Production of Extracellular Polysaccharides by CAP Mutants of Cryptococcus neoformans

ABSTRACT The human pathogen Cryptococcus neoformans causes meningoencephalitis. The polysaccharide capsule is one of the main virulence factors and consists of two distinct polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM). How capsular polysaccharides are synthesized, transported, and assembled is largely unknown. Previously, it was shown that mutations in the CAP10, CAP59, CAP60, and CAP64 genes result in an acapsular phenotype. Here, it is shown that these acapsular mutants do secrete GalXM and GXM-like polymers. GXM and GalXM antibodies specifically reacted with whole cells and the growth medium of the wild type and CAP mutants, indicating that the capsule polysaccharides adhere to the cell wall and are shed into the environment. These polysaccharides were purified from the medium, either with or without anion-exchange chromatography. Monosaccharide analysis of polysaccharide fractions by gas-liquid chromatography/mass spectrometry showed that wild-type cells secrete both GalXM and GXM. The CAP mutants, on the other hand, were shown to secrete GalXM and GXM-like polymers. Notably, the GalXM polymers were shown to contain glucuronic acid. One-dimensional 1H nuclear magnetic resonance confirmed that the CAP mutants secrete GalXM and also showed the presence of O-acetylated polymers. This is the first time it is shown that CAP mutants secrete GXM-like polymers in addition to GalXM. The small amount of this GXM-like polymer, 1 to 5% of the total amount of secreted polysaccharides, may explain the acapsular phenotype.

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Extracellular polysaccharides from suspension cultures of Nicotiana plumbaginifolia

10.26686/wgtn.12597827.v1 ◽

2020 ◽

Author(s):

Ian Sims ◽

A Bacic

Keyword(s):

Uronic Acid ◽

Cell Suspension Cultures ◽

Anion Exchange Chromatography ◽

Nicotiana Plumbaginifolia ◽

Suspension Cultures ◽

Exchange Chromatography ◽

Arabinogalactan Protein ◽

Extracellular Polysaccharides ◽

Selective Precipitation ◽

The Individual

The soluble polymers secreted by cell-suspension cultures of Nicotiana plumbaginifolia contained 78% carbohydrate, 6% protein and 4% inorganic material. The extracellular polysaccharides were separated into three fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7 and the individual polysaccharides in each fraction were then isolated by selective precipitation and enzymic treatment. Monosaccharide and linkage compositions were determined for each polysaccharide after reduction of uronic acid residues and the degree of esterification of the various uronic acid residues in each polysaccharide was determined concurrently with the linkage types. Six components were identified: an arabinoxyloglucan (comprising 34% of the total polysaccharide) and a galactoglucomannan (15%) in the unbound neutral fraction, a type II arabinogalactan (an arabinogalactan-protein, 11%) and an acidic xylan (3%) in the first bound fraction, and an arabinoglucuronomannan (11%) and a galacturonan (26%) in the second bound fraction. © 1995.

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Specific Non-Reducing Ends in Heparins from Different Animal Origins: Building Blocks Analysis Using Reductive Amination Tagging by Sulfanilic Acid

Molecules ◽

10.3390/molecules25235553 ◽

2020 ◽

Vol 25 (23) ◽

pp. 5553

Author(s):

Pierre A. J. Mourier

Keyword(s):

Building Block ◽

Reductive Amination ◽

Building Blocks ◽

Anion Exchange Chromatography ◽

Sulfanilic Acid ◽

Exchange Chromatography ◽

Sulfated Polysaccharides ◽

Liquid Chromatography Mass Spectrometry ◽

Anticoagulant Drugs ◽

Strong Anion Exchange

Heparins are linear sulfated polysaccharides widely used as anticoagulant drugs. Their nonreducing-end (NRE) has been little investigated due to challenges in their characterization, but is known to be partly generated by enzymatic cleavage with heparanases, resulting in N-sulfated glucosamines at the NRE. Uronic NRE (specifically glucuronic acids) have been isolated from porcine heparin, with GlcA-GlcNS,3S,6S identified as a porcine-specific NRE marker. To further characterize NRE in heparinoids, a building block analysis involving exhaustive heparinase digestion and subsequent reductive amination with sulfanilic acid was performed. This study describes a new method for identifying heparin classical building blocks and novel NRE building blocks using strong anion exchange chromatography on AS11 columns for the assay, and ion-pair liquid chromatography-mass spectrometry for building block identification. Porcine, ovine, and bovine intestine heparins were analyzed. Generally, NRE on these three heparins are highly sulfated moieties, particularly with 3-O sulfates, and the observed composition of the NRE is highly dependent on heparin origin. At the highest level of specificity, the isolated marker was only detected in porcine heparin. However, the proportion of glucosamines in the NRE and the proportion of glucuronic/iduronic configurations in the NRE uronic moieties greatly varied between heparin types.

