Synthesis and bioevaluation of laccase substrates and substituted quinolines

Doctor of PhilosophyDepartment of ChemistryDuy H. HuaOur research work is divided into three chapters. In the first chapter, synthesis of substituted phenolic compounds including halogenated di- and trihydroxybenzenes, aminophenols, and substituted di-tert-butylphenols, their redox potential, laccase oxidation, and mosquito anti-larval activities are discussed. The synthesized substituted phenols were found to be the substrates but not the inhibitors of laccase. An inverse correlation between the oxidation potential and the laccase oxidation efficiency of halogenated hydroxybenzenes and aminophenols was established. However, substituted di-tert-butylphenols were found to have anti-larval activities in mosquitoes resulting in the death of the larvae just before reaching pupation. Among the di-tert-butyl phenols studied, water insoluble, 2,4-di-tert-butyl-6-(3-methyl-2-butenyl)phenol (16), 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-methylpropanal oxime (14), and 6,8-di-tert-butyl-2,2-dimethyl-3,4-dihydro-2H-chromene (17) caused the mortility of 98%, 93%, and 92% of Anopheles gambiae larvae in the concentration of 182 nM, 3.4 µM, and 3.7 µM, respectively. In particular, compound 16 had similar anti-larval activities as compared to MON-0585, an anti-larval agent reported by Monsanto in the 70’s. In the second chapter, inhibition of protein kinase C (PKC) phosphorylation by substituted quinolines (PQs) is inverstigated. PQ compounds such as N-(3-aminopropyl)-6-methoxy-4-methyl-5-(3-(trifluormethyl)phenoxy)quinolin-8-amine (PQ1), N-(furan-2-ylmethyl)-6-methoxy-4-methyl)-5-(3-(trifluoromethyl)phenoxy)quinolin-8-amine (PQ11), and 6-methoxy-4-methyl-N-(quinolin-4-ylmethyl)-5-(3-(trifluoromethyl)phenoxy)quinolin-8-amine (PQ15) were found to inhibit PKC phosphorylation with IC50 values of 35 nM, 42.3 nM, and 216.3 nM respectively, among which PQ1 and PQ11 were found to be potent PKC inhibitors as comparable to that of staurosporine (IC50 = 33 nM). In chapter three, the tissue distribution of PQ1 and PQ11 in normal C57BL/6J mice and the effect of PQ1 on the normal tissues of mice were investigated. Substituted quinolines, PQ1 and PQ11 were distributed in the tissues in concentrations that were more than 40 folds of their effective dose. PQ1 and PQ11 were also found to penetrate the blood brain barrier and collect in the tissue in significant amounts. The administration of PQ1 and PQ11 had no effect in the normal behavior of the animals indicating no short term adverse effects. PQ1 was found to increase the expression of survivin, an anti-apoptotic factor and decrease the expression of cleaved caspase-3 and caspase-8, pro-apoptotic proteins. These studies suggests that PQ1 might have anti-apoptotic activities in normal cells, in contrast to the role of PQ1 in cancer cells where it has demonstrated to induce apoptosis. The study also indicated that PQ11 was better metabolized from the tissues over time as compared to PQ1

Human platelet lysate improves human cord blood derived ECFC survival and vasculogenesis in three dimensional (3D) collagen matrices

