Genomic analysis of individual differences in ethanol drinking: evidence for non-genetic factors in C57BL/6 mice

Genetic analysis of factors affecting risk to develop excessive ethanol drinking has been extensively studied in humans and animal models for over 20 years. However, little progress has been made in determining molecular mechanisms underlying environmental or non-genetic events contributing to variation in ethanol drinking. Here, we identify persistent and substantial variation in ethanol drinking behavior within an inbred mouse strain and utilize this model to identify gene networks influencing such "non-genetic" variation in ethanol intake. C57BL/6NCrl mice showed persistent inter-individual variation of ethanol intake in a two-bottle choice paradigm over a three-week period, ranging from less than 1 g/kg to over 14 g/kg ethanol in an 18 h interval. Differences in sweet or bitter taste susceptibility or litter effects did not appreciably correlate with ethanol intake variation. Whole genome microarray expression analysis in nucleus accumbens, prefrontal cortex and ventral midbrain region of individual animals identified gene expression patterns correlated with ethanol intake. Results included several gene networks previously implicated in ethanol behaviors, such as glutamate signaling, BDNF and genes involved in synaptic vesicle function. Additionally, genes functioning in epigenetic chromatin or DNA modifications such as acetylation and/or methylation also had expression patterns correlated with ethanol intake. In verification for the significance of the expression findings, we found that a histone deacetylase inhibitor, trichostatin A, caused an increase in 2-bottle ethanol intake. Our results thus implicate specific brain regional gene networks, including chromatin modification factors, as potentially important mechanisms underlying individual variation in ethanol intake.     

Estimating past and future male loss in three Zambian lion populations

African lions (Panthera leo) are declining continent-wide, with protected area populations subject to a variety of anthropogenic effects. Zambia contains viable lion populations of considerable importance for photographic and hunting tourism, but long-term lion demographic data do not exist to guide recent management directives and population projections under different strategies. We described population size, as well as age and sex structure of lions in 3 Zambian national park populations bordering hunting areas, and found them to be male-depleted relative to other systems. We then estimated rates of adult male loss leading to male depletion in these populations and the effect of different future hunting management options on population characteristics. Predictions from matrix population models constructed within a Bayesian framework confirmed that the observed population structure was likely due to high rates of adult male loss and that instituting age limits on male harvests with quota reductions would reduce male depletion, improve tourism by providing older and more abundant males, and slightly increase population size. Reducing male mortality from wire snare poaching would also result in similar demographic impacts, and in concert with changes in hunting regulations would substantially improve the quality and quantity of adult male lions. However, model results varied depending on whether we assumed historical population stability. Predictions assuming negative historical growth rate indicated that substantially more conservative lion harvest management is warranted. We discuss the relevance of these findings for maintaining viable lion populations in and around protected areas in Zambia. © 2012 The Wildlife Society.     

Use of Chemically Modified Nucleotides to Determine a 62-Nucleotide RNA Crystal Structure: A Survey of Phosphorothioates,Br, Pt and Hg

Abstract

Two important challenges confronting RNA crystallographers are producing crystals and finding isomorphous heavy-atom derivatives. Non-isomorphism can be addressed by determining the phases using the multiwavelength anomalous dispersion (MAD) method. These phases can be greatly improved by combining phases from MAD experiments done on different heavy-atom derivatives. Heavy-atom derivatives can be created by chemically modifying the RNA through covalent attachment of bromine or mercury to C5 of pyrimidines or [Pt(NH₃)₃]²⁺ to N7 of guanine. While phosphorothioates can provide mercury binding sites, disorder can reduce their value for phase determination. The location of these chemical modifications is critical since crystallization of these derivatized RNAs is sensitive to heavy atom induced conformational alterations and crystal packing.     

Relationship between onion thrips (Thrips tabaci) and Stemphylium vesicarium in the development of Stemphylium leaf blight in onion

Stemphylium leaf blight caused by Stemphylium vesicarium and onion thrips (Thrips tabaci) are two common causes of leaf damage in onion production. Onion thrips is known to interact synergistically with pathogens to exacerbate plant disease. However, the potential relationship between onion thrips and Stemphylium leaf blight is unknown. In a series of controlled laboratory and field trials, the relationship between thrips feeding and movement on the development and severity of Stemphylium leaf blight were examined. In laboratory assays, onions (“Avalon” and “Ailsa Craig”) with varying levels of thrips feeding damage were inoculated with S. vesicarium. Pathogen colonisation and leaf dieback were measured after 2 weeks. In pathogen transfer assays, thrips were exposed to S. vesicarium conidia, transferred to onion and leaf disease development was monitored. In field trials, insecticide use was examined as a potential indirect means to reduce Stemphylium leaf blight disease and pathogen colonisation by reducing thrips damage. Results from laboratory trials revealed that a reduction in thrips feeding decreased S. vesicarium colonisation of onion leaves by 2.3–2.9 times, and decreased leaf dieback by 40–50%. Additionally, onion thrips were capable of transferring S. vesicarium conidia to onion plants (albeit at a low frequency of 2–14% of plants inoculated). In field trials, the symptoms and colonisation of Stemphylium leaf blight were reduced by 27 and 17%, respectively with the use of insecticide to control thrips. These results suggest that onion thrips may play a significant role in the development of Stemphylium leaf blight, and thrips control may reduce disease in commercial onion fields.     

