Expression and purification of recombinant tung tree diacylglycerol acyltransferase 2

Diacylglycerol acyltransferases (DGATs) esterify sn-1,2-diacylglycerol with a long-chain fatty acyl-CoA, the last and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. At least 74 DGAT2 sequences from 61 organisms have been identified, but the expression of any DGAT2 as a partial or full-length protein in Escherichia coli had not been reported. The main objective of this study was to express and purify recombinant DGAT2 (rDGAT2) from E. coli for antigen production with a minor objective to compare rDGAT2 expression in yeast. A plasmid was engineered to express tung tree DGAT2 fused to maltose binding protein and poly-histidine (His) affinity tags. Immunoblotting showed that rDGAT2 was detected in the soluble, insoluble, and membrane fractions. The rDGAT2 in the soluble fraction was partially purified by amylose resin, nickel-nitrilotriacetic agarose (Ni-NTA) beads, and tandem affinity chromatography. Multiple proteins co-purified with rDGAT2. Size exclusion chromatography estimated the size of the rDGAT2-enriched fraction to be approximately eight times the monomer size. Affinity-purified rDGAT2 fractions had a yellow tint and contained fatty acids. The rDGAT2 in the insoluble fraction was partially solubilized by seven detergents with SDS being the most effective. Recombinant DGAT2 was purified to near homogeneity by SDS solubilization and Ni-NTA affinity chromatography. Mass spectrometry identified rDGAT2 as a component in the bands corresponding to the monomer and dimer forms as observed by SDS-PAGE. Protein bands with monomer and dimer sizes were also observed in the microsomal membranes of Saccharomyces cerevisiae expressing hemagglutinin-tagged DGAT2. Nonradioactive assay showed TAG synthesis activity of DGAT2 from yeast but not E. coli. The results suggest that rDGAT2 is present as monomer and dimer forms on SDS-PAGE, associated with other proteins, lipids, and membranes, and that post-translational modification of rDGAT2 may be required for its enzymatic activity and/or the E. coli protein is misfolded.     

Individual differences in the effects of mobile phone exposure on human sleep: rethinking the problem

Mobile phone exposure‐related effects on the human electroencephalogram (EEG) have been shown during both waking and sleep states, albeit with slight differences in the frequency affected. This discrepancy, combined with studies that failed to find effects, has led many to conclude that no consistent effects exist. We hypothesised that these differences might partly be due to individual variability in response, and that mobile phone emissions may in fact have large but differential effects on human brain activity. Twenty volunteers from our previous study underwent an adaptation night followed by two experimental nights in which they were randomly exposed to two conditions (Active and Sham), followed by a full‐night sleep episode. The EEG spectral power was increased in the sleep spindle frequency range in the first 30 min of non‐rapid eye movement (non‐REM) sleep following Active exposure. This increase was more prominent in the participants that showed an increase in the original study. These results confirm previous findings of mobile phone‐like emissions affecting the EEG during non‐REM sleep. Importantly, this low‐level effect was also shown to be sensitive to individual variability. Furthermore, this indicates that previous negative results are not strong evidence for a lack of an effect and, given the far‐reaching implications of mobile phone research, we may need to rethink the interpretation of results and the manner in which research is conducted in this field. Bioelectromagnetics 33:86–93, 2012. © 2011 Wiley Periodicals, Inc.     

Single nucleotide polymorphisms in the imprinted bovine insulin-like growth factor 2 receptor gene (IGF2R) are associated with body size traits in Irish Holstein-Friesian cattle

The regulation of the bioavailability of insulin‐like growth factors (IGFs) is critical for normal mammalian growth and development. The imprinted insulin‐like growth factor 2 receptor gene (IGF2R) encodes a transmembrane protein receptor that acts to sequester and degrade excess circulating insulin‐like growth factor 2 (IGF‐II) – a potent foetal mitogen – and is considered an important inhibitor of growth. Consequently, IGF2R may serve as a candidate gene underlying important growth‐ and body‐related quantitative traits in domestic mammalian livestock. In this study, we have quantified genotype–phenotype associations between three previously validated intronic bovine IGF2R single nucleotide polymorphisms (SNPs) (IGF2R:g.64614T>C, IGF2R:g.65037T>C and IGF2R:g.86262C>T) and a range of performance traits in 848 progeny‐tested Irish Holstein‐Friesian artificial insemination sires. Notably, all three polymorphisms analysed were associated (P ≤ 0.05) with at least one of a number of performance traits related to animal body size: angularity, body depth, chest width, rump width, and animal stature. In addition, the C‐to‐T transition at the IGF2R:g.65037T>C polymorphism was positively associated with cow carcass weight and angularity. Correction for multiple testing resulted in the retention of two genotype–phenotype associations (animal stature and rump width). None of the SNPs analysed were associated with any of the milk traits examined. Analysis of pairwise r² measures of linkage disequilibrium between all three assayed SNPs ranged between 0.41 and 0.79, suggesting that some of the observed SNP associations with performance may be independent. To our knowledge, this is one of the first studies demonstrating associations between IGF2R polymorphisms and growth‐ and body‐related traits in cattle. These results also support the increasing body of evidence that imprinted genes harbour polymorphisms that contribute to heritable variation in phenotypic traits in domestic livestock species.     

