Reactivity of modified ribose moieties of guanosine: new cleavage reactions mediated by the IVS of Tetrahymena precursor rRNA.

An RNA molecule consisting of the 5' exon and intervening sequence (IVS) of Tetrahymena precursor rRNA was oxidized with sodium periodate to convert the ribose moiety of the 3' terminal guanosine into a dialdehyde form. The modified RNA undergoes a specific cleavage reaction at the 5' splice site, but has no apparent cyclization activity. This novel reaction mediated by the IVS RNA is pH dependent over the range 6.5-8.5 and leaves a 5' phosphate and a 3'-OH at the newly created termini. The dialdehyde form of monomer guanosine is also capable of causing a specific cleavage reaction at the 5' splice site although the nucleotide is not covalently attached to the IVS RNA in the final product. These and other findings described in this report suggest that the cis diol of the intact ribose moiety of guanosine is not an absolute requirement for the IVS-mediated reactions.     

mRNA acetylated at 2'-OH-groups of ribose residues is functionally active in the cell-free translation system from wheat embryos

Modification (acetylation) of 2'-OH groups of mRNA ribose residues does not result in a loss of their template activity in a cell-free translation system from wheat embryos and in some cases activates inactive mRNAs. Modification of 70–75% of 2'-OH groups makes mRNA stable to the effect of pancreatic ribonuclease.     

Modification of human placental alkaline phosphatase by periodate-oxidized 1,N6-ethenoadenosine monophosphate.

Oxidation of 1,N6-ethenoadenosine monophosphate (epsilon AMP) with periodate cleaved the cis-diol of the ribose ring and resulted in the formation of a dialdehyde derivative (epsilon AMP-dial). At room temperature epsilon AMP-dial was unstable and underwent beta-elimination to give 4',5'-anhydro-1,N6-ethenoadenosine dialdehyde acetal (A epsilon Ado-dial). These nucleotide analogues were found to inactivate human placental alkaline phosphatase in a time- and concentration-dependent manner. epsilon AMP-dial was shown to be an affinity label for the enzyme on the basis of the following criteria. (a) Kinetics of the enzyme activity loss over a wide range of epsilon AMP-dial concentration showed a saturating phenomenon. Removal of the phosphate group made the reagent (A epsilon Ado-dial) become a general chemical modifying reagent. (b) The artificial substrate p-nitrophenyl phosphate gave substantial protection of the enzyme against inactivation. (c) epsilon AMP-dial was a substrate and a partial mixed-type inhibitor for the enzyme. Results of the inhibition and protection studies indicated that the reagent and substrate could combine with the enzyme simultaneously. Besides the phosphate-binding domain, an induced hydrophobic region is proposed for the substrate-binding site for human placental alkaline phosphatase.     

Oxidation and cleavage of 3-aminopyridine adenine dinucleotide phosphate with periodate

Treatment of 3-aminopyridine adenine dinucleotide phosphate with sodium periodate in the neutral pH resulted in oxidation of the ribose linked to 3-aminopyridine and cleavage of the dinucleotide into adenosine- and 3-aminopyridine-containing moieties. Separation of these moieties was afforded by thin-layer chromatography, high-performance liquid chromatography, and fast protein liquid chromatography. From fast atom bombardment mass spectra and nuclear magnetic resonance spectra, the adenosine-containing moiety was identified as 2'-phosphoadenosine 5'-phosphate while the aminopyridine moiety was present in a mixture of the hydrated 3-aminopyridine mononucleotide/nucleoside dialdehyde. Separation of the completely oxidized product by Pharmacia fast protein liquid chromatography gave three major peaks corresponding to 2'-phosphoadenosine 5'-phosphate, 2'-phosphoadenosine 5'-diphosphate and oxidized 3-aminopyridine nucleoside, with minor amount of oxidized 3-aminopyridine mononucleotide. Thus the oxidized 3-aminopyridine adenine dinucleotide phosphate was shown to cleave by two pathways: it may either undergo beta-elimination to give 2'-phosphoadenosine 5'-diphosphate and oxidized 3-aminopyridine nucleoside; or the phosphodiester linkage may be hydrolyzed to give 2'-phosphoadenosine 5'-phosphate and oxidized 3-aminopyridine mononucleotide. The latter compound may further undergo beta-elimination and eventually give oxidized 3-aminopyridine nucleoside. Hydrolysis could be prevented by storing the sample as lyophilized powder, while beta-elimination was diminished by lowering the storage temperature. We found that the lyophilized powder of oxidized 3-aminopyridine adenine dinucleotide phosphate can be stored at -50 degrees C for several months with minimum decomposition.     

