Population genetic analysis of Bromus tectorum (Poaceae) indicates recent range expansion may be facilitated by specialist genotypes

? Premise of the study: The mechanisms for range expansion in invasive species depend on how genetic variation is structured in the introduced range. This study examined neutral genetic variation in the invasive annual grass Bromus tectorum in the Intermountain Western United States. Patterns of microsatellite (SSR) genotype distribution in this highly inbreeding species were used to make inferences about the roles of adaptively significant genetic variation, broadly adapted generalist genotypes, and facultative outcrossing in the recent range expansion of B. tectorum in this region. ? Methods: We sampled 20 individuals from each of 96 B. tectorum populations from historically and recently invaded habitats throughout the region and used four polymorphic SSR markers to characterize each individual. ? Key results: We detected 131 four-locus SSR genotypes; however, the 14 most common genotypes collectively accounted for 79.2% of the individuals. Common SSR genotypes were not randomly distributed among habitats. Instead, characteristic genotypes sorted into specific recently invaded habitats, including xeric warm and salt desert as well as mesic high-elevation habitats. Other SSR genotypes were common across a range of historically invaded habitats. We observed very few heterozygous individuals (0.58%). ? Conclusions: Broadly adapted, generalist genotypes appear to dominate historically invaded environments, while recently invaded salt and warm desert habitats are dominated by distinctive SSR genotypes that contain novel alleles. These specialist genotypes are not likely to have resulted from recombination; they probably represent more recent introductions from unknown source populations. We found little evidence that outcrossing plays a role in range expansion.     

Perinatal Exposure of Mice to the Pesticide DDT Impairs Energy Expenditure and Metabolism in Adult Female Offspring

Dichlorodiphenyltrichloroethane (DDT) has been used extensively to control malaria, typhus, body lice and bubonic plague worldwide, until countries began restricting its use in the 1970s. Its use in malaria control continues in some countries according to recommendation by the World Health Organization. Individuals exposed to elevated levels of DDT and its metabolite dichlorodiphenyldichloroethylene (DDE) have an increased prevalence of diabetes and insulin resistance. Here we hypothesize that perinatal exposure to DDT disrupts metabolic programming leading to impaired metabolism in adult offspring. To test this, we administered DDT to C57BL/6J mice from gestational day 11.5 to postnatal day 5 and studied their metabolic phenotype at several ages up to nine months. Perinatal DDT exposure reduced core body temperature, impaired cold tolerance, decreased energy expenditure, and produced a transient early-life increase in body fat in female offspring. When challenged with a high fat diet for 12 weeks in adulthood, female offspring perinatally exposed to DDT developed glucose intolerance, hyperinsulinemia, dyslipidemia, and altered bile acid metabolism. Perinatal DDT exposure combined with high fat feeding in adulthood further impaired thermogenesis as evidenced by reductions in core temperature and in the expression of numerous RNA that promote thermogenesis and substrate utilization in the brown adipose tissue of adult female mice. These observations suggest that perinatal DDT exposure impairs thermogenesis and the metabolism of carbohydrates and lipids which may increase susceptibility to the metabolic syndrome in adult female offspring.     

Photochemical cross-linking of the Escherichia coli single-stranded DNA-binding protein to oligodeoxynucleotides. Identification of phenylalanine 60 as the site of cross-linking

