Roberts A. Smith

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.

Determination of inorganic phosphate in the presence of labile organic phosphates

Abstract An operationally simple and rapid procedure of orthophosphate determination, based on the formation of a phosphomolybdovanadate complex, followed by its subsequent extraction into butanol, is described. The procedure is particularly advantageous for estimation of orthophosphate in the presence of labile phosphates, and yields stable colors. Conditions affecting the reliability of this procedure are evaluated.

Urinary excretion levels of unconjugated pterins in cancer patients and normal individuals.

Urinary excretion levels of seven unconjugated pterins in healthy individuals and in cancer patients, most of whom were undergoing chemotherapy, were measured utilizing a newly developed high-pressure liquid chromatographic system. Excretion of pterins in the control group appears to be under strict metabolic control as the values obtained were confined within a small range. When the mean excretion levels in control subjects were compared with those in cancer patients, we found a significant increase in the excretion of xanthopterin, neopterin and pterin and a significant decrease in isoxanthopterin by cancer patients. Biopterin levels, on the other hand, were found only slightly but not significantly increased, whereas pterin-6-carboxylic acid and 6-hydroxymethylpterin were found to be excreted in approximately equal amounts in both groups. Urinary excretion levels of pterins were monitored for a period of nine months in a patient being treated with chemotherapy for metastatic ovarian carcinomatosis. We found that the excretion pattern of pterins appeared to correlate with the clinical status of the patient. These results indicate that a definite imbalance in pterin, and possibly folate metabolism, is associated with the presence of malignant diseases.

N5-methyltetrahydrofolate: homocysteine methyltransferase activity in extracts from normal, malignant and embryonic tissue culture cells.

Abstract Malignant cells (J111, L1210, W-256) and human embryonic cells (FL) are unable to survive and grow when homocystine replaces methionine in tissue culture media containing excess vitamin B12 and folic acid. Extracts of these same cells when grown in media containing methionine and more than adequate vitamin B12 and folic acid have diminished N5-methyltetrahydrofolate: homocysteine methyltransferase activities in the absence of added cyanocobalamin when compared with extracts of normal cells (adult rat thymus and liver fibroblasts). Extracts of human monocytic leukemia (J111) and human amnion cells (FL) have normal enzymatic activity in the presence of added cyanocobalamin whereas the rodent malignant cells (W-256 and L1210) have abnormally low activity in the absence or presence of added vitamin B12.

A fluorimetric method for quantitation in the picomole range of N1-methylnicotinamide and nicotinamide in serum

Abstract A method is described for the fluorimetric determination of N 1 -methylnicotinamide in deproteinized serum extract and of nicotinamide after extraction into ethyl acetate from deproteinized serum extract and subsequent conversion to N 1 -methylnicotinamide. N 1 -methylnicotinamide is converted to fluorescent derivatives by treatment with acetophenone in alcoholic KOH followed by addition of 99% formic acid.

Techniques in the detection and characterization of phosphoramidate-containing proteins

Publisher Summary This chapter describes the techniques used in the detection and characterization of phosphoramidate-containing proteins. The more commonly employed assay procedures and isolation methods in the investigations of phosphoproteins involve the use of acid. Several techniques have been devised to detect, quantify, and characterize phosphorylated basic amino acids on proteins. When examining for acid-labile phosphorylation, care must be taken not to perform any procedure at low pH. Several methods have been employed that can detect acid-labile phosphorylation. The chapter outlines the methods that can be used as a general screening for acid-labile phosphates; they do not, however, indicate any specific acid-labile phosphoamino acid. There are numerous polyacrylamide gel electrophoretic systems that can be employed in the investigation of proteins containing acid-labile phosphate and in the determination of P–N and P–O content. Not only must the gel buffers be of neutral or basic pH, but the staining and destaining procedures must also adhere strictly to the absence of acid when dealing with phosphoramidate-containing proteins.

Nicotinamide: A natural inhibitor of tRNA methylase

Abstract A dialyzable tRNA methylase inhibitor has been isolated from rat liver. By chromatographic and electrophoretic data as well as ultraviolet and mass spectral data, the inhibitor is identified as nicotinamide. Kinetic evidence indicates that nicotinamide is an inhibitor of tRNA methylase.

Separation of phosphohydroxyamino acids by high-performance liquid chromatography

Abstract A high-performance liquid chromatography system has been developed which resolves O -phosphoserine, O -phosphothreonine, and O 4 -phosphotyrosine. Separation is performed on an anion-exchange resin eluted with phosphate buffer of low pH. Detection of the amino acid derivatives is accomplished using O -phthalaldehyde in an in-stream fluorometric system. This highly sensitive method has been applied to the detection of phosphohydroxyamino acids in bovine myelin basic protein phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase and in whole bovine myelin phosphorylated by endogenous kinases.

Quantitative determination of pterins in biological fluids by high-performance liquid chromatography

During our continuing study of pteridine metabolism, the need arose for a more rapid and quantitative determination of pterins in biological fluids. By adopting and modifying previously developed techniques, we have obtained a rapid and sensitive method that allows the simultaneous determination of eight different pterins in human urine and blood. When examined over a 10-day period, the levels of pterins excreted by a normal individual averaged the following values expressed in picomoles per mg of creatinine: biopterin, 9104; neopterin, 6018; xanthopterin, 6561; pterin, 1136; isoxanthopterin, 636; pterin-6-carboxylate, 483; and 6-hydroxymethylpterin, 315. Moreover, 6-hydroxymethylpterin and pterin-6-carboxaldehyde were detected for the first time in the blood of normal individuals.

Separation of unconjugated pteridines by high-pressure cation-exchange liquid chromatography

In the course of determining the levels of unconjugated pteridines occurring in various biological fluids, such as urines, plasma and tissue culture media, a method has been developed for the separation and quantitative determination in the picomole range of ten 2-amino-4-hydroxy substituted pteridines. This method involves separation by high-pressure cation-exchange liquid chromatography and fluorescence detection of the eluted compounds at 450 nm. Optimal separation was obtained by isocratic elution with 3 mM phosphoric acid-7% methanol-1% acetonitrile at a flow-rate of 2 ml/min or with 1 mM ammonium dihydrogenphosphate pH 2.8-7% methanol-5% acetonitrile at a flow-rate of 1.5 ml/min. With either solvent, the order of elution of the compounds is: isoxanthopterin, pterin-6-carboxylic acid, xanthopterin, pterin-6-carboxaldehyde, D-erythro-neopterin, L-threo-neopterin, biopterin, 6-hydroxymethylpterin, pterin, 6-methylpterin. In addition, a systemic investigation of the effects of ammonium ion concentration and pH of the solvent as well as column temperature on the separation of these compounds was also conducted.