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Biomedical subjects

R W Trewyn

Publications and source records attributed to R W Trewyn.

At least 37 records · Page 2Linked to original sources
PubMed1985

Hematopoiesis and the inosine modification in transfer RNA.

Human promyelocytic leukemia (HL-60) cells were used to begin to evaluate the role in hematopoiesis of inosine biosynthesis in the tRNA anticodon wobble position; a reaction involving the enzymatic insertion of performed hypoxanthine. Dimethyl sulfoxide (DMSO) and hypoxanthine were found to induce the differentiation of HL-60 cells in a synergistic manner, and the induced differentiation was independent of changes in the purine catabolic enzymes adenosine deaminase and purine nucleoside phosphorylase. The short-term exposure of HL-60 cells to DMSO plus hypoxanthine resulted in enhanced leucine incorporation, and a model is presented showing how the inosine modification reaction in tRNA may be involved. A means by which hypoxanthine insertion into tRNA may modulate the synthesis of regulatory proteins (e.g., lymphokines and cell surface receptors) is also outlined.

Adenosine Deaminase↗
PubMed1985

Inhibition of queuine uptake in cultured human fibroblasts by phorbol-12,13-didecanoate.

The modified base queuine is inserted posttranscriptionally into the first position of the anticodon of tyrosine tRNA, histidine tRNA, asparginine tRNA, and aspartic acid tRNA. Phorbol-12,13-didecanoate (PDD) effects a decrease in the queuine content of tRNA in cultured human foreskin fibroblasts. The present data suggest that this results from a PDD-mediated inhibition of queuine uptake. Nonsaturable uptake was observed for tritiated dihydroqueuine (rQT3) for up to 2 hr at 10 to 1000 nM concentrations, while saturation of uptake was observed after 3 to 4 hr. Lineweaver-Burke analysis of concentration versus uptake revealed biphasic uptake kinetics with high and low Km components of approximately 350 and 30 nM, respectively. Competition by queuine of rQT3 uptake indicated that both compounds have equal affinity for the uptake mechanism. PDD inhibited rQT3 uptake but required 30 to 60 min of exposure before the uptake was completely blocked. The rQT3 efflux rate from cells was found to be 3 to 4 times greater than that of uptake, and PDD also inhibited the efflux reaction. The potential inhibitors furosemide, nitrobenzylthioinosine, ouabain, 7-methylguanine, 7-deazaguanine, guanine, guanosine, adenine, adenosine, hypoxanthine, and epidermal growth factor had no effect on rQT3 uptake. However, dipyridamole was immediately effective at reducing rQT3 uptake.

Biological Transport↗
PubMed1984

Inosine biosynthesis in transfer RNA by an enzymatic insertion of hypoxanthine.

An enzyme was discovered which incorporates hypoxanthine into mature tRNA macromolecules. This enzyme is postulated to be similar to tRNA-guanine ribosyltransferase which inserts 7-(3,4-trans-4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl )-7-deazaguanine into the first position of the anticodon of four tRNAs. The hypoxanthine-incorporating enzyme has been assayed in extracts of rat liver and cultured human leukemia cells and it has been resolved from tRNA-guanine ribosyltransferase by DEAE-cellulose column chromatography. The enzyme assay is based on the incorporation of radiolabeled hypoxanthine into unfractionated heterologous tRNA and the reaction rate is proportional to the amount of added enzyme extract. Hydrolysis of the radiolabeled tRNA and analysis of the nucleoside composition yields inosine (the nucleoside of hypoxanthine) as the only radiolabeled product. It is proposed that the enzyme, a tRNA-hypoxanthine ribosyltransferase, is responsible for the biosynthesis of inosine in the anticodon wobble position of specific tRNAs, resulting in greatly expanded codon recognition by these tRNAs.

Animals↗
PubMed1984

Altered growth properties of normal human cells induced by phorbol 12,13-didecanoate.