Requirement for 7-methylguanosine in translation of globin mRNA in vivo.

The 7-methylguanosine (m7G) residue present in the m7G5' ppp5'X-"CAP" structure of rabbit globin mRNA was removed quantitatively by periodate oxidation followed by beta-elimination in the presence of cyclohexylamine. The RNA thus treated was intact and exhibited no signs of degradation as examined by polyacrylamide gel electrophoresis in formamide. Assay for protein synthesis using a wheat germ cell-free system showed that the globin mRNA lacking m7G had lost most of its messenger activity. Identical treatment, of satellite tobacco necrosis virus (STNV) RNA, which does not contain the 5'-terminal "CAP" structure, resulted in no loss of its mRNA activity. Since the importance of the m7G residue in eukaryotic mRNA has not yet been shown essential for translation in vivo, both untreated and treated globin mRNAs were injected into frog oocytes and their translation into globin was measured at intervals over a ninety-six hour period. Globin mRNA either treated with periodate alone or lacking in m7g altogether were both found to have lost more than 90% of their activity in vivo.     

Fluorescent labeling of fragments of high molecular weight RNA.

We describe a method to fluorescently label microgram quantities of high molecularweight RNA with acriflavine. The method involves hydrolyzing the RNA with HCl at pH 1.0 for 10 min to obtain segments of about 80 nucleotides. The 3′-terminal phosphate is removed from the ribose with alkaline phosphatase, and the terminal ribose is oxidized with periodate to form dialdehydes. Acriflavine is bound to the dialdehyde by the formation of a Schiff's base, and unbound acriflavine is removed by dialysis followed by chromatography on a Sephadex G-25 column eluted with phosphate buffered guanidine-HCl. Human 18 S rRNA bound 0.94 acriflavine molecules per 100 nucleotides and had a fluorescence excitation maximum at 460 nm and an emission maximum at 508 nm. If the hydrolysis step was omitted, this RNA bound only 0.12 acriflavine molecule per 100 nucleotides. Acriflavine-labeled high molecular weight yeast RNA showed a fluorescent intensity which was proportional to RNA concentration to a 1000-fold dilution.     

Specific inhibition of glucosyltransferase of Streptococcus mutans

Clinical dextran, partially oxidized with sodium periodate, acts as a potent inhibitor of the extracellular glucosyltransferases of several cariogenic strains of oral Streptococcus mutans. Preincubation with oxidized dextran resulted in a rapid loss of up to 80% of the ability of the enzyme preparation to synthesize polysaccharide from sucrose, but there was no loss of enzyme activity when the oxidized dextrans were reduced with sodium borohydride before preincubation with enzyme. The presence of unoxidized clinical dextran during the preincubation period afforded the enzymes protection against inhibition by partially-oxidized dextran, but clinical dextran did not readily restore activity when it was added after incubation of the enzyme with oxidized polysaccharide. Fructosyltransferase, and glycogen and starch phosphorylase, activities were not inhibited by oxidized dextran, and the bacterial glucosyltransferases were not inhibited by partially oxidized glycogen and amylose. It is proposed that the potent and specific inhibition of glucosyltransferase by oxidized dextran results from the interaction of dialdehyde groups with reactive functional groups close to the dextran-binding site of the enzyme.     

