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.

Structure function relationships of ENaC and its role in sodium handling.

The epithelial sodium channel (ENaC) in the apical membrane of polarized epithelial cells is the rate-limiting step for Na entry into the cell; in series with the basolateral Na pump, it allows the vectorial transepithelial transport of Na ions. ENaC is expressed in different epithelia like the distal nephron or colon, and the airways epithelium. In the lung ENaC controls the composition and the amount of pulmonary fluid, whereas in the distal nephron ENaC under the control of aldosterone and vasopressin, is essential to adapt the amount of Na+ reabsorbed with the daily sodium intake. Activating mutations of ENaC cause severe disturbances of Na+ homeostasis leading to hypertension in human and in mouse models. Functional expression of ENaC in different cell systems allowed the identification of structural domains of the protein that are essential for channel function and/or modulation of channel activity. Site-directed mutations in specific domains of the channel protein lead to channel hyperactivity or channel loss of function. Knowledge about ENaC structure-function relationships opens new opportunities for development of pharmacological tools for controlling ENaC activity, such as channel activators of potential benefit in the treatment of pulmonary edema, or highly potent ENaC blockers with natriuretic effects.

An Ion Transport-Independent Role for the Cation-Chloride Cotransporter KCC2 in Dendritic Spinogenesis In Vivo.

The neuron-specific K-Cl cotransporter, KCC2, is highly expressed in the vicinity of excitatory synapses in pyramidal neurons, and recent in vitro data suggest that this protein plays a role in the development of dendritic spines. The in vivo relevance of these observations is, however, unknown. Using in utero electroporation combined with post hoc iontophoretic injection of Lucifer Yellow, we show that premature expression of KCC2 induces a highly significant and permanent increase in dendritic spine density of layer 2/3 pyramidal neurons in the somatosensory cortex. Whole-cell recordings revealed that this increased spine density is correlated with an enhanced spontaneous excitatory activity in KCC2-transfected neurons. Precocious expression of the N-terminal deleted form of KCC2, which lacks the chloride transporter function, also increased spine density. In contrast, no effect on spine density was observed following in utero electroporation of a point mutant of KCC2 (KCC2-C568A) where both the cotransporter function and the interaction with the cytoskeleton are disrupted. Transfection of the C-terminal domain of KCC2, a region involved in the interaction with the dendritic cytoskeleton, also increased spine density. Collectively, these results demonstrate a role for KCC2 in excitatory synaptogenesis in vivo through a mechanism that is independent of its ion transport function.

Glucagon-like peptide-1 increases beta-cell glucose competence and proliferation by translational induction of insulin-like growth factor-1 receptor expression.

Glucagon-like peptide-1 (GLP-1) protects beta-cells against apoptosis, increases their glucose competence, and induces their proliferation. We previously demonstrated that the anti-apoptotic effect was mediated by an increase in insulin-like growth factor-1 receptor (IGF-1R) expression and signaling, which was dependent on autocrine secretion of insulin-like growth factor 2 (IGF-2). Here, we further investigated how GLP-1 induces IGF-1R expression and whether the IGF-2/IGF-1R autocrine loop is also involved in mediating GLP-1-increase in glucose competence and proliferation. We show that GLP-1 up-regulated IGF-1R expression by a protein kinase A-dependent translational control mechanism, whereas isobutylmethylxanthine, which led to higher intracellular accumulation of cAMP than GLP-1, increased both IGF-1R transcription and translation. We then demonstrated, using MIN6 cells and primary islets, that the glucose competence of these cells was dependent on the level of IGF-1R expression and on IGF-2 secretion. We showed that GLP-1-induced primary beta-cell proliferation was suppressed by Igf-1r gene inactivation and by IGF-2 immunoneutralization or knockdown. Together our data show that regulation of beta-cell number and function by GLP-1 depends on the cAMP/protein kinase A mediated-induction of IGF-1R expression and the increased activity of an IGF-2/IGF-1R autocrine loop.

The epithelial sodium channel and the control of sodium balance.

