Functional characterization of a Kar3/Vik1-like Kinesin-14 heterodimer from the filamentous multinucleate fungus Ashbya gossypii

Kinesins are motor proteins that convert chemical energy from ATP hydrolysis into mechanical energy used to generate force along microtubules, transporting organelles, vesicles, and proteins within the cell. Kar3 kinesins are microtubule minus-end-directed motors with pleiotropic functions in mating and mitosis of budding and fission yeast. In Saccharomyces cerevisiae, Kar3 is multifunctionalized by two non-catalytic companion proteins, Vik1 and Cik1. A Kar3-like kinesin and a single Vik1/Cik1 ortholog are also expressed by the filamentous fungus Ashbya gossypii, which exhibits different nuclear movement challenges and unique microtubule dynamics from its yeast relatives. We hypothesized that these differences in A. gossypii physiology could translate into interesting and novel differences in its versions of Kar3 and Vik1/Cik1. Presented here is a structural and functional analysis of recombinantly expressed and purified forms of these motor proteins. Compared to the previously published S. cerevisiae Kar3 motor domain structure (ScKar3MD), AgKar3MD displays differences in the conformation of the ATPase pocket. Perhaps it is not surprising then that we observed the maximal microtubule-stimulated ATPase rate (kcat) of AgKar3MD to be approximately 3-fold slower than ScKar3MD, and that the affinity of AgKar3MD for microtubules (Kd,MT) was lower than ScKar3MD. This may suggest that elements that compose the ATPase pocket and that participate in conformational changes required for efficient ATP hydrolysis or products release work differently for AgKar3 and ScKar3. There are also subtle structural differences in the disposition of the secondary structural elements in the small lobe (B1a, B1b, and B1c) at the edge of the motor domain of AgKar3 that may reflect the enhanced microtubule-depolymerization activity that we observed for this motor, or they could relate to its interactions with a different regulatory companion protein than its budding yeast counterpart. Although we were unable to gain experimentally determined high-resolution information of AgVik1, the results of Phyre2-based bioinformatics analyses may provide a structural explanation for the limited microtubule-binding activity we observed. These and other fundamental differences in AgKar3/Vik1 could explain divergent functionalities from the ScKar3/Vik1 and ScKar3/Cik1 motor assemblies.

CHARACTERIZATION OF FILLING MATERIALS FOR EASEL PAINTINGS

Developing appropriate treatments for easel paintings can be complex, as many works are composed of various materials that respond in different ways. When selecting a filling material for these artworks, several properties are investigated including: the need for the infill to react to environmental conditions in a similar manner as the original material; the need for the infill to have good handling properties, adhesion to the original support, and cohesion within the filling material; the ability for the infill to withstand the stress of the surrounding material and; be as flexible as the original material to not cause further damage. Also, changes in colour or mechanical properties should not occur as part of the ageing process. Studies are needed on acrylic-based materials used as infills in conservation treatments. This research examines some of the chemical, physical, and optical changes of eleven filling materials before and after ageing, with the aim to evaluate the overall appropriateness of these materials as infills for easel paintings. The materials examined were three rabbit skin glue (RSG) gessoes, and seven commercially prepared acrylic materials, all easily acquired in North America. Chemical analysis was carried out with Fourier transform infrared (FTIR) spectroscopy and X-ray fluorescence (XRF), pyrolysis gas chromatography-mass spectroscopy (Py-GC/MS), and differential scanning calorimetry (DSC). Overall the compositions of the various materials examined were found to be in agreement with the available literature and previous research. This study also examined characteristics of these materials not described in previous works and, additionally, presented the compositions and behaviour of several commonly used materials with little literature description. After application of an ageing regimen, most naturally aged and artificially aged samples displayed small changes in gloss, colour, thickness, and diffusive behaviour; however, to evaluate these materials fully mechanical testing and environmental studies should be carried out.

