Pyrethroids Differentially Alter Voltage-Gated Sodium Channels from the Honeybee Central Olfactory Neurons

PubMed Central

Kadala, Aklesso; Charreton, Mercedes; Jakob, Ingrid; Cens, Thierry; Rousset, Matthieu; Chahine, Mohamed; Le Conte, Yves; Charnet, Pierre; Collet, Claude

2014-01-01

The sensitivity of neurons from the honey bee olfactory system to pyrethroid insecticides was studied using the patch-clamp technique on central ‘antennal lobe neurons’ (ALNs) in cell culture. In these neurons, the voltage-dependent sodium currents are characterized by negative potential for activation, fast kinetics of activation and inactivation, and the presence of cumulative inactivation during train of depolarizations. Perfusion of pyrethroids on these ALN neurons submitted to repetitive stimulations induced (1) an acceleration of cumulative inactivation, and (2) a marked slowing of the tail current recorded upon repolarization. Cypermethrin and permethrin accelerated cumulative inactivation of the sodium current peak in a similar manner and tetramethrin was even more effective. The slow-down of channel deactivation was markedly dependent on the type of pyrethroid. With cypermethrin, a progressive increase of the tail current amplitude along with successive stimulations reveals a traditionally described use-dependent recruitment of modified sodium channels. However, an unexpected decrease in this tail current was revealed with tetramethrin. If one considers the calculated percentage of modified channels as an index of pyrethroids effects, ALNs are significantly more susceptible to tetramethrin than to permethrin or cypermethrin for a single depolarization, but this difference attenuates with repetitive activity. Further comparison with peripheral neurons from antennae suggest that these modifications are neuron type specific. Modeling the sodium channel as a multi-state channel with fast and slow inactivation allows to underline the effects of pyrethroids on a set of rate constants connecting open and inactivated conformations, and give some insights to their specificity. Altogether, our results revealed a differential sensitivity of central olfactory neurons to pyrethroids that emphasize the ability for these compounds to impair detection and processing of information at several levels of the bees olfactory pathway. PMID:25390654

Compound-Specific Effects of Mutations at Val787 in DII-S6 of Nav1.4 Sodium Channels on the Action of Sodium Channel Inhibitor Insecticides

PubMed Central

von Stein, Richard T.; Soderlund, David M.

2012-01-01

Sodium channel inhibitor (SCI) insecticides are hypothesized to inhibit voltage-gated sodium channels by binding selectively to the slow-inactivated state. Replacement of valine at position 787 in the S6 segment of homology domain II of the rat Nav1.4 sodium channel by lysine (V787K) enchances slow inactivation of this channel whereas replacement by alanine or cysteine (V787A, V787C) inhibits slow inactivation. To test the hypothesis that SCI insecticides bind selectively to the slow-inactivated state, we constructed mutated Nav1.4/V787A, Nav1.4/V787C, and Nav1.4/V787K cDNAs, expressed wildtype and mutated channels with the auxiliary β1 subunit in Xenopus oocytes, and used the two-electrode voltage clamp technique to examine the effects of these mutations on channel inhibition by four SCI insecticides (indoxacarb, its bioactivated metabolite DCJW, metaflumizone, and RH3421). Mutations at Val787 affected SCI insecticide sensitivity in a manner that was independent of mutation-induced changes in slow inactivation gating. Sensitivity to inhibition by 10 μM indoxacarb was significantly increased in all three mutated channels, whereas sensitivity to inhibition by 10 μM metaflumizone was significantly reduced in Nav1.4/V787A channels and completely abolished in Nav1.4/V787K channels. The effects of Val787 mutations on metaflumizone were correlated with the hydrophobicity of the substituted amino acid rather than the extent of slow inactivation. None of the mutations at Val787 significantly affected the sensitivity to inhibition by DCJW or RH3421. These results demonstrate that the impact of mutations at Val787 on sodium channel inhibition by SCI insecticides depends on the specific insecticide examined and is independent of mutation-induced changes in slow inactivation gating. We propose that Val787 may be a unique determinant of metaflumizone binding. PMID:22983119

Modulation of slow inactivation in human cardiac Kv1.5 channels by extra- and intracellular permeant cations

PubMed Central

Fedida, David; Maruoka, Neil D; Lin, Shunping

1999-01-01

The properties and regulation of slow inactivation by intracellular and extracellular cations in the human heart K+ channel hKv1.5 have been investigated. Extensive NH2- and COOH-terminal deletions outside the central core of transmembrane domains did not affect the degree of inactivation. The voltage dependence of steady-state inactivation curves of hKv1.5 channels was unchanged in Rb+ and Cs+, compared with K+, but biexponential inactivation over 10 s was reduced from ∼100% of peak current in Na+ to ∼65% in K+, ∼50% in Rb+ and ∼30% in Cs+. This occurred as a result of a decrease in both fast and slow components of inactivation, with little change in inactivation time constants. Changes in extracellular cation species and concentration (5-300 mM) had only small effects on the rates of inactivation and recovery from inactivation (τrecovery∼1 s). Mutation of residues at a putative regulatory site at R487 in the outer pore mouth did not affect slow inactivation or recovery from inactivation of hKv1.5, although sensitivity to extracellular TEA was conferred. Symmetrical reduction of both intra- and extracellular cation concentrations accelerated and augmented both components of inactivation of K+ (Kd = 34.7 mM) and Cs+ (Kd = 20.5 mM) currents. These effects could be quantitatively accounted for by unilateral reduction of intracellular K+ (Ki+) (Kd = 43.4 mM) or Csi+ with constant 135 mM external ion concentrations. We conclude that inactivation and recovery from inactivation in hKv1.5 were not typically C-type in nature. However, the ion species dependence of inactivation was still closely coupled to ion permeation through the pore. Intracellular ion modulatory actions were more potent than extracellular actions, although still of relatively low affinity. These results suggest the presence of ion binding sites capable of regulating inactivation located on both intracellular and extracellular sides of the pore selectivity filter. PMID:10050000

Inactivation of Gating Currents of L-Type Calcium Channels

PubMed Central

Shirokov, Roman; Ferreira, Gonzalo; Yi, Jianxun; Ríos, Eduardo

1998-01-01

In studies of gating currents of rabbit cardiac Ca channels expressed as α1C/β2a or α1C/β2a/α2δ subunit combinations in tsA201 cells, we found that long-lasting depolarization shifted the distribution of mobile charge to very negative potentials. The phenomenon has been termed charge interconversion in native skeletal muscle (Brum, G., and E. Ríos. 1987. J. Physiol. (Camb.). 387:489–517) and cardiac Ca channels (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1992. J. Gen. Physiol. 99:863–895). Charge 1 (voltage of half-maximal transfer, V1/2 ≃ 0 mV) gates noninactivated channels, while charge 2 (V1/2 ≃ −90 mV) is generated in inactivated channels. In α1C/β2a cells, the available charge 1 decreased upon inactivating depolarization with a time constant τ ≃ 8, while the available charge 2 decreased upon recovery from inactivation (at −200 mV) with τ ≃ 0.3 s. These processes therefore are much slower than charge movement, which takes <50 ms. This separation between the time scale of measurable charge movement and that of changes in their availability, which was even wider in the presence of α2δ, implies that charges 1 and 2 originate from separate channel modes. Because clear modal separation characterizes slow (C-type) inactivation of Na and K channels, this observation establishes the nature of voltage-dependent inactivation of L-type Ca channels as slow or C-type. The presence of the α2δ subunit did not change the V1/2 of charge 2, but sped up the reduction of charge 1 upon inactivation at 40 mV (to τ ≃ 2 s), while slowing the reduction of charge 2 upon recovery (τ ≃ 2 s). The observations were well simulated with a model that describes activation as continuous electrodiffusion (Levitt, D. 1989. Biophys. J. 55:489–498) and inactivation as discrete modal change. The effects of α2δ are reproduced assuming that the subunit lowers the free energy of the inactivated mode. PMID:9607938

Conduction velocity is regulated by sodium channel inactivation in unmyelinated axons innervating the rat cranial meninges

PubMed Central

De Col, Roberto; Messlinger, Karl; Carr, Richard W

2008-01-01

Axonal conduction velocity varies according to the level of preceding impulse activity. In unmyelinated axons this typically results in a slowing of conduction velocity and a parallel increase in threshold. It is currently held that Na+–K+-ATPase-dependent axonal hyperpolarization is responsible for this slowing but this has long been equivocal. We therefore examined conduction velocity changes during repetitive activation of single unmyelinated axons innervating the rat cranial meninges. In direct contradiction to the currently accepted postulate, Na+–K+-ATPase blockade actually enhanced activity-induced conduction velocity slowing, while the degree of velocity slowing was curtailed in the presence of lidocaine (10–300 μm) and carbamazepine (30–500 μm) but not tetrodotoxin (TTX, 10–80 nm). This suggests that a change in the number of available sodium channels is the most prominent factor responsible for activity-induced changes in conduction velocity in unmyelinated axons. At moderate stimulus frequencies, axonal conduction velocity is determined by an interaction between residual sodium channel inactivation following each impulse and the retrieval of channels from inactivation by a concomitant Na+–K+-ATPase-mediated hyperpolarization. Since the process is primarily dependent upon sodium channel availability, tracking conduction velocity provides a means of accessing relative changes in the excitability of nociceptive neurons. PMID:18096592

Role of the Local Anesthetic Receptor in the State-Dependent Inhibition of Voltage-Gated Sodium Channels by the Insecticide Metaflumizone

PubMed Central

von Stein, Richard T.

2012-01-01

Sodium channel inhibitor (SCI) insecticides selectively target voltage-gated sodium (Nav) channels in the slow-inactivated state by binding at or near the local anesthetic receptor within the sodium channel pore. Metaflumizone is a new insecticide for the treatment of fleas on domesticated pets and has recently been reported to block insect sodium channels in the slow-inactivated state, thereby implying that it is also a member of the SCI class. Using the two-electrode voltage-clamp technique, we examined metaflumizone inhibition of rat Nav1.4 sodium channels expressed in Xenopus laevis oocytes. Metaflumizone selectively inhibited Nav1.4 channels at potentials that promoted slow inactivation and shifted the voltage dependence of slow inactivation in the direction of hyperpolarization. Metaflumizone perfusion at a hyperpolarized holding potential also shifted the conductance-voltage curve for activation in the direction of depolarization and antagonized use-dependent lidocaine inhibition of fast-inactivated sodium channels, actions not previously observed with other SCI insecticides. We expressed mutated Nav1.4/F1579A and Nav1.4/Y1586A channels to investigate whether metaflumizone shares the domain IV segment S6 (DIV-S6) binding determinants identified for other SCI insecticides. Consistent with previous investigations of SCI insecticides on rat Nav1.4 channels, the F1579A mutation reduced sensitivity to block by metaflumizone, whereas the Y1586A mutation paradoxically increased the sensitivity to metaflumizone. We conclude that metaflumizone selectively inhibits slow-inactivated Nav1.4 channels and shares DIV-S6 binding determinants with other SCI insecticides and therapeutic drugs. However, our results suggest that metaflumizone interacts with resting and fast-inactivated channels in a manner that is distinct from other compounds in this insecticide class. PMID:22127519

Lidocaine reduces the transition to slow inactivation in Nav1.7 voltage-gated sodium channels

PubMed Central

Sheets, Patrick L; Jarecki, Brian W; Cummins, Theodore R

2011-01-01

BACKGROUND AND PURPOSE The primary use of local anaesthetics is to prevent or relieve pain by reversibly preventing action potential propagation through the inhibition of voltage-gated sodium channels. The tetrodotoxin-sensitive voltage-gated sodium channel subtype Nav1.7, abundantly expressed in pain-sensing neurons, plays a crucial role in perception and transmission of painful stimuli and in inherited chronic pain syndromes. Understanding the interaction of lidocaine with Nav1.7 channels could provide valuable insight into the drug's action in alleviating pain in distinct patient populations. The aim of this study was to determine how lidocaine interacts with multiple inactivated conformations of Nav1.7 channels. EXPERIMENTAL APPROACH We investigated the interactions of lidocaine with wild-type Nav1.7 channels and a paroxysmal extreme pain disorder mutation (I1461T) that destabilizes fast inactivation. Whole cell patch clamp recordings were used to examine the activity of channels expressed in human embryonic kidney 293 cells. KEY RESULTS Depolarizing pulses that increased slow inactivation of Nav1.7 channels also reduced lidocaine inhibition. Lidocaine enhanced recovery of Nav1.7 channels from prolonged depolarizing pulses by decreasing slow inactivation. A paroxysmal extreme pain disorder mutation that destabilizes fast inactivation of Nav1.7 channels decreased lidocaine inhibition. CONCLUSIONS AND IMPLICATIONS Lidocaine decreased the transition of Nav1.7 channels to the slow inactivated state. The fast inactivation gate (domain III–IV linker) is important for potentiating the interaction of lidocaine with the Nav1.7 channel. PMID:21232038

Activity of the anticonvulsant lacosamide in experimental and human epilepsy via selective effects on slow Na+ channel inactivation.

PubMed

Holtkamp, Dominik; Opitz, Thoralf; Niespodziany, Isabelle; Wolff, Christian; Beck, Heinz

2017-01-01

In human epilepsy, pharmacoresistance to antiepileptic drug therapy is a major problem affecting ~30% of patients with epilepsy. Many classical antiepileptic drugs target voltage-gated sodium channels, and their potent activity in inhibiting high-frequency firing has been attributed to their strong use-dependent blocking action. In chronic epilepsy, a loss of use-dependent block has emerged as a potential cellular mechanism of pharmacoresistance for anticonvulsants acting on voltage-gated sodium channels. The anticonvulsant drug lacosamide (LCM) also targets sodium channels, but has been shown to preferentially affect sodium channel slow inactivation processes, in contrast to most other anticonvulsants. We used whole-cell voltage clamp recordings in acutely isolated cells to investigate the effects of LCM on transient Na + currents. Furthermore, we used whole-cell current clamp recordings to assess effects on repetitive action potential firing in hippocampal slices. We show here that LCM exerts its effects primarily via shifting the slow inactivation voltage dependence to more hyperpolarized potentials in hippocampal dentate granule cells from control and epileptic rats, and from patients with epilepsy. It is important to note that this activity of LCM was maintained in chronic experimental and human epilepsy. Furthermore, we demonstrate that the efficacy of LCM in inhibiting high-frequency firing is undiminished in chronic experimental and human epilepsy. Taken together, these results show that LCM exhibits maintained efficacy in chronic epilepsy, in contrast to conventional use-dependent sodium channel blockers such as carbamazepine. They also establish that targeting slow inactivation may be a promising strategy for overcoming target mechanisms of pharmacoresistance. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

Afferent control mechanisms involved in the development of soleus fiber alterations in simulated hypogravity

NASA Astrophysics Data System (ADS)

Shenkman, B. S.; Nemirovskaya, T. L.; Shapovalova, K. B.; Podlubnaya, Z. A.; Vikhliantsev, I. M.; Moukhina, A. M.; Kozlovskaya, I. B.

2007-02-01

It was recently established that support withdrawal (withdrawal of support reaction force) in microgravity provokes a sequence of functional shifts in the activity of motor units (inactivation of slow ones) and peripheral muscle apparatus which lead to the decline of postural muscle contractility and alterations in fiber characteristics. However, mechanisms involved in inactivation of the slow motor units and appropriate slow-twitch muscle fiber disuse under the supportless conditions remained unknown. We show here that artificial inactivation of muscles-antagonists (which are known to be hyperactive during unloading) counteracts some of the unloading-induced events in the rat soleus (fiber size reduction, slow-to-fast fiber-type transition and decline of titin and nebulin content). It was also demonstrated that direct activation of the muscarinic receptors of the neostriatum neurons prevented slow-to-fast fiber-type transformation in soleus of hindlimb suspended rats.

Adaptation of primate vestibuloocular reflex to altered peripheral vestibular inputs. II Spatiotemporal properties of the adapted slow-phase eye velocity

NASA Technical Reports Server (NTRS)

Angelaki, D. E.; Hess, B. J.

1996-01-01

1. The ability of the vestibuloocular reflex (VOR) to undergo adaptive modification after selective changes in the peripheral vestibular system was investigated in rhesus monkeys by recording three-dimensional eye movements before and after inactivation of selective semicircular canals. In the preceding paper we showed that the horizontal VOR gain evoked by passive yaw oscillations after lateral semicircular canal inactivation recovers gradually over time in a frequency-specific manner. Here we present the spatial tuning of the adapted slow-phase eye velocity and describe its spatiotemporal properties as a function of time after canal inactivation. 2. The spatial organization of the VOR was investigated during oscillations at different head positions in the pitch, roll, and yaw planes, as well as in the right anterior/left posterior and left anterior/right posterior canal planes. Acutely after bilateral inactivation of the lateral semicircular canals, a small horizontal response could still be elicited that peaked during rotations in pitched head positions that would maximally stimulate vertical semicircular canals. In addition, the phase of horizontal slow-phase velocity abruptly reversed through 180 degrees at positions close to upright, similarly to torsional slow-phase velocity. These spatial response properties suggest that the small, residual horizontal response components that are present acutely after plugging of both lateral canals originate from vertical semicircular canal signals. 3. As the horizontal response amplitude increased over time, consistent changes were also observed in the spatiotemporal tuning of horizontal slow-phase velocity. 1) The spatiotemporal response properties of horizontal slow-phase velocity acquired noncosine tuning characteristics, primarily in the pitch plane, in the right anterior/left posterior and left anterior/right posterior canal planes. Accordingly, horizontal response amplitude was nonzero during rotation in any head position in these planes and response phase varied significantly as a function of head orientation. 2) The peak horizontal response amplitude shifted spatially over time, such that 5-10 mo after plugging it was maximal during rotations at head positions close to upright. 4. In parallel to these unique spatiotemporal response properties characterizing the adapted horizontal VOR, torsional slow-phase velocity also exhibited small spatiotemporal changes after lateral canal inactivation that tended to precede in time the changes associated with the horizontal response components. In contrast, vertical slow-phase velocity in the plugged animals was unaltered and continued to be characterized by cosine-tuned spatial properties in three dimensions. 5. Recovery of the horizontal response gain during yaw oscillations in upright position, as well as the unique, noncosine spatiotemporal characteristics of the adapted horizontal VOR, were also observed in an animal with all but one vertical semicircular canals inactivated. There was, however, no sign of VOR gain recovery up to 2 mo after all semicircular canals were inactivated. These results suggest that the observed recovery of horizontal VOR is at least partly due to signals originating from the remaining intact vertical canal(s). Even in the presence of a single intact vertical canal, the improvement in horizontal gaze stability is at least partly restored through spatiotemporal changes in the processing of vestibuloocular signals that improve the gain and spatial tuning of horizontal VOR at the expense of temporal response properties.

In Silico Docking and Electrophysiological Characterization of Lacosamide Binding Sites on Collapsin Response Mediator Protein-2 Identifies a Pocket Important in Modulating Sodium Channel Slow Inactivation*

PubMed Central

Wang, Yuying; Brittain, Joel M.; Jarecki, Brian W.; Park, Ki Duk; Wilson, Sarah M.; Wang, Bo; Hale, Rachel; Meroueh, Samy O.; Cummins, Theodore R.; Khanna, Rajesh

2010-01-01

The anti-epileptic drug (R)-lacosamide ((2R)-2-(acetylamino)-N-benzyl-3-methoxypropanamide (LCM)) modulates voltage-gated sodium channels (VGSCs) by preferentially interacting with slow inactivated sodium channels, but the observation that LCM binds to collapsin response mediator protein 2 (CRMP-2) suggests additional mechanisms of action for LCM. We postulated that CRMP-2 levels affects the actions of LCM on VGSCs. CRMP-2 labeling by LCM analogs was competitively displaced by excess LCM in rat brain lysates. Manipulation of CRMP-2 levels in the neuronal model system CAD cells affected slow inactivation of VGSCs without any effects on other voltage-dependent properties. In silico docking was performed to identify putative binding sites in CRMP-2 that may modulate the effects of LCM on VGSCs. These studies identified five cavities in CRMP-2 that can accommodate LCM. CRMP-2 alanine mutants of key residues within these cavities were functionally similar to wild-type CRMP-2 as assessed by similar levels of enhancement in dendritic complexity of cortical neurons. Next, we examined the effects of expression of wild-type and mutant CRMP-2 constructs on voltage-sensitive properties of VGSCs in CAD cells: 1) steady-state voltage-dependent activation and fast-inactivation properties were not affected by LCM, 2) CRMP-2 single alanine mutants reduced the LCM-mediated effects on the ability of endogenous Na+ channels to transition to a slow inactivated state, and 3) a quintuplicate CRMP-2 alanine mutant further decreased this slow inactivated fraction. Collectively, these results identify key CRMP-2 residues that can coordinate LCM binding thus making it more effective on its primary clinical target. PMID:20538611

Reciprocal voltage sensor-to-pore coupling leads to potassium channel C-type inactivation

PubMed Central

Conti, Luca; Renhorn, Jakob; Gabrielsson, Anders; Turesson, Fredrik; Liin, Sara I; Lindahl, Erik; Elinder, Fredrik

2016-01-01

Voltage-gated potassium channels open at depolarized membrane voltages. A prolonged depolarization causes a rearrangement of the selectivity filter which terminates the conduction of ions – a process called slow or C-type inactivation. How structural rearrangements in the voltage-sensor domain (VSD) cause alteration in the selectivity filter, and vice versa, are not fully understood. We show that pulling the pore domain of the Shaker potassium channel towards the VSD by a Cd2+ bridge accelerates C-type inactivation. Molecular dynamics simulations show that such pulling widens the selectivity filter and disrupts the K+ coordination, a hallmark for C-type inactivation. An engineered Cd2+ bridge within the VSD also affect C-type inactivation. Conversely, a pore domain mutation affects VSD gating-charge movement. Finally, C-type inactivation is caused by the concerted action of distant amino acid residues in the pore domain. All together, these data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel. PMID:27278891

Reciprocal voltage sensor-to-pore coupling leads to potassium channel C-type inactivation

NASA Astrophysics Data System (ADS)

Conti, Luca; Renhorn, Jakob; Gabrielsson, Anders; Turesson, Fredrik; Liin, Sara I.; Lindahl, Erik; Elinder, Fredrik

2016-06-01

Voltage-gated potassium channels open at depolarized membrane voltages. A prolonged depolarization causes a rearrangement of the selectivity filter which terminates the conduction of ions - a process called slow or C-type inactivation. How structural rearrangements in the voltage-sensor domain (VSD) cause alteration in the selectivity filter, and vice versa, are not fully understood. We show that pulling the pore domain of the Shaker potassium channel towards the VSD by a Cd2+ bridge accelerates C-type inactivation. Molecular dynamics simulations show that such pulling widens the selectivity filter and disrupts the K+ coordination, a hallmark for C-type inactivation. An engineered Cd2+ bridge within the VSD also affect C-type inactivation. Conversely, a pore domain mutation affects VSD gating-charge movement. Finally, C-type inactivation is caused by the concerted action of distant amino acid residues in the pore domain. All together, these data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel.

Insulin increases excitability via a dose-dependent dual inhibition of voltage-activated K+ currents in differentiated N1E-115 neuroblastoma cells.

PubMed

Lima, Pedro A; Vicente, M Inês; Alves, Frederico M; Dionísio, José C; Costa, Pedro F

2008-04-01

A role in the control of excitability has been attributed to insulin via modulation of potassium (K(+)) currents. To investigate insulin modulatory effects on voltage-activated potassium currents in a neuronal cell line with origin in the sympathetic system, we performed whole-cell voltage-clamp recordings in differentiated N1E-115 neuroblastoma cells. Two main voltage-activated K(+) currents were identified: (a) a relatively fast inactivating current (I(fast) - time constant 50-300 ms); (b) a slow delayed rectifying K(+) current (I(slow) - time constant 1-4 s). The kinetics of inactivation of I(fast), rather than I(slow), showed clear voltage dependence. I(fast) and I(slow) exhibited different activation and inactivation dependence for voltage, and have different but nevertheless high sensitivities to tetraethylammonium, 4-aminopyridine and quinidine. In differentiated cells - rather than in non-differentiated cells - application of up to 300 nm insulin reduced I(slow) only (IC(50) = 6.7 nm), whereas at higher concentrations I(fast) was also affected (IC(50) = 7.7 microm). The insulin inhibitory effect is not due to a change in the activation or inactivation current-voltage profiles, and the time-dependent inactivation is also not altered; this is not likely to be a result of activation of the insulin-growth-factor-1 (IGF1) receptors, as application of IGF1 did not result in significant current alteration. Results suggest that the current sensitive to low concentrations of insulin is mediated by erg-like channels. Similar observations concerning the insulin inhibitory effect on slow voltage-activated K(+) currents were also made in isolated rat hippocampal pyramidal neurons, suggesting a widespread neuromodulator role of insulin on K(+) channels.

The Impact of Cortical Deafferentation on the Neocortical Slow Oscillation

PubMed Central

Lemieux, Maxime; Chen, Jen-Yung; Lonjers, Peter; Bazhenov, Maxim

2014-01-01

Slow oscillation is the main brain rhythm observed during deep sleep in mammals. Although several studies have demonstrated its neocortical origin, the extent of the thalamic contribution is still a matter of discussion. Using electrophysiological recordings in vivo on cats and computational modeling, we found that the local thalamic inactivation or the complete isolation of the neocortical slabs maintained within the brain dramatically reduced the expression of slow and fast oscillations in affected cortical areas. The slow oscillation began to recover 12 h after thalamic inactivation. The slow oscillation, but not faster activities, nearly recovered after 30 h and persisted for weeks in the isolated slabs. We also observed an increase of the membrane potential fluctuations recorded in vivo several hours after thalamic inactivation. Mimicking this enhancement in a network computational model with an increased postsynaptic activity of long-range intracortical afferents or scaling K+ leak current, but not several other Na+ and K+ intrinsic currents was sufficient for recovering the slow oscillation. We conclude that, in the intact brain, the thalamus contributes to the generation of cortical active states of the slow oscillation and mediates its large-scale synchronization. Our study also suggests that the deafferentation-induced alterations of the sleep slow oscillation can be counteracted by compensatory intracortical mechanisms and that the sleep slow oscillation is a fundamental and intrinsic state of the neocortex. PMID:24741059

Selective control of cortical axonal spikes by a slowly inactivating K+ current

PubMed Central

Shu, Yousheng; Yu, Yuguo; Yang, Jing; McCormick, David A.

2007-01-01

Neurons are flexible electrophysiological entities in which the distribution and properties of ionic channels control their behaviors. Through simultaneous somatic and axonal whole-cell recording of layer 5 pyramidal cells, we demonstrate a remarkable differential expression of slowly inactivating K+ currents. Depolarizing the axon, but not the soma, rapidly activated a low-threshold, slowly inactivating, outward current that was potently blocked by low doses of 4-aminopyridine, α-dendrotoxin, and rTityustoxin-Kα. Block of this slowly inactivating current caused a large increase in spike duration in the axon but only a small increase in the soma and could result in distal axons generating repetitive discharge in response to local current injection. Importantly, this current was also responsible for slow changes in the axonal spike duration that are observed after somatic membrane potential change. These data indicate that low-threshold, slowly inactivating K+ currents, containing Kv1.2 α subunits, play a key role in the flexible properties of intracortical axons and may contribute significantly to intracortical processing. PMID:17581873

Removal of sodium inactivation and block of sodium channels by chloramine-T in crayfish and squid giant axons.

PubMed Central

Huang, J. M.; Tanguy, J.; Yeh, J. Z.

1987-01-01

Modification of sodium channels by chloramine-T was examined in voltage clamped internally perfused crayfish and squid giant axons using the double sucrose gap and axial wire technique, respectively. Freshly prepared chloramine-T solution exerted two major actions on sodium channels: (a) an irreversible removal of the fast Na inactivation, and (b) a reversible block of the Na current. Both effects were observed when chloramine-T was applied internally or externally (5-10 mM) to axons. The first effect was studied in crayfish axons. We found that the removal of the fast Na inactivation did not depend on the states of the channel since the channel could be modified by chloramine-T at holding potential (from -80 to -100 mV) or at depolarized potential of -30 mV. After removal of fast Na inactivation, the slow inactivation mechanism was still present, and more channels could undergo slow inactivation. This result indicates that in crayfish axons the transition through the fast inactivated state is not a prerequisite for the slow inactivation to occur. During chloramine-T treatment, a distinct blocking phase occurred, which recovered upon washing out the drug. This second effect of chloramine-T was studied in detail in squid axons. After 24 h, chloramine-T solution lost its ability to remove fast inactivation but retained its blocking action. After removal of the fast Na inactivation, both fresh and aged chloramine-T solutions blocked the Na currents with a similar potency and in a voltage-dependent manner, being more pronounced at lower depolarizing potentials. A similar voltage-dependent block was observed with aged chloramine-T solution in an axon with intact inactivation. In contrast to the action of the fresh solution, the aged chloramine-T solution was found to accelerate the decay of Na currents.These results suggest that chloramine-T solution contains at least two active molecular forms that act at different sites in the Na channel. PMID:2444276

Unfolding and inactivation during thermal denaturation of an enzyme that exhibits phytase and acid phosphatase activities.

PubMed

Wang, Xiao-Yun; Meng, Fan-Guo; Zhou, Hai-Meng

2004-03-01

The thermostability of an enzyme that exhibits phytase and acid phosphatase activities was studied. Kinetics of inactivation and unfolding during thermal denaturation of the enzyme were compared. The loss of phytase activity on thermal denaturation is most suggestive of a reversible process. As for acid phosphatase activities, an interesting phenomenon was observed; there are two phases in thermal inactivation: when the temperature was between 45 and 50 degrees C, the thermal inactivation could be characterized as an irreversible inactivation which had some residual activity and when the temperature was above 55 degrees C, the thermal inactivation could be characterized as an irreversible process which had no residual activity. The microscopic rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method [Adv. Enzymol. Relat. Areas Mol. Biol. 61 (1988) 381]. Fluorescence analyses indicate that when the enzyme was treated at temperatures below 60 degrees C for 60 min, the conformation of the enzyme had no detectable change; when the temperatures were above 60 degrees C, some fluorescence red-shift could be observed with a decrease in emission intensity. The inactivation rates (k(+0)) of free enzymes were faster than those of conformational changes during thermal denaturation at the same temperature. The rapid inactivation and slow conformational changes of phytase during thermal denaturation suggest that inactivation occurs before significant conformational changes of the enzyme, and the active site of this enzyme is situated in a relatively fragile region which makes the active site more flexible than the molecule as a whole.

Regulation of Na+ channel inactivation by the DIII and DIV voltage-sensing domains.

PubMed

Hsu, Eric J; Zhu, Wandi; Schubert, Angela R; Voelker, Taylor; Varga, Zoltan; Silva, Jonathan R

2017-03-06

Functional eukaryotic voltage-gated Na + (Na V ) channels comprise four domains (DI-DIV), each containing six membrane-spanning segments (S1-S6). Voltage sensing is accomplished by the first four membrane-spanning segments (S1-S4), which together form a voltage-sensing domain (VSD). A critical Na V channel gating process, inactivation, has previously been linked to activation of the VSDs in DIII and DIV. Here, we probe this interaction by using voltage-clamp fluorometry to observe VSD kinetics in the presence of mutations at locations that have been shown to impair Na V channel inactivation. These locations include the DIII-DIV linker, the DIII S4-S5 linker, and the DIV S4-S5 linker. Our results show that, within the 10-ms timeframe of fast inactivation, the DIV-VSD is the primary regulator of inactivation. However, after longer 100-ms pulses, the DIII-DIV linker slows DIII-VSD deactivation, and the rate of DIII deactivation correlates strongly with the rate of recovery from inactivation. Our results imply that, over the course of an action potential, DIV-VSDs regulate the onset of fast inactivation while DIII-VSDs determine its recovery. © 2017 Hsu et al.

Inhibition of Neuronal Voltage-Gated Sodium Channels by Brilliant Blue G

PubMed Central

Jo, Sooyeon

2011-01-01

Brilliant blue G (BBG), best known as an antagonist of P2X7 receptors, was found to inhibit voltage-gated sodium currents in N1E-115 neuroblastoma cells. Sodium currents elicited from a holding potential of −60 mV were blocked with an IC50 of 2 μM. Block was enhanced in a use-dependent manner at higher stimulation rates. The voltage-dependence of inactivation was shifted in the hyperpolarizing direction, and recovery from inactivation was slowed by BBG. The most dramatic effect of BBG was to slow recovery from inactivation after long depolarizations, with 3 μM BBG increasing half-time for recovery (measured at −120 mV) from 24 to 854 ms after a 10-s step to 0 mV. These results were mimicked by a kinetic model in which BBG binds weakly to resting channels (Kd = 170 μM) but tightly to fast-inactivated channels (Kd = 5 μM) and even more tightly (Kd = 0.2 μM) to slow-inactivated channels. In contrast to BBG, the structurally related food-coloring dye Brilliant Blue FCF had very little effect at concentrations up to 30 μM. These results show that BBG inhibits voltage-gated sodium channels at micromolar concentrations. Although BBG inhibition of sodium channels is less potent than inhibition of P2X7 receptors, there may be significant inhibition of sodium channels at BBG concentrations achieved in spinal cord or brain during experimental treatment of spinal cord injury or Huntington's disease. Considered as a sodium channel blocker, BBG is remarkably potent, acting with more than 10-fold greater potency than lacosamide, another blocker thought to bind to slow-inactivated channels. PMID:21536754

Field and laboratory investigations of inactivation of viruses (PRD1 and MS2) attached to iron oxide-coated quartz san

USGS Publications Warehouse

Ryan, Joseph N.; Harvey, Ronald W.; Metge, David W.; Elimelech, Menachem; Navigato, Theresa; Pieper, Ann P.

2002-01-01

Field and laboratory experiments were conducted to investigate inactivation of viruses attached to mineral surfaces. In a natural gradient transport field experiment, bacteriophage PRD1, radiolabeled with 32P, was injected into a ferric oxyhydroxide-coated sand aquifer with bromide and linear alkylbenzene sulfonates. In a zone of the aquifer contaminated by secondary sewage infiltration, small fractions of infective and 32P-labeled PRD1 broke through with the bromide tracer, followed by the slow release of 84% of the 32P activity and only 0.011% of the infective PRD1. In the laboratory experiments, the inactivation of PRD1, labeled with 35S (protein capsid), and MS2, dual radiolabeled with 35S (protein capsid) and 32P (nucleic acid), was monitored in the presence of groundwater and sediment from the contaminated zone of the field site. Release of infective viruses decreased at a much faster rate than release of the radiolabels, indicating that attached viruses were undergoing surface inactivation. Disparities between 32P and35S release suggest that the inactivated viruses were released in a disintegrated state. Comparison of estimated solution and surface inactivation rates indicates solution inactivation is ∼3 times as fast as surface inactivation. The actual rate of surface inactivation may be substantially underestimated owing to slow release of inactivated viruses.

Properties of an intermediate-duration inactivation process of the voltage-gated sodium conductance in rat hippocampal CA1 neurons.

PubMed

French, Christopher R; Zeng, Zhen; Williams, David A; Hill-Yardin, Elisa L; O'Brien, Terence J

2016-02-01

Rapid transmembrane flow of sodium ions produces the depolarizing phase of action potentials (APs) in most excitable tissue through voltage-gated sodium channels (NaV). Macroscopic currents display rapid activation followed by fast inactivation (IF) within milliseconds. Slow inactivation (IS) has been subsequently observed in several preparations including neuronal tissues. IS serves important physiological functions, but the kinetic properties are incompletely characterized, especially the operative timescales. Here we present evidence for an "intermediate inactivation" (II) process in rat hippocampal CA1 neurons with time constants of the order of 100 ms. The half-inactivation potentials (V0.5) of steady-state inactivation curves were hyperpolarized by increasing conditioning pulse duration from 50 to 500 ms and could be described by a sum of Boltzmann relations. II state transitions were observed after opening as well as subthreshold potentials. Entry into II after opening was relatively insensitive to membrane potential, and recovery of II became more rapid at hyperpolarized potentials. Removal of fast inactivation with cytoplasmic papaine revealed time constants of INa decay corresponding to II and IS with long depolarizations. Dynamic clamp revealed attenuation of trains of APs over the 10(2)-ms timescale, suggesting a functional role of II in repetitive firing accommodation. These experimental findings could be reproduced with a five-state Markov model. It is likely that II affects important aspects of hippocampal neuron response and may provide a drug target for sodium channel modulation. Copyright © 2016 the American Physiological Society.

Nucleophilic modification of human complement protein C3: correlation of conformational changes with acquisition of C3b-like functional properties.

PubMed

Isenman, D E; Kells, D I; Cooper, N R; Müller-Eberhard, H J; Pangburn, M K

1981-07-21

Inactivation of C3 by enzymatic cleavage, nucleophilic addition, or slow freezing and thawing resulted in the acquisition of similar end-state conformations as judged by near-UV circular dichroism. Although inactivation by the two nonenzymatic processes involves no peptide bond scission, the inactivated C3 resembled C3b in that it possessed a free sulfhydryl group not present in the native protein and an increased surface hydrophobicity as evidenced by enhanced binding of the fluorophore 8-anilino-1-naphthalensulfonate (ANS). The C3b-like functional properties of modified C3 [Pangburn, M. K., & Müller-Eberhard, H. J. (1980) J. Exp. Med. 152, 1102-1114] may thus be understood in terms of the similarity of its conformation to that of C3b. The rate of the conformational change following proteolytic cleavage was fast and appeared to be limited by the rate of the enzymatic reaction. In contrast, the rate of conformational change following addition of methylamine was slow and rate limited by the conformational rearrangement itself, not by the chemical modification. A kinetic analysis of the changes in circular dichroism and ANS fluorescence enhancement suggested that the nucleophilic addition was spectroscopically undetectable and was followed by a minimally biphasic, spectroscopically demonstrable conformational rearrangement. The appearance of C3b-like functional activity in nucleophile-modified C3 largely parallels the time course of the spectroscopically detectable conformational change but is distinctly slower than the rate at which hemolytic activity is lost. While fully transconformed methylamine-inactivated C3 can bind factor B and is susceptible to cleavage by C3b inactivator and its cofactor beta 1H, this cleavage occurs at a substantially slower rate than the equivalent process in C3b. The implications of these findings in terms of the mechanism through which the alterative pathway of complement is initiated are discussed.

Current understanding of the mechanism of action of the antiepileptic drug lacosamide.

PubMed

Rogawski, Michael A; Tofighy, Azita; White, H Steve; Matagne, Alain; Wolff, Christian

2015-02-01

The antiepileptic drug lacosamide [(R)-2-acetamido-N-benzyl-3-methoxypropanamide], a chiral functionalized amino acid, was originally identified by virtue of activity in the mouse and rat maximal electroshock (MES) test. Attention was drawn to lacosamide because of its high oral potency and stereoselectivity. Lacosamide is also active in the 6 Hz seizure model but inactive against clonic seizures in rodents induced by subcutaneous pentylenetetrazol, bicuculline and picrotoxin. It is also ineffective in genetic models of absence epilepsy. At doses greater than those required to confer protection in the MES test, lacosamide inhibits behavioral and electrographic seizures in hippocampal kindled rats. It also effectively terminates seizures in the rat perforant path stimulation status epilepticus model when administered early after the onset of seizures. Lacosamide does not exhibit antiepileptogenic effects in kindling or post-status epilepticus models. The profile of lacosamide in animal seizure and epilepsy models is similar to that of sodium channel blocking antiepileptic drugs, such as phenytoin and carbamazepine. However, unlike these agents, lacosamide does not affect sustained repetitive firing (SRF) on a time scale of hundreds of milliseconds or affect fast inactivation of voltage-gated sodium channels; however, it terminates SRF on a time scale of seconds by an apparent effect on sodium channel slow inactivation. Lacosamide shifts the slow inactivation curve to more hyperpolarized potentials and enhances the maximal fraction of channels that are in the slow inactivated state. Currently, lacosamide is the only known antiepileptic drug in clinical practice that exerts its anticonvulsant activity predominantly by selectively enhancing slow sodium channel inactivation. Copyright © 2014 Elsevier B.V. All rights reserved.

Mycobacteria inactivation using Engineered Water Nanostructures (EWNS).

PubMed

Pyrgiotakis, Georgios; McDevitt, James; Gao, Ya; Branco, Alan; Eleftheriadou, Mary; Lemos, Bernardo; Nardell, Edward; Demokritou, Philip

2014-08-01

Airborne transmitted pathogens such as Mycobacterium tuberculosis (Mtb) cause serious, often fatal infectious disease with enormous global health implications. Due to their unique cell wall and slow growth, mycobacteria are among the most resilient microbial forms. Herein we evaluate the ability of an emerging, chemical-free, nanotechnology-based method to inactivate M. parafortuitum (Mtb surrogate). This method is based on the transformation of atmospheric water vapor into engineered water nano-structures (EWNS) via electrospray. We demonstrate that the EWNS can interact with and inactivate airborne mycobacteria, reducing their concentration levels significantly. Additionally, EWNS can inactivate M. parafortuitum on surfaces eight times faster than the control. The mechanism of mycobacteria inactivation was also investigated in this study. It was demonstrated that the EWNS effectively deliver the reactive oxygen species, encapsulated during the electrospray process, to the bacteria oxidizing their cell membrane resulting into inactivation. Overall, this is a method with the potential to become an effective intervention technology in the battle against airborne infections. This study demonstrates the feasibility of mycobacterium inactivation in airborne form or on contact surfaces using electrospray activated water nano-structures. Given that the method is free of toxic chemicals, this might become an important tool in the prevention of mycobacterial infections, which are notoriously hard to treat. Copyright © 2014 Elsevier Inc. All rights reserved.

Structural model of the open–closed–inactivated cycle of prokaryotic voltage-gated sodium channels

PubMed Central

Bagnéris, Claire; Naylor, Claire E.; McCusker, Emily C.

2015-01-01

In excitable cells, the initiation of the action potential results from the opening of voltage-gated sodium channels. These channels undergo a series of conformational changes between open, closed, and inactivated states. Many models have been proposed for the structural transitions that result in these different functional states. Here, we compare the crystal structures of prokaryotic sodium channels captured in the different conformational forms and use them as the basis for examining molecular models for the activation, slow inactivation, and recovery processes. We compare structural similarities and differences in the pore domains, specifically in the transmembrane helices, the constrictions within the pore cavity, the activation gate at the cytoplasmic end of the last transmembrane helix, the C-terminal domain, and the selectivity filter. We discuss the observed differences in the context of previous models for opening, closing, and inactivation, and present a new structure-based model for the functional transitions. Our proposed prokaryotic channel activation mechanism is then compared with the activation transition in eukaryotic sodium channels. PMID:25512599

Hippocampal A-type current and Kv4.2 channel modulation by the sulfonylurea compound NS5806.

PubMed

Witzel, Katrin; Fischer, Paul; Bähring, Robert

2012-12-01

We examined the effects of the sulfonylurea compound NS5806 on neuronal A-type channel function. Using whole-cell patch-clamp we studied the effects of NS5806 on the somatodendritic A-type current (I(SA)) in cultured hippocampal neurons and the currents mediated by Kv4.2 channels coexpressed with different auxiliary β-subunits, including both Kv channel interacting proteins (KChIPs) and dipeptidyl aminopeptidase-related proteins (DPPs), in HEK 293 cells. The amplitude of the I(SA) component in hippocampal neurons was reduced in the presence of 20 μM NS5806. I(SA) decay kinetics were slowed and the recovery kinetics accelerated, but the voltage dependence of steady-state inactivation was shifted to more negative potentials by NS5806. The peak amplitudes of currents mediated by ternary Kv4.2 channel complexes, associated with DPP6-S (short splice-variant) and either KChIP2, KChIP3 or KChIP4, were potentiated and their macroscopic inactivation slowed by NS5806, whereas the currents mediated by binary Kv4.2 channels, associated only with DPP6-S, were suppressed, and the NS5806-mediated slowing of macroscopic inactivation was less pronounced. Neither potentiation nor suppression and no effect on current decay kinetics in the presence of NS5806 were observed for Kv4.2 channels associated with KChIP3 and the N-type inactivation-conferring DPP6a splice-variant. For all recombinant channel complexes, NS5806 slowed the recovery from inactivation and shifted the voltage dependence of steady-state inactivation to more negative potentials. Our results demonstrate the activity of NS5806 on native I(SA) and possible molecular correlates in the form of recombinant Kv4.2 channels complexed with different KChIPs and DPPs, and they shed some light on the mechanism of NS5806 action. Copyright © 2012 Elsevier Ltd. All rights reserved.

Factors affecting UV/H2O2 inactivation of Bacillus atrophaeus spores in drinking water.

PubMed

Zhang, Yongji; Zhang, Yiqing; Zhou, Lingling; Tan, Chaoqun

2014-05-05

This study aims at estimating the performance of the Bacillus atrophaeus spores inactivation by the UV treatment with addition of H2O2. The effect of factors affecting the inactivation was investigated, including initial H2O2 dose, UV irradiance, initial cell density, initial solution pH and various inorganic anions. Under the experimental conditions, the B. atrophaeus spores inactivation followed both the modified Hom Model and the Chick's Model. The results revealed that the H2O2 played dual roles in the reactions, while the optimum reduction of 5.88lg was received at 0.5mM H2O2 for 10min. The inactivation effect was affected by the UV irradiance, while better inactivation effect was achieved at higher irradiance. An increase in the initial cell density slowed down the inactivation process. A slight acid condition at pH 5 was considered as the optimal pH value. The inactivation effect within 10min followed the order of pH 5>pH 7>pH 9>pH 3>pH 11. The effects of three added inorganic anions were investigated and compared, including sulfate (SO4(2)(-)), nitrate (NO3(-)) and carbonate (CO3(2)(-)). The sequence of inactivation effect within 10min followed the order of control group>SO4(2)(-)>NO3(-)>CO3(2)(-). Copyright © 2014 Elsevier B.V. All rights reserved.

Fiber type conversion alters inactivation of voltage-dependent sodium currents in murine C2C12 skeletal muscle cells.

PubMed

Zebedin, Eva; Sandtner, Walter; Galler, Stefan; Szendroedi, Julia; Just, Herwig; Todt, Hannes; Hilber, Karlheinz

2004-08-01

Each skeletal muscle of the body contains a unique composition of "fast" and "slow" muscle fibers, each of which is specialized for certain challenges. This composition is not static, and the muscle fibers are capable of adapting their molecular composition by altered gene expression (i.e., fiber type conversion). Whereas changes in the expression of contractile proteins and metabolic enzymes in the course of fiber type conversion are well described, little is known about possible adaptations in the electrophysiological properties of skeletal muscle cells. Such adaptations may involve changes in the expression and/or function of ion channels. In this study, we investigated the effects of fast-to-slow fiber type conversion on currents via voltage-gated Na+ channels in the C(2)C(12) murine skeletal muscle cell line. Prolonged treatment of cells with 25 nM of the Ca2+ ionophore A-23187 caused a significant shift in myosin heavy chain isoform expression from the fast toward the slow isoform, indicating fast-to-slow fiber type conversion. Moreover, Na+ current inactivation was significantly altered. Slow inactivation less strongly inhibited the Na+ currents of fast-to-slow fiber type-converted cells. Compared with control cells, the Na+ currents of converted cells were more resistant to block by tetrodotoxin, suggesting enhanced relative expression of the cardiac Na+ channel isoform Na(v)1.5 compared with the skeletal muscle isoform Na(v)1.4. These results imply that fast-to-slow fiber type conversion of skeletal muscle cells involves functional adaptation of their electrophysiological properties.

Sodium channel slow inactivation as a therapeutic target for myotonia congenita

PubMed Central

Novak, Kevin R; Norman, Jennifer; Mitchell, Jacob R; Pinter, Martin J; Rich, Mark M

2014-01-01

Objective Patients with myotonia congenita have muscle hyperexcitability due to loss-of-function mutations in the chloride channel in skeletal muscle, which causes spontaneous firing of muscle action potentials (myotonia), producing muscle stiffness. In patients, muscle stiffness lessens with exercise, a change known as the warm-up phenomenon. Our goal was to identify the mechanism underlying warm up and to use this information to guide development of novel therapy. Methods To determine the mechanism underlying warm-up, we used a recently discovered drug to eliminate muscle contraction, thus allowing prolonged intracellular recording from individual muscle fibers during induction of warm-up in a mouse model of myotonia congenita. Results Changes in action potentials suggested slow inactivation of sodium channels as an important contributor to warm-up. These data suggested enhancing slow inactivation of sodium channels might offer effective therapy for myotonia. Lacosamide and ranolazine enhance slow inactivation of sodium channels and are FDA-approved for other uses in patients. We compared the efficacy of both drugs to mexiletine, a sodium channel blocker currently used to treat myotonia. In vitro studies suggested both lacosamide and ranolazine were superior to mexiletine. However, in vivo studies in a mouse model of myotonia congenita suggested side effects could limit the efficacy of lacosamide. Ranolazine produced fewer side effects and was as effective as mexiletine at a dose that produced none of mexiletine’s hypoexcitability side effects. Interpretation We conclude ranolazine has excellent therapeutic potential for treatment of patients with myotonia congenita. PMID:25515836

[Effect of various factors on ozone inactivating Giardia in water].

PubMed

Ran, Zhi-Lin; Li, Shao-Feng; Huang, Jun-Li; Yuan, Yi-Xing; Cui, Chong-Wei

2010-06-01

In order to study the effect of O3 inactivating Giardia in water, different factors (CT value, pH, temperature, turbidity, organic content and inorganic ions) which might influence the inactivation were investigated by using fluorescence staining method. The results indicated that the whole process of O3 inactivating Giardia could be divided into two periods, the inactivated rate in log phase was significantly faster than it in the slow phase [k1 = (5.64 +/- 0.023) x 10(-1) mg x min, k2 = (2.72 +/- 0.002) x 10(-2) mg x min, k1 > k2]. When the turbidity was 0.1 to 20. 0NTU, temperature was 5 to 35 degrees C, pH was 6.0 to 9.0, HA content was 0.5 to 10.0 mg/L, the turbidity was lower, the higher inactivating ratio could be received. With the increasing of temperature, the inactivating effect was decreased. The ability of O3 inactivating Giardia was stronger under acidic condition than it was in alkali circumstance. When the reaction system contained higher concentration of organics, the competition reaction might take place between Giardia and organics with O3, which might reduce inactivation ratio. The sequence of affecting disinfectant ability of O3 was NO3- > None > SO4(2-) > HCO3-, while inorganic cations (Ca2+, Mg2+ and Cu2+) promoted the inactive reaction to a certain extent. If the CT value of O3 was more than 15.0 min x mg/L, the ratio of inactivation could exceed 99.0% during disinfecting drinking water.

Selective suppression of the slow-inactivating potassium currents by nootropics in molluscan neurons.

PubMed

Bukanova, Julia V; Solntseva, Elena I; Skrebitsky, Vladimir G

2002-09-01

The role of the voltage-gated K+ channels in the effect of some nootropics was investigated. Earlier, the multiple effect of high concentrations of two nootropics, piracetam and its peptide analogue GVS-111 [Seredenin et al. (1995), US Patent No. 5,439,930], on Ca2+ and K+ currents of molluscan neurons was shown [Solntseva et al. (1997), General Pharmacology 29, 85-89]. In the present work, we describe the selective effect of low concentrations of these nootropics as well as vinpocetine on certain types of K+ current. The experiments were performed on isolated neurons of the land snail Helix pomatia using a two-microelectrode voltage-clamp method. The inward voltage-gated Ca2+ current (ICa) and three subtypes of the outward voltage-gated K+ current were recorded: Ca2+-dependent K+ current (IK(Ca)), delayed rectifying current (IKD), and fast-inactivating K+ current (IA). It has been found that I Ca was not changed in the presence of 30 microM vinpocetine, 100 microM piracetam or 10 nM GVS-111, while slow-inactivating, TEA-sensitive IK(Ca) and IKD were inhibited (IK(Ca) more strongly than IKD). In contrast, the fast-inactivating, 4-AP-sensitive K+ current (IA) was not diminished by low concentrations of piracetam and GVS-111, while vinpocetine even augmented it. A possible role of slow-inactivating subtypes of the K+ channels in the development of different forms of dementia is discussed.

Symposium on 'nutritional effects of new processing technologies'. New processing technologies: an overview.

PubMed

Gould, G W

2001-11-01

Most food-preservation techniques act by slowing down or completely inhibiting the growth of micro-organisms. Few techniques act by inactivating them. While heat remains the technique most extensively used for inactivation, there has been increasing interest recently in the development of alternative approaches in response to the desires of consumers for products which are less organoleptically and nutritionally damaged during processing and less reliant on additives than previously. The new approaches, therefore, mostly involve technologies that offer full or partial alternatives to heat for the inactivation of bacteria, yeasts and moulds. They include the application to foods of high hydrostatic pressure, high-voltage electric discharges, high-intensity laser and non-coherent light pulses, 'manothermosonication' (the combination of mild heating with ultrasonication and slightly-raised pressure), and high-magnetic-field pulses. In addition, a number of naturally-occurring antimicrobials, including lysozyme and low-molecular-weight products of micro-organisms are finding increasing use. High pressure is being used commercially to non-thermally pasteurize a number of foods, while the other physical procedures are in various stages of development and commercial evaluation. Possible nutritional consequences have so far been given little attention compared with microbiological ones.

Inactivation of the Lactobacillus leichmannii ribonucleoside triphosphate reductase by 2'-chloro-2'-deoxyuridine 5'-triphosphate: stoichiometry of inactivation, site of inactivation, and mechanism of the protein chromophore formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ashley, G.W.; Harris, G.; Stubbe, J.A.

1988-06-14

The ribonucleoside triphosphate reductase (RTPR) of Lactobacillus leichmannii is inactivated by the substrate analogue 2'-chloro-2'-deoxyuridine 5'-triphosphate (ClUTP). Inactivation is due to alkylation by 2-methylene-3(2H)-furanone, a decomposition product of the enzymic product 3'-keto-2'-deoxyuridine triphosphate. The former has been unambiguously identified as 2-((ethylthio)methyl)-3(2H)-furanone, an ethanethiol trapped adduct, which is identical by /sup 1/H NMR spectroscopy with material synthesized chemically. Subsequent to rapid inactivation, a slow process occurs that results in formation of a new protein-associated chromophore absorbing maximally near 320 nm. The terminal stages of the inactivation have now been investigated in detail. The alkylation and inactivation stoichiometries were studied as amore » function of the ratio of ClUTP to enzyme. The amount of labeling of RTPR increased with increasing ClUTP concentration up to the maximum of approximately 4 labels/RTPR, yet the degree of inactivation did not increase proportionally. This suggests that (1) RTPR may be inactivated by alkylation of a single site and (2) decomposition of 3'-keto-dUTP is not necessarily enzyme catalyzed. The formation of the new protein chromophore was also monitored during inactivation and found to reach its full extent upon the first alkylation . Thus, out of four alkylation sites, only one appears capable of undergoing the subsequent reaction to form the new chromophore. Model studies suggest that the new chromophore is due to addition of an amino group to the 5-position of enzyme-bound furanone, followed by ring opening and tautomerization to give a ..beta..-aminoenone structure.« less

LGI1 antibody encephalitis and psychosis.

PubMed

Wang, Dahai; Hao, Qinjian; He, Lan; Wang, Qiang

2018-05-01

To describe a case of leucine-rich, glioma inactivated 1 antibody-encephalitis presenting with psychosis. Case report. A young man with leucine-rich, glioma inactivated 1-antibody encephalitis initially presented with acute psychotic symptoms, short-term memory loss and faciobrachial dystonic seizures. Magnetic resonance imaging revealed hippocampal lesions. Electroencephalography revealed frontotemporal slowing of background activity. Increased awareness of leucine-rich, glioma inactivated 1-antibody encephalitis may promote early recognition and treatment.

Effects of benzocaine on the kinetics of normal and batrachotoxin-modified Na channels in frog node of Ranvier.

PubMed Central

Schneider, M F; Dubois, J M

1986-01-01

The effects of benzocaine (0.5-1 mM) on normal Na currents, and on Na current and gating charge movement (Q) of batrachotoxin (BTX)-modified Na channels were analyzed in voltage-clamped frog node of Ranvier. Without BTX treatment the decay of Na current during pulses to between -40 and 0 mV could be decomposed into two exponential components both in the absence and in the presence of benzocaine. Benzocaine did not significantly alter the inactivation time constant of either component, but reduced both their amplitudes. The amplitude of the slow inactivating component was more decreased by benzocaine than the amplitude of the fast one, leading to an apparently faster decline of the overall Na current. After removal of Na inactivation and charge movement immobilization by BTX, benzocaine decreased the amplitude of INa with no change in time course. INa, QON, and QOFF were all reduced by the same factor. The results suggest that the rate of reaction of benzocaine with its receptor is slow compared to the rates of channel activation and inactivation. The differential effects of benzocaine on the two components of Na current inactivation in normal channels can be explained assuming two types of channel with different rates of inactivation and different affinities for the drug. PMID:2428413

Simultaneous E. coli inactivation and NOM degradation in river water via photo-Fenton process at natural pH in solar CPC reactor. A new way for enhancing solar disinfection of natural water.

PubMed

Moncayo-Lasso, Alejandro; Sanabria, Janeth; Pulgarin, César; Benítez, Norberto

2009-09-01

Bacteria inactivation and natural organic matter oxidation in river water was simultaneously conducted via photo-Fenton reaction at "natural" pH ( approximately 6.5) containing 0.6 mg L(-1) of Fe(3+) and 10 mg L(-1) of H(2)O(2). The experiments were carried out by using a solar compound parabolic collector on river water previously filtered by a slow sand filtration system and voluntarily spiked with Escherichia coli. Fifty five percent of 5.3 mg L(-1) of dissolved organic carbon was mineralized whereas total disinfection was observed without re-growth after 24h in the dark.

Inactivation and pharmacological properties of sqKv1A homotetramers in Xenopus oocytes cannot account for behavior of the squid "delayed rectifier" K(+) conductance.

PubMed Central

Jerng, Henry H; Gilly, William F

2002-01-01

Considerable published evidence suggests that alpha-subunits of the cloned channel sqKv1A compose the "delayed rectifier" in the squid giant axon system, but discrepancies regarding inactivation properties of cloned versus native channels exist. In this paper we define the mechanism of inactivation for sqKv1A channels in Xenopus oocytes to investigate these and other discrepancies. Inactivation of sqKv1A in Xenopus oocytes was found to be unaffected by genetic truncation of the N-terminus, but highly sensitive to certain amino acid substitutions around the external mouth of the pore. External TEA and K(+) ions slowed inactivation of sqKv1A channels in oocytes, and chloramine T (Chl-T) accelerated inactivation. These features are all consistent with a C-type inactivation mechanism as defined for Shaker B channels. Treatment of native channels in giant fiber lobe neurons with TEA or high K(+) does not slow inactivation, nor does Chl-T accelerate it. Pharmacological differences between the two channel types were also found for 4-aminopyridine (4AP). SqKv1A's affinity for 4AP was poor at rest and increased after activation, whereas 4AP block occurred much more readily at rest with native channels than when they were activated. These results suggest that important structural differences between sqKv1A homotetramers and native squid channels are likely to exist around the external and internal mouths of the pore. PMID:12023225

Mechanism of the modulation of Kv4:KChIP-1 channels by external K+.

PubMed

Kaulin, Yu A; De Santiago-Castillo, J A; Rocha, C A; Covarrubias, M

2008-02-15

In response to a prolonged membrane depolarization, inactivation autoregulates the activity of voltage-gated ion channels. Slow inactivation involving a localized constriction of the selectivity filter (P/C-type mechanism) is prevalent in many voltage-gated K(+) channels of the Kv1 subfamily. However, the generalization of this mechanism to other Kv channel subfamilies has remained uncertain and controversial. In agreement with a "foot-in-the-door" mechanism and the presence of ion-ion interactions in the pore, elevated external K(+) slows the development of P/C-type inactivation and accelerates its recovery. In sharp contrast and resembling the regulation of the hippocampal A-type K(+) current, we found that Kv4.x channels associated with KChIP-1 (an auxiliary subunit) exhibit accelerated inactivation and unaffected recovery from inactivation when exposed to elevated external K(+). This regulation depends on the ability of a permeant ion to enter the selectivity filter (K(+) = Rb(+) = NH4(+) > Cs(+) > Na(+)); and the apparent equilibrium dissociation constant of a single regulatory site is 8 mM for K(+). By applying a robust quantitative global kinetic modeling approach to all macroscopic properties over a 210-mV range of membrane potentials, we determined that elevated external K(+) inhibits unstable closed states outside the main activation pathway and thereby promotes preferential closed-state inactivation. These results suggest the presence of a vestigial and unstable P/C-type mechanism of inactivation in Kv4 channels and strengthen the concept of novel mechanisms of closed-state inactivation. Regulation of Kv4 channel inactivation by hyperkalemia may help to explain the pathophysiology of electrolyte imbalances in excitable tissues.

A new mode of regulation of N-type inactivation in a Caenorhabditis elegans voltage-gated potassium channel.

PubMed

Cai, Shi-Qing; Sesti, Federico

2007-06-22

N-type inactivation in voltage-gated K+ (Kv) channels is a widespread means to modulate neuronal excitability and signaling. Here we have shown a novel mechanism of N-type inactivation in a Caenorhabditis elegans Kv channel. The N-terminal sequence of KVS-1 contains a domain of 22 amino acids that resembles the inactivation ball in A-type channels, which is preceded by a domain of eighteen amino acids. Wild type KVS-1 currents can be described as A-type; however, their kinetics are significantly (approximately 5-fold) slower. When the putative inactivation ball is deleted, the current becomes non-inactivating. Inactivation is restored in non-inactivating channels by diffusion of the missing inactivation domain in the cytoplasm. Deletion of the domain in front of the ball speeds inactivation kinetics approximately 5-fold. We conclude that KVS-1 is the first example of a novel type of Kv channel simultaneously possessing an N-inactivating ball preceded by an N inactivation regulatory domain (NIRD) that acts to slow down inactivation through steric mechanisms.

Tentonin 3/TMEM150c Confers Distinct Mechanosensitive Currents in Dorsal-Root Ganglion Neurons with Proprioceptive Function.

PubMed

Hong, Gyu-Sang; Lee, Byeongjun; Wee, Jungwon; Chun, Hyeyeon; Kim, Hyungsup; Jung, Jooyoung; Cha, Joo Young; Riew, Tae-Ryong; Kim, Gyu Hyun; Kim, In-Beom; Oh, Uhtaek

2016-07-06

Touch sensation or proprioception requires the transduction of mechanical stimuli into electrical signals by mechanoreceptors in the periphery. These mechanoreceptors are equipped with various transducer channels. Although Piezo1 and 2 are mechanically activated (MA) channels with rapid inactivation, MA molecules with other inactivation kinetics have not been identified. Here we report that heterologously expressed Tentonin3 (TTN3)/TMEM150C is activated by mechanical stimuli with distinctly slow inactivation kinetics. Genetic ablation of Ttn3/Tmem150c markedly reduced slowly adapting neurons in dorsal-root ganglion neurons. The MA TTN3 currents were inhibited by known blockers of mechanosensitive ion channels. Moreover, TTN3 was localized in muscle spindle afferents. Ttn3-deficient mice exhibited the loss of coordinated movements and abnormal gait. Thus, TTN3 appears to be a component of a mechanosensitive channel with a slow inactivation rate and contributes to motor coordination. Identification of this gene advances our understanding of the various types of mechanosensations, including proprioception. Copyright © 2016 Elsevier Inc. All rights reserved.

Depolarized inactivation overcomes impaired activation to produce DRG neuron hyperexcitability in a Nav1.7 mutation in a patient with distal limb pain.

PubMed

Huang, Jianying; Yang, Yang; Dib-Hajj, Sulayman D; van Es, Michael; Zhao, Peng; Salomon, Jody; Drenth, Joost P H; Waxman, Stephen G

2014-09-10

Sodium channel Nav1.7, encoded by SCN9A, is expressed in DRG neurons and regulates their excitability. Genetic and functional studies have established a critical contribution of Nav1.7 to human pain disorders. We have now characterized a novel Nav1.7 mutation (R1279P) from a female human subject with distal limb pain, in which depolarized fast inactivation overrides impaired activation to produce hyperexcitability and spontaneous firing in DRG neurons. Whole-cell voltage-clamp recordings in human embryonic kidney (HEK) 293 cells demonstrated that R1279P significantly depolarizes steady-state fast-, slow-, and closed-state inactivation. It accelerates deactivation, decelerates inactivation, and facilitates repriming. The mutation increases ramp currents in response to slow depolarizations. Our voltage-clamp analysis showed that R1279P depolarizes channel activation, a change that was supported by our multistate structural modeling. Because this mutation confers both gain-of-function and loss-of-function attributes on the Nav1.7 channel, we tested the impact of R1279P expression on DRG neuron excitability. Current-clamp studies reveal that R1279P depolarizes resting membrane potential, decreases current threshold, and increases firing frequency of evoked action potentials within small DRG neurons. The populations of spontaneously firing and repetitively firing neurons were increased by expressing R1279P. These observations indicate that the dominant proexcitatory gating changes associated with this mutation, including depolarized steady-state fast-, slow-, and closed-state inactivation, faster repriming, and larger ramp currents, override the depolarizing shift of activation, to produce hyperexcitability and spontaneous firing of nociceptive neurons that underlie pain. Copyright © 2014 the authors 0270-6474/14/3412328-13$15.00/0.

Identification of the Benzyloxyphenyl Pharmacophore: A Structural Unit That Promotes Sodium Channel Slow Inactivation

PubMed Central

2012-01-01

Four compounds that contained the N-benzyl 2-amino-3-methoxypropionamide unit were evaluated for their ability to modulate Na+ currents in catecholamine A differentiated CAD neuronal cells. The compounds differed by the absence or presence of either a terminal N-acetyl group or a (3-fluoro)benzyloxy moiety positioned at the 4′-benzylamide site. Analysis of whole-cell patch-clamp electrophysiology data showed that the incorporation of the (3-fluoro)benzyloxy unit, to give the (3-fluoro)benzyloxyphenyl pharmacophore, dramatically enhanced the magnitude of Na+ channel slow inactivation. In addition, N-acetylation markedly increased the stereoselectivity for Na+ channel slow inactivation. Furthermore, we observed that Na+ channel frequency (use)-dependent block was maintained upon inclusion of this pharmacophore. Confirmation of the importance of the (3-fluoro)benzyloxyphenyl pharmacophore was shown by examining compounds where the N-benzyl 2-amino-3-methoxypropionamide unit was replaced by a N-benzyl 2-amino-3-methylpropionamide moiety, as well as examining a series of compounds that did not contain an amino acid group but retained the pharmacophore unit. Collectively, the data indicated that the (3-fluoro)benzyloxyphenyl unit is a novel pharmacophore for the modulation of Na+ currents. PMID:23259039

Lacosamide

PubMed Central

Curia, Giulia; Biagini, Giuseppe; Perucca, Emilio; Avoli, Massimo

2016-01-01

The mechanism of action of several antiepileptic drugs (AEDs) rests on their ability to modulate the activity of voltage-gated sodium currents that are responsible for fast action potential generation. Recent data indicate that lacosamide (a compound with analgesic and anticonvulsant effects in animal models) shares a similar mechanism. When compared with other AEDs, lacosamide has the unique ability to interact with sodium channel slow inactivation without affecting fast inactivation. This article reviews these findings and discusses their relevance within the context of neuronal activity seen during epileptiform discharges generated by limbic neuronal networks in the presence of chemical convulsants. These seizure-like events are characterized by sustained discharges of sodium-dependent action potentials supported by robust depolarizations, thus providing synchronization within neuronal networks. Generally, AEDs such as phenytoin, carbamazepine and lamotrigine block sodium channels when activated. In contrast, lacosamide facilitates slow inactivation of sodium channels both in terms of kinetics and voltage dependency. This effect may be relatively selective for repeatedly depolarized neurons, such as those participating in seizure activity in which the persistence of sodium currents is more pronounced and promotes neuronal excitation. The clinical effectiveness of lacosamide has been demonstrated in randomized, double-blind, parallel-group, placebo-controlled, adjunctive-therapy trials in patients with refractory partial seizures. Further studies should determine whether the effects of lacosamide in animal models and in clinical settings are fully explained by its selective action on sodium current slow inactivation or whether other effects (e.g. interactions with the collapsin-response mediator protein-2) play a contributory role. PMID:19552484

A qualitative assessment of Toxoplasma gondii risk in ready-to-eat smallgoods processing.

PubMed

Mie, Tanya; Pointon, Andrew M; Hamilton, David R; Kiermeier, Andreas

2008-07-01

Toxoplasma gondii is one of the most common parasitic infections of humans and other warm-blooded animals. In most adults, it does not cause serious illness, but severe disease may result from infection in fetuses and immunocompromised people. Consumption of raw or undercooked meats has consistently been identified as an important source of exposure to T. gondii. Several studies indicate the potential failure to inactivate T. gondii in the processes of cured meat products, This article presents a qualitative risk-based assessment of the processing of ready-to-eat smallgoods, which include cooked or uncooked fermented meat, pâté, dried meat, slow cured meat, luncheon meat, and cooked muscle meat including ham and roast beef. The raw meat ingredients are rated with respect to their likelihood of containing T. gondii cysts and an adjustment is made based on whether all the meat from a particular source is frozen. Next, the effectiveness of common processing steps to inactivate T. gondii cysts is assessed, including addition of spices, nitrates, nitrites and salt, use of fermentation, smoking and heat treatment, and the time and temperature during maturation. It is concluded that processing steps that may be effective in the inactivation of T. gondii cysts include freezing, heat treatment, and cooking, and the interaction between salt concentration, maturation time, and temperature. The assessment is illustrated using a Microsoft Excel-based software tool that was developed to facilitate the easy assessment of four hypothetical smallgoods products.

Role of N-terminal domain and accessory subunits in controlling deactivation-inactivation coupling of Kv4.2 channels.

PubMed

Barghaan, Jan; Tozakidou, Magdalini; Ehmke, Heimo; Bähring, Robert

2008-02-15

We examined the relationship between deactivation and inactivation in Kv4.2 channels. In particular, we were interested in the role of a Kv4.2 N-terminal domain and accessory subunits in controlling macroscopic gating kinetics and asked if the effects of N-terminal deletion and accessory subunit coexpression conform to a kinetic coupling of deactivation and inactivation. We expressed Kv4.2 wild-type channels and N-terminal deletion mutants in the absence and presence of Kv channel interacting proteins (KChIPs) and dipeptidyl aminopeptidase-like proteins (DPPs) in human embryonic kidney 293 cells. Kv4.2-mediated A-type currents at positive and deactivation tail currents at negative membrane potentials were recorded under whole-cell voltage-clamp and analyzed by multi-exponential fitting. The observed changes in Kv4.2 macroscopic inactivation kinetics caused by N-terminal deletion, accessory subunit coexpression, or a combination of the two maneuvers were compared with respective changes in deactivation kinetics. Extensive correlation analyses indicated that modulatory effects on deactivation closely parallel respective effects on inactivation, including both onset and recovery kinetics. Searching for the structural determinants, which control deactivation and inactivation, we found that in a Kv4.2 Delta 2-10 N-terminal deletion mutant both the initial rapid phase of macroscopic inactivation and tail current deactivation were slowed. On the other hand, the intermediate and slow phase of A-type current decay, recovery from inactivation, and tail current decay kinetics were accelerated in Kv4.2 Delta 2-10 by KChIP2 and DPPX. Thus, a Kv4.2 N-terminal domain, which may control both inactivation and deactivation, is not necessary for active modulation of current kinetics by accessory subunits. Our results further suggest distinct mechanisms for Kv4.2 gating modulation by KChIPs and DPPs.

Acidosis Differentially Modulates Inactivation in NaV1.2, NaV1.4, and NaV1.5 Channels

PubMed Central

Vilin, Yury Y.; Peters, Colin H.; Ruben, Peter C.

2012-01-01

NaV channels play a crucial role in neuronal and muscle excitability. Using whole-cell recordings we studied effects of low extracellular pH on the biophysical properties of NaV1.2, NaV1.4, and NaV1.5, expressed in cultured mammalian cells. Low pH produced different effects on different channel subtypes. Whereas NaV1.4 exhibited very low sensitivity to acidosis, primarily limited to partial block of macroscopic currents, the effects of low pH on gating in NaV1.2 and NaV1.5 were profound. In NaV1.2 low pH reduced apparent valence of steady-state fast inactivation, shifted the τ(V) to depolarizing potentials and decreased channels availability during onset to slow and use-dependent inactivation (UDI). In contrast, low pH delayed open-state inactivation in NaV1.5, right-shifted the voltage-dependence of window current, and increased channel availability during onset to slow and UDI. These results suggest that protons affect channel availability in an isoform-specific manner. A computer model incorporating these results demonstrates their effects on membrane excitability. PMID:22701426

Elimination of fast inactivation in Kv4 A-type potassium channels by an auxiliary subunit domain.

PubMed

Holmqvist, Mats H; Cao, Jie; Hernandez-Pineda, Ricardo; Jacobson, Michael D; Carroll, Karen I; Sung, M Amy; Betty, Maria; Ge, Pei; Gilbride, Kevin J; Brown, Melissa E; Jurman, Mark E; Lawson, Deborah; Silos-Santiago, Inmaculada; Xie, Yu; Covarrubias, Manuel; Rhodes, Kenneth J; Distefano, Peter S; An, W Frank

2002-01-22

The Kv4 A-type potassium currents contribute to controlling the frequency of slow repetitive firing and back-propagation of action potentials in neurons and shape the action potential in heart. Kv4 currents exhibit rapid activation and inactivation and are specifically modulated by K-channel interacting proteins (KChIPs). Here we report the discovery and functional characterization of a modular K-channel inactivation suppressor (KIS) domain located in the first 34 aa of an additional KChIP (KChIP4a). Coexpression of KChIP4a with Kv4 alpha-subunits abolishes fast inactivation of the Kv4 currents in various cell types, including cerebellar granule neurons. Kinetic analysis shows that the KIS domain delays Kv4.3 opening, but once the channel is open, it disrupts rapid inactivation and slows Kv4.3 closing. Accordingly, KChIP4a increases the open probability of single Kv4.3 channels. The net effects of KChIP4a and KChIP1-3 on Kv4 gating are quite different. When both KChIP4a and KChIP1 are present, the Kv4.3 current shows mixed inactivation profiles dependent on KChIP4a/KChIP1 ratios. The KIS domain effectively converts the A-type Kv4 current to a slowly inactivating delayed rectifier-type potassium current. This conversion is opposite to that mediated by the Kv1-specific "ball" domain of the Kv beta 1 subunit. Together, these results demonstrate that specific auxiliary subunits with distinct functions actively modulate gating of potassium channels that govern membrane excitability.

Transient inactivation of the anterior cingulate cortex in rats disrupts avoidance of a dynamic object.

PubMed

Svoboda, Jan; Lobellová, Veronika; Popelíková, Anna; Ahuja, Nikhil; Kelemen, Eduard; Stuchlík, Aleš

2017-03-01

Although animals often learn and monitor the spatial properties of relevant moving objects such as conspecifics and predators to properly organize their own spatial behavior, the underlying brain substrate has received little attention and hence remains elusive. Because the anterior cingulate cortex (ACC) participates in conflict monitoring and effort-based decision making, and ACC neurons respond to objects in the environment, it may also play a role in the monitoring of moving cues and exerting the appropriate spatial response. We used a robot avoidance task in which a rat had to maintain at least a 25cm distance from a small programmable robot to avoid a foot shock. In successive sessions, we trained ten Long Evans male rats to avoid a fast-moving robot (4cm/s), a stationary robot, and a slow-moving robot (1cm/s). In each condition, the ACC was transiently inactivated by bilateral injections of muscimol in the penultimate session and a control saline injection was given in the last session. Compared to the corresponding saline session, ACC-inactivated rats received more shocks when tested in the fast-moving condition, but not in the stationary or slow robot conditions. Furthermore, ACC-inactivated rats less frequently responded to an approaching robot with appropriate escape responses although their response to shock stimuli remained preserved. Since we observed no effect on slow or stationary robot avoidance, we conclude that the ACC may exert cognitive efforts for monitoring dynamic updating of the position of an object, a role complementary to the dorsal hippocampus. Copyright © 2017 Elsevier Inc. All rights reserved.

[Alkaline phosphatase in Amoeba proteus].

PubMed

Sopina, V A

2005-01-01

In free-living Amoeba proteus (strain B), 3 phosphatase were found after disc-electrophoresis of 10 microg of protein in PAGE and using 1-naphthyl phosphate as a substrate a pH 9.0. These phosphatases differed in their electrophoretic mobilities - "slow" (1-3 bands), "middle" (one band) and "fast" (one band). In addition to 1-naphthyl phosphate, "slow" phosphatases were able to hydrolyse 2-naphthyl phosphate and p-nitrophenyl phosphate. They were slightly activated by Mg2+, completely inhibited by 3 chelators (EDTA, EGTA and 1,10-phenanthroline), L-cysteine, sodium dodecyl sulfate and Fe2+, Zn2+ and Mn2+ (50 mM), considerably inactivated by orthovanadate, molybdate, phosphatase inhibitor cocktail 1, p-nitrophenyl phosphate, Na2HPO4, DL-dithiothreitol and urea and partly inhibited by H2O2, DL-phenylalanine, 2-mercaptoethanol, phosphatase inhibitor cocktail 2 and Ca2+. Imidazole, L-(+)-tartrate, okadaic acid, NaF and sulfhydryl reagents -p-(hydroxy-mercuri)benzoate and N-ethylmaleimide - had no influence on the activity of "slow" phosphatases. "Middle" and "fast" phosphatases, in contrast to "slow" ones, were not inactivated by 3 chelators. The "middle" phosphatase differed from the "fast" one by smaller resistance to urea, Ca2+, Mn2+, phosphates and H2O2 and greater resistance to dithiothreitol and L-(+)-tartrate. In addition, the "fast" phosphatase was inhibited by L-cysteine but the "middle" one was activated by it. Of 5 tested ions (Mg2+, Cu2+, Mn2+, Ca2+ and Zn2+), only Zn2+ reactivated "slow" phosphatases after their inactivation by EDTA treatment. The reactivation of apoenzyme was only partial (about 35 %). Thus, among phosphatases found in amoebae at pH 9.0, only "slow" ones are Zn-metalloenzymes and may be considered as alkaline phosphatases (EC 3.1.3.1). It still remains uncertain, to which particular phosphatase class "middle" and "fast" phosphatases (pH 9.0) may belong.

Block of human cardiac sodium channels by lacosamide: evidence for slow drug binding along the activation pathway.

PubMed

Wang, Ging Kuo; Wang, Sho-Ya

2014-05-01

Lacosamide is an anticonvulsant hypothesized to enhance slow inactivation of neuronal Na(+) channels for its therapeutic action. Cardiac Na(+) channels display less and incomplete slow inactivation, but their sensitivity toward lacosamide remains unknown. We therefore investigated the action of lacosamide in human cardiac Nav1.5 and Nav1.5-CW inactivation-deficient Na(+) channels. Lacosamide showed little effect on hNav1.5 Na(+) currents at 300 µM when cells were held at -140 mV. With 30-second conditioning pulses from -90 to -50 mV; however, hNav1.5 Na(+) channels became sensitive to lacosamide with IC50 (50% inhibitory concentration) around 70-80 µM. Higher IC50 values were found at -110 and -30 mV. The development of lacosamide block at -70 mV was slow in wild-type Na(+) channels (τ; 8.04 ± 0.39 seconds, n = 8). This time constant was significantly accelerated in hNav1.5-CW inactivation-deficient counterparts. The recovery from lacosamide block at -70 mV for 10 seconds was relatively rapid in wild-type Na(+) channels (τ; 639 ± 90 milliseconds, n = 8). This recovery was accelerated further in hNav1.5-CW counterparts. Unexpectedly, lacosamide elicited a time-dependent block of persistent hNav1.5-CW Na(+) currents with an IC50 of 242 ± 19 µM (n = 5). Furthermore, both hNav1.5-CW/F1760K mutant and batrachotoxin-activated hNav1.5 Na(+) channels became completely lacosamide resistant, indicating that the lacosamide receptor overlaps receptors for local anesthetics and batrachotoxin. Our results together suggest that lacosamide targets the intermediate preopen and open states of hNav1.5 Na(+) channels. Lacosamide may thus track closely the conformational changes at the hNav1.5-F1760 region along the activation pathway.

Block of Human Cardiac Sodium Channels by Lacosamide: Evidence for Slow Drug Binding along the Activation Pathway

PubMed Central

Wang, Sho-Ya

2014-01-01

Lacosamide is an anticonvulsant hypothesized to enhance slow inactivation of neuronal Na+ channels for its therapeutic action. Cardiac Na+ channels display less and incomplete slow inactivation, but their sensitivity toward lacosamide remains unknown. We therefore investigated the action of lacosamide in human cardiac Nav1.5 and Nav1.5-CW inactivation-deficient Na+ channels. Lacosamide showed little effect on hNav1.5 Na+ currents at 300 µM when cells were held at −140 mV. With 30-second conditioning pulses from −90 to −50 mV; however, hNav1.5 Na+ channels became sensitive to lacosamide with IC50 (50% inhibitory concentration) around 70–80 µM. Higher IC50 values were found at −110 and −30 mV. The development of lacosamide block at −70 mV was slow in wild-type Na+ channels (τ; 8.04 ± 0.39 seconds, n = 8). This time constant was significantly accelerated in hNav1.5-CW inactivation-deficient counterparts. The recovery from lacosamide block at −70 mV for 10 seconds was relatively rapid in wild-type Na+ channels (τ; 639 ± 90 milliseconds, n = 8). This recovery was accelerated further in hNav1.5-CW counterparts. Unexpectedly, lacosamide elicited a time-dependent block of persistent hNav1.5-CW Na+ currents with an IC50 of 242 ± 19 µM (n = 5). Furthermore, both hNav1.5-CW/F1760K mutant and batrachotoxin-activated hNav1.5 Na+ channels became completely lacosamide resistant, indicating that the lacosamide receptor overlaps receptors for local anesthetics and batrachotoxin. Our results together suggest that lacosamide targets the intermediate preopen and open states of hNav1.5 Na+ channels. Lacosamide may thus track closely the conformational changes at the hNav1.5-F1760 region along the activation pathway. PMID:24563546

Using advanced oxidation treatment for biofilm inactivation by varying water vapor content in air plasma

NASA Astrophysics Data System (ADS)

Ryota, Suganuma; Koichi, Yasuoka

2015-09-01

Biofilms are caused by environmental degradation in food factories and medical facilities. The inactivation of biofilms involves making them react with chemicals including chlorine, hydrogen peroxide, and ozone, although inactivation using chemicals has a potential problem because of the hazardous properties of the residual substance and hydrogen peroxide, which have slow reaction velocity. We successfully performed an advanced oxidation process (AOP) using air plasma. Hydrogen peroxide and ozone, which were used for the formation of OH radicals in our experiment, were generated by varying the amount of water vapor supplied to the plasma. By varying the content of the water included in the air, the main product was changed from air plasma. When we increased the water content in the air, hydrogen peroxide was produced, while ozone peroxide was produced when we decreased the water content in the air. By varying the amount of water vapor, we realized a 99.9% reduction in the amount of bacteria in the biofilm when we discharged humidified air only. This work was supported by JSPS KAKENHI Grant Number 25630104.

Dopamine Inactivation Efficacy Related to Functional DAT1 and COMT Variants Influences Motor Response Evaluation

PubMed Central

Bender, Stephan; Rellum, Thomas; Freitag, Christine; Resch, Franz; Rietschel, Marcella; Treutlein, Jens; Jennen-Steinmetz, Christine; Brandeis, Daniel; Banaschewski, Tobias; Laucht, Manfred

2012-01-01

Background Dopamine plays an important role in orienting, response anticipation and movement evaluation. Thus, we examined the influence of functional variants related to dopamine inactivation in the dopamine transporter (DAT1) and catechol-O-methyltransferase genes (COMT) on the time-course of motor processing in a contingent negative variation (CNV) task. Methods 64-channel EEG recordings were obtained from 195 healthy adolescents of a community-based sample during a continuous performance task (A-X version). Early and late CNV as well as motor postimperative negative variation were assessed. Adolescents were genotyped for the COMT Val158Met and two DAT1 polymorphisms (variable number tandem repeats in the 3′-untranslated region and in intron 8). Results The results revealed a significant interaction between COMT and DAT1, indicating that COMT exerted stronger effects on lateralized motor post-processing (centro-parietal motor postimperative negative variation) in homozygous carriers of a DAT1 haplotype increasing DAT1 expression. Source analysis showed that the time interval 500–1000 ms after the motor response was specifically affected in contrast to preceding movement anticipation and programming stages, which were not altered. Conclusions Motor slow negative waves allow the genomic imaging of dopamine inactivation effects on cortical motor post-processing during response evaluation. This is the first report to point towards epistatic effects in the motor system during response evaluation, i.e. during the post-processing of an already executed movement rather than during movement programming. PMID:22649558

Inactivation of 1-aminocyclopropane-1-carboxylate oxidase involves oxidative modifications.

PubMed

Barlow, J N; Zhang, Z; John, P; Baldwin, J E; Schofield, C J

1997-03-25

1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the final step in the biosynthesis of the plant signaling molecule ethylene. It is a member of the ferrous iron dependent family of oxidases and dioxygenases and is unusual in that it displays a very short half-life under catalytic conditions, typically less than 20 min, and a requirement for CO2 as an activator. The rates of inactivation of purified, recombinant ACC oxidase from tomato under various combinations of substrates and cofactors were measured. Inactivation was relatively slow in the presence of buffer alone (t1/2 > 1 h), but fast in the presence of ferrous iron and ascorbate (t1/2 approximately 10 min). The rate of iron/ascorbate-mediated inactivation was increased by the addition of ACC, unaffected by the addition of CO2 at saturation (supplied as bicarbonate) but decreased by the addition of catalase or ACC + CO2 at saturation (supplied as bicarbonate). Iron/ascorbate-mediated inactivation was accompanied by partial proteolysis as observed by SDS-PAGE analysis. The fragmentation pattern was altered when ACC was also included, suggesting that ACC can bind to ACC oxidase in the absence of bicarbonate. N-terminal sequencing of fragments resulted in identification of an internal cleavage site which we propose is proximate to active-site bound iron. Thus, ACC oxidase inactivates via relatively slow partial unfolding of the catalytically active conformation, oxidative damage mediated via hydrogen peroxide which is catalase protectable and oxidative damage to the active site which results in partial proteolysis and is not catalase protectable.

