[Cross-over comparison of the pharmacokinetics of estradiol during hormone replacement therapy with estradiol valerate or micronized estradiol]. / Uberkreuz-Vergleich der Pharmakokinetik von Estradiol unter der Hormonsubstitution mit Estradiolvalerat oder mikronisiertem Estradiol.

OBJECTIVE: The aim of this randomized cross-over study was the comparison between a sequential 28-day hormone replacement therapy (HRT) using micronized estradiol and a cyclic 21-day HRT using estradiol valerate with regard to the pharmacokinetics of estradiol. - MATERIAL AND METHODS: Fifty postmenopausal women were randomly assigned to be treated either with Trisequens(R) for 28 days or with Sisare(R) for 21 days. After a wash-out cycle, the women were treated for one cycle with the other preparation in a cross-over fashion. The pharmacokinetic profile of the serum concentrations of estradiol was measured on day 1, 21 and 28 each immediately before and 1, 2, 4, 6, 8, and 10 hours after intake of a tablet, and the AUC (area under the curve) was calculated. - RESULTS: The serum concentrations of estradiol increased from a mean of 10 pg/ml up to 40 pg/ml (Trisequens(R)) and 30 pg/ml (Sisare(R)) on day 1, and to 80 pg/ml (Trisequens(R)) and 60 pg/ml (Sisare(R)) on day 21, and declined to 40 pg/ml (Trisequens(R)) and 10 pg/ml (Sisare(R)) on day 28. The AUC as calculated from both treatment cycles, was significantly higher on day 1, 21, and 28 during treatment with Trisequens(R) than with Sisare(R). This difference was, however, not signifcant on day 1 and 21 of the first treatment cycle. - CONCLUSION: During treatment with 2 mg micronized estradiol the serum concentrations are significantly higher than with 2 mg estradiol valerate. On day 28 of treatment with Sisare(R), the estradiol levels decline to baseline values, while using Trisequens(R) they remain in the range of those measured on day 1.

Genetic rearrangements in the pathogenicity locus of Clostridium difficile strain 8864--implications for transcription, expression and enzymatic activity of toxins A and B.

The pathogenicity locus (PaLoc) of Clostridium difficile isolate 8864 was investigated to locate genetic rearrangements that would explain the exceptional pathogenicity of this particular isolate. Two major changes were defined: an insertion of 1.1 kb between the two genes tcdA and tcdE, coding for the enterotoxin and an accessory protein of unknown function, respectively, and a deletion of 5.9 kb encompassing the 3' ends of tcdA and tcdC. Transcription of the tcdA-E genes is severely affected by both rearrangements, explaining the demonstrated complete lack of TcdA polypeptide. We present a model of coordinate, growth-related transcription of the tcdA-E genes that confirms our previous findings in strain 10463. Recombinant TcdA-8864 had UDP-glucose-glucosyltransferase activity, proving that the N-terminal 698 amino acids of the polypeptide represent the catalytic domain. However, this truncated TcdA molecule lacks a ligand and translocation domain. To assess the catalytic domain of TcdB-8864, the sequence of the 5' end of its gene was determined. TcdB-8864 shows high homology to TcdB-1470 but lower homology to TcdB-10463 within this domain. This fits well with the altered glucosylation specificity of TcdB-8864 (Rac1, Rap2 and Ra1). Having defined the variations of transcription, expression and enzymatic activity of toxins A and B, implications for the pathogenic potential of strain 8864 are discussed.

The C-terminal ligand-binding domain of Clostridium difficile toxin A (TcdA) abrogates TcdA-specific binding to cells and prevents mouse lethality.

We have investigated the ability of a recombinant protein (REP231), derived from Clostridium difficile toxin A C-terminal domain, to protect against toxin A (TcdA) intoxication in vitro and in vivo. REP231 was cloned, expressed and purified by thyroglobulin affinity chromatography, and demonstrated identical binding properties to TcdA. Immunofluorescence experiments and in vitro cytotoxicity assays using mouse teratocarcinoma cells F9 showed that specific binding of TcdA to F9 cells through its C-terminal domain is essential for producing cytotoxic effects. TcdA binding and cytotoxicity was inhibited by REP231 and a monoclonal antibody directed against the C-terminal domain. Toxin B did not bind to F9 cells and was consequently inactive in cytotoxicity assays. Inhibition studies with lectins and a Le(x)-specific antibody supported earlier findings that a terminal galactose is part of the bound saccharide but excluded Le(x) as a receptor for TcdA. Mice immunised with REP231 were protected against a threefold lethal dose of TcdA. Thus, REP231 appeared to be a suitable candidate to develop an alternative therapeutic agent, which is able to neutralise carbohydrate-mediated TcdA binding and might act as a vaccine.

Delineation of the catalytic domain of Clostridium difficile toxin B-10463 to an enzymatically active N-terminal 467 amino acid fragment.

In an attempt to directly approach the postulated toxic domain of Clostridium difficile's TcdB-10463, eight subclones of different size and locations in the N-terminal third of the toxin were generated. Expression of these toxin fragments was checked in Western blots and the enzymatic activity of the expressed proteins was analyzed by glucosylating Ras related small GTP-binding proteins. Two polypeptides of 875 aa (TcdBc1-3) and 557 aa (TcdBc1-H) glucosylated their targets Rho, Rac and Cdc42 with the same activity and specificity as the holotoxin. In comparison 516 aa (TcdBc1-N) and 467 aa (TcdBc1-A) protein fragments exhibited highly reduced activity, while Tcdc1 and TcdB2-3 (aa 1-243 and 244-890, respectively) were enzymatically inactive. Our results indicate that all structures involved in the catalysis are located at several different sites within the 557 aa fully active fragment. The shortest enzymatically still active protein covers aa 1-467 and obviously fulfils all minimal requirements for glucosylation. The data support the postulated three domain model of 'large clostridial cytotoxins'.

