Phenylalanine promotes interaction of transmembrane domains via GxxxG motifs

Interactions of transmembrane helices play a crucial role in the folding and oligomerisation of integral membrane proteins. In order to uncover novel sequence motifs mediating these interactions, we randomised one face of a transmembrane helix with a set of non-polar or moderately polar amino acids. Those sequences capable of self-interaction upon integration into bacterial inner membranes were selected by means of the ToxR/POSSYCCAT system. A comparison between low/medium-affinity and high-affinity sequences reveals that high-affinity sequences are strongly enriched in phenylalanine residues that are frequently observed at the − 3 position of GxxxG motifs, thus yielding FxxGxxxG motifs. Mutation of Phe or GxxxG in selected sequences significantly reduces self-interaction of the transmembrane domains without affecting their efficiency of membrane integration. Conversely, grafting FxxGxxxG onto unrelated transmembrane domains strongly enhances their interaction. Further, we find that FxxGxxxG is significantly over-represented in transmembrane domains of bitopic membrane proteins. The same motif contributes to self-interaction of the vesicular stomatitis virus G protein transmembrane domain. We conclude that Phe stabilises membrane-spanning GxxxG motifs. This is one example of how the role of certain side-chains in helix-helix interfaces is modulated by sequence context.     

Further differentiation of murine double-positive thymocytes is inhibited in adenosine deaminase-deficient murine fetal thymic organ culture

Murine fetal thymic organ culture (FTOC) was used to investigate the mechanism by which a lack of adenosine deaminase (ADA) leads to a failure of T cell production in the thymus. We previously showed that T cell development was inhibited beginning at the CD4(-)CD8(-)CD25(+)CD44(low) stage in ADA-deficient FTOC initiated at day 15 of gestation when essentially all thymocytes are CD4(-)CD8(-). In the present study, we asked whether thymocytes at later stages of differentiation would also be sensitive to ADA inhibition by initiating FTOC when substantial numbers of CD4(+)CD8(+) thymocytes were already present. dATP was highly elevated in ADA-deficient cultures, and the recovery of alphabeta TCR(+) thymocytes was inhibited by 94%, indicating that the later stages of thymocyte differentiation are also dependent upon ADA. ADA-deficient cultures were partially rescued by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or by the use of apoptotic protease-activating factor-1-deficient mice. Rescue was even more dramatic, with 60- to >200-fold increases in the numbers of CD4(+)CD8(+) cells, when FTOC were performed with an inhibitor of adenosine kinase, the major thymic deoxyadenosine phosphorylating enzyme, or with bcl-2 transgenic mice. dATP levels were normalized by treatment with either carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or an adenosine kinase inhibitor, but not in cultures with fetal thymuses from bcl-2 transgenic mice. These data suggest that ADA deficiency leads to the induction of mitochondria-dependent apoptosis as a consequence of the accumulation of dATP derived from thymocytes failing the positive/negative selection checkpoint.     

Animal models of ischemic stroke: balancing experimental aims and animal care

Animal models of ischemic stroke are examples of an induced model that can present challenges from the perspectives of protocol review and animal management. The review presented here will include a brief summary of the current state of knowledge about clinical stroke; a general synopsis of important unanswered research questions that justify use of animal stroke models; an overview of various animal models of ischemic stroke, including strengths and limitations; and a discussion of animal care issues relative to ischemic stroke models. Good communication and interactive education among primary investigators, laboratory animal veterinarians and caretakers, and institutional animal care and use committee members are critical in achieving a balance between research objectives and animal care issues when using animal stroke models.     

Indirect competitive immunoassay for detection of aflatoxin B1 in corn and nut products using the array biosensor

Because of the potential health risks of aflatoxin B₁ (AFB₁), it is essential to monitor the level of this mycotoxin in a variety of foods. An indirect competitive immunoassay has been developed using the NRL array biosensor, offering rapid, sensitive detection and quantification of AFB₁ in buffer, corn and nut products. AFB₁-spiked foods were extracted with methanol and Cy5-anti-AFB₁ added to the resulting sample. The extracted sample/antibody mix was passed over a waveguide surface patterned with immobilized AFB₁. The resulting fluorescence signal decreased as the concentration of AFB₁ in the sample increased. The limit of detection for AFB₁ in buffer, 0.3 ng/ml, was found to increase to between 1.5 and 5.1 ng/g and 0.6 and 1.4 ng/g when measured in various corn and nut products, respectively.     