Studies aiming at the elucidation of the genetic basis of rare monogenic forms of hypertension have identified mutations in genes coding for the epithelial sodium channel ENaC, for the mineralocorticoid receptor, or for enzymes crucial for the synthesis of aldosterone. These genetic studies clearly demonstrate the importance of the regulation of Na(+) absorption in the aldosterone-sensitive distal nephron (ASDN), for the maintenance of the extracellular fluid volume and blood pressure. Recent studies aiming at a better understanding of the cellular and molecular basis of ENaC-mediated Na(+) absorption in the distal part of nephron, have essentially focused on the regulation ENaC activity and on the aldosterone-signaling cascade. ENaC is a constitutively open channel, and factors controlling the number of active channels at the cell surface are likely to have profound effects on Na(+) absorption in the ASDN, and in the amount of Na(+) that is excreted in the final urine. A number of membrane-bound proteases, kinases, have recently been identified that increase ENaC activity at the cell surface in heterologous expressions systems. Ubiquitylation is a general process that regulates the stability of a variety of target proteins that include ENaC. Recently, deubiquitylating enzymes have been shown to increase ENaC activity in heterologous expressions systems. These regulatory mechanisms are likely to be nephron specific, since in vivo studies indicate that the adaptation of the renal excretion of Na(+) in response to Na(+) diet occurs predominantly in the early part (the connecting tubule) of the ASDN. An important work is presently done to determine in vivo the physiological relevance of these cellular and molecular mechanisms in regulation of ENaC activity. The contribution of the protease-dependent ENaC regulation in mediating Na(+) absorption in the ASDN is still not clearly understood. The signaling pathway that involves ubiquitylation of ENaC does not seem to be absolutely required for the aldosterone-mediated control of ENaC. These in vivo physiological studies presently constitute a major challenge for our understanding of the regulation of ENaC to maintain the Na(+) balance.

Neurotrophic activity of human adipose stem cells isolated from deep and superficial layers of abdominal fat.

New approaches to the clinical treatment of traumatic nerve injuries may one day utilize stem cells to enhance nerve regeneration. Adipose-derived stem cells (ASC) are found in abundant quantities and can be harvested by minimally invasive procedures that should facilitate their use in such regenerative applications. We have analyzed the properties of human ASC isolated from the deep and superficial layers of abdominal fat tissue obtained during abdominoplasty procedures. Cells from the superficial layer proliferate significantly faster than those from the deep layer. In both the deep and superficial layers, ASC express the pluripotent stem cell markers oct4 and nanog and also the stro-1 cell surface antigen. Superficial layer ASC induce the significantly enhanced outgrowth of neurite-like processes from neuronal cell lines when compared with that of deep layer cells. However, analysis by reverse transcription with the polymerase chain reaction and by enzyme-linked immunosorbent assay has revealed that ASC isolated from both layers express similar levels of the following neurotrophic factors: nerve growth factor, brain-derived neurotrophic factor and glial-derived neurotrophic factor. Thus, human ASC show promising potential for the treatment of traumatic nerve injuries. In particular, superficial layer ASC warrant further analysis of their neurotrophic molecules.

