Biochemical characterization of the thrombin inhibitor of the tick, Ornithodoros savignyi, and investigation into the expression of its recombinant forms

Mans (2002) hypothesized that the two domains of savignin interact with each other, giving a globular form in the absence of thrombin. Binding of the C-terminal domain of the inhibitor to the fibrinogen-binding site of thrombin leads to the dissociation of the domains. This would yield an extended conformation that would allow binding of the N-terminal residues of the N-terminal domain to thrombin’s active site. To test this hypothesis, both theoretical and experimental approaches were employed to determine the molecular dimensions of uncomplexed savignin. In the theoretical approach, the hydrodynamic radius (Rh) of the extended form of savignin was calculated from the crystal structure data of the thrombin-ornithodorin complex, and found to be 2.319 nm. With the same programme, based on the crystal structure data for bikunin, a protein in which both domains are closely associated, the Rh value for the compact form of savignin was estimated as 1.96 nm. Using the equation that relates Rh to molecular mass, a value of 1.84 nm was calculated for savignin (12 430 Da). In the experimental approach, the SEC of salivary gland extracts, using lysozyme (Rh = 1.99 nm) as standards and chrymotrypsinogen (Rh = 2.31nm) indicated that uncomplexed savignin exists in both the globular and extended conformations. However, the majority of inhibitory activity was associcated with the extended form. Heat stability assays as well as SDS-PAGE experiments indicated the possible existence of the compact form of savignin. Generation of adequate amounts of savignin will allow further structural studies, to determine the structure of savignin in the uncomplexed form and in complex with thrombin. Expression of full length savignin and the separate N- and C-domains will facilitate further kinetic analysis. In the recombinant production of savignin, various factors were investigated: cell-lines, transformation efficiency, induction times, purification strategy and protease cleavage of expressed fusion protein. Even though large quantities of expressed fusion protein were obtained, cleavage of the target protein and its separation from the fusion partner by enzymatic means presented a major hurdle. Expression of Nsav was not observed and is most likely as a result of misfolding of the recombinant form. Due to the probable non-specific cleavage of the fusion protein, switching the prokaryotic expression system to other expression systems, like yeast- or baculovirus-insect cell-expression systems, is warranted.Dissertation (MSc (Biochemistry))--University of Pretoria, 2007.Biochemistryunrestricte

Biochemical and molecular characterization of putative immunoprotective molecules of the soft tick, Ornithodoros savignyi Audouin (1827)

Most studies on innate immunity in ticks have focused on the antimicrobial peptides from hemolymph, such as defensins and lysozyme, while less is known about bacterial recognition molecules, or antimicrobial mechanisms in other tissues. The current study attempted to identify novel antimicrobial mechanisms, with a focus on bacterial recognition by hemolymph proteins and antimicrobial activity in salivary gland extracts. Using bacteria as affinity beads, two high molecular mass molecules (Protein X and Protein Y) have been identified in tick hemolymph. These proteins are thought to interact with the bacterial surface via ionic interactions. Tandem mass spectrometry analysis followed by de novo sequencing indicated that these proteins are novel as no homologs could be identified from sequence databases. In an attempt to clone Protein X, using a degenerate primer obtained from a de novo sequence, an unrelated hemocyte protein was identified. This protein, named savicalin, was shown to belong to the lipocalin family based on bioinformatical analysis. Transcriptional profiling indicated that savicalin is found in hemocytes, midgut and ovaries, but not in the salivary glands. To date, this is the first tick lipocalin not derived from salivary glands. Interestingly, up-regulation of its mRNA transcript in response to bacterial challenge suggests that this protein could be involved in antimicrobial activity. Up-regulation after feeding also suggests a role in the post-feeding development of the tick. Two different approaches were used to purify the Gram-positive antibacterial activity from salivary gland extracts. The first attempt entailed a two-step separation approach. Tricine SDS-PAGE of the active fraction showed 3 components (~20, ~10 and ~7 kDa). BLAST searches using the N-terminal sequences of the latter proteins identified the ~20 kDa protein as savignin, while the other two proteins could not be matched. The second strategy included an ultrafiltration step (10 kDa cut-off) and MS-analysis of the active fraction in this case indicated the presence of various components with molecular masses ranging from 0.99 – 7.182 kDa, with 12 predominant components ranging from 0.99 - 4.448 kDa. Further tandem mass spectrometry analysis of the active fraction revealed the presence of three tick actin-derived fragments. This is of interest as actin fragments have been implicated in innate immunity of other invertebrates. In this study, synthetic peptides corresponding to one of the detected tick actin fragments as well as actin5C (detected in Drosophila hemolymph) were found not to inhibit the growth of Bacillus subtilis when tested up to a concentration of 100 ìg/ml. It is envisaged that future studies of immunoprotective molecules of the tick, O. savignyi, may contribute to the development of novel anti-infective agents and potential targets for anti-tick vaccine design.Thesis (PhD)--University of Pretoria, 2011.Biochemistryunrestricte

