Photodimerization and photocycloaddition of 2-cyclohexenone in confined space of cation-exchanged Y zeolites

Zeolite Y was used in this research. The structure of FAU zeolite is cubic and built from sodalite cage connected via the double 6-membered ring. It forms a three-dimensional network of nearly spherical supercages of about 1.3 nm in diameter connected tetrahedrally to four other supercages through 0.74 nm windows. The charge-compensating cations are mobile and distributed among several types of sites

Preconditioning of Cardiosphere-Derived Cells With Hypoxia or Prolyl-4-Hydroxylase Inhibitors Increases Stemness and Decreases Reliance on Oxidative Metabolism

Cardiosphere-derived cells (CDCs), which can be isolated from heart explants, are a promising candidate cell source for infarcted myocardium regeneration. However, current protocols used to expand CDCs require at least 1 month in vitro to obtain sufficient cells for transplantation. We report that CDC culture can be optimized by preconditioning the cells under hypoxia (2% oxygen), which may reflect the physiological oxygen level of the stem cell niche. Under hypoxia, the CDC proliferation rate increased by 1.4-fold, generating 6 × 10(6) CDCs with higher expression of cardiac stem cell and pluripotency gene markers compared to normoxia. Furthermore, telomerase (TERT), cytokines/ligands involved in stem cell trafficking (SDF/CXCR-4), erythropoiesis (EPO), and angiogenesis (VEGF) were increased under hypoxia. Hypoxic preconditioning was mimicked by treatment with two types of hypoxia-inducible factor (HIF) prolyl-4-hydroxylase inhibitors (PHDIs): dimethyloxaloylglycine (DMOG) and 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetic acid (BIC). Despite the difference in specificity, both PHDIs significantly increased c-Kit expression and activated HIF, EPO, and CXCR-4. Furthermore, treatment with PHDIs for 24 h increased cell proliferation. Notably, all hypoxic and PHDI-preconditioned CDCs had decreased oxygen consumption and increased glycolytic metabolism. In conclusion, cells cultured under hypoxia could have potentially enhanced therapeutic potential, which can be mimicked, in part, by PHDIs

On the pivotal role of PPARa in adaptation of the heart to hypoxia and why fat in the diet increases hypoxic injury

The role of peroxisome proliferator activated alpha (PPARα) -mediated metabolic remodeling in cardiac adaptation to hypoxia has yet to be defined. Here, mice were housed in hypoxia for 3 weeks before in vivo contractile function was measured using cine magnetic resonance (MR) imaging. In isolated, perfused hearts, energetics were measured using 31P MR spectroscopy and glycolysis and fatty acid oxidation were measured using 3H labelling. Compared with normoxic, chow-fed control mouse heart, hypoxia decreased PPARα expression, fatty acid oxidation and mitochondrial UCP3 levels, while increasing glycolysis, all of which served to maintain normal ATP concentrations and thereby ejection fractions. A high-fat diet increased cardiac PPARα expression, fatty acid oxidation and UCP3 levels, with decreased glycolysis. Hypoxia was unable to alter the high PPARα expression or reverse the metabolic changes caused by the high fat diet, with the result that ATP concentrations and contractile function decreased significantly. The adaptive metabolic changes caused by hypoxia in control mouse hearts were found to have already occurred in PPARα-/- mouse hearts, and sustained function in hypoxia despite an inability for further metabolic remodelling. We conclude that decreased cardiac PPARα expression is essential for adaptive metabolic remodelling in hypoxia, but is prevented by dietary fat

Identification of valid housekeeping genes for quantitative RT-PCR analysis of cardiosphere-derived cells preconditioned under hypoxia or with prolyl-4-hydroxylase inhibitors

