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1.
《Journal of molecular biology》2021,433(15):167097
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. 相似文献
2.
Plant somatic cells have the capability to switch their cell fates from differentiated to undifferentiated status under proper
culture conditions, which is designated as totipotency. As a result, plant cells can easily regenerate new tissues or organs
from a wide variety of explants. However, the mechanism by which plant cells have such remarkable regeneration ability is
still largely unknown. In this study, we used a set of meristem-specific marker genes to analyze the patterns of stem cell
differentiation in the processes of somatic embryogenesis as well as shoot or root organogenesis in vitro. Our studies furnish preliminary and important information on the patterns of the de novo stem cell differentiation during various types of in vitro organogenesis. 相似文献
3.
Evangelia Jockers-Wretou Valya Russanova Christo Venkov 《Molecular biology reports》1988,13(3):123-131
In a recent publication the isolation and some characteristics of an anti-histone 3 monoclonal antibody, 1GB3 were described (Muller et al. FEBS Lett. 182: 459–464, 1985). We now report that the epitope recognized is phylogenetically conserved and located in the N-terminal part of H3, most likely between residues 40 and 50. Using the ELISA technique we found this region to be accessible in chromatin to the monoclonal antibody. The effect of non-ionic detergents on the adsorbtion of chromatin on microtiter plates was studied in this context.Immunological analysis of the reaction of the monoclonal antibody with chromatin by immunoinhibition and immunosedimentation shows that the H3 epitope is accessible in both folded and unfolded chromatin fibre as well as in high- and low-molecular weight oligonucleosomes.Abbreviations BSA
Bovine srum albumin
- mab
Monoclonal antibody
- PBS
Phosphate buffered saline
- PMSF
Phenylmethyl sulfonyl fluoride 相似文献
4.
导水管周围灰质(periaqueductal gray,PAG)在疼痛的调控过程中处于一个不可或缺的位置.其不仅是痛觉信息上行传递的重要部位,还是疼痛抑制系统的重要组成部分.在PAG,包括γ-氨基丁酸(γ-aminobutyric acid,GABA)、5-羟色胺(5-hydroxytryptamine,5-HT)和谷氨酸(glutamate,Glu)在内的神经递质以及内源性阿片肽(endogenous opioid peptides,EOP)和内源性大麻素(endocannabinoid,e CB)为代表的神经调质都参与了PAG对疼痛的信息传递以及调节.本文重点综述GABA、5-HT、Glu、EOP和eCB在PAG参与疼痛生理调控机制的研究进展,以期为中枢神经系统的镇痛研究提供一定的理论基础. 相似文献
5.
Veli Isomaa 《Biochimica et Biophysica Acta (BBA)/General Subjects》1981,675(1):9-16
In vitro binding affinities of various progestins to cytosol and nuclear progesterone receptors of rabbit uterus were determined and correlated with the biological potency of these steroids. In addition, cytosol and nuclear progesterone receptor levels were measured after a 5-day administration of different progestins (0.5 mg/kg daily) with variable biologic activites. The receptor levels were compared with the bilological response; the induction of uteroglobin synthesis. Cytosol and nuclear progesterone receptors had identical steroid binding properties (r = 0.98). The correlation between the in vitro binding affinity (cytosol or nuclear) and the in vivo biologic activity of the steroids was good (r = 0.73). After a 5-day treatment with progestins, the nuclear receptor concentration correlated in an inverse manner (r = ?0.84) with the uterine fluid unteroglobin concentration. A similar, but slightly weaker correlation (r = ?0.81) was also found for the cytosol receptor content and uteroglobin secretion. These data indicate that not only nuclear, but also cytosol progesterone receptor levels decrease in the rabbit uterus during chronic hormone action. Decline in the nuclear progesterone receptor content seemed to occur during treatment with all progestational steroids, while onlyi progestins with high biological potency were capable of decreasing the cytosol receptor content. 相似文献
6.
(+)-2,9 alpha-Dimethyl-5-(m-hydroxyphenyl)morphan is the only phenylmorphan analog whose affinity for opioid kappa-receptors is greater than its affinity for opioid mu-receptors. Pharmacologically, the compound is a pure opioid antagonist devoid of agonist activity in in vivo assays of antinociception. The absolute configuration of the compound has been determined to be (1R,5S,9R) from an X-ray crystallographic study of the chloride salt. Thus, the absolute configuration corresponds to that of the atypical opioid agonist (-)-phenylmorphan while the weak atypical agonist (-)-2,9 alpha-dimethyl-5-(m- hydroxyphenyl)morphan corresponds to the potent morphine-like (+)-phenylmorphan. The preferred orientations of the phenyl ring for the two stereoisomers were determined using the molecular mechanics program MM2-87 and found to vary from that of the two parent compounds. The atypical properties of the two 9 alpha-methyl analogs is consistent with an opioid ligand model which proposes that morphine-like properties require a particular range of phenyl orientations. There was good agreement between the structure obtained from X-ray crystallography and computed with the MM2-87 program. 相似文献
7.
Peanut (Arachis hypogaea) agglutinin (PNA) is extensively used as tumour marker as it strongly recognises the cancer specific T antigen (Galβ1→3GalNAc-), but not its sialylated version. However, an additional specificity towards Galβ1→4GlcNAc (LacNAc), which is not tumour specific, had been attributed to PNA. For correct interpretation of lectin histochemical results we examined PNA sugar specificity using naturally occurring or semi-synthetic glycoproteins, matrix-immobilised galactosides and lectin-binding tissue glycoproteins, rather than mono- or disaccharides as ligands. Dot-blots, transfer blots or polystyrene plate coatings of the soluble glycoconjugates were probed with horse-radish peroxidase (HRP) conjugates of PNA and other lectins of known specificity. Modifications of PNA-binding glycoproteins, including selective removal of O-linked oligosaccharides and treatment with glycosidases revealed that Galβ1→4GlcNAc (LacNAc) was ineffective while terminal α-linked galactose (TAG) as well as exposed T antigen (Galβ1→3 GalNAc-) was excellent as sugar moiety in glycoproteins for their recognition by PNA. When immobilised, melibiose was superior to lactose in PNA binding. Results were confirmed using TAG-specific human serum anti-α-galactoside antibody. 相似文献
8.
《Bioscience, biotechnology, and biochemistry》2013,77(8):2224-2227
We isolated a cDNA clone with homology to known desaturase genes from Oblongichytrium sp., recently classified as a new genus of thraustochytrids (Labyrinthulomycetes), and found that it encoded Δ5-desaturase by its heterologous expression in yeast. The enzyme had higher activity toward 20:4n-3 than 20:3n-6, indicating that this Δ5-desaturase can be used in the production of n-3 polyunsaturated fatty acids in transgenic organisms. 相似文献
9.
Minjuan Shen Mingli Lin Mengqi Zhu Wenxin Zhang Danyang Lu Huanhuan Liu Jingjing Deng Kehua Que Xu Zhang 《Biochimica et Biophysica Acta (BBA)/General Subjects》2019,1863(1):167-181
Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering. 相似文献
10.
Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG. A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background. 相似文献