Ligand-mediated protection against phage lysis as a positive selection strategy for the enrichment of epitopes displayed on the surface of E. coli cells

We present a novel strategy, termed CISTEM, which allows direct in vivo screening of polypeptides displayed on the surface of E. coli cells by a combination of ligand-mediated protection and phage-mediated selection. The effectiveness of this new approach was demonstrated by displaying the T7.tag on the surface of E. coli as a fusion with the outer membrane protein A, the receptor for bacteriophage K3. A monoclonal T7.tag antibody was used as protective ligand for T7.tag-displaying cells and phage K3 for the elimination of unprotected cells. When populations of bacteria, containing between 6 to 10,000 cells displaying the T7.tag and approximately 10(8) cells displaying an unrelated OmpA fusion protein, were infected with phage K3, specific and antibody-dependent survival of T7.tag displaying cells was observed, yielding an enrichment factor of up to 10(7)-fold. The CISTEM technology was used to select sequences from a T7.tag-based, randomised library and the results were compared to those obtained from selection by MACS with the same library. Together, these results reveal a novel in vivo screening strategy in which an E. coli phage receptor is used as display plafform and selection is performed in suspension upon addition of a protective ligand and a bacteriophage. Extentions and modifications of the basic strategy should lead to novel applications for the identification of protein-ligand interactions.     

Broad spectrum antiprotozoal agents that inhibit histone deacetylase: structure-activity relationships of apicidin. Part 1

Apicidin, a natural product recently isolated at Merck, inhibits both mammalian and protozoan histone deacetylases (HDACs). The conversion of apicidin, a nanomolar inhibitor of HDACs, into a series of side-chain analogues that display picomolar enzyme affinity is described within this structure-activity study.     

A sequence-based map of Arabidopsis genes with mutant phenotypes

The classical genetic map of Arabidopsis contains 462 genes with mutant phenotypes. Chromosomal locations of these genes have been determined over the past 25 years based on recombination frequencies with visible and molecular markers. The most recent update of the classical map was published in a special genome issue of Science that dealt with Arabidopsis (D.W. Meinke, J.M. Cherry, C. Dean, S.D. Rounsley, M. Koornneef [1998] Science 282: 662-682). We present here a comprehensive list and sequence-based map of 620 cloned genes with mutant phenotypes. This map documents for the first time the exact locations of large numbers of Arabidopsis genes that give a phenotype when disrupted by mutation. Such a community-based physical map should have broad applications in Arabidopsis research and should serve as a replacement for the classical genetic map in the future. Assembling a comprehensive list of genes with a loss-of-function phenotype will also focus attention on essential genes that are not functionally redundant and ultimately contribute to the identification of the minimal gene set required to make a flowering plant.     

Inheritance of methyl-parathion resistance in Nebraska western corn rootworm populations (Coleoptera: Chrysomelidae)

Field populations of western corn rootworm, Diabrotica virgifera virgifera LeConte, were collected from three different sites (York Co., Phelps Co., and Saunders Co.) in Nebraska during 1996. Adult bioassays of these three populations were conducted with different concentrations of methyl-parathion and at a diagnostic concentration (1.0 microg/ml) to determine resistance levels among these populations. Self and reciprocal crosses were made between the two resistant and one susceptible laboratory-reared populations. Dose-responses and dominance ratios calculated for the four reciprocal crosses indicated that resistance was incompletely dominant in both strains, although in one of the strains there was an indication of sex linkage. However, evaluation of native polyacrylamide gels stained for nonspecific esterases and nonspecific esterase activity of parents and F1 progeny of the crosses suggested that esterase inheritance was completely dominant and autosomal. The results of this study were inconclusive with regard to the precise nature of inheritance, because the bioassays and esterase assays could not discriminate between heterozygotes and homozygotes. However, they do provide insight into the potential for developing simple diagnostic assays to assess resistance frequencies. Based on the inheritance studies described in this investigation, we can begin to generate information on specific genetic factors that dictate the evolutionary divergence of discrete resistant populations and facilitate modeling efforts designed to approximate the movement of genes for resistance among populations.     

Comparison of nodulisporic acid analogs in a Lucilia sericata in vitro assay and a Ctenocephalides felis membrane feeding system

A medicinal chemistry program on the nodulisporic acid chemical class, guided by an artificial membrane flea-feeding assay, has recently identified permissive and nonpermissive regions of the pharmacophore for exploitation against fleas. This pathway was validated when several promising compounds from this program were administered orally to dogs at 15.0 mg/kg and found to have >90% flea activity for 2 wk. To determine if a surrogate insect assay would have provided the same guidance, a nodulisporic acid analog series was examined in both a Lucilia sericata larval assay and an artificial membrane flea-feeding assay using Ctenocephalides felis. Results from both insect assays were concordant in that even subtle chemical modification or substitution to the left-hand side of the nodulisporic acid pharmacophore resulted in substantial loss of insecticidal activity. Both assays were also in general agreement that the only modifications to the pharmacophore that did not result in loss of activity occurred to the C-8 side chain on the right-hand side of the molecule. Although there was good agreement between the 2 assays on the general regions of the pharmacophore, there was variance on individual compounds in the mono- and disubstituted amide series from the C-8 side chain. For example, the L. sericata assay showed several analogs from this subclass to possess similar activity to the parent acid, whereas the membrane assay indicated superior activity against fleas relative to the same parent. Consequently, although there was substantial general agreement between the assays, it was concluded that finer optimization of a lead compound should be done against the target parasite, even if it is ex vivo, as early as possible in a medicinal chemistry program.     

