Energy and information in Hodgkin-Huxley neurons

[EN]The generation of spikes by neurons is energetically a costly process and the evaluation of the metabolic energy required to maintain the signaling activity of neurons a challenge of practical interest. Neuron models are frequently used to represent the dynamics of real neurons but hardly ever to evaluate the electrochemical energy required to maintain that dynamics. This paper discusses the interpretation of a Hodgkin-Huxley circuit as an energy model for real biological neurons and uses it to evaluate the consumption of metabolic energy in the transmission of information between neurons coupled by electrical synapses, i.e., gap junctions. We show that for a single postsynaptic neuron maximum energy efficiency, measured in bits of mutual information per molecule of adenosine triphosphate (ATP) consumed, requires maximum energy consumption. For groups of parallel postsynaptic neurons we determine values of the synaptic conductance at which the energy efficiency of the transmission presents clear maxima at relatively very low values of metabolic energy consumption. Contrary to what could be expected, the best performance occurs at a low energy cost.

Adaptive evolution of energy metabolism genes and the origin of flight in bats

Bat flight poses intriguing questions about how flight independently developed in mammals. Flight is among the most energy-consuming activities. Thus, we deduced that changes in energy metabolism must be a primary factor in the origin of flight in bats. The respiratory chain of the mitochondrial produces 95% of the adenosine triphosphate (ATP) needed for locomotion. Because the respiratory chain has a dual genetic foundation, with genes encoded by both the mitochondrial and nuclear genomes, we examined both genomes to gain insights into the evolution of flight within mammals. Evidence for positive selection was detected in 23.08% of the mitochondrial-encoded and 4.90% of nuclear-encoded oxidative phosphorylation (OXPHOS) genes, but in only 2.25% of the nuclear-encoded nonrespiratory genes that function in mitochondria or 1.005% of other nuclear genes in bats. To address the caveat that the two available bat genomes are of only draft quality, we resequenced 77 OXPHOS genes from four species of bats. The analysis of the resequenced gene data are in agreement with our conclusion that a significantly higher proportion of genes involved in energy metabolism, compared with background genes, show evidence of adaptive evolution specific on the common ancestral bat lineage. Both mitochondrial and nuclear-encoded OXPHOS genes display evidence of adaptive evolution along the common ancestral branch of bats, supporting our hypothesis that genes involved in energy metabolism were targets of natural selection and allowed adaptation to the huge change in energy demand that were required during the origin of flight.

Intercellular calcium wave propagation in linear and circuit-like bone cell networks

In the present study, the mechanism of intercellular calcium wave propagation in bone cell networks was identified. By using micro-contact printing and self-assembled monolayer technologies, two types of in vitro bone cell networks were constructed: open-ended linear chains and looped hexagonal networks with precisely controlled intercellular distances. Intracellular calcium responses of the cells were recorded and analysed when a single cell in the network was mechanically stimulated by nano-indentation. The looped cell network was shown to be more efficient than the linear pattern in transferring calcium signals from cell to cell. This phenomenon was further examined by pathway-inhibition studies. Intercellular calcium wave propagation was significantly impeded when extracellular adenosine triphosphate (ATP) in the medium was hydrolysed. Chemical uncoupling of gap junctions, however, did not significantly decrease the transferred distance of the calcium wave in the cell networks. Thus, it is extracellular ATP diffusion, rather than molecular transport through gap junctions, that dominantly mediates the transmission of mechanically elicited intercellular calcium waves in bone cells. The inhibition studies also demonstrated that the mechanical stimulation-induced calcium responses required extracellular calcium influx, whereas the ATP-elicited calcium wave relied on calcium release from the calcium store of the endoplasmic reticulum.

Enzyme Colorimetric Assay Using Unmodified Silver Nanoparticles

Colorimetric assay based on the unique surface plasmon resonance properties of metallic nanoparticles has received considerable attention in bioassay due to its simplicity, high sensitivity, and low cost. Most of colorimetric methods previously reported employed gold nanoparticles (GNPs) as sensing elements. In this work, we develop a sensitive, selective, simple, and label-free colorimetric assay using unmodified silver nanoparticle (AgNP) probes to detect enzymatic reactions. Enzymatic reactions concerning adenosine triphosphate (ATP) dephosphorylation by calf intestine alkaline phosphatase (CLAP) and peptide phosphorylation by protein kinase A (PKA) were studied.

