Zymolyase Treatment Research Articles

Zymolyase Treatment of Saccharomyces cerevisiae Affects Cellular Proteins and Degrades Tyrosyl-DNA Phosphodiesterase I.

Saccharomyces cerevisiae is a genetically tractable, affordable, and extensively documented eukaryotic single-cell model organism. This budding yeast is amenable for the development of genetic and biochemical experiments and is frequently used to investigate the function, activity, and mechanism of mammalian proteins. However, yeast contains a cell wall that hinders select assays including organelle isolation. Lytic enzymes, with Zymolyase as the most effective and frequently used tool, are utilized to weaken the yeast cell wall resulting in yeast spheroplasts. Spheroplasts are easily lysed by, for example, osmotic-shock conditions to isolate yeast nuclei or mitochondria. However, during our studies of the DNA repair enzyme tyrosyl-DNA phosphodiesterase I (Tdp1), we encountered a negative effect of Zymolyase. We observed that Zymolyase treatment affected the steady-state protein levels of Tdp1. This was revealed by inconsistencies in technical and biological replicate lysates of plasmid-born galactose-induced expression of Tdp1. This off-target effect of Zymolyase is rarely discussed in articles and affects a select number of intracellular proteins, including transcription factors and assays such as chromatin immunoprecipitations. Following extensive troubleshooting, we concluded that the culprit is the Ser-protease, Zymolyase B, component of the Zymolyase enzyme mixture that causes the degradation of Tdp1. In this study, we report the protocols we have used, and our final protocol with an easy, affordable adaptation to any assay/protocol involving Zymolyase.

Crosstalk between Saccharomycescerevisiae SAPKs Hog1 and Mpk1 is mediated by glycerol accumulation

Two stress-activated MAP kinase (SAPK) pathways in Saccharomyces cerevisiae respond to osmotic imbalances. The High Osmolarity Glycerol (HOG) pathway is activated in response to hyper-osmotic stress, whereas the Cell Wall Integrity (CWI) pathway is activated in response to hypo-osmotic stress. However, there is also evidence of complex interplay and crosstalk between the two pathways. For example, treatment with zymolyase, a mixture of cell wall degrading enzymes, is known to activate the SAPK Hog1 of the HOG pathway and the SAPK Mpk1 of the CWI pathway sequentially, with Mpk1 activation dependent upon Hog1. Additionally, the PTP2- and PTP3-encoded tyrosine-specific protein phosphatases play a key role in down-regulation of Hog1, but may also down-regulate Mpk1. In this study, we show that hyperactivation of Mpk1 in a ptp2 ptp3 null mutant is an indirect consequence of Hog1 hyperactivation, which induces accumulation of intracellular glycerol and an attendant hypo-osmotic stress. Mpk1 hyperactivity in the absence of PTP2 and PTP3 was suppressed by a hog1 null mutation, or by restoration of osmotic balance with a constitutive form of the glycerol channel Fps1. We found similarly that activation of Mpk1 in response to zymolyase treatment is partly a consequence of Hog1-driven glycerol accumulation. Thus, we have identified two conditions in which glycerol serves as a mediator of crosstalk between the HOG and CWI pathways.

Changes in nucleosome formation at gene promoters in the archiascomycetous yeast Saitoella complicata.

We measured the degree of nucleosome formation at the gene promoters in trichostatin A-treated (1, 2, and 3 µg/mL) cells of the archiascomycete Saitoella complicata and those in enlarged S. complicata cells after zymolyase treatment. TSA-treated and enlarged cells showed similar changes in nucleosome occupancy in five out of six positions in the gene promoters. These results suggest that changes in nucleosome formation at the gene promoters could serve as stress response mechanisms elicited in response to spheroplast (zymolyase treatment) and TSA treatment. In addition, we demonstrated that changes in nucleosome position occurred mainly in cells treated with 1 µg/mL TSA, whereas cells treated with 2 and 3 µg/mL TSA did not exhibit significant changes in the degree of nucleosome formation.

Evaluation of a novel method for measurement of intracellular calcium ion concentration in fission yeast.

