Digoxin has been known as a treatment for chronic heart failure for over 200 years. Its effect on the heart itself has been extensively studied and its inotropic effect well established. The inotropic effect of digoxin is the result of its inhibition of the membrane sodium pump or Na+/K+-ATPase, which plays an important role in maintaining the resting membrane potential across the plasma membrane through constantly pumping Na+ and K+ across the plasma membrane. Na+/K+-ATPase is not found exclusively in heart muscle. It is also found extensively throughout the brain. As digoxin is the drug of choice for pregnant woman with chronic heart failure, this study aimed to examine how digoxin affects brain development and neurons in culture. The well established chicken embryo animal model was used in this study. To probe for deviations from normal brain development, chicken embryos were exposed in ovo. Brains were examined using both transmission and scanning electron microscopy. Microscopy indicated significant damage to the neurons, specifically membranes and mitochondria, as well as cellular death by means of aponecrosis. An unexpected result was premature myelinogenesis in the brain. Chick embryo neurons (CEN) were exposed to digoxin in vitro and cell viability was assessed by performing crystal violet (CV) assays. Results showed that cell number increased over time. This is however, impossible as CEN are non-dividing cells and results were therefore interpreted as an increase in protein synthesis over time, correlating with the myelinogenesis results seen with electron microscopy. To assess membrane integrity, fluorescence microscopy was performed using propidium iodide as stain. Results from this experiment showed a sharp increase in propidium iodide uptake in exposed cells indicative of the membrane damage caused by digoxin. These results also correlated with the aponecrosis seen with electron microscopy, as the nuclei indicated apoptosis while propidium iodide is normally only absorbed by cells undergoing necrosis. Finally, a literature search was conducted to shed some light on the role that digoxin plays in serotonin production and levels in the brain. From the literature it seems that digoxin could increase serotonin production and elevate serotonin levels in the brain, which may influence normal brain development and may therefore play a role in myelinogenesis in the brain. / Dissertation (MSc (Anatomy))--University of Pretoria, 2008. / Anatomy / unrestricted

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Les glycosides cardiaques (GCs) utilisés en clinique contre les maladies cardiaques sont des inhibiteurs de la pompe sodium-potassium-ATPase. Récemment il a été montré que ces composés ont un effet anti-cancéreux. Nous avons étudié les effets de l’UNBS1450, un GC modifié partiellement extrait de la plante Calotropis procera. Nous avons analysé ses effets sur une protéine responsable de la résistance aux traitements anti-cancéreux : Mcl-1. L’UNBS1450 diminue son expression dans différents types de cancer à des concentrations très faibles. Cette modulation est capitale pour l’induction de la mort cellulaire. De plus, l’UNBS1450 diminue l’expression d’une protéine importante dans la prolifération cellulaire : c-Myc, cette modulation intervient dans les mêmes conditions, que celles qui permettent la diminution de Mcl-1. Nous avons observé que l’UNBS1450 est actif dans les cancers du sein, en bloquant le cycle cellulaire et la prolifération. Dans cette étude, nous avons montré que l’UNBS1450 et les CGs en général, d’une part induisent la mort cellulaire en diminuant l’expression de Mcl-1, et d’autre part affecte la prolifération cellulaire en modulant c-Myc / Cardiac glycosides (CGs) are used in clinics to treat heart diseases. They inhibit the sodium-potassium-ATPase. Recently, anti-cancers activities have been ascribed to these compounds. In this study, we investigated the anti-cancer effects of UNBS1450, a compound partially modified from a CG extracted from the plant Calotropis procera. We analyzed its activities against a protein responsible for the chemoresistance of cancer cells: Mcl-1. We found that UNBS1450 decreases the levels of Mcl-1 in many forms of cancer, at very low concentrations. This modulation is important to induce cancer cell death and is common to other CGs. Furthermore, UNBS1450 also decreases the intracellular levels of another protein important for the cancer cell proliferation: c-Myc. This modulation takes place in the same conditions determining Mcl-1 decrease. Finally, we found UNBS1450 active on breast cancer, by blocking the cell cycle and proliferation of the breast cancer cells. In this study, we have shown that UNBS1450 and CGs in general induce cell death by decreasing the expression of Mcl-1 protein and affect cell proliferation with the concomitant decrease of c-Myc protein

