Ernest Borek

Quantitative high-performance liquid chromatography of nucleosides in biological materials☆

A rigorous, comprehensive, and reliable reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the analysis of ribonucleosides in urine (psi, m1A, m1I, m2G, A, m2(2)G). An initial isolation of ribonucleosides with an affinity gel containing an immobilized phenylboronic acid was used to improve selectivity and sensitivity. Response for all nucleosides was linear from 0.1 to 50 nmoles injected and good quantitation was obtained for 25 microliter or less of sample placed on the HPLC column. Excellent precision of analysis for urinary nucleosides was achieved on matrix dependent and independent samples, and the high resolution of the reversed-phase column allowed the complete separation of 9 nucleosides from other unidentified UV absorbing components at the 1-ng level. Supporting experimental data are presented on precision, recovery, chromatographic methods, minimum detection limit, retention time, relative molar response, sample clean-up, stability of nucleosides, boronate gel capacity, and application to analysis of urine from patients with leukemia and breast cancer. This method is now being used routinely for the determination of the concentration and ratios of nucleosides in urine from patients with different types of cancer and in chemotherapy response studies.

The broad spectrum antiviral agent ribavirin inhibits capping of mRNA

Abstract Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a broad spectrum antiviral substance active against a wide range of both DNA and RNA viruses. It is, however, virtually inactive against polio virus. Its pharmacological mechanism of action was obscure. A possible common target for a chemotherapeutic agent in both DNA and RNA viruses is the “capping” reaction of mRNAs which inter alia involves the formation of a guanine pyrophosphate structure at the 5′ terminus by mRNA guanylyl transferase. We have observed that Ribavirin triphosphate is a potent competitive inhibitor of the capping guanylation of viral mRNA. This finding could account for the antiviral potency of the drug against both DNA and RNA viruses and its ineffectiveness against a virus in which the mRNAs derived from them are not capped.

Atypical Transfer RNA's And Their Origin In Neoplastic Cells

Publisher Summary This chapter focuses on the structure and synthesis of transfer RNA (tRNA) and discusses the biochemistry of tRNA methylases. Translation is the most complex molecular mechanism known in the living cell. It requires messenger RNA, which is the simplest of the structures involved, even though it is laden with information. The ribosomes are complex structures composed of RNA and at least a score of different proteins. Transfer RNA is the most complex biomacromolecule known. In addition to these major components, there are ancillary ones— enzymes that transfer amino acids to the transfer RNA. Moreover, there are at least 8 soluble protein factors needed for the completion of the synthesis of a protein. Any or all of these numerous components could be a regulatory factor in protein synthesis. There are three different lines of biological evidence that point to transfer RNA as a regulatory factor. Transfer RNA is a pivotal molecule in protein synthesis. It is the link between the amino acids and the message-bearing nucleic acids. The tRNA methylases are a complex family of enzymes that modify the structure of preformed tRNA by the insertion of methyl groups into specific positions in the four main bases of tRNA. The enzymes are species-specific, organ-specific, base-specific, and even site-specific for particular bases.

Modified Ribonucleosides as Biological Markers for Patients with Small Cell Carcinoma of the Lung

A variety of individual modified ribonucleosides may be elevated in the urine of cancer patients. They can be readily measured quantitatively in a single reversed-phase high-performance liquid chromatographic run. A total of 41 patients with small cell carcinoma of the lung were studied. For 5-ribonucleosides determined in the pretreatment urine of 28 patients, the respective frequency of elevation was directly related to stage of disease. One or more nucleosides were evaluated in the pretreatment urine of 27 out of 28 patients (96%). Included were 11 patients with limited disease and 10 (91%) had 2 or less than 2 nucleosides elevated, whereas 16 out of 17 (94%) with extensive disease had 3 or more elevated. Based on this same discriminant, median survival was significantly extended for patients with 2 or less nucleosides elevated (24 months) in contrast to 3 or more (10 months). Using a single number to represent the summation of equally weighted individual nucleoside values as a composite score, a direct relationship was found between increasing extent of disease or tumor burden. This was in contrast to more variable results for carcinoembryonic antigen analyzed in plasma samples obtained at the same time. When determined serially the composite score paralleled in general the clinical response categories for individual patients.

The Relaxed Control Phenomenon

Publisher Summary This chapter reviews the variety of possible mechanisms for the relaxed control phenomenon, without expressing the prejudices toward any of them. It reviews that a single genetic locus in control of RNA synthesis as the “RC” locus and considered its effectiveness to be “relaxed” in all derivatives of the original mutant regardless of the amino acid deficiency imposed. While all species of RNA accumulated in RC mutants, excess DNA synthesis seemed to be relatively minor during an absolute amino acid starvation. It discusses the concept of relaxed control to the synthesis of DNA as well. Since a significant synthesis of this macromolecule is observed only when a gradual depletion of the amino acid supply occurs, it is our view that its magnitude reflects the adequacy of the amino acid starvation rather than a genetically controlled relaxation. Synthesis of DNA to the completion of its replication cycle in amino acid-starved strains that exhibit stringent control over RNA synthesis is also discussed. New insights and new skills are needed in this field and the solicitation of these is the goal of this chapter. It is hoped that amino acid auxotrophs of mammalian cells have become a reality, the question of the possible existence of a similar control phenomenon for RNA synthesis in mammalian cells may also be open to exploration.

