Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

Structure–antimicrobial activity of some natural isocoumarins and their analogues

Three naturally occurring isocoumarins (paepalantine, paepalantine 9-O-beta-D-glucopyranoside and paepalantine 9-O-beta-D-allopyranosyl(1 --> 6) glucopyranoside) and two semi-synthetic analogues, 9,10-acylated compound and 9-OH-10-methylated compound, structurally similar to paepalantine, were evaluated for antimicrobial activity using a spectrophotometric microdilution technique. The paepalantine was active against S. aureus, S. epidermidis, and E. faecalis while the other four compounds proved ineffective against all microorganisms tested at concentrations of 500 microg/ml. Variations in phenolic substitution at OH-9 and/or OH-10 in the paepalantine molecule resulted in compounds without antimicrobial activity. A combination of structural features, two phenolic groups as cathecolic system, forms an oxygenated system arrangement that may reflect the potentially antimicrobial properties of paepalantine.     

Aminoadamantanes containing monoterpene-derived fragments as potent tyrosyl-DNA phosphodiesterase 1 inhibitors

The ability of a number of nitrogen-containing compounds that simultaneously carry the adamantane and monoterpene moieties to inhibit Tdp1, an important enzyme of the DNA repair system, is studied. Inhibition of this enzyme has the potential to overcome chemotherapeutic resistance of some tumor types. Compound (+)-3c synthesized from 1-aminoadamantane and (+)-myrtenal, and compound 4a produced from 2-aminoadamantane and citronellal were found to be most potent as they inhibited Tdp1 with IC₅₀ values of 6 and 3.5 µM, respectively. These compounds proved to have low cytotoxicity in colon HCT-116 and lung A-549 human tumor cell lines (CC₅₀ > 50 µM). It was demonstrated that compound 4a at 10 µM enhanced cytotoxicity of topotecan, a topoisomerase 1 poison in clinical use, against HCT-116 more than fivefold and to a lesser extent of 1.5 increase in potency for A-549.     

多种天敌控制多种害虫的模糊数学模型

从生态经济学的原理出发，利用模糊集理论，探讨如何评价天敌控制多种害虫的为害作用，提出了多种天敌控制多种害虫的模糊数学模型。同时又得到了天敌对害虫的控制能力C，影响率G，影响强度H，及权数ω(x)的计算公式，以及在天敌作用下安全度的讨论与分析。     

Variation of chemical composition and antioxidant activity of essential oils of Mentha x rotundifolia (L.) Huds. (Lamiaceae) collected from different bioclimatic areas of Tunisia

The variation of the essential oils composition of 10 Tunisian Mentha x rotundifolia (L.) Huds. Populations and their antioxidant activity were assessed. Essential oils showed high percentages of oxygenated monoterpenes and sesquiterpene hydrocarbons. Rotundifolone, p-menthane-1,2,3-triol, β-caryophyllene and germacrene D were identified as main compounds at the species level. A variation in the essential oil composition was observed according to the populations and ecological factors. The populations 7, 8, 9 and 10 from the upper semi-arid bioclimatic zone and situated at high altitudes, exhibited the highest amount of rotundifolone. The populations 1, 2, 3, 4 and 5 from the lower humid showed a rotundifolone/β-caryophyllene/germacrene D chemotype. The population 6, situated at the lowest altitude, was characterized by the highest amount of p-menthane-1,2,3-triol. The level of antioxidant activity of the populations was linked to their chemical composition difference. The highest scavenging activity and the best ability to reduce ferric ions were recorded for the population 10. The most important capacity to inhibit β-carotene bleaching was revealed for the population 8. For all populations, the antioxidant activities were substantial but lower than antioxidant standards used (Trolox and BHT).The populations (7, 8, 9 and 10) from the upper semi-arid showed the best yields of essential oils and the highest contents of rotundifolone. Chemotypes within these populations could be selected for improvement programs.     

Regional differences in the carbon isotopic compositions of teak from two monsoonal regimes of India

Inter and intra-annual carbon isotope compositions (δ¹³C) of several annual growth rings of teak trees from two monsoonal regimes from India were studied and compared with the corresponding oxygen isotopic (δ¹⁸O) variations. In teak from both the regimes, amplitudes of intra-annual δ¹³C were ∼2-3 times lower than that observed in δ¹⁸O. Seasonal cycle with lower δ¹³C values at the middle and higher at ring boundaries was observed for teak from central India, dominated by the southwest monsoon. Positive correlations of intra-annual δ¹³C values with the corresponding δ¹⁸O values of the same rings and with relative humidity (RH) of the concurrent period suggest a dominant role of RH in controlling δ¹³C values of teak from central India. Intra-annual δ¹³C variations of teak from southern India, receiving both the southwest and northeast monsoons, revealed an initial decreasing trend followed by an increasing trend before culminating in depleted ¹³C values at the end of the growing season. No correlation was observed between intra-annual δ¹³C and δ¹⁸O variations of teak trees from southern India. Regional differences in the climatology of δ¹³C of atmospheric CO₂ or the lengths of growing season could be likely reasons for differing intra-annual δ¹³C variations of teak from the two climatic regimes.     

Path analysis of natural selection on plant chemistry: the xylem resin of ponderosa pine

We analyzed the pattern of correlations among fitness components, herbivory, and resin characteristics in a natural all-aged stand of ponderosa pine, to infer the strength and mechanism of natural selection on plant chemistry. Male and female cone production were monitored yearly for 15 years, and levels of herbivory for 9 years in 165 trees. Resin flow rate and monoterpene composition were determined for these same trees. Multiple regression of fitness components on resin characteristics showed significant associations consistent with directional selection for increased resin flow rates and increased proportions of α- and β-pinene, myrcene and terpinolene. However, negative correlations among monoterpene fractions of the resin constrained the overall selection. Selective herbivory by aphids approached statistical significance and monoterpenes showed some (non-significant) effect as deterrents against deer browse. Resin characteristics were not correlated with attack by cone insects or porcupines. However, the association between resin characteristics and fitness is significantly different from that predicted by the path coefficients involving herbivores. Therefore the hypothesis that these herbivores mediate selection on the resin is not supported by the observed pattern of correlations among resin characteristics, herbivory, growth and fecundity. In this population, most of the association between resin characteristics and fitness appears to be mediated by some other factor independent of attack by herbivore species present. Received: 18 March 1996/Accepted: 18 July 1996