Multifunctional label-free electrochemical biosensor based on an integrated aptamer

Aptamers, which are in vitro selected functional oligonucleotides, have been employed to design novel biosensors (i.e., aptasensors) due to their inherent selectivity, affinity, and their multifarious advantages over traditional recognition elements. In this work, we reported a multifunctional reusable label-free electrochemical biosensor based on an integrated aptamer for parallel detection of adenosine triphosphate (ATP) and alpha-thrombin, by using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). A An electrode as the sensing surface was modified with a part DNA duplex which contained a 5'-thiolated partly complementary strand (PCS) and a mixed aptamer (MBA).

Au nanoparticles grafted sandwich platform used amplified small molecule electrochemical aptasensor

We report a sensitively amplified electrochemical aptasensor using adenosine triphosphate (ATP) as a model. ATP is a multifunctional nucleotide thatis most important as a "molecular currency" of intracellular energy transfer. In the sensing process, duplexes consisting of partly complementary strand (PCS1), ATP aptamer (ABA) and another partly complementary strand (PCS2) were immobilized onto Au electrode through the 5'-HS on the PCS1. Meanwhile, PCS2 was grafted with the Au nanoparticles (AuNPs) to amplify the detection signals. In the absence of ATP, probe methylene blue (MB) bound to the DNA duplexes and also bound to guanine bases specifically to produce a strong differential pulse voltammetry (DPV) signal. But when ATP exists, the ABA-PCS2 or ABA-PCS1 part duplexes might be destroyed, which decreased the amount of MB on the electrode and led to obviously decreased DPV signal.

A kinesin motor in a force-producing conformation.

BACKGROUND: Kinesin motors hydrolyze ATP to produce force and move along microtubules, converting chemical energy into work by a mechanism that is only poorly understood. Key transitions and intermediate states in the process are still structurally uncharacterized, and remain outstanding questions in the field. Perturbing the motor by introducing point mutations could stabilize transitional or unstable states, providing critical information about these rarer states. RESULTS: Here we show that mutation of a single residue in the kinesin-14 Ncd causes the motor to release ADP and hydrolyze ATP faster than wild type, but move more slowly along microtubules in gliding assays, uncoupling nucleotide hydrolysis from force generation. A crystal structure of the motor shows a large rotation of the stalk, a conformation representing a force-producing stroke of Ncd. Three C-terminal residues of Ncd, visible for the first time, interact with the central beta-sheet and dock onto the motor core, forming a structure resembling the kinesin-1 neck linker, which has been proposed to be the primary force-generating mechanical element of kinesin-1. CONCLUSIONS: Force generation by minus-end Ncd involves docking of the C-terminus, which forms a structure resembling the kinesin-1 neck linker. The mechanism by which the plus- and minus-end motors produce force to move to opposite ends of the microtubule appears to involve the same conformational changes, but distinct structural linkers. Unstable ADP binding may destabilize the motor-ADP state, triggering Ncd stalk rotation and C-terminus docking, producing a working stroke of the motor.

Overexpression of the cardiac beta(2)-adrenergic receptor and expression of a beta-adrenergic receptor kinase-1 (betaARK1) inhibitor both increase myocardial contractility but have differential effects on susceptibility to ischemic injury.