Covalent immobilization of a glucoamylase to bagasse dialdehyde cellulose

Cellulose fibres from bagasse were oxidized by sodium periodate in sulphuric acid media at positions 2 and 3 of the anhydroglucose unit to produce dialdehyde cellulose. The aldehyde groups of the dialdehyde cellulose were able to react with amino groups of a glucoamylase to form covalent bonds and result in a dialdehyde cellulose immobilized enzyme. The optimum pH of this immobilized enzyme and free enzyme were in the range of 3.0–5.0 and 3.5–5.0, respectively. The optimum temperature for both the free and immobilized enzymes was 60–65 °C. The relative remaining activity of the immobilized enzyme was 36% and its stability was very good, since it could be reused for over 30 cycles. Its activity decreased from the first to the seventh reuse cycles, due to the slow detachment of non-covalently bound enzyme. However, activity tended to stabilize after the seventh cycle of reuse, indicating very stable covalent binding between the enzyme and dialdehyde cellulose.     

The periodate oxidation of sucrose in aqueous N,N-dimethylformamide.

Sucrose has been oxidized with sodium periodate in 0-50% aqueous N,N-dimethylformamide (DMF). In 50% aqueous DMF the reaction is selective for the glucose ring, yielding a dialdehyde. The increased selectivity is not due to conformational factors but is ascribed to the dissociation of water from cyclic periodate ester species which makes the reaction via the acyclic ester on fructose unfavourable.     

A conjugate of 5-fluorouridine-poly(L-lysine) and an antibody reactive with human colon carcinoma

The ribose moiety of 5-fluorouridine (FUR) was oxidized with periodate and the product was bound through a poly(L-lysine) bridge to monoclonal antibodies, denoted SF25MAb, reactive with a human colon carcinoma LS180. The antibody was linked via its polysaccharide (previously oxidized with periodate) to the poly(L-lysine)-drug conjugate. The linking of FUR-poly(L-lysine) to the antibody markedly increased the latter's binding to the tumor cells. A relatively lower increase was also observed with conjugates of nonrelated antibodies, such as anti-hepatitis B surface antigen and anti-epidermal growth factor receptor antibodies. The pharmacological activity of the specific conjugate FUR-poly(L-lysine) -SF25MAb was higher than that of the drug-substituted polymer alone. The poly(L-lysine) bridge caused toxic effects in vivo, even though substituted both by FUR and by antibody. Therefore, the additional unreacted lysyl residues were blocked by succinylation. Partial blocking of free amino groups on the conjugate rendered it nontoxic but decreased its cell-binding capacity, though to a level still higher than that of the original unmodified antibody. The pharmacological activity of the specific conjugate after blocking was also reduced and necessitated prolonged incubation periods or higher concentrations. Following periodate oxidation and reduction, FUR was as effective as the clinically preferred compound 5-fluoro-2'-deoxyuridine in vitro and in vivo, against the LS180 colon carcinoma. Experiments in nude mice, with LS180 tumor subcutaneous xenotransplants, showed that FUR-poly(L-lysine)-SF25MAb (blocked by succinylation) was not toxic and was effective in the retardation of tumor growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reaction of the ribose moiety of adenosine and AMP with periodate and carboxylic acid hydrazides

The dialdehyde (II–V) generated by periodate oxidation of the ribose moiety in adenosine or AMP reacts readily with carboxylic acid hydrazides yielding morpholine derivatives (VI, VII, VIII) which are stable over a wide range of pH and temperature. No side reactions have been observed. This reaction will allow introduction of various substituents into the 3′ end of RNAs, and the resulting modifications would permit investigations on the structure and function of such RNAs.     

