Half Inactivation Research Articles

Enhancing the thermostability of carboxypeptidase A by a multiple computer-aided rational design based on amino acids preferences at β-turns

Carboxypeptidase A (CPA) with efficient hydrolysis ability has shown vital potential in food and biological fields. In addition, it is also the earliest discovered enzyme with Ochratoxin A (OTA) degradation activity. Thermostability plays an imperative role to catalyze the reactions at high temperatures in industry, but the poor thermostability of CPA restricts its industrial application. In order to improve the thermostability of CPA, flexible loops were predicted through molecular dynamics (MD) simulation. Based on the amino acid preferences at β-turns, three ΔΔG-based computational programs (Rosetta, FoldX and PoPMuSiC) were employed to screen three variants from plentiful candidates and MD simulations were then used to verify two potential variants with enhanced thermostability (R124K and S134P). Results showed that compared to the wild-type CPA, the variants S134P and R124K exhibited rise of 4.2 min and 7.4 min in half-life (t1/2) at 45 °C, 3 °C and 4.1 °C in the half inactivation temperature (T5010), in addition to increase by 1.9 °C and 1.2 °C in the melting temperature (Tm), respectively. The mechanism responsible for the enhanced thermostability was elucidated through the comprehensive analysis of molecular structure. This study shows that the thermostability of CPA can be improved by the multiple computer-aided rational design based on amino acid preferences at β-turns, broadening its industrial applicability of OTA degradation and providing a valuable strategy for the protein engineering of mycotoxin degrading enzymes.

State-Dependent Inhibition of Voltage-Gated Sodium Channels in Neuroblastoma Neuro-2A Cells by Arachidonic Acid from Halichondria okadai.

Voltage-gated sodium channels (Nav) are closely associated with epilepsy, cardiac and skeletal muscle diseases, and neuropathic pain. Several toxic compounds have been isolated from the marine sponge Halichondria okadai; however, toxic substances that modulate Nav are yet to be identified. This study aimed to identify Nav inhibitors from two snake venoms and H. okadai using mouse neuroblastoma Neuro-2A cells (N2A), which primarily express the specific Nav subtype Nav1.7, using whole-cell patch-clamp recordings. We successfully isolated arachidonic acid (AA, 1) from the hexane extract of H. okadai, and then the fatty acid-mediated modulation of Nav in N2A was investigated in detail for the first time. Octanoic acid (2), palmitic acid (3), and oleic acid (4) showed no inhibitory activity at 100 μM, whereas AA (1), dihomo-γ-linolenic acid (DGLA, 5), and eicosapentaenoic acid (EPA, 6) showed IC50 values of 6.1 ± 2.0, 58 ± 19, and 25 ± 4.0 μM, respectively (N = 4, mean ± SEM). Structure and activity relationships were investigated for the first time using two ω-3 polyunsaturated fatty acids (PUFAs), EPA (6) and eicosatetraenoic acid (ETA, 7), and two ω-6 PUFAs, AA (1) and DGLA (5), to determine their effects on a resting state, activated state, and inactivated state. Steady-state analysis showed that the half inactivation potential was largely hyperpolarized by 10 μM AA (1), while 50 μM DGLA (5), 50 μM EPA (6), and 10 μM ETA (7) led to a slight change. The percentages of the resting state block were 24 ± 1, 22 ± 1, 34 ± 4, and 38 ± 9% in the presence of AA (1), DGLA (5), EPA (6), and ETA (7), respectively, with EPA (6) and ETA (7) exhibiting a greater inhibition than both AA (1) and DGLA (5), and their inhibitions did not increase in the following depolarization pulses. None of the compounds exhibited the use-dependent block. The half recovery times from the inactivated state for the control, AA (1), DGLA (5), EPA (6), and ETA (7) were 7.67 ± 0.33, 34.3 ± 1.10, 15.5 ± 1.10, 10.7 ± 0.31, and 3.59 ± 0.18 ms, respectively, with AA (1) exhibiting a distinctively large effect. Overall, distributed binding to the resting and the inactivated states of Nav would be significant for the inhibition of Nav, which presumably depends on the active structure of each PUFA.