The distribution of metal and metalloid species in each of the cell compartments is termed as "metallome". It is important to elucidate the molecular mechanism underlying the beneficial or toxic effects exerted by a given metal or metalloid on human health. Therefore, we developed a method to measure intracellular metal ion concentration (particularly, intracellular calcium ion) in fission yeast. We evaluated the effects of nitric acid (HNO3), zymolyase, and westase treatment on cytolysis in fission yeast. Moreover, we evaluated the changes in the intracellular calcium ion concentration in fission yeast in response to treatment with/without micafungin. The fission yeast undergoes lysis when treated with 60% HNO3, which is simpler and cheaper compared to the other treatments. Additionally, the intracellular calcium ion concentration in 60% HNO3-treated fission yeast was determined by inductively coupled plasma atomic emission spectrometry. This study yields significant information pertaining to measurement of the intracellular calcium ion concentration in fission yeast, which is useful for elucidating the physiological or pathological functions of calcium ion in the biological systems. This study is the first step to obtain perspective view on the effect of the metallome in biological systems.

NCW2, a Gene Involved in the Tolerance to Polyhexamethylene Biguanide (PHMB), May Help in the Organisation of β-1,3-Glucan Structure of Saccharomyces cerevisiae Cell Wall.

In the present work, we provide biological evidences supporting the participation of NCW2 gene in the mechanism responsible for cell tolerance to polyhexamethylene biguanide (PHMB), an antifungal agent. The growth rate of yeast cells exposed to this agent was significantly reduced in ∆ncw2 strain and the mRNA levels of NCW2 gene in the presence of PHMB showed a 7-fold up-regulation. Moreover, lack of NCW2 gene turns yeast cell more resistant to zymolyase treatment, indicating that alterations in the β-glucan network do occur when Ncw2p is absent. Computational analysis of the translated protein indicated neither catalytic nor transmembrane sites and reinforced the hypothesis of secretion and anchoring to cell surface. Altogether, these results indicated that NCW2 gene codes for a protein which participates in the cell wall biogenesis in yeasts and that Ncw2p might play a role in the organisation of the β-glucan assembly.

A simple and rapid method for separation and isolation of marine algal species from naturally evolved populations

ABSTRACTTo improve the study of mixed microalgal populations, three naturally evolved marine microalgal cultures were subjected to a light crushing mechanical treatment using a silicon spatula coupled with zymolyase treatment at four concentrations: 5, 10, 20 and 25 U/ml, for 15, 30, 45 and 60 min before being observed under a microscope. The enzyme concentration of 20 U/ml after 45 min reduces the size of macroscopic microalgal aggregates and improves the microscopic observation of the different microalgal species comprising the population. There was no improvement using the higher enzyme concentration. This paper proposes a new approach to the study of naturally evolved microalgal populations which is useful for distinguishing the morphology of the different species present in the population and allowing for the identification by classical keys, and also to obtain a pure culture from an inoculum of mixed species by using a micromanipulator for cell counting.

Synthesis of mannosylinositol phosphorylceramides is involved in maintenance of cell integrity of yeast Saccharomyces cerevisiae.

Complex sphingolipids play important roles in many physiologically important events in yeast Saccharomyces cerevisiae. In this study, we screened yeast mutant strains showing a synthetic lethal interaction with loss of mannosylinositol phosphorylceramide (MIPC) synthesis and found that a specific group of glycosyltransferases involved in the synthesis of mannan-type N-glycans is essential for the growth of cells lacking MIPC synthases (Sur1 and Csh1). The genetic interaction was also confirmed by repression of MNN2, which encodes alpha-1,2-mannosyltransferase that synthesizes mannan-type N-glycans, by a tetracycline-regulatable system. MNN2-repressed sur1Δ csh1Δ cells exhibited high sensitivity to zymolyase treatment, and caffeine and sodium dodecyl sulfate (SDS) strongly inhibited the growth of sur1Δ csh1Δ cells, suggesting impairment of cell integrity due to the loss of MIPC synthesis. The phosphorylated form of Slt2, a mitogen-activated protein (MAP) kinase activated by impaired cell integrity, increased in sur1Δ csh1Δ cells, and this increase was dramatically enhanced by the repression of Mnn2. Moreover, the growth defect of MNN2-repressed sur1Δ csh1Δ cells was enhanced by the deletion of SLT2 or RLM1 encoding a downstream target of Slt2. These results indicated that loss of MIPC synthesis causes impairment of cell integrity, and this effect is enhanced by impaired synthesis of mannan-type N-glycans.

