Cell Wall Fragments Research Articles

Monocyte-Derived Dendritic Cells Pulsed with Aspergillus Fumigatus Components Enhance the Anti-Aspergillus Effect of Human Natural Killer Cells

Abstract 2983Dendritic cells (DCs) are well characterized for their ability to induce adaptive immune responses. To mimic these properties, in vitro generated monocyte-derived DCs (moDCs) have been extensively studied and used as antigen presenting cells, initiating specific T cell immune responses against virus infected and malignant cells. There is increasing evidence that the immunoregulatory effect of DCs is exerted also towards innate immune effector cells, like NK cells. Recently, we have shown a significant anti-Aspergillus fumigatus impact of NK cells, mainly mediated by the release of IFN- γ, which demonstrated direct fungicidal properties. The interest for our study is driven by the high mortality characterizing invasive aspergillosis, an infection of immunosuppressed patients caused mainly by this fungus. To date, it has been shown that NK cells activated by DCs demonstrate increased IFN- γ release, proliferation and cytotoxicity against malignant cells. In our study, we have investigated the potential effect of moDCs, previously pulsed with fungal proteins and with cell wall fragments on NK cells and on their cytotoxicity against A. fumigatus.For the generation of moDCs, we used CD14+ cells isolated from peripheral blood of healthy donors by positive selection with magnetic microbeads. Cells were cultured for 5 days in serum-free GMP grade medium supplemented with GM-CSF and IL-4. On the second day of culture, immature DCs were pulsed either with a β-(1, 3)- glucan–rich alkali-insoluble (AI) cell wall fraction extracted from fungal conidia or from mycelia, inactivated conidia and germ tubes or with the recombinant catalase Cat1 and transglycosidase Crf1. Three hours later, DCs were maturated with TNF- α. Before being used, DC properties were characterized by quantifying the expression of CD1a, CD14, CD40, CD80, CD83, CD86, HLA-I, HLA-DR, CCR7, Dectin-1, TLR-2, TLR-4, IL12p35, IL15, and IFN- γ. Autologous NK cells were obtained by magnetic negative selection of peripheral mononuclear cells and were kept frozen in human serum supplemented with 8% DMSO at −80°C. DC/ NK cell co-cultures were set in unconditioned GMP grade medium on different ratios (1:1, 1:5, 1:10). To evaluate proliferation, NK cells were stained with CFSE. Proliferation was measured at the 7th day of DC / NK co-culture. IFN- γ release by NK cells was quantified by FACS analysis after 16 h of DC / NK co-culture. Finally, the antifungal impact on A. fumigatus germ tubes was evaluated by 2, 3-bis[2-methoxy-4-nitro-5-sulpho-phenyl]2H-tetrazolium-5-carboxanilide (XTT) assays.DCs pulsed with the different fungal stimuli demonstrated a ‘mature' surface profile with negative CD1a & CD14 expression and high positivity of CD40, CD80, CD83, CD86, HLA-I, HLA-DR & CCR7. All DCs produced IL12p35 and IL15, but no IFN- γ. The expression of Dectin-1 was strongly down-regulated (p<.001) by the alkali-insoluble cell wall fraction and by germlings, moderately by conidia (p<.05) and it was unaffected after Cat1 and Crf1 stimulation. This observation suggests a major involvement of Dectin-1 and its ligand, β-(1, 3)-glucan in the interaction of moDCs with A. fumigatus. In contrast, TLR-2 and TLR-4 were not found to be regulated on DCs. Furthermore, DCs induced the proliferation of NK cells, higher DC / NK ratios elicited higher NK cell expansion. DCs stimulated with AI cell wall fractions or germlings provoked a significantly higher proliferation of NK cells in comparison to unstimulated DCs (p<.05). Similarly, significant up-regulation of IFN- γ expression was observed in NK cells co-cultured with DCs previously pulsed with germ tubes and with fungal cell wall fractions, respectively (p<.05). Finally, we revealed significant higher fungal damage in NK cell / DC co-cultures (especially in those primed with fungal cell wall fractions or germlings), compared to NK cell or DC cultures alone (p<.05).To summarize, our study shows for the first time that moDCs pulsed with A. fumigatus cell wall components induce NK cell proliferation and potentiate IFN- γ release and cytotoxicity against this fungus. These results provide evidence for a role of moDCs as activators of NK cells towards an immune response against IA. Disclosures:No relevant conflicts of interest to declare.

