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Isotopic enrichment and receptor-binding analysis of insect and Lec1 cell expressed Avian Thy-1Walton, Wendy Jean. Logan, Timothy M. January 2006 (has links)
Thesis (Ph. D.)--Florida State University, 2006. / Advisor: Timothy M. Logan, Florida State University, College of Arts and Sciences, Program in Molecular Biophysics. Title and description from dissertation home page (viewed June 7, 2006). Document formatted into pages; contains xvi, 97 pages. Includes bibliographical references.
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Chemical and spectroscopic studies of the capsular polysaccharides of some Klebsiella serotypesRavenscroft, Neil January 1988 (has links)
Includes bibliographical references. / As part of an international collaborative programme concerned with the elucidation of the molecular structures of capsular polysaccharides (the K-antigen) produced by strains of the bacterial genus Klebsiella, the capsular material of serotype K71 has been investigated, and that of serotypes K36 and K64 re-examined, by novel enzymic and spectroscopic methods. The cultivation and employment of bacteriophages which are capable of cleaving (by specific glycanase action) the isolated, cognate bacterial polysaccharide in vitro has yielded highly significant oligosaccharides. These may represent the repeating unit in the polysaccharide or be derivatives resulting from conversion of uronic acid to the 4,5-unsaturated analogue where, as found for serotype K64, the mode of cleavage is β-elimination not hydrolysis. The oligosaccharides thus generated have proved to be far more amenable to chemical and spectroscopic studies than their parent polymers, thereby facilitating complete characterisation of the molecular structures of the original polysaccharides. Chemical methods applied to these oligosaccharides included specific degradations by periodate oxidation and acid-, alkali- or enzyme- catalysed hydrolysis, products being identified by methylation analysis (involving the extensive use of gas-liquid chromatography coupled to mass spectrometry) and spectroscopic studies (mass and n.m.r.).
During the course of these investigations it became apparent that the structures of the intact oligosaccharides (containing six or seven sugar residues) could be determined almost entirely from spectroscopic analysis, chiefly by detailed two-dimensional n.m.r. studies involving the use of high field spectrometers and the application of homo- and heteronuclear shift correlated spectroscopy, the sequence of sugar units being confirmed by mass spectrometric analysis of the permethylated derivatives. Methylation analysis of the oligosaccharides derived from Klebsiella serotype K36 proved that the glucuronic acid residue is linked through 0-2, and not 0-4 as published by others; this finding was corroborated during characterisation of the monomeric oligosaccharide by mass- and n.m.r. spectroscopy. Bacteriophage φ64 was shown to cleave the cognate K64 exopolysaccharide by a β-elimination process; the resulting hex-4-enuronic acid, present as a terminal group in the derived oligosaccharide was fully characterised by hydrogenation and g.l.c.-m.s. of acetylated products, and by detailed n.m.r. studies including long-range heteronuclear experiments. Finally the structure of the heptasaccharide repeating unit of the Klebsiella K71 capsular polysaccharide was established by spectroscopic analysis of the oligosaccharides derived by bacteriophage φ71 cleavage of the polymer; features of the proposed structure were confirmed by chemical degradation studies performed on the native polysaccharide.
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Xylo-Oligosaccharides Production from Corn Fiber and In-Vitro Evaluation for Prebiotic EffectSamala, Aditya 14 December 2013 (has links)
Xylooligosaccharides (XOS) are considered to be prebiotics. Prebiotics are defined as non-digestible food ingredients that benefit the host by stimulating the growth and activity of a limited number of bacteria, such as the Bifidobacterium genus, in the colon. Corn fiber separated from distillers dried grains with solubles (DDGS) could be a valuable feedstock for XOS production. The objective of the first chapter was to determine the efficacy for autohydrolysis to produce XOS using fiber separated from DDGS. Fiber was treated with deionized water in a Parr-reactor, at temperatures ranging from 140 to 220 °C to produce XOS. The maximum total yield of XOS in the solution was 18.6 wt% of the corn fiber at 180 °C. The objective of the second chapter was to evaluate and compare the prebiotic effect of XOS produced by autohydrolysis of DDGS fiber (XOS-D) with other substrates (FOS, commercial XOS (XOS-C), xylose, glucose and inulin) on intestinal bacteria, B. adolescentis, B. breve and Lactobacillus brevis. Bacterial growth on XOS-C was comparable with growth on FOS and inulin. XOS-D promoted bacterial growth more than that of control. Prebiotic potential of XOS produced from corn fiber was confirmed. The objective of third chapter is to determine the yield of XOS from corn fiber separated from ground corn flour (FC) and DDGS (FD) at different autohydrolysis temperatures and hold-times. The conditions for maximum XOS production for FD and FC were 180 °C with 20 min hold-time and 190 °C with 10 min hold-time, respectively. The fourth chapter focuses on production of XOS by enzymatic hydrolysis method for XOS production. Endo-1-4-xylanase enzyme was ineffective for corn fiber as well as corn fiber gum (CFG), despite evaluating a multitude of pretreatment methods and processing conditions. We have proposed use of Multifect Pectinase PE and Multifect Xylanase enzymes, based on work from other researchers. For commercial applications such as food industries, XOS would need to be isolated from liquor. The fifth chapter of this study focuses on literature review of purification methods used in XOS purification.
