Unlabeled Uridine Research Articles

Cell Model for Studying Nucleoside Transporters, a Key Component of the Blood‐Testis Barrier

Sertoli cells in the testis represent the principle element of the Blood‐Testis Barrier (BTB). Equilibrative nucleoside transporters (ENTs) are responsible for the transport of nucleosides across rodent BTB and immunohistochemistry suggests that hENT1 is located on the basal membrane and hENT2 is located on the apicolateral membrane of Sertoli cells (Klein et al., 2013). Therefore, the ENTs are of particular interest in studying the disposition of nucleoside reverse transcriptase inhibitors (NRTIs) in the human male genital tract because of their similarity in chemical structure to nucleosides. This study characterized the transport of a specific nucleoside, uridine, to determine the relative roles of ENT1 and ENT2 in nucleoside transport in HeLa S3 cells, a potential model system for the study of ENT selectivity, using the ENT specific inhibitor S‐(4‐nitrobenzyl)‐6‐thioinosine (NBMPR). Uptake of 20 nM [3H]uridine was linear for 7 minutes and 100 nM NBMPR blocked 77% of this uptake, with the remaining uptake blocked by 100 uM NBMPR (to the same level produced by 5 mM unlabeled uridine). ENT1 and ENT2 are known to have different sensitivities for NBMPR and, consistent with this difference, inhibition of [3H]uridine uptake produced by increasing concentration of NBMPR proved to be biphasic, with IC50s of 11.1 nM (ENT1) and 7.2 uM (ENT2). Consequently, uridine uptake measured in the presence of 100 nM NBMPR was taken to represent ENT2‐mediated transport; subtracting that from total uptake represented ENT1‐mediated transport. The kinetics of ENT1‐ and ENT2‐mediated [3H]uridine uptake into HeLa S3 cells showed no difference in Jmax (16.1 and 12.3 fmol cm−2 min−1) and a three‐fold difference in Km (13.3 and 35.2 uM). The resulting 3.5‐fold difference in intrinsic clearance for uridine uptake (i.e., Jmax/Km) for hENT1 and hENT2 mediated transport (1.21 mL cm−2 min−1 and 0.35 mL cm−2 min−1, respectively), accounted for the observed inhibition of uridine transport produced by 100 nM NBMPR, indicating that at low concentrations, hENT1 is primarily responsible for uridine uptake in these cells. Together, these data suggest that HeLa S3 cells are an adequate model for studying the characteristics of nucleoside transporters present in the BTB.Support or Funding InformationSupported by R01GM123643This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Role of Human Nucleoside Transporters In Uptake of 5-Azacitidine, 5-Aza-2′-Deoxycytidine and Gemcitabine

