Amount Of Chitin Research Articles

In vivo and in vitro effects of tebufenozide and 20-hydroxyecdysone on chitin synthesis

Last-instar larvae of the beet armyworm, Spodoptera exigua, fed on diet containing the nonsteroidal molting hormone agonist, tebufenozide, at 1 mg/liter, exhibited a premature induction of chitin synthesis. Treatment with tebufenozide apparently caused normal amounts of chitin to be formed in the cuticle. This effect complemented the precocious induction of larval molting (apolysis) that was observed in response to tebufenozide. Subsequently, normal ecdysis was inhibited and treated larvae died while still within their old cuticle. In another series of experiments, 1-day-old pupae of the European corn borer, Ostrinia nubilalis, were injected with various doses of tebufenozide or 20E. Incorporation of N-[acetyl-1-14C]glucosamine served as a measure of chitin production and was monitored in cultured day-4 and day-5 claspers. Tebufenozide at 100, 200, and 400 ng induced synthesis in day-4 claspers and incorporation was enhanced 8-fold as compared to control claspers. A similar stimulation of chitin synthesis was observed with 2,000 ng of 20E. For day-5 claspers, high doses of tebufenozide inhibited chitin synthesis. Treatment with tebufenozide had a deleterious effect on adult development and even at the lowest dose injected, 10 ng, adult eclosion was completely inhibited. Our current results indicate that in both larvae and pupae/pharate adults, tebufenozide can induce the premature synthesis of chitin as can 20E, but the effective dose of tebufenozide is much lower than that for 20E. The results support the view that tebufenozide, although nonsteroidal in nature, is able to mimic the activity of ecdysteroids in inducing chitin synthesis. However, development in treated insects is abnormal and the newly formed cuticle is imperfect and unable to survive a molt. Arch. Insect Biochem. Physiol. 41:33–41, 1999. © 1999 Wiley-Liss, Inc.

Molecular Cloning and Cell Cycle-dependent Expression of Mammalian CRM1, a Protein Involved in Nuclear Export of Proteins

Crm1 of Schizosaccharomyces pombe, a nuclear protein essential for proliferation and chromosome region maintenance, is a possible target of leptomycin B, an antifungal and antitumor antibiotic with cell cycle-arresting activity. cDNA encoding a human homolog of Crm1 was cloned. Human CRM1 (hCRM1) consisted of 1071 amino acids, of which the sequence showed 52% homology with S. pombe Crm1. hCRM1 weakly complemented the cold-sensitive mutation of S. pombe crm1-809, as did S. pombe crm1+. Overproduction of hCRM1 under the control of a series of nmt1 promoters suppressed cell proliferation in wild-type S. pombe in an expression level-dependent manner. A similar inhibitory effect was also observed for crm1+. Cells overproducing either hCRM1 or S. pombe Crm1 were distinctly larger than uninduced cells and contained compacted and fragmented nuclei. Furthermore, calcofluor staining demonstrated that most of these cells formed two septa per cell and accumulated a large amount of chitin or its related polysaccharides around the septa. Closely similar phenotypes between hCRM1- and S. pombe Crm1-induced cells indicate that the cloned cDNA encodes a functional homolog of S. pombe crm1+. Northern blot analyses with RNAs isolated from synchronized mammalian cells showed that the expression of mammalian CRM1 was initiated in late G1 and reached a peak at G2/M, although its protein level unchanged during the cell cycle. Transient expression of hCRM1 fused to the green fluorescent protein (GFP) in NIH3T3 cells showed that hCRM1 was localized preferentially in the nuclear envelope and was also detectable in the nucleoplasm and the cytoplasm. A crm1 mutation of S. pombe caused nuclear import of a GFP fusion protein containing a nuclear export signal but no change in the distribution of a GFP fusion protein containing a nuclear localization signal. All of these data suggest that CRM1 is a novel cell-cycle regulated gene that is essential for the nuclear export signal-dependent nuclear export of proteins.

ポリウレタン／キチン系複合材料の開発

A novel functional composite by combining chitin and polyurethane elastomers was developed. Diisocyanates were reacted with polymer glycols at 80°C in a nitrogen atmosphere to form NCO-terminated prepolymers. Various amounts of chitin powder were added to a prepolymer with catalyst and mixed thoroughly. Then the reaction mixtures were molded by a press at 70-90°C for 1-7hr. After synthetic conditions were examined, it was found that the proper range of the amount of chitin, the optimum time, temperature, and pressure required for pressure-molding were 60wt%, 4hr, 90°C, and 30MPa, respectively. After screening several polymer glycols and diisocyanates, poly(oxytetramethylene) glycol and hexamethylene diisocyanate were found to be suitable for compatibility with chitin. Swelling, thermal property, tensile property, SEM, and water sorption of polyurethane/chitin composites were measured. The composites were brittle, but the wet composites became elastomeric. The composites had good humid-breathing function.

