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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↗
PubMed1982

Low Temperature-Induced Alterations in the Chloroplast and Microsomal Membranes of Dunaliella salina.

The metabolic regulation of membrane lipid composition has been examined using the cell wall-less, unicellular green alga Dunaliella salina (UTEX 1644) as a model system. Low temperature stress was employed to initiate and study the regulatory response.When cultures growing logarithmically at 30 degrees C were chilled to 12 degrees C, cell division ceased for approximately 100 hours, and then the cells resumed logarithmic growth at a slower rate. The phospholipid, glycolipid and protein content, on a per cell basis, was, in each case, approximately 20% higher in cells grown at 12 degrees C. The volume of the 12 degrees C-acclimated cells was 2.8 times that of 30 degrees C-grown cells. The quantity of chloroplast membrane, as determined by morphometric analysis, was 20% greater, whereas the content of microsomal membrane material was more elevated, being approximately 2.8 times that of 30 degrees C-grown cells.Lipid compositional analyses were carried out on purified chloroplasts and microsomes isolated from Dunaliella grown at 30 and 12 degrees C and also from cells 12 and 60 hours following a shift from 30 to 12 degrees C. In both chloroplast and microsomal phospholipids fatty acid unsaturation increased during acclimation to low temperature. Generally, microsomal phospholipids responded more quickly and to a greater extent than did chloroplast phospholipids. Despite these alterations, little change in the relative proportions of phospholipid classes was observed in either cell fraction.In sharp contrast to the pattern of phospholipid change, chloroplast glycolipids responded to low temperature by significantly increasing the proportion of one specific class, digalactosyl diglycerides, relative to monogalactosyl diglycerides, while showing minimal change in fatty acid distribution within any given glycolipid class.The ease and rapidity with which Dunaliella cells can be manipulated with respect to environmental stress and isolation of intact cell organelles makes it particularly well suited for research on intermembrane lipid dynamics within the plant cell.

Journal Article↗
PubMed2001

Biochemical mechanism of combined effect of ethambutol and sparfloxacin against Mycobacterium smegmatis.

M. smegmatis cells grown in the presence of combination of ethambutol (EMB) and sparfloxacin (SPX) had decreased level of total cellular lipids as compared to control as well as cells grown in the presence of sub-inhibitory concentration (MIC50) of individual drugs. Amongst various phospholipids analyzed, maximum decrease was observed in the content of phosphatidylinositolmannosides (PIMs) of the cells grown in combination of EMB and SPX. In contrast, the subcellular distribution of phospholipids revealed a significant increase in PIMs content of both cell wall and cell membrane of the cells grown in the presence of combination of drugs as compared to control as well as individual drugs. Mycolic acids of M. smegmatis cells were found to be main targets as combination of drugs resulted in significant decrease in total cellular as well as cell wall mycolic acids as compared to control and individual drugs. Changed lipid composition of M. smegmatis cells grown in the presence of MIC50 of EMB, SPX and combination resulted in significant surface changes as was evident from decreased limiting fluorescence (Fmax) intensity of 1-anilinonaphthalene-8-sulfonate (ANS). Thus, the results of this study suggested that ethambutol and sparfloxacin in combination exerted their antimycobacterial effect principally due to their action on phosphatidylinositolmannosides (PIMs) and mycolic acids, which form the permeability barrier of mycobacteria.

Anti-Infective Agents↗
PubMed1983

Acid and alkaline phosphatases of Capnocytophaga species. III. The relationship of the enzymes to the cell wall.

Acid (AcP) and alkaline phosphatase (AlP) were localized by physicobiochemical techniques. Greater than 53% of the phosphatases were detected, following sonication, in a low speed centrifugation pellet while osmotically shocked and spheroplasted Capnocytophaga species released only 9-28% and 11-43% of the cellular phosphatases, respectively. French pressure cell disruption was more effective in releasing the phosphatases. Cell fragments were separated into cell wall, cytoplasmic membrane, and soluble fractions as determined by marker enzyme, chemical composition, transmission electron microscopy, sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) protein analysis, and gel diffusion. AcP and AlP was partitioned between the isolated cell wall (31-46%) and soluble material (33-61%), with greater than 60% of the phosphatases remaining with the cell wall following Triton X-100 treatment. The amount of phosphatase at the surface of intact C. ochracea was quantitated by specific 125I-protein labelling.

