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1.
R Süssmuth 《Mutation research》1980,78(4):301-315
Prophage induction and mutation by alkylaminosulfonates, ethyl aminosulfonate and alkyl methanesulfonates were examined comparatively. Prophage induction was carried out with a lysozyme lysis technique on the lysogenic strain Micrococcus lysodeikticus 53-40 (N5). The sulfonic ester derivatives show a slight lysogenic induction. At higher concentrations their toxicity seems to mask phage detection. Only methyl isopropylaminosulfonate and ethyl aminosulfonate exhibit no or negligible toxic effects, and with these compounds at higher concentrations a strong prophage induction is found. Alkyl sulfonate derivatives induce mutations in the tester strain of Salmonella typhimurium TA1535. Methyl methylaminosulfonate and ethyl N-methyl-N-2-chloroethyl aminosulfonate show a mutagenicity comparable to that of the well-known methyl methanesulfonate or ethyl methanesulfonate. With ethyl aminosulfonate, however, which does not show inactivation, no significant mutagenic effect was observed. DNA alterations were found in the polymerase-deficient strain E. coli P3478. The results of prophage induction and mutagenicity are compared and discussed. 相似文献
2.
The inactivation of the cysteinyl exopeptidases cathepsin H and C by affinity-labelling reagents. 总被引:1,自引:1,他引:0
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An attempt has been made to extend to the cysteinyl exopeptidases cathepsins H and C affinity-labelling approaches shown to be effective with cysteinyl endopeptidases such as cathepsins B and L and the calcium-activated proteinase. This involved the preparation of amino acid and dipeptide derivatives with unblocked N-termini to satisfy the aminopeptidase and dipeptidyl aminopeptidase characteristics of cathepsins H and C respectively. For covalent reactivity, the possibilities examined included diazomethanes (-CHN2), fluoromethanes (-CH2F) and dimethylsulphonium salt [-CH2S+(CH3)2]. A dipeptidylfluoromethane with a free amino group could not be prepared, perhaps due to inherent instability. Cathepsin H was inactivated by 1 microM-H2N-Phe-CH2F (the 'H2N' indicates a free unblocked amino group) (k2 = 1878 M-1.s-1); this reagent was without effect on cathepsins C and B, even at 100-fold this concentration. Analogous selectivity was shown by H2N-Ser(OBzl)-CHN2 and H2N-Phe-CH2S+(CH3)2, members of other classes of covalently binding reagents. For cathepsin C the dipeptide derivatives H2N-Gly-Phe-CHN2 and H2N-Phe-Ala-CH2S+(CH3)2 caused rapid inactivation near 10(-7) M. Higher concentrations inactivated cathepsins H and B, but the rates were slower by two to three orders of magnitude than for cathepsin C. 相似文献
3.
We have synthesized inhibitors of chymotrypsin, based on fluoromethyl ketones, that bind at S and S' subsites. "Small" inhibitors of serine proteases, which have previously been synthesized, only interact with S subsites. The parent compound is Ac-Leu-ambo-Phe-CF2H (1) (Ki = 25 X 10(-6) M). This inhibitor was modified by successively replacing H of the -CF2H group by -CH2CH2CONHCH3, (4), -CH2CH2CONH-Leu-NHMe (5), -CH2CH2CONH-Leu-Val-OEt (6), and -CH2CH2CONH-Leu-Arg-OMe (7). Corresponding Ki values are 7.8 (4), 0.23 (5), 0.21 (6), and 0.014 (7) microM. Extending 5 to 6 by addition of Val-OEt at P3' does not decrease Ki. In contrast, extension of 5 to 7 by incorporating Arg-OMe at P3' decreases Ki approximately 15-fold, suggesting interaction between Arg and the S3' subsite but no corresponding interaction at that subsite with Val. These results are in accordance with results obtained with the homologous family of avian ovomucoid third domain proteins. Proteins with Arg at the P3' position show highly favorable interactions with the protease at the S3' subsite [Park, S. J. (1985) Ph.D. Thesis, Purdue University; M. Laskowski, Jr., personal communication]. These results establish that incorporation of residues which interact with S' subsites significantly increases the efficacy of inhibitors and that valuable information concerning the most effective amino acid composition of small inhibitors can be obtained from the amino acid sequence of protein inhibitors. 相似文献
4.
