Oxidation of Ethanol and Its Derivatives on Well Defined Pt Single Crystal Electrodes Vicinal to Pt(111): A Comparative Study

The oxidation of ethanol (EtOH) at Pt(111) electrodes is dominated by the 4e path leading to acetic acid. The inclusion of surface defects such as those present on stepped surfaces leads to an increase of the reactivity towards the most desirable 12e path leading to CO2 as final product. This path is also favored when the methyl group is more oxidized, as in the case of ethylene glycol (EG) that spontaneously decomposes to CO on Pt(111) electrodes, thus showing a more effective breaking of the C-C bond. Some trends in reactivity can be envisaged when other derivative molecules are compared at well-ordered electrodes. This strategy was used in the past, but the improvement in the electrode pretreatment and the overall information available on the subject suggest that relevant information is still missing.

Is Vietnam the Right Choice for Foreign Investment?: The Case of the European Union’s Slipping Opportunity. ACES Working Papers, 2012

This analysis is broken into three interdependent sections: First, an analysis of the restrictions placed on foreign direct investment in Vietnam captures the current freedoms and inhibitors of investment in Vietnam. Foreign direct investment is defined by the UN as an investment made to acquire a lasting interest in or effective control over an enterprise operating outside of the economy of the investor. Second, a cursory look at the macroeconomic risks, to which investment dollars are susceptible, will paint a realistic portrait of return of foreign investment. Finally, this paper will examine the current, and historical, trade relationship between Vietnam and the European Union, in order to convey that the opportunity for investment in Vietnam remains to be an opportunity for Europe’s developed economies.

The proteasome inhibitor MLN-273 blocks exoerythrocytic and erythrocytic development of Plasmodium parasites.

Protein degradation is regulated during the cell cycle of all eukaryotic cells and is mediated by the ubiquitin-proteasome pathway. Potent and specific peptide-derived inhibitors of the 20S proteasome have been developed recently as anti-cancer agents, based on their ability to induce apoptosis in rapidly dividing cells. Here, we tested a novel small molecule dipeptidyl boronic acid proteasome inhibitor, named MLN-273 on blood and liver stages of Plasmodium species, both of which undergo active replication, probably requiring extensive proteasome activity. The inhibitor blocked Plasmodium falciparum erythrocytic development at an early ring stage as well as P. berghei exoerythrocytic progression to schizonts. Importantly, neither uninfected erythrocytes nor hepatocytes were affected by the drug. MLN-273 caused an overall reduction in protein degradation in P. falciparum, as demonstrated by immunoblots using anti-ubiquitin antibodies to label ubiquitin-tagged protein conjugates. This led us to conclude that the target of the drug was the parasite proteasome. The fact that proteasome inhibitors are presently used as anti-cancer drugs in humans forms a solid basis for further development and makes them potentially attractive drugs also for malaria chemotherapy.

The Plasmodium serine-type SERA proteases display distinct expression patterns and non-essential in vivo roles during life cycle progression of the malaria parasite

Parasite proteases play key roles in several fundamental steps of the Plasmodium life cycle, including haemoglobin degradation, host cell invasion and parasite egress. Plasmodium exit from infected host cells appears to be mediated by a class of papain-like cysteine proteases called 'serine repeat antigens' (SERAs). A SERA subfamily, represented by Plasmodium falciparum SERA5, contains an atypical active site serine residue instead of a catalytic cysteine. Members of this SERAser subfamily are abundantly expressed in asexual blood stages, rendering them attractive drug and vaccine targets. In this study, we show by antibody localization and in vivo fluorescent tagging with the red fluorescent protein mCherry that the two P. berghei serine-type family members, PbSERA1 and PbSERA2, display differential expression towards the final stages of merozoite formation. Via targeted gene replacement, we generated single and double gene knockouts of the P. berghei SERAser genes. These loss-of-function lines progressed normally through the parasite life cycle, suggesting a specialized, non-vital role for serine-type SERAs in vivo. Parasites lacking PbSERAser showed increased expression of the cysteine-type PbSERA3. Compensatory mechanisms between distinct SERA subfamilies may thus explain the absence of phenotypical defect in SERAser disruptants, and challenge the suitability to develop potent antimalarial drugs based on specific inhibitors of Plasmodium serine-type SERAs.

