On the function of proton-gated ion channels ASICs and TRPV1 : a patch-clamp study

AbstractAcidosis is encountered during tissue inflammation and triggers pain in humans. H+-gated ion channels are expressed at high levels in sensory neurons of the peripheral nervous system. Ion channels from two different families present the required pH sensitivity to detect the acidosis associated with peripheral inflammation: Acid-Sensing Ion Channels (ASICs) and the Transient Receptor Potential Vanilloid-1 (TRPV1) channel.ASICs are members of the Degenerin/Epithelial Na+ Channel family of ion channels. Six ASIC subunits have been identified in mammals (ASICla, -lb, -2a, -2b, -3 and -4). ASICs form In-activated voltage-insensitive homo- or heterotrimeric Na+ channels. TRPV1 is a member of the TRP family of ion channels and forms non-selective cation channels that mediate a sustained current. TRPV1 is activated by H+, heat (T>43°C), lipids, capsaicin, voltage and other stimuli. A stimulus can increase TRPV1 response to a different stimulus. For example H+ can shift the capsaicin concentration dependence of TRPV1 to lower values. ASICs and TRPV1 have been shown to be involved in inflammatory pain. Using the patch-clamp technique, we studied different aspects of the function of ASICs and TRPV1 in the physiological context of pain.In the first part of this thesis, we characterize the effect of a temperature increase from 25 to 35°C on the function of ASICs and TRPV1 in transfected CHO cells and primary cultures of rat DRG sensory neurons. ASICs give rise to transient currents while TRPV1 mediates a sustained current. In addition, ASICs and TRPV1 respond to H+ with distinct pH dependences. We assess the relative contribution of ASICs and TRPV1 to H+-evoked electrical signaling in rat DRG neurons and we conclude that ASICs are the most important pH sensors in the pH range 7.4 to 6.0 at 35°C in sensory neurons.ASICs and TRPV1 are expressed in the epithelium lining the lumen of the bladder (urothelium). The Bladder Pain Syndrome/Interstitial Cystitis (BPS/IC) is a painful condition associated with a dysfunction of the urothelial barrier and with inflammation. In the second part of this thesis, we show that human urothelial cells -the cell line TEU2 and primary cultures of human bladder urothelium- express functional ASICs but no functional TRPV1 channels. In addition, we show that the levels of ASIC2 and ASIC3 mRNA are increased in the urothelium of patients suffering from BPS/IC. These data suggest that ASICs are involved in the pathology of BPS/IC.Finally, we demonstrate that APETx2 inhibits the sensory neuron specific voltage-dependent Na+ channel Nav1.8. APETx2 was previously shown to inhibit homo- or heterotrimeric ASIC3- containing channels with IC5o from 0.08 to 1 μΜ. We show that APETx2 also inhibits Nav1.8 with an ICsoof «2.6 μΜ. APETx2 reduces the maximal conductance and induces a depolarizing shift in the voltage dependence of activation of Nav1.8. In current-clamp experiments, APETx2 reduces the number of action potentials (APs) evoked by a current ramp. Nav1.8 mediates most of the current during the AP upstroke and has been shown to be an important mediator of inflammatory pain. The fact that APETx2 inhibits two ion channels involved in inflammatory pain suggests that APETx2 or derivatives may represent novel analgesic compounds.RésuméL'acidose tissulaire est observée durant l'inflammation et entraine la douleur chez l'humain. Des canaux ioniques activés par les protons (H+) sont fortement exprimés dans les neurones sensoriels du système nerveux périphérique. De ceux-ci, les Acid-Sensing Ion Channels [ASICs) et Transient Receptor Potential Vanilloid-1 (TRPV1) présentent une sensibilité adéquate à l'acidité pour servir de détecteurs d'acidose.Les ASICs sont membres de la famille Degenerin/Epithelial Na* Channel. Six sous-unités ASIC ont été identifiées chez les mammifères (ASICla, -lb, -2a, -2b, -3 et -4). Les ASICs forment des canaux sélectifs au Na\ insensibles au voltage et activés par les H+. Les canaux fonctionnels sont des homo- ou hétérotrimères de sous-unités ASIC. TRPV1 est un membre de la famille TRP de canaux ioniques. Les canaux TRPV1 sont activés par les H+, la chaleur (T>43°Ç), les lipides, la capsaicine, le voltage et d'autres stimulus. L'activation de TRPV1 entraine un courant soutenu non-sélectif. Un stimulus peut augmenter la réponse de TRPV1 à un autre stimulus. Les H+ peuvent, par exemple, induire un décalage vers des valeurs plus faibles de la courbe de dépendance à la concentration de TRPV1 pour la capsaicine. Il a été démontré que les ASICs et TRPV1 sont impliqués dans la douleur inflammatoire. En utilisant la technique du patch-clamp, nous avons étudié différents aspects de la fonction des ASICs et de TRPV1 dans des contextes associés à la douleur.Dans la première partie de cette thèse, nous caractérisons l'effet d'une augmentation de température de 25 à 35°C sur la fonction des canaux ASICs et TRPV1, dans des cellules CHO transfectées et dans des cultures primaires de neurones sensoriels (DRG) de rat. L'activation des ASICs entraine l'apparition d'un courant transitoire tandis que l'activation de TRPV1 entraine un courant soutenu. De plus, les ASICs et TRPV1 possèdent des dépendances au pH différentes. Nous évaluons la contribution relative des ASICs et de TRPV1 au signalement électrique induit par les H+ et nous concluons que les ASICs sont les senseurs d'acidité les plus importants dans les neurones sensoriels, dans le domaine de pH de 7.4 à 6.0, à température corporelle.Les ASICs et TRPV1 sont exprimés dans l'épithélium recouvrant l'intérieur de la vessie (l'urothélium). Le Bladder Pain Syndrome/Interstitial Cystitis (BPS/IC) est une condition médicale douloureuse associée à une dysfonction de la barrière urothéliale et à une inflammation. Dans la seconde partie de cette thèse, nous démontrons que des cellules urothéliales (de la lignée cellulaire TEU2) et des cellules provenant de cultures primaires d'épithéliums de vessies humaines expriment des canaux ASIC fonctionnels mais pas de TRPV1 fonctionnels. De plus, nous montrons que le niveau d'expression de ASIC2 et -3 est augmenté dans l'urothélium de la vessie de patients souffrant de BPS/IC. Ces données suggèrent que les ASICs sont impliqués dans la pathologie BPS/IC.Pour finir, nous démontrons que la toxine APETx2 inhibe le canal spécifique aux neurones sensoriels Nav1.8, un membre de la famille des canaux sodiques dépendants du potentiel. Il a été démontré précédemment que la toxine APETx2 inhibe les canaux contenant une ou plusieurs sous-unités ASIC3 avec un ICso entre 0.08 et 1 μΜ. Nous montrons que la toxine APETx2 inhibe Nav1.8 avec un IC50 de «2.6 μΜ. La toxine APETx2 réduit la conductance maximale et induit un décalage de la dépendance au potentiel de Nav1.8 vers des valeurs plus positives. Dans des expériences de courant imposé sur des neurones sensoriels, la toxine APETx2 réduit le nombre de potentiels d'action induits par une rampe de courant. Nav1.8 est responsable de la majeure partie du courant durant la phase ascendante du potentiel d'action et a été démontré comme étant un médiateur important de la douleur inflammatoire. L'inhibition de deux types de canaux, impliqués dans la douleurs inflammatoire, par la toxine APETx2, suggère que cette dernière ou ses dérivés représentent des composés analgésiques prometteurs.