A Direct Demonstration of Closed-State Inactivation of K+ Channels at Low pH

PubMed Central

Claydon, Thomas W.; Vaid, Moni; Rezazadeh, Saman; Kwan, Daniel C.H.; Kehl, Steven J.; Fedida, David

2007-01-01

Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels. Using voltage-clamp fluorimetry we directly determined the fate of Shaker-IR channels at low pH by measuring fluorescence emission from tetramethylrhodamine-5-maleimide attached to substituted cysteine residues in the voltage sensor domain (M356C to R362C) or S5-P linker (S424C). One aspect of the distal S3-S4 linker α-helix (A359C and R362C) reported a pH-induced acceleration of the slow phase of fluorescence quenching that represents P/C-type inactivation, but neither site reported a change in the total charge movement at low pH. Shaker S424C fluorescence demonstrated slow unquenching that also reflects channel inactivation and this too was accelerated at low pH. In addition, however, acidic pH caused a reversible loss of the fluorescence signal (pKa = 5.1) that paralleled the reduction of peak current amplitude (pKa = 5.2). Protons decreased single channel open probability, suggesting that the loss of fluorescence at low pH reflects a decreased channel availability that is responsible for the reduced macroscopic conductance. Inhibition of inactivation in Shaker S424C (by raising external K+ or the mutation T449V) prevented fluorescence loss at low pH, and the fluorescence report from closed Shaker ILT S424C channels implied that protons stabilized a W434F-like inactivated state. Furthermore, acidic pH changed the fluorescence amplitude (pKa = 5.9) in channels held continuously at −80 mV. This suggests that low pH stabilizes closed-inactivated states. Thus, fluorescence experiments suggest the major mechanism of pH-induced peak current reduction is inactivation of channels from closed states from which they can activate, but not open; this occurs in addition to acceleration of P/C-type inactivation from the open state. PMID:17470663

Ternary Kv4.2 channels recapitulate voltage-dependent inactivation kinetics of A-type K+ channels in cerebellar granule neurons.

PubMed

Amarillo, Yimy; De Santiago-Castillo, Jose A; Dougherty, Kevin; Maffie, Jonathon; Kwon, Elaine; Covarrubias, Manuel; Rudy, Bernardo

2008-04-15

Kv4 channels mediate most of the somatodendritic subthreshold operating A-type current (I(SA)) in neurons. This current plays essential roles in the regulation of spike timing, repetitive firing, dendritic integration and plasticity. Neuronal Kv4 channels are thought to be ternary complexes of Kv4 pore-forming subunits and two types of accessory proteins, Kv channel interacting proteins (KChIPs) and the dipeptidyl-peptidase-like proteins (DPPLs) DPPX (DPP6) and DPP10. In heterologous cells, ternary Kv4 channels exhibit inactivation that slows down with increasing depolarization. Here, we compared the voltage dependence of the inactivation rate of channels expressed in heterologous mammalian cells by Kv4.2 proteins with that of channels containing Kv4.2 and KChIP1, Kv4.2 and DPPX-S, or Kv4.2, KChIP1 and DPPX-S, and found that the relation between inactivation rate and membrane potential is distinct for these four conditions. Moreover, recordings from native neurons showed that the inactivation kinetics of the I(SA) in cerebellar granule neurons has voltage dependence that is remarkably similar to that of ternary Kv4 channels containing KChIP1 and DPPX-S proteins in heterologous cells. The fact that this complex and unique behaviour (among A-type K(+) currents) is observed in both the native current and the current expressed in heterologous cells by the ternary complex containing Kv4, DPPX and KChIP proteins supports the hypothesis that somatically recorded native Kv4 channels in neurons include both types of accessory protein. Furthermore, quantitative global kinetic modelling showed that preferential closed-state inactivation and a weakly voltage-dependent opening step can explain the slowing of the inactivation rate with increasing depolarization. Therefore, it is likely that preferential closed-state inactivation is the physiological mechanism that regulates the activity of both ternary Kv4 channel complexes and native I(SA)-mediating channels.

Biophysical characterization and functional consequences of a slowly inactivating potassium current in neostriatal neurons.

PubMed

Gabel, L A; Nisenbaum, E S

1998-04-01

Neostriatal spiny projection neurons can display a pronounced delay in their transition to action potential discharge that is mediated by a slowly developing ramp depolarization. The possible contribution of a slowly inactivating A-type K+ current (IAs) to this delayed excitation was investigated by studying the biophysical and functional properties of IAs using whole cell voltage- and current-clamp recording from acutely isolated neostriatal neurons. Isolation of IAs from other voltage-gated, calcium-independent K+ currents was achieved through selective blockade of IAs with low concentrations (10 microM) of the benzazepine derivative, 6-chloro-7,8-dihydroxy-3-allyl- 1-phenyl-2,3,4,5-tetra-hydro-1H-3-benzazepine (APB; SKF82958) and subsequent current subtraction. Examination of the voltage dependence of activation showed that IAs began to flow at approximately -60 mV in response to depolarization. The voltage dependence of inactivation revealed that approximately 50% of IAs channels were available at the normal resting potential (-80 mV) of these cells, but that only 20% of the channels were available at membrane potentials corresponding to spike threshold (about -40 mV). At these depolarized membrane potentials, the rate of activation was moderately rapid (tau approximately 60 ms), whereas the rate of inactivation was slow (tau approximately 1.5 s). The time course of removal of inactivation of IAs at -80 mV also was relatively slow (tau approximately 1.0 s). The subthreshold availability of IAs combined with its rapid activation and slow inactivation rates suggested that this current should be capable of dampening the onset of prolonged depolarizing responses, but over time its efficacy should diminish, slowly permitting the membrane to depolarize toward spike threshold. Voltage recording experiments confirmed this hypothesis by demonstrating that application of APB at a concentration (10 microM) that selectively blocks IAs substantially decreased the latency to discharge and increased the frequency of firing of neostriatal neurons. The properties of IAs suggest that it should play a critical role in placing the voltage limits on the recurring episodes of subthreshold depolarization which are characteristic of spiny neurons recorded in vivo. However, the voltage dependence and recovery kinetics of inactivation of IAs predict that its effectiveness will vary exponentially with the level and duration of hyperpolarization which precedes depolarizing episodes. Thus long periods of hyperpolarization should increase the availability of IAs and dampen succeeding depolarizations; whereas brief epochs of hyperpolarization should not sufficiently remove inactivation of IAs, thereby reducing its ability to limit subsequent depolarizing responses.

Substituted N-(biphenyl-4'-yl)methyl (R)-2-acetamido-3-methoxypropionamides: potent anticonvulsants that affect frequency (use) dependence and slow inactivation of sodium channels.

PubMed

Lee, Hyosung; Park, Ki Duk; Torregrosa, Robert; Yang, Xiao-Fang; Dustrude, Erik T; Wang, Yuying; Wilson, Sarah M; Barbosa, Cindy; Xiao, Yucheng; Cummins, Theodore R; Khanna, Rajesh; Kohn, Harold

2014-07-24

We prepared 13 derivatives of N-(biphenyl-4'-yl)methyl (R)-2-acetamido-3-methoxypropionamide that differed in type and placement of a R-substituent in the terminal aryl unit. We demonstrated that the R-substituent impacted the compound's whole animal and cellular pharmacological activities. In rodents, select compounds exhibited excellent anticonvulsant activities and protective indices (PI=TD50/ED50) that compared favorably with clinical antiseizure drugs. Compounds with a polar, aprotic R-substituent potently promoted Na+ channel slow inactivation and displayed frequency (use) inhibition of Na+ currents at low micromolar concentrations. The possible advantage of affecting these two pathways to decrease neurological hyperexcitability is discussed.

Slow fusion pore expansion creates a unique reaction chamber for co-packaged cargo

PubMed Central

Bittner, Mary A.; Lawrence, Daniel A.

2017-01-01

A lumenal secretory granule protein, tissue plasminogen activator (tPA), greatly slows fusion pore dilation and thereby slows its own discharge. We investigated another outcome of the long-lived narrow fusion pore: the creation of a nanoscale chemical reaction chamber for granule contents in which the pH is suddenly neutralized upon fusion. Bovine adrenal chromaffin cells endogenously express both tPA and its primary protein inhibitor, plasminogen activator inhibitor 1 (PAI). We found by immunocytochemistry that tPA and PAI are co-packaged in the same secretory granule. It is known that PAI irreversibly and covalently inactivates tPA at neutral pH. We demonstrate with zymography that the acidic granule lumen protects tPA from inactivation by PAI. Immunocytochemistry, total internal reflection fluorescence (TIRF) microscopy, and polarized TIRF microscopy demonstrated that co-packaged PAI and tPA remain together in granules for many seconds in the nanoscale reaction chamber, more than enough time to inhibit tPA and create a new secreted protein species. PMID:28882880

Synergistic Inhibition of Delayed Rectifier K+ and Voltage-Gated Na+ Currents by Artemisinin in Pituitary Tumor (GH3) Cells.

PubMed

So, Edmund Cheung; Wu, Sheng-Nan; Wu, Ping-Ching; Chen, Hui-Zhen; Yang, Chia-Jung

2017-01-01

Artemisinin (ART) is an anti-malarial agent reported to influence endocrine function. Effects of ART on ionic currents and action potentials (APs) in pituitary tumor (GH3) cells were evaluated by patch clamp techniques. ART inhibited the amplitude of delayed-rectifier K+ current (IK(DR)) in response to membrane depolarization and accelerated the process of current inactivation. It exerted an inhibitory effect on IK(DR) with an IC50 value of 11.2 µM and enhanced IK(DR) inactivation with a KD value of 14.7 µM. The steady-state inactivation curve of IK(DR) was shifted to hyperpolarization by 10 mV. Pretreatment of chlorotoxin (1 µM) or iloprost (100 nM) did not alter the magnitude of ART-induced inhibition of IK(DR) in GH3 cells. ART also decreased the peak amplitude of voltage-gated Na+ current (INa) with a concentration-dependent slowing in inactivation rate. Application of KMUP-1, an inhibitor of late INa, was effective at reversing ART-induced prolongation in inactivation time constant of INa. Under current-clamp recordings, ART alone reduced the amplitude of APs and prolonged the duration of APs. Under ART exposure, the inhibitory actions on both IK(DR) and INa could be a potential mechanisms through which this drug influences membrane excitability of endocrine or neuroendocrine cells appearing in vivo. © 2017 The Author(s). Published by S. Karger AG, Basel.

Solution structure and function of the "tandem inactivation domain" of the neuronal A-type potassium channel Kv1.4.

PubMed

Wissmann, Ralph; Bildl, Wolfgang; Oliver, Dominik; Beyermann, Michael; Kalbitzer, Hans-Robert; Bentrop, Detlef; Fakler, Bernd

2003-05-02

Cumulative inactivation of voltage-gated (Kv) K(+) channels shapes the presynaptic action potential and determines timing and strength of synaptic transmission. Kv1.4 channels exhibit rapid "ball-and-chain"-type inactivation gating. Different from all other Kvalpha subunits, Kv1.4 harbors two inactivation domains at its N terminus. Here we report the solution structure and function of this "tandem inactivation domain" using NMR spectroscopy and patch clamp recordings. Inactivation domain 1 (ID1, residues 1-38) consists of a flexible N terminus anchored at a 5-turn helix, whereas ID2 (residues 40-50) is a 2.5-turn helix made up of small hydrophobic amino acids. Functional analysis suggests that only ID1 may work as a pore-occluding ball domain, whereas ID2 most likely acts as a "docking domain" that attaches ID1 to the cytoplasmic face of the channel. Deletion of ID2 slows inactivation considerably and largely impairs cumulative inactivation. Together, the concerted action of ID1 and ID2 may promote rapid inactivation of Kv1.4 that is crucial for the channel function in short term plasticity.

Membrane potential correlates of sensory perception in mouse barrel cortex.

PubMed

Sachidhanandam, Shankar; Sreenivasan, Varun; Kyriakatos, Alexandros; Kremer, Yves; Petersen, Carl C H

2013-11-01

Neocortical activity can evoke sensory percepts, but the cellular mechanisms remain poorly understood. We trained mice to detect single brief whisker stimuli and report perceived stimuli by licking to obtain a reward. Pharmacological inactivation and optogenetic stimulation demonstrated a causal role for the primary somatosensory barrel cortex. Whole-cell recordings from barrel cortex neurons revealed membrane potential correlates of sensory perception. Sensory responses depended strongly on prestimulus cortical state, but both slow-wave and desynchronized cortical states were compatible with task performance. Whisker deflection evoked an early (<50 ms) reliable sensory response that was encoded through cell-specific reversal potentials. A secondary late (50-400 ms) depolarization was enhanced on hit trials compared to misses. Optogenetic inactivation revealed a causal role for late excitation. Our data reveal dynamic processing in the sensory cortex during task performance, with an early sensory response reliably encoding the stimulus and later secondary activity contributing to driving the subjective percept.

Functional integrity of the retrosplenial cortex is essential for rapid consolidation and recall of fear memory.

PubMed

Katche, Cynthia; Dorman, Guido; Slipczuk, Leandro; Cammarota, Martín; Medina, Jorge H

2013-03-15

Memory storage is a temporally graded process involving different phases and different structures in the mammalian brain. Cortical plasticity is essential to store stable memories, but little is known regarding its involvement in memory processing. Here we show that fear memory consolidation requires early post-training macromolecular synthesis in the anterior part of the retrosplenial cortex (aRSC), and that reversible pharmacological inactivation of this cortical region impairs recall of recent as well as of remote memories. These results challenge the generally accepted idea that neocortical areas are slow encoding systems that participate in the retrieval of remote memories only.

S1-S3 counter charges in the voltage sensor module of a mammalian sodium channel regulate fast inactivation.

PubMed

Groome, James R; Winston, Vern

2013-05-01

The movement of positively charged S4 segments through the electric field drives the voltage-dependent gating of ion channels. Studies of prokaryotic sodium channels provide a mechanistic view of activation facilitated by electrostatic interactions of negatively charged residues in S1 and S2 segments, with positive counterparts in the S4 segment. In mammalian sodium channels, S4 segments promote domain-specific functions that include activation and several forms of inactivation. We tested the idea that S1-S3 countercharges regulate eukaryotic sodium channel functions, including fast inactivation. Using structural data provided by bacterial channels, we constructed homology models of the S1-S4 voltage sensor module (VSM) for each domain of the mammalian skeletal muscle sodium channel hNaV1.4. These show that side chains of putative countercharges in hNaV1.4 are oriented toward the positive charge complement of S4. We used mutagenesis to define the roles of conserved residues in the extracellular negative charge cluster (ENC), hydrophobic charge region (HCR), and intracellular negative charge cluster (INC). Activation was inhibited with charge-reversing VSM mutations in domains I-III. Charge reversal of ENC residues in domains III (E1051R, D1069K) and IV (E1373K, N1389K) destabilized fast inactivation by decreasing its probability, slowing entry, and accelerating recovery. Several INC mutations increased inactivation from closed states and slowed recovery. Our results extend the functional characterization of VSM countercharges to fast inactivation, and support the premise that these residues play a critical role in domain-specific gating transitions for a mammalian sodium channel.

Redox modulation of A-type K+ currents in pain-sensing dorsal root ganglion neurons.

PubMed

Hsieh, Chi-Pan

2008-06-06

Redox modulation of fast inactivation has been described in certain cloned A-type voltage-gated K(+) (Kv) channels in expressing systems, but the effects remain to be demonstrated in native neurons. In this study, we examined the effects of cysteine-specific redox agents on the A-type K(+) currents in acutely dissociated small diameter dorsal root ganglion (DRG) neurons from rats. The fast inactivation of most A-type currents was markedly removed or slowed by the oxidizing agents 2,2'-dithio-bis(5-nitropyridine) (DTBNP) and chloramine-T. Dithiothreitol, a reducing agent for the disulfide bond, restored the inactivation. These results demonstrated that native A-type K(+) channels, probably Kv1.4, could switch the roles between inactivating and non-inactivating K(+) channels via redox regulation in pain-sensing DRG neurons. The A-type channels may play a role in adjusting pain sensitivity in response to peripheral redox conditions.

Substituted N-(Biphenyl-4′-yl)methyl (R)-2-Acetamido-3-methoxypropionamides: Potent Anticonvulsants That Affect Frequency (Use) Dependence and Slow Inactivation of Sodium Channels

PubMed Central

2015-01-01

We prepared 13 derivatives of N-(biphenyl-4′-yl)methyl (R)-2-acetamido-3-methoxypropionamide that differed in type and placement of a R-substituent in the terminal aryl unit. We demonstrated that the R-substituent impacted the compound’s whole animal and cellular pharmacological activities. In rodents, select compounds exhibited excellent anticonvulsant activities and protective indices (PI = TD50/ED50) that compared favorably with clinical antiseizure drugs. Compounds with a polar, aprotic R-substituent potently promoted Na+ channel slow inactivation and displayed frequency (use) inhibition of Na+ currents at low micromolar concentrations. The possible advantage of affecting these two pathways to decrease neurological hyperexcitability is discussed. PMID:25004277

Mechanism of Cd2+-coordination during Slow Inactivation in Potassium Channels

PubMed Central

Raghuraman, H.; Cordero-Morales, Julio F.; Jogini, Vishwanath; Pan, Albert C.; Kollewe, Astrid; Roux, Benoît; Perozo, Eduardo

2013-01-01

Summary In K+ channels, rearrangements of the pore outer-vestibule have been associated with C-type inactivation gating. Paradoxically, the crystal structure of Open/C-type inactivated KcsA suggest these movements to be modest in magnitude. Here, we show that under physiological conditions, the KcsA outer-vestibule undergoes relatively large dynamic rearrangements upon inactivation. External Cd2+ enhances the rate of C-type inactivation in an outer-vestibule cysteine mutant (Y82C) via metal-bridge formation. This effect is not present in a non-inactivating mutant (E71A/Y82C). Tandem dimer and tandem tetramer constructs of equivalent cysteine mutants in KcsA and Shaker K+ channels demonstrate that these Cd2+ metal bridges are formed only between adjacent subunits. This is well supported by molecular dynamics simulations. Based on the crystal structure of Cd2+-bound Y82C-KcsA in the closed state, together with EPR distance measurements in the KcsA outer-vestibule, we suggest that subunits must dynamically come in close proximity as the channels undergo inactivation. PMID:22771214

KChIP2 genotype dependence of transient outward current (Ito) properties in cardiomyocytes isolated from male and female mice

PubMed Central

Waldschmidt, Lara; Junkereit, Vera; Bähring, Robert

2017-01-01

The transient outward current (Ito) in cardiomyocytes is largely mediated by Kv4 channels associated with Kv Channel Interacting Protein 2 (KChIP2). A knockout model has documented the critical role of KChIP2 in Ito expression. The present study was conducted to characterize in both sexes the dependence of Ito properties, including current magnitude, inactivation kinetics, recovery from inactivation and voltage dependence of inactivation, on the number of functional KChIP2 alleles. For this purpose we performed whole-cell patch-clamp experiments on isolated left ventricular cardiomyocytes from male and female mice which had different KChIP2 genotypes; i.e., wild-type (KChIP2+/+), heterozygous knockout (KChIP2+/-) or complete knockout of KChIP2 (KChIP2-/-). We found in both sexes a KChIP2 gene dosage effect (i.e., a proportionality between number of alleles and phenotype) on Ito magnitude, however, concerning other Ito properties, KChIP2+/- resembled KChIP2+/+. Only in the total absence of KChIP2 (KChIP2-/-) we observed a slowing of Ito kinetics, a slowing of recovery from inactivation and a negative shift of a portion of the voltage dependence of inactivation. In a minor fraction of KChIP2-/- myocytes Ito was completely lost. The distinct KChIP2 genotype dependences of Ito magnitude and inactivation kinetics, respectively, seen in cardiomyocytes were reproduced with two-electrode voltage-clamp experiments on Xenopus oocytes expressing Kv4.2 and different amounts of KChIP2. Our results corroborate the critical role of KChIP2 in controlling Ito properties. They demonstrate that the Kv4.2/KChIP2 interaction in cardiomyocytes is highly dynamic, with a clear KChIP2 gene dosage effect on Kv4 channel surface expression but not on inactivation gating. PMID:28141821

KChIP2 genotype dependence of transient outward current (Ito) properties in cardiomyocytes isolated from male and female mice.

PubMed

Waldschmidt, Lara; Junkereit, Vera; Bähring, Robert

2017-01-01

The transient outward current (Ito) in cardiomyocytes is largely mediated by Kv4 channels associated with Kv Channel Interacting Protein 2 (KChIP2). A knockout model has documented the critical role of KChIP2 in Ito expression. The present study was conducted to characterize in both sexes the dependence of Ito properties, including current magnitude, inactivation kinetics, recovery from inactivation and voltage dependence of inactivation, on the number of functional KChIP2 alleles. For this purpose we performed whole-cell patch-clamp experiments on isolated left ventricular cardiomyocytes from male and female mice which had different KChIP2 genotypes; i.e., wild-type (KChIP2+/+), heterozygous knockout (KChIP2+/-) or complete knockout of KChIP2 (KChIP2-/-). We found in both sexes a KChIP2 gene dosage effect (i.e., a proportionality between number of alleles and phenotype) on Ito magnitude, however, concerning other Ito properties, KChIP2+/- resembled KChIP2+/+. Only in the total absence of KChIP2 (KChIP2-/-) we observed a slowing of Ito kinetics, a slowing of recovery from inactivation and a negative shift of a portion of the voltage dependence of inactivation. In a minor fraction of KChIP2-/- myocytes Ito was completely lost. The distinct KChIP2 genotype dependences of Ito magnitude and inactivation kinetics, respectively, seen in cardiomyocytes were reproduced with two-electrode voltage-clamp experiments on Xenopus oocytes expressing Kv4.2 and different amounts of KChIP2. Our results corroborate the critical role of KChIP2 in controlling Ito properties. They demonstrate that the Kv4.2/KChIP2 interaction in cardiomyocytes is highly dynamic, with a clear KChIP2 gene dosage effect on Kv4 channel surface expression but not on inactivation gating.

Modulation of neuronal sodium channels by the sea anemone peptide BDS-I

PubMed Central

Liu, Pin; Jo, Sooyeon

2012-01-01

Blood-depressing substance I (BDS-I), a 43 amino-acid peptide from sea anemone venom, is used as a specific inhibitor of Kv3-family potassium channels. We found that BDS-I acts with even higher potency to modulate specific types of voltage-dependent sodium channels. In rat dorsal root ganglion (DRG) neurons, 3 μM BDS-I strongly enhanced tetrodotoxin (TTX)-sensitive sodium current but weakly inhibited TTX-resistant sodium current. In rat superior cervical ganglion (SCG) neurons, which express only TTX-sensitive sodium current, BDS-I enhanced current elicited by small depolarizations and slowed decay of currents at all voltages (EC50 ∼ 300 nM). BDS-I acted with exceptionally high potency and efficacy on cloned human Nav1.7 channels, slowing inactivation by 6-fold, with an EC50 of approximately 3 nM. BDS-I also slowed inactivation of sodium currents in N1E-115 neuroblastoma cells (mainly from Nav1.3 channels), with an EC50 ∼ 600 nM. In hippocampal CA3 pyramidal neurons (mouse) and cerebellar Purkinje neurons (mouse and rat), BDS-I had only small effects on current decay (slowing inactivation by 20–50%), suggesting relatively weak sensitivity of Nav1.1 and Nav1.6 channels. The biggest effect of BDS-I in central neurons was to enhance resurgent current in Purkinje neurons, an effect reflected in enhancement of sodium current during the repolarization phase of Purkinje neuron action potentials. Overall, these results show that BDS-I acts to modulate sodium channel gating in a manner similar to previously known neurotoxin receptor site 3 anemone toxins but with different isoform sensitivity. Most notably, BDS-I acts with very high potency on human Nav1.7 channels. PMID:22442564

THE ACTION OF EXTREME COLD ON LEUKEMIC CELLS OF MICE

PubMed Central

Breedis, Charles

1942-01-01

Suspensions of leukemic cells of mice from three different strains of leukemia were subjected to rapid or slow freezing and rapid or slow thawing. Suspensions rapidly frozen to –196°C. were in all cases innocuous, whereas those frozen slowly were capable of transmitting leukemia. The infectivity of slowly frozen material varied from an estimated 0.0001 per cent to 1 per cent of that of fresh material, and this figure probably represents the percentage of surviving leukemic cells. Particles of spleen and lymph node reacted to slow and rapid freezing in the same manner as suspensions prepared from them. For one of the strains rapid thawing was less injurious than slow thawing; for the other two the rate of thawing seemed to be immaterial. Infectivity was equally well preserved after freezing to –21°C. whether freezing occurred spontaneously after supercooling or was initiated near the freezing point by inoculation with ice, or whether thawing was slow or rapid. Suspensions already slowly frozen at temperatures of –2° or lower, whether spontaneously or by inoculation with ice, could no longer be completely inactivated by subsequent rapid cooling to –196°C. Unfrozen suspensions initially above the freezing point or supercooled to –2°C. or –8°C. and then rapidly cooled to –196°C. were inactivated. This protective action of previous slow freezing was most marked when the initial temperature of the frozen suspension was –15°C. or lower; when it was –2°C. protection was barely detected. These observations indicate that the changes which are peculiar to rapid freezing alone and lead to complete inactivation take place during rapid transition from the liquid to the solid state, in a range of temperature lying between –15°C. and the freezing point. Temperature measurements carried out in this range showed that suspensions were about equally infections whether the temperature at their centers dropped from 0°C. to –15°C. in 30 minutes or in 1 minute; when the drop occurred in 12 seconds or less, the suspensions became innocuous. PMID:19871231

Knockout of the BK β2 subunit abolishes inactivation of BK currents in mouse adrenal chromaffin cells and results in slow-wave burst activity

PubMed Central

Martinez-Espinosa, Pedro L.; Yang, Chengtao; Gonzalez-Perez, Vivian; Xia, Xiao-Ming

2014-01-01

Rat and mouse adrenal medullary chromaffin cells (CCs) express an inactivating BK current. This inactivation is thought to arise from the assembly of up to four β2 auxiliary subunits (encoded by the kcnmb2 gene) with a tetramer of pore-forming Slo1 α subunits. Although the physiological consequences of inactivation remain unclear, differences in depolarization-evoked firing among CCs have been proposed to arise from the ability of β2 subunits to shift the range of BK channel activation. To investigate the role of BK channels containing β2 subunits, we generated mice in which the gene encoding β2 was deleted (β2 knockout [KO]). Comparison of proteins from wild-type (WT) and β2 KO mice allowed unambiguous demonstration of the presence of β2 subunit in various tissues and its coassembly with the Slo1 α subunit. We compared current properties and cell firing properties of WT and β2 KO CCs in slices and found that β2 KO abolished inactivation, slowed action potential (AP) repolarization, and, during constant current injection, decreased AP firing. These results support the idea that the β2-mediated shift of the BK channel activation range affects repetitive firing and AP properties. Unexpectedly, CCs from β2 KO mice show an increased tendency toward spontaneous burst firing, suggesting that the particular properties of BK channels in the absence of β2 subunits may predispose to burst firing. PMID:25267913

Kinetics of veratridine action on Na channels of skeletal muscle

PubMed Central

Sutro, JB

1986-01-01

Veratridine bath-applied to frog muscle makes inactivation of INa incomplete during a depolarizing voltage-clamp pulse and leads to a persistent veratridine-induced Na tail current. During repetitive depolarizations, the size of successive tail currents grows to a plateau and then gradually decreases. When pulsing is stopped, the tail current declines to zero with a time constant of approximately 3 s. Higher rates of stimulation result in a faster build-up of the tail current and a larger maximum value. I propose that veratridine binds only to open channels and, when bound, prevents normal fast inactivation and rapid shutting of the channel on return to rest. Veratridine-modified channels are also subject to a "slow" inactivation during long depolarizations or extended pulse trains. At rest, veratridine unbinds with a time constant of approximately 3 s. Three tests confirm these hypotheses: (a) the time course of the development of veratridine-induced tail currents parallels a running time integral of gNa during the pulse; (b) inactivating prepulses reduce the ability to evoke tails, and the voltage dependence of this reduction parallels the voltage dependence of h infinity; (c) chloramine-T, N-bromoacetamide, and scorpion toxin, agents that decrease inactivation in Na channels, each greatly enhance the tail currents and alter the time course of the appearance of the tails as predicted by the hypothesis. Veratridine-modified channels shut during hyperpolarizations from -90 mV and reopen on repolarization to -90 mV, a process that resembles normal activation gating. Veratridine appears to bind more rapidly during larger depolarizations. PMID:2419478

THE INACTIVATION OF PENICILLINS F, G, K, AND X BY HUMAN AND RABBIT SERUM

PubMed Central

Eagle, Harry

1947-01-01

1. Penicillins F, G, K, and X were all inactivated by human and rabbit serum, but two qualitatively distinct mechanisms were apparently involved. 2. One was a slow inactivation of all four penicillins by a relatively thermostable serum component which was not demonstrably affected by heating for 60 minutes at 56°C. (a) In both human and rabbit serum this general inactivation of penicillin behaved like a pseudo first order reaction, with a velocity constant of 0.05–0.07 for penicillin X, and 0.09–0.11 for penicillins F and G. (b) The percentage of penicillins F, G, and X inactivated per hour was independent of their concentration over the range 0.4 to 50 micrograms per cc., averaging 9.5, 10, and 6.5 per cent, respectively, in human serum, and 9,8.5, and 5 per cent in rabbit serum. (c) The rate of inactivation varied linearly with the concentration of the serum factor. (d) Penicillin X was consistently and significantly less susceptible to inactivation than any of the other penicillins. Although minor differences were observed between F and G, these were not consistent, and are of questionable significance. 3. Superimposed on this slow inactivation of penicillins F, G, K, and X by a thermostable serum component was a much faster inactivation observed only with penicillin K. (a) In both rabbit and human serum, the serum factor responsible for this inactivation was highly thermolabile, and was almost completely destroyed within 5 minutes at 56°C., leaving only a thermostable component, not affected by further heating. (b) The inactivation of K by this thermolabile component was not a first order reaction, but varied with the concentration of both serum and penicillin. At high concentrations of K, the rate of inactivation due to the thermolabile factor was negligible, and penicillin K was destroyed no more rapidly than F, G, or X. The rate of inactivation increased as the concentration of penicillin was reduced. At penicillin K concentrations of 50, 10, 2, and 0.4 micrograms per cc., the hourly destruction in rabbit serum averaged 10, 16, 21, and 54 per cent. The corresponding figures in human serum were 10, 11, 14, and 54 per cent. The reservations entailed by the large serum error at the lower concentrations of penicillin are discussed in the text. 4. The temperature coefficient for the inactivation of penicillin K by fresh human or rabbit serum was 2.5 for each 10°C. No significant inactivation was observed in 24 hours at 20°C.; and this was true also of penicillins F, G, and X. 5. Heparinized plasma was just as active as serum, washed red blood cells had no effect, and the activity of whole blood was referable to its plasma content. 6. The nature of the serum factors responsible for these two types of penicillin inactivation are under present study. 7. The urinary excretion of penicillin is so rapid that the slow destruction of penicillins F, G, and X in the circulating blood as here described is of secondary significance therapeutically. It nevertheless must contribute to their rapid disappearance from the blood; and the fact that X is inactivated more slowly than either F or G could be reflected in higher and more sustained blood levels than are afforded by the latter two species. There are some reports that such is the case (15–17), and the following paper provides further evidence for the superiority of penicillin X in this respect over the other species so far studied. The serum inactivation of penicillin K, at a rate which increases as its concentration falls, should be reflected in significantly lower and more evanescent blood levels than are observed with penicillins F, G, or X. As will be discussed in the following paper, this has been found to be the case, and provides a simple explanation for its paradoxically low therapeutic activity in vivo (8–11). PMID:19871604

Analgesic ineffectiveness of lacosamide after spinal nerve ligation and its sodium channel activity in injured neurons.

PubMed

Hagenacker, T; Schäfer, N; Büsselberg, D; Schäfers, M

2013-07-01

Lacosamide is a novel anti-epileptic drug that enhances the slow- and not fast-inactivating state of voltage-gated sodium channels. Lacosamide has demonstrated analgesic efficacy in several animal studies but preclinical studies on neuropathic pain models are rare, and recent clinical trials showed no superior analgesic effects. Here, we examine whether an acute or chronic administration of lacosamide (3-60 mg/kg, i.p.) attenuates pain behaviour induced by spinal nerve ligation (SNL). To validate the inhibitory efficacy of lacosamide on voltage-gated sodium channels, sodium currents in naïve and SNL-injured dorsal root ganglion (DRG) neurons were recorded using whole-cell patch clamping. Lacosamide only marginally attenuated thermal hyperalgesia, but not tactile allodynia when applied once 7 or 14 days after SNL and showed no analgesic effect when applied daily for 19 days. In naïve neurons, 100 μmol/L lacosamide inhibited sodium channel currents by 58% and enhanced the slow inactivation (87% for lacosamide vs. 47% for control). In contrast, lacosamide inhibited sodium currents in injured DRG neurons by only 15%, while the effects on slow inactivation were diminished. Isolated currents from the NaV 1.8 channel subtype were only marginally changed by lacosamide. The reduced effectiveness of lacosamide on voltage-gated sodium channel currents in injured DRG neurons may contribute to the reduced analgesic effect observed for the SNL model. © 2012 European Federation of International Association for the Study of Pain Chapters.

Inactivation of Cg10062, a cis-3-chloroacrylic acid dehalogenase homologue in Corynebacterium glutamicum, by (R)- and (S)-oxirane-2-carboxylate: analysis and implications.

PubMed

Robertson, Brooklyn A; Johnson, William H; Lo, Herng-Hsiang; Whitman, Christian P

2008-08-19

( R)- and ( S)-oxirane-2-carboxylate were determined to be active site-directed irreversible inhibitors of the cis-3-chloroacrylic acid dehalogenase ( cis-CaaD) homologue Cg10062 found in Corynebacterium glutamicum. Kinetic analysis indicates that the ( R) enantiomer binds more tightly and is the more potent inhibitor, likely reflecting more favorable interactions with active site residues. Pro-1 is the sole site of covalent modification by the ( R) and ( S) enantiomers. Pro-1, Arg-70, Arg-73, and Glu-114, previously identified as catalytic residues in Cg10062, have also been implicated in the inactivation mechanism. Pro-1, Arg-70, and Arg-73 are essential residues for the process as indicated by the observation that the enzymes with the corresponding alanine mutations are not covalently modified by either enantiomer. The E114Q mutant slows covalent modification of Cg10062 but does not prevent it. The results are comparable to those found for the irreversible inactivation of cis-CaaD by ( R)-oxirane-2-carboxylate with two important distinctions: the alkylation of cis-CaaD is stereospecific, and Glu-114 does not take part in the cis-CaaD inactivation mechanism. Cg10062 exhibits low-level cis-CaaD and trans-3-chloroacrylic acid dehalogenase (CaaD) activities, with the cis-CaaD activity predominating. Hence, the preference of Cg10062 for the cis isomer correlates with the observation that the ( R) enantiomer is the more potent inactivator. Moreover, the factors responsible for the relaxed substrate specificity of Cg10062 may account for the stereoselective inactivation by the enantiomeric epoxides. Delineation of these factors would provide a more complete picture of the substrate specificity determinants for cis-CaaD. This study represents an important step toward this goal by setting the stage for a crystallographic analysis of inactivated Cg10062.

Adaptation of primate vestibuloocular reflex to altered peripheral vestibular inputs. I. Frequency-specific recovery of horizontal VOR after inactivation of the lateral semicircular canals

NASA Technical Reports Server (NTRS)

Angelaki, D. E.; Hess, B. J.; Arai, Y.; Suzuki, J.

1996-01-01

1. The adaptive plasticity of the vestibuloocular reflex (VOR) following a selective lesion of the peripheral vestibular organs was investigated in rhesus monkeys whose lateral semicircular canals were inactivated by plugging of the canal lumen in both ears. Gain and phase of horizontal, vertical, and torsional slow-phase eye velocity were determined from three-dimensional eye movement recordings obtained acutely after the plugging operation, as well as in regular intervals up to 10 mo later. 2. Acutely after plugging, horizontal VOR was minimal during yaw rotation with gains of < 0.1 at all frequencies. Horizontal VOR gain gradually increased over time, reaching gains of 0.4-0.5 for yaw oscillations at 1.1 Hz approximately 5 mo after lateral canal inactivation. This response recovery was strongly frequency dependent: horizontal VOR gains were largest at the highest frequency tested and progressively decreased for lower frequencies. Below approximately 0.1 Hz, no consistent horizontal VOR could be elicited even 10 mo after plugging. 3. The frequency-dependent changes in gain paralleled changes in horizontal VOR phase. Below approximately 0.1-0.05 Hz large phase leads were present, similarly as in semicircular canal primary afferents. Smaller phase leads were also present at higher frequencies, particularly at 1.1 Hz (the highest frequency tested). 4. Consistent with the afferent-like dynamics of the adapted horizontal VOR, per- and postrotatory horizontal responses to constant-velocity yaw rotations were short lasting. Time constants of the slow-phase eye velocity envelope of the horizontal postrotatory nystagmus were approximately 2 s. Nonetheless, a consistent horizontal optokinetic afternystagmus was evoked in plugged animals. 5. A torsional component that was absent in intact animals was consistently present during yaw rotation acutely after lateral canal inactivation and remained approximately constant thereafter. The frequency response characteristics of this torsional component resembled those of the adapted horizontal slow-phase responses: gain decreased and large phase leads were introduced at frequencies below approximately 0.05-0.1 Hz. Torsional responses elicited by roll oscillations in supine position, on the other hand, were indistinguishable in their dynamics from intact animals. No consistent vertical nystagmus was elicited during yaw rotation. 6. Our results show that there is a slow, frequency-specific recovery of horizontal VOR after selective inactivation of the lateral semicircular canals. Both the spatial organization and the dynamic properties of the adapted VOR responses are distinctly different from responses in intact animals, suggesting complex changes in the underlying vestibuloocular circuitry.

Null mutation in the rhodopsin kinase gene slows recovery kinetics of rod and cone phototransduction in man

PubMed Central

Cideciyan, Artur V.; Zhao, Xinyu; Nielsen, Lori; Khani, Shahrokh C.; Jacobson, Samuel G.; Palczewski, Krzysztof

1998-01-01

Rhodopsin kinase (RK), a specialized G-protein-coupled receptor kinase expressed in retina, is involved in quenching of light-induced signal transduction in photoreceptors. The role of RK in recovery after photoactivation has been explored in vitro and in vivo experimentally but has not been specifically defined in humans. We investigated the effects on human vision of a mutation in the RK gene causing Oguchi disease, a recessively inherited retinopathy. In vitro experiments demonstrated that the mutation, a deletion of exon 5, abolishes the enzymatic activity of RK and is likely a null. Both a homozygote and heterozygote with this RK mutation had recovery phase abnormalities of rod-isolated photoresponses by electroretinography (ERG); photoactivation was normal. Kinetics of rod bleaching adaptation by psychophysics were dramatically slowed in the homozygote but normal final thresholds were attained. Light adaptation was normal at low backgrounds but became abnormal at higher backgrounds. A slight slowing of cone deactivation kinetics in the homozygote was detected by ERG. Cone bleaching adaptation and background adaptation were normal. In this human in vivo condition without a functional RK and probable lack of phosphorylation and arrestin binding to activated rhodopsin, reduction of photolyzed chromophore and regeneration processes with 11-cis-retinal probably constitute the sole pathway for recovery of rod sensitivity. The role of RK in rods would thus be to accelerate inactivation of activated rhodopsin molecules that in concert with regeneration leads to the normal rate of recovery of sensitivity. Cones may rely mainly on regeneration for the inactivation of photolyzed visual pigment, but RK also contributes to cone recovery. PMID:9419375

Somatic mutations of the histone H3K27 demethylase gene UTX in human cancer.

PubMed

van Haaften, Gijs; Dalgliesh, Gillian L; Davies, Helen; Chen, Lina; Bignell, Graham; Greenman, Chris; Edkins, Sarah; Hardy, Claire; O'Meara, Sarah; Teague, Jon; Butler, Adam; Hinton, Jonathan; Latimer, Calli; Andrews, Jenny; Barthorpe, Syd; Beare, Dave; Buck, Gemma; Campbell, Peter J; Cole, Jennifer; Forbes, Simon; Jia, Mingming; Jones, David; Kok, Chai Yin; Leroy, Catherine; Lin, Meng-Lay; McBride, David J; Maddison, Mark; Maquire, Simon; McLay, Kirsten; Menzies, Andrew; Mironenko, Tatiana; Mulderrig, Lee; Mudie, Laura; Pleasance, Erin; Shepherd, Rebecca; Smith, Raffaella; Stebbings, Lucy; Stephens, Philip; Tang, Gurpreet; Tarpey, Patrick S; Turner, Rachel; Turrell, Kelly; Varian, Jennifer; West, Sofie; Widaa, Sara; Wray, Paul; Collins, V Peter; Ichimura, Koichi; Law, Simon; Wong, John; Yuen, Siu Tsan; Leung, Suet Yi; Tonon, Giovanni; DePinho, Ronald A; Tai, Yu-Tzu; Anderson, Kenneth C; Kahnoski, Richard J; Massie, Aaron; Khoo, Sok Kean; Teh, Bin Tean; Stratton, Michael R; Futreal, P Andrew

2009-05-01

Somatically acquired epigenetic changes are present in many cancers. Epigenetic regulation is maintained via post-translational modifications of core histones. Here, we describe inactivating somatic mutations in the histone lysine demethylase gene UTX, pointing to histone H3 lysine methylation deregulation in multiple tumor types. UTX reintroduction into cancer cells with inactivating UTX mutations resulted in slowing of proliferation and marked transcriptional changes. These data identify UTX as a new human cancer gene.

Somatic mutations of the histone H3K27 demethylase, UTX, in human cancer

PubMed Central

van Haaften, Gijs; Dalgliesh, Gillian L; Davies, Helen; Chen, Lina; Bignell, Graham; Greenman, Chris; Edkins, Sarah; Hardy, Claire; O’Meara, Sarah; Teague, Jon; Butler, Adam; Hinton, Jonathan; Latimer, Calli; Andrews, Jenny; Barthorpe, Syd; Beare, Dave; Buck, Gemma; Campbell, Peter J; Cole, Jennifer; Dunmore, Rebecca; Forbes, Simon; Jia, Mingming; Jones, David; Kok, Chai Yin; Leroy, Catherine; Lin, Meng-Lay; McBride, David J; Maddison, Mark; Maquire, Simon; McLay, Kirsten; Menzies, Andrew; Mironenko, Tatiana; Lee, Mulderrig; Mudie, Laura; Pleasance, Erin; Shepherd, Rebecca; Smith, Raffaella; Stebbings, Lucy; Stephens, Philip; Tang, Gurpreet; Tarpey, Patrick S; Turner, Rachel; Turrell, Kelly; Varian, Jennifer; West, Sofie; Widaa, Sara; Wray, Paul; Collins, V Peter; Ichimura, Koichi; Law, Simon; Wong, John; Yuen, Siu Tsan; Leung, Suet Yi; Tonon, Giovanni; DePinho, Ronald A; Tai, Yu-Tzu; Anderson, Kenneth C; Kahnoski, Richard J.; Massie, Aaron; Khoo, Sok Kean; Teh, Bin Tean; Stratton, Michael R; Futreal, P Andrew

2010-01-01

Somatically acquired epigenetic changes are present in many cancers. Epigenetic regulation is maintained via post-translational modifications of core histones. Here, we describe inactivating somatic mutations in the histone lysine demethylase, UTX, pointing to histone H3 lysine methylation deregulation in multiple tumour types. UTX reintroduction into cancer cells with inactivating UTX mutations resulted in slowing of proliferation and marked transcriptional changes. These data identify UTX as a new human cancer gene. PMID:19330029

A novel Ile1455Thr variant in the skeletal muscle sodium channel alpha-subunit in a patient with a severe adult-onset proximal myopathy with electrical myotonia and a patient with mild paramyotonia phenotype.

PubMed

Bednarz, Marcin; Stunnenberg, Bas C; Kusters, Benno; Kamsteeg, Erik-Jan; Saris, Christiaan G; Groome, James; Winston, Vern; Meola, Giovanni; Jurkat-Rott, Karin; Voermans, Nicol C

2017-02-01

In sodium channelopathies, a severe fixed myopathy caused by a dominant mutation is rare. We describe two unrelated patients with a novel variant, p.Ile1455Thr, with phenotypes of paramyotonia in one case and fixed proximal myopathy with latent myotonia in another. In-vitro whole cell patch-clamp studies show that the mutation slows inactivation and accelerates recovery, in line with other paramyotonia variants with destabilized fast inactivation as pathomechanism. Additionally, p.IleI1455 causes a loss-of-function by reduced membrane insertion, right-shift of activation, and slowed kinetics. Molecular dynamics simulations comparing wild type and mutant Nav1.4 showed that threonine substitution hindered D4S4 mobility in response to membrane depolarization, consistent with effects of the mutation on channel inactivation. The fixed myopathy is likely to be associated to gain-of-function leading to sodium accumulation, regional edema, T-tubular swelling and mitochondrial stress. A possible contribution of the loss-of-function features towards myotonia and myopathy is discussed. Copyright © 2016. Published by Elsevier B.V.

Mesenchymal chemotaxis requires selective inactivation of Myosin II at the leading edge via a non-canonical PLCγ/PKCα pathway

PubMed Central

Asokan, Sreeja B.; Johnson, Heath E.; Rahman, Anisur; King, Samantha J.; Rotty, Jeremy D.; Lebedeva, Irina P.; Haugh, Jason M.; Bear, James E.

2014-01-01

Summary Chemotaxis, migration towards soluble chemical cues, is critical for processes such as wound healing and immune surveillance, and is exhibited by various cell types from rapidly-migrating leukocytes to slow-moving mesenchymal cells. To interrogate the mechanisms involved in mesenchymal chemotaxis, we observed cell migration in microfluidic chambers that generate stable gradients of the chemoattractant PDGF. Surprisingly, we found that pathways implicated in amoeboid chemotaxis, such as PI3K and mTOR signaling, are dispensable for chemotaxis to PDGF. Instead, we find that local inactivation of Myosin IIA, through a non-canonical Ser1/2 phosphorylation of the regulatory light chain, is essential. This site is phosphorylated by PKCα, which is activated by an intracellular gradient of diacylglycerol generated by PLCγ. Using a combination of TIRF imaging and gradients of activators/inhibitors in the microfluidic chambers, we demonstrate that this signaling pathway and subsequent inhibition of Myosin II activity at the leading edge is required for mesenchymal chemotaxis. PMID:25482883

Butyrylcholinesterase for protection from organophosphorus poisons; catalytic complexities and hysteretic behavior

PubMed Central

Masson, Patrick; Lockridge, Oksana

2009-01-01

Butyrylcholinesterase is a promiscuous enzyme that displays complex kinetic behavior. It is toxicologically important because it detoxifies organophosphorus poisons (OP) by making a covalent bond with the OP. The OP and the butyrylcholinesterase are both inactivated in the process. Inactivation of butyrylcholinesterase has no adverse effects. However inactivation of acetylcholinesterase in nerve synapses can be lethal. OP-inhibited butyrylcholinesterase and acetylcholinesterase can be reactivated with oximes provided the OP has not aged. Strategies for preventing the toxicity of OP include a) treatment with an OP scavenger, b) reaction of nonaged enzyme with oximes, c) reactivation of aged enzyme, d) slowing down aging with peripheral site ligands, and e) design of mutants that rapidly hydrolyze OP. Option (a) has progressed through phase I clinical trials with human butyrylcholinesterase. Option (b) is in routine clinical use. The others are at the basic research level. Butyrylcholinesterase displays complex kinetic behavior including activation by positively charged esters, ability to hydrolyze amides, and a lag time (hysteresis) preceding hydrolysis of benzoylcholine and N-methyl indoxyl acetate. Mass spectrometry has identified new OP binding motifs on tyrosine and lysine in proteins that have no active site serine. It is proposed, but not yet proven, that low dose exposure involves OP modification of proteins that have no active site serine. PMID:20004171

Endotoxin inactivation by selected drinking water treatment oxidants.

PubMed

Anderson, William B; Mayfield, Colin I; Dixon, D George; Huck, Peter M

2003-11-01

Exposure to endotoxins in treated drinking water can occur through ingestion, dermal abrasions, inhalation of water vapor, intravenous injection or during dialysis. While the risks associated with endotoxin ingestion and entry through dermal abrasions are not well quantified, adverse effects of intravenous injection and dialysis are well known and some studies indicate that inhalation of moisture-laden air may impact human health. This study quantifies the inactivation of endotoxin derived from Escherichia coli O55:B5 by three substances used either as disinfectants or oxidants in drinking water treatment: chlorine, monochloramine and potassium permanganate. Inactivation rates were found to be 1.4, 1.0 and 0.7 endotoxin units (EU)/mL h, for free chlorine, potassium permanganate and monochloramine, respectively. These rates are relatively slow given that contact times in drinking water distribution systems are typically less than 48 h. While small amounts of endotoxin may be removed by oxidation the observed removals are much less than those provided by physical removal processes. The significance of this finding is important for dialysis considerations but is as yet unclear with regard to inhalation, as the risk of inhaling sufficient quantities of endotoxin-containing aerosolized water droplets to adversely affect human health has not yet been adequately quantified.

Modeling removal of bacteriophages MS2 and PRD1 by dune recharge at Castricum, Netherlands

NASA Astrophysics Data System (ADS)

Schijven, Jack F.; Hoogenboezem, Wim; Hassanizadeh, S. Majid; Peters, Jos H.

1999-04-01

Removal of model viruses by dune recharge was studied at a field site in the dune area of Castricum, Netherlands. Recharge water was dosed with bacteriophages MS2 and PRD1 for 11 days at a constant concentration in a 10- by 15-m compartment that was isolated in a recharge basin. Breakthrough was monitored for 120 days at six wells with their screens along a flow line. Concentrations of both phages were reduced about 3 log10 within the first 2.4 m and another 5 log10 in a linear fashion within the following 27 m. A model accounting for one-site kinetic attachment as well as first-order inactivation was employed to simulate the bacteriophage breakthrough curves. The major removal process was found to be attachment of the bacteriophages. Detachment was very slow. After passage of the pulse of dosed bacteriophages, there was a long tail whose slope corresponds to the inactivation rate coefficient of 0.07-0.09 day-1 for attached bacteriophages. The end of the rising and the start of the declining limbs of the breakthrough curves could not be simulated completely, probably because of an as yet unknown process.

How does KCNE1 regulate the Kv7.1 potassium channel? Model-structure, mutations, and dynamics of the Kv7.1-KCNE1 complex.

PubMed

Gofman, Yana; Shats, Simona; Attali, Bernard; Haliloglu, Turkan; Ben-Tal, Nir

2012-08-08

The voltage-gated potassium channel Kv7.1 and its auxiliary subunit KCNE1 are expressed in the heart and give rise to the major repolarization current. The interaction of Kv7.1 with the single transmembrane helix of KCNE1 considerably slows channel activation and deactivation, raises single-channel conductance, and prevents slow voltage-dependent inactivation. We built a Kv7.1-KCNE1 model-structure. The model-structure agrees with previous disulfide mapping studies and enables us to derive molecular interpretations of electrophysiological recordings that we obtained for two KCNE1 mutations. An elastic network analysis of Kv7.1 fluctuations in the presence and absence of KCNE1 suggests a mechanistic perspective on the known effects of KCNE1 on Kv7.1 function: slow deactivation is attributed to the low mobility of the voltage-sensor domains upon KCNE1 binding, abolishment of voltage-dependent inactivation could result from decreased fluctuations in the external vestibule, and amalgamation of the fluctuations in the pore region is associated with enhanced ion conductivity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Novel carbapenem derivative SF2103A: studies on the mode of beta-lactamase inactivation.

PubMed Central

Yamaguchi, A; Hirata, T; Sawai, T

1984-01-01

A novel carbapenem, SF2103A, is a strong inhibitor of various types of beta-lactamase. Equimolar concentrations of SF2103A completely inactivated the cephalosporinases of Proteus vulgaris and Citrobacter freundii and type Ib and type II penicillinases mediated by R plasmids in a progressive manner. The inactivation of the two penicillinases and P. vulgaris cephalosporinase was apparently irreversible; however, when the inactivated enzymes were separated from excess SF2103A by gel filtration, they showed very slow reactivation. The hydrolysis of SF2103A by these three beta-lactamases was below the limit of detection. It is concluded that SF2103A acts as a tight-binding competitive inhibitor for the penicillinases and P. vulgaris cephalosporinase. In contrast, the inactivation of C. freundii cephalosporinase by SF2103A was evidently reversible. The rate constant of reactivation of the enzyme was compatible with the turnover rate of the enzyme in the steady state of SF2103A hydrolysis. Thus, SF2103A simply acts as a poor substrate for C. freundii cephalosporinase. PMID:6372682

Distinguishing the rates of gene activation from phenotypic variations.

PubMed

Chen, Ye; Lv, Cheng; Li, Fangting; Li, Tiejun

2015-06-18

Stochastic genetic switching driven by intrinsic noise is an important process in gene expression. When the rates of gene activation/inactivation are relatively slow, fast, or medium compared with the synthesis/degradation rates of mRNAs and proteins, the variability of protein and mRNA levels may exhibit very different dynamical patterns. It is desirable to provide a systematic approach to identify their key dynamical features in different regimes, aiming at distinguishing which regime a considered gene regulatory network is in from their phenotypic variations. We studied a gene expression model with positive feedbacks when genetic switching rates vary over a wide range. With the goal of providing a method to distinguish the regime of the switching rates, we first focus on understanding the essential dynamics of gene expression system in different cases. In the regime of slow switching rates, we found that the effective dynamics can be reduced to independent evolutions on two separate layers corresponding to gene activation and inactivation states, and the transitions between two layers are rare events, after which the system goes mainly along deterministic ODE trajectories on a particular layer to reach new steady states. The energy landscape in this regime can be well approximated by using Gaussian mixture model. In the regime of intermediate switching rates, we analyzed the mean switching time to investigate the stability of the system in different parameter ranges. We also discussed the case of fast switching rates from the viewpoint of transition state theory. Based on the obtained results, we made a proposal to distinguish these three regimes in a simulation experiment. We identified the intermediate regime from the fact that the strength of cellular memory is lower than the other two cases, and the fast and slow regimes can be distinguished by their different perturbation-response behavior with respect to the switching rates perturbations. We proposed a simulation experiment to distinguish the slow, intermediate and fast regimes, which is the main point of our paper. In order to achieve this goal, we systematically studied the essential dynamics of gene expression system when the switching rates are in different regimes. Our theoretical understanding provides new insights on the gene expression experiments.

Tetrapentylammonium block of chloramine-T and veratridine modified rat brain type IIA sodium channels

PubMed Central

Ghatpande, A S; Rao, S; Sikdar, S K

2001-01-01

Tetrapentylammonium (TPeA) block of rat brain type IIA sodium channel α subunit was studied using whole cell patch clamp. Results indicate that TPeA blocks the inactivating brain sodium channel in a potential and use-dependent manner similar to that of the cardiac sodium channel. Removal of inactivation using chloramine-T (CT) unmasks a time-dependent block by TPeA consistent with slow blocking kinetics. On the other hand, no time dependence is observed when inactivation is abolished by modification with veratridine. TPeA does not bind in a potential-dependent fashion to veratridine-modified channels and does not significantly affect gating of veratridine-modified channels suggesting that high affinity binding of TPeA to the brain sodium channel is lost after veratridine modification. PMID:11309247

Validation of a patch clamp screening protocol that simultaneously measures compound activity in multiple states of the voltage-gated sodium channel Nav1.2.

PubMed

Liu, Yi; Beck, Edward J; Flores, Christopher M

2011-12-01

Hyperactivity of voltage-gated sodium channels underlies, at least in part, a range of pathological states, including pain and epilepsy. Selective blockers of these channels may offer effective treatment of such disorders. Currently employed methods to screen for sodium channel blockers, however, are inadequate to rationally identify mechanistically diverse blockers, limiting the potential range of indications that may be treated by such agents. Here, we describe an improved patch clamp screening assay that increases the mechanistic diversity of sodium channel blockers being identified. Using QPatch HT, a medium-throughput, automated patch clamp system, we tested three common sodium channel blockers (phenytoin, lidocaine, and tetrodotoxin) with distinct mechanistic profiles at Nav1.2. The single-voltage protocol employed in this assay simultaneously measured the compound activity in multiple states, including the slow inactivated state, of the channel. A long compound incubation period (10 s) was introduced during channel inactivation to increase the probability of identifying "slow binders." As such, phenytoin, which preferentially binds with slow kinetics to the fast inactivated state, exhibited significantly higher potency than that obtained from a brief exposure (100 ms) used in typical assays. This assay also successfully detected the use-dependent block of tetrodotoxin, a well-documented property of this molecule yet unobserved in typical patch clamp protocols. These results indicate that the assay described here can increase the likelihood of identification and mechanistic diversity of sodium channel blockers from a primary screen. It can also be used to efficiently guide the in vitro optimization of leads that retain the desired mechanistic properties. © MARY ANN LIEBERT, INC.

Mechanism of Competitive Inhibition and Destabilization of Acidothermus cellulolyticus Endoglucanase 1 by Ionic Liquids.

PubMed

Summers, Samantha R; Sprenger, K G; Pfaendtner, Jim; Marchant, Jan; Summers, Michael F; Kaar, Joel L

2017-12-07

The ability of ionic liquids (ILs) to solubilize cellulose has sparked interest in their use for enzymatic biomass processing. However, this potential is yet to be realized, primarily because ILs inactivate requisite cellulases by mechanisms that are yet to be identified. We used a combination of enzymology, circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular dynamics (MD) methods to investigate the molecular basis for the inactivation of the endocellulase 1 (E1) from Acidothermus cellulolyticus by the imidazolium IL 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). Enzymatic studies revealed that [BMIM][Cl] inactivates E1 in a biphasic manner that involves rapid, reversible inhibition, followed by slow, irreversible deactivation. Backbone NMR signals of the 40.5 kDa E1 were assigned by triple resonance NMR methods, enabling monitoring of residue-specific perturbations. 1 H- 15 N NMR titration experiments revealed that [BMIM][Cl] binds reversibly to the E1 active site, indicating that reversible deactivation is due to competitive inhibition of substrate binding. Prolonged incubation with [BMIM][Cl] led to substantial global changes in the 1 H- 15 N heteronuclear single quantum coherence NMR and CD spectra of E1 indicative of protein denaturation. Notably, weak interactions between [BMIM][Cl] and residues at the termini of several helices were also observed, which, together with MD simulations, suggest that E1 denaturation is promoted by [BMIM][Cl]-induced destabilization of helix capping structures. In addition to identifying determinants of E1 inactivation, our findings establish a molecular framework for engineering cellulases with improved IL compatibility.

Development and Testing of Enhanced Affinity Reagents for Use in Environmental Detection Assays

DTIC Science & Technology

Current affinity reagent development methodologies generally rely on costly and slow antibody production that is based on animal inoculations with...attenuated, inactivated, or surrogate biothreat agents. Recent literature has demonstrated that the de novo computer design of recombinant affinity

Control of the red tide dinoflagellate Cochlodinium polykrikoides by ozone in seawater.

PubMed

Shin, Minjung; Lee, Hye-Jin; Kim, Min Sik; Park, Noh-Back; Lee, Changha

2017-02-01

The inactivation of C. polykrikoides, a red tide dinoflagellate, by ozonation was investigated in seawater by monitoring numbers of viable and total cells. Parameters affecting the inactivation efficacy of C. polykrikoides such as the ozone dose, initial cell concentration, pH, and temperature were examined. The viable cell number rapidly decreased in the initial stage of the reaction (mostly in 1-2 min), whereas the decrease in total cell number was relatively slow and steady. Increasing ozone dose and decreasing initial cell concentration increased the inactivation efficacy of C. polykrikoides, while increasing pH and temperature decreased the cell inactivation efficacy. The addition of humic acid (a promoter for the ozone decomposition) inhibited the inactivation of C. polykrikoides, whereas bicarbonate ion (an inhibitor for the ozone decomposition) accelerated the C. polykrikoides inactivation. Observations regarding the effects of pH, temperature, humic acid, and bicarbonate ion collectively indicate that the inactivation of C. polykrikoides by ozonation is mainly attributed to oxidative cell damages by molecular ozone, rather than by hydroxyl radical, produced during the ozone decomposition. At high ozone dose (e.g., 5 mg/L), hypobromous acid formed by the reaction of bromide with ozone may partially contribute to cell inactivation. The use of ozone of less than 1 mg/L produced 0.75-2.03 μg/L bromate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Closed-state inactivation involving an internal gate in Kv4.1 channels modulates pore blockade by intracellular quaternary ammonium ions

PubMed Central

Fineberg, Jeffrey D.; Szanto, Tibor G.; Panyi, Gyorgy; Covarrubias, Manuel

2016-01-01

Voltage-gated K+ (Kv) channel activation depends on interactions between voltage sensors and an intracellular activation gate that controls access to a central pore cavity. Here, we hypothesize that this gate is additionally responsible for closed-state inactivation (CSI) in Kv4.x channels. These Kv channels undergo CSI by a mechanism that is still poorly understood. To test the hypothesis, we deduced the state of the Kv4.1 channel intracellular gate by exploiting the trap-door paradigm of pore blockade by internally applied quaternary ammonium (QA) ions exhibiting slow blocking kinetics and high-affinity for a blocking site. We found that inactivation gating seemingly traps benzyl-tributylammonium (bTBuA) when it enters the central pore cavity in the open state. However, bTBuA fails to block inactivated Kv4.1 channels, suggesting gated access involving an internal gate. In contrast, bTBuA blockade of a Shaker Kv channel that undergoes open-state P/C-type inactivation exhibits fast onset and recovery inconsistent with bTBuA trapping. Furthermore, the inactivated Shaker Kv channel is readily blocked by bTBuA. We conclude that Kv4.1 closed-state inactivation modulates pore blockade by QA ions in a manner that depends on the state of the internal activation gate. PMID:27502553

The effects of Thalamic Deep Brain Stimulation on speech dynamics in patients with Essential Tremor: An articulographic study.

PubMed

Mücke, Doris; Hermes, Anne; Roettger, Timo B; Becker, Johannes; Niemann, Henrik; Dembek, Till A; Timmermann, Lars; Visser-Vandewalle, Veerle; Fink, Gereon R; Grice, Martine; Barbe, Michael T

2018-01-01

Acoustic studies have revealed that patients with Essential Tremor treated with thalamic Deep Brain Stimulation (DBS) may suffer from speech deterioration in terms of imprecise oral articulation and reduced voicing control. Based on the acoustic signal one cannot infer, however, whether this deterioration is due to a general slowing down of the speech motor system (e.g., a target undershoot of a desired articulatory goal resulting from being too slow) or disturbed coordination (e.g., a target undershoot caused by problems with the relative phasing of articulatory movements). To elucidate this issue further, we here investigated both acoustics and articulatory patterns of the labial and lingual system using Electromagnetic Articulography (EMA) in twelve Essential Tremor patients treated with thalamic DBS and twelve age- and sex-matched controls. By comparing patients with activated (DBS-ON) and inactivated stimulation (DBS-OFF) with control speakers, we show that critical changes in speech dynamics occur on two levels: With inactivated stimulation (DBS-OFF), patients showed coordination problems of the labial and lingual system in terms of articulatory imprecision and slowness. These effects of articulatory discoordination worsened under activated stimulation, accompanied by an additional overall slowing down of the speech motor system. This leads to a poor performance of syllables on the acoustic surface, reflecting an aggravation either of pre-existing cerebellar deficits and/or the affection of the upper motor fibers of the internal capsule.

Gating current studies reveal both intra- and extracellular cation modulation of K+ channel deactivation

PubMed Central

Wang, Zhuren; Zhang, Xue; Fedida, David

1999-01-01

The presence of permeant ions can modulate the rate of gating charge return in wild-type human heart K+ (hKv1.5) channels. Here we employ gating current measurements in a non-conducting mutant, W472F, of the hKv1.5 channel to investigate how different cations can modulate charge return and whether the actions can be specifically localized at the internal as well as the external mouth of the channel pore. Intracellular cations were effective at accelerating charge return in the sequence Cs+ > Rb+ > K+ > Na+ > NMG+. Extracellular cations accelerated charge return with the selectivity sequence Cs+ > Rb+ > Na+ = NMG+. Intracellular and extracellular cation actions were of relatively low affinity. The Kd for preventing slowing of the time constant of the off-gating current decay (τoff) was 20.2 mM for intracellular Cs+ (Csi+) and 358 mM for extracellular Cs+ (Cso+). Both intracellular and extracellular cations can regulate the rate of charge return during deactivation of hKv1.5, but intracellular cations are more effective. We suggest that ion crystal radius is an important determinant of this action, with larger ions preventing slowing more effectively. Important parallels exist with cation-dependent modulation of slow inactivation of ionic currents in this channel. However, further experiments are required to understand the exact relationship between acceleration of charge return and the slowing of inactivation of ionic currents by cations. PMID:10050001

Na+ channel regulation by Ca2+/calmodulin and Ca2+/calmodulin-dependent protein kinase II in guinea-pig ventricular myocytes†

PubMed Central

Aiba, Takeshi; Hesketh, Geoffrey G.; Liu, Ting; Carlisle, Rachael; Villa-Abrille, Maria Celeste; O'Rourke, Brian; Akar, Fadi G.; Tomaselli, Gordon F.

2010-01-01

Aims Calmodulin (CaM) regulates Na+ channel gating through binding to an IQ-like motif in the C-terminus. Ca2+/CaM-dependent protein kinase II (CaMKII) regulates Ca2+ handling, and chronic overactivity of CaMKII is associated with left ventricular hypertrophy and dysfunction and lethal arrhythmias. However, the acute effects of Ca2+/CaM and CaMKII on cardiac Na+ channels are not fully understood. Methods and results Purified NaV1.5–glutathione-S-transferase fusion peptides were phosphorylated in vitro by CaMKII predominantly on the I–II linker. Whole-cell voltage-clamp was used to measure Na+ current (INa) in isolated guinea-pig ventricular myocytes in the absence or presence of CaM or CaMKII in the pipette solution. CaMKII shifted the voltage dependence of Na+ channel availability by ≈+5 mV, hastened recovery from inactivation, decreased entry into intermediate or slow inactivation, and increased persistent (late) current, but did not change INa decay. These CaMKII-induced changes of Na+ channel gating were completely abolished by a specific CaMKII inhibitor, autocamtide-2-related inhibitory peptide (AIP). Ca2+/CaM alone reproduced the CaMKII-induced changes of INa availability and the fraction of channels undergoing slow inactivation, but did not alter recovery from inactivation or the magnitude of the late current. Furthermore, the CaM-induced changes were also completely abolished by AIP. On the other hand, cAMP-dependent protein kinase A inhibitors did not abolish the CaM/CaMKII-induced alterations of INa function. Conclusion Ca2+/CaM and CaMKII have distinct effects on the inactivation phenotype of cardiac Na+ channels. The differences are consistent with CaM-independent effects of CaMKII on cardiac Na+ channel gating. PMID:19797425

Removal of inactivation causes time-invariant sodium current decays

PubMed Central

1988-01-01

The kinetic properties of the closing of Na channels were studied in frog skeletal muscle to obtain information about the dependence of channel closing on the past history of the channel. Channel closing was studied in normal and modified channels. Chloramine-T was used to modify the channels so that inactivation was virtually removed. A series of depolarizing prepulse potentials was used to activate Na channels, and a -140-mV postpulse was used to monitor the closing of the channels. Unmodified channels decay via a biexponential process with time constants of 72 and 534 microseconds at 12 degrees C. The observed time constants do not depend upon the potential used to activate the channels. The contribution of the slow component to the total decay increases as the activating prepulse is lengthened. After inactivation is removed, the biexponential character of the decay is retained, with no change in the magnitude of the time constants. However, increases in the duration of the activating prepulse over the range where the current is maximal 1-75 ms) produce identical biexponential decays. The presence of biexponential decays suggests that either two subtypes of Na channels are found in muscle, or Na channels can exist in one of two equally conductive states. The time- invariant decays observed suggest that channel closure does not depend upon their past history. PMID:2852208

The effects of crowding agents Dextran-70k and PEG-8k on actin structure and unfolding reaction

NASA Astrophysics Data System (ADS)

Gagarskaia, Iuliia A.; Povarova, Olga I.; Uversky, Vladimir N.; Kuznetsova, Irina M.; Turoverov, Konstantin K.

2017-07-01

Recently, an increasing number of studies on proteins' structure, stability and folding are trying to bring the experimental conditions closer to those existing in a living cell, namely to the conditions of macromolecular crowding. In vitro such conditions are typically imitated by the ;inert; highly water-soluble polymers with different hydrodynamic dimensions. In this work, the effects of crowded milieu on the structure and conformational stability of actin, which is a key component of the muscle contraction system, was examined. The crowded milieu was simulated by high concentrations of PEG-8k or Dextran-70k. It was revealed that both crowding agents decelerated but not inhibited actin unfolding and made a compact state of inactivated actin thermodynamically more favorable in comparison with the unfolded state. At the same time, the high viscosity of the solution of crowding agents slowed down all processes and especially inactivated actin formation, since it involves the interaction of 14-16 partially unfolded actin molecules. The effects of crowding agent were larger when its hydrodynamic dimensions were closer to the size of globular actin.

Molecular Basis of Paralytic Neurotoxin Action on Voltage-Sensitive Sodium Channels

DTIC Science & Technology

1988-10-20

channels bind a scorpion toxins and sea anemone toxins, which act at an extracellular site and spccifically slow Na+ channel inactivation (Catterall, 1980...the molecule by antibodies as well as by polypeptide neurotoxins from scorpions, sea anemones , coral and snail (Catterall, 1980; Strichartz et al

Inactivation of E.coli 0157:H7 in cultivable soil by fast and slow pyrolysis-generated biochars

USDA-ARS?s Scientific Manuscript database

Biochar is a byproduct of incomplete combustion of organic matter, producing a fine, grainy, highly porous material. Benefits of biochar production include generation of bio-fuels, useful soil amendments for fertilizing crops, binding heavy metals, sequestering environmental biocarbon, and reducing...

Inactivation of Mycobacterium avium with free chlorine.

PubMed

Luh, Jeanne; Mariñas, Benito J

2007-07-15

The inactivation kinetics of Mycobacterium avium with free chlorine was characterized by two stages: an initial phase at a relatively fast rate followed by a slower second stage of pseudo first-order kinetics. The inactivation rate of each stage was approximately the same for all experiments performed at a certain condition of pH and temperature; however, variability was observed for the disinfectant exposure at which the transition between the two stages occurred. This variability was not a function of the initial disinfectant concentration, the initial bacterial density, or the bacterial stock. However, the transition to the second stage varied more significantly at high temperatures (30 degrees C), while lower variability was observed at lower temperatures (5 and 20 degrees C). Experiments conducted at pH values in the range of 6-9 revealed that the inactivation of M. avium was primarily due to hypochlorous acid, with little contribution from hypochlorite ion within this pH range. The inactivation kinetics was represented with a two-population model. The activation energies for the resulting pseudo first-order rate constants for the populations with fast and slow kinetics were 100.3 and 96.5 kJ/mol, respectively. The magnitude of these values suggested that for waters of relatively high pH and low temperatures, little inactivation of M. avium would be achieved within treatment plants, providing a seeding source for distribution systems.

Population Density and Moment-based Approaches to Modeling Domain Calcium-mediated Inactivation of L-type Calcium Channels.

PubMed

Wang, Xiao; Hardcastle, Kiah; Weinberg, Seth H; Smith, Gregory D

2016-03-01

We present a population density and moment-based description of the stochastic dynamics of domain [Formula: see text]-mediated inactivation of L-type [Formula: see text] channels. Our approach accounts for the effect of heterogeneity of local [Formula: see text] signals on whole cell [Formula: see text] currents; however, in contrast with prior work, e.g., Sherman et al. (Biophys J 58(4):985-995, 1990), we do not assume that [Formula: see text] domain formation and collapse are fast compared to channel gating. We demonstrate the population density and moment-based modeling approaches using a 12-state Markov chain model of an L-type [Formula: see text] channel introduced by Greenstein and Winslow (Biophys J 83(6):2918-2945, 2002). Simulated whole cell voltage clamp responses yield an inactivation function for the whole cell [Formula: see text] current that agrees with the traditional approach when domain dynamics are fast. We analyze the voltage-dependence of [Formula: see text] inactivation that may occur via slow heterogeneous domain [[Formula: see text

Inactivation of E.coli 0157:H7 in crop soil by amending with fast and slow pryolysis-generated biochars

USDA-ARS?s Scientific Manuscript database

Biochar, generated by pyrolysis (i.e., heating biomass under low-oxygen conditions), results in incomplete combustion, producing a fine, grainy, highly porous material, 200 – 1,000 um in diameter. Benefits of biochar production include generation of bio-fuels, useful soil amendments for fertilizing...

Initial segment Kv2.2 channels mediate a slow delayed rectifier and maintain high frequency action potential firing in medial nucleus of the trapezoid body neurons

PubMed Central

Johnston, Jamie; Griffin, Sarah J; Baker, Claire; Skrzypiec, Anna; Chernova, Tatanya; Forsythe, Ian D

2008-01-01

The medial nucleus of the trapezoid body (MNTB) is specialized for high frequency firing by expression of Kv3 channels, which minimize action potential (AP) duration, and Kv1 channels, which suppress multiple AP firing, during each calyceal giant EPSC. However, the outward K+ current in MNTB neurons is dominated by another unidentified delayed rectifier. It has slow kinetics and a peak conductance of ∼37 nS; it is half-activated at −9.2 ± 2.1 mV and half-inactivated at −35.9 ± 1.5 mV. It is blocked by several non-specific potassium channel antagonists including quinine (100 μm) and high concentrations of extracellular tetraethylammonium (TEA; IC50 = 11.8 mm), but no specific antagonists were found. These characteristics are similar to recombinant Kv2-mediated currents. Quantitative RT-PCR showed that Kv2.2 mRNA was much more prevalent than Kv2.1 in the MNTB. A Kv2.2 antibody showed specific staining and Western blots confirmed that it recognized a protein ∼110 kDa which was absent in brainstem tissue from a Kv2.2 knockout mouse. Confocal imaging showed that Kv2.2 was highly expressed in axon initial segments of MNTB neurons. In the absence of a specific antagonist, Hodgkin–Huxley modelling of voltage-gated conductances showed that Kv2.2 has a minor role during single APs (due to its slow activation) but assists recovery of voltage-gated sodium channels (Nav) from inactivation by hyperpolarizing interspike potentials during repetitive AP firing. Current-clamp recordings during high frequency firing and characterization of Nav inactivation confirmed this hypothesis. We conclude that Kv2.2-containing channels have a distinctive initial segment location and crucial function in maintaining AP amplitude by regulating the interspike potential during high frequency firing. PMID:18511484

Different kinetic properties of two T-type Ca2+ currents of rat reticular thalamic neurones and their modulation by enflurane

PubMed Central

Joksovic, Pavle M; Bayliss, Douglas A; Todorovic, Slobodan M

2005-01-01

Currents arising from T-type Ca2+ channels in nucleus reticularis thalami (nRT) play a critical role in generation of low-amplitude oscillatory bursting involving mutually interconnected cortical and thalamic neurones, and are implicated in the state of arousal and sleep, as well as seizures. Here we show in brain slices from young rats that two kinetically different T-type Ca2+ currents exist in nRT neurones, with a slowly inactivating current expressed only on proximal dendrites, and fast inactivating current predominantly expressed on soma. Nickel was about twofold more potent in blocking fast (IC50 64 μm) than slow current (IC50 107 μm). The halogenated volatile anaesthetic enflurane blocked both currents, but only the slowly inactivating current was affected in voltage-dependent fashion. Slow dendritic current was essential for generation of low-threshold Ca2+ spikes (LTS), and both enflurane and nickel also suppressed LTS and neuronal burst firing at concentrations that blocked isolated T currents. Differential kinetic properties of T currents expressed in cell soma and proximal dendrites of nRT neurones indicate that various subcellular compartments may exhibit different membrane properties in response to small membrane depolarizations. Furthermore, since blockade of two different T currents in nRT neurones by enflurane and other volatile anaesthetics occurs within concentrations that are relevant during clinical anaesthesia, our findings suggest that these actions could contribute to some important clinical effects of anaesthetics. PMID:15845580

Relative insignificance of virus inactivation during aluminum electrocoagulation of saline waters.

PubMed

Tanneru, Charan Tej; Jothikumar, N; Hill, Vincent R; Chellam, Shankararaman

2014-12-16

Combined removal and inactivation of the MS2 bacteriophage from model saline (0-100 mM NaCl) waters by electrochemical treatment using a sacrificial aluminum anode was evaluated. Both chemical and electrodissolution contributed to coagulant dosing since measured aluminum concentrations were statistically higher than purely electrochemical predictions using Faraday's law. Electrocoagulation generated only small amounts of free chlorine in situ but effectively destabilized viruses and incorporated them into Al(OH)3(s) flocs during electrolysis. Low chlorine concentrations combined with virus shielding and aggregation within flocs resulted in very slow disinfection rates necessitating extended flocculation/contact times to achieve significant log-inactivation. Therefore, the dominant virus control mechanism during aluminum electrocoagulation of saline waters is "physical" removal by uptake onto flocs rather than "chemical" inactivation by chlorine. Attenuated total reflectance-Fourier transform infrared spectroscopy provided evidence for oxidative transformations of capsid proteins including formation of oxyacids, aldehydes, and ketones. Electrocoagulation significantly altered protein secondary structures decreasing peak areas associated with turns, bends, α-helices, β-structures, and random coils for inactivated viruses compared with the MS2 stock. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) measurements showed rapid initial RNA damage following a similar trend as plaque assay measurements of infectious viruses. However, ssRNA cleavage measured by qRT-PCR underestimated inactivation over longer durations. Although aluminum electrocoagulation of saline waters disorders virus capsids and damages RNA, inactivation occurs at a sufficiently low rate so as to only play a secondary role to floc-encapsulation during residence times typical of electrochemical treatment.

Experimental burial inhibits methanogenesis and anaerobic decomposition in water-saturated peats.

PubMed

Blodau, Christian; Siems, Melanie; Beer, Julia

2011-12-01

A mechanistic understanding of carbon (C) sequestration and methane (CH(4)) production is of great interest due to the importance of these processes for the global C budget. Here we demonstrate experimentally, by means of column experiments, that burial of water saturated, anoxic bog peat leads to inactivation of anaerobic respiration and methanogenesis. This effect can be related to the slowness of diffusive transport of solutes and evolving energetic constraints on anaerobic respiration. Burial lowered decomposition constants in homogenized peat sand mixtures from about 10(-5) to 10(-7) yr(-1), which is considerably slower than previously assumed, and methanogenesis slowed down in a similar manner. The latter effect could be related to acetoclastic methanogenesis approaching a minimum energy quantum of -25 kJ mol(-1) (CH(4)). Given the robustness of hydraulic properties that locate the oxic-anoxic boundary near the peatland surface and constrain solute transport deeper into the peat, this effect has likely been critical for building the peatland C store and will continue supporting long-term C sequestration in northern peatlands even under moderately changing climatic conditions.

Heat exposure enhances radiosensitivity by depressing DNA-PK kinase activity during double strand break repair.

PubMed

Ihara, Makoto; Takeshita, Satoshi; Okaichi, Kumio; Okumura, Yutaka; Ohnishi, Takeo

2014-03-01

From the role of double strand DNA dependent protein kinase (DNA-PKcs) activity of non-homologous end joining (NHEJ) repair for DNA double strand breaks (DSBs), we aim to define possible associations between thermo-sensitisation and the enzyme activities in X-ray irradiated cells. DNA-PKcs deficient mouse, Chinese hamster and human cultured cells were compared to the parental wild-type cells. The radiosensitivities, the number of DSBs and DNA-PKcs activities after heat-treatment were measured. Both DNA-PKcs deficient cells and the wild-type cells showed increased radiosensitivities after heat-treatment. The wild-type cells have two repair processes; fast repair and slow repair. In contrast, DNA-PKcs deficient cells have only the slow repair process. The fast repair component apparently disappeared by heat-treatment in the wild-type cells. In both cell types, additional heat exposure enhanced radiosensitivities. Although DNA-PKcs activity was depressed by heat, the inactivated DNA-PKcs activity recovered during an incubation at 37 °C. DSB repair efficiency was dependent on the reactivation of DNA-PKcs activity. It was suggested that NHEJ is the major process used to repair X-ray-induced DSBs and utilises DNA-PKcs activity, but homologous recombination repair provides additional secondary levels of DSB repair. The thermo-sensitisation in X-ray-irradiated cells depends on the inhibition of NHEJ repair through the depression of DNA-PKcs activities.

Nucleic acid stains as indicators of Giardia muris viability following cyst inactivation.

PubMed

Taghi-Kilani, R; Gyürék, L L; Millard, P J; Finch, G R; Belosevic, M

1996-06-01

A reliable viability assay for Giardia is required for the development of disinfection process design criteria and pathogen monitoring by water treatment utilities. Surveys of single-staining nucleic acid dyes (stain dead parasites only), and double-staining vital dye kits from Molecular Probes (stain live and dead parasites) were conducted to assess the viability of untreated, heat-killed, and chemically inactivated Giardia muris cysts. Nucleic acid staining results were compared to those of in vitro excystation and animal infectivity. Nucleic acid stain, designated as SYTO-9, was considered the best among the single-staining dyes for its ability to stain dead cysts brightly and its relatively slow decay rate of visible light emission following DNA binding. SYTO-9 staining was correlated to animal infectivity. A Live/Dead BacLight was found to be the better of 2 double-staining viability kits tested. Logarithmic survival ratios based on SYTO-9 and Live/Dead BacLight were compared to excystation and infectivity results for G. muris cysts exposed to ozone or free chlorine. The results indicate that SYTO-9 and Live/Dead BacLight staining is stable following treatment of cysts with chemical disinfectants.

Voltage-gated sodium channels

PubMed Central

Abdelsayed, Mena; Sokolov, Stanislav

2013-01-01

Epilepsy is a brain disorder characterized by seizures and convulsions. The basis of epilepsy is an increase in neuronal excitability that, in some cases, may be caused by functional defects in neuronal voltage gated sodium channels, Nav1.1 and Nav1.2. The effects of antiepileptic drugs (AEDs) as effective therapies for epilepsy have been characterized by extensive research. Most of the classic AEDs targeting Nav share a common mechanism of action by stabilizing the channel’s fast-inactivated state. In contrast, novel AEDs, such as lacosamide, stabilize the slow-inactivated state in neuronal Nav1.1 and Nav1.7 isoforms. This paper reviews the different mechanisms by which this stabilization occurs to determine new methods for treatment. PMID:23531742

Effects of haloperidol on Kv4.3 potassium channels.

PubMed

Lee, Hong Joon; Sung, Ki-Wug; Hahn, Sang June

2014-10-05

Haloperidol is commonly used in clinical practice to treat acute and chronic psychosis, but it also has been associated with adverse cardiovascular events. We investigated the effects of haloperidol on Kv4.3 currents stably expressed in CHO cells using a whole-cell patch-clamp technique. Haloperidol did not significantly inhibit the peak amplitude of Kv4.3, but accelerated the decay rate of inactivation of Kv4.3 in a concentration-dependent manner. Thus, the effects of haloperidol on Kv4.3 were estimated from the integral of the Kv4.3 currents during the depolarization pulse. The Kv4.3 was decreased by haloperidol in a concentration-dependent manner with an IC50 value of 3.6 μM. Haloperidol accelerated the decay rate of Kv4.3 inactivation and activation kinetics in a concentration-dependent manner, thereby decreasing the time-to-peak. Haloperidol shifted the voltage dependence of the steady-state activation and inactivation of Kv4.3 in a hyperpolarizing direction. Haloperidol also caused an acceleration of the closed-state inactivation of Kv4.3. Haloperidol produced a use-dependent block of Kv4.3, which was accompanied by a slowing of recovery from the inactivation of Kv4.3. These results suggest that haloperidol blocks Kv4.3 by both interacting with the open state of Kv4.3 channels during depolarization and accelerating the closed-state inactivation at subthreshold membrane potentials. Copyright © 2014 Elsevier B.V. All rights reserved.

Putting out the fire: what terminates calcium-induced calcium release in cardiac muscle?

PubMed

Stern, Michael D; Cheng, Heping

2004-06-01

The majority of contractile calcium in cardiac muscle is released from stores in the sarcoplasmic reticulum (SR), by a process of calcium-induced calcium release (CICR) through ryanodine receptors. Because CICR is intrinsically self-reinforcing, the stability of and graded regulation of cardiac EC coupling appear paradoxical. It is now well established that this gradation results from the stochastic recruitment of varying numbers of elementary local release events, which may themselves be regenerative, and which can be directly observed as calcium sparks. Ryanodine receptors (RyRs) are clustered in dense lattices, and most calcium sparks are now believed to involve activation of multiple RyRs. This implies that local CICR is regenerative, requiring a mechanism to terminate it. It was initially assumed that this mechanism was inactivation of the RyR, but during the decade since the discovery of sparks, no sufficiently strong inactivation mechanism has been demonstrated in vitro and all empirically determined gating schemes for the RyR give unstable EC coupling in Monte Carlo simulations. We consider here possible release termination mechanisms. Stochastic attrition is the spontaneous decay of active clusters due to random channel closure; calculations show that it is much too slow unless assisted by another process. Calcium-dependent RyR inactivation involving third-party proteins remains a viable but speculative mechanism; current candidates include calmodulin and sorcin. Local depletion of SR release terminal calcium could terminate release, however calculations and measurements leave it uncertain whether a sufficient diffusion resistance exists within the SR to sustain such depletion. Depletion could be assisted by dependence of RyR activity on SR lumenal [Ca(2+)]. There is substantial evidence for such lumenal activation, but it is not clear if it is a strong enough effect to account for the robust termination of sparks. The existence of direct interactions among clustered RyRs might account for the discrepancy between the inactivation properties of isolated RyRs and intact clusters. Such coupled gating remains controversial. Determining the mechanism of release termination is the outstanding unsolved problem of cardiac EC coupling, and will probably require extensive genetic manipulation of the EC coupling apparatus in its native environment to unravel the solution.

