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PubMed1994

Similar regulatory mechanisms despite differences in membrane lipid composition in Acholeplasma laidlawii strains A-EF22 and B-PG9. A multivariate data analysis.

Mycoplasmas are small, cell wall-deficient bacteria. The metabolic regulation of the lipid composition in the membrane of the species Acholeplasma laidlawii, strains A-EF22 and B-JU, is governed mainly by the balance between the potential formation of lamellar and nonlamellar phase structures. However, the regulatory features have not been consistently observed in the B-PG9 strain. A comparison has been performed between the membrane lipid composition for strains A-EF22 and B-PG9, simultaneously changing eight experimental conditions known to affect the regulation and packing properties of the A-EF22 lipids. Multiple regression and partial least-square discriminant analyses of many variables showed: (i) quantitative differences in membrane lipid and protein composition, and in membrane protein molecular masses of the two strains; (ii) different molar fractions of the major polar lipids monoglucosyldiacylglycerol (nonlamellar) and diglucosyldiacylglycerol (lamellar), which were caused by differences in lipid acyl chain length and unsaturation inherent in the strains and by the type of growth medium used; and (iii) similar regulatory mechanisms for changes in the lipid composition under most conditions, responding to the experimentally varied bilayer and nonbilayer properties of the lipid matrix. These regulatory principles are probably valid in other bacteria as well.

Acholeplasma laidlawii↗
PubMed2005

Biosynthesis and cell-wall deposition of a pectin-xyloglucan complex in pea.

Golgi-enriched enzyme preparations prepared from etiolated pea epicotyls incorporated [U-(14)C]galactose from UDP-[U-(14)C]galactose into the 1,4-beta-galactan sidechains of a pectin-xyloglucan complex. This complex could bind to paper and was degraded both by pectin-degrading enzymes and by a xyloglucan-specific endoglucanase. Gel permeation chromatography was used to assess the molecular size of the complex and of enzymically-degraded, galactan-containing fragments of it. Etiolated pea stems were labelled with [U-(14)C]sucrose for 1 h, and the newly-synthesised cell wall polysaccharides were extracted with EDTA or NaOH and fractionated by ion-exchange chromatography. The NaOH-extracted, acidic radioactive polysaccharides obtained in this way were also degraded both by pectin-degrading enzymes and by xyloglucan-specific endoglucanase. Analysis of the radioactive sugar composition indicated that neutral sugars characteristic of both pectin and xyloglucan were present. Analysis of the total non-radioactive, neutral sugar composition of the NaOH-extracted, acidic cell-wall polysaccharides indicated that pectin-xyloglucan complexes were a general feature of the cell wall in this tissue.

Cell Wall↗
PubMed1989

Dendritic cells and scavenger macrophages in chronic inflammatory bowel disease.

We used enzyme (acid phosphatase [AP]) and immunohistochemical techniques and a set of monoclonal antibodies (CD11, CD5, CD4, CD19, CD8, OKIa), including two recently developed antibodies--for example, HECA-452 (specific for an adhesion molecule on high endothelial venules) and RFD1 (specific for 'active' human dendritic cells) to analyse the composition of the gut wall infiltrate of 10 well defined cases of chronic inflammatory bowel disease (CIBD) (six Crohn's disease (CD), four ulcerative colitis (UC]. Two polar forms in a spectrum of gut mononuclear phagocyte types (CD11+) were identified: at the one extreme scavenger macrophages with blunted projections (AP+, Heca-452-, RFD1-) and at the other extreme, dendritic cells with long dendritic cytoplasmic projections (AP-, Heca-452+, RFD1+). Dendritic cells were mainly found in highly organised lymphoid tissue present at the deeper layers in the gut wall (normal gut: underneath the muscularis mucosae and T-cell areas of lymph follicles [25-30 per follicle]; surrounding the broad zone of scavenger macrophages at the bottom of ulcers (CIBD) and fissures (CD) and in the lymphoid aggregates [25-30 dendritic cells per aggregate] adjacent to granulomas (CD]. These observations can be taken as evidence that exaggerated antigen handling and presentation and stimulation of the immune response takes place at these foci. The observation that scavenger macrophages were localised more superficial, as band like zones (normal gut: subepithelial; mainly surrounding ulcers (CIBD) and fissures (CD] can be taken as evidence that at these spots the ingestion and degradation of foreign material takes place.