Study of conformational changes of ASIC1a by voltage-clamp fluorometry

Le fonctionnement du système nerveux est sensible aux variations de la concentration d'acide. Une acidification des tissus peut se produire pendant une activité neuronale intense ou dans des situations physiopathologiques telles que l'inflammation ou les lésions cérébrales. Les canaux ioniques sensibles à l'acide (ASIC) sont activés par acidification et jouent un rôle important dans la détection des changements d'acide. Les ASICs contribuent à la dégénérescence neuronale après une lésion cérébrale, puisque leur inhibition limite la lésion neuronale. L'acidification induite par une inflammation du tissu nerveux conduit à des stimuli de douleur, qui sont détectés par ces canaux. En effet, les toxines qui bloquent spécifiquement les ASICs montrent des effets analgésiques dans des modèles animaux. La structure 3D d'ASIC peut être comparée à une main qui tient une boule entre son pouce et le doigt. Les différents domaines d'ASIC sont appelés doigt, pouce, jointure, boule-ß et paume. Les domaines transmembranaires représentent le poignet de cette main. Mon projet de thèse vise à décrire les mouvements survenant sur ce canal pendant son activité. A cet effet, j'ai utilisé une technique combinée qui permet la mesure des mouvements en temps réel durant l'activité du canal. J'ai montré les réarrangements des domaines extracellulaires pendant l'activité ASIC. Ces mouvements sont transmis au pore du canal, ou ils contrôlent sa fermeture et ouverture. La direction de ces mouvements a été évaluée pour les domaines doigt et jointure, et nous avons montré qu'ils s'éloignent de la boule-ß lors de l'acidification. J'ai également été en mesure de décrire les mouvements qui se produisent dans la poche acidique, une zone qui est considérée comme importante, car elle représente le site de liaison de certaines toxines de venin qui agissent sur les ASICs. J'ai ainsi pu montrer que les domaines doigt et le pouce qui forment la poche acidique se rapprochent l'un de l'autre pendant l'activation du canal. Ces résultats sont en accord avec des observations précédentes réalisées sur les ASICs par d'autres chercheurs. Enfin, cette analyse approfondie permet d'améliorer les connaissances sur le contrôle de l'activité ASIC; de plus, les mécanismes trouvés ici sont probablement partagés entre les canaux de la famille à laquelle appartiennent les ASICs. -- Les acid-sensing ion channels (ASICs) sont des canaux sodiques ouverts par les protons et principalement exprimés dans le système nerveux. Ils sont impliqués dans la détection des protons dans de nombreux états physiologiques et pathologiques comme l'ischémie et la perception de la douleur. La structure cristalline de l'isoforme ASIC1 de poulet a été déterminée dans l'état désensibilisé et ouvert. Les études fonctionnelles indiquent que la protonation des résidus clés dans la boucle extracellulaire déclenche des changements de conformation conduisant à l'ouverture du canal. Cependant, les mécanismes moléculaires qui relient la protonation à l'ouverture et la fermeture du canal n'ont pas encore été clarifiés. Dans cette étude, nous avons utilisé la voltage-clamp fluorimétrie (VCF) pour révéler les mouvements de l'activité associée qui se produisent dans les différents domaines de l'ASICla. Les fluorophores positionnés dans le pouce, la paume, le doigt, l'articulation et dans les domaines de l'entrée du pore extracellulaire ont montré des signaux de VCF liés à des changements de conformation au cours de l'activité du canal. La synchronisation des changements de fluorescence indique une séquence complexe de mouvements en fonction du pH. La cinétique de la fluorescence et des signaux de courant ont été comparés les uns aux autres afin de déterminer si le mouvement détecté par le signal de fluorescence correspond à une transition fonctionnelle définie du canal. Certains des résidus testés se sont révélés être étroitement liés à la désensibilisation du canal ou au rétablissement après la désensibilisation. En outre, nous avons trouvé qu'un tryptophane endogène de la boule-ß diminue le signal de fluorescence des sondes positionnées dans les domaines doigt et jointure. L'augmentation observée de ces signaux indique que ces domaines s'éloignent à une distance à partir de la boucle de la boule-ß. Sur la base de ce principe, nous avons généré des paires Trp-Cys « quencher», dans lequel le Cys est utilisé comme site d'ancrage pour attacher le fluorophore. Ensuite, nous avons évalué les changements de conformation qui se produisent au niveau de la poche acide, une zone importante pour la fonction et la régulation d'ASIC. Les signaux de fluorescence indiquent un mouvement de l'hélice supérieure du pouce vers le doigt et une rotation de la boule-ß dans le sens horaire. L'analyse de la cinétique indique que les mouvements des sous-domaines qui composent la poche acide se produisent pendant la désensibilisation du canal. Mon projet de doctorat représente la première analyse approfondie des changements conformationnels dépendants de l'activité des ASICs et fournit des informations sur les mécanismes de contrôle de l'activité du canal qui sont probablement partagés avec d'autres canaux proches.