Conformational Transition Pathway in the Activation Process of Allosteric Glucokinase

PubMed Central

Shi, Ting; Zhao, Yaxue; Chen, Yingyi; Li, Xiaobai; Liu, Xinyi; Huang, Zhimin; Zhang, Jian

2013-01-01

Glucokinase (GK) is a glycolytic enzyme that plays an important role in regulating blood glucose level, thus acting as a potentially attractive target for drug discovery in the treatment of diabetes of the young type 2 and persistent hyperinsulinemic hypoglycemia of infancy. To characterize the activation mechanism of GK from the super-open state (inactive state) to the closed state (active state), a series of conventional molecular dynamics (MD) and targeted MD (TMD) simulations were performed on this enzyme. Conventional MD simulation showed a specific conformational ensemble of GK when the enzyme is inactive. Seven TMD simulations depicted a reliably conformational transition pathway of GK from the inactive state to the active state, and the components important to the conformational change of GK were identified by analyzing the detailed structures of the TMD trajectories. In combination with the inactivation process, our findings showed that the whole conformational pathway for the activation-inactivation-activation of GK is a one-direction circulation, and the active state is less stable than the inactive state in the circulation. Additionally, glucose was demonstrated to gradually modulate its binding pose with the help of residues in the large domain and connecting region of GK during the activation process. Furthermore, the obtained energy barriers were used to explain the preexisting equilibrium and the slow binding kinetic process of the substrate by GK. The simulated results are in accordance with the recent findings from the mutagenesis experiments and kinetic analyses. Our observations reveal a complicated conformational process in the allosteric protein, resulting in new knowledge about the delicate mechanisms for allosteric biological macromolecules that will be useful in drug design for targeting allosteric proteins. PMID:23409066

Andrographolide sodium bisulphite-induced inactivation of urease: inhibitory potency, kinetics and mechanism.

PubMed

Mo, Zhi-Zhun; Wang, Xiu-Fen; Zhang, Xie; Su, Ji-Yan; Chen, Hai-Ming; Liu, Yu-Hong; Zhang, Zhen-Biao; Xie, Jian-Hui; Su, Zi-Ren

2015-07-16

The inhibitory effect of andrographolide sodium bisulphite (ASB) on jack bean urease (JBU) and Helicobacter pylori urease (HPU) was performed to elucidate the inhibitory potency, kinetics and mechanism of inhibition in 20 mM phosphate buffer, pH 7.0, 2 mM EDTA, 25 °C. The ammonia formations, indicator of urease activity, were examined using modified spectrophotometric Berthelot (phenol-hypochlorite) method. The inhibitory effect of ASB was characterized with IC50 values. Lineweaver-Burk and Dixon plots for JBU inhibition of ASB was constructed from the kinetic data. SH-blocking reagents and competitive active site Ni2+ binding inhibitors were employed for mechanism study. Molecular docking technique was used to provide some information on binding conformations as well as confirm the inhibition mode. The IC50 of ASB against JBU and HPU was 3.28±0.13 mM and 3.17±0.34 mM, respectively. The inhibition proved to be competitive and concentration- dependent in a slow-binding progress. The rapid formation of initial ASB-JBU complex with an inhibition constant of Ki=2.86×10(-3) mM was followed by a slow isomerization into the final complex with an overall inhibition constant of Ki*=1.33×10(-4) mM. The protective experiment proved that the urease active site is involved in the binding of ASB. Thiol reagents (L-cysteine and dithiothreithol) strongly protect the enzyme from the loss of enzymatic activity, while boric acid and fluoride show weaker protection, indicating that the active-site sulfhydryl group of JBU was potentially involved in the blocking process. Moreover, inhibition of ASB proved to be reversible since ASB-inactivated JBU could be reactivated by dithiothreitol application. Molecular docking assay suggested that ASB made contacts with the important sulfhydryl group Cys-592 residue and restricted the mobility of the active-site flap. ASB was a competitive inhibitor targeting thiol groups of urease in a slow-binding manner both reversibly and concentration-dependently, serving as a promising urease inhibitor for the treatment of urease-related diseases.

Optimization of the synthesis process of an iron oxide nanocatalyst supported on activated carbon for the inactivation of Ascaris eggs in water using the heterogeneous Fenton-like reaction.

PubMed

Morales-Pérez, Ariadna A; Maravilla, Pablo; Solís-López, Myriam; Schouwenaars, Rafael; Durán-Moreno, Alfonso; Ramírez-Zamora, Rosa-María

2016-01-01

An experimental design methodology was used to optimize the synthesis of an iron-supported nanocatalyst as well as the inactivation process of Ascaris eggs (Ae) using this material. A factor screening design was used for identifying the significant experimental factors for nanocatalyst support (supported %Fe, (w/w), temperature and time of calcination) and for the inactivation process called the heterogeneous Fenton-like reaction (H2O2 dose, mass ratio Fe/H2O2, pH and reaction time). The optimization of the significant factors was carried out using a face-centered central composite design. The optimal operating conditions for both processes were estimated with a statistical model and implemented experimentally with five replicates. The predicted value of the Ae inactivation rate was close to the laboratory results. At the optimal operating conditions of the nanocatalyst production and Ae inactivation process, the Ascaris ova showed genomic damage to the point that no cell reparation was possible showing that this advanced oxidation process was highly efficient for inactivating this pathogen.

Open channel block of A-type, kv4.3, and delayed rectifier K+ channels, Kv1.3 and Kv3.1, by sibutramine.

PubMed

Kim, Sung Eun; Ahn, Hye Sook; Choi, Bok Hee; Jang, Hyun-Jong; Kim, Myung-Jun; Rhie, Duck-Joo; Yoon, Shin-Hee; Jo, Yang-Hyeok; Kim, Myung-Suk; Sung, Ki-Wug; Hahn, Sang June

2007-05-01

The effects of sibutramine on voltage-gated K+ channel (Kv)4.3, Kv1.3, and Kv3.1, stably expressed in Chinese hamster ovary cells, were investigated using the whole-cell patch-clamp technique. Sibutramine did not significantly decrease the peak Kv4.3 currents, but it accelerated the rate of decay of current inactivation in a concentration-dependent manner. This phenomenon was effectively characterized by integrating the total current over the duration of a depolarizing pulse to +40 mV. The IC50 value for the sibutramine block of Kv4.3 was 17.3 microM. Under control conditions, the inactivation of Kv4.3 currents could be fit to a biexponential function, and the time constants for the fast and slow components were significantly decreased after the application of sibutramine. The association (k+1) and dissociation (k-1) rate constants for the sibutramine block of Kv 4.3 were 1.51 microM-1s-1 and 27.35 s-1, respectively. The theoretical KD value, derived from k-1/k+1, yielded a value of 18.11 microM. The block of Kv4.3 by sibutramine displayed a weak voltage dependence, increasing at more positive potentials, and it was use-dependent at 2 Hz. Sibutramine did not affect the time course for the deactivating tail currents. Neither steady-state activation and inactivation nor the recovery from inactivation was affected by sibutramine. Sibutramine caused the concentration-dependent block of the Kv1.3 and Kv3.1 currents with an IC50 value of 3.7 and 32.7 microM, respectively. In addition, sibutramine reduced the tail current amplitude and slowed the deactivation of the tail currents of Kv1.3 and Kv3.1, resulting in a crossover phenomenon. These results indicate that sibutramine acts on Kv4.3, Kv1.3, and Kv3.1 as an open channel blocker.

High pressure processing's potential to inactivate norovirus and other fooodborne viruses

USDA-ARS?s Scientific Manuscript database

High pressure processing (HPP) can inactivate human norovirus. However, all viruses are not equally susceptible to HPP. Pressure treatment parameters such as required pressure levels, initial pressurization temperatures, and pressurization times substantially affect inactivation. How food matrix ...

Two Ca current components of the receptor current in the electroreceptors of the marine catfish Plotosus.

PubMed

Sugawara, Y

1989-02-01

In the isolated sensory epithelium of the Plotosus electroreceptor, the receptor current has been dissected into inward Ca current, ICa, and superimposed outward transient of Ca-gated K current, IK(Ca). In control saline (170 mM/liter Na), with IK(Ca) abolished by K blockers, ICa declined in two successive exponential phases with voltage-dependent time constants. Double-pulse experiments revealed that the test ICa was partially depressed by prepulses, maximally near voltage levels for the control ICa maximum, which suggests current-dependent inactivation. In low Na saline (80 mM/liter), ICa declined in a single phase with time constants similar to those of the slower phase in control saline. The test ICa was then unaffected by prepulses. The implied presence of two Ca current components, the fast and slow ICa's, were further examined. In control saline, the PSP externally recorded from the afferent nerve showed a fast peak and a slow tonic phase. The double-pulse experiments revealed that IK(Ca) and the peak PSP were similarly depressed, i.e., secondarily to inactivation of the peak current. The steady inward current, however, was unaffected by prolonged prepulses that were stepped to 0 mV, the in situ DC level. Therefore, the fast ICa seems to initiate IK(Ca) and phasic release of transmitter, which serves for phasic receptor responses. The slow ICa may provide persistent active current, which has been shown to maintain tonic receptor operation.

Inactivation of MS2 bacteriophage by streamer corona discharge in water.

PubMed

Lee, Changha; Kim, Jaeeun; Yoon, Jeyong

2011-02-01

Electrical discharge processes are emerging as water treatment technologies applicable to both the degradation of organic contaminants as well as inactivation of pathogens. Particularly as a disinfection technology, electrical discharge processes do not produce toxic byproducts, and effectively inactivate a wide spectrum of microorganisms by multiple lethal actions generated by the formation of plasma channels. This study demonstrates the inactivation of a virus using the streamer corona discharge process (SCDP) with MS2 phage as a surrogate. A rapid inactivation of MS2 phage (i.e., approximately 4 log inactivation in 5 min) was observed in all experimental runs conducted. Discharge conditions such as applied voltage and storage capacitance significantly affected the inactivation efficiency of MS2 phage, whereas the influence of water quality parameters was minor. In order to elucidate the mechanism of MS2 phage inactivation, potentially lethal factors that can be generated by the SCDP were selected, and their roles in the inactivation of MS2 phage were examined. As a result, effects of UV radiation, chemical oxidants, and pulsed electric fields were found to be insignificant. The shockwave generated upon plasma channel formation appears to be the most important factor responsible for MS2 phage inactivation. Copyright © 2010 Elsevier Ltd. All rights reserved.

Inactivation of Byssochlamys nivea ascospores in strawberry puree by high pressure, power ultrasound and thermal processing.

PubMed

Evelyn; Silva, F V M

2015-12-02

Byssochlamys nivea is a mold that can spoil processed fruit products and produce mycotoxins. In this work, high pressure processing (HPP, 600 MPa) and power ultrasound (24 kHz, 0.33 W/mL; TS) in combination with 75°C for the inactivation of four week old B. nivea ascospores in strawberry puree for up to 30 min was investigated and compared with 75°C thermal processing alone. TS and thermal processing can activate the mold ascospores, but HPP-75°C resulted in 2.0 log reductions after a 20 min process. For a 10 min process, HPP-75°C was better than 85°C alone in reducing B. nivea spores (1.4 vs. 0.2 log reduction), demonstrating that a lower temperature in combination with HPP is more effective for spore inactivation than heat alone at a higher temperature. The ascospore inactivation by HPP-thermal, TS and thermal processing was studied at different temperatures and modeled. Faster inactivation was achieved at higher temperatures for all the technologies tested, indicating the significant role of temperature in spore inactivation, alone or combined with other physical processes. The Weibull model described the spore inactivation by 600 MPa HPP-thermal (38, 50, 60, 75°C) and thermal (85, 90°C) processing, whereas the Lorentzian model was more appropriate for TS treatment (65, 70, 75°C). The models obtained provide a useful tool to design and predict pasteurization processes targeting B. nivea ascospores. Copyright © 2015 Elsevier B.V. All rights reserved.

Persistent spatial information in the FEF during object-based short-term memory does not contribute to task performance.

PubMed

Clark, Kelsey L; Noudoost, Behrad; Moore, Tirin

2014-06-01

We previously reported the existence of a persistent spatial signal in the FEF during object-based STM. This persistent activity reflected the location at which the sample appeared, irrespective of the location of upcoming targets. We hypothesized that such a spatial signal could be used to maintain or enhance object-selective memory activity elsewhere in cortex, analogous to the role of a spatial signal during attention. Here, we inactivated a portion of the FEF with GABAa agonist muscimol to test whether the observed activity contributes to object memory performance. We found that, although RTs were slowed for saccades into the inactivated portion of retinotopic space, performance for samples appearing in that region was unimpaired. This contrasts with the devastating effects of the same FEF inactivation on purely spatial working memory, as assessed with the memory-guided saccade task. Thus, in a task in which a significant fraction of FEF neurons displayed persistent, sample location-based activity, disrupting this activity had no impact on task performance.

Interactions among DIV voltage-sensor movement, fast inactivation, and resurgent Na current induced by the NaVβ4 open-channel blocking peptide

PubMed Central

Lewis, Amanda H.

2013-01-01

Resurgent Na current flows as voltage-gated Na channels recover through open states from block by an endogenous open-channel blocking protein, such as the NaVβ4 subunit. The open-channel blocker and fast-inactivation gate apparently compete directly, as slowing the onset of fast inactivation increases resurgent currents by favoring binding of the blocker. Here, we tested whether open-channel block is also sensitive to deployment of the DIV voltage sensor, which facilitates fast inactivation. We expressed NaV1.4 channels in HEK293t cells and assessed block by a free peptide replicating the cytoplasmic tail of NaVβ4 (the “β4 peptide”). Macroscopic fast inactivation was disrupted by mutations of DIS6 (L443C/A444W; “CW” channels), which reduce fast-inactivation gate binding, and/or by the site-3 toxin ATX-II, which interferes with DIV movement. In wild-type channels, the β4 peptide competed poorly with fast inactivation, but block was enhanced by ATX. With the CW mutation, large peptide-induced resurgent currents were present even without ATX, consistent with increased open-channel block upon depolarization and slower deactivation after blocker unbinding upon repolarization. The addition of ATX greatly increased transient current amplitudes and further enlarged resurgent currents, suggesting that pore access by the blocker is actually decreased by full deployment of the DIV voltage sensor. ATX accelerated recovery from block at hyperpolarized potentials, however, suggesting that the peptide unbinds more readily when DIV voltage-sensor deployment is disrupted. These results are consistent with two open states in Na channels, dependent on the DIV voltage-sensor position, which differ in affinity for the blocking protein. PMID:23940261

Block of Inactivation-deficient Na+ Channels by Local Anesthetics in Stably Transfected Mammalian Cells

PubMed Central

Wang, Sho-Ya; Mitchell, Jane; Moczydlowski, Edward; Wang, Ging Kuo

2004-01-01

According to the classic modulated receptor hypothesis, local anesthetics (LAs) such as benzocaine and lidocaine bind preferentially to fast-inactivated Na+ channels with higher affinities. However, an alternative view suggests that activation of Na+ channels plays a crucial role in promoting high-affinity LA binding and that fast inactivation per se is not a prerequisite for LA preferential binding. We investigated the role of activation in LA action in inactivation-deficient rat muscle Na+ channels (rNav1.4-L435W/L437C/A438W) expressed in stably transfected Hek293 cells. The 50% inhibitory concentrations (IC50) for the open-channel block at +30 mV by lidocaine and benzocaine were 20.9 ± 3.3 μM (n = 5) and 81.7 ± 10.6 μM (n = 5), respectively; both were comparable to inactivated-channel affinities. In comparison, IC50 values for resting-channel block at −140 mV were >12-fold higher than those for open-channel block. With 300 μM benzocaine, rapid time-dependent block (τ ≈ 0.8 ms) of inactivation-deficient Na+ currents occurred at +30 mV, but such a rapid time-dependent block was not evident at −30 mV. The peak current at −30 mV, however, was reduced more severely than that at +30 mV. This phenomenon suggested that the LA block of intermediate closed states took place notably when channel activation was slow. Such closed-channel block also readily accounted for the LA-induced hyperpolarizing shift in the conventional steady-state inactivation measurement. Our data together illustrate that the Na+ channel activation pathway, including most, if not all, transient intermediate closed states and the final open state, promotes high-affinity LA binding. PMID:15545401

Inactivation disinfection property of Moringa Oleifera seed extract: optimization and kinetic studies

NASA Astrophysics Data System (ADS)

Idris, M. A.; Jami, M. S.; Hammed, A. M.

2017-05-01

This paper presents the statistical optimization study of disinfection inactivation parameters of defatted Moringa oleifera seed extract on Pseudomonas aeruginosa bacterial cells. Three level factorial design was used to estimate the optimum range and the kinetics of the inactivation process was also carried. The inactivation process involved comparing different disinfection models of Chicks-Watson, Collins-Selleck and Homs models. The results from analysis of variance (ANOVA) of the statistical optimization process revealed that only contact time was significant. The optimum disinfection range of the seed extract was 125 mg/L, 30 minutes and 120rpm agitation. At the optimum dose, the inactivation kinetics followed the Collin-Selleck model with coefficient of determination (R2) of 0.6320. This study is the first of its kind in determining the inactivation kinetics of pseudomonas aeruginosa using the defatted seed extract.

Chimeric Derivatives of Functionalized Amino Acids and α-Aminoamides: Compounds with Anticonvulsant Activity in Seizure Models and Inhibitory Actions on Central, Peripheral, and Cardiac Isoforms of Voltage-gated Sodium Channels

PubMed Central

Torregrosa, Robert; Yang, Xiao-Fang; Dustrude, Erik T.; Cummins, Theodore R.

2015-01-01

Six novel 3″-substituted (R)-N-(phenoxybenzyl) 2-N-acetamido-3-methoxypropionamides were prepared and then assessed using whole-cell, patch-clamp electrophysiology for their anticonvulsant activities in animal seizure models and for their sodium channel activities. We found compounds with various substituents at the terminal aromatic ring that had excellent anticonvulsant activity. Of these compounds, (R)-N-4′-((3″-chloro)phenoxy)benzyl 2-N-acetamido-3-methoxypropionamide ((R)-5) and (R)-N-4′-((3″-trifluoromethoxy)phenoxy)benzyl 2-N-acetamido-3-methoxypropionamide ((R)-9) exhibited high protective indices (PI = TD50/ED50) comparable with many antiseizure drugs when tested in the maximal electroshock seizure test to mice (intraperitoneally) and rats (intraperitoneally, orally). Most compounds potently transitioned sodium channels to the slow-inactivated state when evaluated in rat embryonic cortical neurons. Treating HEK293 recombinant cells that expressed hNav1.1, rNav1.3, hNav1.5, or hNav1.7 with (R)-9 recapitulated the high levels of sodium channel slow inactivation. PMID:25922183

C. botulinum inactivation kinetics implemented in a computational model of a high-pressure sterilization process.

PubMed

Juliano, Pablo; Knoerzer, Kai; Fryer, Peter J; Versteeg, Cornelis

2009-01-01

High-pressure, high-temperature (HPHT) processing is effective for microbial spore inactivation using mild preheating, followed by rapid volumetric compression heating and cooling on pressure release, enabling much shorter processing times than conventional thermal processing for many food products. A computational thermal fluid dynamic (CTFD) model has been developed to model all processing steps, including the vertical pressure vessel, an internal polymeric carrier, and food packages in an axis-symmetric geometry. Heat transfer and fluid dynamic equations were coupled to four selected kinetic models for the inactivation of C. botulinum; the traditional first-order kinetic model, the Weibull model, an nth-order model, and a combined discrete log-linear nth-order model. The models were solved to compare the resulting microbial inactivation distributions. The initial temperature of the system was set to 90 degrees C and pressure was selected at 600 MPa, holding for 220 s, with a target temperature of 121 degrees C. A representation of the extent of microbial inactivation throughout all processing steps was obtained for each microbial model. Comparison of the models showed that the conventional thermal processing kinetics (not accounting for pressure) required shorter holding times to achieve a 12D reduction of C. botulinum spores than the other models. The temperature distribution inside the vessel resulted in a more uniform inactivation distribution when using a Weibull or an nth-order kinetics model than when using log-linear kinetics. The CTFD platform could illustrate the inactivation extent and uniformity provided by the microbial models. The platform is expected to be useful to evaluate models fitted into new C. botulinum inactivation data at varying conditions of pressure and temperature, as an aid for regulatory filing of the technology as well as in process and equipment design.

Inactivation of faecal indicator bacteria in a roof-captured rainwater system under ambient meteorological conditions.

PubMed

Ahmed, W; Richardson, K; Sidhu, J P S; Jagals, P; Toze, S

2014-01-01

In this study, faecal indicator bacteria (FIB) namely Escherichia coli and Enterococcus spp. were seeded into slurries of possum faeces and placed on the roof and in the gutter of a roof-captured rainwater (RCR) system. The persistence of FIB in these circumstances was determined under ambient climatic conditions. FIB persistence was also determined under in situ conditions in tank water using diffusion chambers. The numbers of surviving FIB at different time intervals were enumerated using culture-based methods. Both FIB were rapidly inactivated on the roof under sunlight conditions (T(90) = 2 h) compared with shade conditions (T(90) = 9-53 h). Significant differences were observed between sunlight and shade conditions on the roof for both T90 values of E. coli (P < 0·001) and Enterococcus spp. (P < 0·001). E. coli showed biphasic inactivation patterns under both clean and unclean gutter conditions. Enterococcus spp., however, showed rapid inactivation (T(90) = 2 h for the clean gutter and T(90) = 6 h for the unclean gutter) compared with E. coli (T(90) = 22 h for the clean gutter and T(90) = 20 h for the unclean gutter). Significant differences were also observed between the T(90) values of E. coli and Enterococcus spp. for both clean (P < 0·001) and unclean (P < 0·001) gutters. Both E. coli and Enterococcus spp. showed nonlinear biphasic inactivation in tank water. Significant difference was observed between the T(90) value of E. coli inactivation compared with Enterococcus spp. (P < 0·001) in the tank water. In this study, FIB were observed to survive longer (T(90) = 9-53 h) on the roof under shade conditions compared with sunlight conditions (T(90) = 2 h). If there is a rainfall event within two to three days after the deposition of faecal maters on the roof, it is highly likely that FIB would be transported to the tank water. When introduced into the tank, a relatively slow inactivation process may take place (T(90) = 38-72 h). The presence of FIB in water indicates faecal pollution and potential presence of enteric pathogens. Therefore, the information on the resilience of FIB, as obtained in this study, can be used for indirect assessment of health risks associated with using roof-captured rainwater for potable and nonpotable purposes. © 2013 The Society for Applied Microbiology.

The Nucleus Reuniens Controls Long-Range Hippocampo-Prefrontal Gamma Synchronization during Slow Oscillations.

PubMed

Ferraris, Maëva; Ghestem, Antoine; Vicente, Ana F; Nallet-Khosrofian, Lauriane; Bernard, Christophe; Quilichini, Pascale P

2018-03-21

Gamma oscillations are involved in long-range coupling of distant regions that support various cognitive operations. Here we show in adult male rats that synchronized bursts of gamma oscillations bind the hippocampus (HPC) and prefrontal cortex (mPFC) during slow oscillations and slow-wave sleep, a brain state that is central for consolidation of memory traces. These gamma bursts entrained the firing of the local HPC and mPFC neuronal populations. Neurons of the nucleus reuniens (NR), which is a structural and functional hub between HPC and mPFC, demonstrated a specific increase in their firing before gamma burst onset, suggesting their involvement in HPC-mPFC binding. Chemical inactivation of NR disrupted the temporal pattern of gamma bursts and their synchronization, as well as mPFC neuronal firing. We propose that the NR drives long-range hippocampo-prefrontal coupling via gamma bursts providing temporal windows for information exchange between the HPC and mPFC during slow-wave sleep. SIGNIFICANCE STATEMENT Long-range coupling between hippocampus (HPC) and prefrontal cortex (mPFC) is believed to support numerous cognitive functions, including memory consolidation occurring during sleep. Gamma-band synchronization is a fundamental process in many neuronal operations and is instrumental in long-range coupling. Recent evidence highlights the role of nucleus reuniens (NR) in consolidation; however, how it influences hippocampo-prefrontal coupling is unknown. In this study, we show that HPC and mPFC are synchronized by gamma bursts during slow oscillations in anesthesia and natural sleep. By manipulating and recording the NR-HPC-mPFC network, we provide evidence that the NR actively promotes this long-range gamma coupling. This coupling provides the hippocampo-prefrontal circuit with a novel mechanism to exchange information during slow-wave sleep. Copyright © 2018 the authors 0270-6474/18/383026-13$15.00/0.

Latent injury in frozen-thawed bacteriophage T4Bo.

PubMed Central

Steele, P. R.

1976-01-01

Two interesting new phenomena have been observed in suspensions of T4Bo bacteriophage which were frozen to temperatures below the eutectic temperature of the salt (sodium chloride) in the suspending medium. Approximately 10% of the phage appeared to survive such a phase change as determined by plaque titre. However, exposure of these survivors to ultrasonic vibration or repeated freezing showed them to be hypersensitive and thus latently injured. The hypersensitivity was lost on incubating the phage at 37 degrees C. for 3 hr. Furthermore, following a eutectic phase change, the surviving phage could be inactivated by rapid cooling to -90 degrees C. followed by slow rewarming. Such inactivation cannot be accounted for by accepted theories of freezing injury. PMID:1068188

Voltage-gated sodium channels: pharmaceutical targets via anticonvulsants to treat epileptic syndromes.

PubMed

Abdelsayed, Mena; Sokolov, Stanislav

2013-01-01

Epilepsy is a brain disorder characterized by seizures and convulsions. The basis of epilepsy is an increase in neuronal excitability that, in some cases, may be caused by functional defects in neuronal voltage gated sodium channels, Nav1.1 and Nav1.2. The effects of antiepileptic drugs (AEDs) as effective therapies for epilepsy have been characterized by extensive research. Most of the classic AEDs targeting Nav share a common mechanism of action by stabilizing the channel's fast-inactivated state. In contrast, novel AEDs, such as lacosamide, stabilize the slow-inactivated state in neuronal Nav1.1 and Nav1.7 isoforms. This paper reviews the different mechanisms by which this stabilization occurs to determine new methods for treatment.

Inactivation of A currents and A channels on rat nodose neurons in culture

PubMed Central

1989-01-01

Cultured sensory neurons from nodose ganglia were investigated with whole-cell patch-clamp techniques and single-channel recordings to characterize the A current. Membrane depolarization from -40 mV holding potential activated the delayed rectifier current (IK) at potentials positive to -30 mV; this current had a sigmoidal time course and showed little or no inactivation. In most neurons, the A current was completely inactivated at the -40 mV holding potential and required hyperpolarization to remove the inactivation; the A current was isolated by subtracting the IK evoked by depolarizations from -40 mV from the total outward current evoked by depolarizations from -90 mV. The decay of the A current on several neurons had complex kinetics and was fit by the sum of three exponentials whose time constants were 10- 40 ms, 100-350 ms, and 1-3 s. At the single-channel level we found that one class of channel underlies the A current. The conductance of A channels varied with the square root of the external K concentration: it was 22 pS when exposed to 5.4 mM K externally, the increased to 40 pS when exposed to 140 mM K externally. A channels activated rapidly upon depolarization and the latency to first opening decreased with depolarization. The open time distributions followed a single exponential and the mean open time increased with depolarization. A channels inactivate in three different modes: some A channels inactivated with little reopening and gave rise to ensemble averages that decayed in 10-40 ms; other A channels opened and closed three to four times before inactivating and gave rise to ensemble averages that decayed in 100-350 ms; still other A channels opened and closed several hundred times and required seconds to inactivate. Channels gating in all three modes contributed to the macroscopic A current from the whole cell, but their relative contribution differed among neurons. In addition, A channels could go directly from the closed, or resting, state to the inactivated state without opening, and the probability for channels inactivating in this way was greater at less depolarized voltages. In addition, a few A channels appeared to go reversibly from a mode where inactivation occurred rapidly to a slow mode of inactivation. PMID:2592953

Memory consolidation in human sleep depends on inhibition of glucocorticoid release.

PubMed

Plihal, W; Born, J

1999-09-09

Early sleep dominated by slow-wave sleep has been found to be particularly relevant for declarative memory formation via hippocampo-neocortical networks. Concurrently, early nocturnal sleep is characterized by an inhibition of glucocorticoid release from the adrenals. Here, we show in healthy humans that this inhibition serves to support declarative memory consolidation during sleep. Elevating plasma glucocorticoid concentration during early sleep by administration of cortisol impaired consolidation of paired associate words, but not of non-declarative memory of visuomotor skills. Since glucocorticoid concentration was enhanced only during retention sleep, but not during acquisition or retrieval, a specific effect on the consolidation process is indicated. Blocking mineralocorticoid receptors by canrenoate did not affect memory, suggesting inactivation of glucocorticoid receptors to be the essential prerequisite for memory consolidation during early sleep.

Thermal Inactivation of Feline Calicivirus in Pet Food Processing.

PubMed

Haines, J; Patel, M; Knight, A I; Corley, D; Gibson, G; Schaaf, J; Moulin, J; Zuber, S

2015-12-01

Extrusion is the most common manufacturing process used to produce heat-treated dry dog and cat food (pet food) for domestic use and international trade. Due to reoccurring outbreaks of notifiable terrestrial animal diseases and their impact on international trade, experiments were undertaken to demonstrate the effectiveness of heat-treated extruded pet food on virus inactivation. The impact of extrusion processing in a pet food matrix on virus inactivation has not been previously reported and very few inactivation studies have examined the thermal inactivation of viruses in complex food matrices. The feline calicivirus vaccine strain FCV F-9 was used as a surrogate model RNA virus pathogen. Small-scale heat inactivation experiments using animal-derived pet food raw materials showed that a > 4 log10 reduction (log10 R) in infectivity occurred at 70 °C prior to reaching the minimum extrusion manufacturing operating temperature of 100 °C. As anticipated, small-scale pressure studies at extrusion pressure (1.6 MPa) showed no apparent effect on FCV F-9 inactivation. Additionally, FCV F-9 was shown not to survive the acidic conditions used to produce pet food palatants of animal origin that are typically used as a coating after the extrusion process.

Overcoming Rapid Inactivation of Lung Surfactant: Analogies Between Competitive Adsorption and Colloid Stability

PubMed Central

Zasadzinski, Joseph A.; Stenger, Patrick C.; Shieh, Ian; Dhar, Prajnaparamita

2009-01-01

Lung surfactant (LS) is a mixture of lipids and proteins that line the alveolar air-liquid interface, lowering the interfacial tension to levels that make breathing possible. In acute respiratory distress syndrome (ARDS), inactivation of LS is believed to play an important role in the development and severity of the disease. This review examines the competitive adsorption of LS and surface-active contaminants, such as serum proteins, present in the alveolar fluids of ARDS patients, and how this competitive adsorption can cause normal amounts of otherwise normal LS to be ineffective in lowering the interfacial tension. LS and serum proteins compete for the air-water interface when both are present in solution either in the alveolar fluids or in a Langmuir trough. Equilibrium favors LS as it has the lower equilibrium surface pressure, but the smaller proteins are kinetically favored over multi-micron LS bilayer aggregates by faster diffusion. If albumin reaches the interface, it creates an energy barrier to subsequent LS adsorption that slows or prevents the adsorption of the necessary amounts of LS required to lower surface tension. This process can be understood in terms of classic colloid stability theory in which an energy barrier to diffusion stabilizes colloidal suspensions against aggregation. This analogy provides qualitative and quantitative predictions regarding the origin of surfactant inactivation. An important corollary is that any additive that promotes colloid coagulation, such as increased electrolyte concentration, multivalent ions, hydrophilic non-adsorbing polymers such as PEG, dextran, etc. or polyelectrolytes such as chitosan, added to LS, also promotes LS adsorption in the presence of serum proteins and helps reverse surfactant inactivation. The theory provides quantitative tools to determine the optimal concentration of these additives and suggests that multiple additives may have a synergistic effect. A variety of physical and chemical techniques including isotherms, fluorescence microscopy, electron microscopy and X-ray diffraction show that LS adsorption is enhanced by this mechanism without substantially altering the structure or properties of the LS monolayer. PMID:20026298

Curing conditions to inactivate Trichinella spiralis muscle larvae in ready-to-eat pork sausage

USDA-ARS?s Scientific Manuscript database

Curing processes for ready to eat (RTE) pork products currently require individual validation of methods to demonstrate inactivation of Trichinella spiralis. This is a major undertaking for each process; currently no model of meat chemistry exists that can be correlated with inactivation of Trichin...

Inactivation mechanisms of pathogenic bacteria in several matrixes during the composting process in a composting toilet.

PubMed

Sossou, S K; Hijikata, N; Sou, M; Tezuka, R; Maiga, A H; Funamizu, N

2014-01-01

This study aimed to compare the inactivation rate and the mechanisms of pathogenic bacteria in three matrixes (sawdust, rice husk and charcoal) during the composting process. The inactivation rate was evaluated with Escherichia coli strain and the damaged parts and/or functions were evaluated with three different media. Normalized inactivation rate constant in three media and from three matrixes had no significant difference in each process (pure, 1 month and 2 months). The value in rice husk was relatively increased during 2 months but there was no significant difference. The inactivation rate constants of Tryptic Soy Agar (TSA) and Compact Dry E. coli/Coliform in pure sawdust and rice husk were relatively lower than that of Desoxycholate Agar, but increased in 2 months. This indicated that damaging part was changed from outer membrane to enzymes and metabolisms during the 2-month composting process. In the case of charcoal, only the TSA value in apure matrix was relatively lower than that of others, but it increased in 2 months. This indicated that damaging part was changed from outer membrane and enzyme to metabolisms during the composting process. Composting matrix and composting process did not significantly affect inactivation rate of pathogenic bacteria during the process but affected the damaging part of the bacteria.

Calcineurin Dysregulation Underlies Spinal Cord Injury-Induced K+ Channel Dysfunction in DRG Neurons

PubMed Central

Zemel, Benjamin M.; Brown, Eric V.; Urban, Mark W.; Tymanskyj, Stephen R.; Lepore, Angelo C.

2017-01-01

Dysfunction of the fast-inactivating Kv3.4 potassium current in dorsal root ganglion (DRG) neurons contributes to the hyperexcitability associated with persistent pain induced by spinal cord injury (SCI). However, the underlying mechanism is not known. In light of our previous work demonstrating modulation of the Kv3.4 channel by phosphorylation, we investigated the role of the phosphatase calcineurin (CaN) using electrophysiological, molecular, and imaging approaches in adult female Sprague Dawley rats. Pharmacological inhibition of CaN in small-diameter DRG neurons slowed repolarization of the somatic action potential (AP) and attenuated the Kv3.4 current. Attenuated Kv3.4 currents also exhibited slowed inactivation. We observed similar effects on the recombinant Kv3.4 channel heterologously expressed in Chinese hamster ovary cells, supporting our findings in DRG neurons. Elucidating the molecular basis of these effects, mutation of four previously characterized serines within the Kv3.4 N-terminal inactivation domain eliminated the effects of CaN inhibition on the Kv3.4 current. SCI similarly induced concurrent Kv3.4 current attenuation and slowing of inactivation. Although there was little change in CaN expression and localization after injury, SCI induced upregulation of the native regulator of CaN 1 (RCAN1) in the DRG at the transcript and protein levels. Consistent with CaN inhibition resulting from RCAN1 upregulation, overexpression of RCAN1 in naive DRG neurons recapitulated the effects of pharmacological CaN inhibition on the Kv3.4 current and the AP. Overall, these results demonstrate a novel regulatory pathway that links CaN, RCAN1, and Kv3.4 in DRG neurons. Dysregulation of this pathway might underlie a peripheral mechanism of pain sensitization induced by SCI. SIGNIFICANCE STATEMENT Pain sensitization associated with spinal cord injury (SCI) involves poorly understood maladaptive modulation of neuronal excitability. Although central mechanisms have received significant attention, recent studies have identified peripheral nerve hyperexcitability as a driver of persistent pain signaling after SCI. However, the ion channels and signaling molecules responsible for this change in primary sensory neuron excitability are still not well defined. To address this problem, this study used complementary electrophysiological and molecular methods to determine how Kv3.4, a voltage-gated K+ channel robustly expressed in dorsal root ganglion neurons, becomes dysfunctional upon calcineurin (CaN) inhibition. The results strongly suggest that CaN inhibition underlies SCI-induced dysfunction of Kv3.4 and the associated excitability changes through upregulation of the native regulator of CaN 1 (RCAN1). PMID:28751455

Studies of inactivation mechanism of non-enveloped icosahedral virus by a visible ultrashort pulsed laser

PubMed Central

2014-01-01

Background Low-power ultrashort pulsed (USP) lasers operating at wavelengths of 425 nm and near infrared region have been shown to effectively inactivate viruses such as human immunodeficiency virus (HIV), M13 bacteriophage, and murine cytomegalovirus (MCMV). It was shown previously that non-enveloped, helical viruses such as M13 bacteriophage, were inactivated by a USP laser through an impulsive stimulated Raman scattering (ISRS) process. Recently, enveloped virus like MCMV has been shown to be inactivated by a USP laser via protein aggregation induced by an ISRS process. However, the inactivation mechanism for a clinically important class of viruses – non-enveloped, icosahedral viruses remains unknown. Results and discussions We have ruled out the following four possible inactivation mechanisms for non-enveloped, icosahedral viruses, namely, (1) inactivation due to ultraviolet C (UVC) photons produced by non-linear optical process of the intense, fundamental laser beam at 425 nm; (2) inactivation caused by thermal heating generated by the direct laser absorption/heating of the virion; (3) inactivation resulting from a one-photon absorption process via chromophores such as porphyrin molecules, or indicator dyes, potentially producing reactive oxygen or other species; (4) inactivation by the USP lasers in which the extremely intense laser pulse produces shock wave-like vibrations upon impact with the viral particle. We present data which support that the inactivation mechanism for non-enveloped, icosahedral viruses is the impulsive stimulated Raman scattering process. Real-time PCR experiments show that, within the amplicon size of 273 bp tested, there is no damage on the genome of MNV-1 caused by the USP laser irradiation. Conclusion We conclude that our model non-enveloped virus, MNV-1, is inactivated by the ISRS process. These studies provide fundamental knowledge on photon-virus interactions on femtosecond time scales. From the analysis of the transmission electron microscope (TEM) images of viral particles before and after USP laser irradiation, the locations of weak structural links on the capsid of MNV-1 were revealed. This important information will greatly aid our understanding of the structure of non-enveloped, icosahedral viruses. We envision that this non-invasive, efficient viral eradication method will find applications in the disinfection of pharmaceuticals, biologicals and blood products in the near future. PMID:24495489

Inactivation of Caliciviruses

PubMed Central

Nims, Raymond; Plavsic, Mark

2013-01-01

The Caliciviridae family of viruses contains clinically important human and animal pathogens, as well as vesivirus 2117, a known contaminant of biopharmaceutical manufacturing processes employing Chinese hamster cells. An extensive literature exists for inactivation of various animal caliciviruses, especially feline calicivirus and murine norovirus. The caliciviruses are susceptible to wet heat inactivation at temperatures in excess of 60 °C with contact times of 30 min or greater, to UV-C inactivation at fluence ≥30 mJ/cm2, to high pressure processing >200 MPa for >5 min at 4 °C, and to certain photodynamic inactivation approaches. The enteric caliciviruses (e.g.; noroviruses) display resistance to inactivation by low pH, while the non-enteric species (e.g.; feline calicivirus) are much more susceptible. The caliciviruses are inactivated by a variety of chemicals, including alcohols, oxidizing agents, aldehydes, and β-propiolactone. As with inactivation of viruses in general, inactivation of caliciviruses by the various approaches may be matrix-, temperature-, and/or contact time-dependent. The susceptibilities of the caliciviruses to the various physical and chemical inactivation approaches are generally similar to those displayed by other small, non-enveloped viruses, with the exception that the parvoviruses and circoviruses may require higher temperatures for inactivation, while these families appear to be more susceptible to UV-C inactivation than are the caliciviruses. PMID:24276023

Pressure- and heat-induced inactivation of butyrylcholinesterase: evidence for multiple intermediates and the remnant inactivation process.

PubMed Central

Weingand-Ziade, A; Ribes, F; Renault, F; Masson, P

2001-01-01

The inactivation process of native (N) human butyrylcholinesterase (BuChE) by pressure and/or heat was found to be multi-step. It led to irreversible formation of an active intermediate (I) state and a denatured state. This series-inactivation process was described by expanding the Lumry-Eyring [Lumry, R. and Eyring, H. (1954) J. Phys. Chem. 58, 110-120] model. The intermediate state (I) was found to have a K(m) identical with that of the native state and a turnover rate (k(cat)) twofold higher than that of the native state with butyrylthiocholine as the substrate. The increased catalytic efficiency (k(cat)/K(m)) of I can be explained by a conformational change in the active-site gorge and/or restructuring of the water-molecule network in the active-site pocket, making the catalytic steps faster. However, a pressure/heat-induced covalent modification of native BuChE, affecting the catalytic machinery, cannot be ruled out. The inactivation process of BuChE induced by the combined action of pressure and heat was found to continue after interruption of pressure/temperature treatment. This secondary inactivation process was termed 'remnant inactivation'. We hypothesized that N and I were in equilibrium with populated metastable N' and I' states. The N' and I' states can either return to the active forms, N and I, or develop into inactive forms, N(')(in) and I(')(in). Both active N' and I' intermediate states displayed different rates of remnant inactivation depending on the pressure and temperature pretreatments and on the storage temperature. A first-order deactivation model describing the kinetics of the remnant inactivation of BuChE is proposed. PMID:11368776

Validating the Inactivation Effectiveness of Chemicals on Ebola Virus.

PubMed

Haddock, Elaine; Feldmann, Friederike

2017-01-01

While viruses such as Ebola virus must be handled in high-containment laboratories, there remains the need to process virus-infected samples for downstream research testing. This processing often includes removal to lower containment areas and therefore requires assurance of complete viral inactivation within the sample before removal from high-containment. Here we describe methods for the removal of chemical reagents used in inactivation procedures, allowing for validation of the effectiveness of various inactivation protocols.

Mechanism of HERG potassium channel inhibition by tetra-n-octylammonium bromide and benzethonium chloride

DOE Office of Scientific and Technical Information (OSTI.GOV)

Long, Yan; Lin, Zuoxian; Xia, Menghang

Tetra-n-octylammonium bromide and benzethonium chloride are synthetic quaternary ammonium salts that are widely used in hospitals and industries for the disinfection and surface treatment and as the preservative agent. Recently, the activities of HERG channel inhibition by these compounds have been found to have potential risks to induce the long QT syndrome and cardiac arrhythmia, although the mechanism of action is still elusive. This study was conducted to investigate the mechanism of HERG channel inhibition by these compounds by using whole-cell patch clamp experiments in a CHO cell line stably expressing HERG channels. Tetra-n-octylammonium bromide and benzethonium chloride exhibited concentration-dependentmore » inhibitions of HERG channel currents with IC{sub 50} values of 4 nM and 17 nM, respectively, which were also voltage-dependent and use-dependent. Both compounds shifted the channel activation I–V curves in a hyperpolarized direction for 10–15 mV and accelerated channel activation and inactivation processes by 2-fold. In addition, tetra-n-octylammonium bromide shifted the inactivation I–V curve in a hyperpolarized direction for 24.4 mV and slowed the rate of channel deactivation by 2-fold, whereas benzethonium chloride did not. The results indicate that tetra-n-octylammonium bromide and benzethonium chloride are open-channel blockers that inhibit HERG channels in the voltage-dependent, use-dependent and state-dependent manners. - Highlights: ► Tetra-n-octylammonium and benzethonium are potent HERG channel inhibitors. ► Channel activation and inactivation processes are accelerated by the two compounds. ► Both compounds are the open-channel blockers to HERG channels. ► HERG channel inhibition by both compounds is use-, voltage- and state dependent. ► The in vivo risk of QT prolongation needs to be studied for the two compounds.« less

Incorporation of DPP6a and DPP6K variants in ternary Kv4 channel complex reconstitutes properties of A-type K current in rat cerebellar granule cells.

PubMed

Jerng, Henry H; Pfaffinger, Paul J

2012-01-01

Dipeptidyl peptidase-like protein 6 (DPP6) proteins co-assemble with Kv4 channel α-subunits and Kv channel-interacting proteins (KChIPs) to form channel protein complexes underlying neuronal somatodendritic A-type potassium current (I(SA)). DPP6 proteins are expressed as N-terminal variants (DPP6a, DPP6K, DPP6S, DPP6L) that result from alternative mRNA initiation and exhibit overlapping expression patterns. Here, we study the role DPP6 variants play in shaping the functional properties of I(SA) found in cerebellar granule (CG) cells using quantitative RT-PCR and voltage-clamp recordings of whole-cell currents from reconstituted channel complexes and native I(SA) channels. Differential expression of DPP6 variants was detected in rat CG cells, with DPP6K (41 ± 3%)>DPP6a (33 ± 3%)>DPP6S (18 ± 2%)>DPP6L (8 ± 3%). To better understand how DPP6 variants shape native neuronal I(SA), we focused on studying interactions between the two dominant variants, DPP6K and DPP6a. Although previous studies did not identify unique functional effects of DPP6K, we find that the unique N-terminus of DPP6K modulates the effects of KChIP proteins, slowing recovery and producing a negative shift in the steady-state inactivation curve. By contrast, DPP6a uses its distinct N-terminus to directly confer rapid N-type inactivation independently of KChIP3a. When DPP6a and DPP6K are co-expressed in ratios similar to those found in CG cells, their distinct effects compete in modulating channel function. The more rapid inactivation from DPP6a dominates during strong depolarization; however, DPP6K produces a negative shift in the steady-state inactivation curve and introduces a slow phase of recovery from inactivation. A direct comparison to the native CG cell I(SA) shows that these mixed effects are present in the native channels. Our results support the hypothesis that the precise expression and co-assembly of different auxiliary subunit variants are important factors in shaping the I(SA) functional properties in specific neuronal populations.

Solar disinfection of drinking water contained in transparent plastic bottles: characterizing the bacterial inactivation process.

PubMed

McGuigan, K G; Joyce, T M; Conroy, R M; Gillespie, J B; Elmore-Meegan, M

1998-06-01

A series of experiments is reported to identify and characterize the inactivation process in operation when drinking water, heavily contaminated with a Kenyan isolate of Escherichia coli, is stored in transparent plastic bottles that are then exposed to sunlight. The roles of optical and thermal inactivation mechanisms are studied in detail by simulating conditions of optical irradiance, water turbidity and temperature, which were recorded during a series of solar disinfection measurements carried out in the Kenyan Rift Valley. Optical inactivation effects are observed even in highly turbid water (200 ntu) and at low irradiances of only 10 mW cm-2. Thermal inactivation is found to be important only at water temperatures above 45 degrees C, at which point strong synergy between optical and thermal inactivation processes is observed. The results confirm that, where strong sunshine is available, solar disinfection of drinking water is an effective, low cost method for improving water quality and may be of particular use to refugee camps in disaster areas. Strategies for improving bacterial inactivation are discussed.

Quality-related enzymes in plant-based products: effects of novel food-processing technologies part 3: ultrasonic processing.

PubMed

Terefe, Netsanet Shiferaw; Buckow, Roman; Versteeg, Cornelis

2015-01-01

High-power ultrasound is a versatile technology which can potentially be used in many food processing applications including food preservation. This is part 2 of a series of review articles dealing with the effectiveness of nonthermal food processing technologies in food preservation focusing on their effect on enzymes. Typically, ultrasound treatment alone does not efficiently cause microbial or enzyme inactivation sufficient for food preservation. However, combined with mild heat with or without elevated pressure (P ≤ 500 kPa), ultrasound can effectively inactivate enzymes and microorganisms. Synergistic effects between ultrasound and mild heat have been reported for the inactivation of both enzymes and microorganisms. The application of ultrasound has been shown to enhance the rate of inactivation of quality degrading enzymes including pectin methylesterase (PME), polygalacturonase (PG), peroxidase (POD), polyphenol oxidase (PPO), and lipoxygenase (LOX) at mild temperature by up to 400 times. Moreover, ultrasound enables the inactivation of relatively heat-resistant enzymes such as tomato PG1 and thermostable orange PME at mild temperature conditions. The extent to which ultrasound enhances the inactivation rate depends on the type of enzyme, the medium in which the enzyme is suspended, and the processing condition including frequency, ultrasonic intensity, temperature, and pressure. The physical and chemical effects of cavitation are considered to be responsible for the ultrasound-induced inactivation of enzymes, although the dominant mechanism depends on the structure of the enzyme.

Active α-macroglobulin is a reservoir for urokinase after fibrinolytic therapy in rabbits with tetracycline-induced pleural injury and in human pleural fluids

PubMed Central

Florova, Galina; Azghani, Ali; Karandashova, Sophia; Kurdowska, Anna K.; Idell, Steven

2013-01-01

Intrapleural processing of prourokinase (scuPA) in tetracycline (TCN)-induced pleural injury in rabbits was evaluated to better understand the mechanisms governing successful scuPA-based intrapleural fibrinolytic therapy (IPFT), capable of clearing pleural adhesions in this model. Pleural fluid (PF) was withdrawn 0–80 min and 24 h after IPFT with scuPA (0–0.5 mg/kg), and activities of free urokinase (uPA), plasminogen activator inhibitor-1 (PAI-1), and uPA complexed with α-macroglobulin (αM) were assessed. Similar analyses were performed using PFs from patients with empyema, parapneumonic, and malignant pleural effusions. The peak of uPA activity (5–40 min) reciprocally correlated with the dose of intrapleural scuPA. Endogenous active PAI-1 (10–20 nM) decreased the rate of intrapleural scuPA activation. The slow step of intrapleural inactivation of free uPA (t1/2β = 40 ± 10 min) was dose independent and 6.7-fold slower than in blood. Up to 260 ± 70 nM of αM/uPA formed in vivo [second order association rate (kass) = 580 ± 60 M−1·s−1]. αM/uPA and products of its degradation contributed to durable intrapleural plasminogen activation up to 24 h after IPFT. Active PAI-1, active α2M, and α2M/uPA found in empyema, pneumonia, and malignant PFs demonstrate the capacity to support similar mechanisms in humans. Intrapleural scuPA processing differs from that in the bloodstream and includes 1) dose-dependent control of scuPA activation by endogenous active PAI-1; 2) two-step inactivation of free uPA with simultaneous formation of αM/uPA; and 3) slow intrapleural degradation of αM/uPA releasing active free uPA. This mechanism offers potential clinically relevant advantages that may enhance the bioavailability of intrapleural scuPA and may mitigate the risk of bleeding complications. PMID:23997178

Inhibition of cardiac sodium currents by toluene exposure

PubMed Central

Cruz, Silvia L; Orta-Salazar, Gerardo; Gauthereau, Marcia Y; Millan-Perez Peña, Lourdes; Salinas-Stefanón, Eduardo M

2003-01-01

Toluene is an industrial solvent widely used as a drug of abuse, which can produce sudden sniffing death due to cardiac arrhythmias. In this paper, we tested the hypothesis that toluene inhibits cardiac sodium channels in Xenopus laevis oocytes transfected with Nav1.5 cDNA and in isolated rat ventricular myocytes. In oocytes, toluene inhibited sodium currents (INa+) in a concentration-dependent manner, with an IC50 of 274 μM (confidence limits: 141–407μM). The inhibition was complete, voltage-independent, and slowly reversible. Toluene had no effect on: (i) the shape of the I–V curves; (ii) the reversal potential of Na+; and (iii) the steady-state inactivation. The slow recovery time constant from inactivation of INa+ decreased with toluene exposure, while the fast recovery time constant remained unchanged. Block of INa+ by toluene was use- and frequency-dependent. In rat cardiac myocytes, 300 μM toluene inhibited the sodium current (INa+) by 62%; this inhibition was voltage independent. These results suggest that toluene binds to cardiac Na+ channels in the open state and unbinds either when channels move between inactivated states or from an inactivated to a closed state. The use- and frequency-dependent block of INa+ by toluene might be responsible, at least in part, for its arrhythmogenic effect. PMID:14534149

Connexin40 and connexin43 determine gating properties of atrial gap junction channels.

PubMed

Lin, Xianming; Gemel, Joanna; Glass, Aaron; Zemlin, Christian W; Beyer, Eric C; Veenstra, Richard D

2010-01-01

While ventricular gap junctions contain only Cx43, atrial gap junctions contain both Cx40 and Cx43; yet the functional consequences of this co-expression remain poorly understood. We quantitated the expression of Cx40 and Cx43 and their contributions to atrial gap junctional conductance (g(j)). Neonatal murine atrial myocytes showed similar abundances of Cx40 and Cx43 proteins, while ventricular myocytes contained at least 20 times more Cx43 than Cx40. Since Cx40 gap junction channels are blocked by 2 mM spermine while Cx43 channels are unaffected, we used spermine block as a functional dual whole cell patch clamp assay to determine Cx40 contributions to cardiac g(j). Slightly more than half of atrial g(j) and

Effect of iron salt counter ion in dose-response curves for inactivation of Fusarium solani in water through solar driven Fenton-like processes

NASA Astrophysics Data System (ADS)

Aurioles-López, Verónica; Polo-López, M. Inmaculada; Fernández-Ibáñez, Pilar; López-Malo, Aurelio; Bandala, Erick R.

2016-02-01

The inactivation of Fusarium solani in water was assessed by solar driven Fenton-like processes using three different iron salts: ferric acetylacetonate (Fe(acac)3), ferric chloride (FeCl3) and ferrous sulfate (FeSO4). The experimental conditions tested were [Fe] ≈ 5 mg L-1, [H2O2] ≈ 10 mg L-1 and [Fe] ≈ 10 mg L-1; [H2O2] ≈ 20 mg L-1 mild and high, respectively, and pH 3.0 and 5.0, under solar radiation. The highest inactivation rates were observed at high reaction conditions for the three iron salts tested at pH 5.0 with less than 3.0 kJ L-1 of accumulate energy (QUV) to achieve over 99.9% of F. solani inactivation. Fe(acac)3 was the best iron salt to accomplishing F. solani inactivation. The modified Fermi equation was used to fix the experimental inactivation, data showed it was helpful for modeling the process, adequately describing dose-response curves. Inactivation process using FeSO4 at pH 3.0 was modeled fairly with r2 = 0.98 and 0.99 (mild and high concentration, respectively). Fe(acac)3, FeCl3 and FeSO4 at high concentration (i.e. [Fe] ≈ 10 mg L-1; [H2O2] ≈ 20 mg L-1) and pH 5.0 showed the highest fitting values (r2 = 0.99). Iron salt type showed a remarkable influence on the Fenton-like inactivation process.

S-glutathionylation of an auxiliary subunit confers redox sensitivity to Kv4 channel inactivation.

PubMed

Jerng, Henry H; Pfaffinger, Paul J

2014-01-01

Reactive oxygen species (ROS) regulate ion channels, modulate neuronal excitability, and contribute to the etiology of neurodegenerative disorders. ROS differentially suppress fast "ball-and-chain" N-type inactivation of cloned Kv1 and Kv3 potassium channels but not of Kv4 channels, likely due to a lack of reactive cysteines in Kv4 N-termini. Recently, we discovered that N-type inactivation of Kv4 channel complexes can be independently conferred by certain N-terminal variants of Kv4 auxiliary subunits (DPP6a, DPP10a). Here, we report that both DPP6a and DPP10a, like Kv subunits with redox-sensitive N-type inactivation, contain a highly conserved cysteine in their N-termini (Cys-13). To test if N-type inactivation mediated by DPP6a or DPP10a is redox sensitive, Xenopus oocyte recordings were performed to examine the effects of two common oxidants, tert-butyl hydroperoxide (tBHP) and diamide. Both oxidants markedly modulate DPP6a- or DPP10a-conferred N-type inactivation of Kv4 channels, slowing the overall inactivation and increasing the peak current. These functional effects are fully reversed by the reducing agent dithiothreitol (DTT) and appear to be due to a selective modulation of the N-type inactivation mediated by these auxiliary subunits. Mutation of DPP6a Cys-13 to serine eliminated the tBHP or diamide effects, confirming the importance of Cys-13 to the oxidative regulation. Biochemical studies designed to elucidate the underlying molecular mechanism show no evidence of protein-protein disulfide linkage formation following cysteine oxidation. Instead, using a biotinylated glutathione (BioGEE) reagent, we discovered that oxidation by tBHP or diamide leads to S-glutathionylation of Cys-13, suggesting that S-glutathionylation underlies the regulation of fast N-type inactivation by redox. In conclusion, our studies suggest that Kv4-based A-type current in neurons may show differential redox sensitivity depending on whether DPP6a or DPP10a is highly expressed, and that the S-glutathionylation mechanism may play a previously unappreciated role in mediating excitability changes and neuropathologies associated with ROS.

Inhibition of human Na(v)1.5 sodium channels by strychnine and its analogs.

PubMed

Yuan, Chunhua; Sun, Lirong; Zhang, Meng; Li, Shuji; Wang, Xuemin; Gao, Tianming; Zhu, Xinhong

2011-08-15

Strychnine and brucine from the seeds of the plant Strychnos nux vomica have been shown to have interesting pharmacological effects on several neurotransmitter receptors. In this study, we have characterized the pharmacological properties of strychnine and its analogs on human Na(v)1.5 channels to assess their potential therapeutic advantage in certain arrhythmias. Among the eight alkaloids, only strychnine and icajine exhibited inhibition potency on the Na(v)1.5 channel with the half-maximum inhibition (IC(50)) values of 83.1μM and 104.6μM, respectively. Structure-function analysis indicated that the increased bulky methoxy groups on the phenyl ring or the negatively charged oxygen atom may account for this lack of inhibition on the Na(v)1.5 channel. Strychnine and icajine may bind to the channel by cation-π interactions. The substitution with a large side chain on the phenyl ring or the increased molecular volume may alter the optimized position for the compound close to the binding sites of the channel. Strychnine and icajine bind to the Na(v)1.5 channel with a new mechanism that is different from TTX and local anesthetics. They bind to the outer vestibule of the channel pore with fast association and dissociation rates at resting state. Strychnine and icajine had little effect on steady-state fast inactivation but markedly shifted the slow inactivation of Na(v)1.5 currents toward more hyperpolarized potentials. The property of icajine influencing slow-inactivated state of Na(v)1.5 channel would be potential therapeutic advantages in certain arrhythmias. Copyright © 2011 Elsevier Inc. All rights reserved.

Comparison of Gating Properties and Use-Dependent Block of Nav1.5 and Nav1.7 Channels by Anti-Arrhythmics Mexiletine and Lidocaine

PubMed Central

Wang, Ying; Mi, Jianxun; Lu, Ka; Lu, Yanxin; Wang, KeWei

2015-01-01

Mexiletine and lidocaine are widely used class IB anti-arrhythmic drugs that are considered to act by blocking voltage-gated open sodium currents for treatment of ventricular arrhythmias and relief of pain. To gain mechanistic insights into action of anti-arrhythmics, we characterized biophysical properties of Nav1.5 and Nav1.7 channels stably expressed in HEK293 cells and compared their use-dependent block in response to mexiletine and lidocaine using whole-cell patch clamp recordings. While the voltage-dependent activation of Nav1.5 or Nav1.7 was not affected by mexiletine and lidocaine, the steady-state fast and slow inactivation of Nav1.5 and Nav1.7 were significantly shifted to hyperpolarized direction by either mexiletine or lidocaine in dose-dependent manner. Both mexiletine and lidocaine enhanced the slow component of closed-state inactivation, with mexiletine exerting stronger inhibition on either Nav1.5 or Nav1.7. The recovery from inactivation of Nav1.5 or Nav1.7 was significantly prolonged by mexiletine compared to lidocaine. Furthermore, mexiletine displayed a pronounced and prominent use-dependent inhibition of Nav1.5 than lidocaine, but not Nav1.7 channels. Taken together, our findings demonstrate differential responses to blockade by mexiletine and lidocaine that preferentially affect the gating of Nav1.5, as compared to Nav1.7; and mexiletine exhibits stronger use-dependent block of Nav1.5. The differential gating properties of Nav1.5 and Nav1.7 in response to mexiletine and lidocaine may help explain the drug effectiveness and advance in new designs of safe and specific sodium channel blockers for treatment of cardiac arrhythmia or pain. PMID:26068619

Voltage-dependent neuromodulation of Na+ channels by D1-like dopamine receptors in rat hippocampal neurons.

PubMed

Cantrell, A R; Scheuer, T; Catterall, W A

1999-07-01

Activation of D1-like dopamine (DA) receptors reduces peak Na+ current in acutely isolated hippocampal neurons through phosphorylation of the alpha subunit of the Na+ channel by cAMP-dependent protein kinase (PKA). Here we report that neuromodulation of Na+ currents by DA receptors via PKA is voltage-dependent in the range of -110 to -70 mV and is also sensitive to concurrent activation of protein kinase C (PKC). Depolarization enhanced the ability of D1-like DA receptors to reduce peak Na+ currents via the PKA pathway. Similar voltage-dependent modulation was observed when PKA was activated directly with the membrane-permeant PKA activator DCl-cBIMPS (cBIMPS; 20 microM), indicating that the membrane potential dependence occurs downstream of PKA. PKA activation caused only a small (-2.9 mV) shift in the voltage dependence of steady-state inactivation and had no effect on slow inactivation or on the rates of entry into the fast or slow inactivated states, suggesting that another mechanism is responsible for coupling of membrane potential changes to PKA modulation. Activation of PKC with a low concentration of the membrane-permeant diacylglycerol analog oleylacetyl glycerol also potentiated modulation by SKF 81297 or cBIMPS, and these effects were most striking at hyperpolarized membrane potentials where PKA modulation was not stimulated by membrane depolarization. Thus, activation of D1-like DA receptors causes a strong reduction in Na+ current via the PKA pathway, but it is effective primarily when it is combined with depolarization or activation of PKC. The convergence of these three distinct signaling modalities on the Na+ channel provides an intriguing mechanism for integration of information from multiple signaling pathways in the hippocampus and CNS.

Bacterial spore inactivation induced by cold plasma.

PubMed

Liao, Xinyu; Muhammad, Aliyu Idris; Chen, Shiguo; Hu, Yaqin; Ye, Xingqian; Liu, Donghong; Ding, Tian

2018-04-05

Cold plasma has emerged as a non-thermal technology for microbial inactivation in the food industry over the last decade. Spore-forming microorganisms pose challenges for microbiological safety and for the prevention of food spoilage. Inactivation of spores induced by cold plasma has been reported by several studies. However, the exact mechanism of spore deactivation by cold plasma is poorly understood; therefore, it is difficult to control this process and to optimize cold plasma processing for efficient spore inactivation. In this review, we summarize the factors that affect the resistance of spores to cold plasma, including processing parameters, environmental elements, and spore properties. We then describe possible inactivation targets in spore cells (e.g., outer structure, DNA, and metabolic proteins) that associated with inactivation by cold plasma according to previous studies. Kinetic models of the sporicidal activity of cold plasma have also been described here. A better understanding of the interaction between spores and cold plasma is essential for the development and optimization of cold plasma technology in food the industry.

Inactivation of pathogenic bacteria in food matrices: high pressure processing, photodynamic inactivation and pressure-assisted photodynamic inactivation

NASA Astrophysics Data System (ADS)

Cunha, A.; Couceiro, J.; Bonifácio, D.; Martins, C.; Almeida, A.; Neves, M. G. P. M. S.; Faustino, M. A. F.; Saraiva, J. A.

2017-09-01

Traditional food processing methods frequently depend on the application of high temperature. However, heat may cause undesirable changes in food properties and often has a negative impact on nutritional value and organoleptic characteristics. Therefore, reducing the microbial load without compromising the desirable properties of food products is still a technological challenge. High-pressure processing (HPP) can be classified as a cold pasteurization technique, since it is a non-thermal food preservation method that uses hydrostatic pressure to inactivate spoilage microorganisms. At the same time, it increases shelf life and retains the original features of food. Photodynamic inactivation (PDI) is also regarded as promising approach for the decontamination of food matrices. In this case, the inactivation of bacterial cells is achieved by the cytotoxic effects of reactive oxygens species (ROS) produced from the combined interaction of a photosensitizer molecule, light and oxygen. This short review examines some recent developments on the application of HPP and PDI with food-grade photosensitizers for the inactivation of listeriae, taken as a food pathogen model. The results of a proof-of-concept trial of the use of high-pressure as a coadjutant to increase the efficiency of photodynamic inactivation of bacterial endospores is also addressed.

Slowly inactivating component of Na+ current in peri-somatic region of hippocampal CA1 pyramidal neurons

PubMed Central

Park, Yul Young; Johnston, Daniel

2013-01-01

The properties of voltage-gated ion channels on the neuronal membrane shape electrical activity such as generation and backpropagation of action potentials, initiation of dendritic spikes, and integration of synaptic inputs. Subthreshold currents mediated by sodium channels are of interest because of their activation near rest, slow inactivation kinetics, and consequent effects on excitability. Modulation of these currents can also perturb physiological responses of a neuron that might underlie pathological states such as epilepsy. Using nucleated patches from the peri-somatic region of hippocampal CA1 neurons, we recorded a slowly inactivating component of the macroscopic Na+ current (which we have called INaS) that shared many biophysical properties with the persistent Na+ current, INaP, but showed distinctively faster inactivating kinetics. Ramp voltage commands with a velocity of 400 mV/s were found to elicit this component of Na+ current reliably. INaS also showed a more hyperpolarized I-V relationship and slower inactivation than those of the fast transient Na+ current (INaT) recorded in the same patches. The peak amplitude of INaS was proportional to the peak amplitude of INaT but was much smaller in amplitude. Hexanol, riluzole, and ranolazine, known Na+ channel blockers, were tested to compare their effects on both INaS and INaT. The peak conductance of INaS was preferentially blocked by hexanol and riluzole, but the shift of half-inactivation voltage (V1/2) was only observed in the presence of riluzole. Current-clamp measurements with hexanol suggested that INaS was involved in generation of an action potential and in upregulation of neuronal excitability. PMID:23236005

Defective Fast Inactivation Recovery of Nav1.4 in Congenital Myasthenic Syndrome

PubMed Central

Arnold, W. David; Feldman, Daniel H.; Ramirez, Sandra; He, Liuyuan; Kassar, Darine; Quick, Adam; Klassen, Tara L.; Lara, Marian; Nguyen, Joanna; Kissel, John T.; Lossin, Christoph; Maselli, Ricardo A.

2015-01-01

Objective To describe the unique phenotype and genetic findings in a 57-year-old female with a rare form of congenital myasthenic syndrome (CMS) associated with longstanding muscle fatigability, and to investigate the underlying pathophysiology. Methods We used whole-cell voltage clamping to compare the biophysical parameters of wild-type and Arg1457His-mutant Nav1.4. Results Clinical and neurophysiological evaluation revealed features consistent with CMS. Sequencing of candidate genes indicated no abnormalities. However, analysis of SCN4A, the gene encoding the skeletal muscle sodium channel Nav1.4, revealed a homozygous mutation predicting an arginine-to-histidine substitution at position 1457 (Arg1457His), which maps to the channel’s voltage sensor, specifically D4/S4. Whole-cell patch clamp studies revealed that the mutant required longer hyperpolarization to recover from fast inactivation, which produced a profound use-dependent current attenuation not seen in the wild type. The mutant channel also had a marked hyperpolarizing shift in its voltage dependence of inactivation as well as slowed inactivation kinetics. Interpretation We conclude that Arg1457His compromises muscle fiber excitability. The mutant fast-inactivates with significantly less depolarization, and it recovers only after extended hyperpolarization. The resulting enhancement in its use dependence reduces channel availability, which explains the patient’s muscle fatigability. Arg1457His offers molecular insight into a rare form of CMS precipitated by sodium channel inactivation defects. Given this channel’s involvement in other muscle disorders such as paramyotonia congenita and hyperkalemic periodic paralysis, our study exemplifies how variations within the same gene can give rise to multiple distinct dysfunctions and phenotypes, revealing residues important in basic channel function. PMID:25707578

Potassium channels cloned from neuroblastoma cells display slowly inactivating outward currents in Xenopus oocytes.

PubMed

Ito, Y; Yokoyama, S; Higashida, H

1992-05-22

Messenger RNAs (mRNAs) specific for NGK1 and NGK2 potassium channels were synthesized from complementary DNAs (cDNAs) that had been cloned from mouse neuroblastoma x rat glioma hybrid NG108-15 cells. Outward pottasium currents were evoked by 5 s depolarizing voltage commands in Xenopus oocytes injected with NGK1- or NGK2-specific mRNAs. The NGK1 or NGK2 currents showed different activation and inactivation kinetics, and different pharmacological sensitivities. The threshold potential for activation of the NGK2 current (-14 mV) was more positive than that for the NGK1 (-36 mV). The NGK2 current showed faster inactivation during a 5 s depolarizing pulse than did the NGK1 current. Inactivation was best fit by time constants of 0.37, 1.5 and 19 s for the NGK2 current and 4.4 and 19 s for NGK1. Extracellularly applied tetraethylammonium chloride (TEA) was 1000 times more potent on the NGK2 current than the NGK1 current. Furthermore we examined outward current following co-injection of an equal amount of mRNAs for NGK1 and NGK2. The timecourse of inactivation differed from either alone or from a simple sum of the two individual currents. TEA sensitivity could not be explained by summation of the two homomultimeric channels. These findings suggest that both NGK1 and NGK2 proteins assemble to form heteromultimeric K+ channels in addition to homomultimeric K+ channels. NGK2 channels and the heteromultimeric channels may be responsible for the native transient outward current with slow inactivation in NG108-15 hybrid cells.

Calmodulin and calcium differentially regulate the neuronal Nav1.1 voltage-dependent sodium channel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gaudioso, Christelle; Carlier, Edmond; Youssouf, Fahamoe

2011-07-29

Highlights: {yields} Both Ca{sup ++}-Calmodulin (CaM) and Ca{sup ++}-free CaM bind to the C-terminal region of Nav1.1. {yields} Ca{sup ++} and CaM have both opposite and convergent effects on I{sub Nav1.1}. {yields} Ca{sup ++}-CaM modulates I{sub Nav1.1} amplitude. {yields} CaM hyperpolarizes the voltage-dependence of activation, and increases the inactivation rate. {yields} Ca{sup ++} alone antagonizes CaM for both effects, and depolarizes the voltage-dependence of inactivation. -- Abstract: Mutations in the neuronal Nav1.1 voltage-gated sodium channel are responsible for mild to severe epileptic syndromes. The ubiquitous calcium sensor calmodulin (CaM) bound to rat brain Nav1.1 and to the human Nav1.1 channelmore » expressed by a stably transfected HEK-293 cell line. The C-terminal region of the channel, as a fusion protein or in the yeast two-hybrid system, interacted with CaM via a consensus C-terminal motif, the IQ domain. Patch clamp experiments on HEK1.1 cells showed that CaM overexpression increased peak current in a calcium-dependent way. CaM had no effect on the voltage-dependence of fast inactivation, and accelerated the inactivation kinetics. Elevating Ca{sup ++} depolarized the voltage-dependence of fast inactivation and slowed down the fast inactivation kinetics, and for high concentrations this effect competed with the acceleration induced by CaM alone. Similarly, the depolarizing action of calcium antagonized the hyperpolarizing shift of the voltage-dependence of activation due to CaM overexpression. Fluorescence spectroscopy measurements suggested that Ca{sup ++} could bind the Nav1.1 C-terminal region with micromolar affinity.« less

Reconstruction of the action potential of ventricular myocardial fibres

PubMed Central

Beeler, G. W.; Reuter, H.

1977-01-01

1. A mathematical model of membrane action potentials of mammalian ventricular myocardial fibres is described. The reconstruction model is based as closely as possible on ionic currents which have been measured by the voltage-clamp method. 2. Four individual components of ionic current were formulated mathematically in terms of Hodgkin—Huxley type equations. The model incorporates two voltage- and time-dependent inward currents, the excitatory inward sodium current, iNa, and a secondary or slow inward current, is, primarily carried by calcium ions. A time-independent outward potassium current, iK1, exhibiting inward-going rectification, and a voltage- and time-dependent outward current, ix1, primarily carried by potassium ions, are further elements of the model. 3. The iNa is primarily responsible for the rapid upstroke of the action potential, while the other current components determine the configuration of the plateau of the action potential and the re-polarization phase. The relative importance of inactivation of is and of activation of ix1 for termination of the plateau is evaluated by the model. 4. Experimental phenomena like slow recovery of the sodium system from inactivation, frequency dependence of the action potential duration, all-or-nothing re-polarization, membrane oscillations are adequately described by the model. 5. Possible inadequacies and shortcomings of the model are discussed. PMID:874889

Non-linear pressure/temperature-dependence of high pressure thermal inactivation of proteolytic Clostridium botulinum type B in foods.

PubMed

Maier, Maximilian B; Lenz, Christian A; Vogel, Rudi F

2017-01-01

The effect of high pressure thermal (HPT) processing on the inactivation of spores of proteolytic type B Clostridium botulinum TMW 2.357 in four differently composed low-acid foods (green peas with ham, steamed sole, vegetable soup, braised veal) was studied in an industrially feasible pressure range and temperatures between 100 and 120°C. Inactivation curves exhibited rapid inactivation during compression and decompression followed by strong tailing effects. The highest inactivation (approx. 6-log cycle reduction) was obtained in braised veal at 600 MPa and 110°C after 300 s pressure-holding time. In general, inactivation curves exhibited similar negative exponential shapes, but maximum achievable inactivation levels were lower in foods with higher fat contents. At high treatment temperatures, spore inactivation was more effective at lower pressure levels (300 vs. 600 MPa), which indicates a non-linear pressure/temperature-dependence of the HPT spore inactivation efficiency. A comparison of spore inactivation levels achievable using HPT treatments versus a conventional heat sterilization treatment (121.1°C, 3 min) illustrates the potential of combining high pressures and temperatures to replace conventional retorting with the possibility to reduce the process temperature or shorten the processing time. Finally, experiments using varying spore inoculation levels suggested the presence of a resistant fraction comprising approximately 0.01% of a spore population as reason for the pronounced tailing effects in survivor curves. The loss of the high resistance properties upon cultivation indicates that those differences develop during sporulation and are not linked to permanent modifications at the genetic level.

Determining thermal inactivation of Escherichia coli O157:H7 in fresh compost by simulating early phases of the composting process.

PubMed

Singh, Randhir; Kim, Jinkyung; Shepherd, Marion W; Luo, Feng; Jiang, Xiuping

2011-06-01

A three-strain mixture of Escherichia coli O157:H7 was inoculated into fresh dairy compost (ca. 10(7) CFU/g) with 40 or 50% moisture and was placed in an environmental chamber (ca. 70% humidity) that was programmed to ramp from room temperature to selected composting temperatures in 2 and 5 days to simulate the early composting phase. The surviving E. coli O157:H7 population was analyzed by direct plating and enrichment. Optimal and suboptimal compost mixes, with carbon/nitrogen (C/N) ratios of 25:1 and 16:1, respectively, were compared in this study. In the optimal compost mix, E. coli O157:H7 survived for 72, 48, and 24 h in compost with 40% moisture and for 72, 24, and 24 h with 50% moisture at 50, 55, and 60°C, respectively, following 2 days of come-up time (rate of heating up). However, in the suboptimal compost mix, the pathogen survived for 288, 72, and 48 h in compost with 40% moisture and for 240, 72, 24 h in compost with 50% moisture at the same temperatures, respectively. Pathogen survival was longer, with 5 days of come-up time compared with 2 days of come-up. Overall, E. coli O157:H7 was inactivated faster in the compost with 50% moisture than in the compost with 40% at 55 and 60°C. Both moisture and come-up time were significant factors affecting Weibull model parameters. Our results suggest that slow come-up time at the beginning of composting can extend pathogen survival during composting. Additionally, both the C/N ratio and the initial moisture level in the compost mix affect the rate of pathogen inactivation as well.

Determining Thermal Inactivation of Escherichia coli O157:H7 in Fresh Compost by Simulating Early Phases of the Composting Process ▿

PubMed Central

Singh, Randhir; Kim, Jinkyung; Shepherd, Marion W.; Luo, Feng; Jiang, Xiuping

2011-01-01

A three-strain mixture of Escherichia coli O157:H7 was inoculated into fresh dairy compost (ca. 107 CFU/g) with 40 or 50% moisture and was placed in an environmental chamber (ca. 70% humidity) that was programmed to ramp from room temperature to selected composting temperatures in 2 and 5 days to simulate the early composting phase. The surviving E. coli O157:H7 population was analyzed by direct plating and enrichment. Optimal and suboptimal compost mixes, with carbon/nitrogen (C/N) ratios of 25:1 and 16:1, respectively, were compared in this study. In the optimal compost mix, E. coli O157:H7 survived for 72, 48, and 24 h in compost with 40% moisture and for 72, 24, and 24 h with 50% moisture at 50, 55, and 60°C, respectively, following 2 days of come-up time (rate of heating up). However, in the suboptimal compost mix, the pathogen survived for 288, 72, and 48 h in compost with 40% moisture and for 240, 72, 24 h in compost with 50% moisture at the same temperatures, respectively. Pathogen survival was longer, with 5 days of come-up time compared with 2 days of come-up. Overall, E. coli O157:H7 was inactivated faster in the compost with 50% moisture than in the compost with 40% at 55 and 60°C. Both moisture and come-up time were significant factors affecting Weibull model parameters. Our results suggest that slow come-up time at the beginning of composting can extend pathogen survival during composting. Additionally, both the C/N ratio and the initial moisture level in the compost mix affect the rate of pathogen inactivation as well. PMID:21498743

Calcium Currents of Olfactory Bulb Juxtaglomerular Cells: Profile and Multiple Conductance Plateau Potential Simulation

PubMed Central

Masurkar, Arjun V.; Chen, Wei R.

2011-01-01

The olfactory glomerulus is the locus of information transfer between olfactory sensory neurons and output neurons of the olfactory bulb. Juxtaglomerular cells (JGCs) may influence intraglomerular processing by firing plateau potentials that support multiple spikes. It is unclear what inward currents mediate this firing pattern. In previous work, we characterized potassium currents of JGCs. We focus here on the inward currents using whole cell current clamp and voltage recording in a rat in vitro slice preparation, as well as computer simulation. We first showed that sodium current was not required to mediate plateau potentials. Voltage clamp characterization of calcium current (ICa) determined that ICa consisted of a slow activating, rapidly inactivating (τ10%–90% rise 6–8ms, τinactivation 38–77ms) component Icat1, similar to T-type currents, and a sustained (τinactivation≫500ms) component Icat2, likely composed of L-type and P/Q-type currents. We used computer simulation to test their roles in plateau potential firing. We robustly modeled Icat1 and Icat2 to Hodgkin-Huxley schemes (m3h and m2, respectively) and simulated a JGC plateau potential with 6 conductances: calcium currents as above, potassium currents from our prior study (A-type Ikt1, D-type Ikt2, delayed rectifier Ikt3), and a fast sodium current (INa). We demonstrated that Icat1 was required for mediating the plateau potential, unlike INa and Icat2, and its τinactivation determined plateau duration. We also found that Ikt1 dictated plateau potential shape more than Ikt2 and Ikt3. The influence of these two transient and opposing conductances suggests a unique mechanism of plateau potential physiology. PMID:21704681

Enzyme reactor design under thermal inactivation.

PubMed

Illanes, Andrés; Wilson, Lorena

2003-01-01

Temperature is a very relevant variable for any bioprocess. Temperature optimization of bioreactor operation is a key aspect for process economics. This is especially true for enzyme-catalyzed processes, because enzymes are complex, unstable catalysts whose technological potential relies on their operational stability. Enzyme reactor design is presented with a special emphasis on the effect of thermal inactivation. Enzyme thermal inactivation is a very complex process from a mechanistic point of view. However, for the purpose of enzyme reactor design, it has been oversimplified frequently, considering one-stage first-order kinetics of inactivation and data gathered under nonreactive conditions that poorly represent the actual conditions within the reactor. More complex mechanisms are frequent, especially in the case of immobilized enzymes, and most important is the effect of catalytic modulators (substrates and products) on enzyme stability under operation conditions. This review focuses primarily on reactor design and operation under modulated thermal inactivation. It also presents a scheme for bioreactor temperature optimization, based on validated temperature-explicit functions for all the kinetic and inactivation parameters involved. More conventional enzyme reactor design is presented merely as a background for the purpose of highlighting the need for a deeper insight into enzyme inactivation for proper bioreactor design.

Microbial Inactivation by Ultrasound Assisted Supercritical Fluids

NASA Astrophysics Data System (ADS)

Benedito, Jose; Ortuño, Carmen; Castillo-Zamudio, Rosa Isela; Mulet, Antonio

A method combining supercritical carbon dioxide (SC-CO2) and high power ultrasound (HPU) has been developed and tested for microbial/enzyme inactivation purposes, at different process conditions for both liquid and solid matrices. In culture media, using only SC-CO2, the inactivation rate of E. coli and S. cerevisiae increased with pressure and temperature; and the total inactivation (7-8 log-cycles) was attained after 25 and 140 min of SC-CO2 (350 bar, 36 °C) treatment, respectively. Using SC-CO2+HPU, the time for the total inactivation of both microorganisms was reduced to only 1-2 min, at any condition selected. The SC-CO2+HPU inactivation of both microorganisms was slower in juices (avg. 4.9 min) than in culture media (avg. 1.5 min). In solid samples (chicken, turkey ham and dry-cured pork cured ham) treated with SC-CO2 and SC-CO2+HPU, the inactivation rate of E. coli increased with temperature. The application of HPU to the SC-CO2 treatments accelerated the inactivation rate of E. coli and that effect was more pronounced in treatments with isotonic solution surrounding the solid food samples. The application of HPU enhanced the SC-CO2 inactivation mechanisms of microorganisms, generating a vigorous agitation that facilitated the CO2 solubilization and the mass transfer process. The cavitation generated by HPU could damage the cell walls accelerating the extraction of vital constituents and the microbial death. Thus, using the combined technique, reasonable industrial processing times and mild process conditions could be used which could result into a cost reduction and lead to the minimization in the food nutritional and organoleptic changes.

A modeling approach to estimate the solar disinfection of viral indicator organisms in waste stabilization ponds and surface waters.

PubMed

Kohn, Tamar; Mattle, Michael J; Minella, Marco; Vione, Davide

2016-01-01

Sunlight is known to be a pertinent factor governing the infectivity of waterborne viruses in the environment. Sunlight inactivates viruses via endogenous inactivation (promoted by absorption of solar light in the UVB range by the virus) and exogenous processes (promoted by adsorption of sunlight by external chromophores, which subsequently generate inactivating reactive species). The extent of inactivation is still difficult to predict, as it depends on multiple parameters including virus characteristics, solution composition, season and geographical location. In this work, we adapted a model typically used to estimate the photodegradation of organic pollutants, APEX, to explore the fate of two commonly used surrogates of human viruses (coliphages MS2 and ϕX174) in waste stabilization pond and natural surface water. Based on experimental data obtained in previous work, we modeled virus inactivation as a function of water depth and composition, as well as season and latitude, and we apportioned the contributions of the different inactivation processes to total inactivation. Model results showed that ϕX174 is inactivated more readily than MS2, except at latitudes >60°. ϕX174 inactivation varies greatly with both season (20-fold) and latitude (10-fold between 0 and 60°), and is dominated by endogenous inactivation under all solution conditions considered. In contrast, exogenous processes contribute significantly to MS2 inactivation. Because exogenous inactivation can be promoted by longer wavelengths, which are less affected by changes in season and latitude, MS2 exhibits smaller fluctuations in inactivation throughout the year (10-fold) and across the globe (3-fold between 0 and 60°) compared to ϕX174. While a full model validation is currently not possible due to the lack of sufficient field data, our estimated inactivation rates corresponded well to those reported in field studies. Overall, this study constitutes a step toward estimating microbial water quality as a function of spatio-temporal information and easy-to-determine solution parameters. Copyright © 2015 Elsevier Ltd. All rights reserved.

Radiation resistence of microorganisms from radiation sterilization processing environments

NASA Astrophysics Data System (ADS)

Sabovljev, Svetlana A.; Žunić, Zora S.

The radiation resistance of microorganisms was examined on the samples of dust collected from the radiation sterilization processing environments including assembly, storage, and sterilization plant areas. The isolation of radiation resistant strains was performed by irradiation with screening doses ranging from 10 to 35 kGy and test pieces containing 10 6 to 10 8 CFU in dried serum-broth, representing 100 to 5000 colonies of primary cultures of microorganisms from 7 different sites. In an examination of 16900 colonies of aerobic microorganisms from 3 hygienically controlled production sites and 4 uncontrolled ones, 30 strains of bacteria were isolated. Of those 15 were classified as genus Bacillus, 9 as Micrococcus and 6 as Sarcina. All of the 15 strains of Gram positive sporeforming aerobic rods exhibited an exponential decrease in the surviving fraction as a function of dose, indicating that the inactivation of spores of aerobic rods is a consequence of a single energy deposition into the target. All strains were found to be moderately resistant to radiation with D-6 values (dose required to reduce survival to 6 log cycles) between 18 and 26 kGy. All of the isolated Gram positive cocci showed inactivation curves having a shoulder, indicating that different processes are involved in the inactivation of these cells, e.g. accumulation of sublethal lesions, or final repair capacity of potential lethal lesions. Moderate radiation resistance was observed in 13 strains with D-6 values between 16 to 30 kGy. Two slow-growing, red pigmented strains tentatively classified as genus Micrococcus isolated from uncontrolled sites (human dwellings) were exceptionally resistant with D-6 more than 45 kGy. For hygienically controlled sites, Gram positive spereforming rods composed two thirds of the resistant microflora, while Gram positive cocci comprised one third. For hygienically uncontrolled sites this ratio was reversed. An assumption is made that one isolated strain has grown up from one CFU in the original microflora. In the light of the observed proportion of radiation resistant bacteria in microflora from hygienically controlled radiation sterilization processing environments, and assuming an initial contamination of 50 CFU per product unit, there is no more than one chance of a contaminated item remaining within a population of 10 7 items subjected to sterilization by exposure to a radiation dose of 25 kGy.

Effect of storage conditions in the response of Listeria monocytogenes in a fresh purple vegetable smoothie compared with an acidified TSB medium.