A simple sustained release device for the ethylene binding inhibitor 1-methylcyclopropene

The efficacy of 1-methylcyclopropene (1-MCP) gas to prevent the adverse effects of ethylene is limited by its short-term residual activity in some plants. Development of a simple 1-MCP sustained release device that prolongs 1-MCP exposure is reported herein. Sustained release devices comprised of polyvinylchloride tubes containing 0.1 g SmartFresh(TM) powder (a.i. 3.3% 1-MCP) and 1.25 ml deionised water were used to release 1-MCP into fibreboard cartons containing cut Geraldton waxflower (Chamelaucium uncinatum Schauer) cv. CWA Pink bunches during export shipment by air (107 h) from Australia to the UK. The devices protected flowers against abscission induced by subsequent test exposures to ethylene (1011,mul l(-1), 12 h, 20 degreesC) for 3-5 days after arrival. In contrast, pre-shipment treatments with either a single application of 790 nl l(-1) 1-MCP for 14 h at 2 degreesC or a 0.2 mM Ag+ (as silver thiosulphate; STS) pulse for 14 h at 2 degreesC protected flowers against exogenous ethylene for only 1-2 days of post-export life. However, pre-shipment 1-MCP fumigation was up to about three-fold more effective than either sustained 1-MCP release or pre-shipment STS treatments in reducing floral organ and leaf abscission from bunches during export. Thus, it is suggested that a combination of pre-shipment 1-MCP fumigation before export with sustained 1-MCP release during shipment should maximise efficacy against ethylene-induced waxflower flower abscission. (C) 2004 Elsevier B. V. All rights reserved.

Countering cooperative effects in protease inhibitors using constrained b-strand-mimicking templates in focused combinatorial libraries

A major problem in de novo design of enzyme inhibitors is the unpredictability of the induced fit, with the shape of both ligand and enzyme changing cooperatively and unpredictably in response to subtle structural changes within a ligand. We have investigated the possibility of dampening the induced fit by using a constrained template as a replacement for adjoining segments of a ligand. The template preorganizes the ligand structure, thereby organizing the local enzyme environment. To test this approach, we used templates consisting of constrained cyclic tripeptides, formed through side chain to main chain linkages, as structural mimics of the protease-bound extended beta-strand conformation of three adjoining amino acid residues at the N- or C-terminal sides of the scissile bond of substrates. The macrocyclic templates were derivatized to a range of 30 structurally diverse molecules via focused combinatorial variation of nonpeptidic appendages incorporating a hydroxyethylamine transition-state isostere. Most compounds in the library were potent inhibitors of the test protease (HIV-1 protease). Comparison of crystal structures for five protease-inhibitor complexes containing an N-terminal macrocycle and three protease-inhibitor complexes containing a C-terminal macrocycle establishes that the macrocycles fix their surrounding enzyme environment, thereby permitting independent variation of acyclic inhibitor components with only local disturbances to the protease. In this way, the location in the protease of various acyclic fragments on either side of the macrocyclic template can be accurately predicted. This type of templating strategy minimizes the problem of induced fit, reducing unpredictable cooperative effects in one inhibitor region caused by changes to adjacent enzyme-inhibitor interactions. This idea might be exploited in template-based approaches to inhibitors of other proteases, where a beta-strand mimetic is also required for recognition, and also other protein-binding ligands where different templates may be more appropriate.

