Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species

The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle.As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90⁺ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres.The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.Key words: CD90, mesenchymal stem cells, hair follicle, dog.The hair follicle represents an important stem cell niche in the skin. It contains dermal and epithelial stem populations that display distinct properties and localization. While epithelial stem cells reside in the middle region of the hair follicle outer root sheath (Schneider et al., 2009; Lyle et al., 1998; Cotsarelis et al., 1990), dermal stem cells are located in the dermal sheath (DS) (Jahoda, 2003; Jahoda and Reynolds, 2001).The dermal sheath, or fibrous root sheath, is a layer of dense connective tissue that extends along the hair follicle, from the bulb up to the infundibulum. In the anagen hair follicle, it is comprised of mesenchymal cells located among collagen and elastic fibres.The cells are flat and elongated while collagen fibres form a circular inner layer and a longitudinal outer layer in the lower part of hair follicle (VonTscharner and Suter, 1994; Jahoda et al., 1992). At the base of the hair follicle, the DS is connected to the dermal papilla (Scott et al., 2000). The basement membrane, or glassy membrane, separates the DS from the epithelial component of the hair follicle (Scott et al., 2000).Follicular dermal stem cells have a mesenchymal origin and share many properties common to bone marrow-derived mesenchymal stem cells (MSCs) (Hoogduijn et al., 2006). They express the MSC cell-surface marker CD90, show a high colony forming unit ability and can differentiate into several mesenchymal lineages, such as osteoblasts, adipocytes, chondrocytes and myocytes (Hoogduijn et al., 2006; Jahoda et al., 2003). They also express neuroprogenitor markers (Hoogduijn et al., 2006) and, finally, they can repopulate the haematopoietic system (Lako et al., 2002). In the literature, we can find different information about stem cell localization: the whole dermal sheath, the peri-bulbar dermal sheath, the dermal papilla (Hoogduijn et al., 2006, McElwee et al., 2003, Gharzi et al., 2003, Jahoda et al., 2003.)CD90 (Thy-1) is a small GPI-anchored protein localized in the outer leaflet of the cell membrane (Low and Kincade, 1985). This protein is present in a large number of tissues and cells, even if a great species variation has been described (Mansour Haeryfar, 2004; Tokugawa et al., 1997; McKenzle and Fabre, 1981). CD90 plays a role in cell-cell interaction events, including intracellular adhesion and cell recognition during development (Saalbach et al., 2000; Morris, 1985), and is considered an important stem cell marker; for this last reason it is commonly used to identify mesenchymal stem cells in vitro (Kern et al., 2007; Yoshimura et al., 2006; Le Blanc and Ringdén, 2006; Pittenger et al., 1999). Furthermore, it has been identified in other kinds of stem cells such as haematopoietic progenitor cells (Craig et al., 1993) and hepatic progenitor cells in the human fetal liver (Masson et al., 2006).The hair follicle is the focus of increasing interest because it contains well defined stem cell populations that exhibit various developmental properties. We retain that in dogs, as already demonstrated in other species (Hoogduijn et al., 2006; Zhang et al., 2006; Jahoda et al., 2003; Lako et al., 2002), this organ may be a suitable and accessible source for both epithelial and mesenchymal stem cells that may be isolated and in vitro cultured. Since it is possible to take skin samples without injuring the patient, we chose the hair follicle to study and identify stem cells with the future purpose of using them in regenerative medicine.Dogs are affected by several skin diseases and some of them may be related to alterations of somatic stem cells. We retain that the study of hair follicle stem cell biology may improve our knowledge of etiology and pathogenesis of these skin diseases.In previous works we investigated the stem cells in dog hair follicles; we identified the location of putative epithelial stem cells at the isthmus and described the bulge-like region (Pascucci et al., 2006; Mercati et al., 2008). To the authors’ knowledge, there are no data available neither concerning the localization of DS stem cells nor concerning the expression of CD90 in the hair follicle as regards the canine species. Therefore, in this study, we described the morphological characteristics of DS cells and examined the immunohistochemical localization of CD90 protein in dog hair follicles with both light and transmission electron microscopy. The aim of our study is to observe the dermal sheath cells encompassing the hair follicle and to determine where CD90⁺ cells reside. CD90 is one of the main markers used to identify mesenchymal stem cells and it has been observed in stem cells isolated from the dermal sheath of hair follicles (Hoogduijn et al.,2006). For this reason, we suppose that CD90 protein can help us to identify the hair follicle dermal stem compartment in dog.     

