E. Van den Eeckhout

Factors influencing Agrobacterium tumefaciens-mediated transformation of Artemisia annua L.

Following our previously described Agrobacterium tumefaciens-mediated transformation procedure for Artemisia annua L., we have undertaken several additional experiments to establish the importance of some parameters such as explant type, age of explant source, A. tumefaciens strain and type of binary vector. Several binary vectors were useful for the production of transgenic callus on explants of different ages. In transformed calli, a good correlation between integration and expression of foreign DNA was observed: different assays showed expression of β-Glucuronidase, neomycin phosphotransferase II, superoxide dismutase and bleomycin acetyl transferase. The regeneration of transgenic plants required more restricted conditions. Only with the pTJK136 vector could transgenic plants be obtained from leaf and stem explants from 12- to 18-week-old plants. Co-cultivation for 48 h seemed favorable for the regeneration of transgenic plants. Stable integration and expression of the transgenes was also shown in the progeny.

Flow cytometric procedure to detect apoptosis of bovine polymorphonuclear leukocytes in blood.

A flow cytometric technique was used to detect apoptosis and necrosis of bovine polymorphonuclear neutrophil leukocytes (PMN) using fluorescein isothiocyanate labeled annexin-V and propidium iodide (PI). Isolation of PMN from the blood following lysis by water or NH4Cl resulted in false positive results for apoptosis. Therefore, a method was developed to identify living, apoptotic and necrotic PMN simultaneously in a single 100 microl blood sample. To establish a positive control for PMN apoptosis, the effect of cycloheximide, actinomycin D, diamide, buthionine sulfoximine and sodium arsenite, that have been described to induce apoptosis by various mechanisms was tested. Only actinomycin D induced a significant increase in the percentage of apoptotic PMN after 2 h. Incubation of blood for 6 h with cycloheximide, actinomycin D and buthionine sulfoximine resulted in a significant increase of apoptotic PMN compared to control values. Sodium arsenite, mainly caused necrosis after 6 h of incubation.

Capillary zone electrophoresis and micellar electrokinetic capillary chromatography of some nonsteroidal antiinflammatory drugs (NSAIDs)

The possibilities of capillary electrophoresis (CE) and micellar electrokinetic capillary chromatography (MEKC) were investigated for the qualitative analysis of some non-steroidal antiinflammatory drugs. In CE the influence of the pH of the buffer and its ionic strength were investigated for a test mixture of six compounds. Also the influence of organic modifiers was studied. The best conditions were applied to the separation of 15 drugs. In MEKC the influence of the concentration of SDS in buffers with pH ranges of 8.0-9.0 was investigated. The influence of an organic modifier, namely acetonitrile was discussed, whereby an interesting phenomenon of change in retention behaviour was noted. A combination of CE and MEKC allows the separation of the 15 above-mentioned compounds and forms an interesting alternative to HPLC.

Fast Atom Bombardment and Tandem Mass Spectrometry for the Identification of Nucleoside Adducts with Phenyl Glycidyl Ether

Abstract The present study deals with the use of fast atom bombardment (FAB) in combination with constant neutral loss (CNL) scanning, high resolution mass spectrometry and tandem mass spectrometry (MS-MS) with collisionally activated decomposition (CAD), as complementary methods for the identification and structural analysis of phenyl glycidyl ether-nucleoside adducts. Selective detection of the parent ions of the modified nucleosides at the 1–10 ng level has been achieved by suitably designed CNL scans. The elemental composition of the adducts has been determined by accurate mass measurements. CAD-MS has been carried out on the [M + H]+ and [M - H]− ions to derive structural data on the size and nature of the base, sugar and alkyl substituent. In some cases, information on the alkylation site has been obtained, which is very useful for distinguishing isomeric adducts.

