Opendra K. Sharma

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.

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.

An ATP-dependent inhibition of protein synthesis in ascites cell extracts by wheat germ protein

A high speed supernatant fraction from wheat germ was shown to be a very effective inhibitor of protein synthesis in a cell-free protein synthesizing system from Ehrlich ascites cells. Low concentrations of the extract were equally effective in inhibiting the translation of EMC viral RNA, ascites cell mRNA (exogenous) and endogenous mRNA. The kinetics of inhibition in the presence and absence of pactamycin, as well as the observed inhibition of polyphenylalanine synthesis, indicate that the wheat germ inhibitor acts at the level of elongation. Preincubation of the ascites system with the wheat germ inhibitor in the presence and absence of ATP showed that ATP was required for the development of the inhibition. The inhibitor was partially purified and appears to be a basic protein with a molecular weight of 30 000--40 000. These results are discussed with respect to the hypothesis that this may be another example of a protein kinase-induced inhibition of protein synthesis.

Methylation of transfer RNA during transformation of human lymphocytes by phytohemagglutinin

Abstract Methylation of transfer RNA during phytohemagglutinin induced transformation of human lymphocytes was studied by labeling the tRNA in vivo with (methyl-H 3 )-methionine and measuring the distribution of tritium in the methylated nucleotides. An alteration in the pattern of methylation occurred within hours after PHA-stimulation and this change was maintained through several cell generations. There was a 50 to 94% increase in the relative amount of methylated N 2 -methyl-guanine (1 to 3 hr) and a 40 to 59% decrease in the relative amount of 1-methyladenine (1 to 12 hr). Treatment of the stimulated cells with Actinomycin D prevented the subsequent methylation not tRNA. However, inhibition of protein synthesis by puromycin did not effect the early changes observed in the methylation of tRNA.

Inhibition of ovalbumin mRNA translation by 7-methylguanosine-5′-phosphate

Summary Translation of hen oviduct mRNA to ovalbumin in in vitro cell-free protein synthesizing systems, isolated from wheat germ and Ehrlich ascites cells, was inhibited by 7-methylguanosine-5′-phosphate. Treatment of hen oviduct mRNA with periodate followed by aniline, a process which removes 5′ terminal 7-methylguanosine, resulted in a marked reduction of ovalbumin mRNA activity. Similar treatment of encephalomyocarditis viral RNA did not significantly lower its activity. These results suggest the presence of a “capped 5′ terminus” in ovalbumin mRNA.

Differences in the transfer RNA methyltransferases from normal rat liver and Novikoff hepatoma

Abstract Transfer RNA methyltransferases from normal rat liver and Novikoff hepatoma have been compared by using a variety of tRNAs as substrates, after the enzymes had been precipitated with 60 % (NH 4 ) 2 SO 4 , and fractionated on hydroxylapatite columns. The methyltransferases from rat liver and from Novikoff hepatoma differ in the rate, extent and pattern of methylation of heterologous tRNAs.

A transfer rna-dependent protein synthesizing system from ehrlich ascites extracts.

An homologous cell-free system from Ehrlich ascites cells was made tRNA dependent by passing both the ribosomes and the ammonium sulfate fraction through DEAE-cellulose columns in 0.30 M KCl. Protein synthesis was routinely stimulated 20-50 fold by the addition of 100 mug/ml of ascites cell tRNA in the presence of oviduct mRNA, ascites mRNA, or encephalomyocarditis RNA.

Infidelity of translation of encephalomyocarditis viral RNA with tRNA from human malignant trophoblastic cells

Abstract We have investigated tRNA from the human malignant trophoblastic cells (BeWo cell) and human chorionic tissue for the translation of specific mRNAs, in a tRNA-dependent protein synthesizing system from Ehrlich ascites cells. BeWo cell tRNA and chorionic tRNA supported oviduct mRNA or encephalo-myocarditis (EMC) viral RNA directed amino acid incorporation into polypeptides equally effectively. Polypeptides synthesized with oviduct mRNA and tRNA from both sources were identical upon sodium dodecylsulfate polyacrylamide gel electrophoresis. But the EMC RNA directed polypeptides synthesized with BeWo cell tRNA were different from those synthesized with chorionic tRNA. A polypeptide (molecular weight 58,000) was apparently not synthesized and the synthesis of a faster moving component (molecular weight, 14,000) was enhanced when BeWo cell tRNA was used. These results imply a functional difference in tRNA from human malignant cells compared to their normal counterpart.

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate elevates serum progesterone levels

Summary The phorbol ester 12-O-tetradecanoyl-13-acetate (TPA), the most potent promoter of two-stage carcinogenesis in mouse skin, simulates some of the early effects of several carcinogens in immature chicks. Actinomycin D, ethionine, thioacetamide (non-mutagenic in the Ames Salmonella test) and kepone are known to increase serum progesterone concentrations and induce synthesis of ovalbumin in chicks. Intraperitoneal administration of TPA to chicks caused a 5- to 7-fold elevation of serum progesterone levels and induced ovalbumin synthesis. Phorbol ester, 4α-phorbol-12,13-didecanoate, which is inactive as a mouse skin tumor promoter, did not increase serum progesterone levels nor did it induce ovalbumin synthesis.