Supported bilayer lipid membranes modified with a phosphate ionophore

This article reports the electrical responses of a phosphate ionophore, the cyclic polyamine 3-decyl-1,5,8-triazacyclodecane-2,4-dione (N₃-cyclic amine) incorporated into metal supported bilayer lipid membranes (s-BLM). Teflon coated silver wire was used as a support. In a potentiometric mode, the ionophore had a response that was linearly related to the logarithm of HPO₄²⁻ concentration and was also dependant on pH. Selectivity coefficients for other anions compared to HPO₄²⁻ ions, determined by the separate solution method, fell within the range 1.73 × 10⁻⁴ to 6.38 × 10⁻².     

Solution Structure of Factor I-like Modules from Complement C7 Reveals a Pair of Follistatin Domains in Compact Pseudosymmetric Arrangement

Factor I-like modules (FIMs) of complement proteins C6, C7, and factor I participate in protein-protein interactions critical to the progress of a complement-mediated immune response to infections and other trauma. For instance, the carboxyl-terminal FIM pair of C7 (C7-FIMs) binds to the C345C domain of C5 and its activated product, C5b, during self-assembly of the cytolytic membrane-attack complex. FIMs share sequence similarity with follistatin domains (FDs) of known three-dimensional structure, suggesting that FIM structures could be reliably modeled. However, conflicting disulfide maps, inconsistent orientations of subdomains within FDs, and the presence of binding partners in all FD structures led us to determine the three-dimensional structure of C7-FIMs by NMR spectroscopy. The solution structure reveals that each FIM within C7 contains a small amino-terminal FOLN subdomain connected to a larger carboxyl-terminal KAZAL domain. The open arrangement of the subdomains within FIMs resembles that of first FDs within structures of tandem FDs but differs from the more compact subdomain arrangement of second or third FDs. Unexpectedly, the two C7-FIMs pack closely together with an approximate 2-fold rotational symmetry that is rarely seen in module pairs and has not been observed in FD-containing proteins. Interfaces between subdomains and between modules include numerous hydrophobic and electrostatic contributions, suggesting that this is a physiologically relevant conformation that persists in the context of the parent protein. Similar interfaces were predicted in a homology-based model of the C6-FIM pair. The C7-FIM structures also facilitated construction of a model of the single FIM of factor I.The membrane attack complex (MAC)² is the terminal product of the complement cascade and is therefore a fundamental component of mammalian innate immunity. The formation of this multi-protein complex is triggered by proteolytic cleavage of complement component C5. This is followed swiftly by a remarkable, although little understood, self-assembly process involving multiple sequential protein-protein recognition events. MAC assembly culminates in the formation of a pore traversing the targeted cell membrane (1). Accumulation of multiple MACs in a membrane triggers cell-dependent responses and may result in cell lysis (2). The key to progress in understanding MAC formation will be three-dimensional structural information for each of its component proteins, namely C5b, C6, C7, C8, and C9.Classical, alternative, and lectin pathways of complement activation converge at a step in which C5 is cleaved to release activated C5b. Immediately following C5b formation, C6 and C7 bind sequentially; the C5b6 complex is soluble and relatively stable (3), but soluble C5b67 has a brief half-life and is proposed to attach rapidly to target membrane surfaces (4, 5). Subsequently, C8 binds to the nascent complex, inserting into the target membrane and causing disruptive rearrangements of the lipid bilayer. Finally the mature MAC, C5b6789_n, forms by recruitment of between 10 and 16 copies of C9 that insert in the membrane to form the pore. Notably, once C5b is generated, MAC assembly requires no additional enzymatic triggers; this implies that individual components encompass highly specific, complementary binding sites that become exposed during MAC formation.Complement proteins C6, C7, C8 (α and β subunits), and C9 comprise the “MAC family” (Fig. 1a) (6). Family members share, in addition to a large central membrane attack complex perforin domain (7–9), several tandemly arranged, cysteine-rich modules of less than 80 amino acid residues each. These smaller modules include thrombospondin type I (10), low density lipoprotein receptor class A (11) and modules similar in sequence to epidermal growth factor (Fig. 1a). C6 and C7 each contain an additional four modules at their carboxyl termini: two ∼60-residue complement control protein modules (12, 13), followed by two cysteine-rich modules composed of ∼75 residues each; these are the factor I-like modules (FIMs) (also known as factor I membrane attack complex domains (14, 15)), so named because of their apparent relatedness to an amino-terminal domain of complement factor I (fI) (Fig. 1b).Open in a separate windowFIGURE 1.Modular composition of the proteins of the membrane attack complex (MAC). a, the MAC family of proteins aligned, domain-wise, with C6. b, the domain structure of fI. The heavy chain contains the amino-terminal domains and the light chain comprises a serine protease domain. An intramolecular disulfide bond between light and heavy chain (Cys³⁰⁹–Cys⁴³⁵) and a proposed interdomain disulfide between the amino-terminal region and first low density lipoprotein domain (Cys¹⁵–Cys²³⁷) are shown as diagonal lines. The domains were defined using the SMART data base (16, 17). TSP, thrombospondin type 1; LDL, low density lipoprotein receptor type A; MACPF, membrane attack complex perforin domain; EGF, epidermal growth factor; CCP, complement control protein; FIM, factor I-like module; CD5, CD5-like; SP, serine protease domain.Latent C5 was shown, in vitro, to bind reversibly to both C6 and C7 prior to activation. These interactions are distinct from and precede irreversible binding of C6 and subsequently C7 to C5b (18). It is hypothesized that the C56 and C57 preactivation complexes ensure that C6 and C7 are maintained proximal to C5 in the plasma. This may be significant because activated C5b is labile (19, 20), hence swift assembly of C5b67 is advantageous. Within this preactivation complex, critical interactions occur between the carboxyl-terminal C345C domain of C5, C5-C345C (21), and the carboxyl-terminal FIM pair of both C6 and C7 (22, 23). The involvement of these domains in MAC formation was demonstrated using recombinant proteins, where either C7-FIMs or C5-C345C inhibited the binding of C7 to C5b6 and inhibited complement-mediated erythrocyte lysis (23). The FIMs of C6, however, although shown to promote MAC assembly, do not appear to be essential for MAC formation (22). C7-FIMs have a stronger affinity than C6-FIMs for C5-C345C, suggesting that C7-FIMs may displace C6-FIMs during MAC assembly (23). Thus, interactions between C5- C345C and FIMs are key to the early assembly of MAC, and their structural basis is an important target of investigations.The structure of the C5-C345C domain is well established (24, 25); however, there has been no three-dimensional structural information available for any of the FIMs or for any other domains within C6 or C7. The closely related FIM within fI has been postulated to resemble a follistatin domain (26). Intriguingly, however, disulfide mapping of human C6 isolated from plasma appeared to exclude that possibility (27). The three-dimensional arrangement of the neighboring FIMs, and the extent of interactions between them, has also been a mystery.We previously described a protein construct comprising the carboxyl-terminal pair of FIMs from human C7 (18), which folds homogeneously and binds to C5 in surface plasmon resonance assays. Here we report the solution structure of this consecutive pair of FIMs. This new structure reveals that, despite previous evidence to the contrary, each FIM adopts a follistatin-like fold, and the two FIMs are intimately associated to form a homodimer-like, pseudosymmetrical carboxyl terminus of C7. This work, therefore, serendipitously provides the first published structure of a follistatin-domain pair in the absence of ligand and suggests that conformational changes within FIM pairs accompany ligand binding. Novel structures of the FIMs from both C6 and fI have been modeled based upon our NMR-derived solution structure of the C7-FIMs.     

