The isoxazole derivative Leflunomide (HWA 486) is a novel immunoregulatory and anti-inflammatory drug. Affinity chromatography was used to purify and identify Leflunomide binding proteins, which might play a role as potential cellular targets in the molecular mode of action. The Leflunomide derivative A 0273 was covalently coupled to a Fractogel(R) matrix. This column was used to separate a cytosolic protein extract of the macrophage cell line RAW 264.7 by several selected and specific gradient elution steps. Proteins that were specifically eluted through the active metabolite of Leflunomide, A 1726, were identified by subsequent protein sequence analysis. This allowed us to specify 10 cytosolic proteins, which bind with high affinity to this matrix. Three of them, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and phosphoglycerate mutase belong to the second part of the glycolytic pathway. The binding specificity of these protein/drug interactions was further evaluated using BIAcore(R) analysis. Kd values of glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and lactic dehydrogenase were similar to the Kd value of a known Leflunomide target protein, dihydroorotate dehydrogenase. In order to elucidate the features as well as the overall relevance of these results, cytosolic fractions of three additional cell lines MOLT-4, A20.2J, HeLa were compared using the same chromatographic protocol. The elution profiles as well as subsequent Western blot analyses confirmed the data obtained previously for the macrophage cell line RAW 264.7.

Prp2p, Prp16p, Prp22p, and Prp43p are members of the DEAH-box family of ATP-dependent putative RNA helicases required for pre-mRNA splicing in Saccharomyces cerevisiae. Recently, mammalian homologues of Prp43p and Prp22p have been described, supporting the idea that splicing in yeast and man is phylogenetically conserved. In this study, we show that a murine cell line resistant to the novel immunoregulatory drug Leflunomide (Arava) overexpresses a 135-kDa protein that is a putative DEAH-box RNA helicase. We have cloned the human counterpart of this protein and show that it shares pronounced sequence homology with Prp16p. Apart from its N-terminal domain, which is rich in RS, RD, and RE dipeptides, this human homologue of Prp16p (designated hPrp16p) is 41% identical to Prp16p. Significantly, homology is not only observed within the phylogenetically conserved helicase domain, but also in Prp16p-specific sequences. Immunofluorescence microscopy studies demonstrated that hPrp16p co-localizes with snRNPs in subnuclear structures referred to as speckles. Antibodies specific for hPrp16p inhibited pre-mRNA splicing in vitro prior to the second step. Thus, like its yeast counterpart, hPrp16p also appears to be required for the second catalytic step of splicing. Taken together, our data indicate that the human 135-kDa protein identified here is the structural and functional homologue of the yeast putative RNA helicase, Prp16p.

The combination of two‐dimensional electrophoresis (2‐DE) and subsequent Western blot analysis with antibodies directed against common cellular proteins is a straightforward and reliable method to quickly generate fix points in a protein map. In order to assure high accuracy in the allocation of protein spots, two different replica blotting methods for semidry blotting devices were established. The first of the two was described by Johansson ( Electrophoresis 1987, 8 , 379–383). By systematically changing the direction of the blotting current, proteins were simultaneously transferred from one gel onto two membranes placed at both sides of the gel. However, several modifications of this method were necessary in order to use a semidry blotting device. The second method described here combines the standard blotting procedure with the generation of a ‘contact copy’ from the gel. Both systems offer the possibility to subject one membrane to antibody‐mediated imaging, while the second membrane can be stained with highly sensitive total protein detection procedures. Protein identification is then carried out by comparing the signals on both matrices.

The analysis of bile acids in biological matrices, e.g. gall bladder bile and serum, depends on accurate and reproducible sample pre-treatment techniques. The present study compares published and commonly used protocols with a special focus on the analysis of serum bile acids. Since adsorption of bile acids to serum proteins has a dramatic impact on the recovery rate and the reproducibility of the method used, several deproteination procedures were compared. The combination of given protocols made it possible to develop a very sensitive and efficient sample pre-treatment which is well suited for all chromatographic methods for bile acid analysis.

The effect of the pretreatment with the powder of crataegus oxyacantha on the release of lactate dehydrogenase (LDH) during ischemia and reperfusion was studied in an isolated rat heart model. Male Wistar rats were divided into control and crataegus group (for which the standard diet was mixed with a 2% crataegus powder standardized to 2.2% flavonoids). The investigations started 3 months after commencing the treatment. The hearts were isolated and a retrograde perfusion was performed at constant pressure according to the technique of Langendorff. The experimental protocol comprised 10 min equilibration, according to the technique of Langendorff. The experimental protocol comprised 10 min equilibration, according to the technique of Langendorff. The experimental protocol comprised 10 min equilibration, 110 min occlusion of the left anterior descending coronary artery, and 30 min reperfusion. The coronary effluent was sampled for the LDH determination after 5, 30, 90, 120 and 150 min. The LDH activity, which was initially very low in both groups (control, 16.5 +/- 4.3; crataegus, 26.0 +/- 8.8 mU/min) increased slightly during the ischemia, and very strongly as soon as the heart was reperfused. However, the increase in the crataegus group was significantly lower (1777.3 +/- 451.9 vs control 3795.3 +/- 511.9 mU/min, p = 0.01). At the end of the reperfusion period, LDH activity decreased markedly but did not reach the ischemic values. The attenuation of the LDH release by crataegus pretreatment suggests a preservation of the cell membrane and a protection from myocardial damage.

