Isolation and Characterization of a Polysaccharide Antigen from Propionibacterium acnes Released by a Glycine-Specific Chemical Protein Degradation Procedure

1990 ◽  
Vol 45 (7-8) ◽  
pp. 797-804
Author(s):  
Mary Fran Verostek ◽  
Lee E. Bartholomew ◽  
Peter Weber
Keyword(s):  
Molecular Weight ◽  
Cell Wall ◽  
Propionibacterium Acnes ◽  
Culture Media ◽  
Water Soluble ◽  
High Molecular Weight Fraction ◽  
Mercuric Oxide ◽  
Specific Chemical ◽  
Antigenic Polysaccharide ◽  
Isolation And Characterization

Abstract An acid-labile antigenic polysaccharide has been isolated from both cell walls and culture media of Propionibacterium acnes using a new chemical degradation procedure which liberates protein-bound or associated carbohydrate. Lyophilized cells and culture media were treated with a suspension of mercuric oxide in a solution of alkaline mercuric cyanide for several hours at room temperature liberating water-soluble polysaccharide material. The antigenic polysac- charide was freed of reaction products by alcohol extraction and purified by anion exchange chromatography and gel filtration, resulting in three distinct fractions of acidic polysaccha- rides of apparent molecular weights between 15 - 50 kDa. Sugar analysis showed the polysac- charides to contain fucose, galactose, glucose, mannose, galactosamine. glucosamine, and 2,3- diamino-2.3-dideoxy-D-glucuronic acid. The three fractions also contained amino acids, predominantly glutamic acid, alanine, and glycine, known to be components of P. acnes cell wall peptidoglycan. All three molecular weight fractions reacted with rabbit antisera raised against whole P. acnes cells, with the highest titer for both cell and media-derived polysaccharide material consistently in the high molecular weight fraction. This procedure was also capable of releasing antigenic polysaccharide from tissues of rats administered P. acnes cells or radio- labeled cell wall fragments.

scholarly journals Isolation and characterization of monoclonal antibodies directed against plant plasma membrane and cell wall epitopes: identification of a monoclonal antibody that recognizes extensin and analysis of the process of epitope biosynthesis in plant tissues and cell cultures.

1988 ◽  
Vol 107 (1) ◽  
pp. 163-175 ◽  
Author(s):  
D J Meyer ◽  
C L Afonso ◽  
D W Galbraith
Keyword(s):  
Monoclonal Antibody ◽  
Cell Wall ◽  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Solid Phase ◽  
Culture Media ◽  
Gradient Centrifugation ◽  
Cell Suspension Cultures ◽  
Isolation And Characterization

Membranes from tobacco cell suspension cultures were used as antigens for the preparation of monoclonal antibodies. Use of solid phase and indirect immunofluorescence assays led to the identification of hybridomas producing antibodies directed against cell surface epitopes. One of these monoclonal antibodies (11.D2) was found to recognize a molecular species which on two-dimensional analysis (using nonequilibrium pH-gradient electrophoresis and SDS-PAGE) was found to have a high and polydisperse molecular mass and a very basic isoelectric point. This component was conspicuously labeled by [3H]proline in vivo. The monoclonal antibody cross-reacted with authentic tomato extensin, but not with potato lectin nor larch arabinogalactan. Use of the monoclonal antibody as an immunoaffinity reagent allowed the purification of a tobacco glycoprotein which was identical in amino acid composition to extensin. Finally, immunocytological analyses revealed tissue-specific patterns of labeling by the monoclonal antibody that were identical to those observed with a polyclonal antibody raised against purified extensin. We have concluded that monoclonal antibody 11.D2 recognizes an epitope that is carried exclusively by extensin. Analysis of cellular homogenates through differential and isopycnic gradient centrifugation revealed that biosynthesis of the extensin epitope was found on or within the membranes of the endoplasmic reticulum, Golgi region and plasma membrane. This result is consistent with the progressive glycosylation of the newly-synthesized extensin polypeptide during its passage through a typical eukaryotic endomembrane pathway of secretion. The 11.D2 epitope was not found in protoplasts freshly isolated from leaf tissues. However, on incubation of these protoplasts in appropriate culture media, biosynthesis of the epitope was initiated. This process was not impeded by the presence of chemicals that are reported to be inhibitors of cell wall production or of proline hydroxylation.

