In vitro mammalian metabolism of the mitosis inhibitor zoxamide and the relationship to its in vitro toxicity.

The in vitro mammalian metabolism of the fungicide zoxamide is related to its in vitro mammalian toxicity. After incubation of zoxamide with rat liver microsomes leading to practically 100% metabolism (mostly hydroxylated zoxamide), the cytotoxicity (methyl thiazole tetrazolium (MTT) test) and the mitosis-inhibiting potential (shown by cell count and by cell cycle analysis) for V79 were not distinguishable from those of zoxamide, demonstrating that the hydroxylation of zoxamide did not change the cytotoxicity or mitosis-inhibiting potential as determined by these assays. After incubation of zoxamide with rat liver S9 predominantly leading to conjugation with glutathione, and after incubation of zoxamide with rat liver slices predominantly leading to the glucuronide of the hydroxylated zoxamide, these activities were eliminated demonstrating that the glutathione conjugate and the glucuronide had lost the activities in these assays due either to no intrinsic potential of these conjugates or to their inability to penetrate the plasma membrane of mammalian cells. It is concluded that the metabolic hydroxylation of zoxamide did not change its activity in the assays used for investigating its influence on cell proliferation, cell cycle and cytotoxicity, while the formation of conjugates with glutathione or glucuronic acid led to the apparent loss of these activities. Thus, with zoxamide as a prototype, it was shown that, in principle, mammalian metabolism and its relationship to mammalian detoxication of fungicidal mitosis inhibitors may be reasonably anticipated from in vitro studies. In addition, the results provide a rational for the observed absence of typically mitosis inhibition-associated toxicities of zoxamide in mammals in vivo.

Rapid analysis of taurine in energy drinks using amino acid analyzer and Fourier transform infrared (FTIR) spectroscopy as basis for toxicological evaluation.

So-called energy drinks with very high amounts of taurine (up to 4000 mg/l are usually granted by certificates of exemption) are increasingly offered on the market. To control the currently valid maximum limits of taurine in energy drinks, a simple and rapid analytical method is required to use it routinely in food monitoring. In this article, we describe a fast and efficient analytical method (FTIR-spectroscopy) that is able to reliably characterize and quantify taurine in energy drinks. The determination of taurine in energy drinks by FTIR was compared with amino acid analyzer (ion chromatography with ninhydrin-postcolumn derivatization). During analysis of 80 energy drinks, a median concentration of 3180 mg/l was found in alcohol-free products, 314 mg/l in energy drinks with spirits, 151 mg/l in beer-containing drinks and 305 mg/l in beverages with wine. Risk analysis of these products is difficult due to the lack of valid toxicological information about taurine and its interferences with other ingredients of energy drinks (for example caffeine and alcohol). So far, the high taurine concentrations of energy drinks in comparison to the rest of the diet are scientifically doubtful, as the advertised physiological effects and the value of supplemented taurine are unproven.

Formation of a covalent Hg-Cys-bond during mercurial activation of PMNL procollagenase gives evidence of a cysteine-switch mechanism.

A common method for the activation of mammalian metalloproteinases is the use of mercurial compounds. Activation of PMNL procollagenase by soluble mercurials takes place as a three-step mechanism with a final intermolecular loss of the PRCGVPD autoinhibitor region. In this study covalently bound mercury in the form of mercurial agarose was chosen to probe activation of PMNL procollagenase. Activation was not achieved, since the final intermolecular cleavage with removal of the PRCGVPD motif could not take place. An intermediate form of the enzyme was bound to the column. Its N-terminal sequence determination proved cleavage of the Asp64-Met65 peptide bond leaving the cysteine of the propeptide domain for covalent attachment to the mercurial agarose. This gives further evidence of a cysteine-switch mechanism involving Cys71.

A 25 kDa alpha 2-microglobulin-related protein is a component of the 125 kDa form of human gelatinase.

Besides the monomeric mammalian 95 kDa progelatinase, two additional forms, a disulfide-bridged 220 kDa dimer and a 125 kDa form were isolated from human PMN leukocytes. The 125 kDa progelatinase was identified as a covalently linked, disulfide-bridged heterodimer formed of the monomer with a 25 kDa protein. This 25 kDa protein was isolated from gelatinase bound to the affinity support of gelatin-Sepharose and eluted by DTE-containing buffer. The amino acid sequence of tryptic peptides of this protein revealed homology with an alpha 2-microglobulin-related protein from rats, a protein so far unknown in humans.

Inactivation of human plasma C1-inhibitor by human PMN leucocyte matrix metalloproteinases.

Highly purified human polymorphonuclear (PMN) leucocyte matrix metalloproteinases, collagenase and gelatinase, cleaved human plasma C1-inhibitor at the carboxyl site of Ala439 (P6). This led to a concomitant loss of C1-inhibitor activity. An additional cleavage site, at the carboxyl site of Ser441 (P4), was observed during PMN leucocyte gelatinase-induced inactivation, and a minor fragment of the plasma C1-inhibitor was generated.

