Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer

The coordinated assembly of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62) to form the bacteriophage T4 DNA polymerase holoenzyme is a multistep process. A partially opened toroid-shaped gp45 is loaded around DNA by gp44/62 in an ATP-dependent manner. Gp43 binds to this complex to generate the holoenzyme in which gp45 acts to topologically link gp43 to DNA, effectively increasing the processivity of DNA replication. Stopped-flow fluorescence resonance energy transfer was used to investigate the opening and closing of the gp45 ring during holoenzyme assembly. By using two site-specific mutants of gp45 along with a previously characterized gp45 mutant, we tracked changes in distances across the gp45 subunit interface through seven conformational changes associated with holoenzyme assembly. Initially, gp45 is partially open within the plane of the ring at one of the three subunit interfaces. On addition of gp44/62 and ATP, this interface of gp45 opens further in-plane through the hydrolysis of ATP. Addition of DNA and hydrolysis of ATP close gp45 in an out-of-plane conformation. The final holoenzyme is formed by the addition of gp43, which causes gp45 to close further in plane, leaving the subunit interface open slightly. This open interface of gp45 in the final holoenzyme state is proposed to interact with the C-terminal tail of gp43, providing a point of contact between gp45 and gp43. This study further defines the dynamic process of bacteriophage T4 polymerase holoenzyme assembly.

Interaction of the β sliding clamp with MutS, ligase, and DNA polymerase I

The β and proliferating cell nuclear antigen (PCNA) sliding clamps were first identified as components of their respective replicases, and thus were assigned a role in chromosome replication. Further studies have shown that the eukaryotic clamp, PCNA, interacts with several other proteins that are involved in excision repair, mismatch repair, cellular regulation, and DNA processing, indicating a much wider role than replication alone. Indeed, the Escherichia coli β clamp is known to function with DNA polymerases II and V, indicating that β also interacts with more than just the chromosomal replicase, DNA polymerase III. This report demonstrates three previously undetected protein–protein interactions with the β clamp. Thus, β interacts with MutS, DNA ligase, and DNA polymerase I. Given the diverse use of these proteins in repair and other DNA transactions, this expanded list of β interactive proteins suggests that the prokaryotic β ring participates in a wide variety of reactions beyond its role in chromosomal replication.

Membrane transport mechanisms probed by capacitance measurements with megahertz voltage clamp.

We have used capacitance measurements with a 1-microsecond voltage clamp technique to probe electrogenic ion-transporter interactions in giant excised membrane patches. The hydrophobic ion dipicrylamine was used to test model predictions for a simple charge-moving reaction. The voltage and frequency dependencies of the apparent dipicrylamine-induced capacitance, monitored by 1-mV sinusoidal perturbations, correspond to single charges moving across 76% of the membrane field at a rate of 9500 s-1 at 0 mV. For the cardiac Na,K pump, the combined presence of cytoplasmic ATP and sodium induces an increase of apparent membrane capacitance which requires the presence of extracellular sodium. The dependencies of capacitance changes on frequency, voltage, ATP, and sodium verify that phosphorylation enables a slow, 300- to 900-s-1, pump transition (the E1-E2 conformational change), which in turn enables fast, electrogenic, extracellular sodium binding reactions. For the GAT1 (gamma-aminobutyric acid,Na,Cl) cotransporter, expressed in Xenopus oocyte membrane, we find that chloride binding from the cytoplasmic side, and probably sodium binding from the extracellular side, results in a decrease of membrane capacitance monitored with 1- to 50-kHz perturbation frequencies. Evidently, ion binding by the GAT1 transporter suppresses an intrinsic fast charge movement which may originate from a mobility of charged residues of the transporter binding sites. The results demonstrate that fast capacitance measurements can provide new insight into electrogenic processes closely associated with ion binding by membrane transporters.

Evidence for synaptotagmin as an inhibitory clamp on synaptic vesicle release in Aplysia neurons.

While previous studies have demonstrated that synaptotagmin plays an essential role in evoked neurotransmitter release, it has been difficult to determine whether it acts to facilitate or inhibit release. To address this question, we used acute genetic manipulations to alter the expression of synaptotagmin in Aplysia neurons. Transient overexpression of synaptotagmin in acutely dissected cholinergic neurons and in cultured glutaminergic neurons decreased the amplitude of the excitatory postsynaptic potential (EPSP) by 32% and 26%, respectively. In contrast, treatment of cultured presynaptic neurons with synaptotagmin antisense oligonucleotides increased the amplitude of the EPSP by 50-75%. These results are consistent with a role of synaptotagmin as an inhibitor of release.