Construction and evaluation of a gradient stress model of PC12 cells induced by corticosterone

Objective: A gradient stress model of PC12 cells induced by corticosterone was established to provide a basis for the evaluation and regulation of cell stress. Methods: The effect of corticosterone on cell viability was observed by measuring PC12 cell viability at different concentrations of corticosterone (0~1 000 μmol/L) after different intervention times (8~48 h) to screen the cell models for optimal intervention conditions. Key stress indicators (MDA, SOD, NADH, LDH) were measured spectrophotometrically and microscopically to evaluate the models. Results: When the concentration of corticosterone was below 200 μmol/L and the intervention time was 12 h, the cell viability was below half inactivation rate, which could reduce the confounding factors due to the decrease of cell viability in each group. Compared with the blank control group, corticosterone increased the levels of MDA, NADH and LDH,and decreased the levels of SOD in the model group in a concentration-dependent manner (P＜0.01), which was consistent with the construction of the gradient stress model. Conclusion: A gradient stress injury model of PC12 cells was successfully established, with intervention concentrations of 0 μmol/L, 25 μmol/L, 50 μmol/L, 100 μmol/L, 150 μmol/L and 200 μmol/L corticosterone at an intervention time of 12 h. The degree of stress injury of the cell model was increased gradually, which could be used as a basis and object for conducting cell stress injury assessment and regulation experiments.

FC025: Physiological Role of KV Channel in Ureter Smooth Muscle Cell Investigated Quantitatively by Electrophysiological Modeling

Abstract BACKGROUND AND AIMS Spontaneous peristaltic contraction disorder of the ureter smooth muscle (USM) is an important etiology of congenital giant ureter, congenital ureteropelvic junction deformity and non-obstructive hydronephrosis [1]. The USM cell action potential (AP) plays a key role in commencing USM contraction by allowing influx of Ca2+ from the extra cellular space [2]. Therefore, an in-depth quantitative investigation of the USM cell AP generation will assist in developing better pharmacological therapies for the USM abnormal contraction. The purpose of this in-silico study is to unfold the physiological role of Kv channel in modulating the USM AP generation. METHOD The model consists voltage- dependent Na+ currents (INa), hyperpolarization-activated cyclic nucleotide- gated current (Ih), voltage- dependent Ca2+ currents (ICa), Ca2+ dependent potassium channel (IBKCa) and voltage-dependent potassium channels (IKv) as the components of active electrophysiological characteristics [3]. All ion channels are represented by the Hodgkin–Huxley formalism. The maximum conductance of the is varied to investigate the physiological role of the Kv channel in modulating the USM AP generation. RESULTS The whole cell IKv current is generated by applying the voltage clamp protocol, where the membrane potential is stepped from a holding potential of –80 mV–60 mV for a duration of 500 ms. The half activation potential, half inactivation potential, activation slope factor and inactivation slope factors are: 43 mV, –76.1 mV, 15.4 mV and 6.5 mV, respectively, to fit the Boltzmann function equation. The model reveals that fast activation and inactivation properties are the essential feature of IKv for the USM cell. The resting membrane potential (RMP) is set at –50 mV. The AP was induced in the whole cell model by applying an external stimulus current (10–30 pA), as a brief square pulse of 500 ms duration. It is demonstrated that IKv opposes the Ca2+ current during the first 50 ms of the AP duration and then inactivated. It doesn't contribute to the repolarizing phase of the AP. CONCLUSION The plateau phase of the USM AP shows that the total AP duration much longer than that in other smooth muscle cells. It strongly supports the phenomenon is due to the activation of Ca2+-dependent K+ channel and inactivation of L-type Ca2+ channel. In ureter cells, the IKv does not contribute significantly to the repolarization of the AP, but it may regulate membrane excitability by opposing Ca2+ current activated around the threshold of the AP. Therefore, the agonist and antagonist of the IKv might be useful partially as new pharmacological targets for the USM abnormal contraction.

Characterization of the A-type potassium current in murine gastric fundus smooth muscles.