Cardiac beta(2)-adrenergic receptor (beta(2)AR) overexpression is a potential contractile therapy for heart failure. Cardiac contractility was elevated in mice overexpressing beta(2)ARs (TG4s) with no adverse effects under normal conditions. To assess the consequences of beta(2)AR overexpression during ischemia, perfused hearts from TG4 and wild-type mice were subjected to 20-minute ischemia and 40-minute reperfusion. During ischemia, ATP and pH fell lower in TG4 hearts than wild type. Ischemic injury was greater in TG4 hearts, as indicated by lower postischemic recoveries of contractile function, ATP, and phosphocreatine. Because beta(2)ARs, unlike beta(1)ARs, couple to G(i) as well as G(s), we pretreated mice with the G(i) inhibitor pertussis toxin (PTX). PTX treatment increased basal contractility in TG4 hearts and abolished the contractile resistance to isoproterenol. During ischemia, ATP fell lower in TG4+PTX than in TG4 hearts. Recoveries of contractile function and ATP were lower in TG4+PTX than in TG4 hearts. We also studied mice that overexpressed either betaARK1 (TGbetaARK1) or a betaARK1 inhibitor (TGbetaARKct). Recoveries of function, ATP, and phosphocreatine were higher in TGbetaARK1 hearts than in wild-type hearts. Despite basal contractility being elevated in TGbetaARKct hearts to the same level as that of TG4s, ischemic injury was not increased. In summary, beta(2)AR overexpression increased ischemic injury, whereas betaARK1 overexpression was protective. Ischemic injury in the beta(2)AR overexpressors was exacerbated by PTX treatment, implying that it was G(s) not G(i) activity that enhanced injury. Unlike beta(2)AR overexpression, basal contractility was increased by betaARK1 inhibitor expression without increasing ischemic injury, thus implicating a safer potential therapy for heart failure.

PDLA a potential new potent topical analgesic: a case report.

Polymer D-lactic acid (PDLA) is a hydrogel that has been shown to sequester L-lactate (lactate). This reaction is rapid, spontaneous, and non-enzymatic. Lactate has been shown to have many functions within the nervous system including its use as a secondary fuel to sustain neural activity and as a neuromodulator. In the central nervous system, lactate is produced in glial cells and shuttled to neurons to be used mostly as a fuel. Lactate dehydrogenase (LDH)1 is the predominant LDH isoform within neurons and unlike LDH5, it preferentially converts lactate to pyruvate which can be used to produce adenosine triphosphate (ATP). Considering that lactate is intimately involved in the sustenance of neural activity, PDLA was applied to an open wound and its effects were examined. The results showed that the application of PDLA induced topical analgesia. This may be the first report to demonstrate that sequestering lactate, a source of energy required to sustain the firing of action potentials in neurons, may produce analgesia.

KCNQ Currents and Their Contribution to Resting Membrane Potential and the Excitability of Interstitial Cells of Cajal From the Guinea Pig Bladder

PURPOSE: The presence of novel KCNQ currents was investigated in guinea pig bladder interstitial cells of Cajal and their contribution to the maintenance of the resting membrane potential was assessed. MATERIALS AND METHODS: Enzymatically dispersed interstitial cells of Cajal were patch clamped with K(+) filled pipettes in voltage clamp and current clamp modes. Pharmacological modulators of KCNQ channels were tested on membrane currents and the resting membrane potential. RESULTS: Cells were stepped from -60 to 40 mV to evoke voltage dependent currents using a modified K(+) pipette solution containing ethylene glycol tetraacetic acid (5 mM) and adenosine triphosphate (3 mM) to eliminate large conductance Ca activated K channel and K(adenosine triphosphate) currents. Application of the KCNQ blockers XE991, linopirdine (Tocris Bioscience, Ellisville, Missouri) and chromanol 293B (Sigma) decreased the outward current in concentration dependent fashion. The current-voltage relationship of XE991 sensitive current revealed a voltage dependent, outwardly rectifying current that activated positive to -60 mV and showed little inactivation. The KCNQ openers flupirtine and meclofenamic acid (Sigma) increased outward currents across the voltage range. In current clamp mode XE991 or chromanol 293B decreased interstitial cell of Cajal resting membrane potential and elicited the firing of spontaneous transient depolarizations in otherwise quiescent cells. Flupirtine or meclofenamic acid hyperpolarized interstitial cells of Cajal and inhibited any spontaneous electrical activity. CONCLUSIONS: This study provides electrophysiological evidence that bladder interstitial cells of Cajal have KCNQ currents with a role in the regulation of interstitial cell of Cajal resting membrane potential and excitability. These novel findings provide key information on the ion channels present in bladder interstitial cells of Cajal and they may indicate relevant targets for the development of new therapies for bladder instability.