In an attempt to identify agents that specifically target neuroblastoma (NB) tumour-initiating cells (TIC) we performed drug screens using libraries of bioactive compounds. Cardiac glycosides (CGs) were the largest class of drugs identified with antitumour activity. At high CG doses inhibitory effects on the Na+/K+-ATPase induce cardiotoxicity; therefore, CG analogues were designed in an attempt to separate the effects on NB cells from cardiotoxicity. We identified RIDK34 as our lead compound from a structure-activity-relationship analysis (IC50 8 nM). RIDK34 contains a unique oxime group and shows increasing potency against NB TICs. The Na+/K+-ATPase is a target for the apoptotic activity of digoxin and RIDK34, whereby a signaling cascade involving Src and ERK may induce apoptosis. Furthermore, we predict that signaling activation does not require inactivation of the Na+/K+-ATPase and subsequent deregulation of [Na+]i and [K+]I gradients. Thus CGs and particularly RIDK34 may be expected to display diminished cardiotoxicity and greater therapeutic potential.

In an attempt to identify agents that specifically target neuroblastoma (NB) tumour-initiating cells (TIC) we performed drug screens using libraries of bioactive compounds. Cardiac glycosides (CGs) were the largest class of drugs identified with antitumour activity. At high CG doses inhibitory effects on the Na+/K+-ATPase induce cardiotoxicity; therefore, CG analogues were designed in an attempt to separate the effects on NB cells from cardiotoxicity. We identified RIDK34 as our lead compound from a structure-activity-relationship analysis (IC50 8 nM). RIDK34 contains a unique oxime group and shows increasing potency against NB TICs. The Na+/K+-ATPase is a target for the apoptotic activity of digoxin and RIDK34, whereby a signaling cascade involving Src and ERK may induce apoptosis. Furthermore, we predict that signaling activation does not require inactivation of the Na+/K+-ATPase and subsequent deregulation of [Na+]i and [K+]I gradients. Thus CGs and particularly RIDK34 may be expected to display diminished cardiotoxicity and greater therapeutic potential.

Dissertation (Ph.D.)--University of Toledo, 2008. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title in Ohio LINK ETD Center record : Na/K-ATPase signaling : from bench to bedside. Title from title page of PDF document. Bibliography: p. 99-120.

Thesis (Ph.D.)--Medical University of Ohio, 2005. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Joseph I. Shapiro. Includes abstract. Document formatted into pages: v, 265 p. Title from title page of PDF document. Bibliography: pages 52-59,94-100,129-134,171-176,200-263.