Twenty-one substituted 1,4-naphthoquinones and five 8-quinolinols and copper(II) chelates were tested for antifungal activity against Candida albicans and Trichophyton mentagrophytes. Compounds containing electron-releasing or weak electron-withdrawing groups in the 2 and 3 positions of the 1,4-naphthoquinone ring were the most active against C. albicans at pH 7.0 in the presence of beef serum in the following order: 2-CH3O = 2,3-(CH3O)2 greater than 2-CH3 greater than 2-CH3S greater than 2-NH2 greater than 2,6-(CH3)2. For T. mentagrophytes under the same conditions the inhibitory 1,4-naphthoquinones contained the substituents 2-CH3O greater than 2,3-(CH3O)2 greater than 2-CH2S greater than 2-CH3 greater than 2-CH3(NaHSO3) greater than 2-NH2 greater than 2-C2H5S, 3-CH3 greater than 2,6-(CH3)2 greater than 2,3-CL2 greater than 5,8-(OH)2. 相似文献
5.
The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoacetium catalyzes transfer of the N5-methyl group of (6S)-methyltetrahydrofolate (CH3-H4folate) to the cob(I)amide center of a corrinoid/iron-sulfur protein (CFeSP), forming H4folate and methylcob(III)amide. We have investigated binding of 13C-enriched (6R,S)-CH3-H4folate and (6R)-CH3-H4folate to MeTr by 13C NMR, equilibrium dialysis, fluorescence quenching, and proton uptake experiments. The results described here and in the accompanying paper [Seravalli, J., Shoemaker, R. K., Sudbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 5728-5735] constitute the first evidence for protonation of the pterin ring of CH3-H4folate. The pH dependence of the chemical shift in the 13C NMR spectrum for the N5-methyl resonance indicates that MeTr decreases the acidity of the N5 tertiary amine of CH3-H4folate by 1 pK unit in both water and deuterium oxide. Binding of (6R,S)-CH3H4folate is accompanied by the uptake of one proton. These results are consistent with a mechanism of activation of CH3-H4folate by protonation to make the methyl group more electrophilic and the product H4folate a better leaving group toward nucleophilic attack by cob(I)amide. When MeTr is present in excess over (6R,S)-13CH3-H4folate, the 13C NMR signal is split into two broad signals that reflect the bound states of the two diastereomers. This unexpected ability of MeTr to bind both isomers was confirmed by the observation of MeTr-bound (6R)-13CH3-H4folate by NMR and by the measurement of similar dissociation constants for (6R)- and (6S)-CH3-H4folate diastereomers by fluorescence quenching experiments. The transversal relaxation time (T2) of 13CH3-H4folate bound to MeTr is pH independent between pH 5.50 and 7.0, indicating that neither changes in the protonation state of bound CH3-H4folate nor the previously observed pH-dependent MeTr conformational change contribute to broadening of the 13C resonance signal. The dissociation constant for (6R,S)-CH3-H4folate is also pH independent, indicating that the role of the pH-dependent conformational change is to stabilize the transition state for methyl transfer, and not to favor the binding of CH3-H4folate. 相似文献
6.
