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Schistosoma mansoni : role of antioxidant systems in protection of developmental stages against oxidative killing and the effects of oltipraz on glutathione S-transferaseNare, Bakela January 1991 (has links)
This study shows that resistance to killing by reactive oxygen intermediates (ROI) increases during migration and development in Schistosoma mansoni. Resistance is associated with the protective role of antioxidants as shown by the increased levels of superoxide dismutase and of the glutathione system enzymes. Hydroperoxide-dependent glutathione peroxidase activity was not detectable in newly transformed schistosomula, however the activity was present in the liver stages. The antischistosomal drug oltipraz (OPZ) decreased in an irreversible manner the activity of S. mansoni glutathione S-transferase (GST), an important protective enzyme, both in vivo and in vitro. The inhibition of GST activity was not isoenzyme restricted and was non-competitive with respect to the two substrates essential for GST activity. On the other hand, OPZ treatment increased the levels of mouse (S. mansoni host) liver GST activity in an isoenzyme specific manner, with the $ mu$ class subunit induction accounting for most of the increase. However, mammalian GST activity was inhibited by OPZ in vitro. However, the inhibition of mammalian GST activity was reversible upon addition of dithiol reducing compounds. OPZ inhibited the binding of ($ sp{14}$C) N-ethylmaleimide (specifically alkylates SH groups), suggesting that OPZ interacts with SH-groups of GST to inhibit its enzymatic activity. Another SH-dependent enzyme, hexokinase, from yeast and S. mansoni was reversibly inhibited by OPZ. The oxy-analogue of OPZ, in which the thione sulphur is replaced with oxygen, did not inhibit the enzymatic activity of GST and hexokinase. Many of the biochemical effects of OPZ on S. mansoni and its mammalian hosts may be related to its ability to bind to SH groups and inactivation of the functions of many essential proteins.
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Schistosoma mansoni : role of antioxidant systems in protection of developmental stages against oxidative killing and the effects of oltipraz on glutathione S-transferaseNare, Bakela January 1991 (has links)
No description available.
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Cell-based phenotypic screens to identify modulators of sensitivity to N-methyl-N'-nitro-N-nitrosoguanidinePedley, Nicholas Michael January 2011 (has links)
Defective DNA repair capacity has been shown to be a common feature of cancer, and loss of function mutations in 'stability' genes that normally maintain the integrity of the genome may prove a key rate-limiting step in carcinogenesis. Since even genetically unstable cells require some repair functionality to maintain viability, these cancers likely exhibit an over-reliance on other DNA repair pathways for survival. Therapeutically targeting backup repair processes in such tumours represents a novel means by which to achieve selective tumour toxicity. Full exploitation of these synthetic lethal interactions will require an in-depth knowledge of the genetic basis of DNA repair in combination with an armoury of small molecule inhibitors of cellular targets. To this end, we have designed, optimised and run two high-throughput cell-based screens to identify genes and small molecules that can modulate mismatch repair (MMR) activity. Key to these screening strategies are the resistance of cells with dysfunctional MMR to a range of cytotoxic drugs, including the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). By exploiting this MMR-dependent toxicity we have assayed for siRNA and small molecules that permit the survival of MNNG-treated MMR-proficient cells to levels comparable to MMR-deficient cells, and which therefore represent putative MMR modulating agents. A screen of 571 siRNA for gene depletions that reduce MNNG sensitivity by at least two population standard deviations identified 10 genes of potential interest, and included the four canonical MMR genes, MSH2 (2.87 ± 0.28 (Z ± SE)), MSH6 (4.87 ± 