Rational design of a dual-mode optical and chemical prodrug

Purpose. The purpose of this study is to demonstrate the rational design and behaviour of the first dual mode optical and chemical prodrug, exemplified by an acetyl salicylic acid-based system. Methods. A cyclic 1,4-benzodioxinone prodrug was synthesised by reaction of 3,5-dimethoxybenzoin and acetyl salicoyl chloride with pyridine. After purification by column chromatography and recrystallization, characterization was achieved using infrared and NMR spectroscopies, mass spectrometry, elemental analysis and single crystal X-ray diffraction. Light-triggered drug liberation was characterised via UV-visible spectroscopy following low-power 365 nm irradiation for controlled times. Chemical drug liberation was characterised via UV-visible spectroscopy in pH 5.5 solution. Results. The synthetic method yielded pure prodrug, with full supporting characterisation. Light-triggered drug liberation proceeded at a rate of 8.30 10j2 sj1, while chemical, hydrolytic liberation proceeded independently at 1.89 10j3 sj1. The photochemical and hydrolytic reactions were both quantitative. Conclusions. This study demonstrates the first rational dual-mode optical and chemical prodrug, using acetyl salicylic acid as a model, acting as a paradigm for future dual-mode systems. Photochemical drug liberation proceeds 44 times faster than chemical liberation, suggesting potential use in drug-eluting medical devices where an additional burst of drug is required at the onset of infection.

Pachymedusa dacnicolor tryptophyllin-1 (PdT-1): structural characterization, pharmacological activity and cloning of precursor cDNA.

Tryptophyllins are a heterogenous group of amphibian skin peptides originally identified in skin extracts of Neotropical leaf frogs, Phyllomedusa sp., by chemical means. Until now, biosynthetic precursor structure and biological activity remain unreported. Here we describe the isolation of a novel, post-translationally modified tryptophyllin, Lys-Pro-Hyp-Ala-Trp-Val-Pro.amide (PdT-1), from the skin secretion of the Mexican leaf frog, Pachymedusa dacnicolor. Using a 3'- and 5'-RACE strategy and an in vitro skin cDNA library, the PdT-1-encoding precursor was cloned and found to consist of an open-reading frame of 62 amino acids with a single copy of PdT-1 located towards the C-terminus. A synthetic replicate of PdT-1 was found to be a potent myoactive agent, relaxing mammalian arterial smooth muscle and contracting small intestinal smooth muscle at nanomolar concentrations. PdT-1 is thus the first amphibian skin tryptophyllin to be pharmacologically characterized and the first whose precursor cDNA has been cloned.

A Fabry-Perot Refractometer for Chemical Vapor Sensing by Solid-Phase Microextraction

The contour lithography method [1] is used to improve the fabrication yield of previously demonstrated [2] microfluidic Fabry-Perot (FP) refractive index (RI) sensors. The sensors are then coated with polydimethylsiloxane (PDMS) based polymers to detect vapor analytes by solid-phase microextraction (SPME). Preliminary characterization of devices coated with two different polymers and exposed to xylenes vapors yields a maximum sensitivity of 0.015 nm/ppm and a detection limit below 120 ppm.

Physicochemical characterization of bioactive polyacrylic acid organogels as potential antimicrobial implants for the buccal cavity

This study describes the formulation and physicochemical characterization of poly(acrylic acid) (PAA) organogels, designed as bioactive implants for improved treatment of infectious diseases of the oral cavity. Organogels were formulated containing a range of concentrations of PAA (3-10% w/w) and metronidazole (2 or 5% w/w, representing a model antimicrobial agent) in different nonaqueous solvents, namely, glycerol (Gly), polyethylene glycol (PEG 400), or propylene glycol (PG). Characterization of the organogels was performed using flow rheometry, compressional analysis, oscillatory rheometry, in vitro mucoadhesion, moisture uptake, and drug release, methods that provide information pertaining to the nonclinical and clinical use of these systems. Increasing the concentration of PAA significantly increased the consistency, compressibility, storage modulus, loss modulus, dynamic viscosity, mucoadhesion, and the rate of drug release. These observations may be accredited to enhanced molecular polymer entanglement. In addition, the choice of solvent directly affected the physicochemical parameters of the organogels, with noticeable differences observed between the three solvents examined. These differences were accredited to the nature of the interaction of PAA with each solvent and, importantly, the density of the resultant physical cross-links. Good correlation was observed between the viscoelastic properties and drug release, with the exception of glycerol-based formulations containing 5 and 10% w/w PAA. This disparity was due to excessive swelling during the dissolution analysis. Ideally, formulations should exhibit controlled drug release, high viscoelasticity, and mucoadhesion, but should flow under minimal stresses. Based on these criteria, PEG 400-based organogels composed of 5% or 10% w/w PAA exhibited suitable physicochemical properties and are suggested to be a potentially interesting strategy for use as bioactive implants designed for use in the oral cavity. © 2008 American Chemical Society.

