Susan B. Rivera

Chrysanthemyl diphosphate synthase: Isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium

Chrysanthemyl diphosphate synthase (CPPase) catalyzes the condensation of two molecules of dimethylallyl diphosphate to produce chrysanthemyl diphosphate (CPP), a monoterpene with a non-head-to-tail or irregular c1′-2-3 linkage between isoprenoid units. Irregular monoterpenes are common in Chrysanthemum cinerariaefolium and related members of the Asteraceae family. In C. cinerariaefolium, CPP is an intermediate in the biosynthesis of the pyrethrin ester insecticides. CPPase was purified from immature chrysanthemum flowers, and the N terminus of the protein was sequenced. A C. cinerariaefolium λ cDNA library was screened by using degenerate oligonucleotide probes based on the amino acid sequence to identify a CPPase clone that encoded a 45-kDa preprotein. The first 50 aa of the ORF constitute a putative plastidial targeting sequence. Recombinant CPPase bearing an N-terminal polyhistidine affinity tag in place of the targeting sequence was purified to homogeneity from an overproducing Escherichia coli strain by Ni2+ chromatography. Incubation of recombinant CPPase with dimethylallyl diphosphate produced CPP. The diphosphate ester was hydrolyzed by alkaline phosphatase, and the resulting monoterpene alcohol was analyzed by GC/MS to confirm its structure. The amino acid sequence of CPPase aligns closely with that of the chain elongation prenyltransferase farnesyl diphosphate synthase rather than squalene synthase or phytoene synthase, which catalyze c1′-2-3 cyclopropanation reactions similar to the CPPase reaction.

Interactions between cargo-carrying biomolecular shuttles

Microtubule shuttles propelled by the motor protein kinesin embedded in self-assembled monolayers are being developed for active transport functions in artificial microfluidic systems. As a model system, biotinylated microtubules have been laden with streptavidin-coated particles as cargo. The behaviour of cargo-laden microtubules has been observed using fluorescence microscopy upon activation of kinesin-driven transport processes. Collisions between mobile microtubules and their particulate cargo result in six distinct behaviours: bypass, microtubule bending, particle knock-off, particle transfers between microtubules, co-joining of microtubules to a common particle, and particle-induced severing of microtubules. The distribution of observed events can be described qualitatively on the basis of the mechanics of motor proteins and microtubules, the geometry of the collision events, and the loading rate dependence of the strength of microtubule–particle binding. Implications of the results for the use of motor proteins in active transport and cargo-handling systems for nanomaterials are described.

The monoterpenes of Artemisia tridentata ssp. vaseyana, Artemisia cana ssp. viscidula and Artemisia tridentata ssp. spiciformis.

Monoterpenes from three different members of the Anthemideae family, Artemisia tridentata ssp. vaseyana, Artemisia cana ssp. viscidula and Artemisia tridentata ssp. spiciformis were isolated and their structures determined using spectroscopic techniques. A total of 26 irregular and regular monoterpenes were identified. Among these, 20 had previously been identified in the Anthemideae family. Of the remaining six, four were known, but previously unidentified in this family. 2,2-Dimethyl-6-isopropenyl-2H-pyran, 2,3-dimethyl-6-isopropyl-4H-pyran and 2-isopropenyl-5-methylhexa-trans-3,5-diene-1-ol were isolated from both A. tridentata ssp. vaseyana and A. cana ssp. viscidula. The irregular monoterpene 2,2-dimethyl-6-isopropenyl-2H-pyran has a carbon skeleton analogous to the biologically important triterpene squalene. Two additional irregular monoterpenes, artemisia triene and trans-chrysanthemal were isolated from A. cana ssp. viscidula and lavandulol was isolated from A. tridentata ssp. spiciformis. This is the first time a compound possessing a lavandulyl-skeletal type has been found in the Anthemideae family.

Investigation of the use of chlorine based advanced oxidation in surface water: Oxidation of natural organic matter and formation of disinfection byproducts

Abstract Disinfection byproduct (DBP) formation during the treatment of raw Colorado River Water (CRW) using aqueous chlorine and ultraviolet (UV) light advanced oxidation processes (AOPs) was investigated. Here, CRW was combined with aqueous chlorine from two distinct sources (electrochemically-generated Mixed Oxidant Solution (MOS) and commercial sodium hypochlorite) and then exposed to ultraviolet C (UV-C) and ultraviolet A (UV-A) light. The impact of the treatment process on the structure of (NOM) in the CRW was examined, as well as the resulting production of various halogenated organic Disinfection By-Products (DBPs). Both AOP conditions tested resulted in destruction of chromophoric components of the NOM, while formation of total amounts of haloacetic acids and trihalomethanes was far below the US EPA regulated maximum contaminant level values for these contaminants, even though the UV and chlorine doses used in these studies were much higher than the typical doses used in an actual treatment process.

