Cell Wall

Detection of the Mycobacterium tuberculosis cell wall antigen lipoarabinomannan (LAM) in urine permits diagnoses of tuberculosis (TB) to be made in HIV-infected patients with advanced immunodeficiency. This can be achieved at the point-of-care within just 30 minutes using the Determine TB-LAM, which is a commercially available, lateral-flow urine 'strip test' assay. The assay has been shown to have useful diagnostic accuracy in patients enrolling in antiretroviral treatment services or in HIV-infected patients requiring admission to hospital medical wards in sub-Saharan Africa. Such patients have high mortality risk and have most to gain from rapid diagnosis of TB and immediate initiation of treatment. However, few studies using this assay have yet been reported and many questions remain concerning the correct use of the assay, interpretation of results, the role of the assay as an add-on test within existing diagnostic algorithms and the types of further studies needed. In this paper we address a series of questions with the aim of informing the design, conduct and interpretation of future studies. Specifically, we clarify which clinical populations are most likely to derive benefit from use of this assay and how patients enrolled in such studies might best be characterised. We describe the importance of employing a rigorous microbiological diagnostic reference standard in studies of diagnostic accuracy and discuss issues surrounding the specificity of the assay in different geographical areas and potential cross-reactivity with non-tuberculous mycobacteria and other organisms. We highlight the importance of careful procedures for urine collection and storage and the critical issue of how to read and interpret the test strips. Finally, we consider how the assay could be used in combination with other assays and outline the types of studies that are required to build the evidence base concerning its use.

A serologic survey of anti-Brucella spp. antibodies was undertaken on 2,470 samples of 14 North American marine mammal species collected between 1984-97. Serum or blood from eight species of cetaceans and six species of pinnipeds was sampled from Pacific, Atlantic, and Arctic oceans. Two competitive enzyme-linked immunosorbent assays (C-ELISA's), using specific monoclonal antibodies to Brucella abortus cell wall components, were used to detect anti-Brucella spp. antibodies in the samples. Sera from 33 cetaceans and 61 pinnipeds gave inhibition values, in one or both of the tests, which exceeded the threshold that indicates Brucella spp. exposure in cattle. Seropositive animals were identified from Pacific, Atlantic, and Arctic oceans. While Brucella spp. was not isolated, differences in the response of seropositive cetacean and pinniped sera in the two assays suggest that two antigenically distinct species or biovars of Brucella spp. are present. No pathology consistent with clinical brucellosis was noted in any of the animals tested although detailed examination was not conducted on all carcasses.

for Escherichia coli and Staphylococcus epidermidis. Large intensity differences seen in the tryptophan-associated 1556-cm-' peak appear to be strongly related to Gram type. Unlike E. coli, S. epidermidis possess a very intense peak at 1658 cm-' which varies in intensity with cultural conditions. Spectra excited from E. coil and Enterobacter aerogenes with 200-nm light show peaks which strongly reflect nucleic acid composition, unlike spectra excited at 218 nm. Purified, separated ribosomes of E.

The influence on the adherence of Clostridium difficile to Vero cells of the yeast Saccharomyces boulardii, the yeast fractions (cytoplasm and cell wall) and the culture supernatant was investigated in vitro. C. difficile adherence was significantly inhibited when bacteria were pre-incubated with the whole yeast and the cell wall fraction; this adherence inhibition was dose-dependent. The cell wall fraction also acts upon the target cultured cells inasmuch as the level of adherence was significantly decreased when Vero cells were preincubated with it. The same experiments carried out in the presence of an inhibitor of serine proteases resulted in no inhibition of bacterial adherence. These results suggest that the yeast could inhibit adherence of C. difficile to cells thanks to its proteolytic activity but also through steric hindrance.

Streptococcus mutans is generally considered to be the principal etiological agent for dental caries. Many of the proteins necessary for its colonization of the oral cavity and pathogenesis are exported to the cell surface or the extracellular matrix, a process that requires the assistance of the export machineries. Bioinformatic analysis revealed that the S. mutans genome contains a prsA gene, whose counterparts in other gram positive bacteria, including Bacillus and Lactococcus encode functions involved in protein post-export. In this study, we constructed a PrsA-deficient derivative of S. mutans and demonstrated that the prsA mutant displayed an altered cell wall/ membrane protein profile as well as cell surface related phenotypes, including auto-aggregation, increased surface hydrophobicity, and abnormal biofilm formation. Further analysis revealed that the disruption of the prsA gene resulted in reduced insoluble glucan production by cell surface localized glucosyltransferases, and mutacin as well as cell surface-display of a heterologous expressed GFP fusion to the cell surface protein SpaP. Our study suggested that PrsA in S. mutans encodes functions similar to the ones identified in Bacillus, and thus is likely involved in protein post-export.

Relationships between silage chemical components or organic matter (OM) pepsin-cellulase solubility and in vivo organic matter digestibility (OMD) and D-value (digestible OM content in dry matter) were studied. Twenty-five silages were made from primary growth timothy-meadow fescue swards in 6 years (3-6 silages per year) at 6-10 days intervals. The silages were analyzed for chemical composition, in vitro pepsin-cellulase solubility and in vivo digestibility in sheep. Chemical components of silage were highly correlated with OMD and D-value, but the accuracy of the OMD and D-value prediction equations were not satisfactory for ration formulation. Acid detergent fibre was the best single predictor of chemical parameters explaining 0.80 of the variation in OMD (residual mean square error (RMSE) 27.2 g kg −1). Relationships between crude protein (CP) and lignin were improved, when the effects of year or year × component interaction were included in the model. For CP the intercepts and for lignin the slopes, were different between the years. Pepsin-cellulase solubility was superior to chemical parameters in predicting silage OMD and D-value. The monovariate regression equation between OM solubility and OMD was: OMD (g kg −1) = 97 + 0.87 × OM solubility (R 2 = 0.974; RMSE = 10.8 g kg −1). Prediction of D-value was improved by including ash as a second factor in bivariate regression analysis: D-value (g DOM (kg DM) −1) = 160 + 0.818 × OM solubility −1.