Alternation in cell wall chitin of Zygosaccharomyces rouxii

Chitin was present as a main component occupying about 15% of the cell wall preparation of Zygosaccharomyces rouxii ATCC 52698 grown in a non-salt medium, and a netlike structure spreading on the whole cell surface, that mainly consisted of chitin, was observed by an electron microscope after treating the cells with an excess of Zymolyase. The amounts of chitin, however, remarkably decreased according to the increase in NaCl in the medium. The net like structure was not observed entirely on the cell envelope of the yeast grown in a hypertonic salt medium, and the ratio of chitin contained in the cell walls decreased to 1–2%.

The compositional analysis of recent cephalopod shell carbohydrates by Fourier transform infrared spectrometry and high performance anion exchange-pulsed amperometric detection

The organic matrix of four living cephalopod skeletons was extracted, purified, separated in a soluble and an insoluble fraction. These fractions were submitted to infrared analysis. After a light hydrolysis, their monosaccharide content was determined by HPAE-PAD. The results were treated by principal component analysis. Infrared analysis has been used to estimate the protein/sugar ratios and showed a high amount of chitin within the insoluble fractions. This was corroborated by their high content of glucosamine. The composition of soluble matrices appeared more heterogeneous: the general tendency is an increase of glucosamine from the most mineralized shell (Nautilus) to the non-mineralized one (Loligo), and a decrease in glucose and galactose. These data would be in agreement with the evolutionary tendency towards shell reduction and disappearance among cephalopods.

Chitin preservation in Quaternary Coleoptera: preliminary results

Although many fossil organisms are known to have had chitinous exoskeletons, there is conflicting information concerning chitin preservation in fossils. Remains of arthropods, particularly insects, are relatively common in Cenozoic deposits and provide useful paleoenvironmental information. Experimental studies on the chemistry of modern and fossil arthropod chitin suggest that it may remain sufficiently stable for paleoecologic and paleoclimatic information to be derived from stable isotope analysis. Such techniques might be applied to a wide range of chitinous fossils. Analyses of Quaternary Coleoptera from late-glacial and last interglacial deposits indicate that chitin comprises 10–30% of the dry weight of fossil beetle fragments. These amounts represent half to three-quarters of the amount of chitin reported for a range of modern insects (25–40%). The abundance and quality of chitin preservation has important implications for paleoentomological research.

The onset of the helical arrangement of chitin microfibrils in fruit-body development of Coprinus cinereus

The onset of helical arrangement of chitin microfibrils in the wall was investigated during fruit body development of Coprinus cinereus . The onset was traced back to hyphal knots of 0·1–0·2 mm in diameter, i.e. fruit-body initials in which no differentiation into hyphal tissues was yet discernible. The walls of the hyphal knots were distinguishable from those of hyphae in other developmental phases in that the former contained lower amounts of chitin. The results obtained support our model for the ontogeny of the helical wall structure.

Identification of a Saccharomyces cerevisiae mutation that allows cells to grow without chitin synthase 1 or 2.

Chitin is a component of the yeast cell wall which is localized to the septum between mother and daughter cells. Previous work in Saccharomyces cerevisiae has shown that this organism possesses three chitin synthases, 1, 2, and 3. Disruption experiments have shown that loss of chitin synthase 2 has a more profound effect on cell viability than loss of either of the other two and is lethal in complete media. We report here the finding of an S. cerevisiae strain which does not require the chitin synthase 2 structural gene for viability. We present evidence that there is a gene in this strain which suppresses the lethality of disruption of the chitin synthase 2 structural gene and is genetically distinct from the structural genes for chitin synthase 1 and chitin synthase 2. We show that an S. cerevisiae mutant containing the suppressor and lacking both structural genes for chitin synthase 1 and 2 has normal amounts of chitin in its cell wall. We hypothesize that the suppressor gene encodes or controls the expression of chitin synthase 3.

DIT101 (CSD2, CAL1), a cell cycle-regulated yeast gene required for synthesis of chitin in cell walls and chitosan in spore walls.