We have examined the interaction of methyl isocyanide with the purified component proteins of Azotobacter vinelandii nitrogenase (Av1 and Av2). CH3NC was shown to be a potent reversible inhibitor (Ki = 158 microM) of total electron flow, apparently uncoupling magnesium adenosine 5'-triphosphate hydrolysis from electron transfer to substrate. CH3NC is a substrate (Km = 0.688 mM at Av2/Av1 = 8), and extrapolation of the data indicates that at high enough CH3NC concentration, H2 evolution can be eliminated. The products are methane plus methylamine (six electrons) and dimethylamine (four electrons). There is an excess (relative to methane) of methylamine formed, which may arise by hydrolysis of a two-electron intermediate. A rapid high-performance liquid chromatography/fluorescence method was developed for methylamine determination. The products C2H4 and C2H6 appear to be formed via a reduction followed by an insertion mechanism. CH3NC appears to be reduced at an enzyme state more oxidized than the one responsible for H2 evolution or N2 reduction. Other substrates (C2H2 greater than N2 congruent to azide greater than N2O) all both relieve CH3NC inhibition and inhibit CH3NC reduction. Both effects occur in the same relative order, implying productive (substrate) and nonproductive (inhibitor) modes of binding of CH3NC to the same site. 相似文献
7.
Aerobic degradation of dimethyl sulfide (DMS), previously described for thiobacilli and hyphomicrobia, involves catabolism to sulfide via methanethiol (CH3SH). Methyl groups are sequentially eliminated as HCHO by incorporation of O2 catalyzed by DMS monooxygenase and methanethiol oxidase. H2O2 formed during CH3SH oxidation is destroyed by catalase. We recently isolated Thiobacillus strain ASN-1, which grows either aerobically or anaerobically with denitrification on DMS. Comparative experiments with Thiobacillus thioparus T5, which grows only aerobically on DMS, indicate a novel mechanism for aerobic DMS catabolism by Thiobacillus strain ASN-1. Evidence that both organisms initially attacked the methyl group, rather than the sulfur atom, in DMS was their conversion of ethyl methyl sulfide to ethanethiol. HCHO transiently accumulated during the aerobic use of DMS by T. thioparus but not with Thiobacillus strain ASN-1. Catalase levels in cells grown aerobically on DMS were about 100-fold lower in Thiobacillus strain ASN-1 than in T. thioparus T5, suggesting the absence of H2O2 formation during DMS catabolism. Also, aerobic growth of T. thioparus T5 on DMS was blocked by the catalase inhibitor 3-amino-1,2,4-triazole whereas that of Thiobacillus strain ASN-1 was not. Methyl butyl ether, but not CHCl3, blocked DMS catabolism by T. thioparus T5, presumably by inhibiting DMS monooxygenase and perhaps methanethiol oxidase. In contrast, DMS metabolism by Thiobacillus strain ASN-1 was unaffected by methyl butyl ether but inhibited by CHCl3. DMS catabolism by Thiobacillus strain ASN-1 probably involves methyl transfer to a cobalamin carrier and subsequent oxidation as folate-bound intermediates. 相似文献
8.
S Claudel O Tasseau S Sagan C Grison P Coutrot S Lavielle 《The journal of peptide research》2004,64(5):186-193
Glycine-9 and leucine-10 of substance P (SP) are critical for (NK)-1 receptor recognition and agonist activity. Propsi(Z)-CH=CH(CH3)-CONH)Leu (or Met) and Propsi((E)-CH=CH(CH3)-CONH)Leu (or Met) have been introduced in the sequence of SP, in order to restrict the conformational flexibility of the C-terminal tripeptide, Gly-Leu-Met-NH2, of SP. Propsi((Z)-CH=C(CH2CH(CH3)2)-CONH)Met-NH2, with an isobutyl substituent to mimic the Leu side-chain, was also incorporated in place of the C-terminal tripeptide. The substituted-SP analogs were tested for their affinity to human NK-1 receptor specific binding sites (NK-1M and NK-1m) and their potency to stimulate adenylate cyclase and phospholipase C in Chinese Hamster ovary (CHO) cells transfected with the human NK-1 receptor. The most potent SP analogs [Pro9psi((Z)CH=C(CH3)CONH)Leu10]SP and [Pro9psi ((E)CH=C(CH3)CONH)Leu10]SP, are about 100-fold less potent than SP on both binding sites and second messenger pathways. These vinylogous (Z)- or (E)-CH=C(CH3)- or (Z)-CH=C(CH2CH(CH3)2) moieties hamper the correct positioning of the C-terminal tripeptide of SP within both the NK-1M- and NK-1m-specific binding sites. The origin of these lower potencies is related either to an incorrect peptidic backbone conformation and/or an unfavorable receptor interaction of the methyl or isobutyl group. 相似文献
9.
