共查询到20条相似文献,搜索用时 31 毫秒
1.
2.
Strigolactones' ability to regulate root development may be executed by induction of the ethylene pathway 总被引:1,自引:0,他引:1
Hinanit Koltai 《Plant signaling & behavior》2011,6(7):1004-1005
The newly defined phytohormones strigolactones (SLs) were recently shown to act as regulators of root development. Their positive effect on root-hair (RH) elongation enabled examination of their cross talk with auxin and ethylene. Analysis of wild-type plants and hormone-signaling mutants combined with hormonal treatments suggested that SLs and ethylene regulate RH elongation via a common regulatory pathway, in which ethylene is epistatic to SLs. The SL and auxin hormonal pathways were suggested to converge for regulation of RH elongation; this convergence was suggested to be mediated via the ethylene pathway, and to include regulation of auxin transport.Key words: strigolactone, auxin, ethylene, root, root hair, lateral rootStrigolactones (SLs) are newly identified phytohormones that act as long-distance shoot-branching inhibitors (reviewed in ref. 1). In Arabidopsis, SLs have been shown to be regulators of root development and architecture, by modulating primary root elongation and lateral root formation.2,3 In addition, they were shown to have a positive effect on root-hair (RH) elongation.2 All of these effects are mediated via the MAX2 F-box.2,3In addition to SLs, two other plant hormones, auxin and ethylene, have been shown to affect root development, including lateral root formation and RH elongation.4–6 Since all three phytohormones (SLs, auxin and ethylene) were shown to have a positive effect on RH elongation, we examined the epistatic relations between them by examining RH length.7 Our results led to the conclusion that SLs and ethylene are in the same pathway regulating RH elongation, where ethylene may be epistatic to SLs.7 Moreover, auxin signaling was shown to be needed to some extent for the RH response to SLs: the auxin-insensitive mutant tir1-1,8 was less sensitive to SLs than the wild type under low SL concentrations.7On the one hand, ethylene has been shown to induce the auxin response,9–12 auxin synthesis in the root apex,11,12 and acropetal and basipetal auxin transport in the root.4,13 On the other, ethylene has been shown to be epistatic to SLs in the SL-induced RH-elongation response.7 Therefore, it might be that at least for RH elongation, SLs are in direct cross talk with ethylene, whereas the cross talk between SL and auxin pathways may converge through that of ethylene.7 The reduced response to SLs in tir1-1 may be derived from its reduced ethylene sensitivity;7,14 this is in line with the notion of the ethylene pathway being a mediator in the cross talk between the SL and auxin pathways.The suggested ethylene-mediated convergence of auxin and SLs may be extended also to lateral root formation, and may involve regulation of auxin transport. In the root, SLs have been suggested to affect auxin efflux,3,15 whereas ethylene has been shown to have a positive effect on auxin transport.4,13 Hence, it might be that in the root, the SLs'' effect on auxin flux is mediated, at least in part, via the ethylene pathway. Ethylene''s ability to increase auxin transport in roots was associated with its negative effect on lateral root formation: ethylene was suggested to enhance polar IAA transport, leading to alterations in the quantity of auxin that unloads into the tissues to drive lateral root formation.4 Under conditions of sufficient phosphate, SL''s effect was similar to that of ethylene: SLs reduced the appearance of lateral roots; this was explained by their ability to change auxin flux.3 Taken together, one possibility is that the SLs'' ability to affect auxin flux and thereby lateral root formation in the roots is mediated by induction of ethylene synthesis.To conclude, root development may be regulated by a network of auxin, SL and ethylene cross talk.7 The possibility that similar networks exist elsewhere in the SLs'' regulation of plant development, including shoot architecture, cannot be excluded. 相似文献
3.
4.
5.
6.
