Lipid membranes modulate the structure of islet amyloid polypeptide

The 37-residue islet amyloid polypeptide (IAPP) is thought to play an important role in the pathogenesis of type II diabetes. Despite a growing body of evidence implicating membrane interaction in IAPP toxicity, the membrane-bound form has not yet been well characterized. Here we used circular dichroism (CD) and fluorescence spectroscopy to investigate the molecular details of the interaction of IAPP with lipid membranes of varying composition. In the presence of membranes containing negatively charged phosphatidylserine (PS), we observed significant acceleration in the formation of IAPP aggregates. This acceleration is strongly modulated by the PS concentration and ionic strength, and is also observed at physiologically relevant PS concentrations. CD spectra of IAPP obtained immediately after the addition of membranes containing PS revealed features characteristic of an alpha-helical conformation approximately approximately 15-19 residues in length. After a longer incubation with membranes, IAPP gave rise to CD spectra characteristic of a beta-sheet conformation. Taken together, our CD and fluorescence data indicate that conditions that promote weakly stable alpha-helical conformations may promote IAPP aggregation. The potential roles of IAPP-membrane interaction and the novel membrane-bound alpha-helical conformation in IAPP aggregation are discussed.     

A new ecdysteroid, 2-deoxy-5beta,20-dihydroxyecdysone from the fruits of Diploclisia glaucescens

Chemical investigation of ethyl acetate extract of the fruits of Diploclisia glaucescens of the family Menispermaceae furnished a new ecdysteroid 2-deoxy-5beta,20-dihydroxyecdysone, together with 20-hydroxyecdysone, 3-deoxy-1beta,20-dihydroxyecdysone, 2-deoxy-20-hydroxyecdysone, 24-ethyl-20-hydroxyecdysone (makisterone C). Latter two ecdysteroids are reported first time from the family Menispermaceae.     

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

Förster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo^1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest^3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells⁵, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium⁶, cAMP^7-8, inositol phosphates⁹ and cGMP^10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a β-adrenergic receptor agonist and blocker.     

A quantitative analysis of microalgal lipids for optimization of biodiesel and omega‐3 production

The lipid characteristics of microalgae are known to differ between species and change with growth conditions. This work provides a methodology for lipid characterization that enables selection of the optimal strain, cultivation conditions, and processing pathway for commercial biodiesel production from microalgae. Two different microalgal species, Nannochloropsis sp. and Chlorella sp., were cultivated under both nitrogen replete and nitrogen depleted conditions. Lipids were extracted and fractionated into three major classes and quantified gravimetrically. The fatty acid profile of each fraction was analyzed using GC–MS. The resulting quantitative lipid data for each of the cultures is discussed in the context of biodiesel and omega‐3 production. This approach illustrates how the growth conditions greatly affect the distribution of fatty acid present in the major lipid classes and therefore the suitability of the lipid extracts for biodiesel and other secondary products. Biotechnol. Bioeng. 2013; 110: 2096–2104. © 2013 Wiley Periodicals, Inc.     

MPtopo: A database of membrane protein topology

The reliability of the transmembrane (TM) sequence assignments for membrane proteins (MPs) in standard sequence databases is uncertain because the vast majority are based on hydropathy plots. A database of MPs with dependable assignments is necessary for developing new computational tools for the prediction of MP structure. We have therefore created MPtopo, a database of MPs whose topologies have been verified experimentally by means of crystallography, gene fusion, and other methods. Tests using MPtopo strongly validated four existing MP topology-prediction algorithms. MPtopo is freely available over the internet and can be queried by means of an SQL-based search engine.     

Comparison of naturally acquired antibody responses against the C-terminal processing products of Plasmodium vivax Merozoite Surface Protein-1 under low transmission and unstable malaria conditions in Sri Lanka

We report here, for the first time, a comparison of naturally acquired antibody responses to the 42 and 19 kDa C-terminal processing products of Plasmodium vivax Merozoite Surface Protein-1 assayed by ELISA using p42 and p19 baculovirus-derived recombinant proteins, respectively. Test populations comprised patients with microscopy confirmed acute P. vivax infections from two regions endemic for vivax malaria where low transmission and unstable malaria conditions prevail, and a non-endemic urban area, in Sri Lanka. The antibody prevalence to the two proteins, both at the individual and population levels, tend to respond more to p42 than to p19 in all test areas, where >14% of individuals preferentially recognized p42, compared with <2% for p19. In patients with no previous exposure to malaria, 21% preferentially recognized p42, whereas none exclusively recognized p19. A significantly lower prevalence of anti-p19 IgM, but not anti-p42 IgM, was observed among residents from endemic areas compared with their non-endemic counterparts. Individuals from both endemic areas produced significantly less anti-p19 IgM compared with anti-p42 IgM. IgG1 was the predominant IgG isotype for both antigens in all individuals. With increasing exposure to malaria in both endemic areas, anti-p19 antibody responses were dominated by the functionally important IgG1 and IgG3 isotypes, with a concurrent reduction in IgM that was lacking in the non-endemic residents. This antibody switch was also reflected for PvAMA-1 as we previously reported with the identical battery of sera. In contrast, the antibody switch for p42 was restricted to endemic residents with more extensive exposure. These results suggest that an IgM-dominated antibody response against the p42 polymorphic region in endemic residents may interfere with the development of an IgG-dominated "protective" isotype shift to p19, that may complicate vaccine development.     

