Measurement of glycolysis reactants by high-throughput solid phase extraction with tandem mass spectrometry: Characterization of pyrophosphate-dependent phosphofructokinase as a case study

Glycolysis is a 10-step metabolic pathway involved in producing cellular energy. Many tumors exhibit accelerated glycolytic rates, and enzymes that participate in this pathway are focal points of cancer research. Here, a novel method for the measurement of glycolysis reactants from in vitro samples is presented. Fast and direct measurement is achieved by an automated system that couples on-line solid phase extraction (SPE) with tandem mass spectrometry (MS/MS). The single analytical method enables multiple reactants to be measured concurrently, sustains a cycle time of 8 s, and permits the measurement of up to 10,000 samples per day. Concentration–response curves were conducted using standards for 10 metabolic intermediates, and the results demonstrate that the detection strategy has excellent sensitivity (average limit of detection = 5.4 nM), dynamic range (nanomolar to micromolar), and linear response (average R² = 0.998). To test the analysis method on reactions, pyrophosphate-dependent phosphofructokinase (PPi–PFK) was used as a model system. Data that corroborate the activation and inhibition of PPi–PFK are presented, and the ways in which SPE–MS/MS simplifies experimental design and interpretation are highlighted. In summary, the method for measuring metabolic intermediates described here demonstrates unprecedented speed, performance, and versatility.     

Process optimisation for anion exchange monolithic chromatography of 4.2 kbp plasmid vaccine (pcDNA3F)

Anion exchange monolithic chromatography is increasingly becoming a prominent tool for plasmid DNA purification but no generic protocol is available to purify all types of plasmid DNA. In this work, we established a simple framework and used it to specifically purify a plasmid DNA model from a clarified alkaline-lysed plasmid-containing cell lysate. The framework involved optimising ligand functionalisation temperature (30–80 °C), mobile phase flow rate (0.1–1.8 mL/min), monolith pore size (done by changing the porogen content in the polymerisation reaction by 50–80%), buffer pH (6–10), ionic strength of binding buffer (0.3–0.7 M) and buffer gradient elution slope (1–10% buffer B/min). We concluded that preferential pcDNA3F adsorption and optimum resolution could be achieved within the tested conditions by loading the clarified cell lysate into 400 nm pore size of monolith in 0.7 M NaCl (pH 6) of binding buffer followed by increasing the NaCl concentration to 1.0 M at 3%B/min.     

Modification of gel architecture and TBE/TAE buffer composition to minimize heating during agarose gel electrophoresis

Agarose gel electrophoresis of DNA and RNA is routinely performed using buffers containing either Tris, acetate, and EDTA (TAE) or Tris, borate, and EDTA (TBE). Gels are run at a low, constant voltage (∼10 V/cm) to minimize current and asymmetric heating effects, which can induce band artifacts and poor resolution. In this study, alterations of gel structure and conductive media composition were analyzed to identify factors causing higher electrical currents during horizontal slab gel electrophoresis. Current was reduced when thinner gels and smaller chamber buffer volumes were used, but was not influenced by agarose concentration or the presence of ethidium bromide. Current was strongly dependent on the amount and type of EDTA used and on the concentrations of the major acid–base components of each buffer. Interestingly, resolution and the mobilities of circular versus linear plasmid DNAs were also affected by the chemical form and amount of EDTA. With appropriate modifications to gel structure and buffer constituents, electrophoresis could be performed at high voltages (20–25 V/cm), reducing run times by up to 3-fold. The most striking improvements were observed with small DNAs and RNAs (10–100 bp): high voltages and short run times produced sharper bands and higher resolution.     

A chemical quenching apparatus for studying rapid reactions.

A chemical quench-flow apparatus is described for studying enzymatic reactions with half-lives of 0.005 sec or longer. The syringe pistons are driven by a stepping motor which provides precise control over the volume and rate of flow of reactants. The drive mechanism also ensures a rapid approach to a steady flow velocity and thus minimizes the amount of material used per stroke. Improved mixing efficiency is accomplished by means of ball mixers which utilize the zone of turbulence in the wake of a sphere as the mixing mechanism. The instrument was used to follow the presteady state time course of phosphorylation and dephosphorylation of a microsomal preparation of (Na⁺ + K⁺)-stimulated ATPase.     