The single-stranded DNA-binding proteins from bacteriophage T4, F plasmid, Escherichia coli, and calf thymus can all be covalently cross-linked in vitro to thymine oligonucleotides by irradiating the respective protein-oligonucleotide complexes with ultraviolet light. More extensive studies on the E. coli single-stranded DNA-binding protein (SSB) indicate that this reaction is dependent upon both the length of the oligonucleotide and the dose of ultraviolet irradiation. Using anion-exchange and reverse-phase ion-pairing high-performance liquid chromatography we have isolated a specific cross-linked tryptic peptide comprising residues 57-62 of the SSB protein with the sequence valine-valine-leucine-phenylalanine-glycine-lysine. Solid-phase sequence analysis of the covalent [32P] p(dT)8-peptide complex indicates that phenylalanine 60 is the site of cross-linking. This amino acid is located within the general region of SSB (residues 1-115) that has previously been shown to contain the DNA-binding site (Williams, K. R., Spicer, E. K., LoPresti, M. B., Guggenheimer, R. A., and Chase, J. W. (1983) J. Biol. Chem. 258, 3346-3355). The high-performance liquid chromatography purification procedure we have devised to isolate cross-linked peptide-oligonucleotide complexes should be of general applicability and should facilitate future structure/function studies on other nucleic acid-binding proteins.     

Novel interactions of ESCRT-III with LIP5 and VPS4 and their implications for ESCRT-III disassembly

The AAA+ ATPase VPS4 plays an essential role in multivesicular body biogenesis and is thought to act by disassembling ESCRT-III complexes. VPS4 oligomerization and ATPase activity are promoted by binding to LIP5. LIP5 also binds to the ESCRT-III like protein CHMP5/hVps60, but how this affects its function remains unclear. Here we confirm that LIP5 binds tightly to CHMP5, but also find that it binds well to additional ESCRT-III proteins including CHMP1B, CHMP2A/hVps2-1, and CHMP3/hVps24 but not CHMP4A/hSnf7-1 or CHMP6/hVps20. LIP5 binds to a different region within CHMP5 than within the other ESCRT-III proteins. In CHMP1B and CHMP2A, its binding site encompasses sequences at the proteins' extreme C-termini that overlap with "MIT interacting motifs" (MIMs) known to bind to VPS4. We find unexpected evidence of a second conserved binding site for VPS4 in CHMP2A and CHMP1B, suggesting that LIP5 and VPS4 may bind simultaneously to these proteins despite the overlap in their primary binding sites. Finally, LIP5 binds preferentially to soluble CHMP5 but instead to polymerized CHMP2A, suggesting that the newly defined interactions between LIP5 and ESCRT-III proteins may be regulated by ESCRT-III conformation. These studies point to a role for direct binding between LIP5 and ESCRT-III proteins that is likely to complement LIP5's previously described ability to regulate VPS4 activity.     

Activation of Human VPS4A by ESCRT-III Proteins Reveals Ability of Substrates to Relieve Enzyme Autoinhibition

VPS4 proteins are AAA⁺ ATPases required to form multivesicular bodies, release viral particles, and complete cytokinesis. They act by disassembling ESCRT-III heteropolymers during or after their proposed function in membrane scission. Here we show that purified human VPS4A is essentially inactive but can be stimulated to hydrolyze ATP by ESCRT-III proteins in a reaction that requires both their previously defined MIT interacting motifs and ∼50 amino acids of the adjacent sequence. Importantly, C-terminal fragments of all ESCRT-III proteins tested, including CHMP2A, CHMP1B, CHMP3, CHMP4A, CHMP6, and CHMP5, activated VPS4A suggesting that it disassembles ESCRT-III heteropolymers by affecting each component protein. VPS4A is thought to act as a ring-shaped cylindrical oligomer like other AAA⁺ ATPases, but this has been difficult to directly demonstrate. We found that concentrating His₆-VPS4A on liposomes containing Ni²⁺-nitrilotriacetic acid-tagged lipid increased ATP hydrolysis, confirming the importance of inter-subunit interactions for activity. We also found that mutating pore loops expected to line the center of a cylindrical oligomer changed the response of VPS4A to ESCRT-III proteins. Based on these data, we propose that ESCRT-III proteins facilitate assembly of functional but transient VPS4A oligomers and interact with sequences inside the pore of the assembled enzyme. Deleting the N-terminal MIT domain and adjacent linker from VPS4A increased both basal and liposome-enhanced ATPase activity, indicating that these elements play a role in autoinhibiting VPS4A until it encounters ESCRT-III proteins. These findings reveal new ways in which VPS4 activity is regulated and specifically directed to ESCRT-III polymers.     

