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Item When less is more: shortening the Lpp protein leads to increased vancomycin resistance in Escherichia coli.(Springer Nature Limited, 2023-12-01) Wykes H; Le VVH; Olivera C; Rakonjac JVancomycin is a naturally occurring cell-wall-targeting glycopeptide antibiotic. Due to the low potency of this antibiotic against Gram-negative pathogens, such as Escherichia coli, there is a limited knowledge about interactions between vancomycin and this group of bacteria. Here, we show that an in-frame 63 bp deletion of the lpp gene caused a fourfold increase in vancomycin resistance in E. coli. The resulting protein, LppΔ21, is 21 amino acids shorter than the wild-type Lpp, a helical structural lipoprotein that controls the width of the periplasmic space through its length. The mutant remains susceptible to synergistic growth inhibition by combination of furazolidone and vancomycin; with furazolidone decreasing the vancomycin MIC by eightfold. These findings have clinical relevance, given that the vancomycin concentration required to select the lpp mutation is reachable during typical vancomycin oral administration for treating Clostridioides difficile infections. Combination therapy with furazolidone, however, is likely to prevent emergence and outgrowth of the lpp-mutated Gram-negative coliforms, avoiding exacerbation of the patient's condition during the treatment.Item Synthesis of peptides via cobalt (III) chelates of amino acid methyl esters : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Chemistry at Massey University(Massey University, 1979) Knighton, Derek RobinPEPTIDE SYNTHESIS – THE NEED FOR FURTHER METHODS The rapid development of the field of peptide synthesis provides an ever increasing number of techniques available to the peptide chemist (1, 2, 11). Each technique which has been developed is subject to particular advantages and disadvantages. For example, one of the most widely used methods, solid phase peptide synthesis (3) has several associated problems. Uiese are: diketopiperazine formation (4, 5), lability of the ester linkage to the solid phase (6), slower coupling reactions compared with solution phase syntheses (12), the occurrence of deletion peptides which are difficult to separate from the product (7), chain termination caused by steric hindrance from the resin (8) or the conformation of the peptide (9) and the side reactions which occur during the addition of glutamine and asparagine residues when N,N'-dicyclohexylcarbodiimide (DCC) is used as the coupling reagent (10). Since no one technique has overcome all of the problems associated with peptide synthesis there is a need for continued research in this area. The number of amino acids and their diverse properties results in an enormous variety of possible synthetic peptides. With a large variety of techniques at his disposal the peptide chemist is better equipped to solve particular problems associated with the synthesis of these peptides. This thesis describes the effective use of one more synthesis technique. THE STRATEGY USED IN PEPTIDE SYNTHESIS Current methods of peptide synthesis involve the sequential addition of amino acids from the carboxy to the amino terminal. To ensure only the amino group of the growing peptide chain, N₂-Peptide, is available for reaction, the amino group on the amino acid must first be protected (with group X). The carbonyl on the amino acid is then activated (with group Y) and the protected and activated amino acid is added to the growing peptide chain resulting in a specific condensation reaction. The amino protection is removed and the peptide is then free to be used in another cycle as shown in Figure 1.1.[FROM INTRODUCTION]Item An investigation of the preparation of insoluble cellulose sulphates and their use for the isolation of lipoproteins from serum : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Chemistry at Massey University, New Zealand(Massey University, 1976) Sheerin, Brian EdwinThe aim of this thesis was to prepare an insoluble sulphated polysaccharide and explore its potential as a rapid column chromatographic method for the isolation and separation of lipoproteins directly from serum. Regenerated cellulose crosslinked with formaldehyde was found to be relatively inert towards esterification. A degree of sulphation of 0.5 milliequivalents per gram could be obtained but the product had poor hydraulic properties. Epichlorohydrin crosslinked regenerated cellulose was found to be readily sulphated so long as the matrix was highly crosslinked (50-100% v/w). The reactivity of the matrix was