Decorin regulates endothelial cell-matrix interactions during angiogenesis

Interactions between endothelial cells and the surrounding extracellular matrix are continuously adapted during angiogenesis, from early sprouting through to lumen formation and vessel maturation. Regulated control of these interactions is crucial to sustain normal responses in this rapidly changing environment, and dysfunctional endothelial cell behaviour results in angiogenic disorders. The proteoglycan decorin, an extracellular matrix component, is upregulated during angiogenesis. While it was shown previously that the absence of decorin leads to dysregulated angiogenesis in vivo, the molecular mechanisms were not clear. These abnormal endothelial cell responses have been attributed to indirect effects of decorin; however, our recent data provides evidence that decorin directly regulates endothelial cell-matrix interactions. This data will be discussed in conjunction with findings from previous studies, to better understand the role of this proteoglycan in angiogenesis.Key words: decorin, angiogenesis, motility, α2β1 integrin, insulin-like growth factor I receptor, Rac GTPaseLed by appropriate cues, the vascular system undergoes postnatal remodelling (angiogenesis), to maintain tissue homeostasis. Thus while much of the mature endothelium is quiescent, locally activated endothelial cells re-enter the cell cycle, and assume a motile phenotype essential for sprouting and neo-vessel formation. Concomitantly, the surrounding extracellular matrix (ECM) is significantly altered through de novo protein expression, deposition of plasma components and protease-mediated degradation. The latter liberates cryptic binding sites and sequestered growth factors in addition to intact and degraded ECM components, which themselves possess pro- and anti-angiogenic signalling properties. For supported blood flow, endothelium quiescence and integrity is re-established, and the ECM is organized into mature, cross-linked networks. In short, endothelial cells regulate ECM synthesis, assembly and turnover while the structure and composition of ECM in turn influences cellular phenotype. The ECM therefore, plays a critical role in control of endothelial cell behaviour during angiogenesis.Decorin is a member of the small leucine-rich repeat proteoglycan (SLRP) family, which was first discovered ‘decorating’ collagen I fibrils and was subsequently shown to regulate fibrillogenesis.^1,2 Both the protein core and the single, covalently attached glycosaminoglycan (GAG) moieties of decorin are involved in this function, the relevance of which is demonstrated by the phenotype of the decorin null mouse, which exhibits loose, fragile skin due to dysregulated fibrillogenesis.² Interestingly, a role for decorin in postnatal angiogenesis was also revealed by studies in the decorin null background. Corneal neoangiogenesis was reduced.³ Conversely, neo-angiogenesis was enhanced during dermal wound healing, although surprisingly this led to delayed wound closure.⁴ In this case, skin fragility due to the absence of decorin may have hindered wound closure, despite an increased blood supply. It is apparent however, that decorin plays a role in inflammation-associated angiogenesis. Indeed, endothelial cells undergoing angiogenic morphogenesis in this environment express decorin, while quiescent endothelial cells do not,^3–6 indicating that decorin modulates endothelial cell behaviour specifically during inflammatory-associated remodelling of the vascular system.To understand decorin effects on angiogenic morphogenesis within a minimalist environment, various in vitro models of angiogenesis have been employed (6 Similarly, decorin expression enhanced tube formation on matrigel,⁸ but in other studies utilising this substrate was found to either have no influence⁹ or to inhibit tubulogenesis induced by growth factors.¹⁰ In yet another study, decorin inhibited tube formation when presented as a substrate prior to addition of collagen I.⁷ These contrasting observations may reflect the importance of the micro-environment within which decorin is presented. Alternatively, controversial results could result from different sources of decorin since cell types differ in their post-translational modifications of the GAG moiety. Hence, varying length or sulfation patterns of GAG chains may account for different biological activities of decorin. Discrepancies can also be explained as artefacts due to different purification protocols, such as when denaturing conditions are used to extract decorin from tissue. Taken together however, these observations suggest that decorin is neither a pro- nor an anti-angiogenic factor per se, but rather a regulator of angiogenesis, dependent on local cues for different activities. Further, that decorin is capable of both enhancing and inhibiting tubulogenesis may suggest a role in balancing vessel regression versus persistence. Immature vessels have a period of plasticity prior to maturation, during which they can be remodelled, and either regress, or given the appropriate signals, proceed to maturity.¹¹ As a modulator of tube formation, it is tempting to speculate that decorin could influence the switch from immature to mature vessels, favouring one or the other in conjunction with signals from the local environment.

