The flavoprotein spermine oxidase catalyzes the oxidation of spermine and oxygen to spermidine, 3-aminopropanol, and hydrogen peroxide. To allow mechanistic studies of the enzyme, methods have been developed to obtain large amounts of purified recombinant protein. The enzyme requires co-expression with chaperone proteins GroEL and GroES to remain soluble and active. Purification requires the use of a Ni-NTA and size exclusion column. Human spermine oxidase is a monomer with an extinction coefficient of 14000 M-1cm-1. The kinetic mechanism is ping pong. Therefore, oxygen is bound to the enzyme before spermidine is released. N1-Acetyl spermine is a slow substrate with kcat and kcat/Km values 2 and 3 orders of magnitude smaller than the values for spermine. Spermidine is a competitive inhibitor, and 1,8-diaminooctane (DAO) is an uncompetitive inhibitor. The pH effects indicate that two ionizable groups are present in the kcat/Km profile and one ionizable group is in the kcat profile. The reductive half reaction reveals no phase other than the reduction of the FAD, indicating the probability of a single chemical step. Reduction is not limiting to the overall reaction. Isotope effects were determined; Dkcat at pH 7.5 = 4.1±0.4, pH 8.5 = 2.6±0.01.

The flavoprotein spermine oxidase catalyzes the oxidation of spermine and oxygen to spermidine, 3-aminopropanol, and hydrogen peroxide. To allow mechanistic studies of the enzyme, methods have been developed to obtain large amounts of purified recombinant protein. The enzyme requires co-expression with chaperone proteins GroEL and GroES to remain soluble and active. Purification requires the use of a Ni-NTA and size exclusion column. Human spermine oxidase is a monomer with an extinction coefficient of 14000 M-1cm-1. The kinetic mechanism is ping pong. Therefore, oxygen is bound to the enzyme before spermidine is released. N1-Acetyl spermine is a slow substrate with kcat and kcat/Km values 2 and 3 orders of magnitude smaller than the values for spermine. Spermidine is a competitive inhibitor, and 1,8-diaminooctane (DAO) is an uncompetitive inhibitor. The pH effects indicate that two ionizable groups are present in the kcat/Km profile and one ionizable group is in the kcat profile. The reductive half reaction reveals no phase other than the reduction of the FAD, indicating the probability of a single chemical step. Reduction is not limiting to the overall reaction. Isotope effects were determined; Dkcat at pH 7.5 = 4.1±0.4, pH 8.5 = 2.6±0.01.

No description available.

The aims of this thesis are to design and synthesize non-viral cationic lipid vectors based on spermine, for the intracellular delivery of siRNA (short interfering RNA) and the subsequent siRNA mediated gene silencing. Two parameters were varied: the type of fatty acid and the cationic head-group. Among the symmetrical spermine conjugates, N4,N9-dierucoyl spermine (DES) resulted in higher siRNA delivery compared to N4,N9-dioleoyl spermine (DOS), while enhanced green fluorescent protein (EGFP) silencing in HeLa cells showed that the unsaturated fatty acid conjugates are more efficient than the saturated fatty acid ones, and cell viability was 75%-85% for conjugates with chain length ≥ 18. Two cationic lipids with guanidine head-groups, N1,N12-diamidino-N4,N9-dioleoylspermine and N1,N12-diamidino-N4-linoleoyl-N9-oleoylspermine, were more efficient in EGFP gene silencing compared to cationic lipids with shorter C12 (lauroyl) and very long C22 (erucoyl) chains, with cell viability (64%-83% for chain length ≥ 18). Changing the cationic headgroup to guanidine did not offer a significant advantage in gene silencing over the conjugates with terminal primary amine groups. The asymmetrical N4-linoleoyl-N9-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS) resulted in the best gene silencing, while LigOS (with one lignoceroyl 24:0 chain) resulted in the best siRNA delivery. Conjugates with two unsaturated fatty chains generally resulted in better EGFP gene silencing, while conjugates with one saturated chain and one unsaturated chain resulted in better siRNA delivery. Increasing the chain length also resulted in increased siRNA delivery (cell viabilities of asymmetrical > 74%, LinOS 88%). siRNA lipoplexes prepared using mixtures of LinOS with either cholesterol or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) resulted in increased siRNA delivery, and enhanced EGFP silencing, with LinOS/Chol mixture (1:2 molar ratio) resulting in the highest siRNA delivery and the best gene silencing (EGFP reduced to 20%). Temperature studies of intracellular entry showed that the majority of lipoplexes are internalized by endocytosis, however the majority of gene-silencing occurs due to lipoplexes internalized via another mechanism.

No description available.