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Synthesis, Conformational Analysis And Biophysical Studies Of Oligoarabinan And Oligoarabinomannan GlcolipidsNaresh, Kottari 03 1900 (has links) (PDF)
Mycobacterial infection is a major health concern. High drug resistivity of the mycobacterium is due to its multi-layered, thick hydrophobic waxy cell wall components, consisting of cross-linked peptidoglycan (PG), mycolyl arabinogalactan (mAG) and lipoarabinomannan (LAM) polysaccharides. These polysaccharides are composed of arabinose and galactose in the furanose form and mannose in the pyranose form. The high waxy hydrophobic components of the mycobacterial cell wall acts as a barrier for most hydrophilic antibacterial agents. Enzymes responsible for the biosynthesis of polysaccharides of mAG and LAM are arabinosyl transferase (AraT), galactosyl transferase (GlfT) and mannosyl transferase (ManT). In the absence of furanoside derivatives of D-arabinose and D-galactose in mammalian systems, inhibitors based on these sugars arise an interest. Upon realizing structural characteristics of cell wall polysaccharides, the chemical syntheses of such polysaccharides were reported. Biological studies of synthetic arabinomannan and arabinogalactan oligosaccharides were performed, in order to identify their effects in enzymatic, as well as, mycobacterial growth assays. Chapter 1 of the thesis describes the structural features of mAG and LAM polysaccharides. Chemical synthesis of oligosaccharides related to mycobacterial cell wall components and their effects of mycobacterial growth and enzymatic assays are discussed.
In my research program, synthesis and studies of oligosaccharides pertaining to mycobacterial cell wall components were undertaken. Monovalent and bivalent glycolipids 1 and 2 (Figure 1), containing arabinofuranoside trisaccharide as the sugar head group, were synthesized and their effects on the growth of M. smegmatis strain were studied. In the presence of arabinan glycolipids, retardation of the growth of M. smegmatis was observed and the inhibitory activity was found to be specific with glycolipids containing arabinofuranoside head groups. Glycolipid with maltosyl sugar and arabinofuranoside trisaccharide without lipid chains, did not affect the mycobacterial growth. Continuing the effort, tri- and tetrasaccharide of arabinomannan glycolipids were synthesized and their effects in the mycobacterial growth were studied. It was found that 3 was inhibiting the growth of the mycobacterium, whereas in the case of 4, inhibition was found to be less when compared to 3. Relative inhibitions of mycobacterial growth by synthetic glycolipids 1-4, at a concentration 200 µg/mL, were found to be in a varying degrees, ranging from 16 % in the case of 4 and 65 % in the case of 3.
Figure 1. Molecular structures of arabinan and arabinomannan oligosaccharides 1-7.
Following mycobacterial growth inhibition studies, surface plasmon resonance studies of synthetic oligosaccharides were performed, in order to identify their interactions with mycobacterial cell lysates. Amine tethered glycosides 5-7 (Figure 1) were synthesized and immobilized onto SPR sensor chip through amine coupling methodology. From SPR studies, it was found that the binding affinity was higher with cell lysates from motile strains than non-motile strain. Among various arabinomannans, glycoside 5, presenting two mannose units showed higher affinity than 6 and 7, having no or one mannose unit, respectively. Chapter 2 of the thesis provides details of synthesis, biological and biophysical studies of arabinan and arabinomannan glycolipids.