retained at low levels of crosslinking if hydroxypropyl groups were introduced with propylene oxide. The hydroxypropylated regenerated celluloses were readily sulphated up to four milliequivalents per gram with minimal degradation of the cellulose chain. HP-cellulose 8-50 sulphates proved to be very successful in the selective isolation of the beta lipoproteins (VLDL plus LDL) directly from serum over a wide range of conditions. The conditions necessary for the preparation of pure alpha lipoprotein (HDL) were found to be more critical. Employing conditions so that all of the alpha lipoprotein (HDL) was sequested completely from serum resulted in it being contaminated by other serum proteins. Under other conditions where the alpha lipoprotein (HDL) was obtained without contamination by other serum proteins, it appeared that a small sub-fraction of the alpha lipoprotein remained in the serum.Item A study of lipoprotein lipase and lipases of bovine milk and lactating mammary gland : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry(Massey University, 1974) Deane, Geoffrey BruceNo abstract.Item A study of phospholipid associating peptides : a thesis presented in partial fulfilment of the requirements for the degree of Ph.D. in Chemistry at Massey University(Massey University, 1983) Knighton, Derek RobinThis thesis describes the solid-phase synthesis of a series of 5 peptides and their subsequent purification by conventional chromatography and semipreparative reversed-phase HPLC. The efficiencies of these 2 methods of purification have been compared. The peptides are: peptide 208, VSSLLSSLKEYWSSLKESFS; peptide 199, RALASSLKEYWSSLKESFS; peptide 202, LESFLKSWLSALEQALKA; peptide 203, LESFKVSWLSALEEYTKA; and peptide 209, LESFLLSWLSAKEQALKA. The peptides were chosen so that each would exhibit a slightly different non-polar face when it adopted an α-helical conformation. Peptides 202 and 209 have exactly the same amino acid composition but differ in that a leucine and a lysine residue have changed positions. This results in the non-polar face of peptide 209 containing one less leucine relative to peptide 202. The retentions of the series of peptides in several reversed-phase HPLC systems were measured by gradient elution. These systems utilised the following solvent system: Solvent A = 1% triethylammonium phosphate, pH 3.2, Solvent B = 80% 2-propanol, 20% solvent A. Radial-PAK CN, Radial-PAK C18 and µBondapak alkylphenyl columns were used. When a linear gradient from 0 to 100% Solvent B was used the retention of the peptides on the Radial-PAK CN column were: peptide 202, 54.75; peptide 208, 51.5; peptide 209, 49; peptide 203, 48; and peptide 199, 44; (expressed as a percentage of the gradient). The isocratic elution of the peptides were studied in the same solvent system on a µBondapak alkylphenyl column by varying the organic solvent content of the mobile phase. The retention of the peptides could not be correlated with the total hydrophobicity of the peptides but could be correlated with the total hydrophobicity of the non-polar side of each peptide when in the α-helical conformation. This result suggests that the peptides adopt an α-helical conformation when binding to the reversed-phase and suggest an adsorption rather than a partitioning mode of binding. The isocratic elution of peptide 202 in the same system was studied at 4 different temperatures. Construction of van't Hoff plots allowed the calculation of the standard enthalpies of association of peptide 202 with the reversed phase. The standard enthalpy of association of peptide 202 at 39% Solvent B was -12 kcal/mol. The affinity of the peptides for dimyristoyl phosphatidylcholine (DMPC) was determined by monitoring turbidity clearance and by determining fluorescence emission wavelength changes of the tryptophan residues upon binding of the peptides to phospholipid vesicles. The peptides affinities for DMPC could be correlated with their retention on the HPLC systems detailed above and with their number of cationic residues. Application of this relationship to the total number of synthesised apolipoprotein fragments allows a very accurate division (92% correct) between those fragments which will and those.which will not bind to phosphatidylcholines. This relationship also appears to be applicable to peptides which are not apolipoprotein in origin and may also be useful in modelling β-endorphin - opiate receptor interactions. The hydrophobic effect is discussed in relation to simple systems and to RP-HPLC and phospholipid binding. The conclusion is drawn that the hydrophobic effect is not always entropy driven.