Effects of Cap Analogue or Cap Removal on the Translation of Rat Brain mRNA In Vitro

Abstract: The role of cap structures in the translation of brain mRNA was examined by measuring protein biosynthesis in vitro in wheat germ and reticulocyte systems programmed by mRNA that was either untreated or oxidized by periodate or from which 5'-terminal 7-methylguanosine (m⁷G) was removed by oxidation and β -elimination. In another series of reactions, amino acid incorporation into polypeptides was measured in the absence and in the presence of varying concentrations of the cap analogue 7-methylguanosine 5'-triphosphate (pppm⁷G). The results indicated that any of the above treatments interfered with brain mRNA translation, the degree of inhibition depending on the translation system used, the concentration of mRNA, and the source of initiation factors. Homologous brain initiation factors were superior to reticulocyte factors in providing a partial relief from inhibition of translation caused by these treatments. It was also found that synthesis of the brain-specific protein S-100 was inhibited by β -elimination of mRNA, by pppm⁷G, or by the presence of capped globin mRNA, indicating that the mRNA for this protein was probably capped.     

The derivatization of oxidized polysaccharides for protein immobilization and affinity chromatography.

The present report describes the preparation of modified polysaccharides matrices useful for the synthesis of affinity adsorbents and immobilized proteins. Hydrazido-matrices were synthesized by condensing an excess of the bifunctional reagent, adipic acid dihydrazide, with periodate oxidized cellulose paper, Sephadex, or Sepharose matrices. Ribonucleotide dialdehyde cofactors, glyceraldehyde 3-phosphate, pyridoxal 5'-phosphate and oxidized DNAase B were separately bound to the hydrazido-polymers. Azido-matrices obtained by modification of the hydrazido-derivatives were coupled to specific amino ligands such as amino acids and proteins. Several adsorbents were prepared and used as models for affinity chromatography.     

The derivatization of oxidized polysaccharides for protein immobilization and affinity chromotography

The present report describes the preparation of modified polysaccharides matrices useful for the synthesis of affinity adsorbents and immobilized proteins. Hydrazido-matrices were synthesized by condensing an excess of the bifunctional reagent, adipic acid dihydrazide, with periodate oxidized cellulose paper, Sephadex, or Sepharose matrices. Ribonucleotide dialdehyde cofactors, glyceraldehyde 3-phosphate, pyridoxal 5′-phosphate and oxidized DNAase B were separately bound to the hydrazido-polymers. Azido-matrices obtained by modification of the hydrazido-derivatives were coupled to specific amino ligands such as amino acids and proteins. Several adsorbents were prepared and used as models for affinity chromatography.     

Purification, characterization, and structural elucidation of the active moiety of the previously called "suppressor activating factor (SAF)"

Upon extensive purification of the serum-free supernatant produced by a mutant T cell line (6T-CEM), an immunosuppressive activity was found to reside in an oxidized product of spermine, spermine dialdehyde (SDA). The activity was purified to homogeneity from a serum-free supernatant by using gel filtration chromatography and reverse-phase C18 HPLC. Fast Atom Bombardment (FAB) mass spectral analysis revealed its MW to be 202 and Electron Impact (EI) analysis of the acetylated material identified the purified molecule to be spermine. In the presence of human or rodent plasma, spermine exhibited no immunosuppressive activity up to 2 mg/ml. However, when assayed in the presence of FCS, which contains polyamine oxidase (PAO), spermine is oxidized to its corresponding dialdehyde which is active at 0.1 microM/ml. We have previously described a high molecular weight suppressor activating factor (SAF) found in the serum-containing supernatant of the 6T-CEM cell line. Our preliminary biological data suggest that SDA is probably responsible for the immunosuppressive activities previously observed for the SAF. The strong affinity of SDA for proteins and thiocompounds may account for the apparent high MW previously reported for SAF.     

m-Aminophenylboronate agarose specifically binds capped snRNA and mRNA.

m-Aminophenylboronate-substituted agarose binds specifically RNA chains carrying a mature 5' cap. The binding occurs most effectively at pH greater than 8 and involves diester formation between the negatively charged tetrahedric boronate group and the cisdiol of the ribose of the cap. The positive charge introduced by the m7G methylation is necessary for efficient binding although two closely spaced cis-diol groups alone (as in the cap analog NADH) are sufficient for binding. Non-capped RNA (like poly (U) and rRNA) or decapped RNA are not bound. It is shown that the matrix can be used for the isolation of capped small nuclear RNA and mRNA.     