Table 1

Summary of the key functions of decorin in controlling cell behaviour

The speed of intracellular signal transfer for chloroplast movement

The photoreceptors for chloroplast photorelocation movement have been known, but the signal(s) raised by photoreceptors remains unknown. To know the properties of the signal(s) for chloroplast accumulation movement, we examined the speed of signal transferred from light-irradiated area to chloroplasts in gametophytes of Adiantum capillus-veneris. When dark-adapted gametophyte cells were irradiated with a microbeam of various light intensities of red or blue light for 1 min or continuously, the chloroplasts started to move towards the irradiated area. The speed of signal transfer was calculated from the relationship between the timing of start moving and the distance of chloroplasts from the microbeam and was found to be constant at any light conditions. In prothallial cells, the speed was about 1.0 µm min⁻¹ and in protonemal cells about 0.7 µm min⁻¹ towards base and about 2.3 µm min⁻¹ towards the apex. We confirmed the speed of signal transfer in Arabidopsis thaliana mesophyll cells under continuous irradiation of blue light, as was about 0.8 µm min⁻¹. Possible candidates of the signal are discussed depending on the speed of signal transfer.Key words: Adiantum capillus-veneris, Arabidopsis thaliana, blue light, chloroplast movement, microbeam, red light, signalOrganelle movement is essential for plant growth and development and tightly regulated by environmental conditions.¹ It is well known that light regulates chloroplast movement in various plant species. Chloroplast movement can be separated into three categories, (1) photoperception by photoreceptors, (2) signal transduction from photoreceptor to chloroplasts and (3) movement of chloroplasts and has been analyzed from a physiological point of view.² We recently identified the photoreceptors in Arabidopsis thaliana, fern Adiantum capillus-veneris, and moss Physcomitrella patens. In A. thaliana, phototropin 2 (phot2) mediates the avoidance movement,^3,4 whereas both phototropin 1 (phot1) and phot2 mediate the accumulation response.⁵ A chimeric photoreceptor neochrome 1 (neo1)⁶ was identified as a red/far-red and blue light receptor that mediates red as well as blue light-induced chloroplast movement in A. capillusveneris.⁷ Interestingly, neo1 mediated red and blue light-induced nuclear movement and negative phototropic response of A. capillus-veneris rhizoid cells.^8,9 On the mechanism of chloroplast movement, we also found a novel structure of actin filaments that appeared between chloroplast and the plasma membrane at the front side of moving chloroplast.¹⁰ Recent studies using the technique of microbeam irradiation have revealed that chloroplasts do not have a polarity for light-induced accumulation movement and can move freely in any direction both in A. capillus-veneris prothallial cells and in A. thaliana mesophyll cells.¹¹ However, the signal that may be released from photoreceptors and transferred to chloroplasts remains unknown.To understand the properties of the signal for the chloroplast accumulation response, we examined the speed of signal transfer in dark-adapted A. capillus-veneris gametophyte cells and A. thaliana mesophyll cells by partial cell irradiation with a red and/or blue microbeam of various light intensities for 1 min and the following continuous irradiation, respectively.¹²As shown in Figure 1, the relation between the distance of chloroplasts from the microbeam and the timing when each chloroplast started moving toward the microbeam irradiated area (shown as black dots in Fig. 1) was obtained and plotted. The lag time between the onset of microbeam irradiation and the timing of start moving of chloroplasts is the time period needed for a signal to reach each chloroplast. To obtain more accurate data many chloroplasts at various positions were used. The slope of the approximate line indicates the average speed of the signal transfer. Shown with a protonemal cell at the left side of this figure is an instance where the speed of signal transfer from basal-to-apical (acropetal) direction is obtained.Open in a separate windowFigure 1How to calculate the speed of signal transfer in the basal cell of two-celled protonema of Adiantum capillus-veneris. The relationship between the distance of chloroplast position from the edge of the microbeam to the center of each chloroplast as shown in left side of figure and the timing of chloroplast movement initiated shown as the black dots was obtained. Inclination of the approximate lines connecting dots indicates the speeds of the signal transfer.In protonemal cells, which are tip-growing linear cells, the average speed of signal transfer was about 2.3 µm min⁻¹ from basal-to-apical (acropetal) and about 0.7 µm min⁻¹ from apical-to-basal (basipetal) directions. These values were almost constant irrespective of light intensity, wavelength, irradiation period, and the region of the cell irradiated.¹² The difference of speed between basipetal and acropetal directions may be depending on cell polarity. The signal transfer in prothallial cells of A. capillus-veneris and mesophyll cells of A. thaliana was about 1.0 µm min⁻¹ to any direction, probably because they may not have a polarity comparing to protonemal cells or have a weak polarity if any. Thus, the speed of signal transfer must be conserved in most land plants,¹² if not influenced by strong polarity.

Arabidopsis thaliana overexpressing glycolate oxidase in chloroplasts: H2O2-induced changes in primary metabolic pathways