The leaf economics spectrum (LES) represents a suite of intercorrelated leaf traits concerning construction costs per unit leaf area, nutrient concentrations, and rates of carbon fixation and tissue turnover. Although broad trade-offs among leaf structural and physiological traits have been demonstrated, we still do not have a comprehensive view of the fundamental constraints underlying the LES trade-offs. Here, we investigated physiological and structural mechanisms underpinning the LES by analysing a novel data compilation incorporating rarely considered traits such as the dry mass fraction in cell walls, nitrogen allocation, mesophyll CO2 diffusion and associated anatomical traits for hundreds of species covering major growth forms. The analysis demonstrates that cell wall constituents are major components of leaf dry mass (18-70%), especially in leaves with high leaf mass per unit area (LMA) and long lifespan. A greater fraction of leaf mass in cell walls is typically associated...

Broad-range amplification of bacterial DNA from clinical specimens has proved useful for the diagnosis of various bacterial infections, especially during antimicrobial treatment of the patient. Optimal sample processing protocols for diagnostic broad-range bacterial PCR should release DNA from an array of target organisms with equal efficiencies and wash out inhibitory factors from various sample types without introducing bacterial DNA contamination to the amplification reaction. In the present study, two physical cell wall disintegration methods, bead beating and sonication, for enhanced detection of organisms with difficult-to-lyse cell walls were studied. The analytical sensitivities of several commercially available DNA purification kits, which were used with and without additional cell disintegration steps, were compared by using dilution series of model bacteria. Selected purification methods were used to process routine clinical specimens in parallel with the standard phenol-...

Sortase enzymes are found throughout Gram-positive bacteria and are responsible for the covalent attachment of specific proteins to the cell wall. Through the anchoring of these cell wall proteins, sortase enzymes are important in the ability of several Gram-positive pathogens to cause disease. Previously, deletion of srtA from Streptococcus pneumoniae (the pneumococcus) was shown to disturb the localisation of surface proteins, and decrease bacterial adherence to human pharyngeal cells in vitro. Here we present data demonstrating, for the first time, a role for srtA as a pneumococcal fitness factor in experimental models of pneumonia and bacteraemia. In addition, srtA contributed to nasopharyngeal colonisation in vivo. Furthermore, we find that the contribution of srtA varied between two pneumococcal strains. We show that the known role of srtA in adherence in vitro is dependent on capsule expression, the role of SrtA in adherence to human cells only being apparent in the absence of the pneumococcal capsule. The srtA gene was detected by PCR in all 82 clinical isolates examined and sequencing of the gene from 20 strains showed srtA to be highly conserved. The ubiquitous distribution of srtA was in contrast to the other known pneumococcal sortase genes, srtB, C and D, which were found in only 14 of the 82 tested strains (17%).

Cellulose in the cell wall occurs in microfibrils of various sizes. "Fibre" should refer only to single elongated plant cells, "fibre bundle" to assemblages of fibres. Fibres and bundles can be of various cell types (most sclerenchyma), sizes, volume fractions of cellulose and degree of lignification. Usage of any other terms is liable to lead to confusion. Fibre bundles for industrial use should be defined only by their dimensions, botanical origin and the extraction procedures used.

Potted plants of two cultivars of almond (Amygdalus communis L.), Ramillete and Garrigues, growing under field conditions were subjected to drought by withholding irrigation for 28 days. Stressed plants were reirrigated and the recovery was studied for 8 days. Control plants were drip irrigated daily maintaining the soil matric potential at about -30 kPa. Predawn leaf water potential declined progressively due to water stress effect, reaching -0.80 MPa in Garrigues and -0.98 MPa in Ramillete at the end of the stress period. The reduction in midday leaf conductance in plants under water stress could be related to the reduction in midday leaf water potential. The reductions in leaf conductance values were about 62% and 79% for Garrigues and Ramillete, respectively. Garrigues exhibited osmotic adjustment during the greater part of the stress period and this was sufficient to maintain the leaf turgor. The relationship between leaf conductance and turgor pressure at midday appeared to be nearly linear over the range of turgor pressure observed. Ramillete presented lower leaf water potential at turgor loss point than did Garrigues. Roth cultivars presented high relative apoplastic water contents. The increase in these parameter values by water stress effect observed in Ramillete could indicate changes in cell wall structure. After rewatering, most of the parameters studied recovered quickly, although leaf conductance was delayed.

a glycosylated arginase acting as a fungal lectin from Peltigera canina is able to produce recruitment of cyanobiont Nostoc cells and their adhesion to the hyphal surface. This implies that the cyanobiont would develop organelles to motility toward the chemoattractant. however when visualized by transmission electron microscopy, Nostoc cells recently isolated from P. canina thallus do not reveal any motile, superficial organelles, although their surface was covered by small spindles and serrated layer related to gliding. The use of S-(3,4-dichlorobenzyl)isothiourea, blebbistatin, phalloidin and latrunculin a provide circumstantial evidence that actin microfilaments rather than MreB, the actin-like protein from prokaryota, and probably, an aTPase which develops contractile function similar to that of myosin II, are involved in cell motility. These experimental facts, the absence of superficial elements (fimbriae, pili or flagellum) related to cell movement, and the appearance of sunken cells during of after movement verified by scanning electron microscopy, support the hypothesis that the motility of lichen cyanobionts could be achieved by contraction-relaxation episodes of the cytoskeleton induced by fungal lectin act as a chemoattractant.