Transient outward, or "A-type," currents are rapidly inactivating voltage-gated potassium currents that operate at negative membrane potentials. A-type currents have not been reported in the gastric fundus, a tonic smooth muscle. We used whole cell voltage clamp to identify and characterize A-type currents in smooth muscle cells (SMCs) isolated from murine fundus. A-type currents were robust in these cells with peak amplitudes averaging 1.5 nA at 0 mV. Inactivation was rapid with a time constant of 71 ms at 0 mV; recovery from inactivation at -80 mV was similarly rapid with a time constant of 75 ms. A-type currents in fundus were blocked by 4-aminopyridine (4-AP), flecainide, and phrixotoxin-1 (PaTX1). Remaining currents after 4-AP and PaTX1 displayed half-activation potentials that were shifted to more positive potentials and showed incomplete inactivation. Currents after tetraethylammonium (TEA) displayed half inactivation at -48.1 ± 1.0 mV. Conventional microelectrode and contractile experiments on intact fundus muscles showed that 4-AP depolarized membrane potential and increased tone under conditions in which enteric neurotransmission was blocked. These data suggest that A-type K+ channels in fundus SMCs are likely active at physiological membrane potentials, and sustained activation of A-type channels contributes to the negative membrane potentials of this tonic smooth muscle. Quantitative analysis of Kv4 expression showed that Kcnd3 was dominantly expressed in fundus SMCs. These data were confirmed by immunohistochemistry, which revealed Kv4.3-like immunoreactivity within the tunica muscularis. These observations indicate that Kv4 channels likely form the A-type current in murine fundus SMCs.

Characterization of Compound-Specific, Concentration-Independent Biophysical Properties of Sodium Channel Inhibitor Mechanism of Action Using Automated Patch-Clamp Electrophysiology.

We have developed an automated patch-clamp protocol that allows high information content screening of sodium channel inhibitor compounds. We have observed that individual compounds had their specific signature patterns of inhibition, which were manifested irrespective of the concentration. Our aim in this study was to quantify these properties. Primary biophysical data, such as onset rate, the shift of the half inactivation voltage, or the delay of recovery from inactivation, are concentration-dependent. We wanted to derive compound-specific properties, therefore, we had to neutralize the effect of concentration. This study describes how this is done, and shows how compound-specific properties reflect the mechanism of action, including binding dynamics, cooperativity, and interaction with the membrane phase. We illustrate the method using four well-known sodium channel inhibitor compounds, riluzole, lidocaine, benzocaine, and bupivacaine. Compound-specific biophysical properties may also serve as a basis for deriving parameters for kinetic modeling of drug action. We discuss how knowledge about the mechanism of action may help to predict the frequency-dependence of individual compounds, as well as their potential persistent current component selectivity. The analysis method described in this study, together with the experimental protocol described in the accompanying paper, allows screening for inhibitor compounds with specific kinetic properties, or with specific mechanisms of inhibition.

Modulation of Recombinant Human T-Type Calcium Channels by Δ9-Tetrahydrocannabinolic Acid In Vitro.

Introduction: Low voltage-activated T-type calcium channels (T-type ICa), CaV3.1, CaV3.2, and CaV3.3, are opened by small depolarizations from the resting membrane potential in many cells and have been associated with neurological disorders, including absence epilepsy and pain. Δ9-tetrahydrocannabinol (THC) is the principal psychoactive compound in Cannabis and also directly modulates T-type ICa; however, there is no information about functional activity of most phytocannabinoids on T-type calcium channels, including Δ9-tetrahydrocannabinolic acid (THCA), the natural nonpsychoactive precursor of THC. The aim of this work was to characterize THCA effects on T-type calcium channels. Materials and Methods: We used HEK293 Flp-In-TREx cells stably expressing CaV3.1, 3.2, or 3.3. Whole-cell patch clamp recordings were made to investigate cannabinoid modulation of ICa. Results: THCA and THC inhibited the peak current amplitude CaV3.1 with pEC50s of 6.0±0.7 and 5.6±0.4, respectively. THC (1 μM) or THC produced a significant negative shift in half activation and inactivation of CaV3.1, and both drugs prolonged CaV3.1 deactivation kinetics. THCA (10 μM) inhibited CaV3.2 by 53%±4%, and both THCA and THC produced a substantial negative shift in the voltage for half inactivation and modest negative shift in half activation of CaV3.2. THC prolonged the deactivation time of CaV3.2, while THCA did not. THCA inhibited the peak current of CaV3.3 by 43%±2% (10 μM) but did not notably affect CaV3.3 channel activation or inactivation; however, THC caused significant hyperpolarizing shift in CaV3.3 steady-state inactivation. Discussion: THCA modulated T-type ICa currents in vitro, with significant modulation of kinetics and voltage dependence at low μM concentrations. This study suggests that THCA may have potential for therapeutic use in pain and epilepsy through T-type calcium channel modulation without the unwanted psychoactive effects associated with THC.

