The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers: HYBRID MOLECULES COMPOSED OF ACETYLCHOLINESTERASE AND BUTYRYLCHOLINESTERASE DIMERS ARE UP-REGULATED DURING DEVELOPMENT OF CHICKEN BRAIN*

Acetylcholinesterase (AChE) is anchored onto cell membranes by the transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. In parallel, the PRiMA-linked tetrameric butyrylcholinesterase (BChE) is also found in the brain. A single type of AChE-BChE hybrid tetramer was formed in cell cultures by co-transfection of cDNAs encoding AChE_T and BChE_T with proline-rich attachment domain-containing proteins, PRiMA I, PRiMA II, or a fragment of ColQ having a C-terminal GPI addition signal (Q_N-GPI). Using AChE and BChE mutants, we showed that AChE-BChE hybrids linked with PRiMA or Q_N-GPI always consist of AChE_T and BChE_T homodimers. The dimer formation of AChE_T and BChE_T depends on the catalytic domains, and the assembly of tetramers with a proline-rich attachment domain-containing protein requires the presence of C-terminal “t-peptides” in cholinesterase subunits. Our results indicate that PRiMA- or ColQ-linked cholinesterase tetramers are assembled from AChE_T or BChE_T homodimers. Moreover, the PRiMA-linked AChE-BChE hybrids occur naturally in chicken brain, and their expression increases during development, suggesting that they might play a role in cholinergic neurotransmission.     

Uncoupling protein-3 (UCP3) mRNA expression in reconstituted human muscle after myoblast transplantation in RAG2-/-/gamma c/C5(-) immunodeficient mice

Uncoupling protein-3 (UCP3), which is expressed abundantly in skeletal muscle, is one of the carrier proteins dissipating the transmitochondrial electrochemical gradient as heat and has therefore been implicated in the regulation of energy metabolism. Myoblasts or differentiated muscle cells in vitro expressed little if any UCP3, compared with the levels detected in biopsies of skeletal muscle. In the present report, we sought to investigate UCP3 mRNA expression in human muscle generated by myoblast transplantation in the skeletal muscle of an immunodeficient mouse model. Time course experiments demonstrated that 7-8 weeks following transplantation fully differentiated human muscle fibers were formed. The presence of differentiated human muscle fibers was assessed by quantitative PCR measurement of the human alpha-actin mRNA together with immunohistochemical staining using specific antibodies for spectrin and the slow adult myosin heavy chain. Interestingly, we found that the expression of UCP3 mRNA was dependant on human muscle differentiation and that the UCP3 mRNA level was comparable with that found in human muscle biopsies. Moreover, the human UCP3 (hUCP3) promoter seems to be fully functional, since triiodothyronine treatment of the mice not only stimulated the mouse UCP3 (mUCP3) mRNA expression but also strongly stimulated the hUCP3 mRNA expression in human fibers formed after myoblast transplantation. To our knowledge, this is the first time that primary myoblasts could be induced to express the UCP3 gene at a level comparable of that found in human muscle fibers.     

Expression and purification of recombinant cynomolgus monkey cholesteryl ester transfer protein from Chinese hamster ovary cells

Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl ester from high- and low-density lipoproteins to triglyceride-rich lipoproteins, and reciprocally mediates triglyceride transfer. The gene for cynomolgus monkey CETP was expressed in serum-free CHO culture with 2g/ml insulin as its only exogenous protein supplement. Cell growth was facilitated by immobilizing the CHO cells in alginate beads. Recombinant CETP (rCETP) was purified 176-fold with a three-step protocol resulting in a 60% final yield as measured by a fluorescent CETP activity assay. Typically, 3.4 mg of rCETP was purified from 1700 ml of media by affinity-gel chromatography involving Reactive Red 120 (RR120) followed by concanavalin A Sepharose 4B and rechromatography on RR120. SDS-PAGE shows a single broad band ofM _r , ranging from 68,000 to 74,000 which immunoreacts in Western blot analysis. Amino acid analysis and protein sequencing of the purified protein agree with the theoretical amino acid composition and sequence of cynomolgus CETP.     

Bilateral Song Convergence in a Passerine Hybrid Zone: Genetics Contribute in One Species Only

Hybridization can drive the convergence of territorial and sexual signals. However, non-genetic processes such as competition, environment matching, or cultural transmission, also generate this pattern. We investigated the effect of hybridization on song convergence between two interspecifically territorial warblers in a moving hybrid zone. We confirmed song convergence in each species. Using an AFLP-based genetic index, we detected an effect of genetics on song convergence in Hippolais polyglotta, the expanding species. Evidence was weaker for H. icterina, the receding species. In moving zones, introgression is expected to be larger in the expanding species than in the receding. Thus, the asymmetric contribution of the genetic index to convergence was consistent with expectations for genetically determined traits in moving hybrid zones, and the observed introgression pattern of AFLP markers. However, the geographical location of individuals had an effect on song variation too when genetics was accounted for, suggesting that convergence also has non-genetic explanations. We examine the possible role of alternative processes to that of hybridization and discuss their conflicting effects on reinforcement and hybrid zone dynamics.     