K D Asmus S Nigam 《International journal of radiation biology and related studies in physics, chemistry, and medicine》1976,29(3):211-219
Absolute rate-constants for the reaction of the nitroxyl free radicals TAN and TMPN with radiation-chemically-formed radicals and ions have been determined. k(TAN + X) (in M(-1) sec(-1)=4-0 X 10(9) (for X = OH-), 2-9 X 10(10) (eaq-), 8-0 X 10(9) (H-), 7-2 X 10(8) (-CH2OH), 4-0 X 10(8) (CH3CHOH), 4-3 X 10(8) ((CH3)2COH) 2-8 X 10(8) (-CH2(CH3)2COH), 5-9 X 10(7) (glucose radical), 4-0 X 10(8) (c-C5H9-), and k(TMPN + X)=3-4 X 10(9) (OH-), 7-8 X 10(9) (eq-), 4-9 X 10(9) (H-), 4-4 X 10(8) (-CH2OH), 4-9 X 10(8) (CH3CHOH), 3-6 X 10(8) ((CH3)2COH), 1-5 X 10(8) (-CH2(CH3)2COH), 4-9 X 10(7) (glucose radical), 4-3 X 10(8) (c-C5H9-). Direct measurements by means of a pulse-radiolysis conductivity technique were based on the formation and destruction of charged species in these reactions within certain pH ranges. It is indicated that the radiosensitizing nitroxyls undergo both redox and addition reactions. 相似文献
10.
It is shown by ESR method that 8-methoxypsoralen (8-MOP) under low temperature UV-irradiation has no influence on free radical formation of saturated fatty acids (FA). Under the same conditions the quantum yield of free radical photogeneration for unsaturated FA (oleate and limolenate) after addition of 8-MOP increased 7.5-15 times. The ESR spectrum of free radicals generated after interaction of 8-MOP with polyunsaturated FA, may be presented as superposition of signals-CH-(CH = CH)2-, -CH2CHCH2- and -CH2CH2. The molecular mechanism of photosensitized reactions is discussed. 相似文献
11.
A series of chemically modified mutants (CMMs) of subtilisin B. lentus (SBL) were generated employing the combination of site-directed mutagenesis and chemical modification. This strategy entails the mutation of a selected active site residue to cysteine and its subsequent modification with a methanethiosulfonate reagent CH3SO2S-R, where R may be infinitely variable. The present study was undertaken to evaluate the changes in specificity and pH-activity profiles that could be induced by modification of S156C and S166C in the S1 pocket of SBL with a representative range of side chain modifications, namely R=-CH3, -CH2C6H5, -CH2CH2NH3+ and CH2CH2SO3 . The side chain of S156C is surface exposed and well solvated while that of S166C points into the pocket. Kinetic evaluation of the CMMs with suc-AAPF-pNA as substrate showed that the kcat/K(M)s changed very little for the S156C CMMs, but varied by up to 11-fold for the S166C CMMs. pH-Activity profiles were also determined, and showed that a negatively or positively charged side chain modification increased or decreased respectively, the pKa of the catalytic triad histidine for both modification sites but with more dramatic changes for the interior pointing S166C than for the solvent exposed S156C site. As an additional probe of altered specificity, inhibition of the CMMs by a representative series of 5 boronic acid transition state analogue inhibitors was determined. The K(I)s observed ranged from a 3.5-fold improvement over the WT value, to a 12-fold decrease in binding. Overall, greater variability in all the parameters measured, activity, pKa, and boronic acid binding resulted from modification at the inward pointing 166 site than at the solvent-exposed 156 site. 相似文献
12.
