Covalent binding of dinuclear metal carboxylate compounds to purine DNA nucleobases has been shown to be a source of anticancer activity, and has resulted in intense research to understand the coordination of metal complexes to DNA. This investigation focuses on the formation of dirhenium metal:dinucleotide complexes of purine nucleobases. To our knowledge, complexes formed by the reaction of dinuclear rhenium metal compounds with dinucleotides have not been reported in the literature.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Nucleic acid aptamers are "short single-stranded DNA- or RNA-based oligonucleotides that can selectively bind to small molecular ligands or protein targets with high affinity and specificity, when folded into their unique three-dimensional structure" (1). Aptamers have shown promising ability for detection in the subnanomolar range of nucleic acid targets with specificity down to single nucleotide variations (2). The selectivity, low limit of detection, and cost-effectiveness make these nucleic acid aptamers optimal bio-sensors. Previous work by our group has been done to optimize the signal of the dapoxyl-binding aptamer (DAP), which is a light-up aptamer. Here, we propose to organize this aptamer into a split-aptamer system and determine the limit of detection and selectivity for a target gene in Mycobacterium tuberculosis.

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Orientador: Gilberto de Nucci / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T06:11:56Z (GMT). No. of bitstreams: 1 Lorenzetti_Raquel_D.pdf: 2089930 bytes, checksum: f6ed8b98e678ffdfc6051c645efc8b4b (MD5) Previous issue date: 2007 / Resumo: No presente trabalho foi padronizado um novo método para a dosagem da atividade de fosfodiesterase in vitro, por HPLC-MS/MS. Este novo método conseguiu apresentar exatidão, precisão, sensibilidade e rapidez nas análises; monitorando os nucleotídeos (AMP, GMP, AMPc e GMPc). O desenvolvimento de novos fármacos derivados de um protótipo aponta para a obtenção de moléculas com um melhor perfil farmacocinético ou uma melhor relação estrutura-atividade. Atualmente o sildenafil é considerado o principal fármaco para o tratamento de disfunção erétil. Neste trabalho, avaliaram-se novos compostos denominados análogos de sildenafil (carbonato de lodenafil, dímero uréia e dímero uretana). Os análogos foram analisados quanto à atividade em PDE5 e agregação plaquetária humana, in vitro. Foi determinada a estabilidade destes compostos, em meio ácido e plasma humano, in vitro, além de seus possíveis metabólitos em microssomas e hepatócitos de rato in vitro, e os seus parâmetros farmacocinéticos via intravenosa e oral, em cão, in vivo. Os resultados mostraram que os análogos de sildenafil inibem a atividade de PDE e não inibem a agregação plaquetária do mesmo modo que o sildenafil in vitro, no entanto potencializam a ação do doador de NO (SNP). Os análogos de sildenafil foram estáveis em meio ácido e em plasma humano. No ensaio de metabolização, os dímeros uréia e uretana não foram metabolizados, entretanto o carbonato de lodenafil foi metabolizado principalmente em lodenafil, in vitro. O carbonato de lodenafil é rapidamente biotransformado em lodenafil, após administração v.i. e v.o. em cão. Concluiu-se que este trabalho apresenta um novo método de dosagem de PDEs e uma nova perspectiva terapêutica para a disfunção erétil, representada pelo carbonato de lodenafil, o qual inibe concentração-dependente a atividade de PDE5 / Abstract: In the present work a new method for the dosage of the activity of phosphodiesterase was standardized in vitro, for HPLC-MS/MS. This new method obtained to present exactness, precision, sensitivity, and rapidity in the analyses; monitoring the nucleotides (AMP, GMP, cAMP and cGMP). The development of new drug derived from an archetype points with respect to the molecule attainment with one better pharmacokinetic profile or one better relation structure-activity. Currently the sildenafil is considered the main drug for the treatment of erectile dysfunction. In this work, we evaluate new analogous called composites of sildenafil (carbonate of lodenafil, dimer urea and dimer uretana). The analogous ones had been analyzed how much the activity in PDE5 and platelet aggregation human being, in vitro. The stability of these composites was determined, in human acid way and plasma, in vitro, beyond its possible metabolites in microsomes and hepatocytes of rat in vitro, and its pharmacokinetic profile after intravenous and oral, in dog, in vivo. The results had shown that the analogous of sildenafil inhibit the activity of PDE and they do not inhibit the platelet aggregation in a similar way that the sildenafil in vitro, however potencializam the action of the giver of NO (SNP). The analogous ones of sildenafil are presented steady in human acid way and plasma. In the metabolization assay, metabolization of dimer urea and dimer uretana was not observed, however the lodenafil carbonate was metabolizado mainly in lodenafil, in vitro. The lodenafil carbonate quickly is biotransformation in lodenafil, after administration v.i. and v.o. in dog. We conclude that this work presents a new method for analyze activity of PDEs and a new therapeutically perspective for the erectile dysfunction, represented for lodenafil carbonate, which inhibits concentration-dependent the activity of PDE5 / Doutorado / Doutor em Farmacologia