Made available in DSpace on 2015-05-14T12:59:34Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2299557 bytes, checksum: 01deb75646894a343a19a53f79cf2519 (MD5) Previous issue date: 2012-02-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Ouabain (OUA), a potent inhibitor of the Na+,K+-ATPase pump, was identified as an endogenous substance of human plasma. In recent years, ouabain was shown to affect various immunological processes. Mechanisms that involve cellular differentiation, proliferation, activation and migration, as well as inflammatory mediators release, are activated during inflammation and homeostasis is usually reestablished. This study demonstrated the modulatory ability of OUA on inflammatory process. Aim: This study aimed to evaluate ouabain immunomodulatory role on acute inflammatory process using a murine model. Methods: Initially, a dose and time-response curve was performed with OUA (0.10 mg/kg, 0.31 mg/kg and 0.56 mg/kg) intraperitoneally administered on the paw edema induced by zymosan (10 mg/mL). Mice were also intraperitoneally (i.p.) stimulated with zymosan (2 mg/mL). After 4h, the peritoneal fluid was removed for total and differential cell counts. Neutrophils and macrophages population, as well as cell viability, were analyzed using an annexin KIT by flow cytometry. The concentrations of the cytokines IL-1β, TNF-α, IL-6 and IL-10 in peritoneal lavage fluids were assayed using ELISA kit. Ouabain influence in the vascular permeability increase was determined using evans blue dye. OUA, in vitro, influence on nitric oxide (NO) production was also studied. Results: It was observed that OUA 0,56 mg/kg injected for three consecutive days prevented zymosan edema formation . After induction of inflammation, treatment with OUA led to a 42% reduction in the total cell numbers in the peritoneal cavity, as a reflex of the inhibition of polymorphonuclear leukocytes (54%), which was not due to cell apoptosis. Ouabain also decreased zymosan-induced plasma exudation (33%). Furthermore, OUA decreased the levels of TNF-α (64%) and IL-1β (63%), without interference on IL-6 and IL-10 levels. It was also demonstrated, using peritoneal macrophages, that ouabain did not interfere on LPS induced NO production. Conclusion: Ouabain modulated the acute inflammatory response induced by zymosan. However, further studies are necessary to elucidate the mechanisms involved / A ouabaína (OUA), um potente inibidor da Na+/K+-ATPase, foi identificada como uma substância endógena presente no plasma humano. Nos últimos anos, foi evidenciado que a OUA é capaz de interferir em diversos aspectos do sistema imunológico. Durante o processo inflamatório, são ativados mecanismos que envolvem a diferenciação, proliferação, ativação e migração celular, além da liberação de mediadores inflamatórios, e geralmente, ocorre o retorno à homeostasia. Este trabalho demonstrou a capacidade moduladora da OUA no processo inflamatório. Objetivo: Avaliar o papel imunomodulador da OUA na inflamação aguda em modelo murino. Métodos: Inicialmente, foi realizada uma curva de tempo e dose-resposta com a OUA (0,10 mg/kg; 0,31 mg/kg e 0,56 mg/kg) administrada de forma intra-peritoneal (i.p.) no modelo de edema de pata induzido por zimosan (10mg/mL). Os camundongos também foram estimulados com zimosan i.p.(2mg/ml). Após 4h, o fluido peritoneal foi removido para a contagem total e diferencial das células. Foi realizada a análise das populações de neutrófilos e macrófagos, além da viabilidade celular utilizando o kit anexina por citometria de fluxo. As concentrações das citocinas IL-1β, TNF-α, IL-6 e IL-10 no fluido peritoneal foram testadas por ELISA. Foi determinada a interferência do tratamento com a OUA no aumento da permeabilidade vascular. Também foi estudado, in vitro, o efeito de diferentes concentrações de OUA (10 nM, 100 nM e 1000 nM) na produção de óxido nítrico (NO). Resultados: No modelo do edema de pata, observamos que são necessários três dias consecutivos de tratamento na dose de 0,56 mg/kg para que a atividade anti-inflamatória da OUA seja identificada. No modelo de peritonite induzida por zimosan, o pré-tratamento com a OUA reduziu em 42% no número total de células na cavidade peritoneal, como um reflexo da inibição de leucócitos polimorfonucleares (54%). No entanto, este fenômeno não esta associado a apoptose destas células. A OUA também diminuiu o extravasamento de plasma induzido por zimosan (33%) e reduziu os níveis de TNF-α (64%) e IL-1β (63%), sem alterar os níveis de IL-6 e IL-10. Na cultura de macrófagos peritoneais, a OUA não interferiu na produção de NO. Conclusão: Este conjunto de dados sugere que a OUA possui uma atividade anti-inflamatória. No entanto, mais estudos são necessários para elucidar os mecanismos envolvidos.

