Proteomic analysis of the Arabidopsis thaliana cell wall

With the completion of the Arabidopsis genome, many hypothetical proteins have been predicted without any information on their expression, subcellular localisation and function. We have performed proteomic analysis of proteins sequentially extracted from enriched Arabidopsis cell wall fractions and separated by two-dimensional gel electrophoresis (2-DE). The proteins were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry and genomic database searches. This is part of a targeted exercise to establish the entire Arabidopsis secretome database. We report evidence for new proteins of unknown function whose existence had been predicted from genomic sequences and, furthermore, localise them to the cell wall. In addition, we observed an unexpected presence in the cell wall preparations of proteins whose known biochemical activity has never been associated with this compartment hitherto. We discuss the implications of these findings and present results suggesting a possible involvement of cell wall kinases in plant responses to pathogen attack.     

Optimization of Biocompatibility for a Hydrophilic Biological Molecule Encapsulation System

Despite considerable advances in recent years, challenges in delivery and storage of biological drugs persist and may delay or prohibit their clinical application. Though nanoparticle-based approaches for small molecule drug encapsulation are mature, encapsulation of proteins remains problematic due to destabilization of the protein. Reverse micelles composed of decylmonoacyl glycerol (10MAG) and lauryldimethylamino-N-oxide (LDAO) in low-viscosity alkanes have been shown to preserve the structure and stability of a wide range of biological macromolecules. Here, we present a first step on developing this system as a future platform for storage and delivery of biological drugs by replacing the non-biocompatible alkane solvent with solvents currently used in small molecule delivery systems. Using a novel screening approach, we performed a comprehensive evaluation of the 10MAG/LDAO system using two preparation methods across seven biocompatible solvents with analysis of toxicity and encapsulation efficiency for each solvent. By using an inexpensive hydrophilic small molecule to test a wide range of conditions, we identify optimal solvent properties for further development. We validate the predictions from this screen with preliminary protein encapsulation tests. The insight provided lays the foundation for further development of this system toward long-term room-temperature storage of biologics or toward water-in-oil-in-water biologic delivery systems.     

Hydrocarbon metal sulphide complexes : VI. A new type of rearrangement of a cyclopentadienylruthenium complex

The complexes C₅H₅Ru(CO)₂SR (R = C₆F₅ or CH₃) undergo photochemical conversion to binuclear and trinuclear derivatives which exhibit isomerism. A novel rearrangement has been detected for [C₅H₅Ru(CO)(SC₆F₅)]₂.     

Steady-state and dynamic rheology of poly(1-olefin) melts

The steady-state and dynamic melt rheology for a series of poly(1-olefins) has been investigated. The series includes poly(1-butene), poly(1-hexene), poly(1-heptene), poly(1-octene), Poly(1-undecene), poly(1-tridecene), poly(1-hexadecene), and poly(1-octadecene). The flow behavior was investigated by use of a Weissenberg rheogoniometer. Measurements on poly(1-butene) were also made using an Instron capillary rheometer. The empirical relationship developed by Cox and Merz was obeyed for the entire series of poly(1-olefins) at all temperatures investigated. Graessley's theory was used to calculate the flow curves for the poly(1-olefins) from the measured molecular weight distributions. The purpose was to investigate the effect of polymer composition on the shear rate dependence of viscosity. It was found that all experimental flow curves except those for poly(1-hexene) can be fitted with the calculated curves from the individual molecular weight distributions. The conclusion is made that flow curves of poly(1-olefins) depend predominately on molecular weight distribution and are essentially independent of side-chain length even for poly(1-olefins) with pendant groups as long as 16 carbon atoms. The low-shear limiting Newtonian viscosity η₀ for all poly(1-olefins) was expressed by, η₀ = KM? or by η₀ = K′P? where M?_w is the weight-average molecular weight and P?_w is the weight-average degree of polymerization. The K and K′ values obtained decrease systematically as the side chain is increased.     

Four complementary theoretical approaches for the analysis of NMR paramagnetic relaxation

Four theoretical and computational approaches used at the University of Michigan to analyze NMR paramagnetic relaxation enhancement (NMR-PRE) are described. The primary objective of the theory is to describe the relationship of the NMR-PRE phenomenon to the electron spin hamiltonian and the spin energy level structure when zero field splitting interactions are significant. Four formulations of theory are discussed: (1) spin dynamics simulation; (2) the laboratory frame "constant H(S)" formulation; (3) the Molecular Frame "constant H(S)" formulation; and (4) the zfs-limit "constant H(S)" formulation. No single theoretical approach describes all important aspects of the relaxation mechanism in a fully satisfactory way. We use the four formulations in a complementary manner to provide as complete a picture of the relaxation mechanism as possible. We also discuss the integration of NMR-PRE theory and recently developed theory of electron spin relaxation which accounts for effects of the permanent zfs hamiltonian.     

