Nanomedicines for active targeting: physico-chemical characterization of paclitaxel-loaded anti-HER2 immunonanoparticles and in vitro functional studies on target cells.

Paclitaxel (Tx)-loaded anti-HER2 immunonanoparticles (NPs-Tx-HER) were prepared by the covalent coupling of humanized monoclonal anti-HER2 antibodies (trastuzumab, Herceptin) to Tx-loaded poly (dl-lactic acid) nanoparticles (NPs-Tx) for the active targeting of tumor cells that overexpress HER2 receptors. The physico-chemical properties of NPs-Tx-HER were compared to unloaded immunonanoparticles (NPs-HER) to assess the influence of the drug on anti-HER2 coupling to the NP surface. The immunoreactivity of sulfo-MBS activated anti-HER2 mAbs and the in vitro efficacy of NPs-Tx-HER were tested on SKOV-3 ovarian cancer cells that overexpress HER2 antigens. Tx-loaded nanoparticles (NPs-Tx) obtained by a salting-out method had a size of 171+/-22 nm (P.I.=0.1) and an encapsulation efficiency of about of 78+/-10%, which corresponded to a drug loading of 7.8+/-0.8% (w/w). NPs-Tx were then thiolated and conjugated to activated anti-HER2 mAbs to obtain immunonanoparticles of 237+/-43 nm (P.I.=0.2). The influence of the activation step on the immunoreactivity of the mAbs was tested on SKOV-3 cells using 125I-radiolabeled mAbs, and the activity of the anti-HER2 mAbs was minimally affected after sulfo-MBS functionalization. Approximately 270 molecules of anti-HER2 mAbs were bound per nanoparticle. NPs-Tx-HER exhibited a zeta potential of 0.2+/-0.1 mV. The physico-chemical properties of the Tx-loaded immunonanoparticles were very similar to unloaded immunonanoparticles, suggesting that the encapsulation of the drug did not influence the coupling of the mAbs to the NPs. No drug loss was observed during the preparation process. DSC analysis showed that encapsulated Tx is in an amorphous or disordered-crystalline phase. These results suggest that Tx is entrapped in the polymeric matrix and not adsorbed to the surface of the NPs. In vitro studies on SKOV-3 ovarian cancer cells demonstrated the greater cytotoxic effect of NPs-Tx-HER compared to other Tx formulations. The results showed that at 1 ng Tx/ml, the viability of cells incubated with drug encapsulated in NP-Tx-HER was lower (77.32+/-5.48%) than the viability of cells incubated in NPs-Tx (97.4+/-12%), immunonanoparticles coated with Mabthera, as irrelevant mAb (NPs-Tx-RIT) (93.8+/-12%) or free drug (92.3+/-9.3%).

Modulatory effects of acid-sensing ion channels on action potential generation in hippocampal neurons.

Extracellular acidification has been shown to generate action potentials (APs) in several types of neurons. In this study, we investigated the role of acid-sensing ion channels (ASICs) in acid-induced AP generation in brain neurons. ASICs are neuronal Na(+) channels that belong to the epithelial Na(+) channel/degenerin family and are transiently activated by a rapid drop in extracellular pH. We compared the pharmacological and biophysical properties of acid-induced AP generation with those of ASIC currents in cultured hippocampal neurons. Our results show that acid-induced AP generation in these neurons is essentially due to ASIC activation. We demonstrate for the first time that the probability of inducing APs correlates with current entry through ASICs. We also show that ASIC activation in combination with other excitatory stimuli can either facilitate AP generation or inhibit AP bursts, depending on the conditions. ASIC-mediated generation and modulation of APs can be induced by extracellular pH changes from 7.4 to slightly <7. Such local extracellular pH values may be reached by pH fluctuations due to normal neuronal activity. Furthermore, in the plasma membrane, ASICs are localized in close proximity to voltage-gated Na(+) and K(+) channels, providing the conditions necessary for the transduction of local pH changes into electrical signals.

IL-7/anti-IL-7 mAb complexes restore T cell development and induce homeostatic T Cell expansion without lymphopenia.