Genetic Ablation of NCX1.1 Na+-dependent Inactivation Impacts Cardiac Action Potential and Ca2+ Transient

The cardiac Na+-Ca2+ exchanger (NCX) influences Ca2+ homeostasis therefore cardiac contractility, by extruding one Ca2+ for every three Na+ ions entering the myocyte. Intracellular Na inhibits NCX activity causing an inactivation process known as Na-dependent inactivation. Modulation of NCX by Na is assumed to be physiologically irrelevant, as relatively high [Na] is required for inactivation (half inactivation ∼15-20 mM). To challenge this view, we used CRISPR technology to genetically modify NCX1.1 to remove the Na dependent inactivation from NCX via K229Q mutation. This mutation exclusively removes Na+-dependent inactivation without affecting other regulatory properties (Ca2+ and pH) of NCX (Matsouka et al, JGP 109:273-86, 1997). Adult ventricular myocytes were isolated from K229Q and WT mice. We used the patch clamp technique to measure action potential duration (APD) and NCX current. Compared to WT, the APD of homozygous K229Q myocytes was prolonged (APD90 ms: K229Q 104±25, n=5; WT 43±8, n=3; 35°C, 1Hz). Nickel sensitive NCX currents were measured in the presence of 25 mM intracellular Na+ to promote inactivation. NCX current density was larger in K229Q homozygous mice (pA/pF: K229Q 0.98±0.14 n=5; and WT 0.55±0.08 n=5; 30°C, 50 mV). Ca2+ transients were recorded from field stimulated WT and K229Q myocytes loaded with the Ca2+ sensitive dye Fluo-4, and paced from 0.2 to 5 Hz, 35°C. The Ca2+ transient peak to half time decay was 160±10 ms (n=8) in WT and 205±16 ms (n=11) in K229Q myocytes (1 Hz). These results suggest the Na+-dependent inactivation, heretofore thought of as physiologically irrelevant, actually has an impact on cardiac myocytes properties.

Abstract 191: Difference in Calcium Sensitivity Between Right and Left Ventricles With Lower Expression of Calcium Binding Proteins in Right Ventricular Myocytes

Left ventricle (LV) and right ventricle (RV) have distinctive structural and functional characteristics as well as heterogeneous physiological properties. The RV is exposed to a relatively low pulmonary vasculature, which results in less mechanical afterload. Consistent with these physiological differences, the RV has a thinner free wall than the LV, and the movement of its contraction is geometrically different. Despite these definite differences, the studies of basic excitation-contraction coupling and calcium homeostasis of RV has been less studied than in LV. To establish the interventricular difference, we evaluated the basic electrophysiological and calcium-contractile properties of myocyte with or without β-adrenergic stimulation. Analyses of contraction and Ca 2+ signaling and action potential duration (APD) in isolated RV myocytes showed more prominent APD prolongation with less significant changes in sarcomere shortening and calcium transient, implying less efficient E-C coupling RV myocytes. To investigate the prolonged APD of RV, we measured transient outward potassium (Ito) current and L-type calcium channel (LTCC) current of two ventricles. Although the peak current of two ion channel was not different significantly in two ventricle, the half inactivation voltage of RV Ito was smaller than LV. Comparing with LV, RV myocytes showed round peak, slower early relaxation, and faster late relaxation, suggesting the difference of calcium sensitivity between two ventricles. To investigate the difference, we examined the expression level of calcium-binding proteins that regulate myofilament activities. Using immunoblotting from enriched protein of myofilament fraction, we found that the calcium binding proteins such as troponin I were lower in RV. Taken together, our results suggest that calcium binding proteins of RV was differ from that of LV, which induce the modified calcium sensitivity. We confirmed that reduced calcium binding proteins induced the decrease of calcium sensitivity and modified E-C coupling using Bers’ mathematical rat cardiomyocyte model. The calcium sensitivity of RV is a clue to explain the different physiological properties of the RV.

Biophysical and Pharmacological Profiling of Multiple Voltage-Gated Sodium Channel Subtypes on QPatch II