EFFECT OF XANTHOHUMOL ON BREWING YEAST CELLS

Xanthohumol, a prenylated chalcone from hops and beer, is a phenolic compound that has received considerable attention in recent years. This compound has a range of interesting biological properties that may have therapeutic utility: anti-inflammatory, antioxidant, antilipoperoxidative activities as well as antiangiogenic, antiproliferative and apoptotic effects, mainly assessed in vitro studies that reasonably suggest a potential chemopreventive activity. Phenomenon how xanthohumol affects brewing yeast’s metabolism, yeast viability and vitality were studied during the production of a xanthohumol enriched beer (10 mg/L xanthohumol). The results showed that yeast viability was slightly decreased by xanthohumol, but on the other hand yeast vitality in the xanthohumol enriched brewing trials was slightly better. The content of higher alcohols and esters was similar to the control in all the xanthohumol enriched brewing trials. In laboratory scale experiment about effect of xanthohumol on brewing yeast growing was done. No effect of xanthohumol on yeast growth was detected. Due to the fact that yeast viability was slightly decreased by xanthohumol, experiment with zymolyase was done. Yeast cells cultivated with xanthohumol are significantly more sensitive to zymolyase treatment.

RNA Preparation of Saccharomyces cerevisiae Using the Digestion Method may Give Misleading Results

Zymolyase (lyticase) is used for cell wall digestion in yeast experiments and is needed for incubation processes under moderate experimental conditions. This has been thought to cause unfavorable effects, and many researchers are aware that the enzyme method is unsuitable for RNA preparation following several reports of stress responses to the enzyme process. However, RNA preparation with enzyme digestion continues to be used. This may be because there have been insufficient data directly comparing RNA preparation conditions with previous studies. We investigated the influence of enzyme processes in RNA preparation using a DNA microarray, and compared superoxide dismutase (SOD) activities with a non-treated control and the results of previous research. Gene expressions were commonly changed by enzyme processes, and SOD activities increased only during short-term incubation. Meanwhile, both SOD gene expressions and SOD activity during RNA preparation indicated different results than gained under conditions of long-term incubation. These results suggest that zymolyase treatment surely influences gene expressions and enzyme activity, although the effect assumed by previous studies is not necessarily in agreement with that of RNA preparation.

Ethanol induces calcium influx via the Cch1-Mid1 transporter in Saccharomyces cerevisiae

Yeast suffers from a variety of environmental stresses, such as osmotic pressure and ethanol produced during fermentation. Since calcium ions are protective for high concentrations of ethanol, we investigated whether Ca(2+) flux occurs in response to ethanol stress. We find that exposure of yeast to ethanol induces a rise in the cytoplasmic concentration of Ca(2+). The response is enhanced in cells shifted to high-osmotic media containing proline, galactose, sorbitol, or mannitol. Suspension of cells in proline and galactose-containing media increases the Ca(2+) levels in the cytoplasm independent of ethanol exposure. The enhanced ability for ethanol to induce Ca(2+) flux after the hypertonic shift is transient, decreasing rapidly over a period of seconds to minutes. There is partial recovery of the response after zymolyase treatment, suggesting that cell wall integrity affects the ethanol-induced Ca(2+) flux. Acetate inhibits the Ca(2+) accumulation elicited by the ethanol/osmotic stress. The Ca(2+) flux is primarily via the Cch1 Ca(2+) influx channel because strains carrying deletions of the cch1 and mid1 genes show greater than 90% reduction in Ca(2+) flux. Furthermore, a functional Cch1 channel reduced growth inhibition by ethanol.

Ether–zymolyase ascospore isolation procedure: an efficient protocol for ascospores isolation in Saccharomyces cerevisiae yeast

Here we describe a new procedure for ascospore isolation from cultures containing a majority of unsporulated vegetative cells of Saccharomyces cerevisiae. The EZ ascospore isolation procedure relies on the combination of two conventional protocols, diethyl ether treatment and modified zymolyase treatment, allowing a significant increase in the efficiency of ascospore isolation and consequently enabling a large number of meiotic offspring to be efficiently obtained and screened, thus improving the efficacy of genetic research and the genetic selection of S. cerevisiae strains.

