Binding of MutS protein to oligonucleotides containing a methylated or an ethylated guanine residue, and correlation with mutation frequency

The MutS-based mismatch repair (MMR) system has been conserved from prokaryotes to humans, and plays important roles in maintaining the high fidelity of genomic DNA. MutS protein recognizes several different types of modified base pairs, including methylated guanine-containing base pairs. Here, we looked at the relationship between recognition and the effects of methylating versus ethylating agents on mutagenesis, using a MutS-deficient strain of E. coli. We find that while methylating agents induce mutations more effectively in a MutS-deficient strain than in wild-type, this genetic background does not affect mutagenicity by ethylating agents. Thus, the role of E. coli MMR with methylation-induced mutagenesis appears to be greater than ethylation-induced mutagenesis. To further understand this difference an early step of repair was examined with these alkylating agents. A comparison of binding affinities of MutS with O⁶-alkylated guanine base paired with thymine, which could lead to transition mutations, versus cytosine which could not, was tested. Moreover, we compared binding of MutS to oligoduplexes containing different base pairs; namely, O⁶-MeG:T, O⁶-MeG:C, O⁶-EtG:T, O⁶-EtG:C, G:T and G:C. Dissociation constants (K_d), which reflect the strength of binding, followed the order G:T- > O⁶-MeG:T- > O⁶-EtG:T- = O⁶-EtG:C- ≥ O⁶-MeG:C- > G:C. These results suggest that a thymine base paired with O⁶-methyl guanine is specifically recognized by MutS and therefore should be removed more efficiently than a thymine opposite O⁶-ethylated guanine. Taken together, the data suggest that in E. coli, the MMR system plays a more significant role in repair of methylation-induced lesions than those caused by ethylation.     

Structure of the serine-containing capsular poly-saccharide K40 antigen from Escherichia coli O8:K40:H9

The structure of the K40 antigenic capsular polysaccharide (K40 antigen) of E. coli O8:K40:H9 was elucidated by determination of the composition, ¹H- and ¹³C-n.m.r. spectroscopy, periodate oxidation and Smith degradation, and methylation analysis. The K40 polysaccharide consists of [(O-β- -glucopyranosyluronic acid)-(1→4)-O-(2-acetamido-2-deoxy-- -glucopyranosyl)-(1→6)-O-(2-acetamido-2-deoxy-- -glucopyranosyl)-(1→4)] repeating units. All of the glucuronic acid residues are substituted amidically with -serine.     

Characterization of a respiratory mutant of Escherichia coli with reduced uptake of aminoglycoside antibiotics

A strain of Escherichia coli (NSW77) which is partially resistant to streptomycin was isolated by selecting for growth on plates supplemented with 12.5 μg/ml streptomycin, a concentration which completely inhibits growth of wild-type strains. The low-level resistance of the mutant appears to result from a reduced ability to accumulate streptomycin intracellularly. In addition, the mutant strain is unable to use succinate for growth because of a defective respiratory chain. Thus, membranes of the mutant strain were found to have approximately half the NADH and D-lactate oxidase activity of the parent strain. Succinate oxidase activity was reduced more drastically, to a level of 7% that of the parent strain. Moreover, membranes of the mutant were found to contain demethyl-menaquinone and, in place of ubiquinone, a structural analogue, 2-octaprenyl-3-methyl-6-methoxy-1,4 benzoquinone. The mutation responsible for both the Suc⁻ phenotype and partial resistance to streptomycin was found to be located near minute 15 on the bacterial chromosome. Both the biochemical and genetic evidence suggests that the mutation in strain NSW77 resides in the ubi F gene. Another previously characterized ubi F strain was also found to have a reduced capacity to take up an aminoglycoside antibiotic (gentamicin). These results suggest that the respiratory defects in ubi F strains are responsible for the reduced capacity of such strains to accumulate aminoglycosides.     

