Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

Changes in cardiac aldosterone and its synthase in rats with chronic heart failure: an intervention study of long-term treatment with recombinant human brain natriuretic peptide

The physiological mechanisms involved in isoproterenol (ISO)-induced chronic heart failure (CHF) are not fully understood. In this study, we investigated local changes in cardiac aldosterone and its synthase in rats with ISO-induced CHF, and evaluated the effects of treatment with recombinant human brain natriuretic peptide (rhBNP). Sprague-Dawley rats were divided into 4 different groups. Fifty rats received subcutaneous ISO injections to induce CHF and the control group (n=10) received equal volumes of saline. After establishing the rat model, 9 CHF rats received no further treatment, rats in the low-dose group (n=8) received 22.5 μg/kg rhBNP and those in the high-dose group (n=8) received 45 μg/kg rhBNP daily for 1 month. Cardiac function was assessed by echocardiographic and hemodynamic analysis. Collagen volume fraction (CVF) was determined. Plasma and myocardial aldosterone concentrations were determined using radioimmunoassay. Myocardial aldosterone synthase (CYP11B2) was detected by quantitative real-time PCR. Cardiac function was significantly lower in the CHF group than in the control group (P<0.01), whereas CVF, plasma and myocardial aldosterone, and CYP11B2 transcription were significantly higher than in the control group (P<0.05). Low and high doses of rhBNP significantly improved hemodynamics (P<0.01) and cardiac function (P<0.05) and reduced CVF, plasma and myocardial aldosterone, and CYP11B2 transcription (P<0.05). There were no significant differences between the rhBNP dose groups (P>0.05). Elevated cardiac aldosterone and upregulation of aldosterone synthase expression were detected in rats with ISO-induced CHF. Administration of rhBNP improved hemodynamics and ventricular remodeling and reduced myocardial fibrosis, possibly by downregulating CYP11B2 transcription and reducing myocardial aldosterone synthesis.

Hormone therapy with tamoxifen reduces plasma levels of NT-B-type natriuretic peptide but does not change ventricular ejection fraction after chemotherapy in women with breast cancer

The objective of this study was to evaluate the effect of tamoxifen on the plasma concentration of NT-pro-B-type natriuretic peptide (NT-proBNP) in women undergoing chemotherapy for breast cancer and to correlate changes in NT-proBNP with the left ventricular ejection fraction (LVEF). Over a period of 12 months, we followed 60 women with a diagnosis of breast cancer. The patients were separated into a group that received only chemotherapy (n=23), a group that received chemotherapy + tamoxifen (n=21), and a group that received only tamoxifen (n=16). Plasma levels of NT-proBNP were assessed at 0 (T0), 6 (T6), and 12 (T12) months of treatment, and echocardiography data were assessed at T0 and T12. Plasma NT-proBNP levels were increased in the chemotherapy-only group at T6 and T12, whereas elevated NT-proBNP levels were only found at T6 in the chemotherapy + tamoxifen group. At T12, the chemotherapy + tamoxifen group exhibited a significant reduction in the peptide to levels similar to the group that received tamoxifen alone. The chemotherapy-only group exhibited a significant decrease in LVEF at T12, whereas the chemotherapy + tamoxifen and tamoxifen-only groups maintained levels similar to those at the beginning of treatment. Treatment with tamoxifen for 6 months after chemotherapy significantly reduced the plasma levels of NT-proBNP and did not change LVEF in women with breast cancer.

Peptide separation of commercial fermented milk during refrigerated storage

Milk is an important source of bioactive compounds. Many of these compounds are released during fermentation and refrigerated storage. The aim of this study was to determine the release of peptides by lactic acid bacteria in commercial fermented milk during refrigerated storage. The size and profile of peptides were analyzed by polyacrylamide gel electrophoresis and sizeexclusion HPLC. During electrophoresis, it was observed that the peptides were released from caseins, whereas β-lactoglobulin was the whey protein with the highest degradation. HPLC analysis confirmed the pattern of peptide formation observed in electrophoresis. Two fractions lower than 2 kDa with aromatic amino acids in their structure were separated. These results were consistent with those reported for structures of peptides with antihypertensive activity. Therefore, the presence of aromatic amino acids in the peptide fractions obtained increases the likelihood of finding peptides with such activity in refrigerated commercial fermented milk. In conclusion, during cold storage, peptides with different molecular weights are released and accumulated. This could be due to the action of proteinases and peptidases of the proteolytic system in lactic acid bacteria.

