检测脱氨基速率的超灵敏方法及应用

脱氨基被认为是引起细胞突变的主要因素,如果这些脱氨基的产物不被修复,将引起转换(transition)突变.为了理解DNA结构和其化学活性的关系,介绍一种新的灵敏的遗传学方法,它应用在DNA特定点的脱氨基速率的测定.这种方法基于M13mp2噬菌体内的1acZα基因中的CCC脯氨酸密码子的反转突变,即每个脱氨基事件表现为在白色菌斑背景中的一个蓝色菌斑,其灵敏度可达10⁵ M13mp2 DNA分子中检验出一个脱氨基事件.此外,该法可以计算脱氨基动力学速率常数和反应活化能.     

Chemical modification of bacitracin A and the biological activity of modified bacitracins

The single side chain amino group of the D-ornithine residue in bacitracin seems to be important for the antibacterial activity of the molecule, since, acetylation, formylation, carbamylation and deamination of the antibiotic caused 90–92% loss of antibacterial activity. In contrast, nearly 80–91% of the antibacterial activity of the parent antibiotic was retained after the esterification, amide formation and acid-chloride formation of the α—and Y -carboxyl groups of D-asparagine and D-glutamic acid residues of the antibiotic, respectively.     

An efficient pipeline for ancient DNA mapping and recovery of endogenous ancient DNA from whole‐genome sequencing data

Ancient DNA research has developed rapidly over the past few decades due to improvements in PCR and next‐generation sequencing (NGS) technologies, but challenges still exist. One major challenge in relation to ancient DNA research is to recover genuine endogenous ancient DNA sequences from raw sequencing data. This is often difficult due to degradation of ancient DNA and high levels of contamination, especially homologous contamination that has extremely similar genetic background with that of the real ancient DNA. In this study, we collected whole‐genome sequencing (WGS) data from 6 ancient samples to compare different mapping algorithms. To further explore more effective methods to separate endogenous DNA from homologous contaminations, we attempted to recover reads based on ancient DNA specific characteristics of deamination, depurination, and DNA fragmentation with different parameters. We propose a quick and improved pipeline for separating endogenous ancient DNA while simultaneously decreasing homologous contaminations to very low proportions. Our goal in this research was to develop useful recommendations for ancient DNA mapping and for separation of endogenous DNA to facilitate future studies of ancient DNA.     

The gastrointestinal tract: A major site of antioxidant action?

Diets rich in fruits and vegetables delay the onset of many age-related diseases, and contain a complex mixture of antioxidants (including ascorbate, carotenoids, vitamin E and other phenolics such as the flavonoids). However, diet also contains pro-oxidants, including iron, copper, H₂O₂, haem, lipid peroxides and aldehydes. Nitrite is frequently present in diet, leading to generation of reactive nitrogen species in the stomach. In considering the biological importance of dietary antioxidants, attention has usually focussed on those that are absorbed through the gastrointestinal tract into the rest of the body. In the present paper we develop the argument that the high levels of antioxidants present in certain foods (fruits, vegetables, grains) and beverages (e.g. green tea) play an important role in protecting the gastrointestinal tract itself from oxidative damage, and in delaying the development of stomach, colon and rectal cancer. Indeed, carotenoids and flavonoids do not seem to be as well absorbed as vitamins C and E. Hence their concentrations can be much higher in the lumen of the GI tract than are ever achieved in plasma or other body tissues, making an antioxidant action in the GI tract more likely. Additional protective mechanisms of these dietary constituents (e.g. effects on intercellular communication, apoptosis, cyclooxygenases and telomerase) may also be important.     

Subcellular localization of the APOBEC3 proteins during mitosis and implications for genomic DNA deamination

Humans have seven APOBEC3 DNA cytosine deaminases. The activity of these enzymes allows them to restrict a variety of retroviruses and retrotransposons, but may also cause pro-mutagenic genomic uracil lesions. During interphase the APOBEC3 proteins have different subcellular localizations: cell-wide, cytoplasmic or nuclear. This implies that only a subset of APOBEC3s have contact with nuclear DNA. However, during mitosis, the nuclear envelope breaks down and cytoplasmic proteins may enter what was formerly a privileged zone. To address the hypothesis that all APOBEC3 proteins have access to genomic DNA, we analyzed the localization of the APOBEC3 proteins during mitosis. We show that APOBEC3A, APOBEC3C and APOBEC3H are excluded from condensed chromosomes, but become cell-wide during telophase. However, APOBEC3B, APOBEC3D, APOBEC3F and APOBEC3G are excluded from chromatin throughout mitosis. After mitosis, APOBEC3B becomes nuclear, and APOBEC3D, APOBEC3F and APOBEC3G become cytoplasmic. Both structural motifs as well as size may be factors in regulating chromatin exclusion. Deaminase activity was not dependent on cell cycle phase. We also analyzed APOBEC3-induced cell cycle perturbations as a measure of each enzyme’s capacity to inflict genomic DNA damage. AID, APOBEC3A and APOBEC3B altered the cell cycle profile, and, unexpectedly, APOBEC3D also caused changes. We conclude that several APOBEC3 family members have access to the nuclear compartment and can impede the cell cycle, most likely through DNA deamination and the ensuing DNA damage response. Such genomic damage may contribute to carcinogenesis, as demonstrated by AID in B cell cancers and, recently, APOBEC3B in breast cancers.     

