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PubMed2005

Mass spectrometric identification of modified urinary nucleosides used as potential biomedical markers by LC-ITMS coupling.

In diseases accompanied by strong metabolic disorders, like cancer and AIDS, modifying enzymes are up- or down-regulated. As a result, many different types of metabolic end-products, including abnormal amounts of modified nucleosides, are found in urine. These nucleosides are degradation products of an impaired ribonucleic acid (RNA) metabolism, which affects the nucleoside pattern in urine. In several basic experiments we elucidated the fragmentation pathways of 16 characteristic nucleosides and six corresponding nucleic bases that occur in urine using electrospray ionization ion trap MS(5) (ESI-ITMS) experiments operated in positive ionization mode. For urinary nucleoside analysis, we developed an auto-LC-MS3 method based on prepurification via boronate gel affinity chromatography followed by reversed phase chromatography. For this purpose, an endcapped LiChroCART Superspher RP 18 column with a gradient of ammonium formate and a methanol-water mixture was used. This method gives a limit of detection of between 0.1 and 9.6 pmol for 15 standard nucleosides, depending on the basicity of the nucleoside. Overall, the detection of 36 nucleosides from urine was feasible. It was shown that this auto-LC-MS3 method is a valuable tool for assigning nucleosides from complex biological matrices, and it may be utilized in the diagnosis of diseases associated with disorders in RNA metabolism.

Biomarkers↗
PubMed1990

Chromatographic characterization of nitrobenzylthioinosine binding proteins in band 4.5 of human erythrocytes: purification of a 40 kDa truncated nucleoside transporter.

DEAE-column-purified band 4.5 polypeptides of human erythrocyte membranes are mostly glucose transporters with nucleoside transporters as a minor component. The purpose of the present work was to differentially identify and isolate the nucleoside transporters in band 4.5 free from glucose transporters. Equilibrium binding studies demonstrated that the band 4.5 preparation binds nibrobenzylthioinosine (NBTI), a potent nucleoside transport inhibitor, at two distinct sites, one with a high affinity (dissociation constant, KD of 1 nM) with a small capacity, BT (0.4 nmol/mg protein), and the other with a low affinity (KD of 15 microM) with a large BT (14-16 nmol/mg protein). The BT of the low-affinity site was equal to that of the cytochalasin B binding site in the preparation. A gel-filtration chromatography of band 4.5 photolabeled with [3H]NBTI and [3H]cytochalasin B identified three polypeptides of apparent Mr 55,000, 50,000 and 40,000. Of these, the 55 kDa polypeptide was specifically labeled by cytochalasin B (p55GT), indicating that it is a glucose transporter. Both the 50 and 40 kDa polypeptides were labeled with NBTI at low ligand concentrations (less than 0.1 microM), which was abolished by an excess (20 microM) of nitrobenzylthioguanosine, indicating that they are two forms (p50NT and p40NT, respectively) of the high affinity NBTI binding protein or nucleoside transporter. At higher (not less than 10 microM) NBTI concentrations, however, p55GT was also labeled with NBTI, indicating that the low-affinity NBTI binding is due to a glucose transporter. Treatment of band 4.5 with trypsin reduced the p50NT labeling with a concomitant and stoichiometric increase in the p40NT NBTI labeling without affecting the high-affinity NBTI binding of the preparation. These findings indicate that the nucleoside transporter is slightly smaller in mass than the glucose transporter and that trypsin digestion produces a truncated nucleoside transporter of apparent Mr 40,000 which retains the high-affinity NBTI binding activity of intact nucleoside transporter. Both p55GT and p50 NT were coeluted in a major protein fraction, P1 in the chromatography, while p40NT was eluted separately as a minor protein fraction, P1a. All three polypeptides formed mixed dimers, which were eluted in a fraction PO. We have purified and partially characterized the truncated nucleoside transporter, p40NT. The purified p40NT may be useful for biochemical characterization of the nucleoside transporter.