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Composition of the protoplast membrane from Saccharomyces cerevisiae

Biochemical Journal ◽

10.1042/bj1080401 ◽

1968 ◽

Vol 108 (3) ◽

pp. 401-412 ◽

Cited By ~ 101

Author(s):

R. P. Longley ◽

A. H. Rose ◽

B. A. Knights

Keyword(s):

Saccharomyces Cerevisiae ◽

Magnesium Chloride ◽

Membrane Lipids ◽

Dry Weight ◽

Exponential Phase ◽

Gas Liquid Chromatography ◽

Liquid Chromatography Mass Spectrometry ◽

Sterol Esters ◽

Whole Cells ◽

Fatty Acid Compositions

1. Protoplasts of Saccharomyces cerevisiae N.C.Y.C. 366 were prepared by incubating washed exponential-phase cells in buffered mannitol (0·8m) containing 10mm-magnesium chloride and snail gut juice (about 8mg. of protein/ml. of reaction mixture). Protoplast membranes were obtained by bursting protoplasts in ice-cold phosphate buffer (pH7·0) containing 10mm-magnesium chloride. 2. Protoplast membranes accounted for 13–20% of the dry weight of the yeast cell. They contained on a weight basis about 39% of lipid, 49% of protein, 6% of sterol (assayed spectrophotometrically) and traces of RNA and carbohydrate (glucan+mannan). 3. The principal fatty acids in membrane lipids were C16:0, C16:1 and C18:1 acids. Whole cells contained a slightly greater proportion of C16:0 and a somewhat smaller proportion of C18:1 acids. Membrane and whole-cell lipids included monoglycerides, diglycerides, triglycerides, sterols, sterol esters, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol+phosphatidylserine. Phosphorus analyses on phospholipid fractions from membranes and whole cells showed that membranes contained proportionately more phosphatidylethanolamine and phosphatidylinositol+phosphatidylserine than whole cells, which in turn were richer in phosphatidylcholine. Phospholipid fractions from membranes and whole cells had similar fatty acid compositions. 4. Membranes and whole cells contained two major and three minor sterol components. Gas–liquid chromatography, mass spectrometry and u.v. and i.r. spectra indicated that the major components were probably Δ5,7,22,24(28)-ergostatetraen-3β-ol and zymosterol. The minor sterol components in whole cells were probably episterol (or fecosterol), ergosterol and a C29 di-unsaturated sterol. 5. Defatted whole cells contained slightly more glutamate and ornithine and slightly less leucine and isoleucine than membranes. Otherwise, no major differences were detected in the amino acid compositions of defatted whole cells and membranes.

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The compositional analysis of bacterial extracellular polysaccharides by high-performance anion-exchange chromatography

Analytical Biochemistry ◽

10.1016/0003-2697(91)90270-4 ◽

1991 ◽

Vol 199 (1) ◽

pp. 68-74 ◽

Cited By ~ 51

Author(s):

Anthony J. Clarke ◽

Vivian Sarabia ◽

Wendy Keenleyside ◽

P. Ronald MacLachlan ◽

Chris Whitfield

Keyword(s):

Anion Exchange ◽

High Performance ◽

Compositional Analysis ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Extracellular Polysaccharides

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Yeast Pab1 interacts with Rna15 and participates in the control of the poly(A) tail length in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.17.7.3694 ◽

1997 ◽

Vol 17 (7) ◽

pp. 3694-3701 ◽

Cited By ~ 78

Author(s):

N Amrani ◽

M Minet ◽

M Le Gouar ◽

F Lacroute ◽

F Wyers

Keyword(s):