VERNALIZATION INSENSITIVE 3 (VIN3) encodes a PHD domain chromatin remodelling protein that is induced in response to cold and is required for the establishment of the vernalization response in Arabidopsis thaliana.1 Vernalization is the acquisition of the competence to flower after exposure to prolonged low temperatures, which in Arabidopsis is associated with the epigenetic repression of the floral repressor FLOWERING LOCUS C (FLC).2,3 During vernalization VIN3 binds to the chromatin of the FLC locus,1 and interacts with conserved components of Polycomb-group Repressive Complex 2 (PRC2).4,5 This complex catalyses the tri-methylation of histone H3 lysine 27 (H3K27me3),4,6,7 a repressive chromatin mark that increases at the FLC locus as a result of vernalization.4,7–10 In our recent paper11 we found that VIN3 is also induced by hypoxic conditions, and as is the case with low temperatures, induction occurs in a quantitative manner. Our experiments indicated that VIN3 is required for the survival of Arabidopsis seedlings exposed to low oxygen conditions. We suggested that the function of VIN3 during low oxygen conditions is likely to involve the mediation of chromatin modifications at certain loci that help the survival of Arabidopsis in response to prolonged hypoxia. Here we discuss the implications of our observations and hypotheses in terms of epigenetic mechanisms controlling gene regulation in response to hypoxia.Key words: arabidopsis, VIN3, FLC, hypoxia, vernalization, chromatin remodelling, survival 相似文献
7.
8.
9.
The pattern of salicylic acid (SA)-induced production of reactive oxygen species (ROS) and nitric oxide (NO) were different in the apex of adventitious roots in wild-type and in the ethylene-insensitive Never ripe (Nr) mutants of tomato (Solanum lycopersicum L. cv Ailsa Craig). ROS were upregulated, while NO remained at the control level in apical root tissues of wildtype plants exposed to sublethal concentrations of SA. In contrast, Nr plants expressing a defective ethylene receptor displayed a reduced level of ROS and a higher NO content in the apical root cells. In wild-type plants NO production seems to be ROS(H2O2)-dependent at cell death-inducing concentrations of SA, indicating that ROS and NO may interact to trigger oxidative cell death. In the absence of significant ROS accumulation, the increased NO production caused moderate reduction in cell viability in root apex of Nr plants exposed to 10−3 M SA. This suggests that a functional ethylene signaling pathway is necessary for the control of ROS and NO production induced by SA.Key words: ethylene receptor mutant, never ripe, nitric oxide, reactive oxygen species, root apex, salicylic acid, tomatoSeveral signal molecules, including salicylic acid (SA) have been implicated in the response of plants to biotic1–3 and abiotic stressors.4–6 SA was identified as a central regulator of local defense against (hemi)biotophic pathogens inducing a hypersensitive response (HR), which is characterized by the development of lesions that restrict pathogen spread. It has also emerged as a possible signaling component involved in the activation of certain plant defense responses in non-infected part of the plants establishing the systemic acquired resistance (SAR).7The SA-induced biotic and abiotic stress adaptation most likely involves reactive oxygen species (ROS) and nitric oxide (NO) in primary signaling events that activate multiple signal transduction pathways. SA-induced ROS is required for the activation of antioxidant defense mechanisms4 and if the generation of ROS exceeds the capacity of antioxidant systems, the cells die.8 NO is another important player that is required for the induction of defense mechanisms9 or for ROS-induced cell death.10Accumulation of SA, and two other plant hormones, ethylene (ET) and jasmonic acid (JA) are intimately associated with the initiation or spread of cell death. In HR SA and ROS have been proposed to be on a positive feedback loop that amplifies signals and leads to programmed cell death (PCD). Ethylene caused increased spreading of cell death, while lesion containment can be achieved by JA through decreasing the sensitivity of the cells to ethylene and through the suppression of SA biosynthesis and signaling.8Ethylene evolution is associated with diverse physiological processes such as leaf and flower senescence, abscission of organs and fruit ripening.11 The biosynthesis of ethylene is stimulated by a variety of abiotic and biotic stress factors. Ethylene overproducing mutants (eto1 and eto3) of Arabidopsis were found to be more sensitive to O3, an abiotic stressor which induces ROS-dependent cell death.12 Cadmium-induced cell death was also accompanied by increased production of ethylene and simultaneously by H2O2 accumulation in tomato cell suspension, and based on the effect of specific inhibitors of ethylene biosynthesis and action the authors concluded that the cell death process required H2O2 production and a functional ethylene signaling pathway.13 Ethylene signaling is also required for the susceptible disease response of tomato plants infected with Xanthomonas campestris pv vesicatoria.14 It was found that the accumulation of SA and increased production of ethylene were important components of the disease symptoms of this pathogen in wild-type plants, while in Never ripe (Nr) mutants, which have a non-functional ethylene receptor, the infected plants failed to accumulate SA, produced less ethylene, and the leaves exhibited reduced necrotic lesions.It has been also shown that SA enhances NO synthesis in a dose-dependent manner.15 ROS, such as ·O2− and H2O2 as well as NO can act together in the cell death regulation and propagation.8,16 The compartment-specific (down)regulation of ROS can be controlled by NO, accordingly, ROS and NO homeostasis may be essential for the induction or for the avoidance of cell death. 相似文献
10.