Translation initiator EIF4G1 mutations in familial Parkinson disease

Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.     

Limited influence of local and landscape factors on finescale gene flow in two pond‐breeding amphibians

Dispersal and gene flow within animal populations are influenced by the composition and configuration of the landscape. In this study, we evaluated hypotheses about the impact of natural and anthropogenic factors on genetic differentiation in two amphibian species, the spotted salamander (Ambystoma maculatum) and the wood frog (Lithobates sylvaticus) in a commercial forest in central Maine. We conducted this analysis at two scales: a local level, focused on factors measured at each breeding pond, and a landscape level, focused on factors measured between ponds. We investigated the effects of a number of environmental factors in six categories including Productivity, Physical, Land Composition, Land Configuration, Isolation and Location. Embryos were sampled from 56 spotted salamander breeding ponds and 39 wood frog breeding ponds. We used a hierarchical Bayesian approach in the program GESTE at each breeding pond and a random forest algorithm in conjunction with a network analysis between the ponds. We found overall high genetic connectivity across distances up to 17 km for both species and a limited effect of natural and anthropogenic factors on gene flow. We found the null models best explained patterns of genetic differentiation at a local level and found several factors at the landscape level that weakly influenced gene flow. This research indicates multiscale investigations that incorporate local and landscape factors are valuable for understanding patterns of gene flow. Our findings suggest that dispersal rates in this system are high enough to minimize genetic structuring and that current forestry practices do not significantly impede dispersal.     

An investigation of dye reduction by food-borne bacteria

S.A. LEAROYD, R.G. KROLL AND C.F. THURSTON. 1992. The rates of reduction of seven redox dyes by 13 bacterial strains were measured and found to vary greatly between different bacterium/dye combinations. Phenazine ethosulphate and toluidine blue were the most rapidly reduced dyes by the majority of bacteria and resorufin and 2-hydroxy-1,4-naphthoquinone were reduced slowly, if at all. There was also considerable variation in the rates of reduction with any single dye/organism combination. Glucose stimulated the rates of endogenous dye reduction in about half of the organisms. For Bacillus cereus, Pseudomonas fluorescens and Escherichia coli , dye reduction was stimulated by a range of exogenous substrates but lactose, the primary available carbon and energy source in milk, had little effect. In Lactococcus lactis , dye reduction was stimulated by sugars but not by organic acids. Oxygen successfully competed with dye reduction in organisms containing respiratory chains, but with membrane fractions, dye reduction was more rapid than oxygen consumption. All the organisms showed little cytosolic dye reduction, except L. lactis which showed substantial rates of reduction of some dyes by this fraction. With the membrane fraction of E. coli and Ps. fluorescens , cyanide inhibited NADH and succinate-dependent dye reduction, Antimycin A inhibited lactate and succinate and rotenone had no significant effect, but inhibition was not always observed with membrane from both organisms.     

Bub1 and Bub3 promote the conversion from monopolar to bipolar chromosome attachment independently of shugoshin

The eukaryotic spindle assembly checkpoint (SAC) delays anaphase in the presence of chromosome attachment errors. Bub3 has been reported to be required for SAC activity in all eukaryotes examined so far. We find that Bub3, unlike its binding partner Bub1, is not essential for the SAC in fission yeast. As Bub3 is needed for the efficient kinetochore localization of Bub1, and of Mad1, Mad2 and Mad3, this implies that most SAC proteins do not need to be enriched at the kinetochores for the SAC to function. We find that Bub3 is also dispensable for shugoshin localization to the centromeres, which is the second known function of Bub1. Instead, Bub3, together with Bub1, has a specific function in promoting the conversion from chromosome mono‐orientation to bi‐orientation.