The molecular basis for biological inactivation of nucleic acids. The action of methylating agents on the ribonucleic acid-containing bacteriophage R17

1. The inactivation of an RNA-containing bacteriophage after reaction with four methylating agents was studied. Measurements of the extent of methylation of the RNA and of the nature and amounts of the various reaction products were made. In experiments with dimethyl sulphate and methyl methanesulphonate inactivation can be quantitatively accounted for by methylation at two of the positions involved in hydrogen bonding: N-1 of adenine and N-3 of cytosine. In experiments with N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine methylation at N-1 of adenine and N-3 of cytosine accounts for only about one-half of the observed inactivation. Scission of the RNA chain during reaction accounts for a further 20% of the inactivation. To account for the remainder it seems necessary to postulate that formation of O(6)-methylguanine constitutes a lethal lesion. 2. Breaks in the RNA chain formed on reaction with the nitroso derivatives presumably result from methylation of the phosphate diester group followed by hydrolysis of the unstable triester thus formed.     

The reaction of alkylating agents with bacteriophage R17. Biological effects of phosphotriester formation

The extent of biological inactivation and of the degradation of the RNA after reaction of bacteriophage R17 with ethyl methanesulphonate, isopropyl methanesulphonate and N-ethyl-N-nitrosourea was studied. Formation of breaks in the RNA chain probably results from hydrolysis of phosphotriesters formed in the alkylation reactions. Near neutral pH the ethyl and isopropyl phosphotriesters are sufficiently stable for the kinetics of the hydrolysis reaction to be followed. Results indicate that the rate of hydrolysis increases rapidly as the pH is raised. The evidence shows that a phosphotriester group does not itself constitute a lethal lesion. The extent of phosphotriester formation by the different agents is discussed in terms of reaction mechanism.     

The inactivation of bacteriophage R17 by ethylating agents: the lethal lesions.

The biological inactivation of bacteriophage R17 by ethyl methanesulphonate (EMS) and N-ethyl-N-nitrosourea (ENUA) has been studied. At the mean lethal dose for the first compound 8 moles ethyl are bound/mole RNA and with the nitroso compound 3.5 moles ethyl are bound. Analysis of the amounts of the different ethylated derivatives formed shows that the toxicity of the sulphonate can be accounted for by the formation of 3-ethylcytosine, O6-ethylguanine, 1-ethyladenine and chain breaks produced on the hydrolysis of ethyl phosphotriesters. With the nitroso derivative on the other hand, the sum of chain breaks and of bases alkylated on a position involved in specific hydrogen bonding between base pairs only accounts for 65% of the observed toxicity. The possibility that 3-ethyladenine may constitute a lethal lesion is discussed.     

The dependence of the mutagenicity of methanesulphonic acid esters in S. typhimurium TA100 on the alkylation mechanism

Four different model nucleophiles, 4-(p-nitrobenzyl)pyridine (NBP), N-methylmercaptoimidazole (MMI), trifluoroacetic acid (TFA) and H2O were tested with 22 methanesulphonates of widely varying structures for their suitability to predict mutagenic activities in S. typhimurium TA100. The soft nucleophiles NBP (N-alkylation) and MMI (S-alkylation) revealed as highly sensitive for SN2 reactivities whereas TFA (solvolysis at the O-atom) and H2O (hydrolysis) were very sensitive for SN1 reactivities. No correlation between the NBP or the MMI test and the Ames test was found. Quite good correlations could be demonstrated for the TFA test and the hydrolysis rates: with rising activities in the TFA solvolysis the mutagenic potencies were increasing up to a maximum at i-propyl methanesulphonate. After that due to the fast hydrolysis the mutagenicities were decreasing again despite increasing TFA solvolysis rates. In general the secondary methanesulphonates exerted high SN1 reactivities and distinct mutagenic activities, whereas the primary compounds showed no or very low SN1 reactivities and low mutagenic potentials. The "activated" compounds cyclopropylmethyl methanesulphonate, benzyl methanesulphonate and allyl methanesulphonate exerted high SN1 and SN2 reactivities. Methyl methanesulphonate displayed a high mutagenicity in spite of its lack in SN1 reactivity. This is probably due to the induction of the error prone repair (pkM 101 plasmid in TA100). The relation between the alkylating reactivities (SN1 and SN2) and the molecular mechanisms leading to back mutation is discussed.     