Modulation of muscle contraction by a FMRFamide-related peptide

A FMRFamide-like neuropeptide with the sequence "DRNFLRF-NH2" was recently isolated from pericardial organs of crayfish (Mercier et aI., Peptides, 14, 137-143, 1993). This neuropeptide, referred to as "DF2'" has already been shown to elicit cardioexcitation and to enhance synaptic transmission at neuromuscular junctions. Possible effects ofDF2 on muscle were investigated using superficial extensor muscles of the abdomen of the crayfish, Procambarus clar/ai. These muscles are of the tonic type and generate slow contractions that affect posture. DF2, at concentrations of 10-8 M or higher, increased muscle tonus and induced spontaneous, rhythmic contractions. These effects were antagonized by 5 rnM Mn2+ but not by lO-7M tetrodotoxin (TTX). Thus, they represent direct actions on muscle cells (rather than effects on motor neurons) and are likely to involve calcium influx. In contrast, deep abdominal extensor muscles, responsible for rapid swimming movements, and superficial flexor muscles do not generate contractions in response to the peptide. 2 Spontaneous contractions were also induced in the superficial extensor muscles by decreasing the temperature to II-13°C. Such contractions were also TTX-insensitive and they were antagonized by adding calcium channel blockers (Mn2+, Cd2+ or Ni2+) or by removing calcium from the bathing solution. This suggests that the spontaneous contractions depend on an influx of calcium from the extracellular solution. N-type and L-type voltage dependent calcium channel blockers did not reduce the effect of the peptide or the spontaneous contractions suggesting that calcium influx is not through N- or L-type calcium channels.

Unique and unifying themes in the mechanisms regulating the expression of the arabidopsis thaliana PR-1 and Solanum tuberosum PR-10a inducible defense genes

Arabidopsis is a model plant used to study disease resistance; Solanum tuberosum or potato is a crop species. Both plants possess inducible defense mechanisms that are deployed upon recognition of pathogen invasion. Transcriptional reprogramming is crucial to the activation of defense responses. The Pathogenesis-Related (PR) genes are activated in these defense programs. Expression of Arabidopsis PR-l and potato PR-10a serve as markers for the deployment of defense responses in these plants. PR-l expression indicates induction of systemic acquired resistance (SAR). Activation of SAR requires accumulation of salicylic acid (SA), in addition to the interaction of the non-expressor of pathogenesis-related genes I (NPRI), with the TGA transcription factors. The PR-10a is activated in response to pathogen invasion, wounding and elicitor treatment. PR-10a induction requires recruitment of the Whirly I (Whyl) activator to the promoter. This locus is also negatively regulated by the silencer element binding factor (SEBF). We established that both the PR-l and PR-10a are occupied by repressors under non-inducing conditions. TGA2 was found to be a constitutive resident and repressor of PR-l, which mediates repression by forming an oligomeric complex on the promoter. The DNA-binding activity of this oligomer required the TGA2 N-terminus (NT). Under resting conditions we determined that the PR-10a is bound by a repressosome containing SEBF and curiously the activator Pto interacting protein 4 (Pti4). In the context of this repressosome, SEBF is responsible for PR-10a binding, yet rWe also showed that PR-l and PR-10a are activated by different means. In PR-l activation the NPRI NT domain alleviates TGA2-mediated repression by interacting with the TGA2 NT. TGA2 remains at the PR-l but adopts a dimeric conformation and forms an enhanceosome with NPRl. In contrast, the PR-10a is activated by evicting the repressosome and recruiting Why! to the promoter. These results advance our understanding of the mechanisms regulating PR-l and PR-10a expression under resting and inducing conditions. This study also revealed that the means of regulation for related genes can differ greatly between model and crop s

Mode of action of FMRFamide-like peptide on drosophila body wall muscle conractions