PubMed

González-Tejedor, Gerardo A; Garre, Alberto; Esnoz, A; Artés-Hernández, F; Fernández, P S

2018-06-01

In this study, growth and/or inactivation of Listeria monocytogenes 4032 at different inoculum levels in a vegetable smoothie with purple colour, (previously heat stabilised at 95 °C for 3 min) was evaluated. Growth/inactivation was compared with acidified TSB medium at the same pH level with HCl. Samples were stored at different temperatures (5, 10, 15 and 25 °C). All the smoothies stored at 15 and 25 °C showed growth up to 7.5-8.0 log CFU/mL and at 10 °C growth was only observed at the highest inoculum level. Growth was only observed at 25 °C in acidified TSB. In the case of the smoothies inoculated and stored at 5 °C, L. monocytogenes was not able to grow but survived for a long period of time, whereas at the lower inocula at 10 °C they presented a slow inactivation for an extended time. Acidified TSB inoculated and stored showed inactivation at 5, 10 and 15 °C. Best inactivation modelling alternatives are proposed. The differences between the smoothie and TSB medium about growth or survival in this study, even at relatively low pH values, were due to the favorable nutritional composition of the smoothie compared to a laboratory medium. Results in this study can allow to design safe conditions for smoothie production. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Complex formation between alpha-chymotrypsin and block copolymers based on ethylene and propylene oxide, induced by high pressure].

PubMed

Topchieva, I N; Sorokina, E M; Kurganov, B I; Zhulin, V M; Makarova, Z G

1996-06-01

A new method of formation of non-covalent adducts based on an amphiphilic diblock copolymer of ethylene and propylene oxides with molecular mass of 2 kDa and alpha-chymotrypsin (ChT) under high pressure, has been developed. The composition of the complexes corresponds to seven polymer molecules per one ChT molecule in the pressure range of 1.1 to 400 MPa. The complexes fully retain the catalytic activity. Kinetic constants (Km and kcat) for enzymatic hydrolysis of N-benzoyl-L-tyrosine ethyl ester catalyzed by the complexes are identical with the corresponding values for native ChT. Analysis of kinetics of thermal inactivation of the complexes revealed that the constant of the rate of the slow inactivation step is markedly lower than for ChT.

Calcineurin Dysregulation Underlies Spinal Cord Injury-Induced K+ Channel Dysfunction in DRG Neurons.

PubMed

Zemel, Benjamin M; Muqeem, Tanziyah; Brown, Eric V; Goulão, Miguel; Urban, Mark W; Tymanskyj, Stephen R; Lepore, Angelo C; Covarrubias, Manuel

2017-08-23

Dysfunction of the fast-inactivating Kv3.4 potassium current in dorsal root ganglion (DRG) neurons contributes to the hyperexcitability associated with persistent pain induced by spinal cord injury (SCI). However, the underlying mechanism is not known. In light of our previous work demonstrating modulation of the Kv3.4 channel by phosphorylation, we investigated the role of the phosphatase calcineurin (CaN) using electrophysiological, molecular, and imaging approaches in adult female Sprague Dawley rats. Pharmacological inhibition of CaN in small-diameter DRG neurons slowed repolarization of the somatic action potential (AP) and attenuated the Kv3.4 current. Attenuated Kv3.4 currents also exhibited slowed inactivation. We observed similar effects on the recombinant Kv3.4 channel heterologously expressed in Chinese hamster ovary cells, supporting our findings in DRG neurons. Elucidating the molecular basis of these effects, mutation of four previously characterized serines within the Kv3.4 N-terminal inactivation domain eliminated the effects of CaN inhibition on the Kv3.4 current. SCI similarly induced concurrent Kv3.4 current attenuation and slowing of inactivation. Although there was little change in CaN expression and localization after injury, SCI induced upregulation of the native regulator of CaN 1 (RCAN1) in the DRG at the transcript and protein levels. Consistent with CaN inhibition resulting from RCAN1 upregulation, overexpression of RCAN1 in naive DRG neurons recapitulated the effects of pharmacological CaN inhibition on the Kv3.4 current and the AP. Overall, these results demonstrate a novel regulatory pathway that links CaN, RCAN1, and Kv3.4 in DRG neurons. Dysregulation of this pathway might underlie a peripheral mechanism of pain sensitization induced by SCI. SIGNIFICANCE STATEMENT Pain sensitization associated with spinal cord injury (SCI) involves poorly understood maladaptive modulation of neuronal excitability. Although central mechanisms have received significant attention, recent studies have identified peripheral nerve hyperexcitability as a driver of persistent pain signaling after SCI. However, the ion channels and signaling molecules responsible for this change in primary sensory neuron excitability are still not well defined. To address this problem, this study used complementary electrophysiological and molecular methods to determine how Kv3.4, a voltage-gated K + channel robustly expressed in dorsal root ganglion neurons, becomes dysfunctional upon calcineurin (CaN) inhibition. The results strongly suggest that CaN inhibition underlies SCI-induced dysfunction of Kv3.4 and the associated excitability changes through upregulation of the native regulator of CaN 1 (RCAN1). Copyright © 2017 the authors 0270-6474/17/378257-17$15.00/0.

The S4–S5 Linker Acts as a Signal Integrator for hERG K+ Channel Activation and Deactivation Gating

PubMed Central

Ng, Chai Ann; Perry, Matthew D.; Tan, Peter S.; Hill, Adam P.; Kuchel, Philip W.; Vandenberg, Jamie I.

2012-01-01

Human ether-à-go-go-related gene (hERG) K+ channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in cardiac repolarisation. The slow activation and deactivation kinetics are regulated in part by the S4–S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4–S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4–S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4–S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4–S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4–S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel. PMID:22359612

Influenza Vaccine Manufacturing: Effect of Inactivation, Splitting and Site of Manufacturing. Comparison of Influenza Vaccine Production Processes

PubMed Central

Kon, Theone C.; Onu, Adrian; Berbecila, Laurentiu; Lupulescu, Emilia; Ghiorgisor, Alina; Kersten, Gideon F.; Cui, Yi-Qing; Amorij, Jean-Pierre; Van der Pol, Leo

2016-01-01

The aim of this study was to evaluate the impact of different inactivation and splitting procedures on influenza vaccine product composition, stability and recovery to support transfer of process technology. Four split and two whole inactivated virus (WIV) influenza vaccine bulks were produced and compared with respect to release criteria, stability of the bulk and haemagglutinin recovery. One clarified harvest of influenza H3N2 A/Uruguay virus prepared on 25.000 fertilized eggs was divided equally over six downstream processes. The main unit operation for purification was sucrose gradient zonal ultracentrifugation. The inactivation of the virus was performed with either formaldehyde in phosphate buffer or with beta-propiolactone in citrate buffer. For splitting of the viral products in presence of Tween®, either Triton™ X-100 or di-ethyl-ether was used. Removal of ether was established by centrifugation and evaporation, whereas removal of Triton-X100 was performed by hydrophobic interaction chromatography. All products were sterile filtered and subjected to a 5 months real time stability study. In all processes, major product losses were measured after sterile filtration; with larger losses for split virus than for WIV. The beta-propiolactone inactivation on average resulted in higher recoveries compared to processes using formaldehyde inactivation. Especially ether split formaldehyde product showed low recovery and least stability over a period of five months. PMID:26959983

Nondeterministic computational fluid dynamics modeling of Escherichia coli inactivation by peracetic acid in municipal wastewater contact tanks.

PubMed

Santoro, Domenico; Crapulli, Ferdinando; Raisee, Mehrdad; Raspa, Giuseppe; Haas, Charles N

2015-06-16

Wastewater disinfection processes are typically designed according to heuristics derived from batch experiments in which the interaction among wastewater quality, reactor hydraulics, and inactivation kinetics is often neglected. In this paper, a computational fluid dynamics (CFD) study was conducted in a nondeterministic (ND) modeling framework to predict the Escherichia coli inactivation by peracetic acid (PAA) in municipal contact tanks fed by secondary settled wastewater effluent. The extent and variability associated with the observed inactivation kinetics were both satisfactorily predicted by the stochastic inactivation model at a 95% confidence level. Moreover, it was found that (a) the process variability induced by reactor hydraulics is negligible when compared to the one caused by inactivation kinetics, (b) the PAA dose required for meeting regulations is dictated equally by the fixed limit of the microbial concentration as well as its probability of occurrence, and (c) neglecting the probability of occurrence during process sizing could lead to an underestimation of the PAA dose required by as much as 100%. Finally, the ND-CFD model was used to generate sizing information in the form of probabilistic disinfection curves relating E. coli inactivation and probability of occurrence with the average PAA dose and PAA residual concentration at the outlet of the contact tank.

Carvacrol modulates voltage-gated sodium channels kinetics in dorsal root ganglia.

PubMed

Joca, Humberto Cavalcante; Vieira, Daiana Cardoso Oliveira; Vasconcelos, Aliny Perreira; Araújo, Demetrius Antônio Machado; Cruz, Jader Santos

2015-06-05

Recent studies have shown that many of plant-derived compounds interact with specific ion channels and thereby modulate many sensing mechanisms, such as nociception. The monoterpenoid carvacrol (5-isopropyl-2-methylphenol) has an anti-nociceptive effect related to a reduction in neuronal excitability and voltage-gated Na(+) channels (NaV) inhibition in peripheral neurons. However, the detailed mechanisms of carvacrol-induced inhibition of neuronal NaV remain elusive. This study explores the interaction between carvacrol and NaV in isolated dorsal root ganglia neurons. Carvacrol reduced the total voltage-gated Na(+) current and tetrodotoxin-resistant (TTX-R) Na(+) current component in a concentration-dependent manner. Carvacrol accelerates current inactivation and induced a negative-shift in voltage-dependence of steady-state fast inactivation in total and TTX-R Na(+) current. Furthermore, carvacrol slowed the recovery from inactivation. Carvacrol provoked a leftward shift in both the voltage-dependence of steady-state inactivation and activation of the TTX-R Na(+) current component. In addition, carvacrol-induced inhibition of TTX-R Na(+) current was enhanced by an increase in stimulation frequency and when neurons were pre-conditioned with long depolarization pulse (5s at -50 mV). Taken all results together, we herein demonstrated that carvacrol affects NaV gating properties. The present findings would help to explain the mechanisms underlying the analgesic activity of carvacrol. Copyright © 2015 Elsevier B.V. All rights reserved.

Analogies and differences among bacterial and viral disinfection by the photo-Fenton process at neutral pH: a mini review.

PubMed

Giannakis, Stefanos

2017-12-19

Over the last years, the photo-Fenton process has been established as an effective, green alternative to chemical disinfection of waters and wastewaters. Microorganisms' inactivation is the latest success story in the application of this process at near-neutral pH, albeit without clearly elucidated inactivation mechanisms. In this review, the main pathways of the combined photo-Fenton process against the most frequent pathogen models (Escherichia coli for bacteria and MS2 bacteriophage for viruses) are analyzed. Firstly, the action of solar light is described and the specific inactivation mechanisms in bacteria (internal photo-Fenton) and viruses (genome damage) are presented. The contribution of the external pathways due to the potential presence of organic matter in generating reactive oxygen species (ROS) and their effects on microorganism inactivation are discussed. Afterwards, the effects of the gradual addition of Fe and H 2 O 2 are assessed and the differences among bacterial and viral inactivation are highlighted. As a final step, the simultaneous addition of both reagents induces the photo-Fenton in the bulk, focusing on the differences induced by the homogeneous or heterogeneous fraction of the process and the variation among the two respective targets. This work exploits the accumulated evidence on the mechanisms of bacterial inactivation and the scarce ones towards viral targets, aiming to bridge this knowledge gap and make possible the further application of the photo-Fenton process in the field of water/wastewater treatment.

Pulsed electric field processing of different fruit juices: impact of pH and temperature on inactivation of spoilage and pathogenic micro-organisms.

PubMed

Timmermans, R A H; Nierop Groot, M N; Nederhoff, A L; van Boekel, M A J S; Matser, A M; Mastwijk, H C

2014-03-03

Pulsed electrical field (PEF) technology can be used for the inactivation of micro-organisms and therefore for preservation of food products. It is a mild technology compared to thermal pasteurization because a lower temperature is used during processing, leading to a better retention of the quality. In this study, pathogenic and spoilage micro-organisms relevant in refrigerated fruit juices were studied to determine the impact of process parameters and juice composition on the effectiveness of the PEF process to inactivate the micro-organisms. Experiments were performed using a continuous-flow PEF system at an electrical field strength of 20 kV/cm with variable frequencies to evaluate the inactivation of Salmonella Panama, Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae in apple, orange and watermelon juices. Kinetic data showed that under the same conditions, S. cerevisiae was the most sensitive micro-organism, followed by S. Panama and E. coli, which displayed comparable inactivation kinetics. L. monocytogenes was the most resistant micro-organism towards the treatment conditions tested. A synergistic effect between temperature and electric pulses was observed at inlet temperatures above 35 °C, hence less energy for inactivation was required at higher temperatures. Different juice matrices resulted in a different degree of inactivation, predominantly determined by pH. The survival curves were nonlinear and could satisfactorily be modeled with the Weibull model. Copyright © 2013 Elsevier B.V. All rights reserved.

Feasibility of the silver-UV process for drinking water disinfection.

PubMed

Butkus, Michael A; Talbot, Mark; Labare, Michael P

2005-12-01

A synergistic effect between cationic silver and UV radiation (silver-UV disinfection) has been observed that can appreciably enhance inactivation of viruses. The purpose of this work was to assess the feasibility of this technique for drinking water disinfection and evaluate the effects of selected impurities, found in fresh water, and common parameters on inactivation of the coliphage MS-2 with the silver-UV process. Turbidity (kaolin), calcium hardness, carbonate alkalinity, and pH did not significantly degrade inactivation. Inactivation was reduced in the presence of chloride, at concentrations greater than 30 mg/L, and in water samples with UV-254 absorbance values greater than ca. 0.1 cm(-1). Inactivation of MS-2 with silver-UV disinfection was also reduced at high phosphate concentrations (above ca. 5 mM). Silver-UV inactivation of MS-2 increased with increases in temperature between 10 and 20 degrees C. Silver-UV inactivation of MS-2 was increased by greater than 1-log over UV alone, in two untreated fresh water sources, which indicates that silver-UV may be a viable treatment technology. An assessment of operation and management costs suggests that an increase in inactivation of MS-2 with silver-UV disinfection could be economically beneficial.

Physiology of the Inactivation of Vegetative Bacteria by Thermal Treatments: Mode of Action, Influence of Environmental Factors and Inactivation Kinetics

PubMed Central

Condón, Santiago; Mañas, Pilar

2017-01-01

Heat has been used extensively in the food industry as a preservation method, especially due to its ability to inactivate microorganisms present in foods. However, many aspects regarding the mechanisms of bacterial inactivation by heat and the factors affecting this process are still not fully understood. The purpose of this review is to offer a general overview of the most important aspects of the physiology of the inactivation or survival of microorganisms, particularly vegetative bacteria, submitted to heat treatments. This could help improve the design of current heat processes methods in order to apply milder and/or more effective treatments that could fulfill consumer requirements for fresh-like foods while maintaining the advantages of traditional heat treatments. PMID:29189748

Myostatin signals through Pax7 to regulate satellite cell self-renewal

DOE Office of Scientific and Technical Information (OSTI.GOV)

McFarlane, Craig; Department of Biological Sciences, University of Waikato, Hamilton; Hennebry, Alex

2008-01-15

Myostatin, a Transforming Growth Factor-beta (TGF-{beta}) super-family member, has previously been shown to negatively regulate satellite cell activation and self-renewal. However, to date the mechanism behind Myostatin function in satellite cell biology is not known. Here we show that Myostatin signals via a Pax7-dependent mechanism to regulate satellite cell self-renewal. While excess Myostatin inhibited Pax7 expression via ERK1/2 signaling, an increase in Pax7 expression was observed following both genetic inactivation and functional antagonism of Myostatin. As a result, we show that either blocking or inactivating Myostatin enhances the partitioning of the fusion-incompetent self-renewed satellite cell lineage (high Pax7 expression, lowmore » MyoD expression) from the pool of actively proliferating myogenic precursor cells. Consistent with this result, over-expression of Pax7 in C2C12 myogenic cells resulted in increased self-renewal through a mechanism which slowed both myogenic proliferation and differentiation. Taken together, these results suggest that increased expression of Pax7 promotes satellite cell self-renewal, and furthermore Myostatin may control the process of satellite cell self-renewal through regulation of Pax7. Thus we speculate that, in addition to the intrinsic factors (such as Pax7), extrinsic factors both positive and negative in nature, will play a major role in determining the stemness of skeletal muscle satellite cells.« less

Predicting Bacillus coagulans spores inactivation in tomato pulp under nonisothermal heat treatments.

PubMed

Zimmermann, Morgana; Longhi, Daniel A; Schaffner, Donald W; Aragão, Gláucia M F

2014-05-01

The knowledge and understanding of Bacillus coagulans inactivation during a thermal treatment in tomato pulp, as well as the influence of temperature variation during thermal processes are essential for design, calculation, and optimization of the process. The aims of this work were to predict B. coagulans spores inactivation in tomato pulp under varying time-temperature profiles with Gompertz-inspired inactivation model and to validate the model's predictions by comparing the predicted values with experimental data. B. coagulans spores in pH 4.3 tomato pulp at 4 °Brix were sealed in capillary glass tubes and heated in thermostatically controlled circulating oil baths. Seven different nonisothermal profiles in the range from 95 to 105 °C were studied. Predicted inactivation kinetics showed similar behavior to experimentally observed inactivation curves when the samples were exposed to temperatures in the upper range of this study (99 to 105 °C). Profiles that resulted in less accurate predictions were those where the range of temperatures analyzed were comparatively lower (inactivation profiles starting at 95 °C). The link between fail prediction and both lower starting temperature and magnitude of the temperature shift suggests some chemical or biological mechanism at work. Statistical analysis showed that overall model predictions were acceptable, with bias factors from 0.781 to 1.012, and accuracy factors from 1.049 to 1.351, and confirm that the models used were adequate to estimate B. coagulans spores inactivation under fluctuating temperature conditions in the range from 95 to 105 °C. How can we estimate Bacillus coagulans inactivation during sudden temperature shifts in heat processing? This article provides a validated model that can be used to predict B. coagulans under changing temperature conditions. B. coagulans is a spore-forming bacillus that spoils acidified food products. The mathematical model developed here can be used to predict the spoilage risk following thermal process deviations for tomato products. © 2014 Institute of Food Technologists®

High pressure processing and its application to the challenge of virus-contaminated foods

USDA-ARS?s Scientific Manuscript database

High pressure processing (HPP) is an increasingly popular non-thermal food processing technology. Study of HPP’s potential to inactivate foodborne viruses has defined general pressure levels required to inactivate hepatitis A virus, norovirus surrogates, and human norovirus itself within foods such...

Evaluation of the factors controlling the time-dependent inactivation rate coefficients of bacteriophage MS2 and PRD1

USGS Publications Warehouse

Anders, R.; Chrysikopoulos, C.V.

2006-01-01

Static and dynamic batch experiments were conducted to study the effects of temperature and the presence of sand on the inactivation of bacteriophage MS2 and PRD1. The experimental data suggested that the inactivation process can be satisfactorily represented by a pseudo-first-order expression with time-dependent rate coefficients. The time-dependent rate coefficients were used to determine pertinent thermodynamic properties required for the analysis of the molecular processes involved in the inactivation of each bacteriophage. A combination of high temperature and the presence of sand appears to produce the greatest disruption to the surrounding protein coat of MS2. However, the lower activation energies for PRD1 indicate a weaker dependence of the inactivation rate on temperature. Instead, the presence of air-liquid and air-solid interfaces appears to produce the greatest damage to specific viral components that are related to infection. These results indicate the importance of using thermodynamic parameters based on the time-dependent inactivation model to better predict the inactivation of viruses in groundwater. ?? 2006 American Chemical Society.

Derivation of Hodgkin-Huxley equations for a Na+ channel from a master equation for coupled activation and inactivation

NASA Astrophysics Data System (ADS)

Vaccaro, S. R.

2016-11-01

The Na+ current in nerve and muscle membranes may be described in terms of the activation variable m (t ) and the inactivation variable h (t ) , which are dependent on the transitions of S4 sensors of each of the Na+ channel domains DI to DIV. The time-dependence of the Na+ current and the rate equations satisfied by m (t ) and h (t ) may be derived from the solution to a master equation that describes the coupling between two or three activation sensors regulating the Na+ channel conductance and a two-stage inactivation process. If the inactivation rate from the closed or open states increases as the S4 sensors activate, a more general form of the Hodgkin-Huxley expression for the open-state probability may be derived where m (t ) is dependent on both activation and inactivation processes. The voltage dependence of the rate functions for inactivation and recovery from inactivation are consistent with the empirically determined expressions and exhibit saturation for both depolarized and hyperpolarized clamp potentials.

Inactivation of enveloped and non-enveloped viruses in the process of chemical treatment and gamma irradiation of bovine-derived grafting materials.

PubMed

Lee, Kwang-Il; Lee, Jung-Soo; Jung, Hong-Hee; Lee, Hwa-Yong; Moon, Seong-Hwan; Kang, Kyoung-Tak; Shim, Young-Bock; Jang, Ju-Woong

2012-01-01

Xenografts, unlike other grafting products, cannot be commercialized unless they conform to stringent safety regulations. Particularly with bovine-derived materials, it is essential to remove viruses and inactivate infectious factors because of the possibility that raw materials are imbrued with infectious viruses. The removal of the characteristics of infectious viruses from the bovine bone grafting materials need to be proved and inactivation process should satisfy the management provision of the Food and Drug Administration (FDA). To date, while most virus inactivation studies were performed in human allograft tissues, there have been almost no studies on bovine bone. To evaluate the efficacy of virus inactivation after treatment of bovine bone with 70% ethanol, 4% sodium hydroxide, and gamma irradiation, we selected a variety of experimental model viruses that are known to be associated with bone pathogenesis, including bovine parvovirus (BPV), bovine herpes virus (BHV), bovine viral diarrhea virus (BVDV), and bovine parainfluenza-3 virus (BPIV-3). The cumulative virus log clearance factor or cumulative virus log reduction factor for the manufacturing process was obtained by calculating the sum of the individual virus log clearance factors or log reduction factors determined for individual process steps with different physicochemical methods. The cumulative log clearance factors achieved by three different virus inactivation processes were as follows: BPV ≥ 17.73, BHV ≥ 20.53, BVDV ≥ 19.00, and BPIV-3 ≥ 16.27. On the other hand, the cumulative log reduction factors achieved were as follows: BPV ≥ 16.95, BHV ≥ 20.22, BVDV ≥ 19.27, and BPIV-3 ≥ 15.58. Treatment with 70% ethanol, 4% sodium hydroxide, or gamma irradiation was found to be very effective in virus inactivation, since all viruses were at undetectable levels during each process. We have no doubt that application of this established process to bovine bone graft manufacture will be effective and essential. © 2012 John Wiley & Sons A/S.

Stevia rebaudiana Bertoni as a natural antioxidant/antimicrobial for high pressure processed fruit extract: processing parameter optimization.

PubMed

Barba, Francisco José; Criado, María Nieves; Belda-Galbis, Clara Miracle; Esteve, María José; Rodrigo, Dolores

2014-04-01

Response surface methodology was used to evaluate the optimal high pressure processing treatment (300-500 MPa, 5-15 min) combined with Stevia rebaudiana (Stevia) addition (0-2.5% (w/v)) to guarantee food safety while maintaining maximum retention of nutritional properties. A fruit extract matrix was selected and Listeria monocytogenes inactivation was followed from the food safety point of view while polyphenoloxidase (PPO) and peroxidase (POD) activities, total phenolic content (TPC) and antioxidant capacity (TEAC and ORAC) were studied from the food quality point of view. A combination of treatments achieved higher levels of inactivation of L. monocytogenes and of the oxidative enzymes, succeeding in completely inactivating POD and also increasing the levels of TPC, TEAC and ORAC. A treatment of 453 MPa for 5 min with a 2.5% (w/v) of Stevia succeeded in inactivating over 5 log cycles of L. monocytogenes and maximizing inactivation of PPO and POD, with the greatest retention of bioactive components. Copyright © 2013 Elsevier Ltd. All rights reserved.

Strong and Consistently Synergistic Inactivation of Spores of Spoilage-Associated Bacillus and Geobacillus spp. by High Pressure and Heat Compared with Inactivation by Heat Alone ▿ †

PubMed Central

Olivier, S. A.; Bull, M. K.; Stone, G.; van Diepenbeek, R. J.; Kormelink, F.; Jacops, L.; Chapman, B.

2011-01-01

The inactivation of spores of four low-acid food spoilage organisms by high pressure thermal (HPT) and thermal-only processing was compared on the basis of equivalent thermal lethality calculated at a reference temperature of 121.1°C (Fz121.1°C, 0.1 MPa or 600 MPa) and characterized as synergistic, not different or protective. In addition, the relative resistances of spores of the different spoilage microorganisms to HPT processing were compared. Processing was performed and inactivation was compared in both laboratory and pilot scale systems and in model (diluted) and actual food products. Where statistical comparisons could be made, at least 4 times and up to around 190 times more inactivation (log10 reduction/minute at FTz121.1°C) of spores of Bacillus amyloliquefaciens, Bacillus sporothermodurans, and Geobacillus stearothermophilus was achieved using HPT, indicating a strong synergistic effect of high pressure and heat. Bacillus coagulans spores were also synergistically inactivated in diluted and undiluted Bolognese sauce but were protected by pressure against thermal inactivation in undiluted cream sauce. Irrespective of the response characterization, B. coagulans and B. sporothermodurans were identified as the most HPT-resistant isolates in the pilot scale and laboratory scale studies, respectively, and G. stearothermophilus as the least in both studies and all products. This is the first study to comprehensively quantitatively characterize the responses of a range of spores of spoilage microorganisms as synergistic (or otherwise) using an integrated thermal-lethality approach (FTz). The use of the FTz approach is ultimately important for the translation of commercial minimum microbiologically safe and stable thermal processes to HPT processes. PMID:21278265

Strong and consistently synergistic inactivation of spores of spoilage-associated Bacillus and Geobacillus spp. by high pressure and heat compared with inactivation by heat alone.

PubMed

Olivier, S A; Bull, M K; Stone, G; van Diepenbeek, R J; Kormelink, F; Jacops, L; Chapman, B

2011-04-01

The inactivation of spores of four low-acid food spoilage organisms by high pressure thermal (HPT) and thermal-only processing was compared on the basis of equivalent thermal lethality calculated at a reference temperature of 121.1°C (F(z)(121.1)(°)(C, 0.1 MPa or 600 MPa)) and characterized as synergistic, not different or protective. In addition, the relative resistances of spores of the different spoilage microorganisms to HPT processing were compared. Processing was performed and inactivation was compared in both laboratory and pilot scale systems and in model (diluted) and actual food products. Where statistical comparisons could be made, at least 4 times and up to around 190 times more inactivation (log(10) reduction/minute at F(T)(z)(121.1)(°)(C)) of spores of Bacillus amyloliquefaciens, Bacillus sporothermodurans, and Geobacillus stearothermophilus was achieved using HPT, indicating a strong synergistic effect of high pressure and heat. Bacillus coagulans spores were also synergistically inactivated in diluted and undiluted Bolognese sauce but were protected by pressure against thermal inactivation in undiluted cream sauce. Irrespective of the response characterization, B. coagulans and B. sporothermodurans were identified as the most HPT-resistant isolates in the pilot scale and laboratory scale studies, respectively, and G. stearothermophilus as the least in both studies and all products. This is the first study to comprehensively quantitatively characterize the responses of a range of spores of spoilage microorganisms as synergistic (or otherwise) using an integrated thermal-lethality approach (F(T)(z)). The use of the F(T)(z) approach is ultimately important for the translation of commercial minimum microbiologically safe and stable thermal processes to HPT processes.

High pressure processing and its application to the challenge of virus-contaminated foods.

PubMed

Kingsley, David H

2013-03-01

High pressure processing (HPP) is an increasingly popular non-thermal food processing technology. Study of HPP's potential to inactivate foodborne viruses has defined general pressure levels required to inactivate hepatitis A virus, norovirus surrogates, and human norovirus itself within foods such as shellfish and produce. The sensitivity of a number of different picornaviruses to HPP is variable. Experiments suggest that HPP inactivates viruses via denaturation of capsid proteins which render the virus incapable of binding to its receptor on the surface of its host cell. Beyond the primary consideration of treatment pressure level, the effects of extending treatment times, temperature of initial pressure application, and matrix composition have been identified as critical parameters for designing HPP inactivation strategies. Research described here can serve as a preliminary guide to whether a current commercial process could be effective against HuNoV or HAV.

Enzymatic degradation of somatostatin by rat plasma and hypothalamus.

PubMed

Dupont, A; Alvarado-Urbina, G; Côté, J; Labrie, F

1978-10-01

A highly sensitive and specific radioimmunoassay for somatostatin has been used to study inactivation of the neurohormone by plasma and hypothalamic peptidase(s). Specificity of the inactivation process was indicated by the absence of interference by addition of luteinizing hormone releasing hormone, thyrotropin-releasing hormone, oxytocin, or substance P. The inactivating ability of hypothalamic tissue and plasma was destroyed by heating and the protease inhibitor benzamidine prevented plasma activity, thus suggesting the enzymatic nature of the processes involved. The present data suggest that the inactivation of somatostatin by hypothalamus and plasma could be an important factor in the regulation of circulating somatostatin levels.

Inactivation of Salmonella spp. in ground chicken using High Pressure Processing

USDA-ARS?s Scientific Manuscript database

High pressure processing (HPP) is a safe and effective process for improving the microbial safety and shelf-life of foods. Salmonella is a common contaminant in poultry meat and is frequently responsible for foodborne illness associated with contaminated poultry meat. In this study the inactivation...

Inactivation of Salmonella and Listeria in ground chicken breast meat during thermal processing.

PubMed

Murphy, R Y; Marks, B P; Johnson, E R; Johnson, M G

1999-09-01

Thermal inactivation of six Salmonella spp. and Listeria innocua was evaluated in ground chicken breast and liquid medium. Survival of Salmonella and Listeria was affected by the medium composition. Under the same thermal process condition, significantly more Salmonella and Listeria survived in chicken breast meat than in 0.1% peptone-agar solution. The thermal lethality of six tested Salmonella spp. was additive in chicken meat. Survival of Listeria in chicken meat during thermal processing was not affected by the presence of the six Salmonella spp. Sample size and shape affected the inactivation of Salmonella and Listeria in chicken meat during thermal processing.

Molecular basis of tactile specialization in the duck bill.

PubMed

Schneider, Eve R; Anderson, Evan O; Mastrotto, Marco; Matson, Jon D; Schulz, Vincent P; Gallagher, Patrick G; LaMotte, Robert H; Gracheva, Elena O; Bagriantsev, Sviatoslav N

2017-12-05

Tactile-foraging ducks are specialist birds known for their touch-dependent feeding behavior. They use dabbling, straining, and filtering to find edible matter in murky water, relying on the sense of touch in their bill. Here, we present the molecular characterization of embryonic duck bill, which we show contains a high density of mechanosensory corpuscles innervated by functional rapidly adapting trigeminal afferents. In contrast to chicken, a visually foraging bird, the majority of duck trigeminal neurons are mechanoreceptors that express the Piezo2 ion channel and produce slowly inactivating mechano-current before hatching. Furthermore, duck neurons have a significantly reduced mechano-activation threshold and elevated mechano-current amplitude. Cloning and electrophysiological characterization of duck Piezo2 in a heterologous expression system shows that duck Piezo2 is functionally similar to the mouse ortholog but with prolonged inactivation kinetics, particularly at positive potentials. Knockdown of Piezo2 in duck trigeminal neurons attenuates mechano current with intermediate and slow inactivation kinetics. This suggests that Piezo2 is capable of contributing to a larger range of mechano-activated currents in duck trigeminal ganglia than in mouse trigeminal ganglia. Our results provide insights into the molecular basis of mechanotransduction in a tactile-specialist vertebrate.

Molecular basis of tactile specialization in the duck bill

PubMed Central

Anderson, Evan O.; Mastrotto, Marco; Matson, Jon D.; Schulz, Vincent P.; Gallagher, Patrick G.; LaMotte, Robert H.; Gracheva, Elena O.; Bagriantsev, Sviatoslav N.

2017-01-01

Tactile-foraging ducks are specialist birds known for their touch-dependent feeding behavior. They use dabbling, straining, and filtering to find edible matter in murky water, relying on the sense of touch in their bill. Here, we present the molecular characterization of embryonic duck bill, which we show contains a high density of mechanosensory corpuscles innervated by functional rapidly adapting trigeminal afferents. In contrast to chicken, a visually foraging bird, the majority of duck trigeminal neurons are mechanoreceptors that express the Piezo2 ion channel and produce slowly inactivating mechano-current before hatching. Furthermore, duck neurons have a significantly reduced mechano-activation threshold and elevated mechano-current amplitude. Cloning and electrophysiological characterization of duck Piezo2 in a heterologous expression system shows that duck Piezo2 is functionally similar to the mouse ortholog but with prolonged inactivation kinetics, particularly at positive potentials. Knockdown of Piezo2 in duck trigeminal neurons attenuates mechano current with intermediate and slow inactivation kinetics. This suggests that Piezo2 is capable of contributing to a larger range of mechano-activated currents in duck trigeminal ganglia than in mouse trigeminal ganglia. Our results provide insights into the molecular basis of mechanotransduction in a tactile-specialist vertebrate. PMID:29109250

Sprint training shortens prolonged action potential duration in postinfarction rat myocyte: mechanisms.

PubMed

Zhang, X Q; Zhang, L Q; Palmer, B M; Ng, Y C; Musch, T I; Moore, R L; Cheung, J Y

2001-05-01

Two electrophysiological manifestations of myocardial infarction (MI)-induced myocyte hypertrophy are prolongation of action potential duration (APD) and reduction of transient outward current (I(to)) density. Because high-intensity sprint training (HIST) ameliorated myocyte hypertrophy and improved myocyte Ca(2+) homeostasis and contractility after MI, the present study evaluated whether 6-8 wk of HIST would shorten the prolonged APD and improve the depressed I(to) in post-MI myocytes. There were no differences in resting membrane potential and action potential amplitude (APA) measured in myocytes isolated from sham-sedentary (Sed), MI-Sed, and MI-HIST groups. Times required for repolarization to 50 and 90% APA were significantly (P < 0.001) prolonged in MI-Sed myocytes. HIST reduced times required for repolarization to 50 and 90% APA to values observed in Sham-Sed myocytes. The fast and slow components of I(to) were significantly (P < 0.0001) reduced in MI-Sed myocytes. HIST significantly (P < 0.001) enhanced the fast and slow components of I(to) in MI myocytes, although not to levels observed in Sham-Sed myocytes. There were no significant differences in steady-state I(to) inactivation and activation parameters among Sham-Sed, MI-Sed, and MI-HIST myocytes. Likewise, recovery from time-dependent inactivation was also similar among the three groups. We suggest that normalization of APD after MI by HIST may be mediated by restoration of I(to) toward normal levels.

Inter-subunit interactions across the upper voltage sensing-pore domain interface contribute to the concerted pore opening transition of Kv channels.

PubMed

Shem-Ad, Tzilhav; Irit, Orr; Yifrach, Ofer

2013-01-01

The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.

Perspectives of high power ultrasound in food preservation

NASA Astrophysics Data System (ADS)

Evelyn; Silva, F. V. M.

2018-04-01

High Power ultrasound can be used to alter physicochemical properties and improve the quality of foods during processing due to a number of mechanical, chemical, and biochemical effects arising from acoustic cavitation. Cavitation creates pressure waves that inactivate microbes and de-agglomerate bacterial clusters or release ascospores from fungal asci. Bacterial and heat resistant fungal spores’ inactivation is a great challenge in food preservation due to their ability to survive after conventional food processing, causing food-borne diseases or spoilage. In this work, a showcase of application of high power ultrasound combined with heat or thermosonication, to inactivate bacterial spores i.e. Bacillus cereus spores in beef slurry and fungal spores i.e. Neosartorya fischeri ascospores in apple juice was presented and compared with thermal processing. Faster inactivation was achieved at higher TS (24 KHz, 0.33 W/g or W/mL) temperatures. Around 2 log inactivation was obtained for B. cereus spores after1 min (70 °C) and N. fischeri ascospores after 30 min (75 °C). Thermal treatments caused <1 log in B. Cereus after 2 min (70 °C) and no inactivation in N. Fischeri ascospores after 30 min (80 °C). In conclusion, temperature plays a significant role for TS spore inactivation and TS was more effective than thermal treatment alone. The mould spores were more resistant than the bacterial spores.

Avibactam and Inhibitor-Resistant SHV β-Lactamases

PubMed Central

Winkler, Marisa L.; Papp-Wallace, Krisztina M.; Taracila, Magdalena A.

2015-01-01

β-Lactamase enzymes (EC 3.5.2.6) are a significant threat to the continued use of β-lactam antibiotics to treat infections. A novel non-β-lactam β-lactamase inhibitor with activity against many class A and C and some class D β-lactamase variants, avibactam, is now available in the clinic in partnership with ceftazidime. Here, we explored the activity of avibactam against a variety of characterized isogenic laboratory constructs of β-lactamase inhibitor-resistant variants of the class A enzyme SHV (M69I/L/V, S130G, K234R, R244S, and N276D). We discovered that the S130G variant of SHV-1 shows the most significant resistance to inhibition by avibactam, based on both microbiological and biochemical characterizations. Using a constant concentration of 4 mg/liter of avibactam as a β-lactamase inhibitor in combination with ampicillin, the MIC increased from 1 mg/liter for blaSHV-1 to 256 mg/liter for blaSHV S130G expressed in Escherichia coli DH10B. At steady state, the k2/K value of the S130G variant when inactivated by avibactam was 1.3 M−1 s−1, versus 60,300 M−1 s−1 for the SHV-1 β-lactamase. Under timed inactivation conditions, we found that an approximately 1,700-fold-higher avibactam concentration was required to inhibit SHV S130G than the concentration that inhibited SHV-1. Molecular modeling suggested that the positioning of amino acids in the active site of SHV may result in an alternative pathway of inactivation when complexed with avibactam, compared to the structure of CTX-M-15–avibactam, and that S130 plays a role in the acylation of avibactam as a general acid/base. In addition, S130 may play a role in recyclization. As a result, we advance that the lack of a hydroxyl group at position 130 in the S130G variant of SHV-1 substantially slows carbamylation of the β-lactamase by avibactam by (i) removing an important proton acceptor and donator in catalysis and (ii) decreasing the number of H bonds. In addition, recyclization is most likely also slow due to the lack of a general base to initiate the process. Considering other inhibitor-resistant mechanisms among class A β-lactamases, S130 may be the most important amino acid for the inhibition of class A β-lactamases, perhaps even for the novel diazabicyclooctane class of β-lactamase inhibitors. PMID:25691639

Conceptual model and experimental framework to determine the contributions of direct and indirect photoreactions to the solar disinfection of MS2, phiX174, and adenovirus.

PubMed

Mattle, Michael J; Vione, Davide; Kohn, Tamar

2015-01-06

Sunlight inactivates waterborne viruses via direct (absorption of sunlight by the virus) and indirect processes (adsorption of sunlight by external chromophores, which subsequently generate reactive species). While the mechanisms underlying these processes are understood, their relative importance remains unclear. This study establishes an experimental framework to determine the kinetic parameters associated with a virus' susceptibility to solar disinfection and proposes a model to estimate disinfection rates and to apportion the contributions of different inactivation processes. Quantum yields of direct inactivation were determined for three viruses (MS2, phiX174, and adenovirus), and second-order rate constants associated with indirect inactivation by four reactive species ((1)O2, OH(•), CO3(•-), and triplet states) were established. PhiX174 exhibited the greatest quantum yield (1.4 × 10(-2)), indicating that it is more susceptible to direct inactivation than MS2 (2.9 × 10(-3)) or adenovirus (2.5 × 10(-4)). Second-order rate constants ranged from 1.7 × 10(7) to 7.0 × 10(9) M(-1) s(-1) and followed the sequence MS2 > adenovirus > phiX174. A predictive model based on these parameters accurately estimated solar disinfection of MS2 and phiX174 in a natural water sample and approximated that of adenovirus within a factor of 6. Inactivation mostly occurred by direct processes, though indirect inactivation by (1)O2 also contributed to the disinfection of MS2 and adenovirus.

Weed seed inactivation in soil mesocosms via biosolarization with mature compost and tomato processing waste amendments.

PubMed

Achmon, Yigal; Fernández-Bayo, Jesús D; Hernandez, Katie; McCurry, Dlinka G; Harrold, Duff R; Su, Joey; Dahlquist-Willard, Ruth M; Stapleton, James J; VanderGheynst, Jean S; Simmons, Christopher W

2017-05-01

Biosolarization is a fumigation alternative that combines passive solar heating with amendment-driven soil microbial activity to temporarily create antagonistic soil conditions, such as elevated temperature and acidity, that can inactivate weed seeds and other pest propagules. The aim of this study was to use a mesocosm-based field trial to assess soil heating, pH, volatile fatty acid accumulation and weed seed inactivation during biosolarization. Biosolarization for 8 days using 2% mature green waste compost and 2 or 5% tomato processing residues in the soil resulted in accumulation of volatile fatty acids in the soil, particularly acetic acid, and >95% inactivation of Brassica nigra and Solanum nigrum seeds. Inactivation kinetics data showed that near complete weed seed inactivation in soil was achieved within the first 5 days of biosolarization. This was significantly greater than the inactivation achieved in control soils that were solar heated without amendment or were amended but not solar heated. The composition and concentration of organic matter amendments in soil significantly affected volatile fatty acid accumulation at various soil depths during biosolarization. Combining solar heating with organic matter amendment resulted in accelerated weed seed inactivation compared with either approach alone. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

The comprehensive electrophysiological study of curcuminoids on delayed-rectifier K+ currents in insulin-secreting cells.

PubMed

Kuo, Ping-Chung; Yang, Chia-Jung; Lee, Yu-Chi; Chen, Pei-Chun; Liu, Yen-Chin; Wu, Sheng-Nan

2018-01-15

Curcumin (CUR) has been demonstrated to induce insulin release from pancreatic β-cells; however, how curcuminoids (including demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC)) exert any possible effects on membrane ion currents inherently in insulin-secreting cells remains largely unclear. The effects of CUR and other structurally similar curcuminoids on ion currents in rat insulin-secreting (INS-1) insulinoma cells were therefore investigated in this study. The effects of these compounds on ionic currents and membrane potential were studied by patch-clamp technique. CUR suppressed the amplitude of delayed-rectifier K + current (I K(DR) ) in a time-, state- and concentration-dependent manner in these cells and the inhibition was not reversed by diazoxide, nicorandil or chlorotoxin. The value of dissociation constant for CUR-induced suppression of I K(DR) in INS-1 cells was 1.26μM. Despite the inability of CUR to alter the activation rate of I K(DR) , it accelerated current inactivation elicited by membrane depolarization. Increasing CUR concentrations shifted the inactivation curve of I K(DR) to hyperpolarized potential and slowed the recovery of I K(DR) inactivation. CUR, DMC, and BDMC all exerted depressant actions on I K(DR) amplitude to a similar magnitude, although DMC and BDMC did not increase current inactivation clearly. CUR slightly suppressed the peak amplitude of voltage-gated Na + current. CUR, DMC and BDMC depolarized the resting potential and increased firing frequency of action potentials. The CUR-mediated decrease of I K(DR) and the increase of current inactivation also occurred in βTC-6 INS-1 cells. Taken these results together, these effects may be one of the possible mechanisms contributing their insulin-releasing effect. Copyright © 2017 Elsevier B.V. All rights reserved.

Biophysical mechanism of spike threshold dependence on the rate of rise of the membrane potential by sodium channel inactivation or subthreshold axonal potassium current

PubMed Central

Wester, Jason C.

2013-01-01

Spike threshold filters incoming inputs and thus gates activity flow through neuronal networks. Threshold is variable, and in many types of neurons there is a relationship between the threshold voltage and the rate of rise of the membrane potential (dVm/dt) leading to the spike. In primary sensory cortex this relationship enhances the sensitivity of neurons to a particular stimulus feature. While Na+ channel inactivation may contribute to this relationship, recent evidence indicates that K+ currents located in the spike initiation zone are crucial. Here we used a simple Hodgkin-Huxley biophysical model to systematically investigate the role of K+ and Na+ current parameters (activation voltages and kinetics) in regulating spike threshold as a function of dVm/dt. Threshold was determined empirically and not estimated from the shape of the Vm prior to a spike. This allowed us to investigate intrinsic currents and values of gating variables at the precise voltage threshold. We found that Na+ inactivation is sufficient to produce the relationship provided it occurs at hyperpolarized voltages combined with slow kinetics. Alternatively, hyperpolarization of the K+ current activation voltage, even in the absence of Na+ inactivation, is also sufficient to produce the relationship. This hyperpolarized shift of K+ activation allows an outward current prior to spike initiation to antagonize the Na+ inward current such that it becomes self-sustaining at a more depolarized voltage. Our simulations demonstrate parameter constraints on Na+ inactivation and the biophysical mechanism by which an outward current regulates spike threshold as a function of dVm/dt. PMID:23344915

Effect of tyrphostin AG879 on Kv4.2 and Kv4.3 potassium channels

PubMed Central

Yu, Haibo; Zou, Beiyan; Wang, Xiaoliang; Li, Min

2015-01-01

Background and Purpose A-type potassium channels (IA) are important proteins for modulating neuronal membrane excitability. The expression and activity of Kv4.2 channels are critical for neurological functions and pharmacological inhibitors of Kv4.2 channels may have therapeutic potential for Fragile X syndrome. While screening various compounds, we identified tyrphostin AG879, a tyrosine kinase inhibitor, as a Kv4.2 inhibitor from. In the present study we characterized the effect of AG879 on cloned Kv4.2/Kv channel-interacting protein 2 (KChIP2) channels. Experimental Approach To screen the library of pharmacologically active compounds, the thallium flux assay was performed on HEK-293 cells transiently-transfected with Kv4.2 cDNA using the Maxcyte transfection system. The effects of AG879 were further examined on CHO-K1 cells expressing Kv4.2/KChIP2 channels using a whole-cell patch-clamp technique. Key Results Tyrphostin AG879 selectively and dose-dependently inhibited Kv4.2 and Kv4.3 channels. In Kv4.2/KChIP2 channels, AG879 induced prominent acceleration of the inactivation rate, use-dependent block and slowed the recovery from inactivation. AG879 induced a hyperpolarizing shift in the voltage-dependence of the steady-state inactivation of Kv4.2 channels without apparent effect on the V1/2 of the voltage-dependent activation. The blocking effect of AG879 was enhanced as channel inactivation increased. Furthermore, AG879 significantly inhibited the A-type potassium currents in the cultured hippocampus neurons. Conclusion and Implications AG879 was identified as a selective and potent inhibitor the Kv4.2 channel. AG879 inhibited Kv4.2 channels by preferentially interacting with the open state and further accelerating their inactivation. PMID:25752739

Effect of tyrphostin AG879 on Kv 4.2 and Kv 4.3 potassium channels.

PubMed

Yu, Haibo; Zou, Beiyan; Wang, Xiaoliang; Li, Min

2015-07-01

A-type potassium channels (IA) are important proteins for modulating neuronal membrane excitability. The expression and activity of Kv 4.2 channels are critical for neurological functions and pharmacological inhibitors of Kv 4.2 channels may have therapeutic potential for Fragile X syndrome. While screening various compounds, we identified tyrphostin AG879, a tyrosine kinase inhibitor, as a Kv 4.2 inhibitor from. In the present study we characterized the effect of AG879 on cloned Kv 4.2/Kv channel-interacting protein 2 (KChIP2) channels. To screen the library of pharmacologically active compounds, the thallium flux assay was performed on HEK-293 cells transiently-transfected with Kv 4.2 cDNA using the Maxcyte transfection system. The effects of AG879 were further examined on CHO-K1 cells expressing Kv 4.2/KChIP2 channels using a whole-cell patch-clamp technique. Tyrphostin AG879 selectively and dose-dependently inhibited Kv 4.2 and Kv 4.3 channels. In Kv 4.2/KChIP2 channels, AG879 induced prominent acceleration of the inactivation rate, use-dependent block and slowed the recovery from inactivation. AG879 induced a hyperpolarizing shift in the voltage-dependence of the steady-state inactivation of Kv 4.2 channels without apparent effect on the V1/2 of the voltage-dependent activation. The blocking effect of AG879 was enhanced as channel inactivation increased. Furthermore, AG879 significantly inhibited the A-type potassium currents in the cultured hippocampus neurons. AG879 was identified as a selective and potent inhibitor the Kv 4.2 channel. AG879 inhibited Kv 4.2 channels by preferentially interacting with the open state and further accelerating their inactivation. © 2015 The British Pharmacological Society.

Kv4 channels underlie A-currents with highly variable inactivation time courses but homogeneous other gating properties in the nucleus tractus solitarii.

PubMed

Strube, Caroline; Saliba, Layal; Moubarak, Estelle; Penalba, Virginie; Martin-Eauclaire, Marie-France; Tell, Fabien; Clerc, Nadine

2015-04-01

In the nucleus of the tractus solitarii (NTS), a large proportion of neurones express transient A-type potassium currents (I KA) having deep influence on the fidelity of the synaptic transmission of the visceral primary afferent inputs to second-order neurones. Up to now, the strong impact of I KA within the NTS was considered to result exclusively from its variation in amplitude, and its molecular correlate(s) remained unknown. In order to identify which Kv channels underlie I KA in NTS neurones, the gating properties and the pharmacology of this current were determined using whole cell patch clamp recordings in slices. Complementary information was brought by immunohistochemistry. Strikingly, two neurone subpopulations characterized by fast or slow inactivation time courses (respectively about 50 and 200 ms) were discriminated. Both characteristics matched those of the Kv4 channel subfamily. The other gating properties, also matching the Kv4 channel ones, were homogeneous through the NTS. The activation and inactivation occurred at membrane potentials around the threshold for generating action potentials, and the time course of recovery from inactivation was rapid. Pharmacologically, I KA in NTS neurones was found to be resistant to tetraethylammonium (TEA), sea anemone toxin blood-depressing substance (BDS) and dendrotoxin (DTX), whereas Androctonus mauretanicus mauretanicus toxin 3 (AmmTX3), a scorpion toxin of the α-KTX 15 family that has been shown to block all the members of the Kv4 family, inhibited 80 % of I KA irrespectively of its inactivation time course. Finally, immunohistochemistry data suggested that, among the Kv4 channel subfamily, Kv4.3 is the prevalent subunit expressed in the NTS.

Characteristics of action potentials and their underlying outward currents in rat taste receptor cells.

PubMed

Chen, Y; Sun, X D; Herness, S

1996-02-01

1. Taste receptor cells produce action potentials as a result of transduction mechanisms that occur when these cells are stimulated with tastants. These action potentials are thought to be key signaling events in relaying information to the central nervous system. We explored the ionic basis of action potentials from dissociated posterior rat taste cells using the patch-clamp recording technique in both voltage-clamp and current-clamp modes. 2. Action potentials were evoked by intracellular injection of depolarizing current pulses from a holding potential of -80 mV. The threshold potential for firing of action potentials was approximately -35 mV; the input resistance of these cells averaged 6.9 G omega. With long depolarizing pulses, two or three action potentials could be elicited with successive attenuation of the spike height. Afterhyperpolarizations were observed often. 3. Both sodium and calcium currents contribute to depolarizing phases of the action potential. Action potentials were blocked completely in the presence of the sodium channel blocker tetrodotoxin. Calcium contributions could be visualized as prolonged calcium plateaus when repolarizing potassium currents were blocked and barium was used as a charge carrier. 4. Outward currents were composed of sustained delayed rectifier current, transient potassium current, and calcium-activated potassium current. Transient and sustained potassium currents activated close to -30 mV and increased monotonically with further depolarization. Up to half the outward current inactivated with decay constants on the order of seconds. Sustained and transient currents displayed steep voltage dependence in conductance and inactivation curves. Half inactivation occurred at -20 +/- 3.1 mV (mean +/- SE) with a decrease of 11.2 +/- 0.5 mV per e-fold. Half maximal conductance occurred at 3.6 +/- 1.8 mV and increased 12.2 +/- 0.6 mV per e-fold. Calcium-activated potassium current was evidenced by application of apamin and the use of calcium-free bathing solution. It was most obvious at more depolarized holding potentials that inactivated much of the transient and sustained outward currents. 5. Potassium currents contribute to both the repolarization and afterhyperpolarization phases of the action potential. These currents were blocked by bath application of tetraethylammonium, which also substantially broadened the action potential. Application of 4-aminopyridine was able to selectively block transient potassium currents without affecting sustained currents. This also broadened the action potential as well as eliminated the afterhyperpolarization. 6. A second type of action potential was observed that differed in duration. These slow action potentials had t1/2 durations of 9.6 ms compared with 1.4 ms for fast action potentials. Input resistances of the two groups were indistinguishable. Approximately one-fourth of the cells eliciting action potentials were of the slow type. 7. Cells eliciting fast action potentials had large outward currents capable of producing a quick repolarization, whereas cells with slow action potentials had small outward currents by comparison. The average values of fast cells were 2,563 pA and 1.4 ms compared with 373 pA and 9.6 ms for slow cells. Current and duration values were related exponentially. No significant difference was noted for inward currents. 8. These results suggest that many taste receptor cells conduct action potentials, which may be classified broadly into two groups on the basis of action potential duration and potassium current magnitude. These groups may be related to cell turnover. The physiological role of action potentials remains to be elucidated but may be important for communication within the taste bud as well as to the afferent nerve.

High pressure processing inactivates human norovirus within oysters

USDA-ARS?s Scientific Manuscript database

Consumption of raw bivalve mollusks can result in norovirus infection. One potential intervention for virus-contaminated shellfish is high pressure processing (HPP). Currently HPP is known to inactivate Vibrio bacteria, hepatitis A virus, and murine norovirus within oysters. To evaluate the potentia...

BmP02 Atypically Delays Kv4.2 Inactivation: Implication for a Unique Interaction between Scorpion Toxin and Potassium Channel

PubMed Central

Wu, Bin; Zhu, Yan; Shi, Jian; Tao, Jie; Ji, Yonghua

2016-01-01

BmP02, a short-chain peptide with 28 residues from the venom of Chinese scorpion Buthus martensi Karsch, has been reported to inhibit the transient outward potassium currents (Ito) in rat ventricular muscle cells. However, it remains unclear whether BmP02 modulates the Kv4.2 channel, one of the main contributors to Ito. The present study investigated the effects of BmP02 on Kv4.2 kinetics and its underlying molecular mechanism. The electrophysiological recordings showed that the inactivation of Kv4.2 expressed in HEK293T cells was significantly delayed by BmP02 in a dose-response manner with EC50 of ~850 nM while the peak current, activation and voltage-dependent inactivation of Kv4.2 were not affected. Meanwhile, the recovery from inactivation of Kv4.2 was accelerated and the deactivation was slowed after the application of BmP02. The site-directed mutagenesis combined with computational modelling identified that K347 and K353, located in the turret motif of the Kv4.2, and E4/E5, D20/D21 in BmP02 are key residues to interact with BmP02 through electrostatic force. These findings not only reveal a novel interaction between Kv4.2 channel and its peptidyl modulator, but also provide valuable information for design of highly-selective Kv4.2 modulators. PMID:27690098

Visual ecology and potassium conductances of insect photoreceptors.

PubMed

Frolov, Roman; Immonen, Esa-Ville; Weckström, Matti

2016-04-01

Voltage-activated potassium channels (Kv channels) in the microvillar photoreceptors of arthropods are responsible for repolarization and regulation of photoreceptor signaling bandwidth. On the basis of analyzing Kv channels in dipteran flies, it was suggested that diurnal, rapidly flying insects predominantly express sustained K(+) conductances, whereas crepuscular and nocturnally active animals exhibit strongly inactivating Kv conductances. The latter was suggested to function for minimizing cellular energy consumption. In this study we further explore the evolutionary adaptations of the photoreceptor channelome to visual ecology and behavior by comparing K(+) conductances in 15 phylogenetically diverse insects, using patch-clamp recordings from dissociated ommatidia. We show that rapid diurnal flyers such as the blowfly (Calliphora vicina) and the honeybee (Apis mellifera) express relatively large noninactivating Kv conductances, conforming to the earlier hypothesis in Diptera. Nocturnal and/or slow-moving species do not in general exhibit stronger Kv conductance inactivation in the physiological membrane voltage range, but the photoreceptors in species that are known to rely more on vision behaviorally had higher densities of sustained Kv conductances than photoreceptors of less visually guided species. No statistically significant trends related to visual performance could be identified for the rapidly inactivating Kv conductances. Counterintuitively, strong negative correlations were observed between photoreceptor capacitance and specific membrane conductance for both sustained and inactivating fractions of Kv conductance, suggesting insignificant evolutionary pressure to offset negative effects of high capacitance on membrane filtering with increased conductance. Copyright © 2016 the American Physiological Society.

Inactivation of Escherichia coli, Saccharomyces cerevisiae, and Lactobacillus brevis in Low-fat Milk by Pulsed Electric Field Treatment: A Pilot-scale Study

PubMed Central

Han, Bok Kung; Choi, Hyuk Joon; Kang, Shin Ho; Baick, Seung Chun

2015-01-01

We investigated the effects of a pulsed electric field (PEF) treatment on microbial inactivation and the physical properties of low-fat milk. Milk inoculated with Escherichia coli, Saccharomyces cerevisiae, or Lactobacillus brevis was supplied to a pilot-scale PEF treatment system at a flow rate of 30 L/h. Pulses with an electric field strength of 10 kV/cm and a pulse width of 30 μs were applied to the milk with total pulse energies of 50-250 kJ/L achieved by varying the pulse frequency. The inactivation curves of the test microorganisms were biphasic with an initial lag phase (or shoulder) followed by a phase of rapid inactivation. PEF treatments with a total pulse energy of 200 kJ/L resulted in a 4.5-log reduction in E. coli, a 4.4-log reduction in L. brevis, and a 6.0-log reduction in S. cerevisiae. Total pulse energies of 200 and 250 kJ/L resulted in greater than 5-log reductions in microbial counts in stored PEF-treated milk, and the growth of surviving microorganisms was slow during storage for 15 d at 4℃. PEF treatment did not change milk physical properties such as pH, color, or particle-size distribution (p<0.05). These results indicate that a relatively low electric-field strength of 10 kV/cm can be used to pasteurize low-fat milk. PMID:26877640

Inactivation of Escherichia coli, Saccharomyces cerevisiae, and Lactobacillus brevis in Low-fat Milk by Pulsed Electric Field Treatment: A Pilot-scale Study.

PubMed

Lee, Gun Joon; Han, Bok Kung; Choi, Hyuk Joon; Kang, Shin Ho; Baick, Seung Chun; Lee, Dong-Un

2015-01-01

We investigated the effects of a pulsed electric field (PEF) treatment on microbial inactivation and the physical properties of low-fat milk. Milk inoculated with Escherichia coli, Saccharomyces cerevisiae, or Lactobacillus brevis was supplied to a pilot-scale PEF treatment system at a flow rate of 30 L/h. Pulses with an electric field strength of 10 kV/cm and a pulse width of 30 μs were applied to the milk with total pulse energies of 50-250 kJ/L achieved by varying the pulse frequency. The inactivation curves of the test microorganisms were biphasic with an initial lag phase (or shoulder) followed by a phase of rapid inactivation. PEF treatments with a total pulse energy of 200 kJ/L resulted in a 4.5-log reduction in E. coli, a 4.4-log reduction in L. brevis, and a 6.0-log reduction in S. cerevisiae. Total pulse energies of 200 and 250 kJ/L resulted in greater than 5-log reductions in microbial counts in stored PEF-treated milk, and the growth of surviving microorganisms was slow during storage for 15 d at 4℃. PEF treatment did not change milk physical properties such as pH, color, or particle-size distribution (p<0.05). These results indicate that a relatively low electric-field strength of 10 kV/cm can be used to pasteurize low-fat milk.

Impact of pH and Total Soluble Solids on Enzyme Inactivation Kinetics during High Pressure Processing of Mango (Mangifera indica) Pulp.

PubMed

Kaushik, Neelima; Nadella, Tejaswi; Rao, P Srinivasa

2015-11-01

This study was undertaken with an aim to enhance the enzyme inactivation during high pressure processing (HPP) with pH and total soluble solids (TSS) as additional hurdles. Impact of mango pulp pH (3.5, 4.0, 4.5) and TSS (15, 20, 25 °Brix) variations on the inactivation of pectin methylesterase (PME), polyphenol oxidase (PPO), and peroxidase (POD) enzymes were studied during HPP at 400 to 600 MPa pressure (P), 40 to 70 °C temperature (T), and 6- to 20-min pressure-hold time (t). The enzyme inactivation (%) was modeled using second order polynomial equations with a good fit that revealed that all the enzymes were significantly affected by HPP. Response surface and contour models predicted the kinetic behavior of mango pulp enzymes adequately as indicated by the small error between predicted and experimental data. The predicted kinetics indicated that for a fixed P and T, higher pulse pressure effect and increased isobaric inactivation rates were possible at lower levels of pH and TSS. In contrast, at a fixed pH or TSS level, an increase in P or T led to enhanced inactivation rates, irrespective of the type of enzyme. PPO and POD were found to have similar barosensitivity, whereas PME was found to be most resistant to HPP. Furthermore, simultaneous variation in pH and TSS levels of mango pulp resulted in higher enzyme inactivation at lower pH and TSS during HPP, where the effect of pH was found to be predominant than TSS within the experimental domain. Exploration of additional hurdles such as pH, TSS, and temperature for enzyme inactivation during high pressure processing of fruits is useful from industrial point of view, as these parameters play key role in preservation process design. © 2015 Institute of Food Technologists®

Bacteria, mould and yeast spore inactivation studies by scanning electron microscope observations.

PubMed

Rozali, Siti N M; Milani, Elham A; Deed, Rebecca C; Silva, Filipa V M

2017-12-18

Spores are the most resistant form of microbial cells, thus difficult to inactivate. The pathogenic or food spoilage effects of certain spore-forming microorganisms have been the primary basis of sterilization and pasteurization processes. Thermal sterilization is the most common method to inactivate spores present on medical equipment and foods. High pressure processing (HPP) is an emerging and commercial non-thermal food pasteurization technique. Although previous studies demonstrated the effectiveness of thermal and non-thermal spore inactivation, the in-depth mechanisms of spore inactivation are as yet unclear. Live and dead forms of two food spoilage bacteria, a mould and a yeast were examined using scanning electron microscopy before and after the inactivation treatment. Alicyclobacillus acidoterrestris and Geobacillus stearothermophilus bacteria are indicators of acidic foods pasteurization and sterilization processes, respectively. Neosartorya fischeri is a phyto-pathogenic mould attacking fruits. Saccharomyces cerevisiae is a yeast with various applications for winemaking, brewing, baking and the production of biofuel from crops (e.g. sugar cane). Spores of the four microbial species were thermally inactivated. Spores of S. cerevisiae were observed in the ascus and free form after thermal and HPP treatments. Different forms of damage and cell destruction were observed for each microbial spore. Thermal treatment inactivated bacterial spores of A. acidoterrestris and G. stearothermophilus by attacking the inner core of the spore. The heat first altered the membrane permeability allowing the release of intracellular components. Subsequently, hydration of spores, physicochemical modifications of proteins, flattening and formation of indentations occurred, with subsequent spore death. Regarding N. fischeri, thermal inactivation caused cell destruction and leakage of intracellular components. Both thermal and HPP treatments of S. cerevisiae free spores attacked the inner membrane, altering its permeability, and allowing in final stages the transfer of intracellular components to the outside. The spore destruction caused by thermal treatment was more severe than HPP, as HPP had less effect on the spore core. All injured spores have undergone irreversible volume and shape changes. While some of the leakage of spore contents is visible around the deformed but fully shaped spore, other spores exhibited large indentations and were completely deformed, apparently without any contents inside. This current study contributed to the understanding of spore inactivation by thermal and non-thermal processes. Copyright © 2017 Elsevier B.V. All rights reserved.

High pressure inactivation of human norovirus-like particles: evidence that the capsid of human norovirus is highly pressure resistant

USDA-ARS?s Scientific Manuscript database

High pressure processing (HPP) is a promising non-thermal technology to inactivate foodborne viruses. However, the effectiveness of HPP on inactivating human norovirus (HuNoV), the leading cause of acute gastroenteritis, is unknown because it cannot be propagated in cell culture. Therefore, developi...

Inactivation of virus in intravenous immunoglobulin G using solvent/detergent treatment and pasteurization.

PubMed

Aghaie, A; Pourfatollah, A A; Bathaie, S Z; Moazzeni, S M; Khorsand Mohammad Pour, H; Sharifi, Z

2008-01-01

The safety of plasma derived medicinal products, such as immunoglobulin, depends on viral inactivation steps that are incorporated into the production process. Several attempts have been made to validate the effectiveness of these inactivation methods against a range of physio-chemically diverse viruses. Treatment with solvent/detergent (S/D) and pasteurization (P) has been continuously used in our IgG production and these methods were analysed in this study as models of viral inactivation. Bovine Viral Diarrhoea Virus (BVDV), Herpes Simplex Virus (HSV) and Vesicular Stomatitis Virus (VSV) were employed as models of HCV, HBV and HIV respectively. Polio and Reo viruses also were used as stable viruses to chemical substances. The infectivity of a range of viruses before and after treatment with two methods of viral inactivation was measured by end point titration and their effectiveness expressed as Logarithmic Reduction Factors (LRF). Solvent/detergent treatment reduced the amount of enveloped viruses by 5-6 logs. The reduction factor was between 5-6 logs for all viruses used in the pasteurization process. A final log reduction factor was obtained as the sum of the two individual methods. Both inactivation methods have advantages and disadvantages with respect to their ability to inactivate viruses. Thus,combination of two robust virus inactivation steps, solvent/detergent and pasteurization, increases the safety margin of immunoglobulin preparations.

Factors affecting inactivation of Moraxell-Acinetobacter cells in an irradiation process. [/sup 137/Cs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Firstenberg-Eden, R.; Rowley, D.B.; Shattuck, G.E.

1980-09-01

The effect of various stages of the irradiation processing of beef on the injury and inactivation of radiation-resistant Moraxella-Acinetobactor cells was studied. Moraxella-Acinetobacter cells were more resistant to heat inactivation and injury when heated in meat with salts (0.75% NaCl and 0.375% sodium tripolyphosphate) than in meat without salts. These salts had no effect on radiation resistance. Heated cells were more sensitive to radiation inactivation and injury than unheated cells. After repair, the cells regained their resistance to both NaCl and irradiation. Freezing and storage at -40/sup 0/C for 14 days had only a slight effect on either unstressed ormore » heat-stressed cells.« less

Commercial thermal process for inactivating Salmonella Poona on surfaces of whole fresh cantaloupes

USDA-ARS?s Scientific Manuscript database

Outbreaks of salmonellosis by Salmonella Poona and listeriosis by Listeria monocytogenes have been associated with the consumption of cantaloupes. Commercial washing processes for cantaloupes are limited in their ability to inactivate and/or remove these human pathogens. Our objective was to devel...

Commercial thermal process for inactivating Salmonella Poona on surfaces of whole fresh cantaloupes

USDA-ARS?s Scientific Manuscript database

Outbreaks of salmonellosis by Salmonella Poona and listeriosis by Listeria monocytogenes have been associated with the consumption of cantaloupes. Commercial washing processes for cantaloupes are limited in their ability to inactivate and/or remove this human pathogen. Our objective was to develop...

Evaluation of eco-friendly zwitterionic detergents for enveloped virus inactivation.

PubMed

Conley, Lynn; Tao, Yinying; Henry, Alexis; Koepf, Edward; Cecchini, Douglas; Pieracci, John; Ghose, Sanchayita

2017-04-01

Inclusion of a detergent in protein biotherapeutic purification processes is a simple and very robust method for inactivating enveloped viruses. The detergent Triton X-100 has been used for many years and is part of the production process of several commercial therapeutic proteins. However, recent ecological studies have suggested that Triton X-100 and its break-down products can potentially behave as endocrine disrupters in aquatic organisms, raising concerns from an environmental impact perspective. As such, discharge of Triton X-100 into the waste water treatment plants is regulated in some jurisdictions, and alternative detergents for viral inactivation are required. In this work, we report on the identification and evaluation of more eco-friendly detergents as viable replacements for Triton X-100. Five detergent candidates with low to moderate environmental impact were initially identified and evaluated with respect to protein stability, followed by proof-of-concept virus inactivation studies using a model enveloped virus. From the set of candidates lauryldimethylamine N-oxide (LDAO) was identified as the most promising detergent due to its low ecotoxicity, robust anti-viral activity (LRV >4 at validation set-point conditions with X-MuLX), and absence of any negative impact on protein function. This detergent exhibited effective and robust virus inactivation in a broad range of protein concentrations, solution conductivities, pHs, and in several different cell culture fluid matrices. The only process parameter which correlated with reduced virus inactivation potency was LDAO concentration, and then only when the concentration was reduced to below the detergent's critical micelle concentration (CMC). Additionally, this work also demonstrated that LDAO was cleared to below detectable levels after Protein A affinity chromatography, making it suitable for use in a platform process that utilizes this chromatographic mode for protein capture. All these findings suggest that LDAO may be a practical alternative to Triton X-100 for use in protein therapeutic production processes for inactivating enveloped viruses. Biotechnol. Bioeng. 2017;114: 813-820. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Supercritical carbon dioxide process for pasteurization of fruit juices

USDA-ARS?s Scientific Manuscript database

Supercritical carbon dioxide (SCCO2) nonthermal processing inactivates microorganisms in juices using non-toxic and non-reactive CO2. However, data is lacking on the inactivation of E. coli K12 and L. plantarum in apple cider using pilot plant scale SCCO2 equipment. For this study, pasteurized pres...

Inactivation of salmonella in shell eggs by hot water immersion and its effect on quality

USDA-ARS?s Scientific Manuscript database

Thermal inactivation kinetics of heat resistant strains of Salmonella Enteritidis in shell eggs processed by hot water immersion were determined, and the effects of the processing on egg quality were evaluated. Shell eggs were inoculated with a composite of heat resistant Salmonella Enteritidis (SE)...

High pressure inactivation of Brettanomyces bruxellensis in red wine.

PubMed

van Wyk, Sanelle; Silva, Filipa V M

2017-05-01

Brettanomyces bruxellensis ("Brett") is a major spoilage concern for the wine industry worldwide, leading to undesirable sensory properties. Sulphur dioxide, is currently the preferred method for wine preservation. However, due to its negative effects on consumers, the use of new alternative non-thermal technologies are increasingly being investigated. The aim of this study was to determine and model the effect of high pressure processing (HPP) conditions and yeast strain on the inactivation of "Brett" in Cabernet Sauvignon wine. Processing at 200 MPa for 3 min resulted in 5.8 log reductions. However higher pressure is recommended to achieve high throughput in the wine industry, for example >6.0 log reductions were achieved after 400 MPa for 5 s. The inactivation of B. bruxellensis is pressure and time dependent, with increased treatment time and pressure leading to increased yeast inactivation. It was also found that yeast strain had a significant effect on HPP inactivation, with AWRI 1499 being the most resistant strain. The Weibull model successfully described the HPP "Brett" inactivation. HPP is a viable alternative for the inactivation of B. bruxellensis in wine, with the potential to reduce the industry's reliance on sulphur dioxide. Copyright © 2016 Elsevier Ltd. All rights reserved.

Final Report for the Center for Advanced Processing and Packaging Studies (CAPPS)

DTIC Science & Technology

2010-11-30

were almost completely inactivated (less than 1 log survivor) at pH 7.0 and 4 oC. 2. Inactivation of norovirus in strawberry puree. We have...determined the effectiveness of norovirus inactivation in strawberry puree. Viruses were inoculated into strawberry puree at final concentration of 107...higher pressure (450MPa) and lower temperature (4oC). 3. The effect of pH on the inactivation of norovirus in strawberry puree. The natural pH of

Lethality and injuring the effect of compression and decompression rates of high hydrostatic pressure on Escherichia coli O157:H7 in different matrices

NASA Astrophysics Data System (ADS)

Syed, Qamar Abbas; Buffa, Martin; Guamis, Buenaventura; Saldo, Jordi

2013-03-01

The effect of compression and decompression rates of high hydrostatic pressure (HHP) on Escherichia coli O157:H7 was investigated. Samples of orange juice, skimmed milk and Tris buffer were inoculated with E. coli O157:H7 and subjected to 600 MPa for 3 min at 4°C with fast, medium and slow compression and decompression. Analyses immediately after HHP treatment revealed that E. coli in milk and juice treated with fast compression suffered more than slow compression rates. Slow decompression resulted in higher inactivation of E. coli in all matrices. After overnight storage, highest stress-recovery (1.19 log cfu/mL) was observed in Tris buffer. Healthy cells were<1 log cfu/mL in milk and buffer samples, but no growth was detected in orange juice for any of the treatments immediately after HHP. After 15 days at 4°C, E. coli cells in skimmed milk and Tris buffer recovered significantly, whereas the recovery of sublethally injured cells was inhibited in orange juice.

A high-performance doped photocatalysts for inactivation of total coliforms in superficial waters using different sources of radiation.

PubMed

Claro, Elis Marina Turini; Bidoia, Ederio Dino; de Moraes, Peterson Bueno

2016-07-15

Photocatalytic water treatment has a currently elevated electricity demand and maintenance costs, but the photocatalytic water treatment may also assist in overcoming the limitations and drawbacks of conventional water treatment processes. Among the Advanced Oxidation Processes, heterogeneous photocatalysis is one of the most widely and efficiently used processes to degrade and/or remove a wide range of polluting compounds. The goal of this work was to find out a highly efficient photocatalytic disinfection process in superficial water with different doped photocatalysts and using three sources of radiation: mercury vapor lamp, solar simulator and UV-A LED. Three doped photocatalysts were prepared, SiZnO, NSiZnO and FNSiZnO. The inactivation efficiency of each synthesized photocatalysts was compared to a TiO2 P25 (Degussa(®)) 0.5 g L(-1) control. Photolysis inactivation efficiency was 85% with UV-A LED, which is considered very high, demanding low electricity consumption in the process, whereas mercury vapor lamp and solar simulator yielded 19% and 13% inactivation efficiency, respectively. The best conditions were found with photocatalysts SiZnO, FNSiZnO and NSiZnO irradiated with UV-A LED, where efficiency exceeded 95% that matched inactivation of coliforms using the same irradiation and photocatalyst TiO2. All photocatalysts showed photocatalytic activity with all three radiation sources able to inactivate total coliforms from river water. The use of UV-A LED as the light source without photocatalyst is very promising, allowing the creation of cost-effective and highly efficient water treatment plants. Copyright © 2016 Elsevier Ltd. All rights reserved.

Surface water disinfection by chlorination and advanced oxidation processes: Inactivation of an antibiotic resistant E. coli strain and cytotoxicity evaluation.

PubMed

Miranda, Andreza Costa; Lepretti, Marilena; Rizzo, Luigi; Caputo, Ivana; Vaiano, Vincenzo; Sacco, Olga; Lopes, Wilton Silva; Sannino, Diana

2016-06-01

The release of antibiotics into the environment can result in antibiotic resistance (AR) spread, which in turn can seriously affect human health. Antibiotic resistant bacteria have been detected in different aquatic environments used as drinking water source. Water disinfection may be a possible solution to minimize AR spread but conventional processes, such as chlorination, result in the formation of dangerous disinfection by-products. In this study advanced oxidation processes (AOPs), namely H2O2/UV, TiO2/UV and N-TiO2/UV, have been compared with chlorination in the inactivation of an AR Escherichia coli (E. coli) strain in surface water. TiO2 P25 and nitrogen doped TiO2 (N-TiO2), prepared by sol-gel method at two different synthesis temperatures (0 and -20°C), were investigated in heterogeneous photocatalysis experiments. Under the investigated conditions, chlorination (1.0 mg L(-1)) was the faster process (2.5 min) to achieve total inactivation (6 Log). Among AOPs, H2O2/UV resulted in the best inactivation rate: total inactivation (6 Log) was achieved in 45 min treatment. Total inactivation was not observed (4.5 Log), also after 120 min treatment, only for N-doped TiO2 synthesized at 0°C. Moreover, H2O2/UV and chlorination processes were evaluated in terms of cytotoxicity potential by means of 3-(4,5-dime-thylthiazol-2-yl)-2,5-diphenylte-trazolium colorimetric test on a human-derived cell line and they similarly affected HepG2 cells viability. Copyright © 2016 Elsevier B.V. All rights reserved.

Sterilization of liquid foods by pulsed electric fields-an innovative ultra-high temperature process.

PubMed

Reineke, Kai; Schottroff, Felix; Meneses, Nicolas; Knorr, Dietrich

2015-01-01

The intention of this study was to investigate the inactivation of endospores by a combined thermal and pulsed electric field (PEF) treatment. Therefore, self-cultivated spores of Bacillus subtilis and commercial Geobacillus stearothermophilus spores with certified heat resistance were utilized. Spores of both strains were suspended in saline water (5.3 mS cm(-1)), skim milk (0.3% fat; 5.3 mS cm(-1)) and fresh prepared carrot juice (7.73 mS cm(-1)). The combination of moderate preheating (70-90°C) and an insulated PEF-chamber, combined with a holding tube (65 cm) and a heat exchanger for cooling, enabled a rapid heat up to 105-140°C (measured above the PEF chamber) within 92.2-368.9 μs. To compare the PEF process with a pure thermal inactivation, each spore suspension was heat treated in thin glass capillaries and D-values from 90 to 130°C and its corresponding z-values were calculated. For a comparison of the inactivation data, F-values for the temperature fields of both processes were calculated by using computational fluid dynamics (CFD). A preheating of saline water to 70°C with a flow rate of 5 l h(-1), a frequency of 150 Hz and an energy input of 226.5 kJ kg(-1), resulted in a measured outlet temperature of 117°C and a 4.67 log10 inactivation of B. subtilis. The thermal process with identical F-value caused only a 3.71 log10 inactivation. This synergism of moderate preheating and PEF was even more pronounced for G. stearothermophilus spores in saline water. A preheating to 95°C and an energy input of 144 kJ kg(-1) resulted in an outlet temperature of 126°C and a 3.28 log10 inactivation, whereas nearly no inactivation (0.2 log10) was achieved during the thermal treatment. Hence, the PEF technology was evaluated as an alternative ultra-high temperature process. However, for an industrial scale application of this process for sterilization, optimization of the treatment chamber design is needed to reduce the occurring inhomogeneous temperature fields.

The detrimental influence of bacteria (E. coli, Shigella and Salmonella) on the degradation of organic compounds (and vice versa) in TiO2 photocatalysis and near-neutral photo-Fenton processes under simulated solar light.

PubMed

Moncayo-Lasso, Alejandro; Mora-Arismendi, Luis Enrique; Rengifo-Herrera, Julián Andrés; Sanabria, Janeth; Benítez, Norberto; Pulgarin, César

2012-05-01

TiO2 photocatalytic and near-neutral photo-Fenton processes were tested under simulated solar light to degrade two models of natural organic matter - resorcinol (R) (which should interact strongly with TiO2 surfaces) and hydroquinone (H) - separately or in the presence of bacteria. Under similar oxidative conditions, inactivation of Escherichia coli, Shigella sonnei and Salmonella typhimurium was carried out in the absence and in the presence of 10 mg L(-1) of R and H. The 100% abatement of R and H by using a TiO2 photocatalytic process in the absence of bacteria was observed in 90 min for R and in 120 min for H, while in the presence of microorganisms abatement was only of 55% and 35% for R and H, respectively. Photo-Fenton reagent at pH 5.0 completely removed R and H in 40 min, whereas in the presence of microorganisms their degradation was of 60% to 80%. On the other hand, 2 h of TiO2 photocatalytic process inactivated S. typhimurium and E. coli cells in three and six orders of magnitude, respectively, while S. sonnei was completely inactivated in 10 min. In the presence of R or H, the bacterial inactivation via TiO2 photocatalysis was significantly decreased. With photo-Fenton reagent at pH 5 all the microorganisms tested were completely inactivated in 40 min of simulated solar light irradiation in the absence of organics. When R and H were present, bacterial photo-Fenton inactivation was less affected. The obtained results suggest that in both TiO2 and iron photo-assisted processes, there is competition between organic substances and bacteria simultaneously present for generated reactive oxygen species (ROS). This competition is most important in heterogeneous systems, mainly when there are strong organic-TiO2 surface interactions, as in the resorcinol case, suggesting that bacteria-TiO2 interactions could play a key role in photocatalytic cell inactivation processes.

Dynamic modulation of spike timing-dependent calcium influx during corticostriatal upstates

PubMed Central

Evans, R. C.; Maniar, Y. M.

2013-01-01

The striatum of the basal ganglia demonstrates distinctive upstate and downstate membrane potential oscillations during slow-wave sleep and under anesthetic. The upstates generate calcium transients in the dendrites, and the amplitude of these calcium transients depends strongly on the timing of the action potential (AP) within the upstate. Calcium is essential for synaptic plasticity in the striatum, and these large calcium transients during the upstates may control which synapses undergo plastic changes. To investigate the mechanisms that underlie the relationship between calcium and AP timing, we have developed a realistic biophysical model of a medium spiny neuron (MSN). We have implemented sophisticated calcium dynamics including calcium diffusion, buffering, and pump extrusion, which accurately replicate published data. Using this model, we found that either the slow inactivation of dendritic sodium channels (NaSI) or the calcium inactivation of voltage-gated calcium channels (CDI) can cause high calcium corresponding to early APs and lower calcium corresponding to later APs. We found that only CDI can account for the experimental observation that sensitivity to AP timing is dependent on NMDA receptors. Additional simulations demonstrated a mechanism by which MSNs can dynamically modulate their sensitivity to AP timing and show that sensitivity to specifically timed pre- and postsynaptic pairings (as in spike timing-dependent plasticity protocols) is altered by the timing of the pairing within the upstate. These findings have implications for synaptic plasticity in vivo during sleep when the upstate-downstate pattern is prominent in the striatum. PMID:23843436

Reactive species modify NaV1.8 channels and affect action potentials in murine dorsal root ganglia neurons

PubMed Central

Schink, Martin; Leipolcf, Enrico; Schirmeyer, Jana; Schönherr, Roland; Hoshi, Toshinori; Heinemann, Stefan H.

2016-01-01

Dorsal root ganglia (DRG) neurons are important relay stations between the periphery and the central nervous system and are essential for somatosensory signaling. Reactive species are produced in a variety of physiological and pathophysiological conditions and are known to alter electric signaling. Here we studied the influence of reactive species on the electrical properties of DRG neurons from mice with the whole-cell patch-clamp method. Even mild stress induced by either low concentrations of chloramine-T (10 µM) or low-intensity blue-light irradiation profoundly diminished action potential frequency but prolonged single action potentials in wild-type neurons. The impact on evoked action potentials was much smaller in neurons deficient of the tetrodotoxin (TTX)-resistant voltage-gated sodium channel NaV1.8 (NaV1.8−/−), the channel most important for the action potential upstroke in DRG neurons. Low concentrations of chloramine-T caused a significant reduction of NaV1.8 peak current and at higher concentrations progressively slowed down inactivation. Blue light had a smaller effect on amplitude but slowed down NaV1.8 channel inactivation. The observed effects were less apparent for TTX-sensitive NaV channels. NaV1.8 is an important reactive-species-sensitive component in the electrical signaling of DRG neurons, potentially giving rise to loss-of-function and gain-of-function phenomena depending on the type of reactive species and their effective concentration and time of exposure. PMID:26383867

Reactive species modify NaV1.8 channels and affect action potentials in murine dorsal root ganglion neurons.

PubMed

Schink, Martin; Leipold, Enrico; Schirmeyer, Jana; Schönherr, Roland; Hoshi, Toshinori; Heinemann, Stefan H

2016-01-01

Dorsal root ganglion (DRG) neurons are important relay stations between the periphery and the central nervous system and are essential for somatosensory signaling. Reactive species are produced in a variety of physiological and pathophysiological conditions and are known to alter electric signaling. Here we studied the influence of reactive species on the electrical properties of DRG neurons from mice with the whole-cell patch-clamp method. Even mild stress induced by either low concentrations of chloramine-T (10 μM) or low-intensity blue light irradiation profoundly diminished action potential frequency but prolonged single action potentials in wild-type neurons. The impact on evoked action potentials was much smaller in neurons deficient of the tetrodotoxin (TTX)-resistant voltage-gated sodium channel NaV1.8 (NaV1.8(-/-)), the channel most important for the action potential upstroke in DRG neurons. Low concentrations of chloramine-T caused a significant reduction of NaV1.8 peak current and, at higher concentrations, progressively slowed down inactivation. Blue light had a smaller effect on amplitude but slowed down NaV1.8 channel inactivation. The observed effects were less apparent for TTX-sensitive NaV channels. NaV1.8 is an important reactive-species-sensitive component in the electrical signaling of DRG neurons, potentially giving rise to loss-of-function and gain-of-function phenomena depending on the type of reactive species and their effective concentration and time of exposure.

Effect of protein load on stability of immobilized enzymes.

PubMed

Fernandez-Lopez, Laura; Pedrero, Sara G; Lopez-Carrobles, Nerea; Gorines, Beatriz C; Virgen-Ortíz, Jose J; Fernandez-Lafuente, Roberto

2017-03-01

Different lipases have been immobilized on octyl agarose beads at 1mg/g and at maximum loading, via physical interfacial activation versus the octyl layer on the support. The stability of the preparations was analyzed. Most biocatalysts had the expected result: the apparent stability increased using the highly loaded preparations, due to the diffusional limitations that reduced the initial observed activity. However, lipase B from Candida antarctica (CALB) was significantly more stable using the lowly loaded preparation than the maximum loaded one. This negative effect of the enzyme crowding on enzyme stability was found in inactivations at pH 5, 7 or 9, but not in inactivations in the presence of organic solvents. The immobilization using ethanol to reduce the immobilization rate had no effect on the stability of the lowly loaded preparation, while the highly loaded enzyme biocatalysts increased their stabilities, becoming very similar to that of the lowly loaded preparation. Results suggested that CALB molecules immobilized on octyl agarose may be closely packed together due to the high immobilization rate and this produced some negative interactions between immobilized enzyme molecules during enzyme thermal inactivation. Slowing-down the immobilization rate may be a solution for this unexpected problem. Copyright © 2016 Elsevier Inc. All rights reserved.