Blood Proteins↗
PubMed1987

Trypanosoma cruzi adenine nucleoside phosphorylase. Purification and substrate specificity.

An adenine nucleoside phosphorylase has been partially purified from extracts of epimastigotes of the Peru strain of Trypanosoma cruzi, the causative agent of Chagas' disease. The purification procedure separated this enzyme from the three other nucleoside-cleaving enzymes found in extracts. The adenine nucleoside phosphorylase, which efficiently cleaved 5'-deoxy-5'-methylthioadenosine (MTA), had a particle weight of 68,000 and exhibited a broad pH optimum between pH 6 and 8. In addition to MTA, the purified enzyme cleaved and synthesized adenosine and 2'-deoxyadenosine with high efficiency. This contrasts to the enzyme from S-180 cells which has been reported to cleave adenosine poorly and not to cleave 2'-deoxyadenosine. Several observations suggested that the three substrates, MTA, adenosine and 2'-deoxyadenosine, use a common catalytic site: (a) all served as alternate-substrate inhibitors exhibiting mutually competitive inhibition with Ki values equivalent to their respective Km values, (b) 5'-chloroformycin A exhibited a competitive Ki value of 4 microM with each nucleoside substrate, and (c) the Km value of phosphate derived from initial velocity studies (180 +/- 20 microM) was independent of the nucleoside substrate. Substrate specificity studies in both the synthesis and cleavage direction indicated that the enzyme had a broad specificity for bases and nucleosides. For the synthesis of nucleosides, the enzyme demonstrated a preference for an amino group in the position equivalent to the 6 position of purine. Compounds containing a hydroxyl group in this position were not substrates. Although a hydrogen or methyl group could substitute for a 6-amino group, a marked decrease in substrate efficiency was observed with these compounds. Alterations in the purine ring led to decreases in the maximal velocity values as evidenced by the substrate or nonsubstrate properties of 1-, 3-, and 7-deazaadenine and 4-aminopyrazolo[3,4-d]pyrimidine. The Km values for 5-methylthioribose 1-phosphate, ribose 1-phosphate and 2'-deoxyribose 1-phosphate with adenine serving as acceptor were 21, 150 and 370 microM. For nucleoside cleavage, the T. cruzi enzyme catalyzed the phosphorolysis of a variety of 5'-substituted adenine-containing nucleosides including those possessing 5'-hydrogen-, hydroxyl-, halogeno-, alkylthio-, amino- and azido-moieties. Inclusion of an ionized group in the 5'-position, such as 5'-carboxy-5'-deoxyadenosine or AMP, precluded substrate activity. 3'-Deoxyadenosine, arabinosyladenine and alpha-adenosine did not serve as substrates.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine↗
PubMed2005

Age-dependence of urinary normal and modified nucleosides in childhood as determined by reversed-phase high-performance liquid chromatography.

Modified nucleosides have been characterized as tumor markers for a number of malignant diseases. In order to use these markers in children, the age-dependence of the nucleoside levels in healthy children has to be established and taken into account in diagnostic decisions. In this study, the levels of 12 normal and modified nucleosides in urine of 166 healthy children and adolescents with an age between 1 day and 19 years are determined by reversed-phase HPLC, and age-dependent reference ranges are defined. The urinary nucleoside concentrations are related to the creatinine concentrations, which allows the use of randomly collected urine samples. All nucleoside levels in urine of children decrease with age, most pronounced during the first 4 years of life, and the age-dependence of the reference values of the individual nucleosides can be approximated by a mathematical function y = b(0) + b(1) (1/x) with the regression coefficients b(0) and b(1,) the nucleoside levels y and the age x between 1 year and 19 years. In the very young children, the shifts in the nucleoside concentrations are more differentiated. Starting with low levels on the first day of life, the concentrations of all studied nucleosides rise up to an age of 1-2 months, when they reach their absolute maximum for all age periods, and then decrease.

