Quantitative analysis of cytokinins in plants by liquid chromatography–single-quadrupole mass spectrometry

A sensitive method for the quantitative analysis of all natural isoprenoid cytokinins in plant material by electrospray single-quadrupole mass spectrometry is presented. A baseline chromatographic separation of 20 non-derivatised naturally occurring cytokinins has been developed. Precise analyses of O-glucoside and ribonucleotide fractions were also performed by the high-performance liquid chromatography–mass spectrometry (HPLC–MS) but run separately from the basic cytokinin metabolites. Using post-column splitting, the flux from narrow-bore (2.1 mm i.d.) reversed-phase liquid chromatography column was simultaneously introduced into the diode array and mass detector. Optimal conditions, including final flow rate, desolvation temperature, desolvation gas flow, capillary and cone voltage for effective ionisation in the electrospray ion source were found. When low cone voltage (20 V) was applied, all studied cytokinins were determined in aqueous methanol as dominant quasi-molecular ions of [M+H]⁺ with limits of detection ranging between 10 and 50 fmol. For routine analysis a linearity range between 25 (75) fmol and 100 pmol was obtained. Developed liquid chromatography–mass spectrometry (LC–MS) method in selective ion monitoring mode was employed to quantify cytokinin species in tobacco BY-2 suspension culture and poplar leaves (Populus×canadensis Moench, cv Robusta).

Purified plant cell (BY-2) and plant tissue (poplar leaves) extracts were obtained by using two different ion-exchange chromatography steps, in combination with immunoaffinity purification using a broad-spectrum monoclonal anti-cytokinin antibody. The antibody strongly recognises the presence of N⁶-substituent on purine skeleton and thus does not bind adenine and related compounds. The presence of authentic cytokinins in the extracts quantified by LC–MS was further verified by enzyme-linked immunosorbent assays (ELISAs) with prior LC preparation. The combination of liquid chromatography–single-quadrupole mass spectrometry with immunoaffinity chromatography offers an efficient and elegant method for detection and quantification of cytokinin metabolites.     

Chemistry and biology of dihydroisoxazole derivatives: selective inhibitors of human transglutaminase 2

3-halo-4,5-dihydroisoxazoles are attractive warheads for the selective inhibition of nucleophilic active sites in biological systems. A series of 3-bromo-4,5-dihydroisoxazole compounds were prepared and tested for their ability to irreversibly inhibit human transglutaminase 2 (TG2), an enzyme that plays an important role in the pathogenesis of diverse disorders including Celiac Sprue and certain types of cancers. Several compounds showed high specificity for human TG2 (k(inh)/K(I) > 2000 min(-1)M(-1)) but essentially no reactivity (k < 1 min(-1)M(-1)) toward physiological thiols such as glutathione. The pharmacokinetic and pharmacodynamic properties of a prototype dihydroisoxazole inhibitor, 1b, were evaluated; in mice the compound showed good oral bioavailability, short serum half-life and efficient TG2 inhibition in small intestinal tissue, and low toxicity. It also showed excellent synergism with N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU, carmustine) against refractory glioblastoma tumors in mice. A fluorescent dihydroisoxazole inhibitor 5 facilitated microscopic visualization of TG2 endocytosis from the extracellular surface of HCT-116 cells. Together, these findings demonstrate the promise of dihydroisoxazole compounds as probes for the biology of TG2 and its role in human disease.     

Further Evidence against the Possibility of Faster-than-Light Particles

Arguments are added to the growing number of arguments against the possibility of faster-than-light particles. Causal loops with faster-than-light particles are discussed in the framework of non-quantum special relativity. Thought experiments with steady currents of faster-than-light particles show that the reinterpretation principle is incompatible with the causality principle. If both principles are used consequently paradoxial situations arise. These are visualized utilizing Brehme diagrams. The final conclusion is that particles cannot travel on a macroscopic scale faster than light if space and time are homogeneous and if the relativity and causality principles are valid.     

