Kinetic Characterisation of a Single Chain Antibody against the Hormone Abscisic Acid: Comparison with Its Parental Monoclonal

A single-chain Fv fragment antibody (scFv) specific for the plant hormone abscisic acid (ABA) has been expressed in the bacterium Escherichia coli as a fusion protein. The kinetics of ABA binding have been measured using surface plasmon resonance spectrometry (BIAcore 2000) using surface and solution assays. Care was taken to calculate the concentration of active protein in each sample using initial rate measurements under conditions of partial mass transport limitation. The fusion product, parental monoclonal antibody and the free scFv all have low nanomolar affinity constants, but there is a lower dissociation rate constant for the parental monoclonal resulting in a three-fold greater affinity. Analogue specificity was tested and structure-activity binding preferences measured. The biologically-active (+)-ABA enantiomer is recognised with an affinity three orders of magnitude higher than the inactive (-)-ABA. Metabolites of ABA including phaseic acid, dihydrophaseic acid and deoxy-ABA have affinities over 100-fold lower than that for (+)-ABA. These properties of the scFv make it suitable as a sensor domain in bioreporters specific for the naturally occurring form of ABA

Uninvited Guests: Traditional Insect Repellents in Estonia used Against the Clothes Moth Tineola bisselliella, Human Flea Pulex irritons and Bedbug Cimex lectularius

Extensive folklore records from pre-modern Estonia give us an excellent opportunity to study a variety of local plant knowledge and plant use among the peasantry in various parts of the country. One important biocultural domain where plant knowledge has been crucial was in the various methods of combating different ectoparasites that cohabited and coexisted with humans and their domestic animals. Some of these methods were widely known (world-wide, Eurasia, Europe, Baltic Rim), while others were more local. Here we discuss ways of reducing clothes moths Tineola bisselliella (Hummel) (Lepidoptera: Tineidae), human fleas Pulex irritons L. (Siphonaptera: Pulicidae) and bedbugs Cimex lectularius L. (Hemiptera: Cimicidae) with the help of plants. Various taxa used as traditional repellents have been identified. The use of plants as repellents and their toxic principles are also discussed from a comparative perspective

Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca

Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The enzyme was overexpressed in Escherichia coli at up to 14% of total soluble protein and purified to homogeneity in three steps. CYP154H1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida DSM 50198 as surrogate electron transfer partners. In biocatalytic reactions with different aliphatic and aromatic substrates of varying size, the enzyme converted small aromatic and arylaliphatic compounds like ethylbenzene, styrene, and indole. Furthermore, CYP154H1 also accepted different arylaliphatic sulfides as substrates chemoselectively forming the corresponding sulfoxides and sulfones. The enzyme is moderately thermostable with an apparent melting temperature of 67°C and exhibited still 90% of initial activity after incubation at 50°C

Detection and Alignment of 3D Domain Swapping Proteins Using Angle-Distance Image-Based Secondary Structural Matching Techniques

This work presents a novel detection method for three-dimensional domain swapping (DS), a mechanism for forming protein quaternary structures that can be visualized as if monomers had “opened” their “closed” structures and exchanged the opened portion to form intertwined oligomers. Since the first report of DS in the mid 1990s, an increasing number of identified cases has led to the postulation that DS might occur in a protein with an unconstrained terminus under appropriate conditions. DS may play important roles in the molecular evolution and functional regulation of proteins and the formation of depositions in Alzheimer's and prion diseases. Moreover, it is promising for designing auto-assembling biomaterials. Despite the increasing interest in DS, related bioinformatics methods are rarely available. Owing to a dramatic conformational difference between the monomeric/closed and oligomeric/open forms, conventional structural comparison methods are inadequate for detecting DS. Hence, there is also a lack of comprehensive datasets for studying DS. Based on angle-distance (A-D) image transformations of secondary structural elements (SSEs), specific patterns within A-D images can be recognized and classified for structural similarities. In this work, a matching algorithm to extract corresponding SSE pairs from A-D images and a novel DS score have been designed and demonstrated to be applicable to the detection of DS relationships. The Matthews correlation coefficient (MCC) and sensitivity of the proposed DS-detecting method were higher than 0.81 even when the sequence identities of the proteins examined were lower than 10%. On average, the alignment percentage and root-mean-square distance (RMSD) computed by the proposed method were 90% and 1.8Å for a set of 1,211 DS-related pairs of proteins. The performances of structural alignments remain high and stable for DS-related homologs with less than 10% sequence identities. In addition, the quality of its hinge loop determination is comparable to that of manual inspection. This method has been implemented as a web-based tool, which requires two protein structures as the input and then the type and/or existence of DS relationships between the input structures are determined according to the A-D image-based structural alignments and the DS score. The proposed method is expected to trigger large-scale studies of this interesting structural phenomenon and facilitate related applications