The β-sliding clamp directs the localization of HdaA to the replisome in Caulobacter crescentus.

The initiation of chromosome replication is tightly regulated in bacteria to ensure that it takes place only once per cell cycle. In many proteobacteria, this process requires the ATP-bound form of the DnaA protein. The regulatory inactivation of DnaA (RIDA) facilitates the conversion of DnaA-ATP into replication-inactive DnaA-ADP, thereby preventing overinitiation. Homologues of the HdaA protein, together with the β-clamp of the DNA polymerase (DnaN), are required for this process. Here, we used fluorescence resonance energy transfer experiments to demonstrate that HdaA interacts with DnaN in live Caulobacter crescentus cells. We show that a QFKLPL motif in the N-terminal region of HdaA is required for this interaction and that this motif is also needed to recruit HdaA to the subcellular location occupied by the replisome during DNA replication. An HdaA mutant protein that cannot colocalize or interact with DnaN can also not support the essential function of HdaA. These results suggest that the recruitment of HdaA to the replisome is needed during RIDA in C. crescentus, probably as a means to sense whether chromosome replication has initiated before DnaA becomes inactivated. In addition, we show that a conserved R145 residue located in the AAA+ domain of HdaA is also needed for the function of HdaA, although it does not affect the interaction of HdaA with DnaN in vivo. The AAA+ domain of HdaA may therefore be required during RIDA after the initial recruitment of HdaA to the replisome by DnaN.

Molecular basis of messenger RNA recognition by the specific bacterial repressing clamp RsmA/CsrA.

Proteins of the RsmA/CsrA family are global translational regulators in many bacterial species. We have determined the solution structure of a complex formed between the RsmE protein, a member of this family from Pseudomonas fluorescens, and a target RNA encompassing the ribosome-binding site of the hcnA gene. The RsmE homodimer with its two RNA-binding sites makes optimal contact with an 5'-A/UCANGGANGU/A-3' sequence in the mRNA. When tightly gripped by RsmE, the ANGGAN core folds into a loop, favoring the formation of a 3-base-pair stem by flanking nucleotides. We validated these findings by in vivo and in vitro mutational analyses. The structure of the complex explains well how, by sequestering the Shine-Dalgarno sequence, the RsmA/CsrA proteins repress translation.

Disaggregating chaperones: an unfolding story.

Stress, molecular crowding and mutations may jeopardize the native folding of proteins. Misfolded and aggregated proteins not only loose their biological activity, but may also disturb protein homeostasis, damage membranes and induce apoptosis. Here, we review the role of molecular chaperones as a network of cellular defenses against the formation of cytotoxic protein aggregates. Chaperones favour the native folding of proteins either as "holdases", sequestering hydrophobic regions in misfolding polypeptides, and/or as "unfoldases", forcibly unfolding and disentangling misfolded polypeptides from aggregates. Whereas in bacteria, plants and fungi Hsp70/40 acts in concert with the Hsp100 (ClpB) unfoldase, Hsp70/40 is the only known chaperone in the cytoplasm of mammalian cells that can forcibly unfold and neutralize cytotoxic protein conformers. Owing to its particular spatial configuration, the bulky 70 kDa Hsp70 molecule, when distally bound through a very tight molecular clamp onto a 50-fold smaller hydrophobic peptide loop extruding from an aggregate, can locally exert on the misfolded segment an unfolding force of entropic origin, thus destroying the misfolded structures that stabilize aggregates. ADP/ATP exchange triggers Hsp70 dissociation from the ensuing enlarged unfolded peptide loop, which is then allowed to spontaneously refold into a closer-to-native conformation devoid of affinity for the chaperone. Driven by ATP, the cooperative action of Hsp70 and its co-chaperone Hsp40 may thus gradually convert toxic misfolded protein substrates with high affinity for the chaperone, into non-toxic, natively refolded, low-affinity products. Stress- and mutation-induced protein damages in the cell, causing degenerative diseases and aging, may thus be effectively counteracted by a powerful network of molecular chaperones and of chaperone-related proteases.