Antibacterial properties of Ag-doped hydroxyapatite layers prepared by PLD method

Thin hydroxyapatite (HA), silver-doped HA and silver layers were prepared using a pulsed laser deposition method. Doped layers were ablated from silver/HA targets. Amorphous and crystalline films of silver concentrations of 0.06 at.%, 1.2 at.%, 4.4 at.%, 8.3 at.% and 13.7 at.% were synthesized. Topology was studied using scanning electron microscopy and atomic force microscopy. Contact angle and zeta potential measurements were conducted to determine the wettability, surface free energy and electric surface properties. In vivo measurement (using Escherichia coli cells) of antibacterial properties of the HA, silver-doped HA and silver layers was carried out. The best antibacterial results were achieved for silver-doped HA layers of silver concentration higher than 1.2 at.%.     

Synthesis and anthelmintic activity of benzopyrano[2,3-c]pyrazol-4(2H)-one derivatives

Molecular Diversity - A series of benzopyrano[2,3-c]pyrazol-4(2H)-one derivatives were synthesized from readily available 1-phenyl- and 1-methyl-1H-pyrazol-3-ols by sequentially employing...     

The use of atomic force microscopy for imaging the surfaces of polyamide, 6

The surface morphologies of PA 6 resulting from the use of various processing methods were studied by tapping mode atomic force microscopy. Three PA 6 samples: (1) a thin film, spin coated on a silicon wafer, (2) a freestanding film, i.e. a foil and (3) a monofilament, show definite morphological differences revealing typical supramolecular structures. The thin film having thickness of app. 35 nm is a good example of the initial step of spherulite formation where the sheaf development is still prominent. In an area of 100 μm² 1-4 spherulites can be detected which are typical of crystallization from the solution. The annealing (vacuum, 195°C, 3.5h) causes additional crystallization, which leads to a radial coordination and enlargement of spherulites to app. 50% in diameter and up to 40% in height. The morphology of foil (thickness of 100 μm) can be interpreted as a system of spherulites formed from the melt, and a typical fibrillar structure is observed on the surface of monofilament.     

Interaction of acid and alkali treated titanium with dynamic simulated body environment

Interaction of acid and acid+alkali treated titanium samples with simulated body fluid was studied. In case of alkali treated titanium, the dynamic arrangement of the test enabled the detection of primary calcium and phosphate ion adsorption from the solution and later apatite crystal growth (XRD). The induction time for crystal growth was 24.2±0.3 h. On acid-only treated titanium no crystal growth was detected. The calcium phosphate adsorption layer formed on the acid treated samples was detectable by XPS only, however it differed from that one formed on the acid+alkali treated samples. The adsorption layer formed on the acid+alkali treated samples contained larger amount of calcium, especially in the shortest exposure times. Charging of the apatite crystallites during the XPS measurement enabled the determination their Ca/P ratio separately from Ca/P ratio of the adsorption layers. XPS and EDS analyses indicated that the spherulitic crystallites consisted of carbonated hydroxyapatite with the Ca/P ratio close to that one of the stoichiometric hydroxyapatite. It is proposed that the adsorption layer formed spontaneously and immediately on the acid+alkali treated titanium can provide an ideal interface between the metal implant and the apatite cement line, the first structure formed by osteoblast cells during the formation of the new bone on foreign surfaces. This revised version was published online in August 2006 with corrections to the Cover Date.     

Thermodynamics of non-bridging oxygen in silica bio-compatible glass-ceramics

Correlations between the structural properties of Na₂O-CaO-SiO₂ glasses characterized by the activity of oxygen ions and the bioactivity were examined by comparing the compositional dependence of the structural parameters calculated on the basis of a thermodynamic consideration with that of the bioactivity. A simple model of characterizing the glass structure by considering the bridging and non-bridging oxygen ions was employed as the first step for this purpose. Further detailed thermodynamic analysis on the anionic constitution in the glass was performed and the compositional dependences of the relative proportions of bridging, non-bridging and free oxygen ions were calculated. The bioactive region corresponded to the compositional region characterized by the higher relative proportion of non-bridging oxygen ions with co-existing an appreciable concentration of bridging oxygen ions, suggesting a possible important role of the non-bridging oxygen ions on the surface chemical process of bone-like apatite layer formation. This revised version was published online in July 2006 with corrections to the Cover Date.