Abstract Lysyl hydroxylase (E.C. 1.14.11.4, protocollagen-lysine 2-oxoglutarate 5-dioxygenase, PLOD) catalyses the formation of hydroxylysine in collagens and other proteins with collagen-like sequences. The hydroxylysine residues participate in the formation of collagen crosslinks and serve as attachment sites for carbohydrate units. The importance of lysine hydroxylation is demonstrated by the critical manifestations found in patients with the type VI variant of the Ehlers-Danlos syndrome, which is caused by a deficiency in lysyl hydroxylase activity. Lysyl hydroxylase requires Fe2+, 2-oxoglutarate, O2 and ascorbate. The binding site for the C-5 carboxyl group of 2-oxoglutarate is characterized here by site-directed mutagenesis. Two conserved and one non-conserved amino acid residues at the possible binding site in human lysyl hydroxylase 1 were converted individually to alanine or lysine and the mutant polypeptides were expressed in insect cells. Mutation of arginine-700 to alanine inactivated the enzyme completely, whereas mutation of the other two residues had only a minor effect. In addition, the Km of the arginine-700 to lysine mutant polypeptide for 2-oxoglutarate was increased 10-fold. The results thus indicate that this conserved arginine is the residue that binds the C-5 carboxyl group of 2-oxoglutarate in lysyl hydroxylases. A novel human lysyl hydroxylase isoenzyme, termed lysyl hydroxylase 3, was identified, cloned and characterized here. The overall amino acid sequence identity between the novel human lysyl hydroxylase isoenzyme and the other human lysyl hydroxylase isoenzymes is about 60%. The highest expression levels of the mRNA for lysyl hydroxylase 3 among the tissues studied were found in the placenta, pancreas and heart. The novel isoenzyme was expressed as a recombinant protein in insect cells, and the protein was shown to function as a lysyl hydroxylase in vitro hydroxylation experiments using short synthetic peptides as substrates. No differences in catalytic properties were found between the recombinant lysyl hydroxylases 3 and 1. The structure of the human gene for lysyl hydroxylase 3 was determined in the last part of this work. The gene is shown to be only 11.6 kb in size and to contain 19 exons. Transcription was found to be initiated at multiple sites, and the introns contained 15 full-length Alu retroposons or partial Alu fragments of more than 100 bp. The present characterization of the exon-intron organization of the gene will provide a basis for further studies to determine whether there is any genetic disease that is attributable to mutations in this gene.

Prolyl Hydroxylase Domain 2 (PHD2) has been identified as a key oxygen sensor in humans along with Factor Inhibiting Hypoxia Inducible Factor (FIH). As such PHD2 and FIH play critical roles in myriad pathways of medical relevance by hydroxylation of their target substrate hypoxia inducible factor (HIF), a transcription factor responsible for the regulation of over 100+ genes. With such critical roles in human physiology the ability to selectively regulate these two enzymes could potentially lead the way for novel therapeutic treatments of a vast array of disease states from cancer to myocardial infarction. We report on three classes of iron chelators which show promise for independent regulation of the HIF hydroxylases. Compounds representing the pyrones/pyridinones, pyridines and catechols were tested and found to have differential impacts on PHD2 and FIH under the same experimental conditions. The mode of inhibition is the result of binding to the active site iron and is supported by UV-visible and electroparamagnetic resonance spectroscopy. PHD2 at the current time does not have a well resolved mechanistic understanding regarding its catalytic cycle and subsequent rate determining steps. We have employed pH, solvent isotope, and X-ray absorption studies in an effort to gain further understanding regarding PHD2's overall mechanism. Our data support that dissociation of an iron(II)-OH2 bond centered about the active site contributes to a portion of the overall rate determining steps in the catalytic reaction of PHD2 that activates oxygen and ends with the production of hydroxylated substrate.

No description available.