Reactive oxygen species (ROS) represent both toxic by-products of aerobic metabolism as well as signaling molecules in processes like growth regulation and defense pathways. The study of signaling and oxidative-damage effects can be separated in plants expressing glycolate oxidase in the plastids (GO plants), where the production of H₂O₂ in the chloroplasts is inducible and sustained perturbations can reproducibly be provoked by exposing the plants to different ambient conditions. Thus, GO plants represent an ideal non-invasive model to study events related to the perception and responses to H₂O₂ accumulation. Metabolic profiling of GO plants indicated that under high light a sustained production of H₂O₂ imposes coordinate changes on central metabolic pathways. The overall metabolic scenario is consistent with decreased carbon assimilation, which results in lower abundance of glycolytic and tricarboxylic acid cycle intermediates, while simultaneously amino acid metabolism routes are specifically modulated. The GO plants, although retarded in growth and flowering, can complete their life cycle indicating that the reconfiguration of the central metabolic pathways is part of a response to survive and thus, to adapt to stress conditions imposed by the accumulation of H₂O₂ during the light period.Key words: Arabidopsis thaliana, H₂O₂, oxidative stress, reactive oxygen species, signalingReactive oxygen species (ROS) are key molecules in the regulation of plant development, stress responses and programmed cell death. Depending on the identity of ROS species or its subcellular production site, different cellular responses are provoked.¹ To assess the effects of metabolically generated H₂O₂ in chloroplasts, we have recently generated Arabidopsis plants in which the peroxisomal GO was targeted to chloroplasts.² The GO overexpressing plants (GO plants) show retardation in growth and flowering time, features also observed in catalase, ascorbate peroxidase and MnSOD deficient mutants.^3–5 The analysis of GO plants indicated that H₂O₂ is responsible for the observed phenotype. GO plants represent an ideal non-invasive model system to study the effects of H₂O₂ directly in the chloroplasts because H₂O₂ accumulation can be modulated by growing the plants under different ambient conditions. By this, growth under low light or high CO₂ concentrations minimizes the oxygenase activity of RubisCO and thus the flux through GO whereas the exposition to high light intensities enhances photorespiration and thus the flux through GO.Here, we explored the impact of H₂O₂ production on the primary metabolism of GO plants by assessing the relative levels of various metabolites by gas chromatography coupled to mass spectrometry (GC-MS)⁶ in rosettes of plants grown at low light (30 µmol quanta m⁻² s⁻¹) and after exposing the plants for 7 h to high light (600 µmol quanta m⁻² s⁻¹). The results obtained for the GO5 line are shown in After 1 h at 30 µEAfter 7 h at 600 µEAlanine0.88 ± 0.052.83 ± 0.68Asparagine1.39 ± 0.123.64 ± 0.21Aspartate0.88 ± 0.031.65 ± 0.10GABA1.14 ± 0.051.13 ± 0.05Glutamate0.97 ± 0.041.51 ± 0.07Glutamine1.06 ± 0.111.87 ± 0.06Glycine1.23 ± 0.070.30 ± 0.02Isoleucine3.52 ± 0.403.00 ± 0.15Leucine1.36 ± 0.220.57 ± 0.06Lysine1.49 ± 0.130.38 ± 0.02Methionine0.96 ± 0.054.54 ± 0.51Phenylalanine0.95 ± 0.030.94 ± 0.04Proline1.32 ± 0.221.60 ± 0.13Serine1.05 ± 0.041.49 ± 0.15Threonine4.74 ± 0.175.51 ± 0.34Valine0.91 ± 0.130.29 ± 0.02Citrate/Isocitrate0.65 ± 0.020.64 ± 0.022-oxoglutarate0.95 ± 0.110.76 ± 0.05Succinate0.78 ± 0.040.72 ± 0.02Fumarate0.64 ± 0.030.31 ± 0.01Malate0.74 ± 0.030.60 ± 0.02Pyruvate1.19 ± 0.280.79 ± 0.04Ascorbate1.13 ± 0.142.44 ± 0.45Galactonate-γ-lactone1.81 ± 0.401.62 ± 0.28Fructose1.20 ± 0.130.37 ± 0.01Glucose1.38 ± 0.170.30 ± 0.01Mannose0.90 ± 0.271.34 ± 0.28Sucrose1.04 ± 0.070.49 ± 0.02Fructose-6P0.82 ± 0.151.20 ± 0.15Glucose-6P0.87 ± 0.061.25 ± 0.183-PGA1.13 ± 0.110.35 ± 0.02DHAP1.38 ± 0.091.26 ± 0.08Glycerate0.99 ± 0.040.67 ± 0.01Glycerol1.07 ± 0.041.12 ± 0.05Shikimate1.18 ± 0.040.35 ± 0.01Salicylic acid1.04 ± 0.180.66 ± 0.18Open in a separate windowPlants were grown at 30 µmol m⁻² sec⁻¹ (30 µE). The samples were collected 1 h after the onset of the light period and after 7 h of exposure to 600 µmol m⁻² sec⁻¹ (600 µE), respectively. The values are relative to the respective wild-type (each metabolite = 1) and represent means ± SE of four determinations of eight plants. (*) indicates the value is significantly different from the respective wild-type as determined by the Student''s t test (p < 0.05).At the beginning of the light period in low light conditions, some significant deviations in the levels of metabolites tested were observed in GO plants when compared to the wild-type (2 the transgenic GO activity is sufficient to induce a characteristic metabolic phenotype (Fig. 1). The levels of the tricarboxylic acid (TCA) cycle intermediates, citrate/isocitrate, succinate, fumarate and malate were lower in the GO plants (7 In consequence, OAA might not freely enter the TCA cycle and is redirected to the synthesis of Lys, Thr and Ile, which accumulate in the GO plants (Open in a separate windowFigure 1Simplified scheme of the primary metabolism showing the qualitative variations in metabolite abundance in GO plants obtained by GC-MS analysis (2 Blue boxes indicate a significant increase in the content of the particular metabolite compared to the wild-type, while red boxes indicate a significant decrease. Metabolites without boxes have not been determined. The arrows do not always indicate single steps. Adapted from Baxter et al., 2007.High light treatment induced massive changes in the metabolic profile of GO plants (Fig. 1). The OAA-derived amino acids Asp, Asn, Thr, Ile and Met as well as the 2-oxoglutarate-derived amino acids Glu and Gln accumulated. On the contrary, the levels of the Pyr-derived amino acids Val and Leu and the OAA-derived amino acid Lys decreased. A rational explanation for these metabolic changes is difficult to assess, but these changes could be a consequence of a metabolic reconfiguration in response to high light leading to required physiological functions and thus ensuring continued cellular function and survival, e.g., production of secondary metabolites to mitigate photooxidative damage. The higher levels of Glu observed in the GO plants could be attributed to alternative pathways of glyoxylate metabolism that may occur during photorespiration.⁸ It has been shown earlier that isocitrate derived from glyoxylate and succinate is decarboxylated by cytosolic isocitrate dehydrogenase producing 2-oxoglutarate and further glutamate.⁸In GO plants grown under low light conditions (minimized photorespiratory conditions), the levels of Gly were similar to those of the wild-type whereas, after exposure to high light (photorespiratory conditions), the Gly levels were extremely low, indicating that the GO activity diverts a significant portion of flux from the photorespiratory pathway (7 and also the levels of the lipoic acid-containing subunits of the pyruvate- and 2-oxoglutarate dehydrogenases were shown to be significantly reduced under oxidative stress conditions.^9,10 Similarly, the contents of the soluble sugars sucrose, fructose and glucose and those of 3-PGA and glycerate were lower. In addition, the GO plants showed an impairment in the accumulation of starch under high light conditions, a feature that was not observed if the plants were grown under non-photorespiratory conditions.²Together, these results indicate that the low photosynthetic carbon assimilation in the GO plants exposed to high light is most probably due to enhanced photoinhibition,² the repression of genes encoding photosynthetic components by H₂O₂,^11–13 and the direct damage or inhibition of enzyme activities involved in CO₂ assimilation and energy metabolism by H₂O₂.^7,10,14,15 Moreover, Scarpeci and Valle¹³ showed that in plants treated with the superoxid anion radical producing methylviologen (MV) most of the genes involved in phosphorylytic starch degradation, e.g., the trioseP/Pi translocator and genes involved in starch and sucrose synthesis were repressed, while genes involved in hydrolytic starch breakdown and those involved in sucrose degradation were induced. In line with this, the contents of carbohydrates were also lower in MV-treated plants. Together, these observations can also explain the lower growth rates of the GO plants in conditions where the oxygenase activity of RubisCO becomes important and thus, the flux through GO increases.²The levels of shikimate were lower in GO plants (2^,16 and the low levels of substrates available, as anthocyanins are ultimately synthesized from photosynthates and the GO plants showed a diminished photosynthetic performance.²As expected, the levels of ascorbate and its precursor, galactonate-γ-lactone, were enhanced in the GO plants clearly showing the activation of the cellular antioxidant machinery (10 described the metabolic response to oxidative stress of heterotrophic Arabidopsis cells treated with menadione, which also generates superoxide anion radicals. This oxidative stress was shown to induce metabolic inhibition of flux through the TCA cycle and sectors of amino acid metabolism together with a diversion of carbon into the oxidative pentose phosphate pathway.Signaling and oxidative-damage effects are difficult to separate by manipulating the enzymes of antioxidant systems. In this regard, the GO plants represent a challenging inducible model that avoid acclimatory and adaptative effects. Moreover, it is possible to control the H₂O₂ production in the chloroplasts of GO plants without inducing oxidative damage by changing the conditions of growth.² Further exploration of metabolic changes imposed by different ROS at the cellular and whole organ levels will allow to address many intriguing questions on how plants can rearrange metabolism to cope with oxidative stresses.     