AbstractAbstract 28845-Azacitidine is a cytidine analog, and is used for the treatment of myelodysplastic syndromes. Cellular uptake of nucleosides and nucleoside analogs are mediated by human concentrative nucleoside transporters (hCNTs) and equilibrative nucleoside transporters (hENTs). The objective of this study was to assess the transportability of 5-azacitidine, 5-aza-2'-deoxycytidine and gemcitabine in Saccharomyces cerevisiae yeast and Xenopus laevis oocytes producing individual recombinant hNTs.The relative abilities of the hNT proteins to interact with 5-azacitidine, 5-aza-2'-deoxycytidine and gemcitabine were assessed by determining the inhibition of uptake of 1 μM [3H]uridine by recombinant hENT1, hENT2, hCNT1, hCNT2 and hCNT3 produced in yeast in the presence of varying concentrations (0.3 to 3000 μM) of the compounds. Excess unlabeled uridine (10 mM) was used as the inhibitor control. All three compounds exhibited lower Ki values (3-26 μM) for the inhibition of hCNT1 and hCNT3 than for the other hNTs (Ki > 500 μM), indicating that hCNT1 and hCNT3 had higher affinities for all the tested compounds than the other hNTs.Inhibition of hENT3 and hENT4 was examined with oocytes producing recombinant hENT3 or hENT4 using 10 μM [3H]uridine (hENT3) or [3H]adenosine (hENT4) in transport medium in the presence or absence of 3 mM non-radioactive uridine, adenosine, 5-azacitidine, 5-aza-2'-deoxycytidine or gemcitabine at a 10 min flux interval. The extent of hENT3 inhibition was highest for 5-azacitidine (65%) and uridine (56%), followed by 5-aza-2'-deoxycytidine (37%) and gemcitabine (9%). The hENT3 inhibition by 5-azacitidine (65%) was significantly greater than that by 5-aza-2'-deoxycytidine (37%) (P < 0.005). Inhibition of hENT4 was 26% by 5-azacitidine, 34% by 5-aza-2'-deoxycytidine and 19% by gemcitabine, while unlabeled adenosine caused 89% inhibition, suggesting that 5-azacitidine, 5-aza-2'-deoxycytidine and gemcitabine at 3 mM are weak inhibitors of hENT4.Direct uptake of [14C]5-azacitidine, [3H]5-aza-2'-deoxycytidine and [3H]gemcitabine at 20 μM for 30 min by all seven hNTs was measured with oocytes producing the individual hNTs. Basal (non-mediated) fluxes in water-injected oocytes (negative control) were low for all three compounds, indicating only limited entry into cells by simple diffusion. [3H]Uridine (20 μM) was used as a positive control substrate for hCNT1, hCNT2, hCNT3, hENT1, hENT2 and hENT3 and [3H]adenosine (20 μM) was used as a positive control substrate for hENT4 to confirm the functional activity of recombinant hNTs. Fluxes were determined in the presence and absence of excess (3 mM) unlabelled adenosine (hENT4) or uridine (other hNTs). In this direct uptake study, 5-azacitidine was transported by all seven transporters, with hCNT3 showing the highest rate of transport. In marked contrast, only hENT1 and hENT2 showed notable transport of 5-aza-2'-deoxycytidine, with substantially lower fluxes than 5-azacitidine. Although hCNT1 and hCNT3 exhibited high apparent affinities for 5-aza-2'-deoxycytidine in the inhibition study, 5-aza-2'-deoxycytidine was not transported by either transporter. Gemcitabine was transported by six of the seven transporters (hENT1, hENT2, hENT3, hENT4, hCNT1 and hCNT3), with little activity observed for hCNT2. hCNT3 also showed the highest rate of uptake of gemcitabine.In conclusion, we report for the first time that 5-azacitidine was transported by all seven hNTs (i.e., hENT1, hENT2, hENT3, hENT4, hCNT1, hCNT2 and hCNT3), with hCNT3 exhibiting the highest activity. In marked contrast, only hENT1 and hENT2 showed notable transport of 5-aza-2'-deoxycytidine, with substantially lower fluxes than 5-azacitidine. Individual hNTs transported 5-azacitidine and gemcitabine to broadly similar extents except for hCNT2, which exhibited little activity with gemcitabine. With multiple NTs involved in 5-azacitidine transport, polymorphisms in individual hNTs are unlikely to impact overall 5-azacitidine uptake into cells. Disclosures:No relevant conflicts of interest to declare.

Structure Determination of the Top‐Loop of the Conserved 3′‐Terminal Secondary Structure in the Genome of Flaviviruses

To what extent small differences in RNA sequences (mutations) can have a profound impact on biology remains an intriguing question. This effect can be studied by using untranslated RNA regions as a model. We have studied the influence of mutations on the structure of an RNA hairpin that occurs in the 3'-untranslated region (UTR) of Flaviviridae, and is known to have a large impact on the vector dependency of flaviviruses. Three related RNA sequences were studied by NMR spectroscopy. The selected sequences represent each one of the three clusters in the flavivirus genes (mosquito-borne, tick-borne, and no-known-vector viruses). A new strategy was used to obtain chemical shift signatures of carbonyl atoms in unlabeled uridine nucleobases to characterize their involvement in hydrogen bonding. Clear differences occur in the structures and stacking pattern of the three RNA hairpins. The observed differences cannot be predicted based on sequence analysis. A different biology can be correlated with a different RNA tertiary structure. The underlying biological mechanism, however, remains to be studied.