The CESA gene superfamily of Arabidopsis and other seed plants comprises the CESA family, which encodes the catalytic subunits of cellulose synthase, and eight families of CESA-like (CSL) genes whose functions are largely unknown. The CSL genes have been proposed to encode processive beta-glycosyl transferases that synthesize noncellulosic cell wall polysaccharides. BLAST searches of EST and shotgun genomic sequences from the moss Physcomitrella patens (Hedw.) B.S.G. were used to identify genes with high similarity to vascular plant CESAs, CSLAs, CSLCs, and CSLDs. However, searches using Arabidopsis CSLBs, CSLEs, and CSLGs or rice CSLFs or CSLHs as queries identified no additional CESA superfamily members in P. patens, indicating that this moss lacks representatives of these families. Intron insertion sites are highly conserved between Arabidopsis and P. patens in all four shared gene families. However, phylogenetic analysis strongly supports independent diversification of the shared families in mosses and vascular plants. The lack of orthologs of vascular plant CESAs in the P. patens genome indicates that the divergence of mosses and vascular plants predated divergence and specialization of CESAs for primary and secondary cell wall syntheses and for distinct roles within the rosette terminal complexes. In contrast to Arabidopsis, the CSLD family is highly represented among P. patens ESTs. This is consistent with the proposed function of CSLDs in tip growth and the central role of tip growth in the development of the moss protonema.

The Arabidopsis seed coat epidermis undergoes a complex process of differentiation that includes the biosynthesis and secretion of large quantities of pectinaceous mucilage, cytoplasmic rearrangement, and secondary cell wall biosynthesis. Mutations in MUM4 (MUCILAGE-MODIFIED4) lead to a decrease in seed coat mucilage and incomplete cytoplasmic rearrangement. We show that MUM4 encodes a putative NDP-l-rhamnose synthase, an enzyme required for the synthesis of the pectin rhamnogalacturonan I, the major component of Arabidopsis mucilage. This result suggests that the synthesis of monosaccharide substrates is a limiting factor in the biosynthesis of pectinaceous seed coat mucilage. In addition, the reduced cytoplasmic rearrangement observed in the absence of a key enzyme in pectin biosynthesis in mum4 mutants establishes a causal link between mucilage production and cellular morphogenesis. The cellular phenotype seen in mum4 mutants is similar to that of several transcription factors (AP2 [APETALA2], TTG1 [TRANSPARENT TESTA GLABRA1], TTG2 MYB61, and GL2 [GLABRA2]). Expression studies suggest that MUM4 is developmentally regulated in the seed coat by AP2, TTG1, and GL2, whereas TTG2 and MYB61 appear to be regulating mucilage production through alternate pathway(s). Our results provide a framework for the regulation of mucilage production and secretory cell differentiation.

The only currently available vaccine against tuberculosis (TB) is Mycobacterium bovis Bacille Calmette-Guerin (BCG), which has inconsistent efficacy to protect against the disease in adults. M. tuberculosis (MTB) cell wall components have been implicated in the pathogenicity of TB and therefore have been a prime target for the identification and characterization of cell wall proteins with potential application in vaccine development. In this regard, proteoliposomes (PLs) derived from mycobacteria containing lipids and cell wall proteins could be potential vaccine candidates against TB. In the present study PLs derived from BCG were prepared. These homogeneous population of spherical microparticles was then immunized into Balb/c mice. Sera of immunized animals showed high IgG response and strong cross-reactivity against different MTB antigens.These results showed that BCG PLs could be potential vaccine candidates against TB.

Staphylococcus epidermidis, a Gram-positive, coagulase-negative bacterium is a predominant inhabitant of human skin and mucous membranes. Recently, however, it has become one of the most important agents of hospital-acquired bacteriemia, as it has been found to be responsible for surgical wound infections developed in individuals with indwelling catheters or prosthetic devices, as well as in immunosupressed or neutropenic patients. Despite their medical significance, little is known about proteolytic enzymes of S. epidermidis and their possible contribution to the bacterium's pathogenicity; however, it is likely that they function as virulence factors in a manner similar to that proposed for the proteases of Staphylococcus aureus. Here we describe the purification of a cell wall-associated cysteine protease from S. epidermidis, its biochemical properties and specificity. A homology search using N-terminal sequence data revealed similarity to staphopain A (ScpA) and staphopain B (SspB), cysteine proteases from S. aureus. Moreover, the gene encoding S. epidermidis cysteine protease (Ecp) and a downstream gene coding for a putative inhibitor of the protease form an operon structure which resembles that of staphopain A in S. aureus. The active cysteine protease was detected on the bacterial cell surface as well as in the culture media and is apparently produced in a growth phase-dependent manner, with initial expression occurring in the mid-logarithmic phase. This enzyme, with elastinolytic properties, as well as the ability to cleave a 1 PI, fibrinogen and fibronectin, may possibly contribute to the invasiveness and pathogenic potential of S. epidermidis.