Indirect effects of tending ants on holm oak volatiles and acorn quality

The indirect effect of ants on plants through their mutualism with honeydew-producing insects has been extensively investigated. Honeydew-producing insects that are tended by ants impose a cost on plant fitness and health by reducing seed production and/or plant growth. This cost is associated with sap intake and virus transmissions but may be overcompesated by tending ants if they deter or prey on hebivorous insects. The balance between cost and benefits depends on the tending ant species. In this study we report other indirect effects on plants of the mutualism between aphids and ants. We have found that two Lasius ant species, one native and the other invasive, may change the composition of volatile organic compounds (VOCs) of the holm oak (Quercus ilex) blend when they tend the aphid Lachnus roboris. The aphid regulation of its feeding and honeydew production according to the ant demands was proposed as a plausible mechanism that triggers changes in VOCs. Additionally, we now report here that aphid feeding, which is located most of the time on acorns cap or petiole, significantly increased the relative content of linolenic acid in acorns from holm oak colonized by the invasive ant. This acid is involved in the response of plants to insect herbivory as a precursor or jasmonic acid. No effect was found on acorn production, germination or seedlings quality. These results suggest that tending-ants may trigger the physiological response of holm oaks involved in plant resistance toward aphid herbivory and this response is ant species-dependent.Key words: tended aphid, invasive ants, linolenic acid, jasmonic acid, monoterpene emissionsTo achieve an indirect effect it is necessary to have a minimun of three species, two focal species that interact directly and an associate species whose presence promotes an indirect effect on one or both focal species. In general, indirect effects of a third species are defined by how and to what degree a pairwise species interaction is influenced by the presence and density of this third species.¹ There are several examples of interactions presenting indirect effects: apparent competition,¹ facilitation,² tri-trophic level interactions,³ cascading effects⁴ and exploitative competition. 5 But, indirect effects have been studied most extensively in the context of trophic cascades when top predators are removed⁶ or added⁷ and in the context of mutualisms.^8–10 Usually, indirect effects are investigated as changes in abundance of the focal species occur. However, indirect effects may result in biologically significant changes in a species that are not reflected only to its abundance.¹¹ There are many examples of changes in physiology, behavior, morphology and/or genotypic composition of the focal species.^11,12 These changes on density and/or morphological, physiological and behavioral traits of the focal species are not mutually exclusive, and all can act at the same time.¹³ The magnitude and direction of both direct and indirect effects should influence the relative resilience of communities to perturbation, which in turn will affect species coexistence and community evolution.¹⁴ In this regard, indirect effects had been postulated as one of the main forces structuring communities² and shaping the evolution of communities.¹⁴In terrestrial communities ants interact with plants both directly and indirectly. They can disperse or consume seeds, feed from specialized plant structures such as food bodies and extrafloral nectaries, act as or deter pollinitators, prey on herbivorous insects and/or develop mutualisms with honeydew-producing insects indirectly modifying plant fitness.^15–17 Additionally, through their nesting activities in soil, ants increase soil nutrient content available to plants, may change water infiltration and soil holding-capacity and modify biodiversity and abundance of soil organisms related to the decomposition process.^18,19 As a consequence of their activities, ants may thus change behavior, density, physiology or fitness of other species.^12,22,23 In the case of ants that tend honeydew-producing insects, evidence shows that their attention may change some traits of insect life history, 22 their abundance or physiology.¹⁸ For the plant, the net outcome of the mutualism between ants and honeydew-producing insects will depend on the balance between the costs for plant fitness via consumption of plant sap and transmission of plant pathogens and the benefit of ants deterring herbivorous insects.^18,23 As a consequence, plant seed production, pod production or even plant growth may decrease when the cost of honeydew-producing insects exceed the benefit provided by tending ants.^18,23Recently, we have described the changes that two tending ant species may exert indirectly on monoterpene emissions of holm oak (Quercus ilex) saplings through its mutualism with Lachnus roboris aphids.²⁴ One of these tending ant species was Lasius neglectus, an invasive ant species that displaces the local ant Lasius grandis. We found that aphids feeding on holm oak increased the emission of total volatile organic carbon (VOCs) by 31%. In particular, aphids feeding elicited the emission of a new monoterpene, Δ³-carene, and increased the emission of myrcene (mean ± SE; sapling alone: 0.105 ± 0.011 µg g⁻¹ h⁻¹; sapling plus not tended aphid: 0.443 ± 0.057 µg g⁻1 h⁻1) and γ-terpinene (sapling alone: 0.0013 ± 0.0001; sapling plus not tended aphid: 0.0122 ± 0.0022 µg g⁻1 h⁻1) (Mann-Whitney, sapling alone vs. sapling plus not tended aphids, U_4,4 = 0, p < 0.05 for both compounds). Changes of VOC emission in response to aphid infestation were noticed also in boreal trees.²⁴ When the aphids became tended by the invasive ant, L. neglectus, VOCs emissions increased only 19% because myrcene, the main compound of the blend, decreased significantly (25 When our data was recalculated on leaf area basis (nmol m⁻² s⁻¹), the general pattern was the same independently of the units, but the differences among treatments were not statistically significant (26 These slight differences in the statitiscal significance of the differences of VOC emissions depending on the reference unit may be due to differences in leaf morphology, i.e., changes of leaf area and mass. However, in our study, all holm oaks showed a similar leaf morphology among treatments (Kruskal-Wallis, leaf mass: H_3,20 = 2.16, p = 0.53; leaf area: H_3,20 = 2.64, p = 0.45) (24^,27 This lack of consistence of aphid effect on leaf area and mass limits the development of a clear pattern linking aphids feeding, leaf area or mass and VOC emissions. On the other hand, to achieve statistical significance of emitted VOCs among treatments, values should differ strongly given the high variability of VOC emission within treatments.²⁶ Under this scenario, we recommend giving the values of leaf morphology and to give VOC emissions on both unit bases to facilite comparisons among different studies.