The Y-procedure: How to extract the chemical transformation rate from reaction-diffusion data with no assumption on the kinetic model

The paper presents a new method to extract the chemical transformation rate from reaction–diffusion data with no assumption on the kinetic model (“kinetic model-free procedure”). It is a new non-steady-state kinetic characterization procedure for heterogeneous catalysts. The mathematical foundation of the Y-procedure is a Laplace-domain analysis of the two inert zones in a TZTR followed by transposition to the Fourier domain. When combined with time discretization and filtering the Y-procedure leads to an efficient practical method for reconstructing the concentration and reaction rate in the active zone. Using the Y-procedure the concentration and reaction rate of a non-steady state catalytic process can be determined without any pre-assumption regarding the type of kinetic dependence. The Y-procedure is the basis for advanced software for non-steady state kinetic data interpretation. The Y-procedure can be used to relate changes in the catalytic reaction rate and kinetic parameters to changes in the surface composition (storage) of a catalyst.

Antimicrobial/cytolytic peptides from the venom of the North African scorpion, Androctonus amoreuxi: Biochemical and functional characterization of natural peptides and a single site-substituted analog

The venoms of scorpions are complex cocktails of polypeptide toxins that fall into two structural categories: those that contain cysteinyl residues with associated disulfide bridges and those that do not. As the majority of lethal toxins acting upon ion channels fall into the first category, most research has been focused there. Here we report the identification and structural characterization of two novel 18-mer antimicrobial peptides from the venom of the North African scorpion, Androctonus amoreuxi. Named AamAP1 and AamAP2, both peptides are C-terminally amidated and differ in primary structure at just two sites: Leu?Pro at position 2 and Phe?Ile at position 17. Synthetic replicates of both peptides exhibited a broad-spectrum of antimicrobial activity against a Gram-positive bacterium (Staphylococcus aureus), a Gram-negative bacterium (Escherichia coli) and a yeast (Candida albicans), at concentrations ranging between 20µM and 150µM. In this concentration range, both peptides produced significant degrees of hemolysis. A synthetic replicate of AamAP1 containing a single substitution (His?Lys) at position 8, generated a peptide (AamAP-S1) with enhanced antimicrobial potency (3-5µM) against the three test organisms and within this concentration range, hemolytic effects were negligible. In addition, this His?Lys variant exhibited potent growth inhibitory activity (ID(50) 25-40µm) against several human cancer cell lines and endothelial cells that was absent in both natural peptides. Natural bioactive peptide libraries, such as those that occur in scorpion venoms, thus constitute a unique source of novel lead compounds with drug development potential whose biological properties can be readily manipulated by simple synthetic chemical means.

Development and characterization of a lux-modified 2,4-dichlorophenol-degrading Burkholderia sp. RASC

lux-marked biosensors for assessing the toxicity and bioremediation potential of polluted environments may complement traditional chemical techniques. luxCDABE genes were introduced into the chromosome of the 2,4-dichlorophenol (2,4-DCP)-mineralizing bacterium, Burkholderia sp. RASC c2, by biparental mating using the Tn4431 system. Experiments revealed that light output was constitutive and related to cell biomass concentration during exponential growth. The transposon insertion was stable and did not interrupt 2,4-DCP-degradative genes, and expression of luxCDABE did not constitute a metabolic burden to the cell. A bioluminescence response was detectable at sublethal 2,4-DCP concentrations: at <10.26 microg ml(-1), bioluminescence was stimulated (e.g. 218% of control), but at concentrations >60 microg ml(-1) it declined to <1%. Investigating the effect of [14C]-2,4-DCP concentration on the evolution of 14CO2 revealed that, for initial concentrations of 2.5-25 microg ml(-1), approximately equals 55% of the added 14C was mineralized after 24 h compared with

Characterization of the six glycosyltransferases involved in the biosynthesis of Yersinia enterocolitica serotype O:3 lipopolysaccharide outer core

