A new mechanism of acquisition of drug resistance by partial duplication of topoisomerase I.
(17/1610)
Topoisomerase (topo)-I targeting antitumor agents are very effective in vivo against various human cancers. The indolocarbazole compound 6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13-(beta-D-glucopyranosyl)- 5H-indolo[2,3-alpha]pyrrolo-[3,4-c]carbazole-5,7(6H)-dione (NB-506) is a potent inhibitor of the religation step of topo I reaction, like camptothecin (CPT). We established a NB-506-resistant cell line from murine leukemia cell line P388. This resistant cell line, P388/F11, exhibited 73-fold higher resistance to NB-506 and 3.5-fold higher cross-resistance to CPT than the parental cell line. No induction of cleavable complex formations induced by NB-506 and CPT were detected by K-SDS precipitation assays in P388/F11 cells. Analysis of nuclear extracts from P388/F11 cells revealed that the relaxation activity of topo I was one-quarter of that of the parental cells, and that the activity was resistant to induction of DNA cleavage by these drugs. Furthermore, Western blot and Northern blot analyses showed the expression of an abnormal-sized 170-kDa topo I protein and its 6.0-kb transcript and the absence of the normal topo I protein and transcript in P388/F11 cells. Analyses of the structure of the abnormal topo I transcript by reverse transcription-PCR and direct sequencing methods revealed that a large portion of the gene from codon 21 to codon 609 was duplicated in its coding region. This internal duplication resulted in in-frame fusion and, thus, production of a partially duplicated protein of 1357 amino acids. Finally, we expressed and purified the recombinant P388/F11 topo I in a baculovirus system. P388/F11 topo I showed similar catalytic activity to wild-type topo I, but reduced sensitivities to NB-506 and CPT. These results show that the altered sensitivity of duplicated topo I is involved in the NB-506 resistance of P388/F11 cells and indicate a novel resistant mechanism which involves duplication of the topo I gene. (+info)
Catabolite control of Escherichia coli regulatory protein BglG activity by antagonistically acting phosphorylations.
(18/1610)
In bacteria various sugars are taken up and concomitantly phosphorylated by sugar-specific enzymes II (EII) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The phosphoryl groups are donated by the phosphocarrier protein HPr. BglG, the positively acting regulatory protein of the Escherichia coli bgl (beta-glucoside utilization) operon, is known to be negatively regulated by reversible phosphorylation catalyzed by the membrane spanning beta-glucoside-specific EIIBgl. Here we present evidence that in addition BglG must be phosphorylated by HPr at a distinct site to gain activity. Our data suggest that this second, shortcut route of phosphorylation is used to monitor the state of the various PTS sugar availabilities in order to hierarchically tune expression of the bgl operon in a physiologically meaningful way. Thus, the PTS may represent a highly integrated signal transduction network in carbon catabolite control. (+info)
Intercalation into DNA is not required for inhibition of topoisomerase I by indolocarbazole antitumor agents.
(19/1610)
The DNA-intercalating antitumor drug NB-506 is a potent topoisomerase poison currently undergoing phase I/II clinical trials. It contains a planar indolocarbazole chromophore substituted with a glucose residue. Up until now, it was thought that intercalation of the drug into DNA was essential for the stabilization of topoisomerase I-DNA covalent complexes. But, in the present study, we show that a regio-isomeric form of NB-506 has lost its capacity to intercalate into DNA, but remains an extremely potent topoisomerase I poison. The new analogue contains two hydroxyl groups at positions 2,10 instead of positions 1,11 in NB-506. The relocation of the two OH groups reduces considerably the strength of binding to DNA and prevents the drug from intercalating into the DNA double helix. However, the topoisomerase I inhibition capacity of the new analogue remains very high. The two drug isomers are equally potent at maintaining the integrity of the topoisomerase I-DNA covalent complexes, but stimulate cleavage at different sites on DNA. NB-506 stabilizes topoisomerase I preferentially at sites having a pyrimidine (T or C) and a G on the 5' and 3' sides of the cleaved bond, respectively. The 2,10-isomer induces topoisomerase I-mediated cleavage only at TG sites and, thus, behaves exactly as the reference topoisomerase I poison camptothecin. Finally, cytotoxicity measurements performed with a panel of murine and human cancer cell lines reveal that the newly designed drug is considerably (up to 100-fold) more toxic to tumor cells than the parent drug NB-506. We conclude that the DNA-binding and topoisomerase I poisoning activities of NB-506 can be viewed as two separate mechanisms. (+info)
The staurosporine-like compound L-753,000 (NB-506) potentiates the neurotrophic effects of neurotrophin-3 by acting selectively at the TrkA receptor.