Table 1

Means and standard error of the emission rates of the main compounds emitted by Quercus ilex saplings (n = 4 for T1 and T2 and n = 8 for T3) infested with untended aphids (T1) or infested with aphids tended by the native ant Lasius grandis (T2) or by the invasive ant Lasius neglectus (T3)

Peptidoglycan Fine Structure of the Radiotolerant Bacterium Deinococcus radiodurans Sark

Peptidoglycan from Deinococcus radiodurans was analyzed by high-performance liquid chromatography and mass spectrometry. The monomeric subunit was: N-acetylglucosamine–N-acetylmuramic acid–l-Ala–d-Glu-(γ)–l-Orn-[(δ)Gly-Gly]–d-Ala–d-Ala. Cross-linkage was mediated by (Gly)₂ bridges, and glycan strands were terminated in (1→6)anhydro-muramic acid residues. Structural relations with the phylogenetically close Thermus thermophilus are discussed.The gram-positive bacterium Deinococcus radiodurans is remarkable because of its extreme resistance to ionizing radiation (14). Phylogenetically the closest relatives of Deinococcus are the extreme thermophiles of the genus Thermus (4, 11). In 16S rRNA phylogenetic trees, the genera Thermus and Deinococcus group together as one of the older branches in bacterial evolution (11). Both microorganisms have complex cell envelopes with outer membranes, S-layers, and ornithine-Gly-containing mureins (7, 12, 19, 20, 22, 23). However, Deinococcus and Thermus differ in their response to the Gram reaction, having positive and negative reactions, respectively (4, 14). The murein structure for Thermus thermophilus HB8 has been recently elucidated (19). Here we report the murein structure of Deinococcus radiodurans with similar detail.D. radiodurans Sark (23) was used in the present study. Cultures were grown in Luria-Bertani medium (13) at 30°C with aeration. Murein was purified and subjected to amino acid and high-performance liquid chromatography (HPLC) analyses as previously described (6, 9, 10, 19). For further analysis muropeptides were purified, lyophilized, and desalted as reported elsewhere (6, 19). Purified muropeptides were subjected to plasma desorption linear time-of-flight mass spectrometry (PDMS) as described previously (1, 5, 16, 19). Positive and negative ion mass spectra were obtained on a short linear ²⁵²californium time-of-flight instrument (BioIon AB, Uppsala, Sweden). The acceleration voltage was between 17 and 19 kV, and spectra were accumulated for 1 to 10 million fission events. Calibration of the mass spectra was done in the positive ion mode with H⁺ and Na⁺ ions and in the negative ion mode with H⁻ and CN⁻ ions. Calculated m/z values are based on average masses.Amino acid analysis of muramidase (Cellosyl; Hoechst, Frankfurt am Main, Germany)-digested sacculi (50 μg) revealed Glu, Orn, Ala, and Gly as the only amino acids in the muramidase-solubilized material. Less than 3% of the total Orn remained in the muramidase-insoluble fraction, indicating an essentially complete solubilization of murein.Muramidase-digested murein samples (200 μg) were analyzed by HPLC as described in reference 19. The muropeptide pattern (Fig. ​(Fig.1)1) was relatively simple, with five dominating components (DR5 and DR10 to DR13 [Fig. 1]). The muropeptides resolved by HPLC were collected, desalted, and subjected to PDMS. The results are presented in Table ​Table11 compared with the m/z values calculated for best-matching muropeptides made up of N-acetylglucosamine (GlucNAc), N-acetylmuramic acid (MurNAc), and the amino acids detected in the murein. The more likely structures are shown in Fig. ​Fig.1.1. According to the m/z values, muropeptides DR1 to DR7 and DR9 were monomers; DR8, DR10, and DR11 were dimers; and DR12 and DR13 were trimers. The best-fitting structures for DR3 to DR8, DR11, and DR13 coincided with muropeptides previously characterized in T. thermophilus HB8 (19) and had identical retention times in comparative HPLC runs. The minor muropeptide DR7 (Fig. ​(Fig.1)1) was the only one detected with a d-Ala–d-Ala dipeptide and most likely represents the basic monomeric subunit. The composition of the major cross-linked species DR11 and DR13 confirmed that cross-linking is mediated by (Gly)₂ bridges, as proposed previously (20). Open in a separate windowFIG. 1HPLC muropeptide elution patterns of murein purified from D. radiodurans. Muramidase-digested murein samples were subjected to HPLC analysis, and the A₂₀₄ of the eluate was recorded. The most likely structures for each muroeptide as deduced by PDMS are shown. The position of residues in brackets is the most likely one as deduced from the structures of other muropeptides but could not be formally demonstrated. R = GlucNac–MurNac–l-Ala–d-Glu-(γ)→.