Inhibition of protein interactions with the beta(2) sliding clamp of Escherichia coli DNA polymerase III by peptides from beta(2)-binding proteins

The sliding clamp of the Escherichia coli replisome is now understood to interact with many proteins involved in DNA synthesis and repair. A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding clamp, a homodimer of the beta subunit, at a single site on the dimer. The numerous beta(2)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence. To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(2) site, synthetic peptides derived from the putative beta(2)-binding motifs were assessed for their abilities to inhibit protein-beta(2) interactions, to bind directly to beta(2), and to inhibit DNA synthesis in vitro. A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/D)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(2) in various stages or circumstances of DNA replication and repair.

A study of stray load losses in core clamp plates of salient pole synchronous machines

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Nouveaux indices de suppression de la lipolyse par l'insuline déterminés lors de l'hyperglycémie provoquée par voie orale : comparaisons avec le clamp euglycémique-hyperinsulinémique et les paramètres métaboliques chez les femmes

Résumé : Une dysrégulation de la lipolyse des tissus adipeux peut conduire à une surexposition des tissus non-adipeux aux acides gras non-estérifiés (AGNE), qui peut mener à un certain degré de lipotoxicité dans ces tissus. La lipotoxicité constitue, par ailleurs, l’une des causes majeures du développement de la résistance à l’insuline et du diabète de type 2. En plus de ses fonctions glucorégulatrices, l’insuline a pour fonction d’inhiber la lipolyse et donc de diminuer les niveaux d’AGNE en circulation, prévenant ainsi la lipotoxicité. Il n’y a pas d’étalon d’or pour mesurer la sensibilité de la lipolyse à l’insuline. Le clamp euglycémique hyperinsulinémique constitue la méthode étalon d’or pour évaluer la sensibilité du glucose à l’insuline mais il est aussi utilisé pour mesurer la suppression de la lipolyse par l’insuline. Par contre, cette méthode est couteuse et laborieuse, et ne peut pas s’appliquer à de grandes populations. Il existe aussi des indices pour estimer la fonction antilipolytique de l’insuline dérivés de l’hyperglycémie provoquée par voie orale (HGPO), un test moins dispendieux et plus simple à effectuer à grande échelle. Cette étude vise donc à : 1) Étudier la relation entre les indices de suppressibilité des AGNE par l’insuline dérivés du clamp et ceux dérivés de l’HGPO; et 2) Déterminer laquelle de ces mesures corrèle le mieux avec les facteurs connus comme étant reliés à la dysfonction adipeuse : paramètres anthropométriques et indices de dysfonction métabolique. Les résultats montrent que dans le groupe de sujets étudiés (n=29 femmes, 15 témoins saines et 14 femmes avec résistance à l’insuline car atteintes du syndrome des ovaires polykystiques), certains indices de sensibilité à l’insuline pour la lipolyse dérivés de l’HGPO corrèlent bien avec ceux dérivés du clamp euglycémique hyperinsulinémique. Parmi ces indices, celui qui corrèle le mieux avec les indices du clamp et les paramètres anthropométriques et de dysfonction adipeuse est le T50[indice inférieur AGNE] (temps nécessaire pour diminuer de 50% le taux de base – à jeun – des AGNE). Nos résultats suggèrent donc que l’HGPO, facile à réaliser, peut être utilisée pour évaluer la sensibilité de la lipolyse à l’insuline. Nous pensons que la lipo-résistance à l’insuline peut être facilement quantifiée en clinique humaine.

Enhanced Glucose-induced Intracellular Signaling Promotes Insulin Hypersecretion: Pancreatic Beta-cell Functional Adaptations In A Model Of Genetic Obesity And Prediabetes.