Landscape genetics of a tropical rescue pollinator

Pollination services are increasingly threatened by the loss and modification of natural habitats, posing a risk to the maintenance of both native plant biodiversity and agricultural production. In order to safeguard pollination services, it is essential to examine the impacts of habitat degradation on the population dynamics of key pollinators and identify potential “rescue pollinators” capable of persisting in these human-altered landscapes. Using a landscape genetic approach, we assessed the impact of landscape structure on genetic differentiation in the widely-distributed tropical stingless bee Trigona spinipes (Apidae: Meliponini) across agricultural landscape mosaics composed of coffee plantations and Atlantic forest fragments in southeastern Brazil. We genotyped 115 bees at 16 specific and highly polymorphic microsatellite loci, developed using next-generation sequencing. Our results reveal that T. spinipes is capable of dispersing across remarkably long distances, as we did not find genetic differentiation across a 200 km range, nor fine-scale spatial genetic structure. Furthermore, gene flow was not affected by forest cover, land cover, or elevation, indicating that reproductive individuals are able to disperse well through agricultural landscapes and across altitudinal gradients. We also found evidence of a recent population expansion, suggesting that this opportunistic stingless bee is capable of colonizing degraded habitats. Our results thus suggest that T. spinipes can persist in heavily-altered landscapes and can be regarded as a rescue pollinator, potentially compensating for the decline of other native pollinators in degraded tropical landscapes.     

Histone deacetylase inhibition decreases preference without affecting aversion for nicotine

Mutations in the parkin gene cause early-onset, autosomal recessive Parkinson's disease. Parkin functions as an E3 ubiquitin ligase to mediate the covalent attachment of ubiquitin monomers or linked chains to protein substrates. Substrate ubiquitination can target proteins for proteasomal degradation or can mediate a number of non-degradative functions. Parkin has been shown to preserve mitochondrial integrity in a number of experimental systems through the regulation of mitochondrial fission. Upon mitochondrial damage, parkin translocates to mitochondria to mediate their selective elimination by autophagic degradation. The mechanism underlying this process remains unclear. Here, we demonstrate that parkin interacts with and selectively mediates the atypical poly-ubiquitination of the mitochondrial fusion factor, mitofusin 1, leading to its enhanced turnover by proteasomal degradation. Our data supports a model whereby the translocation of parkin to damaged mitochondria induces the degradation of mitofusins leading to impaired mitochondrial fusion. This process may serve to selectively isolate damaged mitochondria for their removal by autophagy.     

Does Manganese Protect Cultured Human Skin Fibroblasts Against Oxidative Injury by Uva, Dithranol and Hydrogen Peroxide?

Reactive oxygen species (ROS) are involved in the mechanism of photoaging and carcinogenesis. Skin is endowed with antioxidant enzymes including superoxide dismutases (SOD): cytosolic copper zinc SOD and mitochondrial manganese SOD. The aim of our study was to estimate the protective effect of manganese against oxidative injury on cultured human skin fibroblasts. Dithranol, hydrogen peroxide and UV-A radiation (375 nm) were employed as oxidative stressors. The supply of manganese chloride produced an increase in cellular content of this element up to 24 fold without concomitant elevation of MnSOD activity. Nevertheless, manganese protects cells against two of the three ROS generating systems assessed, namely hydrogen peroxyde and UV-A. This protective effect depends on the concentration of manganese in the medium, 0.1 mM and 0.2 mM protect against UVA cytotoxicity, only 0.2 mM protects against H₂O₂ cytotoxicity.     

Groundwater promotes emergence of asporogenic mutants of emetic Bacillus cereus

Bacillus cereus is a ubiquitous endospore-forming bacterium, which mainly affects humans as a food-borne pathogen. Bacillus cereus can contaminate groundwater used to irrigate food crops. Here, we examined the ability of the emetic strain B. cereus F4810/72 to survive abiotic conditions encountered in groundwater. Our results showed that vegetative B. cereus cells rapidly evolved in a mixed population composed of endospores and asporogenic variants bearing spo0A mutations. One asporogenic variant, VAR-F48, was isolated and characterized. VAR-F48 can survive in sterilized groundwater over a long period in a vegetative form and has a competitive advantage compared to its parental strain. Proteomics analysis allowed us to quantify changes to cellular and exoproteins after 24 and 72 h incubation in groundwater, for VAR-F48 compared to its parental strain. The results revealed a significant re-routing of the metabolism in the absence of Spo0A. We concluded that VAR-F48 maximizes its energy use to deal with oligotrophy, and the emergence of spo0A-mutated variants may contribute to the persistence of emetic B. cereus in natural oligotrophic environments.