Infarction irreversibly damages the heart, with formation of an akinetic scar that may lead to heart failure. Endogenous cardiac stem cells (CSCs) are a promising candidate cell source for restoring lost tissue and thereby preventing heart failure. CSCs may be isolated in vitro, via the formation of cardiospheres, to give cardiosphere-derived cells (CDCs). Although qRT-PCR analyses of CDCs have been performed, no justification for the selection of the housekeeping gene has been published. Here, we evaluated the most suitable housekeeping gene for RNA expression analysis in CDCs cultured under normoxia, hypoxia or with prolyl-4-hydroxylase inhibitors (PHDIs), from both neonatal and adult rats, to determine the effects of ageing and different culture conditions on the stability of the housekeeping gene for CDCs. Six candidate housekeeping genes, [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (Actb), hypoxanthine phosphoribosyltransferase 1 (HPRT-1), beta-2-microtubulin (β2M), 60S acidic ribosomal protein large P1 (RPLP-1) and TATA box binding protein (Tbp)] were evaluated in this study. Analysis using geNorm and NormFinder revealed that GAPDH was the most constant housekeeping gene among all genes tested under normoxia for both neonatal and adult CDCs, whereas Actb was the most stable housekeeping gene under hypoxia. For the PHDI-treated CDCs, overall, GADPH, Actb and β2M were more consistently expressed, whereas HPRT-1, RPLP-1 and Tbp showed unstable expression. The ranking for β2M, HPRT-1 and RPLP-1 stability was different for neonatal and adult cells, indicating that expression of these genes was age-dependent. Lastly, independent of age or culture conditions, Tbp was the least stable housekeeping gene. In conclusion, a combination of Actb and GADPH gave the most reliable normalization for comparative analyses of gene transcription in neonatal and adult rat CDCs preconditioned by hypoxia or PHDIs

Intermolecular photoreactions and selectivity studies in confined space of cation-exchanged y zeolites

Photochemistry in organized assemblies has attracted considerable attention because of their potential use in controlling photophysical and photochemical behaviour of organic molecules in a confined space. Conversion of a starting material to product in a photoreaction involves selectivity by the reaction cavity to the specified product. For solid and rigid media like zeolite, the size of the reaction cavity plays an important role in products selectivity. The surface of NaY zeolite was first studied with paramagnetic probe using Electron Spin Resonance spectroscopy (ESR). Two favourable active sites were identified. The study of a confine space reaction was first studied in the photosensitization of triethylamine by acetophenone in NaY zeolite. ESR result showed that radical cation of amine dimer was formed inside zeolite resulted from the confinement effect of the zeolite Y supercage. Ultraviolet (UV) irradiation of acetophenone in toluene solution results in photochemical hydrogen abstraction and yielded a mixture of both symmetric (1,2-diphenylethane and 1,2-diphenylethyl alcohol) and asymmetric (1,2-diphenylpinacol) coupling products. These were identified and characterized by gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR). With the introduction of NaY zeolite, high yield of asymmetric product, 1,2-diphenylpinacol was observed. It further proved the confinement effect played by the zeolite produced a drastic change in product selectivity compared to homogenous reaction. Photodimerization of 2-cyclohexenone in various cation-exchanged Y zeolites were also studied in solid state and zeolite-solvent slurries. Both the reactions showed a great reversal of head-to-tail (HT) cyclohexenone dimer, to head-to-head (HH) cyclohexenone dimer with increasing pattern from LiY to CsY zeolite. The study of regioselectivity in the photocycloaddition of 2-cyclohexenone to vinyl acetate was also carried out in zeolite slurries, in which the result showed a drastically change of product yield compared to the homogeneous reaction. However, the cationexchanged zeolites failed to control the selectivity. This is explained by the passive cavity effect of zeolite