Modular synthesis of diphospholipid oligosaccharide fragments of the bacterial cell wall and their use to study the mechanism of moenomycin and other antibiotics

We present a flexible, modular route to Glc NAc–Mur NAc-oligosaccharides that can be readily converted into peptidoglycan (PG) fragments to serve as reagents for the study of bacterial enzymes that are targets for antibiotics. Demonstrating the utility of these synthetic PG substrates, we show that the tetrasaccharide substrate lipid IV ( 3), but not the disaccharide substrate lipid II ( 2), significantly increases the concentration of moenomycin A required to inhibit a prototypical PG-glycosyltransferase (PGT). These results imply that lipid IV and moenomycin A bind to the same site on the enzyme. We also show the moenomycin A inhibits the formation of elongated polysaccharide product but does not affect length distribution. We conclude that moenomycin A blocks PG-strand initiation rather than elongation or chain termination. Synthetic access to diphospholipid oligosaccharides will enable further studies of bacterial cell wall synthesis with the long-term goal of identifying novel antibiotics.

Anisotropy of cell wall polymers in branches of hardwood and softwood: a polarized FTIR study

The mechanical and physical properties of wood fibres are dependent on the organisation of their constituent polymers (cellulose, hemicellulose and lignin). Fourier Transform Infrared (FTIR) microscopy was used to examine the anisotropy of the main wood polymers in isolated cell wall fragments from branches of maple and Serbian spruce. Polarised FTIR measurements indicated an anisotropy, i.e. orientation of the cellulose microfibrils that was more or less parallel to the longitudinal axis of the cell wall. The hemicelluloses, glucomannan and xylan appeared to have a close link to the orientation of the cellulose and, thus, an orientation more parallel to the axis of the cell wall. An important result is that, in both maple and spruce samples, lignin was found to be organised in a parallel way in relation to the longitudinal cell wall axis, as well as to the cellulose. The results show that, despite the different lignin precursors and the different types of hemicelluloses in these two kinds of wood, lignin has a similar orientation, when it comes to the longitudinal axis of the cell wall.

Antiviral activity of Lactobacillus brevis towards herpes simplex virus type 2: Role of cell wall associated components

The aim of this research was to study the mechanisms of Lactobacillus brevis antiviral activity towards HSV-2 and to identify the bacterial components responsible for the inhibiting effect. Bacterial extract and cell walls were prepared by lysozyme digestion of L. brevis cells untreated or treated with LiCl to remove S-layer proteins. Bacterial extract and cell wall fragments showed a dose dependent inhibitory effect on HSV-2 multiplication. In order to characterize the inhibitory activity of L. brevis, the bacterial extract was subjected to different physical and chemical treatments. The inhibitory activity was resistant to high temperature and proteases digestion and appeared to be associated with compounds with a molecular weight higher than 10 kDa. DNA, RNA and lipids isolated from bacterial cells were devoid of inhibitory effect. The antiviral activity of both bacterial extract and cell wall fragments obtained from L. brevis cells after the S-layer removal was significantly reduced compared to untreated cells suggesting that the inhibitory activity is likely due to a heat-resistant non-protein cell surface bacterial component.

The continuing debate over the structure of cellulose: historical perspective and outlook

