Evaluation of two immunoassay procedures for drug testing in hair samples.

A preliminary initial enzyme-linked immunosorbent assay (LUCIO-Direct ELISA kit) and a preliminary DRI enzyme immunoassay were evaluated for drug detection in head hair with respect to lowered cutoff values recommended in Germany for the control of abstinence in cases of re-granting of drivers' licences. Following drug classes were included: cannabinoids, opiates, cocaine like substances, amphetamine, methamphetamine (and methylenedioxyamphetamines), methadone, and benzodiazepines. 759 analyses were performed using LUCIO-Direct ELISA kits and 936 analyses using DRI enzyme immunoassay tests. Sample size for each drug group and immunoassay test reached from 74 to 178. The LUCIO-Direct ELISA kit revealed a sensitivity of 91% for amphetamine up to 98% for methadone (methamphetamine 92%, cocaine 94%, opiates 94%, benzodiazepines 96%) and values of specificity of 72% for methadone up to 89% for amphetamine and benzodiazepines. The test was not useful for a preliminary screening for tetrahydrocannabinol (sensitivity of 65%) in consideration of a suggested cutoff of 0.02 ng/mg. The DRI enzyme immunoassay test was only useful for morphine and cocaine testing at low recommended new cutoff values (0.1 ng/mg) revealing sensitivities of 94% and 99%, respectively.

The nuclear envelope in the plant cell cycle: structure, function and regulation.

BACKGROUND: Higher plants are, like animals, organisms in which successful completion of the cell cycle requires the breakdown and reformation of the nuclear envelope in a highly controlled manner. Interestingly, however, while the structures and processes appear similar, there are remarkable differences in protein composition and function between plants and animals. SCOPE: Recent characterization of integral and associated components of the plant nuclear envelope has been instrumental in understanding its functions and behaviour. It is clear that protein interactions at the nuclear envelope are central to many processes in interphase and dividing cells and that the nuclear envelope has a key role in structural and regulatory events. CONCLUSION: Dissecting the mechanisms of nuclear envelope breakdown and reformation in plants is necessary before a better understanding of the functions of nuclear envelope components during the cell cycle can be gained.

Short cut of protein purification by integration of cell-disrupture and affinity extraction.

Screening strategies based on functional genomics require the isolation of gene products of several hundred cDNA clones in a fast and versatile manner. Conventional purification strategies will fail to accomplish this goal within a reasonable time frame. In order to short-cut these procedures, we have developed a combination of cell disintegration and affinity technique for rapid isolation and purification. For our purpose, tagged proteins have been produced in yeast by fusing the FLAG-sequence adjacent to the 5' end of cDNAs coding for the respective protein. The example of an over-expressed FLAG-tagged fusion protein, human serum albumin (HSA), was released into the cytoplasm. Detection and purification of the FLAG-fusion protein were carried out by using a mouse monoclonal antibody directed against the FLAG-peptide. For purification purposes, the antibody was immobilized on PROSEP magnetic glass beads. These magnetic glass beads with 500 microns diameter have been investigated for disintegration of yeast and simultaneous capturing of the target protein. After 60 s, 90% of the maximal disintegration level was achieved when a ratio of 20 microliters yeast cell suspension and 100 microliters glass are vortexed. After a wash step, the FLAG-fusion proteins have been eluted with chelating agents such as EDTA. The short-cut procedure has been compared to a conventional purification strategy using an affinity chromatography process. Due to the highly favorable binding characteristics of the applied immunoaffinity sorbent the yield observed in batch operation was 90% and purity in the range of 70-80%.

Agonistic and synergistic activity of tamoxifen in a yeast model system.

