BALB/c-nu裸小鼠繁殖中杂合仔鼠剔除时间对纯合裸仔鼠生长发育的影响

目的研究屏障设施下裸小鼠繁殖生产时,在不同时间段剔除杂合仔鼠对纯合裸仔鼠的成活率及生长发育情况的影响。方法选取25窝头胎生BALB/c-nu新生裸小鼠按剔除杂合仔鼠的时间不同分成五组,以24h剔除组为对照比较纯合裸仔鼠生长发育及死亡率、离乳率的差异性,统计分析其生长发育过程中的体重增长等。结果剔除组生长发育及死亡率与对照组之间均存在差异性,其中14d剔除组和21d剔除组差异极显著。结论剔除杂合仔鼠的时间对纯合裸仔鼠的成活率及生长发育情况的有很大影响。为了保证SPF级裸鼠生长发育要求,生产繁殖出合格的实验用裸小鼠,剔除全部杂合仔鼠的最佳时间应该在新生仔鼠出生24h内,超过3d则会极大地影响纯合裸小鼠的正常生长发育。     

Effects of 2-Deoxygalactose on Auxin-Induced Growth and Levels of UDP-Sugars in Higher Plants

Galactose inhibited the IAA-induced elongation of cells in segmentsof oat coleoptiles but not in segments of azuki bean and cucumberstems. In contrast, its analogs, 2-deoxygalactose and 2-deoxyglucose,inhibited the IAA-induced elongation of cells in these planttissues. In segments of cucumber stems, 2-deoxygalactose inhibitedformation of cell-wall polysaccharides with little effect onrespiration in terms of either reduced consumption of O₂ orlevels of ATP. 2-Deoxygalactose and 2-deoxyglucose caused arapid decrease in the levels of UTP and UDP-sugars with a concomitantincrease in levels of UDP in cucumber segments. These resultssuggest that 2-deoxysugars inhibit synthesis of cell-wall polysaccharidesby decreasing the levels of UDP-sugars, thereby inhibiting IAA-inducedelongation of cells in cucumber segments. 2-Deoxysugars can,therefore, be used as potential inhibitors with which to assessthe role of UDP-sugars in the IAA-induced elongation of cellsin dicotyledonous plants. (Received September 27, 1990; Accepted February 5, 1991)     

Higher Growth Hormone Levels are Associated with Erectile Dysfunction in Male Patients With Acromegaly

Objective: To investigate in vivo correlates of erectile dysfunction (ED) in male patients with acromegaly.Methods: Fifty-one male patients with acromegaly were assessed by the International Index of Erectile Function-5 and Acromegaly Quality of Life (Acro-QoL) questionnaires. The measurement of serum nitric oxide (NO) were performed in patients and age-matched nonacromegalic controls.Results: Among 51 patients analyzed, 32 (62.7%) had ED. Patients with ED showed lower Acro-QoL scores regarding global (69.8 ± 17.7 versus 79.4 ± 11.2; P = .035) and personal relationship dimensions (59.6 ± 22.1 versus 76.8 ± 17.6; P = .012) than non-ED patients. ED patients were older (44.5 ± 11.2 years versus 33.2 ± 8.5 years; P = .04) and showed higher growth hormone (GH) levels (15.5 μg/L [interquartile range of 9.5 to 34.5 μg/L] versus 5.9 μg/L [interquartile range of 3.4 to 13.9 μg/L]; P = .001) compared to non-ED patients. The cutoff values for identifying ED were 7.9 μg/L for random GH and 5.3 μg/L for GH nadir after oral administration of 75 g of glucose. There was no significant difference in total testosterone levels between the two groups (6.36 ± 4.24 nmol/L versus 9.54 ± 5.50 nmol/L; P = .299). The NO levels in patients with acromegaly were significantly lower than those in nonacromegalic controls (8.77 ± 1.78 μmol/L versus 19.19 ± 5.02 μmol/L, respectively; P = .049). Furthermore, the NO levels were even lower in ED patients than those in non-ED patients (5.14 ± 0.98 μmol/L versus 12.09 ± 3.44 μmol/L; P = .027).Conclusion: Our study showed that ED is prevalent in male acromegalic patients and may be associated with systemic endothelial dysfunction induced by excessive GH. Further studies investigating the mechanism of GH and ED are required.Abbreviations: Acro-QoL = Acromegaly Quality of Life; ED = erectile dysfunction; FSH = follicle-stimulating hormone; GH = growth hormone; IGF-1 = insulin-like growth factor 1; IIEF-5 = international index of erection function-5; LH = luteinizing hormone; MRI = magnetic resonance imaging; NO = nitric oxide; OGTT = oral glucose tolerance test; QoL = quality of life; ROC = receiver operating characteristic     