Amelioratory effects of testosterone treatment on cognitive performance deficits induced by soluble Aβ1–42 oligomers injected into the hippocampus

This study was undertaken to investigate the protective effects and potential mechanism of testosterone (T) on cognitive performance in adult male rats given bilateral intrahippocampal injections of beta amyloid 1–42 oligomers (Aβ1–42) combined with gonadectomy (Aβ + GDX). A series of experiments were designed to verify the optimal administration time and dose of T and to explore its potential protective mechanisms on spatial ability in Aβ + GDX rats in the Morris water maze test. Aβ1–42 was injected only once two weeks before testing, while T and the androgen receptor (AR) antagonist flutamide (F) were administered daily beginning 2 days before and throughout the 6 days of testing. The Aβ1–42 injection and GDX individually impaired cognitive performance, and the combination of these treatments was additive, leading to even greater impairment. The serum T level peaked at 48 h after administration. T doses ranging from 0.25 to 1.00 mg corresponding to serum T levels of 4.5–21.35 ng/ml improved the spatial ability. Animals administered 0.75 mg of T corresponding to the serum T level of 15.2 ng/ml had the most significantly improved behavioral performances. However, higher T doses of 1.50 and 2.00 mg resulting in serum T levels of 34.8 and 45 ng/ml, respectively, impaired the behavioral performances. F had no effect on the serum T level and spatial ability, but it blocked the activational effect of T. These findings indicate that the effect of T on behavioral performances is partly mediated through ARs.     

The effect of fulvic acid on pre‐ and postaggregation state of Aβ17–42: Molecular dynamics simulation studies

Alzheimer's disease (AD), a neurodegenerative disorder, is directly related to the aggregation of Aβ peptides. These peptides can self-assemble from monomers to higher oligomeric or fibrillar structures in a highly ordered and efficient manner. This self-assembly process is accompanied by a structural transition of the aggregated proteins from their normal fold into a predominantly β-sheet secondary structure. 14 ns molecular dynamics simulation revealed that fulvic acid interrupted the dimer formation of Aβ_17–42 peptide while in its absence Aβ_17–42 dimer formation occurred at ~ 12 ns. Additionally, fulvic acid disrupted the preformed Aβ_17–42 trimer in a very short time interval (12 ns). These results may provide an insight in the drug design against Aβ_17–42 peptide aggregation using fulvic acid as lead molecule against Aβ_17–42 mediated cytotoxicity and neurodegeneration.     

Complete microscale profiling of tumor microangiogenesis: A microradiological methodology reveals fundamental aspects of tumor angiogenesis and yields an array of quantitative parameters for its characterization

Complete profiling would substantially facilitate the fundamental understanding of tumor angiogenesis and of possible anti-angiogenesis cancer treatments. We developed an integrated synchrotron-based methodology with excellent performances: detection of very small vessels by high spatial resolution (~ 1 μm) and nanoparticle contrast enhancement, in vivo dynamics investigations with high temporal resolution (~ 1 ms), and three-dimensional quantitative morphology parametrization by computer tracing. The smallest (3–10 μm) microvessels were found to constitute > 80% of the tumor vasculature and exhibit many structural anomalies. Practical applications are presented, including vessel microanalysis in xenografted tumors, monitoring the effects of anti-angiogenetic agents and in vivo detection of tumor vascular rheological properties.     

Ulminium diluviale Unger : historique de la découverte et nouvelle étude

A fossil tree was discovered during the 16th century at Jáchymov (Bohemia). The wood was first named by Unger, in 1842, Ulminium diluviale. But it belongs to the Lauraceae family and Felix, in 1883, named it Laurinoxylon diluviale. The authors give the history and the geological setting of the area and describe the anatomy of the wood. The diagnosis of the genus Laurinoxylon Felix, 1883. is emended as follows: heteroxylous fossil wood with average sized solitary vessels or in radial groups; perforation plates simple and sometimes scalariform; intervascular pits alternate and moderately large; thyloses present. Paratracheal parenchyma. Uni to five seriate rays, slightly heterocellular and less than 1 mm high; ray-vessel pits large often stretched. Libriform or with radial pits fibres. Oil cells or mucilage (idioblasts) present. The diagnosis of the species Laurinoxylon diluviale (Unger) Felix, 1883. is also emended. Heteroxylous fossil wood with distinct growth rings; late wood poorly developed with vessels of diameter distinctly smaller as compared to the early wood and with smaller diameter fibres. Diffuse to semiporous vessels, solitary or in radial groups of two to seven , nine to 16 pores/mm²; tangential diameter 100 to 154 μm in early wood and 44 to 72 μm in late wood; vessel length 300 to 550 μm; perforation plates simple and scalariforme (6–12 bars); intervascular pits alternate, rounded (diameter 7–10 μm) or elliptic (long axes × short axes: 10–15 μm × 7–10 μm); thylosis present. Paratracheal parenchyma in more or less complete rows (1–2 cells wide) around the vessels. Heterocellular rays (1–(3) rows of upright cells), of one to five, more frequently three to four cells wide (80%); two to 36 cells high (60 to 820 μm); six to seven rays per tangential millimetre; vessels-rays pits sometimes large, stretched horizontally to vertically. Fibres of 15 to 25 μm in diameter; cell walls of 2–3 μm thick; pits not seen. Oil cells (idioblasts) at the ray margins; 27–60 μm in tangential diameter; 50–80 μm in radial diameter; 72–140 μm high; density of zero to 18 per transversal square millimetres depending on the observed area.     

