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PubMedDate not supplied

Quantitative determination of carboxymethyl chitin in polymer-coated liposomes.

A simple turbidity method for the quantitative determination of carboxymethyl chitin (CM-chitin) in coated liposomes has been developed. Isopropanol:buffer (1:1 v/v) was selected as a good solvent system for dimyristoyl-, dipalmitoyl-, or egg phosphatidylcholines but not CM-chitin. The turbidity of CM-chitin in this solvent system was spectrophotometrically determined at 400 nm without significant interference by these phosphatidylcholines. The dependence of turbidity on the CM-chitin concentration, pH, and ionic strength were determined. It was found that analyses of the coating efficiency of dipalmitoylphosphatidylcholine liposomes by CM-chitin were reproducible within the specified ranges of formulation conditions and enabled differentiation of the effect of order of CM-chitin addition on the coating efficiency.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1995

Development of a chitin assay for the quantification of fungus.

Chitin, a unique structural polysaccharide found in fungi and arthropods, is not produced by vertebrates. Thus, the potential applications of a specific and sensitive assay for chitin are numerous, including the evaluation of the extent of fungal keratitis. Chitin is a homopolymer of beta (1, 4) linked D-N-acetylglucosamine. We have developed a simple and reproducible assay for chitin and applied it to Candida albicans cultures. The assay involves homogenization of the culture and treatment with 21.1 M KOH to remove soluble materials, including proteins. This base treatment also deacetylates the chitin to the glucosamine polymer, chitosan. Chitosan is hydrolyzed by 0.5 M H2SO4 to glucosamine monomers which are then deaminated by the addition of NaNO2 to the acid solution. The resulting 2,5-anhydromannose is reduced by NaB[3H]4 to 1-[3H] 2,5-anhydromannitol. This radiolabelled sugar is isolated by paper chromatography and quantified via liquid scintillation. The sensitivity of this assay is assessed by comparison of colony forming units (CFU's) with a glucosamine standard. A typical run of the assay detects 53.1 CFU/c.p.m., and 356,000 c.p.m. per nanomole of N-acetylglucosamine. The specificity of the assay is very high because of the unique nature of chitin. This method of chitin determination may be a useful alternative method for future investigations involving the study of fungal infections in mammalian tissues.

Candida albicans↗
PubMed2000

Oral administration of chitin down-regulates serum IgE levels and lung eosinophilia in the allergic mouse.

Previous studies showed that local macrophages phagocytose nonantigenic chitin particles (1-10 micrometer polymers of N-acetyl- d -glucosamine) through mannose receptors and produce IL-12, IL-18, and TNF-alpha. These cytokines lead to the production of IFN-gamma by NK cells. To determine whether chitin could down-regulate Th2 responses, chitin was given orally (8 mg/day for 3 days before and 13 days during ragweed allergen immunization) in BALB/c and C57BL/6 mice. These ragweed-immunized mice were given ragweed intratracheally on day 11. Three days after the challenge, the immunized mice with saline (controls) showed increases in serum IgE levels and lung eosinophil numbers. The chitin treatment resulted in decreases of these events in both strains. To dissect the inhibitory mechanisms of Th2 responses, spleen cells (4 x 106 cells/ml) isolated from the ragweed-immunized mice (controls) were cultured in the presence of ragweed and/or chitin for 3 days (recall responses). Ragweed alone stimulated the production of IL-4 (0.6 ng/ml), IL-5 (20 U/ml), and IL-10 (3.2 ng/ml), but not IFN-gamma. Ragweed/chitin stimulation resulted in significant decreases of IL-4, IL-5, and IL-10 levels and the production of IFN-gamma (48 U/ml). Moreover, spleen cells isolated from the chitin-treated mice showed ragweed-stimulated IFN-gamma production (15 U/ml) and significantly lower levels of the Th2 cytokines, suggesting that the immune responses were redirected toward a Th1 response. Collectively, these results indicate that chitin-induced innate immune responses down-regulate Th2-facilitated IgE production and lung eosinophilia in the allergic mouse.

