During meiosis, reciprocal exchange between homologous chromosomes occurs as a result of crossovers (COs). CO frequency varies within genomes and is subject to genetic, epigenetic and environmental control. As robust measurement of COs is limited by their low numbers, typically 1-2 per chromosome, we adapted flow cytometry for use with Arabidopsis transgenic fluorescent protein-tagged lines (FTLs) that express eCFP, dsRed or eYFP fluorescent proteins in pollen. Segregation of genetically linked transgenes encoding fluorescent proteins of distinct colors can be used to detect COs. The fluorescence of up to 80,000 pollen grains per individual plant can be measured in 10-15 min using this protocol. A key element of CO control is interference, which inhibits closely spaced COs. We describe a three-color assay for the measurement of CO frequency in adjacent intervals and calculation of CO interference. We show that this protocol can be used to detect changes in CO frequency and interference in the fancm zip4 double mutant. By enabling high-throughput measurement of CO frequency and interference, these methods will facilitate genetic dissection of meiotic recombination control.
GABA(A) receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die. As many anaesthetics act via GABA(A) receptors, the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients.
Immunization with Schistosoma mansoni soluble antigen preparations protects non-obese diabetic (NOD) mice against the development of type 1 diabetes. These preparations have long been known to induce Th2 responses in vitro and in vivo. Recently, two separate groups have reported that ?-1, a well-characterized glycoprotein in S. mansoni soluble egg antigens (SEA), which with IL-4 inducing principle of S. mansoni eggs (IPSE/?-1) is one of the two major glycoproteins secreted by live eggs, is a major SEA component responsible for this effect. We found that ?-1 induces Foxp3 as well as IL-4 expression when injected in vivo. We confirmed that ?-1 conditions DCs to drive Th2 responses and further demonstrated that ?-1 induces Foxp3(+) T cells from NOD mouse naïve T cells. In contrast, IPSE/?-1 did not drive Foxp3 responses. The in vitro development of Foxp3-expressing T cells by ?-1 was TGF-?- and retinoic acid-dependent. Our work, therefore, identifies ?-1 as an important factor for the induction of Foxp3(+) T cells by SEA in NOD mice.
Radix Angelica sinensis is a Chinese medicinal herb that has been used extensively in the East for the treatment of cardiovascular diseases (CVDs). Angiogenesis plays an important role in the pathogenesis of CVDs. We hypothesized that Radix A. sinensis may contain angiogenesis modulators. In the current study, we investigated the effects of a volatile oil of Radix A. sinensis (VOAS) and n-butylidenephthalide (BP), one of the bioactive components in VOAS, on angiogenesis in vitro and in vivo. The results suggested that VOAS exerted anti-angiogenic effects by inhibiting human umbilical vein endothelial cell proliferation, migration and capillary-like tube formation on Matrigel. BP was also shown to be anti-angiogenic and its mechanisms were through inhibition of cell cycle progression and induction of apoptosis. Western blotting analysis indicated that the anti-angiogenic actions of BP were associated with the activation of p38 and ERK 1/2 but not SAPK/JNK and Akt signaling pathways. Further investigations showed that BP inhibited endothelial sprouting in an ex vivo mouse aortic ring model and was a potent inhibitor of the development of zebrafish subintestinal vessels in vivo. Our data using the volatile oil contrast with previous findings, which showed an aqueous extract of Radix A. sinensis was pro-angiogenic. This highlights the importance of identifying pro- and anti-angiogenic substances in Radix A. sinensis, not only for the development of novel angiogenesis modulators for the treatment of CVDs, but also to ensure the proper use of Radix A. sinensis as a nutraceutical.
Genome-wide active DNA demethylation in primordial germ cells (PGCs), which reprograms the epigenome for totipotency, is linked to changes in nuclear architecture, loss of histone modifications, and widespread histone replacement. Here, we show that DNA demethylation in the mouse PGCs is mechanistically linked to the appearance of single-stranded DNA (ssDNA) breaks and the activation of the base excision repair (BER) pathway, as is the case in the zygote where the paternal pronucleus undergoes active DNA demethylation shortly after fertilization. Whereas BER might be triggered by deamination of a methylcytosine (5mC), cumulative evidence indicates other mechanisms in germ cells. We demonstrate that DNA repair through BER represents a core component of genome-wide DNA demethylation in vivo and provides a mechanistic link to the extensive chromatin remodeling in developing PGCs.