Laboratory Biosecurity: A Survey of the U.S. Bioscience Community

Laboratory biosecurity practices, or measures to prevent the theft or sabotage of biological research materials, must coexist with biosafety. Within the United States, laboratory biosecurity, for a list of select agents, has been regulated through several Codes of Federal Regulation. In 2004 and 2005, Sandia National Laboratories conducted a survey of the U.S. bioscience community in conjunction with Reed Research Group, to assess the extent biosecurity is implemented in laboratories and the relationship between biosecurity and biosafety and good laboratory practices in regulated select and non-select agent laboratories. This paper describes the results of this survey.

Incorporation of bioactive materials into integrated systems

Sandia is exploring two classes of integrated systems involving bioactive materials: 1) microfluidic systems that can be used to manipulate biomolecules for applications ranging from counter-terrorism to drug delivery systems, and 2) fluidic systems in which active biomolecules such as motor proteins provide specific functions such as active transport. An example of the first class involves the development of a reversible protein trap based on the integration of the thermally-switchable polymer poly(N-isopropylacrylamide)(PNIPAM) into a micro-hotplate device. To exemplify the second class, we describe the technical challenges associated with integrating microtubules and motor proteins into microfluidic systems for: 1) the active transport of nanoparticle cargo, or 2) templated growth of high-aspect ratio nanowires. These examples illustrate the functions of bioactive materials, synthesis and fabrication issues, mechanisms for switching surface chemistry and active transport, and new techniques such as the interfacial force microscope (IFM) that can be used to characterize bioactive surfaces.

Material control and accountability for laboratory biosecurity

Laboratory biosecurity refers to a suite of measures to protect legitimate stocks of dangerous pathogens and toxins from being acquired by individuals with malicious intent. Intentional misuse presents a different problem than accidental misuse, which is the subject of laboratory biosafety. In particular, the material control and accountability (MC&A) component of laboratory biosecurity addresses an insider threat. MC&A directly involves the personnel who work with pathogens and toxins that could be attractive to bioterrorists. Control of material comprises both procedural and physical measures for both material and MC&A-relevant information. Accountability requires a one-to-one correspondence between materials and people, together with a system of records, reporting and audit. Key elements of MC&A should be understood intellectually, rather than being applied simply as a prescriptive list of security measures. MC&A measures reflect an additional stewardship responsibility that technical people accept in working with material that could harm others if used improperly.

Stability of Chemically Crosslinked Microtubules

Microtubules (MT) are dynamic protein-based polymers with numerous applications in materials science including the active transport of nanomaterials and as templates for biomimetic materials synthesis. Some of these applications require that the dynamic nature of the MT be suppressed, and in this report we will discuss the preparation and stability of chemically crosslinked microtubules (CLMTs). MT reaction with gluteraldehyde results in the formation of protein dimers and higher molecular weight oligimers as observed by gel electrophoresis, confirming the formation of covalent inter-protein linkages. While extensive crosslinking was found to destabilize MTs and inactivate them with regards to kinesin binding, moderate amounts of crosslinking lead to CLMTs that had functional lifetimes of at least twice that of uncrosslinked MTs. Further studies demonstrated that CLMTs exhibited a wider thermal stability window and were far more resistant to metal-ion induced depolymerization than uncrosslinked MTs.

Conjugation and transport of quantum dots and nanoparticles by microtubule-associated kinesin motor proteins

In contrast to synthetic materials that passively assemble to form static structures through equilibrium dynamics, Nature has evolved complex, nonequilibrium strategies for the active assembly, reconfiguration and trafficking of nanomaterials. Microtubule-associated kinesin motor proteins play important roles in cell division, intracellular transport, and material assembly/reconfiguration in eukaryotic organisms. The primary goals of our work are to (1) integrate kinesin-microtubule shuttles for transporting synthetic nanoparticles, and (2) utilize the unique properties of these molecular shuttles to design dynamic and adaptive materials at synthetic interfaces. Towards this end, we are currently developing schemes by which kinesin-microtubule complexes are utilized in the transport of inorganic nanoparticles. Microtubule copolymers have been prepared from unlabeled and biotinylated tubulin and conjugated with streptavidin-coated gold nanoparticles and CdSe quantum dots. A variety of composite structures, ranging from centrosome-like assemblies to fully decorated microtubules, have been observed, and appear to be dependent on both the degree of biotinylation as well as the ratio of microtubules to quantum dots. Further, we have observed that these nanoparticle-laden microtubules can be transported by surface bound kinesins with an efficiency comparable to dye-labeled microtubules. To further facilitate microtubule-based nanoparticle transport, we have modified the microtubule polymerization and composition to create unique areas for attachment of nanoparticles and increase the efficacy for nanoparticle transport. Overall, these data demonstrate the ability to efficiently assemble and transport inorganic nanoparticle arrays using biological transport mechanisms, and provide a platform for the further development of dynamic nanomaterials based on these strategies.