The crystalline structure of native cellulose, with its unique microfibrillar morphology resulting from a complex biogenesis mechanism, has not only been the most studied subject in polymer science, but is still generating great interest along with lively debates and controversies. This issue of Cellulose features such a debate in the form of two articles dealing with the conformational analysis of the glucosidic bond in the cellulose chains. This debate is but the latest incident in a long string of events, which represent the foundation of our current understanding of this important material. This makes this an opportune moment to reflect on some of the major milestones in the history of cellulose structure evaluation, which also often involved debates and controversies. It was in 1913, i.e. close to one century ago, that Nishikawa and Ono showed the first diffraction patterns of two cellulose-based plant materials, namely bamboo and hemp, taking advantage of the discovery of the interference phenomena of X-rays reported 1 year earlier by Laue, Friedrich and Knipping. Following this early report, diffraction diagrams of a large number of cellulose materials were duly recorded and subjected to analysis, as it was felt that the knowledge of the crystalline structure of cellulose was of great importance for the development and improvement of cellulose-based products. While recording diffraction patterns of these specimens, it was soon realized that even the X-ray data sets of the most crystalline samples remained limited and it was therefore hard to deduce from them alone the required structural details. It is only when the diffraction data could be complemented by the occurrence of new techniques that substantial advances toward the structural description of cellulose could be made. The sequential emergence of such techniques explains the succession of steps leading to the present understanding. In the progress toward the molecular description of the crystal structure of cellulose from X-ray diffraction data, a first landmark was the incorporation of the stereochemistry of the glucosyl moieties in the molecular models built by, among others, Meyer and Mark (1928), later revised by Meyer and Misch (1937), who presented a model where the crystal of cellulose consisted of two antiparallel chains. This last model, which remained the established standard for at least two decades, was confronted with a series of conflicting data, resulting from new techniques. In 1958, the recording of electron diffraction diagrams of frozen Valonia cell wall fragments allowed Honjo and Watanabe to obtain diffraction data sets far better H. Chanzy—affiliated with the Universite Joseph Fourier and member of the Institut de Chimie Moleculaire de Grenoble.

Neglected Food Bubbles: The Espresso Coffee Foam.

Coffee beverage known as espresso, must be topped by a velvety thick, reddish-brown foam called crema, to be considered properly prepared and to be appreciated by connoisseurs. In spite of the relevant role played by crema as a quality marker, espresso coffee foam has not yet been the subject of detailed investigations. Only recently, some aspects of the Physics and Chemistry behind the espresso coffee foam have attracted the attention of scientists. In addition to sharing several characteristics with other food foams like beer foam, for instance, the espresso coffee foam may contain solid particles (minute coffee cell-wall fragments), it is subjected to a remarkable temperature gradient and its continuous phase is an oil in water emulsion rendering it a very complex system to be studied. Moreover, in the typical regular espresso coffee cup volume (serving) of 25–30 mL, crema represents at least 10% of the total volume, and this is a limitation in obtaining experimental data by conventional instruments. The present work is aimed at reviewing the literature on espresso coffee foam. The traditional espresso brewing method will be briefly described with emphasis on the steps particularly relevant to foam formation and stabilization. In addition to present up-dated experimental data on surface properties at solid/beverage and air/beverage interface, recent advances on the espresso foam formation mechanism, as well as on foam stability, will be critically examined. The key role played by carbon dioxide generated by roasting and the effects of low and high-molecular-weight coffee compounds in promoting/inhibiting the espresso coffee foam will be discussed and emphasized.

Peptide mimics of peptidoglycan are vaccine candidates and protect mice from infection with Staphylococcus aureus

Staphylococcus aureus drug resistance to antibiotics is a serious situation that has drawn greater attention to immunotherapy and prophylaxis. Peptidoglycan (PGN) is a common and conserved component of the cell wall of Gram-positive bacteria such as S. aureus. However, PGN, as a thymus-independent antigen, cannot be considered a vaccine candidate because of its very weak immunogenicity. In this study we have attempted to enhance the immunogenicity of PGN by identifying a PGN peptide mimic sequence that would act as a thymus-dependent antigen. Several peptide sequences were obtained from a phage display peptide library using a mAb against S. aureus PGN, and a 12-mer linear single peptide (Sp-31) and a four-branch multiple antigen peptide (MAP) (MAP-P31) were synthesized. Both Sp-31 and MAP-P31 were shown to bind directly to anti-PGN mAb and a polyclonal antibody against S. aureus. These peptides could also inhibit the binding of PGN to a mAb against PGN. Furthermore, MAP-P31 was able to provoke an effective secondary antibody response in mice to PGN and to cell-wall fragments isolated from S. aureus, Escherichia coli, Staphylococcus epidermidis and Pseudomonas aeruginosa by sonication. In addition, the MAP-P31 antiserum showed a potent bactericidal or bacteriostatic activity against S. aureus in the presence and absence of complement in vitro. Importantly, immunization with MAP-P31 significantly prolonged the survival and enhanced bacterial clearance in BALB/c mice challenged with live S. aureus. In addition, the serum IgG-type antibodies against PGN persisted in mice, demonstrating that MAP-P31, as a peptide mimicking epitopes on PGN, provokes an effective secondary or memory antibody response, which can only be induced by a thymus-dependent antigen and which protects against infection with S. aureus. These results suggest that MAP-31 may be a novel vaccine candidate against S. aureus.