DNA甲基化与植物的生长发育

文章就DNA甲基化与植物生长发育的关系、催化甲基化的酶、5－甲基胞嘧啶在植物基因组中的分布、甲基化的发生和遗传的研究进展作了介绍。     

Cytosine Methylation Dysregulation in Neonates Following Intrauterine Growth Restriction

Background

Perturbations of the intrauterine environment can affect fetal development during critical periods of plasticity, and can increase susceptibility to a number of age-related diseases (e.g., type 2 diabetes mellitus; T2DM), manifesting as late as decades later. We hypothesized that this biological memory is mediated by permanent alterations of the epigenome in stem cell populations, and focused our studies specifically on DNA methylation in CD34+ hematopoietic stem and progenitor cells from cord blood from neonates with intrauterine growth restriction (IUGR) and control subjects.

Methods and Findings

Our epigenomic assays utilized a two-stage design involving genome-wide discovery followed by quantitative, single-locus validation. We found that changes in cytosine methylation occur in response to IUGR of moderate degree and involving a restricted number of loci. We also identify specific loci that are targeted for dysregulation of DNA methylation, in particular the hepatocyte nuclear factor 4α (HNF4A) gene, a well-known diabetes candidate gene not previously associated with growth restriction in utero, and other loci encoding HNF4A-interacting proteins.

Conclusions

Our results give insights into the potential contribution of epigenomic dysregulation in mediating the long-term consequences of IUGR, and demonstrate the value of this approach to studies of the fetal origin of adult disease.     

Alu RNP and Alu RNA regulate translation initiation in vitro

Alu elements are the most abundant repetitive elements in the human genome; they emerged from the signal recognition particle RNA gene and are composed of two related but distinct monomers (left and right arms). Alu RNAs transcribed from these elements are present at low levels at normal cell growth but various stress conditions increase their abundance. Alu RNAs are known to bind the cognate proteins SRP9/14. We purified synthetic Alu RNP, composed of Alu RNA in complex with SRP9/14, and investigated the effects of Alu RNPs and naked Alu RNA on protein translation. We found that the dimeric Alu RNP and the monomeric left and right Alu RNPs have a general dose-dependent inhibitory effect on protein translation. In the absence of SRP9/14, Alu RNA has a stimulatory effect on all reporter mRNAs. The unstable structure of sRight RNA suggests that the differential activities of Alu RNP and Alu RNA may be explained by conformational changes in the RNA. We demonstrate that Alu RNPs and Alu RNAs do not stably associate with ribosomes during translation and, based on the analysis of polysome profiles and synchronized translation, we show that Alu RNP and Alu RNA regulate translation at the level of initiation.     

Gene-Specific DNA Methylation Association with Serum Levels of C-Reactive Protein in African Americans

A more thorough understanding of the differences in DNA methylation (DNAm) profiles in populations may hold promise for identifying molecular mechanisms through which genetic and environmental factors jointly contribute to human diseases. Inflammation is a key molecular mechanism underlying several chronic diseases including cardiovascular disease, and it affects DNAm profile on both global and locus-specific levels. To understand the impact of inflammation on the DNAm of the human genome, we investigated DNAm profiles of peripheral blood leukocytes from 966 African American participants in the Genetic Epidemiology Network of Arteriopathy (GENOA) study. By testing the association of DNAm sites on CpG islands of over 14,000 genes with C-reactive protein (CRP), an inflammatory biomarker of cardiovascular disease, we identified 257 DNAm sites in 240 genes significantly associated with serum levels of CRP adjusted for age, sex, body mass index and smoking status, and corrected for multiple testing. Of the significantly associated DNAm sites, 80.5% were hypomethylated with higher CRP levels. The most significant Gene Ontology terms enriched in the genes associated with the CRP levels were immune system process, immune response, defense response, response to stimulus, and response to stress, which are all linked to the functions of leukocytes. While the CRP-associated DNAm may be cell-type specific, understanding the DNAm association with CRP in peripheral blood leukocytes of multi-ethnic populations can assist in unveiling the molecular mechanism of how the process of inflammation affects the risks of developing common disease through epigenetic modifications.     

DNA Sequence Explains Seemingly Disordered Methylation Levels in Partially Methylated Domains of Mammalian Genomes

For the most part metazoan genomes are highly methylated and harbor only small regions with low or absent methylation. In contrast, partially methylated domains (PMDs), recently discovered in a variety of cell lines and tissues, do not fit this paradigm as they show partial methylation for large portions (20%–40%) of the genome. While in PMDs methylation levels are reduced on average, we found that at single CpG resolution, they show extensive variability along the genome outside of CpG islands and DNase I hypersensitive sites (DHS). Methylation levels range from 0% to 100% in a roughly uniform fashion with only little similarity between neighboring CpGs. A comparison of various PMD-containing methylomes showed that these seemingly disordered states of methylation are strongly conserved across cell types for virtually every PMD. Comparative sequence analysis suggests that DNA sequence is a major determinant of these methylation states. This is further substantiated by a purely sequence based model which can predict 31% (R²) of the variation in methylation. The model revealed CpG density as the main driving feature promoting methylation, opposite to what has been shown for CpG islands, followed by various dinucleotides immediately flanking the CpG and a minor contribution from sequence preferences reflecting nucleosome positioning. Taken together we provide a reinterpretation for the nucleotide-specific methylation levels observed in PMDs, demonstrate their conservation across tissues and suggest that they are mainly determined by specific DNA sequence features.     

