No description available.

No description available.

<p>The peritrophic matrix (PM) lines the insect midgut and is composed of chitin and protein. It is required for organization of digestion and for protection of epithelial cells from mechanical damage, pathogens, and toxins. The PM of <i>Mamestra configurata</i> (Lepidoptera: Noctuidae), bertha armyworm, a serious pest of cruciferous oilseed rape, was studied. The multilayered PM is delaminated from the anterior midgut epithelium during molting Phase II by periodic pulses and degraded during the molting Phase I stage. These events are controlled by chitin synthase-B, and chitinolytic enzymes, such as chitinase and β-<i>N</i>-acetylglucosaminidase. Eighty-two PM proteins were identified and classified as: i) peritrophins, ii) enzymes and iii) other proteins. Peritrophins were further classified as simple, binary, complex and repetitive according to their structural organization and phylogenetic analysis of peritrophin A domains. The expression of most genes encoding PM proteins was specific to the midgut and independent of larval feeding status, developmental stage, or PM formation.</p> <p>This study includes the first report of chitin deacetylase (CDA) activity in the insect midgut suggesting that the PM may contain chitosan. Digestive enzymes, such as insect intestinal lipases (IILs) and serine proteases were also associated with the PM. The IIL genes differed in their expression during larval development; however, serine protease genes were expressed continuously and serine protease activity was present in the midgut of feeding and nonfeeding stages. <i>M. configurata</i> IIM4, a complex peritrophin, was susceptible to degradation by Mamestra configurata nucleopolyhedrovirus-A challenge, as the first evidence of IIM degradation by an alphabaculovirus enhancin. <i>M. configurata</i> IIM2, a binary peritrophin, was unaffected by baculoviral challenge and such resistance of an IIM has not been reported previously. The current study is also the first demonstration of silencing by RNA interference (RNAi) of any gene encoding a PM protein, in this case <i>M. configurata</i> CDA1 (McCDA1) and McPM1. In addition, both <i>in vitro</i> and <i>per os</i> feeding experiments revealed <i>McCDA1</i> silencing starting at 24 or 36 hours posttreatment, as one of the most successful demonstrations of RNAi in a lepidopteran.</p>

<p>The peritrophic matrix (PM) lines the insect midgut and is composed of chitin and protein. It is required for organization of digestion and for protection of epithelial cells from mechanical damage, pathogens, and toxins. The PM of <i>Mamestra configurata</i> (Lepidoptera: Noctuidae), bertha armyworm, a serious pest of cruciferous oilseed rape, was studied. The multilayered PM is delaminated from the anterior midgut epithelium during molting Phase II by periodic pulses and degraded during the molting Phase I stage. These events are controlled by chitin synthase-B, and chitinolytic enzymes, such as chitinase and β-<i>N</i>-acetylglucosaminidase. Eighty-two PM proteins were identified and classified as: i) peritrophins, ii) enzymes and iii) other proteins. Peritrophins were further classified as simple, binary, complex and repetitive according to their structural organization and phylogenetic analysis of peritrophin A domains. The expression of most genes encoding PM proteins was specific to the midgut and independent of larval feeding status, developmental stage, or PM formation.</p> <p>This study includes the first report of chitin deacetylase (CDA) activity in the insect midgut suggesting that the PM may contain chitosan. Digestive enzymes, such as insect intestinal lipases (IILs) and serine proteases were also associated with the PM. The IIL genes differed in their expression during larval development; however, serine protease genes were expressed continuously and serine protease activity was present in the midgut of feeding and nonfeeding stages. <i>M. configurata</i> IIM4, a complex peritrophin, was susceptible to degradation by Mamestra configurata nucleopolyhedrovirus-A challenge, as the first evidence of IIM degradation by an alphabaculovirus enhancin. <i>M. configurata</i> IIM2, a binary peritrophin, was unaffected by baculoviral challenge and such resistance of an IIM has not been reported previously. The current study is also the first demonstration of silencing by RNA interference (RNAi) of any gene encoding a PM protein, in this case <i>M. configurata</i> CDA1 (McCDA1) and McPM1. In addition, both <i>in vitro</i> and <i>per os</i> feeding experiments revealed <i>McCDA1</i> silencing starting at 24 or 36 hours posttreatment, as one of the most successful demonstrations of RNAi in a lepidopteran.</p>