Administration, Oral↗
PubMed1992

A hemagglutinating substance in chitin.

Chitin from crustacean shells has often been used to isolate and purify plant lectins that have an affinity for poly-N-acetylglucosamine (poly-GlcNAc). When we used washed chitin from crab shells as an affinity medium to isolate a lectin from Pinus strobus L. (eastern white pine) ovules, we found that a substance having a strong capacity to agglutinate red blood cells was eluted from the chitin during a weak acid desorption step. The chitin agglutinin is a complex structure containing protein and poly-GlcNAc. Chitin samples from four biochemical suppliers were tested; all contained the elutable agglutinin. Acid (0.05 N HCl or 0.1 N acetic acid) appears to hydrolyze the material from the solid chitin. NaOH at 0.5 N does not remove the agglutinin. Since agglutination is the assay used to monitor lectin purification, care must be taken to avoid the native agglutinin if chitin is used as an affinity matrix.

Acetylglucosamine↗
PubMed1990

Visualization of chitin-protein layer formation in Ascaris lumbricoides egg-shells.

The formation of chitin-protein layer in Ascaris lumbricoides egg-shells was studied using the method of chitin structures visualization by means of derivatives of stilbene-disulfonic acid (Blankophor, Bayer, FRG). Intensive formation of chitin structures in A. lumbricoides egg-shell occurred only in fertilized females in a very short portion of uterus from the site of connection of the oviduct with the uterus up to 25-30 mm distally, i.e. in the section forming about one fifth or less than one sixth of length of adult female uterus. Already 3 mm from the beginning of uterus there was a thin layer on the surface of the fertilized eggs which contained chitin detectable by Blankophor visualization, whereas the chitin-protein layer on the shells could not be demonstrated histologically. The thickness of this chitin-protein complex increased distally. At 25 mm from the beginning of uterus the chitin layer was already complete and its thickness did not increase any more.

Animals↗
PubMed1990

Effect of chitin heparinoids on the activation of peritoneal macrophages and on the production of monokines in mice.

Sulfonated derivatives of chitin which showed anticoagulant activity (chitin heparinoids) were studied with regard to the activation of mouse peritoneal macrophages and the production of monokines. In comparison with 70% deacetylated chitin (DAC-70), which was the most adjuvant-active derivative of chitin, all chitin heparinoids were less effective for the augmentation of cytolytic activity of peritoneal macrophages. The number of macrophages was hardly increased or decreased by intraperitoneal injection of chitin heparinoids, and the activity of circulating colony-stimulating factor was not changed by their treatment. Only N-sulfonated DAC-70 stimulated the production of interleukin-1 by thioglycolate-induced peritoneal macrophages in vitro. However, its effect was weaker than that of DAC-70. Chitin heparinoids showed no or weak mitogenic activity on normal mouse spleen cells.

Adjuvants, Immunologic↗
PubMed1986

Chitin synthetase 2, a presumptive participant in septum formation in Saccharomyces cerevisiae.

Strains containing a disrupted structural gene for chitin synthetase (chs1::URA3) are defective in chitin synthetase 1 (Chs1) activity but contain normal amounts of chitin (Bulawa, C.E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, L., and Robbins, P. W. (1986) Cell 46, 213-225). We have now detected in such strains a new chitin synthetase activity (Chs2), at levels about 5% of those of Chs1 in wild-type cells. Thus, Chs2 is presumably the physiological agent for chitin deposition in strains with a disrupted CHS1 gene and probably also in wild-type strains. Chs1 and Chs2 share certain properties, such as stimulation by N-acetylglucosamine and by partial proteolysis. They differ sharply, however, in divalent cation specificity and in pH optimum. Chs2 also shows less sensitivity than Chs1 to inhibition by polyoxin D or sodium chloride, a property that was used to demonstrate the presence of Chs2 in wild-type extracts. As in the case of Chs1, most of the Chs2 activity was found to be associated with the plasma membranes. This finding, together with the apparent zymogenic nature of Chs2, is consistent with the hypothesis, previously put forward for Chs1, that localized deposition of chitin is attained by activation of the zymogen form at a specific time and place. Function and significance of the two chitin synthetases are discussed in connection with fungal morphogenesis and evolution.