Esterification Rates of Main Organic Acids in Wines

The esterification rates (ratio of the concentration of an acid in the neutral ethyl ester form to total concentration of the acid) of main organic acids in wines were determined to study the extent of ethyl ester formation of organic acids. The esterification rates ranged from zero to 24.6%. The averaged values of table wines were from 6 to 16% for acetic and lactic acid, from 0.3 to 3.6% for succinic and malic acid, and from zero to 0.1% for tartaric acid. Sherries had higher esterification rates, about 1.6 to 6 times larger, than table wines. It was found that storage time and temperature influence the formation of ethyl esters, and it was suggested that the aging period required for the ester equilibrium is about one year for acetic and lactic acid, and more than two years for succinic, malic and tartaric acid. The possibility and the procedure to control wine quality during the aging process were discussed.     

Dopa decarboxylase exhibits low pH half-transaminase and high pH oxidative deaminase activities toward serotonin (5-hydroxytryptamine)

Dopa decarboxylase (DDC) catalyzes not only the decarboxylation of L-aromatic amino acids but also side reactions including half-transamination of D-aromatic amino acids and oxidative deamination of aromatic amines. The latter reaction produces, in equivalent amounts, an aromatic aldehyde or ketone (depending on the nature of the substrate), and ammonia, accompanied by O(2) consumption in a 1 : 2 molar ratio with respect to the products. The kinetic mechanism and the pH dependence of the kinetic parameters have been determined in order to obtain information on the chemical mechanism for this reaction toward 5-hydroxytryptamine (5-HT). The initial velocity studies indicate that 5-HT and O(2) bind to the enzyme sequentially, and that D-Dopa is a competitive inhibitor versus 5-HT and a noncompetitive inhibitor versus O(2). The results are consistent with a mechanism in which 5-HT binds to DDC before O(2). The pH dependency of log V for the oxidative deaminase reaction shows that the enzyme possesses a single ionizing group with a pK value of approximately 7.8 that must be unprotonated for catalysis. In addition to an ionizing residue with a pK value of 7.9 similar to that found in the V profile, the (V/K)(5-HT) profile exhibits a pK value of 9.8, identical to that of free substrate. This pK was therefore tentatively assigned to the alpha-amino group of 5-HT. No titratable ionizing residue was detected in the (V/K)(O2) profile, in the pH range examined. Surprisingly, at pH values lower than 7, where oxidative deamination does not occur to a significant extent, a half-transamination of 5-HT takes place. The rate constant of pyridoxamine 5'-phosphate formation increases below a single pK of approximately 6.7. This value mirrors the spectrophotometric pK(spec) of the shift 420-384 nm of the external aldimine between DDC and 5-HT. Nevertheless, the analysis of the reaction of DDC with 5-HT under anaerobic conditions indicates that only half-transamination occurs with a pH-independent rate constant over the pH range 6-8.5. A model accounting for these data is proposed that provides alternative pathways leading to oxidative deamination or half-transamination.     

Evolution of the AID/APOBEC family of polynucleotide (deoxy)cytidine deaminases

The AID/APOBEC family (comprising AID, APOBEC1, APOBEC2, and APOBEC3 subgroups) contains members that can deaminate cytidine in RNA and/or DNA and exhibit diverse physiological functions (AID and APOBEC3 deaminating DNA to trigger pathways in adaptive and innate immunity; APOBEC1 mediating apolipoprotein B RNA editing). The founder member APOBEC1, which has been used as a paradigm, is an RNA-editing enzyme with proposed antecedents in yeast. Here, we have undertaken phylogenetic analysis to glean insight into the primary physiological function of the AID/APOBEC family. We find that although the family forms part of a larger superfamily of deaminases distributed throughout the biological world, the AID/APOBEC family itself is restricted to vertebrates with homologs of AID (a DNA deaminase that triggers antibody gene diversification) and of APOBEC2 (unknown function) identifiable in sequence databases from bony fish, birds, amphibians, and mammals. The cloning of an AID homolog from dogfish reveals that AID extends at least as far back as cartilaginous fish. Like mammalian AID, the pufferfish AID homolog can trigger deoxycytidine deamination in DNA but, consistent with its cold-blooded origin, is thermolabile. The fine specificity of its mutator activity and the biased codon usage in pufferfish IgV genes appear broadly similar to that of their mammalian counterparts, consistent with a coevolution of the antibody mutator and its substrate for the optimal targeting of somatic mutation during antibody maturation. By contrast, APOBEC1 and APOBEC3 are later evolutionary arrivals with orthologs not found in pufferfish (although synteny with mammals is maintained in respect of the flanking loci). We conclude that AID and APOBEC2 are likely to be the ancestral members of the AID/APOBEC family (going back to the beginning of vertebrate speciation) with both APOBEC1 and APOBEC3 being mammal-specific derivatives of AID and a complex set of domain shuffling underpinning the expansion and evolution of the primate APOBEC3s.