Antimicrobial Action of Hydrolyzed Chitosan against Spoilage Yeasts and Lactic Acid Bacteria of Fermented Vegetables

2002 ◽  
Vol 65 (5) ◽  
pp. 828-833 ◽  
Author(s):  
TONY SAVARD ◽  
CAROLE BEAULIEU ◽  
ISABELLE BOUCHER ◽  
CLAUDE P. CHAMPAGNE
Keyword(s):  
Molecular Weight ◽  
Cell Wall ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Culture Media ◽  
Antimicrobial Properties ◽  
Spoilage Yeasts ◽  
Entire Cell ◽  
Fermented Vegetables ◽  
Chitosan Lactate

The antimicrobial properties of various chitosan-lactate polymers (ranging from 0.5 to 1.2 MDa in molecular weight) against two yeasts isolated from fermented vegetables and against three lactic acid bacteria from a mixed starter for sauerkraut on methylene blue agar (MBA) and in vegetable juice medium (VJM) were investigated. Chitosan-lactate reduced the growth of all microorganisms in solid (MBA) as well as in liquid (VJM) medium. In MBA, a concentration of 5 g/liter was needed to inhibit the growth of Saccharomyces bayanus, while 1 g/liter was sufficient to inhibit the growth of Saccharomyces unisporus. Lactic acid bacteria were also inhibited in this range of concentrations. The low-molecular-weightchitosan-lactateDP3 (0.5 kDa) was most efficient in solid medium (MBA), and inhibitory activities decreased with increasing hydrolysate lengths. In liquid medium (VJM), 0.5 g of chitosan-lactate per liter reduced the growth rates for both yeasts, but 10 g/liter was insufficient to prevent yeast growth. Intermediate-molecular-weight chitosan-lactate (5 kDa) was more efficient than chitosan of low molecular weight. Native chitosan (1.2 MDa) showed no inhibition in either medium. Microscopic examination of S. unisporus Y-42 after treatment with chitosan-lactate DP25 showed agglutination of a refractive substance on the entire cell wall, suggesting an interaction between chitosan and the cell wall. When chitosanase was added to the culture media containing chitosan-lactate, refractive substances could not be observed.

scholarly journals Construction of soil defined media using quantitative exometabolomic analysis of soil metabolites

2017 ◽  
Author(s):  
Stefan Jenkins ◽  
Tami L Swenson ◽  
Rebecca Lau ◽  
Andrea Rocha ◽  
Alex Aaring ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Weight ◽  
Organic Carbon ◽  
Quantitative Information ◽  
Water Soluble ◽  
Gas Chromatography Mass Spectrometry ◽  
Isolation And Characterization ◽  
Defined Media ◽  
Chromatography Mass Spectrometry

Exometabolomics enables analysis of metabolite utilization of low molecular weight organic substances by soil isolates. Environmentally-based defined media are needed to examine ecologically relevant patterns of substrate utilization. Here, we describe an approach for the construction of defined media using untargeted characterization of water soluble soil metabolites. To broadly characterize soil metabolites, both liquid chromatography mass spectrometry (LC/MS) and gas chromatography mass spectrometry (GC/MS) were used. With this approach, 96 metabolites were identified, including amino acids, amino acid derivatives, sugars, sugar alcohols, mono- and di-carboxylic acids, osmolytes, nucleobases, and nucleosides. From this pool of metabolites, 25 were quantified. Water soluble organic carbon was fractionated by molecular weight and measured to determine the fraction of carbon accounted for by the quantified metabolites. This revealed that, much like soil microbial community structures, these soil metabolites have an uneven quantitative distribution, with a single metabolite, trehalose accounting for 9.9 percent of the (< 1 kDa) water extractable organic carbon. This quantitative information was used to formulate two soil defined media (SDM), one containing 23 metabolites (SDM1) and one containing 46 (SDM2). To evaluate SDM for supporting the growth of bacteria found at this field site, we examined the growth of 30 phylogenetically diverse soil isolates obtained using standard R2A medium. The simpler SDM1 supported the growth of up to 13 isolates while the more complex SDM2 supported up to 25 isolates. One isolate, Pseudomonas corrugata strain FW300-N2E2 was selected for a time-series exometabolomics analysis to investigate SDM1 substrate preferences. Interestingly, it was found that this organism preferred lower-abundance substrates such as guanine, glycine, proline and arginine and glucose and did not utilize the more abundant substrates maltose, mannitol, trehalose and uridine. These results demonstrate the viability and utility of using exometabolomics to construct a tractable environmentally relevant media. We anticipate that this approach can be expanded to other environments to enhance isolation and characterization of diverse microbial communities.