Rapid, quantitative high-performance liquid column chromatography of pseudouridine

Abstract A rapid, precise, and accurate chromatographic method for the determination of pseudo-uridine (ψ) in urine by high-performance liquid chromatography (HPLC) has been developed. The ribonucleosides were first isolated with an affinity gel containing immobilized phenylboronic acid. The response for ψ was linear well above and below the range necessary to determine urinary ψ. Good precision was obtained for both matrix-dependent and matrix-independent samples. Supporting experimental data are presented on precision, recovery, chromatographic methods, sample cleanup and application to the analysis of urine samples from normal males and females, and patients with advanced colon cancer. In a comparison of 40 normals with 10 colon cancer patients, 9 of the 10 patients had a ψ: creatinine (Cr) ratio greater than x + 2σ for the normal population. This HPLC method is now being used extensively in our laboratory as a routine method for determination of ψ in urine from patients with various types of cancer and in chemotherapy response studies. Data are presented on the dynamics of ψ excretion by normal males and females. When the excretion of ψ was normalized with the excretion of creatinine, it was noted that samples collected at random have the same ψ: Cr ratio value as for the 24-h total collection, thus, allowing the use of random samples. The constancy of the ψ: Cr ratio implies that RNA turnover is constant and ψ excretion is independent of diet. Base values are presented for the ψ: Cr

Quantitative gas-liquid chromatography of neutral sugars in human serum glycoproteins. Fucose, mannose, and galactose as predictors in ovarian and small cell lung carcinoma.

A precise and accurate gas-liquid chromatographic (GLC) method has been developed for the quantitative analysis of the neutral sugars L-fucose (6-deoxygalactose), mannose, galactose, and glucose in ethanol precipitates of human serum proteins. The chromatographic conditions and sample preparation resulted in short analysis time (20 min per run) and made routine analyses practicable (twelve samples per day). The alditol acetate derivatization yielded single derivatives for each sugar. Complete separation was achieved on a 2.0 m X 2 mm I.D. column with 2.0% Silar-7CP on Chromosorb W AW 80--100 mesh. The results of hydrolysis showed that the release of fucose and galactose preceded the release of mannose. Hydrolysis with AG 50 W-X8 (H+) ion-exchange resin in 0.5 N HCl at 100 degrees for 7 h optimized glycosidic bond cleavage with only minimal destruction of fucose, mannose and galactose. A combination of strong cation- and anion-exchange resin columns was used to remove chromatographic background of peptides, amino acids, amino sugars, and inorganic ions. An average R.S.D. of less than 4% with recovery of greater than 86% for the three sugars was achieved. The homogeneity of the chromatographic peaks for the neutral sugars of normal human serum glycoproteins was confirmed by GLC--mass spectrometry. Significantly elevated ratios of fucose, galactose, and mannose to serum protein were observed for patients with small cell lung and ovarian carcinomas.

Transfer RNA methylases during sea urchin embryogenesis

Methylation of tRNA in sea urchin Strongylocentrotus purpuratus, during embryogenesis was studied by labeling with [Me-3H]methionine. The changes in methylated bases in tRNA following fertilization were not uniform. The N2-monomethylguanine was reduced to half and methylated uridylic acids increased 3-fold. Extracts of eggs and of embryos of various stages after fertilization were active in incorporating methyl groups from S-adenosyl-l-[Me-14C]methionine into Escherichia coli B tRNA.

Deficiency of the DNA of Micrococcus radiodurans in methyladenine and methylcytosine.

Abstract Micrococcus radiodurans , an extraordinarily radiation-resistant organism, was found to have no detectable methylated bases in its DNA nor any DNA methyltransferase activity. The DNA can receive methyl groups in vitro from enzymes from heterologous sources. It thus may serve as ‘methyl-deficient’ DNA.

Regulation of the tRNA methyltransferases in normal and neoplastic tissues

Abstract The tRNA methyltransferases are under the control of several regulatory systems. The known mechanisms are hormonal, an unrelated enzyme system glycine methyltransferase, which competes for SAM, and natural inhibitors. The glycine methyltransferase is either absent or its activity is profoundly lowered in embryonic and tumor tissue. Novel tRNAs which are qualitatively different from those in normal tissue have been found in every tumor examined.