TABLE 1

Calculated and measured m/z values for the molecular ions of the major muropeptides from D. radiodurans

Cell Biology of Mitotic Recombination

Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination.Homologous recombination (HR) is defined as the homology-directed exchange of genetic information between two DNA molecules (Fig. 1). Mitotic recombination is often initiated by single-stranded DNA (ssDNA), which can arise by several avenues (Mehta and Haber 2014). They include the processing of DNA double-strand breaks by 5′ to 3′ resection, during replication of damaged DNA, or during excision repair (Symington 2014). The ssDNA is bound by replication protein A (RPA) to control its accessibility to the Rad51 recombinase (Sung 1994, 1997a; Sugiyama et al. 1997; Morrical 2014). The barrier to Rad51-catalyzed recombination imposed by RPA can be overcome by a number of mediators, such as BRCA2 and Rad52, which serve to replace RPA with Rad51 on ssDNA, and the Rad51 paralogs Rad55-Rad57 (RAD51B-RAD51C-XRCC2-XRCC3) and the Psy3-Csm2-Shu1-Shu2 complex (SHU) (RAD51D-XRCC2-SWS1), which stabilize Rad51 filaments on ssDNA (see Sung 1997b; Sigurdsson et al. 2001; Martin et al. 2006; Bernstein et al. 2011; Liu et al. 2011; Qing et al. 2011; Amunugama et al. 2013; Zelensky et al. 2014). The Rad51 nucleoprotein filament catalyzes the invasion into a homologous duplex to produce a displacement loop (D-loop) (Fig. 1). At this stage, additional antirecombination functions are exerted by Srs2 (FBH1, PARI), which dissociates Rad51 filaments from ssDNA, and Mph1 (FANCM), which disassembles D-loops (see Daley et al. 2014). Upon Rad51-catalyzed strand invasion, the ATP-dependent DNA translocase Rad54 enables the invading 3′ end to be extended by DNA polymerases to copy genetic information from the intact duplex (Li and Heyer 2009). Ligation of the products often leads to joint molecules (JMs), such as single- or double-Holliday junctions (s/dHJs) or hemicatenanes (HCs), which must be processed to allow separation of the sister chromatids during mitosis. JMs can be dissolved by the Sgs1-Top3-Rmi1 complex (STR) (BTR, BLM-TOP3α-RMI1-RMI2) (see Bizard and Hickson 2014) or resolved by structure-selective nucleases, such as Mus81-Mms4 (MUS81-EME1), Slx1-Slx4, and Yen1 (GEN1) (see Wyatt and West 2014). Mitotic cells favor recombination events that lead to noncrossover events likely to avoid potentially detrimental consequences of loss of heterozygosity and translocations.Open in a separate windowFigure 1.Primary pathways for homology-dependent double-strand break (DSB) repair. Recombinational repair of a DSB is initiated by 5′ to 3′ resection of the DNA end(s). The resulting 3′ single-stranded end(s) invade an intact homologous duplex (in red) to prime DNA synthesis. For DSBs that are repaired by the classical double-strand break repair (DSBR) model, the displaced strand from the donor duplex pairs with the 3′ single-stranded DNA (ssDNA) tail at the other side of the break, which primes a second round of DNA synthesis. After ligation of the newly synthesized DNA to the resected 5′ strands, a double-Holliday junction (dHJ) intermediate is generated. The dHJ can be either dissolved by branch migration (indicated by arrows) into a hemicatenane (HC) leading to noncrossover (NCO) products or resolved by endonucleolytic cleavage (indicated by triangles) to produce NCO (positions 1, 2, 3, and 4) or CO (positions 1, 2, 5, and 6) products. Alternatively to the double-strand break repair (DSBR) pathway, the invading strand is often displaced after limited synthesis and the nascent complementary strand anneals with the 3′ single-stranded tail of the other end of the DSB. After fill-in synthesis and ligation, this pathway generates NCO products and is referred to as synthesis-dependent strand annealing (SDSA).

