CTCF cis-Regulates Trinucleotide Repeat Instability in an Epigenetic Manner: A Novel Basis for Mutational Hot Spot Determination

At least 25 inherited disorders in humans result from microsatellite repeat expansion. Dramatic variation in repeat instability occurs at different disease loci and between different tissues; however, cis-elements and trans-factors regulating the instability process remain undefined. Genomic fragments from the human spinocerebellar ataxia type 7 (SCA7) locus, containing a highly unstable CAG tract, were previously introduced into mice to localize cis-acting “instability elements,” and revealed that genomic context is required for repeat instability. The critical instability-inducing region contained binding sites for CTCF—a regulatory factor implicated in genomic imprinting, chromatin remodeling, and DNA conformation change. To evaluate the role of CTCF in repeat instability, we derived transgenic mice carrying SCA7 genomic fragments with CTCF binding-site mutations. We found that CTCF binding-site mutation promotes triplet repeat instability both in the germ line and in somatic tissues, and that CpG methylation of CTCF binding sites can further destabilize triplet repeat expansions. As CTCF binding sites are associated with a number of highly unstable repeat loci, our findings suggest a novel basis for demarcation and regulation of mutational hot spots and implicate CTCF in the modulation of genetic repeat instability.     

Lipid peroxidation is a consequence of elicitor activity

Elicitor-active preparations from the fungal pathogen of bean Colletotrichum lindemuthianum stimulated the accumulation of products characteristic of lipid peroxidation in treated bean tissues. Bean suspension cells treated with crude and purified elicitors accumulated `lipofuscin-like pigment' (LEP) and malondialdehyde. The accumulation of LFP after about 6 h of treatment coincided with the onset of visible browning and production of the bean phytoalexins kievitone, phaseollin, and phaseollinisoflavan. The induction of phytoalexins and accumulation of LFP were also triggered by treatments with generators of activated oxygen species, xanthine:xanthine oxidase and Fe:ethylenediaminedi-o-hydroxyphenylacetic acid. These data suggest that generation of active oxygen species may be involved in lipid peroxidation triggered by elicitors.     

Reprint of: Saprolegnia strains isolated from river insects and amphipods are broad spectrum pathogens

Saprolegnia species are destructive pathogens to many aquatic organisms and are found in most parts of the world. Reports based on phylogenetic analysis suggest that Saprolegnia strains isolated from aquatic animals such as crustaceans and frogs are close to Saprolegnia strains isolated from infected fish or fish eggs and vice versa. However, it has often been assumed that host specificity occurs for each individual isolate or strain. Here we demonstrate that Saprolegnia spp. can have multiple hosts and are thus capable of infecting different aquatic organisms. Saprolegnia delica, Saprolegnia hypogyna, and 2 strains of Saprolegnia diclina were isolated from aquatic insects and amphipods while S. delica, Saprolegnia ferax, Pythium pachycaule, and a Pythium sp. were isolated from the water of a medium to fast flowing river. The ITS region of the rRNA gene was sequenced for all isolates. In challenge experiments, all four isolates from insects were found to be highly pathogenic to eggs of Atlantic salmon (Salmo salar) and embryos of the African clawed frog (Xenopus laevis). We found that Saprolegnia spp. isolated from salmon eggs were also able to successfully establish infection in nymphs of stonefly (Perla bipunctata) and embryos of X. laevis). These results suggest that Saprolegnia spp. are capable of infecting multiple hosts, which may give them an advantage during seasonal variation in their natural environments.     

γδT Cells Are Prevalent in the Proximal Aorta and Drive Nascent Atherosclerotic Lesion Progression and Neutrophilia in Hypercholesterolemic Mice

Unique innate immunity-linked γδT cells have been seen in early human artery lesions, but their role in lesion development has received little attention. Here we investigated whether γδT cells modulate atherogenesis in apolipoprotein E-deficient (ApoE KO) mice. We found that γδT cell numbers were markedly increased in the proximal aorta of ApoE-deficient vs. wild-type mice during early atherogenesis, particularly in the aortic root and arch, where they comprised most of the T cells and lesion progression is most rapid. γδT cells infiltrated intimal lesions in ApoE KO mice, but only the adventitia in wild-type mice, and were more prevalent than CD4⁺ T cells in early nascent lesions, as evaluated by en face confocal microscopy. These aortic γδT cells produced IL-17, but not IFN-γ, analyzed by ex vivo FACS. Furthermore, aortic arch lipid accumulation correlated strongly with abundance of IL-17-expressing splenic γδT cells in individual ApoE KO mice. To investigate the role of these γδT cells in early atherogenesis, we analyzed ApoE/γδT double knockout (DKO) compared to ApoE KO mice. We observed reduced early intimal lipid accumulation at sites of nascent lesion formation, both in chow-fed (by 40%) and Western diet-fed (by 44%) ApoE/γδT DKO mice. In addition, circulating neutrophils were drastically reduced in these DKO mice on Western diet, while expansion of inflammatory monocytes and splenic Th1 or Th17 lymphocytes was not affected. These data reveal, for the first time, a pathogenic role of γδT cells in early atherogenesis in ApoE KO mice, by mechanisms likely to involve their IL-17 production and induction of neutrophilia. Targeting γδT cells thus might offer therapeutic benefit in atherosclerosis or other inflammatory vascular diseases.     