The reaction products obtained from mixtures of 5'-GMP and platinum(IV) compounds with formula Pt(IV)Cl4(LL) and Pt(IV)Cl2(OH)2(LL) (LL representing two monodentate or one bidentate amine ligand) have been characterized by proton NMR spectroscopy. The amines used are NH3, H2N-CH2-CH2-NH2 (ethylenediamine, en), H2N-CH2-C(CH3)2-CH2-NH2 (2,2-dimethyl-1,3-diaminopropane, dmdap), and HC(CH3)2-NH2 (isopropylamine, ipa). Conditions varied during the reaction are pH (values of 4, 7, and 10), effect of visible light, and addition of vitamin C as a reducing agent. In all cases, the major product appeared to be the bis(5'-GMP)(LL)Pt(II) compound. The pH effect is limited; i.e., at pH 4 the reactions proceed somewhat faster than at neutral pH, while at pH 10 slower reactions occur. The illumination with visible light also induces only slight differences in the yields of the products. On the other hand, when vitamin C is present, the reactions proceed quite rapidly, resulting in the same main product but in higher yields (up to 80%). The facts that apparently no Pt(IV) adducts with 5'-GMP can be observed under these conditions and that the major products are bis(5'-GMP)(LL)Pt(II) compounds clearly support the hypothesis that the antitumor activity of certain platinum(IV) compounds is based upon in vivo reduction to the corresponding platinum(II) compounds. 相似文献
13.
Banerjee SR Maresca KP Stephenson KA Valliant JF Babich JW Graham WA Barzana M Dong Q Fischman AJ Zubieta J 《Bioconjugate chemistry》2005,16(4):885-902
Hydrazinonicotinamide (HYNIC) forms stable coordination complexes with Tc-99m when reacted with Tc(V)oxo species such as Tc-mannitol or other Tc-polyhydric complexes. However, radio-HPLC of [Tc-For-MLFK-HYNIC] labeled via Tc-polyhydric ligands demonstrated multiple radiochemical species each with unique biodistribution patterns. This is likely due to the fact that Tc can bind to the hydrazino moiety, as well as polyhydric ligands, in a variety of coordination geometries. Tridentate ligands, such as bis(mercaptoethyl)methylamine (NS2), may constrain the possible coordination geometries and improve overall stability. To investigate this, we synthesized NS2, converted the [Tc-mannitol-For-MLFK-HYNIC] to the corresponding NS2-containing complex [Tc-NS2-For-MLFK-HYNIC], and compared its infection imaging and biodistribution properties with [Tc-mannitol-For-MLFK-HYNIC]. Conversion to the NS2 complex was confirmed by HPLC which showed a single unique hydrophobic species with retention time greater than the [Tc-mannitol-For-MLFK-HYNIC] complex. Imaging experiments with both preparations were performed in rabbits with E. coli infections in the left thigh. Tissue radioactivity measurements demonstrated that compared to Tc-mannitol-peptide, accumulation of Tc-NS2-peptide was lower in blood, heart, and normal muscle and higher in spleen, infected muscle, and pus (p < 0.01). These results indicate that the Tc-NS2-peptide complex is chemically more homogeneous and exhibits improved infection localization and biodistribution properties. In an effort to model the interactions of the metal-HYNIC core with NS2 and related ligand types, the reactions of [ReCl3(NNC5H4NH)(NHNC5H4N)] and [99TcCl3(NNC5H4NH)(NHNC5H4N)], effective structural analogues for the [M(NNC5H4NH(x))2] core, with NS2, C5H3N-2,6-(CH2SH)2, O(CH2CH2SH)2, and S(CH2CH2SH)2 were investigated and the compounds [M[CH3N(CH2CH2S)2](NNC5H4N)(NHNC5H4N] (M = 99Tc (5a), Re (5b)), [Re[C5H3N-2,6-(CH2S)2](NNC5H4N)(NHNC5H4N)].CH2Cl2.0.5MeOH (7), [Re[SCH2CH2)2O] (NNC5H4N)(NHNC5H4N)] (8), and [Re[(SCH2CH2)2S](NNC5H4NH)(NHNC5H4N)]Cl (9) were isolated. Similarly, the reaction of [ReCl3(NNC5H4NH)(NHNC5H4N)] with the bidentate