T-type Ca2+ channels, SK2 channels and SERCAs gate sleep-related oscillations in thalamic dendrites.

T-type Ca2+ channels (T channels) underlie rhythmic burst discharges during neuronal oscillations that are typical during sleep. However, the Ca2+-dependent effectors that are selectively regulated by T currents remain unknown. We found that, in dendrites of nucleus reticularis thalami (nRt), intracellular Ca2+ concentration increases were dominated by Ca2+ influx through T channels and shaped rhythmic bursting via competition between Ca2+-dependent small-conductance (SK)-type K+ channels and Ca2+ uptake pumps. Oscillatory bursting was initiated via selective activation of dendritically located SK2 channels, whereas Ca2+ sequestration by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) and cumulative T channel inactivation dampened oscillations. Sk2-/- (also known as Kcnn2) mice lacked cellular oscillations, showed a greater than threefold reduction in low-frequency rhythms in the electroencephalogram of non-rapid-eye-movement sleep and had disrupted sleep. Thus, the interplay of T channels, SK2 channels and SERCAs in nRt dendrites comprises a specialized Ca2+ signaling triad to regulate oscillatory dynamics related to sleep.

Sustaining sleep spindles through enhanced SK2-channel activity consolidates sleep and elevates arousal threshold.

Sleep spindles are synchronized 11-15 Hz electroencephalographic (EEG) oscillations predominant during nonrapid-eye-movement sleep (NREMS). Rhythmic bursting in the reticular thalamic nucleus (nRt), arising from interplay between Ca(v)3.3-type Ca(2+) channels and Ca(2+)-dependent small-conductance-type 2 (SK2) K(+) channels, underlies spindle generation. Correlative evidence indicates that spindles contribute to memory consolidation and protection against environmental noise in human NREMS. Here, we describe a molecular mechanism through which spindle power is selectively extended and we probed the actions of intensified spindling in the naturally sleeping mouse. Using electrophysiological recordings in acute brain slices from SK2 channel-overexpressing (SK2-OE) mice, we found that nRt bursting was potentiated and thalamic circuit oscillations were prolonged. Moreover, nRt cells showed greater resilience to transit from burst to tonic discharge in response to gradual depolarization, mimicking transitions out of NREMS. Compared with wild-type littermates, chronic EEG recordings of SK2-OE mice contained less fragmented NREMS, while the NREMS EEG power spectrum was conserved. Furthermore, EEG spindle activity was prolonged at NREMS exit. Finally, when exposed to white noise, SK2-OE mice needed stronger stimuli to arouse. Increased nRt bursting thus strengthens spindles and improves sleep quality through mechanisms independent of EEG slow waves (<4 Hz), suggesting SK2 signaling as a new potential therapeutic target for sleep disorders and for neuropsychiatric diseases accompanied by weakened sleep spindles.

Blocking VLDL secretion causes hepatic steatosis but does not affect peripheral lipid stores or insulin sensitivity in mice