Continuing the synthesis and studies with arabinose oligosaccharides, a linear tetra-, hexa and octasaccharide glycolipids, containing α-(1→5) linkages (10-12), as well as, a branched heptasaccharide containing α-(1→2) and α-(1→5) linkages (14) between the arabinofuranoside units (Figure 2) were synthesized. In addition to glycolipids, oligosaccharides without alkyl chains (8, 9 and 13) were also prepared. Synthesis was performed using trichloroacetimidate and
Figure 2. Molecular structures of linear and branched arabinan derivatives 8-14.
thioglycosides as glycosyl donors. Synthesis of linear oligosaccharide derivatives 8-12 was achieved by iterative glycosylation and deprotection strategies. Branched heptasaccharide derivatives 13 and 14 were synthesized by using block glycosylation method, wherein two fold excess of arabinose disaccharide was reacted with a suitably protected arabinose trisaccharide. Upon synthesis, molecular modeling studies were performed to identify the conformational behavior of arabinan glycolipids. Conformational studies were performed in three steps, namely, (i) dihedral scan (ii) conformational search and (iii) molecular dynamics. Dihedral scan was performed to assess favorable torsion angles at each glycosidic linkage with respect to overall conformation of the molecule. Monte-Carlo conformational search was performed to obtain the lowest energy structure of arabinan glycolipids. Relative orientations of lipidic portions and sugar portions were identified for linear and branched arabinan glycolipids. The least energy conformations of 10, 11, 12 and 14 are shown in Figure 3. In the case of linear molecules 10, 11 and 12, alkyl chains and arabinofuranoside portion did not phase segregate, whereas in the case of branched glycolipid 14, the alkyl chains were observed to move away from the sugar moieties. Molecular dynamic calculations were performed for the lowest energy structure, in order to evaluate the torsion angles in the trajectory.
Following the synthesis and conformational analysis of the arabinan glycolipids, surface plasmon resonance studies were performed to assess their interactions with a host protein, namely, pulmonary surfactant protein-A (SP-A). For the interaction studies, SP-A was immobilized on to the CM-5 sensor chip using amine coupling method. Varying concentrations of arabinan glycolipids 10, 11, 12 and 14 and oligosaccharides 8, 9 and 13 were used as analytes. Responses from the surface of SP-A were subtracted from that of ethanolamine to eliminate the non-specific interactions. Primary sensorgrams were fitted using 1:1 Langmuir model to obtain the kinetic parameters of the interactions. Specificities and relative binding affinities of arabinan oligosaccharides interacting with SP-A are presented in Table 1. The affinities between
Figure 3. Lowest energy structures of glycolipids 10, 11, 12 and 14 derived from molecular modeling studies.
arabinan oligosaccharides and SP-A were found in the range of 4.9-47x103 M-1. Among the series, branched arabinan oligosaccharides 13 and 14 showed higher Ka values than the linear arabinan glycolipids. The association rate constants (kon) were generally higher for the oligosaccharides without lipidic chain, whereas, the dissociation rate constants (koff) were slower with oligosaccharides having lipidic chains. Faster kon was also associated with a faster koff for oligosaccharides without the lipidic chains. For the glycolipids, a relatively slower koff was found to be the trend. In the case of branched heptasaccharide derivatives, glycolipid 14 showed higher binding constant than heptasaccharide with a thiocresyl group at the reducing end 13. Chapter 3 of the thesis presents the synthesis, conformational analysis and SPR studies of linear and branched arabinan glycolipids.
Table 1. Kinetic parameters of the interactions between arabinose derivatives 8-14 and SP-A.
Compound kon (M-1s-1) kd (s-1) (104) Ka (M-1) (10-3) χ2
12 3.9 7.91 4.9 8.3
11 1.5 3.98 3.77 2.9
10 0.384 0.22 17.5 6.7
14 27.3 5.79 47.2 4.5
8 11.3 6.14 18.4 2.3
9 23.3 11.6 20.1 2.4
13 53.6 17.9 29.9 5.4
Upon assessing the biophysical studies of the α-arabinofuranoside glycolipids, an effort was undertaken to prepare glycolipids containing β-arabinofuranoside linkages and to study their conformational and biophysical properties. Arabinan glycolipids 15 and 16 (Figure 4), containing β-(1→2), β-(1→3) and β-(1→5) linkages between furanoside units were synthesized to compare the properties with the corresponding synthetic α-arabinan glycolipids. Incorporation of β-arabinofuranoside linkages in 15 and 16 was achieved using low temperature activation of silyl substituted glycosyl donor 17 (Figure 4), with NIS and AgOTf. The configurations in 15 and 16 were confirmed through 1H-1H COSY, 1H-13C HMQC NMR techniques. During the synthesis of 15 and 16, stereoselective incorporation of two β-Araf linkages on a single furanoside unit was achieved for the first time. Conformational studies of 15 and 16 were conducted similar to α-arabinan glycolipids, as above, to identify most favorable conformations of inter-ring, as well as, overall conformation of the molecule. The interactions between the SP-A and β-arabinofuranoside glycolipids 15 and 16 were also assessed with the aid of SPR technique. The analysis showed that the affinities of glycolipids 15 and 16 to SP-A were found to be relatively lower when compared to α-arabinofuranoside glycolipids. Synthesis and studies of β-arabinofuranoside glycolipids are described in chapter 4 of the thesis.