IL-7, a member of the common gamma-chain family of cytokines, is essential for B and T lymphocyte development and homeostasis of mature T cell subsets. Thus, naive and memory T cells are both dependent on IL-7 for survival and homeostatic proliferation under lymphopenic conditions. In line with prior findings with IL-2, we show in this study that the biological activity of IL-7 in vivo is greatly increased by association with anti-IL-7 mAb. Under in vivo conditions, IL-7/mAb complexes displayed 50- to 100-fold higher activity than free IL-7 and induced massive expansion of pre-B cells. IL-7/mAb complexes also increased thymopoiesis in normal mice and restored thymopoeisis in IL-7-deficient mice. For mature T cells, IL-7/mAb complexes induced marked homeostatic proliferation of both naive and memory CD4(+) and CD8(+) cell subsets even under normal T cell-replete conditions. Finally, IL-7/mAb complexes were able to enhance the magnitude of the primary response of Ag-specific naive CD8(+) cells. The strong stimulatory activity of IL-7/mAb complexes could be useful for treatment of immunodeficiency and cancer.

RpoN (sigma54) controls production of antifungal compounds and biocontrol activity in Pseudomonas fluorescens CHA0.

Pseudomonas fluorescens CHA0 is an effective biocontrol agent of root diseases caused by fungal pathogens. The strain produces the antibiotics 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT) that make essential contributions to pathogen suppression. This study focused on the role of the sigma factor RpoN (sigma54) in regulation of antibiotic production and biocontrol activity in P. fluorescens. An rpoN in-frame-deletion mutant of CHAO had a delayed growth, was impaired in the utilization of several carbon and nitrogen sources, and was more sensitive to salt stress. The rpoN mutant was defective for flagella and displayed drastically reduced swimming and swarming motilities. Interestingly, the rpoN mutant showed a severalfold enhanced production of DAPG and expression of the biosynthetic gene phlA compared with the wild type and the mutant complemented with monocopy rpoN+. By contrast, loss of RpoN function resulted in markedly lowered PLT production and plt gene expression, suggesting that RpoN controls the balance of the two antibiotics in strain CHA0. In natural soil microcosms, the rpoN mutant was less effective in protecting cucumber from a root rot caused by Pythium ultimum. Remarkably, the mutant was not significantly impaired in its root colonization capacity, even at early stages of root infection by Pythium spp. Taken together, our results establish RpoN for the first time as a major regulator of biocontrol activity in Pseudomonas fluorescens.

ACID-SENSING ION CHANNELS: functional and molecular characterization in putative nociceptors, and modulation by nerve injury and serine protease

