scholarly journals The separation of β-glucanases produced by Cytophaga johnsonii and their role in the lysis of yeast cell walls

1970 ◽  
Vol 120 (1) ◽  
pp. 67-78 ◽  
Author(s):  
J. S. D. Bacon ◽  
A. H. Gordon ◽  
D. Jones ◽  
Irene F. Tayor ◽  
D. M. Webley
Keyword(s):  
Yeast Cell ◽  
Cell Walls ◽  
Culture Fluid ◽  
The Other ◽  
Yeast Cells ◽  
Wall Structure ◽  
Enzyme Preparations ◽  
End Products ◽  
Isolated Cell

1. When Cytophaga johnsonii was grown in the presence of suitable inducers the culture fluid was capable of lysing thiol-treated yeast cell walls in vitro. 2. Autoclaved or alkali-extracted cells, isolated cell walls and glucan preparations made from them were effective inducers, but living yeast cells or cells killed by minimal heat treatment were not. 3. Chromatographic fractionation of lytic culture fluids showed the presence of two types of endo-β-(1→3)-glucanase and several β-(1→6)-glucanases; the latter may be induced separately by growing the myxo-bacterium in the presence of lutean. 4. Extensive solubilization of yeast cell walls was obtained only with preparations of one of these glucanases, an endo-β-(1→3)-glucanase producing as end products mainly oligosaccharides having five or more residues. Lysis by the other endo-β-(1→3)-glucanase was incomplete. 5. The β-(1→6)-glucanases produced a uniform thinning of the cell walls, and mannan–peptide was found in the solution. 6. These results, and the actions of the enzyme preparations on a variety of wall-derived preparations made from baker's yeast, are discussed in the light of present conceptions of yeast cell-wall structure.

Effects of an exogenous fibrolytic enzyme preparation on in vitro ruminal fermentation of three forages and their isolated cell walls

2008 ◽  
Vol 145 (1-4) ◽  
pp. 109-121 ◽  
Author(s):  
M.J. Ranilla ◽  
M.L. Tejido ◽  
L.A. Giraldo ◽  
J.M. Tricárico ◽  
M.D. Carro
Keyword(s):  
Cell Walls ◽  
Enzyme Preparation ◽  
Ruminal Fermentation ◽  
Isolated Cell

scholarly journals THE PARTICULATE HYDROLASES OF MACROPHAGES

1963 ◽  
Vol 118 (6) ◽  
pp. 1009-1020 ◽  
Author(s):  
Zanvil A. Cohn ◽  
Edith Wiener
Keyword(s):  
Acid Phosphatase ◽  
Yeast Cell ◽  
Cell Walls ◽  
Hydrolytic Enzymes ◽  
Neutral Red ◽  
Biochemical Properties ◽  
Extracellular Medium ◽  
Cell Content ◽  
Prolonged Incubation

The influence of phagocytosis on the morphological and biochemical properties of macrophage hydrolase-containing granules has been studied in vitro. Following the uptake of large numbers of heat-killed bacteria, an intracellular rearrangement of hydrolytic enzymes occurred. This was associated with the solubilization of 50 to 60 per cent of the total cell content of acid phosphatase, cathepsin, lysozyme, beta glucuronidase, acid ribonuclease, and acid desoxyribonuclease and with a corresponding decrease in granule-bound enzyme. With more prolonged incubation the majority of the soluble intracellular pool of acid ribonuclease and lysozyme was lost to the extracellular medium. No change in the total content of any of the hydrolases was noted during 180 minutes of incubation in vitro. The morphological fate of the granules was studied by a histochemical method for acid phosphatase. After the phagocytosis of yeast cell walls there was a disappearance of acid phosphatase-positive granules and an accumulation of reaction product about the ingested particle. Experiments employing macrophages which were supravitally stained with neutral red also demonstrated the loss of neutral red-positive granules and the accumulation of the dye about the yeast cell walls. These results strongly suggest that lysis of macrophage granules occurs following phagocytosis and that a portion of the granule contents are then resegregated within the newly formed phagocytic vacuole.

scholarly journals Staphylococcus aureus cell wall structure and dynamics during host-pathogen interaction