Figure 4. Molecular structures of β-arabinofuranoside glycolipids 15 and 16.
In summary, the present thesis describes synthesis, conformational and biophysical studies of synthetic arabinan and arabinomannan glycolipids. Monovalent and bivalent arabinan, tri- and tetrasaccharide arabinomannan glycolipids were synthesized and their effects in the mycobacterial growth were studied. It was found that arabinan and arabinomannan glycolipids inhibited the growth of the mycobacterium. The inhibitory activity is specific with the arabinan and arabinomannan glycolipids and the glycolipids with higher arabinose composition were found to be better inhibitors for mycobacterial growth. The interactions of mycobacterial cell lysates with arabinomannan compounds were evaluated through SPR technique. Linear tetra-, hexa-, octa- and branched heptasaccahride arabinan glycolipids containing α-Araf linkages between furanoside units were synthesized. Molecular modeling studies of arabinan glycolipids were performed, in order to identify their lowest energy conformations. Biophysical studies of linear and branched arabinan glycolipids were conducted to assess their interactions with pulmonary surfactant protein-A (SP-A) through surface plasmon resonance technique. Syntheses, conformational and biophysical studies were extended further to β-arabinofuranoside glycolipids. Overall, the thesis provides synthesis, conformational, biological and biophysical studies of a series of lipoarabinomannan oligosaccharides. The results provide a possibility to evolve newer types of glycolipids that can act as inhibitors of mycobacterial growth.
(For structural formula pl see the hard copy)
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Separation and detection of cellooligosaccharides on cellulose thin-layer chromatographySookavatana, Narumon 11 June 2001 (has links)
Linear oligosacchardies of 1,4 linked β-D-glucopyranose are commonly
referred to as cellodextrins (CD) or cellooligosaccharides (CO). They are of interest
to those working in disciplines involving cellulose chemistry because they are often
used as model substrates for cellulose itself. They are of interest to food scientists
and nutritionists because they are easily incorporated into foods as non-digestible
oligosaccharides, a category of food ingredients that is thought to be beneficial lo
human health. The intent of the research presented in this thesis was to evaluate the
potential of using cellulose supports for the chromatographic separation of soluble
CDs differing in their degree of polymerization (DP; a numerical value indicating
the number of glucose substituents per molecule). Soluble CDs range in DP from 2
to 8. Thin layer chromatography (TLC), using cellulose-coated TLC plates, was
used as a model chromatographic system.
Mixed CD preparations, containing CDs ranging in DP from 2 to 8 were
prepared by incomplete acid-catalyzed hydrolysis of cellulose. The DP profiles of
the different CD preparations were qualitatively demonstrated by TLC using silica-coated
plates, an organic solvent-based mobile phase, and a standard carbohydrate
visualizing reagent (p-anisaldehye in sulfuric acid). CD-preparations were then
chromatographed on cellulose-coated TLC plates. Visualization of the
chromatographed CDs was accomplished using a silver nitrate-sodium hydroxide
reagent system, a reducing-sugar visualizing reagent. The silver nitrate-sodium
hydroxide system was found to be the most appropriate, based on detection limits,
simplicity and safety, of the several visualization reagents tested.
Eight different mobile phases, all aqueous-based, were tested as potential
solvents for the resolution of CDs, differing in DP, on cellulose-coated tlc plates at
room temperature. The optimum solvent was found to be 60% ethanol/40% water.
This solvent clearly resolved CDs of DP 3, 4 and 5. CD preparations
chromatographed with the same mobile phase, but with silica-coated TLC plates,
were not resolved. These combined results suggest that the TLC system with the
cellulose stationary phase behaves similar to an affinity system, since silica and
cellulose are both relatively hydrophilic stationary phases (i.e. both systems are
typically considered examples of normal phase adsorption chromatography). The
results further illustrate that cellulose supports have potential for use in the
preparation of CDs of defined DP. / Graduation date: 2002
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Human conjunctival mucinsEllingham, Roger Bruce January 2000 (has links)
No description available.
