Resistance of African green monkey kidney cell lines to actinomycin D: drug uptake in 37 RC cells after persistent inhibition of transcription.
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37 RC cells, a cultured line derived from African green monkey kidneys, survived long treatments with actinomycin D (AMD; 0.1 to 0.5 mug/ml) under strong inhibition of ribonucleic acid synthesis and blocking of cell division. One aspect of the complex cellular response to this treatment was a progressive lowering of the influx rate of AMD and, consequently, of its endocellular concentration, leading to a late resurgence of transcription. Overall protein synthesis decreased in AMD-treated cells, but more of the residual protein was exported to the cell surface, a fact associated with the development of numerous strands of endoplasmic reticulum and Golgi bodies in the cytoplasm. The lowering of AMD influx during the treatment was not simply due to the decay of protein synthesis, and there was no evidence for a carrier-mediated transport of the drug. It was paralleled by, but seemingly not related to, modifications in cellular microtubules and microfilaments. The rate of AMD influx was restored to levels comparable to those of untreated cells by short exposure to ethylenediaminetetraacetic acid and trypsin. It is concluded that the changes in plasma membrane of 37 RC cells, creating an obstacle to the influx of AMD after long treatment with this drug, probably consist of an accumulation and/or a different distribution of glycoproteins or other surface components on the cell surface. (+info)
Isolation of an Escherichia coli mutant deficient in glutathione synthesis.
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A mutant of Escherichia coli that contains essentially no detectable glutathione has been isolated. The mutant contains a very low level of the enzyme glutathione synthetase and accumulates lambda-glutamyl cysteine at a concentration approximately equal to the level of glutathione found in its parent. No significant differences in growth were observed between the mutant and its parent. However, the activity of at least one enzyme was found to be affected by the absence of glutathione; the specific activity of the B1 subunit of ribonucleoside diphosphate reductase was greatly reduced. The possibility that the decreased B1 activity is due to a mutation in the structural gene coding for B1 or its regulatory gene could be eliminated. This suggests that one role of glutathione in the cell is to maintain at least this one protein in an active state. We propose the designation gshB for the gene coding for glutathione synthetase. (+info)
Phosphorylation of uridine and cytidine nucleoside analogs by two human uridine-cytidine kinases.
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Uridine-cytidine kinases (UCK) have important roles for the phosphorylation of nucleoside analogs that are being investigated for possible use in chemotherapy of cancer. We have cloned the cDNA of two human UCKs. The approximately 30-kDa proteins, named UCK1 and UCK2, were expressed in Escherichia coli and shown to catalyze the phosphorylation of Urd and Cyd. The enzymes did not phosphorylate deoxyribonucleosides or purine ribonucleosides. UCK1 mRNA was detected as two isoforms of approximately 1.8 and approximately 2.7 kb. The 2.7-kb band was ubiquitously expressed in the investigated tissues. The band of approximately 1.8 kb was present in skeletal muscle, heart, liver, and kidney. The two isoforms of UCK2 mRNA of 1.2 and 2.0 kb were only detected in placenta among the investigated tissues. The genes encoding UCK1 and UCK2 were mapped to chromosome 9q34.2-9q34.3 and 1q22-1q23.2, respectively. We tested 28 cytidine and uridine nucleoside analogs as possible substrates of the enzymes. The enzymes phosphorylated several of the analogs, such as 6-azauridine, 5-fluorouridine, 4-thiouridine, 5-bromouridine, N(4)-acetylcytidine, N(4)-benzoylcytidine, 5-fluorocytidine, 2-thiocytidine, 5-methylcytidine, and N(4)-anisoylcytidine. The cloning and recombinant expression of the two human UCKs will be important for development of novel pyrimidine ribonucleoside analogs and the characterization of their pharmacological activation. (+info)
Epstein-Barr virus encoded nuclear protein EBNA-3 binds a novel human uridine kinase/uracil phosphoribosyltransferase.
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BACKGROUND: Epstein-Barr virus (EBV) infects resting B-lymphocytes and transforms them into immortal proliferating lymphoblastoid cell lines (LCLs) in vitro. The transformed immunoblasts may grow up as immunoblastic lymphomas in immuno-suppressed hosts. RESULTS: In order to identify cellular protein targets that may be involved in Epstein-Barr virus mediated B-cell transformation, human LCL cDNA library was screened with one of the transformation associated nuclear antigens, EBNA-3 (also called EBNA-3A), using the yeast two-hybrid system. A clone encoding a fragment of a novel human protein was isolated (clone 538). The interaction was confirmed using in vitro binding assays. A full-length cDNA clone (F538) was isolated. Sequence alignment with known proteins and 3D structure predictions suggest that F538 is a novel human uridine kinase/uracil phosphoribosyltransferase. The GFP-F538 fluorescent fusion protein showed a preferentially cytoplasmic distribution but translocated to the nucleus upon co-expression of EBNA-3. A naturally occurring splice variant of F538, that lacks the C-terminal uracil phosphoribosyltransferase part but maintain uridine kinase domain, did not translocate to the nucleus in the presence of EBNA3. Antibody that was raised against the bacterially produced GST-538 protein showed cytoplasmic staining in EBV negative Burkitt lymphomas but gave a predominantly nuclear staining in EBV positive LCL-s and stable transfected cells expressing EBNA-3. CONCLUSION: We suggest that EBNA-3 by direct protein-protein interaction induces the nuclear accumulation of a novel enzyme, that is part of the ribonucleotide salvage pathway. Increased intranuclear levels of UK/UPRT may contribute to the metabolic build-up that is needed for blast transformation and rapid proliferation. (+info)
The complete genomic sequence of Mycoplasma penetrans, an intracellular bacterial pathogen in humans.