ligands pyridine-2-methanethiol and 3-(trimethlysilyl)pyridine-2-thiol led to the isolation of [ReCl(C5H4N-2-CH2S) (NNC5H4N)(NHNC5H4N)] (10) and [Re(2-SC5H3N-3-SiMe3)2 (NNC5H4N)(NHNC5H4N)] (11), respectively, while reaction with N-methylimidazole-2-thiol yielded the binuclear complex [Re(OH)Cl(SC3H2N2CH3)2(NNC5H4N)2 (NHNC5H4N)2] (12). The analogous metal-(HYNIC-OH) precursor, [ReCl3[NNC5H3NH(CO2R)] [NHNC5H3N(CO2R)]] (R = H, 13a; R = CH3, 13b) has been prepared and coupled to lysine to provide [RCl3[NNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)] [NHNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)]].2HCl (14.2HCl), while the reaction of the methyl ester 13b with 2-mercaptopyridine yields [Re(2-SC5H4N)2[NNC5H3N(CO2Me)][NHNC5H3N(CO2Me)]] (15). While the chemical studies confirm the robustness of the M-HYNIC core (M = Tc, Re) and its persistence in ligand substitution reactions at adjacent coordination sites of the metal, the isolation of oligomeric structures and the insolubility of the peptide conjugates of 13, 14, and 15 underscore the difficulty of characterizing these materials on the macroscopic scale, an observation relevant to the persistent concerns with reagent purity and identity on the tracer level. 相似文献
14.
Propineb, a dithiocarbamate fungicide, was studied by using the sperm morphology assay in (C57BL6 male X C3H female) F1 mice. At all dose levels, no statistically significant increase in the percentage of sperm abnormalities was observed. Methyl methanesulfonate (MMS) and 2-acetylaminofluorene (2-AAF), which were tested as positive controls, induced a dose-effect-related increase in teratospermia. 相似文献
15.
Monosaccharides, disaccharides, and trisaccharides were tested as inhibitors of the in vitro growth of Plasmodium falciparum (strain FCB). While certain monosaccharides (N-acetyl-D-glucosamine, D-mannose, and 3-O-methyl-D-glucose) proved to exhibit a toxic or reversibly retarding effect on the intraerythrocytic development of the parasite, the corresponding alpha- or beta-methylglycosides did not. Several methylglycosides, synthetic di- and tri-saccharides, and artificial blood group antigens were further tested for inhibitory effects on invasion of host red blood cells in vitro. The synthetic disaccharides beta DGlcNAc(1----4) alpha DManOMe and beta DGlcNAc(1----4) DGlcNAc (chitobiose) were good inhibitors of invasion at 10 mM concentration, whereas beta DGal(1----4)beta DGlcNAcOMe was negligibly inhibitory. The inhibition rate of N-acetyl-D-glucosamine, beta-glycosidically linked to bovine serum albumin (BSA) by an alipathic spacer, -(CH2)8CO-, was not enhanced, compared to the corresponding hapten, beta DGlcNAcO(CH2)8COOCH3. The inhibition rates of blood group A- and B-trisaccharide haptens, which were inhibitors of invasion, were also not significantly enhanced when coupled to BSA by way of the corresponding amide spacer, -(CH2)2NHCO(CH2)7CO-. A remarkable enhancement of the inhibition rate was, however, observed when beta DGal(1----3) alpha DGalNAcO(CH2)2NHCO(CH2)7COOCH3 (T-hapten) was coupled to BSA. A clear-cut decrease in the inhibition rates of different beta-glycosides of N-acetyl-D-glucosamine, beta DGlcNAcOR, was observed, depending on the nature of the aglycon R(p-nitrophenyl greater than -(CH2)8COOCH3 greater than -(CH2)2NHCO(CH2)2COOCH3 greater than -CH3). Also, p-nitrophenyl-alpha-D-glucopyranoside was a much better inhibitor of invasion than the corresponding methyl glycoside, alpha DGlcOMe, which was not inhibitory. The properties of the aglycon spacer, used for the covalent attachment of the carbohydrate to the carrier protein, may thus be crucial for the outcome of the inhibition rate. 相似文献
16.
