Real-time analysis of the inside-out regulation of lymphocyte function-associated antigen-1 revealed similarities to and differences from very late antigen-4

Ten years ago, we introduced a fluorescent probe that shed light on the inside-out regulation of one of the major leukocyte integrins, very late antigen-4 (VLA-4, CD49d/CD29). Here we describe the regulation of another leukocyte integrin, lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18) using a novel small fluorescent probe in real time on live cells. We found that multiple signaling mechanisms regulate LFA-1 conformation in a manner analogous to VLA-4. LFA-1 can be rapidly activated by Gα(i)-coupled G protein-coupled receptors (GPCRs) and deactivated by Gα(s)-coupled GPCRs. The effects of Gα(s)-coupled GPCR agonists can be reversed in real time by receptor-specific antagonists. The specificity of the fluorescent probe binding has been assessed in a competition assay using the natural LFA-1 ligand ICAM-1 and the LFA-1-specific α I allosteric antagonist BIRT0377. Similar to VLA-4 integrin, modulation of the ligand dissociation rate can be observed for different LFA-1 affinity states. However, we also found a striking difference in the binding of the small fluorescent ligand. In the absence of inside-out activation ligand, binding to LFA-1 is extremely slow, at least 10 times slower than expected for diffusion-limited binding. This implies that an additional structural mechanism prevents ligand binding to inactive LFA-1. We propose that such a mechanism explains the inability of LFA-1 to support cell rolling, where the absence of its rapid engagement by a counterstructure in the inactive state leads to a requirement for a selectin-mediated rolling step.     

NMR structure of a 4 x 4 nucleotide RNA internal loop from an R2 retrotransposon: identification of a three purine-purine sheared pair motif and comparison to MC-SYM predictions

The NMR solution structure is reported of a duplex, 5'GUGAAGCCCGU/3'UCACAGGAGGC, containing a 4 × 4 nucleotide internal loop from an R2 retrotransposon RNA. The loop contains three sheared purine-purine pairs and reveals a structural element found in other RNAs, which we refer to as the 3RRs motif. Optical melting measurements of the thermodynamics of the duplex indicate that the internal loop is 1.6 kcal/mol more stable at 37°C than predicted. The results identify the 3RRs motif as a common structural element that can facilitate prediction of 3D structure. Known examples include internal loops having the pairings: 5'GAA/3'AGG, 5'GAG/3'AGG, 5'GAA/3'AAG, and 5'AAG/3'AGG. The structural information is compared with predictions made with the MC-Sym program.     

<Emphasis Type="Italic">pc8.1</Emphasis>, a major QTL for pigment content in pepper fruit,is associated with variation in plastid compartment size

Studies on the genetic control of pigment content in pepper fruit have focused mainly on monogenic mutations leading to changes in fruit color. In addition to the qualitative variation in fruit color, quantitative variation in pigment content and color intensity exists in pepper giving rise to a range of color intensities. However, the genetic basis for this variation is poorly understood, hindering the development of peppers that are rich in these beneficial compounds. In this paper, quantitative variation in pigment content was studied in a cross between a dark-green Capsicum annuum pepper and a light-green C. chinense pepper. Two major pigment content QTLs that control chlorophyll content were identified, pc8.1 and pc10.1. The major QTL pc8.1, also affected carotenoid content in the ripe fruit. However, additional analyses in subsequent generations did not reveal a consistent effect of this QTL on carotenoid content in ripe fruit. Confocal microscopy analyses of green immature fruits of the parents and of near-isogenic lines for pc8.1 indicated that the QTL exerts its effect via increasing chloroplast compartment size in the dark-green genotypes, predominantly in a fruit-specific manner. Metabolic analyses indicated that in addition to chlorophyll, chloroplast-associated tocopherols and carotenoids are also elevated. Future identification of the genes controlling pigment content QTLs in pepper will provide a better understanding of this important trait and new opportunities for breeding peppers and other Solanaceae species with enhanced nutritional value.     

Rapid evolution of the compact and unusual mitochondrial genome in the ctenophore, Pleurobrachia bachei

Ctenophores are one of the most basally branching lineages of metazoans with the largest mitochondrial organelles in the animal kingdom. We sequenced the mitochondrial (mtDNA) genome from the Pacific cidipid ctenophore, Pleurobrachia bachei. The circular mitochondrial genome is 11,016 nts, with only 12 genes, and one of the smallest metazoan mtDNA genomes recorded. The protein coding genes are intronless cox1-3, cob, nad1, 3, 4, 4L and 5. The nad2 and 6 genes are represented as short fragments whereas the atp6 gene was found in the nuclear genome. Only the large ribosomal RNA subunit and two tRNAs were present with possibly the small subunit unidentifiable due to extensive fragmentation. The observed unique features of this mitochondrial genome suggest that nuclear and mitochondrial genomes have evolved at very different rates. This reduced mtDNA genome sharply contrasts with the very large sizes of mtDNA found in other basal metazoans including Porifera (sponges), and Placozoa (Trichoplax).     

