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Peptide microarrays displaying biologically active small synthetic peptides in a high-density format provide an attractive technology to probe complex samples for the presence and/or function of protein analytes. We present a new approach for manufacturing functional peptide microarrays for molecular immune diagnostics. Our method relies on the efficiency of site-specific solution-phase coupling of biotinylated synthetic peptides to NeutrAvidin (NA) and localized microdispensing of peptide-NA-complexes onto activated glass surfaces. Antibodies are captured in a sandwich manner between surface immobilized peptide probes and fluorescence-labeled secondary antibodies. Our work includes a total of 54 peptides derived from immunodominant linear epitopes of the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein, and three domains of the Human coronavirus polymerase polyprotein and their cognate mAbs. By using spacer molecules of different type and length for NA-mediated peptide presentation, we show that the incorporation of a minimum spacer length is imperative for antibody binding, whereas the peptide immobilization direction has only secondary importance for antibody affinity and binding. We further demonstrate that the peptide array is capable of detecting low-picomolar concentrations of mAbs in buffered solutions and diluted human serum with high specificity
Peptide microarrays with site-specifically immobilized synthetic peptides for antibody diagnostics
(2006)
Peptide microarrays bear the potential to discover molecular recognition events on protein level, particularly in the field of molecular immunology, in a manner and with an efficiency comparable to the performance of DNA microarrays. We developed a novel peptide microarray platform for the detection of antibodies in liquid samples. The system comprises site-specific solution phase coupling of biotinylated peptides to NeutrAvidin, localized microdispensing of peptide-NeutrAvidin conjugates onto activated glass slides and a fluorescence immuno sandwich assay format for antibody capture and detection. Our work includes synthetic peptides deduced from amino acid sequences of immunodominant linear epitopes, such as the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein and three domains of the Human coronavirus 229E polymerase polyprotein. We demonstrate that our method produces peptide arrays with excellent spot morphology which are capable of specific and sensitive detection of monoclonal antibodies from fluid samples.
Seasonal, host sex and age-related variations in helminth egg and coccidian oocyst counts were investigated in a naturally infected wild bushbuck (Tragelaphus scriptus) population in Queen Elizabeth National Park, western Uganda from April 2000 to February 2002. The prevalence and mean intensity quantified as the number of eggs and oocysts per gram of faeces were taken as a measure of parasite burdens. Host sex and age-related differences in prevalence values were not found but the overall prevalence of Eimeria sp. was significantly higher during the rainy season, and peak counts were recorded either during or soon after a peak rainfall. A similar trend was observed for Moniezia spp., although the results were marginally not significant. There were also no significant differences in mean intensity values, relative to host sex, age or season.
Bushbuck (Tragelaphus scriptus) often deposit faeces at specific localised defecation sites (LDS). We tested whether LDS have a function in the context of parasite avoidance. In a population of bushbuck in Queen Elizabeth National Park, Uganda, seven radio-collared individuals were observed. We recorded feeding behaviour inside and outside LDS. Furthermore, pasture contamination with gastro-intestinal tract parasites inside and outside LDS was examined. There were significant differences between the expected and the observed feeding rates inside LDS, but, contrary to our prediction, the bushbuck increased their feeding rate inside LDS. There was no significant difference in the parasite contamination of pastures inside and outside LDS. We discuss the hypothesis that LDS mainly serve a social function in bushbuck communities, whereas parasite avoidance seems to play a minor or no role
Biochemical and physiological studies of Arabidopsis thaliana Diacylglycerol Kinase 7 (AtDGK7)
(2006)
A family of diacylglycerol kinases (DGK) phosphorylates the substrate diacylglycerol (DAG) to generate phosphatidic acid (PA) . Both molecules, DAG and PA, are involved in signal transduction pathways. In the model plant Arabidopsis thaliana, seven candidate genes (named AtDGK1 to AtDGK7) code for putative DGK isoforms. Here I report the molecular cloning and characterization of AtDGK7. Biochemical, molecular and physiological experiments of AtDGK7 and their corresponding enzyme are analyzed. Information from Genevestigator says that AtDGK7 gene is expressed in seedlings and adult Arabidopsis plants, especially in flowers. The AtDGK7 gene encodes the smallest functional DGK predicted in higher plants; but also, has an alternative coding sequence containing an extended AtDGK7 open reading frame, confirmed by PCR and submitted to the GenBank database (under the accession number DQ350135). The new cDNA has an extension of 439 nucleotides coding for 118 additional amino acids The former AtDGK7 enzyme has a predicted molecular mass of ~41 kDa and its activity is affected by pH and detergents. The DGK inhibitor R59022 also affects AtDGK7 activity, although at higher concentrations (i.e. IC50 ~380 µM). The AtDGK7 enzyme also shows a Michaelis-Menten type saturation curve for 1,2-DOG. Calculated Km and Vmax were 36 µM 1,2-DOG and 0.18 pmol PA min-1 mg of protein-1, respectively, under the assay conditions. Former protein AtDGK7 are able to phosphorylate different DAG analogs that are typically found in plants. The new deduced AtDGK7 protein harbors the catalytic DGKc and accessory domains DGKa, instead the truncated one as the former AtDGK7 protein (Gomez-Merino et al., 2005).
