S-adenosylmethionine decarboxylase from Leishmania donovani. Molecular, genetic, and biochemical characterization of null mutants and overproducers. (73/752)

The polyamine biosynthetic enzyme, S-adenosylmethionine decarboxylase (ADOMETDC) has been advanced as a potential target for antiparasitic chemotherapy. To investigate the importance of this protein in a model parasite, the gene encoding ADOMETDC has been cloned and sequenced from Leishmania donovani. The Delta adometdc null mutants were created in the insect vector form of the parasite by double targeted gene replacement. The Delta adometdc strains were incapable of growth in medium without polyamines; however, auxotrophy could be rescued by spermidine but not by putrescine, spermine, or methylthioadenosine. Incubation of Delta adometdc parasites in medium lacking polyamines resulted in a drastic increase of putrescine and glutathione levels with a concomitant decrease in the amounts of spermidine and the spermidine-containing thiol trypanothione. Parasites transfected with an episomal ADOMETDC construct were created in both wild type and Delta adometdc parasites. ADOMETDC overexpression abrogated polyamine auxotrophy in the Delta adometdc L. donovani. In addition, ADOMETDC overproduction in wild type parasites alleviated the toxic effects of 5'-(((Z)-4-amino-2-butenyl)methylamino)-5'-deoxyadenosine (MDL 73811), but not pentamidine, berenil, or methylglyoxyl bis(guanylhydrazone), all inhibitors of ADOMETDC activities in vitro. The molecular, biochemical, and genetic characterization of ADOMETDC establishes that it is essential in L. donovani promastigotes and a potential target for therapeutic validation.  (+info)

Actions of the selective protein kinase C inhibitor PKC412 on B-chronic lymphocytic leukemia cells in vitro. (74/752)

BACKGROUND AND OBJECTIVES: The staurosporine derivative PKC412 (CGP41251) is a more selective inhibitor of the conventional isoforms of protein kinase C (PKC) than is the parent compound. In addition to its growth inhibitory properties, PKC412 reverses the efflux function of the multidrug resistance (MDR)-1 gene product, P-glycoprotein (P-gp). DESIGN AND METHODS: The in vitro actions of PKC412 were investigated in peripheral blood lymphocytes (PBL) from 4 normal volunteers, B-cell isolates from 3 normal tonsils and 31 patients with B-cell chronic lymphocytic leukemia (B-CLL). Following incubation with PKC412 for 2 days, the viability of B-CLL cells was decreased relative to that of controls (63+/-23% at 1 micromole/L; 52+/-30% at 10 micromole/L; n=20). Normal PBL were significantly more resistant to the drug (91+/-5% viable cells at 1 micromole/L; 73+/-18% at 10 micromole/L; n=4). Thirteen of the B-CLL patients were treated with oral PKC412 in a phase II trial. RESULTS: PKC activity in malignant cells from these patients showed a reduction post-treatment of 25-96% of their respective pre-treatment levels. Morphologic analysis, as well as in situ assay for DNA strand breaks (TUNEL assay) showed that B-CLL cells were killed by an apoptotic mechanism. In B-CLL cells the mean IC50, for PKC412, as measured by the reduction of 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), was 2.1 micromol/L in 16 samples in which the IC50 were below the maximum concentration of PKC412 used for the assay. In tonsillar B-cells, the mean IC50 was 11 micromol/L whereas PBL cells were resistant. Four of eight and 1/3 B-CLL samples that were resistant to chlorambucil and fludarabine, respectively, were sensitive to PKC412. In 15/31 B-CLL samples a dose-dependent reversal of P-gp-mediated drug efflux by PKC412 was observed. A statistically significant correlation (p<0.001) was observed between P-gp protein expression as measured by FACScan analysis and the reversal of efflux activity by either PKC412 or verapamil. PKC412 increased the sensitivity of B-CLL cells to 2'-chlorodeoxyadenosine and chlorambucil. INTERPRETATION AND CONCLUSIONS: This study establishes the in vitro cytotoxic and multidrug resistance (MDR) modulatory properties of PKC412 towards malignant cells from B-CLL patients. The direct antitumor activity combined with the potential for P-gp modulation make PKC412 an attractive drug for the treatment of malignancies expressing the MDR phenotype, or in combination with conventional drugs.  (+info)