The liver secretes triglyceride-rich VLDLs, and the triglycerides in these particles are taken up by peripheral tissues, mainly heart, skeletal muscle, and adipose tissue. Blocking hepatic VLDL secretion interferes with the delivery of liver-derived triglycerides to peripheral tissues and results in an accumulation of triglycerides in the liver. However, it is unclear how interfering with hepatic triglyceride secretion affects adiposity, muscle triglyceride stores, and insulin sensitivity. To explore these issues, we examined mice that cannot secrete VLDL [due to the absence of microsomal triglyceride transfer protein (Mttp) in the liver]. These mice exhibit markedly reduced levels of apolipoprotein B-100 in the plasma, along with reduced levels of triglycerides in the plasma. Despite the low plasma triglyceride levels, triglyceride levels in skeletal muscle were unaffected. Adiposity and adipose tissue triglyceride synthesis rates were also normal, and body weight curves were unaffected. Even though the blockade of VLDL secretion caused hepatic steatosis accompanied by increased ceramides and diacylglycerols in the liver, the mice exhibited normal glucose tolerance and were sensitive to insulin at the whole-body level, as judged by hyperinsulinemic euglycemic clamp studies. Normal hepatic glucose production and insulin signaling were also maintained in the fatty liver induced by Mttp deletion. Thus, blocking VLDL secretion causes hepatic steatosis without insulin resistance, and there is little effect on muscle triglyceride stores or adiposity

Effect of rufinamide on gating properties of voltage-gated sodium channel Na(v)1.7

Background: Voltage-gated sodium channels (Nav1.x) are important players in chronic pain. A particular interest has grown in Nav1.7, expressed in nociceptors, since mutations in its gene are associated to two inherited pain syndromes or insensitivity to pain. Rufinamide, a drug used to treat refractory epilepsy such as the Lennox-Gastaut syndrome, has been shown to reduce the number of action potentials in cortical neurons without completely blocking Na channels. Aim: The goal of this study was to investigate the effect of rufinamide on Nav1.7 current. Methods and results: Whole-cell patch clamp experiments were performed using HEK293 cells stably expressing Nav1.7. Rufinamide significantly decreased peak sodium current by 28.3, 21.2 and 12.5% at concentrations of 500, 100 and 50μM respectively (precise EC50 could not be calculated since higher rufinamide concentrations could not be achieved in physiological buffer solution). No significant difference on the V1/2 of voltage-dependence of activation was seen; however a shift in the steady-state inactivation curve was observed (-82.6 mV to -88.8 mV and -81.8 to -87.6 mV for 50 and 100 μM rufinamide respectively, p <0.005). Frequency-dependent inhibition of Nav1.7 was also influenced by the drug. One hundred μM rufinamide reduced the peak sodium current (in % of the peak current taken at the first sweep of a train of 50) from 90.8 to 80.8% (5Hz), 88.7 to 71.8% (10 Hz), 69.1 to 49.2% (25 Hz) and 22.3 to 9.8% (50 Hz) (all p <0.05). Onset of fast inactivation was not influenced by the drug since no difference in the time constant of current decay was observed. Conclusion: In the concentration range of plasma level in human treated for epilepsy, 15 μM, rufinamide only minimally blocks Nav1.7. However, it stabilizes the inactivated state and exerts frequencydependent inhibition of Nav1.7. These pharmacological properties may be of use in reducing ectopic discharges as a causal and symptom related contributor of neuropathic pain syndrome.

The human CFTR protein expressed in CHO cells activates aquaporin-3 in a cAMP-dependent pathway: study by digital holographic microscopy.

The transmembrane water movements during cellular processes and their relationship to ionic channel activity remain largely unknown. As an example, in epithelial cells it was proposed that the movement of water could be directly linked to cystic fibrosis transmembrane conductance regulator (CFTR) protein activity through a cAMP-stimulated aqueous pore, or be dependent on aquaporin. Here, we used digital holographic microscopy (DHM) an interferometric technique to quantify in situ the transmembrane water fluxes during the activity of the epithelial chloride channel, CFTR, measured by patch-clamp and iodide efflux techniques. We showed that the water transport measured by DHM is fully inhibited by the selective CFTR blocker CFTRinh172 and is absent in cells lacking CFTR. Of note, in cells expressing the mutated version of CFTR (F508del-CFTR), which mimics the most common genetic alteration encountered in cystic fibrosis, we also show that the water movement is profoundly altered but restored by pharmacological manipulation of F508del-CFTR-defective trafficking. Importantly, whereas activation of this endogenous water channel required a cAMP-dependent stimulation of CFTR, activation of CFTR or F508del-CFTR by two cAMP-independent CFTR activators, genistein and MPB91, failed to trigger water movements. Finally, using a specific small-interfering RNA against the endogenous aquaporin AQP3, the water transport accompanying CFTR activity decreased. We conclude that water fluxes accompanying CFTR activity are linked to AQP3 but not to a cAMP-stimulated aqueous pore in the CFTR protein.