Table 1.

Evolutionary conservation of homologous recombination proteins between Saccharomyces cerevisiae and Homo sapiens

De novo mammalian prion synthesis

Prions are responsible for a heterogeneous group of fatal neurodegenerative diseases. They can be sporadic, genetic, or infectious disorders involving post-translational modifications of the cellular prion protein (PrP^C). Prions (PrP^Sc) are characterized by their infectious property and intrinsic ability to convert the physiological PrP^C into the pathological form, acting as a template. The “protein-only” hypothesis, postulated by Stanley B. Prusiner, implies the possibility to generate de novo prions in vivo and in vitro. Here we describe major milestones towards proving this hypothesis, taking into account physiological environment/s, biochemical properties and interactors of the PrP^C.Key words: prion protein (PrP), prions, amyloid, recombinant prion protein, transgenic mouse, protein misfolding cyclic amplification (PMCA), synthethic prionPrions are responsible for a heterogeneous group of fatal neurodegenerative diseases (1 They can be sporadic, genetic or infectious disorders involving post-translational modifications of the cellular prion protein (PrP^C).² Prions are characterized by their infectious properties and by their intrinsic ability to encipher distinct biochemical properties through their secondary, tertiary and quaternary protein structures. In particular, the transmission of the disease is due to the ability of a prion to convert the physiological PrP^C into the pathological form (PrP^Sc), acting as a template.³ The two isoforms of PrP appear to be different in terms of protein structures, as revealed by optical spectroscopy experiments such as Fourier-transform infrared and circular dichroism.⁴ PrP^C contains 40% α-helix and 3% β-sheet, while the pathological isoform, PrP^Sc, presents approximately 30% α-helix and 45% β-sheet.^4,5 PrP^Sc differs from PrP^C because of its altered physical-chemical properties such as insolubility in non-denaturing detergents and proteinases resistance.^2,6,7

Table 1

The prion diseases

Improved Molecular Detection of Angiostrongylus cantonensis in Mollusks and Other Environmental Samples with a Species-Specific Internal Transcribed Spacer 1-Based TaqMan Assay

Angiostrongylus cantonensis is the most common cause of human eosinophilic meningitis. Humans become infected by ingesting food items contaminated with third-stage larvae that develop in mollusks. We report the development of a real-time PCR assay for the species-specific identification of A. cantonensis in mollusk tissue.Angiostrongylus cantonensis is the most common agent associated with eosinophilic meningitis in humans. Young adult worms develop in the brains of rodents and are carried to pulmonary arteries to reach sexual maturity. Eggs are laid in lung tissues, and first-stage (L1) larvae break into air spaces, migrate to the trachea, are swallowed, and are passed with rodent feces. The L1 larvae must infect mollusks to develop into third-stage (L3) larvae; L3 is the infective stage for rodents and other mammals. Humans become infected by ingesting raw produce contaminated with L3 larvae or infected raw or undercooked mollusks or paratenic hosts. The immature worms remain in the human brain, creating tissue damage and inflammation (2, 19, 21).A. cantonensis is endemic in Southeast Asia, parts of the Caribbean, and the Pacific Islands, including Hawaii (7, 12, 15-17). The worm has been detected in host animals in Louisiana (5, 14) and in one human patient from New Orleans (18), but it is currently unclear to what extent the nematode has spread into other U.S. states (8, 9). Ascertaining the geographic presence of the parasite is important to manage and prevent new cases of eosinophilic meningitis associated with ingestion of infective larvae (12, 18).Detection of A. cantonensis in mollusks can be performed by releasing the larvae from the tissue with pepsin digestion (11). However, that procedure requires access to living mollusks, which complicates analysis of large numbers of samples. After a recent outbreak of angiostrongyliasis in Hawaii (12), we developed a conventional PCR assay and applied it to survey the Hawaiian mollusk population using frozen tissue (20). That PCR assay, as well as morphological identification using pepsin digestion, can only identify the larvae on the superfamily level, so additional molecular work is required for species-specific classification. Here we describe a new real-time PCR assay that allows for a direct detection of A. cantonensis at the species level.The 18S rRNA gene is too conserved among nematode species to allow species-specific detection. The first and second internal transcribed spacers (ITS1 and ITS2) are comparatively more variable than the rRNA coding regions and have thus been used for differentiation of closely related species (1, 4, 6, 10, 22, 23). We PCR amplified and sequenced ITS1 from A. costaricensis (two laboratory strains from Costa Rica and Brazil), A. vasorum (from naturally infected hosts in United Kingdom), and A. cantonensis from three geographical regions (one laboratory strain from Japan plus nine environmental isolates from Hawaii and New Orleans, LA) to assess the variability of this potential PCR target. The oligonucleotide primers used were AngioF1674 (5′-GTCGTAACAAGGTATCTGTAGGTG-3′) and 58SR4 (5′-TAGCTGCGTTTTTCATCGATA-3′). The reaction mixtures contained 0.4 μM each primer and AmpliTaq Gold PCR master mix (Applied Biosystems, Foster City, CA) and were cycled 45 times at 94°C for 30 s, 65°C for 30 s, and 72°C for 1 min. PCR products were cloned into pCR2.1 vectors using the TOPO cloning technique (Invitrogen, Carlsbad, CA) and sequenced on both strands as described elsewhere (20).The sequence analysis revealed high interspecific and low intraspecific variability. A TaqMan assay targeting ITS1 was then designed using Primer Express version 2.3 (Applied Biosystems, Foster City, CA). The real-time PCR assay was performed in a 20-μl total volume containing Platinum qPCR Supermix (Invitrogen, Carlsbad, CA), 0.2 μM (each) primers AcanITS1F1 (5′-TTCATGGATGGCGAACTGATAG-3′) and AcanITS1R1 (5′-GCGCCCATTGAAACATTATACTT-3′), and 0.05 μM the TaqMan probe AcanITS1P1 (5′-6-carboxyfluorescein-ATCGCATATCTACTATACGCATGTGACACCTG-BHQ-3′). The standard cycling conditions for TaqMan assays were used (i.e., 40 cycles of 95°C for 15 s and 60°C for 1 min).We evaluated the real-time PCR assay with a set of 26 Parmarion martensi slugs from Hawaii. Seventeen slugs were positive for L3 larvae as determined by pepsin digestion, and nine slugs were negative. DNA was extracted from approximately 25 mg of tissue of each slug using the DNeasy tissue and blood DNA extraction kit (Qiagen, Inc., Valencia, CA). The real-time PCR performed on this set of samples returned an identical result to the morphological analysis. The real-time PCR amplified only DNA from A. cantonensis and did not react with DNA from other nematode species (Table ​(Table1).1). The detection limit of the assay was determined by serially diluting a recombinant plasmid containing the ITS1 sequence to less than 1 copy per μl of sample. The real-time PCR reliably detected down to 10 plasmid copies in the reaction.