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The complete genomic sequence of an intracellular bacterial pathogen, Mycoplasma penetrans HF-2 strain, was determined. The HF-2 genome consists of a 1 358 633 bp single circular chromosome containing 1038 predicted coding sequences (CDSs), one set of rRNA genes and 30 tRNA genes. Among the 1038 CDSs, 264 predicted proteins are common to the Mycoplasmataceae sequenced thus far and 463 are M.penetrans specific. The genome contains the two-component system but lacks the essential cellular gene, uridine kinase. The relatively large genome of M.penetrans HF-2 among mycoplasma species may be accounted for by both its rich core proteome and the presence of a number of paralog families corresponding to 25.4% of all CDSs. The largest paralog family is the p35 family, which encodes surface lipoproteins including the major antigen, P35. A total of 44 genes for p35 and p35 homologs were identified and 30 of them form one large cluster in the chromosome. The genetic tree of p35 paralogs suggests the occurrence of dynamic chromosomal rearrangement in paralog formation during evolution. Thus, M.penetrans HF-2 may have acquired diverse repertoires of antigenic variation-related genes to allow its persistent infection in humans. (+info)
Control of pyrimidine biosynthesis in human lymphocytes. Inhibitory effect of guanine and guanosine on induction of enzymes for pyrimidine biosynthesis de novo in phytohemagglutinin-stimulated lymphocytes.
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Human peripheral lymphocytes were incubated with Phaseolus vulgaris phytohemagglutinin. The induction of glutamine-utilizing carbamyl phosphate synthetase (EC 2.7.2.5) and aspartate transcarbamylase (EC 2.1.3.2) for pyrimidine biosynthesis de novo and the induction of uridine kinase were observed as described previously (Ito, K., and Uchino, H. (1971) J. Biol. Chem. 246, 4060-4065; Ito, K., and Uchino, H. (1973) J. Biol. Chem. 248, 389-392; Lucas, Z.J. (1967) Science 156, 1237-1240). By the addition of 1 mM guanine to the culture, the induction of the former two enzymes was inhibited, while that of uridine kinase was not, and even accelerated. An increase in the rate of [14C] bicarbonate incorporation into the acid-soluble uridine nucleotides via the de novo pathway for pyrimidine biosynthesis after phytohemagglutinin stimulation was inhibited by guanine, the incorporation rate being almost at the level of the control culture without phytohemagglutinin. Guanosine had a similar effect on pyrimidine biosynthesis. The induction of the three enzymes mentioned above was completely inhibited by adenine (1 mM). Guanine and guanosine seem to have a unique inhibitory effect on the induction of glutamine-utilizing carbamyl phosphate synthetase and aspartate transcarbamylase. (+info)
Analysis of single nucleotide polymorphisms in uridine/cytidine kinase gene encoding metabolic enzyme of 3'-ethynylcytidine.
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We investigated single nucleotide polymorphisms (SNPs) in uck2 gene encoding metabolic enzyme of 3'-ethynylcytidine (ECyd) which were associated with drug response of ECyd, and the newly synthesized antitumor ribonucleoside analog. We analized that on exon-intron junction and exon region to affect the qualitative alteration of gene product directly in ECyd sensitive and resistant human cancer cell lines. As the results, cSNP and sSNP were detected in exon 4. In the promoter region, 3 SNPs were detected. Our data seem to be able to give an important knowledge, when ECyd is applied clinically. (+info)
Structural basis for the specificity, catalysis, and regulation of human uridine-cytidine kinase.
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Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine and cytidine and activates pharmacological ribonucleoside analogs. Here we present the crystal structures of human UCK alone and in complexes with a substrate, cytidine, a feedback inhibitor, CTP or UTP, and with phosphorylation products, CMP and ADP, respectively. Free UCK takes an alpha/beta mononucleotide binding fold and exists as a homotetramer with 222 symmetry. Upon inhibitor binding, one loop region was loosened, causing the UCK tetramer to be distorted. Upon cytidine binding, a large induced fit was observed at the uridine/cytidine binding site, which endows UCK with a strict specificity for pyrimidine ribonucleosides. The first UCK structure provided the structural basis for the specificity, catalysis, and regulation of human uridine-cytidine kinase, which give clues for the design of novel antitumor and antiviral ribonucleoside analogs that inhibit RNA synthesis. (+info)