Isolation and Characterization of Secretory Vesicles in Germinated Pollen of Lilium longiflorum

1971 ◽  
Vol 8 (2) ◽  
pp. 331-351 ◽  
Author(s):  
W. J. VAN DER WOUDE ◽  
D. J. MORRÉ ◽  
C. E. BRACKER
Keyword(s):  
Cell Wall ◽  
Secretory Pathway ◽  
Periodic Acid ◽  
Lilium Longiflorum ◽  
Water Soluble ◽  
Hot Water ◽  
Secretory Vesicles ◽  
Vesicle Membrane ◽  
Isolation And Characterization ◽  
Germinated Pollen

Secretory vesicles containing polysaccharide were isolated from germinated pollen of Lilium longiflorum and characterized by biochemical and ultrastructural investigation. Pollen tubes exhibit a secretory pathway in which the vesicles concentrated in the tube apex are produced by the Golgi apparatus and contributed to the cell wall at the apex upon fusion of the vesicle membrane with the plasma membrane. Secretory vesicles were isolated by a method involving the size discrimination of cytoplasmic components using Millipore filters. Cells were disrupted under conditions which minimized membrane vesiculation. Identification was made by electron-microscopic comparison of the periodic acid-silver hexamine (PASH) reactivities of in situ and isolated secretory vesicles. The secretory vesicles contained polysaccharides which were high in galacturonic acid and similar in sugar composition to those of the hot-water-soluble fraction of pollen tube cell wall. A hot-water-insoluble, non-cellulosic glucan was the major component of the cell wall. Less than 7% of the wall was cellulosic. Chitin was absent. Similarities in the ultrastructure and PASH staining of apical secretory vesicles and an amorphous component of the cell wall support a precursor-product relationship between these 2 cell components. Ultrastructural investigations revealed complexes of the endoplasmic reticulum (ER) associated with electron-translucent regions of cytoplasm, suggesting a possible function of the ER in cell wall formation. Additionally, patterns of PASH staining show that changes in polysaccharides occur in secretory vesicles after vesicles have been formed by dictyosomes. Therefore, secretory vesicles may have a role in polysaccharide synthesis as well as in membrane and product transport.

scholarly journals The isolation and characterization of the high-molecular-weight glycoprotein from pig colonic mucus

1978 ◽  
Vol 173 (2) ◽  
pp. 569-578 ◽  
Author(s):  
T Marshall ◽  
A Allen
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Quaternary Structure ◽  
Water Soluble ◽  
Isolation And Characterization ◽  
Nuclease Digestion ◽  
Non Covalent Interactions ◽  
Covalent Interactions ◽  
Equilibrium Centrifugation

1. A high-molecular-weight glycoprotein constitutes over 80% by weight of the total glycoprotein from water-soluble pig colonic mucus. 2. It was isolated from from nucleic acid and non-covalently bound protein by nuclease digestion followed by equilibrium centrifugation in a CsCl gradient. 3. The glycoprotein has the following composition by weight: fucose 10.4%; glucosamine 23.9%; galactosamine 8.3%; sialic acid 9.9%; galactose 20.8%; sulphate 3.0%; protein 13.3%; moisture about 10%. 4. The native glycoprotein has the high mol.wt. of 15×10(6). 5. Reduction of the native glycoprotein with 2-mercaptoethanol results in a glycoprotein of mol.wt. 6×10(6). 6. Pronase digestion removes 29% of the protein (3% of the glycoprotein) but none of the carbohydrate. 7. The molecular weight of the Pronase-digested glycoprotein is 1.5×10(6), which is halved to 0.76×10(6) on reduction with 2-mercaptoethanol. 8. The contribution of non-covalent interactions, disulphide bridges and the non-glycosylated peptide core to the quaternary structure of the glycoprotein are discussed and compared with the known structure of pig gastric glycoportein.