S. M. Morris O. E. Domon R. L. Kodell L. J. McGarrity D. R. Stegall 《Cell biology and toxicology》1988,4(3):281-294
To determine the relationships between the induction of specific biological responses and exposure to DNA-damaging agents, human teratocarcinoma-derived cells were exposed to either ethyl methanesulfonate or to methyl methanesulfonate, and sister chromatid exchange, cellular proliferation and relative cloning ability measured. SCE increased while cellular proliferation and relative cloning ability each decreased in a concentration-dependent manner. Methyl methanesulfonate was consistently more efficient in inducing biological responses than was ethyl methanesulfonate. When the individual responses were compared, the decrease in cellular proliferation paralleled the reduction in cloning efficiency. A strong correlation was also observed between the reduction in relative cloning ability and sister chromatid exchange frequency. Because these relationships are similar to those previously described in other mammalian cell lines, the observations in our study suggest that the P3 cell line is an appropriate choice for modeling effects of toxicant exposure in human cells.Abbreviations AGT
average generation time
- BUdR
5-bromodeoxyuridine
- CHO
Chinese hamster ovary
- EMS
ethyl methanesulfonate
- ENU
N-ethyl-N-nitrosourea
- MMS
methyl methanesulfonate
- MNU
N-methyl-Nnitrosourea
- SCE
sister chromatid exchange 相似文献
17.
Eugene F DeRose Dawei Li Thomas Darden Scott Harvey Fred W Perrino Roel M Schaaper Robert E London 《Biochemistry》2002,41(1):94-110
The DNA polymerase III holoenzyme (HE) is the primary replicative polymerase of Escherichia coli. The epsilon subunit of the HE complex provides the 3'-exonucleolytic proofreading activity for this enzyme complex. epsilon consists of two domains: an N-terminal domain containing the proofreading exonuclease activity (residues 1-186) and a C-terminal domain required for binding to the polymerase (alpha) subunit (residues 187-243). Multidimensional NMR studies of (2)H-, (13)C-, and (15)N-labeled N-terminal domains (epsilon186) were performed to assign the backbone resonances and measure H(N)-H(N) nuclear Overhauser effects (NOEs). NMR studies were also performed on triple-lableled [U-(2)H,(13)C,(15)N]epsilon186 containing Val, Leu, and Ile residues with protonated methyl groups, which allowed for the assignment of H(N)-CH(3) and CH(3)-CH(3) NOEs. Analysis of the (13)C(alpha), (13)C(beta), and (13)CO shifts, using chemical shift indexing and the TALOS program, allowed for the identification of regions of the secondary structure. H(N)-H(N) NOEs provided information on the assembly of the extended strands into a beta-sheet structure and confirmed the assignment of the alpha helices. Measurement of H(N)-CH(3) and CH(3)-CH(3) NOEs confirmed the beta-sheet structure and assisted in the positioning of the alpha helices. The resulting preliminary characterization of the three-dimensional structure of the protein indicated that significant structural homology exists with the active site of the Klenow proofreading exonuclease domain, despite the extremely limited sequence homology. On the basis of this analogy, molecular modeling studies of epsilon186 were performed using as templates the crystal structures of the exonuclease domains of the Klenow fragment and the T4 DNA polymerase and the recently determined structure of the E. coli Exonuclease I. A multiple sequence alignment was constructed, with the initial alignment taken from the previously published hidden Markov model and NMR constraints. Because several of the published structures included complexed ssDNA, we were also able to incorporate an A-C-G trinucleotide into the epsilon186 structure. Nearly all of the residues which have been identified as mutators are located in the portion of the molecule which binds the DNA, with most of these playing either a catalytic or structural role. 相似文献
18.