We have synthesized a new class of photoaffinity analogs, 2',3'-O-(2,4,6-trinitrophenyl)-8-azido-ATP, -ADP and -AMP (TNP- 8N₃ATP, -ADP and -AMP), and their radiolabeled derivatives, and characterized their interaction with the sarcoplasmic reticulum Ca²⁺-ATPase. The TNP-8N₃-nucleotides were synthesized from ATP in three steps involving bromination in the 8-position of the adenine ring followed by displacement with an azido group and then trinitrophenylation of the resulting 8N₃-nucleotide with TNBS. Inclusion of the oxidizing agent, DTNB, in the final reaction was found to be necessary to prevent reduction of the azido group by the released sulfite anion and also elevated the yield of trinitrophenylation to about 80%. Purity was determined spectrophotometrically, as well as by anion exchange TLC and reversed phase HPLC. In the dark, the compounds were found to display most of the features of the parent TNP-nucleotides and interacted with the Ca²⁺-ATPase in a similar way. When activated by illumination, the probes were specifically incorporated into SR vesicles with high efficiency at alkaline pH. The site of labeling was identified as being on the A₁ tryptic fragment.

Properties of the nucleotide binding site of the Ca²⁺-ATPase of skeletal muscle sarcoplasmic reticulum have been investigated. The study centred around interaction of the high affinity ATP analog, 2'-3'-0-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate, (TNP-ATP), with the Ca²⁺-ATPase. Defined fractions of the sarcoplasmic reticulum (SR), corresponding to the terminal cisternae (TC) and light SR (LSR), were isolated. The TC were shown to have distinctive morphological characteristics that differ from the LSR. The TC vesicles contained electron dense intravesicular material representative of Ca²⁺ binding proteins, and visible membranous "feet" structures, which are reported to interconnect with the transverse tubule. Functional characterisation of the isolated fractions provided evidence for the predominant localisation of Ca²⁺ release channels in TC, and concentration of Ca²⁺-ATPase molecules in LSR. These conclusions were based on the following observations: (a) decreased Ca²⁺ transport of TC versus LSR; ruthenium red, a Ca²⁺ channel blocker, enhanced Ca²⁺ transport and pumping efficiency in TC, (b) higher Ca²⁺-ATPase activity for LSR in the presence and absence of ionophore, (c) rapid Ca²⁺ efflux from TC which is inhibited by ruthenium red. Of special interest was the characterisation of the TC and LSR with respect to turnover-dependent TNP-ATP fluorescence. Fluorescence observed for TC was approximately 65% of that for LSR. This phenomenon may be attributable to either the decreased Ca²⁺ ATPase content of the TC vesicles or open Ca²⁺ release channels. Hence the TNP-ATP fluorescence characteristics appear to reflect the morphological and functional subspecialisation of the defined SR fractions.

