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.     

The steroidal glycoalkaloid α-tomatine

The literature relating to chemical, biochemical and biological aspects of the steroidal glycoalkaloid, α-tomatine, is reviewed. The alkaloid, which can be used as a starting compound for the synthesis of steroidal hormones, is toxic to a wide range of living organisms. The significance of tomatine to plants which elaborate it is discussed and some possible uses of the compound are mentioned.     

Molecular Mechanisms of Translation Initiation in Mammals Studied by in vitroReconstruction of Initiation Complexes

Papers on the mechanisms of translation initiation in mammals studied by reconstruction of initiation complexes from individual components are reviewed. The author points to the constraints of this approach and to the pitfalls ignoring which one might come to erroneous conclusions and even artifacts. In addition, some methods employed in the field as well as some technical problems are discussed in the paper, together with the means of obviating them. The review could be a guidebook for newcomers into this quite labor-consuming field.     

Complementarity of regulation for the two glutamine synthetases from Bacillus caldolyticus, an extreme thermophile

Chromatophores from Rhodopseudomonas capsulata cells grown semiaerobically in the dark oxidize NADH, succinate, and dichlorophenolindophenol. In the presence of N₃^? these activities are inhibited, but light induces oxidation of dichlorophenolindophenol with O₂ as a terminal electron acceptor. Cyanide also inhibits electron transport but much higher concentrations are required to inhibit the photooxidation than the dark oxidation. The photooxidation was studied in a mutant strain of Rhodopseudomonas capsulata (YIV) which cannot grow anaerobically in the light, but similarly to the wild type, grows in the presence of oxygen. Chromatophores from YIV mutant catalyze photophosphorylation and dark oxidation activities with the same properties as those of the wild type. However, the rate of photooxidation in the mutant is only one-third that of the wild type. Based on the differential inhibitor sensitivity and on the mutation it is suggested that the photooxidase is different from the two respiratory oxidases and that this photooxidation activity might be essential for growth of the cells under anaerobic conditions in the light.     

Growth and shaping of the fin and limb buds

The development of the fin and limb buds involves a balance of centrifugal (active) and centripetal (passive) mechanical forces, the first of which acts to move the walls of these structures away from each other and the second of which holds them together. When the volume of the mesodermal core increases, the generated force meets with the resistance of the basal membrane, and as a result, the limb bud has a tendency to acquire a cylindrical shape. Collagen fibers, individual mesenchymal cells, and their groups hold together the dorsal and the ventral wall of the limb bud, prevent the movement of these walls away from each other, and in this way direct bud growth along the proximodistal and the anteroposterior axes. The balance of the forces which stretch the ectodermal layer and those which constrain it has also been observed in the development of other body parts.     

Effect of the time of day on central and peripheral fatigue during 2-min maximal voluntary contractions in persons with multiple sclerosis: gender differences

There is a lack of data on fatigue changes within 24 h among patients with multiple sclerosis. The purpose of this study was to evaluate the effect of time of day on central and peripheral fatigue during a continuous 2-min maximal voluntary contraction of the quadriceps muscle in women and men with multiple sclerosis (MS). We studied age-matched MS patients (range, 40–50 years). The inclusion criteria for patients were: a Kurtzke Expanded Disability Status score and a Fatigue Severity Scale score. We found a significant gender difference in central activation ratio (CAR) in the evening. At the end of the 2-min maximal voluntary contraction (MVC), the voluntary torque decreased by about 65% in men and women with MS in both the morning and evening. We also observed that, in women, CAR decreased markedly during the first 30 s in the evening test. The most interesting finding of our study is that central fatigue increased, whereas peripheral fatigue decreased markedly in the evening only in women. It remains unclear why women’s central fatigue is greater in the evening than in the morning.     

Nearest Neighbor Classification Rule for Mixtures of Discrete and Continuous Random Variables

In this paper very simple nonparametric classification rule for mixtures of discrete and continuous random variables is described. It is based on the method of nearest neighbor proposed by Cover and Hart (1967). The bounds on the limit of the nearest neighbor rule risks are given. Both lower and upper bound depend on the Bayes risk and the loss function. Finally the method is compared with other existing methods on some practical data set.