Grazing experiments were performed with the zooplankters Daphnia pulex and Daphnia magna feeding on nitrogen-and phosphorus-limited cells of two green algae (Chlamydomonas reinhardtii and Selenastrum cupricornutum). To analyze the role of the cell wall structure in digestibility of the algae by Daphnia, the same experiments were carried out with both wild-type C. reinhardtii and a cell wall-deficient mutant.

Le septembre 2008 Titre : Caractérisation moléculaire et physiologique des Facteurs de Réponse à l'Ethylène (ERF) chez la tomate (Solanum lycopersicon) JURY Mr. Jean-Claude PECH Président (INP-ENSAT) Mr. Christophe ROTHAN Rapporteur (INRA Bordeaux) Mr. Guy ALBAGNAC Rapporteur (INRA Montpellier) Mme. Mathilde CAUSSE Examinatrice (INRA Avignon) Mr. Masaru OHME-TAKAGI Examinateur (AIST Tsukuba) Mr. Farid REGAD Examinateur (INP-ENSAT) Mr. Mondher BOUZAYEN Directeur de thèse (INP-ENSAT) Mr. Xavier BERTHET Membre invité (NINSAR Barcelone) Ecole doctorale : Biologie Santé et Biotechnologie Unité de recherche : UMR990 INP-ENSAT/INRA Génomique et Biotechnologie des Fruits Directeur de Thèse : Pr. Mondher BOUZAYEN Je remercie Mondher BOUZ AYEN et Masaru OHME-T AKAGI pour m'avoir acceuilli, encadré et fait confiance pendant ce travail de thèse. Je remercie Farid REGAD pour les discussions scientifiques ainsi que son soutien jusqu'à très tard dans la nuit. Je remercie Jean-Claude PECH et Alain LATCHE pour avoir échangé scientifiquement avec moi. Un merci tout spéciale à Corinne, Isabelle, Simone, Marie, Salma et Benoît pour tous les moments de sciences (ou pas) partagés. Merci à Prassad, Ravi, Wangshu et Lanlan d'avoir participé à cette aventure. Un grand merci à Hélène, Carole, Lydie, Olivie r et Dominique pour la transformation et le suivi des plantes transgéniques (et ce n'est pas peu dire). Un grand merci à l'ensemble des membres du laboratoire pour tous les moments partagés, de sciences, et de convivialité : Christian, Je tiens également à remercier tous les membres du Research Institute of Genome-based Biofactory Miho Ikeda, Akira Iwase, Yoshimi Umemura, Tomotsugu Koyama, Masahito Shikata, Kyoko Matsui, Nobut aka Mitsuda, Naoko Kaneta , sans qui cette thèse n'aurait pas eu le même parfum. ありがとう. Merci aussi à Toshi et Hiroshi Ez ura de m'avoir acceuilli. Merci à mes proches qui m'ont soutenu pendant ces années, mes parents et mon frère. Enfin, je tiens à remercier tout particulièrement Laëtitia qui m'a suivi au bout du monde (au sens propre et au sens figuré), pour son soutien permanent et sans qui ce travail aurait été un peu différent. (2007): Isolation and characterization of a Vitis vinifera transcription factor, VvWRKY1, and its effect on responses to fungal pathogens in transgenic tobacco plants J Exp Bot.;58 (8):

There are scarce data connecting water relations in Azospirillum-inoculated wheat suffering drought during anthesis with the yield and mineral content of grains. Azospirillum brasilense Sp245-inoculated seeds of Triticum aestivum 'Pro INTA Oasis' were sown in nonirrigated and control plots. Water potential, water content, and relative water content were determined on flag leaves. Plant water status was calculated from pressure-volume curves. At maturity, grain yield and its components were determined. P, Ca, Mg, K, Fe, Cu, and Zn were determined in dried grains. Even though the cultivar underwent osmotic adjustment, significantly higher water content, relative water content, water potential, apoplastic water fraction, and lower cell wall modulus of elasticity values were obtained in Azospirillum-inoculated plants suffering drought. Grain yield loss to drought was 26.5% and 14.1% in noninoculated and Azospirilluminoculated plants, respectively. Grain Mg and K diminished in nonirrigated, noninoculated plots. However, grains harvested from Azospirillum-inoculated plants had significantly higher Mg, K, and Ca than noninoculated plants. Neither drought nor inoculation changed grain P, Cu, Fe, and Zn contents. A better water status and an additional "elastic adjustment" in Azospirillum-inoculated wheat plants could be crucial in promoting higher grain yield and mineral quality at harvest, particularly when drought strikes during anthesis.