0.06), MLH1 (3.42 ± 0.43) and PMS2 (3.36 ± 0.44). TDG represented an unexpected hit that decreased MNNG sensitivity by 2.55 ± 0.04 population standard deviations. However, clonogenic survival experiments found TDG depletion to be contextual synthetic lethal within an MMR-null background when treated with MNNG, reducing HCT116 clonogenicity by 37% (p < 0.001). Moreover, TDG knockdown increased the number of 53 binding protein 1 (53bp1) foci in MMR-proficient cells by 40% and MMR–deficient cells by 27% following MNNG exposure (p < 0.001). Combined with a failure to replicate the primary screen result, the role for TDG in the response to MNNG could be explained solely through its established role as a member of the base excision repair pathway. A second screen of the NCI Diversity I and II small molecule libraries (n=1786) was conducted to identify putative MMR inhibitors. Subsequent analysis revealed NSC197049 to increase cellular viability of MNNG treated cells by 3.60±0.32 population standard deviations and was successfully validated as a hit. Co-treatment of NSC197049 with MNNG conferred dose-dependent chemoprotection independently of MMR status and cell line, an effect that was lost if NSC197049 was pre- or post-treated. The protection was associated with a reduction in MNNG-dependent 53bp1 foci of 60% in MMR proficient cells and 15% in MMR deficient cells (p < 0.001), together with a marked reduction of > 80% in subG1 content at 48 hours post-MNNG that was independent of MMR status. Interestingly, the characteristic G2/M arrest of MNNG-treated MMR-proficient cells remained intact (~40% arrested). Taken together, these observations are not consistent with NSC197049 acting as an inhibitor of MMR. Dithiolthiones have been described as chemoprotective agents that induce antioxidant defences, whilst we have found NSC197049 phenocopies known antioxidants ascorbic acid and glutathione in protecting against MNNG-induced toxicity. NSC197049 may therefore act by bolstering cellular antioxidant defences. The precise mechanism may be novel, since the proto-typical dithiolthione, Oltipraz, failed to be protective in this study. In summary, we have confirmed that MMR is the primary determinant of MNNG sensitivity, and found that TDG is unlikely to be involved in MMR. We have also identified a novel chemoprotective small molecule that is unlikely to represent an MMR inhibitor, but that might be useful in cancer chemoprevention.
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Hypertension et régulation de l'expression moléculaire de l'angiotensinogène par la ribonucléoprotéine hétérogène nucléaire KAbdo, Shaaban 06 1900 (has links)
Le diabète est une maladie chronique dont la principale
caractéristique est un niveau plasmatique élevé de glucose, qui est causé
soit par un défaut dans la production d’insuline, l’action de l’insuline, ou
les deux à la fois. Plusieurs études ont démontré que l’hyperglycémie
chronique peut mener à la dysfonction et même la défaillance de
plusieurs organes, dont le coeur, le système vasculaire, les yeux et les
reins, se traduisant par des infarctus du myocarde, des accidents
cérébro-vasculaires et des complications rétinales et rénales,
respectivement. La néphropathie diabétique (DN) est la principale cause
de déficience rénale et affecte près de 25-40% des patients diabétiques.
La DN est invariablement associée à un risque élevé d’accident cérébrovasculaire
et de dysfonction cardivasculaire. L’angiotensinogène (Agt) est
l’unique précurseur de tous les types d’angiotensines. En plus du
système rénine-angiotensine (RAS) sytémique, le rein possède son
propre système intrarénal et exprime tous les composants du RAS. L’Agt
est fortement exprimé dans les cellules du tubule proximal rénal (RPTC)
et y est converti en angiotensine II (AngII), le peptide biologiquement actif
du RAS. Les patients diabétiques présentent de hauts niveaux d’AngII et
une augmentation de l’expression des gènes du RAS, suggérant que
l’activation du RAS intrarénal joue un rôle important dans la progression
de la DN. Les mécanismes qui contrôlent la régulation du niveau rénal
d’Agt par l’hyperglycémie et l’insuline demeurent mal compris.