A mutant screen has been designed to isolate mutants in Saccharomyces cerevisiae deficient in spore wall dityrosine. As shown by electron microscopy, most of the mutant spores lacked only the outermost, dityrosine-rich layer of the spore wall. Mutant dit101, however, was additionally lacking the chitosan layer of the spore wall. Chemical measurements showed that this mutant does not synthesize chitosan during sporulation. The mutant spores were viable but sensitive to lytic enzymes (glusulase or zymolyase). Unlike most of the dit-mutants, dit101 did show a distinctive phenotype in vegetative cells: they grew normally but contained very little chitin and were therefore resistant to the toxic chitin-binding dye, Calcofluor White. The cells showed barely detectable staining of the walls with Calcofluor White or primulin. The decrease in the amount of chitin in vegetative cells and the absence of chitosan in spores suggested that the mutant dit101 could be defective in a chitin synthase. Indeed, a genomic yeast clone harboring the gene, CSD2, sharing significant sequence similarity with yeast chitin synthases I and II (C. E. Bulawa (1992), Mol. Cell. Biol. 12, 1764-1776), complemented our mutant and was shown to correspond to the chromosomal locus of dit101. Thus, the mutations dit101 and csd2 (and probably also call; M. H. Valdivieso et al., (1991), J. Cell Biol. 114, 101-109) were shown to be allelic. The gene was mapped to chromosome II and was located about 3 kb distal of GAL1. Using this DNA clone, a transcript of about 3500-4000 nucleotides was detected. Comparing RNA isolated from vegetative cells and from sporulating cells at different times throughout the sporulation process, no significant differences in DIT101 transcript levels could be detected indicating absence of sporulation-specific transcriptional regulation. However, the amount of DIT101 transcript changed significantly at different stages of the mitotic cell cycle, peaking after septum formation, but before cytokinesis. As most of the chitin synthesis of vegetative cells occurs at this stage of the cell division cycle, chitin synthesis mediated by DIT101 could be primarily regulated at the level of transcription in vegetatively growing cells.

CSD2, CSD3, and CSD4, genes required for chitin synthesis in Saccharomyces cerevisiae: the CSD2 gene product is related to chitin synthases and to developmentally regulated proteins in Rhizobium species and Xenopus laevis.

In Saccharomyces cerevisiae, chitin forms the primary division septum and the bud scar in the walls of vegetative cells. Three chitin synthetic activities have been detected. Two of them, chitin synthase I and chitin synthase II, are not required for synthesis of most of the chitin present in vivo. Using a novel screen, I have identified three mutations, designated csd2, csd3, and csd4, that reduce levels of chitin in vivo by as much as 10-fold without causing any obvious perturbation of cell division. The csd2 and csd4 mutants lack chitin synthase III activity in vitro, while csd3 mutants have wild-type levels of this enzyme. In certain genetic backgrounds, these mutations cause temperature-sensitive growth on rich medium; inclusion of salts or sorbitol bypasses this phenotype. Gene disruption experiments show that CSD2 is nonessential; a small amount of chitin, about 5% of the wild-type level, is detected in the disruptants. DNA sequencing indicates that the CSD2 protein has limited, but statistically significant, similarity to chitin synthase I and chitin synthase II. Other significant similarities are to two developmental proteins: the nodC protein from Rhizobium species and the DG42 protein of Xenopus laevis. The relationship between the nodC and CSD2 proteins suggests that nodC may encode an N-acetylglucosaminyltransferase that synthesizes the oligosaccharide backbone of the nodulation factor NodRm-1.

Ultrastructural detection of polysaccharides in the cell walls of two members of the Hyphocytriales

Cytochemical methods that employed observation of lectin- or enzyme-gold complexes with an electron microscope were used to study the occurrence and distribution of sugars and polysaccharides in the cell walls of Rhizidiomyces apophysatus and Hyphochytrium catenoides , two fungi belonging to the class Hyphochytriomycetes. Both fungi have the polysaccharides cellulose and chitin in their cell walls and a minor component that contains n -acetyl- d -galactosamine. The discharge tubes emanating from sporangia of R. apophysatus stained positively for chitin but were negative for cellulose and the N -acetyl- d -galactosamine containing compound. Whereas the cell walls of developing sporangia of H. catenoides labelled strongly for cellulose, the results indicated they contained only trace amounts of chitin. The rhizomycelial walls of H. catenoides had a composition similar to that of the sporangia except for the septa that appeared to contain more sites indicative of chitin. The significance of the results relative to earlier studies of these fungi and generalizations with respect to cell wall chemistry and systematic relationships is discussed.