Cations, Divalent↗
PubMed1995

Purification and characterization of chitin deacetylase from Absidia coerulea.

Chitin deacetylase, which releases the acetyl groups of glycol chitin was purified from a fungus, Absidia coerulea, and characterized. The enzyme was purified 516-fold to homogeneity by means of 65-80% ammonium sulfate precipitation followed by chromatography on Butyl Toyoperal-650M, Gigapite (hydroxyapatite), and DEAE Toyopearl-650M. It had an apparent molecular weight of 75 kDa both on sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration chromatography, indicating that the enzyme exists as a monomer. The amino-terminal sequence was determined to be Gly-Glu-Tyr-Trp-Gln-Ser-Phe-. The enzyme is active on chitooligosaccharides with more than two N-acetylglucosamine residues (chitobiose) and is able to convert the nascent chitin synthesized by chitin synthase to chitosan in vitro. When O-hydroxyethylated chitin (glycol chitin) was used as a substrate, the optimum pH for enzyme activity was 5.0 and the optimum temperature was 50 degrees C. The enzyme was heat-stable and strongly inhibited by Fe3+. Furthermore, chitin deacetylase was demonstrated to be localized near the inner face of the cell wall (periplasmic space) in the mycelia by using immunoelectron microscopy.

Amidohydrolases↗
PubMed2001

Chitin synthesis and inhibition: a revisit.

Chitin is an abundant biologically important aminopolysaccharide composed of N-acetyl-D-glucosamine units. Individual polymers, which are synthesized intracellularly by chitin synthase (CS), a membrane-bound glycosyl transferase, are translocated across the plasma membrane and coalesce to form rigid crystallites. These crystallites, inter alia, are integral parts of septa and cell walls in yeast and filamentous fungi, respectively, and of cuticles in invertebrates, notably crustaceans and insects. Despite decades of intensive research, many events associated with the complexity of chitin formation and deposition are still obscure, or only partially understood. The list includes the hormonal control of CS at the transcriptional and translational levels as well as the post-translational CS packaging; trafficking and guidance of CS clusters to proper sites in the cells and their intricate insertion into the plasma membranes; activation of the catalytic step and its control or modulation; and translocation of chitin chains across cell membranes, their orientation, fibrillogenesis and association with other extracellular structural components such as polysaccharides (fungi) and cuticular proteins (insects). Also the precise biochemical lesions inflicted by CS inhibitors, such as the acylurea insect growth regulators, are largely unclear. The recent isolation and sequencing of insect CS genes should help in elucidating various aspects of chitin biochemistry and inhibition. In particular, the large number of transmembrane segments, characteristic of the insect CS, are speculated to be involved in chitin translocation and are expected to shed light on the mode of action of acylurea insecticides.

Animals↗
PubMed1996

chs-4, a class IV chitin synthase gene from Neurospora crassa.