The action of mono- and di-functional sulphur mustards on the ribonucleic acid-containing bacteriophage μ2

Bacteriophage μ2 is inactivated by both mono- and di-functional sulphur mustards at relatively low extents of alkylation. No degradation of alkylated RNA was detected. Cross-linking of RNA to protein was observed with the difunctional agent, but this reaction was only a minor contribution to the inactivation. Analyses of the reaction products in bacteriophage RNA showed that, at the mean lethal doses, more than one mono-alkylation of guanine had occurred but the sum total of other types of RNA alkylation was close to a single event. The results therefore suggest that inactivation results from the mono-alkylation of adenine or cytosine. In experiments with the difunctional agent cross-linking of RNA bases or of RNA to protein also prevented replication, the existence of these reactions accounting for the greater sensitivity of the bacteriophage to this agent.     

单克隆抗体S2C4对2型志贺毒素及其亚型毒性的中和作用

纯化的2型志贺毒素(Shiga toxin 2,Stx2)经福尔马林脱毒后免疫BALB/c小鼠制备Stx2单克隆抗体,用体外中和试验对具有中和活性的阳性抗体克隆进行初筛,对所获得的中和抗体的重、轻链同种型及结合特异性进行鉴定,其中和保护作用通过体内、体外中和试验加以验证,最后,中和抗体对Stx2亚型Stx2c和Stx2vha的中和谱用体内中和试验验证．结果显示,12株抗Stx2的阳性抗体克隆中,只有1株具有中和活性,命名为S2C4,其重、轻链同种型为G₁/κ,其靶分子为Stx2的A亚单位,与Stx2的B亚单位或Stx1不结合．在体外中和试验中S2C4可有效中和Stx2对Vero细胞的杀伤作用,同样,S2C4可中和致死量的Stx2及其亚型Stx2c和Stx2vha对小鼠的毒性作用．该抗体有望成为治疗产志贺毒素大肠杆菌感染的候选分子．     

The stability of rat liver ribonucleic acid in vivo after methylation with methyl methanesulphonate or dimethylnitrosamine

1. RNA was isolated from rat liver at selected times after the intraperitoneal injection of either [¹⁴C]methyl methanesulphonate (50mg/kg) or [¹⁴C]dimethylnitrosamine (2mg/kg). These doses were chosen to minimize effects due to toxicity. 2. Two methods of extraction and purification of RNA were used and an analysis of the radioactivity present was made by column chromatography of acid hydrolysates of the purified RNA. 3. The extent of methylation of guanine, the principal site of alkylation in rat liver RNA, was determined at times up to 14 days after injection. Although dimethylnitrosamine is a potent liver carcinogen and methyl methanesulphonate is not carcinogenic to rat liver, the rate of disappearance of 7-methylguanine from RNA was similar for both compounds, with a half-life of about 3.5 days. 4. An estimate of the biological half-life of rRNA was made by using [³H]orotic acid. A half-life of 5 days was obtained and this was not affected by injecting animals with unlabelled methyl methanesulphonate at the same dosage of 50mg/kg used in the studies of RNA methylation. 5. After administration of labelled orotic acid, reutilization of labelled RNA degradation products probably results in an overestimation of the biological half-life for rRNA. It is suggested that non-toxic doses of methylating agents such as methyl methanesulphonate and dimethylnitrosamine may prove to be a more effective way of accurately estimating the biological turnover of RNA species.     