Family-based association studies of lipid gene polymorphisms in coronary artery disease

Dysfunction of lipid-metabolizing proteins is implicated in the pathogenesis of coronary artery disease. Single nucleotide polymorphisms in genes that encode sterol regulatory binding protein-la, adenosine triphosphate binding cassette-A1, hepatic lipase, lipoprotein lipase, and cholesteryl ester transfer protein were assessed as potential markers of disease susceptibility in a family-based study of 1,012 patients from 386 families. Association between single nucleotide polymorphisms and coronary artery disease was tested by the combined transmission disequilibrium test/sib transmission disequilibrium test and pedigree disequilibrium test. After Bonferroni's correction, the pedigree disequilibrium test demonstrated significant excess transmission (p < 0.0083) to affected patients of the hepatic lipase -514 T allele, which suggests that this may constitute a novel disease-susceptibility locus. (c) 2005 Elsevier Inc. All rights reserved.

The effect of polyphosphate kinase gene deletion on polyhydroxyalkanoate accumulation and carbon metabolism in Pseudomonas putida KT2440.

The primary enzyme involved in polyphosphate (polyP) synthesis, polyP kinase (ppk), has been deleted in Pseudomonas putida KT2440. This has resulted in a threefold to sixfold reduction in polyhydroxyalkanoate (PHA) accumulation compared with the wild type under conditions of nitrogen limitation, with either temperature or oxidative (H2O2) stress, when grown on glucose. The accumulation of PHA by Δppk mutant was the same as the wild type under nitrogen-limiting growth conditions. There was no difference in polyP levels between wild-type and Δppk strains under all growth conditions tested. In the Δppk mutant proteome, polyP kinase (PPK) was undetectable, but up-regulation of the polyp-associated proteins polyP adenosine triphosphate (ATP)/nicotinamide adenine dinucleotide (NAD) kinase (PpnK), a putative polyP adenosine monophosphate (AMP) phosphotransferase (PP_1752), and exopolyphosphatase was observed. Δppk strain exhibited significantly retarded growth with glycerol as carbon and energy source (42 h of lag period compared with 24 h in wild-type strain) but similar growth to the wild-type strain with glucose. Analysis of gene transcription revealed downregulation of glycerol kinase and the glycerol facilitator respectively. Glycerol kinase protein expression was also downregulated in the Δppk mutant. The deletion of ppk did not affect motility but reduced biofilm formation. Thus, the knockout of the ppk gene has resulted in a number of phenotypic changes to the mutant without affecting polyP accumulation.

Nuclear and mitochondrial control of the apoptosis machinery regulates neural stem cell fate

Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2014

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.

Structural organization of alpha-subunit from purified and microsomal toad kidney (Na+ + K+)-ATPase as assessed by controlled trypsinolysis

The membrane organization of the alpha-subunit of purified (Na+ + K+)-ATPase ((Na+ + K+)-dependent adenosine triphosphate phosphorylase, EC 3.6.1.3) and of the microsomal enzyme of the kidney of the toad Bufo marinus was compared by using controlled trypsinolysis. With both enzyme preparations, digestions performed in the presence of Na+ yielded a 73 kDa fragment and in the presence of K+ a 56 kDa, a 40 kDa and small amounts of a 83 kDa fragment from the 96 kDa alpha-subunit. In contrast to mammalian preparations (Jørgensen, P.L. (1975) Biochim. Biophys. Acta 401, 399-415), trypsinolysis of the purified amphibian enzyme led to a biphasic loss of (Na+ + K+)-ATPase activity in the presence of both Na+ and K+. These data could be correlated with an early rapid cleavage of 3 kDa from the alpha-subunit in both ionic conditions and a slower degradation of the remaining 93 kDa polypeptide. On the other hand, in the microsomal enzyme, a 3 kDa shift of the alpha-subunit could only be produced in the presence of Na+. Our data indicate that (1) purification of the amphibian enzyme with detergent does not influence the overall topology of the alpha-subunit but produces a distinct structural alteration of its N-terminus and (2) the amphibian kidney enzyme responds to cations with similar conformational transitions as the mammalian kidney enzyme. In addition, anti alpha-serum used on digested enzyme samples revealed on immunoblots that the 40 kDa fragment was better recognized than the 56 kDa fragment. It is concluded that the NH2-terminal of the alpha-subunit contains more antigenic sites than the COOH-terminal domain in agreement with the results of Farley et al. (Farley, R.A., Ochoa, G.T. and Kudrow, A. (1986) Am. J. Physiol. 250, C896-C906).