In Saccharomyces cerevisiae, most of the cellular chitin is produced by chitin synthase III, which requires the product encoded by the CSD2/CAL1/DIT101/KT12 gene. We have identified, isolated and structurally characterized as CSD2/CAL1/DIT101/KT12 homologue in the filamentous ascomycete Neurospora crassa and have used a "reverse genetics" approach to determine its role in vivo. The yeast gene was used as a heterologous probe for the isolation of a N. crassa gene(designated chs-4) encoding a polypeptide belonging to a class of chitin synthases which we have designated class IV. The predicted polypeptide encoded by this gene is highly similar to those of S. cerevisiae and Candida albicans. N. crassa strains in which chs-4 had been inactivated by the Repeat-Induced point mutation (RIP) process grew and developed in a normal manner under standard growth conditions. However, when grown in the presence of sorbose (a carbon source which induces morphological changes accompanied by elevated chitin content), chitin levels in the chs-4RIP strain were significantly lower than those observed in the wild type. We suggest that CHS4 may serve as an auxiliary enzyme in N. crassa and that, in contrast to yeasts, it is possible that filamentous fungi may have more than one class IV chitin synthase.

Acetylglucosamine↗
PubMed2004

Chemical synthesis of UDP-4-O-methyl-GlcNAc, a potential chain terminator of chitin synthesis.

Chitin synthase converts uridine diphosphoryl-N-acetylglucosamine (UDP-GlcNAc) to chitin (poly-beta-(1-->4)-GlcNAc). During polymerization, elongation occurs at the 4-OH (nonreducing) terminus of the growing chitin chain. Blockage of the 4-OH via incorporation of UDP-N-acetyl-4-O-methylglucosamine (UDP-4-OMe-GlcNAc, 3) can potentially terminate chitin polymerization, and represents a novel strategy for chitin synthase inhibition. The chemical synthesis of 3 and preliminary evaluation of its possible incorporation by chitin synthase are reported herein.

Carbohydrate Sequence↗
PubMed2003

Relation between cell wall chitin content and susceptibility to amphotericin B in Kluyveromyces, Candida and Schizosaccharomyces species.

Yeast strains belonging to the genera Candida, Kluyveromyces and Schizosaccharomyces were tested for their susceptibility (or resistance) to amphotericin B (AmB) in relation to their cell wall chitin content. Results showed that membrane sterol contents did not enable us to explain resistance or susceptibility of these yeasts to AmB. Indeed, we noted that resistant strains were as rich in ergosterol as sensitive strains. The suppression of the wall of yeasts induced an increase in susceptibility to AmB. Strains with high cell wall chitin content were more sensitive to this polyenic antifungal agent than strains with low chitin content. Growth of the yeasts in the presence of chitin induced a resistance of the yeasts to AmB. Similar results were obtained after treatment of the cells by chitinase. In contrast, growth of the yeasts in the presence of chitin synthase activators induced high susceptibility to AmB. Yeast cell wall chitin is an aminopolysaccharide, usually at low concentrations. In Schizosaccharomyces pombe its presence was not established. This polymer is associated with glucans in the wall matrix of the lateral wall and in the budding scars. Even at low content, this polymer seems to play an essential role in the sensitivity (or resistance) of yeast cells to AmB.

Amphotericin B↗
PubMed2003

Deletion of BGL2 results in an increased chitin level in the cell wall of Saccharomyces cerevisiae.

It is shown that the deletion of BGL2 gene leads to increase in chitin content in the cell wall of Saccharomyces cerevisiae. A part of the additional chitin can be removed from the bgl2Delta cell wall by alkali or trypsin treatment. Chitin synthase 1 (Chs1) activity was increased by 60 % in bgl2Delta mutant. No increase in chitin synthase 3 (Chs3) activity in bgl2Delta cells was observed, while they became more sensitive to Nikkomycin Z. The chitin level in the cell walls of a strain lacking both BGL2 and CHS3 genes was higher than that in chs3Delta and lower than that in bgl2Delta strains. Together these data indicate that the deletion of BGL2 results in the accumulation and abnormal incorporation of chitin into the cell wall of S. cerevisiae, and both Chs1 and Chs3 take part in a response to BGL2 deletion in S. cerevisiae cells.

Aminoglycosides↗
PubMed1996

Chs1p and Chs3p, two proteins involved in chitin synthesis, populate a compartment of the Saccharomyces cerevisiae endocytic pathway.