The tumor promoter phorbol 12,13-didecanoate (PDD) significantly altered the growth properties of early passage normal human skin cells in vitro in culture medium supplemented with elevated concentrations of selected amino acids. Continuous treatment of cells with 10(-7) or 10(-8) M PDD resulted in a 5 to 10-fold increase in saturation density at early passages followed by a long-term two- to fourfold increase. The PDD-treated cultures remained in exponential growth at cell densities greater than 10-fold higher than the control cultures. Removal of PDD from the culture medium while the cells were at a high cell density resulted in a return to near-normal saturation density by the subsequent passage. Anchorage independent growth of normal human cells in methylcellulose was also promoted by PDD in a dose dependent manner, with prior subculturing in the presence of PDD being required for maximal colony formation. The structural analog 4 alpha-phorbol 12,13-didecanoate failed to elicit similar cellular responses.

Carcinogens↗
PubMed1984

Inosine biosynthesis in transfer RNA: a postulated role in immune regulation.

A hypothesis is put forth describing a role in immune regulation for inosine biosynthesis in the anticodon of tRNA. The enzymatic insertion of hypoxanthine into the tRNA wobble base position is predicted to be a control point for the translation of proteins and peptides required for normal immune function. The substrate for inosine biosynthesis in tRNA, hypoxanthine, is an intermediate in the purine catabolic pathway, and defects in this pathway are associated with inherited immunodeficiency diseases. Therefore, a role for aberrant inosine biosynthesis in tRNA is postulated in causing the immunodeficient conditions, and it may be a relevant molecular defect in leukemia as well.

Antibody Formation↗
PubMed1984

Adenine nucleotide metabolism and compartmentalization in isolated adult rat heart cells.

The metabolism and intracellular compartmentalization of adenine nucleotides in a preparation of adult rat heart myocytes showing good morphology, viability, and tolerance to calcium ion has been examined by high performance liquid chromatography. These myocytes contain an average of 23 nmol adenine nucleotide per milligram protein which is about 60% of the adenine nucleotide content of intact rat heart tissue. The loss of adenine nucleotide occurs during the incubation and washing steps that increase the yield of viable cells, rather than during the collagenase perfusion. An analysis of cellular compartments shows that the adenine nucleotide of the cell consists of 17 nmol adenine nucleotide in the cytosol, 5 nmol in the mitochondria, and 1.3 nmol adenosine diphosphate bound to myofibrils per milligram cell protein. Myocytes lose both adenosine triphosphate and adenine nucleotide when incubated anaerobically in the absence of glucose, and the lost adenine nucleotide can be accounted for as increased inosine, adenosine, and inosine monophosphate. Myocytes that contain less than 0.1 nmol of cytosol adenosine triphosphate per milligram cell protein maintain an intact sarcolemma, but are unable to carry out anaerobic glycolysis. Reoxygenation of anaerobic cells results in restoration of energy charge and a net resynthesis of about 2 nmol adenine nucleotide per milligram protein. Adenosine and inosine monophosphate decrease on reaeration of anaerobic cells, whereas inosine levels increase. When iodoacetate is added to block glycolysis, the decline in adenine nucleotide and production of inosine monophosphate are accelerated and there is no resynthesis of adenine nucleotide when anaerobic cells are reoxygenated . Large accumulations of inosine monophosphate are also seen in myocytes treated with an uncoupler of oxidative phosphorylation.

Adenine Nucleotides↗
PubMed1984

Relationship between a tumor promoter-induced decrease in queuine modification of transfer RNA in normal human cells and the expression of an altered cell phenotype.

With normal human skin fibroblasts in culture, a transient decrease in queuine modification of tRNA precedes a phorbol ester tumor promoter-induced 5- to 10-fold increase in saturation density. Subsequently, an increase in the queuine content of cellular tRNA (to levels comparable to those in untreated cultures) precedes a decrease in saturation density. This reversal of the phorbol ester-induced alteration in tRNA modification occurs in the continued presence of the tumor promoter, and it parallels an increased ability of the cells to salvage queuine from catabolized endogenous tRNA. Addition of exogenous queuine concurrently with the tumor promoter at early passage significantly inhibits the increase in saturation density. The results suggest a role for the decrease in queuine modification of tRNA in mediating the phenotypic change induced by the tumor promoter.