Enzymatic characterization and homology model of a catalytically active recombinant West Nile virus NS3 protease

West Nile Virus (WNV) is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection causes severe neurological disease and fatalities in both human and animal hosts. The West Nile viral protease (NS2B-NS3) is essential for post-translational processing in host-infected cells of a viral polypeptide precursor into structural and functional viral proteins, and its inhibition could represent a potential treatment for viral infections. This article describes the design, expression, and enzymatic characterization of a catalytically active recombinant WNV protease, consisting of a 40-residue component of cofactor NS2B tethered via a noncleavable nonapeptide (G(4)SG(4)) to the N-terminal 184 residues of NS3. A chromogenic assay using synthetic para-nitroanilide (pNA) hexapeptide substrates was used to identify optimal enzyme-processing conditions (pH 9.5, I < 0.1 M, 30% glycerol, 1 mM CHAPS), preferred substrate cleavage sites, and the first competitive inhibitor (Ac-FASGKR- H, IC50 &SIM; 1 μM). A putative three-dimensional structure of WNV protease, created through homology modeling based on the crystal structures of Dengue-2 and Hepatitis C NS3 viral proteases, provides some valuable insights for structure-based design of potent and selective inhibitors of WNV protease.

Development and senescence of Grevillea 'Sylvia' inflorescences, flowers and flower parts

To characterise the physiology of development and senescence for Grevillea 'Sylvia'. oral organs, respiration, ethylene production and ACC concentrations in harvested flowers and flower parts were measured. The respiration rate of harvested inflorescences decreased over time during senescence. In contrast, both ethylene production and ACC concentration increased. Individual flowers, either detached from cut inflorescences held in vases at 20degreesC or detached from in planta inflorescences at various stages of development, had similar patterns of change in ACC concentration and rates of respiration and ethylene production as whole inflorescences. The correlation between ACC concentration and ethylene production by individual flowers detached from cut inflorescences held in vases was poor (r(2)=0.03). The isolated complete gynoecium (inclusive of the pedicel) produced increasing amounts of ethylene during development. Further sub-division of flower parts and measurement of their ethylene production at various stages of development revealed that the distal part of the gynoecium (inclusive of the stigma) had the highest rate of ethylene production. In turn, anthers had higher rates of ethylene production and also higher ACC concentrations than the proximal part of the gynoecium (inclusive of the ovary). Rates of ethylene production and ACC concentrations for tepal abscission zone tissue and adjacent central tepal zone tissue were similar. ACC concentration in pollen was similar to that in senescing perianth tissue. Overall, respiration, ethylene and ACC content measurements suggest that senescence of G. 'Sylvia' is non-climacteric in character. Nonetheless, the phytohormone ethylene is produced and evidently mediates normal flower development and non-climacteric senescence processes.

Blockade of the C5a receptor fails to protect against experimental autoimmune encephalomyelitis in rats

Complement activation contributes to inflammation and tissue damage in human demyelinating diseases and in rodent models of demyelination. Inhibitors of complement activation ameliorate disease in the rat model antibody-dependent experimental autoimmune encephalomyelitis and rats unable to generate the membrane attack complex of complement develop inflammation without demyelination. The role of the highly active chemotactic and anaphylactic complement-derived peptide C5a in driving inflammation and pathology in rodent models of demyelination has been little explored. Here we have used a small molecule C5a receptor antagonist, AcF-[OPdChaWR], to examine the effects of C5a receptor blockade in rat models of brain inflammation and demyelination. C5a receptor antagonist therapy completely blocked neutrophil response to C5a in vivo but had no effect on clinical disease or resultant pathology in either inflammatory or demyelinating rat models. We conclude that C5a is not required for disease induction or perpetuation in these strongly complement-dependent disease models.

The role of the complement system in ischemia-reperfusion injury

Ischemia-reperfusion (I/R) injury is a common clinical event with the potential to seriously affect, and sometimes kill, the patient. Interruption of blood supply causes ischemia, which rapidly damages metabolically active tissues. Paradoxically, restoration of blood flow to the ischemic tissues initiates a cascade of pathology that leads to additional cell or tissue injury. I/R is a potent inducer of complement activation that results in the production of a number of inflammatory mediators. The use of specific inhibitors to block complement activation has been shown to prevent local tissue injury after I/R. Clinical and experimental studies in gut, kidney, limb, and liver have shown that I/R results in local activation of the complement system and leads to the production of the complement factors C3a, C5a, and the membrane attack complex. The novel inhibitors of complement products may find wide clinical application because there are no effective drug therapies currently available to treat I/R injuries.

