The tomato terpene synthase gene family

Compounds of the terpenoid class play numerous roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of cultivated tomato (Solanum lycopersicum) contains 44 terpene synthase (TPS) genes, including 29 that are functional or potentially functional. Of these 29 TPS genes, 26 were expressed in at least some organs or tissues of the plant. The enzymatic functions of eight of the TPS proteins were previously reported, and here we report the specific in vitro catalytic activity of 10 additional tomato terpene synthases. Many of the tomato TPS genes are found in clusters, notably on chromosomes 1, 2, 6, 8, and 10. All TPS family clades previously identified in angiosperms are also present in tomato. The largest clade of functional TPS genes found in tomato, with 12 members, is the TPS-a clade, and it appears to encode only sesquiterpene synthases, one of which is localized to the mitochondria, while the rest are likely cytosolic. A few additional sesquiterpene synthases are encoded by TPS-b clade genes. Some of the tomato sesquiterpene synthases use z,z-farnesyl diphosphate in vitro as well, or more efficiently than, the e,e-farnesyl diphosphate substrate. Genes encoding monoterpene synthases are also prevalent, and they fall into three clades: TPS-b, TPS-g, and TPS-e/f. With the exception of two enzymes involved in the synthesis of ent-kaurene, the precursor of gibberellins, no other tomato TPS genes could be demonstrated to encode diterpene synthases so far.     

X-ray structural studies of quinone reductase 2 nanomolar range inhibitors

Quinone reductase 2 (QR2) is one of two members comprising the mammalian quinone reductase family of enzymes responsible for performing FAD mediated reductions of quinone substrates. In contrast to quinone reductase 1 (QR1) which uses NAD(P)H as its co‐substrate, QR2 utilizes a rare group of hydride donors, N‐methyl or N‐ribosyl nicotinamide. Several studies have linked QR2 to the generation of quinone free radicals, several neuronal degenerative diseases, and cancer. QR2 has been also identified as the third melatonin receptor (MT3) through in cellulo and in vitro inhibition of QR2 by traditional MT3 ligands, and through recent X‐ray structures of human QR2 (hQR2) in complex with melatonin and 2‐iodomelatonin. Several MT3 specific ligands have been developed that exhibit both potent in cellulo inhibition of hQR2 nanomolar, affinity for MT3. The potency of these ligands suggest their use as molecular probes for hQR2. However, no definitive correlation between traditionally obtained MT3 ligand affinity and hQR2 inhibition exists limiting our understanding of how these ligands are accommodated in the hQR2 active site. To obtain a clearer relationship between the structures of developed MT3 ligands and their inhibitory properties, in cellulo and in vitro IC₅₀ values were determined for a representative set of MT3 ligands (MCA‐NAT, 2‐I‐MCANAT, prazosin, S26695, S32797, and S29434). Furthermore, X‐ray structures for each of these ligands in complex with hQR2 were determined allowing for a structural evaluation of the binding modes of these ligands in relation to the potency of MT3 ligands.     

Proactive strategies to avoid infectious disease

Infectious disease exerts a large selective pressure on all organisms. One response to this has been for animals to evolve energetically costly immune systems to counter infection, while another--the focus of this theme issue--has been the evolution of proactive strategies primarily to avoid infection. These strategies can be grouped into three types, all of which demonstrate varying levels of interaction with the immune system. The first concerns maternal strategies that function to promote the immunocompetence of their offspring. The second type of strategy influences mate selection, guiding the selection of a healthy mate and one who differs maximally from the self in their complement of antigen-coding genes. The third strategy involves two classes of behaviour. One relates to the capacity of the organisms to learn associations between cues indicative of pathogen threat and immune responses. The other relates to prevention and even treatment of infection through behaviours such as avoidance, grooming, quarantine, medicine and care of the sick. In humans, disease avoidance is based upon cognition and especially the emotion of disgust. Human disease avoidance is not without its costs. There is a propensity to reject healthy individuals who just appear sick--stigmatization--and the system may malfunction, resulting in various forms of psychopathology. Pathogen threat also appears to have been a highly significant and unrecognized force in shaping human culture so as to minimize infection threats. This cultural shaping process--moralization--can be co-opted to promote human health.     