TheSaccharomycesSUN gene,UTH1, is involved in cell wall biogenesis

Deletion of the Saccharomyces gene, UTH1, a founding member of the SUN family of fungal genes, has pleiotropic effects. Several phenotypes of Deltauth1 cells including their decreased levels of mitochondrial proteins, their impaired autophagic degradation of mitochondria, and their increased viability in the presence of mammalian BAX, a proapoptotic regulator localized to the mitochondria, have prompted others to propose that the Uth1p functions primarily at the mitochondria. In this report, we show that cells lacking UTH1 have more robust cell walls with higher levels of beta-d-glucan that allows them to grow in the presence of calcofluor white or sodium dodecyl sulfate, two reagents known to perturb the yeast cell wall. Moreover, these Deltauth1 cells are also significantly more resistant to spheroplast formation induced by zymolyase treatment than their wild-type counterparts. Surprisingly, our data suggest that several of the enhanced growth phenotypes of Deltauth1 cells, including their resistance to BAX-mediated toxicity, arise from a strengthened cell wall. Therefore, we propose that Uth1p's role at the cell wall and not at the mitochondria may better explain many of its effects on yeast physiology and programmed cell death.

The High Osmotic Response and Cell Wall Integrity Pathways Cooperate to Regulate Transcriptional Responses to Zymolyase-induced Cell Wall Stress in Saccharomyces cerevisiae

The adaptation of Saccharomyces cerevisiae to situations in which cell wall integrity is seriously compromised mainly involves the cell wall integrity (CWI) pathway. However, in a recent work ( Bermejo, C., Rodriguez, E., García, R., Rodríguez-Peña, J. M., Rodríguez de la Concepción, M. L., Rivas, C., Arias, P., Nombela, C., Posas, F., and Arroyo, J. (2008) Mol. Biol. Cell 19, 1113-1124 ) we have demonstrated the co-participation of the high osmotic response (HOG) pathway to ensure yeast survival to cell wall stress mediated by zymolyase, which hydrolyzes the beta-1,3 glucan network. Here we have characterized the role of both pathways in the regulation of the overall yeast transcriptional responses to zymolyase treatment using whole genome expression profiling. A main group of yeast genes is dependent on both MAPKs, Slt2 and Hog1, for their induction. The transcriptional activation of these genes depends on the MAPKKK Bck1, the transcription factor Rlm1, and elements of the sho1 branch of the HOG pathway, but not on the sensors of the CWI pathway. A second group of genes is dependent on Slt2 but not Hog1 or Pbs2. However, the induction of these genes is dependent on upstream elements of the HOG pathway such as Sho1, Ste50, and Ste11, in accordance with a sequential activation of the HOG and CWI pathways. Zymolyase also promotes an osmotic-like transcriptional response with the activation of a group of genes dependent on elements of the Sho1 branch of HOG pathway but not on Slt2, with the induction of many of them dependent on Msn2/4. Additionally, in the absence of Hog1, zymolyase induces an alternative response related to mating and filamentation as a consequence of the cross-talk between these pathways and the HOG pathway. Finally, in the absence of Slt2, zymolyase increases the induction of genes associated with osmotic adaptation with respect to the wild type, suggesting an inhibitory effect of the CWI pathway over the HOG pathway. These studies clearly reveal the complexity of the signal transduction machinery responsible for regulating yeast adaptation responses to cell wall stress.

Amiodarone induces stress responses and calcium flux mediated by the cell wall inSaccharomyces cerevisiae

We used a proteomic approach to study effects of amiodarone on cells of the yeast Saccharomyces cerevisiae. Amiodarone has been shown to have antifungal activity in vitro and causes a massive increase in cytoplasmic calcium levels ([Ca2+]cyt). Proteomic analysis of cells exposed to amiodarone show that this drug elicits stress responses and points to involvement of proteins associated with the cell wall. We tested several of those proteins for involvement in the Ca2+ flux. In particular, the amiodarone-induced Ca2+ flux was decreased in bgl2Delta cells, which have altered levels of beta-glucan and chitin. The involvement of the cell wall in the Ca2+ flux induced by amiodarone treatment was tested by addition of yeast cell-wall components. While mannan inhibited the rise in [Ca2+]cyt, beta-glucan potentiated the Ca2+ flux by 4.5-fold, providing evidence that the cell wall is directly involved in controlling this Ca2+ flux. This conclusion is corroborated by the inhibition of the Ca2+ flux by calcofluor, which is known to bind to cell-wall chitin and inhibit cell growth. Zymolyase treatment altered the kinetics of amiodarone-induced calcium flux and uncoupled the inhibitory effect of calcofluor. These effects demonstrate that the cell-wall beta-glucan regulates calcium flux elicited by amiodarone.