Neuropeptides are the largest group of signalling chemicals that can convey the information from the brain to the cells of all tissues. DPKQDFMRFamide, a member of one of the largest families of neuropeptides, FMRFamide-like peptides, has modulatory effects on nerve-evoked contractions of Drosophila body wall muscles (Hewes et aI.,1998) which are at least in part mediated by the ability of the peptide to enhance neurotransmitter release from the presynaptic terminal (Hewes et aI., 1998, Dunn & Mercier., 2005). However, DPKQDFMRFamide is also able to act directly on Drosophila body wall muscles by inducing contractions which require the influx of extracellular Ca 2+ (Clark et aI., 2008). The present study was aimed at identifying which proteins, including the membrane-bound receptor and second messenger molecules, are involved in mechanisms mediating this myotropic effect of the peptide. DPKQDFMRFamide induced contractions were reduced by 70% and 90%, respectively, in larvae in which FMRFamide G-protein coupled receptor gene (CG2114) was silenced either ubiquitously or specifically in muscle tissue, when compared to the response of the control larvae in which the expression of the same gene was not manipulated. Using an enzyme immunoassay (EIA) method, it was determined that at concentrations of 1 ~M- 0.01 ~M, the peptide failed to increase cAMP and cGMP levels in Drosophila body wall muscles. In addition, the physiological effect of DPKQDFMRFamide at a threshold dose was not potentiated by 3-lsobutyl-1-methylxanthine, a phosphodiesterase inhibitor, nor was the response to 1 ~M peptide blocked or reduced by inhibitors of cAMP-dependent or cGMP-dependent protein kinases. The response to DPKQDFMRFamide was not affected in the mutants of the phosholipase C-~ (PLC~) gene (norpA larvae) or IP3 receptor mutants, which suggested that the PLC-IP3 pathway is not involved in mediat ing the peptide's effects. Alatransgenic flies lacking activity of calcium/calmodul in-dependent protein kinase (CamKII showed an increase in muscle tonus following the application of 1 JlM DPKQDFMRFamide similar to the control larvae. Heat shock treatment potentiated the response to DPKQDFMRFamide in both ala1 and control flies by approximately 150 and 100 % from a non heat-shocked larvae, respectively. Furthermore, a CaMKII inhibitor, KN-93, did not affect the ability of peptide to increase muscle tonus. Thus, al though DPKQDFMRFamide acts through a G-protein coupled FMRFamide receptor, it does not appear to act via cAMP, cGMP, IP3, PLC or CaMKl1. The mechanism through which the FMRFamide receptor acts remains to be determined.

The Arabidopsis NPR1 Protein Is a Receptor for the Plant Defense Hormone Salicylic Acid

Systemic Acquired Resistance (SAR) is a type of plant systemic resistance occurring against a broad spectrum of pathogens. It can be activated in response to pathogen infection in the model plant Arabidopsis thaliana and many agriculturally important crops. Upon SAR activation, the infected plant undergoes transcriptional reprogramming, marked by the induction of a battery of defense genes, including Pathogenesis-related (PR) genes. Activation of the PR-1 gene serves as a molecular marker for the deployment of SAR. The accumulation of a defense hormone, salicylic acid (SA) is crucial for the infected plant to mount SAR. Increased cellular levels of SA lead to the downstream activation of the PR-1 gene, triggered by the combined action of the Non-expressor of Pathogenesis-related Gene 1 (NPR1) protein and the TGA II-clade transcription factor (namely TGA2). Despite the importance of SA, its receptor has remained elusive for decades. In this study, we demonstrated that in Arabidopsis the NPR1 protein is a receptor for SA. SA physically binds to the C-terminal transactivation domain of NPR1. The two cysteines (Cys521 and Cys529), which are important for NPR1’s coactivator function, within this transactivation domain are critical for the binding of SA to NPR1. The interaction between SA and NPR1 requires a transition metal, copper, as a cofactor. Our results also suggested a conformational change in NPR1 upon SA binding, releasing the C-terminal transactivation domain from the N-terminal autoinhibitory BTB/POZ domain. These results advance our understanding of the plant immune function, specifically related to the molecular mechanisms underlying SAR. The discovery of NPR1 as a SA receptor enables future chemical screening for small molecules that activate plant immune responses through their interaction with NPR1 or NPR1-like proteins in commercially important plants. This will help in identifying the next generation of non-biocidal pesticides.

Modeling short-range ballistic missile defense and Israel's Iron Dome system

This paper develops a model of short-range ballistic missile defense and uses it to study the performance of Israel’s Iron Dome system. The deterministic base model allows for inaccurate missiles, unsuccessful interceptions, and civil defense. Model enhancements consider the trade-offs in attacking the interception system, the difficulties faced by militants in assembling large salvos, and the effects of imperfect missile classification by the defender. A stochastic model is also developed. Analysis shows that system performance can be highly sensitive to the missile salvo size, and that systems with higher interception rates are more “fragile” when overloaded. The model is calibrated using publically available data about Iron Dome’s use during Operation Pillar of Defense in November 2012. If the systems performed as claimed, they saved Israel an estimated 1778 casualties and $80 million in property damage, and thereby made preemptive strikes on Gaza about 8 times less valuable to Israel. Gaza militants could have inflicted far more damage by grouping their rockets into large salvos, but this may have been difficult given Israel’s suppression efforts. Counter-battery fire by the militants is unlikely to be worthwhile unless they can obtain much more accurate missiles.