Loss of LORELEI function in the pistil delays initiation but does not affect embryo development in Arabidopsis thaliana

Double fertilization, uniquely observed in plants, requires successful sperm cell delivery by the pollen tube to the female gametophyte, followed by migration, recognition and fusion of the two sperm cells with two female gametic cells. The female gametophyte not only regulates these steps but also controls the subsequent initiation of seed development. Previously, we reported that loss of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein, in the female reproductive tissues causes a delay in initiation of seed development. From these studies, however, it was unclear if embryos derived from fertilization of lre-5 gametophytes continued to lag behind wild-type during seed development. Additionally, it was not determined if the delay in initiation of seed development had any lingering effects during seed germination. Finally, it was not known if loss of LORELEI function affects seedling development given that LORELEI is expressed in eight-day-old seedlings. Here, we showed that despite a delay in initiation, lre-5/lre-5 embryos recover, becoming equivalent to the developing wild-type embryos beginning at 72 hours after pollination. Additionally, lre-5/lre-5 seed germination, and seedling and root development are indistinguishable from wild-type indicating that loss of LORELEI is tolerated, at least under standard growth conditions, in vegetative tissues.Key words: LORELEI, glycosylphosphatidylinositol (GPI)-anchored protein, embryogenesis, DD45, seed germination, primary and lateral root growth, seedling developmentDouble fertilization is unique to flowering plants. Upon female gametophyte reception of a pollen tube, the egg and central cells of the female gametophyte fuse with the two released sperm cells to form zygote and endosperm, respectively and initiate seed development.¹ The female gametophyte controls seed development by (1) repressing premature central cell or egg cell proliferation until double fertilization is completed,^1–3 (2) supplying factors that mediate early stages of embryo and endosperm development^1,4,5 and (3) regulating imprinting of genes required for seed development.^1,6The molecular mechanisms underlying female gametophyte control of early seed development are poorly understood. Although much progress has been made in identifying genes and mechanisms by which the female gametophyte represses premature central cell or egg cell proliferation until double fertilization is completed and regulates imprinting of genes required for seed development,^1,6 only a handful of female gametophyte-expressed genes that affect early embryo^7,8 and endosperm⁹ development after fertilization have been characterized. This is particularly important given that a large number of female gametophyte-expressed genes likely regulate early seed development.⁵We recently reported on a mutant screen for plants with reduced fertility and identification of a mutant that contained a large number of undeveloped ovules and very few viable seeds.¹⁰ TAIL-PCR revealed that this mutant is a new allele of LORELEI(LRE) [At4g26466].^10,11 Four lre alleles have been reported;¹¹ so, this mutant was designated lre-5.¹⁰ The Arabidopsis LORELEI protein contains 165 amino acids and possesses sequence features indicative of a glycosylphosphatidylinositol (GPI)-anchor containing cell surface protein. GPI-anchors serve as an alternative to transmembrane domains for anchoring proteins in cell membranes and GPI-anchored proteins participate in many functions including cell-cell signaling.¹²     

Strigolactones' ability to regulate root development may be executed by induction of the ethylene pathway

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.     