In young Arabidopsis seedlings, retrograde signaling from plastids regulates the expression of photosynthesis-associated nuclear genes in response to the developmental and functional state of the chloroplasts. The chloroplast-located PPR protein GUN1 is required for signalling following disruption of plastid protein synthesis early in seedling development before full photosynthetic competence has been achieved. Recently we showed that sucrose repression and the correct temporal expression of LHCB1, encoding a light-harvesting chlorophyll protein associated with photosystem II, are perturbed in gun1 mutant seedlings.1 Additionally, we demonstrated that in gun1 seedlings anthocyanin accumulation and the expression of the “early” anthocyanin-biosynthesis genes is perturbed. Early seedling development, predominantly at the stage of hypocotyl elongation and cotyledon expansion, is also affected in gun1 seedlings in response to sucrose, ABA and disruption of plastid protein synthesis by lincomycin. These findings indicate a central role for GUN1 in plastid, sucrose and ABA signalling in early seedling development.Key words: ABA, ABI4, anthocyanin, chloroplast, GUN1, retrograde signalling, sucroseArabidopsis seedlings develop in response to light and other environmental cues. In young seedlings, development is fuelled by mobilization of lipid reserves until chloroplast biogenesis is complete and the seedlings can make the transition to phototrophic growth. The majority of proteins with functions related to photosynthesis are encoded by the nuclear genome, and their expression is coordinated with the expression of genes in the chloroplast genome. In developing seedlings, retrograde signaling from chloroplasts to the nucleus regulates the expression of these nuclear genes and is dependent on the developmental and functional status of the chloroplast. Two classes of gun (genomes uncoupled) mutants defective in retrograde signalling have been identified in Arabidopsis: the first, which comprises gun2–gun5, involves mutations in genes encoding components of tetrapyrrole biosynthesis.2,3 The other comprises gun1, which has mutations in a nuclear gene encoding a plastid-located pentatricopeptide repeat (PPR) protein with an SMR (small MutS-related) domain near the C-terminus.4,5 PPR proteins are known to have roles in RNA processing6 and the SMR domain of GUN1 has been shown to bind DNA,4 but the specific functions of these domains in GUN1 are not yet established. However, GUN1 has been shown to be involved in plastid gene expression-dependent,7 redox,4 ABA1,4 and sucrose signaling,1,4,8 as well as light quality and intensity sensing pathways.9–11 In addition, GUN1 has been shown to influence anthocyanin biosynthesis, hypocotyl extension and cotyledon expansion.1,11 相似文献
11.
12.
Hyun-Myung Oh Ilnam Kang Steve Ferriera Stephen J. Giovannoni Jang-Cheon Cho 《Journal of bacteriology》2010,192(18):4796-4797
Here we announce the complete genome sequence of Croceibacter atlanticus HTCC2559T, which was isolated by high-throughput dilution-to-extinction culturing from the Bermuda Atlantic Time Series station in the Western Sargasso Sea. Strain HTCC2559T contained genes for carotenoid biosynthesis, flavonoid biosynthesis, and several macromolecule-degrading enzymes. The genome confirmed physiological observations of cultivated Croceibacter atlanticus strain HTCC2559T, which identified it as an obligate chemoheterotroph.The phylum Bacteroidetes comprises 6 to ∼30% of total bacterial communities in the ocean by fluorescence in situ hybridization (8-10). Most marine Bacteroidetes are in the family Flavobacteriaceae, most of which are aerobic respiratory heterotrophs that form a well-defined clade by 16S rRNA phylogenetic analyses (4). The members of this family are well known for degrading macromolecules, including chitin, DNA, cellulose, starch, and pectin (17), suggesting their environmental roles as detritus decomposers in the ocean (6). Marine Polaribacter and Dokdonia species in the Flavobacteriaceae have also shown to have photoheterotrophic metabolism mediated by proteorhodopsins (11, 12).Several strains of the family Flavobacteriaceae were isolated from the Sargasso Sea and Oregon coast, using high-throughput culturing approaches (7). Croceibacter atlanticus HTCC2559T was cultivated from seawater collected at a depth of 250 m from the Sargasso Sea and was identified as a new genus in the family Flavobacteriaceae based on its 16S rRNA gene sequence similarities (6). Strain HTCC2559T met the minimal standards for genera of the family Flavobacteriaceae (3) on the basis of phenotypic characteristics (6).Here we report the complete genome sequence of Croceibacter atlanticus HTCC2559T. The genome sequencing was initiated by the J. Craig Venter Institute as a part of the Moore Foundation Microbial Genome Sequencing Project and completed in the current announcement. Gaps among contigs were closed by Genotech Co., Ltd. (Daejeon, Korea), using direct