Mercurial activation of human PMN leucocyte type IV procollagenase (gelatinase).

Autoproteolytic activation and processing of human polymorphonuclear leucocyte (PMNL) type IV procollagenase (gelatinase) was initiated by HgCl2 and was investigated by kinetic analysis and N-terminal sequence determination of the reaction products. In the first instance the propeptide domain was lost by subsequent cleavage of the Asp15-Leu16, Glu40-Met41, Leu52-Leu53 and Ala74-Met75 peptide bonds. The PRCGVPD sequence motif (residues Pro78-Asp84), which is conserved in all metalloproteinases and expected to be relevant for latency, remained uncleaved at the activated enzyme. The generated intermediate was further processed by three C-terminal cleavages. The Glu666-Leu667, Ala506-Glu507 and Ala398-Leu399 bonds were hydrolysed successively. From the fragmentation products we were able to conclude that three released fragment peptides contained unpaired free cysteine with the residues Cys497, Cys653, Cys683. Cleavage of the first C-terminal peptide bond resulted in the loss of one of the conserved Cys residues of the hemopexin-like domain, whereas the Cys residue of the PRCGVPD motif was retained at the fully active enzyme. The possibility of an entirely different activation mechanism for PMNL type IV procollagenase is discussed.

A sandwich enzyme immunoassay for the determination of neutrophil lipocalin in body fluids.

Human neutrophil lipocalin was purified from human buffycoat. A polyclonal antibody was obtained by immunisation of rabbits. The antibody reacted with the free lipocalin as well as with the PMNL-gelatinase bound protein. This antibody was used to establish a sensitive sandwich-ELISA for the determination of the protein in body fluids using the biotin/streptavidin system. The mean intra-assay C.V. was 2.3% and the mean inter-assay C.V. 6.7%. The recovery in human plasma was determined to be 98.8%. The ELISA allowed the determination of the protein in the concentration range 0.2-25 micrograms/l. Measurement of the neutrophil lipocalin concentration showed that human plasma of healthy donors contained 9.7 +/- 81 micrograms/l (n = 122) and that the concentrations in serum were significantly higher (P < 0.001) with 133 +/- 90 micrograms/l (n = 122). Neutrophil lipocalin was also found in the urine of healthy donors (8.1 micrograms/l; n = 9). Very high concentrations of this lipocalin were found in the synovial fluids of patients suffering from inflammatory rheumatoid arthritis (1.7 +/- 1.4 mg/l; n = 37).

Determination of metalloproteinases, plasminogen-activators and their inhibitors in the synovial fluids of patients with rheumatoid arthritis during chemical synoviorthesis.

The concentrations of matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-8 (MMP-8), matrix metalloproteinase-9 (MMP-9), lactoferrin and urokinase plasminogen activator (uPA), tissue-type plasminogen activator (tPA) and the inhibitors, tissue inhibitor of metalloproteinase-1 (TIMP-1), plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor (PAI-2), and alpha2-macroglobulin in the synovial fluids of patients with rheumatoid arthritis was determined before and during chemical synoviorthesis with a sodium salt of the fatty acids from cod-liver oil (Varicocid). Synovial fluids were obtained before treatment from 37 patients with rheumatoid arthritis and, in most cases, at 8 and 24 h after injection of the agent. Well-established ELISAs were used to determine the amounts of all proteins. All patients with rheumatoid arthritis revealed very high levels of metalloproteinases (about 1-15 mu g/ml) in their synovial fluids. During the inflammation inducing treatment the granulocyte enzymes increased. In contrast to this, the level of MMP-1 decreased. All granulocyte-derived enzymes were strongly correlated with each other, whereas their dependence on the granulocyte count was only weak. uPA and PAI-2 showed good correlations with the granulocytes-derived enzymes, but were also only weakly correlating with the cell counts. t-PA was not detected by the ELISA used. The proteases, MMP-8, MMP-9 and uPA were increased 8 h after the treatment, whereas the specific inhibitors TIMP-1, PAI-1 and PAI-2 showed significant changes only 24 h after the injection. Matrix metalloproteinases are important factors in the pathogenesis of rheumatoid arthritis. The inflammatory activity in the joint could be better correlated to the granulocyte enzymes than to the granulocyte counts. The levels of uPA and PAI-2 are also parallel to the granulocyte enzyme levels and might underly the same regulatory mechanism.

The human neutrophil lipocalin supports the allosteric activation of matrix metalloproteinases.