The photosensitive microvilli of Drosophila photoreceptors R1-R6 are not aligned in parallel over the entire length of the visual cells. In the distal half of each cell, the microvilli are slightly tilted toward one side and, in the proximal half, extremely toward the opposite side. This phenomenon, termed rhabdomere twisting, has been known for several decades, but the developmental and cell biological basis of rhabdomere twisting has not been studied so far. We show that rhabdomere twisting is also manifested as molecular polarization of the visual cell, because phosphotyrosine- containing proteins are selectively partitioned to different sides of the rhabdomere stalk in the distal. and proximal sections of each R1-R6 photoreceptor. Both the asymmetrical segregation of phosphotyrosine proteins and the tilting of the microvilli occur shortly before eclosion of the flies, when eye development in all other aspects is considered to be essentially complete. Establishment of rhabdomere twisting occurs in a light-independent manner, because phosphotyrosine staining is unchanged in dark-reared wild-type flies and in mutants with defects in the phototransduction cascade, ninaE(17) and norpA(P24). We conclude that antiphosphotyrosine immunofluorescence can be used as a light microscopic probe for the analysis of rhabdomere twisting and that microvilli tilting represents a type of planar cell polarity that is established by an active process in the last phase of photoreceptor morphogenesis, just prior to eclosion of the flies.
Because of its high reaction rate and specificity, the enzyme superoxide dismutase (SOD) offers great potential for the sensitive quantification of superoxide radicals in electrochemical biosensors. In this work, monomeric mutants of human Cu,Zn-SOD were engineered to contain one or two additional cysteine residues, which could be used to bind the protein to gold surfaces, thus making the use of promotor molecules unnecessary. Six mutants were successfully designed, expressed, and purified. All mutants bound directly to unmodified gold surfaces via the sulfur of the cysteine residues and showed a quasireversible, direct electron transfer to the electrode. Thermodynamic and kinetic parameters of the electron transfer were characterized and showed only slight variations between the individual mutants. For one of the mutants, the interaction with the superoxide radical was studied in more detail. For both partial reactions of the dismutation, an interaction between protein and radical could be shown. In an amperometric biosensorial approach, the SOD-mutant electrode was successfully applied for the detection of superoxide radicals. In the oxidation region, the electrode surpassed the sensitivity of the commonly used cytochrome c electrodes by similar to 1 order of magnitude while not being limited by interferences, but the electrode did not fully reach the sensitivity of dimeric Cu,Zn-SOD immobilized on MPA-modified gold
1. The in situ abundance, biomass and mean cell volume of Actinophrys sol (Sarcodina: Heliozoa), the top predator in an extremely acidic German mining lake (Lake 111; pH 2.65), were determined over three consecutive years (spring to autumn, 2001-03). 2. Actinophrys sol exhibited pronounced temporal and vertical patterns in abundance, biomass and mean cell volume. Increasing from very low spring densities, maxima in abundance and biomass were observed in late June/early July and September. The highest mean abundance recorded during the study was 7 x 10(3) Heliozoa L-1. Heliozoan abundance and biomass were higher in the epilimnion than in the hypolimnion. Actinophrys sol cells from this acidic lake were smaller than individuals of the same species found in other aquatic systems. 