The solution structure of an oligonucleotide duplex containing a 2'-deoxyadenosine-3-(2-hydroxyethyl)- 2'-deoxyuridine base pair determined by NMR and molecular dynamics studies. (75/752)

Determination of the solution structure of the duplex d(GCAAGTC(HE)AAAACG)*d(CGTTTTAGACTTGC) containing a 3-(2-hydroxyethyl)-2'-deoxyuridine*deoxyadenine (HE*A) base pair is reported. The three-dimensional solution structure, determined starting from 512 models via restrained molecular mechanics using inter-proton distances and torsion angles, converged to two final families of structures. For both families the HE and the opposite A residues are intrahelical and in the anti conformation. The hydroxyethyl chain lies close to the helix axis and for one family the hydroxyl group is above the HE*A plane and in the other case it is below. These two models were used to start molecular dynamic calculations with explicit solvent to explore the hydrogen bonding possibilities of the HE*A base pair. The dynamics calculations converge finally to one model structure in which two hydrogen bonds are formed. The first is formed all the time and is between HEO4 and the amino group of A, and the second, an intermittent one, is between the hydroxyl group and the N1 of A. When this second hydrogen bond is not formed a weak interaction CH...N is possible between HEC7H2 and N1A21. All the best structures show an increase in the C1'-C1' distance relative to a Watson-Crick base pair.  (+info)

Prediction of gene function in methylthioadenosine recycling from regulatory signals. (76/752)

The S-box transcription termination control system, first identified in Bacillus subtilis, is used for regulation of gene expression in response to methionine availability. The presence of the S-box motif provided the first indication that the ykrTS and ykrWXYZ genes could play a role in recycling of 5'-methylthioadenosine, a by-product of polyamine biosynthesis that can be converted to methionine. In this study we demonstrate a role for the ykrTS and ykrWXYZ gene products in this pathway.  (+info)

Simultaneous expression of hCNT1-CFP and hENT1-YFP in Madin-Darby canine kidney cells. Localization and vectorial transport studies. (77/752)

To test the hypothesis that human concentrative and equilibrative nucleoside transporters (hCNT1 and hENT1) are present on the apical and basolateral membrane, respectively, we constructed a Madin-Darby canine kidney (MDCK) cell line that simultaneously and stably expresses recombinant hCNT1 and hENT1 gene products tagged with CFP and YFP fluorescent proteins, respectively. Using a confocal microscope, both hCNT1-CFP and hENT1-YFP were found to be distributed uniformly on the plasma membrane of undifferentiated MDCK cells. Upon differentiation of the MDCK cells on Transwell filter inserts, hCNT1-CFP was visualized exclusively on the apical membrane, whereas hENT1-YFP appeared predominantly on the basolateral membrane. As differentiation proceeded, there was an increase in alkaline phosphatase activity, and activity of hENT1 in the apical compartment decreased while hCNT1 activity remained constant. These results suggest that, on differentiation, hENT1 is sorted to the basolateral membrane. This was confirmed when the hCNT1-mediated uptake of [(3)H]uridine from the apical compartment of the differentiated cells was found to be approximately 20-fold higher and that for hENT1 was approximately 4-fold lower than the corresponding uptake from the basal compartment. As observed in vivo, the net transport of [(3)H]adenosine was from the apical to the basal compartment, whereas that for (14)C-deoxyadenosine was from the basal to the apical compartment. In summary, we have shown for the first time that hCNT1 and hENT1 are expressed in polarized MDCK cells on the apical and basolateral membrane, respectively, allowing vectorial transport in both directions depending on the relative activity (ratio of maximal transporter activity to affinity) of each transporter for their substrates.  (+info)

5'-Esters of 2'-deoxyadenosine and 2-chloro-2'-deoxyadenosine with cell differentiation-provoking agents. (78/752)