Modulation of the epithelial sodium channel by serine proteases

Abstract The epithelial sodium channel (ENaC) is composed of three homologous subunits α, ß, and γ. This channel is involved in the regulation of sodium balance, which influences the periciliary liquid level in the lung, and blood pressure via the kidney. ENaC expressed in Xenopus laevis oocytes is preferentially and rapidly assembled into heteromeric αßγ complexes. Expression of homomeric α or heteromeric αß and αγ complexes lead to channel expression at the cell surface wíth low activities. Recent studies have demonstrated that α and γ (but not ß) ENaC subunits undergo proteolytic cleavage by endogenous proteases (i.e. furin) correlating with increased channel activity. We therefore assayed the full-length subunits and their cleavage products at the cell surface, as well as in the intracellular pool for all homo- and heteromeric combínations (α, ß, γ, ßγ, αß, αγ, ßγ and αßγ) and measured the corresponding channel activities as amiloride-sensitive sodíum transport (INa). We showed that upon assembly, cleavage of the y ENaC subunit ís responsible for increasing INa. We further demonstrated that in disease states such as cystic fibrosis (CF) where there is disequilibrium in the proteaseprotease inhibitor balance, ENaC is over-activated by the serine protease elastase (NE). We demonstrated that elevated NE concentrations can cleave cell surface expressed γ ENaC (but not α, or ß ENaC), suggesting a causal relationship between γ ENaC cleavage and ENaC activation, taking place at the plasma membrane. In addition, we demonstrated that the serine protease inhibitor (serpin) serpinH1, which is co-expressed with ENaC in the distal nephron is capable of inhibiting the channel by preventing cleavage of the γ ENaC subunit. Aldosterone mediated increases in INa aze known to be inhibted by TGFß. TGFß is also known to increase serpinHl expression. The demonstrated inhibition of γ ENaC cleavage and channel activation by serpinH1 may be responsible for the effect of TGFß on aldosterone stimulation in the distal nephron. In summary, we show that cleavage of the γ subunit, but not the α or ß subunit is linked to channel activation in three seperate contexts. Résumé Le canal épithélial à sodium (ENaC) est constitué de trois sous-unités homologues α, ß, and γ. Ce canal est impliqué dans le maintien de la balance sodique qui influence le niveau du liquide périciliaire du poumon et la pression sanguine via le rein. Dans les ovocytes de Xenopus laevis ENaC est préférentiellement et rapidement exprimé en formant un complexe hétéromérique αßγ. En revanche, l'expression homomérique de α ou hétéromérique des complexes αß et αγ conduit à une expression à la surface cellulaire d'un canal ENaC ne possédant qu'une faible activité. Des études récentes ont mis en évidence que les sous-unités α et γ d'ENaC (mais pas ß) sont coupées par des protéases endogènes (les farines) et que ces clivages augmentent l'activité du canal. Nous avons donc analysé, aussi bien à la surface cellulaire que dans le cytoplasme, les produits des clivages de combinaison homo- et hétéromérique des sous-unités d'ENaC (α, ß, γ, ßγ, αß, αγ, ßγ et αßγ). En parallèle, nous avons étudié l'activité correspondante à ces canaux par la mesure du transport de sodium sensible à l'amiloride (INa). Nous avons montré que lors de l'assemblage des sous-unités d'ENaC, le clivage de γ correspond à l'augmentation de INa. Nous avons également mis en évidence que dans une maladie telle que la fibrose cystique (CF) caractérisée par un déséquilibre de la balance protéase-inhibiteur de protéase, ENaC est suractivé par une sérine protéase nommée élastase (NE). L'augmentation de la concentration de NE clive γ ENaC exprimé à la surface cellulaire (mais pas α, ni ß ENaC) suggérant une causalité entre le clivage d'ENaC et son activation à la membrane plasmique. De plus, nous avons démontré que l'inhibiteur de sérine protéase (serpin) serpinH1, qui est co-exprimé avec ENaC dans le néphron distal, inhibe l'activité du canal en empêchant le clivage de la sous-unité γ ENaC. Il est connu que le INa induit par l'aldostérone peut être inhibé par TGFß. Or TGFß augmente l'expression de serpinH1. L'inhibition du clivage de γ ENaC et de l'activation du canal par la serpinH1 que nous avons mis en évidence pourrait ainsi être responsable de l'effet de TGFß sur la stimulation du courant par l'aldostérone dans le néphron distal. En résumé, nous avons montré que le clivage de la sous-unité γ, mais pas des sous-unités α et ß, est lié à l'activation du canal dans trois contextes distincts. Résumé tout public Le corps humain est composé d'environ 10 000 milliards de cellules et d'approximativement 60% d'eau. Les cellules du corps sont les unités fondamentales de la vie et elles sont dépendantes de certains nutriments et molécules. Ces nutriments et molécules sont dissous dans l'eau qui est présente dans et hors des cellules. Le maintien d'une concentration adéquate - de ces nutriments et de ces molécules dans l'eau à l'intérieur et à l'extérieur des cellules est -..essentiel pour leur survie. L'eau hors des cellules est nommée le fluide extracellulaire et peut être subdivisée en fluide interstitiel, qui se trouve autour des cellules, et en plasma, qui est le fluide des vaisseaux sanguins. Les fluides, les nutriments et les molécules sont constamment échangés entre les cellules, le fluide interstitiel, et le plasma. Le plasma circule dans le système circulatoire afin de distribuer les nutriments et molécules dans tout le corps et afin d'enlever les déchets cellulaires. Le rein joue un rôle essentiel dans la régulation du volume et de la concentration du plasma en éliminant sélectivement les nutriments et les molécules via la formation de l'urine. L'être humain possède deux reins, constitués chacun d'environ 1 million de néphrons. Ces derniers sont responsables de réabsorber et de sécréter sélectivement les nutriments et les molécules. Le canal épithélial à sodium (ENaC) est localisé à la surface cellulaire des néphrons et est responsable de la réabsorption du sodium (Na+). Le Na+ est présent dans quasiment toute la nourriture que nous mangeons et représente, en terme de molécule, 50% du sel de cuisine. Si trop de sodium est consommé, ENaC est inactif, si bien que le Na+ n'est pas réabsorbé et quitte le corps par l'urine. Ce mécanisme permet d'éviter que la concentration plasmatique de Na+ ne devienne trop grande, ce qui résulterait en une augmentation de la pression sanguine. Si trop peu de Na+ est consommé, ENaC réabsorbe le Na+ de l'urine primaire ce qui permet de conserver la concentration de Na+ et de prévenir une diminution de la pression sanguine par une perte de Na+. ENaC est aussi présent dans les cellules des poumons qui sont les organes permettant la respiration. La respiration est aussi essentielle pour la survie des cellules. Les poumons ne doivent pas contenir trop de liquide afin de permettre la respiration, mais en même temps ils ne doivent pas non plus être trop secs. En effet, ceci tuerait les cellules et empêcherait aussi la respiration. ENaC permet de maintenir un niveau d'humidité approprié dans les poumons en absorbant du Na+ ce qui entraîne un mouvement osmotique d'eau. L'absorption de sodium par ENaC ~ est augmentée par les protéases (in vitro et ex vivo). Les protéases sont des molécules qui peuvent couper d'autres molécules à des endroits précis. Nous avons démonté que certaines protéases augmentent l'absorption de Na+ en coupant ENaC à des endroits spécifiques. L'inhibition de ces protéases diminue le transport de Na+ et empêche le clivage d'ENaC. Dans certaines maladies telle que la mucoviscidose, des protéases sont suractivées et augmentent l'activité d'ENaC de manière inappropriée conduisant à une trop forte absorption de Na+ et à un déséquilibre de la muqueuse des poumons. Cette étude est donc particulièrement importante dans le cadre de la recherche thérapeutique de ce genre de maladie.