Selective lignin downregulation leads to constitutive defense response expression in alfalfa (Medicago sativa L.)

• Downregulation of hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) in alfalfa (Medicago sativa) reduces lignin levels and improves forage quality and saccharification efficiency for bioethanol production. However, the plants have reduced stature. It was previously reported that HCT-down-regulated Arabidopsis have impaired auxin transport, but this has recently been disproved. • To address the basis for the phenotypes of lignin-modified alfalfa, we measured auxin transport, profiled a range of metabolites including flavonoids and hormones, and performed in depth transcriptome analyses. • Auxin transport is unaffected in HCT antisense alfalfa despite increased flavonoid biosynthesis. The plants show increased cytokinin and reduced auxin levels, and gibberellin levels and sensitivity are both reduced. Levels of salicylic, jasmonic and abscisic acids are elevated, associated with massive upregulation of pathogenesis and abiotic stress-related genes and enhanced tolerance to fungal infection and drought. • We suggest that HCT downregulated alfalfa plants exhibit constitutive activation of defense responses, triggered by release of bioactive cell wall fragments and production of hydrogen peroxide as a result of impaired secondary cell wall integrity.

Characterization of the Medicago truncatula cell wall proteome in cell suspension culture upon elicitation and suppression of plant defense

In addition to establishing methods for proteome analysis of cell wall proteins (CWPs) for the model plant Medicago truncatula, this work highlights the presence of several protein classes in cell culture. Using a combination of two-dimensional gel electrophoresis (2D-PAGE) and/or liquid chromatography–tandem mass spectrometry (LC–MS/MS), we established the proteome reference map of M. truncatula cell wall proteins. CWPs extracted from purified cell wall fragments resulted in the identification of 46 (2D-PAGE) and 65 (LC–MS/MS) proteins, respectively, with a total of 111 proteins. The identified proteins are involved in various processes, including cell wall modifications, signaling, defense mechanisms, membrane transport, protein synthesis and processing. Further, we conducted comparative proteome analysis to identify changes in protein composition during interaction of M. truncatula cell suspension culture with a pathogen-derived yeast elicitor (YE) and suppressor using Sinorhizobium meliloti LPS. 2D-PAGE analysis for the CWPs after YE and LPS treatment resembled the proteome map of YE alone, with a few up-regulated proteins involved in defense, and in the case of the LPS-treated cell wall proteome, there was no significant difference observed. Using this approach, proteins involved in defense, such as l-ascorbate peroxidase, specifically targeted proteins to the cell wall during defense, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and proteins that play an important role during growth and development were identified. Also, some defense-related proteins were absent in the same gel after YE treatment, suggesting that oxidant protection is regulated by these proteins.

Enhanced disease resistance in transgenic carrot (Daucus carota L.) plants over-expressing a rice cationic peroxidase