Obesity is associated with insulin resistance and is known to be a risk factor for type-2 diabetes. In obese individuals, pancreatic beta-cells try to compensate for the increased insulin demand in order to maintain euglycemia. Most studies have reported that this adaptation is due to morphological changes. However, the involvement of beta-cell functional adaptations in this process needs to be clarified. For this purpose, we evaluated different key steps in the glucose-stimulated insulin secretion (GSIS) in intact islets from female ob/ob obese mice and lean controls. Obese mice showed increased body weight, insulin resistance, hyperinsulinemia, glucose intolerance and fed hyperglycemia. Islets from ob/ob mice exhibited increased glucose-induced mitochondrial activity, reflected by enhanced NAD(P)H production and mitochondrial membrane potential hyperpolarization. Perforated patch-clamp examination of beta-cells within intact islets revealed several alterations in the electrical activity such as increased firing frequency and higher sensitivity to low glucose concentrations. A higher intracellular Ca(2+) mobilization in response to glucose was also found in ob/ob islets. Additionally, they displayed a change in the oscillatory pattern and Ca(2+) signals at low glucose levels. Capacitance experiments in intact islets revealed increased exocytosis in individual ob/ob beta-cells. All these up-regulated processes led to increased GSIS. In contrast, we found a lack of beta-cell Ca(2+) signal coupling, which could be a manifestation of early defects that lead to beta-cell malfunction in the progression to diabetes. These findings indicate that beta-cell functional adaptations are an important process in the compensatory response to obesity.

Epicardial And Pericardial Fat In Type 2 Diabetes: Favourable Effects Of Biliopancreatic Diversion.

Ectopic fat is often identified in obese subjects who are susceptible to the development of type 2 diabetes mellitus (T2DM). The ectopic fat favours the decrease in insulin sensitivity (IS) and adiponectin levels. We aimed to evaluate the effect of biliopancreatic diversion (BPD) on the accumulation of ectopic fat, adiponectin levels and IS in obese with T2DM. A nonrandomised controlled study was performed on sixty-eight women: 19 lean-control (23.0 ± 2.2 kg/m(2)) and 18 obese-control (35.0 ± 4.8 kg/m(2)) with normal glucose tolerance and 31 obese with T2DM (36.3 ± 3.7 kg/m(2)). Of the 31 diabetic women, 20 underwent BPD and were reassessed 1 month and 12 months after surgery. The subcutaneous adipose tissue, visceral adipose tissue, epicardial adipose tissue and pericardial adipose tissue were evaluated by ultrasonography. The IS was assessed by a hyperglycaemic clamp, applying the minimal model of glucose. One month after surgery, there was a reduction in visceral and subcutaneous adipose tissues, whereas epicardial and pericardial adipose tissues exhibited significant reduction at the 12-month assessment (p < 0.01). Adiponectin levels and IS were normalised 1 month after surgery, resembling lean-control values and elevated above the obese-control values (p < 0.01). After 12 months, the improvement in IS and adiponectin was maintained, and 17 of the 20 operated patients exhibited fasting glucose and glycated haemoglobin within the normal range. After BPD, positive physiological adaptations occurred in grade I and II obese patients with T2DM. These adaptations relate to the restoration of IS and decreased adiposopathy and explain the acute (1 month) and chronic (12 months) improvements in the glycaemic control.

Biópsia hepática por laparotomia paracostal em bovinos e búfalos

A técnica de biópsia hepática em ruminantes tem importante valor no diagnóstico clínico de doenças tóxicas e metabólicas, em especial nos desequilíbrios minerais. As técnicas mais comumente utilizadas restringem análises devido ao limitado volume de tecido obtido. No presente trabalho, avaliou-se o uso de uma técnica de biópsia hepática por laparotomia paracostal em bovinos e búfalos. Foram utilizados 10 bovinos e 10 búfalos hígidos. Os animais foram mantidos em estação, sedados com xilazina e infiltrados localmente com lidocaína e epinefrina. O acesso à cavidade abdominal foi realizado por meio de uma incisão dorso-ventral de 15cm no flanco direito, iniciada ventralmente (cerca de 4-5cm) ao processo transverso da 2a ou 3a vértebra lombar e situada caudalmente (cerca de 4cm) e paralelamente à 13a costela, obtendo-se visualização do fígado. Foi então realizado pinçamento do bordo caudal do órgão com pinça Doyen para remoção de fragmento hepático (2 a 4g). Procedeu-se o fechamento da cavidade abdominal como de rotina. Foram analisados os parâmetros bioquímicos e hematológicos antes do procedimento (tempo zero) e após 24 horas, 48 horas, 5 dias e 10 dias após a biópsia. Todas as variáveis bioquímicas estudadas retornaram aos valores basais 5 e 10 dias após o procedimento nos bovinos e búfalos, respectivamente. O tempo médio de cirurgia por animal foi de 25 minutos. A biópsia hepática por laparotomia paracostal demonstrou ser uma técnica eficaz e de baixo risco à saúde dos animais, permitindo a coleta de suficiente quantidade de tecido hepática para realização de múltiplas análises.