Melanoma is a cancer of the pigment producing cells, melanocytes, and is the most serious type of skin cancer. Cancer is a condition in which one type of cell grows without limit in a disorganized fashion, disrupting and replacing normal tissues and their functions. Normal melanocytes reside in the outer layer of the skin and produce a brown pigment called melanin, which is responsible for skin color. Melanoma occurs when melanocytes become cancerous, grow, and invade other tissues. Pro-opiomelanocortin (POMC) is a precursor polypeptide of 241 amino acids and the prohormone of various neuropeptide, including corticotropin (ACTH), £\-melanocyte-stimulating hormone (£\-MSH), and £]-endorphin (£]-EP). Recently, we demonstrated that systemic POMC overexpression potently suppresses the growth and metastasis of B16-F10 melanoma in vitro and in vivo. However, despite potent inhibition of tumor proliferation and angiogenesis, B16-F10 melanoma still managed to survive after POMC gene therapy. The underlying survival mechanism of B16-F10 melanoma remains unclear. Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix transcription factor that plays a key role not only in melanin synthesis, but also in melanocyte development and survival. Besides, MITF binds to the hypoxia-inducible factor-1£\ (HIF-1£\) promoter to stimulate its transcriptional activity. In this study, we investigate the influence of POMC gene delivery on the pro-survival MITF/HIF-1£\ pathway in B16-F10 melanoma cells. Quantitative RT-PCR and western blot analysis revealed that POMC gene delivery increased the MITF mRNA and protein level in B16-F10 melanoma cells. Besides, POMC gene delivery significantly enhanced the HIF-1£\-driven luciferase activities in melanoma cells. By transfection and puromycin selection, we generated and characterized a MITF-knockdown B16-F10 melanoma cells (MITF KD) stably expressing short hairpin RNA against MITF. The growth, invasion, and colonies formation of MITF-KD were similar to those of vector control. However, implantation of MITF-KD cells led to melanoma with significantly reduced tumor size compared with those in mice implanted with vector control cells. Histological analysis revealed a significant reduction of CD31-positive blood vessels in implantation of MITF-KD cells-treated tumors, which was accompanied with a decrease in Ki-67-positive proliferating cells and an increase in TUNEL-positive apoptotic cells. Moreover, POMC-mediated upregulation of MITF and HIF-1 £\ was significantly attenuated in MITF KD-B16-F10 cells. Acetylsalicylic acid (aspirin; ASA) is widely used as an analgesic/antipyretic drug. ASA exhibits a wide range of biological effects, including preventative effects against heart attack, stroke, and the development of some types of cancer. In our study, we found ASA enhanced cell proliferation. However, in invasion test, ASA had no effect on cell migration. POMC gene delivery elevated the mRNA and protein level of hemeoxygenase-1 (HO-1), a downstream effector of HIF-1£\ pathway and an enzyme catalyzing the converting reaction of heme to carbon monoxide, ion and biliverdine. Inhibition of HO-1 activities augmented the inhibitory effect of POMC gene delivery on proliferation, migration and anchorage-independent growth of B16-F10 melanoma cells. These studies indicated that activation of MITF/HIF-1£\/HO-1 indeed contributes to melanoma survival after POMC gene delivery.

Objective: Heme oxygenase-1 (HO-1) has been well established as a cytoprotective molecule, and has been shown to exert cardioprotective effects in both hypertension and cardiac hypertrophy. However, the precise mechanism of the cardioprotective effect of HO-1 has yet to be fully elucidated. The natriuretic peptide system (NPS) is also a key player in cardiovascular homeostasis and tissue dynamics, and has also been shown to be cardioprotective in a variety of pathologic conditions. This study examined the effect of high dietary salt treatment in genetic models of HO-1, and assessed the expression of the NPS in the left ventricle (LV), in order to gain insight into the relationship between varying levels of HO-1 expression with the development of cardiac hypertrophy and the expression of the NPS. Methods: Age-matched 12-week old male HO-1 knockout (HO-1-/-) and HO-1 cardiomyocyte-specific transgenic overexpressing (HO-1Tg) mice were treated with either normal salt (NS; 0.8%) or high salt (HS; 8.0%) chow for 5 weeks. LV mRNA expression was determined using quantitative real-time RT-PCR. Results: HO-1-/- mice fed HS diet had significantly higher left ventricle-to-body weight ratio (LV/BW) compared to HO-1+/+ mice fed NS diet. HO-1-/- mice had significantly reduced expression of the NPS compared to controls, and these mice did not exhibit a salt-induced increase in ANP expression. HS treatment had no effect on LV/BW in HO-1Tg mice compared to controls. HO-1Tg mice had significantly higher ANP and BNP expression compared to controls. Conclusions: The presence of HO-1 is required for normal salt-induced changes in the local cardiac NPS. HO-1 ablation resulted in significantly lower mRNA expression of the NPS, whereas HO-1 overexpression resulted in higher mRNA expression of the NPS. These data indicate that the detrimental effect of reduced HO-1 expression and the cardioprotective effect of increased HO-1 expression may be due, in part, to altered expression of the NPS. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2009-10-20 09:15:20.541

Thesis (M.S.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Thesis advisor: Dr. William Durante. "December 2008" Includes bibliographical references.