Studies on the Inhibition of Human Hypoxia Inducible Factor (HIF) Hydroxylases

Hypoxia inducible factor (HIF) is an α/β-heterodimeric transcription factor that regulates cellular responses to hypoxia in metazoans. The activity and stability of the HIF-a subunits are regulated by prolyl and asparaginyl hydroxylation. Human HIF-lα prolyl hydroxylation occurs at Pro402 and Pro564 while asparaginyl hydroxylation occurs at Asn803. Asparaginyl hydroxylation blocks the HIF-la interaction with the p300/CBP eo-activator, thus inhibiting HIF transcriptional activation. Prolyl hydroxylation promotes the HIF-la interaction with the von Hippel-Lindau (VHL) ubiquitin E3 ligase complex and targets it for proteasomal degradation. HIF prolyl hydroxylation is catalysed by the PHDs (prolyl hydroxylase domain) 1, 2 and 3, whereas asparaginyl hydroxylation is catalysed by FIH (factor inhibiting HIF). Both the PHDs and FIH are Fe(II) and 2-oxoglutarate (20G) dependent oxygenases, which utilise oxygen as a co-substrate. In hypoxia, the activity of the PHDs and FIH are suppressed, thus enabling HIF-a subunits to form a productive transcriptional complex. There is widespread interest in developing HIF hydroxylase inhibitors for the treatment of ischemic/hypoxic diseases. The extent to which HIF-lα prolyl and asparaginyl hydroxylation are differentially regulated by chemical reagents is an important question. This thesis describes the development of methods employing immunoblotting and HIF hydroxy-residue specific antibodies to enable the simultaneous measurement of the effects of chemical inhibition at all three HIF-la hydroxylation sites in cells. The findings reveal that HIF prolyl hydroxylation is substantially more sensitive than asparaginyl hydroxylation to inhibition by iron chelators and transition metal ions, in contrast to predictions from in vitro studies. Studies on a range of 20G analogue inhibitors resulted in the identification of several cell- permeable PHD specific inhibitors as well as an FIH specific inhibitor that is active in cells. Excessive accumulation of R-2-hydroxyglutarate (R-2HG) III mutated isocitrate dehydrogenase (IDH)-mediated cancers has led R-2HG to be recognised as an 'oncometabolite'. The newly developed antibody assays were used to investigate the effects of cell-permeable 2HG derivatives on the activity of the HIF hydroxylases in cells. This indicated that direct R-2HG inhibition of PHDs does not play a role in mutated IDH-mediated tumourgenesis. The PHDs have been proposed to play a general role as metabolic sensors besides their function as intracellular oxygen sensors. The Krebs cycle metabolite 20G (a eo- substrate of HIP hydroxylases) was therefore investigated as a potential regulator of the PHDs. The cell-based results demonstrate that 20G elevation results in HIF-a induction, a mechanism suggested to be, at least in part, through PHD inhibition as supported by in vitro and cell-based results. This thesis also describes the first attempt to apply a chemical-genetic approach to functional studies of the PHD isoforms. The in vitro results demonstrate the feasibility of selective inhibition of PHD2 by employing small-molecule-sensitive PHD2 variants. However, attempts to test this approach in mammalian cells have not been successful to date due to the lack of a suitable cell-system. Work on PHD inhibition then describes the development of a new class of diacylhydrazine-based PHD inhibitors. Findings show that some of these compounds are capable of binding to the PHD2 active site and simultaneously inducing the binding of a second iron to PHD2. The reported PHD inhibitor, aspirin metabolite 2,3- dihydroxybenzoylglycine (DHBG), was unexpectedly found to exhibit a concentration- dependent dichotomous effect on HIF stabilisation and HIF suppression in VHL-competent cells. DHBG and other aspirin metabolites including gentisuric acid (GUA) were subsequently found to suppress HIF-la and HIF-2a levels in renal carcinoma RCC4 VHL- defective cells, suggesting that therapeutic effects of aspirin in cancers may involve potential regulation of HIF-a activity.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

