Towards a membrane proteome in Drosophila: a method for the isolation of plasma membrane

<p>Abstract</p> <p>Background</p> <p>The plasma membrane (PM) is a compartment of significant interest because cell surface proteins influence the way in which a cell interacts with its neighbours and its extracellular environment. However, PM is hard to isolate because of its low abundance. Aqueous two-phase affinity purification (2PAP), based on PEG/Dextran two-phase fractionation and lectin affinity for PM-derived microsomes, is an emerging method for the isolation of high purity plasma membranes from several vertebrate sources. In contrast, PM isolation techniques in important invertebrate genetic model systems, such as <it>Drosophila melanogaster</it>, have relied upon enrichment by density gradient centrifugation. To facilitate genetic investigation of activities contributing to the content of the PM sub-proteome, we sought to adapt 2PAP to this invertebrate model to provide a robust PM isolation technique for <it>Drosophila</it>.</p> <p>Results</p> <p>We show that 2PAP alone does not completely remove contaminating endoplasmic reticulum and mitochondrial membrane. However, a novel combination of density gradient centrifugation plus 2PAP results in a robust PM preparation. To demonstrate the utility of this technique we isolated PM from fly heads and successfully identified 432 proteins using MudPIT, of which 37% are integral membrane proteins from all compartments. Of the 432 proteins, 22% have been previously assigned to the PM compartment, and a further 34% are currently unassigned to any compartment and represent candidates for assignment to the PM. The remainder have previous assignments to other compartments.</p> <p>Conclusion</p> <p>A combination of density gradient centrifugation and 2PAP results in a robust, high purity PM preparation from <it>Drosophila</it>, something neither technique can achieve on its own. This novel preparation should lay the groundwork for the proteomic investigation of the PM in different genetic backgrounds in <it>Drosophila</it>. Our results also identify two key steps in this procedure: The optimization of membrane partitioning in the PEG/Dextran mixture, and careful choice of the correct lectin for the affinity purification step in light of variations in bulk membrane lipid composition and glycosylation patterns respectively. This points the way for further adaptations into other systems.</p

Comparative proteomic analysis of pathogenic and non-pathogenic strains from the swine pathogen Mycoplasma hyopneumoniae

<p>Abstract</p> <p>Background</p> <p><it>Mycoplasma hyopneumoniae </it>is a highly infectious swine pathogen and is the causative agent of enzootic pneumonia (EP). Following the previous report of a proteomic survey of the pathogenic 7448 strain of swine pathogen, <it>Mycoplasma hyopneumoniae</it>, we performed comparative protein profiling of three <it>M. hyopneumoniae </it>strains, namely the non-pathogenic J strain and the two pathogenic strains 7448 and 7422.</p> <p>Results</p> <p>In 2DE comparisons, we were able to identify differences in expression levels for 67 proteins, including the overexpression of some cytoadherence-related proteins only in the pathogenic strains. 2DE immunoblot analyses allowed the identification of differential proteolytic cleavage patterns of the P97 adhesin in the three strains. For more comprehensive protein profiling, an LC-MS/MS strategy was used. Overall, 35% of the <it>M. hyopneumoniae </it>genome coding capacity was covered. Partially overlapping profiles of identified proteins were observed in the strains with 81 proteins identified only in one strain and 54 proteins identified in two strains. Abundance analysis of proteins detected in more than one strain demonstrates the relative overexpression of 64 proteins, including the P97 adhesin in the pathogenic strains.</p> <p>Conclusions</p> <p>Our results indicate the physiological differences between the non-pathogenic strain, with its non-infective proliferate lifestyle, and the pathogenic strains, with its constitutive expression of adhesins, which would render the bacterium competent for adhesion and infection prior to host contact.</p