sequencing of combinatorial PCR products (16). The HTCC2559T genome was analyzed with a genome annotation system based on GenDB (14) at Oregon State University and with the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (15, 16).The HTCC2559T genome is 2,952,962 bp long, with 33.9 mol% G+C content, and there was no evidence of plasmids. The number of protein-coding genes was 2,715; there were two copies of the 16S-23S-5S rRNA operon and 36 tRNA genes. The HTCC2559T genome contained genes for a complete tricarboxylic acid cycle, glycolysis, and a pentose phosphate pathway. The genome also contained sets of genes for metabolic enzymes involved in carotenoid biosynthesis and also a serine/glycine hydroxymethyltransferase, which is often associated with the assimilatory serine cycle (13). The potential for HTCC2559T to use bacterial type III polyketide synthase (PKS) needs to be confirmed because this organism had a naringenin-chalcone synthase (CHS) or chalcone synthase (EC 2.3.1.74), a key enzyme in flavonoid biosynthesis. CHS initiates the addition of three molecules of malonyl coenzyme A (malonyl-CoA) to a starter CoA ester (e.g., 4-coumaroyl-CoA) (1) and takes part in a few bacterial type III polyketide synthase systems (1, 2, 5, 18).The complete genome sequence confirmed that strain HTCC2559T is an obligate chemoheterotroph because no genes for phototrophy were found. As expected from physiological characteristics (6), the HTCC2559T genome contained a set of genes coding for enzymes required to degrade high-molecular-weight compounds, including peptidases, metallo-/serine proteases, pectinase, alginate lyases, and α-amylase. 相似文献
13.
14.
15.
16.
Olga V Dyachenko Taras V Shevchuk Leo Kretzner Yaroslav I Buryanov Steven S Smith 《Epigenetics》2010,5(7):569-572
Non-CG methylation is well characterized in plants where it appears to play a role in gene silencing and genomic imprinting. Although strong evidence for the presence of non-CG methylation in mammals has been available for some time, both its origin and function remain elusive. In this review we discuss available evidence on non-CG methylation in mammals in light of evidence suggesting that the human stem cell methylome contains significant levels of methylation outside the CG site.Key words: non-CG methylation, stem cells, Dnmt1, Dnmt3a, human methylomeIn plant cells non-CG sites are methylated de novo by Chromomethylase 3, DRM1 and DRM2. Chromomethylase 3, along with DRM1 and DRM2 combine in the maintenance of methylation at symmetric CpHpG as well as asymmetric DNA sites where they appear to prevent reactivation of transposons.1 DRM1 and DRM2 modify DNA de novo primarily at asymmetric CpH and CpHpH sequences targeted by siRNA.2Much less information is available on non-CG methylation in mammals. In fact, studies on mammalian non-CG methylation form a tiny fraction of those on CG methylation, even though data for cytosine methylation in other dinucleotides, CA, CT and CC, have been available since the late 1980s.3 Strong evidence for non-CG methylation was found by examining either exogenous DNA sequences, such as plasmid and viral integrants in mouse and human cell lines,4,5 or transposons and repetitive sequences such as the human L1 retrotransposon6 in a human embryonic fibroblast cell line. In the latter study, non-CG methylation observed in L1 was found to be consistent with the capacity of Dnmt1 to methylate slippage intermediates de novo.6Non-CG methylation has also been reported at origins of replication7,8 and a region of the human myogenic gene Myf3.9 The Myf3 gene is silenced in non-muscle cell lines but it is not methylated at CGs. Instead, it carries several methylated cytosines within the sequence CCTGG. Gene-specific non-CG methylation was also reported in a study of lymphoma and myeloma cell lines not expressing many B lineage-specific genes.10 The study focused on one specific gene, B29 and found heavy CG promoter methylation of that gene in most cell lines not expressing it. However, in two other cell lines where the gene was silenced, cytosine methylation was found almost exclusively at CCWGG sites. The authors provided evidence suggesting that CCWGG methylation was sufficient for silencing the B29 promoter and that methylated probes based on B29 sequences had unique gel shift patterns compared to non-methylated but otherwise identical sequences.10 The latter finding suggests that the presence of the non-CG methylation causes changes in the proteins able to bind the promoter, which could be mechanistically related to the silencing seen with this alternate methylation.Non-CG methylation is rarely seen in DNA isolated from cancer patients. However, the p16 promoter region was reported to contain both CG and non-CG methylation in breast tumor specimens but lacked methylation at these sites in normal breast tissue obtained at mammoplasty.11 Moreover, CWG methylation at the CCWGG sites in the calcitonin gene is not found in normal or leukemic lymphocyte