Human Erythrocyte Nucleoside Transporter ENT1 Functions at Ice-cold Temperatures

The functionality of human erythrocyte nucleoside transporter ENT1 was examined at ice-cold temperatures (ICT; measured temperature, 0.5-0.7 degrees C) using rightside-out membrane vesicles (ROVs). The uptake of uridine, an ENT1 substrate, showed saturation kinetics and was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBMPR), a specific ENT1 inhibitor, at both 23 degrees C and ICT. [3H]Uridine uptake was markedly trans-stimulated by preloading ROVs with unlabeled uridine or ribavirin, another ENT1 substrate, and the overshoot phenomenon was observed at ICT. Similarly, [3H]ribavirin uptake was markedly trans-stimulated by unlabeled ribavirin or uridine at ICT. The trans-stimulated uptake of [3H]uridine at ICT was inhibited by ENT1 inhibitors/substrates such as NBMPR, dipyridamole, adenosine, and ribavirin in a concentration-dependent manner. The inhibition of [3H]uridine uptake by NBMPR and dipyridamole at ICT was also observed in intact red blood cells. Like uridine uptake, [3H]D-glucose uptake by ROVs, which is mediated by facilitative glucose transporter GLUT1, was trans-stimulated by unlabeled D-glucose at ICT, and the overshoot phenomenon was observed. In contrast, the ability of ATP-dependent transport of 5-(and-6)-carboxy-2',7'-dichlorofluorescein via multidrug resistance-associated protein 5 in inside-out membrane vesicles disappeared at ICT. These results clearly indicate that human erythrocyte transporters such as ENT1 function even at very low temperatures near 0 degrees C. The significance of these findings in transporter research is discussed.

Substituted Cysteine Accessibility Method Analysis of Human Concentrative Nucleoside Transporter hCNT3 Reveals a Novel Discontinuous Region of Functional Importance within the CNT Family Motif (G/A)XKX3NEFVA(Y/M/F)

The human SLC28 family of integral membrane CNT (concentrative nucleoside transporter) proteins has three members, hCNT1, hCNT2, and hCNT3. Na(+)-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 mediates transport of both pyrimidine and purine nucleosides utilizing Na(+) and/or H(+) electrochemical gradients. These and other eukaryote CNTs are currently defined by a putative 13-transmembrane helix (TM) topology model with an intracellular N terminus and a glycosylated extracellular C terminus. Recent mutagenesis studies, however, have provided evidence supporting an alternative 15-TM membrane architecture. In the absence of CNT crystal structures, valuable information can be gained about residue localization and function using substituted cysteine accessibility method analysis with thiol-reactive reagents, such as p-chloromercuribenzene sulfonate. Using heterologous expression in Xenopus oocytes and the cysteineless hCNT3 protein hCNT3C-, substituted cysteine accessibility method analysis with p-chloromercuribenzene sulfonate was performed on the TM 11-13 region, including bridging extramembranous loops. The results identified residues of functional importance and, consistent with a new revised 15-TM CNT membrane architecture, suggest a novel membrane-associated topology for a region of the protein (TM 11A) that includes the highly conserved CNT family motif (G/A)XKX(3)NEFVA(Y/M/F).

A Proton-mediated Conformational Shift Identifies a Mobile Pore-lining Cysteine Residue (Cys-561) in Human Concentrative Nucleoside Transporter 3

The concentrative nucleoside transporter (CNT) protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 mediates transport of a broad range of purine and pyrimidine nucleosides and nucleoside drugs, whereas hCNT1 and hCNT2 are pyrimidine and purine nucleoside-selective, respectively. All three hCNTs are Na(+)-coupled. Unlike hCNT1/2, however, hCNT3 is also capable of H(+)-mediated nucleoside cotransport. Using site-directed mutagenesis in combination with heterologous expression in Xenopus oocytes, we have identified a C-terminal intramembranous cysteine residue of hCNT3 (Cys-561) that reversibly binds the hydrophilic thiol-reactive reagent p-chloromercuribenzene sulfonate (PCMBS). Access of this membrane-impermeant probe to Cys-561, as determined by inhibition of hCNT3 transport activity, required H(+), but not Na(+), and was blocked by extracellular uridine. Although this cysteine residue is also present in hCNT1 and hCNT2, neither transporter was affected by PCMBS. We conclude that Cys-561 is located in the translocation pore in a mobile region within or closely adjacent to the nucleoside binding pocket and that access of PCMBS to this residue reports a specific H(+)-induced conformational state of the protein.