Effects of pH and Repellent Tactic Stimuli on Protein Methylation Levels in Escherichia coli

Intracellular pH (pH_int) and extracellular pH (pH_ext) of Escherichia coli were measured at 12-s time resolution by ³¹P-nuclear magnetic resonance: a sudden neutral-to-acid shift in pH_ext (e.g., from 7.0 to 5.6) caused a transient failure of homeostasis, with pH_int decreasing by about 0.4 unit in ca. 30 s and then returning to its original value (ca. 7.5) over a period of several minutes. Membrane proton conductance was estimated to be 20 pmol s⁻¹ cm⁻² pH unit⁻¹. Addition of the membrane-permeant weak acid benzoate at constant pH_ext also caused a lowering of pH_int; at high concentrations it generated an inverted transmembrane pH gradient (ΔpH). The buffering capacity of the cells was estimated by such experiments to be ca. 50 mM per pH unit. Effects of pH-related stimuli on the methyl-accepting chemotaxis proteins (MCPs) were examined: the steady-state methylation of MCP I was found to decrease when pH_int was lowered by weak acid addition or when pH_ext was lowered. The extent of demethylation in the latter case was too great to be explained by imperfect steady-state homeostasis; a small but reproducible undershoot in methylation level correlated with the observed short-term homeostatic failure. MCP II underwent smaller and more complex changes than MCP I, in response to pH-related stimuli. The methylation level of MCP I could not, by any condition tested, be driven below a limit of ca. 15% of the control level (unstimulated cells at pH_ext 7.0). The weak-acid concentration needed to reach that limit was dependent on pH_ext, as would be expected on the basis of ΔpH-driven concentrative effects. The potency ranking of weak acids was the same with respect to lowering pH_int, demethylating MCP I, and causing repellent behavioral responses. The data are consistent with a model whereby MCP I and hence tactic behavior are sensitive to both pH_int and pH_ext. Evidence is presented that pH_int may also have a direct (non-MCP-related) effect on motor function. Comparison of methyl-³H- and ³⁵S-labeled MCP I revealed that in both unstimulated and repellent-stimulated cells the major species did not carry methyl label, yet it had an electrophoretic mobility that indicated that it was more positively charged than the unmethylated form observed in methyltransferase mutants, and it was susceptible to base hydrolysis. This suggests that a substantial fraction of MCP I molecules is methylated or otherwise modified but neither exchanges methyl label nor undergoes reverse modification by repellent stimuli.     

Sequence conservation in Alu evolution

A statistical analysis of a set of genomic human Alu elements is based on a published alignment and a recent classification of these sequences. After separation of the Alu sequences into families, the consensus sequences of these families are determined, using the correct weighting of the unidirectional decay of CG-dinucleotides. For, the tenfold greater mutation rate at CG's requires separate consideration of an independent clock at every stage of analysis. The distributions of the substitutions with respect to the new consensus sequences, taking the CG and the non-CG-nucleotide positions separately, lie far closer to the expected distributions than the total diversity. Computer analysis of the folding of RNAs derived from these sequences indicates that RNA secondary structure is conserved among Alu families, suggesting its importance for Alu proliferation and/or function. The folding pattern, further substantiated by a number of compensatory mutations, includes secondary structure domains which are homologous to those observed in 7SL RNA and a defined region of interaction between the two Alu subunits. These results are consistent with a model in which a small number of conserved Alu master genes give rise via retroposition to the numerous copies of Alu pseudogenes, that then diversify by random substitution. The master genes appeared at different periods during evolution giving rise to different families of Alu sequences.     

Non-Thermal Plasmas Affect Plant Growth and DNA Methylation Patterns in Glycine max

Journal of Plant Growth Regulation - Non-thermal plasmas (NTP) are partially ionized gases that represent a promising technology for seed treatment to enhance seed health while promoting...     