DNA obtained from patients.12 Further, in DNA obtained from breast cancer patients, MspI sites that are refractory to digestion by MspI and thus candidates for CHG methylation were found to carry CpG methylation.13 Their resistance to MspI restriction was found to be caused by an unusual secondary structure in the DNA spanning the MspI site that prevents restriction.13 This latter observation suggests caution in interpreting EcoRII/BstNI or EcoRII/BstOI restriction differences as due to CWG methylation, since in contrast to the 37°C incubation temperature required for full EcoRII activity, BstNI and BstOI require incubation at 60°C for full activity where many secondary structures are unstable.The recent report by Lister et al.14 confirmed a much earlier report by Ramsahoye et al.15 suggesting that non-CG methylation is prevalent in mammalian stem cell lines. Nearest neighbor analysis was used to detect non-CG methylation in the earlier study on the mouse embryonic stem (ES) cell line,15 thus global methylation patterning was assessed. Lister et al.14 extend these findings to human stem cell lines at single-base resolution with whole-genome bisulfite sequencing. They report14 that the methylome of the human H1 stem cell line and the methylome of the induced pluripotent IMR90 (iPS) cell line are stippled with non-CG methylation while that of the human IMR90 fetal fibroblast cell line is not. While the results of the two studies are complementary, the human methylome study addresses locus specific non-CG methylation. Based on that data,14 one must conclude that non-CG methylation is not carefully maintained at a given site in the human H1 cell line. The average non-CG site is picked up as methylated in about 25% of the reads whereas the average CG methylation site is picked up in 92% of the reads. Moreover, non-CG methylation is not generally present on both strands and is concentrated in the body of actively transcribed genes.14Even so, the consistent finding that non-CG methylation appears to be confined to stem cell lines,14,15 raises the possibility that cancer stem cells16 carry non-CG methylation while their nonstem progeny in the tumor carry only CG methylation. Given the expected paucity of cancer stem cells in a tumor cell population, it is unlikely that bisulfite sequencing would detect non-CG methylation in DNA isolated from tumor cells since the stem cell population is expected to be only a very minor component of tumor DNA. Published sequences obtained by bisulfite sequencing generally report only CG methylation, and to the best of our knowledge bisulfite sequenced tumor DNA specimens have not reported non-CG methylation. On the other hand, when sequences from cell lines have been reported, bisulfite-mediated genomic sequencing8 or ligation mediated PCR17 methylcytosine signals outside the CG site have been observed. In a more recent study plasmid DNAs carrying the Bcl2-major breakpoint cluster18 or human breast cancer DNA13 treated with bisulfite under non-denaturing conditions, cytosines outside the CG side were only partially converted on only one strand18 or at a symmetrical CWG site.13 In the breast cancer DNA study the apparent CWG methylation was not detected when the DNA was fully denatured before bisulfite treatment.13In both stem cell studies, non-CG methylation was attributed to the Dnmt3a,14,15 a DNA methyltransferase with similarities to the plant DRM methyltransferase family19 and having the capacity to methylate non-CG sites when expressed in Drosophila melanogaster.15 DRM proteins however, possess a unique permuted domain structure found exclusively in plants19 and the associated RNA-directed non-CG DNA methylation has not been reproducibly observed in mammals despite considerable published20–23 and unpublished efforts in that area. Moreover, reports where methylation was studied often infer methylation changes from 5AzaC reactivation studies24 or find that CG methylation seen in plants but not non-CG methylation is detected.21,22,25,26 In this regard, it is of interest that the level of non-CG methylation reported in stem cells corresponds to background non-CG methylation observed in vitro with human DNA methyltransferase I,27 and is consistent with the recent report that cultured stem cells are epigenetically unstable.28The function of non-CG methylation remains elusive. A role in gene expression has not been ruled out, as the studies above on Myf3 and B29 suggest.9,10 However, transgene expression of the bacterial methyltransferase M.EcoRII in a human cell line (HK293), did not affect the CG methylation state at the APC and SerpinB5 genes29 even though the promoters were symmetrically de novo methylated at mCWGs within each CCWGG sequence in each promoter. This demonstrated that CG and non-CG methylation are not mutually exclusive as had been suggested by earlier reports.9,10 That observation is now extended to the human stem cell line methylome where CG and non-CG methylation co-exist.14 Gene