Chitin is an important reinforcing component in load-bearing structures in many organisms such as insects and crustaceans (i.e. shrimps, lobsters, crabs etc.). It is of increasing interest for use in packaging materials as well as in biomedical applications. Furthermore, biological materials may inspire the development of new man-made material concepts. Chitinmolecules are crystallized in extended chain conformations to form nanoscale fibrils of about 3 nm in diameter. In the present study, novel materialshave been developed based on a new type of chitin nanofibers prepared from the lobster exoskeleton. Improved understanding about effects of chitin from crustaceans and chitin material preparation on structure is provided through Atomic Force Microscopy(AFM) (paper I&amp;II), Scanning Transmission Electron Microscopy(STEM) (paper I&amp;II), X-Ray Diffraction (XRD), Intrinsic Viscosity, solid state 13C Nuclear Magnetic Resonance (NMR) (paper II), Field Emission Scanning Electron Microscopy(FE-SEM) (paper I, II, III, IV &amp; V), Ultraviolet-Visible Spectrophotometryand Dynamic Light Scattering (DLS) (paper III). The presence of protein was confirmed through colorimetric method(paper I &amp; II). An interesting result from the thesis is the new features of chitin nanofiber including small diameter, high molar mass or nanofiber length,and high purity. The structure and composition of the nanofibers confirms this (paper I &amp; II). Furthermore, the structure and properties of the corresponding materials confirm the uniqueness of the present nanofibers: chitin membrane (I &amp; II), polymer matrix composites (III),and hydrogels (paper IV). Improved mechanical properties compared with typical data from the literature were confirmed for chitin nanofiber membranes in paper II, chitin-chitosan polymer matrix composites in paper III, and chitin hydrogel in paper IV. Mechanical tests included dynamic mechanical analysis and uniaxial tensile tests. Mechanical properties of chitin hydrogels were evaluated based onrheological and compression properties (paper IV). The values were the highest reported for this kind of chitin material. Furthermore, the relationships between materials structure and properties were analyzed. For membranes and polymer matrix nanocomposites, the degree of dispersion is an important parameter. For the hydrogels, the preparation procedure is very simple and has interesting practical potential. Chitin-binding characteristics of cuticular proteins areinteresting fornovel bio-inspired material development. In the present work(paper V), chitin nanofibers with newfeaturesincluding high surface area and low protein content were combined with resilin-like protein possessing the chitin-binding characteristics. Hydrated chitin-resilin nanocomposites with similar composition as in rubber-like insect cuticles were prepared. The main objective was to improve understanding on the role of chitin-binding domain on mechanical properties. Resilin is a rubber-like protein present in insects. The exon I (comprising 18 N-terminal elastic repeat units) together with or without the exon II (a typical cuticular chitin-binding domain) from the resilin gene CG15920 found in Drosophila melanogasterwere cloned and the encoded proteins were expressed as soluble products in Escherichia coli.Resilin-like protein with chitin-binding domain (designated as ResChBD) adsorbedin significant amount to chitin nanofiber surface andprotein-bound cuticle-like soft nanocomposites were formed. Although chitin bindingwas taking place only in proteinswith chitin-binding domain, the global mechanical behavior of the hydrated chitin-resilin nanocomposites was not so sensitive to this chitin-resilin interaction. In summary, chitin is an interesting material component with high potential as mechanical reinforcement in a variety of nanomaterials. The present study reports the genesisof novel chitin nanofibers and outlines the basic relationships between structure and properties for materials based on chitin. Future work should be directed towards both bio-inspired studies of the nanocomposite chitin structures in organisms, as well as the industrial applications of chitin waste from the food industry. Chitin nanofibers can strengthen the properties of materials, andprovide optical transparency as well as biological activities such as antimicrobial properties. / <p>QC 20141110</p>

Thesis (Dr. P.H.)--University of Texas Health Science Center at Houston, School of Public Health, 1988. / Includes bibliographical references (leaves 158-179).

No description available.