Conserved Glutamate Residues Are Critically Involved in Na+/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter 1 (hCNT1)

Human concentrative nucleoside transporter 1 (hCNT1), the first discovered of three human members of the SLC28 (CNT) protein family, is a Na+/nucleoside cotransporter with 650 amino acids. The potential functional roles of 10 conserved aspartate and glutamate residues in hCNT1 were investigated by site-directed mutagenesis and heterologous expression in Xenopus oocytes. Initially, each of the 10 residues was replaced by the corresponding neutral amino acid (asparagine or glutamine). Five of the resulting mutants showed unchanged Na+-dependent uridine transport activity (D172N, E338Q, E389Q, E413Q, and D565N) and were not investigated further. Three were retained in intracellular membranes (D482N, E498Q, and E532Q) and thus could not be assessed functionally. The remaining two (E308Q and E322Q) were present in normal quantities at cell surfaces but exhibited low intrinsic transport activities. Charge replacement with the alternate acidic amino acid enabled correct processing of D482E and E498D, but not of E532D, to cell surfaces and also yielded partially functional E308D and E322D. Relative to wild-type hCNT1, only D482E exhibited normal transport kinetics, whereas E308D, E308Q, E322D, E322Q, and E498D displayed increased K50(Na+) and/or Km(uridine) values and diminished Vmax(Na+) and Vmax(uridine) values. E322Q additionally exhibited uridine-gated uncoupled Na+ transport. Together, these findings demonstrate roles for Glu-308, Glu-322, and Glu-498 in Na+/nucleoside cotransport and suggest locations within a common cation/nucleoside translocation pore. Glu-322, the residue having the greatest influence on hCNT1 transport function, exhibited uridine-protected inhibition by p-chloromercuriphenyl sulfonate and 2-aminoethyl methanethiosulfonate when converted to cysteine.

Glucosylation of glycosylphosphatidylinositol membrane anchors: identification of uridine diphosphate-glucose as the direct donor for side chain modification in Toxoplasma gondii using carbohydrate analogues.

Toxoplasma gondii is an obligate intracellular parasite of the phylum apicomplexa and a common and often life-threatening opportunistic infection associated with AIDS. A family of parasite-specific glycosylphosphatidylinositols containing a novel glucosylated side chain has been shown to be highly immunogenic in humans (Striepen et al. (1997) J. Mol. Biol. 266, 797-813). In contrast to trypanosomes in T. gondii side chain modification takes place before addition to protein in the endoplasmic reticulum. The biosynthesis of these modifications was studied in an in vitro system prepared from hypotonically lysed T. gondii parasites. Radiolabeled glucose-containing glycosylphosphatidylinositol precursors were synthesized by T. gondii membrane preparations upon incubation with uridine diphosphate-[3H]glucose. Synthesis of glucosylated glycolipids took place only in the presence of exogenous uridine diphosphate-glucose and was stimulated by unlabeled uridine diphosphate-glucose in a dose-dependent manner. In contrast to glycosylphosphatidylinositol mannosylation, glucosylation was shown to be insensitive to amphomycin treatment. In addition, the glucose analogue 2-deoxy-D-glucose was used to trace the glycosylphosphatidylinositol glucosylation pathway. Detailed analysis of glycolipids synthesized in vitro in the presence of UDP and GDP derivatives of D-glucose and 2-deoxy-D-glucose ruled out an involvement of dolichol phosphate-glucose and demonstrates direct transfer of glucose from uridine diphosphate-glucose.

Time-Dependent RNA Synthesis in Early Bovine Embryos Derived from In-Vitro Fertilization.

RNA synthesis in bovine embryos produced in vitro was investigated to clarify changes in transcriptional activity. Overall 1003 follicular oocytes were matured and fertilized in vitro and subsequently cultured with a cumulus cell monolayer in TCM 199 with 5% fetal bovine serum (FBS). Embryos were transferred to TCM199 supplemented with 100 μCi/ml of [5-3H]uridine at 26, 35, 43, 50, 68 and 116 h post insemination (hpi) and cultured for 8 h at 39 C. Treated embryos were washed in PBS and the zona pellucida weakened with 5% proteinase K followed by fixation in acetic-alcohol. Preparations were air-dried, dipped in K5 emulsion, and exposed at 4 C. After 4.5 days exposure, the preparations were developed in D19 and the density of 3H-uridine incorporation was analyzed by image analysis (NIH-157 analysis system). 3H-Uridine incorporation into nuclei was observed in 43% of 2-cell embryos at 26 hpi although the level was low. The change of 3H-uridine incorporation level was not consistent until 4-cell stage and thereafter the level increased in a time-dependent manner but not dependent on cell number. Incorporation of 3H-uridine into nucleoli was observed from 50 hpi. Twenty-two embryos at 5-cell to morula were incubated with labelled and unlabelled uridine as negative control showed no incorporation of 3H-uridine. These results show that the transcription of early bovine embryos fertilized in vitro is already active in 2-cell embryos at 26 hpi, the level increases in a time-dependent manner after 5-cell stage and is not dependent on the cell number.