Early‐life patterns of growth are linked to levels of phenotypic trait covariance and postfledging mortality across avian species

Life history studies have established that trade‐offs between growth and survival are common both within and among species. Identifying the factor(s) that mediate this trade‐off has proven difficult, however, especially at the among‐species level. In this study, we examined a series of potentially interrelated traits in a community of temperate‐zone passerine birds to help understand the putative causes and consequences of variation in early‐life growth among species. First, we examined whether nest predation risk (a proven driver of interspecific variation in growth and development rates) was correlated with species‐level patterns of incubation duration and nestling period length. We then assessed whether proxies for growth rate covaried with mean trait covariance strength (i.e., phenotypic correlations ( ^rp), which can be a marker of early‐life stress) among body mass, tarsus length, and wing length at fledging. Finally, we examined whether trait covariance strength at fledging was related to postfledging survival. We found that higher nest predation risk was correlated with faster skeletal growth and that our proxies for growth corresponded with increased trait covariance strength ( ^rp), which subsequently, correlated with higher mortality in the next life stage (postfledging period). These results provide an indication that extrinsic pressures (nest predation) impact rates of growth, and that there are costs of rapid growth across species, expressed as higher mean ^rp and elevated postfledging mortality. The link between higher levels of trait covariance at fledging and increased mortality is unclear, but increased trait covariance strength may reflect reduced phenotypic flexibility (i.e., phenotypic canalization), which may limit an organism''s capacity for coping with environmental or ecological variability.     

Cloning, sequence analysis, and overexpression of Escherichia coli folK, the gene coding for 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase.

The gene coding for the Escherichia coli enzyme 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase has been cloned and sequenced. This gene, designated folK, codes for a protein of 159 amino acids, including an amino-terminal methionine. The protein was overexpressed in E. coli MC4100 by cloning the gene behind the lacUV5 promoter in a high-copy-number plasmid. The enzyme was purified to homogeneity. Amino-terminal analysis of the purified protein showed that the amino-terminal methionine had been removed. The compositional molecular mass (17,945 Da) was identical to the molecular mass determined by mass spectrometry. The enzyme was observed to have a large number of proline residues and migrated anomalously in sodium dodecyl sulfate-polyacrylamide gels, with an apparent molecular mass of 23,000 Da.     

Vitamin E alleviates non-alcoholic fatty liver disease in phosphatidylethanolamine N-methyltransferase deficient mice

Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC), mainly in the liver. Pemt^?/? mice are protected from high-fat diet (HFD)-induced obesity and insulin resistance, but develop severe non-alcoholic fatty liver disease (NAFLD) when fed a HFD, mostly due to impaired VLDL secretion. Oxidative stress is thought to be an essential factor in the progression from simple steatosis to steatohepatitis. Vitamin E is an antioxidant that has been clinically used to improve NAFLD pathology. Our aim was to determine whether supplementation of the diet with vitamin E could attenuate HFD-induced hepatic steatosis and its progression to NASH in Pemt^?/? mice. Treatment with vitamin E (0.5?g/kg) for 3?weeks improved VLDL-TG secretion and normalized cholesterol metabolism, but failed to reduce hepatic TG content. Moreover, vitamin E treatment was able to reduce hepatic oxidative stress, inflammation and fibrosis. We also observed abnormal ceramide metabolism in Pemt^?/? mice fed a HFD, with elevation of ceramides and other sphingolipids and higher expression of mRNAs for acid ceramidase (Asah1) and ceramide kinase (Cerk). Interestingly, vitamin E supplementation restored Asah1 and Cerk mRNA and sphingolipid levels. Together this study shows that vitamin E treatment efficiently prevented the progression from simple steatosis to steatohepatitis in mice lacking PEMT.