In Defense of Welfarism

This paper characterizes welfarist social evaluation in a multi-profile setting where, in addition to multiple utility profiles, it is assumed that there are several profiles of non-welfare information. We prove new versions of the welfarism theorems in this alternative framework, and we illustrate that a very plausible and weak anonymity property is sufficient to generate anonymous social-evaluation orderings.

Étude de l'activité anti-cancéreuse du PCK3145, un peptide dérivé de PSP-94, sur les cancers hématologiques

Le PCK3145 est un peptide de 15 acides aminés inhibant la sécrétion de MMP-9 et démontrant une activité anti-tumorale contre le cancer de la prostate. Comme les cancers hématologiques sécrètent MMP-9, nous avons donc évalué l’effet du PCK3145 sur ces cancers. Nous avons démontré que les lignées humaines de lymphome non- Hodgkinien (LNH) SR et de myélome multiple RPMI-8226 ainsi que la lignée murine de mastocytome P815 ont une prolifération réduite suite à une exposition au PCK3145. Ce peptide diminue également la clonogénicité de ces cellules. In vivo, le PCK3145 diminue significativement la croissance des tumeurs sous-cutanées P815 comparativement au PBS (p<0.001) et aux peptides contrôles (« scrambled peptide » (p<0.05) et PCK5266 (p<0.01)). De plus, le traitement au PCK3145 diminue le nombre de métastases au niveau du foie par rapports aux contrôles (p<0.05). Les niveaux de MMP-9 dans le sang des souris traitées au PCK3145 sont similaires à ceux dans le sang des souris sans tumeur. Par contre, chez les souris recevant le PBS ou le « scrambled peptide », les niveaux de MMP-9 étaient significativement plus élevés que dans les souris sans tumeur et les souris traitées au PCK3145 (p<0.05). De surcroît, dans un modèle de xénogreffe, le PCK3145 diminue significativement la croissance des lymphomes SR par rapport au PBS (p<0.01) et au « scrambled peptide » (p<0.001). Ces résultats indiquent que le PCK3145 possède une activité anti-tumorale et pourrait représenter un agent intéressant pour le traitement de plusieurs cancers hématologiques.

Élucidation des mécanismes moléculaires impliqués dans l’expression aberrante du récepteur au peptide insulinotropique glucose-dépendant (GIP) dans les tumeurs du cortex de la glande surrénale

Les tumeurs du cortex surrénalien sont variées et fréquentes dans la population. Bien que des mutations aient été identifiées dans certains syndromes familiaux, les causes génétiques menant à la formation de tumeur du cortex surrénalien ne sont encore que peu connues. Un sous-type de ces tumeurs incluent les hyperplasies macronodulaires et sont pressenties comme la voie d’entrée de la tumorigenèse du cortex surrénalien. L’événement génétique le plus fréquemment observé dans ces tumeurs est l’expression aberrante d’un ou plusieurs récepteurs couplés aux protéines G qui contrôle la production de stéroïdes ainsi que la prolifération cellulaire. L’événement génétique menant à l’expression aberrante de ces récepteurs est encore inconnu. En utilisant le récepteur au peptide insulinotropique dépendant du glucose (GIP) comme modèle, cette étude se propose d’identifier les mécanismes moléculaires impliqués dans l’expression aberrante du récepteur au GIP (GIPR) dans les tumeurs du cortex surrénalien. Une partie clinique de cette étude se penchera sur l’identification de nouveaux cas de tumeurs surrénaliennes exprimant le GIPR de façon aberrante. Les patients étudiés seront soumis à un protocole d’investigation in vivo complet et les tumeurs prélevées seront étudiées extensivement in vitro par RT-PCR en temps réel, culture primaire des tumeurs, immunohistochimie et biopuces. Le lien entre le GIP et la physiologie normal sera également étudiée de cette façon. Une autre partie de l’étude utilisera les nouvelles techniques d’investigation à grande échelle en identifiant le transcriptome de différents cas de tumeurs exprimant le GIPR de façon aberrante. L’importance fonctionnelle des gènes identifiée par ces techniques sera confirmée dans des modèles cellulaires. Cette étude présente pour la première des cas de tumeurs productrices d’aldostérone présentant des réponses aberrantes, auparavant confinées aux tumeurs productrice de cortisol ou d’androgènes surrénaliens. Le cas probant présenté avait une production d’aldostérone sensible au GIP, le GIPR était surexprimé au niveau de l’ARNm et un fort marquage a été identifié dans la tumeur spécifiquement. Dans les surrénales normales, cette étude démontre que le GIP est impliqué dans le contrôle de la production d’aldostérone. Ces résultats ont été confirmés in vitro. Finalement, le profilage à grande échelle des niveaux d’expression de tous les gènes du génome a permis d’isoler une liste de gènes spécifiquement liés à la présence du GIPR dans des hyperplasies du cortex surrénalien. Cette liste inclus la périlipine, une protéine de stockage des lipides dans les adipocytes et la glande surrénale, dont l’expression est fortement réprimée dans les cas GIP-dépendant. Des études dans un modèle cellulaire démontrent que la répression de ce gène par siRNA est suffisante pour induire l’expression du récepteur au GIP et que cette protéine est impliquée dans la stimulation de la stéroïdogénèse par le GIP. En alliant des méthodes d’investigation in vivo de pointe à des techniques in vitro avancée, cette étude offre de nouveaux regards sur les liens entre le GIP et la physiologie de la glande surrénale, que ce soit dans des conditions normales ou pathologiques.