expression at the APC locus was likewise unaffected by transgene expression of M.EcoRII. In those experiments genome wide methylation of the CCWGG site was detected by restriction analysis and bisulfite sequencing,29 however stem cell characteristics were not studied.Many alternative functions can be envisioned for non-CG methylation, but the existing data now constrains them to functions that involve low levels of methylation that are primarily asymmetric. Moreover, inheritance of such methylation patterns requires low fidelity methylation. If methylation were maintained with high fidelity at particular CHG sites one would expect that the spontaneous deamination of 5-methylcytosine would diminish the number of such sites, so as to confine the remaining sites to those positions performing an essential function, as is seen in CG methylation.30–33 However, depletion of CWG sites is not observed in the human genome.34 Since CWG sites account for only about 50% of the non-CG methylation observed in the stem cell methylome14 where methylated non-CG sites carry only about 25% methylation, the probability of deamination would be about 13% of that for CWG sites that are subject to maintenance methylation in the germ line. Since mutational depletion of methylated cytosines has to have its primary effect on the germ line, if the maintenance of non-CG methylation were more accurate and more widespread, one would have had to argue that stem cells in the human germ lines lack CWG methylation. As it is the data suggests that whatever function non-CG methylation may have in stem cells, it does not involve accurate somatic inheritance in the germ line.The extensive detail on non-CG methylation in the H1 methylome14 raises interesting questions about the nature of this form of methylation in human cell lines. A key finding in this report is the contrast between the presence of non-CG methylation in the H1 stem cell line and its absence in the IMR90 human fetal lung fibroblast cell line.14 This suggests that it may have a role in the origin and maintenance of the pluripotent lineage.14By analogy with the well known methylated DNA binding proteins specific for CG methylation,35 methylated DNA binding proteins that selectively bind sites of non-CG methylation are expected to exist in stem cells. Currently the only protein reported to have this binding specificity is human Dnmt1.36–38 While Dnmt1 has been proposed to function stoichiometrically39 and could serve a non-CG binding role in stem cells, this possibility and the possibility that other stem-cell specific non-CG binding proteins might exist remain to be been explored.Finally, the nature of the non-CG methylation patterns in human stem cell lines present potentially difficult technical problems in methylation analysis. First, based on the data in the H1 stem cell methylome,40 a standard MS-qPCR for non-CG methylation would be impractical because non-CG sites are infrequent, rarely clustered and are generally characterized by partial asymmetric methylation. This means that a PCR primer that senses the 3 adjacent methylation sites usually recommended for MS-qPCR primer design41,42 cannot be reliably found. For example in the region near Oct4 (Chr6:31,246,431), a potential MS-qPCR site exists with a suboptimal set of two adjacent CHG sites both methylated on the + strand at Chr6:31,252,225 and 31,252,237.14,40 However these sites were methylated only in 13/45 and 30/52 reads. Thus the probability that they would both be methylated on the same strand is about 17%. Moreover, reverse primer locations containing non-CG methylation sites are generally too far away for practical bisulfite mediated PCR. Considering the losses associated with bisulfite mediated PCR43 the likelihood that such an MS-qPCR system would detect non-CG methylation in the H1 cell line or stem cells present in a cancer stem cell niche44,45 is very low.The second difficulty is that methods based on the specificity of MeCP2 and similar methylated DNA binding proteins for enriching methylated DNA (e.g., MIRA,46 COMPARE-MS47) will discard sequences containing non-CG methylation since they require cooperative binding afforded by runs of adjacent methylated CG sites for DNA capture. This latter property of the methylated cytosine capture techniques makes it also unlikely that methods based on 5-methylcytosine antibodies (e.g., meDIP48) will capture non-CG methylation patterns accurately since the stem cell methylome shows that adjacent methylated non-CG sites are rare in comparison to methylated CG sites.14In summary, whether or not mammalian stem cells in general or human stem cells in particular possess functional plant-like methylation patterns is likely to continue to be an interesting and challenging question. At this point we can conclude that the non-CG patterns reported in human cells appear to differ significantly from the non-CG patterns seen in plants, suggesting that they do not have a common origin or function. 相似文献
17.