Résumé Les canaux ioniques ASICs (acid-sensing ion channels) appartiennent à la famille des canaux ENaC/Degenerin. Pour l'instant, quatre gènes (1 à 4) ont été clonés dont certains présentent des variants d'épissage. Leur activation par une acidification rapide du milieu extracellulaire génère un courant entrant transitoire essentiellement sodique accompagné pour certains types d'ASICs d'une phase soutenue. Les ASICs sont exprimés dans le système nerveux, central (SNC) et périphérique (SNP). On leur attribue un rôle dans l'apprentissage, la mémoire et l'ischémie cérébrale au niveau central ainsi que dans la nociception (douleur aiguë et inflammatoire) et la méchanotransduction au niveau périphérique. Toutefois, les données sont parfois contradictoires. Certaines études suggèrent qu'ils sont des senseurs primordiaux impliqués dans la détection de l'acidification et la douleur. D'autres études suggèrent plutôt qu'ils ont un rôle modulateur inhibiteur dans la douleur. De plus, le fait que leur activation génère majoritairement un courant transitoire alors que les fibres nerveuses impliquées dans la douleur répondent à un stimulus nocif avec une adaptation lente suggère que leurs propriétés doivent être modulés par des molécules endogènes. Dans une première partie de ma thèse, nous avons abordé la question de l'expression fonctionnelle des ASICs dans les neurones sensoriels primaires afférents du rat adulte pour clarifier le rôle des ASICs dans les neurones sensoriels. Nous avons caractérisé leurs propriétés biophysiques et pharmacologiques par la technique du patch-clamp en configuration « whole-cell ». Nous avons pu démontrer que près de 60% des neurones sensoriels de petit diamètre expriment des courants ASICs. Nous avons mis en évidence trois types de courant ASIC dans ces neurones. Les types 1 et 3 ont des propriétés compatibles avec un rôle de senseur du pH alors que le type 2 est majoritairement activé par des pH inférieurs à pH6. Le type 1 est médié par des homomers de la sous-unité ASIC1 a qui sont perméables aux Ca2+. Nous avons étudié leur co-expression avec des marqueurs des nocicepteurs ainsi que la possibilité d'induire une activité neuronale suite à une acidification qui soit dépendante des ASICs. Le but était d'associer un type de courant ASIC avec une fonction potentielle dans les neurones sensoriels. Une majorité des neurones exprimant les courants ASIC co-expriment des marqueurs des nocicepteurs. Toutefois, une plus grande proportion des neurones exprimant le type 1 n'est pas associée à la nociception par rapport aux types 2 et 3. Nous avons montré qu'il est possible d'induire des potentiels d'actions suite à une acidification. La probabilité d'induction est proportionnelle à la densité des courants ASIC et à l'acidité de la stimulation. Puis, nous avons utilisé cette classification comme un outil pour appréhender les potentielles modulations fonctionnelles des ASICs dans un model de neuropathie (spared nerve injury). Cette approche fut complétée par des expériences de «quantitative RT-PCR ». En situation de neuropathie, les courants ASIC sont dramatiquement changés au niveau de leur expression fonctionnelle et transcriptionnelle dans les neurones lésés ainsi que non-lésés. Dans une deuxième partie de ma thèse, suite au test de différentes substances sécrétées lors de l'inflammation et l'ischémie sur les propriétés des ASICs, nous avons caractérisé en détail la modulation des propriétés des courants ASICs notamment ASIC1 par les sérines protéases dans des systèmes d'expression recombinants ainsi que dans des neurones d'hippocampe. Nous avons montré que l'exposition aux sérine-protéases décale la dépendance au pH de l'activation ainsi que la « steady-state inactivation »des ASICs -1a et -1b vers des valeurs plus acidiques. Ainsi, l'exposition aux serine protéases conduit à une diminution du courant quand l'acidification a lieu à partir d'un pH7.4 et conduit à une augmentation du courant quand l'acidification alleu à partir d'un pH7. Nous avons aussi montré que cette régulation a lieu des les neurones d'hippocampe. Nos résultats dans les neurones sensoriels suggèrent que certains courants ASICs sont impliqués dans la transduction de l'acidification et de la douleur ainsi que dans une des phases du processus conduisant à la neuropathie. Une partie des courants de type 1 perméables au Ca 2+ peuvent être impliqués dans la neurosécrétion. La modulation par les sérines protéases pourrait expliquer qu'en situation d'acidose les canaux ASICs soient toujours activables. Résumé grand publique Les neurones sont les principales cellules du système nerveux. Le système nerveux est formé par le système nerveux central - principalement le cerveau, le cervelet et la moelle épinière - et le système nerveux périphérique -principalement les nerfs et les neurones sensoriels. Grâce à leur nombreux "bras" (les neurites), les neurones sont connectés entre eux, formant un véritable réseau de communication qui s'étend dans tout le corps. L'information se propage sous forme d'un phénomène électrique, l'influx nerveux (ou potentiels d'actions). A la base des phénomènes électriques dans les neurones il y a ce que l'on appelle les canaux ioniques. Un canal ionique est une sorte de tunnel qui traverse l'enveloppe qui entoure les cellules (la membrane) et par lequel passent les ions. La plupart de ces canaux sont normalement fermés et nécessitent d'être activés pour s'ouvrire et générer un influx nerveux. Les canaux ASICs sont activés par