Effect of water velocity on the uptake of polychlorinatedd biphenyls (PCBs) by silicone rubber (SR) and low-density polyethylene (LDPE) passive samplers: an assessment of the efficiency of performance reference compounds (PRCs) in river-like flow conditions

One aim of this study is to determine the impact of water velocity on the uptake of indicator polychlorinated biphenyls (iPCBs) by silicone rubber (SR) and low-density polyethylene (LDPE) passive samplers. A second aim is to assess the efficiency of performance reference compounds (PRCs) to correct for the impact of water velocity. SR and LDPE samplers were spiked with 11 or 12 PRCs and exposed for 6 weeks to four different velocities (in the range of 1.6 to 37.7 cm s−1) in river-like flow conditions using a channel system supplied with river water. A relationship between velocity and the uptakewas found for each iPCB and enables to determine expected changes in the uptake due to velocity variations. For both samplers, velocity increases from 2 to 10 cm s−1, 30 cm s−1 (interpolated data) and 100 cm s−1 (extrapolated data) lead to increases of the uptake which do not exceed a factor of 2, 3 and 4.5, respectively. Results also showed that the influence of velocity decreased with increasing the octanol-water coefficient partition (log Kow) of iPCBs when SR is used whereas the opposite effect was observed for LDPE. Time-weighted average (TWA) concentrations of iPCBs in water were calculated from iPCB uptake and PRC release. These calculations were performed using either a single PRC or all the PRCs. The efficiency of PRCs to correct the impact of velocity was assessed by comparing the TWA concentrations obtained at the four tested velocities. For SR, a good agreement was found among the four TWA concentrations with both methods (average RSD b 10%). Also for LDPE, PRCs offered a good correction of the impact of water velocity (average RSD of about 10 to 20%). These results contribute to the process of acceptance of passive sampling in routine regulatory monitoring programs.