Adolescent↗
PubMed2002

Synthesis of oxathiolane imidazole nucleosides.

Nucleosides have been of great interest since their strong antiviral activities were discovered. 1,3-Oxathiolane ring system has been known for many years, but it is in recent years that the ring has been used as the sugar ring in nucleoside analogs (Synthesis (1991) 1046; J. Am. Chem. Soc. 113 (1991) 9377; Tetrahedron Lett. 35 (1994) 4739). Besides, bredinin is a natural nucleoside antibiotic with imidazole moiety and there are some other studies reported on nucleosides with the imidazole group (Biorg. Med. Chem. 7 (1999) 481; Biorg. Med. Chem. 7 (1999) 1617; Nucleosides Nucleotides 18 (1999) 331). These findings make the imidazole group interesting as the base of a nucleoside. In this study, in order to find out the structure-activity relationships of L-oxathiolanyl nucleosides, L-oxathiolanyl imidazole nucleosides 7 and 8 were synthesized, via novel intermediates 2-6, which were then tested for anti-HIV activity (Antivir. Res. 1-11 (1994) 25) in human peripheral blood mononuclear (PBM) cells, the synthesized nucleosides did not show significant activity up to 100 microM against HIV-1.

Anti-HIV Agents↗
PubMed1997

Role of nucleosides and nucleotides in the immune system, gut reparation after injury, and brain function.

Emerging evidence indicates the importance of nucleosides and nucleotides in the maintenance of functions of the bone marrow hematopoietic cells, intestinal mucosa, and the brain, which have limited de novo synthesis of purine and pyrimidine bases. We have found that nucleosides and nucleotides stimulate hemopoieses and increase peripheral neutrophil counts in mice treated with cyclophosphoamide. Intraperitoneal administration of nucleosides and nucleotides decreased bacterial translocation, the number of colony-forming units, and increased survival against methicillin-resistant Staphylococcus aureus. In vitro immune studies in mice showed that nucleosides and nucleotides increase the delayed-type cutaneous hypersensitivity and the popliteal lymph node blastogenic response to antigens, allogens, and mitogens. Both intraperitoneal and oral administration of nucleosides and nucleotides reduced endotoxin-induced bacterial translocation and improved injury to the gut in protein-deficient mice. However, oral administration of nucleosides and nucleotides in experimental colitis resulted in a worsening of colitic conditions and increased interleukin-8 and tumor necrosis factor-alpha concentrations in inflamed colonic portions, indicating the pro-inflammatory activities of nucleosides and nucleotides. Memory-deficient senescence-accelerated mice and mice with dementia showed improved memory with dietary nucleosides and nucleotides supplementation. These results indicate that supplementation with nucleosides and nucleotides is beneficial to the functions of the system and the brain. However, beneficial effects to the gut appear to depend on the type of damage sustained by the gut.

Animals↗
PubMed2005

Synthesis of nucleoside boranophosphoramidate prodrugs conjugated with amino acids.

[structure: see text] Nucleoside boranophosphates and nucleoside amino acid phosphoramidates have been shown to be potent antiviral and anticancer agents with the potential to act as nucleoside prodrugs. A combination of these two types of compounds results in a boranophosphoramidate linkage between the nucleoside and amino acid. This new class of potential prodrugs is expected to possess advantages conferred by both types of parent compounds. Two approaches, specifically the H-phosphonate and oxathiaphospholane approaches, are described here to synthesize nucleoside boranophosphoramidate prodrugs conjugated with amino acids. The H-phosphonate approach involves a key intermediate, silylated nucleoside amino acid phosphoramidite 6, prepared from a series of reactions starting from nucleoside H-phosphonate in the presence of condensing reagent DPCP. Due to the lengthy procedure and the difficulties in removing DPCP from the final products, we switched to the oxathiaphospholane approach in which the DBU-assisted oxathiaphospholane ring-opening process constituted a key step for the generation of nucleoside amino acid boranophosphoramidates 24. We demonstrate that this key step did not cause any measurable C-racemization of boranophosphorylated amino acids 22. Diastereomers of compounds 24a-f were separated by RP-HPLC. An "adjacent"-type mechanism is proposed to explain the diastereomer ratio in the final products obtained via the oxathiaphospholane approach. A tentative assignment of configuration for the diastereomers was carried out based on the mechanism, molecular modeling, and (1)H NMR. Conclusively, the oxathiaphospholane methodology proved to be more facile and efficient than H-phosphonate chemistry in the preparation of the nucleoside amino acid boranophosphoramidate analogues that are promising as a new type of antiviral prodrugs.