l'acidification et sont exprimés dans tout le système nerveux. Cette acidification a lieu notamment lors d'une attaque cérébrale (ischémie cérébrale) ou lors de l'inflammation. Les expériences sur les animaux ont montré que les canaux ASICs avaient entre autre un rôle dans la mort des neurones lors d'une attaque cérébrale et dans la douleur inflammatoire. Lors de ma thèse je me suis intéressé au rôle des ASICs dans la douleur et à l'influence des substances produites pendant l'inflammation sur leur activation par l'acidification. J'ai ainsi pu montrer chez le rat que la majorité des neurones sensoriels impliqués dans la douleur ont des canaux ASICs et que l'activation de ces canaux induit des potentiels d'action. Nous avons opéré des rats pour qu'ils présentent les symptômes d'une maladie chronique appelée neuropathie. La neuropathie se caractérise par une plus grande sensibilité à la douleur. Les rats neuropathiques présentent des changements de leurs canaux ASICs suggérant que ces canaux ont une peut-être un rôle dans la genèse ou les symptômes de cette maladie. J'ai aussi montré in vitro qu'un type d'enryme produit lors de l'inflammation et l'ischémie change les propriétés des ASICs. Ces résultats confirment un rôle des ASICs dans la douleur suggérant notamment un rôle jusque là encore non étudié dans la douleur neuropathique. De plus, ces résultats mettent en évidence une régulation des ASICs qui pourrait être importante si elle se confirmait in vivo de part les différents rôles des ASICs. Abstract Acid-sensing ion channels (ASICs) are members of the ENaC/Degenerin superfamily of ion channels. Their activation by a rapid extracellular acidification generates a transient and for some ASIC types also a sustained current mainly mediated by Na+. ASICs are expressed in the central (CNS) and in the peripheral (PNS) nervous system. In the CNS, ASICs have a putative role in learning, memory and in neuronal death after cerebral ischemia. In the PNS, ASICs have a putative role in nociception (acute and inflammatory pain) and in mechanotransduction. However, studies on ASIC function are somewhat controversial. Some studies suggest a crucial role of ASICs in transduction of acidification and in pain whereas other studies suggest rather a modulatory inhibitory role of ASICs in pain. Moreover, the basic property of ASICs, that they are activated only transiently is irreconcilable with the well-known property of nociception that the firing of nociceptive fibers demonstrated very little adaptation. Endogenous molecules may exist that can modulate ASIC properties. In a first part of my thesis, we addressed the question of the functional expression of ASICs in adult rat dorsal root ganglion (DRG) neurons. Our goal was to elucidate ASIC roles in DRG neurons. We characterized biophysical and pharmacological properties of ASIC currents using the patch-clamp technique in the whole-cell configuration. We observed that around 60% of small-diameter sensory neurons express ASICs currents. We described in these neurons three ASIC current types. Types 1 and 3 have properties compatible with a role of pH-sensor whereas type 2 is mainly activated by pH lower than pH6. Type 1 is mediated by ASIC1a homomultimers which are permeable to Ca 2+. We studied ASIC co-expression with nociceptor markers. The goal was to associate an ASIC current type with a potential function in sensory neurons. Most neurons expressing ASIC currents co-expressed nociceptor markers. However, a higher proportion of the neurons expressing type 1 was not associated with nociception compared to type 2 and -3. We completed this approach with current-clamp measurements of acidification-induced action potentials (APs). We showed that activation of ASICs in small-diameter neurons can induce APs. The probability of AP induction is positively correlated with the ASIC current density and the acidity of stimulation. Then, we used this classification as a tool to characterize the potential functional modulation of ASICs in the spared nerve injury model of neuropathy. This approach was completed by quantitative RT-PCR experiments. ASICs current expression was dramatically changed at the functional and transcriptional level in injured and non-injured small-diameter DRG neurons. In a second part of my thesis, following an initial screening of the effect of various substances secreted during inflammation and ischemia on ASIC current properties, we characterized in detail the modulation of ASICs, in particular of ASIC1 by serine proteases in a recombinant expression system as well as in hippocampal neurons. We showed that protease exposure shifts the pH dependence of ASIC1 activation and steady-state inactivation to more acidic pH. As a consequence, protease exposure leads to a decrease in the current response if ASIC1 is activated by a pH drop from pH 7.4. If, however, acidification occurs from a basal pH of 7, protease-exposed ASIC1a shows higher activity than untreated ASIC1a. We provided evidence that this bi-directional regulation of ASIC1a function also occurs in hippocampal neurons. Our results in DRG neurons suggest that some ASIC currents are involved in the transduction of peripheral acidification and pain. Furthermore, ASICs may participate to the processes leading to neuropathy. Some Ca 2+-permeable type 1 currents may be involved in neurosecretion. ASIC modulation by serine proteases may be physiologically relevant, allowing ASIC activation under sustained slightly acidic conditions.

