Phase I and pharmacokinetic study of the topoisomerase II catalytic inhibitor fostriecin.
We conducted a phase I and pharmacokinetic study of the topoisomerase II catalytic inhibitor fostriecin. Fostriecin was administered intravenously over 60 min on days 1-5 at 4-week intervals. Dose was escalated from 2 mg m(-2) day(-1) to 20 mg m(-2) day(-1) in 20 patients. Drug pharmacokinetics was analysed with high performance liquid chromatography with UV-detection. Plasma collected during drug administration was tested in vitro for growth inhibition of a teniposide-resistant small-cell lung cancer (SCLC) cell line. The predominant toxicities were elevated liver transaminases (maximum common toxicity criteria (CTC) grade 4) and serum creatinine (maximum CTC grade 2). These showed only a limited increase with increasing doses, often recovered during drug administration and were fully reversible. Duration of elevated alanine-amino transferase (ALT) was dose-limiting in one patient at 20 mg m(-2). Other frequent toxicities were grade 1-2 nausea/vomiting, fever and mild fatigue. Mean fostriecin plasma half-life was 0.36 h (initial; 95% CI, 0-0.76 h) and 1.51 h (terminal; 95% CI, 0.41-2.61 h). A metabolite, most probably dephosphorylated fostriecin, was detected in plasma and urine. No tumour responses were observed, but the plasma concentrations reached in the patients were insufficient to induce significant growth inhibition in vitro. The maximum tolerated dose (MTD) has not been reached, because drug supply was stopped at the 20 mg m(-2) dose level. However, further escalation seems possible and is warranted to achieve potentially effective drug levels. Fostriecin has a short plasma half-life and longer duration of infusion should be considered. (+info)
RESPONSIVE-TO-ANTAGONIST1, a Menkes/Wilson disease-related copper transporter, is required for ethylene signaling in Arabidopsis.
Ethylene is an important regulator of plant growth. We identified an Arabidopsis mutant, responsive-to-antagonist1 (ran1), that shows ethylene phenotypes in response to treatment with trans-cyclooctene, a potent receptor antagonist. Genetic epistasis studies revealed an early requirement for RAN1 in the ethylene pathway. RAN1 was cloned and found to encode a protein with similarity to copper-transporting P-type ATPases, including the human Menkes/Wilson proteins and yeast Ccc2p. Expression of RAN1 complemented the defects of a ccc2delta mutant, demonstrating its function as a copper transporter. Transgenic CaMV 35S::RAN1 plants showed constitutive expression of ethylene responses, due to cosuppression of RAN1. These results provide an in planta demonstration that ethylene signaling requires copper and reveal that RAN1 acts by delivering copper to create functional hormone receptors. (+info)
Oxidative stress markers in preovulatory follicular fluid in humans.
Intensified peroxidation in the Graafian follicle may be a factor compromising the normal development of the oocyte. The aim of this study was to measure concentrations of three oxidative stress markers: conjugated dienes, lipid hydroperoxides and thiobarbituric acid-reactive substances, in preovulatory follicular fluids and sera of 145 women attending an in-vitro fertilization programme, and to correlate these concentrations with pregnancy outcome. Determinations were conducted either with or without an antioxidant (10 microM butylated hydroxytoluene) and an iron chelate (10 microM deferoxamine mesylate) to examine peroxidation associated with the methods used. Concentrations of conjugated dienes, lipid hydroperoxides and thiobarbituric acid-reactive substances in follicular fluid were all significantly lower than those in serum, both in the presence or absence of the antioxidant and iron chelate. These concentrations did not correlate with pregnancy outcome. In conclusion, the intensity of peroxidation in the Graafian follicle is much lower than that in serum. This gradient is the result of the lower rate of initiation of peroxidation in the follicular fluid, suggestive of the presence of efficient antioxidant defence systems in the direct milieu of the oocyte before ovulation. The concentrations of investigated oxidative stress markers in follicular fluid do not reflect the reproductive potential of oocytes. (+info)
A role for coenzyme M (2-mercaptoethanesulfonic acid) in a bacterial pathway of aliphatic epoxide carboxylation.