Spermine (Spm), a potent bactericidal polyamine, exerts a strong synergistic effect with β-lactams against methicillin-resistant Staphylococcus aureus (MRSA) in a pH-dependent manner. At high pH (>8) Spm is a potent nucleophile, and able to form Spm-β-lactam adduct. At physiological pH (or lower), Spm carries positive charges, and can bind to DNA through charge interactions. The potential of Spm interfering with cell wall was first investigated. A spontaneous mutant of MRSA Mu50 selected for Spm resistance conferred resistance to Spm/β-lactam synergy. This phenotype was due to the presence of a 7-bp deletion in pbpB as identified by genome resequencing and confirmed by complementation. Analysis of cell wall composition by HPLC revealed the combination of Spm and β-lactam can reduce the cross-linkage of peptidoglycan. These two lines of evidence suggest Spm may perturb cell wall integrity in favor of β-lactam efficacy with PBPs as a promising target. However, from the results of microarray analysis and fluorescent Bocillin labeling, Spm did not appear to suppress the PBPs expression or alter their interactions with β-lactams. Next, transcriptome analyses reveal the genes responsive to the synergy effect overlap extensively with those to high Spm challenge, implying the enhanced detrimental effect of Spm facilitated by β-lactams in inhibition on cell growth. In particular the induction of iron transport and reduction of energy production under synergy were depicted in this study, and high dose Spm was found to turn off the SigB regulon. Of interest, the tetM gene encoding a ribosomal protection protein for tetracycline (Tc) resistance exhibited the most significant fold change and high signals by both high and low dose Spm. Further analysis by qRT-PCR demonstrated the tetM expression was specifically induced by Tc and Spm to a comparable level but not by other polyamines, suggesting a similar mode of action by Spm and Tc in interactions with the ribosome to initiate tetM induction. Collectively, these data indicated the role of Spm could be multifarious with more than one target, and a combination of Spm and β-lactams may inhibit growth of MRSA in a more complicated manner than just potentiating β-lactam inhibition on PBPs.

Prostate cancer is the second leading cancer in men. A large amount of polyamines are synthesised in the human prostate and are involved in prostate cell growth and its physiological functions. The content of intracellular polyamines is closely related to cell growth. An increase in cell growth is accompanied by a rise of intracellular polyamine content, and a depletion of intracellular polyamine pools can cause growth arrest or cell death. Therefore, maintaining polyamine concentrations is critical to the cell. Spermidine/spermine N1-acetyltransferase (SSAT) is the first and rate-limiting enzyme in the polyamine catabolic pathway. SSAT gene is highly inducible, with many stimuli including polyamine analogues and some anticancer drugs producing dramatic increases in activity. Many studies have focussed on polyamine analogues as inducers of SSAT activity as increases in SSAT are associated with a growth inhibition in many tumour cells. However, the mechanisms of this inhibition are not fully understood with respect to polyamine content. Additionally, in vivo results in SSAT transgenic mice studies are contradictory. For example, prostate carcinogenesis is reduced in TRAMP mice but Apcmin/+ mice show a promoted intestinal tumorigenesis. It is thus necessary to characterise the regulation of polyamine content and metabolism by SSAT in prostate cancer cells. The aim of the present study was to characterise the role of SSAT in both the growth of LNCaP prostate carcinoma cells and the response of these cells to anticancer drugs. Our hypothesis is that increased SSAT activity will inhibit cell growth and that this is associated with a decrease of intracellular polyamine pools. Furthermore, if SSAT induction is an essential part of the response of cancer cells to anticancer drugs, then altered SSAT activity should affect sensitivity of the cells to the drugs. The present study used a cell culture model of human prostate cancer: LNCaP wild type (WT) and SSAT cDNA transfected prostate carcinoma cell lines. The expression of SSAT in the transfected cell line (SSAT- & SSAT+) was controlled through the “Tet-off” system. This model system provided a background for comparison of effects under basal (WT), low (SSAT-), and high (SSAT+) SSAT activity. Due to our interest in acetylpolyamine derivatives and their low concentrations in cells, a new method for quantifying polyamine concentrations was developed using liquid chromatography-mass spectrometry (LC-MS). This method was highly sensitive and can detect polyamines about 250 fold lower than HPLC, as well as N-acetylpolyamines and N1,N12-diacetylspermine. In addition, a variety of methods were utilised to measure cell growth, enzyme activity, protein expression, polyamine efflux and apoptosis, which includes enzyme assays, western blot, radiochemical labelled assays, flow cytometry, spectrophotometry and fluorescent microscopy. A stable increase in SSAT activity was inhibitory to the cell growth. This inhibition was associated with significant changes in the activity of the polyamine pathway. The alterations included an increase in ODC, APAO, and SMO activity; an accumulation of intracellular N1-acetylspermidine and putrescine; a decrease in intracellular spermidine and spermine; an increased polyamine flux and efflux; and an increase in apoptosis. Combination treatment to the cells with DFMO and MDL72527 partially restored the growth of SSAT+ cells. The original contribution of this study to the field is that the cells with a higher SSAT activity are less sensitive to aspirin and 5-FU, and the sensitivity increased while the overexpressed SSAT activity decreased. The growth inhibition was associated with a depletion of total intracellular polyamine pools by the drug treatments. Moreover, to our knowledge, it is first time that the extracellular polyamine concentrations were quantified by LC-MS in human tumour cells. Overall, an increase in SSAT activity led to an inhibition of prostate cancer cell growth, and vice versa. Thereby, this study suggests that SSAT is a potential target for novel drug discovery for cancer chemotherapy or chemoprevention. For example, a combination treatment could be designed that acts as an inducer of SSAT activity in tumour cells, leading to an inhibition of the cell growth in the first place and increased sensitivity to cytotoxic agents. This would then be followed by an agent to decrease SSAT activity when the sensitivity of cancer cells to the cytotoxic treatment was optimal.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (p. [117]-123).

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Thesis (doctoral)--Utrecht, 1984.