The first direct evaluation of the two-active site mechanism for chitin synthase

Chitin synthase polymerizes UDP-GlcNAc to form chitin (poly-beta(1,4)-GlcNAc) and is essential for fungal cell wall biosynthesis. The alternating orientation of the GlcNAc residues within the chitin chain has led to the proposal that chitin synthase possesses two active sites. We report the results of the first direct test of this possibility. Two simple uridine-derived dimeric inhibitors are shown to exhibit 10-fold greater inhibition than a monomeric control, consistent with the presence of two active sites. This observation has important implications for the development of antifungal agents, as well as the understanding of polymerizing glycosyltransferases.     

Arrested chloride abstraction from trans-RuCl2(DMeOPrPE)2 with TlPF6; formation of a 1-D coordination polymer having unusual octahedral coordination around thallium(I)

An "arrested" chloride abstraction occurs in the reaction of trans-RuCl(2)(DMeOPrPE)(2) with TlPF(6); the product is a 1-D coordination polymer in which the Tl(I) centers have an unusual octahedral coordination geometry with a stereochemically active 6s(2) lone pair.     

Measurement of enzyme kinetics using microscale steady-state kinetic analysis

This paper describes a new technique--microscale steady-state kinetic analysis (microSKA)--that enables the rapid and parallel analysis of enzyme kinetics. Rather than physically defining a microscopic reactor through microfabrication, we show how the relative rates of reaction and transport in a macroscopic flow chamber, where the enzyme is immobilized on one wall of the chamber, results in the confinement of an enzyme-catalyzed reaction to a microscopic reactor volume adjacent to this wall. This volume has linear dimensions that are orders of magnitude smaller than the physical dimensions of the system (i.e., micrometer vs millimeter). Conversion within this volume is monitored at steady state as a function of position, rather than time. In this way, limitations due to reactor dead time and mixing are avoided. We use microSKA to determine kinetic parameters for the alkaline phosphatase-catalyzed de-phosphorylation of nonfluorescent methylumbelliferyl phosphate (MUP) to fluorescent 7-hydroxy-4-methylcoumarin (HMC) at two different values of pH. Kinetic parameters measured with microSKA are in good agreement with values obtained using conventional methods, if one takes into account effects of immobilization on enzyme activity. This technique provides a rapid and simple method for determining enzyme kinetics using small amounts of sample material and may be useful for applications in proteomics, drug discovery, biocatalyst development, and clinical diagnostics.     

The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae is a dinuclear metallohydrolase

The Zn K-edge extended X-ray absorption fine structure (EXAFS) spectra, of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae have been recorded in the presence of one or two equivalents of Zn(II) (i.e. [Zn_(DapE)] and [ZnZn(DapE)]). The Fourier transforms of the Zn EXAFS are dominated by a peak at ca. 2.0 A, which can be fit for both [Zn_(DapE)] and [ZnZn(DapE)], assuming ca. 5 (N,O) scatterers at 1.96 and 1.98 A, respectively. A second-shell feature at ca. 3.34 A appears in the [ZnZn(DapE)] EXAFS spectrum but is significantly diminished in [Zn_(DapE)]. These data show that DapE contains a dinuclear Zn(II) active site. Since no X-ray crystallographic data are available for any DapE enzyme, these data provide the first glimpse at the active site of DapE enzymes. In addition, the EXAFS data for DapE incubated with two competitive inhibitors, 2-carboxyethylphosphonic acid and 5-mercaptopentanoic acid, are also presented.     

Soliton self-frequency shift in a short tapered air-silica microstructure fiber

We report a soliton self-frequency shift of more than 20% of the optical frequency in a tapered air-silica microstructure fiber that exhibits a widely flattened large anomalous dispersion in the near infrared. Remarkably, the large frequency shift was realized in a fiber of length as short as 15 cm, 2 orders of magnitude shorter than those reported previously with similar input pulse duration and pulse energies, owing to the small mode size and the large and uniform dispersion in the tapered fiber. By varying the power of the input pulses, we generated compressed sub-100-fs soliton pulses of ~1-nJ pulse energy tunable from 1.3 to 1.65 mum with greater than 60% conversion efficiency.