Productivity Change in Taiwan's Farmers' Credit Unions: A Nonparametric Risk-Adjusted Malmquist Approach

This article proposes an extended three-stage DEA methodology similar to Fried et al. (2002) to improve the measurement of productivity growth then the assumption of free disposability of undesirable outpu t does not apply. A directional distance function is used to construct adjusted Malmquist-Luenberger productivity indexes which simultaneously account for the impacts of undesirable outputs, environmental variables, and statistical noise. Panel data for 264 farmers' credit unions (FCUs) in Taiwan covering the 1998-2000 period are employed to illustrate the advantages of this method. On average, the productivity of Taiwan's FCUs is found to have deteriorated over the 1998-2000 period. Although an improvement in efficiency has been observed, the major reason for the deterioration is found to be due to the regression of techno logy.Malmquist-Luenberger productivity index, three-stage DEA, undesirable outputs, directional distance function, Agricultural Finance, Productivity Analysis,

Mechanism of thermal field and electric field in resistive random access memory using the high/low-k side wall structure

In the Internet of things (IoT) era, low power consumption memory will be a critical issue for further device development. Among many kinds of next-generation memories, resistive random access memory (RRAM) is considered as having the most potential due to its high performance. To prevent unrecoverable hard break-down of a RRAM device, the RRAM should be collocated with a transistor for external current compliance. With decreasing device cell size, however, the operating voltage of the transistor will become smaller and smaller. Previous study has determined that the forming voltage of RRAM increases when device cell size is reduced, which is a very crucial issue especially when the device is scaled down. We have proposed a high-k sidewall spacer structure in RRAM to solve the dilemma of increasing forming voltages for device cell scaling down. Based on the COMSOL-simulated electrical field distributions in the high-k RRAM. In addition, thermal conductivity of sidewall spacer influenced resistive switching behavior. Suitable thermal conductivity of sidewall materials can enhance resistive switching behavior. Please click Additional Files below to see the full abstract

In Situ Confocal Raman Mapping Study of a Single Ti-Assisted ZnO Nanowire

In this work, we succeeded in preparing in-plane zinc oxide nanowires using a Ti-grid assisted by the chemical vapor deposition method. Optical spatial mapping of the Confocal Raman spectra was used to investigate the phonon and geometric properties of a single ZnO nanowire. The local optical results reveal a red shift in the non-polar E2 high frequency mode and width broadening along the growth direction, reflecting quantum-confinement in the radial direction

The potential of selected macroalgal species for treatment of AMD at different pH ranges in temperate regions

The metal bioaccumulation potential of selected macroalgae species as different pH ranges were study for usage as part of a possible secondary passive AMD treatment technology in algae ponds in temperate regions during winter months. . Two separate studies were conducted to determine the suitability of macroalgae for passive treatment when metabolic processes in macrophytes and microrganisms in constructed wetlands decreases due to seasonal changes. In the field study, the bioconcentration of metals (mg/kg dry weight) measured in the macroalgae mats were in the following order: site 1. Oedegonium crassum Al > Fe > Mn > Zn; site 2. Klebsormidium klebsii, Al > Fe > Mn > Zn; site 3. Microspora tumidula, Fe > Al > Mn > Zn and site 4. M. tumidula, Fe > Mn > Al > Z. In the laboratory study, cultured macroalgae K. klebsii, O. crassum and M. tumidula isolated from the field sampling sites were exposed to three different pH values (3, 5 and 7), while bioaccumulation of the metals, Al, Fe, Mn and Zn and glutathione-S-transferase (GST) activity was measured in the different algae species at a constant water temperature of 14 ˚C. . Bioaccumulation of Al was the highest for O. crassum followed by K. klebsii and M. tumidula (p < 0.0001). From the study it was evident that the highest metal bioaccumulation occurred in the macroalgae O. crassum at all three tested pH values under constant low water temperature.http://www.elsevier.com/locate/watreshb201