Synthesis and antibacterial activity of novel neamine derivatives

Synthesis and activity of derivatives at the O5 or O6 positions of 1-N-((S)-4-amino-2-hydroxybutyryl)-3′,4′-dideoxyneamine, which is the neamine moiety of arbekacin, were reported. Among these results, the 5-O-aminoethylaminocarbonyl derivative showed effective activity against Staphylococcus aureus expressing a bifunctional aminoglycoside-modifying enzyme AAC(6′)-APH(2″).     

Synthesis of methylated chitosan containing aromatic moieties: Chemoselectivity and effect on molecular weight

N-Arylated chitosans were synthesized via Schiff bases formed by the reaction between the primary amino group of chitosan with aromatic aldehydes followed by reduction of the Schiff base intermediates with sodium cyanoborohydride. Treatment of chitosan containing N,N-dimethylaminobenzyl and N-pyridylmethyl substituents with iodomethane under basic conditions led to quaternized N-(4-N,N-dimethylaminobenzyl) chitosan and quaternized N-(4-pyridylmethyl) chitosan. Methylation occurred at either N,N-dimethylaminobenzyl and N-pyridylmethyl groups before the residual primary amino groups of chitosan GlcN units were substituted. The total degree of quaternization of each chitosan varied depending on the extent of N-substitution (ES) and the sodium hydroxide concentration used in methylation. Increasing ES increased the total degree of quaternization but reduced attack at the GlcN units. N,N-dimethylation and N-methylation at the primary amino group of chitosan decreased at higher ES’s. Higher total degrees of quaternization and degrees of O-methylation resulted when higher concentrations of sodium hydroxide were used. The molecular weight of chitosan before and after methylation was determined by gel permeation chromatography under mild acidic condition. The methylation of the N,N-dimethylaminobenzyl derivative with iodomethane was accompanied by numerous backbone cleavages and a concomitant reduction in the molecular weight of the methylated product was observed. The antibacterial activity of water-soluble methylated chitosan derivatives was determined using Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria; minimum inhibitory concentrations (MIC) of these derivatives ranged from 32 to 128 μg/mL. The presence of the N,N-dimethylaminobenzyl and N-pyridylmethyl substituents on chitosan backbone after methylation did not enhance the antibacterial activity against S. aureus. However, N-(4-N,N-dimethylaminobenzyl) chitosan with degree of quaternization at the aromatic substituent and the primary amino group of chitosan of 17% and 16–30%, respectively, exhibited a slightly increased antibacterial activity against E. coli.     

Bacillussubtilis holo-cytochrome c-550 can be synthesised in aerobic Escherichia coli

Bacillus subtilis membrane-bound holo-cytochrome c-550 was found to be expressed from the structural gene cloned on a plasmid vector in aerobically grown Escherichia coli and exhibited normal biochemical properties. This occurs despite the lack of endogenous eytochrome c and suggests that eytochrome c-heme lyase activity is also present in aerobic E. coli. The membrane topology of B. subtilis eytochrome c-550 was studied using fusions to alkaline phosphatase (PhoA). The results show that the heme domain (at least when fused to PhoA) can be translocated as apo-cytochrome and confirm that the N-terminal part of the cytochrome functions as both export signal and membrane anchor for the C-tenninal heme domain. A model for the organisation of B. subtilis cytochrome c-550 in the cytoplasmic membrane is presented.     

Investigation of antibacterial mode of action for traditional and amphiphilic aminoglycosides

Aminoglycoside represents a class of versatile and broad spectrum antibacterial agents. In an effort to revive the antibacterial activity against aminoglycoside resistant bacteria, our laboratory has developed two new classes of aminoglycoside, pyranmycin and amphiphilic neomycin (NEOF004). The former resembles the traditional aminoglycoside, neomycin. The latter, albeit derived from neomycin, appears to exert antibacterial action via a different mode of action. In order to discern that these aminoglycoside derivatives have distinct antibacterial mode of action, RNA-binding affinity and fluorogenic dye were employed. These studies, together with our previous investigation, confirm that pyranmycin exhibit the traditional antibacterial mode of action of aminoglycosides by binding toward the bacterial rRNA. On the other hand, the amphiphilic neomycin, NEOF004 disrupts the bacterial cell wall. In a broader perspective, it verifies that structurally modified neomycin can exert different antibacterial mode of action leading to the revival of activity against aminoglycoside resistant bacteria.     