Le but global de cette thèse est de mieux comprendre les
mécanismes moléculaires qui contrôlent l’expression du gène Agt chez la
souris Akita (un modèle murin de diabète de type 1). Dans cette optique,
la première partie de la thèse se concentre sur deux facteurs de
transcription de la famille des ribonucléoprotéines nucléaires hétérogènes
(hnRNP). Chan et collaborateurs ont déjà identifié 2 protéines nucléaires
hnRNP F et hnRNP K, de 48kD et 70kD respectivement. HnRNP F et
hnRNP K forment un hétérodimère et se lient à l’élément de réponse à
l’insuline (IRE) présent dans le promoteur du gène Agt du rat et inhibent
la transcription du gène Agt in vitro. Afin de déterminer si hnRNP F / K
sont responsables de l’inhibition de l’expression rénale de Agt par
l’insuline in vivo, nous avons étudié des souris Akita males traités ou non
avec des implants d’insuline pour une période de 4 semaines. Des souris
non-Akita males ont été employées comme contrôles. Les souris Akita
développent de l’hypertension et de l’hypertrophie rénale. Le traitement à
l’insuline rétablit les niveaux de glucose plasmatiques et la pression
systolique (SBP), et atténue l’hypertrophie rénale, l’albuminurie (ratio
albumine/créatinine urinaire, ACR) et les niveaux urinaires d’Agt et AngII
chez les souris Akita. De plus, le traitement à l’insuline inhibe l’expression
rénale du gène Agt, tout en augmentant l’expression des gènes hnRNP
F, hnRNP K et ACE2 (enzyme de conversion de l’angiotensine-2). Dans
des RPTC in vitro, l’insuline inhibe Agt, mais stimule l’expression de
hnRNP F et hnRNP K en présence de hautes concentrations de glucose,
et ce via la voie de signalisation MAPK p44/42 (protéine kinase activée
par un mitogène). La transfection avec des petits ARN interférents
(siRNA) contre hnRNP F et hnRNP K prévient l’inhibition de l’expression
d’Agt par l’insuline dans les RPTC. Cette étude démontre bien que
l’insuline prévient l’hypertension et atténue les dommages rénaux
observés chez les souris Akita diabétiques, en partie grâce à la
suppression de la transcription rénale de Agt, via une augmentation de
l’expression de hnRNP F et hnRNP K.
La seconde partie de cette thèse change de focus et se tourne
vers le facteur Nrf2 (nuclear factor erythroid 2-related factor 2). Nrf2 est
un facteur de transcription qui contrôle les gènes de la réponse
antioxydante cellulaire en réponse au stress oxydant ou aux
électrophiles. Le but de cette étude est d’examiner l’impact de la
surexpression de la catalase (Cat) dans les RPTC sur l’expression du
gène Agt via Nrf2 et sur le développement de l’hypertension et des
dommages rénaux résultants chez les souris diabétiques Akita
transgéniques (Tg). Nos études ont démontré que la surexpression de
Cat dans les souris Akita Cat-Tg normalise la SBP, atténue les
dommages rénaux et inhibe l’expression des gènes Nrf2 et Agt dans les
RPTC. In vitro, le glucose élevé (HG) et l’oltipraz (un activateur de Nrf2)
stimulent l’expression de Nrf2 et Agt, et cet effet peut être bloqué par la
trigonelline (inhibiteur de Nrf2), des siRNA contre Nrf2, des antioxydants
ou des inhibiteurs pharmacologiques NF-κB et MAPK p38. La
suppression de sites de réponse à Nrf2 présents dans le promoteur du
gène Agt du rat abolit la stimulation par l’oltipraz. Finalement, des souris
males adultes non-transgéniques traitées avec l’oltipraz montrent une
augmentation de l’expression de Nrf2 et Agt dans leurs RPTC et cette
augmentation peut être normalisée par la trigonelline. Ces données
permettent d’identifier un nouveau mécanisme d’action de Nrf2, par la
stimulation du gène Agt intrarénal et l’activation du RAS, qui induisent
l’hypertension et les dommages rénaux par le glucose élevé et les
espèces réactives de l’oxygène chez les souris diabétiques. Nos
conclusions permettent de démontrer que l’insuline induit l’expression de
hnRNP F et hnRNP K, qui jouent ensuite un rôle protecteur en prévenant
l’hypertension. La surexpression de la catalase dans les RPTC vient
quant à elle atténuer l’activation de Nrf2 et ainsi réduit la SBP chez les
souris Akita. / Diabetes mellitus is a chronic metabolic disorder characterized by
high plasma glucose caused by an impairment of insulin production,
insulin action or both. Accumulating evidence has shown that chronic
hyperglycemia can lead to dysfunction and failure of multiple organs
including the heart, vascular system, eyes, and kidneys resulting in