Effect of somatostatin on duodenal glucose absorption in man.

OBJECTIVE: The hyperglycemic hyperinsulinemic clamp technique using intraduodenally infused glucose is an attractive tool for studying postprandial glucose metabolism under strictly controlled conditions. Because it requires the use of somatostatin (SST), we examined, in this study, the effect of SST on intestinal glucose absorption. CONTEXT: Twenty-six normal volunteers were given a constant 3-h intraduodenal infusion of glucose (6 mg.kg(-1).min(-1)) labeled with [2-(3)H]glucose for glucose absorption measurement. During glucose infusion, 19 subjects received iv SST at doses of 10-100 ng.kg(-1).min(-1) plus insulin and glucagon, and seven subjects were studied under control conditions. In the controls, glucose was absorbed at a rate that, after a 20-min lag period, equaled the infusion rate. RESULTS: With all the doses of SST tested, absorption was considerably delayed but equaled the rate of infusion after 3 h. At that time, only 5 +/- 2% of the total amount of infused glucose was unabsorbed in the control subjects vs. 36 +/- 2% (P < 0.001) in the SST-infused subjects. In the latter, the intraluminal residue was almost totally absorbed within 40 min of the cessation of SST infusion. At the lowest dose of SST tested (10 ng.kg(-1).min(-1)), suppression of insulin secretion was incomplete. CONCLUSION: These properties of SST hamper the use of intraduodenal hyperglycemic hyperinsulinemic clamps as a tool for exploring postprandial glucose metabolism.

The fourth transmembrane segment of the Na,K-ATPase alpha subunit: a systematic mutagenesis study.

The Na,K-ATPase is a major ion-motive ATPase of the P-type family responsible for many aspects of cellular homeostasis. To determine the structure of the pathway for cations across the transmembrane portion of the Na,K-ATPase, we mutated 24 residues of the fourth transmembrane segment into cysteine and studied their function and accessibility by exposure to the sulfhydryl reagent 2-aminoethyl-methanethiosulfonate. Accessibility was also examined after treatment with palytoxin, which transforms the Na,K-pump into a cation channel. Of the 24 tested cysteine mutants, seven had no or a much reduced transport function. In particular cysteine mutants of the highly conserved "PEG" motif had a strongly reduced activity. However, most of the non-functional mutants could still be transformed by palytoxin as well as all of the functional mutants. Accessibility, determined as a 2-aminoethyl-methanethiosulfonate-induced reduction of the transport activity or as inhibition of the membrane conductance after palytoxin treatment, was observed for the following positions: Phe(323), Ile(322), Gly(326), Ala(330), Pro(333), Glu(334), and Gly(335). In accordance with a structural model of the Na,K-ATPase obtained by homology modeling with the two published structures of sarcoplasmic and endoplasmic reticulum calcium ATPase (Protein Data Bank codes 1EUL and 1IWO), the results suggest the presence of a cation pathway along the side of the fourth transmembrane segment that faces the space between transmembrane segments 5 and 6. The phenylalanine residue in position 323 has a critical position at the outer mouth of the cation pathway. The residues thought to form the cation binding site II ((333)PEGL) are also part of the accessible wall of the cation pathway opened by palytoxin through the Na,K-pump.

Moderate amounts of fructose consumption impair insulin sensitivity in healthy young men: a randomized controlled trial.

