Cloning and sequence analysis of Galleria mellonella juvenile hormone binding protein--a search for ancestors and relatives

Juvenile hormone binding proteins (JHBPs) serve as specific carriers of juvenile hormone (JH) in insect hemolymph. As shown in this report, Galleria mellonella JHBP is encoded by a cDNA of 1063 nucleotides. The pre-protein consists of 245 amino acids with a 20 amino acid leader sequence. The concentration of the JHBP mRNA reaches a maximum on the third day of the last larval instar, and decreases five-fold towards pupation. Comparison of amino acid sequences of JHBPs from Bombyx mori, Heliothis virescens, Manduca sexta and G. mellonella shows that 57 positions out of 226 are occupied by identical amino acids. A phylogeny tree was constructed from 32 proteins, which function could be associated to JH. It has three major branches: (i) ligand binding domains of nuclear receptors, (ii) JHBPs and JH esterases (JHEs), and (iii) hypothetical proteins found in Drosophila melanogaster genome. Despite the close positioning of JHEs and JHBPs on the tree, which probably arises from the presence of a common JH binding motif, these proteins are unlikely to belong to the same family. Detailed analysis of the secondary structure modeling shows that JHBPs may contain a beta-barrel motif flanked by alpha-helices and thus be evolutionary related to the same superfamily as calycins.     

Liquid chromatography analysis of enrofloxacin and ciprofloxacin in chicken blood spotted on filter-paper disks

A simple, low-cost, sensitive and selective LC method was developed for the determination of enrofloxacin and ciprofloxacin in chicken blood. The method was applied to whole blood from a chicken using dried blood spots on filter paper disks. The detection limits of enrofloxacin and ciprofloxacin (100 microl of whole blood on a disk) were 0.005 and 0.01 microg/ml, respectively. The whole procedure was verified in intra-laboratory studies (recoveries of both compounds were above 90%), and its applicability was tested with blood from the chicken receiving enrofloxacin in a single oral dose at a level of 10 mg/kg body mass. The method permits the use of a small volume of blood from a chicken and should be useful for pharmacokinetic studies.     

QTL mapping of partial resistance in winter wheat to Stagonospora nodorum blotch.

Stagonospora nodorum blotch is an important foliar and glume disease in cereals. Inheritance of resistance in wheat appears to be quantitative. To date, breeding of partially resistant cultivars has been the only effective way to combat this pathogen. The partial resistance components, namely length of incubation period, disease severity, and length of latent period, were evaluated on a population of doubled haploids derived from a cross between the partially resistant Triticum aestivum 'Liwilla' and susceptible Triticum aestivum 'Begra'. Experiments were conducted in a controlled environment and the fifth leaf was examined. Molecular analyses were based on bulked segregant analyses using 240 microsatellite markers. Four QTLs were significantly associated with partial resistance components and were located on chromosomes 2B, 3B, 5B, and 5D. The percentage of phenotypic variance explained by a single QTL ranged from 14 to 21% for incubation period, from 16 to 37% for disease severity, and from 13 to 28% for latent period,     

Construction and characterization of a bacterial artificial chromosome (BAC) library of hexaploid wheat (Triticum aestivum L.) and validation of genome coverage using locus-specific primers.

A BAC library of hexaploid wheat was constructed using the spring wheat cultivar Triticum aestivum L. 'Glenlea'. Fresh shoot tissue from 7- to 10-day-old seedlings was used to obtain HMW DNA. The library was constructed using the HindIII site of pIndigoBAC-5 and the BamHI site of pIndigoBAC-5 and pECBAC1. A total of 12 ligations were used to construct the entire library, which contains over 650 000 clones. Ninety-six percent of the clones had inserts. The insert size ranged from 5 to 189 kb with an average of 79 kb. The entire library was gridded onto 24 high-density filters using a 5 x 5 array. A subset of these membranes was hybridized with two intergenic chloroplast probes and the percentage of clones containing chloroplast DNA (cpDNA) was calculated to be 2.2%. The genome coverage was estimated to be 3.1 x haploid genome equivalents, giving a 95.3% probability of identifying a clone corresponding to any wheat DNA sequence. BAC pools were constructed and screened using markers targeting the Glu-B1 locus (1BL), the hardness loci (5AS, 5BS, 5DS), the leaf rust resistance locus Lr1 (5DL), and the major fusarium head blight QTL locus located on 3BS. These markers were either locus-specific amplicons or microsatellites. A total of 49 BAC clones were identified for 14 markers giving an average of 3.5 clones/marker, thereby corroborating the estimated 3.1x genome coverage. An example using the gene encoding the HMW glutenin Bx7 is illustrated.     

Biological Consequences of the Incorporation of Amphiphilic Amino Acids into Opioid Peptide Sequences

Hydrophobic and aromatic interactions are most critical for membrane peptide receptor-ligand complex stability. We have hypothesized that proper location of hydrophilic counterparts to lipophilic and/or aromatic residues may stabilize complexation with the receptor pocket. In this work, we are presenting the biological consequences of introduction of a hydroxymethyl group into the -position of phenylalanine or tyrosine residues of enkephalin or deltorphin analogues. The consequences of such a modification are strongly dependent on the position of the primary amino acid in the peptide chain.     

Isolation and purification of proteins from the symbiosome membrane of yellow lupine root nodules

A unique feature of the symbiotic association between legume plants and rhizobia is the plant-derived membrane which separates the symbionts within root nodule; this membrane is termed the peribacteroid membrane (PBM). Although this membrane plays a vital role in facilitating transport and other processes in nodules, little is known about the proteins that are associated with and are an integral part of it. The objective of this work was to apply modern methods of protein purification to the characterisation of proteins of peribacteroid membrane from nodules of yellow lupine (Lupines luteus). The 17-kDa protein was isolated from purified peribacteroid membrane using size exclusion and ion exchange chromatography (FPLC). The N-terminal amino acid sequence of this protein was determined; the sequence does not match any of the previously reported lupine and other legume sequences. Following detergent solubilisation of purified peribacteroid membrane, integral proteins of 15 to 20 kDa were purified by size exclusion chromatography.     

Activities of a Mechanosensitive Ion Channel in anE. Coli Mutant Lacking the Major Lipoprotein

Summary The activity of the mechanosensitive (MS) ion channels in membrane patches, excised fromE. coli spheroplasts, was analyzed using the patch-clamp technique. Outer membranes from a mutant lacking the major lipoprotein (Lpp) and its wildtype parent were examined. The MS-channel activities in the wild-type membrane rarely revealed substates at the time resolution used. These channels showed a stretch sensitivity indicated by the IISP (the suction for ane-fold increase in channel open probability) of 4.9 mm Hg suction. The MS-channel activities oflpp included a prominent substate and showed a weaker mechano-sensitivity with an 1/S _p of 10.0 mm Hg. Whereas small amphipaths (chlorpromazine, trinitrophenol) or a larger amphipath (lysolecithin) all activated the MS channel in the wild-type membrane under minimal suction, only the larger lysolecithin could activate the MS channel in thelpp membranes. After lysolecithin addition, thelpp membrane became more effective in transmitting the stretch force to the MS channel, as indicated by a steepening of the Boltzmann curve. We discuss one interpretation of these results, in which the major lipoprotein serves as a natural amphipath inserted in the inner monolayer and the loss of this natural amphipath makes the bilayer less able to transmit the gating force.