myocardial infarction, stroke, and retinal and renal complications,
respectively. Diabetic nephropathy (DN) is the leading cause of end-stage
renal disease affecting approximately 25–40% of diabetic patients. DN is
invariably associated with an increased risk of stroke and cardiovascular
dysfunction. Angiotensinogen (Agt) is the sole precursor for all types of
angiotensins. In addition to systemic renin-angiotensin system (RAS), all
the components of the intrarenal RAS are expressed in the kidney. Agt is
highly expressed in the renal proximal tubular cells (RPTCs) and
converted into biologically active angiotensin II (Ang II). In Diabetics,
intrarenal Ang II level and RAS gene expression are upregulated,
suggesting that intrarenal RAS activation plays an important role in the
progression of DN. The mechanism (s) underlying the regulation of renal
Agt by hyperglycemia and insulin are not completely understood. The
overall aim of this thesis is to understand the molecular mechanism(s)
that regulate renal Agt gene expression in an Akita mouse (a mouse
model of type 1 diabetes). For this purpose, the first part of this thesis
focuses on two transcription factors from the heterogenous nuclear
ribonucleoprotein (hnRNPs) family. Previously, Chan’s group identified
two nuclear proteins hnRNP F and hnRNP K of 48kD and 70kD,
respectively. hnRNP F and hnRNP K form a heterodimer and bind to the
insulin-responsive element (IRE) in the rat Agt gene promoter inhibiting
Agt gene transcription in vitro. To determine whether hnRNP F / K
mediate insulin inhibition of renal Agt expression in vivo, we used adult
male Akita mice treated ± insulin implants for 4 weeks. Non-Akita mice
served as controls. The Akita mice developed hypertension and exhibited
renal hypertrophy. Insulin treatment normalized plasma glucose levels
and systolic blood pressure (SBP), attenuated renal hypertrophy,
decreased urinary albumin/creatinine ratio (ACR) and urinary Agt and
Ang II levels in Akita mice. Furthermore, insulin treatment inhibited renal
Agt expression but enhanced hnRNP F, hnRNP K and angiotensinconverting
enzyme-2 (ACE2) expression. In vitro, insulin inhibited Agt but
stimulated hnRNP F and hnRNP K expression in high-glucose media via
p44/42 mitogen-activated protein kinase signaling in RPTCs. Transfection
with hnRNP F and hnRNP K small interfering RNAs (siRNA) prevented
the insulin inhibition of Agt expression in RPTCs. This study
demonstrates that insulin prevents hypertension and attenuates kidney
injury, at least in part, through suppressing renal Agt transcription via
upregulation of hnRNP F and hnRNP K expression in diabetic Akita mice.
In the second part of the thesis we focused on the nuclear factor
erythroid 2-related factor 2 (Nrf2). Nrf2 is a transcription factor that
regulates cellular antioxidant gene defense against oxidative stress or
electrophiles. The purpose of this study is to investigate the impact of the
overexpressing catalase (Cat) in RPTCs on Agt gene expression via
Nrf2and the resulting effects on the development of hypertension and
renal injury in diabetic Akita transgenic (Tg) mice. Our studies
demonstrate that Cat overexpression normalizes SBP, attenuates renal
injury, and inhibits RPTC Nrf2 and Agt gene expression in the Akita Cat-
Tg compared to Akita mice. In vitro, high glucose (HG) and Oltipraz
stimulated Nrf2 and Agt gene expression; these changes were blocked by
Trigonelline (an inhibitor of Nrf2), siRNA against Nrf2, antioxidants, or
pharmacological inhibitors of NF-kB and p38 mitogen-activated protein
kinase. Moreover, deletion of Nrf2-responsive elements in the rat Agt
gene promoter abolishes the stimulatory effect of Oltipraz. Finally,non
transgenic adult male mice treated with the Nrf2 activator Oltipraz,
upregulated Nrf2 and Agt expression in mouse RPTs, an effect that was
normalized by Trigonelline. These data identify a novel mechanism via
which Nrf2 mediates the stimulation of intrarenal Agt gene expression and
activates the RAS through whichHG/reactive oxygen species (ROS)
induce hypertension and renal injury in diabetic mice. Our findings
demonstrate that the insulin induced hnRNP F and hnRNP K gene
expression play a protective role in the preventing hypertension. Catalase
overexpression, in RPT's, attenuates Nrf2 activation and lowers the SBP
in Akita mice.
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