Chitin, a homopolymer of β (1-4)-linked N-acetylglucosamine, is one of the most abundant biopolymers in nature. It is widely distributed in the exoskeleton of crustaceans and insects, in the cell walls of most fungi and some algae. Chitin is an extremely insoluble material with limited industrial applicability. The deacetylated derivative of chitin, chitosan, is a water soluble cationic biopolymer having a broad range of applications (Hirano, 1999). Chitosan is naturally found in the cell wall of Zygomycetes, in the ascospore of Saccharomyces cerevisiae (Briza et al., 1988) and in the cyst wall of Entamoeba invadens (Das et al., 2006). Chitosan biosynthesis requires the coordinated action of chitin synthase (E.C.2.4.1.16) and chitin deacetylase (E.C.3.5.1.41) (Davis & Bartnicki, 1984). Chitin synthase polymerizes N-acetyl glucosamine precursor molecules into chitin and chitin deacetylase catalyzes the deacetylation of the nascent chitin chains. The chitin deacetylase enzymes are members of the family 4 of carbohydrate esterases (CE-4s) as defined by the CAZY database [http://afmb.cnrs-mrs.fr/~cazy/CAZY] (Couthino et al., 1999), which includes several members sharing a conserved region in the primary structure assigned as the NodB homology domain(Caufrier et al., 2003) or polysaccharide deacetylase domain. Chitin deacetylase was first identified and partially purified from extracts of the fungus Mucor rouxii. Since then, chitin deacetylase has been purified from several fungi and chitin deacetylase open reading frames have been cloned from a few microorganisms including M. rouxii (Kafetzopoulos et al., 1993), Colletotrichum lindemuthianum (Tokuyasu et al., 1999; Shresta et al., 2004), Phycomyces blakesleeanus (GenBank AB046690), Schizophillum commune (GenBank AF271216), Blumeria graminis (GenBank AAK84438), Saccharomyces cerevisiae (Christodoulidou et al., 1999) and Schizosaccharomyces pombe (Matsuo et al., 2005). The structure and the catalytic mechanism of chitin deacetylase from C. lindemuthianum were recently studied (Blair et al., 2006). Chitin deacetylase plays a role in the cell wall biosynthesis in M. rouxii and Absidia coerulea (Gao et al., 1995). In C. lindemuthianum and Aspergillus nidulans, it was suggested that chitin deacetylase participates in plant-pathogen interactions to promote plant invasion (Tsigos et al., 2000). In S. cerevisiae, chitin deacetylase is essential for the ascospore cell wall rigidity and the resistance against lytic enzymes (Christodoulidou et al., 1996). The use of chitin deacetylase enzyme for the industrial deacetylation of chitin awaked a great interest. Different fungal strains were screened and compared for their ability to produce a chitin deacetylase secreted, active on insoluble substrates and showing low inhibition with acetate, a product of reaction. Rhizopus circinans proved to be a good chitin deacetylase producer with the targeted characteristics. The second part of the work was to isolate the cDNA encoding for the chitin deacetylase of R. circinans. The native enzyme was purified to homogeneity for sequencing the N-Terminal extremity. The enzyme was purified in only two steps from the culture supernatant of R. circinans. Then, the purified enzyme was sequenced and the first nine amino acids were identified. In the same way, a R. circinans cDNA library was also constructed. The cDNA library was screened using two approaches: on the one hand with radiolabeled homologous probe and on the other hand by PCR with primers designed for the 5 extremity, on the basis of the deduced sequence of the N-Terminal extremity of the native enzyme and for the 3 extremity, from the deduced R. oryzae chitin deacetylase. Two cDNA sequences (D2 and I3/2) with homology to fungal chitin deacetylase genes were isolated with the radiolabeled probe and one sequence (RC+) by PCR approach. The sequences were analyzed and characterized. The three sequences possessed several characteristics of chitin deacetylase sequence: homology with known chitin deacetylase cDNA, the presence of the deacetylase polysaccharide domain, and the same potential glycosylation sites than M. rouxii chitin deacetylase. The cDNA D2, I3/2 and RC (RC sequence is the mature protein sequence of RC+ sequence) were expressed in the yeast Pichia pastoris to confirm their potential chitin deacetylase activity. Numerous constructions were tested. A poly-histidine tag was cloned to facilitate the further purification of the recombinant enzyme. Only the RC sequence showed a high chitin deacetylase activity. Several hypotheses were emitted to explain the low chitin deacetylase activity level measured with the inserts D2 and I3/2. The recombinant RC protein was purified to homogeneity in one step, and partially characterized.

The control of synthesis of the cell-wall polymer chitin has been implicated as a crucial event in yeast - hyphal dimorphism of the pathogenic fungus, <i>Candida albicans</i>. This thesis presents evidence that suggests that the activity of the zymogenic enzyme, chitin synthase, is modulated by an endogenous activatory metalloprotease <i>in vitro</i>. The proposed activating protease is characterised and partially purified. A comparison of the intracellular proteolytic activity of yeast and hyphal cells suggest that hyphal cell may be nutrient-stressed compared to yeast cells.

Thesis (Ph. D.)--University of Washington. / Bibliography: l. 262-284.