Serosurvey of free-ranging Amur tigers in the Russian Far East

Wild Amur tigers (Panthera tigris altaica, n=44) from the Russian Far East were tested for antibodies to feline leukemia virus, feline corona virus (FCoV), feline immunodeficiency virus, feline parvovirus (FPV), canine distemper virus (CDV), Toxoplasma gondii, and Bartonella henselae. Antibodies to FCoV, CDV, FPV, and T. gondii were detected in 43, 15, 68, and 42% of tigers, respectively. No differences were detected in antibody prevalence estimates between tigers captured as part of a research program and those captured to mitigate human-tiger conflicts. Domestic dogs (Canis familiaris) were tested as a potential source for CDV; 16% were vaccinated against CDV and 58% of unvaccinated dogs were antibody positive for CDV. A high percentage of tigers were exposed to potential pathogens that could affect the survival of this species. We recommend continued monitoring of wild tigers throughout Asia, development of standardized sampling and postmortem examination procedures, and additional research to better understand potential domestic and wild animal sources for these pathogens.     

Implication of proteins containing tetratricopeptide repeats in conditional virulence phenotypes of Legionella pneumophila

Legionella pneumophila, the causative agent of Legionnaires' disease, is a ubiquitous freshwater bacterium whose virulence phenotypes require a type IV secretion system (T4SS). L. pneumophila strain JR32 contains two virulence-associated T4SSs, the Dot/Icm and Lvh T4SSs. Defective entry and phagosome acidification phenotypes of dot/icm mutants are conditional and reversed by incubating broth-grown stationary-phase cultures in water (WS treatment) prior to infection, as a mimic of the aquatic environment of Legionella. Reversal of dot/icm virulence defects requires the Lvh T4SS and is associated with a >10-fold induction of LpnE, a tetratricopeptide repeat (TPR)-containing protein. In the current study, we demonstrated that defective entry and phagosome acidification phenotypes of mutants with changes in LpnE and EnhC, another TPR-containing protein, were similarly reversed by WS treatment. In contrast to dot/icm mutants for which the Lvh T4SS was required, reversal for the ΔlpnE or the ΔenhC mutant required that the other TPR-containing protein be present. The single and double ΔlpnE and ΔenhC mutants showed a hypersensitivity to sodium ion, a phenotype associated with dysfunction of the Dot/Icm T4SS. The ΔlpnE single and the ΔlpnE ΔenhC double mutant showed 3- to 9-fold increases in translocation of Dot/Icm T4SS substrates, LegS2/SplY and LepB. Taken together, these data identify TPR-containing proteins in a second mechanism by which the WS mimic of a Legionella environmental niche can reverse virulence defects of broth-grown cultures and implicate LpnE and EnhC directly or indirectly in translocation of Dot/Icm T4SS protein substrates.     

Hydrophobic imbalance in the cytoplasmic domain of phospholamban is a determinant for lethal dilated cardiomyopathy

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) and its regulatory partner phospholamban (PLN) are essential for myocardial contractility. Arg(9) → Cys (R9C) and Arg(14) deletion (R14del) mutations in PLN are associated with lethal dilated cardiomyopathy in humans. To better understand these mutations, we made a series of amino acid substitutions in the cytoplasmic domain of PLN and tested their ability to inhibit SERCA. R9C is a complete loss-of-function mutant of PLN, whereas R14del is a mild loss-of-function mutant. When combined with wild-type PLN to simulate heterozygous conditions, the mutants had a dominant negative effect on SERCA function. A series of targeted mutations in this region of the PLN cytoplasmic domain ((8)TRSAIRR(14)) demonstrated the importance of hydrophobic balance in proper PLN regulation of SERCA. We found that Arg(9) → Leu and Thr(8) → Cys substitutions mimicked the behavior of the R9C mutant, and an Arg(14) → Ala substitution mimicked the behavior of the R14del mutant. The results reveal that the change in hydrophobicity resulting from the R9C and R14del mutations is sufficient to explain the loss of function and persistent interaction with SERCA. Hydrophobic imbalance in the cytoplasmic domain of PLN appears to be a predictor for the development and progression of dilated cardiomyopathy.     

Equol production changes over time in postmenopausal women

Equol (EQ) is produced by intestinal bacteria from the soy isoflavone daidzein (DE) in 30%-60% of the population and is believed to provide benefits from soy intake. A robust EQ status definition is lacking, and it is uncertain whether EQ is formed consistently within an individual and ceases upon oral antibiotic treatment. In a randomized, double-blind, placebo-controlled soy intervention trial with 350 postmenopausal women, DE and EQ were analyzed by liquid chromatography/tandem mass spectrometry at baseline and every 6 months over 2.5 years in overnight urine, spot urine and plasma. Equol production changes and status (remaining an EQ producer or nonproducer or changing towards an EQ producer or nonproducer) were assessed. Equol status was determined most dependably by overnight urine applying as cutoff a ratio of EQ/DE≥0.018 with a DE threshold ≥2 nmol/mg creatinine: the soy and placebo groups had approximately 30% consistent EQ producers during the study, but 14% and 35%, respectively, changed EQ status (mean 1.4-1.7 times), while 27% and 17%, respectively, had antibiotic treatment (P<.01 for inverse association). No significant trend in change of EQ production or status was observed when overnight urine was limited to collections closest to before and after antibiotic treatment. Similarly, antibiotic type or class, duration, dose or time between antibiotic treatment and overnight urine collection showed no consistent influence on EQ production. Equol production can markedly change intraindividually over 2.5 years, and antibiotic treatment impacts it inconsistently. Factors other than antibiotic treatment must be considered as causes for EQ production changes.     

Medicinal chemistry approaches to avoid aldehyde oxidase metabolism

Aldehyde oxidase (AO) is a molybdenum-containing enzyme distributed throughout the animal kingdom and capable of metabolising a wide range of aldehydes and N-heterocyclic compounds. Although metabolism by this enzyme in man is recognised to have significant clinical impact where human AO activity was not predicted by screening in preclinical species, there is very little reported literature offering real examples where drug discoverers have successfully designed away from AO oxidation. This article reports on some strategies adopted in the Pfizer TLR7 agonist programme to successfully switch off AO metabolism that was seen principally in the rat.