A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

The secondary structure of gap junctions. Influence of isolation methods and proteolysis.

Paired intercellular transmembrane channels, termed connexons, comprised of hexameric assemblies of gap junction protein, were isolated and purified from rat liver by exploiting their resistance to either Sarkosyl detergent solubilization or alkali extraction. The secondary structures of the gap junction proteins prepared by these methods were compared by circular dichroism (CD) spectroscopy. Both the spectra and the calculated net secondary structures of the proteins obtained by the two isolation methods were different. The protein isolated by the Sarkosyl treatment was found to be approximately 50% alpha-helical, while protein isolated by alkali extraction had a lower helix content (approximately 40%). In both types of preparations, however, the helical content of the gap junction protein was sufficiently large to be consistent with an all-helical model for the membrane-spanning parts of the structure. CD spectroscopy was also used to examine the effects of proteolytic digestion of the cytoplasmic domain on the net secondary structure of the detergent-treated gap junction protein. The membrane-bound fragments had a slightly higher proportion of their residues that were alpha-helical in nature, suggesting that the transmembrane and/or intra-gap domains are indeed enriched in this type of secondary structure. This information constrains the range of models which can be realistically proposed for the channel structure.     

Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei

Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series.     

Plasmonic Enhancement by a Continuous Gold Underlayer: Application to SERS Sensing

Plasmonics - In this paper, we report on an improved enhancement of the surface-enhanced Raman scattering (SERS) effect. Such improvement is obtained by using a continuous gold film (underlayer),...     

Structural changes in GroEL effected by binding a denatured protein substrate

In the absence of nucleotides or cofactors, the Escherichia coli chaperonin GroEL binds select proteins in non-native conformations, such as denatured glutamine synthetase (GS) monomers, preventing their aggregation and spontaneous renaturation. The nature of the GroEL-GS complexes thus formed, specifically the effect on the conformation of the GroEL tetradecamer, has been examined by electron microscopy. We find that specimens of GroEL-GS are visibly heterogeneous, due to incomplete loading of GroEL with GS. Images contain particles indistinguishable from GroEL alone, and also those with consistent identifiable differences. Side-views of the modified particles reveal additional protein density at one end of the GroEL-GS complex, and end-views display chirality in the heptameric projection not seen in the unliganded GroEL. The coordinate appearance of these two projection differences suggests that binding of GS, as representative of a class of protein substrates, induces or stabilizes a conformation of GroEL that differs from the unliganded chaperonin. Three-dimensional reconstruction of the GroEL-GS complex reveals the location of the bound protein substrate, as well as complex conformational changes in GroEL itself, both cis and trans with respect to the bound GS. The most apparent structural alterations are inward movements of the apical domains of both GroEL heptamers, protrusion of the substrate protein from the cavity of the cis ring, and a narrowing of the unoccupied opening of the trans ring.     

Trichothecenes and zearalenone production by fusarium species isolated from Argentinean black beans

Isolates of Fusarium species obtained from freshly harvested bean grains for human consumption collected from different Argentinean regions, were investigated for their ability to biosynthesise trichothecenes and zearalenone either on rice grains or beans. Low incidence of toxigenic fungi was observed. These mycotoxigenic species produced several toxins when grown on rice but none or little amount when cultured on beans. The results of this report suggest that contamination of Argentinean beans with Fusarium mycotoxins will not be common and therefore people would be at low mycotoxicosis risk through consumption of beans.     

Targeting dendritic cells for priming cellular immune responses

The cardinal role of dendritic cells (DC) in priming adaptive immunity and in orchestrating immune responses against all classes of pathogens and also against tumors is well established. Their unique potential both to maintain self-tolerance and to initiate protective immune responses against foreign and/or dangerous structures is based on the functional diversity and flexibility of these cells. Tissue DC lining antigenic portals such as mucosal surfaces and the skin are specialized to take up a wide array of compounds including proteins, lipids, carbohydrates, glycoproteins, glycolipids and oligonucleotides, particles carrying such structures and apoptotic or necrotic cells. This process is facilitated by specialized receptors with high endocytic capacity, which provides potential targets for delivering designed molecules. The best route for targeting B- and/or T cell epitopes, however, is still the subject of intense investigation. Immature DC, which reside in various tissues, can be activated by pathogens, stress and inflammation or modified metabolic products, which induce mobilization of cells to draining lymph nodes where they act as highly potent professional antigen presenting cells. This is brought about by the ability to present their accumulated intracellular content for both CD4+ helper (Th) and CD8+ cytotoxic/cytolytic T lymphocytes (Tc/CTL). Engulfed proteins are processed intracellularly and their peptide fragments are transported to the cell surface in the context of major histocompatibility complex encoded class I and II molecules for presentation to Th cells and CTLs, respectively. The T cell priming capacity of DC, however, depends not only on antigen presentation but also on other features of DC. Human monocyte-derived DC provide an excellent tool to study the internalizing, antigen-presenting and T cell-activating functions of DC at their immature and activated differentiation states. These biological activities of DC, however, are highly dependent on their migratory potential from the peripheral non-lymphoid tissues to the lymph nodes, on the expression of adhesion molecules, which support the interaction of DC with T lymphocytes, and the cytokines secreted by DC, which polarize immune responses to Th1-mediated cellular or Th2-mediated antibody responses. These results altogether demonstrate that monocyte-derived DC are useful candidates for in vitro or in vivo targeting of antigens to induce efficient adaptive immune responses against pathogens and also against tumors.