Modulation of Yeast Sln1 Kinase Activity by the Ccw12 Cell Wall Protein

The yeast Sln1p sensor kinase is best known as an osmosensor involved in the regulation of the hyperosmolarity glycerol mitogen-activated protein kinase cascade. Down-regulation of Sln1 kinase activity occurs under hypertonic conditions and leads to phosphorylation of the Hog1p mitogen-activated protein kinase and increased osmotic stress-response gene expression. Conditions leading to kinase up-regulation include osmotic imbalance caused by glycerol retention in the glycerol channel mutant, fps1 (Tao, W., Deschenes, R. J., and Fassler, J. S. (1999) J. Biol. Chem. 274, 360-367). The hypothesis that Sln1p kinase activity is responsive to turgor was first suggested by the increased Sln1p kinase activity in mutants lacking Fps1p in which glycerol accumulation leads to water uptake. Also consistent with the turgor hypothesis is the observation that reduced turgor caused by treatment of cells with nystatin, a drug that increases membrane permeability and causes cell shrinkage, reduced Sln1p kinase activity (Tao, W., Deschenes, R. J., and Fassler, J. S. (1999) J. Biol. Chem. 274, 360-367; Reiser, V., Raitt, D. C., and Saito, H. (2003) J. Cell Biol. 161, 1035-1040). The turgor hypothesis is revisited here in the context of the identification and characterization of the cell wall gene, CCW12, as a determinant of Sln1p activity. Results of this analysis suggest that the activity of the plasma membrane localized Sln1p is affected by the presence or absence of specific outer cell wall proteins and that this effect is independent of turgor.

Characterization of the transcriptional response to cell wall stress in Saccharomyces cerevisiae.

The cell wall perturbants Calcofluor white and Zymolyase activate the Pkc1-Rho1-controlled Slt2p MAP kinase pathway in Saccharomyces cerevisiae. A downstream transcription factor of this pathway, Rlm1p, is known to control expression of about 20 cell wall-related genes. Global transcript analysis of Calcofluor white and Zymolyase treatment was performed to determine whether cell wall stress affects transcription of these and other genes. Transcript profiles were analysed using two recently developed algorithms, viz. REDUCE, which correlates upstream regulatory motifs with expression, and Quontology, which compares expression of genes from functional groups with overall gene expression. Both methods indicated upregulation of Rlm1p-controlled cell wall genes and STRE-controlled genes, and downregulation of ribosomal genes and rRNA genes. Comparison of these expression profiles with the published profiles of two constitutively active upstream activators of the Slt2p-MAP kinase pathway, viz. Pkc1-R398A and Rho1-Q68A, revealed significant similarity. In addition, a new putative regulatory motif, CCC(N)(10)GGC, was found. In Zymolyase-treated cells a regulatory site was identified, ATGACGT, which resembles the AFT/CRE binding site. Interestingly, Sko1p, a downstream regulator of the high osmolarity pathway is known to bind to the AFT/CRE binding site, suggesting a possible role for the Hog1 pathway in the response to cell wall stress. Finally, using REDUCE, an improved version of the Rlm1 binding motif, viz. TA(W)(4)TAGM, was discovered. We propose that this version can be used in combination with REDUCE as a sensitive indicator of cell wall stress. Taken together, our data indicate that cell wall stress results in activation of various signalling pathways including the cell wall integrity pathway.

Shochu brewing characteristics and properties of a trichothecin-resistant shochu yeast mutant

An antibiotic-resistant strain of Saccharomyces cerevisiae was isolated from shochu yeast. Three mutants were used for shochu brewing and gave higher ethanol productivities than the parent. The mutants were resistant to cycloheximide, cerulenin, trichothecin and other organic compounds such as lauric acid. In the presence of 20% (v/v) ethanol, the viability of the mutants was 87–96%, but that of the parent was 77%. Zymolyase treatment for 3 h, decreased the viability of the parent by 44% but that of the mutants only by 11–32%. Thus the higher ethanol productivity of these mutants is related to their high ethanol tolerance and resistance to various organic compounds.