Strigolactones as mediators of plant growth responses to environmental conditions

Strigolactones (SLs) have been recently identified as a new group of plant hormones or their derivatives thereof, shown to play a role in plant development. Evolutionary forces have driven the development of mechanisms in plants that allow adaptive adjustments to a variety of different habitats by employing plasticity in shoot and root growth and development. The ability of SLs to regulate both shoot and root development suggests a role in the plant''s response to its growth environment. To play this role, SL pathways need to be responsive to plant growth conditions, and affect plant growth toward increased adaptive adjustment. Here, the effects of SLs on shoot and root development are presented, and possible feedback loops between SLs and two environmental cues, light and nutrient status, are discussed; these might suggest a role for SLs in plants'' adaptive adjustment to growth conditions.Key words: strigolactones, light, nutrient status, root, shoot, branching, lateral roots, root hairsStrigolactones (SLs) are carotenoid-derived terpenoid lactones suggested to stem from the carotenoid pathway¹ via the activity of various oxygenases.^2,3 SLs production has been demonstrated in both monocotyledons and eudicotyledons (reviewed in ref. ⁴), suggesting their presence in many plant species.⁵ SLs are synthesized mainly in the roots and in some parts of the stem and then move towards the shoot apex (reviewed ref. ⁷).^6,8,9SLs were first characterized more than 40 years ago as germination stimulants of the parasitic plants Striga and Orobanche and later, as stimulants of arbuscular mycorrhiza hyphal branching as well (reviewed in ref. ^{4, 10–13}). Recently, SLs or derivatives thereof, have been identified as a new group of plant hormones, shown to play a role in inhibition of shoot branching,^2,3,8,9 thereby affecting shoot architecture; more recently they have also been shown to affect root growth by affecting auxin efflux.¹⁴Plants have developed mechanisms that allow adaptive adjustments to a variety of different habitats by employing plasticity in their growth and development.¹⁵ Shoot architecture is affected by environmental cues, such as light quality and quantity and nutrient status.^16–19 Root-system architecture and development are affected by environmental conditions such as nutrient availability (reviewed in ref. ^{20, 21}). At the same time, plant hormones are known to be involved in the regulation of plant growth, development and architecture (reviewed in ref. ^22–24) and to be mediators of the effects of environmental cues on plant development; one classic example is auxin''s role in the plant''s shade-avoidance response (reviewed in ref. ²⁵).The ability of SLs to regulate shoot and root development suggests that these phytohormones also have a role in the plant''s growth response to its environment. To play this putative role, SL pathways need to be responsive to plant growth conditions, and affect plant growth toward enhancing its adaptive adjustment. The present review examines the SLs'' possible role in adaptive adjustment of the plant''s response to growth conditions, by discussing their effect on plant development and the possible associations and feedback loops between SLs and two environmental cues: light and nutrient status.     

Human non-CG methylation: Are human stem cells plant-like?

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 replication^7,8 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 published^20–23 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 design^41,42 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 niche^44,45 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.     

Neutralization of Clostridium difficile toxin A using antibody combinations

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 issue^19,20 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.     

