Mutagenicity of dichlorvos and methyl methanesulphonate for Escherichia coli WP2 and some derivatives deficient in DNA repair

The mutagenic and lethal action of methyl methanesulphonate (MMS) and dichlorvos (DDVP) has been studied on Escherichia coli WP2 and some derivatives deficient in DNA repair genes. The exrA⁺ and recA⁺ alleles were necessary for significant mutagenesis by either compound, and the uvrA gene affected neither the lethal nor mutagenic responses. Increased sensitivity to both compounds was shown by the exrA and uvrAexrA strains and in a more pronounced way by the uvrApolA, recA, and uvrAexrApolA strains.Bacteria deficient at the polA locus were 2 and 3 times more mutable by DDVP and MMS respectively, consistent with the hypothesis that the absence of the polA system for the repair of single-strand gaps results in a greater proportion of the total repair being channelled through the error-prone exrA⁺/recA⁺-dependent system. Single-strand breaks were detectable by alkaline sucrose gradient centrifugation after both MMS and DDVP treatment of polA bacteria. Thus in all the tests carried out, both compounds showed similar patterns of activity, and the results are consistent with their known ability to alkylate DNA. The chief differences were quantitative; sensitivity increases were far more pronounced with MMS which was also a far more potent mutagen than DDVP.     

Prenylated p-coumarates from Werneria stuebelii

The aerial parts of Werneria stuebelii afforded in addition to β-cedrene and bicyclogermacrene eight derivatives of methyl p-coumarate, all being formed by prenylation. The structures were elucidated by their molecular formulae and their ¹H NMR spectra. The chemotaxonomy of the Andean genus Werneria is discussed briefly.     

Enzymatic removal of a cephalosporin methyl ester blocking group

Over 7000 microorganisms were screened to find an enzyme source for the hydrolysis of a C₄ methyl ester blocking group on 7-aminodesacetoxycephalosporanic acid (7-ADCA). Only one culture, Streptomyces capillispira Mertz and Higgens nov. sp., produced an enzyme that catalysed the reaction. Enzyme synthesis in a defined mineral salts medium was repressed by NH₃ and amino acids. Under optimum fermentation conditions, the maximum rate of substrate hydrolysis was 6 × 10^?10 mol min^?1 mg^?1 cell. The enzyme was recovered from the mycelia and partially purified by gel filtration. Kinetic studies by pH-stat titration indicated that the pH optimum was 7.5–8.5, the temperature optimum was 25–30°C, and the substrate K_m value was 2.3 mg ml^?1. The reaction products, 7-ADCA and methanol, were weak competitive inhibitors of the enzyme with K₁ values of 6.63 and 0.188 mg ml^?1, respectively. The enzyme also hydrolysed cefaclor and cephalexin methyl esters but did not hydrolyse cephalosporin ethyl esters. With further improvements in enzyme yields and stability, enzymatic deblocking of cephalosporins could provide an alternative to chemical deblocking processes.     

Synthesis and properties of methyl 9,12,15-octadecatrienoate geometric isomers

The eight geometrically isomeric methyl 9,12,15-octadecatrienoates were prepared by using the Wittig reaction to couple cis- or trans-3-hexyenyltriphenylphosphonium bromide and methyl 12-oxo-cis- or trans-9-dodecenoate. Pairs of geometric triene isomers formed were separated by partial silver resin chromatography. Physical constants including melting points, percent trans by infrared, equivalent chain lengths (ECL), and ¹³C nuclear magnetic resonance (NMR) chemcial shifts are tabulated for the individual isomers.     

Effect of N-nitroso-N-methylurea on viability and mutagenic response of repair-deficient strains of Escherichia coli

Various E. coli mutants, deficient in DNA repair, differed in their response to increasing concentrations of N-nitroso-N-methylurea (NMU).Loss of viability due to exposure to NMU was greatest in those strains with a reduced capacity for repair of single-strand breaks. Viability of wild-type and uvrA^? strains was not affected by NMU concentrations up to 3.0 mM. Some loss of viability occurred, at the higher NMU concentrations, in both strains carrying exrA^? while strains carrying uvrA^?polA^? or recA^? were the most sensitive. The results support the hypothesis that the lethal effect of NMU on repair-deficient E. coli was due to its ability to induce single-strand breaks.Induction of mutations by NMU was observed in all the strains used and the results suggested that NMU damage per se was the major mutational event. The dose response curve for induction of revertants by NMU was, however, influenced by the repair system(s) present. The number of revertants scored at the higher NMU concentrations was greater in those strains lacking the recA and polA dependent repair functions than in the wild-type strain. However, at NMU concentrations below 2.0 mM the numbers of revertants induced in exrA^? carrying strains, prossessing accurate rec-dependent repair, were lower than the comparable wild-type values. The evidence suggests that the uvrA gene product also acts on some, possibly non-mutagenic, types of NMU damage and that error-prone repair of these lesions increases the number of potential revertants.     