The immunomodulatory effect of Schistosoma mansoni antigens has often been attributed to interaction with PRR expressed on APC. Our previous work has shown that S. mansoni-soluble egg antigen (SEA) can induce, together with a Th2 response, TGF-?-dependent Foxp3 expression in naïve CD4(+) T cells from NOD mice. We found that SEA can directly upregulate the expression of surface-bound TGF-? in purified CD4(+) T cells in the absence of accessory cell interactions. In this study, we show that the C-type lectin receptors DEC-205 and galectin-3 were involved in the direct interaction between S. mansoni antigens and CD4(+) T cells. SEA was able to enhance CD4(+) T-cell secretion of bioactive TGF-? in response to TLR2 ligand stimulation, in the absence of APC. We also show that TLR2 expressed on CD4(+) T cells was important for the Foxp3 expression induced by SEA.
We have shown that Schistosoma mansoni egg soluble antigen (SEA) prevents diabetes in the nonobese diabetic (NOD) mouse inducing functional changes in antigen presenting cells (APCs) and expanding T helper (Th) 2 and regulatory T cell (Treg) responses. A Th2 response to S. mansoni infection or its antigens is key to both the establishment of tolerance and successfully reproduction in the host. More recently we demonstrated that SEA treatment upregulates bioactive TGFbeta on T cells with consequent expansion of Foxp3+ Tregs, and these cells might be important in SEA-mediated diabetes prevention together with Th2 cells. In this study we profile further the phenotypic changes that SEA induces on APCs, with particular attention to cytokine expression and markers of macrophage alternative activation. Our studies suggest that TGFbeta from T cells is important not just for Treg expansion but also for the successful Th2 response to SEA, and therefore, for diabetes prevention in the NOD mouse.
Schistosoma mansoni soluble egg antigens (SEA) profoundly regulate the infected hosts immune system. We previously showed that SEA prevents type 1 diabetes in NOD mice and that splenocytes from SEA-treated mice have reduced ability to transfer diabetes to NOD.scid recipients. To further characterize the mechanism of diabetes prevention we examined the cell types involved and showed that CD25(+) T-cell depletion of splenocytes from SEA-treated donors restored their ability to transfer diabetes. Furthermore, SEA treatment increased the number and proportional representation of Foxp3(+) T cells in the pancreas of NOD mice. We have used in vitro systems to analyze the effect of SEA on the development of NOD Foxp3(+) T cells. We find that SEA can induce Foxp3 expression in naïve T cells in a TGF-beta-dependent manner. Foxp3 induction requires the presence of DC, which we also show are modified by SEA to upregulate C-type lectins, IL-10 and IL-2. Our studies show that SEA can have a direct effect on CD4(+) T cells increasing expression of TGF-beta, integrin beta8 and galectins. These effects of SEA on DC and T cells may act in synergy to induce Foxp3(+) Treg in the NOD mouse.
Meiosis is a specialized eukaryotic cell division that generates haploid gametes required for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover (CO). Meiotic CO frequency varies along the physical length of chromosomes and is determined by hierarchical mechanisms, including epigenetic organization, for example methylation of the DNA and histones. Here we investigate the role of DNA methylation in determining patterns of CO frequency along Arabidopsis thaliana chromosomes. In A. thaliana the pericentromeric regions are repetitive, densely DNA methylated, and suppressed for both RNA polymerase-II transcription and CO frequency. DNA hypomethylated methyltransferase1 (met1) mutants show transcriptional reactivation of repetitive sequences in the pericentromeres, which we demonstrate is coupled to extensive remodeling of CO frequency. We observe elevated centromere-proximal COs in met1, coincident with pericentromeric decreases and distal increases. Importantly, total numbers of CO events are similar between wild type and met1, suggesting a role for interference and homeostasis in CO remodeling. To understand recombination distributions at a finer scale we generated CO frequency maps close to the telomere of chromosome 3 in wild type and demonstrate an elevated recombination topology in met1. Using a pollen-typing strategy we have identified an intergenic nucleosome-free CO hotspot 3a, and we demonstrate that it undergoes increased recombination activity in met1. We hypothesize that modulation of 3a activity is caused by CO remodeling driven by elevated centromeric COs. These data demonstrate how regional epigenetic organization can pattern recombination frequency along eukaryotic chromosomes.
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