Excitation dynamics in Photosystem I from Chlamydomonas reinhardtii. Comparative studies of isolated complexes and whole cells

Identical time-resolved fluorescence measurements with ~ 3.5-ps resolution were performed for three types of PSI preparations from the green alga, Chlamydomonas reinhardtii: isolated PSI cores, isolated PSI–LHCI complexes and PSI–LHCI complexes in whole living cells. Fluorescence decay in these types of PSI preparations has been previously investigated but never under the same experimental conditions. As a result we present consistent picture of excitation dynamics in algal PSI. Temporal evolution of fluorescence spectra can be generally described by three decay components with similar lifetimes in all samples (6–8 ps, 25–30 ps, 166–314 ps). In the PSI cores, the fluorescence decay is dominated by the two fastest components (~ 90%), which can be assigned to excitation energy trapping in the reaction center by reversible primary charge separation. Excitation dynamics in the PSI–LHCI preparations is more complex because of the energy transfer between the LHCI antenna system and the core. The average trapping time of excitations created in the well coupled LHCI antenna system is about 12–15 ps longer than excitations formed in the PSI core antenna. Excitation dynamics in PSI–LHCI complexes in whole living cells is very similar to that observed in isolated complexes. Our data support the view that chlorophylls responsible for the long-wavelength emission are located mostly in LHCI. We also compared in detail our results with the literature data obtained for plant PSI.     

Surface-dependent coagulation enzymes. Flow kinetics of factor Xa generation on live cell membranes

The initial surface reactions of the extrinsic coagulation pathway on live cell membranes were examined under flow conditions. Generation of activated coagulation factor X (fXa) was measured on spherical monolayers of epithelial cells with a total surface area of 41-47 cm(2) expressing tissue factor (TF) at >25 fmol/cm(2). Concentrations of reactants and product were monitored as a function of time with radiolabeled proteins and a chromogenic substrate at resolutions of 2-8 s. At physiological concentrations of fVIIa and fX, the reaction rate was 3.05 +/- 0.75 fmol fXa/s/cm(2), independent of flux, and 10 times slower than that expected for collision-limited reactions. Rates were also independent of surface fVIIa concentrations within the range 0.6-25 fmol/cm(2). The transit time of fX activated on the reaction chamber was prolonged relative to transit times of nonreacting tracers or preformed fXa. Membrane reactions were modeled using a set of nonlinear kinetic equations and a lagged normal density curve to track the expected surface concentration of reactants for various hypothetical reaction mechanisms. The experimental results were theoretically predicted only when the models used a slow intermediate reaction step, consistent with surface diffusion. These results provide evidence that the transfer of substrate within the membrane is rate-limiting in the kinetic mechanisms leading to initiation of blood coagulation by the TF pathway.     

Conversion of waste produced by the deodorization of palm oil as feedstock for the production of biodiesel using a catalyst prepared from waste material

The distillate produced by deodorization of palm oil (DDPO) is a waste that corresponds to 4% of the product formed in this process. DDPO is 83% free of fatty acids (FFA), making it a good material for biodiesel production. In this paper, a catalyst prepared from a waste material, Amazon flint kaolin, was used for the esterification of DDPO with methanol. Leached metakaolin treated at 950 °C and activated with 4 M sulfuric acid (labeled as MF9S4) offered maximum esterification activity (92.8%) at 160 °C with a DDPO:methanol molar ratio of 1:60 and a 4-h reaction time. The influences of reaction parameters, such as the molar ratio of the reactants, alcohol chain length, temperature, time and the presence of glycerides and unsaponifiable matter, have also been investigated. Based on the catalytic results, esterification of DDPO using MF9S4 can be a cheaper alternative for production of sustainable fuels.     