Human cord blood (CB) is enriched in circulating endothelial colony forming cells (ECFCs) that display high proliferative potential and in vivo vessel forming ability. Since diminished ECFC survival is known to dampen the vasculogenic response in vivo, we tested how long implanted ECFC survive and generate vessels in three-dimensional (3D) type I collagen matrices in vitro and in vivo. We hypothesized that human platelet lysate (HPL) would promote cell survival and enhance vasculogenesis in the 3D collagen matrices. We report that the percentage of ECFC co-cultured with HPL that were alive was significantly enhanced on days 1 and 3 post-matrix formation, compared to ECFC alone containing matrices. Also, co-culture of ECFC with HPL displayed significantly more vasculogenic activity compared to ECFC alone and expressed significantly more pro-survival molecules (pAkt, p-Bad and Bcl-xL) in the 3D collagen matrices in vitro. Treatment with Akt1 inhibitor (A-674563), Akt2 inhibitor (CCT128930) and Bcl-xL inhibitor (ABT-263/Navitoclax) significantly decreased the cell survival and vasculogenesis of ECFC co-cultured with or without HPL and implicated activation of the Akt1 pathway as the critical mediator of the HPL effect on ECFC in vitro. A significantly greater average vessel number and total vascular area of human CD31(+) vessels were present in implants containing ECFC and HPL, compared to the ECFC alone implants in vivo. We conclude that implantation of ECFC with HPL in vivo promotes vasculogenesis and augments blood vessel formation via diminishing apoptosis of the implanted ECFC

iPSC-Derived Vascular Cell Spheroids as Building Blocks for Scaffold-Free Biofabrication

Recently a protocol is established to obtain large quantities of human induced pluripotent stem cells (iPSC)-derived endothelial progenitors, called endothelial colony forming cells (ECFC), and of candidate smooth-muscle forming cells (SMFC). Here, the suitability for assembling in spheroids, and in larger 3D cell constructs is tested. iPSC-derived ECFC and SMFC are labeled with tdTomato and eGFP, respectively. Spheroids are formed in ultra-low adhesive wells, and their dynamic proprieties are studied by time-lapse microscopy, or by confocal microscopy. Spheroids are also tested for fusion ability either in the wells, or assembled on the Regenova 3D bioprinter which laces them in stainless steel micro-needles (the “Kenzan” method). It is found that both ECFC and SMFC formed spheroids in about 24 h. Fluorescence monitoring indicated a continuous compaction of ECFC spheroids, but stabilization in those prepared from SMFC. In mixed spheroids, the cell distribution changed continuously, with ECFC relocating to the core, and showing pre-vascular organization. All spheroids have the ability of in-well fusion, but only those containing SMFC are robust enough to sustain assembling in tubular structures. In these constructs a layered distribution of alpha smooth muscle actin-positive cells and extracellular matrix deposition is found. In conclusion, iPSC-derived vascular cell spheroids represent a promising new cellular material for scaffold-free biofabrication

Isoflavone metabolism in domestic cats (Felis catus): comparison of plasma metabolites detected after ingestion of two different dietary forms of genistein and daidzein

Some felid diets contain isoflavones but the metabolic capacity of cats toward isoflavones is relatively unknown, despite the understanding that isoflavones have divergent biological potential according to their metabolite end products. The objective of this study was to determine the plasma metabolites detectable in domestic cats after exposure to 2 different dietary forms of isoflavones, either as a soy extract tablet ( n = 6) or as part of a dietary matrix ( n = 4). Serial blood samples were collected after isoflavone exposure to identify the plasma metabolites of each cat. Genistein was detected in its unconjugated form or as a monosulfate. Daidzein was detected as both a mono- and disulfate as well as in its unconjugated form. Other daidzein metabolites detected included equol mono- and disulfate, dihydrodaidzein, and O -desmethylangolensin. No β -glucuronide metabolites of either isoflavone were detected. Equol was produced in markedly fewer cats after ingestion of a soy extract tablet as a single oral bolus compared with cats consuming an isoflavone-containing diet. The detectable metabolites of the isoflavones, genistein and daidzein, in domestic cat plasma after dietary ingestion has been described in the present study for the first time. The metabolic capacity for isoflavones by domestic cats appears to be efficient, with only minimal proportions of the ingested amount detected in their unconjugated forms. This has implications for the potential of isoflavones to exert physiological activity in the domestic cat when consumed at concentrations representative of typical dietary intake

Analysis of phenolic compounds from different morphological parts of Helichrysum devium by liquid chromatography with on-line UV and electrospray ionization mass spectrometric detection