Carcinogens↗
PubMed1983

The 5-methylcytosine content of DNA from human tumors.

The over-all 5-methylcytosine (m5C) content of DNA from normal tissues varies considerably in a tissue-specific manner. By high-performance liquid chromatography, we have examined the m5C contents of enzymatic digests of DNA from 103 human tumors including benign, primary malignant and secondary malignant neoplasms. The diversity and large number of these tumor samples allowed us to compare the range of DNA methylation levels from neoplastic tissues to that of normal tissues from humans. Most of the metastatic neoplasms had significantly lower genomic m5C contents than did most of the benign neoplasms or normal tissues. The percentage of primary malignancies with hypomethylated DNA was intermediate between those of metastases and benign neoplasms. These findings might reflect an involvement of extensive demethylation of DNA in tumor progression. Such demethylation could be a source of the continually generated cellular diversity associated with cancer.

5-Methylcytosine↗
PubMed1983

Differential excretion of modified nucleosides in adult acute leukemia.

Excretion of modified nucleosides in urine was measured in 23 adults with acute leukemia to determine correlation of nucleoside excretion with disease activity. In addition, differences in excretion between patients with acute lymphoblastic leukemia (ALL) and patients with acute myeloid leukemia (AML) were established. Six modified nucleosides were resolved and quantitated by reversed-phase high-performance liquid chromatography (HPLC). Patients with ALL at initial diagnosis or in relapse had significantly higher concentrations of 1-methylinosine and N2,N2-dimethylguanosine in their urine compared to patients in remission (p less than 0.01, p less than 0.05, respectively). One patient with ALL was followed with serial nucleoside determinations over a period of 18 mo; nucleoside excretion correlated closely with disease activity. Nucleoside excretion in patients with AML did not change significantly with disease activity. Considering only those patients at initial diagnosis or in relapse, excretion of 1-methylinosine and N2,N2-dimethylguanosine was significantly higher in ALL than in AML (p less than 0.01, p less than 0.05, respectively). Thus, urinary excretion of 1-methylinosine and N2,N2-dimethylguanosine by adults with acute leukemia may prove to be valuable clinically in following disease activity in patients with ALL and in distinguishing patients with ALL from those with AML.

Adenosine↗
PubMed1983

Urinary excretion of modified nucleosides in chronic myelogenous leukemia.

Urinary excretion of modified nucleosides was measured in 15 patients with Philadelphia chromosome-positive CML to determine the correlation with activity of this disease. Resolution and quantitation of seven nucleosides were accomplished with reversed-phase HPLC. Patients in the stable phase of CML had excretion levels one to two times normal, whereas patients in the blastic phase showed elevations up to 12 times normal. The nucleosides showing the most significant differences in excretion between stable phase and blastic phase were 1-methylinosine, pseudouridine, and N2,N2-dimethylguanosine (p less than 0.01, p less than 0.001, and p less than 0.01, respectively). Nucleoside excretion was also determined in patients with bacterial pneumonia and urinary tract infection for comparison. Serial nucleoside determinations were made in two patients with CML and found to correlate closely with disease activity. The degree of elevation and the correlation with disease activity suggest the potential value of urinary nucleoside quantitation in monitoring patients with CML; in particular, nucleoside excretion may be useful in detecting early blastic transformation.

Adult↗
PubMed1983

Relationship of urinary excretion of modified nucleosides to disease status in childhood acute lymphoblastic leukemia.

The levels of urinary excretion of five modified nucleosides were quantitated by high-performance liquid chromatography for 15 normal children and 24 children with acute lymphoblastic leukemia (ALL). Excretion of each nucleoside decreased linearly with age when quantitation was based on urine creatinine content. Patients with childhood ALL at initial diagnosis or in relapse had significantly higher concentrations of 1-methylinosine, N2,N2-dimethylguanosine, 1-methylguanosine, and pseudouridine in their urine when compared to the concentrations in either patients in remission (P less than 0.001, P less than 0.001, P less than 0.01, and P less than 0.05, respectively) or normal controls (P less than 0.001, P less than 0.02, P less than 0.01, and P less than 0.001, respectively). Excretion of 2-pyridone-5-carboxamide-N'-ribofuranoside did not show significant differences. Urinary excretion of 1-methylinosine demonstrated a positive linear relationship with the percentage of blast cells in the bone marrow [correlation coefficient (r) = 0.90]; the other nucleosides had lower degrees of correlation. In comparison, the absolute blast cell count in the peripheral blood showed less correlation to the percentage of blast cells in the bone marrow (r = 0.47) than did four of the five nucleosides. The data demonstrate that excretion of modified nucleosides reflects disease activity in childhood ALL and that the urinary nucleosides could be useful clinical markers for this disease.