Transcriptional activity in in vitro produced bovine two- and four-cell embryos.

The objectives of this study on in vitro produced bovine two- and four-cell embryos were (1) to investigate the uptake of 3H-uridine through the plasma membrane, (2) to characterize the pattern of RNA synthesis during the second cell cycle, and (3) to measure the incorporation of 3H-uridine into de novo synthesized RNA. A total of 200 embryos were incubated with 3H-uridine for 15, 30, 60 (two- and four-cell embryos), 120 (four-cell embryos), 180 (two-cell embryos), and 240 min (two- and four-cell embryos), respectively. 3H-uridine uptake reached a maximum by 30 min in two-cell embryos, whereas four-cell embryos reached a maximum at 120 min. A total of 440 two-cell embryos were isolated 27-33 hr postinsemination (hpi), and 90 of these were incubated for 10 hr with 3H-uridine (200 microCi/ml). The remainder were incubated with 3H-uridine for 3 hr starting at 0-3 (n = 54), 3-6 (n = 75), 6-9 (n = 77), or 9-12 (n = 77) hr after cleavage to the two-cell stage. Control two-cell embryos (n = 67) were incubated with 3H-uridine supplemented with 5 mg/ml of unlabelled uridine for 10 hr (inhibition control), or they were incubated with 3H-uridine for 10 hr and RNase treated (100 micrograms/ml) post fixation (RNase control). Subsequently, the embryos were processed for autoradiography. The long-term incubation revealed transcription (autoradiographically labelled nuclei) in a total of 77% of the two- and four-cell embryos. No transcription was observed in any of the 3 hr incubation groups. The RNase control embryos lacked labelling of the nuclei, whereas the inhibition control embryos only showed markedly reduced labelling. Finally, total RNA extraction was performed on a total of 336 two-cell embryos that were incubated with 3H-uridine or 3H-uridine supplemented with unlabelled uridine for 2, 5, or 10 hr. It was possible to detect an increasing amount of labelled RNA after the 2, 5, and 10 hr incubation periods, and it was possible to inhibit this incorporation competitively. Together the data demonstrate a low level of transcription during the second cell cycle without a well-defined transcriptional peak.

Initiation of transcription and nucleologenesis in equine embryos

The time of activation of the embryonic genome (maternal-embryonic transition) in equine embryos was investigated by assessing incorporation of 3H-uridine and nucleolar development. In Experiment 1, embryos were recovered from the oviduct (n = 15) and the uterus (n = 3). Recovered embryos were assessed for morphologic development and quality score. Recovered embryos with less than 8 cells (two cells, n = 4; four cells, n = 5; five cells, n = 2) were incubated with 3H-uridine (560 microCi/ml) for 10 hr, while eight-cell embryos (n = 2), morulae (n = 2), and blastocysts (n = 3) were incubated with 280 microCi/ml for 0.5-1 hr. At the end of incubation, embryos were washed twice in PBS with 10% FBS and incubated for 30 min with 2.5 mg/ml of unlabelled uridine. Embryos were spread onto glass slides, dipped into emulsion, and exposed for 8 d, then developed and counterstained with Giemsa and propidium iodide. Embryos at the blastocyst, morula, eight-cell, and five-cell stages incorporated 3H-uridine into their cell nuclei as detected by autoradiography. In a second experiment, nucleologenesis in equine embryos was examined by transmission electron microscopy. Nucleoli or nucleolar precursors were found in 12 of 23 embryos examined. Most embryos in the four- to six-cell stage (n = 7) had nucleolar precursor bodies (npb) consisting of homogeneous fibrillar structures. Two five- to six-cell embryos also possessed reticulated nucleoli with both fibrillar and granular components as did all eight-cell embryos (n = 3). Nucleoli in one morula and one blastocyst were reticulated with prominent granular components, fibrillar components, and apparent fibrillar centers. These results indicate that incorporation of 3H-uridine and the formation of functional nucleoli with typical fibrillar and granular components occurs between the four- to eight-cell stage in equine embryos.