Adult↗
PubMed2001

Developmental regulation of pectic epitopes during potato tuberisation.

We show, by immunogold labelling, that potato (Solanum tuberosum L. cv Karnico) pectic epitopes are developmentally regulated within regions of the stolon, in addition to showing tissue-specific differences in abundance and localisation. The (1-->4)-beta-D-galactan and (1-->5)-alpha-arabinan epitopes demarcate two distinct zones within stolons; galactans are enriched in primary walls of elongating cells proximal to the stolon hook, whilst arabinans predominate in younger cells distal to the hook. Low-methoxyl homogalacturonan epitopes are concentrated in the middle lamella and show a proximo-distal gradient in stolons similar to that of galactans, whilst high-methoxyl homogalacturonan is uniformly abundant. Calcium pectate is restricted to the middle lamella at cell corners and pit fields. Calcium-binding sites are uniformly present in stolon cell walls, but their total density is reduced and they become localised to a few cell corners in mature tubers, as determined by image-electron energy loss spectroscopy. During the transition from elongation growth to isodiametric expansion during tuberisation of the stolon hook, there were no detectable changes in pectic epitope abundance or localisation. As tubers matured, all epitopes increased in abundance in parenchymal cell walls, except for calcium pectate. We conclude that potentially significant changes in pectic composition occur as young cells distal to the stolon hook move into the zone of cell elongation proximal to the hook.

Antibodies, Monoclonal↗
PubMedDate not supplied

[Localization of catalysis enzyme systems that degrade higher plants' cell wall polysaccharides. Pectinases (review)].

This paper reviews recent data on the locations and compositions of enzyme systems catalyzing the cleavage of cellulose, hemicelluloses, and pectin polysaccharides in higher plants. The physiological functions and physicochemical properties of certain enzymes degrading pectin polymers in higher plants, fungi, and bacteria are described.

Carbohydrate Sequence↗
PubMed1964

IDENTIFICATION OF SPECIES OF ACTINOMYCES.

Georg, Lucille K. (Communicable Disease Center, Atlanta, Ga.), Gordon W. Robertstad, and Sherry A. Brinkman. Identification of species of Actinomyces. J. Bacteriol. 88:477-490. 1964.-Four unusual isolates of Actinomyces species were compared with six control strains of well-identified Actinomyces species. Their oxygen requirements and their morphological and biochemical characteristics were determined. Final identifications were confirmed by cell-wall analyses for amino acid and carbohydrate composition and by agar-gel techniques. Two strains, A-13-R and A-13-S, isolated from the same cow head were shown to represent rough and smooth variants of A. bovis. The strains were not only morphologically distinct, but had different biochemical reactions and antigenic makeup. The two other strains, X407 and X573, isolated from human pleural fluid and a lung abscess, respectively, were shown to represent a new Actinomyces species.

Actinomyces↗
PubMed1970

Structure des parois cellulaires des nocardia. I-isolement et composition des parois de Nocardia kirovani.

Nocardia kirovani cell walls possess a lipidic fraction, a lipolysaccharidic fraction and a peptidoglycan. The lipidic part is composed of glycerides, C(16) and C(18) free fatty acids, nocardic acids and of a carotenoid pigment. The lipopolysaccharidic part is composed of an arabinogalactan esterified by nocardic acids. The glycan strand consists of alternating 1-->4 linked N-acetylglucosamine and N-glycolylmuramic acid residues substituted by a peptide subunit in which the aminoacids Ala, Glu, meso-Dap are present in the molar proportions 1,7:1,2:1.

Journal Article↗
PubMedDate not supplied

[The effect of the composition of the medium on the concentration of free calcium ions in the cells of the follicular wall in the common frog and in the clawed toad].