Exacerbated brain damage, edema and inflammation in type-2 diabetic mice subjected to focal ischemia

One of the limiting factors in stroke therapeutic development is the use of animal models that do not well represent the underlying medical conditions of patients. In humans, diabetes increases the risk of stroke incidence as well as post-stroke mortality. To understand the mechanisms that render diabetics to increased brain damage, we evaluated the effect of transient middle cerebral artery occlusion in adult db/db mice. The db/db mouse is a model of type-2 diabetes with four times higher blood sugar than its normoglycemic genetic control(db/+ mouse). Following transient middle cerebral artery occlusion, the db/db mice showed significantly higher mortality, bigger infarcts, increased cerebral edema, worsened neurological status compared to db/+ mice. The db/db mice also showed significantly higher post-ischemic inflammatory markers (ICAM1(+) capillaries, extravasated macrophages/neutrophils and exacerbated proinflammatory gene expression) compared to db/+ mice. In addition, the post-ischemic neuroprotective heat-shock chaperone gene expression was curtailed in the db/db compared to db/+ mice.     

Discrimination of mumps virus small hydrophobic gene deletion effects from gene translation effects on virus virulence

Deletion of the small hydrophobic (SH) protein of certain paramyxoviruses has been found to result in attenuation, suggesting that the SH protein is a virulence factor. To investigate the role of the mumps virus (MuV) SH protein in virulence, multiple stop codons were introduced into the open reading frame (ORF) of a MuV molecular clone (r88-1961(SHstop)), preserving genome structure but precluding production of the SH protein. No differences in neurovirulence were seen between the wild-type and the SH(stop) viruses. In contrast, upon deletion of the SH gene, significant neuroattenuation was observed. These data indicate that the MuV SH protein is not a neurovirulence factor and highlight the importance of distinguishing gene deletion effects from protein-specific effects.     

Social stress and recovery: implications for body weight and body composition

Social stress resulting from dominant-subordinate relationships is associated with body weight loss and altered body composition in subordinate (SUB) male rats. Here, we extend these findings to determine whether stress-induced changes in energy homeostasis persist when the social stress is removed, and the animal is allowed to recover. We examined body weight (BW), body composition, and relevant endocrine measures after one or two cycles of 14 days of social stress, each followed by 21 days of recovery in each rat's individual home cage. SUB lost significantly more BW during social housing in a visible burrow system (VBS) compared with dominant (DOM) animals. Weight loss during social stress was attributable to a decrease in adipose tissue in DOM and SUB, with an additional loss of lean tissue in SUB. During both 21-day recovery periods, DOM and SUB regained lost BW, but only SUB were hyperphagic. Following recovery, SUB had a relatively larger increase in adipose tissue and plasma leptin compared with DOM, indicating that body composition changes were dependent on social status. Control animals that were weight matched to SUB or male rats exposed to the VBS environment without females, and that did not form a social hierarchy, did not exhibit changes in body composition like SUB in the VBS. Therefore, chronic social stress causes social status-dependent changes in BW, composition and endocrine measures that persist after repeated stress and recovery cycles and that may ultimately lead to metabolic disorders and obesity.     

Kinetic and Structural Characterization of the Interaction between the FMN Binding Domain of Cytochrome P450 Reductase and Cytochrome c

Cytochrome P450 reductase (CPR) is a diflavin enzyme that transfers electrons to many protein partners. Electron transfer from CPR to cyt c has been extensively used as a model reaction to assess the redox activity of CPR. CPR is composed of multiple domains, among which the FMN binding domain (FBD) is the direct electron donor to cyt c. Here, electron transfer and complex formation between FBD and cyt c are investigated. Electron transfer from FBD to cyt c occurs at distinct rates that are dependent on the redox states of FBD. When compared with full-length CPR, FBD reduces cyt c at a higher rate in both the semiquinone and hydroquinone states. The NMR titration experiments reveal the formation of dynamic complexes between FBD and cyt c on a fast exchange time scale. Chemical shift mapping identified residues of FBD involved in the binding interface with cyt c, most of which are located in proximity to the solvent-exposed edge of the FMN cofactor along with other residues distributed around the surface of FBD. The structural model of the FBD-cyt c complex indicates two possible orientations of complex formation. The major complex structure shows a salt bridge formation between Glu-213/Glu-214 of FBD and Lys-87 of cyt c, which may be essential for the formation of the complex, and a predicted electron transfer pathway mediated by Lys-13 of cyt c. The findings provide insights into the function of CPR and CPR-cyt c interaction on a structural basis.