A simple and rapid method has been used for the screening and identification of the main phenolic compounds from Helichrysum devium using high-performance liquid chromatography with on-line UV and electrospray ionization mass spectrometric detection (LC-DAD/ESI-MS(n)). The total aerial parts and different morphological parts of the plant, namely leaves, flowers and stems, were analyzed separately. A total of 34 compounds present in the methanolic extract from Helichrysum devium were identified or tentatively characterized based on their UV and mass spectra and retention times. Three of these compounds were positively identified by comparison with reference standards. The phenolic compounds included derivatives of quinic acid, O-glycosylated flavonoids, a caffeic acid derivative and a protocatechuic acid derivative. The characteristic loss of 206 Da from malonylcaffeoyl quinic acid was used to confirm the malonyl linkage to the caffeoyl group. This contribution presents one of the first reports on the analysis of phenolic compounds from Helichrysum devium using LC-DAD/ESI-MS(n) and highlights the prominence of quinic acid derivatives as the main group of phenolic compounds present in these extracts. We also provide evidence that the methanolic extract from the flowers was significantly more complex when compared to that of other morphological parts.info:eu-repo/semantics/publishedVersio

Mitogen-Activated Protein Kinase 14 Promotes AKI

An improved understanding of pathogenic pathways may identify novel acute kidney injury (AKI) therapeutic approaches. Unbiased LC-MS/MS protein expression profiling combined with focused data mining identified MAP3K14 and non-canonical NFκB activation at the crossroads of the enriched pathways MAPK, ubiquitin-mediated proteolysis, chemokines, NFκB and apoptosis in the kidney cortex of experimental toxic AKI. In AKI the upstream kinase MAP3K14, the NFκB DNA binding heterodimer RelB/NFκB2, and proteins involved in NFκB2 p100 ubiquitination and proteasomal processing to p52, such as Ube2m and cullin1 were up-regulated. Immunohistochemistry localized MAP3K14 expression to tubular cells in experimental and human AKI. In vivo evidence of MAP3K14 activation in experimental folic acid-induced AKI consisted of NFκB2 p100 processing to p52, nuclear location and DNA binding of RelB and NFκB2. MAP3K14 activity-deficient aly/aly mice were protected from kidney dysfunction, inflammation and apoptosis in AKI induced by folic acid and from lethality in cisplatin-induced AKI. MAP3K14 siRNA targeting in cultured tubular cells decreased inflammation and cell death. Bone marrow transplantation experiments where consistent with a protective effect of renal cell MAP3K14 targeting. Cell culture and in vivo studies identified chemokines MCP-1, RANTES and CXCL10 as MAP3K14 targets in tubular cells, thus identifying potential mediators of the deleterious effect of MAP3K14 in kidney injury. In conclusion, MAP3K14 promotes kidney injury through promotion of inflammation and cell death and is a promising novel therapeutic target

Peroxynitrate formed during a transient episode of brain ischemia increases endothelium-derived hyperpolarization-type dilations in thromboxane/prostaglandin receptor stimulated rat cerebral arteries