Intestinal Uptake of Uridine in Suckling Rats: Mechanism and Ontogeny

Nucleosides, essential substrates for a variety of intracellular metabolic reactions, are obtained from dietary and endogenous sources. Nucleotides (which dephosphorylate to nucleosides prior to intestinal absorption) are present in milk and have trophic effects on the developing gastrointestinal tract. The mechanism of transport of nucleosides in the developing intestine of suckling rats is unknown. To address this issue, we therefore examined uridine uptake in rat everted intestinal sacs. In suckling rats (15-17 days old), tissue uptake of low (5-microM) and high (60 microM) concentrations of [3H]-uridine was linear for up to 2 min of incubation. Initial rate of uptake of [3H]-uridine was (i) not significantly different in the jejunum and the ileum; (ii) greater in the presence of Na+, than other cations; (iii) saturable as a function of concentration with a Vmax of 21,044 +/- 2,302 pmol/g tissue wet wt/30 sec and an apparent Km of 33.8 +/- 10.1 microM; (iv) inhibited by high concentration (500 microM) of unlabeled uridine and other nucleosides; (v) temperature-dependent; (vi) energy-dependent; and (vii) pH-sensitive. Developmental maturation was associated with a progressive decrease in the Vmax of the uridine transport process (21,044 +/- 2,302, 14,651 +/- 1,679, and 8,461 +/- 1,369 pmol/g tissue wet wt/30 sec for suckling, weanling, and adult rats, respectively) and a progressive increase in the apparent Km of the uptake system (33.8 +/- 10.1, 55.6 +/- 13.1, and 61.7 +/- 14.5 microM for suckling, weanling, and adult rats, respectively). We concluded that uptake of uridine by the developing intestine of suckling rats involves a carrier-mediated system, which is energy- and temperature-dependent, and requires extracellular sodium. Furthermore, the uptake process was found to undergo clear ontogenic changes with maturation.

Nucleoside transport in brush border membrane vesicles from human kidney

The goal of this study was to elucidate the mechanisms of nucleoside transport in the brush border membrane of the human kidney. [ 3H]Uridine was transported into brush border membrane vesicles (BBMV) from human kidney via Na +-independent and Na +-dependent processes. The Na +-dependent transport was saturable ( K m = 4.76 ± 0.39 μM; V max = 6.42 ± 0.17pmol/mg proteins per s) and was trans-stimulated by unlabeled uridine. Structural analogs of uridine (100 μM), 2′-deoxyuridine (2-dU) and dideoxyuridine (ddU), significantly inhibited Na +-uridine uptake into BBMV. Previous studies have suggested that Na +-nucleoside co-transport occurs via two major systems (Vijayalakshmi et al. (1988) J. Biol Chem. 263, 19419–19423). One system (cit) is generally pyrimidine-selective; thymidine serves as a model substrate. The other system (cif) is generally purine-selective; formycin B serves as a model substrate. Uridine and adenosine are substrates of both systems. Thymidine and cytidine (100 μM), but not formycin B (100 μM) inhibited Na +-uridine uptake. In addition, [ 3H]thymidine exhibited an Na +-driven overshoot phenomenon whereas [ 3H]formycin B did not. Na +-thymidine uptake was inhibited by (100 μM) adenosine, uridine, guanosine, but not by formycin B and inosine. Further studies demonstrated that guanosine trans-stimulated thymidine uptake suggesting that guanosine and thymidine share a common transporter in the human renal BBMV. A different pattern was identified in BBMV from the rabbit kidney where both [ 3H]thymidine and [ 3H]formycin B as well as [ 3H]uridine exhibited a transient Na +-driven overshoot phenomenon. Collectively, these data suggest that in rabbit renal BBMV both cif and cit systems are present whereas in human renal BBMV, there appears to be a single concentrative Na +-nucleoside cotransport system that interacts with uridine, cytidine, thymidine, adenosine and guanosine but not with formycin B and inosine. The system is similar to the previously described cit system except that guanosine is also a substrate.