The concentration of intracellular free calcium ions in the follicle wall cells and in the follicle cells of Rana temporaria in Ringer solution is 150 +/- 10 and in the follicle wall cells of Xenopus laevis, 220 +/- 10 nM. In a chloride-free saline, its concentration in the same cells is 2.5-3 times that in Ringer solution. Voltage-dependent Ca(2+)-channel blockers diltiazem and verapamil (100 microM) reduce the level of intracellular free calcium ions in R. temporaria follicle wall cells cultivated in a chloride-free saline to 170 +/- 20 nM, which practically does not differ from the level in Ringer solution. Inhibitors (100 microM) decrease the rate of "spontaneous" maturation of R. temporaria follicle-enclosed oocytes both in chloride-free and Ringer solutions. It was concluded that an increased level of intracellular free calcium ions in the follicle cells, among other factors, may determine the stimulating effect of the medium (Ringer or chloride-free solution) on "spontaneous" maturation of follicle-enclosed amphibian oocytes. Voltage-dependent calcium channels appear to be involved in Ca2+ influx into the cells.

Animals↗
PubMed2003

Requirement for kasB in Mycobacterium mycolic acid biosynthesis, cell wall impermeability and intracellular survival: implications for therapy.

Mycobacterium tuberculosis infects one-third of the world's population and causes two million deaths annually. The unusually low permeability of its cell wall contributes to the ability of M. tuberculosis to grow within host macrophages, a property required for pathogenesis of infection. Mycobacterium marinum is an established model for discovering genes involved in mycobacterial infection. Mycobacterium marinum mutants with transposon insertions in the beta-ketoacyl-acyl carrier protein synthase B gene (kasB) grew poorly in macrophages, although growth in vitro was unaffected. Detailed analyses by thin-layer chromatography, nuclear magnetic resonance (NMR), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, infrared spectroscopy, and chemical degradations showed that the kasB mutants synthesize mycolic acids that are 2-4 carbons shorter than wild type; the defect was localized to the proximal portion of the meromycolate chain. In addition, these mutants showed a significant (approximately 30%) reduction in the abundance of keto-mycolates, with a slight compensatory increase of both alpha- and methoxy-mycolates. Despite these small changes in mycolate length and composition, the kasB mutants exhibited strikingly altered cell wall permeability, leading to a marked increase in susceptibility to lipophilic antibiotics and the host antimicrobial molecules defensin and lysozyme. The abnormalities of the kasB mutants were fully complemented by expressing M. tuberculosis kasB, but not by the closely related gene kasA. These studies identify kasB as a novel target for therapeutic intervention in mycobacterial diseases.

Animals↗
PubMed1992

A repetitive proline-rich protein from the gymnosperm douglas fir is a hydroxyproline-rich glycoprotein.

Intact cell elution of suspension cultures derived from Douglas fir, Pseudotsuga menziesii (Mirbel) Franco, yielded two extensin monomers, the first hydroxyproline-rich glycoproteins (HRGPs) to be isolated from a gymnosperm. These HRGPs resolved on Superose-6 gel filtration. The smaller monomer was compositionally similar to angiosperm extensins like tomato P1. The larger monomer had a simple composition reminiscent of repetitive proline-rich proteins (RPRPs) from soybean cell walls and contained proline, hydroxyproline, and sugar; hence designated a proline-hydroxyproline-rich glycoprotein (PHRGP). The simple composition of the PHRGP implied a periodic structure which was confirmed by the simple chymotryptic map and 45-residue partial sequence of the major proline-hydroxyproline-rich glycoprotein chymotryptide 5: Lys-Pro-Hyp-Val-Hyp-Val-Ile-Pro-Pro-Hyp-Val-Val-Lys-Pro-Hyp-Hyp-Val- Tyr-Lys-Pro-Hyp-Val-Hyp-Val-Ile-Pro-Pro-Hyp-Val-Val-Lys-Pro-Hyp-Hyp- Val-Tyr-Lys-Ile-Pro-Pro(Hyp)-Val-Ile-Lys-Pro. Proline-hydroxyproline-rich glycoprotein chymotryptide 5 contained an 18-residue tandem repeat devoid of tetra(hydroxy)-proline or serine; it also contained two instances of the five-residue motif Hyp-Hyp-Val-Tyr-Lys and five of the general Pro-Pro-X-X-Lys motif, thereby establishing its homology with typical angiosperm RPRPs and extensins from tomato, petunia, carrot, tobacco, sugar beet, and Phaseolus. Unlike the nonglycosylated soybean RPRP, the highly purified Douglas fir PHRGP was lightly glycosylated, confirmed by a quantitative hydroxyproline glycoside profile, indicating that extensins can range from highly glycosylated hydroxyproline to little or no glycosylated hydroxyproline. Comparison of extensin sequence data strongly indicates that a major determinant of hydroxyproline glycosylation specificity is hydroxyproline contiguity: extensins with tetrahydroxyproline blocks are very highly arabinosylated (>90% hydroxyproline glycosylated), tri- and dihydroxyproline are less so, and single hydroxyproline residues perhaps not at all. Despite high yields of extensins eluted from intact cells, the Douglas fir cell wall itself was hydroxyproline poor yet remarkably rich in protein (>20%), again emphasizing the existence of other structural cell wall proteins that are neither HRGPs nor glycine-rich proteins.