Yersinia enterocolitica (Ye) is a gram-negative bacterium; Ye serotype O:3 expresses lipopolysaccharide (LPS) with a hexasaccharide branch known as the outer core (OC). The OC is important for the resistance of the bacterium to cationic antimicrobial peptides and also functions as a receptor for bacteriophage phiR1-37 and enterocoliticin. The biosynthesis of the OC hexasaccharide is directed by the OC gene cluster that contains nine genes (wzx, wbcKLMNOPQ, and gne). In this study, we inactivated the six OC genes predicted to encode glycosyltransferases (GTase) one by one by nonpolar mutations to assign functions to their gene products. The mutants expressed no OC or truncated OC oligosaccharides of different lengths. The truncated OC oligosaccharides revealed that the minimum structural requirements for the interactions of OC with bacteriophage phiR1-37, enterocoliticin, and OC-specific monoclonal antibody 2B5 were different. Furthermore, using chemical and structural analyses of the mutant LPSs, we could assign specific functions to all six GTases and also revealed the exact order in which the transferases build the hexasaccharide. Comparative modeling of the catalytic sites of glucosyltransferases WbcK and WbcL followed by site-directed mutagenesis allowed us to identify Asp-182 and Glu-181, respectively, as catalytic base residues of these two GTases. In general, conclusive evidence for specific GTase functions have been rare due to difficulties in accessibility of the appropriate donors and acceptors; however, in this work we were able to utilize the structural analysis of LPS to get direct experimental evidence for five different GTase specificities.

Structure and thermal properties of salicylate-based-protic ionic liquids as new heat storage media. COSMO-RS structure characterization and modeling of heat capacities

During this research, we present a study on the thermal properties, such as the melting, cold crystallization, and glass transition temperatures as well as heat capacities from 293.15 K to 323.15 K of nine in-house synthesized protic ionic liquids based on the 3-(alkoxymethyl)-1H-imidazol-3-ium salicylate ([H-Im-C₁OC_n][Sal]) with n = 3–11. The 3D structures, surface charge distributions and COSMO volumes of all investigated ions are obtained by combining DFT calculations and the COSMO-RS methodology. The heat capacity data sets as a function of temperature of the 3-(alkoxymethyl)-1H-imidazol-3-ium salicylate are then predicted using the methodology originally proposed in the case of ionic liquids by Ge et al. 3-(Alkoxymethyl)-1H-imidazol-3-ium salicylate based ionic liquids present specific heat capacities higher in many cases than other ionic liquids that make them suitable as heat storage media and in heat transfer processes. It was found experimentally that the heat capacity increases linearly with increasing alkyl chain length of the alkoxymethyl group of 3-(alkoxymethyl)-1H-imidazol-3-ium salicylate as was expected and predicted using the Ge et al. method with an overall relative absolute deviation close to 3.2% for temperatures up to 323.15 K.

Formation and characterization of carbon-radical precursors in char steam gasification

Highly reactive radicals play an important role in high-temperature gasification processes. However, the effect of radicals on gasification has not been systematically investigated. In the present study, the formation of carbon-radical precursors using atomic radicals such as OH, O, and H and molecules such as H₂ and O₂ was characterized, and the effect of the precursors on the adsorption step of steam char gasification was studied using quantum chemistry methods. The results revealed that the radicals can be chemisorbed exothermically on char active sites, and the following order of reactivity was observed: O > H₂ > H > OH > O ₂. Moreover, hydrogen bonds are formed between steam molecules and carbon-radical complexes. Steam molecule adsorption onto carbon-O and carbon-OH complexes is easier than adsorption onto clean carbon surfaces. Alternatively, adsorption on carbon-O₂, carbon-H₂, and carbon-H complexes is at the same level with that of clean carbon surfaces; thus, OH and O radicals accelerate the physical adsorption of steam onto the char surface, H radical and O₂ and H₂ molecules do not have a significant effect on adsorption. © 2010 American Chemical Society.

Prevotella denticola Lipopolysaccharide from a Cystic Fibrosis Isolate Possesses a Unique Chemical Structure

We report the first complete structural characterization of the lipopolysaccharide (LPS) from a cystic fibrosis (CF) clinical isolate of Prevotella denticola (B003V1S1X). Chemical, spectroscopic, and spectrometric analyses revealed a unique rough-type LPS (LOS) structure. The structure has a highly negatively charged heptasaccharide core region containing hexoses, with the first two sugars, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and mannose, highly phosphorylated. Furthermore, the lipid A moiety has the typical structure for the genus Prevotella, and was also highly phosphorylated.