Intrahepatic Glissonian Approach for Pure Laparoscopic Left Hemihepatectomy

Background: Recent advances in laparoscopic devices and experience with advanced techniques have increased the indications for laparoscopic liver. Aim: The aim of this work was to present a video with technical aspects of a pure laparoscopic left hemi-hepatectomy (segments 2, 3, and 4) by using the intrahepatic Glissonian approach and control of venous outflow without hilar dissection or the Pringle maneuver. Patient and Method: A 63-year-old woman with a 5-cm solitary liver metastasis was referred for treatment. Four trocars were used. The left lobe was pulled upward and the lesser omentum was divided, exposing Arantius' ligament. This ligament is a useful landmark for the identification of the main left Glissonian pedicle. A small anterior incision was made in front of the hilum, and a large clamp was introduced behind the Arantius' ligament toward the anterior incision, allowing control of the left main sheath. Ischemic discoloration of the left liver was achieved and marked with cautery. The vascular clamp was replaced by a stapler. If ischemic delineation was coincident with a previously marked area, the stapler was fired. The left hepatic vein was dissected and encircled. Parenchymal transection and vascular control of the hepatic veins were accomplished with a Harmonic scalpel and an endoscopic stapling device, as appropriate. All these steps were performed without the Pringle maneuver and without hand assistance. Results: Operative time was 220 minutes with minimum blood loss. Hospital stay was 4 days. Pathology showed free surgical margins. The patient is alive with no signs of recurrence 18 months after the operation. Conclusion: Totally laparoscopic left hemihepatectomy is safe and feasible in selected patients and should be considered for patients with benign or malignant liver neoplasms. The described technique, with the use of the intrahepatic Glissonian approach and control of venous outflow, may facilitate laparoscopic left hemihepatectomy by reducing the technical difficulties in pedicle control and may decrease bleeding during liver transection.

Fretting fatigue in overhead conductors: Rig design and failure analysis of a Grosbeak aluminium cable steel reinforced conductor

The performance optimisation of overhead conductors depends on the systematic investigation of the fretting fatigue mechanisms in the conductor/clamping system. As a consequence, a fretting fatigue rig was designed and a limited range of fatigue tests was carried out at the middle high cycle fatigue regime in order to access an exploratory S-N curve for a Grosbeak conductor, which was mounted on a mono-articulated aluminium clamping system. Subsequent to these preliminary fatigue tests, the components of the conductor/clamping system, such as ACSR conductor, upper and lower clamps, bolt and nuts, were subjected to a failure analysis procedure in order to investigate the metallurgical free variables interfering on the fatigue test results, aiming at the optimisation of the testing reproducibility. The results indicated that the rupture of the planar fracture surfaces observed in the external At strands of the conductor tested under lower bending amplitude (0.9 mm) occurred by fatigue cracking (I mm deep), followed by shear overload. The V-type fracture surfaces observed in some At strands of the conductor tested under higher bending amplitude (1.3 mm) were also produced by fatigue cracking (approximately 400 mu m deep), followed by shear overload. Shear overload fracture (45 degrees fracture surface) was also observed on the remaining At wires of the conductor tested under higher bending amplitude (1.3 mm). Additionally, the upper and lower Al-cast clamps presented microstructure-sensitive cracking, which was folowed by particle detachment and formation of abrasive debris on the clamp/conductor tribo-interface, promoting even further the fretting mechanism. The detrimental formation of abrasive debris might be inhibited by the selection of a more suitable class of as-cast At alloy for the production of clamps. Finally, the bolt/nut system showed intense degradation of the carbon steel nut (fabricated in ferritic-pearlitic carbon steel, featuring machined threads with 190 HV), with intense plastic deformation and loss of material. Proper selection of both the bolt and nut materials and the finishing processing might prevent the loss in the clamping pressure during the fretting testing. It is important to control the specification of these components (clamps, bolt and nuts) prior to the start of large scale fretting fatigue testing of the overhead conductors in order to increase the reproducibility of this assessment. (c) 2008 Elsevier Ltd. All rights reserved.