Substrate Selection for Fundamental Studies of Electrocatalysts and Photoelectrodes: Inert Potential Windows in Acidic,Neutral, and Basic Electrolyte

The selection of an appropriate substrate is an important initial step for many studies of electrochemically active materials. In order to help researchers with the substrate selection process, we employ a consistent experimental methodology to evaluate the electrochemical reactivity and stability of seven potential substrate materials for electrocatalyst and photoelectrode evaluation. Using cyclic voltammetry with a progressively increased scan range, we characterize three transparent conducting oxides (indium tin oxide, fluorine-doped tin oxide, and aluminum-doped zinc oxide) and four opaque conductors (gold, stainless steel 304, glassy carbon, and highly oriented pyrolytic graphite) in three different electrolytes (sulfuric acid, sodium acetate, and sodium hydroxide). We determine the inert potential window for each substrate/electrolyte combination and make recommendations about which materials may be most suitable for application under different experimental conditions. Furthermore, the testing methodology provides a framework for other researchers to evaluate and report the baseline activity of other substrates of interest to the broader community.     

Enhancement of TCE Attenuation in Soils by Natural Amendments

The objective of this study was to identify low-cost natural amendments that could be used as carbon sources and sustain a bioactive zone to promote biodegradation of TCE in contaminated shallow groundwater. The natural amendments were compared based on their geophysical characteristics as well as TCE adsorption capacities. The amendments studied included low-cost natural and agricultural materials such as eucalyptus tree mulch, pine bark mulch, muck from the Florida Everglades, SRS wetland peat, commercial compost, and peat humus. These natural substrates have relatively high organic fractions that can retard the movement of TCE while serving as carbon sources. Batch sorption studies were conducted to determine the sorption and retardation characteristics of the amendments for TCE. The experimental results were analyzed in relation to the geophysical characteristics of the amendments and compared with those of natural soils.     

DNA sequence context as a determinant of the quantity and chemistry of guanine oxidation produced by hydroxyl radicals and one-electron oxidants

DNA sequence context has emerged as a critical determinant of the location and quantity of nucleobase damage caused by many oxidizing agents. However, the complexity of nucleobase and 2-deoxyribose damage caused by strong oxidants such as ionizing radiation and the Fenton chemistry of Fe2+-EDTA/H2O2 poses a challenge to defining the location of nucleobase damage and the effects of sequence context on damage chemistry in DNA. To address this problem, we developed a gel-based method that allows quantification of nucleobase damage in oxidized DNA by exploiting Escherichia coli exonuclease III to remove fragments containing direct strand breaks and abasic sites. The rigor of the method was verified in studies of guanine oxidation by photooxidized riboflavin and nitrosoperoxycarbonate, for which different effects of sequence context have been demonstrated by other approaches (Margolin, Y., Cloutier, J. F., Shafirovich, V., Geacintov, N. E., and Dedon, P. C. (2006) Nat. Chem. Biol. 2, 365-366). Using duplex oligodeoxynucleotides containing all possible three-nucleotide sequence contexts for guanine, the method was used to assess the role of DNA sequence context in hydroxyl radical-induced guanine oxidation associated with gamma-radiation and Fe2+-EDTA/H2O2. The results revealed both differences and similarities for G oxidation by hydroxyl radicals and by one-electron oxidation by riboflavin-mediated photooxidation, which is consistent with the predominance of oxidation pathways for hydroxyl radicals other than one-electron oxidation to form guanine radical cations. Although the relative quantities of G oxidation produced by hydroxyl radicals were more weakly correlated with sequence-specific ionization potential than G oxidation produced by riboflavin, damage produced by both hydroxyl radical generators and riboflavin within two- and three-base runs of G showed biases in location that are consistent with a role for electron transfer in defining the location of the damage products. Furthermore, both gamma-radiation and Fe2+-EDTA/H2O2 showed relatively modest effects of sequence context on the proportions of different damage products sensitive to E. coli formamidopyrimidine DNA glycosylase and hot piperidine, although GT-containing sequence contexts displayed subtle biases in damage chemistry (formamidopyrimidine DNA glycosylase/piperidine ratio). Overall, the results are consistent with the known chemistry of guanine oxidation by hydroxyl radical and demonstrate that charge migration plays a relatively minor role in determining the location and chemistry of hydroxyl radical-mediated oxidative damage to guanine in DNA.