Effects of subinhibitory dose of amphotericin B on cell wall biosynthesis in Candida Albicans

Subinhibitory doses of amphotericin B in the culture medium of Candida albicans modified yeast cell wall synthesis. Analysis of isolated cell walls showed a decrease in mannose and an increase in amino acid and glucosamine levels. After fractionation of the cell wall by ethylenediamine or pronase digestion, study of the fractions corroborated an outer phosphopeptidomann decrease and an enrichment of matrix constituants. Determination of the amount of chitin showed stimulation of synthesis of this amino polysaccharide in yeasts grown in the presence of amphotericin B. Decrease in phosphopeptidomannans and high production of other cell wall constituents are probably consequences of modification of the lipidic environment of membrane-bound enzymes by the antifungal polyene action.

Isolation and characterization of Saccharomyces cerevisiae mutants resistant to Calcofluor white.

Calcofluor is a fluorochrome that exhibits antifungal activity and a high affinity for yeast cell wall chitin. We isolated Saccharomyces cerevisiae mutants resistant to Calcofluor. The resistance segregated in a Mendelian fashion and behaved as a recessive character in all the mutants analyzed. Five loci were defined by complementation analysis. The abnormally thick septa between mother and daughter cells caused by Calcofluor in wild-type cells were absent in the mutants. The Calcofluor-binding capacity, observed by fluorescence microscopy, in a S. cerevisiae wild-type cells during alpha-factor treatment was also absent in some mutants and reduced in others. Staining of cell walls with wheat germ agglutinin-fluorescein complex indicated that the chitin uniformly distributed over the whole cell wall in vegetative or in alpha-factor-treated cells was almost absent in three of the mutants and reduced in the two others. Cell wall analysis evidenced a five- to ninefold reduction in the amount of chitin in mutants compared with that in the wild-type strain. The total amounts of cell wall mannan and beta-glucan in wild-type and mutant strains were similar; however, the percentage of beta-glucan that remained insoluble after alkali extraction was considerably reduced in mutant cells. The susceptibilities of the mutants and the wild-type strains to a cell wall enzymic lytic complex were rather similar. The in vitro levels of chitin synthase 2 detected in all mutants were similar to that in the wild type. The significance of these results is discussed in connection with the mechanism of chitin synthesis and cell wall morphogenesis in S. cerevisiae.

Digestibility of chitin in cod,Gadus morhua, in vivo

Sixteen cod,Gadus morhua (L.), were individually fed a single ration of shrimps,Crangon allmanni. Four fish were killed and examined 6, 12, 24 and 48 h after the fish had been fed. Chitinase activities were measured in the extracts of stomach contents, stomach tissue, pyloric caecae, intestinal contents and intestinal tissue. The level of enzyme activity in different parts of the digestive tract was shown to be dependent on the phase of the digestive process. High concentrations of the chitin degradation product N-acetyl-D-glucosamine were determined in the stomach and in the intestinal contents. Based on the chitin concentration in the food organisms and the individual food uptake, the amount of chitin consumed by each fish could be calculated. Only up to 9% of the ingested chitin was recovered from the intestinal contents of the fish at any given time after feeding (6, 12, 24 and 48 h). In addition, only 2.4% of the chitin consumed with the food could be recovered in the collected faeces of the fish. The 4 cod killed 48 h after feeding had completely emptied their stomach. Chitin digestion in these fish was calculated to have been 90%.

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.

Mesophilic chitin-degrading anaerobes isolated from an estuarine environment

Eight strains of obligately anaerobic, mesophilic, chitinolytic bacteria were isolated from the sediment of an estuarine environment. The isolates were rod-shaped, Gram-negative, and formed terminal spherical spores that swelled the sporangium. The major products from the fermentation of chitin were: acetate, ethanol, formate, CO 2, H 2 and ammonia. Growth of the isolates was possible at pH values ranging from 5.0–9.0. During the fermentation of chitin, N-acetylglucosamine accumulated in the culture fluids and was not metabolized. No organic compounds other than chitin and its oligomers could be demonstrated to support growth of the isolates. Hydrolysis of chitin proceeded at a relatively low rate and was incomplete. Approximately 65% of the initial amount of chitin was hydrolyzed during a period of 5–10 days. Supplementation of the medium with yeast extract, casamino acids or peptone did not enhance the rate of chitin hydrolysis, but reducing agents such as Na 2S 2O 4 and Ti (III)-NTA markedly stimulated the rate of chitin fermentation. The ecological implications of the high degree of substrate specialization are discussed.