Plant class III peroxidases are involved in numerous responses related to pathogen resistance including controlling hydrogen peroxide (H(2)O(2)) levels and lignin formation. Peroxidases catalyze the oxidation of organic compounds using H(2)O(2) as an oxidant. We examined the mechanisms of disease resistance in a transgenic carrot line (P23) which constitutively over-expresses the rice cationic peroxidase OsPrx114 (previously known as PO-C1) and which exhibits enhanced resistance to necrotrophic foliar pathogens. OsPrx114 over-expression led to a slight enhancement of constitutive transcript levels of pathogenesis-related (PR) genes. These transcript levels were dramatically increased in line P23 compared to controls [GUS construct under the control of 35S promoter (35S::GUS)] when tissues were treated with cell wall fragments of the fungal pathogen Sclerotinia sclerotiorum (SS-walls), and to a lesser extent with 2,6-dichloroisonicotinic acid. There was no basal increase in basal H(2)O(2) levels in tissues of the line P23. However, during an oxidative burst response elicited by SS-walls, H(2)O(2) accumulation was reduced in line P23 despite, typical media alkalinization associated with oxidative burst responses was observed, suggesting that OsPrx114 was involved in rapid H(2)O(2) consumption during the oxidative burst response. Tap roots of line P23 had increased lignin formation in the outer periderm tissues, which was further increased during challenge inoculation with Alternaria radicina. Plant susceptibility to a biotrophic pathogen, Erysiphe heraclei, was not affected. Disease resistance to necrotrophic pathogens in carrot as a result of OsPrx114 over-expression is manifested through increased PR transcript accumulation, rapid removal of H(2)O(2) during oxidative burst response and enhanced lignin formation.

IL-32γ and Streptococcus pyogenes cell wall fragments synergise for IL-1-dependent destructive arthritis via upregulation of TLR-2 and NOD2

ObjectiveTo investigate the potential synergism between interleukin (IL) 32γ and Streptococcus pyogenes cell wall (SCW) fragments in the development of destructive arthritis.MethodsAn adenoviral vector encoding human IL-32γ (AdIL-32γ) was constructed...

Programmed Cell Death and Adaptation: Two Different Types of Abiotic Stress Response in a Unicellular Chlorophyte

Eukaryotic microalgae are highly suitable biological indicators of environmental changes because they are exposed to extreme seasonal fluctuations. The biochemical and molecular targets and regulators of key proteins involved in the stress response in microalgae have yet to be elucidated. This study presents morphological and biochemical evidence of programmed cell death (PCD) in a low temperature strain of Chlorella saccharophila induced by exposure to NaCl stress. Morphological characteristics of PCD, including cell shrinkage, detachment of the plasma membrane from the cell wall, nuclear condensation and DNA fragmentation, were observed. Additionally, a significant production of H(2)O(2) and increase in caspase 3-like activity were detected. We demonstrated that singly applied environmental stresses such as warming or salt stress trigger a pathway of PCD. Intriguingly, the prior application of salt stress seems to reduce heat shock-induced cell death significantly, suggesting a combined effect which activates a defense mechanism in algal cells. These results suggest that C. saccharophila can undergo PCD under stress conditions, and that this PCD shares several features with metazoan PCD. Moreover, the simultaneous exposure of this unicellular chlorophyte to different abiotic stresses results in a tolerance mechanism.

Caracterización de levaduras Debaryomyces hansenii para el control biológico de la podredumbre azul del limón mexicanoCharacterization of yeast Debaryomyces hansenii for the biological control of blue mold decay of Mexican lemon

Twelve yeast Debaryomyces hansenii strains were characterized for their ability to inhibit in vitro growth of Penicillium italicum, production of lytic enzymes (β-1.3-glucanase, chitinase and protease) and postharvest biological control of blue mold decay (caused by P. italicum) on Mexican lemon fruits. The D. hansenii strains inhibited in vitro the growth of P. italicum in different pHs (4.2, 4.6 y 5.0), effect related to killer toxins. The enzyme activity (of β-1.3-glucanase, chitinase and protease) was detected on Yeast Nitrogen Base (YNB) medium inoculated with cell wall fragments of P. italicum, the range enzymatic activity of all strains was between 9 and 13 days after inoculated culture medium. All D. hansenii strains significantly reduced blue mold on Mexican lemon during fifteen days of storage at 13 °C, but the LL1 strain was distinctly more effective under these conditions. These results showed that D. hansenii is an alternative for biological control of blue mold decay of Mexican lemon fruits.