Aim Increased thromboxane A2 and peroxynitrite are hallmarks of cerebral ischemia/reperfusion (I/R). Stimulation of thromboxane/prostaglandin receptors (TP) attenuates endothelium-derived hyperpolarization (EDH). We investigated whether EDH-type middle cerebral artery (MCA) relaxations following TP stimulation are altered after I/R and the influence of peroxynitrite. Methods Vascular function was determined by wire myography after TP stimulation with the thromboxane A2 mimetic 9,11-Dideoxy-9α,11α-methano-epoxy prostaglandin F2α (U46619) in MCA of Sprague-Dawley rats subjected to MCA occlusion (90 min)/reperfusion (24 h) or sham operation, and in non-operated (control) rats. Some rats were treated with saline or the peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron (III) (20 mg kg-1). Protein expression was evaluated in MCA and in human microvascular endothelial cells submitted to hypoxia (overnight)/reoxygenation (24 h) (H/R) using immunofluorescence and immunoblotting. Results In U46619-preconstricted MCA, EDH-type relaxation by the proteinase-activated receptor 2 agonist serine–leucine–isoleucine–glycine–arginine–leucine–NH2 (SLIGRL) was greater in I/R than sham rats due to an increased contribution of small-conductance calcium-activated potassium channels (SKCa), which was confirmed by the enlarged relaxation to the SKCa activator N-cyclohexyl-N-2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine. I/R and H/R induced endothelial protein tyrosine nitration and filamentous-actin disruption. In control MCA, either cytochalasin D or peroxynitrite disrupted endothelial filamentous-actin and augmented EDH-type relaxation. Furthermore, peroxynitrite decomposition during I/R prevented the increase in EDH-type responses. Conclusion Following TP stimulation in MCA, EDH-type relaxation to SLIGRL is greater after I/R due to endothelial filamentous-actin disruption by peroxynitrite, which prevents TP-induced block of SKCa input to EDH. These results reveal a novel mechanism whereby peroxynitrite could promote postischemic brain injury

Analysis of isoflavones and flavonoids in human urine by UHPLC

A rapid, ultra high-performance liquid chromatographic (UHPLC) method has been developed and validated for simultaneous identification and analysis of the isoflavones genistein, daidzein, glycitin, puerarin, and biochanin A, and the flavonoids (±)-catechin, (−)-epicatechin, rutin, hesperidin, neohesperidin, quercitrin, and hesperetin in human urine. Urine samples were incubated with β-glucuronidase/sulfatase. UHPLC was performed with a Hypersil Gold (50 × 2.1 mm, 1.9 μm) analytical column. Elution was with a gradient prepared from aqueous trifluoroacetic acid (0.05%) and acetonitrile. UV detection was performed at 254 and 280 nm. The calibration curves were indicative of good linearity (r2 ≥ 0.9992) in the range of interest for each analyte. LODs ranged between 15.4 and 107.0 ng mL−1 and 3.9 and 20.4 ng mL−1 for flavonoids and isoflavones, respectively. Intra-day and inter-day precision (C.V., %) was less than 3.9% and 3.8%, respectively, and accuracy was between 0.03% and 5.0%. Recovery was 70.35–96.58%. The method is very rapid, simple, and reliable, and suitable for pharmacokinetic analysis. It can be routinely used for simultaneous determination of these five isoflavones and seven flavonoids in human urine. The method can also be applied to studies after administration of pharmaceutical preparations containing isoflavones and flavonoids to humans

Dextran and Polymer Polyethylene Glycol (PEG) Coating Reduce Both 5 and 30 nm Iron Oxide Nanoparticle Cytotoxicity in 2D and 3D Cell Culture

Superparamagnetic iron oxide nanoparticles are widely used in biomedical applications, yet questions remain regarding the effect of nanoparticle size and coating on nanoparticle cytotoxicity. In this study, porcine aortic endothelial cells were exposed to 5 and 30 nm diameter iron oxide nanoparticles coated with either the polysaccharide, dextran, or the polymer polyethylene glycol (PEG). Nanoparticle uptake, cytotoxicity, reactive oxygen species (ROS) formation, and cell morphology changes were measured. Endothelial cells took up nanoparticles of all sizes and coatings in a dose dependent manner, and intracellular nanoparticles remained clustered in cytoplasmic vacuoles. Bare nanoparticles in both sizes induced a more than 6 fold increase in cell death at the highest concentration (0.5 mg/mL) and led to significant cell elongation, whereas cell viability and morphology remained constant with coated nanoparticles. While bare 30 nm nanoparticles induced significant ROS formation, neither 5 nm nanoparticles (bare or coated) nor 30 nm coated nanoparticles changed ROS levels. Furthermore, nanoparticles were more toxic at lower concentrations when cells were cultured within 3D gels. These results indicate that both dextran and PEG coatings reduce nanoparticle cytotoxicity, however different mechanisms may be important for different size nanoparticles