South Africa has one of the richest and diverse floras in the world with over 30000 species of higher plants. There are approximately 3000 species of medicinal plants in South Africa. The discovery of active compounds in medicinal plants plays a strategic role in the phytochemical investigation of crude plant extracts. Secondary metabolites of medicinal plants are a major source of drugs for the treatment of various health disorders. Cardiac glycosides are one of the subgroups of steroids modified from terpenoids. The existence of cardiac glycosides in some plant species often indicates toxicity. Among the contagious elements, diseases caused by viruses are one of the major causes of death, disability, and social and economic disruption for millions of people. Viruses also cause many important plant diseases and are responsible for huge losses in crop production and quality in all parts of the world. According to literature, plants containing cardiac glycosides demonstrate potential for the discovery of more effective new drugs in the treatment of infection especially viral contagions. The main objectives of this study were to evaluate the antiviral (HSV-2 and PVYN), antibacterial and cytotoxic activities of South African plants containing cardiac glycosides. Furthermore, isolation and identification of compounds with an emphasis on cardiac glycosides was carried out. Eight plants known to contain cardiac glycoside compounds were selected. Leaves of Gomphocarpus fruticosus, Nerium oleander, Cotyledon orbiculata, the fruits and leaves of Strophanthus speciosus, the bulbs of Bowiea volubilis and Merwilla plumbea were selected to examine their antimicrobial activities, toxicity, antiviral and to isolate potential pure compounds. The ethanol extracts of all selected plants were screened for antibacterial activity against two Gram-positive (Staphylococcus aureus and Enterococcus faecalis) and two Gram-negative (Escherichia coli and Klebsiella pneumonia) pathogens. Plant extracts showed promising antibacterial action against Gram-positive bacteria. The most active extract against both Gram-positive bacteria was C. orbiculata with the MIC value of 1.25 mg/ml. Cell toxicity was monitored by determining the effect of the ethanolic crude extracts on human embryonic kidney cell line (HEK 293) using the XTT method. All extracts exhibited high toxic effects with IC50 < 100 μg/ml on the tested cell line. The XTT assay was used to determine of the antiviral activity of crude ethanolic extracts on the kidney epithelial cells of African Green Monkey (Vero). The results revealed that the crude ethanolic extracts of all iv selected plants exhibited a cytotoxic effect on Vero cells at concentrations lower than their EC50. Consequently, the determination of antiviral activity of the selected plant extracts was not successful. Based on the chromatographic and bioassay results from the six plants selected for this study, the fruit extract of S. speciosus, belonging to the Apocynaceae family, was chosen for the isolation of compounds, particularly cardiac glycosides. All plant extracts were tested for antibacterial activity against E. coli (Gram-negative) and E. faecalis (Gram-positive) by using bioautography. The general TLC test showed more variety of compounds in the fruit extract of S. speciosus. The results of the bioassay showed promising activity of the fruit extract of S. speciosus and the bulb extract of B. volubilis against Gram-positive bacteria. The chromatographic investigation of the S. speciosus fruit extract led to the isolation of three pure compounds including a cardiac glycoside. The three compounds were identified based on NMR (1D and 2D) and HRMS. The isolated compounds were identified as: a triterpene (ursolic acid methyl ester), a sugar: myo-inositol methyl ester and an unidentified cardiac glycoside. According to literature, it is the first report of the isolation of ursolic acid methyl ester and myo-inositol-methyl ether from S. speciosus fruit extract. The isolated cardiac glycoside exhibited no inhibitory activity at 1.25 mg/ml (the highest concentration tested) against all four tested bacteria (S. aureus, E. faecalis, E. coli and K. pneumonia). The cytotoxicity and anti-HSV-2 screening of the isolated cardenolide demonstrated the highly toxic effect of this compound on the HEK 293 cell line with 4.62 μg/ml IC50 value and < 25 μg/ml IC50 of Vero cell line. No evidence could be found in the literature of the cytotoxic activity of cardiac glycoside compounds on the HEK 293 cell line. The ethanolic extracts of all plant extracts and isolated cardenolide were tested against the PVYN in vivo and in vitro. The results revealed that the high concentration (50 mg/ml) of M. plumbea, N. oleander, B. volubilis (fresh bulb), C. orbiculata and isolated cardenolide reduced the PVYN symptoms on tobacco plants in an in vivo experiment. In in vitro analysis, the high concentration (50 mg/ml) of S. speciosus (leaves & fruits), and especially M. plumbea (dry bulb) showed significant antiphytoviral activity. In vivo and in vitro results demonstrated that M. plumbea has potential antiphytoviral activity. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Plant Science / Unrestricted

The aim of this study has been to explore the site specific phosphorylation pattern of rat heart glycogen synthase paying particular attention to phosphorylations that are important to the in vivo control of enzyme activity. This problem has been approached using techniques of immuneprecipitation of 32P labeled synthase from hormonally responsive, freshly isolated adult rat cardiomyocytes. Identification of the active subunit of rat heart glycogen synthase was accomplished by immuneprecipitating synthase from 32P-labeled cardiomyocytes and performing Western blot analysis on DEAE-cellulose fractions containing synthase activity. Using these methods, glycogen synthase activity has been localized to a protein of 88,000 daltons. Reverse phase HPLC analysis of synthase tryptic peptides from either hormone responsive cardiomyocytes or synthase treated in vitro with cAMP-dependent protein kinase and protein phosphatase-1 (PP-1) resulted in finding six reproducible peaks of phosphopeptides. The incorporation of radioactivity into peaks 1 and 2 was associated with both the treatment of cardiomyocytes with epinephrine and the in vitro phosphorylation of rat heart synthase with cAMP-dependent protein kinase. These same two peaks are selectively dephosphorylated when cAMP-dependent kinase treated synthase is incubated with protein phosphatase-1. This dephosphorylation of peaks 1 and 2 are coincident with the conversion of synthase from the D to the I form. Peak 3 is dephosphorylated upon treatment of cardiomyocytes with insulin and hyperphosphorylated in cardiomyocytes derived from alloxan diabetic animals. Taken together these results demonstrates the direct relationship between the phosphopeptides in peaks 1 and 2 and the inhibition of synthase activity in response to epinephrine treatment in the cell. This inhibition can be explained by the activity of cAMP-dependent protein kinase which can duplicate the intracellular, epinephrine-stimulated synthase phosphopeptide pattern. This inhibition can be relieved in vitro by protein phosphatase-1 which dephosphorylates peaks 1 and 2. The effect of insulin and alloxan diabetes is localized to peak 3 whose phosphorylation is unaffected in vitro by either cAMP-dependent protein kinase or protein phosphatase-1.