The role of integrins in glioma biology and anti-glioma therapies.

The tumor environment is critical for tumor maintenance and progression. Integrins are a large family of cell surface receptors mediating the interaction of tumor cells with their microenvironment and play important roles in glioma biology, including migration, invasion, angiogenesis and tumor stem cell anchorage. Here, we review preclinical and clinical data on integrin inhibition in malignant gliomas. Various pharmacological approaches to the modulation of integrin signaling have been explored including antibodies and peptide-based agents. Cilengitide, a cyclic RGD-mimetic peptide of αvβ3 and αvβ5 integrins is in advanced clinical development in glioblastoma. Cilengitide had only limited activity as a single agent in glioblastoma, but, when added to standard radiochemotherapy, appeared to prolong progression-free and overall survival in patients with newly diagnosed glioblastomas and methylation of the promoter of the O⁶ methylguanine methyltransferase (MGMT) gene. MGMT gene promoter methylation in turn predicts benefit from alkylating chemotherapy. A phase III randomized clinical trial in conjunction with standard radiochemotherapy in newly diagnosed glioblastoma patients with MGMT gene promoter methylation has recently completed accrual (EORTC 26071-22072). A companion trial explores a dose-escalated regimen of cilengitide added to radiotherapy plus temozolomide in patients without MGMT gene promoter methylation. Promising results in these trials would probably result in a broader interest in integrins as targets for glioma therapy and hopefully the development of a broader panel of anti-integrin agents.

Effects of verapamil and ryanodine on activity of the embryonic chick heart during anoxia and reoxygenation.

Perturbations of the trans-sarcolemmal and sarcoplasmic Ca2+ transport contribute to the abnormal myocardial activity provoked by anoxia and reoxygenation. Whether Ca2+ pools of the extracellular compartment and sarcoplasmic reticulum (SR) are involved to the same extent in the dysfunction of the anoxic-reoxygenated immature heart has not been investigated. Spontaneously contracting hearts isolated from 4-day-old chick embryos were submitted to repeated anoxia (1 min) followed by reoxygenation (5 min). Heart rate, atrioventricular propagation velocity, ventricular shortening, velocities of contraction and relaxation, and incidence of arrhythmias were studied, recorded continuously. Addition of verapamil (10 nM), which blocks selectively sarcolemmal L-type Ca2+ channels, was expected to protect against excessive entry of extracellular Ca2+, whereas addition of ryanodine (10 nM), which opens the SR Ca2+ release channel, was expected to increase cytosolic Ca2+ concentration. Verapamil (a) had no dromotropic effect by contrast to adult heart, (b) attenuated ventricular contracture induced by repeated anoxia, (c) shortened cardioplegia induced by reoxygenation, and (d) had remarkable antiarrhythmic properties during reoxygenation specially. On the other hand, ryanodine potentiated markedly arrhythmias both during anoxia and at reoxygenation. Thus despite its immaturity, the SR seems to be functional early in the developing chick heart and involved in the reversible dysfunction induced by anoxia-reoxygenation. Moreover, Ca2+ entry through L-type channels appears to worsen arrhythmias especially during reoxygenation. These findings show that the Ca2+-handling systems involved in irregular activity in immature heart, such as the embryonic chick heart, may differ from those in the adult.

The quest for surrogate markers of angiogenesis: a paradigm for translational research in tumor angiogenesis and anti-angiogenesis trials.

Inhibition of tumor angiogenesis suppresses tumor growth and metastatic spreading in many experimental models, suggesting that anti-angiogenic drugs may be used to treat human cancer. During the past decade more than eighty molecules that showed anti-angiogenic activity in preclinical studies were tested in clinical cancer trials, but most of them failed to demonstrate any measurable anti-tumor activity and none have been approved for clinical use. Recent results stemming from trials with anti-VEGF antibodies, used alone or in combination with chemotherapy, suggest that systemic anti-angiogenic therapy may indeed have a measurable impact on cancer progression and patient survival. From the clinical studies it became nevertheless clear that the classical endpoints used in anti-cancer trials do not bring sufficient discriminative power to monitor the effects of anti-angiogenic drugs. It is therefore necessary to identify and validate molecular, cellular and functional surrogate markers of angiogenesis to monitor activity and efficacy of anti-angiogenic drugs in patients. Availability of such markers will be instrumental to re-evaluate the role of tumor angiogenesis in human cancer, to identify new molecular targets and drugs, and to improve planning, monitoring and interpretation of future studies. Future anti-angiogenesis trials integrating biological endpoints and surrogate markers or angiogenesis will require close collaboration between clinical investigators and laboratory-based researchers.