Hydrogen atom abstraction from HOOOCl by chlorine atom and OH radical

Gas-phase reactions of HOOOCl with both Cl atom and OH radical are investigated using ab initio methods. The structures of all reactants, products, intermediates, and transition states have been optimized and characterized with the quadratic configuration interaction (QCISD) method. The overall mechanism for the Cl + HOOOCl and OH + HOOOCl reaction is the formation of HCl + O₂ + ClO and H₂O + O₂ + ClO, respectively. The rate-limiting step in each reaction is the abstraction of hydrogen from HOOOCl by either Cl or OH radicals and the barrier height is predicted to be 1.9 kcal mol⁻¹ and 8.1 kcal mol⁻¹ for abstraction by Cl atom and OH radical, respectively. Since both barriers for hydrogen abstraction are high, the reaction is suggested to be slow. These results also suggest that an atmospheric removal mechanism for HOOOCl may result from reaction with Cl atoms rather than with OH radicals, and that photolysis of HOOOCl may be the major removal mechanism for the intermediate.     

Rapid mixing methods for exploring the kinetics of protein folding

Information on the time-dependence of molecular species is critical for elucidating reaction mechanisms in chemistry and biology. Rapid flow experiments involving turbulent mixing of two or more solutions continue to be the main source of kinetic information on protein folding and other biochemical processes, such as ligand binding and enzymatic reactions. Recent advances in mixer design and detection methods have opened a new window for exploring conformational changes in proteins on the microsecond time scale. These developments have been especially important for exploring early stages of protein folding.     

Effective rate models for the analysis of transport-dependent biosensor data.

Optical biosensors, including the BIACORE, provide an increasingly popular method for determining reaction rates of biomolecules. In a flow chamber, with one reactant immobilized on a chip on the sensor surface, a solution containing the other reactant (the analyte) flows through the chamber. The time course of binding of the reactants is monitored. Scientists using the BIACORE to understand biomolecular reactions need to be able to separate intrinsic reaction rates from the effects of transport in the biosensor. For a model to provide a useful basis for such an analysis, it must reflect transport accurately, while remaining simple enough to couple with a routine for estimating reaction rates from BIACORE data. Models have been proposed previously for this purpose, consisting of an ordinary differential equation with 'effective rate coefficients' incorporating reaction and transport parameters. In this paper we investigate both the theoretical basis and numerical accuracy of these and related models.     

Temperature manifold for a stopped-flow machine to allow measurements from −10 to +40 °C

Conducting enzymatic stopped-flow experiments at temperatures far removed from ambient can be very problematic because extremes in temperature (<10 °C or >30 °C) can damage the machine or the enzyme. We have devised a simple manifold that can be attached to most commercial stopped-flow systems that is independently heated or cooled separate from the main stopped-flow system. Careful calibration of the flow circuit allows the sample to be heated or cooled to the measurement temperature (−8 to +40 °C) 1 to 2 s before mixing in the reaction chamber. This approach allows measurements at temperatures where the stopped flow or the protein is normally unstable. To validate the manifold, we investigated the well-defined ATP-induced dissociation of rabbit muscle myosin subfragment 1 (S1) from its complex with pyrene-labeled actin. This process has both temperature-dependent and -independent components. Use of ethylene glycol allowed us to measure the reaction below 0 °C and up to 42 °C, and as expected the second-order rate constant (K₁k₊₂) and the maximum rate of dissociation (k₊₂) both increased with temperature, whereas 1/K₁ is unaffected by the change in temperature.     