Potent induction of the adaptive response by a weak mutagen, methyl iodide, in Escherichia coli

Methyl iodide (MeI), a weakly mutagenic and highly chemoselective chemicals, was tested for its abilities to induced the adaptive and SOS responses in E. coli CSH26/pMCP1000 (alkA′-lacZ′) and CSH26/psK1002 (umuC′-lacZ′). MeI induced the adaptive response effectively but gave a very weak SOS response. Its potent ability in inducing the adaptive response was also demonstrated by adaptation to both the mutagenic and killing effects of N-methyl-N-nitrosourea (MNU) in E. coli WP2 cells. Simultaneous treatment with MeI in a non-growth medium slightly increased the mutagenicity of MNU, probably as a result of depletion of the repair enzyme, O⁶-methylguanine-DNA methyltransferase, which is constitutively present in the cells. As MeI itself proved to be only weakly mutagenic, a small part of the adaptive response which we have observed may involve indirect methylation of the repair enzyme by methyl transfer from MeI-induced O⁶-methylguanine residues in DNA. But the extent of the induced adaptive response seems to be much higher than would be expected from the observed weak mutagenicity of MeI. It is therefore suggested that the mechanism of induction of the adaptive response may involve direct methylation of the O⁶-methylguanine-DNA methyltransferase itself.     

Assignment of the H-N.M.R. spectra of heparin and heparan sulphate

Resonances from the main repeating unit of heparan, →4)-β- -GlcA-(1→4)-- -GlcNAc-(1→, have been assigned by using a sample of the capsular polysaccharide of E. coli K5. Comparison of the spectra of heparan sulphate samples before and after O- and/or N-desulphation, with re-N-acetylation or re-N-sulphation, allowed assignment of some of the H-1 doublets in terms of sequence effects. Chemical shifts for H-1 of unsulphated uronic acid residues are influenced by 6-sulphation of the nearest neighbor GlcN on the reducing side; those of GlcN residues vary according to whether they have IdoA or GlcA as the nearest neighbour on the reducing side. The H-1 doublets due to residues in the binding sequence for antithrombin have been assigned by comparison of the spectra of heparins having high and low affinities for immobilised antithrombin.     

Transformation of steroids by Bacillus strains isolated from the foregut of water beetles (Coleoptera:Dytiscidae): I. Metabolism of androst-4-en-3,17-dione (AD)

Two Bacillus strains were isolated from the foregut of the water beetle Agabus affinis (Payk.) and tested for their steroid transforming ability. After incubation with androst-4-en-3,17-dione (AD), 13 different transformation products were detected. AD was hydroxylated at C₆, C₇, C₁₁ and C₁₄, resulting in formation of 6β-, 7-, 11- and 14-hydroxy-AD. One strain also produced small amounts of 6β,14-dihydroxy-AD. Partly, the 6β-hydroxy group was further oxidized to the corresponding 6-oxo steroids. In addition, a specific reduction of the Δ⁴-double bond was observed, leading to the formation of 5-androstane derivatives. In minor yields the carbonyl functions at C₃ and C₁₇ were reduced leading to the formation of 3ξ-OH or 17β-OH steroids. EI mass spectra of the trimethylsilyl and O-methyloxime trimethylsilyl ether derivatives of some transformation products are presented for the first time.     