Acid Phosphatase↗
PubMed1994

[Isolation and analysis of lipid antigen from M. tuberculosis cell walls].

A method has been developed for isolation of an antigen based on nonpolar lipids from M. tuberculosis cell walls, its specific and nonspecific fractions separated, and composition determined by thin-layer chromatography. Ninety-two blood sera of tuberculous patients and 50 ones from healthy donors were examined in enzyme immunoassay with this antigen. Use of this antigen in serologic tests helped detect in patients' sera antibodies to mycobacterial lipids.

Antigens, Bacterial↗
PubMed1989

Gardnerella vaginalis has a gram-positive cell-wall ultrastructure and lacks classical cell-wall lipopolysaccharide.

Gardnerella vaginalis has a very thin cell wall with a characteristic gram-negative staining pattern and an apparent lamellar structure when viewed at an oblique angle by electronmicroscopy. Examination at right angles to the cell-wall plane and by freeze-etching showed absence of an outer membrane or any other lamellar structure. Cell-wall extracts made by methods specific for lipopolysaccharide (LPS) gave negative reactions by silver staining and for endotoxin in the limulus amoebocyte lysate assay. 2-Keto-3-deoxy-D-manno-2-octonoic acid (KDO), heptose and hydroxy fatty acids specific for LPS were not detected in the extracts. G. vaginalis cell walls are unequivocally gram-positive in their ultrastructural characteristics and chemical composition.

Cell Wall↗
PubMed2005

Detection of conjugated dienoic fatty acids in human vascular smooth muscle cells treated with conjugated linoleic acid.

Conjugated linoleic acids (CLA) have attracted scientific interest due to their potential beneficial effects on atherosclerosis. Recent studies demonstrated that conjugated metabolites of CLA are found in tissues of CLA-fed animals and cultured cells treated with CLA. This observation has gained in importance since it has recently been shown that these metabolites of CLA exert specific biological activities. Therefore, the present study aimed to explore the potential formation of metabolites of cis-9, trans-11 CLA, trans-10, cis-12 CLA and trans-9, trans-11 CLA in cells of the vascular wall, which has not yet been shown. Examination of fatty acid composition of total cell lipids using Ag+-HPLC, GC-FID and GC-MS analysis revealed a significant isomer-specific formation of conjugated metabolites of CLA such as CD16:2, CD20:2 and CD22:2 in human coronary artery smooth muscle cells treated with various CLA isomers. Different CD16:2/CLA ratios between various CLA isomers as observed in the present study indicate that fatty acid metabolism is differently affected by the configuration of the double bonds. In conclusion, the observation from the present study suggests that the effects of CLA in vascular cells might not only be mediated by CLA itself but also by its conjugated metabolites. Future studies using highly purified conjugated metabolites of CLA are necessary to study their role in mediating biological effects of CLA in cell culture systems.

Animals↗
PubMed2003

Lignins and lignocellulosics: a better control of synthesis for new and improved uses.

The composition and structure of lignified walls has a dramatic impact on the technological value of raw materials. The chemical flexibility of the secondary cell wall has been demonstrated and it is now possible to develop strategies to optimize its composition through genetic engineering. Thanks to functional genomics, new target genes of both plant and microbial origin are rapidly becoming available for this purpose and their use will open new avenues for producing tailor-made plant products with improved properties. Moreover, the major proportion of terrestrial plant biomass comprises lignified cell walls and this reservoir of carbon should be increasingly exploited for the production of chemicals and energy within the context of sustainable development. For example, the design of plants suitable for downstream conversion processes, such as the production of bioethanol, and the exploitation of microorganisms and microbial enzymes for biomass pretreatments or for the production of novel chemicals.

Biomass↗
PubMed1988

Phagocytosis of heat-killed blastospores of Candida albicans by human monocyte beta-glucan receptors.