Notch signaling inhibits hepatocellular carcinoma following inactivation of the RB pathway

PubMed Central

Viatour, Patrick; Ehmer, Ursula; Saddic, Louis A.; Dorrell, Craig; Andersen, Jesper B.; Lin, Chenwei; Zmoos, Anne-Flore; Mazur, Pawel K.; Schaffer, Bethany E.; Ostermeier, Austin; Vogel, Hannes; Sylvester, Karl G.; Thorgeirsson, Snorri S.; Grompe, Markus

2011-01-01

Hepatocellular carcinoma (HCC) is the third cancer killer worldwide with >600,000 deaths every year. Although the major risk factors are known, therapeutic options in patients remain limited in part because of our incomplete understanding of the cellular and molecular mechanisms influencing HCC development. Evidence indicates that the retinoblastoma (RB) pathway is functionally inactivated in most cases of HCC by genetic, epigenetic, and/or viral mechanisms. To investigate the functional relevance of this observation, we inactivated the RB pathway in the liver of adult mice by deleting the three members of the Rb (Rb1) gene family: Rb, p107, and p130. Rb family triple knockout mice develop liver tumors with histopathological features and gene expression profiles similar to human HCC. In this mouse model, cancer initiation is associated with the specific expansion of populations of liver stem/progenitor cells, indicating that the RB pathway may prevent HCC development by maintaining the quiescence of adult liver progenitor cells. In addition, we show that during tumor progression, activation of the Notch pathway via E2F transcription factors serves as a negative feedback mechanism to slow HCC growth. The level of Notch activity is also able to predict survival of HCC patients, suggesting novel means to diagnose and treat HCC. PMID:21875955

Inactivation of Staphylococcus aureus and Salmonella enteritidis in tryptic soy broth and caviar samples by high pressure processing.

PubMed

Fioretto, F; Cruz, C; Largeteau, A; Sarli, T A; Demazeau, G; El Moueffak, A

2005-08-01

We studied the action of high pressure processing on the inactivation of two foodborne pathogens, Staphylococcus aureus ATCC 6538 and Salmonella enteritidis ATCC 13076, suspended in a culture medium and inoculated into caviar samples. The baroresistance of the two pathogens in a tryptic soy broth suspension at a concentration of 10(8)-10(9) colony-forming units/ml was tested for continuous and cycled pressurization in the 150- to 550-MPa range and for 15-min treatments at room temperature. The increase of cycle number permitted the reduction of the pressure level able to totally inactivate both microorganisms in the tryptic soy broth suspension, whereas the effect of different procedure times on complete inactivation of the microorganisms inoculated into caviar was similar.

Centromere inactivation on a neo-Y fusion chromosome in threespine stickleback fish

PubMed Central

Cech, Jennifer N.; Peichel, Catherine L.

2016-01-01

Having one and only one centromere per chromosome is essential for proper chromosome segregation during both mitosis and meiosis. Chromosomes containing two centromeres are known as dicentric and often mis-segregate during cell division, resulting in aneuploidy or chromosome breakage. Dicentric chromosome can be stabilized by centromere inactivation, a process which re-establishes monocentric chromosomes. However, little is known about this process in naturally occurring dicentric chromosomes. Using a combination of fluorescence in situ hybridization (FISH) and immunoflourescence combined with FISH (IF-FISH) on metaphase chromosome spreads, we demonstrate that centromere inactivation has evolved on a neo-Y chromosome fusion in the Japan Sea threespine stickleback fish (Gasterosteus nipponicus). We found that the centromere derived from the ancestral Y chromosome has been inactivated. Our data further suggest that there have been genetic changes to this centromere in the two million years since the formation of the neo-Y chromosome, but it remains unclear whether these genetic changes are a cause or consequence of centromere inactivation. PMID:27553478

Heme impairs the ball-and-chain inactivation of potassium channels.

PubMed

Sahoo, Nirakar; Goradia, Nishit; Ohlenschläger, Oliver; Schönherr, Roland; Friedrich, Manfred; Plass, Winfried; Kappl, Reinhard; Hoshi, Toshinori; Heinemann, Stefan H

2013-10-15

Fine-tuned regulation of K(+) channel inactivation enables excitable cells to adjust action potential firing. Fast inactivation present in some K(+) channels is mediated by the distal N-terminal structure (ball) occluding the ion permeation pathway. Here we show that Kv1.4 K(+) channels are potently regulated by intracellular free heme; heme binds to the N-terminal inactivation domain and thereby impairs the inactivation process, thus enhancing the K(+) current with an apparent EC50 value of ∼20 nM. Functional studies on channel mutants and structural investigations on recombinant inactivation ball domain peptides encompassing the first 61 residues of Kv1.4 revealed a heme-responsive binding motif involving Cys13:His16 and a secondary histidine at position 35. Heme binding to the N-terminal inactivation domain induces a conformational constraint that prevents it from reaching its receptor site at the vestibule of the channel pore.

Pathogens Inactivated by Low-Energy-Electron Irradiation Maintain Antigenic Properties and Induce Protective Immune Responses

PubMed Central

Fertey, Jasmin; Bayer, Lea; Grunwald, Thomas; Pohl, Alexandra; Beckmann, Jana; Gotzmann, Gaby; Casado, Javier Portillo; Schönfelder, Jessy; Rögner, Frank-Holm; Wetzel, Christiane; Thoma, Martin; Bailer, Susanne M.; Hiller, Ekkehard; Rupp, Steffen; Ulbert, Sebastian

2016-01-01

Inactivated vaccines are commonly produced by incubating pathogens with chemicals such as formaldehyde or β-propiolactone. This is a time-consuming process, the inactivation efficiency displays high variability and extensive downstream procedures are often required. Moreover, application of chemicals alters the antigenic components of the viruses or bacteria, resulting in reduced antibody specificity and therefore stimulation of a less effective immune response. An alternative method for inactivation of pathogens is ionizing radiation. It acts very fast and predominantly damages nucleic acids, conserving most of the antigenic structures. However, currently used irradiation technologies (mostly gamma-rays and high energy electrons) require large and complex shielding constructions to protect the environment from radioactivity or X-rays generated during the process. This excludes them from direct integration into biological production facilities. Here, low-energy electron irradiation (LEEI) is presented as an alternative inactivation method for pathogens in liquid solutions. LEEI can be used in normal laboratories, including good manufacturing practice (GMP)- or high biosafety level (BSL)-environments, as only minor shielding is necessary. We show that LEEI efficiently inactivates different viruses (influenza A (H3N8), porcine reproductive and respiratory syndrome virus (PRRSV), equine herpesvirus 1 (EHV-1)) and bacteria (Escherichia coli) and maintains their antigenicity. Moreover, LEEI-inactivated influenza A viruses elicit protective immune responses in animals, as analyzed by virus neutralization assays and viral load determination upon challenge. These results have implications for novel ways of developing and manufacturing inactivated vaccines with improved efficacy. PMID:27886076

Slow Bursting Neurons of Mouse Cortical Layer 6b Are Depolarized by Hypocretin/Orexin and Major Transmitters of Arousal

PubMed Central

Wenger Combremont, Anne-Laure; Bayer, Laurence; Dupré, Anouk; Mühlethaler, Michel; Serafin, Mauro

2016-01-01

Neurons firing spontaneously in bursts in the absence of synaptic transmission have been previously recorded in different layers of cortical brain slices. It has been suggested that such neurons could contribute to the generation of alternating UP and DOWN states, a pattern of activity seen during slow-wave sleep. Here, we show that in layer 6b (L6b), known from our previous studies to contain neurons highly responsive to the wake-promoting transmitter hypocretin/orexin (hcrt/orx), there is a set of neurons, endowed with distinct intrinsic properties, which displayed a strong propensity to fire spontaneously in rhythmic bursts. In response to small depolarizing steps, they responded with a delayed firing of action potentials which, upon higher depolarizing steps, invariably inactivated and were followed by a depolarized plateau potential and a depolarizing afterpotential. These cells also displayed a strong hyperpolarization-activated rectification compatible with the presence of an Ih current. Most L6b neurons with such properties were able to fire spontaneously in bursts. Their bursting activity was of intrinsic origin as it persisted not only in presence of blockers of ionotropic glutamatergic and GABAergic receptors but also in a condition of complete synaptic blockade. However, a small number of these neurons displayed a mix of intrinsic bursting and synaptically driven recurrent UP and DOWN states. Most of the bursting L6b neurons were depolarized and excited by hcrt/orx through a direct postsynaptic mechanism that led to tonic firing and eventually inactivation. Similarly, they were directly excited by noradrenaline, histamine, dopamine, and neurotensin. Finally, the intracellular injection of these cells with dye and their subsequent Neurolucida reconstruction indicated that they were spiny non-pyramidal neurons. These results lead us to suggest that the propensity for slow rhythmic bursting of this set of L6b neurons could be directly impeded by hcrt/orx and other wake-promoting transmitters. PMID:27379007

Human norovirus inactivation in oysters by high hydrostatic pressure processing: A randomized double-blinded study

USDA-ARS?s Scientific Manuscript database

This randomized, double-blinded, clinical trial assessed the effect of high hydrostatic pressure processing (HPP) on genogroup I.1 human norovirus (HuNoV) inactivation in virus-seeded oysters when ingested by subjects. The safety and efficacy of HPP treatments were assessed in three study phases wi...

Characterization of drinking water treatment for virus risk assessment.

PubMed

Teunis, P F M; Rutjes, S A; Westrell, T; de Roda Husman, A M

2009-02-01

Removal or inactivation of viruses in drinking water treatment processes can be quantified by measuring the concentrations of viruses or virus indicators in water before and after treatment. Virus reduction is then calculated from the ratio of these concentrations. Most often only the average reduction is reported. That is not sufficient when treatment efficiency must be characterized in quantitative risk assessment. We present three simple models allowing statistical analysis of series of counts before and after treatment: distribution of the ratio of concentrations, and distribution of the probability of passage for unpaired and paired water samples. Performance of these models is demonstrated for several processes (long and short term storage, coagulation/filtration, coagulation/sedimentation, slow sand filtration, membrane filtration, and ozone disinfection) using microbial indicator data from full-scale treatment processes. All three models allow estimation of the variation in (log) reduction as well as its uncertainty; the results can be easily used in risk assessment. Although they have different characteristics and are present in vastly different concentrations, different viruses and/or bacteriophages appear to show similar reductions in a particular treatment process, allowing generalization of the reduction for each process type across virus groups. The processes characterized in this paper may be used as reference for waterborne virus risk assessment, to check against location specific data, and in case no such data are available, to use as defaults.

Electrochemically assisted photocatalysis: Highly efficient treatment using thermal titanium oxides doped and non-doped electrodes for water disinfection.

PubMed

Dos Santos, Andreia Betina Kreuser; Claro, Elis Marina Turini; Montagnolli, Renato Nallin; Cruz, Jaqueline Matos; Lopes, Paulo Renato Matos; Bidoia, Ederio Dino

2017-12-15

Electrochemically assisted photocatalysis (by electronic drainage) is a highly promising method for disinfection of water. In this research, the efficiency of photolytic oxidation using UV-A radiation and electrochemically assisted photocatalysis (with electric potential of 1.5 V) was studied by using electrodes prepared by thermal treatment and doped with silver, for inactivation of Escherichia coli and Staphylococcus aureus. The Chick-Watson microorganism inactivation model was applied and the electrical energy consumption of the process was calculated. It was observed no significant inactivation of microorganisms when UV-A light or electric potential were applied separately. However, the electrochemically assisted photocatalytic process, with Ag-doped electrode completely inactivated the microbial population after 10 (E. coli) and 60 min (S. aureus). The best performing non-doped electrodes achieved 52.74% (E. coli) and 44.09% (S. aureus) inactivation rates after 60 min. Thus, electrochemically assisted photocatalytic activity was not only effective for the inactivation of microorganisms, but also notably low on electrical energy consumption during the treatment due to small current and low electric potential applied. Copyright © 2017 Elsevier Ltd. All rights reserved.

Transport, anoxia and energy control on anaerobic respiration and methanogenesis in anoxic peat soils

NASA Astrophysics Data System (ADS)

Bonaiuti, Simona; Blodau, Christian; Knorr, Klaus-Holger

2017-04-01

In deep and permanently water saturated peat deposits, extremely low diffusive transport and concomitant build-up of metabolic end-products, i.e of dissolved inorganic carbon (DIC) and methane (CH4), have been found to slow-down anaerobic respiration and methanogenesis. Such accumulation of DIC and CH4 lowers the Gibbs free energy yield of terminal respiration and methanogenesis, which can inhibit the course of anaerobic metabolic processes. In particular, this affects terminal steps of the breakdown of organic carbon (C), such as methanogenesis, acetogenesis and fermentation processes, which occur near thermodynamic minimum energy thresholds. This effect is thus of critical importance for the long-term C sequestration, as the slow-down of decomposition ultimately regulates the long-term fate of C in deep peat deposits. The exact controls of this observed slow-down of organic matter mineralization are not yet fully understood. Moreover, altered patterns of water or gas transport due to predicted changes in climate may affect these controls in peat soils. Therefore, the aim of this study was to investigate how burial of peat leads to an inactivation of anaerobic decomposition and to investigate the effects of advective water transport and persistently anoxic conditions on anaerobic decomposition, temporal evolution of thermodynamic energy yields to methanogenesis and methanogenic pathways. To this end, we conducted a column experiment with homogenized, ombrotrophic peat over a period of 300 days at 20˚ C. We tested i) a control treatment under diffusive transport only, ii) an advective flow treatment with a flow of 10 mm d-1, and iv) an anoxic treatment to evaluate changes in decomposition in absence of oxygen in the unsaturated zone of the cores. A slow-down of anaerobic respiration and methanogenesis generally set in at larger depths after 150 days at CH4 concentrations of 0.6-0.9 mmol L-1 and DIC concentrations of 6-12 mmol L-1. This effect occurred at higher concentration levels and faster than previously observed. Advective water transport effectively extended the zone of methanogenesis down to 40 cm depth until inhibiting conditions were reached, although net turnover at greater depths was not affected. Strictly anoxic conditions in the unsaturated zone, where diffusive transport is high, had little effect on accelerating anaerobic decomposition. The slow-down of net production rates of CO2 and CH4 agreed well with the decline over time of Gibbs free energies available to methanogenesis, supporting a thermodynamic constraint on decomposition in deeper peat deposits. Keywords: Peatlands; Anaerobic decomposition; Methanogenesis; Production rates; Advection; Anoxia; Thermodynamic calculations.

Rapid water disinfection using vertically aligned MoS 2 nanofilms and visible light

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Chong; Kong, Desheng; Hsu, Po -Chun

Here, solar energy is readily available in most climates and can be used for water purification. However, solar disinfection of drinking water (SODIS) mostly relies on ultraviolet light, which represents only 4% of total solar energy, and this leads to slow treatment speed. The development of new materials that can harvest visible light for water disinfection, and speed up solar water purification, is therefore highly desirable. Here, we show that few-layered vertically aligned MoS 2 (FLV-MoS 2) films can be used to harvest the whole spectrum of visible light (~ 50% of solar energy) and achieve highly efficient water disinfection.more » The bandgap of MoS 2 was increased from 1.3 eV to 1.55 eV by decreasing the domain size, which allowed the FLV-MoS 2 to generate reactive oxygen species (ROS) for bacterial inactivation in water. The FLV-MoS 2 showed ~15 times better log inactivation efficiency of indicator bacteria compared to bulk MoS 2, and much faster inactivation of bacteria under both visible light and sunlight illumination compared to widely used TiO 2. Moreover, by using a 5 nm copper film on top of the FLV-MoS 2 as a catalyst to facilitate electron-hole pair separation and promote the generation of ROS, the disinfection rate was further increased 6 fold. With our approach, we achieved water disinfection of >99.999% inactivation of bacteria in 20 minutes with a small amount of material (1.6 mg/L) under simulated visible light.« less

Rapid water disinfection using vertically aligned MoS 2 nanofilms and visible light

DOE PAGES

Liu, Chong; Kong, Desheng; Hsu, Po -Chun; ...

2016-08-15

Here, solar energy is readily available in most climates and can be used for water purification. However, solar disinfection of drinking water (SODIS) mostly relies on ultraviolet light, which represents only 4% of total solar energy, and this leads to slow treatment speed. The development of new materials that can harvest visible light for water disinfection, and speed up solar water purification, is therefore highly desirable. Here, we show that few-layered vertically aligned MoS 2 (FLV-MoS 2) films can be used to harvest the whole spectrum of visible light (~ 50% of solar energy) and achieve highly efficient water disinfection.more » The bandgap of MoS 2 was increased from 1.3 eV to 1.55 eV by decreasing the domain size, which allowed the FLV-MoS 2 to generate reactive oxygen species (ROS) for bacterial inactivation in water. The FLV-MoS 2 showed ~15 times better log inactivation efficiency of indicator bacteria compared to bulk MoS 2, and much faster inactivation of bacteria under both visible light and sunlight illumination compared to widely used TiO 2. Moreover, by using a 5 nm copper film on top of the FLV-MoS 2 as a catalyst to facilitate electron-hole pair separation and promote the generation of ROS, the disinfection rate was further increased 6 fold. With our approach, we achieved water disinfection of >99.999% inactivation of bacteria in 20 minutes with a small amount of material (1.6 mg/L) under simulated visible light.« less

Mechanism of hERG channel block by the psychoactive indole alkaloid ibogaine.

PubMed

Thurner, Patrick; Stary-Weinzinger, Anna; Gafar, Hend; Gawali, Vaibhavkumar S; Kudlacek, Oliver; Zezula, Juergen; Hilber, Karlheinz; Boehm, Stefan; Sandtner, Walter; Koenig, Xaver

2014-02-01

Ibogaine is a psychoactive indole alkaloid. Its use as an antiaddictive agent has been accompanied by QT prolongation and cardiac arrhythmias, which are most likely caused by human ether a go-go-related gene (hERG) potassium channel inhibition. Therefore, we studied in detail the interaction of ibogaine with hERG channels heterologously expressed in mammalian kidney tsA-201 cells. Currents through hERG channels were blocked regardless of whether ibogaine was applied via the extracellular or intracellular solution. The extent of inhibition was determined by the relative pH values. Block occurred during activation of the channels and was not observed for resting channels. With increasing depolarizations, ibogaine block grew and developed faster. Steady-state activation and inactivation of the channel were shifted to more negative potentials. Deactivation was slowed, whereas inactivation was accelerated. Mutations in the binding site reported for other hERG channel blockers (Y652A and F656A) reduced the potency of ibogaine, whereas an inactivation-deficient double mutant (G628C/S631C) was as sensitive as wild-type channels. Molecular drug docking indicated binding within the inner cavity of the channel independently of the protonation of ibogaine. Experimental current traces were fit to a kinetic model of hERG channel gating, revealing preferential binding of ibogaine to the open and inactivated state. Taken together, these findings show that ibogaine blocks hERG channels from the cytosolic side either in its charged form alone or in company with its uncharged form and alters the currents by changing the relative contribution of channel states over time.

Effect of process temperature, pH and suspended solids content upon pasteurization of a model agricultural waste during thermophilic aerobic digestion.

PubMed

Ugwuanyi, J O; Harvey, L M; McNeil, B

1999-09-01

Thermophilic aerobic digestion(TAD), or liquid composting, is a versatile new process for the treatment and stabilization of high strength wastes of liquid or, perhaps more importantly, slurry consistency. The pattern of inactivation of various pathogenic and indicator organisms was studied using batch digestions under conditions that may be expected to be found in full-scale TAD processes. Rapid inactivation of test populations occurred within the first 10 min from the start of digestion. The inactivation rate was slightly lower when digestions were conducted below 60 degrees C. In some instances, a 'tail' was apparent, possibly indicating the survival of relatively resistant sub-populations particularly in the case of Serratia marcescens and Enterococcus faecalis, or of clumping or attachment of cells to particulate materials. The effect of pH on the inactivation of the test populations depended on the temperature of digestion, but varied with the test population. At 55 degrees C Escherichia coli was more sensitive to temperature effects at pH 7 than at pH 8, but was more sensitive at pH 8, 60 degrees C. The reverse was the case at 60 degrees C for Ent. faecalis. An increase in the solid content of the digesting waste caused a progressive increase in the protection of test organisms from thermal inactivation. Challenging a TAD process with test strains allows (via estimation of D-values) a quantification of the cidal effects of such processes, with a view to manipulating process variables to enhance such effects.

Inactivation characteristics of ozone and electrolysis process for ballast water treatment using B. subtilis spores as a probe.

PubMed

Jung, Youmi; Yoon, Yeojoon; Hong, Eunkyung; Kwon, Minhwan; Kang, Joon-Wun

2013-07-15

Since ballast water affects the ocean ecosystem, the International Maritime Organization (IMO) sets a standard for ballast water management and might impose much tighter regulations in the future. The aim of this study is to evaluate the inactivation efficiency of ozonation, electrolysis, and an ozonation-electrolysis combined process, using B. subtilis spores. In seawater ozonation, HOBr is the key active substance for inactivation, because of rapid reactivity of ozone with Br(-) in seawater. In seawater electrolysis, it is also HOBr, but not HOCl, because of the rapid reaction of HOCl with Br(-), which has not been recognized carefully, even though many electrolysis technologies have been approved by the IMO. Inactivation pattern was different in ozonation and electrolysis, which has some limitations with the tailing or lag-phase, respectively. However, each deficiency can be overcome with a combined process, which is most effective as a sequential application of ozonation followed by electrolysis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Non-random X chromosome inactivation in an affected twin in a monozygotic twin pair discordant for Wiedemann-Beckwith syndrome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oestavik, R.E.; Eiklid, K.; Oerstavik, K.H.

1995-03-27

Wiedemann-Beckwith syndrome (WBS) is a syndrome including exomphalos, macroglossia, and generalized overgrowth. The locus has been assigned to 11p15, and genomic imprinting may play a part in the expression of one or more genes involved. Most cases are sporadic. An excess of female monozygotic twins discordant for WBS have been reported, and it has been proposed that this excess could be related to the process of X chromosome inactivation. We have therefore studied X chromosome inactivation in 13-year-old monozygotic twin girls who were discordant for WBS. In addition, both twins had Tourette syndrome. The twins were monochorionic and therefore themore » result of a late twinning process. This has also been the case in previously reported discordant twin pairs with information on placentation. X chromosome inactivation was determined in DNA from peripheral blood cells by PCR analysis at the androgen receptor locus. The affected twin had a completely skewed X inactivation, where the paternal allele was on the active X chromosome in all cells. The unaffected twin had a moderately skewed X inactivation in the same direction, whereas the mother had a random pattern. Further studies are necessary to establish a possible association between the expression of WBS and X chromosome inactivation. 18 refs., 2 figs., 1 tab.« less

Bacteria and fungi inactivation by photocatalysis under UVA irradiation: liquid and gas phase.

PubMed

Rodrigues-Silva, Caio; Miranda, Sandra M; Lopes, Filipe V S; Silva, Mário; Dezotti, Márcia; Silva, Adrián M T; Faria, Joaquim L; Boaventura, Rui A R; Vilar, Vítor J P; Pinto, Eugénia

2017-03-01

In the last decade, environmental risks associated with wastewater treatment plants (WWTPs) have become a concern in the scientific community due to the absence of specific legislation governing the occupational exposure limits (OEL) for microorganisms present in indoor air. Thus, it is necessary to develop techniques to effectively inactivate microorganisms present in the air of WWTPs facilities. In the present work, ultraviolet light A radiation was used as inactivation tool. The microbial population was not visibly reduced in the bioaerosol by ultraviolet light A (UVA) photolysis. The UVA photocatalytic process for the inactivation of microorganisms (bacteria and fungi, ATCC strains and isolates from indoor air samples of a WWTP) using titanium dioxide (TiO 2 P25) and zinc oxide (ZnO) was tested in both liquid-phase and airborne conditions. In the slurry conditions at liquid phase, P25 showed a better performance in inactivation. For this reason, gas-phase assays were performed in a tubular photoreactor packed with cellulose acetate monolithic structures coated with P25. The survival rate of microorganisms under study decreased with the catalyst load and the UVA exposure time. Inactivation of fungi was slower than resistant bacteria, followed by Gram-positive bacteria and Gram-negative bacteria. Graphical abstract Inactivation of fungi and bacteria in gas phase by photocatalitic process performed in a tubular photoreactor packed with cellulose acetate monolith structures coated with TiO 2 .

Vasopressin Analogue DDAVP in Diabetes Insipidus: Clinical and Laboratory Studies

PubMed Central

Edwards, C. R. W.; Kitau, M. J.; Chard, T.; Besser, G. M.

1973-01-01

In seven patients with cranial diabetes insipidus an analogue of vasopressin, DDAVP, produced an antidiuresis lasting up to 20 hours after a single intranasal dose. Lysine vasopressin (LVP) in the same dose produced a less potent antidiuresis which lasted for only three to four hours. The plasma half life of DDAVP was 7·8 and 75·5 min for the fast and slow phases, compared with 2·5 and 14·5 min for LVP. Radioiodine-labelled DDAVP was not destroyed by incubation with late pregnancy plasma, which contains an enzyme that inactivates vasopressin. The slow metabolic clearance of DDAVP, its absorption through the nasal mucosa, and its lack of side effects make this the ideal drug for the treatment of vasopressin-sensitive diabetes insipidus. Patients usually require 10 to 20 μg DDAVP given intranasally twice daily for good clinical control of their diabetes insipidus. PMID:4581079

[Phosphatase activity in Amoeba proteus at pH 9.0].

PubMed

Sopina, V A

2007-01-01

In the free-living amoeba Amoeba proteus (strain B), after PAAG disk-electrophoresis of the homogenate supernatant, at using 1-naphthyl phosphate as a substrate and pH 9.0, three forms of phosphatase activity were revealed; they were arbitrarily called "fast", "intermediate", and "slow" phosphatases. The fast phosphatase has been established to be a fraction of lysosomal acid phosphatase that preserves some low activity at alkaline pH. The question as to which particular class the intermediate phosphatase belongs to has remained unanswered: it can be both acid phosphatase and protein tyrosine phosphatase (PTP). Based on data of inhibitor analysis, large substrate specificity, results of experiments with reactivation by Zn ions after inactivation with EDTA, other than in the fast and intermediate phosphatases localization in the amoeba cell, it is concluded that only slow phosphatase can be classified as alkaline phosphatase (EC 3.1.3.1).

2-Thioxanthines Are Mechanism-based Inactivators of Myeloperoxidase That Block Oxidative Stress during Inflammation*

PubMed Central

Tidén, Anna-Karin; Sjögren, Tove; Svensson, Mats; Bernlind, Alexandra; Senthilmohan, Revathy; Auchère, Francoise; Norman, Henrietta; Markgren, Per-Olof; Gustavsson, Susanne; Schmidt, Staffan; Lundquist, Stefan; Forbes, Louisa V.; Magon, Nicholas J.; Paton, Louise N.; Jameson, Guy N. L.; Eriksson, Håkan; Kettle, Anthony J.

2011-01-01

Myeloperoxidase (MPO) is a prime candidate for promoting oxidative stress during inflammation. This abundant enzyme of neutrophils uses hydrogen peroxide to oxidize chloride to highly reactive and toxic chlorine bleach. We have identified 2-thioxanthines as potent mechanism-based inactivators of MPO. Mass spectrometry and x-ray crystal structures revealed that these inhibitors become covalently attached to the heme prosthetic groups of the enzyme. We propose a mechanism whereby 2-thioxanthines are oxidized, and their incipient free radicals react with the heme groups of the enzyme before they can exit the active site. 2-Thioxanthines inhibited MPO in plasma and decreased protein chlorination in a mouse model of peritonitis. They slowed but did not prevent neutrophils from killing bacteria and were poor inhibitors of thyroid peroxidase. Our study shows that MPO is susceptible to the free radicals it generates, and this Achilles' heel of the enzyme can be exploited to block oxidative stress during inflammation. PMID:21880720

Unfair competition governs the interaction of pCPI-17 with myosin phosphatase (PP1-MYPT1)

PubMed Central

Filter, Joshua J; Williams, Byron C; Eto, Masumi; Shalloway, David; Goldberg, Michael L

2017-01-01

The small phosphoprotein pCPI-17 inhibits myosin light-chain phosphatase (MLCP). Current models postulate that during muscle relaxation, phosphatases other than MLCP dephosphorylate and inactivate pCPI-17 to restore MLCP activity. We show here that such hypotheses are insufficient to account for the observed rapidity of pCPI-17 inactivation in mammalian smooth muscles. Instead, MLCP itself is the critical enzyme for pCPI-17 dephosphorylation. We call the mutual sequestration mechanism through which pCPI-17 and MLCP interact inhibition by unfair competition: MLCP protects pCPI-17 from other phosphatases, while pCPI-17 blocks other substrates from MLCP’s active site. MLCP dephosphorylates pCPI-17 at a slow rate that is, nonetheless, both sufficient and necessary to explain the speed of pCPI-17 dephosphorylation and the consequent MLCP activation during muscle relaxation. DOI: http://dx.doi.org/10.7554/eLife.24665.001 PMID:28387646

Flow cytometric evaluation of physico-chemical impact on Gram-positive and Gram-negative bacteria

PubMed Central

Fröhling, Antje; Schlüter, Oliver

2015-01-01

Since heat sensitivity of fruits and vegetables limits the application of thermal inactivation processes, new emerging inactivation technologies have to be established to fulfill the requirements of food safety without affecting the produce quality. The efficiency of inactivation treatments has to be ensured and monitored. Monitoring of inactivation effects is commonly performed using traditional cultivation methods which have the disadvantage of the time span needed to obtain results. The aim of this study was to compare the inactivation effects of peracetic acid (PAA), ozonated water (O3), and cold atmospheric pressure plasma (CAPP) on Gram-positive and Gram-negative bacteria using flow cytometric methods. E. coli cells were completely depolarized after treatment (15 s) with 0.25% PAA at 10°C, and after treatment (10 s) with 3.8 mg l−1 O3 at 12°C. The membrane potential of CAPP treated cells remained almost constant at an operating power of 20 W over a time period of 3 min, and subsequently decreased within 30 s of further treatment. Complete membrane permeabilization was observed after 10 s O3 treatment, but treatment with PAA and CAPP did not completely permeabilize the cells within 2 and 4 min, respectively. Similar results were obtained for esterase activity. O3 inactivates cellular esterase but esterase activity was detected after 4 min CAPP treatment and 2 min PAA treatment. L. innocua cells and P. carotovorum cells were also permeabilized instantaneously by O3 treatment at concentrations of 3.8 ± 1 mg l−1. However, higher membrane permeabilization of L. innocua and P. carotovorum than of E. coli was observed at CAPP treatment of 20 W. The degree of bacterial damage due to the inactivation processes is highly dependent on treatment parameters as well as on treated bacteria. Important information regarding the inactivation mechanisms can be obtained by flow cytometric measurements and this enables the definition of critical process parameters. PMID:26441874

High pressure thermal inactivation of Clostridium botulinum type E endospores – kinetic modeling and mechanistic insights

PubMed Central

Lenz, Christian A.; Reineke, Kai; Knorr, Dietrich; Vogel, Rudi F.

2015-01-01

Cold-tolerant, neurotoxigenic, endospore forming Clostridium (C.) botulinum type E belongs to the non-proteolytic physiological C. botulinum group II, is primarily associated with aquatic environments, and presents a safety risk for seafood. High pressure thermal (HPT) processing exploiting the synergistic effect of pressure and temperature can be used to inactivate bacterial endospores. We investigated the inactivation of C. botulinum type E spores by (near) isothermal HPT treatments at 300–1200 MPa at 30–75°C for 1 s to 10 min. The occurrence of heat and lysozyme susceptible spore fractions after such treatments was determined. The experimental data were modeled to obtain kinetic parameters and represented graphically by isoeffect lines. In contrast to findings for spores of other species and within the range of treatment parameters applied, zones of spore stabilization (lower inactivation than heat treatments alone), large heat susceptible (HPT-induced germinated) or lysozyme-dependently germinable (damaged coat layer) spore fractions were not detected. Inactivation followed first order kinetics. Dipicolinic acid release kinetics allowed for insights into possible inactivation mechanisms suggesting a (poorly effective) physiologic-like (similar to nutrient-induced) germination at ≤450 MPa/≤45°C and non-physiological germination at >500 MPa/>60–70°C. Results of this study support the existence of some commonalities in the HPT inactivation mechanism of C. botulinum type E spores and Bacillus spores although both organisms have significantly different HPT resistance properties. The information presented here contributes to closing the gap in knowledge regarding the HPT inactivation of spore formers relevant to food safety and may help industrial implementation of HPT processing. The markedly lower HPT resistance of C. botulinum type E spores compared with the resistance of spores from other C. botulinum types could allow for the implementation of milder processes without endangering food safety. PMID:26191048

Inherited pain: sodium channel Nav1.7 A1632T mutation causes erythromelalgia due to a shift of fast inactivation.

PubMed

Eberhardt, Mirjam; Nakajima, Julika; Klinger, Alexandra B; Neacsu, Cristian; Hühne, Kathrin; O'Reilly, Andrias O; Kist, Andreas M; Lampe, Anne K; Fischer, Kerstin; Gibson, Jane; Nau, Carla; Winterpacht, Andreas; Lampert, Angelika

2014-01-24

Inherited erythromelalgia (IEM) causes debilitating episodic neuropathic pain characterized by burning in the extremities. Inherited "paroxysmal extreme pain disorder" (PEPD) differs in its clinical picture and affects proximal body areas like the rectal, ocular, or jaw regions. Both pain syndromes have been linked to mutations in the voltage-gated sodium channel Nav1.7. Electrophysiological characterization shows that IEM-causing mutations generally enhance activation, whereas mutations leading to PEPD alter fast inactivation. Previously, an A1632E mutation of a patient with overlapping symptoms of IEM and PEPD was reported (Estacion, M., Dib-Hajj, S. D., Benke, P. J., Te Morsche, R. H., Eastman, E. M., Macala, L. J., Drenth, J. P., and Waxman, S. G. (2008) NaV1.7 Gain-of-function mutations as a continuum. A1632E displays physiological changes associated with erythromelalgia and paroxysmal extreme pain disorder mutations and produces symptoms of both disorders. J. Neurosci. 28, 11079-11088), displaying a shift of both activation and fast inactivation. Here, we characterize a new mutation of Nav1.7, A1632T, found in a patient suffering from IEM. Although transfection of A1632T in sensory neurons resulted in hyperexcitability and spontaneous firing of dorsal root ganglia (DRG) neurons, whole-cell patch clamp of transfected HEK cells revealed that Nav1.7 activation was unaltered by the A1632T mutation but that steady-state fast inactivation was shifted to more depolarized potentials. This is a characteristic normally attributed to PEPD-causing mutations. In contrast to the IEM/PEPD crossover mutation A1632E, A1632T failed to slow current decay (i.e. open-state inactivation) and did not increase resurgent currents, which have been suggested to contribute to high-frequency firing in physiological and pathological conditions. Reduced fast inactivation without increased resurgent currents induces symptoms of IEM, not PEPD, in the new Nav1.7 mutation, A1632T. Therefore, persistent and resurgent currents are likely to determine whether a mutation in Nav1.7 leads to IEM or PEPD.

Factors Affecting Bacterial Inactivation during High Hydrostatic Pressure Processing of Foods: A Review.

PubMed

Syed, Qamar-Abbas; Buffa, Martin; Guamis, Buenaventura; Saldo, Jordi

2016-01-01

Although, the High Hydrostatic Pressure (HHP) technology has been gaining gradual popularity in food industry since last two decades, intensive research is needed to explore the missing information. Bacterial inactivation in food by using HHP applications can be enhanced by getting deeper insights of the process. Some of these aspects have been already studied in detail (like pressure, time, and temperature, etc.), while some others still need to be investigated in more details (like pH, rates of compression, and decompression, etc.). Selection of process parameters is mainly dependent on type of matrix and target bacteria. This intensive review provides comprehensive information about the variety of aspects that can determine the bacterial inactivation potential of HHP process indicating the fields of future research on this subject including pH shifts of the pressure treated samples and critical limits of compression and decompression rates to accelerate the process efficacy.

High pressure inactivation of human norovirus virus-like particles: evidence that the capsid of human norovirus is highly pressure resistant

USDA-ARS?s Scientific Manuscript database

Human norovirus (NoV) is the leading cause of non-bacterial acute gastroenteritis epidemics worldwide. High pressure processing (HPP) has been considered a promising non-thermal processing technology to inactivate food- and water-borne viral pathogens. Due to the lack of an effective cell culture fo...

Photodynamic Inactivation of Mammalian Viruses and Bacteriophages

PubMed Central

Costa, Liliana; Faustino, Maria Amparo F.; Neves, Maria Graça P. M. S.; Cunha, Ângela; Almeida, Adelaide

2012-01-01

Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i) summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii) discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process. PMID:22852040

Mechanistic Aspects of Adenovirus Serotype 2 Inactivation with Free Chlorine ▿ †

PubMed Central

Page, Martin A.; Shisler, Joanna L.; Mariñas, Benito J.

2010-01-01

Free chlorine is an effective disinfectant for controlling adenoviruses in drinking water, but little is known about the underlying inactivation mechanisms. The objective of this study was to elucidate the molecular components of adenovirus type 2 (Ad2) targeted by free chlorine during the inactivation process. The effects of free chlorine treatment on several Ad2 molecular components and associated life cycle events were compared to its effect on the ability of adenovirus to complete its life cycle, i.e., viability. Free chlorine treatment of Ad2 virions did not impair their ability to interact with monoclonal antibodies specific for hexon and fiber proteins of the Ad2 capsid, as measured by enzyme-linked immunosorbent assays, nor did it impair their interaction with recombinant, purified Coxsackie-adenovirus receptor (CAR) proteins in vitro. Free chlorine-treated Ad2 virions also retained their ability to bind to CAR receptors on A549 cell monolayers, despite being unable to form plaques, suggesting that free chlorine inactivates Ad2 by inhibiting a postbinding event of the Ad2 life cycle. DNA isolated from Ad2 virions that had been inactivated by free chlorine was able to be amplified by PCR, indicating that genome damage was not the cause of inactivation. However, inactivated Ad2 virions were unable to express E1A viral proteins during infection of A549 host cells, as measured by using immunoblotting. Collectively, these results indicate that free chlorine inactivates adenovirus by damaging proteins that govern life cycle processes occurring after host cell attachment, such as endocytosis, endosomal lysis, or nuclear delivery. PMID:20305026

Effects of PEF and heat pasteurization on PME activity in orange juice with regard to a new inactivation kinetic model.

PubMed

Agcam, E; Akyıldız, A; Evrendilek, G Akdemir

2014-12-15

The inactivation kinetics of pectin methyl esterase (PME) during the shelf life (4°C-180 days) of freshly squeezed orange juice samples processed by both pulsed electric fields (PEF) and heat pasteurization (HP) was evaluated in the study. The PME inactivation level after the PEF (25.26 kV/cm-1206.2 μs) and HP (90°C-20s) treatments were 93.8% and 95.2%, respectively. The PME activity of PEF-processed samples decreased or did not change, while that of HP samples increased during storage (p<0.01). A kinetic model was developed expressing PME inactivation as a function of the PEF treatment conditions, and this enabled the estimation of the reaction rate constant (587.8-2375.4s(-1)), and the time required for a 90% reduction (De, 3917.7-969.5s). Quantification of the increase in PEF energy to ensure a ten-fold reduction in De (ze, 63.7 J), activation electric fields (-921.2 kV cm(-1)mol(-1)), and electrical activation energy (12.9 kJ mol(-1)) was also carried out. Consequently, PEF processing was very effective for the inactivation of PME and for providing stability of orange juice during storage. Copyright © 2014. Published by Elsevier Ltd.

Antigenic characterization of a formalin-inactivated poliovirus vaccine derived from live-attenuated Sabin strains.

PubMed

Tano, Yoshio; Shimizu, Hiroyuki; Martin, Javier; Nishimura, Yorihiro; Simizu, Bunsiti; Miyamura, Tatsuo

2007-10-10

A candidate inactivated poliovirus vaccine derived from live-attenuated Sabin strains (sIPV), which are used in the oral poliovirus vaccine (OPV), was prepared in a large-production scale. The modification of viral antigenic epitopes during the formalin inactivation process was investigated by capture ELISA assays using type-specific and antigenic site-specific monoclonal antibodies (MoAbs). The major antigenic site 1 was modified during the formalin inactivation of Sabin 1. Antigenic sites 1-3 were slightly modified during the formalin inactivation of Sabin 2 strain. Sites 1 and 3 were altered on inactivated Sabin 3 virus. These alterations were different to those shown by wild-type Saukett strain, used in conventional IPV (cIPV). It has been previously reported that type 1 sIPV showed higher immunogenicity to type 1 cIPV whereas types 2 and 3 sIPV induced lower level of immunogenicity than their cIPV counterparts. Our results suggest that the differences in epitope structure after formalin inactivation may account, at least in part, for the observed differences in immunogenicity between Sabin and wild-type inactivated poliovaccines.

Effective virus inactivation and removal by steps of Biotest Pharmaceuticals IGIV production process

PubMed Central

Dichtelmüller, Herbert O.; Flechsig, Eckhard; Sananes, Frank; Kretschmar, Michael; Dougherty, Christopher J.

2012-01-01

The virus validation of three steps of Biotest Pharmaceuticals IGIV production process is described here. The steps validated are precipitation and removal of fraction III of the cold ethanol fractionation process, solvent/detergent treatment and 35 nm virus filtration. Virus validation was performed considering combined worst case conditions. By these validated steps sufficient virus inactivation/removal is achieved, resulting in a virus safe product. PMID:24371563

Sterilization of liquid foods by pulsed electric fields–an innovative ultra-high temperature process

PubMed Central

Reineke, Kai; Schottroff, Felix; Meneses, Nicolas; Knorr, Dietrich

2015-01-01

The intention of this study was to investigate the inactivation of endospores by a combined thermal and pulsed electric field (PEF) treatment. Therefore, self-cultivated spores of Bacillus subtilis and commercial Geobacillus stearothermophilus spores with certified heat resistance were utilized. Spores of both strains were suspended in saline water (5.3 mS cm−1), skim milk (0.3% fat; 5.3 mS cm−1) and fresh prepared carrot juice (7.73 mS cm−1). The combination of moderate preheating (70–90°C) and an insulated PEF-chamber, combined with a holding tube (65 cm) and a heat exchanger for cooling, enabled a rapid heat up to 105–140°C (measured above the PEF chamber) within 92.2–368.9 μs. To compare the PEF process with a pure thermal inactivation, each spore suspension was heat treated in thin glass capillaries and D-values from 90 to 130°C and its corresponding z-values were calculated. For a comparison of the inactivation data, F-values for the temperature fields of both processes were calculated by using computational fluid dynamics (CFD). A preheating of saline water to 70°C with a flow rate of 5 l h−1, a frequency of 150 Hz and an energy input of 226.5 kJ kg−1, resulted in a measured outlet temperature of 117°C and a 4.67 log10 inactivation of B. subtilis. The thermal process with identical F-value caused only a 3.71 log10 inactivation. This synergism of moderate preheating and PEF was even more pronounced for G. stearothermophilus spores in saline water. A preheating to 95°C and an energy input of 144 kJ kg−1 resulted in an outlet temperature of 126°C and a 3.28 log10 inactivation, whereas nearly no inactivation (0.2 log10) was achieved during the thermal treatment. Hence, the PEF technology was evaluated as an alternative ultra-high temperature process. However, for an industrial scale application of this process for sterilization, optimization of the treatment chamber design is needed to reduce the occurring inhomogeneous temperature fields. PMID:25999930

High-pressure processing of apple juice: kinetics of pectin methyl esterase inactivation.

PubMed

Riahi, Esmaeil; Ramaswamy, Hosahalli S

2003-01-01

High-pressure (HP) inactivation kinetics of pectin methyl esterase (PME) in apple juice were evaluated. Commercial PME was dispensed in clarified apple juice, sealed in dual peel sterilizable plastic bags, and subjected to different high-pressure processing conditions (200-400 MPa, 0-180 min). Residual enzyme activity was determined by a titration method estimating the rate of free carboxyl group released by the enzyme acting on pectin substrate at pH 7.5 (30 degrees C). The effects of pressure level and pressure holding time on enzyme inactivation were significant (p < 0.05). PME from the microbial source was found to be more resistant (p < 0.05) to pressure inactivation than PME from the orange peel. Almost a full decimal reduction in the activity of commercial PME was achieved by HP treatment at 400 MPa for 25 min. Inactivation kinetics were evaluated on the basis of a dual effect model involving a pressure pulse effect and a first-order rate model, and the pressure sensitivity of rate constants was modeled by using the z-value concept.

Numerical evaluation of lactoperoxidase inactivation during continuous pulsed electric field processing.

PubMed

Buckow, Roman; Semrau, Julius; Sui, Qian; Wan, Jason; Knoerzer, Kai

2012-01-01

A computational fluid dynamics (CFD) model describing the flow, electric field and temperature distribution of a laboratory-scale pulsed electric field (PEF) treatment chamber with co-field electrode configuration was developed. The predicted temperature increase was validated by means of integral temperature studies using thermocouples at the outlet of each flow cell for grape juice and salt solutions. Simulations of PEF treatments revealed intensity peaks of the electric field and laminar flow conditions in the treatment chamber causing local temperature hot spots near the chamber walls. Furthermore, thermal inactivation kinetics of lactoperoxidase (LPO) dissolved in simulated milk ultrafiltrate were determined with a glass capillary method at temperatures ranging from 65 to 80 °C. Temperature dependence of first order inactivation rate constants was accurately described by the Arrhenius equation yielding an activation energy of 597.1 kJ mol(-1). The thermal impact of different PEF processes on LPO activity was estimated by coupling the derived Arrhenius model with the CFD model and the predicted enzyme inactivation was compared to experimental measurements. Results indicated that LPO inactivation during combined PEF/thermal treatments was largely due to thermal effects, but 5-12% enzyme inactivation may be related to other electro-chemical effects occurring during PEF treatments. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

Inactivation of influenza A virus H1N1 by disinfection process.

PubMed

Jeong, Eun Kyo; Bae, Jung Eun; Kim, In Seop

2010-06-01

Because any patient, health care worker, or visitor is capable of transmitting influenza to susceptible persons within hospitals, hospital-acquired influenza has been a clinical concern. Disinfection and cleaning of medical equipment, surgical instruments, and hospital environment are important measures to prevent transmission of influenza virus from hospitals to individuals. This study was conducted to evaluate the efficacy of disinfection processes, which can be easily operated at hospitals, in inactivating influenza A virus H1N1 (H1N1). The effects of 0.1 mol/L NaOH, 70% ethanol, 70% 1-propanol, solvent/detergent (S/D) using 0.3% tri (n-butyl)-phosphate and 1.0% Triton X-100, heat, and ethylene oxide (EO) treatments in inactivating H1N1 were determined. Inactivation of H1N1 was kinetically determined by the treatment of disinfectants to virus solution. Also, a surface test method, which involved drying an amount of virus on a surface and then applying the inactivation methods for 1 minute of contact time, was used to determine the virucidal activity. H1N1 was completely inactivated to undetectable levels in 1 minute of 70% ethanol, 70% 1-propanol, and solvent/detergent treatments in the surface tests as well as in the suspension tests. H1N1 was completely inactivated in 1 minute of 0.1 mol/L NaOH treatment in the suspension tests and also effectively inactivated in the surface tests with the log reduction factor of 3.7. H1N1 was inactivated to undetectable levels within 5 minutes, 2.5 minutes, and 1 minute of heat treatment at 70, 80, and 90 degrees C, respectively in the suspension tests. Also, H1N1 was completely inactivated by EO treatment in the surface tests. Common disinfectants, heat, and EO tested in this study were effective at inactivating H1N1. These results would be helpful in implementing effective disinfecting measures to prevent hospital-acquired infections. Copyright 2010 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

Inactivation of Bacteria in Oil Field Injected Water by a Pulsed Plasma Discharge Process

NASA Astrophysics Data System (ADS)

Xin, Qing; Li, Zhongjian; Lei, Lecheng; Yang, Bin

2016-09-01

Pulsed plasma discharge was employed to inactivate bacteria in the injection water for an oil field. The effects of water conductivity and initial concentration of bacteria on elimination efficiency were investigated in the batch and continuous flow modes. It was demonstrated that Fe2+ contained in injection water could enhance the elimination efficiency greatly. The addition of reducing agent glutathione (GSH) indicated that active radicals generated by pulsed plasma discharges played an important role in the inactivation of bacteria. Moreover, it was found that the microbial inactivation process for both batch and continuous flow mode well fitted the model based on the Weibull's survival function. supported by Zhejiang Province Welfare Technology Applied Research Project of China (No. 2014C31137), National Natural Science Foundation of China (Nos. 21436007 and U1462201), and the Fundamental Research Funds for the Central Universities of China (No. 2015QNA4032)

Method for determining virus inactivation during sludge treatment processes.

PubMed Central

Traub, F; Spillmann, S K; Wyler, R

1986-01-01

A simple and reliable method is described which allows determination of virus inactivation rates during sludge treatment processes in situ. Bacteriophage f2 was adsorbed onto an electropositive membrane filter which was then sandwiched between two polycarbonate membranes with pores smaller than the virus diameter. The resulting sandwich was fixed in an open filter holder, and several such devices were connected before being exposed in sludge-digesting tanks. The device described prevented uncontrolled virus escape, but allowed direct contact of the various inactivating or stabilizing substances present in the environment tested with the virus adsorbed to the carrier membrane. After exposure to an environment, the surviving fraction of virus was eluted from the inner filter and determined by plaque counting. By using polycarbonate membranes without pores for sandwiching, the influence of temperature alone on virus inactivation could be measured. Thermophilic fermentation at 60 degrees C and at 65 kPa pressure led to a bacteriophage f2 titer reduction of 3.5 log10 units per h, whereas during thermophilic digestion at 54.5 degrees C titers decreased 1.2 log10 units per h. During mesophilic digestion an inactivation rate of only 0.04 log10 units per h was observed. Under these latter conditions, temperature had only a minor effect (19%) on virus inactivation, whereas at 54.5 degrees C during thermophilic digestion heat accounted for 32% of the total inactivation, and during thermophilic fermentation at 60 degrees C temperature and pressure were 100% responsible for virus denaturation. PMID:3532955

Cell inactivation, repair and mutation induction in bacteria after heavy ion exposure: results from experiments at accelerators and in space.

PubMed

Horneck, G; Schafer, M; Baltschukat, K; Weisbrod, U; Micke, U; Facius, R; Bucker, H

1989-01-01

To understand the mechanisms of accelerated heavy ions on biological matter, the responses of spores of B. subtilis to this structured high LET radiation was investigated applying two different approaches. 1) By the use of the Biostack concept, the inactivation probability as a function of radial distance to single particles' trajectory (i.e. impact parameter) was determined in space experiments as well as at accelerators using low fluences of heavy ions. It was found that spores can survive even a central hit and that the effective range of inactivation extends far beyond impact parameters where inactivation by delta-ray dose would be effective. Concerning the space experiment, the inactivation cross section exceeds those from comparable accelerator experiments by roughly a factor of 20. 2) From fluence effect curves, cross sections for inactivation and mutation induction, and the efficiency of repair processes were determined. They are influenced by the ions characteristics in a complex manner. According to dependence on LET, at least 3 LET ranges can be differentiated: A low LET range (app. < 200 keV/micrometers), where cross sections for inactivation and mutation induction follow a common curve for different ions and where repair processes are effective; an intermediate LET range of the so-called saturation cross section with negligible mutagenic and repair efficiency; and a high LET range (>1000 keV/micrometers) where the biological endpoints are majorly dependent on atomic mass and energy of the ion under consideration.

[Substrate specifity in Amoeba proteus].

PubMed

Sopina, V A

2006-01-01

Three different phosphatases ("slow", "middle" and "fast") were found in Amoeba proteus (strain B) after PAGE and a subsequent gel staining in 1-naphthyl phosphate containing incubation mixture (pH 9.0). Substrate specificity of these phosphatases was determined in supernatants of homogenates using inhibitors of phosphatase activity. All phosphatases showed a broad substrate specificity. Of 10 tested compounds, p-nitrophenyl phosphate was a preferable substrate for all 3 phosphatases. All phosphatases were able to hydrolyse bis-p-nitrophenyl phosphate and, hence, displayed phosphodiesterase activity. All phosphatases hydrolysed O-phospho-L-tyrosine to a greater or lesser degree. Only little differences in substrate specificity of phosphatases were noticed: 1) "fast" and "middle" phosphatases hydrolysed naphthyl phosphates and O-phospho-L-tyrosine less efficiently than did "slow" phosphatase; 2) "fast" and "middle" phosphatases hydrolysed 2- naphthyl phosphate to a lesser degree than 1-naphthyl phosphate 3) "fast" and "middle" phosphatases hydrolysed O-phospho-L-serine and O-phospho-L-threonine with lower intensity as compared with "slow" phosphatase; 4) as distinct from "middle" and "slow" phosphatases, the "fast" phosphatase hydrolysed glucose-6-phosphate very poorly. The revealed broad substrate specificity of "slow" phosphatase together with data of inhibitory analysis and results of experiments with reactivation of this phosphatase by Zn2+-ions after its inactivation by EDTA strongly suggest that only the "slow" phosphatase is a true alkaline phosphatase (EC 3.1.3.1). The alkaline phosphatase of A. proteus is secreted into culture medium where its activity is low. The enzyme displays both phosphomono- and phosphodiesterase activities, in addition to supposed protein phosphatase activity. It still remains unknown, to which particular phosphatase class the amoeban "middle" and "fast" phosphatases (pH 9.0) may be assigned.

Quantifying Ca2+ release and inactivation of Ca2+ release in fast- and slow-twitch muscles.

PubMed

Barclay, C J

2012-12-01

The aims of this study were to quantify the Ca(2+) release underlying twitch contractions of mammalian fast- and slow-twitch muscle and to comprehensively describe the transient inactivation of Ca(2+) release following a stimulus. Experiments were performed using bundles of fibres from mouse extensor digitorum longus (EDL) and soleus muscles. Ca(2+) release was quantified from the amount of ATP used to remove Ca(2+) from the myoplasm following stimulation. ATP turnover by crossbridges was blocked pharmacologically (N-benzyl-p-toluenesulphonamide for EDL, blebbistatin for soleus) and muscle heat production was used as an index of Ca(2+) pump ATP turnover. At 20°C, Ca(2+) release in response to a single stimulus was 34 and 84 μmol (kg muscle)(-1) for soleus and EDL, respectively, and increased with temperature (30°C: soleus, 61 μmol kg(-1); EDL, 168 μmol kg(-1)). Delivery of another stimulus within 100 ms of the first produced a smaller Ca(2+) release. The maximum magnitude of the decrease in Ca(2+) release was greater in EDL than soleus. Ca(2+) release recovered with an exponential time course which was faster in EDL (mean time constant at 20°C, 32.1 ms) than soleus (65.6 ms) and faster at 30°C than at 20°C. The amounts of Ca(2+) released and crossbridge cycles performed are consistent with a scheme in which Ca(2+) binding to troponin-C allowed an average of ∼1.7 crossbridge cycles in the two muscles.

Quantifying Ca2+ release and inactivation of Ca2+ release in fast- and slow-twitch muscles

PubMed Central

Barclay, C J

2012-01-01

The aims of this study were to quantify the Ca2+ release underlying twitch contractions of mammalian fast- and slow-twitch muscle and to comprehensively describe the transient inactivation of Ca2+ release following a stimulus. Experiments were performed using bundles of fibres from mouse extensor digitorum longus (EDL) and soleus muscles. Ca2+ release was quantified from the amount of ATP used to remove Ca2+ from the myoplasm following stimulation. ATP turnover by crossbridges was blocked pharmacologically (N-benzyl-p-toluenesulphonamide for EDL, blebbistatin for soleus) and muscle heat production was used as an index of Ca2+ pump ATP turnover. At 20°C, Ca2+ release in response to a single stimulus was 34 and 84 μmol (kg muscle)−1 for soleus and EDL, respectively, and increased with temperature (30°C: soleus, 61 μmol kg−1; EDL, 168 μmol kg−1). Delivery of another stimulus within 100 ms of the first produced a smaller Ca2+ release. The maximum magnitude of the decrease in Ca2+ release was greater in EDL than soleus. Ca2+ release recovered with an exponential time course which was faster in EDL (mean time constant at 20°C, 32.1 ms) than soleus (65.6 ms) and faster at 30°C than at 20°C. The amounts of Ca2+ released and crossbridge cycles performed are consistent with a scheme in which Ca2+ binding to troponin-C allowed an average of ∼1.7 crossbridge cycles in the two muscles. PMID:23027818

Modulation of hERG potassium channel gating normalizes action potential duration prolonged by dysfunctional KCNQ1 potassium channel

PubMed Central

Zhang, Hongkang; Zou, Beiyan; Yu, Haibo; Moretti, Alessandra; Wang, Xiaoying; Yan, Wei; Babcock, Joseph J.; Bellin, Milena; McManus, Owen B.; Tomaselli, Gordon; Nan, Fajun; Laugwitz, Karl-Ludwig; Li, Min

2012-01-01

Long QT syndrome (LQTS) is a genetic disease characterized by a prolonged QT interval in an electrocardiogram (ECG), leading to higher risk of sudden cardiac death. Among the 12 identified genes causal to heritable LQTS, ∼90% of affected individuals harbor mutations in either KCNQ1 or human ether-a-go-go related genes (hERG), which encode two repolarizing potassium currents known as IKs and IKr. The ability to quantitatively assess contributions of different current components is therefore important for investigating disease phenotypes and testing effectiveness of pharmacological modulation. Here we report a quantitative analysis by simulating cardiac action potentials of cultured human cardiomyocytes to match the experimental waveforms of both healthy control and LQT syndrome type 1 (LQT1) action potentials. The quantitative evaluation suggests that elevation of IKr by reducing voltage sensitivity of inactivation, not via slowing of deactivation, could more effectively restore normal QT duration if IKs is reduced. Using a unique specific chemical activator for IKr that has a primary effect of causing a right shift of V1/2 for inactivation, we then examined the duration changes of autonomous action potentials from differentiated human cardiomyocytes. Indeed, this activator causes dose-dependent shortening of the action potential durations and is able to normalize action potentials of cells of patients with LQT1. In contrast, an IKr chemical activator of primary effects in slowing channel deactivation was not effective in modulating action potential durations. Our studies provide both the theoretical basis and experimental support for compensatory normalization of action potential duration by a pharmacological agent. PMID:22745159

Expression and function of a CP339,818-sensitive K+ current in a subpopulation of putative nociceptive neurons from adult mouse trigeminal ganglia

PubMed Central

Sforna, Luigi; D'Adamo, Maria Cristina; Servettini, Ilenio; Guglielmi, Luca; Pessia, Mauro; Franciolini, Fabio

2015-01-01

Trigeminal ganglion (TG) neurons are functionally and morphologically heterogeneous, and the molecular basis of this heterogeneity is still not fully understood. Here we describe experiments showing that a subpopulation of neurons expresses a delayed-rectifying K+ current (IDRK) with a characteristically high (nanomolar) sensitivity to the dihydroquinoline CP339,818 (CP). Although submicromolar CP has previously been shown to selectively block Kv1.3 and Kv1.4 channels, the CP-sensitive IDRK found in TG neurons could not be associated with either of these two K+ channels. It could neither be associated with Kv2.1 channels homomeric or heteromerically associated with the Kv9.2, Kv9.3, or Kv6.4 subunits, whose block by CP, tested using two-electrode voltage-clamp recordings from Xenopus oocytes, resulted in the low micromolar range, nor to the Kv7 subfamily, given the lack of blocking efficacy of 3 μM XE991. Within the group of multiple-firing neurons considered in this study, the CP-sensitive IDRK was preferentially expressed in a subpopulation showing several nociceptive markers, such as small membrane capacitance, sensitivity to capsaicin, and slow afterhyperpolarization (AHP); in these neurons the CP-sensitive IDRK controls the membrane resting potential, the firing frequency, and the AHP duration. A biophysical study of the CP-sensitive IDRK indicated the presence of two kinetically distinct components: a fast deactivating component having a relatively depolarized steady-state inactivation (IDRKf) and a slow deactivating component with a more hyperpolarized V1/2 for steady-state inactivation (IDRKs). PMID:25652918

Plasma Decontamination: A Case Study on Kill Efficacy of Geobacillus stearothermophilus Spores on Different Carrier Materials.

PubMed

Semmler, Egmont; Novak, Wenzel; Allinson, Wilf; Wallis, Darren; Wood, Nigel; Awakowicz, Peter; Wunderlich, Joachim

2016-01-01

A new technology to the pharmaceutical field is presented: surface decontamination by plasmas The technology is comparable to established barrier systems like e-beam, volatile hydrogen peroxide, or radiation inactivation of microbiological contaminations. This plasma technology is part of a fully automated and validated syringe filling line at a major pharmaceutical company and is in production operation. Incoming pre-sterilized syringe containers ("tubs") are processed by plasma, solely on the outside, and passed into the aseptic filling isolator upon successful decontamination. The objective of this article is to present the operating principles and develop and establish a validation routine on the basis of standard commercial biological indicators. Their decontamination efficacies are determined and correlated to the actual inactivation efficacy on the pharmaceutical packaging material.The reference setup is explained in detail and a short presentation of the cycle development and the relevant plasma control parameters is given, with a special focus on the in-process monitor determining the cycle validity. Different microbial inactivation mechanisms are also discussed and evaluated for their contribution and interaction to enhance plasma decontamination. A material-dependent inactivation behavior was observed. In order to be able to correlate the tub surface inactivation of Geobacillus stearothermophilus endospores to metallic biological indicators, a comparative study was performed. Through consistently demonstrating the linear inactivation behavior between the different materials, it becomes possible to develop an effective and time-saving validation scheme. The challenge in new decontamination systems lies in a thorough validation of the inactivation efficacy under different operating regimes. With plasma, as an ionized gas, a new barrier concept is introduced into pharmaceutical aseptic processing of syringes. The presented system operates in vacuum and only decontaminates the outer surface of pre-sterilized syringe containers ("tubs"), before they are transferred into the aseptic area. The plasma does not penetrate into the tub. This article discusses the phase from development and test germ selection, across the identified sporicidal mechanisms, to a proposal for a validation scheme on the basis of commercially available biological indicators. A special focus is placed on an extensive investigation to establish a link between the tub surface microbial kill (polystyrene and Tyvek(and (2)) ) and biological indicator inactivation (stainless steel). Additionally, a rationale is developed on how an optical in-process monitor can be applied to establish a validatable limit on the base of the predetermined inactivation data of Geobacillus stearothermophilus endospores. © PDA, Inc. 2016.

Inactivation and removal of influenza A virus H1N1 during the manufacture of plasma derivatives.

PubMed

Jeong, Eun Kyo; Sung, Hark Mo; Kim, In Seop

2010-11-01

Although transmission of pandemic influenza A virus H1N1 2009 is still occurring globally, little has been reported about how this outbreak has affected the safety of plasma derivatives. To evaluate the safety of plasma derivatives, dedicated virus clearance processes used during their production were investigated for their effectiveness in eliminating this virus of recent concern. In this study, influenza A virus H1N1 strain A/NWS/33 (H1N1) was chosen as a surrogate. H1N1 was completely inactivated by fraction IV fractionation as well as pasteurization during the manufacture of albumin. H1N1 was also effectively removed into the precipitate by fraction III fractionation and completely inactivated by low pH incubation as well as pasteurization during the manufacture of intravenous immunoglobulin. H1N1 was completely inactivated within 1 min of solvent/detergent treatment using 0.3% tri (n-butyl) phosphate and 1.0% Triton X-100 and also completely inactivated within 10 min of dry-heat treatment at 98 °C during the manufacture of factor VIII. H1N1 was completely removed by virus filtration process using Viresolve NFP filter and also completely inactivated by pasteurization during the manufacture of anti-thrombin III. These results indicate that all the virus clearance processes commonly used have sufficient H1N1 reducing capacity to achieve a high margin of safety. Copyright © 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

Molecular modeling study of the induced-fit effect on kinase inhibition: the case of fibroblast growth factor receptor 3 (FGFR3)

NASA Astrophysics Data System (ADS)

Li, Yan; Delamar, Michel; Busca, Patricia; Prestat, Guillaume; Le Corre, Laurent; Legeai-Mallet, Laurence; Hu, RongJing; Zhang, Ruisheng; Barbault, Florent

2015-07-01

Tyrosine kinases are a wide family of targets with strong pharmacological relevance. These proteins undergo large-scale conformational motions able to inactivate them. By the end of one of these structural processes, a new cavity is opened allowing the access to a specific type of inhibitors, called type II. The kinase domain of fibroblast growth factor receptor 3 (FGFR3) falls into this family of kinases. We describe here, for the first time, its inactivation process through target molecular dynamics. The transient cavity, at the crossroad between the DFGout and Cα helix out inactivation is herein explored. Molecular docking calculations of known ligands demonstrated that type II inhibitors are able to interact with this metastable transient conformation of FGFR3 kinase. Besides, supplemental computations were conducted and clearly show that type II inhibitors drive the kinase inactivation process through specific stabilization with the DFG triad. This induced-fit effect of type II ligands toward FGFR3 might be extrapolated to other kinase systems and provides meaningful structural information for future drug developments.

Conventional and advanced oxidation processes used in disinfection of treated urban wastewater.

PubMed

Rodríguez-Chueca, J; Ormad, M P; Mosteo, R; Sarasa, J; Ovelleiro, J L

2015-03-01

The purpose of the current study is to compare the inactivation of Escherichia coli in wastewater effluents using conventional treatments (chlorination) and advanced oxidation processes (AOPs) such as UV irradiation, hydrogen peroxide (H2O2)/solar irradiation, and photo-Fenton processes. In addition, an analysis of the operational costs of each treatment is carried out taking into account the optimal dosages of chemicals used. Total inactivation of bacteria (7.5 log) was achieved by means of chlorination and UV irradiation. However, bacterial regrowth was observed 6 hours after the completion of UV treatment, obtaining a disinfection value around 3 to 4 log. On the other hand, the combination H2O2/solar irradiation achieved a maximum inactivation of E. coli of 3.30 ± 0.35 log. The photo-Fenton reaction achieved a level of inactivation of 4.87 ± 0.10 log. The order of disinfection, taking into account the reagent/cost ratio of each treatment, is as follows: chlorination > UV irradiation > photo-Fenton > H2O2/sunlight irradiation.

Inhibition of recombinant Ca(v)3.1 (alpha(1G)) T-type calcium channels by the antipsychotic drug clozapine.

PubMed

Choi, Kee-Hyun; Rhim, Hyewhon

2010-01-25

Low voltage-activated T-type calcium channels are involved in the regulation of the neuronal excitability, and could be subject to many antipsychotic drugs. The effects of clozapine, an atypical antipsychotic drug, on recombinant Ca(v)3.1 T-type calcium channels heterologously expressed in human embryonic kidney 293 cells were examined using whole-cell patch-clamp recordings. At a standard holding potential of -100 mV, clozapine inhibited Ca(v)3.1 currents with an IC(50) value of 23.7+/-1.3 microM in a use-dependent manner. However, 10 microM clozapine inhibited more than 50% of the Ca(v)3.1 currents in recordings at a more physiologically relevant holding potential of -75 mV. Clozapine caused a significant hyperpolarizing shift in the steady-state inactivation curve of the Ca(v)3.1 channels, which is presumably the main mechanism accounting for the inhibition of the Ca(v)3.1 currents. In addition, clozapine slowed Ca(v)3.1 deactivation and inactivation kinetics but not activation kinetics. Clozapine-induced changes in deactivation and inactivation rates of the Ca(v)3.1 channel gating would likely facilitate calcium influx via Ca(v)3.1 T-type calcium channels. Thus, clozapine may exert its therapeutic and/or side effects by altering cell's excitability and firing properties through actions on T-type calcium channels.

Dependency of Calcium Alternans on Ryanodine Receptor Refractoriness

PubMed Central

Alvarez-Lacalle, Enric; Cantalapiedra, Inma R.; Peñaranda, Angelina; Cinca, Juan; Hove-Madsen, Leif; Echebarria, Blas

2013-01-01

Background Rapid pacing rates induce alternations in the cytosolic calcium concentration caused by fluctuations in calcium released from the sarcoplasmic reticulum (SR). However, the relationship between calcium alternans and refractoriness of the SR calcium release channel (RyR2) remains elusive. Methodology/Principal Findings To investigate how ryanodine receptor (RyR2) refractoriness modulates calcium handling on a beat-to-beat basis using a numerical rabbit cardiomyocyte model. We used a mathematical rabbit cardiomyocyte model to study the beat-to-beat calcium response as a function of RyR2 activation and inactivation. Bi-dimensional maps were constructed depicting the beat-to-beat response. When alternans was observed, a novel numerical clamping protocol was used to determine whether alternans was caused by oscillations in SR calcium loading or by RyR2 refractoriness. Using this protocol, we identified regions of RyR2 gating parameters where SR calcium loading or RyR2 refractoriness underlie the induction of calcium alternans, and we found that at the onset of alternans both mechanisms contribute. At low inactivation rates of the RyR2, calcium alternans was caused by alternation in SR calcium loading, while at low activation rates it was caused by alternation in the level of available RyR2s. Conclusions/Significance We have mapped cardiomyocyte beat-to-beat responses as a function of RyR2 activation and inactivation, identifying domains where SR calcium load or RyR2 refractoriness underlie the induction of calcium alternans. A corollary of this work is that RyR2 refractoriness due to slow recovery from inactivation can be the cause of calcium alternans even when alternation in SR calcium load is present. PMID:23390511

Ranolazine vs phenytoin: greater effect of ranolazine on the transient Na(+) current than on the persistent Na(+) current in central neurons.

PubMed

Terragni, Benedetta; Scalmani, Paolo; Colombo, Elisa; Franceschetti, Silvana; Mantegazza, Massimo

2016-11-01

Voltage-gated Na(+) channels (NaV) are involved in pathologies and are important targets of drugs (NaV-blockers), e.g. some anti-epileptic drugs (AEDs). Besides the fast inactivating transient Na(+) current (INaT), they generate a slowly inactivating "persistent" current (INaP). Ranolazine, a NaV-blocker approved for treatment of angina pectoris, is considered a preferential inhibitor of INaP and has been proposed as a novel AED. Although it is thought that classic NaV-blockers used as AEDs target mainly INaT, they can also reduce INaP. It is important to disclose specific features of novel NaV-blockers, which could be necessary for their effect as AEDs in drug resistant patients. We have compared the action of ranolazine and of the classic AED phenytoin in transfected cells expressing the neuronal NaV1.1 Na(+) channel and in neurons of neocortical slices. Our results show that the relative block of INaT versus INaP of ranolazine and phenytoin is variable and depends on Na(+) current activation conditions. Strikingly, ranolazine blocks with less efficacy INaP and more efficacy INaT than phenytoin in conditions mimicking pathological states (i.e. high frequency firing and long lasting depolarizations). The effects are consistent with binding of ranolazine to both open/pre-open and inactivated states; larger INaT block at high stimulation frequencies is caused by the induction of a slow inactivated state. Thus, contrary than expected, ranolazine is not a better INaP blocker than phenytoin in central neurons, and phenytoin is not a better INaT blocker than ranolazine. Nevertheless, they show a complementary action and could differentially target specific pathological dysfunctions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Rapid water disinfection using vertically aligned MoS2 nanofilms and visible light

NASA Astrophysics Data System (ADS)

Liu, Chong; Kong, Desheng; Hsu, Po-Chun; Yuan, Hongtao; Lee, Hyun-Wook; Liu, Yayuan; Wang, Haotian; Wang, Shuang; Yan, Kai; Lin, Dingchang; Maraccini, Peter A.; Parker, Kimberly M.; Boehm, Alexandria B.; Cui, Yi

2016-12-01

Solar energy is readily available in most climates and can be used for water purification. However, solar disinfection of drinking water mostly relies on ultraviolet light, which represents only 4% of the total solar energy, and this leads to a slow treatment speed. Therefore, the development of new materials that can harvest visible light for water disinfection, and so speed up solar water purification, is highly desirable. Here we show that few-layered vertically aligned MoS2 (FLV-MoS2) films can be used to harvest the whole spectrum of visible light (∼50% of solar energy) and achieve highly efficient water disinfection. The bandgap of MoS2 was increased from 1.3 to 1.55 eV by decreasing the domain size, which allowed the FLV-MoS2 to generate reactive oxygen species (ROS) for bacterial inactivation in the water. The FLV-MoS2 showed a ∼15 times better log inactivation efficiency of the indicator bacteria compared with that of bulk MoS2, and a much faster inactivation of bacteria under both visible light and sunlight illumination compared with the widely used TiO2. Moreover, by using a 5 nm copper film on top of the FLV-MoS2 as a catalyst to facilitate electron-hole pair separation and promote the generation of ROS, the disinfection rate was increased a further sixfold. With our approach, we achieved water disinfection of >99.999% inactivation of bacteria in 20 min with a small amount of material (1.6 mg l-1) under simulated visible light.

Genes That Escape X-Inactivation in Humans Have High Intraspecific Variability in Expression, Are Associated with Mental Impairment but Are Not Slow Evolving

PubMed Central

Zhang, Yuchao; Castillo-Morales, Atahualpa; Jiang, Min; Zhu, Yufei; Hu, Landian; Urrutia, Araxi O.; Kong, Xiangyin; Hurst, Laurence D.

2013-01-01

In female mammals most X-linked genes are subject to X-inactivation. However, in humans some X-linked genes escape silencing, these escapees being candidates for the phenotypic aberrations seen in polyX karyotypes. These escape genes have been reported to be under stronger purifying selection than other X-linked genes. Although it is known that escape from X-inactivation is much more common in humans than in mice, systematic assays of escape in humans have to date employed only interspecies somatic cell hybrids. Here we provide the first systematic next-generation sequencing analysis of escape in a human cell line. We analyzed RNA and genotype sequencing data obtained from B lymphocyte cell lines derived from Europeans (CEU) and Yorubans (YRI). By replicated detection of heterozygosis in the transcriptome, we identified 114 escaping genes, including 76 not previously known to be escapees. The newly described escape genes cluster on the X chromosome in the same chromosomal regions as the previously known escapees. There is an excess of escaping genes associated with mental retardation, consistent with this being a common phenotype of polyX phenotypes. We find both differences between populations and between individuals in the propensity to escape. Indeed, we provide the first evidence for there being both hyper- and hypo-escapee females in the human population, consistent with the highly variable phenotypic presentation of polyX karyotypes. Considering also prior data, we reclassify genes as being always, never, and sometimes escape genes. We fail to replicate the prior claim that genes that escape X-inactivation are under stronger purifying selection than others. PMID:24023392

Inhibitory effects of hesperetin on Kv1.5 potassium channels stably expressed in HEK 293 cells and ultra-rapid delayed rectifier K(+) current in human atrial myocytes.

PubMed

Wang, Huan; Wang, Hong-Fei; Wang, Chen; Chen, Yu-Fang; Ma, Rong; Xiang, Ji-Zhou; Du, Xin-Ling; Tang, Qiang

2016-10-15

In the present study, the inhibitory effects of hesperetin (HSP) on human cardiac Kv1.5 channels expressed in HEK 293 cells and the ultra-rapid delayed rectifier K(+) current (Ikur) in human atrial myocytes were examined by using the whole-cell configuration of the patch-clamp techniques. We found that hesperetin rapidly and reversibly suppressed human Kv1.5 current in a concentration dependent manner with a half-maximal inhibition (IC50) of 23.15 μΜ with a Hill coefficient of 0.89. The current was maximally diminished about 71.36% at a concentration of 300μM hesperetin. Hesperetin significantly positive shifted the steady-state activation curve of Kv1.5, while negative shifted the steady-state inactivation curve. Hesperetin also accelerated the inactivation and markedly slowed the recovery from the inactivation of Kv1.5 currents. Block of Kv1.5 currents by hesperetin was in a frequency dependent manner. However, inclusion of 30μM hesperetin in pipette solution produced no effect on Kv1.5 channel current, while the current were remarkable and reversibly inhibited by extracellular application of 30μM hesperetin. We also found that hesperetin potently and reversibly inhibited the ultra-repaid delayed K(+) current (Ikur) in human atrial myocytes, which is in consistent with the effects of hesperetin on Kv1.5 currents in HEK 293 cells. In conclusion, hesperetin is a potent inhibitor of Ikur (which is encoded by Kv1.5), with blockade probably due to blocking of both open state and inactivated state channels from outside of the cell. Copyright © 2016 Elsevier B.V. All rights reserved.

Conserved Kv4 N-terminal domain critical for effects of Kv channel-interacting protein 2.2 on channel expression and gating.

PubMed

Bähring, R; Dannenberg, J; Peters, H C; Leicher, T; Pongs, O; Isbrandt, D

2001-06-29

Association of Kv channel-interacting proteins (KChIPs) with Kv4 channels leads to modulation of these A-type potassium channels (An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S., and Rhodes, K. J. (2000) Nature 403, 553-556). We cloned a KChIP2 splice variant (KChIP2.2) from human ventricle. In comparison with KChIP2.1, coexpression of KChIP2.2 with human Kv4 channels in mammalian cells slowed the onset of Kv4 current inactivation (2-3-fold), accelerated the recovery from inactivation (5-7-fold), and shifted Kv4 steady-state inactivation curves by 8-29 mV to more positive potentials. The features of Kv4.2/KChIP2.2 currents closely resemble those of cardiac rapidly inactivating transient outward currents. KChIP2.2 stimulated the Kv4 current density in Chinese hamster ovary cells by approximately 55-fold. This correlated with a redistribution of immunoreactivity from perinuclear areas to the plasma membrane. Increased Kv4 cell-surface expression and current density were also obtained in the absence of KChIP2.2 when the highly conserved proximal Kv4 N terminus was deleted. The same domain is required for association of KChIP2.2 with Kv4 alpha-subunits. We propose that an efficient transport of Kv4 channels to the cell surface depends on KChIP binding to the Kv4 N-terminal domain. Our data suggest that the binding is necessary, but not sufficient, for the functional activity of KChIPs.

Combined pressure-thermal inactivation effect on spores in lu-wei beef--a traditional Chinese meat product.

PubMed

Wang, B-S; Li, B-S; Du, J-Z; Zeng, Q-X

2015-08-01

This study investigated the inactivation effect and kinetics of Bacillus coagulans and Geobacillus stearothermophilus spores suspended in lu-wei beef by combining high pressure (500 and 600 MPa) and moderate heat (70 and 80 °C or 80 and 90 °C). During pressurization, the temperature of pressure-transmitting fluid was tested with a K-type thermocouple, and the number of surviving cells was determined by a plate count method. The pressure come-up time and corresponding inactivation of Bacillus coagulans and G. stearothermophilus spores were considered during the pressure-thermal treatment. For the two types of spores, the results showed a higher inactivation effect in phosphate buffer solution than that in lu-wei beef. Among the bacteria evaluated, G. stearothermophilus spores had a higher resistance than B. coagulans spores during the pressure-thermal processing. One linear model and two nonlinear models (i.e. the Weibull and log-logistic models) were fitted to the survivor data to obtain relevant kinetic parameters, and the performance of these models was compared. The results suggested that the survival curve of the spores could be accurately described utilizing the log-logistic model, which produced the best fit for all inactivation data. The compression heating characteristics of different pressure-transmitting fluids should be considered when using high pressure to sterilize spores, particularly while the pressure is increasing. Spores can be inactivated by combining high pressure and moderate heat. The study demonstrates the synergistic inactivation effect of moderate heat in combination with high pressure in real-life food. The use of mathematical models to predict the inactivation for spores could help the food industry further to develop optimum process conditions. © 2015 The Society for Applied Microbiology.

Hierarchical equivalence of somatosensory areas I and II for tactile processing in the cerebral cortex of the marmoset monkey.

PubMed

Zhang, H Q; Zachariah, M K; Coleman, G T; Rowe, M J

2001-05-01

Responsiveness of the first somatosensory area (SI) of the cerebral cortex was investigated in the marmoset monkey (Callithrix jacchus) in association with cooling-induced, reversible inactivation of the second somatosensory area, SII. The aim was to determine whether SI responsiveness to peripheral tactile stimulation depends on SII and therefore whether SI and SII in the marmoset occupy hierarchically equivalent positions in a parallel organizational scheme for thalamocortical tactile processing as appears to be the case in nonprimate mammals. Inactivation of SII was achieved when the temperature over SII was lowered to < or =12 degrees C, as indicated by abolition of the SII-evoked potentials generated by brief tap stimuli to the hand or foot, and by abolition of tactile responses in single SII neurons located at the margin beneath the block. The effect of SII inactivation on SI-evoked potentials was examined in 16 experiments by simultaneous recording of the SI- and SII-evoked potentials. SI-evoked potentials were never abolished and remained unaffected in 11 cases. In three experiments there was a small reduction in amplitude and inconsistent effects in the remaining two. Responsiveness to controlled tactile stimuli was examined quantitatively in 31 individual SI neurons of different functional classes before, during, and after the inactivation of SII. Tactile responsiveness in individual SI neurons was never abolished by SII inactivation, remaining unchanged in 20 neurons (65%) while undergoing some reduction in the remaining 11 SI neurons (35%). This reduction of tactile responsiveness in one-third of SI neurons is most likely attributable to a removal of a facilitatory influence emanating from SII, based on the observation that background activity of the affected neurons was also reduced. Furthermore, phase locking of SI responses to vibrotactile stimulation was unchanged when SII was inactivated. The retention of responsiveness in SI neurons when SII was inactivated by cooling in the marmoset demonstrates that tactile inputs can reach SI without traversing an indirect, serially organized path through SII. The present results, together with our previous observations that responsiveness in the majority of SII neurons survived SI inactivation, demonstrate that there is a parallel organization of the SI and SII areas for tactile processing in the marmoset monkey and that SI and SII occupy hierarchically equivalent positions in a parallel processing network. There is therefore no longer justification for the view that there are fundamental differences in the organization of thalamocortical tactile processing for SI and SII between simian primates, in general, and other mammals.

Feasibility of utilizing bioindicators for testing microbial inactivation in sweetpotato purees processed with a continuous-flow microwave system.

PubMed

Brinley, T A; Dock, C N; Truong, V-D; Coronel, P; Kumar, P; Simunovic, J; Sandeep, K P; Cartwright, G D; Swartzel, K R; Jaykus, L-A

2007-06-01

Continuous-flow microwave heating has potential in aseptic processing of various food products, including purees from sweetpotatoes and other vegetables. Establishing the feasibility of a new processing technology for achieving commercial sterility requires evaluating microbial inactivation. This study aimed to assess the feasibility of using commercially available plastic pouches of bioindicators containing spores of Geobacillius stearothermophilus ATCC 7953 and Bacillus subtilis ATCC 35021 for evaluating the degree of microbial inactivation achieved in vegetable purees processed in a continuous-flow microwave heating unit. Sweetpotato puree seeded with the bioindicators was subjected to 3 levels of processing based on the fastest particles: undertarget process (F(0) approximately 0.65), target process (F(0) approximately 2.8), and overtarget process (F(0) approximately 10.10). After initial experiments, we found it was necessary to engineer a setup with 2 removable tubes connected to the continuous-flow microwave system to facilitate the injection of indicators into the unit without interrupting the puree flow. Using this approach, 60% of the indicators injected into the system could be recovered postprocess. Spore survival after processing, as evaluated by use of growth indicator dyes and standard plating methods, verified inactivation of the spores in sweetpotato puree. The log reduction results for B. subtilis were equivalent to the predesigned degrees of sterilization (F(0)). This study presents the first report suggesting that bioindicators such as the flexible, food-grade plastic pouches can be used for microbial validation of commercial sterilization in aseptic processing of foods using a continuous-flow microwave system.

Combined Effect of Ultrasound and Mild Temperatures on the Inactivation of E. coli in Fresh Carrot Juice and Changes on its Physicochemical Characteristics.

PubMed

Pokhrel, Prashant Raj; Bermúdez-Aguirre, Daniela; Martínez-Flores, Héctor E; Garnica-Romo, M Guadalupe; Sablani, Shyam; Tang, Juming; Barbosa-Cánovas, Gustavo V

2017-10-01

The combination of ultrasound and mild temperatures to process fruits and vegetables juices is a novel approach that is showing promising results for microbial inactivation and preservation of bioactive compounds and sensory attributes. This study centers on investigating the inactivation of Escherichia coli (ATCC 11755) in carrot juice as a result of the combined effect of ultrasound (24 kHz frequency, 120 μm, and 400 W) with temperature (50, 54, and 58 °C) and processing time (0 to 10 min). In addition, the possible changes in physicochemical properties and the retention of bioactive compounds after processing were analyzed. Microbial inactivation with ultrasound treatment at 50 °C resulted in 3.5 log reduction after 10 min, whereas at 54 °C almost 5 log reduction was attained in the same period of time; meanwhile, for treatment at 58 °C, no viable cells were detected (>5 log reduction) after 2 min. There was no significant difference (P > 0.05) on pH (6.80 to 6.82), °Brix (8.0 to 8.5), titratable acidity (0.29% to 0.30%), total carotenoid (1774 to 1835 μg/100 mL), phenolic compounds (20.19 to 20.63 μg/mL), ascorbic acid (4.8 mg/100 mL), and color parameters between fresh and ultrasound treated samples at the studied temperatures. To predict the inactivation patterns, observed values were tested using 3 different general models: first-order, Weibull distribution, and biphasic. The Weibull and biphasic models show good correlation for inactivation under all processing conditions. Results show ultrasound in combination with mild temperature could be effectively used to process fresh carrot juice providing a safe product without affecting physicochemical characteristics. The combination of ultrasound and mild temperatures is effective in reducing microbial load in carrot juice to safe levels. This combination would be beneficial in the industrial processing of carrot juice without altering the quality attributes or bioactive compounds. © 2017 Institute of Food Technologists®.

Optimising the inactivation of grape juice spoilage organisms by pulse electric fields.

PubMed

Marsellés-Fontanet, A Robert; Puig, Anna; Olmos, Paola; Mínguez-Sanz, Santiago; Martín-Belloso, Olga

2009-04-15

The effect of some pulsed electric field (PEF) processing parameters (electric field strength, pulse frequency and treatment time), on a mixture of microorganisms (Kloeckera apiculata, Saccharomyces cerevisiae, Lactobacillus plantarum, Lactobacillus hilgardii and Gluconobacter oxydans) typically present in grape juice and wine were evaluated. An experimental design based on response surface methodology (RSM) was used and results were also compared with those of a factorially designed experiment. The relationship between the levels of inactivation of microorganisms and the energy applied to the grape juice was analysed. Yeast and bacteria were inactivated by the PEF treatments, with reductions that ranged from 2.24 to 3.94 log units. All PEF parameters affected microbial inactivation. Optimal inactivation of the mixture of spoilage microorganisms was predicted by the RSM models at 35.0 kV cm(-1) with 303 Hz pulse width for 1 ms. Inactivation was greater for yeasts than for bacteria, as was predicted by the RSM. The maximum efficacy of the PEF treatment for inactivation of microorganisms in grape juice was observed around 1500 MJ L(-1) for all the microorganisms investigated. The RSM could be used in the fruit juice industry to optimise the inactivation of spoilage microorganisms by PEF.