Voltage - gated sodium channels (VGSC) are responsible for the initiation and propagation of action potentials in excitable cells. VGSC have been identified as excellent drug targets for treatment of pain, epilepsy and to other neurological disorders. Early compounds, however, were developed using empirical approaches. The identification of the molecular identity of VGSC in combination with technological advances, such as the automated patch clamp technique, provide the basis for a rational design of subtype – selective compounds. To date, 9 functional mammalian isoforms (NaV1.1–1.9) have been described in the literature. The various subtypes differ in their expression pattern and exhibit distinct biophysical and pharmacological profiles. All have in common that they produce a transient inward current in response to membrane depolarization. During this process the VGSC transitions from a closed to and open into an inactivated state. Interestingly, inhibitor compounds often exhibit different pharmacological profiles dependent upon the ion channel conformational state. In the present study, the second generation QPatch (QPatch II; Sophion Bioscience) was used in combination with adaptive voltage protocols to investigate state-dependent inhibition of tetrodotoxin (TTX) and tetracaine on 8 different VGSC subtypes (NaV1.1-8). A first step was to determine the half inactivation potential V½(inactivation) for each individual cell. This value was then used during the next steps as preconditioning pulse. Such an adaptive protocol allowed to determine IC50 values for both the closed and the inactivated state and reduce heterogeneity of the cells. Both IC50 values and biophysical parameters of the different subtypes align well with literature values.

Communication—Accessing Stability of Oxidase-Based Biosensors via Stabilizing the Advanced H2O2 Transducer

Operational stability of biosensors is of particular importance especially for wearable devices. Prussian Blue (PB) based advanced hydrogen peroxide transducer, 1000 times more active and selective than platinum, is deposited onto screen-printed structures and stabilized with nickel hexacyanoferrate (NiHCF), both in open circuit mode. Operational stability of PB-NiHCF bilayer based biosensors and labile lactate oxidase is significantly improved in terms of twice longer half inactivation and ≈3.5 times lower inactivation constant. The dynamic range of PB-NiHCF based biosensors is similar to it for conventional PB based ones, which allows using the former for similar purposes drastically improving their performance characteristics.

0218 : Multiple components of voltage-activated calcium current in rat pulmonary vein cardiomyocytes

Ectopic activity arising from cardiomyocytes (CM) in the pulmonary vein (PV) plays a prominent role in the onset of atrial fibrillation in humans. Norepinephrine induces an automatic activity occurring in bursts in rat PV CM (but not in left atria; LA) which depends on Ca channels. Thus, the aim of this study was to compare the Ca current I Ca in rat PV and LA CM (and for comparison, left ventricle – LV) from adult male Wistar rats. Whole cell I Ca (2mM CaCl 2 ) was recorded with classical Na + - and K + -free solutions. Peak I Ca density at +15mV is significantly higher in PV (4.64±0.20pA/pF, n=56) than in LA CM (3.44±0.19pA/pF, n=25, P<0.01) and identical to that of LV CM (5.00±0.39 pA/pF, n=25). The IV curve is biphasic in PV and LA CM due to an I Ca T-like current present between -55 and -30mV representing respectively 10% and 5% of maximum peak I Ca . Although it is insensitive to nifedipine (NIF, 5 and 10μM), increased by CaCl 2 (2 to 5mM) and suppressed by its removal, the lack of effect of NiCl 2 (40μM) and of TTA-A2 (10 and 100nM) suggest that Ca v 3.1 and Ca v 3.2 expressed in the rat heart are not involved in this fast activating and inactivating current. Moreover, its blockade by 10μM TTX and its mid inactivation potential of –85 mV strongly suggest that this I Ca TTX is due to Ca permeation through Na channels. A second TTX-insensitive low threshold activated I Ca with slower kinetics is also present (half inactivation at –65mV). NIF (5μM) inhibited the major high-threshold I Ca component activating at ~-30mV with the sequence LV (-80.0±5.0%, n=6) > LA (–71.0±5.6%, n=5) ≥ PV (-63.4±3.05%, n=9) and NiCl 2 (40μM) with PV (–78.6±4.8%, n=9) > LA (-69.9±4.2%, n=6) > LV (-62.2±3.9%, n=6), suggesting that part of the current flows through Ca v 3.2 channels. However, TTAA2 (10nM) was found not so specific of Ca v 3.x as it partially inhibited I Ca in both PV and LV CM and decreased NIF efficacy. In conclusion, at least three components of Ca current are present in rat PV and LA CM. The author hereby declares no conflict of interest

Calmodulin kinase II inhibitor regulates calcium homeostasis changes caused by acute β-adrenergic receptor agonist stimulation in mouse ventricular myocytes.