Journal Article↗
PubMed2005

Microscale multilayer cocultures for biomimetic blood vessels.

Living tissues have complex and well-organized microstructures. Although microtechnology has been used to create in vivo-like cell microstructures in vitro, most available microscale systems are two-dimensional, and few three-dimensional (3D) systems have been explored. This article demonstrates a 3D hierarchical biomimetic multilayer microsystem created by a generally applicable technique. The technique employs layer-by-layer microfluidics to build layers of cells and biopolymers in microchannels, allowing controlled patterning of cells and their microenvironments in the x, y, and z-dimension. As a prototype, a multilayer system was created using three vascular cell types within heterogeneous types of biopolymers to mimic the structure and composition of a blood vessel wall. The effects of matrix composition and multilayer configurations on 3D cell-cell interactions and cell biology were revealed. Cell migration in the z-dimension, matrix remodeling, intercellular adhesion molecule expression and actin organization were examined under different 3D coculture conditions. A more biomimetic coculture was found to reproduce a more stable structure and in vivo-like function. This approach provides a method to fabricate microscale hierarchical "neotissues" with 3D configurations of matrix materials and multiple cell types, and an in vitro cell coculture model to understand 3D processes of cell-cell and cell-matrix interactions.

Actin Cytoskeleton↗
PubMed1999

Analysis of peptidoglycan structure from vegetative cells of Bacillus subtilis 168 and role of PBP 5 in peptidoglycan maturation.

The composition and fine structure of the vegetative cell wall peptidoglycan from Bacillus subtilis were determined by analysis of its constituent muropeptides. The structures of 39 muropeptides, representing 97% of the total peptidoglycan, were elucidated. About 99% analyzed muropeptides in B. subtilis vegetative cell peptidoglycan have the free carboxylic group of diaminopimelic acid amidated. Anhydromuropeptides and products missing a glucosamine at the nonreducing terminus account for 0.4 and 1.5%, respectively, of the total muropeptides. These two types of muropeptides are suggested to end glycan strands. An unexpected feature of B. subtilis muropeptides was the occurrence of a glycine residue in position 5 of the peptide side chain on monomers or oligomers, which account for 2.7% of the total muropeptides. This amount is, however, dependent on the composition of the growth media. Potential attachment sites for anionic polymers to peptidoglycan occur on dominant muropeptides and account for 2.1% of the total. B. subtilis peptidoglycan is incompletely digested by lysozyme due to de-N-acetylation of glucosamine, which occurs on 17.3% of muropeptides. The cross-linking index of the polymer changes with the growth phase. It is highest in late stationary phase, with a value of 33.2 or 44% per muramic acid residue, as determined by reverse-phase high-pressure liquid chromatography or gel filtration, respectively. Analysis of the muropeptide composition of a dacA (PBP 5) mutant shows a dramatic decrease of muropeptides with tripeptide side chains and an increase or appearance of muropeptides with pentapeptide side chains in monomers or oligomers. The total muropeptides with pentapeptide side chains accounts for almost 82% in the dacA mutant. This major low-molecular-weight PBP (DD-carboxypeptidase) is suggested to play a role in peptidoglycan maturation.

Alanine↗
PubMed1987

Structure of Plant Cell Walls : XIX. Isolation and Characterization of Wall Polysaccharides from Suspension-Cultured Douglas Fir Cells.