OBJECTIVE: Adverse effects of hypercaloric, high-fructose diets on insulin sensitivity and lipids in human subjects have been shown repeatedly. The implications of fructose in amounts close to usual daily consumption, however, have not been well studied. This study assessed the effect of moderate amounts of fructose and sucrose compared with glucose on glucose and lipid metabolism. RESEARCH DESIGN AND METHODS: Nine healthy, normal-weight male volunteers (aged 21-25 years) were studied in this double-blind, randomized, cross-over trial. All subjects consumed four different sweetened beverages (600 mL/day) for 3 weeks each: medium fructose (MF) at 40 g/day, and high fructose (HF), high glucose (HG), and high sucrose (HS) each at 80 g/day. Euglycemic-hyperinsulinemic clamps with [6,6]-(2)H(2) glucose labeling were used to measure endogenous glucose production. Lipid profile, glucose, and insulin were measured in fasting samples. RESULTS: Hepatic suppression of glucose production during the clamp was significantly lower after HF (59.4 ± 11.0%) than HG (70.3 ± 10.5%, P < 0.05), whereas fasting glucose, insulin, and C-peptide did not differ between the interventions. Compared with HG, LDL cholesterol and total cholesterol were significantly higher after MF, HF, and HS, and free fatty acids were significantly increased after MF, but not after the two other interventions (P < 0.05). Subjects' energy intake during the interventions did not differ significantly from baseline intake. CONCLUSIONS: This study clearly shows that moderate amounts of fructose and sucrose significantly alter hepatic insulin sensitivity and lipid metabolism compared with similar amounts of glucose.

Surgery for ruptured thoracic and thoraco-abdominal aortic aneurysms.

OBJECTIVE: To assess the outcome of patients with ruptured descending thoracic and thoracoabdominal aortic aneurysms undergoing emergency repair, in comparison to elective surgery for chronic lesions. METHODS: A prospective study of 100 consecutive patients operated upon the descending aorta (1-8 segments) using proximal unloading and distal protection with partial cardiopulmonary bypass, heparin surface-coated perfusion equipment and low systemic heparinization (loading dose 100 IU/kg, activated coagulation time > 180 s), staged cross-clamping, sealed grafts and graft inclusion. RESULTS: Arteriosclerotic lesions were present in 53/100 patients (53%) for all, 30/53 (56%) for chronic, and 21/33 (63%) for ruptured, aneurysms (NS). Dissecting lesions were found in 38/100 patients (38%) for all, 20/53 (38%) for chronic, and 8/33 (24%) for ruptured aneurysms (NS). Preoperative hematocrit was 38 +/- 6% for all, 40 +/- 5% for chronic, and 33 +/- 5% for ruptured aneurysmal patients (P < 0.001 ruptured versus chronic). The extent of aortic repair (1-8 segments) was 3.3 +/- 1.6 for all, 3.5 +/- 1.5 for chronic, and 3.2 +/- 1.4 for ruptured, aneurysms (NS). Transdiaphragmatic repair was performed in 51/100 (51%) of all, 28/53 (53%) of chronic, and 17/33 (51%) of ruptured aneurysms (NS). Aortic cross-clamp time was 38 +/- 21 min for all, 39 +/- 24 min for chronic, and 38 +/- 17 min for ruptured, aneurysmal patients (NS). The amount of red cells washed and autotransfused was 2792 +/- 2239 ml in all, 3143 +/- 2531 ml in chronic, and 2074 +/- 1350 ml in ruptured, aneurysmal patients (P < 0.025). The amount of packed red cells required was 2181 +/- 1830 ml for all, 1736 +/- 1333 ml for chronic, and 2947 +/- 2395 ml for ruptured aneurysmal patients (P < 0.010). Thirty-day mortality was 9/100 (9%) for all, 3/53 (6%) for chronic, and 5/33 (15%) for ruptured aneurysmal patients (NS). Parapareses/plegias occurred in 9/100 (9%) of all, 6/53 (11%) of chronic, and 3/33 (9%) of ruptured, aneurysmal patients (NS). Stepwise regression analysis identified aortic cross-clamp time as a predictor of early mortality (P = 0.002) and parapareses and paraplegias (P = 0.001). Age (P = 0.001), extent of repair (P = 0.008) and preoperative hematocrit (P = 0.001) were predictors for homologous transfusion requirements. CONCLUSION: Emergency repair of ruptured descending thoracic and thoracoabdominal aortic aneurysms can be achieved with acceptable results.

Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project.

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.