The alpha(2)delta auxiliary subunit reduces affinity of omega-conotoxins for recombinant N-type (Ca(v)2.2) calcium channels

The omega-conotoxins from fish-hunting cone snails are potent inhibitors of voltage-gated calcium channels. The omega-conotoxins MVIIA and CVID are selective N-type calcium channel inhibitors with potential in the treatment of chronic pain. The beta and alpha(2)delta-1 auxiliary subunits influence the expression and characteristics of the alpha(1B) subunit of N-type channels and are differentially regulated in disease states, including pain. In this study, we examined the influence of these auxiliary subunits on the ability of the omega-conotoxins GVIA, MVIIA, CVID and analogues to inhibit peripheral and central forms of the rat N-type channels. Although the beta3 subunit had little influence on the on- and off-rates of omega-conotoxins, coexpression of alpha(2)delta with alpha(1B) significantly reduced on- rates and equilibrium inhibition at both the central and peripheral isoforms of the N-type channels. The alpha(2)delta also enhanced the selectivity of MVIIA, but not CVID, for the central isoform. Similar but less pronounced trends were also observed for N-type channels expressed in human embryonic kidney cells. The influence of alpha(2)delta was not affected by oocyte deglycosylation. The extent of recovery from the omega-conotoxin block was least for GVIA, intermediate for MVIIA, and almost complete for CVID. Application of a hyperpolarizing holding potential ( - 120 mV) did not significantly enhance the extent of CVID recovery. Interestingly, [R10K] MVIIA and [O10K] GVIA had greater recovery from the block, whereas [K10R] CVID had reduced recovery from the block, indicating that position 10 had an important influence on the extent of omega-conotoxin reversibility. Recovery from CVID block was reduced in the presence of alpha(2)delta in human embryonic kidney cells and in oocytes expressing alpha(1B-b). These results may have implications for the antinociceptive properties of omega-conotoxins, given that the alpha(2)delta subunit is up-regulated in certain pain states.

Determination of alpha-conotoxin binding modes on neuronal nicotinic acetylcholine receptors

alpha-Conotoxins, from cone snails, and alpha-neurotoxins, from snakes, are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) that have overlapping binding sites in the ACh binding pocket. These disulphide-rich peptides are used extensively as tools to localize and pharmacologically characterize specific nAChRs subtypes. Recently, a homology model based on the high-resolution structure of an ACh binding protein (AChBP) allowed the three-fingered alpha-neurotoxins to be docked onto the alpha7 nAChR. To investigate if alpha-conotoxins interact with the nAChR in a similar manner, we built homology models of human alpha7 and alpha3beta2 nAChRs, and performed docking simulations of alpha-conotoxins ImI, PnIB, PnIA and MII using the program GOLD. Docking revealed that alpha-conotoxins have a different mode of interaction compared with alpha-neurotoxins, with surprisingly few nAChR residues in common between their overlapping binding sites. These docking experiments show that Imi and PnIB bind to the ACh binding pocket via a small cavity located above the beta9/beta10 hairpin of the (+)alpha7 nAChR subunit. Interestingly, PnIB, PnIA and MII were found to bind in a similar location on alpha7 or alpha3beta2 receptors mostly through hydrophobic interactions, while ImI bound further from the ACh binding pocket, mostly through electrostatic interactions. These findings, which distinguish alpha-conotoxin and alpha-neurotoxin binding modes, have implications for the rational design of selective nAChR antagonists. Copyright (C) 2004 John Wiley Sons, Ltd.