Mesophilic chitin-degrading anaerobes isolated from an estuarine environment

Eight strains of obligately anaerobic, mesophilic, chitinolytic bacteria were isolated from the sediment of an estuarine environment. The isolates were rod-shaped, Gram-negative, and formed terminal spherical spores that swelled the sporangium. The major products from the fermentation of chitin were: acetate, ethanol, formate, CO2, H2 and ammonia. Growth of the isolates was possible at pH values ranging from 5.0–9.0. During the fermentation of chitin, N-acetylglucosamine accumulated in the culture fluids and was not metabolized. No organic compounds other than chitin and its oligomers could be demonstrated to support growth of the isolates. Hydrolysis of chitin proceeded at a relatively low rate and was incomplete. Approximately 65% of the initial amount of chitin was hydrolyzed during a period of 5–10 days. Supplementation of the medium with yeast extract, casamino acids or peptone did not enhance the rate of chitin hydrolysis, but reducing agents such as Na2S2O4 and Ti (III)-NTA markedly stimulated the rate of chitin fermentation. The ecological implications of the high degree of substrate specialization are discussed.

Alterations in the biosynthesis of chitin and glucan in the slime mutant of Neurospora crassa

The slime mutant of Neurospora crassa lacks both chitin and insolubleβ(1 → 3)glucans either cellbound or secreted to the medium. Failure to synthesize both polymers is not due to deficiencies in the precursor sugar nucleotides, UDP-GlcNAc and UDP-Glc, since these are present in higher amounts in the mutant than in the wild type. The slime mutant also contains higher levels of chitin synthetase than the wild type, but lacks detectable glucan synthetase activity. an os-1 mutant grown in hypertonic medium contained reduced amounts of chitin and glucan, but its glucan and chitin synthetase activities were nearly normal. It is concluded that the absence of a cell wall in the slime mutant is due to a mutational event(s) which affects the mechanism of activation or delivery of chitin synthetase to the cell surface and the formation of a functionalβ(1 → 3)glucan synthetase. This mutational event must be different in the os-1 strain.

The growth of rainbow trout ( Salmo gairdneri) given diets containing chitin and its relationship to chitinolytic enzymes and chitin digestibility

The growth of rainbow trout fed diets containing 4, 10 and 25% chitin, over a 12-week period, was significantly depressed ( P < 0.001) when compared with controls fed diets containing 25% starch. There was no difference in growth rate between control fish and those fed diets containing 10% N-acetyl glucosamine (GlcNAc). Isotopically labelled amino sugars were shown to be oxidized when injected intraperitoneally into rainbow trout. Relatively high levels were found of chitinase activity in the stomachs and of chitobiase in the intestines. These enzyme activities were similar in all the trout, irrespective of the amount of chitin in their diets, except that chitobiase in the intestines of fish fed diets containing GlcNAc showed higher levels of activity than the controls ( P < 0.05). Chitin was not significantly digested when fed at 10 and 30% of the diet but the apparent digestibility of pre-cooked starch was 50% when fed at either 15 or 25%, on the basis of the inert indicator (Cr 2O 3) method. Chitinase and chitobiase activities were not reduced in fish fed diets containing 10% chitin and an antibiotic (Tribrissen ®) although the alimentary microflora were completely eliminated. Enzyme activities were not enhanced when live chitinolytic bacteria ( Vibrio alginolyticus) were incorporated into diets with 10% chitin; these bacteria were only recovered from the intestine. The evidence indicates an endogenous origin of chitinolytic enzymes in the trout gastro-intestinal tract. The presence of either antibiotic or bacteria in the diet had no effect on the digestibility of chitin.

Cell wall composition and protoplast regeneration in Candida albicans.

The transition of blastospores to the mycelial phase in Candida albicans was induced after the blastospores were kept at 4 degrees C for several hours and then transferred to a fresh medium prewarmed at 37 degrees C. Glucan was the most abundant polymer in the wall in the two morphogenetic forms but the amount of chitin was higher in the mycelial form than in blastospores. Efficient protoplasting required reducing agents and proteases together with beta-glucanases (zymolyase). Protein synthesis in regenerating protoplasts was initiated after about 30 min. Chitin synthetase, initially very low, was incorporated in important amounts into cell membranes mainly in a zymogenic state. After a few hours chitin was the most abundant polymer found in the aberrant wall of the regenerating protoplast.