The bacterial metabolism of short-chain aliphatic alkenes occurs via oxidation to epoxyalkanes followed by carboxylation to beta-ketoacids. Epoxyalkane carboxylation requires four enzymes (components I-IV), NADPH, NAD(+), and a previously unidentified nucleophilic thiol. In the present work, coenzyme M (2-mercaptoethanesulfonic acid), a compound previously found only in the methanogenic Archaea where it serves as a methyl group carrier and activator, has been identified as the thiol and central cofactor of aliphatic epoxide carboxylation in the Gram-negative bacterium Xanthobacter strain Py2. Component I catalyzed the addition of coenzyme M to epoxypropane to form a beta-hydroxythioether, 2-(2-hydroxypropylthio)ethanesulfonate. Components III and IV catalyzed the NAD(+)-dependent stereoselective dehydrogenation of R- and S-enantiomers of 2-(2-hydroxypropylthio)ethanesulfonate to form 2-(2-ketopropylthio)ethanesulfonate. Component II catalyzed the NADPH-dependent cleavage and carboxylation of the beta-ketothioether to form acetoacetate and coenzyme M. These findings evince a newfound versatility for coenzyme M as a carrier and activator of alkyl groups longer in chain-length than methane, a function for coenzyme M in a catabolic pathway of hydrocarbon oxidation, and the presence of coenzyme M in the bacterial domain of the phylogenetic tree. These results serve to unify bacterial and Archaeal metabolism further and showcase diverse biological functions for an elegantly simple organic molecule. (+info)
Importance of the beta12-beta13 loop in protein phosphatase-1 catalytic subunit for inhibition by toxins and mammalian protein inhibitors.
Type-1 protein serine/threonine phosphatases (PP1) are uniquely inhibited by the mammalian proteins, inhibitor-1 (I-1), inhibitor-2 (I-2), and nuclear inhibitor of PP1 (NIPP-1). In addition, several natural compounds inhibit both PP1 and the type-2 phosphatase, PP2A. Deletion of C-terminal sequences that included the beta12-beta13 loop attenuated the inhibition of the resulting PP1alpha catalytic core by I-1, I-2, NIPP-1, and several toxins, including tautomycin, microcystin-LR, calyculin A, and okadaic acid. Substitution of C-terminal sequences from the PP2A catalytic subunit produced a chimeric enzyme, CRHM2, that was inhibited by toxins with dose-response characteristics of PP1 and not PP2A. However, CRHM2 was insensitive to the PP1-specific inhibitors, I-1, I-2, and NIPP-1. The anticancer compound, fostriecin, differed from other phosphatase inhibitors in that it inhibited wild-type PP1alpha, the PP1alpha catalytic core, and CRHM2 with identical IC(50). Binding of wild-type and mutant phosphatases to immobilized microcystin-LR, NIPP-1, and I-2 established that the beta12-beta13 loop was essential for the association of PP1 with toxins and the protein inhibitors. These studies point to the importance of the beta12-beta13 loop structure and conformation for the control of PP1 functions by toxins and endogenous proteins. (+info)
Degradation of trichloroethene by a linear-plasmid-encoded alkene monooxygenase in Rhodococcus corallinus (Nocardia corallina) B-276.