Stephen J Demarest Mangala Hariharan Marikka Elia Jared Salbato Ping Jin Colin Bird Jay M Short Bruce E Kimmel Michael Dudley Gary Woodnutt Geneviève Hansen 《MABS-AUSTIN》2010,2(2):190-198
The pathogenicity of Clostridium difficile (C. difficile) is mediated by the release of two toxins, A and B. Both toxins contain large clusters of repeats known as cell wall binding (CWB) domains responsible for binding epithelial cell surfaces. Several murine monoclonal antibodies were generated against the CWB domain of toxin A and screened for their ability to neutralize the toxin individually and in combination. Three antibodies capable of neutralizing toxin A all recognized multiple sites on toxin A, suggesting that the extent of surface coverage may contribute to neutralization. Combination of two noncompeting antibodies, denoted 3358 and 3359, enhanced toxin A neutralization over saturating levels of single antibodies. Antibody 3358 increased the level of detectable CWB domain on the surface of cells, while 3359 inhibited CWB domain cell surface association. These results suggest that antibody combinations that cover a broader epitope space on the CWB repeat domains of toxin A (and potentially toxin B) and utilize multiple mechanisms to reduce toxin internalization may provide enhanced protection against C. difficile-associated diarrhea.Key words: Clostridium difficile, toxin neutralization, therapeutic antibody, cell wall binding domains, repeat proteins, CROPs, mAb combinationThe most common cause of nosocomial antibiotic-associated diarrhea is the gram-positive, spore-forming anaerobic bacillus Clostridium difficile (C. difficile). Infection can be asymptomatic or lead to acute diarrhea, colitis, and in severe instances, pseudomembranous colitis and toxic megacolon.1,2The pathological effects of C. difficile have long been linked to two secreted toxins, A and B.3,4 Some strains, particularly the virulent and antibiotic-resistant strain 027 with toxinotype III, also produce a binary toxin whose significance in the pathogenicity and severity of disease is still unclear.5 Early studies including in vitro cell-killing assays and ex vivo models indicated that toxin A is more toxigenic than toxin B; however, recent gene manipulation studies and the emergence of virulent C. difficile strains that do not express significant levels of toxin A (termed “A− B+”) suggest a critical role for toxin B in pathogenicity.6,7Toxins A and B are large multidomain proteins with high homology to one another. The N-terminal region of both toxins enzymatically glucosylates small GTP binding proteins including Rho, Rac and CDC42,8,9 leading to altered actin expression and the disruption of cytoskeletal integrity.9,10 The C-terminal region of both toxins is composed of 20 to 30 residue repeats known as the clostridial repetitive oligopeptides (CROPs) or cell wall binding (CWB) domains due to their homology to the repeats of Streptococcus pneumoniae LytA,11–14 and is responsible for cell surface recognition and endocytosis.12,15–17C. difficile-associated diarrhea is often, but not always, induced by antibiotic clearance of the normal intestinal flora followed by mucosal C. difficile colonization resulting from preexisting antibiotic resistant C. difficile or concomitant exposure to C. difficile spores, particularly in hospitals. Treatments for C. difficile include administration of metronidazole or vancomycin.2,18 These agents are effective; however, approximately 20% of patients relapse. Resistance of C. difficile to these antibiotics is also an emerging issue19,20 and various non-antibiotic treatments are under investigation.20–25Because hospital patients who contract C. difficile and remain asymptomatic have generally mounted strong antibody responses to the toxins,26,27 active or passive immunization approaches are considered hopeful avenues of treatment for the disease. Toxins A and B have been the primary targets for immunization approaches.20,28–33 Polyclonal antibodies against toxins A and B, particularly those that recognize the CWB domains, have been shown to effectively neutralize the toxins and inhibit morbidity in rodent infection models.31 Monoclonal antibodies (mAbs) against the CWB domains of the toxins have also demonstrated