Candida albicans is an opportunistic human pathogen with a cell wall that is qualitatively similar in carbohydrate composition to zymosan. Monolayers of human peripheral blood monocytes ingested 2.5 x 10(6)-2.5 x 10(7) heat-killed C. albicans blastospores in a dose-related manner. The percentage of monocytes ingesting at least one C. albicans reached a near plateau level of 68 +/- 6% (mean +/- SD, n = 3) when 2.5 x 10(7) particles per ml were incubated with monocytes for 30 min. Pretreatment of monocytes with 10 ng/ml to 100 micrograms/ml of soluble yeast beta-glucan inhibited C. albicans ingestion in a dose-dependent manner without affecting Fc-mediated ingestion of IgG-coated sheep erythrocytes (EsIgG). Pretreatment of monocytes with 100 micrograms/ml of soluble yeast beta-glucan inhibited the ingestion of 1 or more C. albicans by 80 +/- 11% (mean +/- SD, n = 4), with 50% inhibition occurring with approximately 7 micrograms/ml of soluble beta-glucan. Incubation of monocytes with 100 micrograms/ml to 1000 micrograms/ml of soluble yeast mannan resulted in no significant inhibition of C. albicans ingestion. The pretreatment of monolayers of monocytes for 30 min with 1 microgram/ml to 50 micrograms/ml of affinity-purified trypsin resulted in a dose-dependent inhibition of C. albicans ingestion with 10 micrograms/ml of trypsin inhibiting the percentage of monocytes ingesting 1 or more organisms by 71 +/- 6%. The inhibitory effects of soluble yeast beta-glucan and trypsin pretreatment on the ingestion of heat-killed C. albicans are comparable to the inhibitory effect of these agents on monocyte phagocytosis of zymosan and glucan particles. This relationship indicates that C. albicans contain a ligand that is recognized by the monocyte beta-glucan receptor of human monocytes.

Candida albicans↗
PubMed1976

[Nutritional study of the membranes of S. cerevisiae. 1. Chemical composition and viscosity].

After an enzymic auto-degradation of their cytoplasma, the residue of the yeast (S. cerevisiae) contains a thick cell wall and a thin plasma membrane. These total membranes have the following composition in dry substance: proteins = 20,5%, lipids = 31,5%, carbohydrates = 42,0%, and ash = 3,7%. About 85% of carbohydrates (glycans) are easily hydrolysable by chemical method and should be digestible by the non-ruminant species. Proteins, contain 6,9% of lysine, 6,35% of threonine and have only one serious deficit, that of methionine which is 57%. These proteins seem to be resistant to the Maillard reaction. The percentage of in vitro digestible lysine increases when the membranes have undergone a heating of sufficient intensity. The methods of the treatment may give to the membranes an important apparent viscosity. These membranes could play the part of thickening and gelifiant agent in food technology. They also might constitute an interesting source of proteins because of its concentration in lysine and threonine.

Amino Acids↗
PubMed1982

Taxonomic relationships of Geotrichum flavo-brunneum.

Geotrichum flavo-brunneum Miller, Giddens et Foster shows both basidiomycetous (viz. the presence of urease and the high % G + C of DNA) and ascomycetous (viz, the carbohydrate composition) affinities. The study of the cell-wall ultrastructure, the hyphal branching and the Diazonium Blue B colouration points to a relationship with anamorphs of Ascomycetes.

Geotrichum↗
PubMed1984

Structure of the cell surface of the yeast Candida tropicalis and its relation to hydrocarbon transport.

The surface structure of the hydrocarbon-utilizing yeast Candida tropicalis was investigated by scanning and transmission electron microscopy (SEM and TEM respectively). The sample preparation technique was based on a rapid cryofixation without any addition of cryoprotectants. In subsequently freeze-dried samples the surface structure was analysed by scanning electron microscopy. Thin sections were prepared from freeze substituted samples. Both techniques revealed hair-like structures at the surface of hydrocarbon-grown cells. The hairy surface structure of the cells was less expressed in glucose-grown cells and it was absent completely after proteolytic digestion of the cells. When cells were incubated with hexadecane prior to cryofixation a contrast-rich region occurred in the hair fringe of thin sections as revealed by TEM. Since these structures were characteristic for hexadecane-grown cells and could not be detected in glucose-grown or protease-treated cells it was concluded that they originate from hexadecane adhering to the cell surface and are functionally related to hexadecane transport. The structure of the surface and its relation to hydrocarbon transport are discussed in view of earlier results on the chemical composition of the surface layer of the cell wall.

Candida↗
PubMed1975

[Biological activity of bacterial peptidoglycan (mucopeptide) (author's transl)].

