Weighing in on a Timeless controversy

Timeless (Tim) and Period (Per) are coordinately synthesized interacting proteins that in response to positional/environmental cues comigrate to the nucleus as obligate heterodimers where they act to suppress their own gene expression as part of the circadian rhythm network in Drosophila. There has been considerable controversy about the structural nature of Tim because of somewhat conflicting results generated by the automated threading algorithm 3D-PSSM. With use of a computer-assisted but largely manual approach, it is shown here that Tim is composed of repetitive structural elements and that those elements comprise two ARM repeat domains, validating the essence of the original 3D-PSSM prediction. Eleven individual ARM structural units are assigned, and a phylogenetic analysis showing their relatedness to one another is performed. In addition, there appears to be a small domain of prenyltransferase-like repeats C-terminal to the second ARM domain, which went undetected in previous analyses. Although we cannot know the precise conformation it adopts until a structure is solved, Tim emerges here as clearly a member of the helical repeat protein superfamily. Given its role in periodic nuclear translocation, Tim may, therefore, have a functional analogy with the photoperiod-responsive protein Phor1 and other karyopherin-like molecules.     

Sequence variation in NPC1L1 and association with improved LDL-cholesterol lowering in response to ezetimibe treatment

Niemann-Pick C1-like 1 (NPC1L1) is an intestinal cholesterol transporter and the molecular target of ezetimibe, a cholesterol absorption inhibitor demonstrated to reduce LDL-cholesterol (LDL-C) both as monotherapy and when co-administered with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins). Interestingly, significant interindividual variability has been observed for rates of intestinal cholesterol absorption and LDL-C reductions at both baseline and post ezetimibe treatment. To test the hypothesis that genetic variation in NPC1L1 could influence the LDL-C response to ezetimibe, we performed extensive resequencing of the gene in 375 apparently healthy individuals and genotyped hypercholesterolemic patients from clinical trial cohorts. No association was observed between NPC1L1 single-nucleotide polymorphism and baseline cholesterol. However, significant associations to LDL-C response to treatment with ezetimibe were observed in patients treated with ezetimibe in two large clinical trials. Our data demonstrate that DNA sequence variants in NPC1L1 are associated with an improvement in response to ezetimibe pharmacotherapy and suggest that detailed analysis of genetic variability in clinical trial cohorts can lead to improved understanding of factors contributing to variable drug response.     

Nitration of gamma-tocopherol prevents its oxidative metabolism by HepG2 cells

Gamma-tocopherol (gammaT) is one of the major forms of vitamin E consumed in the diet. Previous reports have suggested increased levels of nitrated gamma-tocopherol (5-NO2-gammaT) in smokers and individuals with conditions associated with elevated nitrative stress. The monitoring of 5-NO2-gammaT and its possible metabolite(s) may be a useful marker of reactive nitrogen species generation in vivo. The major pathway for the metabolism of gammaT is the cytochrome P450 dependent oxidation to its water-soluble metabolite gamma-CEHC, which is excreted in urine. In order to determine if 5-NO2-gammaT could be metabolised via the same route and detected in urine we developed a sensitive gas chromatography-mass spectrometry assay for 5-NO2-gamma-CEHC. 5-NO2-gamma-CEHC was synthesised and its structure confirmed by proton nuclear magnetic resonance and mass spectrometry. While gamma-CEHC was abundant in urine from healthy volunteers, as well as patients with coronary heart disease and type 2 diabetes, 5-NO2-gamma-CEHC was undetectable (limit of detection of 5 nM). To understand this observation we examined the uptake and metabolism of gammaT and 5-NO2-gammaT by HepG2 cells. gammaT was readily incorporated into cells and metabolised to gamma-CEHC over a period of 48 hours. In contrast, 5-NO2-gammaT was poorly incorporated into HepG2 cells and not metabolised to 5-NO2-gamma-CEHC over the same time period. We conclude that nitration of gammaT prevents its incorporation into liver cells and therefore its metabolism to the water-soluble metabolite. Whether 5-NO2-gammaT could be metabolised via other pathways in vivo requires further investigation.     

Abeta42 is essential for parenchymal and vascular amyloid deposition in mice

Considerable circumstantial evidence suggests that Abeta42 is the initiating molecule in Alzheimer's disease (AD) pathogenesis. However, the absolute requirement for Abeta42 for amyloid deposition has never been demonstrated in vivo. We have addressed this by developing transgenic models that express Abeta1-40 or Abeta1-42 in the absence of human amyloid beta protein precursor (APP) overexpression. Mice expressing high levels of Abeta1-40 do not develop overt amyloid pathology. In contrast, mice expressing lower levels of Abeta1-42 accumulate insoluble Abeta1-42 and develop compact amyloid plaques, congophilic amyloid angiopathy (CAA), and diffuse Abeta deposits. When mice expressing Abeta1-42 are crossed with mutant APP (Tg2576) mice, there is also a massive increase in amyloid deposition. These data establish that Abeta1-42 is essential for amyloid deposition in the parenchyma and also in vessels.     

Effects of alpha-synuclein immunization in a mouse model of Parkinson's disease

Abnormal folding of alpha-synuclein (alpha-syn) is thought to lead to neurodegeneration and the characteristic symptoms of Lewy body disease (LBD). Since previous studies suggest that immunization might be a potential therapy for Alzheimer's disease, we hypothesized that immunization with human (h)alpha-syn might have therapeutic effects in LBD. For this purpose, halpha-syn transgenic (tg) mice were vaccinated with halpha-syn. In mice that produced high relative affinity antibodies, there was decreased accumulation of aggregated halpha-syn in neuronal cell bodies and synapses that was associated with reduced neurodegeneration. Furthermore, antibodies produced by immunized mice recognized abnormal halpha-syn associated with the neuronal membrane and promoted the degradation of halpha-syn aggregates, probably via lysosomal pathways. Similar effects were observed with an exogenously applied FITC-tagged halpha-syn antibody. These results suggest that vaccination is effective in reducing neuronal accumulation of halpha-syn aggregates and that further development of this approach might have a potential role in the treatment of LBD.     