Amides↗
PubMed2004

Nucleoside transport at the blood-testis barrier studied with primary-cultured sertoli cells.

Nucleosides are essential for nucleotide synthesis in testicular spermatogenesis. In the present study, the mechanism of the supply of nucleosides to the testicular system across the blood-testis barrier was studied using primary-cultured Sertoli cells from rats and TM4 cells from mice. Uptake of uridine by these cells was time- and concentration-dependent. Uridine uptake was decreased under Na(+)-free conditions, and the system was presumed to be high affinity, indicating an Na(+)-dependent concentrative nucleoside transporter (CNT) is involved. On the other hand, nitrobenzylthioinosine, a potent inhibitor of Na(+)-independent equilibrative nucleoside transporters (ENTs), inhibited uridine uptake by the Sertoli cells in a concentration-dependent manner. Expression of nucleoside transporters ENT1, ENT2, ENT3, CNT1, CNT2, and CNT3 was detected in Sertoli cells by reverse transcriptase-polymerase chain reaction analysis. Inhibition studies of the uptake of uridine by various nucleosides both in the presence and absence of Na(+) indicated that the most of those expressed nucleoside transporters, ENTs and CNTs, are involved functionally. These results demonstrated that Sertoli cells are equipped with multiple nucleoside transport systems, including ENT1, ENT2, and CNTs, to provide nucleosides for spermatogenesis.

Algorithms↗
PubMed1976

Thymidine uptake and utilization in Escherichia coli: a new gene controlling nucleoside transport.

A commonly used strain of Escherichia coli K-12 was shown to be deficient in the transport of a number of nucleosides, including thymidine. Thymidine incorporation was unaffected. Strain AB2497 exhibited a strikingly lower thymidine pulse-label incorporation at low (less than 1 mug/ml) thymidine concentrations than do many other strains. The deficiency appeared to be due to mutation in a single gene. This gene, which we designated nup (for nucleoside uptake), is located at 10 to 13 min on the E. coli linkage map. In nup+ strains, the transport of a given nucleoside was relatively insensitive to large excesses of other nucleosides but was competitively inhibited by the same nucleoside. Mutants deficient inthymidine kinase are deficient in thymidine uptake but normal in deoxyadenosine uptake. A two-step model for nucleoside transport is presented in which the first step, utilizing the nup gene product, is a nonspecific translocation of nucleoside to the interior of the cell. In the second step, the individual nucleosides are modified by cellular enzymes (e.g., nucleosides kinases) facilitate accumulation.

Adenosine↗
PubMed1996

Nucleoside-nucleotide free diet protects rat colonic mucosa from damage induced by trinitrobenzene sulphonic acid.

