The present program of research, therefore, was designed to attempt to gain new insights into the possible etiology of inflammatory states - for when new information concerning the etiology of these diseases states becomes available, more meaningful screening procedures for anti-inflammatory drugs will certainly be devised. Two preliminary studies have been published.

No description available.

In the presented studies, chitin synthase containing nanoparticles (chitosomes) from the yeast Saccharomyces cerevisiae lacking the chs3 gene were investigated. Two step centrifugations using sucrose gradients led to considerable purity of the chitosomal complexes. Chitin synthase I activity was determined via a previously described ELISA based WGA assay and a novel assay using the Streptomyces chitin binding protein CHB1, which provided good tools to follow the purification procedure. In collaboration, it could be shown that the complexes produce fibers in the presence of the substrates uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) and N-acetylglucosamine (GlcNAc) and this reaction was inhibited by addition of chitin synthase inhibitor nikkomycin Z. These results demonstrate for the first time that CSI containing chitosomes can be gained. Investigation of the purified nanocomplexes with CSI activity led to the additional conclusion that proteins of the glycolytic pathway such as glyceraldehyde-3-phosphate (GAPDH isoform Tdh3), enolase (Eno1), pyruvate decarboxylase (Pdc1) and pyruvate kinase (Pyk1) are also concentrated around the peak of CSI activity. The presence of these proteins in the pure chitosomes was further verified via testing for their individual enzymatic activities and by antibody studies. The relative levels of GAPDH, Pdc1 and Pyk1 were found to be higher in comparison to enolase; however GAPDH and Pdc1 proteins had a broad distribution across the purification gradient and were also found in neighboring fractions of peak of CSI activity. In addition to these, two high molecular weight proteins showing similarity to glucan synthase and fatty acid synthase were also found in such fractions as analyzed via MALDI-MS. In future it will be worthwhile to ascertain the active functional relationships among the different proteins found in chitosomal preparations using immuno fluorescence co-localization studies.

The overall aim of this study was to provide new and updated information about subterranean termite morphology and digestive physiology. Scanning electron microscopy was used to obtain high resolution images of morphological features of the termite cuticle not discernable by light microscopy. In addition, digital scanning electron micrographs clearly show the appearance of wood particles recovered from the termite digestive tract at different stages of digestion. The ability of termites to obtain and conserve nitrogen in their diet was demonstrated by establishing whether the insect can digest chitin, as well as from determination of levels of soluble proteins and uric acid in the feces. Finally, a technique for determining the efficiency of cellulosic food digestion was tested. Scanning electron microscope images of alate abdomens showed similar appearance of cuticular structures between two subterranean termite species. In addition, the high magnification of electron microscopy allowed for identification of a pair of cuticular structures in the vicinity of the female genitalia that had not been reported from previous light microscope studies. Scanning electron microscopy also revealed the appearance of wood undergoing degradation as it traverses worker termite digestive tract, showing detail not possible from light microscope images. Adult workers of a native termite species were found to produce the enzymes needed to digest chitin, a nitrogen containing polysaccharide ingested by termites. Specific activity of chitinase was subsequently determined to establish the efficiency of chitin digestion for the termite species tested. As termites are coprophagous, significant levels of proteins measured from the feces demonstrated an additional potential dietary nitrogen source. Uric acid was generally found to be absent from termite feces, however. Digestive efficiency of wood cellulose could not be determined for Formosan termites due to the inability to obtain sufficient feces for quantification of undigested food contained in it. Previous studies of Formosan termite digestive efficiency did not address difficulty in obtaining feces for assay. Therefore, interpretation of data from earlier studies of Formosan termite digestive efficiency should be viewed with caution.

No description available.

