Characterization of five polyamine oxidase isoforms in Arabidopsis thaliana.

The genome of Arabidopsis thaliana contains five genes (AtPAO1 to AtPAO5) encoding polyamine oxidase (PAO) which is an enzyme responsible for polyamine catabolism. To understand the individual roles of the five AtPAOs, here we characterized their tissue-specific and space-temporal expression. AtPAO1 seems to have a specific function in flower organ. AtPAO2 was expressed in shoot meristem and root tip of seedlings, and to a higher extent in the later growth stage within restricted parts of the organs, such as shoot meristem, leaf petiole and also in anther. The expression of AtPAO3 was constitutive, but highest in flower organ. AtPAO3 promoter activity was detected in cotyledon, distal portion of root, boundary region of mature rosette leaf and in filaments of flower. AtPAO4 was expressed at higher level all over young seedlings including roots, and in the mature stage its expression was ubiquitous with rather lower level in stem. AtPAO5 expression was observed in the whole plant body throughout various growth stages. Its highest expression was in flowers, particularly in sepals, but not in petals. Furthermore, we determined the substrate specificity of AtPAO1 to AtPAO4. None of the AtPAO enzymes recognized putrescine (Put). AtPAO2 and AtPAO3 showed almost similar substrate recognition patterns in which the most preferable substrate is spermidine (Spd) followed by less specificity to other tetraamines tested. AtPAO4 seemed to be spermine (Spm)-specific. More interestingly, AtPAO1 preferred thermospermine (T-Spm) and norspermine (NorSpm) to Spm, but did not recognize Spd. Based on the results, the individual function of AtPAOs is discussed.

Membrane cartridges for endotoxin removal from interferon preparations.

The suitability of membrane cartridges for the removal of endotoxin from both distilled water and interferon preparations was examined. The endotoxin concentrations were reduced to 4.0 and 7.3 EU/ml, respectively, when about 4000 ml of distilled water with 20 and 28 EU/ml were passed through the deoxycholate and chitosan immobilized membrane cartridges. When 200 ml of interferon preparation with endotoxin concentration more than 80 EU/ml and pH 3.9 were applied to a deoxycholate immobilized membrane cartridge at a flow-rate of 9 ml/min, the endotoxin concentration was reduced to less than 10 EU/ml. However, if an interferon preparation of 450 ml, with more than 80 EU/ml of endotoxin and pH 3.9 was applied to the chitosan immobilized membrane cartridge at a flow-rate of 18 ml/min, the endotoxin concentration was reduced to less than 10 EU/ml.

A new type of chemically bonded phase for reversed-phase HPLC.

A new type of ether-bonded packing for reversed-phase HPLC (RP-HPLC) was synthesized by reacting 1-octanol with beta-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, followed by coupling the product onto porous silica. The prepared packing was characterized by elemental analysis, solid-state 13C NMR, and Fourier transform infrared (FT-IR) spectroscopy. Chromatographic evaluations were performed by using a mixture of organic compounds as the analyte and methanol-water as binary mobile phase. The influence of the composition of organic modifier on the retention behavior of basic compounds was studied. The hydrolytic stability of the packing between pH 2.5-7.5 was also investigated. The results showed that the new stationary phase has excellent chromatographic properties and good hydrolytic stability.

[Direct enantioseparation of pantoprazole sodium by high performance liquid chromatography on teicoplanin-bonded chiral stationary phase].

Teicoplanin-bonded chiral stationary phase was prepared and used for the direct chiral separation of racemic pantoprazole sodium by reversed-phase high performance liquid chromatography (HPLC). The influences of organic modifiers, the composition of mobile phase, the flow rate and column temperature on enantioseparation were studied. It was found that methanol was preferred than acetonitrile when used as organic modifier. With the increase of column temperature, chiral solutes were less retained and the separation factor value decreased indicating that lower temperature was better for the enantioseparation. Better resolution was obtained at lower flow rates as a result of minimization of the resistance of mass transfer. Based on these experimental data, the chiral HPLC method for the analysis of pantoprazole sodium on prepared chiral column (150 mm x 4.6 mm i. d., 5 microm) was established with simplicity and good reproducibility, by using methanol-water (35 : 65, v/v) as mobile phase at a flow rate of 0.6 mL/min and 20 degrees C.

[Direct enantiomeric separation of pantoprazole sodium by high performance liquid chromatography].