cAMP-elevating agents such as adenosine and epinephrine (after binding to beta-adrenergic receptor) contribute to local vascular dilation and some of these dilations are endothelium-dependent. Previous intracellular Ca 2+ imaging studies in mouse microvessel endothelial cells reported that addition of adenosine or epinephrine induced a Ca2+ influx which is blocked by CNG channel blockers such as L-cis-diltiazem or LY83583. Inside-out patch clamp studies confirmed the existence of a cAMP-activated current in endothelial cells, strongly suggesting a functional role of CNG, in particular CNGA2, channels in endothelial cells. The current study went further to show that similar Ca2+ influx in response to adenosine or epinephrine occurred in endothelial cells in freshly isolated mouse aortic strips and was again blocked by L-cis-diltiazem. By measuring the isometric force developed in mouse aortic strips, we showed that CNGA2 channel-mediated Ca2+ influx in endothelial cells contributed to the endothelium-dependent vascular dilatation in response to adenosine and epinephrine. / In conclusion, cyclic nucleotides playa vital role in the regulation of intracellular Ca2+ concentration in vascular cells and Jurket T cells. / In Jurkat T cells, cyclic nucleotides regulated Ca2+ mobilization in a different way. Fluorescence-imaging studies showed that cGMP inhibited store-operated Ca2+ influx and histamine-induced Ca 2+ rise in Jurkat T cells through activation of PKG. / Thromboxane A2 (TxA2)-induced smooth muscle contraction has been implicated in cardiovascular, renal and respiratory diseases. This contraction can partly be attributed to TxA2-induced Ca2+ influx, which activates the Ca2+-calmodulin-MLCK pathway. This study aims to identify the channels that mediate TxA2-induced Ca2+ influx in vascular smooth muscle cells. Application of U-46619, a thromboxane A2 mimic, resulted in a constriction in endothelium-denuded small mesenteric artery segments. The constriction relied on the presence of extracellular Ca2+, because removal of extracellular Ca2+ abolished the constriction. This constriction was partially inhibited by a L-type Ca2+ channel inhibitor nifedipine (0.5-1 muM). The remaining component was inhibited by L-cis-diltiazem, a selective inhibitor for CNG channels, in a dose-dependent manner, Another CNG channel blocker LY83583 [6-(phenylamino)-5,8-quinolinedione] had similar effect. In primary cultured smooth muscle cells derived from rat aorta, application of U46619 (100 nM) induced a rise in cytosolic Ca2+, which was inhibited by L-cis-diltiazem. Immunoblot experiments confirmed the presence Of CNGA2 protein in vascular smooth muscle cells, These data suggest a functional role of CNG channels in U-46619-induced Ca 2+ influx and contraction of smooth muscle cells. / Leung, Yuk Ki. / "August 2010." / Adviser: Yao Xiaoxiang. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 116-132). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / Typescript. Vita. Includes bibliographical references (l. 140-148). Also available on the Internet.

In mammals, newly synthesized proteins destined for secretion are translocated cotranslationally into the lumen of the Endoplasmic Reticulum (ER). Once inside, these nascent polypeptide chains are bound by a lumenal ER protein called BiP (Immunoglobulin Binding Protein) or Grp 78 (Glucose Regulated Protein 78). It is hypothesized that this binding is necessary to protect the nascent chains until they are properly folded or assembled with other subunits. When the proteins are folded and assembled, they are released from BiP by a process that is dependent on ATP hydrolysis. Since ATP is synthesized mainly in the mitochondria, we hypothesized that there must be an ATP transporter in the ER which would allow the protein mediated transport of ATP from the cytosol into the ER lumen. We studied the transport of ATP in vitro and found that ATP enters the lumen of the ER in a saturable manner with a Kmapp~3μM. ATP transport is dependent on time, protein, and vesicle integrity, it is also inhibited by the general anion transport inhibitor, 4,4' diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). We also found that the transport was inhibited by membrane impermeable protein modifying agents such as N-ethlymaleamide (NEM) and Pronase when added to intact ER vesicles. These results suggest that the transport is mediated by a protein with an active cytoplasmic face. Using monoclonal and polyclonal antibodies to BiP and Grp94 (another resident ER protein) and U.V. crosslinking, we demonstrated that after transport of ATPα32P into intact vesicles, radiolabeled BiP and Grp94 could be immunoprecipitated. We also found that labeling of lumenal proteins with ATP is dependent on the transport of ATP. Finally using ATP labeled with 35S, we concluded that BiP was able to bind intact ATP and we confirmed earlier work that BiP was thiophosphorylated while Grp94 is not. The second area of study involves processes that occur further along the secretory pathway in the Golgi apparatus. It was known from previous work that the nucleotide sugar substrates necessary for the synthesis of the linkage region, UDP-xylose (UDP-Xyl), UDP-galactose (UDP-Gal) and UDP-glucuronic acid (UDP-GlcA) were transported into the Golgi apparatus from the cytosol via protein mediated transporters. In order to eventually purify one of these transporter proteins, we wanted to reconstitute their activities. We were able to reconstitute the activities that exhibited kinetic parameters and inhibitor sensitivities very similar to those seen in intact Golgi vesicles. In the case of UDP-xylose it was necessary to prepare the liposomes using endogenous Golgi lipids in order to get transport activity similar to that seen in the intact Golgi vesicles. This suggested a specific lipid requirement for the UDP-xylose transporter. These transporters seem to be antiporters, whereby the nucleotide sugar enters the lumen of the Golgi coupled to the equimolar exit of the corresponding nucleoside monophosphate (Hirschberg, C.B. and Snider, M.D. 1987). We also showed that we could reproduce the hypothesized antiporter system in the reconstituted proteoliposomes by preloading the proteoliposomes with the putative antiporter molecule UMP. The rationale for developing the reconstituted system is eventually to use this system to purify one of these nucleotide sugar translocators. In the last set of studies, I have shown that this reconstituted system can be used to monitor the purification of the UDP-galactose translocator. Using column chromatography we were able to purify this membrane translocator protein 45,000 fold from a rat liver homogenate.