Epithelial Sodium Channel-Mediated Sodium Transport Is Not Dependent on the Membrane-Bound Serine Protease CAP2/Tmprss4.

The membrane-bound serine protease CAP2/Tmprss4 has been previously identified in vitro as a positive regulator of the epithelial sodium channel (ENaC). To study its in vivo implication in ENaC-mediated sodium absorption, we generated a knockout mouse model for CAP2/Tmprss4. Mice deficient in CAP2/Tmprss4 were viable, fertile, and did not show any obvious histological abnormalities. Unexpectedly, when challenged with sodium-deficient diet, these mice did not develop any impairment in renal sodium handling as evidenced by normal plasma and urinary sodium and potassium electrolytes, as well as normal aldosterone levels. Despite minor alterations in ENaC mRNA expression, we found no evidence for altered proteolytic cleavage of ENaC subunits. In consequence, ENaC activity, as monitored by the amiloride-sensitive rectal potential difference (ΔPD), was not altered even under dietary sodium restriction. In summary, ENaC-mediated sodium balance is not affected by lack of CAP2/Tmprss4 expression and thus, does not seem to directly control ENaC expression and activity in vivo.

ENaC activity in collecting ducts modulates NCC in cirrhotic mice.

Cirrhosis is a frequent and severe disease, complicated by renal sodium retention leading to ascites and oedema. A better understanding of the complex mechanisms responsible for renal sodium handling could improve clinical management of sodium retention. Our aim was to determine the importance of the amiloride-sensitive epithelial sodium channel (ENaC) in collecting ducts in compensate and decompensate cirrhosis. Bile duct ligation was performed in control mice (CTL) and collecting duct-specific αENaC knockout (KO) mice, and ascites development, aldosterone plasma concentration, urinary sodium/potassium ratio and sodium transporter expression were compared. Disruption of ENaC in collecting ducts (CDs) did not alter ascites development, urinary sodium/potassium ratio, plasma aldosterone concentrations or Na,K-ATPase abundance in CCDs. Total αENaC abundance in whole kidney increased in cirrhotic mice of both genotypes and cleaved forms of α and γ ENaC increased only in ascitic mice of both genotypes. The sodium chloride cotransporter (NCC) abundance was lower in non-ascitic KO, compared to non-ascitic CTL, and increased when ascites appeared. In ascitic mice, the lack of αENaC in CDs induced an upregulation of total ENaC and NCC and correlated with the cleavage of ENaC subunits. This revealed compensatory mechanisms which could also take place when treating the patients with diuretics. These compensatory mechanisms should be considered for future development of therapeutic strategies.

Activity of daptomycin- and vancomycin-loaded poly-epsilon-caprolactone microparticles against mature staphylococcal biofilms.