A Study of the Interactions Between Milk Proteins and Soy Proteins

This research investigates the protein interactions that occur when soy protein is added to milk and subjected to renneting or heating. Milk was fortified with 20% soy protein and enzymic coagulation studied at 35°C at various pH\u27s and CaCl2 levels. The first part deals with the interaction between milk and soy proteins during rennet-induced milk coagulation. The first goal was to determine how soy proteins affected milk coagulation. The effects of native versus heat-denatured soy proteins on rennet coagulation time and curd firmness were compared. lmmunogold labeling along with transmission electron microscopy was used to identify and localfze soy proteins in coagulated milk. Partitioning of ß-conglycinin and glycinin, the two main soy protein fractions, between cheese and whey was determined by electrophoresis. Soy proteins affected milk coagulation to the greatest extent at pH 6.6. Both heat-denatured and native soy proteins increased rennet coagulation time. Only heat-denatured soy proteins affected final curd firmness. Most of ß-conglycinin was lost in whey, whereas glycinin was retained in curd. Soy proteins existed in the curd as aggregates that were less electron dense than casein micelles. At pH 6.6, heat-denatured soy proteins were fibrous and adhered to the surfaces of casein micelle, preventing direct micelle-micelle contact. This would delay aggregation rate and decrease curd firmness by decreasing the number and strength of links between casein micelles. Native soy proteins did not bind to the casein micelles but rather were physically trapped within curd. Their effect of delaying aggregation is thought to be a function of their binding of calcium. Adding CaCl2 or lowering the pH to 6.3 or 6.0 helped restore coagulation properties. The second goal was to determine what heat-induced interaction occurs between milk and soy proteins, specifically between κ-casein and glycinin. Both κ-casein and glycinin are heat labile and form insoluble aggregates when heated. When glycinin and κ-casein were heated together, some acidic polypeptides of glycinin crosslinked with κ-casein via disulfide linkages. However, when disulfide linkage was prevented by adding ß-mercaptoethanol , non-covalent interactions between κ-casein and both acidic and basic polypeptides of glycinin occurred that prevented the heat precipitation of glycinin. This non-covalent interaction between glycinin polypeptides and κ-casein may explain why the heat-treated soy proteins became attached to the surfaces of casein micelles during rennet coagulation of milk

Role of plastid transglutaminase in LHCII polyamination and thylakoid electron and proton flow

Transglutaminases function as biological glues in animal cells, plant cells and microbes. In energy producing organelles such as chloroplasts the presence of transglutaminases was recently confirmed. Furthermore, a plastidial transglutaminase has been cloned from maize and the first plants overexpressing tgz are available (Nicotiana tabacum TGZ OE). Our hypothesis is that the overexpression of plastidal transglutaminase will alter photosynthesis via increased polyamination of the antenna of photosystem II. We have used standard analytical tools to separate the antenna from photosystem II in wild type and modified plants, 6 specific antibodies against LHCbs to confirm their presence and sensitive HPLC method to quantify the polyamination level of these proteins. We report that bound spermidine and spermine were significantly increased (∼80%) in overexpressors. Moreover, we used recent advances in in vivo probing to study simultaneously the proton and electron circuit of thylakoids. Under physiological conditions overexpressors show a 3-fold higher sensitivity of the antenna down regulation loop (qE) to the elicitor (luminal protons) which is estimated as the ΔpH component of thylakoidal proton motive force. In addition, photosystem (hyper-PSIIα) with an exceptionally high antenna (large absorption cross section), accumulate in transglutaminase over expressers doubling the rate constant of light energy utilization (Kα) and promoting thylakoid membrane stacking. Polyamination of antenna proteins is a previously unrecognized mechanism for the modulation of the size (antenna absorption cross section) and sensitivity of photosystem II to down regulation. Future research will reveal which peptides and which residues of the antenna are responsible for such effects

A dynamic framework based on local Zernike Moment and motion history image for facial expression recognition

A dynamic descriptor facilitates robust recognition of facial expressions in video sequences. The current two main approaches to the recognition are basic emotion recognition and recognition based on facial action coding system (FACS) action units. In this paper we focus on basic emotion recognition and propose a spatio-temporal feature based on local Zernike moment in the spatial domain using motion change frequency. We also design a dynamic feature comprising motion history image and entropy. To recognise a facial expression, a weighting strategy based on the latter feature and sub-division of the image frame is applied to the former to enhance the dynamic information of facial expression, and followed by the application of the classical support vector machine. Experiments on the CK+ and MMI datasets using leave-one-out cross validation scheme demonstrate that the integrated framework achieves a better performance than using individual descriptor separately. Compared with six state-of-arts methods, the proposed framework demonstrates a superior performance