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Studies on the expression, purification, and synthetic utility of recombinant yeast #beta#-1,4-mannosyltransferaseRevers, Leigh January 1996 (has links)
No description available.
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Evaluation of sample preparation techniques for MALDI-TOF-MS analysis of oligosaccharidesLiti, Samone January 2016 (has links)
The aim of this study was to optimize the sample preparation and methods for analysis of oligosaccharides of hyaluronic acid with MALDI- TOF-MS. The analysis was carried out on an Autoflex speed MADLI-TOF- MS instrument with both linear and reflectron mode. Matrices used in this study were 2,5-DHB and Super-DHB and type of matrix was chosen depending on the size of the analyzed oligosaccharides. The application of sample and matrix on the target that gave the most homogenous crystallization was sandwich and the laser power in the MALDI was kept at 65 %. Since it is known that salts and buffers interfere with the analysis, sample clean-up such as solid phase extraction (SPE) in pipette tips and dialysis was performed. SPE worked best for low mass oligosaccharides and provided high intensity and little noise. With SPE a concentration of the analyte could be done which was the advantage over dialysis. Dialysis worked well for larger oligosaccharides and mixtures of different sized oligosaccharides. Another way of using MALDI for biomolecule analysis is with TLC-MALDI. A fast and accurate separation was achieved and analysis could be done directly from the plate. The optimized methods were evaluated according to linearity and precision, LOD, mass accuracy and matrix stability. The linearity and precision was good in a higher concentration range (50 μg/mL and higher), but the test for limit-of-detection (LOD) indicated that concentrations from 20-30 μg/mL could be analyzed with no interference from the background. The mass accuracy was within the acceptable limits according to Bruker Daltonics when a mass calibration was done for each analyzed sample. The stability of the matrix in solution was difficult to study because of the day-to-day variation in intensities given by the MALDI-TOF-MS technique.
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Molecular structure of exudate gums with special reference to gums of the Sterculia genusSanderson, George R. January 1981 (has links)
The term 'gum', in its broadest sense, refers to both hydrophobic and hydrophilic substances of high molecular weight which usually exhibit colloidal properties when dispersed in an appropriate solvent. Hydrophobic substances often called gums include high molecular weight hydrocarbons and other petroleum products, rubbers, certain synthetic polymers and resinous saps which often exude from evergreens. More specifically, the term gum applies to plant polysaccharides or their derivatives which are dispersible in either cold or hot water to produce viscous solutions or suspensions. As much as three-quarters of the dry weight of plants may be polysaccharide and, consequently, such substances are of wide occurrence. The most important gums, however, are those which are readily obtainable in large amounts from the plant. Some of these gums are used industrially and, indeed, many have been known since ancient times. One of the chief sources of such polysaccharides is seaweed which furnishes agar, algin and carrageenin while seed gums, such as gum guar and locust bean gum, are also important, particularly from the point of view that the plant which produces the seeds is often grown extensively as a food crop. In contrast to these naturally occurring gums, other gums are obtained from cellulose, one of the main components of the plant cell wall, and starch, a food reserve polysaccharide, by esterification and etherification. Commercially, however, the most important gums are plant exudates and most plant families have been found to include species which exude gums to a greater or lesser degree. In this context, the term 'exudate gum' strictly refers to those commercially important gums which exude in copious amounts from shrubs or low-growing trees, forming, on exposure to the atmosphere, glossy nodules or flakes which are usually brown or yellow in colour. These gum producing trees grow predominantly in Africa or Asia indicating the climatic requirements for their growth.
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Glycoprocessing in classical galactosaemia / Barry Denison Lewis.Lewis, Barry Denison. January 1997 (has links)
Addendum pasted inside the back end-paper. / x, 179 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis presents a hypothesis that there are abnormalities of N-glycosylation in classical galactosaemia and that these abnormalities could contribute to the long-term complications. The aim of the thesis is to characterise and model N-glycosylation in skin fibroblasts from patients with galactosaemia. The study identifies a disturbance in the synthesis and processing of dolichol-linked oligosaccharides. It is anticipated that the serum glycoproteins in untreated galactosaemia may contain N-glycans that are partly absent or truncated. / Thesis (M.D.)--University of Adelaide, Dept. of Paediatrics, 1997
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