STUDIES ON THE INHIBITION OF HUMAN HYPOXIA INDUCIBLE FACTOR (HIF) HYDROXYLASES

Hypoxia inducible factor (HIF) is an &alpha;/&beta;-heterodimeric transcription factor that regulates cellular responses to hypoxia in metazoans. The activity and stability of the HIF-&alpha; subunits are regulated by prolyl and asparaginyl hydroxylation. Human HIF-1&alpha; prolyl hydroxylation occurs at Pro402 and Pro564 while asparaginyl hydroxylation occurs at Asn803. Asparaginyl hydroxylation blocks the HIF-1&alpha; interaction with the p300/CBP co-activator, thus inhibiting HIF transcriptional activation. Prolyl hydroxylation promotes the HIF-1&alpha; interaction with the von Hippel-Lindau (VHL) ubiquitin E3 ligase complex and targets it for proteasomal degradation. HIF prolyl hydroxylation is catalysed by the PHDs (prolyl hydroxylase domain) 1, 2 and 3, whereas asparaginyl hydroxylation is catalysed by FIH (factor inhibiting HIF). Both the PHDs and FIH are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases, which utilise oxygen as a co-substrate. In hypoxia, the activity of the PHDs and FIH are suppressed, thus enabling HIF-&alpha; subunits to form a productive transcriptional complex. There is widespread interest in developing HIF hydroxylase inhibitors for the treatment of ischemic/hypoxic diseases. The extent to which HIF-1&alpha; prolyl and asparaginyl hydroxylation are differentially regulated by chemical reagents is an important question. This thesis describes the development of methods employing immunoblotting and HIF hydroxy-residue specific antibodies to enable the simultaneous measurement of the effects of chemical inhibition at all three HIF-1&alpha; hydroxylation sites in cells. The findings reveal that HIF prolyl hydroxylation is substantially more sensitive than asparaginyl hydroxylation to inhibition by iron chelators and transition metal ions, in contrast to predictions from in vitro studies. Studies on a range of 2OG analogue inhibitors resulted in the identification of several cell-permeable PHD specific inhibitors as well as an FIH specific inhibitor that is active in cells. Excessive accumulation of R-2-hydroxyglutarate (R-2HG) in mutated isocitrate dehydrogenase (IDH)-mediated cancers has led R-2HG to be recognised as an 'oncometabolite'. The newly developed antibody assays were used to investigate the effects of cell-permeable 2HG derivatives on the activity of the HIF hydroxylases in cells. This indicated that direct R-2HG inhibition of PHDs does not play a role in mutated IDH-mediated tumourgenesis. The PHDs have been proposed to play a general role as metabolic sensors besides their function as intracellular oxygen sensors. The Krebs cycle metabolite 2OG (a co-substrate of HIF hydroxylases) was therefore investigated as a potential regulator of the PHDs. The cell-based results demonstrate that 2OG elevation results in HIF-&alpha; induction, a mechanism suggested to be, at least in part, through PHD inhibition as supported by in vitro and cell-based results. This thesis also describes the first attempt to apply a chemical-genetic approach to functional studies of the PHD isoforms. The in vitro results demonstrate the feasibility of selective inhibition of PHD2 by employing small-molecule-sensitive PHD2 variants. However, attempts to test this approach in mammalian cells have not been successful to date due to the lack of a suitable cell-system. Work on PHD inhibition then describes the development of a new class of diacylhydrazine-based PHD inhibitors. Findings show that some of these compounds are capable of binding to the PHD2 active site and simultaneously inducing the binding of a second iron to PHD2. The reported PHD inhibitor, aspirin metabolite 2,3-dihydroxybenzoylglycine (DHBG), was unexpectedly found to exhibit a concentration-dependent dichotomous effect on HIF stabilisation and HIF suppression in VHL-competent cells. DHBG and other aspirin metabolites including gentisuric acid (GUA) were subsequently found to suppress HIF-1α and HIF-2α levels in renal carcinoma RCC4 VHL-defective cells, suggesting that therapeutic effects of aspirin in cancers may involve potential regulation of HIF-&alpha; activity. </p

Probing replacement of pyrophosphate via click chemistry; synthesis of UDP-sugar analogues as potential glycosyl transferase inhibitors

A series of potential UDP-sugar mimics were readily synthesised by copper(I) catalysed modified Huisgen cycloaddition of the corresponding a-propargyl glycosides with 5-azido uridine in aqueous solution. None of the compounds accessed displayed significant inhibitory activity at concentrations of up to 4.5 mM in an assay against bovine milk b-1,4-galactosyltransferase