Ca(2+)/calmodulin-dependent kinase II (CaMKII) is an important regulatory molecule under chronic β-adrenergic receptor agonist stimulation but cardiac diseases also occur when β-adrenergic elevated acutely in the circulation, of which the most harmful is lethal arrhythmia. The purpose of this study was to explore the effects of acute isoproterenol (ISO) stimulation on intracellular calcium handling and evaluate whether CaMKII inhibitor may change the effects caused by isoproterenol. Mouse ventricular myocytes were acutely isolated by enzymatic method and divided into four groups: control group, ISO group, KN-93 group, ISO + KN-93 group. The whole-cell patch clamp was used to study the effect of ISO and KN-93 on L-type calcium current (IL-Ca) in isolated ventricular myocytes. The technology of laser scanning confocal microscopy was used to record cardiomyocyte calcium transients after ISO and KN-93 were used. ISO significantly increased current density of IL-Ca (p < 0.01) and decreased the half activation voltage (p < 0.01), half inactivation voltage (p < 0.01), and the recovery time constant (p < 0.01). In the presence of CaMKII inhibitor, KN-93 decreased the increased current density of IL-Ca (p < 0.05), increased the reduced half activation voltage caused by ISO (p < 0.01), and prolonged the shortened recovery time constant caused by ISO (p < 0.01). In addition, KN-93 alone can change the activation, inactivation, and recovery kinetics of L-type calcium channels. Moreover, ISO significantly increased the Ca(2+) transient amplitude during both stimulation frequencies (0.5 Hz: p < 0.01, 1 Hz: p < 0.01) and was easy to induce calcium disorders; in the presence of KN-93, these changes were weakened (0.5 Hz: p < 0.05, 1 Hz: p < 0.05). Therefore, changes of the calcium homeostasis in cardiomyocytes caused by ISO can be adjusted by KN-93, thus KN-93 plays a vital role in regulating calcium homeostasis changes caused by ISO.

Mechanisms of ion channel in irritable bowel syndrome caused by depression induced abnormal colon motility in rats

Objective To investigate mechanisms of ion channel in irritable bowel syndrome (IBS) caused by depression induced abnormal colon motility in rats. Methods Adult male SD SPF rats were divided into control group and depression group, 15 rats in each group. The depression models were induced by chronic unpredictable mild stress stimulation. After 28 days, the success of depression model was confirmed by open field test (OFT) and fluid consumption test (FCT). The gastrointestinal reaction was determined by clinical symptoms, fecal character and times.Abdominal contraction reflex threshold was calculated to evaluate the changes of visceral sensitivity after colorectal distention and to determine the incidence of IBS. Single rat colonic myocytes were isolated by enzyme method. The changes of L-type calcium current (ICa-L) and kinetics properties were recorded via whole-cell patch clamp technique. The t test was performed for comparison between two groups and Pearson method was for correlation analysis. Results Compared with control group, the body weight, OFT and FCT of the rats in depression group were significantly decreased. The rats of depression group have severe body weakness symptoms, the particle number and water content of fecal increasing, capacity threshold significantly decreased and which was negative correlated with OFT and FCT (r=-0.89 and 0.91, both P<0.01). The incidence of IBS was 14/15. Compared with control group, ICa-Lof colonic myocytes of rats in depression group was significantly increased, ICa-Lof control group was (-21.31±4.79) pA and that of depression group was (-33.52±4.45) pA, and which was positive correlated with the particle number and water content of fecal of depression rats (r=0.91 and 0.91, both P<0.01). Moreover the current-voltage (I-V) curve of control group was at middle and of depression group was at the bottom. The steady-state activation curve of ICa-L of depression group markedly left shifted, and the maximum half activated voltages decreased from (-7.96±5.95) mV of control group to (-14.81±3.33) mV. Moreover, recovery of inactivation of ICa-Lwas fast, the recovery time of maximum half inactivation was shorten from (393.28±41.79) ms of control group to (163.29±27.34) ms, 58.48% in advance. Conclusions Depression could induce obvious IBS in gastrointestinal of rats. The mechanisms maybe depression causes ICa-Lof colonic myocytes abnormally increasing, steady-state activation gate open in advance, accelerate recovery time of inactivation and ion channel remodeling of ICa-L. Key words: Depression; Colonic smooth muscle cells; L-type calcium current/Kinetics; Irritable bowel syndrome; Electrical remodeling

The Capacity of Lignin to Enhance the Phthorimaea opercullela Granulovirus (PhopGV) Stability in Laboratory Condition