The background of agonist/antagonist behaviour of the non-steroidal antiestrogen tamoxifen is still not fully understood. Depending on cell type, its activities range from full agonistic to antagonistic in different tissues. We investigated the transactivational properties of tamoxifen in a basic yeast model system which reconstitutes ligand-dependent human estrogen receptor-alpha (hER alpha) gene activation. Tamoxifen exerted low agonist activity in this system compared to 17 beta-estradiol (E2). Efficiencies and potencies of several isomers were calculated by fitting experimental data with a logistic dose-response function. Cis-, trans- and cis-transtamoxifen and trans-4-hydroxytamoxifen (4-OHT) showed comparable efficiencies and potencies in yeast. When subeffective doses of trans-, cis-/trans-, or trans-4-OH tamoxifen were combined with increasing 17 beta-estradiol concentrations, even a synergistic increase in efficiencies could be observed. Interestingly, the cis-isomer did not show this synergistic effect. Mutation of the N-terminus of the estrogen receptor changed the transactivational behaviour of tamoxifen and abolished the synergistic action with 17 beta-estradiol. Except for 4-OHT, the potencies of the investigated isomers, defined as ligand concentrations with half-maximal response, highly correlated with the binding affinities to hER alpha. Therefore, cis-, trans-, and cis-/trans-tamoxifen could be regarded as full agonists in yeast, while 4-OHT was regarded as a partial antagonist in yeast. Furthermore, these results indicate that the functional difference between trans-tamoxifen and trans-4-OHT is not due to their different affinities for the receptor protein.

Quantitative assessment of complex formation of nuclear-receptor accessory proteins.

Like other nuclear receptors, steroid hormone receptors form large protein hetero-complexes in their inactive, ligand-friendly state. Several heat-shock proteins, immunophilins and others have been identified as members of these highly dynamic complexes. The interaction kinetics and dynamics of hsp90, hsp70, p60 (Hop), FKBP52, FKBP51, p48 (Hip) and p23 have been assessed by a biosensor approach measuring the complex formation in real time. A core chaperone complex has been reconstituted from p60, hsp90 and hsp70. p60 forms a molecular bridge between hsp90 and hsp70 with an affinity in the range of 10(5) M(-1). Dynamics of hsp90-p60 complex formation is modulated by ATP through changes in the co-operativity of interaction. At low protein concentrations ATP stabilizes the complex. Binding of p23 to hsp90 did not change the affinity of the hsp90-p60 complex and the stabilizing effect of ATP. Saturation of the p48-hsp70 interaction could not be achieved, suggesting multiple binding sites. A picture of the protein complex, including stoichiometric coefficients, co-operativity of interaction and equilibrium-binding constants, has been formed.

Affinity chromatography of human estrogen receptor-alpha expressed in Saccharomyces cerevisiae. Combination of heparin- and 17beta-estradiol-affinity chromatography.

Estrogen receptor-alpha is a member of the nuclear hormone receptor superfamily and is considered as a very important regulatory protein. Human estrogen receptor-alpha has been cloned into Saccharomyces cerevisiae as a fusion to ubiquitin and expression is controlled by a metallothionin promotor. Pilot scale quantities of receptor have been produced by a yeast strain transformed with expression plasmid YEpE13 [Graumann et al., J. Steroid Biochem. Mol. Biol. 57 (1996) 293] in a 14 l stirred tank reactor. The yeast extract contained 2-4 pmol of receptor protein per mg total protein. A purification scheme has been developed using heparin-affinity chromatography combined with affinity chromatography with immobilized 17beta-estradiol 17-hemisuccinate. Heparin-affinity chromatography was very efficient to remove host cell protein. Accompanying proteins that stabilize unoccupied receptor have not been dissociated during elution. The receptor could be purified 5-10-fold in ligand-free state. In contrast to previous reports, we did not find a difference of the binding affinity of liganded and unliganded receptor for heparin immobilized onto Sepharose. The unoccupied receptor could be further purified 100-fold with ligand-affinity chromatography using 17beta-estradiol 17-hemisuccinate-bovine serum albumin-Sepharose. The receptor could be kept in its native state, although saturated with 17beta-estradiol. The purification sequence allows an efficient production of receptor. Further improvement of productivity can be only accomplished by increasing the expression level.

Monitoring of estrogen mimics by a recombinant yeast assay: synergy between natural and synthetic compounds?

Properties of mixtures of compounds exhibiting estrogenic potential have been questioned in the past. Synergistic effects of endocrine disrupters have been proposed, but could never be confirmed. In this study, the transactivational potential of xenoestrogens and phytoestrogens has been evaluated in a yeast test system. Pesticides such as endosulfan, dieldrin, atrazine, and the main metabolites, desethylatrazine and desisopropylatrazine, have been tested and their behavior as mixtures is compared to the behavior of the single compounds. Our results are in contrast to a report (Tran et al., 1996) on the inhibitive effects of xenoestrogens on 17 beta-estradiol-dependent transactivation. Phytoestrogens have been investigated in a similar manner. A synergistic effect could not be confirmed for both, xenoestrogens and phytoestrogens. These compounds are either weak estrogens or completely lack estrogenic potential. Their endocrine disrupting potential in more complex systems must be therefore attributed to other molecular mechanisms such as to metabolic modification or interference with steroidogenesis. This study shows that yeast systems are useful tools for monitoring pure estrogenic properties.