Klebsiella serotype-13 capsular polysaccharide: primary structure and depolymerization by a bacteriophage-borne glycanase

Periodate oxidation and Smith degradation, methylation analysis including uronic acid degradation, partial hydrolysis with acid, bacteriophage degradation, and p.m.r. spectroscopy have been used to elucidate the primary structure of the Klebsiella serotype-13 capsular polysaccharide. The polymer consists of pentasaccharide repeating-units comprising a 4)-beta-D-Manp-(1 leads to 4)-alpha-D-Glcp-(1 leads to 3)-beta-D-Glcp-(1 leads to chain with a 3,4-O-(1-carboxyethylidene)-beta-D-Galp-(1 leads to 4)-alpha-D-GlcAp-(1 leads to branch at position 3 of the mannose. It is shown that there is a glycanase activity associated with particles of Klebsiella bacteriophage No. 13, which catalyses hydrolysis of chain beta-D-Glcp-(1 leads to 4)-beta-D-Manp linkages in the type-13 polysaccharide. The chemical basis of some serological cross-reactions of the Klebsiella K13 antigen is discussed.     

Leakage induced in Escherichia coli cells by A protein-RNA complexes from bacteriophage f2

Complexes of f2 phage RNA and its A protein, or maturation protein, transfect Escherichia coli cells much better than does protein-free RNA. We used these complexes to introduce the bacteriophage f2 lysis gene into cells. The A protein-RNA complex was found to kill cells, probably by causing them to leak large macromolecules. Previously induced beta-galactosidase leaked from cells treated either with the A protein-RNA complex or with lethal but noninfectious complexes that had been treated with formaldehyde. This observation was consistent with an earlier finding that formaldehyde-treated f2 RNA stimulates the in vitro synthesis of a lysis protein. The complexes did not stimulate the rate of leakage of beta-galactosidase from a streptomycin-resistant mutant known to be lysis defective. On the other hand, the rate of leakage was increased in a double mutant resistant to both streptomycin and rifampin and which is lysed normally by f2 bacteriophage.     

The interaction of the 7,8-dihydrodiol-9, 10-oxides of benzo(a) pyrene with bacteriophages R17 and T7

Dose-survival curves for bacteriophages R17 and T7 treated with the syn- and anti-isomers for 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene in 0.02 M phosphate buffer, pH 7.0, have been determined. In both cases the anti-isomer proved to be the more toxic: mean lethal dose for R17; syn- 3 μg/ml, anti- 2 μg/ml: and for T7, syn- 3 μg/ml, anti- 0.3 μg/ml. With both reagents reaction with bacteriophage or loss by solvolysis were complete within minutes. Physico-chemical studies of the RNA failed to detect any degradation 1 and 24 h after the addition of the reagents to the bacteriophage and no change in survival of the bacteriophage occurred during this period. In experiments with bacteriophage T7 and T7-DNA reaction did not, in the first hour, introduce any significant number of alkali-labile sites in the nucleic acid. These results suggest that no reaction occurs with the phosphate groups of the nucleic acids. Following the initial loss of infectivity when bacteriophage T7 was treated with the syn-isomer there was a further, progressive loss of biological activity over 4 days which was associated with the development of alkali labile lesions. It seems probable that these latter effects are due to the loss of alkylated bases from the DNA, a process similar to the depurination reactions observed following the reaction of DNA with e.g. methylating agents.     

Characterization of segmented double-helical RNA from bacteriophage phi6

The nucleic acid component of bacteriophage φ6 is characterized as a double stranded RNA molecule with a buoyant density of 1.605 g/cm³ and nucleotide composition of C, 27.3%; A, 21.8%; G, 28.9%; and U, 22.0%. The hyperchromicity profile in 0.1 × SSC (SSC is 0.15 m-NaCl, 0.015 m-sodium citrate) demonstrated a rapid increase with a T_m value of 91 °C. The nucleic acid was resistant to degradation by DNase, spleen phosphodiesterase and pancreatic RNase in 2 × SSC buffer but sensitive to degradation by venom phosphodiesterase, pancreatic RNase in 0.01 × SSC and hydrolysis in KOH. Three distinct double stranded RNA species of 2.2, 2.8 and 4.5 × 10⁶ daltons were observed after rate zonal centrifugation, polyacrylamide gel electrophoresis and electron microscopy. This communication therefore presents data establishing a new class of double stranded RNA bacteriophage.     