(20/1610)
K-252b, a member of the staurosporine family of protein kinase inhibitors, selectively potentiates the activation of the nerve growth factor receptor, TrkA, by a nonpreferred ligand, neurotrophin-3 (NT-3), in a variety of cell types. At higher (micromolar) concentrations of K-252b, an inhibitory effect occurs because of the inhibitory action of K-252b on the Trk kinase. By examining analogs of K-252b, we identified the compound L-753,000 (NB-506), which potentiates the action of NT-3 on TrkA but is devoid of the inhibitory action of K-252b. L-753,000 was effective at nanomolar concentrations in a Chinese hamster ovary cell line that expressed TrkA but was devoid of p75, the low-affinity neurotrophin receptor. L-753,000 also potentiated the activation of mitogen-activating protein kinase signaling (downstream from Trk activation) by NT-3 in this cell line. Although L-753,000, like K-252b, had a negligible effect in the absence of NT-3, the compound was found to potentiate NT-3-induced survival in both rat and chick primary cultures of dissociated dorsal root ganglia (DRG) and on neurite outgrowth of chick DRG explants. Unlike K-252b, which at micromolar concentrations inhibits the survival response of NT-3 in dissociated rat DRG, L-753,000 continued to potentiate the actions of NT-3 up to a concentration of 10 microM. Furthermore, the compound, unlike K-252b, did not inhibit an unrelated protein kinase, protein kinase C, at concentrations up to 10 microM. Because L-753, 000 selectively potentiates the NT-3-induced stimulation of TrkA without inhibiting Trks and other protein kinases, it represents a novel class of selective modifiers of neurotrophin actions. (+info)
Structure elucidation of Sch 20562, a glucosidic cyclic dehydropeptide lactone--the major component of W-10 antifungal antibiotic.
(21/1610)
A novel bacterium designated as Aeromonas sp. W-10 produces the antibiotic W-10 complex which comprises of two major and several minor components. The two major components from this complex, Sch 20562 (1) and Sch 20561 (1a), are of biological interest in view of their potent antifungal activity. The chemical degradation studies utilized for the assignment of structure 1 for Sch 20562 are described here. Some of the noteworthy diversity of structural features in this glucosidic cyclic dehydrononapeptide lactone 1 are: an N-terminal (D)-beta-hydroxymyristyl unit, three D-amino acid units, two (E)-alpha-aminocrotonyl units, and an O-alpha-D-glucosyl-N-methyl-L-allo-threonine unit. The structure determination of 1 utilized the selective cleavage of the dehydropeptide units by ozonolysis to form fragments that were sequenced by mass spectrometry. The stereochemistry of the amino acid units were assigned by isolation of the free amino acids from the hydrolysates of the fragments. The stereochemistry of the alpha-aminocrotonyl units and the glucosidic linkage were assigned by nmr spectroscopy and molecular rotation data. (+info)
A novel Sinorhizobium meliloti operon encodes an alpha-glucosidase and a periplasmic-binding-protein-dependent transport system for alpha-glucosides.