Inhibitory activity of prostaglandin E2 production by the synthetic 2′-hydroxychalcone analogues: Synthesis and SAR study

A series of 2′-hydroxychalcones has been synthesized and screened for their in vitro inhibitory activities of cyclooxygenase-2 catalyzed prostaglandin production from lipopolysaccharide-treated RAW 264.7 cells. Structure–activity relationship study suggested that inhibitory activity against prostaglandin E₂ production was governed to a greater extent by the substituent on B ring of the chalcone, and most of the active compounds have at least two methoxy or benzyloxy groups on B ring. The relationship between chalcone structures and their PGE₂ inhibitory activities was also interpreted by docking study on cyclooxygenase-2.     

Proteomic analysis of responses to osmotic stress in laboratory and sake-brewing strains of Saccharomyces cerevisiae

The difference in responses to osmotic stress between the laboratory and sake-brewing strains of Saccharomyces cerevisiae at the translational level was compared by two-dimensional polyacrylamide gel electrophoresis. Proteins, whose production was significantly changed by the osmotic stress, were identified by peptide mass fingerprinting. In the laboratory strain, translation of Hor2p, the protein responsible for glycerol biosynthesis, and Ald6p, related to acetate biosynthesis, was induced under high osmotic pressure conditions. In addition, production of proteins related to translation and stress response was also changed under this condition. On the other hand, in the sake-brewing strain, translation of Hor2p, Hsp26p, and some stress-related proteins was upregulated. The change in the production of enzymes related to glycolysis and ethanol formation was small; however, the production of enzymes related to glycerol formation increased in both strains. These results suggest that enhancement of glycerol formation due to enhancement of the translation of proteins, such as Hor2p, is required for growth of S. cerevisiae under high osmotic pressure condition. This is the first report on the analysis of responses of a sake-brewing strain to high osmotic pressure stress based on proteomics.     