Substrate Efflux Propensity Is the Key Determinant of Ca2+-independent Phospholipase A-β (iPLAβ)-mediated Glycerophospholipid Hydrolysis

The A-type phospholipases (PLAs) are key players in glycerophospholipid (GPL) homeostasis and in mammalian cells; Ca²⁺-independent PLA-β (iPLAβ) in particular has been implicated in this essential process. However, the regulation of this enzyme, which is necessary to avoid futile competition between synthesis and degradation, is not understood. Recently, we provided evidence that the efflux of the substrate molecules from the bilayer is the rate-limiting step in the hydrolysis of GPLs by some secretory (nonhomeostatic) PLAs. To study whether this is the case with iPLAβ as well, a mass spectrometric assay was employed to determine the rate of hydrolysis of multiple saturated and unsaturated GPL species in parallel using micelles or vesicle bilayers as the macrosubstrate. With micelles, the hydrolysis decreased with increasing acyl chain length independent of unsaturation, and modest discrimination between acyl positional isomers was observed, presumably due to the differences in the structure of the sn-1 and sn-2 acyl-binding sites of the protein. In striking contrast, no significant discrimination between positional isomers was observed with bilayers, and the rate of hydrolysis decreased with the acyl chain length logarithmically and far more than with micelles. These data provide compelling evidence that efflux of the substrate molecule from the bilayer, which also decreases monotonously with acyl chain length, is the rate-determining step in iPLAβ-mediated hydrolysis of GPLs in membranes. This finding is intriguing as it may help to understand how homeostatic PLAs are regulated and how degradation and biosynthesis are coordinated.     

The primary structure of the coat protein of the broad-host-range RNA bacteriophage PRR1.

The complete amino acid sequence of the coat protein of RNA bacteriophage PRR1 is presented. After thermolysin digestion, 26 peptides were isolated, covering the complete coat protein chain. Their alignment was established in part using automated Edman degradation on the intact protein, in part with overlapping peptides obtained by enzymic hydrolysis with trypsin, pepsin, subtilisin and Staphylococcus aureus protease, and by chemical cleavage with cyanogen bromide and N-bromosuccinimide. To obtain the final overlaps, a highly hydrophobic, insoluble tryptic peptide was sequenced for seven steps by the currently used manual dansyl-Edman degradation procedure, which was slightly modified for application on insoluble peptides. PRR1 coat protein contains 131 amino acids, corresponding to a molecular weight of 14534. It is highly hydrophobic, and the residues with ionizable side chains are distributed unevenly: acidic residues are absent in the middle third of the sequence, whereas a clustering of basic residues occurs between positions 44 and 62. PRR1 coat protein was compared with the coat proteins of RNA coliphages MS2 and Q beta, and the minimum mutation distance was calculated for both comparisons. It is highly probable that PRR1. Q beta and MS2 share a common ancestor. The basic region present in the three coat proteins is recognized as an essential structural feature of RNA phage coat proteins.     

Physiological modification of alkylating agent induced mutagenesis. II. Influence of the numbers of chromosome replicating forks and gene copies on the frequency of mutations induced in Escherichia coli.

The frequency of reversions induced in Escherichia coli K-12 trpA58 by any of five different monofunctional alkylating agents increased as the growth rate of the organism was raised prior to mutagen treatment. The increase in mutation frequency did not correlate with growth rate-dependent changes in cell area or total cellular protein and DNA. After treatment of cells with N-methyl-N-nitrosourea (MNUA), no growth rate-dependent change was observed in the total DNA alkylation or percentage of O6-methylguanine present in the DNA extracted. The frequency of reversions induced by one mutagen, methyl methanesulphonate (MMS), increased in proportion to the average number of trpA gene copies per cell, whereas the frequency of reversions induced by the other compounds was dependent on the average number of chromosome replicating forks per cell. This difference was attributed to the different ratios of DNA base alkylation products observed, formed after treatment with MMS, an SN2-type reagent, or after treatment with the SN1-type reagents ethyl methanesulphonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), MNUA and N-ethyl-N-nitrosourea (ENUA). Possible reasons for the dependence of mutation frequency on the number of replicating forks per cell are discussed.     