The Effects of Commercials on Children’s Perceptions of Gender Appropriate Toy Use

Sixty-two first and second grade students (28 boys, 34 girls) were exposed to one of three commercial videotapes in which either all-boys (traditional condition) or all-girls (nontraditional) were playing with a toy. Participants in the control condition were exposed to nontoy commercials. After exposure to one of the conditions participants performed a toy sort where they were asked if six toys, including the two manipulated toys, were “for boys, girls, or both boys and girls.” Participants in the nontraditional condition were more likely to report that the manipulated toys were for both boys and girls than were participants in the traditional condition, who were more likely to report that the manipulated toys were for boys. This effect was stronger for boys than for girls.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45638/1/11199_2005_Article_1195.pd

Metabolomics reveals a link between homocysteine and lipid metabolism and leukocyte telomere length: the ENGAGE consortium

Telomere shortening has been associated with multiple age-related diseases such as cardiovascular disease, diabetes, and dementia. However, the biological mechanisms responsible for these associations remain largely unknown. In order to gain insight into the metabolic processes driving the association of leukocyte telomere length (LTL) with age-related diseases, we investigated the association between LTL and serum metabolite levels in 7,853 individuals from seven independent cohorts. LTL was determined by quantitative polymerase chain reaction and the levels of 131 serum metabolites were measured with mass spectrometry in biological samples from the same blood draw. With partial correlation analysis, we identified six metabolites that were significantly associated with LTL after adjustment for multiple testing: lysophosphatidylcholine acyl C17:0 (lysoPC a C17:0, p-value = 7.1 × 10−6), methionine (p-value = 9.2 × 10−5), tyrosine (p-value = 2.1 × 10−4), phosphatidylcholine diacyl C32:1 (PC aa C32:1, p-value = 2.4 × 10−4), hydroxypropionylcarnitine (C3-OH, p-value = 2.6 × 10−4), and phosphatidylcholine acyl-alkyl C38:4 (PC ae C38:4, p-value = 9.0 × 10−4). Pathway analysis showed that the three phosphatidylcholines and methionine are involved in homocysteine metabolism and we found supporting evidence for an association of lipid metabolism with LTL. In conclusion, we found longer LTL associated with higher levels of lysoPC a C17:0 and PC ae C38:4, and with lower levels of methionine, tyrosine, PC aa C32:1, and C3-OH. These metabolites have been implicated in inflammation, oxidative stress, homocysteine metabolism, and in cardiovascular disease and diabetes, two major drivers of morbidity and mortality

Monitoring the effect of upwelling on the chlorophyll a distribution in the Gulf of Finland (Baltic Sea) using remote sensing and in situ data

The spatio-temporal variability of chlorophyl a (Chl a) caused by a sequence of upwelling events in the Gulf of Finland in July–August 2006 was studied using remote sensing data and field measurements. Spatial distributions of sea surface temperature (SST) and Chl a concentration were examined using MODIS and MERIS data respectively. The MERIS data were processed with an algorithm developed by the Free University of Berlin (FUB) for case 2 waters. Evaluation of MERIS Chl a versus in situ Chl a showed good correlation (r2 = 0.67), but the concentration was underestimated. The linear regression for a 2 h window was applied to calibrate MERIS Chl a. The spatio-temporal variability exhibited the clear influence of upwelling events and related filaments on Chl a distribution in the western and central Gulf. The lowest Chl a concentrations were recorded in the upwelled water, especially at the upwelling centres, and the highest concentrations (13 mg m−3) were observed about two weeks after the upwelling peak along the northern coast. The areas along the northern coast of upwelled water (4879 km2) on the SST map, and increased Chl a (5526 km2) two weeks later, were roughly coincident. The effect of upwelling events was weak in the eastern part of the Gulf, where Chl a concentration was relatively consistent throughout this period