Myosin light chain kinases: Division of work in cell migration

Cell motility is a highly coordinated multistep process. Uncovering the mechanism of myosin II (MYO2) activation responsible for the contractility underlying cell protrusion and retraction provides clues on how these complementary activities are coordinated. Several protein kinases have been shown to activate MYO2 by phosphorylating the associated myosin light chain (MLC). Recent work suggests that these MLC kinases are strategically localized to various cellular regions during cell migration in a polarized manner. This localization of the kinases together with their specificity in MLC phosphorylation, their distinct enzymatic properties and the distribution of the myosin isoforms generate the specific contractile activities that separately promote the cell protrusion or retraction essential for cell motility.Key words: myosin, MLCK, ROK, MRCK, phosphorylation, cell migrationCell movement is a fundamental activity underlying many important biological events ranging from embryological development to immunological responses in the adult. A typical cell movement cycle entails polarization, membrane protrusion, formation of new adhesions, cell body translocation and finally rear retraction.¹ A precise temporal and spatial coordination of these separate steps that take place in different parts of the cell is important for rapid and efficient movement.²One major event during eukaryotic cell migration is the myosin II (MYO2)-mediated contraction that underlies cell protrusion, traction and retraction.^1,3 An emerging theme from collective findings is that there are distinct myosin contractile modules responsible for the different functions which are separately regulated by local myosin regulatory light chain (MLC) kinases. These kinases contribute to contractile forces that connect adhesion, protrusion and actin organization.² Unraveling the regulation of these contractile modules is therefore pivotal to a better understanding of the coordination mechanism.At the lamellipodium, the conventional calcium/calmodulin-dependent myosin light chain kinase (MLCK) has been shown to play an essential role in a Rac-dependent lamellipodial extension.⁴ Inhibition of calmodulin or MLCK activity by specific photoactivatable peptides in motile eosinophils effectively blocks lamellipodia extension and net movement.⁵ Furthermore, there is a strong correlation between activated MLCK and phosphorylated MLC within the lamellipodia of Ptk-2 cells as revealed by fluorescence resonance energy transfer (FRET) analysis.⁶ More recent studies showed MLCK to regulate the formation of focal complexes during lamellipodia extension.^7,8 Functionally, MLCK is thought to play a critical role in the environment-sensing mechanism that serves to guide membrane protrusion. It mediates contraction that exerts tension on integrin-extracellular matrix (ECM) interaction, which, depending on the rigidity of the substratum, will lead to either stabilization of adhesion resulting in protrusion or destabilization of attachment seen as membrane ruffling on non-permissive surfaces.^8,9As a Rho effector, Rho-associated kinase (ROK/ROCK/Rho-kinase) has been shown to regulate stress fibers and focal adhesion formation by activating myosin, an effect that can be blocked by the specific ROK inhibitor Y-27632.^10,11 Myosin activation by ROK is the effect of two phosphorylation events: the direct phosphorylation on MLC and the inhibition of myosin phosphatase through phosphorylation of its associated myosin-binding subunit (MBS).¹¹ Consistent with this notion of a localization-function relationship, ROK and MBS, which can interact simultaneously with activated RhoA,¹¹ have been shown to colocalize on stress fibers.^12,13 In migrating cells, Rho and ROK activities have been mostly associated with the regulation of tail retraction, as inhibition of their activities often results in trailing tails due to the loss of contractility specifically confined to the cell rear.^14,15 Tail retraction requires high contractile forces to overcome the strong integrin-mediated adhesion established at the rear end, an event which coincides with the strategic accumulation of highly stable and contractile stress fibers that assemble at the posterior region of migrating cells.MRCK was previously shown to phosphorylate MLC and promote Cdc42-mediated cell protrusion.¹⁶ More recently, it was found to colocalize extensively with and regulate the dynamics of a specific actomyosin network located in the lamella and cell center, in a Cdc42-dependent manner but independent of MLCK and ROK.¹⁷ The lamellar actomyosin network physically overlaps with, but is biochemically distinct from the lamellipodial actin meshwork.^9,18 The former network consists of an array of filaments assembled in an arrangement parallel to the leading edge, undergoing continuous retrograde flow across the lamella, with their disassembly occurring at the border of the cell body zone sitting in a deeper region.^17–19 Retrograde flow of the lamellar network plays a significant role in cell migration as it is responsible for generating contractile forces that support sustained membrane protrusion and cell body advancement.^17–19It is therefore conceivable that these three known MLC kinases are regulated by different signaling mechanisms at different locations and on different actomyosin contractile modules. The coordination of the various modules will ensure persistent directional migration (Figure 1). Phosphorylation of MLC by PAK and ZIP kinase has also been reported, but their exact roles in this event have yet to be determined.^20,21 It is also noteworthy that individual kinases can work independently of each other, as amply shown by evidence from inhibitor treatments. This is particularly true for MRCK in the lamella, whose activity on lamellar actomyosin flow is not affected by ML7 and Y-27632, the inhibitors of MLCK and ROK respectively.