Qualitation and Quantitation on Microplasma Jet for Bacteria Inactivation.

PubMed

Du, ChangMing; Liu, Ya; Huang, YaNi; Li, ZiMing; Men, Rui; Men, Yue; Tang, Jun

2016-01-06

In this work, a self-made microplasma jet system was used to conduct the qualitation and quantitation of inactivation with Escherichia coli as the target bacteria. The logarithmic concentration and the size of antimicrobial rings served as the evaluation parameters, respectively. The effect of various parameters on inactivation effect was studied. The results showed that the majority of bacteria had been inactivated in 30 s. The inactivation effect enhanced and then weakened with the increase of air flow rate, and receded as the extension of treatment distance. The effect with different carrier gases showed as follows: oxygen > air > nitrogen > argon. Meanwhile, the effect of different components of microplasma was studied in the optimum conditions (The flow rate was 5 L/min; inactivation distance was 2 cm). The results showed that electrically neutral active species was the main factor of inactivation rather than heating effect, ultraviolet radiation and charged particles. Finally the experiments of thallus change proved that microplasma jet had etching effect on cell membrane. It also found that microplasma could degrade organic material like protein. Furthermore, the images of scanning electron microscope (SEM) revealed the change of cell morphology step by step in the whole process of inactivation.

Qualitation and Quantitation on Microplasma Jet for Bacteria Inactivation

NASA Astrophysics Data System (ADS)

Du, Changming; Liu, Ya; Huang, Yani; Li, Ziming; Men, Rui; Men, Yue; Tang, Jun

2016-01-01

In this work, a self-made microplasma jet system was used to conduct the qualitation and quantitation of inactivation with Escherichia coli as the target bacteria. The logarithmic concentration and the size of antimicrobial rings served as the evaluation parameters, respectively. The effect of various parameters on inactivation effect was studied. The results showed that the majority of bacteria had been inactivated in 30 s. The inactivation effect enhanced and then weakened with the increase of air flow rate, and receded as the extension of treatment distance. The effect with different carrier gases showed as follows: oxygen > air > nitrogen > argon. Meanwhile, the effect of different components of microplasma was studied in the optimum conditions (The flow rate was 5 L/min inactivation distance was 2 cm). The results showed that electrically neutral active species was the main factor of inactivation rather than heating effect, ultraviolet radiation and charged particles. Finally the experiments of thallus change proved that microplasma jet had etching effect on cell membrane. It also found that microplasma could degrade organic material like protein. Furthermore, the images of scanning electron microscope (SEM) revealed the change of cell morphology step by step in the whole process of inactivation.

Qualitation and Quantitation on Microplasma Jet for Bacteria Inactivation

PubMed Central

Du, ChangMing; Liu, Ya; Huang, YaNi; Li, ZiMing; Men, Rui; Men, Yue; Tang, Jun

2016-01-01

In this work, a self-made microplasma jet system was used to conduct the qualitation and quantitation of inactivation with Escherichia coli as the target bacteria. The logarithmic concentration and the size of antimicrobial rings served as the evaluation parameters, respectively. The effect of various parameters on inactivation effect was studied. The results showed that the majority of bacteria had been inactivated in 30 s. The inactivation effect enhanced and then weakened with the increase of air flow rate, and receded as the extension of treatment distance. The effect with different carrier gases showed as follows: oxygen > air > nitrogen > argon. Meanwhile, the effect of different components of microplasma was studied in the optimum conditions (The flow rate was 5 L/min; inactivation distance was 2 cm). The results showed that electrically neutral active species was the main factor of inactivation rather than heating effect, ultraviolet radiation and charged particles. Finally the experiments of thallus change proved that microplasma jet had etching effect on cell membrane. It also found that microplasma could degrade organic material like protein. Furthermore, the images of scanning electron microscope (SEM) revealed the change of cell morphology step by step in the whole process of inactivation. PMID:26732987

Biocontrol interventions for inactivation of foodborne pathogens on produce

USDA-ARS?s Scientific Manuscript database

Post-harvest interventions for control of foodborne pathogens on minimally processed foods are crucial for food safety. Biocontrol interventions have the primary objective of developing novel antagonists in combinations with physical and chemical interventions to inactivate pathogenic microbes. Ther...

Reduction of spiked porcine circovirus during the manufacture of a Vero cell-derived vaccine.

PubMed

Lackner, Cornelia; Leydold, Sandra M; Modrof, Jens; Farcet, Maria R; Grillberger, Leopold; Schäfer, Birgit; Anderle, Heinz; Kreil, Thomas R

2014-04-11

Porcine circovirus-1 (PCV1) was recently identified as a contaminant in live Rotavirus vaccines, which was likely caused by contaminated porcine trypsin. The event triggered the development of new regulatory guidance on the use of porcine trypsin which shall ensure that cell lines and porcine trypsin in use are free from PCV1. In addition, manufacturing processes of biologicals other than live vaccines include virus clearance steps that may prevent and mitigate any potential virus contamination of product. In this work, artificial spiking of down-scaled models for the manufacturing process of an inactivated pandemic influenza virus vaccine were used to investigate inactivation of PCV1 and the physico-chemically related porcine parvovirus (PPV) by formalin and ultraviolet-C (UV-C) treatment as well as removal by the purification step sucrose gradient ultracentrifugation. A PCV1 infectivity assay, using a real-time PCR infectivity readout was established. The formalin treatment (0.05% for 48h) showed substantial inactivation for both PCV1 and PPV with reduction factors of 3.0log10 and 6.8log10, respectively, whereas UV-C treatment resulted in complete PPV (≥5.9log10) inactivation already at a dose of 13mJ/cm but merely 1.7log10 at 24mJ/cm(2) for PCV1. The UV-C inactivation results with PPV were confirmed using minute virus of mice (MVM), indicating that parvoviruses are far more sensitive to UV-C than PCV1. The sucrose density gradient ultracentrifugation also contributed to PCV1 clearance with a reduction factor of 2log10. The low pH treatment during the production of procine trypsin was investigated and showed effective inactivation for both PCV1 (4.5log10) and PPV (6.4log10). In conclusion, PCV1 in general appears to be more resistant to virus inactivation than PPV. Still, the inactivated pandemic influenza vaccine manufacturing process provides for robust virus reduction, in addition to the already implemented testing for PCV1 to avoid any contaminations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Analysis of the negative inotropic effect of acetylcholine on frog atrial fibres.

PubMed

Nargeot, J; Garnier, D; Rougier, O

1981-03-01

Voltage-clamp experiments have been performed on frog atrial preparations in order to study the mechanism of the inotropic effect of acetylcholine (ACh) at various concentrations. The amplitude of the slow inward current (Is) is reduced even at low ACh concentrations; such low concentrations have little or no effect on potassium permeability. Dose-effect relationships for Is inhibition (Is/Is max) by ACh show a half amplitude dose (K0.5 around 8 X 10(-8) M ACh. The reduction of Is is attributed largely to a decrease of the maximal conductance of the slow channel (gs). Steady-state activation and inactivation parameters are not affected by ACh. Experiments in a Na-free solution (Na replaced by Li ions) or in a Ca-free solution (with EGTA) indicate that the "slow sodium current" is more sensitive to ACh than the "slow Ca current", although these two currents both seem to flow through the slow channel. The decrease of the phasic component of contraction observed in the presence of ACh is very well correlated with the decrease of Is (K0.5 = 8 X 10(-8) M ACh), while the increase of the tonic tension may be related to the outward potassium current induced by high concentrations of ACh. The significant difference between the half amplitude dose (K0.5) observed in the dose effect curves with ACh for Is inhibition (K0.5 = 8 X 10(-8) M) and for ACh-induced extra-current (K0.5 - 10(-6) M) may indicate the presence of two muscarinic receptors.

Robustness of solvent/detergent treatment of plasma derivatives: a data collection from Plasma Protein Therapeutics Association member companies.

PubMed

Dichtelmüller, Herbert O; Biesert, Lothar; Fabbrizzi, Fabrizio; Gajardo, Rodrigo; Gröner, Albrecht; von Hoegen, Ilka; Jorquera, Juan I; Kempf, Christoph; Kreil, Thomas R; Pifat, Dominique; Osheroff, Wendy; Poelsler, Gerhard

2009-09-01

Solvent/detergent (S/D) treatment is an established virus inactivation technology that has been applied in the manufacture of medicinal products derived from human plasma for more than 20 years. Data on the inactivation of enveloped viruses by S/D treatment collected from seven Plasma Protein Therapeutics Association member companies demonstrate the robustness, reliability, and efficacy of this virus inactivation method. The results from 308 studies reflecting production conditions as well as technical variables significantly beyond the product release specification were evaluated for virus inactivation, comprising different combinations of solvent and detergent (tri(n-butyl) phosphate [TNBP]/Tween 80, TNBP/Triton X-100, TNBP/Na-cholate) and different products (Factor [F]VIII, F IX, and intravenous and intramuscular immunoglobulins). Neither product class, process temperature, protein concentration, nor pH value has a significant impact on virus inactivation. A variable that did appear to be critical was the concentration of solvent and detergent. The data presented here demonstrate the robustness of virus inactivation by S/D treatment for a broad spectrum of enveloped test viruses and process variables. Our data substantiate the fact that no transmission of viruses such as human immunodeficiency virus, hepatitis B virus, hepatitis C virus, or of other enveloped viruses was reported for licensed plasma derivatives since the introduction of S/D treatment.

Systematic approach to in-depth understanding of photoelectrocatalytic bacterial inactivation mechanisms by tracking the decomposed building blocks.

PubMed

Sun, Hongwei; Li, Guiying; Nie, Xin; Shi, Huixian; Wong, Po-Keung; Zhao, Huijun; An, Taicheng

2014-08-19

A systematic approach was developed to understand, in-depth, the mechanisms involved during the inactivation of bacterial cells using photoelectrocatalytic (PEC) processes with Escherichia coli K-12 as the model microorganism. The bacterial cells were found to be inactivated and decomposed primarily due to attack from photogenerated H2O2. Extracellular reactive oxygen species (ROSs), such as H2O2, may penetrate into the bacterial cell and cause dramatically elevated intracellular ROSs levels, which would overwhelm the antioxidative capacity of bacterial protective enzymes such as superoxide dismutase and catalase. The activities of these two enzymes were found to decrease due to the ROSs attacks during PEC inactivation. Bacterial cell wall damage was then observed, including loss of cell membrane integrity and increased permeability, followed by the decomposition of cell envelope (demonstrated by scanning electronic microscope images). One of the bacterial building blocks, protein, was found to be oxidatively damaged due to the ROSs attacks, as well. Leakage of cytoplasm and biomolecules (bacterial building blocks such as proteins and nucleic acids) were evident during prolonged PEC inactivation process. The leaked cytoplasmic substances and cell debris could be further degraded and, ultimately, mineralized with prolonged PEC treatment.

Trehalose and sorbitol alter the kinetic pattern of inactivation of glutamate dehydrogenase during drying in levitated microdroplets.

PubMed

Lorenzen, Elke; Lee, Geoffrey

2013-12-01

A single-droplet acoustic levitator was used to determine the drying rate and the kinetics of inactivation of glutamate dehydrogenase in the presence of added trehalose or sorbitol. The solution was also spray dried under the same process condition of drying gas temperature on a bench-top machine. Both trehalose and sorbitol delay the point of onset of enzyme inactivation which lies after the critical point of drying. Both carbohydrates also reduce the apparent rate constant of inactivation calculated during the subsequent inactivation phase. The carbohydrates stabilise, therefore, the enzyme during droplet drying and particle formation mainly during the falling rate drying period. There is no difference between the stabilising effects of the two carbohydrates when examined as levitated single droplets. This suggests the importance of water replacement as a stabilising mechanism in the levitated droplets/particles. On spray drying, the trehalose stabilises the enzyme better than does the sorbitol at a drying gas (outlet) temperature of 60°C. This suggests glass formation with the trehalose but not the sorbitol during the very rapid drying process of small-atomised droplets in the spray dryer. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.

Inactivation of Escherichia coli in water by pulsed dielectric barrier discharge in coaxial reactor.

PubMed

Hernández-Arias, A N; Rodríguez-Méndez, B G; López-Callejas, R; Alcántara-Díaz, D; Valencia-Alvarado, R; Mercado-Cabrera, A; Peña-Eguiluz, R; Muñoz-Castro, A E; Barocio, S R; de la Piedad-Beneitez, A

2012-09-01

An experimental study of ATCC (American Type Culture Collection) 8739 Escherichia coli bacteria inactivation in water by means of pulsed dielectric barrier discharge (PDBD) atmospheric pressure plasmas is presented. Plasma is generated by an adjustable power source capable of supplying high voltage 25 kV pulses, ∼30 μs long and at a 500 Hz frequency. The process was conducted in a ∼152 cm(3) cylindrical stainless steel coaxial reactor, endowed with a straight central electrode and a gas inlet. The bacterial concentration in water was varied from 10(3) up to 10(8) E. coli cells per millilitre. The inactivation was achieved without gas flow in the order of 82% at 10(8) colony-forming units per millilitre (CFU mL(-1)) concentrations in 600 s. In addition, oxygen was added to the gas supply in order to increase the ozone content in the process, raising the inactivation percentage to the order of 90% in the same treatment time. In order to reach a higher efficiency however, oxygen injection modulation is applied, leading to inactivation percentages above 99.99%. These results are similarly valid for lower bacterial concentrations.

High Pressure Inactivation of HAV within Mussels

USDA-ARS?s Scientific Manuscript database

The potential of hepatitis A virus (HAV) to be inactivated within Mediterranean mussels (Mytilus galloprovincialis) and blue mussels (Mytilus edulis) by high pressure processing was evaluated. HAV was bioaccumulated within mussels to approximately 6-log10 PFU by exposure of mussels to HAV-contamina...

Evaluation of the U.S. Department of Agriculture's egg pasteurization processes on the inactivation of high pathogenicity avian influenza virus and velogenic Newcastle disease virus in processed egg products

USDA-ARS?s Scientific Manuscript database

High pathogenicity avian influenza virus (HPAIV) A/chicken/Pennsylvania/1370/1983 (H5N2), and velogenic Newcastle disease virus (vNDV) AMPV-1/California/212676/2002 were inoculated into various egg products then heat treated at various temperatures for 0 to 30 min to determine thermal inactivation p...

Inactivation of dinoflagellate Scripsiella trochoidea in synthetic ballast water by advanced oxidation processes.

PubMed

Yang, Zhishan; Jiang, Wenju; Zhang, Yi; Lim, T M

2015-01-01

Ship-borne ballast water contributes significantly to the transfer of non-indigenous species across aquatic environments. To reduce the risk of bio-invasion, ballast water should be treated before discharge. In this study, the efficiencies of several conventional and advanced oxidation processes were investigated for potential ballast water treatment, using a marine dinoflagellate species, Scripsiella trochoidea, as the indicator organism. A stable and consistent culture was obtained and treated by ultraviolet (UV) light, ozone (O3), hydrogen peroxide (H2O2), and their various combinations. UV apparently inactivated the cells after only 10 s of irradiation, but subsequently photo-reactivation of the cells was observed for all methods involving UV. O3 exhibited 100% inactivation efficiency after 5 min treatment, while H2O2 only achieved maximum 80% inactivation in the same duration. Combined methods, e.g. UV/O3 and UV/H2O2, were found to inhibit photo-reactivation and improve treatment efficiency to some degree, indicating the effectiveness of using combined treatment processes. The total residual oxidant (TRO) levels of the methods were determined, and the results indicated that UV and O3 generated the lowest and highest TRO, respectively. The synergic effect of combined processes on TRO generation was found to be insignificant, and thus UV/O3 was recommended as a potentially suitable treatment process for ballast water.

Inactivation of Candida glabrata by a humid DC argon discharge afterglow: dominant contributions of short-lived aqueous active species

NASA Astrophysics Data System (ADS)

Xiong, Qing; Liu, Hongbin; Lu, Weiping; Chen, Qiang; Xu, Le; Wang, Xia; Zhu, Qunlin; Zeng, Xue; Yi, Ping

2017-05-01

Plasma medicine applications are currently attracting significant interest all over the world. Bactericidal treatments of Candida glabrata cultured in saline suspension are performed in this study by a room-temperature reactive afterglow of a DC-driven argon discharge. Water vapor was added to the discharge to study the inactivation contributions of reactive hydrolytic species including OH and H2O2 transporting along the gas flow to the treated solutions. The inactivation results indicate that the dominant roles in the bactericidal treatments are played by the short-lived aqueous active species, but not the stable species like H2O2aq (aq indicates an aqueous species). Further analysis shows that the ·OHaq radicals play an important role in the inactivation process. The ·OHaq radicals in the suspension are mostly produced from the direct dissolution of the OH species in the reactive afterglow. With the increase of added water vapor content, the ·OHaq production increases and enhances the inactivation efficiency of C. glabrata. Furthermore, it is found that the ambient air diffusion shows essential effects on the bactericidal activity of the remote humid argon discharge. Higher bactericidal effects can be obtained in open-space treatments compared to in a controlled Ar + H2O gas atmosphere. Key active air-byproduct species are believed to be generated in the suspension during the treatments and contributing to the inactivation process. Based on chemical analysis, the peroxynitrous acid ONOOHaq is considered as the key antimicrobial air-byproduct species. These results indicate the important dependence of plasma biomedical effects on the processing environment, which finally relates to the critical contributions of the key reactive species formed therein.

Activity map of the tammar X chromosome shows that marsupial X inactivation is incomplete and escape is stochastic

PubMed Central

2010-01-01

Background X chromosome inactivation is a spectacular example of epigenetic silencing. In order to deduce how this complex system evolved, we examined X inactivation in a model marsupial, the tammar wallaby (Macropus eugenii). In marsupials, X inactivation is known to be paternal, incomplete and tissue-specific, and occurs in the absence of an XIST orthologue. Results We examined expression of X-borne genes using quantitative PCR, revealing a range of dosage compensation for different loci. To assess the frequency of 1X- or 2X-active fibroblasts, we investigated expression of 32 X-borne genes at the cellular level using RNA-FISH. In female fibroblasts, two-color RNA-FISH showed that genes were coordinately expressed from the same X (active X) in nuclei in which both loci were inactivated. However, loci on the other X escape inactivation independently, with each locus showing a characteristic frequency of 1X-active and 2X-active nuclei, equivalent to stochastic escape. We constructed an activity map of the tammar wallaby inactive X chromosome, which identified no relationship between gene location and extent of inactivation, nor any correlation with the presence or absence of a Y-borne paralog. Conclusions In the tammar wallaby, one X (presumed to be maternal) is expressed in all cells, but genes on the other (paternal) X escape inactivation independently and at characteristic frequencies. The paternal and incomplete X chromosome inactivation in marsupials, with stochastic escape, appears to be quite distinct from the X chromosome inactivation process in eutherians. We find no evidence for a polar spread of inactivation from an X inactivation center. PMID:21182760

High hydrostatic pressure inactivation of Lactobacillus plantarum cells in (O/W)-emulsions is independent from cell surface hydrophobicity and lipid phase parameters

NASA Astrophysics Data System (ADS)

Kafka, T. A.; Reitermayer, D.; Lenz, C. A.; Vogel, R. F.

2017-07-01

Inactivation efficiency of high hydrostatic pressure (HHP) processing of food is strongly affected by food matrix composition. We investigated effects of fat on HHP inactivation of spoilage-associated Lactobacillus (L.) plantarum strains using defined oil-in-water (O/W)-emulsion model systems. Since fat-mediated effects on HHP inactivation could be dependent on interactions between lipid phase and microbial cells, three major factors possibly influencing such interactions were considered, that is, cell surface hydrophobicity, presence and type of surfactants, and oil droplet size. Pressure tolerance varied noticeably among L. plantarum strains and was independent of cell surface hydrophobicity. We showed that HHP inactivation of all strains tended to be more effective in presence of fat. The observation in both, surfactant-stabilized and surfactant-free (O/W)-emulsion, indicates that cell surface hydrophobicity is no intrinsic pressure resistance factor. In contrast to the presence of fat per se, surfactant type and oil droplet size did not affect inactivation efficiency.

A unified partial likelihood approach for X-chromosome association on time-to-event outcomes.

PubMed

Xu, Wei; Hao, Meiling

2018-02-01

The expression of X-chromosome undergoes three possible biological processes: X-chromosome inactivation (XCI), escape of the X-chromosome inactivation (XCI-E), and skewed X-chromosome inactivation (XCI-S). Although these expressions are included in various predesigned genetic variation chip platforms, the X-chromosome has generally been excluded from the majority of genome-wide association studies analyses; this is most likely due to the lack of a standardized method in handling X-chromosomal genotype data. To analyze the X-linked genetic association for time-to-event outcomes with the actual process unknown, we propose a unified approach of maximizing the partial likelihood over all of the potential biological processes. The proposed method can be used to infer the true biological process and derive unbiased estimates of the genetic association parameters. A partial likelihood ratio test statistic that has been proved asymptotically chi-square distributed can be used to assess the X-chromosome genetic association. Furthermore, if the X-chromosome expression pertains to the XCI-S process, we can infer the correct skewed direction and magnitude of inactivation, which can elucidate significant findings regarding the genetic mechanism. A population-level model and a more general subject-level model have been developed to model the XCI-S process. Finite sample performance of this novel method is examined via extensive simulation studies. An application is illustrated with implementation of the method on a cancer genetic study with survival outcome. © 2017 WILEY PERIODICALS, INC.

Method for flow cytometric monitoring of Renibacterium salmoninarum inactivation

USGS Publications Warehouse

Pascho, R.J.; Ongerth, J.E.

2000-01-01

The slow growth of Renibacterium salmoninarum limits the usefulness of culture as a research tool. Development of a 2-color flow cytometric assay to quantify the proportions of live and dead R. salmoninarum in a test population is described. Bacteria were simultaneously stained with fluorescein isothiocyanate-conjugated immunoglobulin and exposed to the exclusion dye propidium iodide. Propidium iodide red fluorescence profiles of control groups of untreated and killed R. salmoninarum were compared with those for bacteria exposed to chlorine. Bacterial inactivation was based on mean red fluorescence intensity, and analyzed by high-red fluorescence intensity (HRFI) and curve subtraction (CS) analyses. When the concentration of R. salmoninarum was 8.65 x 106 bacteria ml-1 and the bacteria exposed to chlorine at 1 mg l-1 for periods from 1 to 20 min (high-Rs assessment), the mean red fluorescence intensity of the profile for each chlorine-exposure group was higher than that for the untreated control (p < 0.0001). When the concentration of R. salmoninarum was reduced to 1.76 x 106 bacteria ml-1 and exposed to 0.8 mg l-1 free chlorine level for periods from 20 s to 5 min (reduced-Rs assessment), the mean red fluorescence intensities of the exposure groups were higher than that for the untreated control only when the R. salmoninarum was exposed to chlorine for at least 1 min (p ??? 0.01). On the basis of red fluorescence intensity, the proportion of dead cells generally increased with the duration of chlorine exposure. Whereas the rates of inactivation derived from the HRFI and CS analyses did not correlate with the duration of exposure in the high-Rs assessment (r2 ??? 0.27), there was a correlation between these estimates and the duration of exposure in the reduced-Rs assessment (r2 ??? 0.92). Because of the rapid loss of culturable R. salmoninarum in both assessments following chlorine exposure, neither the duration of exposure nor the inactivation estimates correlated with bacteriological culture (r2 ??? 0.22). In both assessments, there was a correlation between the estimates of inactivation based upon HRFI and CS analyses (r2 > 0.99). These results suggest that flow cytometry can be used as a supplementary or alternative method to bacteriological culture for monitoring the inactivation of R. salmoninarum.

Modelling the influence of total suspended solids on E. coli removal in river water.

PubMed

Qian, Jueying; Walters, Evelyn; Rutschmann, Peter; Wagner, Michael; Horn, Harald

2016-01-01

Following sewer overflows, fecal indicator bacteria enter surface waters and may experience different lysis or growth processes. A 1D mathematical model was developed to predict total suspended solids (TSS) and Escherichia coli concentrations based on field measurements in a large-scale flume system simulating a combined sewer overflow. The removal mechanisms of natural inactivation, UV inactivation, and sedimentation were modelled. For the sedimentation process, one, two or three particle size classes were incorporated separately into the model. Moreover, the UV sensitivity coefficient α and natural inactivation coefficient kd were both formulated as functions of TSS concentration. It was observed that the E. coli removal was predicted more accurately by incorporating two particle size classes. However, addition of a third particle size class only improved the model slightly. When α and kd were allowed to vary with the TSS concentration, the model was able to predict E. coli fate and transport at different TSS concentrations accurately and flexibly. A sensitivity analysis revealed that the mechanisms of UV and natural inactivation were more influential at low TSS concentrations, whereas the sedimentation process became more important at elevated TSS concentrations.

Studying the non-thermal plasma jet characteristics and application on bacterial decontamination

NASA Astrophysics Data System (ADS)

Al-rawaf, Ali F.; Fuliful, Fadhil Khaddam; Khalaf, Mohammed K.; Oudah, Husham. K.

2018-04-01

Non-thermal atmospheric-pressure plasma jet represents an excellent approach for the decontamination of bacteria. In this paper, we want to improve and characterize a non-thermal plasma jet to employ it in processes of sterilization. The electrical characteristics was studied to describe the discharge of the plasma jet and the development of plasma plume has been characterized as a function of helium flow rate. Optical emission spectroscopy was employed to detect the active species inside the plasma plume. The inactivation efficiency of non-thermal plasma jet was evaluated against Staphylococcus aureus bacteria by measuring the diameter of inhibition zone and the number of surviving cells. The results presented that the plasma plume temperature was lower than 34° C at a flow rate of 4 slm, which will not cause damage to living tissues. The diameter of inhibition zone is directly extended with increased exposure time. We confirmed that the inactivation mechanism was unaffected by UV irradiation. In addition, we concluded that the major reasons for the inactivation process of bacteria is because of the action of the reactive oxygen and nitrogen species which formed from ambient air, while the charged particles played a minor role in the inactivation process.

The nucleus of the optic tract. Its function in gaze stabilization and control of visual-vestibular interaction

NASA Technical Reports Server (NTRS)

Cohen, B.; Reisine, H.; Yokota, J. I.; Raphan, T.

1992-01-01

1. Electrical stimulation of the nucleus of the optic tract (NOT) induced nystagmus and after-nystagmus with ipsilateral slow phases. The velocity characteristics of the nystagmus were similar to those of the slow component of optokinetic nystagmus (OKN) and to optokinetic after-nystagmus (OKAN), both of which are produced by velocity storage in the vestibular system. When NOT was destroyed, these components disappeared. This indicates that velocity storage is activated from the visual system through NOT. 2. Velocity storage produces compensatory eye-in-head and head-on-body movements through the vestibular system. The association of NOT with velocity storage implies that NOT helps stabilize gaze in space during both passive motion and active locomotion in light with an angular component. It has been suggested that "vestibular-only" neurons in the vestibular nuclei play an important role in generation of velocity storage. Similarities between the rise and fall times of eye velocity during OKN and OKAN to firing rates of vestibular-only neurons suggest that these cells may receive their visual input through NOT. 3. One NOT was injected with muscimol, a GABAA agonist. Ipsilateral OKN and OKAN were lost, suggesting that GABA, which is an inhibitory transmitter in NOT, acts on projection pathways to the brain stem. A striking finding was that visual suppression and habituation of contralateral slow phases of vestibular nystagmus were also abolished after muscimol injection. The latter implies that NOT plays an important role in producing visual suppression of the VOR and habituating its time constant. 4. Habituation is lost after nodulus and uvula lesions and visual suppression after lesions of the flocculus and paraflocculus. We postulate that the disappearance of vestibular habituation and of visual suppression of vestibular responses after muscimol injections was due to dysfacilitation of the prominent NOT-inferior olive pathway, inactivating climbing fibers from the dorsal cap to nodulouvular and flocculoparafloccular Purkinje cells. The prompt loss of habituation when NOT was inactivated, and its return when the GABAergic inhibition dissipated, suggests that although VOR habituation can be relatively permanent, it must be maintained continuously by activity of the vestibulocerebellum.

Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET

PubMed Central

Kubota, Tomoya; Durek, Thomas; Dang, Bobo; Finol-Urdaneta, Rocio K.; Craik, David J.; Kent, Stephen B. H.; French, Robert J.; Bezanilla, Francisco; Correa, Ana M.

2017-01-01

Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating. PMID:28202723

Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET.

PubMed

Kubota, Tomoya; Durek, Thomas; Dang, Bobo; Finol-Urdaneta, Rocio K; Craik, David J; Kent, Stephen B H; French, Robert J; Bezanilla, Francisco; Correa, Ana M

2017-03-07

Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.

Development of lacosamide for the treatment of partial-onset seizures

PubMed Central

Doty, Pamela; Hebert, David; Mathy, Francois-Xavier; Byrnes, William; Zackheim, James; Simontacchi, Kelly

2013-01-01

Lacosamide is an antiepileptic drug (AED) available in multiple formulations that was first approved in 2008 as adjunctive therapy for partial-onset seizures (POS) in adults. Unlike traditional sodium channel blockers affecting fast inactivation, lacosamide selectively enhances sodium channel slow inactivation. This mechanism of action results in stabilization of hyperexcitable neuronal membranes, inhibition of neuronal firing, and reduction in long-term channel availability without affecting physiological function. Lacosamide has a well-characterized and favorable pharmacokinetic profile, including a fast absorption rate, minimal or no interaction with cytochrome P-450 izoenzymes, and a low potential for drug–drug interactions. Lacosamide clinical development included three placebo-controlled, double-blind, randomized trials conducted in more than 1300 patients, each demonstrating safety and efficacy of lacosamide compared to placebo as adjunctive therapy for adults with POS. The clinical use of lacosamide may broaden, pending results of trials evaluating its use as monotherapy for POS in adults, as treatment for epilepsy in pediatric subjects, and as adjunctive treatment for uncontrolled primary generalized tonic–clonic seizures in those with idiopathic generalized epilepsy. PMID:23859801

Suicide inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-halocatechols

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bartels, I.; Knackmuss, H.J.; Reineke, W.

The inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-chloro- and 3-fluorocatechol and the iron-chelating agent Tiron (catechol-3,5-disulfonate) was studied. Whereas inactivation by Tiron is an oxygen-independent and mostly reversible process, inactivation by the 3-halocatechols was only observed in the presence of oxygen and was largely irreversible. The rate constants for inactivation (K/sub 2/) were 1.62 x 10/sup -3/ sec/sup -1/ for 3-chlorocatechol and 2.38 x 10/sup -3/ sec/sup -1/ for 3-fluorocatechol. The inhibitor constants (K/sub i/) were 23 ..mu..M for 3-chlorocatechol and 17 ..mu..M for 3-fluorocatechol. The kinetic data for 3-fluorocatechol could only be obtained in the presencemore » of 2-mercaptoethanol. Besides inactivated enzyme, some 2-hydroxyhexa-2,4-dienoic acid as the actual suicide product of meta-cleavage. A side product of 3-fluorocatechol cleavage is a yellow compound with the spectral characteristics of a 2-hydroxy-6-oxohexa-2,4-dienoci acid indicating 1,6-cleavage. Rates of inactivation by 3-fluorocatechol were reduced in the presence of superoxide dismutase, catalase, formate, and mannitol, which implies that superoxide anion, hydrogen peroxide, and hydroxyl radical exhibit additional inactivation. 64 references.« less

STAT3 Controls the Long-Term Survival and Phenotype of Repair Schwann Cells during Nerve Regeneration.

PubMed

Benito, Cristina; Davis, Catherine M; Gomez-Sanchez, Jose A; Turmaine, Mark; Meijer, Dies; Poli, Valeria; Mirsky, Rhona; Jessen, Kristjan R

2017-04-19

After nerve injury, Schwann cells convert to a phenotype specialized to promote repair. But during the slow process of axonal regrowth, these repair Schwann cells gradually lose their regeneration-supportive features and eventually die. Although this is a key reason for the frequent regeneration failures in humans, the transcriptional mechanisms that control long-term survival and phenotype of repair cells have not been studied, and the molecular signaling underlying their decline is obscure. We show, in mice, that Schwann cell STAT3 has a dual role. It supports the long-term survival of repair Schwann cells and is required for the maintenance of repair Schwann cell properties. In contrast, STAT3 is less important for the initial generation of repair Schwann cells after injury. In repair Schwann cells, we find that Schwann cell STAT3 activation by Tyr705 phosphorylation is sustained during long-term denervation. STAT3 is required for maintaining autocrine Schwann cell survival signaling, and inactivation of Schwann cell STAT3 results in a striking loss of repair cells from chronically denervated distal stumps. STAT3 inactivation also results in abnormal morphology of repair cells and regeneration tracks, and failure to sustain expression of repair cell markers, including Shh, GDNF, and BDNF. Because Schwann cell development proceeds normally without STAT3, the function of this factor appears restricted to Schwann cells after injury. This identification of transcriptional mechanisms that support long-term survival and differentiation of repair cells will help identify, and eventually correct, the failures that lead to the deterioration of this important cell population. SIGNIFICANCE STATEMENT Although injured peripheral nerves contain repair Schwann cells that provide signals and spatial clues for promoting regeneration, the clinical outcome after nerve damage is frequently poor. A key reason for this is that, during the slow growth of axons through the proximal parts of injured nerves repair, Schwann cells gradually lose regeneration-supporting features and eventually die. Identification of signals that sustain repair cells is therefore an important goal. We have found that in mice the transcription factor STAT3 protects these cells from death and contributes to maintaining the molecular and morphological repair phenotype that promotes axonal regeneration. Defining the molecular mechanisms that maintain repair Schwann cells is an essential step toward developing therapeutic strategies that improve nerve regeneration and functional recovery. Copyright © 2017 Benito, Davis et al.

Effects of UV/Ag-TiO2/O3 advanced oxidation on unicellular green alga Dunaliella salina: implications for removal of invasive species from ballast water.

PubMed

Wu, Donghai; You, Hong; Du, Jiaxuan; Chen, Chuan; Jin, Darui

2011-01-01

The UV/Ag-TiO2/O3 process was investigated for ballast water treatment using Dunaliella salina as an indicator. Inactivation curves were obtained, and the toxicity of effluent was determined. Compared with individual unit processes using ozone or UV/Ag-TiO2, the inactivation efficiency of D. salina by the combined UV/Ag-TiO2/O3 process was enhanced. The presence of ozone caused an immediate decrease in chlorophyll a (chl-a) concentration. Inactivation efficiency and ch1-a removal efficiency were positively correlated with ozone dose and ultraviolet intensity. The initial total residual oxidant (TRO) concentration of effluent increased with increasing ozone dose, and persistence of TRO resulted in an extended period of toxicity. The results suggest that UV/Ag-TiO2/O3 has potential for ballast water treatment.

In situ studies of microbial inactivation during high pressure processing

NASA Astrophysics Data System (ADS)

Maldonado, Jose Antonio; Schaffner, Donald W.; Cuitiño, Alberto M.; Karwe, Mukund V.

2016-01-01

High pressure processing (HPP) has been shown to reduce microbial concentration in foods. The mechanisms of microbial inactivation by HPP have been associated with damage to cell membranes. The real-time response of bacteria to HPP was measured to elucidate the mechanisms of inactivation, which can aid in designing more effective processes. Different pressure cycling conditions were used to expose Enterobacter aerogenes cells to HPP. Propidium iodide (PI) was used as a probe, which fluoresces after penetrating cells with damaged membranes and binding with nucleic acids. A HPP vessel with sapphire windows was used for measuring fluorescence in situ. Membrane damage was detected during pressurization and hold time, but not during depressurization. The drop in fluorescence was larger than expected after pressure cycles at higher pressure and longer times. This indicated possible reversible disassociation of ribosomes resulting in additional binding of PI to exposed RNA under pressure and its release after depressurization.

Using ultrasound technology for the inactivation and thermal sensitization of peroxidase in green coconut water.

PubMed

Rojas, Meliza Lindsay; Trevilin, Júlia Hellmeister; Funcia, Eduardo Dos Santos; Gut, Jorge Andrey Wilhelms; Augusto, Pedro Esteves Duarte

2017-05-01

Green coconut water has unique nutritional and sensorial qualities. Despite the different technologies already studied, its enzymatic stability is still challenging. This study evaluated the use of ultrasound technology (US) for inactivating/sensitizing coconut water peroxidase (POD). The effect of both US application alone and as a pre-treatment to thermal processing was evaluated. The enzyme activity during US processing was reduced 27% after 30min (286W/L, 20kHz), demonstrating its high resistance. The thermal inactivation was described by the Weibull model under non-isothermal conditions. The enzyme became sensitized to heat after US pre-treatment. Further, the use of US resulted in more uniform heat resistance. The results suggest that US is a good technology for sensitizing enzymes before thermal processing (even for an enzyme with high thermal resistance). Therefore, the use of this technology could decrease the undesirable effects of long times and/or the high temperatures of the conventional thermal processing. Copyright © 2016 Elsevier B.V. All rights reserved.

Bacterial spore inactivation at 45-65 °C using high pressure processing: study of Alicyclobacillus acidoterrestris in orange juice.

PubMed

Silva, Filipa V M; Tan, Eng Keat; Farid, Mohammed

2012-10-01

High pressure processing (HPP) is a new non-thermal technology commercially used to pasteurize fruit juices and extend shelf-life, while preserving delicate aromas/flavours and bioactive constituents. Given the spoilage incidents and economic losses due to Alicyclobacillus acidoterrestris in the fruit juice industry, the use of high pressure (200 MPa - 600 MPa) in combination with mild temperature (45 °C-65 °C) for 1-15 min, to inactivate these spores in orange juice were investigated. As expected, the higher the temperature, pressure and time, the larger was the A. acidoterrestris inactivation. The survival curves were described by the first order Bigelow model. For 200 MPa, D(45 °C) = 43.9 min, D(55 °C) = 28.8 min, D(65 °C) = 5.0 min and z-value = 21.3 °C. At 600 MPa, D(45 °C) = 12.9 min, D(55 °C) = 7.0 min, D(65 °C) = 3.4 min and z-value = 34.4 °C. Spores were inactivated at 45 °C and 600 MPa, and at 65 °C only 200 MPa was needed to achieve reduction in spore numbers. Results demonstrated that HPP allowed A. acidoterrestris spore inactivation at lower temperatures (45-65 °C) than conventional thermal processing (85-95 °C) without pressure, yielding a fresher and higher quality preserved food. Copyright © 2012 Elsevier Ltd. All rights reserved.

INACTIVATION OF BIOLOGICAL CONTAMINANTS IN DRINKING WATER IN RESPONSE TO A TERRORIST ATTACK

EPA Science Inventory

In the event of an intentional biological contamination of a drinking water treatment and distribution system, information on the removal and/or inactivation of known biological agents by drinking water treatment processes is needed. This project was initiated to determine what i...

Gating transitions in the selectivity filter region of a sodium channel are coupled to the domain IV voltage sensor.

PubMed

Capes, Deborah L; Arcisio-Miranda, Manoel; Jarecki, Brian W; French, Robert J; Chanda, Baron

2012-02-14

Voltage-dependent ion channels are crucial for generation and propagation of electrical activity in biological systems. The primary mechanism for voltage transduction in these proteins involves the movement of a voltage-sensing domain (D), which opens a gate located on the cytoplasmic side. A distinct conformational change in the selectivity filter near the extracellular side has been implicated in slow inactivation gating, which is important for spike frequency adaptation in neural circuits. However, it remains an open question whether gating transitions in the selectivity filter region are also actuated by voltage sensors. Here, we examine conformational coupling between each of the four voltage sensors and the outer pore of a eukaryotic voltage-dependent sodium channel. The voltage sensors of these sodium channels are not structurally symmetric and exhibit functional specialization. To track the conformational rearrangements of individual voltage-sensing domains, we recorded domain-specific gating pore currents. Our data show that, of the four voltage sensors, only the domain IV voltage sensor is coupled to the conformation of the selectivity filter region of the sodium channel. Trapping the outer pore in a particular conformation with a high-affinity toxin or disulphide crossbridge impedes the return of this voltage sensor to its resting conformation. Our findings directly establish that, in addition to the canonical electromechanical coupling between voltage sensor and inner pore gates of a sodium channel, gating transitions in the selectivity filter region are also coupled to the movement of a voltage sensor. Furthermore, our results also imply that the voltage sensor of domain IV is unique in this linkage and in the ability to initiate slow inactivation in sodium channels.

Myosin Light Chain Kinase and the Role of Myosin Light Chain Phosphorylation in Skeletal Muscle

PubMed Central

Stull, James T.; Kamm, Kristine E.; Vandenboom, Rene

2011-01-01

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca2+/calmodulin-dependent serine-threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca2+ binding to calmodulin forming a (Ca2+)4•calmodulin complex sufficient for activation with a diffusion limited, stoichiometic binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca2+ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca2+/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca2+-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue. PMID:21284933

Long Non-coding RNAs in the X-inactivation Center

PubMed Central

Kalantry, Sundeep

2014-01-01

The X-inactivation center is a hotbed of functional long non-coding RNAs in eutherian mammals. These RNAs are thought to help orchestrate the epigenetic transcriptional states of the two X-chromosomes in females as well as of the single X-chromosome in males. To balance X-linked gene expression between the sexes, females undergo transcriptional silencing of most genes on one of the two X-chromosomes in a process termed X-chromosome inactivation. While one X-chromosome is inactivated, the other X-chromosome remains active. Moreover, with a few notable exceptions, the originally established epigenetic transcriptional profiles of the two is maintained as such through many rounds of cell division, essentially for the life of the organism. The stable divergent transcriptional fates of the two X-chromosomes, despite residing in a shared nucleoplasm, make X-inactivation a paradigm of epigenetic transcriptional regulation. Originally proposed in 1961 by Mary Lyon, the X-inactivation hypothesis has been validated through much experimentation over the last fifty years. In the last 25 years, the discovery and functional characterization has firmly established X-linked long non-coding RNAs as key players in choreographing X-chromosome inactivation. PMID:24297756

Inactivation gating determines nicotine blockade of human HERG channels.

PubMed

Wang, H Z; Shi, H; Liao, S J; Wang, Z

1999-09-01

We have previously found that nicotine blocked multiple K+ currents, including the rapid component of delayed rectifier K+ currents (IKr), by interacting directly with the channels. To shed some light on the mechanisms of interaction between nicotine and channels, we performed detailed analysis on the human ether-à-go-go-related gene (HERG) channels, which are believed to be equivalent to the native I(Kr) when expressed in Xenopus oocytes. Nicotine suppressed the HERG channels in a concentration-dependent manner with greater potency with voltage protocols, which favor channel inactivation. Nicotine caused dramatic shifts of the voltage-dependent inactivation curve to more negative potentials and accelerated the inactivation process. Conversely, maneuvers that weakened the channel inactivation gating considerably relieved the blockade. Elevating the extracellular K+ concentration from 5 to 20 mM increased the nicotine concentration (by approximately 100-fold) needed to achieve the same degree of inhibition. Moreover, nicotine lost its ability to block the HERG channels when a single mutation was introduced to a residue located after transmembrane domain 6 (S631A) to remove the rapid channel inactivation. Our data suggest that the inactivation gating determines nicotine blockade of the HERG channels.

The use of chlorine dioxide for the inactivation of copepod zooplankton in drinking water treatment.

PubMed

Lin, Tao; Chen, Wei; Cai, Bo

2014-01-01

The presence of zooplankton in drinking water treatment system may cause a negative effect on the aesthetic value of drinking water and may also increase the threat to human health due to they being the carriers of bacteria. Very little research has been done on the effects of copepod inactivation and the mechanisms involved in this process. In a series of bench-scale experiments we used a response surface method to assess the sensitivity of copepod to inactivation when chlorine dioxide (ClO₂) was used as a disinfectant. We also assessed the effects of the ClO₂dosage, exposure time, organic matter concentration and temperature. Results indicated that the inactivation rate improved with increasing dosage, exposure time and temperature, whereas it decreased with increasing organic matter concentration. Copepod inactivation was more sensitive to the ClO₂dose than that to the exposure time, while being maintained at the same Ct-value conditions. The activation energy at different temperatures revealed that the inactivation of copepods with ClO₂was temperature-dependent. The presence of organic matter resulted in a lower available dose as well as a shorter available exposure time, which resulted in a decrease in inactivation efficiency.

Kinetics of inactivation of indicator pathogens during thermophilic anaerobic digestion.

PubMed

Popat, Sudeep C; Yates, Marylynn V; Deshusses, Marc A

2010-12-01

Thermophilic anaerobic sludge digestion is a promising process to divert waste to beneficial use, but an important question is the required temperature and holding time to achieve a given degree of pathogen inactivation. In this study, the kinetics of inactivation of Ascaris suum and vaccine strain poliovirus type 1 (PVS-1), selected as indicators for helminth ova and enteric viruses respectively, were determined during anaerobic digestion at temperatures ranging from 51 to 56 °C. Inactivation of both indicator organisms was fast with greater than two log reductions achieved within 2 h for A. suum and three log reductions for PVS-1, suggesting that the current U.S. regulations are largely conservative. The first-order inactivation rate constants k followed Arrhenius relationship with activation energies of 105 and 39 KJ mol(-1) for A. suum and PVS-1, respectively indicating that A. suum was more sensitive to temperature. Although inactivation was fast, the presence of compounds in the sludge that are known to be protective of pathogen inactivation was observed, suggesting that composition-dependent time-temperature relationships are necessary. Copyright © 2010 Elsevier Ltd. All rights reserved.

Molecular mechanism of acetylcholine receptor-controlled ion translocation across cell membranes

PubMed Central

Cash, Derek J.; Hess, George P.

1980-01-01

Two molecular processes, the binding of acetylcholine to the membrane-bound acetylcholine receptor protein and the receptor-controlled flux rates of specific inorganic ions, are essential in determining the electrical membrane potential of nerve and muscle cells. The measurements reported establish the relationship between the two processes: the acetylcholine receptor-controlled transmembrane ion flux of 86Rb+ and the concentration of carbamoylcholine, a stable analog of acetylcholine. A 200-fold concentration range of carbamoylcholine was used. The flux was measured in the millisecond-to-minute time region by using a quench flow technique with membrane vesicles prepared from the electric organ of Electrophorus electricus in eel Ringer's solution at pH 7.0 and 1°C. The technique makes possible the study of the transmembrane transport of specific ions, with variable known internal and external ion concentrations, in a system in which a determinable number of receptors is exposed to a known concentration of ligand. The response curve of ion flux to ligand was sigmoidal with an average maximum rate of 84 sec-1. Carbamoylcholine induced inactivation of the receptor with a maximum rate of 2.7 sec-1 and a different ligand dependence so that it was fast relative to ion flux at low ligand concentration but slow relative to ion flux at high ligand concentration. The simplest model that fits the data consists of receptor in the active and inactive states in ligand-controlled equilibria. Receptor inactivation occurs with one or two ligand molecules bound. For channel opening, two ligand molecules bound to the active state are required, and cooperativity results from the channel opening process itself. With carbamoylcholine, apparently, the equilibrium position for the channel opening step is only one-fourth open. The integrated rate equation, based on the model, predicts the time dependence of receptor-controlled ion flux over the concentration range of carbamoylcholine investigated. The values of the constants in the rate equation form the basis for predicting receptor-controlled changes in the transmembrane potential of cells and the conditions leading to transmission of signals between cells. PMID:6928684

MS2 and ΦX174 inactivation by high frequency ultrasound

NASA Astrophysics Data System (ADS)

Manariotis, I. D.; Syngouna, V.; Chrysikopoulos, C. V.

2012-04-01

Biocolloid inactivation in water with the use of ultrasound can be quite effective, because the implosion of cavitation bubbles can generate high temperatures and pressures at the heart of collapsed bubbles. Biocolloid inactivation by cavitation takes place due to a combination of simultaneously acting processes involving mechanical effects (caused by turbulence generation, microstreaming, liquid circulation currents, and shear stresses), chemical effects of cavitation (generation of active free radicals), and heat effects (generation of local hot spots). Generally, the mechanical effects are more responsible for biocolloid disinfection, whereas the chemical and heat effects play only a supporting role. The present study focuses on inactivation of MS2 and ΦΧ174 at three different relatively high frequencies (i.e. 582, 862, and 1142 kHz). The experimental results indicate that, for all three frequencies and power input of 133 W, both phages were at least 90% inactivated after 60 min of sonication.

Strategy to inactivate Clostridium perfringens spores in meat products.

PubMed

Akhtar, Saeed; Paredes-Sabja, Daniel; Torres, J Antonio; Sarker, Mahfuzur R

2009-05-01

The current study aimed to develop an inactivation strategy for Clostridium perfringens spores in meat through a combination of spore activation at low pressure (100-200 MPa, 7 min) and elevated temperature (80 degrees C, 10 min); spore germination at high temperatures (55, 60 or 65 degrees C); and inactivation of germinated spores with elevated temperatures (80 and 90 degrees C, 10 and 20 min) and high pressure (586 MPa, at 23 and 73 degrees C, 10 min). Low pressures (100-200 MPa) were insufficient to efficiently activate C. perfringens spores for germination. However, C. perfringens spores were efficiently activated with elevated temperature (80 degrees C, 10 min), and germinated at temperatures lethal for vegetative cells (>or= 55 degrees C) when incubated for 60 min with a mixture of L-asparagine and KCl (AK) in phosphate buffer (pH 7) and in poultry meat. Inactivation of spores (approximately 4 decimal reduction) in meat by elevated temperatures (80-90 degrees C for 20 min) required a long germination period (55 degrees C for 60 min). However, similar inactivation level was reached with shorter germination period (55 degrees C for 15 min) when spore contaminated-meat was treated with pressure-assisted thermal processing (568 MPa, 73 degrees C, 10 min). Therefore, the most efficient strategy to inactivate C. perfringens spores in poultry meat containing 50 mM AK consisted: (i) a primary heat treatment (80 degrees C, 10 min) to pasteurize and denature the meat proteins and to activate C. perfringens spores for germination; (ii) cooling of the product to 55 degrees C in about 20 min and further incubation at 55 degrees C for about 15 min for spore germination; and (iii) inactivation of germinated spores by pressure-assisted thermal processing (586 MPa at 73 degrees C for 10 min). Collectively, this study demonstrates the feasibility of an alternative and novel strategy to inactivate C. perfringens spores in meat products formulated with germinants specific for C. perfringens.

Concepts and Principles of Photodynamic Therapy as an Alternative Antifungal Discovery Platform

PubMed Central

Dai, Tianhong; Fuchs, Beth B.; Coleman, Jeffrey J.; Prates, Renato A.; Astrakas, Christos; St. Denis, Tyler G.; Ribeiro, Martha S.; Mylonakis, Eleftherios; Hamblin, Michael R.; Tegos, George P.

2012-01-01

Opportunistic fungal pathogens may cause superficial or serious invasive infections, especially in immunocompromised and debilitated patients. Invasive mycoses represent an exponentially growing threat for human health due to a combination of slow diagnosis and the existence of relatively few classes of available and effective antifungal drugs. Therefore systemic fungal infections result in high attributable mortality. There is an urgent need to pursue and deploy novel and effective alternative antifungal countermeasures. Photodynamic therapy (PDT) was established as a successful modality for malignancies and age-related macular degeneration but photodynamic inactivation has only recently been intensively investigated as an alternative antimicrobial discovery and development platform. The concept of photodynamic inactivation requires microbial exposure to either exogenous or endogenous photosensitizer molecules, followed by visible light energy, typically wavelengths in the red/near infrared region that cause the excitation of the photosensitizers resulting in the production of singlet oxygen and other reactive oxygen species that react with intracellular components, and consequently produce cell inactivation and death. Antifungal PDT is an area of increasing interest, as research is advancing (i) to identify the photochemical and photophysical mechanisms involved in photoinactivation; (ii) to develop potent and clinically compatible photosensitizers; (iii) to understand how photoinactivation is affected by key microbial phenotypic elements multidrug resistance and efflux, virulence and pathogenesis determinants, and formation of biofilms; (iv) to explore novel photosensitizer delivery platforms; and (v) to identify photoinactivation applications beyond the clinical setting such as environmental disinfectants. PMID:22514547

Thermal and non-thermal surface treatments of fresh cantaloupes and other melons for inactivating human pathogens

USDA-ARS?s Scientific Manuscript database

Outbreaks of salmonellosis by Salmonella Poona and listeriosis by Listeria monocytogenes have been associated with the consumption of cantaloupes. Commercial washing processes for cantaloupes are limited in their ability to inactivate and/or remove this human pathogen. Our objective was to develop...

Inactivation of Lactobacillus plantarum in apple cider using radio frequency electric fields

USDA-ARS?s Scientific Manuscript database

Radio frequency electric fields (RFEF) processing is effective at inactivating Gram negative bacteria in fruit juices at moderately low temperatures, but has yet to be shown to be effective at reducing Gram positive bacteria. Lactobacillus plantarum ATCC 49445, a Gram positive bacterium, was inocula...

High pressure treatment of human norovirus-like particles: factors affecting destruction efficacy

USDA-ARS?s Scientific Manuscript database

Human norovirus (HuNoV) is the leading cause of foodborne diseases worldwide. High pressure processing (HPP) is considered a promising non-thermal technology to inactivate viral pathogens in foods. However, the effectiveness of HPP on inactivating HuNoV remains poorly understood because it cannot be...

Hot water surface pasteurization for inactivating Salmonella on surfaces of mature green tomatoes

USDA-ARS?s Scientific Manuscript database

Outbreaks of salmonellosis have been associated with the consumption of tomatoes contaminated with Salmonella. Commercial washing processes for tomatoes are limited in their ability to inactivate and/or remove this human pathogen. Our objective was to develop a hot water surface pasteurization pro...

Evaluation of Filtration and UV Disinfection for Inactivation of Viruses in Non-Community Water Systems in Minnesota

EPA Science Inventory

This study evaluated filtration and disinfection processes for removal and inactivation of pathogens in non-community water systems (NCWS) in two surface water supplies. Pretreatment systems included 1) pressure sand filtration, and 2) granular activated carbon adsorption, and 3...

Mechanism of block by tedisamil of transient outward current in human ventricular subepicardial myocytes

PubMed Central

Wettwer, Erich; Himmel, Herbert M; Amos, Gregory J; Li, Qi; Metzger, Franz; Ravens, Ursula

1998-01-01

Tedisamil is a new antiarrhythmic drug with predominant class III action. The aim of the present study was to investigate the blocking pattern of the compound on the transient outward current (Ito) in human subepicardial myocytes isolated from explanted left ventricles. Using the single electrode whole cell voltage clamp technique, Ito was analysed after appropriate voltage inactivation of sodium current and block of calcium current.Tedisamil reduced the amplitude of peak Ito, but did not affect the amplitude of non-inactivating outward current. The drug accelerated the apparent rate of Ito inactivation. The reduction in time constant of Ito inactivation depended on drug concentration, the apparent IC50 value was 4.4 μM.Tedisamil affected Ito amplitude in a use-dependent manner. After 2 min at −80 mV, maximum block of Ito was reached after 4–5 clamp steps either at the frequency of 0.2 or 2 Hz, indicating that the block was not frequency-dependent in an experimentally relevant range. Recovery from block was very slow and proceeded with a time constant of 12.1±1.8 s. Also in the presence of drug, a fraction of channels recovered from inactivation with a similar time constant as in control myocytes (i.e. 81±40 ms and 51±8 ms, respectively, n.s.).From the onset of fractional block of Ito by tedisamil during the initial 60 ms of a clamp step, we calculated k1=9×106 mol−1 s−1 for the association rate constant, and k2=23 s−1 for the dissociation rate constant. The resulting apparent KD was 2.6 μM and is similar to the IC50 value.The effects of tedisamil on Ito could be simulated by assuming a four state channel model where the drug binds to the channel in an open (activated) conformation. It is concluded that in human subepicardial myocytes tedisamil is an open channel blocker of Ito and that this effect probably contributes to the antiarrhythmic potential of this drug. PMID:9831899

Evaluation of Different Dose-Response Models for High Hydrostatic Pressure Inactivation of Microorganisms

PubMed Central

2017-01-01

Modeling of microbial inactivation by high hydrostatic pressure (HHP) requires a plot of the log microbial count or survival ratio versus time data under a constant pressure and temperature. However, at low pressure and temperature values, very long holding times are needed to obtain measurable inactivation. Since the time has a significant effect on the cost of HHP processing it may be reasonable to fix the time at an appropriate value and quantify the inactivation with respect to pressure. Such a plot is called dose-response curve and it may be more beneficial than the traditional inactivation modeling since short holding times with different pressure values can be selected and used for the modeling of HHP inactivation. For this purpose, 49 dose-response curves (with at least 4 log10 reduction and ≥5 data points including the atmospheric pressure value (P = 0.1 MPa), and with holding time ≤10 min) for HHP inactivation of microorganisms obtained from published studies were fitted with four different models, namely the Discrete model, Shoulder model, Fermi equation, and Weibull model, and the pressure value needed for 5 log10 (P5) inactivation was calculated for all the models above. The Shoulder model and Fermi equation produced exactly the same parameter and P5 values, while the Discrete model produced similar or sometimes the exact same parameter values as the Fermi equation. The Weibull model produced the worst fit (had the lowest adjusted determination coefficient (R2adj) and highest mean square error (MSE) values), while the Fermi equation had the best fit (the highest R2adj and lowest MSE values). Parameters of the models and also P5 values of each model can be useful for the further experimental design of HHP processing and also for the comparison of the pressure resistance of different microorganisms. Further experiments can be done to verify the P5 values at given conditions. The procedure given in this study can also be extended for enzyme inactivation by HHP. PMID:28880255

Inactivation of viruses by pasteurization at 60 °C for 10 h with and without 40% glucose as stabilizer during a new manufacturing process of α2-Macroglobulin from Cohn Fraction IV.

PubMed

Huangfu, Chaoji; Ma, Yuyuan; Jia, Junting; Lv, Maomin; Zhu, Fengxuan; Ma, Xiaowei; Zhao, Xiong; Zhang, Jingang

2017-03-01

Pasteurization is regularly used to inactivate viruses for the safety of plasma derivatives. Influence of pasteurization at 60 °C for 10 h on α2-Macroglobulin activity and virus inactivation were studied. With 40% sugar as stabilizers more than 70% α2-Macroglobulin activity was reserved after pasteurization compared with 20% in control. Glucose presented a better activity protection effect than sucrose and maltose. By pasteurization without stabilizer the virus titers of pseudorabies virus, Sindbis virus, porcine parvovirus and encephalomyocarditis virus were reduced more than 5.88 log 10 , 7.50 log 10 , 4.88 log 10 , and 5.63 log 10 respectively within 2 h. By pasteurization with 40% glucose vesicular stomatitis virus was inactivated more than 5.88 log 10 within 1 h. Only 2.71 log 10 reduction was achieved for encephalomyocarditis virus after 10 h. 40% glucose protected α2-M activity and viruses simultaneously from pasteurization. Other viral inactivation methods need to be incorporated to ensure viral safety of this manufacturing process of α2-Macroglobulin. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Response surface methodology as a tool for modeling and optimization of Bacillus subtilis spores inactivation by UV/ nano-Fe0 process for safe water production.

PubMed

Yousefzadeh, Samira; Matin, Atiyeh Rajabi; Ahmadi, Ehsan; Sabeti, Zahra; Alimohammadi, Mahmood; Aslani, Hassan; Nabizadeh, Ramin

2018-04-01

One of the most important aspects of environmental issues is the demand for clean and safe water. Meanwhile, disinfection process is one of the most important steps in safe water production. The present study aims at estimating the performance of UV, nano Zero-Valent Iron particles (nZVI, nano-Fe 0 ), and UV treatment with the addition of nZVI (combined process) for Bacillus subtilis spores inactivation. Effects of different factors on inactivation including contact time, initial nZVI concentration, UV irradiance and various aerations conditions were investigated. Response surface methodology, based on a five-level, two variable central composite design, was used to optimize target microorganism reduction and the experimental parameters. The results indicated that the disinfection time had the greatest positive impact on disinfection ability among the different selected independent variables. According to the results, it can be concluded that microbial reduction by UV alone was more effective than nZVI while the combined UV/nZVI process demonstrated the maximum log reduction. The optimum reduction of about 4 logs was observed at 491 mg/L of nZVI and 60 min of contact time when spores were exposed to UV radiation under deaerated condition. Therefore, UV/nZVI process can be suggested as a reliable method for Bacillus subtilis spores inactivation. Copyright © 2018. Published by Elsevier Ltd.

Electromagnetic energy and food processing.

PubMed

Mudgett, R

1988-01-01

The use of electromagnetic energy in food processing is reviewed with respect to food safety, nutritional quality, and organoleptic quality. The effects of nonionizing radiation sources such as microwave and radio-frequency energy and ionizing radiation sources, e.g. radioactive cobalt-60 and caesium-137, on the inactivation of microbes and nutrients are compared with those of conventional heating processes both in terms of their kinetic behavior and their mechanisms of interaction with foods. The kinetics of microwave and conventional thermal inactivation are considered for a generalized nth-order model based on time and temperature conditions. However, thermal inactivation effects are often modeled by 1st-order kinetics. Microbial and nutrient inactivation by ionizing sources are considered for a 1st-order model based on radiation dose. Both thermal and radiation resistance concepts are reviewed and some typical values of radiation resistance are given for sensitive vegetative bacterial cells, yeasts, and molds and for resistant bacterial spores and viruses. Nonionizing microwave energy sources are increasingly used in home and industrial food processing and are well-accepted by the American public. But, despite recent Food and Drug Administration approval of low and intermediate ionizing radiation dose levels for grains and other plants products and the fact that irradiated foods are sold in more than 20 countries of the world, public fears in the U.S. about nuclear energy may limit the role of ionizing radiation in food processing and preservation and may also limit the use of nuclear fuels as an alternate source of electrical energy.

Current Strategies in the Modification of PLGA-based Gene Delivery System.

PubMed

Ramezani, Mohammad; Ebrahimian, Mahboubeh; Hashemi, Maryam

2017-01-01

Successful gene therapy has been limited by safe and efficient delivery of nucleic acid to the target cells. Poly (d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) are able to deliver drugs and genes efficiently. This formulation has several advantages in comparison with other formulations including improvement in solubility, stability, controlling of degradation and release of the entrapped agents. For application of PLGA as a gene carrier, there exist many challenges. PLGA NPs could protect the encapsulated DNA from in vivo degradation but the DNA release is slow and the negative charge acts as a barrier to DNA incorporation and delivery. Also, during the preparation process, DNA could be exposed to high shear stress and organic solvents which could result in its inactivation. Moreover, PLGA NPs could be modified with different agents to reduce cytotoxicity, to enhance delivery efficiency and to target specific tissues/cells. This review summarizes different methods used for the preparation of PLGA NPs as gene carriers and recent strategies for the modification of PLGA particles applied in gene therapy. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Disinfection of wastewater effluents with the Fenton-like process induced by electromagnetic fields.

PubMed

Rodríguez-Chueca, J; Mediano, A; Ormad, M P; Mosteo, R; Ovelleiro, J L

2014-09-01

This research work is focused on the application and assessment of effectiveness of the Fenton-like processes induced by radiofrequency for the inactivation of faecal bacteria (Escherichia coli and Enterococcus sp.) present in treated urban wastewater effluents. Fenton processes were carried out at near neutral pH (pH 5) with different iron sources, such as iron salts (ferric chloride, 5, 50 and 100 mg/L Fe(3+)), magnetite (1 g/L) and clay (80 g/L), hydrogen peroxide (25 mg/L) and in absence and presence of radiofrequency. Two different electromagnetic field intensities (1.57 and 3.68 kA/m) were used in Fenton processes induced by radiofrequency. Different agents used in the Fenton processes induced by electromagnetic fields (iron source, hydrogen peroxide and RF) were analyzed individually and in combination under the same experimental conditions. First assays of ferromagnetic material/H2O2/radiofrequency processes achieved promising results in terms of bacterial inactivation. For instance, Fe(3+)/H2O2/Radiofrequency achieved a maximum level of E. coli inactivation of 3.55 log after 10 min of treatment. These results are higher than those obtained in absence of radiofrequency. The thermal activation of iron atoms allows the Fenton reaction to intensify, increasing the final yield of the treatment. On the other hand, different behavior was observed in the inactivation of E. coli and Enterococcus sp. due to the structural differences between Gram-negative and Gram-positive bacteria. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterization of long-term extension of isolated cell walls from growing cucumber hypocotyls

NASA Technical Reports Server (NTRS)

Cosgrove, D. J.

1989-01-01

Walls from frozen-thawed cucumber (Cucumis sativus L.) hypocotyls extend for many hours when placed in tension under acidic conditions. This study examined whether such "creep" is a purely physical process dependent on wall viscoelasticity alone or whether enzymatic activities are needed to maintain wall extension. Chemical denaturants inhibited wall creep, some acting reversibly and others irreversibly. Brief (15 s) boiling in water irreversibly inhibited creep, as did pre-incubation with proteases. Creep exhibited a high Q10 (3.8) between 20 degrees and 30 degrees C, with slow inactivation at higher temperatures, whereas the viscous flow of pectin solutions exhibited a much lower Q10 (1.35). On the basis of its temperature sensitivity, involvement of pectic gel-sol transitions was judged to be of little importance in creep. Pre-incubation of walls in neutral pH irreversibly inactivated their ability to creep, with a half-time of about 40 min. At 1 mM, Cu2+, Hg2+ and Al3+ were strongly inhibitory whereas most other cations, including Ca2+, had little effect. Sulfhydryl-reducing agents strongly stimulated creep, apparently by stabilizing wall enzyme(s). The physical effects of these treatments on polymer interactions were examined by Instron and stress-relaxation analyses. Some treatments, such as pH and Cu2+, had significant effects on wall viscoelasticity, but others had little or no apparent effect, thus implicating an enzymatic creep mechanism. The results indicate that creep depends on relatively rugged enzymes that are firmly attached to or entangled in the wall. The sensitivity of creep to SH-reducing agents indicates that thiol reduction of wall enzymes might provide a control mechanism for endogenous cell growth.

A functional and thromboelastometric-based micromethod for assessing crotoxin anticoagulant activity and antiserum relative potency against Crotalus durissus terrificus venom.

PubMed

Prezoto, B C; Tanaka-Azevedo, A M; Marcelino, J R; Tashima, A K; Nishiduka, E S; Kapronezai, J; Mota, J O; Rocha, M M T; Serino-Silva, C; Oguiura, N

2018-06-15

The assessment of the capacity of antivenoms to neutralize the lethal activity of snake venoms still relies on traditional rodent in vivo lethality assay. ED 50 and LD 50 assays require large quantities of venoms and antivenoms, and besides leading to animal suffering. Therefore, in vitro tests should be introduced for assessing antivenom neutralizing capacity in intermediary steps of antivenom production. This task is facilitated when one key lethal toxin is identified. A good example is crotoxin, a β-neurotoxin phospholipase A 2 -like toxin that presents anticoagulant activity in vitro and is responsible for the lethality of venoms of Crotalus durissus snakes. By using rotational thromboelastometry, we reported recently one sensitive coagulation assay for assessing relative potency of the anti-bothropic serum in neutralizing procoagulant activity of Bothrops jararaca venom upon recalcified factor-XII-deficient chicken plasma samples (CPS). In this study, we stablished conditions for determining relative potency of four batches of the anti-crotalic serum (ACS) (antagonist) in inactivating crotoxin anticoagulant activity in CPS (target) simultaneously treated with one classical activator of coagulation (agonists). The correlation coefficient (r) between values related the ACS potency in inactivating both in vitro crotoxin anticoagulant activity and the in vivo lethality of whole venom (ED 50 ) was 0.94 (p value < 0.05). In conclusion, slowness in spontaneous thrombin/fibrin generation even after recalcification elicit time lapse sufficient for elaboration of one dose-response curve to pro- or anti-coagulant agonists in CPS. We propose this methodology as an alternative and sensitive assay for assessing antivenom neutralizing ability in plasma of immunized horses as well as for in-process quality control. Copyright © 2018 Elsevier Ltd. All rights reserved.

Modulation of voltage-gated Na+ and K+ channels by pumiliotoxin 251D: a "joint venture" alkaloid from arthropods and amphibians.

PubMed

Vandendriessche, Thomas; Abdel-Mottaleb, Yousra; Maertens, Chantal; Cuypers, Eva; Sudau, Alexander; Nubbemeyer, Udo; Mebs, Dietrich; Tytgat, Jan

2008-03-01

Certain amphibians provide themselves with a chemical defense by accumulating lipophilic alkaloids into skin glands from dietary arthropods. Examples of such alkaloids are pumiliotoxins (PTXs). In general, PTXs are known as positive modulators of voltage-gated sodium channels (VGSCs). Unlike other PTXs, PTX 251D does not share this characteristic. However, mice and insect studies showed that PTX 251D is highly toxic and to date the basis of its toxicity remains unknown. In this work, we searched for the possible target of PTX 251D. The toxin was therefore made synthetically and tested on four VGSCs (mammalian rNa(v)1.2/beta(1), rNa(v)1.4/beta(1), hNa(v)1.5/beta(1) and insect Para/tipE) and five voltage-gated potassium channels (VGPCs) (mammalian rK(v)1.1-1.2, hK(v)1.3, hK(v)11.1 (hERG) and insect Shaker IR) expressed heterologously in Xenopus laevis oocytes, using the two-electrode voltage clamp technique. PTX 251D not only inhibited the Na(+) influx through the mammalian VGSCs but also affected the steady-state activation and inactivation. Interestingly, in the insect ortholog, the inactivation process was dramatically affected. Additionally, PTX 251D inhibited the K(+) efflux through all five tested VGPCs and slowed down the deactivation kinetics of the mammalian VGPCs. hK(v)1.3 was the most sensitive channel, with an IC(50) value 10.8+/-0.5 microM. To the best of our knowledge this is the first report of a PTX affecting VGPCs.

Particle effects on ultraviolet disinfection of coliform bacteria in recycled water.

PubMed

Jolis, D; Lam, C; Pitt, P

2001-01-01

Pilot- and bench-scale coliform inactivation tests with UV irradiation were used to show how suspended solids remaining in filtered secondary effluent affect the efficiency of the UV disinfection process. Observed kinetic inactivation rates decreased with increasing suspended particle sizes of 7 microm or larger present in tertiary effluent. First-order inactivation rates estimated from collimated beam dose-response curves for discrete ranges of UV doses were substantially different, which should caution researchers not to compare inactivation data obtained with largely dissimilar UV doses or suspended particle distributions. A dose of approximately 800 J/m2 was identified as the minimum dose that will consistently meet the California wastewater reclamation coliform criterion when applied to in-line filtration effluent.

Silver nanoparticle-E. coli colloidal interaction in water and effect on E. coli survival.

PubMed

Dror-Ehre, A; Mamane, H; Belenkova, T; Markovich, G; Adin, A

2009-11-15

Silver nanoparticles exhibit antibacterial properties via bacterial inactivation and growth inhibition. The mechanism is not yet completely understood. This work was aimed at elucidating the effect of silver nanoparticles on inactivation of Escherichia coli, by studying particle-particle interactions in aqueous suspensions. Stable, molecularly capped, positively or negatively charged silver nanoparticles were mixed at 1 to 60microgmL(-1) with suspended E. coli cells to examine their effect on inactivation of the bacteria. Gold nanoparticles with the same surfactant were used as a control, being of similar size but made up of a presumably inert metal. Log reduction of 5log(10) and complete inactivation were obtained with the silver nanoparticles while the gold nanoparticles did not show any inactivation ability. The effect of molecularly capped nanoparticles on E. coli survival was dependent on particle number. Log reduction of E. coli was associated with the ratio between the number of nanoparticles and the initial bacterial cell count. Electrostatic attraction or repulsion mechanisms in silver nanoparticle-E. coli cell interactions did not contribute to the inactivation process.

Thermal and high pressure inactivation kinetics of blueberry peroxidase.

PubMed

Terefe, Netsanet Shiferaw; Delon, Antoine; Versteeg, Cornelis

2017-10-01

This study for the first time investigated the stability and inactivation kinetics of blueberry peroxidase in model systems (McIlvaine buffer, pH=3.6, the typical pH of blueberry juice) during thermal (40-80°C) and combined high pressure-thermal processing (0.1-690MPa, 30-90°C). At 70-80°C, the thermal inactivation kinetics was best described by a biphasic model with ∼61% labile and ∼39% stable fractions at temperature between 70 and 75°C. High pressure inhibited the inactivation of the enzyme with no inactivation at pressures as high as 690MPa and temperatures less than 50°C. The inactivation kinetics of the enzyme at 60-70°C, and pressures higher than 500MPa was best described by a first order biphasic model with ∼25% labile fraction and 75% stable fraction. The activation energy values at atmospheric pressure were 548.6kJ/mol and 324.5kJ/mol respectively for the stable and the labile fractions. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Effect of water content and temperature on inactivation kinetics of myrosinase in broccoli (Brassica oleracea var. italica).

PubMed

Oliviero, T; Verkerk, R; Van Boekel, M A J S; Dekker, M

2014-11-15

Broccoli belongs to the Brassicaceae plant family consisting of widely eaten vegetables containing high concentrations of glucosinolates. Enzymatic hydrolysis of glucosinolates by endogenous myrosinase (MYR) can form isothiocyanates with health-promoting activities. The effect of water content (WC) and temperature on MYR inactivation in broccoli was investigated. Broccoli was freeze dried obtaining batches with WC between 10% and 90% (aw from 0.10 to 0.96). These samples were incubated for various times at different temperatures (40-70°C) and MYR activity was measured. The initial MYR inactivation rates were estimated by the first-order reaction kinetic model. MYR inactivation rate constants were lower in the driest samples (10% WC) at all studied temperatures. Samples with 67% and 90% WC showed initial inactivation rate constants all in the same order of magnitude. Samples with 31% WC showed intermediate initial inactivation rate constants. These results are useful to optimise the conditions of drying processes to produce dried broccoli with optimal MYR retention for human health. Copyright © 2014 Elsevier Ltd. All rights reserved.

Thermal inactivation of Salmonella enteritidis PT 30 in almond kernels as influenced by water activity.

PubMed

Villa-Rojas, Rossana; Tang, Juming; Wang, Shaojin; Gao, Mengxiang; Kang, Dong-Hyun; Mah, Jae-Hyung; Gray, Peter; Sosa-Morales, Maria Elena; López-Malo, Aurelio

2013-01-01

Salmonellosis outbreaks related to consumption of raw almonds have encouraged the scientific community to study the inactivation kinetics of pathogens in this dry commodity. However, the low moisture content of the product presents a challenge for thermal control, because the time required to achieve the desired thermal inactivation of microorganisms increases sharply with reduced moisture content and water activity. In this study, we explored and modeled the heat inactivation of Salmonella enterica serovar Enteritidis PT 30 in almond cultivar 'Nonpareil' kernel flour at four water activity (a(w)) values (0.601, 0.720, 0.888, and 0.946) using four temperatures for each a(w). The results showed that the inactivation was well fitted by both Weibull distribution (R(2) = 0.93 to 1.00) and first-order kinetics (R(2) = 0.82 to 0.96). At higher a(w) values, the rate of inactivation increased and less time was needed to achieve the required population reduction. These results suggest that, to avoid deterioration of product quality, shorter process times at lower temperatures may be used to achieve desired inactivation levels of Salmonella Enteritidis PT 30 by simply increasing the moisture content of almonds. These goals could be achieved with the use of existing procedures already practiced by the food industry, such as washing or prewetting scalding before heat inactivation.

Enhancing Food Processing by Pulsed and High Voltage Electric Fields: Principles and Applications.

PubMed

Wang, Qijun; Li, Yifei; Sun, Da-Wen; Zhu, Zhiwei

2018-02-02

Improvements in living standards result in a growing demand for food with high quality attributes including freshness, nutrition and safety. However, current industrial processing methods rely on traditional thermal and chemical methods, such as sterilization and solvent extraction, which could induce negative effects on food quality and safety. The electric fields (EFs) involving pulsed electric fields (PEFs) and high voltage electric fields (HVEFs) have been studied and developed for assisting and enhancing various food processes. In this review, the principles and applications of pulsed and high voltage electric fields are described in details for a range of food processes, including microbial inactivation, component extraction, and winemaking, thawing and drying, freezing and enzymatic inactivation. Moreover, the advantages and limitations of electric field related technologies are discussed to foresee future developments in the food industry. This review demonstrates that electric field technology has a great potential to enhance food processing by supplementing or replacing the conventional methods employed in different food manufacturing processes. Successful industrial applications of electric field treatments have been achieved in some areas such as microbial inactivation and extraction. However, investigations of HVEFs are still in an early stage and translating the technology into industrial applications need further research efforts.

Isolation, Culture, and Motility Measurements of Epidermal Melanocytes from GFP-Expressing Reporter Mice.

PubMed

Dagnino, Lina; Crawford, Melissa

2018-03-27

In this article, we provide a method to isolate primary epidermal melanocytes from reporter mice, which also allow targeted gene inactivation. The mice from which these cells are isolated are bred into a Rosa26 mT/mG reporter background, which results in GFP expression in the targeted melanocytic cell population. These cells are isolated and cultured to >95% purity. The cells can be used for gene expression studies, clonogenic experiments, and biological assays, such as capacity for migration. Melanocytes are slow moving cells, and we also provide a method to measure motility using individual cell tracking and data analysis.

Treatment alternatives of slaughterhouse wastes, and their effect on the inactivation of different pathogens: a review.

PubMed

Franke-Whittle, Ingrid H; Insam, Heribert

2013-05-01

Slaughterhouse wastes are a potential reservoir of bacterial, viral, prion and parasitic pathogens, capable of infecting both animals and humans. A quick, cost effective and safe disposal method is thus essential in order to reduce the risk of disease following animal slaughter. Different methods for the disposal of such wastes exist, including composting, anaerobic digestion (AD), alkaline hydrolysis (AH), rendering, incineration and burning. Composting is a disposal method that allows a recycling of the slaughterhouse waste nutrients back into the earth. The high fat and protein content of slaughterhouse wastes mean however, that such wastes are an excellent substrate for AD processes, resulting in both the disposal of wastes, a recycling of nutrients (soil amendment with sludge), and in methane production. Concerns exist as to whether AD and composting processes can inactivate pathogens. In contrast, AH is capable of the inactivation of almost all known microorganisms. This review was conducted in order to compare three different methods of slaughterhouse waste disposal, as regards to their ability to inactivate various microbial pathogens. The intention was to investigate whether AD could be used for waste disposal (either alone, or in combination with another process) such that both energy can be obtained and potentially hazardous materials be disposed of.

Treatment alternatives of slaughterhouse wastes, and their effect on the inactivation of different pathogens: A review

PubMed Central

2013-01-01

Slaughterhouse wastes are a potential reservoir of bacterial, viral, prion and parasitic pathogens, capable of infecting both animals and humans. A quick, cost effective and safe disposal method is thus essential in order to reduce the risk of disease following animal slaughter. Different methods for the disposal of such wastes exist, including composting, anaerobic digestion (AD), alkaline hydrolysis (AH), rendering, incineration and burning. Composting is a disposal method that allows a recycling of the slaughterhouse waste nutrients back into the earth. The high fat and protein content of slaughterhouse wastes mean however, that such wastes are an excellent substrate for AD processes, resulting in both the disposal of wastes, a recycling of nutrients (soil amendment with sludge), and in methane production. Concerns exist as to whether AD and composting processes can inactivate pathogens. In contrast, AH is capable of the inactivation of almost all known microorganisms. This review was conducted in order to compare three different methods of slaughterhouse waste disposal, as regards to their ability to inactivate various microbial pathogens. The intention was to investigate whether AD could be used for waste disposal (either alone, or in combination with another process) such that both energy can be obtained and potentially hazardous materials be disposed of. PMID:22694189

Sequential and Simultaneous Applications of UV and Chlorine for Adenovirus Inactivation.

PubMed

Rattanakul, Surapong; Oguma, Kumiko; Takizawa, Satoshi

2015-09-01

Adenoviruses are water-borne human pathogens with high resistance to UV disinfection. Combination of UV treatment and chlorination could be an effective approach to deal with adenoviruses. In this study, human adenovirus 5 (HAdV-5) was challenged in a bench-scale experiment by separate applications of UV or chlorine and by combined applications of UV and chlorine in either a sequential or simultaneous manner. The treated samples were then propagated in human lung carcinoma epithelial cells to quantify the log inactivation of HAdV-5. When the processes were separate, a fluence of 100 mJ/cm(2) and a CT value of 0.02 mg min/L were required to achieve 2 log inactivation of HAdV-5 by UV disinfection and chlorination, respectively. Interestingly, synergistic effects on the HAdV-5 inactivation rates were found in the sequential process of chlorine followed by UV (Cl2-UV) (p < 0.05, ANCOVA) in comparison to the separate processes or the simultaneous application of UV/Cl2. This implies that a pretreatment with chlorine may increase the sensitivity of the virus to the subsequent UV disinfection. In conclusion, this study suggests that the combined application of UV and chlorine could be an effective measure against adenoviruses as a multi-barrier approach in water disinfection.

Inactivation of HAV and norovirus surrogates within raw shellfish and other foods

USDA-ARS?s Scientific Manuscript database

High pressure processing can inactivate hepatitis A virus, (HAV) and the human norovirus surrogates, feline calicivirus (FCV) and murine norovirus (MNV), in foods such as oysters, strawberries, and green onions. A 5-min 400-Megapascals (MPa) treatment at 5 degrees C and a 1–min 400-MPa treatment at ...

Temperature effects for high pressure processing of Picornaviruses

USDA-ARS?s Scientific Manuscript database

Investigation of the effects of pre-pressurization temperature on the high pressure inactivation for single strains of aichivirus (AiV), coxsackievirus A9 (CAV9) and B5 (CBV5) viruses, as well as human parechovirus -1 (HPeV), was performed. For CAV9, an average 1.99 log10 greater inactivation was ...

High pressure treatment of human norovirus virus-like particles: factors affecting destruction efficacy

USDA-ARS?s Scientific Manuscript database

Human norovirus (NoV) accounts for more than 90% of nonbacterial gastroenteritis. To date, the efficacy of human NoV inactivation interventions cannot be accurately evaluated because the virus is nonculturable. In this study, we aimed to estimate inactivation of human NoV by high pressure processing...

Viologen-modified electrodes for protection of hydrogenases from high potential inactivation while performing H2 oxidation at low overpotential.

PubMed

Oughli, Alaa A; Vélez, Marisela; Birrell, James A; Schuhmann, Wolfgang; Lubitz, Wolfgang; Plumeré, Nicolas; Rüdiger, Olaf

2018-06-08

In this work we present a viologen-modified electrode providing protection for hydrogenases against high potential inactivation. Hydrogenases, including O2-tolerant classes, suffer from reversible inactivation upon applying high potentials, which limits their use in biofuel cells to certain conditions. Our previously reported protection strategy based on the integration of hydrogenase into redox matrices enabled the use of these biocatalysts in biofuel cells even under anode limiting conditions. However, mediated catalysis required application of an overpotential to drive the reaction, and this translates into a power loss in a biofuel cell. In the present work, the enzyme is adsorbed on top of a covalently-attached viologen layer which leads to mixed, direct and mediated, electron transfer processes; at low overpotentials, the direct electron transfer process generates a catalytic current, while the mediated electron transfer through the viologens at higher potentials generates a redox buffer that prevents oxidative inactivation of the enzyme. Consequently, the enzyme starts the catalysis at no overpotential with viologen self-activated protection at high potentials.

Epac-induced ryanodine receptor type 2 activation inhibits sodium currents in atrial and ventricular murine cardiomyocytes.

PubMed

Valli, Haseeb; Ahmad, Shiraz; Sriharan, Sujan; Dean, Lydia D; Grace, Andrew A; Jeevaratnam, Kamalan; Matthews, Hugh R; Huang, Christopher L-H

2018-03-01

Acute RyR2 activation by exchange protein directly activated by cAMP (Epac) reversibly perturbs myocyte Ca 2+ homeostasis, slows myocardial action potential conduction, and exerts pro-arrhythmic effects. Loose patch-clamp studies, preserving in vivo extracellular and intracellular conditions, investigated Na + current in intact cardiomyocytes in murine atrial and ventricular preparations following Epac activation. Depolarising steps to varying test voltages activated typical voltage-dependent Na + currents. Plots of peak current against depolarisation from resting potential gave pretreatment maximum atrial and ventricular currents of -20.23 ± 1.48 (17) and -29.8 ± 2.4 (10) pA/μm 2 (mean ± SEM [n]). Challenge by 8-CPT (1 μmol/L) reduced these currents to -11.21 ± 0.91 (12) (P < .004) and -19.3 ± 1.6 (11) pA/μm 2 (P < .04) respectively. Currents following further addition of the RyR2 inhibitor dantrolene (10 μmol/L) (-19.91 ± 2.84 (13) and -26.6 ± 1.7 (17)), and dantrolene whether alone (-19.53 ± 1.97 (8) and -27.6 ± 1.9 (14)) or combined with 8-CPT (-19.93 ± 2.59 (12) and -29.9 ± 2.5(11)), were indistinguishable from pretreatment values (all P > .05). Assessment of the inactivation that followed by applying subsequent steps to a fixed voltage 100 mV positive to resting potential gave concordant results. Half-maximal inactivation voltages and steepness factors, and time constants for Na + current recovery from inactivation in double-pulse experiments, were similar through all the pharmacological conditions. Intracellular sharp microelectrode membrane potential recordings in intact Langendorff-perfused preparations demonstrated concordant variations in maximum rates of atrial and ventricular action potential upstroke, (dV/dt) max . We thus demonstrate an acute, reversible, Na + channel inhibition offering a possible mechanism for previously reported pro-arrhythmic slowing of AP propagation following modifications of Ca 2+ homeostasis, complementing earlier findings from chronic alterations in Ca 2+ homeostasis in genetically-modified RyR2-P2328S hearts. © 2017 The Authors. Clinical and Experimental Pharmacology and Physiology Published by John Wiley & Sons Australia, Ltd.

Physiological and ultrastructural characterisation of a desiccation-tolerant filmy fern, Hymenophyllum caudiculatum: Influence of translational regulation and ABA on recovery.

