The biosynthesis of transfer RNA in insects. II. Isolation of transfer RNA precursors from the posterior silk gland of Bombyx mori.
The occurrence of precursors to tRNA in the post-polysomal fraction of the posterior silk gland of Bombyx mori was demonstrated by pulse-chase labeling and DNA-RNA hybridization competition experiments. These precursors had molecular sizes ranging from 4S to 5S on polyacrylamide gel electrophoresis. Analysis of the incorporation of the methyl group from [methyl-14C]methionine revealed that a radioactive peak on polyacrylamide gel appeared in the 4.5S region during brief labeling. This suggested that some methylation occurred at the 4.5S precursor step. (+info)
[3H]gemcitabine uptake by nucleoside transporters in a human head and neck squamous carcinoma cell line.
Cellular uptake of many chemotherapeutic nucleoside analogs is dependent on the activity of a family of nucleoside transport proteins located in the cell plasma membrane. In the present study, we examined the role of these transporters in the accumulation of gemcitabine by a human head and neck squamous carcinoma cell line. The uptake of [3H]gemcitibine was compared with that of [3H]uridine and [3H]formycin B in the parent cell line (HN-5a) and in a gemcitabine-resistant variant (GEM-8e). The HN-5a and GEM-8e cells were similar in their transport characteristics and expressed predominantly the es (equilibrative, inhibitor-sensitive) transporter subtype; less than 10% of the influx of [3H]formycin B or [3H]uridine was mediated by the ei (equilibrative inhibitor-resistant) system, and there was no evidence for Na+-dependent nucleoside transporters. [3H]Gemcitabine (10 microM) entered these cells via both the es and ei transporters with an initial rate of uptake similar to that seen with the use of [3H]formycin B or [3H]uridine. In addition, ATP-replete cells accumulated significantly less [3H]gemcitabine than did ATP-depleted cells, which is indicative of an active efflux mechanism for gemcitabine. These results show that gemcitabine is a substrate for both the es and ei nucleoside transporters of HN-5a and GEM-8e cells and that gemcitabine resistance of the GEM-8e cells cannot be attributed to changes in transporter activity. Further studies to define the characteristics of the putative efflux mechanism are clearly warranted because this system has the potential to significantly affect the clinical efficacy of gemcitabine. (+info)
Nucleoside-3'-phosphotriesters as key intermediates for the oligoribonucleotide synthesis. III. An improved preparation of nucleoside 3'-phosphotriesters, their 1H NMR characterization and new conditions for removal of 2-cyanoethyl group.
An improved procedure for the transformation of 5'-O-monomethoxytrityl-2'-O-acetyl-3'-phosphates of uridine la, inosine ib and 6-N-benzoyladenosine lc into corresponding 3'/2,2,2-trichloroethyl, 2-cyanoethyl/-phosphates iiaic is reported. H NMR characterization of nucleoside 3'-phosphotriesters is presented. New conditions i.e. anhydrous triethylamine-pyridine treatment have been found for the selective removal of 2-cyanoethyl group from nucleoside 3'-phosphotriesters in the presence of neighbouring 2'-O-acetyl one. (+info)
Binding of neuronal ELAV-like proteins to the uridine-rich sequence in the 3'-untranslated region of tumor necrosis factor-alpha messenger RNA.
Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that is involved in the pathogenesis of several human CNS disorders. The AU-rich element (ARE) in the 3'-untranslated region (UTR) of TNF-alpha mRNA is implicated in post-transcriptional control of TNF-alpha. In this study, we showed that a human neuronal ELAV-like protein binds to the ARE in the 3'-UTR of TNF-alpha mRNA. The protein binds to the uridine stretch in AUUUA pentanucleotides inside the ARE in the 3'-UTR of TNF-alpha mRNA. The TNF-alpha mRNA-binding region in the protein appears to be identical to the c-myc and IL-3 mRNA-binding regions. Moreover, this study showed that in vitro treatment of neuroblastoma cells with interleukin-4 (IL-4), which inhibits TNF-alpha production, reduced the expression of the neuronal ELAV-like proteins. These results suggest that the expression of neuronal ELAV-like proteins may be closely associated with the expression of TNF-alpha in neuronal cells. (+info)
Use of tracheal organ cultures in toxicity testing.
Fragments of tracheal epithelium alone or in continuity with connective tissues, can be maintained in culture medium and used for short term or long term studies of toxicity of a variety of chemicals. Large numbers of uniform cultures are prepared with the aid of a slicing device or by application of simple method for dissecting sheets of epithelium free from underlying cartilage. The cultures may be placed in an exposure chamber-incubator mounted on a microscope stage and monitored continually for ciliostasis and exfoliation of cells. Morphology is further studied by fixation of selected specimens and preparation for light microscopy and electron microscopy. Synthetic functions are evaluated by autoradiographic measurement of incorporation of radioactive precursors into macromolecules and other dynamic features are indirectly assessed by histochemical and histoenzymatic methods. Short-term studies using these several techniques have shown that ciliostasis does not correlate with cell injury in all instances, and a long-term study has demonstrated dose dependence of a cytotoxic agent when duration of culture viability is measured. The method lends itself to a broad range of investigations in which dose, period of exposure, and role of cofactors must be independently and quantitatively assessed. (+info)
Biochemical studies of isolated hamster tracheal epithelium.
