Comparative studies of the endonucleases from two related Xenopus laevis retrotransposons, Tx1L and Tx2L: target site specificity and evolutionary implications

In the genome of the South African frog, Xenopus laevis, there are two complex families of transposable elements, Tx1 and Tx2, that have identical overall structures, but distinct sequences. In each family there are approximately 1500 copies of an apparent DNA-based element (Tx1D and Tx2D). Roughly 10% of these elements in each family are interrupted by a non-LTR retrotransposon (Tx1L and Tx2L). Each retrotransposon is flanked by a 23-bp target duplication of a specific D element sequence. In earlier work, we showed that the endonuclease domain (Tx1L EN) located in the second open reading frame (ORF2) of Tx1L encodes a protein that makes a single-strand cut precisely at the expected site within its target sequence, supporting the idea that Tx1L is a site-specific retrotransposon. In this study, we express the endonuclease domain of Tx2L (Tx2L EN) and compare the target preferences of the two enzymes. Each endonuclease shows some preference for its cognate target, on the order of 5-fold over the non- cognate target. The observed discrimination is not sufficient, however, to explain the observation that no cross-occupancy is observed – that is, L elements of one family have never been found within D elements of the other family. Possible sources of additional specificity are discussed. We also compare two hypotheses regarding the genome duplication event that led to the contemporary pseudotetraploid character of Xenopus laevis in light of the Tx1L and Tx2L data. This revised version was published online in July 2006 with corrections to the Cover Date.     

Epididymal SPAM1 is a marker for sperm maturation in the mouse

Sperm adhesion molecule 1 (SPAM1), is a glycosyl phoshatidylinositol-linked sperm membrane protein that is dually expressed in testis and epididymis. Epididymal SPAM1 is secreted in all three regions of the epididymis in all mammalian species studied, including humans. It shares the same molecular mass and neutral hyaluronidase activity as the testicular and sperm isoforms that are responsible for the penetration of the cumulus during fertilization. Using wild-type (W/T) sperm and those from mice homozygous for either a null (Spam1-/-) or mutant Spam1 allele, which results in decreased mRNA and protein, we demonstrate that sperm binding of epididymal SPAM1 occurs in vitro after exposure to W/T sperm-free epididymal luminal fluid (ELF). Binding or adsorption that occurred after incubation at room temperature or 32 degrees C was detected immunocytochemically and confirmed quantitatively using flow cytometry. The localization of SPAM1 on the plasma membrane of Spam1-null sperm mimicked that seen in the W/T. The remarkable increase in binding on W/T caudal sperm indicates that they are not fully saturated with SPAM1 during storage, and suggests that uptake of epididymal SPAM1 in vivo augments testicular SPAM1. Spam1-null sperm exposed to W/T ELF for 45-60 min during in vitro capacitation to allow epididymal SPAM1 binding showed a highly significant (P < 0.001) increase in cumulus penetration after 6-7 h compared to those incubated in ELF from null males. Similarly, the number of cumulus-free oocytes was also highly significantly greater (P < 0.001) than that for sperm capacitated in W/T SPAM1-antibody-inhibited ELF. Because epididymal SPAM1 uptake significantly increases cumulus penetration, we conclude that it is a marker of sperm maturation.     

Genetic and Pathogenic Characterization of Different Stagonospora sp. Isolated from Bindweed