Elucidating the specificity of binding of sulfonylurea herbicides to acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) is the target for the sulfonylurea herbicides, which act as potent inhibitors of the enzyme. Chlorsulfuron (marketed as Glean) and sulforneturon methyl (marketed as Oust) are two commercially important members of this family of herbicides. Here we report crystal structures of yeast AHAS in complex with chlorsulfuron (at a resolution of 2.19 Angstrom), sulforneturon methyl (2.34 Angstrom), and two other sulfonylureas, metsulfuron methyl (2.29 Angstrom) and tribenuron methyl (2.58 Angstrom). The structures observed suggest why these inhibitors have different potencies and provide clues about the differential effects of mutations in the active site tunnel on various inhibitors. In all of the structures, the thiamin diphosphate cofactor is fragmented, possibly as the result of inhibitor binding. In addition to thiamin diphosphate, AHAS requires FAD for activity. Recently, it has been reported that reduction of FAD can occur as a minor side reaction due to reaction with the carbanion/enamine of the hydroxyethyl-ThDP intermediate that is formed midway through the catalytic cycle. Here we report that the isoalloxazine ring has a bent conformation that would account for its ability to accept electrons from the hydroxyethyl intermediate. Most sequence and mutation data suggest that yeast AHAS is a high-quality model for the plant enzyme.

Structure-activity relationships for a new family of sulfonylurea herbicides

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) catalyzes the first common step in branched-chain amino acid biosynthesis. The enzyme is inhibited by several chemical classes of compounds and this inhibition is the basis of action of the sulfonylurea and imidazolinone herbicides. The commercial sulfonylureas contain a pyrimidine or a triazine ring that is substituted at both meta positions, thus obeying the initial rules proposed by Levitt. Here we assess the activity of 69 monosubstituted sulfonylurea analogs and related compounds as inhibitors of pure recombinant Arabidopsis thaliana AHAS and show that disubstitution is not absolutely essential as exemplified by our novel herbicide, monosulfuron (2-nitro-N-(4'-methyl-pyrimidin-2'-yl) phenyl-sulfonylurea), which has a pyrimidine ring with a single meta substituent. A subset of these compounds was tested for herbicidal activity and it was shown that their effect in vivo correlates well with their potency in vitro as AHAS inhibitors. Three-dimensional quantitative structure-activity relationships were developed using comparative molecular field analysis and comparative molecular similarity indices analysis. For the latter, the best result was obtained when steric, electrostatic, hydrophobic and H-bond acceptor factors were taken into consideration. The resulting fields were mapped on to the published crystal structure of the yeast enzyme and it was shown that the steric and hydrophobic fields are in good agreement with sulfonylurea-AHAS interaction geometry.

Coconut (Cocos nucifera) in vitro ecology: Modifications of headspace and medium additives can optimize somatic embryogenesis

Of those explants tested, immature zygotic embryo tissues proved to be the best for initiating callus with potential for somatic embryogenesis. Slicing of this tissue and use of the central sections (near to and including the meristematic tissue) gave the best embryogenic response. Slices that were placed under illumination necrosed more rapidly and to a greater degree than those incubated in the dark. Explant slice necrosis could be prevented or severely retarded by the addition of activated charcoal into the medium. Washing the explants for short periods of time prior to culture was also found to improve callus production. Prolonged washing resulted in low rates of callus production. In an attempt to prevent ethylene accumulation in the culture vessel headspace, AVG, an ethylene biosynthesis inhibitor and STS, a chemical which reduces the physiological action of ethylene, were successfully used to promote somatic embryogenesis. Spermidine, putrescine and spermine, polyamines that are known to delay plant senescence and promote somatic embryogenesis in some plant species, enhanced the rate of somatic embryogenesis when they were introduced into the callus induction medium. The use of polyethylene glycol in combination with abscisic acid helped promote somatic embryo formation and maturation as well as the subsequent formation of plantlets. The use of all of these improvements together has created a new and improved protocol for coconut somatic embryogenesis. This new protocol puts significant emphasis on improving the in vitro ecology of the explant, callus and somatic embryogenic tissues.