Theory and application of fluorescence homotransfer to melittin oligomerization.

Fluorescence homotransfer (electronic energy transfer between identical fluorophores) has the potential to quantitate the number of subunits in membrane protein oligomers. Homotransfer strongly depolarizes fluorescence emission as a result of intermolecular excitation energy exchange between an initially excited, oriented molecule and a randomly oriented neighbor. We have theoretically treated fluorescein labeled subunits in an oligomer as a cluster of molecules that can exchange excitation energy back and forth among the subunits within that group. We find that the larger the number of subunits, the more depolarized is the emission. The general equations to calculate the expected anisotropy for complexes composed of varying numbers of labeled subunits are presented. Self-quenching of fluorophores, orientation, and changes in lifetime are also discussed and/or considered. To test this theory, we have specifically labeled melittin on its N-terminal with fluorescein and monitored its monomer to tetramer equilibrium both in solution and in lipid bilayers. The calculated anisotropies are close to the experimental values when non-fluorescent fluorescein dimers are taken into account. Our results show that homotransfer may be a promising method to study membrane-protein oligomerization.     

GT-Miner: a graph-theoretic data miner, viewer, and model processor

Inexpensive computational power combined with high-throughput experimental platforms has created a wealth of biological information requiring analytical tools and techniques for interpretation. Graph-theoretic concepts and tools have provided an important foundation for information visualization, integration, and analysis of datasets, but they have often been relegated to background analysis tasks. GT-Miner is designed for visual data analysis and mining operations, interacts with other software, including databases, and works with diverse data types. It facilitates a discovery-oriented approach to data mining wherein exploration of alterations of the data and variations of the visualization is encouraged. The user is presented with a basic iterative process, consisting of loading, visualizing, transforming, and then storing the resultant information. Complex analyses are built-up through repeated iterations and user interactions. The iterative process is optimized by automatic layout following transformations and by maintaining a current selection set of interest for elements modified by the transformations. Multiple visualizations are supported including hierarchical, spring, and force-directed self-organizing layouts. Graphs can be transformed with an extensible set of algorithms or manually with an integral visual editor. GT-Miner is intended to allow easier access to visual data mining for the non-expert.     

Effects of Tithonia diversifolia (Hemsl.) A. Gray Extract on Adipocyte Differentiation of Human Mesenchymal Stem Cells

Tithonia diversifolia (Hemsl.) A. Gray (Asteraceae) is widely used in traditional medicine. There is increasing interest on the in vivo protective effects of natural compounds contained in plants against oxidative damage caused from reactive oxygen species. In the present study the total phenolic and flavonoid contents of aqueous, methanol and dichloromethane extracts of leaves of Tithonia diversifolia (Hemsl.) A. Gray were determined; furthermore, free radical scavenging capacity of each extract and the ability of these extracts to inhibit in vitro plasma lipid peroxidation were also evaluated. Since oxidative stress may be involved in trasformation of pre-adipocytes into adipocytes, to test the hypothesis that Tithonia extract may also affect adipocyte differentiation, human mesenchymal stem cell cultures were treated with Tithonia diversifolia aqueous extract and cell viability, free radical levels, Oil-Red O staining and western bolt analysis for heme oxygenase and 5''-adenosine monophoshate-activated protein kinase were carried out. Results obtained in the present study provide evidence that Tithonia diversifolia (Hemsl.) A. Gray exhibits interesting health promoting properties, resulting both from its free radical scavenger capacity and also by induction of protective cellular systems involved in cellular stress defenses and in adipogenesis of mesenchymal cells.     

The evolutionary history of Cytochrome P450 genes in four filamentous Ascomycetes

Background  

The Cytochrome P450 system is important in fungal evolution for adapting to novel ecological niches. To elucidate the evolutionary process of cytochrome P450 genes in fungi with different life styles, we studied the patterns of gene gains and losses in the genomes of four filamentous Ascomycetes, including two saprotrophs (Aspergillus nidulans (AN) and Neurospora crassa (NC)) and two plant pathogens (Fusarium graminearum (FG) and Magnaporthe grisea (MG)).     