Differential and combined effects of 0.25 and 0.50% antibiotics (ampicillin, neomycin, furadentine) and alkylating agents (ethyl methanesulfonate, methyl ethanesulfonate, methyl methanesulfonate) were assayed on Phaseolus vulgaris L. (2 n = 22) at the M2 generation for chlorophyll mutations. The general types scored were Albino, Xantha, Virescens and Maculata. Yellowish-green leaves having red mid-veins and veinlets were observed only amongst the progeny raised after treatment with 0.25% ethyl methanesulfonate or 0.25% methyl ethanesulfonate + 0.25% ampicillin. The frequency of chlorophyll mutation after combined treatments in general was higher than after differential treatments. Methyl methanesulfonate among alkanesulfonates and neomycin among antibiotics induced higher frequencies of chlorophyll mutations. No chlorophyll mutant was produced by ampicillin.Although antibiotics induced a lower frequency of chlorophyll mutation than alkylating agents, the frequency and pattern of spectra of chlorophyll mutants showed an action of antibiotics in inducing mutation similar to that of alkylating agents. Therefore, it is considered that antibiotics are potential mutagens. 相似文献
19.
Wines BD Powell MS Parren PW Barnes N Hogarth PM 《Journal of immunology (Baltimore, Md. : 1950)》2000,164(10):5313-5318
The CH2-CH3 interface of the IgG Fc domain contains the binding sites for a number of Fc receptors including Staphylococcal protein A and the neonatal Fc receptor (FcRn). It has recently been proposed that the CH2-CH3 interface also contains the principal binding site for an isoform of the low affinity IgG Fc receptor II (Fc gamma RIIb). The Fc gamma RI and Fc gamma RII binding sites have previously been mapped to the lower hinge and the adjacent surface of the CH2 domain although contributions of the CH2-CH3 interface to binding have been suggested. This study addresses the question whether the CH2-CH3 interface plays a role in the interaction of IgG with Fc gamma RI and Fc gamma RIIa. We demonstrate that recombinant soluble murine Fc gamma RI and human Fc gamma RIIa did not compete with protein A and FcRn for binding to IgG, and that the CH2-CH3 interface therefore appears not to be involved in Fc gamma RI and Fc gamma RIIa binding. The importance of the lower hinge was confirmed by introducing mutations in the proposed binding site (LL234,235AA) which abrogated binding of recombinant soluble Fc gamma RIIa to human IgG1. We conclude that the lower hinge and the adjacent region of the CH2 domain of IgG Fc is critical for the interaction between Fc gamma RIIa and human IgG, whereas contributions of the CH2-CH3 interface appear to be insignificant. 相似文献
20.
The substrate specificity of N-acetylhexosaminidase (E.C. 3.2.1.51) from Aspergillus oryzae was examined using p-nitrophenyl 6-O-sulfo-N-acetyl-beta-D-glucosaminide (6-O-sulfo-GlcNAc-O-pNP) as the glycosyl donor and a series of beta-d-glucopyranosides and N-acetyl-beta-D-glucosaminides with variable aglycons at the anomeric positions as the acceptors. When beta-D-glucopyranosides with methyl (CH(3)), allyl (CH(2)CHCH(2)), and phenyl (C(6)H(5)) groups at the reducing end were used as the acceptors, this enzyme transferred the 6-O-sulfo-GlcNAc moiety in the donor to the location of O-4 in these glycosyl acceptors with a high regioselectivity, producing the corresponding 6-O-sulfo-N-acetylglucosaminyl beta-D-glucopyranosides. However, beta-D-glucopyranose lacking aglycon was a poor substrate for transglycosylation. This A. oryzae enzyme could also accept various N-acetyl-beta-D-glucosaminides carrying hydroxyl (OH), methyl (CH(3)), propyl (CH(2)CH(2)CH(3)), allyl (CH(2)CHCH(2)) and p-nitrophenyl (pNP; C(6)H(4)-NO(2)) groups at their aglycons, yielding 6-O-sulfo-N-acetylglucosaminyl-beta(1-->4)-disaccharide products. 相似文献