The aim of the present study was to develop novel daptomycin-loaded poly-epsilon-caprolactone (PCL) microparticles with enhanced antibiofilm activity against mature biofilms of clinically relevant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) and polysaccharide intercellular adhesin-positive Staphylococcus epidermidis. Daptomycin was encapsulated into PCL microparticles by a double emulsion-solvent evaporation method. For comparison purposes, formulations containing vancomycin were also prepared. Particle morphology, size distribution, encapsulation efficiency, surface charge, thermal behavior, and in vitro release were assessed. All formulations exhibited a spherical morphology, micrometer size, and negative surface charge. From a very early time stage, the released concentrations of daptomycin and vancomycin were higher than the minimal inhibitory concentration and continued so up to 72 hours. Daptomycin presented a sustained release profile with increasing concentrations of the drug being released up to 72 hours, whereas the release of vancomycin stabilized at 24 hours. The antibacterial activity of the microparticles was assessed by isothermal microcalorimetry against planktonic and sessile MRSA and S. epidermidis. Regarding planktonic bacteria, daptomycin-loaded PCL microparticles presented the highest antibacterial activity against both strains. Isothermal microcalorimetry also revealed that lower concentrations of daptomycin-loaded microparticles were required to completely inhibit the recovery of mature MRSA and S. epidermidis biofilms. Further characterization of the effect of daptomycin-loaded PCL microparticles on mature biofilms was performed by fluorescence in situ hybridization. Fluorescence in situ hybridization showed an important reduction in MRSA biofilm, whereas S. epidermidis biofilms, although inhibited, were not eradicated. In addition, an important attachment of the microparticles to MRSA and S. epidermidis biofilms was observed. Finally, all formulations proved to be biocompatible with both ISO compliant L929 fibroblasts and human MG63 osteoblast-like cells.

Attenuated Levels of Hippocampal Connexin 43 and its Phosphorylation Correlate with Antidepressant- and Anxiolytic-Like Activities in Mice.

Clinical and preclinical studies have implicated glial anomalies in major depression. Conversely, evidence suggests that the activity of antidepressant drugs is based, at least in part, on their ability to stimulate density and/or activity of astrocytes, a major glial cell population. Despite this recent evidence, little is known about the mechanism(s) by which astrocytes regulate emotionality. Glial cells communicate with each other through gap junction channels (GJCs), while they can also directly interact with neurons by releasing gliotransmitters in the extracellular compartment via an hemichannels (HCs)-dependent process. Both GJCs and HCs are formed by two main protein subunits: connexins (Cx) 30 and 43 (Cx30 and Cx43). Here we investigate the role of hippocampal Cx43 in the regulation of depression-like symptoms using genetic and pharmacological approaches. The first aim of this study was to evaluate the impact of the constitutive knock-down of Cx43 on a set of behaviors known to be affected in depression. Conversely, the expression of Cx43 was assessed in the hippocampus of mice subjected to prolonged corticosterone (CORT) exposure, given either alone or in combination with an antidepressant drug, the selective serotonin reuptake inhibitor fluoxetine. Our results indicate that the constitutive deficiency of Cx43 resulted in the expression of some characteristic hallmarks of antidepressant-/anxiolytic-like behavioral activities along with an improvement of cognitive performances. Moreover, in a new cohort of wild-type mice, we showed that CORT exposure elicited anxiety and depression-like abnormalities that were reversed by chronic administration of fluoxetine. Remarkably, CORT also increased hippocampal amounts of phosphorylated form of Cx43 whereas fluoxetine treatment normalized this parameter. From these results, we envision that antidepressant drugs may exert their therapeutic activity by decreasing the expression and/or activity of Cx43 resulting from a lower level of phosphorylation in the hippocampus.

Involvement of a transforming-growth-factor-beta-like molecule in tumor-cell-derived inhibition of nitric-oxide synthesis in cerebral endothelial cells.

Nitric oxide (NO) has been shown to exert cytotoxic effects on tumor cells. We have reported that EC219 cells, a rat-brain-microvessel-derived endothelial cell line, produced NO through cytokine-inducible NO synthase (iNOS), the induction of which was significantly decreased by (a) soluble factor(s) secreted by DHD/PROb, an invasive sub-clone of a rat colon-carcinoma cell line. In this study, the DHD/PROb cell-derived NO-inhibitory factor was characterized. Northern-blot analysis demonstrated that the induction of iNOS mRNA in cytokine-activated EC219 cells was decreased by PROb-cell-conditioned medium. When DHD/PROb cell supernatant was fractionated by affinity chromatography using Con A-Sepharose or heparin-Sepharose, the NO-inhibitory activity was found only in Con A-unbound or heparin-unbound fractions, respectively, indicating that the PROb-derived inhibitory factor was likely to be a non-glycosylated and non-heparin-binding molecule. Pre-incubation of DHD/PROb-cell supernatant with anti-TGF-beta neutralizing antibody completely blocked the DHD/PROb-derived inhibition of NO production by EC219 cells. Addition of exogenous TGF-beta 1 dose-dependently inhibited NO release by EC219 cells. The presence of active TGF-beta in the DHD/PROb cell supernatant was demonstrated using a growth-inhibition assay. Moreover, heat treatment of medium conditioned by the less invasive DHD/REGb cells, which constitutively secreted very low levels of active TGF-beta, increased both TGF-beta activity and the ability to inhibit NO production in EC219 cells. Thus, DHD/PROb colon-carcinoma cells inhibited NO production in EC219 cells by secreting a factor identical or very similar to TGF-beta.