(22/1610)
The most abundant carbon source transported into legume root nodules is photosynthetically produced sucrose, yet the importance of its metabolism by rhizobia in planta is not yet known. To identify genes involved in sucrose uptake and hydrolysis, we screened a Sinorhizobium meliloti genomic library and discovered a segment of S. meliloti DNA which allows Ralstonia eutropha to grow on the alpha-glucosides sucrose, maltose, and trehalose. Tn5 mutagenesis localized the required genes to a 6.8-kb region containing five open reading frames which were named agl, for alpha-glucoside utilization. Four of these (aglE, aglF, aglG, and aglK) appear to encode a periplasmic-binding-protein-dependent sugar transport system, and one (aglA) appears to encode an alpha-glucosidase with homology to family 13 of glycosyl hydrolases. Cosmid-borne agl genes permit uptake of radiolabeled sucrose into R. eutropha cells. Analysis of the properties of agl mutants suggests that S. meliloti possesses at least one additional alpha-glucosidase as well as a lower-affinity transport system for alpha-glucosides. It is possible that the Fix+ phenotype of agl mutants on alfalfa is due to these additional functions. Loci found by DNA sequencing to be adjacent to aglEFGAK include a probable regulatory gene (aglR), zwf and edd, which encode the first two enzymes of the Entner-Doudoroff pathway, pgl, which shows homology to a gene encoding a putative phosphogluconolactonase, and a novel Rhizobium-specific repeat element. (+info)
Stability and binding properties of wild-type and c17s mutated human sterol carrier protein 2.
(23/1610)
The temperature- and solvent-induced denaturation of both the SCP2 wild-type and the mutated protein c71s were studied by CD measurements at 222 nm. The temperature-induced transition curves were deconvoluted according to a two-state mechanism resulting in a transition temperature of 70.5 degrees C and 59.9 degrees C for the wild-type and the c71s, respectively, with corresponding values of the van't Hoff enthalpies of 183 and 164 kJ/mol. Stability parameters characterizing the guanidine hydrochloride denaturation curves were also calculated on the basis of a two-state transition. The transitions of the wild-type occurs at 0.82 M GdnHCl and that of the c71s mutant at 0.55 M GdnHCl. These differences in the half denaturation concentration of GdnHCl reflect already the significant stability differences between the two proteins. A quantitative measure are the Gibbs energies DeltaG(0)(D)(buffer) at 25 degrees C of 15.5 kJ/mol for the wild-type and 8.0 kJ/mol for the mutant. We characterized also the alkyl chain binding properties of the two proteins by measuring the interaction parameters for the complex formation with 1-O-Decanyl-beta-D-glucoside using isothermal titration microcalorimetry. The dissociation constants, K(d), for wild-type SCP2 are 335 microM at 25 degrees C and 1.3 mM at 35 degrees C. The corresponding binding enthalpies, DeltaH(b), are -21. 5 kJ/mol at 25 degrees C and 72.2 kJ/mol at 35 degrees C. The parameters for the c71s mutant at 25 degrees C are K(d)=413 microM and DeltaH(b)=16.6 kJ/mol. These results suggest that both SCP2 wild-type and the c71s mutant bind the hydrophobic compound with moderate affinity. (+info)
Identification of glutathionyl-3-hydroxykynurenine glucoside as a novel fluorophore associated with aging of the human lens.
(24/1610)
A novel fluorophore was isolated from human lenses using high performance liquid chromatography (HPLC). The new fluorophore was well separated from 3-hydroxykynurenine glucoside (3-OHKG) and its deaminated isoform, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-glucoside, which are known UV filter compounds. The new compound exhibited UV absorbance maxima at 260 and 365 nm, was fluorescent (Ex(360 nm)/Em(500 nm)), and increased in concentration with age. Further analysis of the purified compound by microbore HPLC with in-line electrospray ionization mass spectrometry revealed a molecular mass of 676 Da. This mass corresponds to that of an adduct of GSH with a deaminated form of 3-OHKG. This adduct was synthesized using 3-OHKG and GSH as starting materials. The synthetic glutathionyl-3-hydroxykynurenine glucoside (GSH-3-OHKG) adduct had the same HPLC elution time, thin-layer chromatography R(F) value, UV absorbance maxima, fluorescence characteristics, and mass spectrum as the lens-derived fluorophore. Furthermore, the (1)H and (13)C NMR spectra of the synthetic adduct were entirely consistent with the proposed structure of GSH-3-OHKG. These data indicate that GSH-3-OHKG is present as a novel fluorophore in aged human lenses. The GSH-3-OHKG adduct was found to be less reactive with beta-glucosidase compared with 3-OHKG, and this could be due to a folded conformation of the adduct that was suggested by molecular modeling. (+info)