scholarly journals Comparison of Cell Wall Components in Normal and Disordered Juice Vesicles of Grapefruit

1990 ◽  
Vol 115 (2) ◽  
pp. 281-287 ◽  
Author(s):  
Yong-Soo Hwang ◽  
D.J. Huber ◽  
L.G. Albrigo
Keyword(s):  
Molecular Weight ◽  
Cell Wall ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Cell Wall Composition ◽  
Water Soluble ◽  
Citrus Paradisi ◽  
Composition And Structure ◽  
The Difference ◽  
Wall Components

Cell wall composition and structure were examined in visually normal (N), granulated (G), and collapsed (VC) juice vesicles of `Marsh Seedless' grapefruit (Citrus paradisi) Macf.). According to gel-filtration data, VC appeared to be associated with a modification of water-soluble (WSP) and chelate-soluble (CSP) pectin molecular weight (Mr); small-Mr pectins increased, whereas large-J4. pectins decreased. The difference in M= of pectins did not appear to be mediated by polygalacturonases. Molecular weight of hemicelluloses did not differ. Granulated vesicles contained about two times more structural polysaccharides (pectins, hemicelhdose, and cellulose) than N vesicles, although hemicellulose and pectin M= modification were absent. Ion-exchange profiles of WSP, CSP, and hemicelhrlose fractions of VC and G vesicles were not different from those of N vesicles. Individual cells in vesicles with G and these vesicles themselves were much larger than those of N vesicles, whereas cells in VC were partially or completely collapsed.

scholarly journals Molecular Weight and Degree of Methoxylation in Cell Wall Polyuronide during Softening in Pear and Apple Fruit

1992 ◽  
Vol 117 (4) ◽  
pp. 600-606 ◽  
Author(s):  
H. Yoshioka ◽  
K. Aoba ◽  
Y. Kashimura
Keyword(s):  
Molecular Weight ◽  
Cell Wall ◽  
Cell Walls ◽  
Soluble Fraction ◽  
Apple Fruit ◽  
Water Soluble ◽  
Pear Fruit ◽  
Water Soluble Fraction ◽  
High Degree ◽  
Soluble Fractions

The concentrations of water-soluble polyuronides in apples [Malus domestica Borkh.) and pears (Pyrus communis L.) increased, but those of EDTA- and HCl-soluble polyuronides decreased during softening. Total polyuronide content decreased slightly during softening in both fruits. Depolymerization of polyuronides was observed only in the water-soluble fraction in pear fruit during softening, concomitant with an increase in polygalacturonase (PG) activity. No detectable depolymerization was observed in any of the polyuronide fractions during softening of apple fruit nor was any PG activity detected. The polyuronide fractions extracted from pear and apple cell walls contained various amounts of methoxyl groups. Polyuronides with a high degree of methoxylation were preferentially lost from EDTA- and HCl-soluble polyuronides during softening of both fruit. The water-soluble polyuronide had a lower degree of methoxylation than those lost in the EDTA- and HCl-soluble fractions. These results suggest de-esterification of polyuronides with a high degree of methoxylation rather than the depolymerization of polyuronides in the solubilization of polyuronides during ripening of apples and pears.