A brief survey on the ultrastructure, the chemical composition and the immunological properties of bacterial cell wall peptidoglycan (mucopeptide) is presented. This paper deals with the various recently discovered biological activities of peptidoglycan. These could be divided into three different groups, namely: 1. Endotoxin-like properties: pyrogenicity, induction of the local Shwartzman reaction, increase in non-specific resistance to bacterial infection, release of 5-hydroxytryptamine from rabbit platelets, complement activation,gelation of amebocyte lysate. 2. Inflammatory reactions of skin and internal organs, aggressin activity (virulence factor), inhibition of phagocytosis of bacteria by granulocytes and macrophages, inhibition of growth of cell cultures, cytotoxity to granulocytes and macrophages. 3. Potentiation of humoral and cellular immune responses (adjuvant), enhancement of tumor defense in experimental animals. The potential mechanisms of action of peptidoglycan are discussed and attention is focused on the implications of the various peptidoglycan activities for medicine.

Amino Acid Sequence↗
PubMed1997

Inheritance, gene expression, and lignin characterization in a mutant pine deficient in cinnamyl alcohol dehydrogenase.

We have discovered a mutant loblolly pine (Pinus taeda L.) in which expression of the gene encoding cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) is severely reduced. The products of CAD, cinnamyl alcohols, are the precursors of lignin, a major cell wall polymer of plant vascular tissues. Lignin composition in this mutant shows dramatic modifications, including increased incorporation of the substrate of CAD (coniferaldehyde), indicating that CAD may modulate lignin composition in pine. The recessive cad-n1 allele, which causes this phenotype, was discovered in a tree heterozygous for this mutant allele. It is inherited as a simple Mendelian locus that maps to the same genomic region as the cad locus. In mutant plants, CAD activity and abundance of cad RNA transcript are low, and free CAD substrate accumulates to a high level. The wood of the mutant is brown, whereas the wood in wild types is nearly white. The wood phenotype resembles that of brown midrib (bm) mutants and some transgenic plants in which xylem is red-brown due to a reduction in CAD activity. However, unlike transgenics with reduced CAD, the pine mutant has decreased lignin content. Wood in which the composition of lignin varies beyond previous expectations still provides vascular function and mechanical support.

Alcohol Oxidoreductases↗
PubMed1998

Molecular cloning and DNA sequencing of the Staphylococcus aureus UDP-N-acetylmuramyl tripeptide synthetase (murE) gene, essential for the optimal expression of methicillin resistance.

The Tn551 insertion in mutant RUSA235 of a highly methicillin resistant Staphylococcus aureus strain results in drastic reduction in the level of methicillin resistance and abnormalities, both in the composition of the peptidoglycan and of the cell wall precursor pool. Cloning and sequencing of the inactivated gene indicates that it is the murE gene of Staphylococcus aureus.

Amino Acid Sequence↗
PubMed1978

Adsorption of cyanophage AS-1 to unicellular cyanobacteria and isolation of receptor material from Anacystis nidulans.

Cells of unicellular cyanobacteria of typological group Ia, containing approximately 50 mol% guanine + cytosine (G+C) in their DNA (R. Y. Stanier, R. Kunisawa, M. Mandel, and G. Cohen-Bazire, Bacteriol. Rev. 35:171-205, 1971), were susceptible to infection by the cyanophage AS-1. Cyanobacteria of the same typological group, containing approximately 65 mol% G+C in their DNA, did not adsorb the cyanophage AS-1 or adsorbed it at a low rate. AS-1 was not propagated by any of the investigated strains with a high G+C content in their DNA. However, cells of strains 6907 and 6911 were lysed by cyanophage AS-1. A comparison of the host range of this phage with the lipopolysaccharide composition of host and non-host cell walls suggests that lipopolysaccharides are involved in the adsorption process. About 8 microgram of lipopolysaccharide per ml from host strains inactivated 50% of the particles of a solution containing 100 PFU/ml after 60 min of incubation at 30 degrees C. Material with receptor activity was extracted from the host strain Anacystis nidulans KM. The extract was purified of glycolipids and pigments, and a fraction showing receptor activity was isolated. This fraction contained three polypeptides of molecular weights between 54,000 and 64,000. Heat and protease treatment of whole cells and of isolated receptor material decreased the receptor activity. The fluorescence intensity of A. nidulans cells labeled with 1-anilino-8-naphthalene sulfonate was increased when AS-1 was adsorbed to these cells. The participation of lipopolysaccharides and proteins in the formation of the receptor complex is discussed.

