Biomedical subjects

M Sievertzon

Publications and source records attributed to M Sievertzon.

3 recordsLinked to original sources

PubMed2001

cDNA microarray analysis of small plant tissue samples using a cDNA tag target amplification protocol.

Microarray technology is becoming an important comprehensive tool to study gene expression in plants. However, the use of this technology is limited by the large amount of sample tissue needed for microarray analysis. Generally, 50-200 microg of total RNA and 1-2 microg of mRNA is required for each hybridisation, which is equivalent to 50-100 mg of plant tissue. This requirement for large amounts of starting material severely constrains the use of microarrays for transcript profiling in specific tissues and cell types during plant development. Here we report on a robust and reliable target amplification method that enables transcript profiling from sub-mg amounts of plant tissue. Using 0.1 microg of total RNA we show that twofold expression differences are possible to distinguish with 99% confidence. We also demonstrate the application of this method in an analysis of secondary phloem development in hybrid aspen using defined tissue sections, corresponding to 2-4 cell layers with a fresh weight of approximately 0.5 mg.

DNA, Complementary↗

PubMed1994

Synthesis of fluorescent derivatives of 7-methylguanine through reaction with 2-aryl-substituted malondialdehydes: analysis by HPLC with fluorescence detection.

Fluorescent derivatives of 7-methylguanine were prepared through reaction with 2-aryl-substituted-malondialdehydes and analysed by reversed-phase HPLC with fluorescence detection. Reaction of carbons 1 and 3 of the malondialdehyde molecule at the N1 and N2 positions of 7-methylguanine yielded fluorescent tricyclic structures. Two novel fluorescent derivatives of 7-MeG were obtained, namely, 7-(3,4-dimethoxyphenyl)-10-oxo-1-methyl-9,10-dihydropyrimido[1,2- alpha]purine (yield 15-34%) and 7-(1-naphthyl)-10-oxo-1-methyl-9,10- dihydropyrimido[1,2-alpha]purine (yield 56-70%) after reaction with 3,4-dimethoxyphenylmalondialdehyde and 1-naphthylmalondialdehyde, respectively which were characterized by IR, NMR, MS and UV and fluorescence spectroscopy. The fluorescence intensity of the derivatives was found to be 10-20-fold higher than the intrinsic fluorescence of 7-methylguanine. Concentration versus fluorescence intensity curves exhibit linearity in the picomole to nanomole range. The 2-aryl-substituted malondialdehydes were used to analyse the concentration of 7-methylguanine in neutral hydrolysates obtained from calf thymus DNA samples alkylated with dimethyl sulfate. The results obtained indicate their potential as reagents for the analysis of alkylated guanines in biological samples. Molecular modeling calculations were carried out to generate lowest energy spatial configurations. The results obtained indicated that the aryl-substituents on the malondialdehyde moiety do not lie in the same plane as the tricyclic moiety of the fluorescent derivatives with implications for their fluorescence properties.

Chromatography, High Pressure Liquid↗

PubMedDate not supplied

Comparative immunization study using RNA and DNA constructs encoding a part of the Plasmodium falciparum antigen Pf332.

Development of nucleic acid-based vaccines against parasitic diseases shows great promise, although certain concerns about safety aspects of conventional DNA vaccines have been raised. This study presents a comparison of antibody responses induced in mice by DNA and RNA-based immunization with vectors encoding a part of the P. falciparum antigen Pf332. Two types of plasmids were used, one conventional DNA plasmid containing a cytomegalovirus promoter and one suicidal DNA plasmid encoding the Semliki Forest virus (SFV) replicase. RNA, encoding the SFV replicase and the relevant antigen, was delivered either as naked RNA or packaged in SFV suicide particles. In general, the antibody responses induced by the DNA plasmids were low and peaking after three injections, the conventional plasmid giving the highest responses. Also the RNA delivered in SFV particles consistently induced antibody responses, although comparatively low. Analyses of the ratio of immunoglobulin (Ig)G1/IgG2a subclasses in the responses indicated that all plasmids resulted in a bias for a Th2-type of response, while the SFV-particles elicited a Th1 type of response. Importantly, all these immunogens induced an immunological memory, which could be efficiently activated by a booster injection with the corresponding protein, with unchanged patterns of IgG subclasses.

Animals↗