Cyanide utilization in Pseudomonas fluorescens NCIMB 11764 occurs via oxidative conversion to carbon dioxide and ammonia, the latter satisfying the nitrogen requirement. Substrate attack is initiated by an enzyme referred to as cyanide oxygenase (CNO), previously shown to require components in both high (H) (>30 kDa) and low (L) (<10 kDa) molecular weight cell fractions. In this study, tetrahydrobiopterin (H4biopterin) was identified as a cofactor in fraction L, thus making CNO appear as a pterin- dependent hydroxylase. CNO was purified 150-fold (specific activity 0.9 U/mg) and quantitatively converted cyanide to formate and ammonia as reaction products. When coupled with formate dehydrogenase, the complete enzymatic system for cyanide oxidation to carbon dioxide and ammonia was reconstituted. CNO was found to be an aggregate of known enzymes that included NADH oxidase (Nox), NADH peroxidase (Npx), cyanide dihydratase (CynD) and carbonic anhydrase (CA). A complex multi-step reaction mechanism is proposed in which Nox generates hydrogen peroxide which in turn is utilized by Npx to catalyze the oxygenation of cyanide to formamide accompanied by the consumption of one and two molar equivalents of oxygen and NADH, respectively. The further hydrolysis of formamide to ammonia and formate is thought to be mediated by CynD. The role of H4biopterin and of the enzyme CA in the proposed process remains unclear, but the involvement of each in reactive oxygen and radical chemistry is consistent with the proposed formation of such species in the catalytic process. H4biopterin may additionally serve as a protein stabilizing agent along with a protein co-purifying with CynD identified as elongation factor Tu, a known chaperone. At least two of the CNO components (Nox and CynD) are complex oligomeric proteins whose apparent association with Npx and CA appears to be favored in bacterial cells induced with cyanide allowing their purification in toto as a multiprotein enzyme complex.

Liu, Jian. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 127-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed on June 24, 2009). Includes bibliographical references (p. 99-116).