In order to analyse bile acids in biological matrices, e.g. rat bile and human serum, high performance liquid chromatography (HPLC) was coupled to continuous-flow fast atom bombardment mass spectrometry (CFFAB-MS). A gradient elution system which had already proved to be well suited for the quantitative determination of conjugated bile acids in bile was modified to allow HPLC-CFFAB-MS-coupling. Due to the sensitivity of this coupling method it is possible to obtain more information about the biliary bile acid pattern and species-specific secondary bile acids. Furthermore, we were able to identify obviously unknown bile acid species in rat bile which most likely classify as mono-oxo and di-oxo-taurocholates (MW 513 Da, 511 Da) and mono-oxo-glycocholates (MW 463 Da). In the present study we show that using this system it is possible to determine both conjugated and unconjugated as well as sulfated bile acids, without time consuming group separation and derivatization, from rat bile and human serum. In addition, it is suggested that the method presented here should be considered for use in routine analysis.

The influence of several important experimental conditions on the thin layer chromatographic separation of bile acids has been evaluated. The major focus of the study was the influence of the temperature, ionic strength, and apparent pH of the eluent, rather than its composition. The effect of adding modifiers to the eluent or support were also tested. With silica gel supports, reduction of the temperature to 5-degrees-C had an overall beneficial effect on resolution and band shape, and so improved quantification of the chromatograms, whereas increasing the apparent pH generally had an unfavorable effect. Separations on reversed phase materials were more dependent on the ionic strength and less influenced by variations in the apparent pH. Reduced temperature also had beneficial effect on separations on reversed phase materials. The influence of temperature and the other conditions should, therefore, be intensively considered with regard to critical separation problems in thin layer chromatography.

Glycosyl-phosphatidylinositol-anchored membrane proteins (GPI-proteins) are normally identified either by cleavage of the lipid anchor using (glycosyl)phosphatidylinositol-specific phospholipases C or D (GPI-PLs) or by metabolic labeling of the lipid moiety with specific building blocks. Therefore, methods for discrimination between transmembrane proteins and GPI-proteins on the basis of their physicochemical properties are desirable. Here we are presenting a selective extraction method for typical well-characterized mammalian GPI-proteins, e.g., acetylcholine esterase, alkaline phosphatase, 5′-nucleotidase, and lipoprotein lipase, using a derivative of taurocholate. The results were compared to those obtained with well-characterized transmembrane proteins, e.g., insulin receptor and hydroxymethyl glutaryl coenzyme A-reductase, glucose transporters, or aminopeptidase M and several commercially available detergents. With regard to total membrane proteins, it was possible to selectively enrich GPI-proteins up to 8- to 14-fold by using concentrations between 0.1 and 0.3% of 4′-NH2-amino-7β-benzamido-taurocholic acid (BATC). In addition, the cleavage specificity and efficiency of (G)PI-PLs were increased in the presence of identical concentrations of BATC compared to commonly used detergents, e.g., Nonidet P-40. Therefore, the present study shows that the use of BATC facilitates the identification of glycosyl-phosphatidylinositol-anchored membrane proteins.

Cholic acid (1) has been coupled via ω-aminoalkoxy spacer at C-3 to chlorambucil (3), HR 780 (4) and oxaproline peptide (5). Drug conjugates 9, 11, 18 exhibit strong affinity to specific bile acid transport systems.

Evaluation of the yield of expression of exogeneous protein in transformed Escherichia coli cells by means of one-dimensional SDS-PAGE often leads to overestimation and miscalculation. For example, it is possible that proteins of similar size comigrate and thus mask the overexpressed product band. Therefore, two-dimensional electrophoresis was used to analyze two types of recombinant fusion proteins, i.e., a beta-galactosidase insulin fusion protein and a interleukin II insulin fusion protein, directly after fermentation. We found that production scale expression products show charge and size heterogeneity. The heterogeneous protein spots were characterized by subsequent blotting onto Immobilon membrane and by N-terminal sequencing. Some of the separated spots were either N-terminally blocked or already degraded to some extent. The integrity of the actual product component of the fusion protein was examined with a C-terminus-specific antibody and by Western blot analysis of the 2D gels.

Methodology for the preparation of 7α-12α-dihydroxy-3β- (2) and 7α,12α-dihydroxy-3α-(2-hydroxyethoxy)-5β-cholanic acid (3) is described. Nucleophilic displacement of the 3-mesylate of unprotected cholic acid with ethylene glycol led to the 3β-isomer whereas the 3α-isomer was synthesized via the 7,12-diacetyl protected 3-allyl ether of methyl cholate. Only the 3α-isomer 3 is recognized by the ileal bile acid transport system with affinity comparable to cholic acid.

In evaluating the purification of genetically engineered human insulin there is no plausible correlation between the yield of expression as determined by SDS-PAGE (taking into account all normally occurring losses during the several purification steps) and the actual yield, i.e., the final pure product. The aim of our work was to develop a fast, accurate, and reproducible method for the quantification of the initial yield of the intact insulin fusion protein directly after fermentation and without prior purification of the fermentation product. Therefore, a protocol for efficient tryptic digestion of protease-resistant inclusion bodies was established. The resulting crude peptide mixture was oxidized by performic acid and the insulin A-chain, which contains no cleavage side for trypsin, was quantified by HPLC in the form of a tetrasulfonate derivative to reduce artefacts due to free -SH groups. Compared with SDS-PAGE, this procedure allows sensitive monitoring of possible degradation at the C-terminus. Furthermore, quantification of expression products on the basis of the present method will provide better correlation between initial and actual yield.