Wild Type Strain ◽

Tail Length ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Wild Type ◽

Cleavage And Polyadenylation ◽

Two Hybrid ◽

Polyadenylation Factor

In Saccharomyces cerevisiae, the single poly(A) binding protein, Pab1, is the major ribonucleoprotein associated with the poly(A) tails of mRNAs in both the nucleus and the cytoplasm. We found that Pab1 interacts with Rna15 in two-hybrid assays and in coimmunoprecipitation experiments. Overexpression of PAB1 partially but specifically suppressed the rna15-2 mutation in vivo. RNA15 codes for a component of the cleavage and polyadenylation factor CF I, one of the four factors needed for pre-mRNA 3'-end processing. We show that Pab1 and CF I copurify in anion-exchange chromatography. These data suggest that Pab1 is physically associated with CF I. Extracts from a thermosensitive pab1 mutant and from a wild-type strain immunoneutralized for Pab1 showed normal cleavage activity but a large increase in poly(A) tail length. A normal tail length was restored by adding recombinant Pab1 to the mutant extract. The longer poly(A) tails were not due to an inhibition of exonuclease activities. Pab1 has previously been implicated in the regulation of translation initiation and in cytoplasmic mRNA stability. Our data indicate that Pab1 is also a part of the 3'-end RNA-processing complex and thus participates in the control of the poly(A) tail lengths during the polyadenylation reaction.

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Identification of Lithocholic Acid and Measurement of Other Bile Acids in Serum of Healthy Humans

Clinical Chemistry ◽

10.1093/clinchem/19.2.248 ◽

1973 ◽

Vol 19 (2) ◽

pp. 248-252 ◽

Cited By ~ 13

Author(s):

E D Pellizzari ◽

F S O’Neil ◽

R W Farmer ◽

L F Fabre

Keyword(s):

Bile Acids ◽

Healthy Subjects ◽

Lithocholic Acid ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Gas Liquid Chromatography ◽

Serum Bile Acid ◽

Healthy Humans ◽

Hydrolysis Of ◽

Fasting Subject

Abstract Combined gas—liquid chromatography mass-spectroscopy was used to identify lithocholic acid and confirm the presence of other bile acids in serum of a healthy fasting subject. GLC was used to measure deoxycholic (DCA), chenodeoxycholic (CDCA), and cholic (CA) acids in sera. Before analysis, serum bile acids were purified by (a) enzymatic hydrolysis of conjugates, (b) anion-exchange chromatography, (c) alumina adsorption chromatography, and (d) GLC of methyl trifluoroacetate derivatives on QF-1. Recovery of bile acids (determined by adding [14C]cholic acid to each sample), after correction for loss during purification, was 63-83%. Fasting values for 28 healthy subjects were: 1.4-46.5 (av 7.1), 1.4-49.6 (12.8), and 1.4-46.0 (16.0) µg/100 ml for DCA, CDCA, and CA, respectively. Traces of lithocholic acid were found in 20% of the cases studied. The smaller ranges we found for serum bile acid concentrations in a healthy fasting population are attributed to the careful health-screening of subjects and improved techniques.

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Streptococcus iniae Capsule Impairs Phagocytic Clearance and Contributes to Virulence in Fish

Journal of Bacteriology ◽

10.1128/jb.01175-06 ◽

2006 ◽

Vol 189 (4) ◽

pp. 1279-1287 ◽

Cited By ~ 54

Author(s):

Jeffrey B. Locke ◽

Kelly M. Colvin ◽

Anup K. Datta ◽

Silpa K. Patel ◽

Nandita N. Naidu ◽

...

Keyword(s):