3. We determined the growth rate of A. sol using all potential prey items available in, and isolated and cultured from, Lake 111. Prey items included: single-celled and filamentous bacteria of unknown taxonomic affinity, the mixotrophic flagellates Chlamydomonas acidophila and Ochromonas sp., the ciliate Oxytricha sp. and the rotifers Elosa worallii and Cephalodella hoodi. Actinophrys sol fed over a wide-size spectrum from bacteria to metazoans. Positive growth was not supported by all naturally available prey. Actinophrys sol neither increased in cell number (k) nor biomass (k(b)) when starved, with low concentrations of single-celled bacteria or with the alga Ochromonas sp. Positive growth was achieved with single- celled bacteria (k = 0.22 +/- 0.02 d(-1); k(b) = -0.06 +/- 0.02 d(-1)) and filamentous bacteria (k = 0.52 +/- < 0.01 d(- 1); k(b) = 0.66 d(-1)) at concentrations greater than observed in situ, and the alga C. acidophila (up to k = 0.43 +/- 0.03 d(-1); k(b) = 0.44 +/- 0.04 d(-1)), the ciliate Oxytricha sp. (k = 0.34 +/- 0.01 d(-1)) and in mixed cultures containing rotifers and C. acidophila (k = 0.23 +/- 0.02-0.32 +/- 0.02 d(-1); maximum k(b) = 0.42 +/- 0.05 d(-1)). The individual- and biomass-based growth of A. sol was highest when filamentous bacteria were provided. 4. Existing quantitative carbon flux models for the Lake 111 food web can be updated in light of our results. Actinophrys sol are omnivorous predators supported by a mixed diet of filamentous bacteria and C. acidophila in the epilimnion. Heliozoa are important components in the planktonic food webs of 'extreme' environments
The occurrence of two individuals of Zu cristatus at 2 m depth in coastal waters of the Gulf of Castellamare (Tyrrhenian Sea, Italy) together with records of this rare pan-Oceanic mesopelagic species is reported. Analyses of two mitochondrial genes (12 s and 16 s; 936 bp) revealed a 2.6% sequence divergence between Mediterranean and Pacific (Japanese) samples of the species.
Sucrose synthase (Susy) is a key enzyme of sucrose metabolism, catalysing the reversible conversion of sucrose and UDP to UDP-glucose and fructose. Therefore, its activity, localization and function have been studied in various plant species. It has been shown that Susy can play a role in supplying energy in companion cells for phloem loading (Fu and Park, 1995), provides substrates for starch synthesis (Zrenner et al., 1995), and supplies UDP-glucose for cell wall synthesis (Haigler et al., 2001). Analysis of the Arabidopsis genome identifies six Susy isoforms. The expression of these isoforms was investigated using promoter-reporter gene constructs (GUS) and real time RT-PCR. Although these isoforms are closely related at the protein level they have radically different spatial and temporal patterns of expression in the plant with no two isoforms showing the same distribution. More than one isoform is expressed in all organs examined. Some of them have high but specific expression in particular organs or developmental stages whilst others are constantly expressed throughout the whole plant and across various stages of development. The in planta function of the six Susy isoforms were explored through analysis of T-DNA insertion mutants and RNAi lines. Plants without the expression of individual isoforms show no differences in growth and development, and are not significantly different from wild type plants in soluble sugars, starch and cellulose contents under all growth conditions investigated. Analysis of T-DNA insertion mutant lacking Sus3 isoform that was exclusively expressed in stomata cells only had a minor influence on guard cell osmoregulation and/or bioenergetics. Although none of the sucrose synthases appear to be essential for normal growth under our standard growth conditions, they may be necessary for growth under stress conditions. Different isoforms of sucrose synthase respond differently to various abiotic stresses. It has been shown that oxygen deprivation up regulates Sus1 and Sus4 and increases total Susy activity. However, the analysis of the plants with reduced expression of both Sus1 and Sus4 revealed no obvious effects on plant performance under oxygen deprivation. Low temperature up regulates Sus1 expression but the loss of this isoform has no effect on the freezing tolerance of non acclimated and cold acclimated plants. These data provide a comprehensive overview of the expression of this gene family which supports some of the previously reported roles for Susy and indicates the involvement of specific isoforms in metabolism and/or signalling.