Osteogenic properties of a short BMP‐2 chimera peptide

Bone morphogenetic proteins (BMPs) play a key role in bone and cartilage formation. For these properties, BMPs are employed in the field of tissue engineering to induce bone regeneration in damaged tissues. To overcome drawbacks due to the use of entire proteins, synthetic peptides derived from their parent BMPs have come out as promising molecules for biomaterial design. On the structural ground of the experimental BMP‐2 receptor complexes reported in the literature, we designed three peptides, reproducing the BMP‐2 region responsible for the binding to the type II receptor, ActRIIB. These peptides were characterized by NMR, and the structural features of the peptide–receptor binding interface were highlighted by docking experiments. Peptide–receptor binding affinities were analyzed by means of ELISA and surface plasmon resonance techniques. Furthermore, cellular assays were performed to assess their osteoinductive properties. A chimera peptide, obtained by combining the sequence portions 73–92 and 30–34 of BMP‐2, shows the best affinity for ActRIIB in the series and represents a good starting point for the design of new compounds able to reproduce osteogenic properties of the parent BMP‐2. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Evidence for a Second, High Affinity G�¦� Binding Site on G��i1(GDP) Subunits

It is well known that Gα_i1(GDP) binds strongly to Gβγ subunits to form the Gα_i1(GDP)-Gβγ heterotrimer, and that activation to Gα_i1(GTP) results in conformational changes that reduces its affinity for Gβγ subunits. Previous studies of G protein subunit interactions have used stoichiometric amounts of the proteins. Here, we have found that Gα_i1(GDP) can bind a second Gβγ subunit with an affinity only 10-fold weaker than the primary site and close to the affinity between activated Gα_i1 and Gβγ subunits. Also, we find that phospholipase Cβ2, an effector of Gβγ, does not compete with the second binding site implying that effectors can be bound to the Gα_i1(GDP)-(Gβγ)₂ complex. Biophysical measurements and molecular docking studies suggest that this second site is distant from the primary one. A synthetic peptide having a sequence identical to the putative second binding site on Gα_i1 competes with binding of the second Gβγ subunit. Injection of this peptide into cultured cells expressing eYFP-Gα_i1(GDP) and eCFP-Gβγ reduces the overall association of the subunits suggesting this site is operative in cells. We propose that this second binding site serves to promote and stabilize G protein subunit interactions in the presence of competing cellular proteins.The plasma membranes of cells are organized as a series of protein-rich and lipid-rich domains (1–3). Many of the protein-rich domains, in particular those organized by caveolin proteins, are thought to be complexes of functionally related proteins that transduce extracellular signals (2). There is increasing evidence that heterotrimeric G proteins exist in pre-formed membrane complexes with their receptors and their intracellular effectors (4–8).The G protein signaling system is initiated when an extracellular agonist binds to its specific G protein-coupled receptor (for review see Refs. 9–12). The ligand-bound receptor will then catalyze the exchange of GTP for GDP on the Gα subunit in the G protein heterotrimer. In the basal state, Gα(GDP) binds strongly to Gβγ, but in the GTP-bound state this affinity is reduced, allowing Gα(GTP) and Gβγ subunits to individually bind to a host of specific intracellular enzymes and change their catalytic activity.Although the interactions between G protein subunits have been studied extensively in vitro, their behavior in cells may differ. For example, in pure or semi-pure systems, activation of Gα(GDP) sufficiently weakens its affinity for Gβγ resulting in dissociation (13). However, in cells separation of the heterotrimer is observed under some circumstances, but not others (7, 14–17). The reason for these differences in behavior is not clear. There are four families of Gα subunits that each contain several members, and, additionally, there are many subtypes of Gβγ subunits (18). It is possible that differences in dissociation behavior reflect differences in affinity between G protein subunit subtypes (19), the presence of various protein partners, and/or differences in post-synthetic modifications of the subunits (20).The mechanism that allows activated G proteins to remain bound is not apparent from the crystal structure (21, 22). If G protein subunits do not dissociate in cells, then their interaction must change in such a manner as to expose the effector interaction site(s). We have found that phospholipase Cβ1 (PLCβ1),⁴ an important effector of Gα_q (23), is bound to Gα_q prior to activation and throughout the activation cycle (6) implying that Gα_q(GDP) interacts with PLCβ1 in a non-functional manner.We have evidence that signaling complexes are stabilized by a series of secondary interactions. Using purified proteins and model membranes, we have found that membranes of the Gα_q-Gβγ/PLCβ1/RGS4 signaling system have secondary, weaker binding sites to members of this signaling system in addition to their high affinity site(s) to their functional partner(s). We speculate that secondary contacts allow for self-scaffolding of signaling proteins. To understand the nature of these secondary contacts, we have studied the ability of the Gα_i1(GDP)-Gβγ heterotrimer to remain complexed through the activation cycle (24). Here, we present evidence that Gα_i1(GDP) has two distinct Gβγ binding sites that only differ in affinity by an order of magnitude and may allow for continued association between the subunits upon activation. We also find that this site plays an important role in stabilizing G protein associations in cells and provides a mechanism of self-scaffolding.     