Les statines sont des inhibiteurs sélectifs de la 3-hydroxy-3-méthylglutaryl-coenzyme A réductase, utilisées pour diminuer la biosynthèse du cholestérol. Ces molécules possèdent en plus de leur capacité à réduire le cholestérol des effets pléiotropiques comme les propriétés anti-inflammatoires et anti-oxydantes. Les cucurbitacines sont des triterpènes dérivés des plantes, ayant des propriétés biologiques diverses comme les effets anti-inflammatoires et anticancéreux, associées toutefois à une toxicité élevée. Les dérivés de thiazole sont des molécules contenant un noyau hétérocyclique formé de trois atomes de carbones, un atome de sulfure et un atome d'azote, disposant des effets anti-inflammatoires importantes. Les cyclooxygénases et les hème-oxygénases jouent un rôle dans l'inflammation et le stress oxydatif et sont les cibles des statines et des cucurbitacines in vitro. Les dérivés de thiazole peuvent inhiber plutôt l'activité enzymatique des cyclooxygénases. Le but de ma thèse est d'étudier les effets de ces 3 molécules in vivo et d'analyser si possible les mécanismes impliqués, comme par exemple pour les statines. Pour cela, nous avons eu recours chez les souris C57BL/6 au modèle d'inflammation de la poche à air-stérile injecté par le zymosan.Nous avons d'abord montré que le traitement des souris avec l'atorvastatine pendant 10 jours a réduit la migration des cellules dans l'exsudat de la poche à air ainsi que l'expression de gènes des marqueurs pro-inflammatoires tels que les cytokines, les chimiokines, la cyclooxygénase-2 et la nitric oxide synthase -II. Le taux de la prostaglandine E2 et de l'interleukine-6 a été également réduit. L'inhibition de l'hème-oxygénase-1 par son inhibiteur sélectif, tin protoporphyrine, a partiellement réduit l'effet inhibiteur de l'atorvastatine sur la migration des cellules et sur certaines cytokines suggérant un rôle important de la l'hème-oxygénase-1 dans les propriétés anti-inflammatoires in vivo.En parallèle, nous avons utilisé ce modèle animal tester l'effet de la cucurbitacine E sur l'inflammation et évaluer le rôle d'encapsulation in vivo dans des liposomes à base de phosphatidylcholine. Nous avons démontré que la cucurbitacine E libre (12.5 μg/poche ou 25 μg/poche) a tendance à diminuer l'interleukine-6, l'hème oxygénase-1 et la cyclooxygénase-2 alors que la cucurbitacine E encapsulée (12.5 μg/poche) a significativement réduit la prostaglandine E2, l'interleukine-6 et le taux de nitrite sans affecter le niveau d'ARNm de la cyclooxygénase-2 et l'hème oxygénase-1. Nos résultats suggèrent que l'incorporation de la cucurbitacine E dans des liposomes améliore son effet anti-inflammatoire et réduit sa cytotoxicité.Finalement, deux dérivés de thiazole qui diffèrent dans leur structure par la présence d'un groupement butyle- ou benzyle- sur leur chaîne latérale, ont été explorés dans ce modèle. Nous avons montré que le dérivé de thiazole contenant le groupe benzyle est sélectif de la cyclooxygénase-2 dans les macrophages et le modèle in vivo chez la souris.En résumé, mon travail de thèse met en évidence in vivo les effets anti-inflammatoires des statines et le rôle de l'hème oxygénase-1, et permet en plus la caractérisation des effets anti-inflammatoires des cucurbitacines et des dérivés de thiazole. L'ensemble de ces résultats renforcent les effets anti-inflammatoires de ces substances et démontre in vivo leur effet et les suggèrent comme molécules anti-inflammatoires. / Statins are selective competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase used to lower cholesterol biosynthesis and have multiple pleiotropic effects beyond lowering cholesterol such as anti-inflammatory and anti-oxidant properties. Cucurbitacins are triterpenoid derived from plants and exhibit potential anti-inflammatory and anticancer properties though they have a high cytotoxicity. Thiazole derivatives are molecules containing heterocyclic ring with three carbons, one sulfur and one nitrogen atom, with important anti-inflammatory activities. Cyclooxygenases and heme-oxygenases play a role in inflammation and oxidative stress and are targets for statins or cucurbitacins in vitro. Thiazole derivatives are potential inhibitors of cyclooxygenases. The aim of my thesis is to investigate the anti-inflammatory effect of these three compounds in vivo and attempt to analyze the mechanisms involved, mainly for statins. Thus we set up an animal model of inflammation in C57BL/6 corresponding to the zymosan -injected dorsal sterile air pouch.First we have shown that treatment of mice with atorvastatin for 10 days reduced cell migration in the exudate of the air pouch as well as the expression of inflammatory markers such as cytokines, chemokines, cyclooxygenase-2 and nitric oxide synthase -II. The synthesis of prostaglandin E2 and interleukin-6 was also reduced. Inhibition of heme-oxygenase-1 by the selective inhibitor tin protoporphyrin partially diminished the inhibitory effect of statins on cell migration and some cytokines suggesting a significant role of HO-1 in the anti-inflammatory properties for atorvastatin in vivo.For cucurbitacins, we used the same animal model to investigate the effect of this substance in vivo and further assess the beneficial effect of encapsulating cucurbitacin in phosphatidylcholine-liposomes. Free cucurbitacin E (12.5 μg/pouch or 25 μg/pouch) tended to increase interleukin-6, decrease heme oxygenase-1 and cyclooxygenase-2 whereas cucurbitacin E loaded liposomes (12.5 μg/pouch) significantly reduced prostaglandin E2, interleukin-6 and nitrite without affecting cyclooxygenase-2 and heme oxygenase-1 mRNA levels. We demonstrated that the incorporation of Cuc E into liposomes enhances its anti-inflammatory effect and reduces its cytotoxicity.Finally, we used the dorsal air pouch model to investigate the anti-inflammatory effect of two thiazole derivatives that differ in their side chain by the presence of butyl or benzyl group. In addition to analyzing their effect on cyclooxygenase activity in human blood platelets, on recombinant COX-1 and in culture macrophage cell lines, we demonstrated their capacity to block cyclooxygenase-dependent prostaglandin synthesis in the lumen of the air pouch.In summary, my thesis presents in vivo evidence for the anti-inflammatory effects of atorvastatin dependent on HO-1 activity. My studies characterized the anti-inflammatory effects of cucurbitacins and thiazole derivatives. All these results support the anti-inflammatory effects of these substances and suggested them as potential anti-inflammatory substances.