Transcription analysis of the genes tcdA-E of the pathogenicity locus of Clostridium difficile.

To analyse the transcription pattern of the five tcdA-E genes of the pathogenicity locus (PaLoc) of Clostridium difficile a protocol was established to purify RNA from strain VPI10463. Transcription analysis of the five tcdA-E genes showed that they were all transcribed. In the early exponential phase, a high level of tcdC and low levels of tcdA,B,D,E transcripts were detectable; this was inverted in the stationary phase, suggesting that TcdC might have a negative influence on transcription of the other genes. Three transcription initiation sites, one for tcdA and two for tcdB were determined by primer extension analysis. Readthrough transcripts from outside the locus were not obtainable, so that parts of the transcription of tcdD, tcdB, tcdA and tcdC must occur by monocistronic transcription. Within the locus all possible intergenic readthrough transcripts were detectable except that between tcdC and tcdA, a stretch of DNA interrupted by a functional transcription terminator. Thus we found mono- and polycistronic transcription of tcdA and tcdB to occur which should lead to production of a surplus of tcdA over tcdB transcripts. This would explain the surplus of TcdA over TcdB expression observed in vitro. Due to its basic nature and similarity to BcnA of Clostridium perfringens and to Orf-22 of Clostridium botulinum, TcdD is most probably a regulatory protein with DNA-binding properties. On the basis of the presented study we discuss a model for the growth-phase-related, coordinate regulation of toxin expression wherein tcdC has a negative and tcdD a positive regulatory function on transcription of the tcdD,B,E and tcdA genes.

Definition of the single integration site of the pathogenicity locus in Clostridium difficile.

We determined the nucleotide sequence 3.8 kb upstream and 5.2 kb downstream of the toxin genes A and B of Clostridium difficile. Nine ORFs were discovered. Based on PCR-directed approaches, two were attributed to the pathogenicity locus (PaLoc). The other seven were found in every C. difficile isolate obtained from the human gastrointestinal tract, respectless of their toxinogenicity. The ORFs cdu1 and cdu2/2' upstream of the PaLoc displayed similarity to repressors of Gram-positive bacteria (cdu1), and to an Na+/H+ antiporter described for Enterococcus hirae (cdu2/2'). Downstream of the locus a putative ABC transporter (cdd2-4) was identified. With a set of three paired primers used in polymerase chain reactions we succeeded in delineating the PaLoc. Sequencing of the appropriate stretch of DNA in C. difficile VPI10463 and four additional toxinogenic strains proved a high conservation of the borders of the PaLoc in all these strains. Our data define the locus as a distinct genetic element. Comparing the sequences of five toxinogenic and five non-toxinogenic strains the integration site of the PaLoc was defined. This showed that a stretch of 115 bp found in non-toxinogenic strains is replaced by the 19-kb locus in toxinogenic strains. Analysis of the boundary sequences showed that the locus is obviously not a mobile genetic element by itself. Instead we propose that it is the independent pathogenic part of a more extended genetic element associated with virulence. The 115 bp of non-toxinogenic strains replaced by the locus in toxinogenic strains carry the putative transcription terminator of the cdu1, a predicted repressor protein. A possible polar effect of the loss of this terminator on transcription of the TcdABCDE genes is discussed. Such an effect would explain the unidirectional insertion of the PaLoc at a single site of the C. difficile genome and might give a rationale for the development of the disease which is induced after antibiotical treatment.

Modification of DNA bases by anthralin and related compounds.

Modification of bases in calf thymus DNA by treatment with the antipsoriatic drug anthralin was studied. The products of DNA bases were identified and their yields measured by gas chromatography-mass spectrometry with selected ion monitoring. Treatment of calf thymus DNA with anthralin significantly enhanced the amount of modified bases above control levels. Purine bases were modified to products identical with those known to be typical of DNA damage induced by hydroxyl radicals. The yields of Fapy-adenine, 8-hydroxyadenine, Fapy-guanine, and 8-hydroxyguanine were maximally increased at an anthralin concentration of 75 microM. A variety of structural analogues of anthralin were also tested at 75 microM were either weaker or stronger hydroxylating agents. It is likely that damage to DNA bases induced by anthrones contributes to their antiproliferative activity. The pharmacological implications of these characteristics of the action of anthralin on DNA bases are discussed.

Antipsoriatic anthrones with modulated redox properties. 2. Novel derivatives of chrysarobin and isochrysarobin--antiproliferative activity and 5-lipoxygenase inhibition.

A novel series of 2- and 3-substituted 1,8-dihydroxy-9(10H)-anthracenones were synthesized and tested for their inhibitory activity against 5-lipoxygenase (5-LO) in bovine polymorphonuclear leukocytes and the growth of human keratinocytes. Structure-activity relationships are discussed with respect to the following redox properties of the compounds: reactivity against 2,2-diphenyl-1-picrylhydrazyl, generation of hydroxyl radicals as measured by deoxyribose degradation, and inhibition of lipid peroxidation in model membranes. Inhibition of cell proliferation seemed to be related to these properties, whereas 5-LO inhibition was not. Within a class of structural analogs the activity against 5-LO, which was markedly improved as compared to that of the antipsoriatic drug anthralin, correlated well with the overall lipophilicity. Even though a number of compounds in this series enhanced oxidative damage to nonlipid molecules such as deoxyribose, their antioxidant properties predominate in membrane lipids. Among the prooxidant compounds were also the most potent antiproliferative agents (IC50 values in the 10(-7) M range).