Phenylacetic and retinoic acids are carboxyacidic cell differentiating agents displaying anticancer activities. We report on a new class of compounds including the 5'-esters of 2'-deoxyadenosine (dA) or 2-chloro-2'-deoxyadenosine (cladribine, 2CdA) and the aforementioned acids. The rationale behind the synthesis of these esters was that if they are hydrolyzed inside the lymphoid cells, either dA will be removed from the intracellular environment by deamination, or 2CdA will be phosphorylated and accumulated. In either case targetted delivery of the differentiating agent to the lymphoid cells may be envisaged. The said compounds were synthesized by the Mitsunobu procedure employing triphenylphosphine and azadicarboxylic acid esters, and their stability was tested against various esterases. Esters of dA and 2CdA with phenylacetic acids were found to be resistant to enzymatic hydrolysis, whereas those with retinoic acids were efficiently hydrolyzed by commercially available hepatic esterase as well as by esterases present in the blood plasma and in diluted human lymphocyte lysate. Susceptibility to enzymatic hydrolysis was found to be a prerequisite of cytotoxic and/or differentiating activity of these esters in leukemic cell lines.  (+info)

Three-dimensional organization of active rRNA genes within the nucleolus. (79/752)

In this work, we have localized transcribing rRNA genes at the ultrastructural level and described their three-dimensional organization within the nucleolus by electron tomography. Isolated nucleoli, which exhibit a reduced transcriptional rate, were used to determine the sites of initial BrUTP incorporation (i.e. rRNA synthesis by the transcriptional machinery). Using pulse-chase experiments with BrUTP and an elongation inhibitor, cordycepin, it was possible to precisely localize the initial sites of BrUTP incorporation. Our data show that BrUTP incorporation initially takes place in the fibrillar centers and that elongating rRNAs rapidly enter the surrounding dense fibrillar component. Furthermore, we investigated the spatial arrangement of RNA polymerase I molecules within the whole volume of the fibrillar centers. Electron tomography was performed on thick sections of cells that had been labeled with anti-RNA polymerase I antibodies prior to embedding. Detailed tomographic analyses revealed that RNA polymerase I molecules are mainly localized within discrete clusters. In each of them, RNA polymerase I molecules were grouped as several coils, 60 nm in diameter. Overall, these findings have allowed us to propose a model for the three-dimensional organization of transcribing rDNA genes within the nucleolus.  (+info)

Adenosine kinase inhibition promotes survival of fetal adenosine deaminase-deficient thymocytes by blocking dATP accumulation. (80/752)

Thymocyte development past the CD4(-)CD8(-) stage is markedly inhibited in adenosine deaminase-deficient (ADA-deficient) murine fetal thymic organ cultures (FTOCs) due to the accumulation of ADA substrates derived from thymocytes failing developmental checkpoints. Such cultures can be rescued by overexpression of Bcl-2, suggesting that apoptosis is an important component of the mechanism by which ADA deficiency impairs thymocyte development. Consistent with this conclusion, ADA-deficient FTOCs were partially rescued by a rearranged T cell receptor beta transgene that permits virtually all thymocytes to pass the beta-selection checkpoint. ADA-deficient cultures were also rescued by the adenosine kinase inhibitor 5'-amino-5'-deoxyadenosine (5'A5'dAdo), indicating that the metabolite responsible for the inhibition of thymocyte development is not adenosine or deoxyadenosine, but a phosphorylated derivative of an ADA substrate. Correction of ADA-deficient FTOCs by 5'A5'dAdo correlated with reduced accumulation of dATP, implicating this compound as the toxic metabolite. In ADA-inhibited FTOCs rescued with a Bcl-2 transgene, however, dATP levels were superelevated, suggesting that cells failing positive and negative selection continued to contribute to the accumulation of ADA substrates. Our data are consistent with dATP-induced mitochondrial cytochrome c release followed by apoptosis as the mechanism by which ADA deficiency leads to reduced thymic T cell production.  (+info)