BACKGROUND: Growing evidence suggests that intestinal recovery from injury induced by radiation, endotoxin, and protein deficiency is improved by the ingestion of nucleosides and nucleotides. AIM: This study examined the effect of dietary nucleosides and nucleotides supplementation on trinitrobenzene sulphonic acid induced colonic damage in experimental colitis. METHODS: Sprague-Dawley rats were randomised into two groups and fed nucleic acid free 20% casein diet (control) or this diet supplemented with 0.5% nucleoside-nucleotide mixture for four weeks. On the second week, colonic inflammation was induced in rats by intracolonic administration of 0.25 ml of 50% ethanol containing 25 mg of trinitrobenzene sulphonic acid. Additionally, other sets of rats were treated with 0.25 ml of 50% ethanol, 25 mg of trinitrobenzene sulphonic acid in 0.25 ml saline, or 0.25 ml of 0.9% saline. RESULTS: After two weeks, colon weight, macroscopic and microscopic damage scores, were significantly greater (p < 0.05) in the nucleoside-nucleotide supplemented group compared with the non-supplemented control groups. The same variables seen in the trinitrobenzene sulphonic acid-ethanol group fed nucleoside-nucleotide free diet were greater (p < 0.05) than in the rest of the groups fed nucleoside-nucleotide free diet and treated with ethanol, trinitrobenzene sulphonic acid in saline, or saline. Histologically, segmental ulceration and inflammation associated with significantly increased infiltration of polymorphonuclear leucocytes, macrophages, lymphocytes, fibroblasts were observed in the supplemented group compared with the controls. In the nucleoside-nucleotide supplemented group the epithelial damage, mucosal erosion, oedema, and coagulative necrosis of the muscularis propria was more extensive in comparison to the non-supplemented control groups. CONCLUSIONS: This study suggests that dietary nucleosides and nucleotides may aggravate colonic damage and inflammation in chemically induced experimental colitis in rats; and that nucleoside-nucleotide free diet combined with other pharmacological agents may offer a better response.

Animals↗
PubMed2006

Characterization of the rat Na+/nucleoside cotransporter 2 and transport of nucleoside-derived drugs using electrophysiological methods.

The Na(+)-dependent nucleoside transporter 2 (CNT2) mediates active transport of purine nucleosides and uridine as well as therapeutic nucleoside analogs. We used the two-electrode voltage-clamp technique to investigate rat CNT2 (rCNT2) transport mechanism and study the interaction of nucleoside-derived drugs with the transporter expressed in Xenopus laevis oocytes. The kinetic parameters for sodium, natural nucleosides, and nucleoside derivatives were obtained as a function of membrane potential. For natural substrates, apparent affinity (K(0.5)) was in the low micromolar range (12-34) and was voltage independent for hyperpolarizing membrane potentials, whereas maximal current (I(max)) was voltage dependent. Uridine and 2'-deoxyuridine analogs modified at the 5-position were substrates of rCNT2. Lack of the 2'-hydroxyl group decreased affinity but increased I(max). Increase in the size and decrease in the electronegativity of the residue at the 5-position affected the interaction with the transporter by decreasing both affinity and I(max). Fludarabine and formycin B were also transported with higher I(max) than uridine and moderate affinity (102 +/- 10 and 66 +/- 6 microM, respectively). Analysis of the pre-steady-state currents revealed a half-maximal activation voltage of about -39 mV and a valence of about -0.8. K(0.5) for Na(+) was 2.3 mM at -50 mV and decreased at hyperpolarizing membrane potentials. The Hill coefficient was 1 at all voltages. Direct measurements of radiolabeled nucleoside fluxes with the charge associated showed a ratio of two positive inward charges per nucleoside, suggesting a stoichiometry of two Na(+) per nucleoside. This discrepancy in the number of Na(+) molecules that bind rCNT2 may indicate a low degree of cooperativity between the Na(+) binding sites.

Animals↗
PubMed2002

Nucleosides are efficiently absorbed by Na(+)-dependent transport across the intestinal brush border membrane in veal calves.

