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Stefan Werner

Publications and source records attributed to Stefan Werner.

9 recordsLinked to original sources
PubMed2007

HPLC determinations of enantiomeric ratios.

Evaporative light-scattering detection (ELSD) is widely applied in the HPLC analysis of organic compounds lacking a UV chromophore. However, this detection method is generally unsuitable for determination of enantiomeric ratios (er). The er calculated from a UV trace and an ELS trace of the same compound differs significantly. Because of the nonlinear concentration response of the ELS detector, a compound with an er of 95:5 appears to be enantiomerically pure by ELS detection. It is possible to obtain a calibration curve and to calculate a correction factor, but this procedure is time consuming and therefore not very practical for routine analyses. In contrast, a charged aerosol detector allows a very accurate determination of the enantiomeric ratios. Like the ELS detection, the CA detection is independent of the chromophore properties of the substrate. Therefore, we recommend the use of CA instead of ELS detection for determination of the enantiomeric ratios of non-UV active compounds.

Chromatography, High Pressure Liquid↗
PubMed2006

Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A.

Earlier attempts to express peptides longer than 20 aa on the surface of tobamoviruses such as tobacco mosaic virus have failed. Surprisingly, we found that a functional fragment of protein A (133 aa) can be displayed on the surface of a tobamovirus as a C-terminal fusion to the coat protein via a 15-aa linker. The macromolecular nature of these nanoparticles allowed the design of a simple protocol for purification of mAbs with a recovery yield of 50% and > 90% product purity. The extremely dense packing of protein A on the nanoparticles (> 2,100 copies per viral particle) results in an immunoadsorbent with a binding capacity of 2 g mAb per g. This characteristic, combined with the high level of expression of the nanoparticles (> 3 g adsorbent per kg of leaf biomass), provides a very inexpensive self-assembling matrix that could meet the criteria for a single-use industrial immunoadsorbent for antibody purification.

Antibodies, Monoclonal↗
PubMed2006

Use of transcriptional synergy to augment sensitivity of a splicing reporter assay.

A primary limitation in the development and use of screens to identify factors that regulate mammalian pre-mRNA splicing has been the development of sensitive reporter assays. Alternative splicing typically involves relatively small (< 10-fold) changes in isoform ratios. Therefore, reporter constructs designed to allow direct analysis of isoform expression historically have at most a 10-fold window of discrimination between a positive signal and background. Here we describe the design and application of a reporter cell line that makes use of the phenomenon of transcriptional synergy to amplify the detection of changes in splicing, such that a three- to five-fold change in splicing pattern is observed as a 30- to 50-fold change in GFP expression. Using this cell line we have identified two small molecules, from a library of approximately 300 synthetic compounds, that can induce partial repression of a variable exon from the CD45 gene. We propose that the concept of transcription-based amplification of signal will allow the development of true high-throughput screening approaches to identify effectors of mammalian alternative splicing.

Alternative Splicing↗
PubMed2006

A screening method for chiral selectors that does not require covalent attachment.

A high-throughput screening protocol is proposed for chiral selector discovery. It is modeled after the protocol for biological screening of candidate drugs from chemical libraries. The procedure works based on target distribution between an aqueous phase and an organic phase. The target may be a racemate or separate enantiomers. Screening for noncovalent intermolecular association between target and candidate selectors is carried out by partitioning experiments in the presence and absence of the candidate chiral selectors in the organic phase (plasticized poly(vinyl chloride)). The partition ratio measurement uses 96-well plates for high throughput. The feasibility of this approach is validated by working with a known target/chiral selector pair, N-(3,5-dinitrobenzoyl)-alpha-phenylglycine and 2,2,2-trifluoro-1-(9-anthryl)ethanol. The validated protocol is applied to a small library of 12 cyclopropyl dipeptide isosteres. Eight bind the racemic target, econazole. Among them, one has measurable chiral selectivity. The advantage of the method is that it does not require the covalent attachment of either the analyte or the selector, and the required amount of the potential chiral selector is about 100 mug.

Anthracenes↗
PubMed2004

Expression of the fibroblast growth factor-2 isoforms and the FGF receptor 1-4 transcripts in the rat model system of Parkinson's disease.

Basic fibroblast growth factor (FGF)-2 occurs in different isoforms representing different translation products of a single mRNA. We have previously shown that the high molecular weight FGF-2 isoforms (21, 23 kD) stimulated survival- and neurite-promoting activities and protective effects on cultured embryonic dopaminergic (DA) neurons of the substantia nigra (Neuroscience 100 (2000) 73). In this study the expression of FGF-2 isoforms in the striatum and substantia nigra was analyzed by Western blot in adult intact rats and following complete unilateral 6-hydroxydopamine (6-OHDA) lesion. In intact rats, all three FGF-2 isoforms (18, 21, 23 kD) are expressed. Neurotoxin-mediated lesion of nigral DA neurons revealed no change of the FGF-2 isoform expression pattern in the nigrostriatal system. Additionally, the FGF receptors 1, 2 and 3 are expressed in these tissues and displayed no alterations after 6-OHDA injection as demonstrated by RT-PCR. The presence of all three FGF-2 isoforms and the FGFR 1-3, together with the previous demonstrated neurotrophic effects of FGF-2 on dopaminergic neurons, suggest a physiological function of the FGF-2 isoforms in the nigrostriatal system.

Animals↗
PubMed2003

Fluorous dienophiles are powerful diene scavengers in Diels-Alder reactions.

[reaction: see text] Three fluorous dienophiles have been synthesized, and their value in scavenging an excess diene after Diels-Alder reactions is shown. The resulting fluorous derivatives are separated by solid-phase extraction on fluorous silica gel (FSPE). The fluorous [1,2,4]triazoline-3,5-dione 10 reacted with most dienes within seconds or minutes.

Aldehydes↗
PubMed2002

Chitin synthesis during in planta growth and asexual propagation of the cellulosic oomycete and obligate biotrophic grapevine pathogen Plasmopara viticola.

PCR amplification of two CHS gene fragments of the obligate biotroph Plasmopara viticola, the causal agent of downy mildew of grapevine, is described. While one fragment shows homology to fungal class IV chitin synthases, the other fragment groups with other oomycete chitin synthases to form a novel class of chitin synthases most closely related to class I-III. RT-PCR experiments indicate that PvCHS1 is constitutively expressed, whereas PvCHS2 is specifically transcribed in sporangiophores and sporangia. Analyses of wheat germ agglutinin labeling patterns by confocal laser scanning microscopy show that chitin is present on the surface of hyphal cell walls during in planta growth, and of sporangiophores and sporangia.

Chitin↗
PubMedDate not supplied

Solution-phase synthesis of a tricyclic pyrrole-2-carboxamide discovery library applying a stetter-Paal-Knorr reaction sequence.

The solution-phase synthesis of a discovery library of 178 tricyclic pyrrole-2-carboxamides was accomplished in nine steps and seven purifications starting with three benzoyl-protected amino acid methyl esters. Further diversity was introduced by two glyoxaldehydes and 41 primary amines. The combination of Pauson-Khand, Stetter, and microwave-assisted Paal-Knorr reactions was applied as a key sequence. The discovery library was designed with the help of QikProp 2.1, and physicochemical data are presented for all pyrroles. Library members were synthesized and purified in parallel and analyzed by LC/MS. Selected compounds were fully characterized.

Amides↗