Compensatory payments and vasectomy acceptance in urban Sri Lanka

The effects of different levels of compensatory payment for vasectomy on sterilization acceptance were examined in 496 vasectomized men in urban Sri Lanka. The results indicate that compensatory payments significantly enhanced the participation of economically poor men in vasectomy programs, especially those who had already achieved a large family size. The proportion of poor acceptors (those with a monthly income of Rs 1000 or less) increased with higher levels of payment; the acceptor's level of education was negatively correlated with the compensation amount, and the mean age of the youngest child was higher among those who received higher payments. 60% of vasectomy acceptors reported using contraception immediately before the vasectomy, suggesting a high level of motivation not to have another child. Respondents cited high effectiveness, no extra expense, and no side effects for their wives as the main reasons for selecting vasectomy over other means of contraception, regardless of the amount of payment received. Only 5% said cash payment was an important reason for choosing vasectomy, and this response did not vary significantly by level of payment. There was no influence of payment level on postoperative complications or satisfaction. While compensatory payments significantly enhanced the participation of poor men in vasectomy programs, they were not effective in attracting poorer men with few children or those whose last child was relatively young. Overall, this study's findings suggest that the decision to provide compensatory payments and how much to provide should be based on economic and political factors, not on the grounds that higher compensatory payments have led to the recruitment of ineligible men or that the promotion of vasectomy has been at the expense of a loss in the quality of services provided.     

Role of the amino latch of staphylococcal alpha-hemolysin in pore formation: a co-operative interaction between the N terminus and position 217

Staphylococcal alpha-hemolysin (alphaHL) is a beta barrel pore-forming toxin that is secreted by the bacterium as a water-soluble monomeric protein. Upon binding to susceptible cells, alphaHL assembles via an inactive prepore to form a water-filled homoheptameric transmembrane pore. The N terminus of alphaHL, which in the crystal structure of the fully assembled pore forms a latch between adjacent subunits, has been thought to play a vital role in the prepore to pore conversion. For example, the deletion of two N-terminal residues produced a completely inactive protein that was arrested in assembly at the prepore stage. In the present study, we have re-examined assembly with a comprehensive set of truncation mutants. Surprisingly, we found that after truncation of up to 17 amino acids, the ability of alphaHL to form functional pores was diminished, but still substantial. We then discovered that the mutation Ser(217) --> Asn, which was present in our original set of truncations but not in the new ones, promotes complete inactivation upon truncation of the N terminus. Therefore, the N terminus of alphaHL cannot be critical for the prepore to pore transformation as previously thought. Residue 217 is involved in the assembly process and must interact indirectly with the distant N terminus during the last step in pore formation. In addition, we provide evidence that an intact N terminus prevents the premature oligomerization of alphaHL monomers in solution.     

Cell electrospinning: a unique biotechnique for encapsulating living organisms for generating active biological microthreads/scaffolds

Jet-based technologies are increasingly being explored as potential high-throughput and high-resolution methods for the manipulation of biological materials. Previously shown to be of use in generating scaffolds from biocompatible materials, we were interested to explore the possibility of using electrospinning technology for the generation of scaffolds comprised of living cells. For this, it was necessary to identify appropriate parameters under which viable threads containing living cells could be produced. Here, we describe a method of electrospinning that can be used to deposit active biological threads and scaffolds. This has been achieved by use of a coaxial needle arrangement where a concentrated living biosuspension flows through the inner needle and a medical-grade poly(dimethylsiloxane) (PDMS) medium with high viscosity (12,500 mPa s) and low electrical conductivity (10-15 S m-1) flows through the outer needle. Using this technique, we have identified the operational conditions under which the finest cell-bearing composite microthreads are formed. Collected cells that have been cultured, postelectrospinning, have been viable and show no evidence of having incurred any cellular damage during the bionanofabrication process. This study demonstrates the feasibility of using coaxial electrospinning technology for biological and biomedical applications requiring the deposition of living cells as composite microthreads for forming active biological scaffolds.