The lab persistence of Phthorimaea opercullela granulovirus (PhopGV) with and without lignin for egg bioassay with different five virus concentrations (lab experiment- 60, 10, 1.67, 0.28 and 0.046 LE/l 20- 0.015432 LE/l ) to determine the inactivation curve under artificial UVlight, assess the effect and verify the protective capacity of lignin with PhopGV for virus preparations suspensions of raw PhopGV alone and mixed with lignin (1%) used as UV protectants was conducted in simply completely randomised design in the laboratory condition. The LC50 value for PhopGV in lab condition was 1x10-3 LE/l with the slope of the probit regression line 0.51. Similarly, half inactivation speed with lignin was 4.58 days compared to 0.559 days without lignin in lab condition. Inactivation speed was decreased by 87.8 % in case of lignin applied in lab condition. So, the suppression of PTM with virus combined with lignin gave cost effective measures through the use of low dosages. In lab experiment, the control mortality of 19.6 % was probable for all the controls of lignin present and absent. This revealed that there was no effect of lignin alone on target insects’ survival.Int J Appl Sci Biotechnol, Vol 3(2): 256-260 DOI: http://dx.doi.org/10.3126/ijasbt.v3i2.12481

Site-Directed Mutagenesis of a Neutral Phytase from &lt;i&gt;Bacillus amyloliquefaciens&lt;/i&gt;: Influencing Activity and Stability

In order to improve the activity and stability of phytase fromBacillus amyloliquefaciens, site-directed mutagenesis has been performed base on the previous recombinantE.coliBL21 harboring the expression vector ofphyC. Mutation residues were chosen based on the sequence alignments and structure analysis of neutral phytsaes from different microorganisms. Site-directed mutagenesis techniques were used to get three mutants (D148E/H149R, Q67E/N68R, and D191E), then the mutants were expressed and purified. Enzymatic characters of different mutants were investigated. The results indicated that the optimum pH of all mutants were 7.0, and the optimum temperature were between 65 °C–70 °C. The maximum specific activity of mutant D148E/H149E was 27.84 U/mg which was 2.19 times than that of the wild-type phytase. The half inactivation temperature of D191E was 4.5 °C higher than that of the wild-type phytase. Fluorescence emission spectra showed that slight differences were among the structures of the mutant phytases. The phytases described here which have increased activity and thermostability may have promosing potential as feed additives in animal diets.

The hypotensive agent dodoneine inhibits L-type Ca2+ current with negative inotropic effect on rat heart

Agelanthus dodoneifolius is one of the medicinal plants used in African pharmacopeia and traditional medicine for the treatment of cardiovascular diseases. A chemical analysis has identified one of the active principles: Dodoneine (Ddn). It is a new dihydropyranone which exerts hypotensive and vasorelaxant effects on rat. Since the mechanism of the hypotensive effect is unknown, we performed a variety of preclinical and mechanistic studies to characterize the specific cardiac effect of Ddn at tissue (ex-vivo) and cellular levels (in-vitro) in order to determine a molecular target. Ddn effects were evaluated in an isolated rat heart preparation using Langendorff retrograde perfusion and then, the effects of Ddn were characterized in freshly dissociated cardiac ventricular myocytes using the whole-cell patch-clamp configuration. Ex-vivo, Ddn produced a dose-dependent negative inotropic effect with an IC50 value of 10µM without changed heart rate. 100µM Ddn decreased left ventricular developed pressure of about 40%. In isolated cardiac myocytes, Ddn reduced ICa,L density of about 30% with an IC50 value estimated at 3µM. Ddn did not change current–voltage relation but it shifted the inactivation curve toward negative potentials and modified the half inactivation potentials. Furthermore, Ddn induced a phasic-dependent blocking on ICa,L. This study demonstrates that the hypotensive property of dodoneine is likely associated with a negative inotropic effect and the blockade of the L-type calcium channels.