The partial purification and characterization of cell wall polysaccharides isolated from suspension-cultured Douglas fir (Pseudotsuga menziesii) cells are described. Extraction of isolated cell walls with 1.0 m LiCl solubilized pectic polysaccharides with glycosyl-linkage compositions similar to those of rhamnogalacturonans I and II, pectic polysaccharides isolated from walls of suspension-cultured sycamore cells. Treatment of LiCl-extracted Douglas fir walls with an endo-alpha-1,4-polygalacturonase released only small, additional amounts of pectic polysaccharide, which had a glycosyl-linkage composition similar to that of rhamnogalacturonan I. Xyloglucan oligosaccharides were released from the endo-alpha-1,4-polygalacturonase-treated walls by treatment with an endo-beta-1,4-glucanase. These oligosaccharides included hepta- and nonasaccharides similar or identical to those released from sycamore cell walls by the same enzyme, and structurally related octa- and decasaccharides similar to those isolated from various angiosperms. Finally, additional xyloglucan and small amounts of xylan were extracted from the endo-beta-1,4-glucanase-treated walls by 0.5 n NaOH. The xylan resembled that extracted by NaOH from dicot cell walls in that it contained 2,4- but not 3,4-linked xylosyl residues. In this study, a total of 15% of the cell wall was isolated as pectic material, 10% as xyloglucan, and less than 1% as xylan. The noncellulosic polysaccharides accounted for 26% of the cell walls, cellulose for 23%, protein for 34%, and ash for 5%, for a total of 88% of the cell wall. The cell walls of Douglas fir were more similar to dicot (sycamore) cell walls than to those of graminaceous monocots, because they had a predominance of xyloglucan over xylan as the principle hemicellulose and because they possessed relatively large amounts of rhamnogalacturonan-like pectic polysaccharides.

Journal Article↗
PubMed2001

A novel carbohydrate-binding protein is a component of the plant cell wall-degrading complex of Piromyces equi.

The recycling of photosynthetically fixed carbon by the action of microbial plant cell wall hydrolases is a fundamental biological process that is integral to one of the major geochemical cycles and, in addition, has considerable industrial potential. Enzyme systems that attack the plant cell wall contain noncatalytic carbohydrate-binding modules (CBMs) that mediate attachment to this composite structure and play a pivotal role in maximizing the hydrolytic process. Anaerobic fungi that colonize herbivores are the most efficient plant cell wall degraders known, and this activity is vested in a high molecular weight complex that binds tightly to the plant cell wall. To investigate whether plant cell wall attachment is mediated by noncatalytic proteins, a cDNA library of the anaerobic fungus Piromyces equi was screened for sequences that encode noncatalytic proteins that are components of the cellulase-hemicellulase complex. A 1.6-kilobase cDNA was isolated encoding a protein of 479 amino acids with a M(r) of 52548 designated NCP1. The mature protein had a modular architecture comprising three copies of the noncatalytic dockerin module that targets anaerobic fungal proteins to the cellulase-hemicellulase complex. The two C-terminal modules of NCP1, CBM29-1 and CBM29-2, respectively, exhibit 33% sequence identity with each other but have no homologues in protein data bases. A truncated form of NCP1 comprising CBM29-1 and CBM29-2 (CBM29-1-2) and each of the two individual copies of CBM29 bind primarily to mannan, cellulose, and glucomannan, displaying the highest affinity for the latter polysaccharide. CBM29-1-2 exhibits 4-45-fold higher affinity than either CBM29-1 or CBM29-2 for the various ligands, indicating that the two modules, when covalently linked, act in synergy to bind to an array of different polysaccharides. This paper provides the first report of a CBM-containing protein from an anaerobic fungal cellulase-hemicellulase complex. The two CBMs constitute a novel CBM family designated CBM29 whose members exhibit unusually wide ligand specificity. We propose, therefore, that NCP1 plays a role in sequestering the fungal enzyme complex onto the plant cell wall.

Amino Acid Sequence↗
PubMed1982

Studies on the Pectic Substances of Plant Cell Walls: III. DEGRADATION OF CARROT ROOT CELL WALLS BY ENDOPECTATE LYASE PURIFIED FROM ERWINIA AROIDEAE.