Inhibition of tumor angiogenesis by non-steroidal anti-inflammatory drugs: emerging mechanisms and therapeutic perspectives.

Chronic intake of non steroidal anti-inflammatory drugs (NSAIDs) is associated with a reduced risk of developing gastrointestinal tumors, in particular colon cancer. Increasing evidence indicates that NSAID exert tumor-suppressive activity on pre-malignant lesions (polyps) in humans and on established experimental tumors in mice. Some of the tumor-suppressive effects of NSAIDs depend on the inhibition of cyclooxygenase-2 (COX-2), a key enzyme in the synthesis of prostaglandins and thromboxane, which is highly expressed in inflammation and cancer. Recent findings indicate that NSAIDs exert their anti-tumor effects by suppressing tumor angiogenesis. The availability of COX-2-specific NSAIDs opens the possibility of using this drug class as anti-angiogenic agents in combination with chemotheapy or radiotherapy for the treatment of human cancer. Here we will briefly review recent advances in the understanding of the mechanism by which NSAIDs suppress tumor angiogenesis and discuss their potential clinical application as anti-cancer agents.

Immune sensing of nucleic acids in inflammatory skin diseases.

Endosomal and cytosolic nucleic acid receptors are important immune sensors required for the detection of infecting or replicating viruses. The intracellular location of these receptors allows viral recognition and, at the same time, avoids unnecessary immune activation to self-nucleic acids that are continuously released by dying host cells. Recent evidence, however, indicates that endogenous factors such as anti-microbial peptides have the ability to break this protective mechanism. Here, we discuss these factors and illustrate how they drive inflammatory responses by promoting immune recognition of self-nucleic acids in skin wounds and inflammatory skin diseases such as psoriasis and lupus.

Brain glucose sensing and neural regulation of insulin and glucagon secretion.

Glucose homeostasis requires the tight regulation of glucose utilization by liver, muscle and white or brown fat, and glucose production and release in the blood by liver. The major goal of maintaining glycemia at ∼ 5 mM is to ensure a sufficient flux of glucose to the brain, which depends mostly on this nutrient as a source of metabolic energy. This homeostatic process is controlled by hormones, mainly glucagon and insulin, and by autonomic nervous activities that control the metabolic state of liver, muscle and fat tissue but also the secretory activity of the endocrine pancreas. Activation or inhibition of the sympathetic or parasympathetic branches of the autonomic nervous systems are controlled by glucose-excited or glucose-inhibited neurons located at different anatomical sites, mainly in the brainstem and the hypothalamus. Activation of these neurons by hyper- or hypoglycemia represents a critical aspect of the control of glucose homeostasis, and loss of glucose sensing by these cells as well as by pancreatic β-cells is a hallmark of type 2 diabetes. In this article, aspects of the brain-endocrine pancreas axis are reviewed, highlighting the importance of central glucose sensing in the control of counterregulation to hypoglycemia but also mentioning the role of the neural control in β-cell mass and function. Overall, the conclusions of these studies is that impaired glucose homeostasis, such as associated with type 2 diabetes, but also defective counterregulation to hypoglycemia, may be caused by initial defects in glucose sensing.

Ligands for pheromone-sensing neurons are not conformationally activated odorant binding proteins.

Pheromones form an essential chemical language of intraspecific communication in many animals. How olfactory systems recognize pheromonal signals with both sensitivity and specificity is not well understood. An important in vivo paradigm for this process is the detection mechanism of the sex pheromone (Z)-11-octadecenyl acetate (cis-vaccenyl acetate [cVA]) in Drosophila melanogaster. cVA-evoked neuronal activation requires a secreted odorant binding protein, LUSH, the CD36-related transmembrane protein SNMP, and the odorant receptor OR67d. Crystallographic analysis has revealed that cVA-bound LUSH is conformationally distinct from apo (unliganded) LUSH. Recombinantly expressed mutant versions of LUSH predicted to enhance or diminish these structural changes produce corresponding alterations in spontaneous and/or cVA-evoked activity when infused into olfactory sensilla, leading to a model in which the ligand for pheromone receptors is not free cVA, but LUSH that is "conformationally activated" upon cVA binding. Here we present evidence that contradicts this model. First, we demonstrate that the same LUSH mutants expressed transgenically affect neither basal nor pheromone-evoked activity. Second, we compare the structures of apo LUSH, cVA/LUSH, and complexes of LUSH with non-pheromonal ligands and find no conformational property of cVA/LUSH that can explain its proposed unique activated state. Finally, we show that high concentrations of cVA can induce neuronal activity in the absence of LUSH, but not SNMP or OR67d. Our findings are not consistent with the model that the cVA/LUSH complex acts as the pheromone ligand, and suggest that pheromone molecules alone directly activate neuronal receptors.