PubMed

Garcés, M; Ulloa, M; Miranda, A; Bravo, L A

2018-03-01

The filmy fern Hymenophyllum caudiculatum can lose 60% of its relative water content, remain dry for some time and recover 88% of photochemical efficiency after 30 min of rehydration. Little is known about the protective strategies and regulation of the cellular rehydration process in this filmy fern species. The aim of this study was to characterise the filmy fern ultrastructure during a desiccation-rehydration cycle, and measure the physiological effects of transcription/translation inhibitors and ABA during desiccation recovery. Confocal and transmission electron microscopy were used to compare changes in structure during fast or slow desiccation. Transcription (actinomycin D) and translation (cycloheximide) inhibitors and ABA were used to compare photochemical efficiency during desiccation recovery. Cell structure was conserved during slow desiccation and rehydration, constitutive properties of the cell wall, allowing invagination and folding of the membranes and an important change in chloroplast size. The use of a translational inhibitor impeded recovery of photochemical efficiency during the first 80 min of rehydration, but the transcriptional inhibitor had no effect. Exogenous ABA delayed photochemical inactivation, and endogenous ABA levels decreased during desiccation and rehydration. Frond curling and chloroplast movements are possible strategies to avoid photodamage. Constitutive membrane plasticity and rapid cellular repair can be adaptations evolved to tolerate a rapid recovery during rehydration. Further research is required to explore the importance of existing mRNAs during the first minutes of recovery, and ABA function during desiccation of H. caudiculatum. © 2017 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.

[Development and validation of an analytical method to quantify residues of cleaning products able to inactivate prion].

PubMed

Briot, T; Robelet, A; Morin, N; Riou, J; Lelièvre, B; Lebelle-Dehaut, A-V

2016-07-01

In this study, a novel analytical method to quantify prion inactivating detergent in rinsing waters coming from the washer-disinfector of a hospital sterilization unit has been developed. The final aim was to obtain an easy and functional method in a routine hospital process which does not need the cleaning product manufacturer services. An ICP-MS method based on the potassium dosage of the washer-disinfector's rinsing waters was developed. Potassium hydroxide is present on the composition of the three prion inactivating detergent currently on the French market. The detergent used in this study was the Actanios LDI(®) (Anios laboratories). A Passing and Bablok regression compares concentrations measured with this developed method and with the HPLC-UV manufacturer method. According to results obtained, the developed method is easy to use in a routine hospital process. The Passing and Bablok regression showed that there is no statistical difference between the two analytical methods during the second rinsing step. Besides, both methods were linear on the third rinsing step, with a 1.5ppm difference between the concentrations measured for each method. This study shows that the ICP-MS method developed is nonspecific for the detergent, but specific for the potassium element which is present in all prion inactivating detergent currently on the French market. This method should be functional for all the prion inactivating detergent containing potassium, if the sensibility of the method is sufficient when the potassium concentration is very low in the prion inactivating detergent formulation. Copyright © 2016. Published by Elsevier Masson SAS.

Randomized, double-blinded clinical trial for human norovirus inactivation in oysters by high hydrostatic pressure processing.

PubMed

Leon, Juan S; Kingsley, David H; Montes, Julia S; Richards, Gary P; Lyon, G Marshall; Abdulhafid, Gwen M; Seitz, Scot R; Fernandez, Marina L; Teunis, Peter F; Flick, George J; Moe, Christine L

2011-08-01

Contamination of oysters with human noroviruses (HuNoV) constitutes a human health risk and may lead to severe economic losses in the shellfish industry. There is a need to identify a technology that can inactivate HuNoV in oysters. In this study, we conducted a randomized, double-blinded clinical trial to assess the effect of high hydrostatic pressure processing (HPP) on Norwalk virus (HuNoV genogroup I.1) inactivation in virus-seeded oysters ingested by subjects. Forty-four healthy, positive-secretor adults were divided into three study phases. Subjects in each phase were randomized into control and intervention groups. Subjects received Norwalk virus (8FIIb, 1.0 × 10(4) genomic equivalent copies) in artificially seeded oysters with or without HPP treatment (400 MPa at 25°C, 600 MPa at 6°C, or 400 MPa at 6°C for 5 min). HPP at 600 MPa, but not 400 MPa (at 6° or 25°C), completely inactivated HuNoV in seeded oysters and resulted in no HuNoV infection among these subjects, as determined by reverse transcription-PCR detection of HuNoV RNA in subjects' stool or vomitus samples. Interestingly, a white blood cell (granulocyte) shift was identified in 92% of the infected subjects and was significantly associated with infection (P = 0.0014). In summary, these data suggest that HPP is effective at inactivating HuNoV in contaminated whole oysters and suggest a potential intervention to inactivate infectious HuNoV in oysters for the commercial shellfish industry.

Effective inactivation of a wide range of viruses by pasteurization.

PubMed

Gröner, Albrecht; Broumis, Connie; Fang, Randel; Nowak, Thomas; Popp, Birgit; Schäfer, Wolfram; Roth, Nathan J

2018-01-01

Careful selection and testing of plasma reduces the risk of blood-borne viruses in the starting material for plasma-derived products. Furthermore, effective measures such as pasteurization at 60°C for 10 hours have been implemented in the manufacturing process of therapeutic plasma proteins such as human albumin, coagulation factors, immunoglobulins, and enzyme inhibitors to inactivate blood-borne viruses of concern. A comprehensive compilation of the virus reduction capacity of pasteurization is presented including the effect of stabilizers used to protect the therapeutic protein from modifications during heat treatment. The virus inactivation kinetics of pasteurization for a broad range of viruses were evaluated in the relevant intermediates from more than 15 different plasma manufacturing processes. Studies were carried out under the routine manufacturing target variables, such as temperature and product-specific stabilizer composition. Additional studies were also performed under robustness conditions, that is, outside production specifications. The data demonstrate that pasteurization inactivates a wide range of enveloped and nonenveloped viruses of diverse physicochemical characteristics. After a maximum of 6 hours' incubation, no residual infectivity could be detected for the majority of enveloped viruses. Effective inactivation of a range of nonenveloped viruses, with the exception of nonhuman parvoviruses, was documented. Pasteurization is a very robust and reliable virus inactivation method with a broad effectiveness against known blood-borne pathogens and emerging or potentially emerging viruses. Pasteurization has proven itself to be a highly effective step, in combination with other complementary safety measures, toward assuring the virus safety of final product. © 2017 The Authors Transfusion published by Wiley Periodicals, Inc. on behalf of AABB.

Functional imaging reveals rapid reorganization of cortical activity after parietal inactivation in monkeys

PubMed Central

Wilke, Melanie; Kagan, Igor; Andersen, Richard A.

2012-01-01

Impairments of spatial awareness and decision making occur frequently as a consequence of parietal lesions. Here we used event-related functional MRI (fMRI) in monkeys to investigate rapid reorganization of spatial networks during reversible pharmacological inactivation of the lateral intraparietal area (LIP), which plays a role in the selection of eye movement targets. We measured fMRI activity in control and inactivation sessions while monkeys performed memory saccades to either instructed or autonomously chosen spatial locations. Inactivation caused a reduction of contralesional choices. Inactivation effects on fMRI activity were anatomically and functionally specific and mainly consisted of: (i) activity reduction in the upper bank of the superior temporal sulcus (temporal parietal occipital area) for single contralesional targets, especially in the inactivated hemisphere; and (ii) activity increase accompanying contralesional choices between bilateral targets in several frontal and parieto-temporal areas in both hemispheres. There was no overactivation for ipsilesional targets or choices in the intact hemisphere. Task-specific effects of LIP inactivation on blood oxygen level-dependent activity in the temporal parietal occipital area underline the importance of the superior temporal sulcus for spatial processing. Furthermore, our results agree only partially with the influential interhemispheric competition model of spatial neglect and suggest an additional component of interhemispheric cooperation in the compensation of neglect deficits. PMID:22562793

Mechanism-based inactivation of human cytochrome P450 enzymes: strategies for diagnosis and drug-drug interaction risk assessment.

PubMed

Venkatakrishnan, K; Obach, R S; Rostami-Hodjegan, A

2007-01-01

Among drugs that cause pharmacokinetic drug-drug interactions, mechanism-based inactivators of cytochrome P450 represent several of those agents that cause interactions of the greatest magnitude. In vitro inactivation kinetic data can be used to predict the potential for new drugs to cause drug interactions in the clinic. However, several factors exist, each with its own uncertainty, that must be taken into account in order to predict the magnitude of interactions reliably. These include aspects of in vitro experimental design, an understanding of relevant in vivo concentrations of the inactivator, and the extent to which the inactivated enzyme is involved in the clearance of the affected drug. Additionally, the rate of enzyme degradation in vivo is also an important factor that needs to be considered in the prediction of the drug interaction magnitudes. To address mechanism-based inactivation for new drugs, various in vitro experimental approaches have been employed. The selection of approaches for in vitro kinetic characterization of inactivation as well as in vitro-in vivo extrapolation should be guided by the purpose of the exercise and the stage of drug discovery and development, with an increase in the level of sophistication throughout the research and development process.

Mobile genetic element proliferation and gene inactivation impact over the genome structure and metabolic capabilities of Sodalis glossinidius, the secondary endosymbiont of tsetse flies

PubMed Central

2010-01-01

Background Genome reduction is a common evolutionary process in symbiotic and pathogenic bacteria. This process has been extensively characterized in bacterial endosymbionts of insects, where primary mutualistic bacteria represent the most extreme cases of genome reduction consequence of a massive process of gene inactivation and loss during their evolution from free-living ancestors. Sodalis glossinidius, the secondary endosymbiont of tsetse flies, contains one of the few complete genomes of bacteria at the very beginning of the symbiotic association, allowing to evaluate the relative impact of mobile genetic element proliferation and gene inactivation over the structure and functional capabilities of this bacterial endosymbiont during the transition to a host dependent lifestyle. Results A detailed characterization of mobile genetic elements and pseudogenes reveals a massive presence of different types of prophage elements together with five different families of IS elements that have proliferated across the genome of Sodalis glossinidius at different levels. In addition, a detailed survey of intergenic regions allowed the characterization of 1501 pseudogenes, a much higher number than the 972 pseudogenes described in the original annotation. Pseudogene structure reveals a minor impact of mobile genetic element proliferation in the process of gene inactivation, with most of pseudogenes originated by multiple frameshift mutations and premature stop codons. The comparison of metabolic profiles of Sodalis glossinidius and tsetse fly primary endosymbiont Wiglesworthia glossinidia based on their whole gene and pseudogene repertoires revealed a novel case of pathway inactivation, the arginine biosynthesis, in Sodalis glossinidius together with a possible case of metabolic complementation with Wigglesworthia glossinidia for thiamine biosynthesis. Conclusions The complete re-analysis of the genome sequence of Sodalis glossinidius reveals novel insights in the evolutionary transition from a free-living ancestor to a host-dependent lifestyle, with a massive proliferation of mobile genetic elements mainly of phage origin although with minor impact in the process of gene inactivation that is taking place in this bacterial genome. The metabolic analysis of the whole endosymbiotic consortia of tsetse flies have revealed a possible phenomenon of metabolic complementation between primary and secondary endosymbionts that can contribute to explain the co-existence of both bacterial endosymbionts in the context of the tsetse host. PMID:20649993

Nonthermal pasteurization of beer by high pressure processing: modelling the inactivation of saccharomyces cerevisiae ascospores in different alcohol beers

NASA Astrophysics Data System (ADS)

Milani, Elham A.; Silva, Filipa V. M.

2016-10-01

The industrial production of beer ends with a process of thermal pasteurization. In this research, the nonthermal pasteurization of beer by high pressure processing (HPP) was carried out. First, the effect of alcohol content on Saccharomyces cerevisiae ascospore inactivation at 400 MPa was studied. The number of ascospores in 0.0%, 4.8%, and 7.0% alc/vol beers for 10 min processing time decreased by 3.1, 4.9, and ≥ 6.0 log, respectively. The Weibull model fitted the ascospore inactivation by HPP in 0.0%, 4.8%, and 7.0% alc/vol beers. At 400 MPa, 7.2 s could ensure the minimum pasteurization of beers and for 600 MPa 5 s were enough for ≥ 7 log reductions. The overall flavour of HPP vs. untreated beers was evaluated for a lager and an ale, with no significant differences between the untreated and HPP beers. Thus, nonthermal HPP is a feasible technology to pasteurize beer with different alcohol contents without heat.

Sterilization by oxygen plasma

NASA Astrophysics Data System (ADS)

Moreira, Adir José; Mansano, Ronaldo Domingues; Andreoli Pinto, Terezinha de Jesus; Ruas, Ronaldo; Zambon, Luis da Silva; da Silva, Mônica Valero; Verdonck, Patrick Bernard

2004-07-01

The use of polymeric medical devices has stimulated the development of new sterilization methods. The traditional techniques rely on ethylene oxide, but there are many questions concerning the carcinogenic properties of the ethylene oxide residues adsorbed on the materials after processing. Another common technique is the gamma irradiation process, but it is costly, its safe operation requires an isolated site and it also affects the bulk properties of the polymers. The use of a gas plasma is an elegant alternative sterilization technique. The plasma promotes an efficient inactivation of the micro-organisms, minimises the damage to the materials and presents very little danger for personnel and the environment. Pure oxygen reactive ion etching type of plasmas were applied to inactivate a biologic indicator, the Bacillus stearothermophilus, to confirm the efficiency of this process. The sterilization processes took a short time, in a few minutes the mortality was complete. In situ analysis of the micro-organisms' inactivating time was possible using emission spectrophotometry. The increase in the intensity of the 777.5 nm oxygen line shows the end of the oxidation of the biologic materials. The results were also observed and corroborated by scanning electron microscopy.

Slow diffusion by Markov random flights

NASA Astrophysics Data System (ADS)

Kolesnik, Alexander D.

2018-06-01

We present a conception of the slow diffusion processes in the Euclidean spaces Rm , m ≥ 1, based on the theory of random flights with small constant speed that are driven by a homogeneous Poisson process of small rate. The slow diffusion condition that, on long time intervals, leads to the stationary distributions, is given. The stationary distributions of slow diffusion processes in some Euclidean spaces of low dimensions, are presented.

Choosing the Active X: The Human Version of X Inactivation.

PubMed

Migeon, Barbara R

2017-12-01

Humans and rodents differ in how they carry out X inactivation (XI), the mammalian method to compensate for the different number of X chromosomes in males and females. Evolutionary changes in staging embryogenesis and in mutations within the XI center alter the process among mammals. The mouse model of XI is predicated on X counting and subsequently choosing the X to 'inactivate'. However, new evidence suggests that humans initiate XI by protecting one X in both sexes from inactivation by XIST, the noncoding RNA that silences the inactive X. This opinion article explores the question of how the active X is protected from silencing by its own Xist locus, and the possibility of different solutions for mouse and human. Copyright © 2017 Elsevier Ltd. All rights reserved.

The effect of processing on veterinary residues in foods.

PubMed

Moats, W A

1999-01-01

Heat stability of antibiotics in foods to cooking has been determined by a variety of methods. These include heating in such liquid media as milk, water, buffers and meat extracts, and in solids such as buffered meat homogenates and various sausages. Inactivation of incurred residues in tissues and eggs was also studied. Time and temperature of heating were more easily controlled in liquid media, but results in actual meat products are more indicative of actual cooking processes. Ordinary cooking procedures for meat, even to "well-done", cannot be relied on to inactivate even the more heat sensitive compounds such as penicillins and tetracyclines. More severe heating as for canning or prolonged cooking with moist heat can inactivate the more heat sensitive compounds. The relevance to food safety is uncertain since the nature of the degradation products is unknown in most cases.

One-step global parameter estimation of kinetic inactivation parameters for Bacillus sporothermodurans spores under static and dynamic thermal processes.

PubMed

Cattani, F; Dolan, K D; Oliveira, S D; Mishra, D K; Ferreira, C A S; Periago, P M; Aznar, A; Fernandez, P S; Valdramidis, V P

2016-11-01

Bacillus sporothermodurans produces highly heat-resistant endospores, that can survive under ultra-high temperature. High heat-resistant sporeforming bacteria are one of the main causes for spoilage and safety of low-acid foods. They can be used as indicators or surrogates to establish the minimum requirements for heat processes, but it is necessary to understand their thermal inactivation kinetics. The aim of the present work was to study the inactivation kinetics under both static and dynamic conditions in a vegetable soup. Ordinary least squares one-step regression and sequential procedures were applied for estimating these parameters. Results showed that multiple dynamic heating profiles, when analyzed simultaneously, can be used to accurately estimate the kinetic parameters while significantly reducing estimation errors and data collection. Copyright © 2016 Elsevier Ltd. All rights reserved.

Experimental and numerical study of heterogeneous pressure-temperature-induced lethal and sublethal injury of Lactococcus lactis in a medium scale high-pressure autoclave.

PubMed

Kilimann, K V; Kitsubun, P; Delgado, A; Gänzle, M G; Chapleau, N; Le Bail, A; Hartmann, C

2006-07-05

The present contribution is dedicated to experimental and theoretical assessment of microbiological process heterogeneities of the high-pressure (HP) inactivation of Lactococcus lactis ssp. cremoris MG 1363. The inactivation kinetics are determined in dependence of pressure, process time, temperature and absence or presence of co-solutes in the buffer system namely 4 M sodium chloride and 1.5 M sucrose. The kinetic analysis is carried out in a 0.1-L autoclave in order to minimise thermal and convective effects. Upon these data, a deterministic inactivation model is formulated with the logistic equation. Its independent variables represent the counts of viable cells (viable but injured) and of the stress-resistant cells (viable and not injured). This model is then coupled to a thermo-fluiddynamical simulation method, high-pressure computer fluid dynamics technique (HP-CFD), which yields spatiotemporal temperature and flow fields occurring during the HP application inside any considered autoclave. Besides the thermo-fluiddynamic quantities, the coupled model predicts also the spatiotemporal distribution of both viable (VC) and stress-resistant cell counts (SRC). In order to assess the process non-uniformity of the microbial inactivation in a 3.3-L autoclave experimentally, microbial samples are placed at two distinct locations and are exposed to various process conditions. It can be shown with both, experimental and theoretical models that thermal heterogeneities induce process non-uniformities of more than one decimal power in the counts of the viable cells at the end of the treatment. (c) 2006 Wiley Periodicals, Inc.

Efficacy of low-temperature high hydrostatic pressure processing in inactivating Vibrio parahaemolyticus in culture suspension and oyster homogenate.

PubMed

Phuvasate, Sureerat; Su, Yi-Cheng

2015-03-02

Culture suspensions of five clinical and five environmental Vibrio parahaemolyticus strains in 2% NaCl solution were subjected to high pressure processing (HPP) under various conditions (200-300MPa for 5 and 10 min at 1.5-20°C) to study differences in pressure resistance among the strains. The most pressure-resistant and pressure-sensitive strains were selected to investigate the effects of low temperatures (15, 5 and 1.5°C) on HPP (200 or 250MPa for 5 min) to inactivate V. parahaemolyticus in sterile oyster homogenates. Inactivation of V. parahaemolyticus cells in culture suspensions and oyster homogenates was greatly enhanced by lowering the processing temperature from 15 to 5 or 1.5°C. A treatment of oyster homogenates at 250MPa for 5 min at 5°C decreased the populations of V. parahaemolyticus by 6.2logCFU/g for strains 10290 and 100311Y11 and by >7.4logCFU/g for strain 10292. Decreasing the processing temperature of the same treatment to 1.5°C reduced all the V. parahaemolyticus strains inoculated to oyster homogenates to non-detectable (<10CFU/g) levels. Factors including pressure level, processing temperature and time all need to be considered for developing effective HPP for eliminating pathogens from foods. Further studies are needed to validate the efficacy of the HPP (250MPa for 5 min at 1.5°C) in inactivating V. parahaemolyticus cells in whole oysters. Copyright © 2014 Elsevier B.V. All rights reserved.

Inactivation of pathogenic bacteria inoculated onto a Bacto™ agar model surface using TiO2-UVC photocatalysis, UVC and chlorine treatments.

PubMed

Yoo, S; Ghafoor, K; Kim, S; Sun, Y W; Kim, J U; Yang, K; Lee, D-U; Shahbaz, H M; Park, J

2015-09-01

The aim of this study was to study inactivation of different pathogenic bacteria on agar model surface using TiO2-UV photocatalysis (TUVP). A unified food surface model was simulated using Bacto(™) agar, a routinely used microbial medium. The foodborne pathogenic bacteria Escherichia coli K12 (as a surrogate for E. coli O157:H7), Salmonella Typhimurium, Staphylococcus aureus and Listeria monocytogenes were inoculated onto the agar surface, followed by investigation of TUVP-assisted inactivation and morphological changes in bacterial cells. The TUVP process showed higher bacterial inactivation, particularly for Gram-negative bacteria, than UVC alone and a control (dark reaction). A TUVP treatment of 17·2 mW cm(-2) (30% lower than the UVC light intensity) reduced the microbial load on the agar surface by 4·5-6·0 log CFU cm(-2). UVC treatment of 23·7 mW cm(-2) caused 3·0-5·3 log CFU cm(-2) reduction. The use of agar model surface is effective for investigation of bacterial disinfection and TUVP is a promising nonthermal technique. The results showing effects of photocatalysis and other treatments for inactivation of bacterial pathogens on model surface can be useful for applying such processes for disinfection of fruit, vegetables and other similar surfaces. © 2015 The Society for Applied Microbiology.

Disruption of the IS6-AID linker affects voltage-gated calcium channel inactivation and facilitation.

PubMed

Findeisen, Felix; Minor, Daniel L

2009-03-01

Two processes dominate voltage-gated calcium channel (Ca(V)) inactivation: voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). The Ca(V)beta/Ca(V)alpha(1)-I-II loop and Ca(2+)/calmodulin (CaM)/Ca(V)alpha(1)-C-terminal tail complexes have been shown to modulate each, respectively. Nevertheless, how each complex couples to the pore and whether each affects inactivation independently have remained unresolved. Here, we demonstrate that the IS6-alpha-interaction domain (AID) linker provides a rigid connection between the pore and Ca(V)beta/I-II loop complex by showing that IS6-AID linker polyglycine mutations accelerate Ca(V)1.2 (L-type) and Ca(V)2.1 (P/Q-type) VDI. Remarkably, mutations that either break the rigid IS6-AID linker connection or disrupt Ca(V)beta/I-II association sharply decelerate CDI and reduce a second Ca(2+)/CaM/Ca(V)alpha(1)-C-terminal-mediated process known as calcium-dependent facilitation. Collectively, the data strongly suggest that components traditionally associated solely with VDI, Ca(V)beta and the IS6-AID linker, are essential for calcium-dependent modulation, and that both Ca(V)beta-dependent and CaM-dependent components couple to the pore by a common mechanism requiring Ca(V)beta and an intact IS6-AID linker.

Disruption of the IS6-AID Linker Affects Voltage-gated Calcium Channel Inactivation and Facilitation

PubMed Central

Findeisen, Felix

2009-01-01

Two processes dominate voltage-gated calcium channel (CaV) inactivation: voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). The CaVβ/CaVα1-I-II loop and Ca2+/calmodulin (CaM)/CaVα1–C-terminal tail complexes have been shown to modulate each, respectively. Nevertheless, how each complex couples to the pore and whether each affects inactivation independently have remained unresolved. Here, we demonstrate that the IS6–α-interaction domain (AID) linker provides a rigid connection between the pore and CaVβ/I-II loop complex by showing that IS6-AID linker polyglycine mutations accelerate CaV1.2 (L-type) and CaV2.1 (P/Q-type) VDI. Remarkably, mutations that either break the rigid IS6-AID linker connection or disrupt CaVβ/I-II association sharply decelerate CDI and reduce a second Ca2+/CaM/CaVα1–C-terminal–mediated process known as calcium-dependent facilitation. Collectively, the data strongly suggest that components traditionally associated solely with VDI, CaVβ and the IS6-AID linker, are essential for calcium-dependent modulation, and that both CaVβ-dependent and CaM-dependent components couple to the pore by a common mechanism requiring CaVβ and an intact IS6-AID linker. PMID:19237593

When the Lyon(ized chromosome) roars: ongoing expression from an inactive X chromosome.

PubMed

Carrel, Laura; Brown, Carolyn J

2017-11-05

A tribute to Mary Lyon was held in October 2016. Many remarked about Lyon's foresight regarding many intricacies of the X-chromosome inactivation process. One such example is that a year after her original 1961 hypothesis she proposed that genes with Y homologues should escape from X inactivation to achieve dosage compensation between males and females. Fifty-five years later we have learned many details about these escapees that we attempt to summarize in this review, with a particular focus on recent findings. We now know that escapees are not rare, particularly on the human X, and that most lack functionally equivalent Y homologues, leading to their increasingly recognized role in sexually dimorphic traits. Newer sequencing technologies have expanded profiling of primary tissues that will better enable connections to sex-biased disorders as well as provide additional insights into the X-inactivation process. Chromosome organization, nuclear location and chromatin environments distinguish escapees from other X-inactivated genes. Nevertheless, several big questions remain, including what dictates their distinct epigenetic environment, the underlying basis of species differences in escapee regulation, how different classes of escapees are distinguished, and the roles that local sequences and chromosome ultrastructure play in escapee regulation.This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'. © 2017 The Author(s).

Effective Thermal Inactivation of the Spores of Bacillus cereus Biofilms Using Microwave.

PubMed

Park, Hyong Seok; Yang, Jungwoo; Choi, Hee Jung; Kim, Kyoung Heon

2017-07-28

Microwave sterilization was performed to inactivate the spores of biofilms of Bacillus cereus involved in foodborne illness. The sterilization conditions, such as the amount of water and the operating temperature and treatment time, were optimized using statistical analysis based on 15 runs of experimental results designed by the Box-Behnken method. Statistical analysis showed that the optimal conditions for the inactivation of B. cereus biofilms were 14 ml of water, 108°C of temperature, and 15 min of treatment time. Interestingly, response surface plots showed that the amount of water is the most important factor for microwave sterilization under the present conditions. Complete inactivation by microwaves was achieved in 5 min, and the inactivation efficiency by microwave was obviously higher than that by conventional steam autoclave. Finally, confocal laser scanning microscopy images showed that the principal effect of microwave treatment was cell membrane disruption. Thus, this study can contribute to the development of a process to control food-associated pathogens.

Combined pressure-thermal inactivation kinetics of Bacillus amyloliquefaciens spores in egg patty mince.

PubMed

Rajan, S; Ahn, J; Balasubramaniam, V M; Yousef, A E

2006-04-01

Bacillus amyloliquefaciens is a potential surrogate for Clostridium botulinum in validation studies involving bacterial spore inactivation by pressure-assisted thermal processing. Spores of B. amyloliquefaciens Fad 82 were inoculated into egg patty mince (approximately 1.4 x 10(8) spores per g), and the product was treated with combinations of pressure (0.1 to 700 MPa) and heat (95 to 121 degrees C) in a custom-made high-pressure kinetic tester. The values for the inactivation kinetic parameter (D), temperature coefficient (zT), and pressure coefficient (zP) were determined with a linear model. Inactivation parameters from the nonlinear Weibull model also were estimated. An increase in process pressure decreased the D-value at 95, 105, and 110 degrees C; however, at 121 degrees C the contribution of pressure to spore lethality was less pronounced. The zP-value increased from 170 MPa at 95 degrees C to 332 MPa at 121 degrees C, suggesting that B. amyloliquefaciens spores became less sensitive to pressure changes at higher temperatures. Similarly, the zT-value increased from 8.2 degrees C at 0.1 MPa to 26.8 degrees C at 700 MPa, indicating that at elevated pressures, the spores were less sensitive to changes in temperature. The nonlinear Weibull model parameter b increased with increasing pressure or temperature and was inversely related to the D-value. Pressure-assisted thermal processing is a potential alternative to thermal processing for producing shelf-stable egg products.

Combination of endolysins and high pressure to inactivate Listeria monocytogenes.

PubMed

van Nassau, Tomas J; Lenz, Christian A; Scherzinger, Anna S; Vogel, Rudi F

2017-12-01

Outbreaks of listeriosis are often related to the consumption of low-processed ready-to-eat food products (e.g. soft cheeses or smoked fish) contaminated with Listeria monocytogenes. Traditional preservation techniques, such as heat treatment, cannot eliminate Listeria from these products without strongly affecting the quality of the foods. We therefore investigated the use of endolysin (PlyP40, Ply511, or PlyP825) in combination with high hydrostatic pressure processing to kill L. monocytogenes in buffer. The results demonstrated a more than additive effect when both treatments were combined. For example, whereas 0.16 μg/mL PlyP825 or 300 MPa (1 min, 30 °C) applied individually reduced the cell count by 0.2 and 0.3 log cfu, respectively, a combined treatment resulted in a reduction of 5.5 log cfu. Similar results were obtained for the other endolysins combined with high pressure processing. We also showed that the synergistic inactivation of cells by endolysin and HHP is possible at a pressure level of only 200 MPa (2 min, 30 °C). Thus, the application of endolysins did not only substantially increase the bactericidal effect of high pressure, but it also enabled the inactivation of bacterial cells at much lower pressure levels. This shows the potential of using such combined processes for the inactivation of L. monocytogenes and food preservation. Copyright © 2017 Elsevier Ltd. All rights reserved.

A review of cyanobacteria and cyanotoxins removal/inactivation in drinking water treatment.

PubMed

Westrick, Judy A; Szlag, David C; Southwell, Benjamin J; Sinclair, James

2010-07-01

This review focuses on the efficiency of different water treatment processes for the removal of cyanotoxins from potable water. Although several investigators have studied full-scale drinking water processes to determine the efficiency of cyanotoxin inactivation, many of the studies were based on ancillary practice. In this context, "ancillary practice" refers to the removal or inactivation of cyanotoxins by standard daily operational procedures and without a contingency operational plan utilizing specific treatment barriers. In this review, "auxiliary practice" refers to the implementation of inactivation/removal treatment barriers or operational changes explicitly designed to minimize risk from toxin-forming algae and their toxins to make potable water. Furthermore, the best drinking water treatment practices are based on extension of the multibarrier approach to remove cyanotoxins from water. Cyanotoxins are considered natural contaminants that occur worldwide and specific classes of cyanotoxins have shown regional prevalence. For example, freshwaters in the Americas often show high concentrations of microcystin, anatoxin-a, and cylindrospermopsin, whereas Australian water sources often show high concentrations of microcystin, cylindrospermopsin, and saxitoxins. Other less frequently reported cyanotoxins include lyngbyatoxin A, debromoaplysiatoxin, and beta-N-methylamino-L-alanine. This review focuses on the commonly used unit processes and treatment trains to reduce the toxicity of four classes of cyanotoxins: the microcystins, cylindrospermopsin, anatoxin-a, and saxitoxins. The goal of this review is to inform the reader of how each unit process participates in a treatment train and how an auxiliary multibarrier approach to water treatment can provide safer water for the consumer.

Comparison of Thermal and Non-Thermal Processing of Swine Feed and the Use of Selected Feed Additives on Inactivation of Porcine Epidemic Diarrhea Virus (PEDV).

PubMed

Trudeau, Michaela P; Verma, Harsha; Sampedro, Fernando; Urriola, Pedro E; Shurson, Gerald C; McKelvey, Jessica; Pillai, Suresh D; Goyal, Sagar M

2016-01-01

Infection with porcine epidemic diarrhea virus (PEDV) causes diarrhea, vomiting, and high mortality in suckling pigs. Contaminated feed has been suggested as a vehicle of transmission for PEDV. The objective of this study was to compare thermal and electron beam processing, and the inclusion of feed additives on the inactivation of PEDV in feed. Feed samples were spiked with PEDV and then heated to 120-145°C for up to 30 min or irradiated at 0-50 kGy. Another set of feed samples spiked with PEDV and mixed with Ultracid P (Nutriad), Activate DA (Novus International), KEM-GEST (Kemin Agrifood), Acid Booster (Agri-Nutrition), sugar or salt was incubated at room temperature (~25°C) for up to 21 days. At the end of incubation, the virus titers were determined by inoculation of Vero-81 cells and the virus inactivation kinetics were modeled using the Weibull distribution model. The Weibull kinetic parameter delta represented the time or eBeam dose required to reduce virus concentration by 1 log. For thermal processing, delta values ranged from 16.52 min at 120°C to 1.30 min at 145°C. For eBeam processing, a target dose of 50 kGy reduced PEDV concentration by 3 log. All additives tested were effective in reducing the survival of PEDV when compared with the control sample (delta = 17.23 days). Activate DA (0.81) and KEM-GEST (3.28) produced the fastest inactivation. In conclusion, heating swine feed at temperatures over 130°C or eBeam processing of feed with a dose over 50 kGy are effective processing steps to reduce PEDV survival. Additionally, the inclusion of selected additives can decrease PEDV survivability.

Inactivation and regrowth of multidrug resistant bacteria in urban wastewater after disinfection by solar-driven and chlorination processes.

PubMed

Fiorentino, Antonino; Ferro, Giovanna; Alferez, María Castro; Polo-López, Maria Inmaculada; Fernández-Ibañez, Pilar; Rizzo, Luigi

2015-07-01

Solar disinfection and solar-driven advanced oxidation processes (AOPs) (namely H2O2/sunlight, TiO2/sunlight, H2O2/TiO2/sunlight, solar photo-Fenton) were evaluated in the inactivation of indigenous antibiotic-resistant bacteria (ARB) in real urban wastewater. A multidrug resistant (MDR) Escherichia coli strain isolated from the effluent of the biological process of an urban wastewater treatment plant was the target ARB. The higher inactivation rates (residual density under detection limit, 2 CFUm L(-1)) were achieved with H2O2/TiO2/sunlight (cumulative energy per unit of volume (QUV) in the range 3-5 kJ L(-1), depending on H2O2/TiO2 ratio) and H2O2/sunlight (QUV of 8 kJ L(-1)) processes. All investigated processes did not affect antibiotic resistance of survived colonies. Moreover, H2O2/sunlight was compared with conventional chlorination process to evaluate bacterial regrowth potential and particularly the proportion of indigenous MDR E. coli with respect to total indigenous E. coli population. Chlorination (1.0 mg Cl2 L(-1)) was more effective than H2O2/sunlight (50 mg H2O2 L(-1)) to achieve total inactivation of MDR E. coli (15 min Vs 90 min) but less effective in controlling their regrowth (24 h Vs 48 h). Interestingly, the percentage of MDR E. coli in H2O2/sunlight treated samples decreased as incubation time increased; the opposite was observed for chlorinated samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Irreversible modification of sodium channel inactivation in toad myelinated nerve fibres by the oxidant chloramine-T.

PubMed Central

Wang, G K

1984-01-01

The effects of externally applied chloramine-T on the excitability of single toad myelinated nerve fibres were studied. Chloramine-T is a mild oxidant which reacts specifically with the cysteine and methionine residues of proteins. Chloramine-T prolongs the action potential of a single myelinated fibre by more than 1000-fold. This effect is concentration- and time-dependent; higher concentrations and longer incubation times increase prolongation. Under voltage-clamp conditions, sodium channel inactivation is markedly inhibited by chloramine-T while sodium channel activation remains normal. Prolonged depolarization of the membrane leads to a maintained sodium current. The maintained sodium currents show activation kinetics, dependence on membrane potential, and reversal potentials which are similar to those of normal, inactivating sodium currents in untreated fibres. Both the maintained and the peak sodium currents are equally inhibited by tetrodotoxin. After partial removal of sodium inactivation by brief exposures to chloramine-T, the voltage dependence of the steady-state sodium current inactivation (h infinity) is shifted in the depolarized direction by about 20 mV, even after correction for the non-inactivating component contributed by the maintained current. The phenomena described here imply that cysteine or methionine residues are critical for the sodium channel inactivation processes. The two different modifications of inactivation, its removal shown by the maintained current, and the shift in the voltage-dependence of the remaining inactivatable channels, reveal that at least two separate residues are modified by chloramine-T. PMID:6321714

Covalent Allosteric Inactivation of Protein Tyrosine Phosphatase 1B (PTP1B) by an Inhibitor-Electrophile Conjugate.

PubMed

Punthasee, Puminan; Laciak, Adrian R; Cummings, Andrea H; Ruddraraju, Kasi Viswanatharaju; Lewis, Sarah M; Hillebrand, Roman; Singh, Harkewal; Tanner, John J; Gates, Kent S

2017-04-11

Protein tyrosine phosphatase 1B (PTP1B) is a validated drug target, but it has proven difficult to develop medicinally useful, reversible inhibitors of this enzyme. Here we explored covalent strategies for the inactivation of PTP1B using a conjugate composed of an active site-directed 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxide inhibitor connected via a short linker to an electrophilic α-bromoacetamide moiety. Inhibitor-electrophile conjugate 5a caused time-dependent loss of PTP1B activity consistent with a covalent inactivation mechanism. The inactivation occurred with a second-order rate constant of (1.7 ± 0.3) × 10 2 M -1 min -1 . Mass spectrometric analysis of the inactivated enzyme indicated that the primary site of modification was C121, a residue distant from the active site. Previous work provided evidence that covalent modification of the allosteric residue C121 can cause inactivation of PTP1B [Hansen, S. K., Cancilla, M. T., Shiau, T. P., Kung, J., Chen, T., and Erlanson, D. A. (2005) Biochemistry 44, 7704-7712]. Overall, our results are consistent with an unusual enzyme inactivation process in which noncovalent binding of the inhibitor-electrophile conjugate to the active site of PTP1B protects the nucleophilic catalytic C215 residue from covalent modification, thus allowing inactivation of the enzyme via selective modification of allosteric residue C121.

Inactivation of Pathogens in Feces by Desiccation and Urea Treatment for Application in Urine-Diverting Dry Toilets

PubMed Central

Philippi, Luiz Sérgio; Vinnerås, Björn

2013-01-01

Ecological sanitation technologies can be effective in providing health and environmental pollution control if they can efficiently reduce the pathogenicity of microorganisms carried in fecal material to safe levels. This study evaluated the sanitizing effects of different additives for dry treatment of feces from urine-diverting dry toilets, based on inactivation of Enterococcus faecalis, Salmonella enterica serovar Typhimurium, bacteriophages MS2 and ΦX, and Ascaris suum. The additives, ash (A) and oyster shell (O) in different amounts and urea (U) to optimize the process, were compared with no additive, solely urea, and sawdust as controls (C) and were covered ([x%O:A]) or uncovered (x%O:A). The main inactivation factors found were desiccation, ammonia content, and pH. S. Typhimurium and E. faecalis were more affected by the ammonia content. A combination of neutral to high pH and desiccation was most effective for inactivation of MS2, and desiccation was most effective for inactivation of ΦX and A. suum. The inactivation rate was modeled for all combinations studied. The most promising treatments were [150%O:A+U], 150%O:A+U, and 150%O:A. According to the models, these could inactivate, for example, 7 log10 units of all bacteria and bacteriophages within 83, 125, and 183 days, respectively. The inactivation of A. suum was modeled, albeit the measured decay in egg viability was low. PMID:23335764

X inactivation in a mammal species with three sex chromosomes.

PubMed

Veyrunes, Frédéric; Perez, Julie

2018-06-01

X inactivation is a fundamental mechanism in eutherian mammals to restore a balance of X-linked gene products between XY males and XX females. However, it has never been extensively studied in a eutherian species with a sex determination system that deviates from the ubiquitous XX/XY. In this study, we explore the X inactivation process in the African pygmy mouse Mus minutoides, that harbours a polygenic sex determination with three sex chromosomes: Y, X, and a feminizing mutant X, named X*; females can thus be XX, XX*, or X*Y, and all males are XY. Using immunofluorescence, we investigated histone modification patterns between the two X chromosome types. We found that the X and X* chromosomes are randomly inactivated in XX* females, while no histone modifications were detected in X*Y females. Furthermore, in M. minutoides, X and X* chromosomes are fused to different autosomes, and we were able to show that the X inactivation never spreads into the autosomal segments. Evaluation of X inactivation by immunofluorescence is an excellent quantitative procedure, but it is only applicable when there is a structural difference between the two chromosomes that allows them to be distinguished.

Living with an imperfect cell wall: compensation of femAB inactivation in Staphylococcus aureus.

PubMed

Hübscher, Judith; Jansen, Andrea; Kotte, Oliver; Schäfer, Juliane; Majcherczyk, Paul A; Harris, Llinos G; Bierbaum, Gabriele; Heinemann, Matthias; Berger-Bächi, Brigitte

2007-09-04

Synthesis of the Staphylococcus aureus peptidoglycan pentaglycine interpeptide bridge is catalyzed by the nonribosomal peptidyl transferases FemX, FemA and FemB. Inactivation of the femAB operon reduces the interpeptide to a monoglycine, leading to a poorly crosslinked peptidoglycan. femAB mutants show a reduced growth rate and are hypersusceptible to virtually all antibiotics, including methicillin, making FemAB a potential target to restore beta-lactam susceptibility in methicillin-resistant S. aureus (MRSA). Cis-complementation with wild type femAB only restores synthesis of the pentaglycine interpeptide and methicillin resistance, but the growth rate remains low. This study characterizes the adaptations that ensured survival of the cells after femAB inactivation. In addition to slow growth, the cis-complemented femAB mutant showed temperature sensitivity and a higher methicillin resistance than the wild type. Transcriptional profiling paired with reporter metabolite analysis revealed multiple changes in the global transcriptome. A number of transporters for sugars, glycerol, and glycine betaine, some of which could serve as osmoprotectants, were upregulated. Striking differences were found in the transcription of several genes involved in nitrogen metabolism and the arginine-deiminase pathway, an alternative for ATP production. In addition, microarray data indicated enhanced expression of virulence factors that correlated with premature expression of the global regulators sae, sarA, and agr. Survival under conditions preventing normal cell wall formation triggered complex adaptations that incurred a fitness cost, showing the remarkable flexibility of S. aureus to circumvent cell wall damage. Potential FemAB inhibitors would have to be used in combination with other antibiotics to prevent selection of resistant survivors.

Decentralised black water treatment by combined auto-thermal aerobic digestion and ammonia - A pilot study optimising treatment capacity.

PubMed

Nordin, A; Göttert, D; Vinnerås, B

2018-02-01

Partial heating of black water by auto-thermal aerobic digestion was combined with the addition of 1% w/w urea and monitoring of pathogens and indicator organisms over a 21-day period. After initial mixing, the 160 m 3 black water (60 m 3 heated and 100 m 3 non-heated) was left undisturbed. The urea was confirmed to be fully degraded into ammonia (5.1 g N L -1 ) first after 14 days, while the pH stabilised at around 9.2 after one week. The initial temperature of 17 °C fell by 6 °C during the study. E. coli and Salmonella spp., which are sensitive to ammonia, were inactivated during the first few days of the study, despite the urea only being partly hydrolysed. At day 14, f-RNA bacteriophages could also no longer be detected. The more persistent somatic coliphages, Enterococcus spp. and Ascaris eggs, showed significant but slow inactivation. The treatment proved to be efficient with regards to salmonella, which is a target pathogen in the Swedish context, but for parasite egg inactivation a higher temperature was required. The treatment would benefit from more frequent stirring to speed up the hydrolysis of urea and thus improve treatment efficiency. The alternative treatment scheme could increase capacity by 2.4 times, albeit with a 40% higher cost per volume due to the increased use of urea. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Schizophrenia-Associated Kv11.1-3.1 Isoform Results in Reduced Current Accumulation during Repetitive Brief Depolarizations

PubMed Central

Heide, Juliane; Mann, Stefan A.; Vandenberg, Jamie I.

2012-01-01

Recent genome wide association studies identified a brain and primate specific isoform of a voltage-gated potassium channel, referred to as Kv11.1-3.1, which is significantly associated with schizophrenia. The 3.1 isoform replaces the first 102 amino acids of the most abundant isoform (referred to as Kv11.1-1A) with six unique amino acids. Here we show that the Kv11.1-3.1 isoform has faster rates of channel deactivation but a slowing of the rates of inactivation compared to the Kv11.1-1A isoform. The Kv11.1-3.1 isoform also has a significant depolarizing shift in the voltage-dependence of steady-state inactivation. The consequence of the altered gating kinetics is that there is lower current accumulation for Kv11.1-3.1 expressing cells during repetitive action potential firing compared to Kv11.1-1A expressing cells, which in turn will result in longer lasting trains of action potentials. Increased expression of Kv11.1-3.1 channels in the brain of schizophrenia patients might therefore contribute to disorganized neuronal firing. PMID:23029143

Muscle inactivation of mTOR causes metabolic and dystrophin defects leading to severe myopathy

PubMed Central

Risson, Valérie; Mazelin, Laetitia; Roceri, Mila; Sanchez, Hervé; Moncollin, Vincent; Corneloup, Claudine; Richard-Bulteau, Hélène; Vignaud, Alban; Baas, Dominique; Defour, Aurélia; Freyssenet, Damien; Tanti, Jean-François; Le-Marchand-Brustel, Yannick; Ferrier, Bernard; Conjard-Duplany, Agnès; Romanino, Klaas; Bauché, Stéphanie; Hantaï, Daniel; Mueller, Matthias; Kozma, Sara C.; Thomas, George; Rüegg, Markus A.; Ferry, Arnaud; Pende, Mario; Bigard, Xavier; Koulmann, Nathalie

2009-01-01

Mammalian target of rapamycin (mTOR) is a key regulator of cell growth that associates with raptor and rictor to form the mTOR complex 1 (mTORC1) and mTORC2, respectively. Raptor is required for oxidative muscle integrity, whereas rictor is dispensable. In this study, we show that muscle-specific inactivation of mTOR leads to severe myopathy, resulting in premature death. mTOR-deficient muscles display metabolic changes similar to those observed in muscles lacking raptor, including impaired oxidative metabolism, altered mitochondrial regulation, and glycogen accumulation associated with protein kinase B/Akt hyperactivation. In addition, mTOR-deficient muscles exhibit increased basal glucose uptake, whereas whole body glucose homeostasis is essentially maintained. Importantly, loss of mTOR exacerbates the myopathic features in both slow oxidative and fast glycolytic muscles. Moreover, mTOR but not raptor and rictor deficiency leads to reduced muscle dystrophin content. We provide evidence that mTOR controls dystrophin transcription in a cell-autonomous, rapamycin-resistant, and kinase-independent manner. Collectively, our results demonstrate that mTOR acts mainly via mTORC1, whereas regulation of dystrophin is raptor and rictor independent. PMID:20008564

Development of lacosamide for the treatment of partial-onset seizures.

PubMed

Doty, Pamela; Hebert, David; Mathy, Francois-Xavier; Byrnes, William; Zackheim, James; Simontacchi, Kelly

2013-07-01

Lacosamide is an antiepileptic drug (AED) available in multiple formulations that was first approved in 2008 as adjunctive therapy for partial-onset seizures (POS) in adults. Unlike traditional sodium channel blockers affecting fast inactivation, lacosamide selectively enhances sodium channel slow inactivation. This mechanism of action results in stabilization of hyperexcitable neuronal membranes, inhibition of neuronal firing, and reduction in long-term channel availability without affecting physiological function. Lacosamide has a well-characterized and favorable pharmacokinetic profile, including a fast absorption rate, minimal or no interaction with cytochrome P-450 izoenzymes, and a low potential for drug-drug interactions. Lacosamide clinical development included three placebo-controlled, double-blind, randomized trials conducted in more than 1300 patients, each demonstrating safety and efficacy of lacosamide compared to placebo as adjunctive therapy for adults with POS. The clinical use of lacosamide may broaden, pending results of trials evaluating its use as monotherapy for POS in adults, as treatment for epilepsy in pediatric subjects, and as adjunctive treatment for uncontrolled primary generalized tonic-clonic seizures in those with idiopathic generalized epilepsy. © 2013 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals Inc. on behalf of The New York Academy of Sciences.

Calcium current in type I hair cells isolated from the semicircular canal crista ampullaris of the rat.

PubMed

Almanza, Angélica; Vega, Rosario; Soto, Enrique

2003-12-24

The low voltage gain in type I hair cells implies that neurotransmitter release at their afferent synapse should be mediated by low voltage activated calcium channels, or that some peculiar mechanism should be operating in this synapse. With the patch clamp technique, we studied the characteristics of the Ca(2+) current in type I hair cells enzymatically dissociated from rat semicircular canal crista ampullaris. Calcium current in type I hair cells exhibited a slow inactivation (during 2-s depolarizing steps), was sensitive to nimodipine and was blocked by Cd(2+) and Ni(2+). This current was activated at potentials above -60 mV, had a mean half maximal activation of -36 mV, and exhibited no steady-state inactivation at holding potentials between -100 and -60 mV. This data led us to conclude that hair cell Ca(2+) current is most likely of the L type. Thus, other mechanisms participating in neurotransmitter release such as K(+) accumulation in the synaptic cleft, modulation of K(+) currents by nitric oxide, participation of a Na(+) current and possible metabotropic cascades activated by depolarization should be considered.

Pathogenic psychrotolerant sporeformers: an emerging challenge for low-temperature storage of minimally processed foods.

PubMed

Markland, Sarah M; Farkas, Daniel F; Kniel, Kalmia E; Hoover, Dallas G

2013-05-01

Sporeforming bacteria are a significant problem in the food industry as they are ubiquitous in nature and capable of resisting inactivation by heat and chemical treatments designed to inactivate them. Beyond spoilage issues, psychrotolerant sporeformers are becoming increasingly recognized as a potential hazard given the ever-expanding demand for refrigerated processed foods with extended shelf-life. In these products, the sporeforming pathogens of concern are Bacillus cereus, Bacillus weihenstephanensis, and Clostridium botulinum type E. This review article examines the foods, conditions, and organisms responsible for the food safety issue caused by the germination and outgrowth of psychrotolerant sporeforming pathogens in minimally processed refrigerated foods.

Dual mechanism of integrin αIIbβ3 closure in procoagulant platelets.

PubMed

Mattheij, Nadine J A; Gilio, Karen; van Kruchten, Roger; Jobe, Shawn M; Wieschhaus, Adam J; Chishti, Athar H; Collins, Peter; Heemskerk, Johan W M; Cosemans, Judith M E M

2013-05-10

Inactivation of integrin αIIbβ3 reverses platelet aggregate formation upon coagulation. Platelets from patient (Scott) and mouse (Capn1(-/-) and Ppif(-/-)) blood reveal a dual mechanism of αIIbβ3 inactivation: by calpain-2 cleavage of integrin-associated proteins and by cyclophilin D/TMEM16F-dependent phospholipid scrambling. These data provide novel insight into the switch mechanisms from aggregating to procoagulant platelets. Aggregation of platelets via activated integrin αIIbβ3 is a prerequisite for thrombus formation. Phosphatidylserine-exposing platelets with a key role in the coagulation process disconnect from a thrombus by integrin inactivation via an unknown mechanism. Here we show that αIIbβ3 inactivation in procoagulant platelets relies on a sustained high intracellular Ca(2+), stimulating intracellular cleavage of the β3 chain, talin, and Src kinase. Inhibition of calpain activity abolished protein cleavage, but only partly suppressed αIIbβ3 inactivation. Integrin αIIbβ3 inactivation was unchanged in platelets from Capn1(-/-) mice, suggesting a role of the calpain-2 isoform. Scott syndrome platelets, lacking the transmembrane protein TMEM16F and having low phosphatidylserine exposure, displayed reduced αIIbβ3 inactivation with the remaining activity fully dependent on calpain. In platelets from Ppif(-/-) mice, lacking mitochondrial permeability transition pore (mPTP) formation, agonist-induced phosphatidylserine exposure and αIIbβ3 inactivation were reduced. Treatment of human platelets with cyclosporin A gave a similar phenotype. Together, these data point to a dual mechanism of αIIbβ3 inactivation via calpain(-2) cleavage of integrin-associated proteins and via TMEM16F-dependent phospholipid scrambling with an assistant role of mPTP formation.

Non-thermal plasma for inactivated-vaccine preparation.

PubMed

Wang, Guomin; Zhu, Ruihao; Yang, Licong; Wang, Kaile; Zhang, Qian; Su, Xia; Yang, Bing; Zhang, Jue; Fang, Jing

2016-02-17

Vaccines are of great importance in controlling the spread of infectious diseases in poultry farming. The safety and efficacy of vaccines are also essential. To explore the feasibility of a novel technology (non-thermal plasma) in inactivated vaccine preparation, an alternating current atmospheric pressure non-thermal plasma (NTP) jet with Ar/O2/N2 as the operating gas was used to inactivate a Newcastle disease virus (NDV, LaSota) strain and H9N2 avian influenza virus (AIV, A/Chicken/Hebei/WD/98) for vaccine preparation. The results showed that complete inactivation could be achieved with 2 min of NTP treatment for both NDV and AIV. Moreover, a proper NTP treatment time is needed for inactivation of a virus without destruction of the antigenic determinants. Compared to traditional formaldehyde-inactivated vaccine, the vaccine made from NDV treated by NTP for 2 min (NTP-2 min-NDV-vaccine) could induce a higher NDV-specific antibody titer in specific pathogen-free (SPF) chickens, and the results of a chicken challenge experiment showed that NTP-2 min-NDV-vaccine could protect SPF chickens from a lethal NDV challenge. Vaccines made from AIV treated by NTP for 2 min (NTP-2 min-AIV-vaccine) also showed a similar AIV-specific antibody titer compared with traditional AIV vaccines prepared using formaldehyde inactivation. Studies of the morphological changes of the virus, chemical analysis of NDV allantoic fluid and optical emission spectrum analysis of NTP suggested that reactive oxygen species and reactive nitrogen species produced by NTP played an important role in the virus inactivation process. All of these results demonstrated that it could be feasible to use non-thermal NTP as an alternative strategy to prepare inactivated vaccines for Newcastle disease and avian influenza. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of blue or violet light on the inactivation of Staphylococcus aureus by riboflavin-5'-phosphate photolysis.

PubMed

Wong, Tak-Wah; Cheng, Chien-Wei; Hsieh, Zong-Jhe; Liang, Ji-Yuan

2017-08-01

The light sensitive compound riboflavin-5'-phosphate (or flavin mononucleotide, FMN) generates reactive oxygen species (ROS) upon photo-irradiation. FMN is required by all flavoproteins because it is a cofactor of biological blue-light receptors. The photochemical effects of FMN after irradiation by blue or violet light on the inactivation of Staphylococcus aureus strains, including a methicillin-resistant strain (MRSA), were investigated in this study. Upon blue- or violet-light photo-treatment, FMN was shown to inactivate S. aureus due to the generated ROS. Effective bacterial inactivation can be achieved by FMN photolysis without an exogenous electron provider. Inactivation rates of 94.9 and 95.2% in S. aureus and MRSA, respectively, can be reached by blue light irradiation (2.0mW/cm 2 ) with 120μM FMN for 120min. A lower FMN concentration and a shorter time are required to reach similar effects by violet light irradiation. Inactivation rates of 96.3 and 97.0% in S. aureus and MRSA, respectively, can be reached by violet light irradiation (1.0mW/cm 2 ) with 30μM FMN for 30min. The sensitivity of the inherent photosensitizers is lower under blue-light irradiation. A long exposure photolytic treatment of FMN by blue light is required to inactivate S. aureus. Violet light was found to be more efficient in S. aureus inactivation at the same radiant intensity. FMN photolysis with blue or violet light irradiation enhanced the inactivation rates of S. aureus and MRSA. FMN photochemical treatment could be a supplemental technique in hygienic decontamination processes. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of surface pretreatment of TiO2 films on interfacial processes leading to bacterial inactivation in the dark and under light irradiation

PubMed Central

Rtimi, Sami; Nesic, Jelena; Pulgarin, Cesar; Sanjines, Rosendo; Bensimon, Michael; Kiwi, John

2015-01-01

Evidence is presented for radio-frequency plasma pretreatment enhancing the amount and adhesion of TiO2 sputtered on polyester (PES) and on polyethylene (PE) films. Pretreatment is necessary to attain a suitable TiO2 loading leading to an acceptable Escherichia coli reduction kinetics in the dark or under light irradiation for PES–TiO2 and PE–TiO2 samples. The amount of TiO2 on the films was monitored by diffuse reflectance spectroscopy and X-ray fluorescence. X-ray electron spectroscopy shows the lack of accumulation of bacterial residues such as C, N and S during bacterial inactivation since they seem to be rapidly destroyed by TiO2 photocatalysis. Evidence was found for Ti4+/Ti3+ redox catalysis occurring on PES–TiO2 and PE–TiO2 during the bacterial inactivation process. On PE–TiO2 surfaces, Fourier transform infrared spectroscopy (ATR-FTIR) provides evidence for a systematic shift of the na(CH2) stretching vibrations preceding bacterial inactivation within 60 min. The discontinuous IR-peak shifts reflect the increase in the C–H inter-bond distance leading to bond scission. The mechanism leading to E. coli loss of viability on PES–TiO2 was investigated in the dark up to complete bacterial inactivation by monitoring the damage in the bacterial outer cell by transmission electron microscopy. After 30 min, the critical step during the E. coli inactivation commences for dark disinfection on 0.1–5% wt PES–TiO2 samples. The interactions between the TiO2 aggregates and the outer lipopolysaccharide cell wall involve electrostatic effects competing with the van der Waals forces. PMID:25657831

Effect of surface pretreatment of TiO2 films on interfacial processes leading to bacterial inactivation in the dark and under light irradiation.

PubMed

Rtimi, Sami; Nesic, Jelena; Pulgarin, Cesar; Sanjines, Rosendo; Bensimon, Michael; Kiwi, John

2015-02-06

Evidence is presented for radio-frequency plasma pretreatment enhancing the amount and adhesion of TiO2 sputtered on polyester (PES) and on polyethylene (PE) films. Pretreatment is necessary to attain a suitable TiO2 loading leading to an acceptable Escherichia coli reduction kinetics in the dark or under light irradiation for PES-TiO2 and PE-TiO2 samples. The amount of TiO2 on the films was monitored by diffuse reflectance spectroscopy and X-ray fluorescence. X-ray electron spectroscopy shows the lack of accumulation of bacterial residues such as C, N and S during bacterial inactivation since they seem to be rapidly destroyed by TiO2 photocatalysis. Evidence was found for Ti(4+)/Ti(3+) redox catalysis occurring on PES-TiO2 and PE-TiO2 during the bacterial inactivation process. On PE-TiO2 surfaces, Fourier transform infrared spectroscopy (ATR-FTIR) provides evidence for a systematic shift of the na(CH2) stretching vibrations preceding bacterial inactivation within 60 min. The discontinuous IR-peak shifts reflect the increase in the C-H inter-bond distance leading to bond scission. The mechanism leading to E. coli loss of viability on PES-TiO2 was investigated in the dark up to complete bacterial inactivation by monitoring the damage in the bacterial outer cell by transmission electron microscopy. After 30 min, the critical step during the E. coli inactivation commences for dark disinfection on 0.1-5% wt PES-TiO2 samples. The interactions between the TiO2 aggregates and the outer lipopolysaccharide cell wall involve electrostatic effects competing with the van der Waals forces.

Food processing by high hydrostatic pressure.

PubMed

Yamamoto, Kazutaka

2017-04-01

High hydrostatic pressure (HHP) process, as a nonthermal process, can be used to inactivate microbes while minimizing chemical reactions in food. In this regard, a HHP level of 100 MPa (986.9 atm/1019.7 kgf/cm 2 ) and more is applied to food. Conventional thermal process damages food components relating color, flavor, and nutrition via enhanced chemical reactions. However, HHP process minimizes the damages and inactivates microbes toward processing high quality safe foods. The first commercial HHP-processed foods were launched in 1990 as fruit products such as jams, and then some other products have been commercialized: retort rice products (enhanced water impregnation), cooked hams and sausages (shelf life extension), soy sauce with minimized salt (short-time fermentation owing to enhanced enzymatic reactions), and beverages (shelf life extension). The characteristics of HHP food processing are reviewed from viewpoints of nonthermal process, history, research and development, physical and biochemical changes, and processing equipment.

Inactivation and unfolding of protein tyrosine phosphatase from Thermus thermophilus HB27 during urea and guanidine hydrochloride denaturation.

PubMed

Wang, Yejing; He, Huawei; Liu, Lina; Gao, Chunyan; Xu, Shui; Zhao, Ping; Xia, Qingyou

2014-01-01

The effects of urea and guanidine hydrochloride (GdnHCl) on the activity, conformation and unfolding process of protein tyrosine phosphatase (PTPase), a thermostable low molecular weight protein from Thermus thermophilus HB27, have been studied. Enzymatic activity assays showed both urea and GdnHCl resulted in the inactivation of PTPase in a concentration and time-dependent manner. Inactivation kinetics analysis suggested that the inactivation of PTPase induced by urea and GdnHCl were both monophasic and reversible processes, and the effects of urea and GdnHCl on PTPase were similar to that of mixed-type reversible inhibitors. Far-ultraviolet (UV) circular dichroism (CD), Tryptophan and 1-anilinonaphthalene -8-sulfonic acid (ANS) fluorescence spectral analyses indicated the existence of a partially active and an inactive molten globule-like intermediate during the unfolding processes induced by urea and GdnHCl, respectively. Based on the sequence alignment and the homolog Tt1001 protein structure, we discussed the possible conformational transitions of PTPase induced by urea and GdnHCl and compared the conformations of these unfolding intermediates with the transient states in bovine PTPase and its complex structures in detail. Our results may be able to provide some valuable clues to reveal the relationship between the structure and enzymatic activity, and the unfolding pathway and mechanism of PTPase.

Inactivation and Unfolding of Protein Tyrosine Phosphatase from Thermus thermophilus HB27 during Urea and Guanidine Hydrochloride Denaturation

PubMed Central

Liu, Lina; Gao, Chunyan; Xu, Shui; Zhao, Ping; Xia, Qingyou

2014-01-01

The effects of urea and guanidine hydrochloride (GdnHCl) on the activity, conformation and unfolding process of protein tyrosine phosphatase (PTPase), a thermostable low molecular weight protein from Thermus thermophilus HB27, have been studied. Enzymatic activity assays showed both urea and GdnHCl resulted in the inactivation of PTPase in a concentration and time-dependent manner. Inactivation kinetics analysis suggested that the inactivation of PTPase induced by urea and GdnHCl were both monophasic and reversible processes, and the effects of urea and GdnHCl on PTPase were similar to that of mixed-type reversible inhibitors. Far-ultraviolet (UV) circular dichroism (CD), Tryptophan and 1-anilinonaphthalene -8-sulfonic acid (ANS) fluorescence spectral analyses indicated the existence of a partially active and an inactive molten globule-like intermediate during the unfolding processes induced by urea and GdnHCl, respectively. Based on the sequence alignment and the homolog Tt1001 protein structure, we discussed the possible conformational transitions of PTPase induced by urea and GdnHCl and compared the conformations of these unfolding intermediates with the transient states in bovine PTPase and its complex structures in detail. Our results may be able to provide some valuable clues to reveal the relationship between the structure and enzymatic activity, and the unfolding pathway and mechanism of PTPase. PMID:25255086

A Causal Role for the Cortical Frontal Eye Fields in Microsaccade Deployment

PubMed Central

Dash, Suryadeep; Lomber, Stephen G.

2016-01-01

Microsaccades aid vision by helping to strategically sample visual scenes. Despite the importance of these small eye movements, no cortical area has ever been implicated in their generation. Here, we used unilateral and bilateral reversible inactivation of the frontal eye fields (FEF) to identify a cortical drive for microsaccades. Unexpectedly, FEF inactivation altered microsaccade metrics and kinematics. Such inactivation also impaired microsaccade deployment following peripheral cue onset, regardless of cue side or inactivation configuration. Our results demonstrate that the FEF provides critical top-down drive for microsaccade generation, particularly during the recovery of microsaccades after disruption by sensory transients. Our results constitute the first direct evidence, to our knowledge, for the contribution of any cortical area to microsaccade generation, and they provide a possible substrate for how cognitive processes can influence the strategic deployment of microsaccades. PMID:27509130

A Causal Role for the Cortical Frontal Eye Fields in Microsaccade Deployment.

PubMed

Peel, Tyler R; Hafed, Ziad M; Dash, Suryadeep; Lomber, Stephen G; Corneil, Brian D

2016-08-01

Microsaccades aid vision by helping to strategically sample visual scenes. Despite the importance of these small eye movements, no cortical area has ever been implicated in their generation. Here, we used unilateral and bilateral reversible inactivation of the frontal eye fields (FEF) to identify a cortical drive for microsaccades. Unexpectedly, FEF inactivation altered microsaccade metrics and kinematics. Such inactivation also impaired microsaccade deployment following peripheral cue onset, regardless of cue side or inactivation configuration. Our results demonstrate that the FEF provides critical top-down drive for microsaccade generation, particularly during the recovery of microsaccades after disruption by sensory transients. Our results constitute the first direct evidence, to our knowledge, for the contribution of any cortical area to microsaccade generation, and they provide a possible substrate for how cognitive processes can influence the strategic deployment of microsaccades.

Natural inactivation of Escherichia coli in anaerobic and reduced groundwater.

PubMed

Lisle, J T

2016-06-01

Inactivation rates of Escherichia coli in groundwater have most often been determined in aerobic and oxidized systems. This study examined E. coli inactivation rates in anaerobic and extremely reduced groundwater systems that have been identified as recharge zones. Groundwater from six artesian wells was diverted to above-ground, flow-through mesocosms that contained laboratory grown E. coli in diffusion chambers. All groundwater was anaerobic and extremely reduced (ORP < -300 mV). Cells were plated onto mTEC agar during 21-day incubation periods. All data fit a bi-phasic inactivation model, with >95% of the E. coli population being inactivated <11·0 h (mean k = 0·488 ±0·188 h(-1) ). The groundwater geochemical conditions enhanced the inactivation of E. coli to rates approx. 21-fold greater than previously published inactivation rate in groundwater (mean k = 0·023 ± 0·030 h(-1) ). Also, mTEC agar inhibits E. coli growth following exposure to anaerobic and reduced groundwater. Aquifer recharge zones with geochemical characteristics observed in this study complement above-ground engineered processes (e.g. filtration, disinfection), while increasing the overall indicator micro-organism log-reduction rate of a facility. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Quality-related enzymes in fruit and vegetable products: effects of novel food processing technologies, part 1: high-pressure processing.

PubMed

Terefe, Netsanet Shiferaw; Buckow, Roman; Versteeg, Cornelis

2014-01-01

The activity of endogenous deteriorative enzymes together with microbial growth (with associated enzymatic activity) and/or other non-enzymatic (usually oxidative) reactions considerably shorten the shelf life of fruits and vegetable products. Thermal processing is commonly used by the food industry for enzyme and microbial inactivation and is generally effective in this regard. However, thermal processing may cause undesirable changes in product's sensory as well as nutritional attributes. Over the last 20 years, there has been a great deal of interest shown by both the food industry and academia in exploring alternative food processing technologies that use minimal heat and/or preservatives. One of the technologies that have been investigated in this context is high-pressure processing (HPP). This review deals with HPP focusing on its effectiveness for controlling quality-degrading enzymes in horticultural products. The scientific literature on the effects of HPP on plant enzymes, mechanism of action, and intrinsic and extrinsic factors that influence the effectiveness of HPP for controlling plant enzymes is critically reviewed. HPP inactivates vegetative microbial cells at ambient temperature conditions, resulting in a very high retention of the nutritional and sensory characteristics of the fresh product. Enzymes such as polyphenol oxidase (PPO), peroxidase (POD), and pectin methylesterase (PME) are highly resistant to HPP and are at most partially inactivated under commercially feasible conditions, although their sensitivity towards pressure depends on their origin as well as their environment. Polygalacturonase (PG) and lipoxygenase (LOX) on the other hand are relatively more pressure sensitive and can be substantially inactivated by HPP at commercially feasible conditions. The retention and activation of enzymes such as PME by HPP can be beneficially used for improving the texture and other quality attributes of processed horticultural products as well as for creating novel structures that are not feasible with thermal processing.

Modulation of the binding of basic fibroblast growth factor and heparanase activity by purified λ-carrageenan oligosaccharides.

PubMed

Niu, Ting-Ting; Zhang, Dong-Sheng; Chen, Hai-Min; Yan, Xiao-Jun

2015-07-10

Inhibitors of angiogenesis and tumor metastasis are increasingly emerging as promising agents for cancer therapy. Here, we report λ-carrageenan oligosaccharides (λ-COs), highly-sulfated oligosaccharides acting as a basic fibroblast growth factor (bFGF) antagonist and heparanase inhibitor. λ-COs with degree of polymerization (DP) from 2 to 8 degraded by λ-carrageenase were separated and purified. The structures were identified by mass spectrometry. The activities of λ-COs are closely related with DP. λ-COs showed no cytotoxicity, but inactivated bFGF-induced cell proliferation; among them, λ-carraheptaose showed highest capability. Only λ-carraheptaose can effectively bind to bFGF. Binding kinetics showed that λ-carraheptaose and suramin had different binding modes, i.e., suramin displayed a fast association and fast dissociation, but λ-carraheptaose exhibited a slow association and slow dissociation. In addition, λ-COs showed the highest heparanase inhibitory ability and abolished the endothelial cell invasion. Thus, λ-COs may provide a tool to develop of new carbohydrate-based therapeutics against cancer and angiogenesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Investigations of blue light-induced reactive oxygen species from flavin mononucleotide on inactivation of E. coli.

PubMed

Liang, Ji-Yuan; Cheng, Chien-Wei; Yu, Chin-Hao; Chen, Liang-Yü

2015-02-01

The micronutrients in many cellular processes, riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) are photo-sensitive to UV and visible light for generating reactive oxygen species (ROS). Produced from phosphorylation of riboflavin, FMN is more water-soluble and rapidly transformed into free riboflavin after ingestion. This study investigated the application of visible blue light with FMN to development of an effective antimicrobial treatment. The photosensitization of bacterial viability with FMN was investigated by light quality, intensity, time, and irradiation dosage. The blue light-induced photochemical reaction with FMN could inactivate Escherichiacoli by the generated ROS in damaging nucleic acids, which was validated. This novel photodynamic technique could be a safe practice for photo-induced inactivation of environmental microorganism to achieve hygienic requirements in food processing. Copyright © 2015 Elsevier B.V. All rights reserved.

New mechanistically based model for predicting reduction of biosolids waste by ozonation of return activated sludge.

PubMed

Isazadeh, Siavash; Feng, Min; Urbina Rivas, Luis Enrique; Frigon, Dominic

2014-04-15

Two pilot-scale activated sludge reactors were operated for 98 days to provide the necessary data to develop and validate a new mathematical model predicting the reduction of biosolids production by ozonation of the return activated sludge (RAS). Three ozone doses were tested during the study. In addition to the pilot-scale study, laboratory-scale experiments were conducted with mixed liquor suspended solids and with pure cultures to parameterize the biomass inactivation process during exposure to ozone. The experiments revealed that biomass inactivation occurred even at the lowest doses, but that it was not associated with extensive COD solubilization. For validation, the model was used to simulate the temporal dynamics of the pilot-scale operational data. Increasing the description accuracy of the inactivation process improved the precision of the model in predicting the operational data. Copyright © 2014 Elsevier B.V. All rights reserved.

Changes of contractile responses due to simulated weightlessness in rat soleus muscle

NASA Astrophysics Data System (ADS)

Elkhammari, A.; Noireaud, J.; Léoty, C.

1994-08-01

Some contractile and electrophysiological properties of muscle fibers isolated from the slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) muscles of rats were compared with those measured in SOL muscles from suspended rats. In suspendede SOL (21 days of tail-suspension) membrane potential (Em), intracellular sodium activity (aiNa) and the slope of the relationship between Em and log [K]o were typical of fast-twitch muscles. The relation between the maximal amplitude of K-contractures vs Em was steeper for control SOL than for EDL and suspended SOL muscles. After suspension, in SOL muscles the contractile threshold and the inactivation curves for K-contractures were shifted to more positive Em. Repriming of K-contractures was unaffected by suspencion. The exposure of isolated fibers to perchlorate (ClO4-)-containing (6-40 mM) solutions resulted ina similar concentration-dependent shift to more negative Em of activation curves for EDL and suspended SOL muscles. On exposure to a Na-free TEA solution, SOL from control and suspended rats, in contrast to EDL muscles, generated slow contractile responses. Suspended SOL showed a reduced sensitivity to the contracture-producing effect of caffeine compared to control muscles. These results suggested that the modification observed due to suspension could be encounted by changes in the characteristics of muscle fibers from slow to fast-twitch type.

Caloric restriction induces energy-sparing alterations in skeletal muscle contraction, fiber composition and local thyroid hormone metabolism that persist during catch-up fat upon refeeding

PubMed Central

De Andrade, Paula B. M.; Neff, Laurence A.; Strosova, Miriam K.; Arsenijevic, Denis; Patthey-Vuadens, Ophélie; Scapozza, Leonardo; Montani, Jean-Pierre; Ruegg, Urs T.; Dulloo, Abdul G.; Dorchies, Olivier M.

2015-01-01

Weight regain after caloric restriction results in accelerated fat storage in adipose tissue. This catch-up fat phenomenon is postulated to result partly from suppressed skeletal muscle thermogenesis, but the underlying mechanisms are elusive. We investigated whether the reduced rate of skeletal muscle contraction-relaxation cycle that occurs after caloric restriction persists during weight recovery and could contribute to catch-up fat. Using a rat model of semistarvation-refeeding, in which fat recovery is driven by suppressed thermogenesis, we show that contraction and relaxation of leg muscles are slower after both semistarvation and refeeding. These effects are associated with (i) higher expression of muscle deiodinase type 3 (DIO3), which inactivates tri-iodothyronine (T3), and lower expression of T3-activating enzyme, deiodinase type 2 (DIO2), (ii) slower net formation of T3 from its T4 precursor in muscles, and (iii) accumulation of slow fibers at the expense of fast fibers. These semistarvation-induced changes persisted during recovery and correlated with impaired expression of transcription factors involved in slow-twitch muscle development. We conclude that diminished muscle thermogenesis following caloric restriction results from reduced muscle T3 levels, alteration in muscle-specific transcription factors, and fast-to-slow fiber shift causing slower contractility. These energy-sparing effects persist during weight recovery and contribute to catch-up fat. PMID:26441673

How do children fall asleep? A high-density EEG study of slow waves in the transition from wake to sleep.

PubMed

Spiess, Mathilde; Bernardi, Giulio; Kurth, Salome; Ringli, Maya; Wehrle, Flavia M; Jenni, Oskar G; Huber, Reto; Siclari, Francesca

2018-05-17

Slow waves, the hallmarks of non-rapid eye-movement (NREM) sleep, are thought to reflect maturational changes that occur in the cerebral cortex throughout childhood and adolescence. Recent work in adults has revealed evidence for two distinct synchronization processes involved in the generation of slow waves, which sequentially come into play in the transition to sleep. In order to understand how these two processes are affected by developmental changes, we compared slow waves between children and young adults in the falling asleep period. The sleep onset period (starting 30s before end of alpha activity and ending at the first slow wave sequence) was extracted from 72 sleep onset high-density EEG recordings (128 electrodes) of 49 healthy subjects (age 8-25). Using an automatic slow wave detection algorithm, the number, amplitude and slope of slow waves were analyzed and compared between children (age 8-11) and young adults (age 20-25). Slow wave number and amplitude increased linearly in the falling asleep period in children, while in young adults, isolated high-amplitude slow waves (type I) dominated initially and numerous smaller slow waves (type II) with progressively increasing amplitude occurred later. Compared to young adults, children displayed faster increases in slow wave amplitude and number across the falling asleep period in central and posterior brain regions, respectively, and also showed larger slow waves during wakefulness immediately prior to sleep. Children do not display the two temporally dissociated slow wave synchronization processes in the falling asleep period observed in adults, suggesting that maturational factors underlie the temporal segregation of these two processes. Our findings provide novel perspectives for studying how sleep-related behaviors and dreaming differ between children and adults. Copyright © 2018 Elsevier Inc. All rights reserved.

Dynamical characterization of inactivation path in voltage-gated Na+ ion channel by non-equilibrium response spectroscopy

PubMed Central

Pal, Krishnendu; Gangopadhyay, Gautam

2016-01-01

ABSTRACT Inactivation path of voltage gated sodium channel has been studied here under various voltage protocols as it is the main governing factor for the periodic occurrence and shape of the action potential. These voltage protocols actually serve as non-equilibrium response spectroscopic tools to study the ion channel in non-equilibrium environment. In contrast to a lot of effort in finding the crystal structure based molecular mechanism of closed-state(CSI) and open-state inactivation(OSI); here our approach is to understand the dynamical characterization of inactivation. The kinetic flux as well as energetic contribution of the closed and open- state inactivation path is compared here for voltage protocols, namely constant, pulsed and oscillating. The non-equilibrium thermodynamic quantities used in response to these voltage protocols serve as improved characterization tools for theoretical understanding which not only agrees with the previously known kinetic measurements but also predict the energetically optimum processes to sustain the auto-regulatory mechanism of action potential and the consequent inactivation steps needed. The time dependent voltage pattern governs the population of the conformational states which when couple with characteristic rate parameters, the CSI and OSI selectivity arise dynamically to control the inactivation path. Using constant, pulsed and continuous oscillating voltage protocols we have shown that during depolarization the OSI path is more favored path of inactivation however, in the hyper-polarized situation the CSI is favored. It is also shown that the re-factorisation of inactivated sodium channel to resting state occurs via CSI path. Here we have shown how the subtle energetic and entropic cost due to the change in the depolarization magnitude determines the optimum path of inactivation. It is shown that an efficient CSI and OSI dynamical profile in principle can characterize the open-state drug blocking phenomena. PMID:27367642

Low pH inactivation for xenotropic gamma retrovirus in recombinant human TNF-α receptor immunoglobulin G and mechanism of inactivation.

PubMed

Ma, Rong; Cui, Xiaolan

2014-01-01

CHO-derived recombinant proteins for human therapeutic are used commonly. There are noninfectious endogenous retroviruses in CHO cells. Validation study for inactivation process is required. Murine xenotropic gamma retrovirus (X-MulV) is a model virus in validation study. In our previous study, optimum conditions for X-MulV inactivation were sifted. In this study, we performed a further research on low pH inactivation for evaluation of X-MulV clearance in manufacturing of recombinant human TNF-α receptor immunoglobulin G fusion proteins (rhTNF-α) for injection. Cell-based infectivity assay was used for the evaluation of X-MulV clearance. RhTNF-α were spiked with X-MulV and were inactivated at pH 3.60 ∼ 3.90, 25 ± 2 °C, and 0 ∼ 240 min, respectively. Samples incubated at the conditions for 15 ∼ 180 min were not inactivated effectively. For 4 h incubation, log10 reductions were achieved 5.0 log10. Biological activity of rhTNF-α incubated at pH 3.60, 25 °C for 4 h, which was assayed on murine L929 fibroblasts cells, was not affected by low pH. Env gene of X-MulV, which was detected by conventional PCR method for the first time, was not detected after incubation at pH 3.60, and it may be the mechanism of low pH inactivation. Copyright © 2013. Published by Elsevier Ltd.

Fast but fleeting: adaptive motor learning processes associated with aging and cognitive decline.

PubMed

Trewartha, Kevin M; Garcia, Angeles; Wolpert, Daniel M; Flanagan, J Randall

2014-10-01

Motor learning has been shown to depend on multiple interacting learning processes. For example, learning to adapt when moving grasped objects with novel dynamics involves a fast process that adapts and decays quickly-and that has been linked to explicit memory-and a slower process that adapts and decays more gradually. Each process is characterized by a learning rate that controls how strongly motor memory is updated based on experienced errors and a retention factor determining the movement-to-movement decay in motor memory. Here we examined whether fast and slow motor learning processes involved in learning novel dynamics differ between younger and older adults. In addition, we investigated how age-related decline in explicit memory performance influences learning and retention parameters. Although the groups adapted equally well, they did so with markedly different underlying processes. Whereas the groups had similar fast processes, they had different slow processes. Specifically, the older adults exhibited decreased retention in their slow process compared with younger adults. Within the older group, who exhibited considerable variation in explicit memory performance, we found that poor explicit memory was associated with reduced retention in the fast process, as well as the slow process. These findings suggest that explicit memory resources are a determining factor in impairments in the both the fast and slow processes for motor learning but that aging effects on the slow process are independent of explicit memory declines. Copyright © 2014 the authors 0270-6474/14/3413411-11$15.00/0.

Thermal inactivation kinetics of hepatitis A virus in homogenized clam meat (Mercenaria mercenaria).

PubMed

Bozkurt, H; D'Souza, D H; Davidson, P M

2015-09-01

Epidemiological evidence suggests that hepatitis A virus (HAV) is the most common pathogen transmitted by bivalve molluscs such as clams, cockles, mussels and oysters. This study aimed to generate thermal inactivation kinetics for HAV as a first step to design adequate thermal processes to control clam-associated HAV outbreaks. Survivor curves and thermal death curves were generated for different treatment times (0-6 min) at different temperatures (50-72°C) and Weibull and first-order models were compared. D-values for HAV ranged from 47·37 ± 1·23 to 1·55 ± 0·12 min for the first-order model and 64·43 ± 3·47 to 1·25 ± 0·45 min for the Weibull model at temperatures from 50 to 72°C. z-Values for HAV in clams were 12·97 ± 0·59°C and 14·83 ± 0·0·28°C using the Weibull and first-order model respectively. The calculated activation energies for the first-order and Weibull model were 145 and 170 kJ mole(-1) respectively. The Weibull model described the thermal inactivation behaviour of HAV better than the first-order model. This study provides novel and precise information on thermal inactivation kinetics of HAV in homogenized clams. This will enable reliable thermal process calculations for HAV inactivation in clams and closely related seafood. © 2015 The Society for Applied Microbiology.

Physicochemical changes and microbial inactivation after high-intensity ultrasound processing of prebiotic whey beverage applying different ultrasonic power levels.

PubMed

Guimarães, Jonas T; Silva, Eric Keven; Alvarenga, Verônica O; Costa, Ana Letícia R; Cunha, Rosiane L; Sant'Ana, Anderson S; Freitas, Monica Q; Meireles, M Angela A; Cruz, Adriano G

2018-06-01

In this work, we investigated the effects of the ultrasonic power (0, 200, 400 and 600 W) on non-thermal processing of an inulin-enriched whey beverage. We studied the effects of high-intensity ultrasound (HIUS) on microbial inactivation (aerobic mesophilic heterotrophic bacteria (AMHB), total and thermotolerant coliforms and yeasts and molds), zeta potential, microstructure (optical microscopy, particle size distribution), rheology, kinetic stability and color. The non-thermal processing applying 600 W of ultrasonic power was comparable to high-temperature short-time (HTST) treatment (75 °C for 15 s) concerning the inactivation of AMHB and yeasts and molds (2 vs 2 log and 0.2 vs 0.4 log, respectively), although HIUS has reached a lower output temperature (53 ± 3 °C). The HIUS was better than HTST to improve beverage kinetic stability, avoiding phase separation, which was mainly attributed to the decrease of particles size, denaturation of whey proteins and gelation of polysaccharides (inulin and gellan gum). Thus, non-thermal processing by HIUS seems to be an interesting technology for prebiotic dairy beverages production. Copyright © 2018 Elsevier B.V. All rights reserved.

Ventral, but not dorsal, hippocampus inactivation impairs reward memory expression and retrieval in contexts defined by proximal cues.

PubMed

Riaz, Sadia; Schumacher, Anett; Sivagurunathan, Seyon; Van Der Meer, Matthijs; Ito, Rutsuko

2017-07-01

The hippocampus (HPC) has been widely implicated in the contextual control of appetitive and aversive conditioning. However, whole hippocampal lesions do not invariably impair all forms of contextual processing, as in the case of complex biconditional context discrimination, leading to contention over the exact nature of the contribution of the HPC in contextual processing. Moreover, the increasingly well-established functional dissociation between the dorsal (dHPC) and ventral (vHPC) subregions of the HPC has been largely overlooked in the existing literature on hippocampal-based contextual memory processing in appetitively motivated tasks. Thus, the present study sought to investigate the individual roles of the dHPC and the vHPC in contextual biconditional discrimination (CBD) performance and memory retrieval. To this end, we examined the effects of transient post-acquisition pharmacological inactivation (using a combination of GABA A and GABA B receptor agonists muscimol and baclofen) of functionally distinct subregions of the HPC (CA1/CA3 subfields of the dHPC and vHPC) on CBD memory retrieval. Additional behavioral assays including novelty preference, light-dark box and locomotor activity test were also performed to confirm that the respective sites of inactivation were functionally silent. We observed robust deficits in CBD performance and memory retrieval following inactivation of the vHPC, but not the dHPC. Our data provides novel insight into the differential roles of the ventral and dorsal HPC in reward contextual processing, under conditions in which the context is defined by proximal cues. © 2017 Wiley Periodicals, Inc.

Inactivation of Salmonella enterica by UV-C Light Alone and in Combination with Mild Temperatures

PubMed Central

Gayán, E.; Serrano, M. J.; Raso, J.; Álvarez, I.

2012-01-01

The aim of this investigation was to study the efficacy of the combined processes of UV light and mild temperatures for the inactivation of Salmonella enterica subsp. enterica and to explore the mechanism of inactivation. The doses to inactivate the 99.99% (4D) of the initial population ranged from 18.03 (Salmonella enterica serovar Typhimurium STCC 878) to 12.75 J ml−1 (Salmonella enterica serovar Enteritidis ATCC 13076). The pH and water activity of the treatment medium did not change the UV tolerance, but it decreased exponentially by increasing the absorption coefficient. An inactivating synergistic effect was observed by applying simultaneous UV light and heat treatment (UV-H). A less synergistic effect was observed by applying UV light first and heat subsequently. UV did not damage cell envelopes, but the number of injured cells was higher after a UV-H treatment than after heating. The synergistic effect observed by combining simultaneous UV and heat treatment opens the possibility to design combined treatments for pasteurization of liquid food with high UV absorptivity, such as fruit juices. PMID:23001665

Nonthermal dielectric-barrier discharge plasma-induced inactivation involves oxidative DNA damage and membrane lipid peroxidation in Escherichia coli.