In Saccharomyces cerevisiae, the synthesis of chitin, a cell-wall polysaccharide, is temporally and spatially regulated with respect to the cell cycle and morphogenesis. Using immunological reagents, we found that steady-state levels of Chs1p and Chs3p, two chitin synthase enzymes, did not fluctuate during the cell cycle, indicating that they are not simply regulated by synthesis and degradation. Previous cell fractionation studies demonstrated that chitin synthase I activity (CSI) exists in a plasma membrane form and in intracellular membrane-bound particles called chitosomes. Chitosomes were proposed to act as a reservoir for regulated transport of chitin synthase enzymes to the division septum. We found that Chs1p and Chs3p resided partly in chitosomes and that this distribution was not cell cycle regulated. Pulse-chase cell fractionation experiments showed that chitosome production was blocked in an endocytosis mutant (end4-1), indicating that endocytosis is required for the formation or maintenance of chitosomes. Additionally, Ste2p, internalized by ligand-induced endocytosis, cofractionated with chitosomes, suggesting that these membrane proteins populate the same endosomal compartment. However, in contrast to Ste2p, Chs1p and Chs3p were not rapidly degraded, thus raising the possibility that the temporal and spatial regulation of chitin synthesis is mediated by the mobilization of an endosomal pool of chitin synthase enzymes.

Cell Compartmentation↗
PubMed1999

The Candida albicans CHS4 gene complements a Saccharomyces cerevisiae skt5/chs4 mutation and is involved in chitin biosynthesis.

The Candida albicans CHS4 gene encoding chitin synthase 4 has been isolated using the Saccharomyces cerevisiae CHS4/SKT5 gene as a probe. The gene contains a 2061 bp open reading frame capable of encoding a protein of 687 amino acids (76053 Da). No intron was observed in the gene. Disruption of CHS4 in C. albicans yielded a Calcofluor-resistant phenotype, indicating that Chs4p contributes to chitin biosynthesis. Consistent with this, overexpression of Chs4p under the regulation of the ScGAL1 promoter enhanced chitin synthase 3 activity in S. cerevisiae 7- to 38-fold. In addition, chs3 and chs4 null mutants were significantly defective in Calcofluor white staining and their chitin content was 10% of that of the parental strain. Chs4p of C. albicans and S. cerevisiae showed 61% identity in the C-terminal half of the proteins and that region of C. albicans Chs4p complemented the Chs4p function of a mutant of S. cerevisiae resistant to Calcofluor white. Therefore, it appears that Chs4p is involved in chitin synthase 3 activity by combining with Chs3p to interact synergistically in chitin biosynthesis.

Amino Acid Sequence↗
PubMed1997

Increase in chitin as an essential response to defects in assembly of cell wall polymers in the ggp1delta mutant of Saccharomyces cerevisiae.

The GGP1/GAS1 gene codes for a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae. The ggp1delta mutant shows morphogenetic defects which suggest changes in the cell wall matrix. In this work, we have investigated cell wall glucan levels and the increase of chitin in ggp1delta mutant cells. In these cells, the level of alkali-insoluble 1,6-beta-D-glucan was found to be 50% of that of wild-type cells and was responsible for the observed decrease in the total alkali-insoluble glucan. Moreover, the ratio of alkali-soluble to alkali-insoluble glucan almost doubled, suggesting a change in glucan solubility. The increase of chitin in ggp1delta cells was found to be essential since the chs3delta ggp1delta mutations determined a severe reduction in the growth rate and in cell viability. Electron microscopy analysis showed the loss of the typical structure of yeast cell walls. Furthermore, in the chs3delta ggp1delta cells, the level of alkali-insoluble glucan was 57% of that of wild-type cells and the alkali-soluble/alkali-insoluble glucan ratio was doubled. We tested the effect of inhibition of chitin synthesis also by a different approach. The ggp1delta cells were treated with nikkomycin Z, a well-known inhibitor of chitin synthesis, and showed a hypersensitivity to this drug. In addition, studies of genetic interactions with genes related to the construction of the cell wall indicate a synthetic lethal effect of the ggp1delta kre6delta and the ggp1delta pkc1delta combined mutations. Our data point to an involvement of the GGP1 gene product in the cross-links between cell wall glucans (1,3-beta-D-glucans with 1,6-beta-D-glucans and with chitin). Chitin is essential to compensate for the defects due to the lack of Ggp1p. Moreover, the activities of Ggp1p and Chs3p are essential to the formation of the organized structure of the cell wall in vegetative cells.