Genetic variation among 38 isolates of Stagonospora sp. and 10 isolates of Septoria sp. from bindweed was studied using (a) restriction fragment length plymorphism (RFLP) analysis of the internal transcribed spacer (ITS) region, and (b) random amplified polymorphic DNA (RAPD) PCR analysis. RFLP analysis revealed three types of fragment patterns among the isolates. A total of 26 distinct groups, based on common fragment patterns, were identified using cluster analysis of the RAPD-PCR data. When the grouping results of the two methods were compared, the fragment pattern types and clusters were generally in agreement. The degree of pathogenicity of six genetically characterized isolates of Stagonospora sp. was assessed on three ecotypes of field bindweed (Convolvulus arvensis). Disease symptoms were observed with all isolates on all ecotypes, but only Stagonospora convolvuli strain LA39, a potential biocontrol agent, showed a high degree of pathogenicity on all ecotypes. A mixture of two Stagonospora sp. enhanced the mean necrotic leaf area on bindweed from 33.9 and 39.0% (when applied alone) to 64.9% applied together at the same final concentration of 5 X 106 spores ml -1 . Molecular methods were used to identify the two pathogens. Both were present on the same plant when applied together, but never found in the same lesion.     

Chikungunya virus impacts the diversity of symbiotic bacteria in mosquito vector

Mosquitoes transmit numerous arboviruses including dengue and chikungunya virus (CHIKV). In recent years, mosquito species Aedes albopictus has expanded in the Indian Ocean region and was the principal vector of chikungunya outbreaks in La Reunion and neighbouring islands in 2005 and 2006. Vector‐associated bacteria have recently been found to interact with transmitted pathogens. For instance, Wolbachia modulates the replication of viruses or parasites. However, there has been no systematic evaluation of the diversity of the entire bacterial populations within mosquito individuals particularly in relation to virus invasion. Here, we investigated the effect of CHIKV infection on the whole bacterial community of Ae. albopictus. Taxonomic microarrays and quantitative PCR showed that members of Alpha‐ and Gammaproteobacteria phyla, as well as Bacteroidetes, responded to CHIKV infection. The abundance of bacteria from the Enterobacteriaceae family increased with CHIKV infection, whereas the abundance of known insect endosymbionts like Wolbachia and Blattabacterium decreased. Our results clearly link the pathogen propagation with changes in the dynamics of the bacterial community, suggesting that cooperation or competition occurs within the host, which may in turn affect the mosquito traits like vector competence.     

Paradoxical Condensation of Copper with Elevated ��-Amyloid in Lipid Rafts under Cellular Copper Deficiency Conditions: IMPLICATIONS FOR ALZHEIMER DISEASE*