Perception of the Arabidopsis Danger Signal Peptide 1 Involves the Pattern Recognition Receptor AtPEPR1 and Its Close Homologue AtPEPR2

Plasma membrane-borne pattern recognition receptors, which recognize microbe-associated molecular patterns and endogenous damage-associated molecular patterns, provide the first line of defense in innate immunity. In plants, leucine-rich repeat receptor kinases fulfill this role, as exemplified by FLS2 and EFR, the receptors for the microbe-associated molecular patterns flagellin and elongation factor Tu. Here we examined the perception of the damage-associated molecular pattern peptide 1 (AtPep1), an endogenous peptide of Arabidopsis identified earlier and shown to be perceived by the leucine-rich repeat protein kinase PEPR1. Using seedling growth inhibition, elicitation of an oxidative burst and induction of ethylene biosynthesis, we show that wild type plants and the pepr1 and pepr2 mutants, affected in PEPR1 and in its homologue PEPR2, are sensitive to AtPep1, but that the double mutant pepr1/pepr2 is completely insensitive. As a central body of our study, we provide electrophysiological evidence that at the level of the plasma membrane, AtPep1 triggers a receptor-dependent transient depolarization through activation of plasma membrane anion channels, and that this effect is absent in the double mutant pepr1/pepr2. The double mutant also fails to respond to AtPep2 and AtPep3, two distant homologues of AtPep1 on the basis of homology screening, implying that the PEPR1 and PEPR2 are responsible for their perception too. Our findings provide a basic framework to study the biological role of AtPep1-related danger signals and their cognate receptors.

GanedenBC30 cell wall and metabolites: anti-inflammatory and immune modulating effects in vitro.

BackgroundThis study was performed to evaluate anti-inflammatory and immune modulating properties of the probiotic, spore-forming bacterial strain: Bacillus coagulans: GBI-30, (PTA-6086, GanedenBC30TM). In addition, cell wall and metabolite fractions were assayed separately to address whether biological effects were due to cell wall components only, or whether secreted compounds from live bacteria had additional biological properties. The spores were heat-activated, and bacterial cultures were grown. The culture supernatant was harvested as a source of metabolites (MTB), and the bacteria were used to isolate cell wall fragments (CW). Both of these fractions were compared in a series of in vitro assays.ResultsBoth MTB and CW inhibited spontaneous and oxidative stress-induced ROS formation in human PMN cells and increased the phagocytic activity of PMN cells in response to bacteria-like carboxylated fluorospheres. Both fractions supported random PMN and f-MLP-directed PMN cell migration, indicating a support of immune surveillance and antibacterial defense mechanisms. In contrast, low doses of both fractions inhibited PMN cell migration towards the inflammatory mediators IL-8 and LTB4. The anti-inflammatory activity was strongest for CW, where the PMN migration towards IL-8 was inhibited down to dilutions of 1010.Both MTB and CW induced the expression of the CD69 activation marker on human CD3- CD56+ NK cells, and enhanced the expression of CD107a when exposed to K562 tumor cells in vitro.The fractions directly modulated cytokine production, inducing production of the Th2 cytokines IL-4, IL-6, and IL-10, and inhibiting production of IL-2.Both fractions further modulated mitogen-induced cytokine production in the following manner: Both fractions enhanced the PHA-induced production of IL-6 and reduced the PHA-induced production of TNF-alpha. Both fractions enhanced the PWM-induced production of TNF-alpha and IFN-gamma. In addition, MTB also enhanced both the PHA- and the PWM-induced expression of IL-10.ConclusionThe data suggest that consumption of GanedenBC30TM may introduce both cell wall components and metabolites that modulate inflammatory processes in the gut. Both the cell wall and the supernatant possess strong immune modulating properties in vitro. The anti-inflammatory effects, combined with direct induction of IL-10, are of interest with respect to possible treatment of inflammatory bowel diseases as well as in support of a healthy immune system.

