The PDZ-interacting domain of cystic fibrosis transmembrane conductance regulator is required for functional expression in the apical plasma membrane

Polarization of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel to the apical plasma membrane in epithelial cells is critical for vectorial chloride transport. Previously, we reported that the C terminus of CFTR constitutes a PDZ-interacting domain that is required for CFTR polarization to the apical plasma membrane and interaction with the PDZ domain-containing protein EBP50 (NHERF). PDZ-interacting domains are typically composed of the C-terminal three to five amino acids, which in CFTR are QDTRL. Our goal was to identify the key amino acid(s) in the PDZ-interacting domain of CFTR with regard to its apical polarization, interaction with EBP50, and ability to mediate transepithelial chloride secretion. Point substitution of the C-terminal leucine (Leu at position 0) with alanine abrogated apical polarization of CFTR, interaction between CFTR and EBP50, efficient expression of CFTR in the apical membrane, and chloride secretion. Point substitution of the threonine (Thr at position -2) with alanine or valine had no effect on the apical polarization of CFTR, but reduced interaction between CFTR and EBP50, efficient expression of CFTR in the apical membrane as well as chloride secretion. By contrast, individual point substitution of the other C-terminal amino acids (Gln at position -4, Asp at position -3 and Arg at position -1) with alanine had no effect on measured parameters. We conclude that the PDZ-interacting domain, in particular the leucine (position 0) and threonine (position -2) residues, are required for the efficient, polarized expression of CFTR in the apical plasma membrane, interaction of CFTR with EBP50, and for the ability of CFTR to mediate chloride secretion. Mutations that delete the C terminus of CFTR may cause cystic fibrosis because CFTR is not polarized, complexed with EBP50, or efficiently expressed in the apical membrane of epithelial cells.     

The plastid of Toxoplasma gondii is divided by association with the centrosomes

Apicomplexan parasites harbor a single nonphotosynthetic plastid, the apicoplast, which is essential for parasite survival. Exploiting Toxoplasma gondii as an accessible system for cell biological analysis and molecular genetic manipulation, we have studied how these parasites ensure that the plastid and its 35-kb circular genome are faithfully segregated during cell division. Parasite organelles were labeled by recombinant expression of fluorescent proteins targeted to the plastid and the nucleus, and time-lapse video microscopy was used to image labeled organelles throughout the cell cycle. Apicoplast division is tightly associated with nuclear and cell division and is characterized by an elongated, dumbbell-shaped intermediate. The plastid genome is divided early in this process, associating with the ends of the elongated organelle. A centrin-specific antibody demonstrates that the ends of dividing apicoplast are closely linked to the centrosomes. Treatment with dinitroaniline herbicides (which disrupt microtubule organization) leads to the formation of multiple spindles and large reticulate plastids studded with centrosomes. The mitotic spindle and the pellicle of the forming daughter cells appear to generate the force required for apicoplast division in Toxoplasma gondii. These observations are discussed in the context of autonomous and FtsZ-dependent division of plastids in plants and algae.     

Toxicity of diatomaceous earth to red flour beetles and confused flour beetles (Coleoptera: Tenebrionidae): effects of temperature and relative humidity

Red flour beetles, Tribolium castaneum (Herbst), and confused flour beetles, Tribolium confusum (DuVal), were exposed for 8-72 h to diatomaceous earth (Protect-It) at 22, 27, and 32 degrees C and 40, 57, and 75% RH (9 combinations). Insects were exposed to the diatomaceous earth at 0.5 mg/cm2 on filter paper inside plastic petri dishes. After exposure, beetles were held for 1 wk without food at the same conditions at which they were exposed. Mortality of both species after initial exposure was lowest at 22 degrees C but increased as temperature and exposure interval increased, and within each temperature decreased as humidity increased. With 2 exceptions, all confused flour beetles were still alive after they were exposed at 22 degrees C, 57 and 75% RH. Mortality of both species after they were held for 1 wk was greater than initial mortality for nearly all exposure intervals at each temperature-humidity combination, indicating delayed toxic effects from exposure to diatomaceous earth. For both species, the relationship between mortality and exposure interval for initial and 1-wk mortality was described by linear, nonlinear, quadratic, and sigmoidal regression. Mortality of confused flour beetles was lower than mortality of red flour beetles exposed for the same time intervals for 46.7% of the total comparisons at the various temperature-relative humidity combinations.     

