biased toward either of the two parental species. We used a Bayesian approach to estimate the position and effect of transmission ratio distorting loci (TRDLs) in the distorted regions of each parental linkage map. The relative viability of TRDL alleles ranged from 0.20 to 0.72. Contrary to expectation, heterospecific (donor) alleles of TRDLs were favored as often as recurrent alleles in both backcrosses, suggesting that positive and negative heterospecific interactions affect introgression rates in this wide interspecific pedigree.     

Sulfur stable isotopes separate producers in marine food-web analysis

Ecological applications of stable isotope analysis rely on different producers having distinct isotopic ratios to trace energy and nutrient transfer to consumers. Carbon (C) and nitrogen (N) are the usual elements analysed. We tested the hypothesis that producers unable to be separated using C and N would be separated by sulphur (S), by reviewing estuarine and marine food web studies using all three elements (total of 836 pairwise comparisons between producers). S had a wider range of values across all producers than C and N (S: 34.4, C: 23.3, N: 18.7), and a higher mean difference among producers (S: 9.3, C: 6.5, N: 3.3). We varied from 1 to 10 the distance producers must be apart to be considered separate. For each of these gap distances, S-separated producers tied on C and N in 40% or more of cases. Comparing the three elements individually, S had fewer tied pairs of producers for any gap distance than C or N. However, S also has higher within-producer variability. Statistical tests on simulated data showed that this higher variability caused S to be less effective than C for analysing differences among mean producer values, yet mixing models showed that S had the smallest confidence intervals around mean estimates of source contributions to consumers. We also examined the spatial and temporal scales over which S isotope signatures of the saltmarsh plant Spartina alterniflora varied. Differences between samples taken within tens of metres were smallest, but between samples hundreds of metres apart were as different as samples thousands of kilometres apart. The time between samples being taken did not influence S signatures. Overall, the use of S is recommended because it has a high probability of distinguishing the contribution of different producers to aquatic food webs. When two elements are employed, the combination of S and C separates more producers than any other combination.     

Insights from animal models on the origins and progression of retinoblastoma

The RB gene was discovered 20 years ago because of its role in the childhood eye cancer retinoblastoma. However, surprisingly little progress was made in defining the role of RB protein in the retina. In the last two years, new models exploiting conditional deletion of the mouse Rb gene have altered this picture radically. These models provide insight into the first Rb function, the cell of origin of retinoblastoma, the window during which Rb acts, distinct cell-specific defenses against Rb loss, the number and type of post-Rb lesions required for transformation, why pediatric tumors exist, the controversial role of the p53 pathway in retinoblastoma, and the reason why the disease is virtually unique to humans. Two years have dramatically improved our understanding of Rb function in the tissue that gave us this important tumor suppressor.     

Relative and absolute quantitative expression profiling of cytochromes P450 using isotope-coded affinity tags

The development of a novel method for absolute quantification of proteins based on isotope-coded affinity tagging using ICAT reagents is described. The method exploits synthetic peptide standards to determine protein content at the femtomole level in biological samples. The approach is generally applicable to any subset of proteins, but is particularly appropriate for quantitative analysis of multiple, closely related isoforms, and for hydrophobic proteins that are poorly represented in 2-D gels. Relative and absolute quantification techniques are applied to an important group of microsomal metabolic enzymes, the cytochromes P450 (P450), which are critical in determining the disposition, safety and efficacy of drugs in man. Measurement of the P450 induction profile in response to chemicals is a fundamental aspect of drug safety evaluation and is currently achieved by low-throughput methods employing poorly discriminatory antibodies or substrates. Tagging technology is shown to supersede conventional methods for P450 profiling in terms of discriminatory power and throughput, exemplified by the simultaneous detection of distinct induction profiles for cyp2c subfamily members in response to phenobarbitone: cyp2c29 expression, but not cyp2c40 or cyp2c50, was induced threefold by treatment. This technology should abbreviate the drug development pathway, and provide a widely applicable, rapid means of quantifying proteins.     

Monoamine oxidase A gene polymorphisms and enzyme activity associated with risk of gout in Taiwan aborigines

Taiwanese aborigines have a high prevalence of hyperuricemia and gout. Uric acid levels and urate excretion have correlated with dopamine-induced glomerular filtration response. MAOs represent one of the major renal dopamine metabolic pathways. We aimed to identify the monoamine oxidase A (MAOA, Xp11.3) gene variants and MAO-A enzyme activity associated with gout risk. This study was to investigate the association between gout and the MAOA single-nucleotide polymorphisms (SNPs) rs5953210, rs2283725, and rs1137070 as well as between gout and the COMT SNPs rs4680 Val158Met for 374 gout cases and 604 controls. MAO-A activity was also measured. All three MAOA SNPs were significantly associated with gout. A synonymous MAOA SNP, rs1137070 Asp470Asp, located in exon 14, was associated with the risk of having gout (P = 4.0 × 10^?5, adjusted odds ratio 1.46, 95% confidence intervals [CI]: 1.11–1.91). We also showed that, when compared to individuals with the MAOA GAT haplotype, carriers of the AGC haplotype had a 1.67-fold (95% CI: 1.28–2.17) higher risk of gout. Moreover, we found that MAOA enzyme activity correlated positively with hyperuricemia and gout (P for trend = 2.00 × 10^?3 vs. normal control). We also found that MAOA enzyme activity by rs1137070 allele was associated with hyperuricemia and gout (P for trend = 1.53 × 10^?6 vs. wild-type allele). Thus, our results show that some MAOA alleles, which have a higher enzyme activity, predispose to the development of gout.