Purification and characterization of Ts15, the first member of a new alpha-KTX subfamily from the venom of the Brazilian scorpion Tityus serrulatus

Voltage-gated potassium channel toxins (KTxs) are basic short chain peptides comprising 23-43 amino acid residues that can be cross-linked by 3 or 4 disulfide bridges. KTxs are classified into four large families: alpha-, beta-, gamma- and kappa-KTx. These peptides display varying selectivity and affinity for K(v) channel subtypes. In this work, a novel toxin from the Tityus serrulatus venom was isolated, characterized and submitted to a wide electrophysiological screening on 5 different subtypes of Nay channels (Na(V)1.4; Na(V)1.5; Na(V)1.6; Na(V)1.8 and DmNa(V)1) and 12 different subtypes of Kv channels (K(V)1.1 - K(V)1.6; K(V)2.1; K(V)3.1; K(V)4.2; K(V)4.3; Shaker IR and ERG). This novel peptide, named Ts15, has 36 amino acids, is crosslinked by 3 disulfide bridges, has a molecular mass of 3956 Da and pI around 9. Electrophysiological experiments using patch clamp and the two-electrode voltage clamp techniques show that Ts15 preferentially blocks K(V)1.2 and K(V)1.3 channels with an IC(50) value of 196 +/- 25 and 508 +/- 67 nM, respectively. No effect on Na(V) channels was observed, at all tested concentrations. Since Ts15 shows low amino acid identity with other known KTxs, it was considered a bona fide novel type of scorpion toxin. Ts15 is the unique member of the new alpha-Ktx21 subfamily and therefore was classified as alpha-Ktx21.1. (C) 2011 Elsevier Ltd. All rights reserved.

Characterization of calcium-activated chloride channels in patches excised from the dendritic knob of mammalian olfactory receptor neurons

We investigated the properties of calcium-activated chloride channels in inside-out membrane patches from the dendritic knobs of acutely dissociated rat olfactory receptor neurons. Patches typically contained large calcium-activated currents, with total conductances in the range 30-75 nS. The dose response curve for calcium exhibited an EC50 of about 26 mu M. In symmetrical NaCl solutions, the current-voltage relationship reversed at 0 mV and was linear between -80 and +70 mV. When the intracellular NaCl concentration was progressively reduced from 150 to 25 mM, the reversal potential changed in a manner consistent with a chloride-selective conductance. Indeed, modeling these data with the Goldman-Hodgkin-Katz equation revealed a P-Na/P-Cl of 0.034. The halide permeability sequence was P-Cl > P-F > P-I > P-Br indicating that permeation through the channel was dominated by ion binding sites with a high field strength. The channels were also permeable to the large organic anions, SCN-, acetate(-), and gluconate(-), with the permeability sequence P-Cl > P-SCN > gluconaie. Significant permeation to gluconate ions suggested that the channel pore had a minimum diameter of at least 5.8 Angstrom.

Are there functional P2X receptors on cell bodies in intact dorsal root ganglia of rats?

P2X purinoceptors have been suggested to participate in transduction of painful stimuli in nociceptive neurons. In the current experiments, ATP (1-10 mM), alpha,beta-methylene-ATP (10-30 mu M) and capsaicin (10 nM-1 mu M) were applied to neurons impaled with high resistance microelectrodes in rat dorsal root ganglia (L4 and L5) isolated in vitro together with the sciatic nerve and dorsal roots. The agonists were either bath applied or focally applied using a picospritzer. GABA (100 mu M) and 40-80 mM K+ solutions gave brisk responses when applied by either technique. Only three of 22 neurons with slowly conducting axons (C cells) showed evidence of P2X-purinoceptor-mediated responses. Only two of 13 cells which responded to capsaicin (putative nociceptors), and none of 29 cells with rapidly conducting axons (A cells), responded to the purinergic agonists. When acutely dissociated dorsal root ganglion cells were studied using patch-clamp techniques, all but four of 30 cells of all sizes responded with an inward current to either ATP or alpha,beta-methylene-ATP (both 100 mu M). Our data suggest that few sensory cell bodies in intact dorsal root ganglia express functional purinoceptors. (C) 1998 IBRO. Published by Elsevier Science Ltd.