Capsular Polysaccharide ◽

Critical Role ◽

Indirect Evidence ◽

Mass Spectrometry Analysis ◽

Extracellular Polysaccharides ◽

Streptococcus Iniae ◽

Gas Liquid Chromatography ◽

Wild Type

ABSTRACT Surface capsular polysaccharides play a critical role in protecting several pathogenic microbes against innate host defenses during infection. Little is known about virulence mechanisms of the fish pathogen Streptococcus iniae, though indirect evidence suggests that capsule could represent an important factor. The putative S. iniae capsule operon contains a homologue of the cpsD gene, which is required for capsule polymerization and export in group B Streptococcus and Streptococcus pneumoniae. To elucidate the role of capsule in the S. iniae infectious process, we deleted cpsD from the genomes of two virulent S. iniae strains by allelic exchange mutagenesis to generate the isogenic capsule-deficient ΔcpsD strains. Compared to wild-type S. iniae, the ΔcpsD mutants had a predicted reduction in buoyancy and cell surface negative charge. Transmission electron microscopy confirmed a decrease in the abundance of extracellular capsular polysaccharide. Gas-liquid chromatography-mass spectrometry analysis of the S. iniae extracellular polysaccharides showed the presence of l-fucose, d-mannose, d-galactose, d-glucose, d-glucuronic acid, N-acetyl-d-galactosamine, and N-acetyl-d-glucosamine, and all except mannose were reduced in concentration in the isogenic mutant. The ΔcpsD mutants were highly attenuated in vivo in a hybrid striped bass infection challenge despite being more adherent and invasive to fish epithelial cells and more resistant to cationic antimicrobial peptides than wild-type S. iniae. Increased susceptibility of the S. iniae ΔcpsD mutants to phagocytic killing in whole fish blood and by a fish macrophage cell line confirmed the role of capsule in virulence and highlighted its antiphagocytic function. In summary, we report a genetically defined study on the role of capsule in S. iniae virulence and provide preliminary analysis of S. iniae capsular polysaccharide sugar components.

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Gas-Liquid Chromatography-Mass Spectrometry Investigation of Tropane Alkaloids in Hyoscyamus albus L. from Morocco

Zeitschrift für Naturforschung C ◽

10.1515/znc-2012-9-1003 ◽

2012 ◽

Vol 67 (9-10) ◽

pp. 461-465 ◽

Cited By ~ 3

Author(s):

Ahmed El Bazaoui ◽

My Ahmed Bellimam ◽

Ibn Toumert Lançar ◽

Abdelmajid Soulaymani

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Tropane Alkaloids ◽

Gas Liquid Chromatography ◽

Liquid Chromatography Mass Spectrometry ◽

Plant Organs ◽

Chromatography Mass Spectrometry ◽

New Compounds ◽

First Time ◽

Hyoscyamus Albus

Thirty-four alkaloids were identified in the organs of Hyoscyamus albus L. by gas-liquid chromatography-mass spectrometry (GLC-MS). Eight new compounds for the roots, eleven for the stems, twelve for the leaves, nineteen for the flowers, and seven for the seeds were detected. The alkaloids 5-(2-oxopropyl)-hygrine (8) and phygrine (20) are new for this species and 3-(hydroxyacetoxy)tropane (9), 6,7-dehydro-3-phenylacetoxytropane (15), 3-(2’-phenylpropionyloxy) tropane (17), 6,7-dehydro-3-apotropoyloxytropane (18), 3-(3’-methoxytropoyloxy)tropane (23), and aponorscopolamine (25) are described for the first time for the genus Hyoscyamus. Hyoscyamine was the main alkaloid in the plant organs

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Characterization of extracellular polysaccharides from suspension cultures of apple (Malus domestica)

10.26686/wgtn.12597845.v1 ◽

2020 ◽

Author(s):

S Reid ◽

Ian Sims ◽

LD Melton ◽

AM Gane

Keyword(s):

Monosaccharide Composition ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Neutral Fraction ◽

Extracellular Polysaccharides ◽

Neutral Sugar ◽

Diffusion Test ◽

Selective Precipitation ◽

Acidic Fraction

The polymers secreted by suspension-cultured apple cells were composed of 85% carbohydrate (76% neutral sugar and 9% uronic acid) and 15% w/w protein. The extracellular polysaccharides (ECPs) contain 23% XG and 59% AGPs. The monosaccharide composition of the ECPs consisted of Gal, Ara, Glc and Xyl, with smaller amounts of Rha, Fuc and Man. Fractionation of the ECPs by anion-exchange chromatography yielded an unbound neutral fraction and a bound acidic fraction. Monosaccharide and linkage compositions of each fraction were determined. The neutral fraction (48% recovered carbohydrate) was composed of xyloglucan (XG;>90 mol%) which was purified by selective precipitation with Fehling's solution to yield pure XG. The purified XG had a Glc:Xyl:Gal:Fuc ratio of 4.0:2.5:0.8:0.5; the XG was not O-acetylated. The structure of the secreted XG was similar to that extracted from apple-pomace. The acidic fraction (52% recovered carbohydrate) was composed primarily of arabinogalactan-proteins (AGPs) as detected by the β-glucosyl Yariv diffusion test. The AGP had a Gal:Ara ratio of 1.3: 1.0. Minor amounts of arabinan, xylan and mannan were also detected in the ECPs. This study is the first examination of the polysaccharides secreted by apple cells grown in suspension culture.

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