Disease avoidance as a functional basis for stigmatization

Stigmatization is characterized by chronic social and physical avoidance of a person(s) by other people. Infectious disease may produce an apparently similar form of isolation-disease avoidance-but on symptom remission this often abates. We propose that many forms of stigmatization reflect the activation of this disease-avoidance system, which is prone to respond to visible signs and labels that connote disease, irrespective of their accuracy. A model of this system is presented, which includes an emotional component, whereby visible disease cues directly activate disgust and contamination, motivating avoidance, and a cognitive component, whereby disease labels bring to mind disease cues, indirectly activating disgust and contamination. The unique predictions of this model are then examined, notably that people who are stigmatized evoke disgust and are contaminating. That animals too show avoidance of diseased conspecifics, and that disease-related stigma targets are avoided in most cultures, also supports this evolutionary account. The more general implications of this approach are then examined, notably how it can be used to good (e.g. improving hygiene) or bad (e.g. racial vilification) ends, by yoking particular labels with cues that connote disease and disgust. This broadening of the model allows for stigmatization of groups with little apparent connection to disease.     

Accuracies of genomic breeding values in American Angus beef cattle using K-means clustering for cross-validation

Background

Genomic selection is a recently developed technology that is beginning to revolutionize animal breeding. The objective of this study was to estimate marker effects to derive prediction equations for direct genomic values for 16 routinely recorded traits of American Angus beef cattle and quantify corresponding accuracies of prediction.

Methods

Deregressed estimated breeding values were used as observations in a weighted analysis to derive direct genomic values for 3570 sires genotyped using the Illumina BovineSNP50 BeadChip. These bulls were clustered into five groups using K-means clustering on pedigree estimates of additive genetic relationships between animals, with the aim of increasing within-group and decreasing between-group relationships. All five combinations of four groups were used for model training, with cross-validation performed in the group not used in training. Bivariate animal models were used for each trait to estimate the genetic correlation between deregressed estimated breeding values and direct genomic values.

Results

Accuracies of direct genomic values ranged from 0.22 to 0.69 for the studied traits, with an average of 0.44. Predictions were more accurate when animals within the validation group were more closely related to animals in the training set. When training and validation sets were formed by random allocation, the accuracies of direct genomic values ranged from 0.38 to 0.85, with an average of 0.65, reflecting the greater relationship between animals in training and validation. The accuracies of direct genomic values obtained from training on older animals and validating in younger animals were intermediate to the accuracies obtained from K-means clustering and random clustering for most traits. The genetic correlation between deregressed estimated breeding values and direct genomic values ranged from 0.15 to 0.80 for the traits studied.

Conclusions

These results suggest that genomic estimates of genetic merit can be produced in beef cattle at a young age but the recurrent inclusion of genotyped sires in retraining analyses will be necessary to routinely produce for the industry the direct genomic values with the highest accuracy.     

Evidence that a copper-metallothionein complex is responsible for fluorescence in acid-secreting cells of the Drosophila stomach

Copper cells were originally identified in Drosophila midgut epithelium by their striking orange fluorescence in copper-fed larvae. Here, we examined copper cell fluorescence in light of the previous observations that (1) a similar fluorescent signal in yeast is produced by a complex between copper and metallothionein, and (2) metallothionein is expressed constitutively in the copper cell region and inducibly in other regions of the Drosophila midgut. Pulse-feeding experiments with 1 mM CuCl2 revealed that fluorescence appeared rapidly in copper cells (<5 min) and slowly in other cells of the midgut (days), suggesting a constitutive cofactor in the former and an inducible cofactor in the latter. Fluorescence was also detected in Drosophila S2 tissue culture cells after induction of metallothionein synthesis by addition of CuCl2 to the growth medium. Thus, fluorescence coincided spatially and temporally with the expression of metallothionein. Fluorescence was also linked to the acid-secreting activity of copper cells. Fluorescence was not observed when acid secretion was inhibited by a mutation in the alpha spectrin gene and acidification was blocked in copper-fed wild-type larvae. However, acidification was restored after a 1-day chase period in which the fluorescent signal became sequestered within a vesicular compartment. We therefore conclude that copper cell fluorescence is most probably attributable to a cytoplasmic copper-metallothionein complex, suggesting an unanticipated role for metallothionein in acid-secreting cells.     