Na+-dependent methyl β-thiogalactoside transport in Salmonella typhimurium

We have studied the role of sodium ions in methyl β-thiogalactoside (TMG) transport via the melibiose permease (TMG II) in SalmonellaTMG uptake via TMG Il in anaerobic, starved and metabolically poisoned cells is dependent on an inward-directed Na⁺ gradient.Cells which have been partially depleted of endogenous substrates show H⁺ extrusion upon sodium-stimulated TMG influx.Measurements of the electrochemical H⁺ gradient in cells, starved in different ways for endogenous substrates, suggest that this proton extrusion is probably not linked to the actual translocation mechanism but is the result of metabolism induced by TMG plus Na⁺ uptake.     

Origin of the methyl carbon of methyl coniine in Conium maculatum

Studies with [methyl-¹⁴C]-l-methionine have established that the methyl carbon of l-methionine can act as a precursor of the N-methyl group of methyl coniine in Conium maculatum.     

Mechaism of Arthrobacter sialophilus neuraminidase: The binding of substrates and transition-state analogs

2-Deoxy-2,3-dehydro-N-acetylneuraminic acid and its methyl ester are competitive inhibitors of Arthrobacter sialophilus neuraminidase with K_i = 1.4 × 10^?6$\text{M}$ and 4.8 × 10^?5$\text{M}$, respectively. The K_m for the substrate, N-acetylneuraminlactose, is 1.0 × 10^?3$\text{M}$. These data, taken together with the conformation of these compounds, indicate that these compounds are transition-state analogs of the enzyme. These results also suggest that the substrate upon binding to neuraminidase is distorted to a conformation approaching that of a half-chair.     

Genetic analysis of X-linked mutagen-sensitive mutants of Drosophila melanogaster

Mutants at 2 new loci which control mutagen-sensitivity are described. Mutants at both loci are female-sterile and are hypersensitive to killing by MMS; neither increases the frequency of sex-linked recessive lethals. A screen of previously described female-sterile and meotic mutants has revealed that a number of these are also sensitive to mutagens. In addition, several new mutants have been identified on the basis of sensitivity to either HN2 or MMS. An anlysis of complementation data suggests that all of the X-linked genes controlling sensitivity to MMS may now have been identified. Among the new mei-41 alleles are mutants which show verly little meiotic nondisjunction or loss. Cytogenetic mapping of previously known mutants is also described. The mutants mus(1)104^D1 and mei-41^D5 are located in th eregion 14B13±?14D1,2 on the polytene chromosome map, and they map very close to each other genetically. Cytogenetically mus(1)101^D1 is between salivary chromosome bands 12A6,7 and 12D3, mus(1)103^D1 is between bands 12A1,2 and 12A6,7, and mus(1)-109^A1 is in section 8F3-9A2.     

Mutagen sensitivity of Drosophila melanogaster. I. Isolation and preliminary characterization of a methyl methanesulphonate-sensitive strain

Sensitivity to the monofunctional alkylating agent methyl methanesulfonate (MMS) has been tested as a selection technique to isolate mutant strains which can provide insights into the genetic control of DNA replication, DNA repair and recombination in the complex eucaryote, Drosophila melanogaster. The successful isolation of an X-linked MMS-sensitive strain, mut^s, has suggested that mutagen sensitivity is a feasible methodology for the selection of mutant strains of Drosophila which will be useful in the genetic and biochemical analysis of these cellular functions. Preliminary characterization of this mutant strain indicates that: (A) it is extremely sensitive to killing by MMS; (B) it is more mutable by MMS than the parent wildtype strain; and (C) it appears to possess mutator gene activity.     

Catabolism of 6-aminolaevulinic acid in etiolated Euglena gracilis

[4-¹⁴C]-δ-Aminolaevulinic acid (δ-ALA) was employed to study the specificity of δ-ALA incorporation into chlorophyll in etiolated Euglena gracilis. Degradation of chlorophyll a to acetate and propionate, obtained from ring B, indicated that, although some of the radioactivity was incorporated, it did not result from incorporation of intact δ-ALA.     