The pyrolysis of switchgrass (Panicum virgatum) of the cultivar, ''Cave-in-Rock'' harvested at three stages of physiological maturity was studied in a PY-GC/MS system at the 600-1050 8C temperature range. Under these conditions, the decomposition was complete within 20 s yielding char, and two sets of pyrolysis gas, condensable and non-condensable. The former consisted of acetaldehyde (CH 3 CHO), acetic acid (CH 3 COOH) and higher molecular weight compounds possibly from the hydroxyl group and from the methoxy groups of the cell wall components. The non-condensable gases were mainly CO, CO 2 and C 1 -C 3 hydrocarbons. For these, there was a 900 8C temperature boundary where dramatic change occurred in their evolution rates. Below this temperature, CO 2 decreased but CO and the C 1 -C 3 hydrocarbons increased almost linearly with temperature. Above this temperature boundary, the hydrocarbons leveled off but there was a rapid rise in CO and CO 2 evolution at a constant CO/CO 2 ratio. These suggest the appearance of secondary or tertiary pyrolysis reactions involving rearrangement and release of CO and hydrocarbons prior to this temperature boundary and the release of CO and CO 2 from the tightly bond oxygen functionalities including C-C bonds thereafter. At <750 8C, there were modest increases in condensable gas yield and decrease in non-condensable gas due to differences in plant maturity at harvest. However, the effect of switchgrass physiological maturity on gas yield was statistically insignificant at high temperatures. The energy content of the non-condensable gas measured was about 68% of the gross energy content of the biomass for the early harvest crop and 80% for the mature crop. The activation energy for the decomposition, estimated assuming first order reaction kinetics, showed a linear increase with plant physiological maturity. The results demonstrate that physiological maturity at harvest of switchgrass biomass can result in different concentrations of pyrolysis products at different temperatures. These results also demonstrate the need for additional research with a broader array of herbaceous biomass materials to develop a better understanding of the synergies of crop cultivation, harvesting and processing of dedicated herbaceous biomass energy crops during their thermochemical conversion. #

Slow-growing bacteria and biofilms are notoriously tolerant to antibiotics. Oritavancin is a lipoglycopeptide with multiple mechanisms of action that contribute to its bactericidal action against exponentially growing gram-positive pathogens, including the inhibition of cell wall synthesis and perturbation of membrane barrier function. We sought to determine whether oritavancin could eradicate cells known to be tolerant to many antimicrobial agents, that is, stationary-phase and biofilm cultures of Staphylococcus aureus in vitro. Oritavancin exhibited concentration-dependent bactericidal activity against stationaryphase inocula of methicillin-susceptible S. aureus (MSSA) ATCC 29213, methicillin-resistant S. aureus (MRSA) ATCC 33591, and vancomycin-resistant S. aureus (VRSA) VRS5 inoculated into nutrient-depleted cation-adjusted Mueller-Hinton broth. As has been described for exponential-phase cells, oritavancin induced membrane depolarization, increased membrane permeability, and caused ultrastructural defects including a loss of nascent septal cross walls in stationary-phase MSSA. Furthermore, oritavancin sterilized biofilms of MSSA, MRSA, and VRSA at minimal biofilm eradication concentrations (MBECs) of between 0.5 and 8 g/ml. Importantly, MBECs for oritavancin were within 1 doubling dilution of their respective planktonic broth MICs, highlighting the potency of oritavancin against biofilms. These results demonstrate a significant activity of oritavancin against S. aureus in phases of growth that exhibit tolerance to other antimicrobial agents.

The present study was conducted to screen microorganisms that produce phospholipase D (PLD), and we especially focused on the strains having high transphosphatidylation activity. Eighty bacterial strains were isolated from soil samples by a screening method utilizing a preliminary selection medium with phosphatidylcholine (PC) as the sole carbon source. The culture supernatants were then assayed for PLD activity. The finding of dual PLD activities in cultures revealed that the hydrolytic and transphosphatidylation activities were correlated. Consequently, six strains were selected as stably producing PLD enzyme(s) during continuous subcultures. The culture supernatants of selected strains synthesized phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine from PC with high conversion rates. These isolated strains will be made available to carry out phospholipid modification through the efficient transphosphatidylation activity of the PLD that they produce.

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Plant cell walls are composed primarily of cellulose, hemicelluloses, lignins, and pectins. Of these components, lignins exhibit unique chemistry and physiological functions. Although lignins can be used as a product feedstock or as a fuel, lignins are also generally seen as a barrier to efficient enzymatic breakdown of biomass to sugars. Indeed, many pretreatment strategies focus on removing a significant fraction of lignin from biomass to better enable saccharification. In order to better understand the fate of biomass lignins that remain with the solids following dilute acid pretreatment, we undertook a structural investigation to track lignins on and in biomass cell walls. SEM and TEM imaging revealed a range of droplet morphologies that appear on and within cell walls of pretreated biomass; as well as the specific ultrastructural regions that accumulate the droplets. These droplets were shown to contain lignin by FTIR, NMR, antibody labeling, and cytochemical staining. We provide evidence supporting the idea that thermochemical pretreatments reaching temperatures above the range for lignin phase transition cause lignins to coalesce into larger molten bodies that migrate within and out of the cell wall, and can redeposit on the surface of plant cell walls. This decompartmentalization and relocalization of lignins is likely to be at least as important as lignin removal in the quest to improve the digestibility of biomass for sugars and fuels production.

As the protein sequence and structure databases expand, the relationships between proteins, the notion of protein superfamily, and the driving forces of evolution are better understood. Key steps of the synthesis of the bacterial cell wall peptidoglycan are revisited in light of these advances. The reactions through which the D-alanyl-D-alanine depeptide is formed, utilized, and hydrolyzed and the sites of action of the glycopeptide and /3-lactam antibiotics illustrate the concept according to which new enzyme functions evolve as a result of tinkering of existing proteins. This occurs by the acquisition of local structural changes, the fusion into multimodular polypeptides, and the association into multiprotein complexes.