Breaking the light and heavy chain linkage of human immunoglobulin G1 (IgG1) by radical reactions

We report that the production of hydrogen peroxide by radical chain reductions of molecular oxygen into water in buffers leads to hinge degradation of a human IgG1 under thermal incubation conditions. The production of the hydrogen peroxide can be accelerated by superoxide dismutase or redox active metal ions or inhibited by free radical scavengers. The hydrogen peroxide production rate correlates well with the hinge cleavage. In addition to radical reaction mechanisms described previously, new degradation pathways and products were observed. These products were determined to be generated via radical reactions initiated by electron transfer and addition to the interchain disulfide bond between Cys(215) of the light chain and Cys(225) of the heavy chain. Decomposition of the resulting disulfide bond radical anion breaks the C-S bond at the side chain of Cys, converting it into dehydroalanine and generating a sulfur radical adduct at its counterpart. The hydrolysis of the unsaturated dehydropeptides removes Cys and yields an amide at the C terminus of the new fragment. Meanwhile, the competition between the carbonyl (-C(α)ONH-) and the side chain of Cys allows an electron transfer to the α carbon, forming a new intermediate radical species (-(·)C(α)(O(-))NH-) at Cys(225). Dissociative deamidation occurs along the N-C(α) bond, resulting in backbone cleavage. Given that hydrogen peroxide is a commonly observed product of thermal stress and plays a role in mediating the unique degradation of an IgG1, strategies for improving stability of human antibody therapeutics are discussed.     

An assay for phosphotriester formation in the reaction of alkylating agents with deoxyribosenucleic acid in vitro and in vivo.

Preliminary studies in vitro using bacteriophage T7-DNA have shown that breaks formed in the DNA on the alkaline hydrolysis of apurinic sites and phosphotriesters can be distinguished from each other by measuring the extent of degradation of the DNA immediately after adding NaOH to 0.1 M and after incubating for 1 h in 0.5 M NaOH. This method has then been applied to the study of the formation and stability of phosphotriesters invivo. Methyl phosphotriesters formed in liver DNA following injection of mice with N-methyl-N-nitrosourea (MNUA) disappear with time (50% in 4-5 days). The concentration of ethyl phosphotriesters in liver DNA formed by injecting mice with N-ethyl-N-nitrosourea (ENUA) does not appear to decrease with time. Results of experiments on injecting methyl methane-sulphonate (MMS), ethyl methanesulphonate (EMS) and dimethyl sulphate (DMS) are also reported. The method described does not require the use of radioactively labelled reagents.     

Differences in the patterns of methylation in rat liver ribosomal ribonucleic acid after reaction in vivo with methyl methanesulphonate and NN-dimethylnitrosamine

1. rRNA was isolated from rat liver at short intervals after the intraperitoneal injection of [(14)C]methyl methanesulphonate (50mg/kg) or NN-di[(14)C]methylnitrosamine (2mg/kg). These doses were chosen to minimize the effects of toxicity. 2. The following methods of hydrolysis of [(14)C]methylated rRNA were employed: enzymic digestion to nucleosides at pH8; alkaline hydrolysis and conversion into nucleosides; acid hydrolysis to bases. 3. The methylation products were analysed by chromatography on columns of Dowex-50 (H(+) form) and Dowex-50 (NH(4) (+) form). 4. With both methylating agents the principal product of methylation was 7-methylguanine. Differences were obtained, however, in the molar proportions of the minor bases 3-methylcytosine, 1-methyladenine and 7-methyladenine. Methylation at the O-6 position of guanine was a significant feature of rRNA obtained from the NN-di[(14)C]methylnitrosamine-treated animals but was not detected in rRNA after treatment with [(14)C]methyl methanesulphonate.     