Experimental epithelioid cell granuloma formation in Lewis rats induced by injection of cell wall fragments derived from alpha-streptococcus--etiologic relationship between bacterial cell wall and sarcoid granuloma

We observed the presence of epithelioid granulomas in follicles near lacunae in the tonsils from patients with active sarcoidosis. Bacteria isolated in these tissues were mostly alpha-streptococci. The present study was undertaken to determine whether alpha-streptococcus can induce granuloma formation. Streptococcal cell wall (SCW) fragments were injected into the foot pads of female Lewis rats. Epithelioid granulomas were abundantly formed in popliteal lymph nodes when SCW aqueous suspension was injected four times. SCW antigens were detected in macrophages in the granuloma but not in epithelioid cells, by immunoperoxidase method. These findings suggest that macrophages transform into epithelioid cells after phagocytosis and digestion of SCW, and that components of bacterial cell wall such as SCW may induce the granuloma formation in sarcoidosis.

Role of the cytoplasmic pattern recognition receptor Nod2 in Coxiella burnetii infection

Q fever is caused by Coxiella burnetii, an obligate intracellular bacterium, which survives in an acidic vacuole and escapes killing by preventing phagosome maturation [1]. In acute Q fever, granuloma formation is dependent on migration of leucocytes through the vascular endothelium. During chronic Q fever, granulomas are lacking and monocytes do not migrate across the endothelium [1], suggesting that host resistance against C. burnetii requires innate immune components. Toll-like receptors (TLRs) are pattern recognition receptors important in C. burnetii recognition. Although TLR4 ⁄) and control mice have a similar pattern of tissue infection and clearance, granuloma formation and cytokine response are controlled, in part, by TLR4 [1]. TLR2 is also involved in granuloma formation but seems dispensable for bacterial clearance [2]. The obligate intracellular lifestyle of C. burnetii drew our attention to elucidate the contribution of another family of proteins implicated in microbial detection, the cytoplasmic nucleotide-binding oligomerization domain (Nod) proteins, and more specifically Nod2, in innate immunity to C. burnetii. Nod2 contains leucine-rich repeats similar to those found in TLRs and is thought to be responsible for recognition of peptidoglycans from Gram positive and Gram negative bacteria through direct or indirect interaction with muramyl dipeptide. Nod2 signalling contributes to inflammatory response via activation of the transcription factor NF-jB, which is necessary for clearing pathogens. The intracellular localisation of Nod2 and C. burnetii makes Nod2 a potential candidate for C. burnetii recognition through leaking into the cytoplasm of muramyl dipeptide-containing cell wall fragments. Bone marrow-derived macrophages (BMDM) from control and Nod2 ⁄) mice [3] were incubated with C. burnetii for 4 h and cultured for 12 days. Infection was measured by determining the number of C. burnetii DNA copies as previously described [2]. Uptake and survival of C. burnetii in BMDM did not depend on Nod2 as DNA copy number was similar in control and Nod2 ⁄) BMDM (data not shown). Next, we tested whether the production of cytokines ⁄ chemokines by C. burnetii-stimulated BMDM required Nod2. We assessed the expression of genes encoding pro-inflammatory (TNF) and immunoregulatory cytokines (IL-10). BMDM were stimulated for 8 h with C. burnetii and gene expression was monitored by quantitative RTPCR [2]. In Nod2 ⁄) BMDM, although TNF transcripts were induced, they were significantly reduced compared with control macrophages (Fig. 1a). This result agrees with other studies showing that TNF and IL-12p40 levels are reduced in the absence of Nod2 after stimulation with Mycobacterium tuberculosis [4]. Note that IL10 was not affected by the absence of Nod2 (Fig. 1a). IL-10 is known to interfere with macrophage activation by stimulating the replication of C. burnetii through decreasing TNF production and blocking phagosomal maturation [1]. IL-10 is also associated with the chronic evolution of Q fever [1]. The absence of IL-10 modulation observed in Nod2 ⁄) BMDM is consistent with these data, as we did not observe any increase in bacterial replication. We also measured the expression of RANTES and MCP-1, two chemokines modulated in response to C. burnetii [5]. RANTES expression was fourfold increased in Nod2 ⁄) BMDM whereas MCP-1 expression was decreased by 40% (Fig. 1a). It is likely that RANTES and MCP-1 play a role in Q fever pathophysiology, as their production is Corresponding author and reprint requests: B. Desnues, CNRS UMR 6236, Faculte de Medecine de la Timone, 27 Bd J. Moulin, 13385 Marseille Cedex 05, France. E-mail: benoit.desnues@univmed.fr