2021 ◽  
Vol 17 (3) ◽  
pp. e1009468
Author(s):  
Joshua A. F. Sutton ◽  
Oliver T. Carnell ◽  
Lucia Lafage ◽  
Joe Gray ◽  
Jacob Biboy ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Walls ◽  
Ex Vivo ◽  
Cell Wall Structure ◽  
Wall Structure ◽  
Bacterial Cells ◽  
Human Macrophages ◽  
Structure And Dynamics

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which correlated with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease.

scholarly journals Pathway of Glucose Catabolism by Strain VeGlc2, an Anaerobe Belonging to the Verrucomicrobiales Lineage of Bacterial Descent

1998 ◽  
Vol 64 (12) ◽  
pp. 4830-4833 ◽  
Author(s):  
Peter H. Janssen
Keyword(s):  
Electron Transport ◽  
Enzyme Activities ◽  
The Other ◽  
Difference Spectra ◽  
Glucose Catabolism ◽  
Cell Extracts ◽  
In Vitro Measurements ◽  
End Products ◽  
Reductive Pathway

ABSTRACT Strain VeGlc2, an anaerobic ultramicrobacterium belonging to theVerrucomicrobiales lineage of bacterial descent, fermented glucose to acetate, propionate, succinate, and CO2. The distribution of radiolabel in the fermentation end products produced from position-labelled glucose and in vitro measurements of enzyme activities in crude cell extracts prepared from glucose-grown cells showed that glucose was metabolized via the Embden-Meyerhof-Parnas pathway. The 6-phosphofructokinase (EC 2.7.1.90 ) activity required pyrophosphate as the phosphoryl donor, and ATP could not replace pyrophosphate. The other enzyme activities were those of a classical Embden-Meyerhof-Parnas pathway. 14CO2 was incorporated into propionate and succinate, suggesting that a carboxylation reaction rather than a transcarboxylation reaction was involved in the reductive pathway leading to succinate and propionate. Difference spectra showed that a type b cytochrome was present, which could be involved in electron transport in the reductive pathway.

Studies on the Specific Fibrinolytic Effect of Human Extrinsic (Tissue-Type) Plasminogen Activator in Human Blood and in Various Animal Species in Vitro

1981 ◽  
Vol 46 (02) ◽  
pp. 561-565 ◽  
Author(s):  
C Korninger ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Culture Fluid ◽  
The Other ◽  
Tissue Type ◽  
Chain Molecule ◽  
Autologous Plasma ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryHuman extrinsic (tissue-type) plasminogen activator (EPA) was highly purified from the culture fluid of a human melanoma cell line, both as a one-chain or as a two-chain molecule. Its specific fibrinolytic effect on human whole blood clots or plasma clots with different degrees of fibrin crosslinking was evaluated in an in vitro system, composed of a 125I-fibrin labeled clot, hanging in circulating human plasma. After infusion of EPA (30 IU per ml over 3 hrs), non-crosslinked clots lysed more extensively (75-100 percent in 5 hrs) than totally-crosslinked clots (50-65 percent), and no difference was found between one-chain or two-chain EPA. The extent of lysis of totally-crosslinked human or animal plasma clots hanging in autologous plasma induced by EPA varied markedly from one species to the other. When 90 IU of EPA were infused over 3 hrs, crosslinked human plasma clots dissolved for over 95 percent within 5 hrs. Under comparable conditions, the degree of lysis was 80 percent in primate plasma (cynomolgus fascicularis), 60 percent in cat and rabbit plasma, 30 percent in dog plasma and only 10 percent in rat plasma. Systemic activation of the fibrinolytic system in the circulating plasmas was minor and dose-dependent in all species, but complete fibrinogen breakdown was not observed in any species following infusion of up to 90 IU EPA per ml plasma.It is concluded that the human system is more susceptible to EPA induced fibrinolysis than the other animal systems which were investigated, and that even totally-crosslinked clots can be lysed after infusion of EPA.