Atrial Selectivity in Sodium Channel Block by Amiodarone

Introduction: Na+ channel blockers are usually applied to atrial fibrillation (AF), but may sometimes cause cardiac contractile dysfunction. However, amiodarone, a multi-channel blocker with Na+ channel block, does not induce cardiac dysfunction. In this study we tested the hypothesis that Na+ channel block by amiodarone is selective in atrial myocytes (AM) compared to ventricular myocytes (VM). Methods and Results: Na+ currents (INa) and resting membrane potentials (RMPs) were measured using whole cell patch-clamp technique in isolated rabbit AM and VM. Amiodarone inhibited INa in AM (IC50: 1.4 ± 0.3μM; n=8) much more than in VM (40.4 ± 11.9μM; n=7; P<0.01). Amiodarone at 10μM dramatically shifted steady state inactivation curve to hyperpolarized direction in AM (−19.6 ± 2.1mV shift; n=12) compared to VM (−6.3 ± 0.8mV shift; n=13; P<0.01). In mexiletine, there was no significant difference in INa inhibition between AM and VM. The shifts of inactivation curves by mexiletine at 10μM were comparable in AM and VM. RMPs in AM (−75.0 ± 1.3mV; n=4) were more depolarized than in VM (−82.1 ± 1.1mV; n=9; P<0.01). In the absence of drugs, the half inactivation voltage in AM (V1/2: −89.7 ± 0.9mV; n=19) was 12.5 mV more negative than VM (−77.2 ± 0.6mV; n=20; P<0.01). Furthermore, we evaluated the effects of amiodarone and mexiletine on conduction velocity (CV) in Langendorff-perfused rabbit hearts by optical mapping system. The decrease of CV by amiodarone at 5μM was significantly larger in atrium (−34.3 ± 5.6%; n=5) compared to ventricle (−4.8 ± 1.0%; n=5; P<0.01). However, the reduction of CV by mexiletine at 5uM in atrium was smaller than in ventricle. Conclusion: Amiodarone preferentially inhibits INa of AM compared to VM. This atrial-selective Na+ channel block by amiodarone may contribute to treating AF without affecting ventricular contractility.

Characterization of a cold-adapted glutathione synthetase from the psychrophile Pseudoalteromonas haloplanktis

Glutathione (GSH) biosynthesis occurs through two ATP-dependent reactions, usually involving distinct enzymes; in the second step of this process, catalysed by glutathione synthetase (GshB), GSH is formed from γ-glutamylcysteine and glycine. A recombinant form of GshB from the cold-adapted source Pseudoalteromonas haloplanktis (rPhGshB) was purified and characterised. The enzyme formed a disulfide adduct with β-mercaptoethanol, when purified in the presence of this reducing agent. The homotetrameric form of rPhGshB observed at high protein concentration disassembled into two homodimers at low concentration. A new method for directly determining the rPhGshB activity was developed, based on [γ-(32)P]ATP hydrolysis coupled to the GSH synthesis. The ATPase activity required the presence of both γ-glutamylcysteine and glycine and its optimum was reached in the 7.4-8.6 pH range; a divalent cation was absolutely required for the activity, whereas monovalent cations were dispensable. rPhGshB was active at low temperatures and had a similar affinity for ATP (K(m) 0.26 mM) and γ-glutamylcysteine (K(m) 0.25 mM); a lower affinity was measured for glycine (K(m) 0.75 mM). The oxidised form of glutathione (GSSG) acted as an irreversible inhibitor of rPhGshB (K(i) 10.7 mM) and formed disulfide adducts with the enzyme. rPhGshB displayed a great temperature-dependent increase in its activity with an unusually high value of energy of activation (75 kJ mol(-1)) for a psychrophilic enzyme. The enzyme was moderately thermostable, its half inactivation temperature being 50.5 °C after 10 min exposure. The energy of activation of the heat inactivation process was 208 kJ mol(-1). To our knowledge, this is the first contribution to the characterization of a GshB from cold-adapted sources.

Purification and the Secondary Structure of Fucoidanase from Fusarium sp. LD8

The fucoidanase from Fusarium sp. (LD8) was obtained by solid-state fermentation. The fermented solid medium was extracted by citric acid buffer, and the extracts were precipitated by acetone and purified by Sephadex G-100 successively. The results showed that the specific fucoidanase activity of purified enzyme was 22.7-fold than that of the crude enzyme. The recovery of the enzyme was 23.9%. The purified enzyme gave a single band on SDS-PAGE gel, and the molecular weight of fucoidanase was about 64 kDa. The isoelectric point of the enzyme was 4.5. The enzyme properties were also studied. The results showed that the optimum temperature and pH were 60°C and 6.0, respectively; the temperature of half inactivation was 50°C, and the most stable pH for the enzyme was 6.0. K M, and the V max of the enzyme was 8.9 mg·L−1 and 2.02 mg·min−1 ·mL−1 by using fucoidan from Fucus vesiculosus as substrate. The compositions of the secondary structure of fucoidanase were estimated by FTIR, the second derivative spectra, and the curve-fitting analysis of the amide I bands in their spectra. The results showed that β-sheet was the dominant component (58.6%) and α-helix was the least (12%); the content of β-turn and random coil were 15.39% and 14.5%, respectively.