neutralizing capabilities; however, their activity in cell-based assays is significantly weaker than that observed for polyclonal antibody mixtures.33–36We investigated the possibility of creating a cocktail of two or more neutralizing mAbs that target the CWB domain of toxin A with the goal of synthetically re-creating the superior neutralization properties of polyclonal antibody mixtures. Using the entire CWB domain of toxin A, antibodies were raised in rodents and screened for their ability to neutralize toxin A in a cell-based assay. Two mAbs, 3358 and 3359, that (1) both independently demonstrated marginal neutralization behavior and (2) did not cross-block one another from binding toxin A were identified. We report here that 3358 and 3359 use differing mechanisms to modify CWB-domain association with CHO cell surfaces and combine favorably to reduce toxin A-mediated cell lysis. 相似文献
18.
Fetal cells migrate into the mother during pregnancy. Fetomaternal transfer probably occurs in all pregnancies and in humans the fetal cells can persist for decades. Microchimeric fetal cells are found in various maternal tissues and organs including blood, bone marrow, skin and liver. In mice, fetal cells have also been found in the brain. The fetal cells also appear to target sites of injury. Fetomaternal microchimerism may have important implications for the immune status of women, influencing autoimmunity and tolerance to transplants. Further understanding of the ability of fetal cells to cross both the placental and blood-brain barriers, to migrate into diverse tissues, and to differentiate into multiple cell types may also advance strategies for intravenous transplantation of stem cells for cytotherapeutic repair. Here we discuss hypotheses for how fetal cells cross the placental and blood-brain barriers and the persistence and distribution of fetal cells in the mother.Key Words: fetomaternal microchimerism, stem cells, progenitor cells, placental barrier, blood-brain barrier, adhesion, migrationMicrochimerism is the presence of a small population of genetically distinct and separately derived cells within an individual. This commonly occurs following transfusion or transplantation.1–3 Microchimerism can also occur between mother and fetus. Small numbers of cells traffic across the placenta during pregnancy. This exchange occurs both from the fetus to the mother (fetomaternal)4–7 and from the mother to the fetus.8–10 Similar exchange may also occur between monochorionic twins in utero.11–13 There is increasing evidence that fetomaternal microchimerism persists lifelong in many child-bearing women.7,14 The significance of fetomaternal microchimerism remains unclear. It could be that fetomaternal microchimerism is an epiphenomenon of pregnancy. Alternatively, it could be a mechanism by which the fetus ensures maternal fitness in order to enhance its own chances of survival. In either case, the occurrence of pregnancy-acquired microchimerism in women may have implications for graft survival and autoimmunity. More detailed understanding of the biology of microchimeric fetal cells may also advance progress towards cytotherapeutic repair via intravenous transplantation of stem or progenitor cells.Trophoblasts were the first zygote-derived cell type found to cross into the mother. In 1893, Schmorl reported the appearance of trophoblasts in the maternal pulmonary vasculature.15 Later, trophoblasts were also observed in the maternal circulation.16–20 Subsequently various other fetal cell types derived from fetal blood were also found in the maternal circulation.21,22 These fetal cell types included lymphocytes,23 erythroblasts or nucleated red blood cells,24,25 haematopoietic progenitors7,26,27 and putative mesenchymal progenitors.14,28 While it has been suggested that small numbers of fetal cells traffic across the placenta in every human pregnancy,29–31 trophoblast release does not appear to occur in all pregnancies.32 Likewise, in mice, fetal cells have also been reported in maternal blood.33,34 In the mouse, fetomaternal transfer also appears to occur during all pregnancies.35 相似文献
19.
20.
A role for SR proteins in plant stress responses 总被引:1,自引:0,他引:1
Paula Duque 《Plant signaling & behavior》2011,6(1):49-54