High Differentiation among Eight Villages in a Secluded Area of Sardinia Revealed by Genome-Wide High Density SNPs Analysis

To better design association studies for complex traits in isolated populations it''s important to understand how history and isolation moulded the genetic features of different communities. Population isolates should not “a priori” be considered homogeneous, even if the communities are not distant and part of a small region. We studied a particular area of Sardinia called Ogliastra, characterized by the presence of several distinct villages that display different history, immigration events and population size. Cultural and geographic isolation characterized the history of these communities. We determined LD parameters in 8 villages and defined population structure through high density SNPs (about 360 K) on 360 unrelated people (45 selected samples from each village). These isolates showed differences in LD values and LD map length. Five of these villages show high LD values probably due to their reduced population size and extreme isolation. High genetic differentiation among villages was detected. Moreover population structure analysis revealed a high correlation between genetic and geographic distances. Our study indicates that history, geography and biodemography have influenced the genetic features of Ogliastra communities producing differences in LD and population structure. All these data demonstrate that we can consider each village an isolate with specific characteristics. We suggest that, in order to optimize the study design of complex traits, a thorough characterization of genetic features is useful to identify the presence of sub-populations and stratification within genetic isolates.     

Synthesis of pyrrolo[3,2-h]quinolinones with good photochemotherapeutic activity and no DNA damage

In the search for new photochemotherapeutic agents, a series of derivatives of the ring system pyrrolo[3,2-h]quinoline—bioisosters of the angular furocoumarin angelicin—were synthesized through a four-step synthetic approach, in reasonable overall yields. Eight of the synthesized derivatives showed a remarkable phototoxicity against a panel of four human tumor cell lines and a great dose UV-A dependence, reaching IC₅₀ values at submicromolar level. The mode of cellular death photoinduced by pyrrolo[3,2-h]quinolines was evaluated through a series of flow cytometric analysis and other tests were performed to clarify their mechanism of action.     

Multiorgan Detection and Characterization of Protease-Resistant Prion Protein in a Case of Variant CJD Examined in the United States

Background

Variant Creutzfeldt–Jakob disease (vCJD) is a prion disease thought to be acquired by the consumption of prion-contaminated beef products. To date, over 200 cases have been identified around the world, but mainly in the United Kingdom. Three cases have been identified in the United States; however, these subjects were likely exposed to prion infection elsewhere. Here we report on the first of these subjects.

Methodology/Principal Findings

Neuropathological and genetic examinations were carried out using standard procedures. We assessed the presence and characteristics of protease-resistant prion protein (PrP^res) in brain and 23 other organs and tissues using immunoblots performed directly on total homogenate or following sodium phosphotungstate precipitation to increase PrP^res detectability. The brain showed a lack of typical spongiform degeneration and had large plaques, likely stemming from the extensive neuronal loss caused by the long duration (32 months) of the disease. The PrP^res found in the brain had the typical characteristics of the PrP^res present in vCJD. In addition to the brain and other organs known to be prion positive in vCJD, such as the lymphoreticular system, pituitary and adrenal glands, and gastrointestinal tract, PrP^res was also detected for the first time in the dura mater, liver, pancreas, kidney, ovary, uterus, and skin.

Conclusions/Significance

Our results indicate that the number of organs affected in vCJD is greater than previously realized and further underscore the risk of iatrogenic transmission in vCJD.