Adsorption↗
PubMed1995

Mechanisms of membrane toxicity of hydrocarbons.

Microbial transformations of cyclic hydrocarbons have received much attention during the past three decades. Interest in the degradation of environmental pollutants as well as in applications of microorganisms in the catalysis of chemical reactions has stimulated research in this area. The metabolic pathways of various aromatics, cycloalkanes, and terpenes in different microorganisms have been elucidated, and the genetics of several of these routes have been clarified. The toxicity of these compounds to microorganisms is very important in the microbial degradation of hydrocarbons, but not many researchers have studied the mechanism of this toxic action. In this review, we present general ideas derived from the various reports mentioning toxic effects. Most importantly, lipophilic hydrocarbons accumulate in the membrane lipid bilayer, affecting the structural and functional properties of these membranes. As a result of accumulated hydrocarbon molecules, the membrane loses its integrity, and an increase in permeability to protons and ions has been observed in several instances. Consequently, dissipation of the proton motive force and impairment of intracellular pH homeostasis occur. In addition to the effects of lipophilic compounds on the lipid part of the membrane, proteins embedded in the membrane are affected. The effects on the membrane-embedded proteins probably result to a large extent from changes in the lipid environment; however, direct effects of lipophilic compounds on membrane proteins have also been observed. Finally, the effectiveness of changes in membrane lipid composition, modification of outer membrane lipopolysaccharide, altered cell wall constituents, and active excretion systems in reducing the membrane concentrations of lipophilic compounds is discussed. Also, the adaptations (e.g., increase in lipid ordering, change in lipid/protein ratio) that compensate for the changes in membrane structure are treated.

Bacteria↗
PubMed2002

Remodeling of small intramyocardial coronary arteries distal to a severe epicardial coronary artery stenosis.

OBJECTIVE: Impaired coronary blood flow (CF) or flow reserve with incomplete and delayed recovery of left ventricular (LV) function after revascularization is common in severe ischemic LV dysfunction. The underlying mechanism is not fully known. We studied structural changes of small intramyocardial coronary arteries (SIMCAs) in a pig model of chronic coronary stenosis, testing the hypothesis that microvascular remodeling develops distally to a severe epicardial coronary artery stenosis. METHODS AND RESULTS: A total of 24 pigs were studied in 3 groups. Left anterior descending coronary stenosis was created to reduce CF by a mean of approximately 30%, producing severe regional systolic dysfunction without infarction. The stenosis was maintained for 7 days in 6 pigs (Group 1) and for 4 weeks in 12 pigs (Group 2). The control group (Group 3) consisted of 6 pigs with the same surgical procedures but without stenosis. The wall thickness (WTa) and lumen (L) diameter of SIMCA were measured, and the ratio of WTa/L and lumen area/total vessel area (% lumen) were calculated. The composition of the arterial wall was studied with cell proliferation markers Ki67 and BrdU. The immediate reduction in CF after the creation of the stenosis was similar in both study groups, but after the first week, CF decreased significantly (P<0.05) when the stenosis was maintained (group 2). The left anterior descending stenosis caused regional LV dysfunction in all pigs (groups 1 and 2). After 4 weeks of stenosis with chronic myocardial hibernation (group 2), but not after 1 week (group 1), WTa and WTa/L increased and L decreased significantly in the chronic hibernating region located distally to the stenosis, compared with both the control (group 3) and the normal region in the same pig. The mean % lumen of SIMCA per pig correlated with the CF reduction (r=0.92, P<0.001) and with myocardial fibrosis (r=0.82, P<0.01) in the 4-week stenosis group. Ki67- and BrdU-positive cells were increased in the wall of SIMCA in Group 1 and 2 compared with the control group (P<0.01 for each). The proliferated cells were stained positively with smooth muscle alpha-actin antibody. CONCLUSION: In the chronic ischemic, hibernating myocardial region distal to a flow-limiting epicardial coronary stenosis, the small intramyocardial coronary arteries undergo remodeling, with an increase in wall thickness and a decrease in lumen. These structural changes may further restrict blood flow to ischemic myocardium and may account for the pathophysiologic impairment of CF or flow reserve after revascularization, which leads to delayed or incomplete recovery of myocardial function.

Animals↗