Impact of food source on survival of red flour beetles and confused flour beetles (Coleoptera: Tenebrionidae) exposed to diatomaceous earth

A series of experiments was conducted to determine the effect of a flour food source on survival of red flour beetle, Tribolium castaneum (Herbst), and confused flour beetle, Tribolium confusum (DuVal), exposed to the labeled rate (0.5 mg/cm2) of Protect-It, a marine formulation of diatomaceous earth. Beetles were exposed at 27 degrees C, and 40, 57, and 75% RH in 62-cm2 petri dishes. When beetles were exposed for 1 or 2 d in dishes with the labeled rate (0.5 mg/cm2, or 31 mg per dish) of diatomaceous earth or in dishes containing flour at varying levels from 0 to 200 mg mixed with the labeled rate of diatomaceous earth, survival of both species increased as the amount of flour increased, and quickly plateaued at levels approaching 100%. In a second set of experiments, beetles were transferred to dishes containing flour at varying levels from 0 to 200 mg after they were exposed for 1 or 2 d in dishes with the labeled rate of diatomaceous earth alone. There were no significant differences in beetle survival among the levels of flour, however, survival in dishes with flour was usually greater than survival in dishes with diatomaceous earth alone. In a third test, beetles were exposed for 1, 2, and 3 d in dishes with either the labeled rate of diatomaceous earth alone (clean dishes), dishes with diatomaceous earth and empty straws, or dishes with diatomaceous earth and approximately 300 mg of flour packed in the straws. Survival was not significantly different between clean dishes or dishes with straws, but survival in dishes containing the straws with flour was usually 100%, regardless of exposure interval. In all experiments, confused flour beetles were less susceptible to diatomaceous earth than red flour beetles. In addition, survival was negatively related to exposure interval and positively related to relative humidity.     

Polyploidy induces centromere association

Many species exhibit polyploidy. The presence of more than one diploid set of similar chromosomes in polyploids can affect the assortment of homologous chromosomes, resulting in unbalanced gametes. Therefore, a mechanism is required to ensure the correct assortment and segregation of chromosomes for gamete formation. Ploidy has been shown to affect gene expression. We present in this study an example of a major effect on a phenotype induced by ploidy within the Triticeae. We demonstrate that centromeres associate early during anther development in polyploid species. In contrast, centromeres in diploid species only associate at the onset of meiotic prophase. We propose that this mechanism provides a potential route by which chromosomes can start to be sorted before meiosis in polyploids. This explains previous reports indicating that meiotic prophase is shorter in polyploids than in their diploid progenitors. Even artificial polyploids exhibit this phenotype, suggesting that the mechanism must be present in diploids, but only expressed in the presence of more than one diploid set of chromosomes.     

A logical sequence search for S100B target proteins

The EF-hand calcium-binding protein S100B has been shown to interact in vitro in a calcium-sensitive manner with many substrates. These potential S100B target proteins have been screened for the preservation of a previously identified consensus sequence across species. The results were compared to known structural and in vitro properties of the proteins to rationalize choices for potential binding partners. Our approach uncovered four oligomeric proteins tubulin (alpha and beta), glial fibrillary acidic protein (GFAP), desmin, and vimentin that have conserved regions matching the consensus sequence. In the type III intermediate filament proteins (GFAP, vimentin, and desmin), this region corresponds to a portion of a coiled-coil (helix 2A), the structural element responsible for their assembly. In tubulin, the sequence matches correspond to regions of alpha and beta tubulin found at the alpha beta tubulin interface. In both cases, these consensus sequence matches provide a logical explanation for in vitro observations that S100B is able to inhibit oligomerization of these proteins.     

Design, synthesis and biological evaluation of pyridine-phenylpiperazines: a novel series of potent and selective alpha1a-adrenergic receptor antagonist

Beginning from the screening hit and literature alpha1-adrenergic compounds, a hybridized basic skeleton A was proposed as the pharmacophore for potent and selective alpha1a-AR antagonists. Introduction of a hydroxy group to increase the flexibility afforded B which served as the screening model and resulted in the identification of the second-generation lead 1. Using the Topliss approach, a number of potent and selective alpha1a-AR antagonists were discovered. In all cases, binding affinity and selectivity at the alpha1a-AR of S-hydroxy enantiomers were higher than the R-hydroxy enantiomers. As compared to the des-hydroxy analogues, the S-hydroxy enantiomers displayed comparable potency and better selectivity at alpha1a-AR. The S-hydroxy enantiomer 17 (Ki = 0.79 nM; alpha1b/alpha1a = 800; alpha1d/alpha1a = 104) was slightly less potent but much more selective at alpha1a-AR than tamsulosin (Ki = 0.13 nM, alpha1b/alpha1a = 15, alpha1d/alpha1a = 1.4). Compound 17 displayed higher selectivity in inhibiting rat prostate contraction over rat aorta contraction and also exhibited a higher degree of uroselectivity than tamsulosin in the anesthetized dog model.     

Mutagenesis of the proposed iron-sulfur cluster binding ligands in Escherichia coli biotin synthase

Biotin synthase (BioB) is a member of a family of enzymes that includes anaerobic ribonucleotide reductase and pyruvate formate lyase activating enzyme. These enzymes all use S-adenosylmethionine during turnover and contain three highly conserved cysteine residues that may act as ligands to an iron-sulfur cluster required for activity. Three mutant enzymes of BioB have been made, each with one cysteine residue (C53, 57, 60) mutated to alanine. All three mutant enzymes were inactive, but they still exhibited the characteristic UV-visible spectrum of a [2Fe-2S]2+ cluster similar to that of the wild-type enzyme.