Tumor-associated embryonic antigen-expressing vaccines that target CCR6 elicit potent CD8+ T cell-mediated protective and therapeutic antitumor immunity

Despite its potency, the wider use of immunotherapy for B cell malignancies is hampered by the lack of well-defined tumor-specific Ags. In this study, we demonstrate that an evolutionarily conserved 37-kDa immature laminin receptor protein (OFA-iLRP), a nonimmunogenic embryonic Ag expressed by a variety of tumors, is rendered immunogenic if targeted to the APCs using the CCR6 ligands MIP3alpha/CCL20 and mDF2beta. The CCR6 targeting facilitated efficient Ag cross-presentation and induction of tumor-neutralizing CTLs. Although the Ag targeting alone, without activation of dendritic cells (DCs), is proposed to induce tolerance, and MIP3alpha does not directly activate DCs, the MIP3alpha-based vaccine efficiently induced protective and therapeutic antitumor responses. The responses were as strong as those elicited by the OFA-iLRP fusions with moieties that activated DCs and Th1-type cytokine responses, mDF2beta, or mycobacterial Hsp70 Ag. Although the same cDNA encodes the dimerized high-affinity mature 67-kDa mLRP that is expressed in normal tissues to stabilize the binding of laminin to cell surface integrins, the vaccines expressing OFA-iLRP elicited long-term protective CD8(+) T cell-mediated memory responses against syngeneic B cell lymphoma, indicating the potential application of these simple vaccines as preventive and therapeutic formulations for human use.     

Proteomic analysis of the effects and interactions of sleep deprivation and aging in mouse cerebral cortex

The cellular and molecular processes that underlie the drives and functions of sleep have been the topic of many studies in the last few decades. Discovery-based techniques, such as cDNA microarrays, have increasingly been utilized in conjunction with sleep deprivation paradigms to examine the molecular mechanisms and functions of sleep. These studies have helped to validate and expand existing hypotheses, such as those on the roles of sleep in synaptic plasticity and in energy metabolism. The mechanisms underlying the highly prevalent changes in sleep architecture with age are not known, but likely reflect fundamental changes in the molecular basis of circadian timing and sleep homeostatic processes. We decided to explore the effects and interactions of sleep deprivation and aging utilizing the proteomic technique of difference in gel electrophoresis (DIGE). DIGE, which utilizes cyanine dye labeling of samples, allows for the comparison of multiple experimental groups within and across gels. In this study, we compared cerebral cortex tissue from young (2.5 months) and old (24 months) mice that had been sleep deprived for 6 h to tissue from undisturbed young and old control animals. Following DIGE, automatic image matching and spot identification, and statistical analysis, 43 unique proteins were identified. The proteins were grouped into seven functional classes based on published characteristics: cell signaling, cytoskeletal, energy metabolism, exocytosis, heat shock proteins, mRNA processing/trafficking, and serum proteins. The identity and characteristics of these proteins relevant to sleep and aging are discussed.     

Enhanced detection of human immunodeficiency virus type 1 (HIV-1) Nef-specific T cells recognizing multiple variants in early HIV-1 infection

A human immunodeficiency virus (HIV)-preventive vaccine will likely need to induce broad immunity that can recognize antigens expressed within circulating strains. To understand the potentially relevant responses that T-cell based vaccines should elicit, we examined the ability of T cells from early infected persons to recognize a broad spectrum of potential T-cell epitopes (PTE) expressed by the products encoded by the HIV type 1 (HIV-1) nef gene, which is commonly included in candidate vaccines. T cells were evaluated for gamma interferon (IFN-gamma) secretion using two peptide panels: subtype B consensus (CON) peptides and a novel peptide panel providing 70% coverage of PTE in subtype B HIV-1 Nef. Eighteen of 23 subjects' T cells recognized HIV-1 Nef. In one subject, Nef-specific T cells were detected with the PTE but not with the CON peptides. The greatest frequency of responses spanned Nef amino acids 65 to 103 and 113 to 147, with multiple epitope variants being recognized. Detection of both the epitope domain number and the response magnitude was enhanced using the PTE peptides. On average, we detected 2.7 epitope domains with the PTE peptides versus 1.7 domains with the CON peptides (P = 0.0034). The average response magnitude was 2,169 spot-forming cells (SFC)/10(6) peripheral blood mononuclear cells (PBMC) with the PTE peptides versus 1,010 SFC/10(6) PBMC with CON peptides (P = 0.0046). During early HIV-1 infection, Nef-specific T cells capable of recognizing multiple variants are commonly induced, and these responses are readily detected with the PTE peptide panel. Our findings suggest that Nef responses induced by a given vaccine strain before HIV-1 exposure may be sufficiently broad to recognize most variants within subtype B HIV-1.