Studies examining the immune response upon exposure to chitin microparticles in living models have reached drastically differing conclusions, and the reason remains unclear. One notable issue between the experiments is that they have not characterized their chitin preparations for degree of acetylation. They all use different chitin processing methods prior to administration, which could potentially be the source of the variance between studies. Chitin and chitosan preparations specified in the literature and several novel preparations were analyzed for degree acetylation (DA) using High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). It was found that autoclaving and sonication processing steps do not have a significant influence on degree of acetylation. Chitin and chitosan preparations were used to create a dose-response curve of DA compared to cytokine elicitation from THP-1 monocytes, and it was found that the initial response was dominated by TNF (similar to previous studies), though after 12 hours showed a tip toward the start of an IL-1β-dominated Th17 effector response. This study also confirmed that immunostimulatory effects can occur from chitin and chitosan particles at orparticles, which would have long residence times in air, might be implicated in initiating allergic or asthmatic processes.

LysM-domain containing (LysMD) proteins are widespread in nature and associated with host-pathogen interactions, often-binding peptidoglycan and chitin. However, the functions of mammalian LysMD proteins have not been fully defined. Chitin, a major component of fungal cell walls, has been associated with allergic disorders such as asthma. However, chitin recognition by mammals remains enigmatic at best. The principal receptor(s) on epithelial cells for chitin recognition remain to be determined. In this study, we demonstrate that LYSMD3 is expressed on the surface of human airway epithelial cells. Interestingly, LYSMD3 is able to bind chitin and β-glucan as well as fungal spores. Knockdown and knockout of LYSMD3 markedly impaired chitin and fungi-induced inflammatory cytokine production in lung epithelial cells. Antagonization of LYSMD3 ectodomain by soluble LYSMD3 protein, multiple ligands, or antibody against LYSMD3 all significantly blocked chitin signaling. Taken together our study identifies LYSMD3 as a mammalian pattern recognition receptor (PRR) for chitin and is required for the epithelial inflammatory response to chitin and fungal spores. / Doctor of Philosophy / Chitin is the main ingredient in the crustacean shells (e.g. crab, shrimp, lobster). It can also be found in fungal cell walls and insect exoskeletons like house dust mites and cockroaches. Many people are allergic to seafood, fungal spores, house dust mites, and cockroach. These allergies are thought to be driven at least partially by a response to chitin. However, how mammals sense and response to chitin is largely unknown. In plants, LysM-domain (LysMD, chitin binding domain) containing receptors are the primary receptors for chitin. These receptors can bind directly to chitin and/or mediate the innate immune response against chitin-containing pathogens such as fungi. Mammals also have LysMD containing proteins, but the functions of these proteins are unclear. In this study, we demonstrate that human LYSMD3 is a novel receptor for chitin. LYSMD3 is essential for chitin recognition and chitin induced inflammatory responses by airway epithelial cells. Our characterization of LYSMD3 as the elusive human epithelial cell receptor for chitin, resolves a long-standing mystery and provides a new insight into the context of innate immunity in mammals against chitin-containing organisms and allergic inflammation.

No description available.

As the two most abundant natural polymers on earth, cellulose and chitin have attracted increasing attention as a source of renewable energy and functional materials. Thin films of cellulose and chitin are useful for studying interactions of these materials with other natural and synthetic molecules via techniques such as quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR). Because of the difficulty of extracting native cellulose, regenerated cellulose (RC), sulfated nanocrystalline cellulose (SNC), and desulfated nanocrystalline cellulose (DNC) thin films are often studied in its place. In this work, QCM-D solvent exchange studies showed that water contents of RC, SNC and DNC films were proportional to the film thickness (d). Accessibility and degradation of the films was further analyzed via substrate exposure to cellulase. Cellulase adsorption onto RC films was independent of d, whereas cellulase adsorption onto SNC and DNC films increased with d. Enhanced access to guest molecules for SNC and DNC films relative to RC films revealed they are more porous. The porosity of these cellulose films aided in understanding the observed differences of xyloglucan (XG) adsorption onto their surfaces. Xyloglucan adsorption onto RC, SNC, and DNC was studied by QCM-D and SPR. The amount of adsorbed XG increased in the order RC < SNC < DNC. XG adsorption onto RC films was independent of d, whereas XG adsorption was weakly dependent upon d for SNC films and strongly dependent upon d for DNC films. However, XG adsorbed onto "monolayer" thin films of RC, SNC, and DNC in approximately the same amount. These results suggested that the morphology and surface charge of the cellulose substrate had a limited effect upon XG adsorption and that accessible surface area of the cellulose film may be the factor leading to apparent differences in XG adsorption for different surfaces. The porosity and surface charge of SNC films presented a unique opportunity to examine polyelectrolyte adsorption and subsequent dewatering of the SNC substrate. The adsorption of a series of cationically derivatized dextran (cDex) polyelectrolytes with various degrees of substitution (DS) onto SNC was studied using QCM-D and SPR. As the hydrophobic character of the cDex samples increased, the water content of the adsorbed cDex layer decreased. For cDex with the greatest hydrophobic content, nearly 50% by mass of the initial water present in the porous SNC film was removed upon cDex adsorption. This study indicated that the water content of the film could be tailored by controlling the DS and hydrophobic character of the polyelectrolyte. This work also presents the first report of smooth, homogeneous, ultrathin chitin films, opening the door to surface studies of binding interactions, adsorption kinetics, and enzymatic degradation. The chitin films were formed by spincoating trimethylsilyl chitin onto gold or silica substrates, followed by regeneration to a chitin film. The utility of these chitin films as biosensors was evident from QCM-D and SPR studies that revealed bovine serum albumin adsorbed as a monolayer. / Ph. D.