TABLE 1.

Comparison of conventional and real-time PCR for detection of Angiostrongylus cantonensis in mollusks and nematode samples

Tomato BRI1 and systemin wound signalling

Brassinosteroids (BRs) are perceived by Brassinosteroid Insensitive 1 (BRI1), that encodes a leucine-rich repeat receptor kinase. Tomato BRI1 has previously been implicated in both systemin and BR signalling. The role of tomato BRI1 in BR signalling was confirmed, however it was found not to be essential for systemin/wound signalling. Tomato roots were shown to respond to systemin but this response varied according to the species and growth conditions. Overall the data indicates that mutants defective in tomato BRI1 are not defective in systemin-induced wound signalling and that systemin perception can occur via a non-BRI1 mechanism.Key words: tomato BRI1, brassinosteroids, systemin, wound signallingBrassinosteroids (BRs) are steroid hormones that are essential for normal plant growth. The most important BR receptor in Arabidopsis is BRASSINOSTERIOD INSENSITIVE 1 (BRI1), a serine/threonine kinase with a predicted extracellular domain of ∼24 leucine-rich repeats (LRRs).^1,2 BRs bind to BRI1 via a steroid-binding domain that includes LRR 21 and a so-called “island” domain.^2,3 In tomato a BRI1 orthologue has been identified that when mutated, as in the curl3 (cu3) mutation, results in BR-insensitive dwarf plants.⁴ Tomato BRI1 has also been purified as a systemin-binding protein.⁵ Systemin is an eighteen amino acid peptide, which is produced by post-translational cleavage of prosystemin. Systemin has been implicated in wound signalling and is able to induce the production of jasmonate, protease inhibitors (PIN) and rapid alkalinization of cell suspensions (reviewed in ref. ⁶).To clarify whether tomato BRI1 was indeed a dual receptor it was important to first confirm its role in BR signalling. Initially this was carried out by genetic complementation of the cu3 mutant phenotype.⁷ Overexpression of tomato BRI1 restored the dwarf phenotype and BR sensitivity and normalized BR levels (35S:TomatoBRI1 complemented lineWt^*cu3^*6-deoxocathasterone5669646766-deoxoteasteronend47483-dehydro-6-deoxoteasterone8762696-deoxotyphasterolnd5884226-deoxocastasterone1,7556,24726,210castasterone25563717,428brassinolidendndndOpen in a separate windowBR content ng/kg fw.^*Montoya et al.⁴ nd, not detected.To show the role of tomato BRI1 in systemin signalling tomato BR mutants and the complemented line were tested for their systemin response. Tomato cu3 mutants were shown not to be defective in systemin-induced proteinase inhibitor (PIN) gene induction, nor were they defective in PIN gene induction in response to wounding. Cell suspensions made from cu3 mutant tissue exhibited an alkalinization of culture medium similar to wild-type cell suspension. These data taken together indicated that BRI1 was not essential for systemin signalling. However, Scheer et al.⁸ demonstrated that the overexpression of tomato BRI1 in tobacco suspension cultures results in an alkalinization in response to systemin, which was not observed in untransformed cultures. This suggests that BRI1 is capable of eliciting systemin responsiveness and that in tomato BRI1 mutants another mechanism is functioning to enable systemin signalling.Root elongation is a sensitive bioassay for BR action with BRs inhibiting root growth. Solanum pimpinellifolium roots elongate in response to systemin, in a BRI1-dependent fashion. In Solanum lycopersicum root length was reduced in response to systemin and BR and jasmonate synthesis mutants indicated that the inhibition did not require jasmonates or BRs. Normal ethylene signalling was required for the root response to systemin. When a tobacco, Nicotiana benthamiana, BRI1 orthologue was transformed into cu3 both the dwarfism and systemin-induced root elongation was restored to that of wild type. Tobacco plants however do not respond to systemin. This is puzzling as the introduction of tomato BRI1 into tobacco enabled systemin responsiveness.⁸ Further investigation as to how tomato BRI1 elicits this response is therefore required.Systemin has been demonstrated to bind to two tomato proteins BRI1/SR160⁵ and SBP50.⁹ The data presented by Holton et al.⁷ indicates that tomato BRI1 is not essential for systemin-induced wound responses and that a non-BRI1 pathway is present that is able to facilitate a systemin response. Whether this is via a related LRR receptor kinase or by another protein remains to be elucidated.