The human neutrophil lipocalin (HNL), a member of the large family of lipocalins that exhibit various physiological functions, is coexpressed in granulocytes with progelatinase B (MMP-9). Part of it is covalently bound to the proenzyme and therefore may play a possible role in the activation process of promatrix metalloproteinases. We now report that HNL is able to accelerate the direct activation of promatrix metalloproteinases slightly. A significant enhancement of the activity could be demonstrated for the HgCl2- and the plasma kallikrein-induced activation of all three secretory forms of proMMP-9 and of proMMP-8. The same activating effects were exerted by HNL isolated from granulocytes as well as by the recombinant forms expressed by the yeast Pichia pastoris or by Escherichia coli. This demonstrates that the carbohydrate moiety is not essential for the biological activity of HNL. Activation and activity enhancement are obviously mediated by entrapping the remaining N-terminal sequence residues of the partially truncated proenzyme into the hydrophobic binding pocket of the HNL. In conclusion these results document that HNL can exert an enzyme-activating effect in the regulation of inflammatory and pathophysiological responses of granulocytes in the physiological activation of MMPs that have been subject to limited proteolytic processing.

[Analysis of alpha-amylase activity during pregnancy and labor]. / Bestimmungen der Alpha-Amylase-Aktivität während der Schwangerschaft und unter der Geburt.

Analyses were applied by the authors to 278 samples of maternal serum, 106 amniotic fluid samples, and 81 samples of umbilical vein serum, since no complex study into alpha-amylase activity in the context of pregnancy and labour had been found in the literature. Rise in amylase activity in amniotic fluid, depending on length of gestation, had been one of the points made elsewhere in a publication and was confirmed by the authors' experiments (17th to 22nd weeks of pregnancy: means = 1,8 +/- 0,8 AU/1; 37th to 41st weeks of pregnancy: means = 10,3 +/- 6,0 AU/1). Somewhat relaxed relationships between amylase activity in maternal serum, on the one hand, and age of the pregnant woman as well as activity in amniotic fluid, on the other, were found to be without any clinical relevance. Additional correlations were not detected, not even those with complications in the course of pregnancy and in labour. Global measurement of amylase activity is of no importance to obstetric diagnosis.

A highly sensitive immunoenzymometric assay for the determination of angiogenin.

A polyclonal antibody to human recombinant angiogenin was prepared in rabbits using a Pam3CysSerGly conjugate. The antibody was then used to develop the first highly sensitive enzyme-labelled immunometric assay for this vascularisation inducing and tumour associated protein. The assay was suitable for quantification of angiogenin in body fluids between 2.5 and 0.05 micrograms/l. The mean intra-assay imprecision was 6.0% and the inter-assay imprecision 7.9%. Angiogenin in human plasma was found to lie in the range of 0.38 to 0.11 mg/l with a mean of 0.25 +/- 0.07 mg/l.

Evidence for the tissue inhibitor of metalloproteinases-1 (TIMP-1) in human polymorphonuclear leukocytes.

A tissue inhibitor of metalloproteinases (TIMP)-1 was isolated from human polymorphonuclear leukocytes (PMNL) in a complex with latent 95-kDa gelatinase (matrixmetalloproteinase, MMP-9). It was separated from the enzyme by gel filtration in the presence of SDS. Using a competitive ELISA procedure, we determined that 10% of the isolated gelatinase was complexed with TIMP-1. The presence of the inhibitor in isolated PMNL could also be demonstrated by indirect immunofluorescence using a specific antibody against TIMP-1. Cellular mRNA was isolated from PMNL, which were highly purified by separation via formylMet-Leu-Pro-stimulated chemotactic migration in a Boyden chamber. Using reverse-transcription PCR and Northern blotting, TIMP-1 mRNA was shown to be present in PMNL, suggesting that these cells are also capable of synthesizing TIMP-1.

Preparation of active recombinant TIMP-1 from Escherichia coli inclusion bodies and complex formation with the recombinant catalytic domain of PMNL-collagenase.

TIMP-1 is a member of the family of tissue inhibitors of metalloproteinases involved in regulating the activity of extracellular matrix degrading metalloproteinases. The TIMP-1 cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) amplification of the corresponding mRNA from human fibroblasts. Cloning and expression of the TIMP-1 cDNA were performed in Escherichia coli. In the host vector system chosen, rTIMP-1 is stored intracellularly in its denatured, insoluble form in inclusion bodies. We report a new method for the purification and renaturation of rTIMP-1 from E. coli inclusion bodies to an active inhibitor of matrix metalloproteinases (80% yield), presumably containing the correct assignment of the six disulfide bonds. A resin with the covalently bound recombinant catalytic domain of the PMNL-collagenase as the affinity ligand provided an effective means for the separation of correctly folded, active rTIMP-1 from inactive forms with mismatched disulfides. TIMP-1 and TIMP-2, the two most extensively examined members of the family of tissue inhibitors of metalloproteinases, are known to form a complex with the activated forms of most matrix metalloproteinases and the latent forms of the 92-kDa and 72-kDa gelatinases, respectively. In this study, we report on the complex formation of the recombinant catalytic domain of the PMNL-collagenase with TIMP-1, nonglycosylated recombinant TIMP-1, and recombinant TIMP-2. The Ki values for the different inhibitors were determined in a kinetic assay using a fluorogenic substrate peptide. In this assay, rTIMP-2 had a more effective inhibitory capability against the recombinant catalytic domain of the PMNL-collagenase than TIMP-1.(ABSTRACT TRUNCATED AT 250 WORDS)