Slowed Transcription and Rapid Messenger RNA Turnover Contribute to a Decline in Synthesis of Ovine Trophoblast Protein-i during in Vitro Culture1

Ovine trophoblast protein-1 (oTP-1) is produced in massive amounts by conceptuses during the Day-12-20-period of early pregnancy. The rate of production of oTP-1 declines during culture of conceptuses, however, suggesting a role for critical intrauterine factors for continued production. The present study was conducted to define the mechanism responsible for the in vitro decline in oTP-1 synthesis. Over 24 h culture, synthesis of oTP-1 was initially rapid but then declined such that there was little increase in either the protein or its antiviral activity after 12 h. By contrast, the release of total protein into the medium continued at an approximately linear rate for the entire 24-h period of culture. By using in situ hybridization to tissue sections and dot-blot analysis of tissue extracts with labeled cDNA probes, it was shown that the quantity of oTP-1 mRNA fell 3- to 5-fold during culture, whereas the amount of actin mRNA remained relatively constant. To determine whether this selective fall in levels of oTP-1 mRNA resulted from decreased transcription rate and/or high turnover rate of existing mRNA, conceptuses were either provided continuously with [3H]uridine for 24 h or labeled for 9 h and then exposed to medium enriched in unlabeled uridine and cytidine for a further 15 h. Specific incorporation of 3H into oTP-1 mRNA was assessed by hybridization to excess oTP-1 cDNA immobilized on nitrocellulose membranes. In the continuous labeling study, 3H in total RNA increased at an approximately linear rate for at least 18 h, whereas the content of 3H in oTP-1 mRNA peaked at 9 h and declined about 10-fold by 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of transcription-pausing in vivo in the trp operon leader region.

To determine whether RNA polymerase pauses during transcription in vivo, we have examined transcripts of the trp operon leader regions of Serratia marcescens and Escherichia coli. Labeled RNAs synthesized in E. coli strains containing plasmids bearing wild-type or mutant trp leader regions of S. marcescens or E. coli were isolated by hybridization and analyzed by polyacrylamide gel electrophoresis. The labeled RNAs synthesized in vivo on the S. marcescens wild-type and deletion mutant plasmids were the same size as the in vitro pause and leader transcripts. Hybridization of the presumed in vivo pause RNAs, and control in vitro pause RNAs, to M13 phage DNA containing a trp leader region deletion followed by treatment with S1 nuclease produced identical protected RNA species, proving that the in vitro and in vivo RNAs were identical. The amount of labeled pause RNAs relative to leader RNAs decreased following a chase with unlabeled uridine. E. coli RNAs identical to the previously characterized in vitro pause and leader transcripts were demonstrated by electrophoretic band position and fingerprint analysis. The finding that transcription pausing occurs in vivo is consistent with the view that transcription pausing and ribosome release of paused transcription complexes are responsible for the coupling of translation with transcription that is crucial to attenuation.

Enhanced delivery to target cells by heat-sensitive immunoliposomes.

Heat-sensitive immunoliposomes are capable of releasing the entrapped content at the target cell surface upon a brief heating to the phase transition temperature of the liposome membrane. In this study we have examined the delivery efficiency of drugs entrapped in heat-sensitive immunoliposomes. Immunoliposomes composed of dipalmitoyl phosphatidylcholine with entrapped [3H]uridine were incubated with target cells at 4 degrees C. The cell-liposome mixture was then heated to 41 degrees C and the uptake of [3H]uridine into the intracellular pool of phosphorylated uridine-containing molecules was measured. The immunoliposomes showed maximal release of the uridine at 41 degrees C, the phase transition temperature of dipalmitoyl phosphatidylcholine liposomes. The largest accumulation of [3H]uridine in the target cells also took place at 41 degrees C. The initial level of uptake of [3H]uridine released from immunoliposomes by heating was greatly enhanced over that observed for free [3H]uridine and [3H]uridine released from liposomes without attached antibody. The nucleoside uptake inhibitors nitrothiobenzylinosine, dipyridamole, and unlabeled uridine were able to inhibit uptake of [3H]uridine released from immunoliposomes. This supports the hypothesis that the enhanced uptake is due to a heat-induced release of [3H]uridine at the cell surface followed by transport and phosphorylation of [3H]uridine by the target cells. These results indicate the feasibility of using the heat-sensitive immunoliposomes as a target-specific drug delivery system.

Uridine transport and metabolism in the central nervous system.