Rhodococcus corallinus (formerly Nocardia corallina) B-276, isolated with propene as sole carbon and energy source, is able to oxidize trichloroethene (TCE). Glucose- or propene-grown R. corallinus B-276 cells exhibited no difference in TCE degradation efficiency. TCE degradation was found to be growth-phase-dependent and maximum rates were monitored with stationary-phase cells. K(m) and Vmax values for TCE degradation of R. corallinus B-276 grown in nutrient broth medium in the presence of glucose were 187 microM and 2.4 nmol min-1 (mg protein)-1, respectively. Escherichia coli recombinants harbouring and expressing the alkene monooxygenase genes of R. corallinus B-276 exhibited the ability to degrade TCE. This result provides clear evidence that the alkene monooxygenase of R. corallinus B-276 catalyses TCE oxidation. R. corallinus B-276 was shown to contain four linear plasmids, pNC10 (70 kb), pNC20 (85 kb), pNC30 (185 kb) and pNC40 (235 kb). The observation that pNC30-deficient strains had lost the ability to grow on propene suggested that the genes of the propene degradation pathway are encoded by the linear plasmid pNC30. Southern blot analysis with cloned alkene monooxygenase genes from R. corallinus B-276 revealed a positive hybridization signal with the linear plasmid pNC30. This result clearly shows that the alkene monooxygenase is encoded by the linear plasmid pNC30. Eleven short-chain-alkene-oxidizing strains were screened for the presence of linear plasmids. Among these, four propene-oxidizing Rhodococcus strains and one ethene-oxidizing Mycobacterium strain were found to contain linear megaplasmids. Southern blot analysis with the alkene monooxygenase revealed positive signals with linear plasmids of two propene-oxidizing Rhodococcus ruber strains. These results indicate that homologous alkene monooxygenases are encoded by linear plasmids in R. ruber strains. (+info)
Characterization of the gene cluster involved in isoprene metabolism in Rhodococcus sp. strain AD45.
The genes involved in isoprene (2-methyl-1,3-butadiene) utilization in Rhodococcus sp. strain AD45 were cloned and characterized. Sequence analysis of an 8.5-kb DNA fragment showed the presence of 10 genes of which 2 encoded enzymes which were previously found to be involved in isoprene degradation: a glutathione S-transferase with activity towards 1,2-epoxy-2-methyl-3-butene (isoI) and a 1-hydroxy-2-glutathionyl-2-methyl-3-butene dehydrogenase (isoH). Furthermore, a gene encoding a second glutathione S-transferase was identified (isoJ). The isoJ gene was overexpressed in Escherichia coli and was found to have activity with 1-chloro-2,4-dinitrobenzene and 3,4-dichloro-1-nitrobenzene but not with 1, 2-epoxy-2-methyl-3-butene. Downstream of isoJ, six genes (isoABCDEF) were found; these genes encoded a putative alkene monooxygenase that showed high similarity to components of the alkene monooxygenase from Xanthobacter sp. strain Py2 and other multicomponent monooxygenases. The deduced amino acid sequence encoded by an additional gene (isoG) showed significant similarity with that of alpha-methylacyl-coenzyme A racemase. The results are in agreement with a catabolic route for isoprene involving epoxidation by a monooxygenase, conjugation to glutathione, and oxidation of the hydroxyl group to a carboxylate. Metabolism may proceed by fatty acid oxidation after removal of glutathione by a still-unknown mechanism. (+info)
N-Acetylation of xenobiotic-derived cysteine S-conjugates is a key step in the mercapturic acid pathway. The aim of this study was to investigate the N-acetylation of haloalkene-derived S-haloalkyl and S-haloalkenyl cysteine S-conjugates by porcine kidney cysteine S-conjugate N-acetyltransferase (NAcT). A radioactive assay for the quantification of NAcT activity was developed as a new method for partial purification of the enzyme, which was necessitated by the substantial loss of activity during the immunoaffinity chromatography method. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane-sulfonate, rather than N,N-bis[3-gluconamidopropyl]deoxycholamide, was used to solubilize the NAcT from porcine kidney microsomes in the revised procedure. The partially purified NAcT was free of detectable aminoacylase activity. Although low acetyl-coenzyme A hydrolase activity was observed, its effect on the assay was minimized by addition of excess acetyl-coenzyme A in the NAcT assay mixture. Attempts to separate the residual hydrolase activity from NAcT by different chromatographic procedures were either unsuccessful or lead to inactivation of NAcT. Most of the cysteine S-conjugates studied were N-acetylated by NAcT. Although the apparent K(m) values for the cysteine S-conjugates studied differed by a factor of approximately 2.5 (124-302 microM), a greater than 15-fold difference in the apparent V(max) (0.75-15.6 nmol/h) and V(max)/K(m) (0.008-0.126 x 10(-3) l h(-1)) values was observed. These data show that a range of haloalkene-derived cysteine S-conjugates serve as substrates for pig kidney NAcT. The significant differences in cytotoxicity of these conjugates may be a result of more variable deacetylation rates of the corresponding mercapturates. (+info)