Chitin in shrimp waste is tightly associated with proteins, lipids, pigments and mineral deposits. Therefore, these source materials have to be pretreated to remove these components. For a long time, chemical process has been used widely for extraction of chitin from shrimp waste. The chemical process however led to severe environmental damage and low chitin quality. The biological process has been shown promising to replace the harsh chemical process to reduce the environment impact. In our previous study chitin recovery from sterilized shrimp waste by Lactobacillus plantarum NCDN4 was investigated. However in large scale it is uneconomical to sterilize the shrimp waste. For that reason, in this study the microbial process using Lactobacillus plantarum NCDN4 for chitin recovery from unsterilezed shrimp waste has been investigated. Factors affecting the demineralization by this strain such as inoculum size, glucose concentration, initial pH, NaCl concentration and fermentation time were investigated. It was found that when unsterilized shrimp waste fermented with 20% L. plantarum inoculum, 12,5% glucose, and pH 6 for 4 days at 30oC, 99. 28% emineralization and 48.65% deproteination could be achieved. The ash and protein content of fermented residues were 1.33% and 22.46% respectively. Compared to sterilized condition the efficiency of demineralization and deproteination was similar. / Chitin trong phế liệu tôm liên kết chặt chẽ với protein, sắc tố và khoáng. Do vậy để thu được chitin cần có các bước tiền xử lí để loại các thành phần không phải chitin ra. Phương pháp hóa học được sử dụng rộng rãi từ lâu để tiền xử lí chitin. Tuy nhiên do phương pháp hóa học gây hại cho môi trường và tạo ra chitin chất lượng thấp, các nhà khoa học nỗ lực nghiên cứu tìm ra các phương pháp thay thế. Phương pháp sinh học được xem là rất khả quan để thay thế phương pháp hóa học. Trong nghiên cứu trước của chúng tôi, quá trình lên men phế liệu tôm thanh trùng bằng Lactobacillus plantarum NCDN4 đã được nghiên cứu. Tuy nhiên việc thanh trùng phế liệu tôm không kinh tế. Trong nghiên cứu này quá trình lên men phế liệu tôm không thanh trùng bằng Lactobacillus plantarum NCDN4 đã được khảo sát. Các yếu tố như tỷ lệ giống, nồng độ đường glucose, nồng độ NaCl, pH ban đầu của môi trường lên men và thời gian lên men đã được nghiên cứu. Kết quả cho thấy ở điều kiện 20% giống theo thể tích, 12,5% dịch đường glucose, 2% muối theo khối lượng, pH ban đầu 6, sau 5 ngày lên men lượng khoáng và protein trong nguyên liệu giảm tương ứng 99.28% và 48.65%. Lượng khoáng và protein còn lại tương ứng 1.33% và 22.46% (tính theo trọng lượng khô). So với phế liệu tôm không thanh trùng, hiệu quả loại khoáng và protein là tương đương.