The effect of a cross-bridging thiol reagent on the catecholamine fluxes of adrenal medulla vesicles

The thiol groups of the vesicular protein of bovine adrenal medulla were allowed to react with the bifunctional thiol reagent bis-(N-maleimidomethyl) ether and with the monofunctional thiol reagent N-ethylmaleimide, and the ATP-dependent and -independent catecholamine fluxes of the modified preparations were studied. 1. During the initial phase of the reaction bis-(N-maleimidomethyl) ether blocks twice as many thiol groups as does N-ethylmaleimide at equimolar concentrations. 2. Labelling of the bis-(N-maleimidomethyl) ether-protein compound with [(14)C]-cysteine shows that 70-80% of the blocked thiol groups are interconnected by the bifunctional thiol reagent. 3. At a low extent of reaction (1.5mol of thiol groups/10(6)g of protein) the catecholamine efflux is diminished. If more than 2mol of thiol groups/10(6)g of protein are blocked, the efflux is enhanced whichever thiol reagent is applied. 4. If 2-4mol of thiol groups/10(6)g of protein are blocked the inhibition of the catecholamine influx increases linearly with the proportion of the thiol groups blocked. 5. ATP protects the catecholamine influx and the adenosine triphosphatase activity against bis-(N-maleimidomethyl) ether poisoning somewhat less effectively than against N-ethylmaleimide poisoning.     

Genotoxicity of monofunctional methanesulphonates in the SOS chromotest as a function of alkylation mechanisms. A comparison with the mutagenicity in S. typhimurium TA100

17 monofunctional methanesulphonates of widely varying structures were investigated in the SOS chromotest using the E. coli strain PQ37. All compounds tested were positive in this assay. The monofunctional methanesulphonates in general possess low SOSiP values. Five of the compounds tested i.e. iBMS, NpMS, 2 PhPMS, PkMS and 1,3-DC12PMS (for abbreviations see Table 1) did not show increasing beta-galactosidase activity and both the positive induction factors and the positive SOSiP values resulted from the toxicity correction as performed according to Quillardet and Hofnung (1985). In general methanesulphonates with a higher SN1 reactivity, in particular the secondary compounds, showed clear genotoxic activities whereas those possessing low SN1 reactivities (primary compounds) induced a low SOS repair indicating that the alkylation of O-atoms in the DNA bases contributes more to the induction of SOS repair in strain PQ37 than N-alkylations. The only exception was methyl methanesulphonate (MMS) which possessed a very high SN2 reactivity but a rather low SN1 reactivity. It had the highest SOSiP value of all tested methanesulphonates. No dependence of the genotoxicity on the SN2 reactivity could be found in this series. In general the phenyl-substituted methanesulphonates showed higher SOSiP values, which is presumably due to their relatively high SN1 reactivities and their relatively long life times in aqueous systems. There is a clear relationship between SN1 reactivities and the SOSiP values: the SOSiP values increase with rising SN1 reactivities reaching a maximum at iPMS after which the genotoxicities decrease due to the decreasing life times. The compounds with very high SN1 reactivities also possess very high hydrolysis rates. A good correlation could be established between the mutagenicities in S. typhimurium TA100 and the SOS chromotest (strain PQ37). Only 4 small deviations from this correlation could be found. The reasons for these deviations are discussed.     

Remediation of Explosive-Contaminated Soils: Alkaline Hydrolysis and Subcritical Water Degradation

Alkaline hydrolysis and subcritical water degradation were investigated as ex-situ remediation processes to treat explosive-contaminated soils from military training sites in South Korea. The addition of NaOH solution to the contaminated soils resulted in rapid degradation of the explosives. The degradation of explosives via alkaline hydrolysis was greatly enhanced at pH ≥12. Estimated pseudo-first-order rate constants for the alkaline hydrolysis of 2,4-dinitrotoluene (DNT), 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in contaminated soil at pH 13 were (9.6?±?0.1)×10^?2, (2.2?±?0.1)×10^?1, and (1.7?±?0.2)×10^?2 min^?1, respectively. In the case of subcritical water degradation, the three explosives were completely removed at 200–300°C due to oxidation at high temperatures and pressures. The degradation rate increased as temperature increased. The pseudo-first-order rate constants for DNT, TNT, and RDX at 300°C were (9.4?±?0.8)×10^?2, (22.8?±?0.3)×10^?2, and (16.4?±?1.0)×10^?2, respectively. When the soil-to-water ratio was more than 1:5, the extent of alkaline hydrolysis and subcritical water degradation was significantly inhibited.