Impaired aldehyde dehydrogenase 1 subfamily member 2A-dependent retinoic acid signaling is related with a mesenchymal-like phenotype and an unfavorable prognosis of head and neck squamous cell carcinoma.

BACKGROUND: An inverse correlation between expression of the aldehyde dehydrogenase 1 subfamily A2 (ALDH1A2) and gene promoter methylation has been identified as a common feature of oropharyngeal squamous cell carcinoma (OPSCC). Moreover, low ALDH1A2 expression was associated with an unfavorable prognosis of OPSCC patients, however the causal link between reduced ALDH1A2 function and treatment failure has not been addressed so far. METHODS: Serial sections from tissue microarrays of patients with primary OPSCC (n = 101) were stained by immunohistochemistry for key regulators of retinoic acid (RA) signaling, including ALDH1A2. Survival with respect to these regulators was investigated by univariate Kaplan-Meier analysis and multivariate Cox regression proportional hazard models. The impact of ALDH1A2-RAR signaling on tumor-relevant processes was addressed in established tumor cell lines and in an orthotopic mouse xenograft model. RESULTS: Immunohistochemical analysis showed an improved prognosis of ALDH1A2(high) OPSCC only in the presence of CRABP2, an intracellular RA transporter. Moreover, an ALDH1A2(high)CRABP2(high) staining pattern served as an independent predictor for progression-free (HR: 0.395, p = 0.007) and overall survival (HR: 0.303, p = 0.002), suggesting a critical impact of RA metabolism and signaling on clinical outcome. Functionally, ALDH1A2 expression and activity in tumor cell lines were related to RA levels. While administration of retinoids inhibited clonogenic growth and proliferation, the pharmacological inhibition of ALDH1A2-RAR signaling resulted in loss of cell-cell adhesion and a mesenchymal-like phenotype. Xenograft tumors derived from FaDu cells with stable silencing of ALDH1A2 and primary tumors from OPSCC patients with low ALDH1A2 expression exhibited a mesenchymal-like phenotype characterized by vimentin expression. CONCLUSIONS: This study has unraveled a critical role of ALDH1A2-RAR signaling in the pathogenesis of head and neck cancer and our data implicate that patients with ALDH1A2(low) tumors might benefit from adjuvant treatment with retinoids.

Imaging the time-integrated cerebral metabolic activity with subcellular resolution through nanometer-scale detection of biosynthetic products deriving from (13)C-glucose.

Glucose is the primary source of energy for the brain but also an important source of building blocks for proteins, lipids, and nucleic acids. Little is known about the use of glucose for biosynthesis in tissues at the cellular level. We demonstrate that local cerebral metabolic activity can be mapped in mouse brain tissue by quantitatively imaging the biosynthetic products deriving from [U-(13)C]glucose metabolism using a combination of in situ electron microscopy and secondary ion mass-spectroscopy (NanoSIMS). Images of the (13)C-label incorporated into cerebral ultrastructure with ca. 100nm resolution allowed us to determine the timescale on which the metabolic products of glucose are incorporated into different cells, their sub-compartments and organelles. These were mapped in astrocytes and neurons in the different layers of the motor cortex. We see evidence for high metabolic activity in neurons via the nucleus (13)C enrichment. We observe that in all the major cell compartments, such as e.g. nucleus and Golgi apparatus, neurons incorporate substantially higher concentrations of (13)C-label than astrocytes.