In previous work, a comparatively high capacity for Na(+)-dependent transport of nucleosides across the intestinal brush border membrane (BBM) was observed in dairy cows, which might be related to digestion of the large amount of nucleic acids present in ruminal microorganisms in the ruminant small intestine. If this were the case, the capacity for Na(+)-dependent intestinal nucleoside transport should be much lower in veal calves, in which only small amounts of nucleic acids, nucleotides, and nucleosides reach the small intestine via the milk replacer. To test this hypothesis, we investigated Na(+)-dependent transport of 3H-labeled thymidine and guanosine across the BBM using BBM vesicles (BBMV) isolated from the small intestine of veal calves. In the presence of a transmembrane Na+ gradient both substrates were transported against a concentration gradient. Inhibitory studies showed that thymidine and guanosine are transported by two different transporters with overlapping substrate specificity, one accepting predominantly pyrimidine nucleosides (N2) and one accepting particularly purine nucleosides (N1). Nucleoside transport was inhibited by glucose along the whole small intestine. Maximal transport rates similar to those in dairy cows were obtained for the proximal, mid-, and distal small intestine. These findings suggest that the high absorptive capacity for nucleosides is a genetically fixed property in the bovine small intestine, which is already present in the preruminant state of veal calves. It may contribute to the high digestibility of nucleic acids observed by others in veal calves receiving milk replacer supplemented with RNA. Its main function may be the efficient absorption of nucleosides resulting from the digestion of nucleic acids associated with desquamated enterocytes. Due to the limited de novo synthesis of nucleotides in enterocytes intracellular uptake of nucleosides across the BBM may contribute to nucleic acid synthesis in enterocytes and thus may have a trophic effect on the intestinal epithelium.

Animals↗
PubMed2000

A Na(+)-dependent nucleoside transporter in microglia.

In the central nervous system, HIV-1 has a defined tropism for brain macrophages and microglia. Nucleoside analog drugs such as zidovudine improve the clinical and neuropsychological functions in HIV-demented patients. Multiple carrier-mediated transport systems can play an important role in the membrane permeation of nucleosides and nucleoside analog drugs in a number of cells. The purpose of this project was to characterize the uptake properties of the pyrimidine nucleoside probe thymidine by a continuous rat microglia cell line (MLS-9) grown as a monolayer on an impermeable substratum. Approximately 50% of thymidine (10 microM) uptake by the monolayer cells was found to be Na(+) dependent. Kinetics of specific thymidine uptake showed a single saturation system (K(m) = 44 microM at 37 degrees C) and a Na(+)/thymidine stoichiometry of 2:1. Pyrimidine and purine nucleoside probes (50 microM) exerted a competitive inhibitory effect on specific thymidine uptake with K(i) values of 40, 38, 45, and 39 microM for adenosine, uridine, guanosine, and cytidine, respectively. In addition, nucleoside analog drugs significantly decreased specific thymidine uptake, with IC(50) values of 135.1 microM for abacavir and 0.6 microM for zidovudine, which inhibited in a noncompetitive manner. These results suggest that a Na(+)-dependent nucleoside transport system is present in rat microglia and that long-range interactions between antiretroviral nucleoside analog drugs and the nucleoside substrates may occur at the transporter sites.

Adenosine↗
PubMed2000

Electrophysiological analysis of the substrate selectivity of a sodium-coupled nucleoside transporter (rCNT1) expressed in Xenopus laevis oocytes.