Electronic spectra and fluorescence properties of multichromophoric sulfonylureas

The multichromophoric character of two sulfonylurea herbicides, SMT and BNS, has been investigated in its manifestations in the electronic absorption spectra and in some fluorescence properties through a combined experimental and theoretical approach. After a theoretical analysis of the most stable structures of these flexible systems, the UV absorption spectra of the two multichromophoric compounds have been analysed both experimentally and theoretically, and most transitions have been assigned to individual chromophores, also by comparison with four suitable reference compounds (5-8). Finally, some experimental information concerning the fluorescence spectra and quantum yields have been analysed with reference to the contributions from single fluorophores and the role of low-lying n → π^∗ states.     

The kinetics and specificity of electron transfer from cytochromes and copper proteins to P700

The rates of electron transfer to P700 from plastocyanin and cytochrome f have been compared with those from three other c-type cytochromes and azurin, a copper protein resembling plastocyanin. Three different disruptive techniques were used to expose P700; digitonin, Triton X-100 and sonication. The following rate constants were measured at 25 °C, pH 7.0, with digitonin-treated chloroplasts: plastocyanin, 8 · 10⁷ M^?1 · s^?1; red-algal cytochrome c-553, 1.9 · 10⁷ M^?1 · s^?1; Pseudomonas cytochrome c-551, 8 · 10⁶ M^?1 · s^?1; azurin, ? 3 · 10⁵ M^?1 · s^?1; cytochrome f, ? 2 · 10⁴ M^?1 · s^?1; mammalian cytochrome c, ? 2 · 10⁴ M^?1 · s^?1. For electron transfer from plastocyanin, the effects of ionic strength, pH and temperature were also studied, and saturation effects found in earlier work were avoided by a full consideration of the various secondary reactions and inclusion of superoxide dismutase. The relative rates are discussed in relation to photosynthetic electron transport.     

Surface lipids of Sphaerotheca fuliginea spores

The major components (50%) of the surface lipid extract of fungal spores (5.6% of dry spore wt) of Sphaerotheca fuliginea are esters of primary alcohols and fatty acids. Esters (15%) of primary alcohols and a Δ^2t acid are present. The major acid moieties of the alkyl esters are C₂₂ and C₂₄ and of the Δ^2t alkyl ester is Δ^2t C₂₂; for both classes eicosanol is the major primary alcohol. The major ester of each class was concluded to be eicosanyl docosanoate and eicosanyl trans-2-docosenoate. Minor components are saturated and Δ^2t methyl and diol diesters and free fatty acids. The major acid moieties of the diol diesters are C₂₂ and C₂₄ and the major diol is 1,12-dodecanediol.     

Biosynthesis of the sesquiterpenoid,capsidiol, in sweet pepper fruits inoculated with fungal spores

Capsicum frutescens fruits inoculated with spore suspensions of Monilinia fructicola incorporated 1–4% of sodium acetate-[2-¹⁴C] or RS-mevalonolactone-[2-¹⁴C] into the phytoalexin, capsidiol. Labelled capsidiol was characterized by GC-RC, TLC-RC, gel chromatography (in conjunction with liquid scintillation counting) and GC-MS. The mode of incorporation of sodium acetate-[1,2-¹³C₂] into capsidiol, as indicated by the pattern of ¹³C-¹³C coupling from ¹³C NMR data, supports the hypothesis that the angular methyl group of the capsidiol skeleton arises by migration from the C-10 position of a eudesmane-type intermediate.     

The aggregation-attachment pheromone of the tropical bont tick Amblyomma variegatum Fabricius (Acari,Ixodidae): Isolation,identification and action of its components

Combining a new in vitro bioassay with the analytical method capillary gas chromatography-mass spectrometry, the aggregation-attachment pheromone produced by fed males of the tropical bont tick Amblyomma variegatum was shown to consist of o-nitrophenol, methyl salicylate and pelargonic acid in the approximate amounts of 2/1/8 × 10^?6 g/tick. A synthetic pheromone blend composed of those three volatile compounds evoked an aggregation response in unfed males and females in a bioassay comparable to the response to a natural pheromone source. Of the individual components. only o-nitrophenol induced a significant, although not complete aggregation response. Methyl salicylate and pelargonic acid contribute to complete pheromone activity, but induce no aggregation response at all, when offered separately.     