The mechanisms by which uridine enters and leaves brain, choroid plexus, and cerebrospinal fluid (CSF) were investigated in the isolated choroid plexus in vitro and by injecting [3H]uridine intravenously and intraventricularly. Consistent with its postulated role in transporting uridine from blood into CSF, the isolated rabbit choroid plexus concentrated uridine with 10 microM uridine in the medium. [3H]Uridine, with and without unlabeled uridine, was infused at a constant rate into conscious adult rabbits. At 180 min, [3H]uridine entered CSF, choroid plexus, and brain more rapidly than mannitol. In brain, approximately 60-80% of the nonvolatile radioactivity was [3H]uridine phosphates. The addition of 2.1 mmol/kg of unlabeled uridine to the infusion syringe decreased the relative entry of [3H]uridine into brain by approximately 75% due mainly to the decreased formation of [3H]uridine phosphates and increased formation of [3H]uracil. Two hours after the intraventricular injection of [3H]uridine, [3H]uridine was cleared from CSF more rapidly than mannitol, in part to brain, where approximately 75% of the [3H]uridine was converted to [3H]uridine phosphates. The intraventricular injection of 42 mumol unlabeled uridine with the [3H]uridine decreased the phosphorylation of [3H]uridine in brain significantly and also decreased the clearance of [3H]uridine from the CSF. From blood, uridine enters CSF and the extracellular space of brain. Uridine then can enter brain cells, be phosphorylated to uridine phosphates, and subsequently incorporated into RNA or be catabolized to uracil.

Cell division does not affect Sendai virus genome replication in persistently infected BHK cells.

The extent of Sendai virus genome replication in persistently infected BHK cells actively growing or at confluence was followed by estimation of the [3H]uridine incorporated into intracellular nucleocapsid RNA. First, we showed that, in the presence of actinomycin D, actively growing persistently infected cells were taking up threefold more [3H]uridine than resting cells. This higher uptake exhibited by growing cells was observed neither in persistently infected cells in the absence of actinomycin D, nor in acutely infected cells in the presence of actinomycin D. Assuming that the cellular pool of unlabelled uridine stays constant, we used a correction factor for this difference in [3H]uridine uptake and estimated [3H]uridine incorporation in nucleocapsid RNA, normalizing the data either to the amount of cell or of viral template. Results showed that the viral genome replication, expressed either way, was not significantly influenced by cell growth conditions.

Encephalomyocarditis virus replication complexes preferentially utilizing nucleoside diphosphates as substrates for viral RNA synthesis. Nucleotide kinases specifically associated with the complex channel RNA precursor.

Replication complexes (RC) of the encephalomyocarditis (EMC) virus were shown previously to contain components that exhibit marked preference for nucleoside diphosphates over nucleoside triphosphates (NTP) as substrates for viral RNA synthesis [Koonin and Agol (1983), Virology 129, 309-318]. These NDP-preferring components have now been found to posses the following properties. When RC preparations were fractionated by sucrose density gradient centrifugation, the fractions containing NDP-preferring components exhibited a considerably higher nucleotide kinase activity as compared to either the fractions containing NTP-preferring components or corresponding fractions from mock-infected cells. When NDP-preferring RC were incubated with ADP and three other NTP, very low concentrations of endogenously generated ATP ensured a greater rate of RNA synthesis than did much higher concentrations of exogenous ATP. When an equimolar mixture of differently labelled UDP and UTP was used as a substrate for NDP-preferring RC, the label from UDP predominated in the newly synthesized RNA, even though the UDP-derived UTP constituted a minor portion of the total UTP pool. When labelled UDP was diluted with unlabelled uridine nucleotides, unlabelled UTP proved to be far less efficient than unlabelled UDP in diminishing the specific radioactivity of UMP incorporated into RNA by NDP-preferring RC. These data are interpreted in the sense that the NTP generated by the built-in nucleotide kinase system are not freed into the external milieu but rather form a separate pool preferentially used for synthesis of viral RNA by NDP-preferring RC. It is suggested that this functional compartmentation of NTP may be significant for the replication of viral RNA in vivo.

High resolution autoradiographical detection of RNA in the interchromatin granules of DRB-treated cells

Isolated rat liver cells were pulse-labelled with tritiated uridine and post-incubated in the presence of an excess of unlabelled uridine and of adenosine analog DRB (5-6-dichloro-1-β- d-ribofuranosyl benzimidazole). Nuclear radioactivity was detected with high resolution autoradiography. A significant labelling of the interchromatin granules was revealed in these conditions. Pretreatments of cells with low doses of actinomycin D in order to preferentially inhibit ribosomal RNA (rRNA) synthesis prevented the labelling of the interchromatin granules during subsequent DRB treatments. These observations indicate that in DRB-treated cells, the interchromatin granules are sites of transfer or of accumulation of nucleolar RNA. Our results are discussed in connection with our knowledge of the action of DRB on RNA metabolism in mammalian cells and with recent data concerning the still enigmatic interchromatin granules which are present in the nuclei of most cells.