Dengue Dynamics in Binh Thuan Province,Southern Vietnam: Periodicity,Synchronicity and Climate Variability

Background

Dengue is a major global public health problem with increasing incidence and geographic spread. The epidemiology is complex with long inter-epidemic intervals and endemic with seasonal fluctuations. This study was initiated to investigate dengue transmission dynamics in Binh Thuan province, southern Vietnam.

Methodology

Wavelet analyses were performed on time series of monthly notified dengue cases from January 1994 to June 2009 (i) to detect and quantify dengue periodicity, (ii) to describe synchrony patterns in both time and space, (iii) to investigate the spatio-temporal waves and (iv) to associate the relationship between dengue incidence and El Niño-Southern Oscillation (ENSO) indices in Binh Thuan province, southern Vietnam.

Principal Findings

We demonstrate a continuous annual mode of oscillation and a multi-annual cycle of around 2–3-years was solely observed from 1996–2001. Synchrony in time and between districts was detected for both the annual and 2–3-year cycle. Phase differences used to describe the spatio-temporal patterns suggested that the seasonal wave of infection was either synchronous among all districts or moving away from Phan Thiet district. The 2–3-year periodic wave was moving towards, rather than away from Phan Thiet district. A strong non-stationary association between ENSO indices and climate variables with dengue incidence in the 2–3-year periodic band was found.

Conclusions

A multi-annual mode of oscillation was observed and these 2–3-year waves of infection probably started outside Binh Thuan province. Associations with climatic variables were observed with dengue incidence. Here, we have provided insight in dengue population transmission dynamics over the past 14.5 years. Further studies on an extensive time series dataset are needed to test the hypothesis that epidemics emanate from larger cities in southern Vietnam.     

Identification and characterization of Cu(2)O- and ZnO-binding polypeptides by Escherichia coli cell surface display: toward an understanding of metal oxide binding

We have used the FliTrx cell surface display system to identify disulfide-constrained dodecapeptides binding to the semiconducting metal oxides Cu(2)O and ZnO. Sequence analysis of the inserts revealed that the two populations exhibit similar, yet subtly different patterns of amino acid usage. Both sets of binders were enriched in arginine, tryptophan, and glycine with a higher degree of positional preference in the case of Cu(2)O binders. Tyrosine, proline, and serine were underrepresented in both populations. Peptides binding electrodeposited Cu(2)O or ZnO with high avidity could be subdivided into two classes based on pI and hydrophilicity. In the hydrophilic and positively charged Class I binders, the Arg-X-X-Arg tetrapeptide appears to be implicated in metal oxide binding, whereas Arg-Arg and Arg-Lys pairs allow for discrimination between Cu(2)O and ZnO. Molecular dynamics simulations of the disulfide-constrained peptides suggest that the aforementioned motifs are important to properly orient two basic residues that are likely to contact the metal oxides. The implications of our results in understanding the rules governing the interaction between peptides and inorganic compounds and in their use for the design of hybrid nanoarchitectures are discussed.     

Long-size isoelectricfocusing (IEF) in flexible silicone tubes: an application to semi-preparative fractionation of proteins

Soluble proteins extracted from porcine brains were subjected to a series of optional fractionation steps on various chromatographic media including a novel device for semi-preparative isoelectrofocusing (IEF) carried in a flexible silicone tube. The dimensions of the IEF granulated gel beds can be varied from 40 to 75 cm (length) and 0.4-1.6 cm (diameter) which are dependent on the protein's concentration. An average optimal focalisation time of proteins is dependent of the tube length, its diameter and complexity of proteins' mixtures but it is usually reached during 15,000-30,000 Vh. A series of sequential protein's fractionation techniques including semi-preparative IEF carried in the flexible silicone tube with the following dimensions: 75 cm in length and 1.6 cm in diameter permitted for observation and partial characterisation of several proteins whose expression levels are specifically high in the brain.     

A synthetic weak neurotoxin binds with low affinity to Torpedo and chicken alpha7 nicotinic acetylcholine receptors.

Weak neurotoxins from snake venom are small proteins with five disulfide bonds, which have been shown to be poor binders of nicotinic acetylcholine receptors. We report on the cloning and sequencing of four cDNAs encoding weak neurotoxins from Naja sputatrix venom glands. The protein encoded by one of them, Wntx-5, has been synthesized by solid-phase synthesis and characterized. The physicochemical properties of the synthetic toxin (sWntx-5) agree with those anticipated for the natural toxin. We show that this toxin interacts with relatively low affinity (K(d) = 180 nm) with the muscular-type acetylcholine receptor of the electric organ of T. marmorata, and with an even weaker affinity (90 microm) with the neuronal alpha7 receptor of chicken. Electrophysiological recordings using isolated mouse hemidiaphragm and frog cutaneous pectoris nerve-muscle preparations revealed no blocking activity of sWntx-5 at microm concentrations. Our data confirm previous observations that natural weak neurotoxins from cobras have poor affinity for nicotinic acetylcholine receptors.