Killer Toxins of Yeasts: Inhibitors of Fermentation and Their Adsorption

1987 ◽  
Vol 50 (3) ◽  
pp. 234-238 ◽  
Author(s):  
FERDINAND RADLER ◽  
MANFRED SCHMITT
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Yeast Cell ◽  
Cell Walls ◽  
Killer Toxin ◽  
Yeast Cells ◽  
Toxin Concentration ◽  
Commercial Preparation ◽  
Killer Toxins ◽  
Original Amount

The killer toxin (KT 28), a glycoprotein of Saccharomyces cerevisiae strain 28, was almost completely adsorbed by bentonite, when applied at a concentration of 1 g per liter. No significant differences were found between several types of bentonite. Killer toxin KT 28 is similarly adsorbed by intact yeast cells or by a commercial preparation of yeast cell walls that has been recommended to prevent stuck fermentations. An investigation of the cell wall fractions revealed that the toxin KT 28 was mainly adsorbed by mannan, that removed the toxin completely. The alkali-soluble and the alkali-insoluble β-1,3- and β-1,6-D-glucans lowered the toxin concentration to one tenth of the original amount. The killer toxin of the type K1 of S. cerevisiae was adsorbed much better by glucans than by mannan.

scholarly journals Two Novel Techniques for Determination of Polysaccharide Cross-Links Show that Crh1p and Crh2p Attach Chitin to both β(1-6)- and β(1-3)Glucan in the Saccharomyces cerevisiae Cell Wall

2009 ◽  
Vol 8 (11) ◽  
pp. 1626-1636 ◽  
Author(s):  
Enrico Cabib
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Acetic Acid ◽  
Yeast Cell ◽  
Cell Walls ◽  
Double Mutant ◽  
The Other ◽  
Cross Links

ABSTRACT Previous work, using solubilization of yeast cell walls by carboxymethylation, before or after digestion with β(1-3)- or β(1-6)glucanase, followed by size chromatography, showed that the transglycosylases Crh1p and Crh2p/Utr2p were redundantly required for the attachment of chitin to β(1-6)glucan. With this technique, crh1Δ crh2Δ mutants still appeared to contain a substantial percentage of chitin linked to β(1-3)glucan. Two novel procedures have now been developed for the analysis of polysaccharide cross-links in the cell wall. One is based on the affinity of curdlan, a β(1-3)glucan, for β(1-3)glucan chains in carboxymethylated cell walls. The other consists of in situ deacetylation of cell wall chitin, generating chitosan, which can be extracted with acetic acid, either directly (free chitosan) or after digestion with different glucanases (bound chitosan). Both methodologies indicated that all of the chitin in crh1Δ crh2Δ strains is free. Reexamination of the previously used procedure revealed that the β(1-3)glucanase preparation used (zymolyase) is contaminated with a small amount of endochitinase, which caused erroneous results with the double mutant. After removing the chitinase from the zymolyase, all three procedures gave coincident results. Therefore, Crh1p and Crh2p catalyze the transfer of chitin to both β(1-3)- and β(1-6)glucan, and the biosynthetic mechanism for all chitin cross-links in the cell wall has been established.

The structure of the yeast cell wall - II. Degradative studies with enzymes

1958 ◽  
Vol 149 (936) ◽  
pp. 425-440 ◽  
Keyword(s):  
Cell Wall ◽  
Phosphorus Content ◽  
Large Fraction ◽  
Helix Pomatia ◽  
Yeast Cells ◽  
Reaction Products ◽  
Enzyme Preparations ◽  
Phosphate Ions ◽  
Insoluble Glucan ◽  
Isolated Cell

In contrast to the use by earlier investigators of alkali in degrading the walls of yeast cells, various enzymes have now been employed for this purpose, the reaction products being characterized with the aid of chromatography. Isolated cell walls were dissolved completely by enzyme preparations from Helix pomatia and to various extents up to about 50% by enzymes present in malt, by crystalline trypsin or by crystalline papain. In the case of the malt and snail enzymes, the rapidity of the reactions was directly proportional to the phosphorus content of the cell wall. Both glucose and N-acetylglucosamine were formed using either the malt or the snail enzymes, but no dialyzable products were detected following the action of trypsin or papain. In all cases the non-dialyzable products included both mannan and glucan fractions. Using papain, an initial phase of rapid reaction led to almost complete loss of the negative charge associated with phosphate ions. Effectively the whole of the phosphate thereby dissolved was subsequently recovered in the form of a complex with mannan and protein, which therefore probably forms part of the wall surface. On the basis of such observations, the major constituents of the wall are envisaged as consisting of an insoluble glucan matrix ( ca . 50%) attached by means of a protein ‘cement’ ( ca . 7%) to mannan ( ca . 20%) and also to soluble glucan ( ca . 10%). From the magnitude of the charge density at both the inner and outer surfaces of the wall, it is tentatively concluded that a relatively large fraction of each is occupied by mannan and a comparatively small fraction by protein.

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