Evaluation of liquid chromatography—thermospray mass spectrometry in the determination of some phenylglycidyl ether-2′-deoxynucleoside adducts

Abstract The adducts formed between 2′-deoxyadenosine (dAdo), 2′-deoxycytidine (dCyd) and 2′-deoxyuridine (dUrd) and phenyl glycidyl ether (PGE) were analysed by HPLC and LC—thermospray (TSP)-MS. Good results were obtained on a 10 RP Select B column (12.5 cm × 4 mm I.D.) using 0.1 M NH4OAcCH3OH at a flow-rate of 0.8 ml/min. The mass spectra of the 2′-deoxynucleoside—PGE adducts, obtained under LC-TSP-MS conditions were all characterized by the presence of the protonated molecule [MH]+ and [BH + H]+ ions. The PGE-dCyd adduct underwent hydrolytic deamination to the corresponding PGE-dUrd adduct. There was an indication that this process of hydrolytic deamination also took place in the TSP interface. Localization of the alkylation site was possible in the PGE-dUrd adduct by the presence of an RDA rearrangement leading to a fragment ion at m/z 194. Preliminary sensitivity studies on PGE-dUrd showed a detection limit of 500 pg (signal-to-noise ratio = 2) in multiple ion monitoring at m/z 263 and 379.

Radioimmunoassay of bromperidol and haloperidol in human and canine plasma

SummaryIn order to check the specificity of a radioimmunological assay for haloperidol and bromperidol reported by Michiels et al. (1) and commercialized by IRE (Fleurus, Belgium) #, the assay was performed on canine and human plasma samples with and without extraction. After a single dose of bromperidol in dogs, plasma levels obtained with and without extraction were similar. In man the plasma levels for bromperidol after a single administration were markedly lower after extraction. The same was true for haloperidol plasma levels after chronic dosing in man. The findings suggest that the direct radioimmunoassay of haloperidol or bromperidol with the commercialized kit, lacks specificity for humans, possibly because of the presence of one or more polar metabolites.

The impact of hydrogen on the ductility loss of bainitic Fe–C alloys

The influence of hydrogen on the mechanical properties of generic lab-cast Fe–C bainitic alloys is studied by tensile tests on notched samples. The bainitic microstructure is induced in a 0.2% C and 0.4% C Fe–C alloy by an appropriate heat treatment. The hydrogen embrittlement susceptibility is evaluated by mechanical tests on both in situ hydrogen pre-charged and uncharged specimens. The observed ductility loss of the materials is correlated with the present amount of hydrogen and the hydrogen diffusion coefficient. In addition to the correlation between the amount of hydrogen and the hydrogen-induced ductility loss, the hydrogen diffusion during the tensile test, quantified by the hydrogen diffusion distance during the test, appears to be of major importance as well.

Phenylglycidyl ether adducts of 2′-deoxycytidine and 2′-deoxyadenosine: Stability in solution and structure analysis by electrospray tandem mass spectrometry

The adducts of phenylglycidyl ether with 2′-deoxyadenosine (dAdo) and 2′-deoxycytidine (dCyd) exhibit structural modifications. The N-1 adduct of dAdo underwent rearrangement to the N-6 adduct; the N-3 adduct of dCyd was deaminated to the corresponding 2′-deoxyuridine adduct. These structural modifications were studied by using liquid chromatography-electrospray tandem mass spectrometry, and kinetic data for both reactions are presented. The low energy (+) collision-activated dissociation spectra of the dAdo adducts allow the two positional isomers N-1 versus N-6 to be distinguished. The structure of the latter is independently proven by an extended NMR study. For the dCyd and 2′-deoxyuridine adducts, information about the alkylation site is found in the (−) collision-activated dissociation spectra. These spectra show the presence of an unexpected N-4-alkylated dCyd in addition to the two epimeric N-3 adducts.

Evaluation of different packings for high-performance liquid chromatographic analysis of alkyl lysophospholipids

Abstract The analysis of the alkyl lysophospholipid 1-octadecyl-2-0-methyl- d , l -glycero-3-phosphorylcholine is currently under investigation because of its anticancer activity. The chromatographic behaviour of this compound and its 1-hexadecyl-2-0-methyl- d , l -glycero-3-phosphorylcholine homologue, which is used as an internal standard for pharmacokinetic studies, on various liquid chromatography packings gave rise to many problems. The retention and elution characteristics of both ether phospholipids were studied on silica, straight polyethyleneglycol-coated silica, reversed-phase materials, base-deactivated reversed-phase silica and polymeric resins.

Mutagenicity in Salmonella assays of cyclohexane epoxide derivatives

15 Cyclohexane epoxide derivatives were synthesized and compared for direct mutagenicity and bacterial toxicity using Salmonella typhimurium strain TA100 in the liquid suspension and spot-test version of the Ames procedure. While no general correlations could be established for position and stereochemistry of the hydroxylated derivatives, an increase in mutagenicity was noted for the presence of electron-withdrawing groups and unsaturation in conjugation with the oxirane groups.