Nucleoside transporters that mediate cellular uptake of therapeutic nucleoside analogs are major determinants of the pharmacokinetic properties of these compounds. Understanding the substrate selectivity of these transporters is critical in the development of therapeutic nucleoside analogs with optimal pharmacokinetic properties, including high oral bioavailability and tissue-specific distribution. In general, substrate selectivity of nucleoside transporters has been evaluated indirectly by inhibition studies. The purpose of this study was to directly measure the transport of nucleoside analogs by the sodium-coupled pyrimidine-selective transporter rCNT1 using electrophysiology methods. We used a two-electrode voltage clamp assay to investigate the substrate selectivity of rCNT1; 19 structurally diverse nucleosides and nucleoside analogs were studied. Uridine-induced currents in voltage-clamped oocytes expressing rCNT1 were sodium-, voltage-, and concentration-dependent (K(0.5) = 21 microM), and were blocked by adenosine. Uridine-induced currents increased approximately 5-fold upon hyperpolarization of membrane potential from -10 to -150 mV. Uridine, thymidine, and cytidine induced currents in rCNT1-expressing oocytes, whereas guanosine, inosine, and adenosine did not. Uridine, deoxyuridine, and cytidine analogs with modifications at the 3-, 4-, or 5-position were found to be substrates of rCNT1, whereas uridine and cytidine analogs modified at the 6-position were not. In addition, it was found that the 5'-hydroxyl group of the sugar is not required for transport by rCNT1. These results enhance our understanding of the structural basis for substrate selectivity of nucleoside transporters and should prove useful in the development of therapeutic nucleoside analogs.

Animals↗
PubMed1981

The fine specificity of immune suppression to individual nucleosides.

The fine specificity of immune suppression was further studied in a murine experimental model in which the induction and expression of suppression by individual nucleoside-bound spleen cells were examined. The experiments were undertaken: a) to determine whether all 4 nucleosides have the ability to elicit suppressor T cells; b) to examine the specificity of cross-suppression induced by individual nucleosides; and c) to establish that nucleoside-specific suppression is mediated by T cells bearing nucleoside-specific receptors. The results show that, of the 4 nucleosides studied, only guanosine and thymine riboside were capable of eliciting immune suppression. Thymine riboside-coupled spleen cells induced cross-suppression to all 4 nucleosides. Furthermore, the nonresponsive state generated by guanosine-spleen cells was found to be transferable and mediated by nucleoside-binding T cells. The ability of only select nucleosides to elicit immune suppression is discussed in the context of recent evidence demonstrating the selective activation of T cell subsets by intramolecular antigenic determinants.

Animals↗
PubMed1995

Time-dependent antinociceptive interactions between opioids and nucleoside transport inhibitors.

Endogenous purinergic systems are important in spinal mechanisms of antinociception. Antinociception induced by spinal mu opioid receptor-selective agonists, in particular, appears to be mediated in part by opioid-stimulated adenosine release. Nucleoside transport system(s) have been implicated both in adenosine release and in its reuptake at spinal sites. The present investigations were designed to determine the significance of nucleoside transport system(s) inhibition in vivo in antinociception induced by opioids administered intrathecally in mice. Dilazep, but not dipyridamole or s (4-nitrobenzyl)-6-thioinosine, nucleoside transport system(s) inhibitors, induced time- and dose-dependent antinociception in the tail-flick test, putatively via spinal adenosine reuptake inhibition. Each nucleoside transport system(s) inhibitor, at doses that have no significant effects alone, enhanced adenosine-mediated antinociception when coadministered intrathecally. Concurrent treatment of mice with opioid receptor-selective agonists and nucleoside transport system(s) inhibitors had varying effects on antinociception, depending on the timing of the nucleoside transport inhibitor. In general, antinociception induced by mu opioid receptor-selective agonists was inhibited by pretreatment, was not affected after coadministration and was enhanced by post-treatment, with nucleoside transport system(s) inhibitors. In contrast, antinociception induced by delta opioid receptor-selective agonists was enhanced by nucleoside transport system(s) inhibitors in all treatment protocols. These results provide in vivo evidence that alterations in adenosine movements into or out of spinal neurons via the nucleoside transport systems can induce antinociception and enhance or inhibit opioid-mediated antinociception. These data also support the hypothesis that adenosine plays significant but independent roles in antinociception induced by mu and delta opioid receptor-selective agonists.

Adenosine↗
PubMed1993

An immunohistochemical analysis for cancer of the esophagus using monoclonal antibodies specific for modified nucleosides.