Dietary carbohydrates are a group of chemically defined substances with a range of physical and physiological properties and health benefits. As with other macronutrients, the primary classification of dietary carbohydrate is based on chemistry, that is character of individual monomers, degree of polymerization (DP) and type of linkage (a or b), as agreed at the Food and Agriculture Organization/World Health Organization Expert Consultation in 1997. This divides carbohydrates into three main groups, sugars (DP 1-2), oligosaccharides (short-chain carbohydrates) (DP 3-9) and polysaccharides (DPX10). Within this classification, a number of terms are used such as mono-and disaccharides, polyols, oligosaccharides, starch, modified starch, non-starch polysaccharides, total carbohydrate, sugars, etc. While effects of carbohydrates are ultimately related to their primary chemistry, they are modified by their physical properties. These include water solubility, hydration, gel formation, crystalline state, association with other molecules such as protein, lipid and divalent cations and aggregation into complex structures in cell walls and other specialized plant tissues. A classification based on chemistry is essential for a system of measurement, predication of properties and estimation of intakes, but does not allow a simple translation into nutritional effects since each class of carbohydrate has overlapping physiological properties and effects on health. This dichotomy has led to the use of a number of terms to describe carbohydrate in foods, for example intrinsic and extrinsic sugars, prebiotic, resistant starch, dietary fibre, available and unavailable carbohydrate, complex carbohydrate, glycaemic and whole grain. This paper reviews these terms and suggests that some are more useful than others. A clearer understanding of what is meant by any particular word used to describe carbohydrate is essential to progress in translating the growing knowledge of the physiological properties of carbohydrate into public health messages.

The infectious and diagnostic stage of Giardia lamblia (also known as G. intestinalis or G. duodenalis) is the cyst. The Giardia cyst wall contains fibrils of a unique b-1,3-linked N-acetylgalactosamine (GalNAc) homopolymer and at least three cyst wall proteins (CWPs) composed of Leu-rich repeats (CWP LRR) and a C-terminal conserved Cys-rich region (CWP CRR). Our goals were to dissect the structure of the cyst wall and determine how it is disrupted during excystation. The intact Giardia cyst wall is thin (,400 nm), easily fractured by sonication, and impermeable to small molecules. Curled fibrils of the GalNAc homopolymer are restricted to a narrow plane and are coated with linear arrays of oval-shaped protein complex. In contrast, cyst walls of Giardia treated with hot alkali to deproteinate fibrils of the GalNAc homopolymer are thick (,1.2 mm), resistant to sonication, and permeable. The deproteinated GalNAc homopolymer, which forms a loose lattice of curled fibrils, is bound by native CWP1 and CWP2, as well as by maltose-binding protein (MBP)-fusions containing the full-length CWP1 or CWP1 LRR. In contrast, neither MBP alone nor MBP fused to CWP1 CRR bind to the GalNAc homopolymer. Recombinant CWP1 binds to the GalNAc homopolymer within secretory vesicles of Giardia encysting in vitro. Fibrils of the GalNAc homopolymer are exposed during excystation or by treatment of heat-killed cysts with chymotrypsin, while deproteinated fibrils of the GalNAc homopolymer are degraded by extracts of Giardia cysts but not trophozoites. These results show the Leu-rich repeat domain of CWP1 is a lectin that binds to curled fibrils of the GalNAc homopolymer. During excystation, host and Giardia proteases appear to degrade bound CWPs, exposing fibrils of the GalNAc homopolymer that are digested by a stage-specific glycohydrolase.

UV-B radiation (280-320 nm) is harmful to living organisms and has detrimental effects on plant growth, development and physiology. In this work we examined some mechanisms involved in plant responses to UV-B radiation. Seedlings of quinoa (Chenopodiurn quinoa Willd.) were exposed to variable numbers of UV-B radiation doses, and the effect on cotyledons was studied. We analyzed (1) cotyledons anatomy and chloroplasts ultrastructure; (2) peroxidase activity involved in the lignification processes; and (3) content of photosynthetic pigments, phenolic compounds and carbohydrates. Exposure to two UV-B doses induced an increase in the wall thickness of epidermal cells, which was associated with lignin deposition and higher activity of the peroxidase. The chloroplast ultrastructure showed an appearance typical of plants under shade conditions, likely in response to reduced light penetration into the mesophyll cells due to the screening effect of epidermal lignin deposition. Exposure to UV-B radiation also led to (1) enhancement in the level of phenolics, which may serve a protective function; (2) strong increase in the fructose content, a fact that might be related to higher requirement of erythrose-4P as a substrate for the synthesis of lignin and phenolics; and (3) reduction in the chlorophyll concentration, evidencing alteration in the photosynthetic system. We propose that the observed lignin deposition in epidermal tissues of quinoa is a resistance mechanism against UV-B radiation, which allows growing of this species in Andean highlands.

Drought and salinity are environmental constraints that affect crop yields worldwide. In nature, both stresses are multifaceted problems that are usually associated with other adverse circumstances which limit plant performance such as water shortage and nutrient deficits. In order to assess common features of both stresses, the effects of mannitol-induced osmotic stress were monitored using two Phaseolus vulgaris cultivars, Cv. 'Flamingo' (tolerant) and Cv. 'Coco Blanc' (sensitive) which differed in their drought and salinity tolerance. Growth, water relations, organic and inorganic compound accumulation and soluble protein contents were measured in leaves and nodules of these N 2 -fixing plants. The aim of the present study was to check whether osmotic stress tolerance is associated with accumulation of some of these compounds either in leaves, nodules or both organs. At the whole-plant level, Cv. 'Flamingo' showed a better maintenance of plant biomass and shoot water status. At the cell level, this was related to a better osmotic adjustment ability both in leaves and nodules and also to a better adjustment of the cell wall elasticity. At the metabolic level, the contrasting accumulation of the different amino acids in nodules of each cultivar suggested that amino acids pathways can be regulated to different degrees under stress conditions. At the metabolic level, it seems that symbiosis in the sink organ (the nodule) plays a crucial role in conferring drought and salinity tolerance in the common bean.