Adolescent↗
PubMed1981

Modulation of Platelet thromboxane A2 and arterial prostacyclin by dietary vitamin E.

Platelets from vitamin E-deficient and vitamin E-supplemented rats generate the same amount of thromboxane A2 (TxA2) when they are incubated with unesterified arachidonic acid. Platelets from vitamin E-deficient rats produced more TxA2 than platelets from vitamin E-supplemented rats when the platelets are challenged with collagen. Arterial tissue from vitamin E-deficient rats generates less prostacyclin (PGI2) than arterial tissue from vitamin E- supplemented rats. The vitamin E effect with arterial tissue is observed when the tissue is incubated with and without added unesterified arachidonic acid. These data show that arterial prostacyclin synthesis is diminished in vitamin E-deficient rats. Vitamin E, in vivo, inhibits platelet aggregation both by lowering platelet TxA2 and by raising arterial PGI2.

Adenosine Diphosphate↗
PubMed1981

An improved rapid assay for S-adenosyl-L-homocysteine hydrolase.

A coupled enzyme system was devised to assay S-adenosyl-L-homocysteine hydrolase activity spectrophotometrically and to stain the hydrolase selectively in polyacrylamide gels. The assay procedure monitors the formation of uric acid derived from the catabolism of the adenosine moiety of S-adenosylhomocysteine. The staining procedure allows the determination of the molecular weight of the hydrolase when extracts are electrophoresed on polyacrylamide gradient gels and compared to standard of known molecular weight. The specificity of the enzyme for the homocysteine moiety was also investigated by testing modified compounds as substrates. All the analogs tested were inactive as substrates for hydrolysis, indicating a strict specificity.

Adenosylhomocysteinase↗
PubMed1980

Vitamin E and the inhibition of platelet lipoxygenase.

Lipoxygenase was assayed by the formation of hydroxy-eicosatetraenoic acid (HETE) from arachidonic acid in human and rabbit washed platelets. Platelets from vitamin E-supplemented rabbits had much less lipoxygenase activity than platelets from either vitamin E-deficient or normal rabbits. Human and rabbit platelets preincubated with vitamin E had lowered lipoxygenase activity. These data show that vitamin E inhibits platelet lipoxygenase. Vitamin E and vitamin E acetate, in vitro, were equally effective inhibitors of lipoxygenase. Tween 20, in vitro, was a highly effective inihbitor of lipoxygenase. These data show that vitamin E functions, in vitro, as a surfactant in the inhibition of platelet lipoxygenase.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1979

Karyotype and tumorigenicity of 1-methylguanine-transformed Chinese hamster cells.

Chinese hamster embryo cells transformed with the tRNA catabolite 1-methylguanine were characterized by Giemsa-banded karyotyping and by their tumorigenic potency in athymic nude mice. All seven 1-methylguanine-transformed cell lines were hyperdiploid with a modal chromosome number of 23. Three of these lines had an additional marker chromosome derived from the long (q) arm of chromosome no. 4, and they had alterations of chromosome no. 5 as well. Two of these three cell lines were tumorigenic. Nonrandom chromosome changes were observed in the other four 1-methylguanine-transformed cell lines, which included the addition of all or a portion of chromosome no. 6. One of these cell lines was also tumorigenic in nude mice, Specific cytogenetic changes were observed in most 1-methylguanine-transformed populations in contrast to the karyotypic heterogeneity of a benzo[a]pyrene-transformed cell line.

Animals↗