Regulation of human estrogen receptor by phytoestrogens in yeast and human cells.

Phytoestrogens are defined as plant substances that are structurally or functionally similar to estrogen. They are present in many foods and their higher consumption in certain populations has been correlated with protection against many diseases including coronary heart disease, breast cancer and endometrial and ovarian cancer. In this report, ten phytoestrogens with diverse chemical structures were studied for their binding to the human estrogen receptor and their transcription activation properties in yeast and mammalian cells. Our results showed that some of these compounds bind with relatively high affinity to the estrogen receptor and activate the receptor in the yeast and mammalian cell system. In addition, none of these compounds showed anti-estrogenic activity. We conclude that the yeast system accurately predicts the estrogenic activity of compounds with diverse chemical structures in mammalian cells. In addition, our data with phytoestrogens that do not show transcription activation properties raise the possibility that these compounds may exert their biological effects through pathways different from the classical estrogen signalling mechanism.

Structural and functional analysis of N-terminal point mutants of the human estrogen receptor.

Twenty N-terminal point mutations of the human estrogen receptor (hER) were constructed as ubiquitin fusion products and expressed under the control of the copper regulated promoter CUP1 in Saccharomyces cerevisiae. The objective of these studies was to overexpress hER in yeast and also to evaluate the functional properties of the N-terminal variants of hER. Fusion of the C-terminus of ubiquitin to the N-terminus of other proteins has been shown to increase the level of protein expression in yeast. Ubiquitin C-terminal hydrolases (UCHs) in yeast efficiently and precisely cleave at the junction with ubiquitin and render free hER with desired amino termini. The variant hER proteins, that were generated by mutating the N-terminus of hER, showed enormous differences in receptor protein levels and transactivation potential. All variant hER proteins were synthesized as 66 kDa species as identified by Western blotting with the exception of the proline-containing variant (Pro-ER). The UB-Pro-ER variant was cleaved inefficiently by UCHs in yeast. The UB-Pro-hER [correction of UB-Pro-hEr] variant also exhibited a different DNA band-shift profile compared to those of the other receptor variants and the wild-type. Val-, Thr-, and Lys-ER did not express, as measured by enzyme-immunoassay and Western blotting; nor did they transactivate a beta-galactosidase reporter gene in yeast. However, the Glu-ER was 50% more active in transactivation as compared to the wild-type. The results of the receptor content, DNA binding properties and transactivation analysis in yeast demonstrate that the N-terminal residue plays an important role in the structure and function of hER.

Laboratory-scale production and purification of recombinant HIV-1 reverse transcriptase.

HIV-1 reverse transcriptase from the HIV-1 strain WMF 1.13 was expressed in Escherichia coli JM 105 using a pKK233-2 vector. The bacteria were cultivated in a 20-l fermentor with 14-l net volume using M9ZB medium containing bactotryptone and yeast extract. After induction of reverse transcriptase (RT) expression by addition of isopropyl-beta-D-thiogalactopyranoside the enzyme concentration was monitored. Both soluble and inclusion-body deposited RT were detected by Western blots. Inclusion-body formation was confirmed by transmission electron microscopy. Further purification of soluble and insoluble RT was investigated. After cell desintegration by enzymatic treatment combined with osmotic shock and centrifugation, the supernatant was desalted by size-exclusion chromatography and further purified by DEAE-Sepharose FF, AF-Heparin Toyopearl 650 M and Fractogel EMD TMAE 650 (S). The results of the purification steps were monitored by SDS-PAGE with silver staining, non-radioactive RT assay and protein determination with Coomassie Blue. The sediment was extracted with 6 M GuHCl and after clarification and conventional refolding, treated in the same manner as soluble RT. This method is well suited for studying fermentation conditions as well as purification conditions. The RT is expressed in approximately equal amounts as soluble and insoluble enzyme.