Green synthesis of silver nanoparticles using cellulose extracted from an aquatic weed; water hyacinth

As part of the desire to save the environment through “green” chemistry practices, we herein report an environmentally benign synthesis of silver nanoparticles (Ag-NPs) using cellulose extracted from an environmentally problematic aquatic weed, water hyacinth (WH), as both reducing and capping agent in an aqueous medium. By varying the pH of the solution and reaction time, the temporal evolutions of the optical and morphological properties of the as-synthesised Ag-NPs were investigated. The as-synthesised cellulose capped silver nanoparticles (C–Ag-NPs) were characterised using Fourier transform infrared spectroscopy (FTIR), ultraviolet–visible spectroscopy (UV–vis), transmission electron microscopy (TEM) and high resolution transmission electron microscopy (HRTEM). The maximum surface plasmon resonance (SPR) peak decreased as the pH increased indicating that an increase in the pH of the solution favoured the formation of smaller particles. In addition, instantaneous change in the colour of the solution from colourless to brown within 5 min at pH 11 showed that the rate of reduction is faster at this pH compared to those at lower pH. The TEM micrographs showed that the materials are small, highly monodispersed and spherical in shape. The average particle mean diameters were calculated to be 5.69 ± 5.89 nm, 4.53 ± 1.36 nm and 2.68 ± 0.69 nm nm at pH 4, 8 and 11 respectively. The HRTEM confirmed the crystallinity of the material while the FTIR spectra confirmed the capping of the as-synthesised Ag-NPs by the cellulose. It has been shown therefore that based on this synthetic method, this aquatic plant can be used to the advantage of mankind.     

Extraction,characterization and spontaneous emulsifying properties of pectin from sugar beet pulp

The effects of organic acid extractants on the yield and characteristics of pectin from sugar beet pulp were investigated with citric acid, malic acid and lactic acid at different pH (1.5 and 2.0) and time (1 h and 2 h). The results demonstrated that the yields of pectins were directly correlated with the decrease of pH and reaction time, and the optimum yield of 17.2% was obtained at pH 1.5 and 2 h. Furthermore, the acid type also affected the physicochemical characteristics of pectin, especially on the esterification degree (42–71), galacturonic acid content (60.2–77.8%), emulsion activity (35.2–40.1%) and emulsion stability (62.1–79.4%), and a relatively single pectin mainly consisted of homogalacturonan could be obtained under a suitable reaction condition, which was an excellent crude material for the production of emulsion activity.     

Kinetic investigation of recombinant human hyaluronidase PH20 on hyaluronic acid

The kinetic investigation of hyaluronidases using physiologically relevant hyaluronic acid (HA or hyaluronan) substrate will provide useful and important clues to their catalytic behavior and function in vivo. We present here a simple and sensitive method for kinetic measurement of recombinant human hyaluronidase PH20 (rHuPH20) on HA substrates with sizes ranging from 90 to 752 kDa. The method is based on 2-aminobenzamide labeling of hydrolyzed HA products combined with separation by size exclusion–ultra performance liquid chromatography coupled with fluorescence detection. rHuPH20 was found to follow Michaelis–Menten kinetics during the initial reaction time. Optimal reaction rates were observed in the pH range of 4.5–5.5. The HA substrate size did not have significant effects on the initial rate of the reaction. By studying HA substrates of 215, 357, and 752 kDa, the kinetic parameters K_m, V_max, and k_cat were determined to be 0.87–0.91 mg/ml, 1.66–1.74 nM s⁻¹, and 40.5–42.4 s⁻¹, respectively. This method allows for direct measurement of kinetics using physiologically relevant HA substrates and can be applied to other hyaluronidase kinetic measurements.     

Real-time optical studies of respiratory Complex I turnover

Reduction of Complex I (NADH:ubiquinone oxidoreductase I) from Escherichia coli by NADH was investigated optically by means of an ultrafast stopped-flow approach. A locally designed microfluidic stopped-flow apparatus with a low volume (0.2 μl) but a long optical path (10 mm) cuvette allowed measurements in the time range from 270 μs to seconds. The data acquisition system collected spectra in the visible range every 50 μs. Analysis of the obtained time-resolved spectral changes upon the reaction of Complex I with NADH revealed three kinetic components with characteristic times of < 270 μs, 0.45–0.9 ms and 3–6 ms, reflecting reduction of different FeS clusters and FMN. The rate of the major (τ = 0.45–0.9 ms) component was slower than predicted by electron transfer theory for the reduction of all FeS clusters in the intraprotein redox chain. This delay of the reaction was explained by retention of NAD⁺ in the catalytic site. The fast optical changes in the time range of 0.27–1.5 ms were not altered significantly in the presence of 10-fold excess of NAD⁺ over NADH. The data obtained on the NuoF E95Q variant of Complex I shows that the single amino acid replacement in the catalytic site caused a strong decrease of NADH binding and/or the hydride transfer from bound NADH to FMN.