Aminoglycosides↗
PubMed2000

WdCHS3, a gene that encodes a class III chitin synthase in Wangiella (Exophiala) dermatitidis, is expressed differentially under stress conditions.

Class III chitin synthases are important for hyphal growth in some filamentous fungi but are not found in yeasts. Using a specific PCR product that encodes a portion of the class III chitin synthase of W. dermatitidis as a probe, we isolated the chitin synthase gene, WdCHS3, from this polymorphic melanized pathogen of humans. Northern blotting showed that WdCHS3 was highly expressed under stress conditions, such as the shift of cells to temperatures commensurate with infection, or to conditions that induce cellular morphogenesis in this fungus. Analysis of the 5' upstream sequence of WdCHS3 provided evidence for a negative regulatory element at between -780 and -1600 bp. Western blotting indicated that the production of the WdChs3p was temperature dependent and temporally regulated. Disruption of WdCHS3 in a wild-type strain and in two temperature-sensitive morphological mutants resulted in significantly reduced chitin synthase activities but did not obviously affect their morphologies, growth rates, chitin contents, or virulence. This paradox suggested that the contributions of the high levels of WdCHS3 gene expression and WdChs3p production in strains subjected to stress reside in unknown or unexamined parts of the life cycle of this ecologically poorly known member of the Fungi Imperfecti. Nonetheless, this report presents the first evidence that transcription of a chitin synthase gene is regulated by a negative regulatory element in its 5' upstream sequence.

Amino Acid Sequence↗
PubMed1997

CHS5, a gene involved in chitin synthesis and mating in Saccharomyces cerevisiae.

The CHS5 locus of Saccharomyces cerevisiae is important for wild-type levels of chitin synthase III activity. chs5 cells have reduced levels of this activity. To further understand the role of CHS5 in yeast, the CHS5 gene was cloned by complementation of the Calcofluor resistance phenotype of a chs5 mutant. Transformation of the mutant with a plasmid carrying CHS5 restored Calcofluor sensitivity, wild-type cell wall chitin levels, and chitin synthase III activity levels. DNA sequence analysis reveals that CHS5 encodes a unique polypeptide of 671 amino acids with a molecular mass of 73,642 Da. The predicted sequence shows a heptapeptide repeated 10 times, a carboxy-terminal lysine-rich tail, and some similarity to neurofilament proteins. The effects of deletion of CHS5 indicate that it is not essential for yeast cell growth; however, it is important for mating. Deletion of CHS3, the presumptive structural gene for chitin synthase III activity, results in a modest decrease in mating efficiency, whereas chs5delta cells exhibit a much stronger mating defect. However, chs5 cells produce more chitin than chs3 mutants, indicating that CHS5 plays a role in other processes besides chitin synthesis. Analysis of mating mixtures of chs5 cells reveals that cells agglutinate and make contact but fail to undergo cell fusion. The chs5 mating defect can be partially rescued by FUS1 and/or FUS2, two genes which have been implicated previously in cell fusion, but not by FUS3. In addition, mating efficiency is much lower in fus1 fus2 x chs5 than in fus1 fus2 x wild type crosses. Our results indicate that Chs5p plays an important role in the cell fusion step of mating.

Amino Acid Sequence↗