Rôles des kinases IKK et IKK-related dans les maladies inflammatoires chroniques : implications dans l’athérosclérose et la réponse hypoxique

L’inflammation est un procédé complexe qui vise l’élimination de l’agent causal de dommages tissulaires en vue de faciliter la réparation du tissu affecté. La persistance de l’agent causal ou l’incapacité à résoudre l’inflammation mène à un dérèglement homéostatique chronique qui peut avoir une incidence sur la morbidité et la mortalité. L’athérosclérose est une condition inflammatoire chronique des vaisseaux sanguins dont l’origine est multifactorielle. L’hypertension et l’état infectieux représentent respectivement des facteurs de risque classiques et émergents du développement de cette maladie. Les fondements initiaux de l’inflammation font intervenir l’immunité innée, la première ligne de défense dont disposent les cellules pour répondre à un signal de danger. Le but de cette thèse est d’examiner le rôle pro-inflammatoire d’une famille de kinases essentielles à l’immunité innée, soit celle des kinases de IkappaB (IKK) et des kinases IKK-related. Les kinases IKKalpha et IKKbeta forment le complexe IKK avec la molécule adaptatrice NEMO/IKKgamma. Ce complexe est chargé d’effectuer la phosphorylation de l’inhibiteur de NF-kappaB, IkappaBalpha, ce qui mène à sa dégradation et à la libération du facteur de transcription NF-kappaB. Nous montrons que le peptide vasoactif angiotensine II (AngII) induit l’activité phosphotransférase d’IKKbeta dans les VSMC par immunoprécipitation de NEMO puis essai kinase in vitro. Grâce à une approche ARN interférence (ARNi) dirigée contre IKK, nous montrons que cette kinase est responsable de la phosphorylation de p65/RelA. Nous montrons que le mécanisme d’induction de NF-kappaB par l’AngII est atypique, puisqu’il ne module pas IkappaBalpha, et montrons à l’aide d’inhibiteurs pharmacologiques que l’activation de p65 est indépendante des voies MEK-ERK-RSK, PI3K et de la transactivation du récepteur de l’EGF. Les kinases IKK-related Tank-binding kinase 1 (TBK1) et IKK-i sont quant à elles principalement activées suite à une infection bactérienne ou virale. Ces kinases phosphorylent directement le facteur de transcription interferon regulatory factor (IRF)-3. Nous montrons que le cytomégalovirus humain, un pathogène associé à l’athérosclérose, a la capacité d’induire l’activation de TBK1 dans les VSMC. L’usage d’ARNi dirigé contre TBK1 et IKKi montre que les 2 kinases sont impliquées dans l’activation d’IRF-3. De plus, nous montrons à l’aide d’une lignée de VSMC exprimant une version dominante négative d’IRF-3 que ce dernier est essentiel à la synthèse des chimiokines RANTES et IP-10, tel qu’analysé par RT-PCR. Par ailleurs, il a récemment été montré que les kinases IKK-related étaient étroitement liées à la transformation oncogénique, et que TBK1 était pro-angiogénique. Or, l’angiogenèse est le plus souvent modulée par la réponse hypoxique qui est d’ailleurs commune à la majorité des processus inflammatoires. Le facteur de transcription hypoxia inducible factor (HIF)-1 module l’angiogenèse, l’inflammation et la survie cellulaire. Nous montrons à l’aide de cellules Tbk1 et Ikbke -/- et d’une approche lentivirale que TBK1 est spécifiquement impliquée dans l’induction traductionnelle de HIF-1alpha en condition de stress hypoxique. L’expression de TBK1 est induite sous ces conditions, et cette kinase module la phosphorylation de ERK, RSK, Akt et TSC1. Les résultats originaux présentés dans cette thèse montrent donc que les kinases IKK et IKK-related exercent leurs actions pro-inflammatoires par des mécanismes distincts.