Biotransformation of coal by ligninolytic Streptomyces

1988 ◽  
Vol 34 (5) ◽  
pp. 667-674 ◽  
Author(s):  
Rajinder K. Gupta ◽  
Jennifer K. Spiker ◽  
Don L. Crawford
Keyword(s):  
Molecular Weight ◽  
Culture Media ◽  
Water Soluble ◽  
Medium Ph ◽  
Agar Culture ◽  
Coal Solubilization ◽  
Heat Stable ◽  
Coal Particles ◽  
Lignite Coal ◽  
Solubilizing Activity

The chemistry of coal liquefaction by Streptomyces viridosporus, Streptomyces setonii and Streptomyces badius was studied. These ligninolytic actinomycetes solubilized lignite and bituminous coals when coal particles were placed on the surfaces of mycelial lawns on agar culture media. On agar, liquefaction proceeded with the formation of clear to black water-soluble products. Up to 40% of some coals was solubilized in 3 weeks. Lignite coals were also solubilized in liquid shake flask cultures. Coal solubilization by S. viridosporus was oxidative, as shown by Fourier transform infrared spectroscopy and elemental analysis. Compared to the starting coals, the soluble products were enriched in oxygen, nitrogen, sulfur, and ash, but were lower in hydrogen relative to carbon. Insoluble coal residues were less altered than the liquefied products, but they were higher in ash content than the starting coals. Chemical analysis of culture filtrates showed that some low molecular weight products were released, but most of the soluble products were acid precipitable and probably polymeric. Lignite coal solubilization by the S. viridosporus did not require induction and in liquid media solubilization was always accompanied by a significant increase in medium pH. When medium pH was not allowed to become basic, extracellular coal solubilizing activity was negligible. Extracellular solubilizing activity was heat stable, and ultrafiltration experiments showed that the solubilizing agent was of less than 10 000 molecular weight. Coal solubilizing activity of S. viridosporus culture supernatants could be eliminated by lowering their pH, but the activity could be recovered by raising the pH back up to above neutral. Phosphate and Tris buffers of varying strength and pH showed that nonbiological solubilization of lignite coal occurred under basic conditions. The combined data show that biological lignite coal solubilization by these Streptomyces is mediated by alkaline products that the cells release into the growth medium. These findings confirm and expand an earlier report by others that coal solubilization by Streptomyces setonii may be nonenzymatic.

Studies on the Functionality of Newly Synthesized Fibrinogen after Treatment of Acute Myocardial Infarction with Streptokinase, Increase in the Rate of Fibrinopeptide Release

1993 ◽  
Vol 70 (06) ◽  
pp. 0978-0983 ◽  
Author(s):  
Edelmiro Regano ◽  
Virtudes Vila ◽  
Justo Aznar ◽  
Victoria Lacueva ◽  
Vicenta Martinez ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Molecular Weight ◽  
Steady State ◽  
High Molecular Weight ◽  
Coronary Arteries ◽  
Risk Index ◽  
Weight Fraction ◽  
High Molecular Weight Fraction ◽  
Fibrinopeptide A

SummaryIn 15 patients with acute myocardial infarction who received 1,500,000 U of streptokinase, the gradual appearance of newly synthesized fibrinogen and the fibrinopeptide release during the first 35 h after SK treatment were evaluated. At 5 h the fibrinogen circulating in plasma was observed as the high molecular weight fraction (HMW-Fg). The concentration of HMW-Fg increased continuously, and at 20 h reached values higher than those obtained from normal plasma. HMW-Fg represented about 95% of the total fibrinogen during the first 35 h. The degree of phosphorylation of patient fibrinogen increased from 30% before treatment to 65% during the first 5 h, and then slowly declined to 50% at 35 h.The early rates of fibrinopeptide A (FPA) and phosphorylated fibrinopeptide A (FPAp) release are higher in patient fibrinogen than in isolated normal HMW-Fg and normal fibrinogen after thrombin addition. The early rate of fibrinopeptide B (FPB) release is the same for the three fibrinogen groups. However, the late rate of FPB release is higher in patient fibrinogen than in normal HMW-Fg and normal fibrinogen. Therefore, the newly synthesized fibrinogen clots faster than fibrinogen in the normal steady state.In two of the 15 patients who had occluded coronary arteries after SK treatment the HMW-Fg and FPAp levels increased as compared with the 13 patients who had patent coronary arteries.These results provide some support for the idea that an increased synthesis of fibrinogen in circulation may result in a procoagulant tendency. If this is so, the HMW-Fg and FPAp content may serve as a risk index for thrombosis.

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