Montane speciation patterns in Ithomiola butterflies (Lepidoptera: Riodinidae): are they consistently moving up in the world?

Tropical lowland areas have often been seen as the centres of terrestrial species proliferation, but recent evidence suggests that young species may be more frequent in montane areas. Several montane speciation modes have been proposed, but their relative frequencies and predominant evolutionary sequence remain unclear because so few biogeographic and phylogenetic studies have tested such questions. I use morphological data to generate a phylogenetic hypothesis for all 11 species of the riodinid butterfly genus Ithomiola (Riodininae: Mesosemiini: Napaeina). These species are shown here to be all strictly geographically and elevationally allo- or parapatrically distributed with respect to their closest relatives in lowland and montane regions throughout the Neotropics. The overwhelming pattern in Ithomiola is of repeated upward parapatric speciation across an elevational gradient, and the genus appears to provide the clearest example to date of vertical montane speciation. All of the young derived species are montane and all of the old basal species are confined to the lowlands, supporting the hypothesis of montane regions largely as 'species pumps' and lowland regions as 'museums'. Possible reasons for the post-speciation maintenance of parapatric ranges in Ithomiola are discussed.     

Genetic dissection of the zebrafish retinal stem-cell compartment

In a large-scale forward-genetic screen, we discovered that a limited number of genes are required for the regulation of retinal stem cells after embryogenesis in zebrafish. In 18 mutants out of almost 2000 F2 families screened, the eye undergoes normal embryonic development, but fails to continue growth from the ciliary marginal zone (CMZ), the post-embryonic stem-cell niche. Class I-A mutants (5 loci) display lower amounts of proliferation in the CMZ, while nearly all cells in the retina appear differentiated. Class I-B mutants (2 loci) have a reduced CMZ with a concomitant expansion in the retinal pigmented epithelium (RPE), suggesting a common post-embryonic stem cell is the source for these neighboring cell types. Class II encompasses three distinct types of mutants (11 loci) with expanded CMZ, in which the progenitor population is arrested in the cell cycle. We also show that in at least one combination, the reduced CMZ phenotype is genetically epistatic to the expanded CMZ phenotype, suggesting that Class I genes are more likely to affect the stem cells and Class II the progenitor cells. Finally, a comparative mapping analysis demonstrates that the new genes isolated do not correspond to genes previously implicated in stem-cell regulation. Our study suggests that embryonic and post-embryonic stem cells utilize separable genetic programs in the zebrafish retina.     

The dimensionality of genetic variation for wing shape in Drosophila melanogaster

Absolute constraints are limitations on genetic variation that preclude evolutionary change in some aspect of the phenotype. Absolute constraints may reflect complete absence of variation, lack of genetic variation that extends the range of phenotypes beyond some limit, or lack of additive genetic variation. This last type of absolute constraint is bidirectional, because the mean cannot evolve to be larger or smaller. Most traits do possess genetic variation, so bidirectional absolute constraints are most likely to be detected in a multivariate context, where they would reflect combinations of traits, or dimensions in phenotype space that cannot evolve. A bidirectional absolute constraint will cause the additive genetic covariance matrix (G) to have a rank less than the number of traits studied. In this study, we estimate the rank of the G-matrix for 20 aspects of wing shape in Drosophila melanogaster. Our best estimates of matrix rank are 20 in both sexes. Lower 95% confidence intervals of rank are 17 for females and 18 for males. We therefore find little evidence of bidirectional absolute constraints. We discuss the importance of this result for resolving the relative roles of selection and drift processes versus constraints in the evolution of wing shape in Drosophila.     

Colonization, dispersal, and hybridization influence phylogeography of North Atlantic sea urchins (Strongylocentrotus droebachiensis)

We used frequency-based and coalescent-based phylogeographic analysis of sea urchin (Strongylocentrotus droebachiensis) mitochondrial DNA (mtDNA) sequences and previously published microsatellite data to understand the relative influence of colonization and gene flow from older (north Pacific) and younger (northeast Atlantic) sea urchin populations on genetic variation in the northwest Atlantic. We found strong evidence of survival of northwestern Atlantic populations in local Pleistocene glacial refugia: most haplotypes were the same as or closely related to Pacific haplotypes, with deep gene genealogies that reflect divergence times within the northwestern Atlantic that are much older than the last glacial maximum. We detected gene flow across the North Atlantic in the form of haplotypes shared with or recently descended from European populations. We also found evidence of significant introgression of haplotypes from a closely related species (S. pallidus). The relative magnitude of gene flow estimated by coalescent methods (and the effective population size differences among oceanic regions) depended on the genetic marker used. In general, we found very small effective population size in the northeastern Atlantic and high trans-Arctic gene flow between the Pacific and northwestern Atlantic. Both analyses suggested significant back-migration to the Pacific. However, microsatellites more strongly reflected older Pacific migration (with similar effective population sizes across the Arctic), whereas mtDNA sequences appeared to be more sensitive to recent trans- Atlantic dispersal (with larger differences in effective population size). These differences across marker types might have several biological or methodological causes, and they suggest caution in interpretation of the results from a single locus or class of markers.