Time-resolved fluoroimmunoassay of plasma and urine O-desmethylangolensin

We present a method for the determination of the phytoestrogen metabolite O-desmethylangolensin (O-DMA) in plasma (serum) and in urine. O-DMA is a metabolite of daidzein, which occurs in soybeans. It has been suggested that isoflavones may afford protection against breast and prostate cancer and therefore, also the metabolites are of interest. The method is based on time-resolved fluoroimmunoassay (TR–FIA) using a europium chelate as a label. After the synthesis of 4′′-O-carboxymethyl-O-DMA, this compound is coupled to bovine serum albumin, and then used as antigen in immunization of rabbits. The tracers with the europium chelate are synthesized using the same 4′′-O-derivative of the -methyldeoxybenzoin. After enzymatic hydrolysis and ether extraction the immunoassay is carried out by time resolved fluoroimmunoassay (TR–FIA). Cross-reactivity was tested with angolensin, dihydrogenistein, dihydrodaidzein, equol, 6′-OH-angolensin, trans-4-OH-equol, 6′-OH-O-DMA, cis-4-OH-equol and 5-OH-equol. The antiserum cross-reacted only with angolensin. This cross-reactivity seems not to influence the results, which were highly specific. Plasma samples are hydrolyzed and extracted. Urine samples are analyzed directly after hydrolysis without extraction. The correlation coefficient between the plasma TR–FIA results and the GC–MS results was high; r value was 0.985. The correlation coefficient between the urine TR–FIA results and the GC–MS results was high over the entire range of concentrations (0–1500 nmol/l); r value was 0.976, but lower in the low concentration range (0–100 nmol/l), i.e. value was 0.631. The intra-assay coefficients of variation (CVs) for plasma O-DMA concentrations and for urine O-DMA concentrations at three different concentrations varied 2.8–7.7 and 3.0–6.0%, respectively and the inter-assay CVs varied 3.8–8.9 and 4.4–6.6%, respectively. The working range of the plasma and urine O-DMA assays was 0.5–512 nmol/l.     

Regioselective enzymatic aminoacylation of lobucavir to give an intermediate for lobucavir prodrug

Synthesis of lobucavir prodrug, L-valine, [(1S,2R,3R)-3-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (BMS 233866), requires regioselective coupling of one of the two hydroxyl groups of lobucavir (BMS 180194) with valine. Either hydroxyl group of lobucavir could be selectively aminoacylated with valine by using enzymatic reactions. N-[(Phenylmethoxy)carbonyl]-L-valine, [(1R,2R,4S)-2-(2-amino-6-oxo-1H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester (3, 82.5% yield), was obtained by selective hydrolysis of N,N′-bis[(phenylmethoxy)carbonyl]bis[L-valine], O,O′-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester (1) with lipase M, and L-valine, [(1R,2R,4S)-2-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (4, 87% yield) was obtained by hydrolysis of bis[L-valine], O,O′-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester, dihydrochloride (2), with lipase from Candida cylindracea. The final intermediate for lobucavir prodrug, N-[(phenylmethoxy)carbonyl]-L-valine, [(1S,2R,4R)-3-(2-amino-6-oxo-1H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester (5), could be obtained by transesterification of lobucavir using ChiroCLEC™ BL (61% yield), or more selectively by using immobilized lipase from Pseudomonas cepacia (84% yield).     

Diplatinum(III) complexes with bridging 1-methylcytosinate ligands and variable axial ligands, including guanine nucleobases

A series of diplatinum(III) complexes derived from cis-(NH₃)₂Pt^II and the model nucleobase 1-methylcytosine (1-MeC) has been prepared and X-ray structurally characterized, all of which contain two anionic base ligands (1-MeC⁻) in a head–tail (ht) arrangement: ht-cis-[(ONO₂)(NH₃)₂Pt(1-MeC⁻-N3,N4)₂Pt(NH₃)₂(ONO₂)](NO₃)₂·HNO₃·3H₂O (2b), ht-cis-[(NO₂) (NH₃)₂ Pt(1-MeC⁻-N3,N4)₂Pt(NH₃)₂(OH₂)](ClO₄)₃·3.5H₂O (3), ht-cis-[(OH₂)(NH₃)₂Pt(1-MeC⁻-N3,N4)₂Pt(NH₃)₂(OH₂)](ClO₄)₄·H₂O (4b), and ht-cis-[(9-EtGH-N7)(NH₃)₂Pt(1-MeC⁻-N3,N4)₂Pt (NH₃)₂(9-EtGH-N7)](NO₃)₄·9H₂O (7b) (9-EtGH=9-ethylguanine). Several other compounds, differing in the nature of the axial ligands, have been isolated and or observed in solution by ¹H and ¹⁹⁵Pt NMR spectroscopy. The chemistry of these diplatinum(III) compounds is dominated by facile substitution reactions of the axial ligands. Of particular interest in this context is the ready reaction of 2b or 3 with guanine nucleobases. Since similar compounds are not obtained with any of the other common nucleobases, 2b and 3 can be considered guanine-specific chemical probes.     