¹⁷ These findings further indicate that although both ROK and MRCK have been shown to upregulate phosphorylated MLC levels by inhibiting the myosins phosphatases,^11,22 they are likely to act as genuine MLC kinases themselves, without the need of MLCK as previously suggested.¹¹Open in a separate windowFigure 1Upper panel depicts a model for the specific activation of the different MLC kinases at various locations in the cell. In response to upstream signals, MLC kinases MLCK, MRCK and ROK are activated and localized to different regions. In the case of MRCK and ROK, the interaction of the GTP-bound Rho GTPase binding domain will determine the specific action of the downstream kinase, resulting in actomyosin contractility at different locations. The coordination of these signalling events is crucial for directional cell migration. Lower panel shows a typical front-rear location for Myosin 2A and 2B in a migrating U2OS cell.In conjunction with their differences in localization, the three MLC kinases show apparent individual preferences and specificity towards the MYO2 isoforms that they associate with. The two major MYO2 isoforms MYO2A and 2B are known to have distinct intracellular distributions that are linked to their individual functions (Figure 1).^23,24 In motile cells, MYO2A localization that is skewed towards the protruding cell front is consistent with it being the major myosin 2 component of the lamellar filaments regulated by MRCK as well as its regulation by MLCK in lamellipodial contraction.^8,17,19 In contrast, the enrichment of MYO2B at retracting cell rear conforms well with the requirement of thick and stable stress fibers capable of causing tail contraction and prevention of protrusion under the control of Rho/ROK signaling.^23,25 The selection for MYO2B filaments in the cell rear stems from their more contractile and stable nature compared with MYO2A, a consequence of their higher time-averaged association with actin.^26,27 Conversely, the lower tension property of MYO2A filaments suggests that they are more dynamic in nature,^26,27 a characteristic which fits well with the dynamic actomyosin activities at the leading edge and lamella that regulate protrusion.It deserves special mention that the three MLC kinases display subtle differences in their specificity towards MLC. While MLCK and MRCK phosphorylate only a single Ser19 site (monophosphorylation),^18,28 ROK is able to act on both Thr18 and Ser19 residues causing diphosphorylation of MLC,²⁹ MLCK only causes diphosphorylation when present at higher concentrations.³⁰ By further increasing its actin-activated ATPase activity, diphosphorylation of MLC has been shown to induce a higher myosin activation and filament stability.^30–32 The use of specific antibodies that can differentiate between the two populations of phosphorylated MLC has been instrumental in revealing their localization and correlation with the activity of the MLC kinases. The emerging picture from these experiments is that mono and diphosphorylated MLC exhibit distinct distributions in migrating cells, with the monophosphorylated MLC localized more towards the protrusive region, while the diphosphorylated form is more enriched at the posterior end.^21,33 Taking into account their biochemical properties, the polarized distributions of these differentially phosphorylated MLC coincide functionally with the segregation of the MYO2 isoforms and their corresponding regulators. These findings provide further support for the existence of segregated contractile modules in migrating cell and their distinctive regulation.The mechanisms that determine the specific segregation of the contractile modules and their regulation are unclear. However, some clues have emerged from recent studies. It has been shown that the C-terminal coiled-coil region of MYO2B is important for determining its localization in cell rear²⁵ and which requires Rho/ROK activity as their inhibition resulted in the loss of this specific localization.²³ Correspondingly, the inhibition of MRCK activity resulted in the loss of lamella-localized MYO2A.¹⁷ These findings suggest that activation of MYO2 filaments by their upstream regulators is important for their functional segregation and maintenance. It is noteworthy that both ROK and MRCK have distinct regulatory domains including the pleckstrin homology domains which have been shown to be essential for their localization, a process which may involve myosin interaction and lipid-dependent targeting as has been respectively shown for ROK and MRCK.^11,13,16 Further, the specificity of MRCK for lamellar actomyosin is believed to be largely determined by the two proteins it forms a complex with: the adaptor LRAP35a, and the MYO2-related MYO18A. Activation of MYO18A by MRCK, a process bridged by LRAP35a, is a crucial step which facilitates MRCK regulation on lamellar MYO2A.¹⁷The mechanisms responsible for segregating the contractile modules and their regulators may also comprise a pathway that parallels the microtubule-modulatory Par6/aPKC/GSK3β signalling pathway which regulates cellular polarization. This notion is supported by both Cdc42 and Rho being common upstream regulators of these two pathways.³⁴ GTPase activation may determine the localized activities of the separate contractile modules and create an actomyosin-based asymmetry across the cell body, which together with the microtubule-based activities, result in the formation of a front-back axis important for directional movement. The involvement of MRCK in MTOC reorientation and nuclear translocation events,³⁵ and our unpublished observation that LRAP35a has a GSK3β-dependent microtubule stabilizing function are supportive of a possible cross-talk between these two pathways.In conclusion, the complex regulation of contractility in cell migration emphasizes the importance of the localization, specificity and enzymatic properties of the different MLC kinases and myosin isoforms involved. The initial excitement and confusion caused by the emergence of the different MLC kinases are fading, being now overtaken by the curiosity about how they cooperate and are coordinated while promoting cell motility.     