Pectate lyase was isolated from the cell extract of Erwinia aroideae. The enzyme was further purified to a high degree by a procedure involving ammonium sulfate fractionation and chromatography on CM-Sephadex C-50 and on Sephadex G-200. The enzyme attacked its substrate in an endo fashion and was more active on the sodium salt of acid-insoluble polygalacturonate or pectic acid than it was on the methoxylated pectin. The enzyme had an optimum pH at 9.3, was stimulated by calcium ions, and was completely inhibited by ethylenediaminetetraacetic acid. In addition, the reaction products showed an absorption maximum between 230 and 235 nm and reacted with thiobarbituric acid. These results indicate that the purified enzyme is an endopectate lyase. The endopectate lyase also had the ability to solubilize effectively the pectic fraction from the cell walls of carrot (Daucus carota) root tissue. The enzyme released 30.5% of the wall as soluble products and also liberated all of the galacturonic acid present in the walls. The total neutral sugars released by the enzyme were 10.6% of the walls, which corresponded to 71.5% of noncellulosic neutral sugars. The soluble products were separated into five fractions by DEAE-Sephadex A-50 column chromatography. Based on the analysis of sugar composition of each fraction, the pectic fraction of carrot cell wall is presented.

Journal Article↗
PubMed2004

Bacillus vietnamensis sp. nov., a moderately halotolerant, aerobic, endospore-forming bacterium isolated from Vietnamese fish sauce.

Five strains of Gram-positive, endospore-forming, moderately halotolerant bacteria were studied taxonomically. Four were isolated from Vietnamese fish sauce and one from the Gulf of Mexico. Phylogenetic analysis based on 16S rRNA gene sequences showed that these strains clustered within the radiation of the genus Bacillus but separately from recognized Bacillus species. DNA G+C composition of the isolates ranged from 43 to 44 mol%. Strains 15-1(T) and NRRL B-14850 showed high levels of DNA-DNA relatedness (82-100 %) to each other and to the other strains isolated here; they displayed low levels of DNA-DNA relatedness (<29 %) to the type strains of selected recognized Bacillus species. They grew in 15 % NaCl and optimally in 1 % NaCl, which is characteristic of moderately halotolerant bacteria. The isolates grew at pH 6.5 to 10.0 but not at pH 6.0. Their cell walls contained meso-diaminopimelic acid. The major isoprenoid quinone was MK-7 and the principal cellular fatty acids were anteiso-C(15 : 0), iso-C(15 : 0) and anteiso-C(17 : 0). Based on these results, the strains tested were regarded as members of a novel Bacillus species for which the name Bacillus vietnamensis sp. nov. is proposed. The type strain is 15-1(T) (=JCM 11124(T)=NRIC 0531(T)=NRRL 23890(T)).

Animals↗
PubMed1964

ORIGIN AND PROPERTIES OF NATURALLY OCCURRING HAPTEN FROM ESCHERICHIA COLI.

Anacker, R. L. (National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratory, Hamilton, Mont.), R. A. Finkelstein, W. T. Haskins, M. Landy, K. C. Milner, E. Ribi, and P. W. Stashak. Origin and properties of naturally occurring hapten from Escherichia coli. J. Bacteriol. 88:1705-1720. 1964.-Haptens found in preparations of endotoxin and in fractions of disrupted cells, particularly one termed "native hapten," which appeared to be associated with the protoplasm of cells rather than with cell walls, have been further investigated with a view to establishing their origin and composition as well as their host-reactive properties. For this purpose, cells from a smooth strain of Escherichia coli O111:B4 were either extracted directly or disrupted and separated into cell-wall and protoplasmic fractions. Haptens were obtained by gel filtration of endotoxins, by trichloroacetic acid extraction of protoplasm, and by a mild acid hydrolysis of endotoxin. Several lines of evidence indicated that native hapten originated in the protoplasm rather than by autolysis or degradation of cell-wall endotoxin during procedures employed in disruption. In gel diffusion and quantitative precipitin tests, no hapten was identical with endotoxin, but native hapten was serologically the most complex of the haptens and precipitated the most antibody. Native and acid haptens, on a weight basis, fixed about 1% of the quantity of complement fixed by homologous endotoxin. Haptens did not stimulate the production of antibodies in mice or rabbits and did not elicit endotoxic host reactions. Chemically, native hapten differed from endotoxin and from acid hapten in that it lacked phosphorus, heptose, long-chain fatty acids, and 2-keto-3-deoxyoctonate. These substances did not appear to be determinants of antigenic specificity, but they may provide necessary bonds for assembling hapten-like units into fully antigenic and toxic macromolecules.

Animals↗