The epithelial lining of respiratory air passageways is a primary target tissue for toxicity and carcinogenesis in man and in animal models of human disease. The importance of this target tissue was the basis for development of methods to study its biochemistry, and with this information to distinguish the unique properties of this tissue from properties common to all cell types. Biochemical methods employed labeling of macromolecules in isolated hamster treacheas during brief (less than 4 hr) incubation in vitro. Studies of RNA metabolism in isolated tracheas demonstrated a pattern of maturation of ribosomal RNA like that shown for other cell types. Alterations in RNA metabolism were observed in isolated tracheas obtained from vitamin A-deficient hamsters and hamsters previously treated by intratracheal administration of benzo[a]pyrene (BP) plus ferric oxide (Fe2O3) in vivo. Studies with toyocamycin, actinomycin D, and alpha-amanitin, all inhibitors of RNA metabolism, were performed to characterize the class of RNA molecules with a decreased proportion of labeling in tracheas from vitamin A deficient hamsters. In another series of experiments, BP was shown to bind to DNA in epithelial cells of isolated tracheas. The quantity of BP binding was increased by prior intratracheal treatment of hamsters with BP plus Fe2O3 in vivo, this induced binding was inhibited by addition of 7,8-benzoflavone to the incubation medium. Increased BP binding was also observed in isolated tracheas from hamsters believed to be in states of increased susceptibility to respiratory carcinogenesis in vivo. The results show that biochemical studies are feasible with this tissue. Furthermore, a number of questions of importance with regard to this target epithelium are best studied directly in its constituent cells. (+info)
The uridine in "U-turn": contributions to tRNA-ribosomal binding.
"U-turns" represent an important class of structural motifs in the RNA world, wherein a uridine is involved in an abrupt change in the direction of the polynucleotide backbone. In the crystal structure of yeast tRNAPhe, the invariant uridine at position 33 (U33), adjacent to the anticodon, stabilizes the exemplar U-turn with three non-Watson-Crick interactions: hydrogen bonding of the 2'-OH to N7 of A35 and the N3-H to A36-phosphate, and stacking between C32 and A35-phosphate. The functional importance of each noncanonical interaction was determined by assaying the ribosomal binding affinities of tRNAPhe anticodon stem and loop domains (ASLs) with substitutions at U33. An unsubstituted ASL bound 30S ribosomal subunits with an affinity (Kd = 140+/-50 nM) comparable to that of native yeast tRNAPhe (Kd = 100+/-20 nM). However, the binding affinities of ASLs with dU-33 (no 2'-OH) and C-33 (no N3-H) were significantly reduced (2,930+/-140 nM and 2,190+/-300 nM, respectively). Surprisingly, the ASL with N3-methyluridine-33 (no N3-H) bound ribosomes with a high affinity (Kd = 220+/-20 nM). In contrast, ASLs constructed with position 33 uridine analogs in nonstacking, nonnative, and constrained conformations, dihydrouridine (C2'-endo), 6-methyluridine (syn) and 2'O-methyluridine (C3'-endo) had almost undetectable binding. The inability of ASLs with 6-methyluridine-33 and 2'O-methyluridine-33 to bind ribosomes was not attributable to any thermal instability of the RNAs. These results demonstrate that proton donations by the N3-H and 2'OH groups of U33 are not absolutely required for ribosomal binding. Rather, the results suggest that the overall uridine conformation, including a dynamic (C3'-endo > C2'-endo) sugar pucker, anti conformation, and ability of uracil to stack between C32 and A35-phosphate, are the contributing factors to a functional U-turn. (+info)
The mismatch repair protein, hMLH1, mediates 5-substituted halogenated thymidine analogue cytotoxicity, DNA incorporation, and radiosensitization in human colon cancer cells.
Deficiency in DNA mismatch repair (MMR) is found in some hereditary (hereditary nonpolyposis colorectal cancer) and sporadic colon cancers as well as other common solid cancers. MMR deficiency has recently been shown to impart cellular resistance to multiple chemical agents, many of which are commonly used in cancer chemotherapy. It is therefore of interest to find an approach that selectively targets cells that have lost the ability to perform MMR. In this study, we examine the response of MMR-proficient (hMLH1+) and MMR-deficient (hMLH1-) colon carcinoma cell lines to the halogenated thymidine (dThd) analogues iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd) before and after irradiation. These dThd analogues are used clinically as experimental sensitizing agents in radioresistant human cancers, and there is a direct correlation between the levels of dThd analogue DNA incorporation and tumor radiosensitization. In contrast to the well-characterized, marked increase in cytotoxicity (> 1 log cell kill) found with 6-thioguanine exposures in HCT116/3-6 (hMLH1+) cells compared to HCT116 (hMLH1-) cells, we found only modest cytotoxicity (10-20% cell kill) in both cell lines when treated with IdUrd or BrdUrd for 1 population doubling. Upon further analysis, the levels of halogenated dThd analogues in DNA were significantly lower (two to three times lower) in HCT116/3-6 cells than in HCT116 cells, and similar results were found in Mlh1+/+ spontaneously immortalized murine embryonic fibroblasts and fibroblasts from Mlh1 knockout mice. As a result of the higher levels of the dThd analogue in DNA, there was an increase in radiation sensitivity in HCT116 cells but not in HCT116/3-6 cells after pretreatment with IdUrd or BrdUrd when compared to treatment with radiation alone. Additionally, we found no differences in the cellular metabolic pathways for dThd analogue DNA incorporation because the enzyme activities of dThd kinase and thymidylate synthase, as well as the levels of triphosphate pools, were similar in HCT116 and HCT116/3-6 cells. These data suggest that the hMLH1 protein may participate in the recognition and subsequent removal of halogenated dThd analogues from DNA. Consequently, whereas MMR-deficient cells and tumor xenografts have shown intrinsic resistance to a large number of chemotherapeutic agents, the 5-halogenated dThd analogues appear to selectively target such cells for potential enhanced radiation sensitivity. (+info)