User Interfaces and Difference Visualizations for Alternatives

Designers often create multiple iterations to evaluate alternatives. Todays computer-based tools do not support such easy exploration of a design space, despite the fact that such support has been advocated. This dissertation is centered on this. I begin by investigating the effectiveness of various forms of difference visualizations and support for merging changes within a system targeted at diagrams with node and edge attributes. I evaluated the benefits of the introduced difference visualization techniques in two user studies. I found that the basic side-by-side juxtaposition visualization was not effective and also not well received. For comparing diagrams with matching node positions, participants preferred the side-by-side option with a difference layer. For diagrams with non-matching positions animation was beneficial, but the combination with a difference layer was preferred. Thus, the difference layer technique was useful and a good complement to animation. I continue by investigating if explicit support for design alternatives better supports exploration and creativity in a generative design system. To investigate the new techniques to better support exploration, I built a new system that supports parallel exploration of alternative designs and generation of new structural combinations. I investigate the usefulness of my prototype in two user studies and interviews. The results and feedback suggest and confirm that supporting design alternatives explicitly enables designers to work more creatively. Generative models are often represented as DAGs (directed acyclic graphs) in a dataflow programming environment. Existing approaches to compare such DAGs do not generalize to multiple alternatives. Informed by and building on the first part of my dissertation, I introduce a novel user interface that enables visual differencing and editing alternative graphsspecifically more than two alternatives simultaneously, something that has not been presented before. I also explore multi-monitor support to demonstrate that the difference visualization technique scales well to up to 18 alternatives. The novel jamming space feature makes organizing alternatives on a 23 monitor system easier. To investigate the usability of the new difference visualization method I conducted an exploratory interview with three expert designers. The received comments confirmed that it meets their design goals

The physiological importance of photosynthetic ferredoxin NADP+ oxidoreductase (FNR) isoforms in wheat

Ferredoxin NADP+ oxidoreductase (FNR) enzymes catalyse electron transfer between ferredoxin and NADPH. In plants, a photosynthetic FNR (pFNR) transfers electrons from reduced ferredoxin to NADPH for the final step of linear electron flow, providing reductant for carbon fixation. pFNR is also thought to play important roles in two different mechanisms of cyclic electron flow around photosystem I; and photosynthetic reductant is itself partitioned between competing linear, cyclic, and alternative electron flow pathways. Four pFNR protein isoforms in wheat that display distinct reaction kinetics with leaf-type ferredoxin have previously been identified. It has been suggested that these isoforms may be crucial to the regulation of reductant partition between carbon fixation and other metabolic pathways. Here the 12 cm primary wheat leaf has been used to show that the alternative N-terminal pFNRI and pFNRII protein isoforms have statistically significant differences in response to the physiological parameters of chloroplast maturity, nitrogen regime, and oxidative stress. More specifically, the results obtained suggest that the alternative N-terminal forms of pFNRI have distinct roles in the partitioning of photosynthetic reductant. The role of alternative N-terminal processing of pFNRI is also discussed in terms of its importance for thylakoid targeting. The results suggest that the four pFNR protein isoforms are each present in the chloroplast in phosphorylated and non-phosphorylated states. pFNR isoforms vary in putative phosphorylation responses to physiological parameters, but the physiological significance requires further investigation

Assessing and Interpreting the Within-Body Biogeography of Human Microbiome Diversity

A human body hosts a relatively independent microbiome including five major regional biomes (i.e., airway, oral, gut, skin, and urogenital). Each of them may possess different regional characteristics with important implications to our health and diseases (i.e., so-termed microbiome associated diseases). Nevertheless, these regional microbiomes are connected with each other through diffusions and migrations. Here, we investigate the within-body (intra-individual) distribution feature of microbiome diversity via diversity area relationship (DAR) modeling, which, to the best of our knowledge, has not been systematically studied previously. We utilized the Hill numbers for measuring alpha and beta-diversities and built 1,200 within-body DAR models with to date the most comprehensive human microbiome datasets of 18 sites from the human microbiome project (HMP) cohort. We established the intra-DAR profile (z-q pattern: the diversity scaling parameter z of the power law (PL) at diversity order q = 0–3), intra-PDO (pair-wise diversity overlap) profile (g-q), and intra-MAD (maximal accrual diversity) profile (Dmax-q) for the within-body biogeography of the human microbiome. These profiles constitute the “maps” of the within-body biogeography, and offer important insights on the within-body distribution of the human microbiome. Furthermore, we investigated the heterogeneity among individuals in their biogeography parameters and found that there is not an “average Joe” that can represent majority of individuals in a cohort or population. For example, we found that most individuals in the HMP cohort have relatively lower maximal accrual diversity (MAD) or in the “long tail” of the so-termed power law distribution. In the meantime, there are a small number of individuals in the cohort who possess disproportionally higher MAD values. These findings may have important implications for personalized medicine of the human microbiome associated diseases in practice, besides their theoretical significance in microbiome research such as establishing the baseline for the conservation of human microbiome