Synthesis of compounds having antimicrobial activity from alginate

Compounds having antimicrobial activity were synthesized from sodium alginate, the main constituent of brown algae. Sodium alginate was oxidized with sodium periodate to get alginate dialdehyde (ADA). FTIR spectrum of the ADA gave very small peak characteristic for aldehyde groups at 1720 cm⁻¹, indicating that the aldehyde group is masked somehow. It may be hydrated, involving at hemiacetal formation or hemialdol, similar to cellulose dialdehyde. Two methods were used for the condensation of ADA with o-phenylenediamine analogs to obtain the final products. The first method was stirring at room temperature and the second method was heating in microwave. The microwave method gave higher yield and shorter reaction time than the other method. The condensation reaction is considered as a shiff-base formation and the proposed mechanism was suggested. The condensation products were characterized by FTIR and UV spectra. The antimicrobial potency for five of these products in addition to the used alginate and to the precursor amines was evaluated against four pathogenic fungi and six pathogenic bacteria species.     

Characteristics of antimicrobial fibers prepared with wood periodate oxycellulose

A kind of antimicrobial fibers, a composite of Chitosan-Dialdehyde Cellulose (C-DAC) fibers, was prepared by using commercially elemental chlorine-free (ECF) bleached kraft softwood cellulose fibers oxidized by periodate and then further grafted by chitosan with different molecular weights. The characteristics of the C-DAC fibers and physical properties as well as antimicrobial activities of the handsheet made of C-DAC fibers were measured. The results show that the dry and rewet tensile indices, antimicrobial activity against Staphylococcus aureus and Escherichia coli, can be improved significantly as the molecular weight of grafted chitosan on the dialdehyde cellulose fibers decreases.     

Adequate system for studying translation initiation on the human retrotransposon L1 mRNA in vitro

The L1 retrotransposon codes for a unique bicistronic mRNA, which serves as a transposition intermediate and as a template for the synthesis of two proteins. According to preliminary data, the translation of both cistrons is initiated by a noncanonical mechanism. The L1 mRNA was translated in rabbit reticulocyte lysate (RRL), a standard system widely used to study the eukaryotic mechanisms of protein synthesis. Translation yielded not only the expected products, but also several products of aberrant translation initiation on internal AUG codons. Such products are not generated during in vivo translation of the L1 mRNA. When RRL was supplemented with a cytoplasmic extract of HeLa cells, the aberrant products were not synthesized, while the first cistron was translated with the same efficiency. The efficiency of translation of the second cistron became substantially lower, corresponding to the situation in vivo. These and other experiments clearly demonstrated that the new combined system RRL + HeLa is far more adequate for studying the mechanisms of translation initiation than the standard RRL system.     

Translational properties of mHNA, a messenger RNA containing anhydrohexitol nucleotides

Short messenger RNAs (mRNAs) with hexitol residues in two codons were constructed and their properties were studied in an Escherichia coli in vitro translation system. The replacement of the natural ribonucleotides of mRNA in the AUG start codon and the UUC second codon by hexitol nucleotides did not influence the main steps of translation, as indicated by the same level of binding of mRNA with or without hexitol residues under P-site conditions, and the same yield of tRNA binding to the P- and A-sites. Moreover, both peptide formation and translocation took place on mRNAs with hexitol residues. The presence of an A-type messenger hexitol nucleic acid (mHNA)-transfer RNA (tRNA) duplex is important for efficient translation and the 2'-OH function in mRNA is not necessary for binding and movement through the ribosome. Groove shape recognition of the codon-anticodon complex, more than hydrogen-bond interactions of ribose residues in mRNA, is an important factor for correct translation.