Redox-active copper is implicated in the pathogenesis of Alzheimer disease (AD), β-amyloid peptide (Aβ) aggregation, and amyloid formation. Aβ·copper complexes have been identified in AD and catalytically oxidize cholesterol and lipid to generate H₂O₂ and lipid peroxides. The site and mechanism of this abnormality is not known. Growing evidence suggests that amyloidogenic processing of the β-amyloid precursor protein (APP) occurs in lipid rafts, membrane microdomains enriched in cholesterol. β- and γ-secretases, and Aβ have been identified in lipid rafts in cultured cells, human and rodent brains, but the role of copper in lipid raft amyloidogenic processing is presently unknown. In this study, we found that copper modulates flotillin-2 association with cholesterol-rich lipid raft domains, and consequently Aβ synthesis is attenuated via copper-mediated inhibition of APP endocytosis. We also found that total cellular copper is associated inversely with lipid raft copper levels, so that under intracellular copper deficiency conditions, Aβ·copper complexes are more likely to form. This explains the paradoxical hypermetallation of Aβ with copper under tissue copper deficiency conditions in AD.Imbalance of metal ions has been recognized as one of the key factors in the pathogenesis of Alzheimer disease (AD).² Aberrant interactions between copper or zinc with the β-amyloid peptide (Aβ) released into the glutamatergic synaptic cleft vicinity could result in the formation of toxic Aβ oligomers and aggregation into plaques characteristic of AD brains (reviewed in Ref. 1). Copper, iron, and zinc are highly concentrated in extracellular plaques (2, 3), and yet brain tissues from AD (4–6) and human β-amyloid precursor protein (APP) transgenic mice (7–10) are paradoxically copper deficient compared with age-matched controls. Elevation of intracellular copper levels by genetic, dietary, and pharmacological manipulations in both AD transgenic animal and cell culture models is able to attenuate Aβ production (7, 9, 11–15). However, the underlying mechanism is at present unclear.Abnormal cholesterol metabolism is also a contributing factor in the pathogenesis of AD. Hypercholesterolemia increases the risk of developing AD-like pathology in a transgenic mouse model (16). Epidemiological and animal model studies show that a hypercholesterolemic diet is associated with Aβ accumulation and accelerated cognitive decline, both of which are further aggravated by high dietary copper (17, 18). In contrast, biochemical depletion of cholesterol using statins, inhibitors of 3-hydroxy-3-methyglutaryl coenzyme A reductase, and methyl-β-cyclodextrin, a cholesterol sequestering agent, inhibit Aβ production in animal and cell culture models (19–25).Cholesterol is enriched in lipid rafts, membrane microdomains implicated in Aβ generation from APP cleavage by β- and γ-secretases. Recruitment of BACE1 (β-secretase) into lipid rafts increases the production of sAPP_β and Aβ (23, 26). The β-secretase-cleaved APP C-terminal fragment (β-CTF), and γ-secretase, a multiprotein complex composed of presenilin (PS1 or PS2), nicastrin (Nct), PEN-2 and APH-1, colocalize to lipid rafts (27). The accumulation of Aβ in lipid rafts isolated from AD and APP transgenic mice brains (28) provided further evidence that cholesterol plays a role in APP processing and Aβ generation.Currently, copper and cholesterol have been reported to modulate APP processing independently. However, evidence indicates that, despite tissue copper deficiency, Aβ·Cu²⁺ complexes form in AD that catalytically oxidize cholesterol and lipid to generate H₂O₂ and lipid peroxides (e.g. hydroxynonenal and malondialdehyde), which contribute to oxidative damage observed in AD (29–35). The underlying mechanism leading to the formation of pathological Aβ·Cu²⁺ complexes is unknown. In this study, we show that copper alters the structure of lipid rafts, and attenuates Aβ synthesis in lipid rafts by inhibition of APP endocytosis. We also identify a paradoxical inverse relationship between total cellular copper levels and copper distribution to lipid rafts, which appear to possess a privileged pool of copper where Aβ is more likely to interact with Cu²⁺ under copper-deficiency conditions to form Aβ·Cu²⁺ complexes. These data provide a novel mechanism by which cellular copper deficiency in AD could foster an environment for potentially adverse interactions between Aβ, copper, and cholesterol in lipid rafts.     

Evolutionary insights into primate skeletal gene regulation using a comparative cell culture model

The evolution of complex skeletal traits in primates was likely influenced by both genetic and environmental factors. Because skeletal tissues are notoriously challenging to study using functional genomic approaches, they remain poorly characterized even in humans, let alone across multiple species. The challenges involved in obtaining functional genomic data from the skeleton, combined with the difficulty of obtaining such tissues from nonhuman apes, motivated us to consider an alternative in vitro system with which to comparatively study gene regulation in skeletal cell types. Specifically, we differentiated six human (Homo sapiens) and six chimpanzee (Pan troglodytes) induced pluripotent stem cell lines (iPSCs) into mesenchymal stem cells (MSCs) and subsequently into osteogenic cells (bone cells). We validated differentiation using standard methods and collected single-cell RNA sequencing data from over 100,000 cells across multiple samples and replicates at each stage of differentiation. While most genes that we examined display conserved patterns of expression across species, hundreds of genes are differentially expressed (DE) between humans and chimpanzees within and across stages of osteogenic differentiation. Some of these interspecific DE genes show functional enrichments relevant in skeletal tissue trait development. Moreover, topic modeling indicates that interspecific gene programs become more pronounced as cells mature. Overall, we propose that this in vitro model can be used to identify interspecific regulatory differences that may have contributed to skeletal trait differences between species.