Assessment of the chemosensitizing activity of TAT-RasGAP317-326 in childhood cancers.

Although current anti-cancer protocols are reasonably effective, treatment-associated long-term side effects, induced by lack of specificity of the anti-cancer procedures, remain a challenging problem in pediatric oncology. TAT-RasGAP317-326 is a RasGAP-derived cell-permeable peptide that acts as a sensitizer to various anti-cancer treatments in adult tumor cells. In the present study, we assessed the effect of TAT-RasGAP317-326 in several childhood cancer cell lines. The RasGAP-derived peptide-induced cell death was analyzed in several neuroblastoma, Ewing sarcoma and leukemia cell lines (as well as in normal lymphocytes). Cell death was evaluated using flow cytometry methods in the absence or in the presence of the peptide in combination with various genotoxins used in the clinics (4-hydroperoxycyclophosphamide, etoposide, vincristine and doxorubicin). All tested pediatric tumors, in response to at least one genotoxin, were sensitized by TAT-RasGAP317-326. The RasGAP-derived peptide did not increase cell death of normal lymphocytes, alone or in combination with the majority of the tested chemotherapies. Consequently, TAT-RasGAP317-326 may benefit children with tumors by increasing the efficacy of anti-cancer therapies notably by allowing reductions in anti-cancer drug dosage and the associated drug-induced side effects.

Brain glucose sensing in homeostatic and hedonic regulation.

Glucose homeostasis as well as homeostatic and hedonic control of feeding is regulated by hormonal, neuronal, and nutrient-related cues. Glucose, besides its role as a source of metabolic energy, is an important signal controlling hormone secretion and neuronal activity, hence contributing to whole-body metabolic integration in coordination with feeding control. Brain glucose sensing plays a key, but insufficiently explored, role in these metabolic and behavioral controls, which when deregulated may contribute to the development of obesity and diabetes. The recent introduction of innovative transgenic, pharmacogenetic, and optogenetic techniques allows unprecedented analysis of the complexity of central glucose sensing at the molecular, cellular, and neuronal circuit levels, which will lead to a new understanding of the pathogenesis of metabolic diseases.

Furosemide stimulation of parathormone in humans: role of the calcium-sensing receptor and the renin-angiotensin system.

Interactions between sodium and calcium regulating systems are poorly characterized but clinically important. Parathyroid hormone (PTH) levels are increased shortly after furosemide treatment by an unknown mechanism, and this effect is blunted by the previous administration of a calcimimetic in animal studies. Here, we explored further the possible underlying mechanisms of this observation in a randomized crossover placebo-controlled study performed in 18 human males. Volunteers took either cinacalcet (60 mg) or placebo and received a 20 mg furosemide injection 3 h later. Plasma samples were collected at 15-min intervals and analyzed for intact PTH, calcium, sodium, potassium, magnesium, phosphate, plasma renin activity (PRA), and aldosterone up to 6 h after furosemide injection. Urinary electrolyte excretion was also monitored. Subjects under placebo presented a sharp increase in PTH levels after furosemide injection. In the presence of cinacalcet, PTH levels were suppressed and marginal increase of PTH was observed. No significant changes in electrolytes and urinary excretion were identified that could explain the furosemide-induced increase in PTH levels. PRA and aldosterone were stimulated by furosemide injection but were not affected by previous cinacalcet ingestion. Expression of NKCC1, but not NKCC2, was found in parathyroid tissue. In conclusion, our results indicate that furosemide acutely stimulates PTH secretion in the absence of any detectable electrolyte changes in healthy adults. A possible direct effect of furosemide on parathyroid gland needs further studies.