DNA strand breakage caused by dichlorvos, methyl methanesulphonate and iodoacetamide in Escherichia coli and cultured Chinese hamster cells

The DNA damaging properties of dichlorvos (2,2 dichlorovinyl dimethyl phosphate), methyl methanesulphonate (MMS) and iodoacetamide (IAA) have been studied, using alkaline sucrose sedimentation. In a strain of E. coli deficient in DNA polymerase I (polA) both dichlorvos and MMS caused random strand breakage, MMS being about twice as efficient as dichlorvos on a molar basis. In pol⁺ bacteria, DNA strand breaks or alkali labile bonds were detected following treatment with roughly five-fold higher concentrations of MMS but at similar high concentrations of dichlorvos there was an all or none breakdown of DNA molecules to fragments of very low molecular weight which correlated well with lethality.DNA synthesized after treatment of pol⁺ and polA bacteria with MMS was of low molecular weight, indicating the presence of discontinuities. With dichlorvos, the effect was much smaller.Apparent all-or-none DNA breakdown was also found when the polA strain of E. coli was treated with low concentrations of iodoacetamide, an agent that does not detectably alkylate DNA. At higher concentrations the breakdown was suppressed and random strand breakage occurred instea. These effects did not occurr with pol⁺ bacteria and correlated well with the greater sensitivity to iodoacetamide of the polA strain in survival experiments. We suggest that the major DNA damage resulting from treatment with iodoacetamide and dichlorvos arises indirectly through alkylation of other cellular constituents and consequent uncontrolled nuclease attack on the DNA. Discontinuities in newly synthesized DNA and mutagenesis following dichlorvos treatment, however, presumably result from direct alkylation of DNA.Strand breakage caused by dichlorvos and MMS in Chinese hamster cells tended to correlate with the extent to which these agents alkylate DNA, but survivval tended to correlate with the alkylation of protein.     

Genetic Recombination — Understanding the Mechanism : by H.L.K. Whitehouse Wiley; Chichester,New York, 1982

Interaction of peanut agglutinin with MeUmbβGalβ(1→3)GalNAc was followed with the stopped-flow technique. The mechanism is a simple bimolecular association with k₊ = 7.1 × 10³ M^?1. s^?1 and k_? = 0.24 s^?1 at 25°C. The very slow dissociation rate of the complex strongly supports earlier conclusions that the combining site of peanut agglutinin is complementary to the Galβ(1→3)GalNac structure.     

Biosynthetic relationship between tetrahydroanthracene and anthraquinone in Aloe saponaria

The incorporation of Me¹⁴COONa into aloesaponol I, laccaic acid D methyl ester and aloesaponarin I was demonstrated. The biosynthetic relation between aloesaponol I and aloesaponarin I was established, but incorporation of aloesaponol I into laccaic acid D methyl ester, or vice versa was not demonstrated and this result was confirmed by an investigation using labelled laccaic acid D methyl (¹⁴CH₃) ester. It was possible to show that aloesaponol I and laccaic acid D methyl ester were biosynthesized in parallel in Aloe saponaria.     

Light-dependent rubidium transport in intact Halobacterium halobium cells

The uptake of rubidium in intact Halobacterium halobium cells was followed, and found to be light-dependent. The exchange process is slow, the steady-state rate of ⁸⁶Rb⁺/Rb⁺ exchange being given by k = 6.3 · 10^?4 min^?1. Starved cells exhibited a faster rate than unstarved cells. The influx of ⁸⁶Rb⁺ was almost completely blocked in the presence of proton conductors (CCCP, FCCP, and SF 6847), and was sensitive to the presence of the permeant cation TPMP⁺. Valinomycin very slightly increased the rate of uptake, while 1 · 10^?6 M nigericin showed significant inhibition. On the other hand, release of ⁸⁶Rb⁺ was not light-dependent, although still affected by uncouplers, TPMP⁺, and nigericin. These experimental observations may be explained in terms of a passive flux driven by an electrical potential difference, and influenced by positive isotope interaction within the membrane. In carefully matched influx-efflux studies, the extent of the positive isotope interaction was measured. Using the formal treatment of Kedem and Essig, the ratio (exchange resistance)/(resistance to net flow) for ⁸⁶Rb⁺ was found to be 1.7.