1,10-Phenanthroline-5,6-dione as a building block for the synthesis of homo- and heterometallic complexes

1,10-Phenanthroline-5,6-dione (C₁₂H₆N₂O₂ (1)) reacts with V(η⁶-mesitylene)₂ and Ti(η⁶-toluene)₂ affording coordination compounds of general formula M(O,O′---C₁₂H₆N₂O₂)₃ (M=Ti (2); M=V (3)) which further react with TiCl₄ or TiCp₂(CO)₂ yielding the tetrametallic species M(O,O′---C₁₂H₆N₂O₂---N,N′)₃(M′L_n)₃ (M=V, M′L_n=TiCl₄ (4); M=Ti, M′L_n=TiCp₂ (5); M=V, M′L_n=TiCp₂ (6)). The complex salt [Fe(N,N′---C₁₂H₆N₂O₂)₃][PF₆]₂ (7) has been obtained from iron(II) chloride tetrahydrate and 1 in the presence of NH₄PF₆. The reaction of 7 with TiCp₂(CO)₂ affords the tetrametallic derivative [Fe(N,N′---C₁₂H₆N₂O₂---O,O′)₃(TiCp₂)₃][PF₆]₂ (8). TiCl₂(THF)₂ reacts with MCp₂(O,O′---C₁₂H₆N₂O₂) to give MCp₂(O,O′---C₁₂H₆N₂O₂---N,N′)TiCl₂ (M=Ti (9); M=V (10)). By reaction of TiCp₂(O,O′---C₁₂H₆N₂O₂---N,N′)TiCl₂ (9) with C₁₂H₆N₂O₂, the bimetallic derivative TiCp₂(O,O′---C₁₂H₆N₂O₂---N,N′)TiCl₂(O,O′---C₁₂H₆N₂O₂) (11) has been prepared, which readily adds to TiCl₄, to give the trimetallic titanium derivative TiCp₂(O,O′---C₁₂H₆N₂O₂---N,N′)TiCl₂(O,O′---C₁₂H₆N₂O₂---N,N′)TiCl₄ (12). VCp₂(O,O′---C₁₂H₆N₂O₂---N,N′)TiCl₂ (10) reacts with the tris-chelate iron(II) cation 7 affording the heptametallic cationic complex [Fe(N,N′---C₁₂H₆N₂O₂---O,O′)TiCl₂(N,N′---C₁₂H₆N₂O₂---O,O′)VCp₂]₃ ⁺² isolated as the hexafluorophosphate 13.     

Unique mode of acetylation of oligosaccharides in aqueous two-phase system by Trichoderma reesei acetyl esterase

Trichoderma reesei RUT C-30 acetyl esterase, known to catalyze transacetylation reactions in water/vinyl acetate two-phase mixtures, was studied with respect to regioselectivity of acetylation of oligosaccharides in aqueous environment. Using series of oligosaccharides and their methyl glycosides, it was found that the enzyme catalyzes an efficient acetylation at O-3 position of the non-reducing terminal units of gluco-, xylo- and manno-oligosaccharides and a less efficient acetylation of O-2 position of the reducing end units of gluco- and xylo-oligosaccharides. The axial hydroxyl group at O-2 position of the reducing end mannose in mannooligosaccharides was not recognized by the enzyme and its acetylation was not observed. The structure of isolated transacetylation products was established by NMR, ESI-MS analysis and on the basis on their resistance towards action of glycosidases acting from the non-reducing end of oligosaccharides. The position of acetylation allowed deduce on some of the structural requirements of the enzyme for the acetyl group acceptors. T. reesei RUT C-30 acetyl esterase was also found to be capable of liberation of acetyl groups from terminal units of oligosaccharides, which speaks for its classification as an exo-acting acetyl esterase.     