Cell Migration from Baby to Mother

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 progenitors^7,26,27 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     

Hypoxia: A novel function for VIN3

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     

Roles of calcineurin B-like protein-interacting protein kinases in innate immunity in rice

Cytosolic free Ca²⁺ mobilization induced by microbe/pathogen-asssociated molecular patterns (MAMPs/PAMPs) plays key roles in plant innate immunity. However, components involved in Ca²⁺ signaling pathways still remain to be identified and possible involvement of the CBL (calcineurin B-like proteins)-CIPK (CBL-interacting protein kinases) system in biotic defense signaling have yet to be clarified. Recently we identified two CIPKs, OsCIPK14 and OsCIPK15, which are rapidly induced by MAMPs, involved in various MAMP-induced immune responses including defense-related gene expression, phytoalexin biosynthesis and hypersensitive cell death. MAMP-induced production of reactive oxygen species as well as cell browning were also suppressed in OsCIPK14/15-RNAi transgenic cell lines. Possible molecular mechanisms and physiological functions of the CIPKs in plant innate immunity are discussed.Key words: PAMPs/MAMPs, calcium signaling, CBL-CIPK, hypersensitive cell death, reactive oxygen speciesCa²⁺ plays an essential role as an intracellular second messenger in plants as well as in animals. Several families of Ca²⁺ sensor proteins have been identified in higher plants, which decode spatiotemporal patterns of intracellular Ca²⁺ concentration.^1,2 Calcineurin B-Like Proteins (CBLs) comprise a family of Ca²⁺ sensor proteins similar to both the regulatory β-subunit of calcineurin and neuronal Ca²⁺ sensors of animals.^3,4 Unlike calcineurin B that regulates protein phosphatases, CBLs specifically target a family of protein kinases referred to as CIPKs (CBL-Interacting Protein Kinases).⁵ The CBL-CIPK system has been shown to be involved in a wide range of signaling pathways, including abiotic stress responses such as drought and salt, plant hormone responses and K+ channel regulation.^6,7Following the recognition of pathogenic signals, plant cells initiate the activation of a widespread signal transduction network that trigger inducible defense responses, including the production of reactive oxygen species (ROS), biosynthesis of phytoalexins, expression of pathogenesis-related (PR) genes and reorganization of cytoskeletons and the vacuole,⁸ followed by a form of programmed cell death known as hypersensitive response (HR).^9,10 Because complexed spatiotemporal patterns of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) have been suggested to play pivotal roles in defense signaling,^1,9 multiple Ca²⁺ sensor proteins and their effectors should function in defense signaling pathways. Although possible involvement of some calmodulin isoforms^11–13 and the calmodulin-domain/calcium-dependent protein kinases (CDPKs)^14–19 has been suggested, other Ca²⁺-regulated signaling components still remain to be identified. No CBLs or CIPKs had so far been implicated as signaling components in innate immunity.