BACKGROUND: Modified nucleosides such as 1-methyl-adenosine and pseudouridine exist as minute components of transfer ribonucleic acid (tRNA) and are excreted in the urine in large amounts in the presence of malignancy. Although use of these modified nucleosides as tumor markers has long been studied and many reports have detailed their relationship with malignant tumors and the urinary excretion of various modified nucleosides, there have been no reports on modified nucleosides in esophageal carcinoma. METHODS: Monoclonal antibody patterns against 1-methyladenosine and pseudouridine were studied in esophageal carcinoma, freshly resected esophageal carcinoma tissue specimens fixed in 10% neutral formaldehyde solution, embedded in paraffin, and sectioned for immunohistochemical study. Inhibition enzyme-linked immunosorbent assay (ELISA) was used to examine urinary excretion of these modified nucleosides in patients with esophageal carcinoma. RESULTS: Although rare in normal esophageal epithelium, these modified nucleosides were strongly stained in esophageal carcinoma cells. Most carcinoma cells exhibited a cytoplasmic pattern, although some cells at the infiltrating edge displayed a nuclear pattern. These modified nucleosides were intensely imaged in 11 of 12 cultured esophageal cell lines, the exception being one line that had a much longer doubling time. Using ELISA, urinary excretion of these modified nucleosides was found to be significantly higher in patients with esophageal carcinoma than in healthy subjects; such excretion correlated with carcinoma size and stage and tended to decrease after treatment. CONCLUSIONS: These findings indicate that the modified nucleosides 1-methyladenosine and pseudouridine may be useful as tumor markers for esophageal carcinoma.

Adenosine↗
PubMed1979

The enzymatic preparation of [alpha-(32)P]nucleoside triphosphates, cyclic [32P] AMP, and cyclic [32P] GMP.

A method has been developed for the enzymatic preparation of alpha-(32)P-labeled ribo- and deoxyribonucleoside triphosphates, cyclic [(32)P]AMP, and cyclic [(32)P]GMP of high specific radioactivity and in high yield from (32)Pi. The method also enables the preparation of [gamma-(32)P]ATP, [gamma-(32)P]GTP, [gamma-(32)P]ITP, and [gamma-(32)P]-dATP of very high specific activity and in high yield. The preparation of the various [alpha-(32)P]nucleoside triphosphates relies on the phosphorylation of the respective 3'-nucleoside monophosphates with [gamma-(32)P]ATP by polynucleotide kinase and a subsequent nuclease reaction to form [5'-(32)P]nucleoside monophosphates. The [5'-(32)P]nucleoside monophosphates are then converted enzymatically to the respective triphosphates. All of the reactions leading to the formation of [alpha-(32)P]nucleoside triphosphates are carried out in the same reaction vessel, without intermediate purification steps, by the use of sequential reactions with the respective enzymes. Cyclic [(32)P]AMP and cyclic [(32)P]GMP are also prepared enzymatically from [alpha-(32)P]ATP or [alpha-(32)P]GTP by partially purified preparations of adenylate or guanylate cyclases. With the exception of the cyclases, all enzymes used are commerically available. The specific activity of (32)P-labeled ATP made by this method ranged from 200 to 1000 Ci/mmol for [alpha-(32)P]ATP and from 5800 to 6500 Ci/mmol for [gamma-(32)P]ATP. Minor modifications of the method should permit higher specific activities, especially for the [alpha-(32)P]nucleoside triphosphates. Methods for the use of the [alpha-(32)P]nucleoside phosphates are described for the study of adenylate and guanylate cyclases, cyclic AMP- and cyclic GMP phosphodiesterase, cyclic nucleotide binding proteins, and as precursors for the synthesis of other (32)P-labeled compounds of biological interest. Moreover, the [alpha-(32)P]nucleoside triphosphates prepared by this method should be very useful in studies on nucleic acid structure and metabolism and the [gamma-(32)P]nucleoside triphosphates should be useful in the study of phosphate transfer systems.

Adenosine Triphosphate↗