Anthocyanins with an unusual acylation pattern from stem of Allium victorialis

Two novel anthocyanins have been isolated from the stem of Allium victorialis. By means of chemical degradation and spectroscopy, especially homo- and hetero-nuclear two-dimensional NMR techniques, the structures were determined to be cyanidin 3-O-(3″,6″-O-dimalonyl-β-glucopyranoside) (76.6%) and cyanidin 3-O-(3″,O-malonyl-β-glucopyranoside) (13.8%). This is the first report of acylation of the 3-position in the sugar moiety of any anthocyanin. The stability of malonyl substitution in the 3″-position on glucose is higher than the corresponding 6″-malonylation.     

Inhibition of hexose transport and abrogation of pH homeostasis in the intraerythrocytic malaria parasite by an O-3-hexose derivative

An O-3-hexose derivative, shown previously to inhibit a malaria parasite hexose transporter expressed in Xenopus oocytes as well as to suppress the multiplication of parasites, both in vitro and in vivo, was shown here to block the uptake of hexose sugars into isolated blood-stage parasites. This led to a decline in ATP levels and the loss of intracellular pH control. The results are consistent with those obtained with the cloned transporter. They support the notion that the transporter mediates uptake of glucose into the intraerythrocytic parasite and provide further support for the view that it is a suitable antimalarial drug target.     

Hydrogels of N-acylchitosans and their cellulose composites generated from the aqueous alkaline solutions

Hydrogels of N-acetyl and N-propionylchitosans were prepared form aqueous solutions of sodium N-acylchitosan salts (alkaline N-acylchitosans) and sodium N-acylchitosan xanthate [O-(sodium thio)thiocarbonyl N-acylchitosan], respectively, by standing at room temperature and on heating. Novel hydrogels of N-acetylchitosan-cellulose and N-propionylchitosan-cellulose composites were also prepared from sodium cellulose xanthate [O-(sodiumthio)thiocarbonyl cellulose] solutions mixed with sodium N-acylchitosan salts and with sodium N-acylchitosan xanthates, respectively.     

Direct expression in Escherichia coli and characterization of bovine adrenodoxins with modified amino-terminal regions.

Four forms of bovine adrenodoxin with modified amino-termini obtained by direct expression of cDNAs in Escherichia coli are Ad(Met¹), Ad(Met⁻¹), Ad(Met⁻¹²), and Ad(Met⁶). The shoulder numbers represent this site of translation initiator Met at the amino-termini. The adrenodoxins, except for Ad(Met⁻¹), were purified from the cell lysate and the ratios of A₄₁₄-to-A₂₇₆ of the purified proteins were over 0.92. NADPH-cytochrome c reductase activities of the three forms of adrenodoxin in the presence of adrenodoxin reductase were the same as that of purified bovine adrenocortical adrenodoxin. However, as cytochrome P-450_SCC reduction catalyzed by Ad(Met⁰) was about 60% or that by Ad(Met¹), the contribution of the amino-terminal region for the electron transfer or binding to cytochrome P-450_SCC would need to be considered.     

Guar gum derivatives. Part I: Preparation and properties

O-(2-Hydroxyethyl), O-(2-hydroxypropyl) and O-carboxymethyl derivatives of guar gum have been prepared under different experimental conditions. Several properties such as moisture regain, rate of hydration, solubility, viscosity and rheology of these derivatives have been studied. The properties depend upon polysaccharide chain length, and the nature and degree of chemical modification. The effect of alkali and alkaline hydrogen peroxide on the properties of guar gum have also been studied.