The Pneumocystis carinii major surface glycoprotein (MSG): its potential involvement in the pathophysiology of pneumocystosis.

The major antigens of rat Pneumocystis carinii are 45, 60 and 116 kDa, and those of human P. carinii 20, 40, 66, and 95 kDa [1]. The predominant band on acrylamide gels is a surface antigen, which varies in size (95–140 kDa) depending on the host and method of preparation. This antigen is referred to by a variety of names, including gpA, gp95, gp116, gp120 and MSG. In the following the latter term will be used for this antigen. MSG is encoded by a family of approximately 100 genes, each of which is responsible for the expression of a different isoform of MSG [2, 3]. Human MSG is 95 kDa, slightly smaller than the corresponding rat antigen [4,5]. Polyclonal and monoclonal antibodies directed against rat and human P. carinii MSG show that species-specific as well as antigenic determinants are shared and that individual isolates of human and rat P. carinii are antigenically different [4–8]. Biochemical studies indicate that MSG is an acidic glycoprotein composed of a protein core and a carbohydrate portion. N -Linked carbohydrates rich in mannose, glucose, and N -acetylglucosamine residues represent one tenth of the MSG mass [4,9]. Expression of recombinant MSG has largely been unsuccessful. Rat P. carinii MSG has been recombinantly expressed using the Escherichia coli or baculovirus expression system, but differs in glycosylation from native MSG [10]. The difficulty of expressing human recombinant MSG is believed to rely on critical upstream conserved sequences not yet understood. Native MSG preparations from all species are obtained by solubilization of P. carinii organisms with zymolyase and purification utilizing high performance liquid chromatography [11]. Zymolyase hydrolyzes linear glucose polymers with β-1,3-linkages [12]. Studies of the cyst wall have demonstrated that zymolyase treatment removes …

Zymolyase treatment exposes p55 antigen of Pneumocystis carinii.

Rats exposed to Pneumocystis carinii mount antibody responses to a broad band migrating on western blot with an apparent molecular weight of 45-55 kDa. One antigen within this band, designated p55, is uniformly recognized by P. carinii exposed rats. Although the gene encoding the p55 antigen had been previously cloned, the location of this antigen within the organism was unknown. Prior attempts to localize the protein were unsuccessful. A monospecific polyclonal antiserum raised against a carboxyl-terminal 15-oligomer peptide yielded specific reactivity with a single 55 kDa band on a western blot of P. carinii. Using this antiserum, little to no reactivity could be detected with P. carinii organisms by immunofluorescence assay (IFA). However, zymolyase treatment of P. carinii dramatically increased the intensity and proportion of organisms reactive by IFA. Zymolyase, an enzyme with beta-1,3 glucanase activity, has previously been shown to remove the electron dense outer layer of the P. carinii cell wall, exposing an electron lucent layer. Immunoelectron microscopy performed on zymolyase treated organisms showed the majority of labeling occurs within the cell wall.

Study of supramolecular structures released from the cell wall of Candida albicans by ethylenediamine treatment.

Candida albicans cell wall components were analyzed by ethylenediamine (EDA) treatment. Based on their different solubility properties, the cell wall components produced three fractions (A, B, and C). Fractions B (EDA-soluble, water-insoluble) and C (EDA-insoluble) contained glucan, chitin, and protein in different proportions. After zymolyase (mainly a beta-glucanase complex) or chitinase treatment of fractions B and C, more polysaccharides and proteins were solubilized by a second EDA treatment, suggesting that the solubility of the polymers in EDA depends on the degree of polymer interactions. Western blot analysis using two monoclonal antibodies (1B12 and 4C12) revealed electrophoretic patterns that were similar in mycelial and yeast morphologies, except that in material obtained from mycelial walls, an additional band was detected with MAb 1B12. Fluorescence microscopy of cell wall fractions treated with FITC-labeled Con-A, Calcofluor white, and FITC-labeled agglutinin showed that glucan and mannoproteins are uniformly distributed in fractions B and C, while chitin is restricted to distinct patches. Transmission electron microscopy demonstrated that fraction C maintained the original shape of the cells, with an irregular thickness generally wider than the walls. When fraction C was treated with chitinase, the morphology was still present and was maintained by an external glucan layer, with an internal expanded fibrillar material covering the entire cellular lumen. Degradation of the glucan skeleton of fraction C with zymolyase resulted in the loss of the morphology.