PubMed

Joshi, Suresh G; Cooper, Moogega; Yost, Adam; Paff, Michelle; Ercan, Utku K; Fridman, Gregory; Friedman, Gary; Fridman, Alexander; Brooks, Ari D

2011-03-01

Oxidative stress leads to membrane lipid peroxidation, which yields products causing variable degrees of detrimental oxidative modifications in cells. Reactive oxygen species (ROS) are the key regulators in this process and induce lipid peroxidation in Escherichia coli. Application of nonthermal (cold) plasma is increasingly used for inactivation of surface contaminants. Recently, we reported a successful application of nonthermal plasma, using a floating-electrode dielectric-barrier discharge (FE-DBD) technique for rapid inactivation of bacterial contaminants in normal atmospheric air (S. G. Joshi et al., Am. J. Infect. Control 38:293-301, 2010). In the present report, we demonstrate that FE-DBD plasma-mediated inactivation involves membrane lipid peroxidation in E. coli. Dose-dependent ROS, such as singlet oxygen and hydrogen peroxide-like species generated during plasma-induced oxidative stress, were responsible for membrane lipid peroxidation, and ROS scavengers, such as α-tocopherol (vitamin E), were able to significantly inhibit the extent of lipid peroxidation and oxidative DNA damage. These findings indicate that this is a major mechanism involved in FE-DBD plasma-mediated inactivation of bacteria.

Nonthermal Dielectric-Barrier Discharge Plasma-Induced Inactivation Involves Oxidative DNA Damage and Membrane Lipid Peroxidation in Escherichia coli▿

PubMed Central

Joshi, Suresh G.; Cooper, Moogega; Yost, Adam; Paff, Michelle; Ercan, Utku K.; Fridman, Gregory; Friedman, Gary; Fridman, Alexander; Brooks, Ari D.

2011-01-01

Oxidative stress leads to membrane lipid peroxidation, which yields products causing variable degrees of detrimental oxidative modifications in cells. Reactive oxygen species (ROS) are the key regulators in this process and induce lipid peroxidation in Escherichia coli. Application of nonthermal (cold) plasma is increasingly used for inactivation of surface contaminants. Recently, we reported a successful application of nonthermal plasma, using a floating-electrode dielectric-barrier discharge (FE-DBD) technique for rapid inactivation of bacterial contaminants in normal atmospheric air (S. G. Joshi et al., Am. J. Infect. Control 38:293-301, 2010). In the present report, we demonstrate that FE-DBD plasma-mediated inactivation involves membrane lipid peroxidation in E. coli. Dose-dependent ROS, such as singlet oxygen and hydrogen peroxide-like species generated during plasma-induced oxidative stress, were responsible for membrane lipid peroxidation, and ROS scavengers, such as α-tocopherol (vitamin E), were able to significantly inhibit the extent of lipid peroxidation and oxidative DNA damage. These findings indicate that this is a major mechanism involved in FE-DBD plasma-mediated inactivation of bacteria. PMID:21199923

A user-friendly and scalable process to prepare a ready-to-use inactivated vaccine: the example of heartwater in ruminants under tropical conditions.

PubMed

Marcelino, Isabel; Lefrançois, Thierry; Martinez, Dominique; Giraud-Girard, Ken; Aprelon, Rosalie; Mandonnet, Nathalie; Gaucheron, Jérôme; Bertrand, François; Vachiéry, Nathalie

2015-01-29

The use of cheap and thermoresistant vaccines in poor tropical countries for the control of animal diseases is a key issue. Our work aimed at designing and validating a process for the large-scale production of a ready-to-use inactivated vaccine for ruminants. Our model was heartwater caused by the obligate intracellular bacterium Ehrlichia ruminantium (ER). The conventional inactivated vaccine against heartwater (based on whole bacteria inactivated with sodium azide) is prepared immediately before injection, using a syringe-extrusion method with Montanide ISA50. This is a fastidious time-consuming process and it limits the number of vaccine doses available. To overcome these issues, we tested three different techniques (syringe, vortex and homogenizer) and three Montanide ISA adjuvants (50, 70 and 70M). High-speed homogenizer was the optimal method to emulsify ER antigens with both ISA70 and 70M adjuvants. The emulsions displayed a good homogeneity (particle size below 1 μm and low phase separation), conductivity below 10 μS/cm and low antigen degradation at 4 °C for up to 1 year. The efficacy of the different formulations was then evaluated during vaccination trials on goats. The inactivated ER antigens emulsified with ISA70 and ISA70M in a homogenizer resulted in 80% and 100% survival rates, respectively. A cold-chain rupture assay using ISA70M+ER was performed to mimic possible field conditions exposing the vaccine at 37 °C for 4 days before delivery. Surprisingly, the animal survival rate was still high (80%). We also observed that the MAP-1B antibody response was very similar between animals vaccinated with ISA70+ER and ISA70M+ER emulsions, suggesting a more homogenous antigen distribution and presentation in these emulsions. Our work demonstrated that the combination of ISA70 or ISA70M and homogenizer is optimal for the production of an effective ready-to-use inactivated vaccine against heartwater, which could easily be produced on an industrial scale. Copyright © 2014 Elsevier Ltd. All rights reserved.

A causal relationship between face-patch activity and face-detection behavior.

PubMed

Sadagopan, Srivatsun; Zarco, Wilbert; Freiwald, Winrich A

2017-04-04

The primate brain contains distinct areas densely populated by face-selective neurons. One of these, face-patch ML, contains neurons selective for contrast relationships between face parts. Such contrast-relationships can serve as powerful heuristics for face detection. However, it is unknown whether neurons with such selectivity actually support face-detection behavior. Here, we devised a naturalistic face-detection task and combined it with fMRI-guided pharmacological inactivation of ML to test whether ML is of critical importance for real-world face detection. We found that inactivation of ML impairs face detection. The effect was anatomically specific, as inactivation of areas outside ML did not affect face detection, and it was categorically specific, as inactivation of ML impaired face detection while sparing body and object detection. These results establish that ML function is crucial for detection of faces in natural scenes, performing a critical first step on which other face processing operations can build.

[Inactivation of Mycobacteria mucogenicum in drinking water: chlorine resistance and mechanism analysis].

PubMed

Zheng, Qi; Chen, Chao; Zhang, Xiao-Jian; Lu, Pin-Pin; Liu, Yuan-Yuan; Chen, Yu-Qiao

2013-02-01

In recent years, chlorine-resistant bacteria were detected in drinking water distribution systems which threatened the drinking water safety. Our group detected one strain named Mycobacteria mucogenicum from the drinking water distribution system of a city in south China. This paper studied chlorine resistance and mechanism of Mycobacteria mucogenicum. Inactivation experiments of one strain Mycobacteria mucogenicum were conducted with free chlorine, monochloramind and chlorine dioxide. The CT values of 99.9% inactivation by free chlorine, monochloramine and chlorine dioxide were detected as (76.25 +/- 47.55)mg.min.L-1, (1396 +/-382)mg.min.L-1, (13.5 +/- 4.9) mg.min L-1. Using transmission electronmicroscopy (TEM) observed the inactivation process of Mycobacteria mucogenicum. The bacteria surface hydrophobic of Mycobacteria mucogenicum was 37.2%. Mycobacteria mucogenicum has a higher hydrophobicity than other bacteria which prevented the diffusion of chlorine into cells. Mycobacteria mucogenicum is more resistant to chorine than other bacteria.

Electron beam inactivation of selected microbial pathogens and indicator organisms in aerobically and anaerobically digested sewage sludge.

PubMed

Praveen, Chandni; Jesudhasan, Palmy R; Reimers, Robert S; Pillai, Suresh D

2013-09-01

Microbial pathogens in municipal sewage sludges need to be inactivated prior to environmental disposal. The efficacy of high energy (10 MeV) e-beam irradiation to inactivate a variety of selected microbial pathogens and indicator organisms in aerobically and anaerobically digested sewage sludge was evaluated. Both bacterial and viral pathogens and indicator organisms are susceptible to e-beam irradiation. However, as expected there was a significant difference in their respective e-beam irradiation sensitivity. Somatic coliphages, bacterial endospores and enteric viruses were more resistant compared to bacterial pathogens. The current US EPA mandated 10 kGy minimum dose was capable of achieving significant reduction of both bacterial and viral pathogens. Somatic coliphages can be used as a microbial indicator for monitoring e-beam processes in terms of pathogen inactivation in sewage sludges. Copyright © 2013 Elsevier Ltd. All rights reserved.

Acute toxicity and inactivation tests of CO2 on invertebrates in drinking water treatment systems.

PubMed

Yin, Wen-Chao; Zhang, Jin-Song; Liu, Li-Jun; Zhao, Jian-Shu; Li, Tuo

2011-01-01

In addition to the esthetic problem caused by invertebrates, researchers are recently starting to be more aware of their potential importance in terms of public health. However, the inactivation methods of invertebrates which could proliferate in drinking water treatment systems are not well developed. The objective of this study is to assess the acute toxicity and inactivation effects of CO2 on familiar invertebrates in water treatment processes. The results of this study revealed that CO2 has a definite toxicity to familiar invertebrates. The values of 24-h LC50 (median lethal concentration) were calculated for each test with six groups of invertebrates. The toxicity of CO2 was higher with increasing concentrations in solution but was lower with the increase in size of the invertebrates. Above the concentration of 1,000 mg/L for the CO2 solution, the 100% inactivation time of all the invertebrates was less than 5 s, and in 15 min, the inactivation ratio showed a gradient descent with a decline in concentration. As seen for Mesocyclops thermocyclopoides, by dosing with a sodium bicarbonate solution first and adding a dilute hydrochloric acid solution 5 min later, it is possible to obtain a satisfactory inactivation effect in the GAC (granular activated carbon) filters.

Kinetics of Hydrothermal Inactivation of Endotoxins ▿

PubMed Central

Li, Lixiong; Wilbur, Chris L.; Mintz, Kathryn L.

2011-01-01

A kinetic model was established for the inactivation of endotoxins in water at temperatures ranging from 210°C to 270°C and a pressure of 6.2 × 106 Pa. Data were generated using a bench scale continuous-flow reactor system to process feed water spiked with endotoxin standard (Escherichia coli O113:H10). Product water samples were collected and quantified by the Limulus amebocyte lysate assay. At 250°C, 5-log endotoxin inactivation was achieved in about 1 s of exposure, followed by a lower inactivation rate. This non-log-linear pattern is similar to reported trends in microbial survival curves. Predictions and parameters of several non-log-linear models are presented. In the fast-reaction zone (3- to 5-log reduction), the Arrhenius rate constant fits well at temperatures ranging from 120°C to 250°C on the basis of data from this work and the literature. Both biphasic and modified Weibull models are comparable to account for both the high and low rates of inactivation in terms of prediction accuracy and the number of parameters used. A unified representation of thermal resistance curves for a 3-log reduction and a 3 D value associated with endotoxin inactivation and microbial survival, respectively, is presented. PMID:21193667

Inactivation of avirulent Yersinia pestis on food and food contact surfaces by ultraviolet light and freezing.

PubMed

Sommers, Christopher H; Sheen, Shiowshuh

2015-09-01

Yersinia pestis, the causative agent of plague, can occasionally be contracted as a naso-pharyngeal or gastrointestinal illness through consumption of contaminated meat. In this study, the use of 254 nm ultraviolet light (UV-C) to inactivate a multi-isolate cocktail of avirulent Y. pestis on food and food contact surfaces was investigated. When a commercial UV-C conveyor was used (5 mW/cm(2)/s) 0.5 J/cm(2) inactivated >7 log of the Y. pestis cocktail on agar plates. At 0.5 J/cm(2), UV-C inactivated ca. 4 log of Y. pestis in beef, chicken, and catfish, exudates inoculated onto high density polypropylene or polyethylene, and stainless steel coupons, and >6 log was eliminated at 1 J/cm(2). Approximately 1 log was inactivated on chicken breast, beef steak, and catfish fillet surfaces at a UV-C dose of 1 J/cm(2). UV-C treatment prior to freezing of the foods did not increase the inactivation of Y. pestis over freezing alone. These results indicate that routine use of UV-C during food processing would provide workers and consumers some protection against Y. pestis. Published by Elsevier Ltd.

Microwave as an emerging technology for the treatment of biohazardous waste: A mini-review.

PubMed

Zimmermann, Klaus

2017-05-01

Microwave is an emerging technology to treat biohazardous waste, including material from healthcare facilities. A screen of the peer-reviewed literature shows that only limited information may be found in this area of work and, furthermore, analysis of the references reveals that sometimes not all necessary aspects for the appropriate use of the technology are considered. Very often conventional microwave technology is applied for the inactivation of pathogens, which might make sense for certain applications but, on the other hand, may lead to the misbelief that microwave systems cannot be used for the inactivation of a solid "dry" waste. However, conventional microwave units have no means to control the inactivation process, and especially moisture content. But there are a few sophisticated microwave technologies with appropriate measurements allowing a validated inactivation of biohazardous materials. These technologies are an effective tool for inactivation and some of them are commercially available. It must also be considered that the waste should be preferably inactivated either directly at the place where it is generated or biohazardous waste should be transported only in closed systems. Moreover, microwave technology presents a possibility to save energy costs in comparison to the more widely used autoclaves. This mini-review will discuss important aspects for the use of microwave technology for the treatment of biohazardous waste.

The role of slow and persistent TTX-resistant sodium currents in acute tumor necrosis factor-α-mediated increase in nociceptors excitability

PubMed Central

Gudes, Sagi; Barkai, Omer; Caspi, Yaki; Katz, Ben; Lev, Shaya

2014-01-01

Tetrodotoxin-resistant (TTX-r) sodium channels are key players in determining the input-output properties of peripheral nociceptive neurons. Changes in gating kinetics or in expression levels of these channels by proinflammatory mediators are likely to cause the hyperexcitability of nociceptive neurons and pain hypersensitivity observed during inflammation. Proinflammatory mediator, tumor necrosis factor-α (TNF-α), is secreted during inflammation and is associated with the early onset, as well as long-lasting, inflammation-mediated increase in excitability of peripheral nociceptive neurons. Here we studied the underlying mechanisms of the rapid component of TNF-α-mediated nociceptive hyperexcitability and acute pain hypersensitivity. We showed that TNF-α leads to rapid onset, cyclooxygenase-independent pain hypersensitivity in adult rats. Furthermore, TNF-α rapidly and substantially increases nociceptive excitability in vitro, by decreasing action potential threshold, increasing neuronal gain and decreasing accommodation. We extended on previous studies entailing p38 MAPK-dependent increase in TTX-r sodium currents by showing that TNF-α via p38 MAPK leads to increased availability of TTX-r sodium channels by partial relief of voltage dependence of their slow inactivation, thereby contributing to increase in neuronal gain. Moreover, we showed that TNF-α also in a p38 MAPK-dependent manner increases persistent TTX-r current by shifting the voltage dependence of activation to a hyperpolarized direction, thus producing an increase in inward current at functionally critical subthreshold voltages. Our results suggest that rapid modulation of the gating of TTX-r sodium channels plays a major role in the mediated nociceptive hyperexcitability of TNF-α during acute inflammation and may lead to development of effective treatments for inflammatory pain, without modulating the inflammation-induced healing processes. PMID:25355965

Origin and evolution of the long non-coding genes in the X-inactivation center.

PubMed

Romito, Antonio; Rougeulle, Claire

2011-11-01

Random X chromosome inactivation (XCI), the eutherian mechanism of X-linked gene dosage compensation, is controlled by a cis-acting locus termed the X-inactivation center (Xic). One of the striking features that characterize the Xic landscape is the abundance of loci transcribing non-coding RNAs (ncRNAs), including Xist, the master regulator of the inactivation process. Recent comparative genomic analyses have depicted the evolutionary scenario behind the origin of the X-inactivation center, revealing that this locus evolved from a region harboring protein-coding genes. During mammalian radiation, this ancestral protein-coding region was disrupted in the marsupial group, whilst it provided in eutherian lineage the starting material for the non-translated RNAs of the X-inactivation center. The emergence of non-coding genes occurred by a dual mechanism involving loss of protein-coding function of the pre-existing genes and integration of different classes of mobile elements, some of which modeled the structure and sequence of the non-coding genes in a species-specific manner. The rising genes started to produce transcripts that acquired function in regulating the epigenetic status of the X chromosome, as shown for Xist, its antisense Tsix, Jpx, and recently suggested for Ftx. Thus, the appearance of the Xic, which occurred after the divergence between eutherians and marsupials, was the basis for the evolution of random X inactivation as a strategy to achieve dosage compensation. Copyright © 2011. Published by Elsevier Masson SAS.

Membrane Permeabilization in Relation to Inactivation Kinetics of Lactobacillus Species due to Pulsed Electric Fields

PubMed Central

Wouters, Patrick C.; Bos, Ad P.; Ueckert, Joerg

2001-01-01

Membrane permeabilization due to pulsed electric field (PEF) treatment of gram-positive Lactobacillus cells was investigated by using propidium iodide uptake and single-cell analysis with flow cytometry. Electric field strength, energy input, treatment time, and growth phase affected membrane permeabilization of Lactobacillus plantarum during PEF treatment. A correlation between PEF inactivation and membrane permeabilization of L. plantarum cells was demonstrated, whereas no relationship was observed between membrane permeabilization and heat inactivation. The same results were obtained with a Lactobacillus fermentum strain, but the latter organism was more PEF resistant and exhibited less membrane permeabilization, indicating that various bacteria have different responses to PEF treatment. While membrane permeabilization was the main factor involved in the mechanism of inactivation, the growth phase and the acidity of the environment also influenced inactivation. By using flow cytometry it was possible to sort cells in the L. plantarum population based on different cell sizes and shapes, and the results were confirmed by image analysis. An apparent effect of morphology on membrane permeabilization was observed, and larger cells were more easily permeabilized than smaller cells. In conclusion, our results indicate that the ability of PEF treatment to cause membrane permeabilization is an important factor in determining inactivation. This finding should have an effect on the final choice of the processing parameters used so that all microorganisms can be inactivated and, consequently, on the use of PEF treatment as an alternative method for preserving food products. PMID:11425727

How the early sporulation sigma factor sigmaF delays the switch to late development in Bacillus subtilis.

PubMed

Karmazyn-Campelli, Céline; Rhayat, Lamya; Carballido-López, Rut; Duperrier, Sandra; Frandsen, Niels; Stragier, Patrick

2008-03-01

Sporulation in Bacillus subtilis is a primitive differentiation process involving two cell types, the forespore and the mother cell. Each cell implements two successive transcription programmes controlled by specific sigma factors. We report that activity of sigma(G), the late forespore sigma factor, is kept in check by Gin, the product of csfB, a gene controlled by sigma(F), the early forespore sigma factor. Gin abolishes sigma(G) transcriptional activity when sigma(G) is artificially synthesized during growth, but has no effect on sigma(F). Gin interacts strongly with sigma(G) but not with sigma(F) in a yeast two-hybrid experiment. The absence of Gin allows sigma(G) to be active during sporulation independently of the mother-cell development to which it is normally coupled. Premature sigma(G) activity leads to the formation of slow-germinating spores, and complete deregulation of sigma(G) synthesis is lethal when combined with gin inactivation. Gin allows sigma(F) to delay the switch to the late forespore transcription programme by preventing sigma(G) to take over before the cell has reached a critical stage of development. A similar strategy, following a completely unrelated route, is used by the mother cell.

Isoflurane modulates neuronal excitability of the nucleus reticularis thalami in vitro.

PubMed

Joksovic, Pavle M; Todorovic, Slobodan M

2010-06-01

The thalamus has a key function in processing sensory information, sleep, and cognition. We examined the effects of a common volatile anesthetic, isoflurane, on modulation of neuronal excitability in reticular thalamic nucleus (nRT) in intact brain slices from immature rats. In current-clamp recordings, isoflurane (300-600 micromol/L) consistently depolarized membrane potential, decreased input resistance, and inhibited both rebound burst firing and tonic spike firing modes of nRT neurons. The isoflurane-induced depolarization persisted not only in the presence of tetrodotoxin, but after replacement of Ca(2+) with Ba(2+) ions in external solution; it was abolished by partial replacement of extracellular Na(+) ions with N-methyl-D-glucamine. In voltage-clamp recordings, we found that isoflurane slowed recovery from inactivation of T-type Ca(2+) current. Thus, at clinically relevant concentrations, isoflurane inhibits neuronal excitability of nRT neurons in developing brain via multiple ion channels. Inhibition of the neuronal excitability of thalamic cells may contribute to impairment of sensory information transfer in the thalamocortical network by general anesthetics. The findings may be important for understanding cellular mechanisms of anesthesia, such as loss of consciousness and potentially damaging consequences of general anesthetics on developing mammalian brains.

The effect of oxaloacetic acid on tyrosinase activity and structure: Integration of inhibition kinetics with docking simulation.

PubMed

Gou, Lin; Lee, Jinhyuk; Hao, Hao; Park, Yong-Doo; Zhan, Yi; Lü, Zhi-Rong

2017-08-01

Oxaloacetic acid (OA) is naturally found in organisms and well known as an intermediate of citric acid cycle producing ATP. We evaluated the effects of OA on tyrosinase activity and structure via integrating methods of enzyme kinetics and computational simulations. OA was found to be a reversible inhibitor of tyrosinase and its induced mechanism was the parabolic non-competitive inhibition type (IC 50 =17.5±0.5mM and K i =6.03±1.36mM). Kinetic measurements by real-time interval assay showed that OA induced multi-phasic inactivation process composing with fast (k 1 ) and slow (k 2 ) phases. Spectrofluorimetry studies showed that OA mainly induced regional changes in the active site of tyrosinase accompanying with hydrophobic disruption at high dose. The computational docking simulations further revealed that OA could interact with several residues near the tyrosinase active site pocket such as HIS61, HIS259, HIS263, and VAL283. Our study provides insight into the mechanism by which energy producing intermediate such as OA inhibit tyrosinase and OA is a potential natural anti-pigmentation agent. Copyright © 2017 Elsevier B.V. All rights reserved.

Inflammation-induced increase in nicotinic acetylcholine receptor current in cutaneous nociceptive DRG neurons from the adult rat.

PubMed

Zhang, X-L; Albers, K M; Gold, M S

2015-01-22

The goals of the present study were to determine (1) the properties of the nicotinic acetylcholine receptor (nAChR) currents in rat cutaneous dorsal root ganglion (DRG) neurons; (2) the impact of nAChR activation on the excitability of cutaneous DRG neurons; and (3) the impact of inflammation on the density and distribution of nAChR currents among cutaneous DRG neurons. Whole-cell patch-clamp techniques were used to study retrogradely labeled DRG neurons from naïve and complete Freund's adjuvant inflamed rats. Nicotine-evoked currents were detectable in ∼70% of the cutaneous DRG neurons, where only one of two current types, fast or slow currents based on rates of activation and inactivation, was present in each neuron. The biophysical and pharmacological properties of the fast current were consistent with nAChRs containing an α7 subunit while those of the slow current were consistent with nAChRs containing α3/β4 subunits. The majority of small diameter neurons with fast current were IB4- while the majority of small diameter neurons with slow current were IB4+. Preincubation with nicotine (1 μM) produced a transient (1 min) depolarization and increase in the excitability of neurons with fast current and a decrease in the amplitude of capsaicin-evoked current in neurons with slow current. Inflammation increased the current density of both slow and fast currents in small diameter neurons and increased the percentage of neurons with the fast current. With the relatively selective distribution of nAChR currents in putative nociceptive cutaneous DRG neurons, our results suggest that the role of these receptors in inflammatory hyperalgesia is likely to be complex and dependent on the concentration and timing of acetylcholine release in the periphery. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Inactivation of Bacillus anthracis Spores by a Combination of Biocides and Heating under High-Temperature Short-Time Pasteurization Conditions ▿

PubMed Central

Xu, Sa; Labuza, Theodore P.; Diez-Gonzalez, Francisco

2008-01-01

The milk supply is considered a primary route for a bioterrorism attack with Bacillus anthracis spores because typical high-temperature short-time (HTST) pasteurization conditions cannot inactivate spores. In the event of intentional contamination, an effective method to inactivate the spores in milk under HTST processing conditions is needed. This study was undertaken to identify combinations and concentrations of biocides that can inactivate B. anthracis spores at temperatures in the HTST range in less than 1 min. Hydrogen peroxide (HP), sodium hypochlorite (SH), and peroxyacetic acid (PA) were evaluated for their efficacy in inactivating spores of strains 7702, ANR-1, and 9131 in milk at 72, 80, and 85°C using a sealed capillary tube technique. Strains ANR-1 and 9131 were more resistant to all of the biocide treatments than strain 7702. Addition of 1,260 ppm SH to milk reduced the number of viable spores of each strain by 6 log CFU/ml in less than 90 and 60 s at 72 and 80°C, respectively. After neutralization, 1,260 ppm SH reduced the time necessary to inactivate 6 log CFU/ml (TTI6-log) at 80°C to less than 20 s. Treatment of milk with 7,000 ppm HP resulted in a similar level of inactivation in 60 s. Combined treatment with 1,260 ppm SH and 1,800 ppm HP inactivated spores of all strains in less than 20 s at 80°C. Mixing 15 ppm PA with milk containing 1,260 ppm SH resulted in TTI6-log of 25 and 12 s at 72 and 80°C, respectively. TTI6-log of less than 20 s were also achieved at 80°C by using two combinations of biocides: 250 ppm SH, 700 ppm HP, and 150 ppm PA; and 420 ppm SH (pH 7), 1,100 ppm HP, and 15 ppm PA. These results indicated that different combinations of biocides could consistently result in 6-log reductions in the number of B. anthracis spores in less than 1 min at temperatures in the HTST range. This information could be useful for developing more effective thermal treatment strategies which could be used in HTST milk plants to process contaminated milk for disposal and decontamination, as well as for potential protective measures. PMID:18390680

Inactivation of Bacillus anthracis spores by a combination of biocides and heating under high-temperature short-time pasteurization conditions.

PubMed

Xu, Sa; Labuza, Theodore P; Diez-Gonzalez, Francisco

2008-06-01

The milk supply is considered a primary route for a bioterrorism attack with Bacillus anthracis spores because typical high-temperature short-time (HTST) pasteurization conditions cannot inactivate spores. In the event of intentional contamination, an effective method to inactivate the spores in milk under HTST processing conditions is needed. This study was undertaken to identify combinations and concentrations of biocides that can inactivate B. anthracis spores at temperatures in the HTST range in less than 1 min. Hydrogen peroxide (HP), sodium hypochlorite (SH), and peroxyacetic acid (PA) were evaluated for their efficacy in inactivating spores of strains 7702, ANR-1, and 9131 in milk at 72, 80, and 85 degrees C using a sealed capillary tube technique. Strains ANR-1 and 9131 were more resistant to all of the biocide treatments than strain 7702. Addition of 1,260 ppm SH to milk reduced the number of viable spores of each strain by 6 log CFU/ml in less than 90 and 60 s at 72 and 80 degrees C, respectively. After neutralization, 1,260 ppm SH reduced the time necessary to inactivate 6 log CFU/ml (TTI6-log) at 80 degrees C to less than 20 s. Treatment of milk with 7,000 ppm HP resulted in a similar level of inactivation in 60 s. Combined treatment with 1,260 ppm SH and 1,800 ppm HP inactivated spores of all strains in less than 20 s at 80 degrees C. Mixing 15 ppm PA with milk containing 1,260 ppm SH resulted in TTI6-log of 25 and 12 s at 72 and 80 degrees C, respectively. TTI6-log of less than 20 s were also achieved at 80 degrees C by using two combinations of biocides: 250 ppm SH, 700 ppm HP, and 150 ppm PA; and 420 ppm SH (pH 7), 1,100 ppm HP, and 15 ppm PA. These results indicated that different combinations of biocides could consistently result in 6-log reductions in the number of B. anthracis spores in less than 1 min at temperatures in the HTST range. This information could be useful for developing more effective thermal treatment strategies which could be used in HTST milk plants to process contaminated milk for disposal and decontamination, as well as for potential protective measures.

Phasic Firing in Vasopressin Cells: Understanding Its Functional Significance through Computational Models

PubMed Central

MacGregor, Duncan J.; Leng, Gareth

2012-01-01

Vasopressin neurons, responding to input generated by osmotic pressure, use an intrinsic mechanism to shift from slow irregular firing to a distinct phasic pattern, consisting of long bursts and silences lasting tens of seconds. With increased input, bursts lengthen, eventually shifting to continuous firing. The phasic activity remains asynchronous across the cells and is not reflected in the population output signal. Here we have used a computational vasopressin neuron model to investigate the functional significance of the phasic firing pattern. We generated a concise model of the synaptic input driven spike firing mechanism that gives a close quantitative match to vasopressin neuron spike activity recorded in vivo, tested against endogenous activity and experimental interventions. The integrate-and-fire based model provides a simple physiological explanation of the phasic firing mechanism involving an activity-dependent slow depolarising afterpotential (DAP) generated by a calcium-inactivated potassium leak current. This is modulated by the slower, opposing, action of activity-dependent dendritic dynorphin release, which inactivates the DAP, the opposing effects generating successive periods of bursting and silence. Model cells are not spontaneously active, but fire when perturbed by random perturbations mimicking synaptic input. We constructed one population of such phasic neurons, and another population of similar cells but which lacked the ability to fire phasically. We then studied how these two populations differed in the way that they encoded changes in afferent inputs. By comparison with the non-phasic population, the phasic population responds linearly to increases in tonic synaptic input. Non-phasic cells respond to transient elevations in synaptic input in a way that strongly depends on background activity levels, phasic cells in a way that is independent of background levels, and show a similar strong linearization of the response. These findings show large differences in information coding between the populations, and apparent functional advantages of asynchronous phasic firing. PMID:23093929

High-Pressure Inactivation of Rotaviruses: Role of Treatment Temperature and Strain Diversity in Virus Inactivation

PubMed Central

Araud, Elbashir; DiCaprio, Erin; Yang, Zhihong; Li, Xinhui; Lou, Fangfei; Hughes, John H.; Chen, Haiqiang

2015-01-01

Rotavirus (RV) is the major etiological agent of acute gastroenteritis in infants worldwide. Although high-pressure processing (HPP) is a popular method to inactivate enteric pathogens in food, the sensitivity of different virus strains within same species and serotype to HPP is variable. This study aimed to compare the barosensitivities of seven RV strains derived from four serotypes (serotype G1, strains Wa, Ku, and K8; serotype G2, strain S2; serotype G3, strains SA-11 and YO; and serotype G4, strain ST3) following high-pressure treatment. RV strains showed various responses to HPP based on the initial temperature and had different inactivation profiles. Ku, K8, S2, SA-11, YO, and ST3 showed enhanced inactivation at 4°C compared to 20°C. In contrast, strain Wa was not significantly impacted by the initial treatment temperature. Within serotype G1, strain Wa was significantly (P < 0.05) more resistant to HPP than strains Ku and K8. Overall, the resistance of the human RV strains to HPP at 4°C can be ranked as Wa > Ku = K8 > S2 > YO > ST3, and in terms of serotype the ranking is G1 > G2 > G3 > G4. In addition, pressure treatment of 400 MPa for 2 min was sufficient to eliminate the Wa strain, the most pressure-resistant RV, from oyster tissues. HPP disrupted virion structure but did not degrade viral protein or RNA, providing insight into the mechanism of viral inactivation by HPP. In conclusion, HPP is capable of inactivating RV at commercially acceptable pressures, and the efficacy of inactivation is strain dependent. PMID:26187961

Familial skewed X inactivation: a molecular trait associated with high spontaneous-abortion rate maps to Xq28.

PubMed Central

Pegoraro, E; Whitaker, J; Mowery-Rushton, P; Surti, U; Lanasa, M; Hoffman, E P

1997-01-01

We report a family ascertained for molecular diagnosis of muscular dystrophy in a young girl, in which preferential activation (> or = 95% of cells) of the paternal X chromosome was seen in both the proband and her mother. To determine the molecular basis for skewed X inactivation, we studied X-inactivation patterns in peripheral blood and/or oral mucosal cells from 50 members of this family and from a cohort of normal females. We found excellent concordance between X-inactivation patterns in blood and oral mucosal cell nuclei in all females. Of the 50 female pedigree members studied, 16 showed preferential use (> or = 95% cells) of the paternal X chromosome; none of 62 randomly selected females showed similarly skewed X inactivation was maternally inherited in this family. A linkage study using the molecular trait of skewed X inactivation as the scored phenotype localized this trait to Xq28 (DXS1108; maximum LOD score [Zmax] = 4.34, recombination fraction [theta] = 0). Both genotyping of additional markers and FISH of a YAC probe in Xq28 showed a deletion spanning from intron 22 of the factor VIII gene to DXS115-3. This deletion completely cosegregated with the trait (Zmax = 6.92, theta = 0). Comparison of clinical findings between affected and unaffected females in the 50-member pedigree showed a statistically significant increase in spontaneous-abortion rate in the females carrying the trait (P < .02). To our knowledge, this is the first gene-mapping study of abnormalities of X-inactivation patterns and is the first association of a specific locus for recurrent spontaneous abortion in a cytogenetically normal family. The involvement of this locus in cell lethality, cell-growth disadvantage, developmental abnormalities, or the X-inactivation process is discussed. Images Figure 4 Figure 7 PMID:9245997

Influence of heat inactivation of human serum on the opsonization of Streptococcus mutans.

PubMed

Moore, M A; Hakki, Z W; Gregory, R L; Gfell, L E; Kim-Park, W K; Kowolik, M J

1997-12-15

Phagocytosis of bacteria, such as Streptococcus mutans, is important to host defense. One mechanism by which phagocytosis can be enhanced is by antibody or complement-mediated opsonization of bacteria. Many studies utilize opsonization of bacteria to enhance a cellular response, but little information has been found examining methodology or validity of the opsonization process following the denaturization of the serum. Human serum was inactivated by heat in order to disrupt the classical and alternative pathways of the complement cascade. S. mutans isolated from human subjects were opsonized with heat-inactivated human serum before exposing them to viable neutrophils in vitro. Luminol-dependent chemiluminescence (CL) was used to measure neutrophil activation. Human serum used to opsonize the bacteria was denatured by incubation at 57 degrees C for intervals of 30 and 60 min to inactivate complement. The results from the opsonization data indicated that there was significantly increased CL with 60-min inactivation of the serum (34% increase in mean integration mV.min; p < or = 0.05) over the nonopsonized control. This indicated a successful opsonization of the bacteria. In addition, the data demonstrate that the inactivation of serum requires a minimum of 60 min at 57 degrees C to disrupt the complement cascade, while 30- and 15-min inactivations produced no significant increase in CL activity over the control. Standard sandwich ELISA assays, detecting complement binding to S. mutans, confirmed successful heat inactivation of serum showing a significant decrease (p < or = 0.001) in complement binding to S. mutans after 30 min, but could not explain the increased CL response after 60-min heat deactivation of the serum.

Comparison of Thermal and Non-Thermal Processing of Swine Feed and the Use of Selected Feed Additives on Inactivation of Porcine Epidemic Diarrhea Virus (PEDV)

PubMed Central

Trudeau, Michaela P.; Verma, Harsha; Sampedro, Fernando; Urriola, Pedro E.; Shurson, Gerald C.; McKelvey, Jessica; Pillai, Suresh D.; Goyal, Sagar M.

2016-01-01

Infection with porcine epidemic diarrhea virus (PEDV) causes diarrhea, vomiting, and high mortality in suckling pigs. Contaminated feed has been suggested as a vehicle of transmission for PEDV. The objective of this study was to compare thermal and electron beam processing, and the inclusion of feed additives on the inactivation of PEDV in feed. Feed samples were spiked with PEDV and then heated to 120–145°C for up to 30 min or irradiated at 0–50 kGy. Another set of feed samples spiked with PEDV and mixed with Ultracid P (Nutriad), Activate DA (Novus International), KEM-GEST (Kemin Agrifood), Acid Booster (Agri-Nutrition), sugar or salt was incubated at room temperature (~25°C) for up to 21 days. At the end of incubation, the virus titers were determined by inoculation of Vero-81 cells and the virus inactivation kinetics were modeled using the Weibull distribution model. The Weibull kinetic parameter delta represented the time or eBeam dose required to reduce virus concentration by 1 log. For thermal processing, delta values ranged from 16.52 min at 120°C to 1.30 min at 145°C. For eBeam processing, a target dose of 50 kGy reduced PEDV concentration by 3 log. All additives tested were effective in reducing the survival of PEDV when compared with the control sample (delta = 17.23 days). Activate DA (0.81) and KEM-GEST (3.28) produced the fastest inactivation. In conclusion, heating swine feed at temperatures over 130°C or eBeam processing of feed with a dose over 50 kGy are effective processing steps to reduce PEDV survival. Additionally, the inclusion of selected additives can decrease PEDV survivability. PMID:27341670

Pathogen Inactivated Plasma Concentrated: Preparation and Uses

DTIC Science & Technology

2004-09-01

REPORT DATE 01 SEP 2004 2 . REPORT TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Pathogen Inactivated Plasma Concentrated: Preparation...Concentrated: Preparation and Uses 22 - 2 RTO-MP-HFM-109 Results: Both UVC and ozone yielded a PPV logarithmic reduction factor (LRF) of 6, for a...technology to be marketed; the industry name is Plas+SD [ 2 ]. This process functions by attacking the lipid sheathes that surround enveloped viruses

β1 subunit stabilises sodium channel Nav1.7 against mechanical stress.

PubMed

Körner, Jannis; Meents, Jannis; Machtens, Jan-Philipp; Lampert, Angelika

2018-06-01

The voltage-gated sodium channel Nav1.7 is a key player in neuronal excitability and pain signalling. In addition to voltage sensing, the channel is also modulated by mechanical stress. Using whole-cell patch-clamp experiments, we discovered that the sodium channel subunit β1 is able to prevent the impact of mechanical stress on Nav1.7. An intramolecular disulfide bond of β1 was identified to be essential for stabilisation of inactivation, but not activation, against mechanical stress using molecular dynamics simulations, homology modelling and site-directed mutagenesis. Our results highlight the role of segment 6 of domain IV in fast inactivation. We present a candidate mechanism for sodium channel stabilisation against mechanical stress, ensuring reliable channel functionality in living systems. Voltage-gated sodium channels are key players in neuronal excitability and pain signalling. Precise gating of these channels is crucial as even small functional alterations can lead to pathological phenotypes such as pain or heart failure. Mechanical stress has been shown to affect sodium channel activation and inactivation. This suggests that stabilising components are necessary to ensure precise channel gating in living organisms. Here, we show that mechanical shear stress affects voltage dependence of activation and fast inactivation of the Nav1.7 channel. Co-expression of the β1 subunit, however, protects both gating modes of Nav1.7 against mechanical shear stress. Using molecular dynamics simulation, homology modelling and site-directed mutagenesis, we identify an intramolecular disulfide bond of β1 (Cys21-Cys43) which is partially involved in this process: the β1-C43A mutant prevents mechanical modulation of voltage dependence of activation, but not of fast inactivation. Our data emphasise the unique role of segment 6 of domain IV for sodium channel fast inactivation and confirm previous reports that the intracellular process of fast inactivation can be modified by interfering with the extracellular end of segment 6 of domain IV. Thus, our data suggest that physiological gating of Nav1.7 may be protected against mechanical stress in a living organism by assembly with the β1 subunit. © 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.

Variants of β-Lactamase KPC-2 That Are Resistant to Inhibition by Avibactam

PubMed Central

Papp-Wallace, Krisztina M.; Winkler, Marisa L.; Taracila, Magdalena A.

2015-01-01

KPC-2 is the most prevalent class A carbapenemase in the world. Previously, KPC-2 was shown to hydrolyze the β-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam. In addition, substitutions at amino acid position R220 in the KPC-2 β-lactamase increased resistance to clavulanic acid. A novel bridged diazabicyclooctane (DBO) non-β-lactam β-lactamase inhibitor, avibactam, was shown to inactivate the KPC-2 β-lactamase. To better understand the mechanistic basis for inhibition of KPC-2 by avibactam, we tested the potency of ampicillin-avibactam and ceftazidime-avibactam against engineered variants of the KPC-2 β-lactamase that possessed single amino acid substitutions at important sites (i.e., Ambler positions 69, 130, 234, 220, and 276) that were previously shown to confer inhibitor resistance in TEM and SHV β-lactamases. To this end, we performed susceptibility testing, biochemical assays, and molecular modeling. Escherichia coli DH10B carrying KPC-2 β-lactamase variants with the substitutions S130G, K234R, and R220M demonstrated elevated MICs for only the ampicillin-avibactam combinations (e.g., 512, 64, and 32 mg/liter, respectively, versus the MICs for wild-type KPC-2 at 2 to 8 mg/liter). Steady-state kinetics revealed that the S130G variant of KPC-2 resisted inactivation by avibactam; the k2/K ratio was significantly lowered 4 logs for the S130G variant from the ratio for the wild-type enzyme (21,580 M−1 s−1 to 1.2 M−1 s−1). Molecular modeling and molecular dynamics simulations suggested that the mobility of K73 and its ability to activate S70 (i.e., function as a general base) may be impaired in the S130G variant of KPC-2, thereby explaining the slowed acylation. Moreover, we also advance the idea that the protonation of the sulfate nitrogen of avibactam may be slowed in the S130G variant, as S130 is the likely proton donor and another residue, possibly K234, must compensate. Our findings show that residues S130 as well as K234 and R220 contribute significantly to the mechanism of avibactam inactivation of KPC-2. Fortunately, the emergence of S130G, K234R, and R220M variants of KPC in the clinic should not result in failure of ceftazidime-avibactam, as the ceftazidime partner is potent against E. coli DH10B strains possessing all of these variants. PMID:25666153

Variants of β-lactamase KPC-2 that are resistant to inhibition by avibactam.

PubMed

Papp-Wallace, Krisztina M; Winkler, Marisa L; Taracila, Magdalena A; Bonomo, Robert A

2015-07-01

KPC-2 is the most prevalent class A carbapenemase in the world. Previously, KPC-2 was shown to hydrolyze the β-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam. In addition, substitutions at amino acid position R220 in the KPC-2 β-lactamase increased resistance to clavulanic acid. A novel bridged diazabicyclooctane (DBO) non-β-lactam β-lactamase inhibitor, avibactam, was shown to inactivate the KPC-2 β-lactamase. To better understand the mechanistic basis for inhibition of KPC-2 by avibactam, we tested the potency of ampicillin-avibactam and ceftazidime-avibactam against engineered variants of the KPC-2 β-lactamase that possessed single amino acid substitutions at important sites (i.e., Ambler positions 69, 130, 234, 220, and 276) that were previously shown to confer inhibitor resistance in TEM and SHV β-lactamases. To this end, we performed susceptibility testing, biochemical assays, and molecular modeling. Escherichia coli DH10B carrying KPC-2 β-lactamase variants with the substitutions S130G, K234R, and R220M demonstrated elevated MICs for only the ampicillin-avibactam combinations (e.g., 512, 64, and 32 mg/liter, respectively, versus the MICs for wild-type KPC-2 at 2 to 8 mg/liter). Steady-state kinetics revealed that the S130G variant of KPC-2 resisted inactivation by avibactam; the k2/K ratio was significantly lowered 4 logs for the S130G variant from the ratio for the wild-type enzyme (21,580 M(-1) s(-1) to 1.2 M(-1) s(-1)). Molecular modeling and molecular dynamics simulations suggested that the mobility of K73 and its ability to activate S70 (i.e., function as a general base) may be impaired in the S130G variant of KPC-2, thereby explaining the slowed acylation. Moreover, we also advance the idea that the protonation of the sulfate nitrogen of avibactam may be slowed in the S130G variant, as S130 is the likely proton donor and another residue, possibly K234, must compensate. Our findings show that residues S130 as well as K234 and R220 contribute significantly to the mechanism of avibactam inactivation of KPC-2. Fortunately, the emergence of S130G, K234R, and R220M variants of KPC in the clinic should not result in failure of ceftazidime-avibactam, as the ceftazidime partner is potent against E. coli DH10B strains possessing all of these variants. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Suicide Inhibition of Cytochrome P450 Enzymes by Cyclopropylamines via a Ring-opening Mechanism: Proton-Coupled Electron Transfer Makes a Difference

NASA Astrophysics Data System (ADS)

Zhang, Xiaoqian; Li, Xiao-Xi; Liu, Yufang; Wang, Yong

2017-01-01

N-benzyl-N-cyclopropylamine (BCA) has been attracting great interests for decades for its partial suicide inactivation role to cytochrome P450 (P450) via a ring-opening mechanism besides acting as a role of normal substrates. Understanding the mechanism of such partial inactivation is vital to the clinical drug design. Thus, density functional theoretical (DFT) calculations were carried out on such P450-catalyzed reactions, not only on the metabolic pathway, but on the ring-opening inactivation one. Our theoretical results demonstrated that, in the metabolic pathway, besides the normal carbinolamine, an unexpected enamine was formed via the dual hydrogen abstraction (DHA) process, in which the competition between rotation of the H-abstracted substrate radical and the rotation of hydroxyl group of the protonated Cpd II moiety plays a significant role in product branch; In the inactivation pathway, the well-noted single electron transfer (SET) mechanism-involved process was invalidated for its high energy barrier, a proton-coupled electron transfer (PCET(ET)) mechanism plays a role. Our results are consistent with other related theoretical works on heteroatom-hydrogen (X-H, X = O, N) activation and revealed new features. The revealed mechanisms will play a positive role in relative drug design.

Influence of yogurt fermentation and refrigerated storage on the stability of protein toxin contaminants.

PubMed

Jackson, Lauren S; Triplett, Odbert A; Tolleson, William H

2015-06-01

Dairy products sold in a ready-to-eat form present the risk that adulterants persisting through manufacturing, storage, and distribution would reach consumers. Pathogenic microbes, including shigatoxigenic strains of Escherichia coli and the toxins they produce, are common food safety hazards associated with dairy products. Ricin and abrin are plant-derived ribosome-inactivating protein toxins related to the shiga-like toxins produced by E. coli. Limited information exists on the effects of manufacturing processes on the stabilities of these heat-resistant ribosome-inactivating proteins in the presence of foods. The goal of this study was to determine how typical yogurt manufacturing and storage processes influence ribosome-inactivating protein toxins. Ricin and abrin were added to skim or whole milk and batch pasteurized. Complete inactivation of both toxins was observed after 30 minutes at 85 °C. If the toxins were added after pasteurization, the levels of ricin and abrin in yogurt and their cytotoxic activities did not change significantly during fermentation or refrigerated storage for 4 weeks. The activities of ricin and abrin were inhibited by skim milk, nonfat yogurt, whole milk, and whole milk yogurt. The results showed minimal effects of the toxins on yogurt pH and %titratable acidity but inhibitory effects of yogurt on toxin activity. Published by Elsevier Ltd.

Ultra-Slow Dielectric Relaxation Process in Polyols

NASA Astrophysics Data System (ADS)

Yomogida, Yoshiki; Minoguchi, Ayumi; Nozaki, Ryusuke

2004-04-01

Dielectric relaxation processes with relaxation times larger than that for the structural α process are reported for glycerol, xylitol, sorbitol and their mixtures for the first time. Appearance of this ultra-slow process depends on cooling rate. More rapid cooling gives larger dielectric relaxation strength. However, relaxation time is not affected by cooling rate and shows non-Arrhenius temperature dependence with correlation to the α process. It can be considered that non-equilibrium dynamic structure causes the ultra-slow process. Scale of such structure would be much larger than that of the region for the cooperative molecular orientations for the α process.

Reading strategies of fast and slow readers.

PubMed

Haberlandt, K F; Graesser, A C; Schneider, N J

1989-09-01

In three subject-paced experiments we evaluated reading patterns at the word, line, and sentence level for fast and slow readers. A moving-window method was used to collect word reading times for natural texts. At the word level, reading times of word N were influenced by features of word N-1 for fast readers but not for slow readers. The lag effect exhibited by fast readers indicates that they continue to process a word when it is no longer in view, thus limiting the notion of immediate processing. Contrary to our initial expectation that fast readers would process only a single new argument from a sentence, whereas slow readers would process several new arguments, we found that both reader groups adopted a many-argument strategy. However, fast and slow readers differed in terms of the text units (lines vs. sentences) defining the new-argument effects: Fast readers exhibited greater new-argument effects relative to lines, whereas slow readers exhibited greater new-argument effects relative to sentences. Specifically, slow readers integrated the new arguments primarily at the end of the sentence, whereas fast readers did so at line boundaries. These results are discussed in terms of a buffer-and-integrate model of reading comprehension.

Iberiotoxin-sensitive and -insensitive BK currents in Purkinje neuron somata

PubMed Central

Benton, Mark D.; Lewis, Amanda H.; Bant, Jason S.

2013-01-01

Purkinje cells have specialized intrinsic ionic conductances that generate high-frequency action potentials. Disruptions of their Ca or Ca-activated K (KCa) currents correlate with altered firing patterns in vitro and impaired motor behavior in vivo. To examine the properties of somatic KCa currents, we recorded voltage-clamped KCa currents in Purkinje cell bodies isolated from postnatal day 17–21 mouse cerebellum. Currents were evoked by endogenous Ca influx with approximately physiological Ca buffering. Purkinje somata expressed voltage-activated, Cd-sensitive KCa currents with iberiotoxin (IBTX)-sensitive (>100 nS) and IBTX-insensitive (>75 nS) components. IBTX-sensitive currents activated and partially inactivated within milliseconds. Rapid, incomplete macroscopic inactivation was also evident during 50- or 100-Hz trains of 1-ms depolarizations. In contrast, IBTX-insensitive currents activated more slowly and did not inactivate. These currents were insensitive to the small- and intermediate-conductance KCa channel blockers apamin, scyllatoxin, UCL1684, bicuculline methiodide, and TRAM-34, but were largely blocked by 1 mM tetraethylammonium. The underlying channels had single-channel conductances of ∼150 pS, suggesting that the currents are carried by IBTX-resistant (β4-containing) large-conductance KCa (BK) channels. IBTX-insensitive currents were nevertheless increased by small-conductance KCa channel agonists EBIO, chlorzoxazone, and CyPPA. During trains of brief depolarizations, IBTX-insensitive currents flowed during interstep intervals, and the accumulation of interstep outward current was enhanced by EBIO. In current clamp, EBIO slowed spiking, especially during depolarizing current injections. The two components of BK current in Purkinje somata likely contribute differently to spike repolarization and firing rate. Moreover, augmentation of BK current may partially underlie the action of EBIO and chlorzoxazone to alleviate disrupted Purkinje cell firing associated with genetic ataxias. PMID:23446695

Antagonism of Lidocaine Inhibition by Open-Channel Blockers That Generate Resurgent Na Current

PubMed Central

Bant, Jason S.; Aman, Teresa K.; Raman, Indira M.

2013-01-01

Na channels that generate resurgent current express an intracellular endogenous open-channel blocking protein, whose rapid binding upon depolarization and unbinding upon repolarization minimizes fast and slow inactivation. Na channels also bind exogenous compounds, such as lidocaine, which functionally stabilize inactivation. Like the endogenous blocking protein, these use-dependent inhibitors bind most effectively at depolarized potentials, raising the question of how lidocaine-like compounds affect neurons with resurgent Na current. We therefore recorded lidocaine inhibition of voltage-clamped, tetrodotoxin-sensitive Na currents in mouse Purkinje neurons, which express a native blocking protein, and in mouse hippocampal CA3 pyramidal neurons with and without a peptide from the cytoplasmic tail of NaVβ4 (the β4 peptide), which mimics endogenous open-channel block. To control channel states during drug exposure, lidocaine was applied with rapid-solution exchange techniques during steps to specific voltages. Inhibition of Na currents by lidocaine was diminished by either the β4 peptide or the native blocking protein. In peptide-free CA3 cells, prolonging channel opening with a site-3 toxin, anemone toxin II, reduced lidocaine inhibition; this effect was largely occluded by open-channel blockers, suggesting that lidocaine binding is favored by inactivation but prevented by open-channel block. In constant 100 μM lidocaine, current-clamped Purkinje cells continued to fire spontaneously. Similarly, the β4 peptide reduced lidocaine-dependent suppression of spiking in CA3 neurons in slices. Thus, the open-channel blocking protein responsible for resurgent current acts as a natural antagonist of lidocaine. Neurons with resurgent current may therefore be less susceptible to use-dependent Na channel inhibitors used as local anesthetic, antiarrhythmic, and anticonvulsant drugs. PMID:23486968

Phosphorylation of light-harvesting complex II and photosystem II core proteins shows different irradiance-dependent regulation in vivo. Application of phosphothreonine antibodies to analysis of thylakoid phosphoproteins.

PubMed

Rintamäki, E; Salonen, M; Suoranta, U M; Carlberg, I; Andersson, B; Aro, E M

1997-11-28

An immunological approach using a polyclonal phosphothreonine antibody is introduced for the analysis of thylakoid protein phosphorylation in vivo. Virtually the same photosystem II (PSII) core phosphoproteins (D1, D2, CP43, and the psbH gene product) and the light-harvesting chlorophyll a/b complex II (LHCII) phosphopolypeptides (LHCB1 and LHCB2), as earlier identified by radiolabeling experiments, were recognized in both pumpkin and spinach leaves. Notably, the PSII core proteins and LHCII polypeptides were found to have a different phosphorylation pattern in vivo with respect to increasing irradiance. Phosphorylation of the PSII core proteins in leaf discs attained the saturation level at the growth light intensity, and this level was also maintained at high irradiances. Maximal phosphorylation of LHCII polypeptides only occurred at low light intensities, far below the growth irradiance, and then drastically decreased at higher irradiances. These observations are at variance with traditional studies in vitro, where LHCII shows a light-dependent increase in phosphorylation, which is maintained even at high irradiances. Only a slow restoration of the phosphorylation capacity for LHCII polypeptides at the low light conditions occurred in vivo after the high light-induced inactivation. Furthermore, if thylakoid membranes were isolated from the high light-inactivated leaves, no restoration of LHCII phosphorylation took place in vitro. However, both the high light-induced inactivation and low light-induced restoration of LHCII phosphorylation seen in vivo could be mimicked in isolated thylakoid membranes by incubating with reduced and oxidized dithiothreitol, respectively. We propose that stromal components are involved in the regulation of LHCII phosphorylation in vivo, and inhibition of LHCII phosphorylation under increasing irradiance results from reduction of the thiol groups in the LHCII kinase.

Living with an imperfect cell wall: compensation of femAB inactivation in Staphylococcus aureus

PubMed Central

Hübscher, Judith; Jansen, Andrea; Kotte, Oliver; Schäfer, Juliane; Majcherczyk, Paul A; Harris, Llinos G; Bierbaum, Gabriele; Heinemann, Matthias; Berger-Bächi, Brigitte

2007-01-01

Background Synthesis of the Staphylococcus aureus peptidoglycan pentaglycine interpeptide bridge is catalyzed by the nonribosomal peptidyl transferases FemX, FemA and FemB. Inactivation of the femAB operon reduces the interpeptide to a monoglycine, leading to a poorly crosslinked peptidoglycan. femAB mutants show a reduced growth rate and are hypersusceptible to virtually all antibiotics, including methicillin, making FemAB a potential target to restore β-lactam susceptibility in methicillin-resistant S. aureus (MRSA). Cis-complementation with wild type femAB only restores synthesis of the pentaglycine interpeptide and methicillin resistance, but the growth rate remains low. This study characterizes the adaptations that ensured survival of the cells after femAB inactivation. Results In addition to slow growth, the cis-complemented femAB mutant showed temperature sensitivity and a higher methicillin resistance than the wild type. Transcriptional profiling paired with reporter metabolite analysis revealed multiple changes in the global transcriptome. A number of transporters for sugars, glycerol, and glycine betaine, some of which could serve as osmoprotectants, were upregulated. Striking differences were found in the transcription of several genes involved in nitrogen metabolism and the arginine-deiminase pathway, an alternative for ATP production. In addition, microarray data indicated enhanced expression of virulence factors that correlated with premature expression of the global regulators sae, sarA, and agr. Conclusion Survival under conditions preventing normal cell wall formation triggered complex adaptations that incurred a fitness cost, showing the remarkable flexibility of S. aureus to circumvent cell wall damage. Potential FemAB inhibitors would have to be used in combination with other antibiotics to prevent selection of resistant survivors. PMID:17784943

Effect of pharmacological inactivation of nucleus reticularis tegmenti pontis on saccadic eye movements in the monkey.

PubMed

Kaneko, Chris R S; Fuchs, Albert F

2006-06-01

The superior colliculus (SC) provides signals for the generation of saccades via a direct pathway to the brain stem burst generator (BG). In addition, it sends saccade-related activity to the BG indirectly through the cerebellum via a relay in the nucleus reticularis tegmenti pontis (NRTP). Lesions of the oculomotor vermis, lobules VIc and VII, and inactivation of the caudal fastigial nucleus, the cerebellar output nucleus to which it projects, produce saccade dysmetria but have little effect on saccade peak velocity and duration. We expected similar deficits from inactivation of the NRTP. Instead, injections as small as 80 nl into the NRTP first slowed ipsiversive saccades and then gradually reduced their amplitudes. Postinjection saccades had slower peak velocities and longer durations than preinjection saccades with similar amplitudes. Contraversive saccades retained their normal kinematics. When the gains of ipsiversive saccades to 10 degrees target steps had fallen to their lowest values (0.28 +/- 0.19; mean +/- SD; n = 10 experiments), the gains of contraversive saccades to 10 degrees target steps had decreased very little (0.82 +/- 0.11). Eventually, ipsiversive saccades did not exceed 5 degrees , even to 20 degrees target steps. Moreover, these small remaining saccades apparently were made with considerable difficulty because their latencies increased substantially. When ipsiversive saccade gain was at its lowest, the gain and kinematics of vertical saccades to 10 degrees target steps exhibited inconsistent changes. We argue that our injections did not compromise the direct SC pathway. Therefore these data suggest that the cerebellar saccade pathway does not simply modulate BG activity but is required for horizontal saccades to occur at all.

Effect of Pharmacological Inactivation of Nucleus Reticularis Tegmenti Pontis on Saccadic Eye Movements in the Monkey

PubMed Central

Kaneko, Chris R. S.; Fuchs, Albert F.

2006-01-01

The superior colliculus (SC) provides signals for the generation of saccades via a direct pathway to the brain stem burst generator (BG). In addition, it sends saccade-related activity to the BG indirectly through the cerebellum via a relay in the nucleus reticularis tegmenti pontis (NRTP). Lesions of the oculomotor vermis, lobules VIc and VII, and inactivation of the caudal fastigial nucleus, the cerebellar output nucleus to which it projects, produce saccade dysmetria but have little effect on saccade peak velocity and duration. We expected similar deficits from inactivation of the NRTP. Instead, injections as small as 80 nl into the NRTP first slowed ipsiversive saccades and then gradually reduced their amplitudes. Postinjection saccades had slower peak velocities and longer durations than preinjection saccades with similar amplitudes. Contraversive saccades retained their normal kinematics. When the gains of ipsiversive saccades to 10° target steps had fallen to their lowest values (0.28 ± 0.19; mean ± SD; n = 10 experiments), the gains of contraversive saccades to 10° target steps had decreased very little (0.82 ± 0.11). Eventually, ipsiversive saccades did not exceed 5°, even to 20° target steps. Moreover, these small remaining saccades apparently were made with considerable difficulty because their latencies increased substantially. When ipsiversive saccade gain was at its lowest, the gain and kinematics of vertical saccades to 10° target steps exhibited inconsistent changes. We argue that our injections did not compromise the direct SC pathway. Therefore these data suggest that the cerebellar saccade pathway does not simply modulate BG activity but is required for horizontal saccades to occur at all. PMID:16467420

ARF6 directs axon transport and traffic of integrins and regulates axon growth in adult DRG neurons.

PubMed

Eva, Richard; Crisp, Sarah; Marland, Jamie R K; Norman, Jim C; Kanamarlapudi, Venkateswarlu; ffrench-Constant, Charles; Fawcett, James W

2012-07-25

Integrins are involved in axon growth and regeneration. Manipulation of integrins is a route to promoting axon regeneration and understanding regeneration failure in the CNS. Expression of α9 integrin promotes axon regeneration, so we have investigated α9β1 trafficking and transport in axons and at the growth cone. We have previously found that α9 and β1 integrins traffic via Rab11-positive recycling endosomes in peripheral axons and growth cones. However, transport via Rab11 is slow, while rapid transport occurs in vesicles lacking Rab11. We have further studied α9 and β1 integrin transport and traffic in adult rat dorsal root ganglion axons and PC12 cells. Integrins are in ARF6 vesicles during rapid axonal transport and during trafficking in the growth cone. We report that rapid axonal transport of these integrins and their trafficking at the cell surface is regulated by ARF6. ARF6 inactivation by expression of ACAP1 leads to increased recycling of β1 integrins to the neuronal surface and to increased anterograde axonal transport. ARF6 activation by expression of the neuronal guanine nucleotide exchange factors, ARNO or EFA6, increases retrograde integrin transport in axons and increases integrin internalization. ARF6 inactivation increases integrin-mediated outgrowth, while activation decreases it. The coordinated changes in integrin transport and recycling resulting from ARF6 activation or inactivation are the probable mechanism behind this regulation of axon growth. Our data suggest a novel mechanism of integrin traffic and transport in peripheral axons, regulated by the activation state of ARF6, and suggest that ARF6 might be targeted to enhance integrin-dependent axon regeneration after injury.

Molecular and functional differences in voltage-activated sodium currents between GABA projection neurons and dopamine neurons in the substantia nigra

PubMed Central

Ding, Shengyuan; Wei, Wei

2011-01-01

GABA projection neurons (GABA neurons) in the substantia nigra pars reticulata (SNr) and dopamine projection neurons (DA neurons) in substantia nigra pars compacta (SNc) have strikingly different firing properties. SNc DA neurons fire low-frequency, long-duration spikes, whereas SNr GABA neurons fire high-frequency, short-duration spikes. Since voltage-activated sodium (NaV) channels are critical to spike generation, the different firing properties raise the possibility that, compared with DA neurons, NaV channels in SNr GABA neurons have higher density, faster kinetics, and less cumulative inactivation. Our quantitative RT-PCR analysis on immunohistochemically identified nigral neurons indicated that mRNAs for pore-forming NaV1.1 and NaV1.6 subunits and regulatory NaVβ1 and Navβ4 subunits are more abundant in SNr GABA neurons than SNc DA neurons. These α-subunits and β-subunits are key subunits for forming NaV channels conducting the transient NaV current (INaT), persistent Na current (INaP), and resurgent Na current (INaR). Nucleated patch-clamp recordings showed that INaT had a higher density, a steeper voltage-dependent activation, and a faster deactivation in SNr GABA neurons than in SNc DA neurons. INaT also recovered more quickly from inactivation and had less cumulative inactivation in SNr GABA neurons than in SNc DA neurons. Furthermore, compared with nigral DA neurons, SNr GABA neurons had a larger INaR and INaP. Blockade of INaP induced a larger hyperpolarization in SNr GABA neurons than in SNc DA neurons. Taken together, these results indicate that NaV channels expressed in fast-spiking SNr GABA neurons and slow-spiking SNc DA neurons are tailored to support their different spiking capabilities. PMID:21880943

Simultaneous control of microorganisms and disinfection by-products by sequential chlorination.

PubMed

Chen, Chao; Zhang, Xiao-Jian; He, Wen-Jie; Han, Hong-Da

2007-04-01

To introduce a new sequential chlorination disinfection process in which short-term free chlorine and chloramine are sequentially added. Pilot tests of this sequential chlorination were carried out in a drinking water plant. The sequential chlorination disinfection process had the same or better efficiency on microbe (including virus) inactivation compared with the free chlorine disinfection process. There seemed to be some synergetic disinfection effect between free chlorine and monochloramine because they attacked different targets. The sequential chlorination disinfection process resulted in 35.7%-77.0% TTHM formation and 36.6%-54.8% THAA5 formation less than the free chlorination process. The poorer the water quality was, the more advantage the sequential chlorination disinfection had over the free chlorination. This process takes advantages of free chlorine's quick inactivation of microorganisms and chloramine's low disinfection by-product (DBP) yield and long-term residual effect, allowing simultaneous control of microbes and DBPs in an effective and economic way.

Modeling of pathogen survival during simulated gastric digestion.

PubMed

Koseki, Shige; Mizuno, Yasuko; Sotome, Itaru

2011-02-01

The objective of the present study was to develop a mathematical model of pathogenic bacterial inactivation kinetics in a gastric environment in order to further understand a part of the infectious dose-response mechanism. The major bacterial pathogens Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. were examined by using simulated gastric fluid adjusted to various pH values. To correspond to the various pHs in a stomach during digestion, a modified logistic differential equation model and the Weibull differential equation model were examined. The specific inactivation rate for each pathogen was successfully described by a square-root model as a function of pH. The square-root models were combined with the modified logistic differential equation to obtain a complete inactivation curve. Both the modified logistic and Weibull models provided a highly accurate fitting of the static pH conditions for every pathogen. However, while the residuals plots of the modified logistic model indicated no systematic bias and/or regional prediction problems, the residuals plots of the Weibull model showed a systematic bias. The modified logistic model appropriately predicted the pathogen behavior in the simulated gastric digestion process with actual food, including cut lettuce, minced tuna, hamburger, and scrambled egg. Although the developed model enabled us to predict pathogen inactivation during gastric digestion, its results also suggested that the ingested bacteria in the stomach would barely be inactivated in the real digestion process. The results of this study will provide important information on a part of the dose-response mechanism of bacterial pathogens.

Modeling of Pathogen Survival during Simulated Gastric Digestion ▿

PubMed Central

Koseki, Shige; Mizuno, Yasuko; Sotome, Itaru

2011-01-01

The objective of the present study was to develop a mathematical model of pathogenic bacterial inactivation kinetics in a gastric environment in order to further understand a part of the infectious dose-response mechanism. The major bacterial pathogens Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. were examined by using simulated gastric fluid adjusted to various pH values. To correspond to the various pHs in a stomach during digestion, a modified logistic differential equation model and the Weibull differential equation model were examined. The specific inactivation rate for each pathogen was successfully described by a square-root model as a function of pH. The square-root models were combined with the modified logistic differential equation to obtain a complete inactivation curve. Both the modified logistic and Weibull models provided a highly accurate fitting of the static pH conditions for every pathogen. However, while the residuals plots of the modified logistic model indicated no systematic bias and/or regional prediction problems, the residuals plots of the Weibull model showed a systematic bias. The modified logistic model appropriately predicted the pathogen behavior in the simulated gastric digestion process with actual food, including cut lettuce, minced tuna, hamburger, and scrambled egg. Although the developed model enabled us to predict pathogen inactivation during gastric digestion, its results also suggested that the ingested bacteria in the stomach would barely be inactivated in the real digestion process. The results of this study will provide important information on a part of the dose-response mechanism of bacterial pathogens. PMID:21131530

Heat inactivation of Salmonella spp. in fresh poultry compost by simulating early phase of composting process.

PubMed

Singh, R; Kim, J; Jiang, X

2012-05-01

The purpose of this study was to determine the effect of moisture on thermal inactivation of Salmonella spp. in poultry litter under optimal composting conditions. Thermal inactivation of Salmonella was studied in fresh poultry compost by simulating early phase of composting process. A mixture of three Salmonella serotypes grown in Tryptic soy broth with rifampin (TSB-R) was inoculated in fresh compost with 40 or 50% moisture at a final concentration of c. 7 log CFU g(-1). The inoculated compost was kept in an environmental chamber which was programmed to rise from room temperature to target composting temperatures in 2 days. In poultry compost with optimal moisture content (50%), Salmonella spp. survived for 96, 72 and 24 h at 50, 55 and 60°C, respectively, as compared with 264, 144 and 72 h at 50, 55 and 60°C, respectively, in compost with suboptimal moisture (40%). Pathogen decline was faster during the come-up time owing to higher ammonia volatilization. Our results demonstrated that Salmonella spp. survived longer in fresh poultry compost with suboptimal moisture of 40% than in compost with optimal moisture of 50% during thermophilic composting. High nitrogen content of the poultry compost is an additional factor contributing to Salmonella inactivation through ammonia volatilization during thermal exposure. This research validated the effectiveness of the current composting guidelines on Salmonella inactivation in fresh poultry compost. Both initial moisture level and ammonia volatilization are important factors affecting microbiological safety and quality of compost product. © 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Antimicrobial Activity of Nisin and Natamycin Incorporated Sodium Caseinate Extrusion-Blown Films: A Comparative Study with Heat-Pressed/Solution Cast Films.

PubMed

Colak, Basak Yilin; Peynichou, Pierre; Galland, Sophie; Oulahal, Nadia; Prochazka, Frédéric; Degraeve, Pascal

2016-05-01

Antimicrobial edible films based on sodium caseinate, glycerol, and 2 food preservatives (nisin or natamycin) were prepared by classical thermomechanical processes. Food preservatives were compounded (at 65 °C for 2.5 min) with sodium caseinate in a twin-screw extruder. Anti-Listeria activity assays revealed a partial inactivation of nisin following compounding. Thermoplastic pellets containing food preservatives were then used to manufacture films either by blown-film extrusion process or by heat-press. After 24 h of incubation on agar plates, the diameters of K. rhizophila growth inhibition zones around nisin-incorporated films prepared by solution casting (control), extrusion blowing or heat pressing at 80 °C for 7 min of nisin-containing pellets were 15.5 ± 0.9, 9.8 ± 0.2, and 8.6 ± 1.0 mm, respectively. Since heat-pressing for 7 min at 80 °C of nisin-incorporated pellets did not further inactivate nisin, this indicates that nisin inactivation during extrusion-blowing was limited. Moreover, the lower diameter of the K. rhizophila growth inhibition zone around films prepared with nisin-containing pellets compared to that observed around films directly prepared by solution casting confirms that nisin inactivation mainly occurred during the compounding step. Natamycin-containing thermoplastic films inhibited Aspergillus niger growth; however, by contrast with nisin-containing films, heat-pressed films had higher inhibition zone diameters than blown films, therefore suggesting a partial inactivation of natamycin during extrusion-blowing. © 2016 Institute of Food Technologists®

Inactivation of marine heterotrophic bacteria in ballast water by an Electrochemical Advanced Oxidation Process.

PubMed

Moreno-Andrés, Javier; Ambauen, Noëmi; Vadstein, Olav; Hallé, Cynthia; Acevedo-Merino, Asunción; Nebot, Enrique; Meyn, Thomas

2018-05-03

Seawater treatment is increasingly required due to industrial activities that use substantial volumes of seawater in their processes. The shipping industry and the associated management of a ship's ballast water are currently considered a global challenge for the seas. Related to that, the suitability of an Electrochemical Advanced Oxidation Process (EAOP) with Boron Doped Diamond (BDD) electrodes has been assessed on a laboratory scale for the disinfection of seawater. This technology can produce both reactive oxygen species and chlorine species (especially in seawater) that are responsible for inactivation. The EAOP was applied in a continuous-flow regime with real seawater. Natural marine heterotrophic bacteria (MHB) were used as an indicator of disinfection efficiency. A biphasic inactivation kinetic model was fitted on experimental points, achieving 4-Log reductions at 0.019 Ah L -1 . By assessing regrowth after treatment, results suggest that higher bacterial damages result from the EAOP when it is compared to chlorination. Furthermore, several issues lacking fundamental understanding were investigated such as recolonization capacity or bacterial community dynamics. It was concluded that, despite disinfection processes being effective, there is not only a possibility for regrowth after treatment but also a change on bacterial population diversity produced by the treatment. Finally, energy consumption was estimated and indicated that 0.264 kWh·m -3 are needed for 4.8-Log reductions of MHB; otherwise, with 0.035 kWh·m -3 , less disinfection efficiency can be obtained (2.2-Log red). However, with a residual oxidant in the solution, total inactivation can be achieved in three days. Copyright © 2018 Elsevier Ltd. All rights reserved.

Factors affecting thermal resistance of Salmonella enterica serovar enteritidis ODA 99-30581-13 in shell egg contents and use of heat-ozone combinations for egg pasteurization.

PubMed

Perry, Jennifer J; Yousef, Ahmed E

2013-02-01

Infection of laying hens with Salmonella enterica serovar Enteritidis leads to deposition of the pathogen into the albumen or yolk of forming eggs. Heat treatment can inactivate internalized Salmonella Enteritidis in shell eggs, but factors such as the nature and location of contamination may influence the efficacy of thermal treatments. In the current research, natural contamination was mimicked by introducing small inocula of Salmonella Enteritidis into different locations of shell eggs and incubating inoculated eggs. These pathogen-containing eggs were heated at 57°C for 40 min, and temperature within eggs was monitored at the locations of inocula. Comparison of inactivation at equivalent internal temperatures revealed similar levels of lethality regardless of inoculum location. Refrigeration between incubation and heat treatment did not increase thermal resistance of cells in albumen but decreased cell inactivation in yolk. Sequential application of heat and gaseous ozone allows for the development of a process capable of decontaminating shell eggs with minimal thermal treatment and impact on egg quality. Inoculated eggs were subjected to (i) an immersion heating process similar to that used in commercial pasteurization or (ii) immersion heating, at reduced duration, followed by vacuum (50.8 kPa) and treatment with ozone gas (maximum 160 g/m(3)) under pressure (∼187.5 kPa). All treatments tested produced greater than 5-log inactivation, which is required for "pasteurization" processes. Differences were observed in the visual quality of eggs depending on treatment parameters. Application of ozone subsequent to heating allows for a significant reduction in heating time without decreasing process lethality.

Inactivation of Escherichia coli Endotoxin by Soft Hydrothermal Processing▿

PubMed Central

Miyamoto, Toru; Okano, Shinya; Kasai, Noriyuki

2009-01-01

Bacterial endotoxins, also known as lipopolysaccharides, are a fever-producing by-product of gram-negative bacteria commonly known as pyrogens. It is essential to remove endotoxins from parenteral preparations since they have multiple injurious biological activities. Because of their strong heat resistance (e.g., requiring dry-heat sterilization at 250°C for 30 min) and the formation of various supramolecular aggregates, depyrogenation is more difficult than sterilization. We report here that soft hydrothermal processing, which has many advantages in safety and cost efficiency, is sufficient to assure complete depyrogenation by the inactivation of endotoxins. The endotoxin concentration in a sample was measured by using a chromogenic limulus method with an endotoxin-specific limulus reagent. The endotoxin concentration was calculated from a standard curve obtained using a serial dilution of a standard solution. We show that endotoxins were completely inactivated by soft hydrothermal processing at 130°C for 60 min or at 140°C for 30 min in the presence of a high steam saturation ratio or with a flow system. Moreover, it is easy to remove endotoxins from water by soft hydrothermal processing similarly at 130°C for 60 min or at 140°C for 30 min, without any requirement for ultrafiltration, nonselective adsorption with a hydrophobic adsorbent, or an anion exchanger. These findings indicate that soft hydrothermal processing, applied in the presence of a high steam saturation ratio or with a flow system, can inactivate endotoxins and may be useful for the depyrogenation of parenterals, including end products and medical devices that cannot be exposed to the high temperatures of dry heat treatments. PMID:19502435

Immunolocalization of tripeptidyl peptidase II, a cholecystokinin-inactivating enzyme, in rat brain.

PubMed

Facchinetti, P; Rose, C; Rostaing, P; Triller, A; Schwartz, J C

1999-01-01

Tripeptidyl peptidase II (EC 3.4.14.10) is a serine peptidase apparently involved in the inactivation of cholecystokinin octapeptide [Rose C. et al. (1996) Nature 380, 403-409]. We have compared its distribution with that of cholecystokinin in rat brain, using a polyclonal antibody raised against a highly purified preparation for immunohistochemistry at the photon and electron microscope levels. Tripeptidyl peptidase II-like immunoreactivity was mostly detected in neurons, and also in ependymal cells and choroid plexuses, localizations consistent with a possible participation of the peptidase in the inactivation of cholecystokinin circulating in the cerebrospinal fluid. Immunoreactivity was mostly detected in cell bodies, large processes and, to a lesser extent, axons of various neuronal populations. Their localization, relative to that of cholecystokinin terminals, appears to define three distinct situations. The first corresponds to neurons with high immunoreactivity in areas containing cholecystokinin terminals, as in the cerebral cortex or hippocampal formation, where pyramidal cell bodies and processes surrounded by cholecystokinin axons were immunoreactive. A similar situation was encountered in many other areas, namely along the pathways through which cholecystokinin controls satiety, i.e. in sensory vagal neurons, the nucleus tractus solitarius and hypothalamic nuclei. The second situation corresponds to cholecystokinin neuronal populations containing tripeptidyl peptidase II-like immunoreactivity, as in neurons of the supraoptic or paraventricular nuclei, axons in the median eminence or nigral neurons. In both situations, localization of tripeptidyl peptidase II-like immunoreactivity is consistent with a role in cholecystokinin inactivation. The third situation corresponds to areas with mismatches, such as the cerebellum, a region devoid of cholecystokinin, but in which Purkinje cells displayed high tripeptidyl peptidase II-like immunoreactivity, possibly related to a role in the inactivation of neuropeptides other than cholecystokinin. Also, some areas with cholecystokinin terminals, e.g., the molecular layer of the cerebral cortex, were devoid of tripeptidyl peptidase II-like immunoreactivity, suggesting that processes other than cleavage by tripeptidyl peptidase II may be involved in cholecystokinin inactivation. Tripeptidyl peptidase II-like immunoreactivity was also detected at the ultrastructural level in the cerebral cortex and hypothalamus using either immunoperoxidase or silver-enhanced immunogold detection. It was mainly associated with the cytoplasm of neuronal somata and dendrites, often in the vicinity of reticulum cisternae, Golgi apparatus or vesicles, and with the inner side of the dendritic plasma membrane. Hence, whereas a fraction of tripeptidyl peptidase II-like immunoreactivity localization at the cellular level is consistent with its alleged function in cholecystokinin octapeptide inactivation, its association with the outside plasma membrane of neurons remains to be confirmed.

Ebola Virus and Marburg Virus in Human Milk Are Inactivated by Holder Pasteurization.

PubMed

Hamilton Spence, Erin; Huff, Monica; Shattuck, Karen; Vickers, Amy; Yun, Nadezda; Paessler, Slobodan

2017-05-01

Potential donors of human milk are screened for Ebola virus (EBOV) using standard questions, but testing for EBOV and Marburg virus (MARV) is not part of routine serological testing performed by milk banks. Research aim: This study tested the hypothesis that EBOV would be inactivated in donor human milk (DHM) by standard pasteurization techniques (Holder) used in all North American nonprofit milk banks. Milk samples were obtained from a nonprofit milk bank. They were inoculated with EBOV (Zaire strain) and MARV (Angola strain) and processed by standard Holder pasteurization technique. Plaque assays for EBOV and MARV were performed to detect the presence of virus after pasteurization. Neither EBOV nor MARV was detectable by viral plaque assay in DHM or culture media samples, which were pasteurized by the Holder process. EBOV and MARV are safely inactivated in human milk by standard Holder pasteurization technique. Screening for EBOV or MARV beyond questionnaire and self-deferral is not needed to ensure safety of DHM for high-risk infants.

Inactivation of Primate Prefrontal Cortex Impairs Auditory and Audiovisual Working Memory.

PubMed

Plakke, Bethany; Hwang, Jaewon; Romanski, Lizabeth M

2015-07-01

The prefrontal cortex is associated with cognitive functions that include planning, reasoning, decision-making, working memory, and communication. Neurophysiology and neuropsychology studies have established that dorsolateral prefrontal cortex is essential in spatial working memory while the ventral frontal lobe processes language and communication signals. Single-unit recordings in nonhuman primates has shown that ventral prefrontal (VLPFC) neurons integrate face and vocal information and are active during audiovisual working memory. However, whether VLPFC is essential in remembering face and voice information is unknown. We therefore trained nonhuman primates in an audiovisual working memory paradigm using naturalistic face-vocalization movies as memoranda. We inactivated VLPFC, with reversible cortical cooling, and examined performance when faces, vocalizations or both faces and vocalization had to be remembered. We found that VLPFC inactivation impaired subjects' performance in audiovisual and auditory-alone versions of the task. In contrast, VLPFC inactivation did not disrupt visual working memory. Our studies demonstrate the importance of VLPFC in auditory and audiovisual working memory for social stimuli but suggest a different role for VLPFC in unimodal visual processing. The ventral frontal lobe, or inferior frontal gyrus, plays an important role in audiovisual communication in the human brain. Studies with nonhuman primates have found that neurons within ventral prefrontal cortex (VLPFC) encode both faces and vocalizations and that VLPFC is active when animals need to remember these social stimuli. In the present study, we temporarily inactivated VLPFC by cooling the cortex while nonhuman primates performed a working memory task. This impaired the ability of subjects to remember a face and vocalization pair or just the vocalization alone. Our work highlights the importance of the primate VLPFC in the processing of faces and vocalizations in a manner that is similar to the inferior frontal gyrus in the human brain. Copyright © 2015 the authors 0270-6474/15/359666-10$15.00/0.

The temporal structure of behaviour and sleep homeostasis.

PubMed

Vyazovskiy, Vladyslav V; Tobler, Irene

2012-01-01

The amount and architecture of vigilance states are governed by two distinct processes, which occur at different time scales. The first, a slow one, is related to a wake/sleep dependent homeostatic Process S, which occurs on a time scale of hours, and is reflected in the dynamics of NREM sleep EEG slow-wave activity. The second, a fast one, is manifested in a regular alternation of two sleep states--NREM and REM sleep, which occur, in rodents, on a time scale of ~5-10 minutes. Neither the mechanisms underlying the time constants of these two processes--the slow one and the fast one, nor their functional significance are understood. Notably, both processes are primarily apparent during sleep, while their potential manifestation during wakefulness is obscured by ongoing behaviour. Here, we find, in mice provided with running wheels, that the two sleep processes become clearly apparent also during waking at the level of behavior and brain activity. Specifically, the slow process was manifested in the total duration of waking periods starting from dark onset, while the fast process was apparent in a regular occurrence of running bouts during the waking periods. The dynamics of both processes were stable within individual animals, but showed large interindividual variability. Importantly, the two processes were not independent: the periodic structure of waking behaviour (fast process) appeared to be a strong predictor of the capacity to sustain continuous wakefulness (slow process). The data indicate that the temporal organization of vigilance states on both the fast and the slow time scales may arise from a common neurophysiologic mechanism.

Towards Binding Mechanism of Cu2+ on Creatine Kinase from Pelodiscus sinensis: Molecular Dynamics Simulation Integrating Inhibition Kinetics Study.

PubMed

Cai, Yan; Lee, Jinhyuk; Wang, Wei; Park, Yong-Doo; Qian, Guo-Ying

2017-01-01

Cu2+ is well known to play important roles in living organisms having bifacial distinction: essential microelement that is necessary for a wide range of metabolic processes but hyper-accumulation of Cu2+ can be toxic. The physiological function of Cu2+ in ectothermic animals such as Pelodiscus sinensis (Chinese soft-shelled turtle) has not been elucidated. In this study, we elucidated effect of Cu2+ on the energy producing metabolic enzyme creatine kinase (CK), which might directly affect energy metabolism and homeostasis of P. sinensis. We first conducted molecular dynamics (MD) simulations between P-CK and Cu2+ and conducted the inactivation kinetics including spectrofluorimetry study. MD simulation showed that Cu2+ blocked the binding site of the ATP cofactor, indicating that Cu2+ could directly inactivate P-CK. We prepared the muscle type of CK (P-CK) and confirmed that Cu2+ conspicuously inactivated the activity of P-CK (IC50 = 24.3 μM) and exhibited non-competitive inhibition manner with creatine and ATP in a first-order kinetic process. This result was well matched to the MD simulation results that Cu2+-induced non-competitive inactivation of P-CK. The spectrofluorimetry study revealed that Cu2+ induced tertiary structure changes in PCK accompanying with the exposure of hydrophobic surfaces. Interestingly, the addition of osmolytes (glycine, proline, and liquaemin) effectively restored activity of the Cu2+-inactivated P-CK. Our study illustrates the Cu2+-mediated unfolding of P-CK with disruption of the enzymatic function and the protective restoration role of osmolytes on P-CK inactivation. This study provides information of interest on P-CK as a metabolic enzyme of ectothermic animal in response to Cu2+ binding. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Ftx is a non-coding RNA which affects Xist expression and chromatin structure within the X-inactivation center region.

PubMed

Chureau, Corinne; Chantalat, Sophie; Romito, Antonio; Galvani, Angélique; Duret, Laurent; Avner, Philip; Rougeulle, Claire

2011-02-15

X chromosome inactivation (XCI) is an essential epigenetic process which involves several non-coding RNAs (ncRNAs), including Xist, the master regulator of X-inactivation initiation. Xist is flanked in its 5' region by a large heterochromatic hotspot, which contains several transcription units including a gene of unknown function, Ftx (five prime to Xist). In this article, we describe the characterization and functional analysis of murine Ftx. We present evidence that Ftx produces a conserved functional long ncRNA, and additionally hosts microRNAs (miR) in its introns. Strikingly, Ftx partially escapes X-inactivation and is upregulated specifically in female ES cells at the onset of X-inactivation, an expression profile which closely follows that of Xist. We generated Ftx null ES cells to address the function of this gene. In these cells, only local changes in chromatin marks are detected within the hotspot, indicating that Ftx is not involved in the global maintenance of the heterochromatic structure of this region. The Ftx mutation, however, results in widespread alteration of transcript levels within the X-inactivation center (Xic) and particularly important decreases in Xist RNA levels, which were correlated with increased DNA methylation at the Xist CpG island. Altogether our results indicate that Ftx is a positive regulator of Xist and lead us to propose that Ftx is a novel ncRNA involved in XCI.

Subcellular mechanism of Escherichia coli inactivation during electrochemical disinfection with boron-doped diamond anode: A comparative study of three electrolytes.

PubMed

Long, Yujiao; Ni, Jinren; Wang, Zuhui

2015-11-01

Although the identification of effective oxidant species has been extensively studied, yet the subcellular mechanism of bacterial inactivation has never been clearly elucidated in electrochemical disinfection processes. In this study, subcellular mechanism of Escherichia coli inactivation during electrochemical disinfection was revealed in terms of comprehensive factors such as cell morphology, total organic components, K(+) leakage, membrane permeability, lipid peroxidation, membrane potential, membrane proteins, intracellular enzyme, cellular ATP level and DNA. The electrolysis was conducted with boron-doped diamond anode in three electrolytes including chloride, sulfate and phosphate. Results demonstrated that cell inactivation was mainly attributed to damage to the intracellular enzymatic systems in chloride solution. In sulfate solution, certain essential membrane proteins like the K(+) ion transport systems were eliminated. Thus, the pronounced K(+) leakage from cytosol resulted in gradual collapse of the membrane potential, which would hinder the subcellular localization of cell division-related proteins as well as ATP synthesis and thereby lead to the bacterial inactivation. Remarkable lipid peroxidation was observed, while the intracellular damage was negligible. In phosphate solution, the cells sequentially underwent overall destruction as a whole cell with no captured intermediate state, during which the organic components of the cells were mostly subjected to mineralization. This study provided a thorough insight into the bacterial inactivation mechanism on the subcellular level. Copyright © 2015 Elsevier Ltd. All rights reserved.