Investigation into the intragranular structures of microcrystalline cellulose and pre-gelatinised starch

The internal structures of commercial spheronised microcrystalline cellulose (s-MCC) and pre-gelatinised starch (PGS) granules were investigated, using a range of methods. Results from scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM) revealed hierarchical structures, with dimensions ranging from nanometres to a few micrometres, for both materials. Residual fragments of plant cell walls, consisting largely of crystalline fibrillar bundles, were indicated within s-MCC granules, while PGS granules appeared to consist of densely packed spherical features. The lack of any obvious regular periodicity associated with the intragranular sub-structures was entirely consistent with the power-law behaviour of the small-angle X-ray scattering (SAXS) patterns from these materials. The presence of intragranular porosity was inferred from TEM, AFM and N(2)-adsorption measurements, while the ability to deform these structures was clearly indicated by the irregular force-displacement curves recorded by AFM on the granule surfaces. Hence, the intragranular sub-structures observed for s-MCC and PGS appeared to be consistent with the possibility of entire granules undergoing affine deformation during compaction. Since this mechanism was postulated to explain changes in SAXS patterns from these materials following compaction, as reported elsewhere, the work reported here provides a considerably stronger basis for using 2D-SAXS to investigate powder compaction behaviour.

Enhancement of Diosgenin Production in Dioscorea zingiberensis Cell Culture by Oligosaccharide Elicitor from its Endophytic Fungus Fusarium oxysporum Dzf17

Diosgenin accumulation in cell suspension cultures of Dioscorea zingiberensis C. H. Wright was enhanced by treatment with saccharide elicitors from its endophytic fungus Fusarium oxysporum isolate Dzf17. The crude oligosaccharide was prepared by partial acid hydrolysis of the isolated Dzf17 fungal cell wall fragments. Optimal elicitation of diosgenin production by the isolated Dzf17 oligosaccharide in cell suspension culture was achieved when the oligosaccharide was added to the medium at a concentration of 30 mg/L after 16-day's continuous cell suspension culture, and the cells were cultured for another 8 days before harvesting. By using these optimal conditions, the diosgenin yield of the cultured cells reached its maximum of 5.25 mg/L, which was over a three-fold increase.

Broad-spectrum disease resistance to necrotrophic and biotrophic pathogens in transgenic carrots (Daucus carota L.) expressing an Arabidopsis NPR1 gene

The development of transgenic plants highly resistant to a range of pathogens using traditional signal gene expression strategies has been largely ineffective. Modification of systemic acquired resistance (SAR) through the overexpression of a controlling gene such as NPR1 (non-expressor of PR genes) offers an attractive alternative for augmenting the plants innate defense system. The Arabidopsis (At) NPR1 gene was successfully introduced into 'Nantes Coreless' carrot under control of a CaMV 35S promoter and two independent transgenic lines (NPR1-I and NPR1-XI) were identified by Southern and Northern blot hybridization. Both lines were phenotypically normal compared with non-transformed carrots. Northern analysis did not indicate constitutive or spontaneous induction in carrot cultures of SAR-related genes (DcPR-1, 2, 4, 5 or DcPAL). The duration and intensity of expression of DcPR-1, 2 and 5 genes were greatly increased compared with controls when the lines were treated with purified cell wall fragments of Sclerotinia sclerotiorum as well as with 2,6-dichloroisonicotinic acid. The two lines were challenged with the necrotrophic pathogens Botrytis cinerea, Alternaria radicina and S. sclerotiorum on the foliage and A. radicina on the taproots. Both lines exhibited 35-50% reduction in disease symptoms on the foliage and roots when compared with non-transgenic controls. Leaves challenged with the biotrophic pathogen Erysiphe heraclei or the bacterial pathogen Xanthomonas hortorum exhibited 90 and 80% reduction in disease development on the transgenic lines, respectively. The overexpression of the SAR controlling master switch in carrot tissues offers the ability to control a wide range of different pathogens, for which there is currently little genetic resistance available.