A Potential Role for CHH DNA Methylation in Cotton Fiber Growth Patterns

DNA methylation controls many aspects of plant growth and development. Here, we report a novel annual growth potential change that may correlate with changes in levels of the major DNA demethylases and methyltransferases in cotton ovules harvested at different times of the year. The abundances of DNA demethylases, at both the mRNA and protein levels, increased significantly from February to August and decreased during the remainder of the 12-month period, with the opposite pattern observed for DNA methyltransferases. Over the course of one year, substantial changes in methylcytosine content was observed at certain CHH sites (H = A, C, or T) in the promoter regions of the ETHYLENE RESPONSIVE FACTOR 6 (ERF6), SUPPRESSION OF RVS 161 DELTA 4 (SUR4) and 3-KETOACYL-COA SYNTHASE 13 (KCS13), which regulate cotton fiber growth. Three independent techniques were used to confirm the annual fluctuations in DNA methylation. Furthermore, in homozygous RNAi lines specifically targeting REPRESSOR OF SILENCING 1 (ROS1, a conserved DNA demethylase domain), promotion of DNA methylation significantly reduced fiber growth during August.     

Effects of High Levels of DNA Adenine Methylation on Methyl-Directed Mismatch Repair in ESCHERICHIA COLI

Two methods were used in an attempt to increase the efficiency and strand selectivity of methyl-directed mismatch repair of bacteriophage lambda heteroduplexes in E. coli. Previous studies of such repair used lambda DNA that was only partially methylated as the source of methylated chains. Also, transfection was carried out in methylating strains. Either of these factors might have been responsible for the incompleteness of the strand selectivity observed previously. In the first approach to increasing strand selectivity, heteroduplexes were transfected into a host deficient in methylation, but no changes in repair frequencies were observed. In the second approach, heteroduplexes were prepared using DNA that had been highly methylated in vitro with purified DNA adenine methylase as the source of methylated chains. In heteroduplexes having a repairable cI/+ mismatch, strand selectivity was indeed enhanced. In heteroduplexes with one chain highly methylated and the complementary chain unmethylated, the frequency of repair on the unmethylated chain increased to nearly 100%. Heteroduplexes with both chains highly methylated were not repaired at a detectable frequency. Thus, chains highly methylated by DNA adenine methylase were refractory to mismatch repair by this system, regardless of the methylation of the complementary chain. These results support the hypothesis that methyl-directed mismatch repair acts to correct errors of replication, thus lowering the mutation rate.     

马哈利樱桃矮化砧的DNA甲基化水平及模式分析

该研究利用MSAP技术,对25株矮化马哈利樱桃和25株半矮化马哈利樱桃进行甲基化水平和模式分析,以探讨其矮化的表观性状与其基因组甲基化修饰的关系。结果表明:(1)从64对引物中筛选出15对引物,在半矮化组中共扩增4 577个条带,其中半甲基化336个,全甲基化1 274个;在矮化组中共扩增4 444个条带,其中半甲基化349个,全甲基化1 383个;t检验和方差分析表明,矮化组与半矮化组在总甲基化水平和全甲基化水平上差异极显著,在半甲基化水平上差异显著,矮化组甲基化水平高于半矮化组。(2)半矮化组单态性位点23个,多态性位点136个;矮化组单态性位点17个,多态性位点142个,表明矮化组多态性高于半矮化组。(3)多态性类型分析表明,矮化组出现A4类型的频率较半矮化组高,A2类型的频率较半矮化组低,即矮化组中发生超甲基化的位点多于半矮化组,且‘马哈利’基因组甲基化多态性位点主要发生在双链内侧甲基化位点以及超甲基化位点上。研究认为,马哈利樱桃矮化和半矮化的基因组甲基化水平及模式存在差异,马哈利砧木的矮化性状与其基因组甲基化修饰有关。     

A Proof-of-Principle Demonstration of a Novel Microarray-Based Method for Quantifying DNA Methylation Levels

Demethylation of CD11a (ITGAL; GeneID:3683; HGNC: 6148) and CD70 (TNFSF7; GeneID:970; HGNC:11937) regulatory regions in CD4⁺ T cells contributes to the development of autoreactivity and autoantibody overstimulation in systemic lupus erythematosus (SLE). In this study, we present a novel approach for measuring the methylation status of CD11a and CD70 promoter sequences. The procedure combines the standard method of bisulfite conversion of methylated CpG pairs with high-throughput oligonucleotide microarray-based technology that allows for rapid quantification of deoxycytosine and deoxymethylcytosine content in bisulfite-treated DNA samples. The microarrays were first used to generate a standard curve from fully methylated and fully unmethylated DNA samples using a one-dimensional linear regression equation that calculated fluorescence emission as a function of methylation levels. The methylation status of the CD70 and CD11a promoters in SLE and control CD4⁺ T cell samples were measured, and the microarray prediction was found to be highly accurate when compared to bisulfite sequencing. Furthermore, the microarrays were able to detect differences in the methylation status between SLE patient and healthy control samples. These results indicate that our new microarray-based assay could prove to be a highly reliable, rapid, and cost effective diagnostic and prognostic test for SLE.