The impact of sugar (17.6-31.2%) and fat (8.7-15.8%) levels on cookie structure was studied. Cookie diameter increased and its height decreased with increasing sugar or fat levels. X-ray microfocus computed tomography porosities and cell sizes increased with fat level, but cell size distribution, cell wall thickness and distribution were not affected by fat level, indicating that fat primarily incorporates air. In contrast, the sugar level influenced porosity, cell size, cell wall thickness and their relative distributions. Thus, the sucrose level, probably by affecting dough viscosity during baking, largely influences the baked cookie structure. Cell and cell wall anisotropy measurements indicated that the inner orientation of cells and cell walls probably depends on the horizontal spread behaviour, rather than on the maximum cookie height and collapse. Finally, the surface cracking pattern was determined by sugar level, rather than by structural collapse at the end of baking.

PE and PPE proteins appear to be important for virulence and immunopathogenicity in mycobacteria, yet the functions of the PE/PPE domains remain an enigma. To decipher the role of these domains, we have characterized the triacylglycerol (TAG) hydrolase LipY from Mycobacterium tuberculosis, which is the only known PE protein expressing an enzymatic activity. The overproduction of LipY in mycobacteria resulted in a significant reduction in the pool of TAGs, consistent with the lipase activity of this enzyme.

Two proteins from the eggshell of Rhodnius prolixus were isolated, characterized and named Rp30 and Rp45 according to their molecular masses. Purified proteins were used to obtain specific antiserum which was later used for immunolocalization. The antiserum against Rp30 and Rp45 detected their presence inside the follicle cells, their secretion and their association with oocyte microvilli. Both proteins are expressed during the final stage of vitellogenesis, preserved during embryogenesis and discarded together with the eggshell. The amino terminals were sequenced and both proteins were further cloned using degenerated primers. The amino acid sequences appear to have a tripartite arrangement with a highly conserved central domain which presents a repetitive motif of valine-proline-valine (VPV) at intervals of 15 amino acid residues. Their amino acid sequence showed no similarity to any known eggshell protein. The expression of these proteins was also investigated; the results demonstrated that this occurred strictly in choriogenic follicles. Antifungal activity against Aspergillus niger was found to be associated with Rp45 but not with Rp30. A. niger exposed to Rp45 protein induced growth inhibition and several morphological changes such as large vacuoles, swollen mitochondria, multi-lamellar structures and a disorganized cell wall as demonstrated by electron microscopy analysis. r

Although numerous physiological studies have addressed the interactions between brassinosteroids and auxins, little is known about the underlying molecular mechanisms. Using an Affymetrix GeneChip representing approximately 8,300 Arabidopsis genes, we studied comprehensive transcript profiles over 24 h in response to indole-3-acetic acid (IAA) and brassinolide (BL). We identified 409 genes as BL inducible, 276 genes as IAA inducible, and 637 genes in total. These two hormones regulated only 48 genes in common, suggesting that most of the actions of each hormone are mediated by gene expression that is unique to each. IAA-up-regulated genes were enriched in genes regulated in common. They were induced quickly by IAA and more slowly by BL, suggesting divergent physiological roles. Many were early auxin-inducible genes and their homologs, namely SAUR, GH3, and IAA. The comprehensive comparison also identified IAA-and BL-specific genes, which should help to elucidate the specific actions of each hormone. The identified genes were classified using hierarchical clustering based on the similarity of their responses to the two hormones. Gene classification also allowed us to analyze the frequency of cis-elements. The TGTCTC element, a core element of the previously reported auxin response element, was not enriched in genes specifically regulated by IAA but was enriched in the 59-flanking region of genes up-regulated by both IAA and BL. Such gene classification should be useful for predicting the functions of unknown genes, to understand the roles of these two hormones, and the promoter analysis should provide insight into the interaction of transcriptional regulation by the two hormones. fax 81-45-503-9492.

By means of alkaline extraction of the cell walls of the yeasts Saccharomyces cermisiue and Candida albicans, water-insoluble glucans were obtained. Methylation analysis and 13C-nmr investigation in dimethyl sulfoxide solution revealed the similar chemical structure of these glucans, being composed of p(1 + 3) glycosidically linked D-glucopyranmyl units with a small amount of p(1 4 6) linkages. More detailed study in dilute alkaline solutions and in the gel state a t neutral pH, however, showed that an ordered helical conformation of the glucan chain is less stable in the case of the S. cereuisiae glucan in comparison with that of C. albicans. Measurements of the shift of the absorption maximum of the glucan complexes with Congo Red also demonstrated such difference. The S. cereuisiue glucan was also inable to form a gel at neutral pH. The difference in stability of helical conformation of the glucans is explained on the basis of the methylation analysis, so that the S. cereoisiue glucan possesses longer side chains, which hinder its adoption of a stable helical conformation.