Chemical modification of nodulisporic acid A: preliminary structure-activity relationships

Medicinal chemistry efforts were initiated to identify the key constituents of the nodulisporic acid A (1) pharmacophore that are integral to its potent insecticidal activity. New semisynthetic derivatives delineated 1 into 'permissive' and 'nonpermissive' regions and led to the discovery of new nodulisporamides with significantly improved flea efficacy.     

The TITAN5 gene of Arabidopsis encodes a protein related to the ADP ribosylation factor family of GTP binding proteins

The titan (ttn) mutants of Arabidopsis exhibit dramatic alterations in mitosis and cell cycle control during seed development. Endosperm development in these mutants is characterized by the formation of giant polyploid nuclei with enlarged nucleoli. Embryo development is accompanied by significant cell enlargement in some mutants (ttn1 and ttn5) but not others (ttn2 and ttn3). We describe here the molecular cloning of TTN5 using a T-DNA-tagged allele. A second allele with a similar phenotype contains a nonsense mutation in the same coding region. The predicted protein is related to ADP ribosylation factors (ARFs), members of the RAS family of small GTP binding proteins that regulate various cellular functions in eukaryotes. TTN5 is most closely related in sequence to the ARL2 class of ARF-like proteins isolated from humans, rats, and mice. Although the cellular functions of ARL proteins remain unclear, the ttn5 phenotype is consistent with the known roles of ARFs in the regulation of intracellular vesicle transport.     

Evidence of Field-Evolved Resistance to Bifenthrin in Western Corn Rootworm (Diabrotica virgifera virgifera LeConte) Populations in Western Nebraska and Kansas

Pyrethroid insecticides have been used to control larvae or adults of the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, a key pest of field corn in the United States. In response to reports of reduced efficacy of pyrethroids in WCR management programs in southwestern areas of Nebraska and Kansas the present research was designed to establish a baseline of susceptibility to the pyrethroid insecticide, bifenthrin, using susceptible laboratory populations and to compare this baseline with susceptibility of field populations. Concentration-response bioassays were performed to estimate the baseline susceptibility. From the baseline data, a diagnostic concentration (LC₉₉) was determined and used to test adults of both laboratory and field populations. Larval susceptibility was also tested using both laboratory and field populations. Significant differences were recorded in adult and larval susceptibility among WCR field and laboratory populations. The highest LC₅₀ for WCR adults was observed in populations from Keith 2 and Chase Counties, NE, with LC₅₀s of 2.2 and 1.38 μg/vial, respectively, and Finney County 1, KS, with 1.43 μg/vial, as compared to a laboratory non-diapause population (0.24 μg/vial). For larvae, significant differences between WCR field and laboratory populations were also recorded. Significant differences in mortalities at the diagnostic bifenthrin concentration (LC₉₉) were observed among WCR adult populations with western Corn Belt populations exhibiting lower susceptibility to bifenthrin, especially in southwestern Nebraska and southwestern Kansas. This study provides evidence that resistance to bifenthrin is evolving in field populations that have been exposed for multiple years to pyrethroid insecticides. Implications to sustainable rootworm management are discussed.     

Wavelength,dose, skin type and skin model related radical formation in skin

The exposure to sun radiation is indispensable to our health; however, a long-term and high exposure could lead to cell damage, erythema, premature skin aging, and promotion of skin tumors. An underlying pathomechanism is the formation of free radicals which may induce oxidative stress at elevated concentrations. Different skin models, such as porcine-, murine-, human- ex vivo skin, reconstructed human skin (RHS) and human skin in vivo, were investigated during and after irradiation using X- and L-band EPR spectroscopy within different spectral regions (UVC to NIR). The amount of radical formation was quantified with the spin probe PCA and the radical types were measured ex vivo with the spin trap DMPO. The radiation dose influences the types of radicals formed in the skin. While reactive oxygen species (ROS) are always pronounced at low doses, there is an increase in lipid oxygen species (LOS) at high doses. Furthermore, the radical types arise independent from the irradiation wavelength, whereas the general amount of radical formation differs with the irradiation wavelength. Heat pre-stressed porcine skin already starts with higher LOS values. Thus, the radical type ratio might be an indicator of stress and the reversal of ROS/LOS constitutes the point where positive stress turns into negative stress.Compared to light skin types, darker types produce less radicals in the ultraviolet, similar amounts in the visible and higher ones in the infrared spectral region, rendering skin type-specific sun protection a necessity.     

Detecting Nucleotide Additivity from Direct Sequences is a SNAP: An Example from Sidalcea (Malvaceae)

Abstract: Superimposed nucleotide additivity patterns (SNAPs) were detected from direct sequences of the nuclear ribosomal DNA internal transcribed spacers in a complex of perennial Sidalcea species (Malvaceae) from the Pacific Northwest of the United States. Although only 2.8 % sequence variation exists among the eight ITS accessions, parsimony analysis identified two distinct lineages within this complex consistent with known ploidy levels. Six SNAPs were identified in a known tetraploid S. virgata, suggesting allopolyploid origins from diploid S. virgata and one of two hexaploid Sidalcea species. A dosage effect detected at all six SNAP sites is consistent with the unequal sized parental genomes of allopolyploid S. virgata.