Thermal destabilization of rhodopsin and opsin by proteolytic cleavage in bovine rod outer segment disk membranes

The G-protein coupled receptor, rhodopsin, consists of seven transmembrane helices which are buried in the lipid bilayer and are connected by loop domains extending out of the hydrophobic core. The thermal stability of rhodopsin and its bleached form, opsin, was investigated using differential scanning calorimetry (DSC). The thermal transitions were asymmetric, and the temperatures of the thermal transitions were scan rate dependent. This dependence exhibited characteristics of a two-state irreversible denaturation in which intermediate states rapidly proceed to the final irreversible state. These studies suggest that the denaturation of both rhodopsin and opsin is kinetically controlled. The denaturation of the intact protein was compared to three proteolytically cleaved forms of the protein. Trypsin removed nine residues of the carboxyl terminus, papain removed 28 residues of the carboxyl terminus and a portion of the third cytoplasmic loop, and chymotrypsin cleaved cytoplasmic loops 2 and 3. In each of these cases the fragments remained associated as a complex in the membrane. DSC studies were carried out on each of the fragmented proteins. In all of the samples the scan rate dependence of the Tm indicated that the transition was kinetically controlled. Trypsin-proteolyzed protein differed little from the intact protein. However, the activation energy for denaturation was decreased when cytoplasmic loop 3 was cleaved by papain or chymotrypsin. This was observed for both bleached and unbleached samples. In the presence of the chromophore, 11-cis-retinal, the noncovalent interactions among the proteolytic fragments produced by papain and chymotrypsin cleavage were sufficiently strong such that each of the complexes denatured as a unit. Upon bleaching, the papain fragments exhibited a single thermal transition. However, after bleaching, the chymotrypsin fragments exhibited two calorimetric transitions. These data suggest that the loops of rhodopsin exert a stabilizing effect on the protein.     

Are subspecies useful in evolutionary and conservation biology?

The taxonomic rank of subspecies remains highly contentious, largely because traditional subspecies boundaries have sometimes been contradicted by molecular phylogenetic data. The most complete meta-analysis to date, for instance, found that only 3% of traditional avian subspecies represented distinct phylogenetic lineages. However, the global generality of this phenomenon remains unclear due to this previous study's narrow geographic focus on continental Nearctic and Palearctic subspecies. Here, we present a new global analysis of avian subspecies and show that 36% of avian subspecies are, in fact, phylogenetically distinct. Among biogeographic realms we find significant differences in the proportion of subspecies that are phylogenetically distinct, with Nearctic/Palearctic subspecies showing significantly reduced levels of differentiation. Additionally, there are differences between island and continental subspecies, with continental subspecies significantly less likely to be genetically distinct. These results indicate that the overall level of congruence between taxonomic subspecies and molecular phylogenetic data is greater than previously thought. We suggest that the widespread impression that avian subspecies are not real arises from a predominance of studies focusing on continental subspecies in North America and Eurasia, regions which show unusually low levels of genetic differentiation. The broader picture is that avian subspecies often provide an effective short-cut for estimating patterns of intraspecific genetic diversity, thereby providing a useful tool for the study of evolutionary divergence and conservation.     

Oviposition Behavior of the Eastern Spruce Budworm Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae)

We documented 13 behaviors associated with oviposition in unfertilized and fertilized spruce budworm moths, Choristoneura fumiferana, by videotaping active moths with a macro lens. Apart from resting, the most pronounced behaviors were probing, drumming + probing, and egg laying. Probing was a bending of the abdomen and extension of the ovipositor to touch the substrate in a rocking, back and forth motion. Drumming + probing involved tapping of the substrate with the pro- and mesothocacic legs with concurrent bending of the abdomen as above. Egg laying was the actual process of egg deposition onto the needle surface of a balsam fir twig. Both the frequency and the duration of these behavioral elements varied depending on the mating status of the female. Unfertilized females exhibited a higher frequency of probing and egg laying. The duration of probing was longer in unfertilized females, while drumming + probing and egg laying were longer for fertilized females. This study is the basis for future work on the chemosensilla associated with the perception of host-plant surface chemicals by ovipositing females.     

Cholesteryl ester transfer protein and hyperalphalipoproteinemia in Caucasians

It is unclear whether cholesteryl ester transfer protein (CETP) contributes to high density lipoprotein cholesterol (HDL-C) levels in hyperalphalipoproteinemia (HALP) in Caucasians. Moreover, even less is known about the effects of hereditary CETP deficiency in non-Japanese. We studied 95 unrelated Caucasian individuals with HALP. No correlations between CETP concentration or activity and HDL-C were identified. Screening for CETP gene defects led to the identification of heterozygosity for a novel splice site mutation in one individual. Twenty-five heterozygotes for this mutation showed reduced CETP concentration (-40%) and activity (-50%) and a 35% increase of HDL-C compared with family controls. The heterozygotes presented with an isolated high HDL-C, whereas the remaining subjects exhibited a typical high HDL-C/low-triglyceride phenotype. The increase of HDL-C in the CETP-deficient heterozygotes was primarily attributable to increased high density lipoprotein containing apolipoprotein A-I and A-II (LpAI:AII) levels, contrasting with an increase in both high density lipoprotein containing apolipoprotein A-I only and LpAI:AII in the other group. This study suggests the absence of a relationship between CETP and HDL-C levels in Caucasians with HALP. The data furthermore indicate that genetic CETP deficiency is rare among Caucasians and that this disorder presents with a phenotype that is different from that of subjects with HALP who have no mutation in the CETP gene.