OBJECTIVES: To determine the stability/reversibility and mechanism of monensin adaptation in monensin-treated cattle isolates compared with reference bacterial isolates, exposed in vitro to high monensin concentrations. METHODS: We evaluated the potential for cattle-origin strains of Clostridium perfringens, Enterococcus faecium and Enterococcus faecalis exposed to monensin in vivo (in vivo monensin-exposed isolates) to maintain or achieve the ability to grow in the presence of high monensin concentrations (in vitro monensin-adapted isolates). Twenty-one consecutive subcultures of the in vitro monensin-adapted strains were performed, and monensin MICs were determined for the 3rd, 7th, 14th and 21st subcultures (subcultured isolates). SDS-PAGE and transmission electron microscopy (TEM) were used to determine protein expression and visualize extracellular morphology changes. RESULTS: Monensin-non-exposed isolates did not display monensin adaptation during in vitro monensin exposure. I...

Rubber wood (Hevea brasiliensis) esteri®ed with acetic anhydride, maleic anhydride and phthalic anhydride was studied for moisture adsorption behaviour. FTIR spectroscopic technique was used to analyse the chemical changes in the wood through the correspondent reactions. Spectra indicated increasing intensity of the C@O and C-O stretching bands, and reduction in O-H stretching vibrations that re¯ects formation of less hygroscopic ester bonds with hydroxyl groups of cell wall polymers. Moisture adsorption isotherms show low equilibrium moisture content in esteri®ed wood at all relative humidity values as compared to a control. The adsorption data was analysed by applying Hailwood-Horrobin theory which separated the adsorbed water into hydrated water and dissolved water corresponding to mono-layer and multi-layer adsorption. Curve for M h and M s were similar in pattern for esteri®ed wood and control with different magnitude. Low M h and M s in esteri®ed wood indicate reduction in moisture adsorption sites. Inaccessible fraction of wood for water vapour was increased by 43%, 32% and 26% in acetic, maleic and phthalic anhydride treated wood, respectively, compared to the control. Acetic anhydride treatment was found most effective in reducing hygroscopicity of wood.

A novel endophytic actinobacterium, designated strain EUM 378T, was isolated from the surface-sterilized root tissue of Eucalyptus microcarpa, a eucalyptus tree known as Grey Box. Phylogenetic evaluation based on 16S rRNA gene sequence analysis, including alignment with taxon-specific 16S rRNA gene signature nucleotides, placed this isolate as a member of the family Nocardioidaceae. Strain EUM 378T showed &gt;5.5 % 16S rRNA gene sequence divergence from other members of this family and was related most closely to Actinopolymorpha alba YIM 48868T (94.2 %) and Actinopolymorpha singaporensis IM 7744T (94.4 %). This Gram-positive, aerobic actinobacterium has well-developed substrate mycelia that fragment into small rods. Chemotaxonomic data revealed that the cell wall contains ll-diaminopimelic acid, ribose, glucose and rhamnose. MK-10(H6) is the predominant menaquinone. Chemotaxonomic and phylogenetic evidence confirmed that strain EUM 378T represents a novel species of a new genus, fo...

Background: Seed germination is of immense significance for agriculture and has been studied for centuries. Yet, our understanding of the molecular mechanisms underlying regulation of dormancy and germination is still in its infancy. Gibberellins are the key phytohormones that promote germination, and the DELLA protein RGL2 is the main signalling intermediate involved in this response. Germination is completely inhibited if functional RGL2 is overexpressed and/or stabilized; however, the molecular mechanisms of RGL2 function are still largely unknown. We therefore attempted to shed light onto some of the genetic events downstream of RGL2. Results: Gene ontology of the transcriptome differentially regulated by RGL2, as well as extensive cross-comparison with other available microarray data indicates that RGL2-mediated inhibition of germination causes seeds to enter a state of dormancy. RGL2 also appears to differentially regulate a number of transcription factors, many of which are known to be involved in light-or phytohormone-mediated aspects of germination. A promoter analysis of differentially expressed genes identified an enrichment of several motifs that can be bound by specific transcription factors, for example GAMYB, ARF1, or Dof-type zinc fingers. We show that Dof-binding motifs indeed play a role in RGL2-mediated transcription. Using Chromatin Immunoprecipitation (ChIP), we show that RGL2 directly downregulates at least one cell wall modifying enzyme, which is predicted to constrain cell growth thereby leading to inhibition of seed germination.

Although lignins play important roles in plants, they often represent an obstacle to the utilization of plant biomass in different areas: pulp industry, forage digestibility. The recent characterization of different lignification genes has stimulated research programmes aimed at modifying the lignin profiles of plants through genetic engineering (antisense and sense suppression of gene expression). The first transgenic plants with a modification of monomeric composition of lignins and lignin content have been recently obtained. Down regulation of the OMT gene induces dramatic reduction of syringyl units. CAD down regulated plants exhibit a unusual red phenotype associated with the developing xylem and several chemical modifications of their lignins including an increase in cinnamaldehydes in the polymer structure. Interestingly this novel lignin is removed more easily during the pulping process. In both OMT and CAD down regulated plants no changes in phenotypic characteristics such as growth architecture and morphology were observed. More recent experiments have shown that a reduction of CCR activity determines specific changes in the coloration of the xylem area suggesting significant chemical modifications which are currently being studied. These different results show that it is possible to manipulate lignins through targeted genetic transformation of plants and that lignins exhibit a relative flexibility of their chemical structure. Future developments should probe the impact of down regulating the expression of other recently characterized lignification genes such as F5H and CCoAOMT and also of a combination of genes in order to tailor lignins more adapted to specific purposes. In addition to biotechnological applications which should provide important economical benefits for utilization of wood in the pulp industry, genetic engineering of lignins offer very promising perspectives for the understanding of lignin synthesis, structure and properties.