By using the Kromasil CHI-TBB chiral stationary phase, the racemic mixture of pantoprazole sodium was resolved in the normal phase mode. The influences of the composition of the mobile phase, the content of acidic and basic additives and the flow rate on this enantiomer separation were studied. The chiral high performance liquid chromatographic method for the analysis of pantoprazole sodium was established with high efficiency and good repeatability, by using hexane-isopropanol-acetic acid mixture (95:5:0.1, v/v) as the mobile phase at a flow rate of 2.0 mL/min at 25 degrees C.

[Chiral separation of beta-receptor blockers and analogs on novel norvancomycin-bonded chiral stationary phase].

Enantiomeric separation of racemates of 8 beta-receptor blockers and analogs was performed on a novel norvancomycin-bonded chiral stationary phase (NVC-CSP) in polar organic mode using high performance liquid chromatography. The influences of mobile phase composition, acetic acid and triethylamine contents, temperature and flow rate on enantiomeric separation were studied. It was found experimentally that with the increase of the fraction of methanol in the mobile phase, the same tendency was found for all the chiral solutes studied, i. e., the decrease in retention factor (kappa) and the increase in the enantioselectivity factor (alpha). With the increase of column temperature, most solutes were less retained and the alpha value decreased accordingly. Better resolutions were obtained at lower flow rates as a result of the minimization of the resistance to mass-transfer. Better separation results and suitable retentions for most solutes were obtained when a mixture of acetonitrile-methanol-acetic acid-triethylamine (60: 40: 0.4: 0.2, v/v) was used as mobile phase at a flow rate of 0.6 mL/min, and the column temperature was set at 25 degrees C. Conclusions can be drawn from the experimental data that pi-pi complexation, dipole-stacking interactions were engaged in the process of enantiomeric separation, while hydrogen bonding interaction between solutes and CSP was commonly non-enantioselective, although it was mainly responsible for the increased retention in the acetonitrile-predominant mobile phases.

Chiral separation of enantiomers of amino acid derivatives by high-performance liquid chromatography on a norvancomycin-bonded chiral stationary phase.

A novel norvancomycin-bonded chiral stationary phase (NVC-CSP) was synthesized by using the chiral selector of norvancomycin. The chiral separation of enantiomers of several dansyl-amino acids by high-performance liquid chromatography (HPLC) in the reversed-phase mode is described. The effects of some parameters, such as organic modifier concentration, column temperature, pH and flow rate of the mobile phase, on the retention and enantioselectivity were investigated. The study showed that ionic, as well as hydrophobic interactions were engaged between the analyte and macrocycle in this chromatographic system. Increasing pH of buffers usually improved the chiral resolution for dansyl-alpha-amino-n-butyric acid (Dns-But), dansyl-methionine (Dns-Met) and dansyl-threonine (Dns-Thr), but not for dansyl-glutamic acid (Dns-Glu) which contains two carboxylic groups in its molecular structure. The natural logarithms of selectivity factors (lnalpha) of all the investigated compounds depended linearly on the reciprocal of temperature (1/T), most processes of enantioseparation were controlled enthalpically. Interestingly, the process of enantioseparaton for dansyl-threonine was enthalpy-controlled at pH of 3.5, while at pH of 7.0, it was entropy-controlled according to thermodynamic parameters Delta(R,S)DeltaH degrees and Delta(R,S)DeltaS degrees afforded by Van't Hoff plots. In order to get baseline separation for all the solutes researched, norvancomycin was also used as a chiral mobile phase additive. In combination with the NVC-CSP, remarkable increases in enanselectivity were observed for all the compounds, as the result of a "synergistic" effect.

[Simple preparation and evaluations of amide-octyl-bonded phase].

A simple preparation process of amide-octyl-bonded phase (AOBP) is described. In the process aminopropyltriethoxysilane first reacted with octanoyl chloride and then the obtained intermediate was bonded onto porous silica. Characterization of prepared packing was carried out with elemental analysis, solid-state nuclear magnetic resonance (13C-NMR) and Fourier transform infrared (FTIR) spectroscopy. Chromatographic evaluations were carried out by testing with a mixture including acidic, basic and neutral organic compounds using methanol-water as binary mobile phase. The results show that the stationary phase has excellent chromatographic properties and resistance to hydrolysis between pH 2.5-7.5. It can be used for the efficient separation of basic compounds.